Mutation Research 761 (2014) 1–9

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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

Lack of genotoxic potential of ZnO nanoparticles in in vitro and in vivo tests Jee Young Kwon a,b,1 , Seung Young Lee c,d,1 , Preeyaporn Koedrith e , Jong Yun Lee c , Kyoung-Min Kim f , Jae-Min Oh f , Sung Ik Yang g , Meyoung-Kon Kim h , Jong Kwon Lee i , Jayoung Jeong i , Eun Ho Maeng c , Beam Jun Lee d,∗∗ , Young Rok Seo a,∗ a

Department of Life Science, Institute of Environmental Medicine, Dongguk University, Seoul, South Korea Department of Biomedical Science, School of Medicine, Kyung Hee University, Seoul, South Korea c Korea Testing and Research Institute, Seoul, South Korea d College of Veterinary Medicine Chungbuk National University, Cheongju, Chungcheongbuk-do, South Korea e Faculty of Environment and Resource Studies, Mahidol University, Phuttamonthon District, NakhonPathom 73170, Thailand f Department of Chemistry and Medical Chemistry, College of Science and Technology, Yonsei University, Wonju, Gangwondo, South Korea g Department of Applied Chemistry, Kyung Hee University, Yongin, South Korea h Department of Biochemistry & Molecular Biology, Korea University College of Medicine, Seoul, South Korea i Toxicological Research Division, National Institute of Food and Drug Safety Evaluation (NIFDS), Ministry of Food and Drug Safety (MFDS), Chungcheongbuk-do, South Korea b

a r t i c l e

i n f o

Article history: Received 25 March 2013 Received in revised form 8 January 2014 Accepted 12 January 2014 Available online 22 January 2014 Keywords: Genotoxicity test Organization for Economic Cooperation and Development test guideline Good laboratory practice Zinc oxide nanoparticles

a b s t r a c t The industrial application of nanotechnology, particularly using zinc oxide (ZnO), has grown rapidly, including products such as cosmetics, food, rubber, paints, and plastics. However, despite increasing population exposure to ZnO, its potential genotoxicity remains controversial. The biological effects of nanoparticles depend on their physicochemical properties. Preparations with well-defined physicochemical properties and standardized test methods are required for assessing the genotoxicity of nanoparticles. In this study, we have evaluated the genotoxicity of four kinds of ZnO nanoparticles: 20 nm and 70 nm size, positively or negatively charged. Four different genotoxicity tests (bacterial mutagenicity assay, in vitro chromosomal aberration test, in vivo comet assay, and in vivo micronucleus test, were conducted, following Organization for Economic Cooperation and Development (OECD) test guidelines with good laboratory practice (GLP) procedures. No statistically significant differences from the solvent controls were observed. These results suggest that surface-modified ZnO nanoparticles do not induce genotoxicity in in vitro or in vivo test systems. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Application of nanotechnology has increased in various industries for the last decade. A various type of nanoparticles including carbon black, carbon nanotubes, TiO2 and ZnO are used in cosmetics, electrical appliances and plastic wares [1]. Roco estimated that the demand for nanoparticles in the market will reach up to 1 trillion US dollars by 2015 [2]. With increasing in various kinds of manufactured nanoparticles continuously, the risk of human exposure to nanoparticles through ingestion, inhalation and

∗ Corresponding author. Tel.: +82 2 2260 3321; fax: +82 2 2290 1392. ∗∗ Corresponding author. E-mail address: [email protected] (Y.R. Seo). 1 These authors contributed equally to this work and should be considered co-first authors. 1383-5718/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2014.01.005

dermal absorption also has increased. Nanoparticles have physicochemical properties and high reactivity due to their unique nano scale, thereby they may cause unexpected hazards for human health [3,4]. The Organization for Economic Co-operation and Development (OECD) also has taken notice of human health and environment safety issues associated with nanoparticles since 2005 [5]. For efficiently and effectively addressing the safety issues of nanoparticles, the Working Party on Manufactured Nanomaterials (WPMN) was established in 2006 [5]. They selected thirteen kinds of representative manufactured nanoparticles, aluminum oxide, cerium oxide, dendrimers, fullerences (C60), gold nanoparticles, iron nanoparticles, multi-walled carbon nanotubes (MWCNTs), nanoclays, silicon dioxide, silver nanoparticles, single-walled carbon nanotubes (SWCNTs), titanium dioxide and zinc oxide for evaluating the toxicological effect [5].

