In vitro and in vivo genotoxicity of silver nanoparticles

Mutation Research 749 (2012) 60–69

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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

In vitro and in vivo genotoxicity of silver nanoparticles Manosij Ghosh a , Manivannan J a,b , Sonali Sinha a , Anirban Chakraborty c , Sanjaya Kumar Mallick d , Maumita Bandyopadhyay e , Anita Mukherjee a,b,∗ a

Cell Biology and Genetic Toxicology Laboratory, Centre of Advanced Study, Department of Botany, University of Calcutta 35 Ballygunge Circular Road, Kolkata, India Department of Genetics, University of Calcutta 35 Ballygunge Circular Road, Kolkata, India Department of Chemical Engineering, University of Calcutta, India e Centre of Advanced Study, Department of Botany, University of Calcutta 35 Ballygunge Circular Road, Kolkata, India d BD Biosciences, India b c

a r t i c l e

i n f o

Article history: Received 25 November 2011 Received in revised form 9 August 2012 Accepted 24 August 2012 Available online 31 August 2012 Keywords: Apoptosis Cytotoxicity Flow cytometry Genotoxicity ROS Necrosis

a b s t r a c t The biocidal effect of silver nanoparticles (Ag-np) has resulted in their incorporation into consumer products. While the population exposed to Ag-np continues to increase with ever new applications, Ag-np remains a controversial research area with regard to their toxicity in biological systems. Here a genotoxic and cytotoxic approach was employed to elucidate the activity of Ag-np in vitro and in vivo. Characterization of Ag-np using scanning electron microscopy revealed a size range of 90–180 nm. Cytotoxic potential of Ag-np was evaluated in human lymphocytes via cell viability assay (Trypan blue dye exclusion method, MTT and WST assay). The uptake and incorporation of Ag-np into the lymphocytes was confirmed by flow cytometry. Additionally apoptosis (AnnexinV-FITC–PI staining) and DNA strand breaks (comet assay) in human lymphocytes revealed that Ag-np at concentration 25 ␮g/ml can cause genotoxicity. In vivo experiments on plants (Allium cepa and Nicotiana tabacum) and animal (Swiss albino male mice) showed impairment of nuclear DNA. Induction of oxidative stress was also studied. The DNA damage and chromosomal aberrations raise the concern about the safety associated with applications of the Ag-np. A single ip administration of Ag-np gave a significant (P ≤ 0.05) increase in the frequency of aberrant cells and Tail DNA percent at concentrations 10 mg/kg body weight and above. Results of comet assay in A. cepa and N. tabacum demonstrated that the genotoxic effect of Ag-np was more pronounced in root than shoot/leaf of the plants. The present study indicated a good correlation between the in vitro and in vivo experiments. Therefore the biological applications employing Ag-np should be given special attention besides adapting the antimicrobial potential. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The increasing use of nanomaterials in consumer and industrial products has aroused global concern regarding their possible adverse effects on biological systems. The factors which might have an impact on interactions between the different types of nanomaterials and biomolecules have remarkably increased though not solved [1]. The safety/toxicity aspects of nanomaterials have lagged far behind the rate at which they are being produced. This can be attributed to the lack of any guidelines and the absence of a consensus among researchers on experimental protocols or study designs in this field, as well as the unique properties of nanoscale materials,

∗ Corresponding author at: Cell Biology and Genetic Toxicology Laboratory, Centre of Advanced Study, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, India. Tel.: +91 9831061998; fax: +91 033 24614849. E-mail addresses: [email protected] (M. Ghosh), [email protected], [email protected] (A. Mukherjee). 1383-5718/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2012.08.007

which cause problems during the toxicological assessment of novel nanomaterials [2]. Many classes of silver nanoparticles (Ag-np) have been synthesized and widely applied for their bactericidal and viricidal properties [3,4]. Worldwide an extensive growth is projected in the Ag-np market [5] and it is unclear whether the exposure of humans, animals and plants to Ag-np through industrial or domestic waste could produce harmful biological responses [6]. There are a significant number of studies on the genotoxicity and cytotoxicity of silver nanoparticles on mammalian and human cell lines [7–18] and a few reports on plant systems [19,20]. Majority of nanotoxicity research has focused on cell culture systems and to assess nanotoxicity accurately. Fischer and Chan [21] in their review article mentioned the need for in vivo studies. Therefore, we examined the genotoxic potential of Ag-np in both in vitro (in human lymphocyte) and in vivo (mouse bone marrow cells, Allium and Nicotiana) systems. Apart from animal system, plants are an important component of the ecosystem that needs to be included when evaluating the overall toxicological impact of the nanoparticles in the

