Metal Ions in Toxicology: Effects, Interactions, Interdependencies

Volume METAL IONS
8 IN LIFE SCIENCES
Metal Ions in Toxicology: Effects, Interactions,

Interdependencies
Editors Astrid Sigel,1 Helmut Sigel,1 and Roland K. O. Sigel 2
1
Department of Chemistry, Inorganic Chemistry, University of Basel, CH-4056 Basel, Switzerland
2
Institute of Inorganic Chemistry, University of Zürich, CH-8057 Zürich, Switzerland
About this Book

Volume 8, solely devoted to the toxicology of metals and metalloids as well as their compounds, focuses on human health. Not
surprisingly, all related research areas are rapidly developing due to the role of metals and metalloids in the environment, for
the work place, for food and water supply, etc. Written by 40 internationally recognized experts, the 14 stimulating chapters
provide an authoritative and timely resource for scientists working in the wide range from analytical, physical, inorganic, and
environmental biochemistry all the way through to toxicology, physiology, and medicine.
Metal Ions in Toxicology: Effects, Interactions, Interdependencies highlights, supported by nearly 1900 references, in an
authoritative and timely manner the principles of risk assessment regarding the effects of metals on human health. It examines how
metal ions and their compounds affect the pulmonary, cardiovascular, gastrointestinal (including liver), hematological, immune,
and neurological systems, the kidney, skin and eyes, as well as human reproduction and development. MILS-8 terminates with the
role of metal ions as endocrine disrupters, in genotoxicity, and in cancer risk.
About the Series

Metal Ions in Life Sciences links coordination chemistry and biochemistry in their widest sense and thus increases our
understanding of the relationship between the chemistry of metals and life processes. The series reflects the interdisciplinary
nature of Biological Inorganic Chemistry and coordinates the efforts of scientists in fields like biochemistry, inorganic
chemistry, coordination chemistry, molecular and structural biology, enzymology, environmental chemistry, physiology,
toxicology, biophysics, pharmacy, and medicine. Consequently, the volumes are an essential source for researchers active in
these and related fields as well as teachers preparing courses, e.g., in Bioinorganic Chemistry.
About the Editors

Astrid Sigel has studied languages and was an editor of the Metal Ions in Biological Systems series (until Volume 44) and
also of the Handbooks on Toxicity of Inorganic Compounds (1988), on Metals in Clinical and Analytical Chemistry (1994;
both with H. G. Seiler) and on Metalloproteins (2001; with Ivano Bertini).
Helmut Sigel is Emeritus Professor (2003) of Inorganic Chemistry at the University of Basel, Switzerland, and a previous
editor of the MIBS series until Volume 44. He serves on various editorial and advisory boards, published over 300 articles on
metal ion complexes of nucleotides, coenzymes, and other ligands of biological relevance, and lectured worldwide. He was
named Protagonist in Chemistry (2002) by ICA (issue 339); among further honors are the P. Ray Award (Indian Chemical
Society, of which he is also an Honorary Fellow), the Alfred Werner Prize (Swiss Chemical Society), a Doctor of Science
honoris causa degree (Kalyani University, India), appointments as Visiting Professor (e.g., Austria, China, Japan, Kuwait,
UK) and Endowed Lectureships.
Roland K. O. Sigel is Associate Professor (2009) of Inorganic Chemistry at the University of Zürich, Switzerland; from 2003
to 2008 he was endowed with a Förderungsprofessur of the Swiss National Science Foundation. He received his doctoral
degree summa cum laude (1999) from the University of Dortmund, Germany, working with Bernhard Lippert. Thereafter he
spent nearly three years at Columbia University, New York, USA, with Anna Marie Pyle (now Yale University). During the
six years abroad he received several prestigious fellowships from various sources, and he was awarded the EuroBIC Medal
in 2008 and the Alfred Werner Prize (SCS) in 2009. His research focuses on the structural and catalytic role of metal ions in
ribozymes, especially group II introns, and on related topics. He was also an editor of Volumes 43 and 44 of the MIBS series.
ISSN 1559-0836
DOI 10.1039/9781849730914
ISBN 978-1-84973-091-4
9 781849 730914
www.rsc.org/books
METAL IONS
IN LIFE SCIENCES
VOLUME 8
Metal Ions in Toxicology:

Effects, Interactions, Interdependencies
METAL IONS
IN LIFE SCIENCES
edited by
Astrid Sigel,(1) Helmut Sigel,(1) and Roland K. O. Sigel(2)
(1)
Department of Chemistry
Inorganic Chemistry
University of Basel
Spitalstrasse 51
CH-4056 Basel, Switzerland
(2)
Institute of Inorganic Chemistry
University of Zürich
Winterthurerstrasse 190
CH-8057 Zürich, Switzerland
VOLUME 8

The figure on the dust cover shows Table 1 of Chapter 3 by Moiz Mumtaz,
Hugh Hansen, and Hana R. Pohl.
ISBN: 978-1-84973-091-4
ISSN: 1559-0836
DOI: 10.1039/9781849732116
A catalogue record for this book is available from the British Library
r Royal Society of Chemistry 2011
All rights reserved
Apart from fair dealing for the purposes of research for non-commercial purposes or
for private study, criticism or review, as permitted under the Copyright, Designs
and Patents Act 1988 and the Copyright and Related Rights Regulations 2003, this
publication may not be reproduced, stored or transmitted, in any form or by any means,
without the prior permission in writing of The Royal Society of Chemistry or the
copyright owner, or in the case of reproduction in accordance with the terms of licences
issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of
the licences issued by the appropriate Reproduction Rights Organization outside the
UK. Enquiries concerning reproduction outside the terms stated here should be sent to
The Royal Society of Chemistry at the address printed on this page.
The RSC is not reponsible for individual opinions expressed in this work.
Published by The Royal Society of Chemistry,

Thomas Graham House, Science Park, Milton Road,
Cambridge CB4 0WF, UK
Registered Charity Number 207890
For further information see our web site at www.rsc.org

Historical Development and Perspectives
of the Series
Metal Ions in Life Sciences*
It is an old wisdom that metals are indispensable for life. Indeed, several
of them, like sodium, potassium, and calcium, are easily discovered in liv-
ing matter. However, the role of metals and their impact on life remained
largely hidden until inorganic chemistry and coordination chemistry
experienced a pronounced revival in the 1950s. The experimental and the-
oretical tools created in this period and their application to biochemical
problems led to the development of the field or discipline now known as
Bioinorganic Chemistry, Inorganic Biochemistry, or more recently also
often addressed as Biological Inorganic Chemistry.
By 1970 Bioinorganic Chemistry was established and further promoted by
the book series Metal Ions in Biological Systems founded in 1973 (edited by
H.S., who was soon joined by A.S.) and published by Marcel Dekker, Inc.,
New York, for more than 30 years. After this company ceased to be a family
endeavor and its acquisition by another company, we decided, after having
edited 44 volumes of the MIBS series (the last two together with R.K.O.S.)
to launch a new and broader minded series to cover today’s needs in the Life
Sciences. Therefore, the Sigels new series is entitled
Metal Ions in Life Sciences.
After publication of the first four volumes (2006–2008) with John Wiley &
Sons, Ltd., Chichester, UK, we are happy to join forces now in this still new
endeavor with the Royal Society of Chemistry, Cambridge, UK; a most
experienced Publisher in the Sciences.
*
Reproduced with some alterations by permission of John Wiley & Sons, Ltd.,
Chichester, UK (copyright 2006) from pages v and vi of Volume 1 of the series Metal
Ions in Life Sciences (MILS-1).
vi PERSPECTIVES OF THE SERIES
The development of Biological Inorganic Chemistry during the past 40

years was and still is driven by several factors; among these are (i) the
attempts to reveal the interplay between metal ions and peptides, nucleo-
tides, hormones or vitamins, etc., (ii) the efforts regarding the understanding
of accumulation, transport, metabolism and toxicity of metal ions, (iii) the
development and application of metal-based drugs, (iv) biomimetic synth-
eses with the aim to understand biological processes as well as to create
efficient catalysts, (v) the determination of high-resolution structures of
proteins, nucleic acids, and other biomolecules, (vi) the utilization of pow-
erful spectroscopic tools allowing studies of structures and dynamics, and
(vii), more recently, the widespread use of macromolecular engineering to
create new biologically relevant structures at will. All this and more is and
will be reflected in the volumes of the series Metal Ions in Life Sciences.
The importance of metal ions to the vital functions of living organisms,
hence, to their health and well-being, is nowadays well accepted. However,
in spite of all the progress made, we are still only at the brink of under-
standing these processes. Therefore, the series Metal Ions in Life Sciences
will endeavor to link coordination chemistry and biochemistry in their
widest sense. Despite the evident expectation that a great deal of future
outstanding discoveries will be made in the interdisciplinary areas of science,
there are still ‘‘language’’ barriers between the historically separate spheres
of chemistry, biology, medicine, and physics. Thus, it is one of the aims of
this series to catalyze mutual ‘‘understanding’’.
It is our hope that Metal Ions in Life Sciences proves a stimulus for new
activities in the fascinating ‘‘field’’ of Biological Inorganic Chemistry. If so, it
will well serve its purpose and be a rewarding result for the efforts spent by
the authors.
Astrid Sigel, Helmut Sigel Roland K. O. Sigel

Department of Chemistry Institute of Inorganic Chemistry
Inorganic Chemistry University of Zürich
University of Basel CH-8057 Zürich
CH-4056 Basel Switzerland
Switzerland
October 2005
and October 2008
Preface to Volume 8
The preceding Volume 7, Organometallics in Environment and Toxicology, is

somewhat related to the present one, although it concentrates on organo-
metallic compounds. The volume at hand, however, focuses on the effect of
metals and metalloids on human health. Volume 8 opens with three general
chapters, beginning with the aim to understand combined effects of metal
co-exposure in ecotoxicology. Indeed, it is a particular challenge to assess the
potential of deleterious biological effects occurring from environmental
exposure, including work place, food and water supply, to metal mixtures.
Therefore, Chapters 2 and 3 are devoted to the risk assessment of metals and
metalloids for humans and the underlying principles. Considering that a
variety of health risks exist, agencies have provided health-based guidance
values to prevent the occurrence of adverse health effects in humans, though
it is clear that in the future new and innovative interdisciplinary approaches
and shared technologies between consortia are needed.
Chapters 4 through 11 describe and summarize how metal ions, metal
compounds, and metalloids affect the pulmonary and cardiovascular sys-
tems, the gastrointestinal system including the liver, the kidney, the hema-
tological system, the immune system, skin and eyes, and the neurological
system as well as human reproduction and development. Indeed, many metal
ions and their compounds (As, Cd, Cr, Cu, Hg, Li, Ni, Pb, V) exert a wide
variety of adverse effects including their influence on male and female
subfertility or fertility, on abortions, malformations, birth defects, and
developmental effects, which occur mainly in the central nervous system.
Metal Ions in Life Sciences, Volume 8 Edited by Astrid Sigel, Helmut Sigel, and Roland K. O. Sigel
Published by the Royal Society of Chemistry, www.rsc.org DOI: 10.1039/9781849732116FP007
viii PREFACE TO VOLUME 8
Are cadmium and other heavy metal compounds acting as endocrine

disrupters? This question is addressed in Chapter 12: The realization that
cadmium compounds and other heavy metals are capable of activating the
estrogen receptor has not only spawned extensive research, but has also
raised concerns about their role as risk factors in hormone-related cancers
and other endocrine disorders. Indeed, despite existing inconsistencies, the
available evidence forces the conclusion that cadmium and certain other
heavy metals should be regarded as estrogen mimicks.
The two terminating Chapters 13 and 14 are devoted to the genotoxicity of
metal ions and their role in human cancer development. Special attention is
paid to the underlying chemical mechanisms and in Chapter 13 the geno-
toxicity of metal ions is defined as the damage to cellular DNA with genetic
consequences. Chapter 14 focuses on metallic agents that are known to be
human carcinogens, that is, on arsenic, beryllium, cadmium, chromium(VI),
nickel, and their compounds. It covers further probable and possible human
metallic carcinogens, like inorganic lead compounds, cisplatin (cis-diammi-
nedichloroplatinum(II)), indium phosphide, and certain cobalt compounds;
potential mechanisms of metal carcinogenesis are discussed.
Special thanks go to Dr. Hana R. Pohl (Agency for Toxic Substances and
Disease Registry, U.S. Department of Health and Human Services, Atlanta,
GA, USA) for her help in initiating this volume and the valuable advice
provided.
Astrid Sigel
Helmut Sigel
Roland K. O. Sigel
Contents
HISTORICAL DEVELOPMENT
AND PERSPECTIVES OF THE SERIES v
PREFACE TO VOLUME 8 vii
CONTRIBUTORS TO VOLUME 8 xvii
TITLES OF VOLUMES 1–44 IN THE
METAL IONS IN BIOLOGICAL SYSTEMS SERIES xxi
CONTENTS OF VOLUMES IN THE
METAL IONS IN LIFE SCIENCES SERIES xxiii
1 UNDERSTANDING COMBINED EFFECTS FOR METAL

CO-EXPOSURE IN ECOTOXICOLOGY 1
Rolf Altenburger
Abstract 2
1. Ecotoxicity from Mixture Exposure 2
2. Combination Effect Analysis 6
3. Interactions During Exposure 13
4. Joint Action in Toxicodynamics 17
5. Interaction with Organic Compounds 21
6. Outlook 24
Acknowledgments 25
Abbreviations 25
References 25
Published by the Royal Society of Chemistry, www.rsc.org DOI: 10.1039/9781849732116FP009
x CONTENTS
2 HUMAN RISK ASSESSMENT OF HEAVY METALS:

PRINCIPLES AND APPLICATIONS 27
Jean-Lou C. M. Dorne, George E. N. Kass, Luisa R. Bordajandi,
Billy Amzal, Ulla Bertelsen, Anna F. Castoldi, Claudia Heppner,
Mari Eskola, Stefan Fabiansson, Pietro Ferrari, Elena Scaravelli,
Eugenia Dogliotti, Peter Fuerst, Alan R. Boobis, and Philippe
Verger
Abstract 28
1. Introduction 29
2. Principles of Chemical Risk Assessment 29
3. Toxicology of Heavy Metals 34
4. Analytical Techniques and Exposure Assessment of Heavy
Metals 39
5. Applications to the Human Risk Assessment of Heavy
Metals and Metalloids 43
6. Conclusions and Future Perspectives 53
Acknowledgments 54
Abbreviations and Definitions 54
References 54
3 MIXTURES AND THEIR RISK ASSESSMENT IN

TOXICOLOGY 61
Moiz M. Mumtaz, Hugh Hansen, and Hana R. Pohl
Abstract 62
1. Introduction 62
2. Predictions of Toxicity Outcomes 64
3. Weight-of-Evidence Evaluations 66
4. Experimental Validations 68
5. Conclusion 77
Abbreviations 77
References 77
4 METAL IONS AFFECTING THE PULMONARY AND

CARDIOVASCULAR SYSTEMS 81
Massino Corradi and Antonio Mutti
Abstract 82
1. Introduction 83
2. Aluminum 83
3. Arsenic 84
CONTENTS xi
4. Beryllium 85
5. Copper 86
6. Cadmium 87
7. Chromium 89
8. Cobalt 92
9. Lead 93
10. Manganese 95
11. Nickel 97
12. Zinc 98
13. Concluding Remarks 99
References 100
5 METAL IONS AFFECTING THE GASTROINTESTINAL

SYSTEM INCLUDING THE LIVER 107
Declan P. Naughton, Tamás Nepusz, and Andrea Petroczi
Abstract 108
1. Introduction 108
2. Exposure to Metal Ions in the Gastrointestinal Tract and
Liver 110
3. Estimation of Toxicity Associated with Metal Ions in the
Gastrointestinal Tract and Liver 117
4. Metal Ion-Molecular Interactions: Effects on Oxidative
Damage 123
5. Concluding Remarks and Future Directions 127
Abbreviations 127
References 128
6 METAL IONS AFFECTING THE KIDNEY 133

Bruce A. Fowler
Abstract 133
1. Introduction 134
2. Exposure to Metal Ions in Air, Food, and Water 134
3. Transport of Metals/Metalloids in the Circulation 135
4. Mechanisms of Metal and Metalloid Uptake by
the Kidney 136
5. Effects of Metals/Metalloids on the Kidney 136
6. Mechanisms of Renal Cell Injury 137
7. Renal Biomarkers 137
8. Metal/Metalloid Interactions in the Kidney 138
xii CONTENTS

Abbreviations 139
References 139
7 METAL IONS AFFECTING THE HEMATOLOGICAL

SYSTEM 143
Nickolette Roney, Henry G. Abadin, Bruce Fowler, and
Hana R. Pohl
Abstract 144
1. Exposure to Metals and Their Mixtures 144
2. Metals Affecting the Hematological System 145
3. Binary Interactions of Metals and Hematological
Effects 147
4. Interaction of Metals with other Chemicals 152
5. Conclusions 153
Abbreviations 153
References 153
8 METAL IONS AFFECTING THE IMMUNE SYSTEM 157

Irina Lehmann, Ulrich Sack, and Jörg Lehmann
Abstract 157
1. Introduction 158
2. Immunotoxicity and Immunomodulation 159
3. Effect of Heavy Metals on Innate Immunity 160
4. Effect of Heavy Metals on Adaptive Immunity 161
5. Mechanisms of Heavy Metal-Induced
Immunotoxic/Immunomodulatory Effects 166
6. Influence of Heavy Metals on the Resistance Toward
Infections 170
7. Chronic Inflammation and Autoimmunity 173
8. Concluding Remarks 176
Acknowledgments 177
References 178
9 METAL IONS AFFECTING THE SKIN AND EYES 187

Alan B. G. Lansdown
Abstract 188
1. Introduction 188
CONTENTS xiii
2. Metal Ions and Metal Ion Gradients in the Physiology

and Homeostasis of Mammalian Skin 190
3. Xenobiotic Metal Ions 205
4. Carcinogenicity of Metal Ions in the Skin 222
5. The Eye 224
6. General Conclusions 228
Abbreviations 229
References 230
10 METAL IONS AFFECTING THE NEUROLOGICAL

SYSTEM 247
Hana R. Pohl, Nickolette Roney, and Henry G. Abadin
Abstract 248
1. Exposure to Metals and Their Mixtures 248
2. Metals Affecting the Neurological System 249
3. Interaction of Metals and Neurological Effects 253
4. Interactions of Metals with Other Chemicals 256
5. Conclusions 259
Abbreviations 260
References 260
11 METAL IONS AFFECTING REPRODUCTION AND

DEVELOPMENT 263
Pietro Apostoli and Simona Catalani
Abstract 264
1. Introduction 265
2. Time and Duration of Exposure 267
3. Mechanisms of Action 269
4. Reproductive Effects 270
5. Abortions and Other Pregnancy Effects 280
6. Prenatal Exposure and Developmental Effects 283
7. Early Postnatal Exposure and Developmental
Effects 288
8. Concluding Remarks and Needs for Further
Research 293
Abbreviations 294
References 295
xiv CONTENTS
12 ARE CADMIUM AND OTHER HEAVY METAL

COMPOUNDS ACTING AS ENDOCRINE DISRUPTERS? 305
Andreas Kortenkamp
Abstract 306
1. Introduction 306
2. A Model for Estrogen Receptor Activation by Cadmium 307
3. Cadmium Exposure and Cancer Risks in Endocrine-
Sensitive Tissues 308
4. In Vivo Studies of Estrogenic Effects of Cadmium 310
5. Cadmium and Other Heavy Metals in In Vitro Cell-Based
Assays of Estrogenicity 311
6. Weight of Evidence and Implications for Human Risk
Assessment 313
Abbreviations 315
References 315
13 GENOTOXICITY OF METAL IONS: CHEMICAL

INSIGHTS 319
Wojciech Bal, Anna Maria Protas, and Kazimierz S.
Kasprzak
Abstract 320
1. Introduction 321
2. Overview of Chemical and Biochemical Processes Leading
to Genotoxic Lesions 322
3. Mechanisms of Metal Ion Genotoxicity 330
4. Genotoxic Properties of Selected Metals 336
5. Critical Overview of the Experimental Methods for
Studying the Genotoxic Potential of Metals 354
Acknowledgments 358
Abbreviations 358
References 359
14 METAL IONS IN HUMAN CANCER DEVELOPMENT 375

Erik J. Tokar, Lamia Benbrahim-Tallaa, and Michael
P. Waalkes
Abstract 376
1. Introduction 376
CONTENTS xv
2. Known Human Metallic Carcinogens 380

3. Probable and Possible Metallic Carcinogens 388
4. Potential Mechanisms of Metallic Carcinogens 391
5. Periods of Particular Sensitivity to Inorganic Carcinogens 395
6. Future Issues in Metal Carcinogenesis 396
Acknowledgments 397
References 397
SUBJECT INDEX 403

Contributors to Volume 8
Numbers in parentheses indicate the pages on which the authors’

contributions begin.
Henry G. Abadin Agency for Toxic Substances and Disease Registry

(ATSDR), US Dept. of Health and Human Services, Division of Toxicol-
ogy, 1600 Clifton Road, Atlanta, GA 30333, USA (143, 247)
Rolf Altenburger Department of Bioanalytical Ecotoxicology, UFZ Helm-

holtz Centre for Environmental Research, Permoserstrasse 15, D-04318
Leipzig, Germany, Fax: +49-341-235-2401 orolf.altenburger@ufz.de4 (1)
Billy Amzal European Food Safety Authority, Unit on Food Con-

taminants, Largo N. Palli 5/A, I-43100 Parma, Italy (27)
Pietro Apostoli Department of Experimental and Applied Medicine, Unit

of Occupational Medicine and Industrial Hygiene, University of Brescia,
P.le Spedali Civili, 1, I-25123 Brescia, Italy oapostoli@med.unibs.it4 (263)
Wojciech Bal Institute of Biochemistry and Biophysics, Polish Academy of

Sciences, Pawinskiego 5A, PL-02-106 Warsaw, Poland and Central Institute
for Labour Protection, National Research Institute, Czerniakowska 16, PL-
00-701 Warsaw, Poland owbal@ibb.waw.pl4 (319)
Lamia Benbrahim-Tallaa IARC Monographs Section, International

Agency for Research on Cancer, Lyon, France (375)
Ulla Bertelsen European Food Safety Authority, Unit on Food Con-

Alan R. Boobis Imperial College, Department of Experimental Medicine

and Toxicology, Burlington Danes, Hamersmith Campus, Du Cane Road,
London, W12 ONN, UK (27)
xviii CONTRIBUTORS TO VOLUME 8
Luisa R. Bordajandi European Food Safety Authority, Unit on Food

Contaminants, Largo N. Palli 5/A, I-43100 Parma, Italy (27)
Anna F. Castoldi European Food Safety Authority, Unit on Food Con-

Simona Catalani Department of Experimental and Applied Medicine,

Unit of Occupational Medicine and Industrial Hygiene, University of
Brescia, P.le Spedali Civili, 1, I-25123 Brescia, Italy ocatalani@med.
unibs.it4 (263)
Massimo Corradi Department of Clinical Medicine, Nephrology and

Health Sciences, University of Parma, Via Gramsci 14, I-43100 Parma, Italy
omassimo.corradi@unipr.it4 (81)
Eugenia Dogliotti Istituto Superiore di Sanità, Viale Regina Elena, 299,

I-00161 Rome, Italy (27)
Jean-Lou C. M. Dorne European Food Safety Authority, Unit on Food

Contaminants, Largo N. Palli 5/A, I-43100 Parma, Italy
ojean-lou.dorne@efsa.europa.eu4 (27)
Mari Eskola European Food Safety Authority, Unit on Food Contaminants,

Largo N. Palli 5/A, I-43100 Parma, Italy (27)
Stefan Fabiansson European Food Safety Authority, Unit on Food Con-

Pietro Ferrari European Food Safety Authority, Unit on Food Con-

Bruce A. Fowler Agency for Toxic Substances and Disease Registry

(ATSDR), US Dept. of Health and Human Services, Division of Toxicology
and Environmental Medicine, 1600 Clifton Road, Atlanta, GA 30341, USA,
Fax: +1-770-488-4178 obfowler@cdc.gov4 (133, 143)
Peter Fuerst Chemical and Veterinary Analytical Institute, Muensterland-

Emscher-Lippe (CVUA-MEL), Joseph-Königstrasse 40, D-48147 Münster,
Germany (27)
Hugh Hansen Agency for Toxic Substances and Disease Registry

ogy, 1600 Clifton Road, F-62, Atlanta, GA 30333, USA (61)
CONTRIBUTORS TO VOLUME 8 xix
Claudia Heppner European Food Safety Authority, Unit on Food Con-

Kazimierz Kasprzak Laboratory for Comparative Carcinogenesis,

National Cancer Institute at Frederick, Bldg 538, Room 205E, Frederick,
MD 21702-1201, USA okasprkaz@ncifcrf.gov4 (319)
George E. N. Kass European Food Safety Authority, Unit on Food

Contaminants, Largo N. Palli 5/A, I-43100 Parma, Italy (27)
Andreas Kortenkamp School of Pharmacy, Department of Toxicology,

University of London, 29-39 Brunswick Square, London, WC1N 1AX, UK,
Fax: +44-20-7753-5908 oandreas.kortenkamp@pharmacy.ac.uk4 (305)
Alan B. G. Lansdown Chemical Pathology, Division of Investigative Med-

icine, The Faculty of Medicine, Imperial College, London, Charing Cross
Campus, London, W6 8RP, UK oa.lansdown@imperial.ac.uk4 (187)
Irina Lehmann Department of Environmental Immunology, Helmholtz

Centre for Environmental Research-UFZ, Permoserstrasse 15, D-04318
Leipzig, Germany, Fax: +49-341-235-1787 oirina.Lehmann@ufz.de4 (157)
Jörg Lehmann Department of Environmental Immunology, Helmholtz

Leipzig, Germany, Fax: +49-341-235-1787 (157)
Moiz M. Mumtaz Agency for Toxic Substances and Disease Registry

ogy, 1600 Clifton Road, F-62, Atlanta, GA 30333, USA, Fax: +1-770-488-
4178 omgm4@cdc.gov4 (61)
Antonio Mutti Department of Clinical Medicine, Nephrology and Health

Sciences, University of Parma, Via Gramsci 14, I-43100 Parma, Italy
oantonio.mutti@unipr.it4 (81)
Declan P. Naughton School of Life Sciences, Kingston University, Penrhyn

Road, Kingston upon Thames, Surrey, KT1 2EE, UK, Tel: +44-208-417-
7097 od.naughton@kingston.ac.uk4 (107)
Tamás Nepusz School of Life Sciences, Kingston University, Penrhyn

Road, Kingston upon Thames, Surrey, KT1 2EE, UK, Tel: +44-208-417-
7097 ot.nepusz@kingston.ac.uk4 (107)
xx CONTRIBUTORS TO VOLUME 8
Andrea Petroczi School of Life Sciences, Kingston University, Penrhyn

Road, Kingston upon Thames, Surrey, KT1 2EE, UK Tel: +44-208-417-
7097 oa.petroczi@kingston.ac.uk4 (107)
Hana R. Pohl Agency for Toxic Substances and Disease Registry (ATSDR),
US Dept. of Health and Human Services, Division of Toxicology, 1600
Clifton Road, F-62, Atlanta, GA 30333, USA, Fax: +1-770-488-4178
ohpohl@cdc.gov4 (61, 143, 247)
Anna Maria Protas Central Institute for Labour Protection, National

Research Institute, Czerniakowska 16, PL-00-701 Warsaw, Poland
oannaprotas@ibb.waw.pl4 (319)
Nickolette Roney Agency for Toxic Substances and Disease Registry

ogy, 1600 Clifton Road, F-62, Atlanta, GA 30333, USA (143, 247)
Ulrich Sack Department of Environmental Immunology, Helmholtz

Leipzig, Germany, Fax: +49-341-235-1787 (157)
Elena Scaravelli European Food Safety Authority, Unit on Food Con-

Erik J. Tokar Inorganic Carcinogenesis Section, Laboratory of Compara-

tive Carcinogeneses, National Cancer Institute at NIEHS, 111 Alexander
Drive, Mail Drop F0-09, Research Triangle Park, NC 27709, USA (375)
Philippe Verger World Health Organization, Department of Food Safety

and Zoonoses, 20 Avenue Appia, CH-1211 Geneva, Switzerland (27)
Michael P. Waalkes Inorganic Carcinogenesis Section, Laboratory of

Comparative Carcinogeneses, National Cancer Institute at NIEHS, 111
Alexander Drive, Mail Drop F0-09, Research Triangle Park, NC 27709,
USA owaalkes@niehs.nih.gov4 (375)
Titles of Volumes 1–44 in the
Metal Ions in Biological Systems Series
edited by the SIGELs
and published by Dekker/Taylor & Francis (1973–2005)
Volume 1: Simple Complexes

Volume 2: Mixed-Ligand Complexes
Volume 3: High Molecular Complexes
Volume 4: Metal Ions as Probes
Volume 5: Reactivity of Coordination Compounds
Volume 6: Biological Action of Metal Ions
Volume 7: Iron in Model and Natural Compounds
Volume 8: Nucleotides and Derivatives: Their Ligating Ambivalency
Volume 9: Amino Acids and Derivatives as Ambivalent Ligands
Volume 10: Carcinogenicity and Metal Ions
Volume 11: Metal Complexes as Anticancer Agents
Volume 12: Properties of Copper
Volume 13: Copper Proteins
Volume 14: Inorganic Drugs in Deficiency and Disease
Volume 15: Zinc and Its Role in Biology and Nutrition
Volume 16: Methods Involving Metal Ions and Complexes in Clinical
Chemistry
Volume 17: Calcium and Its Role in Biology
Volume 18: Circulation of Metals in the Environment
Volume 19: Antibiotics and Their Complexes
Volume 20: Concepts on Metal Ion Toxicity
Volume 21: Applications of Nuclear Magnetic Resonance to Paramagnetic
Species
Volume 22: ENDOR, EPR, and Electron Spin Echo for Probing
Coordination Spheres
Volume 23: Nickel and Its Role in Biology
xxii VOLUMES IN THE MIBS SERIES
Volume 24: Aluminum and Its Role in Biology

Volume 25: Interrelations among Metal Ions, Enzymes, and Gene
Expression
Volume 26: Compendium on Magnesium and Its Role in Biology, Nutrition,
and Physiology
Volume 27: Electron Transfer Reactions in Metalloproteins
Volume 28: Degradation of Environmental Pollutants by Microorganisms
and Their Metalloenzymes
Volume 29: Biological Properties of Metal Alkyl Derivatives
Volume 30: Metalloenzymes Involving Amino Acid-Residue and Related
Radicals
Volume 31: Vanadium and Its Role for Life
Volume 32: Interactions of Metal Ions with Nucleotides, Nucleic Acids,
and Their Constituents
Volume 33: Probing Nucleic Acids by Metal Ion Complexes of Small
Molecules
Volume 34: Mercury and Its Effects on Environment and Biology
Volume 35: Iron Transport and Storage in Microorganisms, Plants,
and Animals
Volume 36: Interrelations between Free Radicals and Metal Ions in
Life Processes
Volume 37: Manganese and Its Role in Biological Processes
Volume 38: Probing of Proteins by Metal Ions and Their
Low-Molecular-Weight Complexes
Volume 39: Molybdenum and Tungsten. Their Roles in Biological Processes
Volume 40: The Lanthanides and Their Interrelations with Biosystems
Volume 41: Metal Ions and Their Complexes in Medication
Volume 42: Metal Complexes in Tumor Diagnosis and as Anticancer Agents
Volume 43: Biogeochemical Cycles of Elements
Volume 44: Biogeochemistry, Availability, and Transport of Metals in the
Environment
Contents of Volumes in the
Metal Ions in Life Sciences Series
edited by the SIGELs
Volumes 1–4
published by John Wiley & Sons, Ltd., Chichester, UK (2006–2008)
<www.Wiley.com/go/mils>
and from Volume 5 on
by the Royal Society of Chemistry, Cambridge, UK (since 2009)
<www.rsc.org/shop/books/series/85.asp?seriesid=85>
Volume 1: Neurodegenerative Diseases and Metal Ions

1. The Role of Metal Ions in Neurology. An Introduction
Dorothea Strozyk and Ashley I. Bush
2. Protein Folding, Misfolding, and Disease
Jennifer C. Lee, Judy E. Kim, Ekaterina V. Pletneva,
Jasmin Faraone-Mennella, Harry B. Gray, and Jay R. Winkler
3. Metal Ion Binding Properties of Proteins Related to
Neurodegeneration
Henryk Kozlowski, Marek Luczkowski, Daniela Valensin, and
Gianni Valensin
4. Metallic Prions: Mining the Core of Transmissible Spongiform
Encephalopathies
David R. Brown
5. The Role of Metal Ions in the Amyloid Precursor Protein and in
Alzheimer’s Disease
Thomas A. Bayer and Gerd Multhaup
6. The Role of Iron in the Pathogenesis of Parkinson’s Disease
Manfred Gerlach, Kay L. Double, Mario E. Götz,
Moussa B. H. Youdim, and Peter Riederer
xxiv CONTENTS OF MILS VOLUMES
7. In Vivo Assessment of Iron in Huntington’s Disease and Other

Age-Related Neurodegenerative Brain Diseases
George Bartzokis, Po H. Lu, Todd A. Tishler, and Susan Perlman
8. Copper-Zinc Superoxide Dismutase and Familial Amyotrophic
Lateral Sclerosis
Lisa J. Whitson and P. John Hart
9. The Malfunctioning of Copper Transport in Wilson and Menkes
Diseases
Bibudhendra Sarkar
10. Iron and Its Role in Neurodegenerative Diseases
Roberta J. Ward and Robert R. Crichton
11. The Chemical Interplay between Catecholamines and Metal Ions
in Neurological Diseases
Wolfgang Linert, Guy N. L. Jameson, Reginald F. Jameson, and
Kurt A. Jellinger
12. Zinc Metalloneurochemistry: Physiology, Pathology, and Probes
Christopher J. Chang and Stephen J. Lippard
13. The Role of Aluminum in Neurotoxic and Neurodegenerative
Processes
Tamás Kiss, Krisztina Gajda-Schrantz, and Paolo F. Zatta
14. Neurotoxicity of Cadmium, Lead, and Mercury
Hana R. Pohl, Henry G. Abadin, and John F. Risher
15. Neurodegerative Diseases and Metal Ions. A Concluding Overview
Dorothea Strozyk and Ashley I. Bush
Subject Index
Volume 2: Nickel and Its Surprising Impact in Nature
1. Biogeochemistry of Nickel and Its Release into the Environment

Tiina M. Nieminen, Liisa Ukonmaanaho, Nicole Rausch, and
William Shotyk
2. Nickel in the Environment and Its Role in the Metabolism of Plants
and Cyanobacteria
Hendrik Küpper and Peter M. H. Kroneck
3. Nickel Ion Complexes of Amino Acids and Peptides
Teresa Kowalik-Jankowska, Henryk Kozlowski, Etelka Farkas, and
Imre Sóvágó
4. Complex Formation of Nickel(II) and Related Metal Ions with Sugar
Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids
Roland K. O. Sigel and Helmut Sigel
5. Synthetic Models for the Active Sites of Nickel-Containing Enzymes
Jarl Ivar van der Vlugt and Franc Meyer
CONTENTS OF MILS VOLUMES xxv
6. Urease: Recent Insights in the Role of Nickel

Stefano Ciurli
7. Nickel Iron Hydrogenases
Wolfgang Lubitz, Maurice van Gastel, and Wolfgang Gärtner
8. Methyl-Coenzyme M Reductase and Its Nickel Corphin Coenzyme
F430 in Methanogenic Archaea
Bernhard Jaun and Rudolf K. Thauer
9. Acetyl-Coenzyme A Synthases and Nickel-Containing Carbon
Monoxide Dehydrogenases
Paul A. Lindahl and David E. Graham
10. Nickel Superoxide Dismutase
Peter A. Bryngelson and Michael J. Maroney
11. Biochemistry of the Nickel-Dependent Glyoxylase I Enzymes
Nicole Sukdeo, Elisabeth Daub, and John F. Honek
12. Nickel in Acireductone Dioxygenase
Thomas C. Pochapsky, Tingting Ju, Marina Dang, Rachel Beaulieu,
Gina Pagani, and Bo OuYang
13. The Nickel-Regulated Peptidyl-Prolyl cis/trans Isomerase SlyD
Frank Erdmann and Gunter Fischer
14. Chaperones of Nickel Metabolism
Soledad Quiroz, Jong K. Kim, Scott B. Mulrooney, and
Robert P. Hausinger
15. The Role of Nickel in Environmental Adaptation of the Gastric
Pathogen Helicobacter pylori
Florian D. Ernst, Arnoud H. M. van Vliet, Manfred Kist,
Johannes G. Kusters, and Stefan Bereswill
16. Nickel-Dependent Gene Expression
Konstantin Salnikow and Kazimierz S. Kasprzak
17. Nickel Toxicity and Carcinogenesis
Kazimierz S. Kasprzak and Konstantin Salnikow
Subject Index
Volume 3: The Ubiquitous Roles of Cytochrome P450 Proteins
1. Diversities and Similarities of P450 Systems: An Introduction

Mary A. Schuler and Stephen G. Sligar
2. Structural and Functional Mimics of Cytochromes P450
Wolf-D. Woggon
3. Structures of P450 Proteins and Their Molecular Phylogeny
Thomas L. Poulos and Yergalem T. Meharenna
4. Aquatic P450 Species
Mark J. Snyder
xxvi CONTENTS OF MILS VOLUMES
5. The Electrochemistry of Cytochrome P450

Alan M. Bond, Barry D. Fleming, and Lisandra L. Martin
6. P450 Electron Transfer Reactions
Andrew K. Udit, Stephen M. Contakes, and Harry B. Gray
7. Leakage in Cytochrome P450 Reactions in Relation to Protein
Structural Properties
Christiane Jung
8. Cytochromes P450. Structural Basis for Binding and Catalysis
Konstanze von König and Ilme Schlichting
9. Beyond Heme-Thiolate Interactions: Roles of the Secondary
Coordination Sphere in P450 Systems
Yi Lu and Thomas D. Pfister
10. Interactions of Cytochrome P450 with Nitric Oxide and Related
Ligands
Andrew W. Munro, Kirsty J. McLean, and Hazel M. Girvan
11. Cytochrome P450-Catalyzed Hydroxylations and
Epoxidations
Roshan Perera, Shengxi Jin, Masanori Sono, and John H. Dawson
12. Cytochrome P450 and Steroid Hormone Biosynthesis
Rita Bernhardt and Michael R. Waterman
13. Carbon-Carbon Bond Cleavage by P450 Systems
James J. De Voss and Max J. Cryle
14. Design and Engineering of Cytochrome P450 Systems
Stephen G. Bell, Nicola Hoskins, Christopher J. C. Whitehouse, and
Luet L. Wong
15. Chemical Defense and Exploitation. Biotransformation of
Xenobiotics by Cytochrome P450 Enzymes
Elizabeth M. J. Gillam and Dominic J. B. Hunter
16. Drug Metabolism as Catalyzed by Human Cytochrome
P450 Systems
F. Peter Guengerich
17. Cytochrome P450 Enzymes: Observations from the Clinic
Peggy L. Carver
Subject Index
Volume 4: Biomineralization. From Nature to Application
1. Crystals and Life: An Introduction

Arthur Veis
2. What Genes and Genomes Tell Us about Calcium Carbonate
Biomineralization
Fred H. Wilt and Christopher E. Killian
CONTENTS OF MILS VOLUMES xxvii
3. The Role of Enzymes in Biomineralization Processes

Ingrid M. Weiss and Fre´de´ric Marin
4. Metal–Bacteria Interactions at Both the Planktonic Cell and
Biofilm Levels
Ryan C. Hunter and Terry J. Beveridge
5. Biomineralization of Calcium Carbonate. The Interplay with
Biosubstrates
Amir Berman
6. Sulfate-Containing Biominerals
Fabienne Bosselmann and Matthias Epple
7. Oxalate Biominerals
Enrique J. Baran and Paula V. Monje
8. Molecular Processes of Biosilicification in Diatoms
Aubrey K. Davis and Mark Hildebrand
9. Heavy Metals in the Jaws of Invertebrates
Helga C. Lichtenegger, Henrik Birkedal, and
J. Herbert Waite
10. Ferritin. Biomineralization of Iron
Elizabeth C. Theil, Xiaofeng S. Liu, and
Manolis Matzapetakis
11. Magnetism and Molecular Biology of Magnetic Iron
Minerals in Bacteria
Richard B. Frankel, Sabrina Schübbe, and
Dennis A. Bazylinski
12. Biominerals. Recorders of the Past?
Danielle Fortin, Sean R. Langley, and Susan Glasauer
13. Dynamics of Biomineralization and Biodemineralization
Lijun Wang and George H. Nancollas
14. Mechanism of Mineralization of Collagen-Based Connective
Tissues
Adele L. Boskey
15. Mammalian Enamel Formation
Janet Moradian-Oldak and Michael L. Paine
16. Mechanical Design of Biomineralized Tissues. Bone and Other
Hierarchical Materials
Peter Fratzl
17. Bioinspired Growth of Mineralized Tissue
Darilis Suárez-González and William L. Murphy
18. Polymer-Controlled Biomimetic Mineralization of Novel
Inorganic Materials
Helmut Cölfen and Markus Antonietti
Subject Index
xxviii CONTENTS OF MILS VOLUMES
Volume 5: Metallothioneins and Related Chelators
1. Metallothioneins: Historical Development and Overview

Monica Nordberg and Gunnar F. Nordberg
2. Regulation of Metallothionein Gene Expression
Kuppusamy Balamurugan and Walter Schaffner
3. Bacterial Metallothioneins
Claudia A. Blindauer
4. Metallothioneins in Yeast and Fungi
Benedikt Dolderer, Hans-Jürgen Hartmann, and
Ulrich Weser
5. Metallothioneins in Plants
Eva Freisinger
6. Metallothioneins in Diptera
Silvia Atrian
7. Earthworm and Nematode Metallothioneins
Stephen R. Stürzenbaum
8. Metallothioneins in Aquatic Organisms: Fish, Crustaceans, Molluscs,
and Echinoderms
Laura Vergani
9. Metal Detoxification in Freshwater Animals. Roles of
Metallothioneins
Peter G. C. Campbell and Landis Hare
10. Structure and Function of Vertebrate Metallothioneins
Juan Hidalgo, Roger Chung, Milena Penkowa, and
Milan Vasˇák
11. Metallothionein-3, Zinc, and Copper in the Central
Nervous System
Milan Vasˇák and Gabriele Meloni
12. Metallothionein Toxicology: Metal Ion Trafficking and Cellular
Protection
David H. Petering, Susan Krezoski, and
Niloofar M. Tabatabai
13. Metallothionein in Inorganic Carcinogenesis
Michael P. Waalkes and Jie Liu
14. Thioredoxins and Glutaredoxins. Functions and Metal Ion
Interactions
Christopher Horst Lillig and Carsten Berndt
15. Metal Ion-Binding Properties of Phytochelatins and
Related Ligands
Aure´lie Devez, Eric Achterberg, and Martha Gledhill
Subject Index
CONTENTS OF MILS VOLUMES xxix
Volume 6: Metal-Carbon Bonds in Enzymes and Cofactors
1. Organometallic Chemistry of B12 Coenzymes

Bernhard Kräutler
2. Cobalamin- and Corrinoid-Dependent Enzymes
Rowena G. Matthews
3. Nickel-Alkyl Bond Formation in the Active Site of Methyl-Coenzyme
M Reductase
Bernhard Jaun and Rudolf K. Thauer
4. Nickel-Carbon Bonds in Acetyl-Coenzyme A Synthases/Carbon
Monoxide Dehydrogenases
Paul A. Lindahl
5. Structure and Function of [NiFe]-Hydrogenases
Juan C. Fontecilla-Camps
6. Carbon Monoxide and Cyanide Ligands in the Active Site of
[FeFe]-Hydrogenases
John W. Peters
7. Carbon Monoxide as Intrinsic Ligand to Iron in the Active Site of
[Fe]-Hydrogenase
Seigo Shima, Rudolf K. Thauer, and Ulrich Ermler
8. The Dual Role of Heme as Cofactor and Substrate in the Biosynthesis
of Carbon Monoxide
Mario Rivera and Juan C. Rodriguez
9. Copper-Carbon Bonds in Mechanistic and Structural Probing of
Proteins as well as in Situations where Copper Is a Catalytic or
Receptor Site
Heather R. Lucas and Kenneth D. Karlin
10. Interaction of Cyanide with Enzymes Containing
Vanadium and Manganese, Non-Heme Iron,
and Zinc
Martha E. Sosa-Torres and Peter M. H. Kroneck
11. The Reaction Mechanism of the Molybdenum Hydroxylase
Xanthine Oxidoreductase: Evidence against the Formation
of Intermediates Having Metal-Carbon Bonds
Russ Hille
12. Computational Studies of Bioorganometallic Enzymes and
Cofactors
Matthew D. Liptak, Katherine M. Van Heuvelen, and
Thomas C. Brunold
Subject Index
Author Index of Contributors to MIBS-1 to MIBS-44 and MILS-1
to MILS-6
xxx CONTENTS OF MILS VOLUMES
Volume 7: Organometallics in Environment and Toxicology
1. Roles of Organometal(loid) Compounds in Environmental

Cycles
John S. Thayer
2. Analysis of Organometal(loid) Compounds in Environmental and
Biological Samples
Christopher F. Harrington, Daniel S. Vidler, and Richard O. Jenkins
3. Evidence for Organometallic Intermediates in Bacterial Methane
Formation Involving the Nickel Coenzyme F430
Mishtu Dey, Xianghui Li, Yuzhen Zhou, and Stephen W. Ragsdale
4. Organotins. Formation, Use, Speciation, and Toxicology
Tamas Gajda and Attila Jancsó
5. Alkyllead Compounds and Their Environmental Toxicology
Henry G. Abadin and Hana R. Pohl
6. Organoarsenicals: Distribution and Transformation in the
Environment
Kenneth J. Reimer, Iris Koch, and William R. Cullen
7. Organoarsenicals. Uptake, Metabolism, and Toxicity
Elke Dopp, Andrew D. Kligerman, and Roland A. Diaz-Bone
8. Alkyl Derivatives of Antimony in the Environment
Montserrat Filella
9. Alkyl Derivatives of Bismuth in Environmental and Biological Media
Montserrat Filella
10. Formation, Occurrence and Significance of Organoselenium and
Organotellurium Compounds in the Environment
Dirk Wallschläger and Jörg Feldmann
11. Organomercurials. Their Formation and Pathways in the
Environment
Holger Hintelmann
12. Toxicology of Alkylmercury Compounds
Michael Aschner, Natalia Onishchenko, and Sandra Ceccatelli
13. Environmental Bioindication, Biomonitoring, and Bioremediation of
Organometal(loid)s
John S. Thayer
14. Methylated Metal(loid) Species in Humans
Alfred V. Hirner and Albert W. Rettenmeier
Subject Index
Volume 8: Metal Ions in Toxicology:

(this book)
CONTENTS OF MILS VOLUMES xxxi
Volume 9: Structural and Catalytic Roles of Metal Ions in RNA

(in press)
1. Metal Ion Binding to RNA

Pascal Auffinger, Neena Grover, and Eric Westhof
2. Methods to Detect and Characterize Metal Ion Binding to RNA
Miche`le C. Erat and Roland K. O. Sigel
3. Importance of Diffuse Metal Ion Binding to RNA
Zhi-Jie Tan and Shi-Jie Chen
4. RNA Quadruplexes
Kangkan Halder and Jörg S. Hartig
5. The Roles of Metal Ions in Regulation by Riboswitches
Adrian Ferre´-D’Amare´ and Wade C. Winkler
6. Metal Ions: Supporting Actors in the Playbook of Small Ribozymes
Alexander E. Johnson-Buck, Sarah E. McDowell, and Nils G. Walter
7. Multiple Roles of Metal Ions in Large Ribozymes
Daniela Donghi and Joachim Schnabl
8. The Spliceosome and Its Metal Ions
Samuel E. Butcher
9. The Ribosome: A Molecular Machine Powered by RNA
Krista Trappl and Norbert Polacek
10. Metal Ion Requirements in Artificial Ribozymes that Catalyze
Aminoacylations and Redox Reactions
Hiroaki Suga, Kazuki Futai, and Koichiro Jin
11. Metal Ion Binding and Function in Natural and Artificial
Small RNA Enzymes from a Structural Perspective
Joseph E. Wedekind
12. Binding of Kinetically Inert Metal Ions to RNA: The Case of
Platinum(II)
Erich G. Chapman, Alethia A. Hostetter, Maire F. Osborn,
Amanda L. Miller, and Victoria J. DeRose
Volume 10: Interplay between Metal Ions and Nucleic Acids

(in preparation)
Comments and suggestions with regard to contents, topics, and the like for
future volumes of the series are welcome.
Met. Ions Life Sci. 2011, 8, 1–26
1
Understanding Combined Effects for Metal
Co-Exposure in Ecotoxicology
Rolf Altenburger
UFZ Helmholtz Centre for Environmental Research, Department of Bioanalytical
Ecotoxicology, Permoserstrasse 15, D-04318 Leipzig, Germany
<rolf.altenburger@ufz.de>
ABSTRACT 2
1. ECOTOXICITY FROM MIXTURE EXPOSURE 2
1.1. Occurrence of Chemical Mixtures in the Environment 2
1.2. Observational Evidence for Combined Effects 4
1.3. The Synergism Hypothesis 5
2. COMBINATION EFFECT ANALYSIS 6
2.1. Reference Models 6
2.2. Empirical and Mechanistic Approaches 9
2.3. Study Design and Assessment Issues 11
3. INTERACTIONS DURING EXPOSURE 13
3.1. Bioavailability 13
3.2. Uptake and Kinetics 14
3.3. The Biotic Ligand Model 16
4. JOINT ACTION IN TOXICODYNAMICS 17
4.1. Mechanisms of Action 17
4.2. Modes of Action 19
5. INTERACTION WITH ORGANIC COMPOUNDS 21
5.1. Metals and Polyaromatic Hydrocarbons 21
5.2. Metals and Other Organic Compounds 22
6. OUTLOOK 24
Published by the Royal Society of Chemistry, www.rsc.org DOI: 10.1039/978184973211600001
2 ALTENBURGER
ACKNOWLEDGMENTS 25
ABBREVIATIONS 25
REFERENCES 25
ABSTRACT: Organisms in the environment experience exposure to mixtures of metals

as a rule rather than an exception. Observational as well as experimental evidence
shows that such co-exposure may give rise to combined effects that are different from
what can be attributed to considering the effects of chemicals one by one. The two
established reference models, concentration addition and response addition, there-
fore derive explicit expectations of a joint effect from the biological activities of the
mixture constituents. The current empirical evidence of metal mixture effects in various
mainly aquatic species shows, that while the reference models provide reasonable tools
for analyzing combined effects, their actual predictions for binary mixtures compared
to what has been observed show often somewhat less than additive combined effects.
As the bioavailability of metals is governed by several environmental factors as well as
biosystem properties, the different processes involved provide ample opportunities for
interaction which may confound non-interactive combined effects. The biotic ligand
model offers scope to address these issues on a more mechanism-focussed basis in the
near future. Furthermore, the toxicodynamics of metals is highly compound-specific,
considering the various specific metalloid transporters, regarding the essential functions
of metals in metabolism and taking account of the organisms’ efforts to maintain
homeostasis for some metals. This and the diversity of already known molecular
interferences with cellular metabolism offer scope to unravel potentially adverse
interactive outcomes. Thus, for improving our predictability of combined effects from
metal co-exposure, we require more quantitative insight into and models for the pro-
cesses governing the toxicokinetics and dynamics of metals in environmental
organisms.
KEYWORDS: bioavailability . biotic ligand model . combined effects . concentration

addition . interaction . mixture toxicity . response addition . transition metals
1. ECOTOXICITY FROM MIXTURE EXPOSURE
1.1. Occurrence of Chemical Mixtures in the Environment

Organismic life develops in an environment that to a substantial amount is
composed of metals. Evolution has taken advantage of metals by utilizing
some elements for essential biological functions such as transport of oxy-
gen, stabilization of macromolecular structures or participation in enzy-
matic processes. Excess metal exposure of organisms may, however, cause
deleterious effects due to the reactive properties of metals. Occurrence of
metals in the environment is in mixtures of varying composition and
concentrations due to site specific geogenic backgrounds. Many forms of
life have developed means and mechanisms to discriminate between

UNDERSTANDING COMBINED EFFECTS FOR METAL CO-EXPOSURE 3
essential and non-essential metals and keep metal levels balanced and
regulated. The task to assess the potential of deleterious biological effects
occurring from environmental exposure to metal mixtures is therefore a
particular challenge as essentiality, regulatory and stress responses have to
be accounted for.
Anthropogenic activities such as mining, smelters, or fertilizer production
lead to substantial point source releases of metals into the environment.
Together with diffuse sources of pollution generated from a products life
cycle, e.g., fertilizer application, wheathering of surfaces, battery deposition
and the like, this results in increased exposure of organisms to metals in the
environment. Moreover, there are intentional emissions, when, e.g., pest
control mixtures of biocides are used in antifouling products to prevent ship
hulls from biofouling processes. In this particular case specific metal mix-
tures such as copper and booster biocide formulations containing zinc
pyrithione or zineb are of importance.
Typical or exemplary emission patterns may be described for certain
geogenic conditions or for specific processes such as mining or smelting and
the respective tailing and solid waste handling. Also, for formulated pro-
ducts such as biocides on ship hulls distinct emitted mixtures may be iden-
tified. Imission patterns may be deduced from monitoring data of terrestrial
and aquatic media. These tend to be site-specific though, of course, specific
processes such as mining may lead to typical and site-independent con-
taminant mixtures downstream of point sources. Thus, it can be anticipated
that co-occurrence of metals due to anthropogenic activities is the rule rather
than the exception, though the composition of such mixtures will vary in
space and time [24].
When it comes to biologically accessible and available concentrations of
metals in the environment, biomonitoring efforts reveal that many metals
can be detected in organisms despite of not being easily taken up into bodies
as charged chemical species. Thus, body burden or internal exposure of
organisms can be expected and has been described using accumulation
biomonitors to happen against multiple metal compounds. The degree,
composition and concentrations may be species-dependent and will not be
deducible in a straightforward manner from prevalent ambient concentra-
tions as uptake and distribution processes are highly variable between spe-
cies and environmental conditions. Nevertheless, we can conclude that
organisms in the environment have to cope with metal exposure that occurs
in mixtures which may vary over time in composition and concentration.
Moreover, due to the essentiality of some components, a mere exclusion
strategy for metals from internal compartments is not a viable option, but
instead organisms have to allocate resources for maintaining a regulated
balance [24].

4 ALTENBURGER
1.2. Observational Evidence for Combined Effects

The conventional evidence-based assessment of the effects of metal exposure
in organisms is commonly related to individual compounds. Accidental
exposure events or other extreme exposure scenarios such as the disposal of
mining waste or tailing from dump sites could be addressed using this
reductionistic approach.
Observational evidence as well as intentional experimentation using
designed metal mixtures has shed doubt that the single substance approach
can account for all types of effects due to mixtures occurring in the envir-
onment. Furthermore, existing evidence demonstrates that joint exposure to
mixture may lead to effects that are different from that of single compounds
[1]. Such effects are typically called combined or joint effects. The major task
in attributing the biological effects to individual as opposed to mixture
exposure requires accounting for the variability of the observed biological
responses. Mixture exposure does not necessarily translate into combined
effects when there are odd effect ratios. Vice versa, effects allocated to
observe single compounds may in fact be evoked by co-exposure if relevant
components are analytically overlooked [2].
If the effect elucidated by one or more of the components becomes
enhanced or weakened due to co-exposure, we may call it more or less active
than an individual compounds activity, while by contrast, if only the activity
due to one component is retrieved despite of a mixture exposure, the
situation may be assigned as inertism. Inertism is conceptually used in most
product formulations and, e.g., the components added to an active ingre-
dient in pesticide products are subsequently called inerts. If the effect of co-
exposure of metals or compounds leads to an effect undetected before for
any of the components, this will typically be called coalism [3]. Any devia-
tion of mixture effects from the effects provoked by single chemicals, here
metals, may thus be considered with respect to their degree as well as type,
i.e., quantitatively or qualitatively.
There is now plenty of experimental evidence that any metal mixture may
give rise to combination effects rather than being explained by only one of the
components [1] where terms like additive and non-additive play a central role.
It has to be said though that the response observation, the dose-response
functions of the components, and the mixture ratio are major factors regarding
the mixture outcome. Therefore, experimental settings using designed mixtures
can only provide proof-of-principle evidence that may be limited in inference
when it comes to assess the site-specific occurrence of metal mixtures as we will
see later. However, given the variability in composition of metal mixtures and
the potentially resulting combined effects, provision of observational evidence
for anticipated exposure will be a viable option only in selected cases. The
alternative is to strive for extrapolative models.

1.3. The Synergism Hypothesis

The single most often used term for describing the observational or
experimental findings from a mixture effect study is synergism. The term in
its etymological Greek origin sunergóz, synergos literally means ergos ¼
work and syn ¼ together, hence working together. As such, it is assigning an
effect output that is believed to be causally related to the exposure input of
the mixture. While some of the authors who use the term synergism would
agree with such a purely descriptive connotation, most papers imply a
meaning that either refers to an unexpected observation for a mixture effect
in a qualitative sense or to an increased output following mixture exposure
as compared against a null hypothesis. We immediately see that without an
explicit definition of what is actually meant for specified circumstances,
retrieval of an assessment statement for a given mixture observation, such as
synergism, alone is meaningless and may give rise to confusion rather than
improve our understanding. There are several reviews and monograph
contributions available that illustrate the same point for other commonly
used terms in the description of combined effects from mixture exposure,
such as potentiation, antagonism, interaction, additivity, multiplication,
independence, synergy, and the like [4].
In order to avoid terminological confusion, it can be said that observation-
based statements on the specific outcome of a mixture exposure situation
require a comparison with an explicit hypothesis. Typically, such a hypothesis
will be derived from what is known or extrapolated for the biological effects
of the individual constituents of the mixture under consideration. A funda-
mental issue that is often raised in this context relates to the level of under-
standing or assumptive statements on the mechanisms behind observable joint
effects. This perception originates in the plausible notion that any mixture
exposure situation might offer the potential for interaction of components
during the various toxicokinetic and toxicodynamic processes as listed in
Table 1 and illustrated later in this chapter. The need and opportunity for that
matter to gather this type of information and actually accommodate for it,
crucially depends on the purpose of the mixture study.
Table 2 tries to distinguish major purposes. Clearly, if understanding
of a joint action from a mixture exposure situation is the objective as may be
the case in developing a multi-drug treatment, we intend to characterize the
molecular mechanisms and therapeutic modes of biological action through
focusing on drug targets and drug pathway interaction studies. By contrast,
accounting for mixture exposure in an environmental quality standard for-
mulation would require the deconstruction of complex environmental mix-
tures through identification of major contributing components. When
safeguarding against unintended effects is the purpose, say, avoiding toxic
environmental side-effects in the application of biocides, the description of

6 ALTENBURGER
Table 1. Processes of interaction.
Level of Processes of Information

consideration interaction required Output
Chemical Speciation, Chemical Chemical species

structures dissociation properties
Milieu Chelation, Environmental Bioaccessible
sorption, variables concentration
speciation
Uptake Competition Transporters, rate Bioavailable
constants concentration
Chemical Reaction, binding Moleculare targets Primary
biosystem interaction
Mode of action Similar/dissimilar Joint action Toxicity
pathway
Apical response Correlated effects Sensitivity Joint effects
Table 2. Scope of environmental mixture studies.
Objective Focus Intention
Understanding joint Toxicant/target Characterization of

action interactions toxicant/ mechanisms and modes
co-solute interactions of (inter)action
Prioritization in Qualitative contributions Identification of toxic
environmental quality under multiple stress components
setting, remedial action exposure
Safeguarding against Quantitative assessment Description of combined
effects unintended effects
Risk management/ Extrapolation concepts Prediction of mixture
regulation toxicity
combined effects even needs to become quantitative. Further, if in risk

management the intention is to derive predictive statements on expectable
combined effects for mixtures of potential concern, then we would need to
employ plausible reasonable worst case concepts as a basis for extrapolation.
2. COMBINATION EFFECT ANALYSIS
2.1. Reference Models

Central to dealing productively with possible combination effects from
mixture exposure is the formulation of a plausible and explicit null

hypothesis. In other words what do we expect to happen if we consider a

given mixture of compounds? Interaction is not a sufficient answer as any
resulting observation would fit with this assumption and as we cannot dis-
prove this; our hypothesis would thus be indiscriminate and trivial.
Out of 100 years of scientific debate, two reference models have evolved as
null hypothesis that may be used, concentration addition and response
addition (see Table 3). Concentration addition as a model has been for-
mulated by Loewe and Muischnek back in 1926 [5]. It is easily illustrated by
the thought experiment of a sham combination, whereby one considers the
expectation of providing n times 1/nth of the concentration of a component
for which we know the effect of the concentration sum. Clearly, we would
expect to retrieve the same effect as for the undivided sample, i.e., the effect
remains constant. Formally, this is expressed as the sum of the concentra-
tions of our components as present in the mixture over their individual effect
concentrations because the same effect level should be constant. Basically, in
this context we consider mixtures as dilutions of one and the same com-
pound. The major requirement lies in the need to provide reliable estimates
of effect concentrations for the mixture components regarding the relevant
effect level. This is typically to be met by studying dilution series and explicit
modelling of concentration response functions.
Response addition as the second basic reference model has been intro-
duced by Bliss [6]. It derives from the idea of independent events leading to
an overall sum effect. This may be illustrated by thinking of subsequent
throws of nails at balloons and observing the joint effect on their intactness.
Table 3. Reference models for calculating expected combined effects for chemical
mixtures.
Concentration Addition (LOEWE Additivity)

Suggested for: same site of action;
similar mode of action
Formula:
Binary c1/ECx,1+c2/ECx,2 ¼ 1
n 1
P pi
Multiple ECxmix ¼ 1
i¼1 Fi ðxi Þ
Response Addition (BLISS independence, independent action, effect multiplication)

Suggested for: different sites of action
dissimilar modes of action
Formula:
Binary E(c1,2) ¼ E(c1)+E(c2)E(c1) E(c2)
Qn
Multiple X ¼ 1 ð1 Fi ðpi ðECxmix ÞÞÞ
i¼1

8 ALTENBURGER
Mathematically, the fractional effects for the individual treatments are

multiplied. There are different means to do this as displayed in Table 3 with
the formula for the binary and multiple mixtures.
Both models are established in the literature under various synonyms and
can be considered as fundamental null hypothesis; hence, we call them
reference models. In principle, they allow the calculation of a combined
effect for a defined biological response for binary and multiple mixtures.
Both reference models consider quantitative assessments for a predefined
biological response, based on information of the efficacy of the components
for the same response. One will find supporters and contesters for both
reference models in many different biological disciplines as well as believers
that declare only one of the models as universally valid. A good deal of
debate has circled around the notion that the suitability of these reference
models is linked to the site, mechanism or mode of action of the mixture
components. Often concentration addition is thought to be the appro-
priate reference model when the mixture components act through the
same principle, be that the site, mechanism or mode of action, while for
response addition the governing idea of statistically independent responses
is felt to be adequately reflected if the mixture components act in a dis-
similar way.
However, as the formula are non-responsive to any assumptions related to
biological reasoning plausible as they may be, they may simply be used for
providing a stringent and straightforward null hypothesis generation. Fur-
thermore, often you may find that calculated combined effects do not differ
much for either model [7] despite of the detail of argument and information
that you may have available from a mechanistic perspective. However, more
critical for the practical use of either concept may be practical issues, such as
the need to estimate the effects for each component at low doses, the lim-
itations to use a specific experimental design, the steepness of the dose-
response relationships, or the demand to provide a reasonable worst case
assumption.
Moreover, it has to be said that there are variants of the reference models.
Response addition, e.g., from its statistical background regarding combi-
nation effects as independent events from the mixture components, has also
been formulated with an additional term of correlated responses. This in
turn, however, yields the model descriptive because correlated responses [8]
cannot be predicted most of the time but only be described after a mixture
response observation. Similarly, for concentration addition, interaction
terms have been provided (e.g., [3]) which again is a way to describe a
mixture response deviating from the simple null hypothesis assumption
through data fitting. Moreover, there is the suggestion for multiple mixtures
composed of similar and dissimilar acting components to use both reference
models in a stepwise manner. Here, the expected combined effect is

calculated first for those components that are thought to act similarly by
applying the concentration addition model and subsequently these groups
are considered for the overall effect using the model of response addition [9].
Finally, we should be aware that there are many suggestions for calculus and
display out in the literature, typically called by specific names, that in the one
or the other way fall back on the described reference models of either con-
centration addition or response addition [3,4,8].
2.2. Empirical and Mechanistic Approaches

Looking into the available empirical evidence for combined effect observa-
tions from metal mixture exposure there has been one comprehensive review
provided by Norwood and colleagues [1]. These authors collated, inspected,
recalculated, and summarized reported experimental evidence of metal
mixture effects on aquatic biota. They analyzed some 100 original com-
munications beginning from the mid 70ies, and a total of 22 different
metals were included in the analysis of 249 mixtures and their combined
effects on 77 different aquatic species. The mixtures so far being investigated
experimentally for combination effects are biased towards binary mixtures,
with some ternary and only a few other multiple mixtures. Also, from
the 22 different metals included in mixture investigations, Zn, Cu(II), Cd,
Hg, and Ni account for over 80% of the metals employed in all studies
reported.
The species employed stem from systematically diverse groups though not
surprisingly there is a bias for organisms with well established standard test
protocols, e.g., for algae, fish, and invertebrates. Different test protocols
imply different testing conditions with respect to media composition,
exposure duration, and effect observation. Media composition varied from
natural fresh- and saltwater to artificial media. Biological responses
observed covered various effects from short-term functional responses such
as sodium flux rate or photosynthetic rate to structural responses on his-
topathological observation levels or community structure measures. Also,
different life and development stages have been investigated for mixture
effects [1].
Given this heterogeneity in our evidence base, the general trends that have
been elucidated are quite striking (Table 4). A little more than a quarter of
the mixtures are being assessed in the original communication as being in
agreement with the idea of an additive combination effect, while for another
quarter more than additive effects are described. The remaining almost half
of observations claimed to have detected less than additive combination
effects. This result is more or less reproduced with a more stringent reana-
lysis of the data performed by Norwood and colleagues [1].

10 ALTENBURGER
Table 4. Summary of published observations for metal mixture effects (modified

after [1])
No. of
metals in Less than Strictly More than Total Could
mixture additive additive additive tests not test
2 69 42 45 156 14
3 7 6 5 18 4
4 1 0 0 1 2
5 3 0 3 6 2
6 1 3 2 6 1
7 0 0 0 0 1
8 1 1 0 2 0
10 0 0 1 1 1
11 1 0 0 1 0
Total 89 58 63 210 12
Percent 42.4 27.6 30.0 100.0 5.7
Technically, a comparative analysis is demanding as authors use different

dose references and models for the calculation of expected combination
effects for their assessment. While the former typically comprises nominal
ambient water concentrations but also measured pore water or sediment
concentrations, the latter includes not only the above outlined two reference
models but various calculus rules plus the comparison of the mixture
response against the most effective component. In particular, it is difficult to
define a stringent way to decide on the significance of a deviation between an
observed combined effect and the calculated expected effect. Thus, the
findings of an individual study may be assessed differently with different
approaches but the overall picture on combined effects from metal mixture
exposure in aquatic biota should be taken as a clear hint that, in contrast to
what is known for many mixtures of organic compounds, there seems to be a
tendency to overestimate mixture effect outcomes when using a default
additivity assumption. For risk regulation this is good news, as it allows
utilizing the additivity model as a reasonable worst case reference model
with some confidence. For our mechanistic understanding and toxicological
interpretation, however, this result needs to be reflected and discussed, as
will be undertaken in the following. The major conclusion of many authors
is that interaction between metals in mixtures occurs and in particular the
processes leading to alterations in bioavilability are suspected to play a
crucial role in understanding observed non-additive combination effects.
After going into some basics of mixture study design, we will therefore
illuminate our current understanding of the different processes affecting
possible metal interactions.

2.3. Study Design and Assessment Issues

A lesson to be learned from current empirical evidence is that the quality of
outcome and its usefulness greatly depend on the choice of an experimental
design that is adequate to the purpose of the studies. Many studies published
up to this day can greatly improve to this end. We have already developed
the idea to distinguish between different purposes in dealing with mixture
exposure (Table 1). When we now consider study design for mixture studies
the purpose of a study is of course the single most important driving factor.
Given that our resources are always limited, Table 5 illustrates the options
for the simple case of a binary mixture for which an experimenter in prin-
ciple could opt in devising an experiment. Clearly, if the intention is to
describe all possible interactions for one situation it would be optimal to
cover the theoretical response surface maximally, that is, to employ a so-
called composite design using various mixture ratios and total concentra-
tions. The other extreme would be that our purpose is to decide on the
predictivity of the reference models of either concentration or response
addition. In this case a ray design, using a fixed mixture ratio and varying
only the overall concentration would suit the purpose best.
Next, it seems to become common sense that effect estimations for che-
micals should be based on dilution series testing rather than repeated testing
of benchmark concentrations. There are elegant ways available to combine
experimental repeats with adapting concentration spacing to the steepness of
the concentration response relationship of interest in order to achieve
maximum information. Most importantly, however, researchers should use
Table 5. Illustration of experimental design strategies for studying a binary mixture

of substances (S1, S2) at various concentrations (C1,. . .,C6).
[C1]S1 [C2]S1 [C3]S1 [C4]S1 [C5]S1 [C6]S1
[C1]S2 ’J ’$ ’$
[C2]S2 J
[C3]S2 J $
[C4]S2 ’ $ ’ J ’ $
[C5]S2 J
[C6]S2 $ J
Theoretical design points for binary mixtures with identical number of observations
according to:
’ n*n design
J ray design
$ composite design
Reproduced by permission from [23], copyright 2003.

12 ALTENBURGER
an explicit concentration-response model. There are several models, parti-

cularly those of non-linear regression, available for these purposes (e.g.,
[10]), many of which can also be found in graphical or statistical software
packages. Also, to improve our ability to independently repeat and improve
combined effect assessments, researchers should take the effort to report all
estimated parameters for the used model rather than only the derived EC50
values and provide statements on the quality of the model fit to the data.
More difficult is the demand to give indication on the variance of one’s
findings. If we believe in non-linear concentration response relationships, no
straightforward variance estimates are available for this type of responses.
Instead, approximated error estimates have to be used, and the researcher
typically has to rely on what is being made available by the software tools
used. Thus, a critical appraisal during data inspection and general expertise
is a valuable information.
Techniques to calculate expectable combined effects from the activity
observed for the individual components are legion. Bödeker et al. [8]
introduced a classification that distinguishes graphical methods such as the
classical isobologramm that work without explicit calculus from indices for
point estimates such as provided by the summation of toxic units from
calculation models that are capable to estimate full concentration response
surfaces as descriptive input-output functions or even fully parametrized
models. They are typically based on either reference model, namely con-
centration addition or response addition, but provide different means for
estimating the expected combination effects. The choice of an adequate
model again depends on the purpose of a particular investigation and may
need either sufficient expertise or collaboration.
Another issue that requires attention in the design of a mixture study is the
selection of a means to decide whether or not an observed effect is con-
sidered to deviate substantially from what is expected on the basis of the
components activities. Clearly, one would not expect an ideal match between
expectation and observation, due to variance of responses. The most strin-
gent way to address this issue would be the performance of a significance
test, again there are no standard solutions available and the problem is far
from trivial as estimation of component activity and mixture activity require
subsequent testing. Moreover, in many settings it is not clear which reference
model is adequate to use because mode-of-action information may not be
available or ambiguous, and thus, we may start right away with two different
expectations and no means to validate either of them. Therefore, we often
find rules of thumb in place to decide whether or not a deviation from either
additivity model is strong enough to speak of sub- or superadditivity,
interaction, or synergism and antagonism. Many papers that went into
pattern recognition instead of trying to elucidate the specifics of a particular
mixture for this reason, rather quantify the difference between expected and

observed combination effect and leave it to the user to decide on the rele-
vance of a deviation for a particular purpose. The advantage is easily seen:
In one case we may be able to identify a 1.2-fold deviation from our mixture
toxicity expectation as significant while in another case this is true for a
2-fold deviation only. An observation of, say, 1.5 higher activity for the
observed compared to the expected mixture toxicity would now result in an
additivity statement in the one and a synergy statement in the other case.
Again, there are various means available to give a number to such differ-
ences, such as the additivity, combination or magnification index, index on
prediction quality, and many more.
Finally, it has to be said that many investigations on the combined effects
of mixtures do design the mixture ratios studied experimentally in a way that
each of the components can be expected to contribute equally to the overall
effect. A typical example would be an equitoxic ratio, whereby the mixture is
composed from the ratio of the components, e.g., the individual EC50s. If
tested as a dilution series this would gain the ray design illustrated in Table 5.
For studying interactions between components more systematically as is
needed for instance in product design or deconstructing an environmentally
prevalent mixture, odd mixture ratios will have to be considered. In such
cases, it may be useful to examine whether or not the additivity null
hypothesis would lead to combined effects that can be differentiated from
the individual substance effects. Provided the biological activities of the
individual components are available as explicit concentration response
functions, this is easy to achieve through simulation of all kinds of mixture
ratios and response levels of interest. This approach is useful to learn about
the expected sensitivity of a combination effect as well as identifying an
optimal design for an experiment.
Thus, we see that in order to deduce a reasonable answer from a mixture
experiment on combined effects, it requires some efforts in experimental
design next to providing a clear hypothesis. In the following, we will now try
to summarize the current knowledge on interactions of metals during
exposure and effect propagation that might help to improve our under-
standing on deviations from additive combined effects.
3. INTERACTIONS DURING EXPOSURE

3.1. Bioavailability
It has been known for a long time that the milieu conditions play a major
role in determining the apparent toxic effect of metals on organisms. Water
chemistry with factors such as pH, water hardness or the occurrence of other

14 ALTENBURGER
ions in the exposure medium will influence the redox state and speciation of
the metals. In consequence, the prevalent metal species will determine the
potential for molecular interactions such as sorption or reactivity and thus
subsequently also determine the toxic properties. Next to speciation also
complexation or chelation of cations by organic substances such as humic
acids or polymers such as polyphosphates may affect apparent biological
outcomes from metal exposure. For the combined effect of metal mixtures
all these processes may be regarded as potential confounders for the preci-
sion of predicting the combined effect of a metal mixture from the compo-
nents activity as each metal will be affected differently by changes in any of
these factors [25].
One way to experimentally deal with the milieu dependence of apparent
metal toxicity is strict control and standardization of the exposure situation
in the experimental set-up. However, in the environment and thus, in site-
specific assessment, this will neither be reasonable nor possible at all times.
Alternatively, one can try and provide adequate consideration of the major
influences through modelling. The free ion activity model is historically one
of the more successful attempts to capture the influence of milieu factors on
the toxicity of metals. The basic assumption being that it is the free ion that
eventually determines the biological effect of metals and if therefore the
ambient concentration of a metal can be corrected for the other metal spe-
cies, the resultant toxicity should be an expectable value purely dependent on
the concentration of the free ion. To our knowledge this concept has not
been extended to the study of metal mixtures though. An application that
seems straight at hand to that end, would be to check apparent deviations of
mixture effects from the additivity hypothesis by calculating the free ion
concentration from the ambient concentrations of the metals in the different
experiments of individual component and mixture testing.
For metal mixtures, the ambient concentration in any case seems to be an
unreliable indicator for an expectable combined effect. A logic alternative
would thus appear to head for estimates of internal concentrations or bio-
logical doses as a basis for a toxicity assessment which might be less prone to
confounding factors.
3.2. Uptake and Kinetics

In contrast to uptake of many xenobiotic organic substances, even non-
essential metals as prevalently charged species are not able to passively pass
through cell membranes, but seem to enter cells and tissues actively via the
various ion transporters and ion channel proteins invented during evolution.
There is a high number and variability in transporter proteins to be
acknowledged at least at the level of larger systematic units. Ion transporter

proteins for instance make up for more than 40% of all transporter types in
primates, while accounting for only 12% in plants or less than 2% in pro-
tozoa [11]. It is therefore not too surprising that the uptake kinetics of metals
observed for organisms and cells are specific for individual metals and vary
greatly between species. The subsequent distribution, metabolism, and fate
of the intracellular metals also show patterns related to biological sys-
tematics rather than to common chemical features. For instance, many plant
species are believed to take up arsenic compounds via phosphate transpor-
ters and have the capability to methylate and further metabolize intracellular
arsenic resulting in the production of less toxic forms such as arsenosugars,
lipids or peptides [26]. Active or facilitated transport of cationic metals is a
feature commonly described in heterotrophic systems where often metals
seem to compete for transporters that regulate cation homeostasis or for
specific functions of essential metals such as neuron activation. The phe-
nomenon of competition of essential and non-essential metals for cation
uptake transporter sites is known as molecular mimicry [12].
A fundamental difference between essential and non-essential metals with
regard to intoxication events appears to be the dependence of the internal
concentration on the bioavailable fraction and the exposure duration for the
latter, signifying a lack of sufficient homeostatic control mechanisms. By
contrast, for essential metals most cells seem capable of maintaining a
narrow range of intracellular concentrations.
For the metal mixtures this situation renders combined effects as vul-
nerable to the mixture type and organism considered. And indeed, many
authors believe that non-additive metal mixture effects may be attributed to
interactions during uptake and bioaccumulation which seems plausible
considering what is known about metal uptake via specific sites and
mechanisms. Borgmann and colleagues [13] have undertaken the effort to
summarize the current status of addressing the toxicity from metal mixture
exposure based on modelling bioaccumulation. In principle, they suggest a
simplification in the study of all conceivable interaction types to a few
classes, namely competitive, anti-competitive, and non-competitive inhibi-
tion. Experimental studies undertaking to distinguish these enzymatic
interaction types do best to choose an n*n design (see Table 5), i.e., vary the
mixture ratio and run dilution series at various fixed concentrations of the
second metal. Bear in mind that a simple competition of toxic metals for a
binding site is a type of interaction that would be covered by the reference
model of concentration addition, and would therefore be regarded as zero
interaction.
It is obvious that this effort becomes laborious when advancing to mul-
tiple mixtures. For combined effect assessment this approach offers the
opportunity to derive effect predictions from tissue concentrations or
internal dose rather than ambient concentrations. The drawbacks, however,

16 ALTENBURGER
are also numerous. For one we have to acknowledge that a total internal
metal concentration may not be as informative as it is for many organic
compounds, since subsequent chemical speciation or internal sequestration
may determine the compounds capability to provoke harmful events. For
example, cellular defence systems like metallothioneins show compound and
biological species-specific potencies to deactivate intracellular free metal
ions. Also, organisms have evolved an array of means to sequester or
eliminate higher concentrations of metals, e.g., through formation of com-
plexes with polyphosphates or organic compounds, sequestration into plant
vacuoles, or excretion out of cells and tissues.
Furthermore, the primary site and events of toxic action at least for
short-term effects may already occur during uptake. An approach that links
our current bioavailability and sorption understanding with a simplified
toxicity perception is the so-called biotic ligand model. It has recently
become very successful and popular in individual metal short-term eco-
toxicity assessment.
3.3. The Biotic Ligand Model

The biotic ligand model (BLM) is a formalized way to incorporate the
impact of water chemistry on metal speciation, the availability of free metal
ions in the water phase when accounting for organic and inorganic metal
complexation, and the binding to a biotic ligand in competition to other
ions. The biotic ligand is typically thought of as a membrane-related mac-
romolecular structure such as a transporter protein. If that structure has a
relevant biological function, e.g., transport of essential metals, then it can be
regarded as a primary site of toxic action and it may be used to model short-
term toxic effects [14]. An example would be hypocalcemia believed to be
caused through blockage of Ca uptake, e.g., by Co, Zn or Cd. A conceptual
sketch summarizing the different aspects of the BLM is provided in Figure 1.
Historically, this approach has been developed for mono- and divalent
metals and in particular Cu, Ag, Ni with fish as receptor species in mind, i.e.,
the cation transporters at the gill surface are envisaged as primary biotic
ligands [14].
As the BLM for toxicity assessment is relating a calculated free metal
concentration to a toxic effect concentration it should also be able to be used
for a mixture of metals that target the same biotic ligand [15]. Different
approaches and simulation studies to this end have been performed, that
sometimes confuse toxic units with toxicity. Based on such misconception of
a linear relationship between substance concentration and biological
response non-additive mixture behavior has been predicted but anyway
no observations are available so far. In general, up to now there is

Figure 1. Biotic ligand model (BLM) – conceptual sketch. Reproduced from [14]
with permission of Elsevier, copyright (2002).
unfortunately very little experimental data showing how this approach could
improve the predictability of combined effects of metals. By contrast, the-
oretical arguments highlighting several limitations have been raised [13]. For
example, a major model assumption lies in the consideration of one biotic
ligand only, while of course we know that typically there are several cation
transporters expressed and affected by metals in biomembranes. While the
arguments have their virtue, in combination toxicology of metal mixtures we
would be very fortunate if we could get a better grip on some of the variance
producing factors, as this would greatly help to improve their predictability.
I would therefore opt for performing clarifying mixture studies based on
BLM modelling.
4. JOINT ACTION IN TOXICODYNAMICS

4.1. Mechanisms of Action
Formulating a reasonable hypothesis for the expectable combined effects of
a mixture of metals in a biosystem of interest can be improved with
knowledge on whether the mixture components show similar or dissimilar
toxicodynamic behavior. Currently, three major principles are discussed as
mechanisms of metal toxicity, namely competitive binding at membrane
transporters, metal-induced oxidative stress and direct dysfunctional inter-
action with biological macromolecules. While the former is often linked to a
short-term apical effect, the later two are seen in the context of long-term
and irreversible damage such as carcinogenicity.
Regarding the potency of metals to compete with one another for uptake
using either transporters or ionophores, we now know that organisms have

18 ALTENBURGER
evolved several means to help maintain cellular metal homeostasis

and support functionality of essential metals [11]. Thus, we have to
acknowledge cell- and tissue-type specific expression and abundance of
different uptake proteins for mono- and polyvalent cations and anions.
Together, with the compound specific influence of prevalent environmental
conditions on metal oxidations state and speciation, a simple dilution
concept in predicting mixture effects seems therefore limited in scope at
this level.
With respect to the pathways of metal-induced oxidative stress current
knowledge comprises different processes [16]. While compounds like iron,
copper, cobalt, chromium, nickel, and vanadium seem capable of superoxide
and hydroxyl radical formation through Fenton-type reactions, depletion of
glutathione and bonding to sulfhydryl groups of proteins is another
mechanism of elucidating oxidative stress for a separate group of metals
including mercury, cadmium, and nickel. Arsenic, next to binding to thiol
groups in macromolecules, is also connected to a separate route of hydrogen
peroxide formation in cells. Moreover, metals such as cadmium or arsenic
when present in cells have been linked with inhibition of DNA repair
mechanisms that will be activated after gene activation due to exposure to
oxidative stress. The picture becomes even more complicated when adding
metal-induced involvement of nitric oxide or the antioxidant effects such as
resulting from zinc exposure.
Considering the rich coordination chemistry of metals, it comes as no big
surprise that there is a diversity of modifications described for different
biological macromolecules resulting from direct interaction between metals
and macromolecules. For example, types of DNA damages caused by spe-
cific metal exposure include base modifications, cross-linking, strand scis-
sion, and depurination.
In summary, we may conclude that we know that organisms and cells offer
several targets prone to interaction with metals that do not seem mutually
exclusive, which at the same time through their diversity offer scope for
specific interaction profiles of metals. Importantly, cells can be expected to
vary in their interaction properties depending on their type and develop-
mental status. It is thus difficult to suggest a uniform hypothesis of
expectable combined effects for metal mixture exposures at the level of
primary molecular interactions. A recent study of Vandenbrouck and col-
leagues [17] on binary mixtures of nickel/cadmium and nickel/lead, respec-
tively, observing gene transcription and physiological costs in daphnids,
provided evidence along this line of thinking. Using an equitoxic design
for the mixture study, they observed additional affected pathways after
mixture treatment, which points to an interaction at the molecular scale
leading to novel response qualities, in the present case to additionally
affected pathways.

4.2. Modes of Action

The chain of events that lead from a molecular interaction of a metal with a
biosystem to an adverse biological outcome may simplify the above sketched
picture. Most organisms and thus their cellular systems seem to have typical
reaction patterns upon metal exposure that may perhaps best be envisaged
from the perspective of essential metals. Cellular homeostasis is maintained
for essential metals by means of regulating uptake and utilization. During
low availability, metabolism will regulate towards most efficient uptake of
the required metals, while during higher than optimal metal presence,
sequestration and elimination mechanisms will become more prominent.
There is evidence for several compounds that the concentration of free ions
in the cytosolic environment is maintained at very low levels, e.g., by
shielding the charge, e.g., through weakly chelating compounds. Metal-
protein interactions with specific proteins perform essential functions in
uptake, storage and elimination of metals. For iron for instance, we know
specific transporters such as the divalent metal transporter 1 (DMT1), fer-
ritin as a storage protein, and other iron-carrying proteins such as trans-
ferrin, ferroportin or haphaestin, the latter catalyzing the oxidation of Fe21
to Fe31 [16]. Thus, exposure to metals may be regarded as a stress situation,
whereby the biosystem will react with regulated responses up to the point
where the system is overloaded either due to a too high concentration of
bioavailable free ions or a too long exposure duration to a metal.
For combined effects from metal mixture exposure this should mean that, as
specific effects at the molecular level will converge into general stress
responses, there should be a reasonable chance to estimate the joint effects
from what can be seen for the individual components at the same level. We are
not aware of any examples that have tested this hypothesis quantitatively, but
qualitative examples are present in the literature. Demuynck and coworkers
[18] investigated the metallothionein response in the earthworm Eisenia fetida
upon co-exposure with Cd and Zn. Figure 2 displays some of their findings.
The investigation describes that zinc concentrations in the body of earthworms
were constant irrespective of the offered range of ambient soil zinc con-
centrations and the occurrence of varying co-exposure with cadmium. Thus,
for the element zinc, regulation seemed to be functional in a wide concentra-
tion range. By contrast, for the non-essential metal cadmium the body con-
centration did increase with the ambient concentration offered and only at low
cadmium concentrations (8 mg/kg dry soil) or high zinc concentrations
(1500 mg/kg dry soil) co-exposure to zinc slightly reduced the bioaccumulation
of cadmium. Metallothionein gene expression studied as mRNA quantifica-
tion with a specific probe for Cd-binding isoforms showed increased expres-
sion at higher cadmium exposures only, indicating that the system tries to
counteract the increasing internal body burden of cadmium by sequestration.

20 ALTENBURGER
Figure 2. Metal co-exposure and metal homeostasis in earthworms. Reproduced

from [18] with permission from Elsevier, copyright (2007).
Toxic effect would thus be expected to occur irrespective of whether it

is due to single or mixture exposure if via competitive action to essential
elements, their homeostasis is perturbed or if stress responses such as
metallothionein expression, glutathione production or DNA repair are
overloaded. It seems, that when we have measures for quantifying such
processes as, e.g., the iron balance disturbance or the change in cell redox
status, it should be possible to formulate a refined hypothesis for expectable
combined effects for metal mixtures based on the above discussed reference
models.

Figure 2. Continued.
5. INTERACTION WITH ORGANIC COMPOUNDS
5.1. Metals and Polyaromatic Hydrocarbons

Co-occurrence of chemicals in the environment is not restricted to metals but
organic compounds such as polyaromatic hydrocarbons (PAHs) derived
from natural or anthropogenic sources can be found in mixtures with metals
in almost any environment. Sediment and soil systems are often in the focus
when reflecting upon the likeliness of resultant co-exposure of organisms
against metals and PAH-type compounds. Polyaromatic hydrocarbon
compounds, as organic chemicals in general, differ fundamentally in their
behavior and effects in the environment as a result of the different chemical
properties. This is evident with respect to the susceptibility for transforma-
tion reactions that may lead to complete mineralization of compounds, as
well as if one considers the importance of partitioning-driven bioavailability,

22 ALTENBURGER
uptake, and toxic processes. Combined effects in organisms from co-expo-

sure to metals and PAHs have therefore been a subject of observational and
experimental studies coming from different angles [19,21].
Observations of combined effects from simultaneous joint exposure
against various metals and PAHs have been reported to occur for an array of
organisms, considering different biological responses and exposure settings.
Moreover, it seems that despite their fundamental differences in chemical
properties, the above introduced concepts of concentration addition and
response addition are of use in assessing the magnitude of observable
combined effects (cf., e.g., an early work [19]). Like for metal mixtures,
investigations have frequently reported deviations from the model-based
combined effect expectations. No clear pattern with respect to deviation
types or magnitudes, structural properties or biological responses has yet
emerged.
From consideration of photochemical processes, interesting hypotheses
are derived which may lead to a better understanding and thus, to an
improved predictability of the biological outcomes of mixture exposure
against metals and PAHs. Many PAH-type compounds are able to absorb
ultraviolet radiation upon which the compounds reactivity in biological
systems are driven by either photosensitization or photomodification [20].
Both processes typically enhance the otherwise mainly baseline or narcotic
type of effects. Interestingly in our context, many of these photoenhanced
effects can be seen as caused by oxidative stress. The means of provoking
oxidative stress differ for the different processes and compounds (Figure 3)
but regarding the combination effects with metals they might provide a clue
where to search for compound interaction. Wang et al. [21] provided evi-
dence, based on early work from the same group, that the type of interaction
between a metal and a PAH compound may depend on the mixture ratio as
well as the redox activity of the metal in question. With higher redox activity
and concentration of a metal the notion of coupled redox-cycling processes
seems to explain observed increased joint toxicities. This hypothesis, inves-
tigated with quinone-type PAH photomodification products and some
metals of varying redox activity, could be a starting point for more sys-
tematic evaluations in other biosystems.
5.2. Metals and Other Organic Compounds

Not only polyaromatic hydocarbons co-occur with metals in the environ-
ment. Three further examples will be provided: Surfactants are probably the
group of organic chemicals most abundantly emitted into freshwaters via
wastewater effluents. In conjunction with metals, it has been shown that
surfactants by complexation may reduce availability and thus biological

superoxide
generating
systems As5+ As3+
+e- SOD O2
.-
O2 O2 H2O2 H2O
GSH GSSG
Hg2+ Cd2+
As3+
Fe3+ Cu2+ Co2+ NADP+ NADPH
Cr6+ Ni2+ V5+ 4+
V Cr5+ V5+ Cr6+
.
hydroxyl radical OH
singlet oxygen 1O2 lipid peroxidation
. .
metal-peroxo [Me-OO] LOO LO
cancer DNA damage As3+ Cd2+

gene activation
DNA repair
mechanisms
repaired DNA
Figure 3. Interaction of metals contributing to oxidative stress. Reproduced from

[16] with permission from Bentham Science Publishers, copyright (2005).
effects of metals, though for the case of chlorophenol and iron complexes,
there is evidence that complexation may also help to shield the electronic
charge and thus increase uptake and subsequently the internal biological
dose. Also, when surfactants such as in the case of some cationic quaternary
ammonium compounds elucidate biological effects at low concentrations
themselves, different principles seem to govern the overall response.
From the multitude of organic chemicals known to be present in the
environment, particularly those that are purposefully used and emitted have
been studied for their joint effects with metals. Biocide products, for instance
the active ingredients of ship antifoulings that are made from copper,
organozinc or organtin compounds, are often used in conjunction with
organic compounds. Right from their intentional use it may be concluded
that the environmental activity in terms of biological efficacy towards a
wider spectrum of species is increased.
Other biologically highly active compounds such as pesticides co-occur as
mixtures after field spray drift or runoff events and have been shown as

24 ALTENBURGER
being potent to elucidate combined effects. Barata and colleagues [22] in a

study on lethal effects and feeding impairment in daphnids upon joint
exposure against several mixtures of metals and phyrethroid insecticides
showed the predictivity of the above outlined reference concepts as well as
the deviations that may be found. From their observations they deduce that
despite a specific molecular interaction between a compound and the bio-
logical system, the overall adverse outcome of a joint exposure might be well
described by either reference model. A major conclusion from their study,
that may therefore be of general interest, was support for a previously purely
theoretical notion, namely, that it would be useful to calculate combined
effect expectations using both reference models in parallel to provide a
‘prediction window’ for what can be expected from the individual com-
pounds activities, irrespective of a specific mode of action.
6. OUTLOOK
Understanding the combined effects for metal co-exposure in ecotoxicology

has been shown to be a challenging and striving research effort, where much
detail work for specific purposes will no doubt continuously have to be
performed. With the goal to improve the usefulness of predictive models that
– based on our understanding of the environmental behavior and effects of
individual compounds – allow qualitatively predicting and quantitatively
calculating joint effects, we envisage some major tasks.
In order to clarify the picture where the knowledge of bioavailable metal
concentrations or internal dose levels would help to better predict combined
effects, the existing models and measuring techniques should be used for
carefully designed generic mixture studies. As for the role of understanding
the mechanisms of interaction of metal mixture or perhaps rather their
toxicity pathways, we would need more evidence that strive for pattern
description while for highly specific case studies we would expect the iden-
tification and quantification of processes that improve a mechanism-based
model building.
Given the modern multivariate biological detection tools, such as trans-
criptomic or metabolomic assays, it seems within reach to achieve more
knowledge on where combined effects become different from noise and
accessible for assessment. For general assessment it occurs that in hypothesis
formulation it could be useful to pragmatically calculate a prediction win-
dow by using both reference models, concentration and response addition,
and care most about combined effect deviations that are significant for both
expectations.
Finally, we have to state that evidence is much smaller when it comes to
multiple mixtures, where for using the model of response addition, excellent

description of individual concentration response relationships will be

required. Moreover, up to now, we have just started understanding com-
bined effects from simultaneous exposure while time varying and sequential
exposure are awaiting our scientific curiosity.
ACKNOWLEDGMENTS
The HGF programme topic CITE provided resources for this work.
ABBREVIATIONS
BLM biotic ligand model
Ci concentration of substance i
DMT1 divalent metal transporter 1
E(ci) defined biological effect of a given concentration for
substance i
EC50 concentration of a chemical at which 50% of a defined
biological effect is estimated to occur
PAH polyaromatic hydrocarbon
pyrithione 2-mercaptopyridine N-oxide ( ¼ 1-thiol-pyridine N(1)oxide)
zineb zinc ethylene bis(dithiocarbamate)
REFERENCES
1. W. P. Norwood, U. Borgmann, D. G. Dixon and A. Wallace, Human Ecol. Risk
Assess., 2003, 9, 795–811.
2. R. Altenburger, H. Walter and M. Grote, Environ. Sci. Technol., 2004, 38, 6353–
6362.
3. W. R. Greco, G. Bravo and J. C. Parsons, Pharmacol. Rev., 1995, 47, 331–385.
4. W. Bödeker, R. Altenburger, M. Faust and L. H. Grimme, Arch. Complex
Environ. Studies, 1992, 4, 45–53.
5. S. Loewe and H. Muischnek, Arch. Naunyn-Schmiedebergs Arch. Exp. Pathol.
Pharmakol., 1926, 114, 313–326.
6. C. I. Bliss, Ann. Appl. Biol., 1939, 26, 585–615.
7. K. Drescher and W. Boedeker, Biometrics, 1995, 51, 716–730.
8. W. Bödeker, R. Altenburger, M. Faust and L. H. Grimme, Nachrichtenblatt des
Deutschen Pflanzenschutzdienstes (Braunschweig), 1990, 42, 70–78.
9. R. Altenburger, H. Schmitt and G. Schüürmann, Environ. Toxicol. Chem., 2005,
24, 324–333.
10. M. Scholze, W. Boedeker, M. Faust, T. Backhaus, R. Altenburger and
L. H. Grimme, Environ. Toxicol. Chem., 2001, 20, 448–457.

26 ALTENBURGER
11. Q. Ren and I. T. Paulsen, PLoS Computational Biology, 2005, 1, 190–201.

12. N. Balatori, Environ. Health Perspec., 2002, 110 (Suppl. 5), 689–694.
13. U. Borgmann, W. P. Norwood and D. G. Dixon, Human Ecol. Risk Assess.,
2008, 14, 266–289.
14. P. R. Paquin, J. W. Gorsuch, S. Apte, G. E. Batley, K. C. Bowles,
P. G. C. Campbell, C. G. Delos, D. M. Di Toro, R. L. Dwyer, F. Galvez, R. W.
Gensemer, G. G. Goss, C. Hogstran, C. R. Janssen, J. C. McGeer, R. B. Naddy,
R. C. Playle, R. C. Santore, U. Schneider, W. A. Stubblefield, C. M. Wood and
K. B. Wu, Comp. Biochem. Physiol. C, 2002, 133, 3–35.
15. D. M. Di Toro, H. E. Allen, H. L. Bergmann, J. S. Meyer, P. R. Paquin and
R. C. Santore, Environ. Toxicol. Chem., 2001, 20, 2383–2396.
16. M. Valko, H. Morris and M. T. D. Cronin, Curr. Med. Chem., 2005, 12,
1161–1208.
17. T. Vandenbrouck, A. Soetaert, K. van der Ven, R. Blust and W. De Coen,
Aquatic Toxicol., 2009, 92, 18–29.
18. S. Demuynck, F. Gruminaux, V. Mottier, D. Schkorski, S. Lemière and
A. Lepêtre, Comp. Biochem. Physiol. C, 2007, 145, 658–668.
19. D. de Zwart and W. Sloff, Bull. Environ. Contam. Toxicol., 1987, 38, 345–351.
20. PAHs: An Ecotoxicological Perspective, Ed. P. E. T. Douben, Wiley, Hoboken,
Chichester, UK, 2003, pp. 1–392.
21. W. Wang, M. A. Lampi, X. -D. Huang, K. Gerhardt, D. G. Dixon and
B. M. Greenberg, Environ. Toxicol., 2008, 24, 166–177.
22. C. Barata, S. J. Baird, A. J. A. Nogueira, A. M. V. M. Soares and M. C. Riva,
Aquatic Toxicol., 2006, 78, 1–14.
23. R. Altenburger, M. Nendza and G. Schüürmann, Environ. Toxicol. Chem., 2003,
22, 1900–1915.
24. B. Markert, in Ecotoxicology. Ecological Fundamentals, Chemical Exposure, and
Biological Effects, Ed. G. Schüürmann and B. Markert, John Wiley & Sons, New
York, USA, 1998, pp. 165–222.
25. P. G. C. Campbell and A. Tessier, in Ecotoxicology. A Hierachical Treatment,
Ed. M. C. Newman and C. H. Jagoe, CRC Lewis, Boca Raton, USA, 1996,
pp.11–58.
26. A. A. Meharg and J. Hartley-Whitaker, New Phytol., 2002, 154, 29–43.

2
Human Risk Assessment of Heavy Metals:
Principles and Applications
Jean-Lou C. M. Dorne, 1* George E. N. Kass,1 Luisa R. Bordajandi,1
Billy Amzal, 1 Ulla Bertelsen,1 Anna F. Castoldi,1 Claudia Heppner,1
Mari Eskola, 1 Stefan Fabiansson,1 Pietro Ferrari,1 Elena Scaravelli,1
Eugenia Dogliotti, 2 Peter Fuerst, 3 Alan R. Boobis 4 and
Philippe Verger 5
1
European Food Safety Authority, Largo N. Palli 5, I-43100 Parma, Italy
2
Istituto Superiore di Sanita, Viale Regina Elena 299, I-00161 Rome, Italy
3
Chemical and Veterinary Analytical Institute, Munsterland-Emscher-Lippe (CVUA-MEL),
Joseph-Königstrasse 40, D-48147 Münster, Germany
4
Imperial College, Department of Experimental Medicine and Toxicology, Burlington Danes,
Hamersmith Campus, Du Cane Road, London, W12 ONN, UK
5
World Health Organisation, Department of Food Safety and Zoonoses, 20 Avenue Appia,
CH-1211 Geneva, Switzerland
<jean-lou.dorne@efsa.europa.eu>
ABSTRACT 28
1. INTRODUCTION 29
2. PRINCIPLES OF CHEMICAL RISK ASSESSMENT 29
2.1. Risk Assessment of Non-Genotoxic and Genotoxic
Carcinogens 30
2.2. The Four Pillars of Risk Assessment 33
3. TOXICOLOGY OF HEAVY METALS 34
3.1. General Principles 34
3.2. Toxicokinetics 35
28 DORNE et al.
3.2.1. Absorption, Distribution, Metabolism, and Excretion

of Heavy Metals and Metalloids 35
3.2.2. Physiologically-Based and Population-Based
Toxicokinetic Models 36
3.3. Toxicodynamics 37
3.4. Selected Molecular Mechanisms of Action: Epigenetic
Mechanisms of Carcinogenicity 38
4. ANALYTICAL TECHNIQUES AND EXPOSURE
ASSESSMENT OF HEAVY METALS 39
4.1. Analytical Techniques for the Detection of Heavy Metals
and Metalloids in Biological Samples 39
4.2. Data Sources for the Estimation of Human Dietary Exposure 40
4.3. Combining Occurrence and Consumption Data in Humans
for Exposure Assessment 42
5. APPLICATIONS TO THE HUMAN RISK ASSESSMENT OF
HEAVY METALS AND METALLOIDS 43
5.1. Hazard Identification and Characterization 44
5.1.1. Cadmium 44
5.1.2. Lead 44
5.1.3. Methylmercury 45
5.1.4. Uranium 45
5.1.5. Arsenic 46
5.2. Exposure Assessment of Heavy Metals and Metalloids 47
5.2.1. Cadmium 47
5.2.2. Lead 47
5.2.3. Methylmercury 48
5.2.4. Uranium 48
5.2.5. Arsenic 49
5.3. Risk Characterization of Heavy Metals and Metalloids 50
5.3.1. Cadmium 50
5.3.2. Lead 51
5.3.3. Mercury 51
5.3.4. Uranium 52
5.3.5. Arsenic 52
6. CONCLUSIONS AND FUTURE PERSPECTIVES 53
ACKNOWLEDGMENTS 54
ABBREVIATIONS AND DEFINITIONS 54
REFERENCES 55
ABSTRACT: Humans are exposed to a number of ‘‘heavy metals’’ such as cadmium,

mercury and its organic form methylmercury, uranium, lead, and other metals as well
as metalloids, such as arsenic, in the environment, workplace, food, and water supply.
Exposure to these metals may result in adverse health effects, and national and

HUMAN RISK ASSESSMENT OF HEAVY METALS: PRINCIPLES 29
international health agencies have methodologies to set health-based guidance values

with the aim to protect the human population. This chapter introduces the general
principles of chemical risk assessment, the common four steps of chemical risk assess-
ment: hazard identification, hazard characterization, exposure assessment, risk char-
acterization, and toxicokinetic and toxicity aspects. Finally, the risk assessments
performed by international health agencies such as the World Health Organisation, the
Environmental Protection Agency of the United States, and the European Food Safety
Authority are reviewed for cadmium, lead, mercury, uranium, and arsenic.
KEYWORDS: arsenic . cadmium . lead . mercury . risk assessment . toxicokinetics .

toxicity . uranium
1. INTRODUCTION
Humans are exposed to a range of ‘‘heavy metals’’ such as cadmium, mer-

cury and its organic form methylmercury (CH3-Hg), uranium, lead, and
other metals as well as metalloids, such as arsenic, in the environment,
workplace, food and water supply. In history, a plethora of epidemiological,
toxicological and molecular evidence from all around the globe has shown a
variety of health risks to human populations associated with environmental,
occupational, and dietary exposure to such metals. Consequently, health
agencies have been setting health-based guidance values to prevent the
occurrence of adverse health effects in humans.
The aim of this chapter is first to introduce the four steps of chemical risk
assessment for non-genotoxic and genotoxic carcinogens, namely hazard
identification, hazard characterization, exposure assessment, and risk
characterization. The toxicology and risk assessment performed by inter-
national health agencies on cadmium, lead, mercury, uranium, and arsenic
are reviewed together with potential future developments.
2. PRINCIPLES OF CHEMICAL RISK ASSESSMENT

Risk has been defined as a function of hazard and exposure. The Interna-
tional Program on Chemical Safety (IPCS) of the World Health Organisa-
tion (WHO) has defined hazard as ‘‘the inherent property of an agent or
situation having the potential to cause adverse effects when an organism,
system or (sub)population is exposed to that agent’’ and risk as ‘‘the
probability of an adverse effect in an organism, system or (sub)population
caused under specified circumstances by exposure to an agent’’ [1]. The
qualification and quantification of hazard and risk are the corner stones of

30 DORNE et al.
the risk assessment paradigm. In terms of food safety, the European Union
has defined ‘‘hazard’’ as a biological, chemical or physical agent in, or
condition of, food and ‘‘risk’’ as a function of the probability of an adverse
health effect and the severity of that effect, consequential to a hazard [2].
2.1. Risk Assessment of Non-Genotoxic and Genotoxic

Carcinogens
Risk assessment of chemicals in humans relies on a mechanistic assumption
that such chemicals may either be genotoxic or non-genotoxic. Genotoxic
carcinogens and their metabolites are assumed to act via a mode of action
that involves a direct and potentially irreversible DNA-covalent binding
whereas non-genotoxic carcinogens or their metabolites are assumed to act
via an epigenetic mode of action without covalent binding to DNA. In terms
of risk assessment, a linear low dose-response relationship for life time
exposure with no threshold or a dose without a potential effect is usually
assumed for genotoxic carcinogens whereas a threshold level of exposure,
below which no significant effects are induced, is assumed for non-genotoxic
carcinogens (and for almost all non-cancer endpoints). For the latter, this
implies that homeostatic mechanisms are able to balance biological pertur-
bations produced by low levels of intake, and that structural or functional
changes leading to adverse effects, which may include cancer, would be
observed only at higher intakes [3,4].
Worldwide, the risk assessment of genotoxic carcinogens is performed
using one of the three major methods namely linear extrapolation from high
dose animal studies to low exposures in humans, the threshold of tox-
icological concern and the margin of exposure approach.
The linear extrapolation (LE) approach has been used by the US Envir-
onmental Protection Agency (US-EPA), Norway and in the European
Union for industrial chemicals, non-threshold carcinogens and for carci-
nogens for which the mode of action is unclear. LE often involves modelling
of dose-response data from high dose carcinogenicity studies in animals
using the lower end of the observed range of tumor incidences. Hence, a risk
estimate of cancer for low dose life time exposure in humans (1 in 105 or 106)
can be derived and often LE has involved the lower 95% confidence interval
of the bench mark dose (BMD) producing a 10%, 5%, 1% increase in tumor
incidence compared to background incidences (BMDL10, BMDL05,
BMDL01) from mostly animal data or on rare occasions human epide-
miological data when available. Overall, LE provides estimates of the pos-
sible range of cancer risk associated with lifetime exposure to a particular
concentration of a genotoxic carcinogen in food, air or from other exposure

routes (e.g., a risk of 0–1 in a million). LE has limitations in the fact that the
potency of the carcinogen in animals is assumed to relate directly to the
potency in humans and such assumptions are still not supported by sub-
stantive data [5]. In addition, considerable uncertainty is introduced by the
extent to which it is often necessary to extrapolate to human exposure levels.
The threshold of toxicological concern (TTC) was originally proposed by
Cramer et al. [6] to establish exposure thresholds predicted to be without
adverse effects based on the distribution of potencies of a large number of
compounds. One of the main advantages of the TTC approach is that low
exposure risk can be evaluated without the need for chemical-specific data
from animal toxicity studies as proposed in a TTC decision tree by Kroes
et al. [7]. From this analysis, threshold values for three groups of non-geno-
toxic chemicals were proposed according to their toxicity in relation to
human exposure and expressed in mg/kg b.w./day for a 60 kg adult with
group I (30) (low), group II (9) (intermediate), and group III (1.5) (high)
[5,7,8]. However, this approach is not relevant to heavy metals since metals
were excluded when the TTCs were derived [3,8].
The margin of exposure (MOE) approach was introduced after an inter-
national conference organized by the International Life Sciences Institute
(ILSI), the Joint Food and Agricultural Organization of the United Nations/
WHO (FAO/WHO) Expert Committee on Food Additives (JECFA),
and the scientific committee of the European Food Safety Authority (EFSA)
[9–11]. The MOE is defined as the ratio of a specified point on a dose-
response curve for adverse effects obtained in animal experiments (in the
absence of human epidemiological data) and human intake data. Like for
the LE approach, the preferred reference points describing the dose-response
relationship are the BMD and BMDL. Overall, the Scientific Committee of
EFSA considered that an MOE of 10,000 or more, based on a BMDL10
derived from animal cancer bioassay data and taking into account the
uncertainties in the interpretation, ‘‘would be of low concern from a public
health point of view and might reasonably be considered as a low priority for
risk management actions’’ [9]. EFSA has recently conducted a risk assess-
ment for the metalloid arsenic using this approach [12] (see Sections 5.1.5
and 5.2.5).
For non-genotoxic carcinogens, threshold levels of toxicity are defined as
‘‘without appreciable health risk’’ when consumed every day or weekly for a
lifetime such as the acceptable/tolerable daily intake (ADI/TDI) or provi-
sional tolerable weekly intake (PTWI) used in Europe and by the WHO, the
tolerable daily intake or tolerable concentration in Canada or the ‘reference
dose’ (RfD) in the United States by the US Environmental Protection
Agency (EPA) and the Agency for Toxic Substances and Disease Registry
(ATSDR) [13,14]. Despite the nomenclature differences, these health-based
guidance values are all determined by dividing a surrogate for the threshold

32 DORNE et al.
determined from chronic/subchronic animal studies using the most sensitive

species (usually mouse, rat, rabbit or dog), such as the no observed adverse
effect level (NOAEL) or the BMDL 95% lower confidence limit, by a default
uncertainty factor (UF) of a 100-fold [15]. The BMD is defined as a dose
level, derived from the estimated dose-response curve, associated with a
specified change in response, the benchmark response (BMR) (e.g., 0.1%,
1%, 5% or 10% incidence). The BMD limit (BMDL) is the lower confidence
bound, and is often used as the reference point. e.g., for a BMR of 5%, the
BMDL05 can be interpreted as a dose for which the response is likely to be
smaller than 5% and for which the term ‘‘likely’’ is defined by the statistical
confidence level, usually 95% confidence [16].
The 100-fold uncertainty factor has been further split to allow for dif-
ferences in toxicokinetics (TK), relating the external dose to the internal
dose: i.e., absorption, distribution, metabolism, and excretion, and in tox-
icodynamics (TD), relating the concentration of the proximate toxicant
(parent compound, metabolite or both) in the target organ(s) and the sen-
sitivity of the target organ(s) itself [17,18]. Renwick [17] proposed TK and
TD values of 4 and 2.5 for interspecies differences and even values of 3.16 for
human variability. These were derived from the analysis of a small database
describing interspecies differences, expressed as the ratio between the animal
species and humans for TK processes and parameters (e.g., liver weight, liver
blood flow, renal blood flow, absorption, elimination) as well as for TD
sensitivity to a chemical (e.g., sedation, pain relief) [19]. The subdivision was
subsequently adopted by the IPCS workshop on the derivation of guidance
values [20]. The main aim of this subdivision was to allow for chemical-
specific TK and ideally to derive chemical-specific adjustment factors
(CSAFs) [21,22]. Further refinements have been developed using the ther-
apeutic drug database and include pathway-related uncertainty factors (PR-
UFs) as an intermediate option between CSAFs and the UF when the
pathway of metabolism is known but compound-specific TK data are not
available. These have been derived for human variability in TK for phase I,
phase II, and renal excretion in subgroups of the human population and
interspecies variability [18,22–25] for test species for CYP1A2, glucur-
onidation, and renal excretion [15,26–29].
Ideally, CSAF or a physiologically-based toxicokinetic model (PB-TK)
when compound-specific data are available as recommended by the WHO
[22] and this approach has been recently explored by the panel on con-
taminants in the food chain (CONTAM) of the EFSA for the risk assess-
ment of cadmium in food for which a PB-PK model together with human
BMDL was used to set a PTWI for humans (see Section 5).
Beyond the mechanistic assumptions of genotoxicity and thresholded
toxicity, the application of the four pillars of risk assessment is common and
summarized below.

2.2. The Four Pillars of Risk Assessment

The four steps of the risk assessment, namely (1) hazard identification,
(2) hazard characterization, (3) exposure assessment, and (4) risk char-
acterization have enabled scientists and public health agencies to protect
consumers and the environment from adverse health effects that may result
from acute and chronic chemical exposure [30].
1. Hazard identification has been defined as ‘‘the identification of biolo-

gical chemical and physical agents capable of causing adverse health
effects and which may be present in a particular food or group of
foods’’. The main purpose of hazard identification applied to metals is
to evaluate the weight of evidence for adverse health effects, based on an
assessment of all the available data regarding toxicity and mode of
action (non-genotoxic/genotoxic) of the particular metal. In practice, a
review of studies regarding the mode of action (evidence for muta-
genicity, genotoxicity), the TK of the metal (absorption, distribution,
metabolism, and excretion), the nature of any toxicity or adverse health
effect occurring, and the affected (target) cell(s)/organ(s)/tissue(s) site
(TD) is performed. Toxicological studies in animals (mainly mouse, rat,
rabbit, and dog) play a critical role in hazard identification and ideally
use international guidelines and good laboratory practices (GLPs) and
include acute (single dose studies), sub-chronic (repeated dose studies:
28–90 days) and chronic studies (up to 2-year study) and/or more
specific endpoints (reproductive and developmental toxicity, neuro-
toxicity, immunotoxicity. . .) [31]. However, in the case of most heavy
metals (cadmium, mercury, methylmercury, lead) and metalloids
(arsenic), epidemiological human data were available and these have
been used to select critical studies for the setting of health-based gui-
dance values. For uranium, the results of chronic/sub-chronic (28–90
days) studies from the most sensitive species were selected.
2. Hazard characterization (also known as dose–response assessment)
constitutes ‘‘the qualitative and/or quantitative evaluation of the nature
of the adverse health effects associated with biological, chemical and
physical agents which may be present in food’’ [32]. Currently, the
BMD approach is preferred to the NOAEL/LOAEL approach because
it makes extended use of the dose-response data from studies in the
most sensitive species of experimental animals or from observational
epidemiological studies to estimate the shape of the overall dose-
response relationship for a particular endpoint so that both genotoxic
and non-genotoxic carcinogens can be assessed. In practice, the iden-
tification of the reference point (NOAEL/LOAEL or BMD/BMDL)
constitutes a basis for the risk characterization of a particular chemical.

34 DORNE et al.
An important distinction between thresholded toxicants and genotoxic

carcinogens is that the ADI/TDI for the former is derived in this step
usually by applying UFs whereas the MOE is derived in the risk
characterization part taking into account the human exposure.
3. Exposure assessment is ‘‘the qualitative and/or quantitative evaluation of
the likely intake of biological, chemical and physical agents via food as
well as exposure from other sources if relevant’’. For chemical con-
taminants in the food and the feed chain, exposure assessment integrates
the occurrence and the concentrations of the compound in the human
diet measured using validated analytical techniques and the human
consumption patterns for the different food categories available. Addi-
tionally, a range of intake/exposure scenarios are taken into account so
that special subgroups of the population that may be at either high
dietary exposure or high consumers are taken into account [4,32].
4. Risk characterization is the final step and represents ‘‘the qualitative
and/or quantitative estimation, including attendant uncertainties, of
the probability of occurrence and severity of known or potential
adverse health effects in a given population based on hazard identi-
fication, hazard characterization and exposure assessment’’ [32]. In
practice, risk characterization integrates the hazard identification and
characterization, leading to a health-based guidance value PTWI/TDI
and the human exposure, estimated from either a deterministic or
probabilistic method to conclude on the likelihood of adverse effects
for public health. In contrast, for genotoxic carcinogens, the MOE is
calculated in this step by dividing the point of departure, often a
BMDL, with the human exposure. Currently, an MOE of 10,000 is
considered of low public health concern but the interpretation has also
to be taken on a case by case basis. In summary, from the identifi-
cation and characterization of the toxicological effects (dose-response)
of a chemical a health-based guidance value is derived. Using vali-
dated analytical techniques, the amount of the chemical is measured in
a biological matrix (water, food, air, etc.) and combined with the
human consumption (via oral route or inhalation) of the biological
matrix to estimate human exposure. Exposure is then related to the
health-based guidance value to characterize the potential risk of
adverse health effects in humans after acute or chronic exposure [4].
3. TOXICOLOGY OF HEAVY METALS
3.1. General Principles

The old adage by Paracelsus stipulates ‘‘Sola dosis fecit venenum – it is only
the dose which makes a chemical a poison’’ and applies to heavy metals and

metalloids since such substances are undesirable in food and the environ-
ment. In principle, the toxicity of chemicals including metals arises from two
basic processes: what the body does to the chemical (toxicokinetics, TK) and
what the chemical does to the body (toxicodynamics, TD) [4].
3.2. Toxicokinetics
TK involves the translation of the external dose of a chemical to an internal
dose leading to overall elimination from the body, i.e., absorption from the
site of exposure, often the gastrointestinal tract, distribution in body fluids/
tissues, metabolism to biologically inactive/active metabolites and ultimately
excretion in the urine/feces. Potential bioaccumulation in tissues of either the
parent compound or metabolites is an important aspect for TK and depends
on the absorption, distribution, metabolism, and excretion of the com-
pound. The biological half-life of the compound and its lipophilicity provide
good descriptors as to whether it will bioaccumulate or not. Although some
adjustment factors can be used to translate TK parameters from animals to
humans, only human toxicokinetics is addressed in this section.
3.2.1. Absorption, Distribution, Metabolism, and Excretion of

Heavy Metals and Metalloids
The main absorption routes of heavy metals are usually oral and inhalation.
Absorption from dermal exposure can still exist but at very limited level
(e.g., about 0.1% for uranium). The solubility of the metal forms is highly
influencing the absorption fraction, in either oral or pulmonary routes. Non-
soluble forms have generally a very limited absorption (below 1%) range of
values for various heavy metals absorbed via both routes. Oral absorption is
very variable ranging from 1–10% for cadmium, 10–50% for lead, 1–30%
for methylmercury, 1–6% for uranium, 40–100% for soluble forms of
arsenic [12,33,34].
Transport and distribution models are not always clear-cut for heavy
metals. For most of them, heavy metals get rapidly attached to blood cells
once absorbed. Blood (via erythrocyte binding) and plasma are typically the
main transport routes. Metabolic pathways for most heavy metals and
metalloids are generally complex and multiple and not always identified. For
example, accumulation of uranium in tissues may not be constant over time
during chronic exposure and can significantly accumulate in non-target
organism such as brain and teeth [33]. Furthermore, high inter-individual
variability is observed in human susceptibility and has been attributed to
genetic polymorphism in the enzymes associated with the metal metabolism,
especially in the case of arsenic [34]. Most of the studies dealing with this

36 DORNE et al.
genetic basis of variability in the human metabolism of arsenic concentrate

on the polymorphisms of arsenic-methyltransferases and glutathione-S-
transferases (mainly omega 1 and omega 2 isoforms) [34]. For most metals,
long-term accumulation occurs to a large proportion in the kidney for As,
Cd, and mercury, and in blood for lead, whereas uranium accumulates in
most organs and is released via the urinary route.
Most heavy metals are excreted via the kidney in the urine, and to a much
lesser extent by the gastrointestinal tract. The half-life, which characterizes
the elimination of heavy metals from the body, varies widely between metals.
It can be larger than 10–12 years for cadmium and lead, with inter-individual
variability of about 30% [35], 4 days for arsenic, 60 days for mercury and 0.5
to 1 year for uranium.
3.2.2. Physiologically-Based and Population-Based Toxicokinetic

Models
For most chemical compounds, the TK of heavy metals can be assessed

using compartment models such as the physiologically-based TK model
(PBTK) or the population TK models.
The PBTK models describe in more details the metabolic pathways and
allow the calculation of heavy metal concentrations in the main organs in the
body. On the other hand, the numerous parameters require substantial
parameter information and make any statistical evaluation and fitting more
difficult. It generally requires thorough sensitivity analysis and model vali-
dations. They usually provide estimates of the main TK parameters for a
typical individual, for a given body weight. Conversely, they are usually not
suitable to assess inter-individual variability of those parameters because
models become computationally too intricate. PBTK models have been built
and used in humans for most heavy metals, such as arsenic [36–38], cadmium
(e.g., 8-compartment model in [39,40]), lead [41], methylmercury [42],
chromium and uranium [43].
An alternative to PBTK models is a population approach such as popu-
lation TK models, which are usually simpler (one or two compartments),
focused on the main elimination routes of the compound, and making rough
and global assumptions on other pathways of elimination. In case of poor
prior knowledge, this approach allows a simplified and parsimonious
description of the compound’s elimination, hence enabling more sophisti-
cated statistical evaluations (such as the estimation of population varia-
bility). Population models are therefore often an interesting option in the
area of human risk assessment, as they can provide a more precise and
reliable estimate of chemical-specific UFs [24]. However, in some cases
where the simple toxicokinetic assumptions are not met (like zero-order

absorption or linearity), such an approach could lead to models with poor

fits and high residuals. Moreover, such an approach does not allow for the
evaluation of a compound’s concentration in all organs. Recent population
models for heavy metals have been developed, e.g., for cadmium a
1-compartment model [35] and for arsenic a TKTD model [44]. The choice
between PBTK and population-based TK models depends on the precise
aim of the model and on the available data.
3.3. Toxicodynamics
Toxicological effects may occur when the toxic species, which either is the
parent compound or one or more of its metabolites, reaches a critical target
within the body. The cells in our body are equipped with a range of powerful
defence and repair mechanisms, and toxicity is only observed once this
protective barrier has been overwhelmed. The key defence mechanisms
comprise among others, small antioxidant molecules such as ascorbic acid
and a-tocopherol, the tripeptide glutathione (GSH) and a range of anti-
oxidant enzymes such as superoxide dismutases, catalase, GSH transferases
and GSH peroxidases [45]. Our cells are therefore well equipped to deal with
toxic compounds that induce conditions of oxidative stress. Indeed, the
majority of toxic drugs and environmental compounds and the effects
caused by ionizing and non-ionizing radiation, through the direct generation
of oxygen-based (ROS) (e.g., superoxide anion radical or hydroxyl radical)
or nitrogen-based free radicals (RNS) (e.g., peroxynitrite) or through the
depletion of cellular thiols via oxidation or conjugation, lead to conditions
of oxidative stress. These result in direct or indirect damage to cellular
proteins, phospholipids, and nucleic acids and in turn to a spectrum of
cellular effects ranging from cancer to cell death.
Toxic (non-essential) metals have been shown to induce conditions of
oxidative stress either through their ability to undergo redox-cycling and
generate ROS such as superoxide or as a consequence of enhanced pro-
duction of ROS by damaged mitochondria. For example, lead is able to
generate ROS [46] and similarly, enhanced formation of ROS from mito-
chondria occurs in cells exposed to arsenic [47], probably as a result of the
ability of the metal ion to bind to protein thiol groups and induce mito-
chondrial damage through opening of the mitochondrial permeability
transition pore [48].
A unique feature of toxic metals is the ability of the complexes, formed
between the metal ion and the nucleophilic sites on cellular proteins, to
mimic endogenous substrates or conformations. This property is responsible
for the selective transport of metal ions into or across cells and to interfere
with the functioning of target enzymes [49]. An example of this ionic

38 DORNE et al.
mimicry is the ability of lead to activate protein kinase C by acting as a

surrogate for the enzyme’s normal activator, Ca21 [50]. Perturbations in cell
signaling in response to oxidative stress can lead to changes in cell pro-
liferation and cell differentiation, but from a toxicological point of view,
changes in cell survival signals, such as the growth factor-dependent phos-
phoinositide 3-kinase pathway, play a critical role in the development of a
number of diseases such as cancer [51]. When damage to the cell becomes
excessive, generally as a consequence of damage to mitochondria, cell death
pathways are activated, and these typically take the form of apoptosis,
autophagic cell death or necrosis [52,53].
3.4. Selected Molecular Mechanisms of Action: Epigenetic

Mechanisms of Carcinogenicity
A growing body of evidence indicates that epigenetic alterations, including
DNA methylation and histone modification, contribute to the toxicity of
heavy metals. For instance, cadmium can affect both gene transcription and
translation through the induction of ROS in mitochondria. This causes a
perturbation of cellular redox homeostasis thereby affecting a large set of
transcription factors characterized by reactive cysteines. The comprehensive
analysis of gene expression of human cell lines exposed to non-toxic doses of
cadmium confirmed the induction of cell protection and damage control
genes, such as metallothionein (MT), antioxidant and heat shock proteins,
and revealed several other alterations in genes involved in signaling and
metabolism (reviewed in [54]). Moreover, by inducing oxidative modifica-
tion of proteins cadmium can also target these proteins to degradation.
The key role of epigenetic events in toxicity is similarly well documented for
arsenic (reviewed in [55]). Inorganic arsenic induces hypermethylation of
DNA gene promoters, as shown for the tumor suppressor gene p53, both in
cells in vitro and in subjects exposed to arsenic-contaminated drinking water.
Chronic exposure to arsenic may also lead to loss of global DNA methylation
due to S-adenosylmethionine (SAM) depletion as well as to alteration of
global histone H3 methylation. The alteration of specific histone methylations
represents both gene silencing and activation marks. Arsenic is a carcinogen
with transplacental activity and several studies report alteration of genetic
programming following prenatal exposure that could impact tumor formation
much later in adulthood (reviewed in [56]). Arsenic exposure in utero exa-
cerbated skin cancer response in adulthood in association with distortion of
tumor stem cell dynamics [57]. Finally, in newborns from mothers exposed to
inorganic arsenic through contaminated water in Thailand, altered transcript
profiles in cord blood were reported including changes of stress-related genes
and breast cancer/estrogen-signature genes [58].

By modulation of gene expression and signal transduction heavy metals

may affect cell proliferation, differentiation, apoptosis, and other cellular
activities, thus contributing to carcinogenicity.
4. ANALYTICAL TECHNIQUES AND EXPOSURE

ASSESSMENT OF HEAVY METALS
4.1. Analytical Techniques for the Detection of Heavy

Metals and Metalloids in Biological Samples
The occurrence data for heavy metals in food are usually obtained from
routine monitoring programs conducted at the level of a specific country to
check the compliance for which maximum levels are laid down in legislation.
In Europe, the implementation of the Rapid Alert System (RASFF) for
Food and Feed in Europe has provided a helpful tool to perform systematic
monitoring of specific notifications regarding heavy metals that may be
above maximum levels in food and feed.
To obtain reliable occurrence data on heavy metals in food, the avail-
ability of suitable analytical methods for their determination is of utmost
importance. The complexity of food samples, together with the low con-
centrations at which heavy metals occur, requires sensitive, selective, and
reliable analytical techniques, which can also be applied to biological and
environmental samples. Usually, the analytical methods comprise a sample
preparation step involving the digestion (mineralization) or dry ashing of the
sample, followed by the instrumental determination. Atomic absorption
spectrometry (AAS), either flame AAS (F-AAS) or graphite furnace AAS
(GF-AAS), as well as inductively coupled plasma atomic emission spectro-
metry (ICP-AES), inductively coupled plasma-optical emission spectroscopy
(ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS)
are techniques commonly used for measuring trace metals in food samples,
and vary widely in cost, ease of operation, and analytical performance such
as LODs, linear range, and robustness.
The instrumental techniques applied for the determination of trace con-
centrations of natural uranium include radiometric methods (g-spectrometry,
a-spectrometry, and b-counting) and mass spectrometric (MS) methods
(secondary ion MS, thermal ionization MS, and especially ICP-MS), which
are more sensitive for long-lived radionuclides such as uranium [59,60].
For metals such as arsenic and mercury, speciation is an important
characteristic that provides information on the chemical form present in the
samples, crucial to accurately assess the toxicity. In those cases, additional
steps to separate the different species before detection are needed, such as

40 DORNE et al.
pre-concentration, extraction, and separation. The latter is usually

performed using well established separation techniques such as gas chro-
matography (GC), liquid chromatography (LC), and lately capillary elec-
trophoresis (CE), coupled to selective elemental detection systems. For
arsenic speciation, the most commonly used methods involve LC separation
followed by ICP-MS or AAS [61,62]. In the case of mercury speciation, a
number of analytical methods have been proposed for the determination of
methylmercury, including GC coupled with atomic fluorescence spectro-
metry (GC-AFS) and LC coupled to ICP-MS [63]. Sample preparation still
remains in many cases the bottleneck of the whole analytical procedure. The
selection of the sample preparation methods depends on the matrix and the
analyte. Currently, sample preparation methods tend to move towards more
environmental friendly approaches (less consumption of organic solvents),
to miniaturization, automatization, and ideally to on-line coupling with the
final instrumental determination. This will lead to extracts that are less
manipulated by the analyst, decreasing the probability of experimental
errors. Solid phase extraction (SPE), pressurized liquid extraction (PLE),
microwave assisted extraction (MAE), and solid phase micro-extraction
(SPME), are some of the extraction techniques that fulfil some of the above
mentioned requirements and offer high throughput and the possibility of
on-line coupling with the separation/detection instrumental techniques [64].
The sampling of food for the analysis of metals requires specific precautions
in order to avoid contamination or losses during handling, storage, and
transport to the laboratory. Sampling methods and detailed performance
criteria to be fulfilled by the methods of analysis for cadmium, lead, and
mercury used by the laboratories are laid down in Regulation (EC) No 333/
2007. These performance criteria include recovery ranges, limits of detection
(LOD), limits of quantification (LOQ), and precision requirements. The need
for contamination control together with technological advances will lead to
the development and implementation of effective and efficient analytical
methods, including both sample preparation and final instrumental determi-
nation. The implementation of quality assurance and quality control (QA/
QC) measures are also of utmost importance to ensure reliable occurrence
data on contaminants and decrease the uncertainty of the measurements.
4.2. Data Sources for the Estimation of Human Dietary

Exposure
The estimation of human dietary exposure from food and water corresponds
to the third pillar of risk assessment. This step combines dietary consump-
tion data with occurrence data of heavy metals, i.e., the concentration of a

heavy metal obtained through analytical methods. Ideally, such concentra-

tions are available for a comprehensive and consistent list of food categories
but in practice, these conditions are rarely met.
The most commonly used information on consumption data is derived
from dietary surveys, usually conducted at the national level on a repre-
sentative sample of individuals. In general, these surveys provide estimates
of consumption over a limited time frame, and not on lifetime consumption.
The various dietary assessment instruments can focus on a ‘short term’
diet, usually covering a period that ranges from one day to a few days, in
the case of one administration versus replicate administrations of 24-hour
dietary recalls, food records or weighed records [65]. Such data should be
harmonized to be used for international risk assessment and the easiest
way for such an objective is grouping the food consumed at national level
into broad categories at regional level. The Concise European Food Con-
sumption Database established by EFSA to support exposure assessments
in the EU [66] is compiling data from European countries based on this
principle. Currently, 20 countries provided national food consumption
data in the adult population and to optimize the degree of comparability
between these dietary estimates, consumption data have been aggregated in
15 broad food groups and 29 subcategories. Other surveys exist based on
food frequency questionnaires, dietary history questionnaires or household
purchases which cover a longer period of time in terms of dietary habits.
They are often defined as providing information on habitual diet [67]
making it difficult to quantify individual consumptions [22]. Ultimately,
regional food consumption surveys performed with similar methodologies
would allow a better picture of the dietary habits all around the world.
In parallel, local food consumption surveys, also performed using inter-
nationally recognized methodologies would aim in describing dietary
patterns of local populations in view of the protection of particular groups
at risk.
For heavy metals, most of the analytical data available for risk assessment
are customarily produced to check for regulatory compliance to specific
norm values. Other data exist which are specifically generated for risk
assessment purpose and are particularly useful for estimating the dietary
exposure to heavy metals. They are generated using the so called ‘‘Total Diet
Study’’ approach [20]. Total diet studies consist in the analysis of the con-
centration of various chemicals in food sampled on the market and prepared
to account for the potential increase or decrease in centration during the
home cooking process. The samples of a considered food category are
pooled in order to be representative of an average contamination and to
increase the cost effectiveness. These data provide risk assessors with a
realistic picture of the distribution and trend for chemicals under
consideration.

