ROS-mediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells

Toxicology in Vitro 25 (2011) 231–241

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ROS-mediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells Ritesh K. Shukla a,c, Vyom Sharma a, Alok K. Pandey a, Shashi Singh b, Sarwat Sultana c, Alok Dhawan a,⇑ a

Nanomaterial Toxicology Group, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O. Box 80, Lucknow 226 001, Uttar Pradesh, India b Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research (CSIR), Uppal Road, Hyderabad 500 007, Andhra Pradesh, India c Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110 062, India

a r t i c l e

i n f o

Article history: Received 19 August 2010 Accepted 11 November 2010 Available online 17 November 2010 Keywords: TiO2 nanoparticles Genotoxicity Reactive oxygen species Oxidative stress Human epidermal cells

a b s t r a c t Titanium dioxide nanoparticles (TiO2 NPs) are among the top five NPs used in consumer products, paints and pharmaceutical preparations. Since, exposure to such nanoparticles is mainly through the skin and inhalation, the present study was conducted in the human epidermal cells (A431). A mild cytotoxic response of TiO2 NPs was observed as evident by the MTT and NR uptake assays after 48 h of exposure. However, a statistically significant (p < 0.05) induction in the DNA damage was observed by the Fpgmodified Comet assay in cells exposed to 0.8 lg/ml TiO2 NPs (2.20 ± 0.26 vs. control 1.24 ± 0.04) and higher concentrations for 6 h. A significant (p < 0.05) induction in micronucleus formation was also observed at the above concentration (14.67 ± 1.20 vs. control 9.33 ± 1.00). TiO2 NPs elicited a significant (p < 0.05) reduction in glutathione (15.76%) with a concomitant increase in lipid hydroperoxide (60.51%; p < 0.05) and reactive oxygen species (ROS) generation (49.2%; p < 0.05) after 6 h exposure. Our data demonstrate that TiO2 NPs have a mild cytotoxic potential. However, they induce ROS and oxidative stress leading to oxidative DNA damage and micronucleus formation, a probable mechanism of genotoxicity. This is perhaps the first study on human skin cells demonstrating the cytotoxic and genotoxic potential of TiO2 NPs. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction The use of nanotechnology has seen an exponential growth in the areas of health care, consumer products, clothes, electronics, sporting goods etc. (Thomas et al., 2006; Singh et al., 2009). This is due to the unique properties of nanomaterials (e.g. chemical, mechanical, optical, magnetic, and biological) which make them desirable for commercial and medical applications (Oberdorster et al., 2005; Nel et al., 2006; Jin et al., 2008). According to a recent survey, the number of nanotechnology-based consumer products available in the world market has crossed the 1000 mark (PEN, 2009). TiO2 NPs are among the most commonly used metal oxide NPs in industrial products, such as cosmetics, sunscreens, food products, paints and drugs. TiO2 NPs have a clear transparent appearance upon topical application compared to the micron-scale particles which leave a white residue on the skin. They also increase UV protection capability of sunscreens (Kiss et al., 2008; Barnard, 2010). The increased use of NPs is a matter of a great con⇑ Corresponding author. Tel.: +91 522 2230749; fax: +91 522 2628227/+91 522 2611547. E-mail addresses: [email protected], [email protected] (A. Dhawan). 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.11.008

cern among health and environmental scientists due to their potential human and environmental risk (Oberdorster et al., 2005; Handy et al., 2008; Dhawan and Sharma, 2010; Hu and Gao, 2010). Among the possible exposure routes, inhalation and skin contact are considered most important for NPs. The toxic effects of NPs can be attributed to the small size and hence large surface area, thereby increasing chemical reactivity and penetration in the living cells (Donaldson et al., 2000; Gojova et al., 2007; Medina et al., 2007; Pan et al., 2009). TiO2 NPs have also been shown to produce reactive oxygen species (ROS) leading to the toxicity (Gurr et al., 2005; Wang et al., 2007; Kang et al., 2008; Barnard, 2010). Skin is the largest organ of the body and could serve an important portal route for entry of nanoparticles in the human body. Studies have shown that NPs penetrate the outermost layer of skin cells (Lademann et al., 1999; Bennat and Muller-Goymann, 2000; Tinkle et al., 2003). TiO2 NPs have been reported to elicit various adverse cellular effects including oxidative stress and DNA damage (Gurr et al., 2005; Hussain et al., 2005; Jeng and Swanson, 2006; Wang et al., 2007, 2009; Vamanu et al., 2008; Falck et al., 2009). Therefore, in the present study an attempt was made to assess the uptake and cellular toxicity including genotoxic potential of TiO2 NPs in human epidermal cells (A431) as well as to understand its possible mechanism.

