Toxicology 315 (2014) 86–91

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Genotoxicity of silver and titanium dioxide nanoparticles in bone marrow cells of rats in vivo a,∗ ´ Małgorzata M. Dobrzynska , Aneta Gajowik a , Joanna Radzikowska a , Anna Lankoff b,d , c Maria Duˇsinská , Marcin Kruszewski b,e a

National Institute of Public Health – National Institute of Hygiene, Warsaw, Poland Institute of Nuclear Chemistry and Technology, Warsaw, Poland c Norwegian Institute for Air Research, Kjeller, Norway d Jan Kochanowski University, Department of Radiobiology and Immunology, Kielce, Poland e Institute of Rural Health, Lublin, Poland b

a r t i c l e

i n f o

Article history: Received 15 October 2013 Received in revised form 25 November 2013 Accepted 27 November 2013 Available online 7 December 2013 Keywords: Nanoparticles Micronuclei Comet assay DNA damage Erythrocytes Leukocytes

a b s t r a c t Although nanomaterials have the potential to improve human life, their sideline effects on human health seem to be inevitable and still remain unknown. This study aimed to investigate the cytotoxicity and genotoxicity of titanium dioxide (TiO2 ) and silver (Ag) nanoparticles (NPs) at different doses and particle sizes to bone marrow cells. Both types of nanoparticles were chosen due to their wide applications of them in consumer products. Rats were injected intravenously with a single dose of 5 or 10 mg/kg bw of 20 nm AgNPs or with 5 mg/kg bw 200 nm AgNPs or with 5 mg/kg bw 21 nm TiO2 NPs. The samples were taken at 24 h, 1 week and 4 weeks following the exposure. Micronucleus test and the Comet assay were used to detect DNA damage. Neither AgNPs nor TiO2 NPs caused cytotoxicity to bone marrow red and white cells. The polychromatic erythrocytes are the main target of both nanoparticles. A single exposure to AgNPs induced significantly enhanced frequency of micronuclei not only at 24 h after exposure, but also 1 and 4 weeks later, whereas single exposure to TiO2 NPs showed positive effect at 24 h only. Negative responses were shown in reticulocytes (micronuclei) and in leukocytes (Comet assay) of bone marrow. Results indicated that different bone marrow cells display different susceptibility toward genotoxicity mediated by both investigated nanoparticles. The use of materials containing nanoparticles and the potential health implication of them should be monitored. © 2013 Published by Elsevier Ireland Ltd.

1. Introduction Nanotechnology is currently an area of interest of many scientists due to wide variety of potential applications including biomedical, optical and electronic fields. Nanomaterials exhibit unusual biological, chemical and physical properties, and their use constitutes new opportunities. The number of applications of nanomaterials, including nanoparticles (NPs), in many fields of human life is still growing and hundreds of everyday use products containing nanoparticles are available nowadays on the market. These materials include cosmetics, sunscreens, paints and coatings, catalysts and lubricants, water treatments, textiles and sport items,

∗ Corresponding author at: Department of Radiation Hygiene and Radiobiology, National Institute of Public Health – National Institute of Hygiene, 24 Chocimska Street, 00-791 Warsaw, Poland. Tel.: +48 22 5421253; fax: +48 22 5421309. E-mail addresses: [email protected], ´ [email protected] (M.M. Dobrzynska). 0300-483X/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.tox.2013.11.012

medical and health care products, food products and packing and many others (PEN, 2005; Surendiran et al., 2009). Although, nanoparticles have the potential to improve the environment and lives of people, their interaction with environment is inevitable and the consequences of their use for human health and ecosystem are still not well characterized or unknown. Silver nanoparticles (AgNPs) are widely used in medicine, physics, material sciences and chemistry (Yang et al., 2007). Silver vessels were used since ancient times to preserve wine and water. Until the advent of antibiotics, silver compounds were used against wound infection, especially in burned patients (Chen and Schluesener, 2008; Edwards-Jones, 2009). Due to antibacterial properties silver is used in a variety of applications including dental materials, catheters and burn wound. They have been used for treatment of a range of diseases including malaria, lupus, tuberculosis, typhoid, tetanus and cancer (FDA, 1999). Many medical products are coated or embedded with nanosilvers, for example contraceptive devices, surgical instruments, bone prostheses, dental alloys (Cheng et al., 2004; Cohen et al., 2007; Yang and

