How Nano Destroys Your Genetic Code PDF

Nanotoxicology, 2013; Early Online, 1–46
© 2013 Informa UK, Ltd.

ISSN: 1743-5390 print / 1743-5404 online
DOI: 10.3109/17435390.2013.773464
Mechanisms of genotoxicity. A review of in vitro and in vivo studies

with engineered nanoparticles
1 2 3 3,4 5 1
Zuzana Magdolenova , Andrew Collins , Ashutosh Kumar , Alok Dhawan , Vicki Stone , & Maria Dusinska
1 2
NILU-Norwegian Institute for Air Research, MILK, Health Effects Laboratory, Kjeller, Norway, Department of Nutrition,
3
Faculty of Medicine, University of Oslo, Oslo, Norway, Institute of Life Sciences, School of Science and Technology,
4
Ahmedabad University, Ahmedabad, Gujarat, India, Nanomaterial Toxicology Group, CSIR-Indian Institute of
Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13
5
Toxicology Research, Lucknow, Uttar Pradesh, India and Heriot-Watt University, Edinburgh, UK
Abstract or surface structure in the nanoscale". The term "nanoscale" is

only.
Engineered nanoparticles (NPs) are widely used in different defined as size range from approximately 1 to 100 nm
technologies but their unique properties might also cause (http://cdb.iso.org). "Nanomaterial" means a natural, inci-
adverse health effects. In reviewing recent in vitro and in vivo dental or manufactured material containing particles, in an
genotoxicity studies we discuss potential mechanisms of unbound state or as an aggregate or as an agglomerate and
genotoxicity induced by NPs. Various factors that may influence where, for 50% or more of the particles in the number size
genotoxic response, including physico-chemical properties and distribution, one or more external dimensions is in the size
experimental conditions, are highlighted. From 4346 articles on
range 1–100 nm. This definition covers materials where the
NP toxicity, 112 describe genotoxicity studies (94 in vitro, 22 in
specific surface area by volume of the material is greater than
vivo). The most used assays are the comet assay (58 in vitro, 9
2 3
60 m /cm (http://ec.europa.eu/environment/chemicals/
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in vivo), the micronucleus assay (31 in vitro, 14 in vivo), the

nanotech/index.htm#definition). As the definition of nano-
chromosome aberrations test (10 in vitro, 1 in vivo) and the
material is broad, in this review we considered NPs only. NPs
bacterial reverse mutation assay (13 studies). We describe are increasingly being used in many areas, but their
advantages and potential problems with different methods and possible impact on human health is not well studied or
suggest the need for appropriate methodologies to be used for understood. The properties of the NPs, for which they are used
investigation of genotoxic effects of NPs, in vitro and in vivo. in products, could also be potentially harmful to biological
Keywords: nanoparticle toxicity, DNA damage, DNA oxidation, systems and cause adverse effects (Chan 2006; Dusinska et al.
mechanisms of genotoxicity, primary genotoxicity, secondary
2011). Features such as NP size, shape, surface properties,
genotoxicity, nanoparticle properties, nanotoxicology
composition, solubility, aggregation/ agglomeration, particle
uptake, the presence of mutagens and transition metals
affiliated with the particles (Schins 2002; Stone et al. 2010) can
influence the mechanisms of genotoxicity, both primary and
secondary (Kisin et al. 2007). Unfortunately, there is still a lack
Introduction
of information about
Nanoparticles (NPs) have unique, potentially beneficial human exposure and possible adverse health effects of NPs. A
properties and nanotechnology is considered to be the better understanding of how properties of NPs define their
technology of the future. The definition of ‘Nanoparticle’ given interactions with cells, tissues and organs is a scientific
in the PAS71 document developed by the British Standards challenge that must be addressed for the safe use of NPs.
Institution is "A particle having one or more dimensions of the Toxicity testing of NPs using existing in vitro and in vivo
order of 100 nm or less" (PAS71 2005). NPs are also called models is a difficult task as there are so many different classes
ultrafine particles by some toxicologists (USEPA 2007), aitken of NPs with various characteristics that can contrib-ute to
mode and nucleation mode particles by atmospheric scientists toxicity by many diverse mechanisms (Vega-Villa et al. 2008).
(Kulmala 2004; NRC 1983) and engineered NPs by material Underlying mechanisms of NP toxicity are oxidative stress,
scientists (Oberdörster et al. 2005a,b). The International inflammation, immunotoxicity and genotoxicity (Dusinska et
Organization for Standardization defines the term al. 2012b).
"nanomaterial" as "material with any exter-nal dimensions in A literature search on genotoxicity of NPs was performed in
the nanoscale or having internal structure the PubMed database from year 2000 to 2012 (February)
Correspondence: Maria Dusinska, NILU-Norwegian Institute for Air Research, CEE, Health Effects Group, Instituttvn. 18, N-2027 Kjeller, Norway.
E-mail: maria.dusinska@nilu.no
(Received 23 December 2011; accepted 31 January 2013)
Z. Magdolenova et al.
using key words: NPs and toxicity, in vitro toxicity and NPs, in (diameter between 8 and 10 nm), while larger NPs such as 15–
vivo toxicity and NPs, NPs and genotoxicity, ultrafine particles 60 nm SiC-NPs may only have access to the DNA in dividing
and genotoxicity. From 4346 articles on NP toxicity, 112 cells, during mitosis when the nuclear membrane dissolves
publications describing experimental studies on NP (Singh et al. 2009; Liang et al. 2008). For instance, after in
genotoxicity (94 in vitro, 22 in vivo) were selected (Table I). vitro exposure TiO 2 NPs (Shukla et al. 2011a), Ag NPs
Studies investigating the environmental impact of NPs are not (Hackenberg et al. 2011b; Asharani et al. 2009) and ZnO NPs
included in this review. From the published literature, 67 (Hackenberg et al. 2011a) have been found in the cell nucleus.
genotoxicity studies used the comet assay (58 in vitro, 9 in Even larger intranuclear aggregates of TiO 2 NPs were observed
vivo), 44 the micronucleus assay (31 in vitro, 14 in vivo), 11 in the nucleus (the mean size of studied TiO 2 NPs was 285 ±
the chromosome aberrations test (10 in vitro, 1 in vivo) and 13 52 nm and in particular cases, aggregates could reach
the bacterial reverse mutation assay (Figure 1). In addition, diameters up to 2000 nm) (Hackenberg et al. 2010). These
some studies used other genotoxicity assays, such as HPRT authors observed on human nasal mucosa cells in vitro no
gene mutation, sister chromatid exchange, mRNA expression genotoxicity of TiO2 NPs (Hackenberg et al. 2010) but a
of DNA base exci-sion repair (BER) genes, detection of 8-oxo- positive effect of ZnO (Hackenberg et al. 2011d) with the
7,8-dihydrogua-nine (8-oxoG), in vivo DNA deletion, CII comet assay and also of Ag NPs (Hackenberg et al. 2011b)
only.
mutation analysis, the g-H2AX assay and pulsed field gel with the chromosome aberration assay and the comet assay.
electrophoresis. Large NP aggregates can even deform the nucleus, as
TiO2, iron and silver NPs are the most used NPs in shown by transmission electron microscopy (TEM) observa-
genotoxicity studies (Table I). After them, a higher number of tions in vitro in the Chinese hamster ovary cells CHO-KI study
publications describe genotoxic impact of fullerenes, silica, of Di Virgilio et al. (2010). Aggregates of TiO2 NPs induced
carbon black, zinc, gold NPs. Some studies investigate the the formation of cellular vesicles which impressed on the
effect of multiwalled carbon nanotube (MWCNT), poly-mer nucleus and modified its form. Deforma-tion of nuclear shape
NPs and single-walled carbon nanotube (SWCNT). In addition, could unfavourably affect the process of mitosis, physically
few or single publications can be found on geno-toxicity of preventing the correct segregation of chromosomes, and the
quantum dots, Co or CoCr, Pt, CuO, CeO2, rare metal and correct functioning of the mitotic spindle and its components.
metal oxide and alumina NPs. However, when looking on The NP aggregates could also mechanically damage the
carbon NPs together (WCNT, MWCNT, Fuller-enes, Carbon chromosomes. Di Virgilio et al. (2010) showed an increase in
black) they are the second most used after TiO 2 NPs. frequencies of micronuclei (MN) and sister chromatid
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exchanges after TiO2 NP exposure. However, no NPs were

Particle toxicology and consequent health effects of asbes- observed in nuclei.
tos fibres and coal dust serve as historical reference points for
the development of nanotoxicology concepts (Vega-Villa et al. Direct primary genotoxicity
2008). Several reviews dealing with NP genotoxi-city have
been published (Gonzalez et al. 2008; Stone et al. 2009; Singh Direct interaction of NPs with DNA or chromosomes
et al. 2009; Landsiedel et al. 2009; Ng et al. 2010; Karlsson NPs that are present in the nucleus (entering either by
2010; Donaldson et al. 2010; Xie et al. 2011). In updating this penetration via nuclear pores or during mitosis) might directly
fast-changing research area, we concentrate in particular on interact with DNA organised in chromatin or chromosomes
methods of investigation, mechanisms of gen-otoxicity, and the depending on the phase of cell cycle.
conditions that influence experimental results. During interphase NPs could interact or bind with DNA
molecules and influence DNA replication and transcription of
DNA into RNA. NPs could mechanically disturb the processes
or chemically bind to DNA molecules. To examine interactions
Mechanisms of NP-induced genotoxicity between NPs and DNA, some computational as well as
The mechanisms of NP genotoxicity are still not well under- experimental studies have been performed. Using
stood and it is often not clear if an effect on DNA is nano- computational methods, Jin et al. (2011) found that strong
specific. Genotoxicity may be produced by direct interaction of interactions between Al12X (X = Al, C, N, P) NPs and DNA
NPs with the genetic material, or by indirect damage from NP- bases/base pairs are expected. They suggested that Al NPs
induced reactive oxygen species (ROS), or by toxic ions might cause structural damage and affect DNA stability. An
released from soluble NPs (Kisin et al. 2007; Barnes et al. experimental study by An et al. (2010) showed an interaction
2008). Secondary genotoxicity can be a result of oxidative between carbon NPs and DNA in vivo in Escherichia coli.
DNA attack by ROS via activated phagocytes (neutrophils, Carbon NPs were bound to single-stranded DNA and
macrophages) during NP-elicited inflammation (Stone et al. incorporated into DNA duplex structures, probably during
2009). DNA replication. This suggests that carbon NPs could disturb
NPs that cross cellular membranes may be able to reach the DNA replication (An et al. 2010).
nucleus through diffusion across the nuclear membrane or During mitosis NPs could interact with chromosomes
transportation through the nuclear pore complexes, and interact causing clastogenic or aneugenic effects. NPs might introduce
directly with DNA (Figure 2). As discussed by Barillet et al. breaks into chromosomes or disturb the process of mitosis,
(2010), in vitro studies show that NPs of smaller size may mechanically or by chemical binding (Figure 3).
reach the nucleus via nuclear pores
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Table I. Current review of NP genotoxicity studies (+ positive; negative; ± equivocal).

Physico-chemical Result of NP properties, Genotoxicity Treatment
Nanoparticle characterisation characterisation NP preparation testing method conditions Result Cells/organism Reference
SWCNTs 5 and 10 mg/mL for 24 h
SWCNT (COCC, Size (TEM) Diameters: 8 nm, NP sonication six times Comet assay + Primary mouse embryo Yang et al. 2009
Chinese Academy length <5 mm intermittently fibroblasts
of Science, Rope-shaped (30 sec every 2 min)
Chengdu) Shape (TEM) C >99.99%
Chemical composition
(purity) (Raman
spectroscopy)
2
SWCNT BET surface area 731 ± 2 m /g Declared primary Comet assay 3 h of incubation, FE1-MutaTM mouse Jacobsen et al. 2008
(EliCarb) Pore size 15 nm particle diameter: 100 mg SWCNT/mL lung epithelial cell line
ICP-MS ~95% C, 2% Fe, 0.9–1.7 nm, length
<0.001% Co, Ni, Mn <1 mm
Nd Comet assay (FPG) Pos. Ctrl: Carbon +
Size distributions in 32–60, 280–417, and Following suspending CII mutation black (Printex 90) ±
water 2256 nm in water and media and
Size in media sonicating, shape and
size distributions were
analysed by TEM and
dynamic light
scattering – DLS
HiPco SWCNT Chemical analysis 99.7 wt% elemental C, SWCNT (purified Comet assay 0, 12, 24, 48 or At 3 h, + only at V79 (Chinese hamster Kisin et al. 2007
2 2
(CNI, Inc., (NMAM method nr. 0.23 wt% Fe by acid treatment, 96 mg/cm for 3 or 24 h 96 mg/cm lung fibroblast line)
Houston, TX) 5040 and ICP-AES) containing only At 24 h, + at
2
>99% of C content was 0.23% of Fe). ‡48 mg/cm
Purity (TGA-DSC, TPO, Produced by HiPco MN 0, 12, 24, 48 or V79 (Chinese hamster
2 lung fibroblast line)
NIR, Raman accountable to SWCNT technique were first 96 mg/cm for 24 h
spectroscopy) characterised Ames test 0, 60, 120 or S. typhimurium strains
Diameter distribution 0.4–1.2 nm After ultrasonitation
(Raman spectroscopy) (30 sec, 3 ), SWCNT 240 mg/plate YG1024/YG1029
were analysed with
scanning electron
microscopy (SEM)
and TEM
Specific surface area 2
1040 m /g
Dispersion in About 83%
suspension (SEM) 1–3 mm
Lenght (TEM)
SWCNTs, Acceptable DLS data Comet assay After 3 h instillation, + BAL cells Jacobsen et al. 2009
Genotoxicity of nanoparticles
could not be obtained, 54 mg/mouse (broncho-alveolar
(complex morphology lavage fluid from
and bundling of apolipoprotein E
SWCNT) knockout mouse,
SWCNT (Thomas Gas exchange 2
731 ± 2 m /g Described by the ApoE / )
8-oxodG and dG (HPLC Investigated effects in Both doses + in Female Fisher 344 rats Folkmann et al.
Swan and Co Ltd., surface areas (Jacobsen et al. 2008b) manufacturer: primary with electrochemical liver, lung and colon liver and lung, in from Taconic 2009
Consett, UK) Average pore sizes 15 nm (Jacobsen et al. particle size of 0.9– and UV detection) tissues, 24 h after colon mucosa (Ry, Denmark)
were 0 and 15 nm 2008b) 1.7 nm and a fibre intragastric
length <1 mm administration
Particle size in In saline solution were The particles were Doses: 0.064 and
suspensions (DLS) 195, 797 and 5457 nm suspended in either 0.64 mg/kg body weight
Table I. (Continued).

(low dose); the particle saline or corn oil, by mRNA expression of (bw) suspended in
size in the highest dose sonication (70 W, HO1, MUTYH, NEIL1, saline or corn oil; n =
could not be 42 kHz) in a 5-day NUDT1 and OGG1 8), saline solution
determined period for 10 h each day (control; n = 10) or corn
In corn oil: 34 and and again 30 min oil (control; n = 10)
178 nm (low dose) and before administration
1015 nm (high dose)
Transition metals 2% Fe and traces of Co, OGG1 repair activity
Ni and Mn
PAHs 417 ng/g of the U.S. EPA
priority PAH
compounds
MWCNTs 1, 20, 40 mg/mL, 4 h
MWCNT Particle size in 110–170 Suspended in MilliQ Comet assay + A549 (human lung Karlsson et al. 2008
3
(Sigma-Aldrich) powder (5–9 10 ) nm and sonicated for epithelial cell line)
2 20 sec
Surface area in Nd Suspended in medium Comet assay (FPG)
powder and sonicated
TEM 100–200
3
(3–7 10 ) nm
3
DLS (size of particles 300 (6 10 ) nm
and agglomerates in
medium)
Zeta potential
MWCNT (Facultes Outer diameter 11.3 nm Characteristics were MN in vivo 3 days after + Type II pneumocytes Muller et al. 2008
universitaires Length 0.7 mm described previously intratracheal (AT-II)
Notre-Dame de la Carbon content 98% NP heated (200 C/2 h) administration of
Paix (Namur, Remains Traces of Co and before use MWCNT (0.5 or
Belgium)) Fe catalysts 2 mg/rat)
DLS measurements Aggregates diameter Suspended in a 0.9% CB MN In vitro exposure to + RLE (rat lung epithelial
of suspension for of~ 1 mm saline solution with MWCNT cell line)

in vivo study 1% of Tween 80 (10, 25, 50 mg/mL)
MN with FISH In vitro exposure + MCF-7 (human
To MWCNT epithelial cell line)
(10, 25, 50 mg/mL
Baytubes Purity >95% C (no CHA 2.5, 5, 10 mg/mL, 4 and V79 (Chinese hamster Wirnitzer et al. 2009
macrosized free amorphous C), 18 h, ± S9 mix, lung fibroblast line)
MWCNT
agglomerates 50–5000 mg/plate,
(Bayer Size 0.1–3 mm Ames test S. typhimurium (strains
MaterialScience) 48 h, ±S9 mix TA 1535, TA 100, TA
1537, TA 98, TA 102)
Sonicated 2 5 min in
deionised water, mixed
until used
Size distribution
(by laser diffraction)

