ISSN: 2577-0489
Research Article International Journal of Global Advanced Materials & Nanotechnology Evaluation of Silver Nanoparticles: In Vitro Assays for Cyto/Genotoxicity in Cell Lines and Ecotoxicity on Zebrafish Guilger M1, Pascoli M2, Pasquoto-Stigliani T1, Rheder DT3, Costa TG1, Maruyama CR2, BileskyJosé N1, Fraceto LF2, Carvalho CS3 and Lima R1,3* Laboratory of Bioactivity Assessment and Toxicology of Nanomaterials, University of Sorocaba, Sorocaba, Sao Paulo, Brazil 2 Laboratory of Environmental Nanotechnology, Department of Environmental Engineering, Sao Paulo State University (UNESP), Sorocaba, Sao Paulo, Brazil 3 Federal University of Sao Carlos, Sorocaba campus, Sorocaba, Brazil
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Correspondence: Lima R, LABiToN - Laboratory of Bioactivity Assessment and Toxicology of Nanomaterials,
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University of Sorocaba, Rodovia Raposo Tavares, km 92, 18023-000, Sorocaba, Sao Paulo, Brazil, Tel: +55 15 991117751; E-mail:
[email protected]
Rec date: Mar 20, 2018; Acc date: Apr 10, 2018; Pub date: Apr 15, 2018
Abstract Silver nanoparticles (AgNPs) are used as biocides or incorporated into several products due to their antimicrobial activity. However, when released on aquatic or terrestrial environment these nanomaterials can interact with organic compounds and have their properties and toxicity levels changed, possibly affecting non-target organisms. This work evaluated AgNPs on their physicochemical characteristics when dispersed in different media, toxicity on cell lines and ecotoxicity on Allium cepa and Zebrafish (Danio rerio). Nanoparticles were characterized through dynamic light scattering and nanoparticles tracking analysis, with the first technique comparing dispersion in water, culture medium, exposed to cells and after contact with fish. Cytotoxicity and genotoxicity were evaluated through tetrazolium reduction (MTT) and comet assay. Ecotoxicity was evaluated through Allium cepa and biochemical and comet assays with zebrafish. The biochemical assays were performed in order to evaluate the oxidative stress by the activities of catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) in organs of the fish, while DNA damage was evaluated through comet assay of gill and blood cells. Differences were observed in physicochemical characteristics of the nanoparticles dispersed in water, culture medium and in the presence and absence of cells. In the cytotoxicity and genotoxicity assays, different responses were associated with physicochemical variations. Changes in oxidative stress enzymes were observed in the organs and an increased DNA damage in the gills. Different responses revealed adverse effects of the AgNPs on exposed cells and organisms. However, health and environmental risks remain little understood, in a way that further work is needed. Keywords: Silver nanoparticles; Cytotoxicity; Genotoxicity; Oxidative stress; Zebrafish Abbreviations: ATCC: American Type Culture Collection; DMEM: Dulbecco’s Modified Eagle Medium; MEM-α:
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Minimum Essential Eagle Medium Alpha; PDI: Polydispersity Index; DLS: Dynamic Light Scattering; NTA: Nanoparticle Tracking Analysis; MTT: Tetrazolium Reduction Method; MI: Mitotic Index; AI: Alteration Index; RI: Relative Indices, ASTM: American Society for Testing and Materials; CAT: Catalase; GPx: Glutathione Peroxidase; GST: Glutathione S-Transferase; SOD: Superoxide Dismutase; ROS: Reactive Oxygen Species; GSH: Reduced Glutathione; GR: Glutathione Reductase
Introduction The antimicrobial activity of silver has been recognized for a long time, with compounds based on silver having been used to control microorganisms since the 19th century [1]. With the development of nanotechnology, the use of nanometric silver enhances its antimicrobial activity, due to changes in its physicochemical characteristics, such as a higher surface area, resulting in greater exposure of microorganisms [2,3]. Due to their effective antimicrobial properties, AgNPs have been applied as broad spectrum biocides against antibiotic resistant bacteria [4,5]. Materials used in the home and in hospitals contain AgNPs to prevent the proliferation of pathogenic microorganisms [6]. As a result, this nanomaterial stands out among the ten commercially engineered nanomaterials with the greatest global production [7,8]. The mode of action of AgNPs has still not been fully elucidated, but it is believed that after contact with bacteria, they interact with membrane proteins and penetrate the cells [9]. It has been reported that the nanoparticles bind to the surface of the cell membrane and alter functions such as permeability and respiration and can penetrate cells and interact with structures containing sulfur and phosphorus components, such as DNA, releasing ions that amplify the bactericidal activity [10,11]. Despite the importance of the antimicrobial use of AgNPs, the production and use of this nanomaterial can result in its release into the environment, where the elemental silver and silver ions can affect non-target organisms including animals, plants, non-pathogenic microorganisms from biogeochemical processes and humans [12-14]. Cleveland et al. [15] evaluated the release of AgNPs from products including socks, bandage material and a toy bear. The products were submerged in marine water for 2 h (socks) or 12 h (bandage and the bear), after which the socks showed release of about 95% of the silver nanoparticles, while the bandage and the bear released 99% and 82%, respectively. Therefore, the increasing