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Among various kinds of nanoparticles, ZnO is widely used in numerous materials and products including cement, ceramics, cosmetics, food, glass, rubber, paints, pigments, plastics and sealants [6]. To date, there are several reports on the cytotoxic and genotoxic effect of ZnO nanoparticles in vitro and in vivo system [7,8]. However, these nanotoxicology-based studies reported numerous conflicting results due to slight differences in the physico-chemical features of nanoparticles used under studies [9]. Several studies have demonstrated that the differences were generated by the size and surface charge of nanoparticles [10–12]. The first step of nanomaterials’ study on toxicity or biological behavior is the comprehensive understanding on their physicochemical properties through characterizing various parameters. Particularly in genotoxicity tests, validated and standardized methods should be used for objective evaluation. The WPMN of OECD proposed a guideline on the nanomaterials’ characterization. They recommended primary characterization of 9 parameters including crystallinity, particle size, surface chemistry of single particle, surface charge, etc., and secondary analysis depending on the experimental conditions. In this study, we mainly focused on the two physico-chemical properties, particle size and surface charge. Generally, the nanopowder products such as ZnO maintain their primary particle size in nanoscale; however, they can make agglomerates or large secondary particles depending on suspending media. The general definition on nanomaterial is the substance having dimension less than 100 nm at least to one direction. Therefore, many reports dealt with study on nanomaterials under the criteria of primary particle size evaluated by the microscopy although the secondary particle size in suspension measured by light scattering method reveal large size. In this study, we also took the primary particle size of nanomaterials as criteria and selected two distinguishable primary particle sizes of 20 and 100 nm. The surface charge of nanomaterials was prepared to have either (+) or (−) charge by modifying the surface with an appropriate coating agent, citrate (for negative) and l-serine (for positive). In the present study, we performed four kinds of genotoxicity tests using four types of well-characterized ZnO nanoparticles, having 20 or 70 nm of primary particle size and (+) or (−) surface charge – 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, 70 nm (−) charge – to determine their genotoxic effects. The bacterial mutation assay, in vitro chromosomal aberration test and in vivo micronucleus test, were performed following OECD test guidelines with the good laboratory practice (GLP). The in vivo comet assay was also conducted according to the international validation study guideline. 2. Materials and methods All the animals were cared in accordance to “Guide for the Care and Use of Laboratory Animals” issued by the Animal Care and Use Committee of the National Veterinary Research and Quarantine Service. 2.1. Nanoparticles preparation ZnO nanoparticles with a primary particle size of 20 and 70 nm were purchased from Sumitomo (Japan) and American Elements (USA), respectively. ZnO nanoparticles were suspended in 20 mM HEPES buffer (pH 7.0) (Sigma) containing 1% sodium citrate (Sigma) for surface modification of ZnO nanoparticles with negative charge. In order to modify ZnO nanoparticles with positive charge, ZnO nanoparticles were suspended in 20 mM HEPES buffer (pH 6.0) containing 1% l-serine (Sigma). 2.2. Nanoparticle characterization The size of single nanoparticles (primary particle size) of four different nanoparticles were investigated with scanning electron microscopy (SEM: Hitachi, SU-70) at the Kangneung branch of the Korea Basic Science Institute (KBSI). ZnO nanoparticles in powder state were directly located on the carbon tape and subjected to SEM study. The average particle size and standard deviation was obtained by 300 randomly selected particles from SEM images. In order to evaluate the primary particle size of ZnO nanoparticles in aqueous suspension, ZnO nanoparticles were suspended in deionized water, phosphate buffered saline (PBS) and minimum essential media

