Mechanisms of genotoxicity. A review ofin vitroandin vivostudies with engineered nanoparticles

Zuzana Magdolenova; Andrew Collins; Ashutosh Kumar; Alok Dhawan; Vicki Stone; Maria Dusinska

Nanomaterials (NMs) are of significant relevance due to their unique physicochemical properties, which have been extensively exploited for widespread applications in human healthcare and consumer goods, such as cosmetics and textiles [...]

Nanotoxicology evaluates the relationship between the structure properties of nanoparticles and toxic hazard, which is of considerable importance prior to clinical application of nanosystems. In spite of the introduction of nanotoxicology in regulatory affairs and the elucidation of some physicochemical structure – toxicity relationships by means of in vitro and in vivo research of nanomedicines, the acquired knowledge still does not allow the prediction of chronic toxicity, especially subtle ways of cell function impairment such as carcinogenesis in humans. This review focuses on potential nanoparticle toxicity after dermal or inhalation exposure. Special emphasis is given to intravenous administration of nanoparticles and the related hematotoxicity. Here, we discuss the main factors, affecting the toxicity of nanoparticles for medical application, namely size, surface charge, chemical composition and the interaction with biological matrices.

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 Zuzana Magdolenova1, Andrew Collins2, Ashutosh Kumar3, Alok Dhawan3,4, Vicki Stone5, & Maria Dusinska1 Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. 1 NILU-Norwegian Institute for Air Research, MILK, Health Effects Laboratory, Kjeller, Norway, 2Department of Nutrition, Faculty of Medicine, University of Oslo, Oslo, Norway, 3Institute of Life Sciences, School of Science and Technology, Ahmedabad University, Ahmedabad, Gujarat, India, 4Nanomaterial Toxicology Group, CSIR-Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India and 5Heriot-Watt University, Edinburgh, UK or surface structure in the nanoscale". The term "nanoscale" is deﬁned as size range from approximately 1 to 100 nm (http://cdb.iso.org). "Nanomaterial" means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1–100 nm. This deﬁnition covers materials where the speciﬁc surface area by volume of the material is greater than 60 m2/cm3 (http://ec.europa.eu/environment/chemicals/ nanotech/index.htm#deﬁnition). As the deﬁnition of nanomaterial is broad, in this review we considered NPs only. NPs are increasingly being used in many areas, but their possible impact on human health is not well studied or understood. The properties of the NPs, for which they are used in products, could also be potentially harmful to biological systems and cause adverse effects (Chan 2006; Dusinska et al. 2011). Features such as NP size, shape, surface properties, 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 of information about human exposure and possible adverse health effects of NPs. A better understanding of how properties of NPs deﬁne their interactions with cells, tissues and organs is a scientiﬁc challenge that must be addressed for the safe use of NPs. Toxicity testing of NPs using existing in vitro and in vivo models is a difﬁcult task as there are so many different classes of NPs with various characteristics that can contribute to toxicity by many diverse mechanisms (Vega-Villa et al. 2008). Underlying mechanisms of NP toxicity are oxidative stress, inﬂammation, immunotoxicity and genotoxicity (Dusinska et al. 2012b). A literature search on genotoxicity of NPs was performed in the PubMed database from year 2000 to 2012 (February) Abstract Engineered nanoparticles (NPs) are widely used in different technologies but their unique properties might also cause adverse health effects. In reviewing recent in vitro and in vivo genotoxicity studies we discuss potential mechanisms of genotoxicity induced by NPs. Various factors that may inﬂuence genotoxic response, including physico-chemical properties and experimental conditions, are highlighted. From 4346 articles on NP toxicity, 112 describe genotoxicity studies (94 in vitro, 22 in vivo). The most used assays are the comet assay (58 in vitro, 9 in vivo), the micronucleus assay (31 in vitro, 14 in vivo), the chromosome aberrations test (10 in vitro, 1 in vivo) and the bacterial reverse mutation assay (13 studies). We describe advantages and potential problems with different methods and suggest the need for appropriate methodologies to be used for investigation of genotoxic effects of NPs, in vitro and in vivo. Keywords: nanoparticle toxicity, DNA damage, DNA oxidation, mechanisms of genotoxicity, primary genotoxicity, secondary genotoxicity, nanoparticle properties, nanotoxicology Introduction Nanoparticles (NPs) have unique, potentially beneﬁcial properties and nanotechnology is considered to be the technology of the future. The deﬁnition of ‘Nanoparticle’ given in the PAS71 document developed by the British Standards Institution is "A particle having one or more dimensions of the order of 100 nm or less" (PAS71 2005). NPs are also called ultraﬁne particles by some toxicologists (USEPA 2007), aitken mode and nucleation mode particles by atmospheric scientists (Kulmala 2004; NRC 1983) and engineered NPs by material scientists (Oberdörster et al. 2005a,b). The International Organization for Standardization deﬁnes the term "nanomaterial" as "material with any external dimensions in the nanoscale or having internal structure 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)  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only.  Z. Magdolenova et al. using key words: NPs and toxicity, in vitro toxicity and NPs, in vivo toxicity and NPs, NPs and genotoxicity, ultraﬁne particles and genotoxicity. From 4346 articles on NP toxicity, 112 publications describing experimental studies on NP genotoxicity (94 in vitro, 22 in vivo) were selected (Table I). Studies investigating the environmental impact of NPs are not included in this review. From the published literature, 67 genotoxicity studies used the comet assay (58 in vitro, 9 in vivo), 44 the micronucleus assay (31 in vitro, 14 in vivo), 11 the chromosome aberrations test (10 in vitro, 1 in vivo) and 13 the bacterial reverse mutation assay (Figure 1). In addition, some studies used other genotoxicity assays, such as HPRT gene mutation, sister chromatid exchange, mRNA expression of DNA base excision repair (BER) genes, detection of 8-oxo-7,8-dihydroguanine (8-oxoG), in vivo DNA deletion, CII mutation analysis, the g-H2AX assay and pulsed ﬁeld gel electrophoresis. TiO2, iron and silver NPs are the most used NPs in genotoxicity studies (Table I). After them, a higher number of publications describe genotoxic impact of fullerenes, silica, carbon black, zinc, gold NPs. Some studies investigate the effect of multiwalled carbon nanotube (MWCNT), polymer NPs and single-walled carbon nanotube (SWCNT). In addition, few or single publications can be found on genotoxicity of quantum dots, Co or CoCr, Pt, CuO, CeO2, rare metal and metal oxide and alumina NPs. However, when looking on carbon NPs together (WCNT, MWCNT, Fullerenes, Carbon black) they are the second most used after TiO2 NPs. Particle toxicology and consequent health effects of asbestos ﬁbres and coal dust serve as historical reference points for the development of nanotoxicology concepts (VegaVilla et al. 2008). Several reviews dealing with NP genotoxicity have been published (Gonzalez et al. 2008; Stone et al. 2009; Singh et al. 2009; Landsiedel et al. 2009; Ng et al. 2010; Karlsson 2010; Donaldson et al. 2010; Xie et al. 2011). In updating this fast-changing research area, we concentrate in particular on methods of investigation, mechanisms of genotoxicity, and the conditions that inﬂuence experimental results. Mechanisms of NP-induced genotoxicity The mechanisms of NP genotoxicity are still not well understood and it is often not clear if an effect on DNA is nanospeciﬁc. Genotoxicity may be produced by direct interaction of NPs with the genetic material, or by indirect damage from NP-induced reactive oxygen species (ROS), or by toxic ions released from soluble NPs (Kisin et al. 2007; Barnes et al. 2008). Secondary genotoxicity can be a result of oxidative DNA attack by ROS via activated phagocytes (neutrophils, macrophages) during NP-elicited inﬂammation (Stone et al. 2009). NPs that cross cellular membranes may be able to reach the nucleus through diffusion across the nuclear membrane or transportation through the nuclear pore complexes, and interact directly with DNA (Figure 2). As discussed by Barillet et al. (2010), in vitro studies show that NPs of smaller size may reach the nucleus via nuclear pores (diameter between 8 and 10 nm), while larger NPs such as 15–60 nm SiC-NPs may only have access to the DNA in dividing cells, during mitosis when the nuclear membrane dissolves (Singh et al. 2009; Liang et al. 2008). For instance, after in vitro exposure TiO2 NPs (Shukla et al. 2011a), Ag NPs (Hackenberg et al. 2011b; Asharani et al. 2009) and ZnO NPs (Hackenberg et al. 2011a) have been found in the cell nucleus. Even larger intranuclear aggregates of TiO2 NPs were observed in the nucleus (the mean size of studied TiO2 NPs was 285 ± 52 nm and in particular cases, aggregates could reach diameters up to 2000 nm) (Hackenberg et al. 2010). These authors observed on human nasal mucosa cells in vitro no genotoxicity of TiO2 NPs (Hackenberg et al. 2010) but a positive effect of ZnO (Hackenberg et al. 2011d) with the comet assay and also of Ag NPs (Hackenberg et al. 2011b) with the chromosome aberration assay and the comet assay. Large NP aggregates can even deform the nucleus, as shown by transmission electron microscopy (TEM) observations in vitro in the Chinese hamster ovary cells CHOKI study of Di Virgilio et al. (2010). Aggregates of TiO2 NPs induced the formation of cellular vesicles which impressed on the nucleus and modiﬁed its form. Deformation of nuclear shape could unfavourably affect the process of mitosis, physically preventing the correct segregation of chromosomes, and the correct functioning of the mitotic spindle and its components. The NP aggregates could also mechanically damage the chromosomes. Di Virgilio et al. (2010) showed an increase in frequencies of micronuclei (MN) and sister chromatid exchanges after TiO2 NP exposure. However, no NPs were observed in nuclei. Direct primary genotoxicity Direct interaction of NPs with DNA or chromosomes NPs that are present in the nucleus (entering either by penetration via nuclear pores or during mitosis) might directly interact with DNA organised in chromatin or chromosomes depending on the phase of cell cycle. 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 between NPs and DNA, some computational as well as experimental studies have been performed. Using computational methods, Jin et al. (2011) found that strong interactions between Al12X (X = Al, C, N, P) NPs and DNA bases/base pairs are expected. They suggested that Al NPs might cause structural damage and affect DNA stability. An experimental study by An et al. (2010) showed an interaction between carbon NPs and DNA in vivo in Escherichia coli. Carbon NPs were bound to single-stranded DNA and incorporated into DNA duplex structures, probably during DNA replication. This suggests that carbon NPs could disturb DNA replication (An et al. 2010). During mitosis NPs could interact with chromosomes causing clastogenic or aneugenic effects. NPs might introduce breaks into chromosomes or disturb the process of mitosis, mechanically or by chemical binding (Figure 3). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. Current review of NP genotoxicity studies (+ positive; Physico-chemical Result of Nanoparticle characterisation characterisation SWCNTs SWCNT (COCC, Chinese Academy of Science, Chengdu) SWCNT (EliCarb) Size (TEM) Shape (TEM) Chemical composition (purity) (Raman spectroscopy) BET surface area Pore size ICP-MS Size distributions in water Size in media HiPco SWCNT (CNI, Inc., Houston, TX) SWCNT (Thomas Swan and Co Ltd., Consett, UK) Genotoxicity testing method Treatment conditions Result Cells/organism + Primary mouse embryo ﬁbroblasts Yang et al. 2009 FE1-MutaTM mouse lung epithelial cell line Jacobsen et al. 2008 V79 (Chinese hamster lung ﬁbroblast line) Kisin et al. 2007 Diameters: 8 nm, length <5 mm Rope-shaped C >99.99% NP sonication six times intermittently (30 sec every 2 min) Comet assay 5 and 10 mg/mL for 24 h 731 ± 2 m2/g 15 nm ~95% C, 2% Fe, <0.001% Co, Ni, Mn Nd 32–60, 280–417, and 2256 nm Declared primary particle diameter: 0.9–1.7 nm, length <1 mm Comet assay 3 h of incubation, 100 mg SWCNT/mL Comet assay (FPG) CII mutation Pos. Ctrl: Carbon black (Printex 90) Comet assay 0, 12, 24, 48 or 96 mg/cm2 for 3 or 24 h MN 0, 12, 24, 48 or 96 mg/cm2 for 24 h V79 (Chinese hamster lung ﬁbroblast line) Ames test 0, 60, 120 or 240 mg/plate S. typhimurium strains YG1024/YG1029 Comet assay After 3 h instillation, 54 mg/mouse 8-oxodG and dG (HPLC with electrochemical and UV detection) Investigated effects in liver, lung and colon tissues, 24 h after intragastric administration Doses: 0.064 and 0.64 mg/kg body weight Chemical analysis (NMAM method nr. 5040 and ICP-AES) 99.7 wt% elemental C, 0.23 wt% Fe Purity (TGA-DSC, TPO, NIR, Raman spectroscopy) Diameter distribution (Raman spectroscopy) >99% of C content was accountable to SWCNT Speciﬁc surface area Dispersion in suspension (SEM) Lenght (TEM) Acceptable DLS data could not be obtained, (complex morphology and bundling of SWCNT) 1040 m2/g About 83% Gas exchange surface areas Average pore sizes were 0 and 15 nm 731 ± 2 m2/g (Jacobsen et al. 2008b) 15 nm (Jacobsen et al. 2008b) Particle size in suspensions (DLS) In saline solution were 195, 797 and 5457 nm 0.4–1.2 nm Following suspending in water and media and sonicating, shape and size distributions were analysed by TEM and dynamic light scattering – DLS SWCNT (puriﬁed by acid treatment, containing only 0.23% of Fe). Produced by HiPco technique were ﬁrst characterised After ultrasonitation (30 sec, 3), SWCNT were analysed with scanning electron microscopy (SEM) and TEM Reference + ± At 3 h, + only at 96 mg/cm2 At 24 h, + at ‡48 mg/cm2 1–3 mm Described by the manufacturer: primary particle size of 0.9– 1.7 nm and a ﬁbre length <1 mm The particles were suspended in either + Both doses + in liver and lung, in colon mucosa BAL cells (broncho-alveolar lavage ﬂuid from apolipoprotein E knockout mouse, ApoE / ) Female Fisher 344 rats from Taconic (Ry, Denmark) Jacobsen et al. 2009 Folkmann et al. 2009 Genotoxicity of nanoparticles SWCNTs, negative; ± equivocal). NP properties, NP preparation  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle Physico-chemical characterisation Result of characterisation NP properties, NP preparation Genotoxicity testing method Treatment conditions (low dose); the particle size in the highest dose could not be determined In corn oil: 34 and 178 nm (low dose) and 1015 nm (high dose) 2% Fe and traces of Co, Ni and Mn 417 ng/g of the U.S. EPA priority PAH compounds saline or corn oil, by sonication (70 W, 42 kHz) in a 5-day period for 10 h each day and again 30 min before administration mRNA expression of HO1, MUTYH, NEIL1, NUDT1 and OGG1 (bw) suspended in saline or corn oil; n = 8), saline solution (control; n = 10) or corn oil (control; n = 10) Particle size in powder 110–170 (5–9 103) nm Surface area in powder TEM Nd Suspended in MilliQ and sonicated for 2 20 sec Suspended in medium and sonicated Transition metals PAHs MWCNTs MWCNT (Sigma-Aldrich) MWCNT (Facultes universitaires Notre-Dame de la Paix (Namur, Belgium)) DLS (size of particles and agglomerates in medium) Zeta potential Outer diameter Length Carbon content Remains DLS measurements of suspension for in vivo study Cells/organism Reference OGG1 repair activity Comet assay 1, 20, 40 mg/mL, 4 h + A549 (human lung epithelial cell line) Karlsson et al. 2008 Muller et al. 2008 Comet assay (FPG) 100–200 (3–7 103) nm 300 (6 103) nm 11.3 nm 0.7 mm 98% Traces of Co and Fe catalysts Characteristics were described previously NP heated (200 C/2 h) before use MN in vivo 3 days after intratracheal administration of MWCNT (0.5 or 2 mg/rat) + Type II pneumocytes (AT-II) Aggregates diameter of ~ 1 mm Suspended in a 0.9% saline solution with 1% of Tween 80 CB MN In vitro exposure to MWCNT (10, 25, 50 mg/mL) In vitro exposure To MWCNT (10, 25, 50 mg/mL 2.5, 5, 10 mg/mL, 4 and 18 h, ± S9 mix, + RLE (rat lung epithelial cell line) + MCF-7 (human epithelial cell line) MN with FISH Baytubes macrosized MWCNT agglomerates (Bayer MaterialScience) Result Purity >95% C (no free amorphous C), CHA Size 0.1–3 mm Ames test Sonicated 2 5 min in deionised water, mixed until used Size distribution (by laser diffraction) 50–5000 mg/plate, 48 h, ±S9 mix V79 (Chinese hamster lung ﬁbroblast line) S. typhimurium (strains TA 1535, TA 100, TA 1537, TA 98, TA 102) Wirnitzer et al. 2009  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Nanoparticle MWCNT (Tsinghua and Nanfeng Chemical Group Cooperation, China) MWCNT (Lawrence Berkeley National Laboratory, Berkeley, CA) MWCNT (Prof. D.G.Weiss, Rostock University, Germany) Physico-chemical characterisation Purity Aver. diameter (TEM) Length Metal impurities Particle size (TEM) Mean hydrodynamic diameter Zeta potential Fullerenes C60 fullerenes (Sigma -Aldrich) >95% 30 nm <1 mm Ni 3.38% Y 0.67% Fe 0.13% 5–20 nm in diameter, 300–2000 nm in length 401.3 nm 14.4 mV NP properties, NP preparation Genotoxicity testing method Puriﬁcated, UV-sterilised, diluted under ultrasonication Western blot (OGG1, Rad 51 and XRCC4) APRT gene mutation Conc. 0, 5, 100 mg/mL, 2 and 4 h incubation Synthesised by using a chemical vapour deposition gH2AX 0.5, 5 and 20 mg/mL of MWCNT, for 6, 12 and 24 h Result Cells/organism + Mouse embryonic stem cells J11 and APRT±3C4 Zhu et al. 2007 Reference HUVECs Guo et al. 2011 A549 (human lung epithelial cell line) Srivastava et al. 2011 FE1-Muta mouse lung epithelial cell line Jacobsen et al. 2008 Mori et al. 2006 + + (CVD) method NPs suspended in 1640 medium + 10% FCS, ultrasonicated for 30 cycles of 5 sec with 1 sec pause Suspensions prepared freshly before experiment Sterilisation by heating to 120 C, 2 h, suspended in medium, sonicated, diluted in medium; analysed by LAL assay for presence of endotoxin Antioxidant NAC (6 mM) pretreatment CB MN Western blot (P53) BET surface area <<20 m2/g 99% purity Comet assay Pore size 0 nm Comet assay (FPG) ICP-MS >99.9%C Consist of agglomerates of primary particles Declared primary particle size : 0.7 nm Size distributions in water Size in media 150 and 700 nm 311 nm Treatment conditions Following suspending in water and media and sonicating, shape and size distributions were analysed by TEM and DLS 99.5% purity NAC decreased the percentage of lH2AX positive cells 10 and 50 mg/mL, 24 h + Antigenotoxicity effects of dimetylthiourea (DMTU) and NAC 50 mg/mL, 24 h Antigenotoxicity effects of DMTU and NAC DMTU and NAC reduced MN + DMTU and NAC reduced expression level of P53 3 h of incubation, 100 mg C60/mL + CII mutation Pos. Ctrl : Carbon black (Printex 90) Ames test Doses 39.1– 5000 mg/plate, ±S9 mix S. typhimurium (strainsTA100, A1535, TA98, TA1537); E. coli WP2uvrA/pKM101 CHA 313–5000 mg/mL, ±S9 mix, 6 or 24 h treatment CHL/IU (Chinese hamster lung cells) ± Genotoxicity of nanoparticles C60 fullerenes (mixture of C60 and C70 fullerite) (Vitamin C60 BioResearch Corp, Japan) Result of characterisation  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle C60 fullerenes (Materials research electronics corpor., Tucson, AZ) conc. 0.022–110 mg/L suspensions Aqu/nC60 EtOH/nC60 Physico-chemical characterisation Particle size distribution, particle diameter (DLS) Zeta potential of nC60 colloids (phase analysis light scattering) Particle size distribution (particle diameter) Zeta potential of nC60 colloids Result of characterisation 178.6 nm 13.5 mV NP properties, NP preparation nC60 colloids were prepared from powdered C60 (>99% purity) Genotoxicity testing method Treatment conditions Result 0.022–110 mg/L, 3 and 6h + 0.42–2100 mg/L, 3 and 6 h + gpt delta mutagenicity assay 0.1–30 mg/mL, 3 days exposure Comet assay After 3 h instillation, 54 mg/mouse 8-oxodG and dG (HPLC with electrochemical and UV detection) Investigated effects in liver, lung and colon tissues, 24 h after intragastric administration Doses: 0.064 and 0.64 mg/kg bw suspended in saline or corn oil; n = 8, saline solution (control; n = 10) or corn oil (control; n = 10) Comet assay + gpt delta transgenic mouse embryonic ﬁbroblasts Xu et al. 2009 ± BAL cells (broncho-alveolar lavage ﬂuid obtained from apolipoprotein E knockout mouse, ApoE / ) Female Fisher 344 rats Jacobsen et al. 2009 31.6 mV (99.5% purity) C60 suspension was 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/ aggregates >1 mm C60 fullerenes (Sigma-Aldrich, Brøndby, Denmark) Gas exchange surface areas Average pore sizes Particle size in suspensions (DLS) <20 m2/g (Jacobsen et al. 2008b) 0 nm (Jacobsen et al. 2008b) In the saline solution were 407 nm in the low dose and 621 and 5117 nm in the high dose In corn oil: 234 nm (low dose); 40, 713 and 3124 nm (high dose) None None Described by the manufacturer: 99.9% pure preparation, primary particle size of 0.7 nm The particles were suspended in either saline or corn oil, by sonication (70 W, 42 kHz) in a 5-day period for 10 h each day and again 30 min before administration Diameters 50 and 33 nm CMC-Na and Tween 80 used as dispersants, for preparation see Shinohara et al. 2009 C60 fullerenes Reference Dhawan et al. 2006 Before the experiment, particle size distribution and zeta potential of nC60 colloids were analysed 121.6 nm C60 fullerenes (SES Research) Transition metals PAHs Aqueous suspension with carboxymethylcellulose sodium (CMC-Na) and Tween 80, respectively (DLS) Zeta potential 39 mV Cells/organism Human lymphocytes and 14 mV mRNA expression of HO1, MUTYH, NEIL1, NUDT1, and OGG1 OGG1 repair activity Both doses + in liver, highest dose + in lung in colon mucosa + OGG1in liver Ames test 50–1000 mg/plate; dark or irradiation; ±S9 mix S. typhimurium (strains TA98, TA100, A1535, A153, E. coli WP2uvrA/pKM101) CHA in vitro 12.5–200 mg/mL; dark or irradiation; presence or absence S9 mix CHL/IU (Chinese hamster lung cells) Folkmann et al. 2009 Shinohara et al. 2009  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Nanoparticle