M. Ghosh et al. / Mutation Research 749 (2012) 60–69

environment. The plants selected for the study (Allium cepa and Nicotiana tabacum) are efficient test systems for screening chemicals and in situ monitoring for genotoxicity of environmental contaminants [22–26]. They have been used successfully in our laboratory as a biomarker of genotoxicity of nanomaterials [27,28]. Ag-np (≤100 nm) was purchased and characterized using transmission electron microscopy (TEM), scanning electron microscope (SEM) and X-ray diffraction (XRD), diffraction light scattering (DLS) and UV visible spectrophotometry for size and dispersion. Prior to its genotoxicity evaluation, the nanoparticle was dispersed in solution by sonication. For in vitro study, human lymphocytes were used for cytotoxicity (trypan blue dye exclusion, MTT and WST assays) and genotoxicity (comet assay) studies. In addition, flow cytometry was performed to measure the mode of cell death and uptake of Agnp in human lymphocytes. TEM was performed to study structural modifications and uptake of Ag-np in cells. The in vitro results were further substantiated by in vivo studies. Bone marrow cells of mice and root or leaf tissues of Allium and Nicotiana plants were used in the comet assay. The study provides evidence of genotoxicity of Ag-np in plant and mammalian systems and the results demonstrate that there is good correlation between the in vitro and in vivo effects of Ag-np. 2. Materials and methods 2.1. Ag-np preparation Ag-np was obtained from Sigma–Aldrich, St. Louis, MO, USA. The physical characteristics of the particles according to the manufacturers data are; size (≤100 nm), purity (99.5%), trace metal basis, surface area (5.0 m2 /g), density (10.49 g/cc). Agnps were suspended in PBS or water and dispersed by ultrasonic vibrations (100 W, 30 kHz) for 30 min. Ag-np were serially diluted at different concentrations according to the test performed. 2.2. Characterization of Ag-np Ag-np powder was characterized using transmission electron microscopy (Jeol JEM-2100 LaB6, 200 kV), scanning electron microscopy, energy-dispersive X-ray spectroscopy (EDX) (Hitachi S-415A Electron Microscope, Tokyo, Japan at 25 kV) and by X-ray diffraction (XRD) analysis. The spectra were recorded in a PW. 3040/60 PANalytical X-ray diffractometer Almelo, Netherlands (Cu K␣ radiation,  1.54443) running at 45 kV and 30 mA. The diffracted intensities were recorded from 2◦ to 99◦ 2 angles. Zeta potential of Ag-np in solution was measured by laser diffractometry using a Nano Size Particle Analyzer (Zen 1600 Malvern, USA). Absorption maxima of Ag-np in solution were scanned by UV–Vis spectrophotometer (Beckman Coulter DU 700, California, USA) for agglomeration at the wavelength of 200–800 nm. 2.3. In vitro cytotoxicity and genotoxicity of Ag-np 2.3.1. Cell preparation and materials Human blood was obtained from 3 healthy male volunteers between 20 and 30 years of age (non smokers and not under any medications), after their consent. The lymphocytes were obtained by centrifuging blood overlaid on Histopaque (Sigma–Aldrich, St. Louis, MO, USA) according to the method of Boyum [29]. Cell viability was checked by Trypan blue exclusion method [30] and was found to be approximately 95%. All experiments were approved by the Research Ethics Committee of the University of Calcutta, Kolkata, India. Lymphocytes were incubated for 3 h at 37 ◦ C in RPMI-1640 media with different concentrations of Ag-np (0, 25, 50, 100, 150 and 200 ␮g/ml). Positive control was maintained with methyl methanesulphonate (MMS, 100 ␮M). 2.3.2. Cytotoxicity of Ag-np in human lymphocytes The effect of Ag-np on membrane integrity was evaluated using Trypan blue exclusion method [30]. Failure to exclude Trypan blue reflects a loss of plasma membrane integrity associated with necrosis. In addition MTT and WST assays were performed to assess the effect on mitochondrial dehydrogenase activity. Cells with treatment concentrations of Ag-np (0, 25, 50, 100, 150 and 200 ␮g/ml) were seeded onto 96-well culture plates (at 1 × 105 cells per well). At the end of exposure, cell culture medium was discarded, and cells washed with PBS twice to remove excess nanoparticles and media. The cells were resuspended in fresh media and were treated with 0.5 mg/ml solution of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT, Sigma–Aldrich, St. Louis, MO, USA; 100 ␮l/well) at 37 ◦ C for 3 h. The number of viable cells was determined by uptake of MTT. For WST (Roche, Lewes, UK) assay similar experimental sets were maintained. After incubation cells were washed twice in PBS