42 DORNE et al.
Occurrence data should ideally be consistent in terms of the analytical

methods employed, and provide information on the average and/or the
extreme occurrence of heavy metals in an exhaustive and consistent list of
food categories representative of the diet of the population. In practice, these
conditions are rarely met. Departures from these requirements are likely to
raise concerns on the accuracy of exposure assessment calculations. In
international investigations, careful evaluations on the comparability of
figures produced at the country level need to be performed. In addition,
special efforts are needed to handle non-detect values, i.e., samples for which
the concentration is below the limit of detection/quantification. Data of this
nature are typically left-censored (see Abbreviations and Definitions) [68].
The approach applied can have a great impact on the dietary estimates of the
heavy metal under assessment. Deterministic and probabilistic approaches
have been introduced to deal with the statistical handling of laboratory data
[69]. The comparative performance of these methods varies depending on the
pre-defined scenarios and the variables (sample size, frequency of non-
detects, and departure of empirical values from known statistical distribu-
tions) [70]. In food safety, the most commonly used method is currently the
substitution of results below the LOD/LOQ by half of the value of LOD or
LOQ or to estimate upper (setting all values at the LOD/LOQ at that value)
and lower (setting all values at the LOD/LOQ to zero) boundaries [20].
4.3. Combining Occurrence and Consumption Data in

Humans for Exposure Assessment
Dietary exposure assessment is generally recognized as a tiered approach.
The first steps should be based on conservative and cost-effective methods
and only when necessary, refinements should be performed. Data on food
consumption and chemical occurrence are usually combined using either a
deterministic approach, also called ‘‘point estimate’’, or a probabilistic
approach [65].
The ‘‘point estimate’’ approach is based on the selection of a fixed level in
the distribution of consumption multiplied by a fixed value chosen from the
distribution of concentration. The value of contamination could be the 95th
percentile or the maximum authorized levels in the regulation (food addi-
tives) or an average summary value (mean or median) of the occurrence data
(contaminants such as heavy metals, pesticide residues). Values of the same
nature are used from consumption distributions, so that often combinations
of different average/high values from the consumption and concentration
sides are used to evaluate various risk scenarios. This method does not

reflect the exposure of the overall population, but it is often considered the
most appropriate for screening purposes [71]. In practice, the fixed levels
utilized to calculate a ‘‘point estimate’’ are generally chosen assuming a
conservative scenario, thus being on the safe side when determining the
absence of safety concern. For example, the combination of highest levels of
residues with highest percentiles of food consumption is usually referred to
as ‘‘worst case scenario’’.
Conversely, probabilistic approaches use the full distributions of occur-
rence and consumption data, thus exploiting the variability in both quan-
tities. These probabilistic methods result in more realistic pictures, often
expressed in terms of a range of possible exposure values, thus incorporating
an estimation of the uncertainty associated with exposure estimates, pro-
vided reliable data is available together with the relevant modelling tools.
A variety of empirical, semi-parametric and parametric models have been
described, depending on whether the actual data set is used (non-parametric
approach), or parameters of a theoretical statistical distribution (log-
normal, Weibull, exponential) are estimated before data use (parametric
approach).
When assessing the potential health impact of the consumption of food
containing heavy metals, two main aspects have to be taken into account:
The external dose which can be expressed as the amount of chemical ingested
and the internal dose corresponding to the TK of the compound. Applying
key parameters such as the biological half-life, bioavailability, clearance, and
tissue concentrations to the ingested amounts of a heavy metal, a PB-TK or
a population-based TK model can reduce the uncertainty in the exposure
estimates since the variability in internal dose and its time-dependency are
taken into account (see Section 3) [72].
5. APPLICATIONS TO THE HUMAN RISK

ASSESSMENT OF HEAVY METALS AND
METALLOIDS
This section aims to summarize the hazard identification and characteriza-

tion, exposure assessment and risk characterization steps for cadmium, lead,
methylmercury, uranium, and the metalloid arsenic. For readability and
conciseness, each step considers only the most recent risk assessments per-
formed by international agencies such as the JECFA (FAO/WHO), EPA,
ASDTR, the European Commission’s Scientific Committee for Food (SCF),
and the panel on contaminants in the food chain of the European Food
Safety Authority.

44 DORNE et al.
5.1. Hazard Identification and Characterization

5.1.1. Cadmium
Historically, the most relevant and sensitive endpoint for cadmium toxicity
is an increased risk for potential renal damage and biomarkers of the renal
function from human studies that are excreted in the urine, i.e., b-2 micro-
globulin, have been used to set its PTWI. The JECFA evaluated cadmium in
1988 and set a PTWI of 7 mg/kg b.w. using a 10% prevalence rate of b-2
microglobulinemia in humans, assuming an absorption rate of 5%, a daily
excretion of 0.005% of the body load concentration (reflecting its long half-
life) corresponding to 50 mg/g renal cortex over a 50-year period [73]. This
value was confirmed by the SCF in 1995 and the following JECFA assess-
ments [74]. In 2008, the ATSDR established a minimal risk level for chronic
oral exposure of 0.1 mg/kg/day based on multiple approach namely NOAEL/
LOAEL values and BMD modelling for increased prevalence of b-2
microglobulinemia [75]. For the recent EFSA assessment, the CONTAM
panel developed a PB-TK model from human PB-TK data together with a
human BMD/BMDL derived from a meta-analysis of published studies
relating urinary cadmium and b-2 microglobulin (TD). The PTWI of 2.5 mg/
kg b.w. for cadmium was derived from the human BMDL, a CSAF for
human variability in TD and a back-translation using the human PB-TK
model [33,35].
5.1.2. Lead
Historically, international agencies have used adverse neurodevelopmental

effects of lead in children using intelligence quotients (IQ) as the critical
endpoint to derive a PTWI. In 1992, the SCF endorsed the JECFA PTWI
derived in 1986 of 25 mg/kg b.w. per week which was based on an analysis
relating lead blood concentrations and children’s IQ scores [76]. Recent
studies have shown that children with lifetime average lead concentrations
between 50 and 99 mg/L scored 4.9 points lower on full-scale IQ tests com-
pared with children who had lifetime average blood lead concentrations
o50 mg/L [77]. In adults, lead exposure has been shown to be linked to
neuro-motor disturbances [78], elevated blood pressure [79], and chronic
renal disease (decrease in glomerular filtration rate) [80]. The most recent
assessment by EFSA was based on a dose-response modelling of the meta-
analysis relating lead blood concentrations and its effects on children’s full
scale IQ) by Lanphear et al. [81]. A BMDL01 (for a decrease in IQ of 1
point) of 12 mg B-Pb/L was derived as a reference point concentration when
assessing the risk of intellectual deficits in children. In adults, EFSA also
identified a BMD-01 (for the mean annual increase of SBP by 1%) for

systolic blood pressure of 36 mg/L and a BMDL10 for chronic kidney disease
of 15 mg/L [82].
5.1.3. Methylmercury
Historically, human developmental neurotoxicity has provided the basis for
setting the health-based guidance values for methylmercury by different
regulatory agencies from 1950s and 1970s. The critical data sets relate to
poisoning episodes in Japan and Iraq, or to more recent large scale epide-
miological studies relating childhood development and neurotoxicity in
relation to in utero exposure (reviewed in [83]). In 1972, the WHO estab-
lished a TWI of 3.3 mg methylmercury/kg b.w. based on the data from Japan
[84] which was then lowered to a PTWI of 1.6 mg/kg b.w. from the growing
epidemiological evidence of neurodevelopmental risks to fetuses and chil-
dren from longitudinal studies in the Faroe and Seychelle islands. The latter
studies used methylmercury in maternal hair as the critical biomarker dose.
Hair concentrations of 14 mg/kg were first related to a maternal blood
concentration of 0.056 mg/L and to a daily intake of methylmercury of
1.5 mg/kg b.w that would be expected to have no appreciable adverse effects
on children. A total UF of 6.4 was applied to give a PTWI of 1.6 mg/kg b.w.
per week.
This PTWI of 1.6 mg/kg b.w. per week was also considered by EFSA in its
2004 risk assessment of methylmercury [85]. In 1995, the US-EPA set a RfD
of 0.1 mg methylmercury/kg b.w. per day based on a study in Iraqi children
who were exposed to methylmercury in utero. In a later evaluation [86], the
BMDL05 from the Faroes study was used to set a maternal daily intake of
about 1 mg/kg b.w. per day and a composite UF of 10 (intra-human varia-
bility and data gaps) to derive an identical RfD of 0.1 mg/kg b.w. per day.
5.1.4. Uranium
Nephrotoxicity is the most sensitive endpoint for uranium chemical toxicity

both in experimental animals and humans. In 1989, the US-EPA established
an RfD of 3 mg/kg b.w. per day for uranium (soluble salts) based on a 30-day
oral study in rabbits using a LOAEL of 2.8 mg uranium/kg b.w. per day for
initial body weight loss and moderate nephrotoxicity [87]. A LOAEL of
0.06 mg uranium/kg b.w. per day for nephrotoxicity based on a 91-day oral
study in male rats was taken as the key study by the WHO and a UF of 100
was applied to derive a TDI of 0.6 mg/kg b.w. per day [88,89]. The ATSDR
set a minimal risk level of 2 mg/kg b.w. per day for intermediate duration
(15–364 days) of uranium ingestion by applying an UF of 30 (3 for using the
LOAEL and 10 for human variability) to a LOAEL from a 91-day oral
study in rabbits of 0.05 mg uranium/kg b.w. per day for nephrotoxicity [90].

46 DORNE et al.
Most recently, EFSA endorsed the 1998 WHO TDI for soluble uranium of
0.6 mg/kg b.w. per day [34] after a thorough examination of the recent tox-
icokinetic and toxicological database which did not provide evidence for a
new TDI.
5.1.5. Arsenic
Toxicity of arsenic is complex because of the presence of inorganic and

organic species and the large number of toxic endpoints. Inorganic arsenic is
recognized to be much more toxic than its organic forms. In 1989, a JECFA
evaluation [91,92] confirmed their provisional maximum TDI (PMTDI)
derived in 1983 for inorganic arsenic of 2 mg/kg b.w. and converted to a
PTWI of 15 mg/kg b.w. The PTWI was based on human dose-response data
from Nova Scotians relating skin lesions and arsenic concentrations in
contaminated well water.
The US-EPA derived a RfD of 0.3 mg/kg b.w. per day based on a human
NOAEL of 0.8 mg/kg b.w. per day relating skin lesions in Taiwan and
inorganic arsenic concentrations by applying a UF of 3 to account for
sensitive subjects and the lack of data on reproductive toxicity [93,94]. In
2005, the US-EPA used lung and bladder cancer as endpoints with ED01
values for inorganic arsenic in drinking water estimated at 79–96 mg/L for
lung cancer risk, and at 304–474 mg/L for bladder cancer risk [95]. The
National Research Council (NRC) [96,97] has estimated ED01 (i.e., 1%
effective dose, which according to the NRC is the concentration of arsenic in
drinking water that is associated with a 1% increase in the excess risk) for
various studies using different statistical models. Under different modelling
approaches, the ED01 values for lung cancer estimated for the southwestern
Taiwanese population ranged from 33 to 94 mg/L and for the Chilean
population from 5 to 27 mg/L. For bladder cancer, the ED01 values for the
southwestern Taiwanese population ranged from 102 to 443 mg/L based on a
1% increase relative to the background cancer mortality in the US [97],
whilst the previous estimations, in which the reference was the background
cancer mortality in Taiwan, were 404 to 450 mg/L [96].
Studies presented in [98] established a chronic oral minimal risk to
humans (MRL) of 0.3 mg/kg b.w. per day, applying a similar approach to
that of the US-EPA RfD, based on the NOAEL for skin lesions of
0.8 mg/kg b.w. per day. The recent EFSA risk assessment has used the MOE
approach using dose-response data from key epidemiological studies
(skin, lung, and bladder cancers, skin lesions) and selected a benchmark
response of 1% extra risk together with a range of benchmark dose
lower confidence limit (BMDL01) values between 0.3 and 8 mg/kg b.w. per
day [12].

5.2. Exposure Assessment of Heavy Metals and Metalloids

5.2.1. Cadmium
Only deterministic exposure assessments were performed for cadmium at

regional or international level. In 2006, the JECFA [99] used the GEMS/
Food regional diets and the regional average concentrations of cadmium to
conclude in a mean dietary exposure ranging from 2.8 to 4.2 mg/kg b.w./week
with a value of 3.8 mg/kg b.w./week for the European region. The earlier
European Community SCOOP study [100] showed a mean dietary exposure
ranging from 0.7 to 2.9 mg/kg b.w./day. More recently the EFSA assessed
cadmium dietary exposure based on the occurrence data and the con-
sumption data as reported in the EFSA’s Concise European Food Con-
sumption Database. The mean dietary exposure across European countries
was estimated to be 2.3 mg/kg b.w. per week (range from 1.9 to 3.0 mg/kg b.w.
per week). This difference between JECFA and EFSA might indicate that a
refined assessment based on more disaggregated and representative samples
can result in lower estimates of cadmium exposure from food. EFSA also
estimated the high exposure to cadmium which resulted in a value of 3.0 mg/
kg b.w. per week (range from 2.5 to 3.9 mg/kg b.w. per week). Due to their
high consumption of cereals, nuts, oilseeds and pulses, vegetarians have a
higher dietary exposure of up to 5.4 mg/kg b.w. per week. Regular consumers
of bivalve molluscs and wild mushrooms were also found to have higher
dietary exposures of 4.6 and 4.3 mg/kg b.w. per week, respectively.
In the US, based on the data from a Total Diet Study carried out by the
US Federal Drug Administration (FDA) in 2003 [101], the US FDA con-
cluded in a dietary of 1.5 mg/kg b.w./week. This difference emphasizes the
interest of TDS for estimating the mean dietary exposure based on more
accurate occurrence data.
5.2.2. Lead
The situation for lead exposure is complicated by the fact that key measures
aimed at reducing the release of lead from anthropogenic sources, including
the phasing out of leaded petrol, have led to a major reduction in lead levels
in the environment over the past 50 years. Consequently, blood lead levels in
the general population have decreased from 150–330 mg/L in the 1960’s to
around 15 mg/L [102]. The JECFA evaluated lead at its 53th meeting (WHO,
1999, http://www.inchem.org/documents/jecfa/jeceval/jec_1260.htm). The
exposure assessment focused on the contribution from the diet based on the
WHO GEMS Food regional diets and on levels of occurrence for lead in
food. The JECFA proposed a simple Monte-Carlo simulation to estimate
the dietary exposure in various regions related to frequently consumed

48 DORNE et al.
foods. This simulation was based on estimates of mean intakes in the United
States converted to distributions by assuming that they follow a log normal
distribution with a geometric mean equal to 0.76 times the arithmetic mean
and a geometric standard deviation of 0.76. It resulted in an overall dietary
exposure ranging from about 7 to 30 mg/day/person (about 1 to 4 mg/kg b.w.
per week assuming 60 kg b.w.). The Committee noted that since the model
was based on data for one country, results do not reflect any geographic
difference in lead concentrations. Moreover, summing distributions does not
account for correlations in the consumption of particular foods, in that high
consumption of one food may tend to be accompanied by high consumption
of another. Such correlations would require access to raw data on con-
sumption, which are not usually published.
EFSA performed a deterministic assessment of lead dietary exposure for
adults. In the case of average adult consumers, lead dietary exposure ranges
from 0.36 to 1.24 mg/kg b.w. per day, with major contribution from the
consumption of cereal products, potatoes, leafy vegetables, and tap water.
For children aged 1–3 years mean lead dietary exposure range from 1.10 to
3.10 mg/kg b.w. per day. Compared to dietary exposure, non-dietary expo-
sure to lead is likely to be of minor importance for the general population in
the EU. However, house dust, soil and lead in paints on toys can be an
important source of exposure to lead for children due to their tendency to
ingest soil and mouth toys [82].
5.2.3. Methylmercury
The JECFA assessed the dietary exposure to methylmercury by combining the
mean level of occurrence with the mean consumption for fish and other
seafood from the GEMS Food regional diets. This deterministic assessment
resulted in exposure values ranging from 0.3 to 1.5 mg/kg b.w./week [74,103].
Similarly for the EU, the EFSA reported the mean weekly estimated dietary
exposure would be between 0.1 to 1.0 mg/kg b.w. of mercury from fish and
seafood products. Consequently, the exposure of a fraction of the population
is likely to be above the health based guidance value of 1.6 mg/kg b.w./week,
and in its opinion, the EFSA CONTAM panel performed a probabilistic
analysis of the likelihood of exceeding the PTWIs using the French con-
tamination data as reported to SCOOP in combination with the distribution
of fish and seafood product consumption in France. The probability for a
population to reach an exposure over the available health based guidance
value was calculated to be 1.2% for adults and 11.3% for children [85].
5.2.4. Uranium
EFSA recently estimated the total uranium exposure by multiplying

occurrence values (mg/L for water and mg/kg for foods) by consumption

values (g/day). Values of individual body weight of participants in the

Concise European Food Consumption Database were used to express ura-
nium exposure in mg/kg b.w. per day. In order to provide summary figures of
uranium exposure in Europe, the median of 19 country-specific uranium
exposure values calculated for all water-based products and food were
reported according to four different exposure scenarios. These scenarios
were determined using combinations of average and 95th percentile values of
occurrence and consumption figures. Notably, scenario 1 used mean values
for dietary consumption in conjunction with water and food mean occur-
rence values, scenario 2 used 95th percentile consumption and mean
occurrence values, scenario 3 used mean consumption and 95th percentile
occurrence values, and scenario 4 used 95th percentile consumption and
occurrence values. The median overall lower- and upper-bound dietary
exposure to uranium across European countries is between 0.050 and
0.085 mg/kg b.w. per day. This figure comprises around 0.04 mg/kg b.w. per
day from water (tap and bottled) and water-based products (tea, coffee,
beer, and soft drinks). For high consumers the median country-specific
overall dietary exposure to uranium was estimated to be between 0.09 and
0.14 mg/kg b.w. per day, 0.082 mg/kg b.w. per day coming from water and
water-based products [34].
Two specific subgroups of the population were looked at in more detail.
As a very conservative scenario, it can be assumed that the population of
some local communities with high uranium concentrations in their water
supply can be exposed at the 95th percentile concentration level for life-time.
At the same time there might be high consumers of water among these
subpopulations at the 95th percentile consumption level. In such a situation,
water could contribute 0.36 mg/kg b.w. per day as a median across the
countries studied, and a country maximum of 0.51 mg/kg b.w. per day.
Contribution from food is not considered likely at the 95th percentile con-
centration level of uranium at the same time, but more likely at the mean
concentration level of 0.040 mg/kg b.w. per day and possibly 0.066 mg/kg b.w.
per day in a high consumption scenario. Thus, also in such a situation the
TDI would not be exceeded even if the estimated exposure would be in that
case more than 10 times higher than the median value. This example shows
that in certain situations a worst case scenario could be useful in reinsuring
the risk managers about the absence of safety concern [34].
5.2.5. Arsenic
The European Commission Scientific Cooperation project calculated a mean

dietary exposure to total arsenic in the adult population in three European
countries with complete dietary studies of between 37 and 66 mg/day with an
estimated seafood contribution in excess of 50% [100]. In the United States,

50 DORNE et al.
dietary exposure ranged from 2 mg/day in infants to 92 mg/day in 60–65-year-

old men [104]. From a toxicological point of view the amount of inorganic
arsenic is considered the most important. Tao and Bolger [104] assumed that
10% of the total arsenic in seafood was inorganic and that 100% of the
arsenic in all other foods was inorganic and average daily exposure to inor-
ganic arsenic ranged from 1.3 mg in infants to 12.5 mg in 60–65-year-old men.
The worldwide JECFA assessment estimated total arsenic dietary expo-
sure to range from below 10 mg/day to 200 mg/day and emphasized that these
values are not only reflective of different dietary habits but mirror important
variations in assumptions used to calculate them [73]. The recent EFSA
assessment, estimated dietary exposure to inorganic arsenic using a deter-
ministic approach and several assumptions. The amount of inorganic arsenic
was assumed to be 0.03 mg/kg in fish, 0.1 mg/kg in other seafood, and to
represent 70% of the total arsenic measured in other food categories. For
food consumption, the EFSA concise database was used and several sce-
narios were elaborated and resulted in a mean exposure ranging from 0.13 to
0.56 mg/kg b.w./day and in a dietary exposure at the 95th percentile ranging
from 0.37 to 1.22 mg/kg b.w./day. Consumer groups with higher inorganic
arsenic exposure levels such as high consumers of algae-based products
could be exposed up to 4.03 mg/kg b.w. per day. Infants fed only on cow’s
milk formula reconstituted with water containing arsenic at the average
European concentration level have intakes of inorganic arsenic that are
about 3-fold higher than those of breast-fed infants.
5.3. Risk Characterization of Heavy Metals and Metalloids

5.3.1. Cadmium
Risk characterization was performed by JECFA which concluded that an
excess prevalence of renal tubular dysfunction would not be expected to
occur if urinary cadmium concentration remains o2.5 mg/g creatinine since
the PTWI of 7 mg/kg b.w. per week would not be exceeded. This was based
on an estimation of cadmium intake of ranging from 2.8 to 4.2 mg/kg b.w.
per week, which equates to 40–60% of the PTWI of 7 mg/kg b.w. per week
[74,105]. Back in 1981, the US Environmental Agency published an assess-
ment on health effects of cadmium. The dietary exposure for most Amer-
icans was estimated to be 10–50 mg/day and the threshold level was set at
200 mg cadmium/g wet human renal cortex, it was estimated that an intake of
200 mg/day would result in reaching the threshold after 50 years exposure.
Later, the EPA [106] set an RfD of 0.5 mg Cd/kg b.w. per day for water and a
RfD for cadmium in food of 1 mg/kg b.w. per day. The assessment of the
Joint Research Centre [107] identified the LOAEL for Cd-U to be Z2 mg/g

creatinine based on low molecular weight proteinuria and bone changes.

They found the margins of safety (MOS) between the LOAEL and the
predicted exposure to be Z3 for more than 50% of the population and o1.0
for 5% of the population (both smokers and non-smokers).
A MOS of 3 or more is considered as sufficiently protective for the general
population. In the most recent risk assessment of cadmium, the EFSA
opinion, the mean exposure for adults across Europe was found to be close
to, or slightly exceeding the new TWI of 2.5 mg/kg b.w. (as established in the
scientific opinion) and vegetarians, children, smokers, and people living in
highly contaminated areas may exceed the TWI by about twofold [33].
Although the risk for adverse effects on kidney function at an individual
level is very low, it was recommended that the current exposure to cadmium
at the population level should be reduced [33].
5.3.2. Lead
EFSA recently re-investigated the PTWI of 25 mg/kg b.w. set by the SCF and
JECFA. As a basis for its risk assessment procedure, EFSA performed a
BMD analysis on the three key toxicological endpoints for lead, namely
developmental neurotoxicity in young children, cardiovascular toxicity and
nephrotoxicity in adults. The following benchmark dose (lower limits)
(BMDLs) were derived from blood lead levels (B-Pb) for developmental
neurotoxicity: BMDL01, 12 mg/L; cardiovascular toxicity: BMDL01, 34 mg/L;
nephrotoxicity: BMDL10, 15 mg/L (B-Pb). The dietary lead intakes predicted
from toxicokinetic models to yield the BMDL01 for developmental neuro-
toxicity, cardiovascular toxicity and BMDL10 for nephrotoxicity were 0.50,
1.50 and 0.63 mg/kg b.w./day, respectively. Based on these results, EFSA
concluded that the PTWI of 25 mg/kg b.w. set by the SCF is no longer
appropriate [82].
5.3.3. Mercury
The EFSA’s CONTAM Panel used the intake estimates from the SCOOP
data and the JECFA PTWI of 1.6 mg/kg b.w. per week [85]. The panel
concluded that mercury intake in Europe was very variable between coun-
tries depending on fish consumption but in most cases mean intakes were
below the PTWI. There were indications, however, that proportions of
young children might exceed the PTWI and that adults with high fish con-
sumption would have intakes above the PTWI. Data quality at the Eur-
opean level was not sufficient to assess the size of these population groups
and it was recommended to perform specific intake studies on methylmer-
cury, especially for women of childbearing age and children and that such
exposure should be minimized [85].

52 DORNE et al.
On request of the Codex Commission on Food Additives and Con-

taminants, the JECFA examined the dietary impact of the current guideline
levels of methylmercury in fish (1.0 and 0.5 mg/kg in predatory and non-
predatory fish, respectively on exposure and risk) [103]. The Committee
compared the current situation with a scenario where no guideline levels
were in effect or were enforced. More complete exposure data in Europe
including internal dose levels would allow direct comparison of exposure
with the dose-effect relationships, which are the basis for the hazard char-
acterization [108]. JECFA concluded that, for the general population, the
setting of maximum levels for methylmercury in fish was not an effective
mean to reduce exposure. Because most marketed seafood contains mercury
concentrations below the maximum levels, excluding the food items con-
taining this contaminant at the high end of a log-normal distribution of
concentrations would not significantly diminish average exposure. The
impact of reducing exposure to predatory fish would be greater for women
of childbearing age because predatory fish make up a larger proportion of
their diets than in the case of children, and is a larger vector of exposure for
those that would exceed the PTWI (23% for children versus 70% for
women). Hence, JEFCA concluded that advice targeted at population
subgroups that might be at methylmercury exposure greater than the PTWI
and potentially at risk could effectively lower such exposure. Another
recommendation was to weight the risks and benefits in any advice aimed at
different subpopulations.
5.3.4. Uranium
In 2009, EFSA evaluated whether dietary exposure to uranium in foodstuffs

and water (tap and bottled) and water-based drinks would pose a health risk
to consumers in Europe. For most of the population, including the worst case
scenario (i.e., high consumption of highly contaminated food and water), the
estimated exposure to uranium was below the TDI, and considered not to
pose any significant health risk. Nevertheless, for infants fed with infant
formula reconstituted with water containing uranium, the exposure (expres-
sed on a body weight basis) was estimated to exceed that of adults by up to
threefold. Such exposure was recommended to be avoided [34].
5.3.5. Arsenic
The EFSA estimates of human dietary exposure to inorganic arsenic

(see Section 5.2.5) in Europe were within the range of the BMDL01
values identified with little or no MOE and thus concluding that the pos-
sibility of a risk to high consumers cannot be excluded. Consumer groups

with the highest exposure levels included high consumers of rice,

algae-based products and children whereas breast-fed and formula-fed
infants below 6 months of age had the lowest estimated dietary exposure.
Organic sources of arsenic such as arsenobetaine from fish and most
seafood, was widely assumed to be of no toxicological concern whereas
arsenosugars and arsenolipids could not be considered because of lack of
toxicological data. Overall, EFSA recommended a reduction of such dietary
exposure to inorganic arsenic and the need to produce speciation data
for different food commodities to refine risk assessment, support dietary
exposure assessment and dose-response data for the possible health
effects [12].
6. CONCLUSIONS AND FUTURE PERSPECTIVES
This chapter has highlighted the principles and applications of chemical risk
assessment in humans for heavy metals and metalloids. Further research is
needed regarding the complex and multiple metabolic routes and accumu-
lation organs and the multiple long-term health effects of these metals. More
complete exposure assessments allow to describe the dietary exposure as a
dynamic process determined by the accumulation phenomenon due to suc-
cessive dietary intakes and by the toxicokinetics ruling the elimination
process in between intakes. This has been expressed in a recent study on
methylmercury [108].
Toxicological as well as modeling tools are also of critical interest to
estimate the inter-individual variability in susceptibility to toxicity. This
includes further research on genetic polymorphism, TK and TD modelling,
the use of OMICs (genomics, proteomics, metabolomics) to depict mole-
cular mode of actions and develop biomarkers, as well as statistical meth-
odologies to model such complex dynamic systems (e.g., the use of Bayesian
methods, non-linear mixed effects models, etc.). A relevant example for such
models is the EFSA risk assessment for cadmium for which a human BMD/
BMDL was derived from a meta-analysis of published studies relating
urinary cadmium and biomarkers of renal effects (b-2 microglobulin)
without the need to extrapolate from animals to humans. The PTWI was
then derived using a PB-TK model and a chemical specific adjustment
factor for cadmium variability in TD without the need of the 100-fold
uncertainty factor [33,35,109]. Depending on data availability, these
approaches will prove useful to risk assessors to provide more transparent
science-based risk assessment that integrate quantitative descriptors
regarding variability and uncertainty in TK and TD of single toxicants and
chemical mixtures [3,69].

54 DORNE et al.
ACKNOWLEDGMENTS
The authors would like to thank the members of the working groups on
mercury (EFSA, 2004), cadmium (EFSA, 2009), uranium (EFSA, 2009),
arsenic (EFSA, 2009), and lead (EFSA, 2010). The views presented in this
review are those of the authors’ only; they do not reflect the views of the
European Food Safety Authority, the Istituto Superiore de Sanita, the
Chemisches Landes- und Staatliches Veterinäruntersuchungsamt, the
Imperial College London, or the World Health Organisation.
ABBREVIATIONS AND DEFINITIONS
Left-censored Conducting dietary exposure assessment consists in

data combining deterministically or probabilistically food
consumption figures with occurrence of a given chemical
substance in a number of food categories. The occur-
rence data reported to be below the limit of detection
(LOD) of the analytical method are commonly called
left-censored data. The statistical treatment of those
values is likely to have a critical influence on the results
of the assessment.
AAS atomic absorption spectrometry
AFS atomic fluorescence spectrometry
ATSDR Agency for Toxic Substances and Disease Registry
B-Pb Pb in blood
b.w. body weight
Cd-U Cd in urine
CE capillary electrophoresis
EC European Commission
ED effective dose
EU European Union
F-AAS flame atomic absorption spectrometry
FDA Food and Drug Administration
GC gas chromatography
GEMS Groundwater Environmental Monitoring System
GF-AAS graphite furnace atomic absorption spectrometry
GSH glutathione (reduced)
ICP-AES inductively coupled plasma atomic emission
spectrometry
ICP-MS inductively coupled plasma mass spectrometry
ICP-OES inductively coupled plasma optical emission
spectrometry

IQ intelligence quotient
LC liquid chromatography
LOD limit of detection
LOQ limit of quantification
MAE microwave-assisted extraction
MOS margin of safety
MRL minimal risk limit
MS mass spectrometry
MT metallothionein
NRC National Research Council
PLE pressurized liquid extraction
PMTDI provisional maximum tolerable daily intake
QA quality assurance
QC quality control
RASFF Rapid Alert System for Food and Feed
RNS reactive nitrogen species/nitrogen-based radicals
ROS reactive oxygen species/oxygen-based radicals
SAM S-adenosylmethionine
SBP systolic blood pressure
SCF Scientific Committee for Food
SCOOP Scientific Cooperation on Questions Related to Food
SPE solid phase extraction
SPME solid phase micro-extraction
TDS Total Diet Study
TWI tolerable weekly intake
US-EPA United States-Environmental Protection Agency
REFERENCES
1. WHO, Project to update the principles and methods for the assessment of che-
micals in food, 2008, http://www.who.int/ipcs/food/principles/en/.
2. EC (European Commission), REGULATION (EC) No 178/2002 OF THE
EUROPEAN PARLIAMENT AND OF THE COUNCIL laying down the
general principles and requirements of food law, establishing the European Food
Safety Authority and laying down procedures in matters of food safety., 2002,
http://eurlex.europa.eu/pri/en/oj/dat/2002/l031/l03120020201en00010024.pdf.
3. J. L. Dorne, Toxicology, 2010, 268, 156–164.
4. J. L. C. M. Dorne, L. R. Bordajandi, B. Amzal, P. Ferrari and P. Verger, Trac-
Trends in Analytical Chemistry, 2009, 28, 695.
5. I. Pratt, S. Barlow, J. Kleiner and J. C. Larsen, Mutat. Res., 2009, 678, 113.
6. G. M. Cramer, R. A. Ford and R. L. Hall, Food Cosmetics Toxicol., 1978, 16,
255.

56 DORNE et al.
7. R. Kroes, A. G. Renwick, M. Cheeseman, J. Kleiner, I. Mangelsdorf,

A. Piersma, B. Schilter, J. Schlatter, F. van Schothorst, J. G. Vos and G.
Wurtzen, Food Chem. Toxicol., 2004, 42, 65.
8. A. G. Renwick, Toxicol. Appl. Pharmacol., 2005, 207, 585.
9. EFSA (European Food Safety Authority), The EFSA Journal, 2005, 282, 1.
10. S. Barlow, A. G. Renwick, J. Kleiner, J. W. Bridges, L. Busk, E. Dybing,
L. Edler, G. Eisenbrand, J. Fink-Gremmels, A. Knaap, R. Kroes, D. Liem, D. J.
G. Muller, S. Page, V. Rolland, J. Schlatter, A. Tritscher, W. Tueting and
G. Wurtzen, Food Chem. Toxicol., 2006, 44, 1636.
11. J. O’Brien, A. G. Renwick, A. Constable, E. Dybing, D. J. G. Muller,
J. Schlatter, W. Slob, W. Tueting, J. van Benthem, G. M. Williams and A.
Wolfreys, Food Chem. Toxicol., 2006, 44, 1613.
13. M. L. Dourson, S. F. Felter and D. Robinson, Human and Ecological Risk
Assessment, 1997, 3, 579.
14. R. Truhaut, Food Addit. Contam, 1991, 8, 151.
15. J. L. C. M. Dorne and A. G. Renwick, Toxicol. Sci., 2005, 86, 20.
17. A. G. Renwick, Food Additives and Contaminants, 1993, 10, 275.
18. J. L. C. M. Dorne, K. Walton and A. G. Renwick, Food Chem. Toxicol., 2001,
39, 681.
19. J. L. C. M. Dorne, J. Appl. Toxicol., 2007, 27, 411.
20. WHO, International Programme on Chemical Safety: Chemical-specific Adjust-
ment Factors for Interspecies Differences and Human Variability: Guidance
Document for Use of Data in Dose/concentration Response Assessment, 2005.
http://www.who.int/ipcs/methods/harmonization/areas/uncertainty/en/
index.html
43, 203.
22. WHO, Exposure Assessment for Chemicals in Food, Report of the FAO/WHO
Workshop, Annapolis, Maryland, USA, 2005.
39, 1153.
24. J. L. C. M. Dorne, K. Walton, W. Slob and A. G. Renwick, Food Chem.
Toxicol., 2002, 40, 1633.
25. A. G. Renwick, J. L. C. M. Dorne and K. Walton, Human Ecol. Risk Assess.,
2001, 7, 165.
26. K. Walton, J. L. Dorne and A. G. Renwick, Food Chem. Toxicol., 2001, 39,
1175.
27. K. Walton, J. L. Dorne and A. G. Renwick, Food Chem. Toxicol., 2001, 39, 667.
28. K. Walton, J. L. C. M. Dorne and A. G. Renwick, Human Ecol. Risk Assess.,
2001, 7, 181.
29. K. Walton, J. L. C. M. Dorne and A. G. Renwick, Food Chem. Toxicol., 2004,
42, 261.
30. C. Svendsen, A. M. Ragas and J. L. C. M. Dorne, in Health Benefits of Organic
Foods: Effects of the Environment, CABI Publisher, 2008, Vol. 119, Chapter 6.

31. OECD, OECD Guidelines for testing of chemicals: Full list of test guidelines,
2009.
32. CAC, Codex Alimentarius Commission Procedural Manual, 16th edn., Joint
FAO/WHO Food Standards Programme, 2006..
35. B. Amzal, B. Julin, M. Vahter, A. Wolk, G. Johanson and A. Akesson, Environ.
Health Persp., 2009, 117, 1293.
36. C. M. Liao, H. H. Shen, C. L. Chen, L. I. Hsu, T. L. Lin, S. C. Chen and
C. J. Chen, J. Hazardous Materials, 2009, 165, 652.
37. S. Mann, P. O. Droz and M. Vahter, Toxicol. Appl. Pharmacol., 1996, 140, 471.
38. D. H. Yu, Chemosphere, 1999, 39, 2737.
39. G. L. Diamond, W. C. Thayer and H. Choudhury, J. Toxicol. Environ. Health-
Part A, 2003, 66, 2141.
40. B. Engstrom and G. Nordberg, Toxicology, 1978, 9, 195.
41. J. G. Pounds and R. W. Leggett, Environ. Health Persp., 1998, 106, 1505.
42. J. F. Young, W. D. Wosilait and R. H. Luecke, J. Toxicol. Environ. Health-Part A,
2001, 63, 19.
43. E. J. O’Flaherty, Crit. Rev. Toxicol., 1998, 28, 271.
44. M. Begum and P. K. Sen, Environmetrics, 2007, 18, 515.
45. B. Halliwell and J. M. Gutteridge, in Free Radicals in Biology and Medicine,
Oxford Biosciences, 4th edn., 2007, pp. 1.
46. M. F. Casado, A. L. Cecchini, A. N. C. Simao, R. D. Oliveira and R. Cecehini,
Food Chem. Toxicol., 2007, 45, 945.
47. P. Watcharasit, A. Thiantanawat and J. Satayavivad, J. Appl. Toxicol., 2008,
28, 466.
48. N. Larochette, D. Decaudin, E. Jacotot, C. Brenner, I. Marzo, S. A. Susin,
N. Zamzami, Z. H. Xie, J. Reed and G. Kroemer, Exper. Cell Res., 1999, 249, 413.
49. C. C. Bridges and R. K. Zalups, Toxicol. Appl. Pharmacol., 2005, 204, 274.
50. J. Markovac and G. W. Goldstein, Nature, 1988, 334, 71.
51. P. X. Liu, H. L. Cheng, T. M. Roberts and J. J. Zhao, Nature Reviews Drug
Discovery, 2009, 8, 627.
52. S. Gupta, G. E. N. Kass, E. Szegezdi and B. Joseph, J. Cell. Molec. Med., 2009,
13, 1004.
53. G. E. N. Kass, Chemico-Biological Interactions, 2006, 163, 145.
54. P. Joseph, Toxicol. Appl.Pharmacol., 2009, 238, 272.
55. K. Salnikow and A. Zhitkovich, Chem. Res. Toxicol., 2008, 21, 28.
56. M. Vahter, Ann. Rev. Nutrition, 2009, 29, 381.
57. M. P. Waalkes, J. Liu, D. R. Germolee, C. S. Trempus, R. E. Cannon,
E. J. Tokar, R. W. Tennant, J. M. Ward and B. A. Diwan, Cancer Research,
2008, 68, 8278.
58. R. C. Fry, P. Navasumrit, C. Valiathan, J. P. Svensson, B. J. Hogan, M. Luo,
S. Bhattacharya, K. Kandjanapa, S. Soontararuks, S. Nookabkaew, C. Mahi-
dol, M. Ruchirawat and L. D. Samson, PLOS Genetics, 2007, 3, 2180.
59. C. C. May, P. J. Worsfold and M. J. Keith-Roach, Trends Anal. Chem., 2008,
27, 160.

58 DORNE et al.
60. X. Hou and P. Roos, Anal. Chim. Acta, 2008, 608, 105.
61. S. G. Capar, W. R. Mindak and J. Cheng, Anal. Bioanal. Chem., 2007, 389, 159.
62. P. Kuban, R. Guchardi and P. C. Hauser, Trends Anal. Chem., 2005, 24, 192.
63. D.-Y. Yang, H.-Y. T. Truong, Y. W. Chen and N. Belzile, Anal. Chim. Acta,
2009, 633, 157.
64. K. Ridgway, S. P. D. Lalljie and R. M. Smith, J. Chromatography A, 2007,
1153, 36.
65. R. Kroes, D. Muller, J. Lambe, M. R. Lowik, K. J. van, J. Kleiner, R. Massey,
S. Mayer, I. Urieta, P. Verger and A. Visconti, Food Chem.Toxicol., 2002,
40, 327.
66. EFSA (European Food Safety Authority), Food Consumption Database, 2008.
http://www.efsa.europa.eu/EFSA/ScientificPanels/efsa_locale-1178620753812_
DATEX.htm
67. W. C. Willett, in Nutritional Epidemiology, 2nd edn. Oxford University Press,
Oxford, 1998.
68. R. W. Hornung and L. D. Reed, Appl. Occup. Environ. Hyg., 1990, 5, 46.
69. D. R. Helsel, in Nondetects and Data Analysis: Statistics for Censored Envir-
onmental Data, John Wiley & Sons, Chichester, 2005.
70. P. Hewett and G. H. Ganser, Ann. Occupat. Hyg., 2007, 51, 611.
71. B. Parmar, P. F. Miller and R. Burt, Regulat. Toxicol. Pharmacol., 1997, 26, 44.
72. J. Tressou, A. Crepet, P. Bertail, M. H. Feinberg and J. C. Leblanc, Food Chem.
Toxicol., 2004, 42, 1349.
73. FAO-WHO, Evaluation of certain food additives and contaminants (Thirty-third
report of the Joint FAO/WHO Expert Committee on Food Additives), TRS 776-
JECFA 33, 1988.
74. FAO-WHO, Evaluation of certain food additives (Sixty-first report of the Joint
FAO/WHO Expert Committee on Food Additives), WHO Technical Report
Series, No. 922, 2004.[2003, TRS 922-JECFA 61], 2004.
75. ATSDR (Agency for Toxic Substances and Disease Registry), Toxicological
Profile for Cadmium (Final Report), NTIS Accession No. PB99-166621, 1999..
76. EC (European Commission), Opinion on the potential risk to health presented by
lead in food and drink, 1992. http://ec.europa.eu/food/fs/sc/scf/reports/scf_
reports_32.pdf
77. T. A. Jusko, C. R. Henderson, B. P. Lanphear, D. A. Cory-Slechta,
P. J. Parsons and R. L. Canfield, Environ. Health Persp., 2008, 116, 243.
78. K. Murata, T. Iwata, M. Dakeishi and K. Karita, J. Occupat. Health, 2009, 51, 1.
79. B. S. Glenn, K. Bandeen-Roche, B. K. Lee, V. M. Weaver, A. C. Todd and
B. S. Schwartz, Epidemiology, 2006, 17, 538.
80. J. J. Fadrowski, A. Navas-Acien, M. Tellez-Plaza, E. Guallar, V. M. Weaver
and S. L. Furth, Arch. Intern. Med., 2010, 170, 75.
81. B. P. Lanphear, R. W. Hornung, J. Khoury, K. Yolton, P. Baghurstl,
R. L. Bellinger, K. N. Dietrich, R. Bornschein, T. Greene, S. J. Rothenberg,
H. L. Needleman, L. Schnaas, J. Graziano and R. Roberts, Environ. Health
Persp.s, 2005, 113, 894.
82. EFSA (European Food Safety Authority), The EFSA Journal, 2010, in
preparation.

83. A. F. Castoldi, C. Johansson, N. Onishchenko, T. Coccini, E. Roda, M. Vahter,

S. Ceccatelli and L. Manzo, Regulatory Toxicology and Pharmacology, 2008, 51,
201.
84. FAO-WHO, Evaluation of mercury, lead, cadmium and the food additives
amaranth, diethylpyrocarbonate, and octyl gallate. FAO Nutrition Meetings
Report Series, No. 51A, 1972, WHO Food Additives Series, No. 4, 1972; FAS 4/
NMRS 51A-JECFA 16. 249-253, 1972..
86. US-EPA (United States-Environmental Protection Agency), Water Quality
Criterion for the Protection of Human Health: Methylmercury, EPA-823-R-01-
001, 2001.
87. US-EPA (United States-Environmental Protection Agency), Integrated Risk
Information System (IRIS) on Uranium, Soluble Salts. National Center for
Environmental Assessment, 1989. http://www.epa.gov/iris/subst/0421.htm
88. WHO, Guidelines for Drinking-Water Quality – Second Edition – Volume 2 –
Health Criteria and Other Supporting Information – Addendum, 1998.
89. WHO, Guidelines for drinking-water quality – Third Edition, 2004.
90. ATSDR (Agency for Toxic Substances and Disease Registry), Agency for Toxic
Substances and Disease Registry. Toxicological profile for uranium, 1999.
91. FAO-WHO, Evaluation of certain food additives and contaminants, WHO Food
Additive Report Series, No. 18, 1983.
92. FAO-WHO, Evaluation of certain food additives and contaminants, WHO Food
Additive Report Series, No. 24, 1989.
Information System (IRIS) on Arsenic, 1998.
Information System (IRIS) on Arsenic, 2001.
95. US-EPA (United States-Environmental Protection Agency), Issue Paper: Inor-
ganic Arsenic Cancer Slope Factor, Final Draft. July 23, 2005 report of the EPA
Intra-Agency Arsenic Cancer Slope Factor Workgroup, 2005.
96. NRC (National Research Council), Arsenic in drinking water, 1999. http://
www.nap.edu/openbook/0309063337/html/R1.html
97. NRC (National Research Council), Arsenic in drinking water 2001 Update, 2001.
http://www.nap.edu/openbook/0309076293/html/R1.html
98. ATSDR (Agency for Toxic Substances and Disease Registry), Toxicological
profile for arsenic, 2007.
99. FAO-WHO, Evaluation of certain food contaminants: 64th report of the Joint
FAO/WHO Expert Committee on Food Additives, WHO Technical Report
Series 930, 2006.
100. EC (European Commission), SCOOP Report of task 3.2.11: ‘‘Assessment of the
dietary exposure to arsenic, cadmium, lead and mercury of the population of the
EU Member States’’, 2004.
101. S. K. Egan, P. M. Bolger and C. D. Carrington, J. Exposure Science Environ-
mental Epidemiology, 2007, 17, 573.
102. CDC, Fourth National Report on Human Exposure to Environmental Chemicals,
2009.

60 DORNE et al.
103. FAO-WHO, Evaluation of certain food additives and contaminants. Sixty-seventh

report of the Joint FAO/WHO Expert Committee on Food Additives, WHO
technical report series 940, 2007.
104. S. S. H. Tao and P. M. Bolger, Food Additives and Contaminants, 1999, 16, 465.
105. FAO-WHO, Evaluation of certain food additives and contaminants (Fifty-fifth
report of the Joint FAO/WHO Expert Committee on Food Additives), TRS 901-
JECFA 55, 2000.
106. US-EPA (United States-Environmental Protection Agency), Drinking Water
Criteria Document on Cadmium. Final Draft, 1985.
107. EC (European Commission), Cadmium metal and cadmium oxide, Summary
Risk Assessment Report, CAS No: 7440-43-9 and CAS No: 1306-19-0 Joint
Research Centre. EINECS No: 231-152-8 and EINECS No: 215-146-2, EUR
23424 EN, 2008.
108. P. Verger, S. Houdart, S. Marette, J. Roosen and S. Blanchemanche, Regulat.
Toxicol. Pharmacol., 2007, 48, 259.
109. EFSA (European Food Safety Authority), Technical report of EFSA prepared
by Assessment Methodology Unit on Meta-analysis of Dose-Effect Relationship
of Cadmium for Benchmark Dose Evaluation, EFSA Scientific Report, 2009, 254,
1–62.

3
Mixtures and Their Risk Assessment in
Toxicology
Moiz M. Mumtaz, Hugh Hansen, and Hana R. Pohl
Agency for Toxic Substances and Disease Registry, U.S. Department of Health and
Human Services, Atlanta GA 30333, USA
<mgm4@cdc.gov>
<hrp1@cdc.gov>
ABSTRACT 62
1. INTRODUCTION 62
2. PREDICTIONS OF TOXICITY OUTCOMES 64
3. WEIGHT-OF-EVIDENCE EVALUATIONS 66
4. EXPERIMENTAL VALIDATIONS 68
4.1. Studying the Integration of Mechanistic Carcinogenicity with
Physiologically-Based Pharmacokinetic/Pharmacodynamic
Modeling for Chemical Mixtures 70
4.2. Studying the Refinement and Development of Methods for
the Toxicity Assessment of Mixtures 71
4.3. Studying Dose-Response Relationships and Repair
Mechanisms in Chemical Mixtures Toxicity 72
4.4. Studying Optimization of Risk Assessment Procedures for
Complex Mixtures 73
4.5. Studying Dermal Absorption of Chemical Mixtures 73
4.6. Modeling Dose-Response Relationships and Interaction
Thresholds 74
4.7. Genetic Aspects 76
5. CONCLUSION 77
62 MUMTAZ, HANSEN, and POHL
ABBREVIATIONS 77
REFERENCES 77
ABSTRACT: For communities generally and for persons living in the vicinity of waste
sites specifically, potential exposures to chemical mixtures are genuine concerns. Such
concerns often arise from perceptions of a site’s higher than anticipated toxicity due to
synergistic interactions among chemicals. This chapter outlines some historical approa-
ches to mixtures risk assessment. It also outlines ATSDR’s current approach to toxicity
risk assessment. The ATSDR’s joint toxicity assessment guidance for chemical mixtures
addresses interactions among components of chemical mixtures. The guidance recom-
mends a series of steps that include simple calculations for a systematic analysis of data
leading to conclusions regarding any hazards chemical mixtures might pose. These con-
clusions can, in turn, lead to recommendations such as targeted research to fill data
gaps, development of new methods using current science, and health education to raise
awareness of residents and health care providers. The chapter also provides examples of
future trends in chemical mixtures assessment.
KEYWORDS: chemical mixtures . innovative methods . risk assessment
1. INTRODUCTION
For many years, the effort to establish toxicity testing for mixtures has
struggled against many challenges. Data gaps in exposure and toxicity data,
experimental design shortcomings, lack of statistical analysis, time limita-
tions, unavailability of funds, and least but not last, a lack of awareness that
chemical exposure is most often to mixtures—not to single chemicals.
Today, communities repeatedly raise chemical exposure concerns at town
hall and community meetings. This has brought new awareness to issues
such as (1) adequate research on mixtures, (2) implementation of the assess-
ment methodologies, and (3) application of technological advancements.
And recently, calls from funding agencies for interdisciplinary collaboration
have further heightened interest in the National Academy of Sciences’ new
approach to advancement of mixtures research [1].
With today’s rapid communication methods, consortia can share methods
and data within and among fields of biological science in ways previously
impossible. Examples are numerous of new and innovative interdisciplinary
approaches and shared technologies [2]. Work has progressed in the con-
fident belief that computational toxicology will become an important tool in
developing modeling approaches, and that it will complement mechanis-
tically-based toxicology studies in solving mixtures risk assessment
problems [3–5]. Such an approach would integrate Monte Carlo simulation,
median effect principle (MEP), and response surface methodology (RSM)

MIXTURES AND THEIR RISK ASSESSMENT IN TOXICOLOGY 63
with physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD)

modeling [6].
But toxicologists have not arrived easily at this long-sought for, mixtures-
development phase. Before toxicology became a formal academic discipline –
when toxicology studies were focused on single chemicals – the nature and
action of chemical mixtures was pursued through a theoretical approach [7].
Bliss [7] in this groundbreaking introduction of the concept of ‘‘joint action’’
or, as commonly used, ‘‘joint toxic action’’, advanced approaches that to date
are an important assessment tool in analyzing and studying mixtures. As has
been known for many decades, humans are exposed to a variety of chemicals
and other pollutants including, but not limited to, pesticides, pharmaceu-
ticals, household products, and food additives. The critical issue, however, is
whether these exposures exceed the body’s ability to detoxify, adapt, or
otherwise compensate to maintain homeostasis and thus to maintain
uncompromised health status [8]. Maintenance of homeostasis should also
include a consideration of biological and physical agents, psychological
stress, and other insults on the organism [2]. But just getting those first steps
completed, which include working Bliss’s concepts into the practical world of
risk assessment, is still ongoing; this has led some recent researchers to
conclude that, for now, given that research resources are limited and the
challenges daunting, the focus in mixtures toxicology should be on priority of
chemical combinations [9].
Yet the last 30 years have not been without considerable progress. In 1983,
the National Research Council (NRC) published Risk Assessment in the
Federal Government: Managing the Process, also known as the ‘‘Red Book’’
[10]. Intended as a clarion call for a new era, the Red Book represented a
shift from science and public policy misuse to well-recognized principles of
action guiding science and policy [11]. Since the Red Book’s publication,
various reviewing groups have raised issues regarding its utility, short-
comings, misuse, and research needs. Still, the paradigm the book estab-
lished continues to provide the only framework for the development of risk
assessment methods in all areas. The Red Book continues to be a useful tool
to organize, present, explain, and interpret data considered in a weight-of-
evidence approach derived from a wide span of disciplines—especially tox-
icology, pharmacology, and epidemiology.
Indeed, the Agency for Toxic Substances and Disease Registry’s
(ATSDR) mixtures approach is a recent confirmation of the enduring
quality of Bliss’s ‘‘joint action’’ and demonstrates the continued use of the
Red Book paradigm [8]. The Red Book approach is adopted as one com-
ponent of a risk analysis that also includes biomedical judgment. Invoking
Maltoni and Selikoff’s [12] admonition that ‘‘Science is necessary but not
sufficient,’’ the ATSDR approach gives equal importance to the policy
nature of risk assessments.

The foundational methodology for the weight-of-evidence (WOE)

approach for chemical interactions also provides a mechanism for prior-
itizing binary mixtures in a way useful to both the health assessor and the
risk manager [13]. In the last decade, both ATSDR and the U.S. Environ-
mental Protection Agency developed chemical mixtures assessment guide-
lines using this weight-of-evidence approach [14,15]. For example, the
ATSDR document entitled Guidance Manual for the Assessment of Joint
Toxic Action of Chemical Mixtures provides guidelines for evaluating the
toxicity of chemical mixtures encountered at hazardous waste sites. It has all
the elements needed for application to other, possibly hazardous environ-
mental exposures. ATSDR has also developed a series of Interaction Profiles
for chemical mixtures. These documents rely on an evaluation of binary
combinations using weight-of-evidence [13], but importantly provide a
detailed bibliography and literature review of selected mixtures; the docu-
ments also highlight mixtures that depart from the assumption of additivity
[16,17].
The following sections of this chapter provide a more in-depth review of
some of the approaches referred to above, including predictions of toxicity
outcomes, weight-of-evidence evaluations, experimental validations, and
future trends.
2. PREDICTIONS OF TOXICITY OUTCOMES
Not only are human populations exposed to chemicals, they carry from birth
a body burden of hundreds of chemicals. The U.S. Centers of Disease
Control and Prevention’s recent biomonitoring of environmental chemicals
report – its fourth survey of human populations across the United States –
documents this [18] and posits that these chemicals occur in our bodies as
mixtures, not as single, stand-alone substances.
Although several methods have been used for the joint toxicity assessment
of chemical mixtures, identification of chemical mixtures of concern remains
the most important first step. This involves the identification of individual
chemicals and their quantification. The second step is to identify those
chemicals that have individually exceeded their allowable concentrations/
levels. Chemicals that have exceeded such limitations are grouped based on
the health effects they cause, and their mode of action. Only then can risk
assessors decide which methods are available to determine ill health effects
and which of those methods are most suitable. This is a long and tedious
effort that involves processing analytical chemistry data, health effects data,
and a toxicological understanding of the consequences of exposure to every
identified chemical.

Principles of Mixtures Evaluation
Mixture
Mixture of Similar
Components
Concern Mixture
Risk
Assessment
Figure 1. Principles of mixture evaluation.
Most risk assessors agree that three different data analysis methods are
available for toxicity assessment of chemical mixtures [14,15]. Risk assessors
can use available data to analyze (Figure 1)
the actual mixture of concern (also called the whole mixture),

a similar mixture, or
the actual mixture’s components.
Before selection of a specific method, however, risk assessors need to

address definitions and need to formulate specific questions – both important
steps. Ideally, to make the final recommendations for public health or reme-
diation actions at sites, risk assessors should process the data for every mixture
assessment through all three methods and integrate the results. But lack of
appropriate data usually deprive risk assessors of this luxury. Most of the time,
only a single method is available and data availability usually determines it.
The ‘‘whole mixture’’ first method is used when the complete mixture of
interest has been tested and ‘‘mixture of concern’’ data are available. This is
the most direct and accurate form of risk assessment, with the least uncer-
tainty in drawing conclusions and making recommendations regarding the
joint toxicity of the mixture.
Often, however, although data might be unavailable for the mixture of
concern, they are available for a ‘‘similar mixture.’’ Ideally, formal analysis
will determine any similarity between the mixture toxicologically tested and

the mixture of concern. Because similarity criteria are often unavailable,

informal analysis must suffice. Yet composition of the mixture still receives
due consideration, as do the mixture’s qualitative and quantitative
aspects. Because no criteria are set for the determination of a similar
mixture, this approach is used on a case-by-case basis or by grouping of
chemicals [19].
The third or ‘‘component’’ method is sometimes referred to as the ‘‘hazard
index’’ approach. It is used if data are available for a mixture’s individual
chemical components but are not available for the whole mixture. This is the
most often-used approach and is based on the concept of potency-weighted
dose or response-addition of the toxicity of the mixture’s chemical compo-
nents. Through this procedure, risk assessors attempt to predict the toxicity
of the whole mixture, had it been tested. But this method does incorporate
dose or response-additivity models and assumptions concerning modes of
action or correlations of tolerances that may not be thoroughly understood.
Thus, the number of mixtures to which this method can be applied is limited.
Using a toxic equivalency factors (TEF) approach, the component method
has nonetheless been applied to some very important classes of environ-
mental contaminants such as the polycyclic aromatic hydrocarbons (PAHs),
the polychlorinated biphenyls (PCBs), and the dioxins [20,21]. A more recent
trend in using this method is toward acquisition of robust mechanistic data
and use of computational tools and models [22,23].
3. WEIGHT-OF-EVIDENCE EVALUATIONS
Depending upon the route(s), duration(s), and the levels of exposure, a
myriad of chemicals can be found in the tissue and fluids of all populations
[18]. Presence of more than a single chemical can lead to interactions that
can enhance, inhibit, or otherwise influence the toxicity of individual che-
micals and thus modify the mixture’s overall toxicity. Presence of multiple
chemicals in specific compartments or within the organs of the body
increases the likelihood of interactions at pharmacokinetic and pharmaco-
dynamic levels. In fact, ample information, supported by varying degrees of
mechanistic understanding, substantiates interactions [16,24–27].
Despite that most toxicologists agree this information should not be dis-
regarded, few agree on its use in joint toxicity assessments. Thus, one of the
many sources of uncertainty in toxicity assessments of mixtures is the
potential significance of interactions. This uncertainty is akin to the several
recognized sources of uncertainties embedded in the risk assessment process
such as extrapolation from species-to-species, high-to-low dose, LOAEL-to-
NOAEL and temporal (e.g., chronic-to-subchronic).

By contrast, the uncertainties associated with interactions—and any

recommendations for appropriate default factors or modifying factors—have
not even been characterized very well. One attempt was to use a framework
for systematically assessing the weight-of-evidence for chemical interactions
[13,25,26,28]. To some extent, this method provides the means for qualitative
assessment of interactions (i.e., whether the mixture is likely to be more or
less toxic than its predicted joint toxicity based just on the assumption of
addition of individual component toxicity). This framework can also be used
to assess the magnitude of the interaction and quantitatively adjust the
hazard index of the mixture using dose-response or dose-severity [13].
Briefly, and at a minimum, the WOE evaluation is a qualitative judgment,
based on empirical observations and mechanistic data. The framework
characterizes the plausibility of joint toxicity of pairs of toxicants (i.e., how a
chemical’s toxicity can be influenced by the presence of a second toxicant). It
yields an alphanumeric scheme that takes into consideration several factors
such as the quality of the data, its mechanistic understanding, its toxicological
significance, and factors such as route and duration of exposure that play a
critical role in the expression of a mixture’s overall toxicity [3,13,25,26].
Consider, for example, a 4-component mixture consisting of lead, man-
ganese, zinc, and copper. An abbreviated version of the alphanumeric will be
used in this illustration. Following a WOE analysis, all the binary toxico-
logical interactions in the published literature can be arranged in a matrix
(Table 1). Each cell in the table represents a summary of a specific pair’s
interactive toxicity. An alphanumeric of 4IC for the influence of Mn on the
toxicity of Pb indicates that the neurotoxicity of these two toxicants will be
greater than potency-weighted dose additive. Despite that the mechanism of
this interaction is very well understood, its significance is poorly known or
not well understood. On the other hand, the influence of Zn on the hema-
topoietic toxicity of Pb yields oIA alphanumeric. This suggests that the
joint hematopoietic toxicity of this pair will be less than potency-weighted
dose additive. This interaction’s mechanism is understood very well, as is its
toxicological significance. An overview of the matrix shows that various
combinations of the toxicants of this mixture interact in multiple target
organs. But mostly, the joint toxicity will be less than potency-weighted dose
additive. Thus, for a risk assessor, the interactions between components of
this mixture will not be of real concern. But had a majority of the cells of the
matrix shown greater than potency-weighted dose additive toxicity, the
mixture’s joint toxicity would be a matter of high concern. This type of
analysis captures uncertainty in the joint toxicity by estimating the incre-
mental shift in toxicity as a result of interactions.
It is imperative, however, to understand the mechanistic and empirical
approaches used to study chemical interactions such as the WOE approach
and validate them through follow up and through experimental studies.

Table 1. Interaction matrix for mixtures of metals.
ON TOXICITY OF
Pb Mn Zn Cu
Pb =IIIC =IIB =IIIC

neurologic hematologic hepatic
EFFECT OF
Mn >IC
neurologic
? ?
Zn <IA <IB
hematologic
? hepatic
Cu <IB <IIA
hematologic
? hematologic
• scoring for mechanistic data (I, II, III)

• scoring for toxicological significance (A, B, C)
• direction of interaction (<, >, =)
Targeted research needs to develop further such generic methods that can be
used to assess toxicity of chemical mixtures that range from food additives
and pharmaceuticals to occupational and environmental exposures.
4. EXPERIMENTAL VALIDATIONS
Resource and time limitations while conducting toxicological studies will

never allow collection of direct information on all the possible mixtures to
which humans or other target species are exposed. Even when a great deal of
information is available, several risk assessment questions remain. Should,
for example, the ‘‘data need’’ realized during the exposure assessment phase
be filled through additional sampling, environmental modeling or other
computational techniques? How can the dose-response and dose-severity
relationships be evaluated? How should the issue of differential dose
response slopes for contaminants be interpreted and resolved? Do all the
toxic effects have thresholds? If so, can the errors associated with these
thresholds be estimated and the consequences of exceeding these thresholds
be measured? All of these questions have a direct and significant effect on the
toxicity assessment of mixtures.

Ideally, all the components of a mixture need to be identified, and their

toxicity experimentally determined or obtained from the literature. Several
testing protocols can obtain appropriate information, but the actual
experimental design depends on the number of the mixture’s chemical
components. The mixture should be tested both at high-effective con-
centration and at low-realistic concentration. If toxic effects and risks posed
by a mixture are to be determined, then the toxicologic evaluation of the
mixtures is carried out by testing the whole mixture through a tier or
screening approach. If determination of a causative agent or agents is
necessary to mitigate exposures to the active ingredient of a mixture or to
identify the pollution source, assessors should use bioassay-directed frac-
tionation. Finally, if tools for predictive values are developed, toxicologic
evaluation of individual components in various combinations can gain
toxicologic knowledge about the mechanism and its mode of action, as well
as mechanisms of interactions between the components and within the
mixture.
Bottoms-up and top-down are two common, generic approaches used to
test chemical mixtures. Initially, in the bottoms-up approach, simple
binary mixtures are studied, systematically including pharmacokinetic and
pharmacodynamic parameters. Assessors can thus gain insight into the
behavior of the two chemicals and the mechanisms by which each influences
the other in a biological system. Following this, a third, fourth, and fifth
chemical are added to the starting binary mixture to build up, through a
series of experimental studies, to a complex chemical mixture. Such studies
help to understand the operative mechanisms and their relevance as xeno-
biotics interact with biological macromolecules or influence effects of each
other. Also, this type of experimental design allows observation and
understanding of the patterns and overlays of mechanisms as a function of
the mixture’s chemical composition. Such studies also help to test hypoth-
eses about the joint toxicity of chemical mixtures and promote the use of
innovative techniques such as physiologically-based pharmacokinetic
modeling.
When the whole mixtures are tested, the components and their relative
quantities might be unknown, but at times they can be divided into other
mixtures that can be examined for their contributions to overall toxicity.
This is known as the top-down or the fractionation approach. It starts with
testing of a complex chemical mixture. In a series of experiments, the
number of chemical components is decreased one at a time until the mixture
is reduced to the simplest chemical mixture – the binary mixture. Such
experiments can be conducted with complex, real-life mixtures of diverse and
even unknown chemical composition. The top-down approach also helps
identify the fraction or the chemical component of a mixture that is most or
least toxic.

Over the past two decades, ATSDR has supported and funded pragmatic
experimental testing of mixtures without compromising the sensitivity or
specificity obtained through classical methods [23,29]. These efforts have
taken into consideration all available options. These options include recently
developed innovative techniques where significant advances have been made
in alternative toxicologic testing methods using in vitro bioassays with var-
ious cell lines, short term in vivo studies, and physiologically-based phar-
macokinetic modeling. This experimental testing program has promoted
ideas of fully utilizing and integrating computational technology, mathe-
matical/statistical modeling, mechanistically-based, short-term toxicology
studies, and cellular and molecular biology methodologies. All of these
studies met the following critical requirements of an efficient experimental
approach or system for chemical mixtures:
relatively simple, short-term, and inexpensive,

the best science,
an understanding of toxic mechanisms,
broad applicability, and
predictive capability.
The primary objectives of this experimental program were to perform

critical syntheses of relevant data and identify generalizable rules for use in
site-specific health assessments of waste sites following exposure to mixtures
of environmental chemicals.
4.1. Studying the Integration of Mechanistic

Carcinogenicity with Physiologically-Based
Pharmacokinetic/Pharmacodynamic Modeling for
Chemical Mixtures
The primary objective of this study was to develop an efficient and scienti-
fically credible approach for the assessment of carcinogenicity of chemical
mixtures found at hazardous waste sites [30]. Cell culture systems and in vivo
animal studies had already provided the necessary mechanistic and phar-
macokinetic information for physiologically-based pharmacokinetic/phar-
macodynamic (PBPK/PD) modeling and biologically-based dose response
(BBDR) modeling. Cytotoxicity as an index and the departure from addi-
tivity in chemical mixtures of frequently co-occurring metals were tested,
using human keratinocytes.
Using individual dose-response curves of arsenic, cadmiun, chromium,
and lead in conjunction with the mixtures of these four metals at seven

different concentrations, we found that six of these concentrations followed

additivity while one concentrations showed a highly significant and antag-
onistic effect. We also studied much-needed quantitative information on
phenotypic and genotypic changes as well as birth, death, senescence, and
mutation rates [31,32]. In a series of in vivo animal studies, we studied the
role of individual chemicals in the overall toxicity of a mixture containing
arsenic, 1,2-dichloroethane, trichloroethylene, and vinyl chloride. Using the
top-down (or the reductionist) approach, we determined that the liver foci
formation could be used as an index to evaluate interactions. The study
concluded that arsenic was the principal component responsible for the
antagonistic interactions observed in this mixture.
4.2. Studying the Refinement and Development of

Methods for the Toxicity Assessment of Mixtures
This study’s primary objective was data generation to compare the advan-
tages and limitations of various models used to evaluate joint toxic action:
evaluate the role of mixing ratio on the toxicant interactions,

identify the importance of the model fitting to the performance of other
models and the validity of the conclusions, and
evaluate dose-dependent interactions, or the extrapolation of interac-
tions at high-dose to low-dose [33].
The mathematical models and approaches used for these in vitro studies
provided useful perspective for the risk assessment of mixtures and sup-
ported the experimental design development for the assessment of toxicant
interactions. To achieve these goals, we conducted a series of experiments
wherein Rhesus monkey kidney cells were exposed to select concentrations
of 0–50 mM HgCl2 or CdCl2, or to both metals. Cadmium and mercury are
known to have similar, but not identical, physicochemical, biochemical, and
toxicological properties. The release of the soluble enzyme lactate dehy-
drogenase (LDH) was measured and expressed as the fraction of the total
LDH activity released. To characterize the interaction, the data were ana-
lyzed graphically and mathematically by using isobolgram, response surface,
linear, and nonlinear models. The isobolographic and response surface
analysis suggested antagonism, while nonlinear models concluded no
interaction. When data from all mixing ratios were modeled together for
binary mixtures of Hg and Cd (20:1, 10:1, 5:1, or 2:1), most models sup-
ported an overall synergistic interaction. Individual dataset analyses
revealed, however, that as the ratio decreased from 20:1 to 2:1, the nature of

the interactions gradually changed from synergy to additivity. This was

consistent among all the models used, but was most clearly revealed by the
linear models; the statistical isobole model was the least revealing.
Risk assessors should recognize and consider the implications of the
underlying assumptions inherent in the various models used to evaluate the
risk from exposure to chemical mixtures. The conclusions of interaction
analysis also depend, in part, on the fit of the model. Thus, the interpretation
of any analysis regarding chemical interactions could be made more reliable
by including information such as plots that illustrate the adequacy of the
data fit [34].
4.3. Studying Dose-Response Relationships and Repair

Mechanisms in Chemical Mixtures Toxicity
The objectives of this study were to examine the dose-response relationships
of specific chemical mixtures to decipher the role of tissue repair in the overall
toxicity of chemical mixtures [35]. Dose-response relationships are widely
used paradigms in predictive toxicology. Classically, only toxic injury is
measured, without any consideration of tissue repair response. We know from
hepatotoxicant studies that cell division and tissue repair accompany toxic
response as a mechanism to combat toxic injury, and that the extent of tissue
repair response determines the final toxicity outcome. Measuring tissue repair
in conjunction with injury as a parallel but opposing biological response to
toxic agents might increase the usefulness of dose-response paradigms in joint
toxicity assessments and in predictive toxicology. In this study, we analyzed
dose-dependent effects of four model hepatotoxicants: trichloroethylene,
thioacetamide, allyl alcohol and chloroform, and their combinations.
The results of these studies showed that up to a threshold, dose-dependent
effects of thioacetamide, trichloroethylene, allyl alcohol, and chloroform
revealed a dose-related stimulation of tissue repair at low doses. Above
threshold level, tissue repair was significantly delayed and attenuated,
leading to the progression of injury. Studies with trichloroethylene revealed
that trichloroacetic acid, a metabolite of trichloroethylene, inhibits the levels
of plasma ALT and SDH, thereby explaining the apparent lack of injury
dose-response. This remains true despite histological studies that show a
dose-dependent increase in the injury. In fact, tissue repair plays a pivotal
role in the final outcome of liver injury following exposure to a binary
mixture of chloroform and allyl alcohol [36]. Studies with a four-component
mixture consisting of trichloroethylene, thioacetamaide, allyl alcohol, and
chloroform indicate that tissue repair plays an important role in the final
outcome of toxicity [37].

4.4. Studying Optimization of Risk Assessment Procedures

for Complex Mixtures
The overall objectives of this study were to develop a protocol using a
battery of cell culture assays (e.g., genotoxicity, immunotoxicity) to inves-
tigate the toxicity of several classes of environmentally important chemical
mixtures. We could then use these methods for assessing risk of complex
mixtures found in the environment or associated with lifestyle and occu-
pational exposures.
Polycyclic aromatic hydrocarbons are some of the most common envir-
onmental mixtures present in air particulate, cigarette smoke, and petroleum
products. Mixtures of PAHs, halogenated aromatic hydrocarbons (HAHs)
present in wood-preserving waste, and halogenated aliphatic hydrocarbons
such as trichloroethylene and vinyl chloride were tested in these cell assays.
Individual chemicals and subsequently, two- and three-component mixtures
were tested. The data thus obtained were analyzed to investigate potential
chemical interactions. The data can be compared to the results obtained
from complex mixtures and mixtures of reconstituted chemicals designed to
replicate the complex mixture. Results from the genotoxicity assay using
binary and tertiary mixtures of PAHs revealed a dose-response gradient with
B(a)P-chrysene mixture that suggested an interaction. Similar studies with
complex mixtures and isolated fractions indicated that the B(a)P content of
the PAH mixture cannot be used to predict genotoxicity. The results from
assays of an extract of a manufactured gas plant residue separated into four
fractions revealed that genotoxicity varied from fraction to fraction. These
methods also afforded a determination of the role of high-molecular weight
PAHs and alkyl-substituted PAHs [38].
4.5. Studying Dermal Absorption of Chemical Mixtures

The objective of this study was to develop an experimentally and computa-
tionally robust approach to assessing the absorption of complex chemical
mixtures when applied topically. This study’s goal was to examine the
influence of the components of a mixture that may not be a primary tox-
icologic concern, such as solvents, surfactants, other inert ingredients. Such
chemical components may nonetheless interact with known toxic compo-
nents to alter their rate and extent of penetration into and absorption from
skin. Although this may occur at different levels of biological complexity,
such interactions can be experimentally studied with in vitro models of
increasing biological complexity. The actual response seen in vivo is then the
sum of all interactions seen between the toxicant of interest and other mixture

components and between all chemicals in the mixture and the biological
components of the skin barrier. In fact, less than 0.2% of applied dose of the
coplanar polychlorinated biphenyl congener 3,3 0 ,4,4 0 ,5 PCB was actually
absorbed. The presence of either sodium lauryl sulfate or methyl nicotinate in
a mixture did not affect the absorption. By contrast, the absorption of
pentachlorophenol (PCP) in ethanol and aqueous ethanol vehicles ranged
from 1.12% of applied dose in ethanol to 14.12% in an aqueous ethanol
vehicle containing sodium lauryl sulfate. The addition of methyl nicotinate to
the aqueous ethanol had a dramatic effect on the shape of the absorption
profile, shifting the peak absorptive flux to much earlier time points. Addi-
tion of sodium lauryl sulfate or methyl nicotinate to a PCP mixture also
shifted the skin deposition profile to an enhanced skin depot formation – an
event which could alter the threshold for cutaneous toxicity [39].
4.6. Modeling Dose-Response Relationships and

Interaction Thresholds
Physiologically-based pharmacokinetic modeling is viewed as a useful tool
to fill data needs in human risk assessment for chemical mixtures. PBPK
modeling can
evaluate tissue dose under various exposure conditions,

predict dose-response curves based on pharmacokinetic/dynamic
mechanisms, and
conduct route, species, and dose extrapolations.
Specifically for mixtures, this modeling can determine interaction

thresholds.
In general, because of the need for a large number of laboratory animals,
experimental determination of low-dose interaction thresholds is not eco-
nomically feasible. An alternative approach was developed for chlorpyrifos
and parathion: the approximation of the interaction threshold was achieved
by developing four individual PBPK models – one for each parent com-
pound and its major metabolite. The estimated levels of the metabolites were
then linked to a model for AChE kinetics, which described enzyme synthesis,
degradation, binding to the metabolites of both chemicals, and aging after
binding. Most of the parameters used in the models were either calculated or
obtained from the literature. A calibrated model was used to simulate the
area under the curve, which was assumed to be directly related to the neuro-
toxicity for a range of doses of the two chemicals using the response addition
approach. Model simulations indicated that additivity occurs at oral dose

Table 2. Physiologically-based pharmacokinetic models’ predictions of interaction

thresholds.
Binary mixtures Thresholds in rats References
Chloroform None established [40]

Trichloroethylene
1,1-Dichloroethylene 4100 ppm for each chemical [41]
Trichloroethylene
Vinyl chloride 430 ppm for each chemical [42]
Trichloroethylene
levels below 0.08 mg/kg body weight of each chemical. At higher doses,
antagonism by enzymatic competitive inhibition takes place. The threshold
was obtained by comparing the levels of simulated mixture response with
levels anticipated from the individual response addition [4].
Similarly, metabolism of volatile organic compound (VOC) mixtures by
P450 enzymes such as CYP2E1 may lead to competitive inhibition.
Mechanistically, additivity is likely at low levels, but competitive interac-
tions are expected under high-exposure conditions when CYP catalytic sites
are saturated. PBPK models were developed for some binary combinations
of VOCs. The results are summarized in Table 2.
More complicated PBPK models were also developed. For example, by
using information on binary interactions among the component chemicals, a
PBPK model predicted toxicokinetic interactions in the quaternary mixture
of benzene, toluene, ethylbenzene, and xylenes (BTEX) following inhalation
exposure [43]. The information on blood levels of interacting chemicals was
based on results in rats [43,44]. All four chemicals are known substrates for
the same CYP2E1 isozyme.
The PBPK analyses of the blood kinetic data from all binary exposure
studies suggest competitive inhibition of hepatic metabolism as the most
plausible mechanism of interaction at high exposures [43]. The study con-
cluded that with increasing mixture complexity, the blood level of a chemical
is increased according to the potency and number of inhibitors, rather than
by modification of the metabolic inhibition constant (Ki) for binary inter-
actions. Later, the PBPK model for BTEX [43] was linked to a PBPK
model for dichloromethane to construct a quinary model for dichloro-
methane and BTEX in rats [45]. The model for dichloromethane and BTEX
was rewritten for humans by substituting the rat physiological and physio-
chemical parameters with human values [46]. Efficient experimental design
resources and animal use are drawbacks to the development of suitable
models. Once a model is developed, however, it can be applied in various
exposure scenarios.

4.7. Genetic Aspects

Genetic variability is another parameter that can influence interactions not
covered in the above-described models. Interspecies differences can be
demonstrated through CYP2E1 enzymes using the above example involving
VOCs and their metabolites metabolism. The human CYP2E1 gene is on
chromosome 10. It is expressed primarily in the human liver, but it is also
found in other organs [47–51]. Several polymorphic sites have been identified
in the CYP2E1 gene. For example, the CYP2E1*1D allele is very rare in
Caucasians, but has been identified in 18% and 12% of Africans and
African-Americans, respectively. In East-Asian and Asian populations, this
allele appears in a range from 16% to 21% [52,53]. Other CYP2E1 gene
polymorphisms show various differences among populations as well.
The indicated differences have a clinical effect; occupational cohorts have
demonstrated a strong association between the frequency of a certain geno-
type of CYP2E1 and the liver toxicity induced by vinyl chloride [54–58].
One of the reported genotypes is linked to the increased production of
chloroethylene oxide and chloroacetaldehyde metabolites. These reactive
metabolites form DNA adducts. Under the right conditions (e.g., in the
presence of DNA repair), genes with reduced repair capacity could initiate
the carcinogenic process. Thus, differences in liver disease incidence do
inhere among exposed populations. This example, pertaining to the
mechanism of action of a single chemical, is applicable to mixtures of che-
micals using the same enzymes as well. Although intraspecies variability has
long been a part of the risk assessment for single chemicals, it has yet to be
implemented in the risk assessment of chemicals mixtures.
Another technique available for mixtures evaluation is quantitative
structure activity relationship (QSAR) modeling. QSAR modeling is a the-
oretical framework that can predict physicochemical, biological, and tox-
icological properties of organic compounds (not applicable to metals). The
model quantifies the association between a chemical substructure of a com-
pound and its potential to exhibit a specified adverse effect [59]. To establish
the relationships, modern QSAR software uses either statistical methods,
knowledge-based expert systems, or a combination of both [59–61]. In gen-
eral, QSAR will not predict mixtures of chemicals. The method can, however,
identify data-rich chemicals that exhibit a ‘‘property-sensitive similarity’’ to
that chemical in a binary combination that lacks proper toxicological
information. The final evaluation, then, would be done on a ‘‘similar mix-
ture’’. QSAR models have been developed for various toxicity endpoints.
QSAR models have also been developed for almost all major human P450
enzymes using in vitro inhibition data or substrate data derived with human
liver microsomal or expressed enzymes [62–64]. These models have been used,
for example, in the pharmacological industry to screen quickly new molecules

before further testing. But the method has several limitations, including a
difficulty in predicting the formation of possible multiple metabolites [65].
5. CONCLUSION
Thus, chemical mixtures toxicity provides opportunity for ingenious use of
current methods in toxicology and opportunities to develop new methods
that can be integrated into risk assessment. It is a developing field that is
attracting attention of researchers, assessors, and residents of potentially
affected communities.
ABBREVIATIONS
AChE acetylcholinesterase
ALT alanine transaminase
ATDSR Agency for Toxic Substances and Disease Registry
B(a)P benzo(a)pyrene
BBDR biologically-based dose-response
BTEX benzene, toluene, ethylbenzene, and xylene
HAH halogenated aromatic hydrocarbon
LDH lactate dehydrogenase
LOAEL lowest observed adverse effect level
MEP median effect principle
NOAEL no observable adverse effect level
NRC National Research Council
PAH polycyclic aromatic hydrocarbon
PBPK/PD physiologically-based pharmacokinetic/pharmacodynamic
PCB polychlorinated biphenyl
PCP pentachlorophenol
QSAR quantitative structure activity relationship
RSM response surface methodology
SDH succinate dehydrogenase
TEF toxic equivalency factor
VOC volatile organic compound
WOE weight-of-evidence
REFERENCES
1. National Academy Press, Toxicity testing in the 21st century: a vision and a
strategy, Washington, DC, 2007, pp 216.

2. W. A. Suk, K. Olden and R. Yang, Environ. Health Persp., 2002, 110 (Suppl. 6),
891–892.
3. H. Hansen, C. T. De Rosa, H. R. Pohl, M. Fay and M. M. Mumtaz, Environ.
Health Persp., 1998, 106(suppl. 6), 1271–1280.
4. H. A. El-Masri, M. M. Mumtaz and M. L. Yushak, Environ. Toxicol. Pharma-
col., 2004, 16, 57–71.
5. E. Demchuk, P. Ruiz, J. D. Wilson, F. Scinicariello, H. R. Pohl, M. Fay,
M. M. Mumtaz, H. Hansen and C. T. De Rosa, Toxicol. Mech. Methods, 2008,
18, 119–135.
6. H. A. El-Masri, K. F. Reardon and R. Yang, Crit. Rev. Toxicol., 1997, 27,
175–197.
7. C. I. Bliss, Ann. Appl. Biol., 1939, 26, 585–615.
8. M. M. Mumtaz, H. A. El-Masri, H. Hansen, S. Wilbur, H. R. Pohl and C. T. De
Rosa, Environ. Epidemiol. Toxicol., 2000, 2, 220–234.
9. V. J. Feron, F. R. Cassee, J. P. Groten, P. W. van Vliet and J. A. van Zorge,
Environ. Health Persp., 2002, 110 (Suppl. 6), 893–899.
10. National Academy Press, Risk assessment in the federal government: managing the
process, National Research Council, National Academy of Sciences, 1983.
11. C. T. De Rosa and H. Hansen, Human Ecol. Risk Assess., 2003, 9, 1219–1228.
12. C. Maltoni and I. J. Selikoff, Ann. NY Acad. Sci., 1988, 534, xvi.
13. M. M. Mumtaz and P. R. Durkin, Toxicol. Ind. Health, 1992, 8, 377–406.
14. US Environmental Protection Agency, Supplementary guidance for conducting
health risk assessment of chemical mixtures, 2000. Available at http://cfpub.
epa.gov/ncea/cfm/recordisplay.cfm?deid ¼ 20533 [accessed February 2010].
15. Agency for Toxic Substances and Disease Registry, Guidance manual for the
assessment of joint toxic action of chemical mixtures. Atlanta, GA, 2004. Available
at http://www.atsdr.cdc.gov/interactionprofiles/ipga.html [accessed March 2010].
16. Agency for Toxic Substances and Disease Registry, Interaction profiles of che-
mical mixtures, Atlanta, GA, 2009. Available at http://www.atsdr.cdc.gov/
interactionprofiles/ipga.html [accessed March 2010].
17. E. Monosson, Environ. Health Persp., 2005, 113(4), 383–390.
18. Centers for Disease Control and Prevention, National Report on Human
Exposure to Environmental Chemicals, 2010. Available at: http://www.cdc.gov/
exposurereport [accessed February 2010].
19. P. Durkin, R. Hertzberg, W. Stiteler and M. M. Mumtaz, Tox. Lett., 1995, 79,
251–264.
20. S. Safe, CRC Crit. Rev. Toxicol., 1990, 21, 51–88.
21. S. Safe, Environ, Health Persp., 1998, 106 (Suppl. 4), 1051–1058.
22. J. P. Groten, W. H. M. Hejine, R. H. Stierum, A. P. Freidig and V. J. Feron,
Environ. Toxicol. Pharmacol., 2004, 18, 185–192.
23. R. S. H. Yang, H. A. El-Masri, R. S. Thomas, I. D. Dobrev, J. E. Dennison,
D. S. Bae, J. A. Campain, K. H. Liao, B. Reisfeld, M. E. Andersen and M. M.
Mumtaz, Environ. Toxicol. Pharmacol., 2004, 18, 65–81.
24. M. M. Mumtaz and R. C. Hertzberg, in Hazard Assessment of Chemicals, Vol. 8,
Ed. J. Saxena, Hemisphere Publishing Corporation, Washington, DC, 1993,
pp. 47–79.

25. H. R. Pohl, N. Roney, S. Wilbur, H. Hansen and C. T. De Rosa, Chemosphere,

2003, 53, 183–197.
26. C. T. De Rosa, H. A. El-Masri, H. R. Pohl, W. Cibulas and M. M. Mumtaz,
J. Toxicol. Environ. Health, Part A., 2004, 67, 1–17.
27. K. Krishnan, S. Haddad, M. Beliveau and R. Tardif, Environ. Health Perspect.,
2002, 110, 989–994.
28. H. R. Pohl and H. G. Abadin, Toxicol. Appl. Pharmacol., 2008, 233, 116–125.
29. R. S. H. Yang and J. Ruckman, Toxicology, 1987, 47, 15–34.
30. D.-S. Bae, C. Gennings, W. H. Carter, Jr., R. S. H. Yang and J. A. Campain,
Toxicol. Sci., 2001, 63, 132–142.
31. D.-S. Bae, W. H. Hanneman, R. S. H. Yang and J. A. Campain, Environ. Health
Persp., 2002, 110(Suppl. 6), 931–941.
32. D.-S. Bae, R. J. Handa, R. S. H. Yang and J. A. Campain, Toxicol. Sci., 2003,
74, 32–42.
33. M. M. Mumtaz, H. El-Masri, D. Chen and J. Pounds, in Metal Ions in Biology
and Medicine, Eds. J.A. Centeno, P. H. Collery, G. Vernet, R. B. Finkelman,
H. Gibb and J. C. Etienne, John Libbey, Paris, Eurotext, 2000, Vol. 6,
pp. 297–299.
34. J. G. Pounds, J. Haider, D. G. Chen and M. M. Mumtaz, Environ. Toxicol.
Pharmacol., 2004, 18, 101–113.
35. M. G. Soni, S. K. Ramiah, M. M. Mumtaz, H. Clewell and H. M. Mehendale,
Reg. Tox. Pharmacol., 1999, 29, 165–174.
36. S. S. Anand, S. N. Murthy, V. S. Vaidya, M. M. Mumtaz and H. M. Mehendale,
Food Chem. Toxicol., 2003, 41, 1123–1132.
37. S. S. Anand and H. M. Mehendale, Environ. Toxicol. Pharmacol., 2004, 18, 149–160.
38. K. C. Donnelly, R. Lingenfelter, L. Cizmas, M. H. Falahatpisheh, Y. Qian,
Y. Tang, S. Garcia, K. Ramos, E. Tiffany-Castiglioni and M. M. Mumtaz,
Environ. Toxicol. Pharmacol., 2004, 18, 135–141.
39. J. E. Riviere, G. Qiao, R. E. Baynes, J. D. Brooks and M. M. Mumtaz, Arch.
Toxicol., 2001, 75, 329–334.
40. K. K. Isaacs, M. V. Evans and T. R. Harris, J. Pharmacokinet. Pharmacodyn.,
2004, 31, 215–242.
41. H. A. El-Masri, J. D. Tessari and R. S. H. Yang, Arch. Toxicol., 1996, 70,
527–539.
42. H. A. Barton, J. R. Creech and C. S. Godin, Toxicol. Appl. Pharmacol., 1995,
130, 237–247.
43. S. Haddad, R. Tardif, G. Charest-Tardif and K. Krishnan, Toxicol. Appl.
Pharmacol., 1999, 161, 249–257.
44. R. Tardif, G. Charest-Tarif, J. Brodeur and K. Krishnan, Toxicol. Appl. Phar-
macol., 1997, 144, 120–134.
45. S. Haddad, G. Charest-Tardif, R. Tardif and K. Krishnan, Toxicol. Appl.
Pharm., 2000, 167, 199–209.
46. S. Haddad, M. Béliveau, R. Tardif and K. Krishnan, Toxicol. Sci., 2001, 63,
125–131.
47. F. Botto, E. Seree, E. el Khayari, G. de Sousa, A. Massacrier, M. Placidi, P. Cau,
W. Pellet, R. Rahmani and Y. Barra, Biochem. Pharmacol., 1994, 48, 1095–1103.

48. T. Goasduff, G. Bellec, Y. Amet, Y. Dreano, J. F. Menez and F. Berthou,

Alcohol, 1996, 13, 301–308.
49. M. Ingelman-Sundberg, I. Johansson, H. Yin, Y. Terelius, E. Eliasson, P. Clot
and E. Albano, Alcohol, 1993, 10, 447–452.
50. A. M. Mostafa, A. B. Abdel-Naim, O. Abo-Salem, A. H. Abdel-Haziz and
F. M. Hamada, Pharmacol. Res., 1999, 40, 195–200.
51. I. Vieira, M. Pasanen, H. Raunio and T. Cresteil, Pharmacol. Toxicol., 1998,
83, 183–187.
52. M.-Y. Lee, N. Mukherjee, A. J. Pakstis, S. Khaliq, A. Mohyuddin, S. Q. Mehdi,
W. C. Speed, J. R. Kidd and K. K. Kidd, The Pharmacogenomics Journal, 2008,
8, 349–356.
53. L. Le Marchand, G. R. Wilkinson and L. R. Wilkens, Cancer Epidemiol. Bio-
markers Prev., 1999, 8, 495–500.
54. C. Y. Huang, K. L. Huang, T. J. Cheng, J. D. Wang and L. L. Hsieh, Arch.
Toxicol., 1997, 71, 482–488.
55. Y. Li, M. J. Marion, A. Rundle and P. W. Brandt-Rauf, Biomarkers, 2003, 8,
408–414.
56. H. I. Hsieh, P. C. Chen, R. H. Wong, J. D. Wang, P. M. Yang and T. J. Cheng,
Toxicology, 2007, 239, 34–44.
57. S. M. Zhu, Z. L. Xia, A. H. Wang, X. F. Ren, J. Jiao, N. Q. Zhao, J. Qian, L. Jin
and D. C. Christiani, Toxicol. Lett., 2008, 178, 88–94.
58. A. H. Wang, S. M. Zhu, Y. L. Qiu, R. Zhu, Y. B. Qu, Y. L. Li, P. W. Brandt-
Rauf and Z. L. Xia, Int. J. Occup. Med. Environ. Health, 2008, 21, 141–146.
59. C. Hansch and A. Leo, Fundamentals and Applications in Chemistry and Biology,
American Chemical Society, Washington, DC, 1995.
60. E. Benfenati and G. Gini, Toxicology, 1997, 119, 213–225.
61. R. Benigni and A. M. Richard, Methods, 1998, 14, 264–276.
62. D. F. V. Lewis, Drug Metab. Ref., 1997, 29, 589–650.
63. D. F. V. Lewis, Biochem. Pharmacol., 2000, 60, 293–306.
64. D. F. V. Lewis, P. J. Eddershaw, M. Diekins, M. H. Tarbit and P. S. Goldfarb,
Chem. Biol. Interact., 1998, 115, 175–199.
65. S. Ekins, Drug Discov. Today, 2004, 9, 276–285.

4
Metal Ions Affecting the Pulmonary and
Cardiovascular Systems
Massimo Corradi and Antonio Mutti
1
Department of Clinical Medicine, Nephrology and Health Sciences, University of Parma,
Via Gramsci 14, I-43100 Parma, Italy
<massimo.corradi@unipr.it>
<antonio.mutti@unipr.it>
ABSTRACT 82
1. INTRODUCTION 83
2. ALUMINUM 83
2.1. Aluminum and Pulmonary Effects 83
2.2. Aluminum and Cardiovascular Effects 84
3. ARSENIC 84
3.1. Arsenic and Pulmonary Effects 84
3.2. Arsenic and Cardiovascular Effects 85
4. BERYLLIUM 85
4.1. Beryllium and Pulmonary Effects 86
4.2. Beryllium and Cardiovascular Effects 86
5. COPPER 86
5.1. Copper and Pulmonary Effects 86
5.2. Copper and Cardiovascular Effects 87
6. CADMIUM 87
6.1. Cadmium and Pulmonary Effects 87
6.2. Cadmium and Cardiovascular Effects 89
7. CHROMIUM 89
7.1. Chromium and Pulmonary Effects 90
82 CORRADI and MUTTI
7.2. Chromium and Cardiovascular Effects 91

8. COBALT 92
8.1. Cobalt and Pulmonary Effects 92
8.2. Cobalt and Cardiovascular Effects 93
9. LEAD 93
9.1. Lead and Pulmonary Effects 94
9.2. Lead and Cardiovascular Effects 94
10. MANGANESE 95
10.1. Manganese and Pulmonary Effects 96
10.2. Manganese and Cardiovascular Effects 96
11. NICKEL 97
11.1. Nickel and Pulmonary Effects 97
11.2. Nickel and Cardiovascular Effects 98
12. ZINC 98
12.1. Zinc and Pulmonary Effects 98
12.2. Zinc and Cardiovascular Effects 99
13. CONCLUDING REMARKS 99
REFERENCES 100
ABSTRACT: Some metals, such as copper and manganese, are essential to life and
play irreplaceable roles in, e.g., the functioning of important enzyme systems. Other
metals are xenobiotics, i.e., they have no useful role in human physiology and, even
worse, as in the case of lead, may be toxic even at trace levels of exposure.
Even those metals that are essential, however, have the potential to turn harmful at very
high levels of exposure, a reflection of a very basic tenet of toxicology – ‘‘the dose makes
the poison.’’
Toxic metal exposure may lead to serious risks to human health. As a result of the
extensive use of toxic metals and their compounds in industry and consumer products,
these agents have been widely disseminated in the environment. Because metals are not
biodegradable, they can persist in the environment and produce a variety of adverse
effects. Exposure to metals can lead to damage in a variety of organ systems and, in some
cases, metals also have the potential to be carcinogenic.
Even though the importance of metals as environmental health hazards is now widely
appreciated, the specific mechanisms by which metals produce their adverse effects have
yet to be fully elucidated. The unifying factor in determining toxicity and carcinogenicity
for most metals is the generation of reactive oxygen and nitrogen species. Metal-mediated
formation of free radicals causes various modifications to nucleic acids, enhanced lipid
peroxidation, and altered calcium and sulfhydryl homeostasis. Whilst copper, chromium,
and cobalt undergo redox-cycling reactions, for metals such as cadmium and nickel the
primary route for their toxicity is depletion of glutathione and bonding to sulfhydryl
groups of proteins.
This chapter attempts to show that the toxic effects of different metallic compounds
may be manifested in the pulmonary and cardiovascular systems. The knowledge of health
effects due to metal exposure is necessary for practising physicians, and should be assessed
by inquiring about present and past occupational history and environmental exposure.

METALS AND THE PULMONARY AND CARDIOVASCULAR SYSTEMS 83
KEYWORDS: cardiovascular apparatus . heart . lung . metals . occupational expo-

sure . pulmonary effects
1. INTRODUCTION
For centuries metallic elements have been known to be capable of causing

health diseases. The pulmonary and cardiovascular effects of metal exposure
depend on the nature of the offending agent, its physicochemical form, the dose,
exposure conditions, and host factors. People are rarely exposed to the pure
elemental form of metals, whereas they are usually exposed to oxides or salts.
The interaction of metallic elements with functional groups on macro-
molecules is an important mechanism for their toxicity and their carcinogeni-
city. Thus, several metal ions react with SH groups, thereby possibly inhibiting
active centers of enzymes. Direct interaction of metal ions with deoxyribonucleic
acid is one of the mechanisms for metal carcinogenesis. Another relevant con-
sequence of metal binding to proteins is the possible acquisition of antigenicity.
This chapter is focused on human pulmonary and cardiovascular effects
induced mainly by occupational exposure to some metallic elements.
2. ALUMINUM
Aluminum is one of the most abundant elements in the earth’s crust;

everyone is exposed to low levels of Al from food, air, water, and soil. In
compounds, Al typically occurs in its +3 oxidation state. There is no known
physiologic role for Al.
2.1. Aluminum and Pulmonary Effects

A number of studies have examined the potential for airborne Al to induce
respiratory effects in chronically exposed workers. Exposure to Al fumes and
dust occurs in potrooms where hot Al metal is recovered from ore, in
foundries where Al alloys are melted and poured into molds, in welding
operations, and the production and use of finely powdered Al. Wheezing,
dyspnea, and/or impaired lung function have been observed in potroom
workers [1–4], foundry workers [5–7], workers exposed to fine Al dust [8,9], in
a worker spray-painting with an Al paint, and welders [10–13], although
other studies have not found a significant effect [14,15]. Case reports provide
suggestive evidence that chronic exposure to Al may cause occupational
asthma [16]. An asthmatic reaction was observed following a bronchial
provocation test in an Al foundry worker [17] and an Al welder [18].

Pulmonary fibrosis has also been observed in workers exposed to fine Al

dust, alumina, or bauxite. However, conflicting reports are available on the
fibrogenic potential of Al. In some cases, the fibrosis was attributed to con-
comitant exposure to other chemicals. For example, pulmonary fibrosis has
been observed in a number of bauxite workers or potroom workers [19–21,8];
in these workers, it is very likely that there was simultaneous exposure to silica
and that the latter was the causative agent rather than Al.
Pulmonary fibrosis has further been observed in an Al arc welder [22], an
Al production worker exposed to Al oxide fumes [23], and in workers in an
unspecified Al industry [24]. There is also some evidence suggesting Al-
induced pneumoconiosis [12], pulmonary alveolar proteinosis [9], interstitial
pneumonia [11], and granulomas [25].
2.2. Aluminum and Cardiovascular Effects

Studies in Al production workers have shown an increased risk of coronary
heart disease [26,27]. This effect might be explained by the possible association
between exposure to air pollutants and the occurrence of coronary heart dis-
ease mediated by an inflammatory response that increases blood coagulation.
It is unlikely that Al per se is responsible for this effect, but more likely that it is
due to air pollutants in general. However, there are studies that do not support
the association between potroom exposure and coronary heart disease [28].
Al may cause anemia through decreased heme synthesis, decreased globulin
synthesis, and increased hemolysis. Al may also have a direct effect on iron
metabolism: It influences absorption of iron via the intestine, it hinders iron
transport in the serum, and it displaces iron by its binding to transferrin.
Patients with anemia from Al toxicity often have increased reticulocyte counts,
decreased mean corpuscular volume, and mean corpuscular hemoglobin [29].
3. ARSENIC
Arsenic is chemically classified as a metalloid, having both properties of a

metal and a nonmetal; however, in toxicology it is frequently referred to as a
metal. In compounds, As typically exists in one of three oxidation states, 3,
+3, and +5. There is no known human physiologic role for As.
3.1. Arsenic and Pulmonary Effects

Inhalation is the primary route of exposure to As in the workplace and
happens in industries such as nonferrous smelting, As production, wood

preservation, glass manufacturing, production and application of As-based

pesticides, and electronics.
A body of epidemiologic evidence supports the carcinogenic effects of As,
showing that occupational exposure to inorganic As is associated with an
increased risk of developing lung cancer [30–34]. Interestingly, epidemiolo-
gical studies, relying on cohort [35] or case-control [36] study designs from
various countries, provided further evidence that ingestion of arsenic via
drinking water increased risk of lung cancer in a dose-response manner.
Chronic respiratory effects of As have been reported primarily as a result
of occupational exposure through inhalation of As-containing dust or
fumes. The respiratory effects included rhinitis, pharyngitis, laryngitis, tra-
cheobronchitis, chronic cough, shortness of breath, and chronic restrictive
and/or obstructive lung diseases [37].
3.2. Arsenic and Cardiovascular Effects

Pericarditis and arrhythmias have been documented as As-induced cardiac
effects [38]. A dose-response relationship has been reported between
cumulative As exposure and electrocardiogram alterations from the endemic
area of arseniasis in southwestern Taiwan [39].
Blackfoot disease, a unique peripheral arterial disease characterized by the
severe systemic arteriosclerosis as well as dry gangrene and spontaneous
amputations of affected extremities at end stages, has been well documented
as a characteristic vascular disease associated with long-term As exposure
[40]. Both ingested and inhaled inorganic As have been related to an increased
mortality from cardiovascular disease, especially ischemic heart disease [41].
The mortality from cardiovascular disease was significantly higher among
residents in endemic areas of Blackfoot disease than among the general
population in Taiwan [42]. The association between long-term exposure to As
and increased mortality from cardiovascular disease has also been reported
among copper-smelter workers in the United States and Sweden, among
chimney sweeps in Sweden and Denmark, among glassblowers in Sweden,
and among workers and neighboring residents of an As refinery in Japan [41].
4. BERYLLIUM
Beryllium is a naturally occurring element found in earth’s surface rocks. It

has two common oxidation states, Be(0) and Be (+2). Because of its high
reactivity, Be is not found as the free metal in nature. The element is not
known to be necessary or useful for animal life.

4.1. Beryllium and Pulmonary Effects

Old reports on accidental and non-accidental exposures show that exposure
to fumes or dusts containing Be is capable of causing chemical pneumonitis
or acute airway irritation [43]. Berylliosis or chronic beryllium disease is well
known for its striking histological and clinical resemblance to sarcoidosis
[44]. Follow-up studies have revealed that individuals with chronic beryllium
disease have a higher incidence of lung cancer than the general population
[45,46]. A thorough review article has recently been published on the car-
cinogenesis of beryllium, which is now assigned to category 1 by the Inter-
national Agency for Research on Cancer [47].
4.2. Beryllium and Cardiovascular Effects

Although research has described the toxic effects of Be on the respiratory
apparatus, less is known about its effects on the cardiovascular apparatus,
including pulmonary blood vessels. Studies performed in animals showed
that Be inhibited vascular endothelial adenosine diphosphatase activity,
prostacyclin production, and nitric oxide release, thus inducing functional
alterations in vascular endothelial cells [48]. These findings show that Be
may impair some vascular endothelial functions and alter the interaction
between platelet and endothelial mediators [48].
5. COPPER
Copper displays four oxidation states: Cu(0), Cu(I), Cu(II), and Cu(III). Cu,
which is essential in all plants and animals, is found in a variety of enzymes,
including the Cu centers of cytochrome c oxidase and superoxide dismutase.
5.1. Copper and Pulmonary Effects

In humans, Cu is a respiratory irritant by inhalation. Workers exposed to Cu
dust report a number of symptoms that are suggestive of respiratory irri-
tation, including coughing, sneezing, and runny nose [49]. In a study of
about 100 workers involved in sieving Cu, lung radiographs revealed linear
pulmonary fibrosis, and in some cases, nodulation [49].
Cu is considered the etiologic agent in the occupational disease referred to
as ‘‘vineyard sprayer’s lung’’. This disease is observed in vineyard workers
spraying an antimildew agent containing 1–2.5% Cu sulfate neutralized with

hydrate lime [50,51]. Common findings (obtained by alveolar lavage and

biopsy) include intraalveolar desquamation of macrophages, formation of
histiocytic and noncaseating granulomas containing inclusions of Cu, and
healing of lesions in the form of fibro-hyaline nodules, very similar to those
found in silicosis [52].
5.2. Copper and Cardiovascular Effects

No studies are available regarding cardiovascular effects in humans or
animals following inhalation exposure to Cu. Case reports of acute Cu
sulfate poisoning have indicated the occurrence of acute hemolysis [53].
Hemolysis has been reported after application of solutions of Cu salts to
large areas of burned skin [54] or after the introduction of Cu from Cu-
containing semipermeable membranes and Cu tubing into the circulation
during hemodialysis [55].
6. CADMIUM
Cadmium is a naturally occurring element, one of the metallic com-
ponents in the earth’s crust and oceans, and present everywhere in our
environment. The most common oxidation state of Cd is +2, though rare
examples of +1 can be found. Cd has no constructive purpose in the human
body.
6.1. Cadmium and Pulmonary Effects

In humans, inhalation exposure to high levels of Cd oxide fumes or dust is
intensely irritating to respiratory tissue, but symptoms can be delayed.
During and immediately after (up to 2 hours) an acute exposure for 5 hours
to 8.63 mg/m3, Beton et al. [56] reported that there were few symptoms,
limited to coughing and slight irritation of the throat and mucosa. From 4 to
10 hours post exposure, influenza-like symptoms began to appear, including
cough, tight chest, pain in the chest on coughing, dyspnea, malaise, ache,
chilling, sweating, shivering, and aching pain in the back and limbs. From 8
hours to 7 days post exposure, more advanced stages of pulmonary response
included severe dyspnea and wheezing, chest pain and precordial constric-
tion, persistent cough, weakness and malaise, anorexia, nausea, diarrhea,
nicturia, abdominal pain, hemoptysis, and prostration.

Longer-term occupational exposure to levels of Cd below those causing

lung inflammation, however, have been reported to cause emphysema in
humans [57]. A significant, dose-dependent excess in the ratio of observed to
expected deaths from bronchitis was found among 6.995 men occupationally
exposed to Cd for an average of 11 years [58]. There is some evidence that Cd
may accelerate the development of emphysema in smokers [59].
Recent studies, that were controlled for smoking, report lung impairment in
Cd-exposed workers [60,61]. Cortona et al. [61] measured respiratory function
parameters in 69 smoking and nonsmoking male subjects who were exposed
to concentrations of 0.008–1.53 mg/m3 of Cd fumes over a period of several
years in a factory that produced Cd alloys. The study found that there were
no significant differences in the lung function tests between workers exposed
to Cd fumes and non-exposed individuals. There was a significant increase in
residual volume of 48% in exposed workers; this effect was notably greater
in those with higher cumulative exposures to Cd. It is uncertain how much of
a factor on the increased residual volume was due to the tendency of smokers
to develop an initial emphysematous alteration in lung tissue due to smoking.
However, owing to the presence of Cd in tobacco smoke, controlling for
smoking habits may lead to underestimation of the actual impact of Cd and
of the Cd-smoking interaction on the respiratory tract.
Davison et al. [62] evaluated the lung function in 101 men who had manu-
factured Cu-Cd alloy in a plant in England for Z1 year since 1926. The exposed
men were compared to controls from the factory’s other seven divisions mat-
ched for age and employment status. Smoking in exposed and control men was
similar. The lung function of 77 of the men occupationally exposed to Cd was
significantly impaired compared to the unexposed controls, with the greatest
abnormalities in the highest-dose group. Forced expiratory volume in one sec-
ond, ratio of forced expiratory volume to forced vital capacity, transfer factor,
or transfer coefficient were significantly lower than expected and radiographic
total lung capacity, residual volume, and the ratio of these two were significantly
higher than expected. The greatest abnormalities were observed in workers with
the highest cumulative exposure and the highest liver Cd levels.
Smith et al. [63] studied the pulmonary function of 17 high-exposure
workers, 12 low-exposure workers, and 17 controls. Workers with high
exposure to Cd had significantly decreased forced vital capacity compared to
low-exposure workers and controls. Chest X-rays indicated mild or mod-
erate interstitial fibrosis in 29% of high-exposure workers. A dose-response
relationship was found between forced vital capacity and urinary Cd, and
with months of exposure to Cd fumes, but not Cd sulfate aerosols. In an
analysis of the smoking habits, there was no significant difference between
the two Cd-exposed groups with respect to the proportion of present or past
cigarette smokers, the intensity or duration of cigarette smoking, or cigar or
pipe smoking habits. The control subjects, however, had a significantly

higher exposure to cigarette smoke than the Cd-exposed workers, with

substantially greater numbers of pack-years (which is calculated by multi-
plying the number of packs of cigarettes smoked per day by the number of
years the person has smoked), cigarettes smoked per day, and years smoked.
A step-down and multiple regression analysis with a dependent variable of
forced vital capacity and the independent variables, age-height, cigarette
pack-years, and urinary cadmium, resulted in no indication that an inter-
action between the independent variables led to the observed relationship
between forced vital capacity and Cd excretion.
Other studies, however, have not shown a Cd-related increase in impaired
respiratory function. Edling et al. [64] studied Swedish workers occupation-
ally exposed to Cd oxide fume from Cd-containing solders. The results from
the lung function analysis showed no significant difference in symptoms or
lung function between the Cd-exposed and the reference group. The exposed
and the reference groups were similar with respect to sex, age, and height.
6.2. Cadmium and Cardiovascular Effects

Inhalation exposure to Cd does not appear to have significant effects on the
cardiovascular system. Most studies of workers occupationally exposed to Cd
have not found Cd-related cardiovascular toxicity. In some studies, the mortality
from cardiovascular disease was lower in the Cd-exposed population. Kazantzis
et al. [65] studied the mortality in a cohort of 6.958 Cd-exposed male workers
with average occupational exposures of 12 years. This was a follow-up study to
the work of Armstrong and Kazantzis [58]. There was a significant deficit in
deaths from cerebrovascular disease among men occupationally exposed to Cd.
There was no significant excess risk from hypertensive or renal disease.
Smith et al. [66] studied 16 male high-exposure production workers and 11
male low-exposure office and supervisory workers for renal function. The
average duration of exposure was 25 years. High-exposure workers were
exposed to Cd oxide concentrations of 0.23–45.2 mg/m3 and Cd sulfide
concentrations of 0.04–1.27 mg/m3. No difference was found in hypertension
between high- and low-exposure workers, adjusted for age and weight or
cigarette smoking. One study found a statistically significant increase in
blood pressure in exposed workers compared to controls [67].
7. CHROMIUM
Cr exhibits a wide range of possible oxidation states. The most common

oxidation states of Cr are +2, +3, and +6, with +3 being the most stable,

+1, +4 and +5 are rare. Trivalent Cr in trace amounts influences sugar and
lipid metabolism in humans, and its deficiency is suspected to cause impaired
glucose tolerance and loss of weight. In contrast, hexavalent Cr is very toxic
and mutagenic when inhaled. Notably, Cr(III) is unable to cross cell mem-
branes, but is able to bind to DNA and proteins and to form DNA-protein
cross-links, whereas Cr(VI) can cross cell membranes through anion chan-
nels, but is unable to bind cell macromolecules. However, Cr(VI) taken up
by cells is rapidly reduced to Cr(III) thereby becoming reactive with either
critical targets (e.g., DNA) or with scavengers (e.g., glutathione).
7.1. Chromium and Pulmonary Effects

Numerous studies of workers chronically exposed to Cr(VI) compounds have
reported nasal septum perforation and other respiratory effects. Workers at
an electroplating facility exposed to 0.0001 0.0071 mg Cr(VI)/m3 for an
average of 26.9 months complained of excessive sneezing, rhinorrhea, and
epistaxis. Many of the workers had ulcerations and/or perforations of the
nasal mucosa [68]. A study based only on questionnaires, which were com-
pleted by 997 Cr platers and 1,117 controls, found a statistically significant
increase in the incidence of chronic rhinitis, rhinitis with bronchitis, and nasal
ulcers and perforations in workers exposed to Cr(VI) in the Cr plating
industry in 54 plants compared to the control population [69].
Increased incidences of nasal septum perforation, nasal septum ulcer, and
nasal obstruction were observed in workers at Cr electroplating facilities
exposed for a mean duration of 6.1 years, as compared to workers at zinc
electroplating facilities [70]. The Cr electroplating workers had 31.7 and 43.9
times greater risks of developing nasal septum ulcers or nasal perforations,
respectively, than the zinc workers. A significant relationship between
duration of exposure and the risk of nasal septum ulcers was also found; the
Cr electroplating workers with a work duration of 49 years had a risk 30.8
times higher than those with a work duration of o2 years. Statistically
significant decreases in vital capacity, forced vital capacity, and forced
expiratory volume in 1 second were also observed in the Cr workers.
Alterations in lung function were also reported in a study of 44 workers at 17
Cr electroplating facilities [71].
A study of respiratory effects, lung function, and changes in the nasal
mucosa in 43 chromium plating workers in Sweden exposed to Cr(VI)
reported respiratory effects at occupational exposure levels of 0.002 mg
chromium(VI)/m3 [72]. Signs and symptoms of adverse nasal effects were
observed and reported at mean exposure levels of 0.002–0.2 mg Cr(VI)/m3.
Nasal mucosal ulceration and septal perforation occurred in individuals

exposed at peak levels of 0.02–0.046 mg Cr(VI)/m3; nasal mucosal atrophy

and irritation occurred in individuals exposed at peak levels of 0.0025–
0.011 mg Cr(VI)/m3. Workers exposed to mean concentrations of 0.002–
0.02 mg Cr(VI)/m3 had slight, transient decreases in forced vital capacity,
forced expired volume in 1 second, and forced mid-expiratory flow during
the workday. Workers exposed to o0.002 mg Cr(VI)/m3 showed no effects
on lung function.
Medical records of 2.307 male workers (all nonsmokers) employed at a
chromate production plant in Baltimore (Maryland) between 1950 and 1974
were evaluated to determine the percentage of workers reporting clinical
symptoms, mean time of employment to first diagnosis of symptoms, and
mean exposure to Cr(VI) at the time of first diagnosis. The most frequently
reported clinical symptoms were irritation and ulcerated nasal septum,
occurring in 68.1 and 62.9% of the cohort, respectively [73].
In a study of 54 male miners in Zimbabwe exposed to chromite ore dust,
decreases in pulmonary function was observed compared to an unexposed
control population [74]. In this same study, no changes in lung function were
observed in a group of 46 male miners working for a company following
industrial hygiene procedures.
In a study of stainless steel workers, no increase was observed in the
incidence of nasal ulceration or nasal symptoms in exposed workers com-
pared to a control population [75]. However, although an exposure-related
increase in the incidence of clinical signs of nasal irritation was not observed,
anterior rhinoscopy revealed a slight increase in the incidence of inflam-
matory changes in the nasal mucosa of workers exposed to Cr(VI) compared
to controls.
Numerous epidemiological studies have been performed on workers
exposed to Cr(VI) to determine its carcinogenicity [76,77]. Major studies
allocating Cr(VI) as a human lung carcinogen have been performed on
workers involved in chromate production, chromate pigment production,
and Cr plating [78–82]. These studies indicate occupational exposure to Cr
as an important risk factor of lung cancer.
7.2. Chromium and Cardiovascular Effects

Information regarding cardiovascular effects in humans after inhalation
exposure to Cr and its compounds is limited. No excess deaths were
observed from cardiovascular diseases and ischemic heart disease in a cohort
of 4.227 stainless steel production workers from 1968 to 1984 when com-
pared to expected deaths based on national rates and matched for age and
sex [80]. In a cohort of 3.408 individuals who had worked in four facilities

that produced Cr compounds from chromite ore in northern New Jersey

some time between 1937 and 1971, where the exposure durations of workers
ranged from o1 to 420 years, no increases in atherosclerotic heart disease
were evident [79].
Cardiovascular function was studied in 230 middle-aged workers involved
in potassium dichromate production who had clinical manifestations of Cr
poisoning (96 with respiratory effects and 134 with gastrointestinal disorders)
and in a control group of 70 healthy workers of similar age. Both groups with
clinical manifestations had changes in the bioelectric and mechanical activity
of the myocardium as determined by electrocardiography, kinetocardio-
graphy, rheocardiography, and ballistocardiography. These changes were
more pronounced in the workers with respiratory disorders due to Cr
exposure than in the workers with Cr-induced gastrointestinal effects. The
changes in the myocardium could be secondary to pulmonary effects and/or
to a direct effect on the blood vessels and myocardium [83].
8. COBALT
Cobalt is an essential metal in several coenzymes and enzymes. Dysfunction

in enzymatic processes, e.g. megalocytic anemia, may, therefore, result from
Co deficiency. The biological activity and toxicity of some metallic elements
is also greatly influenced by their ability to change their redox state by loss or
gain of electrons. Co occurs, next to the metallic state, mainly in the +2 and
+3 oxidation state.
8.1. Cobalt and Pulmonary Effects

Cobalt causes asthma with evidence of the formation of specific IgE anti-
bodies, suggesting that asthmatic reaction to the metal involves an IgE-
mediated response. Among workers exposed to hard metal containing either
Co or nickel as a binding matrix, Co-induced bronchial asthma and nickel-
induced asthma are prevalent [84]. The condition known as hard metal lung
disease has been the subject of renewed interest in recent years, particularly
with regard to the causative role of Co [85]. Hard metal or cemented
tungsten carbide is found in tools used for high-speed cutting, drilling,
grinding and polishing of other metallic elements or hard materials. On the
basis of relatively crude animal data [84] showing little toxicity from the
main constituent of hard metal, i.e., tungsten carbide, the consensus is that
tungsten carbide is not the agent responsible for the fibrosis, but that it is

more probably due to the binding agent, i.e., Co [86–88]. The pneumonitis is
often of the desquamative type, and in the subacute forms it appears to be
mainly characterized by the presence of multinucleated giant cells. Indeed it
has been proposed that giant cell interstitial pneumonitis may be pathog-
nomonic for hard metal exposure [88].
8.2. Cobalt and Cardiovascular Effects

Subsequent to the reports of cardiac failure in drinkers of beer in which Co
was used as an additive, a large number of animal studies have shown that
repeated intramuscular injection of Co in the order of 5 mg/kg to rats pro-
duced myocardial degeneration. According to Kerfoot et al. [89], long-term
inhalation exposure to Co produces electrocardiogram changes in miniature
swine.
Endemic outbreaks of cardiomyopathy with mortality rates up to 50%
have been described among heavy beer drinkers, who consumed beer con-
taining high Co concentrations. Common findings were heart failure,
polycythemia, and thyroid lesions [89,90].
Cases of myocardial toxicity have been associated with industrial exposure
[91]. In a group of 30 hard-metal workers, reduced right ventricular con-
tractility was associated with radiographic pulmonary involvement, sug-
gesting occult cor pulmonale in some of these workers. A subtle, but
significant, inverse relationship between duration of exposure and left ven-
tricular function assessed by radionuclide ventriculography was, however,
also reported [92]. In workers from a Co production plant exposed to var-
ious Co compounds, Doppler echocardiography revealed an association
between cumulative Co exposure and both left ventricular isovolumetric
relaxation time and deceleration time of the velocity of the early rapid filling
wave, indicating altered left ventricular diastole [93,94].
9. LEAD
Lead exists in three oxidation states: Pb(0), Pb(II), and Pb(IV). In the
environment, Pb primarily exists as Pb(II). Pb(IV) is only formed under
extremely oxidizing conditions and inorganic Pb(IV) compounds are not
found under ordinary environmental conditions, while organic compounds
are usually formed with lead at the tetravalent (+4) oxidation state. Metallic
Pb, Pb(0), exists in nature, but its occurrence is rare. There is no known
physiological role for Pb in humans.

9.1. Lead and Pulmonary Effects

Very limited information is available regarding respiratory effects in humans
associated with Pb exposure. A study of 62 male Pb workers in Turkey
reported significant alterations in pulmonary function tests among workers
compared to control subjects [95]. The cohort consisted of 22 battery
workers, 40 exhaust workers, and 24 hospital workers with current blood Pb
levels of 37, 27, and 15 mg/dL, respectively.
9.2. Lead and Cardiovascular Effects

The hypertensive effects of Pb have been extensively documented both in
experimental animals and in workers chronically exposed to high Pb levels
[96]. The association between blood Pb and elevated blood pressure has been
identified not only in cross-sectional, but also in prospective studies that
showed that new cases of hypertension and within-person elevations in blood
pressure levels over follow-up were related to baseline Pb exposure [97,98].
Some studies have demonstrated a dose–response relationship between Pb
exposure and blood pressure [99,100] However, the shape of the dose–
response relationship is not completely characterized, particularly at low
levels of exposure. It is not known what is the lowest level of Pb exposure not
associated with increased blood pressure, although in the available studies
there seems to be no evidence of a threshold effect [101].
Numerous experimental studies in animals have shown irrefutable evi-
dence that chronic exposure to low Pb levels results in arterial hypertension
that persists long after the cessation of Pb exposure. The precise mechanisms
explaining a hypertensive effect of low chronic exposure to environmental
Pb are unknown. An inverse association between estimated glomerular fil-
tration rate and blood Pb has been observed at blood Pb levels o5 mg/dL in
general population studies [102,103], indicating that Pb-induced reductions
in renal function could play a major role in hypertension. Other potential
mechanisms include enhanced oxidative stress [104,105], down-regulation of
nitric oxide [106], stimulation of the renin-angiotensin system [107] and
soluble guanylate cyclase [108,109]. These mechanisms could result in
increased vascular tone and peripheral vascular resistance.
Few cohort studies have evaluated the prospective association of Pb with
clinical cardiovascular outcomes in general population settings. The findings
of the National Health and Nutrition Examination Survey (NHANES) II
and NHANES III Mortality Follow-up studies are remarkable. NHANES
are periodic, standardized surveys designed to provide representative health
data from the U.S. noninstitutionalized population. Despite a marked

decline in Pb levels in U.S. adults, both surveys showed statistically sig-

nificant increases in cardiovascular mortality with increasing blood Pb [110].
In addition, a cross-sectional analysis of NHANES 1999–2002 data identi-
fied an association of blood Pb with the prevalence of peripheral arterial
disease [111]. The British Regional Heart Study [112] and two other small
cohort studies [113] showed positive but nonstatistically significant asso-
ciations of coronary heart disease or stroke incidence with higher Pb levels.
The associations of blood Pb with clinical cardiovascular end points in the
NHANES studies were moderately strong, with a clear dose–response gra-
dient. An unresolved issue is the impact of uncontrolled confounding and
measurement error on the relative risk estimates in studies of Pb and clinical
cardiovascular end points. NHANES studies are adjusted for race, educa-
tion, income, and urban versus rural location, which reduces potential
confounding by socioeconomic status. The validity of occupational studies
of Pb and cardiovascular mortality is limited by several methodologic pro-
blems. The comparison of exposed workers with the general population is
particularly inappropriate for cardiovascular mortality because workers are
healthier and their lifestyles and cardiovascular risk factors are likely to
differ widely from those of the general population. In addition, cardiovas-
cular diseases are associated with prolonged disability and changes in
employment status. Even in studies based on comparisons with unexposed
workers, the selection of healthier individuals at the time of hiring or for
specific jobs within an industry may have resulted in biased estimates of the
association. Correcting the bias introduced by the healthy worker survivor
effect is extremely challenging, and stratifying by duration of employment or
time since hire is unlikely to completely account for this source of bias [114].
Additional limitations include the assignment of Pb exposure based on job
titles and of cardiovascular deaths based on death certificates. Mis-
classification of exposure and outcome may have resulted in further
underestimation of the association of Pb and cardiovascular end points.
Finally, the lack of determinations of established cardiovascular risk factors
and of other occupational exposures may have contributed to uncontrolled
confounding. As a result of these methodological limitations, and despite
many occupational cohort studies published in the literature, available
information on occupational Pb exposure and cardiovascular mortality is
inadequate to infer the presence or absence of a causal relationship.
10. MANGANESE
Manganese is an essential trace nutrient in all forms of life. The most

common oxidation states of Mn are +2, +3, +4, +6, and +7, though

oxidation states from 3 to +7 are observed. Mn21 often competes with

Mg21 in biological systems. Mn(VII) compounds, which are mainly
restricted to the oxide Mn2O7 and compounds of the intensely purple per-
manganate anion MnO4 , are powerful oxidizing agents. Mn is a cofactor of
different enzymes such as oxidoreductases, transferases, hydrolases, lyases,
isomerases, ligases, lectins, and integrins.
10.1. Manganese and Pulmonary Effects

Inhalation exposure to high concentrations of Mn dusts (Mn dioxide
(MnO2) and tri-Mn tetroxide (Mn3O4), also known as Mn tetroxide)
can cause an inflammatory response in the lung (chemical pneumonitis).
But exposure to Mn in nonsoluble form, such as MnO2, may also be harm-
ful for the lung at low exposure levels. An increased morbidity and
mortality rate from pneumonia has been found among workers exposed to
Mn dusts [115].
Studies on populations living in the vicinity of Mn-emitting plants have
also indicated that Mn exposure might affect the respiratory system. In a
recent longitudinal follow-up study on pulmonary function and respiratory
symptoms in 145 miners exposed to Mn and 65 matched controls [116], the
obtained results indicated a synergistic effect of Mn and smoking in the
development of chronic respiratory impairment.
10.2. Manganese and Cardiovascular Effects

During an epidemiological study performed in ferroalloy workers, a
decrease in systolic blood pressure was found [117]. Arterial blood pressure
was measured and compared in three groups of male workers aged 20–59
years at different exposure levels to airborne Mn. The lowest mean values
of the systolic blood pressure were found in workers with the highest
occupational exposure, although this group was comparatively the
oldest. The lowest mean diastolic pressure values were found in control
workers.
Jiang and Zheng [118] studied the potential cardiotoxicity of MnO2
exposure in 656 workers engaged in Mn milling, smelting, and sintering. The
geometric mean of Mn in air was 0.13 mg/m3. Duration of exposure varied
from 0–35 years. There was no increase of abnormal electrocardiograms
between Mn workers and their matched controls. Arterial blood pressure
values showed a greater frequency of low diastolic pressure, but this effect
was highest in young workers with the lowest tenure in the plant.

11. NICKEL
Nickel is a metallic element that is naturally present in the earth’s crust. The
most common oxidation state of nickel is +2 with many Ni complexes
known. Due to unique physical and chemical properties, metallic Ni and its
compounds are widely used in modern industry. The high consumption of
nickel-containing products inevitably leads to environmental pollution by
nickel and its by-products at all stages of production, recycling, and dis-
posal. Ni is a cofactor of several enzymes and therefore should be considered
as an essential element for human physiology.
11.1. Nickel and Pulmonary Effects

A number of human studies have examined the potential of Ni and Ni
compounds to induce respiratory effects. Most of them were cohort mor-
tality studies in Ni-exposed workers. A significant excess of deaths from
nonmalignant respiratory system diseases was found among foundry
workers that was associated with the duration of foundry employment,
regardless of exposure to Ni [119]. Other studies of refinery workers or
workers exposed to Ni alloys have not found increases in deaths from
respiratory disease [120,121]. Two studies of welders also did not find sig-
nificant increases in the risk of nonmalignant respiratory disease deaths
[122,123].
A small number of studies have examined potential respiratory tract
effects, not associated with lethality. Reduced vital capacity and expiratory
flows were observed in stainless steel welders exposed to elevated levels of Ni
and Cr [124]. When the welders were divided into two groups based on
smoking status, only the forced expiratory volume was significantly different
from the referent population, suggesting that current smoking status may
have contributed to the observed effects. The study also found that the
prevalence of chronic bronchitis was higher in both the current smoker and
non-smoker groups, as compared to the referent population. Although this
study provides suggestive evidence of respiratory effects in welders, estab-
lishing a causal relationship between Ni and the observed effects is limited by
co-exposure to Cr and the lack of a comparison group of non-Ni-exposed
welders. Examination of chest radiographs of Ni sinter plant workers
exposed to Ni at concentrations as high as 100 mg/m3 did not reveal an
increase in small irregular opacities, which would be indicative of inflam-
matory or fibrogenic response in the lungs [125].
Another study found an increased risk of moderate pulmonary fibrosis,
after controlling for age and smoking, among Ni refinery workers with

cumulative exposure to soluble Ni or sulfidic Ni [126]. A dose–response

trend was also found for soluble Ni among cases in the three highest
cumulative exposure categories, after adjusting for age, smoking, and
exposure to asbestos.
Asthma induced by occupational exposure to Ni has been documented in
a small number of individuals [127,128], in whom asthma may result from
either primary irritation or an allergic response.
11.2. Nickel and Cardiovascular Effects

No increases in the number of deaths from cardiovascular diseases were
reported in workers exposed to Ni [129,130].
Ni(II) chloride induces coronary vasoconstriction in the dog heart in situ
and in isolated perfused rat heart [131,132]. A critical review of the available
literature suggests that cardiotoxicity or cardiovascular disease in humans is
not a recognized response or outcome [133]. Interestingly, in the above
mentioned incident whereby men were acutely exposed to Ni due to con-
sumption of contaminated drinking water at work, palpitations lasting up to
half a day constituted the extent of heart-related symptoms [134,135].
12. ZINC
Zinc is found in the earth’s surface rocks. Because of its reactivity, Zn metal
is not found as the free element in nature. Zn has two common oxidation
states, Zn(0) and Zn(II). Zn forms a variety of different compounds, such as
Zn chloride, Zn oxide, and Zn sulfate. For humans and animals Zn is an
essential nutrient that plays a role in membrane stability, in over 300
enzymes, and in the metabolism of proteins and nucleic acids.
12.1. Zinc and Pulmonary Effects

Metal fume fever, a well-documented acute disease induced by intense
inhalation of metal oxides, especially Zn, impairs pulmonary function but
does not progress to chronic lung disease [136]. The most prominent
respiratory effects of metal fume fever are substernal chest pain, cough, and
dyspnea [137]. The impairment of pulmonary function is characterized by
reduced lung volumes and a decreased diffusing capacity of carbon mon-
oxide [138,139]. The respiratory effects have been shown to be accompanied

by an increase in bronchiolar leukocytes [140]. The respiratory symptoms

generally disappear in the exposed individual within 1–4 days [141]. Inha-
lation of Zn oxide is most likely to occur in occupational situations where Zn
smelting or welding take place. Ultrafine Zn oxide particles originate from
heating Zn beyond its boiling point in an oxidizing atmosphere. Upon
inhalation, these small particles reach the alveoli and cause inflammation
and tissue damage in the lung periphery [138,142].
A number of studies have measured exposure levels associated with metal
fume fever. Acute experimental exposures to lower concentrations of Zn
oxide and occupational exposures to similar concentrations did not produce
symptoms of metal fume fever [143]. In a single-blind experiment, exposure
of subjects to 3.9 mg Zn/m3 as Zn oxide resulted in sore throat and chest
tightness but no impairment of pulmonary function [140]. It is speculated
that subjects in other studies may have been less susceptible because of the
development of tolerance to Zn [141]. Kuschner et al. [142] exposed a group
of 14 volunteers to a single exposure of varying levels of Zn oxide fume for
15–120 minutes and evaluated the response by bronchoalveolar lavage.
Significant increases were reported in the number of polymorphonuclear
leukocytes and lymphocytes in bronchoalveolar lavage fluid, but not in the
number of macrophages.
12.2. Zinc and Cardiovascular Effects

Only limited information is available regarding cardiovascular effects in
animals following inhalation exposure to Zn. Routine gross and microscopic
examination of the hearts of rats and mice revealed no adverse effects 13
months after exposure to 121.7 mg Zn/m3 as Zn chloride smoke for 1 hour/
day, 5 days/week, for 20 weeks [143]. Similarly, no changes were observed in
the hearts of guinea pigs exposed to 119.3 mg Zn/m3 as Zn chloride smoke
for 1 hour/day, 5 days/week, for 20 weeks, and then observed for an addi-
tional 17 months [141].
13. CONCLUDING REMARKS
Environmental contamination and exposure to metals is a serious growing

problem throughout the world. Human exposure to metals has risen dra-
matically in the last 50 years as a result of an exponential increase in the use
of metals in industrial processes and products. It is possible that low-level
metals exposure contributes much more towards the causation of chronic
disease and impaired functioning than previously thought. However, much

about metals toxicity, such as the genetic factors that may render some
individuals especially vulnerable to metal toxicity, remains a subject of
intense investigation. Genetic differences between individuals can affect the
relative sensitivity of each individual to metal exposure. The complex
interplay between multiple genetic and environmental factors on the affected
genes can be important in determining this sensitivity. If we could identify
and characterize these metal responsive genes, we would increase our
understanding of human metal-induced disease susceptibility. Using the
newly developed methods in microarray technology, it is expected that we
can better understand these relationships.
In this chapter we did not address the pulmonary and cardiovascular
effects of some metals, such as iron, palladium, platinum, and titanium.
Information about these metals can be obtained, however, from detailed
reviews [144–147].
REFERENCES
1. R. Bast-Pettersen, P. A. Drablos, L. O. Goffeng, Y. Thomassen and C. G.
Torres, Am. J. Ind. Med., 1994, 25, 649–662.
2. M. Chan-Yeung, R. Wong, L. MacLean, F. Tan, M. Schulzer, D. Enarson,
A. Martin, R. Dennis and S. Grzybowski, Am. Rev. Respir. Dis., 1983, 127,
465–469.
3. K. Radon, D. Nowak and D. Szadkowski, Occup. Environ. Med., 1999, 56,
468–472.
4. B. G. Simonsson, A. Sjoberg, C. Rolf and B. Haeger-Aronsen, Eur. J. Respir.
Dis., 1985, 66, 105–118.
5. A. al-Masalkhi and S. P. Walton, J. Ky. Med. Assoc., 1994, 92, 59–61.
6. P. S. Burge, J. A. Scott and J. McCoach, Allergy, 2000, 55, 779–780.
7. T. Halatek, M. Trzcinka-Ochocka, W. Matczak and J. Gruchåa, Int. J. Occup.
Med. Environ. Health., 2006, 19, 211–223.
8. P. J. Jederlinic, J. L. Abraham, A. Churg, J. S. Himmelstein, G. R. Epler and
E. A. Gaensler, Am. Rev. Respir. Dis., 1990, 142, 1179–1184.
9. R. R. Miller, A. M. Churg, M. Hutcheon and S. Lom, Am. Rev. Respir. Dis.,
1984, 130, 312–315.
10. C. Abbate, C. Giorgianni, R. Brecciaroli, M. A. Tringali and G. D’Arrigo, Am.
J. Ind. Med., 2003, 44, 400–404.
11. A. Herbert, G. Sterling, J. Abraham and B. Corrin, Hum. Pathol., 1982,
13, 694–699.
12. M. J. Hull and J. L. Abraham, Hum. Pathol., 2002, 33, 819–825.
13. O. Vandenplas, J. P. Delwiche, M. L. Vanbilsen, J. Joly and D. Roosels, Eur.
Respir. J., 1998, 11, 1182–1184.
14. A. W. Musk, N. H. de Klerk, J. R. Beach, L. Fritschi, M. R. Sim, G. Benke,
M. Abramson and J. J. McNeil, Occup. Environ. Med., 2000, 57, 279–283.