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2. Materials and methods 2.1. Chemicals Titanium (IV) oxide nanopowder (99.7%, anatase, CAS No. 131770-0), neutral red dye, low melting point agarose (LMA), ethidium bromide (EtBr), triton X-100, ethyl methanesulfonate (EMS) cytochalasin B and 2, 7-dichlorofluorescein diacetate were purchased from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). formamidopyrimidine DNA glycoslyase (Fpg) Enzyme was obtained from Trevigen, Inc (USA). Normal melting agarose (NMA), ethylenediaminetetraacetic acid (EDTA) disodium salt, and (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) dye were purchased from Himedia Pvt. Ltd. (Mumbai, India). Hydrogen peroxide (H2O2) was purchased from Ranbaxy Fine Chemical Ltd., New Delhi, India. Phosphate buffered saline (Ca2+, Mg2+ free; PBS), Dulbecco’s modified eagle medium (DMEM), trypsin–EDTA, fetal bovine serum (FBS), trypan blue, antibiotic and antimycotic solution (10,000 U/ml penicillin,10 mg/ml streptomycin 25 lg/ml amphotericin-B) were purchased from Life Technologies (India) Pvt. Ltd., (New Delhi, India). Giemsa stain powder was purchased from British Drug Houses Ltd. (Poole, England). All other chemicals were obtained locally and were of analytical reagent grade. Cell culture plastic wares were obtained from Tarsons Products Pvt. Ltd. (Kolkata, India). 2.2. Particle preparation and characterization 2.2.1. Preparation TiO2 NPs were suspended in two different dispersion media Milli-Q water and DMEM supplemented with 10% FBS at a concentration of 160 lg/ml and probe sonicated (Sonics Vibra cell, Sonics & Material Inc., New Town, CT, USA) at 30 W for 10 min (1.5 min pulse on and 1 min pulse off for four times). 2.2.2. Characterization The nanoparticle suspensions were characterized by following method as given below. 2.2.2.1. Transmission electron microscopy. Samples for transmission electron microscopy (TEM) analysis were prepared by drop-coating TiO2 NPs suspension (8 lg/ml) on carbon-coated copper grids. The suspension on the TEM grids was allowed to dry prior to measurement. TEM measurements were performed at an accelerating voltage of 120 kV (Model 1200EX, JEOL Ltd., Tokyo, Japan). 2.2.2.2. Dynamic light scattering. The average hydrodynamic size, size distribution and zeta potential of TiO2 NPs in suspension were determined by dynamic light scattering (DLS) and phase analysis light scattering respectively using a Zetasizer Nano-ZS equipped with 4.0 mW, 633 nm laser (Model ZEN3600, Malvern Instruments Ltd., Malvern, UK). 2.3. Cell culture and exposure to NPs The human epidermal cell line (A431) was obtained from National Centre for Cell Sciences, Pune, India, and cultured in DMEM supplemented with 10% FBS, 0.2% sodium bicarbonate and 10 ml/L antibiotic and antimycotic solution at 37 °C under a humidified atmosphere of 5% CO2/95% air. Stock suspension of TiO2 NPs (160 lg/ml) in DMEM (supplemented with 10% FBS) was serially diluted to concentrations ranging from 80 to 0.008 lg/ml (corresponding to 25–0.0025 lg/cm2 respectively) for cellular uptake, cytotoxicity assays, Comet assay, micronucleus assay, ROS generation and oxidative stress parame-

ters (glutathione and lipid peroxidation). For each experiment, the particle suspension was freshly prepared, diluted to appropriate concentrations and immediately applied to the cells. Culture medium without TiO2 NPs served as the control in each experiment. 2.4. Cellular uptake 2.4.1. Flow cytometry The uptake of NPs using flow cytometry was carried out according to the method developed by Suzuki et al. (2007). 1.0  105cells/ well were seeded in 6-well cell culture plates. After 24 h of seeding, the cells were exposed to TiO2 NPs (0.008, 0.08, 0.8, 8 and 80 lg/ ml) for 6 h. After exposure, the culture medium containing NPs was removed and the cells were harvested using 0.05% trypsin. They were then centrifuged at 250g for 5 min. The supernatant was discarded and the pellet was resuspended in 0.5 ml of PBS. The uptake of particles was determined by flow cytometer (FACS Canto™ II, BD BioSciences, San Jose, CA, USA) equipped with a 488 nm laser. 2.4.2. Transmission electron microscopy (TEM) Human epidermal cells (1  105cells/well) were seeded in 6well cell culture plates. 24 h after seeding, the cells were exposed to TiO2 NPs (8 lg/ml) for 6 h. After exposure the treatment was aspirated and the cells were washed twice with 1 PBS. They were then fixed with 2.5% glutaraldehyde in a 6 well plate, scraped and centrifuged. After washing with 0.1% phosphate buffer the pellet was post fixed in 1% osmium tetraoxide for 3 h. Fixed pellet was then washed and dehydrated through grades (30–100%) of acetone. Sample was infiltrated with AralditeÒ resin overnight at room temperature and finally embedded in pure resin. The blocks were cured at 60 °C for 72 h. After incubation, ultrathin sections (60 nm) were prepared using Reichert-Jung ultra microtome (Vienna, Austria). The sections were stained with uranyl acetate & Reynold’s lead citrate. The grids were examined under a TEM (JEM2100, JEOL Ltd., Tokyo, Japan) operated at an accelerating voltage of 120 kV using a 20 lm aperture. 2.5. Cytotoxicity assays Cytotoxicity of TiO2 NPs was measured by using MTT and NRU assay. Cells (1.0  104cells/well) in 100 ll of DMEM were seeded in 96-well plates. 24 h after seeding, the cells were exposed to different concentrations of TiO2 NPs (0.008, 0.08, 0.8, 8, 80 lg/ml) for three time periods i.e. 6, 24 and 48 h. Nanoparticle interference with the assay systems was checked in parallel as given below: To determine the interaction of the NPs with the assay dye or dye product, three sets of experiments were conducted in parallel with the above viability assays: (1) In a cell free system, NPs were added to DMEM and absorbance was taken at 540 nm (corresponding to Neutral red dye) and 550 nm (corresponding to MTT dye) respectively using SYNERGY-HT multiwell plate reader, Bio-Tek (USA) using KC4 software. DMEM alone served as the control. (2) In a cell free system, NPs were added in DMEM along with the respective dye (MTT or NR) and incubated for 2 h, after which the absorbance was measured as described above. (3) MTT/NRU assay was performed in control cells and NPs were added after the dye product was formed. The absorbance was measured at the respective wavelengths as described above. The difference in the absorbance after incubation with the NPs indicated the effect of the NPs on the assay system, if any.