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and cell viability assays were carried out according to the manufacturer’s instructions. Optical density (OD) was read on iMarkTM Microplate Absorbance Reader (BIO-RAD, California, USA) at 570 nm, and 450 nm for MTT and WST respectively. The interference of nanoparticle in the assays was eliminated by maintaining sample blanks for all the concentrations tested. The OD values obtained from the sample blanks were deducted from the OD values obtained after the assays. The values obtained thereafter have been expressed as percentage compared to control. All experiments were performed at least in triplicate on three separate occasions. Data are presented as mean ± SD. Further analysis was done to distinguish apoptotic and necrotic cells by Annexin V and propidium iodide (PI) incorporation. Lymphocytes (1 × 105 ) were seeded into 3.5 cm Petri dishes (Nunc, Wiesbaden) for 3 h at 37 ◦ C with different concentrations of Ag-np. Cells were washed twice in ice cold PBS to remove excess nanoparticles. The cells were then taken up in 100 ␮l calcium containing binding-buffer [10 mM HEPES (pH 7.4), 140 mM NaCl, 5 mM CaCl2 ] and stained for 15 min, with 5 ␮l Annexin V-FITC (BD Pharmingen, New Jersey, USA) and PI at 1 ␮g/ml. The stained cells were analyzed at an excitation wavelength of 488 nm and emission wavelengths of 530 nm for FITC fluorescence and 610 nm for PI fluorescence by FACS Calibur (BD Biosciences, New Jersey, USA). The percentages of viable (PI−, Annexin−), apoptotic (PI−, Annexin+) and necrotic cells (PI+, Annexin+) were evaluated with the CellQuestPro® software (BD Heidelberg, Germany). 2.3.3. Comet assay in lymphocytes To evaluate the DNA damage by Ag-np in lymphocytes, we performed the alkaline comet assay as described by Singh et al. [31] with minor modifications [27]. For the preparation of slides, approximately 100 ␮l of each cell culture (0, 25, 50, 100, 150 and 200 ␮g/ml) were transferred to microtubes containing 100 ␮l of LMP (low melting point) agarose. 100 ␮l of this suspension was pipetted onto precoated glass slides (normal melting point NMP agarose) and then covered with a cover glass,and the agar was allowed to set on ice. The slides were then placed in lysis solution [2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris–HCl, 1%Triton-X-100, 10% (v/v) DMSO] at 4 ◦ C for 60 min. The lysed cells were rinsed and allowed an unwinding time of 20 min in eletrophoresis buffer (1 mM Na2 EDTA, 300 mM NaOH, pH > 13) before electrophoresis for 30 min at 25 V on ice. The slides were then neutralized with 0.04 M Tris–HCl (pH 7.5) for 10 min and stained with ethidium bromide (2 ␮g/ml) just prior to image analysis. DNA migration was assessed by fluorescence microscopy (Leica,Wetzlar, Germany, excitation filter 515–560 nm and barrier filter of 590 nm) in conjunction with a CCD camera. The images were evaluated by auto-image analysis (Komet version 5.5; Andor Technology, Nottingham, UK). Slides were prepared in triplicates per concentration. To quantify the DNA damage,the median values of comet parameter – tail DNA (%) were scored from each slide and expressed as means for each concentration. Images of 150 (50 × 3) cells per concentration were analyzed. 2.3.4. ROS generation in human lymphocyte cells – DCFDA assay Cells incubated with Ag-np (0, 25, 100 and 200 ␮g/ml; 3 h at 37 ◦ C) were washed twice in PBS to remove excess nanoparticles. The cells were then resuspended in PBS in the order of 105 cells/ml. To the cell suspension DCFDA was added to a final concentration of 25 ␮M and incubated for 30 min at 37 ◦ C, in dark. The cells were then analyzed for ROS generation using BD FACS Aria III flow cytometry (488 nm excitation, 530–540 nm emission). A minimum of 20,000 events were analyzed per sample and the results expressed as fold-change of fluorescence intensity over control. 2.3.5. Uptake of Ag-np in blood cells using flow cytometry Human whole blood cells (105 cells/ml) were incubated with different concentrations of Ag-np (0, 25, 100, 200 and 500 ␮g/ml) for a period of 3 h at 37 ◦ C. Following Ag-np treatment cells were washed in PBS to remove excess nanoparticle. RBC was lysed and cells were fixed. Cells were then analyzed using a flow cytometer (BD Biosciences – FACS Aria III cell sorter, New Jersey, USA). The laser light scattered at narrow angles to the axis of laser beam is called forward-scatter(ed) (FS) light. The laser light scattered at about a 90◦ angle to the axis of the laser beam is called side-scatter(ed) (SS) light. The intensities of FS and SS are proportional to the size of cells and the intracellular density, respectively. The change in intensity of SS over control gave estimate of the uptake of Ag-np. 2.4. In vivo genotoxicity of Ag-np 2.4.1. Effect on bone marrow cells of mice 2.4.1.1. Treatment schedule. The study was conducted on Swiss albino male mice Mus musculus (8–12 weeks old, weighing 25–30 g). The animals were obtained from departmental animal house, housed in polycarbonate cages, bedded with rice husk and acclimatized under laboratory conditions (20–22 ◦ C, humidity 50–60%, 12 h light/dark photoperiod) for at least a week prior to experiment and fed with standard rodent pellet (M/S Hindustan Lever foods, India) and water was provided ad libitum. All the experiments were done in accordance to the University Ethical committee guidelines (University of Calcutta, Kolkata, India). The animals were divided into six groups of 5 male mice, each group implying a particular treatment group viz. I: negative control II: positive control, Single i.p. Injection of mitomycin C (2.50 mg/kg body weight) 18 h before sacrifice

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III: positive control, Single i.p. Injection of Cyclophosphamide (20 mg/kg body weight) 18 h before sacrifice IV to VII: single i.p. Injection of Ag-np (10, 20, 40 and 80 mg/kg body weight) 18 h before sacrifice

