NATIONAL ENVIRONMENTAL HEALTH FORUM

Copper

National Environmental Health Monographs

Metal Series No. 3

1997

Copper
Report of an International Meeting 20-21 June 1996 Brisbane

Edited by Michael R Moore, Paula Imray, Charles Dameron, Phil Callan, Andrew Langley and Sam Mangas

National Environmental Health Forum Monographs Metal Series No. 3

National Environmental Health Forum

1997

Copyright 1997 Department of Human Services Printed by Openbook Publishers. Published by the National Environmental Health Forum. Prepared for publication by Sam Mangas Public and Environmental Health Service Department of Human Services

Price available on application. Minor amendments have been made to pages 8 and 9. The list of 'Published Monographs' has been updated and a disclaimer has been included.

National Library of Australia Cataloguing-in-Publication Copper: report of an international meeting 20-21 June 1996, Brisbane. ISBN 0 642 28009 6. ISSN 1327-4775 1. Copper - Physiological effect - Congresses. 2. Copper - Environmental aspects - Congresses. 3. Copper - Toxicology - Congresses. I. Moore, M. R. (Michael R.). II. National Environmental Health Forum. (Series: National Environmental Health Forum monographs. Metal series ; no. 3). 572.518

Copper

Contents
Preface Acknowledgements Disclaimer Published monographs Copper - A Primer 1. History and background 2. Exposure 3. Homeostasis 4. Copper deficiency 5. Excess of copper 6. Copper and zinc - co-homeostasis Health significance of copper Genetic disorders of copper metabolism and the dual nature of copper in biology 1. Introduction 2. The essential nature of copper 2.1 Nutritional copper deficiency 2.2. Menkes disease and its variants 2.3 The mottled mice 3. The toxic nature of copper 3.1 Excess copper disposed of by biliary excretion 3.2 Wilson disease 3.3 Animal models of WD 4. Molecular and cellular basis of copper transport 4.1 Structural and function of MNK and WND 4.2 Copper resistance is acquired by amplification of MNK 4.3 Do metallothioneins protect against copper toxicity? 5. Conclusions Copper in the Aquatic Environment 1. Analysis of copper 2. Chemical speciation 3. Copper bioavailability and toxicity 4. Water quality criteria and standards 5. Aquatic sediments 6. Conclusions 7 7 7 8 9 9 9 10 10 11 11 12 18 18 19 19 20 22 23 23 23 24 24 24 26 27 27 32 32 33 35 37 38 39

Copper

Evaluation of copper guideline values for drinking-water 1. Introduction 2. Historical perspective 2.1 Aesthetics-based guideline values 2.2 Health-based guideline values 3. Derivation of health-based guideline values 3.1 US Environmental Protection Agency 3.2 World Health Organisation 3.3 Australia and New Zealand 3.4 European Commission 4. Overall evaluation Population exposure to copper in drinking water Abstract 1. Introduction 2. Factors that affect the population exposure to copper in drinking water 2.1 Water composition 2.2 Stagnant contact time 2.3 Age of copper piping 2.4 The design and installation procedures 2.5 The use of copper in the distribution network 2.6 Drinking habits of the population 3 Analysis of water utility monitoring data of copper in drinking water in the USA 4. Conclusions Risk assessment for essential trace elements: A proposed methodology 1. Introduction 2. Risk assessment and IPCS 2.1 IPCS goals in risk assessment methodology 2.2. Concepts and definitions 2.3 Development of tolerable intakes for non-essential chemicals 3. Development of recommended safe and adequate daily dietary intakes 3.1 Comparison of methodologies for derivation of RDAs and TIs 4. Principles and methods for assessing human health risks from exposure to an ETE 4.1 Scientific principles 4.2 The AROI concept in human health risk assessment - A proposed methodology 5. The Deficiency - Toxicity (DT) Model in Environmental Risk Assessment 6. Concluding remarks

43 43 43 44 44 44 44 45 46 46 46 48 48 49 49 49 49 49 50 50 50 50 53 54 54 55 55 55 58 58 58 59 59 62 65 65

Copper

Assessment of the requirement of copper in the nutritional support of the severely ill patient 1. Copper deficiency and treatment 2. Biochemical assessment 2.1 Plasma copper 2.2 Monitoring of neonates 2.3 Acute phase plasma protein effect 2.4 Action limits 2.5 Contra-indication for copper supplementation 2.6 Copper isotope tracer studies 3. Summary and conclusions Copper tailing impacts in coastal ecosystems of Northern Chile: From species to community responses 1. Introduction 2. Study sites and methods 3. Results and discussion 3.1 Copper concentration in sea water and Enteromorpha compressa 3.2 Rocky intertidal species richness future perspectives Wilsons Disease after cloning of the gene 1. Genetics 2. DNA-based diagnosis 3. Pathogenesis 4. Clinical features 5. Hepatic manifestations 6. Hepatic pathology 7. Laboratory diagnosis of Wilson's Disease 7.1 Serum ceruloplasmin 7.2 Urinary copper excretion 7.3 Hepatic copper concentration 7.4 Incorporation of orally administered radiocopper into ceruloplasmin 7.5 Abnormal imaging 8. Diagnostic screening 9. Treatment 9.1 Diet 9.2 Pharmacologic therapy 9.3 Long-term management 9.4 Liver transplantation 3.3 Enteromorpha compressa as a biological model: current status of knowledge and

67 68 68 68 69 69 69 69 70 70 71 71 72 73 73 74 77 81 81 82 82 83 83 84 85 85 86 86 86 87 87 87 87 87 89 89

Copper

Indian Childhood Cirrhosis (ICC) - Revisited 1. Background 2. Copper and ICC 3. Clinical features 4. Diagnosis Workshop 1 - Copper and Health 1. Summary 2. Research recommendations Workshop 2 - Distribution and Metabolism 1. Summary Intracellular protection against copper toxicity in mammals 1. Introduction 2. General mechanisms for metal detoxification 3. Redundancy 4. Metalloregulation 5. The copper detoxification pathway 6. Copper detoxification mechanisms 6.1 Metallothioneins 6.2 Transcriptional regulation of metallothioneins 6.3 Cu-ATPases 6.4 Regulation of the human copper ATPases 7. Conclusion Workshop 3 - Copper and the Environment 1. Summary 2. Aquatic systems 3. Toxicity testing 4. Bioavailability 5. Terrestrial testing 6. Essentiality versus toxicity 7. Bioaccumulation 8. Conclusions and recommendations Participants in the international workshop on copper

91 91 91 92 93 94 94 95 96 96 97 97 97 98 98 99 100 100 100 101 102 103 105 105 105 105 106 106 106 107 107 108

Copper

Preface
The National Environmental Health Forum has been established by the Directors of Environmental Health from each State and Territory and the Commonwealth with a secretariat provided by the Commonwealth Department of Health and Family Services. The National Environmental Health Forum is publishing a range of monographs to give expert advice and guidance on a variety of important and topical environmental health matters. This publication is the third in the metals series. A list of published monographs appears on page viii. Acknowledgements This publication has been made possible by technical assistance and funding provided by each State and Territory Environmental Health Branch and the Commonwealth Department of Health and Family Services. Comment has been provided by reviewers from the Commonwealth, States and Territories. Graphic design and layout assistance have been provided by Sandra Sowerby, Environmental Health Branch, South Australian Health Commission. The South Australian Health Commission library has assisted research and cataloguing for the document. Financial assistance from the International Copper Association is gratefully acknowledged. Disclaimer The papers in these proceedings do not necessarily represent the views of the National Environmental Health Forum, or the Health Departments represented on the Forum, or of the authors. This document has been prepared in good faith exercising due care and attention. However, no representation or warranty, expressed or implied, is made as to the relevance, accuracy, completeness or fitness for purpose of this document in respect of any particular users circumstances. Users of this document should satisfy themselves concerning its application to, and where necessary seek expert advice about, their situation. The NEHF, its participants and the DHS shall not be liable to the purchaser or any other person or entity with respect to any liability, loss or damage caused or alleged to have been caused directly or indirectly by this publication.

Copper

Published monographs

Water Series 1. Guidance for the control of Legionella (1996) 2. Guidance on water quality for heated spas (1996) 3. Guidance on the use of rainwater tanks (1998) Soil Series 1. Health-based soil investigation levels, 2nd edition (1998) 2. Exposure scenarios and exposure settings, 2nd edition (1998) 3. Composite sampling (1996) Metal Series 1. Aluminium, 2nd edition (1998) 2. Zinc (1997) 3. Copper (1997) Air Series 1. Ozone (1997) 2. Benzene (1997) 3. Nitrogen Dioxide (1997) 4. Sulfur dioxide (1999) General Series 1. Pesticide use in schools and school grounds (1997) 2. Paint film components (1998) 3. Guidelines for the control of public health pests Lice, fleas, scabies, bird mites, bedbugs and ticks (1999) 4. National Standard for licensing pest management technicians (1999) Indigenous Environmental Health Series 1. Indigenous Environmental Health No. 1 (1999) Exposure Series 1. Child activity patterns for environmental exposure assessment in the home (1999) Counter Disaster series 1. Floods: An environmental health practitioner's emergency management guide (1999)

Copper

Copper - A Primer
Professor Michael R. Moore NHMRC National Research Centre for Environmental Toxicology The University of Queensland - Griffith University 1. History and background Mankind's use of copper stretches into the mists of antiquity and it probably fair to say that the utilisation of this metal in the bronze age, around 3,000 B.C. heralded the relentless process of development of civilisation as we know it. We continue to use this metal in very great quantities and because of its proximity to man there is the attendant possibility of toxic consequences associated with exposure to it. However, this is not necessarily the appropriate response in viewing the consequences of exposure to this element. Copper is an essential trace element and a co-factor in many enzymic reactions. It is for example, central to the operation of cytochrome oxidase, and haem synthesis (Tephly et al., 1978). In these circumstances the appropriate evaluation of an element of this sort is to consider the balance between its essentiality and the potential for it to become toxic, that is the mechanisms of homeostasis. 2. Exposure Copper exposure in the environment is inevitable. It is estimated that in excess of 75,000 tonnes of copper is released into the atmosphere annually of which a quarter is thought to come from natural sources, whilst the rest is of anthropogenic origin. Many soils contain copper and the copper contents can be supplemented through human activities, industrial and agricultural processes. In terms of bioavailability, it is likely that dissolved copper in water supplies present the most likely source of human exposure to the metal. In general however, copper content of potable water supplies tends to be low around 0.8 mg/L in Australia. Such low values can be greatly increased as the water supplies are soft and acidic. Copper is in general a micro-nutrient, essential for normal growth of lower organisms and higher mammals. Various authorities have suggested dietary allowances of between 1 and 3 mg/day in adults. The WHO has previously suggested that copper intake should not exceed 0.5 mg/kg/body weight. In view of the very large differences in these values, it is hard to escape the conclusion that in certain cultural circumstances, copper intake may well be sub-optimal. (Refer to table 1). Table 1: Mean dietary intake of copper in adults
Country
Australia: male, MB female, MB Denmark, DD Germany, DD Netherlands, DD New Zealand Norway, DD Sweden, MB United Kingdom: male female,TD USA: male, MB female, MB

Mean intake mg/day

1.9 2.2 1.2 0.9 1.5 1.0 1.2 1.6 1.2 1.2 0.9

% of subjects less than 2mg/day

79% 84%

Key: MB - Market basket, DD - Duplicate Diet, TD - Total Diet

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3. Homeostasis Copper is an essential element that can be toxic when exposures exceed physiological needs. This relationship is described by a U-shaped curve expressing risk of deficiency or excess with the central portion of the curve between the arms expressing the range of exposure that is related to optimal function (health). The relationship between intake and health is affected by physiological factors for maintenance of homeostasis and extrinsic factors that affect availability of copper. (Refer to figure 1).

Figure 1: Dose -Response Relationships -Typical responses of an individual exposed to Copper Copper Homeostasis. Essential elements show a U shaped curve lying between deficiency ultimately leading to death-and excess which is toxic and which may also lead to death. The relationship differs from that of toxic elements which are non-essential and show a characteristic sigmoidal curve starting in a region of no-effect and rising to the region of toxicity. The homeostatic model defines the principle of an acceptable range of exposures (acceptable range of oral intake, AROI) for an essential trace element like copper. In the acceptable range, it provides the substrates for expression of the genetic potential of the individual. Environmental levels of copper do not produce adverse effects among the general population or the environment. However, there are individuals or groups with imbalances in relation to other trace elements, or with disorders in homeostatic mechanisms that experience effects, of either deficiency or toxicity, from exposures within the acceptable range. These disorders may be acquired or of genetic origin. 4. Copper deficiency Clinical copper deficiency in adults is rarely found in the general population. However recent dietary surveys show that the mean population intake is suboptimal. In some regions of the world such as Europe and USA intakes are about 20% below the recommended levels. The health consequences of barely adequate intakes remain to be determined.

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Low birth weight infants are particularly at risk for deficiency. Frequent episodes of diarrhoea are another risk factor leading to copper deficiency. Copper deficiency commonly occurs during the recovery from protein energy malnutrition, since these infants grow rapidly and are usually fed diets that supply inadequate copper. Individuals taking supplements of zinc and ascorbic acid are at risk of developing copper deficiency and there are malabsorption states associated with copper deficiency. Patients receiving prolonged intravenous nutrient mixtures which lack sufficient copper may develop symptomatic evidence of copper deficiency. Menkes disease is a rare (~1:200,000) X-linked recessive disorder which results in a defect in the intestinal absorption of copper. This disorder leads to a severe, symptomatic, fatal deficiency state even at copper intakes above the AROI. Copper deficiency has been implicated as a possible risk factor in the pathogenesis of cardiovascular disease. When copper homeostatic control is defective and/or copper intake is excessive, copper toxicity may occur - this is an infrequent occurrence. 5. Excess of copper There is little doubt that acute toxicity associated with copper exposure is largely associated with instance of attempted suicide or accidental oral exposure to the metal. The features most commonly reported are metallic taste in the mouth, gastric pain, headache, nausea, dizziness and diarrhoea with massive gastrointestinal bleeding. In addition to these gastrointestinal effects, tachycardia respiratory difficulty, haematuria and liver and kidney failure have been reported. In general however, the more likely situation are the problems associated with copper deficiency which has been linked to anaemia neutropenia and bone abnormality. Where there is upset of copper homeostasis such as the changes associated with genetic conditions, there can be both resultant copper deficit and copper excess. The most common reason for copper accumulation is chronic liver disease and choleostasis but in these circumstances the accumulation of copper is an epiphenomenon associated with the inability of the liver to clear the metal and consequent accumulation in the tissues. 6. Copper and zinc - co-homeostasis Another reason for alteration in copper homeostasis is related to the co-absorption of the metal with other essential metals. The most common of these are the inter-relationships between increased zinc intake and copper deficiency. Disproportionate intake of zinc in relationship to copper can induce copper deficiency in humans. In these circumstances, there are increased physiological copper requirements. The early work in this area carried out by Reiser et al. (1987) involved copper deficiency produced experimentally in a young man with a diet containing 0.8 mg of copper per day. An interesting change observed in the subject was that of increases in plasma cholesterol and development of ventricular tachycardia which was reversed when the subject was given 4 mg of copper daily. In subsequent experiments by Reiser et al, there was evidence of cardiac dysfunction in 2 out of 24 men given an increased zinc diet which induced copper deficiency. There were some changes in standard markers of copper status such as plasma-copper ceruloplasmine and erythrocyte superoxide dismutase (Uauy et al., 1996). All of these measures normalised after copper supplementation (Uauy & Olivares, 1996). Copper deficiency induced by excessive quantities of zinc in the diet resembles deficiencies induced by diet low on copper. Because of this inhibitory effect of zinc or copper utilisation and in that respect it is interesting to note that there is a relationship between the mortality rate for coronary heart disease and the ratio of zinc to copper in cows milk (Klevay, 1975). It is obvious from this that copper homeostasis requires much clearer definition in respect both of its individual role in metabolism and the concurrent role that might be taken in its interaction with other ions. In particular the ratio between zinc and copper requires further investigation. Lifestyle issues in which the relationship between the internal dose of these metals in organs such as liver, should be related to the dietary ratios of these same metals to establish whether there are endpoints in the process which have clinical significance.
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Copper will continue to be used by man in the foreseeable future. It's physical properties and in particular its cost-effective ability to conduct electricity guarantees a continuing need for its use in our society. Much of the research at the current time would seem to suggest that our principal concerns about the metal should be directed towards the consequences of copper deficiency rather than to the effects of copper excess. The copper-zinc ratio is known to induce dyslipidaemia and thus studies on the cardiac consequences of this and indeed upon cardiac conduction need to be further investigated. This book presents the results of discussions of a workshop held in the National Research Centre for Environmental Toxicology prior to the IPCS Task Group Meeting on Copper. The papers presented are a synthesis of the current thinking on copper essentiality, toxicity and on the disease states associated with these. References Klevay, L. (1975), Coronary heart disease: The zinc/copper hypothesis, Am J Clin Nutr.; vol. 28, pp. 764-774. Reiser, S., Powell, A., Yang, C.Y. and Canary, J.J. (1987), Effect of copper intake on blood cholesterol and its lipoprotein deistribution in Men, Nutr Rep In., vol. 36, pp. 641-9. Tephly, T.R., Wagner, G., Sedman, R. and Piper, W. (1978), Effects of metals on heme biosynthesis and metabolism, Fed Proc., vol. 37, pp. 35-9. Uauy, R. and Olivares M. (1996), Copper nutrition in humans: essentiality and toxicity, Amer J Clin Nutr., vol. 63, no.5.

Health significance of copper

Dr Ricardo Uauy and Dr Manuel Olivares Institute of Nutrition and Food Technology University of Chile Criteria for essentiality of nutrients in humans include the relative composition of the element in the organism, the effects of removing the nutrient from diet on health, and finally the effect of restoring the nutrient in question. Following this approach we find that whole body copper content in the adult ranges from about 80 mg with a range of 50 to 120 mg. The organs with the highest concentration are liver and brain with 5.1 mg and 6.3 mg per gram of wet weight respectively (Cartwright and Wintrobe, 1964). The high liver content is interpreted to serve as a reserve, and the high content of copper in the basal ganglia of the brain is considered to be associated to its role in neurotransmitter metabolism. The other condition is deficiency disease associated with removing copper from the diet; this condition occurs in humans given copper-free parenteral nutrition. For example, parenterally fed subjects exhibit within a few weeks, anaemia and neutropenia unless copper is provided. These abnormalities are reversed by a treatment with copper. Copper is involved in the function of multiple important enzymes, most participate in redox reactions (Linder and Hazegh-Azam, 1996). Cytochrome-c-oxidase is a key enzyme for the electron transport chain in the mitochondria, superoxide dismutase has a role in scavenging of free radicals, lysyl oxidase is crucial for the cross linking of collagen and ceruloplasmin acts as a ferroxidase transforming iron from ferrous to ferric state, a step crucial for the transport of this mineral. Dopamine -monooxygenase is necessary for dopamine and catecholamine metabolism, and tyrosinase for the production of melanin, the skin and hair pigment. Copper is actively absorbed, primarily in the stomach and duodenum. Absorption ranges from 25-60% of intake depending on other dietary components and on the copper intake (Lnnerdal, 1996; Turnlund et al., 1989; Ehrenkranz et al., 1989). The percent of copper absorbed decreases as copper intake is increased (see Table 1), (Turnlund et al., 1989; Ehrenkranz et al., 1989). Zinc, iron, ascorbic acid, calcium and phosphorus, fibre, phytates, sucrose, fructose, molybdenum and cadmium have been
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demonstrated to inhibit copper absorption while animal protein, specifically the amino acid histidine, enhances it (Lnnerdal, 1996). Copper is transferred from the intestinal mucosa to portal blood complexed principally with albumin (Linder and Hazegh-Azam, 1996). Within the hepatocyte, it is bound to metallothionein, superoxide dismutase and other binding proteins (Luza and Speisky, 1996). Copper is secreted from the liver into the blood predominantly bound to ceruloplasmin or complexed with low molecular weight compounds. Cellular uptake in some tissues may be mediated by specific ceruloplasmin receptors on their cell surface (Linder and Hazegh-Azam, 1996). Biliary excretion is the main form of elimination in humans (Linder and Hazegh-Azam, 1996). The main dietary sources of copper are shellfish, fish, liver, meats, nuts and chocolate. A lower concentration is found in legumes, grains, human milk, and especially cow milk (Pennington et al., 1995). Table 1: Effect of dietary copper intake on percentage of copper absorption
Subjects Adults Copper intake (mg/d) 0.785 1.68 7.53 LBW infants Human milk fed Human milk fed Formula fed Formula fed LBW: low birth weight 86.6 28.2 151.7 34.4 188.9 39.4 194.2 27.5 67.2 14.6 57.4 13.1 39.3 21.9 26.5 6.9 59.7 13.6 38.7 10.2 15.4 20.0 20.6 24.1
65

Cu absorption (%) 55.6 0.9 36.3 1.3 12.4 0.9

Net Cu absorption(%) not available not available not available

Copper requirements have been established using different approaches. These include the clinical syndrome of copper deficiency requirement which in this case, is defined as the amount of copper necessary to cure the deficiency. It is also established by evaluating the effect of experimental diets with different copper intakes to determine the lowest intake required to prevent the development of biochemical or functional alterations. Another approach is based on epidemiological studies, measuring copper intake of healthy populations (Olivares and Uauy, 1996a). The large variability of copper intake in healthy populations makes it difficult to employ this methodology to define precise intakes. However this approach provides a reasonable range of values from which to select levels to be tested under controlled experimental studies. The US National Academy of Sciences has recommended that all adults should receive a daily intake of 1.5-3 mg of copper to satisfy physiological requirements (safe and adequate intake range). The corresponding values for infants, children and adolescents are shown in Table 2 (NRC, 1989). The WHO recommendation for infants is 80 g/kg per day (WHO, 1985). More recently the FAO/IAEA/WHO expert consultation has provided recommendations using new data from long term balance studies validated by biochemical markers associated with copper status (FAO/IAEA/WHO, 1996). These values are also included in Table 2. The FAO/IAEA/WHO recommendations for the first time suggest an upper value for acceptable dietary intake based on the very limited data available. The upper value of 0.2 mg per kg of body weight per day should be considered as a no observed effect level rather than a true NOAEL (No-Observed-Adverse-Effect-Level) (FAO/IAEA/WHO, 1996).

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Table 2: Estimated safe and adequate daily dietary copper intake (NRC, 1989) and safe minimum mean copper intake (FAO/IAEA/WHO, 1996)

Copper (mg/day) Age range (y) or state 0-0.25 0.25-0.5 0.5-1 1-3 3-6 6-10 10-12 12-15 15-18 18-60+ 10-12 12-15 15-18 18-60+ Pregnancy Lactation Sex M&F M&F M&F M&F M&F M&F F F F F M M M M NRC 0.4-0.6 0.4-0.6 0.6-0.7 0.7-1.0 1.0-1.5 1.0-2.0 1.5-2.5 1.5-2.5 1.5-2.5 1.5-3.0 1.5-2.5 1.5-2.5 1.5-2.5 1.5-3.0 FAO/IAEA/WHO 0.33-0.55 0.37-0.62 0.60 0.56 0.57 0.75 0.77 1.00 1.15 1.15 0.73 1.00 1.33 1.35 1.15 1.25

Various factors predispose to acquired copper deficiency. Deficit can be the consequence of decreased copper stores at birth, the best example of this is the low birth weight infant (Shaw, 1992). Most of copper accumulation in the fetus occurs during the third trimester of pregnancy, so a preterm newborn will have a lower hepatic copper content compared to a full term infant. In addition, there is a greater need for rapid growth, thus they easily become copper deficient unless extra copper is provided. Another cause of copper deficiency is an inadequate copper supply, this is a very common situation where cow milk and high carbohydrates feeds are given (Cordano et al., 1964). This combination is very frequently found during the recovery from infant malnutrition. Copper deficiency is also observed in cases of total parenteral nutrition with inadequate copper supplementation, or in malabsorption syndromes. Increased requirements are observed in premature or malnourished infants; increased faecal losses are principally observed in cases of chronic or prolonged episodes of diarrhoea (Olivares and Uauy, 1996b). The most frequent clinical manifestations of acquired copper deficiency are anaemia, neutropenia and bone abnormalities that include osteoporosis and fractures (Olivares and Uauy, 1996b). Less frequent signs are hypopigmentation of the hair, hypotonia, impaired growth, increased incidence of infections, alterations of phagocytic capacity of neutrophils, and immune cellular abnormalities. Menkes disease is a rare (1:200,000) X-linked recessive genetic disorder in which deficiency is secondary to a defect in cellular copper uptake and transport. In this condition there is a defect in a membrane protein necessary to transport copper out of the basolateral pole of the intestinal cell (Harris and Gitlin, 1996). This disease is characterised by progressive mental deterioration, hypothermia, skin and hair depigmentation, growth retardation, bone and connective tissue abnormalities (Harris and Gitlin, 1996). Toxicity to copper may occur from eating food, drinking water or breathing air with excessive copper content. A small amount of copper may enter the body by skin contact with copper-containing
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substances. The oral route is the main pathway of exposure to the element. Food and water are the predominant sources of copper intake. Food may account for over 90% of copper intake in adults if water has low copper content (<0.1 mg/L). If water copper content is higher (1-2 mg/L) it may account for up to 50% of total intake. In infants consuming copper supplemented artificial formula, the contribution of water may be less than 10% whereas, if the formula is not fortified with copper, water may contribute over 50% of total copper intake, especially when water copper content is 1-2 mg/L. Acute copper toxicity is infrequent in man, and usually is a consequence of ingesting contaminated foodstuffs or beverages (including drinking water), and from accidental or voluntary ingestion of high quantities of copper salts. Acute symptoms include salivation, epigastric pain, nausea, vomiting and diarrhoea (USEPA, 1985; USEPA, 1987; NIPHEP, 1989). Intravascular haemolytic anaemia, acute liver failure, acute renal failure with tubular damage, shock, coma and death have been observed in severe copper poisoning. There are some reports in humans, suggesting that the consumption of beverages or drinking water contaminated with copper results in nausea, vomiting, and diarrhoea (Wyllie, 1957; Spitalny et al., 1984; Knobeloch et al., 1994). The long term toxicity of copper has been less studied. Chronic toxicity in humans is observed principally in patients with Wilsons disease and from the occurrence of infantile cirrhosis in areas of India (ICC, Indian childhood cirrhosis), and isolated clusters of cases in Germany, Austria and other countries (ICT, idiopathic copper toxicosis) that have been also related to excessive copper intake (Pandit and Bhave 1996, Weiss et al., 1989, Adamson et al., 1992, Horslen et al., 1994). Wilsons disease is an autosomal recessive genetic disorder (1:30,000) characterised by a defect in copper biliary excretion. In this condition the accumulation of copper in liver and brain is associated with altered structure and function of these organs (Harris and Gitlin, 1996; Scheinberg and Sternlieb, 1996). Copper-associated infantile cirrhosis is an extremely rare condition; for ICT the estimated incidence based on prospective data from Germany is 1:500,000 to 1:1,000,000. The data for ICC from India reveal a dramatic decline of this condition. Recent observations from Pune district based on hospital admissions reveal a drop from 47 cases per year in 1980-1983 to 2 cases in 1992-1993 (Pandit and Bhave, 1996). ICC and ICT have been linked to the use of copper containers for storage or heating of infant formula or to the high copper content of well water conducted through corroded copper pipes. In the cases reported in India, copper intake was over 900 g/kg/day, which is over ten times the daily requirement recommended by WHO (ONeill and Tanner, 1989). However, some cases of cirrhosis reported in India, and in other countries have occurred in breast-fed infants or despite the virtual absence of copper in the infants drinking water suggesting an inherited metabolic defect as a cause for this condition (Horslen et al., 1994). Furthermore, an epidemiological study performed in seven towns of Massachusetts, with copper concentrations in the drinking water ranging from 8.4 - 8.8 mg/L, did not show any deaths due to liver disease (Scheinberg and Sternlieb, 1994). A recent publication which systematically evaluated the association of infantile liver disease and copper content of drinking water in the UK did not reveal a connection between these variables (Fewtrell et al., 1996). The familial occurrence and consanguinity in the parents of patients in some cases of infantile cirrhosis strongly suggest a genetic disorder as the aetiology of this disease. A recent report from the province of Tyrol, Austria, reviewed data from 138 infants and young children dying from infantile liver cirrhosis during this century (Mller et al., 1996). Clinical features of the cases were indistinguishable from ICT or ICC, and pedigree analysis of the affected families indicated that the susceptibility to this disease was inherited as an autosomal recessive gene. The sex ratio was 0.5 and parental consanguinity increased the risk. Segregation analysis based on a total of 343 children and 112 cases of childhood cirrhosis favoured a genetic factor in the aetiology of the disease. The evaluation of infant and childhood feeding practices indicated that untinned copper and brass cooking utensils contributed to the development of this disease by providing extremely high dietary copper intakes. The replacement of copper cooking utensils by modern non-copper pots occurring in the past decades has eradicated the disease, such that no cases have been diagnosed after 1974.
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In conclusion, ICC/ICT is a disease of unknown aetiology. The most likely explanation for this condition appears to be a combination of a genetically determined defect in copper metabolism and a high copper intake. The relative contribution of each factor remains to be determined. The range of acceptable intake to prevent copper deficiency and toxicity should be based on the protection of healthy populations and should not be expected to meet requirements or prevent excess of special individuals. Disease conditions or genetic alterations in copper metabolism that determine special sensitivity for excess or deficit deserve the attention of public health authorities based on the relevance of these conditions within a given ecological setting. The upper and lower cut-off points for the range of acceptable oral intakes should be defined using a population-based model for the assessment of health risks associated to deficiency or excess. The lower cut-off point should be sufficient to meet the requirements of most individuals in the population. Similarly the upper end point should protect most individuals from the risk of toxicity (see figure 1). A detailed presentation of this risk assessment model as applicable to essential elements is presented in Dr. Beckings chapter (Becking, 1996).

Figure 1: Model to illustrate distribution of individual copper requirements and toxicity in a normal population and sub groups with genetic abnormalities in copper metabolism. ICC is Indian Childhood Cirrhosis, ICT is Idiopathic Copper Toxicosis. The upper and lower cut-off points for the range of acceptable oral intakes are defined using the population distributions of requirements and toxicity. The lower cut-off point is sufficient to meet the requirements of most individuals in the population while the upper limit prevents toxicit in most individuals.

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References Adamsom, M., Reiner, B., Olson J.L. et al. (1992), Indian childhood cirrhosis in an American child, Gastroenterology, vol. 102, pp. 1771-1777. Becking, G.C. (1996), Risk Assessment for Essential Trace Elements: A Proposed Methodology in press. Cartwright, G.E. and Wintrobe, M.M. (1964), Copper metabolism in normal subjects, Am J Clin Nutr., vol. 14, pp. 224-232. Cordano, A., Baertl, J. and Graham, G.G. (1964), Copper deficiency in infants, Pediatrics, vol. 34, pp. 324-336. Ehrenkranz, R.A., Gettner, P.A., Nelli, C.M., Sherwonit, E.A., Williams, J.E., Ting, B.T.G. and Janghorbani, M. (1989), Zinc and copper nutritional studies in very low birth weight infants: comparison of stable isotopic extrinsic tag and chemical balance methods, Pediatr Res., vol. 26, pp. 298-307. FAO/IAEA/WHO. (1996), Copper In: Trace elements in human nutrition and health, WHO (Geneva), pp. 123-143. Fewtrell, L., Kay, D., Jones, F., Baker, A. and Mowat, A. (1996), Copper in drinking water - an investigation into possible health effects, Public Health, vol. 110, pp. 175-177. Harris, Z.L. and Gitlin J.D. (1996), Genetic and molecular basis for copper toxicity, Am J Clin Nutr., vol. 63, pp. 836S-41S. Horslen, S.P., Tanner, M.S., Lyon, T.D.B., Fell, G.S. and Lowry, M.F. (1994), Copper associated childhood cirrhosis, Gut, vol. 35, pp. 1497-1500. Knobeloch, L., Ziarnik, M., Howard ,J., Theis, B., Farmer, D., Anderson, H. and Proctor, M. (1994), Gastrointestinal upsets associated with ingestion of copper-contaminated water, Environ Health Perspect ., vol. 102, pp. 958-961. Linder, M.C. and Hazegh-Azam, M. (1996), Copper biochemistry and molecular biology, Am J Clin Nutr., vol. 63, pp. 797S-811S. Lnnerdal, B. (1996), Bioavailability of copper, Am J Clin Nutr., vol. 63, pp. 821S-9S. Luza, S.C. and Speisky, H. (1996), Liver copper storage and transport during development: implications for cytotoxicity, Am J Clin Nutr, vol. 63, pp. 812S-20S. Mller, T., Feichtinger, H., Berger, H. and Mller, W. (1996), Endemic Tyrolean cirrhosis: an ecogenetic disorder, Lancet, vol. 347, pp. 877-80. National Institute of Public Health and Environmental Protection (NIPHEP). (1989), Integrated criteria document copper, Appendix to report N 758474009. The Netherlands, Bilthoven: National Institute of Public Health and Environmental Protection. National Research Council (NRC).(1989), Recommended dietary allowances,10th edn, Washington, DC, National Academy Press, pp. 224-30. O'Neill, N.C. and Tanner, M.S. (1989), Uptake of copper from brass vessels by bovine milk and its relevance to Indian childhood cirrhosis, J Pediatr Gastroenterol Nutr., vol. 9, pp. 167-172. Olivares, M. and Uauy, R. (1996a), Limits of metabolic tolerance to copper and biological basis for present recommendations and regulations, Am J Clin Nutr., vol. 63, pp. 846S-52S. Olivares, M. and Uauy, R. (1996b), Copper as an essential nutrient, Am J Clin Nutr., vol. 63, pp. 791S-6S. Pandit, A. and Bhave, S. (1996), Present interpretation of the role of copper in Indian childhood cirrhosis, Am J Clin Nutr., vol. 63, pp. 830S-5S.

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Pennington, J.A.T., Schoen, S.A., Salmon, G.D., Young, B, Johnson, R.D. and Marts, R.W. (1995), Composition of core foods of the US food supply, 1982-1991. III. Copper, manganese, selenium, and iodine, J Food Compos Anal., vol. 8, pp. 171-217. Scheinberg, I.H. and Sternlieb I. (1994), Is non-Indian childhood cirrhosis caused by excess dietary copper?, Lancet, vol. 344, pp.1002-1004. Scheinberg, I.H. and Sternlieb I.(1996), Wilson disease and idiopathic copper toxicosis, Am J Clin Nutr., vol. 63, pp. 842S-5S. Shaw, J.L.C. (1992), Copper deficiency in term and preterm infants. In: Fomon SJ, Zlotkin S, eds. Nutritional anemias. Nestl Nutrition Workshop Series. Vol. 30. New York: Raven Press, pp. 105-19. Spitalny, K.C., Brondum, J., Vogt, R.L., Sargent, H.E. and Kappel, S. (1984), Drinking waterinduced intoxication in a Vermont family, Pediatr., vol.74, pp. 1103-1106. Turnlund, J.R., Keyes, W.R, Anderson, H.L. and Acord, L.L. (1989), Copper absorption and retention in young men at three levels of dietary copper by use of the stable isotope 65Cu, Am J Clin Nutr., vol. 49, pp. 870-8. US Environmental Protection Agency (USEPA).(1985), Drinking water criteria document for copper (final draft). Environmental Criteria and Assessment Office. Cincinnati, OH: Environmental Protection Agency, EPA 600/X-84/190-1. US Environmental Protection Agency (USEPA). (1987), Summary review of the health effects associated with copper. Environmental Criteria and Assessment Office. Cincinnati, OH: Environmental Protection Agency, EPA 600/8-87/001. Weiss, M., Mller-Hcker, J., Wiebecke, B. and Belohradsky, B.H. (1989), First description of Indian childhood cirrhosis in a non-Indian infant in Europe, Acta Paediatr Scand ., vol. 79, pp. 152156. WHO Report of an Expert Committee. (1985), Trace elements in human nutrition. World Health Organ Tech Rep Ser 724. Wyllie, J. (1957), Copper poisoning at a cocktail party, Am J Public Health, vol. 47, p. 617.