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2.5.1. Mitochondrial activity Mitochondrial activity was assessed using the MTT assay according to a modified method of Mosmann (1983). Briefly, cells were exposed to NPs for different time periods (6, 24 and 48 h) and MTT dye (0.5 mg/ml) was added 4 h prior to completion of incubation period into each well. Thereafter, the reaction mixture was carefully taken out and the resulting formazan crystals were solubilized by adding 200 ll of dimethyl sulfoxide (DMSO). The interference of nanoparticles was obviated by centrifuging the plates, transferring the supernatant into a new plate and then measuring the absorbance at 550 nm in a SYNERGY-HT multiwell plate reader, Bio-Tek (USA) using KC4 software.

with 20 lg/ml ethidium bromide (EtBr) and stored at 4 °C in a humidified slide box until scoring. Slides were scored at a final magnification of 400 using an image analysis system (Komet 5.0, Kinetic Imaging, Liverpool, UK) attached to a microscope (DMLB, Leica, Germany) equipped with fluorescence attachment of CCD camera and appropriate filters (N2.1). The Comet parameters used to measure DNA damage in the cells were tail DNA (%) and Olive tail moment (OTM). Images from 50 random cells (25 from each replicate slide) were analyzed for each experiment as recommended by the guidelines (Tice et al., 2000). The experiment (and not the cell) was used as the experimental unit for data analysis.

2.5.2. Neutral red uptake Neutral red uptake assay was conducted according to the method of Borenfreund and Puerner (1985). Briefly, after the exposure, the treatment was discarded and 100 ll of neutral red dye (50 lg/ml) dissolved in serum free medium was added to each well. After incubation at 37 °C for 3 h, cells were washed with a solution of 0.5% formaldehyde and 1% CaCl2. The dye taken up by cells was then dissolved in a solution containing 50% ethanol and 1% acetic acid in Milli-Q water. The interference of nanoparticles was obviated by centrifuging the plates, transferring the supernatant into a new plate and then measuring the absorbance at 540 nm in a SYNERGY-HT multiwell plate reader, Bio-Tek (USA) using KC4 software.

2.7.2. Cytokinesis -block micronucleus (CBMN) assay CBMN assay was carried out according to the method of Fenech (2000). Cells were treated with different concentrations of NPs (0.008, 0.08, 0.8, 8, and 80 lg/ml) for 6 h. DMEM with serum and EMS (6 mM) served as negative and positive controls, respectively. After exposure, the NPs were aspirated, cells washed with medium and grown further for 18 h in 1 ml of fresh complete DMEM medium containing Cytochalasin-B (final concentration 3 lg/ml medium) in a CO2 incubator at 5% CO2.

2.6. Cell viability The cell viability using Trypan blue dye exclusion assay was conducted by the method of Phillips (Philips, 1973) before the start and after the completion of the NPs exposure in the Comet assay. 2.7. Genotoxicity assessment The genotoxic potential of TiO2 NPs was assessed by the Comet assay as well as by cytokinesis-block micronucleus (CBMN) assay. Approximately, 7  104 cells in 1.2 ml of DMEM were seeded in a 12-well cell culture plate. After 24 h, the cells were exposed for 6 h to different concentrations of TiO2 NPs (0.008, 0.08, 0.8, 8, 80 lg/ml). Hydrogen peroxide (25 lM) and Ethyl methanesulfonate (6 mM) were used as positive control for Fpg-Comet assay and CBMN assay, respectively. 2.7.1. Single cell gel electrophoresis/comet assay The formamidopyrimidine DNA glycosylase (Fpg) enzyme is used for the detection of oxidative DNA base damage, in particular, 8-OH guanine (Albertini et al., 2000; Tice et al., 2000). Therefore, Fpg-modified Comet assay was used after treating the cells with TiO2 NPs. Briefly, cells exposed to NPs were washed with serum free medium and harvested with 0.06% trypsin. The cells were re-suspended in DMEM supplemented with 10% fetal bovine serum and slides were prepared by the method as described (Singh et al., 1988) and modified by Bajpayee et al. (2005). Two slides were prepared from each well (one well/concentration) and kept overnight at 4 °C in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris (pH10), with 1% Triton X-100 added just before use). After lysis, slides were washed three times in enzyme buffer (40 mM HEPES, 0.1 M KCl, and 0.5 mM EDTA, pH 8; 0.2 mg/ml BSA) and incubated with a 1:3000 Fpg solution for 30 min at 37 °C. Subsequently, the DNA was subjected to alkaline unwinding for 20 min and electrophoresis was carried out in freshly prepared chilled electrophoresis buffer (1 mM EDTA sodium salt and 300 mM NaOH) at 0.7 V/cm and 300 mA at 4 °C for 30 min. The excess alkali was then neutralized with Tris buffer (400 mM, pH 7.4). The slides were stained