(MEM), respectively. A drop of suspension was loaded on the silicon wafer and dried in air before SEM measurement. Size distribution of ZnO nanoparticles in aqueous suspension was examined with dynamic light scattering method. Each nanoparticles was first dispersed in water in the concentration range of 7–50 ␮g/ml. After vigorous stirring for 1 h, the hydrodynamic size (nm) was measured by a Malvern Zetasizer NanoZS using a DTS0012 cuvette (Malvern) at 25 ◦ C with 3 repeated measurements. The refractive indices of ZnO and water were set to 2.00 and 1.33, respectively. The colloidal property of aqueous ZnO suspension was also evaluated by measuring the surface charge of nanoparticles with electrokinetic method (Malvern Zetasizer Nano ZS instrument). Each ZnO nanoparticles was dispersed in phosphate buffered saline (PBS) in the concentration range of 7–200 ␮g/ml and stirred vigorously for 1 h. The surface zeta ()-potential (mV) was measured at 25 ◦ C with 10 repeated measurements where the refractive indices of ZnO and water were set to 2.00 and 1.33, respectively. 2.3. Bacterial reverse mutation test The bacterial reverse mutation test was conducted in compliance with the Korea Food and Drug Administration Notification No. 2009-116 testing guideline and OECD testing guideline (OECD 471). The tester strains used in this study were Ames Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA in the absence and presence of metabolic activation system. All of the test strains were purchased from Molecular Toxicology, Inc., USA. The metabolic activation system was prepared mixing S9 from Molecular Toxicology, Inc. with Cofactor-I from Waco Pure Chemical Industries, Ltd. (Chuo-ku, Osaka, Japan) giving a final concentration of 10% (v/v) S9. The tester strains were cultured in 2.5% Oxoid nutrient broth No.2 (Oxoid, Ltd., UK) in a 37 ◦ C shaking incubator (120 rpm) for approximately 11 h. The mutagenicity test was performed by mixing one of the tester strains which was cultured overnight, with the test substance in the presence and absence of S9 mixture condition. Subsequently, the mixture was incubated in water bath for 20 min at 37 ◦ C, mixed with top agar containing a minimal amount of histidine-biotin (for Salmonella typhimurium strains) or tryptophan (for Escherichia coli strain) and then poured onto the surface of a gamma-ray sterile Petri dish (Falcon, USA) containing about 15 ml of solidified bottom agar. The finished plates were incubated for 48–72 h at 37 ◦ C. The number of revertant colonies was then counted. All plates were done in triplicate, and the results were tabulated as the mean ± standard deviation for each condition. 2.4. In vitro chromosomal aberration test The in vitro chromosomal aberration test was performed according to the Korea Food and Drug Administration Notification No. 2009-116 testing guideline and OECD testing guideline (OECD 473). The clastogenicity of ZnO nanoparticles was evaluated for its ability to induce chromosomal aberrations in Chinese hamster lung (CHL) fibroblast cells. A clonal sub-line of CHL cells was obtained from ATCC (American Type Culture Collection, USA). The karyotype of CHL cells consisted of 25 chromosomes. The CHL cells were grown in minimum essential medium eagles (MEM) (Gibco BRL Life Tech., Inc., Gaithersburg, USA) supplemented with 10% FBS (Gibco BRL Life Tech., Inc., Gaithersburg, USA), 50 units/ml penicillin and 50 ␮g/ml streptomycin (Gibco BRL Life Tech., Inc., Gaithersburg, USA) at 37 ◦ C in humidified atmosphere containing 5% CO2 . The Mitomycin C (MMC, CAS No. 50-07-7) was used as a positive control in combination with or without S9 mixture. After 22 h incubation, colcemid was added to the cultures at the final concentration of 0.2 ␮g/ml, metaphase cells were harvested by trypsinization and centrifugation. The cells were swollen by adding with hypotonic (0.075 M) KCl solution for 20 min at 37 ◦ C, and washed three times in ice-cold fixative (methanol:glacial acetic acid = 3:1). A few drop of cell pellet suspension were dropped onto pre-cleaned glass microscope slides, and dried in the air. Slides were stained with 5% Giemsa buffered solution. The number of cells with chromosomal aberrations was recorded on 200 well-spread metaphases. The classification of aberration types referred to JEMS-MMS. Aberration frequencies, defined as aberrations observed divided by number of cells counted, were analyzed using Fisher’s exact test with Dunnett’s adjustment and compared with results from the solvent controls. 2.5. In vivo alkaline comet assay The alkaline (pH > 13) comet assay was conducted according to international validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens (version 14.2). Male Crl:CD (SD) rats (6 weeks old) were used for in vivo alkaline comet assay. After a 7 days acclimation, test substance was administered three times by gavage at 0, 24, and 45 h. The test doses (500, 1000, and 2000 mg/kg) were selected by range-finding experiment. The liver and stomach were collected from each animal and maintained in cold mincing buffer (Mg2+ and Ca2+ free Hanks’ Balanced Salt Solution (Gibco, CA, USA) with 20 mM Na EDTA (EDTA) (Sigma, USA) and 10% (v/v) dimethyl-sulfoxide (DMSO) (Sigma)). The liver was minced using fine scissors in cold mincing buffer. The stomach was opened and washed free from food using cold mincing buffer. The forestomach was discarded, then glandular stomach was placed into cold mincing buffer and incubated on ice for 15–30 min. After

J.Y. Kwon et al. / Mutation Research 761 (2014) 1–9 incubation, the surface mucosa was gently scoured using a scrapper. This layer was subsequently discarded and stomach epithelia was carefully scoured with a scrapper to release the cells. The cell suspension was stored on ice for 15–30 s to allow large clumps to settle, and the supernatant was used to prepare comet slides. A aliquot of single cell suspension 10 ␮l was mixed with 0.5% low melting agarose (Invitrogen, USA) and spread on comet assay slide (Travigen, USA). The slides were immersed in cold lysis solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris-base, 10% DMSO, 1% Triton-X (pH 10)) overnight. After this incubation period, slides were placed in electrophoresis solution (0.3 M NaOH, 1 mM EDTA (pH > 13)) for 20 min to allow for DNA unwinding. Subsequently, electrophoresis was conducted at 25 V, 300 mA for 20 min. The slides were immersed in a neutralization solution (0.4 M Tris-base (pH7.5)) for at least 5 min and dehydrated with absolute ethanol to fix. The cells were stained with SYBR Gold (Invitrogen) according to manufacturer’s specifications. All slides were independently coded before the microscopic analysis. The comet was observed via fluorescence microscope (Nikon, Japan) at magnification of 200× and analyzed by COMET ASSAY IV software (Perceptive Instruments, UK). For each sample (animal/tissue), fifty comets per slide were analyzed, with 2 slides scored per sample. A positive response is defined as a statistically significant change in the % tail DNA in at least one dose group in comparison with the vehicle control value using Dunnett’s test (two-sided, P < 0.05) as well as a statistically significant linear Trend test (two-sided, P < 0.05). The positive control should produce a statistically significant increase in Student’s t-test (one-sided, P < 0.025).

