Genotoxic mixtures and dissimilar action: concepts for prediction and assessment

Combinations of genotoxic agents have frequently been assessed without clear assumptions regarding their expected (additive) mixture effects, often leading to claims of synergisms that might in fact be compatible with additivity. We have shown earlier that the combined effects of chemicals, which induce micronuclei (MN) in the cytokinesis-block micronucleus assay in Chinese hamster ovary-K1 cells by a similar mechanism, were additive according to the concept of concentration addition (CA). Here, we extended these studies and investigated for the first time whether valid additivity expectations can be formulated for MN-inducing chemicals that operate through a variety of mechanisms, including aneugens and clastogens (DNA cross-linkers, topoisomerase II inhibitors, minor groove binders). We expected that their effects should follow the additivity principles of independent action (IA). With two mixtures, one composed of various aneugens (colchicine, flubendazole, vinblastine sulphate, griseofulvin, paclitaxel), and another composed of aneugens and clastogens (flubendazole, doxorubicin, etoposide, melphalan and mitomycin C), we observed mixture effects that fell between the additivity predictions derived from CA and IA. We achieved better agreement between observation and prediction by grouping the chemicals into common assessment groups and using hybrid CA/IA prediction models. The combined effects of four dissimilarly acting compounds (flubendazole, paclitaxel, doxorubicin and melphalan) also fell within CA and IA. Two binary mixtures (flubendazole/paclitaxel and flubendazole/doxorubicin) showed effects in reasonable agreement with IA additivity. Our studies provide a systematic basis for the investigation of mixtures that affect endpoints of relevance to genotoxicity and show that their effects are largely additive.     

Characterization of formaldehyde’s genotoxic mode of action by gene expression analysis in TK6 cells

Gene expression analysis has been established as a tool for the characterization of genotoxic mechanisms of chemical mutagens. It has been suggested that expression analysis is capable of distinguishing compounds that cause DNA damage from those that interfere with mitotic spindle function. Formaldehyde (FA) is known to be a DNA-reactive substance which mainly induces chromosomal damage in cultured mammalian cells. However, there has been concern that FA might also induce leukemia-specific aneuploidies, although recent cytogenetic studies excluded a relevant aneugenic potential of FA. We now investigated whether gene expression profiling can be used as a molecular tool to further characterize FA’s genotoxic mode of action and to differentiate between clastogenic and aneugenic activity. TK6 cells were exposed to FA for 4 and 24 h, and changes in gene expression were analyzed using a whole-genome human microarray. Results were compared to the expression profiles of two DNA-damaging clastogens (methyl methanesulfonate and ethyl methanesulfonate) and two aneugens (colcemid and vincristine). The genotoxic activity of FA, MMS and EMS under these conditions was confirmed by comet assay experiments. The gene expression profiles indicated that clastogens and aneugens induce discriminable gene expression patterns. Exposure of TK6 cells to FA led to a discrete gene expression pattern, and all toxicogenomics analyses revealed a closer relationship of FA with clastogens than with aneugens.     

免疫荧光染色微核试验检测非整倍体诱变剂

采用免疫荧光染色技术显示微核中的着丝粒，发现纺锤体毒物秋水仙碱诱发Ｖ７９，ＣＨＬ双核细胞徽核着丝粒阳性率分别在７６Ｖ和６６％以上，长春新碱诱发ＢＡＬＢ／ｃ小鼠骨髓红细胞微核着丝粒阳性率在７０％以上；而染色体断裂剂丝裂霉素Ｃ诱发Ｖ７９，ＣＨＬ双核细胞微核着丝粒阳性率分别低于２７％和２１％，环磷酞胺诱发ＢＡＬＢ／ｃ小鼠骨髓红细胞微核着丝粒阳性率低于１５％表明将免疫荧光染色技术与微核试验相结合，计数微核着丝粒阳性率，能够区分微核主要来源于染色体断裂剂引起的无着丝粒断片或纺锤体毒物造成的滞留在胞质中的整条染色体，从而达到简便、快速检测非整倍体诱变剂的目的．     

非整倍体诱导剂的检测方法及风险评估

非整倍体(aneuploidy)指细胞核内染色体数不是染色体组的整倍数,包括单体、三体、四体、双三体、缺体等,与恶性肿瘤发生密切相关.非整倍体诱导剂(aneugen)指诱导子细胞染色体数目增加或减少,即导致非整倍体形成的物质.现行的遗传毒性试验指导原则要求评估化学物质诱发非整倍体的可能性,但标准的遗传毒性试验组合不包括...     