MWCNT (Tsinghua Purificated, Western blot (OGG1, Conc. 0, 5, 100 mg/mL, + Mouse embryonic stem Zhu et al. 2007
and Nanfeng UV-sterilised, diluted Rad 51 and XRCC4) 2 and 4 h incubation cells J11 and APRT±3C4
Chemical Group under ultrasonication APRT gene mutation +
Cooperation, China) >95% 0.5, 5 and 20 mg/mL of
MWCNT (Lawrence Purity Synthesised by using a gH2AX + HUVECs Guo et al. 2011
Berkeley National Aver. diameter 30 nm chemical vapour MWCNT, for 6, 12 and
Laboratory, (TEM) <1 mm deposition 24 h
Berkeley, CA) Length
Metal impurities Ni 3.38% (CVD) method
Y 0.67%
Fe 0.13% NPs suspended in Antioxidant NAC NAC decreased the
1640 medium + 10% (6 mM) pretreatment percentage of
FCS, ultrasonicated for lH2AX positive
30 cycles of 5 sec with cells
1 sec pause
Suspensions
prepared freshly before
experiment
MWCNT (Prof. Particle size (TEM) 5–20 nm in diameter, Sterilisation by heating CB MN 10 and 50 mg/mL, 24 h + A549 (human lung Srivastava et al.
D.G.Weiss, 300–2000 nm in length to 120 C, 2 h, epithelial cell line) 2011
Rostock Mean hydrodynamic 401.3 nm suspended in medium, Antigenotoxicity effects DMTU and NAC
University, diameter sonicated, diluted in of dimetylthiourea reduced MN
Germany) medium; analysed by (DMTU) and NAC
Zeta potential 14.4 mV LAL assay for presence 50 mg/mL, 24 h
of endotoxin Western blot (P53) Antigenotoxicity effects +
of DMTU and NAC DMTU and NAC
reduced expression
level of P
53
Fullerenes
BET surface area 2 99% purity Comet assay 3 h of incubation, FE1-Muta mouse Jacobsen et al. 2008
C60 fullerenes <<20 m /g
(Sigma -Aldrich) 100 mg C60/mL lung epithelial cell line
Pore size 0 nm Consist of agglomerates Comet assay (FPG) +
>99.9%C of primary particles
ICP-MS Declared primary CII mutation Pos. Ctrl : Carbon black ±
particle size : 0.7 nm (Printex 90)
Size distributions in 150 and 700 nm
water
Size in media 311 nm Following suspending
in water and media and
sonicating, shape and
size distributions were
analysed by TEM and
DLS
C60 fullerenes 99.5% purity Ames test Doses 39.1– S. typhimurium Mori et al. 2006
(mixture of C60 and 5000 mg/plate, ±S9 mix (strainsTA100, A1535,
C70 fullerite) TA98, TA1537); E. coli
(Vitamin WP2uvrA/pKM101
C60 BioResearch
Corp, Japan) 313–5000 mg/mL,
CHA CHL/IU (Chinese
±S9 mix, 6 or 24 h hamster lung cells)
treatment
Z. Magdolenova
C60 fullerenes Particle size 178.6 nm nC60 colloids were Comet assay 0.022–110 mg/L, 3 and + Human lymphocytes Dhawan et al. 2006
(Materials research distribution, particle prepared from 6h
electronics corpor., diameter (DLS) powdered C60
Tucson, AZ) conc. (>99% purity)
0.022–110 mg/L
suspensions
Aqu/nC60 Zeta potential of 13.5 mV Before the experiment,
al.
nC60 colloids particle size distribution
(phase analysis light and zeta potential of
scattering)
EtOH/nC60 Particle size 121.6 nm nC60 colloids were
analysed
0.42–2100 mg/L, +
distribution 3 and 6 h
(particle diameter)
Zeta potential of 31.6 mV
nC60 colloids
(99.5% purity) gpt delta mutagenicity 0.1–30 mg/mL, 3 days + gpt delta transgenic Xu et al. 2009
C60 fullerenes (SES
Research) assay exposure mouse embryonic
C60 suspension was fibroblasts
prepared by long-term
(60 days) stirring in
water and sterilised by
autoclaving
Sonicated
on ice for 30 min
Fullerenes C60 (C60) DLS Majority agglomerates/ After 3 h instillation, ± BAL cells Jacobsen et al. 2009
Comet assay
aggregates >1 mm 54 mg/mouse (broncho-alveolar
Gas exchange surface 2 lavage fluid obtained
C60 fullerenes <20 m /g from apolipoprotein E
knockout mouse,
ApoE / )
Described by the 8-oxodG and dG Investigated effects in Both doses + in Female Fisher 344 rats Folkmann et al.
(Sigma-Aldrich, areas (Jacobsen et al. 2008b) manufacturer: 99.9% (HPLC with liver, lung and colon liver, highest dose 2009
Brøndby, Average pore sizes 0 nm (Jacobsen et al. pure preparation, electrochemical and tissues, 24 h after + in lung
Denmark) Particle size in 2008b) primary particle size UV detection) intragastric in colon mucosa
suspensions (DLS) In the saline solution of 0.7 nm administration + OGG1in liver
were 407 nm in the low The particles were mRNA expression of Doses: 0.064 and
dose and 621 and suspended in either HO1, MUTYH, NEIL1, 0.64 mg/kg bw
5117 nm in the high saline or corn oil, by NUDT1, and OGG1 suspended in saline or
dose sonication (70 W, corn oil; n = 8, saline
In corn oil: 234 nm 42 kHz) in a 5-day OGG1 repair activity solution (control;
(low dose); 40, 713 and period for 10 h each day n = 10) or corn oil
3124 nm (high dose) and again 30 min before (control; n = 10)
Transition metals None administration
PAHs None
C60 fullerenes Aqueous suspension Diameters 50 and CMC-Na and Tween Ames test 50–1000 mg/plate; dark S. typhimurium (strains Shinohara et al.
with carboxy- 33 nm 80 used as dispersants, or irradiation; ±S9 mix TA98, TA100, A1535, 2009
methylcellulose sodium for preparation A153, E. coli
(CMC-Na) and Tween see Shinohara et al. WP2uvrA/pKM101)
80, respectively (DLS) 2009 12.5–200 mg/mL; dark CHL/IU (Chinese
Zeta potential 39 mV and 14 mV CHA in vitro
or irradiation; presence hamster lung cells)
or absence S9 mix

TEM spherical MN in vivo 22–88 mg/kg Male and female mice
(bone marrow cells)
for detailed
characteristics see table
in Shinohara et al. 2009
C60 fullerenes 99.5% purity Ames test Doses 156– S. typhimurium (strains Aoshima et al. 2010
(mixture of C60 and 5000 mg/plate; presence TA100, A1535, TA98,
C70 fullerite) or absence S9 mix TA153); E. coli
(Vitamin WP2uvrA/pKM101
C60 BioResearch Water-soluble polymer CHA ±S9 mix, 6 or 24 h CHL/IU (Chinese
Corp, Japan) (polyvinylpyrrolid) treatment hamster lung cells)
enwrapped fullerenes
C60 fullerenes Mean diameter in the 33 nm NPs dispersed in Comet assay Intratracheally Male rats, lung cells Ema et al. 2012
(Frontier Carbon 0.1% Tween 80 aqueous distilled water instilled, single dose at
Co., Ltd., solution 0.5 or 2.5 mg/kg or
Kitakyushu, Japan) repeated dose at 0.1 or
0.5 mg/kg, once a week
for 5 weeks.
The characterisation containing 0.1% Tween For 3 or 24 h after single
and preparation of the 80 instillation and 3 h after
C60 NPs suspension repeated instillation
reported by Yellow colour For preparation of 32 0.46 mg/L HepG2 (human Matsuda et al. 2011
P-postlabelling
Morimoto et al. (2010)
Aqu/C60 fullerene Colour
UV–Vis absorption See the results suspension see the (bulky DNA adducts) hepato-carcinoma cell
spectra protocol line)
8-oxodG and CdG 0.46 mg/L ± for
(HPLC/MS)
Size distribution 59–241 nm 8-oxodG and for
CdG
The average size: Bacillus subtilis 0.048 mg/L + Bacillus subtilis
117 nm Rec-assay H17 and M45
Size distribution in LB 241–554 nm Umu test 0.43 mg/L + S. typhimurium
(TA1535/pSK1002)
broth and DMEM The average size:
320 nm in LB broth and
330 nm in DMEM
Carbon black NPs (NPCB) Particle diameter 14 nm Comet assay 100 mg/mL, + at ‡3 h exposure A 549 (human lung Mroz et al. 2007,
Genotoxicity of
NP CB (Printex 90)
(Degussa, Frank- Before use, NPs different times 0.5–24 h epithelial cell line) 2008
furt, Germany) suspended in culture
medium and sonicated
for 20 min 100 mg/mL, + at ‡3 h exposure
BaP-NP Carbon 26 mg BaP/g Printex90 Comet assay A 549 (human lung Mroz et al. 2007,
black different times ranging epithelial cell line) 2008
form 30 min to 24 h
Before use, NPs
nanoparticles
suspended in culture
medium and sonicated
for 20 min
Z. Magdolenova et

2
NPCB (Printex 90) Specific surface area 295 m /g Primary particle size Comet assay Eight repeated 72-h + FE1-Muta Mouse lung Jacobsen et al. 2007
(Degussa, Frank- Pycnometric particle 14 nm incubations with epithelial cell line
furt,Germany) density 75 mg/mL
3 Media prepared freshly Comet assay (FPG) carbon black
2.2 g/cm
before
exposure, sonicated lacZ and cII mutation +
al.
weakly +
NPCB (Nano- Size (TEM) 12.3 ± 4.1 nm NP sonication 6 Comet assay 5 and 10 mg/mL for 24 h + Primary mouse embryo Yang et al. 2009
Innovation Co. Shape (TEM) Sphere intermittently (30 sec fibroblasts
Ltd., Shenzhen) Chemical composition C >99.4% every 2 min) Comet assay After 3 h instillation + BAL cells Jacobsen et al. 2009
(purity) (Raman
spectroscopy)
Carbon black, Size distribution One mode around The analysis was often
(suspended in 1.2 mm and a less disturbed 54 mg/mouse (broncho-alveolar
instillation media) frequent mode around by agglomeration lavage fluid obtained
Printex 90 (DLS) 5.5 mm from apolipoprotein E
Carbon Particle size in powder <30 nm knockout mouse,
1, 20, 40 mg/mL, 4 h ApoE / )
Suspended in MilliQ Comet assay A549 (human lung Karlsson et al. 2008
nanopowder and sonicated for 2 20 epithelial cell line)
(Sigma-Aldrich) Surface area in powder Nd sec Comet assay (FPG)
Suspended in medium
TEM 20–40 nm and sonicated
DLS (size of particles 210 nm

and agglomerates in
medium)
Zeta potential 6.9 mV
Carbon NPs Exposure characteris- NPs (~60 nm) generated Comet assay (FPG) Inhalation exposure of in mouse and rat Whole lung tissue of Wessels et al. 2011
tics (mass, particle and by electric spark in vivo
3
mice: 142 mg m , 4 h or SPF free female
surface area discharge 3 days for 4 h; and rat: C57BL/6J mice and
concentrations, size 3
152 mg m , 4 h isolated lung epithelial
distribution) see in cells of male Fisher
table (Wessels et al. F344 rats
2011)
mRNA expression of for all, except for
OGG, POLb, XRCC1, + increase in
APE1, E335 APE1 mRNA
expression in rat
14 nm Carbon Nature of NP and Free and chain Suspended by Comet assay in vivo 0.018, 0.054, 0.162 mg; + BAL (broncho-alveolar Bourdon et al. 2012
black NPs (Printex agglomeration (TEM) agglomerates; size:10 to sonication (8 min: single intratracheal lavage) cells, lung and
90; Evonik/ more than 500 nm 10 sec pulses and 10 sec instillation; 1, 3 and liver of C57BL7/6 mice
Degussa, Germany) Zeta potential 10.7 mV pauses) in 0.9% NaCl 28 days exposure
Hydrodynamic particle Highly agglomerated MiliQ water with 10% Comet assay (FPG) +
BAL fluid
size distributions in in vivo
media (DLS) Comet assay 50 mg/mL of SiCNPs, for + all five tested A549 (human lung Barillet et al. 2010
See results for details
Silicon carbide Shape (TEM) Round shaped Laboratory synthesised,
(SiC) NP, 5 batches controlled size and Si/C 4, 24 or 48 h batches of SiCNPs epithelial cell line)
of NPs differing in ratio

diameters and Si/C Si/C ratios In range 0.8–1.2
ratio Impurities O2 and N Dispersed in sterile
Specific surface areas, 2 H2O by sonication
In range 33–140 m /g
sizes BET 30 min, 4 C, pulsed
Sizes TEM 13–58 nm; 12–45 nm mode (1 sec on/1 sec
off); diluted with FBS,
Zeta potentials In range 22 to
sonicated another
31 mV 30 min; suspensions
Hydrodynamic 100–300 nm diluted in medium
diameters before experiment
For more details
see Barillet et al. (2010)
Iron NPs
Iron NP Fe3O4 Particle size in powder 20–30 nm Suspended in MilliQ Comet assay 1, 20, 40 mg/mL, 4 h A549 (human lung Karlsson et al. 2008,
and sonicated for epithelial cell line) 2009
2 20s
(Sigma-Aldrich) Surface area in powder Nd Suspended in medium Comet assay (FPG) +
and sonicated
TEM 20–40
DLS (size of particles <200 nm
and agglomerates in
medium)
Zeta potential 1.8 mV 1, 20, 40 mg/mL, 4 h
CuZnFe2O4 Particle size in powder 29 nm Suspended in MilliQ Comet assay + A549 (human lung Karlsson et al. 2008
(Sigma-Aldrich) and sonicated for epithelial cell line)
2 20s
Surface area in powder 2 Suspended in medium Comet assay (FPG) +
18 m /g
and sonicated.
TEM 10–100 nm
DLS (size of particles 40–300 nm
and agglomerates in
medium)
Zeta potential 34.2 mV 1, 20, 40 mg/mL, 4 h
Iron particles Particle size in powder 29 nm Suspended in MilliQ Comet assay A549 (human lung Karlsson et al. 2008,
Fe2O3 (Sigma- and sonicated for epithelial cell line) 2009
Aldrich) 2 20s
Surface area in powder 2 Suspended in medium Comet assay (FPG)
40 m /g
and sonicated
TEM 30–60 nm
and agglomerates in
medium)
Zeta potential 17.3 mV After synthesis, nano-g
DMSA- coated Shape Roughly spherical Comet assay 6 Human dermal Auffan et al. 2006
10 –10 1 g/L, 2 and
maghemite (nano-g Fe2O3 was coated with 24 h fibroblasts
Fe2O3) NP DMSA (C4S2O4H6)
(NmDMSA) Diameter size 6 nm
(laboratory 2
BET specific surface 172 m /g
synthesised) area
Colloidal stability of Characterised by TEM,
NmDMSA in biol media X-ray diffraction and
BET method
For concentration
lower than 10 3 g/L,
no destabilisation

NmDMSA size Higher, aggregation
distribution (PCS) readily occurred, 2R
increased to 70 nm
Magnetite NPs NP diameter 9.4 nm Magnetite NPs were MN in vivo Intraperitoneally trea- + Bone marrow cells from Freitas et al. 2002
obtained by chemical ted with magnetic fluid female Swiss mice
15
co-precipitation of Fe containing 5 10 ,
16 17
(II) and Fe (III) ions in 5 10 and 5 10
alkaline medium, and particles/kg; MN
then pre-coated with assay – 24 h after
dodecanoic acid application
followed by an
ethoxylated polyalcohol 78.1–5000 mg/plate
Iron-platinum TEM Diameter: 9 nm Ames test Only in S. typhimurium (strains Maenosono et al.
(FePt) NPs capped pKM101 TA109 without TA98, TA100, A1535, 2007
with ±S9 mix S9 mix was A1537); E. coli
tetramethylammo- weakly + WP2uvrA/pKM101
nium hydroxide X-ray diffractometry Standard deviation of
(laboratory size distribution: 11%
synthesised) Energy-dispersive Crystal structure:
X-ray analysis face-centered cubic
FT-IR Composition: Fe50Pt50,
surface ligand: oleic
acid
CHNS elemental Magnetocrystalline
analyser SQUID anisotropy energy:
3
130 kJ/m
Magnetite nano- Average core particle 8.5 nm MNPs obtained by co- MN in vivo Intravenously treated + Bone marrow cells from Sadeghiani et al.
particles (MNPs) diameter (TEM) precipitation of Fe(II) with 50 mL of the PAMF Swiss mice 2005
surface coated with and Fe(III) ions in containing 0.6
polyaspartic acid alkaline medium. Then 1016 or 1.6
(PAMF) surface-coated with 1016 particle/mL. MN
Silica-coated polyaspartic acid (PA) assay – 1, 7, 15 and
to obtain a stable 30 days after
PAMF. application
Size 50 nm Ames test Conc. 0.25, 0.5 and S. typhimurium Kim et al. 2006
magnetic NPs 1.0 mg sample/plate (strains TA97, TA98,
containing ±S9 mix TA100, TA102)
rhodamine B Concentration 0.25, CHL (Chinese hamster
isothiocyanate
All MNPs @ SiO2 (RITC) CHA
(RITC) [MNPs @ confirmed by TEM 0.5 and 1.0 mg/mL lung fibroblasts)
SiO2(RITC)]
nanoparticles
(MNPs) surface
coated with PAMF
(synthesised)
Fe2O3-NP Surface area (BET) 2
34.39 ± 0.17 m /g Comet assay Fe2O3-NP + at 10 and IMR-90 (human lung Bhattacharya et al.
2
(diameter <100 nm) Zeta potential and 28.68 mV; particle concentration 2, 5, 10, 50 mg/cm in diploid fibroblasts); 2009
2 IMR-90, and in SV-40 virus-
(Sigma-Aldrich) particle size hydrodynamic 50 mg/cm , exposure
distribution (DLS) diameter: ~50 nm time 24 h BEAS-2B at transformed BEAS-2B
2 (human bronchial
Determination of the Fe2O3 – NP contained 50 mg/cm
trace iron elements only Fe(III) without any epithelial cells)
trace of Fe(II). The 8-oxodG (ELISA) IMR-90 human lung
amount of Fe(III) diploid fibroblasts
present in 0.4 mg of
Fe2O3-NP was
equivalent to 935 mM