2.7.2.1. Harvesting and slide preparation. The cells were harvested with 500 ll of trypsin (0.125%) and the cell suspension was centrifuged at 250g for 10 min. Slides were prepared by cytocentrifuge method using a cytospin (Thermo Shandon, UK). 250 ll of cell suspension was loaded in each block of cytofunnel and centrifuged at 250g for 8 min. Two slides (two dots on one slide from each replicate culture) were prepared for each concentration. The slides were fixed in chilled methanol (90%) for 5 min, air-dried and stored till stained. 2.7.2.2. Staining and scoring. The slides stained with 10% Giemsa in Sorenson’s buffer. The buffer-mounted slides were examined for the presence of micronuclei in the binucleate cells using a light microscope (DMLB, Leica, Germany) at 1000 magnification. Two thousand binucleate cells from each concentration (1000 binucleate cells from each slide, 500 cells per dot) were scored. The cytokinesis block proliferation index (CBPI) was also calculated from 500cells/concentration as recommended in the OECD Guideline No. 487 (OECD, 2007) as follows: CBPI = (No. of mononucleate cells + 2  No. of binucleate cells + 3  No. of multinucleate cells)/Total No. of cells. 2.8. Measurement of intracellular reactive oxygen species (ROS) The level of intracellular ROS generation was estimated by the method of Wan et al. (1993) and modified by Wilson et al. (2002) using 2, 7-dichlorofluorescein diacetate (DCFDA) dye. Cells (1  104 cells/well) were seeded in a 96-well black bottom plate. After 24 h, the cells were exposed to TiO2 NPs (0.008, 0.08, 0.8, 8 and 80 lg/ml) for 6 h. The NPs containing medium was aspirated and cells were washed twice with 1 PBS. Thereafter, culture medium containing DCFDA dye (20 lM) was added to each well. The plate was incubated for 30 min at 37 °C and the medium containing DCFDA was discarded. 200 ll of 1 PBS was then added to each well and fluorescence intensity was measured in a SYNERGY-HT multiwell plate reader, Bio-Tek (USA) using KC4 software at excitation and emission wavelengths of 485 and 528 nm, respectively. To assess if the autofluorescence of TiO2 NPs interfere with the DCFDA dye, a set of experiment without cells were conducted in parallel. The qualitative analysis of ROS generation was done using a microscope with fluorescence attachment (Model DMLB Leica, Wetzlar, Germany).

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2.10. Statistical analysis

Percentage ROS generation was calculated as follows, after correcting for background fluorescence:

Results were expressed as mean ± SEM of three experiments and data were analyzed using one way analysis of variance (ANOVA) with Dunnett post hoc test to determine significance relative to control. In all cases, p < 0.05 was considered significant.

Percentage ROS generation ¼ ½ðF485=528sample  F485=528sample blank Þ=ðF485=528control  F485=528control blank Þ  100

3. Results

2.9. Oxidative stress markers

3.1. Particle characterization

Cells at a final density of 6  106 in a 75 cm2 culture flask were exposed to different concentrations of TiO2 NPs (0.008, 0.08, 0.8, 8, and 80 lg/ml) for 6 h. After exposure, the cells were scraped and washed twice with chilled 1 PBS. The cells were centrifuged at 500g and the pellet was re-suspended in 1 PBS and then sonicated at 15 W for 60 s (10 s pulse on and 10 s pulse off for three times) to obtain the cell lysate. Protein content was measured by the method of Bradford (Bradford, 1976) using bovine serum albumin as the standard.

The mean hydrodynamic diameter of TiO2 NPs in Milli Q water as measured by DLS was 124.9 nm (Fig. 1A) and the zeta potential was 17.6 mV (Fig. 1B). The characterization of TiO2 NPs was also done in DMEM supplemented with 10% FBS. In culture medium NPs showed a slight increase in the hydrodynamic size (171.4 nm) with a concomitant decrease in the zeta potential (11.5 mV) (Fig. 1C and D). The average size observed from TEM analysis was 50 nm (Fig. 2).

2.9.1. Glutathione estimation Free sulphydryl content was measured as described by Ellman (1959) and expressed as lmole/mg of protein.

3.2. Cellular uptake 3.2.1. Flow cytometry The uptake of TiO2 NPs in the epidermal cells was assessed using a flow cytometer. The side scatter (SSC) intensity which represents granularity of a cell and forward scatter (FSC) representing the size of cell were analysed as shown in Table 1. The percentage intensity of SSC of cells treated with TiO2 NPs (8 and 80 lg/ml) were significantly (p < 0.05) increased (157, and 885% respectively) as compared to the granularity of control cells (100%) (p < 0.05).

2.9.2. Lipid peroxidation (LPO) assay Lipid peroxidation levels were estimated by lipid hydroperoxide assay kit™ (Cayman Chemical Company, MI, USA) according to manufacturers’ protocol. Briefly, lipid hydroperoxide were extracted from cell lysate into chloroform. A solution containing ferrous ions was then added to cell extract, which on reaction with lipid hydroperoxide yielded ferric ions. After 10 min, absorbance of the resulting ferric ion was detected at 500 nm using thiocyanate ion as the chromogen. 13-Hydroperoxy-octadecadienoic acid (13-HpODE) was used as the standard.

16

Size 124.9 nm

14

14

12

12

Intensity (%)

Intensity (%)

16

3.2.2. Transmission electron microscopy Human epidermal cells exposed to TiO2 NPs showed a significant cellular uptake of the NPs as evident from the TEM micropho-

10 8 6

Size 171.4 nm

10 8 6 4

4 2

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0 0.1

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ζ- potential -11.5 mV

500000 400000 300000 200000

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-200

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Fig. 1. Measurement of size and zeta potential of TiO2 NPs by dynamic light scattering: (A) and (B) in Milli Q water; (C) and (D) in DMEM supplemented with 10% FBS.