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use of products containing nanoparticles necessitates greater knowledge concerning their possible effects in the environment. Once released into the environment, many factors can influence the behavior and toxicity of AgNPs, with exposure to different media and conditions altering their properties [16,17]. The nanoparticles may remain dispersed, agglomerate, aggregate, dissolve or react with other substances, consequently leading to unknown effects. Hence, there is a need for the development of standardized characterization that enable understanding of the speciation of nanomaterials and their products following dispersion in different media [18,19]. Although the quantity of toxicity studies has increased, further research is needed on the effects of AgNPs after release into the environment, such as their physicochemical behavior under different conditions and their genotoxicity in soil and water [20,21]. Important issues with regard to the release of AgNPs into aquatic environments include their hydrodynamic behavior, association with sediments and natural colloidal particles, binding with pollutants, routes of incorporation into biota and possible increase of toxicity [22]. In the environment, AgNPs can release silver ions, undergo changes in their shape, size and surface coating and interact with nucleic acids, lipids and proteins, altering their mechanisms of action [23]. Aquatic organisms can be exposed to AgNPs by their flow through the gills and contact with the external epithelium. The main form of internalization is by endocytosis, whose pathways can lead to compartments such as liposomes and endosomes [22,24]. Given this background, the present work describes the characterization and evaluation of cytotoxicity and genotoxicity of commercial AgNPs (Sigma brand) on different cell lines and the identification of possible alterations of DNA and biochemical processes on adult Zebrafish (Danio rerio). Such studies are vital due to the need for a better understanding of the processes according to which AgNPs could present toxicity, hence contributing to scientific knowledge in this area and
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helping in the establishment of regulatory frameworks for the use of these nanomaterials.
Materials and Methods Biological materials The permanent cell lines 3T3, HeLa, HaCaT, V79and MC3T3, American Type Culture Collection (ATCC), obtained from the Rio de Janeiro cell bank, were used in this work. The cells were previously cultured in Dulbecco’s Modified Eagle Medium (DMEM) (CULTILAB) for 3T3, HeLa, HaCat and V79and Minimum Essential Eagle Medium Alpha (MEM-α) (CULTILAB) for MC-3T3, with incubation at 37 ºC in a humid atmosphere with 5% CO2. Zebrafish were purchased from local suppliers and acclimatized prior to the exposures. They were freely fed with flaked commercial feed containing 35-40% crude protein and feeding was suspended 48 h prior to exposure to the AgNPs.
Characterization and cyto/genotoxicity of the nanoparticles: in vitro analyses employing cell culture Characterization of the nanoparticles in different dispersions: The AgNPs powder used in this study was purchased from Sigma-Aldrich and had a primary particle size of 25 nm and 99.5% purity. In order to identify physicochemical variations of the AgNPs in different suspensions, they were dispersed in water, in the culture media DMEM and MEM-α and in these culture media containing the cell lines 3T3 and HeLa (DMEM) and MC-3T3 (MEM-α) at the concentration of 100 μg/mL (1.2×1012 NPs/mL). Hydrodynamic diameter and polydispersity index (PDI) were determined by the dynamic light scattering (DLS), while the surface charge (zeta potential) was measured by electrophoretic mobility, initially in water and culture media and 24 h after dispersion and exposure to the culture media and cell lines. The values were obtained as the average of 3 readings performed using a Zetasizer ZS90 Nano (Malvern), at a fixed angle of 90º and 25ºC. Physicochemical analyses of the AgNPs dispersed in the water after fish exposure were also performed using the same techniques in order to identify any changes possibly caused by organic matter from the fish. The size distribution and concentration of the nanoparticles (NPs/mL) dispersed in water were determined by the nanoparticle tracking analysis (NTA). The sample was diluted 1000 times and five videos were
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analyzed, each of 60 s duration, with 85.16 ± 7.72 particles per frame.