Genetic disorders of copper metabolism and the dual nature of copper in biology
Julian F.B. Mercer Murdoch Institute, Royal Childrens Hospital, Victoria, Australia 1. Introduction Copper is an essential element which is used as a cofactor for a number of important enzymes including cytochrome-c oxidase, the last enzyme in the electron transport chain. Excess copper is, however, toxic to cells and this dual nature of copper, essential yet toxic, must have required the development of a tightly regulated system of copper homeostasis. Since cytochrome-c oxidase is an enzyme that has been used by all aerobic organisms, the use of copper is an early event in evolution, and one might expect that some of the molecules involved in copper transport would be well conserved. The essentiality and toxicity of copper are graphically demonstrated in two genetic disorders of copper transport. Menkes disease (MD) is an X-linked recessive condition, which causes death in early childhood from copper deficiency. Wilson disease (WD) on the other hand is a copper toxicosis disorder characterised by massive copper accumulation in the liver, with consequent aberrant release of copper and damage to the central nervous system (Danks, 1995). Despite the differences in
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phenotype, these diseases are caused by mutations in closely related genes which encode copper ATPases (Mercer et al., 1993; Chelly et al., 1993; Vulpe et al., 1993; Bull et al., 1993; Tanzi et al., 1993; Yamaguchi et al., 1993). From the structure of the predicted proteins and the pattern of copper disturbance in the two diseases, it appears likely that both function as copper efflux molecules. Very similar molecules have been found to be copper transporters in yeast and bacteria (Fu et al., 1995; Rad et al., 1994; Odermatt et al., 1993). Current research in this area is aimed at elucidating the role of these novel Cu-ATPases in copper transport and the types of mutations which cause the MD and WD. The features shown by these extreme phenotypes are yielding valuable insights into the normal processes of copper requirements and protective mechanism against copper toxicity. This paper will review some of the information coming from these molecular studies and attempt to relate some of this information with older studies on the effects of copper deficiency and toxicity. 2. The essential nature of copper 2.1 Nutritional copper deficiency

Copper was first established as an essential nutrient in the 1920s and 1930s from studies of laboratory and farm animals. Many of the features found in these copper deficient animals can be understood in terms of the reduction in the activity of various copper dependent enzymes, but there are still aspects of copper deficiency that are not fully understood. One of the most sensitive indicators of copper deficiency in animals is the hypopigmentation associated with reduced activity of tyrosinase, a copper dependent enzyme required for melanin formation. In sheep, alternate bands of pigmentation in wool can be produced by adding or withdrawing copper from the diet (Underwood, 1977). Another effect of copper deficiency in sheep is the production of steely wool, that is, wool which has lost the natural crimp. This is important for wool producers since steely wool has low tensile strength and reduced elasticity (Underwood, 1977). Steely hair is a characteristic feature of children with Menkes disease (see below). Copper is required for the normal cross-linking of the sulphydryl groups of keratin, but no enzyme has been associated with this process. Neurological problems are a feature of copper deficiency in developing animals and are pronounced feature of MD. Neonatal ataxia is a defect of myelination found in copper deficient lambs and is also known as swayback. The disease is directly due to deficient myelination in the spinal chord, and there are also lesions in parts of the cerebral white matter and brain stem (Smith et al., 1977). A likely cause of the demylenation is a deficiency in cytochrome-c oxidase. Deficiency of the copper dependent enzyme, lysyl oxidase, causes defective cross linking of collagen and elastin leading to abnormalities of skin, cartilage, arteries and bone. Various degrees of connective tissue abnormalities are found in MD and its variants, and it appears that these genetic defects can produce a more specific connective tissue phenotype than nutritional deficiency. Possible reasons for this will be discussed below. Iron-unresponsive anaemia and neutropenia occur in severe copper deficiency, and part of the explanation is that the copper oxidase, ceruloplasmin is required for oxidation of Fe2+ to Fe3+ for transferrin binding. For example, when plasma ceruloplasmin level fell below 1% of normal in copper deficient pigs, the cell to plasma iron flow was impaired and cellular iron accumulated (Roeser et al., 1970). More recently patients with a genetic disorder aceruloplasminaemia, have a been shown to develop an iron disorder, haemosiderosis (Harris et al., 1995). It has been more difficult to establish the effects of Cu deficiency in humans than in animals, and indeed the very possibility of Cu deficiency in humans was debated in the 1950s and 1960s. As discussed below, the severe effects of Cu deficiency brought about by the genetic defect in MD provided decisive evidence of the effects of copper deficiency in humans. Severe copper deficiency has also been observed in patients receiving total parenteral nutrition with inadequate copper content
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(Karpel and Peden, 1972). The question of whether mild chronic copper deficiency is a problem in humans is still unresolved, and is difficult to answer because of the lack of reliable indicators of marginal copper status (Milne, 1994). 2.2. Menkes disease and its variants

MD is the most severe form of a spectrum of genetic copper deficiency conditions, which all result from mutations in the Menkes disease gene (MNK). Children with MD are copper deficient at birth, because of defective placental transfer of copper and continue to suffer from the effects of the deficiency, since the transfer of copper across the small intestine is very inefficient. The physiological effects of the reduced copper intake are exacerbated by the fact that MNK is required for the transport of copper across epithelial cellular layers and within cellular organelles of most cell types. Hepatocytes are the exception, for in these cells the function of MNK is fulfilled by WND, the protein affected in Wilson disease. As illustrated in Fig 1a, MNK is needed to pump copper from the small intestine into the body. Since this step is blocked, copper accumulates in the mucosal epithelial cells, bound to metallothioneins (MT). MNK is also required to transport copper across the blood brain barrier (Fig 1b), effectively imposing a second block to copper transport at a period of development when the developing brain is especially sensitive to the lack of copper. It is likely that the resulting deficiency of cytochrome oxidase is instrumental in producing the profound neurological defects in this disease; in neonatal mice deficiency of copper has long lasting effects on cytochrome oxidase and some other cuproenzymes in the brain (Prohaska and Bailey, 1993). (In this chapter MNK is used for the gene affected in MD and MNK for its protein product. MNK is also known as ATP7a. WND is used for the gene affected in Wilson disease and WND for the protein. WND is also known as ATP7b).

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Figure 1: Transport of copper out of most cells requires the Menkes gene product, MNK.
(a) In polarized epithelial cells, such as the intestinal enterocytes and the proximal tubules of the kidney, MNK possibly effluxes copper across the basolateral surface of the cell. This process is blocked in MD and copper accumulates in the cell bound to metallothioneins (MT). (b) MNK is needed to efflux copper from the vascular endothelial cells, e.g. (1) into the brain and into capillaries (2) following absorption from the diet. (c) Cultured fibroblasts from MD patients accumulate copper since efflux of copper is defective. Also MNK is required to transport copper into the Golgi for incorporation into the copper dependent enzyme lysyl oxidase, but cytoplasmic enzymes such as superoxide dismutase (SOD) can receive copper directly.
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Paradoxically, copper levels in some organs are elevated in MD patients, for example the kidney copper concentrations are about 5 fold normal. The convoluted tubules in the kidney are involved in copper resorption from the urine and MNK is probably needed to pump copper across the basolateral surface back into the circulation (Fig 1a). When MNK is absent, the copper continues to be absorbed across the apical surface but is trapped in the cell. Fibroblasts cultured from MD patients also accumulate copper and this has been shown to be due to defective efflux (Fig 1c). Measurement of copper efflux from amniotic cells is a reliable method of prenatal diagnosis of MD (Tonneson and Horn 1989). The mildest mutant allele of MNK causes a disease termed occipital horn syndrome (OHS) alternatively known as cutis laxa. OHS is primarily a connective tissue disorder, due to low activity of the copper dependent enzyme, lysyl oxidase (Byers et al., 1980). The neurological defects that are so apparent in MD are only present to a mild degree in this disorder. The prominence of the connective tissue defects suggests that lysyl oxidase is particularly sensitive to the lack of MNK. An explanation for this is that MNK is needed for the transport of copper across an intracellular organelle in which lysyl oxidase receives copper (Das et al., 1995). This may occur in the trans Golgi network and this is where MNK is found in copper resistant CHO cells (Fig 1c and see below). The requirement for an additional MNK-dependent step may render lysyl oxidase very sensitive to reduction in MNK activity (Das et al., 1995). An intermediate phenotype, termed mild Menkes, has more severe mental retardation, but patients survive the critical neonatal period (Danks,1988). The reason for the different phenotypes arising from mutations of MNK is not fully understood, but molecular analysis of the mutations in the various diseases has shown that MD arises when there is little or no activity of MNK. The types of mutations causing MD are deletions of the MNK gene, premature stop codons, major splice site mutations and serious missense mutations (Das et al., 1994). The milder disorders OHS and mild Menkes have been shown to result from less severe splice site mutations which allow some normal MNK mRNA, and presumably protein, to be formed (Das et al., 1995; Kaler et al., 1994). It is possible as with a number of inborn errors of metabolism, that residual enzyme activity of greater than about 5% allows sufficient copper to be transported to permit near normal development. 2.3 The mottled mice

Mutations of the X-linked mottled (Mo) gene in mice produce a range of phenotypes which include close homologues to MD and OHS all with defects of copper transport (Lyon and Searle 1990). The brindled mouse (Mobr) has features closely resembling those seen in MD; the affected male dies at about 14 days after birth, a similar developmental stage at which boys with MD usually die. Features include hypopigmented coat, curly whiskers (like the hair defect in Menkes), connective tissue abnormalities and severe neurological defects. The tissue copper levels are abnormal and like MD patients, copper concentrations are elevated in the small intestine and kidney but the liver and brain are markedly copper deficient (Mercer et al., 1991; Camarakis 1979). The blotchy mutant (Moblo) resembles occipital horn syndrome patients since the principal defects are in the connective tissue, and the mouse survives to adult life but dies prematurely from aortic aneurysms, and has a marked lysyl oxidase deficiency (Rowe et al., 1977). An interesting severe mottled variant is the dappled mutant (Modp) which dies during fetal development (Phillips, 1961). We have been characterising a similar severe allele Mo9H, in which fetal death appears to be caused by connective tissue failure. Multiple fetal defects have been found ranging from abdominal wall rupture , vascular abnormalities and even the necrosis of the entire hind quarter of the fetus (Ambrosini and Mercer, unpublished data). There appears to be no human equivalent of these fetal lethal mutants. Despite the variable phenotypes, it appears likely that all the mottled mice arise from mutations of the Menkes gene homologue and show the range of defects that copper deficiency can produce in the mouse (Levinson et al., 1994, Mercer et al 1994). Fetuses affected by the severe dappled mutation express little or no MNK mRNA, like patients with MD, but for unknown reasons the absence of
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MNK in the mouse results in a more severe phenotype (ie. foetal death) than in humans. The blotchy mouse has a mild splice site mutation of exactly the type reported in OHS patients (Das et al., 1995), thus confirming that milder defects result in predominantly connective tissue abnormalities. We have recently identified a two amino acid deletion in the brindled mouse MNK, in a highly conserved, but not well studied region of MNK (Grimes et al., 1997). Such a mutation is likely to allow production of normal amounts of protein with a greatly reduced activity, and Western blot data using an antiserum specific for MNK have demonstrated that the mutant does have normal amounts of MNK in the kidney. 3. The toxic nature of copper 3.1 Excess copper disposed of by biliary excretion

Oral copper is relatively non-toxic for humans and most animals, although there are marked differences in copper homeostasis between species. Neonatal animals are thought to be more sensitive to excess copper intake. The disposal of excess dietary copper is achieved by regulating the rate of biliary excretion, which is the principal way copper is removed from the body (Evans, 1973). Sheep store large amounts of copper in the liver and are particularly sensitive to copper toxicosis since, unlike most other species, the rate of copper excretion in the bile is not influenced by the concentration of copper in the liver (Danks, 1995). On normal pastures, without excess molybdenum, which is an effective antagonist of copper uptake, sheep will gradually accumulate copper. This is similar to the hepatic accumulation of copper in Wilson disease (Danks, 1995). Some breeds of sheep are avid copper accumulators and will develop copper toxicosis even on normal pastures (MacLachlan and Johnston, 1982). Rodents represent the other extreme of copper adaptation; rats and mice efficiently excrete copper and very high dietary intakes are needed to elevate hepatic copper levels. For example rats need to consume diets containing about 300 times the standard level of copper for several months to raise hepatic concentrations to levels similar to those found in Wilson disease (Haywood 1985). 3.2 Wilson disease

In patients with Wilson disease (WD) the biliary excretion of copper is greatly reduced, and since copper absorption from the diet is unaffected, liver copper levels steadily rise until hepatocytes are killed and the patient enters a phase of terminal liver failure (Danks, 1995). Interestingly a variant form of the disease can present as a neurological condition; copper is thought to be released from necrotic hepatocytes and deposits in the central nervous system, ultimately causing neurological damage, presumably by the promotion of free radical damage to the neurones. The molecular basis of WD has been shown to be a defective copper ATPase, WND, closely related to MNK (Bull et al., 1993; Tanzi et al., 1993). Both molecules have the characteristics of P-type ATPases and have the interesting cluster of copper binding sites in the N-terminal region (see below). It is thought that WND will also be a copper efflux molecule, functioning to deliver copper to the bile perhaps across the biliary cannalicular membrane (see Fig. 3); Since ceruloplasmin levels are low in most patients with WD, WND probably also pumps copper into an intracellular organ (possibly the Golgi apparatus) for incorporation into ceruloplasmin, although this conclusion has been recently disputed (Chowrimootoo 1996). Neurological damage in two other human diseases may involve copper. The genetic disorder, amyotrophic lateral sclerosis, a neurodegenerative condition, has been shown in some cases to be due to mutant forms of copper/zinc superoxide dismutase, which may actually increase free radical formation (Wiedau-Pazos et al., 1996). More recently copper has been implicated as playing a role in the development of Alzheimers disease (Multhaup, 1996), so there is clearly a lot more to learn about the toxic potential of copper in biology.

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3.3

Animal models of WD

As noted above, sheep have very low biliary excretion of copper and accumulate very high levels in the liver, much as is found in WD. This characteristic of sheep possibly represents an adaptation of a species to chronically low copper intakes, the accumulated hepatic copper acting as a reserve when supplies are limited (Weber, 1980). This tendency can be a disadvantage under modern agricultural conditions when copper supplies are adequate leading to copper toxicosis (MacLachlan and Johnston, 1982). The molecular basis of this copper accumulation tendency has not been established. Another animal model of WD is a breed of dog, the Bedlington Terrier, which commonly develops a copper toxicosis with many of the features of WD (Su et al., 1982). Brewer has studied the linkage of DNA markers to copper toxicosis and has concluded that the affected gene is unlikely to be WND. If this proves to be correct then it may suggest that another gene is involved in the hepatic metabolism of copper (Yuzbasiyan, 1993). The mutant LEC rat, has been recently shown to have a partial deletion of the WND homologue, thus establishing it as the first true animal WD model (Wu et al., 1994). Copper accumulates in the liver of the mutant rat due a defect of biliary excretion (Sugawara et al., 1993) and is associated mainly with metallothioneins (Suzuki, 1995). In common with patients with WD, incorporation of copper into ceruloplasmin in the Golgi apparatus is greatly reduced in the mutant (Murata, 1995). The mutant has the unusual feature of developing a high frequency of hepatitis and hepatomas (Fujimoto, 1989), which has been attributed to the high copper concentration in the liver, but recently the tendency to develop hepatomas has been shown to segregate independently of the copper accumulation phenotype (Hattori, 1995). We have been studying a mouse model of WD which has been termed the toxic milk mouse, tx (Rauch, 1983). The mutant mouse accumulates high levels of hepatic copper associated with metallothioneins and the pattern of accumulation is very similar to that seen in the LEC rat (Howell and Mercer, 1994). However, this mouse has the unusual feature that the pups of mutant dams are born copper deficient and the milk produced by the tx dam is copper deficient. The combined effects of this copper deficiency is often fatal to the neonates. Despite this copper deficiency, unexpected in a model of copper toxicosis, we have found a mutation in the WD homologue, showing that this mouse is also a true model of WD (Theophilos et al., 1996). The cause of the copper deficiency in the fetus and milk is not clear, but may be related to the disturbance of copper transport in the liver, but if so then similar copper deficiency would be expected in the LEC rat and WD mothers, and so far this has not been reported. 4. Molecular and cellular basis of copper transport 4.1 Structural and function of MNK and WND

The predicted structure of MNK and WND contains a number of interesting features. Firstly they are clearly a member of the family of cation transporters, P-type ATPases (Pederson and Carafoli, 1987). These are transmembrane proteins which move cations across the membrane by means of conformation changes induced by the binding of cations and hydrolysis of ATP. As part of the reaction cycle, a critical aspartic acid residue becomes phosphorylated, hence the name P-type ATPase. MNK and WND are closely related ATPases and it has been suggested that they form a subgroup of heavy metal transporting P-type ATPases termed CpX type ATPases, somewhat distinct from the more commonly known Ca and Na/K ATPases (Solioz and Vulpe, 1996). They are in fact, more closely related to bacterial copper transporters (Bull and Cox, 1994). It is predicted that most of the molecule is in the cytoplasm, with 6 or 8 transmembrane loops forming a channel through which copper passes following the hydrolysis of ATP (Vulpe et al., 1993; Bull and Cox 1994). A two dimensional representation of MNK, which applies also to WND, is shown in Fig 2. These models are based on the extensively studied calcium ATPases (Brandl et al., 1986). This
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model suggests that these molecules function in copper efflux from the cell and a role in efflux accords with both the phenotype of MD and WD.

Figure 2: Hypothetical arrangement of MNK in the cell membrane. There are eight transmembrane loops (TM 1-8), six potential copper binding sites (Cu 1-6) in the N-terminal region , the amino acids cys.pro.cys (CPC) are found in TM 6 and possibly bind copper in the channel. The position of the aspartic acid which is phosphorylated during the reaction cycle is indicated as D-phos and the ATP binding site is shown as ATP. The arrow heads indicate the regions encoded by each exon, numbered 2 to 23. As illustrated in Fig 1, it is likely that MNK functions as the main copper efflux molecule from most cells except hepatocytes. Little or no MNK mRNA is found in the liver (Vulpe et al., 1993, Paynter et al., 1994). Uptake of copper is normal in cells from MD patients, but efflux is defective (Herd et al., 1987). Thus absorption of copper from the small intestine cannot occur because MNK is required to efflux copper across the basolateral surface of the enterocyte into the blood (Fig 1a); similarly copper accumulates in the kidney proximal tubules of the brindled mouse (Yoshimura, 1994), because MNK is needed to efflux the resorbed copper across the basolateral surface back into the blood. As noted above, the deficiency of copper in the brain is exacerbated since MNK is again required for the efflux of copper from the cytoplasm of the cells which constitute the blood brain barrier into the brain (Fig 1b). Copper is found to accumulate in these vascular endothelial cells in the mottled mouse mutants (Kodama 1993). WND is expressed mainly in the liver, but significant amounts of WND mRNA is found in other tissues where MNK is also expressed. It is not known whether the two proteins have distinct roles in such a situation. As shown in Fig 3, WND probably functions to efflux copper from the hepatocyte into the bile. Copper efflux may be via direct biliary excretion across the cannalicular membrane or via a vesicular efflux system, eg. lysosomes. There is evidence for two modes of copper secretion into the bile (Gross et al., 1989; Dijkstra et al., 1995; Nederbragt, 1989). WND most probably donates copper to ceruloplasmin in the Golgi (Muruta et al., 1995). Ceruloplasmin in turn may function as a copper transporter to peripheral tissues (Lee et al., 1993; Harris and Percival 1989). Presumably the activity of WND is regulated in some way by the copper status of the liver, such that the excretion of copper is increased when the concentration becomes too high. Little is known of such a mechanism, however, our work with MNK in cultured cells is suggesting some interesting possibilities, as is discussed in the next section.

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Figure 3: Efflux of copper from hepatocytes and incorporation of copper into ceruloplasmin (CP) requires WND. For efflux into the bile, WND may be located on the biliary cannalicular membrane or perhaps on the lysosome. CP probably receives copper in the Golgi apparatus, and hence WND is probably also located in this organelle. 4.2 Copper resistance is acquired by amplification of MNK

Cells in culture can be selected for resistance to copper by growth in progressively increased concentrations of the metal ion. In the case of hepatocytes, resistant cells have elevated expression of metallothionein (Freedman et al., 1986). In copper resistant Chinese hamster ovary (CHO) cells, however, metallothionein expression has not been found to contribute to copper resistance. Instead, the Menkes gene homologue is amplified, leading to production of up to 100 fold more MNK (Camakaris et al., 1995). The increased amount of MNK allows the cells to efflux more copper and maintain low intracellular copper levels even in culture media containing high amounts of copper. This is the first direct evidence that MNK can function to efflux copper. Significantly, the increased efflux is only apparent at high concentrations of copper in the medium. In normal media there is little difference between the efflux rates of the most resistant cell, CUR3 and the parental K1 (Camakaris et al., 1995), suggesting that the cell is able to regulate the activity of MNK. We have used these cell lines to determine the intracellular location of MNK, using an antibody against the N-terminal one third of MNK. Our data suggest that the primary location of MNK is in the transGolgi Network, and significantly this localisation may be altered by growth in high copper concentrations, with more MNK appearing on the plasma membrane (Petris et al., 1996). Previous data on the levels of MNK mRNA suggested that amounts of mRNA are not altered by copper deficiency or copper loading (Paynter et al., 1994), so the regulation of cellular copper levels may be mediated by changes in the intracellular localisation of MNK. This may also be the case for WND in the liver.

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4.3

Do metallothioneins protect against copper toxicity?

Copper resistant hepatocytes have elevated amounts of metallothionein (MT) in their cytoplasm (Freedman et al., 1986), but this is not the case in the copper resistant CHO cells. The lack of MT elevation in copper resistant CHO cells may suggest that MTs do not protect all cell types from copper toxicity, however, MT genes are not expressed in the parental CHO cell K1, due to gene methylation, and hence the cell may use amplification of MNK rather than expression of MT to achieve copper resistance. More evidence for the protective effect of MT against copper is the invariant association of high MT levels in cells in which copper has accumulated due to a genetic defect of copper transport. For example in the LEC rat and the toxic milk mouse, both models of WD, the excess hepatic copper is bound to MTs (Suzuki et al., 1993; Koropatnick and Cherian, 1993). Cultured cells from MD patients accumulate copper and this excess copper is bound to MTs (LaBadie et al., 1981). This association of MT with high copper and the ability of copper to induce MT synthesis (Wake and Mercer 1985; Durnam and Palmiter 1981) strongly suggests that MT is protective against copper toxicity. It was surprising, therefore, to find that mice which lack the MT-I and MT-II genes (MT minus mice), did not show increased sensitivity to high levels of copper in the drinking water, but were very sensitive to cadmium (Michalska and Choo 1993; J. Mercer, unpublished data). The non-toxicity of copper for the MT minus mice would be explained if both the normal and MT minus mice have a very effective biliary excretion mechanism and hepatic copper levels were never high enough to cause toxicity. Indeed, in contrast to rats on the same level of copper which accumulate large amounts of hepatic copper (Gross et al., 1989), the hepatic copper in the mice was only marginally increased (J. Mercer, unpublished data). A marked effect of copper toxicity in the absence of metallothionein is observed in a double mutant brindled/ MT minus mouse. The double mutant mice die very early in development (less than 10 days gestation). This is most likely due to copper toxicity, perhaps causing placental failure (Kelley and Palmiter, 1996). Certain tissues including the placenta of the brindled mouse accumulate copper due to defective efflux, like in MD, without MT to complex the copper, cell death may occur. 5. Conclusions The genetic defects of copper transport in humans, MD and Wilson disease have finally provided the key to advancing our understanding of some of the key molecules involved in homeostasis of copper. Moreover the structure of these genes and future molecular and cellular investigations promise to establish the molecular basis of copper homeostasis. Further understanding of the molecules used by mammals to handle copper is coming from the metallothionein minus mouse, a mutant generated by homologous recombination. The power of modern molecular and cellular biological techniques, will provide information which can be integrated with previous knowledge of copper nutrition and toxicological studies to yield a depth of knowledge of copper transport and regulation which has been impossible to obtain previously. Acknowledgements I am grateful to Michelle Winsor for her help with the preparation of the figures.

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References Brandl, C. J., Green, N. M., Korczak, B. and MacLennan, D. H. (1986), Two Ca2+ ATPase genes: homologies and mechanistic implications of deduced amino acid sequences, Cell, vol. 44, pp. 597607. Bull, P. C. and Cox, D. W. (1994), Wilsons disease and Menkes syndrome: new handles on heavymetal transport, Trends in Genetics, vol. 10, pp. 246-252. Bull, P. C., Thomas, G. R., Rommens, J. M., Forbes, J. R. and Cox, D. C. (1993), The Wilsons disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene, Nature Genet., vol. 5, pp. 327-337. Byers, P. H., Siegel, R. C., Holbrook, K. A., Narayanan, A. S., Bornstein, P. and Hall, J. G. (1980), X-linked cutis laxa: defective cross-link formation in collagen due to decreased lysyl oxidase activity, New Eng. J. Med., vol. 303, pp. 61-65. Camakaris, J., Mann, J. R. and Danks, D. M. (1979), Copper metabolism in mottled mouse mutants: copper concentrations in tissues during development, Biochem J., vol. 180, pp. 597-604. Camakaris, J., Petris, M., Bailey, L., Shen, P., Lockhart, P., Glover, T. W., Barcroft, C. L., Patton, J. and Mercer, J. F. B. (1995), Gene amplification of the Menkes (MNK; ATP7A) P-type ATPase gene of CHO cells is associated with copper resistance and enhanced copper efflux, Human Mol. Genet., vol. 4, pp. 2117-2123. Chelly, J., Turmer, Z., Tonnerson, T., Petterson, A., Ishikawa-Brush, Y., Tommerup, N., Horn, N. andMonaco, A. P. (1993), Isolation of a candidate gene for Menkes syndrome that encodes a potential heavy metal binding protein, Nature Genet., vol. 3, pp. 14-19. Chowrimootoo, G. F. E., Ahmed, H. A. and Seymour, C. A. (1996), New insights into the pathogenesis of copper toxicosis in Wilsons disease: evidence for copper incorporation and defective canalicular tranport of ceruloplasmin, Biochem. J., vol. 315, pp. 851-855. Danks, D. M. (1995), Disorders of copper transport. The metabolic and molecular basis of inherited disease. Scriver, Beaudet, Sly and Valle eds. McGraw-Hill. New York. Danks, D. M. (1988), The mild form of Menkes disease: progress report on the original case, J. Med. Genet., vol. 30, pp. 859-864. Das, S., Levinson, B., Vulpe, C., Whitney, S., Gitschier, J. and Packman, S. (1995), Similar splicing mutations of the Menkes/mottled copper-tranporting ATPase gene in occipital horn syndrome and the blotchy mouse, Am. J. Hum. Genet., vol. 56, pp. 570-576. Das, S., Levinson, B., Whitney, S., Vulpe, C., Packman, S. and Gitschier, J. (1994), Diverse mutations in patients with Menkes disease often lead to exon skipping, Am. J. Hum. Genet., vol. 55, pp. 883-889. Dijkstra, M., In t Velt, G., van den Berg, G. J., Muller, M., Kuipers, F. and Vonk, R. J. (1995), Adenosine triphosphate-dependent copper transport in isolated rat liver plasma membranes, J. Clin., Invest, vol. 95, pp. 412-416. Durnam, D. M. and Palmiter, R. D. (1981), Transcriptional regulation of the mouse metallothionein-I gene by heavy metals, J. Biol.Chem., vol. 256, pp. 5712-5716. Evans, G. W. (1973), Copper homeostasis in the mammalian system, Physiological Reviews, vol. 53, pp. 535-569. Freedman, J. H., Weiner, R. J. and Peisach, J. (1986), Resistance to copper toxicity of cultured hepatoma cells, J. Biol. Chem., vol. 261, pp. 11840-8. Fu, D., Beeler, T. J. and Dunn, T. M. (1995), Sequence, mapping and disruption of CCC2, a gene that cross-complements the Ca2+ sensitive phenotype of csg1 mutants and encodes a P- type ATPase belonging to the Cu2+ ATPase subfamily, Yeast, vol. 11, pp. 283-292.
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Fujimoto, Y., Takahasi, H., Dempo, K., Mori, M. and al, e. (1989), Hereditary hepatitis in LEC rats: accumulation of abnormally high ploid nuclei, Cancer Detect. and Prevent., vol. 14, pp. 235-237. Grimes, A., Hearn, C.J., Lockhart, P., Newgreen, D.F. and Mercer, J.F.B. (1997), Molecular basis of the brindled Mouse mutant (Mobr): a murine model of Menkes disease, Human Mol. Genet., vol. 6. In press. Gross, J. B., Jr., Myers, B. M., Kost, L. J., Kuntz, S. M. and LaRusso, N. F. (1989), Biliary copper excretion by hepatocyte lysosomes in the rat. Major excretory pathway in experimental copper overload, J Clin Invest, vol. 83, pp. 30-9. Harris, E. D. and Percival, S. S. (1989), Copper transport: insights into a ceruloplasmin-based delivery system, Adv. Exp. Medicine and Biology, vol. 258, pp. 95-102. Harris, Z. L., Takahashi, Y., Miyajima, H., Serizawa, M., MacGillivray, R. T. and Gitlin, J. D. (1995), Aceruloplasminemia: molecular characterization of this disorder of iron metabolism Proc. Natl. Acad. Sci. USA, vol. 92, pp. 2539-2543. Hattori, A., Sawaki, M., Enomoto, K., Tsuzuki, N., Isomura, H., Kojima, T., Kamibayashi, Y., Sugawara, N., Sugiyama, T. and Mori, M. (1995), The high hepatocarcinogen susceptibility of LEC rats is genetically independent of abnormal copper accumulation in the liverCarcinogenesis, vol. 16, pp. 491-494. Haywood, S. (1985), Copper toxicosis and tolerance in the rat. IChanges in copper content of the liver and kidney, J Pathol., vol. 145, pp. 149-58. Herd, S. M., Camakaris, J., Christofferson, R., Wookey, P. and Danks, D. M. (1987), Uptake and efflux of copper-64 in Menkes disease and normal continuous lymphoid cell lines, Biochem J., vol. 247, pp. 341-7. Howell, J.McC. and Mercer, J. F. B. (1994), The pathology and trace element status of the toxic milk mutant mouse, J. Comp. Path., vol. 110, pp. 37-47. Kaler, S. G., Gallo, L. K., Proud, V. K., Percy, A. K., Mark, Y., Segal, N. A., Goldstein, D. S., Holmes, C. S. and Gahl, W. A. (1994), Occipital horn syndrome and a mild Menkes phenotype associated with splice site mutations at the MNK locus, Nature Genet., vol. 8, pp. 195-202. Karpel, J. T. and Peden, V. H. (1972), Copper deficiency in long-term parenteral nutrition, J. Pediatr., vol. 80, pp. 32-36. Kelley, E. J. and Palmiter, R. D. (1996), A murine model of Menkes syndrome reveals a physiological function of metallothionein, Nature Genetics, vol. 13, pp.219-222. Kodama, H. (1993), Recent developments in Menkes disease, J. Inher. Metab. Disease, vol. 16, pp. 791-799. Koropatnick, J. and Cherian, M. G. (1993), A mutant mouse (tx) with increased hepatic metallothionein stability and accumulation, Biochem. J., vol. 296, pp. 442-449. LaBadie, G. U., Beratis, N. G., Price, P. M. and Hirschhorn, K. (1981), Studies of the copper-binding proteins in Menkes and normal skin fibroblast lysates, J. Cell Physiol., vol. 106, pp. 173-178. Lee, S. H., Lancey, R., Montaser, A., Madani, N. and Linder, M. C. (1993), Ceruloplasmin and copper transport during the latter part of gestation in the rat, Proc. Soc. Exp. Biol. Med., vol. 203, pp. 428-439. Levinson, B., Vulpe, C., Elder, B., Martin, C., Verley, F., Packman, S. and Gitschier, J. (1994), The mottled gene is the mouse homologue of the Menkes disease gene, Nature Genet.,vol. 6, pp. 369-373. Lyon, M. F. and Searle, A. G. (1990), Mo locus, Chromosome X, Genetic Variants and Strains of the Laboratory Mouse, Lyon and Searle ed. Oxford University Press. Oxford. MacLachlan, G. K. and Johnston, W. S. (1982), Copper poisoning in sheep from North Ronaldsay maintained on a diet of terrestrial herbage, Vet Rec., vol. 111, pp. 299-301.
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Mercer, J. F. B., Grimes, A., Ambrosini, L., Lockhart, P., Paynter, J. A., Dierick, H. and Glover, T. W. (1994), Mutations in the murine homologue of the Menkes syndrome gene in dappled and blotchy mice, Nature Genet, vol. 6, pp. 374-378. Mercer, J. F. B., Livingston, J., Hall, B. K., Paynter, J. A., Begy, C., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., Siemenack, D. and Glover, T. W. (1993), Isolation of a partial candidate gene for Menkes syndrome by positional cloning, Nature Genet., vol. 3, pp. 20-25. Mercer, J. F. B., Stevenson, T., Wake, S. A., Mitropoulis, G., Camakaris, J. & Danks, D. M. (1991), Developmental variation in copper, zinc metallothionein mRNA in brindled mutant and nutritionally copper deficient mice Biochim. Biophys. Acta., vol. 1097, pp. 205-211. Michalska, A. E. and Choo, K. H. A. (1993), Targetting and germ line transmission of a null mutation at the metallothionein I and II loci in mouse, Proc. Natl. Acad. Sci. USA, vol. 90, pp. 80888092. Milne, D. B. (1994), Assessment of copper nutritional status, Clin Chem., vol. 40, pp. 1479-84. Multhaup, G., Schlicksupp, A., Hesse, L., Beher, D., Ruppert, T., Masters, C. L. and Beyreuther, K. (1996), The amyloid precursor protein of Alzheimers disease in the reduction of copper (II) to copper(I), Science, vol. 271: 1406-9. Murata, Y., Yamakawa, E., Iizuka, T., Kodama, H., Abe, T., Seki, Y. & Kodama, M. (1995), Failure of copper incorporation into ceruloplasmin in the golgi apparatus of LEC rat hepatocytes, Biochem. Biophys. Res. Comm., vol. 209, pp. 349-355. Nederbragt, H. (1989), Effect of the glutathione-depleting agents diethylmaleate, phorone and buthionine sulfoximine on biliary copper excretion in rats, Biochem. Pharmacol., vol. 38, pp. 33993406. Odermatt, A., Suter, H., Krapf, R. and Solioz, M. (1993), Primary structure of two P-type ATPases involved in copper homeostasis in Enterococcus hirae, J. Biol. Chem., vol. 268, pp. 12775-12779. Paynter, J. A., Grimes, A., Lockhart, P. and Mercer, J. F. B. (1994), Expression of the Menkes gene homologue in mouse tissues: lack of effect of copper on the mRNA levels, FEBS lett., vol. 351, pp. 186-190. Pederson, P. L. and Carafoli, E. (1987), Ion motive ATPases. I. Ubiquity, propertes, and significance to cell function, TIBS, vol. 12, pp. 146-150. Phillips, R. J. S. (1961), Dappled, a new allele at the mottled locus in the house mouse, Genet. Res., vol. 2, pp. 209-295. Petris, M.J., Mercer, J.F.B., Culvenor, J.G., Lockhart, P., Gleeson, P.A., Camakaris, J. (1996), Ligand-regulated transport of the Menkes copper P-Type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking, EMBO J., vol. 15, pp. 6084-6095. Prohaska, J. R. and Bailey, W. R. (1993), Persistent regional changes in brain copper, cuproenzymes and catecholamines following perinatal copper deficiency in mice, J Nutr., vol. 123, pp. 1226-34. Rad, M. R., Kirchrath, L. and Hollenberg, C. P. (1994), A putative P-type Cu2+ transporting ATPase on chromosome II of Saccharomyces cerevisiae, Yeast, vol. 10, pp. 1217-1225. Rauch, H. (1983), Toxic milk, a new mutation affecting copper metabolism in the mouse, J. Hered., vol. 74, pp. 141-144. Roeser, H. P., Lee, G. R., Nacht, S. and Cartwright, G. E. (1970), The role of ceruloplasmin in iron metabolism, J. Clin. Invest., vol. 49, pp. 2408-2417. Rowe, D. W., McGoodwin, E. B., Martin, G. R. and Grahn, D. (1977), Decreased lysyl oxidase activity in the aneurism-prone mottled mouse, J. Biol. Chem., vol. 252, pp. 939-942.