EDX spectral analysis of Purely composed of
surface chemistry 78.7% Fe and 21.3% O2
SEM (morphology) Spherical shape
FePt NPs capped TEM Mean diameter, size Synthesised Ames test 0.1, 0.5 or 1.0 mg/mL of S. typhimurium Maenosono et al.
with 2-aminoetha- distribution, crystal (Kang et al. 2008) NP suspension. (strains TA98, TA100, 2009
nethiol (AET) structure – 3 nm, 14,5%, Presence or absence TA1535, TA1537);
face-centered cubic S9 mix -
E.coli WP2uvrA
XRD CHA False positive? CHL/IU (Chinese
hamster lung
fibroblasts)
Magnetic Fe3O4-MNPs prepared MN in vivo 1.25, 2.50, 3.75 and Bone marrow from Wu et al. 2010
nanoparticles by chemical co- 5.00 g/kg of kunming mice
loaded with precipitation; loaded Fe3O4-MNPs/DNR
daunorubicin with DNR and albumin intraperitoneal
(Fe3O4-MNPs/ (for details see Wu et al. injection, twice in an
DNR) 2010) interval between of 24 h
Iron oxide NP Synthesised (see the Comet assay 100, 200, 1000 ppm, for
Average particle sizes Bare, (3-aminopropyl) / no damage for L-929 (murine Hong et al. 2011
Fe3O4 and shapes (TEM) trimethoxysilane protocol) 24 h bare and TEOS fibroblast line from
Bare and modified (APTMS) and citrate coated Fe3O4 subcutaneous
with various coated: average connective tissue)
fuctional groups (– diameter 10 nm;
OH, COOH, tetraethyl orthosilicate
NH2) by coating (TEOS) and TEOS-
with TEOS, APTMS, APTMS coated: average
TEOS-APTMS:30; diameter 150 nm
citrate: 40 Zeta potential (mV) Bare: 20; TEOS: 30;
APTMS: 25; TEOS- + for APTMS,
TEOS-APTMS and
APTMS: 30; citrate: 40
Maghemite Average diameter MAN: 73.0 ± 3.0 nm citrate coated Fe3O4
For preparation see MN in vivo Mice were Bone marrow Estevanato
(g-Fe2O3) NPs (TEM) (maghemite NPs:8.9 ± the protocol intraperitoneally erythrocytes from et al. 2011
encapsulated 0.1 nm) treated with 100 mL of female Swiss mice
within albumin- ~25% in mass and~7% in NP suspension
based nanospheres Lyophilised MAN for 24 and 48 h, 7,
(magnetic albumin volume fraction dispersed in FBS to 15 and 30 days
nanospheres-MAN) Maghemite content in Magnetocrystalline obtain an aqueous
suspension (5 mg
Genotoxicity
MAN/mL)
the MAN sample anisotropy energy at

this maghemite particle
content is~1.119 eV6
Uncoated Size, morphology and All~10 nm; crystalline CB MN 1–100 mg/mL, for 24 h Only dextran- MCL5 (human Singh et al. 2012
of nanoparticles
Fe2O3 NPs, crystallinity (TEM) core with inverse spinel in 1% serum- coated Fe2O3 was + lymphoblastoid cell
dextran- structure containing medium line)
coated Fe2O3 NPs, Zeta potential
Z. Magdolenova

uncoated For all between Kinetochore staining Increased ratio of
Fe3O4 NPs, -13.9 and 3.3 mV (2–100 mg/mL Kinetochor positive
dextran- dextran-coated Fe2O3) MN
coated Fe3O4 NPs Hydrodynamic particle See the results for Pretreatment with NAC reduced MN
et al.
(Liquids Research, size in the presence of details 2 mM NAC frequency
UK) 1% vs. 10% serum (4 and 50 mg/mL
media or water (DLS) dextran-coated Fe2O3)
Oxidation state of Fe 8-oxoG, TG, 2 and 4 mg/mL + increased level of
(X-ray photoelectron 5-OH-5MeHyd, FapyG, dextran-coated Fe2O3, TG, 8-oxoG, FapyG,
spectroscopy) FapyA (GC-MS) for 24 h in 1% FapyA
serum-containing
medium
2
Magnetite NPs, Size and shape (SEM) See the results for Suspended in FBS- CB MN 1, 10, 50, 100 mg/cm , + for all four types; A549 (human lung Könczöl et al. 2011
4 sizes: NPs (20– details free medium with 1% for 24 h; ROS NAC decreased MN epithelial cell line)
60 nm; Sigma- pen/strep; bath scavenger – NAC formation
Aldrich, Germany), sonicated for 20 min at pretreatment
alveolar fraction 40 C; diluted in
(0.5–1.0 mm), FBS-free medium
respirable fraction
(2–3 mm)
2
Bulk magnetite XRD Comet assay 1, 10, 50, 100 mg/cm , + for all 4 types;
(0.2–10 mm; Alfa for 4 h; ROS scavengers NAC and BHA
Aesar, Germany) Chemical composition – NAC and BHA decreased level of
(bulk magnetite (atomic absorption pretreatment DNA damage
used to generate spectroscopy)
respirable fraction
and alveolar Size distribution
fraction)
Silica-free and Commercially designed mRNA expression (52 For in vitro: 100 mm, for Core NPs were Hep3B cells (in vitro) Hwang do et al.
fluorescent by Biterials; genes, including genes 24 h severely genotoxic 2012
silica-coated cobalt NPs synthesis: see the for DNA damage and to liver tissue; and liver tissue from
ferrite (CoFe2O4) protocol repair) fluorescence NPs mice (in vivo)
NPs showed gene
For in vivo: injected into
expression 90%
mice via tail vein, 24 h
similar to untreated
exposure
liver samples
Co NPs, CoCr NPs
CoCr nanoparticles Size NP 29.5 ± 6.3 nm CoCr NPs of alloy Comet assay Five days in tissue + Primary human dermal Papageorgiou et al.
comparation to Shape Oval generated by a flat pin- culture More damage in fibroblasts 2007
on-plate trib-ometer NPs than in MPs
were characterised for MN 12 h exposure +
micron-sized CoCr (micron sized their size and No significant
(Osprey metals 2.904 ± 1.064 mm) morphology difference between
Ltd.) 3
50–5000 mm /cell for 3 nano and micro
8-oxodG
(immunostaining) and 24 h In the contrary MPs
induced MN

Co NP and washed Analysis of elemental 98.83% of purity Suspended in MilliQ CB MN Concentration At non-cytotoxic Human peripheral Colognato et al.
3 6
Co NP, compared impurity (HPLC-ICPMS water, ultrasonicated 10 –10 concentration blood leukocytes 2008
2+
to Co (University ELAN-DRCII) for15 min, diluted to tested, no
of Modena, Italy) with complete culture genotoxic effects
medium
2+ 2 h, concentration 5
Source of Co – Size of CoNP in water Polydispersed Morphology of CoNP in Comet assay + at ‡5 10
aggregates 100–500 nm water and complete 5 5
cobalt chloride and complete culture 10 ,5 10 ,10
4M
(Alfa) medium (DLS and with median value culture medium were
SEM) CoNP size 246 mn characterised by SEM
distribution in water technique and by NP
tracking analysis
Co NP (University Characteristics in water Aggregation in water NP suspended in water, Comet assay Concentration 1, 3, + Balb/3T3 (mouse Ponti et al. 2009
of Modena, Italy) and medium (SEM) and medium washed by 5 mM (subroxic fibroblast line)
centrifugation concentration,
2+ interpolated from CFE
(to remove Co ),
ultrasonicated for assay), 2 h exposure
2+
(compared to Co ) Size distribution 20–500 nm (peak at 15 min, diluted in Concentration 1, 3, +
medium
(tracking analysis)
2+
80 nm) 5 mM
Release of Co in Time-dependent (subroxic concentration,
medium after 2, 24, 48 h increase interpolated from CFE
incubation (g-counter) assay), 24 h exposure
Co NPs (FUNSOM, Size (TEM) 30–70 nm, with a NP heat sterilised Comet assay Co NPs 1–6 mM, for 4 h + at 3 and 6 mM T cells from human Jiang et al. 2012
China); median size of 50 nm (180 C, 4 h), suspended 2+ peripheral blood
2+ in water at 100 mM; (Co 10–30 mM, for 4 h) 2+
Source of Co – (Co did not
cobalt chloride sonicated intermit-
tently 6 for 2 min; induce DNA
(Sigma-Aldrich) damage)
freshly diluted in
medium
CuO NPs
CuO NP (Sigma- Particle size 42 nm Suspended in MilliQ Comet assay 2, 40, 80 mg/mL, 4 h + A549 (human lung Karlsson et al. 2008,
Aldrich) in powder and sonicated for epithelial cell line) 2009
2 20 sec
Surface area 2 Suspended in medium Comet assay (FPG) +
23 m /g
in powder and sonicated
TEM 20–40 nm
and agglomerates in
medium)
Zeta potential 31 mV
Zinc (ZnO) NPs 1, 20, 40 mg/mL, 4 h
ZnO (Sigma- Particle size in powder 71 nm Suspended in MilliQ Comet assay + A549 (human lung Karlsson et al. 2008
Aldrich) and sonicated for epithelial cell line)
2 20 sec
Surface area in powder 2 Suspended in medium Comet assay (FPG) +
15 m /g
and sonicated

TEM 20–200 nm
and agglomerates in
medium)
Zeta potential 26.9 mV 5 and 10 mg/mL for 24 h
Zinc dioxide NP Size (TEM) 19.6 ± 5.8 nm crystal NP sonication six times Comet assay + Primary mouse embryo Yang et al. 2009
(Nanuo Co. Ltd., structure ZnO >99.9% intermittently fibroblasts
Shenzhen) Shape (TEM) (30 sec every 2 min)
(purity) (Raman
spectroscopy) (purity >99%)
ZnO NP (Sigma- Hydrodynamic 165 nm Comet assay 0.001, 0.008, 0.08, 0.8, + at 0.8 and A431 (human skin cells) Sharma et al. 2009
Aldrich) diameter (DLS) 5 mg/mL for 6 h 5 mg/mL, dose
Zeta potential (DLS) 26 mV Suspended in dependent
Milli-Q water at a
concentration of
80 mg/mL,
Average size (TEM) 30 nm sonicated at 30 W for
(of NP in suspension) 10 min
ZnO NP Size (DLS) at different Particles widely Suspended in EMEM- Comet assay 10, 20, 50, 100 mg/mL, + HEp2 (human Osman et al. 2010
(1314-13-2 Sigma- concentrations and distributed. EBSS medium, probe- for 4 h negroid cervix
Aldrich) timepoints Considerably increased sonicated 30 W, 5 min CB MN 10, 20, 50, 100 mg/mL, + carcinoma cell line)
size as a function of for 2 h
concentration, minimally
increased size as function
of time (see the table,
Osman et al. 2010)
ZnO NP (Sigma- Morphology (TEM) Mainly rod-shaped and (<100 nm, surface area Comet assay Concentration 0.01, 0.1, + at ‡10 mg/mL Human nasal mucosa Hackenberg et al.
2 5, 10 and 50 mg/mL cells (10 donors) 2011a
Aldrich, Steinheim, partially spherical 15–25 m /g)
Germany)
vs. ZnO powder Mean diameter (TEM) Longitudinal NPs suspended in for ZnO powder
(<5 mm) 86 ± 41 nm and lateral sterile dH2O, sonicated
42 ± 21 nm, in specific 60s at 4.2
cases aggregates up to 5 3
10 kJ/m using a
210 104 nm continuous
Intracellular Intracytoplasmatic mode. BSA added for
distribution (TEM) ZnO-NPs were in 10% stabilisation. 10 PBS
of cells. Transfer into added. This stock
nucleus observed in diluted in BEGM
1.5% of cells
Mean diameter of 353 nm
aggregates
Purity Above 99%
Release of Zn2+ ions See table,
Hackenberg et al. 2011a
Comet assay+

ZnO NPs (Sigma- Morphology, diameter Oval shape with a mean (<100 nm, surface area 0.1 and 5 mg/mL; 1, 3D mini organ cultures Hackenberg et al.
2
Aldrich, Germany) (TEM) longitudinal diameter 15–25 m /g, purity 2 and 3 consecutive 1-h of human nasal mucosa 2011d
of 76 nm, mean lateral >99%) periods and 24-h from 10 patients
diameter 53 nm regeneration period
Aggregates (DLS) Mean 354 nm (range NPs suspended in (level of damage
from 190 to 1106 nm) water; sonicated increased after 24 h
120 sec at 4.2 regeneration
5 3 period)
10 kJ/m ; added BSA
and 10 PBS; diluted in
medium
Zeta potential 211.2 Mv
Concentration of 2.8 and 52.7 mmol/mL
2+
Zn ions in after incubation with
supernatant cell 0.1 and 5 mg/mL NPs,
culture medium respectively
ZnO NPs (Top Morphology, size, NPs: thin-slice shaped; Particle stock MN in vivo 1.25, 2.5, 5.0 g/kg bw; for NPs and MPs Peripheral blood of Crl: Li et al. 2011c
Nano Technology agglomeration (TEM, diameter ~50 nm; suspension prepared oral administration by CD-1 (ICR) mice
Co., Ltd., Taiwan) DLS) primary particles were using DMSO (final intragastric gavage; 24,
dominant; DMSO concentration 48, 72 h exposure
hydrodynamic diame- <1% in vivo, <5% Ames test +/- S9 mix for NPs and MPs S. typhimurium (strains
ter 93.35 nm in vitro); diluted with
(vs. ZnO MPs: compacted water + 1%
microparticles – crystals, at least hydroxypropyl methyl TA102, TA100, TA1537,
MPs) 1 dimension >100 nm; cellulose or medium, TA98, TA1535)
hydrodynamic diame- mixed, sonicated, FADU 0.4–160 mg/mL, for 5, + A549 (human lung Moreno-
ter 1226.2 nm (>91.6%), immediately applied
lesser percentage of
smaller particles
ZnO NPs (ZncoxTM Minimal NPs prepared freshly;
10, IBUtec) characterisation stock suspensions 30 and 180 min epithelial cell line) Villanueva et al.
provided by sonicated in a bath 2011
manufacturer 10 min; serially diluted
in water
Cerium oxide (CeO2) NPs
CeO2 (cerium Identity, crystallinity, see the results Synthesised (see the SCE 5 and 10 mg/mL, Human lens epithelial Pierscionek et al.
Geno
oxide, nanoceria) crystalline structure, (Pierscionek et al. 2010) protocol) for 24 h cell line 2010
size, shape (high-
resolution TEM)
Emission and Sonicated (ultrasonic Comet assay
absorption spectra bath) 10 min
(fluorescence and UV
vis spectrophotometer)
of nanoparticles
Silver (Ag) NPs
AgNP (Namatech 52.7–70.9 nm MN in vivo Doses (0, 30, 300 and Bone marrow, Kim et al. 2008
Co., Ltd., Korea) (average 60 nm) 1000 mg/kg/day), oral Sprague–Dawley male
exposure, and female rats

Purity >99.98% 28 days
Coated Characterisation was 25 nm Polysaccharide-coated Immunoblot: 50 mg/mL for 4, 24, Coated more severe Mouse embryonic Ahamed et al. 2008
(polysaccharide previously reported AgNPs – more 48 and 72 h damage than stem cells
surface (Murdock et al. 2007; distributed p53 protein uncoated Ag NPs Mouse embryonic
functionalised) Ag Schrand et al. 2008) fibroblasts
NPs (Dr. Dan Goia, Phospho-p53 (ser15)
clarkson university,
Rad51
NY)
Phospho-H2AX-
Ser-139
Uncoated (non- Characterisation – was 25 nm Uncoated AgNPs tend Immunoblot: 50 mg/mL for 4, 24, Coated more severe Mouse embryonic Ahamed et al. 2008
functionalised Ag previously reported to agglomerate 48 and 72 h damage than stem cells
NPs (Dr.Karl (Murdock et al. 2007; p53 protein uncoated AgNPs
Martin, Schrand et al. 2008) Phospho-p53 (ser15) Mouse embryonic
Novacentrix,
fibroblasts
Austin)
Rad51
AgNP TEM Between 20 and
Phospho-H2AX-Ser-
139
RLS and TEM nanoag, 3.3 Weak Calf thymus DNA Chi et al. 2009
6
50 nm 10 g/mL
AgNO3
(Sinopharm
Chemical
Reagent Co)
nano Ag–CPB TEM Effect of adding CPB on
RLS and TEM nanoag, 3.3 + Calf thymus DNA Chi et al. 2009
6
particle aggregation 10 g/mL
6
AgNO3 (Sinopharm CPB 6.0 10 g/mL
Chemical Comet assay Conc. 25 – 400 mg, 48 h + IMR-90 (human lung Asharani et al. 2009
Reagent Co)
Ag NP – starch Size (TEM and UV 6–20 nm Dissolved in water
capped absorption spectrum) using sonication and Conc. 100 – 200 mg, 48 h diploid fibroblasts)
(synthesised, then in culture medium CB MN + and U251 (human
all chemicals glioblastoma cells)
purchased from
Sigma-Aldrich)
AgNPs coated with Ag NP powder 78.1 nm According to the DNA adducts Ag NPs concentration + A549 (human lung Foldbjerg et al. 2011
0.2% polyvinyl (PW-XRD) 69 ± 3 nm manufacturer: 0–15 mg/mL, for 24 h epithelial cell line)
pyrrolidone Stock solution Spherical, multifaceted spherical shape, 32
P postlabelling antioxidant NAC NAC decreased
(NanoAmor, USA) (MilliQ) (TEM) or slightly elongated 30–50 nm in size. The (10 mM) pretreatment DNA adducts
shapes purity (ICP), and metal formation
Stock solution 121 ± 6 nm contaminants <0.1% Ag
NP solution prepared
(MilliQ) (DLS) 21.8 mV
in ddH2O by
RPMI 1640 media + 1% 149 ± 37 nm sonication,
FBS (DLS) (24 h at centrifugation and
37 C) filtration
Zeta potential 11.6 mV (Foldbjerg et al. 2009)
Morphology (TEM) (<50 nm) Comet assay + at ‡0.1 mg/mL

Ag NP (Sigma– Spherical and Concentration 0.01, 0.1, Human mesenchymal Hackenberg et al.
Aldrich, Steinheim, fractionally elongated 1 and 10 mg/mL, for 1, stem cells 2011b
Germany) 3 and 24 h + at ‡0.1 mg/mL
Mean diameter (TEM) 46 ± 21 nm, usually NPs suspended in CHA Concentration 0.01, 0.1,
found in aggregates sterile dH2O, sonicated 1 and 10 mg/mL, for 1 h
5
60s at 4.2 10 kJ/
3
m using a continuous
Intracellular Small aggregates in mode. BSA added for
distribution cellular compartments stabilisation. 10 PBS
as well as free in the added. This stock
cytoplasm; transfer into diluted in DMEM
nucleus observed
Mean diameter of 404 nm

aggregates (TEM)
Zeta potential of 13.6 mV
NPs suspension
AgNP (and Ag ions) Individual NP size 5–10 nm NP dispersed in Comet assay 0.2 mg/L for 4, 12 and + for Ag NP; Jurkat T cells Eom & Choi 2010
(TEM) tetrahydrofuran, 24 h (or very low) for
Agglomerates (DLS) 28–35 nm sonicated 3 h (to Ag ions
volatilise THF), Expression of p- + for Ag NP;
More details in stirred 3 days, H2AX protein for Ag ions
refilled by H2O Comet assay 50 and 100 mg/mL for +
and filtered
Eom & Choi 2010
Ag NPs Shape Spherical, slightly Synthesised by Human peripheral Flower et al. 2012
agglomerated reduction of 5 min and 3 h blood cells
Size (TEM) 40–60 nm AgNO3 using NaBH4 50 and 100 mg/mL for +
Structure and phase Crystalline,
purity (XRD) face-centered cubic 5 min together with
structure of pure Ag 250 mM of H2O2
Ag NPs Size (TEM) 4–12 nm Different Ames test 0.15–76.8 mg/plate – S. typhimurium (strains Li et al. 2012
(Novacentrix, Agglomeration sizes Up to 30 nm concentrations of NPs S9 mix TA102, TA100, TA1537, TA98, TA1535)
Austin, TX, USA) dispersed in 100 mL MN 10–30 mg/mL + at 25 and
in the water water by vortexing for 30 mg/mL
Size in water 61.2 ± 1.6 nm 5 min and sonicating
for 5 min TK6 (human
Size in culture medium 1608.7 ± 175.4
lymphoblastoid cell
surface. Charge in line)
water surface. charge in
media
Zeta potential: 9.37 mV
8.20 mV
UV–visible Absorbance maximum
spectroscopy at 450 nm, a narrow
peak width at half
maximum
Comet assay Asare et al. 2012
Z.

