Studying the genotoxic effects induced by two kinds of bentonite particles on human B lymphoblast cells in vitro

The aim of the present study was to evaluate the genotoxic effects induced by native and active bentonite particles (BPs) on human B lymphoblast cells using comet assay and cytokinesis-block micronucleus (CBMN) assay in vitro. The cells were exposed to BPs at the concentrations of 30, 60, 120 and 240μg/ml for 24, 48 and 72h, respectively. The quartz contents of native and active BPs were 6.80±0.20 and 6.50±0.10%, respectively. Gypsum and DQ-12 quartz served as negative and positive controls. The results of comet assay showed that DNA damage induced by native and active BPs was significantly higher than that induced by gypsum control (P<0.05 or <0.01), and increased with exposure concentration and duration. When the cells were exposed to BPs at the doses of 120 and 240μg/ml for 72h, DNA damage induced by active BPs and native BPs was significantly higher than that induced by DQ-12 quartz (P<0.01), and DNA damage induced by active BPs enhanced significantly, as compared with native BPs (P<0.01). The results of CBMN assay demonstrated that both native BPs and active BPs could induce significant micronuclei, as compared with gypsum control (P<0.05 or <0.01). However, there was no significant difference of micronucleus frequency (MNF) among native BPs, active BPs and DQ-12 quartz. The water-soluble fractions from two kinds of BPs did not induce significant DNA damage and micronuclei. These findings indicated that the genotoxicity induced by active BPs and native BPs could be detected in comet assay and CBMN assay in vitro, the insoluble particle fractions from BPs may play a main role in the genotoxic effects induced by BPs.     

Genotoxic effects of silver nanoparticles stimulated by oxidative stress in human normal bronchial epithelial (BEAS-2B) cells

Many classes of silver nanoparticles (Ag-NPs) have been synthesized and widely applied, but the genotoxicity of Ag-NPs and the factors leading to genotoxicity remain unknown. Therefore, the purpose of this study is to elucidate the genotoxic effects of Ag-NPs in lung and the role of oxidative stress on the genotoxic effects of Ag-NPs. For this, Ag-NPs were completely dispersed in medium by sonication and filtration. The Ag-NPs dispersed in medium were 43-260nm in size. We observed distinct uptake of Ag-NPs into BEAS-2B cells. The Ag-NPs aggregates were wrapped with an endocytic vesicle within the cytoplasm and nucleus of BEAS-2B cells. In the comet assay and micronucleus (MN) assay for BEAS-2B cells, Ag-NPs stimulated DNA breakage and MN formation in a dose-dependent manner. The genotoxic effect of Ag-NPs was partially blocked by scavengers. In particular, of the scavengers tested, superoxide dismutase most significantly blocked the genotoxic effects in both the cytokinesis-block MN assay and the comet assay. In the modified comet assay, Ag-NPs induced a significant increase in oxidative DNA damage. Furthermore, in the oxidative stress assay, Ag-NPs significantly increased the reactive oxygen radicals. These results suggest that Ag-NPs have genotoxic effects in BEAS-2B cells and that oxidative stress stimulated by Ag-NPs may be an important factor in their genotoxic effects.     

Mitigation by vitamin C of the genotoxic effects of nicotine in mice, assessed by the comet assay and micronucleus induction

Nicotine has been reported to cause acute toxicity and to present long-term risks, such as chromosomal damage and genetic instability. The genotoxicity of nicotine may be mediated partly by an oxidative mechanism. We have evaluated the effects of the antioxidant vitamin C on nicotine-induced genotoxicity in mice. The comet assay and the micronucleus test were used to assess the effects of nicotine (15mg/kg) at different exposure times (2, 4, and 24h in the comet assay; 24h in the micronucleus test). Pretreatment with vitamin C 24h before nicotine exposure strongly protected mice against nicotine-induced DNA damage.     