M.M. Dobrzy´ nska et al. / Toxicology 315 (2014) 86–91

Pon, 2003). AgNPs are the component of room sprays, detergents, wall paints, textiles, clothing, socks, cosmetics (Cheng et al., 2004; Zhang and Sun, 2007). Moreover, AgNPs are used in lining of washing machines, dishwashers and refrigerators (Edwards-Jones, 2009). Titanium dioxide (TiO2 ) NPs are widely used in medicine and in the food-processing industry. They are used to provide whiteness and opacity of products, such as paints, paper, inks, food colorants and tootpastes pigments. In cosmetics particularly in sunscreens, sunblocks and skin care products they help to protect the skin from UV light. Moreover, TiO2 NPs are used in self-cleaning surfaces, as photocatalyst in air and water cleaning (Trouiller et al., 2009). Humans are at increasing risk of exposure to NPs, which may enter the body via different routes, through the skin, lungs or intestinal tract during manufacture, use and disposal of nanoproducts. From the site of deposition, the NPs are translocated to different parts of the body through the circulatory or lymphatic system (Kruszewski et al., 2011). Due to their stability it is anticipated that nanomaterials may remain in the body and in the environment for long period of time. In the environment, NPs could associate with solids or sediments, where they could accumulate and enter the food chain or sources of drinking water. The potential health effects from increasing exposure to NPs are ill defined, however limited data so far suggest potentially toxic effects (Karn et al., 2009). One of the most important impact of NP’s action seems to be ability to cause DNA damage. The effect of DNA damage is usually adverse, affecting the metabolism, cell-cycle arrest or causing cell death. Eukaryote organisms have evolved to develop effective molecular mechanism such as DNA damage response, to detect DNA lesions, signal their presence and promote their repair (Rodriguez-Rocha et al., 2011). Thus, DNA damage may be reversible, but in some cases the repair is inaccurate, resulting in acute adverse effects within hours to weeks or delayed effects within months to years after the exposure. Although DNA damage can cause cell death and eliminate potentially dangerous cells, miss-repaired damage may result in chromosomal damage or mutations. The resulting modification will be transmitted to further generations of cells and may eventually lead to development diseases such as cancer (Jackson and Bartek, 2009). Therefore, the aim of present study was to investigate the cytotoxicity and genotoxicity of titanium dioxide and silver NPs at different doses and particle sizes to erythrocytes and leukocytes of bone marrow. Both kinds of NPs were chosen due to their wide use in consumer products.

2. Materials and methods 2.1. Nanoparticle preparation and characterization Spherical silver nanoparticles with diameters of 20 ± 5 nm and 200 ± 50 nm were purchased from PlasmaChem (Berlin, Germany). NPs stock solutions were prepared by dispersion of 5 mg of NPs in 800 ␮l of 0.9% NaCl solution. The investigated TiO2 NPs, an anatase/rutile powder of 21 nm (nominal size), were nanomaterial type NM-105 kindly provided from the European Commission—Joint Research Center (Ispra, Italy). The material corresponds to a selected sample of a nanomaterial produced by Evonik (Essen, Germany) and marketed as Aeroxide TiO2 P25. Sub-samples of NM-105 were packed under Good Laboratory Practice conditions and preserved under argon in the dark until use. NPs were dispersed in deionized H2 O containing DMSO (50 ␮l DMSO/1 ml H2 O). NPs dispersions were sonicated for 5 min (output 20) in a plastic test tube surrounded by ice before each experiment using a probe