Ag NPs – 20 nm Hydrodynamic sizes of Mean diameter Ag20: NPs dispersed in 12.5, 50 and100 mg/mL, Ag 200 nm caused
Magdolenov
Ntera2 (NT2, human
vs. submicron dispersion (DLS) 154.6 nm dH2O/10 for 24 h higher level of testicular embryonic
200 nm Ag particles Mean diameter Ag200: BSA/10 PBS in damage compared carcinoma) cells,
(Plasmachem 266.2 nm 8:1:1 ratio of to Ag 20 nm in primary testicular cells
GmbH, Germany) 2 mg/mL stocks, NT2 cells from C57BL6 mice of
freshly prepared before WT and 8-oxoG DNA
each exposure or very little glycosylase (Ogg1)
damage in knock-out cells
testicular cells (WT
/
and KO-Ogg1 )
Comet assay (FPG) 12.5, 50 and100 mg/mL, /no increase
for 24 h 0.01–10 mg/mL, for
Ag NPs SEM and TEM analysis Single particle size: Homogenously MN and CB MN + BEAS-2B (human
(Sigma-Aldrich, USA) 100 nm or less; dispersed in 24 h bronchial epithelial
Kim et al. 2011 medium by sonication Comet assay + cells)
(30 min), filtered
Size distribution (DLS) from 43 to 260 nm: (cellulose membrane, Comet assay (FPG +
spherical aggregates, pore size 200 nm), and EndoIII)
about 58.9 nm in size serially diluted Co-treatment ± Genotox effect
scavengers (Man decreased by
0.5 mM, SS 1 mg/mL, scavengers, most
CAT 2 U/mL and SOD effectively by SOD
30 U/mL) in the CBMN
and comet assay
Ag NP- Size distribution (TEM) 3–5 nm, 47.9%; CB MN 20, 40 and 60 mg/mL, + HeLa (human Xu et al. 2012
based hydrogel 5–10 nm, 50.8%; for 24 h epithelial cell line)
(clinically available, 10–30 nm, 1.3% Comet assay 1, 25, 100 mg/mL, + HEK 293 (human Hudecová et al.
Egeta Co., China)
Ag NPs (20 nm; Mean hydrodynamic In water: 313.9 and NP dispersed in water,
Plasmachem diameter in water and 3760 nm vortexed, sonicated for 30 min kidney cell line) 2012b
GmbH, Germany) DMEM (DLS) 3 min, 100 W on ice; Comet assay (hOGG1) 1, 25, 100 mg/mL, +
In DMEM: 33.9 nm, PBS with final 1.5%
225.9 nm and 4050 nm; BSA added. Stock for 30 min
only slight shift in size solution added to 24 h pretreatment with Oxidative DNA
after 30 min incubation medium with 10% FBS
(see table for details)
Zeta potential of NPs In water (without BSA
suspension and PBS supplement): plant extract damage
37.4 ± 2.5 mV (containing diminished by
In water (with BSA and antioxidants such as Gentiana
swertiamarin, asclepiadea extract
mangiferin,
homoorientin)
PBS supplement):
26.7 ± 0.8 mV
Morphology (SEM, Small agglomerates,
TEM) average size~100 nm
Size 20–100 nm
Gold (Au) NPs
DLS analysis Comet assay After 3 h instillation ± Jacobsen et al. 2009

Nanosized gold 2 nm gold NP formed BAL cells
particles stable agglomerates (broncho-alveolar
40–200 nm lavage fluid obtained
from apolipoprotein E
54 mg/mouse knockout mouse,
5 mg/plate; with or ApoE / )
Gold NPs stabilised Stability after light NPs stable. Absorption Synthesised by citrate Ames test Gold NP solution is not S. typhimurium
by citrate ions irradiation and mixing peak at 522 nm, no reduction method without light mutagenic but TA102
with phosphate buffer change. 16 nm irradiation; 48 h photomutagenic due to
3+
(UV–Vis absorption diameter nanospheres, incubation after citrate and Au
spectra and TEM) same size and shape 15 min irradiation
Wang et al. 2011a
A series of four 2 nm core; synthesised Comet assay 100, 123, 148 and + for all four HeLa (human Chompoosor et al.
AuNPs via place exchange of 165 nM for AuNP 1–4, AuNPs; level of epithelial cell line) 2010
(quarternary pentanethiol capped respectively (calculated DNA damage
ammonium AuNPs fabricated by concentration yielding depends on
functionality with Brust- 214 nm/well of hydrophobicity of
varied (C1-C6) Schrifrin reduction intracellular gold); for the ligands
hydrophobic alkyl method 24 h
tail)
AuNPs coated with (for detailed (preparation details gH2AX 0.1 nM, with or without for [EuL]Au with/ MRC5VA (human Lewis et al. 2010
+
cerium [CeL] and characterisation see described in Lewis et al. pEGFP-F plasmid without pEGFP-F epithelial lung
luminescent in Lewis et al. 2010) 2010) vector, for 48 h, NPs + for [CeL] +Au fibroblasts)
europium [EuL] transfected to cells with with/without -
Fugene transfection pEGFP-F
agent
AuNPs (20 nm in Prepared in citrate Comet assay 1 nM, for 72 h + MRC5 (human fetal Li et al. 2011a
diameter) reduction from gold CHA FISH + lung fibroblasts)
salts; spun down to 2D gel electrophoresis Downregulation
remove the citrate;
(expression of repair
added FBS, washed;
protein)
stock in PBS; sterile
filtered; added into Comet assay in vivo Single intratracheal
media
AuNPs (gold colloid Primary particle size: NP suspensions Lung cells of male Schulz et al. 2012
suspensions in instillation of 500 ml per Wistar rats
water; 2, 20 and 200 2, 20, 200 nm adjusted with saline to MN in vivo animal (18 mg NP per Bone marrow cells of
nm) (British Biocell See table in material 36 mg/mL, vortexed; no lung), 3 days exposure male Wistar rats
Interna-tional, and methods for agglomeration observed 8-oxoG, 8-oxoA, R-cdA, 0.0002–0.2 mg/mL, for
Cardiff, UK) detailed properties with the unaided eye
Citrate stabilised Au DLS No aggregation of NPs NPs either utilised as HepG2 (human liver Nelson et al. 2011
NPs – 3 sizes: 10, in media during 24 h received or diluted in S-cdA (LC-MS/MS) 3 and 24 h carcinoma cell line)
30 and 60 nm (NIST (see results) water and utilised as
Standard Reference diluted solutions
Materials Program) Diluted NPs Calf thymus DNA
preincubated for
16–24 h prior to
addition to cells
Pt NPs
Pt NPs Size distribution (TEM) 5–8 nm NPs synthesised Comet assay 10–160 mg/mL, for 48 h + IMR-90 (human lung Asharani et al. 2010
functionalised with Metal weight % 5.9% (see the protocol) CB MN 10–160 mg/mL, for 48 h + diploid fibroblasts
polyvinyl alcohol +A610) and U251
Z.
UV-spectroscopy typical hyperbolic
Magdolenova et al.
(EDX spectrum) curve
PtNPs coated with (for detailed (Preparation details gH2AX 0.1 nM, with or without for [EuL]Pt with/ MRC5VA human Lewis et al. 2010
+
cerium [CeL] and characterisation, see described in Lewis et al. pEGFP-F plasmid vec- without pEGFP-F epithelial lung
luminescent euro- in Lewis et al. 2010) 2010) tor, for 48 h, NPs + Pt with/ fibroblasts
+ for [CeL]
pium [EuL] transfected to cells with without pEGFP-F
Fugene transfection
agent
Quantum dots (QDs)
CdSe/ZnS QDs Gel electrophoresis Incubation with + Supercoiled double Green et al. 2005
(CdSe QDs capped (plasmid nicking supercoiled dsDNA, strands of DNA
with ZnS shell) assay) 0–60 min, in 15-min
(commercially intervals)
available)
In the dark and under
UV excitation
QDs negatively It was not possible to Comet assay After 3 h instillation + BAL cells (broncho- Jacobsen et al. 2009
(ADS620QD) and obtain acceptable DLS 54 mg/mouse alveolar lavage fluid
positively charged data obtained from
(ADS621QD) CdTe apolipoprotein E
QDs knockout mouse,
ApoE / )
CdTe QDs lem = 664 1, 10 and 50 mg/mL, for + HUVEC Wang et al. 2010
stabilised by Mean diameters 3.7 nm
g -H2AX
12 h
mercaptopropionic (fluorescent microscopy and
acid (MPA) Surface coupled with flow cytometry) Antioxidant N-acetyl-l- NAC decreased
(Institute of MPA for water cysteine (NAC) 6 mM lH2AX foci
Macromolecular solubilisation and pretreatment 50 mg/mL formation
Science, Fudan suspended in ddH2O of CdTe QDs, for 6 h
University)
CdSe QDs and Size and shape Dot-like shape, uniform Preparation of QDs: see 500, 1000, + Bone marrow cells of Khalil et al. 2011
CdSe QDs doped (TEM) in size and shape; size the protocol 2000 mg/kg bw; oral albino mice
with 1% cobalt ions, 5.1 ± 0.2 nm MN in vivo administration; 2 and 7
48 nm (pure), QDs suspended in days treatment + Liver cells of albino
surface modified Hydrodynamic
using 58 nm (doped) ultrapure bidistilled mice
diameter;
mercaptoacetic water–Tween DNA fragmentation
acid 80 mixture
DLS 0.0689 (pure), + Liver cells of albino
0.0408 (doped); mice
PDI
25.7 mV (pure) HPLC (8-oxoG and
Zeta potential
2-dG)
Optical properties 37.4 mV (doped)
See the results for
details
Magnetic measurement
Rare metal and metal oxide NPs
Indium oxide Size distribution of Dy2O3: 85.8–1132.3 nm Suspended in Ames test 20–80 mg/plate; Dy2O3: +NP, S. typhimurium Hasegawa et al.
(In2O3), NP (DLS) In2O3: 85.8–2881.3 nm ultrapure presence or absence +MP; In2O3: (strains TA98, TA100, 2012
dysprosium oxide WO3: 106.5–1134.1 nm; water at 0.2 mg/mL, S9 mix +NP, MP; TA1535, TA1537
(Dy2O3), tungsten Mo: 85.8–442.2 nm sonicated 5 min, WO3: +NP, (E. coli WP2uvrA
oxide (WO3) and filtered MP; Mo:
molybdenum (Mo) NP, MP
NP and micro- Size distribution More than 7 mm Cell transformation 0.01–5 mg/mL Dy2O3: +NP, Bhas 42 cells
particles (MP) of NP assay +MP; In2O3: (v-Ha-ras-transfected
(Sigma-Aldrich, Purity +NP, +MP; BALB/c 3T3 cells)
USA) Diameter See table in results for WO3: NP,

Mean diameter in sol detailed characteristics MP; Mo:
Zeta potential of NP and MP NP, MP
Primary crystallite size
Surface area
Impurities
2
Alumina (Al2O3) NPs 1–25 mg/cm , for
Al2O3 NP Shape (TEM) Spherical NP stock suspensions SCE - CHO-K1 (CHO cell line) Di Virgilio et al.
(Sigma-Aldrich) were prepared in PBS, 2 cellular cycles 2010
vortexed for 10 min and
stored at 4 C in the
dark 2
0.5–10 mg/cm , for 24 h + at 0.5–10 mg/mL
Size distribution average particle sizes CB MN
(TEM) 28 ± 19 nm
Specific surface area 2
39 m /g
(BET)
Silica NP
Crystalline UFSiO2 Particle size HPPS: by volume 99% purity CB MN 6, 24 and 48 h with 0– + at ‡30 mg/mL, WIL2-NS cells (human Wang et al. 2007c
(Sigma-Aldrich) distribution in the final 7.21 nm (100%), by 120 mg/mL UFSiO2 24-h treatment B cell lymphoblasts)
extract intensity: 9.08 nm Suspended in culture Comet assay
(HPPS) (71.4%) and 123.21 nm
(28.6%) medium, sonicated. In
Ultrafine quartz For details the final extract was + at 120 mg/mL
measured particle size HPRT gene mutation
distriburtion by the 24 h treatment
HPPS
98%; <5 mm diameter; CB MN 0, 60 and 120 mg/mL, + at 120 mg/mL WIL2-NS cells (human Wang et al. 2007b
(Min-U-Sil 5 silica; see Wang et al. 2007b purity 99.5% for 10 h B cell lymphoblasts)
Silica Corp. a-quartz by X-ray
Berkeley Springs) diffraction
Suspended in media, Comet assay
vortexed, sonicated;
centrifuged; superna-
tant filtered; stored – HPRT gene mutation + (dose dependent)
20 C
SiO2 NPs (Runhe Size (TEM) 20.2 ± 6.4 nm Crystal NP sonication six times Comet assay 5 and 10 mg/mL for 24 h + Primary mouse embryo Yang et al. 2009
Co. Ltd., Shanghai) Shape (TEM) structure SiO2 >99.0% intermittently (30 sec fibroblasts
every 2 min)
(purity) (Raman
spectroscopy)
TMR- and Size 50 ± 3 nm in diameter Laboratory synthesised Comet assay 48, 72 h, concentration - A549 (human lung Jin et al. 2007
4
RuBpy-doped (using a reverse 10 –0.5 mg/mL epithelial cell line)
luminescent silica mictoemulsion
NP (laboratory method),
synthesised) resuspended in PBS PFGE 72 h, concentration
4
10 –0.5 mg/mL
DNA adducts 72 h, concentration
4
10 –0.5 mg/mL
4
Agarose gel 48, 72 h, 10 –
electrophoresis 0.5 mg/mL
Genotoxicityofnanoparticles

DNA repair enzyme 48, 72 h, concentration
activity assay 10 4–0.5 mg/mL
Glantreo (30,80, Nanoparticle disper- See the table Comet assay 3, 6, 24 h 3T3-L1 (mouse Barnes et al. 2008
400 nm) sions (DLS in deionised in Barnes et al. 2008 4 or 40 mg/mL fibroblast line)
SigmaLudox CL water, TEM from
420883, suspensions in water,
SigmaLudox CL- buffer, cell culture
media before drying,
X 420891
zeta potential, pH, PDI)
before and after dialysis
(see the table
in Barnes et al. 2008)
Aerosolised amor- Two particle sizes 37 and 83 nm De novo synthesised MN in vivo (flow 3.7 107, 1.8 – Reticulocytes Sayes et al. 2010
8 3
phous silica (AS) (detailed characterisa- aerosolised AS NPs cytometry) 10 particles/cm , 1 and (peripheral blood) of
NPs tion see Sayes et al. produced in NP reactor 3 day inhalation male rats
2010) exposures (measure-
ment at 24 h post-
exposure)
AS Shape, size and size For results see table Particles Comet assay 100 mg/mL, for 24 h + for all 4 particles, HepG2 (human liver Li et al. 2011b
distribution (TEM) higher damage with carcinoma cell line)
Four sizes, (Si498, Zeta potential, were dispersed by decreasing size
nano-Si68, nano- hydrodynamic sizes sonicator and diluted to
Si43 and nano- (DLS) in water and DMEM before use
Si19) (Jilin media after 10 min and
University) 24 h CBMN: 10–60 mg/mL of
AS three sizes (16, Characterisation and CBMN and FISH- Weak, not A549 (human lung Gonzalez et al. 2010
60 and 104 nm) synthesis method centromeric probing; 16 and 60 nm SiNPs; significant (16 nm epithelial cell line)
see Gonzalez et al. 2010 frequency of mitotic 40–330 mg/mL of SiNPs induced
errors 104 nm SiNPs; for 40 h more MN)
FISH: 16 and 60 nm Weak, not
SiNPs, 40 and significant
Comet assay (FPG) 16 and 60 nm SiNPs, Weak, not
40 and 60 mg/mL, for significant
15 min and 4 h
AS NPs (5 types – DLS analysis in water NP diameters 15– Two of them in powder CB MN 100 mg/mL, 24 h Balb/3T3 (mouse Uboldi et al. 2012
unlabelled; origin: and medium 300 nm. See the table in form, four of them lab 100 mg/mL, 72 h fibroblast line) clone
2 of them OECD list SEM image analysis in results syntetised (see the Cell transformation A31-1-1
of interest, 3 of water protocol for details) assay
them lab Aggregations of 10 and 20 nm Comet assay (FPG) + BEAS-2B (human Gurr et al. 2005
synthesised-JRC) Concentration Of TiO2
TiO2 NP
TiO2 anatase 10 nm
(Hombikat UV100) 1000 nm in diameter 10 mg/mL, 1 h, in bronchial epithelial
and 20 nm Suspended in PBS MN darkness + cells)
(Millennium PC500) ‡200 nm Comet assay (FPG) Concentration of TiO2 BEAS-2B (human Gurr et al. 2005
TiO2 anatase
‡200 nm 10 mg/mL, 1 h, in bronchial epithelial
(Aldrich-Sigma, Suspended in PBS MN darkness cells)
cat. no. T8141)
No aggregation Comet assay (FPG) Gurr et al. 2005

TiO2 anatase 200 nm Conc. TiO2 10 mg/mL, BEAS-2B (human
200 nm (Kanto 1 h, in darkness bronchial epithelial
Chemical, cat. nos. Suspended in PBS MN + cells)
40167-01)
TiO2 rutile 200 nm No aggregation 200 nm Comet assay (FPG) Conc. TiO2 + BEAS-2B (human
(Kanto Chemical, Gurr et al. 2005 10 mg/mL, 1 h, in bronchial epithelial
cat. nos. 40982-30 darkness cells)
Suspended in PBS MN Comet assay 6, 24 and 48 h with 0, + WIL2-NS (human Wang et al. 2007a
TiO2 NPs Particle size HPPS: by volume 99% purity
(Aldrich-Sigma) distriburtion in the 6.57 nm (100%), by 26, 65 lymphoblastoid cells)
final extract measured intensity: 8.2 nm CB MN and 130 mg/mL UF- +
by the HPPS (80.4%) and 196.52 nm TiO2
(19.4%)
Sonicated and HPRT gene mutation +
TiO2 NPs (DuPont) Crystalline structure 79% rutile, 21% anatase
suspended in culture Ames test 100, 333, 1000, S. Typhimurium Warheit et al. 2007
medium
Characterised in its dry
(XRD) native state 3333 and 5000 mg per (strains TA100, TA1535,
BET surface area 38.5 m /g
2 (crystallinity and plate TA98, TA1537); E. coli
surface area) and in WP2uvrA
water and buffered CHA 750, 1250 and CHO cells
analysis solutions (size, size 2500 mg/mL for 4 h
Chemical composition/ distribution, pH and (non-activated test
90 wt% TiO2, 7% chemical reactivity)
purity (X-ray alumina and 1% AS condition); 62.5,
fluorescence) 125 and 250 mg/mL, for
Size and distribution in 140 ± 44 nm 4 h (activated test
condition), and 25,
water (DLS) (aggeregation: in 50 and 100 mg/mL for
water – negative)
20 h (non-activated test
Surface reactivity Chemical reactivity
a condition)
delta b : 0.9 Photoclastogenicity 800–5000 mg/mL, ± UV – CHO-WBL (Chinese Theogaraj et al.
Eight different Size 15–60 nm
(CHA) 2 hamster cell line) 2007
forms of TiO2: light (750 mJ/cm )
anatase (100%), Particle sizes (TEM, See the table
rutile(100%), XRD, XDC) (Theogaraj et al. 2007)
combined anatase
(80%)/rutile(20%),
diff. coated The primary NPs
(trimethoxy (see table) formed
caprylylsilane,
aggregates in the
alumina, silica, dimension
simethicone, of 30–150 nm
dimethicone,
stearic acid, doped
di-iron trioxide)
Commercial-grade Crystal phase (XRD) 70–85% anatase and Note: Nano- Comet assay TiO2 (20, 50 or + Human Peripheral Kang et al. 2008
nano-TiO2 30–15% rutile TiO2 TiO2 suspended in PBS, 100 mg/mL) for 0, 6, blood lymphocytes
Degussa Aeroxide Specific surface area 2 sonicated for 30 min 12 and 24 h
50 m /g before exposure to cells CB MN Nano-TiO2 (20, 50 or +
P25 100 mg/mL), 20 h of
Size incubation