Tumor radiosensitivity prediction by the cytokinesis-block micronucleus assay

An in vivo to in vitro cytokinesis-block micronucleus assay technique using cytochalasin B (Cyt-B) was established in xenografted human and murine tumors, and the correlation between radiosensitivity measured by this assay and that measured by a colony-forming assay was investigated. Tumors were irradiated in situ, excised immediately, and disaggregated to single cells that were plated for the micronucleus and colony-forming assays. Some of the tumor cells were irradiated in vitro rather than in vivo. For the micronucleus assay, Cyt-B (0.5-3 micrograms/ml) was added to dishes soon after plating or in vitro irradiation and the cells were subsequently fixed and stained at intervals (12-144 h). The micronucleus frequency in binucleate cells was evaluated under conditions of maximum yield of the binucleate cells. The micronucleus frequency after irradiation was quite variable depending on the tumor type and the average number of micronuclei per single binucleate cell after 4 Gy ranged from 0.2 to 1.4. The results of in vitro irradiation were not significantly different from those of in vivo irradiation for all tumors. A good correlation was found between the radiosensitivity determined by the micronucleus assay and that found with the colony-forming assay in six human tumors (r = 0.94 approximately 0.98) but not in four murine tumors because of one exceptional tumor. When this tumor was excluded, a correlation was also found for the remaining nine tumors (r = 0.62 approximately 0.96). These results indicated that the cytokinesis-block micronucleus assay has some promise as a rapid predictive assay of radiosensitivity.     

Automated analysis of the cytokinesis-block micronucleus assay for radiation biodosimetry using imaging flow cytometry

The cytokinesis-block micronucleus (CBMN) assay is employed in biological dosimetry to determine the dose of radiation to an exposed individual from the frequency of micronuclei (MN) in binucleated lymphocyte cells. The method has been partially automated for the use in mass casualty events, but it would be advantageous to further automate the method for increased throughput. Recently, automated image analysis has been successfully applied to the traditional, slide-scoring-based method of the CBMN assay. However, with the development of new technologies such as the imaging flow cytometer, it is now possible to adapt this microscope-based assay to an automated imaging flow cytometry method. The ImageStream^X is an imaging flow cytometer that has adequate sensitivity to quantify radiation doses larger than 1 Gy while adding the increased throughput of traditional flow cytometry. The protocol and analysis presented in this work adapts the CBMN assay for the use on the ImageStream^X. Ex vivo-irradiated whole blood samples cultured for CBMN were analyzed on the ImageStream^X, and preliminary results indicate that binucleated cells and MN can be identified, imaged and enumerated automatically by imaging flow cytometry. Details of the method development, gating strategy and the dose response curve generated are presented and indicate that adaptation of the CBMN assay for the use with imaging flow cytometry has potential for high-throughput analysis following a mass casualty radiological event.     

Genotoxicity and mutagenicity of Rosmarinus officinalis (Labiatae) essential oil in mammalian cells in vivo

Rosmarinus officinalis (rosemary) oil is widely used by the cosmetic, food, and pharmaceutical industries as a fragrance component of soaps, creams, lotions, and perfumes. Although it is popular, potential harmful side-effects of the oil have been described. We investigated the genotoxic and mutagenic potential of essential oil of R. officinalis in rodents, using comet, micronucleus and chromosome aberration assays. The animals were treated by gavage with one of three dosages of rosemary oil (300, 1000 or 2000 mg/kg). Liver and peripheral blood cells were collected from Swiss mice 24 h after treatment for the comet assay (genotoxicity endpoint), along with bone marrow cells for the micronucleus test (mutagenicity endpoint). Bone marrow cells were collected from Wistar rats 24 h after oil treatment for the micronucleus and chromosome aberration assays. Based on the comet assay, all three doses of rosemary oil induced significant increases in DNA damage in the mouse cells. There was a significant increase in micronucleated cells and chromosome aberrations only at the two higher doses. We conclude that rosemary essential oil provokes genotoxic and mutagenic effects when administered orally.     

The in vitro micronucleus technique

The study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The micronucleus assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably. Because micronuclei can only be expressed in cells that complete nuclear division a special method was developed that identifies such cells by their binucleate appearance when blocked from performing cytokinesis by cytochalasin-B (Cyt-B), a microfilament-assembly inhibitor. The cytokinesis-block micronucleus (CBMN) assay allows better precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxicity of agents tested or sub-optimal cell culture conditions. The method is now applied to various cell types for population monitoring of genetic damage, screening of chemicals for genotoxic potential and for specific purposes such as the prediction of the radiosensitivity of tumours and the inter-individual variation in radiosensitivity. In its current basic form the CBMN assay can provide, using simple morphological criteria, the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement (nucleoplasmic bridges), cell division inhibition, necrosis and apoptosis. The cytosine-arabinoside modification of the CBMN assay allows for measurement of excision repairable lesions. The use of molecular probes enables chromosome loss to be distinguished from chromosome breakage and importantly non-disjunction in non-micronucleated binucleated cells can be efficiently measured. The in vitro CBMN technique, therefore, provides multiple and complementary measures of genotoxicity and cytotoxicity which can be achieved with relative ease within one system. The basic principles and methods (including detailed scoring criteria for all the genotoxicity and cytotoxicity end-points) of the CBMN assay are described and areas for future development identified.     