The in vitro MN assay in 2011: origin and fate,biological significance,protocols, high throughput methodologies and toxicological relevance

Micronuclei (MN) are small, extranuclear bodies that arise in dividing cells from acentric chromosome/chromatid fragments or whole chromosomes/chromatids lagging behind in anaphase and are not included in the daughter nuclei at telophase. The mechanisms of MN formation are well understood; their possible postmitotic fate is less evident. The MN assay allows detection of both aneugens and clastogens, shows simplicity of scoring, is widely applicable in different cell types, is internationally validated, has potential for automation and is predictive for cancer. The cytokinesis-block micronucleus assay (CBMN) allows assessment of nucleoplasmic bridges, nuclear buds, cell division inhibition, necrosis and apoptosis and in combination with FISH using centromeric probes, the mechanistic origin of the MN. Therefore, the CBMN test can be considered as a “cytome” assay covering chromosome instability, mitotic dysfunction, cell proliferation and cell death. The toxicological relevance of the MN test is strong: it covers several endpoints, its sensitivity is high, its predictivity for in vivo genotoxicity requires adequate selection of cell lines, its statistical power is increased by the recently available high throughput methodologies, it might become a possible candidate for replacing in vivo testing, it allows good extrapolation for potential limits of exposure or thresholds and it is traceable in experimental in vitro and in vivo systems. Implementation of in vitro MN assays in the test battery for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified.     

Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models

Abstract

We explored how to assess the genotoxic potential of nanosize particles with a well validated assay, the in vitro cytochalasin-B micronucleus assay, detecting both clastogens and aneugens. Monodisperse Stöber amorphous silica nanoparticles (SNPs) of three different sizes (16, 60 and 104 nm) and A549 lung carcinoma cells were selected as models. Cellular uptake of silica was monitored by ICP-MS. At non-cytotoxic doses the smallest particles showed a slightly higher fold induction of micronuclei (MNBN). When considering the three SNPs together, particle number and total surface area appeared to account for MNBN induction as they both correlated significantly with the amplitude of the effect. Using nominal or cellular dose did not show statistically significant differences. Likewise, alkaline comet assay and FISH-centromeric probing of MNBN indicated a weak and not statistically significant induction of oxidative DNA damage, chromosome breakage and chromosome loss. This line of investigation will contribute to adequately design and interpret nanogenotoxicity assays.     

Genotoxicity of the isoflavones genistein, daidzein and equol in V79 cells

Hormonally active chemicals in the human diet, such as man-made estrogenic chemicals or plant-derived compounds (phytoestrogens), have become a matter of public concern. A significant part of human exposure to phytoestrogens is attributable to soy isoflavones. Besides their estrogenic properties, soy isoflavones also exert genotoxic actions. In this paper, the micronucleus (MN) assay in V79 cells was used to study chromosomal genotoxicity. Genistein caused a clear dose-related induction of MN within the range of 5-25 microM; MN rates were declining at higher genistein concentrations. This was probably due to cytotoxicity of genistein since reduced neutral red uptake and MTT formation with an IC(50) of about 75 microM occurred. Daidzein induced a comparatively shallow increase in the number of MN between 25 and 100 microM. In contrast, the daidzein metabolite equol caused an increase in the number of MN up to 25 microM with no further increase at higher concentrations. Additional staining with anti-kinetochore (CREST) antibodies served to determine if the micronuclei contain whole chromosomes or acentric fragments. Genistein induced mostly CREST(-) micronuclei, i.e. MN with chromosomal fragments, thus indicative of a clastogenic mode of action. MN induced by high concentrations of daidzein were partly CREST(+) and CREST(-), whilst equol induced mostly CREST(+) micronuclei indicative of an aneugenic action. These results point to a differential genotoxicity of phytoestrogens.     