2.6. In vivo micronucleus test The in vivo micronucleus test was performed in compliance with the Korea Food and Drug Administration Notification No. 2009-116 testing guideline and OECD testing guideline (OECD 474). Out-bred mice of strain ICR, 6–7 weeks old, were used in this study. The test particle was applied orally in three doses in volumes of 10 ml/kg. The test substance was given twice with a 24-h interval and killed by cervical dislocation. Preparation and staining of bone marrow were carried out according to the method of Schmid [13]. All slides were independently coded before the microscopic analysis for micronucleus frequencies. In scoring the preparations, micronuclei were counted in polychromatic and, separately in monochromatic, erythrocytes. The rate of micronucleated cells, expressed in percentage, was based on the total of polychromatic erythrocytes present in the scored optic fields. This mode of scoring, which must always be followed where the test substance markedly influences the proliferation rate in the bone marrow, prevents a distortion of the results by the influx of peripheral blood into the damaged marrow. The scoring of micronucleated normocytes not only serves to recognize the presence of artifacts (which is

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rare in preparations from mouse) but provides additional interesting information on the mode of action of the test substance. Generally, an incidence of more than 1 micronucleated normocyte per thousand polychromatic erythrocytes indicates an effect on cell stages past the S-phase.

3. Results 3.1. Characterization of ZnO nanoparticles in aqueous condition Accurate toxicity evaluation of manufactured nanoparticles requires understanding of nanoparticles behavior under physiological conditions similar to those used for in vitro toxicity studies [14]. In this study, four ZnO nanoparticles were determined to have nano-sized primary particle size with electron microscopy as previously reported [15]. As shown in the inset SEM images of Fig. 1 (inset), all the ZnO nanoparticles have primary particle size below 100 nm and irregular spherical morphology. The average size ± standard deviation of primary particle size calculated from the SEM images was determined to be 35 ± 5, 28 ± 8, 70 ± 19, and 72 ± 11 nm for 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge nanoparticles, respectively. We measured the SEM images of ZnO nanoparticles in dried powder as well as in suspension state, and various media such as deionized water, phosphate buffered saline (PBS), and minimum essential medium (MEM) media. All the SEM images showed the similar primary particle size, homogeneous particle size distribution and clearly distinguishable grain boundary with or without suspension media (supplementary data, Fig. S1). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mrgentox. 2014.01.005. According to our previous study [15], the hydrodynamic size of ZnO nanoparticles in aqueous condition showed a little bit larger

Fig. 1. Zeta potential values for (A) 20 nm (+), (B) 20 nm (−), (C) 70 nm (+), and (D) 70 nm (−) ZnO nanoparticles suspended in phosphate buffered saline (PBS). The inset images are the scanning electron microscopic images of each ZnO nanoparticles in powder state.

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TA1537 and E. coli WP2 uvrA at dose of 5000 ␮g/plate in the presence and absence of S9. Based on the results of the range finding test, 5000 ␮g/plate was determined as the highest concentration. As shown in Table 1, all test strains did not increase in the number of revertant colonies compared to the negative control and solvent control when the bacteria were treated with at 312.5, 625, 1250, 2500 and 5000 ␮g/plate of four kinds of ZnO nanoparticles, 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge, regardless of metabolic activation. In contrast, the positive control substances increased revertant colonies in comparison with negative control and solvent control.

values compared with primary particle size. The size distribution ranges from 200 to 400 nm, 180 to 300 nm, 300 to 900 nm, and 200 to 500 nm for 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge nanoparticles, respectively (supplementary data, Fig. S2). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mrgentox. 2014.01.005. Surface charge of ZnO nanoparticles in PBS suspension was determined to be highly positive or negative for all four different samples (Fig. 1). The average zeta potential values for 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge ZnO nanoparticles in PBS suspension was determined to be +25.9, −38.5, +25.9 and −40.6 mV, respectively, suggesting the colloidal stability.

3.3. In vitro chromosome aberration test The range finding test was conducted to determine test doses of chromosomal aberration test. Consequently, highest dose was determined at 15 ␮g/ml which was shown less than 50% of cytotoxicity in CHL cells (data not shown). The chromosomal aberration test was performed using four kinds of ZnO nanoparticles with 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge at concentration of 3.75, 7.5, and 15 ␮g/ml both

3.2. Bacterial reverse mutation test The results of the preliminary range finding tests (data not shown), four kinds of ZnO nanoparticles, 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge, gave no toxic effect to all test stains S. typhimurium TA98, TA100, TA1535,

Table 1 The numbers of revertant colonies induced by ZnO nanoparticles with 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge in Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and Escherichia coli (WP2uvrA), with and without metabolic activation (S9). Colonies/plate (mean ± S.D.) Tester strain

Dose (␮g/plate)

20 nm (+) − S9 mix

20 nm (−) + S9 mix

− S9 mix

70 nm (+) + S9 mix

70 nm (−)