Evaluation of genotoxic effects in human fibroblasts after intermittent exposure to 50 Hz electromagnetic fields: a confirmatory study

The aim of this investigation was to confirm the main results reported in recent studies on the induction of genotoxic effects in human fibroblasts exposed to 50 Hz intermittent (5 min field on/10 min field off) sinusoidal electromagnetic fields. For this purpose, the induction of DNA single-strand breaks was evaluated by applying the alkaline single-cell gel electrophoresis (SCGE)/comet assay. To extend the study and validate the results, in the same experimental conditions, the potential genotoxicity was also tested by exposing the cells to a 50 Hz powerline signal (50 Hz frequency plus its harmonics). The cytokinesis-block micronucleus assay was applied after 24 h intermittent exposure to both sinusoidal and powerline signals to obtain information on cell cycle kinetics. The experiments were carried out on human diploid fibroblasts (ES-1). For each experimental run, exposed and sham-exposed samples were set up; positive controls were also provided by treating cells with hydrogen peroxide or mitomycin C for the comet or micronucleus assay, respectively. No statistically significant difference was detected in exposed compared to sham-exposed samples in any of the experimental conditions tested (P > 0.05). In contrast, the positive controls showed a statistically significant increase in DNA damage in all cases, as expected. Accordingly, our findings do not confirm the results reported previously for either comet induction or an increase in micronucleus frequency.     

Usefulness of combined in vivo skin comet assay and in vivo skin micronucleus test

We have already found that the in vivo skin comet assay is useful for the evaluation of primary DNA damage induced by genotoxic chemicals in epidermal skin cells. The aim of the present study was to evaluate the sensitivity and specificity of the combined in vivo skin comet assay and in vivo skin micronucleus (MN) test using the same animal to explore the usefulness of the new test method. The combined alkaline comet assay and MN test was carried out with three chemicals: 4-nitroquinoline-1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene (B[a]P). In the first experiment, we compared DNA- and chromosome-damaging effects of 3 [72, 24 and 3 hours (h) before sacrifice] and 4 applications (72, 48, 24 and 3h before sacrifice) of 4NQO, which induces dermal irritancy. The animals were euthanized and their skin was sampled for the combination test. As a result, the 4-application method was able to detect both DNA- and chromosome-damaging potential with a lower concentration; therefore, in the second experiment, MNNG and B[a]P were topically applied four times, respectively. The animals were euthanized, and then their skins were sampled for combination tests. In the alkaline comet assay, significant differences in the percent of DNA (%DNA) in the tail were observed in epidermal skin cells treated with MNNG and B[a]P. In the MN test, an increased frequency of MN cells (%MN) cells was observed by treatment with MNNG; however, there were no significant increases. In contrast, significant differences in %MN were observed by treatment with B[a]P. From these results, we conclude that the combined in vivo skin comet assay and in vivo MN test was useful because it can detect different genotoxicity with the same sampling time and reduce the number of animals used.     

Use of the cytokinesis-block micronucleus method in mouse splenocytes

Mouse splenocytes have been used in the cytokinesis-block method for the evaluation of micronuclei induced by mutagenic agents in vitro as well as in vivo. Stimulation with concanavalin A for 48 h followed by 16-24-h treatment with 5 micrograms/ml cytochalasin B was found to be an optimum condition to obtain micronuclei in the binucleated splenocytes after the cells were cultured in vitro. Under the above conditions splenocytes from mice pretreated with a single i.p. injection of cyclophosphamide gave a significant increase in micronucleus production. This increase was dependent on the dose of cyclophosphamide (r = 0.99). A dose of 50 mg/kg resulted in 22% of the binucleated cells producing micronuclei, more than 20 times the level in the untreated control. The increase was also dependent on the time of cyclophosphamide injection before removal of the spleen. A duration of 4-8 h after cyclophosphamide injection gave rather sharp optimum values for the production of micronuclei. When splenocytes from non-treated mice were treated with mitomycin C together with cytochalasin B in the above in vitro condition, there was a significant increase in micronucleus production in the binucleated cells. It was also dependent on the dose of mitomycin C (r = 0.975) and a dose of 0.5 micrograms/ml resulted in a more than 20-fold increase over the untreated control. Thus, the use of mouse splenocytes in the cytokinesis-block micronucleus assay was shown to be sensitive enough for testing mutagenic agents in vivo as well as in vitro.     

