In vitro and in vivo genotoxicity of silver nanoparticles (original) (raw)

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. (A. Mukherjee). which cause problems during the toxicological assessment of novel 35 nanomaterials [2]. 36 Many classes of silver nanoparticles (Ag-np) have been syn-37 thesized and widely applied for their bactericidal and viricidal 38 properties [3,4]. Worldwide an extensive growth is projected in 39 the Ag-np market [5] and it is unclear whether the exposure of 40 humans, animals and plants to Ag-np through industrial or domes-41 tic waste could produce harmful biological responses [6]. There are 42 a significant number of studies on the genotoxicity and cytotox-43 icity of silver nanoparticles on mammalian and human cell lines 44 [7-18] and a few reports on plant systems [19,20]. Majority of nan-45 otoxicity research has focused on cell culture systems and to assess 46 nanotoxicity accurately. Fischer and Chan [21] in their review arti-47 cle mentioned the need for in vivo studies. Therefore, we examined 48 the genotoxic potential of Ag-np in both in vitro (in human lympho-49 cyte) and in vivo (mouse bone marrow cells, Allium and Nicotiana) 50 systems. Apart from animal system, plants are an important 51 component of the ecosystem that needs to be included when eval-52 uating the overall toxicological impact of the nanoparticles in the 53 1383-5718/$ -see front matter G Model MUTGEN 402222 1-10 2 M. Ghosh et al. / Mutation Research xxx (2012) xxx-xxx environment. The plants selected for the study (Allium cepa and 54 Nicotiana tabacum) are efficient test systems for screening chem-55 icals and in situ monitoring for genotoxicity of environmental 56 contaminants [22-26]. They have been used successfully in our 57 laboratory as a biomarker of genotoxicity of nanomaterials [27,28]. 58 Ag-np (≤100 nm) was purchased and characterized using trans-59 mission electron microscopy (TEM), scanning electron microscope 60 (SEM) and X-ray diffraction (XRD), diffraction light scattering (DLS) 61 and UV visible spectrophotometry for size and dispersion. Prior to 62 its genotoxicity evaluation, the nanoparticle was dispersed in solu-63 tion by sonication. For in vitro study, human lymphocytes were used 64 for cytotoxicity (trypan blue dye exclusion, MTT and WST assays) 65 and genotoxicity (comet assay) studies. In addition, flow cytometry 66 was performed to measure the mode of cell death and uptake of Ag-67 np in human lymphocytes. TEM was performed to study structural 68 modifications and uptake of Ag-np in cells. The in vitro results were 69 further substantiated by in vivo studies. Bone marrow cells of mice 70 and root or leaf tissues of Allium and Nicotiana plants were used in 71 the comet assay. 72 The study provides evidence of genotoxicity of Ag-np in plant 73 and mammalian systems and the results demonstrate that there is 74 good correlation between the in vitro and in vivo effects of Ag-np. 75 2. Materials and methods 76 2.1. Ag-np preparation 77 Ag-np was obtained from Sigma-Aldrich, St. Louis, MO, USA. The physical char-78 acteristics of the particles according to the manufacturers data are; size (≤100 nm), 79 purity (99.5%), trace metal basis, surface area (5.0 m 2 /g), density (10.49 g/cc). Ag-80 nps were suspended in PBS or water and dispersed by ultrasonic vibrations (100 W, 81 30 kHz) for 30 min. Ag-np were serially diluted at different concentrations according 82 to the test performed. 83 2.2. Characterization of Ag-np 84 Ag-np powder was characterized using transmission electron microscopy (Jeol 85 JEM-2100 LaB6, 200 kV), scanning electron microscopy, energy-dispersive X-ray 86 spectroscopy (EDX) (Hitachi S-415A Electron Microscope, Tokyo, Japan at 25 kV) 87 and by X-ray diffraction (XRD) analysis. The spectra were recorded in a PW. 3040/60 88 PANalytical X-ray diffractometer Almelo, Netherlands (Cu K␣ radiation, 1.54443) 89 running at 45 kV and 30 mA. The diffracted intensities were recorded from 2 • to 99 • 90 2Â angles. Zeta potential of Ag-np in solution was measured by laser diffractometry 91 using a Nano Size Particle Analyzer (Zen 1600 Malvern, USA). Absorption maxima 92 of Ag-np in solution were scanned by UV-Vis spectrophotometer (Beckman Coulter 93 DU 700, California, USA) for agglomeration at the wavelength of 200-800 nm. 94 2.3. In vitro cytotoxicity and genotoxicity of Ag-np 95 2.3.1. Cell preparation and materials 96 Human blood was obtained from 3 healthy male volunteers between 20 and 97 30 years of age (non smokers and not under any medications), after their con-98 sent. The lymphocytes were obtained by centrifuging blood overlaid on Histopaque 99 (Sigma-Aldrich, St. Louis, MO, USA) according to the method of Boyum [29]. Cell 100 viability was checked by Trypan blue exclusion method [30] and was found to be 101 approximately 95%. All experiments were approved by the Research Ethics Com-102 concentrations of Ag-np (0, 25, 50, 100, 150 and 200 g/ml). Positive control was 105 maintained with methyl methanesulphonate (MMS, 100 M).