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Smith, R. M., Fraser, F. J. & Russell, G. (1977), Enzootic ataxia in lambs: appearance of lesions in the spinal chord during foetal development, J. Comp. Path., vol. 87, pp. 119-128. Solioz, M. and Vulpe, C. (1996), CPX-Type ATPases: a class of P-type ATPases that pump heavy metals, Trends in Biochem. Sci., vol. 21, pp. 237-241. Su, L.-C., Owen, C. A., Zollman, P. E. and Hardy, R. M. (1982), A defect of biliary excretion of copper in copper-laden Bedlington terriers Am. J. Physiol., vol. 243, pp. G231-G236. Sugawara, N., Li, D., Sugawara, C. and Miyake, H. (1993), Decrease in biliary excretion of copper in Long-Evans Cinnamon (LEC) rats causing spontaneous hepatitis due to a gross accumulation of hepatic copper, Res. Comm. Chem. Path. and Pharmacol., vol. 81, pp.45-52. Suzuki, K. T. (1995), Disordered copper metabolism in LEC rats, an animal model of Wilsons disease: role of metallothionein, Res. Commun. Mol. Path. Pharmacol., vol. 82, pp. 221-239. Suzuki, K. T., Yamamoto, K., Kanno, S., Aoki, Y. and Takeichi, N. (1993), Selective removal of copper bound to metallothionein in the liver of LEC rats by tetrathiomolybdate, Toxicology, vol. 83, pp. 149-158. Tanzi, R. E., Petrukhin, K., Chernov, I., Pellequer, J. L., Wasco, W., Ross, B., Romano, D. M., Parano, E., Pavone, L., Brzustowicz, L. M., Devoto, M., Peppercorn, J., Bush, A. I., Sternlieb, I., Pirastu, M., Gusella, J. F., Evgrafov, O., Penchaszadeh, G. K., Honig, B., Edelman, I. S., Soares, M. B., Scheinberg, I. H. and Gilliam, T. C. (1993), The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene, Nature Genet., vol. 5, pp. 344-350. Theophilos, M. B., Cox, D. W. and Mercer, J. F. B. (1996), The toxic milk mouse is a murine model of Wilsons disease, Hum. Mol. Genet., vol.5, pp. 1619-1624. Tonneson, T. and Horn, N. (1989), Prenatal and postnatal diagnosis of Menkes disease, an inherited disorder of copper metabolism, J. Inher. Metab. Dis., vol. 12, pp. 207-214. Underwood, E. J. Copper.Trace Elements in Human and Animal Nutrition. 1977 Academic Press. New York. Vulpe, C., Levinson, B., Whitney, S., Packman, S. & Gitschier, J. (1993), Isolation of a candidate gene for Menke disease and evidence that it encodes a copper-transporting ATPase, Nature Genet., vol. 3, pp. 7-13. Wake, S. A. & Mercer, J. F. B. (1985), Induction of metallothionein mRNA in rat liver and kidney after copper chloride injection, Biochem. J., vol. 228, pp. 425-432. Weber, K. M., Boston, R. C. and Leaver, D. D. (1980), A kinetic model of copper metabolism in sheep, Aust. J. Agric. Res., vol. 31, pp. 773-790. Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. B., Roe, J. A., Lee, M. K., Valentine, J. S. and Bredesen, D. E. (1996), Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis, Science, vol. 271, pp. 515-8. Wu, J., Forbes, J. R., Chen, H. S. and Cox, D. W. (1994), The LEC rat has a deletion in the copper transporting ATPase homologous to the Wilson disease gene, Nature Genet., vol. 7, pp. 541-545. Yamaguchi, Y., Heiny, M. E. & Gitlin, J. D. (1993), Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease, Biochem. Biophys. Res Comm., vol. 197, pp. 271-277. Yoshimura, N. (1994), Histochemical localization of copper in various organs of brindled mice Pathol. Int., vol. 44, pp. 14-9. Yuzbasiyan, G. V., Wagnitz, S., Blanton, S. H. and Brewer, G. J. (1993), Linkage studies of the esterase D and retinoblastoma genes to canine copper toxicosis: a model for Wilsons disease, Genomics, vol. 15, pp. 86-90.

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Copper in the Aquatic Environment

Dr Herbert E Allen Department of Civil and Environmental Engineering University of Delaware, Newark, Delaware U.S.A. Copper is a ubiquitous trace metal that is required by aquatic organisms. At higher concentrations it is toxic. However, the bioavailability of copper varies greatly depending on the water chemistry. Thus, it is necessary to establish the physical and chemical form of copper in the environment being studied. Aquatic life criteria and standards must reflect these speciation effects to be predictive of ecosystem effects. To evaluate aquatic systems, it is necessary to obtain valid measurements of the concentration of copper in the environmental samples and to compare these to criteria and standards. This paper provides a brief review of the most important factors involved in the evaluation of potential effects of copper in the aquatic environment 1. Analysis of copper Copper concentrations in natural waters are frequently in the low g/L concentration range. To obtain valid data for such low concentrations it is necessary to take special precautions in sampling and analysis programs. The EPA (Prothro, 1993) has summarized the necessary components of the measurement program: 1. Use of clean techniques during each step of the measurement process to avoid contamination. 2. Use of analytical methods that have sufficiently low detection limits. 3. Avoidance of interference in the quantification step. 4. Use of blanks to assess contamination. 5. Use of sample spikes and certified reference materials to assess the effects of contamination and interference on accuracy. 6. Use of replicates to assess precision. Although the importance of none of these items should be underestimated, the first item is of particular importance. This includes such factors as acid washing sample bottles, use of clean hands - dirty hands operating protocols during sampling, and use of laminar flow hoods and benches and clean rooms for laboratory measurements. The analytical method should have a detection limit no greater than one-tenth that of the sample and the concentration found for the blank should not exceed one-tenth that of the blank. The need for these precautions in sampling and analysis for environmental studies was first pointed out by Patterson and Settle (1976) with regard to the analysis of lead. Since then it has become apparent that these considerations must be applied to the sampling and analysis of all trace metals (Bruland, 1983; Nriagu et al., 1993; U.S. EPA, 1995). Although the oceanographic research community quickly realized the importance of using these clean techniques, those concerned with the analysis of freshwaters and effluents were slower to respond. This attitude changed after the publication of Windom and his colleagues (1991) that demonstrated the questionability of the data that had been collected for the U.S. Geological Surveys NASQAN national stream quality network. They found copper concentrations about 3-fold lower, cadmium concentrations 30-fold lower, lead concentrations 100-fold lower and zinc concentrations almost 20fold lower by using appropriate low level sampling and analysis techniques. Significant regulatory and economic issues arise as a consequence of results that are biased high. A summary of the results of analyses for waters of New York Harbour are presented in Table 1 (Battelle Ocean Sciences, 1991).

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The harbour had been routinely sampled and analyzed by the New York City Department of Environmental Protection. Of the six metals studied, for 1987 only for cadmium was the annual average below the value of the Water Quality Criteria. The annual average copper concentration exceeded the average more than four-fold. In the following year, a series of cruises was carried out by the U.S. EPA on the Research Vessel Anderson. Sampling and analysis was carried out by Battelle scientists using low level sampling and analysis techniques. The concentrations of all six elements decreased greatly, as much as 40-fold in the case of cadmium, when the 1988 results are compared to those of 1987. The copper concentrations of the earlier data are, on average, three-fold greater than are the results that were obtained using the clean techniques. As a result of evaluation of the newer data, the potential ecological impacts were reassessed and an unnecessary, costly upgrade of sewage treatment plants discharging to the lower Hudson River and the New York Harbour was not instituted. Table 1: Water Quality Criteria and annual average concentrations using conventional sampling and analysis methods (1987) and low level sampling and analysis methods (1988) for New York Harbour (Battelle Ocean Sciences, 1991).

Metal Cadmium Copper Lead Mercury Nickel Zinc

Water Quality Criteria 1987 g/L 9.3 2.9 8.6 0.025 8.3 86

NYCDEP 1987 g/L 4.3 13 70 0.2 15.6 264

Anderson 1988 g/L 0.11 4.6 3.0 0.015 2.6 10

The use of low level sampling and analysis methods may also be important for the evaluation of industrial discharges. Figure 1 shows the results for copper analyses of paired samples collected for an industrial waste discharge. Laboratory A had been doing the analyses of effluent samples, but did not employ clean techniques in the laboratory. The results of Laboratory B indicated that the level of copper in the discharge was not above the level specified in the discharge permit and that an expensive new outfall was not necessary. The results of Laboratory A were as much as 9-fold greater than were those of laboratory B. 2. Chemical speciation Frequently analyses of chemical species, rather than the total elemental concentration, are required. Both bioavailability and sorption of metals are strongly dependent on the metal species that are present (Allen, 1993). Speciation of trace metals involves determination of the physical and chemical forms of the metal. This includes the evaluation of free metal ions, inorganic and organic complexes, and organometallic compounds. Although strictly not speciation, classification by size, including filtration is often considered speciation. Commonly speciation studies incorporate voltammetric and potentiometric electrochemical methods and chromatography. Batley (1989) has reviewed many of the available methods. Among the most important speciation measurements are determination of free metal ion and assessment of the interaction of metals with organic matter. Free metal ions can be quantified with ion selective electrodes; the copper ion selective electrode can respond to free copper ion concentrations as low as pCu = 19 (Avdeef et al., 1983). Samples are frequently titrated with metal, and the titration is monitored electroanalytically (Batley, 1989).

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Voltammetric techniques, particularly anodic and cathodic stripping voltammetry, have commonly been applied. The methods provide differentiation of weak from stronger ligands and the results of these titrations are usually expressed in terms of the complexation capacity of the sample (Neubecker and Allen, 1983). Cd2+, Cu2+, Pb2+ and other metals can form inner and outer sphere complexes with a number of inorganic ligands, such as OH-, HCO3-, NH3, and organic ligands, such as oxalic acid and EDTA (Stumm and Morgan 1981; Pankow 1991). Stability constants for chemically defined ligands are available in a number of data bases. A particularly good source is the NIST Critical Stability Constants of Metal Complexes Database (U.S. Department of Commerce, 1993). Stability constants for these reactions are well-known and the distribution of species can thus be easily computed using thermodynamic principals.

20

16

12

45 line

0 0 5 10 15 20

Laboratory B
Figure 1: Paired samples of copper in industrial effluent. Laboratory A did not, and Laboratory B did, use clean techniques. Results in g Cu/L. From Skrabal and Allen (unpub., 1993). The soluble metal will be present as the free aquo ion and as metal contained in inorganic and organic complexes. The formation of complexes of a divalent metal ion M2+ with monodentate ligands is expressed by the reaction
j=
O=+

+ i ji

( O= =)+

(1)

If the ionic strength is low, the equilibrium is

ML
= [ ML [M
( 2 n )+

]
n

2+

][ L ]

(2)

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where MLn is the overall formation constant for the complex and the brackets indicate the concentrations of the enclosed species. Typically, a number of complexes are present. Then the total concentration of metal, CM, is equal to the concentration of the free metal ion, plus the concentration of metal contained in all complexed species
C M = [M
2+

] 1+

ML [ L ] n

(3)

For ligands that are not monodentate, and for polynuclear reactions involving more than one metal ion, similar reactions and stability constants can be formulated. Chemical equilibrium computer programs are useful for computing the distribution of species in samples containing defined total concentrations of metal and ligands, if appropriate stability constants are available (Nordstrom et al., 1979). Commonly used programs include MINTEQA2 (Allison et al., 1991) and MINEQL+ (Schecher and McAvoy 1992). The description of metal complexation with natural organic matter (NOM) is much more complicated. NOM is an unresolvable mixture of a very large number of compounds varying in their properties, including their ability to bind metal ions. Several approaches have been proposed for the modeling of metal complexation by NOM and humic substances. These include gaussian distribution models (Perdue and Lytle, 1983) and multiple discrete sites (Fish et al., 1986). Recently, Tipping (1994) has presented a model, using 5 sites, that is able to relatively accurately predict metal and proton binding to naturally occurring organic matter. 3. Copper bioavailability and toxicity The toxicity of copper and other metals is profoundly affected by their chemical form (see reviews by Hodson et al., Luoma, 1983; ODonnel et al., 1985; Flemming and Trevors, 1989). Steemann-Nielsen and Wium-Andersen (1970) recognized that free copper ions, at the level of copper found in natural waters, are very toxic to algae. They reported that the predominant forms of copper are not free copper ions, but are organic complexes and they reported these to be not poisonous to algae. In recently upwelled water, phytoplankton growth was limited and could be enhanced by the addition of a chelator. The understanding of the relationship between speciation and toxicity was greatly enhanced by the work of Sunda and Guillard (1976) who determined copper ion activity using a copper ion selective electrode and found that the algal growth rate was related to the free copper ion activity and not to the total copper concentration, which they had varied independently. The amount of copper added to natural waters that is required to produce a given biological response, for example toxicity or reduction of growth, is different for different waters. An example is shown in Figure 2 for the growth of the alga Selanastrum capricornutum in waters collected in three locations near Chicago, Illinois. Based on total added copper, the amount required to produce a 50 percent reduction in growth rate varied by twenty-fold for the samples. This difference is interpreted as being caused by a difference in the bioavailability of the added copper (Benson et al., 1994). Allen and Brisbin (1980) followed the labile copper concentration during a titration of a sample using anodic stripping voltammetry and evaluated the samples conditional stability constant and complexation capacity. These values were used to compute the concentration of copper not complexed by organic matter. In a separate set of experiments, Selanastrum was grown in a chemically defined medium and the concentration of copper ion required to inhibit the growth rate was computed. They reported that they could predict the growth rate response of the algae in the natural water samples. Verwiej et al. (1992) also found that growth inhibition for Scenedesmus quadricauda was highly correlated with copper detected by electrochemical methods and with calculated free copper. Because organism response can be correlated to the concentration of free copper ions, many investigators have used bioassay methods to assess the concentration of free copper ions in natural waters (Gillespie and Vaccaro, 1978; Sunda and Gillespie, 1979; Allen et al., 1983). Hering et al.
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(1987) have shown that the free copper ion concentration, predicted from a bioassay, agrees with that obtained by chemical measurement. Some investigators have reported that chemical species other than free, ionic copper are toxic. This has recently been critically reviewed by Campbell (1995). Cowan et al. (1986) statistically analyzed available data in terms of the inorganic copper species that were toxic. Other investigators have found that not all organic copper species are non-toxic. Florence and Stauber (1986) and Florence et al. (1992) reported that lipid soluble copper (II) complexes are highly toxic to the marine diatom, Nitzschia closterium. Guy and Kean (1980) and Borgmann and Ralph (1983) found that complexes of copper with synthetic ligands were toxic, but at reduced levels compared to free copper ion. Both Meador (1991) and Tubbing et al. (1994) reported that copper complexed with natural organic matter contributed to toxicity. A mechanistic approach to understanding the influence of solution-phase complexation of the toxicity of metals has been proposed by Pagenkopf (1983). He treated metal ion receptor sites on the gills of fish as chemical entities that compete with ligands in the solution for metal ions such as copper. This allows the prediction of the effect of metal on the organism, based on equilibrium considerations such as those predicted by computer programs such as MINTEQA2 or MINEQL+. This approach also allows prediction of the effects of Ca2+ and H+.

Figure 2 : Effect of added copper on the growth rate of alga Selanastrum capricornutum in samples of three filtered, nutrient fortified waters. The principal receptor site for toxic metals in freshwater fish are gills where Na+ and Ca2+ are transported from the bulk water to the bloodstream by active, energy requiring pumps. The channel or carrier proteins associated with these pumps occur as specific, negatively charged ligands on the gill surface. Thus, the gills of freshwater fish have the two important physiological functions of gas transport (O2, CO2, NH3) and active uptake of ions (Na+, Ca2+) (Wood, 1992). Playle and co-workers (1992, 1993a and b) and MacRae et al. (1996) have treated the specific receptor sites on the gill as competitive ligands for the binding of copper and other metals. They have determined conditional stability constants and site densities for these surface complexation reactions through competitive binding experiments with ligands whose complexation constants are known. MacRae et al. (1996) showed that there is a strong relationship between the extent of saturation of the gill receptor sites and
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mortality. Experiments by these research groups have shown that gills indeed do compete for metals with natural DOM. An important conclusion from the gill complexation model is that given species should not be treated as being bioavailable and other species as being not bioavailable. Rather, the presence of the gill causes chemical re-equilibration in the system. It is the degree to which the gill complexation sites are occupied by metal that determines whether toxicity will occur. 4. Water quality criteria and standards The U.S. Environmental Protection Agencys Water Quality Criteria for Copper (U.S. EPA, 1985) forms the basis for the standards for the protection of aquatic life that are promulgated by most states. The freshwater criteria for a four-day average concentration (in g/L) of copper that is not to be exceeded more than once every three years on the average is given by the expression e(0.8545[ln(hardness)]-1.465). The acute criteria value (in g/L), based on a one-hour average concentration should not exceed the numerical value e(0.9422[ln(hardness)]-1.464) more than once every three years on the average. For hardnesses of 50, 100 and 200 mg/L as CaCO3 the four-day average concentrations of copper are 6.5, 12 and 21 g/L, respectively, and the one-hour average concentrations are 9.2, 18, and 34 g/L. These criteria consider hardness as the only water quality parameter that is to be used in modification of the numeric values. Erickson et al. (1996) determined the effects of various water chemistry parameters on the toxicity of copper to larval fathead minnows. Based on total copper concentrations, they found that increased pH, hardness, dissolved organic matter and suspended solids each caused toxicity to decrease, but alkalinity had no observed effect. The effect of pH on toxicity was greater than that of hardness. They also determined cupric ion activity and found that the toxicity varied significantly when expressed on the basis of cupric ion activity, sometimes more than when the toxicity was expressed on the basis of total copper. This study indicates that a number of water quality parameters should be considered in setting criteria. A single numerical value, even when modified by water hardness, does not provide the predictability of aquatic life effects that are desired for national criteria. Therefore, provision is made for sitespecific modification of the criteria. The Water Effects Ratio (WER) has been recommended to provide site-specific modified criteria (Stephan et al., 1985; U.S. EPA, 1992, 1994; Prothro, 1993). To establish a WER, toxicity tests are conducted in a site water and in a reference (laboratory) water. Reference water tests are used as surrogates for the laboratory tests that were used to derive national criteria. The ratio of the toxicities (WER) is used as a multiplier to adjust the National Water Quality Criteria (NWQC) to account for differences in bioavailability, as measured by toxicity tests, that would be applicable to that site. For example: Site - Specific WQC = NWQC WER = NWQC site - water LC50 reference - water LC50 Carlson et al. (1986) applied this procedure in a a series of stations in a stream receiving industrial and municipal treatment plant effluents and and found mean WERs of 3.9 to 7.0, reflective of reduced bioavailability of copper. Allen and Hansen (1996) have analyzed the WER procedure in terms of the change in speciation that occurs in a sample as metal is added to a sample. They indicated that the fraction of metal that is present in forms having reduced bioavailability decreases as the total concentration of metal increases. This implies that the WER for sensitive organisms will be greater for more sensitive organisms than it will be for less sensitive ones. This is in agreement with the information that has been presented by Brungs et al. (1991). Allen and Hansen (1996) recommended that new Water Quality Criteria based on bioavailable metal be developed. Such criteria would have universal applicability and would obviate the need for such
Copper

(4)

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site-specific modifications as WERs. A recent SETAC Pellston Conference (Bergman and DorwardKing, 1997) recommended that the gill complexation model be used as the basis for a WQC that incorporated bioavailability by coupling biological site of action and aquatic speciation. They recommended that Tippings WHAM model be used to compute chemical speciation in a receiving water. This model should be coupled with MINTEQA2 or another speciation code that incorporate the necessary constants for binding of metal at the gill. 5. Aquatic sediments Metals and anthropogenic organic compounds are often present at elevated levels in sediments. Evaluation of sediment quality is frequently ascertained through bioassays. However, if toxicity is found, the cause must be ascertained to ensure proper disposal or treatment to prevent further contaminant input and to allocate responsibility. This creates a difficult situation because high concentrations of metals do not necessarily lead to toxicity. Figure 3 shows the results of a number of toxicity tests in which cadmium, copper, nickel or zinc, or cadmium and nickel were added to sediments. When the concentration of metal is expressed as mol/g dry weight of sediment there is no relationship between the concentration of added metal and the mortality of organisms. Non-toxic sediments were found to contain as much as 3 orders of magnitude more metal than did some sediments not exhibiting toxicity.

Figure 3 : Toxicity of metals in sediments. Metal concentrations expressed on a dry weight normalized basis. Data courtesy of Dr. Dominic Di Toro, Manhattan College. For example, Di Toro et al. (1990) added cadmium to samples of sediment that did not exhibit toxicity. The amount of cadmium that was required to be added before toxicity was observed differed by more than an order of magnitude for the two sediments and was greater than what is conventionally considered to represent a contaminated sediment. They found that the added cadmium displaced iron in FeS to form CdS thus rendering it non-toxic. No toxicity was found unless the amount of added cadmium exceeded the concentration of available sulfide when both are expressed on a molar basis. Analytical methods for the measurement of acid volatile sulfide (AVS) and the concentration of simultaneously extracted metals (SEM) have been described by Allen et al. (1993). Sulfide is evolved by the addition of cold, dilute acid. It is trapped and quantified to provide the AVS value. The
Copper

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dissolved concentrations of the potentially toxic metal that can displace iron in FeS, which are cadmium, copper, lead, nickel and zinc, are determined in the acid. The SEM value is the sum of these metal concentrations on a mol/g dry weight of sediment basis. The SEM/AVS ratio is useful in prediction of those sediments for which toxicity will not be exhibited. Ratios less than one have not been found to be toxic to organisms. That is, there is not toxicity if the amount of sulfide available to bind metals exceeds the concentration of metals. It should be noted that it is the absence of toxicity that is predicted. No prediction of toxicity is provided by this procedure. This is shown in Figure 4 for the same data set shown in Figure 3. No toxicity is seen for any sediments having SEM/AVS ratios less than 1.

Figure 4 : Toxicity of metals in sediments. Metal concentrations expressed on a SEM/AVS normalized basis. Data are the same as those plotted in Figure 3. Data courtesy of Dr. Dominic Di Toro, Manhattan College. Ankley et al. (1993) found that there appeared to be copper binding in excess of the amount of sulfide present in sediment. This can be clearly seen in Figure 4 which demonstrates that, for some sediments, no toxicity was observed until the SEM/AVS ratio exceeded 5. Mahony et al. (1996) have demonstrated that this additional binding phase for cadmium, copper and lead is the organic matter contained in the sediments. Inclusion of this potential for binding of metals in addition to that of the AVS that is in excess of the SEM (i.e., SEM-AVS) should provide a good estimate of the concentration of metals that could be present in a sediment before toxicity will be observed. 6. Conclusions This review of the literature provides two important conclusions regarding the assessment of metals and potential metal toxicity in aquatic systems: Proper precautions must be taken in the sampling and analysis of environmental samples to ensure accurate results. Much of the existing data is of questionable quality and these results are biased high. Assessment of potential impacts of copper and other metals in water and sediment cannot be judged on the basis of the total concentration of metals. Knowledge of metal speciation and of the total system chemistry is essential.
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It is possible to develop Water Quality Criteria and Sediment Quality Criteria that are predictive of the effects of metals on aquatic life. Such criteria, based on sound chemical and physiological bases, would have broad applicability. Present criteria and evaluations based on observations of effects suffer from not providing the evaluator a technical basis for knowing their applicability to a new situation. Site-specific modification is a less technically acceptable process than is the development of the proposed technically sound evaluative tools. References Allen, H.E. (1993), The significance of trace metal speciation for water, sediment and soil quality standards, Sci. Total Environ., vol. 134, Supplement Part 1, p. 23. Allen, H.E., Blatchley, C. and Brisbin, T.D. (1983), An algal assay method for determination of copper complexation capacity of natural waters, Bull. Environ. Contam. Toxicol., vol. 30, p. 448. Allen, H.E. and Brisbin, T.D. (1980), Prediction of bioavailability of copper in natural waters Thal. Jugosl., vol. 16, p. 331. Allen, H.E. G. Fu and Deng, B (1993), Determination of acid-volatile sulfide (AVS) and simultaneously extracted metals (SEM) for the estimation of potential toxicity in aquatic sediments, Environ. Toxicol. Chem., vol. 12, p.1441. Allen, H.E. and Hansen, D.J. (1996), The importance of trace metal speciation to water quality criteria, J. Water Environ. Res., vol. 68, p. 42. Allison, J.D., Brown, D.S. and Novo-Gradac, K.J. (1991), MINTEQA2/PRODEFA2, a geochemical assessment model for environmental systems: version 3.0 users manual. EPA/600/3-91/021. U.S. Environmental Protection Agency, Washington, DC. Ankley, G.T., Matson,V., Leonard, E., West, C. and Bennett, J. (1993), Predicting the acute toxicity of copper in freshwater sediments: evaluation of the role of acid volatile sulfide, Environ. Toxicol. Chem., vol. 12, p. 312. Avdeef, A., Zalironsky, J. and Stuting, H. (1983), Calibration of copper ion selective electrode response to pCu 19, Anal. Chem., vol. 55, p. 298. Batley, G.E. (ed). (1989), Trace Element Speciation: Analytical Methods and Problems. CRC Press, Boca Raton, FL. Battelle Ocean Sciences. (1991), Results of the Ambient and Municipal Sample Interlaboratory Comparison Study in Ambient Waters and Discharges to New York/New Jersey Harbour. Duxsbury, MA. Benson, W.H., Alberts,J., Allen, H.E., Hunt, C.D.and Newman, M.C. (1994), Synopsis of discussion session on bioavailability of inorganic elements In A Mechanistic Understanding of Bioavailability: Physical, Chemical, and Biological Interactions. Chapter 2. J.L. Hamelink, W.H. Benson, H.L. Bergman and P.F. Landrum (eds) CRC Press, Boca Raton, FL. Bergman, II.L and Dorward-King, E.J. (Eds). (1997), Reassessment of Metals Criteria for Aquatic Life Protection: Priorities for Research and Implementation. Society for Environmental Toxicology and Chemistry, Pensacola, FL. Borgmann, U. and K.M. Ralph, K.M. (1983), Complexation and toxicity of copper and the free metal bioassay technique, Water Res., vol. 17. p. 1697. Bruland, K.W. (1983), Trace elements in seawater., Chem. Oceanogr., vol. 8, p. 157. Brungs, W.A. (1991), Synopsis of Water-Effect Ratios for Heavy Metals as Derived for SiteSpecific Water Quality Criteria. U.S. EPA Contract 68-CO-0070. Campbell, P.G.C. (1995) Interactions between trace metals and aquatic organisms: A critique of the free-ion activity model. In Metal Speciation and Bioavailability in Aquatic Systems.Tessier, A. and Turner, D.R. (eds). Wiley, New York.
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Carlson, A.R., Nelson, H. and Hammermeister, D. (1986), Development and validation of sitespecific water quality criteria for copper, Environ. Toxicol. Chem., vol. 5, p. 997. Cowan, C.E., Jenne, E.A. and Kinnison, R.R. (1986), Methodology for determining the relationship between toxicity and the aqueous speciation of a metal. In Aquatic Toxicity and Environmental Fate: Ninth Volume. ASTM STP 921. T.M. Poston and R. Purdy, eds. American Society for Testing and Materials, Philadelphia p. 463. Di Toro, D.M., Mahony, J.D., Hansen, D.J., Scott, K.J., Hicks, M.B., Mayr, S.M. and Redmond, M.S. (1990), Toxicity of cadmium in sediments: The role of acid volatile sulfide, Environ. Toxicol. Chem. vol. 9, p. 1487. Erickson, R.J., Benoit, D.A., Mattson, V.R., Nelson, Jr, H.P. and Leonard, E.N. (1996), The effects of water chemistry on the toxicity of copper to fathead minnows, Environ. Toxicol. Chem., vol. 15, p. 181. Fish, W., Dzombak, D.A. and Morel, F.M.M. (1986), Metal-humate interactions. 1. Application and comparison of models, Environ. Sci. Technol., vol. 20, p. 676. Flemming, C.A. and Trevors, J.T. (1989), Copper toxicity and chemistry in the environment: A review, Water, Air, Soil Pollut. , vol. 44, pp. 143. Florence, T.M., Powell, H.K.J., Stauber, J.L. and R.M. Town. (1992), Toxicity of lipid-soluble copper (II) complexes to the marine diatom Nitzschia closterium: Amelioration by humic substances, Water Res., vol. 26, p. 1187. Florence, T.M. and Stauber J.L. (1986), Toxicity of copper complexes to the marine diatom, Nitzschia closterium, Aquat. Toxicol., vol. 8, p. 11. Gillespie, P.A. and. Vaccaro, R.F. (1978), A bacterial bioassay for measuring the copper-chelation capacity of seawater, Limnol. Oceanogr., vol. 23, p. 543. Guy, R.D. and Kean, A.R.. (1980), Algae as a chemical speciation monitor - I. A comparison of algal growth and computer calculated speciation, Water Res., vol. 14, p. 891. Hering, J.G., Sunda, W.G., Ferguson, R.L. and F.M.M. Morel, F.M.M. (1987), A field comparison of two methods for the determination of copper complexation: bacterial bioassay and fixed-potential amperometry; Mar. Chem., vol. 20, p. 299. Hodson, P.V., Borgmann, U. and Shear, H. (1979), Toxicity of Copper to Aquatic Biota. In Copper in the Environment. Part II. Health Effects. J.O. Nriagu, ed. Wiley-Interscience, New York. Luoma, S.N. (1983), Bioavailability of trace metals to aquatic organisms - a review, Sci. Total Environ., vol. 28, p. 1. MacRae, R.K., D.E. Smith, D.E., Swoboda-Colberg, N., Meyer, J.S. and Bergman, H.L. (1996), Copper binding affinity of rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis) gills, Environ. Toxicol. Chem., (submitted). Mahony, J.D., Di Toro, D.M., Gonzalez, A.M., Curto, M., Dilg, M., De Rosa, L.D. and L.A. Sparrow, L.A. (1996), Partitioning of metals to sediment organic carbon, Environ. Toxicol. Chem., vol. 15, p. 2187. Meador, J.P. (1991), The interaction of pH, dissolved organic carbon, and total copper in the determination of ionic copper and toxicity, Aquat. Toxicol., vol. 19, p. 13. Neubecker, T.A. and Allen, H.E. (1983), The measurement of complexation capacity and conditional stability constants for ligands in natural waters--a review, Water Res., vol. 17, p. 1. Nordstrom, D.K., Plummer, L.N., Wigley, T.M.L., Wolery, T.J., Ball,W., Jenne, E.A.,. Bassett, R.L.,Crerar, D.A., Florence, T.M., Fritz, B., Hoffman, M., Holdren, Jr, G.R., Lafon, G.M.,Mattigod, S.V., McDuff, R.E., Morel, F., Reddy, M.M., Sposito, G. and Thraikill, J. (1979), Comparison of Computerized Chemical Models for Equilibrium Calculations in Aqueous Systems. In Chemical
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Modeling in Aqueous Systems: Speciation, Sorption, Solubility and Kinetics. E.A. Jenne, ed. Symp. Ser. 93. American Chemical Society, Washington, DC. Nriagu, J.O., Larson, G., Wong, H.K.T. and Azcue, J.M. (1993), A protocol for minimizing contamination in the analysis of trace metals in Great Lakes waters, J. Great Lakes Res., vol. 19, p.175. ODonnel, J.R., Kaplan, B.M. and Allen, H.E. (1985), Bioavailability of Trace Metals in Natural Waters. In Aquatic Toxicology and Hazard Assessment: Seventh Symposium, American Society for Testing and Materials, Philadelphia, PA. Pagenkopf, G.K. (1983), Gill surface interaction model for trace-metal toxicity to fishes: Role of complexation, pH and water hardness, Environ. Sci. Technol., vol. 17, p. 347. Pankow, J. (1991), Aquatic Chemistry Concepts. Lewis Publ., Chelsea, MI. Patterson, C.C. and D.M. Settle. (1976), The reduction of orders of magnitude errors in lead analyses of biological materials and natural waters by evaluating and controlling the extent and sources of industrial lead contamination introduced during sample collecting and analysis. In Accuracy in Trace Analysis: Sampling, Sample Handling, Analysis. P.D. LaFleur (ed). Nat. Bur. Standards. NBS Spec. Pub. 422: 321. Perdue, E.M. and Lytle, C.R. (1983), Distribution model for binding of protons and metal ions by humic substances, Environ. Sci. Technol., vol. 17, p.654. Playle, R.C., Dixon, D.G. and Burnison, D.G. (1993a), Copper and cadmium binding to fish gills: Modification by dissolved organic carbon and synthetic ligands, Can. J. Fish. Aquat. Sci., vol. 50, p. 2667. Playle, R.C., Dixon. D.G. and Burnison, D.G. (1993b), Copper and cadmium binding to fish gills: Estimates of metal-gill stability constants and modelling of metal accumulation, Can. J. Fish. Aquat. Sci., vol. 50, p. 2678. Playle, R.C., Gensemer, R.W. and Dixon, D.G. (1992), Copper accumulation on gills of fathead minnows: Influence of water hardness, complexation and pH of the gill microenvironment, Environ. Toxicol. Chem., vol. 1, p. 381. Prothro, M.G. (1993), Office of Water Policy and Technical Guidance on Interpretation and Implementation of Aquatic Life Metals Criteria. U.S. Environmental Protection Agency, Washington, DC. Schecher, W.D. and McAvoy, D.C. (1992), MINEQL+: A software environment for chemical equilibrium modelling, Computers, Environment and Urban Systems, vol. 16, p. 65. Skrabal, S. A. and Allen, H.E. (1993), Unpublished results. Steemann-Nielsen, E. and Wium-Andersen, S. (1970), Copper as poison in the sea and in freshwater, Mar. Biol., vol. 6, p. 93. Stephan, C.E., Mount, D.I., Hansen, D.J., Gentile, J.H., Chapman, G.A. and W.A. Brungs. (1985), Guidelines for Deriving Numerical National Water Quality Criteria for the Protection of Aquatic Organisms and Their Uses. U.S. EPA, Office of Research and Development. PB85-227049. NTIS, Springfield, VA. Stumm, W. and Morgan, J.J. (1981), Aquatic Chemistry. Wiley, New York. Sunda, W. and R.R.L. Guillard, R.R.L. (1976), The relationship between cupric ion activity and the the toxicity of copper to phytoplankton, J. Mar. Res., vol. 34, p. 511. Sunda, W. and Gillespie, P.A. (1979), The responses of a marine bacterium to cupric ion and its use to estimate cupric ion activity, J. Mar. Res., vol. 37. p. 761.

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Tipping, E. (1994), WHAM - A chemical equilibrium model and computer code for waters, sediments, and soils incorporating a discrete site/electrostatic model of ion-binding by humic substances, Computers Geosci., vol. 20, p. 973. Tubbing, D.M.J., Admiraal,W., Cleven, R.F.M.J., Iqbal, M., van de Meent, D. and Verweij, W. (1994), The contribution of complexed copper to the metabolic inhibition of algae and bacteria in synthetic media and river water, Water Res., vol. 28, p. 37. U.S. Department of Commerce. (1993), NIST Critical Stability Constants of Metal Complexes Database. Version 1.0 Washington, D.C. U.S. EPA. (1985), Ambient Water Quality Criteria for Copper - 1984. Office of Water Regulations and Standards, Washington, D.C. U.S. EPA. (1992), Interim Guidance on Interpretation and Implementation of Aquatic Life Criteria for Metals. Washington, D.C. U.S. EPA. (1994), Interim Guidance on Determination and Use of Water-Effect Ratios for Metals. EPA 823-B-94-001. Washington, D.C. U.S. EPA. (1995), Method 1669: Sampling Ambient Water for Trace Metals at EPA Water Quality Criteria Levels. Verweij, Glazewski, R and De Haan, H. (1992), Speciation of copper in relation to its bioavailability, Chem. Speciation Bioavail., vol. 4, p. 43. Windom, H.L., Byrd, J.T., Smith, Jr, R.G. and Huan, F. (1991), Inadequacy of NASQAN data for assessing metal trends in the nations rivers, Environ. Sci. Technol., vol. 25, p. 1137. Wood, C.M. (1992), Flux measurements as indices of H+and metal effects of freshwater fish, Aquatic Toxicology, vol. 22, pp. 239-264.