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sonicator (Branson, USA) with 420 J total ultrasound energy. One hundred microliters of bovine serum albumin and 100 ␮l of a 10× concentrated phosphate buffered saline solution were given immediately after sonication. Stock solutions were prepared before each experiment. A detailed characteristic of AgNPs and TiO2 NPs was published previously (Lankoff et al., 2012). Nanoparticle size and surface charge analysis were measured by dynamic light scattering (DLS). The system measures multiple angles of light scatter and derives a bulk intensity plot of particle sizes. DLS was performed at 25 ◦ C with a scattering angle of 90◦ on the Zetasizer Nano ZS (Malvern, Malvern Hills, UK). Stock solutions were diluted 1:4 in distilled water and measured in triplicate with 20 sub-runs. Zeta potential measurements for surface charge were performed at 25 ◦ C in a folded capillary cell at 150 V and M3-PALS detection using non-invasive backscatter at 173◦ with an Avalanche photodiode, Q.E. > 50% at 633 nm (Malvern, Malvern Hills, UK). Samples were diluted 1:8 in distilled water and measured in triplicate with 20 sub-runs. Zeta potentials (ZP) were calculated using the Smoluchowski limit for the Henry equation with a setting calculated for practical use (f(ka) = 1.5). The polydispersity index values were also monitored. Polydispersity index is a parameter to define the particle size distribution of nanoparticles obtained from photon correlation spectroscopic analysis. It is a dimensionless number extrapolated from the autocorrelation function and ranges from a value of 0–0.1 for monodispersed particles and up to values of 0.5–0.7. Samples with very broad size distribution have polydispersity index values >0.7. Hydrodynamic diameters of all three types of NPs were as follows: AgNPs 20 nm – 77.29 ± 1.4 nm; AgNPs 200 nm – 333.12 ± 2.5 nm and TiO2 NPs 21 nm – 129.50 ± 2.6 nm. The zetapotentials showed negative and relative similar potentials for all tested NPs (AgNPs 20 nm: – 33.6 mV; AgNPs 200 nm: – 37.5 mV and TiO2 NPs 21 nm: – 33.7 mV). The polydispersity index values for all three types of NPs were as follows: AgNPs 20 nm – 0.295; AgNPs 200 nm – 0.328 and TiO2 NPs 21 nm – 0.168. 2.2. Animals husbandry and administration procedure Adult (14 weeks old) male Wistar rats purchased from Mossakowski Medical Research Center Polish Academy of Science were housed in rodent polyurethane individual cages in stable environmental conditions (temperature 23 ◦ C, humidity 60%, photoperiod 12 h light: 12 h dark), with free access to rodent food and drinking water. After 10 days of acclimatization animals were weighed and randomly divided into experimental and control groups. Animals were intravenously injected (tail vein) with a single dose of 5 mg/kg or 10 mg/kg bw of 20 nm size AgNPs or with 5 mg/kg bw of 200 nm size AgNPs or with 5 mg/kg bw of 21 nm size TiO2 NPs. The solvent control group was injected intravenously with 0.9% NaCl solution, only. Nanoparticles, which enter the body by different routes finally reach the blood circulation system. We decided to choose the intravenous injection to enter NPs directly there. Moreover, this way of administration of NPs is generally believed as the most relevant model to simulate a medical exposure of human. The liquid volume averaged 280–350 ␮l, depending on rat’s body weight. Non-treated group served as negative control. Animals were weighed every week. Groups of 7 animals from experimental and control groups were sacrificed (by Isoflurane inhalation) 24 h, 1 week and 4 weeks after the injections. Material from the same animals was used for all methods. The 3rd Local Ethical Commission for Animal Experiments in Warsaw, Poland approved all procedures and gave permission for this study.

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Table 1 Comet assay in bone marrow leukocytes following single exposure to Ag or TiO2 nanoparticles. Doses

24 h

1 week

Tail moment Negative control Solvent control, NaCl Ag NPs, 5 mg, 20 nm Ag NPs, 10 mg, 20 nm AgNPs, 5 mg, 200 nm TiO2 NPs, 5 mg, 21 nm