Approximately 30 nm
25 nm
Zeta potential in 11.6 1 ± 1.2 mV
culture medium
(pH = 7.5)
The negative surface Had an isoelectric point
charge at physiological of pHIEP = 6.4
pH (pH = 7.5–7.6) 1, 20, 40 mg/mL, 4 h
TiO2 (Sigma- Particle size in powder 63 nm Suspended in MilliQ Comet assay + A549 (human lung Karlsson et al. 2008,
Aldrich) and sonicated for epithelial cell line) 2009
2 20 sec
Surface area in powder 2 Suspended in medium Comet assay (FPG) ±
24 m /g
and sonicated
TEM 20–100 nm
and agglomerates in
medium)
Uf-TiO2 Particle size £20 nm MN 0.5, 1.0, 5 and + at concentration Syrian hamster Rahman et al. 2002
2
£20 nm 10 mg/cm between 0.5 and embryo cells)
2
(vs. fine 5 mg/cm
TiO2 particle size Suspended in PBS for 12, 24, 48, 66 and (>200 nm sized
>200 nm) (Ober- 72 h TiO2 was negative)
dorster, University
of Rochester, NY,
USA)
BET surface area 2 5 nm (99.7% purity) gpt delta mutagenicity 3 days exposure 0.1– + gpt delta transgenic Xu et al. 2009
TiO2 anatase 114.1261 m /g,
2
(determined by ASAP 38.2268 m /g and assay 30 mg/mL mouse primary embryo
2
2020) 8.9146 m /g, fibroblasts
respectively
5 and 40 nm Accelerated surface 40 nm (99.9% purity)
(Sigma-Aldrich and area and porosimetry
Inframat Advanced TiO2 325 mesh (‡99%
Materials LCC, purity)
respectively)
TiO2 325 mesh TiO2 suspended in
(Sigma-Aldrich) distilled water and
sterilised by heating to
120 C for 30 min.
Sonicated on ice for
30 min
TiO2 P25(untreated, Microstructure and Both TiO2 highly P25: particle diameter 8-oxoG Rats were exposed by Female Wistar rats Rehn et al. 2003
hydrophilic aggregation aggregated 20 nm, surface (immuno-cytology) instillation 0.15, 0.3,
surface) or characteristics (TEM) hydrophilic 0.6 and 1.2 mg dust/
TiO2T805(silanised, lung of TiO2
hydrophobic
surface) particles
Aerosil & Silanes, T805: primary particle Test: up to 90 days after
Degussa AG diameter 20 nm, exposure (analysis of
(Hanau-Wolfgang, silanised, hydrophobic single cells in lung
Germany) surface suspended in tissue)
physiological saline

with 0.25% lecithin,
sonicated
Surface area (BET) 2 Comet assay IMR-90 (human lung Bhattacharya et al.
TiO2 NP (anatase, 49.71 ± 0.19 m /g TiO2 concentration 2, 5,
2
; <100 nm) Zeta potential +48.8 mV 10, 50 mg/cm , + diploid fibroblasts), 2009
(Degussa GmbH, and particle size Particle hydrodynamic exposure time 24 h SV-40 virus-
Germany) distribution (DLS) diameter: 91 nm 8-oxodG (ELISA) transformed BEAS-2B
Trace Fe elements (human bronchial
TiO2 NP – absolutely
epithelial cells)
pure
IMR-90 (human lung
EDX spectral analysis of 56% Ti, 41% O2, 3% C
diploid fibroblasts)
surface chemistry
SEM (morphology) Spherical shape 2
+ at 80 mg/cm (24 h
TiO2 NP rutile, Primary size, shape 10 40 nm, NP dispersed in Comet assay TiO2 concentration BEAS-2B (human Falck et al. 2009
2
SiO2 coating needle-like crystals medium, ultrasoni- 1–100 mg/cm , for 24, treatment) and + bronchial epithelial
2
(637262, Sigma- cated 37 kHz for 20 min 48, 72 h at ‡80 mg/cm cells)
Aldrich) (72 h treatment)
Specific surface area 2 CB MN
132 m /g
(BET): Ti, O (<5% Si)
Composition:
NP dispersions (optical Contained nanosized
microscopy, TEM) particles and
agglomerates in size
4.5 mm
Primary size, shape: <25 nm, spherical Comet assay 2 BEAS-2B (human Falck et al. 2009
TiO2 NP anatase TiO2 conc. + at ‡10 mg/cm
2
(637254, Sigma- crystals 1–100 mg/cm , for 24, bronchial epithelial
2
Aldrich) Specific surf. area 222 m /g CB MN 48, 72 h + at 10 and cells)
2
(BET): 60 mg/cm
(72 h treatment)
Composition: Ti, O
NP dispersions (optical Contained nanosized
microscopy, TEM) particles and
aglomerates in size
5.5 mm
Shape (TEM) Complex NP stock suspensions SCE TiO2 concentration + at 1–5 mg/mL CHO-K1 (CHO cell line) Di Virgilio et al.
TiO2 NP (Aldrich)
2
Size distribution average particle were prepared in PBS, 1–25 mg/cm , for two 2010
vortexed for 10 min and cellular cycles
stored at 4 C in the
dark 10–25 mg/mL
(TEM) sizes 20 ± 7 nm cytotoxic
Specific surface area 2 CB MN + at 0.5 and
142 m /g TiO2 concentration
(BET) <25 nm 2 1 mg/mL
0.5–10 mg/cm , for 24 h
TiO2 NP (anatase, Shape (TEM) spherical Comet assay TiO2 concentration Human nasal Hackenberg et al.
Sigma-Aldrich) 10–100 mg/mL, for 24 h mucosa cells 2010
Size (TEM) diameter 15–30 nm NPs suspended in (10 donors)
Aggregations (TEM) Mean sized 285 ± distilled water,
sonicated for 60 sec,
52 nm, diameters up to then added BSA and
2000 nm 10 PBS, diluted with
PBS
Morphology (TEM) Sphere shaped Comet assay
Z. Magdolenova

TiO2 NP (anatase, Diameter (TEM) 15–30 nm Particles dispersed by 20, 50, 100, 200 mg/mL, Human peripheral Hackenberg et al.
Sigma-Aldrich) Aggregations (TEM) 285 ± 52 nm sonication according to for 24 h blood lymphocytes 2011c
Bihari et al. 2008, (10 male donors)
diluted in PBS, added
to cell suspension
TiO2 NP (anatase, Size (DLS) at different Particles widely Suspended in EMEM- Comet assay 10, 20, 50, 100 mg/mL, + HEp2 (human negroid Osman et al. 2010
et al.
1317-70-0 Sigma- concentrations and distributed EBSS medium, probe- for 4 h cervix carcinoma cells)
Aldrich) timepoints Considerably increased sonicated 30 W, 5 min CB MN 10, 20, 50 mg/mL, for 2 h +
size as a function of
concentration, minimally
increased size as a
function of time (see the
table, Osman et al. 2010)
TiO2 Aeroxide Crystal structure Mixture of 75% anatase TiO2 dispersed in Comet assay 5 days in vivo oral + Peripheral blood from Trouiller et al. 2009
un
P25 (Degussa, now and 25% rutile. At least drinking water, exposure 500 mg/kg pregnant C57Bl/6Jp /
un
Evonik 99.5% TiO2 ultrasonicated 15 min p mice
Primary size 21 nm at different MN 50, 100, 250, 500 mg/kg + at 500 mg/kg Peripheral blood
Purity 2 concentrations just g-H2AX 50, 100, 250, 500 mg/kg + at all doses Bone marrow cells
50 ± 15 m /g before use
Specific surface area Agglomerates ranged 8-oxodG (HPLC) 500 mg/kg + Liver
from 21 to 1446 nm,
mean size (70% of
particles) 160 ± 5 nm
Size in water (DLS) About to have a size of DNA deletion assay, 500 mg/kg maternal + Fetus
160 nm in vivo exposure + at ‡8 mg/mL
TiO2 NP (anatase, Mean hydrodynamic In MQ water: 124.9 nm; 99.7% anatase Comet assay Concentration 0.008– A431 (human epider- Shukla et al. 2011a
1317-70-0 Sigma diameter (DLS) in medium: 192.5 nm 80 mg/mL, for 6 h + at ‡0.8 mg/mL mal cell line)
Chemical) Zeta potential (DLS) In MQ water: NPs suspended in water Comet assay (FPG)
17.6 mV; in medium: and DMEM with 10% + at ‡0.8 mg/mL
11.5 mV FBS; probe sonicated at
Average size (TEM) 50 nm 30 W for 10 min MN
0.001–10 mg/mL of –TiO2 alone; –PbAc L02 (human embryo Du et al. 2011
TiO2 NP Primary diameter, 21 nm, 80% anatase Aqueous stock solutions 8-oxodG (HPLC)
P25 (Degussa NRW, composition and and 20% rutile; 49.6 TiO2 and ± 1 mg/mL of alone; hepatocytes)
2 of TiO2 and PbAc were
Germany) alone and surface area (TEM, m /g PbAc, for 24 h
XRD) heat sterilised, stored at
combined with + TiO2 with PbAc
PbAc 4 C; ultrasonicated;
Aggregate size in Average 299 nm
aqueous solution TiO2 solution was
diluted with cultures or –TiO2 alone; –PbAc
Absorptive capacity Dose dependent Western blot (OGG1)
of PbAc PbAc; vortexed
alone;
(for details see table, +TiO2 with PbAc
Du et al. 2011) 1–250 mg/mL, for 2, 4, + An stronger than HepG2 (human lung Petkovic´ et al. 2011
TiO2 NP anatase Size, shape (FEG-SEM); TiO2 An <25 nm NPs suspended in PBS; Comet assay
and rutile sonicated in ultrasonic 24 h Ru carcinoma cell line)
(637254 and Size distribution in TiO2 Ru <100 nm; bath; diluted in complete Comet assay (FPG) 1–250 mg/mL, for 2, 4, + and stronger than
637262 Sigma- medium medium; sonicated 24 h Ru
Aldrich) before use 1–250 mg/mL, for 2, 4, Weakly +
Specific surface area high agglomeration Comet assay (EndoIII)
(BET); crystal phase 24 h comparing to FPG
(XRD); UV–Vis 1–250 mg/mL, for 4, 24 h +
spectroscopy;
mRNA expression of
p53, p21, gadd45a,
2
mdm

TiO2 NP anatase Aggregation state after Variously sized Anatase: 99.7% metals Comet assay 20, 50 and 100 mg/mL, + after 24 and 48 h, Bottlenose dolphin Bernardeschi et al.
and rutile sonication in RPMI by aggregates after basis; size <25 nm; for 4, 24, 48 h at 50 and leukocytes 2010
(1317-70-0 and TEM sonication specific gravity/ 100 mg/mL
3
1317-80-2 Sigma Limited number of density 4 g/cm Rutile:
Aldrich) single particles and/or 99.9% metals basis, size
small aggregates <5000 nm; specific
<100 nm and a large gravity/
3
number of aggregates density 4.26 g/cm NPs
sized from a few to sterilised by heating at
several micrometres 120 C for 2 h,
suspended in RPMI;
bath-type sonicated for
30 min, serially diluted 12.5, 50 and 100 mg/mL,
TiO2 NPs – 21 nm Hydrodynamic sizes of Mean diameter TiO2: NPs dispersed in Comet assay Considerable level Ntera2 (NT2, human Asare et al. 2012
(European dispersion (DLS) 259.0 nm dH2O/10 for 24 h of DNA damage testicular embryonic
Commission, JRC) compared with carcinoma) cells,
controls, however, Primary testicular cells
low from C57BL6 mice of
wild type (WT) and
12.5, 50 and100 mg/mL, knock-out cells
BSA/10 PBS in Comet assay (FPG) /no increase 8-oxoG DNA
8:1:1 ratio of 2 mg/mL for 24 h glycosylase (Ogg1)
stocks, freshly prepared
before each exposure
TiO2 NPs – 33 nm Crystal structure of Anatase Solutions of dispersed Comet assay, in vivo Oral gavage; doses: 40, NP: + bone Male F1 (CBAxB6) mice Sycheva et al. 2011
vs. microsized both TiO2 (light TiO2 prepared with 200, 1000 mg/kg bw; marrow; +
TiO2 particles scattering) distilled water daily for 7 days liver; brain
(MP) – 160 nm MP: + bone
(Sensient Cosmetic
Technologies LCW,
France)
Mean particle size 33.2 ± 16.7 nm for NP;
(electron microscopy) 160 ± 59.4 nm for MP marrow; liver;
brain;
MN in vivo NP: bone mar-
row; MP: + at
1000 mg/kg bw
bone marrow
NP and MP: fore
stomach, colon,
10, 20, 40 mg/mL, testis
TiO2 anatase NPs Crystalline structure 100% anatase NPs suspended in HPRT gene mutation CHO-K1 (CHO cell line) Wang et al. 2011b
(corresponds to the (XRD) DMSO; prior exposure 60 days continuous
source used in vortexed 1 min to fully exposure
studies conducted Purity 99.7% TiO2 resuspend; made fresh Comet assay
by the Finnish Particle size Less than 25 nm weekly
Institute of gH2AX 1–1000 mg/mL + for NPs and MPs, Toyooka et al. 2012
TiO2 NPs appeared to
Occupational
aggregate in culture
Health, Helsinki)
medium
Z.

TiO2 anatase NPs The size distribution NP: 250–650 nm Both NPs and MPs A549 (human lung
(5 nm; Sigma- in DMEM (average: 378 nm) suspended in DMEM at epithelial cell line)
Aldrich, St. Louis) 20 mg/mL,
bath-sonicated 1 min
vs. MPs (<5000 nm; MP: 600–1050 nm BSA-coated NPs: NPs vs. MPs more remarkable in
Wako Pure (average: 773 nm) suspended in 1 mL of NPs
et al.
Chemicals Ind., BSA (5 mg/mL),
Japan) sonicated 1 min; NPs vs. NPs coating Coating attenuated
centrifuged for 10 min, with BSA lH2AX generation
resuspended in
Pretreatment with Generation of
water. This washing
inhibitors (wortmannin lH2AX not affected
repeated 2
and U0126) for 0.5 h
Pretreatment with NAC Wortmannin
for 30 min inhibited
generation of
lH2AX
DSBs (BSFGE) + for NPs and MPs,
more remarkable in
NPs
TiO2 (anatase/ rutile Primary characteristics See tables in results for Dispersion prot. (DP)1: Comet assay 0.12, 0.6, 3, 15, for DP1 in TK6, TK6 (human Magdolenova et al.
powder of 21 nm; detailed properties suspended in 20% FBS 75 mg/cm
2 EUE lymphoblast line); 2012
NM105 EC-JRC, in PBS, sonicated (correspond to 0.57, 2.9, + for DP2 in Cos1, EUE (human
Ispra,Italy; DP1: stability up to 2 15 min, 100 W, added to 14.4, 72.0, EUE embryonic epithelial
Secondary properties of
corresponds to two different NP days; bimodal medium, serially diluted. Comet assay (FPG) 360. 2 mg/mL), cells); Cos-1 monkey
Aeroxide P25, dispersions in DMEM dispersion (RPMI: DP2: suspended in for 2 and for DP1 in TK6 kidney fibroblasts
Evonik) and RPMI media 102 nm,285 nm; medium with 15 mM 24 h + for DP2 in TK6,
DMEM: 112 nm, HEPES without FBS, Cos1
296 nm); sonicated 3 min, 60 W,
DP2: agglomerated vortex 10 sec, aliquoted,
(RPMI: 779 nm;
DMEM:752 nm) 20 C. Thawed,
vortexed 10 sec,
sonicated 1 min 60 W,
added to medium
TiO2 NPs Crystal phase Tetragonal NPs synthesised by Comet assay (neutral) 0.625–20 mg/mL, for 6 h + WISH (human amnion Saquib et al. 2012
identification and sonomechanical epithelial cells)
average crystallite method, see the
size (XRD), size and rutile structure of TiO2; protocol. Powdered
morphology (TEM) 30.6 nm; crystallites NPs suspended in
with polyhedral water; sonicated 15
morphologies min, 40 W
Optical properties Absorption edge
(UV–visible around 280–320 nm
spectroscopy)
Aggregation in 13 and 152 nm
suspension and aggregates in RPMI
secondary particles medium; 380 nm
sizes (DLS) aggregates in water
Functional groups and See results for details
stretching vibrations of
the bonds (FTIR
spectrum)