15. L. Fritschi, J. Beach, M. Sim, M. Abramson, G. Benke, A. W. Musk, N. de

Klerk and J. McNeil, Am. J. Ind. Med., 1999, 35, 491–498.
16. M. J. Abramson, J. H. Wlodarczyk, N. A. Saunders and M. J. Hensley, Am.
Rev. Respir. Dis., 1989, 139, 1042–1057.
17. P. S. Burge, J. A. Scott and J. McCoach, Allergy, 2000, 55, 779–780.
18. O. Vandenplas, J. P. Delwiche, M. L. Vanbilsen, J. Joly and D. Roosels, Eur.
Respir. J., 1998, 11, 1182–1184.
19. P. De Vuyst, P. Dumortier, F. Rickaert, R. Van de Weyer, C. Lenclud and
J. C. Yernault, Eur. J. Respir. Dis., 1986, 68, 131–140.
20. E. Gaffuri, A. Donna, R. Pietra and E. Sabbioni, Med. Lav., 1985, 76, 222–227.
21. B. Gilks and A. Churg, Am. Rev. Respir. Dis., 1987, 136, 176–179.
22. V. Vallyathan, W. M. Bergeron, P. A. Robichaux and J. E. Craighead, Chest,
1982, 81, 372–374.
23. S. Han, U. Sakinci, S. K. Kose and R. Yazkan, Interact. Cardiovasc. Thorac.
Surg., 2004, 3, 79–82.
24. M. Akira, Radiology, 1995, 197, 403–409.
25. P. De Vuyst, P. Dumortier, L. Schanden, M. Estenne, A. Verhest and
J. C. Yernault, Am. Rev. Respir. Dis., 1987, 135, 493–497.
26. A. Rönneberg, Occup. Environ. Med., 1995, 52, 255–261.
27. G. P. Theriault, C. G. Tremblay and B. G. Armstrong, Am. J. Ind. Med., 1988,
6, 659–666.
28. P. Romundstad, A. Andersen and T. Haldorsen, Scand. J. Work. Environ.
Health, 2000, 26, 470–475.
29. V. Khurana, I. S. Gambhir and D. Kishore, Indian J. Med. Sci., 2009, 63, 257–259.
30. S. Tapio and B. Grosche, Mutat. Res., 2006, 612, 215–246.
31. I. Hertz-Picciotto, A. H. Smith, D. Holtzman, M. Lipsett and G. Alexeeff,
Epidemiology, 1992, 3, 23–31.
32. P. E. Enterline, V. L. Henderson and G. M. Marsh, Am. J. Epidemiol., 1987,
125, 929–938.
33. L. Jarup and G. Pershagen, Am. J. Epidemiol., 1991, 134, 545–551.
34. M. Adonis, V. Martinez, P. Maran, D. Berrios and L. Gil, Toxicol. Lett., 2005,
159, 32–37.
35. H. Y. Chiou, S. T. Chiou, Y. H. Hsu, Y. L. Chou, C. H. Tseng, M. L. Wei and
C. J. Chen, Am. J. Epidemiol., 2001, 153, 411–418.
36. C. Ferreccio, C. Gonzalez, V. Milosavjlevic, G. Marshall, A. M. Sancha and
A. H. Smith, Epidemiology, 2000, 11, 673–679.
37. B. Nemery, Eur. Respir. J., 1990, 3, 202–219.
38. I. Mordukhovich, R. O. Wright, C. Amarasiriwardena, E. Baja, A. Baccarelli,
H. Suh, D. Sparrow, P. Vokonas and J. Schwartz, Am. J. Epidemiol., 2009, 170,
739–746.
39. G. P. Zaloga, J. Deal, T. Spurling, J. Richter and B. Chernow, Am. J. Med. Sci.,
1985, 289, 210–214.
40. C. J. Chen, M. M. Wu, S. S. Lee, J. D. Wang, S. H. Cheng and H. Y. Wu,
Arteriosclerosis, 1988, 8, 452–460.
41. R. R. Engel, C. Hopenhayn-Rich, O. Receveur and A. H. Smith, Epidemiol.
Rev., 1994, 16, 184–209.

42. M. M. Wu, T. L. Kuo, Y. H. Hwang and C. J. Chen, Am. J. Epidemiol., 1989,

130, 1123–1132.
43. K. Kreiss, G. A. Day and C. R. Schuler, Annu. Rev. Public. Health, 2007,
28, 259–277.
44. L. H. Santo Tomas, Curr. Opin. Pulm. Med., 2009, 15, 165–169.
45. A. P. Fontenot and M. Amicosante, Semin. Respir. Crit. Care Med., 2008,
29, 662–669.
46. J. K. Wagoner, P. F. Infante and D. L. Bayliss, Environ. Res., 1980, 21, 15–34.
47. P. Wild, E. Bourgkard and C. Paris, Methods Mol. Biol., 2009, 472, 139–167.
48. G. Togna, A. R. Togna, P. Russo and L. Caprino, Toxicol. Appl. Pharmacol.,
1997, 144, 262–267.
49. I. Suciu, L. Prodan, V. Lazar, E. Ilea, A. Cocverla, L. Olinici, A. Paduraru, O.
Zagreanu, P. Lengyel, L. Gurffi and D. Andru, Med. Lav., 1981, 72, 190–197.
50. P. Stark, J. Can. Assoc. Radiol., 1981, 32, 183–184.
51. T. G. Villar and T. Nogueira, Bronchopneumologie, 1980, 30, 61–67.
52. P. Plamenac, Z. Santić, A. Nikulin and H. Serdarević, Eur. J. Respir. Dis., 1985,
67, 50–55.
53. D. Bhowmik, R. Mathur, Y. Bhargava, A. K. Dinda, S. K. Agarwal, S. C.
Tiwari and S. C. Dash, Ren. Fail., 2001, 23, 731–735.
54. N. A. Holtzman, D. A. Elliott and R. H. Heller, N. Engl. J. Med., 1966, 275,
347–352.
55. W. J. Klein, E. N. Metz and A. R. Price, Arch. Intern. Med., 1972, 129, 578–582.
56. D. C. Beton, G. S. Andrews, H. J. Davies, L. Howells and G. F. Smith, Br. J.
Ind. Med., 1966, 23, 292–301.
57. B. J. Lampe, S. K. Park, T. Robins, B. Mukherjee, A. A. Litonjua, C. Amar-
asiriwardena, M. Weisskopf, D. Sparrow and H. Hu, Environ. Health Perspect.,
2008, 116, 1226–1230.
58. B. G. Armstrong and G. Kazantzis, Lancet, 1983, 25, 1425–1427.
59. D. Leduc, P. de Francquen, D. Jacobovitz, R. Vandeweyer, R. Lauwerys and
P. De Vuyst, Thorax, 1993, 48, 570–571.
60. O. Y. Chan, S. C. Poh, H. S. Lee, K. T. Tan and S. F. Kwok, Ann. Acad. Med.
Singapore, 1988, 17, 283–287.
61. G. Cortona, P. Apostoli, F. Toffoletto, A. Baldasseroni, I. Ghezzi, E. Goggi,
S. Fornari and L. Alessio, I.A.R.C. Sci. Publ., 1992, 118, 205–210.
62. A. G. Davison, P. M. Fayers, A. J. Taylor, K. M. Venables, J. Darbyshire, C. A.
Pickering, D. R. Chettle, D. Franklin, C. J. Guthrie and M. C. Scott, Lancet,
1988, 8587, 663–667.
63. T. J. Smith, T. L. Petty, J. C. Reading and A. Lakshminarayan, Am. Rev.
Respir. Dis., 1976, 114, 161–169.
64. C. Edling, C. G. Elinder and E. Randma, Br. J. Ind. Med., 1986, 43, 657–662.
65. G. Kazantzis, T. H. Lam and K. R. Sullivan, Scand. J. Work Environ. Health,
1988, 14, 220–223.
66. T. J. Smith, R. J. Anderson and J. C. Reading, Am. J. Ind. Med., 1980,
1, 319–337.
67. M. J. Thun, A. M. Osorio, S. Schober, W. H. Hannon, B. Lewis and W. Halperin,
Br. J. Ind. Med., 1989, 46, 689–697.

68. N. Williams, Occup. Med. (Lond.)., 1998, 48, 135–137.

69. H. Royle, Environ. Res., 1975, 10, 39–53.
70. H. W. Kuo, J. S. Lai and T. I. Lin, Int. Arch. Occup. Environ. Health, 1997, 70,
272–276.
71. P. Bovet, M. Lob and M. Grandjean, Int. Arch. Occup. Environ. Health, 1977,
40, 25–32.
72. E. Lindberg and G. Hedenstierna, Arch. Environ. Health, 1983, 38, 367–374.
73. H. J. Gibb, P. S. Lees, P. F. Pinsky and B. C. Rooney, Am. J. Ind. Med., 2000,
38, 127–31.
74. E. E. Osim, M. Tandayi, H. M. Chinyanga, H. T. Matarira, K. K. Mudambo
and C. T. Musabayane, Trop. Med. Int. Health, 1999, 4, 621–628.
75. M. Huvinen, J. Uitti, P. Oksa, P. Palmroos and P. Laippala, Occup. Med.
[Lond.], 2002, 52, 203–212.
76. P. Wild, E. Bourgkard and C. Paris, Methods Mol. Biol., 2009, 472, 139–167.
77. I. Bruske-Hohlfeld, Methods Mol. Biol., 2009, 472, 3–23.
78. J. J. Moulin, P. Wild, J. M. Haguenoer, D. Faucon, R. De Gaudemaris, J. M.
Mur, M. Mereau, Y. Gary, J. P. Toamain, Y. Birembaut, M. Blanc, M. P.
Debiolles, D. Jegaden, B. Laterriere, M. Leonard, F. Marini, C. Massardier,
M. Moulin, M. Reure, L. Rigal, G. Robert and M. Viossat, Br. J. Ind. Med.,
1993, 50, 234–243.
79. K. D. Rosenman and M. Stanbury, Am. J. Ind. Med., 1996, 29, 491–500.
80. J. J. Moulin, P. Wild, B. Mantout, M. Fournier-Betz, J. M. Mur and
G. Smagghe, Cancer Causes Control, 1993, 4, 75–81.
81. I. Franchini, F. Magnani and A. Mutti, Scand. J. Work Environ. Health, 1983,
9, 247–252.
82. T. Hara, T. Hoshuyama, K. Takahashi, V. Delgermaa and T. Sorahan, Scand.
J. Work Environ. Health, 2009, Dec 21. [Epub ahead of print].
83. A. M. Klefener, B. M. Stolbun, E. I. Likhacheva and L. N. Beliaeva, Gig. Tr.
Prof. Zabol., 1970, 14, 7–10.
84. D. G. Barceloux, J. Toxicol. Clin. Toxicol., 1999, 37, 201–206.
85. G. L. Finch, M. D. Hoover, F. F. Hahn, K. J. Nikula, S. A. Belinsky, P. J.
Haley and W. C. Griffith, Environ. Health Perspect., 1996, 104, 973–979.
86. M. Goldoni, S. Catalani, G. De Palma, P. Manini, O. Acampa, M. Corradi,
R. Bergonzi, P. Apostoli and A. Mutti, Environ. Health Perspect., 2004,
112, 1293–1298.
87. D. Lison, Toxicol. Appl. Pharmacol., 2000, 15, 173–174.
88. G. Chiappino, Sci. Total. Environ., 1994, 150, 65–68.
89. E. J. Kerfoot, W. G. Fredrick and E. Domeier, Am. Ind. Hyg. Assoc. J., 1975,
36, 17–25.
90. C. S. Alexander, Am. J. Med., 1972, 53, 395–417.
91. Y. Morin and P. Daniel, Can. Med. Assoc. J., 1967, 97, 926–928.
92. S. Alusek, J. Cernohorski and M. Barborik, Vnitr. Lek., 1982, 28, 493–496.
93. S. F. Horowitz, A. Fischbein, D. Matza, J. N. Rizzo, A. Stern, J. Machac and
S. I. Solomon, Br. J. Ind. Med., 1988, 45, 742–746.
94. A. Linna, P. Oksa, K. Groundstroem, M. Halkosaari, P. Palmroos, S. Huikko
and J. Uitti, Occup. Environ. Med., 2004, 61, 877–885.

95. C. Bagci, A. I. Bozkurt, E. A. Cakmak, S. Can and B. Cengiz, Int. J. Clin.

Pract., 2004, 58, 568–572.
96. http://www.atsdr.cdc.gov/csem/lead/docs/lead.pdf
97. B. S. Glenn, W. F. Stewart, J. M. Links, A. C. Todd and B. S. Schwartz,
Epidemiology, 2003, 14, 30–36.
98. L. Muller and T. S. Kristensen, Am. J. Epidemiol., 1992, 136, 1091–1100.
99. S. J. Pocock, A. G. Shaper, D. Ashby, T. Delves and T. P. Whitehead, Br. Med.
J., 1984, 289, 872–874.
100. J. Schwartz, Environ. Health Perspect., 1988, 78, 15–22.
101. I. Hertz-Picciotto and J. Croft, Epidemiol. Rev., 1993, 15, 352–73.
102. http://www.epa.gov/lead/
103. E. B. Ekong, B. G. Jaar and V. M. Weaver, Kidney Int., 2006, 70, 2074–2084.
104. C. S. Riedt, B. T. Buckley, R. E. Brolin, H. Ambia-Sobhan, G. C. Rhoads and
S. A. Shapses, J. Expo. Sci. Environ. Epidemiol., 2009, 19, 90–96.
105. S. J. Stohs and D. Bagchi, Free Radic. Biol. Med., 1995, 18, 321–336.
106. N. D. Vaziri and Y. Ding, Hypertension, 2001, 37, 223–226.
107. M. Carmignani, P. Boscolo, A. Poma and A. R. Volpe, Immunopharmacology,
1999, 44, 105–110.
108. N. Dursun, C. Arifoglu, C. Swer and L. Keskinol, Biol. Trace Elem. Res., 2005,
104, 141–149.
109. F. Farmand, A. Ehdaie, C. K. Roberts and R. K. Sindhu, Environ. Res., 2005,
98, 33–39.
110. S. E. Schober, L. B. Mirel, B. I. Graubard, D. I. Brody and K. M. Flegal,
Environ. Health Perspect., 2006, 114, 1538–1541.
111. P. Muntner, A. Menke, K. B. DeSalvo, F. A. Rabito and V. Batuman, Arch.
Intern. Med., 2005, 165, 2155–2161.
112. S. J. Pocock, A. G. Shaper, D. Ashby, H. T. Delves and B. E. Clayton, Environ.
Health Perspect., 1988, 78, 23–30.
113. D. Kromhout, Environ. Health Perspect., 1988, 78, 43–46.
114. H. M. Arrighi and I. Hertz-Picciotto, Epidemiology, 1994, 5, 189–196.
115. P. Derville, G. Morichaud-Beauchant, M. J. Charpentier and E. Derville, Arch.
Mal. Prof., 1966, 27, 222–224.
116. M. M. Boojar and F. Goodarzi, J. Occup. Environ. Med., 2002, 44, 282–290.
117. M. Sarić and O. Hrustić, Environ. Res., 1975, 10, 314–318.
118. Y. Jiang and W. Zheng, Cardiovasc. Toxicol., 2005, 5, 345–54.
119. R. G. Cornell and J. R. Landis, I.A.R.C. Sci. Publ., 1984, 53, 87–93.
120. V. C. Arena, J. P. Costantino, N. B. Sussman and C. K. Redmond, Am. J. Ind.
Med., 1999, 36, 14–21.
121. R. Egedahl, M. Carpenter and D. Lundell, Occup. Environ. Med., 2001, 58, 711–715.
122. H. S. Shannon, C. Walsh, N. Jadon, J. A. Julian, J. K. Weglo, P. G. Thornhill
and A. G. Cecutti, Toxicol. Ind. Health., 1991, 7, 277–294.
123. J. J. Moulin, T. Clavel, D. Roy, B. Dananchy, N. Marquis, J. Favotte and J. M.
Fontana, Int. Arch. Occup. Environ. Health, 2000, 73, 171–180.
124. A. P. Polednak, Arch. Environ. Health, 1981, 36, 235–242.
125. K. H. Kilburn, R. Warshaw, C. T. Boylen, J. C. Thornton, S. M. Hopfer, F. W.
Sunderman and J. Finklea, Am. J. Ind. Med., 1990, 17, 607–615.

126. D. C. Muir, J. Julian, N. Jadon, R. Roberts, J. Roos, J. Chan, W. Maehle and

W. K. Morgan, Br. J. Ind. Med., 1993, 50, 428–431.
127. S. R. Berge and K. Skyberg, J. Environ. Monit., 2003, 5, 681–688.
128. E. Nieboer, S. L. Evans and J. Dolovich, Br. J. Ind. Med., 1984, 41, 56–63.
129. T. Shirakawa, Y. Kusaka, N. Fujimura, M. Kato, S. Heki and K. Morimoto,
Thorax, 1990, 45, 267–271.
130. J. E. Cox, R. Doll, W. A. Scott and S. Smith, Br. J. Ind. Med., 1981, 38,
235–239.
131. D. L. Cragle, D. R. Hollis, T. H. Newport and C. M. Shy, I.A.R.C. Sci. Publ.,
1984, 53, 57–63.
132. G. M. Rubanyi, L. Ligeti, A. Koller and A. G. Kovách, J. Mol. Cell. Cardiol.,
1984, 16, 533–546.
133. Y. Edoute, P. M. Vanhoutte and G. M. Rubanyi, Mechanisms of Nickel-Induced
Coronary Vasoconstriction in Isolated Perfused Rat Hearts, in Advances in
Environmental Sciences and Technology, Vol. 25, Nickel and Human Health:
Current Perspectives, Ed. E. Nieboer and J. O. Nriagu, Wiley, New York, 1992,
pp 587–602.
134. E. Nieboer and C. G. Fletcher, Nickel Absorption, Toxicology and Carcino-
genesis, in Handbook of Metal-Ligand Interactions in Biological Tissues. Bioi-
norganic Medicine, Ed. G. Berthon, Marcel Dekker, New York,1995, pp. 412–
417, 709–715 and 1014–1019.
135. F. W. Sunderman, B. Dingle, S. M. Hopfer and T. Swift, Am. J. Ind. Med.,
1988, 14, 257–266.
136. J. L. Malo, J. Malo, A. Cartier and J. Dolovich, Eur. Respir. J., 1990, 3, 111–114.
137. L. C. Rohrs, Arch. Ind. Health, 1957, 16, 42–47.
138. C. Vogelmeier, G. Konig, K. Bencze and G. Fruhmann, Chest, 1987, 92,
946–948.
139. J. J. Brown, Br. J. Radiol., 1988, 61, 327–329.
140. H. Marquart, T. Smid, D. Heederik and M. Visschers, Am. J. Ind. Med., 1989,
16, 289–296.
141. T. Gordon, L. C. Chen, J. M. Fine, R. B. Schlesinger, W. Y. Su, T. A. Kimmel
and M. O. Amdur, Am. Ind. Hyg. Assoc. J., 1992, 53, 503–509.
142. W. G. Kuschner, A. D’Alessandro, S. F. Wintermeyer, H. Wong, H. A.
Boushey and P. D. Blanc, J. Investig. Med., 1995, 43, 371–378.
143. T. C. Marrs, H. F. Colgrave, J. A. Edginton, R. F. Brown and N. L. Cross,
Arch. Toxicol., 1988, 62, 123–32.
144. D. Galaris and K. Pantopoulos, Oxidative Stress and Iron Homeostasis:
Mechanistic and Health Aspects, Crit. Rev. Clin. Lab. Sci., 2008, 45, 1–23.
145. A. Dubiella-Jackowska, Z. Polkowska and J. Namienik, Platinum Group Ele-
ments in the Environment: Emissions and Exposure, Rev. Environ. Contam.
Toxicol., 2009, 199, 111–135.
146. J. Kielhorn, C. Melber, D. Keller and I. Mangelsdorf, Palladium. A Review of
Exposure and Effects to Human Health, Int. J. Hyg. Environ. Health, 2002, 205,
417–432.
147. P. J. Borm, R. P. Schins and C. Albrecht, Inhaled Particles and Lung Cancer,
Part B: Paradigms and Risk Assessment, Int. J. Cancer, 2004, 110, 3–14.

Met. Ions Life Sci. 2011, 8, 107–132
5
Metal Ions Affecting the Gastrointestinal
System Including the Liver
Declan P. Naughton, Tamás Nepusz, and Andrea Petroczi
School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames,
Surrey, KT1 2EE, UK
<D.Naughton@kingston.ac.uk>
<T.Nepusz@kingston.ac.uk>
<A.Petroczi@kingston.ac.uk>
ABSTRACT 108
1. INTRODUCTION 108
2. EXPOSURE TO METAL IONS IN THE GASTROINTESTINAL
TRACT AND LIVER 110
2.1. Foodstuffs 110
2.2. Supplements 116
3. ESTIMATION OF TOXICITY ASSOCIATED WITH METAL
IONS IN THE GASTROINTESTINAL TRACT AND LIVER 117
3.1. Absorption and Accumulation of Metal Ions 117
3.2. Estimations of Safe Limits 118
3.3. Cumulative Effects 120
3.4. Metal Ion-Induced Toxicity 122
4. METAL ION-MOLECULAR INTERACTIONS: EFFECTS ON
OXIDATIVE DAMAGE 123
4.1. Introduction. Oxidative Damage in the Gastrointestinal Tract
and Liver 123
4.2. Molecular Mechanisms of Metal Ion-Induced Oxidative
Damage 124
4.3. Therapeutic Implications 126
5. CONCLUDING REMARKS AND FUTURE DIRECTIONS 127
ABBREVIATIONS 127
REFERENCES 128
108 NAUGHTON, NEPUSZ, and PETROCZI
ABSTRACT: In the present context, metal ions can be categorized into several classes
including those that are essential for life and those that have no known biological func-
tion and thus can be considered only as potentially hazardous. Many complexities arise
with regard to metal toxicity and there is a paucity of studies relating to many metals
which are frequent components of the diet. For many people ingestion of mineral sup-
plements is considered a risk-free health choice despite growing evidence to the
contrary.
Numerous approaches have been developed to assess risk associated with ingestion of
metal ions. These include straightforward estimation of safe limits such as oral reference
dose which are often based on data derived from animal experiments. More convoluted
approaches such as the Target Hazard Quotient involve assessment of hazard with fre-
quent exposure over long durations such as a lifetime. The latter calculation also affords
facile consideration of the effects of many metals together. In many cases, rigorous data
are unavailable, hence, large factors of uncertainty are employed to relate risk to humans.
Owing to the nature of metal toxicity, data pertaining to the gastrointestinal tract and
liver are often acquired from diseases of metal homeostasis or episodes of considerable
metal overload. Whilst these studies provide evidence for mechanisms of metal-induced
toxicity such as enhancing oxidative stress, extrapolation of these results to healthy
individuals or patients with chronic inflammatory diseases is not straightforward. In
summary, the diverse nature of metals and their effects on human tissues along with a
paucity of studies on the full range of their effects, warrant further in-depth studies on the
association of metals to ageing, chronic inflammatory diseases, and cancer.
KEYWORDS: chelation . contamination . foodstuffs . gastrointestinal tract . health

hazard . heavy metals . liver . oxidative damage . seafoods . supplements
1. INTRODUCTION
As noted throughout this volume, many metal ions have toxic effects in
humans and these are often dealt with in a dose-dependent manner. Studies
of metal toxicity involve considerations of exposure, acute damage, residual
or accumulated effects and, of course, detoxification. Clearly, the exposure
and uptake of metal ions are key issues when exploring toxicity. In this
respect, the gastrointestinal (GI) tract is particularly susceptible to ingested
substances, especially through the diet. Thus, a considerable part of this
chapter is focused on the toxic ramifications of metals found in foodstuffs.
Of necessity, the type and levels of metals covered will be dictated by this
dietary focus which includes food, drinks, and supplements.
In many countries, major efforts have been made to establish and
implement regulatory bodies to protect citizens from food-borne toxins
including metal ions. For example, in the European Union (EU), the Rapid
Alert System for Food and Feed (RASFF) supports the activities of
member states in controlling food safety and security [1]. Whilst analysis of
the patterns of food notifications for metal contamination provides
Met. Ions Life Sci. 2011, 8, 107–132

METAL IONS AFFECTING THE GASTROINTESTINAL SYSTEM 109
confidence for food consumers, it is notable that many countries do not

benefit from rigorous food testing regimes [2]. Thus, the majority of the
world’s population are inadequately protected against foods contaminated
with metals.
Along with continued efforts to monitor food contamination, many
studies are focused on the estimation of safe intake levels of metals in the
diet [3]. These studies combine data from a large number of sources
including both animal and human studies and these data are often coupled
with many uncertainties. A number of approaches have been proposed to set
safe limits on ingested metals. These range from straight concentrations per
kg of foodstuff to complex equations accounting for food intake over a
lifetime. The highly complex nature of the investigations arises from the
inability to run clinical trial type studies on toxins in humans along
with a paucity of knowledge about lifelong exposure for up to a century.
The key to reliable risk assessment is an accurate estimate of exposure.
Regardless of the approach used to assess potential health hazards, the
exposure (both amount and time or frequency) is the ultimate factor that
determines whether the pre-set upper threshold of safe consumption is
exceeded or not. A further key aspect that is less often studied is the spe-
ciation of metals in a foodstuff (apart from a small number of compounds
such as methylmercury) and consequently these will not be a key aspect of
this review. An additional complication arises from the uncertainties around
the intake of metals and the exact proportions that are absorbed within the
GI tract.
In addition to dietary derived metal ions, the consumption of various
mineral supplements, especially in the western world, is a widespread and
increasing source of metal intake for humans. A comprehensive report on
safe upper levels of vitamins and minerals was prepared under the auspices
of the UK Food Standards Agency by the Expert Group on Vitamins and
Minerals [3]. The report covers key aspects such as deficiency, absorption,
excretion, interactions, and toxicity for the key group of minerals and sup-
plements. For all cases further investigations are required to gain a com-
prehensive understanding of the short- and long-term effects of metal intake
on the GI tract and liver. The weight of evidence compiled to date points
towards detrimental effects arising from the intake of metal ions. However,
this is the case for many classes of essential dietary foodstuffs where intake is
necessary but associated with negative effects on health. Numerous studies
have demonstrated interactions between metal ions and biomolecules or
biological systems. For example, considerable evidence exists for redox-
active metal ions augmenting oxidative stress which is implicated in
inflammatory disorders, the initiation or progression of ageing and a wide
range of diseases including cancer, rheumatoid arthritis, diabetes, cardio-
vascular diseases, and neurodegenerative diseases [4].
Met. Ions Life Sci. 2011, 8, 107–132

A carefully controlled healthy tissue with metals ions packaged in proteins

for storage, transport or enzyme functions, can be subjected to significant
challenges by the introduction of labile heavy metals. Speciation is a key
aspect of detrimental contributions of metal ions in biological systems. As
intake and uptake of essential elements are affected by the species present [5],
it is to be expected that this aspect will influence toxicity, especially by
ingestion of metals in purified supplement form.
2. EXPOSURE TO METAL IONS IN THE

GASTROINTESTINAL TRACT AND LIVER
2.1. Foodstuffs
Considerable, yet insufficient advances have been made over the past decade
in relation to the requirements for nutritional supplements such as minerals
and vitamins. For some supplement categories, a paradigm shift has evolved,
owing to evidence that these seemingly benevolent chemicals have, in fact,
pathological ramifications either through the action of metal ions or via
activation of metals (for example by vitamin C) [3,4,6,7]. In addition to
clinical trial data showing links to disease for iron supplements and Par-
kinson’s disease [8,9], vitamins that formerly held the label of antioxidants
have more recently been referred to as electron transfer agents, owing to lack
of therapeutic efficacy in diseases characterized by oxidative stress. The term
electron transfer agent being applied to reflect the ability to trap a radical for
detoxification by another entity. However, some studies demonstrate
prooxidant activities for vitamins at high concentrations in model systems.
Furthermore, interactions between metal ions and vitamins can enhance
radical activity such as is found in Udenfriend’s system for vitamin C and
ferric ions [7].
Although many metals are required for normal biochemical functions,
many have no identified biochemical function and are associated with det-
rimental activities. In addition, dose considerations for those that have
established health functions need to be considered. Thus, the essential
requirements for vitamins and minerals lie between deficiency and over-
exposure and in contrast, many metals may have unknown detrimental
properties. The rationale behind a common unifying approach of identifying
and quantifying nutrients, xenobiotics, metal contaminants, and pollutants
is attractive. In practice, over the past decade numerous approaches
have been advocated which seldom have underlying commonalities of fun-
damental bases, gold standards, set standard levels, or research balances
between detrimental or beneficial dose responses. Indeed, a blanket
Met. Ions Life Sci. 2011, 8, 107–132

approach covering various chemical categories ranging from essential

nutrient trace metal ions to nonessential hazardous metal ions is likely to
add to the confusion rather than deconvolute and distill the key information.
Enhanced food safety requirements resulting from the globalization of
food supplies are being met in a regional approach which defies the con-
struction of a coherent rational worldwide solution. The Beijing Declaration
on Food Safety [10], instituted during the WHO Forum in 2007, was rapidly
adopted by over 50 countries. These signatory nations promised to
‘‘establish food safety authorities . . . within a comprehensive production-to-
consumption legislative framework’’ for the protection of their citizens.
Within the EU, regulation is implemented via member states and reported
centrally to form the RASFF notifications. The EU has established the
maximum levels (MLs) of metals in key food categories including fish pro-
ducts for Cd, Pb, and Hg [11]. Levels have yet to be established for As. This
approach serves the dual roles of (i) triggering a recall or border rejection for
foodstuffs with levels above the ML, and (ii) estimations of provisional
tolerable weekly intake (PTWI) of these metal ions through a regulated diet.
A recent analysis of the RASFF database over a 64 month period resulted
in a dataset of over 15,000 notifications with many having up to a dozen
variables such as food type, contaminant type, country of origin, producing
country, levels of contaminant, etc. [12]. It is noteworthy that metal con-
taminants stand out as growing in number each year within the RASFF
database indicating a continued and growing problem with global dietary
intake of toxic metals. These data can be represented using descriptive sta-
tistics but are of a complexity that advanced data mining tools are desirable.
Using descriptive statistics, Figure 1 depicts the complexity of food alert
data from the RASFF even after processing into selected categories for 61%
of notifications. The 39% uncategorized notifications include those for less
common reasons. These notifications, numbering over 15,000 during a 64
month period, are dominated by microorganisms (46500), mycotoxins
(44500), aflatoxins (44000) with heavy metals triggering some 1000 noti-
fications. Of the metals classified, 820 (85.7%) notifications were owing to
metals found in food. The remaining 135 notifications were for feed (13,
1.4%), migration of metals from plates, containers or utensils to food (81,
8.5%), and metal fragments found in food (41, 4.3%).
Network maps are based on the presence and strength of relationships
(also called ties, e.g., food alerts) between two nodes (e.g., countries). The
whole network can further be decomposed to reveal their inner structures
(e.g., clustered or layered). Algorithms utilized in decomposition of the
network analysis are: modularity maximization and k-core analysis.
Detailed descriptions are available in [12,13]. The igraph library for network
science (http://igraph.sourceforge.net) was used for graph visualizations
to compute degrees, weighted degrees, and to find partitions with high
Met. Ions Life Sci. 2011, 8, 107–132

Figure 1. Breakdown of RASFF notifications by category between May 2003 and

August 2008.
modularity [14]. As noted above, of some 1000 notifications for metal

contamination, during the period, a wide range of countries are involved as
seen in Figure 2. This complex picture reflects the global nature of the
problem.
With a focus on the key metal contaminants, analysis of the RASFF
database allows an indepth analysis of major food types in which metal
contamination prevails. As shown in Figure 3, notifications in this dataset
were mainly owing to four key metals: Hg (39%), Cd (37%), Pb (18%), and
As (6%). In addition (beyond the analyses presented here), Ni, Cr, Al, Zn,
and Fe contamination were also found among the reasons for notification,
mainly resulting from migration of the metals from containers and/or
utensils. The majority of Hg notifications arise from contaminated swordfish
followed by shark and other fish. In contrast, for Cd the major source of
contamination is shellfish followed by swordfish. For Pb and As con-
tamination occurs in more varied sources with water and seaweed being
predominant for As. Clearly metal contamination, especially for Cd and Hg,
is mainly found in seafood which predisposes nations that rely on this
foodstuff to ingestion. Previously it has been shown that seasonal variations
show a marked increase in metal contamination in seafood in autumn and
winter months. Knowledge about types and levels of metal contaminants
along with categories of seafood, country of origin, and seasonal variations
can aid developing countries to inform their citizens against these hazards.
Met. Ions Life Sci. 2011, 8, 107–132

Figure 2. Network analysis graph of RASFF data plotted as countries reporting

and reported against (arrow direction) for metals in foodstuffs. Darker shaded
nations represent 410 notifications.
Where robust regulatory systems are in place to prevent public exposure

to contaminated food, ingestion of metal ions still occurs but ideally under
the PWTI. Thus, the dietary intakes can be estimated using survey data of
normal diets combined with the levels associated with foodstuffs. Using this
approach the maximum level is set at 100% with the normal adult diet set
against this. As shown in Figure 4, exposure to these key metals usually
remains under 25% of the PWTI. The confidence inspired by these figures is
based upon the assumption that no foodstuffs above the ML are available
on the market. The sampling nature of the RASFF system does not preclude
this hazardous scenario entirely.
Met. Ions Life Sci. 2011, 8, 107–132

114
Met. Ions Life Sci. 2011, 8, 107–132

Figure 3. Analysis of RASFF data plotted as foodstuffs with metal contaminations by metal and food category.
NAUGHTON, NEPUSZ, and PETROCZI
METAL IONS AFFECTING THE GASTROINTESTINAL SYSTEM
115
Met. Ions Life Sci. 2011, 8, 107–132

Figure 4. Dietary intake of As, Cd, Hg, and Pb by mean adult population by food category.
In a recent study by da Silva et al. [15a], levels of estimated exposure to

metals via dietary intake varied widely between countries as expected from
knowledge of variations in regulatory systems and approaches to food
production. From their adaption of an earlier study conducted by Chen and
Gao ([15b] as referenced in [15a]), global exposures were of the order of 153
and 25 micrograms per person per day for Pb and Cd, respectively. Of the
countries studied, exposure levels, as micrograms per person per day, in
Guatemala were highest (Pb, 254; Cd, 29; Hg, 10.8) with China (Pb, 86.3; Cd
13.8; Hg, 10.3), Japan (Pb, 85.0; Cd 29.0), and the UK (Pb, 60.5; Cd 18.9),
coming next especially for dietary exposure to Pb. Levels of exposure in the
USA were considerably below global levels or those for other countries listed
(Pb, 9.80; Cd, 13.0; Hg, 3.2).
2.2. Supplements
Supplements and micronutrients are widely used [16,17] for a variety of
health reasons including balancing the diet, compensating for lack of
nutrition in diet or for exercise [18], improving wellness [19] or mental
conditions [20]. Multimineral supplementation may be used to boost the
immune system and combat infections among the elderly [21] and is widely
used among HIV patients [22]. Multivitamin mineral supplements contribute
to a considerable proportion of nutrient intakes in the United States and
may lead to excessive intakes [17], with a similar pattern recently reported in
10 European countries [23].
Non-prescriptive supplement use has been observed among adolescents
[24], students [25], physically active adults [26], cancer patients [27,28] and
people at high risk of cancer [29], people with chronic diseases [30], and in
the elderly population [31–33], with a notable increase of use in the latter
population [34].
Supplement users frequently act beyond the knowledge or remit of clinical
practitioners with many users acting unilaterally or upon the advice of non-
experts. Several studies have revealed a paucity of fact-based decision
making informing the use of most supplements within the athlete commu-
nities studied [35–37]. This scenario of poorly informed decision making is
likely to be more prevalent in the general population where numerous
supplements are often taken at levels considerably above the recommended
daily allowance (where this is available), despite their documented potential
side effects (e.g., [7,38]).
The potentially harmful effect spreads across the general population,
ranging from the adolescents to the elderly [38–40]. The problem is exem-
plified for those in later life as it is more likely that elderly people are on
Met. Ions Life Sci. 2011, 8, 107–132

prescribed medication, hence for them there is an increased possibility of

disadvantageous or harmful drug interaction [34].
In addition to the intake of metals given above, additional exposure routes
include smoking and exposure from pollution in the environment. These
aspects are not covered in depth within this review as they do not directly
affect the GI tract to the level of ingested foodstuffs and supplements.
Further information on these aspects of exposure to other organs are con-
sidered in accompanying chapters in this book.
3. ESTIMATION OF TOXICITY ASSOCIATED WITH

METAL IONS IN THE GASTROINTESTINAL TRACT
AND LIVER
3.1. Absorption and Accumulation of Metal Ions

As detailed above, total human exposure to metals largely arises unin-
tentionally from a variety of foodstuffs along with ingestion of mineral
supplements. Moreover these routes of ingestion directly affect the GI tract
through initial contact. In broad terms metals can be categorized into groups
depending on their effects and/or uses within the body. At one end of the
scale, redox-active metals such as copper and iron are essential for life but
have been accredited with detrimental effects such as enhancing oxidative
damage or contributing to the development of Parkinson’s disease. In
contrast, the group of heavy metals commonly tested for in foodstuffs
including Hg, Pb, Cd, As, and Sn, have no known function in the body and
thus considerations of ingestion do not require deconvolution of benefits
against detrimental effects. An additional consideration for these heavy
metals is their capacity to be accumulated in tissues such as the liver in a
process termed bioaccumulation.
Owing to a myriad of variables including age, gender, health status, and
pharmacogenetic variations, a pre-determined limiting ingestion dose of
each metal is very difficult to achieve. Owing to the complexity of studies in
this area accompanied by a paucity of human studies, considerable gaps in
knowledge exist ranging from the levels of absorption of individual metal
ions through to the detailed effects on complex biosystems. Presently, the
etiologies of a number of diseases, such as rheumatoid arthritis, are not well
understood. Until proposed connections between disease and metal ions are
fully understood or disregarded, caution should be used when using any
measure of safe intake of metals. For this reason many estimations err on the
cautious side by reducing the amount for safe ingestion by ten fold for each
uncertainty factor.
Met. Ions Life Sci. 2011, 8, 107–132

As a result of the increasing presence of toxic elements in the environment,

humans are subject to health risks from contaminated foodstuffs [15a].
Ingestion of heavy metals may lead to adverse health consequences beyond
and above those arising directly from these metal ions. The toxic effects of
lead, mercury, and cadmium can be inflated and reduced by nutritional
status of the individuals [41]. For example, cadmium interacts with Zn, Cu,
Ag, Hg, Se, Fe, As, and Mg; mercury with Se and Ag, lead with Fe, Zn, Cu,
Se, and Ca, whereas elements interacting with As are Cd, Se, and Zn [42].
The interaction between Cd and Hg may be a particular concern as seafood
is known to be the major food group that is prone to accumulation of these
metal ions, hence, increasing the chances of simultaneous ingestion of both.
This combination has also been dominant among those RASFF notifica-
tions that reported multiple reasons (i.e., level exceeded the ML for both Cd
and Hg), and they were found in various seafoods, mainly swordfish. In
addition to food, tobacco is the other major source of Cd exposure in
humans.
Furthermore, the health benefits of a foodstuff may be compromised if a
heavy metal is present. For example, Cd and Pb in fruit [43], As and Ni in
beverages and nuts, respectively [44]; or V and Mn in wines [45] have led to
the proposal that the presence of these metal ions may negate the anti-
oxidant effect expected from the food. This aspect of metals in foodstuff is
further discussed in Section 4 of this chapter.
3.2. Estimations of Safe Limits

The U.S. Environmental Protection Agency (EPA) defines an oral reference
dose (RfD) as a chemical specific estimate of a daily oral exposure to the
human population (including sensitive subgroups) that is likely to be with-
out an appreciable risk of deleterious effects during a lifetime. The RfD is
established in milligrams ingestible per kilograms of the body weight a day
(mg/kg/day). Unless clinical data are available, RfDs are based on animal
(typically rat) studies. The RfD is calculated using the highest amount
ingested without observed health effect (often called ‘‘no observable adverse
effect level’’, NOAEL) with varying magnitude of uncertainty factors: (i)
interspecies uncertainty factor (UFinter) and (ii) intraspecies uncertainty
factor (UFintra) to account for the assumption that some humans may be
more sensitive to the effects of the chemical than others. Usually a value of
10 is assigned to each unknown factor with a possibility to apply additional
uncertainty factors (UFother) if required. In cases of having information on
the upper safe limit for humans, the interpecies uncertainty factor equals 1
but the intraspecies UF ¼ 10 is retained.
Met. Ions Life Sci. 2011, 8, 107–132

An alternative hazard characterization approach that may offer advan-

tages over the traditional NOAEL is being increasingly used in food risk
assessment and also has been shown to be applicable to pesticides and other
toxins such as mycotoxins and natural toxins [46]. The benchmark dose
(BMD), defined as a dose producing a predetermined incidence rate (e.g.,
5%) of an adverse effect, may be used instead of the NOAEL. The advan-
tages of the BMD approach over and above the traditional NOAEL
approach are captured in (i) more use of dose-response information, (ii) the
lack of limit by the arbitrarily selected experimental doses, and (iii) the use of
lower confidence limit (BMDL) reflects the uncertainty of a study [46].
An alternative approach to establishing the upper safe level is the chronic
oral minimal risk level (MRL), developed by the Agency for Toxic Sub-
stances and Disease Registry (ATSDR) and used jointly with the U.S.
Environmental Protection Agency. The MRL is an estimate of daily expo-
sure without appreciable non-cancerous risk and it is based upon data
revealing the target organ or most probabilistic effect on humans informed
by exposure routes. Both RfDs and MRLs are commonly based on animal
studies and contain some degree of uncertainty owing to the lack of tox-
icological information on the effect of chemicals on people; hence, they are
not absolute values. In keeping with the public health principle of preven-
tion, these estimations reflect a conservative approach to uncertainties and
aim to protect the most vulnerable (e.g., children, elderly, ill). Unlike an RfD
that is established for lifetime exposure, a MRL is developed for three stages
of exposure: acute (o14 days), intermediate (15–365 days) and chronic
(4365 days) with the intention to reflect different scenarios that may happen
at or near hazardous waste sites [47].
Yet another term indicating safe exposure is the upper level (UL),
established by the Scientific Committee on Food (SCF). By definition, the
UL does not differ significantly from the RfD or MRL. It is the highest level
of daily nutrient intake that is likely to pose no risk of adverse health effects
to almost all individuals in the general population. Establishing ULs, key
issues such as evidence (if available) of adverse effects and its mechanism on
humans, especially highly-sensitive subpopulations; causality, integrity,
quality and relevance of experimental data; NOAEL/LOAEL with critical
endpoints, and uncertainty assessment are considered in a semiquantitative
way (SCF Guidelines). As the estimation is mainly based on the same or
similar information (mostly animal studies), the observed large discrepancy
most likely arises from the unknown risk factors.
Practical implications of choosing one or the other method may be sig-
nificant. Whilst the RfD and MRL are typically used in the USA, the
European Food Safety Authority (EFSA) has adopted the UL and BMD
approaches. The different upper levels of safe consumption may be a source
of confusion in public health research, policy, and practice [48]. In summary,
Met. Ions Life Sci. 2011, 8, 107–132

upper safe level estimations are intended to serve as a screening tool to help
public health professionals. In most cases, these values are arithmetically
derived estimates of substance specific levels where adverse health effects are
not likely to happen. As such, these values should not constitute thresholds
for toxicity but rather, be used in health hazard estimation to warrant
attention or further studies.
The NOAEL has been routinely used to derive health based guidance
values such as tolerable daily intake (TDI), acceptable daily intakes (ADI) or
oral reference dose (RfD). Despite the name differences, all are derived by
dividing the NOAEL by some uncertainty factors as follows:
NOAEL
ADIðRfD; TDIÞ ¼ ð1Þ
UFinter UFintra UFother
The RfD is defined as a concentration level of a noncarcinogenic chemical

which can be consumed on a daily basis over a lifetime without adverse
health effect. Similarly, TDI represents a tolerable intake of chemicals
(mainly heavy metals) with known toxic effects for a lifetime. To accom-
modate less harmful substances normally found or even added to foodstuff,
an estimate for the acceptable daily intake has been developed for authorized
food additives and pesticides. ADI is a food specific measure (in mg) and
expressed for body weights (BW). It represents the level at which daily
consumption over a lifetime for an average adult (BW ¼ 60 kg) has been
found safe.
3.3. Cumulative Effects

Attempts have been made to estimate health hazards arising from chemicals
to inform public health authorities and policy makers. The safe limits
detailed above can be used directly to determine whether a foodstuff is
aceptable for human consumption. This use of a cut-off level may have long
term implications depending on duration of use and multiple exposures.
More complex formulae that incorporate duration and multiple exposures
with ingested amount have been derived to assess a cumulative effect – two
of these are the target hazard quotient (THQ) and the provisional tolerable
weekly intake (PTWI). Although the THQ was originally developed by the
U.S. Environmental Protection Agency for estimating health hazards from
environmental pollution/waste sites, it has been adapted to estimate hazards
in foodstuffs such as seafood [49–54], vegetables [55], food crops [56], bev-
erages [45,57], and the whole diet [58]. In general, the THQ is designed to
flag up cases where the inhaled, contacted or ingested amount of toxin
exceeds the maximum amounts tolerable without observed negative health
Met. Ions Life Sci. 2011, 8, 107–132

consequences over prolonged (often lifetime) exposure. To see whether the

THQ value exceeds 1 with the concentration of toxins in air, soil, water or
foodstuff, researchers rely on an established upper safe limit (i.e., RfD,
MLR or UL) and have to make assumptions about the exposure frequency
(how many times per year an average person would come in contact with the
toxin), exposure duration (how many years this contact lasts) and finally,
how long this person will have to live with any health consequences resulting
from the contact with the toxin(s) – the averaging time. One clear benefit of
the THQ is that it can account for long-term frequent exposure, but caution
must be used when applying it to seafood where high levels of metal con-
taminants can be sporadic even in the unlikely event that seafood is con-
sumed by an individual each day. The THQ is calculated using the formula:
EFr EDtot SFI MCSinorg

THQ ¼ 103 ð2Þ
RfD BWa ATn
EFr is the exposure frequency (days/year); EDtot is the exposure duration

(year); SFI is the mass of selected dietary ingested (g/day); MCSinorg is the
concentration of inorganic species in the dietary components (mg/g wet
weight); RfD: oral reference dose (mg/kg/day); BWa: the average adult body
weight; ATn: averaging time for non-carcinogens (day); and 103: the unit
conversion factor [49].
Target cancer risk (TR) is used in place of THQ in case of health risk
estimation from exposure to known carcinogens. TR is calculated as:
EFr EDtot SFI MCSinorg CPS

TR ¼ 103 ð3Þ
BWa ATc
where CPS: carcinogenic potency slope, oral (risk per mg/kg/day) and ATc:
averaging time for carcinogens (25,550 days). As it is an adaptation of the
THQ to carcinogens, the other constituents of the equation are the same as
in equation (2).
With sporadic contamination patterns and/or extreme variations of con-
tamination levels in foodstuff, it is recommended that the chronic daily
intake (CDI) is used to assess health hazards from foodstuffs [57,59]. The
CDI is calculated by first multiplying the contaminant concentration (C) by
the daily intake (DI), which gives the contaminant daily intake. This con-
taminant daily intake is then divided by the body weight (BW) to give the
CDI. Finally, the Hazard Quotient is calculated by dividing the CDI by the
oral reference dose (RfD). Notably, the difference between THQ and HQ is
the ratio between the exposure time (EfrEDtot) and averaging time (ATn).
In case EFrEDtot ¼ ATn, THQ becomes HQ.
Met. Ions Life Sci. 2011, 8, 107–132

The PTWI was developed as a measure of permissible human weekly

exposure to contaminants with cumulative properties in foodstuffs. PTWIs
have been set for Cd, Hg, Pb, Sn, and Al as 0.007 mg/kg BW, 1.6 mg/kg BW
(lowered from 3.3 mg/kg BW recently), 0.025 mg/kg BW, 14 mg/kg BW, and
1 mg/kg BW, respectively. Until a PTWI is established, an inorganic As
PTWI level is used at 15 mg/kg BW [60]. They are largely based on NOAELs
which arise from human studies and are constantly updated reflecting the
term provisional.
Complementing the existing hazard estimations, the threshold of tox-
icological concern (TTC) was proposed for situations where there is a
paucity of information on toxicity of the compound but the risk to human
health is assumed to be low [61,62]. In the absence of toxicological infor-
mation, this approach relies on chemical structure and toxicity data of
structurally related chemicals. The TTC is essentially a substitute for sub-
stance-specific information where there is no appreciable health hazards
expected. As such, the TTC is not to be used for heavy metals or any other
compound that accumulates in the body [63,64], but may be applied to
contact materials (such as tin) [65]. The exclusion of heavy metals is owing to
one or both of the following factors: (i) toxicological data are available to
perform a full chemical-specific risk assessment and/or (ii) the safety factors
used for TTC may not be sufficiently high to account for inter-species
differences.
3.4. Metal Ion-Induced Toxicity

Considerable evidence points to metal ion toxicity originating through
oxidative stress. As detailed in Section 4 below, a large number of the metals
to which humans can be exposed are credited with the generation or
enhancement of free radicals and subsequently free radical damage. Several
authors have proposed the threshold approach whereby the levels and
toxicity of the flux in free radicals surmount the defence mechanisms leading
to a marked elevation in oxidative damage. Alternatively, continued, low
level oxidative damage, whether induced by metal ions or not, may, through
persistant attack, lead to key alterations on cell and tissue function. In
addition to modulating the activity of the immune system, these scenarios of
‘above threshold’ or ‘persistant low level’ attacks (or a blend of both) may be
related to the development and progresssion of chronic inflammatory dis-
eases, cancer, and ageing.
Metal ions, either by direct actions or through associated reaction pro-
ducts such as lipid peroxides, have been shown to affect an enormous
number of biomolecules and systems including DNA, lipid membranes,
proteins, mitochondria, lysosomes, cellular calcium homeostasis, and metal
Met. Ions Life Sci. 2011, 8, 107–132

transport systems [4,66,67]. These interventions can be enhanced during

challenging biochemical events such as hypoxia and hypoxic-reperfusion
injury. Furthermore, interactions with metal homeostasis have been shown
for many heavy metals through a number of mechanisms such as membrane
dysfunction through to alteration of transport routes. Hepatic accumulation
of iron in diseases associated with iron overload is associated with fibrosis
and cirrhosis leading to suggestions that malignancy results from iron
induced insults such as free radical damage [66,67].
4. METAL ION-MOLECULAR INTERACTIONS:

EFFECTS ON OXIDATIVE DAMAGE
4.1. Introduction. Oxidative Damage in the Gastrointestinal

Tract and Liver
Essential metals such as iron and copper have been the focus of decades of
research into their contributions to disease when found at supposedly
abnormal levels or in uncontrolled environments. Beyond metabolic dis-
orders pertaining to metal ion homeostasis, redox-active metals have been
ascribed key roles in the enhancement of oxidative damage, and are asso-
ciated with pathologies such as cancer, inflammation diabetes, liver and
heart disease [68]. Numerous studies reveal the roles of redox active metal
ions as prooxidants where unharnessed labile metal ions can further activate
reactive oxygen and nitrogen species (RONS) through established mechan-
isms. This elevation of activity of less reactive RONS, such as hydrogen
peroxide (H2O2) and superoxide (Od2), to the highly reactive hydroxyl
radical (dOH) can evade the key antioxidant defences of compartmentali-
zation and enzyme deactivation.
As several animal models of gastric inflammation are based upon the
administration of redox-active metals ions, it is not surprising that single
doses of ferrous supplements have been shown to induce oxidative damage
in healthy individuals [69]. Furthermore, patients suffering from Crohn’s
disease exhibited an increase in some clinical symptoms of disease activity on
treatment with ferrous fumarate over one week. Similarly, ferrous fumarate
increased disease activity in patients with inflammatory bowel disease whilst
intravenous iron sucrose increased intravascular oxidative stress [70]. At the
molecular level, iron cosupplementation with vitamin C has been shown to
induce oxidative DNA damage in healthy individuals. Recent analyses of
large literature bases highlight metals as major contributors to the initiation
and progression of a wide range of inflammmatory and neurodegenerative
diseases [7,66,71–73].
Met. Ions Life Sci. 2011, 8, 107–132

Whilst metal ions can exert their toxic effects through a wide range of
mechanisms, the body of literature supporting oxidative mechanisms
through which metal ions exert their toxic effects is vast and includes studies
on Fe, Cu, Cd, Cr, Hg, Ni, V, and Pb [74]. Whilst this body of literature
points to redox-active metal ions as key players in oxidative damage, caution
should be exercised because a great deal has yet to be investigated before we
have an authoritative comprehension of the roles of metals in oxidative
stress. This point is exemplified by the lack of rigorous assays that can
simultaneously detect levels of multiple RONS in a complex biosystem, let
alone capture the complete picture of the potential beneficial and detri-
mental roles played by even one metal ion. Further examples include the
recent advances in understanding the potential beneficial roles of metal
complexes of some dietary components which act as antioxidant enzyme
mimetics, along with the demonstrations that under some conditions vita-
mins may act as prooxidants. In a limited number of reports, speciation data
reveal that contributions of copper ions to oxidative stress are less likely to
occur than was once reported [72]. In contrast, studies in isolated rat
hepatocytes reveal some 1% (9.82.9 micromol/L) of the iron content exists
as chelatable iron [75].
Many of the reports on whether metals contribute to oxidative stress are
focused on limited systems in animals or tissues from healthy individuals.
The outcomes of these studies are less meaningful when transferred to
patients with tissue pathologies such as chronic inflammation/hypoxia.
Thus, it is not surprising, that conflicting literature exists when studies are
undertaken in many different systems at various levels without an author-
itative understanding of the multicomponent complex biosystems being
studied. The very ‘Jekyll and Hyde’ nature of redox-active metal ions bal-
ancing between enhancing oxidative stress through mediating RONS
activities or suppressing oxidative stress and complexes exhibiting anti-
oxidant enzyme mimetics points to the need to ascertain the species present.
Whilst the probability of both beneficial and detrimental species coexists for
a metal ion, no studies have to date fully speciated the metal content of a
complex biosystem.
4.2. Molecular Mechanisms of Metal Ion-Induced Oxidative

Damage
Many of the reported mechanisms of RONS activation include redox-active
metal ions as key components. The Fenton reaction involves the activation
of hydrogen peroxide by ferrous ions to form a highly reactive species often
attributed as the hydroxyl radical (reaction (4)). The reaction depends on the
Met. Ions Life Sci. 2011, 8, 107–132

availability of the lower ferrous oxidation state which may limit it to the
intracellular environment and sites of hypoxia. In addition to the require-
ment for hydrogen peroxide, the type of ferrous complex formed may
enhance the reaction through effects on the redox activity and through steric
and scavenging effects.
FeðIIÞ þ H2 O2 ! OH þ FeðIIIÞ þ OH ð4Þ
In the Haber-Weiss system, co-localisation of superoxide and hydrogen

peroxide are required, along with a metal catalyst, to generate the powerful
oxidant hydroxyl radical (reaction (5)).
metal catalyst
H2 O2 þ O2 ! OH þ O2 þ OH ð5Þ
In addition to direct interactions between RONS and redox-active metal

ions, a number of endogenous and dietary compounds are credited with
enhancing oxidative stress through complexation. These include vitamin C
where interactions with metal ions have been studied in detail. Depending on
conditions, vitamin C may act as a prooxidant via Undenfriend’s system,
resulting in oxidation of biomolecules (Figure 5). In addition, Cu(II)-
mediated oxidation of vitamin C can result in the generation of hydrogen
peroxide from oxygen. Detailed coverage of these reactions are beyond the
scope of this chapter but can be found elsewhere [4,7].
In a model study using a chelating peptide containing tyrosine moieties, a
comparison between an aromatic scavenger demonstrated that the presence
of metal ions greatly enhanced the capture of the highly reactive RONS
H H H
R
R
OH O OH
O O Fe (II) O O H
+ Fe (II) + O2 COOH
O
OH
O H+
O
OH
HO COOH
H H H
R R
O O
O + Fe (II) + H2O
O Fe (II) OH-
O O
O O
Figure 5. Proposed mechanism for Udenfriend’s system.
Met. Ions Life Sci. 2011, 8, 107–132

peroxynitrite [76]. With ferric ions complexed to the scavenger the conjugate
was 7-fold more effective at scavenging peroxynitrite when detected as
nitrated tyrosine. This result testifies the abilities of redox-active metal ions
to enhance localized oxidative stress but also gives an insight to their
potential as antioxidant chelators.
The interactions of metal ions with reductants have been well established
especially for vitamin C which is commonly taken with ferrous supplements.
Udenfriend’s system detailing oxidation of organic molecules through acti-
vation of oxygen with vitamin C and ferrous ion complexes was studied in
the early 1950s (Figure 5). Furthermore, vitamin C can generate hydrogen
peroxide via metal ion-catalyzed oxidation which by further interactions
with the metal ion center can generate the highly reactive hydroxyl radical
[77,78]. While literature reports support both a pro- and antioxidant role for
vitamin C, the divergence of views may reflect varying roles dependent on
environment, dose and related factors such as presence of labile metal ions
and hypoxia-driven reductive metabolism [79–84]. The exemplification of
enhancing oxidative stress through the commonly used supplements vitamin
C and ferrous ions is concerning in light of the development of animal
models of GI inflammation using these constituents.
While a great deal of literature addresses the potential and selected
observed detrimental effects of redox-active metal ions, there is still a con-
siderable divergence of views on the overall significance of these events. This
is especially the case for those metals that are essential nutrients. Biomole-
cules come under frequent attack from oxidants and other damaging species
but repair mechanisms are prolific. Most studies measure limited damage
from oxidants which make it difficult to extrapolate conclusions to real
pathology at the cell, tissue, organ or organism level. In contrast to focused
studies in one tissue or organ in animals or humans, case-controlled studies
that demonstrate adverse effects of supplements or dietary metals in healthy
humans are of major concern. Reports claiming that frequent use of iron
supplements or dietary iron intake can increase the incidence of Parkinson’s
disease should be followed up with trials on the effects on the GI tract [8,9].
Further detailed investigations of the effects of therapeutic doses of iron
supplements are required to determine if gastrointestinal damage occurs [3].
4.3. Therapeutic Implications

Natural and synthetic chelators are known to bind metal ions and affect
their absorption and excretion [85]. Although in its infancy, with respect to
many dietary components, further investigations into species formed by
metal ions with dietary components should illuminate the potential toxic
effects on the GI tract. In addition to the potential detrimental interactions
Met. Ions Life Sci. 2011, 8, 107–132

between metal ions and molecular components highlighted above, recent

reports have demonstrated a number of potential beneficial interactions.
Numerous dietary chelators have been shown to form metal complexes that
exhibit superoxide dismutase activities. These include complexes of luteolin,
rutin, epicatechin, curcumin, and several peptides [86–88]. This leads to the
attractive concept of switching a labile redox-active metal ion from a
potential radical enhancing role to an antioxidant enzyme mimetic upon
chelation by a dietary chelator. However, further work is required to
establish these entities as stable therapeutic agents without side reactions
such as acting as epoxidation catalysts in vivo.
5. CONCLUDING REMARKS AND FUTURE

DIRECTIONS
For decades, efforts have been made to develop a better understanding of

the balance between the nutritional needs for and the harmful effects of
metal ions. Parallel to this, a considerable effort has been made to reduce
and minimize the presence of heavy metals in the environment, including in
foods, to ensure that the unavoidable ingestion stays well below the safe
limit. Different approaches to assessing potential health risks from ingested
chemicals have been developed. Whilst most, or even all, are fit for purpose,
the abundance of slightly differing approaches makes the global harmoni-
zation of food safety and security policies a challenging task.
A paucity of research regarding the harmful effects of metals on the GI
tract, along with the yet unknown interaction of these metals, hindered by
the lack of information from human studies, impede the establishment of
safety guidelines in many cases. The appropriate balance between the
nutritional need and excess ingestion of metal ions, and whether it is
achievable via diets alone, remains a topic in forthcoming research. Future
work is needed in most of the aspects outlined above, in particular to address
the lack of information and ensuring that safe foodstuffs are not only
consumed, but produced globally.
ABBREVIATIONS
ADI acceptable daily intake
ATn averaging time
BMD benchmark dose
Met. Ions Life Sci. 2011, 8, 107–132

BMDL lower confidence limit

BW body weight
CDI chronic daily intake
CPS carcinogenic potency slope
DI daily intake
ED exposure duration
EFr exposure frequency
EFSA European Food Safety Authority
EPA U.S. Environmental Protection Agency
EU European Union
GI gastrointestinal tract
HQ hazard quotient
LOAEL lowest observed adverse effect level
ML maximum level
MRL chronic oral minimal risk level
NOAEL no observable adverse effect level
PTWI provisional tolerable weekly intake
RASFF rapid alert system for food and feed
RfD oral reference dose
RONS reactive oxygen and nitrogen species
SCF Scientific Committee on Food
SFI selected dietary ingested
TDI tolerable daily intake
THQ target hazard quotient
TR target cancer risk
TTC threshold of toxicological concern
UF uncertainty factor
UL upper level
WHO World Health Organization
REFERENCES
1. Rapid Alert System for Food and Feed (RASFF): accessible at http://ec.europa.
eu/food/food/rapidalert/index_en.htm
2. World Health Organization: accessible at http://www.who.int/topics/food_safety/
en/
3. Expert Group on Vitamins and Minerals. Safe upper levels for vitamins and
minerals. Great Britain: Food Standards Agency; 2003: accessible at http://
www.food.gov.uk/multimedia/pdfs/vitmin2003.pdf
4. B. Halliwell and J. M. C. Gutteridge, Free Radicals in Biology and Medicine, 4th
edn., Oxford University Press, Oxford, UK, 2007.
Met. Ions Life Sci. 2011, 8, 107–132

5. J. Davidge and D. R. Williams, in Metal Ions in Biological Systems, Vol. 41 of

Metal Ions and Their Complexes in Medicine, Ed. A. Sigel and H. Sigel, Marcel
Dekker, New York, 2004, pp. 1–39..
6. K. A. Naidu, Nutr. J., 2003, 2, 7.
7. A. E. O. Fisher and D. P. Naughton, Nutr. J., 2005, 4, 14.
8. G. Logroscino, X. Gao, H. L. Chen, A. Wing and A. Ascherio, Am. J. Epid.,
2008, 168, 1381–1388.
9. K. M. Powers, T. Smith-Weller, G. M. Franklin, W. T. Longstreth, P. D.
Swanson and H. Checkoway, Neurol., 2003, 60, 1761–1766.
10. World Health Organization: accessible at http://www.who.int/foodsafety/
fs_management/meetings/forum07/en/index.html
11. Commission Regulation (EC) No 466/2001 setting maximum levels for
certain contaminants in foodstuffs. Accessible at: http://www.caobisco.com/
doc_uploads/legislation/466-2001EN.pdf
12. T. Nepusz, A. Petroczi and D. P. Naughton, PLoS ONE, 2009, 4, e6680.
13. T. Nepusz, A. Petroczi and D. P. Naughton, BioMed Central Public Health, 2008,
8, 308.
14. G. Csardi and T. Nepusz, InterJournal Complex Systems, 2006, 1695.
15. (a) A. L. O. Da Silva, P. R. G. Barrocas, S. C. Jacob and J. C. Moreira, Braz. J.
Plant Physiol., 2005, 17, 79–93; (b) J. Chen and J. Gao, J. Assoc. Offic. Analyt.
Chem., 1993, 76, 1193–1205.
16. G. Block, C. D. Jensen, E. P. Norkus, T. B. Dalvi, L. G. Wong, J. F. McManus
and M. L. Hudes, Nutr. J., 2007, 6, 30.
17. C. L. Rock, Am. J. Clin. Nutr., 2007, 85, 2775–2795.
18. J. O’Dea, Health Educ. Res., 2003, 18, 98–107.
19. M. Nichter and J. J. Thompson, Cult. Med. Psychiat., 2006, 30, 175–222.
20. J. L. Reay, D. O. Kennedy and A. B. Scholey, J. Psychopharmacol., 2005, 19,
357–365.
21. A. I. Stephen and A. Avenell, J. Hum. Nutr. Diet., 2006, 19, 179–190.
22. E. Aghdassi, H. Bondar, I. E. Salit, J. Tinmouth and J. P. Allard, Curr. HIV Res.,
2009, 7, 555–561.
23. G. Skeie, T. Braaten, A. Hjartaker, M. Lentjes, P. Amiano, P. Jakszyn, V. Pala,
A. Palanca, E. M. Niekerk, H. Verhagen, K. Avloniti, T. Psaltopoulou,
M. Niravong, M. Touvier, K. Nimptsch, J. Haubrock, L. Walker, E. A. Spencer,
N. Roswall, A. Olsen, P. Wallstrom, S. Nilsson, C. Casagrande, G. Deharveng,
V. Hellstrom, M. C. Boutron-Ruault, A. Tjonneland, A. M. Joensen, F. Clavel-
Chapelon, A. Trichopoulou, C. Martinez, L. Rodriguez, G. Frasca, C. Sacerdote,
P. H. M. Peeters, J. Linseisen, A. Schienkiewitz, A. A. Welch, J. Manjer,
P. Ferrari, E. Riboli, S. Bingham, D. Engeset, E. Lund and N. Slimani, Eur. J.
Clin. Nutr., 2009, 63, S226–S238.
24. F. C. Papadopoulos, I. Skalkidis, J. Parkkari and E. Petridou, Eur. J. Epid., 2006,
21, 307–313.
25. E. Spencer, A. Bendich and E. Frank, J. Am. Diet. Assoc., 2006, 106, 1975–1983.
26. S. N. Tsang, L. A. Pycz and N. H. Herbold, Topics Clin. Nutr., 2007, 22,
246–257.
Met. Ions Life Sci. 2011, 8, 107–132

27. M. J. Verhoef, L. G. Balneaves, H. S. Boon and A. Vroegindewey, Integr. Cancer

Ther., 2005, 4, 274–286.
28. C. M. Velicer and C. M. Ulrich, J. Clin. Oncol., 2008, 26, 665–673.
29. R. G. Uzzo, J. G. Brown, E. M. Horwitz, A. Hanlon, S. Mazzoni, A. Konski,
R. E. Greenberg, A. Pollack, V. Kolenko and D. Watkins-Bruner, Brit. J. Urol.
Int., 2004, 93, 955–60.
30. R. J. Stratton, Proc. Nutr. Soc., 2000, 59, 469–476.
31. T. A. Arcury, J. G. Grzywacz, R. A. Bell, R. H. Neiberg, W. Lang and
S. A. Quandt, J. Gerontol. B: Psych. Sci. Soc. Sci., 2007, 62, S142–S149.
32. J. P. Kelly, D. W. Kaufman, K. Kelley, L. Rosenberg, T. E. Anderson and
A. A. Mitchell, Arch. Intern. Med., 2005, 165, 281–286.
33. J. S. Marinac, C. L. Buchinger, L. A. Godfrey, J. M. Wooten, C. Sun and
S. K. Willsie, J. Am. Osteopath. Assoc., 2007, 107, 13–23.
34. P. H. Canter and E. Ernst, Drugs Aging, 2004, 21, 597–605.
35. A. Petroczi, D. P. Naughton, J. Mazanov, A. Holloway and J. Bingham, J. Int.
Soc. Sports Nutr., 2007, 4, 19.
36. A. Petroczi, D. P. Naughton, J. Mazanov, A. Holloway and J. Bingham, Nutr. J.,
2007, 6, 34.
37. A. Petroczi, D. P. Naughton, G. Pearce, A. Bloodworth, R. Bailey and
M. McNamee, J. Int. Soc. Sports Nutr., 2008, 5, 22.
38. M. Palmer, C. Haller, P. McKinney, W. Klein-Swartz, A. Tschirgi, S. C. Smolinske,
A. Woolf, B. M. Spraque, R. Ko, G. Everson, L. S. Nelson, T. Dodd-Butera, W. D.
Bartlett and B. R. Landzberg, Lancet, 2003, 361, 101–106.
39. C. Haller, T. Kearney, S. Bent, R. Ko, N. Benowitz and K. Olson, J. Med.
Toxicol., 2008, 4, 84–92.
40. B. B. Timbo, M. P. Ross, P. V. McCarthy and C. T. Lin, J. Am. Diet. Assoc.,
2006, 106, 1966–1974.
41. R. A. Goyer, Am. J. Clin. Nutr., 1995, 61, 646S–S650.
42. I. Zukowska and M. Biziuk, J. Food Sci., 2008, 73, R21–R29.
43. O. Donma and M. Metin Donma, Med. Hypotheses, 2005, 65, 699–702.
44. M. Metin Donma and O. Donma, Med. Hypotheses, 2006, 66, 681.
45. D. P. Naughton and A. Petróczi, Chem. Central J., 2009, 3, 6.
46. S. D. Muri, J. R. Schlatter and B. J. Brüschweiler, Food Chem. Toxicol., 2009, 47,
2906–2925.
47. J. F. Risher and C. T. DeRosa, Hum. Ecol. Risk Assess., 1997, 3, 681–700.
48. P. J. Aggett, Food Nutr. Bull., 2007, 28, S27–S37.
49. M. M. Storelli, Food Chem. Toxicol., 2008, 46, 2782–2788.
50. N. P. C. Tu, N. N. Ha, T. Ikemoto, B. C. Tuyen, S. Tanabe and I. Takeuchi,
Marine Pollut. Bull., 2008, 57, 858–866.
51. M. C. Lin and C. M. Liao, Food Chem. Toxicol., 2008, 46, 701–709.
52. L. C. Chien, T. C. Hung, K. Y. Choang, C. Y. Yeh, P. J. Meng, M. J. Shieh and
B. C. Han, Sci. Total Environ., 2002, 285, 177–185.
53. C. W. Liu, C. P. Liang, F. M. Huang and Y. M. Hsueh, Sci. Total Environ., 2006,
361, 57–66.
54. W. Wang, S. Batterman, S. Chernyak and J. Nriagu, Ecotoxicol. Environ. Safety,
2008, 71, 138–148.
Met. Ions Life Sci. 2011, 8, 107–132

55. X. Wang, T. Sato, B. Xing and S. Tao, Sci. Total Environ., 2005, 350, 28–37.
56. P. Zhuang, M. B. McBride, H. Xia, N. Li and Z. Li, Sci. Total Environ., 2008,
407, 1551–1561.
57. S. C. Sofuoglu and P. Kavcar, J. Hazard Mater., 2008, 158, 392–400.
58. N. Zheng, Q. Wang, Z. Zhang, D. Zheng, Z. Zhang and S. Zhang, Sci. Total
Environ., 2007, 387, 96–104.
59. C. Petito Boyce, A. S. Lewis, E. L. Butler, J. K. Saxe and B. D. Beck, Sci. Total
Environ., 2007, 388, 372–375.
60. Chemical Safety Information from Intergovernmental Organizations: accessible
at http://www.inchem.org/pages/jecfa.html
61. I. C. Munro, A. G. Renwick and B. Danielewska-Nikiel, Toxicol. Lett., 2008,
180, 151–156.
62. R. Kroes, J. Kleiner and A. Renwick, Toxicol. Sci., 2005, 86, 226–230.
63. R. Kroes, A. G. Renwick, M. Cheeseman, J. Kleiner, I. Mangelsdorf, A. Piersma,
B. Schilter, J. Schlatter, F. van Schothorst, J. G. Vos and G. Würtzen, Food
Chem. Toxicol., 2004, 42, 65–83.
64. S. Barlow, Threshold of toxicological concern (TTC). A tool for assessing sub-
stances of unknown toxicity present at low levels in the diet, ILSI Europe Concise
Monograph Series, The International Life Sciences Institute (ILSI) Europe, 2005.
65. Scientific Committee on Consumer Products (SCCP) – Scientific Committee on
Health and Environmental Risks (SCHER) – Scientific Committee on Emerging
and Newly Identified Health Risks (SCENIHR) Draft OPINION ON Use of the
Threshold of Toxicological Concern (TTC) Approach for the Safety Assessment
of Chemical Substances. Preliminary report, agreed by SCHER, SCCP and
SCENIHR on 19 November 2008 by written procedure: accessible at http://
ec.europa.eu/health/ph_risk/committees/documents/sc_o_001.pdf
66. R. S. Britton, Semin. Liver Dis., 1996, 16, 3–12.
67. M. C. Kew, Cancer Lett., 2009, 286, 38–43.
68. C. G. Fraga and P. I. Oteiza, Toxicol., 2002, 180, 23–32.
69. F. J. Troost, W. H. Saris, G. R. Haenen, A. Bast and R. J. Brummer, Am. J.
Physiol. Gastrointest. Liver Physiol., 2003, 285, G354–G359.
70. K. Erichsen, R. J. Ulvik, G. Nysaeter, J. Johansen, J. Ostborg, A. Berstad, R. K.
Berge and T. Hausken, Scand. J. Gastroenterol., 2005, 40, 1058–1065.
71. D. B. Kell, BioMed Central Med. Genomics, 2009, 2, 2.
72. M. Valko, H. Morris and M. T. Cronin, Curr. Med. Chem., 2005, 12, 1161–1208.
73. M. Valko, C. J. Rhodes, J. Moncol, M. Izakovic and M. Mazur, Chem. Biol.
Interact., 2006, 160, 1–40.
74. S. J. Stohs and D. Bagchi, Free Radic. Biol. Med., 1995, 18, 321–336.
75. F. Petrat, U. Rauen and H. de Groot, Hepatol., 1999, 29, 1171–1179.
76. A. E. O. Fisher and D. P. Naughton, Bioorg. Med. Chem. Lett., 2003, 13, 1733–
1735.
77. M. M. Khan and A. E. Martell, J. Amer. Chem. Soc., 1967, 89, 4176–4185.
78. M. M. Khan and A. E. Martell, J. Amer. Chem. Soc., 1967, 89, 7104–7111.
79. K. L. Retsky, M. W. Freeman and B. Frei, J. Biol. Chem., 1993, 268, 1304–1309.
80. I. D. Podmore, H. R. Griffiths, K. E. Herbert, N. Mistry, P. Mistry and J. Lunec,
Nature, 1998, 392, 559–559.
Met. Ions Life Sci. 2011, 8, 107–132

81. K. Chen, J. Suh, A. C. Carr, J. D. Morrow, J. Zeind and B. Frei, Am. J. Physiol.
Endocrinol. Metab., 2000, 279, E1406–E1412.
82. H. R. Griffiths and J. Lunec, Environ. Toxicol. Pharmacol., 2001, 10, 173–182.
83. A. Rehman, C. S. Collis, M. Yang, M. Kelly, A. T. Diplock, B. Halliwell and C.
Rice-Evans, Biochem. Biophys. Res. Commun., 1998, 246, 293–298.
84. D. M. Bailey, S. Raman, J. McEneny, I. S. Young, K. L. Parham, D. A. Hullin,
B. Davies, G. McKeeman, J. M. McCord and M. H. Lewis, Free Radic. Biol.
Med., 2006, 40, 591–600.
85. G. J. Kontoghiorghes and A. Kolnagou, Curr. Med. Chem., 2005, 12, 2695–2709.
86. T. Hague, P. R. Andrews, J. Barker and D. P. Naughton, Behav. Pharmacol.,
2006, 17, 425–430.
87. V. A. Kostyuk, A. I. Potapovich, E. N. Strigunova, T. V. Kostyuk and I. B.
Afanas’ev, Arch. Biochem. Biophys., 2004, 428, 204–208.
88. A. Barik, B. Mishra, L. Shen, H. Mohan, R. M. Kadam, S. Dutta, H. Y. Zhang
and K. I. Priyadarsini, Free Radical. Biol. Med., 2005, 39, 811–822.
Met. Ions Life Sci. 2011, 8, 107–132

Met. Ions Life Sci. 2011, 8, 133–141
6
Metal Ions Affecting the Kidney
Bruce A. Fowler
Division of Toxicology and Environmental Medicine, Agency for Toxic Substances and
Disease Registry, Atlanta GA 30341, USA
<bfowler@cdc.gov>
ABSTRACT 133
1. INTRODUCTION 134
2. EXPOSURE TO METAL IONS IN AIR, FOOD, AND WATER 134
3. TRANSPORT OF METALS/METALLOIDS IN THE
CIRCULATION 135
4. MECHANISMS OF METAL AND METALLOID UPTAKE
BY THE KIDNEY 136
5. EFFECTS OF METALS/METALLOIDS ON THE KIDNEY 136
6. MECHANISMS OF RENAL CELL INJURY 137
7. RENAL BIOMARKERS 137
8. METAL/METALLOID INTERACTIONS IN THE KIDNEY 138
ABBREVIATIONS 139
REFERENCES 139
ABSTRACT: This chapter provides a succinct summary of the nephrotoxic effects of a

number of metals/metalloids on an individual or mixture basis. There is a discussion of
routes of exposure, mechanisms of uptake by renal cells and the potential impact of
nanomaterials on these processes. An emphasis is placed on the toxicity of these metals/
metalloids to individual cell types in the kidney and the application of biomarkers for
the early detection of kidney cell injury prior to the onset of an overt clinical state such
as end-stage renal disease. The issue of interactions between nephrotoxic metals in
134 FOWLER
mixture exposures is discussed in relation to the application of molecular biomarkers

for early detection of renal cell injury.
KEYWORDS: arsenic . biomarkers . cadmium . gallium . inclusion bodies . indium .

lead . mercury . metallic mixtures . metallothionein . selenium . semiconductors
1. INTRODUCTION
The kidney is the major target organ for toxic metals and metalloids since it
concentrates them and urine is usually a major route for excretion from the
body. Humans are exposed to combinations of metals and metalloids and
this increases the likelihood of interactive effects in the kidney [1,2]. As
discussed below, the kidney is a complex and metabolically active organ
which regulates a number of basic biological functions that render it sus-
ceptible to toxic insult from metallics. Renal cell types also vary in their
sensitivity to toxic injury. Proximal tubule cells and endothelial cells of the
renal vasculature are common targets for damage from metals or metalloids
which accumulate in the kidney.
The present review will try to provide an overview of general issues which
influence metal/metalloid nephrotoxicity and then focus on mechanisms of
cell injury, molecular biomarkers for following cell injury, and the appli-
cation of these biomarkers for evaluating interactions among mixtures of
nephrotoxic metals and metalloids in the kidney. The chapter will conclude
with suggestions for how these biomarkers may be used to assist in
mechanism-based risk assessments for metallic mixtures and evaluations of
renal toxicity from exposures to metallic nanoparticles which are of growing
toxicological concern.
2. EXPOSURE TO METAL IONS IN AIR, FOOD, AND

WATER
Humans are exposed to nephrotoxic metallics (e.g., lead, cadmium, arsenic,
mercury, antimony) from a variety of environmental, occupational, and
dietary sources which result in the presence of these elements in blood and
urine among the general U.S. population [3]. In addition, there are growing
occupational and environmental concerns about semiconductor metals such
as gallium and indium from occupational exposures and the issue of
‘‘e-waste’’ related to improper recycling of semiconductor devices such as
computers, solar cells, and cellular telephones. In global terms, major
Met. Ions Life Sci. 2011, 8, 133–141

METAL IONS AFFECTING THE KIDNEY 135
sources of environmental exposures to lead, cadmium, arsenic, and

mercury are coal-fired power plants [4]. Smelters may also be important
local sources of lead, cadmium, and arsenic exposures in air [5,6]. It
should be noted that frequently these sources emit mixtures of these toxic
elements so potential interactions among these elements are a major public
health concern. In terms of diet, certain plants such as rice grown in
paddies irrigated with water contaminated with cadmium or arsenic will also
accumulate these elements [7,8]. Leafy green vegetables such as lettuce,
cabbage, and Swiss chard will also accumulate cadmium from soils
treated with super phosphate fertilizers [9,10]. Shellfish such as
oysters [11] may also be an important dietary source for cadmium. Seafood
(certain large predatory fish species) is a major dietary source for
mercury as methylmercury [4]. Lead is found in a number of food stuffs
such as chocolate [10] and calcium dietary supplements derived from bone
meal [10].
Exposure to lead in drinking water usually occurs from the use of lead
solders in plumbing or water distribution systems [12]. Arsenic may occur in
water systems fed by underground aquifers in rock formations containing
high levels of this element [13]. Similar increases in exposure to mercury and
cadmium may also occur in water systems contaminated with these elements
as a result of rock strata containing them or as a result of the use of mercury
in gold mining operations [4].
3. TRANSPORT OF METALS/METALLOIDS IN THE

CIRCULATION
Once absorbed into the body, the metals of concern may exist in the cir-
culation as ionic species, or bound to serum proteins and red blood cells. The
nature of the binding pattern will in part determine the bioavailability/
deposition of these elements in target tissues. For elements such as lead, the
red cell appears to be the major transport vehicle in the circulation with a
smaller ‘‘diffusible’’ protein-bound fraction that actually transports the lead
into target organ systems. Cadmium is largely found in the circulation
bound to the small metal-binding protein metallothionein, which transports
this element to the kidneys with uptake by proximal tubule cells following
glomerular filtration [14]. Arsenic is methylated in the liver and, depending
upon the methylated species, may bind to both serum proteins and/or red
blood cells. Mercury in the circulation as inorganic mercury will be bound to
serum proteins but methylmercury will be largely bound to the red blood cell
fraction.
Met. Ions Life Sci. 2011, 8, 133–141

136 FOWLER
4. MECHANISMS OF METAL AND METALLOID

UPTAKE BY THE KIDNEY
The mechanisms of metal and metalloid uptake by the kidney will be largely
determined by how they are transported in the circulation. Protein-bound and
low molecular weight species will be handled differently from ionic species in
terms of uptake by kidney tubule cells. In general, metals/metalloids bound to
proteins filtered by the glomeruli will be taken into kidney tubule cells by
endocytosis followed by lysosomal degradation of the carrier protein and release
of the metallic ions such as cadmium. This process was first demonstrated by
Fowler and coworkers [14,15] for cadmium bound to metallothionein.
Brush border membrane vesicle studies [16] showed that radioactive ionic
lead was extensively bound to the surface coat with no detectable active
internal transport. These data suggest that uptake of ionic lead by renal
tubule cells most likely occurs via internalization of the cell membrane
during the process of membrane turnover. Other studies for ionic mercury
(Hg21) as reviewed by Bridges and Zalups [17] showed that mercury was
actively transported on the organic anion transport system of renal proximal
tubule cells via molecular mimicry following binding to cysteine.
5. EFFECTS OF METALS/METALLOIDS ON
THE KIDNEY
As noted above, the kidney is a complex organ with numerous cell types
which vary in their sensitivity to metals/metalloids on an individual or
mixture basis. Anatomically, the kidney can be separated into two main
components: the renal blood vasculature (arteries, arterioles, capillaries
including the glomeruli and veins) and the nephrons which are composed of
several distinct segments (proximal, distal, collecting tubules). Most of the
literature on metals/metalloids alone or as mixtures has been focused on
renal tubular effects and on the proximal tubules in particular. Lead intra-
nuclear inclusion bodies in renal proximal tubule cells of humans [18] or
experimental animals treated with elevated dose levels of lead [19,20] are
regarded as pathognomonic of renal lead toxicity and once formed become
the main intracellular storage site for lead in these tubule cells. Exposure of
rats to mixtures of lead, cadmium, and arsenic in food demonstrated a
marked reduction of the formation of lead inclusions in animals also
receiving cadmium which was associated with a 60% reduction of the renal
lead burden but an additive increase in the excretion of porphyrins in the
urine. These data indicate that while total concentration of lead in the kidney
was decreased, the biologically active fraction available to the heme
Met. Ions Life Sci. 2011, 8, 133–141

biosynthetic pathway was actually increased. Other studies involving com-

bined exposures to Hg(II) and Se(VI) [21] demonstrated the formation of
Hg-Se intranuclear inclusion bodies in renal proximal tubule cells of rats
with attenuation of tubular toxicity. Other combinations of metals such as
the binary III-V semiconductors GaAs [22,23] or InAs [24,25] also demon-
strate additive interactions in the kidney.
More recently, a great deal of interest has been focused on the endothelial
cells of the renal blood vasculature as target cell population for metals such
as cadmium [26]. This is an important aspect of metal/metalloid renal
toxicity since clinically, damage to the renal blood vasculature leading to
interstitial fibrosis is a major cause of end stage renal disease (ESRD).
Similar findings of interstitial fibrosis have been reported following lead [19]
and cadmium [7] exposures.
6. MECHANISMS OF RENAL CELL INJURY
Metal/metalloid-induced oxidative stress in the kidney is generally regarded

as a major underlying mechanism of renal cell injury. In general, metals and
metalloids which produce oxidative stress interact in an additive manner in
mixture situations. This is typically mediated by effects on the mitochondrion
with decreases in respiratory function [20,27], leading to increased production
of H2O2. The extent to which this is true is mediated in part by a number of
factors such as dose, duration of exposure, induction of antioxidant systems
such as GSH, SOD, and metallothionein, age, diet, and genetic inheritance.
The two general mechanisms by which renal cells may die are apoptosis
and necrosis. Apoptosis or programmed cell death is usually observed at
lower dose exposures to metals/metalloids [28]. Higher dose exposures to
metals such as cadmium as the cadmium metallothionein complex [29],
which has been demonstrated to be markedly attenuated by prior treatment
with zinc [14,30] induce the renal metallothionein pool.
7. RENAL BIOMARKERS
As noted above, renal biomarkers of metal/metalloid renal cell injury are of

great potential value in the early detection of ongoing pre-clinical kidney
injury but also of value in detecting whether the effects of combined expo-
sures to mixtures of these agents are additive, synergistic, or antagonistic.
The major classes of current renal biomarkers are proteinuria patterns [7],
porphyrinuria patterns [31,32], and the more recent development of ‘‘omic’’
Met. Ions Life Sci. 2011, 8, 133–141

138 FOWLER
(genomic, proteomic or metabalomic/metabanomic) biomarkers. These

potentially powerful tools require validation via correlation with other
endpoints of cell injury or clinical outcomes in order to be interpreted and to
reach their full potential [33–36].
8. METAL/METALLOID INTERACTIONS IN THE KIDNEY
There are a number of reports of metal/metalloid interactions in the kidney

as discussed above and this section will attempt to recapitulate some of
them. The factorial design leadcadmiumarsenic dietary interaction stu-
dies in rats by Mahaffey and coworkers [37–39] provide clear-cut evidence of
additive interactions among these common toxic elements in this organ
system at ‘‘stressor ‘‘dose levels. Follow-up replicate studies at LOEL dose
levels for these elements in drinking water [40,41] confirmed and extended
these interactions at 30, 90, and 180 day time points. Parallel in vitro studies
[42] confirmed the mechanistic nature of these interactions via direct expo-
sure of renal tubule cells to lead, cadmium, and arsenic combinations using a
factorial design and showed similar relative outcomes as a function of dose
and time. It should be noted that on a molecular level, low dose interactions
between lead cadmium and arsenic and zinc may alter the effects of lead on
sensitive heme biosynthetic pathway enzymes such as ALAD [43–46] and the
intranuclear transport of lead [47,48]. Studies of mercury-selenium interac-
tions in the kidney [21] demonstrated a novel interaction with regard to the
formation of Hg-Se containing intranuclear inclusion bodies and the asso-
ciated attenuation of mercury nephrotoxicity.
More recent in vivo and in vitro studies [22–25,33] involving exposures to
3–5 mm particles of the binary III–V semiconductors gallium arsenide and
indium arsenide demonstrated additive interactions on cells of the renal
proximal tubule cells by a variety of molecular biomarker and morpholo-
gical endpoints.
Gender differences were also noted in studies [33,34] on gallium arsenide
and indium arsenide exposures indicating the need to consider this factor
with regard to risk assessments for exposure to these binary mixtures.

DIRECTIONS
In conclusion, this brief review of the available literature on metal/metalloid

interactions in the kidney should provide good confidence that such
Met. Ions Life Sci. 2011, 8, 133–141

interactions do occur among the most common of these major toxic elements
but that other factors such as age, gender, diet, and genetic inheritance are
important mediating factors that will modulate the nature and extent of
these interactions.
It is also clear that the degree to which it is possible to delineate inter-
actions among mixtures of these elements in the kidney is determined by the
endpoints selected due to the extensive reserve capacity of this organ system.
The growing role of molecular biomarkers for the early detection of renal
cell injury from toxic metals/metalloids on an individual or mixture basis is
of increasing value [33–36]. In this regard, the need for future research to
develop and validate molecular renal biomarkers is clearly an area of great
opportunities and challenges due to the complexity and reserve capacity of
the kidney as an organ system. Another area of needed research with regard
to risk assessment for mixtures of toxic metals/metalloids concerns the
growing incorporation of these elements into binary nanomaterials for a
variety of uses. The formulation of these elements into nanomaterials greatly
changes their bioavailability and capacity to produce toxicity to organs such
as the kidney. This is hence an area that should be given a high research
priority.
ABBREVIATIONS
ALAD d-aminolevulinic acid dehydratase
ESRD end-stage renal disease
GaAs gallium arsenide
InAs indium arsenide
LOEL lowest observed effect level
REFERENCES
1. E. F. Madden and B. A. Fowler, Drug and Chemical Toxicol., 2000, 23, 1–12.
2. G. F. Nordberg, L. Gerhardsson, K. Broberg, M. Mumtaz and B. A. Fowler,
Interactions in Metal Toxicology, in Handbook on the Toxicology of Metals, 3rd
edn., Ed. G. F. Nordberg, B. A. Fowler, M. Nordberg and L. Friberg, Elsevier,
Amsterdam, 2007, pp. 117–145.
3. CDC, Third National Report on Human Exposures to Environmental Chemi-
cals, NCEH Pub. No. 05-0570, Atlanta, GA, 2005.
4. ATSDR, Toxicological Profile for Mercury, Agency for Toxic Substances and
Disease Registry, Atlanta, GA, 1999.
5. P. Hotz, J. P. Buchet, A. Bernard, D. Lison and R. Lauwerys, Lancet, 1999, 354,
1508–1513.
Met. Ions Life Sci. 2011, 8, 133–141

140 FOWLER
6. ATSDR, Toxicological Profile for Arsenic, Agency for Toxic Substances and
7. G. F. Nordberg, M. Nordberg, H. Nogawa and L. Friberg, Cadmium, in
Handbook on the Toxicology of Metals, 3rd edn., Ed. G. F. Nordberg, B. A.
Fowler, M. Nordberg and L. Friberg, Elsevier, Amsterdam, 2007, pp. 445–486.
8. A. K. Chakraborty and K. C. Saha, Indian J. Med. Res., 1987, 85, 326–334.
9. ATSDR, Toxicological Profile for Cadmium, Agency for Toxic Substances and
10. FDA, Total Diet Study, www.fda.gov/Food/FoodSafety/FoodContaminants-
Adulteration/TotalDiet Study/ucm184646.htm.
11. L. I. Bendell, Food Additives and Contaminants, 2009, 2, 131–139.
12. ATSDR, Toxicological Profile for Lead, Agency for Toxic Substances and Dis-
ease Registry, Atlanta, GA, 2007.
13. B. A. Fowler, S. Chou, R. Jones, C. J. Chen, Arsenic, in Handbook on the
Toxicology of Metals, 3rd edn., Ed. G. F. Nordberg, B. A. Fowler, M. Nordberg
and L. Friberg, 2007, Elsevier, Amsterdam, pp. 367– 406.
14. K. S. Squibb, J. W. Ridlington, N. G. Carmichael and B. A. Fowler, Envir-
on.Health Perspect., 1979, 28, 287–296.
15. K. S. Squibb, J. B. Pritchard and B. A. Fowler, J. Pharmacol. Exper. Therap.,
1984, 229, 311–321.
16. W. W. Victery, C. R. Miller and B. A. Fowler, J. Pharmacol. Exp. Therap., 1984,
231, 589–596.
17. C. C. Bridges and R. K. Zalups, Toxicol. Appl. Pharmacol., 2005, 204, 274–308.
18. E. L. Baker, R. A. Goyer, B. A. Fowler, U. Khettry, D. B. Barnard, S. Adler, R.
D. White, R. Babayan and R. G. Feldman, Am. J. Indus. Med., 1980, 1, 139–48.
19. R. A. Goyer and B. C. Rhyne, Int. Rev. Exper. Pathol., 1973, 12, 1–77.
20. B. A. Fowler, C. A. Kimmel, J. S. Woods, E. E. McConnell and L. D. Grant,
Toxicol. Appl. Pharmacol., 1980, 56, 59–77.
21. N. G. Carmichael and B. A. Fowler, J. Env. Pathol. Toxicol., 1979, 3, 399–412.
22. P. L. Goering, R. R. Maronpot and B. A. Fowler, Toxicol. Appl. Pharmacol.,
1988, 92, 179–193.
23. Y. Aoki, M. M. Lipsky and B. A. Fowler, Toxicol. Appl. Pharmacol., 1990, 106,
462–468.
24. E. A. Conner, H. Yamauchi, B. A. Fowler and M. Akkerman, J. Exposure Anal.
Environ. Epidemiol., 1993, 3, 431–440.
25. E. A. Conner, H. Yamauchi and B. A. Fowler, Chem. Biol. Interact., 1995, 96,
273–285.
26. W. C. Prozialeck, J. R. Edwards, D. W. Nebert, J. M. Woods, A. Barchowsky
and W. D. Atchison, Tox. Sci., 2008, 102, 207–218.
27. M. M. Brown, B. C. Rhyne, R. A. Goyer and B. A. Fowler, J. Toxicol. Env.
Health, 1976, 1, 507–516.
28. J. Bustamente, L. Dock, M. Vahter, B. Fowler and S. Orrenius, Toxicology, 1997,
118, 129–136.
29. B. A. Fowler and G. F. Nordberg, Toxicol. Appl. Pharmacol., 1978, 46, 609–624.
30. J. Liu, K. S. Squibb, M. Akkerman, G. F. Nordberg, M. Lipsky and B. A.
Fowler, Renal Failure, 1996, 18, 867–882.
Met. Ions Life Sci. 2011, 8, 133–141

31. J. S. Woods and B. A. Fowler, J. Lab. Clin. Med., 1977, 90, 266–272.
32. B. A. Fowler and J. S. Woods, Exper. Molec. Pathol., 1977, 27, 403–412.
33. B. A. Fowler, E. A. Conner and H. Yamauchi, Toxicol. Appl. Pharmacol., 2005,
206, 121–130.
34. B. A. Fowler, E. A. Conner and H. Yamauchi, Toxicol. Appl. Pharmacol., 2008,
233, 110–115.
35. G. Wang and B. A. Fowler, Toxicol. Appl. Pharmacol., 2008, 233, 92–99.
36. B. A. Fowler, Toxicol. Appl. Pharmacol., 2009, 238, 294–300.
37. K. R. Mahaffey and B. A. Fowler, Environ. Health Perspect., 1977, 19, 165–171.
38. B. A. Fowler and K. R. Mahaffey, Environ. Health Perspect., 1978, 25, 87–90.
39. K. R. Mahaffey, S. G. Capar, B. C. Gladen and B. A. Fowler, J. Lab. Clin. Med.,
1981, 98, 4.
40. B. A. Fowler, M. H. Whittaker, M. Lipsky, G. Wang and X.-Q. Chen, Biometals,
2004, 17, 567–568.
41. B. A. Fowler, E. A. Conner, H. Yamauchi, G. Wang and M. H. Whittaker, Cell
Biol. Toxicol., 2008, 24, S118–119.
42. E. F. Madden, M. Akkerman and B. A. Fowler, J. Biochem. Molec. Toxicol.,
2002, 16, 24–32.
43. P. L. Goering and B. A. Fowler, J. Pharmacol. Exp. Therap., 1984, 231, 66–71.
44. P. L. Goering and B. A. Fowler, J. Pharmacol. Exp. Therap., 1985, 234, 365–371.
45. P. L. Goering and B. A. Fowler, Arch. Biochem. Biophys., 1987, 253, 48–55.
46. P. L. Goering and B. A. Fowler, Biochem. J., 1987, 245, 339–345.
47. P. Mistry, G. W. Lucier and B. A. Fowler, J. Pharmacol. Exp. Therap., 1985, 232,
462–469.
48. P. Mistry, C. Mastri and B. A. Fowler, Biochem. Pharmacol., 1986, 35, 711–713.
Met. Ions Life Sci. 2011, 8, 133–141

Met. Ions Life Sci. 2011, 8, 143–155
7
Metal Ions Affecting the Hematological System
Nickolette Roney , Henry G. Abadin , Bruce Fowler,
and Hana R. Pohl
Agency for Toxic Substances and Disease Registry, U.S. Department of Health
and Human Services, Atlanta GA 30333, USA
<nroney@cdc.gov>
<hga0@cdc.gov>
<bxf9@cdc.gov>
<hrp1@cdc.gov>
ABSTRACT 144
1. EXPOSURE TO METALS AND THEIR MIXTURES 144
2. METALS AFFECTING THE HEMATOLOGICAL SYSTEM 145
2.1. Arsenic 145
2.2. Cadmium 145
2.3. Copper 145
2.4. Lead 146
2.5. Mercury 146
2.6. Tin 146
2.7. Zinc 147
3. BINARY INTERACTIONS OF METALS AND
HEMATOLOGICAL EFFECTS 147
3.1. Arsenic and Cadmium 147
3.2. Cadmium and Lead 148
3.3. Copper and Lead 148
3.4. Copper and Zinc 149
3.5. Iron and Lead 149
3.6. Manganese and Lead 150
144 RONEY, ABADIN, FOWLER, and POHL
3.7. Lead and Arsenic 150

3.8. Selenium and Arsenic 150
3.9. Tin and Zinc 151
3.10. Zinc and Copper 151
3.11. Zinc and Lead 151
4. INTERACTION OF METALS WITH OTHER CHEMICALS 152
4.1. Histidine-Rich Glycoprotein and Zinc 152
4.2. Gemcitabine, Hydroxyurea and Gallium 152
4.3. Chelating Agents 152
5. CONCLUSIONS 153
ABBREVIATIONS 153
REFERENCES 153
ABSTRACT: Many metals are essential elements and necessary for proper biological
function at low intake levels. However, exposure to high intake levels of these metals
may result in adverse effects. In addition, exposures to mixtures of metals may produce
interactions that result in synergistic or antagonistic effects. This chapter focuses on
metals that affect the hematological system and how exposures to mixtures of metals
may contribute to their hematotoxicity. Exposure to arsenic, cadmium, copper, lead,
mercury, tin or zinc has been shown to produce some effect on the hematological sys-
tem. Binary interactions resulting from exposure to combinations of metals may
increase or decrease the hematotoxicity induced by individual metals. For example,
copper, iron, and zinc have been shown to have a protective effect on the hematotoxi-
city of lead. In contrast, co-exposure to manganese may increase the hematotoxicity of
lead.
KEYWORDS: hematology . hematotoxicity . interactions . metals . mixtures
1. EXPOSURE TO METALS AND THEIR MIXTURES
Metals are ubiquitous in our lives and in our environment. Many metals are
essential elements for humans and necessary for proper biological function
at low intake levels. However, exposure to high intake levels of these metals
may result in adverse effects. Because of the pervasiveness of metals in our
environment, it follows that exposures to mixtures of metals is a likely
occurrence. For example, elevated concentrations of arsenic, cadmium,
copper, lead, mercury, and zinc have been identified in the environment near
mining and smelting sites. Also, at hazardous waste sites, copper, lead,
manganese, and zinc frequently co-occur in completed exposure pathways
[1,2]. In addition, co-exposures to metals often occur in occupational set-
tings. This chapter will discuss metal ions that affect the hematological
system and how exposures to metal mixtures may contribute to their
hematotoxicity.
Met. Ions Life Sci. 2011, 8, 143–155

METAL IONS AFFECTING THE HEMATOLOGICAL SYSTEM 145
2. METALS AFFECTING THE HEMATOLOGICAL

SYSTEM
There are a number of metals that affect the hematological system. Dis-
cussed below are specific metals and their associated hematological effects.
2.1. Arsenic
Anemia and leukopenia are commonly reported after ingestion of arsenic in
humans. In animals, dietary studies of arsenic have also reported hemato-
logical and hematopoietic effects, including decreased hematocrit and
increased urinary excretion of porphyrins [3]. The interference of arsenic with
mitochondrial heme-synthesis enzymes is thought to produce the increased
urinary excretion of uroporphyrin. Thus, the hematological effects of arsenic
may be due to both a direct cytotoxic or hemolytic effect on the blood cells
and a suppression of erythropoiesis. It should be noted, however, that
hematological effects are not observed in all cases of arsenic exposure [3].
2.2. Cadmium
Anemia has been reported in humans with chronic dietary exposure to cadmium
[4]. However, other studies have not found a significant relationship between
cadmium exposure and anemia. The conflicting results may be due to differ-
ences in iron levels. Oral cadmium exposure reduces gastrointestinal uptake of
iron, which can result in anemia if dietary intake of iron is low. A number of
animal studies have demonstrated that oral exposure to cadmium frequently
produces anemia, and that additional iron prevents the anemia. However, some
oral animal studies (especially chronic studies) have not seen these hematolo-
gical changes. In addition, hematological effects following inhalation of
cadmium in humans and animals are conflicting. Lowered hemoglobin con-
centrations and decreased packed cell volumes have been observed in some
studies of workers occupationally exposed to cadmium, but not in others. It is
uncertain whether other factors, in addition to reduced gastrointestinal
absorption of iron, such as direct cytotoxicity to marrow or inhibition of heme
synthesis may contribute to the anemia produced after cadmium exposure [4].
2.3. Copper
Data on the effect of copper on the human hematological system is limited
[5]. Workers exposed to copper in air had decreased hemoglobin and
Met. Ions Life Sci. 2011, 8, 143–155

erythrocyte levels. However, these workers may also have been exposed to
other metals. Acute hemolytic anemia and acute intravascular hemolysis
have been reported in humans after ingestion of large amounts of copper
sulfate. In animals, decreased hemoglobin and hematocrit levels have been
observed after exposure to high doses of copper. Excessive levels of copper
inhibit the enzyme glucose-6 phosphatase, which can lead to hemolysis [5].
2.4. Lead
Numerous worker and general population studies have shown that exposure to
lead results in hematological changes [6]. Lead alters the hematological system
by inducing anemia that is microcytic and hypochromic. The anemia induced
by lead is mainly the result of both inhibition of heme synthesis and shortening
of the erythrocyte lifespan. However, lead can also induce the overproduction
of the hormone erythropoietin, leading to inadequate maturation of red cell
progenitors, which can contribute to the anemia. Lead interferes with heme
synthesis by inhibiting the activities of several enzymes, in particular d-ami-
nolevulinic acid dehydratase (ALAD) and ferrochelatase. As a consequence of
these changes, heme biosynthesis is decreased and the activity of d-aminole-
vulinic synthetase (ALAS) is subsequently increased. ALAS is the rate-limiting
enzyme of the heme synthesis pathway which is feedback inhibited by heme.
The final results of these changes in enzyme activities are increased urinary
porphyrins, coproporphyrin, and d-aminolevulinic acid (ALA), increased
blood and plasma ALA, and increased erythrocyte protoporphyrin [6].
2.5. Mercury
Exposure to high concentrations of elemental mercury vapors produces a
syndrome characterized by fatigue, fever, chills, and elevated leukocyte
count [7]. This syndrome is similar to metal fume fever seen after exposures
to other metals. Following acute inhalation exposure to metallic mercury,
moderate-to-high leukocytosis with neutrophilia was observed in a number
of studies. In addition, studies in workers exposed to elemental mercury have
reported decreased ALAD activity in erythrocytes, and significant increase
in a-2-macroglobulin and ceruloplasmin (an a-globulin protein active in the
storage and transport of copper) compared to unexposed workers [7].
2.6. Tin
Exposures to tin compounds have produced hematological changes in ani-
mals [8]. Following exposure to excess dietary tin, signs of anemia including
Met. Ions Life Sci. 2011, 8, 143–155

decreased hematocrit, total erythrocytes, and hemoglobin levels were seen in

animals. Tin affects the metabolism of a number of essential minerals
including iron and copper. The hematological changes seen after exposure to
tin are most likely the result of a reduction of serum iron and copper levels
caused by tin. Indeed, the anemia symptoms seen in an animal (rat) study
were reversed by enriching the diet with iron and copper [8].
2.7. Zinc
The most commonly reported effect following inhalation exposure to zinc
oxide is metal fume fever [9]. One of the characteristics of metal fume fever is
leukocytosis lasting for up to 12 hours after the fever dissipates. Leukocytosis
has been observed in a number of case reports of occupational and experi-
mental exposure of humans to zinc oxide fumes. In addition, oral exposure to
zinc has also produced hematological changes in humans and animals.
Ingestion of zinc supplements has caused anemia, and decreased hematocrit,
serum ferritin, and erythrocyte superoxide dismutase activity in humans. In
addition, supplemental oral zinc exposure in humans has produced increases
in bone-specific alkaline phosphatase levels and extracellular superoxide
dismutase with decreases in mononuclear white cell 5 0 -nucleotidase and
plasma 5 0 -nucleotidase activity. In animals, anemia, decreased hemoglobin,
hematocrit, erythrocyte, and/or leukocyte levels were observed following oral
exposure to zinc compounds. The anemia seen in oral studies is thought to be
caused by zinc-induced copper deficiency. Since dietary zinc strongly affects
copper absorption, a diet high in zinc can result in copper deficiency [9].
3. BINARY INTERACTIONS OF METALS AND

HEMATOLOGICAL EFFECTS
Although people are often exposed to mixtures of metals, information on

whole multi-component mixtures is usually not available. Thus, binary
evaluations of mixture components are used to enable risk assessors to
predict the direction of possible interactions (see Chapter 3 for additional
information). Provided below are summaries of binary interaction studies of
some metals focusing on hematological effects.
3.1. Arsenic and Cadmium

A 10-week dietary study in rats investigated the effects of exposure to
50 ppm arsenic (B2.5 mg As/kg/day) and/or 50 ppm cadmium (B2.5 mg
Met. Ions Life Sci. 2011, 8, 143–155

Cd/kg/day) [10,11]. Both arsenic and cadmium increased the red blood cell
count, and arsenic decreased the hematocrit (cadmium decreased hematocrit
slightly but not significantly). Effects of the mixture were less than additive
on these endpoints. In contrast, cadmium did not affect the arsenic-induced
increase in urinary excretion of coproporphyrin and uroporphyrin.
3.2. Cadmium and Lead

In a 10-weeks dietary study, rats were administered 200 ppm lead (B10 mg/
Pb/kg/day) and/or 50 ppm cadmium (B2.5 mg Cd/kg/day) [10,11]. Cad-
mium inhibited the lead-induced increase in urinary ALA. Increased
excretion of urinary ALA is a result of the effects of lead on heme synthesis.
Cadmium’s amelioration of this effect of lead indicates that cadmium may
inhibit lead’s hematopoietic effects. Decreased hematocrit and hemoglobin
were seen in rats exposed to both metals, but not significantly in those
exposed to either metal alone at the same doses as in the mixture [10–12].
This finding indicates that subthreshold exposures to these metals can, in
combination, result in hematological effects. In contrast, decreases in ery-
throcyte size and hemoglobin content resulting from exposure to the mixture
of lead (B430 mg Pb/kg/day) and cadmium (B7.7 mg Cd/kg/day) appeared
additive in another study in rats as compared with exposure to each metal
alone at the same dose as in the mixture [12]. However, the effects of each
metal alone were not statistically significant and duration of this study (42
days) may have been insufficient to allow full expression of effects on these
hematological endpoints. In addition, small numbers of rats were used in the
experiment.
3.3. Copper and Lead

In humans receiving adequate dietary copper and a low dietary lead intake,
supplemental copper at a level about five times the RDA decreased blood
lead and had a protective effect on lead balance (changed lead balance from
positive to negative) [13].
In a 21-day gavage study in rats, the animals received 10 mg/kg/day lead
alone or lead together with supplemental copper of 2 mg/kg/day [14]. The
group receiving both metals had higher ALAD activity and lower lead body
burden than the group receiving lead alone. However, copper did not affect
the lead-induced decrease in hemoglobin levels in exposed animals. In a
subsequent study, rats were administered 100 ppm lead (B14 mg/kg/day) as
the acetate in drinking water and/or 100 ppm copper (B8.6 mg/kg/day) as
Met. Ions Life Sci. 2011, 8, 143–155

the sulfate in their diet for 30 days [15]. Supplemental copper attenuated
lead-induced hematopoietic effects (inhibition of ALAD in erythrocytes,
increase in zinc protoporphyrin, and increase in urinary ALA).
3.4. Copper and Zinc

When rats were fed a diet with extra zinc at 7,000 ppm (B897 mg/kg/day as
zinc carbonate) with or without supplemental copper at 0.2 mg/day
(B2.3 mg/kg/day as copper sulfate) for 6 weeks, co-exposure to copper
attenuated the zinc-induced decrease in blood hemoglobin [16]. A similar
result was obtained in a 5-week study in rats using high copper intake.
Hemoglobin levels were markedly decreased in rats that were fed diets with
7,500 ppm zinc (B900 mg/kg/day) as compared to the controls [17]. In a
group of rats exposed to zinc together with 200 ppm copper (B24 mg/kg/
day), copper attenuated this effect. A proposed mechanim of action suggests
that copper is an essential part of several enzymes including ceruloplasmin,
which oxidizes ferrous iron to the ferric form. Because only ferric iron is
bound to transferrin and transported to the bone marrow, this transfor-
mation is critically important to provide iron for hemoglobin synthesis [18].
Thus, excess copper may overcome the deficiency caused by excess zinc,
protecting against the hematological effects of zinc.
3.5. Iron and Lead

Results from the NHANES II national survey showed that in children low iron
status increases the lead hematotoxic dose response curves [19] and that iron
deficiency plus elevated lead in blood produce a greater degree of hemato-
toxicity compared with either factor alone [20]. In addition, a study of 299
children from 9 months to 5 years old from an urban area found a significant
negative association between blood lead and dietary iron intake [21]. Serum
ferritin concentrations were also associated with lower blood lead in a popu-
lation of pregnant women in Kosovo (former Yugoslavia) [22]. These results
suggest that dietary iron may inhibit lead absorption. Another study of 319
children ages 1–5 from Sacramento, California, found that iron-deficient
children had an unadjusted geometric mean blood lead of 1 mg/dL higher than
iron-replete children [23]. The difference persisted after adjusting for potential
confounders by multivariate regression; the largest difference in blood lead was
approximately 3 mg/dL and was present among those living in the most con-
taminated areas. While the studies mentioned above point to a link between
iron deficiency and lead poisoning, it is unclear whether there is a causal link or
whether iron deficiency is just a marker of high environmental lead.
Met. Ions Life Sci. 2011, 8, 143–155

Additionally, a longitudinal analysis of 1,275 children whose blood was

screened for lead and complete blood count on two consecutive visits to a
clinic suggested that the risk of subsequent lead poisoning associated with iron
deficiency is 4–5 times greater than the baseline risk of lead poisoning [24].
3.6. Manganese and Lead

In rabbits given a single intravenous injection of lead (1 mg/kg) and/or
manganese (0.28 mg/kg), the half-life of lead in blood was prolonged as
compared with the half-life of lead given without manganese [25]. Lead
inhibited blood ALAD activity and its recovery was slightly prolonged by
manganese over a 123-day period as compared to the group treated with lead
alone. By prolonging the residence of lead in blood, manganese is thought to
prolong the effects of lead on ALAD [25,26].
3.7. Lead and Arsenic

In an intermediate-duration (10 weeks) dietary study in rats, hematocrit was
significantly decreased and hemoglobin was slightly decreased by arsenic
alone, but not by lead alone or the lead-arsenic mixture. The dose of each
metal in the mixture was the same as when the metal was given alone, i.e.,
200 ppm lead (B10 mg Pb/kg/day) and 50 ppm (B2.5 mg As/kg/day) [10,11].
Other endpoints related to arsenic’s hematopoietic effects (urinary uropor-
phyrin and coproporphyrin excretion) indicated additivity or no effect of
lead [11,27]. In a chronic (1–2 years) dietary study, groups of female rats
were exposed either to lead arsenate providing a dose of 10 mg lead arsenate/
day (B18 mg Pb/kg/day and B6.3 mg As/kg/day), lead carbonate or cal-
cium arsenate [28]. The exposure in the last two groups was comparable to
daily doses of lead or arsenic in the lead arsenate group. Splenic hemosi-
derosis (an indication of red cell destruction) was less severe in rats coex-
posed to lead and arsenic than in rats exposed to arsenic alone, indicating a
protective effect of lead to arsenic-induced toxicity. Similarly, lead-induced
decreased hematopoietic activity in the spleen was less pronounced in the
lead arsenate group than in the lead carbonate group.
3.8. Selenium and Arsenic

In residents living in an area of Inner Mongolia with high levels of arsenic in
drinking water, administration of 100–200 mg selenium/day (in the form of
selenium yeast) and exposure to arsenic-free water for 14 months resulted in
Met. Ions Life Sci. 2011, 8, 143–155

a greater improvement in clinical signs and symptoms, liver function, and

electrocardiogram readings as compared to residents administered arsenic-
free water only [29,30]. An improvement in skin lesions was observed in 67
and 21% of the subjects in the selenium-supplemented and control groups,
respectively [30]. Additionally, the levels of arsenic in blood, hair, and urine
were significantly lower after the 14-months period only in the selenium
supplemented group.
3.9. Tin and Zinc

The effect of tin on heme biosynthesis appears to be dependent on the
concentration of zinc [31]. Oral administration of tin can affect heme
synthesis by inhibiting ALAD activity in blood. Zinc is required for ALAD
activity and provides a protective role in heme synthesis by increasing the
activity of ALAD. It is postulated that when the tin and zinc are co-admi-
nistered, these metals are probably attaching to similar binding sites in the
ALAD enzyme [31].
3.10. Zinc and Copper

Excessive dietary zinc has been shown to induce a reversible copper defi-
ciency and anemia in experimental animals [32–36]. Similar effects have been
seen in humans receiving long-term treatment with zinc [37,38]. A reduction
in erythrocyte superoxide dismutase (an index of metabolically available
copper), without a decrease in plasma copper levels, was exhibited following
exposure to high amounts of ingested zinc [39].
3.11. Zinc and Lead

An increase in urinary ALA above the normal range was significantly asso-
ciated with a decrease in the chelatable zinc/lead ratio to 18.45 or less in
children given chelation therapy for lead poisoning [40]. Supplemental zinc
protected against the inhibiting effects of lead on ALAD activity, and against
lead-induced increases in zinc protoporphyrin and urinary ALA excretion in
rats given both metals orally for intermediate durations [14,41–44]. These
protective effects were seen at higher (45–60 mg/dL), but not lower lead doses,
and when basal levels of zinc in the diet were adequate. Another study reported
a significant negative correlation between blood lead levels and ALAD activity
Met. Ions Life Sci. 2011, 8, 143–155

in 143 patients whose unknown exposures resulted in blood lead ranging from
4 to 115 mg/dL [45]. The percentage of activated ALAD that could be obtained
by the in vitro addition of zinc to the patients’ blood was correlated with blood
lead. These results imply a reactivation of lead-inhibited ALAD by zinc.
4. INTERACTION OF METALS WITH OTHER

CHEMICALS
Current literature is data-poor regarding interactions between metals and

other chemicals with regards to the hematological system. Provided below
are some binary interactions between metals and other chemicals, however,
the combinations do not represent exposures commonly encountered by
humans in their environment.
4.1. Histidine-Rich Glycoprotein and Zinc

It was demonstrated that histidine-rich glycoprotein binds heparin thus
diminishing its anticoagulant effect. Zinc increases this interaction in vitro [46].
It is not clear whether the histidine-rich glycoprotein/zinc combination can be
used in vivo because of the potential toxicity at high, clinically relevant, doses.
4.2. Gemcitabine, Hydroxyurea and Gallium

Gemcitabine and hydroxyurea are ribonucleotide reductase inhibitors that
are used to slow down the proliferation of human leukemic cells. Gallium
(administered as gallium nitrate) was demonstrated to act synergistically with
these drugs to further inhibit the cell proliferation in vitro [47]. Gallium can
affect the ribonucleotide reductase either directly by inhibiting the deoxy-
ribonucleotide synthesis of the enzyme or it can block the transport of iron to
the R2 subunit of ribonucleotide reductase. It was postulated that interaction
between gallium and gemcitabine may occur at several different levels.
4.3. Chelating Agents

Drugs such as penicillamine, trientine, ethylenediamine-N,N,N 0 ,N 0 -tetra-
acetate (EDTA), 2,3-dimercaptopropanol (British anti-Lewisite ¼ BAL),
and deferoxamine are used for treatment of systemic toxic effects of metal
exposures/overdoses. It is beyond the scope of this chapter to describe all the
chelators in detail; other text books provide this information [48–50].
Met. Ions Life Sci. 2011, 8, 143–155

However, deferoxamine can be specifically noted here for its use of the
treatment of iron overload [51]. It was reported that the uptake of defer-
oxamine by hepatocytes is several hundred-times quicker than by red blood
cells. It was suggested that low-dose continuous infusion of the chelator is
more beneficial, especially in the high-risk patients with failing heart func-
tion, than intermittent infusions of high-doses.
5. CONCLUSIONS
Exposure to some metals has been shown to cause various adverse effects to
the hematological system. Binary interactions resulting from exposure to
combinations of metals has been shown to result in additive, synergistic, and
antagonistic responses. For example, while individual arsenic and cadmium
exposure has been found to increase red blood cell count, co-exposure to
both metals has shown a less than additive effect on this endpoint. Cadmium
appeared to offset lead’s impact on heme synthesis as evidenced by a
decrease in urinary ALA, however, subthreshold dose levels of lead and
cadmium resulted in decreased hemoglobin and hematocrit when given to
rats as a mixture.
Copper, iron, and zinc have also been shown to have protective effects on
the hematotoxicity of lead, most likely by inhibiting lead absorption and/or
inducing metallothionein which sequestors lead [5,9]. Alternatively, man-
ganese prolonged the residence time of lead in blood, increasing its hema-
totoxicity. Thus, combined exposures to metals may increase or decrease the
hematotoxicity induced by individual metals.
ABBREVIATIONS
ALA d-aminolevulinic acid

ALAS d-aminolevulinic synthetase
NHANES National Health and Nutrition Examination Survey
RDA recommended daily allowance
REFERENCES
1. C. T. De Rosa, H. E. El-Masri, H. Pohl, W. Cibulas and M. M. Mumtaz,
J. Toxicol. Environ. Health, 2004, 7, 339–350.
Met. Ions Life Sci. 2011, 8, 143–155

2. H. R. Pohl, S. Tarkowski, A. Buczynska, M. Fay and C. T. De Rosa, Environ.

Toxicol. Pharmacol., 2008, 25, 283–291.
5. ATSDR, Toxicological Profile for Copper, Agency for Toxic Substances and
Disease Registry Atlanta, GA, 1999.
8. ATSDR, Toxicological Profile for Tin, Agency for Toxic Substances and Disease
Registry, Atlanta, GA, 2005.
9. ATSDR, Toxicological Profile for Zinc, Agency for Toxic Substances and Dis-
10. K. R. Mahaffey and B. A. Fowler, Environ. Health Perspect., 1977, 19, 165–171.
11. K. R. Mahaffey, S. G. Capar, B. C. Gladen and B. A. Fowler, J. Lab. Clin. Med.,
1981, 98, 463–481.
12. D. G. Thawley, R. A. Willoughby, B. J. McSherry, G. K. Mac Leod, K. A.
MacKay and W. R. Michell, Environ. Res., 1977, 14, 463–475.
13. C. Kies and S. W. Ip, Trace Subst. Environ. Health, 1990, 24, 177–184.
14. S. J. S. Flora, V. K. Jain, J. R. Behari and S. K. Tandon, Toxicol. Lett., 1982, 13,
51–56.
15. S. J. S. Flora, R. A. Coulombe, R. P. Sharma and S. K. Tandon, Ecotoxicol.
Environ. Saf., 1989, 18, 75–82.
16. S. E. Smith and E. J. Larson, J. Biol. Chem., 1946, 103, 29–38.
17. A. C. Magee and G. Matrone, J. Nutr., 1960, 72, 233–242.
18. L. Friberg, G. F. Nordberg and V. B. Vouk, Handbook on the Toxicology of
Metals, Vol. II, 2nd edn. Elsevier Science Publishers B.V., Amsterdam, 1986,
p. 242.
19. A. H. Marcus and J. Schwartz, Environ. Res., 1987, 44, 221–227.
20. K. R. Mahaffey and J. L. Annest, Environ. Res., 1986, 41, 327–338.
21. T. A. Hammad, M. Sexton and P. Langenberg, Ann. Epidemiol., 1996, 6, 30–33.
22. J. H. Graziano, D. Popovac, P. Factor-Litvak, P. Shrout, J. Kline, M. J. Mur-
phy, Y. H. Zhao, A. Mehmeti, X. Ahmedi and B. Rajovic, Environ. Health
Perspect., 1990, 89, 95–100.
23. A. Bradman, B. Eskenazi, P. Sutton, M. Athanasoulis and L. R. Goldman,
24. R. O. Wright, S. W. Tsaih, J. Schwartz, A. Spiro, 3rd, K. McDonald, S. T. Weiss
and H. Hu, Epidemiology, 2003, 14(6), 713–718.
25. M. Chiba and M. Kikuchi, Toxicol. Lett., 1984, 20, 143–147.
26. M. Chiba and M. Kikuchi, Toxicol. Appl. Pharmacol., 1984, 73, 388–394.
27. B. A. Fowler and K. R. Mahaffey, Environ. Health Perspect., 1978, 25, 87–90.
28. L. T. Fairhall and J. W. Miller, Public Health Rep., 1941, 56, 1610–1625.
29. W. Wuyi, Y. Linsheng and H. Shaofan, Curr. Sci., 2001, 81, 1215–1218.
Met. Ions Life Sci. 2011, 8, 143–155

30. L. Yang, W. Wang and S. Hou, Environ. Geochem. Health, 2002, 24, 359–374.
31. J. Chmielnicka, G. Zareba, E. Polkowska-Kulesza, M. Najder and A. Korycka,
Biol. Trace Elem. Res., 1993, 36, 73–87.
32. A. C. Magee and G. Matrone, J. Nutr., 1960, 72, 233–242.
33. L. Murthy and H. G. Petering, J. Agric. Food Chem., 1976, 24, 808–811.
34. B. J. O’Dell, Am. J. Clin. Nutr., 1969, 22, 1315–1322.
35. E. J. Underwood, Trace Elements in Human and Animal Nutrition, 4th edn.,
Academic Press, New York, New York, 1977.
36. R. A. Wapnir and C. Balkman, Biol. Trace Elem. Res., 1991, 29, 193–202.
37. K. G. Porter, D. McMaster, M. E. Elmes and A. H. Love, Lancet, 1977, 2, 774.
38. A. S. Prasad, G. J. Brewer, E. B. Schoomaker and P. Rabbani, J. Am. Med. Ass.,
1978, 240, 2166–2168.
39. P. W. F. Fischer, A. Giroux and A. R. L’Abbe, Am. J. Clin. Nutr., 1984, 40, 743–
746.
40. J. J. Chisolm, Jr., in Environmental Lead: Proceedings of the 2nd International
Symposium on Environmental Lead Research, ed. D. R. Lynam, L. G. Piantanida
and J. F. Cole, Academic Press, New York, 1981, pp. 1–7.
41. F. L. Cerklewski and R. M. Forbes, J. Nutr., 1976, 106, 689–696.
42. R. M. El-Gazzar, V. N. Finelli and J. Boiano, Toxicol. Lett., 1978, 1, 227–234.
43. S. J. S. Flora, S. Singh and S. K. Tandon, J. Int. Med. Res., 1989, 17, 68–75.
44. S. J. S. Flora, D. Kumar and D. Gupta, Pharmacol. Toxicol., 1991, 68, 3–7.
45. Y. Mauras and P. Allain, Enzyme, 1979, 24, 181–187.
46. C. L. Fu and M. K. Horne, J. Lab. Clin. Med., 2002, 139, 211–217.
47. M. S. Myette, H. L. Elford and C. R. Chitambar, Cancer Lett., 1998, 129, 199–
204.
48. M. J. Ellenhorn, Medical Toxicology: Diagnosis and Treatment of Human Poi-
soning, 2nd edn., Williams and Wilkins, Baltimore, Maryland, 1997, pp. 1563–
1579.
49. C. S. Homan, G. X. Brogan and R. S. Orava, in Emergency Toxicology, Ed.
P. Viccellio, Lippincott-Raven Publishers, Philadelphia, Pennsylvania, 1998,
pp. 363–378.
50. J. B. Leikin and F. P. Paloucek, Poisoning and Toxicology Handbook, 3rd edn.,
Lexi-Comp, Inc., Hudson, Ohio, 2002, pp. 725–731.
51. J. B. Porter, R. Rafique, S. Srichairatanakool, B. A. Davis, F. T. Shah, T. Hair
and P. Evans, Ann. N.Y. Acad. Sci., 2005, 1054, 155–168.
Met. Ions Life Sci. 2011, 8, 143–155

Met. Ions Life Sci. 2011, 8, 157–185
8
Metal Ions Affecting the Immune System
Irina Lehmann , Ulrich Sack, and Jörg Lehmann
Department of Environmental Immunology, Helmholtz Centre for Environmental Research–
UFZ, Permoserstraße 15, D-04318 Leipzig, Germany
<irina.lehmann@ufz.de>
ABSTRACT 157
1. INTRODUCTION 158
2. IMMUNOTOXICITY AND IMMUNOMODULATION 159
3. EFFECT OF HEAVY METALS ON INNATE IMMUNITY 160
4. EFFECT OF HEAVY METALS ON ADAPTIVE IMMUNITY 161
4.1. Humoral Immune Responses 161
4.2. Cell-Mediated Immune Responses 164
5. MECHANISMS OF HEAVY METAL-INDUCED
IMMUNOTOXIC/IMMUNOMODULATORY EFFECTS 166
5.1. Oxidative Stress 166
5.2. Induction of Apoptosis 167
5.3. Interference with Signalling Pathways 168
6. INFLUENCE OF HEAVY METALS ON THE RESISTANCE
TOWARD INFECTIONS 170
7. CHRONIC INFLAMMATION AND AUTOIMMUNITY 173
8. CONCLUDING REMARKS 176
ACKNOWLEDGMENTS 177
REFERENCES 178
ABSTRACT: Certain heavy metals have been reported to seriously affect the immune
system potentially resulting in a broad range of harmful health effects. Reported
158 I. LEHMANN, U. SACK, and J. LEHMANN
alterations in immune cell function include a variety of affected mechanisms. Thereby,

depending on the particular metal, its concentration, route and duration of exposure,
and biologic availability, the net outcome may be either immunosuppression or stimula-
tion of immune cell activity. Since the key importance of the immune system is protec-
tion of the host against pathogenic agents, an impaired immune competence inevitably
increases the susceptibility to invading pathogens. However, being aware that the
immune system represents a sensitively regulated network of different cells, tissues, and
soluble mediators it has to be stated that any form of dys-regulation may result in
adverse health effects with overstimulation being as harmful as inhibition of functional
activity. Chronic-inflammatory reactions, cancer development, hypersensitivity, allergic
and autoimmune diseases are known consequences of persisting overstimulation. All
these manifestations were already found to be related with heavy metal exposure.
KEYWORDS: adaptive immunity . apoptosis . autoimmunity . B cells . cell-mediated

immune response . humoral immune response . hypersensitivity . inflammation . innate
immunity . MAPK . NK cells . oxidative stress . phagocytes . resistance toward
infection . T cells
1. INTRODUCTION
During the past century, global industrialization has caused a dramatic

contamination of the environment with toxic heavy metals such as mercury,
cadmium or lead. Subsequently, environmental pollution with industrial
heavy metal emissions provides for their continual uptake by humans via
drinking water or via the nutrition chain through contaminated plants or
food products (i.e., milk, meat or fish). Another crucial source for accu-
mulation of heavy metals, in particular cadmium, in men is cigarette
smoking. Therefore, mechanistic studies identifying potential adverse effects
of heavy metals on human health are badly needed. Apart from numerous
toxic effects of heavy metals such as mutagenic, cancerogenic, teratogenic,
reprotoxic, nephrotoxic or neurotoxic effects [1–8], certain heavy metals
have been reported to seriously affect the immune system potentially
resulting in a broad range of harmful health effects such as cancer, auto-
immunity or allergy. Immune system abnormalities including reduced
numbers and/or functional impairment of immune cells (i.e., neutrophiles,
macrophages, NK cells, B and T lymphocytes), altered immunoglobulin
levels or proceeding inflammatory reactions have been shown in industrially
exposed workers as well as environmentally exposed humans (via drinking
water, dust aerosols or contaminated food stuffs) and animals. The fact, that
impairment of immune functions can occur at very low sub-toxic exposure
concentrations [9], actually considered as ‘safe’ levels, highlights the parti-
cular relevance of heavy metal-caused effects on the immune system.
Met. Ions Life Sci. 2011, 8, 157–185

METAL IONS AFFECTING THE IMMUNE SYSTEM 159
Due to the immaturity of the developing immune system in the prenatal

period and at younger ages diseases related with immune malfunction dis-
play higher prevalence. However, it has been shown in different animal
models that xenobiotic exposure may cause immunosuppressive effects in
younger as well as in aged animals [10,11]. It could be postulated that
xenobiotic exposure might cause a higher susceptibility to infection at
younger ages but a higher tendency to autoimmunity and a higher incidence
of tumor diseases in the aged. Although there exists some information on
age-related immunosuppression caused by the loss of essential trace element
metals (e.g., iron or zinc) in the elderly [12,13], so far, very few data is
available on age-related immune effects of toxic heavy metals. Blakeley [14]
studied the effect of Cd on humoral immunity in aged mice and found no
significant influence. However, this author has discussed that immunosup-
pressive effects of cadmium, which have been documented in younger, more
immunologically competent mice, were masked by the natural age-related
immunosuppression. Therefore, immunotoxicological studies are not
recommended in aged experimental animals.
In this review article we summarize the scientific information on adverse
effects of toxic heavy metals onto the immune system available so far.
2. IMMUNOTOXICITY AND IMMUNOMODULATION
Reported alterations in immune cell function include a variety of affected

mechanisms. Thereby, depending on the particular metal, its concentration,
route and duration of exposure, and biologic availability, the net outcome
may be either immunosuppression or stimulation of immune cell activity.
Since the key importance of the immune system is protection of the host
against pathogenic agents, an impaired immune competence inevitably
increases the susceptibility to invading pathogens. However, being aware
that the immune system represents a sensitively regulated network of dif-
ferent cells, tissues, and soluble mediators it has to be stated that any form of
dysregulation may result in adverse health effects with overstimulation being
as harmful as inhibition of functional activity. Chronic inflammatory reac-
tions, cancer development, hypersensitivity, allergic and autoimmune dis-
eases are known consequences of persisting overstimulation. All these
manifestations were already found to be related with heavy metal exposure.
Hence, rather than a loss of cell viability, immunotoxicity represents a
modulation of immune functions including suppression and over-
stimulation. Consequently, solely the estimation of immune cell viability is
not sensitive enough for analyzing metal effects on immune competence. It
was consistently stated that functional studies are more sensitive and
Met. Ions Life Sci. 2011, 8, 157–185

therefore better suitable to unravel immune system disturbance by any

xenobiotic agent [15].
3. EFFECT OF HEAVY METALS ON INNATE IMMUNITY
The innate (unspecific) immune system provides the first line of defense
against invading pathogenic agents, thereby protecting the host against
infectious diseases. Cells of the innate immune response are phagocytic cells
including neutrophiles, macrophages, and dendritic cells, eosinophilic and
basophilic granulocytes, mast cells, and natural killer cells. All these cells
respond to pathogens in a rather unspecific manner leading to a transient
protective immunity of the host. Moreover, cells of the innate immune
system cooperate with cells of the adaptive immune system such as B and T
lymphocytes to induce specific immune reactions that finally combat the
invaded pathogen.
Phagocytosis is the major process to eliminate pathogens and debris.
Phagocytic cells internalize solid particles including bacteria by membrane
inversion and intracellular vesicle formation. Intracellular degradation of
ingested particles is realized by fusion with lysosomes. Effects of exposure to
heavy metals on phagocytic cells have been evaluated in various systems.
Exposure to HgCl2 and CdCl2 reduced the phagocytic activity of bovine
neutrophiles in a dose-dependent manner. Thereby, the different metals
affected neutrophile function unequally. While CdCl2 affected phagocytosis
in bovine neutrophiles at lower doses compared to HgCl2, the latter caused a
more severe inhibition of phagocytosis at concentrations of 10–5 M [16].
Human neutrophiles exposed to lead in vitro showed also a decreased pha-
gocytic activity [17,18].
The reported in vitro results were supported by data obtained in humans
occupationally exposed to heavy metals. A reduced phagocytic activity and
neutrophile chemotaxis was observed in lead-exposed workers (mean blood
lead concentration: 3.06 mmol/L) compared to healthy individuals [19].
Exposure to inorganic mercury was found to be associated with a decreased
neutrophile function in vitro of exposed compared to nonexposed workers
[20,21].
Macrophages are mononuclear phagocytes responsible for numerous
homeostatic, immunological, and inflammatory processes. These cells par-
ticipate both in unspecific immunity against bacterial, viral, and fungal
pathogens and specific immunity via antigen presentation and release of
specific mediators involved in T-cell activation.
Mercury-exposed murine macrophages were found to show an impaired
response to bacterial infection [22]. Inorganic mercury decreased the ability
Met. Ions Life Sci. 2011, 8, 157–185

of macrophages to produce nitric oxide, an important factor in host resis-

tance to bacteria. A strong species sensitivity was found regarding the sen-
sitivity of alveolar macrophages to nickel-copper compounds with the
following strength of effect: dog4rat4mouse [23].
Natural killer (NK) cells are primordial lymphocytes with a cytotoxic
potential involved in innate immune responses. Due to their ability to dis-
play spontaneous cytotoxicity against tumor cells, NK cells play a significant
role as part of the first line in defense against cancer cells [24]. Nickel
compounds were found to suppress NK-cell activity [25–28] and in addition
to induce tumor development [29]. It has been discussed that the cancer-
promoting potential of nickel could be attributed to the impairment of NK-
cell activity following Ni exposure [25,30]. The observation that mice of the
A strain expressing low NK-cell activity show an increase in lung tumor
development following exposure to nickelous acetate [31] may support this
hypothesis.
Beside their anticarcinogenic potential NK cells are also involved in
unspecific immune responses against pathogens. Consequently, functional
impairment of NK-cell activity was found to contribute to altered host
resistance in nickel-treated animals [26–27,32]. In addition to nickel, mer-
cury (MeHg: 120 and 200 ng/mL [33]), cadmium [34–36], lead, and zinc [35]
were also found to impair NK-cell activity. However, conflicting reports
have been given on heavy-metal effects on NK-cell function. Other studies
reported that exposure to lead, chromium, nickel, and cadmium fails to
impair NK-cell activity in rat, mice, and man [37–41].
4. EFFECT OF HEAVY METALS ON ADAPTIVE

IMMUNITY
The dose and the virulence of invaded pathogens for the individual host
decide whether innate immunity can control the infection at local sites or
whether the pathogen is disseminating to other tissues and organs resulting
in systemic infection. In this case, only the adaptive immune response gen-
erating antigen-specific effector and memory T and B cells is capable of
controlling the infection and restoring the integrity of the organism.
4.1. Humoral Immune Responses

B cells are lymphocytes responsible for the production and secretion of
specific antibodies. Following infection, B cells recognizing specific antigenic
Met. Ions Life Sci. 2011, 8, 157–185

determinants (epitops) are selected for clonal expansion and differentiation

to IgG-producing plasma cells. Antibodies secreted by B cells or plasma cells
mediate several antibody-dependent defense mechanisms such as ADCC,
opsonization, and neutralization of viruses or bacterial toxins. Impaired B-
cell response is associated with enhanced susceptibility to bacterial and viral
infections, a higher sensitivity to bacterial toxins and persisting bacterial and
viral infections.
There are a couple of reports demonstrating a significant influence of
heavy metals on B-cell activity and antibody production. However, the
messages drawn from these reports are controversial making it difficult to
conclude a general principle of the effect of heavy metals collectively or a
definite effect of a certain heavy metal on the B-cell response. Most com-
prehensive information is available on the influence of cadmium on the
B-cell response. Therefore, we focus on this heavy metal and mention the
situation for other heavy metals if data are available. All data published on
this matter have to be discussed in the context of the particular model.
Earlier studies suggested an immunosuppressive effect of Cd in rodent
models independently of the application form and route. Mice exposed to
subclinical doses of cadmium chloride (i.e., 3–300 mg/g) for ten weeks via the
oral route and inoculated with antigen six weeks after discontinuance of
exposure had a remarkable decrease in antibody-forming cells, particularly
IgG [42]. This was confirmed in part by Borgman et al. [43] who observed
only a suppression of the very early (day 5 post immunization) antibody
response to sheep red blood cells after administration of 50 ppm Cd via
drinking water. Similar results have been reported by Shippee and coworkers
[44] and Fujimaki [45] for application of 2.8 or 1.8 mg/kg, respectively, via
the subcutaneous route as a single injection. A more differentiated view is
given by Malavé and De Ruffino [46]. These authors found a slightly
increased antibody response in mice exposed to 50 or 200 ppm of Cd as
CdCl2 over 3 to 4 or 9 to 11 weeks but in contrast, exposure of 300 ppm Cd
via drinking water or i.p. injection of 2.5 mg/kg resulted in suppression of the
antibody response. The concept that lower Cd doses had no influence on the
humoral immune response whereas higher doses result in suppression of
antibody secretion was later confirmed by two other groups [47,48].
Other authors did not find any influence of Cd onto the humoral immune
response following administration of CdCl2 via drinking water [49,50]. In a
mouse model of Salmonella Enteritidis (SE) infection, we could show that
pretreatment with Cd had selective effects onto the production of individual
antibody isotypes (N. Hemdan, U. Sack, J. Lehmann, unpublished).
Whereas serum levels of SE-specific IgM antibodies were found to be higher
in Cd-exposed mice, IgA, IgG1, and most apparently IgG2b antibody titres
were significantly reduced when the mice had been pre-treated with Cd. IgM
antibodies can be produced in a T-cell independent fashion, thus it is
Met. Ions Life Sci. 2011, 8, 157–185

assumed that Cd affects the T-cell support for IgG class switching. In con-
trast to our findings earlier reports [51,52] demonstrate the suppressive
influence of Cd selectively on the generation of antibodies against T cell-
independent antigens such as DNP-Ficoll or LPS which belong to the IgM
class.
It is very likely that decreased levels of IgA and IgG antibodies result in
elevated invasion of bacteria at mucosal sites and decreased antibody-
dependent cellular cytotoxicity (ADCC) [35], decreased complement lysis,
and lower capacity of opsonization of salmonellae. Together, these
impairments result in higher extracellular bacterial burden. In this model,
the higher level of SE-specific IgM antibodies is obviously not capable of
compensating for the lag of IgA, IgG1, IgG2b antibodies. IgG2a antibodies
were not affected by cadmium. Interestingly, the production of salmonella-
specific IgG3 antibodies was selectively increased following administration
of the lower of both tested Cd doses (i.e., 0.07 mg/kg), whereas the higher
dose (i.e., 0.7 mg/kg) had no significant influence onto IgG3 production.
Selective toxic effects of Cd and Hg on the biology of murine B lymphocytes
and the secretion of IgG subclasses were also described by Daum et al. [53].
These authors found that IgG3 production was most sensitive to inhibition
by Cd or Hg, followed by IgG1 and IgG2b, less influence was observed by
IgM and IgG2a.
From the reports cited it can be concluded that the influence of Cd onto
the humoral immune response depends predominantly on the metal dose but
also on the route and the time point of administration. In terms of the
antigen-specific IgG response, in most situations Cd had suppressive effects.
One of the most interesting findings is that IgG3 antibody synthesis seems to
be a particularly sensitive target of Cd- and Hg-induced immunomodula-
tion. However, the underlying mechanism for this phenomenon has to be
still elucidated. Although the IgM response does not seem to be a target of
Cd-mediated immunosuppression, this is of lower relevance since most of
the important biological functions of antibodies during the immune response
are mediated by antibodies belonging to the IgG class. These findings from
experimental animal models are in agreement with findings from an epide-
miologic study in humans. In a health survey of school children in heavily
Cd-polluted regions of Eastern Germany increasing body burdens of Cd
were associated consistently with lower total IgG blood levels, while IgM,
IgA, and IgE concentrations were not significantly changed [54].
Results from studies of human peripheral blood lymphocytes in vitro have
indicated that the tendency of Cd to inhibit immunoglobulin production
may be somewhat dependent on individual variability, such that the reaction
of the immune system to low doses of Cd might be modified by an indivi-
dual’s susceptibility. Thus, genetic factors could be of importance for the
observed variability of the immune response to cadmium and these authors
Met. Ions Life Sci. 2011, 8, 157–185

support the hypothesis that differences in the metallothionein inducibility

could play a role [55].
In contrast to cadmium, lead was shown to enhance antibody secretion of
human peripheral blood lymphocytes in vitro [55]. Although in a special model
(guinea pig model of Ascaris suum infection) the specific circulating antibody
level was found to be decreased in mercury-treated animals [56], Hg seems to
have no significant influence on immunoglobulin production, neither in normal
situation nor in autoimmunity [57]. However, two effects especially decribed
for mercury are the elevation of serum IgE levels in Hg-exposed workers or
patients with type 1 allergic diseases such as acute atopic eczema [58,59] and the
association with serum autoantibody titres, in particular antinuclear and
antinucleolar autoantibodies [60]. These observations demonstrate a close
relationship between mercury exposure and immunologic disorders in humans.
4.2. Cell-Mediated Immune Responses

Cell-mediated immune effector mechanisms are realized by T cells. This lym-
phocyte population is activated by antigen-presenting cells (APC) via the pre-
sentation of antigenic peptides in the context of MHC class I (CD81 cytotoxic T
cell) or MHC class II molecules (CD41 helper T cell) and accessory signals such
as cytokines or surface molecules realizing cell-cell contact. Helper T cells
mediate differentiation signals for B cells (e.g., IL-4, IL-6) regulating immu-
noglobulin class switch. There are at least five different subpopulations of CD41
T cells: Th1, Th2, Th17, Th22, and Treg cells. Th1 cells are the predominant
subpopulation of cellular immunity with the major function to activate mac-
rophages through secretion of IFN-g whereas Th2 cells trigger antibody
synthesis through IL-4, IL-5, and IL-6. Th17 cells are also inflammatory T cells,
which are involved in the control of extracellular bacterial pathogens through
the secretion of IL-17, which again is responsible for the differentiation of
neutrophiles. IL-22, the key cytokine of Th22 cells stimulates secretion of
defensins as part of the very first defense line at skin and mucosal sites. Treg cells
are the main players for finalization of the inflammatory response and restoring
the homoeostasis. The well-balanced interaction between these T-cell sub-
populations guarantees an effective immune response and avoids a pathologic
outcome. Impaired T-cell response can be associated with enhanced suscept-
ibility to protozoic, fungal, bacterial, or viral infections as well as tumors.
There are several reports demonstrating a significant influence of heavy
metals on cellular immunity. In general, it seems that lymphocytes, in par-
ticular T cells, are more susceptible to effects of Hg compared to monocytes
[33]. Reflecting the suppressive effect of Cd on the IgG response it can be
concluded that this heavy metal must have an immunosuppressive effect on
Met. Ions Life Sci. 2011, 8, 157–185

Th cells, too, since Th-cell activation is a fundamental requirement for IgG

class switching. Indeed, an immunosuppressive influence of Cd exposure on
T cell-mediated immunity has been consistently documented. Early reports
demonstrated that oral exposure to 30, 300, and 600 mg/g Cd acetate caused
a dose-dependent decrease in delayed-type hypersensitivity which represents
a suitable method for measuring antigen-specific local T-cell activation
in vivo. However, polyclonal activation of spleen-derived T cells from Cd-
exposed mice through T cell mitogens (i.e., Con A, PHA) was enhanced [49].
This result was later confirmed by other groups [48,50,61].
Although there is no doubt about a suppressive influence of Cd on cellular
immunity, only poor data are available on the molecular mechanisms how
Cd is suppressing T cell-mediated immunity. Our group has started two
attempts to identify these mechanisms by means of in vitro and in vivo
approaches. Following in vitro exposure of human peripheral blood mono-
nuclear cells to Cd or Hg we could show that IFN-g concentration in culture
supernatants was significantly higher than IL-4 levels suggesting a bias of the
Th1/Th2 balance due to exposure to Cd or Hg salts [62–64]. In contrast,
Krocova et al. [65] reported that cadmium at a dose of 20 mg/mL (177.9 mM)
preferentially enhances the proliferation of Th2 murine cells activated ex vivo
by Con A. While high Hg doses are highly toxic, exposure to lower and
moderate doses exerts immunomodulatory effects resulting in inclination of
the immune response toward type-2 in anti-CD3/CD28-activated cells due to
induction of IL-4 and IL-10 release. Production of the Th1 cytokine IFN-g as
well as the pro-inflammatory cytokines TNF-a and IL-6 was reduced [63].
These studies let assume that the dose of Cd or Hg as well as the pathway
of immunostimulation may result in controversial T-cell differentiation.
Recent data from our in vivo model of SE infection (described above)
delivered evidence for the dose-dependency of Cd-incuded immunomodu-
latory effects. Moreover, results found in this model did not promote the
assumption that Cd is triggering or suppressing selectively one of both type-
1 or type-2 immune responses. More likely, Cd was inducing different, in
part counter-regulating, immune mechanisms in a dose-dependent manner.
It seems that cadmium and probably also other heavy metals cause a strong
and simultaneous stimulation of several cell types with the consequence of a
dysregulated and finally impaired immune response. For instance, Cd-
exposed SE-infected BALB/c mice had increased levels of serum protein and
spleen mRNA expression of IFN-g and TNF-a levels by day 3 p.i. IFN-g
was reverted thereafter and remained inhibited up to day 30 p.i. similar to
IL-12p40. The inhibition of IFN-g and IL-12p40 during the late acute phase
of SE infection (i.e., day 20–30) coincided with delayed bacterial clearance.
These mice revealed increased serum levels of MCP-1 at day 1 and 3 p.i. and
increased serum IL-6 and IL-10 levels at day 3 p.i. (comparable to controls),
accompanied by an increase in spleen mRNA of IL-4, IL-10, IL-6, and
Met. Ions Life Sci. 2011, 8, 157–185

TGF-b1 up to day 30 p.i. (N. Hemdan, U. Sack, J. Lehmann, unpublished).