1.74 1.83 2.93 1.54 1.03 1.91

± ± ± ± ± ±

1.86 1.23 2.80 2.13 0.67 2.28

Tail DNA% 3.40 3.36 5.72 3.33 2.36 4.61

± ± ± ± ± ±

2.69 1.44 4.25 3.10 0.59 3.54

2.3. Comet assay Cells preparation for the Comet assay was performed according ´ to the method described previously (Dobrzynska, 2005). Briefly, bone marrow was flushed from the femora with RMPI 1640 medium, mixed and swirled to obtain single cells remained in the suspension. Ten microliters of cell suspension were mixed in an Eppendorf tube with 75 ␮l of low melting point agarose (LMPA) for embedding on the slides previously covered with normal melting point agarose (NMPA). After solidification of the agarose at 4 ◦ C another layer of LMPA was added and allowed to solidify at 4 ◦ C again. The slides were immersed in lysing solution overnight at 4 ◦ C. Then slides were incubated in the electrophoresis solution for 20 min to allow the unwinding of DNA. Alkaline electrophoresis was conducted for 20 min at 4 ◦ C, using 24 V and 300 mA. After neutralization, the slides were stained with ethidium bromide (EtBr) and examined under fluorescence microscope. Images of 100 randomly selected cells from each animal were recorded and analyzed ´ et al., 2003). The Comet using CASP image-analysis software (Konca tail moment and percentage of DNA in Comet tail were chosen as parameters for further analysis. The tail moment, which is calculated by multiplying tail distance in pixels by the fraction of DNA in the Comet tail (Singh and Lai, 2009) shows the level of DNA damage. The increased tail moment is an indicator of elevated DNA damage, whereas decreased tail moment shows prevention of DNA damage ´ by applied agent (Dobrzynska and Radzikowska, 2013). 2.4. Micronucleus assay Bone marrow was prepared according to the basic method described by Schmid (1975). Briefly, femora from each animal were removed and bone marrow was flushed from these bones with fetal bovine serum. Cells were centrifuged, and after removing the majority of supernatant, the pellet was resuspended in the remaining liquid. For estimation of induction micronuclei (MN) in polychromatic erythrocytes (PCE), the smears of bone marrow cells were prepared on the microscope slides. Next day, after draining bone marrow cells were stained with solutions of May-Grunwald and Giemsa stains (Schmid, 1975). The proportion of polychromatic/normochromatic erythrocytes (PCE/NCE) were determined

4 weeks

Tail moment 1.88 2.93 4.52 3.02 2.62 3.03

± ± ± ± ± ±

1.27 1.90 2.75 2.81 1.62 2.34

Tail DNA% 5.71 4.88 9.58 6.68 5.88 7.22

± ± ± ± ± ±

2.17 2.48 4.60 4.99 2.82 4.06

Tail moment 0.67 0.97 1.07 3.94 1.95 0.81

± ± ± ± ± ±

0.60 0.62 0.44 2.02 1.46 0.22

Tail DNA% 2.15 2.67 2.39 5.69 4.45 2.71

± ± ± ± ± ±

1.17 1.25 0.59 3.84 2.42 0.87

by analyzing 500 erythrocytes in total. Simultaneously, the frequency of MN was recorded in two thousands of PCE per rat under the light microscope. For estimation of MN in reticulocytes the method of Hayashi et al. (1990) was adapted. Briefly, 25 ␮l of above mentioned cells suspensions was dropped on the microscope slides covered previously with acridine orange aqueous solution and immediately covered with cover slip. Then slides were examined under florescence microscope (Nikon, Japan). Reticulocytes were identified by their reticulum structure and with red fluorescence. The frequencies of micronucleated reticulocytes were recorded based on the observation of thousand cells. 2.5. Statistical analysis One-way analysis of variance (ANOVA) was used to determine significant differences between results of various groups. Fisher’s post hoc test was applied to determine significant changes between groups. P < 0.05 was considered significant, whereas P < 0.001 highly significant. 3. Results For all experiments there were no significant differences between negative and solvent (vehicle) controls, so we present here the comparison of results of experimental groups to negative control group. Tail moments and percentages of DNA in Comet tail of bone marrow leukocytes of rats exposed to silver and titanium dioxide particles are shown in Table 1. The values for exposed animals were sometimes slightly enhanced as compared to control, but results were not statistically significant. Nonetheless, the highest DNA migration was noted after the injection of 5 mg/kg bw AgNPs of 20 nm size. At 24 h and 1 week post exposure the percentage of DNA in Comet tail in this group increased approximately 1.7-fold compared to control group. Results regarding to the frequency of micronuclei in bone marrow polychromatic erythrocytes of bone marrow of rats are shown in Table 2. The percentage of PCE among 500 scored erythrocytes (i.e. polychromatic and normochromatic) varied between 43.54 and 58.58. There was no significant differences between control and

Table 2 Induction of micronuclei in bone marrow polychromatic erythrocytes of male rats following single exposure to Ag or TiO2 nanoparticles. Doses

Percentage of polychromatic erythrocytes ± SD 24 h

Negative control Solvent control, NaCl AgNPs, 5 mg, 20 nm AgNPs, 10 mg, 20 nm AgNPs, 5 mg, 200 nm TiO2 NPs, 5 mg, 21 nm * **

44.80 54.10 51.35 54.30 44.50 43.54

1 week ± ± ± ± ± ±

4.37 12.80 11.88 11.89 11.59 5.19

p < 0.05 compared to control by post hoc Fisher’s test. p < 0.001 compared to control by post hoc Fisher’s test.