Evaluation of cytotoxicity and genotoxicity: The tetrazolium reduction method (MTT) was performed to evaluate cell viability through mitochondrial activity and obtain IC50 values. The cells (3T3, HeLa, HaCat and V79) were incubated in 96-well plates (2×104 cells/well) at 37ºC until adherence, followed by exposure to decreasing concentrations of the AgNPs for 24 h. Then, the culture medium was removed, the cells were washed and 100 μL of MTT solution (5 mg/mL) was added to each well. The cells were incubated for 3 h and were then fixed by adding 100 μL of dimethylsulfoxide. Analysis was performed at 570 nm using an ELISA plate reader (Evolution 201, Thermo Scientific). The genotoxicity of the AgNPs was evaluated through comet assay, based on the methodology of Singh et al. [25]. Cells (3T3, HeLa, HaCat and V79) were exposed to the nanoparticles at concentrations of 0.15×1012, 0.31×1012 and 0.47×1012 NPs/mL for 1 h, followed by homogenization with 0.8% low melting point agarose and application to slides coated with 1.5% normal agarose. Controls were prepared using cells without exposure to the nanoparticles. The slides were analyzed by classifying the degree of damage into categories from 0 to 4, as described by Collins et al. [26].
Evaluation of ecotoxicity Allium cepa assay: Allium cepa assay was adapted from Lima et al. [27]. Previously germinated seeds were exposed for 24 h to the AgNPs at concentrations of 3.16×1012, 1.58×1012, 0.31×1012 and 0.15×1012 NPs/ mL with ultrapure water used as a control. The mitotic index (MI) was calculated by dividing the number of cells in division by the total number of cells. The alteration index (AI) was calculated by dividing the number of cells showing alterations by the total number of cells in division. Relative indices (RI) were calculated by dividing the values obtained for the treatments by the values for the controls. Biochemical analyses and comet assay on Zebrafish: Around 100 fish were stocked in 40 L aquaria at a density of one individual per liter of tap water, with continuous water recirculation (1.2 L h-1), constant aeration, controlled temperature (25 ± 1ºC) and a light: dark photoperiod of 12 h: 12 h. The fish were exposed for 72 h to the AgNPs dispersed
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in tap water at concentrations of 0 (control group, without NPs), 0.012×1012, 0.12×1012 and 1.2×1012 NPs/ mL. The animals were divided into 4 groups, with 15 fish per treatment. The assays were performed in triplicate. The aquaria employed for the trials had a capacity of 3 L and were kept at a controlled temperature (25 ± 1ºC), under constant aeration (>6.0 mg O2 L-1). The animals in the control and exposed groups remained under these conditions for 72 h in a semi-static system. All the procedures were performed according to the standards established by the American Society for Testing and Materials (ASTM) [28] and the experiments were previously approved by the ethics committee of the university (protocol 004/2012). After exposure, animals were anesthetized with benzocaine (250 mg L-1) and were euthanized by spinal cord rupture. Samples of blood and gills were collected and stored in tubes containing fetal bovine serum and 0.5 mol L-1 EDTA, prior to the comet assays. Samples of brain, heart, liver, muscle and skin were collected and stored at -80ºC. For biochemical assays, organ samples were homogenized individually (1:10 w/v) in PBS buffer (pH 7.2) at 10,500 rpm for 2 min, followed by centrifugation at 15,294×g (560R centrifuge, Eppendorf) for 20 min at 4ºC. The supernatants were kept for the analyses. The total protein concentrations were determined by the Bradford method [29]. Comet assay was performed with gill and blood cells according to the method previously described for cell lines, in order to investigate possible genotoxicity. The activity of enzymes involved in oxidative stress in the samples of organs was evaluated. CAT activity, calculated as μmol H2O2 decomposed per mg of protein, was determined following the method described by Aebi [30]. The GPx activity was determined according to the method of Nakamura et al. [31] and was expressed as μmol NADPH oxidized per min per mg of protein. GST activity was determined according to the method of Habig et al. [32] and was expressed as nmol per mg of protein.