− S9 mix

+ S9 mix

− S9 mix

22 28 24 25 23 22 25 348

± ± ± ± ± ± ± ±

6 5 3 9 4 2 3 9

20 22 19 24 22 26 18 326

± ± ± ± ± ± ± ±

2 3 2 3 3 3 5 12

22 18 22 22 19 21 20 330

+ S9 mix

TA98

0 312.5 625 1250 2500 5000 HEPESa Positive control

10 8 12 11 9 6 9 330

± ± ± ± ± ± ± ±

2 2 3 3 1 1 1 16

19 17 17 15 13 12 15 355

± ± ± ± ± ± ± ±

6 1 3 1 2 2 3 41

19 18 15 12 21 16 15 288

± ± ± ± ± ± ± ±

3 2 2 1 4 3 2 21

30 21 28 28 31 29 30 348

± ± ± ± ± ± ± ±

2 4 2 3 6 3 2 9

28 ± 2 34 ± 2 34 ± 6 26 ± 3 28 ± 3 23 ± 2 25 ± 2 288 ± 21

TA100

0 312.5 625 1250 2500 5000 HEPESa Positive control

72 81 80 80 78 67 67 469

± ± ± ± ± ± ± ±

3 6 13 2 7 6 6 33

100 85 87 78 78 94 94 464

± ± ± ± ± ± ± ±

7 14 6 14 11 8 8 34

133 136 140 134 153 141 139 364

± ± ± ± ± ± ± ±

4 17 11 5 6 15 9 30

128 119 135 119 117 111 130 451

± ± ± ± ± ± ± ±

16 19 5 6 3 20 4 26

153 199 193 184 186 159 170 564

± ± ± ± ± ± ± ±

11 15 14 6 23 36 4 30

137 196 197 211 226 231 220 451

± ± ± ± ± ± ± ±

12 16 22 28 25 23 21 26

47 52 51 49 56 54 47 568

± ± ± ± ± ± ± ±

5 6 6 8 4 7 10 48

43 ± 5 40 ± 10 47 ± 10 42 ± 4 53 ± 9 51 ± 2 46 ± 7 473 ± 32

TA1535

0 312.5 625 1250 2500 5000 HEPESa Positive control

10 14 14 14 12 14 7 275

± ± ± ± ± ± ± ±

5 0 3 3 5 1 1 28

12 16 16 14 12 12 12 213

± ± ± ± ± ± ± ±

4 2 3 3 1 1 1 14

11 8 11 11 12 10 10 225

± ± ± ± ± ± ± ±

1 2 2 2 2 3 2 10

19 16 22 16 16 14 18 202

± ± ± ± ± ± ± ±

2 2 3 1 4 3 2 15

14 15 14 20 15 18 16 225

± ± ± ± ± ± ± ±

4 3 4 5 2 3 2 10

12 11 11 13 14 10 10 202

± ± ± ± ± ± ± ±

1 1 0 5 4 1 2 15

12 11 8 10 8 9 9 253

± ± ± ± ± ± ± ±

2 3 3 5 1 2 0 40

14 12 12 14 15 11 12 209

± ± ± ± ± ± ± ±

5 3 1 2 4 4 1 13

TA1537

0 312.5 625 1250 2500 5000 HEPESa Positive control

8 7 5 7 6 5 7 686

± ± ± ± ± ± ± ±

2 1 1 2 1 2 1 33

8 11 9 10 9 9 7 194

± ± ± ± ± ± ± ±

1 4 2 2 2 2 1 7

11 9 9 9 9 8 9 741

± ± ± ± ± ± ± ±

4 3 1 2 3 1 2 38

11 10 11 9 13 12 10 170

± ± ± ± ± ± ± ±

4 2 2 4 3 3 2 12

22 22 20 20 16 14 17 741

± ± ± ± ± ± ± ±

4 2 4 2 4 3 4 38

20 16 19 20 16 12 17 170

± ± ± ± ± ± ± ±

3 2 8 7 2 4 5 12

7 6 9 9 9 8 7 743

± ± ± ± ± ± ± ±

1 1 2 1 1 3 2 34

12 12 11 9 10 10 10 185

± ± ± ± ± ± ± ±

2 2 2 3 1 0 1 15

WP2uvrA

0 312.5 625 1250 2500 5000 HEPESa Positive control

18 19 18 20 23 19 19 343

± ± ± ± ± ± ± ±

5 0 6 3 6 5 4 7

37 35 35 33 37 24 34 330

± ± ± ± ± ± ± ±

8 4 6 7 7 4 4 13

38 39 39 32 34 31 39 423

± ± ± ± ± ± ± ±

3 3 7 5 6 1 2 21

49 56 40 52 48 40 40 255

± ± ± ± ± ± ± ±

5 9 8 7 1 3 4 38

31 24 27 26 23 22 32 423

± ± ± ± ± ± ± ±

5 3 5 5 3 3 5 21

38 39 33 28 23 26 31 255

± ± ± ± ± ± ± ±

10 7 7 8 4 1 6 38

36 38 32 26 33 27 28 437

± ± ± ± ± ± ± ±

5 6 9 8 3 3 10 13

43 33 42 38 38 37 39 257

± ± ± ± ± ± ± ±

5 4 10 2 8 6 6 31

a

Negative charge: HEPES–citrate buffer, positive charge: HEPES–serine buffer.