Mechanisms and risk of chemically induced aneuploidy in mammalian germ cells

Aneuploidy is a pathological condition that affects 35% of human spontaneous abortions and 0.3% of livebirths. In spite of the increasing knowledge about molecular mechanisms of meiosis and chromosome segregation, maternal age remains the only ascertained aetiological factor. Genetically modified mouse models have been produced that show increased incidence of aneuploid gametes or abnormalities in meiotic recombination and synapsis. They suggest that genetic polymorphisms might also be involved in the aetiology of human germ cell aneuploidy. Experimental studies in the mouse have identified chemicals that can induce aneuploidy in male and female germ cells. Compounds affecting spindle assembly/dynamics are potent aneugens for oocytes and less so also for spermatocytes. They are active at acute doses during a short time interval preceding the metaphase-to-anaphase transition. Topoisomerase inhibitors are also meiotic aneugens which act on the recombination process; for the first time, the production of viable aneuploid mouse progeny was shown after paternal treatment with etoposide. A comparison between in vitro and in vivo effects of suspect aneugens demonstrates that there are biological mechanisms protecting mammalian oocytes from acute exposures to exogenous chemicals. Endocrine disruptors are a novel group of compounds that might affect chromosome segregation at meiosis. Data on bisphenol-A suggest that such chemicals could be active at low chronic exposure levels, but this hypothesis needs to be confirmed by further experiments. Experiments on cultured mouse oocytes treated with inhibitors of biochemical reactions involved in the regulation of chromosome segregation point to possible new mechanisms of action of environmental aneugens.     

3-Nitrobenzanthrone (3-NBA) induced micronucleus formation and DNA damage in human hepatoma (HepG2) cells

3-Nitrobenzanthrone (3-NBA), identified in diesel exhaust and in airborne particulate matter, is a potent mutagen in Salmonella, induces micronuclei formation in mice and in human cells and DNA adducts in rats. In the present study, we investigated the genotoxic potency of 3-NBA in human HepG2 cells using the micronucleus (MN) assay and the single cell gel electrophoresis (SCGE). 3-NBA caused a genotoxic effect at concentrations > or =12 nM in both assays. In the micronucleus assay, we found 98.7+/-10.3 MN/1000 BNC at a concentration of 100 nM 3-NBA in comparison to 27.3+/-0.6 MN/1000 BNC with the negative control. At the same concentration, the DNA-migration (SCGE) showed an Olive tail moment (OTM) of 2.7+/-0.45 and %DNA in the tail of 8.28+/-0.76; OTM and %DNA in the tail of cells treated with the negative control were 0.73+/-0.08 and 2.81+/-0.30, respectively. The results are discussed under consideration of former studies.     

Comparison of four different treatment conditions of extended exposure in the in vitro micronucleus assay using TK6 lymphoblastoid cells

In the OECD Guideline 487, a total of four extended exposure treatment conditions are proposed for the in vitro micronucleus (MNvit) assay in the presence and absence of a cytokinesis block and with or without a recovery period. This guideline also states that rodent cell lines and human lymphocytes can be used as shown by many validated studies but that human cell lines such as TK6 and HepG2 are not yet validated. In this present study each extended exposure condition was characterized by investigation using TK6 cells and nine chemicals known to be able to induce micronucleus (MN) in rodent cell lines. The results revealed two concerns: six chemicals did not show significant MN induction in the ‘cytokinesis block without recovery period’; two aneugens showed no dose-dependent cytotoxicity in the ‘cytokinesis block with recovery period’. Further investigation revealed that 3–4 times higher spontaneous MN frequency than that in the other conditions is a possible reason for the low sensitivity, and this high spontaneous MN frequency was not observed in Chinese hamster lung cells under the identical treatment condition. With regard to the two conditions without cytokinesis block, two negative substances were evaluated and found to be negative, suggesting the validity of the TK6 test system for these conditions. Although our findings showed a few concerns for the treatment with cytokinesis block, the TK6 cells were considered to be a reliable cell line to be used for detecting potential inducers of MN in the in vitro micronucleus assay based on the overall results.     

Lack of effect of coumarin on the formation of micronuclei in an in vivo mouse micronucleus assay.

Coumarin was tested for its potential to cause genotoxic effects in mouse bone marrow cells using an in vivo micronucleus assay. Male and female Swiss mice were administered a single oral dose of coumarin at 50, 100 or 200 mg/kg by gavage in corn oil vehicle. Control animals received only the vehicle. Groups of male mice were also administered mitomycin C at 0.75 mg/kg and served as positive controls. At 24 h after treatment, mice from all dose levels, and at 48 h after treatment, mice from the high dose level only were sacrificed. Bone marrow cells were collected and assayed for the presence of micronuclei. Coumarin did not cause any increase in the incidence of micronucleated polychromatic erythrocytes in male or female mice at any of the dose levels, the positive control mitomycin C produced a significant increase. There was no evidence of coumarin or mitomycin C treatment related cytotoxicity to bone marrow cells. The results of this study demonstrate that coumarin is negative in the mouse in vivo micronucleus assay.     