TiO2 NPs (99% Mean hydrodynamic In MQ water: 124.9 nm; NPs suspended in Comet assay 1–80 mg/mL, for 6 h + HepG2 (human liver Shukla et al. 2011b
anatase, Sigma diameter (DLS) in medium: 192.5 nm medium without FBS, (corresponding to carcinoma cell line)
Chemical Co. Ltd., Zeta potential (DLS) In MQ water: probe sonicated 2
0.31–25 mg/cm )
USA) 17.6 mV; in medium: 10 min, diluted in
11.5 mV medium with 10% FBS
PDI In MQ water: 0.18 PDI; Comet assay (FPG) +
in medium: 0.12 PDI
Size (TEM) See Shukla et al. 2011a CB MN 100 mg/mL, for 4, 24, +
TiO2 NPs, five Shape See the table in results NPs dispersed in water Comet assay + A549 (human lung Jugan et al. 2011
types; anatase Crystal phase by sonication 30 min, 4 48 h epithelial cell line)
25 nm (Aerooxide Diameter C 8-oxoG (HPLC-MS/MS) 100 mg/mL, for 4, 24, + at all NPs except
P25,Degussa), Specific surface area
rutile 68 nm
Point of zero charge 48 h anatase140
(ref. 637262, Sigma-
Mean hydrodynamic CB MN 50, 100, 200 mg/mL of
Aldrich), anatase
140 nm (ref. T8141, diameter (DLS) anatase 12 and 25, for
Sigma-Aldrich), Zeta potential (DLS) 24 h
anatase 12 (LFP), NPs agglomeration gH2AX 50, 100, 200 mg/mL of
rutile 20 (LFP) (photon correlation anatase 12 and 25, for
spectroscopy) 24 h
Particle size (TEM) 5–25 nm Activity of DNA repair 100 mg/mL, for 24, 48 h NPs impaired
enzymes – BER and cellular repair
NER (multiplex ability
excision/synthesis) 10 and 50 mg/mL, 24 h
TiO2 NPs (anatase) Sterilisation by heating CB MN + A549 (human lung Srivastava et al.
(Sigma-Aldrich, Mean hydrodynamic 417.7 nm to 120 C, 2 h, suspended Antigenotoxicity effects DMTU and NAC epithelial cell line) 2011
USA) diameter (DLS) in medium, sonicated, of DMTU and NAC reduced MNi
Zeta potential 7.83 mV diluted in medium; Western blot (P53) 50 mg/mL, 24 h +
analysed by LAL assay
Purity 99.7% for presence of Antigenotoxicity effects DMTU and NAC
Specific surface area 200–220 m /g
2 endotoxin of DMTU and NAC reduced expression
53
Crystallographic system Tetragonal level of P
Shape Spherical Comet assay in vivo For single intratracheal
TiO2 NPs (anatase) Secondary particle No significant change NPs dispersed in Lung epithelial cells of Naya et al. 2012
(Ishihara Sangyo size – time during the period disodium phosphate, (JaCVAM protocol) instillation: Sprague-Dawley rats
Kaisha, Ltd., Japan) dependent agitated in bead mill 1.0–5.0 mg/kg bw,
Purity 99.99% with ZrO2 beads, 3 and 24 h exposure
supernatant removed For repeated
2 by centrifugation
BET surface area 316 mg/m instillation:
Secondary diameter of 19 ± 6.7 nm 0.2–1.0 mg/kg bw once
dispersed NPs See the results a week for 5 weeks
Size distributions CB MN 5 mg/mL, for 4 h,
(DLS, TEM)
Polymer NPs z-average diameters 70–180 nm
A family (nine Prepared by wow CHO cell line He et al. 2009
kinds) of PELGE, PDI (PCS) 0.23 emulsion solvent presence or absence of
extraction/ S9 mix
PLGA polymers evaporation technique. SCE 5 mg/mL, for 24 h, response of
Lyophilised NPs were presence or absence of 4 PELGE, weakly +
dissolved in S9 mix clastogenic
physiological saline
response of
and sterilised by
5 PELGE
filtration

PNIPAM The hydrodynamic For detailed results see NPs synthesised by Comet assay 12.5, 25, 100, 200, 400, in both cell lines HaCaT (immortalised Naha et al. 2010
diameters in water, tables Naha et al. 2010 free radical 800 mg/L, for 24, 48 and noncancerous human
DMEM and the polymerisation; 72 h keratinocyte line; and
supplemented media dispersed on ice to SW480 (primary
measured over the ensure good solubility, adenocarcinoma of
temperature range gradually warming colon cell line)
30–38 C them; dispersions
Particle size (TEM) prepared in DMEM
F-12 with FBS
Zeta potential
Polyethyleneimine PEI molecular weight Comet assay 1, 3, 5 mg/mL of PEI; + for both (PEI Human acute Choi et al. 2010
(PEI; 25 kDa) and 25 g/mol 10, 30, 50 mg/mL of more significant T-cell leukaemia
polyamidoamine PAMAM, for 4 h than PAMAM) Jurkat cells
(PAMAM) CB MN 1, 10, 20 mg/mL of PEI; Not significant
G4 dendrimer 10, 100, 200 mg/mL of
PAMAM, for 4 h
Curcumin Homogenous Formulation of NPs MN in vivo Oral administration Bone marrow cells from Dandekar et al.
(97 ± 2.47 nm; PI of published elsewhere 100, 200, 300 mg/kg BW Swiss albine mice 2010
-loaded polymeric 0.14 ± 0.01) population (Dandekar et al. 2009) CHA in vivo once daily over a period Bone marrow cells from
nanoparticles of of particles with of 2 days Holtzman rats
Eudragit S100 encapsulation Bone marrow cells from
efficiency of
Comet assay in vivo
72.81 ± 0.13%
Holtzman rats
Bacterial cellulose TEM Needle shaped BC secreted by G. Ames test Concentration 0.1, S. typhimurium Moreira et al. 2009
(BC) nanofibres xylinus 0.5 or 1.0 mg/mL of NFs (strainsTA97a, TA98,
length: 50–1500 nm; The nanofibres were suspension. Presence TA100, TA102)
or absence of S9 mix
Comet assay Concentration of NFs CHO cell line
width: 3–5 nm produced by acidic 0.1, 0.5 or 1 mg/mL,
and/or ultrasonic 48 h exposure
treatment
Polysaccharide cat- Size (DLS) Mean diameter 64 ± NPs prepared from Comet assay 5000, 2500, 1250, 625, 16HBE14o- (human Merhi et al. 2012
ionic NPs (NP+) 13 nm, PDI: 0.22 maltodextrin, see the 312.5 and 156.25 mg/mL bronchial epithelial cell
Zeta potential +25 ± 1.5 mV protocol Comet assay (FPG) (2604, 1302, 651, 326, with serum, + line)
Shape (TEM) Spherical 163 and 81 without serum
2
MN mg/cm ); for + with serum, +
Size and zeta potential See results for details 3 h; in serum- without serum
free medium or with
10% FCS
in the presence of
serum
Ames test, Bacterial reverse mutation assay; BSFG, Biased sinusoidal field gel electrophoresis;CHA, chromosomal aberration test; FADU, Alkaline DNA unwinding – fluorimetric detection; Comet assay, Alkaline version of the comet assay; Comet
assay (FPG, EndoIII, OGG1), Modified comet assay with DNA repair enzymes; gH2AX, Phosphorylation of histone H2AX; GC-MS, Gas chromatography/mass spectrometry; LC-MS, Liquid chromatography/tandem mass spectrometry; MN,
Micronucleus assay (without cytokinesis block); SCE, Sister chromatid exchange test; QRT-PCR, Quantitative reverse-transcription polymerase chain reaction; PFGE, Pulsed field gel electrophoresis; ICP-MS, Inductively coupled plasma mass
spectroscopy; ICP-AES, Inductively coupled plasma optical emission spectrometry; PAHs, Polycyclic aromatic hydrocarbons; DLS, Dynamic light scattering; PCS, Photon correlations spectroscopy; XRD, X-ray diffractometry; PW-XRD, Powder X-
ray diffraction; HPPS, High-performance particle sizer; XDC, X-ray centrifugation; PDI, Polydispersity index; Nd, Not determined or no data
4346
Number of publications
1204
945
112 94 22 67 44 11 13
and NP and NP and NP and NP and NP and NP and CA and MN and CHA and Ames
Aker on 04/22/13
Toxicity vitro toxicity viv Genotoxicity genotoxicity genotoxicity genotoxicitygenotoxicity

o
toxicity
In In vitro vivo NP NP
genotoxicity
NP
In In
NP genotoxicity
only.
by Oslouniversitetssykehus
Figure 1. Review of literature on NP toxicology (CA: comet assay; MN: micronucleus assay; CHA: chromosomal aberration test; Ames: bacterial reverse
mutation assay).
Nanotoxicology Downloaded from informahealthcare.com
For personal use
NP that do not cross cell membrane NP that cross cell membrane

NP release
M+ transition metals
+
M + or free radicals NP interaction with receptor
M
NP in cytoplasm Diffusion
NP disturb mitochondria Endocytosis
Mitochondria
NP cross nuclear envelope Interphase NP aggregates
through nuclear pores deform nucleus shape

DNA replication
Nucleus
Transcription
Translation
Mitosis
NP get access to Centrosome

nucleus during mitosis
Figure 2. Cellular uptake and access of NPs to nucleus.

Mechanically
Affect replication
By chemical
binding to DNA
Interphase Mechanically
(DNA level) Affect

transcription
By chemical
Affect DNA binding to DNA
Primary interaction structure
(conformation)
Primary direct of NP or its
only.
informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13
genotoxicity component with

DNA Mechanically
Clastogenic effect
(chromosomal
break) By chemical
Mitosis binding to DNA

(chromosomal
break)
Mechanically
Aneugenic effect
(loss of
chromosome) By chemical
binding to DNA
Forpersonal use
Figure 3. Scheme of potential NP-induced primary direct genotoxicity.
Indirect primary genotoxicity associated proteins. NPs can affect any function of the mitotic
apparatus, leading to loss or gain of chromosomes in daughter
To induce genotoxicity, NPs do not need to be in direct contact
cells. Huang et al. (2009) demonstrated evidence in vitro that
Nanotoxicology Downloaded from
with DNA. The following are suggested ways in which NPs

might indirectly induce primary genotoxicity (Figure 4). TiO2 NPs disturb mitosis. Long-term exposure to TiO2 NPs led
to abnormal multipolar spindle formation, chromosomal
alignment and segregation during anaphase and telophase
Interaction with nuclear proteins (involved in (Huang et al. 2009). Gonzalez et al. (2010) investigated
replication, transcription, repair) aneugenic events in human lung epithelial cells A549 that
could result from mitotic spindle defects. Also, interference
NPs can potentially interact not only with DNA but also with
with tubulin polymerisation might lead to aneu-genicity
proteins involved in DNA replication, transcription or repair.
(Gonzalez et al. 2010).
Interactions between NPs and proteins were demonstrated by
some in silico investigations. For instance, the modelling study
by Baweja et al. (2011) showed that C60 fullerene binds with Disturbance of cell cycle checkpoint functions
human DNA topoisomerase II alpha in the ATP binding NPs might interact with and influence the function of protein
domain, which could inhibit the enzyme activity. DNA kinases responsible for regulation of cell cycle events such as
topoisomerase II is involved in modification of DNA topol-ogy. DNA replication and cell division. As shown by Huang et al.
Another in silico study showed that C60 fullerene might (2009), TiO2 NPs deregulated the function of mitotic check-
interact with PMS2, RFC3 and PCNA proteins involved in the point PLK1 protein which controls several processes during
DNA mismatch repair pathway (Gupta et al. 2011). Proteins mitosis, including contractile ring formation and cytokinesis.
could also be inactivated by structural modification, for Disturbance of cytokinesis can also give rise to aneuploid or
instance by oxidation by ROS generated during NP exposure multinucleated cells. An important process in cell cycle
(Jugan et al. 2011). regulation of kinases is their inactivation. Kinases are marked
by polyubiquitin and then degraded by proteasomes. Inter-
Interaction of NPs with the mitotic spindle or action of NPs with proteins involved in this process could
its components – aneugenic effect affect their function. Calzolai et al. (2010) identified a specific
An aneugenic effect could also be caused by interaction of NPs domain of human ubiqutin that interacts with Au NPs.
with the mitotic spindle apparatus, centrioles or their Exposure to nano-TiO2 in vitro influenced ERK signalling
Mechanically
DNA replication
proteins By chemical binding
to proteins
Mechanically Defect in the
Interaction with processes
Transcription proteins Inhibition of of
nuclear proteins
By chemical binding protein activity replication,
to proteins transcription,
Mechanically repair
DNA repair proteins By chemical binding
to proteins
Mechanically
Centrioles By chemical binding
to proteins
only.
Mitotic spindle Mechanically Disturb the

Mitotic spindle
apparatus (microtubules) By chemical binding processes of
cell division
to proteins
Associated Mechanically
Primary indirect
genotoxicity (NP are not Proteins By chemical binding
in direct contact with to proteins
DNA)
Disturbing cell Mechanically Inhibition of Defect in cell
cycle
checkpoints By chemical binding protein activity cycle processes
to proteins
ROS arising from ROS attack on DNA
NP surface
For personal use
Production of ROS
via the Fenton-type

Transition metals reaction
(TM) from NP Attack of TM on
surface DNA
Chemical binding of
ROS produced TM to DNA

by cell ROS attack on DNA
components
(mitochondria) Mechanically Prevent
Interaction with antioxidant

antioxidants By chemical binding function, ROS
to proteins accumulation
ROS produced by ROS attack on DNA
Secondary genotoxicity inflammatory
cells
Figure 4. Scheme of potential NP-induced primary indirect and secondary genotoxicity.
activation, ROS production, the numbers of multinucleated give rise to mutations through mispairing in replication, and
cells and MN (Huang et al. 2009). thus are potentially carcinogenic (Cooke et al. 2003). TiO 2 NPs
induced ROS and oxidative stress leading to oxidative DNA
ROS arising from NP surface damage and micronucleus formation, sug-gesting a probable
NPs can generate ROS in the cells that may cause indirect mechanism of genotoxicity in human skin cells in vitro
oxidative damage to DNA through free radical attack. Silica (Shukla et al. 2011a, b).
and TiO2 NPs were found to generate free radicals in aqueous
suspensions in vitro (Barillet et al. 2010; Shukla et al. 2011a). Transition metals from NP surface
Free radicals may interact with cellular biomolecules including Toxic ions released from soluble NPs may also contribute to
2+ + +
DNA, leading to potentially serious consequences. ROS may DNA damage. Transition metal ions such as Fe , Ag , Cu ,
attack the DNA causing purine-(such as 8-oxoG) and 2+ 5+ 2+
Mn , Cr and Ni which can be released from NPs may
pyrimidine-derived oxidised base lesions and DNA strand contribute to the production of intracellular ROS via the
breaks. DNA base lesions can Fenton-type reaction (Kruszewski et al. 2011). Asharani et al.
(2009) presented a possible chemical reaction of H2O2 with indicator of oxidative DNA attack was detected after Ag NP
+ exposure in several studies in vitro using the comet assay
AgNPs that was estimated to cause formation of Ag in vivo
(Yang et al. 2009; Asharani et al. 2009). Transition metal modified with formamidopyrimidine DNA glycosylase (FPG)
complexes can also bind to DNA bases (Robertazzi & Platts (Magdolenova et al. 2012a; Kim et al. 2011; Asare et al. 2012)
2005). or with the human 8-oxoguanine DNA glycosylase (OGG1)
involved in BER of 8-oxoG (Hudecová et al. 2012b). Fur-
ROS produced by cell components (mitochondria) thermore, DNA repair of 8-oxoG has been studied. For
NPs could interfere with cell components such as mitochon- instance, Folkmann et al. (2009) found increased mRNA
dria and cause damage affecting their functions. In response to expression of DNA glycosylase OGG1 in the liver of C60
stress, mitochondria can generate ROS. For instance, Asharani fullerene-treated rats, but observed no increase in OGG1 repair
et al. (2009) suggested that disruption of the mitochondrial activity. The direct correlation between ROS formation and
respiratory chain by Ag NPs leads to produc-tion of ROS and oxidative DNA damage was shown after exposure to several
interruption of ATP synthesis, which can result in damage to NPs. For instance, in in vitro study Shukla et al. (2011a)
DNA. Furthermore, in in vitro study Ag NPs were detected in suggest that oxidative stress could be an important route by
mitochondria by TEM analysis (Asharani et al. 2009; which TiO2 NPs induce DNA damage. Involvement of the
only.
Kruszewski et al. 2011). ROS pathways in NP-induced genotoxicity was also suggested
by experiments in which cells were preincubated with
Inhibition of antioxidant defence antioxidant before the NP treatment. Guo et al. (2011)
Inhibition of antioxidants in vitro and consequent accumulation observed in human umbilical vein endothelial cells (HUVECs)
of reactive oxygen can potentially lead to DNA damage in vitro that pretreatment with the free radical scavenger N-
(Barillet et al. 2010). Silicon carbide NPs were associated with acetyl-l-cys-teine (NAC) can inhibit the genotoxicity of
depletion of glutathione (the major molecular antioxidant of MWCNTs. Foldbjerg et al. (2011) investigated the effect of Ag
cells) and inactivation of some antioxidant enzymes such as 32
NPs in human lung carcinoma cells (A549) using a P
glutathione reductase and superoxide dismutase (Barillet et al. postlabelling technique. Ag NPs increased the formation of
2010). Similarly, the depletion of glutathione and superoxide bulky DNA adducts, and this effect was inhibited by
dismutase (Sharma et al. 2009) and generation of intracellular antioxidant pre-treatment (Foldbjerg et al. 2011). Similarly, we
ROS in vitro were associated with ZnO NP-mediated DNA found that Ag NPs induce 8-oxoG in primary human peripheral
damage and cytotoxicity (Sharma et al. 2012). lympho-cytes as well as human kidney HEK293 cells
For personal use
(measured with human OGG1) that can be eliminated by

pretreatment of cells using plant extract with antioxidative
properties (Hudecová et al. 2012a, b).
Secondary genotoxicity
ROS produced by inflammatory cells There are obviously several simultaneous mechanisms that
NPs can trigger ROS production in activated phagocytes may lead to genetic changes in human and mammalian cells.
(neutrophils, macrophages). Inflammation in several studies The recent review of Iavicoli et al. (2011) reported that
was associated with genotoxicity. For instance, Trouiller et al. exposure to TiO2 NPs leads to an increase of ROS production
(2009) found that TiO 2 NPs induced an inflammatory reac-tion and cytokines levels, induction of apoptosis and genotoxicity.
and oxidative DNA damage in mice. The oxidative burst Several authors reported from in vitro studies that TiO2 NPs
caused by activation of phagocytes may be a potential induce ROS-mediated induction of DNA damage (Ghosh et al.
explanation for the observed genotoxicity. 2010; Saquib et al. 2012; Table I) and photo-genotoxicity
(Kang et al. 2011). TiO2 NPs and UVA syner-gistically
DNA damage as a result of primary and promoted rapid ROS generation and mitochondrial membrane
secondary genotoxic events due to NP exposure potential collapse in human peripheral lymphocytes, triggering
DNA damage can occur as DNA base modifications (oxida- apoptosis and inducing DNA dam-age. The effect was more
tion), bulky DNA adducts, DNA single strand breaks, DNA pronounced by smaller TiO2 NPs. However, Toyooka et al.
double strand breaks (DSBs), cross links or structural DNA (2012) demonstrated that formation of -H2AX (a biomarker for
changes. Many of these lesions were detected after exposure to
DSBs) by TiO2 NPs was ROS independent and no TiO2 NP-
NPs, including strand breaks, alkali labile sites, oxidised
induced chromosomal aberrations were observed in CHO cells
purines and pyrimidines measured by the comet assay
either in the absence (Warheit et al. 2007) or presence of UV
(Karlsson et al. 2008); DSBs measured by phosphorylation of light (Theogaraj et al. 2007).
histone H2AX (Wang et al. 2010); and DNA adducts measured
32
by P postlabelling (Foldbjerg et al. 2011; Kruszewski et al. DNA damage can be repaired by several repair mechan-
2011). The most investigated DNA damage in relation to NPs isms, including BER, nucleotide excision repair (NER),
is DNA base oxidation, since in many studies an increased homologous recombination and nonhomologous end join-ing
level of ROS was observed after NP exposure. 8-OxoG, the repair (NHEJ). Only a few studies have investigated NPs in
major purine oxidation product pro-duced in DNA during relation to DNA repair. Wojewódzka et al. (2011) found that
oxidative stress, tends to mispair with adenine during treatment with Ag NPs delays repair of X-ray-induced DNA
replication, and is highly mutagenic and thus potentially damage in HepG2 cells. Jugan et al. (2011) investigated the
carcinogenic (Vidal et al. 2001). 8-oxoG as an impact of TiO2 NP exposure on DNA repair in A549 cells,
Cell death Cell
transformation
Accumulation of mutations, Changes in
mutations in important genes gene

expresion
Chromosomal mutations Gene mutations
(deletion, duplication, (inversion, deletion,

inversion, transversion, substitution) Silencing of
aneuploidy, polyploidy) important
genes
Not repaired
Cell cycle arrest (A, B, C, D) Cell cycle arrest
Chromosomal damage DNA damage Non-DNA damaging
(SSB, DSB, base changes