2.4.1.2. Chromosome aberration assay (CA). For bone marrow chromosome analysis, 16.5 h after the administration of the test compounds the animals were injected i.p. with 0.04% of colchicine, on an individual weight basis (1 ml/100 g body weight), and 90 min later they were sacrificed. Bone marrow samples were collected 18 h after exposure to Ag-np, when the cells were in their first metaphase. Bone marrow cells were routinely processed by the standard procedure [32,33]. The animals were sacrificed by cervical dislocation. Bone marrow cells from the femur bones of the same animal were flushed into two centrifuge tubes with phosphate-buffered saline (PBS, pH 7.4). One was used for comet assay and the other was used for CA. For CA, the cells were pelleted by centrifugation, and incubated in 0.075 M KCl for 30 min at 37 ◦ C. This was followed by fixation in ice-cold 3:1 methanol:glacial acetic acid for 10 min. Each bone marrow sample was washed twice in fixative, slides prepared and air dried. The slides were coded and stained in 8% Giemsa in Sorenson’s phosphate buffer (pH 6.8). One hundred well spread metaphase plates were scored from each animal per treatment set, at random. The types of aberrations were scored and recorded strictly in accordance with the method of Tice and Ivett [34]. The metaphase cells were scored at 1000× magnification, with selection being based on uniform staining quality, lack of overlapping chromosomes and chromosome number (40 ± 2 chromosomes). Each chromosome aberration recorded was of the following types: G , G , as chromatid and isochromatid gaps; B , B , as chromatid and chromosome breaks, RR as chromatid rearrangement. Responses were evaluated as the percentage of aberrant damaged metaphase cell (%DC) and as the number of aberrations per cell (CA/cell). Chromatid and chromosome gaps were recorded but were not included in calculations. For a count of the number of CA/cell, chromatid and chromosome breaks, and chromatid rearrangements (dicentric, ring, exchanges) were taken as one, regardless of the number of breakage events involved.

2.4.1.3. Comet assay. The bone marrow cells in PBS were dispersed by gentle pipetting and collected by centrifugation at 1000 rpm for 5 min at 37 ◦ C. Cell pellet was resuspended in PBS and used for further analysis. The methodology was same as described in Section 2.3.3 with modifications [35]. Slides were prepared in duplicate per concentration and images of 200 cells per concentration were analyzed.

2.4.1.4. ROS generation in bone marrow cell – DCFDA assay. Following animal sacrifice by cervical dislocation bone marrow cells from the femur bone were collected in RPMI media. The cells were then centrifuged and the cells were resuspended in PBS in the order of 105 cells/ml. To the cell suspension DCFDA was added to a final concentration of 25 ␮M and incubated for 30 min at 37 ◦ C, in dark. The cells were then analyzed for ROS generation using BD FACS Aria III flow cytometry (488 nm excitation, 530–540 nm emission). A minimum of 20,000 events were analyzed per sample and the results expressed as fold-change of fluorescence intensity over control. 2.4.2. Effect on plant system 2.4.2.1. Treatment of plant test system (A. cepa and N. tabacum). A. cepa L. bulbs were set for rooting in sterilized moist sand in the dark. After 2 days, the bulbs with 2–3 cm long roots were washed in running tap water for 5–10 min and then subjected to treatment. N. tabacum seeds were germinated in garden soil. The plantlets were used when they reached the 4th leaf stage. The roots of the test systems were exposed for a period of 24 h, to different concentrations of Ag-np (0, 25, 50, and 75 ␮g/ml) prepared in water, in well aerated glass vials. The experiments were conducted at room temperature 25 ± 1 ◦ C and the roots and leaves were processed for comet assay.

2.4.2.2. Comet assay in A. cepa and N. tabacum. Alkaline comet assay was performed following an improved and simplified procedure that substituted phosphatebuffered saline with Tris–HCl buffer [36]. Nuclei were isolated from the roots and shoots of A. cepa and N. tabacum belonging to different treatment groups and further processed according to the standardized methods of Gichner et al. [37]. The preparation and processing of slides is same as described earlier except that lysing step is not required for plants. For analysis the median values of comet parameter – tail DNA (%) were scored from each slide and expressed as means for each treatment group. Images of 75 (25 × 3) cells per concentration were analyzed. 2.5. TEM study: uptake and structural alterations Root cells of A. cepa and human blood cells were prepared for TEM analysis to study the effect of Ag-np. Ultramicrotome sections of the samples were evaluated for structural alterations using TEM (Jeol JEM-2100 LaB6, 200 kV). To evaluate the effect of Ag-np, TEM images of treated samples were compared to that of control cells.

2.6. Statistical analysis For statistical analysis median values of each concentration with respect to the comet parameters were calculated. Data are presented as means with standard deviation (mean ± S.D.) and one way analysis of variance (ANOVA) test was done by using Sigma Stats.3 software (SPSS Inc., Chicago, IL, USA). For all statistical analysis the level of significance was established at P ≤ 0.05.