Physico-chemical characterisation TEM Result of characterisation NP properties, NP preparation spherical Genotoxicity testing method Treatment conditions Result Cells/organism Reference MN in vivo 22–88 mg/kg Male and female mice (bone marrow cells) 99.5% purity Ames test Doses 156– 5000 mg/plate; presence or absence S9 mix Aoshima et al. 2010 Water-soluble polymer (polyvinylpyrrolid) enwrapped fullerenes NPs dispersed in distilled water CHA ±S9 mix, 6 or 24 h treatment S. typhimurium (strains TA100, A1535, TA98, TA153); E. coli WP2uvrA/pKM101 CHL/IU (Chinese hamster lung cells) Comet assay Intratracheally instilled, single dose at 0.5 or 2.5 mg/kg or repeated dose at 0.1 or 0.5 mg/kg, once a week for 5 weeks. For 3 or 24 h after single instillation and 3 h after repeated instillation Male rats, lung cells Ema et al. 2012 32 P-postlabelling (bulky DNA adducts) 0.46 mg/L HepG2 (human hepato-carcinoma cell line) Matsuda et al. 2011 8-oxodG and CdG (HPLC/MS) 0.46 mg/L ± for Bacillus subtilis Rec-assay Umu test 0.048 mg/L 8-oxodG and CdG + Comet assay 100 mg/mL, different times 0.5–24 h + at ‡3 h exposure A 549 (human lung epithelial cell line) Mroz et al. 2007, 2008 Comet assay 100 mg/mL, different times ranging form 30 min to 24 h + at ‡3 h exposure A 549 (human lung epithelial cell line) Mroz et al. 2007, 2008 for detailed characteristics see table in Shinohara et al. 2009 C60 fullerenes (mixture of C60 and C70 fullerite) (Vitamin C60 BioResearch Corp, Japan) C60 fullerenes (Frontier Carbon Co., Ltd., Kitakyushu, Japan) Aqu/C60 fullerene Mean diameter in the 0.1% Tween 80 aqueous solution The characterisation and preparation of the C60 NPs suspension reported by Morimoto et al. (2010) Colour UV–Vis absorption spectra containing 0.1% Tween 80 Yellow colour See the results Size distribution 59–241 nm Size distribution in LB The average size: 117 nm 241–554 nm broth and DMEM For preparation of suspension see the protocol 0.43 mg/L for + Bacillus subtilis H17 and M45 S. typhimurium (TA1535/pSK1002) The average size: 320 nm in LB broth and 330 nm in DMEM Particle diameter 14 nm Before use, NPs suspended in culture medium and sonicated for 20 min 26 mg BaP/g Printex90 Before use, NPs suspended in culture medium and sonicated for 20 min Genotoxicity of nanoparticles Carbon black NPs (NPCB) NP CB (Printex 90) (Degussa, Frankfurt, Germany) BaP-NP Carbon black 33 nm  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle NPCB (Printex 90) (Degussa, Frankfurt,Germany) Physico-chemical characterisation Speciﬁc surface area Pycnometric particle density Result of characterisation NP properties, NP preparation Genotoxicity testing method 295 m2/g Primary particle size 14 nm Comet assay 2.2 g/cm3 Media prepared freshly before exposure, sonicated Comet assay (FPG) lacZ and cII mutation Treatment conditions Eight repeated 72-h incubations with 75 mg/mL carbon black + weakly + 5 and 10 mg/mL for 24 h Size (TEM) Shape (TEM) Chemical composition (purity) (Raman spectroscopy) Size distribution (suspended in instillation media) (DLS) 12.3 ± 4.1 nm Sphere C >99.4% NP sonication 6 intermittently (30 sec every 2 min) Comet assay One mode around 1.2 mm and a less frequent mode around 5.5 mm The analysis was often disturbed by agglomeration Comet assay After 3 h instillation 54 mg/mouse Carbon nanopowder Particle size in powder <30 nm Comet assay 1, 20, 40 mg/mL, 4 h (Sigma-Aldrich) Surface area in powder Nd Suspended in MilliQ and sonicated for 2 20 sec Suspended in medium and sonicated 20–40 nm 210 nm Carbon NPs TEM DLS (size of particles and agglomerates in medium) Zeta potential Exposure characteristics (mass, particle and surface area concentrations, size distribution) see in table (Wessels et al. 2011) NPCB (NanoInnovation Co. Ltd., Shenzhen) Carbon black, Printex 90 14 nm Carbon black NPs (Printex 90; Evonik/ Degussa, Germany) Silicon carbide (SiC) NP, 5 batches of NPs differing in Nature of NP and agglomeration (TEM) Zeta potential Hydrodynamic particle size distributions in media (DLS) Shape (TEM) Result Cells/organism Reference + FE1-MutaMouse lung epithelial cell line Jacobsen et al. 2007 + Primary mouse embryo ﬁbroblasts Yang et al. 2009 + BAL cells (broncho-alveolar lavage ﬂuid obtained from apolipoprotein E knockout mouse, ApoE / ) A549 (human lung epithelial cell line) Jacobsen et al. 2009 Karlsson et al. 2008 Comet assay (FPG) 6.9 mV NPs (~60 nm) generated by electric spark discharge Comet assay (FPG) in vivo Inhalation exposure of mice: 142 mg m3, 4 h or 3 days for 4 h; and rat: 152 mg m3, 4 h mRNA expression of OGG, POLb, XRCC1, APE1, E335 Free and chain agglomerates; size:10 to more than 500 nm 10.7 mV Highly agglomerated See results for details Round shaped Suspended by sonication (8 min: 10 sec pulses and 10 sec pauses) in 0.9% NaCl MiliQ water with 10% BAL ﬂuid Laboratory synthesised, controlled size and Si/C ratio Comet assay in vivo 0.018, 0.054, 0.162 mg; single intratracheal instillation; 1, 3 and 28 days exposure Comet assay (FPG) in vivo Comet assay in mouse and rat for all, except for + increase in APE1 mRNA expression in rat + Whole lung tissue of SPF free female C57BL/6J mice and isolated lung epithelial cells of male Fisher F344 rats Wessels et al. 2011 BAL (broncho-alveolar lavage) cells, lung and liver of C57BL7/6 mice Bourdon et al. 2012 A549 (human lung epithelial cell line) Barillet et al. 2010 + 50 mg/mL of SiCNPs, for 4, 24 or 48 h + all ﬁve tested batches of SiCNPs  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Nanoparticle diameters and Si/C ratio Physico-chemical characterisation Si/C ratios Impurities Speciﬁc surface areas, sizes BET Sizes TEM Zeta potentials Hydrodynamic diameters Result of characterisation In range 0.8–1.2 O2 and N In range 33–140 m2/g 13–58 nm; 12–45 nm In range 22 to 31 mV 100–300 nm NP properties, NP preparation Genotoxicity testing method Treatment conditions Result Cells/organism Reference Dispersed in sterile H2O by sonication 30 min, 4 C, pulsed mode (1 sec on/1 sec off); diluted with FBS, sonicated another 30 min; suspensions diluted in medium before experiment For more details see Barillet et al. (2010) Particle size in powder 20–30 nm (Sigma-Aldrich) Surface area in powder Nd TEM DLS (size of particles and agglomerates in medium) Zeta potential Particle size in powder 20–40 <200 nm 1.8 mV 29 nm Surface area in powder 18 m2/g TEM DLS (size of particles and agglomerates in medium) Zeta potential Particle size in powder 10–100 nm 40–300 nm 34.2 mV 29 nm Surface area in powder 40 m2/g TEM DLS (size of particles and agglomerates in medium) Zeta potential Shape 30–60 nm 1580 nm CuZnFe2O4 (Sigma-Aldrich) Iron particles Fe2O3 (SigmaAldrich) DMSA- coated maghemite (nano-g Fe2O3) NP (NmDMSA) (laboratory synthesised) Diameter size BET speciﬁc surface area Colloidal stability of NmDMSA in biol media 17.3 mV Roughly spherical Suspended in MilliQ and sonicated for 2 20s Suspended in medium and sonicated Suspended in MilliQ and sonicated for 2 20s Suspended in medium and sonicated. Suspended in MilliQ and sonicated for 2 20s Suspended in medium and sonicated After synthesis, nano-g Fe2O3 was coated with DMSA (C4S2O4H6) 6 nm 172 m2/g Characterised by TEM, X-ray diffraction and BET method 1, 20, 40 mg/mL, 4 h A549 (human lung epithelial cell line) Karlsson et al. 2008, 2009 A549 (human lung epithelial cell line) Karlsson et al. 2008 1, 20, 40 mg/mL, 4 h A549 (human lung epithelial cell line) Karlsson et al. 2008, 2009 10 6–10 24 h Human dermal ﬁbroblasts Auffan et al. 2006 Comet assay (FPG) Comet assay + 1, 20, 40 mg/mL, 4 h Comet assay (FPG) Comet assay + + Comet assay (FPG) Comet assay 1 g/L, 2 and  For concentration lower than 10 3 g/L, no destabilisation Comet assay Genotoxicity of nanoparticles Iron NPs Iron NP Fe3O4 Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle Physico-chemical characterisation NmDMSA size distribution (PCS) Result of characterisation Magnetite NPs NP diameter Higher, aggregation readily occurred, 2R increased to 70 nm 9.4 nm Iron-platinum (FePt) NPs capped with tetramethylammonium hydroxide (laboratory synthesised) TEM Diameter: 9 nm X-ray diffractometry Standard deviation of size distribution: 11% Crystal structure: face-centered cubic Composition: Fe50Pt50, surface ligand: oleic acid Magnetocrystalline anisotropy energy: 130 kJ/m3 8.5 nm Energy-dispersive X-ray analysis FT-IR CHNS elemental analyser SQUID Magnetite nanoparticles (MNPs) surface coated with polyaspartic acid (PAMF) Silica-coated magnetic NPs containing rhodamine B isothiocyanate (RITC) [MNPs @ SiO2(RITC)] nanoparticles (MNPs) surface coated with PAMF (synthesised) Fe2O3-NP (diameter <100 nm) (Sigma-Aldrich) Average core particle diameter (TEM) NP properties, NP preparation Magnetite NPs were obtained by chemical co-precipitation of Fe (II) and Fe (III) ions in alkaline medium, and then pre-coated with dodecanoic acid followed by an ethoxylated polyalcohol MNPs obtained by coprecipitation of Fe(II) and Fe(III) ions in alkaline medium. Then surface-coated with polyaspartic acid (PA) to obtain a stable PAMF. Size 50 nm All MNPs @ SiO2 (RITC) conﬁrmed by TEM Surface area (BET) Zeta potential and particle size distribution (DLS) Determination of the trace iron elements 34.39 ± 0.17 m2/g 28.68 mV; particle hydrodynamic diameter: ~50 nm Fe2O3 – NP contained only Fe(III) without any trace of Fe(II). The amount of Fe(III) present in 0.4 mg of Fe2O3-NP was equivalent to 935 mM Genotoxicity testing method Treatment conditions MN in vivo Intraperitoneally treated with magnetic ﬂuid containing 5 1015, 5 1016 and 5 1017 particles/kg; MN assay – 24 h after application Ames test 78.1–5000 mg/plate pKM101 ±S9 mix MN in vivo Intravenously treated with 50 mL of the PAMF containing 0.6 1016 or 1.6 1016 particle/mL. MN assay – 1, 7, 15 and 30 days after application Conc. 0.25, 0.5 and 1.0 mg sample/plate ±S9 mix Ames test CHA Concentration 0.25, 0.5 and 1.0 mg/mL Comet assay Fe2O3-NP concentration 2, 5, 10, 50 mg/cm2, exposure time 24 h 8-oxodG (ELISA) Result Cells/organism + Bone marrow cells from female Swiss mice Freitas et al. 2002 S. typhimurium (strains TA98, TA100, A1535, A1537); E. coli WP2uvrA/pKM101 Maenosono et al. 2007 Bone marrow cells from Swiss mice Sadeghiani et al. 2005 S. typhimurium (strains TA97, TA98, TA100, TA102) Kim et al. 2006 Only in TA109 without S9 mix was weakly + + Reference CHL (Chinese hamster lung ﬁbroblasts) + at 10 and 50 mg/cm2 in IMR-90, and in BEAS-2B at 50 mg/cm2 IMR-90 (human lung diploid ﬁbroblasts); SV-40 virustransformed BEAS-2B (human bronchial epithelial cells) IMR-90 human lung diploid ﬁbroblasts Bhattacharya et al. 2009  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Nanoparticle FePt NPs capped with 2-aminoethanethiol (AET) Physico-chemical characterisation Result of characterisation EDX spectral analysis of surface chemistry SEM (morphology) TEM Purely composed of 78.7% Fe and 21.3% O2 Spherical shape Mean diameter, size distribution, crystal structure – 3 nm, 14,5%, face-centered cubic NP properties, NP preparation Synthesised (Kang et al. 2008) XRD Magnetic nanoparticles loaded with daunorubicin (Fe3O4-MNPs/ DNR) Iron oxide NP Fe3O4 Bare and modiﬁed with various fuctional groups (–OH, COOH, NH2) by coating with TEOS, APTMS, TEOS-APTMS:30; citrate: 40 Maghemite (g-Fe2O3) NPs encapsulated within albuminbased nanospheres (magnetic albumin nanospheres-MAN) Average particle sizes and shapes (TEM) Zeta potential (mV) Average diameter (TEM) Bare, (3-aminopropyl) trimethoxysilane (APTMS) and citrate coated: average diameter 10 nm; tetraethyl orthosilicate (TEOS) and TEOSAPTMS coated: average diameter 150 nm Bare: 20; TEOS: 30; APTMS: 25; TEOSAPTMS: 30; citrate: 40 MAN: 73.0 ± 3.0 nm (maghemite NPs:8.9 ± 0.1 nm) Size, morphology and crystallinity (TEM) Zeta potential Magnetocrystalline anisotropy energy at this maghemite particle content is ~1.119 eV6 All ~10 nm; crystalline core with inverse spinel structure Fe3O4-MNPs prepared by chemical coprecipitation; loaded with DNR and albumin (for details see Wu et al. 2010) Synthesised (see the protocol) MN in vivo Comet assay Result 0.1, 0.5 or 1.0 mg/mL of NP suspension. Presence or absence S9 mix False positive? 1.25, 2.50, 3.75 and 5.00 g/kg of Fe3O4-MNPs/DNR intraperitoneal injection, twice in an interval between of 24 h 100, 200, 1000 ppm, for 24 h / no damage for bare and TEOS coated Fe3O4 Cells/organism S. typhimurium (strains TA98, TA100, TA1535, TA1537); E.coli WP2uvrACHL/IU (Chinese hamster lung ﬁbroblasts) Bone marrow from kunming mice Reference Maenosono et al. 2009 Wu et al. 2010 L-929 (murine ﬁbroblast line from subcutaneous connective tissue) Hong et al. 2011 Bone marrow erythrocytes from female Swiss mice Estevanato et al. 2011 MCL5 (human lymphoblastoid cell line) Singh et al. 2012 + for APTMS, TEOS-APTMS and citrate coated Fe3O4 For preparation see the protocol MN in vivo Mice were intraperitoneally treated with 100 mL of NP suspension for 24 and 48 h, 7, 15 and 30 days CB MN 1–100 mg/mL, for 24 h in 1% serumcontaining medium Lyophilised MAN dispersed in FBS to obtain an aqueous suspension (5 mg MAN/mL) Only dextrancoated Fe2O3 was + Genotoxicity of nanoparticles Uncoated Fe2O3 NPs, dextrancoated Fe2O3 NPs, Ames test Treatment conditions CHA ~25% in mass and~7% in volume fraction Maghemite content in the MAN sample Genotoxicity testing method  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle uncoated Fe3O4 NPs, dextrancoated Fe3O4 NPs (Liquids Research, UK) Magnetite NPs, 4 sizes: NPs (20– 60 nm; SigmaAldrich, Germany), alveolar fraction (0.5–1.0 mm), respirable fraction (2–3 mm) Bulk magnetite (0.2–10 mm; Alfa Aesar, Germany) (bulk magnetite used to generate respirable fraction and alveolar fraction) Silica-free and ﬂuorescent silica-coated cobalt ferrite (CoFe2O4) NPs Co NPs, CoCr NPs CoCr nanoparticles comparation to micron-sized CoCr (Osprey metals Ltd.) Physico-chemical characterisation Result of characterisation NP properties, NP preparation Genotoxicity testing method For all between -13.9 and 3.3 mV Hydrodynamic particle size in the presence of 1% vs. 10% serum media or water (DLS) Oxidation state of Fe (X-ray photoelectron spectroscopy) See the results for details Size and shape (SEM) See the results for details 8-oxoG, TG, 5-OH-5MeHyd, FapyG, FapyA (GC-MS) Suspended in FBSfree medium with 1% pen/strep; bath sonicated for 20 min at 40 C; diluted in FBS-free medium XRD CB MN Treatment conditions Kinetochore staining (2–100 mg/mL dextran-coated Fe2O3) Pretreatment with 2 mM NAC (4 and 50 mg/mL dextran-coated Fe2O3) 2 and 4 mg/mL dextran-coated Fe2O3, for 24 h in 1% serum-containing medium 1, 10, 50, 100 mg/cm2, for 24 h; ROS scavenger – NAC pretreatment Result Cells/organism + increased level of TG, 8-oxoG, FapyG, FapyA + for all four types; NAC decreased MN formation A549 (human lung epithelial cell line) Könczöl et al. 2011 Hwang do et al. 2012 Comet assay 1, 10, 50, 100 mg/cm2, for 4 h; ROS scavengers – NAC and BHA pretreatment + for all 4 types; NAC and BHA decreased level of DNA damage mRNA expression (52 genes, including genes for DNA damage and repair) For in vitro: 100 mm, for 24 h Core NPs were severely genotoxic to liver tissue; ﬂuorescence NPs showed gene expression 90% similar to untreated liver samples Hep3B cells (in vitro) + More damage in NPs than in MPs + No signiﬁcant difference between nano and micro Primary human dermal ﬁbroblasts Chemical composition (atomic absorption spectroscopy) Reference Increased ratio of Kinetochor positive MN NAC reduced MN frequency Size distribution Commercially designed by Biterials; NPs synthesis: see the protocol For in vivo: injected into mice via tail vein, 24 h exposure Size NP 29.5 ± 6.3 nm Shape Oval (micron sized 2.904 ± 1.064 mm) CoCr NPs of alloy generated by a ﬂat pin-on-plate tribometer were characterised for their size and morphology Comet assay Five days in tissue culture MN 12 h exposure 8-oxodG (immunostaining) 50–5000 mm3/cell for 3 and 24 h In the contrary MPs induced MN and liver tissue from mice (in vivo) Papageorgiou et al. 2007  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Nanoparticle Physico-chemical characterisation Result of characterisation Co NP and washed Co NP, compared to Co 2+ (University of Modena, Italy) Analysis of elemental impurity (HPLC-ICPMS ELAN-DRCII) 98.83% of purity Source of Co2+ – cobalt chloride (Alfa) Size of CoNP in water and complete culture medium (DLS and SEM) CoNP size distribution in water Polydispersed aggregates 100–500 nm with median value 246 mn Co NP (University of Modena, Italy) Characteristics in water and medium (SEM) Aggregation in water and medium (compared to Co2+) Size distribution (tracking analysis) Release of Co2+ in medium after 2, 24, 48 h incubation (g-counter) Size (TEM) 20–500 nm (peak at 80 nm) Time-dependent increase Co NPs (FUNSOM, China); Source of Co2+ – cobalt chloride (Sigma-Aldrich) CuO NPs CuO NP (Sigma-Aldrich) Particle size in powder 42 nm Surface area in powder TEM DLS (size of particles and agglomerates in medium) Zeta potential 23 m2/g Suspended in MilliQ water, ultrasonicated for15 min, diluted to with complete culture medium Morphology of CoNP in water and complete culture medium were characterised by SEM technique and by NP tracking analysis NP suspended in water, washed by centrifugation (to remove Co 2+), ultrasonicated for 15 min, diluted in medium Genotoxicity testing method Treatment conditions CB MN Concentration 10 3–10 6 Comet assay 2 h, concentration 10 5, 5 10 5, 10 Comet assay NP heat sterilised (180 C, 4 h), suspended in water at 100 mM; sonicated intermittently 6 for 2 min; freshly diluted in medium Comet assay Suspended in MilliQ and sonicated for 2 20 sec Suspended in medium and sonicated Comet assay Suspended in MilliQ and sonicated for 2 20 sec Suspended in medium and sonicated Comet assay Result Cells/organism At non-cytotoxic concentration tested, no genotoxic effects 4 + at ‡5 10 Balb/3T3 (mouse ﬁbroblast line) Ponti et al. 2009 T cells from human peripheral blood Jiang et al. 2012 A549 (human lung epithelial cell line) Karlsson et al. 2008, 2009 A549 (human lung epithelial cell line) Karlsson et al. 2008 5 + + Co NPs 1–6 mM, for 4 h + at 3 and 6 mM (Co2+ 10–30 mM, for 4 h) (Co2+ did not induce DNA damage) Comet assay (FPG) Colognato et al. 2008 M Concentration 1, 3, 5 mM (subroxic concentration, interpolated from CFE assay), 2 h exposure Concentration 1, 3, 5 mM (subroxic concentration, interpolated from CFE assay), 24 h exposure 2, 40, 80 mg/mL, 4 h Reference Human peripheral blood leukocytes + + 20–40 nm 220 nm 31 mV Particle size in powder 71 nm Surface area in powder 15 m2/g Comet assay (FPG) 1, 20, 40 mg/mL, 4 h + + Genotoxicity of nanoparticles Zinc (ZnO) NPs ZnO (SigmaAldrich) 30–70 nm, with a median size of 50 nm NP properties, NP preparation  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle Zinc dioxide NP (Nanuo Co. Ltd., Shenzhen) ZnO NP (SigmaAldrich) ZnO NP (1314-13-2 SigmaAldrich) ZnO NP (SigmaAldrich, Steinheim, Germany) vs. ZnO powder (<5 mm) Physico-chemical characterisation TEM DLS (size of particles and agglomerates in medium) Zeta potential Size (TEM) Shape (TEM) Chemical composition (purity) (Raman spectroscopy) Hydrodynamic diameter (DLS) Zeta potential (DLS) Average size (TEM) (of NP in suspension) Size (DLS) at different concentrations and timepoints Morphology (TEM) Mean diameter (TEM) Intracellular distribution (TEM) Mean diameter of aggregates Zeta potential Purity Release of Zn2+ ions Result of characterisation NP properties, NP preparation Genotoxicity testing method Treatment conditions Result Cells/organism + Primary mouse embryo ﬁbroblasts Yang et al. 2009 A431 (human skin cells) Sharma et al. 2009 HEp2 (human negroid cervix carcinoma cell line) Osman et al. 2010 Human nasal mucosa cells (10 donors) Hackenberg et al. 2011a Reference 20–200 nm 320 nm 26.9 mV 19.6 ± 5.8 nm crystal structure ZnO >99.9% 165 nm 26 mV 30 nm Particles widely distributed. Considerably increased size as a function of concentration, minimally increased size as function of time (see the table, Osman et al. 2010) Mainly rod-shaped and partially spherical Longitudinal 86 ± 41 nm and lateral 42 ± 21 nm, in speciﬁc cases aggregates up to 210 104 nm Intracytoplasmatic ZnO-NPs were in 10% of cells. Transfer into nucleus observed in 1.5% of cells 353 nm NP sonication six times intermittently (30 sec every 2 min) Comet assay 5 and 10 mg/mL for 24 h (purity >99%) Comet assay 0.001, 0.008, 0.08, 0.8, 5 mg/mL for 6 h Comet assay 10, 20, 50, 100 mg/mL, for 4 h 10, 20, 50, 100 mg/mL, for 2 h Suspended in Milli-Q water at a concentration of 80 mg/mL, sonicated at 30 W for 10 min Suspended in EMEM-EBSS medium, probe-sonicated 30 W, 5 min (<100 nm, surface area 15–25 m2/g) CB MN Comet assay Concentration 0.01, 0.1, 5, 10 and 50 mg/mL + at 0.8 and 5 mg/mL, dose dependent + + + at ‡10 mg/mL for ZnO powder NPs suspended in sterile dH2O, sonicated 60s at 4.2 105 kJ/m3 using a continuous mode. BSA added for stabilisation. 