Statistical analysis Statistical analyses of the nanoparticle characterization, the cytotoxicity and genotoxicity evaluations and the comet and biochemical assays with Zebrafish were performed using ANOVA followed by Tukey’s HSD post hoc test. Graph Pad Prism software was used for these
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analyses.
Results and Discussion Characterization and cyto/genotoxicity of the nanoparticles: in vitro analyses employing cell culture The physicochemical characteristics of nanoparticles can change according to the medium of dispersion. Murdock et al. [33] analyzed various nanoparticles by DLS and found that when dispersed they rarely presented the primary particle size, with evidence of agglomeration or even aggregation. The properties and toxic effects of AgNPs are governed by a series of physicochemical characteristics, including mean diameter, morphology, stability and surface charge, which can vary depending on the dispersing medium [34,35]. Considering this issue and the different dispersions applied in the toxicity evaluation assays, the AgNPs were characterized dispersed initially in water and in culture medium supplemented with serum and after 24 h of exposure in culture medium in the presence and absence of cells, as well as in the water obtained after the exposure of fish for 72 h. AgNPs dispersed in water showed mean diameters of 149.23 ± 2.45 nm and 146.28 ± 2.26 nm, using DLS and NTA, respectively. The PDI, indicative of variations in size and dispersion, was 0.41 ± 0.02 and the zeta potential, was -29.53 ± 0.95 mV. These values revealed an increase in the size of the nanoparticles, compared to their primary size (25 nm). The NTA analysis showed a nanoparticle concentration of 1.2×1012 NPs/mL, corresponding to 100 μg/mL. With regard to the dispersions in culture media, initially and after 24 h in the presence and absence of cells, the results revealed changes in physicochemical characteristics of the AgNPs (Figure 1a-c). Studies have shown that dispersion in culture media can result in corona formation, due to the presence of proteins, which can lead to an increase in nanoparticle size. It has also been found that in addition to corona formation and changes in nanoparticle stability, the use of a culture medium with fetal bovine serum can increase the release of ions, resulting in greater toxicity [35,36]. In the case of the cell cultures employing media with 10% fetal bovine serum, with exposure for 24 h at 37oC, changes over time were observed in the size of the
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nanoparticles. This effect was not observed in the MEM-α medium, where the nanoparticles presented the same size after 24 h, compared to the size immediately after dispersion (Figure 1a). In the DMEM medium, there was an increase in size compared to the initial dispersion. For both media, the PDI values decreased after 24 h (Figure 1b). The presence of cells in the medium significantly affected the size of the nanoparticles, while the zeta potential was not altered and only the presence of HeLa cells altered the polydispersity in the DMEM medium (Figure 1a-c). The influence of the dispersion medium on the properties of nanoparticles has been reported in studies showing that after contact with the cell environment, the release of proteins affects physicochemical characteristics [33,35]. Increases in the size of AgNPs, relative to the primary particle size, occurs when they are dispersed in culture media or other biological fluids, probably due to corona formation by the proteins in the media, leading to changes in the properties of the nanoparticles and their toxicity and/or bioavailability [37]. As pointed out by Miclaus et al. [38], there is a need for further studies concerning protein-protein binding, in order to improve understanding of biotransformation and effects on toxicity. It is also possible that the proteins could affect nanoparticle dissolution, acting to protect the cells [39] or even to induce different immune responses [40]. Important parameters to consider in investigation of the toxicity of AgNPs include particle size, dose and route of exposure [41], with dermal and respiratory exposure considered the main routes for entry of AgNPs in animals [42]. The internalization of AgNPs by inhalation can cause toxicity to pulmonary epithelial cells, affecting circulation, central nervous system, liver and skin [43]. The primary intracellular target of nanomaterials is the mitochondria, which results in the generation of reactive oxygen species [44]. In this work, evaluating cytotoxicity and genotoxicity, some cell lines appeared to be more sensitive to AgNPs (V79 and HeLa). Although the data were reported as the concentration of nanoparticles per mL, the values can be compared with those reported in literature in terms of μg/mL, since in the present case, 1.2×1012 nanoparticles / mL corresponded to 100 μg/mL (Figure 1d). IC50 values of 9,273×1011, 6.069×1011, 9.407×1011 and 1,107×1012 NPs/mL obtained for the V79, HeLa, 3T3 and HaCat cells respectively (Figure 1d) were calculate by