Evaluation of copper guideline values for drinking-water

Dr D James Fitzgerald Environmental Health Branch, Public and Environmental Health Service South Australian Health Commission, Adelaide, South Australia 1. Introduction Since 1925, when the US Public Health Service was the first regulatory agency to set a guideline value for copper in drinking-water, various guidelines have been established based on aesthetic and health considerations. With time, as the essential nature of copper in the diet as well as the toxicity of copper became better understood, it became clear that there is a narrow margin between coppers essentiality and its toxicity (Uauy & Olivares, 1996). How this margin can be better defined and a guideline set is currently an important issue for water regulatory authorities. This issue also becomes important because of the economic ramifications for the copper-pipe industry if a guideline is stringent and difficult to meet. This chapter will examine how present guideline values have been derived, and will evaluate the validity of these derivations. Brief discussion only will be given to the aesthetics-based guideline values before focussing on guideline values based upon health considerations. 2. Historical perspective A compilation of some historical information on the guideline values for copper in drinking-water is shown in Table 1.

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2.1

Aesthetics-based guideline values

Due principally to consumer complaints of coppers staining of laundry and sanitary ware, the aesthetics-based guideline value is now generally 1.0 mg/L. However, the set value in Europe is 3.0 mg/L which should not be exceeded during 12 hours of stagnation (the Netherlands operates on 16 hours). At one stage in the USA, the aesthetics-based value was also 3.0 mg/L but this was reduced to the present-day 1.0 mg/L in 1942 on the basis of taste problems. In a range of experimental studies, it has been demonstrated that copper salts dissolved in water can be tasted at levels of 1-2 mg Cu/L (Bguin-Bruhin et al., 1983; Cohen et al., 1960; Uauy, pers. comm.). 2.2 Health-based guideline values

The US Environmental Protection Agency (US-EPA) was the first regulatory agency to set a healthbased guideline value for copper in potable water. Their 1985 proposal of 1.3 mg/L has since been promulgated and forms part of the Lead-Copper Rule (USEPA, 1988, 1991a). In 1993, the World Health Organization (WHO) set a provisional guideline value of 2.0 mg/L, a figure which WHO has recently re-proposed (IPCS, 1996a). This same value has been adopted in Australia and New Zealand (NHMRC, 1996) and is being proposed in the European Commission (EC, 1996). Thus there is a wider move to adopt health-based guidelines for copper in drinking-water. Table 1: Copper guideline values for potable water
Agency/Regulator and Year Aesthetics-based guidelines US Public Health Service, 1925 US Public Health Service, 1942 US Public Health Service, 1962 WHO, 1958, 1996 EEC, 1980 NHMRC - Australia, 1987 Health-based guidelines US-EPA, 1985, 1988, 1991a WHO, 1993, 1996 NHMRC - Australia, 1996b EC, proposed 1.3 mg/L 2.0 mg/L 2.0 mg/L 2.0 mg/L
a

Value

0.2 mg/L 3.0 mg/L 1.0 mg/L 1.0 mg/L 3.0 mg/L 1.0 mg/L

Modified from De Zuane (1990) and Fitzgerald (1997), with references therein and in Fitzgerald (1995), IPCS (1996a). aNHMRC (1987); bNHMRC (1996)

3. Derivation of health-based guideline values 3.1 US Environmental Protection Agency

The approach of the US-EPA has been to consider reports of acute effects of excess copper ingestion (nausea, vomiting, abdominal pains, diarrhoea). Several case reports of high copper levels in beverages and of suicide attempts with copper sulphate were evaluated (USEPA, 1988). The study was chosen in which the lowest acute oral dose was observed, this being a report of Dr John Wyllie published in 1957. In his now much-discussed paper, Dr Wyllie recounted an incident involving a small number of female nurses who had consumed various amounts of an alcohol cocktail prepared in a copper-containing vessel. Presumably, some of the copper had leached into the drink and resulted in acute effects in most of the nurses. A remake of the cocktail and subsequent chemical analysis for metals provided information on the copper exposures most likely experienced by the cases. Wyllie
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estimated that the smallest amount of copper ingested was 5.3 mg (Wyllie, 1957). The US-EPA divided this figure by an uncertainty factor of 2, then again by 2 litres for average daily water consumption, to derive their drinking-water copper guideline value (Maximum Contaminant Level Goal) of 1.3 mg/L (USEPA, 1988). Close scrutiny of the brief Wyllie paper reveals a paucity of information, particularly regarding details of individuals intakes and symptoms. This, together with a number of ambiguities including doubt over the Lowest-Observed-Adverse-Effect Level, has resulted in the conclusion by some that the study is inadequate for the derivation of a guideline value (Fitzgerald, 1995, 1996a, 1996b; Fewtrell and Kay, 1995). Notwithstanding the recognised need for better data, 1.3 mg/L remains the US Congress-mandated guideline for copper in drinking-water under the Safe Drinking Water Act (USEPA, 1991a, 1991b). A recent extensive survey of water utilities covering about half the population in the USA has shown that probably no more than 1% of the population receives water containing copper at a mean value greater than 1.3 mg/L (G Lagos, pers. comm.). No mean value greater than 2 mg/L was recorded. 3.2 World Health Organisation

In contrast to the approach taken by the US-EPA, WHO did not consider acute effects of copper but instead evaluated daily intakes and some chronic exposure circumstances. Firstly, a provisional maximum tolerable daily intake (PMTDI) was set at 0.5 mg/kg body weight, based on the belief that a 10-fold excess of the daily intake of copper considered normal at the time, ie. 10 times 2-3 mg/day, would not elicit any health problems (Becking, 1996; WHO, 1967, 1982). Multiplication of the PMTDI by 60 kg and 0.1 for proportion of intake attributable to water, then division by 2L water intake per day, yielded the guideline value of 1.5 mg/L; this has been rounded to 2 mg/L (Mercier, 1996). WHO recognise the lack of useful data for setting a guideline, and so add the caveat that this guideline value is provisional. [The process of establishing a guideline value for an essential element using dietary intake, multiplication of that value by an arbitrary number and attributing some proportion of intake to water is questionable.] Secondly, WHO considered the results of an industry-conducted chronic copper-gluconate ingestion study in dogs, claiming that the above PMTDI for humans was based on the experiments NoObserved-Adverse-Effect Level (NOAEL) (WHO, 1982; IPCS, 1996a). This, however, is not the case, but the erroneously reported and transcribed NOAEL (5 mgCu/kg/day), together with a safety factor of 10, seemed to corroborate the PMTDI of 0.5 mg/kg (Fitzgerald, 1995; Mercier, 1996). We now know that the supposed 5 mgCu/kg was actually 15 mg copper-gluconate/kg, being 2.1 mgCu/kg (Fitzgerald, 1995; Shanaman et al., 1972). In the IPCS Task Group (June, 1996) which met for the evaluation of health aspects of copper, toxicologists assessed this dog study and concluded that the liver enzyme effects, noted only in 2 of 12 dogs exposed to the highest dose (8.4 mgCu/kg/day), were reversible and not toxicologically significant (IPCS, 1996b). Thirdly, WHO consider the liver pathologies associated with chronic copper exposure, and are especially mindful of .... concern regarding the possible involvement of copper from drinking-water in early childhood liver cirrhosis in bottle-fed infants, ..... (WHO, 1993, p46). With no reference to quantitative or epidemiological data, the text continues: .... a concentration of 2 mg/litre should also contain a sufficient margin of safety for bottle-fed infants, because their copper intake from other sources is usually low (WHO, 1993). Mention is made by WHO of acute gastric effects of copper, though there is some confusion over this. One document states: Acute gastric irritation may be observed in some individuals at concentrations in drinking-water above 3 mg/litre (WHO, 1993, p46). A more recent publication states: The estimated concentration of copper(II) in drinking-water or beverages that can lead to symptoms of this type [ie. acute symptoms] is 30 mg/litre but may vary with the binding and chemical form of copper present.... (IPCS, 1996a, p222). It is likely that the former concentration of 3 mg/L is nearer the mark for the threshold of gastric effects of copper in drinking-water (Spitalny et al., 1984; Knobeloch et al. 1994; Fitzgerald, 1996a; R Uauy, pers. comm.). For typical drinking-water, few
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components would be present that bind copper and reduce its gastric irritancy potential. In some beverages, for example Tyrolean milk mixture prepared in copper vessels, coppers irritancy is prevented even at levels of 30-60 mg/L (Mller et al., 1996; T Mller, pers. comm.). It is interesting that Ramazzini in 1713 had noted the ameliorating properties of milk and whey in treating occupational disease in coppersmiths (Ramazzini, 1713). 3.3 Australia and New Zealand

The health-based drinking-water guideline value for copper in Australia and New Zealand has been set at 2 mg/L. In deriving this value, essentially the WHO approach has been adopted; substituting 70 kg for the average adult weight does not alter the outcome (NHMRC, 1996). Authorities in these countries have concluded that copper levels exceeding 2-3 mg/L will induce symptoms of acute toxicity in some people. In South Australia, a Health Alert of 3 mgCu/L has been proposed (HAWQ, 1994). 3.4 European Commission

Some water reticulation systems in Europe are comprised of lead piping, and consideration is being given as to whether copper provides a safe alternative. The Scientific Advisory Committee of the European Commission recently conducted an assessment of the evidence for the toxicity of copper in drinking-water. This included examination of WHOs guideline development and of a range of animal and human studies. The main conclusions of this committee were: i) that animal data are insufficient, ii) that human experience suggests that 1-2 mg/L will be acceptable, and iii) that the proposed 2 mg/L in the EC Draft Drinking Water Directive should be retained (EC, 1996). 4. Overall evaluation Evaluation of the copper guidelines is an important process. A guideline that is too lax may be insufficiently protective of human health, while one that is too conservative can impose heavy compliance costs. Already in the USA, hundreds of millions of dollars are spent on corrosion control to reduce copper in water supplies to levels below 1.3 mg/L (G Lagos, pers. comm.). The need is for a scientifically-defensible guideline to ensure that large sums of money are not wasted. Therefore, to progress the copper guideline debate, there is a definite requirement for further human data since some of the key data employed for guideline derivation are clearly inadequate. There is a need for prospective epidemiological studies that delineate copper exposure via drinking-water and acute effects of copper. However, such approaches will be plagued by the high background prevalence of gastrointestinal symptoms in the community. Controlled studies with volunteers will also be extremely useful; at least one such project is in progress (R Uauy, pers. comm.). A better understanding of the threshold for acute gastric effects of copper will contribute most significantly to the honing of a health-based drinking-water guideline value for copper. Acknowledgements I thank Dr Ricardo Uauy, Professor Gustavo Lagos, Dr Thomas Mller and Dr Debdas Mukerjee for their sharing of information and unpublished data. Dr Andrew Langley is acknowledged for his comments on the manuscript. References Becking, G. (1996), In: Lagos GE & Cifuentes L.A (eds), Scientific Basis for the Regulation of Copper in Potable Water. Catholic University of Chile, pp 64-67. Bguin-Bruhin, Y., Escher, F., Solms, J. and Roth, H.R. (1983), Threshold concentration of copper in drinking water, Lebensmittel-Wissenschaft und Technologie, vol. 16, pp. 22-26.

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Cohen, J.M., Kamphake, L.J., Harris, E.K. and Woodward R.L (1960), Taste threshold concentrations of metals in drinking water, Journal of the American Water Works Association, vol. 52, pp. 660-670. De Zuane, J. (1990), Handbook of drinking water quality: standards and controls. Van Nostrand Reinhold, New York. EC (1996), Opinion of the Scientific Advisory Committee concerning toxicologically acceptable parametric value for Copper in Drinking Water. Scientific Advisory Committee to examine the toxicity and ecotoxicity of chemical compounds. European Commission, Brussels, Feb.20, 1996, CSTE/96/6/V. Fewtrell, L and Kay, D (1995) Copper in Drinking Water: An Appraisal of Health Effects and Current Standards. Report of the Centre of Research into Environment and Health (CREH), University of Leeds, UK. Fitzgerald, D.J (1995), Copper guideline values for drinking water: Reviews in need of review?, Regulatory Toxicology & Pharmacology., vol. 21, pp. 177-179. Fitzgerald, D.J (1996a), Copper regulatory level in drinking-water as proposed by Sidhu et al., Regulatory Toxicology & Pharmacology, vol. 23, pp. 173-175. Fitzgerald, J (1996b), A critical review of the copper standard in potable water. In: Lagos GE & Cifuentes LA (eds.), Scientific Basis for the Regulation of Copper in Potable Water. Catholic University of Chile, pp. 55-63, G1-G11. Fitzgerald, J (1997), Safety guidelines for copper in water. In: Lonnerdal B and Uauy R (eds.), Essentiality and Toxicity of Copper. American Journal of Clinical Nutrition Supplement, in press. HAWQ (1994), Health Alert for copper in drinking-water. Governmental Standing Committee, Health Aspects of Water Quality. South Australian Health Commission, Adelaide. IPCS (1996a), Guidelines for drinking-water quality, 2nd ed. Vol. 2. Health criteria and other supporting information. International Programme on Chemical Safety, World Health Organization, Geneva. IPCS (1996b), Environmental Health Criteria Document: Copper. International Programme on Chemical Safety, World Health Organization, Geneva; in press. Knobeloch, L., Ziarnik, M., Howard, J., Theis, B., Farmer, D., Anderson, H and Proctor, M. (1994), Gastrointestinal upsets associated with ingestion of copper-contaminated water, Environmental Health Perspectives, vol.102, pp. 958-961. Mercier, M. (1996), Health risk assessment of chemicals with particular reference to copper in drinking water. In: Lagos GE & Cifuentes LA (eds), Scientific Basis for the Regulation of Copper in Potable Water. Catholic University of Chile, pp 11-15, B1-B10. Mller, T., Feichtinger, H., Berger, H. and Mller W (1996) Endemic Tyrolean infantile cirrhosis: an ecogenetic disorder. Lancet 347, 877-880. NHMRC (1987), Guidelines for drinking water quality in Australia. National Health & Medical Research Council, and Australian Water Resources Council. Australian Government Publishing Service, Canberra. NHMRC (1996), Australian drinking water guidelines. National Health & Medical Research Council, and Agricultural & Resource Management Council of Australia and New Zealand. Australian Government Publishing Service, Canberra. Ramazzini B (1713), De Morbis Artificum. In: Wright WC, Diseases of Workers: The Latin text of 1713. The University of Chicago Press, Chicago, 1940. Shanaman, J.E., Wazeter, FX. and Goldenthal, E.I. (1972), One year chronic oral toxicity of copper gluconate, W10219A, in Beagle dogs. Research Report No. 955-0353. Warner-Lambert Res. Inst., Morris Plains, New Jersey.
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Spitalny, K.C., Brondum, J., Vogt, R.L., Sergent, H.E. and Kappel S (1984), Drinking-water-induced copper intoxication in a Vermont family, Pediatrics , vol. 74, pp. 1103-1106. Uauy, R and Olivares, M (1996)(eds.), Copper nutrition in humans: essentiality and toxicity, The American Journal of Clinical Nutrition, vol. 63, pp. 791S-852S. US EPA (1988), Drinking Water Regulations: Maximum contaminant level goals and national primary water regulations for lead and copper, Federal Register , vol. 53, no.160, pp. 31516-31578. US EPA (1991a), Maximum contaminant level goals and national primary drinking water regulations for lead and copper; final rule, Federal Register, vol. 56, no.110, pp. 26460. US EPA (1991b), Drinking water regulations; maximum contaminant level goals and national primary drinking water regulations for lead and copper, Federal Register, vol. 56, no.135, pp. 32112-32113. WHO (1967), Specifications for the Identity and Purity of Food Additives and Their Toxicological Evaluation: Some Emulsifiers and Stabilizers and Certain Other Substances. Tenth Report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, No. 373. World Health Organization, Geneva. WHO (1982), Evaluation of Certain Food Additives and Contaminants. Twenty-Sixth Report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, No. 683, pp 31-32. World Health Organization, Geneva. WHO (1993), Guidelines for Drinking-Water Quality, 2nd ed. Vol.1. Recommendations. World Health Organization, Geneva. Wyllie, J. (1957), Copper poisoning at a cocktail party, The American Journal of Public Health vol. 47, p. 617.

Population exposure to copper in drinking water

Gustavo Lagos Catholic University of Chile Abstract Surface waters used for the production of drinking water contain a median level of 10 g/L of copper. Values above this level (up to a few milligrams/litre) in drinking water reflect either the existence of high copper content in the source water, due for instance to the presence of acidic soils, or the leaching of copper from plumbing fixtures and copper piping during transport and distribution. The exposure of the population to copper from drinking water depends on several factors: the water chemical composition, the stagnant contact time between water and the pipe, the age of the pipe, the installation procedures of the copper pipes, the use of copper pipes in the water distribution networks, and the drinking habits of the population. All these factors, except the last one, are intrinsically considered in the copper monitoring data provided by the American Water Works Association, AWWA, and taken from house water taps by 284 Water Utilities in the USA, which supply water to a population of 105.8 million people. A total of 27,407 samples were taken by these utilities in order to comply with the USEPA regulation about copper and lead in drinking water. The mean concentration of the distribution of copper concentrations of the mentioned population is 0.284 mg/L. It was assumed that the consumption of water is evenly distributed throughout the day, therefore the mean exposure of this population was 0.4 mg/day, assuming a consumption of 1.4 L per adult per day. In a worst case scenario, if all people drank all the water from the first draw in the morning, just after stagnation, then the average concentration ingested would be 0.987 mg/L, and the total mean exposure to copper from drinking water would be 1.38 mg/adult/day.
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1. Introduction Surface waters used for the production of drinking water contain a wide range of copper concentrations that vary between 0.5 and 1000 g/L. A median level of 10 g/L is reported by Davies and Bennet (1985). Values above these levels (up to a few milligrams/litre) in drinking water usually reflect the by-product liberation of copper from plumbing fixtures and copper piping during water transport and distribution. This paper examines the main factors that produce liberation of copper by products to water from copper plumbing pipes. The calculation of the population exposure to copper in drinking water is based on the monitoring data of the American Water Works Association of the USA (AWWA, 1996; Edwards et al., 1996a) generated since 1991. 2. Factors that affect the population exposure to copper in drinking water The exposure of the population to copper from drinking water depends on several factors: the water composition, the stagnant contact time between water and the pipe, the age of the pipe, the installation procedures of the copper pipes, the use of copper pipes in the water distribution networks, and the drinking habits of the population. 2.1 Water composition

The concentration of copper after water transport and distribution in a copper pipe depends on the chemical composition of the water, mainly its pH and alkalinity (Schock and Neff, 1988; Dodrill and Edwards 1994; Schock et al., 1996). Other compositional variables such as the dissolved inorganic carbon, organic substances and other compounds also play a role in copper by-product release (Cruse et al., 1988; Rehring et al., 1994) Dodrill and Edwards (1994) conclude that copper in drinking water exceedance problems, with respect to the United States Environmental Protection Agency, USEPA, action level (when the 90th percentile copper concentration exceeds 1.3 mg/L), are confined mainly to two water characteristics: 1. 2. when pH < 7.0 & alkalinity < 30 mg/L as CaCO3 when pH < 7.8 & alkalinity > 90 mg/L as CaCO3

Accordingly the main strategy for reducing copper by-product release should be to modify pH and/or alkalinity. Also phosphate inhibitors are used in the USA as corrosion inhibitors for iron, copper and lead, and can be effective in reducing copper release only below pH 7.8. Above this value its effects are highly variable (Dodrill and Edwards, 1994). It should be added that the 90th percentile concentration, according to the regulation of the USEPA, is defined as the concentration of the 90th sample, out of a hundred, after the concentration of each sample has been sorted in ascending order. 2.2 Stagnant contact time

The copper content of water that has been stagnant and in contact with a copper pipe increases its concentration to any value generally between 0.1 and 10 mg/L of copper, depending mainly on water composition and on the age of the pipe (Lagos, 1996b; Meyer, 1996). When water is flushed after a stagnation period, a few liters of water can be collected at a high concentration. After flushing water for more than 3 to 5 minutes, the copper concentration falls to the source level, i.e., usually to a few micrograms per liter. 2.3 Age of copper piping

Studies conducted in Germany and in other countries show that for new copper pipes the copper by product release from the pipe to the water, also denominated cuprosolvency, is greater than for old copper pipes. Meyer (1996) showed that the reduction of copper concentration of waters can range
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from 2 to approximately 10 times, when a copper pipe ages. The reduction factor depends mainly on the water composition. The distinction between new pipe and old pipe is not quite that clear cut in terms of time, but data available suggest that usually the concentration of copper in drinking water that has been stagnant in a copper pipe for a few hours, begins to fall after 12 months of use of the pipe and that this reduction after 24 months of pipe use is quite substantial. The reduction of copper by-product release in the case of old pipes can be explained by the build up of a protective layer at the inner surface of the pipe that protects the metal from corrosion. There are numerous studies regarding the nature and properties of copper pipe films, but there are no conclusive findings about the composition of these films and the way that they build up (Lagos, 1996b). 2.4 The design and installation procedures

The liberation of copper by-products from a copper pipe to the water can be influenced by network design parameters such as high velocity flowing water, pipes in contact with different types of soil or concrete, differences in temperature, excessive number of bends in the network, electrical grounding contacts, stresses built up in the piping system, etc (Cruse et al., 1988). Also, copper pipes can be joined by mechanical fittings, by welding, by brazing or by soft soldering (Cruse et al., 1988; Mattson, 1990). Fluxes are used in brazing and soft soldering. Liberation of copper by-products to the water is produced when the materials that constitute the joint come into contact with water and with copper. The result is usually to accelerate the copper pipe corrosion at the joint due to chemical attack by the jointing materials or by galvanic corrosion. Jointing corrosion has been extensively studied and materials and methods are available in many countries in order to prevent it. These developments have taken place mainly during the last decade and they are still not applied in many developing countries. 2.5 The use of copper in the distribution network

The use of copper in plumbing pipes varies widely throughout the world (CDA, 1994). It is more than 90% in the U.S.A. and the U.K., i.e., less than 10% of pipes of other materials are used in plumbing tubes in these countries. It is about 50% in Germany, between 50 and 60% in Spain and in France, close to 40% in Italy, and approximately 12% in Japan. Also, some countries like Germany, preclude the use of copper in plumbing tubes when the water composition is prone to copper by product release, and this may affect less than 5% of the country waters. In other countries, such as the U.S.A., the regulation was required since 1991, that water utilities treat the water, when the copper action level is exceeded, so as to prevent copper by-product release (Lagos, 1996a). 2.6 Drinking habits of the population

As well as the above factors, the drinking habits of the population, i.e., the time at which people drink water from the tap, the selection from the tap or from a kettle, the use of one versus several taps in the house, etc., affect the total copper ingested. 3 Analysis of water utility monitoring data of copper in drinking water in the USA Dodrill and Edwards (1994) have examined data originally compiled during a 1991 AWWA survey, which included 366 utilities that distribute water to 159 million people and which represent more than 60% of the US population. This study considered the effects of pH and of alkalinity, with or without the addition of corrosion inhibitors on 90th percentile lead and copper concentrations and some of its conclusions were described before. Parts of this database, added to information about population per utility, was made available for this study. Also some monitoring data before and after corrosion treatment of the water for some utilities was obtained in order to assess the efficiency of the treatment.
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The data base analysed (Edwards, 1996; AWWA 1996) contains the population data of 284 utilities (out of a total of 366), the number of samples taken by each utility, the 90th percentile, the lowest and the highest copper concentrations measured by each utility, the pH, the alkalinity and also the corrosion treatment status of the waters at the time of monitoring. The 284 utilities supply water to a population of 105.8 million people. A total of 27 407 samples was taken by these utilities in order to comply with the US EPA regulation about copper and lead in drinking water (US EPA, 1991). The F test performed with the original sample (366 utilities) and with the reduced sample (284 utilities) showed that the value of F is 0.965, well outside the critical region defined by F<0.751 & F>1.338. There is 99% confidence that there is no difference between the variances of the two samples (366 = 284) and hence, there is no significant error introduced by the sample reduction. Figure 1 shows the copper concentration distributions for the 284 utilities: highest, 90th percentile, mean and lowest.

Figure 1 : Distribution of minimum, mean, 90% tile and maximum copper monitoring results for 286 AWWA utilities, USA, 1991. The mean concentration of each utility was calculated with the following equation:

mean=

lowest

90th%

) 2*0.9 + (

90th%

highest)

/2*0.1

The results of this calculation were tested with available utility distributions of copper concentrations and it was found that the error was within 2.7%. The mean copper concentration of one utility represents the mean exposure of the population supplied by that utility, provided that the water consumption is spread evenly during the day. The highest concentrations measured are weakly related (correlation coefficient R2 of 0.15) to the number of samples taken by each utility, within the range of samples required by the Lead-Copper Rule (US EPA, 1991). This means that the highest value would not change substantially if more samples were taken. The concentration distribution of utilities include therefore factors such as stagnant contact time, new and old pipes, water composition, and jointing factors, any one of which can cause high copper byproduct release. Figure 2 shows the distribution of mean copper concentrations for the 284 utilities, and also the distribution of mean copper concentrations for 118 utilities (33.3 million people) whose water had not received corrosion treatment before monitoring. It should be added that the remaining 168 utilities had received corrosion treatment in order to reduce iron corrosion. This treatment consisted of pH adjustment and/or addition of phosphate inhibitors. The sample of 118 utilites without corrosion control is not statistically representative of the total sample (F test).

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This shows that iron corrosion control introduces a significant statistical difference between the concentrations of copper present in the water, with and without treatment. Nevertheless, figure 2 shows that the two distributions are very similar especially at the low end of the concentrations.

Figure 2: Mean population exposure to copper from drinking water (analysis of AWWA WITAF project) The mean concentration of the total distribution is 0.284 mg/l, and the maximum mean exposure is 1.96 mg/l. This distribution signifies that 1.9% of the sampled population (total of 105.8 million) is exposed, to an average concentration over 1 mg/L of copper, 0.49% is exposed to more than 1.3 mg/L and none is exposed to more than 2 mg/L. The mean concentration of the no treatment distribution is 0.367 mg/L and the maximum mean exposure is the same as in the total distribution. According to the no treatment distribution, 5.27% of the population is exposed to more than an average concentration of 1 mg/L and 1.04% to more than 1.3 mg/L. In the sample analysed, there are 15 utilities that had their highest reading above 3 mg/L of copper and two of them above 5 mg/L. The utility with highest value had 7.6 mg/L of copper. No estimation of the potential exposure has been made but it is apparent that a population in the thousands has been potentially exposed to concentrations above 3 mg/L at least during certain periods of the day. It should be stressed that these data points were included in the calculation of mean exposure discussed above. From the data of figure 2, and assuming a daily intake of drinking water of 1.4 litres (WHO, 1993), daily intakes of copper in adults will vary between less than 0.1 mg and 2.74 mg per day. The data shown are no longer reproducible because after 1991 the utilities that exceeded the copper action limit defined by EPA began treating the water in order to reduce copper and lead by-product release. Data after this treatment are scarce, but the example of two utilities is worthy of note: the first one (see figure 3) supplies water to a population of 175 thousand people and its 90th percentile concentration before treatment was 2.26 mg/L, with a highest concentration of 4.56 mg/L and a minimum close to zero. The pH before corrosion control was 7.1 and the alkalinity was 268 mg/L as CaCO3 . To meet the action limit the utility raised pH to 7.4 and lowered alkalinity to 98 mg/L. Two subsequent monitoring events conducted after this water quality change was implemented demonstrated that the 90th percentile concentration dropped to 0.31 mg/L, and the highest concentration to 0.6 mg/L. Release of copper by-products was mitigated at all houses. A second utility achieved an overall reduction of 90 to 95% of the release of copper by-products after corrosion water treatment.

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Figure 3: Copper concentration monitoring for one water utility in Colorado, USA, before and after corrosion treatment (AWWA WITAC project database). 4. Conclusions The data base analysed contains the population data of 284 water utilities in the USA that supply water to a population of 105.8 million people. A total of 27407 samples were analysed for copper by these utilities in order to comply with the US EPA regulation about copper and lead in drinking water (US EPA, 1991). The mean copper concentration exposure from drinking water of this population is 0.284 mg/L provided that water consumption is spread evenly during the day. The maximum mean exposure is 1.96 mg/l. Of the sampled population, 1.9% is exposed to an average concentration over 1 mg/L of copper, 0.49% is exposed to more than 1.3 mg/L and none is exposed to more than 2 mg/L. In the case of the waters that are not treated for iron corrosion control, which are supplied to 31.5% of the total population sampled, the mean copper concentration exposure is 0.367 mg/L and the maximum mean exposure is the same as in the total distribution. According to the no treatment distribution, 5.27% of the population is exposed to more than 1 mg/L as an average, and 1.04% to more than 1.3 mg/L. Assuming a daily intake of drinking water of 1.4 litres, daily intakes of copper in adults vary between less than 0.1 mg and 2.74 mg per day for the two copper concentration distributions considered. References American Water Works Association (1996), Database for AWWA WITAF Project: Initial Monitoring Experiences of Large Utilities under USEPAs Lead Copper Rule. Version 2 including modifications of Dodrill and Edwards, Denver CO. CDA Annual data (1994), Copper Brass Bronze: Copper Supply & Consumption in the USA in the period 1973-1993. Copper Development Associaton, USA. Cruse, H., Von Franque, O. and Pomeroy, R. (1988), Corrosion in Potable Water Systems, Chapter 5 of Corrosion in Pipes, Published by the American Water Works Association, pp 317- 416. Davies D.J.A., and Bennet, B.G. (1985), Exposure of man to environmental copper: an exposure commitment assessment, Sci. Total Environ, vol. 46, p. 215-227. Dodrill D. and Edwards M (1994), Corrosion Control on the basis of utility experience, submitted to J. of Am. Waterworks Assoc., December.

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Edwards, M., Patel, S., Dodrill, D.M., Reiber, S. and Perry, S. (1996a), A general framework for corrosion control based on utility experience, in press, AWWA, Denver, CO. Edwards, M., Schock, M.R and Meyer, T.E. (1996b), Alkalinity, pH and copper corrosion byproduct release, submitted to J. AWWA, January. Lagos, G.E. (1996a), Regulatory aspects of copper in drinking water & corrosion of copper plumbing tubes: a review, in press. Catholic University of Chile, 93 pp, 1996. Lagos, G.E. (1996b), Introductory Remarks , published in Scientific basis for the regulation of copper in potable water. Edited by G.E. Lagos and L.A. Cifuentes, Published by the Catholic University of Chile, January. Mattson, E. (1990), Copper and Brass for Plumbing: a Guide for Corrosion Prevention, Procedings of Copper 90 Refining, Fabrication, Market, October, The Institute of Metals. Meyer, E. (1996), Determinants of Copper Intake from water, International Conference on Genetic and Environmental Determinants of Copper Metabolism, NIH, Washington D.C., March 18-20. Rehring, J.P., and Edwards M, (1994), The Effects of NOM and Coagulation on Copper Corrosion, Proceedings 94, National Conference on Environmental Engineering",USA. Schock, M.R., and Neff, C.H. (1988), Trace metal contamination from brass fittings, J. of Am. Waterworks Assoc., vol. 7, p. 47-56. U.S. Environmental Protection Agency (US EPA) (1991) Maximum contaminant level goals and national primary drinking water regulations for lead and copper; final rule. 40 CFR Parts 141 and 142. Fed. Reg. 56:110. WHO (1993), Guidelines for the quality of drinking water, 2nd ed.. Volume 1: Recommendations. World Health Organisation, Geneva.

Risk assessment for essential trace elements: A proposed methodology

Dr George C. Becking International Programme on Chemical Safety World Health Organization 1. Introduction Risk assessment to protect human health and the environment from the adverse effects of chemicals is a scientific activity. It provides a framework for the critical examination of all available scientific data in order to define and characterise dose-response relationships in humans in as quantitative a manner as possible. For non-essential chemicals it has a well developed methodology (NRC, 1983; NRC, 1994; IPCS, 1994) and has been used to develop protective health and environmental-based guidance values within countries (Barnes and Dourson, 1988) and internationally (IPCS, 1987; WHO, 1996). However, in the case of essential trace elements (ETEs) there needs to be a re-examination of the scientific principles and methods used in any risk assessment process for such elements. The development of toxicologically- based guidance values for some ETEs (e.g. zinc) which are very near, or in conflict with, the recommended daily allowance serves to reinforce this view (Smith, 1994). For ETEs we have what has been called a U-shaped dose-response curve, that is, adverse effects may result from deficiency associated with low intakes as well as be associated with high intakes (Goyer, 1994; Bowman and Risher, 1994). There is, therefore, a need to define a tolerable intake (TI) to prevent toxicity as well as the level at which the risk of deficiency for the general population is minimised. As is done for non-essential chemicals, one cannot assume that a zero exposure is without adverse effects.
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It is hoped that this paper can present some ideas on how the scientific principles of toxicology and nutrition can be applied to the evaluation of ETEs. Before proposing a methodology it is important for the reader to know what is actually meant by risk assessment within the International Programme on Chemical Safety (IPCS) and then summarise the proposed methodology considered useful internationally. IPCS in proposing such a methodology has drawn greatly upon earlier efforts to examine this issue (Mertz et al., 1994; Nordberg and Skerfving, 1993). The methodology proposed will be for non-carcinogenic ETEs. 2. Risk assessment and IPCS 2.1 IPCS goals in risk assessment methodology

The IPCS is a scientifically based response to problems caused to human health and the environment by the expanded use of chemicals. It was initiated in 1980 by three cooperating organisations (United Nations Environment Programme, International Labour Organisation and the World Health Organization), to provide scientific support for the development of chemical safety programmes in Member States. Emphasis would be placed on assessing the risks from chemical exposures (eg. zinc, copper, dioxins, etc.) and the development and improvement of the methodology for risk assessment. These are, in fact, two of the six objectives agreed upon by the Cooperating Organisations and Member States in 1980. It is within this framework that IPCS is concerned over the methodology used to assess human health and environmental risks from exposure to ETEs. It is part of the international effort by IPCS to improve the scientific foundation for such assessments which hopefully will result in an harmonisation of risk assessment methodologies worldwide. 2.2. Concepts and definitions

Within IPCS, the four step process first put forward by the US National Academy of Sciences (NRC 1983) is considered an appropriate framework for the evaluation of health and environmental data related to effects of chemical exposures. An adaptation (NRC 1994) of this process is presented in Figure 1. The four steps are still hazard identification, dose-response assessment, exposure assessment, and risk characterisation. An important step included in Figure 1 is the identification of research needs in order to lessen the uncertainty of future evaluations. For ETEs, it is hoped to show this is an essential step for both nutritional and toxicological aspects. Much of the uncertainty with regards to evaluation of ETEs results from an inadequate database and a lack of understanding of mammalian homeostatic mechanisms.

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Figure 1: NAS/NRC risk assessment/management paradigm (NRC 1994). In describing the process in Figure 1, hazard identification and dose-response evaluation involve comparing all available experimental animal and human data and the associated doses, routes and duration of exposure to determine if an agent causes the toxicity in question (neurotoxicity, general systemic toxicity, etc.) and the relevant exposure conditions under which this would occur. Exposure assessment ideally provides an estimate of human exposure from all sources and identifies the human populations exposed and the magnitude and duration of such exposures. Simply stated, risk characterisation is the summation of all the other steps and includes an estimation of the incidence and severity of the adverse effects that are liable to occur in a population or ecosystem due to actual or predicted exposures. It includes a description of all default positions taken and the level of uncertainty within the overall assessment. In the development of guideline values as described by Barnes and Dourson (1988), IPCS (1994) and IPCS (1987), the process is more of a safety assessment than a true probabilistic risk assessment. That is, a threshold is assumed for non-cancer end-points, a no-observable adverse affect level (NOAEL) (or lowest observed adverse effect level, LOAEL) identified and a guidance value developed by the use of uncertainty or modifying factors to reflect the uncertainties in the data base and the severity of the critical effect identified. That is, exposure guidelines for a chemical are developed which are assumed to pose no risk to the population (see Figure 2).

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Figure 2: Comparison of the safety assessment and probability models for assessment risks from chemical exposures. Many definitions and terms have been proposed for the health-based guidance values developed by the process in Figure 2. These include: ADI-acceptable daily intake (IPCS 1987); TI or TDI tolerable or tolerable daily intake (IPCS 1994); PTW(D)I - provisional tolerable weekly (daily) intake (IPCS 1987). These are all defined as estimates of exposure (intake) of a substance over a lifetime that is considered without risk to most of the population. Although tolerable means "tolerated"; not necessarily acceptable (IPCS 1994), the US Environmental Protection Agency prefers to utilise the term RfD - daily oral reference dose (Barnes and Dourson 1988). This is an estimate of a daily exposure (intake) to the human population (including sensitive subgroups) to be without appreciable risk during a lifetime with uncertainty spanning an order of magnitude. All of these estimates are given as mg (g) /kg body weight.