10 PBS added. This stock diluted in BEGM 14.7 mV Above 99% See table, Hackenberg et al. 2011a Comet assay +  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Nanoparticle ZnO NPs (SigmaAldrich, Germany) Physico-chemical characterisation Result of characterisation NP properties, NP preparation Morphology, diameter (TEM) Oval shape with a mean longitudinal diameter of 76 nm, mean lateral diameter 53 nm Mean 354 nm (range from 190 to 1106 nm) (<100 nm, surface area 15–25 m2/g, purity >99%) Aggregates (DLS) Zeta potential Concentration of Zn2+ ions in supernatant cell culture medium ZnO NPs (Top Nano Technology Co., Ltd., Taiwan) Morphology, size, agglomeration (TEM, DLS) (vs. ZnO microparticles – MPs) ZnO NPs (ZncoxTM 10, IBUtec) 2.8 and 52.7 mmol/mL after incubation with 0.1 and 5 mg/mL NPs, respectively NPs: thin-slice shaped; diameter ~50 nm; primary particles were dominant; hydrodynamic diameter 93.35 nm MPs: compacted crystals, at least 1 dimension >100 nm; hydrodynamic diameter 1226.2 nm (>91.6%), lesser percentage of smaller particles Minimal characterisation provided by manufacturer see the results (Pierscionek et al. 2010) Treatment conditions NPs suspended in water; sonicated 120 sec at 4.2 105 kJ/m3; added BSA and 10 PBS; diluted in medium 211.2 Mv Particle stock suspension prepared using DMSO (ﬁnal DMSO concentration <1% in vivo, <5% in vitro); diluted with water + 1% hydroxypropyl methyl cellulose or medium, mixed, sonicated, immediately applied Result 0.1 and 5 mg/mL; 1, 2 and 3 consecutive 1-h periods and 24-h regeneration period Cells/organism Reference 3D mini organ cultures of human nasal mucosa from 10 patients Hackenberg et al. 2011d for NPs and MPs Peripheral blood of Crl: CD-1 (ICR) mice Li et al. 2011c for NPs and MPs S. typhimurium (strains TA102, TA100, TA1537, TA98, TA1535) (level of damage increased after 24 h regeneration period) MN in vivo Ames test 1.25, 2.5, 5.0 g/kg bw; oral administration by intragastric gavage; 24, 48, 72 h exposure +/- S9 mix NPs prepared freshly; stock suspensions sonicated in a bath 10 min; serially diluted in water FADU 0.4–160 mg/mL, for 5, 30 and 180 min Synthesised (see the protocol) SCE Sonicated (ultrasonic bath) 10 min Comet assay 52.7–70.9 nm (average 60 nm) MN in vivo + A549 (human lung epithelial cell line) MorenoVillanueva et al. 2011 5 and 10 mg/mL, for 24 h Human lens epithelial cell line Pierscionek et al. 2010 Doses (0, 30, 300 and 1000 mg/kg/day), oral exposure, Bone marrow, Sprague–Dawley male and female rats Kim et al. 2008 Genotoxicity of nanoparticles Cerium oxide (CeO2) NPs Identity, crystallinity, CeO2 (cerium oxide, nanoceria) crystalline structure, size, shape (highresolution TEM) Emission and absorption spectra (ﬂuorescence and UV vis spectrophotometer) Silver (Ag) NPs AgNP (Namatech Co., Ltd., Korea) Genotoxicity testing method  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle Physico-chemical characterisation Result of characterisation 25 nm Coated (polysaccharide surface functionalised) Ag NPs (Dr. Dan Goia, clarkson university, NY) Characterisation was previously reported (Murdock et al. 2007; Schrand et al. 2008) Uncoated (nonfunctionalised Ag NPs (Dr.Karl Martin, Novacentrix, Austin) Characterisation – was previously reported (Murdock et al. 2007; Schrand et al. 2008) AgNP TEM Between 20 and 50 nm TEM Effect of adding CPB on particle aggregation Size (TEM and UV absorption spectrum) 6–20 nm 25 nm NP properties, NP preparation Purity >99.98% Polysaccharide-coated AgNPs – more distributed Uncoated AgNPs tend to agglomerate Genotoxicity testing method Immunoblot: Treatment conditions 28 days 50 mg/mL for 4, 24, 48 and 72 h p53 protein Phospho-p53 (ser15) Rad51 Phospho-H2AXSer-139 Immunoblot: 50 mg/mL for 4, 24, 48 and 72 h p53 protein Phospho-p53 (ser15) Rad51 Phospho-H2AX-Ser139 RLS and TEM Result Cells/organism Reference Coated more severe damage than uncoated Ag NPs Mouse embryonic stem cells Mouse embryonic ﬁbroblasts Ahamed et al. 2008 Coated more severe damage than uncoated AgNPs Mouse embryonic stem cells Ahamed et al. 2008 Mouse embryonic ﬁbroblasts nanoag, 3.3 10 6 g/mL Weak Calf thymus DNA Chi et al. 2009 AgNO3 (Sinopharm Chemical Reagent Co) nano Ag–CPB AgNO3 (Sinopharm Chemical Reagent Co) Ag NP – starch capped (synthesised, all chemicals purchased from Sigma-Aldrich) AgNPs coated with 0.2% polyvinyl pyrrolidone (NanoAmor, USA) Ag NP powder (PW-XRD) Stock solution (MilliQ) (TEM) Stock solution (MilliQ) (DLS) RPMI 1640 media + 1% FBS (DLS) (24 h at 37 C) Zeta potential Morphology (TEM) 78.1 nm 69 ± 3 nm Spherical, multifaceted or slightly elongated shapes 121 ± 6 nm 21.8 mV 149 ± 37 nm 11.6 mV RLS and TEM nanoag, 3.3 10 6 g/mL CPB 6.0 10 6 g/mL + Calf thymus DNA Chi et al. 2009 Dissolved in water using sonication and then in culture medium Comet assay Conc. 25 – 400 mg, 48 h + Asharani et al. 2009 CB MN Conc. 100 – 200 mg, 48 h + IMR-90 (human lung diploid ﬁbroblasts) and U251 (human glioblastoma cells) According to the manufacturer: spherical shape, 30–50 nm in size. The purity (ICP), and metal contaminants <0.1% Ag NP solution prepared in ddH2O by sonication, centrifugation and ﬁltration (Foldbjerg et al. 2009) (<50 nm) DNA adducts Ag NPs concentration 0–15 mg/mL, for 24 h antioxidant NAC (10 mM) pretreatment + A549 (human lung epithelial cell line) Foldbjerg et al. 2011 32 P postlabelling Comet assay NAC decreased DNA adducts formation + at ‡0.1 mg/mL  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Nanoparticle Physico-chemical characterisation Result of characterisation Mean diameter (TEM) 46 ± 21 nm, usually found in aggregates Intracellular distribution Ag NPs NPs suspended in sterile dH2O, sonicated 60s at 4.2 105 kJ/ m3 using a continuous Small aggregates in cellular compartments as well as free in the cytoplasm; transfer into nucleus observed Mean diameter of aggregates (TEM) 404 nm Individual NP size (TEM) 5–10 nm Agglomerates (DLS) 28–35 nm NP dispersed in tetrahydrofuran, sonicated 3 h (to volatilise THF), stirred 3 days, reﬁlled by H2O and ﬁltered CHA Treatment conditions Concentration 0.01, 0.1, 1 and 10 mg/mL, for 1, 3 and 24 h Concentration 0.01, 0.1, 1 and 10 mg/mL, for 1 h Result Cells/organism Reference Human mesenchymal stem cells Hackenberg et al. 2011b Jurkat T cells Eom & Choi 2010 Human peripheral blood cells Flower et al. 2012 S. typhimurium (strains TA102, TA100, TA1537, TA98, TA1535) Li et al. 2012 + at ‡0.1 mg/mL mode. BSA added for stabilisation. 10 PBS added. This stock diluted in DMEM 13.6 mV Shape Size (TEM) Structure and phase purity (XRD) Ag NPs (Novacentrix, Austin, TX, USA) Genotoxicity testing method Spherical and fractionally elongated Ag NP (Sigma– Aldrich, Steinheim, Germany) Zeta potential of NPs suspension AgNP (and Ag ions) NP properties, NP preparation Size (TEM) UV–visible spectroscopy Up to 30 nm 61.2 ± 1.6 nm 1608.7 ± 175.4 Synthesised by reduction of AgNO3 using NaBH4 Different concentrations of NPs dispersed in 100 mL water by vortexing for 5 min and sonicating for 5 min 0.2 mg/L for 4, 12 and 24 h Expression of pH2AX protein Comet assay 50 and 100 mg/mL for 5 min and 3 h + for Ag NP; (or very low) for Ag ions + for Ag NP; for Ag ions + + Ames test MN 50 and 100 mg/mL for 5 min together with 250 mM of H2O2 0.15–76.8 mg/plate – S9 mix 10–30 mg/mL + at 25 and 30 mg/mL TK6 (human lymphoblastoid cell line) 9.37 mV 8.20 mV Absorbance maximum at 450 nm, a narrow peak width at half maximum Comet assay Asare et al. 2012 Genotoxicity of nanoparticles Agglomeration sizes in the water Size in water Size in culture medium surface. Charge in water surface. charge in media Zeta potential: More details in Eom & Choi 2010 Spherical, slightly agglomerated 40–60 nm Crystalline, face-centered cubic structure of pure Ag 4–12 nm Comet assay  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle Ag NPs – 20 nm vs. submicron 200 nm Ag particles (Plasmachem GmbH, Germany) Physico-chemical characterisation Result of characterisation NP properties, NP preparation Hydrodynamic sizes of dispersion (DLS) Mean diameter Ag20: 154.6 nm Mean diameter Ag200: 266.2 nm NPs dispersed in dH2O/10 BSA/10 PBS in 8:1:1 ratio of 2 mg/mL stocks, freshly prepared before each exposure Genotoxicity testing method 12.5, 50 and100 mg/mL, for 24 h Comet assay (FPG) Ag NPs (Sigma-Aldrich, USA) SEM and TEM analysis Single particle size: 100 nm or less; Kim et al. 2011 Size distribution (DLS) Ag NPbased hydrogel (clinically available, Egeta Co., China) Ag NPs (20 nm; Plasmachem GmbH, Germany) from 43 to 260 nm: spherical aggregates, about 58.9 nm in size Size distribution (TEM) 3–5 nm, 47.9%; 5–10 nm, 50.8%; 10–30 nm, 1.3% Mean hydrodynamic diameter in water and DMEM (DLS) In water: 313.9 and 3760 nm Zeta potential of NPs suspension Morphology (SEM, TEM) In DMEM: 33.9 nm, 225.9 nm and 4050 nm; only slight shift in size after 30 min incubation (see table for details) In water (without BSA and PBS supplement): 37.4 ± 2.5 mV (cellulose membrane, pore size 200 nm), serially diluted Homogenously dispersed in medium by sonication (30 min), ﬁltered Comet assay (FPG and EndoIII) CB MN NP dispersed in water, vortexed, sonicated 3 min, 100 W on ice; PBS with ﬁnal 1.5% BSA added. Stock solution added to medium with 10% FBS Treatment conditions 12.5, 50 and100 mg/mL, for 24 h MN and CB MN Result Cells/organism Ag 200 nm caused higher level of damage compared to Ag 20 nm in NT2 cells Ntera2 (NT2, human testicular embryonic carcinoma) cells, primary testicular cells from C57BL6 mice of WT and 8-oxoG DNA glycosylase (Ogg1) knock-out cells or very little damage in testicular cells (WT and KO-Ogg1 / ) /no increase 0.01–10 mg/mL, for 24 h Comet assay + + Reference BEAS-2B (human bronchial epithelial cells) + Co-treatment ± scavengers (Man 0.5 mM, SS 1 mg/mL, CAT 2 U/mL and SOD 30 U/mL) in the CBMN and comet assay 20, 40 and 60 mg/mL, for 24 h Genotox effect decreased by scavengers, most effectively by SOD + HeLa (human epithelial cell line) Xu et al. 2012 HEK 293 (human kidney cell line) Hudecová et al. 2012b Comet assay 1, 25, 100 mg/mL, for 30 min + Comet assay (hOGG1) 1, 25, 100 mg/mL, for 30 min + 24 h pretreatment with plant extract (containing antioxidants such as swertiamarin, mangiferin, homoorientin) Oxidative DNA damage diminished by Gentiana asclepiadea extract In water (with BSA and PBS supplement): 26.7 ± 0.8 mV Small agglomerates, average size ~100 nm Size 20–100 nm Gold (Au) NPs DLS analysis Comet assay After 3 h instillation ± Jacobsen et al. 2009  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Nanoparticle Physico-chemical characterisation Nanosized gold particles Result of characterisation NP properties, NP preparation Genotoxicity testing method Treatment conditions Result 2 nm gold NP formed stable agglomerates 40–200 nm 54 mg/mouse Gold NPs stabilised by citrate ions Wang et al. 2011a A series of four AuNPs (quarternary ammonium functionality with varied (C1-C6) hydrophobic alkyl tail) AuNPs coated with cerium [CeL]+ and luminescent europium [EuL] Stability after light irradiation and mixing with phosphate buffer (UV–Vis absorption spectra and TEM) NPs stable. Absorption peak at 522 nm, no change. 16 nm diameter nanospheres, same size and shape (for detailed characterisation see in Lewis et al. 2010) AuNPs (20 nm in diameter) Primary particle size: Citrate stabilised Au NPs – 3 sizes: 10, 30 and 60 nm (NIST Standard Reference Materials Program) Pt NPs Pt NPs functionalised with polyvinyl alcohol 2, 20, 200 nm See table in material and methods for detailed properties DLS Size distribution (TEM) Metal weight % No aggregation of NPs in media during 24 h (see results) 5–8 nm 5.9% Reference Synthesised by citrate reduction method Ames test 2 nm core; synthesised via place exchange of pentanethiol capped AuNPs fabricated by BrustSchrifrin reduction method Comet assay 100, 123, 148 and 165 nM for AuNP 1–4, respectively (calculated concentration yielding 214 nm/well of intracellular gold); for 24 h + for all four AuNPs; level of DNA damage depends on hydrophobicity of the ligands HeLa (human epithelial cell line) Chompoosor et al. 2010 (preparation details described in Lewis et al. 2010) gH2AX for [EuL]Au with/ without pEGFP-F + for [CeL]+Au with/without pEGFP-F MRC5VA (human epithelial lung ﬁbroblasts) Lewis et al. 2010 Prepared in citrate reduction from gold salts; spun down to remove the citrate; added FBS, washed; stock in PBS; sterile ﬁltered; added into media NP suspensions Comet assay CHA FISH 2D gel electrophoresis (expression of repair protein) 0.1 nM, with or without pEGFP-F plasmid vector, for 48 h, NPs transfected to cells with Fugene transfection agent 1 nM, for 72 h + + Downregulation MRC5 (human fetal lung ﬁbroblasts) Li et al. 2011a Single intratracheal instillation of 500 ml per animal (18 mg NP per lung), 3 days exposure Lung cells of male Wistar rats Bone marrow cells of male Wistar rats Schulz et al. 2012 0.0002–0.2 mg/mL, for 3 and 24 h HepG2 (human liver carcinoma cell line) Nelson et al. 2011 adjusted with saline to 36 mg/mL, vortexed; no agglomeration observed with the unaided eye NPs either utilised as received or diluted in water and utilised as diluted solutions Diluted NPs preincubated for 16–24 h prior to addition to cells NPs synthesised (see the protocol) Comet assay in vivo MN in vivo 8-oxoG, 8-oxoA, R-cdA, S-cdA (LC-MS/MS) S. typhimurium TA102 Calf thymus DNA Comet assay CB MN 10–160 mg/mL, for 48 h 10–160 mg/mL, for 48 h + + IMR-90 (human lung diploid ﬁbroblasts +A610) and U251 Asharani et al. 2010 Genotoxicity of nanoparticles AuNPs (gold colloid suspensions in water; 2, 20 and 200 nm) (British Biocell International, Cardiff, UK) 5 mg/plate; with or without light irradiation; 48 h incubation after 15 min irradiation Cells/organism BAL cells (broncho-alveolar lavage ﬂuid obtained from apolipoprotein E knockout mouse, ApoE / ) Gold NP solution is not mutagenic but photomutagenic due to citrate and Au3+  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle PtNPs coated with cerium [CeL]+ and luminescent europium [EuL] Physico-chemical characterisation UV-spectroscopy (EDX spectrum) (for detailed characterisation, see in Lewis et al. 2010) Result of characterisation NP properties, NP preparation CdTe QDs stabilised by mercaptopropionic acid (MPA) (Institute of Macromolecular Science, Fudan University) CdSe QDs and CdSe QDs doped with 1% cobalt ions, surface modiﬁed using mercaptoacetic acid (Preparation details described in Lewis et al. 2010) gH2AX 0.1 nM, with or without pEGFP-F plasmid vector, for 48 h, NPs transfected to cells with Fugene transfection agent Gel electrophoresis (plasmid nicking assay) Incubation with supercoiled dsDNA, 0–60 min, in 15-min intervals) Comet assay It was not possible to obtain acceptable DLS data lem = 664 Mean diameters 3.7 nm g-H2AX (ﬂuorescent microscopy and ﬂow cytometry) Surface coupled with MPA for water solubilisation and suspended in ddH2O Size and shape (TEM) Hydrodynamic diameter; DLS PDI Zeta potential Optical properties Magnetic measurement Rare metal and metal oxide NPs Size distribution of Indium oxide NP (DLS) (In2O3), dysprosium oxide (Dy2O3), tungsten oxide (WO3) and molybdenum (Mo) NP and microSize distribution particles (MP) of NP (Sigma-Aldrich, Purity USA) Diameter Treatment conditions Result Cells/organism Reference typical hyperbolic curve Quantum dots (QDs) CdSe/ZnS QDs (CdSe QDs capped with ZnS shell) (commercially available) QDs negatively (ADS620QD) and positively charged (ADS621QD) CdTe QDs Genotoxicity testing method Dot-like shape, uniform in size and shape; size 5.1 ± 0.2 nm 48 nm (pure), 58 nm (doped) Dy2O3: 85.8–1132.3 nm In2O3: 85.8–2881.3 nm WO3: 106.5–1134.1 nm; Mo: 85.8–442.2 nm More than 7 mm See table in results for 1, 10 and 50 mg/mL, for 12 h Antioxidant N-acetyl-lcysteine (NAC) 6 mM pretreatment 50 mg/mL of CdTe QDs, for 6 h Preparation of QDs: see the protocol MN in vivo QDs suspended in ultrapure bidistilled water–Tween 80 mixture DNA fragmentation 0.0689 (pure), 0.0408 (doped); 25.7 mV (pure) 37.4 mV (doped) See the results for details In the dark and under UV excitation After 3 h instillation 54 mg/mouse 500, 1000, 2000 mg/kg bw; oral administration; 2 and 7 days treatment HPLC (8-oxoG and 2-dG) Suspended in ultrapure water at 0.2 mg/mL, sonicated 5 min, ﬁltered Ames test 20–80 mg/plate; presence or absence S9 mix Cell transformation assay 0.01–5 mg/mL MRC5VA human epithelial lung ﬁbroblasts Lewis et al. 2010 + Supercoiled double strands of DNA Green et al. 2005 + BAL cells (bronchoalveolar lavage ﬂuid obtained from apolipoprotein E knockout mouse, ApoE / ) HUVEC Jacobsen et al. 2009 + Bone marrow cells of albino mice Khalil et al. 2011 + Liver cells of albino mice + Liver cells of albino mice for [EuL]Pt with/ without pEGFP-F + for [CeL]+Pt with/ without pEGFP-F + Wang et al. 2010 NAC decreased lH2AX foci formation Dy2O3: +NP, +MP; In2O3: +NP, MP; WO3: +NP, MP; Mo: NP, MP Dy2O3: +NP, +MP; In2O3: +NP, +MP; WO3: NP, S. typhimurium (strains TA98, TA100, TA1535, TA1537 (E. coli WP2uvrA Bhas 42 cells (v-Ha-ras-transfected BALB/c 3T3 cells) Hasegawa et al. 2012  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Nanoparticle Physico-chemical characterisation Result of characterisation Mean diameter in sol Zeta potential Primary crystallite size Surface area Impurities detailed characteristics of NP and MP Alumina (Al2O3) NPs Al2O3 NP Shape (TEM) (Sigma-Aldrich) Silica NP Crystalline UFSiO2 (Sigma-Aldrich) Ultraﬁne quartz (Min-U-Sil 5 silica; Silica Corp. Berkeley Springs) SiO2 NPs (Runhe Co. Ltd., Shanghai) Size distribution (TEM) Speciﬁc surface area (BET) average particle sizes 28 ± 19 nm 39 m2/g Particle size distribution in the ﬁnal extract (HPPS) HPPS: by volume 7.21 nm (100%), by intensity: 9.08 nm (71.4%) and 123.21 nm (28.6%) For details see Wang et al. 2007b Size (TEM) Shape (TEM) Chemical composition (purity) (Raman spectroscopy) Size 20.2 ± 6.4 nm Crystal structure SiO2 >99.0% 50 ± 3 nm in diameter Genotoxicity testing method Treatment conditions Result Cells/organism Reference MP; Mo: NP, MP SCE 1–25 mg/cm2, for 2 cellular cycles - CB MN 0.5–10 mg/cm2, for 24 h + at 0.5–10 mg/mL 99% purity CB MN 6, 24 and 48 h with 0– 120 mg/mL UFSiO2 + at ‡30 mg/mL, 24-h treatment Suspended in culture medium, sonicated. In the ﬁnal extract was measured particle size distriburtion by the HPPS 98%; <5 mm diameter; purity 99.5% a-quartz by X-ray diffraction Suspended in media, vortexed, sonicated; centrifuged; supernatant ﬁltered; stored – 20 C NP sonication six times intermittently (30 sec every 2 min) Comet assay NP stock suspensions were prepared in PBS, vortexed for 10 min and stored at 4 C in the dark Laboratory synthesised (using a reverse mictoemulsion method), resuspended in PBS Di Virgilio et al. 2010 WIL2-NS cells (human B cell lymphoblasts) Wang et al. 2007c WIL2-NS cells (human B cell lymphoblasts) Wang et al. 2007b Primary mouse embryo ﬁbroblasts Yang et al. 2009 A549 (human lung epithelial cell line) Jin et al. 2007 + at 120 mg/mL 24 h treatment HPRT gene mutation CB MN CHO-K1 (CHO cell line) 0, 60 and 120 mg/mL, for 10 h + at 120 mg/mL Comet assay HPRT gene mutation + (dose dependent) Comet assay 5 and 10 mg/mL for 24 h Comet assay 48, 72 h, concentration 10 4–0.5 mg/mL PFGE 72 h, concentration 10 4–0.5 mg/mL 72 h, concentration 10 4–0.5 mg/mL 48, 72 h, 10 4– 0.5 mg/mL DNA adducts Agarose gel electrophoresis + - Genotoxicity of nanoparticles TMR- and RuBpy-doped luminescent silica NP (laboratory synthesised) Spherical NP properties, NP preparation  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle Glantreo (30,80, 400 nm) SigmaLudox CL 420883, SigmaLudox CLX 420891 Aerosolised amorphous silica (AS) NPs AS Four sizes, (Si498, nano-Si68, nanoSi43 and nanoSi19) (Jilin University) AS three sizes (16, 60 and 104 nm) Physico-chemical characterisation Nanoparticle dispersions (DLS in deionised water, TEM from suspensions in water, buffer, cell culture media before drying, zeta potential, pH, PDI) before and after dialysis (see the table in Barnes et al. 2008) Two particle sizes (detailed characterisation see Sayes et al. 2010) Shape, size and size distribution (TEM) Zeta potential, hydrodynamic sizes (DLS) in water and media after 10 min and 24 h Characterisation and synthesis method see Gonzalez et al. 2010 Result of characterisation NP properties, NP preparation Genotoxicity testing method DNA repair enzyme activity assay Comet assay See the table in Barnes et al. 2008 DLS analysis in water and medium SEM image analysis in water Result 48, 72 h, concentration 10 4–0.5 mg/mL 3, 6, 24 h Cells/organism Reference 3T3-L1 (mouse ﬁbroblast line) Barnes et al. 2008 – Reticulocytes (peripheral blood) of male rats Sayes et al. 2010 + for all 4 particles, higher damage with decreasing size HepG2 (human liver carcinoma cell line) Li et al. 2011b Weak, not signiﬁcant (16 nm SiNPs induced more MN) Weak, not signiﬁcant A549 (human lung epithelial cell line) Gonzalez et al. 2010 Balb/3T3 (mouse ﬁbroblast line) clone A31-1-1 Uboldi et al. 2012 BEAS-2B (human bronchial epithelial cells) Gurr et al. 2005 BEAS-2B (human bronchial epithelial cells) Gurr et al. 2005 4 or 40 mg/mL 37 and 83 nm De novo synthesised aerosolised AS NPs produced in NP reactor MN in vivo (ﬂow cytometry) For results see table Particles Comet assay 3.7 107, 1.8 108 particles/cm3, 1 and 3 day inhalation exposures (measurement at 24 h postexposure) 100 mg/mL, for 24 h were dispersed by sonicator and diluted to DMEM before use CBMN and FISHcentromeric probing; frequency of mitotic errors Comet assay (FPG) AS NPs (5 types – unlabelled; origin: 2 of them OECD list of interest, 3 of them lab synthesised-JRC) TiO2 NP TiO2 anatase 10 nm (Hombikat UV100) and 20 nm (Millennium PC500) TiO2 anatase ‡200 nm (Aldrich-Sigma, cat. no. T8141) Treatment conditions CBMN: 10–60 mg/mL of 16 and 60 nm SiNPs; 40–330 mg/mL of 104 nm SiNPs; for 40 h FISH: 16 and 60 nm SiNPs, 40 and 60 mg/mL, for 40 h 16 and 60 nm SiNPs, 40 and 60 mg/mL, for 15 min and 4 h 100 mg/mL, 24 h NP diameters 15– 300 nm. See the table in results Two of them in powder form, four of them lab syntetised (see the protocol for details) CB MN Cell transformation assay 100 mg/mL, 72 h Aggregations of 1000 nm in diameter 10 and 20 nm Comet assay (FPG) Suspended in PBS MN Concentration Of TiO2 10 mg/mL, 1 h, in darkness ‡200 nm Comet assay (FPG) Suspended in PBS MN No aggregation Comet assay (FPG) Concentration of TiO2 10 mg/mL, 1 h, in darkness Weak, not signiﬁcant + + Gurr et al. 2005  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Nanoparticle TiO2 anatase 200 nm (Kanto Chemical, cat. nos. 40167-01) TiO2 rutile 200 nm (Kanto Chemical, cat. nos. 40982-30 Suspended in PBS TiO2 NPs (Aldrich-Sigma) TiO2 NPs (DuPont) Physico-chemical characterisation Genotoxicity testing method Suspended in PBS No aggregation Treatment conditions MN + 200 nm Comet assay (FPG) Comet assay 6, 24 and 48 h with 0, 26, 65 and 130 mg/mL UFTiO2 Gurr et al. 2005 MN Particle size distriburtion in the ﬁnal extract measured by the HPPS HPPS: by volume 6.57 nm (100%), by intensity: 8.2 nm (80.4%) and 196.52 nm (19.4%) Crystalline structure (XRD) 79% rutile, 21% anatase BET surface area analysis Chemical composition/ purity (X-ray ﬂuorescence) Size and distribution in water (DLS) 38.5 m2/g 90 wt% TiO2, 7% alumina and 1% AS Size 140 ± 44 nm (aggeregation: in water – negative) Chemical reactivity delta ba: 0.9 15–60 nm Particle sizes (TEM, XRD, XDC) See the table (Theogaraj et al. 2007) 99% purity CB MN Sonicated and suspended in culture medium Characterised in its dry native state (crystallinity and surface area) and in water and buffered solutions (size, size distribution, pH and chemical reactivity) Result Conc. TiO2 10 mg/mL, 1 h, in darkness Conc. TiO2 10 mg/mL, 1 h, in darkness + Cells/organism Reference BEAS-2B (human bronchial epithelial cells) + BEAS-2B (human bronchial epithelial cells) WIL2-NS (human lymphoblastoid cells) Wang et al. 2007a S. Typhimurium (strains