(chromosome break, modification, cross (methylation, histone
loss of chromosome) links,.. ) modification, protein
NP caused phosphorilation,.. )
damage
Repaired
Cell cycle arrest (BER, NER,

HR, HNEJ)
Normal cell
personaluseonly.
Figure 5. Possible consequences of NP-induced DNA damage including non-DNA changes.

by Oslo universitetssykehus Aker on 04/22/13
For
Nanotoxicology Downloaded from informahealthcare.com
and found that TiO 2 NPs simultaneously damaged DNA and (a) chromosome structure changes (deletions, duplications,
impaired cellular DNA repair through inactivation of BER and inversions and translocations of sections of chromosomes), the
NER pathways. Li et al. (2011a) indicate that the NHEJ consequences depending on the genes that have been altered
pathway might be involved in the repair of DNA damage (Russel 2001); and (b) changes in number of chro-mosomes
induced by Au NPs in lung fibroblasts. (aneuploidy – loss or gain of one or more chromo-somes; or
polyploidy – multiplication of whole sets of chromosomes).
DNA damage that is not repaired leads to mutation
DNA damage that is not repaired, or is misrepaired, can lead to Mutations were detected following NP exposure in vitro in
mutation. This situation may arise if (a) DNA damage caused several studies using the HPRT gene mutation assay (Wang et
by NPs is too extensive and the DNA repair mech-anism is not al. 2007b), the bacterial reverse mutation assay (Hasegawa et
efficient enough to repair all damages (Huang et al. 2009); (b) al. 2012), the micronucleus assay (Muller et al. 2008; Könczöl
DNA damage is not recognised by the repair mechanism; (c) et al. 2011; Di Virgilio et al. 2010), chromo-somal aberrations
the DNA repair mechanism is corrupted (it should be noted that (Hackenberg et al. 2011b) or the sister chromatid exchange test
NPs could potentially modulate the function of repair enzymes (Di Virgilio et al. 2010) (for com-plete list see Table I). The
and thus DNA repair could be defective); (d) this interference cytokinesis block micronucleus (CBMN) assay is able to detect
may also give rise to ‘spontaneous’ mutations resulting from both aneugenic and clasto-genic effects, as MN could represent
errors during DNA replication as a consequence of defective lost chromosomes or chromosome fragments (Dusinska et al.
repair (Figure 5). 2012; Kazimirova et al. 2012). To increase the specificity and
to discriminate between these two events, Gonzalez et al.
DNA base pair substitutions give rise to missense or (2010) used the CBMN assay in vitro using A549 cells in
nonsense mutations and DNA base pair insertions or dele-tions combina-tion with fluorescent in situ hybridisation (FISH)-
give rise to frameshift mutations. If these mutations occur in centro-meric probing. Furthermore, the frequency of mitotic
protein coding regions of genes, coding information may be errors (chromosome loss, mitotic arrest and mitotic slippage)
changed leading to errors in gene expression and thus to as consequences of spindle defects was measured. To dis-
formation of defective or no proteins. Accumulation of tinguish between NPs causing aneugenicity and clastogeni-city,
mutations can result in cell death or cell transformation and measurement of MN in mononucleated cells in addition to
cancer (Sharma et al. 2012). those in binucleated cells in the CBMN assay was sug-gested
DNA damage (such as DSBs) or chromosomal damage as an additional marker (Kazimirova et al. 2012).
(chromosome breaks) can lead to chromosome aberrations:
Methods used for in vitro and in vivo assay has been discussed (Karlson et al. 2004; Karlson 2010;
genotoxicity testing of NPs Stone et al. 2009; Shukla et al. 2011a). The presence of NPs
close to DNA during the comet assay also increases the
Genotoxicity testing of NPs can be carried out in vitro or in
probability for an interaction of NPs with FPG. It has recently
vivo. The in vitro approach is appropriate for testing primary
been shown that the incubation of NPs and ions with FPG
genotoxicity, while in vivo models can also give information
leads to the total loss of the ability of the enzyme to detect
on secondary effects such as inflammation (Dusinska et al.
oxidatively damaged DNA in the comet assay (Kain et al.
2011; Kisin et al. 2007; Vega-Villa et al. 2008; Arora et al.
2012). This disturbance is most likely due to the binding of
2012). Initial testing for genotoxicity is usually done with the
ions to the SH groups at the active site (Kain et al. 2012).
bacterial reverse mutation assay (Ames test) (Warheit et al.
However, in the actual comet assay, FPG and NPs do not
2007). Subsequent tests in cultured mammalian cells (either
interact directly. As we recently showed, the presence of NPs
permanent cell lines or primary cultures) employ various
in the agarose had no effect on the ability of FPG to recognise
endpoints. DNA damage is mea-sured with the comet assay
oxidised bases (Magdolenova et al. 2012).
(Shukla et al. 2011a, b). Mutations are assessed at a specific
locus – often the HPRT (hypoxan-thine
A relatively specific and very sensitive assay for DSBs is
phosphoribosyltransferase) gene (Wang et al. 2007a).
only.
based on the fact that a cellular response to these breaks is the

Chromosomal damage is scored either in mitotic cells as
phosphorylation of one of the core nucleosomal his-tones,
chromosome aberrations or in interphase cells as MN. In vivo,
H2AX. The phosphorylated form is known as -H2AX, and the
DNA damage (Bourdon et al. 2012; Schulz et al. 2012),
concentration in the proximity of a DSB is sufficient to form a
chromosome aberrations (Dandekar et al. 2010) and MN
focus, visible by immunohistochemistry using a fluorescence-
(Estevanato et al. 2011; Li et al. 2011c) can be measured in
tagged antibody to the phosphorylated form. Alternatively,
different tissues, and transgenic rodents are available that allow
-H2AX can be measured by flow cytometry (Ismail et al. 2007;
detection of mutations at a specific locus in cells from different
Lewis et al. 2010).
organs.
The chromosomal aberration (CHA) test identifies agents
The Ames test (bacterial reverse mutation) (Mortelmans
that cause structural chromosome or chromatid breaks,
& Zeiger 2000) is based on induction of back-mutations in a
dicentrics and other abnormal chromosomes, nota-bly
defective histidine gene; reversal of this mutation will enable
translocations which are implicated in the aetiology of various
the bacterium to synthesise histidine and form a visible colony
human genetic diseases and cancers. For in vitro testing, cell
For personal use
when plated in minimal histidine medium. There are concerns

cultures are exposed to the test substance and incubated with a
regarding the suitability of the Ames test for NP testing, as
metaphase-arresting substance (e.g. colce-mid) to accumulate
larger NPs are unable to cross the cell wall. If they do enter the
metaphase cells, which are analysed microscopically
cell, NPs could possibly interfere with histidine synthesis and
(Galloway et al. 1994, 1987; Bonassi et al. 2008; Aoshima et
induce false-negative (down-regulation) or positive (up-
al. 2010).
regulation) results. The Ames test has never-theless been used
MN are formed during anaphase from chromosomal
to assess genotoxicity of a variety of NPs, and has so far given
fragments or whole chromosomes that are left behind when the
largely negative results (Landsiedel et al. 2009; Shinohara et al.
nucleus divides. Excluded from the nuclei of daughter cells,
2009; Mori et al. 2006; Wirnitzer et al. 2009; Di Sotto et al.
they form single or multiple MN in the cytoplasm, detected by
2009; Balasubramanyam et al. 2010; Maenosono et al. 2007,
visual (or automated) examination after staining. Formation of
2009; Yoshida et al. 2009; Kumar et al. 2011a, b).
nucleoplasmic bridges and binucleated cells provides a
complementary assay for chro-mosome rearrangement.
The comet assay (single-cell gel electrophoresis) is one of
Increased assay sensitivity can be obtained (in the in vitro
the most common tests for genotoxicity. Cells are embedded in
assay) by incubating treated cells with cytochalasin B, which
agarose on a microscope slide and lysed in detergent and high
blocks cell division, but not mitosis, so that binucleated cells
salt to remove membranes and soluble cell compo-nents, and
accumulate. Scoring MN only in binucleated cells reduces the
also histones. DNA remains as nucleoids, con-sisting of
likelihood of scoring MN that existed before the treatment. The
supercoiled DNA loops attached to a matrix. DNA breaks relax
MN assay is less time-consuming and more suitable for
supercoiling, and relaxed loops of DNA are able to extend
automation than the CHA assay. Histological staining with
during electrophoresis (normally at high pH), to form a ’comet
labelled DNA probes reduces the risk of falsely identifying NP
tail’, visualised by fluorescence microscopy. Relative tail
aggregates as MN fragments (Laingam et al. 2008; Fenech et
intensity indicates break frequency. As well as DNA breaks,
damaged bases can be detected by incubating nucleoids with al. 2011; Doak et al. 2009; Gonzalez et al. 2011; Li et al.
lesion-specific endonucleases, such as endo-nuclease III and 2012).
FPG that recognise oxidised pyrimidines and purines, The HPRT gene has several features that make it suitable
respectively (Collins et al. 1996; Dusinska & Collins 1996; for assessing HPRT mutations induced by a suspect geno-toxic
Hudecová et al. 2012b). Photogenotoxic effects of NPs have agent such as NPs. It is X-linked, with only one active copy
been measured by the comet assay in combi-nation with per cell, so that a mutation in only one allele is needed for
ultraviolet radiation (Jha 2008). The presence of NPs in the phenotypic expression. HPRT is a purine salvage enzyme,
nucleoid has been reported and the possibility that they might which phosphorylates ’waste’ purines and adds them to the
induce additional DNA damage during the cellular DNA precursor nucleotide pool. It is not an essential
enzyme for the cell, and so HPRT mutant cells survive. After
treatment with the suspect agent, cells are cultured for several Size
generations to dilute out pre-existing enzyme, and then plated Because of their small size, NPs have a much larger surface
in dishes at suitable density in the presence or absence of 6- area per unit mass compared with their parent materials. The
thioguanine, a toxic purine analogue that is taken up by wild- number of atoms at the surface increases exponentially as size
type (WT) cells, which die. HPRT cells survive to form decreases. As a result, NPs are very reactive in biological
colonies, which are scored. The mutant frequency is calculated systems. They have a high surface energy and also are more
from the frequency of mutant colo-nies related to plating toxic, in contrast to larger particles of the same chemical
efficiency (Wang et al. 2007a, 2011). composition (Chan 2006). Thus, evaluating the toxicity of
OECD guidelines exist for several genotoxicity assays in engineered NPs cannot rely on extrapolation of toxicity data
vivo or in vitro: OECD 471 for the bacterial reverse mutation from larger particles, as was shown in several studies
test, OECD 473 for the in vitro CHA test, OECD 474 and 475 (Jacobsen et al. 2008). It is also reported that NPs can disperse
for the mammalian erythrocyte and bone marrow CHA tests, throughout the whole body; however, few of them are reported
to penetrate across different barriers, enter individual cells and
OECD 476 for the in vitro mammalian cell gene mutation test
interact with biomolecules on the cell surface and within the
and OECD 487 draft for the in vitro MN test. OECD guidelines
cells (Chan 2006; McNeil 2005). Different sizes of NPs were
are under preparation for the in vivo comet assay test
tested for the possibility of size-dependent genotoxicity
only.
(http://www.jacvam.jp/en_effort/ en_oecd.html). However,

(Barnes et al. 2008). Gurr et al. (2005) investigated the effect
these guidelines are formulated for testing chemicals, and their
of size on induction of DNA damage by studying different
suitability for NP testing should not be taken for granted, since
the very different physico-chemical properties of NPs can sizes of TiO2 particles (anatase – 10, 20 nm, 200 and >200 nm,
seriously influence their interactions with DNA (Dusinska et al. rutile – 200 nm). The results have shown the higher potency of
2009; Warheit & Donner 2010). 10- and 20-nm-sized TiO2 compared with 200- and >200-nm-
sized TiO2 in inducing oxidative stress in the absence of
photoactivation. However, in this study no data are available
Effect of physico-chemical properties on NP- regarding the characterisation and agglomeration of NPs in
induced genotoxicity phosphate buffered saline (PBS) (Gurr et al. 2005).
Papageorgiou et al. (2007) compared the genotoxic effects of
Although the number of studies on genotoxicity of NPs is NP and micron-sized particles of Co/Cr alloy in human
increasing, many conflicting results are published. This is not fibroblasts. The NPs caused more DNA damage than the
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surprising as detailed characterisation of NPs in many stud-ies micron-sized particles. The authors reported that different
especially in testing media is lacking. NPs are prone to change biological responses of human fibro-blasts to surgical implant
properties in different media and treatment condi-tions, and materials in vitro may be dependent on the particle size
without proper in situ characterisation, it is difficult to compare (Papageorgiou et al. 2007). Xu et al. (2009) demonstrated that
results (Stone et al. 2009; Som et al. 2010; Dusinska et al. TiO2 at nanoscale increased the mutant yield at the gam and
2011). Many different NP character-istics (size, shape, surface redBA loci in MEF cells, while TiO2 at micro-scale had little
properties, composition, solubility, aggregation/agglomeration, effect on mutation induction. Several in vivo and in vitro
NP uptake, presence of muta-gens and transition metals studies consistently show that transition from the micro-scale
affiliated with the NPs, etc.) have to be taken into consideration to nanoscale size range increases toxicity (Kang et al. 2011;
to be able to assess toxic effects. Physico-chemical properties Toyooka et al. 2012). The diameter of inhaled or instilled
of NPs have a strong link to their biological activity and many particles is thus an important factor influencing the toxicity
of them may contribute to adverse health effects (Chan 2006; response (Xu et al. 2009). For example, the DNA-damaging
Vega-Villa et al. 2008). It is still not known which of these potential of amorphous silica tested in four sizes, one micro
properties play the most important roles in NPs’ impact on (498 nm) and three nano (68, 43 and 19 nm), was shown to be
genotoxicity. According to Hansen et al. (2007) and Stone et al. size-dependent. With decreasing particle size, the level of DNA
(2010) the following properties are impor-tant: chemical damage in cells gradually increased (Li et al. 2011b). Different
composition, size, shape, crystal structure, surface area, surface particle sizes may pro-duce different kinetic properties of
chemistry, surface charge, solubility and adhesion, defined as substances and may result in enhanced or reduced uptake,
the force by which the NPs and their components are held distribution, metabolism and elimination (Nohynek et al.
together. Warheit (2008) suggests the following minimum 2008). With reduction in size, the properties can change
properties to be characterised: par-ticle size, size distribution dramatically, regarding for example electrical conductivity,
(wet state) in the relevant medium, surface area (dry state), magnetic characteristics, hardness, active surface area,
crystal structure/crystal-linity, aggregation status in the relevant chemical reactivity and biological activity (Karlsson et al.
medium, composi-tion/surface coatings, surface reactivity, 2008).
method of synthesis, purity of sample. A recent report on
nanomaterials in the context of REACH (Malkiewicz et al. Particle shape vs. chemical composition
2011) stressed the importance of NP surface properties in the The shape and the chemical composition of the NPs play an
appropriate test medium, in addition to the chemical important role in induction of cytotoxicity and genotoxicity.
composition, particle size, shape and size distribution. Several reports have shown the size- and shape-dependent
cytotoxicity of the NPs (Nohynek et al. 2008; Yang et al.
2009). It has also been shown that the shape
of the NPs affects their internalisation into the cells. SWCNTs genotoxicity of ZnO NPs in three-dimensional (3D) mini organ
have different toxicological properties from CB particles cultures of human nasal mucosa differed depending on coating.
because of their different shape (Nohynek et al. 2008). Yang Several characteristics of nanomaterials change depending on
and colleagues have also reported that DNA damage induced the medium and environment. The surface of NPs in a
by carbon nanotubes is due to the mechanical injury and not biological environment is modified by the adsorp-tion of
due to the oxidative effect induced by them (Yang et al. 2009). biomolecules such as proteins, polysaccharides and lipids.
Guo et al. (2011) have pointed out that the length of MWCNTs These interact with the NP surface forming a relatively stable
affects the penetration and toxicity of the MWCNT. It is also ’biomolecular corona’ (Monopoli et al. 2011). Thus, the same
reported that the different particles (MWCNT & asbestos NPs in different experimental environments can give different
fibres) of same shape and size have a similar genotoxic effect outcomes. It is therefore important to test NPs under conditions
(Poland et al. 2008). Chemical composition is important as similar to those of potential human exposure.
well. As suggested by Barillet et al. (2010), the cellular
responses of SiC NPs depend on the Si/C ratio; with varying
Si/C ratios NPs may behave as either Si- or C-based. Agglomeration
The agglomeration potential of NPs is an important feature
only.
Crystalline structure which may influence their behaviour and impact on geno-
Crystalline structure could potentially have impact on gen- toxicity. Highly agglomerated NPs cannot enter the nucleus
otoxicity; regarding TiO2 NPs, different crystalline forms such and mitochondria while NPs that do not agglomerate can be
as rutile, anatase or its combinations in various proportions are distributed all over the cell (Ahamed et al. 2008; Dhawan et al.
used in toxicology studies. Petkovic´ et al. (2011) sug-gested 2009). TiO2 NPs were found to be internalised into human skin
that the different genotoxicity responses induced by anatase epidermal cells or to adhere to the cell membrane, depending
and rutile TiO2 NPs could depend not only on size but also on on their size. NPs of 30–100 nm were found in the cytoplasm,
crystalline structure. vesicles and nucleus, while larger particles (>500 nm)
remained outside the cells (Shukla et al. 2011a). In medium,
Surface area NPs can be dissolved or tend to form agglomerates/aggregates,
The surface area of particles is closely associated with their depending on their surface charge (hydrophilic or
size. The smaller the particles, the larger the surface area per hydrophobic) and interactions with medium (medium pH,
unit mass. The number of surface atoms and molecules salinity, protein content, etc.). The surface can be modified to
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increases exponentially and thus higher chemical reactivity is prevent agglomeration. The chemical and physical behaviour
expected. With larger surface area the number of free radicals of nanomaterials is not well under-stood. Some NPs form
and transient metal ions arising from the NP surface increases aggregates and rapidly drop out of suspension. In that case,
and thus the opportunity for their possible inter-action with constant resuspension is necessary in order to maintain a
cells increases as well. A direct relationship between surface homogeneous solution (Colognato et al. 2008). TiO 2 NPs are
area and ROS formation was observed by Li et al. (2011b). prone to form agglom-erates in solutions (Trouiller et al.
ROS formation and DNA damage were size-dependent and 2009). To understand the size distribution, Gurr et al. (2005)
thus a higher surface area could be an important factor. As total tested particle sizes in culture medium. Surprisingly, 10 and 20
surface area could determine NP genotoxicity, Gonzalez et al. nm TiO2 produced aggregations of 1000 nm in diameter, while
(2010) expressed results with doses as a function of surface the 200 nm particles showed no aggregation. The state of
2
area (m /mL) to compare with expression in mass dose agglomeration of thermoresponsive poly N-
(mg/mL) or particle number/mL. isopropylacrylamide (PNIPAM) NPs was dependent on
temperature and vehicle (water, cell culture medium, presence
Surface properties of fetal bovine serum (FBS)) in which they were prepared
The surface properties, including chemistry and charge, are (Naha et al. 2010). We found that the level of agglomeration of
important factors in determining genotoxicity. Various sur-face NPs has an impact on cyto-toxicity and genotoxicity, with
modifications enable binding of chemical, molecular or other different outcomes of TiO2 exposure depending on the state of
biological entities to NPs (McNeil et al. 2005). Ahamed et al. NP agglomeration and dispersion (Magdolenova et al. 2012).
(2008) suggest on the basis of their experi-ments that
differences in surface chemistry of Ag NP deter-mine
differences in genotoxic effects. Polysaccharide-coated Ag NPs Solubility
induce more severe damage than uncoated Ag NPs. Different Solubility is a key factor in the assessment of the intrinsic/
surface chemistry of the NPs results in their different behaviour extrinsic properties of NPs, as this can increase or decrease the
in solution. The uncoated NPs tend to agglomerate while the bioavailability of NPs to the living system. The solubility of
coated are more dispersed. The surface charge determines NPs can be predicted from their structure and reactive groups
whether NPs can be dissolved in medium or whether they form present on the surface. Some of the NPs are reported to
aggregates; it can also influence their biocompatibility and produce ions in soluble form which are toxic to the cells
ability to traverse bio-logical barriers (McNeil et al. 2005). (Franklin et al. 2007). More soluble NPs, such as ZnO and
FeO, show greater cytotoxicity than insoluble NPs, such as
NP surface modifications and coating influence cytotox-icity CeO2 and TiO2, in human mesothelioma MSTO-211H and
and genotoxicity. Yin et al. (2010) showed that rodent 3T3 fibroblast cells; however, the correlation with the
phenomenon of dissolution was not demonstrated experi- of the quantity of NPs in air, water, soil or any consumer
mentally (Brunner et al. 2006). In contrast to the aforesaid product is a technical challenge due to their tiny size and the
statement, there are reports that the toxic responses observed small quantity present. It is also suggested that the concen-
on exposure to NPs are due to the NPs per se rather than the tration/dose of NPs should not exceed a limit that enhances
ions released from them in different cellular systems and agglomeration. The agglomeration of the NPs affects their
exposure conditions (Gojova et al. 2007; Lin et al. 2009; bioavailability to the cell, thereby leading to false positive/
Sharma et al. 2012). negative results.
Physical or chemical agents or impurities