53.91 58.58 54.23 53.03 53.35 52.23

± ± ± ± ± ±

MN/1000 PCE ± SD 4 weeks

8.67 10.27 4.10 0.99 4.38 12.36

50.74 43.91 49.28 49.48 50.25 52.55

± ± ± ± ± ±

24 h 5.96 4.86 1.96 7.17 8.47 5.58

4.71 5.85 14.88 14.81 15.63 12.36

1 week ± ± ± ± ± ±

1.98 9.88 10.66** 3.27** 5.42** 3.20*

5.79 6.50 9.50 10.43 11.00 6.06

± ± ± ± ± ±

4 weeks 0.57 1.63 5.48* 2.56* 5.26* 2.04

4.84 3.71 6.31 8.44 6.19 5.88

± ± ± ± ± ±

2.83 1.44 2.19 3.30* 3.01 2.30

M.M. Dobrzy´ nska et al. / Toxicology 315 (2014) 86–91 Table 3 Frequency of micronuclei in male rats bone marrow reticulocytes following single exposure to Ag and TiO2 nanoparticles. Doses

MN/1000 in bone marrow reticulocytes ± SD 24 h

Negative control Solvent control, NaCl AgNPs, 5 mg, 20 nm AgNPs, 10 mg, 20 nm AgNPs, 5 mg, 200 nm TiO2 NPs, 5 mg, 21 nm

1.67 1.71 2.44 0.88 1.89 1.71

1 week ± ± ± ± ± ±

0.82 0.95 3.94 0.99 1.69 0.95

2.00 1.83 1.83 0.88 2.30 3.00

± ± ± ± ± ±

4 weeks 1.41 2.16 2.56 1.46 2.23 4.66

2.33 2.20 1.25 3.25 3.25 1.63

± ± ± ± ± ±

1.63 2.04 1.75 2.38 2.60 1.51

exposed groups. However, in AgNPs treated animals a highly significant (p < 0.001) increase in the number of micronuclei per 1000 PCE was observed at 24 h after exposure independent on dose and particle size. A significantly elevated (p < 0.05) frequency of MN was also observed in PCE from rats exposed to TiO2 NPs. One week after the exposure the significantly enhanced as compared to the negative control levels of MN was noted in all AgNPs exposed groups, but not in TiO2 NPs group. Four weeks following the single exposure still the significantly higher frequency of MN was observed only in animals administered to 10 mg/kg AgNPs of 20 nm size. On the contrary, the exposure of rats to AgNPs and TiO2 NPs did not affect the frequency of MN in reticulocytes. There were no significant differences in the frequency of micronuclei between control and exposed groups at any dose nor time points (Table 3). 4. Discussion Genotoxicity studies providing to the estimation of different types of DNA damage after exposure to xenobiotics are important for risk assessment of potential carcinogens. Nanoparticles which are smaller than a hundred nanometer are able to penetrate cells (Park et al., 2007) and to bind macromolecules including protein and DNA (Chen and Mikez, 2005; AshaRani et al., 2009a). Genotoxicity of nanoparticles may result from direct interaction with DNA or from indirect effects such as release of toxic irons from soluble nanoparticles or generation of oxidative stress (Donaldson et al., 2006; Singh et al., 2009; Magdolenova et al., 2013). Generally, several types of nanomaterials were shown to induce a significant increase of DNA damage including elevated MN frequencies. The mechanisms of genotoxic effects of nanomaterials have been described in the recent review articles (Klien and Godnic-Cvar, 2012; Magdolenova et al., 2013; Bartłomiejczyk et al., 2013). There are not many genotoxicity papers with AgNPs and TiO2 NPs, especially regarding to in vivo effects. Moreover, the existing results of genotoxicity studies of AgNPs and TiO2 NPs are conflicting. The differences in cellular responses, observed in different studies, may depend among others on the particle size, degree of aggregation, preparation method and doses. The comparison of results is not easy because of incomplete characterization of NPs and inconsistency in describing preparations methods for NPs dispersion. Nanomaterials have a tendency to agglomerate and might be difficult to disperse properly. So, this is very important from the toxicological point of view indication of nanoscale of particles (Schluesener and Schluesener, 2013). Among all characteristics the most important factor related to the toxicity of NPs their size has been considered (Hubbs et al., 2011). Several studies in vitro reported genotoxicity of titanium dioxide NPs (mainly with a maximum diameter of 20 nm), including induction of MN and DNA strand breaks, in different cell lines such as CHO-K1, TK6, COS-1, WIL2-NS, NIH-3T3, SHE fibroblasts (Rahman et al., 2002; Gurr et al., 2005; Wang et al., 2007; Kang et al., 2008; Falck et al., 2009; Huang et al., 2009; Di Virgilio et al., 2010; Magdolenova et al., 2012). Magdolenova et al. (2012) found that