TA100, TA1535, TA98, TA1537); E. coli WP2uvrA CHO cells Warheit et al. 2007 CHO-WBL (Chinese hamster cell line) Theogaraj et al. 2007 Human Peripheral blood lymphocytes Kang et al. 2008 + HPRT gene mutation + Ames test 100, 333, 1000, 3333 and 5000 mg per plate CHA 750, 1250 and 2500 mg/mL for 4 h (non-activated test condition); 62.5, 125 and 250 mg/mL, for 4 h (activated test condition), and 25, 50 and 100 mg/mL for 20 h (non-activated test condition) Photoclastogenicity (CHA) 800–5000 mg/mL, ± UV light (750 mJ/cm2) Comet assay TiO2 (20, 50 or 100 mg/mL) for 0, 6, 12 and 24 h Nano-TiO2 (20, 50 or 100 mg/mL), 20 h of incubation – The primary NPs (see table) formed aggregates in the dimension of 30–150 nm Crystal phase (XRD) 70–85% anatase and 30–15% rutile TiO2 Speciﬁc surface area 50 m2/g Size Note: NanoTiO2 suspended in PBS, sonicated for 30 min before exposure to cells CB MN + + Genotoxicity of nanoparticles Degussa Aeroxide P25 NP properties, NP preparation 200 nm Surface reactivity Eight different forms of TiO2: anatase (100%), rutile(100%), combined anatase (80%)/rutile(20%), diff. coated (trimethoxy caprylylsilane, alumina, silica, simethicone, dimethicone, stearic acid, doped di-iron trioxide) Commercial-grade nano-TiO2 Result of characterisation  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle TiO2 (SigmaAldrich) Uf-TiO2 £20 nm (vs. ﬁne TiO2 particle size >200 nm) (Oberdorster, University of Rochester, NY, USA) TiO2 anatase Physico-chemical characterisation Result of characterisation NP properties, NP preparation Genotoxicity testing method 63 nm Surface area in powder 24 m2/g TEM DLS (size of particles and agglomerates in medium) Zeta potential 20–100 nm 300 nm Suspended in MilliQ and sonicated for 2 20 sec Suspended in medium and sonicated Comet assay TiO2 P25(untreated, hydrophilic surface) or TiO2 T805(silanised, hydrophobic surface) particles Aerosil & Silanes, Degussa AG (Hanau-Wolfgang, Germany) Microstructure and aggregation characteristics (TEM) 1, 20, 40 mg/mL, 4 h Comet assay (FPG) Reference + A549 (human lung epithelial cell line) Karlsson et al. 2008, 2009 Syrian hamster embryo cells) Rahman et al. 2002 gpt delta transgenic mouse primary embryo ﬁbroblasts Xu et al. 2009 Female Wistar rats Rehn et al. 2003 ± 5.9 mV MN Suspended in PBS Accelerated surface area and porosimetry Cells/organism Had an isoelectric point of pHIEP = 6.4 Particle size £20 nm 5 and 40 nm (Sigma-Aldrich and Inframat Advanced Materials LCC, respectively) TiO2 325 mesh (Sigma-Aldrich) Result Approximately 30 nm 25 nm 11.6 1 ± 1.2 mV Zeta potential in culture medium (pH = 7.5) The negative surface charge at physiological pH (pH = 7.5–7.6) Particle size in powder BET surface area (determined by ASAP 2020) Treatment conditions 114.1261 m2/g, 38.2268 m2/g and 8.9146 m2/g, respectively 5 nm (99.7% purity) 0.5, 1.0, 5 and 10 mg/cm2 for 12, 24, 48, 66 and 72 h gpt delta mutagenicity assay 3 days exposure 0.1– 30 mg/mL 8-oxoG (immuno-cytology) Rats were exposed by instillation 0.15, 0.3, 0.6 and 1.2 mg dust/ lung of TiO2 + at concentration between 0.5 and 5 mg/cm2 (>200 nm sized TiO2 was negative) + 40 nm (99.9% purity) TiO2 325 mesh (‡99% purity) Both TiO2 highly aggregated TiO2 suspended in distilled water and sterilised by heating to 120 C for 30 min. Sonicated on ice for 30 min P25: particle diameter 20 nm, surface hydrophilic T805: primary particle diameter 20 nm, silanised, hydrophobic surface suspended in physiological saline Test: up to 90 days after exposure (analysis of single cells in lung tissue)  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Nanoparticle Physico-chemical characterisation Result of characterisation NP properties, NP preparation Genotoxicity testing method Treatment conditions Result Cells/organism Reference with 0.25% lecithin, sonicated TiO2 NP (anatase, ; <100 nm) (Degussa GmbH, Germany) TiO2 NP rutile, SiO2 coating (637262, SigmaAldrich) Surface area (BET) Zeta potential and particle size distribution (DLS) Trace Fe elements EDX spectral analysis of surface chemistry SEM (morphology) Primary size, shape Speciﬁc surface area (BET): Composition: NP dispersions (optical microscopy, TEM) TiO2 NP anatase (637254, SigmaAldrich) Primary size, shape: Speciﬁc surf. area (BET): Composition: NP dispersions (optical microscopy, TEM) TiO2 NP (Aldrich) Spherical shape 10 40 nm, needle-like crystals Comet assay + 8-oxodG (ELISA) NP dispersed in medium, ultrasonicated 37 kHz for 20 min 132 m2/g Comet assay IMR-90 (human lung diploid ﬁbroblasts), SV-40 virustransformed BEAS-2B (human bronchial epithelial cells) IMR-90 (human lung diploid ﬁbroblasts) Bhattacharya et al. 2009 TiO2 concentration 1–100 mg/cm2, for 24, 48, 72 h + at 80 mg/cm2 (24 h treatment) and + at ‡80 mg/cm2 (72 h treatment) BEAS-2B (human bronchial epithelial cells) Falck et al. 2009 TiO2 conc. 1–100 mg/cm2, for 24, 48, 72 h + at ‡10 mg/cm2 BEAS-2B (human bronchial epithelial cells) Falck et al. 2009 CHO-K1 (CHO cell line) Di Virgilio et al. 2010 Human nasal mucosa cells (10 donors) Hackenberg et al. 2010 CB MN Ti, O (<5% Si) Contained nanosized particles and agglomerates in size 4.5 mm <25 nm, spherical crystals 222 m2/g Ti, O Contained nanosized particles and aglomerates in size 5.5 mm Complex TiO2 concentration 2, 5, 10, 50 mg/cm2, exposure time 24 h Comet assay CB MN NP stock suspensions were prepared in PBS, vortexed for 10 min and stored at 4 C in the dark Size distribution (TEM) Speciﬁc surface area (BET) Shape (TEM) average particle sizes 20 ± 7 nm 142 m2/g spherical <25 nm Size (TEM) diameter 15–30 nm Aggregations (TEM) Mean sized 285 ± 52 nm, diameters up to 2000 nm NPs suspended in distilled water, sonicated for 60 sec, then added BSA and 10 PBS, diluted with PBS Morphology (TEM) Sphere shaped SCE CB MN Comet assay Comet assay TiO2 concentration 1–25 mg/cm2, for two cellular cycles TiO2 concentration 0.5–10 mg/cm2, for 24 h TiO2 concentration 10–100 mg/mL, for 24 h + at 10 and 60 mg/cm2 (72 h treatment) + at 1–5 mg/mL 10–25 mg/mL cytotoxic + at 0.5 and 1 mg/mL Genotoxicity of nanoparticles TiO2 NP (anatase, Sigma-Aldrich) Shape (TEM) 49.71 ± 0.19 m2/g +48.8 mV Particle hydrodynamic diameter: 91 nm TiO2 NP – absolutely pure 56% Ti, 41% O2, 3% C  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle Physico-chemical characterisation Result of characterisation TiO2 NP (anatase, Sigma-Aldrich) Diameter (TEM) Aggregations (TEM) 15–30 nm 285 ± 52 nm TiO2 NP (anatase, 1317-70-0 SigmaAldrich) Size (DLS) at different concentrations and timepoints TiO2 Aeroxide P25 (Degussa, now Evonik Crystal structure Particles widely distributed Considerably increased size as a function of concentration, minimally increased size as a function of time (see the table, Osman et al. 2010) Mixture of 75% anatase and 25% rutile. At least 99.5% TiO2 21 nm 50 ± 15 m2/g Agglomerates ranged from 21 to 1446 nm, mean size (70% of particles) 160 ± 5 nm About to have a size of 160 nm In MQ water: 124.9 nm; in medium: 192.5 nm In MQ water: 17.6 mV; in medium: 11.5 mV 50 nm Primary size Purity Speciﬁc surface area Size in water (DLS) TiO2 NP (anatase, 1317-70-0 Sigma Chemical) Mean hydrodynamic diameter (DLS) Zeta potential (DLS) Average size (TEM) TiO2 NP P25 (Degussa NRW, Germany) alone and combined with PbAc TiO2 NP anatase and rutile (637254 and 637262 SigmaAldrich) Primary diameter, composition and surface area (TEM, XRD) Aggregate size in aqueous solution Absorptive capacity of PbAc Size, shape (FEG-SEM); Size distribution in medium Speciﬁc surface area (BET); crystal phase (XRD); UV–Vis spectroscopy; 21 nm, 80% anatase and 20% rutile; 49.6 m2/g Average 299 nm Dose dependent (for details see table, Du et al. 2011) TiO2 An <25 nm TiO2 Ru <100 nm; high agglomeration NP properties, NP preparation Particles dispersed by sonication according to Bihari et al. 2008, diluted in PBS, added to cell suspension Suspended in EMEM-EBSS medium, probe-sonicated 30 W, 5 min TiO2 dispersed in drinking water, ultrasonicated 15 min at different concentrations just before use 99.7% anatase NPs suspended in water and DMEM with 10% FBS; probe sonicated at 30 W for 10 min Aqueous stock solutions of TiO2 and PbAc were heat sterilised, stored at 4 C; ultrasonicated; TiO2 solution was diluted with cultures or PbAc; vortexed NPs suspended in PBS; sonicated in ultrasonic bath; diluted in complete medium; sonicated before use Genotoxicity testing method Treatment conditions Result 20, 50, 100, 200 mg/mL, for 24 h Comet assay CB MN 10, 20, 50, 100 mg/mL, for 4 h 10, 20, 50 mg/mL, for 2 h Comet assay 5 days in vivo oral exposure 500 mg/kg MN g-H2AX 8-oxodG (HPLC) 50, 100, 250, 500 mg/kg 50, 100, 250, 500 mg/kg 500 mg/kg DNA deletion assay, in vivo Comet assay 500 mg/kg maternal exposure Concentration 0.008– 80 mg/mL, for 6 h + + + at 500 mg/kg + at all doses + + + at ‡8 mg/mL + at ‡0.8 mg/mL MN + at ‡0.8 mg/mL 0.001–10 mg/mL of TiO2 and ± 1 mg/mL of PbAc, for 24 h Reference Hackenberg et al. 2011c HEp2 (human negroid cervix carcinoma cells) Osman et al. 2010 Peripheral blood from pregnant C57Bl/6Jpun/ pun mice Peripheral blood Bone marrow cells Liver Trouiller et al. 2009 + Comet assay (FPG) 8-oxodG (HPLC) Cells/organism Human peripheral blood lymphocytes (10 male donors) –TiO2 alone; –PbAc alone; Fetus A431 (human epidermal cell line) Shukla et al. 2011a L02 (human embryo hepatocytes) Du et al. 2011 HepG2 (human lung carcinoma cell line) Petković et al. 2011 + TiO2 with PbAc Western blot (OGG1) Comet assay Comet assay (FPG) Comet assay (EndoIII) mRNA expression of p53, p21, gadd45a, mdm2 –TiO2 alone; –PbAc alone; +TiO2 with PbAc 1–250 mg/mL, for 2, 4, 24 h 1–250 mg/mL, for 2, 4, 24 h 1–250 mg/mL, for 2, 4, 24 h + An stronger than Ru + and stronger than Ru Weakly + comparing to FPG 1–250 mg/mL, for 4, 24 h +  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Physico-chemical characterisation Result of characterisation NP properties, NP preparation TiO2 NP anatase and rutile (1317-70-0 and 1317-80-2 Sigma Aldrich) Aggregation state after sonication in RPMI by TEM Variously sized aggregates after sonication Limited number of single particles and/or small aggregates <100 nm and a large number of aggregates sized from a few to several micrometres Comet assay 20, 50 and 100 mg/mL, for 4, 24, 48 h + after 24 and 48 h, at 50 and 100 mg/mL Bottlenose dolphin leukocytes Bernardeschi et al. 2010 TiO2 NPs – 21 nm (European Commission, JRC) Hydrodynamic sizes of dispersion (DLS) Mean diameter TiO2: 259.0 nm Anatase: 99.7% metals basis; size <25 nm; speciﬁc gravity/ density 4 g/cm3 Rutile: 99.9% metals basis, size <5000 nm; speciﬁc gravity/ density 4.26 g/cm3 NPs sterilised by heating at 120 C for 2 h, suspended in RPMI; bath-type sonicated for 30 min, serially diluted NPs dispersed in dH2O/10 Comet assay 12.5, 50 and 100 mg/mL, for 24 h Considerable level of DNA damage compared with controls, however, low Asare et al. 2012 BSA/10 PBS in 8:1:1 ratio of 2 mg/mL stocks, freshly prepared before each exposure Solutions of dispersed TiO2 prepared with distilled water Comet assay (FPG) 12.5, 50 and100 mg/mL, for 24 h Ntera2 (NT2, human testicular embryonic carcinoma) cells, Primary testicular cells from C57BL6 mice of wild type (WT) and knock-out cells 8-oxoG DNA glycosylase (Ogg1) Comet assay, in vivo Oral gavage; doses: 40, 200, 1000 mg/kg bw; daily for 7 days Male F1 (CBAxB6) mice Sycheva et al. 2011 CHO-K1 (CHO cell line) Wang et al. 2011b Nanoparticle TiO2 NPs – 33 nm vs. microsized TiO2 particles (MP) – 160 nm (Sensient Cosmetic Technologies LCW, France) Crystal structure of both TiO2 (light scattering) Anatase Mean particle size (electron microscopy) 33.2 ± 16.7 nm for NP; 160 ± 59.4 nm for MP Genotoxicity testing method Treatment conditions 100% anatase Purity Particle size 99.7% TiO2 Less than 25 nm TiO2 NPs appeared to aggregate in culture medium NPs suspended in DMSO; prior exposure vortexed 1 min to fully resuspend; made fresh weekly HPRT gene mutation NP: + bone marrow; + liver; brain Reference 10, 20, 40 mg/mL, 60 days continuous exposure Comet assay gH2AX 1–1000 mg/mL + for NPs and MPs, Toyooka et al. 2012 Genotoxicity of nanoparticles Crystalline structure (XRD) /no increase Cells/organism MP: + bone marrow; liver; brain; NP: bone marrow; MP: + at 1000 mg/kg bw bone marrow NP and MP: fore stomach, colon, testis MN in vivo TiO2 anatase NPs (corresponds to the source used in studies conducted by the Finnish Institute of Occupational Health, Helsinki) Result  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle TiO2 anatase NPs (5 nm; SigmaAldrich, St. Louis) Physico-chemical characterisation The size distribution in DMEM Result of characterisation NP: 250–650 nm (average: 378 nm) MP: 600–1050 nm (average: 773 nm) vs. MPs (<5000 nm; Wako Pure Chemicals Ind., Japan) NP properties, NP preparation Genotoxicity testing method Treatment conditions NPs vs. MPs more remarkable in NPs NPs vs. NPs coating with BSA Pretreatment with inhibitors (wortmannin and U0126) for 0.5 h Pretreatment with NAC for 30 min Coating attenuated lH2AX generation Generation of lH2AX not affected DSBs (BSFGE) TiO2 (anatase/ rutile powder of 21 nm; NM105 EC-JRC, Ispra,Italy; corresponds to Aeroxide P25, Evonik) TiO2 NPs Primary characteristics See tables in results for detailed properties Secondary properties of two different NP dispersions in DMEM and RPMI media DP1: stability up to 2 days; bimodal dispersion (RPMI: 102 nm,285 nm; DMEM: 112 nm, 296 nm); DP2: agglomerated (RPMI: 779 nm; DMEM:752 nm) Crystal phase identiﬁcation and average crystallite size (XRD), size and morphology (TEM) Tetragonal Optical properties (UV–visible spectroscopy) Aggregation in suspension and secondary particles sizes (DLS) Functional groups and stretching vibrations of the bonds (FTIR spectrum) rutile structure of TiO2; 30.6 nm; crystallites with polyhedral morphologies Absorption edge around 280–320 nm 13 and 152 nm aggregates in RPMI medium; 380 nm aggregates in water See results for details Dispersion prot. (DP)1: suspended in 20% FBS in PBS, sonicated 15 min, 100 W, added to medium, serially diluted. DP2: suspended in medium with 15 mM HEPES without FBS, sonicated 3 min, 60 W, vortex 10 sec, aliquoted, 20 C. Thawed, vortexed 10 sec, sonicated 1 min 60 W, added to medium NPs synthesised by sonomechanical method, see the protocol. Powdered NPs suspended in water; sonicated 15 min, 40 W Result Cells/organism Reference A549 (human lung epithelial cell line) Both NPs and MPs suspended in DMEM at 20 mg/mL, bath-sonicated 1 min BSA-coated NPs: suspended in 1 mL of BSA (5 mg/mL), sonicated 1 min; centrifuged for 10 min, resuspended in water. This washing repeated 2 Comet assay Comet assay (FPG) 0.12, 0.6, 3, 15, 75 mg/cm2 (correspond to 0.57, 2.9, 14.4, 72.0, 360.2 mg/mL), for 2 and 24 h Wortmannin inhibited generation of lH2AX + for NPs and MPs, more remarkable in NPs for DP1 in TK6, EUE + for DP2 in Cos1, EUE for DP1 in TK6 + for DP2 in TK6, Cos1 Comet assay (neutral) 0.625–20 mg/mL, for 6 h + TK6 (human lymphoblast line); EUE (human embryonic epithelial cells); Cos-1 monkey kidney ﬁbroblasts Magdolenova et al. 2012 WISH (human amnion epithelial cells) Saquib et al. 2012  Z. Magdolenova et al. Table I. (Continued). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Table I. (Continued). Nanoparticle TiO2 NPs (99% anatase, Sigma Chemical Co. Ltd., USA) Physico-chemical characterisation Mean hydrodynamic diameter (DLS) Zeta potential (DLS) PDI TiO2 NPs, ﬁve types; anatase 25 nm (Aerooxide P25,Degussa), rutile 68 nm (ref. 637262, SigmaAldrich), anatase 140 nm (ref. T8141, Sigma-Aldrich), anatase 12 (LFP), rutile 20 (LFP) TiO2 NPs (anatase) (Sigma-Aldrich, USA) Size (TEM) Shape Crystal phase Diameter Speciﬁc surface area Point of zero charge Mean hydrodynamic diameter (DLS) Zeta potential (DLS) NPs agglomeration (photon correlation spectroscopy) Result of characterisation NP properties, NP preparation In MQ water: 124.9 nm; in medium: 192.5 nm In MQ water: 17.6 mV; in medium: 11.5 mV In MQ water: 0.18 PDI; in medium: 0.12 PDI See Shukla et al. 2011a See the table in results NPs suspended in medium without FBS, probe sonicated 10 min, diluted in medium with 10% FBS Comet assay NPs dispersed in water by sonication 30 min, 4 C CB MN Comet assay CB MN gH2AX Purity BET surface area Secondary diameter of dispersed NPs Size distributions (DLS, TEM) 99.99% 316 mg/m2 19 ± 6.7 nm See the results Polymer NPs A family (nine kinds) of PELGE, z-average diameters 70–180 nm PLGA polymers PDI (PCS) 0.23 7.83 mV 99.7% 200–220 m2/g Tetragonal Spherical No signiﬁcant change during the period 1–80 mg/mL, for 6 h (corresponding to 0.31–25 mg/cm2) Sterilisation by heating to 120 C, 2 h, suspended in medium, sonicated, diluted in medium; analysed by LAL assay for presence of endotoxin Activity of DNA repair enzymes – BER and NER (multiplex excision/synthesis) CB MN Western blot (P53) Result + Cells/organism Reference HepG2 (human liver carcinoma cell line) Shukla et al. 2011b A549 (human lung epithelial cell line) Jugan et al. 2011 A549 (human lung epithelial cell line) Srivastava et al. 2011 + 100 mg/mL, for 4, 24, 48 h + + 100 mg/mL, for 4, 24, 48 h 50, 100, 200 mg/mL of anatase 12 and 25, for 24 h 50, 100, 200 mg/mL of anatase 12 and 25, for 24 h 100 mg/mL, for 24, 48 h + at all NPs except anatase140 10 and 50 mg/mL, 24 h Antigenotoxicity effects of DMTU and NAC 50 mg/mL, 24 h Antigenotoxicity effects of DMTU and NAC + DMTU and NAC reduced MNi + DMTU and NAC reduced expression level of P53 NPs impaired cellular repair ability NPs dispersed in disodium phosphate, agitated in bead mill with ZrO2 beads, supernatant removed by centrifugation Comet assay in vivo (JaCVAM protocol) For single intratracheal instillation: 1.0–5.0 mg/kg bw, 3 and 24 h exposure For repeated instillation: 0.2–1.0 mg/kg bw once a week for 5 weeks Lung epithelial cells of Sprague-Dawley rats Naya et al. 2012 Prepared by wow emulsion solvent extraction/ evaporation technique. Lyophilised NPs were dissolved in physiological saline and sterilised by ﬁltration CB MN 5 mg/mL, for 4 h, presence or absence of S9 mix 5 mg/mL, for 24 h, presence or absence of S9 mix CHO cell line He et al. 2009 SCE response of 4 PELGE, weakly + clastogenic response of 5 PELGE Genotoxicity of nanoparticles 5–25 nm 417.7 nm Treatment conditions Comet assay (FPG) 8-oxoG (HPLC-MS/MS) Particle size (TEM) Mean hydrodynamic diameter (DLS) Zeta potential Purity Speciﬁc surface area Crystallographic system Shape Secondary particle size – time dependent TiO2 NPs (anatase) (Ishihara Sangyo Kaisha, Ltd., Japan) Genotoxicity testing method  Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Nanoparticle PNIPAM Physico-chemical characterisation The hydrodynamic diameters in water, DMEM and the supplemented media measured over the temperature range 30–38 C Particle size (TEM) Result of characterisation NP properties, NP preparation Genotoxicity testing method Treatment conditions For detailed results see tables Naha et al. 2010 NPs synthesised by free radical polymerisation; dispersed on ice to ensure good solubility, gradually warming them; dispersions prepared in DMEM F-12 with FBS Comet assay 12.5, 25, 100, 200, 400, 800 mg/L, for 24, 48 and 72 h PEI molecular weight 25 g/mol Comet assay 1, 3, 5 mg/mL of PEI; 10, 30, 50 mg/mL of PAMAM, for 4 h 1, 10, 20 mg/mL of PEI; 10, 100, 200 mg/mL of PAMAM, for 4 h Oral administration 100, 200, 300 mg/kg BW once daily over a period of 2 days Result in both cell lines Cells/organism Reference HaCaT (immortalised noncancerous human keratinocyte line; and SW480 (primary adenocarcinoma of colon cell line) Naha et al. 2010 Human acute T-cell leukaemia Jurkat cells Choi et al. 2010 Bone marrow cells from Swiss albine mice Bone marrow cells from Holtzman rats Dandekar et al. 2010 Zeta potential Polyethyleneimine (PEI; 25 kDa) and polyamidoamine (PAMAM) G4 dendrimer CB MN Curcumin -loaded polymeric nanoparticles of Eudragit S100 Bacterial cellulose (BC) nanoﬁbres Polysaccharide cationic NPs (NP+) TEM Size (DLS) MN in vivo Homogenous (97 ± 2.47 nm; PI of 0.14 ± 0.01) population of particles with encapsulation efﬁciency of 72.81 ± 0.13% Formulation of NPs published elsewhere (Dandekar et al. 2009) Needle shaped BC secreted by G. xylinus Ames test length: 50–1500 nm; width: 3–5 nm The nanoﬁbres were produced by acidic and/or ultrasonic treatment NPs prepared from maltodextrin, see the protocol Comet assay Zeta potential Mean diameter 64 ± 13 nm, PDI: 0.22 +25 ± 1.5 mV Shape (TEM) Spherical Size and zeta potential in the presence of serum See results for details CHA in vivo + for both (PEI more signiﬁcant than PAMAM) Not signiﬁcant Comet assay in vivo Comet assay Comet assay (FPG) MN Bone marrow cells from Holtzman rats S. typhimurium (strainsTA97a, TA98, TA100, TA102) Concentration 0.1, 0.5 or 1.0 mg/mL of NFs suspension. Presence or absence of S9 mix Concentration of NFs 0.1, 0.5 or 1 mg/mL, 48 h exposure 5000, 2500, 1250, 625, 312.5 and 156.25 mg/mL (2604, 1302, 651, 326, 163 and 81 mg/cm2); for 3 h; in serumfree medium or with 10% FCS Moreira et al. 2009 CHO cell line with serum, + without serum + with serum, + without serum 16HBE14o- (human bronchial epithelial cell line) Merhi et al. 2012 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  Z. Magdolenova et al. Table I. (Continued). Genotoxicity of nanoparticles  4346 Number of publications 1204 945 44 es Am ox i ci ty an d an d N P ge no t ge no t ox i ci ty ci ty P N 13 A H C M an d an d N P ge no t ox i an d ox ic ity In 11 N A C P N P ge no t N P ge no t vo vi 67 22 ox ic ity an d N an d ox ic ity ge no t ox ic ity tro vi In 94 N P P N P In vi vo G en ot to xi ci ty an d an d N P N an d to xi ci ty vi tro To xi ci ty In Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. 112 Figure 1. Review of literature on NP toxicology (CA: comet assay; MN: micronucleus assay; CHA: chromosomal aberration test; Ames: bacterial reverse mutation assay). NP that do not cross cell membrane M+ M+ + M NP that cross cell membrane NP release transition metals or free radicals NP interaction with receptor NP in cytoplasm Diffusion Endocytosis NP disturb mitochondria Mitochondria NP cross nuclear envelope through nuclear pores NP aggregates deform nucleus shape Interphase DNA replication Transcription Nucleus Translation Mitosis NP get access to nucleus during mitosis Centrosome Figure 2. Cellular uptake and access of NPs to nucleus.  