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2.3

Development of tolerable intakes for non-essential chemicals

All of the estimates described in section 2.2 are derived using similar default assumptions and the safety assessment process in Figure 2. The NOAEL or LOAEL is usually determined from data obtained from the most appropriate (to humans) species, or the most sensitive species of experimental animal. However, where human data of adequate scientific quality are available this is used to avoid the uncertainties of interspecies extrapolation. Most often there is much scientific uncertainty with the data available, for example, its relevance to humans (interspecies extrapolation) and the variation in susceptibility of individual humans (intraspecies variability). Other factors leading to the uncertainty include adequacy of the pivotal study, nature of the toxicity (severity) and the adequacy of the overall database. Adjustments are therefore made to the data through the use of uncertainty factors (UFs) which actually reduce the dose rate to account for uncertainties or inadequacies in the scientific database. Traditionally, UF of 10 for intraspecies variability and one of 10 for interspecies differences have been used, UF1 x UF2, for a total of 100 (IPCS 1987). Other UFs ranging from 1-10 have been suggested as modifying factors (MF) for adequacy of data (eg. less than chronic exposure) and severity of endpoint (eg. reproductive versus a general systemic effect which is often reversible). To obtain the TI one then divides the NOAEL or LOAEL by the product of the uncertainty factors (NOAEL or LOAEL UF1 x UF2 x MF). When the product of the factors is greater than 5000 one should question whether the data base is of sufficient quality to make a reasonable estimate of the TI. Where data from humans are used to derive a TI, UFs used are usually not larger than 10 and, in fact, values of 2 or 3 are often used where the data are robust and a good dose-response relationship was reported. 3. Development of recommended safe and adequate daily dietary intakes It is beyond the scope of this paper to discuss in detail the development of Recommended Dietary Allowances (RDAs) - "levels of intake of essential nutrients that, on the basis of scientific knowledge, are judged to be adequate to meet the known nutrient needs of practically all healthy persons" (NRC 1989) or an Estimated Safe and Adequate Dietary Intake (ESADDI). The latter forms interim guidance until adequate data become available to establish an RDA. However, there is merit in reviewing the basic principles for such estimations and compare these to the methodology for estimating an TI. More details on the derivation of RDAs and ESADDIs and a comparison of the methodologies used in estimating both RDAs and TDIs can be found in Mertz et al. (1994) and Bowman and Risher (1994). In summary, the first step is to determine the average requirement (x) assuming a normal distribution within the population, assume a coefficient of variation (usually 15%) and calculate an RDA (RDA = x +2 SD = 1.3 x ). Such data are usually obtained from studies of human populations, although extrapolation from animal experiments has been used to recommend an ESADDI for selenium of 50200 g/day (Mertz et al., 1994). For details on the use of balance studies, human exposure studies and Factorial Estimated Requirements in deriving RDAs the reader is referred to WHO (1996) and Mertz et al. (1994). 3.1 Comparison of methodologies for derivation of RDAs and TIs

A few important differences in the assumptions underlying the derivation of RDAs and TIs are: (1) RDAs assume all intake is from dietary sources, whereas, for TIs all sources of exposure are considered; (ii) in deriving RDAs bioavailability, homeostatic regulatory mechanisms, food consumption and dietary interactions are all considered, whereas, for a TI the toxicity measured is rarely modified by similar considerations; (iii) the RDA is considered protective of 97.5 percent of the population whereas TIs are usually considered more protective. In fact, RfDs (another term for a TI) derived by the US EPA are by definition considered protective of 100 percent of the population
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(Bowman and Risher, 1994); and (iv) the toxic endpoint considered adverse in deriving a TI, particularly from animal experiments, is often much less significant clinically in humans than the known severe clinical manifestations found in human deficiency states for ETEs. 4. Principles and methods for assessing human health risks from exposure to an ETE 4.1 4.1.1 Scientific principles The acceptable range of oral intake for an ETE

For an ETE by definition there are risks associated with both low (deficiency) and high (toxicity) intakes. The relationship between intake of ETEs and risk is best represented by the U-shaped curve as shown in Figure 3. There is a range of intake below which the risk from the adverse effects of deficiency increase in the population and at the upper end of this range the risk from adverse toxic responses increases as exposures increase. The range between point A, the RDA or ESADDI and point B, the TI, can be considered an acceptable range of oral intake (AROI). A similar concept was first proposed by Beaton (1988) and the range was termed "a safe range of intake". Within this range normal homeostasis is maintained. Neither the lower or upper boundary of the AROI should be considered an absolute value, below or above which adverse effects in a population or individual will be initiated. In fact, RDAs and TIs are not guidelines for individuals but for populations.

Figure 3: Percent of population subjected to deficiency and toxic effects according to exposure or intake of the essential trace element (ETE). As intake drops below A (lower limit of the AROI) risk for deficiency increases, reaching 100 percent at extremely low intakes. As intakes increase beyond B a progressively larger proportion of subjects will exhibit effects of toxicity. For ETE the oral route is by far the predominant exposure pathway for humans and for this reason the term AROI is maintained to facilitate comparison of the RDA which only considers oral intakes and the TI for toxicity. The concept of a range of intakes within which normal homeostasis is maintained can be used in situations where inhalation becomes a major exposure pathway, for example, in occupational exposures. One then needs to consider an acceptable range of total intake (ARTI). However, for this paper the AROI will be used and the range suggested would apply to protection of normal, healthy, non-occupationally exposed populations. 4.1.2 Determination of boundaries of AROI In deriving the upper bound (point B, Figure 3) of the AROI the use of UF/MFs to derive a TI from a NOAEL (see section 2.3) needs to be less rigidly applied and other factors such as bioavailability and
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nutrient interactions need to modify the choice of any UF used. Such an approach has been suggested by Abernathy et al. (1993) in the derivation of a RfD for zinc where an UF of 3 rather than the usual factor of 10 when a LOAEL from a human study is used. The lower UF took into account nutritional and energy requirements but no consideration was given to the level of bioavailability or nutrient interactions. It should be noted that this RfD (0.3 mg/kg/day in adults) does not provide the RDA for infants, preadolescent children, or possibly lactating women (Abernathy et al., 1993; Sandstead, 1993). The conflict remaining between the RDA and the US EPA RfD for zinc raises other principles to be considered in assessing the risks from all ETEs. In using even a modified approach to setting TIs for an ETE, toxicologists so used to the principle that zero exposure for toxic materials is to be strived for, fail to consider bioavailability of the ETE and possible nutrient interactions in animal or human studies even in cases where levels well in excess of the RDA were consumed and in a form markedly different than that found in normal diets. Animal experiments and some human controlled studies use pharmacological doses of extremely bioavailable forms (eg. chlorides or gluconates) of the ETE when compared to the human dietary situation. It is essential that toxicologists make better use of the available studies on the ETE intake in normal healthy populations to validate the TI derived from animal experiments. Clearly, from a public health perspective, it would be unwise to define an AROI that is outside the range of customary intakes of healthy populations worldwide. 4.1.3 Clinical equivalency of end-points for deficiency and excess exposure In defining an AROI for an ETE, for example, copper, a key issue is the appropriate assessment of the effects of deficiency and toxicity relevant to human health. The most sensitive indices of copper deficiency or toxicosis are biochemical markers with no clear functional or health significance. On the other extreme, death associated with organ damage induced by excess copper or deficiency are clearly of health significance but are not relevant as sensitive early indicators of health risks in preventive public health programmes. Examples of the type of effects to assess health risks associated with copper exposure need to be described and their health significance (adversity) ascertained. Biochemical changes such as in erythrocyte superoxide dismutase (ESOD) activity as an index of copper deficiency (Uauy et al., 1985) or changes in the plasma Cu/Ceruloplasmin molar ratio as an index of copper excess are sensitive but not significant indicators of health risks. Biological indices of subclinical effects indicating potential adverse effects of copper deficiency or excess on organ function need to be developed. For example, decreased white blood cell phagocytic capacity in the case of deficiency or increased serum aminotransferase or transpeptidase hepatic enzymes in response to excess copper are often used in human studies. The important concept shown by these examples is the principle of comparable effects to define effects of excess and deficiency of copper. A set of theoretical curves developed by Dr G. Nordberg, University of Umea, Sweden, further illustrating this point is given in Figure 4. One should select effects of similar health significance to define the upper and lower ranges of the AROI. In general, the range should be defined by dose-effect (response) to intake/exposure levels that prevent the appearance of subclinical adverse effects. The review of these indices and the corresponding studies should be done by toxicologists and nutritionists familiar with health risk assessment. The combined effort should yield clearly defined critical end-points for the upper and lower ranges of the AROI given the available information. Even if the human information is limited, a starting point in the definition of AROI should be the customary intake/exposure determined in "healthy" populations in various regions of the world.

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Figure 4: Theoretical dose-response curves for various effects occurring in a population at various levels of intake (doses) of an essential trace element. While lethal effects and clinical disease must always be prevented, subclinical effects indicating impairment of organ function are often identified as critical effects. The lower end of the dose-response curve for such critical effects related to deficiency (curve 3) and toxicity (curve 6) define the range of acceptable daily oral intakes. Biochemical effects without functional significance (curves 4 and 5) are considered without health impact and should not be taken as critical effects. 4.1.4 Range of acceptable intake for healthy populations

Another guiding principle in defining the AROI is that the acceptable exposure range should be safe for, but not expected to meet the requirements or prevent excess in, groups at special risk. For example, the copper needs of patients with chronic diarrhoea or of patients on chronic haemodialysis may fall outside the AROI. In both of these cases intakes that are of concern for the "healthy" population may be required to meet the special needs of these patients. Safe copper intake/exposure levels for haemodialysis patients may lead to deficiency in healthy subjects while intakes required by patients with chronic diarrhoea may be excessive for normal subjects. The upper and lower cutoff points for the range of acceptable oral intakes should be defined for population groups. The lower cutoff point should be sufficient to meet the requirements of most individuals in the population. Based on criteria used to define RDAs "most" usually implies 97.5% of the population; if the mean and standard deviation for requirements are known this point is defined by the mean + 2 SD, if the SD is not known a coefficient of variation of 15% is customarily used to account for population variability in requirements for ETEs. Similarly, the upper boundary should protect most individuals from the risk of toxicity. A statistical definition for "most" in this context is lacking, but could be derived based on the mean and distribution of toxic effect doses. Using the traditional toxicological approach, an ED50 can be defined and, based on known variability, or extrapolation of the dose-effect (response) relationship, an ED2.5 determined. Figure 5 summarises the concept of population-derived boundaries for the AROI. Special consideration of upper and lower cutoff points should be made in defining AROI for physiological conditions affecting normal populations, such as infancy, pregnancy, lactation, and aging. In the case of the lower boundary of the

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AROI these are usually considered in setting RDAs but, for the upper cut off point specific values for these population categories is often lacking but should be determined as a research priority.

Figure 5: Ideal model for distribution of intakes to meet nutritional requirements of a health population and prevent toxicity. The lower limit to the AROI should cover the requirements of most (97.5%) of the population while the higher limit of the AROI should protect most of the population from toxic effects. In the case of essential elements it is clearly impossible to assure that all (100%) of any population will be protected from deficiency or excess levels of ETEs such as copper. The range of AROI defined at the international level is not intended to address disease conditions or genetic alterations in copper metabolism which determine special sensitivity to excess or deficient exposures. This would require a change in philosophy of agencies using the US EPA RfD in their public health programmes. The AROI is clearly not intended to meet the special needs of population subgroups with genetic alterations of copper metabolism, such as Menkes syndrome or Wilson's disease, and as more data become available probably idiopathic childhood cirrhosis which most likely has a genetic component in its aetiology (Uauy, personal communication). 4.2 The AROI concept in human health risk assessment - A proposed methodology

A summary of the scientific principles supporting the use of an AROI in the assessment of human health risks from ETEs is given in Table 1. Many of these were agreed upon at a workshop in 1992 (Mertz 1993) and some have been applied in the derivation of an RfD for zinc (Abernathy et al., 1993).

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Table 1: Principles underlying use of the Homeostatic Model in human health risk assessment (1) For all ETEs there is a "zone of safe and adequate exposure" a zone compatible with good health - an acceptable range of oral intake (AROI). (2) Both nutritional scientists and toxicologists must be involved in developing an AROI. (3) Data on toxicity and deficiency should receive equally critical evaluation. (4) The concept of bioavailability (biologically effective dose) should be applied and nutrient interactions considered when known. (5) Chemical species studied and the route and mode of application should be fully described. (6) Biological end-points used to define the lower (RDA) and upper (toxic) boundaries of the AROI should have similar degrees of clinical significance. (7) Safety margins and uncertainty factors are used to determine both boundaries of the range. They are usually higher for overexposure but need to be applied taking nutritional needs into account.
Adapted from Mertz, 1993.

When the principles in Table 1 are applied within the framework shown in Figure 6, it should be possible to provide guidance to all countries on the exposure levels for any ETE which would provide adequate nutrition and be without risk from toxicity. The iterative nature of this proposed scheme makes it possible to identify research needs and accommodate special groups within the population with advisories regarding exposures. It is essential that this scheme be applied by nutritional scientists and toxicologists working together to address such aspects as data quality, critical effects, dose-response, bioavailability and nutrient interactions. It is essential that all scientists involved practice sound scientific judgement during each step and make it clear whenever default positions are taken in lieu of scientific data.

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Figure 6: Application of principles for the assessment of risk from essential trace elements. The scheme in Figure 6 should not be considered a new and novel way of assessing health risks. It really represents the thought process used by those scientifically competent and versed in the procedures used to carry out evaluation of data on ETEs whether from studies on experimental animals or humans. The end result should be a range of recommended exposures fully protective of human health, something a single number for an ETE can never do.

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5. The Deficiency - Toxicity (DT) Model in Environmental Risk Assessment It is well beyond the scope of this paper, and the expertise of the author, to discuss in detail the scientific basis for assessing risks to the various compartments of the environment from chemicals. However, a recently proposed model (van Tilborg, 1995), the Deficiency Toxicity Model (DT) has many features of the AROI model described in section 4 of this paper. Based on the dose-response curves for both deficiency and toxicity in various species within say the aquatic environment, a range termed the "Optimal Concentration band for Essential Elements" (OCEE) can be derived. This range, as for the AROI, has a lower boundary as the point below which risk from deficiency increase and an upper boundary based on increased risks from toxicity. The model, as applied to the fresh surface water ecosystem, is shown in Figure 7.

Figure 7: The DT-model for ecological risk assessment for zinc. The natural boundaries of the concentration band defining optimum conditions for life in fresh surface water are shown and for comparison the natural average zinc concentrations of other typical aqueous habitats are shown. Many of the principles shown in Table 1 apply to the DT Model as well as to human health risk assessment models. In particular, the need for scientists with appropriate training and experience to carry out the evaluations and the need for sound scientific decisions in assessing data quality, doseresponse relationships and in determining the critical effect(s). Of critical importance in applying the DT Model is the relevance to the real environment of the testing procedures used to generate data. Test conditions need to be critically evaluated. Only data generated under conditions considered appropriate to the environmental compartment of concern should be used to set the upper boundary of the OCEE. 6. Concluding remarks The proposed methodology for assessing the health effects of human exposure to ETEs has built upon discussions at national and international workshops and conferences over the last four or five years.

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A synthesis of these discussions into what is hopefully a useful set of principles which can be agreed upon worldwide is presented here. Although the primary focus in this paper was on human health risk assessment, the recently proposed DT Model for ecological risk assessment is based on many of the same scientific principles, modified for the complexity of each environmental compartment. Therefore, it may in the future be possible to develop an integrated methodology for both human health and environmental risk assessments for ETEs. However, the usefulness of such methodology will only be as good as the data available. At present, the data on environmental effects needs to be strengthened and testing strategies developed to provide more relevant data if the DT Model is to become widely utilised. For both the DT Model, and that proposed for the development of an AROI for human health, this paper is simply proposing a scheme where risk assessments will be carried out by scientists (or teams of scientists) expert in risk assessment procedures as well as both the nutritional and toxicological sciences. Given the need to be concerned over the effects of deficiency as well as excess exposures, the proposed scheme is even more multidisciplinary in scope than the one used for non-essential chemicals (Figs.1 and 2). In working together nutritional scientists and toxicologists need to understand the principles underlying the development of both RDAs and TIs in the default positions taken in both cases, and how such assumptions can result in apparent conflicts between the upper and lower boundaries of the AROI. By fostering a closer interaction between scientists in the areas of nutrition and toxicology it is hoped that more adequate scientific data will be developed to address the boundaries of the AROI. However, unless both groups of scientists are willing to use all available scientific data, including that on nutrient interactions and bioavailability, we will continue to have apparent conflicts between the TIs and RDAs. The rigid application of conservative UF/MFs in any evaluation process is no substitute for the use of scientific expert judgement on the complete database when developing public health strategies related to the health and environmental effects from exposure to ETEs. Acknowledgements The assistance in developing these concepts by the participants at a recent IPCS consultation is gratefully acknowledged, namely: Dr C. Boreiko, USA; Mr G. Ethier, Canada; Dr H. Gibb, USA; Dr R. Goyer, USA; Dr W. Mertz, USA; Dr G. Nordberg, Sweden; Dr S. Olin, USA; and Dr R. Uauy, Chile. References Abernathy, C.O., Cantilli, R., Du, J.T. and Levander, O.A. (1993), Essentiality versus toxicity: some considerations in the risk assessment of essential trace elements. In Saxena, J. (ed.). Hazard assessment of chemicals, Vol. 8. Taylor and Francis, Washington, D.C., 81-113. Barnes, D.G. and Dourson, M. (1988), Reference dose (RfD): description and use in health risk assessments, Reg. Toxicol. Pharmacol., vol. 8, pp. 471-486. Beaton, G.H. (1988), Nutrient requirements and population data, Proc. Nutr. Soc., vol. 47, pp. 6378. Bowman, B.A. and Risher, J.F. (1994), Comparison of the methodological approaches used in derivation of recommended dietary allowances and oral reference doses for nutritionally essential elements. In: Risk Assessment of Essential Elements. Mertz, W., Abernathy, C. and Olin, S. eds. ILSI Press, Washington, D.D., 63-73. Goyer, R.A. (1994), Biology and nutrition of essential elements. In: Risk Assessment of Essential Elements. Mertz, W., Abernathy, C. and Olin, S. eds. ILSI Press, Washington, D.D., 13-19. IPCS (1987) , Principles for the safety assessment of food additives and contaminants in food. International Programme on Chemical Safety, Environmental Health Criteria 70. World Health Organization, Geneva.

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IPCS (1994), Assessing human health risks of chemicals: derivation of exposure. International Programme on Chemical Safety, Environmental Health Criteria 170. World Health Organization, Geneva. Mertz, W. (1993), Risk assessment of essential elements, Scand. J. Work Environ. Health, vol. 19, no. 1, p. 112. Mertz, W., Abernathy, C.O. and Olin, S. (1994), Risk Assessment of Essential Elements, ILSI Press, Washington, D.C., 300. Nordberg, G.F. and Skerfving, S. (1993), Editors. Biological monitoring, carcinogenicity and risk assessment of trace elements. Scand. J. Work Environ. Health, 19 Suppl 1: 140. NRC (1983), US National Research Council. Risk Assessment in the Federal Government: Managing the Process. National Academy Press, Washington, D.C. NRC (1989), US National Research Council. Recommended dietary allowances, 10th ed. National Academy Press, Washington, D.C. NRC (1994), US National Research Council. Science and Judgement in Risk Assessment. National Academy Press, Washington, D.C. Sanstead, H.H. (1993), Zinc requirements, the recommended dietary allowance and the reference dose, Scand. J. Work Environ. Health, vol. 19, issue 1, pp. 128-131. Smith, J.C., Jr. (1994), Comparison of reference dose with recommended dietary allowances for zinc: methodologies and levels. In: Risk Assessment of Essential Elements. Mertz, W., Abernathy, C. and Olin, S. eds. ILSI Press, Washington, D.D., 127-143. Uauy, R. Castillo-Duran, C., Fisberg, M., Fernandez, N and Valenzuela, A. (1985), Red cell superoxide dismutase activity as an index of copper nutrition, J. Nutr., vol. 115, pp. 1650-1655. van Tilborg, W.J.M. (1995), Integrated Criteria Document Zinc (ICDZ) - Industry Addendum. Project groep Zink, BMRO-VNO. European Zinc Institute, Brussels., 50. WHO (1996), Guidelines for drinking-water quality, 2nd ed. Volume 2: Health Criteria and other supporting information. World Health Organization, Geneva.

Assessment of the requirement of copper in the nutritional support of the severely ill patient
Dr Gordon S Fell, Dr TDB Lyon Scottish Trace Element Service, Macewen Building Royal Infirmary University and NHS Trust Hospital, Glasgow. Dr W Watson Department of Clinical Physics , Southern General Trust Hospitals, Glasgow. The various stages of the metabolic responses to severe injury were investigated and classified by D P Cuthbertson working in Glasgow over a 60 year period (Barton et al., 1990). The ebb or shock phase of hypo-metabolism is followed by the flow or acute phase of hyper-metabolism during which patients have an obligatory negative nitrogen balance. This is a measure of the catabolic loss of intracellular mass which if unchecked will cause muscle weakness, delayed wound healing and a reduced immune response with increased risk of infection. Increased urinary losses of cell contents also includes major inorganic elements such as K, S, P and essential trace elements such as Zn and Cu. As the patient recovers the anabolic phase of weight regain in adults, and renewed growth in children begins. This is characterised by a positive nitrogen balance and requirement for nutritional support. This must include a supply of all nutrients needed to restore the lost intracellular contents. The severely injured patient may also have a variable period of
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inadequate food intake due to the nature of the injuries, and where intestinal surgery is involved have a degree of malabsorption of diet. For these and other reasons, appropriate nutritional support is now an important part of patient care (Hill, 1994). Complete nutritional support is usually instituted as soon as the patient has been stabilised and it is clear that normal diet cannot be taken at all or in insufficient quantity. Often it is necessary to supply nutrients by intravenous infusion (IVN). This requires suitable forms of energy substrates such as glucose and lipid emulsions, as well as amino acids, minerals and the essential trace elements and vitamins. Ideally all of these nutrients should be infused in the same chemical form and amount as that absorbed into portal blood from good quality oral diet. This is not feasible in practice and the essential trace elements, including copper are given in the form of simple salts. The amount required should ideally be determined for each patient by assessment of tissue concentration and the detection of a developing deficiency by a functional assay of an appropriate intracellular metalloezyme system. Additionally the net input-output balance for each essential trace element should be determined by analysis of all IVN fluids, and excreta. In practice such detailed studies are not possible especially in critically ill patients. A compromise multi-component additive mixture of containing several essential trace elements is used. This additive offers amounts estimated to meet most requirements but not thought liable to cause toxicity (Shenkin et al., 1987). 1. Copper deficiency and treatment Failure to supply sufficient copper in the nutritional regimens some years ago, did result in symptomatic copper deficiency (Jeejbhoy, 1989). Symptoms take several months to develop. This is longer period than it takes for the equivalent zinc deficiency disease to develop, due the reserve store of copper in the normal liver. In adults copper deficiency can present as haematological abnormalities. There is a low neutrophil count (neutropenia) and a mild iron resistant microcytic anaemia. A brisk increase in reticulocyte count is seen when copper is added to the IVN regimen with reversal of the neutropenia and anaemia This condition has also been recorded in a patient with chronic intestinal malabsorption (Bruce et al., 1995). In children, especially low birth weight pre-term babies, there is a particular risk of the development of copper deficiency. (Aggett, 1994). This is caused by the failure of the fetal liver to obtain maternal copper during the last trimester of pregnancy. As well as neutropenia these infants have brittle, easily fractured bones. This is due to impairment of the copper-dependent enzymes required for bone collagen formation. The condition can be prevented by supplementation of diet with adequate amounts of copper. The amount of copper recommended for an adult on IVN is 20 mol per day (1.3 mg Cu). This is more than the 6-10 mol/day needed to maintain overall balance, but is an amount needed to replace losses and is not associated with toxic effects (Cruickshank et al., 1985). A safe and adequatedaily intake of 0.4-0.6 mg/day has been proposed for term neonates. Recommendations vary quite widely, and preterm babies may have higher needs ranging from 20-40 g/kg/day (Tyrala, 1992). 2. Biochemical assessment 2.1 Plasma copper

The variable amount of copper stored in liver acts to maintain plasma copper concentrations even in the face of low copper supply. Measurement of plasma copper or plasma ceruloplasmin although convenient markers of copper nutritional status are a relatively insensitive indices of tissue depletion. In a case study, a 42 year old man with severe intestinal malabsorption had been maintained on home IVN for some 5 years. and had remained well and weight stable. He prepared his own IVN fluids and made the trace element additions. However persistent IV line blockage occurred and led him to reduce the use of the trace element additive mixture. Regular monitoring of this patient revealed that his
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plasma Cu had declined to 6.9 mol/L (reference interval (12-24 mol/L). Oral copper supplements were given (40 mol, 2.5 mg) on alternate days which corrected his impending copper depletion but may have triggered a later symptomatic zinc deficiency (de Caestecker 1985). 2.2 Monitoring of neonates

The limited amount of blood available from a low birth weight preterm baby poses considerable technical problems. However analytical methods have been developed able to determine the concentration of some seven essential elements, including Cu, in only 1-2 ml of blood serum. In a study comparing two different forms of trace element additives we were able to show that the recommended input of 20 g/kg /day maintained satisfactory plasma copper concentration. The changing values of plasma copper in neonates with increasing gestational age (Agett, 1994) makes the interpretation difficult, and requires repeated sampling and knowledge of gestational age. 2.3 Acute phase plasma protein effect

An additional difficulty in the use of plasma copper levels to monitor acutely ill patients, particularly where there is accompanying sepsis and inflammation, is the increased liver synthesis of ceruloplasmin. This copper containing protein accounts for some 80-90% of total plasma copper and increases in plasma about 2-3 days after infection or injury as a positive acute phase protein. Sequential measurements after cardiac surgery or choleocystectomy show the marked variation of both ceruloplasmin and plasma copper with time after surgical incision (Fraser et al., 1989). Such changes are independent of copper dietary intake. A biochemical measure of the acute phase effect is plasma C-reactive protein. (CRP). This protein which is normally present in plasma at low concentrations (<10 mg/L) increases 10-100 fold within 48 hours of infection and/or tissue injury. A study of two groups of lung cancer patients showed that those who were catabolic and weight losing had higher CRP values and higher plasma Cu and ceruloplasmin levels than an equivalent group of weight stable patients. Both groups had similar nutritional intakes. 2.4 Action limits

Although plasma Cu does not reflect short term changes in Cu intake the finding of a persistently low concentration of <5.0 mol/L (30 g/dL) is likely to indicate impending symptomatic Cu deficiency caused by chronic copper depeletion. Since the acute phase response, present in most severely ill patients should normally increase plasma Cu well above the reference interval of 12-22 mol Cu/L is probable that values of < 12 mol Cu/L in plasma reflect an incomplete acute phase response due to some degree of Cu deficiency. However it has been shown that, even when Cu input exceeds Cu losses, plasma Cu concentrations do not change, at least in the short term. Therefore the finding of a value within the reference range for plasma Cu does not exclude some degree of tissue Cu accumulation. 2.5 Contra-indication for copper supplementation

It has been shown by metabolic balance studies in animals and in humans, that the major regulator of copper homeostasis, which keeps tissue concentrations of copper constant in the face of widely varying dietary inputs, is the biliary excretion of excess copper. We have demonstrated by metabolic studies (Cruickshank et al., 1988) that a patient with extensive bile fluid losses was in negative copper balance -8.6 mol (0.55mg) Cu /day in spite of receiving the standard amount of Cu in the IVN regimen. It follows that patients with obstructive liver disease and reduced bile excretion will be liable to accumulate copper. Dosage in such cases should reduced and the patient examined for signs of Cu toxicity. However the storage capacity of the liver for Cu is large, and at the daily inputs routinely used it is unlikely that overt Cu toxicity would develop quickly.
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2.6

Copper isotope tracer studies

Marginal copper depletions will not be detected by determination of plasma Cu concentrations for the reasons given above. Direct determination of liver Cu concentration by analysis of needle biopsy samples is feasible but is an invasive process not undertaken unless other strong clinical indications are present. The kinetics of Cu intestinal absorption, blood transport, tissue uptake and excretion in bile and urine have been investigated in animals and in a few humans by use of radioactive Cu isotopes. These are relatively short-lived radioisotopes (T1/2 24-48 h) and not suitable for administration to infants and women of child bearing age. Recently it has been possible to determine the stable copper isotope 65Cu in blood plasma by an inductively-coupled plasma mass spectrometry method (Lyon and Fell 1990) The ratio of 65Cu : 63Cu can be measured sequentially in blood plasma after oral dosage with 3mg 65 Cu and the appearance curve of the enriched isotope determined (Lyon et al., 1995). This together with data taken from published metabolic models has allowed the construction of a mathematical model of human copper metabolism. The administration of the stable isotope 65Cu to various categories of patients is feasible and could be used to quantify the disturbance of copper metabolism caused by disease. An estimate of the percent intestinal absorption from a single dose (3mg 65Cu ) has shown that in healthy controls a mean of 50% is absorbed. Patients with residual malabsorption due to intestinal disease take up a more variable amount but usually less, at around 30% of the administered dose. By conducting quantitative metabolic balance estimations and using the stable isotope as a tracer, we can fit the results into the mathematical model and make an estimate of the concentration of copper in the liver compartments and in the rest of body. 3. Summary and conclusions The routine provision of 1.2 mg/Cu per day during IVN will prevent overt copper deficiency disease in most adult patients. Copper toxicity is not likely although patients with severe obstructive liver disease need to monitored. In pre term infants an increased amount of copper /kg body weight may be needed to allow for their low liver stores. This element is not always present in adequate amounts in oral feeds or IVN regimens. Monitoring for Cu deficiency in both adults and babies can be based on screening for very low levels of plasma Cu. A finding of repeated values of (<5.0 mol/l) indicates a need for further copper supplementation. There are alterations in the metabolism of copper in the ill patient which are independent of dietary copper supply. Plasma copper concentrations at the lower end of the reference range (<12 mol Cu/L) may mask an underlying tissue copper depletion. Marginal copper depletions may have an adverse effect on antioxidant defences and the immune system. Metabolic balance studies employing stable copper isotopes as a tracer may be required to demonstrate copper depletion in at risk groups. References Aggett, P.J. (1994), Aspects of neonatal metabolism of trace elements, Acta paediatr, Suppl 402, pp. 75-82. Barton, R.N., Frayn, K.N. and Little, R.A. (1990), In Chapter 33. Trauma, Burns and Surgery. The metabolic and molecular basis of acquired disease Vol 1 Eds Cohen RD, Lewis B, Alberti KGMM and Denman AM, Baillere Tindall. Bruce, A., Hayton, H., Broome, E and Lilenbaum, R.C. (1995), Copper deficiency induced anemia and neutropenia secondary to intestinal malabsorption, Amer. J of Haematology, vol. 48, pp. 45-47. Cruickshank, A.M., Rodgers, P, Dunbar P., Fell, G.S. and Shenkin, A (1988), Copper balance in intravenously fed patients, Proc. Nutr. Soc., vol.17. de Caestecker, J.S., Shenkin, A., Fell, G.S., Heading, R.C. (1985), Hazards and benefits in a patient on long term total parenteral nutrtion, Proc Nutr Soc., vol. 45, p. 23 A.
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Fraser, W.D., Taggart, D.P., Fell, G.S., Lyon, T.D.B., Wheatly, D., Garden O.J. and Shenkin, A. (1989), Changes in iron, zinc and copper concentrations in serum and in their binding to transport proteins after choleocystectomy and cardiac surgery, Clincial Chemistry, vol. 35, pp. 2243-2247. Hill, G.L. (1994), The impact of nutritional support on the clinical outcome of the surgical patient, Clinical Nutrition, vol. 13, pp. 331-340. Jeejbhoy, K.N. (1990), Trace elements in total parenteral nutrition in Trace elements in Clinical Medicine, Ed Tomita, H. Springer -Verlag. Lyon, T.D.B and Fell, G.S. (1990), Isotopic composition of copper in serum by inductively coupled plasma mass spectrometry, JAAS, vol. 5, pp. 135-137. Lyon, T.D.B., Fell, G.S., Gaffney, D., McGaw, B.A., Russel, R.I., Park, R.H.R., Beattie, A.D., Curry, G., Crofton, R.J., Gunn, I., Sturniolo, G.S., DInca, R. and Patriarca, M. (1995), Use of a stable copper isotope ( 65 Cu) in the differential diagnosis of Wilsons Disease, Clinical Science, vol. 88, pp. 727- 732. Shenkin, A., Fraser, W.D., McLelland, A.J.D., Fell, G.S. and Garden, O.J. (1987), Maintenance of vitamin and trace element status in intravenous nutrition using a complete nutritive mixture, Journal of Parenteral and Enteral Nutrition, vol. 11, no. 3, pp. 238- 242. Tyrala, E.E. (1992), Trace elements, in Chapter 9 of Intravenous Feeding of the Neonate ed. Yu VH and Macmahon R. publ. Edward Arnold

Copper tailing impacts in coastal ecosystems of Northern Chile: From species to community responses
Professor Juan C Castilla and Dr Juan A. Correa Departamento de Ecologia. Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago, Chile. 1. Introduction At the beginning of the 1980s, the anthropogenic inputs of copper into aquatic ecosystems varied between 34.7 and 190.5 tons/year. Copper mining, smelting, and refinery activities accounted for approximately 14% (Pacyna et al., 1995). Mining copper activities take place in all six continents and support a demand of about 10 million tones of copper per year. Copper mines in South America, mainly in Chile and Peru, account for approximately one third of the total world demand. In Chile, the production of fine copper increased from 691,600 metric tones in 1970 (10.9% of the world production) to 2,219,900 metric tones in 1994, representing 24% of the world production (Anonymous, 1995). Chilean copper mine activities are based on 10 major open or underground mines located at high altitude (usually over 2,000 m above sea level) in the Andes mountains, between 22-34 S. In most of the cases the ore extraction, processing, smelting and tailing dumping take place around the mine pits. Nevertheless, in the past (1938-1990), El Salvador copper mine dumped all its untreated tailings directly to coastal areas in northern Chile (this practice is presently banned according to Chilean environmental legislation). The El Salvador case has been reported in detail elsewhere (Castilla and Nealler, 1978; Castilla, 1983; Paskoff and Petiot, 1990; Vermeer and Castilla, 1991; Castilla, 1996; Correa et al., 1996a). El Salvador copper mine is located approximately 120km from the coastal city of Chanorol. From 1938 to 1975 about 150 million tones of untreated copper tailings generated at this mine (and previously at Potrerillos) were conducted through the Salado River (Fig.1) and dumped directly into the sandy beach of Chanorol Bay, causing notorious beach progradations (Castilla, 1983; Paskoff and
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Petiot, 1990). In 1976 the dumping site at Chanorol was abandoned and a new dumping site was established in a rocky beach known as Caleta Palito. This site is located approximately 10 km north from Chanorol Bay and between 1976 and 1989 received approximately 130 million tones of solids from the mine. In 1990 the El Salvador company built a tailing sedimentation dam away from the coast, in the desertic Pampa Austral. Since then, the so called "aguas claras" or sediment-free "clear waters", with a legal upper limit of 2,000 g/L of total copper, have been continuously driven from the dam, through the El Salado River bed, to Caleta Palito (Castilla, 1996). The clear water is pumped from the dam at a rate of 250-500 L/sec and mixes with water discharges from local towns and small-scale (artisanal) mining operations occurring along the El Salado River bed. The 1976-1989 copper tailing dumping caused severe changes to the ecosystem in Caleta Palito (sandy and rocky shores), characterized by major increase of copper concentration in the sea water, beach progradation and the total elimination of invertebrates and most of the algal species around the dumping site. This lead to a reduction of biodiveresity (Castilla, 1983,1996) and the disruption of trophic chains. Among the most striking features of the impacted areas is the almost absolute dominance of primary substratum by the green alga Enteromorpha compressa (L.) Grev., which extends from Caleta Palito to Caleta La Lancha (Fig. 1) and occupies the entire intertidal fringe. This dominance became apparent shortly after the untreated tailings from El Salvador began pouring into Caleta Palito, and has persisted since then. We are using the El Salvador-Caleta Palito study case as a model, and in this context, the objectives of this paper are to review the previous research on the Palito study case, to up-date information and to describe the future lines of research. This study includes: (a) the present concentration of copper in coastal sea water polluted and unpolluted sites, and in the sentinel green alga E. compressa; (b) differences and trends in species richness at polluted and unpolluted sites and (c) the characteristics and mechanisms by which the green algae E. compressa may be able to resist copper enrichments. 2. Study sites and methods The study sites are located in the desertic region of northern Chile. The El Salvador mine, at 2600 m above sea level (26o 14' S, 69o 37' W), and the El Salvador town (approximately 10,000 habitants), are 120km east from the coastal city of Chanorol Bay. The Salado River bed extends from Los Andes Mountains through the Salado Valley (120km long) and connects with the sea at Chanaral Bay. The scarcity of rains characteristic of the region results in a very limited water flux, mainly from ice fields in the higher mountains, most of which either evaporates or is absorbed before reaching the sea. Floods, known as Bolivian winters, are caused by rain episodes occurring during the summer accompanied by ice melting in the Andes. These floods result in large volumes of water and mud being channelled through the Salado River bed which are discharged directly in to Chanorol Bay. Because Bolivian winters are infrequent and of short duration, the Salado River remains dry most of the time with intermittent, brief flood episodes. The towns of Diego de Almagro (ca. 8,000 inhabitants) and El Salado (ca. 1,200 inhabitants), as well as around 20 small-scale artisanal mining operations present along the Del Salado Valley discharge their waste waters to the Salado River bed. These waste waters join the clear waters from the dam about 10 km before reaching Chanorol city; an artificial canal of approximately 15 km diverts the stream from the the Salado Valley and conducts the water directly to Caleta Palito where it discharges openly on the shore (Fig. 1).