3. Results 3.1. Characterization and uptake of Ag-np Ag-nps were characterized by TEM, SEM and XRD. TEM image (Fig. 1a) of Ag-np revealed the particles to be in the size range of 75–130 nm, with an average size of ∼125 nm. SEM image (Fig. 1b) of the Ag-np revealed an average size of 120 nm; most of the nanoparticles being in the size range of 90–180 nm. The purity of the substance was confirmed by EDX analysis (Fig. 1c). The EDX of the nanoparticle dispersion confirmed the presence of elemental silver. Absence of major peaks of any other elements confirmed the purity of the sample. The exact nature of the silver nanoparticle was achieved by measuring the XRD-spectrum of the samples. Inspection of the XRD patterns of silver nanoparticles reveals the existence of sharp diffraction lines at low angles (2–99◦ ). The silver nanoparticles exhibited peaks of silver at 2 = 38◦ , 44◦ , 64◦ , 78◦ and 81◦ that can be indexed to the (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2) facets of silver, respectively (Fig. 1d). Dynamic light scattering measurements performed on the stock suspension of Ag-np showed a tendency of forming agglomerates. Zeta potential of −4.86 mV (Fig. 1e) revealed the unstable nature of the aqueous suspension. UV visible spectra of Ag-np revealed of absorption maxima between 420 and 440 nm (Fig. 1f). With an increase in the Agnp concentration, the intensity and the maximum wavelength of the peak increased. The large change in the optical spectra implied agglomeration of Ag-np with increase in concentration. 3.2. Uptake of Ag-np in blood cells in vitro Uptake of Ag-np in blood cells in vitro with increase in dose was evident from the flow cytometry analysis (Fig. 2). Change in Side Scattered (SS) light increased in proportion to the concentration of the nanoparticle inside the cells. A ∼1.5-fold increase in side scatter was obsreved at the highest Ag-np concentration (200 ␮g/ml). An increase in Forward Scattered (FS) light was observed with an increase in Ag-np concentration. This could be due to the agglomerates of Ag-np. 3.3. In vitro cytotoxicity and genotoxicity of Ag-np 3.3.1. Cytotoxicity of Ag-np in human lymphocyte The result of cytotoxicity tests revealed dose dependent cytoxicity at all the concentrations tested. The Trypan blue dye exclusion method revealed a dose dependent decrease in viability of cells, significant at 150 ␮g/ml (72.54%) and above. The measurement of the activity of the mitochondrial dehydrogenases by the MTT and WST assays implicated concentration dependent decrease of cell viabilty, significant above 25 and 50 ␮g/ml respectively (Fig. 3). To assess the extent and mode of cell death, annexin-V FITC–PI staining was used. Based on the percentages of unstained cells (live cells), and those with red fluorescence (necrotic cells), green fluorescence (early apoptotic cells), and dual stained cells (late apoptotic cells) were analyzed. Annexin-V staining experiment indicated that a small percentage of cells were undergoing apoptosis at the concentrations (∼1.5 fold) as compared to control (Fig. 4).

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Fig. 1. (a) Transmission electron microscope image of Ag-np. (b) Scanning electron microscope image of Ag-np. (c) EDX analysis of Ag-np. (d) XRD image of Ag-np. (e) Zeta potential of Ag-np in suspension. (f) UV visible spectra of Ag-np suspensions.

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Fig. 2. Analysis of incorporation of Ag-np by flow cytometric light scatter. Blood cells were treated with several doses (0, 25, 100 and 200 ␮g/ml) of Ag-np nanoparticles for 3 h; Dose-dependent comparison of FS or SS intensity, red indicates lymphocyte population.

Fig. 3. MTT and WST-I cell viability assay, cytoxicity induced in Ag-np treated human lymphocyte cells.

Fig. 4. Flow cytometric analysis of annexin V, FITC–PI stained lymphocyte cells showing induction of apoptosis and necrosis at different nanosilver treatment concentrations; representative scatter plots of annexin V, FITC–PI stained lymphocyte cells at (a) control, (b) 25 ␮g/ml Ag-np, (c) 100 ␮g/ml Ag-np and (d) 200 ␮g/ml Ag-np treatment concentrations.

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Table 1 Chromosomal aberrations of mice bone marrow cells following treatment with different doses of Ag-np. Test chemical

Dose (mg/kg)

Total chromosome aberrationsa 

Negative control (distilled water) Ag-np

Mitomycin C

– 10 20 40 80 2.5





% aberrant cellsb 

G

G

B

B

RR

6 7 10 12 8 26

– 1 3 1 3 12

5 41 48 65 36 130

– 4 5 2 1 41

– – – – – 2

2.00 17.07 19.10 23.33 15.66 17.00

± ± ± ± ± ±

0.81 4.6* 1.01* 4.38* 6.48* 1.08*

Number of aberrationc /cell

0.02 0.18 0.21 0.27 0.15 0.59

± ± ± ± ± ±

0.01 0.01* 0.04* 0.03* 0.02* 0.04*

G , G : chromatid and isochromatid gaps; B , B : chromatid and isochoromosome breaks; RR: chromatid rearrangements. a 50 metaphase cells/animal (5 animals/dose). b Percentage of cells with damaged metaphase (excluding gaps). c Number of chromosome aberration/cell (excluding gaps). * P ≤ 0.05.