Thus, the stimulation of the type-1 immune response alone is obviously not
sufficient for an effective immune response.
We could show that Cd also stimulates high amounts of IL-17 and IL-10
suggesting the induction of Th17 and Treg cells. A third aspect was that
immunosuppressive cytokines such as IL-6 or TGF-b were found to be much
less suppressed following Cd exposure compared to SE-infected controls not
pre-treated with Cd. Together, such broad stimulation of the immune system
may prevent effective bacterial clearance. Furthermore, such a situation
might be the basis for development of chronic inflammation. This should be
the subject of further investigations.
5. MECHANISMS OF HEAVY METAL-INDUCED

IMMUNOTOXIC/IMMUNOMODULATORY EFFECTS
A range of different immunomodulatory mechanisms of heavy metals have

been discovered so far. The most important mechanisms are reviewed in the
following paragraphs. But additionally to these well-understood modes of
action there seem to be also other heavy metal-induced mechanisms capable
of contributing indirectly to immunotoxicity. For instance, cadmium com-
petes with iron at the same metal binding sites in the iron transfer proteins
[66–68] which may interfere with immune cell function [69]. While replacing
iron and copper in various cytoplasmic and membrane proteins (e.g., ferri-
tin), cadmium can indirectly increase the amount of unbound free or chelated
copper and iron ions which then participate in oxidative stress via Fenton
reactions [70,71]. Moreover, apoptosis may be induced by cadmium indir-
ectly through formation of non-radical oxidative stress by inhibition of
antioxidant enzymes [72]. Present work is encouraged to identify the influ-
ence of toxic heavy metals in low subtoxic doses on the cytokine network and
the activation or suppression of the different T helper cell subpopulations.
Results from these studies will certainly strengthen the understanding of the
immunotoxic mechanisms of toxic heavy metals during the next years.
5.1. Oxidative Stress

The mechanisms underlying immunotoxic/immunomodulatory effects of
heavy metals are not completely understood, but there are several lines of
evidence that oxidative stress is involved in perturbations of immune cell
function and tissue damage. Oxidative stress is caused by an imbalance
Met. Ions Life Sci. 2011, 8, 157–185

between the production of reactive oxygen radicals (ROS) and antioxidant

defense mechanisms. At physiological concentrations, ROS act as second
messengers involved in the regulation of the activity of several signal
transduction pathways [73], such as mitogen-activated protein kinase
(MAPK) signalling pathways [74]. Among others, regulation of these
pathways is of crucial importance for cell survival. Their sustained activation
was found to be related with carcinogenesis [75].
Certain heavy metals, such as cadmium, nickel, copper, arsenic, mercury,
cobalt, and chromium have been shown to induce oxidative stress by ROS
[76]. Thereby, oxidative stress generation by heavy metals may occur by
different mechanisms. Lead was found to inhibit the activity of the d-ami-
nolevulinic acid dehydratase, an enzyme involved in heme biosynthesis.
Inhibition of this enzyme results in the accumulation of its substrate d-
aminolevulinic acid (d-ALA), that can be rapidly oxidized to generate free
radicals as hydroxyl radical (dOH), superoxide ion (Od2 ), and hydrogen
peroxide (H2O2) [77]. Several studies showed that accumulation of d-ALA
induces ROS generation [78,79]. Arsenic was discussed to produce H2O2
during the oxidation of iAsIII to iASV [78,80]. Yamanaka et al. [81] proposed
the formation of intermediary arsenic species.
Perturbance of antioxidative defense mechanisms may further contribute
to oxidative stress-mediated adverse health effects of heavy metals. Lead,
cadmium, and mercury all have electron-sharing affinities that can result in
formation of covalent attachments. These attachments are mainly formed
between heavy metals and sulfhydryl groups of proteins [82]. Depletion of
protein-bound sulfhydryl groups and thiol group-containing proteins, such
as glutathione (GSH), has been implicated in metal-induced oxidative
damage [83,84], as reported for mercury [85,86] or arsenic [87]. GSH and
other thiol group-containing compounds are important antioxidant com-
pounds that help to protect cells from ROS by acting as electron donors.
Furthermore, oxidative stress seems to be of relevance for disease devel-
opment following low level exposure to these metals. Beside oxidative tissue
damage in the heart, liver, kidney, and brain, ROS-induced dysfunction in
immune competent cells have been described [88–90]. Indirect support for the
involvement of oxidative stress in heavy metal-induced immune system-
derived diseases comes from studies demonstrating beneficial or protective
effects of antioxidants against immunosuppressive or immunotoxic effects [91].
5.2. Induction of Apoptosis

The highly regulated process of programmed cell death that occurs in
multicellular organisms is named apoptosis. This process is considered as a
Met. Ions Life Sci. 2011, 8, 157–185

continuous normal event in the control of cell populations. Apoptosis

essentially occurs when cellular damage, including DNA damage, has
exceeded the capacity for repair. Impairment of apoptotic regulation could
facilitate aberrant cell accumulation, which may be a critical step in malig-
nancy or autoimmunity [92].
Apoptosis can be induced by a variety of xenobiotics, including toxic
metals, resulting in the loss of affected cell population. Low levels (0.6 to
5 mM) of methylmercury and mercury chloride have been shown to induce
apoptosis in human T cells. Induction of reactive oxygen species and resulting
depletion of cellular thiols have been discussed to predispose cells to death
signal activation [93,94]. A further study compared cell death-inducing effects
of mercuric compounds (HgCl2 and methyl-HgCl) in human lymphocytes and
monocytes and described stronger effects in monocytes compared to lym-
phocytes with methyl-HgCl being approximately 5–10 times more potent than
HgCl2 [15]. The authors discussed higher accumulation rates in monocytes
compared to lymphocytes as one possible determinant of cell-specific toxicity.
Methylmercury was reported to induce germ cell apotosis and reproductive
toxicity in Wistar rats and impair spermatogenesis via apoptosis [95].
Although detailed molecular mechanisms are not completely understood,
there is some evidence that different effects may contribute to heavy metal-
induced apoptosis. Mercury was found to rise intracellular Ca21 levels thereby
contributing to the expression of so-called ‘‘death genes’’ and/or the stimulation
of enzymes initiating irreversible degradative changes that lead to cell death [15].
The ability of arsenic trioxide (As2O3) to induce apoptosis in leukemia cells is
utilized by applying this compound as anticancer drug in the treatment of
promyelocytic leukemia. Disruption of mitochondrial transmembrane poten-
tials is the main mechanism of As2O3-induced apoptosis [96]. An involvement of
sulfhydryl (-SH) groups was mentioned in this process. The mode of Hg-
induced apoptosis in human lymphocytes (at 2.5 mM level) involves activation of
polyADP-ribose polymerase, NAD1 depletion, and altered mitochondrial
redox status [97,98]. Generation of ROS and subsequent activation of redox-
sensitive MAPK signalling pathways (see Section 5.3) has been found to play a
role in cadmium, chromium, mercury, and arsenic-induced apotosis [92,99–102].
5.3. Interference with Signalling Pathways

Cells try to defend against heavy metal-induced toxic effects by activating
stress-responsive genes, encoding for proteins involved in repair mechanisms
or metal removal [103–105]. Metals have the capacity to affect the cellular
behavior by influencing intracellular signal transduction. They can directly
induce gene expression through the activation of metal-responsive
Met. Ions Life Sci. 2011, 8, 157–185

transcription factors or can affect cells in a more unspecific way, by inducing

oxidative stress and activating redox-sensitive transcription factors. The
resulting cellular response involves the activation of different, but often
interacting, signal transduction pathways [106]. Thereby, pathways affected
by toxic concentrations may differ from those affected by subtoxic levels [103].
The best characterized transcription factor directly induced by metals is
MTF-1, a metal response element (MRE)-binding protein. Genes, coding for
metal-binding proteins, contain multiple copies of MREs in their promoter
regions [107]. Other genes responding to metal through MREs are genes
coding for acute phase reactant a1-acid glycoprotein and C-reactive protein.
In BALB/c mice, mercury, cadmium, lead, copper, nickel, zinc, and mag-
nesium (in order of responsiveness) were shown to response of these genes
through MREs. Other stress-response genes, such as heme oxygenase or
glutathione S-transferase, enzymes involved in protection against oxidative
stress, are activated via binding of transcription factors to antioxidant
response elements (ARE) located in their promoter region. Factors binding
to ARE sequences may contribute to the response to cadmium [106].
The nuclear factor-kB (NF-kB) is known as a redox-sensitive transcription
factor, its activation has been shown to be related with intracellular GSH
levels. GSH controls and regulates inflammatory processes, depletion of
intracellular GSH levels correlate with NF-kB phosphorylation, increased
NF-kB nuclear binding, and the induction of an inflammatory response
[108,109]. Up-regulation of cellular adhesion molecules and activation of
inflammatory cytokines by metals was shown to be induced via NF-kB.
Treatment of human umbilical vein endothelial cells (HUVECs) with 1mM
NiCl2 or CoCl2 increased NF-kB translocation to the nucleus, the expression
of the adhesion molecule E-selectin on the cell surface membrane and the
production of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8
[110,111]. Cadmium chloride exposure was shown to activate IL-8 expression
in the human intestinal epithelial cell line Caco-2 in a NF-kB-dependent
manner [112]. A copper-dependent activation of NF-kB signalling was
demonstrated in human HepG2 cells [103,113]. Arsenic is a further potent
activator of NF-kB-regulated IL-8 expression [114]. Beside inflammation, the
NF-kB pathway has also been shown to be a prominent factor in cell death/
survival balance. Several studies have demonstrated that low dose cadmium
exposure is associated with NF-kB-dependent induction of apoptosis.
Mitogen-activated kinases (MAPKs) are a family of Ser/Thr kinases that
transfer signals into the nucleus, and have been shown to play a regulatory
role in diverse cellular responses such as inflammation, proliferation, and
programmed cell death [115,116]. Members of the MAPK family are the
extracellular signal-regulated protein kinases (ERK1/2), cJun N-terminal
kinases (JNK), and p38. It has been reported that heavy metals such as
cadmium can activate each MAPK family member simultaneously. Each
Met. Ions Life Sci. 2011, 8, 157–185

subfamily member was shown to be phosphorylated by CdCl2 exposure in a

time (1–9 h) and dose (1–20 mM) dependent manner in the mouse fibroblast
cell line NIH 3T3 [117]. Comparable results were found in the human liver
cell line HepG2 [118], in macrophages [119], renal cells [120], and different
tumor cells [121,122]. In human dendritic cells, exposure to NiSO4 was
found to induce the three MAPK pathways resulting in surface expression of
CD83, CD86, and CCR7 and cell maturation. Activated dendritic cells are
involved in the pathology of nickel-induced allergic contact dermatitis [123].
Similarly to NF-kB, MAPK activation by heavy metal results from oxi-
dative stress and intracellular GSH depletion. Pre-treatment of rat glioma
cells with the antioxidant NAC or GSH was found to prevent GSH deple-
tion and p38 MAPK activation by cadmium [124]. Several studies reported
an activation of a heat shock response via MAPK signalling that may
protect cells from oxidative injury and apoptotic cell death. Kim and
Sharma [85] reported that human breast cancer cells induced Hsp70
expression against arsenic trioxide exposure against arsenic or cadmium
toxicity. Leal et al. [125] described a p38-dependent activation of Hsp27 in
bovine adrenal chromaffin cells in response to cadmium exposure.
Low level heavy metal exposure alone may not be sufficient to cause tissue
damage through inflammatory processes. However, combined exposure to
non-toxic doses of bacterial endotoxin (LPS) in addition to low levels of
heavy metals can evoke serious health problems. At least mercury and nickel
were found to exacerbate p38 MAPK signalling in response to bacterial
endotoxin resulting in overexpression of tumor necrosis factor a (TNF-a)
[85]. We have observed comparable results for cadmium chloride. Cadmium
chloride exposed human peripheral blood mononuclear cells (PBMC)
showed an overexpression of TNF-a in the presence of LPS or heat-killed
Salmonella bacteria, while low doses of the metal alone were without effect
(Figure 1). Excessive increase in TNF-a expression by liver macrophages
(Kupffer cells) was found to be the key mechanism in mercury-induced liver
injury by causing apoptosis of murine hepatocytes [126].
6. INFLUENCE OF HEAVY METALS ON THE

RESISTANCE TOWARD INFECTIONS
There is accumulating evidence for a significant impact of heavy metal

exposure on the resistance toward infection. Thereby, the described reduc-
tion of host defense mechanisms by heavy metal exposure contributes to the
increased susceptibility to pathogenic agents. However, although many
studies have been performed with experimental infection models, extremely
few data are available regarding heavy metal effects on resistance to
Met. Ions Life Sci. 2011, 8, 157–185

A
500
unstimulated
10 pg/ml LPS
400 100 pg/ml LPS
TNF-α production [% control]
1000 pg/ml LPS
300
200
100
0
0,1 1 10 100
CdCl2 [µM]
B
500
unstimulated
10°5 SE
400 10°6 SE
TNF-α production [% control]
10°7 SE
10°8 SE
300
200
100
0
0,1 1 10 100
CdCl2 [µM]
Figure 1. (A) Overexpression of TNF-a in human peripheral blood mononuclear

cells (PBMC) following exposure to cadmium in the presence of lipopolysaccharide
(LPS) or (B) heat-killed Salmonella Enteritidis (SE) bacteria for 24 hours. Cells
exposed to cadmium alone were marked as ‘‘unstimulated’’ samples. Summarized
results from 6 healthy donors are shown with medians and inter quartile ranges.
Met. Ions Life Sci. 2011, 8, 157–185

infections in humans. In lead-exposed workers (lead blood levels 21–90 mg/

dL) more cold and influenza infections were found per year, together with
decreased IgM, IgE, and IgA blood levels [127].
Resistance to experimental infections has consistently shown to be decreased
by cadmium. Cook et al. [128] reported a 1000-fold increased susceptibility of
rats to Escherichia coli exposed to a single dose of 6 mg/kg cadmium acetate.
Comparable results were observed in mice infected with Mycobacterium bovis
[129], cytomegalovirus [130] or herpes simplex virus [50]. We could confirm
these earlier obtained data in mice infected with SE. Mice exposed to cadmium
14 days before infection failed to clear the inoculated bacteria resulting in lethal
outcome at infection doses otherwise being controlled (N. Hemdan, U. Sack, J.
Lehmann, unpublished). Thereby, mice exposed to Cd, but not infected with
SE, did not show mortality. Using lower infection doses it was confirmed that
Cd exposure led to impaired intracellular killing on salmonellae by spleen
macrophages and delayed clearance of bacteria in the liver (Figure 2).
Following inhalation of nickel aerosols an enhancement of respiratory
tract infections was found in mice [32]. An increase in susceptibility to
streptococcal infection was seen in mice exposed to 0.499 mg nickel chloride/
m3, 3-day pre-treatment with nickel chloride (0.5 or 1 mg/kg) decreased the
resistance of mice to experimental Klebsiella pneumonia infection [131].
Both organic and inorganic mercury have been shown to enhance the
susceptibility of mice to encephalomyocarditis virus (EMCV) infection
[132,133]. Prolonged exposure to mercury compounds was observed to
decrease the resistance to bacterial infections in chicken [134]. In guinea pigs, a
slightly increased intensity of Ascaris suum infection was seen following a 28-
day HgCl2 treatment (0.5 mg/kg body weight) [56]. Cupric sulfate (2.08 mg/
kg) administered with the same protocol showed comparable results [135].
Combined exposure to lead acetate (10 mg/kg) and sodium arsenite
(0.5 mg/kg) was found to be additive regarding the resistance against Sta-
phylococcus aureus. Bacterial clearance following multimetal exposure to
lead and arsenic was found to be 11% compared to approximately 30%
following single lead or arsenite exposure [136].
Only few data are available so far concerning heavy metal effects on
resistance to infection in marine organisms. Cadmium exposed marine toads
(Bufo marinus) were shown to have increased rates in trematode infections
[137]. An increased incidence of infectious diseases was also reported from
marine mammals exposed to mercury and zinc [138].
It has been demonstrated that in terms of resistance against infections the
route of administration plays an important role. Cadmium sulfate was found
to increase the mortality of CD-1 mice infected with EMCV [132]. However,
in another study, cadmium acetate exposure via drinking water was capable
of reducing mortality in EMCV-infected Swiss Webster mice compared to
non-exposed mice [139]. A further important point concerns the time of
Met. Ions Life Sci. 2011, 8, 157–185

Figure 2. In a mouse model of Salmonella Enteritidis (SE) infection, exposure of mice

to cadmium prior to infection caused fatal outcome of disease using SE ( 2107 CFU)
or Cd doses (0.7 mg/kg body weight) which were otherwise tolerated by the animals
when applied alternatively (---- BALB/c mice infected with SE; BALB/c mice
treated with Cd; ——— BALB/c mice exposed to Cd followed by infection with SE). If
a lower SE infection dose (5106 CFU) was used, Cd-exposed animals survived the
infection. However, liver and spleen bacterial burdens were significantly higher in Cd-
exposed compared to untreated control mice (J untreated control; K Cd-exposed
mice) indicating an immunosuppressive influence of Cd on the protective immune
response to SE. Obviously, Cd interferes with early innate immune mechanisms
essential for limiting bacteria during the first three days of infection and inducing a
protective type-1 immune response (N. Hemdan, U. Sack, J. Lehmann, unpublished).
heavy metal exposure in relation to pathogen challenge. One single intra-

peritoneal dose of 24 mg/kg lead acetate or nickel chloride enhanced the
resistance of mice against Klebsiella pneumonia when administered 24 hours
before infection, whereas the host resistance was impaired when the same
dose was injected 5 hours after infectious challenge [131].
7. CHRONIC INFLAMMATION AND AUTOIMMUNITY
Heavy metal exposure in most cases proceeds in a chronic manner, resulting

in pathogenic immune responses persisting for months if not years. Sus-
tained activation of cytokine cascades bears diverse health risks and may
result in chronic inflammatory diseases.
Met. Ions Life Sci. 2011, 8, 157–185

Chronic inhalative exposure to nickel dusts or aerosols was found to be

associated with inflammatory manifestations of the respiratory tract such as
asthma, bronchitis, rhinitis, and sinusitis. Persistent airway inflammation
was also observed following cadmium inhalation or intratracheal injection
within experimental models [140,141]. As a main component in tobacco
smoke cadmium is suspected to contribute importantly to tobacco-related
lung diseases, in particular the chronic obstructive pulmonary disease [142].
It has furthermore been discussed that oxidative stress-induced inflamma-
tory reactions resulting from mercury and cadmium exposure may also have
cardiovascular consequences. Mercury was described to have serious vas-
cular effects, including inflammation, thrombosis, and endothelial dys-
function. All together, these effects have been suspected to increase the risk
of hypertension and vascular disease. In rabbits exposed to inhaled mercury
vapor, thrombosis in small and medium calibre arteries, endothelial pro-
liferation and inflammation were found [143]. A further study reported
about associations between hair mercury, urine mercury and cardiovascular
events [144]. Consistently, in humans a relationship between mercury
intoxication and hypertension was shown [145–148]. The role of cadmium in
cardiovascular disease is less convincing than that of mercury. However,
results from animal studies also point to a relationship between cadmium
toxicity and atherosclerosis and increased blood pressure [149,150].
It is a well accepted fact that chronic inflammation increases the risk for
the development of autoimmune reactions. There is strong evidence that
heavy metal exposure indeed can induce autoimmunity in both experimental
animal models as well as in humans. A selection of so far observed asso-
ciations is provided in Table 1.
Kidney diseases are frequently reported autoimmune responses resulting
from heavy metal exposure. Thereby, renal pathology was found to be
associated with the occurrence of anti-laminin autoantibodies (laminin is a
component of the glomerular basement membranes). Production of such
autoantibodies was found in Spague Dawley rats, chronically exposed to
cadmium at 1 mg/kg [151] and in cadmium exposed workers with urinary
concentrations 420 mg Cd/g creatinine [152]. Affected individuals devel-
oped proteinuria, tubulointestitial nephritis, and glomerular damage.
Nephrotic syndrome and immune complex-mediated glomerulonephritis
were also reported from patients receiving gold therapy. Gold salt-con-
taining preparations have been widely used in the treatment of rheumatoid
arthritis. Mild proteinuria was observed in approximately 10% and massive
proteinuria in 1% of patients with rheumatoid arthritis treated with gold
salts [153]. A further metal widely used in medical practice and known to
induce renal complications is mercury. As a component of laxatives, oint-
ments, teething powders, and diuretics mercury was frequently ther-
apeutically administered in the past with the result of autoimmune
Met. Ions Life Sci. 2011, 8, 157–185

complications [153]. Anti-laminin autoantibodies and glomerular damage

were also found in rats, mice, and rabbits exposed to mercury chloride in
experimental models.
Another commonly reported autoimmune manifestation following heavy-
metal exposure is the development of a systemic lupus erythematosus (SLE)
like syndrome. SLE is a systemic chronic inflammatory disease affecting
different tissues in the body including skin, joints, and kidney. The occur-
rence of antinuclear (ANA) or antinucleolar (ANolA) autoantibodies
reacting against structures within the nucleus of the cell (e.g., DNA and
histones) is indicative for this autoimmune disease. It is well established that
mercury induces a SLE-like disease in genetically susceptible mice and rats
characterized by the production of ANolA [154,155]. It has been shown that
most of the mercury-induced ANolA react with fibrillarin, a component of
nucleolar small nuclear ribonucleoprotein (snRNP) particles [156]. Beside
mercury, cadmium, gold, and lead were also found to be associated with
SLE development (see Table 1 [157–169]).
Investigation of autoimmune diseases associated with heavy metal expo-
sure has provided strong evidence that in particular genetic factors play an
important role in terms of susceptibility for the development of exposure-
related pathogenic immune responses to self-antigens. Autoimmune kidney
damage caused by anti-laminin antibodies was found in Spague Dawley rats
chronically exposed to cadmium but not in similarly treated Brown Norway
Table 1. Autoimmune manifestations resulting from metal exposure.
Metal Disease Organism Reference
Cadmium SLE-like syndrome Mice [47]

Autoimmune kidney disease Humans [152,157]
Rats [151]
Copper Adjuvant arthritis Rats [158]
Arthritis, spondylitis Humans [159]
Gold Autoimmune kidney disease Humans [153]
SLE
Autoimmune thromobocytopenia Humans [160,161]
Lead SLE Mice [162]
Mercury SLE-like syndrome Mice, rats [154,155]
Humans [60]
Autoimmune myocarditis Mice [163,164]
Autoimmune kidney disease Mice, rats [165–167]
Humans [157]
Zinc Multiple sclerosis Humans [168,169]
SLE ¼ systemic lupus erythematosus
Met. Ions Life Sci. 2011, 8, 157–185

rats [151]. Comparable results were seen in mice. The development of anti-
nuclear antibodies following cadmium exposure showed strong strain var-
iations with ICR mice being more susceptible than BALB/c mice and C57Bl/
6 mice showing no effect [47]. In patients, gold-induced proteinuria was
associated with HLA-DR3, HLA-B8 [160,170], and HLA-Dw3 [171]. The
development of thrombocytopenia following gold treatment was also related
to HLA-DR3 [160]. Only rats and mice that bear the H-2 haplotype develop
autoimmune responses after treatment with gold [172]. The observed asso-
ciation between susceptibility to heavy metal-induced autoimmune diseases
and genetic factors may explain why not all individuals exposed to heavy
metals develop autoimmunity and why results observed in experimental
animal models sometimes cannot be found in humans.
As a further possible mechanism by which heavy metals, in particular Hg,
may be associated with increased risk of autoimmune disease development,
interactions with triggering events (pathogens, antigens or organ damage)
have been discussed. It has been shown that Hg can accelerate autoimmune
disease (SLE and myocarditis) in mice strains that are not susceptible to Hg-
induced autoimmune dysfunction. In mice inoculated with cardiac myosin
peptide to induce autoimmune myocarditis, pre-treatment with 200 mg/kg
inorganic Hg resulted in an increased incidence and severity of autoimmune
myocarditis. Interestingly, Hg treatment itself had no effect on heart
pathology. Thus, it was suggested that Hg treatment is related with wor-
sening of symptoms rather than initiating disease [163]. Furthermore, the
adjuvant effect of Hg on autoimmune development was found to be asso-
ciated with very low exposures related with body burdens found in humans.
Inorganic Hg doses as low as 20 mg/kg for 2 weeks [173] or about 2 mg/kg for
several months [174] were associated with severe exacerbation of patho-
physiology in autoimmune disease.
Beside genetic factors and a general inflammatory background influences
of heavy metals on the antigen-binding capacity of immunoglobulins [175]
and on physicochemical properties of autoantigens [176] may contribute to
the development of a pathological immune response characterized by acti-
vation of autoreactive T and B lymphocytes. Thus, it can be concluded that
the induction of autoimmune responses by heavy metals is likely the result of
a combination of different factors rather than a single pathomechanism.
8. CONCLUDING REMARKS
Various immune parameters have been shown to be affected by heavy metal

exposure with often resulting serious health consequences. Thus, although
variations in the experimental design, exposure situation, affected organisms
Met. Ions Life Sci. 2011, 8, 157–185

and further factors resulted in different and sometimes conflicting results, in

general there is no concern about strong immunotoxic effects of heavy
metals. Thereby, the immunotoxic potential of metals might be even
underestimated. Although most human and animal exposure to heavy
metals involves mixtures at low environmental levels, most of the tox-
icological studies considered only individual chemicals. Additional studies
considering this issue are required.
ACKNOWLEDGMENTS
This work was partly supported by the EU-integrated project NoMiracle

(Novel Methods for Integrated Risk Assessment of Cumulative Stressors in
Europe).
ADCC antibody-dependent cellular cytotoxicity

ADP adenosine 5 0 -diphosphate
d-ALA d-aminolevulinic acid
ANA antinuclear autoantibodies
ANolA antinucleolar autoantibodies
APC antigen presenting cells
ARE antioxidant response element
B cells lymphocytes responsible for the promotion and secre-
tion of specific antibodies
CCR chemokine receptor
CD cluster of differentiation
Con A concanavalin A
EMCV encephalomyocarditis virus
ERK1/2 extracellular signal-regulated protein kinases 1/2
GSH glutathione
HLA human leucocyte antigen
Hsp heat shock protein
HUVEC human umbilical vein endothelial cells
i.p. intraperitoneal
IFN interferon
Ig immunoglobulin
IL interleukin
JNK cJun N-terminal kinases
Met. Ions Life Sci. 2011, 8, 157–185

LPS lipopolysaccharide
MAPK mitogen-activated proteine kinase
MCP monocyte chemoattractant protein
MHC major histocompatibility complex
MRE metal response element
MTF-1 metal regulatory transcription factor-1
NAC N-acetyl cysteine
NAD1 nicotinamide adenine dinucleotide
NF-kB nuclear factor-kappaB
NK cells natural killer cells
p.i. post infection
p38 MAPK p38 (protein 38 kD)-mitogen-activated protein kinase
PBMC peripheral blood mononuclear cells
PHA phytohemagglutinin
ROS reactive oxygen species
SE Salmonella enterica, spp. enterica, serovar Enteritidis
SLE systemic lupus erythematosus
T cells thymus-derived cells (lymphocytes)
TGF transforming growth factor
Th cells T helper cells
TNF tumor necrosis factor
Treg cells regulatory T cells (lymphocytes)
REFERENCES
1. D. Ibrahim, B. Froberg, A. Wolf and D. E. Rusyniak, Clin. Lab. Med., 2006, 26,
67–97.
2. C. Giaginis, E. Gatzidou and S. Theocharis, Toxicol. Appl. Pharmacol., 2006,
213, 282–290.
3. D. Beyersmann and A. Hartwig, Arch. Toxicol., 2008, 82, 493–512.
4. H. C. Gonick, Indian J. Med. Res., 2008, 128, 335–352.
5. F. O. Johnson and W. D. Atchison, Neurotoxicology, 2009, 30, 761–765.
6. P. Mendola, L. C. Messer and K. Rappazzo, Fertil. Steril., 2008, 89, e81–e94.
7. T. Sanders, Y. Liu, V. Buchner and P. B. Tchounwou, Rev. Environ. Health,
2009, 24, 15–45.
8. J. Thompson and J. Bannigan, Reprod. Toxicol., 2008, 25, 304–315.
9. L. D. Koller, Drug Chem. Toxicol., 1979, 2, 99–110.
10. J. E. Duffy, E. A. Carlson, Y. Li, C. Prophete and J. T. Zelikofft, Ecotoxicology,
2003, 12, 251–259.
11. J. A. Oughton, C. B. Pereira, G. K. DeKrey, J. M. Collier, A. A. Frank and N.
I. Kerkvliet, Fundam. Appl. Toxicol., 1995, 25, 60–69.
12. N. Ahluwalia, M. A. Gordon, G. Handte, M. Mahlon, N. Q. Li, J. L. Beard, D.
Weinstock and A. C. Ross, J. Nutr., 2000, 130, 2378–2383.
Met. Ions Life Sci. 2011, 8, 157–185

13. M. Stefanidou, C. Maravelias, A. Dona and C. Spiliopoulou, Arch. Toxicol.,

2006, 80, 1–9.
14. B. R. Blakley, Can. J. Vet. Res., 1988, 52, 291–292.
15. B. J. Shenker, C. Rooney, L. Vitale and I. M. Shapiro, Immunopharmacol.
Immunotoxicol., 1992, 14, 539–553.
16. G. S. De, J. Bernier, P. Lapierre, M. M. Dufresne, P. Dubreuil and M. Fournier,
Am. J. Vet. Res., 2000, 61, 339–344.
17. M. Governa, M. Valentino and I. Visona, Arch. Toxicol., 1987, 59, 421–425.
18. V. K. Singh, K. P. Mishra, R. Rani, V. S. Yadav, S. K. Awasthi and S. K. Garg,
Immunol. Res., 2003, 28, 151–166.
19. M. Valentino, M. Governa, I. Marchiseppe and I. Visona, Arch. Toxicol., 1991,
65, 685–688.
20. R. C. Perlingeiro and M. L. Queiroz, Int. J. Immunopharmacol., 1994, 16, 1011–
1017.
21. R. C. Perlingeiro and M. L. Queiroz, Hum. Exp. Toxicol., 1995, 14, 281–286.
22. S. H. Kim, V. J. Johnson and R. P. Sharma, Nitric Oxide, 2002, 7, 67–74.
23. J. M. Benson, R. F. Henderson and J. A. Pickrell, J. Toxicol. Environ. Health,
1988, 24, 373–383.
24. R. B. Herberman, Clin. Immunol. Rev., 1981, 1, 1–65.
25. R. J. Smialowicz, R. R. Rogers, M. M. Riddle and G. A. Stott, Environ. Res.,
1984, 33, 413–427.
26. R. J. Smialowicz, R. R. Rogers, M. M. Riddle, R. J. Garner, D. G. Rowe and R.
W. Luebke, Environ. Res., 1985, 36, 56–66.
27. R. J. Smialowicz, R. R. Rogers, D. G. Rowe, M. M. Riddle and R. W. Luebke,
Toxicology, 1987, 44, 271–281.
28. P. J. Haley, G. M. Shopp, J. M. Benson, Y. S. Cheng, D. E. Bice, M. I. Luster, J.
K. Dunnick and C. H. Hobbs, Fundam. Appl. Toxicol., 1990, 15, 476–487.
29. F. W. Sunderman, Jr., Environ. Health Perspect., 1981, 40, 131–141.
30. H. M. Shen and Q. F. Zhang, Environ. Health Perspect., 1994, 102 (Suppl. 1),
275–282.
31. G. D. Stoner, M. B. Shimkin, M. C. Troxell, T. L. Thompson and L. S. Terry,
Cancer Res., 1976, 36, 1744–1747.
32. B. Adkins, Jr., J. H. Richards and D. E. Gardner, Environ. Res., 1979, 20, 33–42.
33. M. Fortier, F. Omara, J. Bernier, P. Brousseau and M. Fournier, J. Toxicol.
Environ. Health, A, 2008, 71, 1327–1337.
34. M. G. Cifone, A. Procopio, T. Napolitano, E. Alesse, G. Santoni and A.
Santoni, Immunopharmacology, 1990, 20, 73–80.
35. N. H. Stacey, J. Toxicol. Environ. Health, 1986, 18, 293–300.
36. N. H. Stacey, G. Craig and L. Muller, Environ. Res., 1988, 45, 71–77.
37. B. Yucesoy, S. Mirshahidi, C. Yucesoy and A. Karakaya, Immunopharmacol.
Immunotoxicol., 1999, 21, 599–607.
38. B. Yucesoy, A. Turhan, M. Ure, T. Imir and A. Karakaya, Immunopharmacol.
Immunotoxicol., 1997, 19, 339–348.
39. I. Kimber, J. A. Jackson and M. D. Stonard, Toxicol. Lett., 1986, 31, 211–218.
40. I. Kimber, M. D. Stonard, D. A. Gidlow and Z. Niewola, Int. Arch. Occup.
Environ. Health, 1986, 57, 117–125.
Met. Ions Life Sci. 2011, 8, 157–185

41. B. A. Neilan, K. O’Neill and B. S. Handwerger, Toxicol. Appl. Pharmacol.,

1983, 69, 272–275.
42. L. D. Koller, J. H. Exon and J. G. Roan, Arch. Environ. Health, 1975, 30,
598–601.
43. R. F. Borgman, B. Au and R. K. Chandra, Int. J. Immunopharmacol., 1986, 8,
813–817.
44. R. L. Shippee, D. H. Burgess, R. P. Ciavarra, R. A. DiCapua and P. E. Stake,
45. H. Fujimaki, Toxicol. Lett., 1985, 25, 69–74.
46. I. Malavé and D. T. de Ruffino, Toxicol. Appl. Pharmacol., 1984, 74, 46–56.
47. M. Ohsawa, K. Takahashi and F. Otsuka, Clin. Exp. Immunol., 1988, 73,
98–102.
48. G. Dan, S. B. Lall and D. N. Rao, Drug Chem. Toxicol., 2000, 23, 349–360.
49. S. Muller, K. E. Gillert, C. Krause, G. Jautzke, U. Gross and T. Diamantstein,
Experientia, 1979, 35, 909–910.
50. P. T. Thomas, H. V. Ratajczak, C. Aranyi, R. Gibbons and J. D. Fenters,
51. H. Fujimaki, Toxicol. Lett., 1985, 24, 21–24.
52. B. R. Blakley and R. S. Tomar, Int. J. Immunopharmacol., 1986, 8, 1009–1015.
53. J. R. Daum, D. M. Shepherd and R. J. Noelle, Int. J. Immunopharmacol., 1993,
15, 383–394.
54. B. Ritz, J. Heinrich, M. Wjst, E. Wichmann and C. Krause, Arch. Environ.
Health, 1998, 53, 272–280.
55. P. Borella and A. Giardino, Environ. Res., 1991, 55, 165–177.
56. Z. Boroskova, J. Soltys and M. Benkova, J. Helminthol., 1995, 69, 187–194.
57. S. Roether, H. Rabbani, H. Mellstedt and M. Abedi-Valugerdi, Scand. J.
Immunol., 2002, 55, 493–502.
58. S. Weidinger, U. Kramer, L. Dunemann, M. Mohrenschlager, J. Ring and
H. Behrendt, J. Allergy Clin. Immunol., 2004, 114, 457–459.
59. D. C. Dantas and M. L. Queiroz, Immunopharmacol. Immunotoxicol., 1997,
19, 383–392.
60. I. A. Silva, J. F. Nyland, A. Gorman, A. Perisse, A. M. Ventura, E. C. Santos,
J. M. Souza, C. L. Burek, N. R. Rose and E. K. Silbergeld, Environ. Health,
2004, 3, 11.
61. H. Fujimaki, F. Shimizu, R. Kawamura and K. Kubota, Toxicol. Lett., 1983,
19, 241–245.
62. N. Y. Hemdan, F. Emmrich, U. Sack, G. Wichmann, J. Lehmann, K. Adham
and I. Lehmann, Toxicology, 2006, 222, 37–45.
63. N. Y. Hemdan, I. Lehmann, G. Wichmann, J. Lehmann, F. Emmrich and
U. Sack, Clin. Exp. Immunol., 2007, 148, 325–337.
64. N. Y. Hemdan, F. Emmrich, S. Faber, J. Lehmann and U. Sack, Ann. NY Acad.
Sci., 2007, 1109, 129–137.
65. Z. Krocova, A. Macela, M. Kroca and L. Hernychova, Toxicol. In Vitro, 2000,
14, 33–40.
66. E. C. Foulkes, Toxicology, 1988, 52, 263–272.
67. S. G. Schäfer and W. Forth, J. Nutr., 1984, 114, 1989–1996.
Met. Ions Life Sci. 2011, 8, 157–185

68. N. Sugawara, B. Q. Chen, C. Sugawara and H. Miyake, Bull. Environ. Contam.

Toxicol., 1988, 41, 50–55.
69. J. D. Kemp, J. Clin. Immunol., 1993, 13, 81–92.
70. M. Watanabe, K. Henmi, K. Ogawa and T. Suzuki, Comp. Biochem. Physiol. C
71. E. Casalino, C. Sblano and C. Landriscina, Arch. Biochem. Biophys., 1997, 346,
171–179.
72. W. Wätjen and D. Beyersmann, Biometals, 2004, 17, 65–78.
73. M. Valko, D. Leibfritz, J. Moncol, M. T. Cronin, M. Mazur and J. Telser, Int.
J. Biochem. Cell Biol., 2007, 39, 44–84.
74. V. J. Thannickal and B. L. Fanburg, Am. J. Physiol. Lung Cell Mol. Physiol.,
2000, 279, L1005–L1028.
Interact., 2006, 160, 1–40.
76. A. Galanis, A. Karapetsas and R. Sandaltzopoulos, Mutat. Res., 2009, 674,
31–35.
77. M. Ahamed and M. K. Siddiqui, Clin. Chim. Acta, 2007, 383, 57–64.
78. M. Hermes-Lima, Free Radic. Biol. Med., 1995, 19, 381–390.
79. E. J. Bechara, Braz. J. Med. Biol. Res., 1996, 29, 841–851.
80. S. J. Flora, S. Bhadauria, G. M. Kannan and N. Singh, J. Environ. Biol., 2007,
28, 333–347.
81. K. Yamanaka, H. Hayashi, M. Tachikawa, K. Kato, A. Hasegawa, N. Oku and
S. Okada, Mutat. Res., 1997, 394, 95–101.
82. N. Ercal, H. Gurer-Orhan and N. Aykin-Burns, Curr. Top. Med. Chem., 2001,
1, 529–539.
83. S. A. Thompson, K. L. Roellich, A. Grossmann, S. G. Gilbert and T. J.
Kavanagh, Immunopharmacol. Immunotoxicol., 1998, 20, 299–314.
84. L. I. Sweet, D. R. Passino-Reader, P. G. Meier and G. M. Omann, Biomarkers,
1999, 237–253.
85. S. H. Kim and R. P. Sharma, Immunopharmacol. Immunotoxicol., 2005, 27,
123–135.
86. R. K. Zalups, D. W. Barfuss and L. H. Lash, Toxicol. Appl. Pharmacol., 1999,
154, 135–144.
87. Y. C. Chen, S. Y. Lin-Shiau and J. K. Lin, J. Cell Physiol., 1998, 177,
324–333.
88. N. Pathak and S. Khandelwal, Toxicology, 2006, 220, 26–36.
89. N. Pathak and S. Khandelwal, Biometals, 2008, 21, 179–187.
90. R. L. Messer, P. E. Lockwood, W. Y. Tseng, K. Edwards, M. Shaw, G. B.
Caughman, J. B. Lewis and J. C. Wataha, J. Biomed. Mater. Res. B Appl.
Biomater., 2005, 75, 257–263.
91. M. J. Fernandez-Cabezudo, M. Y. Hasan, N. Mustafa, R. T. El-Sharkawy, M.
A. Fahim and B. K. Al-Ramadi, Free Radic. Res., 2003, 37, 437–445.
92. S. V. Rana, J. Trace Elem. Med. Biol., 2008, 22, 262–284.
93. B. J. Shenker, T. L. Guo and I. M. Shapiro, Environ. Res., 1998, 77, 149–159.
94. M. J. Whitekus, R. P. Santini, A. J. Rosenspire and M. J. McCabe, Jr.,
J. Immunol., 1999, 162, 7162–7170.
Met. Ions Life Sci. 2011, 8, 157–185

95. S. Homma-Takeda, Y. Kugenuma, T. Iwamuro, Y. Kumagai and N. Shimojo,

Toxicology, 2001, 169, 25–35.
96. X. Cai, Y. L. Shen, Q. Zhu, P. M. Jia, Y. Yu, L. Zhou, Y. Huang, J. W. Zhang,
S. M. Xiong, S. J. Chen, Z. Y. Wang, Z. Chen and G. Q. Chen, Leukemia, 2000,
14, 262–270.
97. T. L. Guo, M. A. Miller, S. Datar, I. M. Shapiro and B. J. Shenker, Toxicol.
Appl. Pharmacol., 1998, 152, 397–405.
98. L. I. Sweet and J. T. Zelikoff, J. Toxicol. Environ. Health B Crit. Rev., 2001, 4,
161–205.
99. J. Kim and R. P. Sharma, Toxicol. Sci., 2004, 81, 518–527.
100. N. M. Kalariya, N. K. Wills, K. V. Ramana, S. K. Srivastava and F. J. van
Kuijk, Exp. Eye Res., 2009, 89, 494–502.
101. Y. H. Han, H. J. Moon, B. R. You, S. Z. Kim, S. H. Kim and W. H. Park,
Anticancer Res., 2009, 29, 3837–3844.
102. S. Datta, S. Mazumder, D. Ghosh, S. Dey and S. Bhattacharya, Toxicol. Appl.
Pharmacol., 2009, 241, 329–338.
103. M. O. Song, J. Li and J. H. Freedman, Physiol. Genomics, 2009, 38, 386–401.
104. C. Urani, V. Calini, P. Melchioretto, F. Morazzoni, C. Canevali and M.
Camatini, Toxicol. In vitro, 2003, 17, 553–559.
105. M. Valko, H. Morris and M. T. Cronin, Curr. Med. Chem., 2005, 12, 1161–
1208.
106. J. M. DeMoor and D. J. Koropatnick, Cell Mol. Biol. (Noisy-le-grand), 2000,
46, 367–381.
107. G. K. Andrews, Prog. Food Nutr. Sci., 1990, 14, 193–258.
108. I. Rahman and W. MacNee, Eur. Respir. J., 2000, 16, 534–554.
109. I. Rahman, B. Mulier, P. S. Gilmour, T. Watchorn, K. Donaldson, P. K. Jeffery
and W. MacNee, Biochem. Pharmacol., 2001, 62, 787–794.
110. M. Goebeler, J. Roth, E. B. Brocker, C. Sorg and K. Schulze-Osthoff, J.
Immunol., 1995, 155, 2459–2467.
111. H. N. Wagner, Jr., J. Nucl. Med., 1997, 38, 241–242.
112. J. S. Hyun, H. Satsu and M. Shimizu, Cytokine, 2007, 37, 26–34.
113. M. K. McElwee, M. O. Song and J. H. Freedman, J. Mol. Biol., 2009, 393,
1013–1021.
114. L. Flohe, R. Brigelius-Flohe, C. Saliou, M. G. Traber and L. Packer, Free
Radic. Biol. Med., 1997, 22, 1115–1126.
115. L. Chang and M. Karin, Nature, 2001, 410, 37–40.
116. H. J. Schaeffer and M. J. Weber, Mol. Cell Biol., 1999, 19, 2435–2444.
117. N. Sugisawa, M. Matsuoka, T. Okuno and H. Igisu, Toxicol. Appl. Pharmacol.,
2004, 196, 206–214.
118. M. C. Escobar, V. Souza, L. Bucio, E. Hernandez, L. E. Gomez-Quiroz and M.
C. Gutierrez Ruiz, Toxicol. Mech. Methods, 2009, 19, 503–509.
119. U. K. Misra, G. Gawdi, G. Akabani and S. V. Pizzo, Cell Signal., 2002, 14, 327–
340.
120. S. Hirano, X. Sun, C. A. DeGuzman, R. F. Ransom, K. R. McLeish, W. E.
Smoyer, E. A. Shelden, M. J. Welsh and R. Benndorf, Am. J. Physiol. Renal
Physiol., 2005, 288, F1133–F1143.
Met. Ions Life Sci. 2011, 8, 157–185

121. J. J. Hung, T. J. Cheng, Y. K. Lai and M. D. Chang, J. Biol. Chem., 1998, 273,
31924–31931.
122. J. S. Lee, M. H. Zhang, E. K. Yun, D. Geum, K. Kim, T. H. Kim, Y. S. Lim and
J. S. Seo, Exp. Mol. Med., 2005, 37, 427–435.
123. F. Boisleve, S. Kerdine-Romer and M. Pallardy, Toxicology, 2005, 206, 233–
244.
124. S. M. Kim, J. G. Park, W. K. Baek, M. H. Suh, H. Lee, S. K. Yoo, K. H. Jung,
S. I. Suh and B. C. Jang, Neurosci. Lett., 2008, 440, 289–293.
125. R. B. Leal, T. Posser, A. P. Rigon, C. S. Oliveira, C. A. Goncalves, D. P. Gelain
and P. R. Dunkley, Toxicology, 2007, 234, 34–43.
126. M. Leist, F. Gantner, I. Bohlinger, G. Tiegs, P. G. Germann and A. Wendel,
Am. J. Pathol., 1995, 146, 1220–1234.
127. U. Ewers, R. Stiller-Winkler and H. Idel, Environ. Res., 1982, 29, 351–357.
128. J. A. Cook, E. O. Hoffmann and N. D. Luzio, Proc. Soc. Exp. Biol. Med., 1975,
150, 741–747.
129. B. E. Bozelka and P. M. Burkholder, J. Reticuloendothel. Soc., 1979, 26, 229–
237.
130. M. J. Daniels, M. G. Menache, G. R. Burleson, J. A. Graham and M. K.
Selgrade, Fundam. Appl. Toxicol., 1987, 8, 443–453.
131. A. Laschi-Loquerie, A. Eyraud, D. Morisset, A. Sanou, P. Tachon, C. Veys-
seyre and J. Descotes, Immunopharmacol. Immunotoxicol., 1987, 9, 235–241.
132. J. H. Gainer, Am. J. Vet. Res., 1977, 38, 869–872.
133. L. D. Koller, Am. J. Vet. Res., 1975, 36, 1501–1504.
134. M. A. Bridger and J. P. Thaxton, Arch. Environ. Contam. Toxicol., 1983, 12, 45–
49.
135. J. Soltys, Z. Boroskova and E. Dvoroznakova, J. Helminthol., 1997, 71, 339–344.
136. B. Bishayi and M. Sengupta, Int. Immunopharmacol., 2006, 6, 454–364.
137. D. Linzey, J. Burroughs, L. Hudson, M. Marini, J. Robertson, J. Bacon, M.
Nagarkatti and P. Nagarkatti, Int. J. Environ. Health Res., 2003, 13, 125–148.
138. P. M. Bennett, P. D. Jepson, R. J. Law, B. R. Jones, T. Kuiken, J. R. Baker, E.
Rogan and J. K. Kirkwood, Environ. Pollut., 2001, 112, 33–40.
139. J. H. Exon, L. D. Koller and N. I. Kerkvliet, Arch. Environ. Health, 1979, 34,
469–475.
140. W. M. Thurlbeck and F. D. Foley, Am. J. Pathol., 1963, 42, 431–441.
141. N. Kirschvink, G. Vincke, L. Fievez, C. Onclinx, D. Wirth, M. Belleflamme, R.
Louis, D. Cataldo, M. J. Peck and P. Gustin, Toxicology, 2005, 211, 36–48.
142. D. M. Mannino, F. Holguin, H. M. Greves, A. Savage-Brown, A. L. Stock and
R. L. Jones, Thorax, 2004, 59, 194–198.
143. J. Wojciechowski and W. Kowalski, Pol. Med. Sci. Hist. Bull., 1975, 15, 255–
260.
144. J. T. Salonen, K. Seppanen, K. Nyyssonen, H. Korpela, J. Kauhanen, M.
Kantola, J. Tuomilehto, H. Esterbauer, F. Tatzber and R. Salonen, Circulation,
1995, 91, 645–655.
145. P. Boffetta, G. Sallsten, M. Garcia-Gomez, V. Pompe-Kirn, D. Zaridze, M.
Bulbulyan, J. D. Caballero, F. Ceccarelli, A. B. Kobal and E. Merler, Occup.
Environ. Med., 2001, 58, 461–466.
Met. Ions Life Sci. 2011, 8, 157–185

146. J. T. Salonen, K. Seppanen, T. A. Lakka, R. Salonen and G. A. Kaplan,

Atherosclerosis, 2000, 148, 265–273.
147. A. B. Kobal, M. Horvat, M. Prezelj, A. S. Briski, M. Krsnik, T. Dizdarevic, D.
Mazej, I. Falnoga, V. Stibilj, N. Arneric, D. Kobal and J. Osredkar, J. Trace
Elem. Med. Biol., 2004, 17, 261–274.
148. A. D. Torres, A. N. Rai and M. L. Hardiek, Pediatrics, 2000, 105, E34.
149. N. W. Revis, A. R. Zinsmeister and R. Bull, Proc. Natl. Acad. Sci. USA, 1981,
78, 6494–6498.
150. S. J. Kopp, H. M. Perry, Jr., E. F. Perry and M. Erlanger, Toxicol. Appl.
Pharmacol., 1983, 69, 149–160.
151. A. Bernard, R. Lauwerys, P. Gengoux, P. Mahieu, J. M. Foidart, P. Druet and
J. J. Weening, Toxicology, 1984, 31, 307–313.
152. A. M. Bernard, H. R. Roels, J. M. Foidart and R. L. Lauwerys, Int. Arch.
Occup. Environ. Health, 1987, 59, 303–309.
153. P. E. Bigazzi, Lupus, 1994, 3, 449–453.
154. P. Griem and E. Gleichmann, Curr. Opin. Immunol., 1995, 7, 831–838.
155. S. Enestrom and P. Hultman, Int. Arch. Allergy Immunol., 1995, 106, 180–203.
156. M. Abedi-Valugerdi, H. Hu and G. Moller, Int. Immunol., 1999, 11, 605–615.
157. J. P. Buchet, H. Roels, A. Bernard and R. Lauwerys, J. Occup. Med., 1980, 22,
741–750.
158. R. Milanino and V. Buchner, Rev. Environ. Health, 2006, 21, 153–215.
159. L. H. El-Sayed, H. M. Ghoneim, M. H. El-Sayed, S. R. Deimian, A. N. Adam,
S. N. bou Rawash, N. M. bou Rawash and P. Ursos, Immunopharmacol.
Immunotoxicol., 2003, 25, 473–490.
160. D. P. Singal, B. Reid, D. Green, M. D’souza, W. G. Bensen and W. W.
Buchanan, Ann. Rheum. Dis., 1990, 49, 582–586.
161. J. D. Adachi, W. G. Bensen, D. P. Singal and P. J. Powers, J. Rheumatol., 1984,
11, 355–357.
162. C. A. Hudson, L. Cao, J. Kasten-Jolly, J. N. Kirkwood and D. A. Lawrence, J.
Toxicol. Environ. Health, 2003, 66, 895–918.
163. E. K. Silbergeld, I. A. Silva and J. F. Nyland, Toxicol. Appl. Pharmacol., 2005,
207, 282–292.
164. N. G. Ilback, U. Lindh, L. Wesslen, J. Fohlman and G. Friman, Biol. Trace
Elem. Res., 2000, 78, 131–147.
165. M. Goldman, P. Druet and E. Gleichmann, Immunol. Today, 1991, 12, 223–227.
166. J. F. Bernaudin, E. Druet, P. Druet and R. Masse, Clin. Immunol. Immuno-
pathol., 1981, 20, 129–135.
167. J. Hua, L. Pelletier, M. Berlin and P. Druet, Toxicology, 1993, 79, 119–129.
168. E. C. Stein, R. B. Schiffer, W. J. Hall and N. Young, Neurology, 1987, 37, 1672–
1677.
169. R. B. Schiffer, Neurology, 1994, 44, 1987–1988.
170. O. Scherak, J. S. Smolen, W. R. Mayr, F. Mayrhofer, G. Kolarz and N. J.
Thumb, J. Rheumatol., 1984, 11, 610–614.
171. M. Hakala, A. H. van Assendelft, J. Ilonen, S. Jalava and A. Tiilikainen, Ann.
Rheum. Dis., 1986, 45, 177–182.
172. J. B. Nielsen and P. Hultman, Ren. Fail., 1999, 21, 343–348.
Met. Ions Life Sci. 2011, 8, 157–185

173. C. S. Via, P. Nguyen, F. Niculescu, J. Papadimitriou, D. Hoover and E. K.

Silbergeld, Environ. Health Perspect., 2003, 111, 1273–1277.
174. K. M. Pollard, D. L. Pearson, P. Hultman, B. Hildebrandt and D. H. Kono,
Environ. Health Perspect., 1999, 107 (Suppl. 5), 729–735.
175. R. Bauer, A. Muller, M. Richter, K. Schneider, J. Frey and W. Engelhardt,
Biochim. Biophys. Acta, 1997, 1334, 98–108.
176. K. M. Pollard, D. K. Lee, C. A. Casiano, M. Bluthner, M. M. Johnston and E.
M. Tan, J. Immunol., 1997, 158, 3521–3528.
Met. Ions Life Sci. 2011, 8, 157–185

Met. Ions Life Sci. 2011, 8, 187–246
9
Metal Ions Affecting the Skin and Eyes
Alan B. G. Lansdown
Chemical Pathology, Faculty of Medicine, Imperial College London, Charing Cross Campus,
London W6 8RP, UK
<a.lansdown@ic.ac.uk>
ABSTRACT 188
1. INTRODUCTION 188
2. METAL IONS AND METAL ION GRADIENTS IN THE
PHYSIOLOGY AND HOMEOSTASIS OF MAMMALIAN
SKIN 190
2.1. Metallothioneins and Metal Carrier Proteins 191
2.2. Growth Factors, Metal Ions, and Repair Systems Following
Injury 195
2.3. Metals with a Minor Trace Metal Value 197
2.3.1. General Aspects 197
2.3.2. Cobalt 198
2.3.3. Chromium 199
2.3.4. Nickel 201
2.3.5. Manganese, Molybdenum, Vanadium, and Tin 202
2.3.6. Silicon 204
3. XENOBIOTIC METAL IONS 205
3.1. Silver, Gold, and Platinum 205
3.2. Lead, Cadmium, and Mercury 210
3.3. Aluminum and Zirconium 213
3.4. Metals in Cosmetics 215
3.5. Miscellaneous Toxic Metals 218
3.6. Metalloids: Arsenic, Antimony, and Others 220
188 LANSDOWN
4. CARCINOGENICITY OF METAL IONS IN THE SKIN 222

5. THE EYE 224
5.1. Trace Metals in Ocular Development and Health 224
5.2. Xenobiotic Ions and Ocular Toxicity 227
6. GENERAL CONCLUSIONS 228
ABBREVIATIONS 229
REFERENCES 230
ABSTRACT: The skin and eyes remain in constant exposure to the surrounding envir-
onment and are subject to accidental, occupational, and biological risks at all times.
Normal development, homeostasis, and repair following injury depend upon appro-
priate levels of calcium, zinc, magnesium, copper, iron, and minute amounts of other
trace metals. Both tissues exist in a permanent state of dynamic equilibrium with the
environment whereby cells lost through natural wear and tear are replaced through
genetically regulated mitotic patterns. Normal functional requirements of the con-
stituent tissues depend on critical balances between trace metals, metal ion gradients,
and specific carrier proteins which are modulated by upregulation of growth factors,
cytokines, hormones, and subcellular regulators acting by autocrine, paracrine, and
endocrine mechanisms. Metal ion gradients in epidermal tissues serve critical functions
in basal cell proliferation, post-mitotic migration, and functional differentiation in nor-
mal homeostasis and in repair following injury. Toxic mechanisms reflect imbalances in
trace metals or interaction between xenobiotic and trace metals through competitive
binding key carrier proteins and metabolic pathways leading to trace metal imbalances
and functional impairment. Alternatively, toxic injuries result through direct cytotoxic
action of metal ions on cell membranes, intercellular communication, RNA and DNA
damage, and mutagenic change. Arsenic is the only primary carcinogen in the skin fol-
lowing ingestion or topical exposure; beryllium, aluminum, and zirconium are a cause
of granuloma. Aluminum as a cause for breast cancer is equivocal. Metal toxicities in
the eye result from direct accidental or occupational exposure and systemic uptake of
neurotoxic metals and their action on the retina and optic nerve. Calcium, zinc, magne-
sium, and iron are essential trace elements in eye development and physiology but sil-
ver, gold, lead, and mercury are absorbed through optic membranes or from the
circulation to accumulate in the vitreous leading to local or systemic action. Lead, mer-
cury, cadmium, aluminum, and other xenobiotic metals are implicated in structural and
physiological damage in the mammalian eye. Thallium shows an affinity for melanin.
KEYWORDS: eye . homeostatic mechanisms . metal carrier proteins . metal ions .

metallothionein . nutrients . skin
1. INTRODUCTION
The skin presents a challenge in the predictive safety evaluation of metals. It

varies greatly in outward appearance, structural integrity, and physiological
function from one part of the body to another according to the age, sex,
race, genotype, geographic location, and the state of nutrition. Human skin
Met. Ions Life Sci. 2011, 8, 187–246

METAL IONS AFFECTING THE SKIN AND EYES 189
is unique in the animal kingdom and has no counterpart in sub-human

species although animal models have provided information on the biological
and biochemical changes involved in cellular regulation, genetic transcrip-
tion, and mitotic control in response to normal wear-and-tear and regen-
eration and repair following physical or toxic injury [1].
The skin and its appendages comprise up to 10% of the total weight of the
body and provide a buffer between an individual and the surrounding
environment. The skin in all parts of the body exists in a perpetual state of
dynamic equilibrium with its surroundings, where cell loss through normal
wear-and-tear processes and through injury is replenished through appro-
priate mitotic activity in epidermal and dermal stem cell populations, cell
differentiation, and functional differentiation [2]. Interaction between con-
stituent tissues of the epidermis and dermis is essential in maintaining
homeostatic balances at all times. The multi-laminate epidermis comprising
20–30% of the total skin thickness is better defined than the vascular dermis
which contains a complex structure of connective and elastic tissue, nerves,
and reticuloendothelial cells. Melanocytes of neural crest origin become
established in the epidermis and extend dendritic cell processes into epi-
dermal cell layers. They give a distinct coloration to the skin through mel-
anin secretion and absorb solar radiation as a protective measure.
Langerhans cells and Merkel cells of presumed reticuloendothelial and
ectodermal origin, respectively, reside in the epidermis and have recently
been characterized by cytochemical markers for cytokines and growth fac-
tors, but their role in epidermal cell kinetics is imperfectly understood [3]. All
cell types in the skin are responsive to or dependent upon metal ions for their
normal physiological function and morphogenic processes. As purer and
specific antisera become available, so a greater understanding is gained of
the importance of trace metals and metal carrier proteins in normal skin
physiology and response to injury. Toxic responses in the skin result from
excesses and imbalances in trace metal concentrations

direct cytopathic effects of metal ions in target cells or tissues
insufficiency of protective mechanisms against toxic influences.
At least ten metallic elements are known to have essential roles in one or
more cell types in mammalian skin as electrolytes, enzyme cofactors, and
structural components [4,5]. Calcium, zinc, magnesium, and copper exhibit
defined gradients with established roles in epidermal proliferation, migration
and functional maturation, whereas iron, cobalt, molybdenum, and silicon
have a more restrictive distribution and are normally present in minute
amounts [6–8]. Normal homeostatic mechanisms and repair systems
following injury rely upon critical metal ion balances, and excesses in zinc,
calcium or copper can be as injurious as too little in causing pathological
Met. Ions Life Sci. 2011, 8, 187–246

190 LANSDOWN
change. Metal carrier proteins exhibit fundamental roles in trace metal

metabolism and in maintenance of ionic balances in skin which play a
central role in mitosis, migration, and functional differentiation in response
to injury [9]. Thus, cysteine-rich metallothioneins (MT) which exist in all
living cells in the body and which bind zinc and copper as essential trace
metals, are sensitive also to induction and binding xenobiotic metals like
cadmium, silver, and mercury. In summary, metal-binding proteins serve the
following functions in dermal physiology
regulation of trace metal ion balances and cytoprotection against toxic

metals
uptake and release of trace metals for physiological function
control of mitotic patterns
genetic regulation and expression of metal binding in response to
intrinsic and environmental factors.
Virtually all metals are known to be contact sensitizers and causes of

allergenic hypersensitivity in predisposed persons.
The eye is a target organ for the action and interaction of many metal ions
but responses vary according to the age of an individual, the route of
exposure, and the sensitivity of integral parts, i.e., cornea, conjunctiva,
vitreous, neuroretina, and pigment epithelium [10]. The lens, corneal epithe-
lium, and eyelids are of ectodermal origin whilst the neuroretina develops
from an out-pouching of the forebrain. The component tissues are dependent
on specific metal ion nutrients in development and function and are subject to
injury through metals and soluble metal compounds in industrial fumes and
vapors, particles in the air and through contaminants in the circulation. The
retina is a potential target for systemic toxicity of neurotoxic metals like lead,
cadmium, and mercury. Systemic exposure to lead, nickel, and cobalt pro-
duces degenerative changes in photoreceptor cells and the ganglion cell layer.
2. METAL IONS AND METAL ION GRADIENTS IN THE

PHYSIOLOGY AND HOMEOSTASIS OF MAMMALIAN
SKIN
The skin is a bilaminate structure with the epidermis and dermis subject to
genetically modulated regulation through hormones, growth factors, cyto-
kines, chalones, and nutritional factors [2,11–13]. Well defined gradients for
calcium, magnesium, zinc, and copper exist in normal epidermal homeo-
stasis, but distribution patterns for these and other trace metals are less
clear in dermal tissues [6,7]. Anatomical and physiological studies show that
Met. Ions Life Sci. 2011, 8, 187–246

the unit mass of skin (epidermis and dermis) remains fairly constant
according to the region of the body and the genotype, race, age, geographic
location, and social status of an individual and that maintenance of mitotic
homeostasis is dependent upon interaction between endogenous and envir-
onmental factors [2]. The dermal portion of the tissue representing between
70–90% of the total skin thickness can vary following injury [14]. Metal ion
distribution is specific in normal skin for the region of the dermis or epi-
dermis and its metabolic state [4,5,15].
Virtually all metal ions necessary for normal tissue homeostasis are
derived from the diet and the absorptive capacity of the gastrointestinal
mucosae is critical in the active or passive transfer of ‘‘free’’ ions to the
systemic circulation [4,16]. Generation and availability of free metal ions is
influenced by microbial flora in the intestine and the presence of phytate,
histidine, plant fibres, and agents like ethylenediaminetetraacetate (EDTA)
which selectively bind trace and xenobiotic ions. Carrier proteins including
MTs, ceruloplasmin, calmodulin, cahederin, S-40 proteins, and ferritin are
variously relevant to the uptake and cytoplasmic regulation of zinc, copper,
calcium, magnesium, and iron in normal and damaged tissues, but experi-
mental evidence illustrates interaction and competitive ionic binding, with
excess of one metal impairing uptake of another [9,17,18].
2.1. Metallothioneins and Metal Carrier Proteins

Metallothioneins illustrate the intrinsic role of metal-binding proteins in the
physiology of the skin, their induction, and clinical significance as cyto-
protectants and modulators of proliferation and differentiation [19,20]. Four
biochemically distinct isoforms having been isolated so far, with MT-1 and
MT-2 expressed mainly in mitotically active stem cells of the epidermal
basement epithelium, MT-3 in neurological tissue, and MT-4 in stratified
epithelia of the skin and tongue. The human genome comprises 12 distinct
MT genes of which only 6 or 7 encode as functional proteins for MT-1 and
-2 proteins. Synthesis is promoted by many factors including exposure to
divalent and transition metal ions like zinc, copper, cadmium, mercury,
silver and gold, hormones, growth factors, cytokines, tumor promoters, UV-
damaged DNA, and a variety of stress factors including local nutrient
deficiencies [7,9,21,22]. Nitric oxide is possibly involved in MT regulation
under conditions of stress [23]. MTs are low molecular weight proteins
containing 61 or 62 amino acids including cysteine residues [24]. MT-1 and -
2 located in stem cell populations of the basal epidermis and outer hair root
sheath modulate zinc as cofactor or metalloenzyme component [7], but act as
metal ion regulators, cytoprotectants, and mitotic promoters [20,25–28]. MT
expression may be specific for the phase in the cell cycle since high levels of
Met. Ions Life Sci. 2011, 8, 187–246

192 LANSDOWN
MT-1 and -2 have been seen in the S-phase and in basaloid cells in epidermal
carcinoma [29]. Deficiency states as seen in MT-null transgenic mice have
been associated with mitotic inhibition, impaired wound repair, and
increased susceptibility to chemical carcinogens like 7,12-dimethylbenz-
antharacene [30].
Experimental studies indicate that MT-1 and -2 are upregulated in epi-
dermal cells in wound margins and correlate with increased local concentra-
tions of zinc [5,31]. Increased MT levels persist through hyperplastic phases
but decline in periods of normalization [24,27]. Depressed expression of the
MT gene in MT-null transgenic mice is consistent with reduced zinc accu-
mulation and impaired mitotic activity following exposure to mitotic pro-
moters and UV irradiation [32]. Wound-bearing rats exposed to topical
application of cadmium salts, showed increased levels of MT-1 and -2 and
zinc in wound margins, but cadmium displaced zinc in the MT complex. At
higher levels local cadmium ‘‘overload’’ saturated the cytoprotective function
of the MT proteins and unbound Cd21 was locally toxic and impaired wound
repair [33,34]. Experimental studies have demonstrated that topical applica-
tion of soluble silver salts to skin wounds induced MT-1 and -2 but con-
comitantly led to local increases in zinc concentration in the wound margin
and improved healing [5,35]. In this model, silver(I) induced MT synthesis
which stimulated local zinc uptake in epidermal stem cells, but since Ag-MT
complexes are more stable, Zn21 was released for participation as enzyme
cofactors or metalloenzyme synthesis in DNA/RNA synthesis and re-epi-
thelialization [7]. Unlike cadmium, silver is nontoxic in injured tissue [36],
Ag1 not bound in a biologically inert MT complex, readily precipitates in
complexes with albumins, macroglobulins or cell debris in wound exudates to
be eliminated in normal healing [37,38]. Excess silver precipitated as silver
sulfide or silver selenide as intercellular deposits or bound lysosomally giving
a cosmetically undesirable skin discoloration but without toxic change [39,40].
At least 50 different calcium binding proteins (CaBP) are expressed in the
mammalian genome [6]. The principle CaBP in the skin include the S-100
proteins located in the cytosol and nucleus of epidermal cells, cytosolic
calmodulins and cahederins which are more specifically expressed as mem-
brane proteins with a role in cell motility and migration. The distribution of
the CaBP in the skin reflect the role of calcium in the postmitotic epidermal
cell migration patterns and functional proliferation. Calcyclin (an S-100
protein) is upregulated in proliferating cells and increased levels of calmo-
dulin occur in postwound skin indicative of the role of calcium in differ-
entiation [5,17]. Immunocytochemistry has demonstrated that calmodulin
levels closely reflect calcium concentrations in epidermal cells, being low in
interphase or resting epithelia and increased in proliferating and differ-
entiating tissues. Calcium and calmodulin exhibit defined gradients through
the epidermis with lowest levels in the basal epidermis and highest levels in
Met. Ions Life Sci. 2011, 8, 187–246

the superficial zone of the granular layer [41,42]. Observations in normal and
injured tissue suggest that CaBP, vitamins C and D and possibly parathyroid
hormone regulate balances between calcium and zinc, and between calcium
and magnesium. Whereas zinc is high in mitotically active cells of epidermal
basal epithelium, fibroblasts, and macrophages, calcium-containing enzymes
act in postmitotic functional differentiation. A high zinc level inhibits some
calcium-promoted events through competitive binding to cytoplasmic cal-
modulin, a reciprocity exists between tissue calmodulin and cAMP levels
and modulation by excess zinc [43]. Magnesium levels are high in the pre-
sence of low calcium and low in foci of high calcium-enzyme activity. Cal-
modulin is sensitive to oxidation and in cation stability, but its capacity to
bind magnesium is equivocal [44,45]. Investigation into cation binding of the
calmodulin molecule suggests that the protein expresses six binding sites of
differing ionic affinity, four bind calcium to variable extent and two which
do not discriminate between calcium and magnesium [46]. Mutated calmo-
dulin molecules have shown that magnesium ion modulates calcium-cal-
modulin complexing and is dependent upon pH, ionic strength, and relative
concentrations of other cations. Terbium(III), a toxic xenobiotic rare earth
metal with strong oxidizing potential, can replace calcium in calmodulin
complexes and has provided useful information in understanding mechan-
isms of cation binding on the calmodulin molecule [47,48].
Ceruloplasmin (CPN) is an endogenous plasma ferroxidase which binds at
least 95% of the plasma copper concentration; it regulates copper transport
and is upregulated in response to tissue injury, chronic inflammation or
hormonal action [49,50]. Copper mobilization from the cellular compartment
for inclusion in cuproenzymes like lysyl oxidase, tyrosinase, and cytochrome
oxidase probably reflects an interaction and dynamic equilibrium between
CPN as a carrier protein and MT as a metal segregating peptide [51]. Similar
metal-binding protein balances probably regulate mobilization of other ions
including zinc, magnesium, calcium, and manganese according to cellular
and physiological needs in the skin and other tissues.
Experimental studies involving intravenous injection of radiolabelled
copper have demonstrated the importance of CPN in copper transport and
storage, and in hereditary copper transport disorders including Wilson’s
disease (autosomal hereditary progressive lenticular degeneration) and
Menkes syndrome. CPN receptors are seen on a variety of cell types, and
investigations have disclosed the peculiar ability of this protein to donate its
copper content to recipient tissue systems [52]. Menkes kinky-hair syndrome
is an X-linked inherited neurological disorder of copper metabolism which
reflects mainly as abnormalities in collagen cross-linking and a failure in
the –S-S– bonding configuration in hair keratin attributable to defects in
lysyl oxidase and tyrosinase (also leading to albinoism) [53,54]. Reduced
neurological myelination is a feature of the various hereditary copper
Met. Ions Life Sci. 2011, 8, 187–246

194 LANSDOWN
deficiency diseases including the ‘‘crinkled mouse’’ an autosomal recessive

trait that manifests by low copper leading to reduction in skin thickness and
defects in hair bulb formation [57]. In this model, administration of dietary
copper reduced the expression of the mutant gene and led to normal hair
development. Improved understanding of the Menkes gene should provide
an understanding of the cellular management of copper and the implications
of copper deficiency in elastin and connective tissue formation in wound
healing and epidermal homeostasis [55–58]. Excess copper, especially in
cases of Wilson’s disease, is toxic and multisystem pathology occurs invol-
ving liver, kidneys, brain, and neurological tissues [59,60]. Skin, hair, and
nail are instrumental in excreting excess copper from the body and the
‘‘green hair’’ syndrome may result, but MT-1 and -2 provide cytoprotection
against excesses in Cu21 as well as in regulating the complex interactions
between Cu21, Zn21, and other divalent ions. Copper and zinc interact to a
large extent in biological systems and many examples exist to demonstrate
that high zinc suppresses copper uptake and metabolism in the intestine,
liver, and skin [58]. Whereas zinc serves as cofactor in numerous metal-
loenzymes involved in collagen and protein breakdown in extracellular
matrices [7], cuproenzymes function in the cross-linking of collagen and
elastic fibres, thereby strengthening dermal structure. This means that in a
tissue like the skin which is subject to fluctuating demands for tissue repair
and remodelling, a correct balance should exist between the two ions to
provide appropriate tissue homeostasis and growth [58]. Bremner and
Beattie [58] also point out that although changes may occur in the cellular
activity of cuproenzymes exposed to zinc, there is no evidence to show that
high zinc displaces copper from these enzymes. The availability of free Cu21
is possibly reduced through the regulatory action of the MT molecule [58].
Iron is a minor but important nutrient in the skin providing a route for
mobilization and intracellular metabolism of oxygen radicals [8]. Much of
the body iron load is strongly bound within the hemoglobin molecule, but
uptake and transport of ferric iron is dependent upon ferritin and trans-
ferrins as carrier proteins [61,62]. Although the ferritin molecule binds up to
4,500 atoms of iron, the molecule is rarely saturated allowing binding of
zinc, copper, cadmium, and beryllium [63,64]; apoferritin binds similar
amounts of these four ions also, some of which are dialyzable in rat liver
homogenates. Chelation of iron to plasma transferrin renders it soluble
under physiological conditions, prevents iron-mediated free radical toxicity,
and facilitates iron transport to cells. X-ray crystallography has shown
transferrins to be complex polypeptides with domains expressing iron-
binding and anionic binding sites [65]. The precise mechanism for transfer
and chelation of iron to transferrin in the intestine is not known, but
interaction with the copper-dependent ferroxidase is suspected [66]. Ferritins
are synthesized in skin fibroblasts in response to oxidative stress. They serve
Met. Ions Life Sci. 2011, 8, 187–246

the dual functions of storing iron and segregating iron for protection of iron-
catalyzed reactive oxygen species [67]. Ferritin gene expression is controlled
at translational level or at a transcriptional level in an ion-dependent man-
ner. The heavy and light chain subunits of mammalian ferritins have been
sequenced by polymerase chain reaction (PCR) to identify amino acid
profiles, which seem to be species-specific. The light chain subunit lacks
ferroxidase activity but is possibly involved in iron nucleation and increased
iron uptake; in contrast, the heavy chain fragment plays a crucial role in
chelating iron through ferroxidase activity. Excess zinc binds to the ferritin
molecule and impairs synthesis in rat liver leading to reduced iron absorp-
tion from the diet [68]. Animals fed high dietary zinc became anemic and
exhibited no compensatory increase in iron uptake. The xenobiotic metals
aluminum and beryllium also bind mammalian ferritins with pathological
consequences [69]. Gastrointestinal absorption of iron is promoted by
vitamins C, B2, B3, B6, and B12 [70].
2.2. Growth Factors, Metal Ions, and Repair Systems

Following Injury
Skin injury resulting in a reduction in cell mass, changes in intercellular
relationships, and structural and functional impairment of the tissue triggers
a sequence of events known colloquially as the wound healing cascade [71].
Any reduction in the efficiency of the epidermal barrier function results in
exposure of deeper tissues to physical trauma and toxic factors in the
environment including xenobiotic metals. Constituent events of the wound
healing cascade require cytological competence of cells in the wound margin
and their capacity to upregulate expression of cytokines, growth factors,
mitotic promoters regulating chemotactic gradients. Wound bed preparation
involving debridement of inhibitors in necrotic and damaged tissue, control
of pathogenic infections and provision of a conducive microenvironment
underlies current clinical strategy for maximizing healing in acute wounds
and burns, and chronic ulcers [72,73]. Nutrition is a key element in wound
bed preparation and recent research has emphasized that sequential events in
tissue repair require a balanced pool of metal ions including calcium, zinc,
magnesium, copper, magnesium, iron, sodium, and potassium in the wound
bed [5,74,75]. As the wound healing cascade progresses, so absolute and
relative concentrations of these ions change to reflect their relevance in
metalloenzymes, electrolytes, and cofactors in key biosynthetic events. Bal-
ances have been demonstrated between metal-carrier and storage proteins
and release of metabolizable ions in response to growth factors, cytokines,
nutrient deficiencies, and hormones [71,76].
Met. Ions Life Sci. 2011, 8, 187–246

196 LANSDOWN
Experimental studies emphasize that whereas zinc, calcium, copper,

magnesium, and iron perform essential functions from hemostasis (phase 1)
through the inflammatory and proliferative phases (phases 2 and 3), and
tissue reorganization and normalization (phase 4), requirements for elements
like manganese and silicon are more limited to periods of connective tissue
formation and tissue reconstitution [5,77]. In each case, local concentrations
of metal-binding and carrier proteins are upregulated to maximize metal ion
uptake and cellular metabolism through the action of hormones, growth
factors, and nutrients possibly through a form of negative feedback
mechanism. Local calcium concentrations increase in post-surgical wounds
through the action and interaction of parathyroid hormone, calcitonin, and
vitamin D (or its metabolized form 1,25-dihydroxycalciferol (vitamin D3))
thereby regulating Ca21 concentrations to satisfy demands in the four
sequential and overlapping phases in the wound healing cascade [6]. Similar
modulation can be expected in the case of zinc, copper, magnesium, and
iron, where deficiencies or imbalances can be expected to lead to chronic or
non-healing wounds [7,78].
Experimental studies in a wound healing model illustrate sequential
changes in calcium, zinc, copper, magnesium, and iron and their relationship
to calmodulin and MT-1 and -2 [5,6]. Thus, injury promoting release of
platelet-derived growth factor (PDGF) signalled a local rise in calmodulin
and tissue calcium in response to the need for calcium as Factor IV, VII,
IX, and X in the conversion of prothrombin to thrombin in hemostasis,
platelet remodelling and aggregation [79]. Subsequently, calcium levels
remained high in keeping with their functional role in activation of phos-
pholipases, protein kinases, and events of the inflammatory and proliferative
phases. PDGF has been shown instrumental in motivating calcium and
establishment of chemotactic pathways for cell migration [80]. In a similar
way, local deficiency in zinc and zinc-dependent mitogenic enzymes possibly
mediated by hormones, interleukin-1 or growth factors upregulate the
expression of MT in metabolically active cells of the wound margin and local
increases in zinc. Like calcium, zinc fulfils multiple roles as metalloenzyme
cofactor, metalloproteinases, and DNA and RNA synthetases through
much of the wound healing programme [81]. Events in regeneration and
reorganization in the epidermis and dermis depend upon appropriate gra-
dients in both metals but regulation and the spatial distribution is expected
to be dependent upon conditions in the local environment created by
interleukin-1 and the various growth factors. A prolonged demand for iron
in the carriage and metabolism of oxygen radicals for collagen synthesis, scar
tissue formation, and immunological competence is expected and experience
shows that in a state of low oxygen tension, dermal angiogenesis is retarded,
granulation tissue formation is reduced and healing results are delayed
[82,83].
Met. Ions Life Sci. 2011, 8, 187–246

Copper levels are low in normal skin but increase in response to demand
for lysyl oxidase and related cuproenzymes in collagenesis in wounded tissue
[84]. High copper in the remodelling phase of wound healing has been
associated with increases in the peptide Gly-(L-His)-(L-Lys) or GHK which
has a Cu21 affinity similar to that of albumin. The GHK-Cu21 complex
underpins chemoattraction for macrophages, angioblasts, and mast cells, as
well as providing antiinflammatory action involving suppression of free
radicals, thromboxane formation, release of oxidizing iron, growth factors
(TGF-b1 and TNF-a), and protein glycation. In angiogenesis increased
superoxide dismutase is accompanied by vascular dilation. Increased copper
is also consistent with increased protein synthesis with cuproenzymes pro-
moting collagen, elastin, metalloproteinases, antiproteases, and proliferation
of fibroblasts, keratinocytes, and nerve fibres. Copper as GHK-Cu21 has
been shown to enhance integrin cytokine expression in proliferating kera-
tinocytes in cell culture and their transformation from pro-mitotic state into
S-phase [85]. Immuno-histochemistry has demonstrated that GHK-Cu21
promoted synthesis of the human stem cell marker p63 which maintains the
survival of these basal keratinocytes. It is now known that a close interaction
exists between the divalent metals calcium, copper, and zinc in the motiva-
tion and modulation of proliferation in the basal cells of the epidermis [56],
and that normal homeostasis and repair following injury are dependent
upon appropriate ionic balances and modulation of metal-binding to histi-
dine- and cysteine-rich proteins.
2.3. Metals with a Minor Trace Metal Value

2.3.1. General Aspects
Normal human skin and repair systems rely upon the availability of at least
fifteen metal elements and deficiencies or imbalances through nutritional
insufficiency or disease are potential causes for functional or pathological
conditions [16]. Metal ion chelators or hereditary metabolic disease states
leading to states of unavailability of trace metal ions as metalloenzymes or
enzyme cofactors are causes of dermatological disorders. Resulting defects
may be multifactorial as in the case of calcium and zinc, or more specific as
for molybdenum or cobalt which serve specific roles as cofactors, growth
modulators or vitamin components [4,74,75,86]. Carrier proteins which are
instrumental in regulating uptake and metabolism of major trace elements
are not specific and interact and bind other ions by a competitive process
[19,20,25].
Analytical studies have revealed a range of trace and xenobiotic metals in
skin extracts, melanin, hair, and nail [87]. The significance of such ions as
Met. Ions Life Sci. 2011, 8, 187–246

198 LANSDOWN
silver, arsenic, tin, lead, cadmium, vanadium, etc., is questionable. None

have an acknowledged trace metal value and all are potentially toxic if
present in excess amounts [15,88]. They may be present bound within the
structure of the hair as excretory products from the circulation or bound to
SH ligands on the hair surface and of minimal toxicological significance.
Alternatively, xenobiotic ions may be chelated or otherwise bound by epi-
dermal keratins or the various metal carrier/binding proteins including MT,
CPN, calmodulin or ferritin [5,17]. Iron (heme) complexes are a sensitive
indicator of trace metal availability and bind many metal ions in organic or
inorganic form [89]. Tin and cobalt with minor trace metal value induce
heme oxidase in target cells or tissues and may impair iron metabolism.
Whereas nutrient requirements for zinc, calcium, iron, copper, and mag-
nesium are defined [6–8], the putative roles of manganese, molybdenum,
cobalt, vanadium, and chromium in skin, nail, and hair growth, and overall
body health are less clear [90]. Much information relating to the role of
minor trace elements in the skin has been derived from experimental studies
in animals, but identification of appropriate markers for deficiency states in
human is unclear [91,92].
2.3.2. Cobalt
Cobalt is an essential part of vitamin B12 (cyanocobalamin) with a central
role in the methylation of homocysteine and its conversion to methionine
[93–95]. The vitamin B12 molecule comprises an atom of cobalt linked to
four reduced pyrole rings and a nucleotide group of six conjugated double
bonds. The cobalt core is resistant to chemical degradation and this accounts
for the irreversible binding of cobalt to amino acids like cysteine and histi-
dine [96,97]. Vitamin B12 also converts L-methylmalonyl-coenzyme A (CoA)
to succinyl-CoA by a separate reaction. Patients deficient in cobalt and
vitamin B12 exhibit anemia, neuropathology, and loss of sensory perception
and dementia [98–100]. Fatty acid metabolism becomes increased as a
consequence of vitamin B12-induced changes in hepatic cytosolic enzymes
and increased activity in the Krebs cycle citrate synthetase and mitochon-
drial cristae [95,101]. Dermal implications of cobalt deficiency include
impaired DNA synthesis, hyperpigmentation, vitiligo, angular stomatitis,
and alterations in hair growth [102]. Mucocutaneous lesions are character-
istic of early signs of the condition.
The mechanisms for hyperpigmentation are not fully understood, but may
be attributable to increased melanogenesis rather than to abnormalities in
melanin synthesis or metabolism of tyrosine and associated enzymes. The
chemistry of mammalian melanin synthesis is complex and views have been
expressed as to the relative contributions of metals including cobalt, copper,
iron, manganese, and zinc which have been detected in sites of active
Met. Ions Life Sci. 2011, 8, 187–246

melanogenesis [103]. It is conceivable that these metal ions catalyze or

contribute in some way to the rearrangement and conversion of dopachrome
from 5,6-dihydroxyindole-2-carboxylic acid in melanin and eumelanin for-
mation, but this is an unlikely explanation of hyperpigmentation in cases of
cobalt deficiency.
Cobalt is not well absorbed across guinea pig skin (and presumably
human skin) following topical application of cobaltous chloride (o1.0%) on
account of the strong binding of the Co21 ion to exposed SH groups on
epidermal keratin of the outer stratum corneum [104,105]. Cobalt is a
common cause of contact allergy through exposures in household deter-
gents, cement, food, hair dyes, ceramics, hard metal alloys, and paints, but
on occasions diagnosis is complicated by co-contamination and cross-sen-
sitization with nickel and chromate [106–108]. ‘‘Hard metal allergies’’ are
diagnosed by recurrent dermatitis, folliculitis or chronic lichenified eczema.
As a feature of the sensitization process, Co21 penetrates reticuloendothelial
Langerhans cells of the epidermis to initiating the sensitization process
[107,109]. Cobalt allergy may be exacerbated by solar radiation, but impli-
cation of cobalt as a photoallergen requires further investigation [110].
Occupational exposure to cobalt in mining, metal refining and extraction,
and production of tungsten carbide hard metals is a profound cause of
respiratory distress and pulmonary granuloma [111,112]. Although Co(II)
salts are genotoxic in vitro and in vivo [114], and subcutaneous injections in
rodents have produced sarcomas at injection sites [113], cobalt is not pre-
sently recognized as a human carcinogen. Cardiomyopathy is a well docu-
mented toxic effect of excessive cobalt consumption in beer drinking
[95,115].
2.3.3. Chromium
Chromium is a minor trace element in the human body with a probable role
in the modulation of glucose and lipid metabolism, and tissue sensitivity to
insulin [116]. Recent studies claim that addition of chromium to the diet of
diabetic patients with low insulin sensitivity, improved glucose tolerance and
tissue sensitivity to endogenous insulin to alleviate diabetic symptoms in
elderly patients with Types I and II diabetes. It is conceivable that chromium
improves insulin binding to cellular receptors by influencing protein phos-
phorylation-dephosphorylation reactions and acts in some way as a glucose
tolerance factor (GTF), but more research is required to investigate the
subcellular metabolism of chromium in target cells [117].
Chromium probably has no direct influence on skin morphology but its
action in alleviating diabetes is expected to benefit wound healing where
patients are subject to delayed and indolent wounds with serious infections
leading to poor quality of life [118].
Met. Ions Life Sci. 2011, 8, 187–246

200 LANSDOWN
Hexavalent chromium ion is appreciably more toxic than the trivalent ion.
Cr(VI) compounds are cytotoxic and mutagenic to cells in culture and have
been reported to evoke chromosomal damage with sister-chromatid
exchanges, impaired DNA and protein synthesis, and abnormal nucleotide
metabolism [119,120]. Modification of membrane-linked enzyme activity
may underpin these toxic changes. Experiments in guinea pigs treated
topically with 51Cr-labelled sodium chromate have shown increased intra-
epidermal retention up to 5-hours followed by a plateau phase, suggesting
that the epidermal barrier function was relatively effective in binding
the ion [104]. Trivalent chromium compounds are more common in nature
and found as contaminants in most organic matter. Whilst there is no evi-
dence to show that Cr(III) is converted to Cr(VI) in biological materials
[121], Hostynek et al. considered that from the dermatological perspective,
Cr(III) is least problematic on account of its low solubility and inability to
penetrate biological membranes [15]. During percutaneous penetration
Cr(VI) is reduced to Cr(III) or is absorbed percutaneously in an unchanged
form to act on stem cells in the basal epidermis or deeper. Penetration of
chromium compounds through human and animal skins varies greatly
according to the chemical properties of the compound applied and the
condition of the skin at the site of contact. Cr(III) compounds ionize to bind
strongly to exposed sulfhydryl residues of cysteine but the keratin-chromium
complex is lost through normal desquamation and is of minimal tox-
icological significance. However, as with many salts of inorganic acids, the
free acid released in the presence of skin moisture, exudates, sweat, and
sebum, is irritant and can influence the epidermal barrier function through
corrosive damage [4].
Occupational exposure to Cr(VI) compounds is an acknowledged indus-
trial hazard [122,123]. Experience has shown that Cr(VI) compounds are 10–
100-fold more toxic than Cr(III) on account of their higher solubility, risks
of allergenicity and contact dermatitis are appreciably higher. In cytogeni-
city tests with human fibroblasts, Cr(VI) compounds exhibited a fourfold
higher incidence of cytotoxicity and genotoxicity. In summary, dermal and
systemic toxicity of chromium salts is now regarded as a measure of their
oxidation state and solubility in body fluids. Studies in a bicycle manu-
facturing plant in Japan have shown high rates of lung cancer in patients
with skin ulceration and perforation of the nasal septum allowing greater
penetration of the mutagenic Cr(VI) [124]. Contact allergy and delayed
hypersensitivity are major toxic hazards with chromium exposure in the
home or occupationally [107]. Chromium allergy is commonly complicated
by allergies to other metals like cobalt and nickel and possible cross-sensi-
tization may occur [125–127]. Standard patch tests provide a convenient and
accurate means of determining chromium allergies, and provide an incentive
for adopting rigorous strategies for health and safety at work promotion.
Met. Ions Life Sci. 2011, 8, 187–246

Estimates suggest that exposure to 450 ppm Cr(VI) is sufficient to evoke

contact allergy, compared to 165,000 ppm for Cr(III) compounds [126].
Manufacturers of paint and clothing materials and tanners exposed to
chromium contamination have drawn up codes of conduct for skin and eye
protection [128]. A further caution is indicated by the observation that
chromium induced skin injuries are exacerbated by alkalis and physical
traumas, but mitigated by addition of dilute (0.35%) iron oxide to cements
or building materials [129].
2.3.4. Nickel
Nickel is listed as a micro-trace metal in the human body with roles
in the regulation of lipid and carbohydrate metabolism [16,130,131] and in
melanogenesis [103,132,133]. According to Anke et al. nickel is present
in all tissues where it performs a central role in dehydrogenase and
transaminase metabolism [134]. It interacts with iron and calcium and in
excess can lead to skeletal deformities and parakeratotic changes in
the skin. Nickel deficiency is reported to be a cause of anemia [134].
Mertz noted that institutional diets were commonly low in nickel but that
normal health diets should contain at least 30 mg/kg [131]. Nickel is
excreted in hair in common with many other metals of trace and xenobiotic
importance, but in cases of diabetes, hair nickel concentrations are raised as
part of a wider spectrum of metal ion disturbances [135]. Elsewhere, a
population study involving 206 children (not knowingly exposed to
nickel in their diets or environments) contained an average of 0.6 mg/g nickel
[136], which might reflect a role for nickel in melanogenesis, but more
probably it is a route of excretion and part of the normal detoxification
process [137].
Human epidermal cell homogenates were shown to absorb nickel ion
strongly, thereby reducing the amount penetrating percutaneously [138].
Ni21 exhibits strong SH complexing, but in cultured keratinocytes at least,
this binding appears to be a reversible process which is inhibited by chelating
agents like EDTA, L-histidine, and penicillamine as used to treat patients
following heavy metal poisoning [139]. Other in vitro studies have demon-
strated that nickel binds preferentially to carbonyl residues in tissue extracts
rather than to other amino acid residues [140], and thus differs from most
other metal cations. In keeping with its profound sensitizing potential and
capacity to evoke delayed hypersensitivity reactions, nickel like chromium
and cobalt is readily absorbed into Langerhans cells as a preliminary to
immunogenic changes [141]. There is evidence that nickel absorbed by
metabolically active cells in the skin exhibits modest capacity to evoke MT
synthesis, but its cytoprotective value of MT against nickel-related derma-
titis or allergic reactions is insignificant [142].
Met. Ions Life Sci. 2011, 8, 187–246

202 LANSDOWN
Despite its probable value as a trace element, nickel is a toxic metal. It is a

profound contact allergen and maybe cross-sensitizer with chromium and
cobalt [107]; it is mutagenic and a presumed cause of human cancer [143].
The carcinogenic effects have been related to its lipid peroxidation properties
and induction of DNA strand gaps and breaks and DNA cross-links. The
carcinogenicity of nickel subsulfide has been established in animals and in
human studies, and several nickel compounds are acknowledged mutagens,
dermal irritants, and contact allergens [106,107,144–148]. Workers exposed
occupationally to nickel oxide exhibited chromosomal aberrations in per-
ipheral lymphocyte cultures with the incidence of chromosomal damage
correlating well with the duration of nickel exposure, serum and hair con-
centrations [149].
Clinical evidence suggests that alloys releasing at least 0.5 mg Ni21 per cm2
in 7 days exudates evoke sensitization [150], but more commonly, nickel
contact allergy is associated with inexpensive jewellery, wrist watches, clasps,
metal buttons, and clothing fasteners where perspiration and skin exudates
trigger ionization of the nickel contaminants. ‘‘Blue jeans button’’ allergy is
a common cause of dermatitis associated with nickel in buttons and snaps on
blue jeans worn in many parts of the world [151]. Rarely, dermatitis with
vitiligo-like depigmentation has been seen in patients following wearing
nickel in spectacle frames [152]. The mechanism for depigmentation is not
known.
2.3.5. Manganese, Molybdenum, Vanadium, and Tin
Normal development and function on the human skin are not commonly
held to depend upon systemic availability of manganese, molybdenum,
vanadium or tin, which have been identified as minor trace metals with
biochemical roles in mucopolysaccharide synthesis, xanthene and aldehyde
oxidases, insulin-mimetic functions, melanogenesis, and general growth,
respectively [87]. Much of this work has been conducted in laboratory
animals, but extrapolation to the human body is equivocal [153].
Manganese is better known for its role in cartilage and bone formation
with deficiencies manifest by defects in alkaline and acid phosphatases,
mucopolysaccharide synthesis, cell proliferation, and growth retardation
[154,155], but other studies suggest that manganese may have a role reg-
ulating glucose metabolism and associated hormonal changes [156,157].
Either mechanism is liable to have an impact on skin where mucopoly-
saccharides and glycosaminoglycans perform essential functions as inter-
cellular ground substance, and where glucose metabolism is essential in
normal homeostasis [158]. Manganese concentrates in mitochondria but
intracellular management of the metal is unclear. In systemic overload
situations, manganese has been associated with oxidative changes, possibly
Met. Ions Life Sci. 2011, 8, 187–246

resulting from interaction and impairment of ferritins or other iron carrier

proteins [159,160].
The nutrient function of molybdenum as a cofactor in xanthene oxidase
and dehydrogenase, aldehyde oxidase, and sulfite oxidase is recognized
through experimental studies in animals and inherited defects in molybde-
num metabolism in humans [161,162]. Impairment of molybdenum avail-
ability through antagonism by the xenobiotic metal tungsten and mutational
defects in sulfite oxidase synthesis may have dermal implication in nitrogen
and copper metabolism [163]. Johnson et al. identified four patients with
defects in molybdenum metabolism and inherent inability to synthesise the
enzymes sulfite oxidase and xanthene oxidase [164]. Mental retardation,
deformities in the structure of the skull, neurological deformities, and dis-
located ocular lenses were associated with deficiency in hepatic molybde-
num, even where serum levels of the metal were normal. The dermatological
implications of low sulfite oxidase in cultured dermal fibroblasts in these
patients is unclear but biochemical analysis of defects in synthesis of the
enzyme suggest that the molybdenum cofactor resides in outer mitochon-
drial membranes and is essential in some way for ‘‘internalization or
packaging of the nascent sulfite oxidase in the inter-membrane spaces’’ [164].
Molybdenum like several other trace metals is seen in melanins but its role in
melanogenesis is unclear [87].
The clinical picture of tin as a trace element in the human body and its
implications in skin morphology are not well documented [88]. Experiments
in laboratory animals claim that tin interacts with zinc by blocking zinc
receptors on carrier proteins [165] and hence, may be a cause of impaired
epidermal homeostasis, but this has not been substantiated. Whereas
experimental evidence shows that tin-deficient diets are a cause of impaired
growth and hair loss in laboratory animals, excessive tin inhibits the uptake
and metabolism of key trace metals like iron, copper, and manganese with
toxic consequences [166]. Tin is not well absorbed from Sn(IV) compounds,
either gastrointestinally or percutaneously. Sn(IV) has a strong tendency to
form coordination complexes with 4, 5, 6, and possibly 8 ligands and is
eliminated in urine [167]. Organotin complexes exhibit low percutaneous
absorption and are a cause of skin and eye irritancy and contact hyper-
sensitivity [15,168]. Methyltin affects mitochondrial oxidative phosphor-
ylation and impairs mitochondrial membranes.
Vanadium is a multivalent metal found as an environmental contaminant;
available evidence suggests that the toxicity of pentavalent compounds is
higher than lower oxidation states, the skin being a target organ. Vanadium
is classified as a micronutrient and claimed to act as an insulin-mimetic agent
with a capacity to inhibit phosphotyrosyl-protein phosphates and reduce
blood glucose in vitro and in vivo through activating insulin-receptor kinase
[169,170]. Vanadium acts intracellularly as an inhibitor of several key
Met. Ions Life Sci. 2011, 8, 187–246

204 LANSDOWN
enzymes including ribonucleases, acid and alkaline phosphatases, and

sodium-potassium ATPase and adenylate cyclase [171–174]. Other aspects of
vanadium cytotoxicity include upregulation of insulin receptor activity,
NADH oxidation in microsomes with increased production of hydrogen
peroxide and superoxides [170,175]. Vanadium pentoxide is a dermal irritant
and the dust is cause of occupational eczematous dermatitis at 6.5 mg/m3,
although percutaneous absorption is negligible [176,177]. Certain soluble
vanadium salts are astringent in contact with the skin and exhibit anti-
perspirant action, however, the suitability of vanadyl oxychloride, chloride,
oxybromide with or without aluminum salts in antiperspirant cosmetics is
questionable [178].
2.3.6. Silicon
Silicon is a metalloid element with limited trace nutrient value in connective
tissue synthesis in the dermis, vascular laminae, and the skeletal system
[179–181]. Experimental evidence suggests that silicon acts as a ‘‘bound
component’’ and is central in glycosaminoglycan synthesis and possibly
cross-linking of collagen fibres [182]. Deprivation of silicon in growing rats
was shown to decrease collagen synthesis through an inhibition of collagenic
enzymes, reduction in hydroxyproline and ornithine aminotransferase [183].
It is unclear whether silicon interacts with copper or iron-dependent enzymes
involved in the hydroxylation of proline or in subsequent stages in col-
lagenesis, but impaired collagen production has major implications both in
bone formation and skin wound repair [184]. The metabolism of silicon in
the human body is not fully understood, but following intestinal absorption
it locates epidermally in the basement membrane of the skin (collagen IV),
dermal connective tissue, and intima of blood vessels. The silicon content of
these tissues declines with advancing age and this may be contributory to the
increased fragility of the skin and impairment in wound healing seen in older
people [185]. Silicon(e)-containing wound dressings have become increas-
ingly popular in the therapy of indolent and difficult-to-heal wounds,
although their mechanism of action is not understood. There is no evidence
to show that bioactive silicon is absorbed from the dressings to participate in
the healing process or in collagenesis or angiogenesis in the wound bed
[186,187]. Silicone sheeting and related materials are beneficial in the therapy
of hypertrophic scars and keloids. Silicone sheeting was shown to inhibit cell
proliferation and contraction with a downregulation of TGF-b2 in fibro-
blast-populated collagen matrix constructs [188], even though silicon-con-
taining wound dressing promoted fibroblast proliferation and upregulated
FGF-b are contradictory to this idea [189]. Silicon is neither cytotoxic or
mutagenic to cells in culture. Other reports claim that silicon(e)s can
improve the quality of the skin and are beneficial in skin cosmetics for rough
Met. Ions Life Sci. 2011, 8, 187–246

skin or treatment of chronic papulo-pustular acne and scar tissue, but the
cytological mechanisms are not understood.
Many other silicon containing products including silicones (polysiloxanes)
and organosilicones used in electronics, medical devices, cosmetics, building,
and clothing come into direct contact with the human skin in normal
usage and, clinical and occupational hazards associated with dermal contact
with most inorganic and organic silicon-containing products are very low.
Respiratory distress and lung cancer are prevalent in workers exposed
chronically to crystalline silica dusts and factory effluents [190]. Silicon as a
trace element is not allergenic in exposed persons. Subcutaneous injection or
instillation of silica or silicone has led to subcutaneous granuloma with the
xenobiotic and inert material being demonstrated microscopically by
polarizable light diffraction or X-ray microanalysis [191]. Subcutaneous
reactions to silicone gels in breast implants have proved contentious, but
after more than 40 years experience it is now clear that the reactions are not
attributable to silicon per se, but to the organic complexes administered.
Evaluation of 35 statistically valid studies has concluded that silicone ‘‘does
not cause disease’’ and that women with breast silicone implants experience
a lower incidence of breast cancer than would otherwise be expected
[192,193].
3. XENOBIOTIC METAL IONS
3.1. Silver, Gold, and Platinum

The precious metals gold, silver, and platinum have an established value as
antibacterial agents in the treatment of human disease. They have no known
trace metal value in the human body but the metals or their soluble com-
pounds come into contact with the skin in daily life and through occupa-
tional exposures; each presents risks of contact allergy and delayed
hypersensitivity in predisposed persons [107]. Contact allergy in jewellery is
attributable commonly to the presence of other metals in the alloys as in
‘‘white gold’’ which is composed of 5–17% nickel, o30% copper with minor
concentrations of palladium and other elements with documented allergenic
properties [107].
The value of silver as a broad spectrum antibiotic and its efficacy in
controlling persistent and life-threatening infections, has led to production
and clinical acceptance of a range of antiseptic wound dressings, medical
devices including catheters, prostheses, sutures, orthopedic pins, dental
products, and hygiene clothing [194]. Contact allergy from silver is a pro-
blem but local discolorations attributable to the deposition of silver
Met. Ions Life Sci. 2011, 8, 187–246

206 LANSDOWN
precipitates in epidermal keratin and wound debris are viewed as a sign of

silver toxicity. However, silver presents minimal toxic risk following topical
application, percutaneous absorption is negligible (except in severely burned
patients treated with silver sulfadiazine, and the therapeutic benefits of silver
vastly outweigh is toxic properties [194–196]. Silver nitrate is used in con-
centrated solutions, sticks, and douches in ablation of skin infections [197],
and Strong Silver Nitrates (Avoca) for removal of scars, excessive granu-
lations, warts, and disfiguring skin lesions. These preparations are astringent
and corrosive through the release of nitric acid, but the damage is local and
controlled. Biologically active silver ion released in the presence of tissue
fluids and secretions avidly complexes with proteins (SH groups), epi-
dermal keratin, and cations (Cl, PO3 4 ) present in sweat and skin secretions,
epidermal debris, and hair. Discolorations due to precipitation of silver
sulfide in surface keratins are lost through normal wear-and-tear and
through normal keratinocyte desquamation with minimal toxic con-
sequence. Experimental studies have demonstrated that Ag1 liberated from
silver nitrate or SSD is absorbed into cells at wound margins where it
induces MT synthesis [198,199]. This is accompanied by increased local
concentrations of mitotically active zinc (Zn21) and possibly Cu21 leading to
improved re-epithelialization in acute/surgical wounds. Ag1 precipitated as
colorless complexes with albumins and macroglobulins is eliminated as
wounds heal with only marginal concentrations absorbed systemically [200].
Ag1-induced MT has a mitogenic role in wound repair possibly mediated
through induction of growth factors and cytokines [9].
Silver is absorbed into the human body through ingestion of contami-
nated food and drinking water or by inhalation of dusts or aerosol disper-
sions. Water purification using silver:copper filters or Katodyne products to
protect against Legionella sp. is expected to be a major source of blood
silver in some communities [201]. About 10% of silver ingested is absorbed
as protein complexes to be distributed to all tissues of the body other than
the brain and central nervous system [202]. The brain and nervous
systems are protected from the toxic action of silver and some other ions
through the efficiency of the blood brain barrier and blood-CSF barrier
[203,204]. In industrial exposures, Ag1 is absorbed through the mouth, nose,
and lungs from metallic dusts, silver nitrate droplets in the atmosphere
and exposure to soluble silver compounds. Blood concentrations of silver
are normally o3 mg/L for most people but may be as high as 20 mg/L
following occupational exposure to silver dusts and residues [205,206].
These concentrations are rarely of toxicological significance in view of
the strong tendency of Ag1 to form inert precipitates with proteins and
inorganic anions. Macrophages perform a central function in mopping up
silver precipitates in wound sites and perineural, connective, and interstitial
tissues.
Met. Ions Life Sci. 2011, 8, 187–246

Argyria and chrysiasis are the principle side effects of excessive occupa-
tional exposure or ingestion of soluble silver and gold compounds, respec-
tively. They commonly occur in areas of skin exposed to solar irradiation but
these long lasting discolorations are not health threatening, and are classified
as undesirable cosmetic changes. In contrast, symptoms of platinosis arising
from chronic exposure to platinum in jewellery are rare and mostly attri-
butable to contact allergy [207]. Argyria and argyrosis resulting from
deposition of silver complexes in dermal tissues of the skin and eye (cornea
and conjunctiva), respectively, are commonly seen in patients consuming
unregulated and unsupervised colloidal silver preparations for gastro-
intestinal infections, allergic rhinitis and unspecified respiratory complaints
[208–211]. Regulatory authorities regularly cite symptoms of argyria in
patients receiving intravenous therapy with silver arsphenamine for syphi-
litic infections in promulgating safety guidelines [212,213]. Silver arsphena-
mine is a dangerous drug but these early studies demonstrated a close
relation between argyremia and severity of skin discoloration, but even now
the minimal amount of blood or tissue silver necessary to manifest as
recognizable argyria is not appreciated [214,215]. The mechanism for argyria
is equivocal but may result from imbalances in the local concentrations of
soluble and insoluble complexes and the availability of free selenium ion
[216,217]. Silver complexes react strongly with selenium to precipitate as
insoluble silver selenide by a reductive process involving lysosomal reductase
and solar energy [218]. The orthorhombic a-form crystals deposit as elec-
tron-dense granules in the papillary dermis close to the basement membranes
and basal epithelia of eccrine sweat glands and hair follicles. X-ray micro-
analysis has demonstrated that the black-brown granules of argyria are
mostly intracellular between connective fibres. The granules show a high
silver, sulfur, and selenium content but may contain trace concentrations of
osmium, lead, mercury, and titanium, possibly resulting from workplace
contaminations [219,220]. Histopathological evidence has disproved earlier
claims that silver is excreted transepidermally from dermal deposits [216],
and is not associated with increased melanogenesis or mitotic activity in
melanocytes. Dopa-reaction for tyrosinase was normal [221]. Silver is
excreted via the hair and nail in argyric patients [214,222].
Argyric changes in human skin are associated with use of silver (and
silver-gold) acupuncture needles commonly used in Eastern countries
[220,223]. Diffuse or macular distribution of blue-black skin discolorations
in patients using the ancient Hari practice for relief of muscular pain,
headaches, and shoulder discomfort were directly related to the number of
needles implanted and the duration of therapy. One patient was reported to
have implanted 2,500 needles into all parts of her body over a 13 year period
[224]! Argyria-like discolorations have also been recorded in patients using
silver acetate lozenges as antismoking remedies [214]. Lozenges containing
Met. Ions Life Sci. 2011, 8, 187–246

208 LANSDOWN
radioactive tracer (110mAg) administered sublingually have been used to

determine whole body silver burdens using neutron activation analysis.
Argyria of head and neck in an otherwise healthy patient was associated
with a body burden of 6.40.2 g silver.
Gold differs chemically from silver and is commonly present as Au(III)
form as in gold chloride (AgCl3) and chloroauric acid (H[AuCl4] 3H2O),
but like silver, Au31 readily complexes with serum proteins and induces and
binds MT-1 and -2 [225]. Principle human exposures to gold include jew-
ellery, occupational exposures, and through intramuscular injection or oral
administration of antiarthritic drugs including sodium aurothiomalate,
auranofin, aurothioglucose, and injectable nanogold preparations [226–228].
Rare incidences of gold toxicity are recorded in patients using silver-gold
acupuncture needles [220]. Risks of hepatic and renal damage do occur in
patients receiving antiarthritic gold therapies and chrysiasis and allergic
dermatitis are documented. Renal problems may be exacerbated by
deposition of immunocomplexes of gold in glomerular blood vessels
[107,229–231]. Whilst chrysiasis may present cosmetically undesirable grey-
purple discoloration of the skin in light-exposed areas as a result of gold
complexes deposited in reticular and papillary dermal regions, it is not life
threatening. Gold allergies manifest as chronic eczematous papular changes
with profound dermal inflammatory cell infiltration (monocytes) and edema
are more serious. They tend to persist after removal of the gold jewellery,
probably on account of retention of Au1-keratin complexes in the stratum
corneum. Rare incidences of skin cancer are reported in patients exposed to
jewellery containing residual radioactivity, and where the b or g emissions
elicit mutagenic and neoplastic changes [232]. One such case concerned a
superficial squamous cell carcinoma in a woman who wore a radioactive
gold ring for more than 30 years. Radiation dose measurements indicated
that the dose to basal skin layer was 2.4 Gy (240 rad) per week. If the woman
wore her wedding ring continually for 37 years she would have received a
maximum dose of approximately 4600 Gy. A case of lymphocytoma cutis
was diagnosed in a lady wearing contaminated gold earrings [233]. Experi-
mental studies in rats have demonstrated that repeated subcutaneous
injection of sodium aurothiomalate led to sarcoma [234].
Chrysiasis is a distinctive and permanent discoloration of the skin attri-
butable to administration of 20 mg/kg gold [235]. Skin biopsies have
shown gold precipitates bound lysosomally in macrophages, epidermal cells,
and Langerhans cells, but in contrast to silver, gold is melanogenic and UV
light may enhance the preferential absorption of the metal by human skin
[236]. However, percutaneous penetration of Au31 through intact skin
is low on account of the biologically active nature of the ion and its strong
binding to SH ligands in epidermal keratin. Experimental studies have
shown that metallic gold implanted subcutaneously ionizes and diffuses into
Met. Ions Life Sci. 2011, 8, 187–246

surrounding tissues to be sequestered by mast cells, macrophages, and

fibroblasts [237]. Injection of gold-protein complexes in chrysotherapy have
been associated with gold excretion in epidermal keratinocytes, hair and nail
[238–240] with gold residues in the skin correlating well with levels injected
[241]. Yellow nails are a feature of gold therapies for rheumatoid arthritis
[242]. An interesting clinical report related to two people wearing gold neck
chains who suffered a lightning strike in the countryside [243]. The gold
necklace in the lady partly evaporated to produce a tattoo-like discoloration
but biopsy after 6 months revealed an accumulation of gold particles in
subcutaneous foreign body reactions comprising fibroblasts, histiocytes,
multinucleate giant cells. EDX elemental analysis confirmed the presence of
gold, silver and copper in the proportions normally present in white
gold [107].
Metallic platinum is used in jewellery, dentistry, photography, and in
chemical and electrical industries. The skin is not a major target organ for
platinum toxicity, and pulmonary toxicity, rhinitis, and delayed hypersen-
sitivity are the principle toxic risks of occupational exposure [244–247].
Occupational exposure to platinum is a cause of dermatitis and ocular
effects, but they are rare and more commonly due to complexes like chloro-
platinate. Roberts conducted scratch tests on 60 platinum workers exposed
to chloroplatinate and observed hypersensitivity reactions but in his view the
scratch test was not a reliable means of confirming allergic responses to
platinum and that the tests may be hazardous or life threatening [244].
Hexachloroplatinate (1 mg/mL) produced an anaphylactic reaction after a
single injection [248]. In predictive dermal irritancy patch tests, insoluble
platinum(II) oxide was not irritant to intact or abraded rabbit skin, but the
two soluble chlorides (PtCl2 and PtCl4) were mildly irritant in applications
of 0.1 g in 0.1 mL water [249]. Symptoms of contact sensitivity to platinum
including pruritis, erythema, urticaria-like symptoms and eczematous reac-
tions were identified in platinum refinery workers by standard prick tests
[107,250,251], but hypersensitivities attributed to rhodium, irridium or pal-
ladium may be implicated.
Cisplatin (CISP), carboplatin, and oxaliplatin have been used in the
chemotherapy of ovarian, pulmonary, and other solid tumors [190,252].
Although structurally similar, they differ in their capacity to inhibit cell
division in target cells and DNA binding [253]. CISP and hydroxyl-
malonatodiamine platinum (MDP) inhibited the growth of melanoma cells
in vitro with MDP exhibiting more selective lethality to melanoma cells than
to neoplastic pulmonary or bone marrow cell lines. Mitotic inhibition was
closely related to the amount of platinum bound to tumor cell DNA. Pt(IV)
oxide or chloride lethality to alveolar macrophages and human lung fibro-
blasts was associated with decreased cellular ATP and incorporation of
14
C-thymidine, 14C-uridine, and 14C-leucine [254]. Experiments with four
Met. Ions Life Sci. 2011, 8, 187–246

210 LANSDOWN
platinum complexes revealed suppression of splenic reactive cells to produce

antibody-producing cells and myelocytic progenitor cells with transitory
immunosuppression and minor hemopoietic toxicity [255], but effects on
Langerhans cells in the skin have not been seen.
3.2. Lead, Cadmium, and Mercury

Lead, cadmium, and mercury are potentially toxic in all tissues in the human
body but the skin, hair, and nail are convenient biomarkers of occupational
or environmental exposure and systemic concentrations [137]. Additionally,
lead and mercury are used in cosmetic preparations and concern has been
raised concerning the systemic safety of lead in hair dyes and mercury as
colorants in tattoos [256–258]. The metals are cytotoxic, mutagenic, and a
cause of chromosomal damage in fibroblasts and/or lymphocytes in vitro
[259,260]. Lead and cadmium are classified as putative human carcinogens
through occupational exposures [261], whereas mercury is a cause of sub-
cutaneous granuloma and sarcomas in animals following repeated sub-
cutaneous injection or intravenous administration [262–264]. Subcutaneous
injections of cadmium are also a cause of sarcoma and histocytic tumors but
evidence of lymphonecrosis in Fischer F344 rats suggests that cadmium may
suppress leukemia [265]. Broken thermometers are a common cause of
mercury toxicity and granuloma and foreign body reactions with dense
fibrosis, inflammatory cell infiltration, and lymphoid hyperplasia have been
reported [266]. These reactions were localized in some cases, whereas other
cases were associated with a more generalized systemic mercury toxicity.
Mercury vapor released from broken thermometers in neonatal incubators
has been shown to cause poisoning in tiny babies [267].
Lead absorption by inhalation, consumption of contaminated food, and
drinking water or percutaneous absorption is commonly monitored by
blood lead and by concentrations in hair or fingernails, but the so-called
lead-line at the margin of the gums and in the teeth in cases is characteristic
of lead poisoning [197]. Epidemiological studies in Spain and Germany
illustrate that metal binding in hair and toenails is a useful index of metal
exposures and human risk. Metal uptake in these tissues varies greatly
according to the age and sex of individuals, hair color, geographic area, and
occupational factors. In 478 Spanish children lead in hair was significantly
higher in girls than boys (10.54 versus 6.55 mg/g), but concentrations
declined with advancing age [268]. Concentrations of lead in hair were higher
in red hair than blond, brown or black, possibly reflecting interactions
between metallic elements in melanogenesis and the SH ligands in hair
keratin. The German survey illustrated a more complex interaction between
lead, cadmium, and trace metals in hair and toenail clippings. In 474
Met. Ions Life Sci. 2011, 8, 187–246

individuals, the relative concentrations of cadmium in toenail clippings and

hair were 547 versus 90 ng/g, compared to average lead levels of 8.5 versus
2.7 mg/g [269,270]. Copper and zinc concentrations were similar in both
tissues but toenail samples showed a good correlations between lead, cad-
mium, copper, and zinc. Schroeder and Nason demonstrated that female
hair contained higher levels of magnesium, copper, and cobalt than men,
and that younger women exhibited more copper, lead, and cadmium [271,
272]. Grey hair in women had more magnesium and less cadmium than in
men, and in men there was more magnesium and less cadmium in black hair.
Zinc levels were lower in blond colored hair in men and women. Nickel,
cadmium, and lead did not accumulate with age. Hair lead was higher in
children from industrial areas than in those from agricultural communities
and closely related to environmental concentrations and occupational
situations [273–277].
Metallic lead is without noticeable effect on intact human skin and lead
acetate used in hair dyes is of minimal toxicological significance; allergies are
rare [277–279]. Experimental studies in rats showed that 1% lead acetate was
non-irritant but retarded hair regrowth without obvious changes in hair
papillae or follicles. In human trials, o0.3% of Pb(IV) is absorbed percu-
taneously despite the strong ion binding to –SH ligands in epidermal keratin
[280]. The greyish precipitates in hair keratin provide the color sought in
using the ‘‘Grecian Formula’’ hair dye formulations. Percutaneous uptake is
higher with inorganic compounds like lead naphthenate and tetra-
ethyllead, and absorption is enhanced by sweating [281,282].
Intracellular toxicity of lead in cell membranes and mitochondria is
attributable to its strong ability to inactivate proteins and enzymes by
modifying their tertiary structure through binding sulfhydryl, amino car-
boxyl, and hydroxyl moieties [283]. Lead is a well known cause of mito-
chondrial swelling and degeneration [284]. Lead also exhibits a profound
influence on iron metabolism through binding and inactivating ferritin and
ferroenzymes leading to microcytic anemia, possibly through ionic compe-
tition and impairment of intracellular iron pathways. Lead is capable of
impairing immune responses through its irreversible cytotoxic action and
membrane disruption in Langerhans cells [285–287]. Immunosuppression in
goats to 2,4-dinitrobenzene was increased and cutaneous hypersensitivity
reactions significantly lower when cadmium or lead and cadmium were
administered compared to lead alone, but the mechanism is not known.
Topical instillation of lead acetate into the lacrymal sac resulted in local
irritancy in guinea pig eyes and a reduction in the number of corneal Lan-
gerhans cells [287]. Chromosomal damage expressed as impaired erythrocyte
porphyrin metabolism, reduced d-aminolevulinic acid dehydratase activity
and chromatid aberrations with increased metaphases and gaps occurred in
women exposed to lead in production of batteries [288]. Lead acetate
Met. Ions Life Sci. 2011, 8, 187–246

212 LANSDOWN
injected subcutaneously led to necrosis and foreign body reaction with

lead deposits identified within collagen bundles by energy-dispersive X-ray
analysis [293].
Clinical studies suggest that percutaneous absorption of bioactive lead
from hair dyes (2% lead acetate) is insufficient to cause lead lines at margin
of gums, headache, irritability, impaired muscle action, neuropathy or other
evidence of lead poisoning [257]. Pb(IV) absorbed from dyes applied to the
scalp was excreted in axillary and pubic hair although poor correlations were
seen between lead excretion and blood levels. Lead is a cumulative poison
and accumulates in bone and tooth leading to impairment in skeletal
development and in calcium metabolism irrespective of its route of uptake
[289–292].
Cadmium also has a strong propensity to interact with bivalent trace
metals like zinc and calcium in soft tissues and bone [33,34,294,295]. Cad-
mium and zinc strongly evoke MT synthesis in epidermal cells and form
stable complexes with it [19,20,296]. At topical doses of 41.0% cadmium
chloride, Cd21 was a dermal irritant known and was readily absorbed for
binding to serum albumins and induction of MT synthesis [295,297]. Skog
and Wahlberg using radioactive tracers demonstrated percutaneous pene-
tration of Cd21 and Hg21 in guinea pig skin and their systemic distribution
[104]. Absorption of Hg21 from mercuric chloride, potassium mercuric
iodide, and methyl mercuric dicyanamide increased with concentration to a
maximum of 3.2–4.5% in 5 hours, presumably to a point of –SH ligand
saturation. In comparison, Cd21 was absorbed from the CdCl2 in a con-
centration-related pattern (0.005–4.870 M) over 5 hours and was lower than
Hg21 uptake. The percutaneous absorption and toxicity of the two metals
was increased in injured skin or where the epidermal barrier function was
stripped prior to application. In vitro studies suggest that Cd21 absorption is
higher from contaminated water than soil on account of its strong keratin
binding capacity [298]. Experimental studies in rats and mice have shown
that percutaneous absorption of Cd21 from CdCl2 is equivalent to about
6,000 ng/g, but at this level hyperkeratosis, acanthosis, and ulcerative
changes developed [294]. Epidermal mitotic indices were increased twofold
indicating a compensatory response in homeostatic mechanisms.
The OSHA considered that percutaneous absorption of cadmium in an
industrial setting was of low toxicological significance, but cadmium is
allergenic in predisposed persons [299]. Epidemiological studies of cera-
micists and dental personnel exposed regularly to metallic cadmium or
soluble cadmium compounds exhibited increased concentrations of the
metal in hair, without obvious toxic changes [300]. A study of 226 school-
children in the Tarragona Province of Spain showed that girls excreted more
cadmium in their hair than boys and levels declined with advancing age
[301]. The color of the hair did not influence the amount of cadmium
Met. Ions Life Sci. 2011, 8, 187–246

absorbed or excreted, but cadmium levels in scalp hair were higher than in
pubic hair following environmental exposure. Excretion patterns of cad-
mium in hair were higher in summer months than in winter [269,270].
Glutathione oxidase may be effective against toxic metals in the skin, thus
Bannai et al. demonstrated that Cd21 induced glutathione oxidase in mac-
rophages through induction of amino acids like cysteine [302], but the
implications of this in skin wound repair where macrophages infiltrate as
part of the inflammatory phase remains to be seen.
Mercury vapor is absorbed percutaneously and buccal contact allergy
though dental amalgams are common occupational hazards for dental
patients and dentists [303,304]. A Spanish study revealed that 3% of 2,592
patients exposed to mercury experienced allergy and more than half reacted
to mercury in antiseptics, cosmetics containing ammoniacal mercury, foot-
wear, and inhalation of mercury vapor from broken thermometers [305].
A Japanese study of 59 cases of the mercuric drug thimerosal ( ¼ thiomersal)
proved to be a major source of contact dermatitis, but inconsistent evidence
was presented to show cross-reactions between mercurichrome and ammo-
niated mercury [306]. Lichenoid drug reactions, patchy alopecia, and sto-
matitis were reported following occupational exposure to mercury in
recycling procedures, but an unusual case of mercury hypersensitivity in a
31-year-old man was recorded where antinuclear antibodies were in the
presence of normal IgE levels [307].
Low levels of Hg21 are absorbed percutaneously through human skin
since much of the ion is bound by epidermal keratin to be lost by natural
skin growth [308]. Volunteers exposed to concentrations of 0.88–2.14 ng/cm3
of mercury for up to 43 minutes absorbed 216–844 ng of Hg21 (equivalent to
0.0101–0.0402 ng Hg21/cm2/min per ng Hg21/cm3 in air). More serious
problems arise in cases of occupational exposure to mercury fulminate
(Hg(ONC)2) in factories manufacturing explosives [309]. This compound is
highly irritant and corrosive to the skin and in 29 male workers exposed
occupationally, chromosomal aberrations including micronucleated cells,
gaps, breaks, and fragmentation were detected in peripheral lymphocytes
[310]. Despite clinical and experimental evidence that mercury is toxic to the
skin and a cause of allergic responses, many mercury-containing products
are still in everyday use [311,312].
3.3. Aluminum and Zirconium