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tographs (Fig. 3). NPs were found to be present in the cytoplasm as well as nucleus. TEM images showed that NPs were found to be distributed inside the cells mostly in cytoplasm, but some of them were also found in the nucleus (Fig. 3). 3.3. Cytotoxicity A statistically significant (p < 0.05) cytotoxic effect of TiO2 NPs in human epidermal cells (A431) was observed in MTT and NRU assays at the two higher concentrations (8 and 80 lg/ml) after 48 h exposure (Fig. 4A and B). The MTT results demonstrated a 73% and 68% reduction (relative to control) while the NR uptake was reduced to 75% and 65.4% at 8 and 80 lg/ml respectively demonstrating a cytotoxic effect of TiO2 NPs. However after 6 and 24 h exposure, no significant cytotoxicity was observed (Fig. 4A and B). There was no major interaction observed between dyes and NPs as in cell free system (data not shown). 3.4. Genotoxicity assessment

Fig. 2. TEM photomicrograph of TiO2 NPs.

Table 1 Cellular uptake by TiO2 NPs after 6 h exposure in human epidermal cells using flow cytometry. Concentration

% Forward scatter (FSC)

% Side scatter (SSC)

Control TiO2 NPs TiO2 NPs TiO2 NPs TiO2 NPs TiO2 NPs

100.00 ± 0.00 100.49 ± 3.00 101.25 ± 2.63 96.90 ± 0.97 93.75 ± 0.18 93.07 ± 1.09

100.00 ± 0.00 102.46 ± 0.48 105.80 ± 2.48 110.67 ± 1.78 257.28 ± 9.25* 985.38 ± 10.21*

(0.008 lg/ml) (0.08 lg/ml) (0.8 lg/ml) (8 lg/ml) (80 lg/ml)

Values represent mean ± S.E. of three experiments. * p < 0.05 when compared to control.

3.4.1. DNA Damage The cell viability in the Comet assay exceeded 90% for all experimental groups before and after the treatment as assessed by Trypan blue dye exclusion assay (data not shown). A significant (p < 0.05) increase in the qualitative and quantitative DNA damage in cells exposed to TiO2 NPs was observed, as evident by the Comet assay parameters viz. Olive tail moment (OTM) and tail DNA (%) respectively, with and without Fpg treatment at 0.8, 8 and 80 lg/ml (Table 2). A statistically significant (P < 0.05) induction in DNA damage was observed after 6 h exposure to 8 and 80 lg/ml concentration of TiO2 NPs as compared to the respective control cells in conventional Comet assay. However, the DNA damage was significantly enhanced even at 0.8 lg/ml and higher concentrations of TiO2 NPs with Fpg treatment. When compared among the groups, Fpg elicited a significantly greater response at the two higher concentrations of TiO2 NPs (8 and 80 lg/ml) as evident by the Comet assay parameters (Table 2). The data was also analyzed in terms of percentage distribution of cells on the basis of OTM. With the increasing concentration of TiO2 NPs, a shift from a low DNA

Fig. 3. TEM photomicrographs of human epidermal cells showing internalization of TiO2 NPs (8 lg/ml) in cytoplasm and nucleus (A) and (B). Arrows indicate the presence of TiO2 NPs inside different cellular organelles.

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120

6 hr

24 hr

48 hr

% MTT Reduction

100 80

*

*

60 40 20 0 Control

0.008

0.08

0.8

8

80

Concentration of TiO2 NPs (µg/ml)

120

6 hr

24 hr

48 hr

% NR Uptake

100

*

80

* 60 40 20 0 Control

0.008

0.08

0.8

8

80

Fig. 5. Effect of TiO2 NPs on percentage distribution of human epidermal cells with respect to OTM (A) Fpg () and (B) Fpg (+).

Concentration of TiO2 NPs (µg/ml) Fig. 4. Concentration and time dependent cytotoxicity of TiO2 NPs in human epidermal cells (A) % MTT reduction, (B) % neutral red uptake.The viability of the control cells was considered as 100%. The Data are expressed as means ± SEM from three independent experiments. *p < 0.05, when compared to control.

damage category (OTM < 1.5), in control, to high DNA damage category (OTM > 2.5) in TiO2 NPs exposed cells, was observed (Fig. 5A and B). 3.4.2. Micronucleus induction A statistically significant (p < 0.05) induction in micronucleus formation was observed in human epidermal cells after 6 h exposure to TiO2 NPs at 0.8 lg/ml (14.67 MN/1000 BNCs), 8 lg/ml (15.67/1000) and 80 lg/ml (16.00/1000) with respect to control (9.33 MN/1000 BNCs; Table 3). 3.5. Measurement of intracellular ROS A significant (p < 0.05) qualitative and quantitative concentration-dependent increase in% ROS generation was observed in the

Table 3 Effect of TiO2 NPs on micronucleus formation in human epidermal cells.

Control EMSa TiO2 NPs (0.008 lg/ ml) TiO2 NPs (0.08 lg/ml) TiO2 NPs (0.8 lg/ml) TiO2 NPs (8 lg/ ml) TiO2 NPs (80 lg/ ml)

No. of MN/1000 binucleated cells

Cytokinesis block proliferation index (CBPI)

9.33 ± 1.00 26.67 ± 0.88*** 11.67 ± 1.20

1.64 1.70 1.70

12.67 ± 0.88

1.72

14.67 ± 1.20*

1.70

15.67 ± 0.88**

1.77

**

1.78

16.00 ± 0.58

Values represent mean S.E. of three experiments for each concentration. a EMS- ethyl methanesulfonate-positive control (6 mM). * p < 0.05 when compared to control. ** p < 0.01 when compared to control. *** p < 0.001 when compared to control.