± ± ± ± ± ± ± ±

1 3 2 3 4 4 2 15

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Table 2 Chromosome analysis of ZnO nanoparticles with 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge in Chinese hamster lung fibroblast cells with and without metabolic activation (S9). (A) Observed cell

Concentration (␮g/ml)

6 h without S9 (−S9)

20 nm (+)

200

20 nm (−)

70 nm (+)

70 nm (−)

% of numerical aberration

% of structural aberration (exclusive to gap)

% of numerical aberration

% of structural aberration (exclusive to gap)

% of numerical aberration

% of structural aberration (exclusive to gap)

% of numerical aberration

% of structural aberration (exclusive to gap)

0 Buffera 3.75 7.5 15 Positive control

0.0 0.0 0.0 1.0 2.0 0.0

0.5 0.0 0.0 0.5 1.0 66.5

0.0 0.0 0.5 1.0 1.5 0.0

0.5 0.0 0.5 0.5 0.5 69.5

0.0 0.5 1.0 1.0 2.5 0.0

0.0 0.5 0.5 1.5 2.5 75.0

0.5 0.5 0.0 0.5 0.0 0.0

0.5 0.0 0.0 0.5 0.5 70.0

Concentration (␮g/ml)

6 h with S9 (+S9)

(B) Observed cell

20 nm (+)

200

a

0 Buffera 3.75 7.5 15 Positive control

20 nm (−)

70 nm (+)

70 nm (−)

% of numerical aberration

% of structural aberration (exclusive to gap)

% of numerical aberration

% of structural aberration (exclusive to gap)

% of numerical aberration

% of structural aberration (exclusive to gap)

% of numerical aberration

% of structural aberration (exclusive to gap)

0.0 0.0 0.5 2.0 3.0 0.0

0.0 0.0 0.0 0.5 1.5 66.0

0.0 0.0 0.5 2.5 1.0 0.5

0.0 0.5 0.0 0.5 0.5 68.0

0.0 1.0 1.0 2.0 2.5 1.5

0.5 0.5 1.0 1.5 2.0 68.0

0.0 0.5 0.5 0.0 0.5 1.5

0.5 1.0 0.5 0.0 0.5 73.0

Negative charge: HEPES–citrate buffer; positive charge: HEPES–serine buffer.

with and without metabolic activation. The metaphase arrested cells with structural aberrations were less than 5%. In addition, the results showed no significant increase in ZnO nanoparticles treatment group compared with negative control and solvent control at three kinds of doses regardless of metabolic activation (Table 2). On the contrary, the positive control substances obviously induced structural aberrations in comparison with negative control and solvent control. 3.4. In vivo alkaline comet assay In order to select test doses of in vivo alkaline comet assay, range finding study was performed in SD rat (data not shown). Based on the range finding study, test doses were determined at 500, 1000, and 2000 mg/kg body weights (B.W). As shown in Fig. 2, tail intensity of liver and stomach single cells treated with ZnO nanoparticles with 20 nm (+) and (−) charge had no significant increase in comparison with solvent control group. The results of 70 nm (+) and (−) charged ZnO nanoparticles also revealed no significant increase in tail intensity as shown in Fig. 3. 3.5. In vivo micronucleus test In a preliminary dose range finding test, acute oral toxicity, death and any obvious abnormality of appearance were not observed at dose ranges from 500 to 2000 mg/kg mice body weights (B.W.). However, among four types of ZnO nanoparticles, significant decrease in body weight was observed at dose of 2000 mg/kg B.W. in ZnO nanoparticles with 20 nm (+) charge group (data not shown). The PCE (polychromatic erythrocyte)/NCE (normochromatic erythrocytes) ratios which are index of bone marrow cytotoxicity showed significantly difference in ZnO nanoparticles

with 20 nm (+) and (−) charge groups. The frequencies of MNPCE (micronucleated polychromatic erythrocyte) were not represented statistical significance and dose-dependent response at any dose on four kinds of ZnO nanoparticles (Table 3). Despite the ZnO nanoparticles with 20 nm (+) and (−) charge showed bone marrow cytotoxicity, the genotoxic potential of four types of ZnO nanoparticles was considered to be negative in this in vivo system. 4. Discussion Using ZnO with distinguishable nano-sized primary particles is necessary for evaluating genotoxicity depending on particle size due to its different physicochemical property upon primary particle size [16]. Numerous studies have demonstrated size-dependent toxicity including mitochondrial damage, oxidative stress, chromosomal and oxidative DNA damage, usually with smaller sized nanoparticles causing significantly more damage than their larger sized nanoparticles [10–12]. In addition, surface charge dependent cellular uptake and toxicity has been shown by other research groups, with cationic nanoparticles being more toxic than their anionic nanoparticles [17–19]. Furthermore, characterization of nanoparticles behavior in aqueous conditions similar with in vitro or in vivo nanotoxicity studies should be evaluated and preserved as their own properties [14]. In this study, we used four different kinds of ZnO nanoparticles with different primary particle size and surface charge. From the electron microscopic study, it was revealed that the primary particle size of four ZnO nanoparticles were determined to be ∼20 nm for both 20 nm (+) charge, 20 nm (−) charge samples and ∼70 nm for 70 nm (+) charge, and 70 nm (−) charge ZnO samples. Although the hydrodynamic size of ZnO in aqueous suspension was evaluated larger than the primary particle size, suggesting the

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J.Y. Kwon et al. / Mutation Research 761 (2014) 1–9

Fig. 2. DNA damage determined by in vivo comet assay in liver and stomach tissues from rats treated with positive charged 20 nm ZnO (A and B) and negative charged 20 nm ZnO (C and D).