Z. Magdolenova et al. Mechanically Affect replication By chemical binding to DNA Interphase (DNA level) Mechanically Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Affect transcription Primary direct genotoxicity Affect DNA structure (conformation) Primary interaction of NP or its component with DNA By chemical binding to DNA Mechanically Clastogenic effect (chromosomal break) Mitosis (chromosomal break) By chemical binding to DNA Mechanically Aneugenic effect (loss of chromosome) By chemical binding to DNA Figure 3. Scheme of potential NP-induced primary direct genotoxicity. Indirect primary genotoxicity To induce genotoxicity, NPs do not need to be in direct contact with DNA. The following are suggested ways in which NPs might indirectly induce primary genotoxicity (Figure 4). Interaction with nuclear proteins (involved in replication, transcription, repair) NPs can potentially interact not only with DNA but also with proteins involved in DNA replication, transcription or repair. 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 human DNA topoisomerase II alpha in the ATP binding domain, which could inhibit the enzyme activity. DNA topoisomerase II is involved in modiﬁcation of DNA topology. Another in silico study showed that C60 fullerene might interact with PMS2, RFC3 and PCNA proteins involved in the DNA mismatch repair pathway (Gupta et al. 2011). Proteins could also be inactivated by structural modiﬁcation, for instance by oxidation by ROS generated during NP exposure (Jugan et al. 2011). Interaction of NPs with the mitotic spindle or its components – aneugenic effect An aneugenic effect could also be caused by interaction of NPs with the mitotic spindle apparatus, centrioles or their associated proteins. NPs can affect any function of the mitotic apparatus, leading to loss or gain of chromosomes in daughter cells. Huang et al. (2009) demonstrated evidence in vitro that TiO2 NPs disturb mitosis. Long-term exposure to TiO2 NPs led to abnormal multipolar spindle formation, chromosomal alignment and segregation during anaphase and telophase (Huang et al. 2009). Gonzalez et al. (2010) investigated aneugenic events in human lung epithelial cells A549 that could result from mitotic spindle defects. Also, interference with tubulin polymerisation might lead to aneugenicity (Gonzalez et al. 2010). Disturbance of cell cycle checkpoint functions NPs might interact with and inﬂuence the function of protein kinases responsible for regulation of cell cycle events such as DNA replication and cell division. As shown by Huang et al. (2009), TiO2 NPs deregulated the function of mitotic checkpoint PLK1 protein which controls several processes during mitosis, including contractile ring formation and cytokinesis. Disturbance of cytokinesis can also give rise to aneuploid or multinucleated cells. An important process in cell cycle regulation of kinases is their inactivation. Kinases are marked by polyubiquitin and then degraded by proteasomes. Interaction of NPs with proteins involved in this process could affect their function. Calzolai et al. (2010) identiﬁed a speciﬁc domain of human ubiqutin that interacts with Au NPs. Exposure to nano-TiO2 in vitro inﬂuenced ERK signalling Genotoxicity of nanoparticles DNA replication proteins Mechanically By chemical binding to proteins Mechanically Interaction with nuclear proteins  Transcription proteins By chemical binding to proteins Inhibition of protein activity Defect in the processes of replication, transcription, repair Mechanically DNA repair proteins By chemical binding to proteins Mechanically Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Centrioles By chemical binding to proteins Mitotic spindle apparatus Mitotic spindle (microtubules) Mechanically By chemical binding to proteins Disturb the processes of cell division Mechanically Associated Proteins Primary indirect genotoxicity (NP are not in direct contact with DNA) Disturbing cell cycle checkpoints ROS arising from NP surface Transition metals (TM) from NP surface ROS produced by cell components (mitochondria) Interaction with antioxidants Secondary genotoxicity ROS produced by inflammatory cells By chemical binding to proteins Mechanically By chemical binding to proteins Inhibition of protein activity Defect in cell cycle processes ROS attack on DNA Production of ROS via the Fenton-type reaction Attack of TM on DNA Chemical binding of TM to DNA ROS attack on DNA Mechanically By chemical binding to proteins Prevent antioxidant function, ROS accumulation ROS attack on DNA Figure 4. Scheme of potential NP-induced primary indirect and secondary genotoxicity. activation, ROS production, the numbers of multinucleated cells and MN (Huang et al. 2009). ROS arising from NP surface NPs can generate ROS in the cells that may cause indirect oxidative damage to DNA through free radical attack. Silica and TiO2 NPs were found to generate free radicals in aqueous suspensions in vitro (Barillet et al. 2010; Shukla et al. 2011a). Free radicals may interact with cellular biomolecules including DNA, leading to potentially serious consequences. ROS may attack the DNA causing purine(such as 8-oxoG) and pyrimidine-derived oxidised base lesions and DNA strand breaks. DNA base lesions can give rise to mutations through mispairing in replication, and thus are potentially carcinogenic (Cooke et al. 2003). TiO2 NPs induced ROS and oxidative stress leading to oxidative DNA damage and micronucleus formation, suggesting a probable mechanism of genotoxicity in human skin cells in vitro (Shukla et al. 2011a, b). Transition metals from NP surface Toxic ions released from soluble NPs may also contribute to DNA damage. Transition metal ions such as Fe2+, Ag+, Cu+, Mn2+, Cr5+ and Ni2+ which can be released from NPs may contribute to the production of intracellular ROS via the Fenton-type reaction (Kruszewski et al. 2011). Asharani et al.  Z. Magdolenova et al. (2009) presented a possible chemical reaction of H2O2 with AgNPs that was estimated to cause formation of Ag+ in vivo (Yang et al. 2009; Asharani et al. 2009). Transition metal complexes can also bind to DNA bases (Robertazzi & Platts 2005). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. ROS produced by cell components (mitochondria) NPs could interfere with cell components such as mitochondria and cause damage affecting their functions. In response to stress, mitochondria can generate ROS. For instance, Asharani et al. (2009) suggested that disruption of the mitochondrial respiratory chain by Ag NPs leads to production of ROS and interruption of ATP synthesis, which can result in damage to DNA. Furthermore, in in vitro study Ag NPs were detected in mitochondria by TEM analysis (Asharani et al. 2009; Kruszewski et al. 2011). Inhibition of antioxidant defence Inhibition of antioxidants in vitro and consequent accumulation of reactive oxygen can potentially lead to DNA damage (Barillet et al. 2010). Silicon carbide NPs were associated with depletion of glutathione (the major molecular antioxidant of cells) and inactivation of some antioxidant enzymes such as glutathione reductase and superoxide dismutase (Barillet et al. 2010). Similarly, the depletion of glutathione and superoxide dismutase (Sharma et al. 2009) and generation of intracellular ROS in vitro were associated with ZnO NP-mediated DNA damage and cytotoxicity (Sharma et al. 2012). Secondary genotoxicity ROS produced by inﬂammatory cells NPs can trigger ROS production in activated phagocytes (neutrophils, macrophages). Inﬂammation in several studies was associated with genotoxicity. For instance, Trouiller et al. (2009) found that TiO2 NPs induced an inﬂammatory reaction and oxidative DNA damage in mice. The oxidative burst caused by activation of phagocytes may be a potential explanation for the observed genotoxicity. DNA damage as a result of primary and secondary genotoxic events due to NP exposure DNA damage can occur as DNA base modiﬁcations (oxidation), bulky DNA adducts, DNA single strand breaks, DNA double strand breaks (DSBs), cross links or structural DNA changes. Many of these lesions were detected after exposure to NPs, including strand breaks, alkali labile sites, oxidised purines and pyrimidines measured by the comet assay (Karlsson et al. 2008); DSBs measured by phosphorylation of histone H2AX (Wang et al. 2010); and DNA adducts measured by 32P postlabelling (Foldbjerg et al. 2011; Kruszewski et al. 2011). The most investigated DNA damage in relation to NPs is DNA base oxidation, since in many studies an increased level of ROS was observed after NP exposure. 8-OxoG, the major purine oxidation product produced in DNA during oxidative stress, tends to mispair with adenine during replication, and is highly mutagenic and thus potentially carcinogenic (Vidal et al. 2001). 8-oxoG as an indicator of oxidative DNA attack was detected after Ag NP exposure in several studies in vitro using the comet assay modiﬁed with formamidopyrimidine DNA glycosylase (FPG) (Magdolenova et al. 2012a; Kim et al. 2011; Asare et al. 2012) or with the human 8-oxoguanine DNA glycosylase (OGG1) involved in BER of 8-oxoG (Hudecová et al. 2012b). Furthermore, DNA repair of 8-oxoG has been studied. For instance, Folkmann et al. (2009) found increased mRNA expression of DNA glycosylase OGG1 in the liver of C60 fullerene-treated rats, but observed no increase in OGG1 repair activity. The direct correlation between ROS formation and oxidative DNA damage was shown after exposure to several NPs. For instance, in in vitro study Shukla et al. (2011a) suggest that oxidative stress could be an important route by which TiO2 NPs induce DNA damage. Involvement of the ROS pathways in NPinduced genotoxicity was also suggested by experiments in which cells were preincubated with antioxidant before the NP treatment. Guo et al. (2011) observed in human umbilical vein endothelial cells (HUVECs) in vitro that pretreatment with the free radical scavenger N-acetyl-l-cysteine (NAC) can inhibit the genotoxicity of MWCNTs. Foldbjerg et al. (2011) investigated the effect of Ag NPs in human lung carcinoma cells (A549) using a 32P postlabelling technique. Ag NPs increased the formation of bulky DNA adducts, and this effect was inhibited by antioxidant pretreatment (Foldbjerg et al. 2011). Similarly, we found that Ag NPs induce 8-oxoG in primary human peripheral lymphocytes as well as human kidney HEK293 cells (measured with human OGG1) that can be eliminated by pretreatment of cells using plant extract with antioxidative properties (Hudecová et al. 2012a, b). There are obviously several simultaneous mechanisms that may lead to genetic changes in human and mammalian cells. The recent review of Iavicoli et al. (2011) reported that exposure to TiO2 NPs leads to an increase of ROS production and cytokines levels, induction of apoptosis and genotoxicity. Several authors reported from in vitro studies that TiO2 NPs induce ROS-mediated induction of DNA damage (Ghosh et al. 2010; Saquib et al. 2012; Table I) and photogenotoxicity (Kang et al. 2011). TiO2 NPs and UVA synergistically promoted rapid ROS generation and mitochondrial membrane potential collapse in human peripheral lymphocytes, triggering apoptosis and inducing DNA damage. The effect was more pronounced by smaller TiO2 NPs. However, Toyooka et al. (2012) demonstrated that formation of -H2AX (a biomarker for DSBs) by TiO2 NPs was ROS independent and no TiO2 NP-induced chromosomal aberrations were observed in CHO cells either in the absence (Warheit et al. 2007) or presence of UV light (Theogaraj et al. 2007). DNA damage can be repaired by several repair mechanisms, including BER, nucleotide excision repair (NER), homologous recombination and nonhomologous end joining repair (NHEJ). Only a few studies have investigated NPs in relation to DNA repair. Wojewódzka et al. (2011) found that treatment with Ag NPs delays repair of X-ray-induced DNA damage in HepG2 cells. Jugan et al. (2011) investigated the impact of TiO2 NP exposure on DNA repair in A549 cells, Genotoxicity of nanoparticles Cell transformation Cell death Changes in gene expresion Accumulation of mutations, mutations in important genes Chromosomal mutations (deletion, duplication, inversion, transversion, aneuploidy, polyploidy) Not repaired (A, B, C, D) Cell cycle arrest Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only.  Gene mutations (inversion, deletion, substitution) Cell cycle arrest DNA damage (SSB, DSB, base modification, cross links,.. ) Chromosomal damage (chromosome break, loss of chromosome) NP caused damage Cell cycle arrest Silencing of important genes Non-DNA damaging changes (methylation, histone modification, protein phosphorilation,.. ) Repaired (BER, NER, HR, HNEJ) Normal cell Figure 5. Possible consequences of NP-induced DNA damage including non-DNA changes. and found that TiO2 NPs simultaneously damaged DNA and impaired cellular DNA repair through inactivation of BER and NER pathways. Li et al. (2011a) indicate that the NHEJ pathway might be involved in the repair of DNA damage induced by Au NPs in lung ﬁbroblasts. DNA damage that is not repaired leads to mutation DNA damage that is not repaired, or is misrepaired, can lead to mutation. This situation may arise if (a) DNA damage caused by NPs is too extensive and the DNA repair mechanism is not efﬁcient enough to repair all damages (Huang et al. 2009); (b) DNA damage is not recognised by the repair mechanism; (c) the DNA repair mechanism is corrupted (it should be noted that NPs could potentially modulate the function of repair enzymes and thus DNA repair could be defective); (d) this interference may also give rise to ‘spontaneous’ mutations resulting from errors during DNA replication as a consequence of defective repair (Figure 5). DNA base pair substitutions give rise to missense or nonsense mutations and DNA base pair insertions or deletions give rise to frameshift mutations. If these mutations occur in protein coding regions of genes, coding information may be changed leading to errors in gene expression and thus to formation of defective or no proteins. Accumulation of mutations can result in cell death or cell transformation and cancer (Sharma et al. 2012). DNA damage (such as DSBs) or chromosomal damage (chromosome breaks) can lead to chromosome aberrations: (a) chromosome structure changes (deletions, duplications, inversions and translocations of sections of chromosomes), the consequences depending on the genes that have been altered (Russel 2001); and (b) changes in number of chromosomes (aneuploidy – loss or gain of one or more chromosomes; or polyploidy – multiplication of whole sets of chromosomes). Mutations were detected following NP exposure in vitro in several studies using the HPRT gene mutation assay (Wang et al. 2007b), the bacterial reverse mutation assay (Hasegawa et al. 2012), the micronucleus assay (Muller et al. 2008; Könczöl et al. 2011; Di Virgilio et al. 2010), chromosomal aberrations (Hackenberg et al. 2011b) or the sister chromatid exchange test (Di Virgilio et al. 2010) (for complete list see Table I). The cytokinesis block micronucleus (CBMN) assay is able to detect both aneugenic and clastogenic effects, as MN could represent lost chromosomes or chromosome fragments (Dusinska et al. 2012; Kazimirova et al. 2012). To increase the speciﬁcity and to discriminate between these two events, Gonzalez et al. (2010) used the CBMN assay in vitro using A549 cells in combination with ﬂuorescent in situ hybridisation (FISH)-centromeric probing. Furthermore, the frequency of mitotic errors (chromosome loss, mitotic arrest and mitotic slippage) as consequences of spindle defects was measured. To distinguish between NPs causing aneugenicity and clastogenicity, measurement of MN in mononucleated cells in addition to those in binucleated cells in the CBMN assay was suggested as an additional marker (Kazimirova et al. 2012).  Z. Magdolenova et al. Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Methods used for in vitro and in vivo genotoxicity testing of NPs Genotoxicity testing of NPs can be carried out in vitro or in vivo. The in vitro approach is appropriate for testing primary genotoxicity, while in vivo models can also give information on secondary effects such as inﬂammation (Dusinska et al. 2011; Kisin et al. 2007; Vega-Villa et al. 2008; Arora et al. 2012). Initial testing for genotoxicity is usually done with the bacterial reverse mutation assay (Ames test) (Warheit et al. 2007). Subsequent tests in cultured mammalian cells (either permanent cell lines or primary cultures) employ various endpoints. DNA damage is measured with the comet assay (Shukla et al. 2011a, b). Mutations are assessed at a speciﬁc locus – often the HPRT (hypoxanthine phosphoribosyltransferase) gene (Wang et al. 2007a). Chromosomal damage is scored either in mitotic cells as chromosome aberrations or in interphase cells as MN. In vivo, DNA damage (Bourdon et al. 2012; Schulz et al. 2012), chromosome aberrations (Dandekar et al. 2010) and MN (Estevanato et al. 2011; Li et al. 2011c) can be measured in different tissues, and transgenic rodents are available that allow detection of mutations at a speciﬁc locus in cells from different organs. The Ames test (bacterial reverse mutation) (Mortelmans & Zeiger 2000) is based on induction of back-mutations in a defective histidine gene; reversal of this mutation will enable the bacterium to synthesise histidine and form a visible colony when plated in minimal histidine medium. There are concerns regarding the suitability of the Ames test for NP testing, as larger NPs are unable to cross the cell wall. If they do enter the cell, NPs could possibly interfere with histidine synthesis and induce false-negative (down-regulation) or positive (up-regulation) results. The Ames test has nevertheless been used to assess genotoxicity of a variety of NPs, and has so far given largely negative results (Landsiedel et al. 2009; Shinohara et al. 2009; Mori et al. 2006; Wirnitzer et al. 2009; Di Sotto et al. 2009; Balasubramanyam et al. 2010; Maenosono et al. 2007, 2009; Yoshida et al. 2009; Kumar et al. 2011a, b). The comet assay (single-cell gel electrophoresis) is one of the most common tests for genotoxicity. Cells are embedded in agarose on a microscope slide and lysed in detergent and high salt to remove membranes and soluble cell components, and also histones. DNA remains as nucleoids, consisting of supercoiled DNA loops attached to a matrix. DNA breaks relax supercoiling, and relaxed loops of DNA are able to extend during electrophoresis (normally at high pH), to form a ’comet tail’, visualised by ﬂuorescence microscopy. Relative tail intensity indicates break frequency. As well as DNA breaks, damaged bases can be detected by incubating nucleoids with lesion-speciﬁc endonucleases, such as endonuclease III and FPG that recognise oxidised pyrimidines and purines, respectively (Collins et al. 1996; Dusinska & Collins 1996; Hudecová et al. 2012b). Photogenotoxic effects of NPs have been measured by the comet assay in combination with ultraviolet radiation (Jha 2008). The presence of NPs in the nucleoid has been reported and the possibility that they might induce additional DNA damage during the assay has been discussed (Karlson et al. 2004; Karlson 2010; Stone et al. 2009; Shukla et al. 2011a). The presence of NPs close to DNA during the comet assay also increases the probability for an interaction of NPs with FPG. It has recently been shown that the incubation of NPs and ions with FPG leads to the total loss of the ability of the enzyme to detect oxidatively damaged DNA in the comet assay (Kain et al. 2012). This disturbance is most likely due to the binding of ions to the SH groups at the active site (Kain et al. 2012). However, in the actual comet assay, FPG and NPs do not interact directly. As we recently showed, the presence of NPs in the agarose had no effect on the ability of FPG to recognise oxidised bases (Magdolenova et al. 2012). A relatively speciﬁc and very sensitive assay for DSBs is based on the fact that a cellular response to these breaks is the phosphorylation of one of the core nucleosomal histones, H2AX. The phosphorylated form is known as -H2AX, and the concentration in the proximity of a DSB is sufﬁcient to form a focus, visible by immunohistochemistry using a ﬂuorescence-tagged antibody to the phosphorylated form. Alternatively, -H2AX can be measured by ﬂow cytometry (Ismail et al. 2007; Lewis et al. 2010). The chromosomal aberration (CHA) test identiﬁes agents that cause structural chromosome or chromatid breaks, dicentrics and other abnormal chromosomes, notably translocations which are implicated in the aetiology of various human genetic diseases and cancers. For in vitro testing, cell cultures are exposed to the test substance and incubated with a metaphase-arresting substance (e.g. colcemid) to accumulate metaphase cells, which are analysed microscopically (Galloway et al. 1994, 1987; Bonassi et al. 2008; Aoshima et al. 2010). MN are formed during anaphase from chromosomal fragments or whole chromosomes that are left behind when the nucleus divides. Excluded from the nuclei of daughter cells, they form single or multiple MN in the cytoplasm, detected by visual (or automated) examination after staining. Formation of nucleoplasmic bridges and binucleated cells provides a complementary assay for chromosome rearrangement. Increased assay sensitivity can be obtained (in the in vitro assay) by incubating treated cells with cytochalasin B, which blocks cell division, but not mitosis, so that binucleated cells accumulate. Scoring MN only in binucleated cells reduces the likelihood of scoring MN that existed before the treatment. The MN assay is less time-consuming and more suitable for automation than the CHA assay. Histological staining with labelled DNA probes reduces the risk of falsely identifying NP aggregates as MN fragments (Laingam et al. 2008; Fenech et al. 2011; Doak et al. 2009; Gonzalez et al. 2011; Li et al. 2012). The HPRT gene has several features that make it suitable for assessing HPRT mutations induced by a suspect genotoxic agent such as NPs. It is X-linked, with only one active copy per cell, so that a mutation in only one allele is needed for phenotypic expression. HPRT is a purine salvage enzyme, which phosphorylates ’waste’ purines and adds them to the cellular DNA precursor nucleotide pool. It is not an essential enzyme for the cell, and so HPRT mutant cells survive. After Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Genotoxicity of nanoparticles treatment with the suspect agent, cells are cultured for several generations to dilute out pre-existing enzyme, and then plated in dishes at suitable density in the presence or absence of 6-thioguanine, a toxic purine analogue that is taken up by wild-type (WT) cells, which die. HPRT cells survive to form colonies, which are scored. The mutant frequency is calculated from the frequency of mutant colonies related to plating efﬁciency (Wang et al. 2007a, 2011). OECD guidelines exist for several genotoxicity assays in vivo or in vitro: OECD 471 for the bacterial reverse mutation test, OECD 473 for the in vitro CHA test, OECD 474 and 475 for the mammalian erythrocyte and bone marrow CHA tests, OECD 476 for the in vitro mammalian cell gene mutation test and OECD 487 draft for the in vitro MN test. OECD guidelines are under preparation for the in vivo comet assay test (http://www.jacvam.jp/en_effort/ en_oecd.html). However, these guidelines are formulated for testing chemicals, and their suitability for NP testing should not be taken for granted, since the very different physicochemical properties of NPs can seriously inﬂuence their interactions with DNA (Dusinska et al. 2009; Warheit & Donner 2010). Effect of physico-chemical properties on NPinduced genotoxicity Although the number of studies on genotoxicity of NPs is increasing, many conﬂicting results are published. This is not surprising as detailed characterisation of NPs in many studies especially in testing media is lacking. NPs are prone to change properties in different media and treatment conditions, and without proper in situ characterisation, it is difﬁcult to compare results (Stone et al. 2009; Som et al. 2010; Dusinska et al. 2011). Many different NP characteristics (size, shape, surface properties, composition, solubility, aggregation/agglomeration, NP uptake, presence of mutagens and transition metals afﬁliated with the NPs, etc.) have to be taken into consideration to be able to assess toxic effects. Physico-chemical properties of NPs have a strong link to their biological activity and many of them may contribute to adverse health effects (Chan 2006; VegaVilla et al. 2008). It is still not known which of these properties play the most important roles in NPs’ impact on genotoxicity. According to Hansen et al. (2007) and Stone et al. (2010) the following properties are important: chemical composition, size, shape, crystal structure, surface area, surface chemistry, surface charge, solubility and adhesion, deﬁned as the force by which the NPs and their components are held together. Warheit (2008) suggests the following minimum properties to be characterised: particle size, size distribution (wet state) in the relevant medium, surface area (dry state), crystal structure/crystallinity, aggregation status in the relevant medium, composition/surface coatings, surface reactivity, method of synthesis, purity of sample. A recent report on nanomaterials in the context of REACH (Malkiewicz et al. 2011) stressed the importance of NP surface properties in the appropriate test medium, in addition to the chemical composition, particle size, shape and size distribution.  