Conditions influencing the results of
Toxicological responses can also change in the presence of
genotoxicity studies
other chemical (polycyclic aromatic hydrocarbons) or phys-
Variability in the results of genotoxicity studies can be ical (UV irradiation) agents (Vevers & Jha 2008). Some NPs
attributed to the source of NPs, the method of preparation or such as ZnO NPs or C60 NPs are able to generate ROS under
synthesis, the dispersion protocol, and variables in exper- irradiation (Hackenberg et al. 2011a; Shinohara et al. 2009).
imental conditions such as physico-chemical specifications Several studies have investigated potential genotoxic effects of
only.
(pH, temperature, presence of impurities or irradiation), NPs under UV irradiation, as well as visible light causing
treatment regime, cell type used, exposure time and dose phototoxicity (Table I). For example, a solution of Au NPs
(Shukla et al. 2011a; Handy et al. 2008, 2012; Vevers & Jha stabilised by citrate ions was not mutagenic but photomuta-
3+
2008; Reeves et al. 2008). genic (light irradiation) due to coexisting citrate and Au ions
present in solution (since Au NPs were not purified) (Wang et
Preparation of the NPs al. 2011a).
Properties of NPs and their genotoxic effect can be influ-enced
by the solvents used for dispersing or dissolving them. Cell type
Solvents differ in their physical and chemical properties. Cytotoxicity of NPs also depends on the cell type used
Factors such as pH, salinity, water hardness, temperature and (Karlsson et al. 2008; Dusinska et al. 2012). Different cell
the presence of dissolved or natural organic particles could types (epithelial, connective, neural, macrophages, etc.) vary in
influence the biological response or toxicity (Handy et al. 2008; their metabolic activities (Vevers & Jha 2008). Cell lines of
Vevers & Jha 2008). Therefore, NPs may behave differently in same or different tissue origin may be less or more suscep-tible
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different solvents (water, culture medium, PBS). This could to NP exposure on account of variation in metabolic pathways,
have pronounced effects on their uptake, cellular localisation cell surface receptors, antioxidant and DNA repair capabilities,
and hence the observed toxic response. For example, Vevers presence of different enzymes/hormones, etc. All these factors
and Jha (2008) examined the effect on cells of well- may affect the behaviour, fate and interac-tion of the NPs with
characterised NPs dispersed in tissue culture medium, PBS or the particular cell type. Also, the interactions of NPs with
water. In medium, NPs can be surrounded by a combination of different cell lines vary because they have different
biomolecules such as proteins, forming a protein corona which internalisation, phagocytosis and cyto-plasmic inclusion
could affect their genotoxic potential (Gonzalez et al. 2010). properties. There are also possible inher-ent differences
NPs are often stabilised with proteins (FBS or bovine serum between cells which are phylogenetically different (for
albumin (BSA)) to prevent their agglomeration. The formation instance, fish cells compared with mammalian cells) (Reeves et
of a protein corona helps NPs to disperse better in the medium. al. 2008). Most studies so far have used human (A549, BEAS,
Sonication can be used to prepare dispersed NPs. As dis-cussed MRC, 16HBE13) or animal (rat, mice hamster) lung cells
in Barillet et al. (2010), the preparation of stable dispersed (V79, BAL, RLE, CHL/IU). Primary cells or cells originating
suspensions of SiC NPs by sonication may pro-mote the rapid from blood and liver are also often used (Table I). It should be
oxidation of Si surface atoms to SiO 2 and consequently may noted that NPs may have different effects on Salmonella
trigger ROS production on their surface. Metal impurities in cursiva or E. coli used in bacterial reverse mutation test due to
the medium can also produce ROS via Fenton-like reactions the bacterial cell wall which could prevent NP penetration
and so it is important to test if their presence is not the cause of (Karlsson 2010) and this test is therefore not suitable for
genotoxicity, rather than the NPs themselves. assessing human-related NP gen-otoxicity (Dusinska et al.
2012; Handy et al. 2012).
Bioavailability and uptake

Concentration of NPs Availability of the NPs to the cell/tissue and their uptake is one
The concentration/dose of the NPs is a crucial part in the of the major factors that can provide important infor-mation
assessment of their cytotoxic and genotoxic potential. NPs about their adverse effects on cellular systems. The exponential
have a tendency to agglomerate in dry form and in suspen-sion, increase in usage of the NP-containing products in daily life
because of van der Waal’s forces. Therefore, it is suggested that has also enhanced the likelihood of their inter-action with the
a parallel control should be run to access the stability of NP individual cell. The fate of the NPs largely depends on
suspension over time. The concentration/ dose of the NPs for behaviour, bioavailability and their interaction with the
an in vitro experiment should mimic the actual exposure to the surrounding medium. Proteins are usually adsorbed on the
human. However, the determination surface of the NPs by different electrostatic, hydrogen
bonding and hydrophobic interactions and affect the dis- such as the comet assay, should be used. Exposure time must
persity, uptake and bioavailability of the particles (Kumar et al. be taken into account while comparing in vivo vs. in vitro
2011a). studies. NPs that do not cross the nuclear envelope via nuclear
NPs in aqueous suspension are dispersed due to the pores only have an opportunity to get into contact with DNA
electrostatic and steric repulsion of the surface charge (pos- during mitosis when the nuclear membrane dissolves, and so
itive/negative) present on the particle. As the surface charges of the time of exposure should be relatively long compared with
the particle skew towards the zero value, the repulsive forces the cell cycle period, to ensure maxi-mum accessibility (Di
between the particles are reduced, which leads to the settling of Virgilio et al. 2010). Furthermore, the presence of proteins that
NPs by gravitational force. Due to agglomeration/ aggregation, adsorb to the NP surface forming a corona (a process known as
the physico-chemical properties such as surface charge, size, opsonisation in medical termi-nology) can affect their
size distribution, surface-to-volume ratio and surface reactivity toxicological effect. A recent study (Walczyk et al. 2010)
of NPs are altered, affecting their uptake, bioavailability and shows that various nanomaterials dispersed in a biofluid
toxicological responses. typically involve a monomeric NP core, with a strongly
Apart from the proteins, several other factors can also associated and relatively stable protein ‘hard corona’
influence the aggregation, uptake and bioavailability of the (consistent with one or two packed proteins layers) coexisting
only.
NPs, such as salt ions, presence of hydrophobic surfactant or with NP multimer–corona complexes (dimers, trimers etc.)
polar groups on the surface of NPs. Trace metal ion speci- present in smaller quantities.
ation, e.g. from metal oxide NPs, might alter the property of Therefore, we need to take into account the fact that
NPs and therefore their uptake, bioavailability and potential mechanisms of NP genotoxicity might differ because of the
toxicity. The detection of NP internalisation in any model different amounts of proteins present in in vivo and in vitro
organism is a crucial step for understanding their behaviour testing systems. While in in vitro systems the concentration of
and toxicity. The commonly used methods for assessment of proteins (FBS, BSA) in NP dispersions and cell cultivation
uptake of NPs in the cells are TEM, confocal and fluores-cence media varies from 0% up to approximately 10%, the presence
microscopy, reflection-based imaging and flow cyto-metry of proteins varies from tissue to tissue in vivo and most
(Shukla et al. 2011a, b; Sharma et al. 2012). importantly the components differ from those used in in vitro
studies. For example, the conflicting results pub-lished on TiO 2
Type of assay, biological system and conditions used NPs might be partially related to the presence or absence of
Assays used to investigate NP genotoxicity can measure proteins (FBS, BSA) as well as to sonication in the TiO 2 NP
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various endpoints. Generally they are methods measuring DNA dispersion protocol. The negative genotoxic results observed
damage and mutations as consequences of unrepaired DNA for instance by Hackenberg et al. (2010, 2011c) were obtrained
damage. For example, the comet assay can detect DNA damage using BSA and sonication in the NP dispersion protocol.
while the HPRT assay measures gene mutations. Large However, negative results were also observed by Warheit et al.
chromosomal abnormalities can be detected by the (2007), even though they did not use any proteins in dispersing
micronucleus or chromosomal aberration assays. In com- the NPs. In this study there is a need to consider also whether
parison with in vitro methods that can detect only primary the genotoxicity assays used (Ames and CHA) are suitable for
genotoxicity, in vivo testing can capture secondary genotoxi- the assessment of genotoxicity of NPs since most of the
city arising due to ROS produced by inflammatory cells. available literature data show negative results using these two
methods (Wirnitzer et al. 2009; Mori et al. 2006; Shinohara et
Recent reports show little correlation between in vitro and in
al. 2009; Aoshima et al. 2010; Kim et al. 2006). In our study,
vivo NP toxicity results (Mahmoudi et al. 2010; Sayes et al.
we compared two dispersion protocols, one with serum in
2007).
stock solution and one without, giving TiO 2 NP dispersions
Additionally, factors such as exposure scenario (dose,
exposure time, cell type, presence of proteins, etc.) could with different stability and agglomeration states, in order to
investigate whether dispersion procedures and presence of
influence the results of genotoxicity testing (Schins 2002;
serum influence NP genotoxicity. We found that the level of
Vevers & Jha 2008; Mahmoudi et al. 2010; Sayes et al. 2007).
agglomeration of NPs and size distribution depend on the
Variation of NP characteristics in different media (for exam-ple
dispersion procedure and use of serum in the stock solution.
lung fluid vs. cell culture medium) should therefore be taken
into consideration, in addition to exposure time (long-term vs. The TiO2 NP dispersion with large agglomerates (3 min
short-term effects) and NP dose (Mahmoudi et al. 2010; Sayes sonication and no serum in stock solution) induced DNA
et al. 2007). In in vitro studies the concentrations may be damage, while the TiO2 NPs dispersed with agglomerates less
excessive compared to in vivo studies, where tar-geted organs than 200 nm (FBS in stock solution and sonication 15 min) had
are probably not exposed to NPs at such high doses. For no effect on genotoxicity (Magdolenova et al. 2012a).
assessment of NPs’ genotoxic potential it is important to use Similarly, Toyooka et al. (2012) found that BSA-surface
also non-cytotoxic concentrations. During cell death (apoptosis coating is related to the generation of -H2AX. For-mation of
or necrosis), formation of DSBs and subsequent increased DSBs (detected by -H2AX) was less extensive with BSA-
DNA fragmentation can occur, emphasising the importance of coated than in uncoated TiO2 NPs.
measuring cytotoxicity together with genotoxicity to prevent
false-positive results. Thus, sensitive methods that can detect In most studies (Di Virgilio et al. 2010; Gurr et al. 2005;
low-level DNA dam-age (a few hundred breaks per cell) and Osman et al. 2010; Reeves et al. 2008) with positive results on
specific DNA lesions, TiO2 NP genotoxicity, no proteins were used (or at least they
were not listed in methods) for the dispersion of NPs. How- standardised and validated (Dusinska et al. 2012; Stone et al.
ever, some of the studies which show positive genotoxicity 2009; Dusinska et al. 2009). As there is currently no specific
results used proteins (Wang et al. 2007; Shukla et al. 2011). regulatory requirement to test NPs for safety, health and
Both the content and the amount of proteins in dispersion environmental impacts (Chan 2006; Dusinska et al. 2011), a
might be important for forming the protein corona. In our study framework for their testing needs to be established.
(Magdolenova et al. 2012a) we used 20% FBS in one of the Last but not least, many studies, both in vitro and in vivo,
dispersion protocols (which gave stable TiO 2 NP disper-sion show positive effects most likely due to the use of concen-
and negative genotoxicity result), while the above-mentioned trations that are not relevant to possible environmental
studies used 5% or 10% FBS in the dispersions. exposure. In many studies a demonstration of genotoxicity
In addition to considering test conditions, it is important to simply reflects cytotoxicity, as excessively high concentra-tions
test for possible interference of NPs with the chosen are used. Thus, cytotoxicity should be an integral part of
genotoxicity assay and to include a sufficient number of genotoxicity testing to avoid false-positive results.
relevant controls and reference materials to avoid false- There are many challenges in nanogenotoxicity due to
positive/negative results (Karlsson 2010; Stone et al. 2009; several possible mechanisms and the diversity of pathways that
Magdolenova et al. 2012b; Sharma et al. 2012; Guadagnini et can lead to a final outcome of mutagenicity. These may involve
universitetssykehus Aker on 04/22/13
al. 2013). The assessment of NP genotoxicity cannot rely only also cell signalling pathways, authophagy and epi-genetic
on one test as there could be several mechanisms leading to changes. To deal with these novel possibilities, new endpoints
mutagenicity. The use of liver micro-somal S9 fraction in some and biomarkers might have to be considered.
studies could affect results, through the presence of additional
proteins as well as the possibility of NPs becoming mutagenic Acknowledgments
after metabolic acti-vation by a mixture of liver enzymes.
Supported by EC FP7 [Health-2007-1.3-4], Contract no:
201335, www.nanotest-fp7.eu.
Final remarks Declaration of interest

Nanotoxicology Downloaded from informahealthcare.com by Oslo
Published genotoxicological data for nanomaterials are dif- The authors report no conflicts of interest. The authors alone
ficult to compare even with the same NPs, since important are responsible for the content and writing of the paper.
information is often missing, especially a detailed charac-
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Nanotoxicology, 2013; Early Online, 1–46

© 2013 Informa UK, Ltd.

Mechanisms of genotoxicity. A review of in vitro and in vivo studies

Abstract or surface structure in the nanoscale". The term "nanoscale" is

in vivo), the micronucleus assay (31 in vitro, 14 in vivo), the

exchanges after TiO2 NP exposure. However, no NPs were

Table I. Current review of NP genotoxicity studies (+ positive; negative; ± equivocal).

Physico-chemical Result of NP properties, Genotoxicity Treatment

of suspension for of~ 1 mm saline solution with MWCNT cell line)

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

DLS (size of particles 210 nm

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

the MAN sample anisotropy energy at

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Mean diameter of 404 nm

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Physico-chemical Result of NP properties, Genotoxicity Treatment

Toxicity vitro toxicity viv Genotoxicity genotoxicity genotoxicity genotoxicitygenotoxicity

NP that do not cross cell membrane NP that cross cell membrane

through nuclear pores deform nucleus shape

NP get access to Centrosome

Figure 2. Cellular uptake and access of NPs to nucleus.

(DNA level) Affect

genotoxicity component with

Mitosis binding to DNA

Figure 3. Scheme of potential NP-induced primary direct genotoxicity.

with DNA. The following are suggested ways in which NPs

DNA repair proteins By chemical binding

Centrioles By chemical binding

Mitotic spindle Mechanically Disturb the

via the Fenton-type

ROS produced TM to DNA

Interaction with antioxidant

Figure 4. Scheme of potential NP-induced primary indirect and secondary genotoxicity.

(measured with human OGG1) that can be eliminated by

mutations in important genes gene

(deletion, duplication, (inversion, deletion,

(SSB, DSB, base changes

Cell cycle arrest (BER, NER,

Figure 5. Possible consequences of NP-induced DNA damage including non-DNA changes.