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genotoxicity of TiO2 NPs (the same as used in our study) measured by the comet assay depends on dispersion of NPs. Two different dispersions showed on one case positive and in another, negative effect on human lymphoblastoid cells TK6. Incidence of DNA damage (Kang et al., 2008; Ghosh et al., 2012) including micronuclei (Kang et al., 2008; Akhal’tseva et al., 2011) were noted also in human lymphocytes and human lymphoblastoid TK6 cell lines (Magdolenova et al., 2012). Some other authors did not confirm the genotoxic effects of TiO2 NPs (Driscoll et al., 1997; Linnainmaa et al., 1997; Warheit et al., 2007). Similarly, in vivo studies showed that TiO2 NPs induced micronuclei and DNA strand breaks in peripheral blood in adult male mice exposed to 500 mg/kg TiO2 NPs of 21 nm size through drinking water for 5 days (Trouiller et al., 2009) and in bone marrow of mice administered by gavage for seven days at 40–100 mg/kg of 33 nm size (Sycheva et al., 2011), but five days inhalation of TiO2 NPs did not induce MN in peripheral blood lymphocytes of mice (Lindberg et al., 2012). Ahamed et al. (2008) on the basis of mouse embryonic cells observations reported that silver nanoparticles are able to cause DNA damage to mammalian cells. AgNPs of 6–20 nm size enhanced frequency of MN and DNA strand breaks in the cell lines glioblastoma and IMR-90 human lung fibroblasts (AshaRani et al., 2009a,b). Smaller size (5 nm) AgNPs induced cytotoxicity and DNA damage measured by the Comet assay in mouse lymphoma cells L5178Y/Tk+/− (Mei et al., 2012). Flower et al. (2012) and Ghosh et al. (2012) noted that AgNPs of size ranged from 40 to 100 nm caused migration DNA of human peripheral blood cells. On the contrary, AgNPs did not induce genotoxicity in human skin keratin nor in lymphocytes in vitro (Singh et al., 2010; Lu et al., 2010; Sarkar et al., 2011) In in vivo study, Song et al. (2012) observed increased frequency of MN in peripheral blood reticulocytes of single exposed mice (3 mg AgNPs/mouse). Other authors did not find toxicity in male and female rat bone marrow after 28-days oral exposure at doses 30–1000 mg/kg/day and there were no statistically significant differences in the number of micronucleated erythrocytes after AgNPs exposure when compared to the control (Kim et al., 2008). Similarly, there were no statistically significant differences in the micronucleated PCE or in ratio of PCE among the total erythrocytes after AgNPs inhalation of rats for 90 days (Kim et al., 2011). Current study was focused on the impact of Ag and TiO2 nanoparticles on the bone marrow, where hematopoiesis takes place. Potential damage to DNA of bone marrow cells may cause in consequence diminished presence or increased level of affected cells in the circulated blood. Our study showed that neither AgNPs nor TiO2 NPs caused cytotoxicity to bone marrow’s red and white blood cells. Moreover, our results with the comet assay did not show significant increase of DNA damage in bone marrow leukocytes of rats. Such results were present at all time points post single treatment to AgNPs and TiO2 NPs. Current results might reflect resistance of genetic material of leukocytes to the action of Ag and TiO2 NPs. As the first sample was taken at 24 h post treatment, it is also likely that quick repair of DNA damage might occur. Although the level of DNA migration induced by AgNPs in bone marrow leukocytes were not significant, mild increase in the % DNA in tail (especially in case of AgNPs of 20 nm size) suggests that these NPs might induce DNA breaks for instance after single application of higher dose or after repeated exposure. Hudecová et al. (2012) with the same 20 nm AgNPs in in vitro study showed that these NPs induce oxidized DNA lesions (measured with OGG1) additionally to strand breaks. In our conditions we measured DNA strand breaks and not oxidized DNA lesions with the comet assay. However Ghosh et al. (2012) observed increased DNA damage in bone marrow cells 18 h post single treatment of mice to 10–20 mg/kg of AgNPs of size smaller than 100 nm. Also induction of DNA strand