Size Because of their small size, NPs have a much larger surface area per unit mass compared with their parent materials. The number of atoms at the surface increases exponentially as size decreases. As a result, NPs are very reactive in biological systems. They have a high surface energy and also are more toxic, in contrast to larger particles of the same chemical composition (Chan 2006). Thus, evaluating the toxicity of engineered NPs cannot rely on extrapolation of toxicity data from larger particles, as was shown in several studies (Jacobsen et al. 2008). It is also reported that NPs can disperse throughout the whole body; however, few of them are reported to penetrate across different barriers, enter individual cells and interact with biomolecules on the cell surface and within the cells (Chan 2006; McNeil 2005). Different sizes of NPs were tested for the possibility of sizedependent genotoxicity (Barnes et al. 2008). Gurr et al. (2005) investigated the effect of size on induction of DNA damage by studying different sizes of TiO2 particles (anatase – 10, 20 nm, 200 and >200 nm, rutile – 200 nm). The results have shown the higher potency of 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 regarding the characterisation and agglomeration of NPs in phosphate buffered saline (PBS) (Gurr et al. 2005). Papageorgiou et al. (2007) compared the genotoxic effects of NP and micron-sized particles of Co/Cr alloy in human ﬁbroblasts. The NPs caused more DNA damage than the micron-sized particles. The authors reported that different biological responses of human ﬁbroblasts to surgical implant materials in vitro may be dependent on the particle size (Papageorgiou et al. 2007). Xu et al. (2009) demonstrated that TiO2 at nanoscale increased the mutant yield at the gam and redBA loci in MEF cells, while TiO2 at micro-scale had little effect on mutation induction. Several in vivo and in vitro studies consistently show that transition from the micro-scale to nanoscale size range increases toxicity (Kang et al. 2011; Toyooka et al. 2012). The diameter of inhaled or instilled particles is thus an important factor inﬂuencing the toxicity response (Xu et al. 2009). For example, the DNA-damaging potential of amorphous silica tested in four sizes, one micro (498 nm) and three nano (68, 43 and 19 nm), was shown to be size-dependent. With decreasing particle size, the level of DNA damage in cells gradually increased (Li et al. 2011b). Different particle sizes may produce different kinetic properties of substances and may result in enhanced or reduced uptake, distribution, metabolism and elimination (Nohynek et al. 2008). With reduction in size, the properties can change dramatically, regarding for example electrical conductivity, magnetic characteristics, hardness, active surface area, chemical reactivity and biological activity (Karlsson et al. 2008). Particle shape vs. chemical composition The shape and the chemical composition of the NPs play an important role in induction of cytotoxicity and genotoxicity. Several reports have shown the size- and shapedependent cytotoxicity of the NPs (Nohynek et al. 2008; Yang et al. 2009). It has also been shown that the shape  Z. Magdolenova et al. Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. of the NPs affects their internalisation into the cells. SWCNTs have different toxicological properties from CB particles because of their different shape (Nohynek et al. 2008). Yang and colleagues have also reported that DNA damage induced by carbon nanotubes is due to the mechanical injury and not due to the oxidative effect induced by them (Yang et al. 2009). Guo et al. (2011) have pointed out that the length of MWCNTs affects the penetration and toxicity of the MWCNT. It is also reported that the different particles (MWCNT & asbestos ﬁbres) of same shape and size have a similar genotoxic effect (Poland et al. 2008). Chemical composition is important as 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. Crystalline structure Crystalline structure could potentially have impact on genotoxicity; regarding TiO2 NPs, different crystalline forms such as rutile, anatase or its combinations in various proportions are used in toxicology studies. Petković et al. (2011) suggested that the different genotoxicity responses induced by anatase and rutile TiO2 NPs could depend not only on size but also on crystalline structure. Surface area The surface area of particles is closely associated with their size. The smaller the particles, the larger the surface area per unit mass. The number of surface atoms and molecules increases exponentially and thus higher chemical reactivity is expected. With larger surface area the number of free radicals and transient metal ions arising from the NP surface increases and thus the opportunity for their possible interaction with cells increases as well. A direct relationship between surface area and ROS formation was observed by Li et al. (2011b). ROS formation and DNA damage were size-dependent and thus a higher surface area could be an important factor. As total surface area could determine NP genotoxicity, Gonzalez et al. (2010) expressed results with doses as a function of surface area (m2/mL) to compare with expression in mass dose (mg/mL) or particle number/mL. Surface properties The surface properties, including chemistry and charge, are important factors in determining genotoxicity. Various surface modiﬁcations enable binding of chemical, molecular or other biological entities to NPs (McNeil et al. 2005). Ahamed et al. (2008) suggest on the basis of their experiments that differences in surface chemistry of Ag NP determine differences in genotoxic effects. Polysaccharidecoated Ag NPs induce more severe damage than uncoated Ag NPs. Different surface chemistry of the NPs results in their different behaviour in solution. The uncoated NPs tend to agglomerate while the coated are more dispersed. The surface charge determines whether NPs can be dissolved in medium or whether they form aggregates; it can also inﬂuence their biocompatibility and ability to traverse biological barriers (McNeil et al. 2005). NP surface modiﬁcations and coating inﬂuence cytotoxicity and genotoxicity. Yin et al. (2010) showed that genotoxicity of ZnO NPs in three-dimensional (3D) mini organ cultures of human nasal mucosa differed depending on coating. Several characteristics of nanomaterials change depending on the medium and environment. The surface of NPs in a biological environment is modiﬁed by the adsorption of biomolecules such as proteins, polysaccharides and lipids. These interact with the NP surface forming a relatively stable ’biomolecular corona’ (Monopoli et al. 2011). Thus, the same NPs in different experimental environments can give different outcomes. It is therefore important to test NPs under conditions similar to those of potential human exposure. Agglomeration The agglomeration potential of NPs is an important feature which may inﬂuence their behaviour and impact on genotoxicity. Highly agglomerated NPs cannot enter the nucleus and mitochondria while NPs that do not agglomerate can be distributed all over the cell (Ahamed et al. 2008; Dhawan et al. 2009). TiO2 NPs were found to be internalised into human skin epidermal cells or to adhere to the cell membrane, depending on their size. NPs of 30–100 nm were found in the cytoplasm, vesicles and nucleus, while larger particles (>500 nm) remained outside the cells (Shukla et al. 2011a). In medium, NPs can be dissolved or tend to form agglomerates/aggregates, depending on their surface charge (hydrophilic or hydrophobic) and interactions with medium (medium pH, salinity, protein content, etc.). The surface can be modiﬁed to prevent agglomeration. The chemical and physical behaviour of nanomaterials is not well understood. Some NPs form aggregates and rapidly drop out of suspension. In that case, constant resuspension is necessary in order to maintain a homogeneous solution (Colognato et al. 2008). TiO2 NPs are prone to form agglomerates in solutions (Trouiller et al. 2009). To understand the size distribution, Gurr et al. (2005) tested particle sizes in culture medium. Surprisingly, 10 and 20 nm TiO2 produced aggregations of 1000 nm in diameter, while the 200 nm particles showed no aggregation. The state of agglomeration of thermoresponsive poly N-isopropylacrylamide (PNIPAM) NPs was dependent on temperature and vehicle (water, cell culture medium, presence of fetal bovine serum (FBS)) in which they were prepared (Naha et al. 2010). We found that the level of agglomeration of NPs has an impact on cytotoxicity and genotoxicity, with different outcomes of TiO2 exposure depending on the state of NP agglomeration and dispersion (Magdolenova et al. 2012). Solubility Solubility is a key factor in the assessment of the intrinsic/ extrinsic properties of NPs, as this can increase or decrease the bioavailability of NPs to the living system. The solubility of NPs can be predicted from their structure and reactive groups present on the surface. Some of the NPs are reported to produce ions in soluble form which are toxic to the cells (Franklin et al. 2007). More soluble NPs, such as ZnO and FeO, show greater cytotoxicity than insoluble NPs, such as CeO2 and TiO2, in human mesothelioma MSTO-211H and rodent 3T3 ﬁbroblast cells; however, the correlation with the Genotoxicity of nanoparticles phenomenon of dissolution was not demonstrated experimentally (Brunner et al. 2006). In contrast to the aforesaid statement, there are reports that the toxic responses observed on exposure to NPs are due to the NPs per se rather than the ions released from them in different cellular systems and exposure conditions (Gojova et al. 2007; Lin et al. 2009; Sharma et al. 2012). Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Conditions inﬂuencing the results of genotoxicity studies Variability in the results of genotoxicity studies can be attributed to the source of NPs, the method of preparation or synthesis, the dispersion protocol, and variables in experimental conditions such as physico-chemical speciﬁcations (pH, temperature, presence of impurities or irradiation), treatment regime, cell type used, exposure time and dose (Shukla et al. 2011a; Handy et al. 2008, 2012; Vevers & Jha 2008; Reeves et al. 2008). Preparation of the NPs Properties of NPs and their genotoxic effect can be inﬂuenced by the solvents used for dispersing or dissolving them. Solvents differ in their physical and chemical properties. Factors such as pH, salinity, water hardness, temperature and the presence of dissolved or natural organic particles could inﬂuence the biological response or toxicity (Handy et al. 2008; Vevers & Jha 2008). Therefore, NPs may behave differently in different solvents (water, culture medium, PBS). This could have pronounced effects on their uptake, cellular localisation and hence the observed toxic response. For example, Vevers and Jha (2008) examined the effect on cells of well-characterised NPs dispersed in tissue culture medium, PBS or water. In medium, NPs can be surrounded by a combination of biomolecules such as proteins, forming a protein corona which could affect their genotoxic potential (Gonzalez et al. 2010). NPs are often stabilised with proteins (FBS or bovine serum albumin (BSA)) to prevent their agglomeration. The formation of a protein corona helps NPs to disperse better in the medium. Sonication can be used to prepare dispersed NPs. As discussed in Barillet et al. (2010), the preparation of stable dispersed suspensions of SiC NPs by sonication may promote the rapid oxidation of Si surface atoms to SiO2 and consequently may trigger ROS production on their surface. Metal impurities in the medium can also produce ROS via Fenton-like reactions and so it is important to test if their presence is not the cause of genotoxicity, rather than the NPs themselves.  of the quantity of NPs in air, water, soil or any consumer product is a technical challenge due to their tiny size and the small quantity present. It is also suggested that the concentration/dose of NPs should not exceed a limit that enhances agglomeration. The agglomeration of the NPs affects their bioavailability to the cell, thereby leading to false positive/ negative results. Physical or chemical agents or impurities Toxicological responses can also change in the presence of other chemical (polycyclic aromatic hydrocarbons) or physical (UV irradiation) agents (Vevers & Jha 2008). Some NPs such as ZnO NPs or C60 NPs are able to generate ROS under irradiation (Hackenberg et al. 2011a; Shinohara et al. 2009). Several studies have investigated potential genotoxic effects of NPs under UV irradiation, as well as visible light causing phototoxicity (Table I). For example, a solution of Au NPs stabilised by citrate ions was not mutagenic but photomutagenic (light irradiation) due to coexisting citrate and Au3+ ions present in solution (since Au NPs were not puriﬁed) (Wang et al. 2011a). Cell type Cytotoxicity of NPs also depends on the cell type used (Karlsson et al. 2008; Dusinska et al. 2012). Different cell types (epithelial, connective, neural, macrophages, etc.) vary in their metabolic activities (Vevers & Jha 2008). Cell lines of same or different tissue origin may be less or more susceptible to NP exposure on account of variation in metabolic pathways, cell surface receptors, antioxidant and DNA repair capabilities, presence of different enzymes/hormones, etc. All these factors may affect the behaviour, fate and interaction of the NPs with the particular cell type. Also, the interactions of NPs with different cell lines vary because they have different internalisation, phagocytosis and cytoplasmic inclusion properties. There are also possible inherent differences between cells which are phylogenetically different (for instance, ﬁsh cells compared with mammalian cells) (Reeves et al. 2008). Most studies so far have used human (A549, BEAS, MRC, 16HBE13) or animal (rat, mice hamster) lung cells (V79, BAL, RLE, CHL/IU). Primary cells or cells originating from blood and liver are also often used (Table I). It should be noted that NPs may have different effects on Salmonella cursiva or E. coli used in bacterial reverse mutation test due to the bacterial cell wall which could prevent NP penetration (Karlsson 2010) and this test is therefore not suitable for assessing human-related NP genotoxicity (Dusinska et al. 2012; Handy et al. 2012). Bioavailability and uptake Concentration of NPs The concentration/dose of the NPs is a crucial part in the assessment of their cytotoxic and genotoxic potential. NPs have a tendency to agglomerate in dry form and in suspension, because of van der Waal’s forces. Therefore, it is suggested that a parallel control should be run to access the stability of NP suspension over time. The concentration/ dose of the NPs for an in vitro experiment should mimic the actual exposure to the human. However, the determination Availability of the NPs to the cell/tissue and their uptake is one of the major factors that can provide important information about their adverse effects on cellular systems. The exponential increase in usage of the NP-containing products in daily life has also enhanced the likelihood of their interaction with the individual cell. The fate of the NPs largely depends on behaviour, bioavailability and their interaction with the surrounding medium. Proteins are usually adsorbed on the surface of the NPs by different electrostatic, hydrogen Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only.  Z. Magdolenova et al. bonding and hydrophobic interactions and affect the dispersity, uptake and bioavailability of the particles (Kumar et al. 2011a). NPs in aqueous suspension are dispersed due to the electrostatic and steric repulsion of the surface charge (positive/negative) present on the particle. As the surface charges of the particle skew towards the zero value, the repulsive forces between the particles are reduced, which leads to the settling of NPs by gravitational force. Due to agglomeration/ aggregation, the physico-chemical properties such as surface charge, size, size distribution, surface-to-volume ratio and surface reactivity of NPs are altered, affecting their uptake, bioavailability and toxicological responses. Apart from the proteins, several other factors can also inﬂuence the aggregation, uptake and bioavailability of the NPs, such as salt ions, presence of hydrophobic surfactant or polar groups on the surface of NPs. Trace metal ion speciation, e.g. from metal oxide NPs, might alter the property of NPs and therefore their uptake, bioavailability and potential toxicity. The detection of NP internalisation in any model organism is a crucial step for understanding their behaviour and toxicity. The commonly used methods for assessment of uptake of NPs in the cells are TEM, confocal and ﬂuorescence microscopy, reﬂection-based imaging and ﬂow cytometry (Shukla et al. 2011a, b; Sharma et al. 2012). Type of assay, biological system and conditions used Assays used to investigate NP genotoxicity can measure various endpoints. Generally they are methods measuring DNA damage and mutations as consequences of unrepaired DNA damage. For example, the comet assay can detect DNA damage while the HPRT assay measures gene mutations. Large chromosomal abnormalities can be detected by the micronucleus or chromosomal aberration assays. In comparison with in vitro methods that can detect only primary genotoxicity, in vivo testing can capture secondary genotoxicity arising due to ROS produced by inﬂammatory cells. Recent reports show little correlation between in vitro and in vivo NP toxicity results (Mahmoudi et al. 2010; Sayes et al. 2007). Additionally, factors such as exposure scenario (dose, exposure time, cell type, presence of proteins, etc.) could inﬂuence the results of genotoxicity testing (Schins 2002; Vevers & Jha 2008; Mahmoudi et al. 2010; Sayes et al. 2007). Variation of NP characteristics in different media (for example lung ﬂuid vs. cell culture medium) should therefore be taken into consideration, in addition to exposure time (longterm vs. short-term effects) and NP dose (Mahmoudi et al. 2010; Sayes et al. 2007). In in vitro studies the concentrations may be excessive compared to in vivo studies, where targeted organs are probably not exposed to NPs at such high doses. For assessment of NPs’ genotoxic potential it is important to use also non-cytotoxic concentrations. During cell death (apoptosis or necrosis), formation of DSBs and subsequent increased DNA fragmentation can occur, emphasising the importance of measuring cytotoxicity together with genotoxicity to prevent false-positive results. Thus, sensitive methods that can detect low-level DNA damage (a few hundred breaks per cell) and speciﬁc DNA lesions, such as the comet assay, should be used. Exposure time must be taken into account while comparing in vivo vs. in vitro studies. NPs that do not cross the nuclear envelope via nuclear pores only have an opportunity to get into contact with DNA during mitosis when the nuclear membrane dissolves, and so the time of exposure should be relatively long compared with the cell cycle period, to ensure maximum accessibility (Di Virgilio et al. 2010). Furthermore, the presence of proteins that adsorb to the NP surface forming a corona (a process known as opsonisation in medical