A significant increase in the number of necrotic cells was observed, at the concentration 25 ␮g/ml and above (Fig. 4). 3.3.2. DNA damage in human lymphocytes Ag-np induces DNA breakage in human lymphocyte cells (Fig. 5). The tail intensity (% tail DNA) was higher than the control at all concentrations. There was a sharp increase at the lowest concentration (25 ␮g/ml) with a gradual decrease with increase in concentration of Ag-np. Responses were statistically significant (P ≤ 0.05) at concentrations 25, 50 and 200 ␮g/ml. The results of MMS (100 ␮M)induced DNA break was ∼ 18 fold higher than the background value (data not shown). 3.3.3. ROS generation in human lymphocytes A significant increase in ROS generation was observed at all the concentrations tested. A ∼3–5 fold increase (Fig. 6) in fluorescence intensity (DCFDA) was observed. 3.4. In vivo genotoxicity of Ag-np 3.4.1. Chromosome aberration and DNA damage in mouse bone marrow cells The comparative data on the percentage of aberrant cells and number of aberration per cell are provided in Table 1. The aberrations scored were mainly found to be of chromatid breaks, while in animals treated with the positive compound (mitomycin C) both chromatid and chromosome type of aberrations were recorded. ANOVA test revealed the frequency of aberrant cells and

Fig. 5. Comet data (% tail DNA) of human lymphocytes treated with different concentrations of nanosilver; *P < 0.05.

Table 2 Comet parameter (% tail DNA) of mice bone marrow cells following treatment with different doses of Ag-np. Test chemical

Dose (mg/kg)

% Tail DNA

Control (water) Ag-np

– 10 20 40 80 20

1.99 6.79 10.17 3.64 4.71 32.53

Cyclophosphamide *

± ± ± ± ± ±

0.43 1.53* 1.06* 0.70 0.65 8.11*

P ≤ 0.05.

the number of breaks per cell to be significantly higher (P ≤ 0.05) than the control. The data of comet assay in mouse bone marrow cells are presented in Table 2. An increase in DNA damage in Ag-np treatment groups were observed. The comet parameter (% tail DNA) showed no further increase in DNA damage beyond the concentration of 20 mg/kg body weight. In the positive control the values of % tail DNA was ∼16 fold higher than the background value. 3.4.2. ROS generation in mouse bone marrow cells ROS generation by Ag-np in bone marrow cells were quantified using flow cytometry. The results indicated of siginificat ROS generation at concentrations 10 and 20 mg/kg body weight. A ∼1.5 and 1.4 fold increase in fluorescence intensity was observed at 10 and 20 mg/kg body weights respectively. ROS generation in the

Fig. 6. Production of ROS in Ag-np treated human lymphocyte cells; *P < 0.05.

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Fig. 7. Comet data (% tail DNA) of nanosilver treated Allium cepa and Nicotiana tabacum root and shoot (24 h treatment); *P < 0.05; in inset images of cells at different concentrations, showing varying extent of DNA damage as analyzed by comet assay.

subsequent concentrations were negligible and comparable to that of control set. 3.4.3. DNA damage in A. cepa and N. tabacum In nuclei isolated from A. cepa shoot, the DNA strand breaks increased and reached plateau at concentrations of 50 ␮g/ml and above. Whereas in roots it increased steadily till 50 ␮g/ml followed by a gradual decrease (Fig. 7). The effects of treatment in shoots were statistically significant (P ≤ 0.05) at concentrations 25 and 50 ␮g/ml. Genotoxicity study in N. tabacum plantlets indicated of a greater extent of DNA damage in roots as compared to that in leaves. Responses were statistically significant at most treatment concentrations (Fig. 7). 3.5. TEM study: uptake and structural alterations Human blood cells (in vitro), incubated with Ag-np was compared to control for structural alterations and Ag-np accumulation. Ag-np treated cells (Fig. 8B and C) show marked difference from control cells (Fig. 8A). Cells incubated with Ag-np (Fig. 8B and C) were characterized by different degrees of deformity (Fig. 8B), damaged cell membrane (Fig. 8B) and vacuolation (Fig. 8C). In A. cepa root, cells from control set show normal cellular organization, with proper nuclear and organellar structures (Fig. 8D). Features like extensive vacuolation, loss of nuclear organization, ruptured plasma membrane and shrinkage of the protoplast (Fig. 8E) were observed in sets that were exposed to Ag-np. Localization of nanoparticles was observed in the vacuoles (Fig. 8E) of the cells. 4. Discussion Ag-np which has antibacterial properties has been integrated into hundreds of consumer products. Although Ag-np belongs to the most often studied ones, the mechanisms of their biological effects are still not fully understood [38]. Like, for the majority of nanoparticle toxicity studies, Ag-np toxicology study has been confined to the classical in vitro toxicity test methods. Recent reviews have concluded that information on the genotoxicity of engineered nanomaterials (ENM) is still inadequate for general conclusions [39–41].