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Figure 1: Map of the study area. Sea water and E. compressa samples were collected in 1994, 1995 and 1996 around the pollution point source at Caleta Palito and at three control sites: a) Caleta Pan de Azucar, approximately 24 km north from Caleta Palito; b) Caleta Guanillo, approximately 45 km north from Caleta Palito and c) Caleta Zenteno, about 68 km south from Caleta Palito. Sampling procedures and analytical methods for sea water and E. compressa were the same as those described by Correa et al. (1996a). Dissolved copper in water samples was quantified by potentiometric stripping analysis in stationary solution, using a computerised Radiometer ISS 820 analyzer. Algal tissue was treated with nitric acid and copper was quantified in a GBC 909 PBT atomic absorption spectrophotometer. Transmission electron microscopy was done with E. compressa collected at Caleta Palito and Caleta Zenteno. Processing of algal tissue was done according to Correa and McLachlan (1994). Intertidal rocky shore monitoring of sessile and mobile invertebrates and macroalgae was done during low tides (0.23-0.30 m) between June 1-5, 1996 at Caleta Palito, Caleta Pan de Azucar, Caleta Zenteno, La Lancha beach, and Caleta Coquimbo (see Castilla, 1996 and Fig. 1). The latter is a much less impacted site, approximately 12 km north from Caleta Palito. Field monitoring was conducted in two rocky platforms, with a slope of 10 - 40 and 30-40m long, selected in each site. The platforms were divided in four intertidal fringes: low, mid-low, mid-upper and upper, as described by Castilla (1981, 1996). Two independent observers recorded the species present in each fringe by walking slowly and counting the species seen in intervals of one minute, with a maximum of five minutes per fringe. 3. Results and discussion 3.1 Copper concentration in sea water and Enteromorpha compressa

Values of dissolved copper in the water (Table 1) varied greatly among the different sampling dates and localities. At Caleta Palito values ranged from 10 g/L in May 1996 to slightly more than 40 g/L in July 1995. Major fluctuations can be detected even within the same year. At the discharge point, for example, values of almost 20 g/L were recorded in April 1996, a fifth of the concentration measured at the same spot two months later. These fluctuations should be expected based on the high
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number and diversity of pollution sources occurring along the El Salado river (see Introduction). In spite of that, copper concentration in the water from those localities not directly impacted by the El Salado river (mainly the control sites of Zenteno and Guanillo) were consistently lower than those in water from Caleta Palito. The lowest value of dissolved copper recorded among the control localities was 0.5 g/L in water from Caleta Pan de Azucar (May 1996), whereas the maximum was 6.5 g/L in water from Caleta Zenteno (April 1995). On the other hand, in water from Caleta Palito the minimum value was 10.0 g/L (April 1995) and the maximum was 203.3 g/L (July 1995). The latter difference likely reflects temporal changes in the degree of pollution of the Salado river inputs into the disposal point. Fluctuations also occurred in the content of copper quantified in the tissues of E. compressa. In spite of that, copper content was consistently higher in tissues from individuals collected at Caleta Palito (range: 34-358 g g-1, April 1995) than in tissues from algae growing in Caleta Zenteno (highest value: 5.7g/g, April 1995) and Guanillo (highest value: 30.6g/g, April 1995). Table 1: Values of dissolved copper in coastal waters and in Enteromorpha compressa from northern Chile. Copper values in algal tissue are based on dry weight. Replicate number is indicated in parenthesis. Standards from the National Research Council of Canada were run simultaneously to water and tissue samples and copper values in the standards never deviated more than 2% from the certified values.
Locality Caleto Palito
1

Date August 1994 August 1994 April 1995 April 1995 April 1995 April 1995 April 1995 July 1995 July 1995 May 1996 May 1996 May 1996

Water (g/L) 26.8 - 31.8 (2) 2.9 - 3.4 (2) 0.9 - 1.5 (4) 1.5 - 2.3 (4) 4.0 - 6.5 (4) 10.0 - 12.8 (4) 13.6 - 19.3 (4) 37.7 - 40.7 (3) 162.9 - 203.3 (2) 0.5 - 0.7 (2) 10.2 - 13.9 (2) 1.9 - 2.0 (2)

E. compressa (g/g) 54.9 - 71.8 (2) 1.9 - 2.1 (2) 26.6 - 30.6 (2) 4.7 - 5.7 (3) 34.9 - 358.3 (3) -

Caleta Zenteno Pan de Azucar Guanillo Caleta Zenteno Caleta Palito1 Caleta Palito2 Caleta Palito1 Caleta Palito2 Pan de Azucar Caleta Palito1 Caleta Zenteno

1. Replicates collected at 50 m south from the discharge point. 2. Replicates collected in front of the discharge point, at the mixture zone between the water from the canal and the sea.

3.2

Rocky intertidal species richness

The rocky intertidal species richness in the horizontal platforms of the five sites monitored ranged between 10 and 64 (Table 2). Caleta La Lancha, the most polluted site, has four species of algae
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(including a bio-film of diatoms), five species of mobile invertebrates and one vertebrate. No sessile invertebrates were recorded. Caleta Palito, also a polluted site, shares ten species with Caleta La Lancha, but additionally shows eight more species. Caleta Coquimbo represents a site with intermediate species richness (35), and the control sites, Caleta P. de Azucar and Caleta Zenteno, are the richest with 53 and 64 species respectively. Table 2: List of species present (+) and absent (-). Presence assessed at four tidal levels using a maximum of 5 minutes recording time and non-destructive sampling.
Sites Species Algae Colpomenia sinuosa Codium dimorphum Glossophora kunthii Petalonia fascia Lessonia nigrescenes Scytosiphon lomentaria Ectocarpus sp. Ralfsia sp. Lingbya sp. Ulva sp. Rama novaezelandiae Cladophoropsis sp. Enteromorpha compressa Enteromorpha linza Schottera nicaeensis Porphyra columbina Hildebrandtia lecannellieri Polysiphonia sp. Centroceras clavulatum Ceramium sp. Rhodoglossum sp. Corallina officinalis Corallina sp. Gymnogongrus sp. Ahnfeltia sp. "lithothamnioides" nd. Gelidium chilense Gelidium lingulatum Halopteris hordacea Bangia sp. Diatoms (biofilm) Invertebrates Sessiles Jehlius cirratus Notochthamalus scabrosus Balanus laevis Austromegabalanus psittacus + + + + Copper + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Caleta Pan de Azucar Caleta Coquimbo Caleta La Lancha Caleta Palito Caleta Zenteno

76

Balanus flosculus Semimytilus algosus Perumytilus purpuratus Phymactis clematis Anthothe chilensis Phymanthea pluvia Anthozoa nd. Porifera nd. Pyura chilensis Serpulidae nd. Mobiles Nodilittorina peruviana Nodilittorina araucana Scurria viridula Scurria d'orbigni Scurria plana Scurria ceciliana Scurria araucana Scurria sp. Scurria parasitica Siphonaria lessoni Fissurella crassa Fissurella limbata Fissurella maxima Chiton granosus Acanthopleura echinata Enoplochiton niger Concholepas concholepas Tegula atra Gastropoda nd. Leptograpsus variegatus Acanthocyclus gayi Petrolistes violaceus Taliepus sp. Betaeus sp. Amphipoda nd. Acarii nd. Heliaster helianthus Patiria chilensis Stichaster striatus Tetrapygus niger Loxechinus albus Nemertini nd. Vertebrates Sicyases sanguineus Microlophus sp.

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + -

+ + + + + -

+ + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -

+ +

+ +

The pattern of species richness in the rocky intertidal platforms described in this paper coincides with that reported by Castilla (1996) for perpendicular intertidal walls (approximately 70-85o) in the same
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geographical area. Nevertheless, as expected, the number of species is much greater in horizontal platforms (range between 10-64, this paper) than in perpendicular walls (range between 6-32), Castilla, 1996). This is particularly true for mobile species of invertebrates. The chlorophycean E. compressa remains as the dominant species covering the primary substratum (rock) on intertidal horizontal platforms at Caleta Palito and Caleta La Lancha. This alga is also found in unpolluted sites (Table 2), but its cover values are extremely low (see also Castilla, 1996). Caleta Palito arose as an interesting monitoring site in this study. In June 1996 the rocky platforms of this site presented four species (at low densities) not recorded before: the alga Ulva sp. (Chorophyta); the barnacles Jehlius cirratus and Austromegabalanus psittacus; and the mussel Semimytilus algosus. They were not found at Caleta La Lancha. These species are abundant in unpolluted sites and therefore, their presence could represent a further step in the direction of natural ecological restoration occurring at Caleta Palito. The same process could eventually follow at Caleta La Lancha. 3.3 Enteromorpha compressa as a biological model: current status of knowledge and future perspectives

Species of Enteromorpha, particularly E. compressa, have been reported as organisms able to resist heavy metal enrichments, including copper. The mechanisms involved in that resistance, however, are poorly understood. One of the few experimental studies available (Reed and Moffat 1983) addressed the hypothesis that copper-enriched environments could lead to the development of copper-tolerant ecotypes. Reed and Moffat (1983) showed higher copper tolerance in plants from a copper-enriched environment (vessel hulls with copper-based anti-fouling paint) than those used as controls, obtained from a site with no history of copper pollution. The presence of the same parental pattern of copper tolerance in the progeny lead to the conclusion that tolerance was an inherited character, a feature that supported the original notion of ecotype development. These ideas were recently re-assessed by Correa et al. (1996a), who used the same experimental approach to compare the in vitro responses to copper enrichments of two intertidal populations of E. compressa. The population growing at Caleta Palito has been exposed to a consistently higher copper concentration than the population of the same species growing in the non-polluted Caleta Zenteno. Inconsistencies between the results from the two studies became apparent. Chilean E. compressa obtained from spores of parents collected in copper-polluted (ie. Caleta Palito) and copper-normal (ie. Caleta Zenteno) localities did not show different tolerances to copper in laboratory assays. These results clearly questioned the generalization by Reed and Moffat (1983) and, together with the results from the parental generation (Correa et al., 1996a), suggested physiological flexibility as the responsible for the observed tolerance responses to the copper by the Chilean species. Further analysis of the experimental information available in the literature indicates the existence of important gaps in our understanding of E. compressa as a biological model in the field of algal-heavy metal interactions. At least two areas of research can be foreseen as important to fully unveil the basis for the successful colonization and persistence of E. compressa in heavy metal-enriched coastal waters. One of them relates to the possible occurrence of species-specific mechanisms for metal tolerance. A number of potential mechanisms have been individualized by various authors (see review by Correa et al., 1996b), although most of them are passive in the sense that they include metal complexation by either non-specific exudates or charged groups, which are normal components of the algal cell walls, like polysaccharide (Veroy et al., 1980). It is unlikely, however, that these general, non-specific processes provide advantages in tolerance only to E. compressa to the extent of determining the characteristic, almost absolute, monopoly of the primary substratum by this alga in polluted areas. (Castilla, 1996). There is, on the other hand, a much more specific mechanism which allows organisms to bind and detoxify metals occurring at concentrations higher than normal and it involves the synthesis of proteins known as metallothioneins. These proteins, which are encoded by nuclear DNA, have been found in a number of animals and plants. Their occurrence in algae is still a matter of discussion (Correa et al., 1996b) and certainly no information regarding metallothionein induction in E. compressa (or in any species of Enteromorpha) is currently available. Indirect cellular
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evidence suggests, however, that this physiological process may be operating in the Chilean E. compressa. Recent unpublished ultrastructure observations have shown the occurrence of intracellular, electron dense deposits in cells of E. compressa from Celeta Palito (Figures 2 and 3), whereas similar deposits are absent in cells from plants collected at Celeta Zenteno. Highly magnified images of the above deposits have discarded their crystalline structure, but rather indicate a finely reticular to homogeneous appearance, suggesting that a proteinaceous matrix may be serving as a metal trap. This hypothesis is further supported by the occurrence of structurally similar, metal containing proteinaceous bodies in microalgae exposed to heavy metals (Silverberg et al., 1976; Daniel and Chamberlain, 1981; Wong et al., 1994). Figure 2: Cross sections through Enteromorpha compressa cells from Caleta Palito. Large electron dense granules (Gr) clearly extravacuolar, with a darker center and a pale periphery.

Figure 3: Spheric granules, with a compacted material homogeneously electron dense, occurring apparently within the vacuole.

The other area in need of experimental research relates to the shift of the ecological relationships among species once an opportunistic alga, like E. compressa, becomes the dominant species. In this context, the situation developed at Caleta Palito and surroundings represents a unique opportunity to characterize the species interplay, starting with E. compressa dominated system and moving into one
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where the primary space is shared at different, and fluctuating, ratios with other algae and invertebrates. It must be understood that E. compressa is a normal component of the algal flora of the region, but remains at very low densities, with a patchy distribution, usually restricted to intertidal pools and shaded walls. In Caleta Palito, on the other hand, E. compressa extends almost homogeneously throughout the intertidal fringes of vertical and horizontal platforms. What is important to consider is that the original, long standing, catastrophic event (solid disposal creating sandy tailing beaches and suspended particulate matter abrading the rocks) that was probably the main cause of the disappearance of algal and invertebrate species from the Palito-La Lancha polluted area (Fig. 1) is no longer present. Thus, the question remaining to be addressed is what are the factors permitting the current dominance by the opportunistic E. compressa and what is the role of this species in the eventual restoration process likely to be operating in the area. Within the above framework, our 3-year research program involves a multidisciplinary approach where molecular biologists, marine microbiologists, analytical chemists, and marine ecologists are jointly tackling the deficiency areas, based on the situation developed in Caleta Palito. Thus, the search for copper binding proteins similar to those reported in some marine microalgae and higher plants is underway. We are conducting our search using wild E. compressa from Caleta Palito and several control sites, as well as thalli incubated at various copper concentrations in order to establish causality between the presence of the metal at concentrations higher than normal and the synthesis of specific proteins. At the same time, pioneering work is being attempted to individualize metallothionein encoding genes in nuclear DNA of E. compressa. In the same context, a complementary set of analyses is being conduced to fully characterise the metal content of the intracellular electron dense granules developed in E. compressa from Caleta Palito. In this case, energy dispersive X-ray microanalysis is being implemented to either detect and quantify copper in the granules, or elucidate the cellular compartment where the metal is accumulated. In order to explain the ecological dominance by E. compressa, several avenues are currently being tested. One of them involves testing the hypothesis that there are no propagules or larvae from other organisms in the coastal waters of Caleta Palito and therefore, E. compressa has no competitors or consumers that may recruit and exert an effect upon its pattern of abundance and distribution. To test the hypothesis, water samples are being collected seasonally, and algae developed after a preestablished culture period are recorded. For primary producers, there is yet another possibility, which is that algae other than E. compressa are present as a bank of microscopic forms, unable to develop due to the presence of E. compressa. To test this, rock samples from Caleta Palito, without E. compressa, are being incubated under laboratory conditions to facilitate the development of other species potentially present. As a complementary set of experiments, we are assessing the level of copper tolerance of the most common algae species which, according to their distribution range should be present in the area of Caleta Palito. In these species, the effect of copper is not only tested on adult individuals, but on all the different stages through their life cycles, including alternate phases. Finally, the sequential colonisation of algae or invertebrate species (ie. other than E. compressa) is being monitored along the impacted coastal area and various field manipulative experiments such as in situ bioassay with mussels and plantonic larvae studies are being implemented to assess their role in the natural restoration processes. Acknowlegements We thank Nelson Lagos, Manuel Varas, Marco Ramirez and Jose Miguel Farina for their help with the field work. This study is part of the research grant N 1196303-1, funded by the International Cooper Association (ICA), through the Centro de Investigaciones Minero Metalurgicas (CIMM), to Dr J.A. Correa, Pontificia Universidad Catolica de Chile. JCC appreciates the financial support from the Corporacion del Cobre de Chile (COCHILCO) which permitted his attendance to the International Workshop on Copper, National Research Centre for Environmental Toxicology (20-21 June 1996), Brisbane, Australia.
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References Anonymous (1995). Chilean mining compendium. Edited by R. Corts. Editec Ltda., Santiago, Chile. pp. 531. Castilla, J.C. and Nealler, E. (1978), Marine environmental impact due to mining activities of El Salvador Copper Mine, Chile Mar. Poll. Bull., vol. 9, no3, pp. 67-70. Castilla, J.C. (1981), Perspectivas de investigacion en estructura y dinamica de comunidades intermareales rocosas de Chile Central. II Depredadores de alto nivel trofico, Medio Ambiente, vol. 5, pp. 190-215. Castilla, J.C. (1983), Environmental impacts in sandy beaches of copper mine tailings at Chanaral,Chile. Mar. Poll. Bull., vol. 14, no.2, pp. 459-464. Castilla, J.C. (1996), Copper mine tailing disposal in Northern Chile rocky shores: Enteromorpha compressa (Chlorophyta) as a sentinel species, Environ. Monitor. Assess., vol. 40, pp.41-54. Correa, J.A. and McLachlan, J. (1994), Endophytic algae of Chondrus crispus (Rhodophyta). V. Fine structure of the infection by Acrochaete operculata (Chlorophyta), Eur. J. Phycol., vol. 29, pp. 3347. Correa, J.A., Gonzalez, P., Sanchez, P., Munoz, J. and Orellana, M.C. (1996a), Copper-algae interactions: inheritance or adaptation?, Environ. Mon. Assess., vol. 39, pp. 41-54. Correa, J.A. Ramirez, M.A., Fatigante, F.A. and Castilla, J.C. (1996b), Copper, macroalgae and the marine environment. The Chanaral case in northern Chile. In: Proceedings of the Symposium on the Biology of Microalgae, Macroalgae and Seagrasses in the western Indian Ocean. M. Bjrk (ed.). In press. Daniel, G.F. and Chamberlain, A.H.L. (1981), Copper immobilization in fouling diatoms, Bot. Mar., vol. 24, pp. 229-243. Pacyna, J.M., Scholtz, M.T. and Li, Y.-F.A. (1995), Global budget of trace metal sources. Environ. Rev., vol. 3, pp. 145-159. Paskoff, R. and Petiot, R. (1990), Coastal progradation as a by-product of human activity: an example from Chanaral Bay, Atacama Desert, Chile, J. Coastal Res. Special, no. 6, pp.91-102. Reed, R.H. and Moffat, L. (1983), Copper toxicity and copper tolerance in Enteromorpha compressa (L.), Grev. J. Exp. Mar. Biol. Ecol., vol. 69, pp. 85-103. Silverberg, B.A. Stokes, P.M. and Ferstenberg, L.B. (1976), Intranuclear complexes in a coppertolerant green alga, J. Cell Biol., vol. 69, pp. 210-214. Vermeer, K. and Castilla, J.C. (1991), High cadmium residues observed during a pilot study in shorebirds and their prey downstream from El Salvador copper mine, Chile, Bull. Environ. Contam. Toxicol., vol. 46, pp. 242-248. Veroy, R.L., Monta o, N., Guzman, L., Laserda, E.C. and Cajipe, G.J. (1980), Studies on the binding of heavy metals to algal polysaccharide from Philippine seaweeds. I. Carrageenan and the binding of lead and cadmium, Bot. Mar., vol. 23, pp. 59-62. Wong, S.L., Nakamoto, L. and Wainwright, J.F. (1994), Identification of toxic metals in affected algal cells in assays of waste waters, J. Appl. Phycol., vol. 6, pp. 405-414.

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Wilsons Disease after cloning of the gene

Dr John L Gollan Director Gastroenterology Division Brigham and Womens Hospital Associate Professor of Medicine, Harvard Medical School, Boston, Massachusetts U.S.A. It is more than 80 years since the London neurologist Samuel Alexander Kinnier Wilson defined the familial syndrome of progressive lenticular degeneration associated with cirrhosis of the liver. Considerable advances have been achieved in elucidating the clinical, biochemical, genetic, and histologic features, as well as the management of patients with this disease (Zucker and Gollan 1996). With the recent cloning of the Wilson's Disease gene, our understanding of the disease has accelerated, although clarification of the pathogenetic defect and the application of genetic screening are among the major problems yet to be resolved. 1. Genetics The gene for Wilson's disease is distributed worldwide, having been demonstrated in virtually all races. Current estimates indicate that the prevalence of the disease is approximately 1 in 30,000 live births, with prevalence rates ranging from 15 to 30 per million (per annum). The gene frequency varies between 0.3 and 0.7%, corresponding to a heterozygote carrier rate of slightly greater than 1 in 100. Genetic studies from a large Israeli-Arab kindred identified a linkage between the Wilson's disease locus and the red cell enzyme esterase D, thereby establishing that the gene mutation responsible for Wilson's disease was located on chromosome 13 (Frydman et al., 1985). Using multipoint linkage techniques, the abnormal gene for Wilson's disease was localised more specifically to 13q14-q21. In 1993, a candidate gene for Wilson's disease (WND) was reported independently by several different groups of investigators, using slightly different strategies for positional cloning (Bull et al., 1993, Petrukhin et al., 1993; Tanzi et al., 1993). The WND gene consists of a transcript of approximately 7.5 kilobases, which is expressed primarily in liver, kidney and placenta; although it also has been detected in heart, brain, lung, muscle and pancreas, albeit at much lower levels. the full-length cDNA sequence of the WND gene (Bull et al., 1995; Tanzi et al., 1993) predicts a 1,411 amino acid protein, which is a member of the cation-transporting P-type ATPase subfamily, highly homologous to the Menkes syndrome gene product and the copper transporting ATPase (cop A) found in copper resistant strains of Enterococcus hirae. From sequence analysis of the cDNA, the WND protein is predicted to possess a metal binding domain (containing 5 specific binding sites), an ATP binding domain, a cation channel and phosphorylation region, and a transduction domain responsible for the conversion of the energy of ATP hydrolysis to cation transport. To date, more than forty disease-specific mutations in the Wilson's disease gene have been identified. The wide spectrum of clinical manifestations in Wilson's disease raises the question as to whether variability exists at the molecular level. The fact that Wilson's disease is linked to chromosome 13 markers in all populations studied suggests that there is a single genetic locus for the disease, and it has been postulated that different mutations at that locus may explain the clinical variability. Indeed, physiological studies employing an animal model of Wilson's disease reveal that a single gene mutation may inhibit copper transport at multiple locations within the cell. Hence, the variety of mutations identified in the Wilson's disease gene potentially may affect copper transport to varying degrees, and at different cellular sites (Schilsky, 1994). However, detailed genetic and epidemiological studies suggest that the variability in clinical expression observed in Wilson's disease patients may not be solely a consequence of allelic heterogeneity, since marked differences in presentation, age of onset and disease course have been observed in family members who have inherited two identical mutant alleles.

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2. DNA-based diagnosis Developments involving the molecular genetics of Wilson's disease have provided a means for carrier detection and early diagnosis (Sternlieb, 1993). In fact, several studies using haplotype analysis of relatives with closely linked markers have permitted precise carrier detection with less than 1 to 2% error. There also is a report of prenatal exclusion of Wilson's disease by analysis of DNA polymorphism in a chorionic-villous biopsy performed at nine weeks' gestation (Cossu et al., 1993). Unfortunately, the use of genetic techniques in the diagnosis of Wilson's disease has significant limitations. Currently, DNA marker studies can be performed only within families, and under circumstances where the diagnosis already has been established definitively in at least one family member by standard biochemical methods. The index patient's DNA is then used as a reference to recognise the disease-carrying chromosomes in other members of the family. However, spontaneous chromosomal rearrangements can cause such markers to be uninformative, thereby limiting the diagnostic reliability. These findings indicate considerable potential difficulties for DNA-based genetic screening, since most patients will possess alleles with two different mutations of the Wilson's disease gene (Schilsky, 1994). Moreover, evaluation for Wilson's disease by DNA market analysis is generally performed only in a few specialised centers and there is a delay before results are obtained. Given the rapidity and accuracy of biochemical analyses in establishing the diagnosis of Wilson's disease, as well as the aforementioned limitations of genetic testing, standard biochemical methods should continue to be utilised in the evaluation of the vast majority of suspected cases (Schilsky, 1994). The most likely application of genetic linkage testing will be in the uncommon situation where biochemical methods do not provide a definitive answer, particularly under circumstances where patients have received prior chelation therapy. In addition, genetic screening of young family members of patients afflicted with the disorder would facilitate early diagnosis and permit initiation of therapy in the presymptomatic state. 3. Pathogenesis It is postulated that the harmful effects of excess copper are mediated by the generation of freeradicals, which deplete cellular stores of glutathione and oxidise lipids, enzymes, and cytoskeletal proteins. Indeed, it has been shown that a number of intracellular systems are disrupted by elevated copper concentrations, including organellar membranes, DNA, microtubules, and various enzymes and proteins, although the principal cellular target of copper toxicity is unknown. In the earliest stages of hepatocellular injury, ultrastructural abnormalities involving the endoplasmic reticulum, mitochondria, peroxisomes and nuclei have been identified (Sternlieb, 1990). These changes, in conjunction with diminished mitochondrial enzyme activities, may be important steps in the pathophysiologic events leading to lipid peroxidation and triglyceride accumulation in the hepatocyte (Sternlieb, 199o). Wilson's disease patients exhibit impaired biliary excretion of copper, which is believed to be the fundamental cause of copper overload. Moreover, the prompt reversal of abnormal copper metabolism in Wilson's disease patients following orthotopic liver transplantation confirms that the primary defect resides in the liver. It has been proposed that the Wilson's disease gene product is responsible for copper secretion from the liver cell, either across the canalicular (apical) membrane of the hepatocyte or into a subcellular compartment that communicates with the bile canaliculus (Tanzi et al., 1993). The latter is consistent with a putative endoplasmic reticulum, Golgi or lysosomal defect underlying the diminished biliary excretion and systemic accumulation of copper observed in patients with Wilson's disease. In addition, in an animal model of Wilson's disease, the Long-Evans Cinnamon rat, excessive hepatic copper accumulation occurs in the setting of diminished biliary excretion. These rodents exhibit impaired entry of copper into the lysosomes, with normal delivery of lysosomal copper to the bile (Schilsky et al., 1994a). The Long-Evans Cinnamon (LEC) rat is a mutant strain of the Long-Evans rat
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which spontaneously develops fulminant hepatitis at 3 to 4 months of age, resulting in a 40% mortality rate. Surviving animals manifest chronic hepatic disease, low serum ceruloplasmin levels, and increased copper concentrations in the liver. Thus, the LEC rat shares many important clinical, biochemical, and histologic features with Wilson's disease, and the recent availability of this animal model likely will provide new insight into the pathogenesis of the human disorder. 4. Clinical features The biochemical defect which leads to the accumulation of copper in Wilson's disease is present at birth; however, clinical symptoms rarely are observed before the age of 5 years. the initial signs of Wilson's disease generally are detected in older children, adolescents, and young adults, although case reports have documented the clinical onset as early as 4 years. Wilson's disease patients typically present with hepatic and/or neurologic dysfunction. In a large series of patients (Sternlieb, 1985), the initial clinical manifestations were hepatic in 42%, neurologic in 34%, psychiatric in 10%, haematologic in 12%, and renal in 1%. Less commonly, patients present with skeletal, cardiac, ophthalmologic, endocrinologic or dermatologic symptoms (Table 1). Approximately 25% of the patients have involvement of two or more organ systems at initial evaluation, although, with the advent of aggressive screening, there has been a significant increase in the number of asymptomatic patients diagnosed. Table 1: Clinical manifestations of Wilson's disease
Hepatic Neurologic Psychiatric Orhthalmologic Hematologic Renal Cardiovascular Musculoskeletal Gastrointestinal Endocrinologic Dermatologic - cirrhosis, chronic active hepatitis, fulminant hepatic failure - bradykinesia, rigidity, tremor, ataxia, dyskinesia, dysarthria, seizures - behavioural disturbances, cognitive impairment, affective disorder, psychosis - Kayser-Fleischer rings, sunflow cataracts - haemolysis, coagulopathy - renal tubular defects, diminished glomerular filtration, nephrolithiasis - cardiomyopathy, arrhythmias, conduction disturbances, autonomic dysfunction - osteomalacia, osteoporosis, degenerative joint disease - cholelithiasis, pancreatitis, spontaneous bacterial peritonitis - amenorrhoea, spontaneous abortion, delayed puberty, gynecomastia - azure lunulae, hyperpigmentation, acanthosis nigricans

Adapted from Zucker SD, Gollan JL. In: Prieto J, Rods J, Shafritz DA, eds. Hepatobiliary Diseases. Springer-Verlag, 1992:809, with permission.

5. Hepatic manifestations Hepatic involvement in Wilson's disease tends to manifest at a younger age (mean of 8 to 12 years) than does neurologic dysfunction, and is nonspecific, mimicking the features of a variety of acute and chronic liver diseases. Three major clinical patterns of liver disease are observed: cirrhosis, chronic active hepatitis and fulminant hepatic failure. In the early asymptomatic phase of Wilson's disease, or in the presence of inactive cirrhosis, liver function tests may be normal or only minimally elevated. In the majority of cases, hepatic injury develops insidiously and, if untreated, pursues a chronic and relentless course to cirrhosis. As estimated 5 to 30% of patients with Wilson's disease exhibit clinical, biochemical and histologic features similar to those observed in chronic active hepatitis (Scott et al., 1978; Schilsky et al., 1991). The diagnosis may be overlooked in these patients, since a significant percentage, almost 50% in one series (Scott et al., 1978), have no evidence of neurologic dysfunction or Kayser-Fleischer rings on ophthalmologic examination. Serum cerulophasmin levels also may be normal in the setting of severe hepatic inflammation. It has been estimated that Wilson's disease represents the underlying aetiology in 5% of patients with idiopathic chronic active hepatitis who are under 35 years of age (Schilsky et
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al., 1991). A distinctive feature of Wilsonian chronic active hepatitis is the relatively modest elevations of serum aminotransferase levels in the presence of severe hepatocellular necrosis and inflammation (Schilsky et al., 1991). More dramatically, Wilson's disease occasionally manifests as fulminant hepatic failure. These patients may be indistinguishable from individuals with viral-induced hepatic necrosis, and many of the biochemical tests used to establish the diagnosis of Wilson's disease are abnormal in patients with other forms of fulminant hepatic failure (McCullough et al., 1983). The clinical features most suggestive of fulminant Wilsonian hepatitis include the presence of intravascular haemolysis, splenomegaly, and Kayser-Fleicher rings. Biochemical markers indicative of Wilson's disease include relatively mild elevations in serum transaminases despite massive hepatic necrosis, hyperbilirubinemia with normal or low alkaline phosphatase levels, and a markedly elevated serum copper concentration (McCullough et al., 1983). The serum level of aspartate aminotransferase (AST) typically is higher than that of alanine aminotransferase (ALT), as a result of the associated haemolysis. Although uncommonly observed in Wilsonian fulminant hepatic failure, Kayser-Fleischer rings are not pathognomonic, since they occasionally are seen in patients with other cholestatic hepatic diseases. The presence of severe coagulopathy and hypercupriuria are not useful in distinguishing Wilsonian from non-Wilsonian hepatic failure (McCullough et al., 1983). Liver biopsy with measurement of quantitative copper may be helpful, although deranged clotting function may preclude this procedure, or necessitate the transjugular approach. If a biopsy specimen is obtained, histologic evidence of cirrhosis (predominantly micronodular) in a young patient with fulminant hepatitis is suggestive of Wilson's disease, as is an elevated hepatic copper content. Wilson's disease patients with acute hepatic failure tend to be young and to have a fulminant clinical course, with survival generally no longer than days to weeks unless hepatic transplantation is performed (McCullough et al., 1983; Mowat 1987). Medical treatment frequently is unsuccessful, particularly when the disorder is associated with haemolysis and renal insufficiency. When transplantation is unavailable for patients it remains imperative to make the diagnosis of Wilson's disease for the purpose of aggressive medical therapy and family screening. 6. Hepatic pathology Abnormal liver histology is evident in biopsy specimens from asymptomatic Wilson's disease patients within the first decade of life. The earliest changes detectable on light microscopy include glycogen deposition in the nuclei of periportal hepatocytes, and moderate fatty infiltration. the lipid droplets, which are composed of triglycerides, progressively increase in number and size, in some cases resembling the steatosis induced by ethanol. The rate of progression of the liver histology from fatty infiltration to cirrhosis is variable, although it tends to occur by one of two general processes, either with or without hepatic inflammation. Some Wilson's disease patients develop a histologic picture that is indistinguishable from chronic active hepatitis (Scott et al., 1978; Schilsky et al., 1991). Pathologic features include mononuclear cell infiltrates, which consist mainly of lymphocytes and plasma cells, piecemeal necrosis extending beyond the limiting plate, parenchymal collapse, bridging hepatic necrosis and fibrosis. If untreated, this may evolve into macronodular cirrhosis or progress rapidly to fulminant hepatitis. The development of cirrhosis also may occur in the absence of significant parenchymal inflammatory infiltrate or necrosis. It is notable that the vast majority of Wilson's disease patients have evidence of fibrosis on liver biopsy, despite widely varying levels and patterns of hepatic inflammation and injury. Hepatocellular carcinoma is uncommonly associated with Wilson's disease, in contrast to hemochromatosis. It has been proposed that the diminished cancer risk is due to the relative dearth of an inflammatory component in the development of most cases of Wilsonian cirrhosis. Indeed, animal studies suggest that copper may exert a protective effect against the development of malignancy. On the other hand, LEC rats exhibit a high incidence of hepatocellular carcinoma in the setting of marked hepatic copper accumulation (Sokol, 1994), and this neoplastic potential is effectively abrogated by
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the administration of D-penicillamine. These observations have led to speculation that the low incidence of hepatocellular carcinoma in Wilson's disease patients is attributable to chelation therapy, as long-term survival is rare in untreated patients. The histochemical staining of liver biopsy specimens for copper is of little diagnostic value in patients with Wilson's disease. This is due to the fact that during the initial stages of copper accumulation, the metal is distributed diffusely in the cytoplasm and frequently is undetectable by rhodamine or rubeanic acid staining. Orcein, which is believed to stain polymerised metallothionein sequestered in lysosomes, exhibits a characteristic granular pattern in only half of patients with early Wilson's disease. As the disease becomes more advanced, copper is sequestered within hepatocyte lysosomes and is detectable by routine histochemical techniques, even though tissue concentrations actually are lower than in earlier stages of the disorder. In contradistinction to Wilson's disease, other conditions in which hepatic copper is elevated (eg., primary biliary cirrhosis, sclerosing cholangitis, biliary atresia, intrahepatic cholestasis of childhood, Indian childhood cirrhosis, and the normal neonate) are nearly always associated with stainable copper. Due to the insensitivity of copper staining techniques, timedependent changes in the distribution of copper within the liver cell, heterogeneity of hepatic copper deposition, and the lack of specificity of hepatocyte copper granules, histochemical staining for copper is unreliable in establishing the diagnosis of Wilson's disease. 7. Laboratory diagnosis of Wilson's Disease The simplest screening procedure includes a slit-lamp examination of the eyes, and measurement of serum ceruloplasmin and transaminase (ALT, AST) levels. If Kayser-Fleischer rings are present on ophthalmologic examination and ceruloplasmin levels are below 20 mg/dL in a patient with neurologic signs or symptoms, the diagnosis of Wilson's disease is established. If a patient is asymptomatic, exhibits isolated liver disease, or lacks corneal rings, the coexistence of a hepatic copper concentration above 250 g/g dry weight and a low serum ceruloplasmin level also is sufficient to make the diagnosis. 7.1 Serum ceruloplasmin