terminology) can affect their toxicological effect. A recent study (Walczyk et al. 2010) shows that various nanomaterials dispersed in a bioﬂuid typically involve a monomeric NP core, with a strongly associated and relatively stable protein ‘hard corona’ (consistent with one or two packed proteins layers) coexisting with NP multimer–corona complexes (dimers, trimers etc.) present in smaller quantities. Therefore, we need to take into account the fact that mechanisms of NP genotoxicity might differ because of the different amounts of proteins present in in vivo and in vitro testing systems. While in in vitro systems the concentration of proteins (FBS, BSA) in NP dispersions and cell cultivation media varies from 0% up to approximately 10%, the presence of proteins varies from tissue to tissue in vivo and most importantly the components differ from those used in in vitro studies. For example, the conﬂicting results published on TiO2 NPs might be partially related to the presence or absence of proteins (FBS, BSA) as well as to sonication in the TiO2 NP dispersion protocol. The negative genotoxic results observed for instance by Hackenberg et al. (2010, 2011c) were obtrained using BSA and sonication in the NP dispersion protocol. However, negative results were also observed by Warheit et al. (2007), even though they did not use any proteins in dispersing the NPs. In this study there is a need to consider also whether the genotoxicity assays used (Ames and CHA) are suitable for the assessment of genotoxicity of NPs since most of the available literature data show negative results using these two methods (Wirnitzer et al. 2009; Mori et al. 2006; Shinohara et al. 2009; Aoshima et al. 2010; Kim et al. 2006). In our study, we compared two dispersion protocols, one with serum in stock solution and one without, giving TiO2 NP dispersions with different stability and agglomeration states, in order to investigate whether dispersion procedures and presence of serum inﬂuence NP genotoxicity. We found that the level of agglomeration of NPs and size distribution depend on the dispersion procedure and use of serum in the stock solution. The TiO2 NP dispersion with large agglomerates (3 min sonication and no serum in stock solution) induced DNA damage, while the TiO2 NPs dispersed with agglomerates less than 200 nm (FBS in stock solution and sonication 15 min) had no effect on genotoxicity (Magdolenova et al. 2012a). Similarly, Toyooka et al. (2012) found that BSAsurface coating is related to the generation of -H2AX. Formation of DSBs (detected by -H2AX) was less extensive with BSA-coated than in uncoated TiO2 NPs. In most studies (Di Virgilio et al. 2010; Gurr et al. 2005; Osman et al. 2010; Reeves et al. 2008) with positive results on TiO2 NP genotoxicity, no proteins were used (or at least they Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Genotoxicity of nanoparticles were not listed in methods) for the dispersion of NPs. However, some of the studies which show positive genotoxicity results used proteins (Wang et al. 2007; Shukla et al. 2011). Both the content and the amount of proteins in dispersion might be important for forming the protein corona. In our study (Magdolenova et al. 2012a) we used 20% FBS in one of the dispersion protocols (which gave stable TiO2 NP dispersion and negative genotoxicity result), while the abovementioned studies used 5% or 10% FBS in the dispersions. In addition to considering test conditions, it is important to test for possible interference of NPs with the chosen genotoxicity assay and to include a sufﬁcient number of relevant controls and reference materials to avoid falsepositive/negative results (Karlsson 2010; Stone et al. 2009; Magdolenova et al. 2012b; Sharma et al. 2012; Guadagnini et al. 2013). The assessment of NP genotoxicity cannot rely only on one test as there could be several mechanisms leading to mutagenicity. The use of liver microsomal S9 fraction in some studies could affect results, through the presence of additional proteins as well as the possibility of NPs becoming mutagenic after metabolic activation by a mixture of liver enzymes.  standardised and validated (Dusinska et al. 2012; Stone et al. 2009; Dusinska et al. 2009). As there is currently no speciﬁc regulatory requirement to test NPs for safety, health and environmental impacts (Chan 2006; Dusinska et al. 2011), a framework for their testing needs to be established. Last but not least, many studies, both in vitro and in vivo, show positive effects most likely due to the use of concentrations that are not relevant to possible environmental exposure. In many studies a demonstration of genotoxicity simply reﬂects cytotoxicity, as excessively high concentrations are used. Thus, cytotoxicity should be an integral part of genotoxicity testing to avoid false-positive results. There are many challenges in nanogenotoxicity due to several possible mechanisms and the diversity of pathways that can lead to a ﬁnal outcome of mutagenicity. These may involve also cell signalling pathways, authophagy and epigenetic changes. To deal with these novel possibilities, new endpoints and biomarkers might have to be considered. Acknowledgments Supported by EC FP7 [Health-2007-1.3-4], Contract no: 201335, www.nanotest-fp7.eu. Final remarks Declaration of interest Published genotoxicological data for nanomaterials are difﬁcult to compare even with the same NPs, since important information is often missing, especially a detailed characterisation of the nanomaterial, and in addition the studies may differ in terms of experimental conditions, purity of NPs, dispersion protocol, stability of the suspension in biological media and other physico-chemical properties (Auffan et al. 2006; Stone et al. 2010). Various other factors, in addition to cellular and particle properties, and exposure scenarios could inﬂuence this apparent lack of consistent results for the toxic effect of NPs (Vevers & Jha 2008). Both primary as well as secondary characterisation of tested NPs are crucial, including in situ characterisation during exposure. It is clear that physical and chemical properties can inﬂuence NP behaviour and may have an impact on genotoxicity; they must therefore be an integral part of genotoxicity testing. This is one of the key aspects of toxicity screening strategies (Oberdörster et al. 2005a,b; Dusinska et al. 2009). The physico-chemical properties that should be considered for assessing toxic effects of nanomaterials include at least chemical composition, particle size, shape, surface properties, size distribution, agglomeration state and crystal structure. Regarding the likelihood of opsonisation (biomolecular corona formation), it is also important to set up experimental conditions that can mimic exposure in humans, preferably using human serum with human cells and medium with chemical composition similar to human plasma. Extrapolation from in vivo studies in nanotoxicity testing is even more challenging than with chemical toxicology, and due to the enormous variety of NPs being produced, alternative in vitro toxicity tests will have to be further considered. Methods for assessment of potential hazard of NPs based on a battery of cytotoxicity and genotoxicity assays need to be The authors report no conﬂicts of interest. The authors alone are responsible for the content and writing of the paper. References Ahamed M, Karns M, Goodson M, Rowe J, Hussain SM, Schlager JJ, et al. 2008. DNA damage response to different surface chemistry of silver nanoparticles in mammalian cells. Toxicol Appl Pharmacol 233:404–410. An H, Liu Q, Ji Q, Jin B. 2010. DNA binding and aggregation by carbon nanoparticles. Biochem Biophys Res Commun 393:571–576. Aoshima H, Yamana S, Nakamura S, Mashino T. 2010. Biological safety of water-soluble fullerenes evaluated using tests for genotoxicity, phototoxicity, and pro-oxidant activity. J Toxicol Sci 35:401–409. Arora S, Rajwade JM, Paknikar KM. 2012. Nanotoxicology and in vitro studies: the need of the hour. Toxicol Appl Pharmacol 258:151–165. Asare N, Instanes C, Sandberg WJ, Refsnes M, Schwarze P, Kruszewski M, et al. 2012. Cytotoxic and genotoxic effects of silver nanoparticles in testicular cells. Toxicology 291:65–72. Asharani PV, Low Kah Mun G, Hande MP, Valiyaveettil S. 2009. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano 3:279–290. Asharani PV, Xinyi N, Hande MP, Valiyaveettil S. 2010. DNA damage and p53-mediated growth arrest in human cells treated with platinum nanoparticles. Nanomedicine (Lond) 5:51–64. Auffan M, Decome L, Rose J, Orsiere T, De Meo M, Briois V, et al. 2006. In vitro interactions between DMSA-coated maghemite nanoparticles and human ﬁbroblasts: a physicochemical and cyto-genotoxical study. Environ Sci Technol 40:4367–4373. Balasubramanyam A, Sailaja N, Mahboob M, Rahman MF, Hussain SM, Grover P. 2010. In vitro mutagenicity assessment of aluminium oxide nanomaterials using the Salmonella/microsome assay. Toxicol In Vitro 24:1871–1876. Barillet S, Jugan ML, Laye M, Leconte Y, Herlin-Boime N, Reynaud C, et al. 2010. In vitro evaluation of SiC nanoparticles impact on A549 pulmonary cells: cyto-, genotoxicity and oxidative stress. Toxicol Lett 198:324–330. Barnes CA, Elsaesser A, Arkusz J, Smok A, Palus J, Leśniak A, et al. 2008. Reproducible comet assay of amorphous silica nanoparticles detects no genotoxicity. Nano Lett 8:3069–3074. Baweja L, Gurbani D, Shanker R, Pandey AK, Subramanian V, Dhawan A. 2011. C60-fullerene binds with the ATP binding domain of human DNA topoiosmerase II alpha. J Biomed Nanotechnol 7:177–178. Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only.  Z. Magdolenova et al. Bernardeschi M, Guidi P, Scarcelli V, Frenzilli G, Nigro M. 2010. Genotoxic potential of TiO2 on bottlenose dolphin leukocytes. Anal Bioanal Chem 396:619–623. Bhattacharya K, Davoren M, Boertz J, Schins RP, Hoffmann E, Dopp E. 2009. Titanium dioxide nanoparticles induce oxidative stress and DNA-adduct formation but not DNA-breakage in human lung cells. Part Fibre Toxicol 6: 17. Bonassi S, Norppa H, Ceppi M, Strömberg U, Vermeulen R, Znaor A, et al. 2008. Chromosomal aberration frequency in lymphocytes predicts the risk of cancer: results from a pooled cohort study of 22 358 subjects in 11 countries. Carcinogenesis 29:1178– 1183. Bourdon JA, Saber AT, Jacobsen NR, Jensen KA, Madsen AM, Lamson JS, et al. 2012. Carbon black nanoparticle instillation induces sustained inﬂammation and genotoxicity in mouse lung and liver. Part Fibre Toxicol 9:5. Brunner TJ, Wick P, Manser P, Spohn P, Grass RN, Limbach LK. 2006. In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica, and the effect of particle solubility. Environ Sci Technol 40:4374–4381. Calzolai L, Franchini F, Gilliland D, Rossi F. 2010. Protein-nanoparticle interaction: identiﬁcation of the ubiquitin-gold nanoparticle interaction site. Nano Lett 10:3101–3105. Chan VS. 2006. Nanomedicine: an unresolved regulatory issue. Regul Toxicol Pharmacol 46:218–224. Chi Z, Liu R, Zhao L, Qin P, Pan X, Sun F, et al. 2009. A new strategy to probe the genotoxicity of silver nanoparticles combined with cetylpyridine bromide. Spectrochim Acta A Mol Biomol Spectrosc 72:577–581. Choi JY, Lee SH, Na HB, An K, Hyeon T, Seo TS. 2010. In vitro cytotoxicity screening of water-dispersible metal oxide nanoparticles in human cell lines. Bioprocess Biosyst Eng 33:21–30. Chompoosor A, Saha K, Ghosh PS, Macarthy DJ, Miranda OR, Zhu ZJ, et al. 2010. The role of surface functionality on acute cytotoxicity, ROS generation and DNA damage by cationic gold nanoparticles. Small 6:2246–2249. Collins AR, Dusinska M, Gedik CM, Stetina R. 1996. Oxidative damage to DNA: do we have a reliable biomarker? Environ Health Perspect 104:465–469. Colognato R, Bonelli A, Ponti J, Farina M, Bergamaschi E, Sabbioni E, et al. 2008. Comparative genotoxicity of cobalt nanoparticles and ions on human peripheral leukocytes in vitro. Mutagenesis 23:377–382. Cooke MS, Evans MD, Dizdaroglu M, Lunec J. 2003. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J 17:1195–1214. Dandekar P, Dhumal R, Jain R, Tiwari D, Vanage G, Patravale V. 2010. Toxicological evaluation of pH-sensitive nanoparticles of curcumin: acute, sub-acute and genotoxicity studies. Food Chem Toxicol 48:2073–2089. Dhawan A, Taurozzi JS, Pandey AK, Shan W, Miller SM, Hashsham SA, et al. 2006. Stable colloidal dispersions of C60 fullerenes in water: evidence for genotoxicity. Environ Sci Technol 40:7394–7401. Dhawan A, Sharma V, Parmar D. 2009. Nanomaterials: a challenge for toxicologists. Nanotoxicology 3:1–9. Di Sotto A, Chiaretti M, Carru GA, Bellucci S, Mazzanti G. 2009. Multi-walled carbon nanotubes: Lack of mutagenic activity in the bacterial reverse mutation assay. Toxicol Lett 184:192–197. Di Virgilio AL, Reigosa M, Arnal PM, Fernández Lorenzo de Mele M. 2010. Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells. J Hazard Mater 177:711–718. Doak SH, Grifﬁths SM, Manshian B, Singh N, Williams PM, Brown AP, et al. 2009. Confounding experimental considerations in nanogenotoxicology. Mutagenesis 24:285–293. Donaldson K, Poland CA, Schins RP. 2010. Possible genotoxic mechanisms of nanoparticles: criteria for improved test strategies. Nanotoxicology 4:414–420. Du H, Zhu X, Fan C, Xu S, Wang Y, Zhou Y. 2011. Oxidative damage and OGG1 expression induced by a combined effect of titanium dioxide nanoparticles and lead acetate in human hepatocytes. Environ Toxicol. [Epub ahead of print]. Dusinska M, Collins AR. 1996. Detection of oxidised purines and UV-induced photoproducts in DNA, by inclusion of lesion-speciﬁc enzymes in the comet assay (single cell gell electrophoresis). ATLA 24:405–411. Dusinska M; NanoTEST consortium. 2009. Testing strategies for the safety of nanoparticles used in medical applications. Nanomedicine (Lond) 4:605–607. Dusinska M, Fjellsbø LM, Magdolenova Z, Ravnum S, Rinna A, Rundén-Pran E. 2011. Safety of Nanoparticles in Medicine. In: Nanomedicine in Health and Disease. USA: Science Publishers, Chap. 11, pp. 203–226. Dusinska M, Rundén-Dusinska M, Rundén-Pran E, Carreira SC, Saunders M. 2012. In vitro and in vivo toxicity test methods. Chapter 4. Critical evaluation of toxicity tests. In: Fadeel B, Pietroiusti A, Shvedova A, editors. Adverse effects of engineered nanomaterials: exposure, toxicology and impact on human health. Elsevier; pp. 63–84. Ema M, Tanaka J, Kobayashi N, Naya M, Endoh S, Maru J, et al. 2012. Genotoxicity evaluation of fullerene C60 nanoparticles in a comet assay using lung cells of intratracheally instilled rats. Regul Toxicol Pharmacol 2:419–424. Eom HJ, Choi J. 2010. p38 MAPK activation, DNA damage, cell cycle arrest and apoptosis as mechanisms of toxicity of silver nanoparticles in Jurkat T cells. Environ Sci Technol 44:8337–8342. Estevanato L, Cintra D, Baldini N, Portilho F, Barbosa L, Martins O, et al. 2011. Preliminary biocompatibility investigation of magnetic albumin nanosphere designed as a potential versatile drug delivery system. Int J Nanomed 6:1709–1717. Falck GC, Lindberg HK, Suhonen S, Vippola M, Vanhala E, Catalán J, et al. 2009. Genotoxic effects of nanosized and ﬁne TiO2. Hum Exp Toxicol 28:339–352. Fenech M, Kirsch-Volders M, Natarajan AT, Surralles J, Crott JW, Parry J, et al. 2011. Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis 26:125–132. Flower NA, Brabu B, Revathy M, Gopalakrishnan C, Raja SV, Murugan SS, et al. 2012. Characterization of synthesized silver nanoparticles and assessment of its genotoxicity potentials using the alkaline comet assay. Mutat Res 742:61–65. Foldbjerg R, Dang DA, Autrup H. 2011. Cytotoxicity and genotoxicity of silver nanoparticles in the human lung cancer cell line, A549. Arch Toxicol 85:743–750. Folkmann JK, Risom L, Jacobsen NR, Wallin H, Loft S, Møller P. 2009. Oxidatively damaged DNA in rats exposed by oral gavage to C60 fullerenes and single-walled carbon nanotubes. Environ Health Perspect 117:703–708. Franklin NM, Rogers NJ, Apte SC, Batley GE, Gadd GE, Casey PS. 2007. Comparativetoxicity of nanoparticulate ZnO, bulkZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata): the importance of particle solubility. Environ Sci Technol 41:8484–8490. Freitas MLL, Silva LP, Azevedo RB, Garcia VAP, Lacava LM, Grisolia CK, et al. 2002. A double-coated magnetite-based magnetic ﬂuid evaluation by cytometry and genetic tests. J Magn Magn Mater 252:396–398. Galloway SM, Armstrong MJ, Reuben C, Colman S, Brown B, Cannon C, et al. 1987. Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals. Environ Mol Mutagen 10(Suppl 10): 1–175. Galloway SM, Aardema MJ, Ishidate M Jr, Ivett JL, Kirkland DJ, Morita T, et al. 1994. Report from working group on in vitro tests for chromosomal aberrations. Mutat Res 312:241–261. Guadagnini R, Halamoda B, Walker L, Pojana G, Magdolenova Z, Bilanicova D, et al. 2013. Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests. Nanotoxicology, Special issue – supplement, in press. Gojova A, Guo B, Kota RS, Rutledge JC, Kennedy IM, Barakat AI. 2007. Induction of inﬂammation in vascular endothelial cells by metal oxide nanoparticles: effect of particle composition. Environ Health Perspect 115:403–409. Gonzalez L, Lison D, Kirsch-Volders M. 2008. Genotoxicity of engineered nanomaterials: a critical review. Nanotoxicology 2:252–273. Gonzalez L, Sanderson BJ, Kirsch-Volders M. 2011. Adaptations of the in vitro MN assay for the genotoxicity assessment of nanomaterials. Mutagenesis 26:185–191. Gonzalez L, Thomassen LC, Plas G, Rabolli V, Napierska D, Decordier I, et al. 2010. Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models. Nanotoxicology 4:82–95. Ghosh M, Bandyopadhyay M, Mukherjee A. 2010. Genotoxicity of titanium dioxide (TiO2) nanoparticles at two trophic levels: plant and human lymphocytes. Chemosphere 81:1253–62. Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Genotoxicity of nanoparticles Green M, Howman E. 2005. Semiconductor quantum dots and free radical induced DNA nicking. Chem Commun 7:121–123. Guo YY, Zhang J, Zheng YF, Yang J, Zhu XQ. 2011. Cytotoxic and genotoxic effects of multi-wall carbon nanotubes on human umbilical vein endothelial cells in vitro. Mutat Res 721:184–191. Gupta SK, Baweja L, Gurbani D, Pandey AK, Dhawan A. 2011. Interaction of C60 fullerene with the proteins involved in DNA mismatch repair pathway. J Biomed Nanotechnol 7:179–180. Gurr JR, Wang AS, Chen CH, Jan KY. 2005. Ultraﬁne titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells. Toxicology 213:66–73. Hackenberg S, Friehs G, Froelich K, Ginzkey C, Koehler C, Scherzed A, et al. 2010. Intracellular distribution, geno- and cytotoxic effects of nanosized titanium dioxide particles in the anatase crystal phase on human nasal mucosa cells. Toxicol Lett 195:9–14. Hackenberg S, Friehs G, Kessler M, Froelich K, Ginzkey C, Koehler C, et al. 2011c. Nanosized titanium dioxide particles do not induce DNA damage in human peripheral blood lymphocytes. Environ Mol Mutagen 52:264–268. Hackenberg S, Scherzed A, Kessler M, Hummel S, Technau A, Froelich K, et al.. 2011b. Silver nanoparticles: evaluation of DNA damage, toxicity and functional impairment in human mesenchymal stem cells. Toxicol Lett 201:27–33. Hackenberg S, Scherzed A, Technau A, Kessler M, Froelich K, Ginzkey C, et al.. 2011a. Cytotoxic, genotoxic and pro-inﬂammatory effects of zinc oxide nanoparticles in human nasal mucosa cells in vitro. Toxicol In Vitro 25:657–663. Hackenberg S, Zimmermann FZ, Scherzed A, Friehs G, Froelich K, Ginzkey C, et al. 2011d. Repetitive exposure to zinc oxide nanoparticles induces DNA damage in human nasal mucosa mini organ cultures. Environ Mol Mutagen 52:582–589. Handy RD, Owen R, Valsami-Jones E. 2008. The ecotoxicology of nanoparticles and nanomaterials: current status, knowledge gaps, challenges, and future needs. Ecotoxicology 17:315–325. Handy RD, van den Brink N, Chappell M, Mühling M, Behra R, Dušinská M, et al. 2012. Practical considerations for conducting ecotoxicity test methods with manufactured nanomaterials: what have we learnt so far? Ecotoxicology 21:933–72. Hansen SF, Larse BH, Olsen SI, Baun A. 2007. Categorization framework to aid hazard identiﬁcation of nanomaterials. Nanotoxicology 1:243–250. Hasegawa G, Shimonaka M, Ishihara Y. 2012. Differential genotoxicity of chemical properties and particle size of rare metal and metal oxide nanoparticles. J Appl Toxicol 32:72–80. He L, Yang L, Zhang ZR, Gong T, Deng L, Gu Z, et al. 2009. In vitro evaluation of the genotoxicity of a family of novel MeO-PEG-poly(D, L-lactic-co-glycolic acid)-PEG-OMe triblock copolymer and PLGA nanoparticles. Nanotechnology 20: 455102. Hong SC, Lee JH, Lee J, Kim HY, Park JY, Cho J, et al. 2011. Subtle cytotoxicity and genotoxicity differences in superparamagnetic iron oxide nanoparticles coated with various functional groups. Int J Nanomedicine 6:3219–3231. http://cdb.iso.org http://ec.europa.eu/environment/chemicals/nanotech/index.htm# deﬁnition http://www.jacvam.jp/en_effort/en_oecd.html Huang S, Chueh PJ, Lin YW, Shih TS, Chuang SM. 2009. Disturbed mitotic progression and genome segregation are involved in cell transformation mediated by nano-TiO2 long-term exposure. Toxicol Appl Pharmacol 241:182–194. Hudecová A, Kusznierewicz B, Hašplová K, Huk A, Magdolenová Z, Miadoková E, et al. 2012a. Gentiana asclepiadea exerts antioxidant activity and enhances DNA repair of hydrogen peroxide- and silver nanoparticles-induced DNA damage. Food Chem Toxicol 50:3352–3359. Hudecová A, Kusznierewicz B, Rundén-Pran E, Magdolenová Z, Hašplová K, Rinna A, et al. 2012b. Silver nanoparticles induce pre-mutagenic DNA oxidation that can be prevented by phytochemicals from Gentiana asclepiadea. Mutagenesis 27:759–769. Hwang do W, Lee DS, Kim S. 2012. Gene expression proﬁles for genotoxic effects of silica-free and silica-coated cobalt ferrite nanoparticles. J Nucl Med 53:106–112. Iavicoli I, Leso V, Fontana L, Bergamaschi A. 2011. Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies. Eur Rev Med Pharmacol Sci 15:481–508. Ismail IH, Wadhra TI, Hammarsten O. 2007. An optimized method for detecting gamma-H2AX in blood cells reveals a signiﬁcant interindividual variation in the gamma-H2AX response among humans. Nucleic Acids Res 35: e36.  