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probit curve. Similar results were found by Mukherjee et al. [45], who reported higher resistance of the HaCat cell line to the same AgNPs. Gliga et al. [46] investigated the cytotoxicity of AgNPs to lung cells (BEAS-2B) and found that the size of the nanoparticles had a substantial influence on their level of toxicity, with the greatest effects in cells exposed to smaller nanoparticles. However, Souza et al. [47] found the opposite trend in terms of toxicity and the size of nanoparticles, with 100 nm AgNPs causing greater cell death than 10 nm nanoparticles, following exposure of CHO-K1 and CHO-XRS5 cells. In addition to factors such as size and morphology, the cytotoxicity of AgNPs varies according to the cell line exposed to them, as well as the synthesis method. Kaur and Tikoo [42] evaluated the metabolic activities of A431 (human epithelial carcinoma), A549 (human lung carcinoma) and RAW264-7 (rat macrophage) cell lines exposed to different AgNPs synthesized using the reducing agents tannic acid and sodium borohydride, obtaining different results according to the cell line studied. It was also shown that different reducing agents could lead to different behavior in terms of nanoparticle agglomeration or aggregation, with consequent effects on nanoparticle toxicity [42]. It is important to highlight that different cell culture media can have different effects on the aggregation and size of nanoparticles and consequently on their toxicity [37,48] as found here, where the physicochemical analyses revealed differences according to the dispersing medium (Figure 1). The results of the genotoxicity assays performed using comet analysis indicated that at the three concentrations, the four cell lines presented damage indices (Figure 1e). At concentrations below 0.5×1012 NPs/mL, the V79 and HeLa cells presented the greatest damage, in agreement with cell viability results, where these cells showed IC50 at lower concentrations (Figure 1d). Previous work has used comet assay to investigate the genotoxic potential of commercial AgNPs in lung and tumor cell lines [6]. Avalos et al. [49] investigated the genotoxicity of AgNPs of different sizes in human cells, using comet assay and observed damage in all the cell types, with pulmonary fibroblast (HPF) cells presenting the highest damage index. The induction of DNA damage by AgNPs may be due to the generation of free radicals
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Figure 1: Analyses of (a) size, (b) polydispersity and (c) zeta potential of nanoparticles dispersed in different media, before and after 24 h of exposure in the presence and absence of cells. (d) Mitochondrial activity and IC50 for V79, HaCat, 3T3and HeLa cells exposed to different concentrations of AgNPs in DMEM medium for 24 h. (e) Comet assays of DNA damage for cells lines exposed for 1 h. Equal numbers indicate similarity and different numbers indicate a significant difference. and oxidative stress, resulting in DNA breaks and a range of damage to cell components [50,51]. In another study, it was found that the release of Ag+ was responsible for the side effects of AgNPs [36].
Evaluation of ecotoxicity Physicochemical characterization of the nanoparticles after the exposure of the fish revealed increases of size and polydispersity, as well as alteration of the zeta potential, which were probably due to agglomeration (Figure 2a-c). At the highest concentration, AgNPs showed the smallest alteration of size and increase of the zeta potential, which could have been due to sedimentation, so that the water contained nonsedimented nanoparticles of smaller diameter. The importance of evaluating nanoparticles in different dispersions is due to the likely changes in their physicochemical characteristics and consequently in their toxicity [48]. Most toxicity assays, whether in vivo or in vitro, are performed using dispersions of the
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nanoparticles in culture media or biological fluids, so it is essential to characterize them under these conditions, in order to identify physicochemical changes and to predict possible effects on toxicity. The same considerations apply when the nanoparticles are dispersed in the environment, where they undergo interactions with dissolved organic matter and solutions with different ionic strengths [52-54]. Yin et al. [55] reported that the water chemistry has an important effect on the aggregation and transformation of AgNPs. The electrolytes and dissolved organic matter present in environmental aquatic systems were found to influence aggregation, with sunlight being important in inducing the fusion, morphological alteration and sedimentation of AgNPs. With regard to genotoxicity, the comet analyses of blood and gill cells of fish showed increased damage in the two cell types, with gill cells showing higher damage indices than the blood cells, at the three exposure
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Figure 2: Physicochemical analysis of nanoparticles after fish exposure for 72 h: (a) size, (b) polydispersity, (c) zeta potential. (d) Comet analysis of blood cells and gill cells of fish exposed for 72 h. (e) Mitotic index and chromosomal aberration index values for Allium cepa roots exposed for 24 h. The data are expressed as medians. Different numbers in each column indicate significant differences among treatments (p