The normal serum concentration of ceruloplasmin is 20 to 40 mg/dL and, while levels are low in the human newborn, they gradually rise during the first two years of life, coincident with the postnatal decline in hepatic copper concentration. Although a decreased ceruloplasmin level per se is not diagnostic of Wilson's disease, approximately 90% of all patients, and 85% of individuals presenting with hepatic manifestations of the disease have levels of this glycoprotein that are below the normal range. Hypoceruloplasminemia occasionally may occur in other hepatic conditions, such as fulminant non-Wilsonian hepatitis, as a consequence of diminished hepatic synthetic function. Patients with nephrotic syndrome, protein-losing enteropathy, malabsorption, or severe malnutrition also may manifest low serum ceruloplasmin levels, although there usually is no diagnostic difficulty in these cases. Difficulty may arise with regard to the 10% of heterozygous carriers of the gene for Wilson's disease who manifest diminished serum levels of ceruloplasmin, yet never develop clinical symptoms or signs of the disease. These individuals, who represent approximately 1 in 2,000 persons in the general population, may present a difficult diagnostic dilemma if they fortuitously develop chronic active hepatitis or cirrhosis (or another aetiology), thereby mimicking the clinical, biochemical and histological features of Wilson's disease. Normal ceruloplasmin concentrations are found in up to 15% of patients with Wilson's disease and active liver involvement (Scott et al., 1978). This presumably is due to increased hepatic synthesis and release of the glycoprotein in response to hepatic inflammation, as ceruloplasmin is an "acute phase reactant". The ceruloplasmin concentration declines to the low levels typically associated with Wilson's disease as the inflammatory activity in the liver abates. A further reduction in the ceruloplasmin level generally follows the initiation of chelation therapy. Plasma concentrations also
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are influenced by a variety of humoral and hormonal agents, and elevated estrogen levels, secondary to pregnancy or exogenous administration, occasionally may elevate previously low ceruloplasmin levels into the normal range. 7.2 Urinary copper excretion

The urinary excretion of copper is greater than 100 g/24 hours (normal: <40 g/24 hours) in most patients with symptomatic Wilson's disease, reflecting increased serum levels of the readily filterable fraction of non-ceruloplasmin copper. In patients with fulminant hepatic necrosis due to Wilson's disease, urinary excretion of the metal may exceed 1,000 g/24 hours, as hepatic copper stores are released into the systemic circulation. Unfortunately, the measurement of urinary copper often is misleading due to inaccuracies in collection and laboratory analysis, and care must be taken to use copper-free containers for storage of the urine samples. It also should be noted that asymptomatic Wilson's disease patients do not necessarily exhibit elevated urinary copper concentrations. Moreover, urinary copper levels may be elevated in a variety of other hepatic disorders including cirrhosis, chronic active hepatitis, and cholestatic disorders such as primary biliary cirrhosis. Thus, the quantification of urinary copper is of little value as a screening test for Wilson's disease; although it may be useful as a means of confirming the diagnosis and in evaluating compliance and the response to chelation therapy. 7.3 Hepatic copper concentration

If Kayser-Fleischer rings or neurologic abnormalities are absent, a liver biopsy for quantitative copper determination is essential to establish the diagnosis of Wilson's disease. Care must be taken to insure that the biopsy needle and specimen container are free from coper contamination. The normal hepatic copper concentration varies from 15 to 55 g/g (0.24 - 0.87mol/g) dry liver. Virtually all untreated patients with Wilson's disease have elevated hepatic copper levels, ranging from 250 to as high as 3,000 g/g dry liver. Values below 250 g/g usually are attributable to the irregular distribution of copper in the liver, particularly in the presence of cirrhosis, when small fragmented biopsy samples are obtained. The finding of a normal hepatic copper concentration effectively excludes the diagnosis of untreated Wilson's disease. However, an elevated liver copper level alone is insufficient to establish the diagnosis of Wilson's disease, since concentrations above 250 g/g may be found in other chronic hepatic disorders (mostly cholestatic), including primary biliary cirrhosis, primary sclerosing cholangitis, extrahepatic biliary obstruction or atresia, chronic active hepatitis, intrahepatic cholestasis of childhood and Indian childhood cirrhosis. These patients are readily distinguished from those with Wilson's disease on the basis of history, physical findings and biochemical testing. Moreover, in the great majority of individuals with prolonged cholestasis, serum ceruloplasmin concentrations are either normal or increased. 7.4 Incorporation of orally administered radiocopper into ceruloplasmin

Rarely, when a diagnostic dilemma remains or liver biopsy is contraindicated (eg. severe coagulopathy), the radiocopper loading test may be useful (Sternlieb and Scheinberg 1979). Serum radioactivity is measured at 1, 2, 4 and 48 hours after oral administration of the radionuclide (2 mg cupric acetate containing 0.3 to 0.5 mCi of 64Cu, mixed in 100 to 150 mL fruit juice or ginger ale). In healthy subjects and in patients with hepatic disorders that mimic Wilson's disease, the plasma concentrations of radiocopper rise rapidly, are maximal within one to two hours, and then fall and rise again over the ensuing 48 hours, as the non-ceruloplasmin bound radiocopper is incorporated into newly synthesised ceruloplasmin in the liver and released into the circulation. Wilson's disease patients, on the other hand, incorporate little or no radiocopper into nascent ceruloplasmin, even in the presence of normal ceruloplasmin concentrations. Heterozygotes have a pattern of incorporation that is intermediate between that of Wilson's disease patients and healthy individuals.
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7.5

Abnormal imaging

Despite advances in computed tomography (CT) and magnetic resonance imaging (MRI), these radiologic modalities have little to offer in the diagnosis or evaluation of hepatic involvement in Wilson's disease. While evidence of chronic liver disease (eg., splenomegaly, heterogeneous liver parenchyma, varices) may be identified in patients with advanced disease, these findings are neither specific nor sensitive for Wilson's disease. 8. Diagnostic screening The diagnostic approach to Wilson's disease must be tailored according to the clinical presentation. It must be emphasised that, in the absence of definitive DNA haplotype analysis, the diagnosis of Wilson's disease should not be based on the results of an individual laboratory test, and can only be established in the setting of confirmatory clinical and biochemical data. Patients with neurologic or psychiatric manifestations should undergo slit-lamp examination of the eyes and serum ceruloplasmin determination. The documentation of Kayser-Fleischer rings and a low serum ceruloplasmin concentration is sufficient to establish the diagnosis, which can be confirmed by the presence of increased 24 hour urinary copper excretion. A liver biopsy with quantification of hepatic copper is essential if either (1) Kayser-Fleischer rings are absent (in order to exclude the possibility that the patient is heterozygous for the gene), or (2) if ceruloplasmin levels are normal (as occurs in up to 15% of cases). It is imperative that all first-degree relatives be screened, particularly siblings. Wilson's disease may be clinically silent even in the presence of significant organ damage; hence, a delay in the diagnosis or in the initiation of therapy may lead to irreversible hepatic and/or neurologic injury. Biochemical screening of children should not be performed prior to 3 or 4 years of age. The evaluation should consist of a careful history and physical examination, serologic tests of liver function, a slit-lamp examination of the eyes, and a serum ceruloplasmin level, with liver biopsy and quantitative hepatic copper determination reserved for diagnostic dilemmas. Once the diagnosis of presymptomatic Wilson's disease is established, lifelong chelation therapy should be commenced immediately. 9. Treatment 9.1 Diet

The ubiquitous presence of copper in most foodstuffs makes stringent dietary copper restriction impractical, although it is suggested that patients avoid eating foods with a high copper content, such as liver, chocolate, nuts, mushrooms, legumes, and shellfish. Some authors also recommend the use of deionised or distilled water if the copper content of the patient's home drinking water exceeds 0.2 ppm, particularly during initial therapy. The use of domestic water softeners should be avoided, since these may substantially increase copper concentrations. 9.2 9.2.1 Pharmacologic therapy Penicillamine

Over the past three decades, it has been well documented that oral D-penicillamine results in complete reversal or alleviation of hepatic, neurologic and psychiatric abnormalities in most patients with Wilson's disease, and this drug remains the "gold-standard" therapy for this disorder. The key to a successful outcome is early diagnosis and treatment, and clinical disease can be prevented indefinitely in asymptomatic patients, provided that they adhere to continuous maintenance therapy. Some individuals demonstrate a dramatic response within weeks of initiating D-penicillamine, while others may exhibit no clinical improvement, or even temporary neurologic deterioration, for several months. The precise mechanisms of action of D-penicillamine remains controversial. Although the logic for the use of this drug in the treatment of Wilson's disease is based on its in vitro copper chelating properties, there is conflicting evidence regarding the ability of penicillamine to "decopper" the liver and other organs. It has been proposed that D-penicillamine also detoxifies the liver by sequestering
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intracellular copper in an innocuous state, either through the direct formation of copper complexes or by the induction of metallothionein synthesis. The fulminant decompensation observed in previously compliant patients who discontinue D-penicillamine therapy for a relatively brief period of time offers support for this hypothesis. Other postulated mechanisms of action of D-penicillamine include inhibition of collagen cross-linking, enhancement of intracellular levels of reduced glutathione, and suppression of inflammation via effects on leukotriene and prostaglandin metabolism. The standard dose of D-penicillamine is 1 to 2 g daily given orally in four divided doses 30 min before meals, although as much as 4 g/day can be administered to critically ill patients for brief periods of time. It is best taken on an empty stomach since food reduces its absorption. Most of the excess liver copper appears to be mobilised within the initial year of therapy, with urinary copper excretion approaching 2 to 5 mg/day during this period. After several years of treatment, urinary copper exretion levels decline to approximately 0.5 to 1.0 mg daily, as hepatic copper concentrations approach normal levels. When symptoms have largely abated and a stable clinical course has been achieved, the maintenance dose of D-penicillamine may be reduced to 1 g daily. A variety of adverse effects of D-penicillamine have been recognised (Zucker and Gollan 1996; Sternlieb and Scheinberg, 1985), although serious complications necessitating discontinuation of the drug are infrequent. Thus, D-penicillamine has been demonstrated to be effective and safe for use in the treatment of Wilson's disease, and remains the first-line drug in this disorder. 9.2.2 Trientine Trientine (triethylene tetramine dihydrochloride) was introduced in 1969 as an alternative chelating agent for cases in which serious toxic reactions to D-penicillamine occur. It has been well-established that 1 to 2 g administered orally in 3 divided doses induces negative copper balance and effects clinical improvements in patients with Wilson's disease. As with penicillamine, trientine should be administered prior to meals, since food interferes with absorption. The exact mechanism of action of this drug remains unknown, although it has been shown both to enhance urinary copper excretion and to decrease intestinal copper absorption. Unlike D-penicillamine, trientine causes the serum copper concentration to rise during cupriuresis, suggesting that the two agents may mobilise copper from different systemic pools. Most of the toxic side-effects necessitating conversion from D-penicillamine to trientine typically subside while on trientine. The exception is elastosis perforans serpiginosa, which may progress in some patients. Symptoms of skin rash, gastrointestinal distress and rhabdomyolysis have been reported in patients with Wilson's disease or primary biliary cirrhosis treated with trientine. Sideroblastic anaemia is the only major side-effect attributed to this medication. Although trientine appears to cause minimal toxicity, it has less of a cupriuretic effect than D-penicillamine, and hence, is currently not recommended as primary therapy. 9.2.3 Zinc The principal mode of action of zinc is postulated to be via the induction of intestinal metallothionein synthesis, which results in the sequestration of copper in intestinal epithelial cells, thereby preventing absorption into the portal circulation and enhancing faecal copper excretion. Zinc also may directly exhibit a protective effect on hepatocytes by inducing the synthesis of metallothionein in these cells. A minimum of 75 mg of zinc sulfate or zinc acetate per day, administered in 2 divided doses between meals, can maintain neutral or negative coper balance, although most studies have administered 50 mg thrice daily. Common side-effects of oral zinc include headaches and gastrointestinal upset, the incidence of which may be reduced by the use of zinc acetate, rather than zinc sulfate. Although longterm followup is limited, major complications have not been reported with the use of zinc for the treatment of Wilson's disease. Due to the slower onset of action as compared with other chelating agents, zinc is not recommended for the initial treatment of symptomatic Wilson's disease. In fact, most of the published experience with zinc therapy has been in patients who previously had been decoppered with D-penicillamine. It has been suggested that zinc may be useful for presymptomatic or pregnant patients, as well as for
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maintenance therapy in individuals who have previously been decoppered with chelating drugs. However, there are data indicating that hepatic copper may continue to accrue in some Wilson's disease patients treated with zinc sulfate alone (Zucker and Gollan, 1996). Additionally, long-term followup studies are needed to determine whether the de-coppered state is sustained with zinc therapy, with monitoring for untoward side-effects. Based on the information currently available, oral zinc should be used as a third-line therapy in the rare patients who develop intolerance to both Dpenicillamine and trientine. There appears to be no synergistic effect of zinc in combination with an additional chelating agent; hence the concomitant administration of penicillamine or trientine and zinc is not recommended. 9.2.4 Thiomolybdates Thiomolybdates appears to lower systemic copper levels by complexing lumenal copper, and thereby inhibiting intestinal absorption. In addition, the portion of the drug that is systemically absorbed may bind excessive serum copper and render it less available for cellular uptake, ultimately resulting in the removal of copper from intracellular stores. Moreover, in contrast to penicillamine and trientine, thiomolybdates exhibit a higher affinity for copper than metallothionein in vitro, suggesting that potentially this drug may be able to more effectively remove copper from the cell. Limited studies of ammonium tetrathiomolybdate (60 to 100 mg daily in two divided doses) in Wilson's disease patients in whom D-penicillamine and/or trientine was poorly tolerated or ineffective demonstrated the drug to be highly successful in lowering hepatic copper concentrations (Zucker and Gollan, 1996). Additional trials in a small number of patients have supported the efficacy and safety of ammonium tetrathiomolybdate in the initial treatment of Wilson's disease, and have further suggested that this medication is less prone to precipitate the neurologic decompensation observed with other chelating agents. Although these results appear promising, thiomolybdates have caused bone marrow suppression. Thus, further investigation is required before the routine use of this drug can be recommended. 9.3 Long-term management

Lifelong chelation therapy, without interruption, is necessary in all Wilson's disease patients. Cessation of therapy may result in rapid and irreversible hepatic and neurologic deterioration. In a study of 11 patients who discontinued treatment, 8 patients died of fulminant hepatitis after an average survival of only 2.6 years. Thus, it is imperative that an alternative agent be administered to any patient who is unable to continue D-penicillamine due to adverse effects (Scheinberg 1987). 9.4 Liver transplantation

Despite advances in medical therapy, significant mortality rates are still observed in specific subsets of patients with Wilson's disease. These individuals, in whom orthotopic liver transplantation has proven most successful, generally present with acute fulminant hepatic failure associated with hemolysis and hypercupremia (either as the initial presentation or due to poor compliance with medical therapy), or with advanced cirrhosis and hepatic insufficiency, unresponsive to an adequate trial of chelation therapy and supportive measures. In the absence of severe hepatic disease, liver transplantation generally is not recommended for the management of refractory extrahepatic manifestations, such as neurologic deterioration. In a series of 55 patients with Wilson's disease who underwent hepatic transplantation, a 79% one-year survival was observed, with an overall survival rate of 72%, at 3 months to 20 years (Schilsky et al., 1994b). Transplant recipients uniformly manifest complete reversal of the underlying defects in copper metabolism, and demonstrate significant improvement in a variety of symptoms and signs of the disease.

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References Bull, P.C., Thomas, G.R., Rommens, J.M., et al. (1993), The Wilson's disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene, Nature Genet, vol.5, p. 327. Cossu, P., Pirastu M., Nucaro, A., et al. (1993), Prenatal diagnosis of Wilson's disease by analysis of DNA polymorphism, N Engl J Med., vol. 327, p. 57. Frydman, M., Bonne-Tamir, B., Farrer, L.A., et al. (1985), Assignment of the gene for Wilson's disease to chromosome 13: linkage to the esterase D locus, Pro Natl Acad Sci. USA., vol. 82, p. 1819. McCullough, A.J., Fleming, R., Thistle, J.L., et al. (1983), Diagnosis of Wilson's disease presenting as fulminant hepatic failure, Gastroenterology, vol. 84, p. 161. Mowat, A.P. (1987), Liver disorders in children: the indications for liver replacement in parenchymal and metabolic diseases, Transplant Proc., vol. 19, p. 3236. Petrukhin, K., Fischer S.G., Pirastu, M., et al. (1993), Mapping, cloning and genetic characterization of the region containing the Wilson disease gene, Nature Genet., vol. 5, p. 338. Scheinberg, I.H., Jaffe, M.E and Sternlieb, I. (1987), The use of trientine in preventing the effects of interrupting penicillamine therapy in Wilson's disease, N Engl J Med., vol. 317, p. 209. Schilsky, M.L., Scheinberg, I.H and Sternlieb, I. (1991), Prognosis of Wilsonian chronic active hepatitis, Gastroenterol., vol. 100, p. 762. Schilsky, M.L., (1994), Identification of the Wilson's disease gene: clues for disease pathogenesis and the potential for molecular diagnosis, Hepatology, vol. 20, p. 529. Schilsky, M.L., Stockert, R.J and Sternlieb, I. (1994a), Pleiotropic effect of the LEC mutation: a rodent model of Wilson's disease, Am J Physiol., vol. 266, p. G907. Schilsky, M.L., Scheinberg, I.H and Sternlieb I. (1994b), Liver transplantation for Wilson's disease: indications and outcome, Hepatology, vol. 19, p. 583. Scott, J., Gollan, J.L., Samourian, S., et al. (1978), Wilson's disease, presenting as chronic active hepatitis, Gastroenterol. vol. 74, p. 645. Sokol, R. J. (1994), At long last: an animal model of Wilson's disease, Hepatology, vol. 20, p. 533. Sternlieb, I., Scheinberg, I.H (1979), The role of radiocopper in the diagnosis of Wilson's disease, Gastroenterol, vol. 77, p. 138. Sternlieb, I. and Scheinberg, I.H. (1985), Wilson's disease. In: Wright R, Millward-Sadler GH, Alberti KGMM, et al, eds. Liver and Biliary Disease. London, W.B. Saunders Company : 949. Sternlieb, I. (1990), Perspectives on Wilson's disease, Hepatology, vol. 12, p. 1234. Sternlieb, I. (1993), The outlook for the diagnosis of Wilson's disease, J. Hepatol. vol.17, p. 263. Tanzi, R.E., Petrukhin, K., Chernov, I., et al. (1993), The Wilson disease gene is a copper transporting ATPase with homology to the Menkes syndrome gene, Nature Genet. vol. 5, p. 344. Zucker, S.D, Gollan, J.L. (1996), Wilson's disease and hepatic copper toxicosis. In: Zakim D, Boyer TD, editors. Hepatology: A Textbook of Liver Disease. Philadelphia: Saunders: 1405.

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Indian Childhood Cirrhosis (ICC) - Revisited

Dr Seema Sethi Department of Pathology Lady Hardinge Medical College & Smt. Sucheta Kripalani Hospital, New Delhi, India 1. Background Indian childhood cirrhosis (ICC) was once the fourth most common cause of death in preschool children in India (Sub-committee of the Indian Council of Medical Research, 1955). Despite its recognition for over a century now, its etiopathogenesis is still an enigma. Much work has been done on ICC but every study seems to be but a drop in the vast ocean of this disease. True is the remark about ICC, "I see sea". It is unusual to find a disease confined to one geographical area. ICC is one such disease. Whether cases described from other countries are similar aetiologically to ICC is as yet unclear. A disease of childhood, most cases occur between 6 months and 3 years. However, it can occur up to 5 years of age (Bhave et al., 1982; Sethi et al., 1993). A male:female ratio of 3:2 has been reported (Sethi et al 1993). A high rate of parental consanguinity and up to 22% of incidence in siblings is known (Pandit and Bhave, 1996). Although various aetiological factors have been proposed in ICC none has been confirmed so far. Toxins, nutritional factors, infections, alpha-1-fetoprotein, alphaantitrypsin, disturbances in disaccharide tolerance, abnormality of tryptophan - niacin pathway and various trace metals like zinc selenium, cadmium, manganese, magnesium and copper have all been implicated as causes of ICC (Sub-commitee of the Indian Council of Medical Research, 1955). 2. Copper and ICC The high hepatic copper content in ICC and early introduction of copper-contaminated animal milk led to the hypothesis that copper is an aetiological agent in ICC (Portmann et al., 1978; Tanner et al., 1979). Traditionally, milk and water are boiled and stored in brass (an alloy of 70% copper and 30% zinc) vessels in India. Milk thus boiled and stored in brass vessels has a sixty fold increase in copper concentration and water about six times (Tanner et al., 1983). Not all patients with ICC received milk which had been stored in brass vessels (Sharda and Bhandari, 1984). In one prospective study no history of use of brass vessels was seen in 56% of cases (Table 1) (Sethi et al., 1993). Occurrence of ICC has been reported in bottle rather than breast-fed children (Tanner et al.,1983). However, ICC has been noted in children breast-fed 6-9 months (Sethi et al., 1993). Other family members and siblings receiving milk from the same source as ICC cases were found to have normal serum and urinary copper levels (Sharda and Bhandari, 1984). Whilst ingestion of large amounts of copper in early infancy may be a factor in the aetiology, it cannot fully explain the disease. In a study conducted in India on a group of 32 children who developed ICC, all had a significantly higher mean serum copper values in comparison with the age-matched control group (Table 2) (Sethi et al., 1993). In another 82 children suffering from ICC liver biopsies revealed raised liver copper concentrations. The liver copper concentrations increased with the severity of the disease (Table 3) (Sethi et al.,1993). Variable serum and hepatic content in the same stage of the disease has been explained on the basis of genetic heterogeneity (Sethi et al.,1993). On the basis of familial occurrence and high consanguinity a genetic aetiology is ICC had been suspected (Agarwal et al., 1979). A pedigree analysis compatible with an autosomal recessive inheritance has been reported (Chandra, 1976). It has been hypothesised that ICC is a genetic alteration in the copper metabolism perhaps related to the binding of copper with metallothionein (Sethi et al., 1993).

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Table 1 : Use of brass utensils in families of 32 ICC cases

Only brass and copper utensils Number of cases Percentage Mean Serum copper values (g/dL) 5 16 142 Mixed use 9 28 148.2 Number use of brass utensils 18 56 123.5

Table 2 : Serum copper levels in ICC

Serum Copper Control ICC Stage I Stage II Stage II
* p<0.01.

No. 10 32 7 17 8

Mean (g/dL) 96* 137.27* 123.2 146.5 187

Range (g/dL) 64-120 70-240 88-172 70-240 164-210

Table 3 : Correlation of histological grades and orcein grades in ICC

Orcein Grade Histological Grade I II III Total 1 4 2 1 7 2 4 20 3 27 3 5 20 12 37 4 1 4 6 11 Total 14 46 22 82

3. Clinical features ICC is generally insidious in onset. The clinical presentation of the disease is divided into 3 stages (Sub-committee of the Indian Council of Medical Research, 1955). In the early stage the symptoms are non-specific like abdominal distension, irregular fever, excessive crying and altered appetite. Jaundice is generally a late feature but may rarely be seen early in the course of the disease. Most patients present in the intermediate or "classical" stage of ICC with jaundice, loss of appetite, distended abdomen with a characteristic firm to hard liver with a "leafy" edge. The child is prone to secondary infections at this stage. The progress is generally relentless and within a few months the child develops the terminal stage with oedema, ascites, splenomegaly and haemorrhagic complications. The liver at this stage may be less palpable than the second stage. The disease generally runs this course and within 6-18 months the patient dies due to hepato-cellular failure leading to haemorrhage and coma or due to intercurrent infections. Spontaneous recovery variously reported from 13% (Sur and Bhatti, 1978) to 30% (Sub-committee of the Indian Council of Medical Research, 1955) in early stages of ICC has been reported but the cause is not known.
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Anaemia is commonly seen in patients of ICC. The standard liver function tests are usually deranged but not specific for differentiation of early ICC from other childhood liver disorders. Serum copper is raised significantly in ICC. Mean copper values rise with the clinical progression of the disease (Sethi et al., 1993; Tanner et al., 1979; Sharda and Bhandari 1984). Serum ceruloplasmin levels are however, normal or raised in contrast to Wilson's disease. Hepatic copper levels are raised. A hepatic copper level >800 g/g dry weight helps to distinguish ICC from other liver disorders occurring at this stage (Pandit and Bhave 1996). 4. Diagnosis Histopathology remains the corner stone of definitive diagnosis of ICC (Pandit and Bhave 1996). The two diagnostic features are : (1) widespread, coarse, dark brown orcein deposits and (2) intralobular, pericellular fibrosis (Pradhan et al 1983). Hepatocytic necrosis (97%) and hyaline (66%) are diagnostic though late features. Portal fibrosis, inflammation and disruption of the limiting plate are seen in most cases but are not specific for ICC. Parenchymal fat is usually absent and cholestasis is a late feature (Pandit and Bhave 1996). The intensity of orcein staining representing copper correlates significantly with the histological grades of the disease (Sethi et al 1993). The histological changes in ICC have been divided in three grades (Sub-committee of the Indian Council of Medical Research, 1955). In the early stage there is cloudy swelling of hepatocytes with portal inflammation. In the second stage there is increased fibrosis and in around the portal tracts leading to pseudo-lobule formation and degeneration of hepatocytes. The last stage is characterised by extensive fibrosis with coarse fibrous bands leading to complete loss of liver architecture and hepatic degeneration. Steroids have been used to allay symptoms in ICC. The copper chelater, D-penicillamine has been claimed to lead to clinical and histological remission in up to 65% of patients (Tanner et al., 1987). This is a single study performed on 29 patients with early ICC. More studies need to be done to definitely determine the role of D-penicillamine in the treatment of ICC. A reduction in the incidence of ICC, has been explained on the basis of the reduction in the use of brass utensils in India (Pandit and Bhave 1996). The possibility of dilution of the genetic pool due to inter-caste marriages in India has not been ruled out. A similar reduction seen in fatal infantile liver cirrhosis in certain regions of Austria has been reported (Mller et al.,1996). Further studies need to be done to determine the possible genetic defect in ICC. With the increasing rarity of the disease, genetic studies on the animal model, the "bedlington terrier" dog, would be useful. References Agarwal, S.S., Lahori, U.C., Mehta, S.K., Smith, D.G. and Bajai, P.C (1979), Inheritance of Indian childhood cirrhosis, Hum Hered., vol. 29, pp. 82-9. Bhave, S., Pandit, A.N., Pradhan, A.N., Sidhaye, D.G., Kantarjian, A., Williams, A., Tablot, I.C. and Tanner, M.S. (1982), Paediatric liver disease in India, Arch Dis Child., vol. 57, pp. 922-8. Chandra, R.K.(1976), ICC geneologic data, alpha-foetoprotein MbsAg & circulating immune complexes, Trans Roy Soc Trop Med Hyg., vol. 70, pp. 296-301. Liver Diseases Sub-committee of the Indian Council of Medical Research. (1955), Infantile cirrhosis of the liver in India, Indian J Med Res., vol. 43, pp.723-47. Mller, T., Feichtinger, H., Berger, H. and Mller, W. (1996), Endemic Tyrolean infantile cirrhosis: an ecogenetic disorder, Lancet, vol. 347, pp. 877-80. Pandit, A., and Bhave, S. (1996), Present interpretation of the role of copper in Indian childhood cirrhosis, Am J Clin Nutr., vol. 63, pp. 830S-5S. Portmann, B., Tanner, M.S., Mowat, A.P and Williams, R (1978), Orcein positive liver deposits in Indian childhood cirrhosis, Lancet, vol. 1, pp. 1338-40.

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Pradhan. A.M., Talbot, I.C and Tanner, M.S. (1983), Indian childhood cirrhosis and other cirrhosis of Indian children, Pediatr Res., vol.17, pp. 435-8. Sethi, S., Grover, S. and Khodaskar, M.B. (1993), Role of copper in Indian childhood cirrhosis. Annals of Trop.Paed.,vol. 13, pp. 3-6. Sharda, B and Bhandari, B (1984), Copper concentration in plasma, cells, liver, urine, hair and nails in hepatobiliary disorders in children, Indian Pediatr., vol. 21, pp. 167-71. Sur, A. and Bhatti, A. (1978), ICC : An inherited disorder of tryptophan metabolism, Brit. Med J., vol. 2, pp. 529-31. Tanner, M.S., Portmann, B., Williams, R., Mowat, A.P., Pandit, A.N., Mills, C.F. and Brenner, I. (1979), Increased hepatic copper concentration in Indian childhood cirrhosis, Lancet, vol. 1, pp. 1203-5. Tanner, M.S., Bhave, S.A., Kantarjian, A.H and Pandit, A.N. (1983), Early introduction of copper contaminated animal milk feeds as possible cause of ICC, Lancet, vol. 2, pp. 992-5. Tanner, M.S., Bhave, S.A., Pradhan, A.M. and Pandit, A.N. (1987), Clinical trials of penicillamine in Indian childhood cirrhosis, Arch Dis Child, vol. 62, pp.118-24.

Workshop 1 - Copper and Health

Chairman: Rapporteur: Participants: Dr G Becking Dr K Buckett Dr S Churches Prof B D Culver Prof H H Dieter Prof G Fell Dr D J Fitzgerald 1. Summary In considering the exposure of people to copper, the human population was divided into three separate groups: populations at risk of toxicity arising from exposure to copper; populations which were at risk of copper deficiency; and general population group. The population at risk to copper was considered to include special groups of people with clinical conditions such as ICC (unknown aetiology), ICT (genetic defect), G-6-PD (GSH-T defect leading to haemolysis) and Wilsons disease (defective ATPase), and groups of people on dialysis who have suffered liver disease. The Workshop participants gave consideration to the inclusion of other groups in this category, including pregnant women, normal infants and Wilson disease heterozygotes but have concluded that there was no scientific evidence which indicates that any of these groups are routinely at risk from copper toxicity. Dr T M Florence Dr R Gaunt Mr M T Gerschel Prof R Goyer Dr P Imray Dr B Markey Prof M Moore Dr E V Ohanian Prof A Oskarsson Prof S Sethi Dr M Taylor Dr R Uauy Mr J Williamson Dr K Bentley Dr L Tomaska

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The population at risk from deficiency was considered to include low birth weight infants, people with malnutrition, people who suffered from chronic enterolosses, people who supplement their diets with zinc or vitamin C, people with Menkes syndrome (genetic disorder) and people who consume some, but not all, high fibre diets. The general population was identified as people who fell into neither of the first two groups. In discussion regarding the levels of copper exposure to the normal population, the Workshop considered a WHO report (Trace Elements in Human Nutrition and Health, 1996) which estimated levels of copper exposure from 136 studies to be (at the 50th percentile): adults children 1.5 mg copper/day 1.3 mg copper/day

It was also noted that, in no case did exposure exceed 6 mg copper/day for adults and 4 mg copper/day in children. The report recommended a copper intake of 1.3 mg copper/day for adults and 0.6 mg copper/day for children. It was noted that the upper limit of intake was recommended to be 13 mg copper/day for adults and 1 mg copper/day for children. However, the Workshop noted that this recommended upper limit of intake was not based on any scientifically-derived NOAEL. Professor Dieter gave a short presentation of a report on childhood cirrhosis in Germany, based on 103 cases collected between 1982 and 1994. Of these, three could be directly attributed to copper exposure. 2. Research recommendations The Workshop on Copper and Health made the following research recommendations which would be considered to advance the information base for copper: To determine the concentration of copper and quality of drinking water that produce acute toxicity (such as gastrointestinal effects) from single and chronic exposures; To undertake studies on ICC populations to determine: genetic component; relationship to ICT and sporadic cases of childhood cirrhosis; possible mechanisms related to basic defect; environmental factors which influence copper exposure; and methods for early diagnosis.

To determine the incidence of genetic disorders of copper homeostasis; To determine factors and quantitative influences which determine the bioavailability of dietary copper including vegetarian diets, levels of iron, and zinc; To determine methodologies for identifying copper excess or deficiency in human populations; To study methods for applying stable isotope technology to define bioavailability and stores of copper; To determine the effect of marginal intake of copper on the prevalence of chronic disease; To determine mechanisms that influence copper homeostasis of absorption, storage and excretion in different age groups; and To determine dietary intakes of copper in populations living in developing countries to evaluate adequacy of those diets relative to copper.

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Workshop 2 - Distribution and Metabolism

Facilitator: Rapporteur: Participants: Dr G Anderson Prof HH Dieter Dr R Gooneratne Mr M Harrison Dr JFB Mercer Dr S Moghaddas Dr R Sadler Dr KH Summer Dr C Dameron Mr P Callan

1. Summary Participants of Workshop 2 - Distribution and Metabolism noted that copper is an essential element which is required for several critical metabolic processes in humans. However, it was also recognised that adverse health effects may result where copper deficiency or chronic copper excess occurs in humans. Manifestations of copper deficiency include anaemia, neutropenia and bone abnormalities, but clinical deficiency is relatively infrequent in humans. Adverse effects of acute copper excess include epigastric pain, tachycardia, haemolytic anaemia, haematuria, liver and kidney failure, and gastrointestinal bleeding. Respiratory effects, including metal fume fever, may result from occupational exposure to high concentrations of copper in the air. Some disorders associated with copper deficit or excess have a well defined genetic basis. These diseases include Menkes disease, which is characterised by deficiencies of copper; and Wilson's disease, which leads to progressive accumulation of copper. Indian Childhood Cirrhosis (ICC) and Idiopathic Copper Toxicosis (ICT), which are also considered to be associated with genetically-based copper sensitivity, are fatal liver conditions in early childhood which relate to copper excess. While there was considerable discussion on the distribution and metabolism of copper in humans, principle discussion of the Workshop focussed on consideration of those biological markers which would be useful in determining copper deficiency or excess in humans. In particular whether it was possible to use biomarkers for identification of marginal adverse and chronic excess effects in susceptible individuals, sub-populations and the general population. It was noted that superoxide dismutase and cytochrome oxidase activity, and the levels of low density lipoprotein and ceruloplasmin in blood changed during copper deficiency and excess. It was agreed that while serum copper and ceruloplasmin levels are useful indicators of moderate to severe copper deficiency, they were not sensitive measures of marginal copper deficiency. Similarly, tyrosinase deficiency which would be expected to lead to hypopigmentation of the skin and hair was not considered as a useful indicator of early exposure as microscopy analysis of the tissue was a time consuming method and that tyrosinase, while being an excellent marker, was very difficult to measure. Overall, the Workshop Group noted that while there were currently several biomarkers to indicate moderate to severe copper deficiency and excess, the Workshop Group was unable to identify biomarkers which could be used to efficiently identify effects, marginal copper deficiencies or excesses.

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Intracellular protection against copper toxicity in mammals

Charles T. Dameron, Shadi Moghaddas and Mark D. Harrison NHMRC National Research Centre for Environmental Toxicology, The University of Queensland and Griffith University.
(Paper presented to Workshop 2 Group)

1. Introduction Transition metals such as copper, molybdenum and zinc are essential to life because of the catalytic and structural roles they play in proteins and other biomolecules. Excessive concentrations of essential and non-essential metal ions like cadmium, mercury and lead can induce toxicities at the cellular, tissue and organ levels. Most organisms utilise a redundant array of cellular mechanisms to limit the toxicity of metal ions. The purpose of this review is to discuss intracellular mechanisms used to metabolise excessive concentrations of copper in mammalian cells. 2. General mechanisms for metal detoxification A selection of the cellular mechanisms utilised by mammalian cells to detoxify metals are illustrated in Figure 1 to demonstrate the scope of available mechanisms. Simplistically, the detoxification systems can be subdivided into mechanisms to reduce metal uptake, and to enhance metal sequestration and export. The reduction of metal importation to limit toxicity can operate through inhibition of the import machinery for the metal or by making the extracellular metal unavailable for absorption. For example, the level of iron taken up by mammalian cells is partially regulated by controlling at a translational level the membrane concentration of the receptor for the iron transport protein ferritin (Klausner and Dancis, 1994; Basilion et al., 1994). The regulation is mediated by intracellular concentration of iron atoms. The utilisation of these extracellular mechanisms will not be discussed in this review. The intracellular chelation or sequestration of metals into relatively innocuous complexes or organelles is a commonly used mechanism to limit their toxicity. In many cases the chelating agents are peptides or proteins that form stable complexes which limit the element's reactivity and/or aid in its excretion. In addition to providing a means of limiting the reactivity and toxicity of essential metals, some complexes appear to serve as storage sites for the metal ions. Sequestration of metals can also be an initial step in a pathway that ultimately leads to exportation of the metal or metalcomplex through a pump mechanism. Alternatively, a sequestered ion may be pumped into a vesicle for storage or extrusion by the vesicle. Metallothioneins (MTs), involved in the sequestration of copper, will be described in detail below.