possible formation of agglomerates, we could observe clear grain boundary between particles from the SEM images. It is worthy to note that the primary particle size and grain boundary ZnO nanoparticles were clearly preserved after dispersing them in various media (deionized water, PBS, and MEM) compared with the powder state ZnO nanoparticles. Therefore, we could suggest that the present surface-modified ZnO nanoparticles preserved their nanoparticulate property in biological assay in terms of primary particle size. Powder state nanoparticles when suspended in aqueous media, especially without dispersing agent, often results in the formation of agglomerates. However, it does not stand for that the characteristic features of materials as nano-sized particles disappear. It has been often reported that powdered ZnO nanoparticles suspended in aqueous media often makes agglomerate showing larger hydrodynamic diameter than primary particle size [20,21]. Our previous result has shown that the average hydrodynamic diameter of ZnO nanoparticles can be larger than 200 nm while their primary particle grain boundary was well preserved in various aqueous media (Fig. S1) [15]. Xia et al. and Sharma et al. who studied the possible toxicity of ZnO nanoparticles also reported that the size of ZnO nanoparticles in aqueous media was larger than 200 nm [20,21]. Recently, Sohaebuddin et al. provided that the particle size of various nanoparticles like TiO2 , SiO2 and carbon nanotubes can be

varied (larger than 100 nm) depending on the type of aqueous media regardless of their primary particle size (smaller than 100 nm) [22]. The zeta potential values (Fig. 1) also revealed that the ZnO nanoparticles utilized in this study had clearly distinguishable surface charge with high colloidal stability. Generally, it is accepted that the colloidal stability increase with the increasing zeta potential values either to negative or positive values, as the electrostatic repulsion between particles becomes stronger. The zeta potential in PBS media was approximately +25 mV for (+) ZnO and below −30 mV for (−) ZnO, thus we could expect high colloidal stability for all four ZnO nanoparticles. In order to investigate a potential genotoxic effect of chemical substances, combinations of several genotoxicity tests have been used extensively. In this present study, we used several genotoxicity tests, covering different endpoints, comprising bacterial mutation assay, in vitro chromosomal aberration test, in vivo comet assay and in vivo micronucleus test under validated and standardizes method with GLP system. The bacterial mutation assay was used to determine the mutagenicity of exogenous substances [23]. It is an essential test within the current battery of assays required for genotoxicity evaluation and has also recently been highlighted as one of the two assays recommended by the UK expert advisory Committee on Mutagenicity

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Fig. 3. DNA damage determined by in vivo comet assay in liver and stomach tissues from rats treated with positive charged 70 nm ZnO (A and B) and negative charged 70 nm ZnO (C and D).

that in parallel have sufficient sensitivity to detect carcinogens and genotoxins while minimizing the risk of false positive in vitro genotoxicity testing results [24]. To date, published reports showed that ZnO nanoparticles induced negative mutagenicity in bacterial mutation assay [25–27]. In corresponding to published literatures, our data using ZnO nanoparticles, 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge, also showed negative results in S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA, with and without S9 mixture (Table 1). Recently, there is a report that Ames test is one of the reliable genotoxicity screen method, however, it does not appear to be suitable for the assessment of nanoparticles. They mentioned that it might be led by nanoparticles uptake capacity of bacterial cells as describing two reasons. Firstly, prokaryotes cannot absorb molecules by engulfing them (endocytosis) and secondly, their cell wall can have a barrier against simple diffusion of nanoparticles into the bacterial cell. Since these reasons, false negative result can be found in bacterial mutation assay using nanoparticles. They also announced that very well dispersed nanoparticles of approximately 20 nm diameter could enter the bacterium [9]. In our study, we evaluated mutagenicity of two different sizes of ZnO nanoparticles such as 20 nm and 70 nm. Our result showed that non-mutagenicity was revealed in both 20 nm and 70 nm ZnO. Consequentially this might be a suitable result for mutagenicity test using ZnO nanoparticles.

In a battery of genotoxicity tests, in vitro chromosomal aberration test is commonly used to identify structural aberration of chromosome [28]. According to our results, ZnO nanoparticles, 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge, did not induce clastogenic effect in CHL cells both with and without S9 mixture (Table 2). In contrast with our results, there is a report that ZnO induced chromosomal aberration in cells. However, they have used only 100 nm sized ZnO as well as evaluated photo-clastogenicity of ZnO in Chinese hamster ovary (CHO) cells [25]. Additionally, as mentioned above, nanotoxicology study results can be affected by physico-chemical properties. Dufour et al. used uncoated ZnO nanoparticles and a different cell line, Chinese Hamster Ovary (CHO) cells, was employed [25]. Another literature reported by Kumari et al. was showed increased chromosomal aberration in Allium cepa (Onion bulb) root cells treated with ZnO nanoparticles [29]. This result might be caused by using different cells. Most of nanotoxicology-based studies have not presented the correlation between in vitro and in vivo genotoxicity tests [30]. However, genotoxicity study using animal model has obvious advantage to evaluate accurate nanotoxicology. For this reason, in vivo genotoxicity tests including comet assay and micronucleus test were conducted simultaneously with in vitro bacteria mutation assay and chromosomal aberration test on ZnO nanoparticles.