Aluminum was introduced into cosmetics more than 50 years ago and as
aluminum chloride, aluminum chlorhydrate or activated aluminum zirco-
nium chlorhydrate is still listed as a safe and effective antiperspirant and
deodorant [313,314]. Aluminum is found widely in the human environment
Met. Ions Life Sci. 2011, 8, 187–246

214 LANSDOWN
in drinking water and food and exposed to aluminum cooking pots and
culinary utensils [315,316]. Most aluminum salts ionize to release Al31 which
is absorbed gastrointestinally, but risks of neuropathy and Alzheimer’s
disease have been associated with uptake of aluminum as used in renal
dialysis [317–320]. In antiperspirants aluminum and zirconium salts are
claimed to cause axillary granulomas [321–325]. Recent studies claimed that
aluminum in antiperspirants is estrogenic and a contributory factor in
human breast cancer [326–328]. Presently, the role of aluminum as a possible
carcinogen is not confirmed experimentally or clinically [316,329–331]. Al31
exhibits extremely low percutaneous penetration in intact skin and absorp-
tion through sweat duct or hair follicles is minimal [332,333]. Aluminum
exhibits stronger binding to human scalp hair than cadmium, copper, lead or
zinc with a ‘‘saturation point of 0.34 mg/L (0.154 mg/g), but of this, 14.5 to
46.5% eluted after treatment [334]. The World Health Organization
reviewed the use of aluminum in drinking water in 1978 and noted that
intestinal absorption was also low in humans, but in animals o1% is taken
up according to the concentration, pH, and presence of competing ions
[335]. Absorbed Al31 readily complexes with serum proteins to be meta-
bolized but experimental observations in rats at least showed that aluminum
is not a cumulative toxin. No adverse effect levels (NOAEL) were estimated
to be 70 mg Al31/kg body weight daily [336], which is greatly in excess of
estimated human exposures in drinking water or food (0.0012.7 mg/L)
[337]. Aluminum is nonmutagenic in bacteria but was shown to induce lipid
peroxidation in cultured human dermal fibroblasts with release of lactic
dehydrogenase [338].
The use and mechanisms of action of aluminum and zirconium in cos-
metics and their putative roles in granuloma formation have led to numerous
clinical and experimental studies. Al31 exhibits a strong capacity to bind
SH ligands in epidermal keratin leading to obstruction of sweat and
sebaceous gland ducts and hair follicles [333,339]. Whilst the epidermal
binding and transitory denaturation of the stratum corneum may be of little
physiological importance, irritancy due to release of anions like Cl is a
disadvantage. Aluminum chlorohydrate is less irritant to most people and
allergic dermatitis is rare [107]. The mechanism of action of Al31 as an
anhidrotic agent is not known and views that the ion might in some way
promote intraductal reabsorption of sweat are not established [340–342].
Methylene blue iontophoresis has been used to demonstrate diffusion of
sweat into periductular areas as evidence of ‘‘changed permeability’’, but
milaria and mild inflammatory changes have been noted in the region of
sweat duct pores [342]. Recent views are that reduction in perspiration by
aluminum salts is largely due to obstruction of the sweat and sebaceous
ducts by aluminum-keratin ‘‘plugs’’, which are lost through normal phy-
siological mechanisms [333,343]. Aluminum nicotinate therapy in patients
Met. Ions Life Sci. 2011, 8, 187–246

with acne-related seborrhoea had no effect on sebum excretion in an 8 weeks

study and there were no obvious changes in the clinical acne [344]. Occa-
sional cases of irritancy of minor toxicological significance are recorded, but
in a controlled study of 65 patients treated with 20% aluminum chloride
hexahydrate daily for one week with axillary hyperhidrosis, the Al31 release
achieved excellent control of sweating without side effects [345].
Transitory inflammatory reactions were reported in rats implanted sub-
cutaneously with powdered alumina (Al2O3) with implant sites becoming
fibrosed [346]. Dermal macrophages sequestered and accumulated alumi-
num particles or insoluble precipitates [347]. A clinical case of granuloma
attributable to aluminum used in a tattoo may have been attributable to a
local response to aluminum precipitates or a rare case of delayed hyper-
sensitivity, but diagnosis was complicated by the presence of titanium resi-
dues in isolates [348]. Other clinical studies have established greater risk of
zirconium as a cause of granuloma formation in antiperspirant and deo-
dorant formulations and in therapy for Rhus dermatititis [349]. (Rhus sp. are
a family of toxic plants including poison ivy, poison oak, and poison sumac
found mainly in the United States and Canada). Rhus dermatitis manifests
clinically as a vesicular eczema, patchy erythema, and local discolorations
[350,351]. Biopsies showed dense and persistent infiltration of epithelioid
cell, lymphocytes, and Langerhans-type cells characteristic of delayed
allergic hypersensitivity reactions [352]. Spectrographic analysis revealed
zirconium lines of 3,391 and 3,438 Å in biopsies of the granulomas. Zirco-
nium granulomas were not reproduced by subsubcutaneous, intracutaneous
or percutaneous injection of 20% zirconium tetraisopropoxide injections in
rabbits except in the presence of hexachlorophene, whereas in similar
experiments, acute inflammatory reactions with tissue necrosis and ulcera-
tion were noted following injection of aluminum salts [321].
3.4. Metals in Cosmetics

Cosmetics are defined as ‘‘articles intended to be rubbed, poured, sprinkled
or sprayed on or otherwise applied to the human body for cleansing,
beautifying, promoting attractiveness or altering the appearance of the
body, teeth or mucus membranes of the oral cavity’’ [314]. At least twenty
different metals have been used at different times in cosmetics, but arsenic,
mercury, and antimony have been removed from permitted lists in many
countries for safety reasons (Table 1). Kohl (containing lead and antimony)
is still used in some Asian and Middle Eastern countries as eye preparations
[353,354]. Antimony had been used for many years as a therapy for tropical
diseases and as an antifungal agent in cot mattresses but was withdrawn in
the 1990s on account of its association with sudden infant death syndrome
Met. Ions Life Sci. 2011, 8, 187–246

216 LANSDOWN
Table 1. Metals used in cosmetics.a
Metal Compound Usage
Aluminum chloride, chlorhydrate, sulfate, antiperspirant

sodium aluminum chlorhydroxy
lactate,
aluminum chlorhydrate propylene
glycol,
sodium aluminum sulfosilicate ultramarine color
(Na7Al6Si6O24S2)
alumina (Al(OH)3 or Al2(OH)6) transluscent color
kaolin, nacrite, dickite powders
(Al2O3 2SiO2 2H2O)
Barium sulfate (blanc fix) transluscent pigment
extender
Bismuth oxychloride (BiOCl) pearl lustre pigment
Calcium carbonate, phosphates, fluoride, Powders, dental products
nitrate
Chromium oxide (Cr2O3), hydrated oxide Pigment
Copper copper peptide complex skin conditioner, repair
Iron oxides (Fe2O3, Fe3O4), sulfate, Pigments
ferroso-ferric oxide
(hydroxy(oxo)iron),
OFe(OH)Fe(OH)FeO), ferric
ferrocyanide, ferric ammonium
ferrocyanide
Lead carbonate (PbCO2 Pb(OH)2) pearl lustre pigment
sulfide hair color
Lithium lithium magnesium silicate binder, bulking agent
Magnesium silicate(talc) (3MgO2 4SiO2 H2O) bulking agent, powders
lauryl ether sufates foaming agent
carbonate, stearates, myristate powders
Manganese manganese violet (NH4MnP2O7) Pigment
Potassium hydroxide, nitrate, citrate bath products, dental
hygiene
Silicon silica, silicates suspending agent, dental
hygiene
methicone, dimethicone abrasives
waxes hydrophobicity in cosmetics,
hair conditioners, etc.
Sodium lauryl sulfate, laureth sulfate surface active agent,
chloride, carbonates, phosphates, bath products
borates, cocoyl isethionate
fluoride toothpastes
Strontium acetate, chloride dental hygiene
Met. Ions Life Sci. 2011, 8, 187–246

Table 1. (Continued ).
Metal Compound Usage
Tin fluoride toothpastes abrasive

oxide nail care
Titanium dioxide white coloring, toothpastes,
UV absorber
Zinc stearate talcum powders
oxide, citrate white color, toothpastes,
nail care,
UV absorber (oxide)
pyrithione antidandruff
Zirconium aluminum zirconium chlorhydrate antiperspirant
a
Compiled from [314].
(SIDS) and through release of the toxic gas stibine (SbH3) [355,356]. The
dermal toxicity and toxic risks associated with barium and bismuth in cos-
metics are minimal on account of the low solubility of the compounds used
and their low percutaneous absorption. Barium sulfide (2%) is listed
amongst depilatory agents but no mention is made of its percutaneous
penetration or capacity to evoke sensitization [107]. Lithium is absorbed
through human skin in the course of therapy for seborrhetic dermatitis [357],
but its toxic action in human skin is not known. Mice dosed with lithium
chloride for 7 days exhibited a downregulation in expression of MT-3,
ATPase, and several polypeptides involved in metal ion homeostasis, and
chemical/electrical gradients across cell membranes [358]. It is unclear
whether Li1 induces MT-1 or -2 synthesis in the skin or influences the
availability of zinc and copper in epidermal homeostasis but dermato-
pathological changes including acneform lesions, pustular psoriatic derma-
titis, follicular hyperkeratosis, and perifollicular inflammation are reported
in patients given lithium therapy for psychiatric problems [359–363].
Lithium salts may influence psoriatic symptoms through release of inflam-
matory mediators with degranulation of psoriatic neutrophils, or through
disturbing dermal trace metal ion balances [364].
Strontium acetate and chloride are minor cosmetic constituents, but in
mammalian systems, Sr21 closely mimics Ca21 in bone development and
homeostasis in hydroxyapatite [4]. Strontium chloride (2%) is used as a mild
abrasive in dentifrices and the sulfide (SrS) is a depilatory in cosmetics
without adverse effects. Percutaneous absorption of Sr21 through intact skin
is low on account of its precipitation in epidermal keratin but some per-
follicular absorption occurs [365,366]. Comparative studies have shown that
only 0.26% strontium was absorbed from strontium nitrate applied to intact
Met. Ions Life Sci. 2011, 8, 187–246

218 LANSDOWN
skin in 6 hours, whereas 35% was absorbed in 30 minutes through abraded

skin, and 50% in 6 hours [367]. The dermal toxicity of strontium is low and
no evidence has been seen to show that it impairs calcium or zinc gradients in
epidermal homeostasis. Hahn demonstrated that strontium is a potent and
selective inhibitor of sensory irritation in the skin without local anaesthetic
effects [368]. To investigate the possible antipruritic effects of topical
strontium salts, 20% strontium nitrate was applied to the forearm of 8
volunteers following intradermal injection of histamine to induce itch sen-
sation [369]. The strontium therapy significantly reduced the magnitude and
duration of the histamine itch without complications.
Titanium oxide is popular in cosmetic products on account of its intense
white appearance, stability and low toxicity, but further experiments are
necessary to show whether absorption or ionization of titanium salts is
influenced by UV light [107,370]. Experimental studies have demonstrated
that inert granules located within the folds of the skin and in hair follicles
without irritancy. No evidence was seen that Ti(IV) interacts with trace
elements in epidermal homeostasis. Titanium dioxide in sun-screen for-
mulations provides an opaque barrier between the sun and the skin and
scatters the light rather than absorbing it.
3.5. Miscellaneous Toxic Metals

The dermal toxicity of the xenobiotic elements beryllium, cerium, palladium,
thallium, and thorium are not well documented. Cerium and palladium are
potential sensitizing agents but their dermal toxicity to the skin is minimal
even though both interact with trace metals like calcium and zinc in epi-
dermal and dermal homeostasis. Beryllium, thallium, and thorium as
encountered in industrial processes are toxic. Thorium (as thorium dioxide)
is used as Thorotrast as a contrast medium in radiology and has been
classified as a human carcinogen [371]. Patients injected with Thorotrast
exhibited an excess of malignant liver tumors, leukemias, and bone cancers
[372,373]. Primary routes of exposure to thorium are by inhalation, intra-
venous injection (of Thorotrast medium), ingestion, and dermal contact.
Uptake of metabolizable thorium by all routes is not well known, but
injection site sarcomas have been reported in experimental animals following
subcutaneous injection. Thorotrast is a cumulative poison that is not readily
cleared from the human body.
Cerium is a rare earth lanthanide element with a strong tendency to
interact with calcium in mammalian systems [374]. Clinical studies have
shown cerium to be minimally toxic on account of the relative insolubility of
phosphate and other salts and the capacity of Ce31 to precipitate with
proteins. However, cerium as Ce(NO3)3 has been found highly beneficial in
Met. Ions Life Sci. 2011, 8, 187–246

treating burn wound patients [375–378]. Ce31 does not readily penetrate cell
membranes, but may act through action on calcium-binding proteins and
enzymes like Ca-Mg-ATPase [378]. Whilst cerium has no obvious influence
on the cytological physiology of epidermal or dermal tissues at wound sites,
it exhibits the unusual capacity of mitigating the immunosuppressive action
of toxic materials released by the action of thermal energy on human tissues.
A low molecular weight burn toxin identified as a lipoprotein complex
(LPC) elaborated by heat energy on cell membranes has been shown to
impair physiological responses in skin cells and to damage cellular ultra-
structure in much the same way as thermal insult [379]. In clinical studies a
low molecular weight burn toxin identified as a suppressor active peptide
(SAP) complex of o5000 Daltons was isolated from plasma in burn patients
and shown to be capable of immunosuppression, inhibiting neutrophil
chemotaxis and hemolysis [380]. Although the action of Ce31 on cells at the
wound margin is not known, the ion does interact with and displace calcium
leading to a mineralization of the eschar that forms across wound surfaces.
This protective eschar is removed surgically with minimal trauma when the
wound is re-epithelialized.
Palladium like cerium is minimally toxic in mammalian tissues and shows
negligible percutaneous penetration through dermal contact with jewellery
or dental appliances. Palladium in white gold, industrial alloys and electrical
appliances may be a cause of metal allergy, it shows chemical similarities to
platinum and has been compared to it following human exposure or in
experimental studies. 103Pd has been employed as a source of radioactivity in
tumor marking without adverse reactions [381]. Contact allergies to palla-
dium do occur according to the route of exposure but diagnoses may be
complicated by palladium and nickel cross-reactions [382,383]. Wahlberg’s
and Bowman’s guinea pig maximization test (GPMT) results suggested that
palladium is a more potent sensitizer than nickel and might even be the
primary sensitizer in humans [383].
Thallium is a toxic metal although at one time it was a treatment of choice
for skin diseases, tuberculosis, and fungal infections [384]. Thallium acetate
was used as a ‘‘temporary’’ depilatory in children with favus and ringworm
infection of the scalp [197]. Thallium acetate is highly soluble in water and
percutaneous penetration through intact skin is high. Thallium compounds
are toxic to the skin following oral or topical exposures and alopecia is a
cardinal sign of thallium poisoning suggesting that follicular cells are targets
for the toxic action of this metal [385,386]. Follicular plugging and atrophy,
cystic dilatation with pustular dermatoses involving the face, eyebrows,
nasolabial folds, and limbs were reported in 5 patients with blood thallium
levels of 4500 mg/100 mL following oral dosage. Nail growth was affected.
Thallium is a cumulative poison and available evidence suggests that it is
transported to target cells by a potassium-pump mechanism, but is less
Met. Ions Life Sci. 2011, 8, 187–246

220 LANSDOWN
readily released than potassium [385]. Other information has shown that
thallium exerts a cytotoxic effect on isolated heart and muscle fibres by
interacting with and displacing K1 [387]. Whereas thallium appears to
mimic potassium in penetrating cell membranes, possibly on account of
similarities in their ionic size, Tl1 exhibits far greater binding than K1 to
organic ligands, mitochondrial membranes, ribosomes, and other sites
usually occupied by potassium. This suggests that the nervous system and
glandular tissues which are physiologically dependent upon K1 are parti-
cularly vulnerable to thallium toxicity [388,389]. Most soft tissues are vul-
nerable to thallium toxicity and experimental studies show that thallium
poisoning leads to parathyroid deficiency, hypocalcemia, and rachitogenic
changes in the skeleton [390].
3.6. Metalloids: Arsenic, Antimony, and Others

The metalloid elements boron, silicon, arsenic, and antimony exhibit some
characteristics of metals and non-metals. Silicon is now recognized as an
essential component of connective tissues in the skin and other organs
[180,181,187]. The position of boron is less clear although it is present in the
human body in trace amounts (o0.08 mg/100 mL) but clinical markers of
boron deficiency are not documented. Boric acid has been used in solutions
(4%), dusting powders, and pastes (ca. 25%), and as a topical antiseptic and
astringent in wound care for many years without serious consequences [391].
Boric acid is readily absorbed through the skin and simple experiments
have demonstrated that it is excreted in the urine within a few minutes of
topical application [392]. On rare occasions, topical boric acid has led to
local edema, exfoliation, and epidermal necrolysis and hypersensitivity
[107,393].
Arsenic and antimony are toxic to the skin and internal organs following
topical, intravenous or oral administration. Arsenic-containing therapeutics
including arsphenamine and Salvarsan have a long history in the treatment
of syphilis and spirochaete infections and diseases associated with protozoan
parasites, whilst antimony has been used in treating patients with bilhartzia
infections [197]. Although most of the arsenical drugs were administered by
intravenous or intramuscular injection, contact dermatitis, and dermatolo-
gical changes have been reported following exposure to arsenic in fungicides,
herbicides, and insecticides [394]. Cutaneous irritancy, pyoderma, and fol-
liculitis result from contact with arsenic trioxide but the dermal toxicology
predominantly relates to the carcinogenicity of the element. Occupational
exposure to arsenicals and medications like Fowler’s Solution (potassium
arsenite) led to keratosis, hyperpigmentation, and to an excess of skin can-
cers. All cell types in the human epidermis are susceptible to neoplastic
Met. Ions Life Sci. 2011, 8, 187–246

change through exposure to arsenic but Langerhans cells seem to be most

vulnerable [395,396]. Langerhans cells degenerate and lose their dendrites
following arsenic trioxide exposure and in cases of Bowen’s disease which
manifests as a highly malignant squamous carcinoma extending into folli-
cles, intraepithelial areas, and through the basement membrane to invade
dermal tissues [397]. The toxic mechanisms underpinning the responses of
Langerhans cells to arsenic are not known, but Birbeck granules are pre-
served despite the degenerative changes in other parts of the cell [398].
Experimental evidence has demonstrated that arsenic promotes neutrophil
chemotaxis and upregulates key growth factors including transforming
growth factor a (TGF-a) and granulocyte macrophage colony stimulating
factor (GMCSF) [399], but that it suppresses DNA synthesis and DNA
repair and elicits chromosomal damage [396,400]. The toxic action of arsenic
on skin cells is influenced by nutritional factors and by hepatitis B surface
antigen [401]. Blackfoot is a non-malignant peripheral vascular disease
leading to gangrene affecting lower limbs and digits which is associated with
arsenic [402]. The disease is endemic in south-east Asian countries where
arsenic contaminates food and water in many areas. Humic acid in drinking
water may be contributory in arsenic toxicity [403].
Although antimony is chemically similar to arsenic, its toxicological
profile is quite different. It is not recognized as a human carcinogen in the
skin or other organs although it may evoke chromosomal damage. Anti-
mony sulfide or stibnite (Sb2S3) was used in eye make-up in view of its
intense black color, and other antimony compounds provide vermillion,
yellow, and blue in industrial pigments. Antimonial drugs (notably Sb(V) –
sodium stibogluconate, meglumine antimoniate) have been used extensively
in tropical countries for control of cutaneous leishmaniasis but their toxicity
in the heart, liver, and kidney at blood levels of r10 mg Sb/m are prohibitory
[404,405]. In the 1990s, antimony became a subject of considerable concern
regarding its possible association with the sudden infant death syndrome.
Antimony trioxide used as a fire-retardant, preservative, and antifungal
agent to control risks of Scopulariopsis brevicaulis in PVC cot mattresses was
claimed to release stibine (SbH3) and other toxic gases from cot mattresses in
the presence of moisture and lead to primary SIDS [356]. Despite intense
debate the association was not proven, no substantive evidence produced to
show that stibine or other gases present in the neonatal environment were
toxic at the concentrations present. Identification of antimony in infants’
hair represented no more than evidence of environmental exposure but not
toxicity [355,406–408]. Unlike arsenic, inorganic trivalent antimony is not
methylated in vivo and is excreted in urine and bile following inhalation or
hair keratin following topical exposure [409,410]. SIDS would seem to be of
multifactorial origin and its association with antimony exposure or stibine
gas as causal factors are not substantiated [411].
Met. Ions Life Sci. 2011, 8, 187–246

222 LANSDOWN
Industrial exposure to antimony and related compounds in metal indus-

tries and in production of hard alloys, flame retardants, and glass is a cause
of skin irritancy, dermatitis, and the so-called ‘‘antimony spots’’ [412].
Antimony spots resemble pox-like inflammatory lesions around sweat and
sebaceous glands with eczema and intense irritation mainly restricted to the
neck wrists, thighs, trunk, lower legs, and scrotum. The condition pre-
dominates in summer months when sweat glands are more active. Nasal
perforations, septal lesions, and ulceration are also reported. Sb(III) com-
pounds are toxic when used topically and hypersensitivity reactions occur
[413]. Workers exposed to trivalent antimony are at greater risk of devel-
oping lung cancer [412–414]. Sb(III) compounds have been shown to cause
cytological damage and clastogenic changes in mice [415], but the carcino-
genicity of antimony is not recognized [261].
4. CARCINOGENICITY OF METAL IONS IN THE SKIN
The skin is not a primary site for metal-induced carcinogenesis following

topical exposure to most metals and metallic compounds on account of the
protective role afforded by epidermal keratins in binding free ions [4,15].
Inorganic arsenic, beryllium, cadmium, hexavalent chromium, nickel, cis-
platin, cobalt sulfate, lead, iron dextran, selenium sulfide, thorium, and
certain related compounds are classified as proven or possible human car-
cinogens on the basis of peer reviews of epidemiological and clinical infor-
mation supported by competent studies in experimental animals [416,417].
Questions remain concerning the carcinogenicity of cobalt-tungsten carbide
and hard metals, aluminum salts used in the cosmetic industry are claimed to
be a cause of breast cancers, but this is unresolved [326–328].
Mechanisms of chemical carcinogenesis in the skin commonly involve at
least two discrete steps
induction of mutagenic changes in target cells – identified by irreversible

cytoplasmic DNA damage and impaired transcription
promotion of genetically altered cells to frank neoplasia [418–420].
Carcinogens like inorganic arsenic are regarded as ‘‘complete’’ carcino-

gens on the basis that they evoke DNA damage and mutagenic change, and
promote synthesis of mitogenic growth factors like TGF-a and GMCSF
leading to solid tumor formation [399]. Both steps are genetically modulated
and cellular responses rely on expression of binding sites on cell membranes
of target cells [419,421,422]. Clinical evidence suggests that mutagenic
changes following exposure to chemical carcinogens like arsenic may persist
Met. Ions Life Sci. 2011, 8, 187–246

for up to 30 years pending promotion by physical or chemical means

[401,423,424].
Hexavalent chromium compounds are respiratory tract carcino-
gens in chromate pigment workers but rare causes of dermal tumors in
tanners, chrome platers, and workers exposed to chromate pigments.
Subcutaneous injection or implantation of chromates are a cause of
injection site cancers in rats [4,190,425]. Whereas trivalent chromium is an
essential trace metal in the human body with a probable role in glucose
metabolism [426,427], hexavalent chromium is mutagenic in several
strains of bacteria and human cell lines [428,429]. Similar patterns of
carcinogenicity are reported with nickel and nickel compounds used in
steel manufacture, alloys, and electroplating [430]. Experimental evidence
suggests that whilst exposures to nickel compounds present low risk and are
‘‘not good skin carcinogens’’ [147], they promote or enhance UV-induced
carcinomas in mice [431]. Experiments in mice have demonstrated also that
the skin is a target for chromium or nickel carcinogenicity following
administration in diet or drinking water, where solar irradiation serves a
promoter role [432]. Nickel subsulfide (Ni3S2) and nickel oxides (NiO and
Ni2O3) are classified as human carcinogens [416]. Dunnick et al. suggested
that more water-insoluble nickel compounds are phagocytosed and are
transferred intracytoplasmically to nuclear membranes where nickel ions
effect irreversible DNA damage [144]. Nickel compounds injected intra-
muscularly or subcutaneously induced sarcomas in mice, but non-solubilized
dust particles phagocytosed by reticuloendothelial cells were redistri-
buted to other soft tissue sites leading to production of secondary or
metastatic tumors [433]. X-ray diffraction studies of the insoluble
crystalline materials in tumors indicated a conversion from the a-Ni3S2 to
the a-Ni7S6 molecular form, although the significance of this is presently
unclear.
Inorganic arsenic compounds (sodium and potassium arsenates, arsenic
trioxide) are unequivocally human skin carcinogens with abundant clinical
evidence showing that skin cancers can arise through topical exposures,
inhalation, arsenical drugs, and chronic consumption of arsenites in food
and drink [202,434]. Exposures to arsenic in drinking water and occupa-
tional procedures have been associated with basal cell carcinomas of the
skin, rare incidences of malignant melanoma, mycosis fungoides, papillo-
mas, and rare Merkel cell tumors [435,436]. Langerhans cells are primarily
responsible for the immunological presentation of tumor-associated antigens
and play a role in the elimination of neoplastic clones [398]. Suppression of
Langerhans cells by UV irradiation increases risks of skin carcinogenesis.
The carcinogenicity of antimony in humans is unproven but it is noteworthy
that antimonial agents have been used in diagnosis of malignant melanomas
and associated lymph nodes [437].
Met. Ions Life Sci. 2011, 8, 187–246

224 LANSDOWN
Other metals including cobalt (as sulfate), cisplatin (Pt(NH3)2Cl2), iron

dextran complex (as ferric hydroxide with dextran), lead and cadmium
compounds, and selenium sulfide might be reasonably anticipated to be
human carcinogens on the basis of occupational surveys and experimental
studies but the human dermatological risk is not established [113,425]. Silica
dust is an occupational carcinogen through chronic inhalation in quarry
workers but epidemiological evidence indicates that prolonged exposure
presents a low risk of skin cancer [438]. Beryllium is one of the most toxic
elements in the periodic table and is identified as a Class A carcinogen fol-
lowing inhalation but it is not carcinogenic to the skin following occupational
exposures. It is a cause of subcutaneous granuloma and sarcoma following
subcutaneous injection in animals [4,416]. Aluminum and zirconium are not
carcinogens but recognized causes of granulomas following subcutaneous
injection [321,325,439]. Changes induced in macrophages and dermal fibro-
blasts progressed to cell death and release of lactic dehydrogenase [324].
Aluminum has a genotoxic profile and is capable of inducing DNA changes
and epigenic effects predisposing to cancer, but evidence that aluminum salts
may induce cancers as metalloestrogens is questionable [326,327,440–442].
5. THE EYE
5.1. Trace Metals in Ocular Development and Health

The eye is vulnerable to toxic change as a result of excesses and imbalances in
trace metals [443–445], and through direct insult and occupational exposures
to xenobiotic metals with deposition of inert precipitates in the cornea and
conjunctiva leading to visual impairment [229,446,447]. Additionally, metals
like lead, cadmium, and mercury impair the pigment epithelium and neuro-
sensory function of the retina through direct cytotoxic action or impairment
in critical trace metal ion gradients or electrolytes (K1 and Na1) [448]. The
eye, like the skin, is dependent upon calcium, copper, zinc, iron, magnesium,
and manganese during embryonic development and postnatal functional
maturation such that deficiencies, metal ion imbalances, and blockage of
essential receptor functions can lead to abnormality or pathological damage
including macular degeneration [445]. Most metals accumulate in tissues of
the eye following dietary administration or direct instillation, but metal-
binding proteins like calmodulin and ceruloplasmin perform central roles in
modulation of calcium, iron, and copper in the cornea and neuroretina [443].
Whilst the human eye is morphologically unique, sufficient physiological
similarities exist in its physiology with other species to permit valid research
in predictive toxicology [449,450]. Of the trace metals, cytosolic and
Met. Ions Life Sci. 2011, 8, 187–246

extracellular Ca21 play central roles in ocular development and in patholo-

gical conditions including macular degeneration and cataractogenesis.
Cytosolic and intracellular Ca21 channels with CaBP are instrumental in
early development of the eye and in vitro studies have demonstrated that
invagination of the optic vesicle and development of the optic cup are
dependent on free Ca21 (probably extracellular) and that deficiencies due to
Ca21 channel antagonists lead to abnormal development [443]. During
postnatal development, Ca21 channels modulate the transformation of
immature radial glial cells into Müller cells of the retina and regulation of
low voltage ionic currents involving Na1 and K1 [444]. Electrophysiological
research has shown that Ca21 channel-mediated currents in the early rabbit
retina develop in relation to K1 currents in the Müller cells. In the normal
eye, L-type Ca21 channels have been shown to regulate secretion of vascular
endothelial growth factor (VEGF) and upregulation of VEGF in retinal
pigment epithelial cells. Studies in patients with choroidal neovasculariza-
tion indicate that Ca21 channels may be contributory factors in determining
macular degeneration [451]. Other electrophysiological studies have
demonstrated that Ca21 channels are modulated by cholinergic mechanisms
and that intracellular calcium levels influence neuronal development in the
retina [452]. Acetylcholine (ACh) analogues led to increases in free cytosolic
Ca21 in many cell types in the retina and ganglionic cells at different stages
in eye development and cellular migration and differentiation were deter-
mined by Ca21 gradients. As in the skin, Mg21 interacts with Ca21 in
normal eye physiology and imbalances between the two ions may underlie
cataractogenesis. Nagai et al. demonstrated that imbalances in the ionic
intracellular environment with increased levels of Ca21 and Na1 coupled
with lower Mg21 and K1 predispose to cataract formation [453]. Using
isolated epithelial cells, ionic imbalances leading to decreases in ATP and
ATPase function were related to Mg21 deficiency and increased nitric oxide
synthase (iNOS) and nitric oxide release. Increased iNOS and lowered ATP
were considered to advance progression in lens opacification and cataracto-
genesis. Similar changes have been seen in lens calcium in response to X-ray
induced cataract where studies in the rabbit have shown marked calcium
accumulation in the lens cortex, with much of the calcium bound in cytosolic
and membrane sites; calcium accumulation increased according to pro-
gression of the cataract and levels of opacity although this may not reflect
inactivation of Ca-ATPase [454].
Xenobiotic ions including Cd21 and Pb21 are toxic to the eye, partly
through direct action on target cells but principally through interaction with
and impairment of Ca21 and other trace metal pathways. Thus, cadmium
accumulating in ocular tissues was shown to disturb critical balances in the
trace elements calcium, copper, and iron, presumably through ionic com-
petition and impairment of key metalloenzymes, but the effects were
Met. Ions Life Sci. 2011, 8, 187–246

226 LANSDOWN
influenced by dietary selenium [455]. Dietary cadmium led to o50%

reduction in ocular Ca21 irrespective of selenium levels and the sele-
noenzyme, glutathione peroxidase. Iron concentrations were increased by
30% in the presence of low selenium but significantly lower in rats fed
selenium supplemented diets. Ocular zinc levels were not significantly
affected by cadmium or selenium, even through Cd21 and Zn21 compete for
binding sites on MT-1 and -2. Cadmium is cytotoxic to the corneal epithe-
lium but its action is reflected by ionic flux through Ca21 channels [456].
Channel blockers have been shown to reduce the pathogenic effects of the
metal on focal damage and denuding of the apical endothelial membrane
whereas a calcium ionophore enhanced pathogenicity. Ca21 channel block-
age impairment of critical balances between Ca21, Mg21, K1, and Na1 are
possibly involved in lead-related ocular damage [457–459]. Lead-related
ocular damage in rats varied according to age and duration of exposure and
the interdependence of retinal cells on Ca21 and other trace metal ions.
Exposure of rods and bipolar cells of the retina to lead resulted in apoptotic
loss, concurrent depression in cGMP phosphodiesterase and mitochondrial
ATP, but a significant increase in Ca21 was noted [459]. Pb21 also led to
significant reduction in Na1/K1-ATPase in isolated retinal cells but this was
antagonized by Na1, potentiated by Mg21, and unaffected by either K1 or
Ca21, suggesting that ionic interaction in lead-induced retinal toxicity is
complex. Lead is a mitochondrial poison and in vitro findings suggest that it
competes with Mg21 binding sites and magnesium-dependent enzymes.
Manganese is essential in development and function of the photoreceptor
cells of the retina and serves specific functions in mitochondrial integrity
[460], but no evidence is seen to suggest that lead, mercury or cadmium
interact with Mn21 in evoking toxic change. Manganese as in Mn-super-
oxide in the mitochondrial matrix relates to protein and glycogen metabo-
lism such that deficiencies in metabolizable Mn21 lead to aberrations in
photoreceptor cell populations and integrity of the retinal pigment epithe-
lium. Low levels in the enzyme have been associated with neural cell loss and
proliferation of Müller-like cells.
Iron is an essential trace metal in the physiological functional develop-
ment of the mammalian eye and cases of anemia in infancy are reported in
which patients have shown loss of vision, angiodysplasia, and altered eye
movement [461]. Additionally, excessive use of drugs like desferroxamine to
reduce systemic iron overload situations as in transfusional hemochroma-
tosis or Eales’s disease, have led to rare cases of retinal damage, pigmentary
and electrophysiological changes leading to blurred vision [462,463].
Macular iron levels increasing with advancing age are causes of retinal
degeneration, macular degeneration, and accumulation of iron-laden foreign
bodies; a condition that has been reproduced in murine hereditary acerulo-
plasminema [461,464]. Retinal dysplasias have also been associated with
Met. Ions Life Sci. 2011, 8, 187–246

deficiencies in other iron carrier proteins including ferritin, transferrin,

and ferroportin leading to iron overload in the eye and other tissues.
In proliferative diabetic retinopathy and intraocular foreign body diseases,
oxidative damage and increased glycoxidation attributable to excess iron
was claimed to be a cause of vitreous liquefaction and retinal detachment
resulting in loss of vision. Cobalt is a minor trace element in the eye, but in
overload situations, this metal is a further cause of retinal damage, char-
acterized by edema and atrophy of nerve fibres and photoreceptor cells
[465,466]. Obliteration of choroidal vessels and toxic damage in the iris and
ciliary body, cataract and purulent endophthalmitis are also reported.
5.2. Xenobiotic Ions and Ocular Toxicity

The tissues of the eye show an unusual capacity to accumulate trace and
xenobiotic metal ions following occupational exposures and dietary intake.
Excessive levels of Cu21, Pb41, and Cd21 have been associated with catar-
actous changes, irritancy, and vitreo-retinal damage [62,467,468]. Cadmium,
lead, thallium, and mercury are principle causes of metal-induced ophthalmic
toxicities in occupational exposures [469–471]. They accumulate in most parts
of the eye, but the retina, pigment epithelium, and choroid are principle target
tissues for these systemic toxins. Lead and mercury are acknowledged neu-
rotoxins whereas cadmium, which accumulates in all tissues of the eye, more
specifically acts on epithelial tissues including the outer lens tissue and cornea
where irreversible changes are attributable to mitochondrial edema [472–474].
Photoreceptor cells, pigment epithelium, and ganglionic cells are variously
vulnerable to toxic metals which in part act through direct cytogenicity,
impaired cell migration, local inflammatory changes, and enzymic inacti-
vation [475–477]. Blood levels in affected patients have shown significant
increases in the xenobiotic ions which may conjugate with amino acids and
proteins to be taken up by target cells [478]. A survey of the experimental
literature provides fragmentary evidence indicating the action of the various
metals in impairing specific enzymes or acting on vulnerable tissues like the
pigment epithelium but in most cases, ocular damage is multifactorial and
observed morphological changes are a reflection of the concentrations of the
metals administered and the time and duration of exposure. Neonatal and
developing tissues are appreciably more vulnerable to toxic metals, whatever
their action. Whilst blindness and cataract are obvious signs of toxicity,
electroretinograms have proved useful in understanding subtle changes and
early manifestations of damage and in the influence of xenobiotic metals on
the cognitive dysfunction of the eye [479,480]. On the other hand, electro-
retinograms have proved useful in substantiating lack of retinal damage but
in patients with visual disturbances due to inert deposits of silver or gold
Met. Ions Life Sci. 2011, 8, 187–246

228 LANSDOWN
sulfides in the cornea or conjunctiva as in argyria and chrysiasis

[229,447,481]. Although gold and silver through occupational exposure,
jewellery, and eye cosmetics deposit in all extraneural tissues of the eye, they
are without toxic influence [4,40,202]. Thalllium is toxic to the eye through
its propensity to bind melanin of the pigment epithelium through its affinity
to free carboxyl ligands. Thallium cations injected into the vitreous humour
penetrated melanocyte cell membranes to interact with melanin granules
leading to degeneration of photoreceptors and other cells of the retina
leading to electrophysiological changes [482,483].
Aluminum was identified as a possible cause of neurological damage and
cause of Alzheimer’s disease although this has never been fully sub-
stantiated. However, experimental evidence has shown that Al31 will induce
damage in the retinal epithelium in the rat and rabbit with destruction of
photoreceptor cells of the inner and outer plexiform layers [483,484]. Fry
and his colleagues were of the opinion that aluminum-induced neurofi-
brillary degeneration in the retina had similarities to Alzheimer-related
changes [484]. Other work has substantiated that Al31 does locate in neu-
rological tissues and can inhibit the vestibule-ocular reflex without frank
astrocyte damage in the brain or amyloid deposits [485].
6. GENERAL CONCLUSIONS
Most metals in the periodic table are capable of influencing the development
or physiological function of the skin and eyes. In each case the changes seen
are specific for each metal and are dependent upon levels of ionization, the
bioactivity of the ions in binding to cellular constituents of the epidermal
barrier layer, percutaneous absorption, and the susceptibility of target cells.
In many situations toxic changes reflect interactions with essential trace
metals, impairment of essential trace metal ion gradients, and inhibition of
key metalloenzyme-modulated events. At present, we have an incomplete
and fragmentary knowledge of the action of many metals on the skin and eye
and much research is required using sensitive markers and diagnostic tools in
identifying early degenerative or functional changes.
In the skin, at least 10 metals are recognized nutrients, but most metals are
capable of inducing contact sensitization in predisposed persons. Langer-
hans cells are instrumental in induction of allergic responses and are targets
for metal toxins. Toxic changes reflect the nature of the bioactivity of metals
ions and their ability to overcome protective mechanisms afforded by MT,
CPN or metal binding proteins in the epidermis and serum. Age, genetical
status, and state of health are important factors influencing metal toxicity in
the skin and eye.
Met. Ions Life Sci. 2011, 8, 187–246

Environmental conditions including temperature, solar irradiation, and

humidity influence the condition of the skin, the functionality of the epi-
dermal glands and the state of hydration of the tissues and their respon-
siveness to metal-induced toxicity [1]. The skin serves a central role in
regulating metals of physiological value and in protecting against the toxic
action of xenobiotic metals with no nutrient value.
ABBREVIATIONS
ACh acetylcholine
ATP adenosine 5 0 -triphosphate
CaBP calcium-binding protein
cAMP adenosine cyclic 3 0 ,5 0 -monophosphate
cGMP guanosine cyclic 3 0 ,5 0 -monophosphate
CISP cisplatin
CoA L-methylmalonyl-coenzyme A
CPN ceruloplasmin
CSF cerebrospinal fluid
EDTA ethylenediamine-N,N,N 0 ,N 0 -tetraacetate
EDX energy-dispersive X-ray analysis
FGF fibroblast growt factor
GHK Gly-L-His-L-Lys (glycyl-L-histidyl-L-lysine)
GMCSF granulocyte macrophage colony stimulating factor
GPMT guinea pig maximization test
GTF glucose tolerance factor
IgE immunoglobulin E
iNOS inducible nitric oxide synthase
LPC lipoprotein complex
MDP hydroxyl-malonatodiamine platinum
MT metallothionein
NADH nicotinamide adenine dinucleotide (reduced)
NOAEL no adverse effect level
OSHA Office of Safety and Health Administration (US)
PCR polymerase chain reaction
PDGF platelet-derived growth factor
SIDS sudden infant death syndrome
SSD silver sulfadiazine
TGF tumor growth factor
TGF-a transforming growth factor a
TNF tumor necrosis factor
VEGF vascular endothelial growth factor
Met. Ions Life Sci. 2011, 8, 187–246

230 LANSDOWN
REFERENCES
1. A. M. Kligman, Curr. Probl. Dermatol., 1978, 7, 1–25.
2. W. S. Bullough, Cancer Res., 1965, 25, 1683–727.
3. P. D. Cruz, in The Biology of the Skin, Ed. R. K. Freinkel and D. T. Woodley,
Parthenon Press, New York, 2001, pp.255–263.
4. A. B. G. Lansdown, CRC Crit. Rev. Toxicol., 1995, 25, 397–462.
5. A. B. G. Lansdown, B. Sampson and A. Rowe, J. Anat., 1999, 195, 375–386.
6. A. B. G. Lansdown, Wound Rep. Regen., 2002, 10, 271–285.
7. A. B. G. Lansdown, U. Mirastschijski, N. Stubbs, E. Scanlon and M. S. Ågren,
Wound Rep. Regen., 2007, 15, 2–16.
8. A. B. G. Lansdown, Int. J. Cosmet. Sci., 2001, 23, 129–137.
9. A. B. G. Lansdown, Wound Rep. Regen., 2002, 10, 130–132.
10. B. Ballantyne, in General and Applied Toxicology, Ed. B. Ballantyne, T. C.
Marrs and T. Syversen, Wiley, Chichester, 2009, Vol. 3, pp. 1183–1249.
11. J. G. Rheinwald and H. Green, Nature, 1977, 265, 421–424.
12. A. N. Kingsnorth and J. Slavin, Br. J. Surg., 1991, 78, 12186–1290.
13. J. Slavin, J. Pathol., 1996, 178, 5–10.
14. T. S. Argyris and T. J. Slaga, CRC Crit. Rev. Toxicol., 1981, 9, 151–200.
15. J. J. Hostýnek, R. S. Hinz, C. R. Lorence, M. Price and R. H. Guy, CRC. Crit.
Rev. Toxicol., 1993, 23, 171–235.
16. E. J. Underwood, Trace Elements in Human and Animal Nutrition, 4th edn.,
Academic Press, New York, 1977.
17. A. B. G. Lansdown, B. Sampson and A. Rowe, 10th International Symposium on
Trace Metals in Man and Animals, Evian, France, 1999.
18. D. Witkowska, L. Sedrowicz, R. Oledzka and A. Bialek, Biometals, 2005, 2,
36–39.
19. I. Bremner and J. H. Beattie, Ann. Rev. Nutr., 1990, 10, 63–83.
20. I. Bremner, Prog. Food Nutr. Sci., 1987, 11, 1–37.
21. A. Ansty, R. Marks, C. Long, H. Navabi, A. Pearse, D. Wynford-Thomas and
B. Jasani, J. Pathol., 1996, 178, 84–88.
22. R. J. Cousins and A. S. Leinart, Fed. Am. Soc. Exp. Biol., 1988, 2, 2884–2890.
23. A. Molinero, J. Carrasco, J. Hernandez and J. Hidalgo, Neurochem. Int., 1998,
33, 559–566.
24. A. T. Miles, G. M. Hawksworth, J. H. Beattie and V. Rodilla, Crit. Rev.
Biochem. Mol. Biol., 2000, 35, 35–70.
25. C. D. Klaassen, Toxicology, 1981, 20, 275–279.
26. M. M. Frings, P. P. Kind, G. Goerz and J. Abel, Clin. Exp. Dermatol., 1989, 14,
434–436.
27. M. Karasawa, N. Nishimura, H. Hishimura, C. Tohyama, H. Hashiba and
T. Kuroki, J. Invest. Dermatol., 1991, 97, 97–100.
28. J. J. Van den Oord and M. De Ley, Arch. Dermat. Res., 1994, 286, 62–68.
29. K. Rossen, T. Haerslev, K. Hou-Jensen and G. K. Jacobsen, Br. J. Dermatol
1997, 136, 30–34.
30. B. Zhang, M. Stoh, N. Nishimura, J. S. Suzuki, H. Sone, Y. Aoki and
C. Tohyama, Cancer Res., 1998, 58, 4044–4046.
Met. Ions Life Sci. 2011, 8, 187–246

31. M. Iwata, T. Takebayashi, H. Ohta, R. E. Acalde, Y. Itano and T. Matsumura,

Histochem. Cell Biol., 1999, 112, 283–290.
32. K. Hanada, D. Sawamura, I. Hasimoto, K. Kida and A. Aguma, J. Invest.
Dermatol., 1998, 110, 259–262.
33. A. B. G. Lansdown and B. Sampson, Lab. Anim. Sci., 1996, 46, 549–554.
34. A. B. G. Lansdown, B. Sampson and A. Rowe, Int. J. Exp. Pathol., 2001, 82,
35–41.
35. A. B. G. Lansdown, B. Sampson, P. Laupattarakasem and A. Vuttiviojana, Br.
J. Dermatol., 1997, 137, 728–735.
36. A. B. G. Lansdown, J. Wound Care, 2002, 11, 125–131.
37. A. B. G. Lansdown, Curr. Probl. Dermatol., 2006, 33, 17–34.
38. A. B. G. Lansdown and A. Williams, J. Wound Care, 2004, 13, 131–136.
39. S. S. Bleehan, D. J. Gould, C. I. Harrington, T. E. Durrent, D. N. Slater and
J. C. E. Underwood, Br. J. Dermatol., 1981, 104, 19–25.
40. A. B. G. Lansdown, ‘‘Pharmacological and Toxicological Profile of Silver as an
Antimicrobial Agent in Medical Devices’’ in Advances in Pharmacological
Sciences, Hindawi, New York, 2010, in press.
41. G. K. Menon, L. F. Price, B. Bannerman, P. M. Elias and K. R. Feingold,
J. Invest. Dermatol., 1994, 102, 789–795.
42. G. K. Menon, P. M. Elias and K. R. Feingold, Br. J. Dermatol., 1992, 130,
139–147.
43. M. K. Heng, M. K. Song and M. C. Y. Heng, Br. J. Dermatol., 1993, 129,
280–285.
44. D. La Fitte, T. O. Tsvetkov, F. Davred, R. Toci, F. Barras, C. Briand, A. A.
Makarov and J. Haiech, Biochim. Biophys. Acta, Proteins and Proteonomics,
2002, 1600, 105–110.
45. S. R. Martin, L. Masino and P. M. Bayley, Protein Sci., 2000, 9, 2477–2488.
46. M. -C. Kilhoffer, D. M. Roberts, A. O. Adibi, D. M. Watteron and J. Haiech,
J. Biol. Chem., 1988, 263, 17023–17029.
47. M.-C. Kilhoffer, J. G. Demaille and D. Gerrard, FEBS Lett., 1980, 116,
269–272.
48. T. J. Haley, N. Komesu, N. Flesher, L. Mavis, J. Cawthorne and H. C. Upton,
Energy Citations Database, 1962, Rep. TID-16385, pp. 21.
49. N. E. Hellman and J. D. Gitlin, Ann. Rev. Nutrit., 2002, 22, 439–458.
50. J. J. Hostýnek, Exogenous Dermatol., 2004, 3, 263–269.
51. M. A. Dunn, M. H. Green and R. M. Leach, Am. J. Physiol., 1991, 261, E115–1125.
52. E. D. Harris, Nutrit. Rev., 2001, 59, 281–285.
53. T. Horn, T. Tønnessen and Z. Tümer, Brain Pathol., 1992, 2, 351–362.
54. J. Camakaris, I. Voskoboinik and J. F. Mercer, Biochem. Biophys. Res. Comm.,
1999, 261, 225–232.
55. H. M. Kagan and W. Li, J. Cell. Biochem., 2003, 88, 660–672.
56. Y. Ito, K. Fukuyama, N. Horie and W. L. Epstein, Mol. Cell. Biochem, 1984,
60, 183–188.
57. L. S. Hurley and L. T. Bell, Proc. Soc. Exp. Med., 1975, 149, 830–834.
58. I. Bremner and J. H. Beattie, Proc. Nutr. Soc., 1995, 54, 489–499.
59. R. F. Pfeffer, Semin. Neurol., 2007, 27, 123–132.
Met. Ions Life Sci. 2011, 8, 187–246

232 LANSDOWN
60. A. Ala, A. P. Walker, J. S. Dooley and M. L. Schilsky, Lancet, 2007, 369, 397–408.
61. R. W. Charlton and T. H. Bothwell, Ann. Rev. Med., 1983, 34, 55–68.
62. H. G. Van Eijk and G. De Jong, Biol. Trace Elem. Res., 1992, 35, 13–24.
63. H. E. Stockinger, K. I. Altman and K. Solomon, Biochim. Biophys. Acta, 1953,
12, 439–444.
64. D. J. Price and J. G. Joshi, J. Biol. Chem., 1983, 258, 10873–10880.
65. E. N. Baker and P. F. Lindley, J. Inorg. Biochem., 1992, 47, 147–160.
66. Z. L. Harris, Y. Takahashi, H. Miyajima, M. Serizawa, R. T. A. MacGillivray
and J. Gitlin, Proc. Nat. Acad. Sci., 1995, 92, 2539–2543.
67. A. Takaesu, K. Watnabe, S. Taki, Y. Sasaki and K. Orino, Acta Veterin. Scand.,
2008, 50, 42–47.
68. C. T. Settlemire and G. Mattrone, J. Nutr., 1967, 92, 153–158.
69. J. Fleming and J. G. Joshi, Proc. Nat. Acad. Sci., 1987, 84, 7866–7870.
70. L. Hallberg, M. Brune and L. Rossander, Int. J. Vitamin Nutr. Res. Suppl.,
1989, 30, 103–108.
71. R. F. Diegelmann and M. C. Evans, Frontiers in BioScience, 2004, 9, 283–289.
72. G. S. Schultz, D. J. Barillo, D. W. Mozingo and G. A. Chin, Int. Wound J.,
2004, 1, 19–32.
73. S. Enoch and K. G. Harding, Wounds, 2003, 15, 213–229.
74. A. B. G. Lansdown, Br. J. Nurs., 2004, 13 (Tissue Viability Suppl. 20),
1199–210.
75. A. B. G. Lansdown, Nutrition and the Healing of Skin Wounds, Wound Care
Society, Educational Booklet, 2001, 8, 1–16.
76. T. K. Hunt and Z. Hussain, in Wound Healing, Biochemical and Clinical
Aspects, Ed. I. K. Cohen, R. F. Diegelmann and W. J. Lindbladt, Saunders,
Philadelphia, 1992, pp. 274–281.
77. I. Bremner and J. H. Beattie, Trace Elements in Man and Animals 10, Spring-
erLink, New York, 2007, Part 4, 961–967.
78. R. L. Ruberg, Surg. Clin. North Am., 1984, 64, 705–14.
79. P. Massini, in Platelets and Thrombosis, Ed. D. C. B. Mills and F. I. Pareti,
Academic Press, Inc., New York, 1977, pp. 33–43.
80. J. E. Rasmussen, Arch. Dermatol., 1988, 114, 443–444.
81. C. J. McClain, J. Am. Coll. Nutr., 1985, 4, 49–64.
82. D. A. Knighton, I. A. Silver and T. K. Hunt, Surgery, 1981, 90, 262–270.
83. M. B. Strauss, Orthopedics, 2002, 25, 303–31.
84. L. Pickart, J. Biomater. Sci. Polym. Ed., 2008, 19, 968–988.
85. Y. A. Kang, H. R. Choi, J. L. Na, M. J. Kim, S. W. Youn, K. H. Kim and K. C.
Park, Arch. Dermatol. Res., 2009, 301, 301–306.
86. E. M. Widdowson, Proc. Nutr. Soc., 1974, 33, 275–291.
87. D. P. Chakraborty, C. Chakraborty, M. Ganguly and A. K. Chakraborty,
Experientia, 1983, 39, 282–283.
88. H. A. Schroeder, J. J. Balassa and I. H. Tipton, J. Chron. Dis., 1964, 17, 483–502.
89. A. Kappas and G. S. Drummond, Envir. Health Perspect., 1984, 57, 301–306.
90. National Research Council, Nutrient Requirements of Laboratory Animals, 4th
edn., National Academy Press, Washington DC, 1995.
91. D. Massey, Nurs. Crit. Care, 2006, 11, 80–85.
Met. Ions Life Sci. 2011, 8, 187–246

92. A. B. G. Lansdown, Scand. J. Lab. Anim. Sci., 1996, 23, 201–209.

93. F. H. Nielsen, J. Nutr., 1996, 126(suppl. 9), 2377S–2385S.
94. Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine,
National Academy Press, Washington, DC, 1998, Section 9, p. 1.
95. C. S. Alexander, Am. J. Med., 1972, 53, 395–416.
96. J. Z. Hearon and D. Burk, J. Nat. Canc, Inst., 1948–1949, 9, 337.
97. G. Schnyder, M. Roffi, Y. Flammer, R. Pin and O. M. Hess, J. Am. Med. Ass.,
2003, 288, 973–979.
98. M. I. Behrens, V. Diaz, C. Vásquez and A. Donoso, Rev. Med. Chil., 2003, 131,
915–919.
99. I. Chanarin, J. Perry and M. Lumb, Lancet, 1974, 1, 1251–1252.
100. E. J. Fine and E. D. Soria, South. Med. J., 1991, 84, 1475–1481.
101. E. P. Frenkel, A. Mukherjee, C. R. Hackenbrock and P. A. Srere, J. Biol.
Chem., 1976, 251, 2147–2154.
102. R. Kannan and M. J. M. Ng, Can. Fam. Physician, 2008, 54, 529–532.
103. S. S. Bleehan, F. J. G. Ebling and R. H. Champion, in Textbook of Derma-
tology, Ed. R. H. Champion, J. L. Burton and F. J. G. Ebling, Blackwell,
London, 1993, Vol. 3, 1561–1662.
104. E. Skog and J. E. Wahlberg, J. Invest. Dermatol., 1964, 43, 187–192.
105. O. Norgaard, Acta Derm. Venerol., 1957, 37, 440–445.
106. T. Menné and E. Nieboer, Endeavour, 1989, 13, 117–122.
107. A. A. Fisher, Contact Dermatitis, 3rd edn., Lea and Febiger, Philadelphia, 1987,
pp. 710–744.
108. A. A. Fisher and I. Rystedt, Contact Dermatitis, 1983, 9, 115–121.
109. I. Juhlin and W. B. Shelley, Acta Derm. Venereol., 1977, 57, 289–96.
110. J. R. Manciet, A. Barrade, F. Janssen and P. Morel, Contact Derm., 1995, 33,
282–284.
111. National Institute of Occupational Safety and Health, DHHS Publication, 1981,
82–107, 1–95.
112. M. Demedts and J. L. Ceippens, Chest, 1989, 95, 2–3.
113. M. De Boeck, M. Kirsch-Volders and D. Lison, Mutat. Res., 2003, 533,
135–152.
114. J. M. Mur, J. J. Moulin, M. P. Charruyer-Seinerra and J. Lafitte, Am. J. Ind.
Med., 1987, 11, 75–81.
115. H. C. Grice, L. C. Munro, G. S. Wiberg and A. G. Heggtveit, Clin. Toxicol.,
1969, 2, 273–287.
116. R. A. Anderson, J. Coll. Nutr., 1998, 17, 485–555.
117. R. A. Anderson, M. M. Polanski, N. A. Bryden, S. J. Bhathena and J. Canary,
Metabolism, 1987, 36, 351–355.
118. M. F. McCarty and E. J. Rubin, Med. Hypotheses, 1984, 13, 139–151.
119. V. Bianchi, R. Dal Toso, P. Debetto, A. G. Levis, S. Luciani, F. Majone and
G. Tamino, Toxicology, 1980, 17, 219–224.
120. T. Wolf, R. Kasemann and H. Ottenwälder, Arch. Toxicol., 1989, 13 (Suppl),
48–51.
121. Toxicology of Metals; Biochemical Aspects, Ed. R. A.Goyer and M. G. Cherian,
Springer, Berlin, 1995.
Met. Ions Life Sci. 2011, 8, 187–246

234 LANSDOWN
122. S. A. Katz and H. Salem, J. Appl. Toxicol., 1993, 13, 217–224.

123. R. E. Bagdon and R. E. Hazen, Environ. Health Perspect., 1991, 92, 111–119.
124. S. Horiguchi, K. Morinaga and G. Endo, Asia Pacif. J. Publ. Health, 1990, 4,
169–174.
125. D. A. Basketter, G. Bratico-Vangosa, W. Kaestner, C. Lally and W. J. Botinck,
Contact Dermatitis, 28, 15–25.
126. J. Nethercott, D. Paustenbach, R. Adams, J. Fowler, J. Marks, C. Morton,
J. Taylor, S. Horowitz and B. Finley, Occup. Environ. Med., 1994, 51, 371–380.
127. D. Burrows, Br. J. Dermatol., 1978, 99, 587–595.
128. Paintmakers Association of Great Britain, Report, 1988, January, pp. 1–26.
129. S. Fregert, B. Gruyberger and E. Sandahl, Contact Dermatitis, 1979, 5, 39–42.
130. F. H. Nielson, in Trace Element Metabolism in Animals, Ed. W. G. Hoekstra,
J. W. Suttie, H. E. Ganther and W. Mertz, University Park Press, Baltimore,
1974, p. 281.
131. W. Mertz, Proc. Nutr. Soc., 1974, 33, 307–313.
132. G. Prota, J. Invest. Dermatol., 1980, 75, 122–127.
133. G. Prota, Med. Res. Rev., 1988, 8, 525–556.
134. M. Anke, B. Groppel, H. Kronemann and M. Grün, IARC Sci. Publ., 1984, 53,
339–365.
135. T. G. Kazi, H. I. Alfridi, N. Kazi, M. B. Arain, N. Jalbani and G. A. Kandhro,
Biol. Trace Elem., 2008, 122, 1–18.
136. V. Bencko, in Hair in Toxicology: An Important Biomonitor, Ed. D. Tobin,
Royal Society of Chemistry, Cambridge, 2005, pp. 159–175.
137. S. N. Kales and D. C. Christiani, in Hair in Toxicology: An Important Biomo-
nitor, Ed. D. Tobin, Royal Society of Chemistry, Cambridge, 2005, pp. 125–158.
138. A. Fullerton and A. Hoelgaard, Br. J. Dermatol., 1988, 119, 675–682.
139. P. J. Kostyniak and T. W. Clarkson, Toxicol. Appl. Pharmacol., 1981, 1, 376–380.
140. D. Spruit, J. W. H. Mali and N. D. Groot, J. Invest. Dermatol., 1965, 44,
103–106.
141. L. A. Van Loon, P. W. Elsas, J. D. Bos, H. C. ten Harkel-Hagenaar and
S. R. Krieg, J. Oral Pathol., 1988, 17, 129–13.
142. F. W. Sunderman and C. B. Fraser, Ann. Clin. Lab. Sci., 1983, 13, 489–495.
143. H. Savolainen, Rev. Environ. Health, 1996, 11, 167–173.
144. J. K. Dunnick, M. R. Elwell, A. E. Radovsky, J. M. Benson, F. F. Hahn,
K. J. Nikula, E. B. Barr and C. H. Hobbs, Cancer Res., 1995, 55, 5251–5226.
145. A. Furst, IARC Sci. Publ., 1984, 53, 245–252.
146. M. Hindsen, M. Bruze and O. B. Christensen, Int. Arch. Allergy Immunol., 1997,
112, 212–217.
147. G. Kazantzis, Ann. Occup. Hyg., 1972, 15, 25–29.
148. A. N. Uddin, F. J. Burns, T. G. Rossman, H. Chen, T. Kluz and M. Costa,
149. V. Senft, F. Losen and M. Tucek, Mutat. Res., 1992, 279, 171–179.
150. T. Menne, Contact Dermatitis, 1980, 6, 337–40.
151. T. Suneja, K. H. Flanagan and D. A. Glaser, Dermatitis, 2007, 18, 208–211.
152. H. I. Kim, D. H. Kim, M. S. Yoon, H. J. Kim and S. Lee, Yonsei Med. J., 1991,
32, 79–81.
Met. Ions Life Sci. 2011, 8, 187–246

153. P. J. Aggett, Proc. Nutr. Soc., 1980, 39, 241–248.

154. R. M. Leach and A.-M. Muenster, J. Nutr., 1962, 78, 51–56.
155. R. M. Leach, A.-M. Muenster and E. M. Wien, Arch. Biochem. Biophys., 1969,
133, 22–28.
156. D. L. Baly, J. S. Schneiderman and A. L. Garcia-Walsh, J. Nutr., 1990, 120,
1075–1079.
157. S. Chang, P. M. Brannon and M. Korc, J. Nutr., 1990, 120, 1228–1234.
158. M. Weitzhandler and M. R. Bernfield, in Wound Healing, Biochemical and
Clinical Aspects, Ed. I. K. Cohen, R. F. Diegelmann and W. J. Lindblad,
Saunders, Philadelphia, 1992, pp. 195–208.
159. L. S. Hurley and C. L. Keen, in Trace Elements in Human and Animal Nutrition,
Ed. W. Mertz, Academic Press, Orlando, FL, 1987, pp. 185–223.
160. C. D. Davis, D. M. Ney and J. L. Greger, J. Nutr., 1990, 120, 507–513.
161. C. F. Mills and G. K. Davis, in Trace Elements in Human and Animal Nutrition,
Ed. W. Mertz, Academic Press, San Diego, 1989, 1, 429–463.
162. K. V. Rajagopalan, Ann. Rev. Nutr., 1988, 8, 401–427.
163. N. N. Abumrad, Bull. N.Y. Acad. Med., 1984, 60, 163–171.
164. J. J. Johnson, W. R. Waud, K. V. Rajagopalan, M. Duran, F. A. Beemer and
S. K. Wadman, Proc. Natl. Acad. Sci., 1980, 77, 3715–3719.
165. K. M. Hambidge, C. E. Casey and N. F. Krebs, in Trace Elements in Human and
Animal Nutrition, Ed. W. Mertz, Academic Press, Orlando, FL, 1986, pp. 1–137.
166. K. Yokoi, M. Kimura and Y. Itokawa, Biol. Trace Elem. Res., 1990, 24,
223–231.
167. V. M. Sardesai, in Introduction to Clinical Nutrition, CRC Press, Boca Raton,
FL, 2003, p. 131.
168. K. A. Winship, Adv. Drug React. Acute Poisoning Rev., 1988, 7, 19–38.
169. J. Meyervitch, P. Rothenberg, Y. Shechter and S. Bonner-Weir, J. Clin. Invest.,
1991, 87, 1286–1294.
170. G. I. Fantus, S. Kadota, B. Deragon, B. Foster and B. I. Posner, Biochemistry,
1989, 28, 8864–8871.
171. R. N. Lindquist, J. L. Lynn and G. E. Lienhard, J. Am. Chem. Soc., 1976, 95,
8762–8768.
172. G. Swarup, S. Cohen and D. L. Gebers, Biochem. Biophys. Res. Commun., 1982,
107, 1104–1109.
173. G. L. Nieder, C. N. Corder and P. A. Culp, Arch. Pharmacol., 1979, 307,
191–198.
174. G. Grupp, I. Grupp, C. L. Johnson, E. T. Wallick and A. Schwartz, Biochem.
Biophys. Res. Somm., 1979, 88, 440–447.
175. M. Rau, M. S. Patole, S. Vijaya, C. K. R. Kurup and T. Ramasarma, Mol. Cell.
Biochem., 1987, 75, 151–159.
176. WHO, Environmental Health Criteria, Geneva, 1988, No 81.
177. D. L. G. Thomas and K. Stiebris, Med. J. Austr., 1956, 1, 607–609.
178. L. S. Meriwether, U.S. Patent Application, 1984, No. 06/259746.
179. Department of Health, Dietary Reference Values for Food Energy and Nutrients
in the United Kingdom, Her Majesty’s Stationary Office, London, 1991, No 4.
180. E. M. Carlisle, Nutr. Rev., 1982, 40, 193–198.
Met. Ions Life Sci. 2011, 8, 187–246

236 LANSDOWN
181. E. M. Carlisle, Sci. Total Environ., 1988, 73, 95–106.

182. K. A. Schwartz, Proc. Nat. Acad. Csi., 1973, 70, 1608–1612.
183. C. D. Seaborn and F. H. Nielson, Biol. Trace Element Res., 2002, 89, 251–261.
184. C. A. Smith and E. J. Wood, Biological Molecules, Springer, New York, 2002,
pp. 72–73.
185. M. Branagan, D. H. Chenery and S. Nicholson, Skin Pharmacol. Skin Physiol.,
2000, 13, 157–164.
186. A. Lassus, J. Int. Med. Res., 1993, 21, 209–215.
187. A. B. G. Lansdown, J. Wound Care, 2007, 16, 404–407.
188. M. A. Kuhn, M. R. Moffit, P. D. Smith, W. G. Lyle, F. Ko, D. D. Meltzer and
M. C. Robson, Int, J. Surg. Invest., 2001, 2, 467–474.
189. M. M. Hanasono, J. Lum, L. A. Carroll, A. A. Mikulec and R. J. Koch, Arch.
Fasc. Plast. Surg., 2004, 6, 88–93.
190. International Agency for Research on Cancer (IARC), Silica and Some Sili-
cates, IARC Monographs on the Chemical Risk of some Chemical to Humans,
Lyon, France, 1987, No 42, pp. 289.
191. L. Pimental, M. Barnadas, D. Vidal, F. Sancho, R. Fontarnau and A. Alomar,
Dermatol., 2002, 205, 162–165.
192. J. K. McLaughlin, L. Lipworth, D. K. Murphy and P. S. Walker, Ann. Plast.
Surg., 2007, 59, 569–580.
193. G. S. Brody, Breast Implants, Safety and Efficacy of Silicone, Medscape CDC
Commentary Series, Springer, New York, 2009, pp. 1–12.
194. A. B. G. Lansdown, CRC Crit. Rev. Toxicol., 2007, 37, 237–250.
195. A. B. G. Lansdown, Silver in Wound Care and Management, Wound Care
Society, Educational Booklet, 2003, Vol. 1, 3–17.
196. A. B. G. Lansdown and A. Williams, J. Wound Care, 2004, 13, 131–136.
197. T. Sollemann, Manual of Pharmacology, Saunders, Philadelphia, 1942, pp.
1102–1109.
198. A. B. G. Lansdown, B. Sampson and A. Rowe, Wound Rep. Regen., 1999, 7,
A306.
199. A. B. G. Lansdown, J. Wound Care., 2002, 11, 173–177.
200. A. B. G. Lansdown, A. Williams, S. Chandler and S. Benfield, J. Wound Care,
2005, 14, 131–137.
201. A. Hambidge, Health Estate, 2001, 55, 23–25.
202. S. I. Rappoport, The Blood Brain Barrier in Physiology and Medicine, Raven
Press, New York, 1976.
203. W. Zheng, Clin. Toxicol., 2001, 39, 711–719.
204. W. Zheng, M. Aschner and J.-F. Ghersi-Egea, Toxicol. Appl. Pharmacol., 2003,
192, 1–11.
205. A. T. Wan, R. Conyers, C. J. Coombs and J. Masterton, Clin. Chem., 1991, 37,
1683–1687.
206. G. D. Di Vincenzo, C. J. Giordano and L. S. Schriever, Int. Arch. Occ. Envir.
Health, 1985, 56, 207–215.
207. J. F. Fowler, J. H. Perryman and B. Quinlan, Dermatitis, 2008, 19, 146–147.
208. Food and Drug Administration (FDA), Fed. Register, 1996, 61, No. 200,
pp. 10.
Met. Ions Life Sci. 2011, 8, 187–246

209. B. Kranke and W. Aberer, Lancet, 2004, 363, 532.

210. S. Barrett, New Engl. J. Med., 2004, 352, 2349–2350.
211. M. C. Fung, M. Weintraub and D. L. Bowen, J. Am. Med. Ass., 1995, 274,
1196–1197.
212. L. E. Gaul and A. H. Staud, J. Am. Med. Ass., 1935, 104, 1387–1390.
213. Argyria, the Pharmacology of Silver, 1st. edn., Ed. W. R. Hill and D. M.
Spillsbury, Williams and Wilkins, Baltimore, 1939..
214. B. W. East, K. Boddy, E. D. Williams and D. MacIntyte, Clin. Exp. Dermatol.,
1980, 5, 305–311.
215. A. Wadhera and M. Fung, Dermatol. Online J., 2005, 11, 12–22.
216. W. R. Buckley and C. J. Terhaar, Trans. St. John’s Hosp. Dermatol. Soc., 1973,
59, 39–44.
217. T. Matsumura, M. Kumakiri, A. Ohkawara, H. Numata and R. Adachi,
J. Dermatol., 1992, 19, 87–93.
218. J. Aaseth, A. Olsen and J. Halse, Scand. J. Clin. Lab. Invest., 1981, 41, 247–251.
219. S. S. Bleehan, D. J. Gould, C. I. Harrington, T. E. Durrent, D. N. Slater and
J. C. E. Underwood, Br. J. Dermatol., 1981, 104, 19–26.
220. H. Suzuki, S. Baba, S. Uchigasaki and M. Murase, J. Am. Acad. Dermatol.,
1993, 29, 833–837.
221. M. R. Okun, L. Edelstein, L. G. Niebauer and G. Hamada, J. Invest. Dermatol.,
1969, 53, 39–45.
222. E. L. Kanabrocki, WHO/IAEA Joint Research Programme on Trace Elements in
Cardiovascular Diseases, IAEA Tech. Rep., 1973, No 157, p. 57.
223. Y. Tanita, T. Kato, K. Hanada and H. Tagami, Arch. Dermatol., 1985, 121,
1550–1552.
224. S. Sato, H. Sueki and A. Nishimura, Br. J. Dermatol., 1999, 140, 158–163.
225. E. Cheriathundam and A. P. Alvares, J. Biochem. Toxicol., 1995, 11, 175–181.
226. W. S. Rapson, Contact Dermatitis, 1985, 13, 56–65.
227. D. T. Felson, J. J. Anderson and R. F. Meenan, Arthritis Rheum., 1990, 33,
1449–1461.
228. D. M. Sagranski and R. A. Greenwald, J. Rheum., 1980, 7, 474–478.
229. D. W. Tierney, J. Am. Optom. Ass., 1988, 59, 960–962.
230. J. M. Pelachyk, W. F. Bergfeld and J. T. Mahon, J. Cutan. Pathol., 1984, 11,
491–494.
231. B. J. Payne and L. Z. Saunders, Vet. Pathol., 1978, 15(suppl. 5), 51–87.
232. M. S. Baptiste, R. Rothenberg, P. C. Nasca, D. T. Janerich, C. D. Stutzman,
K. Rimawi, W. O’Brien and J. Matiszek, J. Am. Acad. Dermatol., 1984, 10,
1019–1023.
233. Y. Kobayashi, H. Nanko, J. Nakamura and M. Mizoguchi, J. Am. Acad.
Dermatol., 1992, 27, 457–458.
234. B. J. Payne, Vet. Pathol., 1978, 15(suppl. 5), 39–50.
235. R. W. Smith, B. Leppard, N. L. Barnett, G. H. Millward-Sadler, F. McCrae and
M. I. D. Cawley, Br. J. Dermatol., 1995, 133, 671–678.
236. P. A. Leonard, F. Moatamed, J. R. Ward, M. W. Piepkorn, E. J. Adams and W.
P. Knibbe, J. Rheum., 1986, 13, 58–64.
237. G. Danscher, Histochem. Cell. Biol., 2002, 117, 447–452.
Met. Ions Life Sci. 2011, 8, 187–246

238 LANSDOWN
238. N. L. Gottlieb, P. M. Smith, N. S. Penneys and E. M. Smith, Arthritis Rheum.,

1974, 17, 56–62.
239. D. Jeffrey, D. F. Biggs, J. S. Percy and A. S. Russell, J. Rheum., 1975, 2, 28–35.
240. C. E. Keen, K. Brady, N. Kirkham and D. A. Levison, Histopathology, 1993,
23, 355–360.
241. N. S. Penneys, K. Kramer and N. L. Gottlieb, J. Invest. Dermatol., 1975, 65,
331–333.
242. M. A. B. Roest and R. Ratnavel, Br. J. Dermatol., 2001, 145, 855–856.
243. L. Jonas, H. Nizze, R. Zimmermann, G. Gross, F. Zack, G. Kröning,
G. Holzhüter and H. J. Haas, Ultrastr. Pathol., 2002, 26, 153–159.
244. A. E. Roberts, Am. Med. Ass. Arch. Ind. Hyg. Occup. Med., 1951, 4, 549–559.
245. J. L. Parrot, Arch. Envir. Health, 1969, 19, 685–691.
246. G. M. Levene, Br. J. Dermatol., 1971, 85, 590–593.
247. A. V. Roshchin, V. G. Veselov and A. I. Panova, J. Hyg. Epidemiol. Immunol.,
1984, 28, 17–24.
248. S. O. Freedman and J. Krupcy, J. Allergy, 1968, 42, 233–237.
249. K. I. Campbell, E. L. George, L. L. Hall and J. F. Stara, Arch, Environ. Health,
1975, 30, 168–170.
250. D. B. Baker, P. H. Gann, S. M. Brooks, J. Gallagher and I. L. Bernstein, Am. J.
Ind. Med., 1990, 18, 653–664.
251. R. D. Murdoch and J. Pepys, Clin. Exp. Dermatol., 1984, 58, 478.
252. J. T. Hartmann and H. P. Lipp, Expert Opin. Pharmacother., 2003, 4, 889–901.
253. M. F. Pera, D. Sessford and J. J. Roberts, Biochem. Pharmacol., 1982, 31,
2273–2278.
254. M. D. Waters, T. O. Vaughan, D. J. Abernethy, H. R. Garland, C. C. Cox and
D. L. Coffin, Environ. Health Perspect., 1975, 12, 45–56.
255. D. Wierda and T. L. Pazdernik, J. Pharmacol. Exp. Therap., 1979, 211, 531–538.
256. A. B. G. Lansdown, Int. J. Cosmet. Sci., 2000, 22, 1667–168.
257. F. N. Marzulli, P. M. Watlington and H. I. Maibach, Curr. Probl. Dermatol.,
1978, 7, 196–204.
258. B. D. G. Morgan, Br. Med. J., 1974, 3, 34–36.
259. M. R. Misra, G. T. Smith and M. P. Waalkes, Toxicology, 1998, 126, 103–114.
260. K. Ohba, Y. Okawa, Y. Matsumoto, Y. Nakamura and H. Ohta, J. Toxicol.
Sci., 2007, 32, 107–109.
261. Dept. of Health and Social Secrity, 7th Annual Report on Carcinogens, National
Institute of Environmental Health Sciences, Research Triangle Park, NC, 1994.
262. C. C. Allen, K. A. Lund and P. Treadwell, Int. J. Dermatol., 1992, 31, 353–354.
263. D. T. Netscher, J. A. Friedland and R. M. Guzewicz, Ann. Plast. Surg., 1991,
26, 592–596.
264. M. P. Bagley, R. A. Schwartz and W. C. Lambert, Arch. Dermatol., 1987, 123,
1560–1561.
265. M. P. Waalkes, S. Rehm, B. Sass, N. Konishi and J. M. Ward, Environ. Res.,
1991, 55, 40–50.
266. P. Sau, G. Solivan and F. B. Johnson, J. Am. Acad. Dermatol., 1991, 25,
915–919.
267. F. Waffarn and J. E. Hodgman, Pediatrics, 1979, 6, 640–642.
Met. Ions Life Sci. 2011, 8, 187–246

268. M. Schumacher, J. L. Domingo, J. M. Llobet and J. Corbella, Sci. Total

Environ., 1991, 104, 167–173.
269. M. Wilhelm, D. Hafner, I. Lombeck and F. K. Ohnesorge, Sci. Total Environ.,
1991, 103, 199–207.
270. M. Wilhelm, I. Lombeck and F. K. Ohnesorge, Sci. Total Envon., 1994, 141,
275–280.
271. H. A. Schroeder and A. P. Nason, J. Invest. Dermatol., 1969, 53, 71–78.
272. H. A. Ragan, Sci. Total Environ., 1983, 28, 317–326.
273. P. J. Landrigan, Toxicol. Ind. Health, 1991, 7, 9–14.
274. G. E. Pierard, J. Cutan. Pathol., 1979, 6, 237–242.
275. I. Thornton, Proceedings of Conference on ‘‘Lead into the Future’’, Inst. Mining
and Metallurgy, Buxton, 1996, pp. 217–236.
276. S. Alam, J. Hammill, K. Karta and I. Charernanyarak, Srinagarind Med. J.,
1996, 11, 101–105.
277. A. B. G. Lansdown, Srinargarind Med. J., 1998, 13, 53–61.
278. S. Fregert, Contact Dermatitis Newsletter, 1973, 13, 352.
279. E. K. Edwards and E. K. Edwards, Cutis, 1982, 30, 629–630.
280. M. R. Moore, P. A. Meredith, W. S. Watson, D. J. Sumner, M. K. Taylor and
A. Goldberg, Fd. Cosmet. Toxicol., 1980, 18, 399–405.
281. S. C. Rastogi and J. Clausen, Toxicology, 1976, 6, 371.
282. S. G. Lilley, T. M. Florence and J. L. Stauber, Sci. Total Environ., 1988, 76,
267–278.
283. J. Amess, in General and Applied Toxicology, Ed. B. Ballantyne, T. C. Marrs and
P. Turner, MacMillan, Basingstoke, Hampshire, 1993, pp. 839–867.
284. S. Piomelli, in Haematology of Infancy and Childhood, 3rd edn., Ed. D. G.
Nathan and F. A. Oski, Saunders, Philadelphia, 1987, 3889–412.
285. C. A. Picut, C. S. Lee and R. M. Lewis, Br. J. Dermatol., 1987, 116, 773–784.
286. S. S. Haneef, D. Swarup, D. Kalicharan and S. K. Dwivedi, Vet. Hum. Toxicol.,
1995, 37, 428–429.
287. T. Niebrój and M. Gruszczyńska, Klin. Oczna, 1989, 91, 54.
288. A. Forni, A. Sciame, P. A. Bertazzi and L. Alessio, Arch. Environ. Health, 1980,
35, 139–146.
289. G. Gabbiani, B. Tuchweber and G. Perrault, Calc. Tiss. Res., 1970, 6, 20–31.
290. J. G. Pounds, G. J. Long and J. F. Rosen, Environ. Health Perspect., 1991, 91,
17–32.
291. J. G. Pounds and J. F. Rosen, Toxicol. Appl. Pharmacol., 1991, 83, 531–545.
292. M. R. Moore, Proc. Nutr. Soc., 1979, 38, 243–250.
293. D. F. Vanderputte, W. A. Jacob and R. E. Van Grieken, Matrix, 1990, 10,
33–37.
294. W. Y. Cheung, Ann. N.Y. Acad. Sci., 1988, 522, 74–87.
295. A. Przelecka, A. M. Kluska and M. Zwierzyk, Histochemistry, 1991, 95, 391–395.
296. D. R. Winge and K. B. Nielson, in Trace Elements in Man and Animals, Ed.
C. Mills, I. Bremner and J. K. Chesters, CAB Publishers, Slough, UK, 1985,
pp. 307–311.
297. G. F. Nordberg and M. Nordberg, Sangyo Ika Daigaku Sasshi, 1987, 9 (suppl),
153–164.
Met. Ions Life Sci. 2011, 8, 187–246

240 LANSDOWN
298. R. C. Wester, H. I. Maibach, L. Sedik, J. Melendres, S. DiZio and M. Wade,

Fundam. Appl. Toxicol., 1992, 19, 1–5.
299. Occupational Safety and Health Administration (USA), Occupational Exposure
to Cadmium, 1992, Section 5.
300. C. A. Bache, D. J. Lisk, J. M. Scarlett and L. G. Carbone, J. Toxicol. Environ.
Health, 1991, 34, 423–431.
301. M. A. Bosque, J. L. Domingo, J. M. Llobet and J. Corbella, Biol.Trace Elem.
Res., 1991, 28, 147–155.
302. S. Bannai, H. Sato, T. Ishii and S. Taketani, Biochim. Biophys. Acta, 1991, 1092,
175–179.
303. J. Bolewska, H. J. Hansen, F. Holmstrup, J. J. Pindborg and M. Stangerup,
Oral Surg. Med. Oral Pathol., 1990, 70, 55–58.
304. M. Bergman, Int. J. Dent., 1990, 40, 4–10.
305. J. de la Cuadra, Ann. Derm. Venereol. Slockh., 1993, 120, 37–42.
306. M. Sato, Nippon Hifuka Gakkai Zasshi, 1989, 99, 15–23.
307. K. Schrallhammer-Benkler, J. Ring, B. Przybilla, M. Meurer and M. Landthaler,
Acta Derm. Venereol. Stockh., 1992, 72, 294–296.
308. J. B. Hursh, T. W. Clarkson, E. F. Miles and L. A. Goldsmith, Arch. Environ.
Health, 1989, 44, 120–127.
309. W. A. Anawar and M. S. Gebel, Mutagenesis, 1991, 6, 189–192.
310. G. Kazantzis, Environ. Health. Perspect., 1981, 40, 143–161.
311. W. Aberer, G. Gerstner and H. Pehamberger, Contact. Dermatitis, 1990, 23,
168–171.
312. W. Aberer, Wien. Klin. Wochenschr., 1996, 108, 98–100.
313. T. Govett and M. G. De Navarre, Am. Perf. Ess. Oil Rev., 1947, 49, 365–368.
314. H. Butler, Poucher’s Perfumes, Cosmetics and Soaps, 10th edn., Kluwer, Dor-
drecht, 2000.
315. K. R. Butterworth, P. N. Drewett, C. D. Springall and S. R. Moorhouse,
Lancet, 1992, 339, 1489.
316. World Health Organization (WHO), Guidelines for Drinking Water Quality, 2nd
edn., Addendum, 1998, Vol. 2.
317. G. B. van der Voet and F. A. de Wolff, Arch. Dermatol., 1984, 55, 168–172.
318. G. M. Berlyne, R. Yagil, J. Ben Ari, G. Weinberger, E. Knopf and G. M.
Danovitch, Lancet, 1972, 1, 564–567.
319. R. L. Bertholf, Crit. Rev. Clin. Lab. Sci., 1987, 25, 195–210.
320. R. Verberckmoes, Lancet, 1972, 1, 750.
321. J. T. Prior and G. A. Cronk, Am. Med. Ass. Arch, Dermatol., 1959, 80, 447–454.
322. L. Rubin, A. H. Slepyan, L. F. Weber and I. Neuhauser, J. Am. Med. Ass., 1956,
162, 953–955.
323. L. F. Weber, I. Nauhauser, L. Rubin, A. H. Slepyan and H. Shellow, J. Am.
Med. Ass., 1956, 16, 65.
324. P. Badenoch-Jones, J. L. Turk and D. Parker, J. Pathol., 1978, 124, 51–62.
325. J. L. Turk, P. Badenoch-Jones and D. Parker, J. Pathol., 1978, 124, 43–49.
326. P. Darbre, J. Inorg. Biochem., 2005, 99, 1912–1919.
327. P. D. Darbre, Best Pract. Re. Clin. Endocrinol. Metab., 2006, 20, 121–143.
328. P. W. Harvey and P. Darbre, J. Appl. Toxicol., 2004, 24, 167–176.
Met. Ions Life Sci. 2011, 8, 187–246

329. M. K. Ward, T. G. Feest, H. A. Ellis, J. S. Parkinson and D. N. S. Kerr, Lancet,

1978, 1, 841–845.
330. J. P. Cruse, M. R. Lewin and C. G. Clark, Lancet, 1978, 1, 1261.
331. V. Parsons, C. Davies, C. Goode, C. Ogg and J. Siddiqui, Br. J. Me., 1971, 4,
273–275.
332. I. H. Blank, J. L. Jones and E. Gould, Proc. Toilet Goods Ass., 1958, 29, 1–4.
333. A. B. G. Lansdown, Br. J. Dermatol., 1973, 89, 67–76.
334. M. Wilhelm, F. K. Ohnesorge, I. Lombeck and D. Hafner, J. Anal. Toxicol.,
1989, 13, 17–21.
335. World Health Organization, Aluminum in Drinking Water, Guidelines for Drinking
Water Quality, Geneva, 1998, Add. Vol. 2. Ref. WHO/SDE/WSH/03.04/53.
336. A. C. Katz, D. W. Frank, M. W. Sauerhoff, G. M. Zwicker and R. I. Freudenthal,
Fd. Cosmet. Toxicol., 1984, 22, 7–9.
337. R. G. Miller, F. C. Kopfler, K. C. Ketly, J. A. Stober and N. S. Ulmer, J. Am.
Water Works Ass., 1984, 76, 84–91.
338. R. Anane and E. E. Creppy, Hum. Exp. Toxicol., 2001, 20, 477–481.
339. I. Lyon and L. M. Klotz, J. Am. Pharm. Ass., 1958, 47, 509–513.
340. H. Tronnier and G. Rentschler, J. Soc. Cosmet. Chem., 1973, 24, 281–290.
341. E. Hölzle and A. M. Kligman, J. Soc. Cosmet. Chem., 1979, 30, 357–367.
342. C. M. Papa and A. M. Kligman, J. Invest. Dermatol., 1967, 49, 139–145.
343. A. B. G. Lansdown, J. Soc. Cosmet. Chem., 1973, 24, 677–684.
344. W. J. Cunliffe, J. L. Burton and S. Shuster, Br. J. Dermatol., 1969, 81, 867.
345. K. F. Scoles, K. D. Crow, J. P. Ellis, R. R. Harman and E. M. Saihan, Br. Med.
J., 1978, 2, 84–85.
346. M. Di Silvestre, S. Guizzardi, N. Bettini, G. Gargiulo and R. Savini, Chir.
Organ. Mov., 1991, 76, 167–172.
347. P. Galle, J. P. Berry and C. Galle, Envir. Health Persp., 1992, 97, 145–147.
348. N. McFadden, T. Lyberg and A. Hensten-Pettersen, J. Am. Acad. Dermatol.,
1989, 20, 903–908.
349. P. J. LoPresti and G. W. Hambrick, Arch. Dermatol., 1965, 92, 188–191.
350. A. M. Kligman, Arch. Dermatol., 1958, 77, 149–180.
351. G. Baler, Arch. Dermatol., 1965, 91, 145–148.
352. W. B. Shelley and H. J. Hurley, Br. J. Dermatol., 1958, 70, 75–101.
353. R. M. Al-Ashban, M. Aslam and A. H. Shah, Public Health, 2004, 118, 292–298.
354. R. Sprinkle, J. Fam. Pract., 1995, 40, 358–362.
355. P. J. Fleming, M. Cooke, S. M. Chantler and J. Golding, Br. Med. J., 1994, 309,
1594–1596.
356. B. A. Richardson, J. Forensic Sci. Soc., 1994, 34, 199–204.
357. J. Boyle, J. L. Burton and J. Faergemann, Br. Med. J., 1986, 292, 28.
358. A. Chetcuti, L. J. Adams, P. B. Mitchell and P. R. Scofield, Psychiatr. Genet.,
2008, 18, 64–72.
359. C. L. Callaway, H. C. Hendre and E. D. Luby, Am. J. Psychiat., 1968, 124,
1124–1125.
360. A. Rifkin, S. B. Kurtin, F. Quaitin and D. F. Klein, Am. J. Psychiat., 1973, 130,
1018–1019.
361. M. C. Heng, Arch. Dermatol., 1982, 118, 246–248.
Met. Ions Life Sci. 2011, 8, 187–246

242 LANSDOWN
362. A. Sparsa and A. Bonnetblanc, Ann. Dermatol. Venereol., 2004, 131, 255–261.
363. S. H. Wakeling, T. Lipscombe, D. I. Orton and P. Merran, Clin. Exp. Der-
matol., 1996, 21, 296–298.
364. F. J. Blomfield and M. M. Young, Br. J. Dermatol., 2006, 109, 9–14.
365. J. E. Wahlberg, Arch. Dermatol., 1968, 97, 336–339.
366. K. Bauerová, Z. Kassai, V. Koprda and M. Harangozó, J. Appl. Toxicol., 2001,
21, 241–243.
367. L. A. Iliyn, A. T. Ivannikov, I. D. Parfenov and V. P. Stolyarov, Heath Phys.,
1975, 29, 75–80.
368. G. S. Hahn, Dermatol. Surg., 2001, 25, 689–694.
369. H. Zhai, W. Hannon, G. S. Hahn, R. A. Harper, A. Pelosi and H. I. Maibach,
Dermatology, 2000, 200, 244–246.
370. A. B. G. Lansdown and A. Taylor, Int. J. Cosmet. Sci., 1997, 19, 167–172.
371. ATSDR, Toxicological Profile for Thorium, US Agency for Toxic Substances
and Disease Registry, Atlanta, GA, 1990, pp. 185.
372. M. Faber, Environ.Res., 1979, 18, 37–43.
373. K. Wegener, Virech. Arch. A Pathol. Anat. Histol., 1979, 38, 245–268.
374. J. B. Lansman, J. Gen. Physiol., 1990, 95, 679–696.
375. W. W. Monafo and V. H. Ayvazian, Surg. Clin. North Am., 1978, 58,
1157–1171.
376. C. L. Fox, W. W. Monafo and V. H. Ayzadian, Surg. Gnec. Obstet., 1977, 144,
668–672.
377. J. Koller and M. Orsag, Acta Chirug. Plast., 1998, 40, 73–75.
378. A. B. G. Lansdown, S. R. Myers, J. A. Clarke and P. O’sullivan, J. Wound
Care., 2003, 12, 113–118.
379. G. A. Schoenenberger, Monogr. Allerg., 1975, 9, 72–139.
380. B. Kremer, G. A. M. Allgower and M. Graf, Intens. Care Med., 1981, 7, 77–87.
381. T. Gebel, H. Lantzsch, K. Plessow and H. Dunkelberg, Mutat. Res., 1997, 389,
183–190.
382. D. Monro-Ashman, D. D. Moore and T. H. Hughes, Trans. St. John’s Hosp.
Dermatol. Soc., 1969, 55, 196.
383. J. E. Wahlberg and A. Bowman, Am. J. Contact. Dermat., 1990, 1, 112.
384. T. Heyl and R. J. Barlow, Br. J. Dermatol., 1989, 121, 787–792.
385. P. J. Gehring and P. B. Hammond, J. Pharmacol. Exp. Therap., 1967, 155,
187–201.
386. R. M. Schwartzman and J. O. Kirschbaum, Invest. Dermatol., 1962, 391, 169–173.
387. M. N. Hughes, W. K. Man and B. C. Whaler, Chem. Biol. Interactions, 1978, 23,
85–97.
388. I. Rusznyak, L. Gyorgy, S. Ormai and T. Millner, Experientia, 1969, 24, 8–10.
389. L. Favari and M. Mourelle, J. Appl. Toxicol., 2006, 5, 32–34.
390. A. Buschke, E. Christeller and L. Loewenstein, Klin. Wochenschr., 1927, 6,
1088–1089.
391. G. B. Skipworth, N. Goldstein and W. P. McBride, Arch. Dermatol., 1967, 95,
83–86.
392. L. Kahlenberg and N. Barwasser, J. Biol. Chem., 1928, 79, 405–408.
393. A. M. Memmesheimer, Deutsch. Med. Wchnschr., 1935, 61, 418.
Met. Ions Life Sci. 2011, 8, 187–246

394. D. J. Birmingham, in Dermatology in General Medicine, Ed. T. Fitzpatrick,

K. A. Arndt, A. Z. Eisen, E. J. Van Scott and J. H. Vaughan, McGraw Hill,
New York, 1971, pp. 1046–1047.
395. J. Cuzick, S. Evans, M. Gilman and D. A. Price Evans, Br. J. Cancer, 1982, 45,
904–911.
396. O. Axelson, J. Toxicol. Environ. Health, 1980, 6, 1229–1235.
397. Y. S. Yu, W. T. Liao and C. Y. Chai, J. Biomed. Sci., 2006, 13, 657–666.
398. B. J. Wang, Y. Y. Lee, C. P. Mak, H. F. Kao, M. L. Hsu and J. R. Hsien,
J. Formos. Med. Ass., 1991, 90, 1093–1098.
399. M. I. Lustier, J. L. Wilmer, D. R. Germolec, J. Spalding, T. Yoshida, K. Gaido,
P. P. Simeonova, F. G. Burleson and A. Bruccoleri, Toxicol. Lett., 1985, 82,
471–476.
400. G. Pershagen, Environ. Health Perspect., 1981, 40, 93–100.
401. Y. M. Hsueh, G. S. Cheng, M. M. Wu, H. S. Yu and C. J. H. Chen, Br. J.
Cancer, 1995, 71, 109–114.
402. S. L. Chen, S. J. Yeh, M. H. Yang and T. H. Lin, Biol. Trace Elem. Res., 1995,
48, 263–274.
403. F. J. Lu, Lancet, 1990, 336, 115–116.
404. J. D. Berman, P. C. Melby and F. A. Neva, Trans. Roy. Soc. Trop. Med., 1989,
83, 784–785.
405. J. D. Chulay, H. C. Spencer and M. Mugambi, Am. J. Trop. Med. Hyg., 1985,
34, 702–709.
406. P. N. Gates, J. B. Pridham and J. A. Webber, Lancet, 1995, 345, 386–387.
407. T. Gebel, S. Kevekordes, J. Schaefer, H. von Platen and H. Dunkelberg, Mutat.
Res. Genet. Toxicol., 1996, 368, 267–274.
408. F. A. De Wolff, Br. Med. J., 1995, 310, 1216–1217.
409. R. Bailly, R. Lauwareys, J. P. Buchet, P. Mahieu and J. Konings, Br. J. Ind.
Med., 1991, 48, 93–97.
410. A. Taylor, Lancet, 1996, 347, 616.
411. S. Chantler, Hospital Update, 1996, 22, 212–216.
412. R. I. MacCallum, J. Environ. Monit., 2005, 7, 1245–1250.
413. K. A. Winship, Adverse Drug React. Acute Poisoning Rev., 1987, 6, 67–90.
414. L. Gerhardsson, D. Brune, G. F. Nordberg and P. O. Webster, Scand. J. Work
Environ. Health, 1982, 8, 201–208.
415. N. Gurnani, A. Sharma and G. Talukder, Biol. Trace Element Res., 1993, 37,
281–292.
416. U.S. Department of Health and Human Services, 11th Report on Carcinogens,
Research Triangle Park, NC, 2005.
417. L. Tomatid, C. Agthe, H. Bartsch, J. Huff, R. Montesano, R. Saracci,
E. Walker and J. Wilbourn, Canc. Res., 1978, 38, 877–885.
418. M. C. Bibby, Br. J. Dermatol., 1981, 104, 485–488.
419. F. Marks and G. Fürstenberger, Br. J. Dermatol., 1986, 1115(suppl. 31),
1–8.
420. T. S. Argyris, J. Invest. Dermatol., 1980, 75, 360–362.
421. K. G. Nelson and T. J. Slaga, Cancer Res., 1982, 42, 4176–4181.
422. L.-S. Lee and I. B. Weinstein, Science, 1978, 202, 313–315.
Met. Ions Life Sci. 2011, 8, 187–246

244 LANSDOWN
423. O. Wong, M. D. Whorrton, D. E. Foliart and R. Lowengart, Int. Arch. Occup.

Health, 1992, 64, 235–241.
424. B. Suárez, G. López-Abnente, C. Martinez, C. Navaro, M. J. Tormo, S. Rosso,
S. Schraub, L. Gáfa, H. Sancho-Garnier, J. Wecsler and R. Zanetti, BMC
Public Health, 2007, 7, 180.
425. International Agency for Research on Cancer, Monographs of the Evaluation of
Carcinogenic Risk of Chemicals to Humans, IARC, Lyon, France, 1990, 49,
pp. 677.
426. W. Mertz, E. E. Roginski and H. A. Schroeder, J. Nutr., 1967, 86, 107–112.
427. W. Mertz, Med. Clin. North Am., 1976, 60, 739–744.
428. L. S. Levy and S. Venitt, Carcinogenesis, 1986, 7, 831–835.
429. C. B. Klein, L. Su, D. Bowser and J. Leszczynska, Environ. Health Persp., 2002,
110, 739–743.
430. A. Anderson, S. R. Burge, A. Engeland and T. Norseth, Occup. Environ. Med.,
1996, 53, 107–113.
431. A. N. Uddin, F. J. Burns, T. G. Rossman, H. Chen, T. Kluz and M. Costa,
432. M. Costa and C. B. Klein, Crit. Rev. Toxicol., 2006, 36, 155–163.
433. A. Oskarssen, Y. Andersson and H. Tjälve, Cancer Res., 1979, 39, 4175–4182.
434. P. Mushak and A. F. Crocetti, Environ. Health Perspect., 1996, 104, 1012–1018.
435. S. H. Tan, S. N. Tham and C. L. Goh, Int. J. Dermatol., 1995, 34, 770–776.
436. M. E. Maloney, Dermatol. Surg., 1996, 22, 301–304.
437. R. A. Scolyer, J. F. Thompson, L. X. Li, A. Beavis, M. Dawson, P. Dobie,
R. Soper, F. Uren, J. R. Stretch, R. Sharma and S. W. McCarthy, Mod. Pathol.,
2004, 17, 1191–1197.
438. R. Z. Karim, R. A. Scolyer, V. S. Yee, J. G. McKinnon, L. X. Li, R. F. Uren,
S. Lam, A. Beavis, M. Dawson, P. Doble, D. S. Hoon and J. F. Thompson, Ann.
Surg., 2008, 247, 1003–1010.
439. T. Partenan, E. Pukkala, H. Vainio, H. Kurppa and H. Koskinen, J. Occup.
Med., 1994, 36, 616–622.
440. J. L. Turk and D. Parker, J. Invest. Dermatol., 1977, 68, 336–340.
441. P. D. Gikas, L. Mansfield and L. Mokbel, Int. J. Fertil. Womans Med., 2005, 49,
2112–214.
442. C. J. Rageth, The Breast, 2004, 14, 85–86.
443. R. C. Brady and S. R. Hilfer, Proc. Natl. Acad. Sci. USA, 1982, 79, 5587–5591.
444. A. Bringmann, S. Schopf and A. Reichenbach, J. Neurophysiol., 2000, 84, 2975–
2983.
445. B. H. Grahn, P. G. Paterson, K. T. Gotteschall-Pass and Z. Zhang, J. Am. Coll.
Nutr., 2001, 20, 106–118.
446. K. D. Rosenman, A. Moss and S. Kon, J. Occup. Envir. Med., 1978, 21,
430–435.
447. C. Hanna, F. T. Fraunfelder and J. Sanchez, Arch. Ophthalm., 1974, 92,
18–22.
448. D. A. Fox, S. D. Rubenstein and P. Hsu, Toxicol. Appl. Pharmacol., 1991, 109,
482–493.
449. H. Kobayashi and S. Kohshima, Nature, 1997, 387, 767–768.
Met. Ions Life Sci. 2011, 8, 187–246

450. L. Z. Bito, Exp. Eye Res., 1984, 39, 807–829.

451. R. Rosenthal, H. Heiman, H. Agostini, G. Martin, L. L. Hansen and O. Strauss,
Mol. Vis., 2007, 13, 443–456.
452. R. O. Wong, J. Neurosci., 1995, 15, 2696–2706.
453. N. Nagai, T. Fukuhata and Y. Ito, Biol. Pharm. Bull., 2007, 30, 6–10.
454. K. R. Hightower, F. J. Giblin and V. N. Reddy, Invest. Ophthalmol. Vis. Sci.,
1983, 24, 1188–1193.
455. I. S. Jamall and H. Roque, Biol. Trace Elem. Res., 1989, 23, 55–63.
456. W. J. Weidner and A. J. Sillman, Arch. Toxicol., 1997, 71, 455–460.
457. D. A. Fox and S. D. Rubinstein, Exp. Eye Res., 1989, 48, 237–249.
458. D. A. Fox and D. Srivastava, Toxicol. Lett, 1995, 82–83, 263–270.
459. D. A. Fox, L. He, A. T. Poblenz, C. J. Medrana, Y. S. Blocker and D. Srivastava,
Toxicol. Lett., 1998, 102–103, 359–361.
460. H. Gong and T. Amemiya, Invest. Ophthalmol. Vis. Sci., 1996, 37, 1967–1974.
461. X. He, P. Hahn, J. Iacovelli, R. Wong, C. King, R. Bhisitkul, M. Massaro-
Giordano and J. L. Dunaief, Prog. Retin. Eye Res., 2007, 26, 649–673.
462. N. S. Konerirajapuram, K. Coral, R. Punitham, T. Sharma, N. Kasinathan and
R. Sivaramakrishnan, Ind. J. Ophthalmol, 2004, 52, 145–148.
463. J. S. Baath, W. C. Lam, M. Kirby and A. Chun, Retina, 2008, 28, 894–899.
464. J. L. Dunaief, Invest. Ophthalmol. Vis. Rers., 2006, 47, 4660–4664.
465. A. Monies and M. Prost, Klin. Oczna, 1994, 96, 135–139.
466. A. Monies and M. Prost, Klin. Oczna, 1994, 96, 141–143.
467. O. Cekic, Br. J. Ophthalmol., 1998, 82, 186–188.
468. D. L. Holness, I. G. Taraschuk and J. R. Nethercott, Arch. Envir. Heath, 1989,
44, 291–297.
469. M. Mulak, M. Misiuk-Hojlo, B. Markuszewski and K. Dembska, Klin. Oczna,
2008, 110, 176–182.
470. B. R. Grubb, G. E. DuVal, J. S. Morris and P. J. Bentley, Toxicol. Appl.
Pharmacol., 1985, 77, 444–450.
471. J. C. Erie, J. A. Butz, J. A. Good, E. A. Erie, M. F. Burritt and J. D. Cameron,
Am. J. Ophthalmol., 2005, 139, 888–893.
472. R. Freedman, L. Olson and B. J. Hoffer, Envir. Health Perspect., 1990, 89,
27–33.
473. M. Yoshizuka, K. J. McCarthy, G. I. Kaye and S. Fujimoto, Anat. Rec., 1990,
227, 138–143.
474. A. M. El-Sherbeeny, J. V. Odom and J. E. Smith, Cut. Ocul. Toxicol., 2006, 25,
173–183.
475. N. K. Will, V. M. Ramanujam, J. Chang, N. Kalariya, J. R. Lewis, T. X. Weng
and F. J. Van Kuijk, Exp. Eye Res., 2008, 96, 41–51.
476. K. Kohler, H. Lilienthal, E. Guenther, G. Winneke and E. Zrenner, Neuro-
toxicol., 1997, 18, 623–632.
477. D. A. Fox and D. B. Farber, Exp. Eye Res., 1988, 46, 597–611.
478. C. C. Bridges, J. R. Battle and R. K. Zalups, Toxicol. Appl. Pharmacol., 2007,
221, 251–260.
479. S. Gitter, A. Pardo, N. Kariv and U. Yinon, Toxicology, 1988, 51, 67–76.
480. A. G. Nagpal and S. E. Brodie, Doc. Ophthalmol., 2009, 118, 163–166.
Met. Ions Life Sci. 2011, 8, 187–246

246 LANSDOWN
481. P. J. Prouse, J. J. Kanski and J. M. Gumpel, Ann. Rheum. Dis., 1981, 4, 564–566.
482. H. Tjälve, M. Nilsson and B. Larsson, Acta Pharmacol. Toxicol. (Copenh.),
182, 51, 147–153.
483. Z. H. Lu, H. Gong and T. Amemiya, Toxicol. Sci., 2002, 66, 253–260.
484. K. R. Fry, D. M. Edwards, K. A. Shaw and C. B. Watt, Neurosci. Lett., 1991,
124, 216–220.
485. O. Mameli, M. A. Caria, P. Melis, P. Zambenedetti, M. Ramila and P. Zatta,
Metab. Brain Dis., 2006, 21, 89–107.
Met. Ions Life Sci. 2011, 8, 187–246

Met. Ions Life Sci. 2011, 8, 247–262
10
Metal Ions Affecting the Neurological System
Hana R. Pohl , Nickolette Roney , and Henry G. Abadin
Agency for Toxic Substances and Disease Registry, U.S. Department of Health and Human
Services, Atlanta GA 30333, USA
<hrp1@cdc.gov>
<nxr6@cdc.gov>
<hga0@cdc.gov>
ABSTRACT 248
1. EXPOSURE TO METALS AND THEIR MIXTURES 248
2. METALS AFFECTING THE NEUROLOGICAL SYSTEM 249
2.1. Aluminum 249
2.1.1. Mechanism of Aluminum Neurotoxicity 249
2.2. Arsenic 250
2.3. Cadmium 250
2.4. Lead 250
2.4.1. Mechanisms of Lead Neurotoxicity 251
2.5. Manganese 251
2.5.1. Mechanism of Manganese Neurotoxicity 252
2.6. Mercury 252
2.6.1. Mechanism of Mercury Neurotoxicity 252
3. INTERACTION OF METALS AND NEUROLOGICAL
EFFECTS 253
3.1. Cadmium and Lead 253
3.2. Copper and Lead 253
3.3. Lead and Arsenic 254
3.4. Manganese and Cadmium 255
3.5. Manganese and Lead 255
248 POHL, RONEY, and ABADIN
3.6. Zinc and Lead 255

4. INTERACTIONS OF METALS WITH OTHER CHEMICALS 256
4.1. Ethanol 256
4.1.1. Cadmium 256
4.1.2. Lead 256
4.1.3. Mercury 257
4.2. Polychlorinated Biphenyls 257
4.2.1. Mercury 257
4.3. Organophosphates 258
4.3.1. Lead 258
4.4. Chelating Agents 259
5. CONCLUSIONS 259
ABBREVIATIONS 260
REFERENCES 260
ABSTRACT: Several individual metals including aluminum, arsenic, cadmium, lead,

manganese, and mercury were demonstrated to affect the neurological system. Metals
are ubiquitous in the environment. Environmental and occupational exposure to one
metal is likely to be accompanied by exposure to other metals, as well. It is, therefore,
expected that interactions or ‘‘joint toxic actions’’ may occur in populations exposed to
mixtures of metals or to mixtures of metals with other chemicals. Some metals seem to
have a protective role against neurotoxicity of other metals, yet other interactions may
result in increased neurotoxicity. For example, zinc and copper provided a protective
role in cases of lead-induced neurotoxicity. In contrast, arsenic and lead co-exposure
resulted in synergistic effects. Similarly, information is available in the current literature
on interactions of metals with some organic chemicals such as ethanol, polychlorinated
biphenyls, and pesticides. In depth understanding of the toxicity and the mechanism of
action (including toxicokinetics and toxicodynamics) of individual chemicals is impor-
tant for predicting the outcomes of interactions in mixtures. Therefore, plausible
mechanisms of action are also described.
KEYWORDS: antagonism . ethanol . interactions . metals . mixtures . neurotoxicity

. PCBs . pesticides . synergism
1. EXPOSURE TO METALS AND THEIR MIXTURES

Metals are commonly used in occupational settings and are common
environmental pollutants. Metals are released to the environment from
burning of fossil fuels, mining, smelting, industrial activities, and from
hazardous waste sites [1,2]. Environmental and/or occupational exposure to
one metal is likely to be accompanied by exposure to other metals as well.
For example, lead, arsenic, cadmium, copper, and zinc are all often found at
elevated concentrations in the environment near mining and smelting sites.
Met. Ions Life Sci. 2011, 8, 247–262

METAL IONS AFFECTING THE NEUROLOGICAL SYSTEM 249
Metals also frequently co-occur in completed exposure pathways at hazar-

dous waste sites [2,3]. This chapter will discuss metal ions that affect the
neurological system and how exposures to combinations of metals may
contribute to their neurotoxicity.
2. METALS AFFECTING THE NEUROLOGICAL

SYSTEM
There are a number of metals that affect the neurological system. Discussed
below are specific metals and their associated neurological effects.
2.1. Aluminum
There is suggestive evidence in humans of a relationship between chronic
exposure to aluminum dust (occupational settings) and subclinical neuro-
logical effects such as impairment on neurobehavioral tests and an increase
of subjective neurological symptoms. Also, in patients with reduced renal
function, prolonged dialysis with aluminum-containing dialysates has pro-
duced a neurotoxicity syndrome (dialysis dementia) characterized by the
gradual loss of motor, speech, and cognitive functions. However, the use-
fulness of these studies in predicting toxicity in the general population is
limited.
It has been proposed that Alzheimer’s disease is associated with aluminum
exposure, however, this association is controversial and there is little con-
sensus regarding current evidence. Oral studies in animals have shown that
the nervous system is a sensitive target of aluminum toxicity, producing
neurotoxicity and neurodevelopmental toxicity such as significant altera-
tions in motor function, sensory function, and cognitive function [4].
2.1.1. Mechanism of Aluminum Neurotoxicity

Neurofibrillary tangles within the brain neurons, resulting from changes in
cytoskeletal proteins, are a characteristic response to aluminum in certain
species and exposure situations. Aluminum also influences calcium home-
ostasis and calcium-dependent processes in the brain. Apoptosis may also
contribute to neurodegeneration. Animal studies indicate that aluminum
exposure can affect permeability of the blood-brain barrier, cholinergic
activity, signal transduction pathways, lipid peroxidation, impair neuronal
Met. Ions Life Sci. 2011, 8, 247–262

glutamate nitric oxide-cyclic GMP pathway, and interfere with the meta-
bolism of essential trace elements [4].
2.2. Arsenic
Arsenic (a metalloid) can cause serious neurological effects, both after
inhalation and oral exposure. Common effects seen in humans orally
exposed to arsenic are peripheral and/or central neuropathy. Exposure to
high levels of arsenic produce mainly central nervous system effects and
exposure to low levels produce mainly peripheral nervous system effects. In
addition, more recent studies have indicated that exposure to arsenic may
produce more subtle neurological effects such as intellectual deficits in
children. The mechanism of arsenic-induced neurological changes has not
been determined. However, some of the neurological effects of high level
oral exposure are thought to be the result of direct cytotoxicity. Also, animal
studies have shown altered neurotransmitter concentrations in some areas of
the brain after oral exposure to arsenic [5].
2.3. Cadmium
Environmental cadmium exposure has been associated with neurobeha-
vioral effects in a few studies that used hair cadmium as an index of expo-
sure. Affected endpoints included verbal IQ in children and disruptive
behavior in young adults. However, due to the limitations within the studies,
their usefulness is limited. Neurotoxicity has been seen in animals exposed
orally to cadmium, producing changes in behavior, decrease in motor
activity, alterations in neurotransmitter levels, histopathological changes in
the brain, and peripheral neuropathy. In addition, cadmium is known to
alter neurotransmitter levels in the brain, and may inhibit calcium entry into
neurons [6].
2.4. Lead
Neurological effects are one of the most sensitive endpoints of lead exposure,
and children are particularly vulnerable. Exposure to high lead levels pro-
duces encephalopathy with signs such as hyperirritability, ataxia, convul-
sions, stupor, and coma. In children, exposure to low lead levels has been
associated with neurobehavioral effects including impaired cognitive ability
Met. Ions Life Sci. 2011, 8, 247–262

and IQ deficits. In lead workers, reported neurobehavioral effects include

malaise, forgetfulness, irritability, lethargy, headache, fatigue, impotence,
decreased libido, dizziness, weakness, and paresthesia. Lead exposure in
workers has also been associated with neuropsychological effects, increased
prevalence and severity of white matter lesions, changes in nerve conduction
velocity and postural balance, and alterations of somatosensory evoked
potentials [7].
2.4.1. Mechanisms of Lead Neurotoxicity
Lead can affect the nervous system by multiple mechanisms. During brain
development, lead interferes with the trimming and pruning of synapses,
migration of neurons, and neuron/glia interactions. At the biochemical level
one of the most important mechanisms of lead toxicity is the mimicking of
calcium action and/or disruption of calcium homeostasis [7]. For example,
lead binds to second messenger calcium receptors such as protein kinase C
(PKC) and calmodulin. The premature activation of PKC by lead may
impair brain microvascular formation and function, and may result in gross
defects in the blood-brain barrier that contribute to acute lead encephalo-
pathy (at high lead exposure levels). Lead also may substitute for zinc in
some enzymes and in zinc-finger proteins. It can also interfere with neural
cell adhesion molecules, which may contribute to learning deficits. The fetus
and infant may have increased vulnerability to lead’s neurotoxicity due in
part to the immaturity of the blood-brain barrier and to the lack of the high-
affinity lead-binding protein in astroglia, which sequester lead. In addition,
lead affects virtually every neurotransmitter system in the brain, including
the glutamatergic, dopaminergic, and cholinergic systems [7].
2.5. Manganese
Inhalation of high levels of manganese (as seen in occupational studies) can
lead to a syndrome of disabling neurological effects in humans called
manganism with symptoms of tremors, difficulty in walking, and facial
muscle spasms [8]. Initial symptoms of manganese toxicity that can progress
into mangansim include irritability, aggressiveness, and hallucinations.
Manganese inhalation may also produce adverse cognitive effects such as
difficulty with concentration and memory problems. Effects similar to the
preclinical neurological effects and mood effects seen in occupational studies
have also been associated with environmental exposures to manganese in air.
In addition, there is evidence that oral exposure to manganese may produce
similar neurological effects as reported for inhalation exposure. Exposure to
Met. Ions Life Sci. 2011, 8, 247–262

excess levels of manganese in drinking water has been associated with subtle
learning and behavioral deficits in children [8].
2.5.1. Mechanism of Manganese Neurotoxicity

A mechanism for the neurotoxicity of manganese has not been clearly
established. A suggested mechanism of manganese neurotoxicity is the
increase in autooxidation or turnover of intracellular catecholamines
including dopamine, norepinephrine, and epinephrine. This leads to the
increased production of free radicals, reactive oxygen species, and other
cytotoxic metabolites, along with a depletion of cellular antioxidant defense
mechanisms. Other potential mechanisms include the potential substitution
for calcium by manganese, the possibility that a transport mechanism for
manganese is linked to the dopamine reuptake carrier, the inhibition of brain
mitochondrial oxidative phosphorylation by manganese, and the involve-
ment of manganese in complex interactions with other minerals [8].
2.6. Mercury
Exposure to mercury produces neurological and behavioral effects in
humans. Adverse neurological effects following acute inhalation of high
concentrations of mercury vapor include a number of cognitive, personality,
sensory, and motor disturbances. The most prominent symptoms include
tremors, irritability, insomnia, memory loss, neuromuscular changes,
headaches, polyneuropathy, and performance deficits in tests of cognitive
function. In addition, chronic inhalation exposures have produced signs of
neurotoxicity including tremors, unsteady walking, irritability, poor con-
centration, short-term memory deficits, tremulous speech, blurred vision,
performance decrements in psychomotor skills, paresthesias, and decreased
nerve conduction. The motor system disturbances are most likely reversible
upon the cessation of mercury exposure. However, the cognitive impair-
ments, primarily memory deficits, may be permanent. Adverse neurological
effects in humans have also been reported after oral exposure to inorganic
mercury salts (usually resulting from the ingestion of therapeutic agents
containing mercurous chloride) and methylmercury [9].
2.6.1. Mechanism of Mercury Neurotoxicity
The high-affinity binding activity of divalent mercuric ion to thiol or sulf-

hydryl groups of proteins is believed to be a major mechanism for the
Met. Ions Life Sci. 2011, 8, 247–262

biological activity of mercury. Damage to neurological tissues may involve

oxidative stress, depolarization of mitochondrial inner membranes leading
to hydrogen peroxide formation, and depleted levels of reduced pyridine
nucleotides. It has been suggested that neurons are particularly sensitive to
mercury because of their low endogenous glutathione content or their
inefficient glutathione reduction activity [9].
3. INTERACTION OF METALS AND NEUROLOGICAL

EFFECTS
Although people are often exposed to mixtures of metals, information on
multi-component mixtures is usually not available. Thus, binary evaluations
of mixture components are used to enable risk assessors to predict the
direction of possible interactions (see Chapter 3 for additional information).
Provided below are summaries of binary interaction studies of some metals
focusing on neurological effects.
3.1. Cadmium and Lead

Co-administration of lead as lead acetate (500 ppm PbE43 mg Pb/kg/day)
and cadmium as cadmium chloride (100 ppm CdE8.6 mg Cd/kg/day) in the
diet of rats for 60 days induced changes in schedule-controlled responding
(lever pressing) and in dopamine and serotonin levels in their brains [10].
When both metals were administered alone, the activity was increased.
However, in a group exposed to both metals, lever pressing did not differ
from the negative control. The authors suggested that a possible mechanism
for neurochemical antagonism between cadmium and lead might involve
the metals interaction associated with the neuronal influx of calcium and the
release of catecholamines. Following the same experimental conditions, the
rats were tested in a Digiscan activity monitor [11]. Lead alone increased
movement and vertical activity, while cadmium alone decreased movement
and increased rest time. Cadmium antagonized lead-induced effects in rats
co-exposed to both metals. The metals did not influence each other’s con-
centration in the brain, but cadmium decreased blood lead levels [11].
3.2. Copper and Lead

In humans receiving adequate dietary copper and a low dietary lead intake,
supplemental copper at a level about five times the RDA decreased blood
Met. Ions Life Sci. 2011, 8, 247–262

lead and had a protective effect on lead balance (i.e., copper changed lead
balance from positive to negative) [12]. A case-control study indicated that
420 years of occupational exposure to copper and lead may increase the risk
of Parkinson’s disease [13,14]. However, this study does not provide infor-
mation suitable for determining the type of interaction between these metals.
In rats receiving both metals orally, supplemental copper generally did not
affect lead absorption or blood and liver, kidney, and bone lead concentra-
tions at lower copper doses (5 ppm) and copper/lead dose ratios [15]. At
higher supplemental copper doses (20 ppm) in rats and higher copper/lead
dose ratios, however, supplemental copper decreased blood, liver, and kidney
concentrations of lead [16,17] in intermediate duration studies. Levels of lead
in brain were not affected [17]. Similarly, the brain levels in rats were not
affected in an intermediate duration study (21 days) of intraperitoneally
injected copper as chloride (at a doseE10 mg/kg/day) and orally adminis-
tered lead as acetate in drinking water (at a doseE20 mg/kg/day) [18]. Both
studies investigated the effect of co-exposure to these metals on neuro-
transmitters in the brain. Copper did not affect the lead-induced decrease in
brain concentration of dopamine in rats following intermediate oral exposure
[17]. Lead did not affect norepinephrine or influence copper’s effect on this
transmitter, and neither metal affected serotonin in this study. In contrast,
lead alone and copper alone increased norepinephrine while the mixture
decreased norepinephrine concentrations in the brains of rats in another
study [18], but the dose of copper was higher and was administered through
intraperitoneal injection, which bypasses homeostatic mechanisms for copper
in the gastrointestinal tract and also potential points of interaction with lead.
3.3. Lead and Arsenic

In children, studies using hair lead and arsenic concentrations as biomarkers
of exposure have reported a potentiating interaction of lead on arsenic-
associated decreases in reading and spelling skills [19].
Following 14 days of gavage administration of lead acetate (74 mg Pb/kg/
day) and sodium arsenite (8.0 mg As/kg/day), lead decreased the arsenic
concentrations in the brain of adult mice, as compared with arsenic alone at
the same dose as in the mixture [20]. Although both metals have been
reported to affect neurotransmitter levels in the brain, the study of joint
action of lead and arsenic showed no apparent influence on this endpoint.
Changes in neurotransmitter concentrations which tended to be the same as
for arsenic alone, or in a few instances, additive as compared with the slight
changes seen with either metal alone at the same dose as in the mixture were
reported in the study. Lead alone had little effect on neurotransmitters [20].
Met. Ions Life Sci. 2011, 8, 247–262

3.4. Manganese and Cadmium

An intraperitoneal administration of manganese (4 mg/kg) enhanced the
uptake of cadmium (0.5 mg/kg) in the brain of male albino rats by 222%
[21]. Further studies showed that the co-exposure to manganese and cad-
mium resulted in synergistic effects, producing greater alterations in the
contents of biogenic amines and 5-hydroxyindole acetic acid than a single
exposure to either of the metals [22].
3.5. Manganese and Lead

Intraperitoneal injection of lead and oral exposure to manganese for 14 days
increased the concentration of lead in whole brain of rats [23]. Intraperitoneal
injection of rats with both metals (5 mg Pb/kg, 6 mg Mn/kg, consecutive)
during gestation and/or lactation also increased lead levels in whole brain of
the pups [24]. In addition, intermediate duration oral exposure via drinking
water to lead (5 ppmE2 mg Pb/kg/day) and intraperitoneal injection of low
(1 mg Mn/kg/day) or high (4 mg Mn/kg/day) doses of manganese produced
higher concentrations of lead in five of seven regions of the brain as com-
pared with lead alone at the same dose as in the mixture [25]. In contrast, lead
increased the levels of manganese in only two of the seven regions and only at
the low dose of manganese. Binding studies in vitro by the same group of
investigators showed that the presence of manganese increased the binding of
lead to brain protein [26)]. The above results clearly indicate that manganese
increases the distribution and/or retention of lead in the brain.
In an oral/intraperitoneal study in adult rats, manganese as manganese
chloride (3 mg Mn/mL drinking water) and/or daily intraperitoneal injections
of lead (5, 8, or 12 mg Pb/kg, as lead acetate) were administered for 14 days
[23]. Neurobehavioral endpoints were assessed on days 7 and 14. The co-
exposure to these metals appeared to adversely affect learning (conditioned
avoidance) in an additive manner. However, antagonism was reported for
other endpoints. Spontaneous motor activity and norepinephrine content of
the brain were slightly increased by each metal alone, but were significantly
decreased by the two metals in co-administration at the same doses as tested
alone [23].
3.6. Zinc and Lead

At higher lead doses in rats, supplemental zinc decreased the gastrointestinal
absorption of lead [27] and decreased blood, bone, liver, kidney, and spleen
Met. Ions Life Sci. 2011, 8, 247–262

concentrations of lead [16,27–30] in intermediate duration studies. Levels of

lead in brain were not affected [29,30], but the evidence in general suggests
that oral co-exposure to zinc at levels significantly above essentiality
decreases lead absorption and body burden at higher lead exposures.
Intermediate duration oral studies in animals report a protective effect of zinc
against inhibition of smooth muscle contractility by lead [31] and on pharyngeal
and laryngeal paralysis caused by lead in young horses [32], and no effect of
zinc on the inhibition of nerve conduction velocity in rabbits [33]. All were high
dose studies, and all have significant limitations in their design, reporting, or
relevance. Nevertheless, these studies do not provide evidence of potentiation,
but rather a protective effect, or no effect, of zinc on lead neurotoxicity.
4. INTERACTIONS OF METALS WITH OTHER

CHEMICALS
Provided below are binary interactions between metals and selected chemi-
cals or groups of chemicals. They represent exposures commonly encoun-
tered by humans in their environment.
4.1. Ethanol
Consumption of alcohol is common in human populations. The prevalence
rate for regular alcohol drinkers in the U.S. is 50% [34]. Thus, co-exposure
to ethanol and metals may play an important role in workers occupationally
exposed to metals.
4.1.1. Cadmium
When rats were administered cadmium together with ethanol, there was a
pronounced increase in cadmium accumulation in various regions of the
brain (e.g., the corpus striatum and cerebral cortex). The cadmium was not
bound to metallothionein, and there was a marked increase in lipid perox-
idation and inhibition of membrane-bound enzymes [35,36].
4.1.2. Lead
In animal studies, daily oral dose of lead (10 mg/kg) and ethanol (10%, v/v
in drinking water) administered for 8 weeks synergistically inhibited blood
ALAD activity, depressed dopamine and 5-hydroxytryptamine levels in rat
Met. Ions Life Sci. 2011, 8, 247–262

brain, increased lead burdens in tissue organs, and elevated blood zinc
protoporphyrin [37]. Similarly, combined exposure to ethanol and lead,
given as lead acetate for 12 weeks, induced changes in spontaneous and
evoked potentials in the brains of young rats [38,39].
4.1.3. Mercury
Ethanol potentiates the toxicity of methylmercury [40–42]. Studies in ani-

mals have shown increased mortality [41] and increased severity together
with decreased time to onset of neurotoxicity (hind-limb ataxia) [41,42],
when methylmercury exposure occurred simultaneously with ethanol
ingestion. Although increased mercury concentrations were observed in the
brain and kidneys, the changes in mercury content were insufficient to fully
explain the observed potentiation of toxicity [41]. The mechanism for the
enhancement of toxicity is unknown. Concomitant exposure to ethanol (5%,
v/v, in drinking water) and mercury (given as mercuric chloride by gavage)
induced changes in rats’ cortical activity [38].
The oxidation of ethanol with concurrent NADPH generation enhances the
reduction of the mercuric ion to metallic mercury, thereby making it more
favorable for permeating the placenta [43]. In summary, ethanol can cause
mercury to distribute more easily across the blood-brain barrier and the
placenta, thereby increasing the risk of mercury toxicity to the mature brain or
the developing neurological system of fetus. Indeed, the authors of one of the
studies concluded that co-exposure to heavy metals (e.g., lead or mercury) and
alcohol consumption may result in more severe neurotoxic outcomes [38].
4.2. Polychlorinated Biphenyls

Polychlorinated biphenyls (PCBs) are a category of chemicals that were
manufactured predominantly for use as coolants and lubricants in electrical
equipment such as capacitors and transformers due to their general chemical
inertness and heat stability. PCBs are complex mixtures of chlorinated
biphenyls that vary in the degree of chlorination. PCBs are persistent in the
environment and bioaccumulate in living organisms. Concerns exist
regarding joint toxic action of PCBs and other neurotoxicants such as
methylmercury via consumption of contaminated food (e.g., milk, fish).
4.2.1. Mercury
Experimental data suggest a possible synergism between PCB mixtures and

methylmercury (an organic form of mercury) in affecting neurological
Met. Ions Life Sci. 2011, 8, 247–262

function and development, but the data are not conclusive. Changes in
neurological function or development from PCBs and methylmercury have
been proposed to at least partly involve disruption of calcium homeostatic
mechanisms in neural cells leading to changes in neurotransmitter release
(e.g., dopamine) or cell damage. Combined in vitro exposure of rat brain
cells (striatal tissue) to a methylmercury and a 1:1 mixture of Aroclor 1254/
1260 appeared to synergistically deplete tissue levels of dopamine [44]. In
contrast, in a mouse study involving gestational and lactational exposure to
Kanechlor 500 and methylmercury, exposure to either agent alone or in
combination did not change several measures of F0- and F1-generation
reproductive performance, neurobehavior of offspring, or prevalence of
developmental anomalies [45].
4.3. Organophosphates
Organophosphate pesticides are widely used inside and outside of human
dwellings. They act on the neuronal membrane level causing changes in the
transport of ions that are reflected by a reduced rate at which depolarization
occurs. They are known to bind to acetylcholinesterase, inhibiting its ability
to hydrolyze the neurotransmitter acetylcholine. The resulting accumulation
of acetylcholine at the nerve endings causes continual neurological
stimulation.
4.3.1. Lead
The related phosphorothioate methyl chlorpyrifos and its oxon are hydro-
lyzed to non-cholinesterase-inhibiting compounds by lead in vitro at pHs in
the range of about 4.5–7.3 [46]. Other related phosphorothioates, methyl
parathion and ronnel, also are hydrolyzed to inactive compounds by lead in
vitro. This mechanism would be protective against the toxicity of organo-
phosphates in vivo. Experimental data in animals suggest the relevance of
this observation. For example, oral pretreatment of young adult rats for 3
months with lead in their drinking water, followed by a single oral dose of
methyl parathion or methyl paraoxon, resulted in increased urinary excre-
tion of a organophosphorus breakdown product that is inactive in choli-
nesterase inhibition [47]. The pretreatment also ameliorated the acute signs
of cholinesterase inhibition caused by the insecticides. In a rat neurodeve-
lopmental study of simultaneous oral exposure to lead (80 or 320 mg/kg) and
dimethoate (a phosphorodithioate) (7 or 28 mg/kg) in which the dams were
treated by gavage during gestation and lactation, followed by direct
Met. Ions Life Sci. 2011, 8, 247–262

treatment of the male offspring for 8 weeks, the joint toxic action of these
agents on electrocorticograms and evoked potentials appeared to be additive
or antagonistic [48]. The study design precludes more definitive conclusions;
there were no effects on brain cholinesterase or clinical signs. A study in rats
treated starting as young adults for 4–12 weeks with lead and dimethoate
reported similar results, with apparent antagonistic activity in the two data
examples provided [49].
4.4. Chelating Agents

Drugs such as penicillamine, trientine, ethylenediamine-N,N,N 0 ,N 0 -tetra-
cetate (EDTA), 2,3-dimercaptopropanol (British anti-Lewisite ¼ BAL) and
desferrioxamine are used for treatment of systemic toxic effects of metal
exposures or genetic disorders (e.g., Wilson’s disease). Metals mobilized by
the drugs in the body are subsequently excreted. Because of the difficulty
these drugs face in crossing the blood-brain barrier, the removal of metals
from the brain may not be effective. It should be noted, however, that BAL
is contraindicated for cases of elemental mercury or methylmercury poi-
soning because it has been demonstrated to increase the concentration of
mercury in the brain. It is beyond the scope of this chapter to describe all the
chelators in detail; other text books provide this information [50–52].
5. CONCLUSIONS
Metal mixtures are encountered on a daily basis in the environment. Health
assessments for exposed populations usually rely on the toxicity of indivi-
dual metals, but as discussed here, metal interactions in a mixture can
influence the toxicity of an individual metal. Consideration of organ and
system toxicity and the mechanisms of toxicity for individual metals within a
mixture are important to understand potential health impacts. Likewise, the
use of essential elements to mitigate health effects (e.g., zinc and iron on
lead) or the use of chelating agents which may at time be contraindicated due
to mobilization and concentration of metals in a target organ.
Aluminum, arsenic, cadmium, lead, manganese, and mercury have been
shown to affect the neurological system. In general, zinc and copper are
protective of the effects of lead. Zinc is in the active site of ALAD and can
play a protective role in lead intoxication by reversing the enzyme-inhibiting
effects of lead. Copper, as well as iron and calcium have been shown to
impede the gastrointestinal absorption of lead. Co-exposure of cadmium
Met. Ions Life Sci. 2011, 8, 247–262

and lead has been shown to result in an antagonistic response while a

potentiating interaction of lead on arsenic neurological effects has been
reported. Co-exposure of manganese and lead resulted in an increased
concentration and retention of lead in the brain of rats and adversely
affected cognitive function in an additive manner in these rats.
Some data also exists for interaction of metals with non-metals (e.g.,
ethanol, PCBs, organophosphate pesticides). Ethanol potentiates the effects
of methylmercury and cadmium and has been shown to synergistically
inhibit ALAD activity in animals co-exposed to lead. Lead hydrolyzes some
organophosphate pesticides to inactive compounds in vitro, and thus, may
afford protection in vivo. It has also been suggested that a synergistic rela-
tionship exists between methylmercury and PCBs on neurological function
and development.
ABBREVIATIONS

Aroclor trade name of PCBs, other trade names are Clophen,
Fenclor, Kaneclor, and Phenoclor
BAL 2,3-dimercaptopropanol (British anti-Lewisite)
cyclic GMP cyclic 3 0 ,5 0 -guanosine monophosphate
Kaneclor trade name for PCBs; see also Aroclor
NADPH nicotinamide adenine dinucleotide phosphate (reduced)
PCBs polychlorinated biphenyls with 2–10 chlorine atoms at
the biphenyl residues
PKC protein kinase C
RDA recommended daily allowance
REFERENCES
1. Textbook of Clinical Occupational and Environmental Medicine, Ed. L. Rosen-
stock and M. R. Cullen, W. B. Saunders Company, Philadelphia, 1994, pp. 729–
764.
2. H. R. Pohl, H. Abadin and J. F. Risher, in Neurodegenerative Diseases and Metal
Ions, Vol. 1 of Metal Ions in Life Sciences, Ed. A. Sigel, H. Sigel and R. K. O.
Sigel, John Wiley & Sons, Chichester, 2006, 395–425.
Met. Ions Life Sci. 2011, 8, 247–262

3. H. R. Pohl, S. Tarkowski, A. Buczynska, M. Fay and C. T. De Rosa, Environ.

4. ATSDR, Toxicological Profile for Aluminum, Agency for Toxic Substances and
8. ATSDR, Toxicological Profile for Manganese, Agency for Toxic Substances and
10. J. R. Nation, C. A. Grover, G. R. Bratto and J. A. Salinas, Neurotoxicol. Ter-
atol., 1990, 12, 99–104(1990).
11. J. R. Nation, G. D. Frye, J. von Stultz and G. R. Bratton, Behavior. Neurosci.,
1989, 103, 1108–1114.
12. C. Kies and S. W. Ip, Trace Subst. Environ. Health, 1990, 24, 177–184.
13. J. M. Gorell, C. C. Johnson, B. A. Rybicki, E. L. Peterson, G. X. Kortsha, G. G.
Brown and R. J. Richardson, Neurology, 1997, 48, 650–658.
14. J. M. Gorell, C. C. Johnson, B. A. Rybicki, E. L. Peterson, G. X. Kortsha, G. G.
Brown and R. J. Richardson, Neurotoxicolog, 1999, 20, 239–248.
16. S. J. S. Flora, V. K. Jain, J. R. Behari and S. K. Tandon, Toxicol. Lett., 1982, 13,
51–56.
17. S. J. S. Flora, R. A. Coulombe, R. P. Sharma and S. K. Tandon, Ecotoxicol.
Environ. Saf., 1989, 18, 75–82.
18. K. M. Malhotra, G. S. Shukla and S. V. Chandra, Arch. Toxicol., 1982, 49, 331–
336.
19. C. Moon, M. Marlowe, J. Stellern and J. Errera, J. Learn. Disabil., 1985, 18, 217–
221.
20. J. J. Mejı́a, F. Dı́az-Barriga, J. Calderón, C. Rios and M. E. Jimenez-Capdeville,
Neurotoxicol. Teratol., 1997, 19, 489–497.
21. S. Lal, S. K. Gupta and S. V. Chandra, Toxicol. Lett., 1980, 5, 203–206.
22. G. S. Shukla and S. V. Chandra, Toxicol. Lett., 1982, 10, 163–168.
23. S. V. Chandra, M. Mohd and D. K. Saxena, Arch. Toxicol., 1981, 49, 49–56.
24. S. V. Chandra, R. C. Murthy and D. K. Saxena, Ind. Health, 1983, 21, 273–279.
25. G. S. Shukla and S. V. Chandra, Arch. Environ. Contam. Toxicol., 1987, 16, 303–
310.
26. K. Kalia, S. V. Chandra and P. N. Viswanathan, Ind. Health, 1984, 22, 207–218.
28. F. L. Cerklewski, J. Nutr., 1979, 109, 1703–1709.
29. R. M. El-Gazzar, V. N. Finelli and J. Boiano, Toxicol. Lett., 1978, 1, 227–234.
30. S. J. S. Flora, S. Singh and S. K. Tandon, J. Int. Med. Res., 1989, 17, 68–75.
Met. Ions Life Sci. 2011, 8, 247–262