Table 2 DNA damage in human epidermal cells after 6 h exposure to TiO2 NPs as evident by the Comet parameters. Concentration

Control Positive control (H2O2 25 lM) TiO2 NPs (0.008 lg/ml) TiO2 NPs (0.08 lg/ml) TiO2 NPs (0.8 lg/ml) TiO2 NPs (8 lg/ml) TiO2 NPs (80 lg/ml)

OTM (arbitrary unit)

Tail DNA (%)

Fpg ()

Fpg (+)

Fpg ()

Fpg (+)

1.20 ± 0.01 4.42 ± 0.20* 1.27 ± 0.05 1.30 ± 0.03 1.43 ± 0.09 1.79 ± 0.08* 1.91 ± 0.04*

1.24 ± 0.04 6.31 ± 0.51*,# 1.44 ± 0.07 1.80 ± 0.11 2.20 ± 0.26* 2.56 ± 0.20*,# 2.95 ± 0.20*,#

9.36 ± 0.69 23.89 ± 0.56* 9.72 ± 0.78 9.76 ± 0.40 11.79 ± 0.94 2.35 ± 0.43* 12.89 ± 0.47*

9.65 ± 0.21 29.58 ± 0.90*,# 11.13 ± 0.38 11.50 ± 0.86 13.29 ± 0.59* 16.07 ± 0.42*,# 20.06 ± 1.13*,#

Values represent mean ± S.E. of three experiments. * p < 0.05 when compared to control using one way ANOVA. # p < 0.05 using when compared to Fpg () at the same concentration using Student ‘t’ test.

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form of fluorescence on treatment with TiO2 NPs (49.2%, 66.7%, 78.1% at 0.8, 8 and 80 lg/ml respectively; Figs. 6 and 7).

237

tions respectively after 6 h exposure to TiO2 NPs in human epidermal cells (Fig. 8A).

3.6. Oxidative stress markers 3.6.1. Effect of TiO2 NPs on glutathione (GSH) content A significant reduction (p < 0.05) in cellular GSH content (15.76% and 23.56%) was observed at 8 and 80 lg/ml concentra200

* 4. Discussion

*

180

*

160

% ROS Generation

3.6.2. Effect of TiO2 NPs on lipid peroxidation (LPO) Cells exposed to TiO2 NPs showed a concentration-dependent statistically significant (p < 0.05) increase in hydroperoxide concentration at 8 lg/ml (60.51%) and 80 lg/ml (82.4%; Fig. 8B).

140 120 100 80 60 40 20 0 Control

0.008

0.08

0.8

8

80

Concentration of TiO2 NPs (µg/ml) Fig. 6. Effect of TiO2 NPs on the generation of reactive oxygen species (ROS) in human epidermal cells. The % ROS generation of the control cells was considered 100%. Data represents mean ± SEM of three experiments.*p < 0.05, compared to control.

TiO2 NPs are widely used in consumer products especially sunscreen and cosmetics. Although several studies have been done to assess their toxicity in mammalian cells, to the best of our knowledge this is the first study showing genotoxicity in human epidermal cells. Since the skin serves as the first portal of entry for NPs used in cosmetics, human epidermal cells (A431) were used as an in vitro model for assessing genotoxicity of TiO2 NPs. Concentrations used in this study are far less than those used in the cosmetics. In the commercial sunscreens being sold in the market, the concentration of TiO2 NPs is between 3% and 15%. Therefore a person applying 5 ml lotion/cream is exposed to 150–750 mg NPs (depending on the concentration in the cream). In our study the highest concentration of TiO2 NPs was 80 lg/ml (0.080 mg/ml) which is far less than the concentrations used in sunscreens (30 mg/ml at the lowest concentration). However considering the fact that some NPs may persist on skin even after the cream is washed off and the fact that TiO2 NPs could gain entry inside the skin in case it is damaged, the concentrations used in the present study are realistic.

Fig. 7. Photomicrographs showing the generation of intracellular reactive oxygen species (ROS) using DCFDA dye in human epidermal cells (A) Control cells; (B–D) Cells exposed to TiO2 NPs (0.08 lg/ml, 8 lg/ml and 80 lg/ml respectively) for 6 h showing increase in fluorescence (Magnification-X200).

Glutathione (µmol/mg protein)

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0.4 0.35

*

0.3

*

0.25 0.2 0.15 0.1 0.05 0

Control

0.008

0.08

0.8

8

80

Hydroperoxide concentration (nmol)

Concentration of TiO2 NPs (µg/ml)

1.6

*

1.4

*

1.2 1 0.8 0.6 0.4 0.2 0

Control

0.008

0.08

0.8

8

80

Concentration of TiO2 NPs (µg/ml) Fig. 8. Effect of TiO2 NPs on the oxidative stress markers in human epidermal cells. (A) Glutathione content, (B) lipid peroxidation. Data represents mean ± SEM of three experiments. *p < 0.05, compared to control.