± ± ± ± ± ± 55.25 56.35 55.55 55.82 54.44 44.63 0.04 0.06 0.00 0.04 0.04 0.22** ± ± ± ± ± ± 0.09 0.15 0.10 0.09 0.08 4.56 1.80 2.17 1.48 1.48 1.31 2.36** ± ± ± ± ± ± 55.76 55.87 53.71 54.54 54.94 44.06 0.03 0.06 0.04 0.04 0.07 0.21** ± ± ± ± ± ± 0.10 0.14 0.12 0.08 0.12 4.40 1.52 1.92 3.10* 3.10** 3.79** 4.09** ± ± ± ± ± ± 55.03 51.63 49.20 46.46 47.61 48.15

**

*

a

Negative charge: HEPES–citrate buffer; positive charge: HEPES–serine buffer. P < 0.05. P < 0.01.

0.07 0.07 0.02 0.06 0.04 0.24** ± ± ± ± ± ± 0.13 0.12 0.09 0.09 0.08 4.31 0.88 1.43 2.08* 2.08** 1.80** 1.38** ± ± ± ± ± ± 52.99 51.72 50.37 49.66 46.23 46.86 0.00 0.08 0.06 0.04 0.04 0.16** ± ± ± ± ± ± 0.05 0.13 0.10 0.09 0.09 4.77 0 HEPESa 500 1000 2000 Positive control

MNPCE/2000PCE’s (mean ± S.D., %) MNPCE/2000PCE’s (mean ± S.D., %)

PCE/(PCE + NCE) (mean ± S.D., %)

MNPCE/2000PCE’s (mean ± S.D., %)

PCE/(PCE + NCE) (mean ± S.D., %)

MNPCE/2000PCE’s (mean ± S.D., %)

PCE/(PCE + NCE) (mean ± S.D., %)

70 nm (−) 70 nm (+) 20 nm (−) 20 nm (+) Dose (mg/kg B.W.)

Table 3 Frequencies of micronucleated PCE (MNPCE) per 2000 PCE in the bone marrow of ICR mice exposed to ZnO nanoparticles with 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge.

1.68 0.96 1.33 1.33 3.73 1.30**

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PCE/(PCE + NCE) (mean ± S.D., %)

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The comet assay has been applied to detect initial DNA damage of single cells in in vitro as well as in vivo systems [31]. In vivo comet assay is engaged public attention as a potential replacement for the in vivo rodent hepatocyte unscheduled DNA synthesis (UDS) assay [32]. Recently, standard guideline of in vivo comet assay has been published by international expert groups for in vivo comet assay [32]. To date, nanotoxicity evaluation using in vivo comet assay following guideline which is approved by international validation study has not been reported yet. In the present study, we have identified genotoxicity of four different kinds of ZnO, 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge, following standard method. As a result, we first clarified that ZnO nanoparticles have no genotoxic effect in the liver and stomach cells (Figs. 2 and 3). Recent article reported by Sharma et al. showed that ZnO nanoparticles can induce oxidative stress, DNA damage and apoptosis in mouse liver after sub-acute oral exposure [33]. In their literature, in vivo comet assay was conducted for assessing DNA damage caused by ZnO nanoparticles. However, they were treated ZnO nanoparticles by oral administration for 14 consecutive days, i.e. the treatment period was longer than our study. Furthermore, contrary to our method, they performed formamidopyrimidine-DNA glycosylase (Fpg)-modified comet assay to determine the induction of oxidized bases. They aimed to investigate the link between ZnO nanoparticles oxidative stress and DNA damage. Their result could give evidences to elucidate the relationship between oxidative stress and DNA damage. However, standardized and approved in vivo comet assay method should be employed to detect an accurate analysis of genotoxicity. In this study, our data showed no DNA damage in in vivo comet assay according to the international validation study guideline. It might be suggested that ZnO nanoparticles might have no genotoxicity. Micronuclei induced by genotoxic carcinogens indicate either numerical or structural chromosomal aberrations [16]. Regarding genotoxicity on ZnO nanoparticles, there are numerous publications on via in vitro micronucleus test [34,35]. On the contrary to this, few in vivo micronucleus test has been conducted [36]. Therefore, in the present study, in vivo micronucleus test was conducted to determine whether ZnO nanoparticles cause abnormalities of chromosome or mitotic apparatus or not in animal model. We found that all four ZnO nanoparticles, 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge, and 70 nm (−) charge, were shown to be non-genotoxic (Table 3). Another genotoxicity investigation by Landsiedel et al. have evaluated potential genotoxicity of ZnO using the mouse bone marrow micronucleus test following OECD test guideline [36]. Similarly to our result, their result also indicated no genotoxicity on ZnO. In conclusion, four kinds of genotoxicity tests, covering different endpoints, were performed to elucidate genotoxicity of ZnO nanoparticles with 20 nm (+) charge, 20 nm (−) charge, 70 nm (+) charge and 70 nm (−) charge. To generate the reliable genotoxicity result, not only nanoparticles with different primary particle size and surface charge but also validated standard protocols were employed in this study. From our data, ZnO nanoparticles would be considered as non-genotoxic substances under the OECD test guideline. However, we should not overlook a possibility of genotoxicity at lower doses than those used in this study. Since nanoparticles can be agglomerated at high dose. Furthermore, there may be a possibility that different exposure route or long-term exposure duration can induce genotoxicity in different kinds of organs.

Acknowledgement This research was supported by a grant (10182MFDS991) from the Ministry of Food and Drug Safety in 2010.

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