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breaks in bone marrow cells of mice exposed within 7 days to TiO2 at 40 and 200 mg/kg bw was also reported by Sycheva et al. (2011). Both investigated NPs induced significant levels of MN in PCE of bone marrow, which may originate from lagging acentric chromosomes or chromatid fragments caused by unrepaired DNA breaks. Our results confirmed mentioned before results from previous in vivo studies on MN induction by TiO2 NPs (Trouiller et al., 2009; Sycheva et al., 2011) and showed first time the positive response of AgNPs on the induction of MN in bone marrow PCE. In addition, in case of AgNPs increased level of MN was observed not only one day after the exposure, but also in later time points. Results showed that the frequency of MN decreased within 4 weeks. At 1 week after the exposure the frequencies of MN were similar in all AgNPs exposed groups, whereas 4 weeks after exposure the enhanced level of MN was observed only in case of the highest dose (10 mg/kg bw) of 20 nm AgNPs. This finding confirms the previous observations that the smaller sized silver nanoparticles were found to be more toxic than larger ones (Panda et al., 2011; Gaiser et al., 2012). The enhanced frequency of MN after one or four weeks following the single exposure, noted in this study, is unusual when investigated agents are quickly metabolized and removed. The current results suggest that silver nanoparticles might be accumulated in the organism in the size and dose dependent manner. Thus they may affect the bone marrow PCE for a longer time. Although NPs are taken up by phagocytic system of liver and spleen leading to its clearance from the systematic circulation (Sadauskas et al., 2007) current results showed the possible presence of AgNPs in the rat’s body up to 4 week after exposure. This is in line with other studies, which reported the accumulation of AgNPs in different organs of laboratory animals (Takanaka et al., 2001; Tang et al., 2008; Sung et al., 2008; Garza-Ocanas et al., 2010; Dziendzikowska et al., 2012). Sung et al. (2009) observed a dose-dependent increase of silver nanoparticle concentration in the blood, indicating a systematic distribution of silver nanoparticles by circulating blood. Moreover, in vitro study on IMR-90 cells treated with AgNPs showed endocytosis of AgNPs in the cells and its presence in the nucleus (AshaRani et al., 2009a). Such event may affect cell division, DNA synthesis and damage as well as chromosomal segregation. NPs of smaller size (8–10 nm) may get to the nucleus via nuclear pores, whereas larger (15–60 nm) remain usually free in cytoplasm during interfase and may have access to the genetic material during cell division when nuclear membrane disappears (Liang et al., 2008; Singh et al., 2009; Barillet et al., 2010). During mitosis NPs might interact with chromosomes leading to clastogenic or aneugenic effects i.e. breaks of chromosomes or disturb mitosis (Magdolenova et al., 2013). Such mechanism of interaction of AgNPs was likely present in our experiment. Reticulocytes are younger stages of red blood cells development. Therefore the absence of MN inside them at 24 h after exposure was expected. The reason of absence of MN in reticulocytes at 1 and 4 weeks while they are present in PCE, seems to be the removing of DNA fragments (i.e. MN) from the red blood cells during further differentiation or elimination of whole cells containing genetic material. The mechanism of micronuclei elimination from cells is not clearly understood. It has been proposed that MN are degraded in situ (Rao et al., 2008; Utani et al., 2010), or the content of MN is diluted during cell division if it is not replicated (Shimizu, 2011) or finally that MN might be removed directly from cells to the outside (Shimizu, 2011). In conclusion, current results indicated that different bone marrow cells display different susceptibility toward genotoxicity mediated by both investigated nanoparticles. Genetic material of bone marrow polychromatic erythrocytes are the main target of both TiO2 and Ag NPs, whereas the negative response have been shown in bone marrow reticulocytes and leukocytes. The impact of AgNPs on the DNA of bone marrow polychromatic erythrocytes

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