Jacobsen NR, Møller P, Jensen KA, Vogel U, Ladefoged O, Loft S, et al. 2009. Lung inﬂammation and genotoxicity following pulmonary exposure to nanoparticles in ApoE-/- mice. Part Fibre Toxicol.12;6:2. doi: 10.1186/1743-8977-6-2. PubMed PMID: 19138394; PubMed Central PMCID: PMC2636756. Jacobsen NR, Pojana G, White P, Møller P, Cohn CA, Korsholm KS, et al. 2008. Genotoxicity, cytotoxicity, and reactive oxygen species induced by single-walled carbon nanotubes and C60 fullerenes in the FE1-mutaTMmouse lung epithelial cells. Environ Mol Mutagen 49:476–487. Jacobsen NR, Saber AT, White P, Møller P, Pojana G, Vogel U, et al. 2007. Increased mutant frequency by carbon black, but not quartz, in the lacZ and cII transgenes of muta mouse lung epithelial cells. Environ Mol Mutagen 48:451–461. Jha AN. 2008. Ecotoxicological applications and signiﬁcance of the comet assay. Mutagenesis 23:207–221. Jiang H, Liu F, Yang H, Li Y. 2012. Effects of cobalt nanoparticles on human T cells in vitro. Biol Trace Elem Res 146:23–29. Jin P, Chen Y, Zhang SB, Chen Z. 2011. Interactions between Al(12)X (X = Al, C, N and P) nanoparticles and DNA nucleobases/base pairs: implications for nanotoxicity. J Mol Model. [Epub ahead of print]. Jin Y, Kannan S, Wu M, Zhao JX. 2007. Toxicity of luminescent silica nanoparticles to living cells. Chem Res Toxicol 20:1126–1133. Jugan ML, Barillet S, Simon-Deckers A, Herlin-Boime N, Sauvaigo S, Douki T, et al. 2011. Titanium dioxide nanoparticles exhibit genotoxicity and impair DNA repair activity in A549 cells. Nanotoxicology. [Epub ahead of print]. Kain J, Karlsson HL, Moller L. 2012. DNA damage induced by micro and nanoparticles - interaction with FPG inﬂuences the detection of DNA oxidation in the comet assay. Mutagenesis 27, 491–500. Kang SJ, Kim BM, Lee YJ, Chung HW. 2008. Titanium dioxide nanoparticles trigger p53-mediated damage response in peripheral blood lymphocytes. Environ Mol Mutagen 49:399–405. Kang SJ, Lee YJ, Kim BM, Choi YJ, Chung HW. 2011. Cytotoxicity and genotoxicity of titanium dioxide nanoparticles in UVA-irradiated normal peripheral blood lymphocytes. Drug ChemToxicol. 34:277–84. Karlsson HL, Cronholm P, Gustafsson J, Möller L. 2008. Copper oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes. Chem Res Toxicol 21:1726– 1732. Karlsson HL, Gustafsson J, Cronholm P, Möller L. 2009. Size-dependent toxicity of metal oxide particles–a comparison between nanoand micrometer size. Toxicol Lett 188:112–118. Karlsson HL. 2010. The comet assay in nanotoxicology research. Anal Bioanal Chem 398:651–666. Kazimirova A, Magdolenova Z, Barancokova M, Staruchova M, Volkovova K, Dusinska M. 2012. Genotoxicity testing of PLGA-PEO nanoparticles in TK6 cells by the comet assay and the cytokinesis-block micronucleus assay. Mutat Res 748:42–47. Khalil WK, Girgis E, Emam AN, Mohamed MB, Rao KV. 2011. Genotoxicity evaluation of nanomaterials: dna damage, micronuclei, and 8-hydroxy-2-deoxyguanosine induced by magnetic doped CdSe quantum dots in male mice. Chem Res Toxicol 24:640–650. Kim HR, Kim MJ, Lee SY, Oh SM, Chung KH. 2011. Genotoxic effects of silver nanoparticles stimulated by oxidative stress in human normal bronchial epithelial (BEAS-2B) cells. Mutat Res 726:129–135. Kim JS, Yoon TJ, Yu KN, Kim BG, Park SJ, Kim HW, et al. 2006. Toxicity and tissue distribution of magnetic nanoparticles in mice. Toxicol Sci 89:338–347. Kim YS, Kim JS, Cho HS, Rha DS, Kim JM, Park JD, et al. 2008. Twenty-eight-day oral toxicity, genotoxicity, and gender-related tissue distribution of silver nanoparticles in Sprague-Dawley rats. Inhal Toxicol 20:575–583. Kisin ER, Murray AR, Keane MJ, Shi XC, Schwegler-Berry D, Gorelik O, et al. 2007. Single-walled carbon nanotubes: genoand cytotoxic effects in lung ﬁbroblast V79 cells. J Toxicol Environ Health Part A 70:2071–2079. Kruszewski M, Brzoska K, Brunborg G, Asare N, Dobrzynska M, Dusinska M, et al. 2011. Toxicity of silver nanomaterials in higher eukaryotes, in advances in moleculartoxicology 5, Chap. 5, pp. 179–218. Kulmala M. 2004. Formation and growth rates of ultraﬁne atmospheric particles: a review of observations. J Aerosol Sci 35:143–176. Kumar A, Pandey AK, Singh SS, Shanker R, Dhawan A. 2011a. A ﬂow cytometric method to assess nanoparticle uptake in bacteria. Cytometry A 79: 707–712. Kumar A, Pandey AK, Singh SS, Shanker R, Dhawan A. 2011b. Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells. Chemosphere 83:1124–1132. Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only.  Z. Magdolenova et al. Könczöl M, Ebeling S, Goldenberg E, Treude F, Gminski R, Gieré R, et al. 2011. Cytotoxicity and genotoxicity of size-fractionated iron oxide (magnetite) in A549 human lung epithelial cells: role of ROS, JNK, and NF-kB. Chem Res Toxicol 24:1460–1475. Laingam S, Froscio SM, Humpage AR. 2008. Flow-cytometric analysis of in vitro micronucleus formation: comparative studies with WIL2-NS human lymphoblastoid and L5178Y mouse lymphoma cell lines. Mutat Res 656:19–26. Landsiedel R, Kapp MD, Schulz M, Wiench K, Oesch F. 2009. Genotoxicity investigations on nanomaterials: methods, preparation and characterization of test material, potential artifacts and limitations– many questions, some answers. Mutat Res 681:241–458. Lewis DJ, Bruce C, Bohic S, Cloetens P, Hammond SP, Arbon D, et al. 2010. Intracellular synchrotron nanoimaging and DNA damage/ genotoxicity screening of novel lanthanide-coated nanovectors. Nanomedicine (Lond) 5:1547–1557. Li CH, Shen CC, Cheng YW, Huang SH, Wu CC, Kao CC, et al. 2011c. Organ biodistribution, clearance, and genotoxicity of orally administered zinc oxide nanoparticles in mice. Nanotoxicology. [Epub ahead of print]. Li JJ, Lo SL, Ng CT, Gurung RL, Hartono D, Hande MP, et al.. 2011a. Genomic instability of gold nanoparticle treated human lung ﬁbroblast cells. Biomaterials 32:5515–5523. Li Y, Chen DH, Yan J, Chen Y, Mittelstaedt RA, Zhang Y, et al. 2012. Genotoxicity of silver nanoparticles evaluated using the Ames test and in vitro micronucleus assay. Mutat Res 745:4–10. Li Y, Sun L, Jin M, Du Z, Liu X, Guo C, et al.. 2011b. Size-dependent cytotoxicity of amorphous silica nanoparticles in human hepatoma HepG2 cells. Toxicol In Vitro. [Epub ahead of print]. Liang XJ, Chen C, Zhao Y, Jia L, Wang PC. 2008. Biopharmaceutics and therapeutic potential of engineered nanomaterials. Curr Drug Metab 9:697–709. Lin W, Xu Y, Huang C, Ma Y, Shannon KB, Chen DR, et al. 2009. Toxicity of nano- and micro-sized ZnO particles in human lungepithelial cells. J Nanopart Res 11:25–39. Maenosono S, Suzuki T, Saita S. 2007. Mutagenicity of water-soluble FePt nanoparticles in Ames test. J Toxicol Sci 32:575–579. Maenosono S, Yoshida R, Saita S. 2009. Evaluation of genotoxicity of amine-terminated water-dispersible FePt nanoparticles in the Ames test and in vitro chromosomal aberration test. J Toxicol Sci 34:349–354. Magdolenova Z, Bilaničová D, Pojana G, Fjellsbø LM, Hudecova A, Hasplova K, et al.. 2012a. Impact of agglomeration and different dispersions of titanium dioxide nanoparticles on the human related in vitro cytotoxicity and genotoxicity. J Environ Monit 14:455–464. Magdolenova Z, Lorenzo Y, Collins A, Dusinska M. 2012b. Can standard genotoxicity tests be applied to nanoparticles? J Toxicol Environ Health A Part A 75:1–7. Mahmoudi M, Simchi A, Imani M, Shokrgozar MA, Milani AS, Häfeli UO, et al. 2010. A new approach for the in vitro identiﬁcation of the cytotoxicity of superparamagnetic iron oxide nanoparticles. Colloids Surf B Biointerfaces 75:300–309. Matsuda S, Matsui S, Shimizu Y, Matsuda T. 2011. Genotoxicity of colloidal fullerene C60. Environ Sci Technol 45:4133–4138. McNeil SE. 2005. Nanotechnology for the biologist. J Leukoc Biol 78:585–94. Merhi M, Dombu CY, Brient A, Chang J, Platel A, Le Curieux F, et al. 2012. Study of serum interaction with a cationic nanoparticle: Implications for in vitro endocytosis, cytotoxicity and genotoxicity. Int J Pharm 423:37–44. Monopoli MP, Walczyk D, Lowry-Campbell A, Elia G, Lynch I, Baldelli Bombelli F, et al. 2011. Physical-chemical aspects of protein corona: relevance to in vitro and in vivo biological impacts of nanoparticles. J Am Chem Soc 133:2525–2534. Moreira S, Silva NB, Almeida-Lima J, Rocha HA, Medeiros SR, Alves Jr C, et al. 2009. BC nanoﬁbres: in vitro study of genotoxicity and cell proliferation. Toxicol Lett 189:235–241. Moreno-Villanueva M, Eltze T, Dressler D, Bernhardt J, Hirsch C, Wick P, et al. 2011. The automated FADU-assay, a potential high-throughput in vitro method for early screening of DNA breakage. ALTEX 28:295–303. Mori T, Takada H, Ito S, Matsubayashi K, Miwa N, Sawaguchi T. 2006. Preclinical studies on safety of fullerene upon acute oral administration and evaluation for no mutagenesis. Toxicology 225:48–54. Mortelmans K, Zeiger E. 2000. The Ames Salmonella/microsome mutagenicity assay. Mutat Res 455:29–60. Mroz RM, Schins RP, Li H, Drost EM, Macnee W, Donaldson K. 2007. Nanoparticle carbon black driven DNA damage induces growth arrest and AP-1 and NFkappaB DNA binding in lung epithelial A549 cell line. J Physiol Pharmacol 58:461–470. Mroz RM, Schins RP, Li H, Jimenez LA, Drost EM, Holownia A, et al. 2008. Nanoparticle-driven DNA damage mimics irradiation-related carcinogenesis pathways. Eur Respir J 31:241–251. Muller J, Decordier I, Hoet PH, Lombaert N, Thomassen L, Huaux F, et al. 2008. Clastogenic and aneugenic effects of multi-wall carbon nanotubes in epithelial cells. Carcinogenesis 29:427–433. Naha PC, Bhattacharya K, Tenuta T, Dawson KA, Lynch I, Gracia A, et al. 2010. Intracellular localisation, geno- and cytotoxic response of polyN-isopropylacrylamide (PNIPAM) nanoparticles to human keratinocyte (HaCaT) and colon cells (SW 480). Toxicol Lett 198:134–143. Naya M, Kobayashi N, Ema M, Kasamoto S, Fukumuro M, Takami S, et al. 2012. In vivo genotoxicity study of titanium dioxide nanoparticles using comet assay following intratracheal instillation in rats. Regul Toxicol Pharmacol 62:1–6. Nelson BC, Petersen EJ, Marquis BJ, Atha DH, Elliott JT, Cleveland D, et al. 2011. NIST gold nanoparticle reference materials do not induce oxidative DNA damage. Nanotoxicology. [Epub ahead of print]. Ng CT, Li JJ, Bay BH, Yung LY. 2010. Current studies into the genotoxic effects of nanomaterials. J Nucleic Acids. Nohynek GJ, Dufour EK, Roberts MS. 2008. Nanotechnology, cosmetics and the skin: is there a health risk? Skin Pharmacol Physiol 21:136–149. NRC. 1983. National Research Council. Risk assessment in the federal government: managing the process. Washington, DC: National Academy Press. Oberdörster G, Maynard A, Donaldson K, Castranova V, Fitzpatrick J, Ausman K, et al. 2005a. ILSI Research Foundation/Risk Science Institute Nanomaterial Toxicity Screening Working Group. Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy. Part Fibre Toxicol. 2:8. 10.1186/1743-8977-2-8 PMCID: PMC1260029. Oberdörster G, Oberdörster E, Oberdörster J. 2005b. Nanotoxicology: an emerging discipline evolving from studies of ultraﬁne particles. Environ Health Perspect 113:823–839. OECD Guideline for testing of chemicals, OECD 475. Mammalian Bone Marrow Chromosome Aberration Test, Adopted: 21st July 1997. OECD Guideline for testing of chemicals, OECD 476. In vitro Mammalian Cell Gene Mutation Test, Adopted 4 April 1984, last updated 21 July 1997. OECD Guideline for testing of chemicals. DRAFT PROPOSAL FOR A NEW GUIDELINE 487. In Vitro Mammalian Cell Micronucleus Test (MNvit), DRAFT TEST GUIDELINE, December 13, 2007 (Version 3). OECD Guideline for testing of chemicals. OECD 471. Bacterial Reverse Mutation Test, Adopted 21st July 1997. OECD Guideline for testing of chemicals. OECD 473. In vitro Mammalian Chromosomal Aberration Test, Adopted 21 July 1997. OECD Guideline for testing of chemicals. OECD 474. Mammalian Erythrocyte Micronucleus Test, Adopted: 21st July 1997. Osman IF, Baumgartner A, Cemeli E, Fletcher JN, Anderson D. 2010. Genotoxicity and cytotoxicity of zinc oxide and titanium dioxide in HEp-2 cells. Nanomedicine (Lond) 5:1193–1203. Papageorgiou I, Brown C, Schins R, Singh S, Newson R, Davis S, et al. 2007. The effect of nano- and micron-sized particles of cobalt-chromium alloy on human ﬁbroblasts in vitro. Biomaterials 28:2946–2958. PAS71. 2005. Publicly Available Speciﬁcation. Vocabulary- Nanoparticles. British Standards Institution (BSI). Petković J, Zegura B, Stevanović M, Drnovšek N, Uskoković D, Novak S, et al. 2011. DNA damage and alterations in expression of DNA damage responsive genes induced by TiO(2) nanoparticles in human hepatoma HepG2 cells. Nanotoxicology 5:341–53. Pierscionek BK, Li Y, Yasseen AA, Colhoun LM, Schachar RA, Chen W. 2010. Nanoceria have no genotoxic effect on human lens epithelial cells. Nanotechnology. 21(3):035102. doi: 10.1088/ 0957-4484/21/3/035102. Epub . PubMed PMID: 19966402. Poland CA, Dufﬁn R, Kinloch I, Maynard A, Wallace WA, Seaton A, et al. 2008. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol 3:423–428. Ponti J, Sabbioni E, Munaro B, Broggi F, Marmorato P, Franchini F, et al. 2009. Genotoxicity and morphological transformation induced by cobalt nanoparticles and cobalt chloride: an in vitro study in Balb/3T3 mouse ﬁbroblasts. Mutagenesis 24:439–445. Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. Genotoxicity of nanoparticles Rahman Q, Lohani M, Dopp E, Pemsel H, Jonas L, Weiss DG, et al. 2002. Evidence that ultraﬁne titanium dioxide induces micronuclei and apoptosis in Syrian hamster embryo ﬁbroblasts. Environ Health Perspect 110:797–800. Reeves JF, Davies SJ, Dodd NJ, Jha AN. 2008. Hydroxyl radicals (*OH) are associated with titanium dioxide (TiO2) nanoparticle-induced cytotoxicity and oxidative DNA damage in ﬁsh cells. Mutat Res 640:113–122. Rehn B, Seiler F, Rehn S, Bruch J, Maier M. 2003. Investigations on the inﬂammatory and genotoxic lung effects of two types of titanium dioxide: untreated and surface treated. Toxicol Appl Pharmacol 189:84–95. Robertazzi A, Platts JA. 2005. Binding of transition metal complexes to guanine and guanine-cytosine: hydrogen bonding and covalent effects. J Biol Inorg Chem 10:854–866. Sadeghiani N, Barbosa LS, Silva LP, Azevedo RB, Morais PC, Lacava ZGM. 2005. Genotoxicity and inﬂammatory investigation in mice treated with magnetite nanoparticles surface coated with polyaspartic acid. J Magn Magn Mater 289:466–468. Saquib Q, Al-Khedhairy AA, Siddiqui MA, Abou-Tarboush FM, Azam A, Musarrat J. 2012. Titanium dioxide nanoparticles induced cytotoxicity, oxidative stress and DNA damage in human amnion epithelial (WISH) cells. Toxicol In Vitro 26:351–361. Sayes CM, Reed KL, Glover KP, Swain KA, Ostraat ML, Donner EM, et al. 2010. Changing the dose metric for inhalation toxicity studies: short-term study in rats with engineered aerosolized amorphous silica nanoparticles. Inhal Toxicol 22:348–354. Sayes CM, Reed KL, Warheit DB. 2007. Assessing toxicity of ﬁne and nanoparticles: comparing in vitro measurements to in vivo pulmonary toxicity proﬁles. Toxicol Sci 97:163–180. Schins RP. 2002. Mechanisms of genotoxicity of particles and ﬁbers. Inhal Toxicol 14:57–78. Schulz M, Ma-Hock L, Brill S, Strauss V, Treumann S, Gröters S, et al. 2012. Investigation on the genotoxicity of different sizes of gold nanoparticles administered to the lungs of rats. Mutat Res 745:51–57. Sharma V, Anderson D, Dhawan A. 2012. Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2). Apoptosis 17:852–870. Sharma V, Shukla RK, Saxena N, Parmar D, Das M, Dhawan A. 2009. DNA damaging potential of zinc oxide nanoparticles in human epidermal cells. Toxicol Lett 185:211–218. Shinohara N, Matsumoto K, Endoh S, Maru J, Nakanishi J. 2009. In vitro and in vivo genotoxicity tests on fullerene C60 nanoparticles. Toxicol Lett 191:289–296. Shukla RK, Kumar A, Gurbani D, Pandey AK, Singh S, Dhawan A. 2011b. TiO(2) nanoparticles induce oxidative DNA damage and apoptosis in human liver cells. Nanotoxicology. [Epub ahead of print]. Shukla RK, Sharma V, Pandey AK, Singh S, Sultana S, Dhawan A. 2011a. ROS-mediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells. Toxicol In Vitro 25:231–241. Singh N, Jenkins GJ, Nelson BC, Marquis BJ, Maffeis TG, Brown AP, et al. 2012. The role of iron redox state in the genotoxicity of ultraﬁne superparamagnetic iron oxide nanoparticles. Biomaterials 33:163–170. Singh N, Manshian B, Jenkins GJ, Grifﬁths SM, Williams PM, Maffeis TG, et al. 2009. NanoGenotoxicology: the DNA damaging potential of engineered nanomaterials. Biomaterials 30:3891–3914. Som C, Berges M, Chaudhry Q, Dusinska M, Fernandes TF, Olsen SI, et al. 2010. The importance of life cycle concepts for the development of safe nanoproducts. Toxicology 269:160–169. Srivastava RK, Rahman Q, Kashyap MP, Lohani M, Pant AB. 2011. Ameliorative effects of dimetylthiourea and N-acetylcysteine on nanoparticles induced cyto-genotoxicity in human lung cancer cells-A549. PLoS One 6: e25767. Stone V, Johnston H, Schins RP. 2009. Development of in vitro systems for nanotoxicology: methodological considerations. Crit Rev Toxicol 39:613–626. Stone V, Nowack B, Baun A, van den Brink N, Kammer F, Dusinska M, et al. 2010. Nanomaterials for environmental studies: classiﬁcation, reference material issues, and strategies for physicochemical characterisation. Sci Total Environ 408:1745–1754. Sycheva LP, Zhurkov VS, Iurchenko VV, Daugel-Dauge NO, Kovalenko MA, Krivtsova EK, et al. 2011. Investigation of genotoxic  and cytotoxic effects of micro- and nanosized titanium dioxide in six organs of mice in vivo. Mutat Res 726:8–14. Theogaraj E, Riley S, Hughes L, Maier M, Kirkland D. 2007. An investigation of the photo-clastogenic potential of ultraﬁne titanium dioxide particles. Mutat Res 634:205–219. Toyooka T, Amano T, Ibuki Y. 2012. Titanium dioxide particles phosphorylate histone H2AX independent of ROS production. Mutat Res 742:84–91. Trouiller B, Reliene R, Westbrook A, Solaimani P, Schiestl RH. 2009. Titanium dioxide nanoparticles induce DNA damage and genetic instability in vivo in mice. Cancer Res 69:8784–8789. Uboldi C, Giudetti G, Broggi F, Gilliland D, Ponti J, Rossi F. 2012. Amorphous silica nanoparticles do not induce cytotoxicity, cell transformation or genotoxicity in Balb/3T3 mouse ﬁbroblasts. Mutat Res 745:11–20. USEPA. 2007. Nanotechnology White Paper. Science Policy Council. United States Environmental Protection Agency. EPA100B-07001. Vega-Villa KR, Takemoto JK, Yáñez JA, Remsberg CM, Forrest ML, Davies NM. 2008. Clinical toxicities of nanocarrier systems. Adv Drug Deliv Rev 60:929–938. Vevers WF, Jha AN. 2008. Genotoxic and cytotoxic potential of titanium dioxide (TiO2) nanoparticles on ﬁsh cells in vitro. Ecotoxicology 17:410–420. Vidal AE, Hickson ID, Boiteux I, Radicella JP. 2001. Mechanism of stimulation of the DNA glycosylase activity of hOGG1 by the major human AP endonuclease: bypass of the AP lyase activity step. Nucleic Acids Res 29:1285–1292. Walczyk D, Bombelli F.B, Monopoli M.P, Lynch I, Dawson K. A. 2010. What the cell “sees" in Bionanoscience. J Am Chem Soc 132:5761–5768. Wang JJ, Sanderson BJ, Wang H. 2007c. Cytotoxicity and genotoxicity of ultraﬁne crystalline SiO2 particulate in cultured human lymphoblastoid cells. Environ Mol Mutagen 48:151–157. Wang JJ, Sanderson BJ, Wang H. 2007a. Cyto- and genotoxicity of ultraﬁne TiO2 particles in cultured human lymphoblastoid cells. Mutat Res 628:99–106. Wang JJ, Wang H, Sanderson BJ. 2007b. Ultraﬁne quartz-induced damage in human lymphoblastoid cells in vitro using three genetic damage end-points. Toxicol Mech Methods 17:223–232. Wang L, Zhang J, Zheng Y, Yang J, Zhang Q, Zhu X. 2010. Bioeffects of CdTe quantum dots on human umbilical vein endothelial cells. J Nanosci Nanotechnol 10:8591–8596. Wang S, Hunter LA, Arslan Z, Wilkerson MG, Wickliffe JK. 2011b. Chronic exposure to nanosized, anatase titanium dioxide is not cytoor genotoxic to Chinese hamster ovary cells. Environ Mol Mutagen 52:614–622. Wang S, Lawson R, Ray PC, Yu H. 2011a. Toxic effects of gold nanoparticles on Salmonella typhimurium bacteria. Toxicol Ind Health 27:547–554. Warheit DB, Donner EM. 2010. Rationale of genotoxicity testing of nanomaterials: regulatory requirements and appropriateness of available OECD test guidelines. Nanotoxicology 4:409–413. Warheit DB, Finlay C, Donner EM, Reed KL, Sayes CM. 2007. Development of a base set of toxicity tests using ultraﬁne TiO2 particles as a component of nanoparticle risk management. Toxicol Lett 171:99–110. Warheit DB. 2008. How meaningful are the results of nanotoxicity studies in the absence of adequate material characterization? Toxicol Sci 101:183–185. Wessels A, Van Berlo D, Boots AW, Gerloff K, Scherbart AM, Cassee FR, et al. 2011. Oxidative stress and DNA damage responses in rat and mouse lung to inhaled carbon nanoparticles. Nanotoxicology 5:66–78. Wirnitzer U, Herbold B, Voetz M, Ragot J. 2009. Studies on the in vitro genotoxicity of baytubes, agglomerates of engineered multi-walled carbon-nanotubes (MWCNT). Toxicol Lett 186:160–165. Wojewódzka M, Lankoff A, Dusinska M, Brunborg G, Czerwińska J, Iwaneńko T, et al. 2011. Treatment with silver nanoparticles delays repair of X-ray induced DNA damage in HepG2 cells. Nukleonika 56: 29–33. Wu W, Chen B, Cheng J, Wang J, Xu W, Liu L, et al. 2010. Biocompatibility of Fe3O4/DNR magnetic nanoparticles in the treatment of hematologic malignancies. Int J Nanomedicine 5:1079–1084. Xie H, Mason MM, Wise JP Sr. 2011. Genotoxicity of metal nanoparticles. Rev Environ Health 26:251–268. Xu A, Chai Y, Nohmi T, Hei TK. 2009. Genotoxic responses to titanium dioxide nanoparticles and fullerene in gpt delta transgenic MEF  Z. Magdolenova et al. Nanotoxicology Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 04/22/13 For personal use only. cells. Part Fibre Toxicol. 6:3. doi: 10.1186/1743-8977-6-3. PubMed PMID: 19154577; PubMed Central PMCID: PMC2650674. Xu L, Li X, Takemura T, Hanagata N, Wu G, Chou LL. 2012. Genotoxicity and molecular response of silver nanoparticle (NP)-based hydrogel. J Nanobiotechnology. [Epub ahead of print]. Yang H, Liu C, Yang D, Zhang H, Xi Z. 2009. Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: the role of particle size, shape and composition. J Appl Toxicol 29:69–78. Yin H, Casey PS, McCall MJ, Fenech M. 2010. Effects of surface chemistry on cytotoxicity, genotoxicity, and the generation of reactive oxygen species induced by ZnO nanoparticles. 21. Langmuir 26:15399–408. Yoshida R, Kitamura D, Maenosono S. 2009. Mutagenicity of watersoluble ZnO nanoparticles in Ames test. J Toxicol Sci 34:119–122. Zhu L, Chang DW, Dai L, Hong Y. 2007. DNA damage induced by multiwalled carbon nanotubes in mouse embryonic stem cells. Nano Lett 7:3592–3597.

RELATED PAPERS

RELATED TOPICS

Log In

Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles

Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles

Related Papers

RELATED PAPERS

RELATED TOPICS