Evaluation of genotoxicity from Nilufer Stream (Bursa/Turkey) water using piscine micronucleus test

In the present study, the in vivo micronucleus (MN) test in fish erythrocytes was used to evaluate the genotoxic potentials of water samples collected from four different sites along the Nilufer Stream which empties into the Marmara Sea on the north-west of Turkey. Nilufer Stream receives discharges of industrial and domestic wastes resulting from industrialization and urbanization activities in Bursa city. Nile tilapias (Oreochromis niloticus) were exposed to collected water samples under laboratory conditions for 3 and 6 days. Micronuclei analyses were carried out in peripheral blood erythrocytes. In addition to micronuclei, other nuclear abnormalities (NAs) such as bi-nucleated cells and binuclei with nucleoplasmic bridge and cells with blebbed, notched and lobbed nuclei, were assessed in the erythrocytes. Chemical analyses were also carried out in the water samples to assess the presence of major pollutants. MN and NA frequencies were significantly elevated in fishes exposed to water from polluted areas compared to those exposed to clean water sample. The results of this study indicate that Nilufer Stream is contaminated with potential genotoxic chemicals and the genotoxicity is possibly related with the industrial, agricultural and domestic activities.     

Evaluation of chemopreventive activity of glutamine by the comet and the micronucleus assay in mice's peripheral blood

This research has evaluated the effects of enteral supplementation of glutamine in clastogens and genotoxic damages caused by the acute administration of cisplatin. For this, it was utilized Swiss mice distributed in eight experimental groups: control, cisplatin, glutamine, in three different doses and the combination of these with cisplatin. The results show that the glutamine was present in neither genotoxic nor mutagenic activity. When in association with glutamine and cisplatin, in simultaneous treatment, it was verified the frequency decreased of micronuclei and comets. The damage reduction percentages to the micronucleus ranged from 95.4 to 91.8% after 24h of administration of these compounds and 76.7 to 56.8% after 48h. In the same time the damage reduction percentages to the comet test ranged from 117.0 to 115.0%. The results suggest that glutamine is capable of preventing genotoxic and mutagenic damage according to the experimental design proposed.     

Frequency and size of micronuclei induced in gill cells of medaka fish (Oryzias latipes) after whole-body exposure to clastogenic chemicals

Medaka fish (Oryzias latipes) were whole-bodily treated with various doses of mitomycin C (MMC), ethylmethanesulfonate (EMS), cyclophosphamide (CP), diethylnitrosamine (DEN), or colchicine (COL) for 24?h, and the frequency of micronucleated cells (MNCs) was measured in the gills at 24 and 48?h after treatment. In the present experiments, MMC, CP, and DEN were recorded as efficient inducers of micronuclei at both sampling times, and none of the MNC frequencies recorded with these agents at 24?h significantly exceeded the corresponding frequency at 48?h. For EMS and COL, positive responses were recorded only 48?h after treatment. By comparison with the time-course data reported for radiation-induced MNCs in the same MN assay system, the clear responses observed at the 48-h time point for all the chemicals used were regarded as evidence of their delayed effects on micronucleus (MN) formation. The mean sizes of micronuclei induced after exposure to COL was significantly larger by a factor 2 as compared with that induced by X-irradiation, whereas those determined for the other four chemicals were almost equal to that induced by X-irradiation. These results demonstrate that the medaka gill-cell MN assay can detect chemically-induced chromosome damage, either directly or after metabolic activation, and spindle malfunction, and provide a basis for further development of the present assay system for testing cytogenetic activities of chemical agents.     