31. P. P. Vassilev, K. Venkova, N. Pencheva, R. Radomirov and D. Staneva-

Stoytcheva, Pharmacol. Toxicol., 1994, 75, 129–135.
32. R. A. Willoughby, E. MacDonald, B. J. McSherry and G. Brown, Can. J. Comp.
Med., 1972, 36, 348–359.
33. E. Hietanen, A. Aitio, U. Koivusaari, J. Kilpio, T. Nevalainen, M. Narhi, H.
Savolainen and H. Vainio, Toxicology, 1982, 25, 113–127.
34. Centers for Disease Control and Prevention (CDC), http://www.cdc.gov/nchs/
fastats/alcohol.htm, accessed Jan. 2010.
35. R. Pal, R. Nath and K. D. Gill, Neurochem. Int., 1993, 23, 451–458.
36. R. Pal, R. Nath and K. D. Gill, Pharmocol. Toxicol., 1993, 73, 209–214.
37. S. J. Flora and S. K. Tandon, Biochem. Pharmacol., 1987, 36, 537–541.
38. Z. Fazakas, Z. Lengyel and L. Nagymajtenyi, Arh. Hig. Rada Toksikol., 2005, 56,
249–256.
39. L. Pecze, A. Papp, L. Institoris, A. Szabo and L. Nagymajtenyi, Ecotoxicol.
Environ. Saf., 2005, 61, 139–144.
40. W. K. Rumbeiha, P. A. Gentry and M. K. Bhatnagar, Vet. Hum. Toxicol., 1992,
34, 21–25.
41. H. Tamashiro, M. Arakaki, H. Akagi, K. Murao, K. Hirayama and M. H.
Smolensky, J. Tox. Environ. Health, 1986, 18, 595–605.
42. C. J. Turner, M. K. Bhatnagar and S. Yamashiro, J. Tox. Environ. Health, 1981,
7, 665–668.
43. A. Khayat and L. Dencker, Int. J. Biol. Res. Pregnancy, 1982, 3, 38–46.
44. J. C. Bemis and R. F. Seegal, Environ. Health Perspect., 1999, 107, 879–885.
45. T. Tanimura, M. Ema and T. Kihara, in Neural and Behavioral Teratology, Ed.
T. V. N. Persuad, University Park Press, Baltimore, MD, 1980, pp. 163–198.
46. J. M. Smolen and A. T. Stone, Environ. Sci. Technol., 1997, 31, 1664–1673.
47. H. J. Hapke, S. Youssef and O. H. Omer, Dtsch. Tieraerztl. Wochenschr., 1978,
85, 441–444.
48. L. Nagymajtenyi, H. Schultz, A. Papp and I. Desi, Neurotoxicology, 1998, 19,
617–622.
49. L. Nagymajtenyi, I. Desi, A. Papp and T. Vezer, Cent. Eur. J. Public Health,
2000, 8, 64–65.
50. M. J. Ellenhorn, Medical Toxicology: Diagnosis and Treatment of Human Poi-
soning, 2nd edn., Williams and Wilkins, Baltimore, MD, 1997, pp. 1563–1579.
51. C. S. Homan, G. X. Brogan and R. S. Orava, in Emergency Toxicology, Ed. P.
Viccellio, Lippincott-Raven Publishers, Philadelphia, PA, 1998, pp. 363–378.
52. J. B. Leikin and F. P. Paloucek, Poisoning and Toxicology Handbook, 3rd edn.,
Lexi-Comp, Inc., Hudson, OH, 2002, pp. 725–731.
Met. Ions Life Sci. 2011, 8, 247–262

Met. Ions Life Sci. 2011, 8, 263–303
11
Metal Ions Affecting Reproduction and
Development
Pietro Apostoli and Simona Catalani
Department of Experimental and Applied Medicine, Unit of Occupational Medicine and
Industrial Hygiene, University of Brescia, P. le Spedali Civili, 1, I-25123 Brescia, Italy
<apostoli@med.unibs.it>
ABSTRACT 264
1. INTRODUCTION 265
2. TIME AND DURATION OF EXPOSURE 267
3. MECHANISMS OF ACTION 269
4. REPRODUCTIVE EFFECTS 270
4.1. Male 270
4.1.1. Arsenic 271
4.1.2. Cadmium 271
4.1.3. Chromium 271
4.1.4. Copper 272
4.1.5. Lead 272
4.1.6. Manganese 274
4.1.7. Mercury 275
4.1.8. Nickel 275
4.1.9. Vanadium 276
4.2. Female 276
4.2.1. Arsenic 277
4.2.2. Cadmium 278
4.2.3. Chromium 278
4.2.4. Lead 278
264 APOSTOLI and CATALANI
4.2.5. Mercury 279

4.3. Conception 279
5. ABORTIONS AND OTHER PREGNANCY EFFECTS 280
6. PRENATAL EXPOSURE AND DEVELOPMENTAL EFFECTS283
6.1. Arsenic 284
6.2. Cadmium 284
6.3. Chromium 285
6.4. Copper 285
6.5. Lead 286
6.6. Lithium 286
6.7. Mercury 286
6.8. Nickel 287
6.9. Vanadium 288
6.10. Other Metals 288
7. EARLY POSTNATAL EXPOSURE AND DEVELOPMENTAL
EFFECTS 288
7.1. Arsenic 289
7.2. Cadmium 290
7.3. Lead 290
7.4. Mercury 291
7.5. Manganese 292
8. CONCLUDING REMARKS AND NEEDS FOR FURTHER
RESEARCH 293
ABBREVIATIONS 294
REFERENCES 295
ABSTRACT: Many metal ions (lead, mercury, arsenic, cadmium, chromium, nickel,
vanadium, copper, lithium) exert a wide variety of adverse effects on reproduction and
development, including influence on male and female subfertility or fertility, abortions,
malformations, birth defects, and effects on the central nervous system. The effects pro-
duced by metal ions depend on several factors, such as timing and duration of expo-
sure, their distribution and accumulation in various organs (e.g., the nervous system),
and on the interference with specific developmental processes. Neonatal and early post-
natal periods are lifespan segments during which sensitivity to metals is high; e.g., lead
toxicity on the developing organism is paradigmatic of related well known and still
open questions. In more recent decades, important mechanisms of action have been
suggested: the endocrine disruption via impact of metal ions on reproductive hormones
and the oxidative stress. While experimental data provide clear evidence of effects of
many metals, human data are scant and traditionally limited to high levels of a few
metal ions, like lead on male fertility. Less documented are reproductive effects for
mercury, manganese, chromium, nickel, and arsenic for the same gender. More com-
plex is the demonstration of effects on female reproduction and on pregnancy. The
action of lead, arsenic, cadmium, chromium, and mercury may in fact be relevant in
several stages, beginning in fetal life, during early development or maturity, and is char-
acterized by subfertility, infertility, intrauterine growth retardation, spontaneous abor-
tions, malformations, birth defects, postnatal death, learning and behavior deficits, and
Met. Ions Life Sci. 2011, 8, 263–303

METAL IONS AFFECTING REPRODUCTION AND DEVELOPMENT 265
premature aging. Also, for females the evidences of specific aspects such as fertility or
abortions are usually higher and clearer from animal experiments than from human
studies.
KEYWORDS: abortions . developmental effects . female fertility . male fertility .

malformation . metal ions
1. INTRODUCTION
Metal ions exert a wide variety of adverse effects on reproduction and
development either directly, at relatively low doses, or indirectly through
systemic toxicity, generally at higher doses. Effects also depend on other
factors, such as time and duration of exposure, distribution or accumulation
in various organs, like the nervous system. Interactions have been docu-
mented between genetic polymorphisms, maternal ability to detoxify and
excrete xenobiotics, and fetal susceptibility to teratogenic metals. The
identification and quantification of adverse effects of metals on human
reproduction and development are affected by many open questions: What
is the exact number of metals involved? What are their sources? What are the
adverse events to be investigated? And first of all what are the mechanisms
of action and damage? This led to a scarcity of information about quanti-
tative dose-response relationships and about no-adverse-effect exposure
thresholds, the two basic parameters for assessing the toxicity and preventive
action for xenobiotics. Furthermore, published studies mostly consider
single metal ions, while environmental and occupational conditions are
characterized by combined exposure to elements and other xenobiotics. The
possible cumulative or additive effects appear to be a key point not only
from a theoretical but also from a practical point of view. Other problems
may arise from the complexity of effects under study and from gender diffe-
rences. Clinical and epidemiological findings related to metal-induced effects
on female reproduction may be influenced by age, ovarian reserve, hormonal
imbalance, male cofactors, and sexually transmitted diseases. Thus, the
environmental or occupational factors interact with a wide, complex, mul-
tiple phase process and it might be difficult, for example, to distinguish the
occupational causes of spontaneous abortion or congenital malformations
from other risk factors.
A further question addresses the possibility to extrapolate the results from
animal studies to humans, since there are structural and functional differ-
ences between the species and the mechanisms of adverse effects are seldom
known. So it appears to be difficult to transfer experimental fetal anomalies
due to metal ions during organogenesis or embryo or fetal lethality or other
Met. Ions Life Sci. 2011, 8, 263–303

developmental effects after exposure during other periods of pregnancy

or development [1]. Teratogenetic bioassays in rodents have yielded
positive results for many metal compounds producing fetal and early
postnatal deaths, as well as malformations such as anencephaly, eye defects,
cleft palate, and skeletal anomalies [2], but they were not confirmed in
humans.
Another introductory remark concerns the gender differences in kinetics,
action, and susceptibility to toxic elements [3]. Toxicokinetics may involve
physical constitution (size, fat), physiology, and metabolizing enzymes.
Lifestyle factors such as working environment, smoking, diet, physical
activity, and cosmetics, as well as stress factors can also have effects [4].
There is increasing evidence for estrogenic effects of cadmium both in vitro
and in vivo, mediated through the androgen receptor [5]. More recent
experimental studies show arsenic-related suppression of spermatogenesis,
probably by affecting pituitary gonadotropins and inhibiting androgen
production and these effects are observed at high arsenic exposure, indi-
cating that arsenic acts as an estrogen [6]. In utero exposure to lead resulted
in more severe immunotoxicity in females than males [7]. On the contrary, an
example of the higher susceptibility of males to methylmercury is the decline
in fertility, female birth ratio, and higher fetal death [8]. A gender difference
in the methylation of inorganic arsenic has been observed, with higher rates
in females [3]. Animal studies are, however, difficult to be interpreted with
regard to the potential risk for humans, starting from exposure conditions
and dose levels, metabolic pathways, targets, and action mechanisms [1].
The occupational and environmental exposures are generally coexposures
to many metal ions and or to other organic compounds; at low levels they
are irregular and the results are difficult to be measured. The effects of
coexposures on the female reproductive function in mice was studied by
Belles, Albina, and Sanchez [9], focusing on the developmental toxicity of
lead nitrate, methylmercury chloride, and sodium arsenite. Maternal toxic
effects were more remarkable in the group concurrently exposed to lead,
mercury, and arsenic than in those given binary combinations of the ele-
ments and in those given singly the metal. These data suggest that at current
doses, the interactive effects of lead and arsenic on mercury-induced devel-
opmental toxicity were not greater than the additive ones. In contrast,
exposure of pregnant mice to lead and arsenic at doses that were practically
nontoxic to dams, but administered concurrently with organic mercury at a
toxic dose, caused additive interactions in maternal toxicity. A synergistic
effect of lead and cadmium on testicular injury in rats has been reported [10].
A protective effect against male reproductive toxicity of cadmium was
observed on the other hand in animals treated with zinc, selenium or with
sulfhydryl containing compounds such as British anti-lewisite (BAL),
cysteine, glutathione, and metallothionein [11].
Met. Ions Life Sci. 2011, 8, 263–303

A classic example of occupational coexposure are welding fumes con-

taining a wide range of biologically active substances such as hexavalent
chromium, nitrous oxide, nickel, ozone, copper, and lead, some of which are
known to be teratogenic and embryotoxic [12]. Inhaled welding fumes were
demonstrated to be relevant for adverse pregnancy outcomes, both from
maternal and paternal exposures [13], due to direct genetic or epigenetic
effects on the germ cell or indirect transmission to the mother via seminal
fluid [14]. Nampoothiri and Gupta [15] observed that coexposure of lead and
cadmium has deleterious effects on ovarian cells and that both in vivo and
in vitro, the decrease in progesterone levels is due to reduced gonadotropin
receptors in granulose cells.
Finally, a relevant role has to be assigned to the perception and evaluation
of reproductive pathology deeply conditioned by cultural, religious, histori-
cal, economical, and social aspects. Abortion or malformations are
generally more easily identified and considered, while effects on male fertility
may be less obvious. In some countries the natural male fertility is well
accepted, in others firmly opposed. In industrialized areas there is a diffuse
tendency to delay parenthood, thus lengthening the preconception period
for potential exposure, with possible effects on both male or female repro-
ductive ability, as well as increasing prenatal and early postnatal cumulative
exposure to toxic metal ions. The perception of exposure too may be dif-
ferent. While a reduction of exposure in working settings, resulting from
technological improvement and preventive measures, is generally recog-
nized, the dispersion of metal ions in the general environment (from traffic,
energy production plants, incinerators) even from low to very low doses, is
perceived as an important constantly increasing public health problem in
industrialized countries and by particular groups of the general population.
The possible role of environmental versus lifestyle factors in determining
sperm number and viability is widely debated. To correctly identify the
factors contributing to the deterioration of human fertility and to prevent its
possible decline, appear therefore very important [16].
2. TIME AND DURATION OF EXPOSURE

The exposure to metal ions should be evaluated as closely as possible to the
‘‘target time’’, i.e., the period during which the metal is toxicologically most
active on the reproductive system. The target time may be specific for each
animal species, organ or tissue, kind of effect, and route of exposure. The
linkage of exposure assessing to effects is often difficult because of multiple
exposures, the latency of effects, and the subtle nature of some outcomes
[17]. The time at which exposure occurs becomes therefore a critical factor.
Met. Ions Life Sci. 2011, 8, 263–303

Temporary infertility can occur in an adult woman following damage to

the ovaries, whereas exposure during childhood might induce sterility by
chemically-induced destruction of germ cells. Furthermore, exposures
in utero may cause improper development of ovarian follicles or permanent
alterations in the reproductive apparatus.
The male reproductive system has sensitive windows for toxic exposure
both during development and adult life. Exposure in utero or during child-
hood may lead later to reproductive failure. Exposure in adulthood may
have direct effects on gamete production, spermiation, maturation of sperm
in the epididymis, ejaculation, accessory organs, seminal fluid, and the testis,
and subsequently hormones and sexual performance. In addition, exposure
to the adult male may affect offspring through exposing the mother and
fetus to contaminated or altered semen.
In an ideal situation, the risk assessment procedure combines information
from adequate indicators of exposure with that from sensitive and effect-
specific indicators. Indicators of exposure are frequently based upon blood
or urine concentrations, not always reflecting the active metal dose. How-
ever, it is difficult to correlate effect indicators with dose indicators to
enable, as above mentioned, a more adequate risk assessment. For this
purpose we can recall the time to pregnancy (TTP) and the sperm chromatin
structure assay (SCSA).
TTP is defined as the number of months of unprotected intercourse that
elapse before conception occurs. It is a composite measure that includes
libido, ovulation, sperm or semen quality, and conceptus survival [18].
Specific examples are given in Section 5.
The SCSA is a measure of the abnormal chromatin of the spermatozoa,
defined as increased susceptibility to acid-induced denaturation in situ and
quantified by flow cytometric measurement of denaturated DNA and native
DNA [19].
Another indicator is the socalled fetoplacental unit considered as a mea-
sure for cadmium toxicity mainly during the third trimester of gestation in
rodents [15]. Following the time of exposure we must consider adaptive
mechanisms like the induction of placental metallothionein or inhibition by
metal ions after binding to sulfhydryl groups, as cadmium with cysteine
residues. Chromium(VI) was transferred readily to a fetal mouse, while there
was little transfer of chromium(III). Later in gestation, both chromium(VI)
and chromium(III) accumulated in calcified areas of the fetal skeleton [20].
Chromium trioxide (at a dose that produced maternal toxicity) caused fetal
death and increased the incidence of cleft palate and skeletal anomalies in
hamsters on day 7–9 of pregnancy, while administrated on day 10–11 it had
minor effects [21].
The toxicological response to toxic effects of certain metal ions depends
on the development at different time of receptors. Epstein and coworkers
Met. Ions Life Sci. 2011, 8, 263–303

[22] studying mice exposed to lead at pre-mating, pre- and postnatally,

observed in pre- and postnatally exposed mice lower brain weight and a
decreased DNA amount per brain, but there was no effect on proteins/brain.
In contrast, pre-mating lead exposure significantly increased brain weight
and protein and lowered DNA per brain.
The time of exposure was investigated by Ronis, Badger, and Shema [23]
with rats exposed to lead acetate, in utero, pre-pubertally or post-pubertally.
The most severe effects were observed in the in utero exposed group, with
delayed vaginal opening and disrupted estrous cycling. These effects suggest
lead actions on the hypothalamic pituitary axis and directly on gonadal
steroid biosynthesis. The major lesson from the elegant study by Gulson et
al. [24] was that it is not only a female’s exposure to lead during pregnancy
that are germane in determining risk to her or to the fetus, but also her past
exposure, represented by the lead stored in deep pools.
3. MECHANISMS OF ACTION
The mechanisms of action involved in the reproductive effects of metal ions
can be divided into two groups, those occurring directly to tissues and
organs of the reproductive system, and those occurring indirectly to the
connected endocrine system.
Elements such as cadmium, mercury, arsenic, lead, manganese, and zinc
affect the endocrine system, producing alterations in several physiological
functions. The endocrine disrupters (EDs) hypothesis implies that low-level
exposure to certain chemicals may contribute to end points such as lowering
of age at menarche, impairment of semen quantity and quality, decreasing
male-to-female sex ratio at birth, increasing rates of hypospadias and
testicular cancer, infertility, spontaneous abortions, and structural and
functional congenital malformations [7]. Some of these adverse health effects
are common to various metal ions, such as the stimulation of progesterone
synthesis produced by cadmium and mercury, or negative effects on sperma-
togenesis produced by arsenic, mercury or lead. The effects and mechanism
of action of metal ions such as endocrine disrupters are discussed in detail in
Chapter 12.
The action of metal ions on sperm viability may be due to an increase in
reactive oxygen species (ROS) and a decrease in the cell antioxidant defence
[25]. Another possible mechanism involves DNA protamine binding. In
mammalian spermatozoa, DNA is tightly packed with protamines in the
nucleus, since lead replaces zinc ions bound to nuclear protamines, impairing
chromatin decondensation during fertilization [26]. It was in addition sug-
gested that lead, at current environmental levels, strongly interferes with the
Met. Ions Life Sci. 2011, 8, 263–303

sperm acrosome reaction, essential for fertilization and may affect the out-
comes of artificial insemination [27].
Teratogenesis, which affects the embryo and fetus, results in malforma-
tions and other adverse responses, although the mechanisms appear to be
uncertain, particularly at the molecular level. Among these the electron
transfer (ET) functionality has been studied [28]. Mechanisms of damage
during development include proliferation (cell division), cell death, cellular
differentiation, biosynthesis, cell-cell or tissue-tissue interactions, and cel-
lular movements. Damage may be related to these kinds of epigenetic injury
or to a direct genetic action on developing tissues [29].
For most metal ions, there is little information about quantitative dose-
response relationships and no-adverse-effect exposure thresholds. The dose-
response trend(s) are based on group basis and there is inadequate evidence
for establishing a quantitative dose-response curve and no-adverse-effect
exposure threshold. In a review on the impact of lead on reproduction, the
risk ratio for infertility increased with increasing levels of lead in blood
(PbB), varying from 1.27 to 1.90, when PbB passed from 100 to Z510 mg/L,
however, without any effect when at least one pregnancy was achieved [30].
Scanty is evidence of cadmium-related effects on semen quality, sex hor-
mones or fertility in human males, allowing a reliable estimate of quanti-
tative dose-response relationship(s) or no-adverse-effect exposure
threshold(s) [31]. Studies in animals showed decreased testes weight and
histopathological changes increasing in severity with an increased dose of
potassium dichromate in drinking water [32].
4. REPRODUCTIVE EFFECTS
4.1. Male
Many evidences indicate that the human male reproductive capacity has
deteriorated during the last five decades in many industrialized countries.
About 16% of couples suffer from fertility problems, half of those may be
due to male factors and in vitro fertilization or intracytoplasmic sperm
injection is therefore increasingly sought [33]. Mammalian male reproduc-
tive function can be affected through a direct effect on the testes and/or
indirectly through the neuroendocrine system and results include altered
genetic material, altered spermatogenesis, pregnancy loss or genetic diseases
in the offspring. Common end points for assessment of male reproductive
function include size of testis, semen quality and motility, secretory function
of the prostate and seminal vesicles, reproductive endocrine function,
impotence or reduced libido, and fertility (Table 1).
Met. Ions Life Sci. 2011, 8, 263–303

Table 1. Main effects of metal ions on sperm production.

As Inhibition of spermatogenesis
Cd Disrupt Sertoli cells; sperm viability; hypoactivated motility
Cr Morphological and functional alterations; sperm death and reduced motility
Cu Abnormal sperms; reduced sperm motility and testis weight
Hg Reduction in sperm motility and sperm count
Mn Stimulation of spermatogenesis; reduction in sperm count and motility
Ni Morphologically abnormal sperm; decrease in sperm count and motility
Pb Morphological and functional alterations of sperm and sperm count
4.1.1. Arsenic
Long-term oral exposure of mice to sodium arsenite in drinking water, in a

dose similar to human exposure through drinking water, resulted in signifi-
cant accumulation of arsenic in the mouse testes, epididymis, seminal vesi-
cles and prostate gland, a decrease in the absolute and relative testicular
weight but not of epididymal and accessory sex organ weights, in a decrease
in sperm count, motility, and morphology, or in changes in testicular
enzymes [34]. Changing arsenic species (MMA or DMA 76 mg/kg/day for at
least 14 weeks) no histological alterations in male reproductive tissues and
no alterations in sperm parameters were observed in male rats [35].
4.1.2. Cadmium
The testis is very sensitive to cadmium and one of the possible explanations
is the characteristic of the blood testis barrier [36].
Traditional studies (large doses, administered by injection) recognized that
cadmium could induce deep and irreversible injury to mammalian testes. It is
characterized by disruption of endothelial cells of microvessels, edema, and
hemorrhage, apparently as a result of a primary disruption in the vascular system.
Cadmium may disrupt Sertoli cell tight-junction-barrier function not only by
decreasing the synthesis and/or expression of proteins, but also by promoting
protein redistribution at the Sertoli-Sertoli cell interface [37,38]. The main-
tenance of acrosome is essential to the functional integrity of sperm and for
response to the appropriate signals of oocytes. In infertile men, increasing serum
cadmium levels were significantly associated with abnormal sperm morphology
and decreased sperm counts, sperm motility, and sperm viability [39].
4.1.3. Chromium
Chronic chromium exposure induces a reversible oxidative stress in the

seminal plasma and sperm, leading to sperm death and reduced motility of
Met. Ions Life Sci. 2011, 8, 263–303

live sperm [40]. Histopathological changes increased in severity with dose.

Mice showed significantly decreased sperm counts and markedly increased
rates of sperm abnormality when treated by a single intraperitoneal dose
(1 mg/kg) of chromic acid: these changes are associated with an increase in
the level of lipid peroxidation and H2O2 [41]. In workers exposed to chro-
mium(VI) for 1–15 years in an electroplating factory [42], significantly
decreased sperm concentration and sperm motility were found. Another
study of male welders, who had a very high blood chromium level
(131.0 52.6 mg/L), showed significantly decreased sperm concentration,
with an inverse correlation between sperm concentration and blood chro-
mium levels [43].
4.1.4. Copper
Rats inhaling CuCl2 had increased incidence of abnormal sperms, reduced
sperm motility and testis weight, but also a reduction in testosterone levels.
Similar results were found after intraperitoneal exposure to rats [44]. Incu-
bation of human spermatozoa with metallic copper resulted in a significant
fall in the percentage of motile sperms [45].
4.1.5. Lead
The assessment of a critical dose for effects on male fertility may be carried
out by measuring the metal concentration in different matrices, usually in
blood. It would be important however to measure the metal nearest the
critical organ tissue. In our experience the lead concentrations in blood,
seminal plasma, and spermatozoa were well correlated (Figure 1).
Effects on human semen by lead have been reported for a long time. They
consist of a decrease in semen volume, a decrease in sperm concentration
and sperm count, a decrease in sperm motility and in the quality of motility,
an increase in abnormal sperm morphology particularly at the head of the
sperm, and impairment of prostate secretory function as indicated by
decreased seminal plasma zinc levels [47,48].
These findings indicate that lead can act directly on the testis, reducing
sperm number, causing peritubular testicular fibrosis, lowering testosterone
synthesis, and disrupting regulation of the luteinizing hormone (LH) [49,50].
Several studies in rats and other rodents indicate that blood lead con-
centrations above 300–400 mg/L are associated with impairment of sper-
matogenesis and reduced concentrations of androgens — although some rat
species and strains seem quite resistant [16].
Met. Ions Life Sci. 2011, 8, 263–303

Figure 1. Scatter plots of the concentration of lead in seminal plasma and spermatozoa
(top panel), spermatozoa and blood (center panel), and seminal plasma and blood
(bottom panel) in a subset of 165 industry workers. All correlations are highly significant.
(Note: 10 mg/dl ¼ 100 mg/L.) Reproduced by permission from [46], copyright (2002).
Met. Ions Life Sci. 2011, 8, 263–303

In more recent years it has been shown that lead may interfere with the
reorganisation and tight packaging of sperm DNA during spermatogenesis —
the chromatin condensation — by competition with zinc on protamine
binding sites. This results in reduced stability of chromatin, an abnormal
structure of which is strongly related to reduced fertility in humans. Indeed,
there is limited evidence that in humans the chromatin structure abnormal-
ities are related to a lower range of blood lead or directly with higher lead
levels within spermatozoa [46].
Historical findings reported that high exposure to lead produced a
decrease of sperm motility in men. The possible mechanisms outlined in a
study of Kasperczyk et al. [25] result from increasing lipid peroxidation in
seminal plasma, as demonstrated by malondialdehyde levels, mainly when
the blood lead concentration exceeded 400 mg/L.
The results of an international study of semen quality in industrial workers
exposed to lead showed that the current exposure in the United Kingdom,
Belgium, and Italy (Project Asclepios) did not demonstrate a high risk to
male fertility at the current occupational exposure levels: adverse effects on
sperm concentration and susceptibility to acid-induced denaturation of sperm
chromatin are unlikely at blood lead concentrations below 450 mg/L [46].
Other data indicate that lead can adversely affect human semen quality even
at blood lead levels o150 mg/L [51,52]. In a study of 123 men who had never
been occupationally exposed to metals, the median (range) blood lead values
were 57 (25–149) mg/L. An increase in blood lead was significantly associated
with decreasing percentages of normal and subnormal sperm, and with
increasing percentages of slow sperm and overly wide sperm. In another study
[27], the seminal plasma lead levels of subjects not occupationally exposed to
lead were found to inversely correlate with the fertilizing capacity of sperm
(sperm acrosome reaction) and the fertilization rate when using in vitro fer-
tilization (IVF), but also with seminal plasma zinc levels. Taken together,
these studies suggest that lead may significantly reduce human semen quality
even at low-level lead exposure, currently common for general populations
worldwide. Some considerations in this regard will be discussed in Section 4.3.
4.1.6. Manganese
The effects of manganese on sperm production are controversial. Experi-
mental data demonstrate that while no significant effects were observed at a
10 mg/kg dose, at a 25 mg/kg dose an increase of LH, FSH, and testosterone
was observed [53]. Lee et al. [54] reported a higher efficiency of spermato-
genesis with an increase of the above mentioned hormones. On the contrary,
in mice orally exposed to manganese acetate a significant decrease in sperm
count and motility was observed, without any effect on fertility [55].
Met. Ions Life Sci. 2011, 8, 263–303

Manganism in humans is commonly accompanied with impotence or

reduced libido, indicating that possible effects on male fertility were, at least
in part, of nervous origin.
4.1.7. Mercury
Many studies documented adverse effects of inorganic mercury and

methylmercury on male fertility, but at concentrations of methylmercury
two- or threefold higher in the testes, Rao and Sharma [56] observed a 20–
30% decrease in testes and cauda epididymides weight, sperm from cauda
reduced in number, viability, and motility, and an altered antioxidant and
steroid metabolizing activity. Studies on monkeys confirmed the effects on
sperm motility and morphology [57] without significant depression of tes-
tosterone or histopathological abnormalities. In rats receiving methylmer-
cury from diet, a decrease of intratesticular testosterone and epididymal
sperm count was found in the high-dose group, but inverse correlation
between fertility and testicular mercury concentration has been found [58].
In mice given drinking water with 4 ppm of mercuric chloride, degenerative
testicular changes, decreased testicular weight, and sperm count were
demonstrated [59]. Cauda epididymal sperm count/motility decreased sig-
nificantly in the mercury-treated rats and qualitative examination of testi-
cular sections revealed a reduction of mature luminal spermatozoa [60].
In workers exposed to mercury, the sex hormone binding globulin level in
serum inversely correlated with exposure duration, whereas no correlation
was observed for serum levels of FSH, LH, and testosterone with respect to
exposure duration or mercury levels in blood and urine [61]. A significant
positive correlation between serum total testosterone (but not its free frac-
tion) and cumulative mercury exposure was found in workers exposed to
mercury vapor for an average of 10 years [62]. No effect on fertility as
assessed by the rate of live births was observed in male workers chronically
exposed to mercury vapor (urinary mercury levels of 5–271 mg/g creatinine)
[63]. In men with suspected infertility, no significant association was found
between semen quality and mercury levels in blood [64], urine or ejaculate
[65]. On the contrary, an increased risk of subfertility resulted from dose-
dependent levels of mercury in hair [66].
4.1.8. Nickel
Animal studies showed adverse effects on the male reproduction following

exposure to nickel sulfate, nickel chloride, or nickel nitrate, including his-
tological alterations in the epididymis and seminal vesicles, decreases in
Met. Ions Life Sci. 2011, 8, 263–303

sperm concentration, motility, and abnormalities. Morphological sperm

head abnormalities and decreased fertility in mice have also been demon-
strated [67]. Other effects include decreased absolute and relative weights of
testes, epididymides, seminal vesicles and prostate gland, decreased sperm
count and motility, and increased abnormal sperm morphology in mice [68].
In a study carried out on a group of welders (with a blood nickel level of
123.3 35.2 mg/L) a significant correlation between the percentage of mor-
phologically abnormal sperm with sperm tail defects and the blood nickel
levels have been observed [43].
4.1.9. Vanadium
Vanadium has been recognized for some decades to adversely affect the male
reproduction of humans and animals through disturbance of spermatogen-
esis, function of spermatozoa (reduced motility and increased number of
dead spermatozoa), and by morphologic disturbances in the seminiferous
epithelium [69].
The results of in vitro and in vivo studies by Chandra et al. [70] demonstrate
that vanadium treatment resulted in a significant dose- and time-dependent
increase in the testicular lipid peroxidation, marked inhibition in the level of
superoxide dismutase and catalase activities, and decreased sperm counts.
4.2. Female
As seen for effects on male reproduction the adverse effects on female fer-
tility is usually limited to animal data and the human studies to pregnancy
loss or alteration [71]. The adverse effects of metal ions on female repro-
duction may arise from their action in different stages (from fetal life to
puberty to pregnancy) and with different entity (from subfertility to infer-
tility, from intrauterine growth retardation to spontaneous abortions, from
malformations, birth defects, postnatal death to behavior deficits).
The pregnancy loss is the end point most frequently used to monitor
effects on female reproductive function, starting from early losses, which
contain a large proportion of chromosomal abnormalities and may repre-
sent 35–40% of human pregnancies. The remaining 10–15% later abortions
are clinically manifest and some have been linked to environmental factors
[72]. Another critical point is the adequate measure of exposure/dose. Using
lead as an example, Ernhart and Greene [73] pointed out some of these
critical aspects such as sampling time and the matrix to be used for dose
determination (maternal or fetal blood, cord blood, or placenta). To better
Met. Ions Life Sci. 2011, 8, 263–303

Table 2. Targeting sites and effects of metal ions on the reproductive function of
females.
Cd Pb Hg Cr As
Ovarian accumulation + + + +
Animal effects:
Oocytes loss + + + – –
Follicular atresia + + + – –
kSteroidogenesis + + + – –
Ovarian cancer – – – – +
Outcome in women:
Disrupted menses + – + + +
Infertility + + – + –
Early menopause – + – + –
k indicates that metal ions with the sign + cause a reduction in steroidogenesis.
assess early exposure to metal ions and the trend of absorption through
pregnancy, at least one sample should be taken during the first trimester and
another during the last 6 weeks of the pregnancy. This will facilitate the
assessment of effects on the various stages of development and organogenesis.
Important information in addition should derive from a combined exam-
ination of metals in cord blood and other matrices such as placenta, mainly to
determine body burden in fetus and accumulation in absorption phase.
Limited is the knowledge about pathogenetic mechanisms, usually clas-
sified as direct, when metal ions interact with specific reproductive target
organs or indirect, when metals interfere with endocrine or other regulatory
systems. The ovaries and ova are susceptible to direct damage by metal ions
for an extended period of time, from meiosis through ovulation. The
potential for disruption of ovarian function by trace metals such as cad-
mium, lead, mercury, chromium, and arsenic is summarized in Table 2.
4.2.1. Arsenic
Arsenic accumulation in the ovary was shown in rats exposed for 28 days to
sodium arsenite in drinking water. They demonstrated reduced ovarian
weights and prolonged estrous cyclicity: these adverse ovarian effects could
be prevented by administration of L-ascorbate or sodium selenite [74].
Transplacental exposure to high levels of arsenic induces ovarian epithe-
lial tumors in mice [75]: interestingly, the ovarian tumors were accompanied
by hyperplasia of the uterus and oviduct, likely via the hypothalamic-
pituitary system.
Met. Ions Life Sci. 2011, 8, 263–303

Reports from the Ukraine, Taiwan, and Bangladesh have linked arsenic-
contaminated drinking water to reproductive disorders in women [76].
4.2.2. Cadmium
Cadmium is the most extensively studied metal with respect to effects on

ovarian function, and many studies demonstrated that it is the target site of
accumulation of the metal, leading to direct adverse effects of reproductive
function [77]. Hemorrhagic necrosis has been demonstrated in hamster
ovaries following injection of cadmium chloride at about 5 mg/kg [78].
Cadmium given prior to mating may lead to dose-dependent sterility, due to
reversible pituitary dysfunction. However, animals may develop tolerance
and remain fertile, with normal fetal outcome and development [79].
The result of exposure in humans was in some studies estimated by
measuring cadmium in ovarian follicular fluid of women undergoing assisted
reproductive technologies to treat infertility. In one study cadmium was
higher in follicular fluid in women who were smokers versus non-smokers
[80]. It was suggested that this accumulation might affect the quality of
oocytes. The results of two studies in which the cadmium levels in follicular
fluid were measured did not support, however, the conclusion about the role
of the metal in fertility reduction [81].
4.2.3. Chromium
A number of reproductive effects (reduction of follicles, of ova/mice,

increased estrous cycle duration, and histological alterations) have been
described in mice exposed to potassium dichromate in drinking water with
an apparent dose relation [82]. A dose-dependent reduction in all stages of
preantral follicles (primordial, primary, and growing) and the number of
oocytes ovulated, as well as a lengthening of the estrous cycle has also been
reported [83]. The data suggest that high levels of chromium might disrupt
menstrual cyclicity, ovulation, and fertility and may potentially cause early
menopause.
4.2.4. Lead
Morphological evidence of direct ovarian effects of lead exposure in mice

has been described and resulted in distraction of small preantral follicles and
Met. Ions Life Sci. 2011, 8, 263–303

increased atresia in ovaries [84]. Furthermore, transplacental exposure to

lead resulted in a reduced number of primordial follicles in female pups.
Lead effects reported in human pregnancies relate mostly to miscarriages,
premature delivery, infant mortality and suggest direct toxicities of placental
or fetal development rather than ovarian effects [85].
Reports related to ovarian lead effects are limited, like the study which
compared lead content in follicular fluid with in vitro effects of lead on
granulose cells collected from women undergoing in vitro fertilization and
embryo transfer. It showed that the lead measured in ovarian follicular fluid
does not appear to be related to progesterone secretion by the ovary [79].
Hu et al. [86] studied pregnancies of women exposed to lead during their
childhood between 1930 and 1944. Among these a proportion of about 30%
of pregnancies ending in spontaneous abortion or stillbirth and 15% among
controls was observed.
Since lead is stored in bone for decades it was hypothesized that de-
mineralization of the skeleton might occur during pregnancy.
4.2.5. Mercury
After chronic ingestion of mercuric chloride, the element was detected within
ovarian follicles and in the corpora lutea of rats, with prolonged estrous
cycles and decreased ovulation [87]. Watanabe and coworkers [88] reported
similar decreases in ovulation in golden hamsters injected with mercury, but
was unable to observe alterations in ovulation for animals treated with the
same amounts of methylmercury chloride. For Heath et al. [89] mercuric
chloride may have disruptive effects in the corpora lutea in rats, while in
hamsters and mice treated with mercuric chloride ovulation of oocytes was
decreased and degenerated without chromosomal aberrations [90].
Earlier studies had noted menstrual cycle changes in women who were
exposed to higher levels of mercury vapor in the workplace [91].
4.3. Conception
Female and male fertility alterations might be considered together at con-
ception time: it is in fact very difficult to exactly identify and examine the
specific component of each of the two genders during this time. Animal
studies demonstrated that paternal exposure can increase the rate of
reproductive pathology, like spontaneous abortions. In humans, however,
this relationship remains unclear, in particular whether paternal exposure
can cause adverse pregnancy outcomes through a direct genetic or epigenetic
Met. Ions Life Sci. 2011, 8, 263–303

effect on the germ cell or indirectly by transmission of metal to the mother

via seminal fluid [14].
Some studies on lead workers indicated that paternal blood lead levels
around 300–400 mg/L may be considered a sort of threshold for increasing
abortions [92], reduced rate of live births [93], and prolonged time to preg-
nancy (TTP) [94]. The odds ratio for fetal loss and congenital malformations
with paternal lead exposure may be increased [95].
A study of 251 Italian men did not find an association between lead
exposure in men and delayed TTP in their wives [96]. The data reported were
included in a larger cross-sectional study of TTP, conducted in four Eur-
opean countries, of 1104 male workers, 638 of whom were exposed to lead
[97]. The mean blood lead level of exposed workers was approximately
300 mg/L in each country. An internal control group was assembled, con-
sisting of lead workers for whom the timing of exposure could not have
affected fertility. Only pregnancies that concluded in live births were inclu-
ded in the analyses. No consistent increase in TTP was found in association
with the blood lead level.
A study from Taiwan [98] showed a clear dose-response trend of pro-
longed time to pregnancy with respect to increasing paternal blood lead
levels; the fecundability ratios (inversely related to TTP) were 0.90, 0.72,
0.52, and 0.40 for blood lead categories o200, 200–290, 300–390, and
Z400 mg/L, respectively, as compared to unexposed men, indicating that
even blood lead levels o300 mg/L may prolong TTP.
In an editorial one year later [99], starting from these conclusions, the
discrepancies between the Taiwan study and European collaborative studies
of the Asclepios project were underlined, and furthermore, in conflict with
Shiau et al. [98], the TTP evidences with the semen studies, the semen
characteristics being the more sensitive parameters of male fecundity. In
addition, there is no evidence of chromatin condensation during sperma-
togenesis (considered as a sensitive effect indicator of metal action) at lead
concentrations lower than 300–400 mg/L. The editorial has concluded that
since the Shiau et al. study does not present sufficient data to modify the
main conclusion from the European studies on the lead effect on male fer-
tility, there is definitely a need to continue this line of research.
5. ABORTIONS AND OTHER PREGNANCY EFFECTS
Abortion is a traditional effect related to exposure to xenobiotics and to

some metal ions, lead and mercury in particular. The literature on high lead
exposure and abortion provides consistent evidence, starting with historical
surveys carried out in Milan by Torelli [100]. In this study, spontaneous
Met. Ions Life Sci. 2011, 8, 263–303

abortions were reported at a relative risk of 5.3, and an infant mortality

more than double compared with the Italian national rate at that time. In
more recent time and with a dose decrease, data available from epidemio-
logical studies and mechanisms remain unclear. A direct teratogenic effect
on the fetus or interference with the maternal/fetal hormones are possible,
combined with vascular effects on the placenta or elevated blood pressure
during pregnancy.
Most of the studies have been biased because of small sample size, poor
ascertainment of outcome, lack of control of confounding factors, and poor
exposure assessment, but for some researchers also when they were ade-
quately accounted for, the association between abortion risk and PbB
remains [101].
Falcon et al. [102] assessed this risk by measuring the relationship between
placental lead concentration and outcomes of pregnancy: higher placental
lead levels correspond to premature rupture of membranes and preterm
pregnancies (40.6% for lead in placenta values above 120 ng/g versus 8.8%
in control placentas). Sallmén et al. [103] summarized the epidemiological
studies on the possible impact of parental occupational exposure to lead or
other metals on spontaneous abortion and stated that no clear conclusion
could be reached.
Several studies have examined reproductive function in populations living
in Bangladesh or India exposed to high levels of arsenic in drinking water
and found increases in spontaneous abortions/stillbirths or preterm births
[104]. Similar results were reported in a study of 533 women from Bangla-
desh by Milton et al. [105] who found a significant association between
arsenic concentrations in the water and spontaneous abortions. He et al.
[106] suggested that arsenic is causative for mammalian spontaneous abor-
tion by placental involvement.
The mutagenic effect of hexavalent chromium previously found in both
somatic and germ cells could be responsible for abortions, which in this case
would be a male-mediated effect. In an earlier study, the same group [107]
examined outcomes in 2520 pregnancies of spouses of metal workers
exposed to hexavalent chromium from 1977 to 1987. The number of spon-
taneous abortions was not higher when compared to controls.
The manganese effects are controversial: A study of male workers mod-
erately exposed to manganese [63] showed a significantly reduced rate of live
births as compared to a control group (39 births observed vs. 70.5 expected),
while in a study on similarly exposed males no effects were demonstrated,
but the blood and urinary levels of the metal were about 50% lower [108].
An increase in the rate of spontaneous abortions (15.9%) was reported
among a group of 356 women who worked in a nickel hydrometallurgy
refining plant in the Arctic region of Russia as compared to the rate (8.5%)
in 342 local female construction workers [109]. The investigators noted that
Met. Ions Life Sci. 2011, 8, 263–303

the nickel-exposed women manually lifted heavy nickel anodes and may
have been exposed to other (confounding) factors such as heat stress,
smoking habits, use of alcohol, and intercurrent disease which were not
adequately controlled. Also the experimental data do not appear to be
conclusive: An increase of spontaneous abortions was observed in female
mice exposed to 160 mg but not to 80 mg/kg/day of nickel chloride in
drinking water on gestational days 2–17 [110].
No effect on fertility, as assessed by the rate of live births, was observed in
male workers chronically exposed to mercury vapor with urinary mercury
ranging from 5 to 270 mg/g creatinine [63]. An increased rate of spontaneous
abortions among wives of workers exposed to mercury vapor was, however,
noted at much higher paternal exposure of urinary mercury levels of
44000 mg/L, but the effects were not significant after controlling for indi-
vidual miscarriage history [111]. In another study of workers exposed to
mercury vapor [112], a trend of an increasing rate of spontaneous abortions
was associated with paternal urinary mercury levels (from 20 to 49 mg/L), but
also in this case the confounding factors such as smoking and alcohol
consumption were not addressed.
In a survey carried out by a questionnaire of more than 500 women who
had worked in a copper and lead smelter and were born between 1930 and
1959, the spontaneous abortion rates were highest when the mother was
employed during pregnancy (13.9%), had been employed before and was
living close to the smelter (17%). The frequency rate was higher too when
the father worked at the smelter (19%) [113]. Other reports provide virtually
no evidence that low-moderate lead exposure is associated with an increased
risk of spontaneous abortion [114].
Moving to other pregnancy effects the Cincinnati Prospective Study
showed that higher prenatal PbB was associated with reduced birth weight
and reduced gestational age [115]. In addition, a decrease in birth length of
2.5 cm per natural log unit of maternal PbB was seen, but only in white
infants. In a later report, the prenatal PbB (mean, 82 mg/L; range, 10–270 mg/
L) was related to lower birth weight [116]. PbBs Z100 mg/L were also sig-
nificantly associated (po0.05) with a decrease in total days of gestation and
an increased risk of preterm and small-for-gestational-age birth in a sample
of 262 mother-infant pairs from the general population in California [117].
In a case-control study of stillbirths, Ihrig et al. [118] included the
assessment of environmental arsenic exposures and analysis of confounders
(race, ethnicity, maternal age, median income, and parity). There was a
statistically significant increase in the risk of stillbirth in the group with the
highest exposure to arsenic. Further analysis showed that the increase was
limited to Hispanic people, possibly because of a genetic impairment in
folate metabolism. However, this study had a small number of cases in the
high exposure group, lacked data on smoking, and did not consider potential
Met. Ions Life Sci. 2011, 8, 263–303

confounding exposures to other chemicals. Correlations between relatively low

levels of arsenic (0.8–2 ppb) and significant increases in spontaneous abortion,
for fetal mortality [119], and stillbirth [120] have been reported. Consistent with
the data on in utero fatalities are data correlating human arsenic exposures with
increases in preterm birth rates and reduced birth weights [121]. Arsenic
accumulation in the placenta has led to the assumption that arsenic might have
deleterious vascular effects, causing placental abnormalities or decreased blood
flow and thus retarded fetal growth [122].
It was shown that exposure to cadmium during gestation is associated
with reduced birth weight and an increase in the number of preterm births,
probably due to the influence of cadmium on the synthesis of some hor-
mones or placental cadmium accumulation [123]. Another proposed
mechanism of cadmium effects on birth weight is induction of metal-
lothionein in the placenta and subsequent chelation of essential trace ele-
ments like zinc and copper [124]. No significant association between
maternal blood cadmium levels and newborn body weight were observed in
women with mean blood cadmium levels from 0.7 to 1.7 mg/L [125,126],
while some studies found an association between cord blood cadmium levels
and decreased birth weight [126,127], but the association was statistically
significant only in the study of Salpietro et al. [128].
The intravenous administration of indium to pregnant animals causes growth
inhibition and death of embryos in hamsters, rats, and mice. A single intrave-
nous administration of indium chloride to pregnant mice on gestation day 7, 8,
or 9 causes embryonic death and fetal weight decrease at Z0.8 mg/kg [129].
Nickel exposure is reported to affect early embryonic events in mice.
Injection of nickel chloride on the first day of pregnancy led to decreased
implantation frequency and reduced litter size, while the administration on
days 2–6 significantly reduced litter sizes but did not modify implantation.
No effect was demonstrated on the length of the estrous cycle or on changes
in the reproductive organs in mice or rats exposed to different species of
nickel [130].
6. PRENATAL EXPOSURE AND DEVELOPMENTAL

EFFECTS
Exposure to metal ions during first gestational periods may result in embryo
or fetal lethality or other severe developmental effects, while during orga-
nogenesis may give fetal anomalies. Methylmercury is clearly teratogenic for
humans, other metal ions such as nickel, arsenic, and lead have less severe
effects, producing fetal and early postnatal deaths, as well as malformations
such as anencephaly, eye defects, cleft palate, and skeletal anomalies [131].
Met. Ions Life Sci. 2011, 8, 263–303

Effects may be epigenetic or directly genetic on developing tissues [132], and

enhanced by changes occurring during the development, which may result in
a modified metabolism of the metal ions itself [133].
6.1. Arsenic
Arsenic induces malformations, especially neural tube defects, in laboratory
animals, and its toxicity has been studied in situations more similar to
human exposures, using broader end points, such as behavioral changes and
gene expression [134].
Human data about congenital malformations or developmental effects
potentially caused by arsenic derive mainly from investigations of popula-
tions living near smelting factories or arsenic-processing plants [135]. In
these studies, however, the analyses gave limited consideration to potential
confounders (e.g., smoking, exposure to other xenobiotics), and no data
related to arsenic exposure alone are available. After evaluating 13 studies of
human populations and 43 laboratory animal or in vitro studies, DeSesso
et al. [136] concluded that neither the human nor the animal studies were of
sufficient robustness. Structural malformations in experimental animals
were induced only when maternal arsenic blood concentrations were very
high and depended on the route of administration under conditions not
usual or relevant for human exposure [137]. No overall association between
arsenic in drinking water and congenital heart defects was detected in a case-
control study in Boston [138], although an association with one specific
lesion (coarctation of the aorta) was noted. A study of 184 women with
neural tube defects in the offspring living in a Texas county bordering
Mexico found that exposure to levels of arsenic in drinking water
(40.010 mg/L, range or upper limit not specified) did not significantly
increase the risk for neural tube defects [139]. Smith et al. [140] reported a
significant increase for lung cancer and bronchiectasy among subjects who
had probable exposure in utero (maternal exposure) or during childhood to
high levels of arsenic (near 0.9 mg/L) in drinking water.
6.2. Cadmium
Cadmium may be fetotoxic, manifested as reduced fetal or pup weights, from
oral exposures prior to and during gestation [141]: malformations of the
skeleton, such as fused lower limbs or absence of one or more limbs, and
delayed ossification of the sternum and ribs; dysplasia of facial bones, pala-
toschisis, sharp angulation of the distal third of the tail, have been found only
Met. Ions Life Sci. 2011, 8, 263–303

in some studies. The most sensitive indicator of developmental toxicity of

cadmium in animals appears, however, to be the neurobehavioral develop-
ment, reduced exploratory locomotor activity, and rotorod performance [142].
Babies from smoking mothers, whose cadmium body burden is higher
than in non-smokers, have reduced weight at birth. Among non-smoking
women, cadmium levels in urine were higher in those with infants of below-
normal birth weight. Data were not confirmed after adjusting urine cad-
mium levels by creatinine and it is difficult to attribute the decreased birth
weight exclusively to cadmium [143]. However, Nishijo et al. [123] found an
inverse correlation between maternal urinary cadmium excretion and
gestational age after adjustment for maternal age.
6.3. Chromium
Reproductive effects have been observed in the offspring of mice exposed to
chromium(III) following oral maternal exposure. Significant decreases in the
relative weights of reproductive tissues (ovaries and uterus) were observed in
the offspring of exposed mice. A significant delay in timing of vaginal
opening was also noted [144]. Cartilage formation in differentiating chick
fibroblast cultures was sensitive to damage by chromium(VI) but remained
unaltered by chromium(III). These data suggest that the developmental
effects of chromium(VI), which are more severe than those of chromium(III),
may result from increased uptake as well as higher direct toxicity [20].
Delayed vaginal opening and decreased relative weights of the uterus,
ovaries, testis, seminal vesicle, and preputial glands were observed in mouse
offspring exposed to potassium dichromate or chromium(III) chloride on
gestational day 12 through lactation day 20 [144]. Little is known about the
developmental effects of chromium in humans or animals. A descriptive
geographical study on congenital malformations in communities around a
site heavily polluted by chromium waste was carried out by Eizaguiree et al.
[145]. The relative risk of congenital malformations for the closest sites
seemed to be markedly lower than for other sites and the relative risk peaked
in the ring 2–4 km away from the polluted site. On the basis of their results
the authors excluded a possible teratogenic effect of chromium.
6.4. Copper
Malformations in mice were observed after oral administration of copper
sulfate in feed [146] and a reduced ossification in rats treated with copper
acetate in drinking water [147].
Met. Ions Life Sci. 2011, 8, 263–303

6.5. Lead
Environmental exposure in areas with lead concentrations of 450 mg/L in
water was associated with increased lead concentrations in cord blood and
placenta, as well as in maternal blood. The evidence that prenatal parental
lead exposure, except at very high levels, causes congenital malformations
remains modest, although studies have linked it to specific malformations of
brain and heart.
Studies in animals indicate that oral lead exposure may impair normal
bone growth and remodelling as indicated by decreased bone density and
bone calcium content, decreased trabecular bone volume, increased bone
resorption activity, and altered growth plate morphology [148,149]. Higher
maternal lead levels have been linked to reduced fetal growth and congenital
malformations, although considerable uncertainty remains regarding the
specific malformations and the dose-response relationships.
6.6. Lithium
Animal studies with lithium using doses comparable to human therapeutic
serum levels have not reported any abnormalities. However, higher doses
have produced exencephaly, skeletal and craniofacial defects and abnorm-
alities of blood vessel development. Experiments with other vertebrates have
shown that lithium affects dorsoventral specification and inhibition of vas-
culogenesis. Both these effects can be prevented by pretreatment with myo-
inositol indicating that lithium interferes with the phosphatidyl inositol
cycle.
Effects seen in animals, such as nephrotoxicity or behavioral alterations in
offspring, have not been confirmed in children of lithium-treated women
[150]. Human data indicate that lithium, at doses typical of the therapeutic
range, might cause developmental toxicity and an increased risk of major
(particularly cardiac) malformations. Because other information on terato-
genic effects is contradictory, it is prudent to exercise caution in treating
pregnant women with lithium [151].
6.7. Mercury
Specific malformations have been induced by administration of a single dose
of methylmercury during organogenesis, including cleft palate, limb mal-
formations, and facial and brain defects, while the developmental effects
include increased fetal death and malformation; decreased fetal weight, fetal
Met. Ions Life Sci. 2011, 8, 263–303

death, maternal toxicity; depressed birth weights. Behavioral changes have

also been documented in other animal models exposed to methylmercury
in utero, as differences in motor coordination, passive avoidance, operant
conditioning, audiogenic seizure response, and ultrasonic vocalizations
[152,153].
At higher doses, fetal viability was the major end point affected, followed
by malformation (primarily craniofacial and central nervous system) at
midrange, and skeletal ossification and variation effects at lower doses,
along with edema and kidney effects [154]. Inorganic mercury exposure
during pregnancy has also been studied in rats and mice, although to less
extent. Prenatal exposure of humans to methylmercury can cause damage to
the CNS resulting in cerebral palsy and mental retardation in the infant.
Classic research indicated that the prenatal stage was roughly three to four
times more sensitive to methylmercury damage than the adult one [155]. This
dose-response relationship for prenatal effects was the first to be described
for human exposure to any environmental chemical. Subtle effects on brain
function (motor function, visual spatial perception, language, attention, and
memory) were found in children exposed prenatally to low levels of mercury.
In addition, prenatal methylmercury exposure was also associated with heart
rate and blood pressure affects [156].
A review showed that many studies on children exposed prenatally to
methylmercury have small sample sizes, various designs and end points, and
show differing results [157].
6.8. Nickel
Malformations after administration of soluble nickel salts have been
reported in hamsters, mice, and rats; anomalies included ocular, skeletal,
and neural defects and were generally observed after a single parenteral
administration. Nickel carbonyl appears to be the most teratogenic of the
nickel compounds after exposure by inhalation. Available animal data
suggest that the developing fetus and neonates are sensitive targets of nickel
toxicity, although effects were often reported at maternally toxic doses [158].
An increase in structural malformations was observed in infants of women
who worked in a nickel hydrometallurgy refining plant [159]. Decreased fetal
body weight was observed in offspring of rats exposed to high levels of nickel
via inhalation during gestation. The available animal data on developmental
toxicity provide suggestive evidence that the developing fetus and the neo-
nates are sensitive targets of nickel toxicity. Vaktskjold et al. [160] found no
adverse effect of maternal exposure to water-soluble nickel during the peri
conception period and early pregnancy.
Met. Ions Life Sci. 2011, 8, 263–303

6.9. Vanadium
Varying chemical species of vanadium could explain the differences found in
several developmental toxicity studies. Sodium orthovanadate (V51) causes
slight fetal growth retardation only in the presence of maternal toxicity [161].
Oral administration of vanadyl (V41) sulfate pentahydrate to pregnant mice
resulted in maternal toxicity, embryotoxicity, and fetotoxicity at all dose
levels tested [162]. Decreased fertility, embryolethality, fetotoxicity, and
teratogenicity have been demonstrated in rats, mice, and hamsters following
vanadate (V51) and vanadyl (V41) administration [163]. The same author
[164] reported reproductive, developmental, and behavioral toxicity, as well
as mitogenic activity affecting the distribution of chromosomes during
mitosis, inducing aneuploidy-related end points.
6.10. Other Metals

After the introduction of catalytic converters in cars, platinum, palladium,
and rhodium have been emitted with exhaust fumes, and increasing levels
have been found in different environmental matrices such as road dusts, soils
along heavily frequented roads, and sediments of urban rivers. Compared
with other heavy metals, the biological availability of platinum, palladium,
and rhodium in some experimental studies on road dusts ranged between
that of cadmium and lead [165], and for platinum compounds effects on rat
ovaries, embryotoxic effects in rat, embryo lethality, and teratogenic effects
have been demonstrated [166].
Maternal exposure to organotin compound such as triphenyltin and
dibutyltin caused embryonic/fetal death, suppression of fetal growth and
cleft palate at maternal toxic doses and reduction of fetal ossification at
doses that are nontoxic to the mother [167].
7. EARLY POSTNATAL EXPOSURE AND

DEVELOPMENTAL EFFECTS
Neonatal and early postnatal periods are lifespan segments during which
sensitivity to toxic agents is high. The postnatal period is characterized by
rapid growth and development, with higher caloric and nutritional
requirements and with the activation of specific metabolic pathways. Neo-
nates differ from adults in metal ion absorption, distribution, metabolism,
and excretion. For example, absorption from the gastrointestinal tract is
Met. Ions Life Sci. 2011, 8, 263–303

influenced by higher neonatal gastric pH and the immaturity of the intestinal

mucus, and gastrointestinal absorption has been shown for certain metals to
be higher in the developing organism than in adults.
Metal ion distribution may also differ, due to the different percentage of total
body water (higher in infants and decreasing till 10–12 years) and to the low
level of plasma proteins, resulting in a higher amount of the diffusible fraction
of metals. The weight and body ratio for each organ must be also considered.
The brain/whole body ratio, for instance, is higher at delivery and decreases in
subsequent years. Many enzymes, such as those active in the kidney for
excretion of metals, are present at birth in lower concentrations and begin to
increase during the first year of life. Another factor of higher susceptibility
during peri-neonatal periods is the relative immaturity of the different organs
or tissues. For example, the blood-brain barrier is not fully developed and the
toxic elements may be transferred to the brain more readily during the first
months of life. High absorption of toxic elements may also be related to
‘‘normal’’ infant behaviors such as mouthing of objects, or ingestion of non-
food materials that may be contaminated. For breast-fed children, the main
source of metals during the neonatal period is maternal milk. The nutritional
habits of the mother and her current or past environmental or occupational
exposure strongly influence the kind and level of xenobiotics excreted by the
milk, caused, in part, by the redistribution of cumulative maternal bone stores.
7.1. Arsenic
Health risks caused by chronic exposure to arsenic-contaminated ground-
water have been recognized in many Asian and Latin American countries.
Calderon et al. [168] examined the effects of chronic exposure to lead,
arsenic, and malnutrition on the neuropsychological development of chil-
dren. After checking for significant potential confounders, verbal IQ
decreased with increasing concentrations of arsenic in urine. Watanabe et al.
[169] reviewed data from an arsenic-contaminated area in Bangladesh and
concluded that although some human data suggest possible effects on
developmental end points, the data are not sufficient to determine whether
arsenic represents a serious developmental risk. A cross-sectional study was
carried out to investigate intellectual function in 201 children of Bangladesh
[170]. Exposure to arsenic was associated with reduced intellectual function
after adjustment for socio demographic covariates. Water arsenic levels were
associated with reduced intellectual function in a dose-response manner (for
water As levels 450 mg/L the performance was significantly lower than with
water As levels o5.5 mg/L). The association was generally stronger for water
arsenic than for urinary arsenic.
Met. Ions Life Sci. 2011, 8, 263–303

Exposure to arsenic from drinking water assumed during early childhood

or in utero was associated with an increased mortality from both malignant
and non-malignant lung disease in young Chilean adults [140]. A follow-up
study, reviewed by Dakeishi et al. [171], reported a lower IQ and higher rate
of severe retardation (IQ below 50) in the children given the arsenic-con-
taminated Morinaga milk. Another follow-up study of children 14–16 years
of age, revealed higher prevalence of physical and mental effects, or CNS
pathology as epilepsy [172].
7.2. Cadmium
Cadmium accumulation in the kidney is responsible for effects such as
nephrotoxicity and osteoporosis which are observed at adult age. Although
transfer to the neonate through the placenta and through breast milk is
limited, teratogenic and developmental effects were observed in experi-
mental animals possibly through disturbance of the serotoninergic system
[173]. Moreover, experimental data in animals suggest that early cadmium
exposure may affect the hypothalamus-pituitary axis at different levels. This
may lead to disorders of the endocrine and/or immune system [174,175].
There are some epidemiological data on children showing that urinary
cadmium levels were associated with alteration of immediate hypersensi-
tivity (specific IgE) [176].
7.3. Lead
Lead is the most intensively studied metal for exposure of neonates and
infants. A lot of studies in children and animals confirmed the adverse effects
of lead exposure on cognition and other neurological functions. This con-
stituted in industrialized countries a priority within the public health pro-
blems in the seventies and eighties of the last century and determined the
normative to reduce lead in gasoline. In animals, observations on the basis of
a broad spectrum of learning and retention models support the hypothesis
that lead-induced neurobehavioral deficits extend long into adulthood,
primarily after preweaning exposure; the evidence is less clear after post-
weaning lead exposure. Effects include altered dendrite development and
synapse formation, changes in hippocampus structure and function, neu-
rochemical alterations, effects on glutamatergic synapses and disruption of
calcium homeostasis in the immature neonates [177,178].
The evidence for lowered cognitive ability in children exposed to lead has
come largely from prospective epidemiological studies. Sciarillo et al. [179]
Met. Ions Life Sci. 2011, 8, 263–303

and Stokes et al. [180] observed an increased incidence of depression,

somatic complaints, and aggressive behavior in 4–5-year-old lead-exposed
children. The NHANES III study, conducted from 1988 to 1994, assessed
the relationship between blood lead concentration and performance among
4853 children in the United States aged 6–16 years [181].
Other studies of neurobehavioral effects and cognitive function of children
exposed postnatally to lead include the one by Tong et al. [182], who
examined 375 children born near a lead smelting town for an association
between environmental lead exposure and children’s intelligence at age 11–
13 years. Verbal, performance, and full-scale IQ were inversely related to
PbB, with no apparent threshold. The expected mean full scale IQ declined
by 3.0 points (95% CI, 0.07–5.93) for an increase in lifetime average blood
lead concentration from 100–200 mg/L. Wasserman et al. [183] conducted a
subsequent investigation of 442 children from a lead-polluted area, com-
paring the relative contribution of prenatal blood lead with that of relative
increases in PbB in either the early (0–2 years) or the later (from 2 years on)
postnatal period to child intelligence measured at ages 3 and 4. This study
confirmed that elevations in both prenatal and postnatal PbB were asso-
ciated with small decrements in the children’s intelligence.
In the past decade, children’s blood lead levels have fallen significantly in a
number of countries, and current mean levels in developed countries are
typically below 50 mg/L. Canfield et al. [184] measured the blood lead con-
centration in 172 children at 6, 12, 18, 24, 36, 48, and 60 months of age and
compared it with the Stanford–Binet Intelligence Scale at the ages of 3 and 5
years. Blood lead concentration was inversely associated with IQ. Lanphear et
al. [185] carried out a pooled analysis to examine the association of intelli-
gence test scores and blood lead concentration. They collected data from 1333
subjects and IQ score was the primary outcome measure. The geometric mean
PbB peaked at 178 mg/L and declined to 94 mg/L by 5–7 years of age. After
adjustment for covariates, the estimated IQ point decrements associated with
an increase in blood lead from 24–100 mg/L, 100–200 mg/L, and 200–300 mg/L
were 3.9, 1.9, and 1.1, respectively. This suggests (Figure 2) that the dose-
response curve is steeper at the lower levels compared with the higher ones.
The scientific committee on neurotoxicology and psychophysiology of
metals of the International Commission on Occupational Health (ICOH)
declared that the action level for children, should be immediately reduced to
a PbB level of 50 mg/L [186].
7.4. Mercury
Children are more sensitive to mercury and are at greater risk than adults
[187]. A recent review [188] asserts that primary mercury exposure locations
Met. Ions Life Sci. 2011, 8, 263–303

Figure 2. Linear models of concurrent blood lead and IQ adjusted for some covari-
ates (maternal education, maternal IQ, and birth weight). (Note: 10 mg/dL ¼ 100 mg/L.)
Reproduced from [185] with permission from Environ. Health Persp., copyright (2005).
are at home, at school, and at other locations such as industrial plants not
adequately controlled or medical facilities.
Nonhuman primates exposed to low levels of methylmercury (50 mg Hg/
kg/day) from birth to 7 years, were followed for various types of behavioral
changes in auditory, somatosensory, and visual function for more than 20
years [189]. In rodent models, behavioral/locomotor alterations were noted
in rats exposed postnatally to moderate doses of methylmercury, and his-
topathology of the brains showed focal cerebellar dysphasia [190]. Exposure
of rats to methylmercury (5 mg/kg/day for 30 days) exclusively during the
postnatal period resulted in severe paralysis of the hind limbs and wide-
spread neuronal degeneration in many areas of the brain [191].
More recently, attention was paid to another kind of organic mercury,
thiomersal (thimerosal, ethylmercury thiosalicylate) used as a preservative in
vaccines and other medical products since the 1930s. Several studies have
examined the correlation of thiomersal-containing vaccines and autism.
Controlled epidemiological studies in Denmark, Sweden, the United King-
dom, and the United States provide no evidence for an association between
thiomersal exposure through vaccination and autism [192,193].
7.5. Manganese
Manganese was associated with neurotoxicity at high levels of exposure,
resulting in tremors and motor dysfunction [194]. In developing infants and
Met. Ions Life Sci. 2011, 8, 263–303

children, high manganese exposure has been associated with behavioral

disinhibition, hyperactive behavior, a slower rate of development, and
diminished intellectual function [195]. Moreover, the environmental blood
levels of manganese have also been associated with poorer learning and
recall, along with other adverse neurodevelopmental effects [196].
8. CONCLUDING REMARKS AND NEEDS FOR

FURTHER RESEARCH
The modern assessment of reproductive and developmental effects of metals
is connoted by a pronounced reduction of occupational exposure, resulting
from technological innovation and from improvement of preventive mea-
sures. The presence of other factors, such as organic compounds, possibly
affecting reproduction has, however, to be taken into account. On the other
hand the dispersion of metal ions, even at low exposure levels, in the general
environment is and shall be better and better recognized, following the
substantial improvement in biological monitoring practices.
Exposure to metals at low doses too, via endocrine disruption for instance,
may determine important end points such as: decreasing age at menarche,
decreasing semen quantity and quality, decreasing male-to-female sex ratio
at birth; increasing rates of hypospadias and testicular cancer, infertility,
spontaneous abortion; and structural and functional congenital malforma-
tions. In addition, more recent experimental studies suggest that the for-
mation of ROS by metal ions and their interaction with the hormonal system
or directly with the reproductive system and embryo seems the most plau-
sible explanation of effect on reproduction and development. However,
linking specific exposures to effects is often difficult because of multiple
exposures, the latency of effects, and the subtle nature of some outcomes.
The timing and duration of exposure are therefore key points and enable
us to classify the effects of metal ions into two major categories: that on the
organ and tissues of the reproductive system with direct or indirect effects on
fertility and ability to carry a pregnancy to full term, and the other, firstly on
the fetus and then to the newborn.
A better knowledge of the mechanisms of action involved and the different
target sites, would be crucial to understand this complex and heterogeneous
phenomenon and will represent one of the main aspect for future research
needs.
The characterization of dose is another critical point: the elemental spe-
ciation, the identification of more adequate matrices, nearest to the critical
organ or tissue, the dose-response relationship and threshold for action,
especially for some elements such as mercury, chromium, arsenic, and
Met. Ions Life Sci. 2011, 8, 263–303

vanadium. In risk assessment, the ideal situation combines adequate expo-

sure indicators with sensitive and specific effect indicators: There are limited
examples of early specific indicators of effects to be put in relation with dose
indicators and this appears to be a priority objective.
At present only a few metal ions such as lead, arsenic, cadmium, and
mercury have been extensively evaluated for their potential effect on
reproduction and development. However, in the general and occupa-
tional environment many other metal ions (chromium, nickel, platinum,
palladium, rhodium, vanadium, antimony) are present and have not
been systematically assessed for their effect on reproductive systems.
Research should be extended to these elements too starting from in vitro and
in vivo studies and moving in a second step to human studies in exposed
groups.
We already emphasized the issue regarding current experimental
and human studies which deal with exposure to a single metal, in
contrast with real environmental and occupational exposure generally
characterized by many metal ions and organic compounds. The possible
synergic (additive, multiplicative) or competitive effects of metal coexposure
remain therefore an intrigant, though cumbersome objective for future
research.
ABBREVIATIONS
BAL British anti-lewisite

CI confidence interval
CNS central nervous system
DMA dimethyl arsenic acid
ED endocrine disrupter
ET electron transfer
FSH follicule stimulating hormone
ICOH International Commission on Occupational Health
IgE immunoglobulin E
IVF in vitro fertilization
LH luteinizing hormone
MMA monomethlyarsonic acid
NHANES National Health and Nutrition Examination Survey
PbB lead in blood
SCSA sperm chromatin structure assay
TTP time to pregnancy
Met. Ions Life Sci. 2011, 8, 263–303

REFERENCES
1. G. Windham and L. Fenster, Fertil. Steril., 2008, 89 (Suppl. 1), 111–116.
2. P. G. Wells, Y. Bhuller, C. S. Chen, W. Jeng, S. Kasapinovic, J. C. Kennedy,
P. M. Kim, R. R. Laposa, G. P. McCallum, C. J. Nicol, T. Parman, M. J. Wiley
and A. W. Wong, Toxicol. Appl. Pharmacol., 2005, 207, S354–S366.
3. M. Vahter, A. Akesson, C. Liden, S. Ceccatelli and M. Berglund, Environ. Res.,
2007, 104, 85–95.
4. A. M. Cummings and R. J. Kavlock, J. Toxicol. Sci., 2004, 29, 167–178.
5. V. J. Nesatyy, B. V. Rutishauser, R. I. Eggen and M. J. Suter, Analyst, 2005,
130, 1087–1097.
6. K. Jana, S. Jana and P. K. Samanta, Reprod. Biol. Endocrinol., 2006, 16, 4–9.
7. I. Iavicoli, L. Fontana and A. Bergamaschi, J. Toxicol. Environ. Health B Crit.
Rev., 2009, 12, 206–223.
8. M. Sakamoto, A. Nakano and H. Akagi, Environ. Res., 2001, 87, 92–98.
9. M. Belles, M. L. Albina and D. J. Sanchez, Arch. Environ. Contam. Toxicol.,
2002, 4, 293–298.
10. D. K. Saxena, R. C. Murthy and C. Singh, Res. Commun. Chem. Pathol.
Pharmacol., 1989, 64, 317–329.
11. B. Pollock and K. L. Machin, Arch. Environ. Contam. Toxicol., 2008, 54, 730–709.
12. R. Quansah and J. J. K. Jaakkola, Int. Arch. Occup. Environ. Health, 2009, 82,
529–537.
13. D. A. Savitz, N. L. Sonnenfeld and A. F. Olshan, Am. J. Ind. Med., 1994, 25,
361–383.
14. R. A. Yazigi, R. R. Odem and K. L. Polakoski, JAMA, 1991, 266, 1956–1959.
15. P. L. Nampoothiri and S. Gupta, J. Biochem. Molecular Toxicology, 2008, 22,
337–343.
16. P. Apostoli, P. Kiss, S. Porru, J. P. Bonde and M. Vanhoorne, Occup. Environ.
Med., 1998, 55, 364–374.
17. P. B. Kaplowitz, E. J. Slora and R. C. Wasserman, Pediatrics, 2001, 108, 347–
353.
18. M. Joffe, Occup. Environ. Med., 1997, 54, 289–295.
19. M. Spanò, H. Kolstad, S. B. Larsen, E. Cordelli, G. Leter, A. Giwercman and
J. P. E. Bonde, Scand. J. Work Environ. Health, 1999, 25 (Suppl. 1), 28–30.
20. B. Danielsson, E. Hassoun and L. Dencker, Arch. Toxicol., 1982, 51, 233–245.
21. T. F. Gale and J. D. Bunch, Teratol., 2005, 19, 81–86.
22. H. T. Epstein, J. T. Newton and K. Fenton, Biol. Neonat., 1999, 75, 272–278.
23. M. J. J. Ronis, T. M. Badger and S. J. Shema, Toxicol. Appl. Pharm., 1996, 136,
361–371.
24. B. L. Gulson, K. J. Mizon, J. M. Palmer, M. J. Korsch, A. J. Taylor and K. R.
Mahaffey, Environ. Health Perspect., 2004, 112, 1499–1507.
25. A. Kasperczyk, S. Kasperczyk, S. Horak, A. Ostazowska, E. Grucka-Mamczar,
E. Romuk, A. Olejek and E. Birkner, Toxicol. Appl. Pharmacol., 2008, 228,
378–384.
26. E. K. Silbergeld, B. Quintanilla-Vega and R. E. Gandley, Adv. Exp. Med. Biol.,
2003, 518, 37–48.
Met. Ions Life Sci. 2011, 8, 263–303

27. S. Benoff, I. R. Hurley, C. Millan, B. Napolitano and G. M. Centola, Fertil.

Steril., 2003, 80, 517–525.
28. P. Kovacic and R. Somanathan, Birth Defects Res. Part C, 2006, 78, 308–325.
29. N. K. Mottet, C. M. Shaw and T. M. Burbacher, Environ. Health Perspec.,
1985, 63, 133–140.
30. D. C. Bellinger, Birth Defects Res. Part A, Clin. Mol. Teratol., 2005, 73,
409–420.
31. G. F. Nordberg, Toxicol. Appl. Pharmacol., 2009, 238, 192–200.
32. M. M. Aruldhas, S. Subramanian, P. Sekhar, G. Vengatesh, P. Govindarajulu
and M. A. Akbarsha, Fertil. Steril., 2006, 86, 1097–1105.
33. J. M. Van Weert, S. Repping, J. W. Van der Steeg, P. Steures, F. Van der Veen
and B. W. Mol, J. Reprod. Med., 2008, 53, 250–256.
34. N. Pant, R. C. Murthy and S. P. Srivastava, Hum. Exp. Toxicol., 2004, 23,
399–403.
35. S. M. Cohen, T. Ohnishi, L. L. Arnold and X. C. Le, Toxicol. Appl. Pharmacol.,
2007, 222, 258–263.
36. E. R. Siu, D. D. Mruk, C. S. Porto and C. Y. Cheng, Toxicol. Appl. Pharmacol.,
2009, 238, 240–249.
37. C. H. Wong, D. D. Mruk, W. Y. Lui and C. Y. Cheng, J. Cell. Sci., 2004, 117,
783–798.
38. C. Fiorini, J. Gilleron, D. Carette, A. Valette, A. Tilloy, S. Chevalier, D. Seg-
retain and G. Pointis, Biochim. Biophys. Acta, 2008, 1778, 56–67.
39. O. Akinloye, A. O. Arowojolu, B. O. Shittu and J. I. Anetor, Reprod. Biol.,
2006, 1, 17–26.
40. S. Subramanian, G. Rajendiran and P. Sekhar, Toxicol. Appl. Pharmacol., 2006,
215, 237–249.
41. U. R. Acharya, M. Mishra, R. R. Tripathy and I. Mishra, Reprod. Toxicol.,
2006, 22, 87–91.
42. H. Li, Q. Chen, S. Li, W. Yao, L. Li, X. Shi, L. Wang, V. Castranova,
V. Vallyathan, E. Ernst and C. Chen, Ann. Occup. Hyg., 2001, 45, 505–511.
43. K. Danadevi, R. Rozati, P. P. Reddy and P. Grover, Reprod. Toxicol., 2003, 17,
451–456.
44. S. Chattopadhyay, S. Ghosh, S. Chaki, J. Debnath and D. Ghosh, J. Toxicol.
Sci., 1999, 24, 425–431.
45. S. Roychoudhury, J. Slivkovf, J. Bulla and P. Massinyi, Foli. Vet., 2008, 2,
64–68.
46. J. P. Bonde, M. Joffe, P. Apostoli, A. Dale, P. Kiss, M. Spano, F. Caruso,
A. Giwercman, L. Bisanti, S. Porru, M. Vanhoorne, F. Comhaire and
W. Zschiesche, Occup. Environ. Med., 2002, 59, 234–242.
47. M. Lerda, Am. J. Ind. Med., 1992, 22, 567–571.
48. B. H. Alexander, H. Checkoway and C. van Netten, Occup. Environ. Med.,
1996, 53, 411–416.
49. M. R. Cullen, R. D. Kayne and J. M. Robins, Arch. Environ. Health., 1984, 39,
431–440.
50. M. Rodamilans, M. J. Martinez-Osaba, J. To-Figueras, F. Rivera-Fillat,
M. Torra, P. Pérez and J. Corbella, Toxicol. Lett., 1988, 42, 285–290.
Met. Ions Life Sci. 2011, 8, 263–303

51. S. Telišman, B. Colak, A. Pizent, J. Jurasović and P. Cvitković, Environ. Res.,

2007, 105, 256–266.
52. J. Jurasović, P. Cvitković and A. Pizent, BioMetals, 2004, 17, 735–743.
53. J. P. Prestifilippo, J. Fernández-Solari, C. Mohn, A. De Laurentiis, S. M.
McCann, W. Dees and V. Rettori, Toxicol. Sci., 2007, 97, 75–80.
54. B. Lee, J. K. Hiney, M. D. Pine, V. K. Srivastava and W. L. Dee, J. Physiol.,
2007, 578, 765–772.
55. T. P. Ponnapakkam, K. S Bailey, K. A. Graves and M. B. Iszard, Reprod.
Toxicol., 2003, 17, 547–551.
56. M. V. Rao and P. S. Sharma, Reprod. Toxicol., 2001, 15, 705–712.
57. M. K. Mohamed, T. M. Burbacher and N. K. Mottet, Pharmacol. Toxicol.,
1987, 60, 29–36.
58. A. S. Friedmann, H. L. Chen, L. D. Rabuck and B. R. Zirkin, Environ. Toxicol.
Chem., 1998, 17, 867–871.
59. O. E. Orisakwe, O. J. Afonne and E. Nwobodo, Eur. J. Obstetr. Gynecol.
Reprod. Biol., 2001, 95, 92–96.
60. M. A. Boujbiha, K. Hamden, F. Guermazi, A. Bouslama, A. Omezzine,
A. Kammoun and A. El Feki, Reprod. Toxicol., 2009, 28, 81–89.
61. A. J. McGregor and H. J Mason, in Heavy Metals in the Environment, CEP
Consultants, Edinburgh, 1991, pp. 375–378.
62. L. Barregärd, G. Lindstedt, A. Schütz and G. Sällsten, Occup. Environ. Med.,
1994, 51, 536–540.
63. R. Lauwerys, H. Roels, P. Genet, G. Toussaint, A. Bouckaert and S. De
Cooman, Am. J. Ind. Med., 1985, 7, 171–176.
64. T. Y. Leung, C. M. Y. Choy and S. F. Yim, Austral. New Zealand J. Obstetr.
Gynaecol., 2001, 41, 75–77.
65. V. Hanf, A. Forstmann, J. E. Costea, G. Schieferstein, I. Fischer and
F. Schweinsberg, Toxicol. Lett., 1996, 88, 227–231.
66. M. D. Dickman, K. M. C. Leung and L. C. L. Koo, Marine Pollut. Bull., 1999,
39, 352–356.
67. K. Doreswamy, B. Shrilatha and T. Rajeshkumar, J. Androl., 2004, 25, 996–
1003.
68. R. Pandey, R. Kumar, R. Singh, D. K. Saxena and S. P. Srivastava, BioMetals,
1999, 12, 339–346.
69. A. V. Roshchin, E. K. Ordzhonikidze and I. V. Shalganova, J. Hyg. Epidemiol.
Microbiol. Immunol., 1980, 24, 377–383.
70. A. K. Chandra, R. Ghosh, A. Chatterjee and M. Sarkar, J. Inorg. Biochem.,
2007, 101, 944–956.
71. U. Fagher, T. Laudanski, A. Schutz, M. Sipowicz and M. Akerlund, Int. J.
Gynaecol. Obstet., 1993, 40, 109–114.
72. B. Runnebaum, T. Rabe and M. Sillem, in Infertility of Gynecological Endo-
crinology and Reproductive Medicine, Ed. B. Runnebaum and T. Rabe, Springer,
New York, 1997, pp. 107–164.
73. C. B. Ernhart and T. Greene, Br. J. Ind. Med., 1992, 49, 11–13.
74. S. Chattopadhyay, S. Ghosh, S. Chaki, J. Debnath and D. Ghosh, J. Toxicol.
Sci., 1999, 24, 425–431.
Met. Ions Life Sci. 2011, 8, 263–303

75. M. P. Waalkes, Mutat. Res., 2003, 10(533), 107–120.

76. S. Chattopadhyay, S. Pal Ghosh, D. Ghosh and J. Debnath, J. Toxicol. Sci.,
2003, 75, 412–422.
77. M. T. Rie, K. A. Lendas and I. P. Callard, Comp. Biochem. Physiol. C Toxicol.
Pharmacol., 2001, 130, 41–51.
78. M. P. Waalkes and S. Rehm, Toxicol., 1998, 126, 173–178.
79. K. Paksy, B. Varga and P. Lazar, Acta Physiol. Hung., 1996, 84, 119–130.
80. M. T. Zenzes, S. Krishnan, B. Krishnan, H. Zhang and R. F. Casper, Fertil.
Steril., 1995, 64, 599–603.
81. E. V. Younglai, W. G. Foster, E. G. Hughes, K. Trim and J. F. Jarrell, Arch.
Environ. Contam. Toxicol., 2002, 43, 121–126.
82. R. C. Murthy, M. Junaid and D. K. Saxena, Toxicol Lett., 1996, 89, 147–154.
83. S. K. Banu, J. B. Samuel, J. A. Arosh, R. C. Burghardt and M. M. Aruldhas,
84. C. Taupeau, J. Poupon, F. Nomé and B. Lefèvre, Reprod. Toxicol., 2001, 15,
385–391.
85. M. Wide, Teratol., 1985, 32, 375–380.
86. H. Hu, L. Pepper and R. Goldman, Am. J. Med., 1991, 20, 723–735.
87. A. Stadnicka, Acta Histochem., 1980, 67, 227–233.
88. T. Watanabe, T. Shimada and A. Endo, Teratology, 1982, 25, 381–384.
89. J. C. Heath, Y. Abdelmageed, T. D. Braden, A. C. Nichols and D. A. Steffy,
Food. Chem. Toxicol., 2009, 47, 1600–1605.
90. W. Shen, Y. Chen, C. Li and Q. Ji, Wei Sheng Yan Jiu, 2000, 29, 75–77.
91. R. Sikorski, T. Juszkiewicz, T. Paszkowski and T. Szprengier-Juszkiewicz, Int.
Arch. Occup. Environ. Health, 1987, 59, 551–557.
92. M. L. Lindbohm, M. Sallmén, A. Anttila, H. Taskinen and K. Hemminki,
Scand. J. Work Environ. Health, 1991, 17, 95–103.
93. J. P. Gennart, J. P. Buchet, H. Roels, P. Ghyselen, E. Ceulemans and
R. Lauwerys, J. Epidemiol., 1992, 135, 1208–1219.
94. M. Sallmén, M. L. Lindbohm, A. Anttila, H. Taskinen and K. Hemminki,
Epidemiol., 2000, 11, 141–147.
95. M. Sallmén, M. L. Lindbohm, A. Anttila, H. Taskinen and K. Hemminki,
J. Epidemiol. Community Health, 1992, 46, 519–522.
96. P. Apostoli, A. Bellini, S. Porru and L. Bisanti, Am. J. Ind. Med., 2000, 38,
310–315.
97. M. Joffe, L. Bisanti, P. Apostoli, P. Kiss, A. Dale, N. Roeleveld, M. L. Lindbohm,
M. Sallmén, M. Vanhoorne and J. P. Bonde, Occup. Environ. Med., 2003,
60, 752–758.
98. C. Y. Shiau, J. D. Wang and P. C. Chen, Occup. Environ. Med., 2004,
61, 915–923.
99. J. P. Bonde and P. Apostoli, Occup. Environ. Med., 2005, 62, 2–3.
100. G. Torelli, Med. Lav., 1930, 3, 110–121.
101. I. Hertz-Picciotto, Am. J. Ind. Med., 2000, 38, 300–309.
102. M. Falcon, P. Vifias and A. Luna, Toxicol., 2003, 185, 59–66.
103. M. Sallmén, A. Anttila, M. L. Lindbohm, P. Kyyrönen, H. Taskinen and
K. Hemminki, J. Occup. Environ. Med., 1995, 37, 931–934.
Met. Ions Life Sci. 2011, 8, 263–303

104. O. S. von Ehrenstein, D. N. Guha Mazumder, M. Hira-Smith, N. Ghosh,

Y. Yuan, G. Windham, A. Ghosh, R. Haque, S. Lahiri, D. Kalman, S. Das and
A. H. Smith, Am. J. Epidemiol., 2006, 163, 662–669.
105. A. H. Milton, W. Smith, B. Rahman, Z. Hasan, U. Kulsum, K. Dear,
M. Rakibuddin and A. Ali, Epidemiol., 2005, 16, 82–86.
106. W. He, R. J. Greenwell, D. M. Brooks, L. Calderón-Garcidueñas, H. D. Beall
and J. D. Coffin, Toxicol. Sci., 2007, 99, 244–253.
107. N. H. Hjollund, J. P. Bonde and K. S. Hansen, Scand. J. Work Environ. Health,
1995, 21, 272–276.
108. J. P. Gennart, J. P. Buchet, H. Roels, P. Ghyselen, E. Ceulemans and
R. Lauwerys, Am. J. Epidemiol., 1992, 135, 1208–1219.
109. V. P. Chashschin, G. P. Artunina and T. Norseth, Sci. Total Environ., 1994,
148, 287–291.
110. E. Berman and B. Rehnberg, Fetotoxic effects of nickel in drinking water in mice,
National Technical Information Service, EPA600183007, PB83225383, Virgi-
nia, USA, Spring Field, 1983.
111. K. H. Alcser, K. A. Brix, L. J. Fine, L. R. Kallenbach and R. A. Wolfe, Am. J.
Ind. Med., 1989, 15, 517–529.
112. S. Cordier, F. Deplan, L. Mandereau and D. Hemon, Br. J. Ind. Med., 1991,
48, 375–381.
113. S. Nordstrom, L. Beckman and I. Nordenson, Hereditas, 1978, 88, 43–46.
114. T. Laudanski, M. Sipowicz, P. Modzelewski, J. Bolinski, J. Szamatowicz,
G. Razniewska and M. Akerlund, Int. J. Gynaecol. Obstet., 1991, 36,
309–315.
115. K. Dietrich, K. Krafft, R. Bornschein, P. B. Hammond, O. Berger, P. A. Succop
and M. Bier, Pediatrics, 1987, 80, 721–730.
116. K. Dietrich, M. D. Ris and P. A. Succop, Neurotoxicol. Teratol., 2001, 23,
511–518.
117. L. L. Jelliffe-Pawlowski, S. Q. Miles, J. G. Courtney, B. Materna and
V. Charlton, J. Perinatol., 2006, 26, 154–162.
118. M. M. Ihrig, S. L. Shalat and C. Baynes, Epidemiol., 1998, 9, 290–294.
119. C. Hopenhayn-Rich, S. R. Browning, I. Hertz-Picciotto, C. Ferreccio, C. Per-
alta and H. Gibb, Environ. Health Perspect., 2000, 108, 667–673.
120. M. Börzsönyi, A. Bereczky, P. Rudnai, M. Csanady and A. Horvath, Arch.
Toxicol., 1992, 66, 77–78.
121. C. Y. Yang, C. C. Chang, S. S. Tsai, H. Y. Chuang, C. K. Ho and T. N. Wu,
Environ. Res., 2003, 91, 29–34.
122. C. Hopenhayn, B. Huang, J. Christian, C. Peralta, C. Ferreccio, R. Atallah and
D. Kalman, Environ. Health Perspect., 2003, 111, 1888–1891.
123. M. Nishijo, H. Nakagawa and R. Honda, Occup. Environ. Med., 2002, 59,
394–396.
124. J. Chmielnicka and B. Sowa, Ecotoxicol. Environ. Saf., 1996, 35, 277–281.
125. G. Mokhtar, E. Hossny, M. el-Awady and M. Zekry, East Mediterr. Health J.,
2002, 8, 254–260.
126. V. Galicia-Garcı́a, M. Rojas-López, R. Rojas, G. Olaiz and C. Rı́os, Toxicol.
Lett., 1997, 91, 57–61.
Met. Ions Life Sci. 2011, 8, 263–303

127. Y. L. Zhang, Y. C. Zhao, J. X. Wang, H. D. Zhu, Q. F. Liu, Y. G. Fan,

N. F. Wang, J. H. Zhao, H. S. Liu, L. Ou-Yang, A. P. Liu and T. Q. Fan,
J. Environ. Sci. Health A Tox. Hazard. Subst. Environ. Eng., 2004, 39,
2507–2515.
128. C. D. Salpietro, S. Gangemi, P. L. Minciullo, S. Briuglia, M. V. Merlino, A.
Stelitano, M. Cristani, D. Trombetta and A. Saija, J. Perinat. Med., 2002, 30,
395–399.
129. M. Nakajima, H. Takahashi, M. Sasaki, Y. Kobayashi, Y. Ohno and M.
Usami, Teratog. Carcinog. Mutagen., 2000, 20, 219–227.
130. NTP, Technical Report on the Toxicology and Carcinogenesis Studies of Nickel
Oxide (CAS No. 1313–99–1) in F344/N Rats and B6C3F1 Mice (inhalation
studies). Research Triangle Park, NC: U.S. Department of Health and
Human Services, Public Health Service, National Institutes of Health, 1996,
No. 451.
131. F. W. Sunderman, Jr., Metal Ions Med. Biol., 1998, 5, 275–279.
132. N. K. Mottet and V. H Perm, in Reproductive and Developmental Toxicity of
Metals, Ed. T. W. Clarkson, G. F. Nordberg and P. R. Sager, Plenum Press,
New York, 1983, pp. 93–126.
133. D. Rice and S. J. Barone, Environ. Health Perspect., 2000, 103, 511–533.
134. A. Wang, S. D. Holladay, D. C. Wolf, S. A. Ahmed and J. L. Robertson, Int. J.
Toxicol., 2006, 25, 319–331.
135. M. Vahter, Basic Clin. Pharmacol. Toxicol., 2008, 102, 204–211.
136. J. M. DeSesso, C. F. Jacobson, A. R. Scialli, C. H. Farr and J. F. Holson,
Reprod. Toxicol., 1998, 12, 385–433.
137. J. F. Holson, D. G. Stump, K. J. Clevidence, J. F. Knapp and C. H. Farr, Food
Chem. Toxicol., 2000, 38, 459–466.
138. S. Zierler, M. Theodore, A. Cohen and K. J. Rothman, Int. J. Epidemiol., 1988,
17, 589–594.
139. J. D. Brender, F. B. Zhan, L. Suarez, P. H. Langlois and K. Moody, J. Occup.
Environ. Med., 2006, 48, 565–572.
140. A. H. Smith, G. Marshall, Y. Yuan, C. Ferreccio, J. Liaw, O. von Ehrenstein,
C. Steinmaus, M. N. Bates and S. Selvin, Environ. Health Perspect., 2006, 114,
1293–1296.
141. K. Kostial, in Reproductive and Developmental Toxicity of Metals, Ed. T. W.
Clarkson, G. F. Nordberg and P. R. Sager, Plenum Press, New York, 1983,
pp. 727–744.
142. B. Barański, Environ. Res., 1987, 42, 54–62.
143. L. Cresta, F. Perdelli, Y. Franco, E. Raffo, M. L. Cristina and F. Diani,
Minerva Ginecol., 1989, 41, 85–88.
144. M. H. Al-Hamood, A. Elbetieha and H. Bataineh, Reprod. Fertil. Dev., 1998,
10, 179–183.
145. D. D. Eizaguirre, C. Rodriguez and G. C. Watt, J. Public. Health Med., 2000,
22, 54–58.
146. M. Lecyk, Zool. Pol., 1980, 28, 101–105.
147. D. S. Haddad, A. al-Alousi and A. H. Kantarjian, Funct. Dev. Morphol., 1991,
1, 17–22.
Met. Ions Life Sci. 2011, 8, 263–303

148. A. Escribano, M. Revilla, E. R. Hernández, C. Seco, J. González-Riola, L. F.

Villa and H. Rico, Calcif. Tissue Int., 1997, 60, 200–203.
149. M. J. Ronis, J. Aronson, G. G. Gao, W. Hogue, R. A. Skinner, T. M. Badger
and C. K. Lumpkin, Jr., Toxicol Sci., 2001, 62, 321–329.
150. J. A. Moore, Reprod. Toxicol., 1995, 9, 175–210.
151. A. Léonard, P. Hantson and G. B. Gerber, Mutat. Res., 1995, 339, 131–137.
152. M. Sakamoto, A. Kakita, K. Wakabayashi, H. Takahashi, A. Nakano and
H. Akagi, Brain Res., 2002, 949, 51–59.
153. F. Y. Doré, S. Goulet, A. Gallagher, P. O. Harvey, J. F. Cantin, T. D’Aigle and
M. E. Mirault, Neurotoxicol. Teratol., 2001, 23, 463–472.
154. P. Grandjean, E. Budtz-Jorgensen, R. F. White, P. J. Jørgensen, P. Weihe,
F. Debes and N. Keiding, Am. J. Epidemiol., 1999, 150, 301–305.
155. T. W. Clarkson, C. Cox and D. O. March, in Measurements of Risks, Ed. G. G.
Berg and H. D. Maillie, Plenum Press, New York, 1981, pp. 111–129.
156. N. Sorensen, K. Murata, E. Budtz-Jorgensen, P. Weihe and P. Grandjean,
Epidemiol., 1999, 10, 370–375.
157. A. F. Castoldi, T. Coccini, S. Ceccatelli and L. Manzo, Brain. Res. Bull., 2001,
55, 197–203.
158. E. Nieboer, F. E. Rossetto and C. R. Menon, in Nickel and Its Role in Biology,
Volume 23 of Metal Ions in Biological Systems, Ed. H. Sigel and A. Sigel,
Marcel Dekker, New York, 1988, pp. 359–402.
159. V. P. Chashschin, G. P. Artunina and T. Norseth, Sci. Total Environ., 1994,
6(148), 287–291.
160. A. Vaktskjold, L. V. Talykova, V. P. Chashchin, J. O. Odland and E. Nieboer,
Am. J. Ind. Med., 2008, 51, 825–833.
161. D. Sanchez, A. Ortega, J. L. Domingo and J. Corbella, Biol. Trace Elem. Res.,
1991, 30, 219–226.
162. J. L. Paternain, J. L. Domingo, M. Gómez, A. Ortega and J. Corbella, J. App.
Toxicol., 1990, 10, 181–186.
163. J. L. Domingo, Reprod. Toxicol., 1996, 10, 175–182.
164. J. L. Domingo, Biol. Trace Elem. Res., 2002, 88, 97–112.
165. S. Zimmermann and B. Sures, Environ. Sci. Pollut. Res. Int., 2004, 11,
194–199.
166. G. V. Karpova, T. G. Borovskaia, E. A. Timina and A. V. Pakhomova, Eksp.
Klin. Farmakol., 2004, 67, 38–40.
167. H. Tryphonas, G. Cooke, D. Caldwell, G. Bondy, M. Parenteau, S. Hayward
and O. Pulido, Food Chem. Toxicol., 2004, 42, 221–235.
168. J. Calderón, M. E. Navarro, M. E. Jimenez-Capdeville, M. A. Santos-Diaz,
A. Golden, I. Rodriguez-Leyva, V. Borja-Aburto and F. Dı́az-Barriga, Environ.
Res., 2001, 85, 69–76.
169. C. Watanabe, T. Inaoka, T. Matsui, K. Ishigaki, N. Murayama and R. Ohtsuka,
J. Environ. Sci. Health A Tox. Hazard Subst. Environ. Eng., 2003, 38, 129–139.
170. G. A. Wasserman, X. Liu, F. Parvez, H. Ahsan, P. Factor-Litvak, A. van Geen,
V. Slavkovich, N. J. LoIacono, Z. Cheng, I. Hussain, H. Momotaj and J. H.
Graziano, Environ. Health Perspect., 2004, 112, 1329–1333.
Met. Ions Life Sci. 2011, 8, 263–303

171. M. Dakeishi, K. Murata and P. Grandjean, Environ. Health, 2006, 5, 31–35.

172. P. Grandjean and K. Murata, Epidemiol., 2007, 18, 25–26.
173. A. Oskarsson, I. Palminger-Hallen, J. Sundberg and K. P. Grawe, Analyst,
1998, 123, 19–23.
174. S. L. Schantz and J. J. Widholm, Environ. Health Perspect., 2001, 109, 1197–
1206.
175. G. Schoeters, E. Den Hond, M. Zuurbier, R. Naginiene, P. Van Den Hazel, N.
Stilianakis, R. Ronchetti and J. G. Koppe, Acta Pædiatr., 2006, 95(Suppl. 453),
50–54.
176. B. Ritz, J. Heinrich, M. Wjst, E. Wichmann and C. Krause, Arch. Environ.
Health, 1998, 53, 272–280.
177. C. D. Toscano and T. R. Guilarte, Brain Res. Brain Res. Rev., 2005, 49, 529–
554.
178. G. Winneke, Neurotoxicol., 1996, 17, 565–580.
179. W. G. Sciarillo, G. Alexander and K. P. Farrell, Am. J. Public Health, 1992, 82,
1356–1360.
180. L. Stokes, R. Letz, F. Gerr, M. Kolczak, F. E. McNeill, D. R. Chettle and W. E.
Kaye, Occup. Environ. Med., 1998, 55, 507–516.
181. B. P. Lanphear, K. Dietrich, P. Auinger and C. Cox, Public. Health Rep., 2000,
115, 521–529.
182. S. Tong, P. Baghurst, A. McMichael, M. Sawyer and J. Mudge, Brit. Med. J.,
1996, 312, 1569–1575.
183. G. A. Wasserman, X. Liu, D. Popovac, P. Factor-Litvak, J. Kline, C. Water-
naux, N. LoIacono and J. H. Graziano, Neurotoxicol. Teratol., 2000, 22, 811–
818.
184. R. L. Canfield, D. A. Kreher, C. Cornwell and C. R. Henderson, Jr., Child
Neuropsychol., 2003, 9, 35–53.
185. B. P. Lanphear, R. Hornung, J. Khoury, K. Yolton, P. Baghurst, D. C.
Bellinger, R. L. Canfield, K. N. Dietrich, R. Bornschein, T. Greene, S. J.
Rothenberg, H. L. Needleman, L. Schnaas, G. Wasserman, J. Graziano and R.
Roberts, Environ. Health Perspect., 2005, 113, 894–899.
186. P. Landrigan, M. Nordberg, R. Lucchini, G. Nordberg, P. Grandjean,
A. Iregren and L. Alessio, Am. J. Ind. Med., 2007, 50, 709–711.
187. H. S. Rogers, J. McCullough, S. Kieszak, K. L. Caldwell, R. L. Jones and
C. Rubin, Clin. Toxicol. (Phila.), 2007, 45, 240–247.
188. R. Lee, D. Middleton, K. Caldwell, S. Dearwent, S. Jones, B. Lewis, C.
Monteilh, M. E. Mortensen, R. Nickle, K. Orloff, M. Reger, J. Risher, H. S.
Rogers and M. Watters, Environ. Health Perspect., 2009, 117, 871–878.
189. D. C. Rice and S. G. Gilbert, Toxicol. Appl. Pharmacol., 1995, 134, 161–169.
190. M. Sakamoto, M. Kubota, S. Matsumoto, A. Nakano and H. Akagi, Environ.
Res., 2002, 90, 185–189.
191. M. Sakamoto, K. Wakabayashi, A. Kakita, T. Hitoshi, T. Adachi and
A. Nakano, Brain Res., 1998, 16, 351–354.
192. S. K. Parker, B. Schwartz, J. Todd and L. K. Pickering, Pediatrics, 2004,
114, 793–804.
193. L. Miller and J. Reynolds, J. Spec. Pediatr. Nurs., 2009, 14, 166–172.
Met. Ions Life Sci. 2011, 8, 263–303

194. D. Mergler, M. Baldwin, S. Bélanger, F. Larribe, A. Beuter, R. Bowler,

M. Panisset, R. Edwards, A. de Geoffroy, M. P. Sassine and K. Hudnell,
Neurotoxicol., 1999, 20, 327–342.
195. M. Bouchard, F. Laforest, L. Vandelac, D. Bellinger and D. Mergler, Environ.
Health Perspect., 2007, 115, 122–127.
196. R. O. Wright, C. Amarasiriwardena, A. D. Woolf, R. Jim and D. C. Bellinger,
Neurotoxicol., 2006, 27, 210–216.
Met. Ions Life Sci. 2011, 8, 263–303

Met. Ions Life Sci. 2011, 8, 305–317
12
Are Cadmium and Other Heavy Metal
Compounds Acting as Endocrine Disrupters?
Andreas Kortenkamp
The School of Pharmacy, University of London, 29-39 Brunswick Square,
London WC1N 1AX, United Kingdom
<andreas.kortenkamp@pharmacy.ac.uk>
ABSTRACT 306
1. INTRODUCTION 306
2. A MODEL FOR ESTROGEN RECEPTOR ACTIVATION BY
CADMIUM 307
3. CADMIUM EXPOSURE AND CANCER RISKS IN
ENDOCRINE-SENSITIVE TISSUES 308
3.1. Breast Cancer 308
3.2. Endometrial Cancer 309
4. IN VIVO STUDIES OF ESTROGENIC EFFECTS OF
CADMIUM 310
4.1. Proliferation of Uterine Tissues 310
4.2. Mammary Gland Development 311
5. CADMIUM AND OTHER HEAVY METALS IN IN VITRO
CELL-BASED ASSAYS OF ESTROGENICITY 311
5.1. Proliferation of Estrogen Receptor-Competent Cells 311
5.2. Estrogen Receptor Activation and Transcriptional Events 312
5.3. Phosphorylation Events in the Wake of Estrogen Receptor
Activation 313
6. WEIGHT OF EVIDENCE AND IMPLICATIONS FOR HUMAN
RISK ASSESSMENT 313
306 KORTENKAMP
ABBREVIATIONS 315
REFERENCES 315
ABSTRACT: Observations of specific interactions of the heavy metal cadmium with

the estrogen receptor have spawned a series of studies to investigate the propensity of
this and other heavy metals to act as estrogen mimicks. There is good evidence that Cd
has the ability to produce estrogenic effects in rodents, including proliferation of the
uterine and mammary tissues. These effects could be suppressed by cotreatment with
specific estrogen receptor antagonists, suggesting mediation via the estrogen receptor.
Epidemiological studies have provided some support for the idea that Cd poses cancer
risks for hormone sensitive tissues, such as the breast and the endometrium. Strikingly,
attempts to demonstrate estrogenic effects of Cd in in vitro assay systems have pro-
duced mixed results. Mitogenic effects on estrogen receptor-competent cells, activation
of estrogen receptor-dependent gene transcription and signalling events associated with
the estrogen receptor were observed in cellular models, but could not be reproduced by
others. Despite these inconsistencies, the available evidence forces the conclusion that
Cd and certain other heavy metals should be regarded as estrogen mimicks. In the con-
text of deterministic risk assessment, this should lend further support for risk reduction
measures by controlling exposure to Cd. However, data suitable for the quantitation of
estrogenic risks, especially in comparison with the established health risks of Cd, are
not yet available. It is recommended to close this knowledge gap with urgency.
KEYWORDS: cadmium . estrogenicity . estrogen mimick . heavy metal . human risk

assessment
1. INTRODUCTION
The realization that cadmium compounds and other heavy metals are cap-
able of activating the estrogen receptor [1] has not only spawned extensive
research into these substances as endocrine disrupters, but has also raised
concerns about their role as risk factors in hormone-related cancers and
other endocrine disorders. In 2003, Johnson and colleagues reported that a
single dose of 5 mg Cd/kg body weight was sufficient to promote prolifera-
tion of the uterine tissue and mammary gland milk ducts in rats, effects
considered to be hallmarks of estrogen action [2]. Given that the total human
intake of Cd from food is currently estimated as 2.8–4.2 mg/kg body weight
per week [3], these observations are provocative. However, attempts to
replicate these findings with Cd doses in the range of mg/kg body weight have
run into difficulties, and contradictory results have been reported about the
ability of Cd and other heavy metals to elicit estrogenic responses in in vitro
cell-based assays.
A critical assessment of the evidence for and against heavy metals as
estrogenic chemicals is therefore timely. By far the most data are available
for Cd compounds, and for this reason, this review will focus on Cd and deal
Met. Ions Life Sci. 2011, 8, 305–317

ARE CADMIUM AND OTHER METALS ENDOCRINE DISRUPTERS? 307
with other heavy metals in a less comprehensive way. To provide an orga-

nizing principle for the considerations that follow, we will first introduce the
model of estrogen receptor activation by Cd that was proposed by Martin
and associates [1]. We will turn to epidemiological studies of associations
between Cd exposure and risks of developing mammary carcinomas and
other endocrine-dependent cancers. Attention will then shift to experimental
studies with laboratory animals. Finally, in vitro work with cellular systems
and studies aimed at elucidating mechanisms important in Cd interactions
with steroidal receptors will be considered. The chapter will end with a
discussion of the implications for human risk assessment.
2. A MODEL FOR ESTROGEN RECEPTOR

ACTIVATION BY CADMIUM
Several isoforms of the estrogen receptor (ER) have been described, the best-
known being ERa and ERb [4]. Although the precise role of these isoforms
remains to be established, it is thought that ERa mediates the cell pro-
liferative actions of the female sex hormone estradiol, while ERb appears to
play a role in anti-mitogenic effects. Both receptors essentially function as
ligand-dependent transcription factors. Upon binding of estradiol to the
monomeric receptor molecule, a complex series of dimerisation events takes
place, with shedding of chaperone proteins. The activated receptor dimer
then exposes several docking sites for accessory proteins that trigger rapid
phosphorylations via the Src kinase, Ras and MAP kinases, and via AKT to
the PI3K signalling pathway [5]. The receptor dimer also translocates to the
cell nucleus where it binds to a specific palindromic DNA sequence termed
the estrogen response element (ERE). Upon DNA binding, steroid receptor
co-activators are recruited which stimulate transcription of specific genes.
As is typical of all members of the nuclear receptor family, both ER
isoforms possess a hormone-binding domain and a DNA-binding domain.
The DNA-binding region of the receptors is formed by tetrahedrical coor-
dination of cysteine residues with Zn21, a so-called zinc finger motif. There
are additional regions in the receptor protein that have enhancing effects on
the transactivation of transcription, and these receptor domains interact
with co-activators.
In studies with the purified ERa protein, the group around Martin [6]
demonstrated that Cd can bind the receptor with high affinity and lead to its
activation. Receptor activation could be abolished by cotreatment with the
specific ERa antagonist ICI 182,780, and this finding suggested that the
metal interacted with the ligand-binding domain of the steroid receptor.
Experiments with receptor proteins that where mutated at specific amino
Met. Ions Life Sci. 2011, 8, 305–317

308 KORTENKAMP
acid residues showed significant reductions in the metal’s binding affinity

with mutated cysteines 381 and 447, glutamic acid 523, histidine 524, and
aspartic acid 538, indicating that these amino acids are possible coordination
sites of cadmium. Although some of these putative coordination sites are in
close proximity to those of estradiol, they are distinct from the docking sites
of the hormone in the ligand binding domain. Stoica, Katzenellenbogen, and
Martin [6] hypothesize that interaction of Cd with these amino acids induces
conformational changes of the receptor very similar to those after binding of
estradiol, however, the details of this model need to be worked out further.
Considering the propensity of Cd to displace Zn from protein coordina-
tion sites, it is to be expected that Cd21 substitutes for Zn21 in the DNA-
binding domain zinc finger motif. Other metals have been shown to displace
Zn in this way, with diminutions of the ability of the DNA-binding domain
to associate with the ERE [7]. However, displacement of Zn by Cd does not
influence the affinity of the receptor to DNA.
These ideas provide a basis for anticipating the biological effects of Cd as
an estrogen mimick. If Cd essentially behaves like estrogen, and considering
that estradiol is a known risk factor in breast cancer and other hormone-
sensitive tissues, then Cd might also be implicated in these cancers, in
addition to its established role as a lung carcinogen. Are such concerns
supported by empirical evidence?
3. CADMIUM EXPOSURE AND CANCER RISKS IN

ENDOCRINE-SENSITIVE TISSUES
3.1. Breast Cancer

With a few exceptions, the number of new breast cancer cases among women
is increasing in almost all Western countries. Although late age at first child
birth and genetics are shown to contribute to the increase in breast cancer,
the sheer number of newly diagnosed cases cannot solely be explained by
these factors. It is suspected that environmental influences, including
exposure to chemicals, also play a role.
There is good evidence that estrogens are strong determinants of breast
cancer risks. This is not limited to natural estrogens formed in a woman’s
body, but extends to synthetic hormones used as pharmaceuticals, such as
those employed for the alleviation of menopausal symptoms [8,9].
Epidemiological studies conducted in occupational settings have provided
the first indications of a role of Cd in breast cancer. Cantor et al. [10]
examined death certificates attributed to breast cancer and made compar-
isons with non-cancer death certificates to analyze whether mortality from
Met. Ions Life Sci. 2011, 8, 305–317

breast cancer was linked with occupational exposure to Cd. Among white
women, Cd exposure was associated with an 8–20% increase in breast cancer
risk. This rose to 50–130% among African-American women. The authors
pointed out that the method of establishing Cd exposure (by occupation
listed on the death certificate) may have led to misclassifications leading to
underestimations of risk. There was no information about other known
breast cancer risk factors, and this could have distorted the results further.
A cohort of Swedish women engaged in metal plating and coating showed
a high standardized incidence ratio of breast cancer [11]. However, plating
and coating exposes workers not only to Cd, but also to hexavalent chro-
mium and organic solvents. Therefore, doubts remain as to whether the
observed increases in breast cancer can be attributed solely to Cd.
McElroy et al. [12] conducted a population-based study of 246 women
with breast cancer and 254 age-matched control subjects suffering from
other cancers, but not breast cancer. Cd levels were measured in the women’s
urine, and telephone interviews were carried out with the aim of collecting
information about other breast cancer risk factors. It was found that women
with the highest creatinine-adjusted urinary Cd levels had twice the breast
cancer risk of those with the lowest Cd levels. These risk estimates were
obtained after adjustment for established breast cancer risk factors (age,
parity, age at first birth, family history of breast cancer, body mass index,
alcohol consumption, menopausal status). A clear association with smoking
was not found, mainly due to the small number of smokers enrolled in the
study. If cadmium was involved in breast cancer, then a link between
smoking and breast cancer would be expected, given that tobacco smoke is a
major source of Cd exposure. On the other hand, smoking increases the
metabolic clearance of estrogens [13] and thus shows ‘‘anti-estrogenic’’
effects. It is unclear what impact this might have on the putative role of Cd in
breast cancer. In any case, studies of the influence of smoking on breast
cancer have produced mixed results [14,15]. The observations by McElroy
and coworkers are indicative of a statistically significant increased breast
cancer risk from Cd. However, as the authors pointed out, it is unclear
whether this association reflects a possible effect of cancer treatment or even
breast cancer itself on Cd body burden or whether it is indicative of an effect
of Cd on the initiation or promotion of tumor growth.
3.2. Endometrial Cancer

Similar to breast cancer, estrogens are also major risk factors in endometrial
cancer [16]. Akesson et al. [13] examined the hypothesis that the estrogenicity
of Cd might contribute to endometrial cancer risks. They found that Cd
intake through food was statistically significantly associated with an
Met. Ions Life Sci. 2011, 8, 305–317

310 KORTENKAMP
increased risk of postmenopausal endometrial cancer. In this study, dietary

Cd intake was estimated by using a food consumption questionnaire.
Among never-smoking women with a low body mass index, the association
was even stronger.
Taken together, the available epidemiological evidence indicates that Cd
might contribute to cancer risks in estrogen-sensitive tissues, possibly by
exerting estrogenic effects.
4. IN VIVO STUDIES OF ESTROGENIC EFFECTS OF

CADMIUM
If Cd has the capability of exerting estrogenic effects, it should stimulate the
proliferation of tissues of the female reproductive tract, specifically the
uterus. Before cell-based in vitro assays for estrogenicity became available,
measurements of proliferative effects in the uterus were the only way of
assessing the actions of estrogen. The assay is commonly conducted with
mice or rats whose ovaries had been removed (ovariectomised rats or mice)
and is still regarded as the ‘‘gold standard’’ for measuring estrogenicity.
Rodents also offer the possibility of investigating the proliferative effects of
estrogens on the mammary gland. Estrogens typically promote the growth
and branching of milk ducts during development. Both assays have been
used to study possible estrogenic effects of Cd.
4.1. Proliferation of Uterine Tissues

Johnson and coworkers [2] first reported strong proliferative effects of Cd in
the uterus of ovariectomized Sprague-Dawley rats. The animals were dosed
intraperitoneally with a single Cd dose of 5 mg/kg body weight and the
uterine response measured 4 days later. A 1.9-fold increase in uterine wet
weight was observed relative to untreated control animals. The proliferative
effects of Cd could be abrogated by co-administration of the ER antagonist
ICI 182,780, suggesting that the responses were mediated by the ERa.
Although the uterotrophic effects of Cd could be confirmed by other
laboratories, the exquisite sensitivity of the uterine tissue to Cd seen by
Johnson et al. could not be reproduced by others. For example, in the hands
of Höfer et al. [17] single intraperitoneal doses of 0.5 and 2 mg Cd to Wistar
rats were needed to obtain statistically significantly elevated uterine weights
relative to untreated controls, i.e., 100 to 400-fold higher than the doses
employed by Johnson and colleagues. Höfer et al. also investigated the
Met. Ions Life Sci. 2011, 8, 305–317

influence of route of administration on uterotrophic effects and failed to

observe any proliferation after oral dosing. They showed that the uterine Cd
levels that result from oral exposure were far lower than those seen after
intraperitoneal administration, explaining the lack of effect after oral
administration. Similarly, Alonso-Gonzalez and coworkers [18] observed
uterotrophic effects of Cd in ovariectomized Balb mice after intraperitoneal
dosing, but only with considerably higher doses and longer treatment
durations than Johnson and colleagues (2 mg/kg body weight, dosed for 5
days a week over 7 weeks). Increases in the uterine weight of the treated
animals were observed with adult mice treated for 7 weeks, and with ovari-
ectomized mice exposed for 4 or 7 weeks, but not with prepubertal mice.
Johnson et al. [2] were able to substantiate the hypothesis that the pro-
liferative effects of Cd in the uterus and the mammary gland went hand in
hand with specific estrogenic effects of the metal compound. Estradiol typically
stimulates the expression of the progesterone receptor and of complement C3,
and these effects were seen in both the uterus and the mammary gland after a
single Cd dose of 5 mg/kg body weight. Höfer et al. [17] also observed C3
induction in the uterine tissue after treatment with Cd, albeit at higher doses.
4.2. Mammary Gland Development

Like estradiol, Cd was able to promote proliferation of ducts in the mam-
mary gland of Sprague-Dawley rats that received a single dose of 5 mg/kg
body weight [2]. Johnson and colleagues [2] showed that Cd increased the
epithelial area and the number of terminal end buds. Alonso-Gonzalez and
coworkers [18] also observed such effects on the mammary gland of Balb
mice, but as before with uterotrophic effects, much higher Cd doses (0.5 and
2 mg) and longer exposure times (4 or 7 weeks) were needed to demonstrate
effects on mammary gland development in mice. Proliferative effects were
only seen in adult or adult ovariectomized mice, but not in prepubertal
animals, where Cd even inhibited mammary gland development.
5. CADMIUM AND OTHER HEAVY METALS IN

IN VITRO CELL-BASED ASSAYS OF
ESTROGENICITY
5.1. Proliferation of Estrogen Receptor-Competent Cells

MCF-7 is an established cell line derived from human mammary epithelia.
They contain the ERa and rely on estrogen for cell division. This property
Met. Ions Life Sci. 2011, 8, 305–317

312 KORTENKAMP
has been exploited for the screening of diverse chemicals for estrogenic
effects. MCF-7 cells are widely used to study a range of biochemical effects
of estrogens.
Garcia-Morales and colleagues [1] were the first to describe mitogenic
effects of Cd on MCF-7 cells. The cells were treated with 1 mM Cd for 6 days
and their number determined. After 4 days of exposure, the cell numbers
equalled those in cultures treated with 1 nM estradiol. Similar results were
obtained by Choe et al. [19], Martinez-Campa et al. [20], and Brama et al.
[21]. Choe et al. [19] observed mitogenic effects at very low Cd concentra-
tions, in the range between 1 and 100 nM, and also reported other metal
compounds, including antimony, barium, chromium(VI), lead, and mer-
cury(II) as promoters of MCF-7 cell division.
However, our own laboratory was unable to reproduce these observations
[22]. In experiments with Cd chloride obtained from three different suppli-
ers, the heavy metal was without detectable proliferative effects when tested
in the range between 10 pM and 10 mM. With T47D cells, another ER-
competent line that responds to estradiol by cell division, Zang et al. [23] also
failed to observe proliferative effects of Cd (measured as increases in DNA
synthesis).
Silva et al. [22] investigated possible joint effects of Cd and estradiol on
cell proliferation of MCF-7 cells and found the metal to dampen the effects
of the hormone.
5.2. Estrogen Receptor Activation and Transcriptional

Events
A variety of in vitro assays are available where ER activation is measured
specifically with reporter gene constructs. The T47D cell line permanently
transformed with a construct linking the ERE with luciferase, is exquisitely
sensitive to estradiol and other estrogenic chemicals. Choe et al. [19] and
Wilson et al. [24] observed inductions of luciferase activity with Cd con-
centrations in the nanomolar range, and these effects could be abrogated by
cotreatment with the ER antagonist ICI 182,780. As with MCF-7 cell pro-
liferation, Choe et al. obtained similar effects with a range of other metal
compounds, including antimony, barium, lithium, chromium(VI), and lead.
However, in our own laboratory [33] Cd-induced ER transcriptional acti-
vation was not observed.
The yeast estrogen screen (YES) is another convenient and widely used
screen for estrogenic activity. It employs yeast cells permanently transfected to
express the human ERa, together with a reporter plasmid that couples the ERE
with b-galactosidase. The YES has been used by a number of laboratories,
Met. Ions Life Sci. 2011, 8, 305–317

including our own [22], to assess the ability of Cd to activate the ERa, with
largely negative results. Le Guevel et al. [25] and Denier et al. [26] did not
observe any estrogenic effect of Cd in the YES. This lack of activity was not
due to problems with cellular uptake of the metal compounds, since toxic
effects on the yeast cells were seen.
In coexposures with estradiol and Cd, Le Guevel et al. [25], Vetillard and
Bailhache [27], and Silva et al. [22] observed that the metal inhibited the
transactivation function of ERa in the YES assay. Interestingly, Denier et al.
[26] reported the opposite effect: in their hands, Cd, although inactive on its
own, sensitized the ERa to the actions of estradiol, leading to significant
downward shifts of threshold concentration of the hormone. This phe-
nomenon was also observed with copper(II) and zinc(II) [28].
5.3. Phosphorylation Events in the Wake of Estrogen

Receptor Activation
In addition to inducing translocation of the activated ER to the cell nucleus,
with consequent promotion of gene transcription, estradiol can also trigger
phosphorylation events in the Src/Ras/Erk1,2 and AKT/PI3K pathways,
within minutes of exposure to ER-competent cells. These endpoints afford
further ways of investigating specific estrogenic effects of Cd. As would be
expected from an ER agonist, Cd was found to induce phosphorylations of
the Erk1,2 kinases in HEK293, HeLa, and HepG2 cells [29]. These effects
could be confirmed by using MCF-7 cells with Cd concentrations of up to
10 mM; in addition, phosphorylations of the AKT kinase were observed
[21,23,30]. All the above phosphorylation events could be suppressed by
cotreatment with the ER antagonist ICI 182,780, suggesting that the ERa
was directly involved [21,30]. In contrast, Silva et al. [22] did not observe
phosphorylations of Erk1,2 in MCF-7 cells after treatment with 0.1 mM Cd
for up to 20 minutes. Phosphorylations of the Src kinase were also not seen.
6. WEIGHT OF EVIDENCE AND IMPLICATIONS FOR

HUMAN RISK ASSESSMENT
The original observation by Garcia-Morales et al. [1] of specific ER acti-

vations by Cd could be confirmed in in vivo experiments with rodents where
Cd treatment led to proliferations of the uterine and mammary gland tissues,
responses regarded as hallmarks of estrogen action. These effects could be
suppressed by cotreatment with a specific ER antagonist, suggesting that
they are mediated by the steroid receptor, and were not the result of other
Met. Ions Life Sci. 2011, 8, 305–317

314 KORTENKAMP
mechanisms. Although there are contradictory results about the potency of

Cd in inducing these estrogenic responses, the in vivo estrogenicity of Cd is
not under dispute. The lack of responses under certain experimental con-
ditions (e.g., after oral delivery of the metal) can be attributed to insufficient
amounts of the metal reaching target tissues.
There are even indications of a possible role of Cd in neoplasias of estrogen-
sensitive tissues such as the breast and endometrium. Whether or not this can
be attributed to estrogenic effects of Cd is unclear, mainly because the ways in
which estradiol itself causes breast or endometrial cancer are not resolved.
While a mechanism for estrogen’s role in the early events leading to breast
cancer is not currently defined, it is widely held that estrogens play a role in
the later stages of the disease by providing mitogenic stimuli for transformed
cells that rely on estrogens for proliferation. Viewed from such a perspective,
the ability of Cd to activate the ER should be regarded as providing biological
plausibility for a role in mammary and endometrial carcinogenesis. The
available epidemiological evidence does not contradict this idea, although
further studies are warranted to substantiate risks. In the intervening time, it is
advisable to treat Cd as if it were involved in breast and endometrial cancer.
While the in vivo effects of Cd have proven to be reproducible, it is striking
that the studies investigating events closer to receptor activation in vitro have
produced such mixed results. Concerning cell proliferation in ER-competent
cells, the majority of published studies describe positive effects of Cd, but a
substantial number of papers report an absence of effects. The same is true
for investigations looking at transcriptional activation of the ERa.
Although the reasons for these discrepancies are currently unclear, the
following factors deserve consideration: Cd may not have been bioactive
where negative results were reported, e.g., due to lack of uptake into cells.
However, this is unlikely considering that with an absence of estrogenic
effects Cd was able to abrogate estradiol-induced estrogenicity. This sup-
pression of estrogenicity could not have been possible had the metal not been
able to enter cells. However, estrogenic effects of cadmium could have been
masked by factors such as intervening cell toxicity or by active detoxification
mechanisms including binding to metallothionein or glutathione, and these
factors deserve serious consideration. It is well established that there are
considerable differences between various MCF-7 cell stocks [31] and it is
conceivable that the cells employed in the studies that have yielded negative
outcomes expressed higher levels of glutathione or metallothioneins, thus
obscuring Cd estrogenicity.
Despite these inconsistencies in detail, negative findings by some groups
do not carry sufficient force to dismiss positive observations of in vitro Cd
estrogenicity by others, especially as these positive reports emanate from
different laboratories and therefore have to be classed as independent
observations. Taken together, the available evidence shows clearly that Cd
Met. Ions Life Sci. 2011, 8, 305–317

has the capacity to activate the ER and to trigger physiological, cellular, and
biochemical events that are characteristic of the actions of estradiol. On
balance therefore, it is prudent to regard Cd as an estrogen mimick.
The question is how this evidence should be treated in human risk
assessment? There are various approaches to chemicals risk assessment in
general [32]. First, risk assessment can be carried out with the aim of pro-
viding trigger values for regulatory action to protect humans from harm, so-
called deterministic risk assessment. In this case, a bias towards conservatism
and worst case assumptions is essential. Considering that the tolerable intake
values pronounced by the World Health Organisation do not signify ‘‘safe’’
exposures, the evidence of Cd estrogenicity should provide strong support for
further measures aimed at minimizing human exposures to Cd.
Second, there is risk assessment aimed at quantifying the magnitude of
impact resulting from certain exposures to chemicals. Such approaches need
to be as accurate as possible in their risk estimates; they tend to utilize
probabilistic methods. A key issue that needs to be resolved in the context of
risk quantitations is whether the estrogenic effects of Cd occur at dose levels
that are lower than those known to be associated with kidney dysfunction or
pulmonary carcinogenesis. Resolution of this question requires dose-
response information from in vivo studies with estrogenicity endpoints, but
such data are not yet available. It is urgent to fill this gap.
ABBREVIATIONS
AKT serine/threonine kinase on the pathway of phosphati-
dylinositol 3-kinase
complement C3 protein of the immune system
ER estrogen receptor
ERE estrogen response element
ICI 182,780 trade name: fulvestrant
MAP kinase mitogen-activated protein kinase
Ras kinase rat sarcoma kinase
Src kinase sarcoma kinase
YES yeast estrogen screen
REFERENCES
1. P. Garcia-Morales, M. Saceda, N. Kenney, N. Kim, D. S. Salomon, M. M.
Gottardis, H. B. Solomon, P. F. Sholler, V. C. Jordan and M. B. Martin, J. Biol.
Chem., 1994, 269, 16896–16901.
Met. Ions Life Sci. 2011, 8, 305–317

316 KORTENKAMP
2. M. D. Johnson, N. Kenney, A. Stoica, L. Hilakivi-Clarke, B. Singh, G. Chepko,

R. Clarke, P. F. Sholler, A. A. Lirio, C. Foss, R. Reiter, B. Trock, S. Paik and M.
B. Martin, Nature Med., 2003, 9, 1081–1084.
3. L. Jarup, M. Berglund, C. G. Elinder, G. Nordberg and M. Vahter, Scand. J.
Work Env. Health, 1998, 24 (suppl. 1), 1–52.
4. K. Pettersson and J. A. Gustafsson, Am. Rev. Physiol., 2001, 63, 165–192.
5. R. O’Lone, M. C. Frith, E. K. Karlsson and U. Hansen, Mol. Endocrinol., 2004,
18, 1859–1875.
6. A. Stoica, B. S. Katzenellenbogen and M. B. Martin, Mol. Endocrinol., 2000, 14,
545–553.
7. P. F. Predki and B. Sarkar, J. Biol. Chem., 1992, 267, 5842–5846.
8. R. C. Travis and T. J. Key, Breast Cancer Res., 2003, 5, 239–247.
9. Million Women Study Collaborators, The Lancet, 2003, 362, 419–427.
10. K. P. Cantor, P. A. Stewart, L. A. Brinton and M. Dosemeci, J. Occup. Med.,
1994, 37, 336–348.
11. G. Ursin, L. Bernstein and M. C. Pike, in Cancer Surveys, Ed. R. Doll, J. F.
Fraumeni and C. S. Muir, Cold Spring Harbour Laboratory Press, Plainview,
NY, 1994, pp. 241–264.
12. J. A. McElroy, M. M. Shafer, A. Trentham-Diez, J. M. Hampton and P. A.
Newcomb, J. Natl. Cancer Inst., 2006, 98, 869–873.
13. A. Akesson, B. Julin and A. Wolk, Cancer Res., 2008, 68, 6435–6441.
14. N. Hamajima, K. Hirose, K. Tajima, T. Rohan, E. E. Calle and C. W. Heath, Br.
J. Cancer, 2002, 87, 1234–1245.
15. C. L. Li, K. E. Malone and J. R. Daling, Cancer Causes Control, 2005, 16, 975–
985.
16. A. Akhmedkhanov, A. Zeleniuch-Jacquotte and P. Toniolo, Ann. N.Y. Acad.
Sci., 2001, 943, 296–315.
17. N. Hõfer, P. Diel, J. Wittsiepe, M. Wilhelm and G. H. Degen, Toxicol. Lett.,
2009, 191, 123–131.
18. C. Alonso-Gonzalez, A. Gonzalez, O. Mazarrasa, A. Guezmes, S. Sanchez-
Mateos, C. Martinez-Campa, S. Cos, E. J. Sanchez-Barcelo and M. D. Media-
villa, J. Pineal Res., 2007, 42, 403–410.
19. S. Y. Choe, S. J. Kim, H. G. Kim, J. H. Lee, Y. Choi, H. Lee and Y. Kim, Sci.
Total Environ., 2003, 312, 15–21.
20. C. Martinez-Campa, C. Alonso-Gonzalez, M. D. Mediavilla, S. Cos, A. Gon-
zalez, S. Ramos and E. J. Sanchez-Barcelo, J. Pineal Res., 2006, 40, 291–296.
21. M. Brama, L. Gnessi, S. Basciani, N. Cerulli, L. Politi, G. Spera, S. Mariani, S.
Cherubini, A. S. d’Abusco, R. Scandurra and S. Migliaccio, Mol. Cell Endocri-
nol., 2007, 264, 102–108.
22. E. Silva, M. J. Lopez-Espinosa, J. M. Molina-Molina, M. Fernández, N. Olea
and A. Kortenkamp, Toxicol. Appl. Pharmacol., 2006, 216, 20–28.
23. Y. Zang, S. Odwin-DaCosta and J. D. Yager, Toxicol. Lett., 2009, 184, 134–138.
24. V. S. Wilson, K. Bobseine and L. E. Gray Jr., Toxicol. Sci., 2004, 81, 69–77.
25. R. Le Guevel, F. G. Petit, P. Le Goff, R. Metivier, Y. Valotaire and F. Pakdel,
Biol. Reprod., 2000, 63, 259–266.
Met. Ions Life Sci. 2011, 8, 305–317

26. X. Denier, J. Couteau, M. Baudrimont, E. M. Hill, J. Rotchell and C. Minier,

Marine Environ. Res., 2008, 66, 108–110.
27. A. Vetillard and T. Bailhache, Biol. Reprod., 2005, 72, 119–126.
28. X. Denier, E. M. Hill, J. Rotchell and C. Minier, Toxicol. In vitro, 2009, 23, 569–
573.
29. M. B. Martin, R. Reiter, T. Pham, Y. R. Avellanet, J. Camera, M. Lahm, E.
Pentecost, K. Pratap, B. A. Gilmore, S. Divekar, R. S. Dagata, J. L. Bull and A.
Stoica, Endocrinology, 2003, 144, 2425–2436.
30. Z. Liu, Y. Xinyuan and Z. A. Shaikh, Toxicol. Appl. Pharmacol., 2008, 228, 286–
294.
31. C. Jones, J. Payne, D. Wells, J. D. Delhanty, S. R. Lakhani and A. Kortenkamp,
Cancer Genet. Cytogenet., 2000, 117, 153–158.
32. G. W. Suter and S. M. Cormier, Integrated Environ. Assess. Monitoring, 2008, 4,
486–489.
33. R. Evans and A. Kortenkamp, unpublished results.
Met. Ions Life Sci. 2011, 8, 305–317

Met. Ions Life Sci. 2011, 8, 319–373
13
Genotoxicity of Metal Ions: Chemical Insights
Wojciech Bal, 1, 2 Anna Maria Protas, 1 and Kazimierz S. Kasprzak 3
1
Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Pawinskiego 5A, PL-02-106 Warsaw, Poland
<wbal@ibb.waw.pl>
2
Central Institute for Labour Protection – National Research Institute,
Czerniakowska 16, PL-00-701 Warsaw, Poland
3
Laboratory for Comparative Carcinogenesis, National Cancer Institute at Frederick,
Bldg 538, Room 205E, Frederick, MD 21702-1201, USA
<kasprzak@mail.nih.gov>
ABSTRACT 320
1. INTRODUCTION 321
2. OVERVIEW OF CHEMICAL AND BIOCHEMICAL
PROCESSES LEADING TO GENOTOXIC LESIONS 322
2.1. Reactivity of Nucleobases 322
2.1.1. Hydrolytic Deamination 322
2.1.2. Alkylation of Nucleobases 322
2.1.3. Reactions of Nucleobases with the Hydroxyl Radical 323
2.2. Reactivity of the DNA Polymer 325
2.3. Major DNA Lesions and Their Repair 327
2.3.1. DNA Strand Breaks 327
2.3.2. Pyrimidine Dimers 328
2.3.3. Base Adducts 328
2.4. Mutations: Permanent Alterations of Genetic Information 330
3. MECHANISMS OF METAL ION GENOTOXICITY 330
3.1. Molecular Targets for Genotoxicity of Metal Ions 331
3.2. Direct Genotoxic Effects of Metal Ions 333
320 BAL, PROTAS, and KASPRZAK
3.3. Indirect Genotoxicity of Metal Ions 334