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Figure 1: Mammalian Metal Detoxification Mechanisms M+ is any metal-ion, essential or nonessential. Exportation of excess metal ions to limit their intracellular toxicity is a ubiquitous process. The cation translocating P-type ATPases are among the more common pumping mechanisms used to transport metal ions out of cells or into organelles. The basic pump design is conserved across all life forms from bacteria to humans and is used for a range of elements, drugs, toxins and proteins. The sequence, presumably the structure, is frequently modified to increase the specificity and efficacy for a given element. In addition to the ATPase pumps some mammalian cells transport transition metalions, like zinc by utilising non-ATPase pumps (Palmiter and Findley, 1995). 3. Redundancy An essential feature of metal ion detoxification pathways is their redundancy. Many detoxification mechanisms are not entirely specific and are utilised against a number of different metal ions. An example of a non-specific detoxification pathway is metallothionein and its function in the sequestration of cadmium, zinc and copper ions. In conjunction with non-specific detoxification there can exist a set of detoxification mechanisms that are entirely specific for a particular metal ion. The Menkes protein, a copper-specific ATPase, is a good example of metal-specific detoxification mechanism. Specialisation of cells within an organism or tissue often leads to different patterns and levels of expression of the detoxification mechanisms available to the cells. Few of these mechanisms have been described in detail and in most cases specificity or lack of specificity is not understood at a molecular level. 4. Metalloregulation Metalloregulation, broadly defined as a cellular response to an intracellular metal ion concentration, is well documented in bacterial, fungal and animal systems. Metalloregulation can be accomplished at transcriptional, translational or enzymatic levels. At a molecular level these mechanisms operate through metal binding proteins. Metalloregulatory proteins serve as conformational switches with the metal cations organising or stabilising an active conformation of the protein (O'Halloran, 1993; Dameron et al., 1991; Dameron et al., 1993). These proteins allow cells to respond biochemically to increases and decreases in the intracellular copper concentration.
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5. The copper detoxification pathway The mammalian copper detoxification pathway contains at least two mechanisms to detoxify excess copper. Firstly, copper can be sequestered by the copper-binding protein metallothionein into an innocuous complex , described below. Secondly, excess copper can be transported out of the cell with the aid of an export pump (Dameron and Harrison, in press). An outline of the metabolic pathway for copper in a mammalian liver cell is shown in Figure 2. The abbreviated pathway description highlights some of the known mechanisms used by mammalian cells. The pathway reflects published mechanisms and should not be regarded as complete or exact since this area of research is evolving rapidly. A hepatic (liver) cell was chosen to highlight the pathway because of the liver's central role in copper metabolism. Not all cells would be expected to metabolise copper in the way the liver cell does and some cells are expected to have pathways that liver cells do not have. In contrast to the specific induction of metallothionein by copper in lower eukaryotes the mammalian metallothionein is induced by copper, cadmium, zinc and other transition metals (Hamer, 1986).

Figure 2: The hepatic copper metabolic pathway in mammals All mammalian metallothioneins are homologous, sharing common metal-binding motifs, and induction mechanisms. Metallothionein's function and structure are described below. The disruption of the metallothionein gene does not lead to a marked increase in copper sensitivity in transgenic mice (Michalaska and Choo, 1993), possibly because the cells can use the murine homolog of the Menkes ATPase pump to detoxify (export) the excess copper (Levinson et al., 1994). Transgenic mice that have had their metallothionein genes deleted are, however, sensitive to cadmium (Michalaska and Choo, 1993; Masters et al., 1994). It is plausible that the mice are cadmium sensitive because their cadmium detoxification pathway, being less redundant, lacks a specific export mechanism and must therefore rely wholly on sequestration by metallothionein. Metals, especially cadmium, can accumulate in mammalian cells bound to metallothionein and/or be sequestered into vesicles. The retention of metals, including copper, is tissue-specific and can induce cellular and tissue damage in cases of extreme overload. The primary route of copper excretion in humans is through the bile and may involve metallothionein (Hamer, 1986). The exact role metallothionein plays in the metabolism and detoxification of metals is still being elucidated.

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6. Copper detoxification mechanisms 6.1 Metallothioneins The principle sequestration molecule for transition metal ions in groups 11 and 12, including Cu(I) and Cd(II), are the cysteine-rich metallothioneins (MT). The cysteinyl sulfurs in these proteins function as ligands for the metals. Ultimately, the metals are bound in polynuclear metal-thiolate clusters (Dameron et al., 1991; Winge et al., 1993; Pickering et al., 1993; Winge et al., 1994). The cysteines in metallothioneins typically account for 20-30% of the amino acids and are arranged in repetitive Cys-Cys, Cys-Xaa-Cys and Cys-Xaa-Xaa-Cys motifs (where Xaa is any amino acid). The structural features of the MTs have been reviewed recently (Kille et al., 1994). Metallothioneins are small, 25-62 amino acid, cysteine-rich proteins that contain very few hydrophobic residues (four examples are highlighted in Table 1) Table 1 : Amino acid sequence of selected metallothioneins

S. cervisiae MTMFSELINFQNEGHECQCQCGSCKNNEQCQKSCSCPTGCNSDDKCPCGNKSEETKKSCCSGK N. crassa MT GDCGCSGASSCNCGSGCSCSNCGSK MouseMT2a MDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCA HumanMT2a MDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCA

Metallothioneins are found in a variety of forms in selected bacteria (Silver, 1994), fungi (Tohoyama et al, 1992; Mehra et al., 1990; Galli et al., 1994; Cervantes and Gutierrez-Corona, 1994), plants (Foley and Singh, 1994; Ledger and Gardner, 1994; Zhou and Goldsbrough, 1994), and animals (Hamer, 1986). The mammalian proteins are very homologous and have 62 amino acids with 20 cysteine residues. The protein is organised into two domains when metals are bound. The mammalian MT will bind a total of seven tetrahedral four coordinate atoms, like Cd(II) and Zn(II), or twelve three coordinate trigonal planar atoms, like Cu(I). The Cu(I) and Cd(II) containing forms of the protein have distinct structures (Kille et al., 1994). Ligation is accomplished entirely by cysteinyl thiolates. As seen in the structures of the rabbit 5Cd2Zn metallothionein solved by NMR (Arseniev et al, 1988) and X-ray crystallography, (Robbins and Stout, 1992) the lack of core hydrophobic residues to stabilise the tertiary structure of MT is compensated for by the formation of a metal thiolate core (Kille et al., 1994; Robbins and Stout, 1992). In the absence of metals to organise their core structure the metallothioneins adopt random coil configurations. The number and type of metal species in the protein dictates the tertiary structure of metallothionein. The structure of the copper form of the mammalian metallothionein has not been determined but it is known to adopt a two domain structure with six trigonally bound Cu(I) atoms in each domain. 6.2 Transcriptional regulation of metallothioneins

Metallothionein synthesis is transcriptionally regulated by metals in all higher eukaryotes (Hamer, 1986), excluding the recently discovered brain MT III (Palmiter et al., 1992) and epithelial MT IVs (Quaife et al., 1994), which have unknown functions. In a given organism or cell type the extent of the metallothionein induction depends on the concentration and species of metal ion and the genetic locus. Some cell types respond more strongly to cadmium and zinc (compared to copper) while others are induced primarily by copper ions.
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The transcriptional regulation of MT synthesis in mammals by copper is analogous to the well studied yeast system but is mechanistically distinct. In animals metalloregulation is accomplished through a series of 5' metal regulatory elements (MREs) that are arranged in tandem repeats. The MREs are not homologous to the 5' regulatory up-stream activation sequences (UAS) in yeast (examples are highlighted in Table 2). Table 2: MRE and UAS DNA Sequences S.cervisiae Upstream Activation Sequence 5'GATGCGTCTTTTCCGCTGAACC3' Mammalian Metal Regulatory Element 5'CTCTGCACTCCGCC3' MREs are conserved in higher mammals (Hamer, 1986). Partial purification, sequential cloning studies (Palmiter, 1994) and overlap of the MREs with basal control elements, especially GC rich Spl-like binding sites (Hamer, 1986), suggest the mammalian mechanism relies on multiple proteins, only one of which is a metal sensor. The yeast regulatory mechanism functions through a single metal-sensing transcription factor. If the mammalian mechanism was analogous to the yeast mechanism described above, the sensing protein would bind copper, or other metals, to form an active transcription factor-complex with an increased affinity for the MRE. Homology-based searches and probes for transcription factors homologous to the yeast factor in higher eukaryotes through conventional molecular biology techniques have not been successful. 6.3 Cu-ATPases

ATPase pumps are ubiquitous, being involved in the movement or translocation of ions such as H+, Na+, K+, Ca++ and a variety of metal ions. The ATPase pumps that translocate transition metal ions are in the P-type family of ATPases (Tsai et al., 1992). The "P" designation stems from the covalent phosphorylation of a conserved aspartic acid residue (single letter amino-acid code P) that is part of their reaction cycle. The P-type cation ATPases, including the copper ATPases, are highly conserved from bacteria to humans (Silver, 1994; Solioz et al., 1994; Silver et al., 1993). The human P-type Cu-ATPases are intimately involved in the metabolism and detoxification of copper ions as evidenced by the effects that defective pumps have on copper metabolism (Camakaris et al., 1995). Defects in one of the human Cu-ATPases (MNK) leads to a fatal copper deficiency disease called Menkes disease. The defect blocks transport of copper across the serosal membrane. Copper accumulates in the intestinal cell as a metallothionein complex and is ultimately lost through the normal sloughing of the intestinal cells. Copper transfer across membranes is also blocked in a number of other tissues. Defects in another human Cu-ATPase (WLD) that is primarily expressed in the liver leads to a copper excretory disorder that manifests as a chronic liver disease called Wilsons disease. Wilsons disease can be treated with chelation therapy. The Menkes and Wilsons proteins have the characteristic elements of a P-type ATPase; a phosphorylation domain, a phosphatase domain, an ATP binding site and a transmembrane cation channel. WLD, the Wilsons ATPase, is 57% identical to the Menkes protein; the homology increases to 79% or greater in the transduction, ion channel, phosphorylation and ATP-binding regions. The putative metal binding sites in MNK and WLD have the characteristic -Cys-Xaa-Xaa-Cys- motifs found in a variety of metal binding proteins including some zinc fingers, the metallothioneins, the copper-regulated yeast transcription factors (ACE1 and AMT) and other metal-binding sites in transition metal ATP translocases. Outside of the metal-binding motif there is no homology between the metal ATP translocase proteins and other metal-binding proteins. The putative metal-binding subdomains in the N-terminus of the human P-type Cu-ATPases have significant homology to the N-terminal metal-binding domains of prokaryotic P-type ATPases utilised for copper, cadmium, mercury and calcium transport. Both MNK and WLD have strong homology to
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CopA, a bacterial copper ATPase (Dameron and Harrison, in press; Vulpe et al., 1993). The close homology between MNK and WLD and CopA suggests that CopA is responsible for the active export of copper from bacterial cells. MNK and WLD contains six repetitive homologous sequences (MNKr1-6) of approximately 70 amino acids, each of which contains a single conserved -Met-XaaCys-Xaa-Xaa-Cys- motif as found in the bacterial ATPases. The regions between these putative subdomains are not conserved in the MNK protein, the Wilsons gene or in the bacterial ATPases. These six putative sub-domains are individually homologous in both sequence and predicted secondary structure to a bacterial mercury detoxification protein, merP. The three-dimensional structure of merP has been determined by NMR spectroscopy (Eriksson and Sahlman, 1993). 6.4 Regulation of the human copper ATPases

The Menkes protein functions to export excess intracellular copper and is postulated to be reversibly metalloregulated through the specialised copper-binding subdomains in the amino terminus of the protein. The metalloregulation couples the cellular export of copper to the intracellular concentration of copper ions. In Chinese Hamster Ovary (CHO) cells amplification of the MNK gene and subsequently of the protein, mRNA and protein levels correlate with the cellular copper resistance (Camakaris et al., 1995). Moreover, the amount of message transcribed and translated is not copper dependent but the activity increases with increasing copper. Thus, the regulation appears to take place at an enzymatic level rather than at a transcriptional or translational level. Activation of the MNK pump is hypothesised to occur through a conformational mechanism analogous to that proposed for the mammalian P-type Ca2+- ATPase. The binding of calmodulin to the regulatory domain of the Ca2+-ATPase induces a conformational change that appears to uncover a sterically-protected aspartic acid residue (Falchetto et al., 1992). Phosphorylation of the protected aspartic acid in the P-type ATPases is required for the export of metal cations. Were MNKr to contain a series of structures or sub-domains with folds analogous to merP, the copper metalloregulation could be afforded by rearrangements of the subdomains, Figure 3. The organisation of MNKr into sub-domains that are rearranged via Cu(I) during activation of MNK would document a new regulatory mechanism.

Figure 3: Activation of the Menkes Protein by Copper-Binding to MNKr1-6 Panel A. depicts the Menkes protein in its inactive state. This corresponds to normal intracellular levels of copper. The six subdomains contain no bound copper. Panel B. depicts the Menkes protein in its active state.
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Increasing intracellular copper levels leads to copper-binding to MNKr1-6 and activation of the ATPase with concomitant copper translocation. 7. Conclusion Living systems have developed an array of mechanisms to maintain intracellular copper levels within the "window" between deficiency and excess. This review has focussed on the means by which cells protect themselves from the potentially cytotoxic effects of excess copper. Mammalian cells possess multiple, redundant mechanisms for detoxifying excess intracellular copper. The recent discovery of the genes responsible for Menkes and Wilsons disease has focussed international interest on the understanding of copper homeostasis in mammalian cells. The role of these proteins in copper homeostasis in vivo is still poorly understood and remains an exciting area of biochemical research. References Arseniev, A., Schultze, P., Worgotter. E., Braun, W., Wagner, G., Vasek, M., Kagi, J.H.R. and Wuthrich, K. (1988), Three-dimensional structure of rabbit liver [Cd7] Metallothionein-2a in aqueous solution determined by nuclear magnetic resonance, J Mol Biol., vol. 201, pp. 637-657. Basilion, J.P., Rouault, T.A., Massinople, C.M., Klausner, R.D. and Burgess, W.H. (1994), The ironresponsive element binding protein: Localisation of the RNA-binding site to the aconitase active-site, Proc Natl Acad Sci USA, vol. 91, pp. 574-8. Camakaris, J., Petris, M.J., Bailey, L., Shen, P., Lockhart, P., Glover, TW., Barcroft, C.L., Patton, J. and Mercer, J.F.B.(1995), Gene amplification of the Menkes (MNK;ATP7A) P-type ATPase gene of CHO cells is associated with copper resistance and enhanced copper efflux, Hum Mole Genetics, vol. 4, pp. 2117-23. Cervantes, C. and Gutierrez-Corona, F. (1994), Copper resistance mechanisms in bacteria and fungi, FEMS Microbiol Rev., vol. 14, pp.121-37. Dameron, C.T., Winge, D.R., George, G.N., Sansone, M., Hu, S. and Hamer, D. (1991), A copperthiolate polynuclear cluster in the ACE1 transcription factor, Proc Natl Acad Sci USA, vol. 88, pp. 6127-31. Dameron, C.T., Arnold, P., Santhanagopalan, V., George, G. and Winge, D.R. (1993), Distinct Metal Binding Configurations in ACE, Biochem., vol. 32, pp. 7294-301. Dameron CT, Harrison MD. Mechanisms for protection against copper toxicity. Am J Clin Nutr. In Press. Eriksson, P.O. and Sahlman, L. (1993), 1H NMR studies of the mercuric ion binding protein MerP: Sequential assignment, secondary structure and global fold of oxidised MerP, Journal of Biomolecular NMR, vol.3, pp. 613-626. Falchetto, R., Vorherr, T. and Carafoli, E. (1992), The calmodulin-binding site of the plasma membrane Ca2+ pump interacts with the transduction domain of the enzyme, Protein Sci., vol. 1, pp.1613-21. Foley, R.C. and Singh, K.B. (1994), Isolation of a vicia faba metallothionein-like gene - expression in foliar trichomes, Plant Mole Bio., vol. 26, pp. 435-44. Galli, U., Schuepp, H., and Brunold, C. (1994), Heavy metal binding by mycorrhizal fungi, Physiologia Plantarum, vol. 92, pp. 364-8. Hamer, D.H. (1986), Metallothionein, Annu Rev Biochem., vol. 55, pp. 913-51. Kille, P., Hemmings, A and Lunney, E.A. (1994), Memories of Metallothionein, Biochim Biophys Acta., vol.1205, pp. 151-61. Klausner, R.D and Dancis, A. (1994), A genetic approach to elucidating eukaryotic iron metabolism, FEBS Lett., vol. 355, pp. 109-13.
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Ledger, S.E and Gardner, R.C. (1994), Cloning and characterization of five cDNAs for genes differentially expressed during fruit development of kiwifruit (actinidia deliciosa var deliciosa), Plant Mole Bio., vol. 25, pp. 877-86. Levinson, B., Vulpe, C., Elder, B., Martin, C., Verley, F., Packman, S. and Gitschier, J. (1994), The mottled gene is the murine homolog of the Menkes disease gene, Nat. Genet., vol. 6 (4, April), pp. 369-373. Masters, B.A., Kelly, E.J., Quaife, C.J., Brinster, R.L. and Palmiter, R.D. (1994), Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium, Proc Natl Acad Sci USA, vol. 91, pp. 584-8. Mehra, R.K., Garey, J.R. and Winge, D.R. (1990), Selective and tandem amplification of a member of the metallothionein gene family in Candida glabrata, J Biol Chem., vol. 265, pp. 6369-75. Michalska, A.E and Choo, K.H. (1993), Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse, Proc Natl Acad Sci USA., vol. 90, pp. 8088-92. O Halloran, T.V. (1993), Transition metals in control of gene expression, Science, vol. 261, pp. 71525. Palmiter, R.D., Findley, S.D., Whitmore, T.E. and Durnam, D.M. (1992), MT III, a brain-specific member of the metallothionein gene family, Proc Natl Acad Sci USA, vol.89, pp. 6333-7. Palmiter, R.D. (1994), Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1, Proc Natl Acad Sci USA, vol. 91, pp.1219-23. Palmiter, R.D. and Findley, S.D. (1995), Clonning and functional characterisation of a mammalian zinc transporter that confers resistane to zinc, EMBO, vol.14:639-49. Pickering, I.J., George, G.N., Dameron, C.T., Kurtz, B., Winge, D.R. and Dance, I.G. (1993), X-ray Absorption Spectroscopy of Cuprous-Thiolate Multinuclear Clusters in Proteins and Model Systems, J Am Chem Soc., vol. 115, pp. 9498-505. Quaife, C.J., Findley, S.D., Erickson, J.C. et al. (1994), Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia, Biochem., vol. 33, pp. 7250-9. Robbins, A.H and Stout, C.D. (1992), Crystal Structure of Metallothionein. In: Stillman M.J., Shaw, C.F. and Suzuki, K.T, eds. Metallothioneins: synthesis, structure and properties of metallothioneins, phytochelatins and metal thiolate complexes. New York, New York: VCH Publishers, Inc., pp. 31-54. Silver, S., Nucifora, G. and Phung, L.T. (1993), Human Menkes X-chromosome disease and the staphylococcal cadmium-resistance ATPase: a remarkable similarity in protein sequences, Molecular Micro., vol. 10, pp. 7-12. Silver, S. and Ji, G. (1994), Newer systems for bacterial resistances to toxic heavy metals, Environ Health Perspect., vol. 102, Suppl. 3, pp. 107-13. Solioz, M., Odermatt, A. and Krapf, R. (1994), Copper pumping ATPases: common concepts in bacteria and man, FEBS Lett., vol. 346, pp. 44-7. Tohoyama, H., Tomoyasu, T., Inouhe, M., Joho, M. and Murayama, T. (1992), The gene for cadmium metallothionein from a cadmium-resistant yeast appears to be identical to CUP1 in a copperresistant strain, Curr Genet., vol. 21, pp.275-80. Tsai, K., Yoon, K.P. and Lynn, A.R. (1992), ATP-dependent cadmium transport by the cadA cadmium resistance determinant in everted membrane vesicles of bacillus subtilis, J Bacteriol., vol.174, pp. 116-21. Vulpe, C., Levinson, B., Whitney, S., Packman, S. and Gitschier, J. (1993), Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase, published erratum appears in Nat Genet., vol. 3, (March 3), p. 273. See comments,. Nat Genet., vol. 3, pp. 7-13.
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Winge, D.R., Dameron, C.T., George, G.N., Pickering, I.J and Dance, I.G. (1993), Cuprous-Thiolate Polymetallic Clusters, in Biology. In: Karlin, K.D and Tyeklar, Z, eds. Bioinorganic Chemistry of Copper. New York, NY: Chapman & Hall :110-23. Winge, D.R., Dameron, C.T. and George, G.N. (1994), The Metallothionein Structural Motif in Gene Expression. In: Eichhorn, G.L, Marzilli, L.G, eds. Advances in Inorganic Biochemistry 10. New Jersey: Prentice-Hall, pp. 1-48. Zhou, J. and Goldsbrough, P.B. (1994), Functional homologs of fungal metallothionein genes from Arabidopsis, Plant Cell, vol. 6, pp. 875-84.

Workshop 3 - Copper and the Environment

Chairman: Rapporteur: Participants: Dr WJ Adams Dr G Batley Prof JC Castilla Dr R Erickson Dr P Glazebrook Dr M Hallman Dr D Harkess Dr P Howe Prof T Hutchinson Prof GE Lagos Dr C Lee Mr R Smith Dr J Stauber Dr JHM Temmink Dr JM Weeks Dr HE Allen Mr DM Wagner

1. Summary To set the scene for issues relating to copper and the environment, Dr Paul Howe, Institute of Terrestrial Ecology, UK, outlined his perceptions of the issues that are the most important topics with regard to an environmental risk assessment of copper. These included: environmental sources of exposure; environmental fate; environmental bioavailability, including analytical techniques, uptake and biomonitoring; and deficiency versus toxicity, including essentiality, homeostatic mechanisms, adaptation, tolerance, and toxicity testing. 2. Aquatic systems Considerable group discussion took place on copper in the aquatic system, including whether it was best to use total or dissolved copper as the measure of exposure, and what criteria should be used on the issue of bioavailability. It was noted that the US EPA has given a clear direction on some of these issues by recommending dissolved copper as a better measure for water bodies, while some total copper is recommended for discharges, though these recommendations still require implementation by individual States in the US. The group also discussed definition for discharge zones, mixing zones, and whole river measurements by area, from both an Australian and North American perspective. 3. Toxicity testing An extensive discussion followed on toxicity testing, including bioassays versus physicochemical screening assays, and it was noted that some regulatory authorities were establishing a compendium of toxicity data, with minimum data set requirements, and that it was possible to set a maximum criterion based on acute toxicity data, and that chronic toxicity data can also go through a similar

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procedure. Finally, it was felt that there was a strong need for a separate set of requirements for plants, as existing requirements did not adequately address this area. 4. Bioavailability Speciation is reasonably understood for copper, with binding to natural organic matter being an important control. The assessment of speciation is very important in understanding the bioavailability of copper in the environment, and necessary for conducting a risk assessment for copper. Dissolved copper should be used to evaluate potential copper bioavailability, while still recognising that it generally overestimated the real value, but is still more appropriate than using total copper as a measure. Procedures are being developed, and should be refined and progressed to give more accurate estimates of copper bioavailability. Copper budget and distribution in the environment needs to be addressed, as does accumulation. The receptor site model approach to the prediction of copper speciation was discussed. This is based upon measured concentrations of dissolved copper and other water quality parameters and the known binding constants for copper at an organisms receptor site, e.g. a gill membrane, which might compete for the copper and therefore will control the bioavailable fraction.. 5. Terrestrial testing Terrestrial testing generally stems from field observations, and currently lacks a framework which reflects the mechanism of interaction between the organism and the environment. There is a need to understand the interactions between metals and soils, soils and water, etc. There is difficulty in setting an acceptable background level for copper, given the variability in naturally occurring copper levels. Speciation and bioavailability are also needed for terrestrial toxicity testing. For example, pH data is needed to allow for partitioning between soil and water to make a model useful. As with aquatic testing, it is rare to see single contaminants, and there is a need to take all compounds into account, as the possibility of synergism and/or antagonism arises with multiple sources and multiple compounds. The EU is publishing protocols for soil toxicity testing, covering several soil types representative of a number of countries, a range of compounds including copper, and a range of test species. There is a need to come to a basic mechanistic understanding on bioavailability in terrestrial soils for environmental risk assessment. The correct chemical measurements are important to give links to biological effects of copper in soils. Given that the range of availabilities and concentrations of copper in soils and sediments is greater than in aquatic systems, it is more important to improve the understanding of bioavailability in the terrestrial environment than in water. 6. Essentiality versus toxicity In terrestrial systems, deficiency exists, but copper can still be found in excess, and hence lead to toxicity. It is important to establish whether such a potential for excess and deficiency to copper exists in aquatic systems. New recovery techniques for copper now makes extraction from older sites economically viable, thus increasing the potential for new environmental exposure to copper. In terrestrial systems, exposure can arise from a variety of sources, including from new and previous mine sites, sewerage waste disposal, water courses and agricultural soil. A range of organisms in the environment can uptake and regulate copper, and due to the range of species and sensitivity to copper, there is in sufficient information available to decide what range of copper is acceptable in the environment.
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There is inadequate understanding of the effects that adaptation can have upon test results. Large increases in tolerance to copper are seen in some species in reproduction toxicity tests, and adaptation of populations can occur over a long time period. There is a need for guidelines for copper and other trace elements present in testing environments that ensure that demands for essential levels are met. 7. Bioaccumulation There are well established tests and surrogates for bioaccumulation of organics, but is there such a range of tests for inorganics? Without information on the mechanisms and biochemistry of bioaccumulation across phyla, the use of overconservative safety factors can lead to regulatory standards below normal environmental levels, or lead to deficiency. 8. Conclusions and recommendations Measures of "total" copper usually overestimate bioavailability considerably in aquatic ecosystems. "Dissolved" copper is a better estimate of bioavailable copper in aquatic systems than total copper, but is still conservative. Chemical speciation of copper is, perhaps, the best way to measure or estimate the fraction of total copper that is bioavailable. State-of-the-art techniques are evolving which allow for measurements of copper speciation in water. In a regulatory context, techniques which demonstrate reduced bioavailability in water, sediments, or soil should be used to set site-specific protective levels. It was agreed that an integrative approach should be used in assessing environmental risk associated with the use and disposal of copper. This integrative approach should consider toxicity, fate and transport, dissolution and transformation rate, and extent (as well as potential) for bioaccumulation and background levels. The currently used tests for evaluating the toxicity of metals to terrestrial species are very poor. The basic mechanisms controlling bioavailability of metals in soils have not been demonstrated. There is a need for tests which include different trophic levels and the tests need to consider factors controlling bioavailability. Bioaccumulation of metals does not follow the partitioning laws that govern the accumulation of nonpolar organics. It is recognised that as the concentrations of copper in water increase, the bioaccumulation factor decreases, and vice versa. Because of the balance between toxicity and essentiality, the philosophy underlying the regulation of copper in the environment should be to protect against adverse effects on biota. Where possible, local/indigenous organisms should be used for site-specific risk assessment purposes.

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Participants in the international workshop on copper

Addresses and titles are those current at the time of the workshop. Apologies are extended to those who may have been omitted from the following list.
Dr William J. Adams ICA Kennecott,Utah Copper, 8315 West 3595 South, PO box 6001, MAGNA, UTAH USA Professor Herb Allen University of Delaware Department of Civil and Environmental Engineering, NEWARK DE 19716, USA Dr Greg Anderson QIMR P.O. Royal Brisbane Hospital, HERSTON 4029, QLD AUSTRALIA Dr Graeme Batley CSIRO Centre for Advanced Analytical Chemistry, Private Mail Bag 7 MENAI 2234, NSW AUSTRALIA Dr George Becking IPCS ICCU PO Box 12233, MD EC-10, Research Triangle Park, NORTH CAROLINA 27709, U.S.A. Dr Keith Bentley Commonwealth DepT. of Health and Family Services GPO Box 9848, CANBERRA 2601, ACT AUSTRALIA Dr Kevin J. Buckett Health Dept. Western Australia Environmental Health Service, PO Box 8172, Stirling St, PERTH 6849, WA AUSTRALIA Mr Phil Callan Commonwealth Dept. of Health & Family Services Healthy Public Policy Unit, GPO Box 9848, CANBERRA 2601, ACT AUSTRALIA Professor J. Carlos Castilla P. Universidad Catlica de Chile Departamento de Ecologia, Ecologia Marina Facultad de Ciencas Biologicas, Casilla 114-D, SANTIAGO, CHILE Dr Sarah Churches EPA Canberra, Hazardous Wate Section, Waste Management Branch, 40 Blackall St, BARTON 2600, ACT AUSTRALIA Mr Aaron Cosier NRCET, 39 Kessels Road Coopers Plains, BRISBANE QLD 4108 AUSTRALIA Professor B. Dwight Culver University of California, Irvine 14 Mendel Court IRVINE, CALIFORNIA 92715, U.S.A. Dr Charles Dameron NRCET 39 Kessels Road Coopers Plains, BRISBANE QLD 4108 AUSTRALIA Professor Hermann H. Dieter Umweltbundesamt Institut fr Wasser, Boden Lufthygiene Bundesgesundheitsamts PO Box 33 00 22, D-14191 BERLIN, GERMANY Dr Melissa Haswell-Elkins NRCET, 39 Kessels Road Coopers Plains, BRISBANE 4108, QLD AUSTRALIA Dr Russell Erickson US EPA 6201 Congdon Boulevard, Deluth, MINNESOTA 55804, USA Professor Gordon S. Fell University of Glasgow Department of Pathological Biochemistry Glasgow Royal Infirmary Castle St, GLASGOW G4 0SF SCOTLAND Dr Jim Fitzgerald South Australian Health Commission Public and Environmental Health Service Environmental Health Branch PO Box 6, RUNDLE MALL ADELAIDE 5001, SA AUSTRALIA Dr T. Mark Florence Centre for Environmental Health Sciences 112 Georges River Crescent, OYSTER BAY 2225, NSW AUSTRALIA Dr Richard Gaunt RTZ Ltd Occupational Health Physician 6 St James Square, LONDON SW1Y 4LD, UNITED KINGDOM Mr M. Thierry Gerschel Trefimetaux - Dev/ Standardisation Manager 11 bis rue de lHotel de Ville, F- 92400 Courbevoie, CEDEX FRANCE Dr Peter Glazebrook CRA Limited 55 Collins St GPO Box 384D, MELBOURNE 3001 VIC AUSTRALIA Dr Ravi Gooneratne AVSG Lincoln University Animal & Veterinary Sciences Group Lincoln University, PO box 84, CANTERBURY NEW ZEALAND Professor Robert A. Goyer University of Western Ontario 6405 Huntingridge Rd., CHAPEL HILL, NC 27514, U.S.A. Dr Mal Hallman Osborne Mines PO Box 5170 MC TOWNSVILLE 4810, QLD AUSTRALIA Dr Donna Harkess Department of Primary Industries & Energy Edmund Barton Building GPO Box 858, Barton, CANBERRA 2601,ACT AUSTRALIA Mr Mark Harrison NRCET 39 Kessels Road Coopers Plains, BRISBANE QLD 4108 AUSTRALIA Dr Paul D. Howe Institute of Terrestrial Ecology Monks Woods Abbots Ripton Huntingdon, CAMBRIDGESHIRE 2LS, UNITED KINGDOM Copper

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Dr Paul D. Howe Institute of Terrestrial Ecology Monks Woods Abbots Ripton Huntingdon, CAMBRIDGESHIRE 2LS, UNITED KINGDOM Dr Paula Imray Queensland Health, Scientific Adviser (Toxicology) Environmental Health Branch GPO Box 48 Charlotte St., BRISBANE 4001 AUSTRALIA Ms Sharon Kratzmann NRCET, 39 Kessels Road Coopers Plains, BRISBANE QLD 4108 AUSTRALIA Professor Gustavo E. Lagos Pontificia Universidad Catlica de Chile Mining Centre Casilla 306, Cod. 105, SANTIAGO 22, CHILE Dr Christopher M. Lee International Copper Association 260 Madison Ave.- 16th Floor, New York , NY 10016, USA Dr Bruce Markey Environment Protection Authority (NSW) Citadel Towers, 799 Pacific Highway PO Box 1135, CHATSWOOD 2057, NSW AUSTRALIA Dr Julian F. B. Mercer Murdoch Institute Royal Childrens Hospital Flemington Rd, PARKEVILLE 3052, VIC AUSTRALIA Dr Shadi Moghaddas NRCET 39 Kessels Road Coopers Plains, BRISBANE 4108, QLD AUSTRALIA Professor Michael R Moore Director, NRCET 39 Kessels Road, Coopers Plains, BRISBANE QLD 4108 AUSTRALIA Mr Jack C. Ng NRCET, 39 Kessels Road Coopers Plains, BRISBANE QLD 4108 AUSTRALIA Dr Edward V. Ohanian Office of Water, US EPA Health & Ecological Criteria Division (4304), 401 M st. SW, WASHINGTON DC ,USA Professor Agneta Oskarsson Swedish University of Agricultural Sciences Faculty of Veterinary Medicine, Dept. of Food Hygiene, PO Box 7009, S-750 07 UPPSALA, SWEDEN Dr Arun Prakash NRCET, 39 Kessels Road Coopers Plains, BRISBANE QLD 4108 AUSTRALIA Ms Lixia Qi NRCET, 39 Kessels Road Coopers Plains, BRISBANE QLD 4108 AUSTRALIA

Dr Ross Sadler QHSS GCL, 39 Kessels Rd, Coopers Plains BRISBANE 4108, QLD AUSTRALIA Dr Seema Sethi Lady Hardenge Medical College & S.M.T. Sucheta Kripalani Hospital, Department of Pathology, NEW DEHLI 11001, INDIA Mr Ross Smith BHP R & D Environment, 41 Goldieslie Rd, INDOORPILLY 4108, QLD AUSTRALIA Dr Jenny Stauber CSIRO Division of Coal and Energy Technology Centre for Advanced Analytical Chemistry, Private Mail Bag 7 BANGOR 2234, NSW AUSTRALIA Dr Karl H. Summer GSF-National Research Centre for Environment and Health Institute of Toxicology, Ingolstdter Landstrae 1, D-85758 NEUHERBERG, GERMANY Dr Michael Taylor NZ Ministry of Health 133 Molesworth St PO bx 5013, WELLINGTON NEW ZEALAND Dr J. Hans M. Temmink Landbouwuniversiteit Agricultural University, Department of Toxicology, PO Box 8000, 6700 EA WACHENINGEN, THE NETHERLANDS Dr Luba Tomaska Commonwealth Dept. of Health & Family Services PO Box 9848, CANBERRA 2601, ACT AUSTRALIA Dr Ricardo Uauy Universidad de Chile (INTA) Instituto de Nutricin y Tecnologa de los Alimentos Casilla 138-11, SANTIAGO DE CHILE, CHILE Mr Andrew M. Wagner Commonwealth Department of Health & Family Services Chemicals Policy & Assessment Unit Mail Drop point 88, GPO Box 9848, CANBERRA 2601 ACT AUSTRALIA Dr Jason M. Weeks Institute of Terrestrial Ecology Monks Woods Abots Ripton Huntingdon, CAMBRIDGESHIRE PE17 2LS, UNITED KINGDOM Mr John Williamson Copper Development Association of Australia M M Kembla Copper Systems, Gloucester Boulevarde PORT KEMBLA 2505, NSW AUSTRALIA

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