Genotoxic potential of quinolone antimicrobials in the in vitro comet assay and micronucleus test

The purpose of this study was to examine the genotoxicity of quinolone antimicrobials. We investigated the genotoxic potential of eight quinolones, namely nalidixic acid (NA), pipemidic acid (PPA), oxolinic acid (OA), piromidic acid (PA), enoxacin (ENX), ofloxacin (OFLX), norfloxacin (NFLX) and ciprofloxacin (CPFX), by the in vitro alkaline single-cell gel electrophoresis (comet) assay at pH>13. WTK-1 cells (mutant p53) were treated with each of the eight quinolones at 62.5-1000 microg/mL for 2, 4 and 20 h. NFLX and CPFX significantly induced DNA damage concentration-dependently after 4 and 20 h treatment, but this damage was recoverable. On the other hand, DNA was not damaged in the cells treated with six other quinolones. In the cells treated with NFLX and CPFX for 20 h, DNA migration was compared by the comet assay at pH 10, 12.1 and >13. The comet assay both at pH 12.1 and >13 showed increased DNA migration, but there was no positive response in the comet assay at pH 10. In the in vitro micronucleus (MN) test, WTK-1 cells were treated with each of four quinolones (NA, PPA, NFLX and CPFX) at 15.63-125 microg/mL for 20 h. NFLX significantly increased MNs in the cells, but no changes were noted in the cells treated with three other quinolones. These results suggest that NFLX and CPFX induced DNA single strand breaks (SSBs), and that NFLX-induced SSBs resulted in chromosome aberrations.     

In vitro genotoxicity of danthron and its potential mechanism

To ascertain the in vitro genotoxicity of danthron and its potential mechanism of action, we performed an Ames test, a cytokinesis-block micronucleus assay and a comet assay in Balb/c 3T3 cells. The Ames test revealed that danthron was mutagenic only toward Salmonella typhimurium strain TA102 in the presence of an exogenous metabolic activation system (S9 mix). Danthron (25, 50 and 100μg/ml) increased the frequencies of micronuclear cells with or without S9 mix, and the comet length, tail length and Olive tail moment in comet assays without S9 mix in a dose-dependent manner. These results demonstrated the in vitro genotoxicity of danthron and that 3T3 cells are capable of activating danthron. When NADP was replaced by NAD in the S9 mix, danthron remained mutagenic toward strain TA102. The addition of dicoumarol, a DT-diaphorase inhibitor, decreased the number of danthron-induced histidine revertants by 35-39%, indicating that DT-diaphorase is involved in the metabolic activation of danthron in the presence of NADH as an electron donor. In 3T3 cells, increases in reactive oxygen species (ROS) formation and 8-hydroxydeoxyguanosine levels as well as a reduction in GSH levels were induced by danthron in a dose-dependent manner, indicating that oxidative stress may be a major contributing pathway in the genotoxicity of danthron.     

Comparative evaluation of the in vitro micronucleus test and the comet assay for the detection of genotoxic effects of X-ray radiation

The genotoxic effects of X-ray radiation on human lymphocytes were measured using the single cell gel electrophoresis (SCGE) assay (comet assay) and the cytokinesis-blocked micronucleus (CBMN) test; both were carried out in vitro on isolated human lymphocytes in order to compare the relationship and sensitivity of these two detecting methods. The radiation-doses were 0.00, 0.02, 0.05, 0.10, 0.25, 0.50, 1.00 and 2.00 Gy. In the comet assay, the average comet length (38.6+/-0.8 microm) of 0.05 Gy was significantly longer than that (29.4+/-1.1 microm) of 0 Gy (P<0.01), moreover, the average comet length increased with the dose of X-ray radiation. In the CBMN, both the average micronucleus rate (MN) and micronucleated cell rate (MNC) of 0.05 Gy were 11.5+/-4.5 per thousand, which showed no difference with that (7.5+/-0.5 per thousand) of 0 Gy (P>0.05). The lowest dose, which induced significant increase of average MN and MNC, was 0.25 Gy. The average MN and MNC rates increased with radiation-dose. The results showed that there was correlation between SCGE and CBMN, and the sensitivity of SCGE was significantly higher than that of CBMN.     