4. GENOTOXIC PROPERTIES OF SELECTED METALS 336
4.1. Arsenic 339
4.2. Beryllium 341
4.3. Cadmium 342
4.4. Chromium 343
4.5. Nickel 346
4.6. Other Metals 348
4.6.1. Cobalt 348
4.6.2. Lead 349
4.6.3. Uranium 350
4.6.4. Platinum 351
4.6.5. Copper and Iron 352
4.7. Mixtures of Metals 353
5. CRITICAL OVERVIEW OF THE EXPERIMENTAL METHODS
FOR STUDYING THE GENOTOXIC POTENTIAL OF
METALS 354
ACKNOWLEDGMENTS 358
ABBREVIATIONS 358
REFERENCES 359
ABSTRACT: The purpose of this review is to provide a reader with a brief account of
current results and views in the area of genotoxicity of metal ions, with a special atten-
tion to underlying chemical mechanisms. The text is divided into six sections. Following
a general introduction in Section 1, Section 2 describes main molecular mechanisms of
formation of genotoxic lesions: hydrolysis, alkylation, and radical reactions of nucleo-
bases and the phosphosugar DNA backbone. The basics of cellular repair of DNA
lesions are also shortly presented. This section serves as a background source for Sec-
tions 3, 4, and 5. Section 3 covers the main mechanisms of metal ion genotoxicity, fol-
lowed by Section 4, which describes genotoxicity of individual metals; i.e., of the
confirmed carcinogens As, Be, Cd, Cr, and Ni, as well as of the suspected carcinogens
Co, Cu, Fe, Pb, Pt, and 238U (also known as depleted uranium). The genotoxicity of
exposures to metal mixtures is also discussed. Section 5 provides a critical overview of
methodologies used for studying mutagenicity and carcinogenicity of metals; the final
Section 6 summarizes the current state and future perspectives of research in genotoxic
mechanisms of metal ions.
KEYWORDS: arsenic . cadmium . carcinogenesis . chromium . genotoxicity .

molecular mechanisms . nickel
Met. Ions Life Sci. 2011, 8, 319–373

GENOTOXICITY OF METAL IONS: CHEMICAL INSIGHTS 321
1. INTRODUCTION
The subject of this chapter is genotoxicity of metal ions, defined as

damage to cellular DNA with genetic consequences. DNA damage is a
broad term, covering all alterations of chemical bonds in DNA. It can be
inflicted either by a direct reaction of metal ions with nuclear DNA, or
indirectly through reactive intermediates generated by metal ions reacting
with other cellular components, or both. If the damage cannot be
repaired by designated cellular systems, there are three general fates for cells
with damaged DNA: necrosis (uncontrolled cell death), apoptosis (con-
trolled cell death), or mutations, resulting from genetic code alterations
fixed in the process of DNA duplication and transmission to daughter cells.
Hence, mutations are often considered as a signature of DNA damage.
We have to remember, however, that the presence of mutations does not
necessarily imply an attack of a metal ion or a metal ion-generated
reactive intermediate on DNA, since other mechanisms of DNA damage
triggered by metal ions are possible as well. According to Ames and
Shigenaga in the DNA of each cell in the human body about 10,000
lesions arise every day from natural sources, such as background ionizing
radiation or reactive oxygen species (ROS) generated in the course of
mitochondrial energy production [1]. Therefore, agents that inhibit DNA
repair, including metal ions, will appear genotoxic, too. Finally, mutageni-
city may result from dysregulation of the cell cycle, including the inhibition
of apoptosis, with no causative relationship of the agent with DNA chem-
istry. It is often very difficult to discriminate between these mechanisms
experimentally.
Metallic elements comprise a large proportion of the periodic table and
are strongly represented in lists of chemical carcinogens. In humans, the
established carcinogenic metals include (in an alphabetical order) As, Be,
Cd, Cr, and Ni. The same metals and several more (e.g., Co, Fe, Pb, Pt, U)
appeared to be carcinogenic in animal experiments. In terms of chemical
reactivities that might underlie their genotoxic properties, these elements
have very little to do with each other, thus enforcing an individual approach
in explaining the mechanisms of their carcinogenicity.
Section 2 of this chapter covers essential facts about general mole-
cular mechanisms of formation and repair of DNA lesions, thereby pro-
viding the reader with the background information for Sections 3, 4,
and 5. Section 3 deals with mechanisms of metal ion genotoxicity, Section 4
describes genotoxicity of individual metals and their mixtures, and
Section 5 provides a critical overview of methodologies used for studies
of mutagenicity and carcinogenicity of metals. The final Section 6
discusses future perspectives of research in genotoxic mechanisms of metal
ions.
Met. Ions Life Sci. 2011, 8, 319–373

2. OVERVIEW OF CHEMICAL AND BIOCHEMICAL

PROCESSES LEADING TO GENOTOXIC LESIONS
Cellular DNA can undergo three general kinds of reactivity which lead to
genotoxic lesions: hydrolysis, alkylation, and radical reactions. The general
reactions most relevant to metal ion genotoxicity are briefly described in this
section. Our account of radical reactions is limited to the hydroxyl radical,
which is a prototypical radical species in biological systems. The information
on the role of metals in the generation of other radicals, such as those derived
from lipids, proteins, amino acids, and other molecules, which may attack
DNA, can be found in numerous other publications. These radicals are often
secondary products of the hydroxyl radical attack on various molecules [2–6],
including DNA itself [7]. They generate many kinds of damage [8], including
the formation of bulky DNA adducts [9]. To complement this picture, the
reverse biochemical processes, namely the repair of damaged DNA are also
described. Effects of individual metal ions on these reactions and on DNA
repair mechanisms are presented in the following sections.
2.1. Reactivity of Nucleobases

This section deals with the reactivity of four canonical DNA bases, the purines
adenine (A) and guanine (G), and the pyrimidines cytosine (C) and thymine
(T), due to their direct relationship with genotoxicity. Reactions of bases
specific to RNA are therefore beyond the present scope (but see Section 3.3).
2.1.1. Hydrolytic Deamination

Cytosine undergoes deamination with the participation of a water molecule
or a hydroxyl ion, yielding uracil [10]. While this reaction is very slow in the
double helix, with a half-time of about 60,000 years at pH 7.4 and 37 1C, it is
accelerated in a single DNA chain to a half-time of about 200 years.
Methylcytosine is only slightly more reactive than cytosine, but provides a
mutational hotspot, believed to be due to poor repair of this modified base
[11]. The deamination of purines is much slower than that of pyrimidines,
and is therefore considered less relevant [12].
2.1.2. Alkylation of Nucleobases
Exocyclic nitrogen and oxygen atoms in nucleobases can undergo alkylation

[13]. The resulting adducts are chemically stable. Figure 1 presents the O6-G
Met. Ions Life Sci. 2011, 8, 319–373

Figure 1. O6-guanine (1) and O4-thymine (2) adducts, major promutagenic oxygen
alkylation lesions of nucleobases. E denotes an electrophile, such as an alkyl group.
Figure 2. N7 guanine alkylation. E denotes an electrophile, such as an alkyl group.
and O4-T adducts, which are particularly important, because they affect the
hydrogen bonding responsible for proper base pairing in DNA. As such,
they can induce mutations upon DNA replication.
The guanine N7 position (Figure 2) is the most nucleophilic one among all
nucleobase heteroatoms in reactions with a vast majority of alkylating
agents [14]. There is a clear relationship between the size of the alkyl sub-
stituent and genotoxicity because the larger moieties introduce more sig-
nificant distortions of the DNA structure. Bifunctional alkylating agents,
capable of cross-linking two nucleobases are the most difficult ones to repair
and are therefore considered to be the most genotoxic ones [10].
Guanine derivatized in this fashion can react further to undergo cycliza-
tion, imidazole ring opening, and other rearrangements. Alkylations of other
nucleobase nitrogens and oxygens can also lead to the hydrolysis of the
nucleobase-deoxyribose (glycosidic) bond and the formation of abasic
(apurinic and apyrimidinic) sites in DNA. The derivatization at N7 and N3
positions of adenine is the most productive in this respect, followed by the
same positions at guanine [13–15].
2.1.3. Reactions of Nucleobases with the Hydroxyl Radical
The hydroxyl radical is one of the most reactive electrophilic chemical

species, forming chemical bonds with a huge variety of targets at nearly
Met. Ions Life Sci. 2011, 8, 319–373

diffusional rates, e.g., B1010 M1s1 for double bond additions and
B109 M1s1 for aliphatic hydrogen abstractions [16]. It is therefore able to
efficiently bind at p electron-rich C5 and C6 carbon atoms of pyrimidines
and C4 and C8 carbon atoms of purines. The hydroxyl radical binding is
largely irreversible.
Accordingly, in thymine the hydroxyl radical assault initially yields
C5 (preferably) and C6 ring carbon radicals, accompanied by the formation
of the methyl radical via proton abstraction from the methyl group, as
shown in Figure 3 [17,18]. Further reactions under anaerobic conditions and
in the presence of thiols [19] lead to thymine glycol as the major product
(Figure 4). This stable modified nucleobase is one of the most relevant
genotoxic DNA lesions [20]. Under oxidative conditions the radicals
bind molecular oxygen, and multistep processes lead to the thymine ring
opening [13].
Early products of cytosine proton abstraction and deamination are not
well known. The hydroxyl radical attaches itself to the C5QC6 double bond,
with a preference for the C5 position, similarly to thymine. The resulting
radicals are converted to various final products, including cytosine glycol, a
precursor to 5-hydroxyuracil, which is formed under anaerobic conditions,
and a variety of other five- and six-membered ring products.
Positions C4, C5, and C8 are targets for the hydroxyl radical assault in
guanine. These radicals can undergo oxidation or reduction, depending on
external conditions. The C8 product, which is the most prominent one,
yields 8-hydroxyguanine (8-oxoguanine) upon oxidation and hydrogen
abstraction (Figure 5). The reduction results in the imidazole ring opening,
Figure 3. The initial steps of the hydroxyl radical reaction with thymine.
Figure 4. The formation of thymine glycol under anaerobic conditions.
Met. Ions Life Sci. 2011, 8, 319–373

Figure 5. The formation of 8-oxo-G (top) and FAPy-G (bottom) upon the radical
assault on guanine, followed by oxidation or reduction, respectively. R denotes the
nucleoside/nucleotide/nucleic acid residue.
yielding the formamidopyrimidine derivative FAPyG. The duality of the 8-

OH-G/8-oxo-G nomenclature reflects the tautomeric equilibrium between
these two forms which is strongly shifted towards the oxo-tautomer under
physiological conditions [21].
8-oxo-G is not a stable and final product of guanine oxidation. It reacts
further, leading to several products, among which spiroiminodihydantoin
(Sp) and guanidinohydantoin (Gh) have recently attracted much attention
due to their very strong mutagenic potential and the confirmed formation in
vivo [22–24]. Because of this instability, the reliability of 8-oxo-2 0 -deoxy-
guanosine as a popular analytical (quantitative) marker of oxidative stress
must be questioned.
The adenine radical chemistry is very similar to that of guanine, except for
the fact that only the C4 and C8 positions are preferred hydroxyl radical
targets. The C4 product can revert to adenine under aerobic conditions,
while the C8 chemistry is analogous to that of guanine, with 8-oxoadenine
and FAPyA as major primary products.
2.2. Reactivity of the DNA Polymer

In addition to nucleobase reactions, the phosphosugar backbone of
DNA is also a target of hydrolytic, alkylating and radical yielding agents.
The spontaneous hydrolysis of the phosphodiester backbone, shown
in Figure 6, is very slow, with the reaction half-time estimated for some
30 million years under physiological conditions [25]. This reaction can be
accelerated by enzymes, phosphodiesterases, and also by complexes of some
Met. Ions Life Sci. 2011, 8, 319–373

Figure 6. The hydrolysis of phosphodiester bonds in DNA.
metal ions, such as Co(III), Cu(II), and lanthanides possessing diesterase

activity [26].
In contrast, the glycosidic bond is the weakest of all DNA bonds. The
half-time for its spontaneous decay, proceeding according to the SN1
mechanism (Figure 7), is 14,700 years for pyrimidines and as little as 730
years for purines [27]. Due to this difference of susceptibilities, this process
of generation of abasic sites is often referred to as depurination [13].
Nearly all DNA double helix heteroatoms, including the phosphodiester
group, can undergo alkylation, but the site, rate, and effectiveness of alky-
lation depend on the match between the target and the alkylating agent.
While guanine N7 is usually the most susceptible alkylation site, some
alkylating agents of a hard (in hard-soft terms) chemical character can also
target phosphate oxygens [14].
The deoxyribose moiety in DNA is also a target for assault of hydroxyl
radicals, in addition to nucleobases. The resulting hydrogen abstraction can
occur at each of the five deoxyribose carbon atoms, depending on their steric
availability. All these events eventually lead to the phosphodiester bond
cleavage [17,28,29]. Under typical cellular conditions 90% of sugar radicals
are scavenged by molecular oxygen in an oxidative pathway, and 10% are
scavenged by cellular thiols, such as GSH [30].
DNA is a conducting biopolymer and charge transport phenomena con-
tribute to the site specificity of damage [31]. These processes enable long
range radical migrations, with oligoguanine sequences serving as preferred
targets because of the lowest oxidation potential of G. Therefore, a DNA
Met. Ions Life Sci. 2011, 8, 319–373

Figure 7. The general acid-catalyzed SN1 mechanism of hydrolysis of a glycosidic

bond in DNA, illustrated for adenine.
lesion may occur away from the site of initial interaction with an oxidant,
adding complexity to relationships between local DNA structures and the
damaging factors, such as metal complexes.
2.3. Major DNA Lesions and Their Repair

2.3.1. DNA Strand Breaks
Single strand breaks (SSB) may be caused by a direct radical assault on the
deoxyribose, or indirectly, due to nucleobase damage, as outlined in Sections
2.1 and 2.2. The DNA chain breaking results in the formation of mono-
phosphate and phosphoglycol ends. This lesion is repaired either directly, or
via the base excision repair (BER) system, which fills up the missing base and
ligates the DNA ends [32]. There are two BER pathways. The short-patch
pathway repairs a single nucleotide, with the participation of the Polb
polymerase. The long-patch pathway repairs two or more nucleotides within
the break area. Polb is probably responsible for the attachment of the first
nucleotide in this pathway, followed by elongation with other DNA poly-
merases and strand ligation with DNA ligases I and IIIa.
Met. Ions Life Sci. 2011, 8, 319–373

Double-strand breaks (DSB) are critical DNA lesions, responsible for

both local and chromosomal scale damage. They are caused by a vast variety
of exogenous and endogenous factors, including ROS, cross-linking
chemical agents, all kinds of cellular stress, malfunction of DNA replication
machinery, and, last but not least, carcinogenic metals [33–35]. The DSB
recognition and repair mechanisms primarily involve the homologous
recombination (HR) and non-homologous end-joining (NHEJ) systems.
These two systems appear to compete for DSB repair, with the balance
depending on the cell type and cell cycle phase [33,36]. Both involve multiple
proteins. Separate sets of proteins are engaged in the repair steps, but there is
some phosphorylation pathway sharing during damage recognition and
repair activation. HR uses the undamaged sister chromatid as a repair
template, therefore the alternative NHEJ plays a major role in the G1 phase
of the cell cycle, when sister chromatids are not present. NHEJ recognizes
DNA breaks and ligates them without a template.
2.3.2. Pyrimidine Dimers

Pyrimidine dimers are typical UV irradiation products. Figure 8 depicts the
most common T-T dimer [37,38]. Their repair involves the nucleotide exci-
sion repair (NER) system. The human NER involves at least 11 compo-
nents, involved in the recognition of the lesion, orchestration of the repair
complex, removal of the lesion by 3 0 and 5 0 ends incision and finally the
resynthesis of the missing oligonucleotide strand by a polymerase, followed
by ligation of the ends [39].
2.3.3. Base Adducts
The base adducts, such as products of alkylation and attachment of carbon-

based radicals, are repaired dependent on the adduct size. Smaller modifica-
tions tend to be recognized by the BER system, while bulkier ones, such as
those produced by polycyclic aromatic hydrocarbons, are subject to the NER
pathway [40,41]. Table 1 summarizes eukaryotic DNA repair pathways [40].
Figure 8. The thymine dimer, formed upon the action of UV radiation.
Met. Ions Life Sci. 2011, 8, 319–373

Table 1. Eukaryotic DNA repair pathways [40].
DNA repair
pathway DNA damage Characterization
Base excision Base modifications Recognizes and removes nucleobases

repair (BER) modified by alkylation, oxidation,
Abasic sites deamination and ring opening.
PARP and Polb polymerases replace
the removed bases.
Direct repair (DR) Base modifications Recognizes and removes the following
nucleobase oxygen modifications:
O4-methyl thymine, O6-
ethylguanine, and O6-
chloroethylguanine. A very rapid
repair (within an hour after the
lesion formation), which proceeds
without breaking the DNA strand.
Nucleotide excision Bulky DNA There are two NER mechanisms: the
repair (NER) adducts transcription-coupled repair and the
global genome repair, the latter is
based on a multiprotein complex,
which removes the oligonucleotide
containing the lesion site, followed
by DNA resynthesis.
Mismatch repair Base mismatches The system is based on a cycle of
(MMR) deletions and insertions of mispaired
bases.
Homologous Single-strand Partially overlapping multiprotein
recombination breaks repair systems correcting many
(HR) lesions, including lethal double
Non-homologous Direct and strand breaks. The HR repair
end-joining secondary process uses sister chromatids as
(NHEJ) double-strand template, while the NHEJ system,
breaks active in the G1 cell cycle phase,
Inter- and ligates broken ends without a
intrastrand template.
cross-links
DNA-protein
cross-links
Base insertions and
deletions
Met. Ions Life Sci. 2011, 8, 319–373

Figure 9. The mispairing of thymine glycol with guanine leading to T-C mutation.
2.4. Mutations: Permanent Alterations of Genetic

Information
Structural alterations in damaged nucleobases affect the base pairing, and
may result in replication errors. Also abasic sites and strand breaks may
induce introduction of erroneous bases into the newly synthesized DNA
strand. Such errors may be fixed in the second replication cycle, resulting in
the established mutation. Individual lesions are correlated with particular
mutation frequencies. For example, the thymine glycol yields a T-C
transition mutation because it can pair with guanine as well as with the
canonical adenine (Figure 9). Likewise, due to base mispairing properties,
the major product of oxidative deamination of cytosine, 5-hydroxyuracil,
causes C-T transition [42]; 8-oxo-guanine, if present in the DNA template
causes G-T transversion mutations, while incorporation of the 8-oxo-
dGTP substrate into DNA results in the A-C transversion [43]. 8-
Hydroxyadenine is capable of producing both A-C and A-G mutations
[44], and guanidinohydantoin and spiroiminodihydantoin are potently
mutagenic, causing both G-T and G-C transversion mutations [22,23].
All these particularly potent promutagenic base products are presented in
Figure 10 [45–47].
3. MECHANISMS OF METAL ION GENOTOXICITY
In order to be mutagenic, rather than necrotic, the DNA damage needs to be

of a limited nature. The assaulted cell must retain its ability to undergo
division, in order to transform DNA lesions into daughter cell mutations
that would eventually lead to cancer. Such non-lethal damage to cell nuclear
DNA can be inflicted by metal ions through direct chemical interactions
Met. Ions Life Sci. 2011, 8, 319–373

Figure 10. Examples of promutagenic products of oxidation of nucleobases.
with the DNA molecule, or indirectly through (i) generation of reactive

species capable of attacking nuclear DNA and free nucleotides, (ii) shifting
the cellular redox balance toward oxidation, and (iii) inhibition of DNA
repair mechanisms. The molecular mechanisms of such actions of metal ions
are described in this section in general terms, followed by a specific account
of reactivity of individual metals in Section 4.
3.1. Molecular Targets for Genotoxicity of Metal Ions

Nuclear chromatin, the main target for genotoxic insults, provides metal
ions with two major types of binding molecules: DNA and nuclear proteins.
Theoretically, the binding by DNA is assured by the phosphate moieties and
nitrogen heteroatoms of nucleobases. But in practice this may be moderated
by base shielding (see Section 3.2). The rich coordination chemistry of
nucleobases, nucleosides, and nucleotides has been described in detail in
many reviews, notably those by Sigel et al. [48,49]. For most metal ions, the
anionic phosphate oxygens are the universal primary binding sites in
nucleotides and nucleic acids. Free nucleobases and nucleosides, lacking
these oxygens, form much weaker complexes and cannot serve as targets for
metal ion binding. Also, it seems that among the free nucleotides only the
triphospho-nucleosides, which are the building blocks for DNA synthesis,
constitute a target relevant to the mechanisms of metal mutagenicity and
carcinogenesis [50].
Met. Ions Life Sci. 2011, 8, 319–373

Metal-phosphate oxygen bonds have mostly ionic character. As a con-

sequence, the majority of carcinogenic metal ions bind at the phosphate
groups with stability constants similar to that of the Mg21 ion, which is
considered to be their physiological partner neutralizing the negative charge
[49]. Taking into account the high intracellular concentration of Mg21, the
coordination of divalent carcinogenic cations, such as Be(II), Cd(II) or
Ni(II), to nucleotide phosphates alone does not seem to be of genotoxic
importance. However, those transition metal ions which have high affinity
for nitrogen ligands, such as Co(II), Ni(II), Cu(II), Cd(II) or Pt(II), interact
with nucleobase ring nitrogen heteroatoms. Among these, the N7 nitrogens
of adenine and guanine form the bonds with such metal ions most effec-
tively, yielding closed macrochelate structures that affect nucleotide con-
formation (Figure 11) [49,51].
The binding of metal ions to nuclear proteins, mainly histones, is governed
by general rules of metal-protein interactions [52]. The binding sites are
provided by side chain ligands of Cys, His, Trp, Phe, Glu, Asp, and in some
Figure 11. A simplified structure of a nucleoside triphosphate-metal ion closed-ring

macrochelate (dGTP used as an example). R denotes a water molecule or an addi-
tional ligand, the metal ion may be 4-, 5-, or 6-coordinate, and the triphosphate
moiety may provide two or three bonds.
Met. Ions Life Sci. 2011, 8, 319–373

cases by deprotonated amides of peptide bonds [5,53,54]. Concerning the

mechanisms of metal genotoxicity, such binding may lead to the formation
of redox active metal centers close to nuclear DNA, generating ROS and
thus, indirectly, facilitating DNA damage. In the cytoplasm, metal ions
encounter a wide spectrum of binding partners, including proteins, peptides,
amino acids, reduced glutathione (GSH), and other molecules. Some of
them may become partners in the formation of genotoxic DNA adducts,
e.g., with Cr(III) [55], and some others may facilitate oxidative DNA
damage [56–58].
3.2. Direct Genotoxic Effects of Metal Ions

The double-helical DNA structure shields the bases effectively from the
surrounding solution, leaving the phosphosugar backbone as the main target
for metal ions. Test tube experiments demonstrate that a vast majority of
metal ions bind to the DNA solely via phosphate moieties. The resulting
binding affinities for most of the divalent cations, including those being
confirmed carcinogens, are similar to that of the physiological Mg21 ion,
analogously to the pattern known for trinucleotides [59–61]. The same
pattern of affinities was seen in experiments using isolated nucleosomes, i.e.,
complexes of DNA with histone proteins, which serve as the basic structural
element of nuclear chromatin [62]. These labile, ionic bonds cannot support,
however, genotoxic adduct formation. Two additional factors are required
for such to occur. Some metal ions form particularly strong bonds with
guanine and adenine N7 nitrogens. Among the toxicologically relevant ones,
Cu(II) and Pt(II) fall into this category. The N7 binding destabilizes the local
DNA structure. In the case of the redox-capable copper, the formation of
such a complex leads to base oxidation, most commonly to the promuta-
genic 8-oxo-G derivative, and indirectly to strand breaks [63,64]. Pt(II) has
not been demonstrated to catalyze redox reactions at DNA, but it is a
kinetically inert metal ion and its interactions with purine N7 nitrogens lead
to the formation of stable genotoxic DNA adducts (see Section 4.6) [65]. The
Cr(III) ion is also kinetically inert and was shown to produce genotoxic
bulky DNA adducts in a form of ternary complexes coupled to DNA via
phosphate oxygens. An additional guanine N7 interaction in such complexes
has also been proposed to account for the sequence specificity of ensuing
DNA lesions (see Section 4.4) [55].
Many metal ion complexes are able to hydrolyze the phosphodiester bond
in DNA, yielding strand breaks. Examples include Ce(IV), lanthanides, and
engineered zinc fingers [66–68]. This kind of reactivity is, however, devel-
oping towards the biotechnology use of DNA engineering. The current state
Met. Ions Life Sci. 2011, 8, 319–373

of knowledge does not indicate any relevance of this otherwise very inter-
esting chemistry for mechanisms of metal genotoxicity.
3.3. Indirect Genotoxicity of Metal Ions

The reactivity of nucleotides can provide indirect mechanisms of metal
genotoxicity. The hydrolysis of the phosphate moiety and base oxidation are
two kinds of toxic reactivity that can result from direct nucleotide-metal ion
interactions. The hydrolysis has been studied very broadly for ATP, and
strong accelerations of this process by metal ion complexation were noted
[69,70]. It is conceivable, while not proved, that some of the carcinogenic
metal ions may complement their toxicity by diminishing the energy pool of
the assaulted cell in this fashion.
The oxidation of the nucleobase in a nucleoside or nucleotide has been
demonstrated in many experiments for redox-capable metal ions, such as
Cu(II), Ni(II) or Co(II). Such oxidation occurs in binary metal ion/
nucleotide systems, as well as in the presence of additional strong bioligands,
such as histidine [71,72]. The oxidation may occur in the coordinated base,
e.g., by a direct action of a metal peroxo species, and also in an uncoordi-
nated base via a diffusible ROS. In the case of Ni(II) the oxidation of dGTP
to promutagenic 8-oxo-dGTP in ternary complexes has been indentified in
vitro and is also likely to occur in vivo. The 8-oxo purine triphosphates were
shown to be introduced into the newly synthesized DNA chain. This pool is
not repaired as efficiently as that of 8-oxoguanine within the DNA, thereby
constituting a serious mechanism of promutagenic DNA damage [73–75].
All kinds of RNA interact with metal ions, and the much more open and
flexible structures (compared to DNA) of these largely single strand nucleic
acids make them much more prone to various kinds of metal-dependent
assault. The hydrolysis of phosphodiester bonds in RNA and its oligo-
ribonucleotide models was demonstrated for very many kinds of metal ions,
including the carcinogenic ones, and their complexes [76,77]. The chemical
properties of these systems, including the sequence specificity of hydrolysis,
suggest that their reactivity may contribute heavily to epigenetic mechanisms
of metal ion genotoxicity, e.g., by leading to the formation of altered protein
sequences without having affected the parent DNA template. The related
research will undoubtedly bloom in the future.
The generation of diffusible DNA-reactive species by metal ions con-
stitutes a major chemical pathway in chemical carcinogenesis. Redox-active
metals, such as nickel and cobalt, can generate such species directly [78]. The
Haber-Weiss/Fenton reactions, yielding hydroxyl radicals from metabolic
hydrogen peroxide, provide a classical and still valid mechanism for such
Met. Ions Life Sci. 2011, 8, 319–373

reactivity. Other mechanisms include generation of metal-peroxo and metal-

oxo species, which can interact with DNA [79]. Metal-derived ROS also
react with other molecular targets, producing secondary radical species,
capable of assaulting DNA. Such intermediate genotoxic species include
lipid peroxides and hydroperoxides, as well as C- and S-centered radicals
from amino acids, peptides, and proteins [80,81].
Certain Ni(II) complexes with oligopeptides can also generate radical
species from molecular oxygen in vitro, but the presence of such species
in vivo has not yet been demonstrated [82,83]. The mechanistic ground for
such generation is provided by the phenomenon of shifting the metal redox
potential by certain organic ligands to values allowing for electron transfer
between the metal and other molecules [5]. For example, Ni(II), which does
not react with O2 or H2O2 as an aqua-cation, becomes reactive towards these
molecules when chelated by tetraglycine or glycylglycylhistidine. The
resulting chain reactions produce radical species originating from the oxygen
substrate and the organic ligand [5]. Such species may be capable of
damaging DNA and histones as observed in test tube experiments [84]. In
cultured cells, H2O2 (the major substrate for these reactions) may be a
metabolic product escaping destruction by antioxidant enzymes inhibited by
the metal [85,86]. In animals, H2O2 as well as other ROS are produced by
inflammatory cells responding to the presence of carcinogenic metal parti-
cles in a tissue [87]. Carcinogenic metal ions can also cause oxidative DNA
damage by depleting low molecular weight cellular antioxidants, such as
glutathione, ascorbic acid, and lipoic acid. As a result, hydrogen peroxide
and other ROS generated as byproducts of the normal cellular metabolism
are not scavenged sufficiently and can elevate the background level of DNA
damage. A further indirect effect of antioxidant depletion is the shift of the
cellular redox balance towards the oxidative conditions. This may lead to
cell cycle arrest and apoptosis, which would prevent carcinogenesis. How-
ever, concurrent mechanisms, such as metallothionein induction by Cd(II),
may overcome this natural protective action, leading to carcinogenesis (see
Section 4.3) [88,89]. A direct catalysis of ascorbate oxidation with ambient
oxygen has been demonstrated in vitro for several metal ions, including
Ni(II), Co(II), Mn(II), Cu(II), and V(V), but not Cr(III), As(III) and As(V)
[90,91]. However, the inhibition of enzymes maintaining the antioxidant
levels or destroying ROS (glutathione peroxidase, catalase, redoxins) is
likely a more universal pathway, available for both redox and non-redox
metals, such as cadmium [45].
As shown in the in vitro and in vivo experiments, the radical species gen-
erated in the presence of metal ions assault DNA to form a variety of base
derivatives, cross-links and degradation products. Many of them have base-
mispairing properties that may lead to mutations, as reviewed in [45]. For
example, the G-T transversion mutation typical for the 8-oxo-guanine
Met. Ions Life Sci. 2011, 8, 319–373

presence in template DNA has been observed in nickel-induced tumors in

rats [92] and the tandem C-C-T-T transition mutation, typical for C-C
cross-links in DNA, has been found in vitro following template DNA
exposure to the Ni(II)-Gly-Gly-His/H2O2 reaction mixture [93]. Certain
products of metal-induced lipid peroxidation could also be capable of
reaching the cell nucleus and forming mutagenic adducts [94]. This scenario,
however, awaits experimental verification.
The inhibition of DNA repair is one of the most universal general
mechanisms of genotoxicity of metal ions. The most complete evidence for
this idea comes from studies of the NER and BER systems, notably by
Hartwig and collaborators [95,96]. The current list of metal ions demon-
strated to interfere with DNA repair includes Ni(II), Cd(II), Co(II), Cr(VI),
As(III), As(V), Cu(II), Cu(I), Pb(II), and Hg(II). This concept provides an
explanation of apparent contradictions, demonstrated in metal carcinogen-
esis studies, like the oxidative damage in cells exposed to non-redox metals,
e.g., cadmium, and the low mutagenicity of most carcinogenic metal ions,
except for chromium(VI). It also accounts for synergies in carcinogenic
effects of coexposures to metal ions and organic carcinogens [96]. The
interactions are on the level of individual proteins of repair cascades, and are
therefore both metal- and system-specific. For example, in the NER system
cadmium and nickel were found to impair DNA damage recognition, cobalt
inhibited the DNA polymerization step, and all of these three metals reduced
the frequency of repair incisions. The repair systems are rich in zinc finger
proteins, which participate in both the recognition of damaged DNA sites
and the formation of multiprotein repair complexes. An assault of carci-
nogenic metals on zinc finger (ZF) moieties can result in metal substitutions
and cysteine oxidations, both leading to the loss of ZF functions. This idea,
illustrated in Figure 12, is well supported by in vitro studies [97,98].
It is also possible that the destruction of cellular ascorbate, observed in
metal-exposed cells [99,100], besides other effects, leads to inhibition of a
novel class of DNA repair enzymes, the alkyl-DNA-dioxygenases (ABH2
and ABH3 in humans). These enzymes are non-heme iron-dependent oxy-
genases, which need ascorbate to maintain iron in the Fe(II) state. Their
inactivation would thus enhance mutagenesis by endogenous DNA alky-
lating agents, such as S-adenosylmethionine [101].
4. GENOTOXIC PROPERTIES OF SELECTED METALS
The aim of this section is to provide a brief overview of specific genotoxic

and mutagenic properties of individual metals and metalloids that may cause
cancer. The World Health Organization’s International Agency for
Met. Ions Life Sci. 2011, 8, 319–373

Figure 12. Three kinds of assault by carcinogenic metals on zinc fingers (ZF). The
interaction may result in isomorphic Zn(II) substitution, such ZF may, however,
exhibit different reactivity, in non-isoformic substitution; impairing the functional
ZF structure; and in oxidation, also impairing the ZF.
Research on Cancer (IARC) rates chemical elements and compounds

according to their carcinogenicity. Group 1 includes confirmed human
carcinogens, and groups 2A and 2B include substances assigned as probable
and possible carcinogens, respectively. Group 3 contains chemicals declared
non-carcinogenic according to the current state of knowledge. Current
classifications and analyses provided by IARC can be freely accessed in the
internet at http://www-cie.iarc.fr/monoeval/grlist.html.
Elements, which are group 1 human carcinogens according to the IARC
classification, are described individually in the alphabetical order (Figure 13).
They include arsenic [102], beryllium [103], cadmium [104], chromium [105],
and nickel [106]. Exposures to all of them result in the increased incidence of
respiratory cancers. In addition, arsenic exposure is a confirmed causative
factor for skin, kidney, liver, and bladder cancer [102,107,108], while cad-
mium exposure is implicated, in the context of cigarette smoking, in carci-
nogenesis of bladder, prostate, and pancreas [109–111].
Several other metallic elements are also covered in this section. They have
not been classified as confirmed human carcinogens by IARC, but at least
some of them are likely to be included in the future, depending on the
accumulation of epidemiological data. The elements discussed include
cobalt, lead, depleted uranium, platinum, copper, and iron (see Figure 14). A
brief account on effects of metal mixtures is also provided.
Met. Ions Life Sci. 2011, 8, 319–373

Figure 13. An overview of genotoxic properties of IARC group 1 metals.
Met. Ions Life Sci. 2011, 8, 319–373

Figure 14. An overview of genotoxic properties of suspected carcinogenic metals.
Several excellent reviews, discussing the field of genotoxicity of metals in

general, or covering individual metals, were published in recent years. In
particular, Beyersmann and Hartwig provided a broad review of genotoxi-
city and carcinogenicity of metals [95], Salnikow and Zhitkovich described
molecular mechanisms in carcinogenesis of arsenic, chromium and nickel
[55], and Arita and Costa covered epigenetic mechanisms in arsenic, cad-
mium, chromium, and nickel carcinogenesis [112].
4.1. Arsenic
Arsenic poses a major health hazard in many geographic locations, due to
the geological contamination of water sources, resulting in oral exposure
[102,113,114]. Another important route of environmental exposure to
arsenic is inhalatory, by breathing dust and fly ash produced antropogeni-
cally by fossil fuel combustion [115]. Other exposures to arsenic are related
to occupation, predominantly metallurgy and wood treatment [114,116].
Inorganic tri- and pentavalent arsenic species predominate in the
Met. Ions Life Sci. 2011, 8, 319–373

environment. At neutral pH they exist largely as arsenous acid, As(OH)3,

and monoprotonated arsenate, HAsO2 4 , respectively. The balance of
arsenite versus arsenate versus organic arsenic derivatives in water and soil
depends on pH, oxygenation, general redox conditions, and bioactivity of
microorganisms [117,118].
Inorganic arsenite and arsenate undergo a complex biotransformation in
the human body, consisting of reduction, methylation, and thioester for-
mation steps, which yield a mixture of species, among which mono-
methylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII) seem
to be particularly relevant toxicologically [118–121]. This biotransformation
occurs largely in the liver, but there is extensive redistribution of arsenicals
throughout the human body, as evidenced by the lack of association between
the route of exposure and the localization of resulting tumors [102,120].
Most chemical studies indicated the absence of a direct interaction
between toxicological arsenic species and DNA, even at very high millimolar
concentrations of the former [118]. In accord with these results, arsenicals
were not found to introduce point mutations into DNA in bacterial and
mammalian mutagenicity assays [95]. An ability of introducing double
strand breaks was, however, attributed to methylated As(III) derivatives in
an in vitro plasmid DNA assay [122]. This activity was proposed to stem
from the redox cycling between As(III) and As(V) methylated species. On
the other hand, inorganic arsenate, despite of being an oxidant, does not
interact with DNA, presumably due to its 2 charge under physiological
conditions.
In contrast to mutagenicity assays, arsenicals cause chromosomal aber-
rations, DNA strand breaks, and other oxidative DNA damage in cell line
experiments at micromolar concentrations. Similar damage is observed in
humans exposed to arsenic and in experimental animals [123]. The oxidative
stress induction is proposed as a general pathway of arsenic genotoxicity in
oxygen-rich cells and tissues, such as lymphocytes [124] and lungs [125]. A
direct ROS generation by the As(III)/As(V) redox pair, perhaps including
methylated species, as well as depletion of cellular reductants, such as GSH,
by such redox pair are cited as possible, while not an exclusive molecular
mechanism. Another, less direct mechanism of ROS generation by arsenicals
consists of impairment of mitochondrial function via direct genotoxicity of
arsenic to mitochondrial DNA [126].
Other research points, however, against direct genotoxicity as a relevant
mechanism in arsenic carcinogenesis. Arsenicals were demonstrated to
interfere with the cell division apparatus at concentrations lower than those
required for significant chromosomal aberrations [127,128]. Also, the epi-
demiology of arsenic carcinogenesis, as well as animal studies suggest that
the presence of other DNA damaging factors, such as UV radiation, is
required for deleterious effects to occur [129]. The co-mutagenicity of
Met. Ions Life Sci. 2011, 8, 319–373

arsenicals with UV radiation and organic agents inducing bulky DNA

lesions, such as PAH, was demonstrated in various in vitro and in vivo test
systems [128–130], leading to the concept of arsenic-dependent inhibition of
the NER, and perhaps other pathways of DNA repair [131–134]. Again,
both direct and indirect molecular mechanisms may be operating here. In
humans, arsenic exposure via drinking water was correlated in a dose-
dependent manner to decreased expression of NER genes, ERCC1, XPB,
and XPF, and diminished repair of lesions in lymphocytes [135]. A possi-
bility of a direct and specific interference of arsenicals with zinc finger
domains of DNA repair proteins was indicated by a chemical study [128].
PARP-1, a poly(ADP-ribosylating) DNA repair enzyme is a likely target for
such activity, as it contains two zinc finger domains and recent studies
demonstrated the interference of arsenicals with its activity. However, one of
these studies detected a general inhibition of poly(ADP-ribosylation) in cells
exposed to arsenicals [136], while another reported an increased specific
poly(ADP-ribosylation) of p53 by inorganic arsenite [137]. It is therefore
clear that we are still rather far from a clear picture of mechanisms involved
in arsenic genotoxicity.
It is interesting to note that arsenic trioxide, As2O3, the solid phase form
of As(OH)3, has been successfully used in medicine to treat acute promye-
locytic leukemia (APL) [138], and the order of efficacy of arsenicals in killing
leukemic cells, presumably via apoptosis [139], is strikingly similar to that
inducing genotoxicity in model systems.
4.2. Beryllium
Exposures to beryllium have an occupational character, and include pre-
dominantly inhalation of Be metal and BeO dusts in the course of manu-
facturing beryllium alloys. The limited evidence available suggests that the
dissolution of both metallic Be and BeO particles occurs intracellularly, at a
rather slow rate, yielding soluble Be(II) species [140,141]. Beryllium, the
prototypic alkaline earth element, shares many chemical properties with its
heavier counterpart magnesium. Its only level of oxidation is 2+, conse-
quently it has no redox chemistry in aqueous solution, and its compounds
have a predominantly ionic character [142]. On the other hand, its small
ionic radius results in a strong ability to polarize oxygen atoms, similarly to
Al(III). As a result, Be(II) ions are amphoteric and tend to form hydroxo
species in the physiological pH range [143]. Not surprisingly, there is no
evidence for direct reactivity of Be(II) compounds towards DNA. Cationic
Be(II) species, including Be21 ions would rather stabilize the DNA double
helix by ionic interactions with the phosphate backbone in the same way as
Met. Ions Life Sci. 2011, 8, 319–373

Mg21 ions do, and anionic hydroxide species would not be able to approach
DNA due to electrostatic repulsion.
In accordance with the chemical properties outlined above, bacterial
assays indicated no or little direct mutagenic potential for Be(II) salts
[144,145]. Some activity could be seen only at very high, toxicologically
improbable Be(II) concentrations, and no clear dose-response correlations
were found [145]. It is likely that these discrepant results may be artifacts
related to the sluggish formation of Be(II) hydroxide species at high
micromolar concentrations of Be(II) salts.
In contrast with bacterial assays, eukaryotic cell line studies demonstrated
such effects as sister chromatid exchanges, chromosomal aberrations, and gene
mutations [144]. Moreover, being a weak mutagen at the most, Be(II) appears
to act as a strong co-mutagen in various assays [145,146]. The downregulation
of DNA repair genes upon Be(II) exposure was also noted [147]. These results
suggest the DNA repair inhibition as a possible central mechanism of ber-
yllium genotoxicity [95]. Based on Be(II) chemistry, the phosphate and car-
boxylate groups in repair proteins are considered as likely targets. However,
specific mechanisms remain to be discovered. A possible presence of an oxi-
dative component in beryllium genotoxicity was also suggested by a recent
study, where antioxidant-fed mice exhibited a lower level of beryllium-induced
chromosomal aberrations compared to untreated controls [148].
4.3. Cadmium
All confirmed and suspected carcinogenic exposures to cadmium are of
inhalatory nature, while severe oral exposures to cadmium of large popu-
lations have not resulted in the excess risk of cancer [149]. Occupational
exposure to cadmium-containing fumes is the basis for the assignment of
cadmium to IARC group 1, however, tobacco smoking is a major non-
occupational source of cadmium in the airways, and therefore its relevance
in cadmium carcinogenesis is probably very high [150,151].
Cadmium compounds invariably contain the Cd(II) ion. The toxic effects
of particulate Cd(II) compounds, e.g., CdO, are fully accountable by the
action of subsequently dissolved Cd(II) ions [152]. As a result, the weak
phosphate binding of Cd21 observed in vitro appears to be the only direct
reactivity between Cd(II) and DNA [153]. As such it is toxicologically
irrelevant. Although the Cd(II) ion is not redox-active, typical products of
oxidative damage to DNA, such as strand breaks or 8-oxo-dG formation
were detected in Cd(II)-exposed cells, but only at high micromolar levels of
intracellular Cd(II), nearing the cytotoxic concentration range [154,155].
Therefore, indirect mechanisms of genotoxicity detectable at much lower
cadmium levels are more relevant in the context of actual procarcinogenic
Met. Ions Life Sci. 2011, 8, 319–373

human exposures, which are chronic, but involve low doses of cadmium
[156].
It is not known how the exposure to Cd(II) ions results in ROS generation,
especially as intracellular Cd(II) ions induce expression of metallothioneins
(MT) [157] and activate biosynthesis of GSH [155], both being major anti-
oxidant systems. The impairment of mitochondrial control of ROS gen-
eration was proposed to be involved, but molecular mechanisms, that might
be operating there, remain to be elucidated.
Apoptosis is a frequent result of cadmium exposure in cell cultures, but
cadmium has also been reported to inhibit apoptosis induced by other toxins
[158,159]. Cell type specificity in the interplay of pro- and antioxidative
processes seems to be the key to solve this apparent contradiction. In an
interesting case of cadmium exposure of RWPE-1 prostate cell cultures a
subset of these cells resisted apoptosis as a result of the elevation of MT level
[88]. DNA lesions could accumulate in such surviving cells, leading to
malignant transformation [89]. Alterations in gene expression patterns due
to low level cadmium exposures are also cell type-specific [89]. More research
is required to elucidate the cause-effect patterns involving these phenomena.
DNA repair inhibition is another, and perhaps more important mechan-
ism of indirect cadmium genotoxicity, which can explain the apparent
contradiction between weak mutagenicity and strong carcinogenicity of
cadmium. Cd(II) was reported to interfere with MMR, NER, and BER,
most likely interfering with the actions of individual repair proteins
[95,96,156].
Interfering with BER, Cd(II) inhibited repair of DNA oxidative damage
products [160,161] by inhibiting proteins such as OGG1, which repairs 8-
oxoguanine lesions [162] or PARP, which orchestrates SSB repair [163]. Its
action on OGG1 appears to be mediated indirectly, while that on PARP may
be direct. Cd(II) ions inhibit the initial step of the NER system, the incision
of the DNA lesion. This suggests the XPA protein, a NER repair complex
initiator to be the cadmium toxicity target [164]. In addition, Cd(II) ions
were demonstrated recently to diminish the nuclear level of XPC, the NER
lesion recognition factor [152]. The MMR inhibition by Cd(II) also involves
a direct interaction with the repair complex, resulting in the decrease of ATP
consumption by MSH6 protein, observed in human cell cultures [165,166].
4.4. Chromium
Chromium is present in the human environment at its chemically stable
levels of oxidation, Cr(0), Cr(III), and Cr(VI), as a result of its technological
usage. The level of oxidation of chromium dictates its biological properties
Met. Ions Life Sci. 2011, 8, 319–373

in a striking fashion. Cr(III) is essential for human life, as part of the still
enigmatic glucose tolerance factor (GTF) [167], while Cr(VI), in the chemical
form of the chromate ion, CrO2 4 , is a definite and complete human carci-
nogen, predominantly by respiratory exposure [105,168]. Chromate intro-
duced orally is not related to respiratory cancer, as opposed to arsenic, but
appears to target internal organs [169,170]. At the physiologic pH there are

two protonic forms of chromate in equilibrium, CrO2 4 and HCrO4 [171].
They mimic physiological sulfate ions in terms of size, shape, and charge,
and are therefore easily incorporated into the cell through sulfate channels
[172]. In contrast with carcinogenic metals described above, chromate is a
confirmed genotoxic agent, exhibiting mutagenicity in bacterial and mam-
malian cell assays [173]. Still, its anionic form precludes its direct interaction
with DNA because of mutual electrostatic repulsion. Thus, intracellular
activation is required for its genotoxic reactivity [55].
According to an established and chemically well documented view, once
inside the cell, chromate undergoes gradual reduction to yield Cr(V) and/or
Cr(IV), and, finally Cr(III) species [174–176,55]. The reduction is exerted by
abundant low molecular weight antioxidants, GSH and cysteine [177], and
ascorbate [178], by redox proteins and even by carbohydrates, which have a
unique ability of stabilizing the Cr(V) species [179]. The radical cascades
resulting from these reactions involve the formation of catalytic quasi-stable
Cr(V) species (e.g., complexed with low molecular weight reductants or their
oxidation products) and of shorter-lived Cr(IV) species [178,180–183]. The
one- and two-electron redox pairs involved include Cr(VI)/Cr(V), Cr(V)/
Cr(IV), and Cr(VI)/Cr(IV) systems [184]. The redox activity of the Cr(III)
ions was also proposed, in the form of a Cr(V)/Cr(III) redox pair [185].
However, it was recently argued that such reactivity may be an artifact,
resulting from Cr(VI) impurities present in commercial Cr(III) samples [95].
Ascorbate was indicated to be the most relevant chromate-reducing agent
under physiological conditions, due to its high tissue levels, comparable to
those of GSH, and the favorable kinetics of chromate reaction [55,186,187].
Ascorbate is a two-electron reducing agent and, consequently, its reaction
with chromate yields mostly Cr(IV) species [178,181]. It is important to note
that the large majority of cell culture studies are apparently conducted under
a deficit of ascorbate, and therefore may yield a biased view of the impor-
tance of other reducing agents, such as low molecular weight thiols, and
consequently, an overestimation of the relevance of Cr(V) in chromate
genotoxicity [55,171].
Oxygen, carbon and, to some extent, also sulfur-centered radicals, which
are formed in the course of the intracellular chromate reduction, can, in
principle, all exert oxidative damage to DNA. Base oxidation, depurination
at guanine residues and DNA cross-links with proteins and radical products
of cellular reductants were detected in various test systems [55,95]. If,
Met. Ions Life Sci. 2011, 8, 319–373

however, ascorbate reduction yielding Cr(IV) is the main pathway of

chromate activation, then SSB, depurination, and 8-oxoguanine formation,
generally attributed to Cr(V) reactivity, would not be preferred, and DSB
should predominate. Current data, however, do not seem to support this
simple picture. The ascorbate-dependent DSB formation was indicated to
occur during the G2 phase of the cell cycle and to depend on the MMR
activity, rather than on a direct chemical assault of reactive chromium on
DNA [188]. On the other hand, the in vitro reactions of nucleotides and
DNA with the chromate/ascorbate and chromate/GSH systems yielded 8-
oxoguanine and, preferably, strongly promutagenic products of its further
oxidation, guanidinohydantoin and spiroiminodihydantoin [189,190].
Anyway, according to a large body of recently acquired data, Cr(III)
species, ultimate stable products of chromate reduction, may provide more
relevant genotoxic lesions than the oxidants produced in the course of the
chromate reduction. The Cr(III) ion is exemplary inert in terms of ligand
exchange reactions, with typical rate constants in the order of days [191].
Therefore, once formed, a DNA lesion containing a Cr(III) species may be
resistant to all kinds of DNA repair just because of its unreactivity.
Ternary DNA adducts containing a Cr(III) ion bridging between the
DNA and a low molecular weight ligand (LMWL) were reported to form
preferentially in chromate-exposed cells, with GSH, Cys, His, and ascorbate
as most frequent LMWLs in these adducts [192,193]. The ascorbate adduct
was found to be the most strongly promutagenic among them [193,194].
These adducts contain phosphate oxygen-bound Cr(III) ions, but the sole
phosphate binding of a non-redox species cannot account for the observed
G/C targeting of mutagenic events [194,195]. Moreover, the phosphate-
Cr(III) binding alone results in a very small distortion of the DNA helix
geometry and is non-mutagenic [196]. Therefore, it is speculated that the
promutagenic Cr(III) adducts contain an additional Cr(III)-guanine N7
bond [195]. There are DNA-protein cross-links formed via the bridging
Cr(III), as well, but they were reported to account for not more than 1% of
the total adducts. These adducts were proposed to be formed by attachment
of proteins to binary Cr(III)-DNA adducts [197]. Their relevance in chro-
mium genotoxicity is not known [55].
The in vitro experiments indicated that the LMWL-Cr(III)-DNA cross-
links were formed differently from those involving proteins: binary LMWL-
Cr(III) complexes were formed first, followed by their attachment to DNA
[198]. The LMWLs detected as ternary partners seem to combine the
intracellular abundance and efficient coordination of octahedral metal ions.
GSH is a versatile chelator [199], and ascorbic acid can chelate metal ions in
solution [200]. Both GSH and ascorbate are millimolar, and individual
amino acids are only somewhat less abundant; among the latter, His and Cys
are the strongest terdentate chelators for octahedral transition metal ions,
Met. Ions Life Sci. 2011, 8, 319–373

such as Cr(III) [201]. It is, however, puzzling, why no adducts with other
equally abundant, and potentially as strongly binding LMWLs, for example
ATP and citrate, were found. It is also interesting to notice that adducts were
formed with reduced, and not oxidized forms of GSH and ascorbate, which
should be directly available to chromium in the course of its reduction
process, especially as both GSSG and dehydroascorbate retain some metal
binding properties of their precursors [174,201].
A direct X-ray spectroscopic and EPR study of Cr(III) speciation in
several cell types, following chromate exposure, indicated a different, or
perhaps complementary view, with Cr(III) ions bound predominantly to
proteins of molecular weight higher than 30 kDa [202]. These complexes
were seen to decompose during cell lysis, yielding a low molecular weight
Cr(III) fraction. Also in this case the actual molecular mechanisms remain to
be elucidated.
So far, we have considered the genotoxicity of the soluble chromate ion.
However, there is evidence available that some poorly soluble chromates are
actually more potent carcinogens than the readily soluble ones [203,204]. In
a very recent study, the genotoxicity of poorly soluble Zn(II), Ba(II), Pb(II)
chromates and of soluble Na(I) chromate was studied under identical con-
ditions in the cell culture [205]. The enhanced clastogenicity of Zn(II) and
Ba(II) chromates compared to Pb(II) and Na(I) chromates was seen, while
the ability of all four compounds tested to induce DSB was similar. These
results add further complexity to mechanisms of chromate genotoxicity
because Zn(II) is an essential metal ion, Ba(II) is toxic, but not carcinogenic,
and Pb(II) is both very toxic and a suspected carcinogen. A non-genotoxic
mechanism for ZnCrO4 has been postulated to account for the above
observations, which includes the induction of chromosomal instability as a
result of its interference with the cell division apparatus, in accordance with
the cell-cycle specific action of ascorbate reported previously [188,206].
Various chromate-dependent lesions activate various DNA repair sys-
tems. DSB activate mismatch repair, Sp and Gh are cleared by the BER
system, and Cr(III)-crosslinked adducts are removed by NER. Therefore,
inhibition of DNA repair by redox reactivity of chromate and/or by an
action of Cr(III) species seems to be a plausible pathway of indirect geno-
toxicity, which, however, remains to be explored.
4.5. Nickel
The firm evidence for nickel carcinogenicity is associated with workplace
exposure [106,207,208]. Ni(II) compounds, both insoluble ones, such as
Ni3S2, NiS, and NiO, and aerosols of soluble Ni(II) salts belong to the IARC
group 1, while metallic nickel dusts are assigned to group 2B [106]. General
Met. Ions Life Sci. 2011, 8, 319–373

populations are exposed to nickel compounds in food, tobacco, and urban

air, but there is no direct epidemiological evidence for health hazards that
would be related to such exposure.
Insoluble, particulate nickel compounds are stronger carcinogens than
soluble ones, because they combine high persistence at the site of exposure in
the airways [209] with a high efficiency of intracellular delivery by phago-
cytosis [210–212]. Recently, the particularly high toxicity of NiO nano-
particles, compared to a typical NiO formulation and to a soluble Ni(II) salt
was reported, likely due to a combination of efficient uptake and fast dis-
solution [213]. The latter process, occurring in lysosomes [211,214,215] is
crucial because the soluble Ni21 (most likely as complexed to intracellular
ligands) is the actual ultimate carcinogen for all kinds of delivery (for review,
see [55,184,207,208]). The intracellular delivery of soluble Ni(II) species
occurs via the divalent metal transporter, DMT-1, which exhibits a broad
metal ion specificity, but is less efficient than phagocytosis [211,214].
Similarly to all carcinogenic metals except of chromium, the bacterial
mutagenicity assays are largely negative for Ni(II) compounds [55,112].
Exposures of cells to high levels of intracellular Ni(II), such as those induced
by phagocytosis of Ni(II)-containing particles, resulted, however, in the
detection of oxidative DNA damage. Its character indicated the involvement
of ROS [79,93,184]. Importantly, G-T transversions, mutations typical for
oxidative damage, were found in both experimental renal tumors induced by
Ni3S2, and in human lung cancers associated with nickel exposure [92,216].
Despite that fact, weak mutagenicity following Ni(II) exposure indicates the
presence of indirect mechanisms of genotoxicity, and epigenetic mechanisms
of carcinogenesis which could involve no genotoxicity at all.
Exposure to high Ni(II) levels leads to alterations of acetylation, methy-
lation, and ubiquitylation of core histones, which may result in the silencing
of cell cycle control genes [217–222]. This activity is assumed, but not fully
proven, to occur on the level of histone-modifying enzymes. Ni(II) ions were
also demonstrated to damage histone H2A directly in cell cultures, by
hydrolytic truncation of the C-terminal H2A octapeptide [223]. Likewise,
histone H2B was found to become oxidized, deamidated, and truncated in
cells cultured with Ni(II) [224]. The presence of truncated H2A in cell
nucleus altered the pattern of expression of numerous genes, including
cancer-related genes [225].
Ni(II) ions also contribute to oxidative stress by depleting cellular stores
of GSH and ascorbate and facilitating the accumulation of Fe(III) in the
cells [100,226–229]. The exact molecular mechanisms of these effects are not
known. Interestingly, the Ni(II)-GSH mixtures demonstrated the ability to
damage plasmid DNA even at a high GSH excess over Ni(II), indicating that
a Ni(II)-GSH complex or an associated Ni(II)-GSSG complex is a poten-
tially genotoxic species [201]. Finally, the depletion by Ni(II) of cellular
Met. Ions Life Sci. 2011, 8, 319–373

ascorbate mimics hypoxia and thus leads to activation of the hypoxia-

inducible factor HIF, which in turn increases the expression of HIF-
dependent genes that is typical for fast growing tumors [230,231]. This
hypoxia-like effect may facilitate selection of a neoplastic phenotype that
can escape apoptosis.
At low concentrations, Ni(II) ions enhance mutagenicity of other carci-
nogens, including UV irradiation, N-methyl-N-nitrosourea and benzo[a]-
pyrene in cell line experiments [232–234]. Ni(II) was specifically
demonstrated to inhibit the XPA protein, which enables the formation of the
NER complex [235].
It seems that exposure to Ni(II) can induce many concurrent pathogenic
intracellular processes [55]. The relative relevance of individual molecular
mechanisms apparently depends on the type of cells and tissues in different
strains of laboratory animals [236]. For example, the ability of Ni(II) to
deplete GSH and to hydrolyze histone H2A depended strongly on the cell
type [223,226–229].
The notion of soluble Ni(II) as the ultimate carcinogen notwithstanding,
the mechanisms also depend on a specific nickel compound. An example for
the latter point is given by phagocytosed Ni3S2 crystals, which start to dis-
solve rapidly, exerting oxidative stress, followed by slow Ni(II) release that
may elicit epigenetic mechanisms [236]. The oxidative burst phase is not seen
for NiO crystals.
The elucidation of molecular mechanisms of Ni(II) genotoxicity and
carcinogenicity depends on the knowledge about its molecular speciation in
the cell. The currently available data indicate that all intracellular Ni(II) is
bound to low and high molecular weight ligands, which can be estimated to
be higher than 20 mM. Preliminary estimates suggested that ATP, His, and
histones may bind the majority of Ni(II) ions in the cell nucleus [237–242].
These data indicate another direction of future research, linking the basic
metabolic features, such as energetic status, of particular cell types with their
susceptibility to Ni(II)-induced carcinogenesis. A protective effect against
Ni3S2 carcinogenesis, observed for Mg(II), Mn(II), and other essential
divalent metals [208,243], can also be expected to find explanation in such
coordination chemistry terms.
4.6. Other Metals

4.6.1. Cobalt
Cobalt is probably close to be included in IARC group 1 [244]. Both,

metallic cobalt dust and soluble Co(II) salts are currently rated as possibly
carcinogenic to humans (group 2B), due to the absence of definitive
Met. Ions Life Sci. 2011, 8, 319–373

epidemiologic data, which in turn are difficult to obtain, because cobalt

exposure is practically always accompanied by exposures to other metallic
carcinogens [245]. Exposure to cobalt metal mixture with tungsten carbide
(hard metal) is rated as probably carcinogenic (group 2A) (see Section 4.7
below). Co(II) is, however, definitely carcinogenic in experimental animals.
Co(II) shares many chemical properties with Ni(II), it is also redox-active
and eagerly forms coordination compounds with bioligands. Not surpris-
ingly, similar molecular mechanisms of genotoxicity were proposed for both
ions.
Regarding indirect genotoxicity mechanisms, the exposure of cells to
Co(II) compounds was reported to result in the induction of a hypoxia-like
response, mentioned in Section 4.5 above, and increased iron overload [246],
alteration of posttranslational histone modifications [247], and inhibition of
the DNA NER system [95,248]. The interference of Co(II) with the zinc
finger function may be involved in the latter process [98,249]. Co(II) expo-
sure of F344/NCr rats, cultured cells, as well as DNA in vitro was also
associated with promutagenic oxidative DNA damage [250–252], and the
rats demonstrated organ specificity of the genotoxic activity of Co(II) ions
[250]. These studies indicate a high probability that Co(II) exposures could
be genotoxic, and consequently, carcinogenic. However, there is one phy-
siological difference between cobalt and nickel, that may account in part for
an apparently lesser Co(II) carcinogenicity, compared to Ni(II). While nickel
is not really essential to humans [207], cobalt is, being part of vitamin B12 in
the form of a Co(I) covalent complex. Although vitamin B12 is taken up as a
whole from the food chain [253] and does not release Co(II) ions readily, one
can speculate on the human body’s defense systems that might be optimized
for cobalt rather than nickel detoxication.
4.6.2. Lead
Lead is a very toxic metal, with the neural and hematopoietic systems as
main targets [254]. It is rated as probably carcinogenic to humans (group
2A) by IARC [255]. Both metallic lead and its compounds are still widely
used in technology, including everyday use products, despite the large
international efforts to downscale its usage. Lead metal water pipes, wall-
painting pigments and tetraethyllead additive to gasoline [256] have been
gradually vanishing from the human environment in most countries. How-
ever, some other products, such as lead-acid car batteries, persist as potential
sources of exposure of the general public, and there is also a significant risk
of industrial pollution (wastewater, incinerators) affecting local commu-
nities. The occupational exposure to lead can also be related to the refining
and manufacturing of other metals, such as copper [257].
Met. Ions Life Sci. 2011, 8, 319–373

The firm evidence for carcinogenicity of lead comes largely from animal
studies, as epidemiological investigations in lead battery and smelter
employees did not provide a clear and consistent picture [225]. The only
existing evidence for humans is clastogenic damage in peripheral blood
leukocytes of persons exposed to lead [258]. In rodents challenged with
Pb(II) compounds, the subtoxic and toxic lead levels were associated with
the increased risk of cancer at various locations, including kidney and brain
[255,259]. Renal cancer was also significantly present in the offspring of mice
exposed orally to Pb(II) [260]. Further experimental evidence for the geno-
toxic potential of lead comes from cell culture and chemical studies. There is
evidence for the direct oxidative base damage in DNA [45,95,261], perhaps
involving the Pb(II)/Pb(IV) redox pair, the formation of strand breaks [262],
as well as DNA repair inhibition as an indirect genotoxic mechanism
[95,96,263]. Pb(II) was also found to be mutagenic in some, but not all assays
[264,265]. Recently, an epigenetic mechanism for Pb(II) mutagenicity was
proposed, based on the interference with the protein kinase C pathway,
perhaps in conjunction with DNA repair control [266].
The Pb(II) ion has a very high affinity for thiols and may bind pre-
ferentially at sulfur-rich clusters [267]. The depletion of thiol-based cellular
antioxidant defense may contribute to indirect induction of oxidative DNA
damage [45]. The Zn(II) substitution in zinc fingers has also been proposed
as epigenetic mechanism for Pb(II) genotoxicity, including transplacental
induction of cancer [254,268,269].
4.6.3. Uranium
All uranium isotopes are radioactive. The natural uranium consists mainly
of the 238U isotope (99.3%), supplemented by 235U (0.7%) and traces of
234
U. The carcinogenicity of this metal has usually been considered with
respect to the neutron, a, and g emissions of the fissible 235U isotope, as the
dominant 238U isotope is only a weak a emitter with a half-life of 4.5 billion
years [270]. However, the recent military applications of depleted uranium
(DU), enriched in the 238U isotope, attracted attention to chemical tox-
icology of this material. The exposed populations include not only the
military personnel, but also non-combatants, due to uranium oxide (mostly
238
UO3) debris deposited in the soil in the areas of combat. The inhalatory
exposure to uranium oxide and the intrabody exposure to embedded DU
fragments are the most significant exposure routes in soldiers [271].
Reports on DU/238UO3 carcinogenesis in humans are not conclusive. This
is not surprising, taken the small cohorts available for systematic screening,
uncertainties about actual exposures, and a short period of observation
[272]. Nevertheless, the data from experimental systems are rather clearly
supporting the notion of DU carcinogenicity, and genotoxic lesions were
Met. Ions Life Sci. 2011, 8, 319–373

also observed in tissues of exposed persons [272]. Laboratory animals

exposed to DU/238UO3 demonstrate genotoxic and clastogenic effects,
including double strand DNA breaks in DU target tissues, such as kidney
and lung [273,274]. Cell culture studies confirm clastogenicity of DU/238UO3
below the cytotoxic levels, that cannot be assigned to DU radioactivity
[275,276]. Molecular mechanisms of DU genotoxicity await elucidation, as
there are only a few studies available in the literature. The uranyl cation
UO2+2 is considered to be the actual genotoxic soluble species in DU che-
mical toxicology [277]. Oxidative DNA damage, including 8-oxoguanine
and single strand DNA breaks by the 238UO2+ 2 /ascorbate system was
reported and interpreted in terms of analogy with chromate [277]. The 8-
oxoguanine formation was also seen upon the action of the uranyl ion in the
absence of ascorbate [278] and in the presence of hydrogen peroxide [279],
strongly suggesting a redox activity of U(VI). Therefore, the actual redox
couples and mechanisms in DU genotoxicity in general await further studies.
4.6.4. Platinum
Bioavailable platinum comes into contact with the human body largely in
the form of Pt(II)-based anticancer drugs. The very action of these medicinal
compounds is based on their direct genotoxicity, via the formation of Pt(II)-
nucleobase adducts which interfere with DNA replication and transcription,
ultimately leading to cancer cell apoptosis or necrosis [280,281]. The extent
of DNA damage correlates directly with their ability to kill cancer cells [282].
Most chemical anticancer drugs can also cause secondary cancers, due to
their genotoxicity, and platinum-based ones also follow this unfortunate rule
[283].
The delicate balance between the anticancer and procarcinogenic action of
platinum is controlled by pharmacokinetics of the Pt(II) drug versus the
tissue- and cell type-specific intracellular metabolism of the drug. The
reactive square-planar Pt(II) unit binds two nucleobase nitrogens in a cis
arrangement, resulting in the formation of several types of adducts. The ones
most common in vivo are 1,2-intrastrand d(GpG) cross-links (Pt-GG) and
d(ApG) cross-links (Pt-AG). These two adducts probably also contribute
the most to genotoxicity, by blocking DNA replication and interfering with
NER DNA repair [280,281]. Minor adducts include 1,3-d(GpNpG) cross-
links, interstrand cross-links, and monoadducts. Bifunctional intrastrand
adducts induce mutations, primarily G-T transversions at d(GpG) sites
and G-A transitions and A-T transversions at d(ApG), d(GpNpG), and
d(GpG) sites [65].
The cellular resistance to platinum drugs involves direct binding to
intracellular thiols, such as GSH and MT [284]. Despite the redox properties
of platinum (a Pt(II)/Pt(IV) redox pair), oxidative damage does not seem to
Met. Ions Life Sci. 2011, 8, 319–373

play a vital role in platinum genotoxicity [285]. The observation of oxidative

stress seems to be limited to particular platinum drug/cell type combina-
tions, thus likely involving the interference by Pt(II) with the thiol meta-
bolism [286].
4.6.5. Copper and Iron
The essential metals copper and iron are rarely considered as agents of
oxidative DNA damage in vivo because they are under strict physiological
control during uptake, transport, and metabolism. There is, however, scat-
tered evidence that such control may be lost locally and/or temporarily due
to a pathology, primarily an oxidative/nitrosative stress, e.g., due to
inflammation or toxic assault, turning endogenous copper and/or iron into
local (ultimate) carcinogens [184]. For example, the altered copper meta-
bolism was implicated in hepatic hyperplasia in a rat model [287]. Also, the
Long-Evans Cinnamon rats, which accumulate copper in their livers, thus
providing a model for Wilson’s disease, exhibited significant reduction in the
BER system activity in hepatocytes, resulting in the accumulation of oxi-
dative DNA damage. It is, however, difficult to ascertain whether the effect
was due to a direct action of copper, or rather to the oxidative stress induced
by copper accumulation [288].
Certain redox-active copper and iron complexes with strong chelators are
carcinogenic when administered in experimental animals, with oxidative
DNA damage as an evident mechanism of genotoxicity. Examples include
Cu(NTA) and Fe(NTA), as well as Fe(EDTA) complexes [289,290].
Therefore, genotoxic properties of copper and iron complexes are almost
certainly related to their redox properties [45]. These two metals are usually
considered as Fenton reagents, generating diffusible hydroxyl radicals via
the respective Cu(I)/Cu(II) and Fe(II)/Fe(III) redox pairs, as supported by
the DNA damage pattern in vitro [291,292]. The Cu(II)/Cu(III) redox pair
can also be activated with certain bioligands [184]. In accordance with the
above remarks, the ability of a ligand to form strong complexes with both
redox couple partners seems to be essential for the carcinogenicity of its
copper or iron complex [293]. Metal oxo species, which would act as loca-
lized rather than diffusible DNA oxidants, are also likely participants in
oxidative DNA damage mechanisms [294]. The cupric or ferrous ion,
adventitiously released from its physiological host protein, may also dock at
DNA or nuclear proteins, such as histones, and form active genotoxic spe-
cies locally [184,295].
The concept of adventitious loss of control over metabolic ions, while
compelling and well supported by their chemical properties in simple model
systems, is rather difficult to confirm in vivo in the presence of a plethora of
Met. Ions Life Sci. 2011, 8, 319–373

co-interacting molecules and processes. Its verification has to await further

technical progress in studying chemical reactions in vivo.
4.7. Mixtures of Metals

The actual exposures in the workplace and, particularly, in the environment
have a mixed character – the carcinogenic metals very often accompany each
other and are associated with non-metallic carcinogens. The troubles
encountered at elucidation of genotoxic and non-genotoxic mechanisms for
a single metal exposure increase greatly for mixed exposures, discouraging
extensive research in this area despite its obvious importance. Nevertheless,
there are some notable exceptions.
Residual oil fly ash (ROFA) is generated in the course of combustion of
heavier fractions of oil products in Diesel car engines and power plants. It
contains variable amounts of bioavailable transition metal ions. The
simultaneous presence of water-leachable iron, vanadium and nickel species,
in various proportions, depending on the oil source and the combustion
process, is a hallmark of this material [296,297]. Other metal ions bioavail-
able from ROFA, usually at much lower levels, include chromium, copper,
manganese, and cobalt [296,298–300]. The water-insoluble ROFA fraction
contains nickel sulfides, although this is usually a minor fraction with respect
to the total ROFA nickel [301]. The exposure of cell lines and laboratory
animals to ROFA resulted in oxidative DNA damage, including the for-
mation of 8-oxoguanine and strand breaks [297–299,302–304]. These studies
implicated vanadium(IV) and vanadium(V) species as the major oxidative
agents. This reproducible observation is very interesting. Indeed, vanadium
has a rich redox chemistry, with V(III), V(IV), and V(V) levels of oxidation
available in a biological setting [305,306]. Vanadium, represented by its main
compound, V2O5, is currently rated as possibly carcinogenic (group 2B)
[307]. In the absence of human carcinogenicity data, this evaluation is based
on animal, cell line, and isolated DNA experiments. The ability of V(V) and
V(IV) compounds to generate radicals, induce DNA strand breaks, chro-
mosomal aberrations, and other genotoxic effects is typical for definite
carcinogenic metals [95]. Vanadium might therefore be genotoxic according
to mechanisms similar to those discussed above. The vanadium and ROFA
issue needs to be investigated in more detail because the exposure to ROFA
may be a potentially serious general health hazard. Interestingly, vanadium
and nickel are also simultaneously present in stainless steel implants, and
they were indicated as two sources of DNA damage, resulting from the
contact of these implants with body fluids [308].
Other interesting metal coexposure issues involve cobalt-containing dusts.
The particulate material containing metallic cobalt and tungsten carbide
Met. Ions Life Sci. 2011, 8, 319–373

(WC) is a group 2A IARC carcinogen, for which a specific mechanism of ROS

generation was proposed, based on solid phase catalysis. The metallic cobalt
particles get oxidized to Co21 ions, and electrons produced in this process are
transferred to WC particles where they help reduce O2, yielding ROS [309].
Cobalt-containing dusts which are generated in technological processes
invariably contain other carcinogenic metals, such as cadmium. The exposure
of workers to Co(II)+Cd(II)+Pb(II) mixed materials resulted in the forma-
tion of genotoxic lesions, such as SSB, in their blood cells. The importance of
this finding stems from the fact that the extent of the damage correlated
equally well with Cd(II) as well as with Co(II) levels, both much lower than
official safe limits [310]. In the follow-up study the coordinated gene expression
response to Co(II)+Cd(II)+Pb(II) exposures was detected [311].
Welders are a professional group exposed to fumes containing multiple
metal compounds, primarily those of chromium and nickel, but also lead
and other suspected carcinogenic metals. Several studies demonstrated a
significant elevation of DNA strand breaks and cross-links in these workers,
but the absence of control groups that would be exposed to single carcino-
genic elements does not allow for an analysis of possible synergies in these
multiple exposures, however, the large proportion of damage appeared to be
correlated with chromate exposure [312–314].
There are few studies devoted to genotoxicity of environmental rather
than occupational metal exposure. In one, the urban population of the city
of Bremen, Germany, was tested for correlations between chromium, cad-
mium, and nickel levels in urine, and lead levels in blood on the one hand,
and on the other the extent of oxidative DNA damage in their lymphocytes.
A significant positive correlation was found only for nickel, and not for
chromium exposure [315]. In the light of these scattered results, the eluci-
dation of mixed exposure effects appears to be one of the most challenging
areas of future research in metal genotoxicity.
5. CRITICAL OVERVIEW OF THE EXPERIMENTAL

METHODS FOR STUDYING THE GENOTOXIC
POTENTIAL OF METALS
Specific genotoxic effects of various metal derivatives that may lead to
mutations and eventually cancer, have been studied in a wide variety of
systems, ranging from cell-free test tube experiments, through bacterial and
cell culture models, to cells and tissues of animals and humans exposed to
metals in vivo and in vitro. Thus, in test tubes, chromatin, genomic DNA
(either linear or circular), selected DNA fragments of various origin
(including genes), or even single nucleotides or nucleosides, were incubated
Met. Ions Life Sci. 2011, 8, 319–373

with various metal compounds, often in the presence of natural oxidants,

followed by analysis of specific effects of DNA damage, such as gross
degradation, single and double strand breaks, depurination, cross-linking of
various types, DNA base oxidation, and others. The fidelity of DNA
synthesis in the presence of metal ions other than the ‘‘native’’ Mg21 was
also studied [316]. The results of these experiments indicated that at least
some types of the observed DNA damage may be subject to erroneous DNA
synthesis [317] and/or repair [318] with possible mutagenic consequences.
Although the test tube systems are unrealistic in modeling the vast com-
plexity of cellular environment and disregard the restraints of cellular metal
uptake (concentration levels), they nonetheless provide valuable information
relative to possible molecular targets and types of interactions of carcino-
genic metal ions with biomolecules in a living cell [45,95].
Many, if not all, genotoxic metal effects observed in the test tube models
have been reproduced in a wide variety of cultured cells with one notable
exception of bacteria. It was expected that metal ions would generate reverse
mutations, e.g., in the Ames’ test, through genotoxic mechanisms, as observed
for organic carcinogens. In contrast, the majority of metal ions appeared not
to induce mutations in various strains of Salmonella typhimurium, Escherichia
coli, or Bacillus subtilis [319,320]. The negative results, however, do not seem
to be related to the absence of the genotoxic potential of the metal as much as
to experimental limitations in controlling the uptake of the metal ions due to
the tight metal homeostasis mechanisms in bacteria [321,322]. As shown in
eukaryotic cells, generation of detectable DNA damage requires a relatively
high intracellular metal concentration [160] that is best achievable through
facultative phagocytosis of fine particles of water-insoluble metal compounds.
The phagocytosed particles undergo chemical solubilization by cellular
components [210–215,323–325], and thus constitute a long-lived internal
source of high local metal ion concentrations. This explains the known higher
genotoxic and carcinogenic potential of water-insoluble sulfides of nickel as
compared with soluble salts of this metal [326,327]. Nevertheless, some strains
of bacteria, e.g., Corynebacterium sp.887, could be mutated with water-
soluble nickel(II) chloride, but only after prolonged (4 days) exposure to high,
sublethal concentrations of this salt [328].
The eukaryotic cell cultures offer more experimental flexibility as to the
exposure conditions (e.g., selection of cell types/lines and culture media,
exposure regime), metal derivatives and their concentration ranges, the
spectrum of DNA/chromatin damage, alternative end points (e.g., trans-
formation to neoplastic phenotype, lethal mutations in protozoan offspring,
chromosomal aberrations), as well as specific mutation types that can be
studied. The variety of cultured cells used in metal research includes cells
originating from different species and tissues (normal and neoplastic), either
transformed to stable (immortal) cell lines or in primary cultures, and the
Met. Ions Life Sci. 2011, 8, 319–373

protozoan Paramecium [327]. Examples of such cells used to study nickel

genotoxicity are of (a) Chinese hamster origin: V79 (lung) [329] and CHO
(ovary) cells, both normal and transgenic [330]; (b) mouse: C3H10T1/2
(fibroblasts) [330], FM3A (mammary carcinoma) [331], L5178Y (lym-
phoma), MutaMouse fibroblasts [332]; (c) rat: NRK6m2 (renal epithelium)
[318], BigBlue rat fibroblasts [332]; (d) human: Jurkat (T-cells) [308], renal
cortex cells [333], gastric and nasal mucosa cells [334], fetal kidney cortex
cells [335], blood and hematopoietic cells [336], and others. Special attention
has been given to peripheral lymphocytes isolated from humans and rodents.
They have been frequently examined for DNA damage and chromosomal
abnormalities following in vitro and in vivo exposures [337,338].
The test tube and cultured cell systems revealed what metals could do to
the genetic material under certain experimental conditions. These systems
could not, however, prove what the metals really do following in vivo
exposures. To find the answer(s) to the latter question, whole-animal
experiments and analysis of relevant human data have been necessary. Thus,
rodents have been given metal compounds orally [338,339], through inha-
lation or intratracheal insufflation [332,340,341], or parenteral injections
[342–345,58], and selected tissues were analyzed at different time points after
treatment for damage to their genetic material. Unfortunately, only single
experiments considered the respiratory tract cells, which are the main target
for metal aerosols toxicity and carcinogenesis in humans [340,346], paying
attention mainly to circulatory white blood cells and bone marrow [337,339].
The results were mixed, often incomparable and confusing, depending on the
model. For example, the effect of inhalation of nickel-containing particles
was tested in Wistar rats where it consisted of chromosomal aberrations in
alveolar macrophages [340], and in transgenic MutaMice and BigBlue rats,
in which DNA damage was found only in the nasal mucosa of mice but not
in the lung cells of either species [332]. Intratracheal instillation of water-
soluble and -insoluble nickel in rats produced chromosomal aberrations and
micronuclei formation in bone marrow cells [341] and generated the pro-
mutagenic DNA base product 8-oxo-dG in the lung tissue [347,348]. Oral
dosing of water-soluble nickel salts to mice caused transient DNA strand
breaks in white blood cells [338], whereas in rats no significant increase in
chromatin damage (micronuclei formation) was noticed in the polychro-
matic erythrocytes of bone marrow [339]. The intraperitoneal and intrave-
nous injections of nickel to rats or mice generated DNA damage in the white
blood cells and lungs [342], sperm heads [343], and kidneys [58]. In humans
occupationally exposed to nickel, increased DNA damage (chromosome
gaps, sister chromatid exchange, micronuclei formation, cross-linking,
strand scission) was found in peripheral lymphocytes [312,313,349,350], but
not in the buccal mucosa smears [351]. Interestingly, increased levels of the
Fpg-sensitive sites, indicative of oxidative DNA damage, were also found in
Met. Ions Life Sci. 2011, 8, 319–373

lymphocytes of humans living in a heavy metal-polluted urban environment

(see Section 4.7) [315].
The overall results of testing the genotoxic potential of metals under var-
ious in vitro and in vivo conditions reveal that a particular metal can damage
DNA in one experimental system, or another, but the question whether or
not the same metal is genotoxic under ‘real life’ conditions (occupational or
environmental) cannot be answered with full confidence. Controlling the
exposure, uptake, and retention of metal derivatives in living cells, crucial for
the generation of detectable genotoxic effect(s), selecting proper end-points,
and considering the often hormetic dose/effect relationship [328] is a difficult
task. Therefore, if studies of metal genotoxicity are to unveil its relevance to
human cancer and other diseases, such studies have to be conducted in
occupationally-exposed humans and consider cells isolated from the target
tissue (e.g., the respiratory tract epithelium). Testing another kind of cells, the
peripheral lymphocytes for example, may produce only a suggestive answer.
Animal models have to be treated with caution due to inter-species differ-
ences [334] and cultured cells models cannot mimic the metabolic and
dynamic complexity of a multicellular organism. Also, it has to be stressed,
that genotoxic effects, even those observed following animal in vivo exposures
may not lead to mutations [332] or cancer [352] in the same tissue.

DIRECTIONS
In Sections 1–5 we provided a brief account of currently available data and

views on metal genotoxicity. Looking at the last 20 years of this area of research
we can notice interesting trends in perception. Studies of cellular and molecular
mechanisms of genotoxicity of individual metals were initially focused on ele-
mentary molecular reactions, such as ROS generation due to redox couples.
Despite the apparently high success of this research in providing explanations
to phenomena observed in experimental animals and exposed humans, the
focus has been shifting towards indirect mechanisms, such as DNA repair
inhibition or cell cycle impairment. Currently, the absence of direct genotoxic or
mutagenic effects in common tests is often taken as evidence for the sole validity
of indirect, nongenotoxic mechanisms. However, as reviewed in Section 5, these
tests are not necessarily reliable for studying mechanistic effects of carcinogenic
metal ions. Also, oxidative reactivity seems to correlate quite well with geno-
toxicity in studies of mixed metal exposures, whereas the state of the art of
individual metal research would suggest cell cycle impairment effects to pre-
dominate. What results is a plethora of conflicting opinions, with only rare
attempts to reconcile them into a more or less unified view.
Met. Ions Life Sci. 2011, 8, 319–373

The attention to individual, possibly carcinogenic metals, also exhibits

trends and modes, which are not necessarily related to their actual relevance.
For example, the number of current studies on depleted uranium, which may
only affect very limited populations, exceeds those on genotoxicity of copper
which is likely related to a vast number of human neoplastic diseases.
In our opinion, two directions are particularly important for the future
development of the field of metal genotoxicity and carcinogenesis. One is the
issue of the relative importance of contributions of individual mechanisms to
the overall genotoxicity of a given element or compound, a sort of quanti-
tative mechanism-effect relationship. Another is the emerging new world of
metal-containing nanomaterials, many of which are composed of definite
carcinogens, e.g., the nano-NiO or cadmium-containing quantum dots to
name just a couple. These materials may combine known genotoxic pathways
related to their elemental compositions with new ones, resulting from their
superior abilities to penetrate tissues, cells, and subcellular compartments.
Altogether, the field of metal ion genotoxicity is probably as far from
mechanistic clarity as it has been before, because the continuous flow of new
data, often contradicting each other, is not accompanied by comprehensive
unifying concepts. At the same time, and perhaps for the same reason, this
field of research is important in terms of identifying and quantifying old and
emerging new hazards for occupationally and environmentally exposed
populations. It is also interesting in terms of novel research developments,
including identification of novel chemical interactions involved in the
mechanisms of metal-induced genotoxic effects.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Yih-Horng Shiao, for critical discussion of
this chapter. This project has been funded in part with U.S. federal funds
from the National Cancer Institute, Center for Cancer Research, National
Institutes of Health under the Intramural Research Program and Contract
N01-CO-12400. The content of this publication does not necessarily reflect
the views or policies of the Department of Health and Human Services, nor
does mention of trade names, commercial products, or organizations imply
endorsement by the United States Government.
ABBREVIATIONS
ADP adenosine 5 0 -diphosphate

APL acute promyelocytic leukemia
Met. Ions Life Sci. 2011, 8, 319–373

ATP adenosine 5 0 -triphosphate

BER base excision repair
CHO Chinese hamster ovary
dGTP 2 0 -deoxyguanosine 5 0 -triphosphate
DMAIII dimethylarsinous acid
DMT divalent metal transporter
DSB double strand break
DU depleted uranium
EPR electron paramagnetic resonance
FAPyG formamidopyrimidine derivative (see Figure 5)
Fpg formamidopyrimidine glycosylase
Gh guanidinohydantoin
GSH glutathione (reduced)
GSSG glutathione (oxidized)
GTF glucose tolerance factor
HIF hypoxia-inducible factor
HR homologous recombination
IARC International Agency for Research on Cancer
LMWL low molecular weight ligand
MMAIII monomethylarsonous acid
MMR mismatch repair
MT metallothionein
NER nucleotide excision repair
NHEJ non-homologous end-joining
NTA nitrilotriacetate
8-oxo-dG 8-oxo-2 0 -deoxyguanosine
PAH polyaromatic hydrocarbons
PARP poly(ADP-ribose) polymerase
ROFA residual oil fly ash
Sp spiroiminodihydantoin
SSB single strand break
WC tungsten carbide
ZF zinc finger
REFERENCES
1. B. N. Ames and M. K. Shigenaga, in Molecular Biology of Free Radical
Scavenging Systems, Ed. J. Scandalios, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1992, pp. 1–22.
Met. Ions Life Sci. 2011, 8, 319–373

2. X. Shi, N. S. Dalal and K. S. Kasprzak, Arch. Biochem. Biophys., 1992, 299,

154–162.
3. X. Shi, N. S. Dalal and K. S. Kasprzak, J. Inorg. Biochem., 1993, 15, 211–225.
4. X. Shi, N. S. Dalal and K. S. Kasprzak, Chem. Res. Toxicol., 1993, 6, 277–283.
5. F. P. Bossu, E. B. Paniago, D. W. Margerum, S. T. Kirksey and J. L. Kurtz,
Inorg. Chem., 1978, 17, 1034–1042.
6. L. J. Marnett, Mutat. Res., 1999, 424, 83–95.
7. R. M. Burger, A. R. Berkowitz, J. Peisach and S. B. Horwitz, J. Biol. Chem.,
1980, 255, 11832–11838.
8. R. De Bont and N. van Larebeke, Mutagenesis, 2004, 19, 169–185.
9. K. Randerath, E. Randerath, C. V. Smith and C. Jian, Chem. Res. Toxicol.,
1996, 9, 247–254.
10. K. S. Gates, Chem. Res. Toxicol., 2009, 22, 1747–1760.
11. E. Lutsenko and A. S. Bhagwat, Mutat. Res., 1999, 437, 11–20.
12. P. Karran and T. Lindahl, Biochemistry, 1980, 19, 6005–6011.
13. K. S. Gates, T. Nooner and S. Dutta, Chem. Res. Toxicol., 2004, 17, 839–856.
14. X. Lu, J. M. Heilman, P. Blans and J. C. Fishbein, Chem. Res. Toxicol., 2005,
18, 1462–1470.
15. L. A. Loeb and B. D. Preston, Ann. Rev. Genet., 1986, 20, 201–230.
16. W. A. Pryor, Free Radical Biol. Med., 1988, 4, 219–223.
17. A. P. Breen and J. A. Murphy, Free Radical Biol. Med., 1995, 18, 1033–1077.
18. M. Kuwahara, Radiat. Phys. Chem., 1991, 37, 691–704.
19. M. Dizdaroglu, P. Jaruga, M. Birincioglu and H. Rodriguez, Free Radical Biol.
Med., 2002, 32, 1102–1115.
20. J. R. Wagner, J. E. van Lier, M. Berger and J. Cadet, J. Am. Chem. Soc., 1994,
116, 2235–2242.
21. P. Cysewski, J. Chem. Soc., Faraday Trans., 1998, 94, 117–125.
22. P. T. Henderson, J. C. Delaney, J. G. Muller, W. L. Neeley, S. R. Tannenbaum,
C. J. Burrows and J. M. Essigmann, Biochemistry, 2003, 42, 9257–9262.
23. S. Delaney, W. L. Neeley, J. C. Delaney and J. M. Essigmann, Biochemistry,
2007, 46, 1448–1455.
24. O. Komyushyna and C. J. Burrows, Biochemistry, 2003, 42, 13008–13018.
25. G. K. Schroeder, C. Lad, P. Wyman, N. H. Williams and R. Wolfenden, Proc.
Natl. Acad. Sci. USA, 2006, 103, 4052–4055.
26. N. H. Williams, B. Takasaki, M. Wall and J. Chin, Acc. Chem. Res., 1999, 32,
485–493.
27. J. T. Stivers and Y. J. Jiang, Chem. Rev., 2003, 103, 2729–2759.
28. W. K. Pogozelski and T. D. Tullius, Chem. Rev., 1998, 98, 1089–1107.
29. B. Balasubramanian, W. K. Pogozelski and T. D. Tullius, Proc. Natl. Acad. Sci.
USA, 1998, 95, 9738–9743.
30. J.-T. Hwang and M. M. Greenberg, J. Am. Chem. Soc., 1999, 121, 4311–
4315.
31. J. C. Genereux and J. K. Barton, Chem. Rev., 2010, 110, 1642–1662.
32. P. Fortini and E. Dogliotti, DNA Repair, 2007, 6, 398–409.
33. T. Ohnishi, E. Mori and A. Takahashi, Mutat. Res., 2009, 669, 8–12.
34. Z. Nagy and E. Soutoglou, Trends Cell Biol., 2009, 19, 617–629.
Met. Ions Life Sci. 2011, 8, 319–373

35. S. Katyal and P. J. McKinnon, Mech. Age. Dev., 2008, 129, 483–491.
36. M. Shrivastav, L. P. De Haro and J. A. Nickoloff, Cell Res., 2008, 18,
134–147.
37. R. P. Sinha and D.-P. Häder, Photochem. Photobiol. Sci., 2002, 1, 225–236.
38. T. Reardon and A. Sancer, Genes Dev., 2003, 17, 2539–2551.
39. V. Mocquet, J. P. Laine, T. Riedl, Z. Yajin, M. Y. Lee and J. M. Egly, EMBO
J., 2008, 27, 155–167.
40. Y. Zhu, J. Hu, Y. Hu and W. Liu, Canc. Treat. Rev., 2009, 35, 590–596.
41. C. Desler, C. Johannessen and L. J. Rasmussen, Chem. Biol. Interact., 2009,
177, 212–217.
42. V. Thiviyanathan, A. Somasunderam, D. E. Volk, T. K. Hazra, S. Mitra and
D. G. Gorenstein, Biochem. Biophys. Res. Commun., 2008, 366, 752–757.
43. K. C. Cheng, D. S. Cahill, H. Kasai, S. Nishimura and L. A. Loeb, J. Biol.
Chem., 1992, 267, 166–172.
44. H. Kamiya, H. Miura, N. Murata-Kamiya, H. Ishikawa, T. Sakaguchi,
H. Inoue, T. Sasaki, C. Masutani, F. Hanaoka, S. Nishimura and E. Ohtsuka,
Nucl. Acids Res., 1995, 23, 2893–2899.
45. K. S. Kasprzak, in Toxicology of Metals, Ed. L. W. Chang, CRC Lewis Pub-
lishers, Boca Raton, 1996, pp. 299–320.
46. M. S. Cooke, M. D. Evans, M. Dizdaroglu and J. Lunec, FASEB J., 2003, 17,
1195–1214.
47. N. Krishnamurthy, X. Zhao, C. J. Burrows and S. S. David, Biochemistry, 2008,
47, 7137–7146.
48. R. K. O. Sigel and H. Sigel, Met. Ions Life Sci., 2007, 2, 109–180.
49. H. Sigel and R. Griesser, Chem. Soc. Rev., 2005, 34, 875–900.
50. M. Shimizu, P. Gruz, H. Kamiya, C. Masutani, Y. Xu, Y. Usui, H. Sugiyama,
H. Harashima, F. Hanaoka and T. Nohmi, Biochemistry, 2007, 46, 5515–5522.
51. H. Sigel and B. Song, Met. Ions Biol. Syst., 1996, 32, 135–205.
52. M. Adamczyk, J. Poznański, E. Kopera and W. Bal, FEBS Lett., 2007, 581,
1409–1416.
53. H. Kozzowski, W. Bal, M. Dyba and T. Kowalik-Jankowska, Coord. Chem.
Rev., 1999, 184, 319–346.
54. M. Rózga, M. Sokozowska, A. M. Protas and W. Bal, J. Biol. Inorg. Chem.,
2007, 12, 913–918.
55. K. Salnikow and A. Zhitkovich, Chem. Res. Toxicol., 2008, 21, 28–44.
56. A. K. Datta, M. Misra, S. L. North and K. S. Kasprzak, Carcinogenesis, 1992,
13, 283–287.
57. K. S. Kasprzak, in Cytotoxic, Mutagenic and Carcinogenic Potential of Heavy
Metals Including Metals Related to Human Environment, Ed. N. Hadjiliadis,
Vol. 26 of NATO ASI (Ser. 2), Kluwer Academic, Dordrecht, The Netherlands,
1997, pp. 73–92.
58. M. Misra, R. Olinski, M. Dizdaroglu and K. S. Kasprzak, Chem. Res. Toxicol.,
1993, 6, 33–37.
59. J. Anastassopoulou and T. Theophanides, Crit. Rev. Oncol. Hematol., 2002, 42,
79–91.
60. F. E. Rossetto and E. Nieboer, J. Inorg. Biochem., 1994, 54, 167–186.
Met. Ions Life Sci. 2011, 8, 319–373

61. V. A. Sorokin, V. A. Valeev, G. O. Gladchenko, I. V. Sysa, Y. P. Blagoi and

I. V. Volchok, J. Inorg. Biochem., 1996, 63, 79–98.
62. E. S. Gelagutashvili, I. V. Mikeladze and N. A. Sapojnikova, J. Inorg. Biochem.,
1997, 65, 159–161.
63. R. Drouin, H. Rodriguez, S.-W. Gao, Z. Gebreyes, T. R. O’Connor, G. P.
Holmquist and S. A. Akman, Free Radical Biol. Med., 1996, 21, 261–273.
64. K. Yamamoto and S. Kawanishi, J. Biol. Chem., 1989, 264, 15435–15440.
65. K. J. Yarema, J. M. Wilson, S. J. Lippard and J. M. Essigmann, J. Mol. Biol.,
1994, 236, 1034–1048.
66. A. Sreedhara and J. A. Cowan, J. Biol. Inorg. Chem., 2001, 6, 337–347.
67. H. Katada and M. Komiyama, ChemBioChem., 2009, 10, 1279–1288.
68. M. A. Camargo, A. Neves, A. J. Bortoluzzi, B. Szpoganicz, A. Martendal,
M. Murgu, F. L. Fischer, H. Terenzi and P. C. Severino, Inorg. Chem., 2008, 47,
2919–2921.
69. H. Nicholas and J. Williams, J. Am. Chem. Soc., 2000, 122, 12023–12024.
70. V. Scheller-Krattiger and H. Sigel, Inorg. Chem., 1986, 25, 2628–2634.
71. M. Je(owska-Bojczuk, P. Kaczmarek, W. Bal and K. S. Kasprzak, J. Inorg.
Biochem., 2004, 98, 1770–1777.
72. M. Je(owska-Bojczuk, P. Kaczmarek, W. Bal and K. S. Kasprzak, J. Inorg.
Biochem., 2005, 99, 737–746.
73. Y. Nakabeppu, Mutat. Res., 2001, 477, 59–70.
74. K. Biazkowski, A. Biazkowska and K. S. Kasprzak, Carcinogenesis, 1999, 20,
1621–1624.
75. E. Speina, K. D. Arczewska, D. Gackowski, M. Zielińska, A. Siomek,
J. Kowalewski, R. Oliński, B. Tudek and J. T. Kuśmierek, J. Natl. Cancer Inst.,
2005, 97, 384–395.
76. M. Laine, K. Ketomäki, P. Poijärvi-Virta and H. Lönnberg, Org. Biomol.
Chem., 2009, 7, 2780–2787.
77. D. Kruschel and R. K. O. Sigel, J. Inorg. Biochem., 2008, 102, 2147–2154.
78. Z. Nackerdien, K. S. Kasprzak, G. Rao, B. Halliwell and M. Dizdaroglu,
Cancer Res., 1991, 51, 5837–5842.
79. K. S. Kasprzak, Cancer Invest., 1995, 13, 411–430.
80. C. Crean, J. Shao, B. H. Yun, N. E. Geacintov and V. Shafirovich, Chemistry,
2009, 15, 10634–10640.
81. E. R. Stadtman, Annu. Rev. Biochem., 1993, 62, 797–821.
82. W. Bal, M. I. Djuran, D. W. Margerum, E. T. Gray, Jr., M. A. Mazid,
R. T. Tom, E. Nieboer and P. J. Sadler, J. Chem. Soc., Chem. Comm., 1994,
1889–1890.
83. N. Cotelle, E. Tremolieres, J. L. Bernier, J. P. Catteau and J. P. Henichart,
J. Inorg. Biochem., 1992, 46, 7–15.
84. K. S. Kasprzak and R. M. Bare, Carcinogenesis, 1989, 10, 621–624.
85. R. E. Rodriguez, M. Misra and K. S. Kasprzak, Toxicology, 1990, 63, 45–52.
86. R. E. Rodriguez and K. S. Kasprzak, in Nickel in Human Health: Current
Perspectives, Ed. E. Nieboer and J. O. Nriagu, J. Wiley & Sons, New York,
1992, pp. 375–385.
Met. Ions Life Sci. 2011, 8, 319–373

87. Z. Zhong, W. Troll, K. L. Koenig and K. Frenkel, Cancer Res., 1990, 50,
7564–7570.
88. W. E. Achanzar, K. B. Achanzar, J. G. Lewis, M. M. Webber and M. P.
Waalkes, Toxicol. Appl. Pharmacol., 2000, 164, 291–300.
89. P. Joseph, Toxicol. Appl. Pharmacol., 2009, 238, 272–279.
90. M. Kaczmarek, R. Cachau, I. A. Topol, K. S. Kasprzak, A. Ghio and
K. Salnikow, Toxicol. Sci., 2009, 107, 394–403.
91. M. Kaczmarek, O. Timofeeva, A. Karaczyn, A. Malyguine, K. S. Kasprzak and
K. Salnikow, Free Rad. Biol. Med., 2007, 42, 1246–1257.
92. K. G. Higinbotham, J. M. Rice, B. A. Diwan, K. S. Kasprzak, C. Reed and
A. O. Perantoni, Cancer Res., 1992, 52, 4747–4751.
93. L. K. Tkeshelashvili, T. M. Reid, T. J. McBride and L. A. Loeb, Cancer Res.,
1993, 53, 4172–4174.
94. K. A. Johnson, M. L. Mierzwa, S. P. Fink and L. J. Marnett, J. Biol. Chem.,
1999, 274, 27112–27118.
95. D. Beyersmann and A. Hartwig, Arch. Toxicol., 2008, 82, 493–512.
96. A. Hartwig and T. Schwerdtle, Tox. Lett., 2002, 127, 47–54.
97. A. Hartwig, Antioxid. Redox Signal., 2001, 3, 625–634.
98. A. Witkiewicz-Kucharczyk and W. Bal, Toxicol. Lett., 2006, 162, 29–42.
99. K. Salnikow, S. Donald, R. Bruick, A. Zhitkovich, J. Phang and K. S.
Kasprzak, J. Biol. Chem., 2004, 279, 40337–40344.
100. K. Salnikow and K. S. Kasprzak, Environ. Health Perspect., 2005, 113, 577–584.
101. B. Sedgwick, P. A. Bates, J. Paik, S. C. Jacobs and T. Lindahl, DNA Repair,
2007, 6, 429–442.
102. IARC, IARC Monogr. Eval. Carcinog. Risks Hum., 2004, 84, 39–267.
107. Y. Yuan, G. Marshall, C. Ferreccio, C. Steinmaus, S. Selvin, J. Liaw, M. N.
Bates and A. H. Smith, Am. J. Epidemiol., 2007, 166, 1381–1391.
108. Y. Yuan, G. Marshall, C. Ferreccio, C. Steinmaus, J. Liaw, M. Bates and A. H.
Smith, Epidemiology, 2010, 21, 103–108.
109. C. Samanic, M. Kogevinas, M. Dosemeci, N. Malats, F. X. Real, M. Garcia-
Closas, C. Serra, A. Carrato, R. Garcı́a-Closas, M. Sala, J. Lloreta, A. Tardón,
N. Rothman and D. T. Silverman, Cancer Epidemiol. Biomarkers Prev., 2006,
15, 1348–1354.
110. M. E. Parent and J. Siemiatycki, Epidemiol. Rev., 2001, 23, 138–143.
111. G. G. Schwartz and I. M. Reis, Cancer Epidemiol. Biomarkers Prev., 2000, 9,
139–145.
112. A. Arita and M. Costa, Metallomics, 2009, 1, 222–228.
113. T. Yoshida, H. Yamauchi and G. Fan Sun, Toxicol. Appl. Pharmacol., 2004,
198, 243–252.
114. C. H. Chou and C. T. De Rosa, Int. J. Hyg. Environ. Health, 2003, 206,
381–386.
Met. Ions Life Sci. 2011, 8, 319–373

115. V. Bencko, J. Rames, E. Fabiánová, J. Pesek and M. Jakubis, Environ. Geo-

chem. Health, 2009, 31 (suppl. 1), 239–243.
116. T. Halatek, H. Sinczuk-Walczak, S. Rabieh and W. Wasowicz, Toxicol. Appl.
Pharmacol., 2009, 239, 193–199.
117. W. R. Cullen and K. J. Reimer, Chem. Rev., 1989, 89, 713–764.
118. D. E. Carter, H. V. Aposhian and A. J. Gandolfi, Toxicol. Appl. Pharmacol.,
2003, 193, 309–334.
119. A. D. Kligerman, C. L. Doerr, A. H. Tennant, K. Harrington-Brock, J. W.
Allen, E. Winkfield, P. Poorman-Allen, B. Kundu, K. Funasaka, B. C. Roop,
M. J. Mass and D. M. DeMarini, Environ. Mol. Mutagen., 2003, 42, 192–205.
120. H. V. Aposhian and M. M. Aposhian, Chem. Res. Toxicol., 2006, 19, 1–15.
121. K. Pi˛atek, T. Schwerdtle, A. Hartwig and W. Bal, Chem. Res. Toxicol., 2008, 21,
600–606.
122. S. Nesnow, B. C. Roop, G. Lambert, M. Kadiiska, R. P. Mason, W. R. Cullen
and M. J. Mass, Chem. Res. Toxicol., 2002, 15, 1627–1634.
123. A. Schoen, B. Beck, R. Sharma and E. Dube, Toxicol. Appl. Pharmacol., 2004,
198, 253–267.
124. C. Soriano, A. Creus and R. Marcos, Mutat. Res., 2008, 646, 1–7.
125. K. T. Kitchin and R. Conolly, Chem. Res. Toxicol., 2010, 23, 327–335.
126. M. A. Partridge, S. X. L. Huang, E. Hernandez-Rosa, M. M. Davidson and
T. K. Hei, Cancer Res., 2007, 67, 5239–5247.
127. T. Ochi, K. Kita, T. Suzuki, A. Rumpler, W. Goessler and K. A. Francesconi,
128. C. B. Klein, J. Leszczynska, C. Hickey and T. G. Rossman, Toxicol. Appl.
Pharmacol., 2007, 222, 289–297.
129. T. G. Rossman, A. N. Uddin and F. J. Burns, Toxicol. Appl. Pharmacol., 2004,
198, 394–404.
130. A. Hartmann and G. Speit, Environ. Mol. Mutagen., 1996, 27, 98–104.
131. D. T. Bau, J. R. Gurr and K. Y. Jan, Carcinogenesis, 2001, 22, 709–716.
132. H. P. Tran, A. S. Prakash, R. Barnard, B. Chiswell and J. C. Ng, Toxicol. Lett.,
2002, 133, 59–67.
133. T. Schwerdtle, I. Walter and A. Hartwig, DNA Repair (Amst.), 2003, 2,
1449–1463.
134. H. Danaee, H. H. Nelson, H. Liber, J. B. Little and K. T. Kelsey, Mutagenesis,
2004, 19, 143–148.
135. A. S. Andrew, J. L. Burgess, M. M. Meza, E. Demindenko, M. G. Waugh, J. W.
Hamilton and M. R. Karagas, Environ. Health Perspect., 2006, 114, 1193–1198.
136. I. Walter, T. Schwerdtle, C. Thuy, J. L. Parsons, G. L. Dianov and A. Hartwig,
DNA Repair (Amst.), 2007, 6, 61–70.
137. E. V. Komissarova and T. G. Rossman, Toxicol. Appl. Pharmacol., 2010, 243,
399–404.
138. L. Leu and L. Mohassel, Am. J. Health Syst. Pharm., 2009, 66, 1913–1918.
139. V. Charoensuk, W. P. Gati, M. Weinfeld and X. C. Le, Toxicol. Appl. Phar-
macol., 2009, 239, 64–70.
140. S. André, H. Métivier, G. Lantenois, M. Boyer, D. Nolibé and R. Masse, Hum.
Toxicol., 1987, 6, 233–240.
Met. Ions Life Sci. 2011, 8, 319–373

141. G. A. Day, M. D. Hoover, A. B. Stefaniak, R. M. Dickerson, E. J. Peterson,

N. A. Esmen and R. C. Scripsick, Exp. Lung Res., 2005, 31, 341–360.
142. D. N. Skilleter, Toxicol. Environ. Chem., 1984, 7, 213–228.
143. D. A. Rozmanov, Y. V. Sidorov and K. A. Burkov, J. Sol. Chem., 2005,
34, 1081–1090.
144. T. Gordon and D. Browser, Mutat. Res, 2003, 533, 99–105.
145. J. Taylor-McCabe, Z. Wang, N. N. Sauer and B. L. Marrone, Proteomics, 2006,
6, 1663–1675.
146. A. L. Brooks, W. C. Griffith, N. F. Johnson, G. L. Finch and R. G. Cuddihy,
Radiat. Res., 1989, 120, 494–507.
147. P. Joseph, T. Muchnok and T. M. Ong, Mol. Carcinog., 2001, 32, 28–35.
148. M. A. Fahmy, N. H. A. Hassan, A. A. Farghaly and E. E. S. Hassan, Mutat.
Res., 2008, 652, 103–111.
149. E. Kellen, M. Zeegers, E. D. Hond and F. Buntinx, Cancer Detect. Prev., 2007,
31, 77–82.
150. M. P. Waalkes, Mutat. Res., 2003, 533, 107–120.
151. C. Samanic, M. Kogevinas, M. Dosemeci, N. Malats, F. X. Real, M. Garcia-
Closas, C. Serra, A. Carrato, R. Garcı́a-Closas, M. Sala, J. Lloreta, A. Tardón,
N. Rothman and D. T. Silverman, Cancer Epidemiol. Biomarkers Prev., 2006,
15, 1348–1354.
152. T. Schwerdtle, F. Ebert, C. Thuy, C. Richter, L. H. F. Mullenders and
A. Hartwig, Chem. Res. Toxicol., 2010, 23, 432–442.
153. Y. Li, Y.-L. Xia, Y. Jiang and X.-P. Yan, Electrophoresis, 2008, 29, 1173–1179.
154. M. V. Mikhailova, N. A. Littlefield, B. S. Hass, L. A. Poirier and M. W. Chou,
Cancer Lett., 1997, 115, 141–148.
155. J. Liu, W. Qu and M. B. Kadiiska, Toxicol. Appl. Pharmacol., 2009, 238,
209–214.
156. G. Bertin and D. Averbeck, Biochimie, 2006, 88, 1549–1559.
157. C. D. Klaassen, J. Liu and B. A. Diwan, Toxicol. Appl. Pharmacol., 2009, 238,
215–220.
158. W. E. Achanzar, M. M. Webber and M. P. Waalkes, Prostate, 2002, 52,
236–244.
159. J. J. Mukherjee, S. K. Gupta and S. Kumar, Chem.-Biol. Interact., 2008, 172,
72–80.
160. H. Dally and A. Hartwig, Carcinogenesis, 1997, 18, 1021–1026.
161. T. Fatur, M. Tusek, I. Falnoga, J. Scancar, T. T. Lach and M. Filipic, Mutat.
Res., 2003, 529, 109–116.
162. R. D. Watkin, T. Nawrot, R. J. Potts and B. A. Hart, Toxicology, 2003, 184,
157–178.
163. A. Hartwig, M. Asmuss, H. Blessing, S. Hoffmann, G. Jahnke, S. Khandelwal,
A. Pelzer and A. Burkle, Food Chem. Toxicol., 2002, 40, 1179–1184.
164. M. Asmuss, L. H. F. Mullenders, A. Elker and A. Hartwig, Carcinogenesis,
2000, 21, 2097–2104.
165. A. Lutzen, S. E. Liberti and L. J. Rasmussen, Biochem. Biophys. Res. Commun.,
2004, 321, 21–25.
166. A. B. Clark and T. A. Kunkel, J. Biol. Chem., 2004, 279, 53903–53906.
Met. Ions Life Sci. 2011, 8, 319–373

167. J. B. Vincent, Proc. Nutr. Soc., 2004, 63, 41–47.

168. Y. Ishikawa, K. Nakagawa, Y. Satoh, T. Kitagawa, H. Sugano, T. Hirano and
E. Tsuchiya, Br. J. Cancer, 1994, 70, 160–166.
169. B. D. Kerger, B. L. Finley, G. E. Corbett, D. G. Dodge and D. J. Paustenbach,
J. Tox. Environ. Health., 1997, 50, 67–95.
170. S. De Flora, M. Iltcheva and R. M. Baransky, Mutat. Res., 2006, 610, 38–47.
171. A. Zhitkovich, Chem. Res. Toxicol., 2005, 18, 3–11.
172. J. Alexander and J. Aaseth, Analyst, 1995, 120, 931–933.
173. De Flora, M. Bagnasco, D. Serra and P. Zanacchi, Mutat. Res., 1990, 238, 99–
172.
174. D. M. Stearns and K. E. Wetterhahn, in Cytotoxic, Mutagenic and Carcinogenic
Potential of Heavy Metals Including Metals Related to Human Environment, Ed.
N. Hadjiliadis, Vol. 26 of NATO ASI (Ser. 2), Kluwer Academic, Dordrecht,
The Netherlands, 1997, pp. 107–121.
175. K. D. Sugden and D. M. Stearns, J. Environ. Pathol. Toxicol. Oncol., 2000, 19,
215–230.
176. K. S. Kasprzak and G. S. Buzard, in Molecular Biology and Toxicology of
Metals, Ed. J. Koropatnick and R. Zalups, Taylor & Francis, London, UK,
2000, pp. 477–527.
177. K. M. Borges, J. S. Boswell, R. H. Liebross and K. E. Wetterhahn, Carcino-
genesis, 1991, 12, 551–561.
178. D. M. Stearns and K. E. Wetterhahn, Chem. Res. Toxicol., 1994, 7, 219–230.
179. R. Codd and P. A. Lay, Chem. Res. Toxicol., 2003, 16, 881–892.
180. R. N. Bose, S. Moghaddas and E. Gelerinter, Inorg. Chem., 1992, 31, 1987–
1994.
181. P. A. Lay and A. Levina, J. Am. Chem. Soc., 1998, 120, 6704–6714.
182. D. M. L. Goodgame and M. A. Joy, Inorg. Chim. Acta, 1987, 135, 115–118.
183. A. Levina, L. Zhang and P. A. Lay, Inorg. Chim., 2003, 42, 767–784.
184. W. Bal and K. S. Kasprzak, Toxicol. Lett., 2002, 127, 55–62.
185. C. T. Dillon, P. A. Lay, A. M. Bonin, M. Cholewa and G. J. F. Legge, Chem.
Res. Toxicol., 2000, 13, 742–748.
186. A. M. Standeven and K. E. Wetterhahn, Carcinogenesis, 1992, 13, 1319–1324.
187. G. Quievryn, J. Messer and A. Zhitkovich, Biochemistry, 2002, 41,
3156–3167.
188. M. Reynolds, L. Stoddard, I. Bespalov and A. Zhitkovich, Nucleic Acids Res.,
2007, 35, 465–476.
189. P. G. Slade, M. K. Hailer, B. D. Martin and K. D. Sugden, Chem. Res. Toxicol.,
2005, 18, 1140–1149.
190. J. N. Gremaud, B. D. Martin and K. D. Sugden, Chem. Res. Toxicol., 2010, 23,
379–385.
191. P. Hunt and H. L. Friedman, Prog. Inorg. Chem., 1983, 30, 359–387.
192. A. Zhitkovich, V. Voitkun and M. Costa, Carcinogenesis, 1995, 16, 907–913.
193. G. Quievryn, E. Peterson, J. Messer and A. Zhitkovich, Biochemistry, 2003, 42,
1062–1070.
194. V. Voitkun, A. Zhitkovich and M. Costa, Nucleic Acids Res., 1998, 26,
2024–2030.
Met. Ions Life Sci. 2011, 8, 319–373

195. A. Zhitkovich, Y. Song, G. Quievryn and V. Voitkun, Biochemistry, 2001, 40,

549–560.
196. S. A. Blankert, V. H. Coryell, B. T. Picard, K. K. Wolf, R. E. Lomas and D. M.
Stearns, Chem. Res. Toxicol., 2003, 16, 847–854.
197. A. Macfie, E. Hagan and A. Zhitkovich, Chem. Res. Toxicol., 2010, 23, 341–
347.
198. A. Zhitkovich, V. Voitkun and M. Costa, Biochemistry, 1996, 35, 7275–7282.
199. A. Kr˛e(el and W. Bal, Acta Biochim. Polon., 1999, 46, 567–580.
200. H. A. Tajmir-Riahi, J. Inorg. Biochem., 1991, 42, 47–55.
201. A. Kr˛e(el, W. Szczepanik, M. Sokozowska, M. Je(owska-Bojczuk and W. Bal,
Chem. Res. Toxicol., 2003, 16, 855–864.
202. A. Levina, H. H. Harris and P. A. Lay, J. Am. Chem. Soc., 2007, 129, 1065–
1075.
203. A. L. Holmes, S. S. Wise and J. P. Wise, Sr., Indian J. Med. Res., 2008, 128, 353–
372.
204. R. S. Luippold, K. A. Mundt, R. P. Austin, E. Liebig, J. Panko, C. Crump, K.
Crump and D. Proctor, Occup. Environ. Med., 2003, 60, 451–457.
205. S. S. Wise, A. L. Holmes, Q. Qin, H. Xie, S. P. Katsifis, W. D. Thompson and J.
P. Wise, Sr., Chem. Res. Toxicol., 2010, 23, 365–372.
206. A. L. Holmes, S. S. C. Pelsue, A. Aboueissa, W. Lingle, J. Salisbury, J. Gal-
lagher and J. P. Wise, Sr., Chem. Res. Toxicol., 2010, 23, 386–395.
207. E. Denkhaus and K. Salnikow, Crit. Rev. Oncol. Hematol., 2002, 42, 35–56.
208. K. S. Kasprzak, F. W. Sunderman, Jr. and K. Salnikow, Mutat. Res., 2003, 533,
67–97.
209. J. K. Dunnick, M. R. Elwell, A. E. Radovsky, J. M. Benson, F. F. Hahn, K. J.
Nikula, E. B. Barr and C. H. Hobbs, Cancer Res., 1995, 55, 5251–5256.
210. M. Costa and H. H. Mollenhauer, Science, 1980, 209, 515–517.
211. M. Costa, J. Simmons-Hansen, C. W. Bedrossian, J. Bonura and R. M.
Caprioli, Cancer Res., 1981, 41, 2868–2876.
212. R. M. Evans, P. J. Davies and M. Costa, Cancer Res., 1982, 42, 2729–2735.
213. M. Horie, K. Nishio, K. Fujita, H. Kato, A. Nakamura, S. Kinugasa, S. Endoh,
A. Miyauchi, K. Yamamoto, H. Murayama, E. Niki, H. Iwahashi, Y. Yoshida
and J. Nakanishi, Chem. Res. Toxicol., 2009, 22, 1415–1426.
214. G. G. Fletcher, F. E. Rosetto, J. D. Turnbull and E. Nieboer, Environ. Health
Perspect., 1994, 102 (suppl. 3), 69–79.
215. A. Longstaff, A. I. T. Walker and R. Jackh, in Nickel in the Human Environ-
ment, Ed. F. W. Sunderman, IARC Scientific Publications, Vol. 53, Lyon, 1984,
pp. 235–244.
216. L. C. Harty, D. G. Guinee, Jr., W. D. Travis, W. P. Bennett, J. Jett, T. V. Coby,
H. Tazelaar, V. Trastek, P. Pairolero, L. A. Liotta, C. C. Harris and N. E.
Caporaso, Cancer Epidemiol. Biomark. Prev., 1996, 5, 997–1003.
217. F. Golebiowski and K. S. Kasprzak, Mol. Cell. Biochem., 2005, 279, 133–139.
218. A. A. Karaczyn, F. Golebiowski and K. S. Kasprzak, Chem. Res. Toxicol.,
2005, 18, 1934–1942.
219. A. A. Karaczyn, F. Golebiowski and K. S. Kasprzak, Exp. Cell. Res., 2006, 312,
3252–3259.
Met. Ions Life Sci. 2011, 8, 319–373

220. L. Broday, W. Peng, M. H. Kuo, K. Salnikow, M. Zoroddu and M. Costa,

Cancer Res., 2000, 60, 238–241.
221. H. Chen, Q. Ke, T. Kluz, Y. Yan and M. Costa, Mol. Cell. Biol., 2006, 26, 3728–
3737.
222. Q. Ke, T. Davidson, H. Chen, T. Kluz and M. Costa, Carcinogenesis, 2006, 27,
1481–1488.
223. A. A. Karaczyn, W. Bal, S. L. North, R. M. Bare, V. M. Hoang, R. J. Fisher
and K. S. Kasprzak, Chem. Res. Toxicol., 2003, 16, 1555–1559.
224. A. A. Karaczyn, F. Golebiowski and K. S. Kasprzak, Chem. Res. Toxicol.,
2005, 18, 1934–1942.
225. A. A. Karaczyn, R. Y. S. Cheng, G. S. Buzard, J. Hartley, D. Esposito and K. S.
Kasprzak, Ann. Clin. Lab. Sci., 2009, 39, 251–262.
226. W. Li, Y. Zhao and I. N. Chou, Toxicology, 1993, 77, 65–79.
227. S. Lynn, F. H. Yew, J.-W. Hwang, M.-J. Tseng and K. Y. Jan, Carcinogenesis,
1994, 15, 2811–2816.
228. W. Li, Y. Zhao and I. N. Chou, Toxicol. Appl. Pharmacol., 1996, 136,
101–111.
229. K. Salnikow, M. Gao, V. Voitkun, X. Huang and M. Costa, Cancer Res., 1994,
54, 6407–6412.
230. K. Salnikow, T. Davidson, Q. Zhang, L. C. Chen, W. Su and M. Costa, Cancer
Res., 2003, 63, 3524–3530.
231. M. Kaczmarek, R. E. Cachau, I. A. Topol, K. S. Kasprzak, A. Ghio and K.
Salnikow, Toxicol Sci., 2009, 107, 394–403.
232. A. Hartwig, L. H. F. Mullenders, R. Schlepegrell, U. Kasten and D. Beyers-
mann, Cancer Res., 1994, 54, 4045–4051.
233. F. Iwitzki, R. Schlepegrell, U. Eichhorn, B. Kaina, D. Beyersmann and A.
Hartwig, Arch. Toxicol., 1998, 72, 681–689.
234. T. Schwerdtle, A. Seidel and A. Hartwig, Carcinogenesis, 2002, 23, 47–53.
235. M. Asmuss, L. H. F. Mullenders, A. Elker and A. Hartwig, Carcinogenesis,
2000, 21, 2097–2104.
236. R. E. Rodriguez, M. Misra, B. A. Diwan, C. W. Riggs and K. S. Kasprzak,
Toxicology, 1996, 107, 131–140.
237. W. Bal, J. Lukszo, K. Biazkowski and K. S. Kasprzak, Chem. Res. Toxicol.,
1998, 11, 1014–1023.
238. W. Bal, R. Liang, J. Lukszo, S.-H. Lee, M. Dizdaroglu and K. S. Kasprzak,
Chem. Res. Toxicol., 2000, 13, 616–624.
239. W. Bal, M. Je(owska-Bojczuk, J. Lukszo and K. S. Kasprzak, Chem. Res.
Toxicol., 1995, 8, 683–692.
240. W. Bal, J. Lukszo and K. S. Kasprzak, Chem. Res. Toxicol., 1996, 9, 435–440.
241. W. Bal, V. Karantza, E. N. Moudrianakis and K. S. Kasprzak, Arch. Biochem.
Biophys., 1999, 364, 161–166.
242. A. Kr˛e(el, W. Szczepanik, M. Sokozowska, M. Je(owska-Bojczuk and W. Bal,
Chem. Res. Toxicol., 2003, 16, 855–864.
243. K. S. Kasprzak, J. M. Ward, L. A. Poirier, D. A. Reichardt, A. C. Denn III and
C. W. Reynolds, Carcinogenesis, 1987, 8, 1005–1011.
Met. Ions Life Sci. 2011, 8, 319–373

245. D. Lison, M. De Boeck, V. Verougstraete and M. Kirsch-Volders, Occup.

Environ. Med., 2001, 58, 619–625.
246. G. S. Kang, Q. Li, H. Chen and M. Costa, Mutat. Res., 2006, 610, 48–55.
247. Q. Li, Q. Ke and M. Costa, Carcinogenesis, 2009, 30, 1243–1251.
248. M. Asmuss, L. H. Mullenders, A. Eker and A. Hartwig, Carcinogenesis, 2000,
21, 2097–2104.
249. E. Kopera, T. Schwerdtle, A. Hartwig and W. Bal, Chem. Res. Toxicol., 2004,
17, 1452–1458.
250. K. S. Kasprzak, T. H. Zastawny, S. L. North, C. W. Riggs, B. A. Diwan, J. M.
Rice and M. Dizdaroglu, Chem. Res. Toxicol., 1994, 7, 329–335.
Interact., 2006, 160, 1–40.
252. S. Kawanishi, S. Inoue and K. Yamamoto, Environ. Health. Perspect., 1994, 102
(suppl. 3), 17–20.
253. B. Kräutler, Biochem. Soc. Trans., 2005, 33, 806–810.
254. E. K. Silbergeld, M. Waalkes and J. M. Rice, Am. J. Ind. Med., 2000, 38, 316–
323.
256. D. Seyferth, Organometallics, 2003, 22, 5154–5178.
257. E. Dynerowicz-Bal, R. Andrzejak, J. Antonowicz-Juchniewicz, M. Siewiński, E.
Sujak and B. Smyk, Med. Pr., 2005, 56, 347–361.
258. J. Palus, K. Rydzynski, E. Dziubaltowska, K. Wyszynska, A. T. Natarajan and
R. Nilsson, Mutat. Res., 2003, 540, 19–28.
259. K. S. Kasprzak, K. L. Hoover and L. A. Poirier, Carcinogenesis, 1985, 6, 279–282.
260. M. P. Waalkes, B. A. Diwan, J. M. Ward, D. E. Devor and R. A. Goyer, Cancer
Res., 1995, 55, 5265–5271.
261. J. L. Yang, L. C. Wang, C. Y. Chang and T. Y. Liu, Environ. Mol. Mutagen.,
1999, 33, 194–201.
262. J. Gastaldo, M. Viau, Z. Bencokova, A. Joubert, A.-M. Charvet, J. Balosso and
N. Foray, Toxicol. Lett., 2007, 173, 201–214.
263. A. Hartwig, R. Schlepegrell and D. Beyersmann, Mutat. Res., 1990, 241, 75–82.
264. N. K. Roy and T. G. Rossman, Mutat. Res., 1992, 298, 97–103.
265. M. E. Ariza and M. V. Williams, Environ. Mol. Mutagen., 1996, 27, 30–33.
266. C.-Y. Wang, Y.-W. Lin and J.-L. Yang, Toxicology, 2008, 250, 55–61.
267. J. S. Magyar, T. C. Weng, C. M. Stern, D. F. Dye, B. W. Rous, J. C. Payne, B.
M. Bridgewater, A. Mijovilovich, G. Parkin, J. M. Zaleski, J. E. Penner-Hahn
and H. A. Godwin, J. Am. Chem. Soc., 2005, 127, 9495–9505.
268. B. Quintanilla-Vega, D. Hoover, W. Bal, E. K. Silbergeld, M. P. Waalkes and L.
Anderson, Am. J. Ind. Med., 2000, 38, 324–329.
269. B. Quintanilla-Vega, D. Hoover, W. Bal, E. K. Silbergeld, M. P. Waalkes and L.
Anderson, Chem. Res. Toxicol., 2000, 13, 594–600.
270. A. Bleise, P. R. Danesi and W. Burkart, J. Environ. Radioact., 2003, 64, 93–112.
271. A. C. Miller and D. McClain, Rev. Environ. Health, 2007, 22, 75–89.
272. M. A. McDiarmid, S. M. Engelhardt, M. Oliver, P. Gucer, P. D. Wilson, R.
Kane, A. Cernich, B. Kaup, L. Anderson, D. Hoover, L. Brown, R. Albertini,
R. Gudi, D. Jacobson-Kram and K. S. Squibb, Health Phys., 2007, 93, 60–73.
Met. Ions Life Sci. 2011, 8, 319–373

273. M. Monleau, M. De Méo, F. Paquet, V. Chazel, G. Duménil and M. Donna-

dieu-Claraz, Toxicol. Sci., 2006, 89, 287–295.
274. Y. Hao, R. Li, Y. Leng, J. Ren, J. Liu, G. Ai G, H. Xu, Y. Su and T. Cheng, J.
Radiat. Res. (Tokyo), 2009, 50, 521–528.
275. C. Darolles, D. Broggio, A. Feugier, S. Frelon, I. Dublineau, M. De Meo and F.
Petitot, Toxicol. Lett., 2010, 192, 337–348.
276. H. Xie, C. LaCerte, W. D. Thompson and J. P. Wise, Sr., Chem. Res. Toxicol.,
2010, 23, 373–378.
277. M. Yazzie, S. L. Gamble, E. R. Civitello and D. M. Stearns, Chem. Res. Tox-
icol., 2003, 16, 524–530.
278. V. S. Smirnova, S. V. Gudkov, I. N. Shtarkman, A. V. Chernikov and V. I.
Bruskov, Biofizika, 2005, 50, 456–463.
279. A. C. Miller, M. Stewart, K. Brooks, L. Shi and N. Page, J. Inorg. Biochem.,
2002, 91, 246–252.
280. C. A. Rabik and M. E. Dolan, Cancer Treatment Rev., 2007, 33, 9–23.
281. S. Chijiwa, C. Masutani, F. Hanaoka, S. Iwai and I. Kuraoka, Carcinogenesis,
2010, 31, 388–393.
282. F. T. Unger, H. A. Klasen, G. Tchartchian, R. L. de Wilde and I. Witte, BMC
Cancer, 2009, 9, 359, DOI:10.1186/1471-2407-9-359.
283. J. M. Kaldor, N. E. Day and K. Hemminki, Eur. J. Cancer Clin. Oncol., 1988,
24, 703–711.
284. M. Satoh, Y. Kondo, M. Mita, I. Nakagawa, A. Naganuma and N. Imura,
Cancer Res., 1993, 53, 4767–4768.
285. L. Migliore, G. Frenzilli, C. Nesti, S. Fortaner and E. Sabbioni, Mutagenesis,
2002, 17, 411–417.
286. S. C. Lim, J. E. Choi, H. S. Kang and H. Si, Int. J. Cancer, 2010, 126,
1582–1595.
287. P. K. Eagon, A. G. Teepe, M. S. Elm, S. D. Tadic, M. J. Epley, B. E. Beiler,
H. Shinozuka and K. N. Rao, Carcinogenesis, 1999, 20, 1091–1096.
288. S. Choudhury, R. Zhang, K. Frenkel, T. Kawamori, F.-L. Chung and R. Roy,
Cancer Res., 2003, 63, 7704–7707.
289. U. Giri, M. Iqbal and M. Athar, Int. J. Oncol., 1999, 14, 799–806.
290. L. Jiang, Y. Zhong, S. Akatsuka, Y. T. Liu, K. K. Dutta, W. H. Lee,
J. Onuki, K. Masumura, T. Nohmi and S. Toyokuni, Cancer Sci., 2006, 97,
1159–1167.
291. L. K. Tkeshelashvili, T. J. McBride, K. Spence and L. A. Loeb, J. Biol. Chem.,
1991, 266, 6401–6406.
292. H. Rodriguez, G. P. Holmquist, R. D’Agostino, Jr., J. Keller and S. A. Akman,
Cancer Res., 1997, 57, 2394–2403.
293. J. R. Landolph, in Interrelations Between Free Radicals and Metal Ions in Life
Processes, Vol. 36 of Metal Ions in Biological Systems, Ed. A. Sigel, H. Sigel,
Marcel Dekker, New York, 1999, pp. 445–483.
294. L. J. Kennedy, K. Moore, Jr., J. L. Caulfield, S. R. Tannenbaum and P. C.
Dedon, Chem. Res. Toxicol., 1997, 10, 386–392.
295. M. Mylonas, G. Malandrinos, J. Plakatouras, N. Hadjiliadis, K. S. Kasprzak,
A. Krezel and W. Bal, Chem. Res. Toxicol., 2001, 14, 1177–1183.
Met. Ions Life Sci. 2011, 8, 319–373

296. K. C. Galbreath, R. L. Schulz, D. L. Toman, C. M. Nyberg, F. E. Huggins, G.

P. Huffman and E. J. Zillioux, J. Air Waste Manag. Assoc., 2005, 55, 309–318.
297. U. P. Kodavanti, R. Hauser, D. C. Christiani, Z. H. Meng, J. McGee, A.
Ledbetter, J. Richards and D. L. Cost, Toxicol. Sci., 1998, 43, 204–212.
298. A. K. Prahalad, J. M. Soukup, J. Inmon, R. Willis, A. J. Ghio, S. Becker and J.
E. Gallagher, Toxicol. Appl. Pharmacol., 1999, 158, 81–91.
299. A. K. Prahalad, J. Inmon, A. J. Ghio and J. E. Gallagher, Chem. Res. Toxicol.,
2000, 13, 1011–1019.
300. J. M. Antonini, M. D. Taylor, S. S. Leonard, N. J. Lawryk, X. Shi, R. W.
Clarke and J. R. Roberts, Mol. Cell. Biochem., 2004, 255, 257–265.
301. S. Pattanaik, F. E. Huggins, G. P. Huffman, W. P. Linak and C. A. Miller,
Environ. Sci. Technol., 2007, 41, 1104–1110.
302. D. L. Costa, J. R. Lehmann, D. Winsett, J. Richards, A. D. Ledbetter and K. L.
Dreher, Toxicol. Sci., 2006, 91, 237–246.
303. M. E. Klein-Patel, G. Diamond, M. Boniotto, S. Saad and L. K. Ryan, Toxicol.
Sci., 2006, 92, 115–125.
304. A. Di Pietro, G. Visalli, F. Munaò, B. Baluce, S. La Maestra, P. Primerano, F.
Corigliano and S. De Flora, Int. J. Hyg. Environ. Health, 2009, 212, 196–208.
305. D. Rehder, in Vanadium and Its Role in Life, Vol. 31 of Metal Ions in Biological
Systems, Ed. A. Sigel and H. Sigel, Marcel Dekker, New York, 1995, pp. 1–43.
306. P. Buglyó, D. C. Crans, E. M. Nagy, R. L. Lindo, L. Yang, J. J. Smee, W. Jin,
L. H. Chi, M. E. Godzala III and G. R. Willsky, Inorg. Chem., 2005, 44, 5416–
5427.
308. M. Caicedo, J. J. Jacobs, A. Reddy and N. J. Hallab, J. Biomed. Mater. Res. A,
2008, 86, 905–913.
309. D. Lison, P. Carbonnelle, L. Mollo, R. Lauwerys and B. Fubini, Chem. Res.
Toxicol., 1995, 8, 600–606.
310. J. G. Hengstler, U. Bolm-Audorff, A. Faldum, K. Janssen, M. Reifenrath, W.
Götte, D. Jung, O. Mayer-Popken, J. Fuchs, S. Gebhard, H. G. Bienfait, K.
Schlink, C. Dietrich, D. Faust, B. Epe and F. Oesch, Carcinogenesis, 2003, 24,
63–73.
311. F. Glahn, W. Schmidt-Heck, S. Zellmer, R. Guthke, J. Wiese, K. Golka, R.
Hergenröder, G. H. Degen, T. Lehmann, M. Hermes, W. Schormann, M.
Brulport, A. Bauer, E. Bedawy, R. Gebhardt, J. G. Hengstler and H. Foth,
Arch. Toxicol., 2008, 82, 513–524.
312. M. Costa, A. Zhitkovich and P. Toniolo, Cancer Res., 1993, 53, 460–463.
313. U. Werfel, V. Langen, I. Eickhoff, J. Schoonbrood, C. Vahrenholz, A.
Brauksiepe, W. Popp and K. Norpoth, Carcinogenesis, 1998, 19, 413–418.
314. G. Iarmarcovai, I. Sari-Minodier, F. Chaspoul, C. Botta, M. De Méo, T.
Orsière, J. L. Bergé-Lefranc, P. Gallice and A. Botta, Mutagenesis, 2005, 20,
425–432.
315. H. Merzenich, A. Hartwig, W. Ahrens, D. Beyersmann, R. Schlepegrell, M.
Scholze, J. Timm and K. H. Jöckel, Cancer Epidemiol. Biomarkers Prev., 2001,
10, 515–522.
316. M. A. Sirover and L. A. Loeb, Science, 1976, 194, 1434–1436.
Met. Ions Life Sci. 2011, 8, 319–373