The findings of the present study demonstrate that TiO2 NPs possess DNA damaging potential in human epidermal cells. These NPs also induce significant concentration dependent depletion of GSH, LPO induction and ROS generation. The direct correlation between ROS generation and oxidative DNA damage further suggests that oxidative stress could act as an important route by which TiO2 NPs induce DNA damage. Prior to studying genotoxicity and oxidative stress potential of TiO2 NPs, they were characterized for their size and zeta potential. The Brunauer Emmett Teller (BET) size of TiO2 NPs claimed by its commercial supplier was 25 nm. However, the size of the NPs was further confirmed by two independent methods: DLS and TEM. The mean hydrodynamic size obtained from DLS was found to be more in culture medium (171.4 nm) as compared to milli Q water (124.9 nm), which might be due to slight increase in agglomeration. However, the size observed by TEM was 50 nm. This difference in size can be attributed to the different principles employed for measurement (i.e. BET, DLS and TEM). TEM gives the direct measurement of particle size, distribution and morphology by image analysis, while DLS measures the size distribution of particles in aqueous state which is usually larger than BET or TEM diameter. DLS measurement gives the idea of stability of nanoparticles in aqueous medium which resembles more to the exposure condition as compared to TEM and BET (Dhawan et al., 2009; Sharma et al., 2009). Agglomeration commonly occurs when particles are introduced into the aqueous medium; it was observed that the particles were stable for longer period in serum than without it. This may be due to the coating of NPs with proteins in serum. Suzuki et al. (2007) have suggested that the determination of granularity of cells through flow cytometry is a good way to eval-

uate the uptake potential of NPs in cells. Our results show that uptake of TiO2 NPs in human epidermal cells is concentrationdependent. Our results are consistent with Suzuki et al. (2007) and Xu et al. (2009), who have reported that higher concentrations of TiO2 NPs resulted in higher granularity in the cells. The results obtained from flow cytometry were also validated by TEM. TiO2 NPs were found to get internalized into the human skin epidermal cells or adhere to the cell membrane depending on their size. Nanoparticles of 30–100 nm were internalized into the cytoplasm, vesicles and also the nucleus, while larger particles (>500 nm) remained outside the cells. Our data demonstrates that TiO2 NPs has mild cytotoxic effect on human epidermal cells (A431). The results from the MTT assay, used for studying the expression of mitochondrial succinate dehydrogenase enzyme, showed that there was a significant reduction in the viability of human skin epidermal cells when exposed to TiO2 NPs at 8 and 80 lg/ ml for 48 h. Earlier studies pertaining to cytotoxic evaluation using MTT assay have been inconclusive with some of them showing a positive response (Jin et al., 2008; Simon-Deckers et al., 2008; Di Virgilio et al., 2010) while others showing a negative response (Park et al., 2007; Wang et al., 2007) in different mammalian cell types. The variability in the results of above mentioned studies may be attributed to the different cell types involved. In addition, the source of NPs and method of treatment preparation may also contribute to the differential response of cellular systems. Therefore to further confirm the cytotoxic potential of TiO2 NPs, NRU assay was also performed. The results were in accordance with the MTT assay. NPs can interfere with the various dyes and their products used in colorimetric and fluorometric assays due to their physical and chemical properties which could lead to misinterpretation of data (Doak et al., 2009; Monteiro-Riviere et al., 2009; Stone et al., 2009; Baer et al., 2010; Dhawan and Sharma, 2010). In the present study, this was obviated by a parallel set of experiments conducted to check the interaction of NPs with MTT, NR and DCFDA dyes. Our data demonstrate that TiO2 NPs do not interact with these dyes used for cytotoxicity assessment as well as for ROS measurement. ROS generation has been proposed as a possible mechanism involved in the toxicity of NPs (Gurr et al., 2005; Long et al., 2006; Nel et al., 2006; Eom and Choi, 2009). TiO2 NPs in aqueous suspension produce free radicals (Hirakawa et al., 2004) which may damage DNA by oxidation, nitration, methylation or deamination reactions (Schins and Knaapen, 2007). The data of the present study demonstrate that TiO2 NPs induce oxidative DNA damage as evident by the results of the Fpg-modified Comet assay. This was also corroborated by the fact that ROS generation was significantly enhanced on exposure to TiO2 NPs. These results are in accordance with Kang et al. (2008) who reported that TiO2 NPs induced ROS generation in human lymphocytes. A statistically significant dose-dependent increase was observed in the MN frequency at 0.8, 8 and 80 lg/ml concentration of TiO2 NPs, further confirming their genotoxic potential. Our data is in accordance with that reported by Gurr et al. (2005) who have shown that 10 lg/ml of TiO2 induced genotoxicity in BEAS 2B cells. However the concentrations at which DNA damage was observed are lower than those reported by Wang et al. (2007) and Falck et al. (2009). These differences might be due to the difference in cell type and culture conditions (Lanone et al., 2009). In this study we also observed a strong correlation between ROS and genotoxicity endpoints such as DNA damage and micronucleus formation. The correlation between these genotoxicity endpoints with ROS indicate that ROS might be responsible for the DNA damage observed in the Comet assay (R2 = 0.99) and MN formation observed in the CBMN assay (R2 = 0.96; Fig. 9A and B). In the present study, glutathione depletion and an increase in the lipid peroxidation levels after exposure to TiO2 NPs was

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18 r² = 0.9906

3

16

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OTM (arbitrary Unit)

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2.5 2 1.5 1

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% ROS Generation

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120

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% ROS Generation

Fig. 9. Regression analyses of % ROS generation with respect to different oxidative stress parameters and DNA damage. Correlation after 6 h exposure to TiO2 NPs with (A) Olive tail moment (OTM) (B) micronucleus (MN) formation (C) glutathione levels and (D) hydroperoxide concentration.

observed in human epidermal cells which indicates oxidative stress. Similar observations have been made by Gurr et al. (2005) who measured MDA level in human bronchial epithelial cells while Jin et al. (2008) and Reeves et al. (2008) have shown that TiO2 NPs induce oxidative stress in mouse fibroblast and fish cells respectively. Our data also showed a good correlation between ROS and oxidative stress markers [ROS vs. GSH (R2 = 0.93) and ROS vs. LPO (R2 = 0.85; Fig. 9C and D)].