Cyto- and genotoxicity of ultrafine TiO2 particles in cultured human lymphoblastoid cells

Titanium dioxide is frequently used in the production of paints, paper, plastics, welding rod-coating material, and cosmetics, because of its low toxicity. However, recent studies have shown that nano-sized or ultrafine TiO(2) (UF-TiO(2)) (<100 nm in diameter) can generate pulmonary fibrosis and lung tumor in rats. Cytotoxicity induced by UF-TiO(2) in rat lung alveolar macrophages was also observed. This generates great concern about the possible adverse effects of UF-TiO(2) for humans. The cytotoxicity and genotoxicity of UF-TiO(2) were investigated using the methyl tetrazolium cytotoxicity (MTT) assay, the population growth assay, the apoptosis assay by flow cytometry, the cytokinesis block micronucleus (CBMN) assay, the comet assay, and the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. WIL2-NS cells were incubated for 6, 24 and 48 h with 0, 26, 65 and 130 microg/ml UF-TiO(2). Significant decreases in viability were seen in the MTT assay at higher doses; for example, 61, 7 and 2% relative viability at 130 microg/ml for 6, 24 and 48-h exposure (P<0.01). A dose-dependent relationship was observed, while a time-dependent relationship was seen only at the highest dose (130 microg/ml) after exposure for 24 and 48 h. Treatment with 130 microg/ml UF-TiO(2) induced approximately 2.5-fold increases in the frequency of micronucleated binucleated cells (P<0.01). In addition, a significant reduction in the cytokinesis block proliferation index was observed by the CBMN assay (P<0.05). In the comet assay, treatment with 65 microg/ml UF-TiO(2) induced approximately 5-fold increases in olive tail moment (P<0.05). In the HPRT mutation assay, treatment with 130 microg/ml UF-TiO(2) induced approximately 2.5-fold increases in the mutation frequency (P<0.05). The results of this study indicate that UF-TiO(2) can cause genotoxicity and cytotoxicity in cultured human cells.     

Genotoxic effects of Roundup on the fish Prochilodus lineatus

Glyphosate-based herbicides, such as Roundup, represent the most extensively used herbicides worldwide, including Brazil. Despite its extensive use, the genotoxic effects of this herbicide are not completely understood and studies with Roundup show conflicting results with regard to the effects of this product on the genetic material. Thus, the aim of this study was to evaluate the genotoxic effects of acute exposures (6, 24 and 96 h) to 10 mg L(-1) of Roundup on the neotropical fish Prochilodus lineatus. Accordingly, fish erythrocytes were used in the comet assay, micronucleus test and for the analysis of the occurrence of nuclear abnormalities and the comet assay was adjusted for branchial cells. The results showed that Roundup produces genotoxic damage in erythrocytes and gill cells of P. lineatus. The comet scores obtained for P. lineatus erythrocytes after 6 and 96 h of exposure to Roundup were significantly higher than respective negative controls. For branchial cells comet scores were significantly higher than negative controls after 6 and 24 h exposures. The frequencies of micronucleus and other erythrocyte nuclear abnormalities (ENAs) were not significantly different between Roundup exposed fish and their respective negative controls, for all exposure periods. In conclusion, the results of this work showed that Roundup produced genotoxic effects on the fish species P. lineatus. The comet assay with gill cells showed to be an important complementary tool for detecting genotoxicity, given that it revealed DNA damage in periods of exposure that erythrocytes did not. ENAs frequency was not a good indicator of genotoxicity, but further studies are needed to better understand the origin of these abnormalities.     