In this study we examined the genotoxic potential of Ag-np in both in vitro (in human lymphocyte) and in vivo (mouse bone marrow cells, Allium and Nicotiana) systems. In TEM and SEM analysis of Ag-np powder, an average particle size of 120 nm was noted. EDX and XRD analysis revealed that the Ag-np sample was devoid of any impurity. DLS and UV visible spectrophotometric study revealed of an unstable suspension with a tendency to form aggregates at higher concentrations. Tracking nanoparticle internalization in cellular systems is of the utmost importance for understanding and correlating the biological effects elicited by them. In the present study, the uptake of Ag-np in blood cells in vitro and incorporation of Ag-np into the cells with increase in concentration was evident from the flow cytometry analysis. The effect of nanoparticle size and shape on cytoand genotoxicity has been reported by several authors [42–44]. Literature survey shows that Ag-nps were cytotoxic [45,46] to mammalian cells. In the present study cytotoxicity was evident in MTT, WST and trypan blue dye exclusion tests. Jiang et al. [43] reported that gold and silver nanoparticles within the 2–100 nm size were found to alter signaling processes essential for basic cell functions including cell death, and 40 and 50 nm nanoparticles demonstrated the greatest effect. To assess the extent and mode of cell death, annexin-V FITC–PI staining was used. Compared to control a small percentage of cells were undergoing apoptosis at the treatment concentration (∼1.5 fold). A significant increase in the number of necrotic cells was observed, at the concentration 25 ␮g/ml and above. Thus from the study we could conclude the major reason for Ag-np induced cell death is necrotic. Previously published data [12] on normal human lung fibroblast cells (IMR-90) and human glioblastoma cells (U251) indicated only a small percentage of cells undergoing apoptosis and necrosis at higher concentrations of Ag-np. We conducted comet assay as conventional testing method [41] in vitro on human lymphocytes. The tail intensity (% tail DNA) was higher at all concentrations. There was a 4-fold increase at the lowest concentration (25 ␮g/ml) with a gradual decrease with increase in concentration of Ag-np. Responses were statistically significant (P ≤ 0.05) at concentrations 25, 50 and 200 ␮g/ml. Significant increase in ROS generation was observed at all concentrations tested. Cytotoxicity and genotoxicity of starch-coated

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Fig. 8. TEM images of ultrathin sections of blood cells (A–C) and plant cells (D and E) showing effect of Ag-np treatment; (A) normal blood cells from control set, (B) Ag-np treatment set showing deformed cells and cells with damaged membrane, (C) Ag-np treated blood cells showing large vacuolation; (D) A. cepa root cells in absence of treatment with normal cellular organization, Well defined nuclear and organellar structures, (E) Ag-np treated A. cepa root cells with extensive vacuolation, loss of nuclear organization, ruptured plasma membrane and shrinkage of the protoplast.

silver nanoparticles was studied using normal human lung fibroblast (IMR-90) and human glioblastoma (U251) cells by Asharani et al. [12,13]. They observed an increase in DNA damage with increase in Ag-np concentration in cancer cells, whereas in the fibroblast cells they found increase in DNA damage only beyond a concentration of 100 ␮g/ml. Their choice of capping agent was done based on the stability of Ag-np in cell culture medium. For risk assessment it should be ecologically relevant to use natural aggregated nanoparticles [47]. Most of the manufactured nanoparticles have not been designed to disperse readily in water, further more they will aggregate in many types of natural water. We demonstrated in vivo genotoxicity of Ag-np in mouse bone marrow cells. Compared to control the percentage of aberrant cells induced by Ag-np (20–80 mg/kg body weight; 7.5–12-fold) was significantly higher. The aberrations scored were mainly found to be of chromatid breaks. Therefore, Ag-np can be classified as a clastogen. In addition, comet assay revealed an increase in DNA strand breaks. The comet parameter (% tail DNA) showed no further increase in DNA damage beyond the concentration of 20 mg/kg. Results of ROS quantification in bone marrow cells by flow cytometry revealed comparable results. Negative genotoxic response was reported in rat bone marrow cells of Sprague-Dawley rats for 28 days by Kim et al. [48]. Their results did not affect the frequency of micronucleated polychromatic erythrocytes. The discrepancy can be best explained on the difference in route of administration and the sampling time. Ag-np have been found to induce micronuclei in CHO cells [48] and BRL 3A rat liver cells [8]. Other nanomaterials – aluminum, cobalt [49], magnetite [50] and ultrafine TiO2 [51] were found to increase the frequency of binucleated MN cells and % Tail DNA [52] in mammalian systems.

Our data on in vivo study on plants are in agreement with the results of in vitro study in lymphocytes. In A. cepa and N. tabacum a higher extent of DNA damage was observed in roots than in the leaves. This could be due to the direct exposure of roots to the treatment chemical. Furthermore lesser amount of nanomaterials would have been translocated to leaf than in roots within the same period of time. An increase in DNA damaging effect of Ag-np was observed at certain concentrations in A. cepa (50 ␮g/ml) as well as in N. tabacum (75 ␮g/ml) roots. There was a gradual decrease in % Tail DNA with increase in Ag-np concentration. This could be attributed to a property of nanomaterials to form agglomerates by virtue of which, with increase in treatment concentration the nanoparticles had a tendency to precipitate. The greater interaction of nanoparticles amongst themselves that could have increased owing to increase in treatment concentration might have limited the free Ag-np from interacting with the test systems. Previous studies in A. cepa have shown that Ag-np could enter plant system, affect cell division exhibiting cytotoxic response and cause chromosomal aberrations [19,20]. A number of studies have also shown that Ag-np treatment induced DNA damaging effects on aquatic organism [20] and plant cells [53] with impairment of celldivision. TEM images of cells treated with Ag-np revealed gross morphological alterations and vacuolation in both A. cepa and human blood cells. Extensive vacuolation, loss of nuclear organization, ruptured plasma membrane and shrinkage of the protoplast could be associated with apoptotic/necrotic [54,55] (in human blood cells) and necrotic/vacuolar (in A. cepa) [56] cell death. The ultra structural alterations in human blood cells could be correlated to the necrotic mode of cell death studied using flow cytometry.