Relationship between DNA damage and micronucleus in mouse liver

To determine the optimum timing of partial hepatectomy (PH) in a previously developed mouse liver micronucleus test (Igarashi and Shimada, 1997), the relation between DNA damage and micronucleus was examined using the in vivo alkaline comet assay and the micronucleus test on the liver of the same individual mouse. Five genotoxic carcinogens, 1-nitropyrene (1-NP) (125 mg/kg), cyclophosphamide (CP) (50 mg/kg), methylmethan sulfonate (MMS) (80 mg/kg), mitomycin C (MMC) (2 mg/kg) and diethylnitrosamine (DEN) (50 mg/kg) were intraperitoneally dosed to each group consisting of 4 male ddY mice. The mice were subjected to PH 3, 8 or 24 hr after dosing of each carcinogen, and comet assay was performed using the removed liver. The regenerated hepatocyte was sampled five days after PH, and the incidence of micronucleus was measured. CP, MMS, MMC and DEN induced DNA damage at 8 and 24 hr after dosing, while 1-NP induced DNA damage only 8 hr after dosing. All five carcinogens induced micronuclei whenever PH was performed. In the case of CP, the peak of DNA damage was 24 hr after dosing and the timing of PH did not remarkably affect the incidence of micronuclei. The other 4 carcinogens showed peak DNA damage at 8 hr and the highest incidence of micronuclei when PH was operated 24 hr after dosing. In conclusion, we are the first to show the relation of induction between DNA damage and micronucleus in the liver from the same mouse, and tentatively showed the optimal timing of PH as 24 hr after dosing.     

Co-assessment of cell cycle and micronucleus frequencies demonstrates the influence of serum on the in vitro genotoxic response to amorphous monodisperse silica nanoparticles of varying sizes

Serum proteins have been shown to modulate the cytotoxic and genotoxic responses to nanomaterials. The aim was to investigate the influence of serum on the induction of micronuclei (MN) by nanoparticles (NPs) of different sizes. Therefore, A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles (SNPs) were used as models. Assessment of the cell viability, cell cycle changes and induction of MN by SNPs ranging from 12 to 174 nm was performed in presence or absence of serum, applying the in vitro flow cytometry-based MN assay. Here, it has been demonstrated that serum has an influence on these end points, with a lower cell viability in absence of serum compared with the presence of serum. Further, cell cycle changes, specifically, G₁ and S-phase arrest, were observed in absence of serum for four out of six SNPs tested. A size-dependent MN induction was observed: larger SNPs being more active in absence of serum. In addition, the serum influence was characterised by a size-dependency for cytotoxic and genotoxic effects, with a higher influence of serum for smaller particles. The data indicate that the in vitro micronucleus assay in presence and absence of serum could be advised for hazard assessment because it demonstrates a higher sensitivity in serum-free conditions than in conditions with serum. However, this recommendation applies only if the cell line used is able to proliferate under serum-free conditions because cell division is a prerequisite for MN expression.     

Evaluation of streptozotocin genotoxicity in rats from different ages using the micronucleus assay

Genotoxic potential of streptozotocin (STZ), a naturally occurring antibiotic, has been reported in rat by employing micronucleus (MN) test as the end of evaluation. Influence of age (neonatal, young and adult) was studied for the induction of genotoxicity in peripheral blood and bone marrow. Studies with 3 different doses (10, 30 and 100mg/kg) and three different time periods (1, 3 and 7 days) were conducted to induce MN in peripheral blood reticulocytes (RETs) and bone marrow polychromatic erythrocytes (PCEs). Bone marrow and peripheral blood were analyzed after 24h of last treatment. The induction of MN was observed in both neonatal and young rat peripheral blood RETs as well as in bone marrow PCEs. Results demonstrate the genotoxic nature of STZ in a dose dependent manner. However, significant induction of MN was observed only in bone marrow PCEs of adult rats. The genotoxic potential of STZ (30 mg/kg x 3 days) was further evaluated using comet assay in both bone marrow cells and peripheral blood lymphocytes of young rats. It was found that STZ induced significant DNA damage in comet assay as well. Our data indicate the suitability of neonatal and young rat over adult rats for peripheral blood MN assay. In conclusion, both MN and comet assay can be used as suitable end-points for genotoxic risk assessment in a regulatory set-ups. This can add further advantage of integrating genotoxicity assessment in routine toxicological studies using young rat as a model.     