Cytokinesis-block micronucleus assay evolves into a "cytome" assay of chromosomal instability, mitotic dysfunction and cell death

The cytokinesis-block micronucleus (CBMN) assay was originally developed as an ideal system for measuring micronuclei (MNi) however it can also be used to measure nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), cell death (necrosis or apoptosis) and nuclear division rate. Current evidence suggests that (a) NPBs originate from dicentric chromosomes in which the centromeres have been pulled to the opposite poles of the cell at anaphase and are therefore indicative of DNA mis-repair, chromosome rearrangement or telomere end-fusions, (b) NPBs may break to form MNi, (c) the nuclear budding process is the mechanism by which cells remove amplified and/or excess DNA and is therefore a marker of gene amplification and/or altered gene dosage, (d) cell cycle checkpoint defects result in micronucleus formation and (e) hypomethylation of DNA, induced nutritionally or by inhibition of DNA methyl transferase can lead to micronucleus formation either via chromosome loss or chromosome breakage. The strong correlation between micronucleus formation, nuclear budding and NPBs (r = 0.75–0.77, P < 0.001) induced by either folic acid deficiency or exposure to ionising radiation is supportive of the hypothesis that folic acid deficiency and/or ionising radiation cause genomic instability and gene amplification by the initiation of breakage–fusion–bridge cycles. In its comprehensive mode, the CBMN assay measures all cells including necrotic and apoptotic cells as well as number of nuclei per cell to provide a measure of cytotoxicity and mitotic activity. The CBMN assay has in fact evolved into a “cytome” method for measuring comprehensively chromosomal instability phenotype and altered cellular viability caused by genetic defects and/or nutrional deficiencies and/or exogenous genotoxins thus opening up an exciting future for the use of this methodology in the emerging fields of nutrigenomics and toxicogenomics and their combinations.     

SFTG international collaborative study on in vitro micronucleus test III. Using CHO cells

In this report, results are presented from an international study of the in vitro micronucleus assay using Chinese hamster ovary cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, cytosine arabinoside, urethane and diethylstilboestrol. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in Chinese hamster ovary cells. Mannitol and urethane were negative, while bleomycin, cytosine arabinoside and diethylstilboestrol induced a dose dependent increase in micronucleated cells. In the presence of cytochalasin B, increases in micronuclei were observed in binucleated as well as mononucleated cells in cultures treated with bleomycin, cytosine arabinoside or diethylstilboestrol. Importantly, all three of these chemicals were detected in each of the different treatment/recovery regimens. No differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. Overall, these results demonstrate the suitability of Chinese hamster ovary cells for the in vitro micronucleus assay.     

Genotoxicity of silver nanoparticles evaluated using the Ames test and in vitro micronucleus assay

Silver nanoparticles (AgNPs) have antimicrobial properties, which have contributed to their widespread use in consumer products. A current issue regarding nanomaterials is the extent to which existing genotoxicity assays are useful for evaluating the risks associated with their use. In this study, the genotoxicity of 5 nm AgNPs was assessed using two standard genotoxicity assays, the Salmonella reverse mutation assay (Ames test) and the in vitro micronucleus assay. Using the preincubation version of the Ames assay, Salmonella strains TA102, TA100, TA1537, TA98, and TA1535 were treated with 0.15-76.8 μg/plate of the AgNPs. Toxicity limited the doses that could be assayed to 2.4-38.4 μg/plate; no increases in mutant frequency over the vehicle control were found for the concentrations that could be assayed. Human lymphoblastoid TK6 cells were treated with 10-30 μg/ml AgNPs, and additional cells were treated with water and 0.73 gy X-rays as vehicle and positive controls. Micronucleus frequency was increased by the AgNP treatment in a dose-dependent manner. At a concentration of 30 μg/ml (with 45.4% relative population doubling), AgNPs induced a significant, 3.17-fold increase with a net increase of 1.60% in micronucleus frequency over the vehicle control, a weak positive response by our criteria. These results demonstrate that the 5 nm AgNP are genotoxic in TK6 cells. Also, the data suggest that the in vitro micronucleus assay may be more appropriate than the Ames test for evaluating the genotoxicity of the AgNPs.     

Use of the cytokinesis-block method for the analysis of micronuclei in V79 Chinese hamster lung cells: results with mitomycin C and cyclophosphamide

The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze micronuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for micronucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 micrograms/ml CYB for varying durations (8-48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for micronucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of micronuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125-1.0 micrograms/ml) and cyclophosphamide (2-12 micrograms/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in micronucleus frequency over controls at the highest concentration tested (1.0 micrograms/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in micronucleus frequency over controls at the highest tested concentration (12 micrograms/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze micronucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.