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In summary, our in vitro and in vivo studies demonstrate that Ag-np is genotoxic to plant and animal system, capable of producing ROS and inducing apoptosis and necrosis. We have observed significant impairment in nuclear DNA and cell function indicating interaction of Ag-np and DNA. Overall data suggests that there is a good relation between the in vivo and in vitro effects concerning Ag-np interaction. Therefore, the environmental risk assessment of nanomaterials could be performed using the existing tiered approach and regulatory framework, but with modifications to methodology including chemical characterization of the materials being used. There are many challenges ahead, and controversies (e.g., reference substances for ecotoxicology), but knowledge transfer from mammalian toxicology, colloid chemistry, as well as material and geological sciences, will enable ecotoxicology studies to move forward in this new multi-disciplinary field [2,21]. Conflict of interest statement There is no conflict of interest. Acknowledgements The authors (AM, MG, MJ) would like to acknowledge the Center for Research in Nanoscience and Nanotechnology (CRNN), University of Calcutta for the financial support and instrumentation facility. The authors would like to thank CSIR [MG- Grant No. 09/028 (0860)/2012 EMR-1; SS - Grant no. 09/028 (0728)/2008-EMR-1], Govt of India and BRNS, Govt of India. TEM facility from SAIF, North East Hill University (NEHU) - Shillong, India and Mr. Joston Nongkynrih (NEHU) is kindly acknowledged. The authors would like to thank the reviewers for their valuable suggestion. References [1] K. Savolainen, H. Alenius, H. Norppa, L. Pylkkänen, T. Tuomi, G. Kasper, Risk assessment of engineered nanomaterials and nanotechnologies—a review, Toxicology 269 (2010) 92–104. [2] A. Dhawan, V. Sharma, Toxicity assessment of nanomaterials: methods and challenges, Anal. Bioanal. Chem. 398 (2010) 589–605. [3] X. Chen, H.J. Schluesener, Nanosilver: a nanoproduct in medical application, Toxicol. Lett. 176 (2008) 1–12. [4] S.J. Klaine, P.J. Alvarez, G.E. Batley, T.F. Fernandes, R.D. Handy, D.Y. Lyon, S. Mahendra, M.J. McLaughlin, J.R. Lead, Nanomaterials in the environment: behavior, fate, bioavailability, and effects, Environ. Toxicol. Chem. 27 (2008) 1825–1851. [5] S.A. Blaser, M. Scheringer, M. Macleod, K. Hungerbühler, Estimation of cumulative aquatic exposure and risk due to silver: contribution of nanofunctionalized plastics and textiles, Sci. Total Environ. 390 (2008) 396–409. [6] G. Oberdorster, V. Stone, K. Donaldson, Toxicology of nanoparticles: a historical perspective, Nanotoxicology 1 (2007) 2–25. [7] L. Braydich-Stolle, S. Hussain, J.J. Schlager, M.C. Hofmann, In vitro cytotoxicity of nanoparticles in mammalian germline stem cells, Toxicol. Sci. 88 (2005) 412–419. [8] S.M. Hussain, K.L. Hess, J.M. Gearhart, K.T. Geiss, J.J. Schlager, In vitro toxicity of nanoparticles in BRL 3A rat liver cells, Toxicol. In Vitro 19 (2005) 975–983. [9] S. Park, Y.K. Lee, M. Jung, K.H. Kim, N. Chung, E.K. Ahn, Y. Lim, K.H. Lee, Cellular toxicity of various inhalable metal nanoparticles on human alveolar epithelial cells, Inhal. Toxicol. 19 (2007) 59–65. [10] S. Arora, J. Jain, J.M. Rajwade, K.M. Paknikar, Cellular responses induced by silver nanoparticles: in vitro studies, Toxicol. Lett. 179 (2008) 93–100. [11] Y.H. Hsin, C.F. Chen, S. Huang, T.S. Shih, P.S. Lai, P.J. Chueh, The apoptotic effect of nanosilver is mediated by a ROS- and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells, Toxicol. Lett. 179 (2008) 130–139. [12] P.V. AshaRani, G.L. Kah Mun, M.P. Hande, S. Valiyaveettil, Cytotoxicity and genotoxicity of silver nanoparticles in human cells, ACS Nano Lett. 2 (2009) 279–290. [13] P.V. Asharani, M.P. Hande, S. Valiyaveettil, Antiproliferative activity of silver nanoparticles, BMC Cell Biol. 10 (2009) 65. [14] K. Kawata, M. Osawa, S. Okabe, In vitro toxicity of silver nanoparticles at noncytotoxic doses to HepG2 human hepatoma cells, Environ. Sci. Technol. 43 (2009) 6046–6051. [15] M.F. Rahman, J. Wang, T.A. Patterson, U.T. Saini, B.L. Robinson, G.D. Newport, R.C. Murdock, J.J. Schlager, S.M. Hussain, S.F. Ali, Expression of genes related to oxidative stress in the mouse brain after exposure to silver-25 nanoparticles, Toxicol. Lett. 187 (2009) 15–21.

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