317. A. P. Grollman and M. Moriya, Trends Genet., 1993, 9, 246–249.

318. S. M. Chiocca, D. A. Sterner, N. W. Biggart and E. C. Murphy, Jr., Mol.
Carcinog., 1991, 4, 61–71.
319. A. Léonard, G. B. Gerber and P. Jacquet, Mutat. Res., 1981, 87, 1–15.
320. P. D. Josephy, J. J. Weadge, J. Meissner and F. Ng, Mutat. Res., 2008, 654, 64–
68.
321. C. M. Moore and J. D. Helmann, Curr. Opin. Microbiol., 2005, 8, 188–195.
322. L. A. Finney and T. V. O’Halloran, Science, 2003, 300, 931–936.
323. M. Costa, M. P. Abbracchio and J. Simmons-Hansen, Toxicol. Appl. Phar-
macol., 1981, 60, 313–323.
324. A. Oskarsson, Y. Andersson and H. Tjälve, Cancer Res., 1979, 39, 4175–4182.
325. K. S. Kasprzak and F. W. Sunderman, Jr., Res. Commun. Chem. Pathol.
Pharmacol., 1977, 16, 95–108.
326. K. S. Kasprzak and K. Salnikow, in Nickel and Its Surprising Impact in Nature,
Vol. 2 of Metal Ions in Life Sciences, Ed. A. Sigel, H. Sigel and R. K. O. Sigel, J.
Wiley & Sons, Chichester, UK, 2007, pp. 619–660.
327. J. Smith-Sonneborn, B. Leibovitz, R. Donathan and G. L. Fisher, Environ.
Mutagen., 1986, 8, 621–626.
328. P. Pikálek and J. Necásek, Folia Microbiol. (Praha), 1983, 28, 17–21.
329. S. Ohshima, Mutagenesis, 2003, 18, 133–137.
330. P. Sen, K. Conway and M. Costa, Cancer Res., 1987, 47, 2142–2147.
331. M. Morita, M. Umeda and H. I. Ogawa, Mutat. Res., 1991, 261, 131–137.
332. C. Mayer, R. G. Klein, H. Wesch and P. Schmezer, Mutat. Res., 1998, 420, 85–
98.
333. L. Maehle, R. A. Metcalf, D. Ryberg, W. P. Bennett, C. C. Harris and A.
Haugen, Cancer Res., 1992, 52, 218–221.
334. B. L. Pool-Zobel, N. Lotzmann, M. Knoll, F. Kuchenmeister, R. Lambertz, U.
Leucht, H. G. Schröder and P. Schmezer, Environ. Mol. Mutagen., 1994, 24, 23–
45.
335. G. Tveito, I. L. Hansteen, H. Dalen and A. Haugen, Cancer Res., 1989, 49,
1829–1835.
336. V. I. Chernenko, L. L. Lukash, L. L. Matsevich, T. P. Kochubeı̆, A. A. Ele-
sichev, E. A. Klimenko and L. N. Gorban’, Tsitol. Genet., 2004, 38, 31–35.
337. P. M’Bemba-Meka, N. Lemieux and S. K. Chakrabarti, Arch. Toxicol., 2007,
81, 89–99.
338. K. Danadevi, R. Rozati, B. Saleha Banu and P. Grover, Food. Chem. Toxicol.,
2004, 42, 751–757.
339. A. R. Oller and G. Erexson, Mutat. Res., 2007, 626, 102–110.
340. D. Chorvatovicová and Z. Kováciková, J. Appl. Toxicol., 1992, 12, 67–68.
341. B. Z. Zhong, Z. Q. Li, G. Y. Ma and B. S. Wang, Prog. Clin. Biol. Res., 1990,
340E, 41–46.
342. Y. Cai and Z. Zhuang, Zhonghua Yu Fang Yi Xue Za Zhi, 1999, 33, 75–77.
343. K. Doreswamy, B. Shrilatha, T. Rajeshkumar and Muralidhara, J. Androl.,
2004, 25, 996–1003.
344. K. S. Kasprzak, B. A. Diwan, J. M. Rice, M. Misra, C. W. Riggs, R. Olinski and
M. Dizdaroglu, Chem. Res. Toxicol., 1992, 5, 809–815.
Met. Ions Life Sci. 2011, 8, 319–373

345. Y. X. Lei, Q. Zhang and Z. X. Zhuang, Mutat. Res., 1995, 329, 197–203.
346. C. Z. Deng, M. P. Fons, J. Rosenblatt, R. A. El-Zein, S. Z. Abdel-Rahman and
T. Albrecht, Environ. Mol. Mutagen., 2006, 47, 150–161.
347. S. Kawanishi, S. Inoue, S. Oikawa, N. Yamashita, S. Toyokuni, M. Kawanishi
and K. Nishino, Free Radic. Biol. Med., 2001, 31, 108–116.
348. S. Kawanishi, S. Oikawa, S. Inoue and K. Nishino, Environ. Health Perspect.,
2002, 110(suppl. 5), 789–791.
349. H. Waksvik, M. Boysen and A. C. Høgetveit, Carcinogenesis, 1984, 5, 1525–
1527.
350. I. N. Perminova, T. A. Sinel’shchikova, N. I. Alekhina, E. V. Perminova and G.
D. Zasukhina, Bull. Exp. Biol. Med., 2001, 131, 367–370.
351. M. Kiilunen, J. Utela, T. Rantanen, H. Norppa, A. Tossavainen, M. Koponen,
H. Paakkulainen and A. Aitio, Ann. Occup. Hyg., 1997, 41, 167–188.
352. R. B. Ciccarelli, T. H. Hampton and K. W. Jennette, Cancer Lett., 1981, 12,
349–354.
Met. Ions Life Sci. 2011, 8, 319–373

Met. Ions Life Sci. 2011, 8, 375–401
14
Metal Ions in Human Cancer Development
Erik J. Tokar, 1 Lamia Benbrahim-Tallaa 2 and
Michael P. Waalkes* 1
1
Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National
Cancer Institute at the National Institute of Environmental Health Sciences, Alexander
Drive, Research Triangle Park, NC 27709, USA
<waalkes@niehs.nih.gov>
2
IARC Monographs Section, International Agency for Research on Cancer, Lyon, France
ABSTRACT 376
1. INTRODUCTION 376
1.1. Background of Metals as Carcinogens 376
1.2. Regulatory Definitions of Carcinogens 379
2. KNOWN HUMAN METALLIC CARCINOGENS 380
2.1. Background: Human Carcinogen Assessment 380
2.2. Recent IARC Evaluation of Metallic Carcinogens 381
2.3. Arsenic and Inorganic Arsenic Compounds 382
2.4. Beryllium and Beryllium Compounds 384
2.5. Cadmium and Cadmium Compounds 385
2.6. Chromium(VI) Compounds 386
2.7. Nickel Compounds 387
3. PROBABLE AND POSSIBLE METALLIC CARCINOGENS 388
3.1. Inorganic and Organic Lead Compounds 388
3.2. Cisplatin 389
3.3. Indium Phosphide 390
3.4. Possible Human Inorganic Carcinogens 390
4. POTENTIAL MECHANISMS OF METALLIC
CARCINOGENS 391
376 TOKAR, BENBRAHIM-TALLAA, and WAALKES
4.1. Possibilities at the Chemical Level 391

4.2. DNA Damage and Oxidative Stress 391
4.3. Epigenetic Mechanisms 393
4.4. Aberrant Gene Expression 393
4.5. Compensatory Hyperplasia 394
5. PERIODS OF PARTICULAR SENSITIVITY TO
INORGANIC CARCINOGENS 395
6. FUTURE ISSUES IN METAL CARCINOGENESIS 396
ACKNOWLEDGMENTS 397
REFERENCES 397
ABSTRACT: Metals have been in the environment during the entire evolution of man
and the use of metals is key to human civilization. None-the-less, several very toxic spe-
cies are included in the metallic elements and compounds either widely used by man
and/or widely found in the human environment. This includes the five metallic agents
considered human carcinogens, namely arsenic and arsenic compounds, beryllium and
beryllium compounds, cadmium and cadmium compounds, chromium(VI) compounds,
and nickel compounds, all of which are proven carcinogens in laboratory animals as
well. There is significant human exposure to these carcinogenic inorganics, either occu-
pationally, through the environment, or both. Inhalation is typical in the workplace
while inhalation or ingestion occurs from environmental sources. Human metallic carci-
nogens frequently cause tumors at the portal of entry and lung cancers are the most
common tumor after inhalation. Agent-specific tumors occur as well, like urinary blad-
der tumors after arsenic exposure, which are due to biokinetics or mechanisms that are
specific to arsenic. Even in their simplest elemental form, metals are not inert, and they
have biological activity. However, it should be kept in mind that these inorganic carci-
nogens, when in the atomic form, cannot be broken down into less toxic subunits, and
this, in part, is why they are so important as environmental human carcinogens. This
chapter focuses on the metallic agents that are known human carcinogens.
KEYWORDS: arsenic . beryllium . cadmium . cancer . chromium . mechanisms .

nickel
1. INTRODUCTION
1.1. Background of Metals as Carcinogens

Metals and inorganics have been in the human environment during the entire
evolution of man. Furthermore, the use of metals has been key to the tech-
nological advancement of mankind. It is hard to envision an advanced human
civilization without the use of metals. None-the-less, several very toxic species
are included in the metallic elements either widely used or widely found in the
human environment. This includes the five metals or inorganic agents that are
Met. Ions Life Sci. 2011, 8, 375–401

METAL IONS IN HUMAN CANCER DEVELOPMENT 377
considered human carcinogens by the World Health Organization’s Interna-

tional Agency for Research on Cancer (IARC) in one form or another [1–5].
These five known human metallic carcinogens include: Arsenic and arsenic
compounds, beryllium and beryllium compounds, cadmium and cadmium
compounds, chromium(VI) compounds, and nickel compounds based on the
most recent summarized update by IARC [5] of its prior evaluations [1–4].
There has been longstanding human evidence for carcinogenic potential for
some of these metallic agents, as for example with arsenic, where there was
early evidence of human carcinogenic activity in the 1880s when arsenic
mixtures used as pharmaceuticals were first associated with human cancers
[6]. Others have been more recently established as carcinogenic in humans,
like cadmium [3], primarily from occupational exposures. All these known
metallic human carcinogens have now been proven to have carcinogenic
activity in laboratory animals [1–5], although for some, like the arsenicals, this
surprisingly came long after they were well established as human carcinogens
[4,5]. This chapter will focus on the metallic agents that are now considered
known human carcinogens (IARC Group 1; see below). There is more limited
evidence for other metals, such as lead and lead compounds [7], and lead and
other probable and possible human carcinogens will be surveyed.
It would be essentially meaningless to classify an agent as carcinogenic to
humans if no people were exposed to that agent. There are clearly significant
human populations that are exposed to these carcinogenic inorganics, either
in occupational settings or through the environment or both [1–5]. Inhala-
tion is the typical route of exposure in the workplace while inhalation or
ingestion are the more common routes of exposure from environmental
sources [5]. For example, it is thought that tens of millions of people are
exposed to unhealthy levels of arsenic in the drinking water worldwide from
naturally occurring sources, and clearly this is a major public health issue in
many countries [4]. Occupational health measures have led to a drastic
lowering of occupational metal exposure in the Western world, although the
high work environment levels can still be found in many developing coun-
tries that disproportionate the balance between the need for rapid indus-
trialization and worker safety.
Metallic human carcinogenic agents will frequently, but not always, cause
tumors at the portal of entry. Hence, lung cancers are common after inha-
lation exposures in humans [5]. Other, agent-specific tumors do occur as
well, which are likely due to unique biokinetics, metabolism or mechanisms.
In this regard, even when in their simplest elemental form, metals are not
inert, and they undergo ‘‘metabolism’’ by cells. For instance, inorganic
arsenic undergoes an elaborate series of enzymatic biomethylations that may
create additional carcinogenic species, such as dimethylarsinic acid
(DMA(V)) which causes tumors in the urinary bladder of rats [8–10]. Tri-
valent methylated species of arsenic are, in fact, thought to have the most
Met. Ions Life Sci. 2011, 8, 375–401

potential as toxicant and possible carcinogens. Metals like cadmium do not

undergo biotransformation as such with additions of carbons or other
organic groups but cadmium ions will, for instance, induce a specific metal
binding protein, namely metallothionein (MT), which normally binds phy-
siologically essential metals like zinc [11]. Cadmium actually greatly
increases MT protein levels and disrupts normal zinc homeostasis. Cad-
mium, by causing MT over-expression, thus disrupts the natural metal
physiology (metabolism) of the cell [11]. In the classical sense, a lack of
catabolic metabolism is clearly the rule with atomic species, and it should be
kept in mind that these inorganic ionic carcinogens, when in atomic form,
cannot be broken down into less toxic subunits. This, in part, is why they are
such important environmental human carcinogens [1–5].
Metals in massive forms, such as large medical implants or indwelling
projectiles or fragments, are a different issue, and cancers occurring at such
metallic foreign bodies have been variously linked to chronic irritation, such
as ‘‘Oppenheimer’’ effect, solid state carcinogenesis, or other issues not
related to composition dissolution [12,13]. However, the interactions of
physiological solutions with metallic particle surfaces, even large particles or
indwelling devices, can be very complex and lead to surface dissolution and
locally high concentrations of metals and local tumors [13]. If two different
types of metallic compounds are in contact in what is in essence a salt
solution, galvanic erosion may well occur, exaggerating surface dissolution
and, hence, potentially inducing tumor formation [13,14]. Chronic inflam-
mation, which lowers local tissue pH, appears to lend itself to enhanced
surface metal release and increases tumor formation as well [14]. However,
these are uncommon types and mixed types of metal exposures, and will not
be covered in depth in this chapter.
Many carcinogenic metals have been used experimentally in classical
‘‘two-stage’’ carcinogenesis test systems. This is where either the test metal or
some other substance (usually a carcinogen itself) is either given as the tumor
initiator (first) or subsequently as the tumor promoter (second). Although
such studies can be illuminating about some aspects of mechanisms, they can
also be very complex and often provide little insight into ‘‘complete’’ car-
cinogenic potential for the metal of concern. So this chapter has generally
only looked at studies in animals that provide evidence of complete carci-
nogenic effects of the metals in question. Therefore, for the sake of clarity, a
complete carcinogen is herein defined as a compound which, when given by
itself, causes a statistically significant increase in tumor incidence in a given
organ or tissue over natural background. There are many negative carcino-
genesis studies in animals with inorganic or metallic agents. Space prohibits
the discussion of all these negative studies even if of high quality. The
interested reader is referred to the relevant IARC evaluation sections for
such studies.
Met. Ions Life Sci. 2011, 8, 375–401

1.2. Regulatory Definitions of Carcinogens

In a regulatory sense, the term carcinogen denotes a substance or a mixture of
substances that induces or increases the incidence of cancer, which is an
uncontrolled growth and spread of cells that can often invade surrounding
tissue and can metastasize to distant sites. Substances that have induced benign
and malignant tumors in well-performed experimental animal studies are con-
sidered to be presumed or suspected human carcinogens unless there is strong
evidence that the mechanism of tumor formation is not relevant for humans.
Rarely substances shown only to be carcinogenic in laboratory animals can be
considered as human carcinogens because of unique circumstances usually
involving mechanisms. Different regulatory agencies approach classification of
a chemical using these fundamental characteristics in a variety of ways.
The global burden of cancer is high, continues to increase, and it is
expected to reach 15 million new cases by 2020 [15]. It is thought that it may
be possible to prevent at least one-third of these new cases through better use
of existing knowledge. It is clear from these data that environmental and
occupational carcinogens play a role in this increasing global cancer burden.
With current trends in demographics and exposure, the burden of cancer has
now been shifting from high-resource countries to low- and medium-
resource countries. Obviously, identifying the causes of human cancer is the
first step in cancer prevention. The rapid industrialization of low- and
medium-resource countries, often at the expense of some occupational
health safeguards common in the high-resource countries, clearly con-
tributes to cancer burden. Occupational health during metal production and
use would clearly be included.
National and international health agencies have established programs
with the aim of identifying agents and exposures that cause human cancer.
These include in the United States the Environmental Protection Agency
(EPA), Food and Drug Administration (FDA), the National Institute of
Occupational Safety and Health (NIOSH), and the National Toxicology
Program (NTP), and, as part of the World Health Organization, the IARC.
These authoritative bodies have, as part of their objective, the preparation
and the publication of critical reviews and evaluations concerning the
potential of human carcinogenicity from exposure to a wide range of che-
micals, mixtures and exposure circumstances. The metallic carcinogens are
among the agents that have been considered over the years.
These programs have developed descriptors of carcinogenic potential for
agents. The descriptors and their respective groupings developed by the
IARC [7] are: Carcinogenic to humans (Group 1); Probably carcinogenic to
humans (Group 2A); Possibly carcinogenic to humans (Group 2B); Not
classifiable as to its carcinogenicity to humans (Group 3); and Probably not
carcinogenic to humans (Group 4). Similarly, the EPA [16] has developed the
Met. Ions Life Sci. 2011, 8, 375–401

following descriptors: carcinogenic to humans; likely to be carcinogenic to

humans; suggestive evidence of carcinogenic potential; inadequate infor-
mation to assess carcinogenic potential; and not likely to be carcinogenic to
humans. The NTP Report on Carcinogens (ROC) [17] uses the following
descriptors for potential agents: known to be a human carcinogen; reason-
ably anticipated to be a human carcinogen. As a result of these critical
reviews and evaluations of evidence on human carcinogenicity, national
health agencies in countries throughout the world are able, on a sound
scientific basis, to take measures to reduce human exposure to workplace
and environmental carcinogens. The overall goal is to reduce human burden
of cancer, including cancers caused by exposure to metals.
2. KNOWN HUMAN METALLIC CARCINOGENS
2.1. Background: Human Carcinogen Assessment

A substance that carries a cancer hazard is a substance that is capable of
causing cancer under some circumstances, while a cancer risk is an estimate
of a carcinogenic effect expected form exposure to a cancer hazard [7]. The
IARC initiated a program to identify and evaluate the carcinogenic risk to
humans of environmental factors, including chemicals, complex mixtures,
occupational exposures, physical and biological agents, and lifestyle factors
and to produce the IARC Monographs on the Evaluation of Carcinogenic
Risks to Humans. The IARC Monographs have reviewed more than 900
agents and have identified more than 400 known, probable and possible
carcinogens. Several metals have been subjects of an IARC Monographs
evaluation, among them, arsenic, beryllium, cadmium, chromium, and
nickel. Although the IARC Monographs have emphasized hazard identifi-
cation, important issues may also involve dose-response assessment within
the range of the available epidemiological data, or it may allow comparison
of the dose-response information from experimental and epidemiological
studies [18]. Each IARC monograph represents the consensus of an inter-
national working group of expert scientists. Over time, the structure of a
Monograph has evolved such that it now includes sections covering: (1)
Exposure data, (2) studies of cancer in humans, (3) studies of cancer in
experimental animals, (4) mechanisms and other data relevant to an eva-
luation of carcinogenicity, (5) summary of data reported, and (6) evalua-
tions. The first four sections provide a critical review of the pertinent
scientific literature. Section 5 includes summaries of the scientific data pre-
sented in sections 1 to 4 and brings forth the studies considered valid to the
final evaluations developed by the working group.
Met. Ions Life Sci. 2011, 8, 375–401

Details for each volume, principles and procedures used to guide the
working group’s evaluations can be found in the preamble to the IARC
Monographs [7]. For each agent being evaluated, separate evaluations of the
evidence of cancer in humans and cancer in experimental animals are made,
with one of four descriptors (sufficient evidence, limited evidence, inade-
quate evidence, or evidence suggesting lack of carcinogenicity; for the defi-
nitions of these terms, see [7]). A preliminary default evaluation is made by
combination of the two partial evaluations of the evidence of cancer in
humans and experimental animal data. The agent will be evaluated as being
carcinogenic to humans (Group 1), probably carcinogenic to humans (Group
2A), possibly carcinogenic to humans (Group 2B), not classifiable as to its
carcinogenicity to humans (Group 3), or probably not carcinogenic to humans
(Group 4). In this regard, the human evidence generally drives an evaluation
of Group 1. To determine whether the default evaluation should be mod-
ified, mechanistic and other relevant data are considered. This determination
considers the strength of the mechanistic evidence and whether the
mechanism operates in humans. The final overall evaluation reflects the
weight of the evidence derived from studies in humans, studies in experi-
mental animals, and mechanistic and other relevant data and is a matter of
scientific judgment. By consideration of all relevant scientific data, the
working group may assign a higher or a lower Group than the default would
dictate.
Another agency, for example the EPA, makes its assessment on the health
hazards of chemical contaminants present in the environment. These cover
cancer and adverse effects other than cancer. The principles the EPA uses in
cancer assessment are discussed in the final guidelines for carcinogen risk
assessment [16]. The NTP publishes the Report on Carcinogens (ROC),
which identifies substances that may pose a carcinogenic hazard to human
health and to which a significant number of people residing in the United
States are exposed. One non-governmental and two Federal scientific
committees review the nominations for listing in or delisting from the ROC.
The director of NTP reviews the three groups’ recommendations and all
public reports before the Secretary of Health and Human Services reviews
and approves the ROC [17].
2.2. Recent IARC Evaluation of Metallic Carcinogens

The IARC very recently undertook the re-review of all the known human
metallic/inorganic carcinogens, including the various relevant forms of
arsenic, beryllium, cadmium, chromium(VI), and nickel [5]. Although this
review has not appeared as a full length monograph, the relevant conclu-
sions concerning grouping of agents as human carcinogens have been
Met. Ions Life Sci. 2011, 8, 375–401

summarized [5]. Thus, this chapter will follow the parameters of IARC when
it comes to defining status of metallic carcinogens [5,7], and draw on the
NTP ROC [17] for supportive material. In addition, this chapter will focus
on metallic agents known to be carcinogenic to humans (Group 1), with a
shorter discussion on metals that are probably carcinogenic to humans
(Group 2A) and a brief summary of agents considered by IARC to be
possibly carcinogenic to humans (Group 2B). The agents are listed by
hazard level and then by alphabetical order.
2.3. Arsenic and Inorganic Arsenic Compounds

Arsenic and inorganic arsenic compounds are considered known human
carcinogens that target multiple sites, including the lung, skin, and urinary
bladder [4,5]. Arsenic is unique among the human metallic carcinogens in
that it targets the human lung both after inhalation or after ingestion [4,5].
Lung cancers have been reported with occupational and non-occupational
(drinking water) exposures to arsenic and inorganic arsenic compounds [4,5].
Epidemiological studies show arsenic exposure through drinking water or by
inhalation can also cause skin and urinary bladder cancers [4,5]. Arsenic is
primarily excreted via the urine after ingestion [4]. There is also more limited
evidence that elevated arsenic in the drinking water is associated with can-
cers of the kidney, liver, and prostate, although various factors prevent a
firm conclusion on these organs as established human target sites [5]. Very
high levels of inorganic arsenic do occur in sub-surface water supplies
leading to high human exposures in some parts of the world, such as parts of
Bangladesh, India, Taiwan, Chile, and Argentina, a fact that was only
recently appreciated [4,5]. It is thought that at least tens of millions of people
are faced with issues arising from high arsenic in the drinking water
worldwide [4].
There is also emerging evidence that inorganic arsenic is a transplacental/
early life carcinogen in humans [19,20]. This comes from populations where
arsenic-contaminated drinking water was replaced with a low arsenic water
at key times to give appropriate comparative sub-populations for the study
of transplacental/early life exposure effects [19,20]. The transplacental/early
life target tissues included the lung, an accepted human target tissue for
arsenic carcinogenesis [5], and the liver, a site for which there is considered
limited evidence [5].
Inorganic arsenic, a metalloid that has several qualities like metals but can
also form bonds with carbon, is enzymatically methylated using S-adeno-
sylmethionine (SAM) as the methyl donor to a monomethylated form,
monomethylarsonic acid and a dimethylated form, DMA(V), in humans and
many animals [4]. Monomethylarsonic acid and DMA(V) are also active
Met. Ions Life Sci. 2011, 8, 375–401

ingredients of some herbicides [5]. On the basis of sufficient evidence of

cancer causation in animals by DMA(V) and inorganic arsenic in animals
(see below) and because monomethylarsonic acid is extensively metabolized
to DMA(V), monomethylarsonic acid was considered possibly carcinogenic
to humans (Group 2B).
The evidence for carcinogenicity in animals for arsenic and arsenic com-
pounds lagged behind human evidence for a very long time. In the last
decade or so, however, there has been a large number of studies that have
come forth that indicate various arsenicals, including inorganic arsenic
compounds and DMA(V) are rodent carcinogens [4,5,10,21].
For inorganic arsenicals, there is now a variety of positive rodent carci-
nogenesis studies with several compounds by various routes [4]. Sodium
arsenate produces lung tumors in mice [22]. Multiple intra-tracheal instil-
lations of calcium arsenate produce lung tumors in hamsters [4]. Perinatal
subcutaneous (sc) injections (maternal and offspring) of arsenic trioxide
produce lung tumors in mice [4]. Transplacental sodium arsenite exposure
via maternal drinking water from gestation day 8 to 18 in mice is carcino-
genic in the male and/or offspring when they became adults, producing
tumors of liver, adrenal and ovary in three separate studies, tumors of the
lung in two studies, and tumors of the uterus in one study [4,21]. These
transplacental studies involve positive tumor results in two different strains
(C3H and CD1) of mice [21]. The lung as a target site in these mouse
transplacental studies is concordant with human targets of arsenic carci-
nogenesis [5], and these mouse data are mutually supportive of the emerging
data that inorganic arsenic exposure is a transplacental/early life human
carcinogen [19,20].
Oral exposure to DMA(V) via the water or by addition to the food of rats
will produce cancer of the urinary bladder [4,8–10]. The urinary bladder is an
important target site in humans exposed to arsenic [5] and as a major
metabolite of inorganic arsenic DMA(V) is often found in the urine at levels
correlated with the original inorganic arsenic exposure [4]. Oral exposure to
DMA(V) in the drinking water produces lung tumors in mice [23,24]. Thus,
DMA(V) can cause lung and urinary bladder tumors, two known target sites
in humans exposed to arsenic [5]. Trimethylarsine oxide, another potential
methylated metabolite of arsenic, in the drinking water of rats produces liver
adenoma [25].
Inhalation of gallium arsenide is carcinogenic to female rats producing
tumors in the lungs and adrenal and preneoplastic lung lesions in male rat
and male and female mice [26]. Despite inadequate evidence for human
carcinogenicity and this rodent study [26], which is considered to constitute
limited evidence for carcinogenicity in experimental animals [27], gallium
arsenide is considered as carcinogenic to humans (Group 1) [27]. The
rationale for this appears mechanism-based, in that at least one possibility is
Met. Ions Life Sci. 2011, 8, 375–401

that inorganic arsenic, which is considered a human carcinogen, could be

released from inhaled gallium arsenide and account for the lung tumors in
rodents [27].
Despite being a simple molecule, arsenic is a very complex carcinogen.
Arsenicals, in all probability, have multiple mechanisms of carcinogenic
action that very likely at least depend on the compound (inorganic or
methylated), and on the site of action. It may be that more than one mode of
action is active in one target site at one time. IARC considers established
mechanistic events to include oxidative DNA damage, genomic instability,
gene amplification, epigenetic effects, and DNA repair leading to muta-
genesis [5]. This list is likely incomplete and additional research will establish
additional modes of action.
2.4. Beryllium and Beryllium Compounds

Beryllium and beryllium compounds are considered known human lung car-
cinogens (IARC Group 1) with studies involving complex exposure to the
metal and its compounds making it impossible to assess their carcinogenicity
separately [2,5]. Epidemiology data links occupational beryllium exposure and
lung cancer [2,5,17]. Occupational groups with longer beryllium exposures or
exposed to higher levels of beryllium show higher risks for development of
cancer, which mechanistically supports beryllium as the causative agent
[2,5,17]. Acute beryllium pneumonitis, considered a clinical marker for acute
high exposure to beryllium, is linked to higher rates of lung cancer [17], again
pointing towards beryllium as the causative agent. There is also considered to
be sufficient evidence of the carcinogenicity of beryllium and beryllium com-
pounds in experimental animals with the lung as a prominent target, con-
cordant with human data [2,5,17]. The animal data includes positive inhalation
or intra-tracheal instillation carcinogenicity studies in rats, mice and monkeys
exposed to beryllium or beryllium compounds [2]. Various compounds of
beryllium also have the capacity of causing osteosarcomas when given by
intravenous injection in rabbits [2], a peculiarity among metallic carcinogens.
The highest levels of beryllium exposure occur in occupational settings,
with the highest potential for exposure in industries like beryllium mining,
beryllium alloy manufacture and beryllium alloy fabrication, among others
[17]. The primary occupational route of exposure to beryllium is inhalation
of dusts or fumes, although some dermal contact does occur [17]. Environ-
mental levels of beryllium can be variable, but are generally not an issue, and
the largest source for the general population would be food or drinking
water [2,17].
The mechanism of action for beryllium as a carcinogen is poorly defined.
Increased beryllium has been found in the lungs of people exposed up to two
Met. Ions Life Sci. 2011, 8, 375–401

decades previously [1], indicating a long residence time at its target site.
Beryllium or beryllium compounds can cause chromosome aberrations,
aneuploidy and damage to DNA [5]. On a cellular level beryllium or ber-
yllium compounds have been shown to induce malignant transformation
and mutations in several test systems [1,2]. Infidelity of DNA synthesis after
binding of beryllium ions to nucleic acids has been reported [17]. Any of
these various modes of action could lead to cancer.
Berylliosis is a chronic lung disease involving immunological reactions to
the metal that varies greatly with the individual but any role for this in cancer
is not defined [2]. Similarly, a contact dermatitis occurs with beryllium [2].
2.5. Cadmium and Cadmium Compounds

Cadmium and cadmium compounds are considered known human carci-
nogens that target the lung [3,5]. There is also now considered some evidence
that cadmium and cadmium compounds can target the prostate and kidney
in exposed human populations [5]. There is also considered some suggestive
evidence for human bladder as a target for cadmium carcinogenesis [17].
Other targets for cadmium in humans such as liver, stomach, and pancreas
are considered equivocal [28]. Based on the fact that a wide variety of cad-
mium compounds show carcinogenic potential, it is suspected that the ionic
cadmium form is the active, carcinogenic species [17,28].
In rodents various cadmium compounds can cause lung cancer after
inhalation. In rats inhalation of cadmium induces lung adenocarcinoma, in
accord with its role in human lung cancer [3,5,28]. Cadmium can also induce
tumors or benign preneoplastic lesions of the rat prostate after injection or
ingestion [2,28], consistent with it possibly acting in the human prostate [5].
Systemic exposures to cadmium in experimental animals have also been
associated with induction of leukemia, lymphoma, and tumors of the
adrenal, lung and liver in rats or mice [3,17,28].
Cadmium salts, like many metals, when given by repository injections (i.e.,
subcutaneously or intramuscularly), will cause local sarcoma formation [3].
This includes even water soluble cadmium salts [3]. This is because the
soluble cadmium salts cause a local reaction (necrosis), and were given at
such levels that they likely precipitated local proteins, creating a local
reservoir of long-lived but eventually bioavailable cadmium. These cad-
mium-induced sarcomas become more aggressive (locally invasive, meta-
static, etc.) if cadmium is given repeatedly in the same location [28]
indicating a role for the metal in progression even for these already malig-
nant tumors.
Benign testicular interstitial (Leydig) cell tumors are induced by high
doses of cadmium by several different routes in rats and mice [2,28]. The
Met. Ions Life Sci. 2011, 8, 375–401

mechanism of formation for these tumors probably requires cadmium-

induced overt collapse of the testicular vasculature and may have little
bearing on human testicular cancer [28]. The reasons for the sensitivity of the
testicular vasculature to cadmium have never really been defined.
Recently, separate studies have suggested that cadmium exposure is
associated with an increased risk of breast cancer [29] or endometrial cancer
[30], although additional data are required to determine if cadmium is the
causal factor of these human cancers. Additionally, there is evidence that
cadmium may act as a metalloestrogen in some systems [31] which could
contribute to breast or uterine cancers. However, others find that human
breast cells can be transformed by cadmium without the involvement of
estrogen receptors (ER) [32] making actions as a metalloestrogen unneces-
sary in some cases.
Occupational exposure to cadmium and cadmium compounds comes from
activities such as lead and zinc smelting, melting or welding cadmium-coated
steel, using cadmium-containing solders, and use, processing or production of
cadmium powders for other applications [17]. The main route of occupational
exposure would be inhalation of cadmium-containing dusts or fumes [17]. In
the general population, cadmium exposure comes mostly from contaminated
food and drinking water consumption, and inhalation of cigarette smoke or
particles from air containing cadmium [17]. It is thought that smoking
cigarettes can double the life-time human body burden of cadmium.
2.6. Chromium(VI) Compounds

Chromium(VI) compounds were also recently reaffirmed by IARC as known
carcinogens (Group 1) [5]. Again complex exposure to the metal and its
compounds was assessed [2,5]. An excess risk of lung cancer has been con-
sistently observed among chromium(VI)-exposed workers, particularly in
chromate production, chromate pigment production, and chromium elec-
troplating [2,5].
It is well-established that in laboratory animals, chromium(VI) com-
pounds have a carcinogenic effect [2,5]. Early studies, as summarized by
IARC [2], showed that inhalation of calcium chromate in mice and sodium
dichromate in rats caused lung cancer. Intratracheal instillation of calcium
chromate, sodium dichromate, calcium chromate, zinc chromate or stron-
tium chromate also causes lung cancer in rats [2]. Several chromium com-
pounds by repository injection (calcium chromate, lead chromate, zinc
chromate, strontium chromate) caused local sarcomas in mice and rats [2].
In a recent well conducted animal bioassay, oral administration of sodium
dichromate to rats and mice was carcinogenic in the oral cavity or gastro-
intestinal tract [33].
Met. Ions Life Sci. 2011, 8, 375–401

Several mechanisms involved in chromium(VI)-induced carcinogenicity

have been identified, including the induction of DNA damage, the genera-
tion of oxidative stress, and aneuploidy leading to cell transformation [2,5].
2.7. Nickel Compounds

Nickel compounds are considered known human carcinogens (Group 1) that
target the lung, nasal cavity, and paranasal sinuses [5]. The lung and upper
respiratory cancers as human target sites are consistent with inhalation of
the nickel compounds [2,5]. In this most recent listing, as with several other
human metallic carcinogens, it was considered that human studies involved
complex exposure to the metal and its compounds thus making it impossible
to assess their carcinogenicity separately [5]. This listing is intended to
include both soluble and insoluble nickel compounds. None-the-less, IARC
retains the grouping of possibly carcinogenic to humans (Group 2B) for
metallic nickel established in 1990 [2].
There is a great number of carcinogenesis studies with nickel compounds
in animals [2] and what follows is only a survey and partial update. In
laboratory animals nickel and various nickel compounds are recognized as
having clear carcinogenic potential, where even early studies showed they
can produce lung tumors after inhalation, in accord with the respiratory
system as a tumor target site in humans [2]. It was also evident early on, and
in a large number of studies, that various nickel compounds will produce
local sarcomas or carcinomas at the site of repository injections in experi-
mental animals [2]. It is likely that mesenchymal stem cells are involved in
the generation of injection site sarcomas common with nickel or indeed
repository injections of various other carcinogenic metals.
In two recent and well-conducted animal bioassays, inhalation of nickel
subsulfide or nickel oxide led to dose-related induction of lung tumors in
rats, including lung carcinoma, and also induced tumors distant to the
portal of entry, namely adrenal tumors [17,34,35]. Nickel acetate, a soluble
nickel salt, can also be an effective transplacental carcinogen after fetal
exposure via the maternal animal in rats [36]. Along with inducing relatively
rare malignant pituitary tumors in the offspring as adults in rats after
transplacental exposure to nickel acetate, this treatment also acts as an
initiator for kidney tumors that can be promoted by sodium barbital given in
adulthood [36].
MT not only binds several primarily transition metals but is thought to
reduce oxidative stress [37,38] because of a large pool of internally oriented
cysteines which provide the metal binding sites and could act as a sink for
oxidants. Production of cellular oxidative stress is considered a possible
mechanism of nickel carcinogenesis [37,38]. However, MT-I/-II (the two
Met. Ions Life Sci. 2011, 8, 375–401

major forms of MT) double knockout mice are no more sensitive to nickel-
induced injection site sarcoma formation than wild-type mice that express
MT normally [38]. Conversely, engineered over-expression of MT, as in MT-
transgenic mice, had no impact on the carcinogenic effects of nickel when
compared to wild-type controls [37]. Thus, it appears that any role MT has
in either binding nickel directly or mitigating nickel-induced oxidative stress
does not alter its carcinogenic potential.
Nickel is widely used in industry because it makes a strong, heat resistant,
hard and corrosion resistant alloy with various other metals, such as
stainless steel and copper-nickel alloys [17]. Occupational nickel exposure is
mainly by inhalation of dust or fumes and to a certain extent by dermal
contact [17]. Nickel exposure in the work place can occur in mining, welding,
smelting, electroplating, etc. [17]. Nickel in the environment is present at low
levels in air, food, and water [17] and is not a major source of exposure
compared to occupational exposure.
3. PROBABLE AND POSSIBLE METALLIC

CARCINOGENS
3.1. Inorganic and Organic Lead Compounds
Inorganic lead compounds were recently elevated to probably carcinogenic to
humans (group 2A) by IARC [7] based on limited evidence in humans and
sufficient evidence in experimental animals. Group 2A listed agents and
below do not specify human target sites. Organic lead compounds were
considered as not classifiable as to their carcinogenicity to humans (Group 3).
In this evaluation it was considered that organic lead compounds, to the
extent that they would be metabolized to ionic lead, would take on the toxic
potential of inorganic lead [7], and thereby presumably the carcinogenic
potential. Metallic lead was not considered in this evaluation [7]. The ROC
similarly considers lead and lead compounds reasonably anticipated to be a
human carcinogen [17].
In experimental animals there is clear evidence (sufficient) of the carci-
nogenicity of inorganic soluble (lead acetate and lead subacetate) and
insoluble (lead phosphate and lead chromate) lead compounds with carci-
nogenic potential in animals [7]. That soluble lead compounds are active
points towards the ionic form of the metal as the probable active species in
carcinogenesis. The animal target sites of inorganic lead were primarily
kidney (adenomas and carcinomas), brain (gliomas), and lung [7]. Inorganic
lead compounds are able to induce tumors after oral exposure or injection
[7]. In one study lead acetate produced renal tumors in the offspring after a
Met. Ions Life Sci. 2011, 8, 375–401

combination of maternal treatment resulting in transplacental/translacta-

tional exposure in mice [39]. The tumors occurred in the absence of chronic
nephropathy, an observation which would make a chronic compensatory
hyperplasia the basis for renal cancer development limited (see Section 4.5).
3.2. Cisplatin
Cisplatin (cis-diamminedichloroplatinum(II)) is a metal complex that is
widely used and highly effective in cancer chemotherapy. None-the-less, this
metallochemotherapeutic is considered probably carcinogenic to humans
(Group 2A) essentially on the basis of sufficient evidence in research animals
that show it has multiple tumor target sites in repeated studies in rats or mice
[1,17,40]. Although case reports in humans exist concerning tumor forma-
tion associated with the use of cisplatin, the metallochemotherapeutic is
almost always used in combination with other drugs in such clinical settings
[1]. The concurrent use of other, often putative carcinogens (irradiation,
alkylating agents, etc.), makes the contribution of a single agent in this sort
of setting impossible to dissect [1].
However, in experimental animals, cisplatin is clearly a complete carci-
nogen, inducing malignant tumors of the hematopoietic system and benign
or malignant liver tumors in rats or mice [1,40]. In strain A mice, a strain
genetically susceptible to lung cancers, even from systemic (non-inhalation)
exposures, cisplatin increases lung tumor incidence and multiplicity [40].
Cisplatin, like nickel, arsenic, and lead, is also a transplacental carcinogen in
rodents [41,42]. Maternal treatment of pregnant mice or rats with cisplatin
causes a complete carcinogenic response in various organs in the offspring as
adults, producing lymphomas and lung and livers tumors [41,42]. Cisplatin
also acts as an effective transplacental initiator of tumors of the skin or
kidney that can be promoted by sodium barbital [42] or 12-O-tetra-
decanoylphorbol-13-acetate [41].
It has long been suspected that MT reduces cisplatin toxicity, but only
until recently was it shown that poor production of this metal binding
protein could render animals susceptible to cisplatin carcinogenesis [40].
Mice with the two major isoforms of MT (MT-I/-II) knocked-out are
deficient in MT production [40], particularly in the liver. When treated with
clinically relevant doses of cisplatin, these MT knockout mice show a
marked, dose-related increase in hepatocellular carcinoma formation com-
pared to similarly treated wild-type mice which produce MT normally [40].
Humans show remarkable variation in MT levels, for unknown reasons, and
there appears to be sub-populations with very low MT expression levels [40].
Such sub-populations, though successfully treated for cancers, may be
hypersensitive to secondary tumor formation years after cisplatin therapy.
Met. Ions Life Sci. 2011, 8, 375–401

3.3. Indium Phosphide

Indium is a post transition metal similar to zinc and, as indium phosphide, is
used as a semiconductor in high frequency electronics because of superior
electron velocity [27]. There is inadequate evidence in humans for carcino-
genic effects of indium phosphide so the strength of this listing lies with a
single comprehensive inhalation study performed by the NTP in male and
female rats and mice that shows, among other things, extraordinarily high
incidences of malignant tumors of the lung [27]. Inhalation of indium
phosphide would be the presumed route of exposure in occupational settings.
The rodent study also showed indium phosphide inhalation increased
adrenal tumors in male and female rats and increased liver tumors in male
and female mice [27]. These tumors occurred even though the animals were
exposed to very low levels of indium phosphide (as low as 0.03 mg/m3) and
even though they were exposed for only 22 weeks and followed for 2 years
[27]. This is a case in point where one well-designed, very high impact study
had a remarkable impact on categorization.
3.4. Possible Human Inorganic Carcinogens

There are several inorganic compounds or collections of compounds that
meet the IARC criteria to qualify as possible human carcinogens. This
usually is on the basis of sufficient work in laboratory animals indicating
carcinogenic potential with inadequate or no human data depending on the
strength of the animal work. Such possible human inorganic carcinogens
include antimony trioxide [43], cobalt and cobalt compounds [44], cobalt
sulfate and other soluble cobalt(II) salts [27], foreign bodies that would
include metallic objects [13], iron dextran complex [1], methylmercury
compounds [3], vanadium pentoxide [27], as well as others.
Cobalt deserves special mention because of the complex nature of the listings.
Again, cobalt and cobalt compounds [44], and cobalt sulfate and other soluble
cobalt(II) salts are both classified as possibly carcinogenic to humans (Group 2B)
[27]. However, it is considered that, as an exposure circumstance, exposure to
cobalt metal with tungsten carbide is probably carcinogenic to humans (Group
2A) [27]. On the other hand, exposure to cobalt metal alone without tungsten
carbide is classified as possibly carcinogenic to humans (Group 2B) [27].
As to other exposures where metal production is involved, both aluminum
production and iron and steel founding are considered exposure circum-
stances leading to human carcinogenesis (Group 1), although the role of
metal exposure is not of definitive causality [1]. Some exposures to mixtures
of metals, as with exposure to welding fumes, are considered possibly car-
cinogenic to humans (Group 2B) [2], but again the contribution to cancer of
non-metallic versus metallic factors in this exposure is not well-defined.
Met. Ions Life Sci. 2011, 8, 375–401

4. POTENTIAL MECHANISMS OF METALLIC

CARCINOGENS
4.1. Possibilities at the Chemical Level

The metallic carcinogens are an interesting study in inorganic biochemistry
when it comes to potential mechanisms on the chemical level. Outside of
perhaps chromium(VI) which directly binds to DNA, chemical mechanisms
for the other inorganic carcinogens must be considered mostly hypothetical.
Adventitious binding, as production of strong adducts to DNA leading to
false reading and eventual mutation, would clearly work with some metal
ions (like cisplatin). However, other metallic carcinogens would not strongly
bind DNA, or at least to the extent to cause mutations. The latter would
include cadmium, nickel, and inorganic arsenic.
Mimicry of ‘‘normal’’ physiological compounds for metallic carcinogens
would include essential trace elements. For example, it is clear that cadmium
can mimic zinc at binding sites in proteins, such as MT [11], or competitively
impact metal response elements [45]. Such mimicry could clearly lead to
protein dysfunction or aberrant gene expression, etc. If protracted or
sequentially ‘‘inauspicious’’ this may lead to cellular transformation.
Generation of cellular oxidants could happen directly or indirectly with
metal carcinogens and is likely a major mechanism with several metallic
carcinogens at the chemical level. Direct redox reaction may occur with
some during their metabolism, such as with arsenic. Depletion of cellular
glutathione (for metabolism or efflux) or displacement of redox active
essentials like iron or copper (perhaps involving binding mimicry) are two
examples [46,47]. Oxidative stress as a mechanism will be covered below.
Disruption of the ‘‘normal’’ metabolism of the cells would include altered
metabolism including enzymatic (e.g., arsenic biomethylation) or cellular
uptake or efflux induced by carcinogenic metals. These could lead to major
changes in enzymatic or transport cofactors which would be channeled into
use for the toxicant rather than normal compounds [47]. This can have
serious manifestations if chronic, including epigenetic cellular effects related
to cancer such as DNA hypomethylation [47].
4.2. DNA Damage and Oxidative Stress

Given that carcinogenesis is a highly complex process involving multiple
stages, it is likely that human carcinogens, including the metals discussed in
this chapter, act through multiple mechanisms and at multiple stages. It is
generally believed that the main mechanisms of action of many metals
involve some sort of formation of reactive oxygen species (ROS), a process
Met. Ions Life Sci. 2011, 8, 375–401

that can lead to oxidative stress (damage) including, probably most

importantly for cancer, DNA nucleotide base modifications, single and
double strand breaks, and DNA-protein cross-linking [46]. For instance,
exposure to arsenic compounds can stimulate ROS formation both in vitro
and in vivo and biomethylation of arsenic varies widely depending on cell
type, even with target cells of carcinogenesis [48]. Indeed, assessment of
oxidative DNA damage (ODD) during chronic inorganic arsenite
exposure of arsenic biomethylation-competent and biomethylation-deficient
human and rodent cells reveals some very important aspects about ODD
and acquired malignant phenotype with inorganic arsenic [48]. The bio-
methylation-competent cells were more rapidly transformed into a malig-
nant phenotype by several metrics (hyper-invasive, increased colony
formation, etc.) and showed a dramatic, time-dependent induction of ODD
during transformation, while the biomethylation-deficient cells, although
eventually transformed, showed essentially no arsenic-induced ODD
formation [48]. Thus, biomethylation product of arsenic is necessary for
arsenic-induced ODD and it appears this product accelerates the transfor-
mation process but is not an absolute requirement [48]. This study clearly
indicates that arsenic has multiple carcinogenic mechanisms and
suggests that the speed at which arsenic carcinogenesis occurs depends on
the number of these mechanisms that may be at play in a given cell at a given
time [48]. Since cells that do not biomethylate did not show DNA damage,
this might point towards other potential mechanisms, possibly including
epigenetic.
The reduction of chromium(VI) and subsequent production of free radi-
cals results in the generation of oxidative stress. Such free radicals, including
ROS, can lead to various types of DNA damage including single and double
strand breaks, and DNA-DNA and DNA-protein cross-links [49], all of
which can directly cause acquisition of cancer phenotype. Chromium can
also interact directly with DNA to form DNA-protein cross-links and
chromium-DNA adducts, the latter being the most abundant form of genetic
lesions induced by chromium [49]. These adducts are responsible for all
mutations generated during the reduction of chromium(VI) and can also
give rise to effects such as aneuploidy and altered gene expression [49].
Nickel and beryllium compounds are thought to produce DNA damage [5]
and probably act, at least in part, by producing oxidative stress.
Cadmium, which is not a redox active metal, can also generate ROS and
subsequent oxidative stress, which may play an important role in cadmium
carcinogenesis. Cadmium-induced oxidative stress leads to DNA strand
breaks, formation of ODD, chromosomal aberrations, and gene mutations
and plays a major role in inhibition of DNA damage repair, induction of
apoptosis, and aberrant gene expression [50]. However, ROS-induced oxi-
dative stress does not play an important role in cadmium-induced malignant
Met. Ions Life Sci. 2011, 8, 375–401

transformation in some cells [51], making the actual significance of oxidative

stress in cadmium carcinogenesis somewhat controversial.
4.3. Epigenetic Mechanisms

Epigenetics is typically defined as the study of heritable changes in gene
function that occur without any direct changes in the DNA sequence.
Alterations in DNA methylation patterns are epigenetic phenomena that
play a significant role in the carcinogenic process via transcriptional inac-
tivation or activation of various cancer-related genes. Found in virtually all
human cancers, changes in DNA methylation are among the most common
epigenetic changes induced by metal carcinogens.
Arsenic exposure can induce DNA hypomethylation (activation) and
hypermethylation (inactivation), both likely playing an important role in
arsenic carcinogenesis. Arsenic undergoes mono- and di-methylation, con-
suming cellular methyl groups in the process. Biomethylation of arsenic
requires SAM. Human prostate epithelial cells transformed by and adapted
to arsenic show signs of methyl depletion (DNA hypomethylation) even
though these cells only very poorly methylate arsenic. During early adap-
tation, homocysteine increases, and SAM decreases along with inter-con-
verting enzymes, indicating reduced conversion of homocysteine to SAM
[47]. SAM loss is directly related to DNA hypomethylation. Additionally,
the transulfuration pathway is activated to increase glutathione production
for arsenic efflux. Thus, arsenic adaptation preserves cells but pre-disposes
to a potential epigenetic mode of carcinogenesis.
Epigenetic changes have also been shown to play an important role in
cadmium and nickel carcinogenesis [28,49]. Indeed, it is believed that the pri-
mary events in nickel carcinogenesis are epigenetic changes, including changes
in DNA methylation, histone modifications, and alterations in gene expression,
including that of various transcription factors and tumor suppressors [49].
4.4. Aberrant Gene Expression

Aberrant gene expression is one of a number of possible interrelated mole-
cular mechanisms with regard to metal-induced carcinogenesis. Recent
developments in assessment of gene expression, such as microarray, have
facilitated the identification of a large number of genes and, consequently,
many potential alterations in gene expression in metals carcinogenesis.
Various genes influenced by metal exposure are possibly involved in carcino-
genesis. Major groups include the proto-oncogenes such as k-ras, c-myc,
c-fos, c-jun, which undergo early transcriptional activation in response to
Met. Ions Life Sci. 2011, 8, 375–401

mitogenic stimuli and are frequently over-expressed in response to metal

exposure. The metal-induced over-expression of one or more of these proto-
oncogenes is common in a wide variety of human and rodent cell lines [52–55].
This over-expression in vitro is analogous to proto-oncogene over-expression
in tumors or cells undergoing oncogenic proliferation and aberrant differ-
entiation. Several genes collectively referred to as ‘‘stress genes’’ are also often
induced by the exposure to carcinogenic metals. Among these genes are those
involved in oxidative stress response, the synthesis of MT [55,56], the
encoding of heat shock proteins [57], and those responsible for glutathione
synthesis and homeostasis [58]. Some genes induced by metal carcinogen
exposure encode transcription factors which can result in transcriptional
deregulation of their target genes. These transcription factors include the
metal regulatory transcription factor 1 (MTF1), activator protein-1 tran-
scription element that is present in the promoter regions of several genes
involved in cell growth and division, and nuclear factor kB [59].
Multiple mechanisms are likely for metal-induced aberrant gene expres-
sions. For example, the induction of genes such as MT by cadmium involves
the binding of the transcription factor MTF1 to the metal response element
sequence present in the promoter regions of these genes [60]. Arsenic-induced
malignant transformation and acquired androgen independence causes Ras
signaling activation in human prostate epithelial cells with increased expres-
sion of unmutated K-Ras, and consequently the downstream MAP kinases A-
Raf and B-Raf and phosphorylated MEK1/2 and Elk1 [61]. Both arsenite and
chromium(VI) have profound but preferential effects on expression of several
inducible genes, including the hormone-regulated phosphoenolpyruvate car-
boxykinase (PEPCK) gene, whose expression is associated with the gluco-
corticoid receptor (GR)-mediated regulatory pathway [62,63]. Arsenic
significantly suppressed both basal and inducible expression of PEPCK
through specific suppression of GR as a transcription factors. In contrast,
chromium(VI) enhanced PEPCK expression via cAMP signaling pathway.
Indirect mechanisms involving secondary messengers such as ROS are
possible. Exposure to arsenic, cadmium, and nickel causes ROS, and
depletes antioxidants [64]. ROS generation can accelerate cell proliferation
[65]. Some carcinogenic metals, like cadmium or arsenic, may constitute a
new class of endocrine disrupters, which activate ERa by a mechanism
involved in the hormone-binding domain of the receptor [66]. Activated ERa
triggers the downstream gene expression and mitogenic signaling [67].
4.5. Compensatory Hyperplasia

Inorganic arsenic exposure is clearly associated with human cancer of the
urinary bladder [5]. IARC considers the inorganic arsenic biomethylation
Met. Ions Life Sci. 2011, 8, 375–401

product that is found in the rodent and human urine, DMA(V), possibly
carcinogenic to humans (Group 2B) based on sufficient evidence of cancer in
animals, specifically urinary bladder cancer in rats [5]. A proposed
mechanism by which protracted oral DMA(V) exposure induces urinary
bladder cancer in the rats is when it reaches the urine it induces urothelial
cytotoxicity, causing subsequent persistent regenerative proliferation, which
ultimately leads to compensatory hyperplasia and eventually cancer [68]. It is
proposed that DMA(V) first requires conversion to a more reactive trivalent
metabolite to then cause oxidative stress in the urothelium [68]. This
mechanism is an example of chronic inorganic exposure leading to com-
pensatory hyperplasia and regenerative proliferation thereby leading to
cancer formation. There is no reason that this rodent mechanism [68] would
not apply to humans.
Compensatory hyperplasia was also thought to be the major route to renal
carcinogenesis after chronic inorganic lead exposure in rodents [39]. Chronic
renal tubular damage would lead to proliferative repair and, in concert with
eventual errors, tumors would be brought out by the continuous need for
damage repair and stimulus of cellular proliferation [39]. However, with the
transplacental/translactational carcinogenesis model in mice, animals are
exposed early on and then develop renal tumors late in life long after lead
exposure has ended [39]. Importantly, the renal tumors develop in the
absence of any chronic nephropathy that would be typical of a tissue
requiring compensatory hyperplasia due to chronic insult [39]. So this
mechanism seems not to apply, or at least not fully, to lead-induced renal
carcinogenesis in rodents.
5. PERIODS OF PARTICULAR SENSITIVITY TO

INORGANIC CARCINOGENS
The perinatal life stage is clearly a time of high sensitivity to chemical carci-
nogenesis because of issues like organogenesis, global proliferative growth, etc.
[69], and it is considered one of the most critical life stages for assessing
accumulated cancer risk in humans [70]. However, the consequences of car-
cinogen exposure during this key period of development are still largely
ignored, as acknowledged many years ago [70]. It is well-established that
perinatal exposures have led to cancer in humans, as with in utero exposure to
diethylstilbestrol [69]. In this regard, two of the known human inorganic
carcinogens [5] have shown carcinogenic activity in mice during adulthood
after transplacental exposure [21,36]. This includes multiple positive carcino-
genesis studies with transplacental inorganic sodium arsenite exposure in mice
(for review see [21]), and a positive transplacental study with nickel in rats [36].
Met. Ions Life Sci. 2011, 8, 375–401

Two probable human carcinogens (IARC Group 2A) have also been
shown to have transplacental/early life activity. This includes tumor for-
mation after combined transplacental/translactational lead exposure
in mice [39], and complete carcinogenesis after maternal cisplatin exposure
in mice [41] or rats [42]. Furthermore, there is now accumulating evidence
that transplacental/early life arsenic exposure is carcinogenic in humans
[19,20].
Humans who are exposed to environmental inorganic carcinogens are
often exposed for their entire life, including during early development. It is
clear that, as with other classes of carcinogens, the perinatal stage may also
be particularly sensitive to metallic carcinogens. Perhaps all metallic carci-
nogens of significant environmental concern should be tested for carcino-
genic potential after transplacental/early life exposure.
6. FUTURE ISSUES IN METAL CARCINOGENESIS
Metallic agents will always be part of the human environment. Public

and occupational health measures have mitigated the very high level
exposures that previously led to frequent clusters of human cancers after
exposures to inorganic carcinogens, at least in developed countries. How-
ever, these agents still remain a threat to human health, as attested by
inorganic arsenic in the drinking water from natural sources, which is
thought to be at clearly unhealthy levels for millions of people world
wide [4]. Such issues as the balance between the need for sufficient drinking
water and natural contamination with known metallic carcinogens need to
be reconciled. Occupational health issues may well have reduced the risk
from metal carcinogenesis in the wealthier countries, but in many less
affluent countries increased or low cost production is bought at a price of
diminished worker safety, including occupational exposures to metallic
carcinogens. These issues will need to be dealt with as we move into the
future.
Although there has been significant progress in our understanding of the
molecular mechanisms and modes of metal-induced carcinogenesis,
many issues remain to be defined. Genetic differences between individuals
likely affect their relative sensitivity to metal exposure. Complex inter-
play between multiple genetic and environmental factors on affected
genes likely will be important in determining final individual sensitivity.
If we can identify and characterize such predisposing factors to metal-
induced cancers we may actually be able to predict the most sensitive
subpopulations and, thereby, protect against and even prevent metal
carcinogenesis.
Met. Ions Life Sci. 2011, 8, 375–401

ACKNOWLEDGMENTS
This work was supported by the Intramural Research Program of the NIH,
National Cancer Institute, Center for Cancer Research and by the Inter-
national Agency for Research on Cancer. Authors declare no conflicts of
interest.

cAMP adenosine 3 0 ,5 0 -cyclic monophosphate
DMA(V) dimethylarsinic acid
ER estrogen receptor
GR glucocorticoid receptor
IARC International Agency for Research on Cancer
MT metallothionein
MTF1 metal regulatory transcription factor 1
NIOSH National Institute of Occupational Safety and Health
NTP National Toxicology Program
ODD oxidative DNA damage
PEPCK phosphoenolpyruvate carboxykinase
ROC Report on Carcinogens
SAM S-adenosylmethionine
sc subcutaneous
U.S. EPA United States Environmental Protection Agency
U.S. FDA United States Food and Drug Administration
REFERENCES
1. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Suppl. 7,
Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs
Volumes 1–42, IARC Press, Lyon, France, 1987, pp. 1–440.
2. Chromium, nickel and welding, Vol. 49 of IARC Monographs on the Evaluation of
Carcinogenic Risks to Humans, IARC Press, Lyon, France, 1990, pp. 1–677.
3. Beryllium,Cadmium, Mercury, and Exposures in the Glass Manufacturing Indus-
try, Vol. 58 of IARC Monographs on the Evaluation of Carcinogenic Risks to
Humans, IARC Press, Lyon, France, 1993, pp. 1–444.
4. Some Drinking-water Disinfectants and Contaminants, Including Arsenic, Arsenic
in Drinking-water, Vol. 84 of IARC Monographs on the Evaluation of Carcino-
genic Risks to Humans, IARC Press, Lyon, France, 2004, pp. 1–267.
Met. Ions Life Sci. 2011, 8, 375–401

5. K. Straif, L. Benbrahim-Tallaa, R. Baan, B. Secretan, F. El Ghissassi, V. Bou-

vard, N. Guha, C. Freeman, L. Galichet, V. Cogliano, U. Heinrich, J. Samet, P.
A. Demers, M. Gerin, J. Siemiatycki, P. Grandjean, A. Aitio, T. Kauppinen, M.
Goldberg, A. Hartwig, H. Muhle, B. Fubini, F. Merletti, M. Ikeda, M. D. Att-
field, K. P. Cantor, B. A. Fowler, R. Henderson, P. F. Infante, A. B. Kane, P. J.
Landrigan, R. Lunn, T. Rossman, L. Stayner, M. P. Waalkes, E. M. Ward and J.
M. Ward, Lancet Oncol., 2009, 10, 453–455.
6. J. Hutchinson, Trans. Path. Soc. London, 1888, 39, 352–363.
7. Inorganic Lead and Organic Lead Compounds, Vol. 87 of IARC Monographs on
the Evaluation of Carcinogenic Risks to Humans, IARC Press, Lyon, France,
2006, pp. 1–506.
8. M. Wei, H. Wanibuchi, S. Yamamoto, W. Li and S. Fukushima, Carcinogenesis,
1999, 20, 1873–1876.
9. M. Wei, H. Wanibuchi, K. Morimura, S. Iwai, K. Yoshida, G. Endo, D. Nakae
and S. Fukushima, Carcinogenesis, 2002, 23, 1387–1397.
10. L. L. Arnold, M. Eldan, A. Nyska, M. van Gemert and S. M. Cohen, Toxicology,
2006, 223, 82–100.
11. G. F. Nordberg, Toxicol. Appl. Pharmacol., 2009, 238, 192–200.
12. B. S. Oppenheimer, E. T. Oppenheimer, I. Danishefsky and A. P. Stout, Cancer
Res., 1956, 16, 439–441.
13. Surgical Implants and Other Foreign Bodies, Vol. 74 of IARC Monographs on the
Evaluation of Carcinogenic Risks to Humans, IARC Press, Lyon, France, 1999,
pp. 1–311.
14. M. P. Waalkes, S. Rehm, K. S. Kasprzak and H. J. Issaq, Cancer Res., 1987, 47,
2445–2450.
15. World Cancer Report, Ed. B. W. Stewart and P. Kleihues, IARC Press, Lyon,
France, 2003, pp. 1–351.
16. U.S. EPA, Guidelines for Carcinogen Risk Assessment, U.S. Environmental
Protection Agency, Washington, DC, EPA/630/P-03/001F, 2005, pp. 1–166.
17. Report on Carcinogens, Eleventh Edition, U.S. Department of Health and Human
Services, Public Health Service, NTP, Research Triangle Park, NC, 2004, pp. 1–276.
18. V. J. Cogliano, R. A. Baan, K. Straif, Y. Grosse, M. B. Secretan, F. El Ghissassi
and P. Kleihues, Environ. Health Perspect., 2004, 112, 1269–1274.
19. A. H. Smith, G. Marshall, Y. Yuan, C. Ferreccio, J. Liaw, O. von Ehrenstein, C.
Steinmaus, M. N. Bates and S. Selvin, Environ. Health Perspect., 2006, 114, 1293–
1296.
20. J. Liaw, G. Marshall, Y. Yuan, C. Ferreccio, C. Steinmaus and A. H. Smith,
Cancer Epidemiol. Biomarkers Prev., 2008, 17, 1982–1987.
21. M. P. Waalkes, J. Liu and B. A. Diwan, Toxicol. Appl. Pharmacol., 2007, 222,
271–280.
22. X. Cui, T. Wakai, Y. Shirai, K. Hatakeyama and S. Hirano, Toxicol. Sci., 2006,
91, 372–381.
23. H. Hayashi, M. Kanisawa, K. Yamanaka, T. Ito, N. Udaka, H. Ohji, K. Okudela,
S. Okada and H. Kitamura, Cancer Lett., 1998, 125, 83–88.
24. A. Kinoshita, H. Wanibuchi, K. Morimura, M. Wei, D. Nakae, T. Arai, O. Minowa,
T. Noda, S. Nishimura and S. Fukushima, Cancer Sci., 2007, 98, 803–814.
Met. Ions Life Sci. 2011, 8, 375–401

25. J. Shen, H. Wanibuchi, E. I. Salim, M. Wei, A. Kinoshita, K. Yoshida, G. Endo

and S. Fukushima, Carcinogenesis, 2003, 24, 1827–1835.
26. NTP Technical Report 492, Toxicology and Carcinogenesis Studies of Gallium
Arsenide (CAS No. 1303-00-0) in F344/N Rats and B6C3F1Mice (Inhalation
Studies), Research Triangle Park, NC, 2000, pp. 1–303.
27. Cobalt in Hard Metals and Cobalt Sulfate, Gallium Arsenide, Indium Phosphide
and Vanadium Pentoxide, Vol. 86 of IARC Monographs on the Evaluation of
Carcinogenic Risks to Humans, IARC Press, Lyon, France, pp. 1–330.
28. M. P. Waalkes, Mutat. Res., 2003, 533, 107–120.
29. J. A. McElroy, M. M. Shafer, A. Trentham-Dietz, J. M. Hampton and P. A.
Newcomb, J. Natl. Cancer Inst., 2006, 98, 869–873.
30. A. Akesson, B. Julin and A. Wolk, Cancer Res., 2008, 68, 6435–6441.
31. C. Byrne, S. D. Divekar, G. B. Storchan, D. A. Parodi and M. B. Martin,
32. L. Benbrahim-Tallaa, E. J. Tokar, B. A. Diwan, A. L. Dill, J. F. Coppin and M.
P. Waalkes, Environ. Health Perspect., 2009, 117, 1847–1852.
33. NTP Technical Report 546, Toxicology and Carcinogenesis Studies of
Sodium Dichromate Dihydrate (CAS No. 7789-12-0) in F344/N Rats and
B6C3F1 Mice (Drinking Water Studies), Research Triangle Park, NC, 2008,
1–192.
34. NTP Technical Report 451, Toxicology and Carcinogenesis Studies of Nickel
Oxide (CAS No. 1313-99-1) in F344 Rats and B6C3F1 Mice (Inhalation Studies),
Research Triangle Park, NC, 1996, 1–381.
35. NTP Technical Report 453, Toxicology and Carcinogenesis Studies of Nickel
Subsulfide (CAS No. 12035-72-2) in F344/N Rats and B6C3Fl Mice (Inhalation
Studies), Research Triangle Park, NC, 1996, 1–365.
36. B. A. Diwan, K. S. Kasprzak and J. M. Rice, Carcinogenesis, 1992, 13, 1351–
1357.
37. M. P. Waalkes, J. Liu, K. S. Kasprzak and B. A. Diwan, Toxicol. Lett., 2004,
153, 357–364.
38. M. P. Waalkes, J. Liu, K. S. Kasprzak and B. A. Diwan, Int. J. Cancer, 2005, 24,
215–220.
39. M. P. Waalkes, B. A. Diwan, J. M. Ward, D. E. Devor and R. A. Goyer, Cancer
Res., 1995, 55, 5265–5271.
40. M. P. Waalkes, J. Liu, K. S. Kasprzak and B. A. Diwan, Int. J. Cancer, 2006,
119, 28–32.
41. B. A. Diwan, L. M. Anderson, S. Rehm and J. M. Rice, Cancer Res., 1993, 53,
3874–3876.
42. B. A. Diwan, L. M. Anderson, J. M. Ward, J. R. Henneman and J. M. Rice,
43. IARC, in Some Organic Solvents, Resin Monomers and Related Compounds,
Pigments and Occupational Exposures in Paint Manufacture and Painting, Vol. 47
of IARC Monographs on the Evaluation of Carcinogenic Risks in Humans, IARC
Press, Lyon, France, 1989, pp. 291–306.
44. Chlorinated Drinking-water; Chlorination By-products; Some Other Halogenated
Compounds; Cobalt and Cobalt Compounds, Vol. 52 of IARC Monographs on the
Met. Ions Life Sci. 2011, 8, 375–401

Evaluation of Carcinogenic Risks to Humans, IARC Press, Lyon, France, 1991,

pp. 1–543.
45. D. P. Giedroc, X. Chen and J. L. Apuy, Antioxid. Redox. Signal., 2001, 3, 577–
596.
Interact., 2006, 160, 1–40.
47. J. F. Coppin, W. Qu and M. P. Waalkes, J. Biol. Chem., 2008, 283, 19342–19350.
48. C. Kojima, D. C. Ramirez, E. J. Tokar, S. Himeno, Z. Drobna, M. Styblo, R. P.
Mason and M. P. Waalkes, J. Natl. Cancer Inst., 2009, 101, 1670–1681.
49. K. Salnikow and A. Zhitkovich, Chem. Res. Toxicol., 2008, 21, 28–44.
50. P. Joseph, Toxicol. Appl. Pharmacol., 2009, 238, 272–279.
51. W. Qu, B. A. Diwan, J. M. Reece, C. D. Bortner, J. Pi, J. Liu and M. P. Waalkes,
Int. J. Cancer, 2005, 114, 346–355.
52. M. K. Abshire, D. E. Devor, B. A. Diwan, J. D. Shaughnessy, Jr. and M. P.
Waalkes, Carcinogenesis, 1996, 17, 1349–1356.
53. W. E. Achanzar, K. B. Achanzar, J. G. Lewis, M. M. Webber and M. P.
54. L. Benbrahim-Tallaa, R. A. Waterland, M. Styblo, W. E. Achanzar, M. M.
Webber and M. P. Waalkes, Toxicol. Appl. Pharmacol., 2005, 206, 288–298.
55. J. Liu, L. Benbrahim-Tallaa, X. Qian, L. Yu, Y. Xie, J. Boos, W. Qu and M. P.
56. R. R. Misra, K. A. Crance, R. M. Bare and M. P. Waalkes, Toxicology, 1997,
117, 99–109.
57. M. J. Lee, H. Nishio, H. Ayaki, M. Yamamoto and K. Sumino, Toxicology,
2002, 174, 109–117.
58. J. D. Eneman, R. J. Potts, M. Osier, G. S. Shukla, C. H. Lee, J. F. Chiu and B. A.
Hart, Toxicology, 2000, 147, 215–228.
59. Z. Yang, S. Yang, S. Y. Qian, J. S. Hong, M. B. Kadiiska, R. W. Tennant, M. P.
Waalkes and J. Liu, Toxicol. Sci., 2007, 98, 488–494.
60. R. M. Muller, H. Taguchi and S. Shibahara, J. Biol. Chem., 1987, 262, 6795–
6802.
61. L. Benbrahim-Tallaa, M. M. Webber and M. P. Waalkes, Environ. Health Per-
spect., 2007, 115, 243–247.
62. J. McCaffrey, C. M. Wolf and J. W. Hamilton, Mol. Carcinog., 1994, 10, 189–
198.
63. J. W. Hamilton, R. C. Kaltreider, O. V. Bajenova, M. A. Ihnat, J. McCaffrey, B.
W. Turpie, E. E. Rowell, J. Oh, M. J. Nemeth, C. A. Pesce and J. P. Lariviere,
64. N. Ercal, H. Gurer-Orhan and N. Aykin-Burns, Curr. Top. Med. Chem., 2001, 1,
529–539.
65. K. Kawata, R. Shimazaki and S. Okabe, Environ. Mol. Mutagen., 2009, 50, 46–
59.
66. M. B. Martin, R. Reiter, T. Pham, Y. R. Avellanet, J. Camara, M. Lahm, E.
Pentecost, K. Pratap, B. A. Gilmore, S. Divekar, R. S. Dagata, J. L. Bull and A.
Stoica, Endocrinology, 2003, 144, 2425–2436.
Met. Ions Life Sci. 2011, 8, 375–401

67. N. Geffroy, A. Guedin, C. Dacquet and P. Lefebvre, Mol. Cell. Endocrinol., 2005,
237, 11–23.
68. S. M. Cohen, T. Ohnishi, L. L. Arnold and X. C. Le, Toxicol. Appl. Pharmacol.,
2007, 222, 258–263.
69. L. M. Anderson, B. A. Diwan, N. T. Fear and E. Roman, Environ. Health
Perspect., 2000, 108, 573–594.
70. L. Tomatis, in Perinatal and Multigenerational Carcinogenesis, Ed. N. P.
Napalkov, J. M. Rice, L. Tomatis and H. Yamasaki, Oxford University Press,
New York, 1989, pp. 1–16.
Met. Ions Life Sci. 2011, 8, 375–401

Met. Ions Life Sci. 2011, 8, 403–422
Subject Index
Note
Entries which are solely for a figure or table are indicated by fig or t, respectively.
A Aluminum
carcinogenicity, 214
Aberrant gene expression, 393, 394 in cosmetics, 213–216t
Abortions, 276, 279–283 effects on
Absorption, 35, 117, 118 cardiovascular system, 84
in infants, 288, 289 eye, 228
mixtures, 73, 74 neurological system, 214, 228, 249, 250
pulmonary system, 83, 84
Acceptable daily intake (ADI), 120
skin, 213–215, 224
Accumulation see Bioaccumulation
exposure, 83, 84, 213, 214, 390
Acetylcholine (ACh), 258 (see also
food contamination, 112
Cholinergic system)
interactions with
Acupuncture needles, 207, 208
calcium, 249
Adaptive immune responses, 161–166
iron, 84, 195
Adenine see Nucleobases mechanisms of action, 214, 215, 249, 250
S-Adenosylmethionine (SAM), 382, 393 oxidation states, 83
ADI (acceptable daily intake), 120 PTWI level (provisional tolerable weekly
Agency for Toxic Substances and Disease intake), 122
Registry (ATSDR), 63, 64, 119 Alzheimer’s disease, 214, 228, 249
ALAD (d-aminolevulinic acid dehydratase), d-Aminolevulinic acid dehydratase see
138, 146, 148–152, 167, 259, 260 ALAD
Algae-based food products, 53 Analytical techniques, 39, 40
Alkylation of DNA, 322, 323, 326 Anemia, 84, 92, 145–147, 151, 198, 201, 211,
Allergies, contact, 199–202, 205, 207–209, 226 (see also Hematological effects)
211, 213, 219 Animal studies, 265, 266 (see also individual
Allyl alcohol, 72 species)
Alopecia, 213, 219 age-related immune effects, 159
404 SUBJECT INDEX
[Animal studies] [Arsenic]

aluminum, 249 immune system, 168–170, 172
arsenic, 145, 250, 284, 383, 384 kidney, 137, 138
beryllium, 86, 384 neurological system, 250, 289
cadmium, 145, 174, 250, 271, 284, 285, 290, pulmonary system, 84, 85
385, 386 reproductive system (female), 277, 278,
chromium, 386 281–283
cisplatin, 389 reproductive system (male), 266, 271
copper, 146 skin, 46, 220, 221
indium, 390 estrogenicity, 266
lead, 94, 136, 286, 290, 388, 389 exposure, 135, 339, 340, 382
lithium, 286 dietary, 49, 50, 52, 53, 112, 114fig,
manganese, 202 115fig, 118
mercury, 164, 287 drinking water, 50, 85, 135, 150, 151,
mercury and ethanol, 257 221, 223, 278, 281, 284, 289, 290,
molybdenum, 203 341, 377, 382, 396
nickel, 98, 275, 276, 356, 387 occupational, 84, 85, 339
tin, 146, 147, 203 perinatal, 38, 283, 284, 289, 290, 382,
uranium, 351 395, 396
zinc, 147, 195 food contamination, 49, 50, 52, 53, 112,
zinc and copper, 151 114fig, 115fig, 118
zinc and lead, 255, 256 genotoxicity, 335, 338fig, 339–341
Antagonism, 71, 253, 255, 259, 260 (see also half-life, 36
Combined effects; Synergism hypothesis) interactions with
Anti-smoking lozenges, 207, 208 cadmium, 136–138, 147, 148
Antiarthritic drugs, 174, 208 cadmium, chromium and lead, 70, 71
Antibodies, 161–164 gallium (gallium arsenide), 137, 138
Anticancer drugs, 168, 351, 352, 389 indium (indium arsenide), 137, 138
Antimony, 215–217 lead, 136–138, 150, 172, 254
carcinogenicity, 223, 390 lead and mercury, 266
estrogenicity, 312 selenium, 150, 151
skin effects, 220–222 in leukemia treatment, 168, 341
Antimony spots, 222 mechanisms of action, 38, 167–170, 221,
Antioxidant response elements (AREs), 169 340, 341, 384, 392–395
Antioxidants, 37, 110, 118, 126, 167, 335, 344 metabolism, 35, 36
Antiperspirants, 204, 213–215 oxidation states, 84
Apoptosis, 137, 166–168, 249, 343 oxidative stress, 18, 37, 167, 340, 392
Aquatic species, effects of metal mixtures, perinatal exposure, 38, 283, 284, 289, 290,
9, 10 382, 395, 396
AREs (antioxidant response elements), 169 risk assessment, 46, 49–50, 52–53, 122
Arsenic toxicokinetic (TK) models, 36, 37
absorption, 35 transport, 135
analytical techniques, 39, 40 uptake in plants, 15
bioaccumulation, 36 Arsinic acid, dimethyl- (DMA(V)), 382, 383,
carcinogenicity, 85, 222, 223, 338fig, 377, 395
378, 382–384 Arsinous acid, dimethyl- (DMA(III)), 340
effects on Arsonic acid, monomethyl- (MMA(V)),
cardiovascular system, 85 382, 383
development, 283, 284, 289, 290 Arsonous acid, monomethyl- (MMA(III)),
hematological system, 145, 147, 148, 340
150, 151 Arthritis see Antiarthritic drugs
Met. Ions Life Sci. 2011, 8, 403–422

SUBJECT INDEX 405
Artificial insemination, effect of lead, Bioavailability, 3, 10, 13–16, 135

270 effect of surfactants, 22, 23
Ascaris suum, 172 Biocide products, 23
Ascorbate, 335, 336, 344–348, 351 Biomarkers
Asthma, 83, 92, 98 hair, nails, skin, 45, 210
ATSDR (Agency for Toxic Substances and renal, 44, 137, 138
Disease Registry), 63, 64, 119 Biotic ligand model (BLM), 16, 17
Autism, 292 Bird studies, mercury, 172
Autoimmunity, 173–176 Birth defects see Teratogenesis
Bismuth, in cosmetics, 216t, 217
B Blackfoot disease, 85, 221
Bladder cancer, 46, 337, 382, 394, 395
B cells, 161–164 Bliss independence see Response addition
Babies see Postnatal exposure Blood see Hematological system
Bacillus subtilis, 355 Blood-brain barrier, 249, 251, 257, 259, 289
Bacteria, genotoxic metal effects, 355 Blood pressure, 44, 89, 94, 96, 174
BAL (British anti-Lewisite) (2,3- BMD (benchmark dose), 32, 119
dimercaptopropanol), 259, 266 Bone cancer, 218, 384
Barium Bone effects see Skeletal effects
and chromium, 346 Boron, 220
in cosmetics, 216t, 217 Bottle-feeding, 50, 52, 53
estrogenicity, 312 Bovine studies, mercury and cadmium, 160
Beer, 93, 199 Bowen’s disease, 221
Benchmark dose (BMD), 32, 119 Breast cancer, 170, 205, 214, 308, 309, 314,
Benzene see BTEX 386
Beryllium Breast-feeding, 53, 289
carcinogenicity, 86, 224, 338fig, 384, 385 Breast implants, 205
effects on British anti-Lewisite see BAL
cardiovascular system, 86 BTEX (benzene, toluene, ethylbenzene, and
pulmonary system, 85 xylene), 75
skin, 218 Burns, and cerium, 219
exposure, 384
genotoxicity, 338fig, 341, 342 C
and iron metabolism, 195
mechanisms of action, 384, 385, 392 CaBPs (calcium-binding proteins), 192–193
oxidation states, 85 Cadmium
Binary interactions (see also Combined absorption, 35, 212
effects; and under individual metals and analytical techniques, 40
metalloids) bioaccumulation, 36, 225, 226, 256, 278,
effects on 283, 290
hematological system, 147–152 carcinogenicity, 224, 308–310, 314, 338fig,
neurological system, 253–256 342–343, 378, 385, 386
empirical evidence, 9, 10 effects of nutritional status, 118
study design, 11 effects on
volatile organic compounds (VOCs), 75 cardiovascular system, 89, 174
Bioaccumulation, 3, 15, 16, 35, 36 development, 284, 285, 290
cadmium, 19, 135, 225, 226, 256, 278, 283, eye, 224–227
290 hematological system, 145, 147, 148
copper, 352 immune system, 160–166, 169–173fig,
lead, 212, 255–257, 279 174–176
mercury, 257, 259 kidney, 136, 137, 174, 290
Met. Ions Life Sci. 2011, 8, 403–422

406 SUBJECT INDEX
[Cadmium] [Calcium]
neurological system, 250 nickel, 201
pulmonary system, 87–89, 174 zinc, 193
reproductive system, 268 Calcium-binding proteins (CaBPs), 192, 193,
reproductive system (female), 277t, 278, 196
283 Calcyclin, 192, 193
reproductive system (male), 266, 271 Calmodulin, 192, 193, 196
skin, 210–213 Cancer, 337, 344, 350, 351, 357
estrogenicity, 266, 306–315 bladder, 46, 337, 382, 394, 395
exposure, 135, 308–310, 342, 386 bone, 218
dietary, 47, 50, 51, 111, 112, 114fig, breast, 170, 205, 214, 308, 309, 314, 386
115fig, 116, 135 endometrial, 309, 310, 314, 386
occupational, 88, 89, 212, 309, 354, 386 kidney (renal), 337, 382, 388, 389, 395
perinatal, 284, 285, 290 leukemia, 168, 210, 218, 341
food contamination, 47, 50, 51, 111, 112, liver, 218, 337, 382
114fig, 115fig, 116 lung, 46, 85, 86, 91, 200, 205, 222, 284, 347,
genotoxicity, 338fig, 339, 342, 343 382, 384
half-life, 36 pancreatic, 337
interactions with prostate, 337, 382
arsenic, 136–138, 147, 148 skin, 38, 208, 220, 222–224, 337, 382
arsenic, chromium and lead, 70, 71 testicular, 385, 386
calcium, 212, 250 uterine, 386
cobalt and lead, 211, 354 Carcinogenicity, 376–396 (see also
copper, 166 Genotoxicity)
ethanol, 256 aluminum, 214
iron, 145, 166 antimony, 223, 390
lead, 136–138, 148, 211, 253, 266, 267 arsenic, 85, 222, 223, 338fig, 377, 378,
manganese, 255 382–384
mercury, 71, 72, 118 beryllium, 86, 224, 338fig, 384, 385
nickel, 18 cadmium, 210, 224, 308–310, 314, 338fig,
selenium, 226, 266 342, 343, 378, 385, 386
zinc, 19–21, 137, 212, 226, 266, 307, 308 chromium, 91, 223, 338fig, 386, 387
mechanisms of action, 38, 136, 166, 167, cisplatin, 224, 389
169, 170, 392–394 cobalt, 224, 349, 353, 354, 390
and metallothionein (see Metallothionein) definitions, 378–380
oxidation states, 87 IARC classification, 336–339, 379–382
oxidative stress, 18, 167 indium, 390
risk assessment, 44, 47, 50, 51, 122, 268, iron, 224, 390
313–315 lead, 210, 224, 388, 389
toxicokinetic (TK) models, 36, 37 mechanisms of action, 30, 167, 222–224,
transport, 135 330–336, 384, 385, 387, 388, 391–395
Calcium, 192, 193 mechanisms of action, epigenetic, 30, 38,
in cosmetics, 216t 39, 393
effects on mercury, 210, 390
eye, 224–226 mixtures, 70, 71, 353, 354
skin (wound healing), 196, 197 nickel, 202, 223, 338fig, 353, 387, 388, 392
interactions with perinatal exposure, 395, 396
aluminum, 249 risk assessment, 30–32, 34, 70, 71, 380, 381
cadmium, 212, 250 selenium, 224
lead, 251 silicon (and silica), 224
magnesium, 193 thorium, 218
Met. Ions Life Sci. 2011, 8, 403–422

SUBJECT INDEX 407
[Carcinogenicity] [Chromium]
tungsten carbide (WC), 349, 353, 354, 390 cardiovascular system, 91, 92
vanadium, 353, 390 development, 285
Carcinogens, complete (definition), 378 pulmonary system, 90, 91
Cardiovascular effects reproductive system (female), 268, 277t,
aluminum, 84 278, 281
arsenic, 85 reproductive system (male), 271, 272,
beryllium, 86 281
cadmium, 89, 174 skin, 198–201
chromium, 91, 92 estrogenicity, 312
cobalt, 93, 199 exposure, prenatal, 285
copper, 87 food contamination, 112
lead, 51, 94, 95 genotoxicity, 333, 335, 338fig, 339, 343–346
manganese, 96 interactions with
mercury, 287 iron, 201
nickel, 98 nickel, 354
selenium and arsenic, 150, 151 zinc, 346
zinc, 99 mechanisms of action, 387, 392, 394
Cataracts, 225, 227 in mixtures, 70, 71
CDI (chronic daily intake), 121 oxidation states, 89, 90, 343, 344
Cell-mediated immune responses, 164–166 oxidative stress, 18, 392
Cell signaling pathways, 38, 167–169 toxicokinetic (TK) models, 36
Cereals, 47, 48 Chronic daily intake (CDI), 121
Cerium, effects on skin, 218, 219 Chronic oral minimal risk level (MRL), 119
Ceruloplasmin (CPN), 193 Circulation, 135 (see also Transport)
Chelating agents, therapeutic, 152, 153, 259 Cisplatin (CISP), 209, 224, 389, 396
Chelation, 125–127, 194, 195, 198, 201, 283, Co-exposure see Combined effects
332, 333, 335, 352 Cobalt
low molecular weight ligands, 345, 346 carcinogenicity, 224, 349, 353, 354, 390
Chemical mixtures see Combined effects effects on
Chemotherapy drugs, 209, 389 cardiovascular system, 93, 199
Children (see also Postnatal exposure; eye, 227
Prenatal exposure) immune system, 169
and arsenic, 53, 254 pulmonary system, 92, 93, 199
and cadmium, 51, 250 skin, 197–200
and iron, 149, 150 exposure, occupational, 92, 93, 199, 354
and lead, 44, 48, 149, 150, 250, 251, 254, genotoxicity, 335, 339fig, 348, 349, 353, 354
279, 290, 291 interactions with
and mercury, 45, 48, 51, 52, 287, 291, 292 cadmium and lead, 211, 354
Chloroethylene see Vinyl chloride tungsten carbide, 92, 93, 349, 353, 354,
Chloroform, 72, 75 390
Chlorophenol, 23 mechanisms of action, 169
pentachlorophenol (PCP), 74 oxidation states, 92
Cholinergic system, 225, 249, 251 (see also oxidative stress, 18
Acetylcholine) Combined effects
Cholinesterase inhibition, 258, 259 mixture evaluation, 64–68
Chromatin, 274, 331–333 Combined effects (joint action), 1–25, 62–77
Chromium (see also individual metals and metalloids
carcinogenicity, 91, 223, 338fig, 386, 387 for particular interactions)
in cosmetics, 216t absorption, dermal, 73, 74
effects on empirical evidence, 9, 10, 68–75
Met. Ions Life Sci. 2011, 8, 403–422

408 SUBJECT INDEX
[Combined effects (joint action)] [Copper]

during exposure, 13–17 mechanisms of action, 125, 149, 169
genetic influences, 76, 77 oxidation states, 86
genotoxicity, 73, 353, 354 oxidative stress, 18, 124
hematological effects, 147–152 Correlated responses, 8
in kidney, 136–138 Corynebacterium sp. 877, 355
metals and organic compounds, 21–24, Cosmetics, 210, 213–218, 221
152, 153, 256–259 Cot death see SIDS
neurological effects, 253–256 CPN (ceruloplasmin), 193
observational evidence, 4 Cumulative effects, 120–122
reference models, 6–9, 12, 71, 72 Cuproenzymes, 193, 194
risk assessment of mixtures, 62–77, 139, Cytokines, 164–166, 169
147–152 Cytosine see Nucleobases
study design, 11–13
synergism hypothesis, 5, 6 D
toxicity assessments, 64–68
toxicodynamics, 17–21 Defence mechanisms, 37
Compensatory hyperplasia, 394, 395 Deferoxamine, 153
Competition, ion uptake, 15, 17, 18 Deodorants, 213, 215
Complete carcinogens, definition, 378 2’-Deoxyguanosine, 8-oxo-, 325
Complexation, 37, 38, 125, 126, 352 (see also Dermatological effects see Skin
Chelation) Developmental effects, 283–293
Concentration addition model, 7–9, 22 arsenic, 283, 284, 289, 290
Concentration-response see Dose-response cadmium, 284, 285, 290
Conception, 279, 280 chromium, 285
Congenital malformations see Teratogenesis co-exposure with ethanol, 257
Contamination of food see Dietary exposure copper, 285
Copper lead, 44, 51, 283, 284, 286, 290, 291
and biotic ligand model, 16 lithium, 286
in cosmetics, 216t manganese, 292, 293
effects on mechanisms of action, 270
cardiovascular system, 87 mercury, 45, 283, 284, 286, 287, 291, 292
development, 285 neurodevelopmental effects, 44, 45, 51, 249
eye, 227 nickel, 283, 284, 287
hematological system, 87, 145, 146, 148, palladium, 288
149, 151 platinum, 288
immune system, 161, 169, 172, 175t postnatal exposure and, 288–293
pulmonary system, 86, 87 prenatal exposure and, 283–288
reproductive system (male), 271, 271t, rhodium, 288
272 tin, 288
skin, 193–199 vanadium, 288
estrogenicity, 313 Diabetes, 199, 201, 227
exposure, prenatal, 285 1,2-Dichloroethane, 71
genotoxicity, 333, 335, 339fig, 352, 353 1,2-Dichloroethylene, 75
interactions with Dietary exposure, 40–43, 110–117, 134, 135
cadmium, 166 aluminum, 112
cadmium and lead, 211 arsenic, 49, 50, 52, 53, 112, 114fig, 115fig,
lead, 148, 149, 253, 254, 259 118
molybdenum, 203 cadmium, 47, 50, 51, 111, 112, 114fig,
tin, 147, 203 115fig, 116
zinc, 147, 149, 151, 194 chromium, 112
Met. Ions Life Sci. 2011, 8, 403–422

SUBJECT INDEX 409
[Dietary exposure] Environmental Protection Agency, US

food contamination monitoring, 39, 40, (EPA), 118, 379–381
108, 109 Enzyme inhibition, 83, 86, 256, 258, 259, 335,
food risk assessment, 33, 34, 117–122 336
iron (contamination), 112 by lead, of ALAD, 146, 148–152, 167, 259,
lead, 47, 48, 51, 111, 112, 114fig, 115fig, 260
116 by vanadium, 203, 204, 276
mercury, 48, 51, 52, 111, 112, 114fig, EPA (US Environmental Protection Agency),
115fig, 116 118, 379–381
nickel, 112 Epigenetic mechanisms
uranium, 52 of carcinogenicity, 30, 38, 39, 393
zinc (contamination), 112, 151 of mutagenicity, 350
2,3-Dimercaptopropanol (British anti- Escherichia coli, 172, 355
Lewisite (BAL)), 259, 266 Essential elements see Trace metals
Dimethylarsinic acid (DMA(V)), 382, 383, Estrogen receptor activation, 307, 308, 312,
395 313
Dimethylarsinous acid (DMA(III)), 340 Estrogen receptors (ERs), 307, 308, 386
Dioxins, 66, 112t Estrogenicity
Distribution see Bioaccumulation; Transport aluminum, 214, 224
DMA(III) (dimethylarsinous acid), 340 arsenic, 266
DMA(V) (dimethylarsinic acid), 382, 383, 395 cadmium, 266, 306–315, 386
DNA damage see Genotoxicity other metals/metalloids, 312, 313
DNA methylation, 38, 393 Ethanol, 256, 257
DNA mutations, 330 Ethylbenzene see BTEX
DNA reactivity Ethylenediamine-N,N,N 0 ,N 0 -tetraacetate
DNA backbone, 325–327 (EDTA), 201, 259, 352
nucleobases, 322–325 Ethylmercury thiosalicylate (thiomersal), 213,
DNA repair, 327–329 292
DNA repair inhibition, 336, 341, 342, 343, European Union (EU), 30, 39, 41, 111
346, 349 Excretion, 36
DNA strand breaks, 327, 328, 333, 340 Experimental design, 5, 6, 8, 11–13, 68–70
Dose-response assessment see Hazard Experimental methods, 68–70, 354–357
characterization Exposure, 158, 159, 293, 377 (see also Dietary
Dose-response (concentration-response) exposure; Environmental exposure;
relationships, 12, 72, 74, 75 Occupational exposure; Postnatal
Double strand breaks (DSBs), 328 exposure; Prenatal exposure; and
individual metals and metalloids)
E interactions during, 13–17
response of organisms to, 19
Eales’ disease, 226 time and duration, 267–269
Earthworm studies, cadmium and zinc, Exposure assessment, 34, 39–43, 47–50, 276,
19–21 277
EDTA (ethylenediamine-N,N,N 0 ,N 0 - Eye, 190, 207, 224–228
tetraacetate), 201, 259, 352
Elimination (excretion), 36 F
Endocrine disrupters, 269, 306–315, 394
Endometrial cancer, 309, 310, 314, 386 Female fertility see Reproductive system,
Environmental exposure, 2, 3, 134, 135, 144, female
248, 249, 267 (see also individual metals Fenton reaction, 124, 125, 334
and metalloids) Ferritin, 149, 194, 195, 211, 227
polychlorinated biphenyls (PCBs), 257 Fertility see Reproductive system
Met. Ions Life Sci. 2011, 8, 403–422

410 SUBJECT INDEX
Fingernails see Nails Glutathione oxidase, 213

Fish, 48–52, 111, 112, 118, 135 Gly-L-His-L-Lys see GHK
Food contamination see Dietary exposure Gold
Foreign bodies, metal, 378 (see also effects on
Acupuncture needles) eye, 227, 228
Formula see Milk formula immune system, 175, 176
Free ion activity model, 14 kidney, 174, 208
Fruit, 118 liver, 208
skin, 205, 208, 209
G radioactivity, 208
therapy, 208, 209
Gallium Growth factors, 195–197
and arsenic (gallium arsenide), 137, 138, GSH see Glutathione
383, 384 Guanine see Nucleobases
hematological effects, 152 Guanosine, 8-oxo-2’-deoxy-, 325
Gastrointestinal tract Guidance values, health-based, 31, 32, 44–53,
absorption of metal ions, 117, 118 120–122
exposure to metal ions, 110–117 Guinea pig studies
oxidative damage, 123, 124 cadmium, 212
Gemcitabine, and gallium, 152 chromium, 200
Gender differences, 138, 211, 212, 266 cobalt, 199
Gene transcription, 38, 169, 307, 312, 313, lead, 211
393, 394 mercury, copper, 172
Genetic variability, 35, 36, 76, 163, 164, 175,
176, 210, 282, 309 (see also Gender H
differences; Inter-individual variability)
Genotoxicity (DNA damage), 30, 320–358 Haber-Weiss system, 125, 334
arsenic, 335, 338fig, 339–341 Hair, 45, 210–213 (see also Alopecia)
beryllium, 338fig, 341, 342 Hair dyes, 210–212
cadmium, 338fig, 339, 342, 343 Half-life, 36
chromium, 333, 335, 338fig, 339, 343–346 Hamster studies
cobalt, 335, 339fig, 348, 349, 353, 354 cadmium, 278
copper, 333, 335, 339fig, 352, 353 indium, 283
experimental methods, 354–357 mercury, 279
iron, 339fig, 352, 353 nickel, 287
lead, 339fig, 349, 350 vanadium, 288
manganese, 335 Hard metal see Tungsten carbide
mechanisms of action (general), 322–330 Hard metal lung disease, 92, 93
mechanisms of action (metal ion), 330–336, Hazard characterization, 33, 34, 44–46, 119
340–354, 391–393 Hazard, definition, 29, 30
mixtures, 73, 76, 353, 354 Hazard estimation, cumulative effects, 120–122
nickel, 334–336, 338fig, 339, 346–348, 353, Hazard identification, 33, 44–46
356 Hazard index, 66
platinum, 333, 339fig, 351, 352 Hazard quotient (HQ), 121
risk assessment, 30–32, 34 Hazardous waste sites, 70, 71, 144, 248, 249
uranium, 339fig, 350, 351 Health-based guidance values, 31, 32, 44–53,
vanadium, 335, 353 120–122
GHK (Gly-L-His-L-Lys (glycyl-L-histidyl-L- Heart see Cardiovascular effects
lysine)), 197 Hematological effects, 145–153
Glutathione (GSH), 167, 169, 170, 253, 266, arsenic, 145, 147, 148, 150, 151
314, 343, 345–347, 393 binary interactions, 147–152
Met. Ions Life Sci. 2011, 8, 403–422

SUBJECT INDEX 411
[Hematological effects] [Immune system]

cadmium, 145, 147, 148 nickel, 161, 169, 170, 172–174
copper, 87, 145, 146, 148, 149, 151 zinc, 161, 169, 172, 175t
gallium, 152 humoral immune responses, 161–164
iron, 149, 150 innate immunity, 160, 161
lead, 146, 148–152, 254 mechanisms of action, 163, 165–170, 176
manganese, 150 Immunomodulation, 159, 160, 166–170
mercury, 146 Immunotoxicity, 159, 160, 166–170
metals and organic compounds, 152, 153 Implants, metal, 353, 378
mixtures, 147–152 Implants, silicone gel, 205
single metals, 145–147 Indium
tin, 146, 147, 151 and arsenic (indium arsenide), 137, 138
zinc, 147, 149, 151, 152 carcinogenicity, 390
Hepatoxicity, 72, 76 (see also Liver) reproductive effects, 283
Histidine-rich glycoprotein, and zinc, 152 Infants see Postnatal exposure
Histone modification, 38, 347–349, 393 Inflammation, chronic, 166, 173–176
Homeostatic mechanisms, 15, 18–20, Innate immune system, 160, 161
190–205, 212, 225–227, 251, 258 Insecticides, phyrethroid, 24
HQ (hazard quotient), 121 Insulin, 199
Humic acid, 14, 221 Insulin mimicry, 203
Humoral immune responses, 161–164 Inter-individual variability, 35, 36, 76, 163,
Hydrogen peroxide, 123–126 164, 175, 176, 210, 265, 309, 389 (see also
Hydrolysis, 322, 325, 326, 334 Gender differences)
Hydroxyl radicals, 124–126 Interaction thresholds, 74, 75
and DNA, 323–326 Interactions see Combined effects
Hydroxyurea, and gallium, 152 Interdependencies between metal ions see
Hyperplasia, compensatory, 394, 395 individual metals, interactions with
Hypertension, 44, 89, 94, 174 International Agency for Research on Cancer
see IARC
I International Program on Chemical Safety
(IPCS), 29
IARC (International Agency for Research on Intraspecies variability see Inter-individual
Cancer) variability
classification of carcinogens, 336–339, 377, Ion transport, 14, 15, 19, 35, 135, 193–195
379, 381, 382 (see also Metal-binding (carrier)
monographs, 380, 381 proteins)
Immune system, 158–177 effect of organophosphates, 258
adaptive immunity, 161–166 IPCS (International Program on Chemical
autoimmunity, 173–176 Safety), 29
cell-mediated immunity, 164–166 IQ (intelligence quotient), 44, 250, 289–292t
effects of Iron (see also Hematological effects)
arsenic, 168–170, 172 carcinogenicity, 224, 390
cadmium, 160–166, 169–172, 173fig, complexes as indicator of trace metal
174–176 availability, 198
cobalt, 169 in cosmetics, 216t
copper, 161, 169, 172, 175t effects on
gold, 175, 176 eye, 226, 227
lead, 160, 161, 164, 169, 172, 173, 175, gastrointestinal system, 123, 124
211, 266 hematological system, 149, 150
mercury, 160, 161, 164, 165, 168, 170, liver, 123
172–176 skin, 194, 195, 198, 199
Met. Ions Life Sci. 2011, 8, 403–422

412 SUBJECT INDEX
[Iron (see also Hematological effects)] [Lead]

exposure analytical techniques, 40
occupational, 390 bioaccumulation, 36
postnatal, 226 carcinogenicity, 210, 224, 388, 389
food contamination, 112 in cosmetics, 216t
genotoxicity, 339fig, 352, 353 effects of nutritional status, 118
interactions with effects on
aluminum, 84, 195 cardiovascular system, 94, 95
beryllium, 195 development, 283, 284, 286, 290, 291
cadmium, 145, 166 eye, 224–227
chlorophenol, 23 hematological system, 146, 148–152, 254
chromium, 201 immune system, 160, 161, 164, 169, 172,
lead, 149, 150, 211 173, 175, 211, 266
nickel, 201 kidney, 44, 45, 136, 137, 254
tin, 147, 198, 203 liver, 254
zinc, 195 neurodevelopment, 44
oxidative stress, 18, 123, 124 neurological system, 250, 251
oxidative stress catalysis, 124–126 pulmonary system, 94
and Parkinson’s disease, 117, 126 reproductive system, 269
transport, 19, 194, 195 reproductive system (female), 277t,
278–282
J reproductive system (male), 269, 270,
271t, 272–274, 280
skin, 210–212
Jewellery, 202, 205, 207–209, 219
estrogenicity, 312
Joint action see Combined effects
exposure, 135, 349
dietary, 47, 48, 51, 111, 112, 114fig,
K 115fig, 116, 135
occupational, 94, 251, 274, 282, 349, 350
Kidney prenatal, 251, 291, 396
bioaccumulation in, 36, 290 exposure assessment, 47, 48
effects of food contamination, 47, 48, 51, 111, 112,
arsenic, cadmium and lead, 138 114fig, 115fig, 116
cadmium, 136, 137, 174, 290 genotoxicity, 339fig, 349, 350
gallium arsenide, 137, 138 half-life, 36
gold, 174, 208 interactions with
indium arsenide, 137, 138 arsenic, 136–138, 150, 172, 254
lead, 44, 45, 51, 136, 137, 254 arsenic and mercury, 266
mercury and selenium, 137, 138 cadmium, 136–138, 148, 211, 253, 266,
uranium, 45, 46 267
mechanisms of action, 136, 137 calcium, 251
Kidney cancer, 337, 382, 388, 389, 395 chromium, 346
Kidney disease, autoimmune, 174, 175, 175t cobalt and cadmium, 211, 354
Kinky hair syndrome see Menkes syndrome copper, 148, 149, 253, 254, 259
Klebsiella pneumonia, 172, 173 ethanol, 256, 257
iron, 149, 150, 211
L manganese, 150, 255
nickel, 18
Langerhans cells, 221, 223, 228 organophosphates, 258, 259
Lead zinc, 138, 151, 152, 251, 255, 256, 259,
absorption, 35, 210, 212 274
Met. Ions Life Sci. 2011, 8, 403–422

SUBJECT INDEX 413
[Lead] Malformations see Teratogenesis

mechanisms of action, 136, 167, 169, 251 Mammary gland development, 311, 312
in mixtures, 70, 71, 354 Manganese
oxidation states, 93 in cosmetics, 216t
oxidative stress, 37, 167 effects on
perinatal exposure, 283, 284, 286, 290, 291, cardiovascular system, 96
396 development, 292, 293
risk assessment, 44, 45, 47, 48, 51, 122 eye, 226
toxicokinetic (TK) models, 36 hematological system, 150
Leukemia, 168, 210, 218, 341 neurological system, 251, 252
Levels, estimation of safe, 31, 32, 44–53, 109, pulmonary system, 96
111, 118–122 reproductive system (male), 271, 271t,
Linear extrapolation, 30, 31 274, 275, 281
Lipid peroxidation, 249, 256, 272, 274, 276 skin, 198, 199, 202, 203
Lithium exposure, early postnatal, 292, 293
in cosmetics, 216t genotoxicity, 335
effects on interactions with
development, 286 cadmium, 255
skin, 217 lead, 150, 255
estrogenicity, 312 nickel, 348
prenatal exposure, 286 tin, 203
Liver, 352 mechanisms of action, 252
absorption of metal ions, 117, 118 oxidation states, 95, 96
effects of as trace metal, 198, 202, 203
lead, 254 in wine, 118
mercury, 170 Manganism, 251, 275
selenium and arsenic, 150, 151 MAPK (mitogen-activated protein kinase)
exposure to metal ions, 110–117 signaling, 167–170
genetic differences, 76 Margin of exposure (MOE), 31, 34
injury vs. repair, 72 Margin of safety (MOS), 51
oxidative damage, 123, 124 Marine organisms, resistance to infection,
Liver cancer, 218, 337, 382 172
Loewe additivity see Concentration addition MCF-7 cells, effects of cadmium, 311, 312,
Lung see Pulmonary effects 314
Lung cancer, 46, 85, 86, 91, 200, 205, 222, Mechanisms of action, 17, 18, 122–126 (see
284, 347, 382, 384 also Oxidative stress; and individual
Lupus erythematosus, systemic (SLE), 175, metals and metalloids)
176 carcinogenicity, 30, 167, 222–224, 330–336,
384, 385, 387, 388, 391–395
M carcinogenicity (epigenetic), 30, 38, 39, 393
epigenetic, 30, 38, 39, 350, 393
Macromolecules, 16–18, 83 genotoxicity (general), 322–330
Macrophages, 160, 161 genotoxicity (metal ion), 330–336, 340–354,
Magnesium 391–393
in cosmetics, 216t immune system effects, 163, 165–170,
effects on the eye, 225 176
interactions with mutagenicity, 340–342, 345, 348, 350
cadmium and lead, 211 neurotoxicity, 249–253, 258
calcium, 193 renal cell injury, 137
nickel, 348 repair, 72, 195–197, 327–329
Male fertility see Reproductive system, male reproductive effects, 269, 270
Met. Ions Life Sci. 2011, 8, 403–422

414 SUBJECT INDEX
[Mechanisms of action] Metabolism, metal, 35, 36, 377, 378, 391

in skin, 195–197, 201, 221 calcium, 212
of uptake by the kidney, 136 copper, 147, 193, 194, 203, 352, 353
Menkes syndrome, 193, 194 effect of
Mercury (and methylmercury) aluminum, 84, 250
absorption, 35, 212, 213 cobalt, 198
analytical techniques, 39, 40 lead, 211, 212
bioaccumulation, 36 molybdenum, 203
carcinogenicity, 210, 390 tin, 147, 198, 203
effects of nutritional status, 118 iron, 84, 147, 198, 211, 352, 353
effects on manganese, 203
cardiovascular system, 174 silicon, 204
development, 283, 284, 286, 287, 291, Metal-binding (carrier) proteins, 190–195,
292 224–227 (see also Transport)
eye, 224, 227 Metal fume fever, 98, 99, 147
hematological system, 146 Metal implants and foreign bodies, 353, 378
immune system, 160, 161, 164, 165, 168, (see also Acupuncture needles)
170, 172, 174–176 Metal mixtures see Combined effects (joint
kidney, 174, 175 action)
liver, 170 Metal response elements (MREs), 169
neurological system, 252, 253 Metalloestrogens see Estrogenicity
reproductive system (female), 266, 277t, Metalloids, 220–222 (see also Antimony;
279, 282 Arsenic; Boron; Silicon)
reproductive system (male), 266, 271t, Metallothionein (MT), 153, 190–195
275, 282 and cadmium, 192, 212, 266, 283, 314,
skin, 210, 212, 213 335, 343, 378, 394
estrogenicity, 312 with ethanol, 256
exposure in kidney, 135–137
dietary, 48, 51, 52, 111, 112, 114fig, with zinc, 19–21
115fig, 116, 135 and gold, 208
environmental, 135, 210, 213 and nickel, 201, 387, 388
occupational, 213, 275 and platinum/cisplatin, 351, 389
postnatal, 210, 291, 292 and silver, 192, 206
prenatal, 45, 257, 283, 284, 286, 287 and zinc, 191, 192, 212
food contamination, 48, 51, 52, 111, 112, with cadmium, 19–21
114fig, 115fig, 116 Methionine, S-adenosyl- (SAM), 382,
half-life, 36 393
interactions with Methylmercury see Mercury
British anti-Lewisite (BAL), 259 Micronutrients see Trace metals
cadmium, 71, 72, 118 Milk formula, 52, 53
ethanol, 257 Mimicry
lead and arsenic, 266 of essential metal ions, 15, 37, 38, 251,
polychlorinated biphenyls (PCBs), 257, 391
258 of estrogen (see Estrogenicity)
selenium, 137, 138 Mineral supplements, 109, 110, 116, 117
mechanisms of action, 136, 163, 165, 167, Minimal risk level (MRL), 119
169, 170, 252, 253 Miscarriages, 276, 279–283
oxidative stress, 18, 167 Mixture ratios, 13, 22, 71, 72
risk assessment, 45, 48, 51, 52, 122 Mixtures see Combined effects (joint action)
toxicokinetic (TK) models, 36 MMA(III) (monomethylarsonous acid),
transport, 135 340
Met. Ions Life Sci. 2011, 8, 403–422

SUBJECT INDEX 415
MMA(V) (monomethlyarsonic acid), 382, Mutagenicity, mechanisms, 340–342, 345,

383 348, 350
Models Mutations, genetic, 330, 335, 336, 340, 357
pharmacodynamic (PBPD), 70, 71 Mycobacterium bovis, 172
pharmacokinetic (toxicokinetic) Mycotoxins, 112t
(PBPK, PBTK), 36, 37, 70, 71
Models, combination effect analysis N
(reference models), 6–9, 12, 71, 72
Modes of action, 19–21 (see also Mechanisms Nails (finger- and toe-), 209–211
of action) Nanomaterials, 139, 208, 347, 358
MOE (margin of exposure), 31, 34 National Toxicology Program Report on
Molecular mechanisms see Mechanisms of Carcinogens (ROC), 380, 381
action Natural killer cells (NK cells), 161
Molecular mimicry see Mimicry Neonatal exposure see Postnatal exposure
Molybdenum, 197, 198, 202, 203 Nephrotoxicity see Kidney
Monkey studies, mercury, 275 Neurodevelopmental effects, 44, 45, 51, 249
Monkey studies, mercury and cadmium, Neurological effects, 248–260 (see also
71 Wilson’s disease)
Monomethylarsonic acid (MMA(V)), 382, aluminum, 214, 228, 249, 250
383 arsenic, 250, 289
Monomethylarsonous acid (MMA(III)), cadmium, 250
340 cadmium and lead, 253
MOS (margin of safety), 51 copper and lead, 253, 254
Mouse studies lead, 44, 250, 251, 290, 291
arsenic, 271, 277 lead and arsenic, 254
cadmium, 159, 162–165, 169, 170, 172, 173, manganese, 251, 252, 292, 293
176, 310, 311 manganese and cadmium, 255
chromium, 272, 278, 285 manganese and lead, 255
copper, 169, 194, 285 mechanisms of action, 249–253, 258
gold, 176 mercury, 45, 252, 253, 287, 292
indium, 283 zinc and lead, 255, 256
lead, 169, 278, 279, 350 Neurotransmitters, 250, 251, 253, 254, 256,
258
lead and arsenic, 254, 266
Newborns see Postnatal exposure
lithium, 217
NF-kB (nuclear factor-kappaB), 169
magnesium, 169
Nickel
mercury, 169, 172, 175, 176, 266, 275
and biotic ligand model, 16
metal response elements, 169
carcinogenicity, 202, 223, 338fig, 353, 387,
metallothionein, 192 388, 392
nickel, 161, 169, 172, 223, 282, 283, effects on
287 cardiovascular system, 98
PCBs and mercury, 258 development, 283, 284, 287
vanadium, 288 immune system, 161, 169, 170, 172–174
zinc, 99, 169 pulmonary system, 92, 97, 98
MREs (metal response elements), 169 reproductive system (female), 281–283
MRL (minimal risk level), 119 reproductive system (male), 271t, 275,
MT see Metallothionein 276
Multimineral supplements, 116, 117 skin, 199–202, 219
Multiple sclerosis, 175t exposure, 346, 347, 388
Multivitamin supplements, 116, 117 occupational, 97, 98, 202, 276, 281, 282,
Mushrooms, 47 287, 388
Met. Ions Life Sci. 2011, 8, 403–422

416 SUBJECT INDEX
[Nickel] [Occupational exposure]

prenatal, 283, 284, 287, 395 nickel, 97, 98, 174, 202, 276, 281, 282, 287,
food contamination, 112 388
genotoxicity, 334–336, 338fig, 339, platinum, 209
346–348, 353, 356 silicon, 224
interactions with silver, 206, 207
cadmium, 18 zinc, 99
cadmium and lead, 211 Ocular effects, 190, 207, 224–228
calcium, 201 Oilseeds, 47
chromium, 354 Oral minimal risk level, chronic (MRL), 119
iron, 201 Oral reference dose (RfD), 118–120
lead, 18 Organic compounds, interaction with metals,
mechanisms of action, 169, 170, 201, 387, 21–24, 152, 153, 256–259
388, 392, 393 Organophosphates, 258, 259
in nuts, 118 Osteosarcomas, 384
oxidation states, 97 Oxidative stress (damage), 18, 37, 38, 122,
oxidative stress, 18 391–393
as trace metal, 201, 202 arsenic, 340, 392
Nitrogen-based radicals see Reactive nitrogen cadmium, 392, 393
species chromium, 392
NK cells (natural killer cells), 161 copper, 124
NOAEL (no adverse effect level), 118–120 in eye, 227
Non-genotoxic carcinogens, 30–32 (see also in gastrointestinal tract, 123, 124
Epigenetic mechanisms) in immune system, 166, 167
NTP Report on Carcinogens (ROC), 380, 381 interaction of metals, 23fig
Nuclear factor-kappaB (NF-kB), 169 iron, 123, 124, 227
Nucleobases in kidney, 137
mutations, 330 in liver, 123, 124
oxidation, 334 molecular mechanisms, 124, 126
reactivity, 322–325 nickel, 347
Null hypothesis, combination effect analysis, 8-oxo-dG as marker, 325
6–9 photochemical, 22
Nutrients see Trace metals 8-Oxo-dG (8-oxo-2 0 -deoxyguanosine), as
Nutritional status, 118, 195 marker for oxidative stress, 325
Nuts, 47, 118 Oxygen-based radicals see Reactive oxygen
species
O
P
Occupational exposure, 267, 377, 396
aluminum, 83, 84, 249, 390 PAHs (polyaromatic hydrocarbons), 21, 22,
antimony, 222 73
arsenic, 84, 85, 339 Palladium, 218, 219, 288
beryllium, 341, 384 Pancreatic cancer, 337
cadmium, 88, 89, 174, 212, 309, 354, 386 Parkinson’s disease, 117, 126, 254
chromium, 90–92, 200, 223, 272, 281, 309 PBPK/PD (physiologically-based
cobalt, 92, 93, 199, 354 pharmacokinetic/pharmacodynamic)
copper, 86, 87, 145, 146, 282 models, 70, 71, 74, 75
iron, 390 PBTK (physiologically-based toxicokinetic)
lead, 94, 172, 251, 273, 274, 282, 349, 350 models, 36, 37
manganese, 96 PCBs (polychlorinated biphenyls), 74, 257,
mercury, 213, 275, 282 258
Met. Ions Life Sci. 2011, 8, 403–422

SUBJECT INDEX 417
PCP see Pentachlorophenol Pregnancy effects, 280–283 (see also Prenatal

Penicillamine, 201, 259 exposure)
Pentachlorophenol (PCP), 74 (see also Prenatal exposure, 268, 283–288, 395, 396
Chlorophenol) arsenic, 38, 283, 284, 395, 396
Perinatal exposure, 267, 395, 396 (see also cadmium, 284, 285
Postnatal exposure; Prenatal exposure) chromium, 285
Pesticides, 23, 24, 258 cisplatin, 396
Phagocytes, 160, 161, 355 copper, 285
Phosphorylation, cadmium-induced, 313 ethanol co-exposure, 257
Photochemical processes, 22 lead, 251, 283, 284, 286, 291, 396
Phyrethroid insecticides, 24 lithium, 286
Physiologically-based pharmacodynamic mercury, 45, 257, 283, 284, 286, 287
models (PBPD), 70, 71 nickel, 283, 284, 287, 395
Physiologically-based pharmacokinetic palladium, 288
(toxicokinetic) models (PBPK, PBTK), platinum, 288, 396
36, 37, 70, 71, 74, 75 rhodium, 288
Placenta, 257, 283 tin, 288
Platinum vanadium, 288
carcinogenicity, 224, 389 Probabilistic approach (dietary exposure
effects on assessment), 43
development, 288 Prostate cancer, 337, 382
skin, 205, 209, 210 Proto-oncogenes, 393, 394
exposure PTWI (provisional tolerable weekly intake),
occupational, 209 111, 113, 115fig, 120, 122
prenatal, 288, 396 Pulmonary effects (see also Lung cancer)
genotoxicity, 333, 339fig, 351, 352 aluminum, 83, 84
Point estimation (dietary exposure arsenic, 84, 85
assessment), 42, 43 beryllium, 85
Polyaromatic hydrocarbons (PAHs), 21, 22, cadmium, 87–89, 174
73 chromium, 90, 91
Polychlorinated biphenyls (PCBs), 74, 257, cobalt, 92, 93, 199
258 copper, 86, 87
Population-based toxicokinetic models, 36, lead, 94
37 manganese, 96
Postnatal exposure, 288–293, 395, 396 nickel, 92, 97, 98
arsenic, 38, 50, 53, 289, 290 zinc, 98, 99
cadmium, 290 Pulses, 47
iron, 226 Pyrimidine dimers, repair of, 328
lead, 251, 290–292, 396
manganese, 292, 293
mercury, 210, 291, 292 Q
sudden infant death syndrome (SIDS), 215,
216, 221 QSAR (quantitative structure activity
uranium, 52 relationship) modeling, 76, 77
Potassium, in cosmetics, 216t
Potatoes, 48 R
Potentiation, 254, 257 (see also Combined
effects; Synergism hypothesis) Rabbit studies
Prediction window, 24 aluminum, 228
Predictions of toxicity see Risk assessment; calcium, 225
Toxicity assessment manganese and lead, 150
Met. Ions Life Sci. 2011, 8, 403–422

418 SUBJECT INDEX
[Rabbit studies] Reference dose (RfD), 118–120

mercury, 174, 175 Reference models, combination effect
uranium, 45 analysis, 6–9, 12, 71, 72
Racial differences, 282, 309 (see also Genetic Regulatory bodies, 379, 380
variability) Renal biomarkers, 44, 137, 138
Radical reactions, 124–126, 323–326 (see also Renal cancer, 337, 382, 388, 389, 395
Oxidative stress; Reactive oxygen Renal effects see Kidney
species) Repair mechanisms, 72, 195–197, 327–329, 336
Radioactivity Report on Carcinogens (National Toxicology
gold, 208 Program ), 380, 381
palladium, 219 Reproductive system, 264–294
uranium, 350 effects of
RASSFF (rapid alert system for food and arsenic, 266, 271, 277, 277t, 278, 281–283
feed), 111–114 cadmium, 266, 268, 271, 277t, 278, 283
Rat studies chromium, 268, 271, 272, 277t, 278, 281
aluminum, 214, 215, 228 copper, 271t, 272
arsenic, 277 indium, 283
BTEX, 75 lead, 269, 270, 271t, 272–274, 277t,
cadmium, 310, 311 278–282
cadmium and arsenic, 147, 148, 172, manganese, 271t, 274, 275, 281
174–176, 210 mercury, 266, 271t, 275, 277t, 279, 282
cadmium and ethanol, 256 nickel, 271t, 275, 276, 281–283
cadmium and lead, 148, 253 vanadium, 276
chromium, 223 exposure, time and duration, 267–269
cobalt, 86, 349 female, 268, 276–283
copper, 272, 285, 352 male, 268, 270–276, 279, 280
copper and lead, 148, 149, 254 mechanisms of action, 269, 270
copper and zinc, 149 risk assessment, 265–269
gold, 176 Residual oil fly ash (ROFA), 353
indium, 283 Resistance toward infection, 170–173
iron, 124 Response addition model, 7–9, 22
lead, 211, 225, 272, 350 RfD (oral reference dose), 118–120
lead and arsenic, 150 Rhodium, 288
lead and ethanol, 256, 257 Rice, 53, 135
manganese and cadmium, 255 Risk assessment, 29–34, 40–53, 313–315 (see
manganese and lead, 255 also individual metals and metalloids)
mercury, 168, 175, 275, 279, 292 carcinogens, 30–32, 34, 70, 71, 380, 381
mercury and selenium, 137 definitions, 29, 30
metallothionein, 192 exposure assessment, 34, 40–43, 47–50,
nickel, 287, 356 117, 118, 276, 277
organophosphates and lead, 258, 259 genotoxicity, 30–32, 34
PCBs and mercury, 258 hazard characterization, 33, 34, 44, 46,
platinum, 288 120–122
selenium, 226 hazard identification, 33, 44–46
silicon, 204 heavy metals and metalloids, 43–53
vanadium, 288 mixtures, 62–77, 139
zinc, 99 reproductive effects, 265–269
zinc and lead, 151 Risk characterization, 34, 50–53
Reactive nitrogen species (RNS), 37, 123–126 Risk, definition, 29, 30
Reactive oxygen species (ROS), 37, 123–126, RNA, interaction with metal ions, 334
167, 335, 340, 343, 354, 391, 392, 394 RNS (reactive nitrogen species), 37, 123–126
Met. Ions Life Sci. 2011, 8, 403–422

SUBJECT INDEX 419
ROC (National Toxicology Program Report Silver

on Carcinogens), 380, 381 and biotic ligand model, 16
Rodent studies (see also individual species) effects on
cadmium, 163 eye, 227, 228
mercury, 160, 161, 163 skin, 192, 205–208
nickel and copper, 161 exposure, 206, 207
ROFA (residual oil fly ash), 353 interactions with
RONS (reactive oxygen and nitrogen selenium, 207
species), 123–126 (see also RNS; ROS) zinc, 192
ROS (reactive oxygen species), 37, 123–126, and metallothionein, 192, 206
167, 335, 340, 343, 354, 391, 392, Single strand breaks (SSBs), 327
394 Sites of toxic action, 16
Skeletal effects, 201, 212, 220, 284, 286
S Skin, 188–224
absorption of mixtures, 73, 74
Safe intake levels, 31, 32, 44–53, 109, 111, carcinogenesis in, 222–224
118–122 effects of
Salmonella, 112t aluminum, 213–215, 224
Enteritidis, 171, 173fig antimony, 220–222
typhimurium, 355 arsenic, 46, 220, 221
SAM (S-adenosylmethionine), 382, 393 boron, 220
Sample analysis, 39, 40 cadmium, 210–213
Scopulariopsis brevicaulis, 221 chromium, 199–201
SCSA (sperm chromatin structure assay), cobalt, 197–200
268 gold, 205, 207–209
Seafood, 48–50, 52, 112, 118, 135 (see also lead, 210–212
Fish; Shellfish) manganese, 202, 203
Selenium mercury, 210, 213
carcinogenicity, 224 molybdenum, 202, 203
effects on nickel, 201, 202
kidney, 137, 138 platinum, 205, 208–210
liver, 151 selenium and arsenic, 150, 151
skin, 151 silicon, 196, 204, 205, 220
interactions with silver, 192, 205–208
arsenic, 150, 151 strontium, 217, 218
cadmium, 226, 266 thallium, 218–220
mercury, 137, 138 thorium, 218
silver, 207 tin, 202, 203
zinc, 226 trace metals, 197–205
Semiconductors, 134, 137, 138, 390 vanadium, 202–204
Shellfish, 47, 112, 135 xenobiotic metal ions, 205–222
Short-term toxic effects, 16, 17 zinc, 191, 192, 198, 199
SIDS (sudden infant death syndrome), 215, zirconium, 213–215, 224
216, 221 overview, 188, 190, 197, 198
Signaling pathways, 38, 167–169 repair following injury, 195–197
Silica, carcinogenicity, 224 Skin cancer, 38, 208, 220, 222–224, 337,
Silicon, 204, 205 382
carcinogenicity, 224 SLE (systemic lupus erythematosus),
in cosmetics, 216t 175
skin effects, 196, 204, 205, 220 Smoking, 51, 88, 89, 96, 97, 118, 278,
Silicone, 204, 205 285, 309, 337, 386
Met. Ions Life Sci. 2011, 8, 403–422

420 SUBJECT INDEX
Sodium, in cosmetics, 216t Time to pregnancy (TTP), 268

Solvents, effect on dermal absorption, Tin
73, 74 in cosmetics, 217t
Speciation, 13, 14, 16, 39, 40, 110, 346, effects on
348 development, 288
Sperm chromatin structure assay (SCSA), hematological system, 146, 147,
268 151
Stainless steel implants, 353 skin, 202, 203
Stibine (SbH3), 217, 221 interactions with
Stillbirths, 279, 281–283 copper, 147, 203
Stress genes, 394 iron, 147, 198, 203
Strontium, 216t, 217, 218 manganese, 203
Study design, 5, 6, 8, 11–13, 68–70 zinc, 151, 203
Study methods, 68–70, 354–357 prenatal exposure, 288
Styphylococcus aureus, 172 PTWI level (provisional tolerable weekly
Sudden infant death syndrome (SIDS), intake), 122
215–217, 221 as trace metal, 203
Supplements, 109, 110, 116, 117 Titanium, 215, 217t, 218
Surfactants, 22, 23, 73, 74 TNF-a (tumor necrosis factor a), 170,
Synergism hypothesis, 5, 6 171fig
Systemic lupus erythematosus (SLE), Tobacco see Smoking
175 Toenails see Nails
Tolerable daily intake (TDI), 120
T Toluene see BTEX
Total diet studies, 41
T cells, 164–166 Toxicity assessment, 64–66, 71, 72,
Target cancer risk (TR), 121 117–122
Target hazard quotients (THQs), 120, Toxicity threshold levels, 31, 32, 44–53,
121 109, 111, 118–122
Tattoos, 210, 215 Toxicodynamics (TD), 6, 17–21, 32,
TDI (tolerable daily intake), 120 37, 38
Teratogenesis, 266, 281, 283–290 Toxicokinetics (TK), 32, 35–37, 266
mechanisms of action, 270 TR (target cancer risk), 121
Terbium, 193 Trace metals, 189–191, 195–205, 210,
Testicular cancer, 385, 386 211, 218, 224–227, 250
Thallium Transcription factors, 169, 394
eye effects, 227, 228 Transcription, gene, 38, 169, 307, 312,
skin effects, 218–220 313, 393, 394
Thioacetamide, 72 Transferrins, 194
Thiomersal (thimerosal, ethylmercury Transport, 14, 15, 19, 35, 135, 193–195
thiosalicylate), 213, 292 (see also Metal-binding (carrier)
Thorium, 218 proteins)
THQs (target hazard quotients), 120, effect of organophosphates, 258
121 Trichloroethylene, 71, 72, 75
Threshold levels of toxicity, 31, 32, Trientine, 259
44–53, 109, 111, 118–122 TTC (threshold of toxicological concern),
Threshold of toxicological concern (TTC), 31, 122
31, 122 TTP (time to pregnancy), 268, 280
Thresholds, interaction, 74, 75 Tungsten, 203
Thymine see Nucleobases Tungsten carbide (WC), 92, 93, 199, 349,
Time of exposure, 172, 173, 267–269 353, 354, 390
Met. Ions Life Sci. 2011, 8, 403–422

SUBJECT INDEX 421
U W
Udenfriend’s system, 125, 126 Water, as exposure medium, 13, 14
Uncertainty factors (UFs), 32, 117, 118 Water, metal ions in drinking
Upper levels (ULs), 119 aluminum, 214
Uptake of metal ions, 14–16, 136 arsenic, 50, 85, 135, 150, 151, 221, 223,
(see also Absorption; Bioaccumulation; 278, 281, 284, 289, 290, 341, 377,
Transport) 382, 396
Uranium cadmium, 135
absorption, 35 lead, 48, 135
analytical techniques, 39 manganese, 252
bioaccumulation, 35, 36 mercury, 135
exposure, 48, 49, 52, 350 uranium, 49, 52
genotoxicity, 339fig, 350, 351 Weight-of-evidence (WOE) evaluations,
half-life, 36 66–68
kidney effects, 45, 46 WHO (World Health Organization), 29
risk assessment, 45, 46, 48, 49, 52 (see also IARC (International Agency
toxicokinetic (TK) models, 36 for Research on Cancer))
US Environmental Protection Agency Wilson’s disease, 193, 194, 352
(EPA), 118, 379–381 Wine, 118
Uterine cancer, 386 Worst case scenarios, 43
Uterine tissues, effects of cadmium, 310, Wound healing, 195–197
311
X
V
Xenobiotic metal ions, 205–222, 225, 227,
Vaccination and autism, 292 228
Vanadium
Xylene see BTEX
carcinogenicity, 353, 390
effects on
development, 288
Y
reproductive system (male), 276
skin, 202–204 Yeast estrogen screen (YES), 312, 313
genotoxicity, 335, 353
oxidative stress, 18 Z
prenatal exposure, 288
as trace metal, 198, 203, 204 Zinc
in wine, 118 in cosmetics, 217t
Vegetables, leafy, 48, 135 effects on
Vegetarians, 47, 51 cardiovascular system, 99
Vineyard sprayer’s lung, 86, 87 hematological system, 147, 149, 151,
Vinyl chloride, 71, 75, 76 152
Virus immune system, 161, 169, 172, 175t
herpes simplex, 172 pulmonary system, 98, 99
cytomegalo-, 172 skin, 191, 192, 198, 199
encephalomyocarditis, 172 estrogenicity, 313
Vitamin B12, 198, 349 food contamination, 112
Vitamin C, 125, 126 interactions with
Vitamin supplements, 109, 110, 116, cadmium, 19–21, 137, 212, 226, 266,
117 307, 308
Volatile organic compounds (VOCs), 75 cadmium and lead, 138, 211
Met. Ions Life Sci. 2011, 8, 403–422

422 SUBJECT INDEX
[Zinc] [Zinc]
calcium, 193 silver, 192
chromium, 346 tin, 151, 203
copper, 147, 149, 151, 194 mechanisms of action, 169
iron, 195 and metallothionein, 19–21, 191, 192, 212
lead, 138, 151, 152, 251, 255, 256, 259, oxidation states, 98
274 Zinc fingers (ZF), 336, 337fig, 341, 349, 350
selenium, 226 Zirconium, 213–215, 217t, 224
Met. Ions Life Sci. 2011, 8, 403–422

Metal Ions in Toxicology: Effects, Interactions, Interdependencies

Uploaded by

Copyright:

Available Formats

You might also like

Metal Ions in Toxicology: Effects, Interactions, Interdependencies

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Metal Ions in Toxicology: Effects, Interactions, Interdependencies

Uploaded by

Copyright:

Available Formats

Volume METAL IONS

Metal Ions in Toxicology: Effects, Interactions,

About this Book

About the Series

About the Editors

Metal Ions in Toxicology:

Metal Ions in Toxicology:

r Royal Society of Chemistry 2011

All rights reserved

Published by The Royal Society of Chemistry,

Registered Charity Number 207890

For further information see our web site at www.rsc.org

The development of Biological Inorganic Chemistry during the past 40

Astrid Sigel, Helmut Sigel Roland K. O. Sigel

The preceding Volume 7, Organometallics in Environment and Toxicology, is

Are cadmium and other heavy metal compounds acting as endocrine

1 UNDERSTANDING COMBINED EFFECTS FOR METAL

2 HUMAN RISK ASSESSMENT OF HEAVY METALS:

3 MIXTURES AND THEIR RISK ASSESSMENT IN

4 METAL IONS AFFECTING THE PULMONARY AND

5 METAL IONS AFFECTING THE GASTROINTESTINAL

6 METAL IONS AFFECTING THE KIDNEY 133

9. Concluding Remarks and Future Directions 138

7 METAL IONS AFFECTING THE HEMATOLOGICAL

8 METAL IONS AFFECTING THE IMMUNE SYSTEM 157

9 METAL IONS AFFECTING THE SKIN AND EYES 187

2. Metal Ions and Metal Ion Gradients in the Physiology

10 METAL IONS AFFECTING THE NEUROLOGICAL

11 METAL IONS AFFECTING REPRODUCTION AND

12 ARE CADMIUM AND OTHER HEAVY METAL

13 GENOTOXICITY OF METAL IONS: CHEMICAL

14 METAL IONS IN HUMAN CANCER DEVELOPMENT 375

2. Known Human Metallic Carcinogens 380

SUBJECT INDEX 403

Numbers in parentheses indicate the pages on which the authors’

Henry G. Abadin Agency for Toxic Substances and Disease Registry

Rolf Altenburger Department of Bioanalytical Ecotoxicology, UFZ Helm-

Billy Amzal European Food Safety Authority, Unit on Food Con-

Pietro Apostoli Department of Experimental and Applied Medicine, Unit

Wojciech Bal Institute of Biochemistry and Biophysics, Polish Academy of

Lamia Benbrahim-Tallaa IARC Monographs Section, International

Ulla Bertelsen European Food Safety Authority, Unit on Food Con-

Alan R. Boobis Imperial College, Department of Experimental Medicine

Luisa R. Bordajandi European Food Safety Authority, Unit on Food

Anna F. Castoldi European Food Safety Authority, Unit on Food Con-

Simona Catalani Department of Experimental and Applied Medicine,

Massimo Corradi Department of Clinical Medicine, Nephrology and

Eugenia Dogliotti Istituto Superiore di Sanità, Viale Regina Elena, 299,

Jean-Lou C. M. Dorne European Food Safety Authority, Unit on Food

Mari Eskola European Food Safety Authority, Unit on Food Contaminants,

Stefan Fabiansson European Food Safety Authority, Unit on Food Con-

Pietro Ferrari European Food Safety Authority, Unit on Food Con-

Bruce A. Fowler Agency for Toxic Substances and Disease Registry

Peter Fuerst Chemical and Veterinary Analytical Institute, Muensterland-

Hugh Hansen Agency for Toxic Substances and Disease Registry

Claudia Heppner European Food Safety Authority, Unit on Food Con-

Kazimierz Kasprzak Laboratory for Comparative Carcinogenesis,

George E. N. Kass European Food Safety Authority, Unit on Food