Schins and Knaapen (2007) reported that reactive oxygen species (ROS) play a major role in the genotoxicity of nanoparticles. This may be due to their surface properties, the presence of transition metals and lipid peroxidation (Schins and Knaapen, 2007). The data from the present study demonstrated that TiO2 NPs induce ROS and oxidative stress leading to genotoxicity in human epidermal cells. This probable primary genotoxicity mechanism may further trigger signal transduction pathways leading to apoptosis or

TiO2 NPs Lipid Peroxidation

us

cle

Nu

ROS Oxidative Stress

Mitochondria

Membrane Damage

DNA Damage Apoptosis Cell Death

Fig. 10. Possible mechanism of TiO2 NPs induced genotoxicity in human epidermal cells.

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cause interferences with normal cellular processes thereby causing cell death. The possible schematic mechanism of TiO2 NPs induced cellular toxicity in human epidermal cells under in vitro condition is depicted in Fig. 10. The DNA damage, micronucleus formation and oxidative stress markers raise concern about the safety associated with applications of TiO2 NPs in consumer products. However more studies need to be conducted in vivo to fully understand the mechanism of TiO2 NPs toxicity. Conflict of interest There is no conflict of interest. Acknowledgement The authors wish to thank Dr. K.C. Gupta, Director, IITR for his support. The authors gratefully acknowledge the funding from CSIR, New Delhi under its network project (NWP35), Supra institutional Project (SIP-08), the Department of Science and TechnologyUK India Education and Research Initiative (UKIERI) Research Award (DST/INT/UKIERI/SA/P-10/2008) and DST-NSTI grant (SR/ S5/NM-01/2007). RKS thanks University Grant Commission (UGC), New Delhi for the award of Senior Research Fellowship. VS. (Senior Research Fellow) acknowledge the financial support by Council of Scientific and Industrial Research (New Delhi). The authors are thankful to Dr. Renu Paschricha, National Chemical Laboratory, Pune for carrying out the transmission electron microscopy of nanoparticles. References Albertini, R.J., Anderson, D., Douglas, G.R., Hagmar, L., Hemminki, K., Merlo, F., Natarajan, A.T., Norppa, H., Shuker, D.E.G., Tice, R., Waters, M.D., Aitio, A., 2000. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. Mutat. Res. Rev. Mutat. Res. 463, 111–172. Baer, D.R., Gaspar, D.J., Nachimuthu, P., Techane, S.D., Castner, D.G., 2010. Application of surface chemical analysis tools for characterization of nanoparticles. Anal. Bioanal. Chem. 396, 983–1002. Bajpayee, M., Pandey, A.K., Parmar, D., Mathur, N., Seth, P.K., Dhawan, A., 2005. Comet assay responses in human lymphocytes are not influenced by the menstrual cycle: a study in healthy Indian females. Mutat. Res. 565, 163– 172. Barnard, A.S., 2010. One-to-one comparison of sunscreen efficacy, aesthetics and potential nanotoxicity. Nat. Nanotechnol. 5, 271–274. Bennat, C., Muller-Goymann, C.C., 2000. Skin penetration and stabilization of formulations containing microfine titanium dioxide as physical UV filter. Int. J. Cosmet. Sci. 22, 271–283. Borenfreund, E., Puerner, J.A., 1985. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24, 119–124. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Dhawan, A., Sharma, V., 2010. Toxicity assessment of nanomaterials: methods and challenges. Anal. Bioanal. Chem. 398, 589–605. Dhawan, A., Sharma, V., Parmar, D., 2009. Nanomaterials: a challenge for toxicologists. Nanotoxicology 3, 1–9. Di Virgilio, A.L., Reigosa, M., Arnal, P.M., de Mele, M.F.L., 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, S.H., Griffiths, S.M., Manshian, B., Singh, N., Williams, P.M., Brown, A.P., Jenkins, G.J., 2009. Confounding experimental considerations in nanogenotoxicology. Mutagenesis 24, 285–293. Donaldson, K., Stone, V., Gilmour, P.S., Brown, D.M., Macnee, W., 2000. Ultrafine particles: Mechanisms of lung injury. Philos. Trans. R. Soc. A: Math. Phys. Eng. Sci. 358, 2741–2749. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70– 77. Eom, H.J., Choi, J., 2009. Oxidative stress of silica nanoparticles in human bronchial epithelial cell, Beas-2B. Toxicol. In Vitro 23, 1326–1332. Falck, G.C., Lindberg, H.K., Suhonen, S., Vippola, M., Vanhala, E., Catalan, J., Savolainen, K., Norppa, H., 2009. Genotoxic effects of nanosized and fine TiO2. Hum. Exp. Toxicol. 28, 339–352. Fenech, M., 2000. The in vitro micronucleus technique. Mutat. Res. 455, 81–95.

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