Genotoxicity evaluation of metformin and glimepiride by micronucleus assay in exfoliated urothelial cells of type 2 diabetes mellitus patients

Micronucleus (MN) assay was performed on the exfoliated urothelial cells to detect the genotoxic effects of the anti-hyperglycemic drugs, metformin and glimepiride in T2DM patients and to use it as a biomarker for DNA damage by assessing the frequency of micronuclei in the exfoliated urothelial cells. A total of 201 subjects (147 T2DM patients & 54 Normal cases) were selected from diverse age groups (25–75 years) and the mean MN frequency was examined per 1000 cells in all the subjects. Relative to the control group (5.02 ± 1.01), an increased MN frequency was observed in females (26.15 ± 2.15) when compared to males (23.08 ± 2.09) in T2DM patients. Further analysis showed that there was a profound increase in the number of MN in the patients using metformin alone (23.02 ± 4.44), or combination of metformin & glimepiride (24.98 ± 2.87) than to the subjects using glimepiride alone (17.52 ± 3.28). It has been proven by this simple, reliable and non-invasive method that metformin has a potential role in causing genotoxicity and that the MN observed in exfoliated urothelial cells could be used as a reliable biomarker in monitoring the genotoxic risk of the anti-hyperglycemic drugs.     

The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP Carcinogenicity Database

The comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology. Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by DNA single strand breaks induced chemically and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts. The mouse or rat organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Therefore, organ-specific genotoxicity was necessary but not sufficient for the prediction of organ-specific carcinogenicity. It would be expected that DNA crosslinkers would be difficult to detect by this assay, because of the resulting inhibition of DNA unwinding. The proportion of 10 DNA crosslinkers that was positive, however, was high in the gastrointestinal mucosa, stomach, and colon, but less than 50% in the liver and lung. It was interesting that the genotoxicity of DNA crosslinkers could be detected in the gastrointestinal organs even though the agents were administered intraperitoneally. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative nongenotoxic (Ames test-negative) carcinogens. The Ames test is generally used as a first screening method to assess chemical genotoxicity and has provided extensive information on DNA reactivity. Out of 208 chemicals studied, 117 are Ames test-positive rodent carcinogens, 43 are Ames test-negative rodent carcinogens, and 30 are rodent noncarcinogens (which include both Ames test-positive and negative noncarcinogens). High positive response ratio (110/117) for rodent genotoxic carcinogens and a high negative response ratio (6/30) for rodent noncarcinogens were shown in the comet assay. For Ames test-negative rodent carcinogens, less than 50% were positive in the comet assay, suggesting that the assay, which detects DNA lesions, is not suitable for identifying nongenotoxic carcinogens. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. This assay had a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens, suggesting that the comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic chemicals. For chemicals whose in vivo genotoxicity has been tested in multiple organs by the comet assay, published data are summarized with unpublished data and compared with relevant genotoxicity and carcinogenicity data. Because it is clear that no single test is capable of detecting all relevant genotoxic agents, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. The conventional micronucleus test in the hematopoietic system is a simple method to assess in vivo clastogenicity of chemicals. Its performance is related to whether a chemical reaches the hematopoietic system. Among 208 chemicals studied (including 165 rodent carcinogens), 54 rodents carcinogens do not induce micronuclei in mouse hematopoietic system despite the positive finding with one or two in vitro tests. Forty-nine of 54 rodent carcinogens that do not induce micronuclei were positive in the comet assay, suggesting that the comet assay can be used as a further in vivo test apart from the cytogenetic assays in hematopoietic cells. In this review, we provide one recommendation for the in vivo comet assay protocol based on our own data.     

Effects of the mycotoxin citrinin on micronucleus formation in a cytokinesis-block genotoxicity assay in cultured human lymphocytes

Some mycotoxins produced by microfungi are capable of causing disease and death in animals and humans. In the present study, the mycotoxin citrinin (CTN) was evaluated for its genotoxic effects to human peripheral blood lymphocytes from six different individuals. Lymphocyte cultures were treated for 48 h with CTN at six different concentrations between 10 and 100 microM. Lymphocyte cultures were also incubated with 0.1 microM mitomycin c (MMC) as a positive control, and 0.5% absolute ethanol as a vehicle control.CTN caused a significant concentration-dependent increase in micronucleus (MN) frequency in human lymphocytes. At the 60 microM, 80 microM and 100 microM concentrations, CTN was found to induce MN in cytokinesis-blocked lymphocytes in comparison with negative controls (P = 0.014). All the CTN concentrations also led to a clear decrease in the percentages of binucleated/mononucleated cells (P = 0.014). These results indicate that CTN at high concentrations is genotoxic in cultured human lymphocytes.