In vitro clastogenicity and phototoxicity of fullerene (C(60)) nanomaterials in mammalian cells.

Carbon nanomaterials such as carbon nanotubes, graphene, and fullerenes (C(60)) are widely used in industry. Because of human health concerns, their toxic potential has been examined in vivo and in vitro. Here we used mammalian cells to examine the in vitro clastogenicity as well as the phototoxicity of C(60). While C(60) induced no structural chromosome aberrations in CHL/IU cells at up to 5mg/ml (the maximum concentration tested), it significantly induced polyploidy at 2.5 and 5mg/ml with and without metabolic activation. In BALB 3T3 cells, C(60) showed no phototoxic potential but the anatase form of titanium oxide did. Since insoluble nanomaterials cause polyploidy by blocking cytokinesis rather than by damaging DNA, we concluded that the polyploidy induced by C(60) in CHL/IU cells was probably due to non-DNA interacting mechanisms.

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 in vivo skin comet assay to evaluate the DNA-damaging potential of chemicals applied to the skin.

The aim of the present study was to evaluate both sensitivity and specificity of an in vivo skin comet assay using chemically treated, hairless mouse dorsal skin as a model. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.0125-0.2%), 4-nitroquinoline-1-oxide (4NQO, 0.01-0.25%), mitomycin C (MMC, 0.0125-0.05%), benzo[a]pyrene (B[a]P, 0.25-2%), and 7,12-dimethylbenz[a]anthracene (DMBA, 0.25-1%) were each applied once to the dorsal skin of hairless male mice; after 3h, epidermal skin cells were isolated, and the alkaline comet assay was performed. The assay was performed after 24h for only the B[a]P and DMBA. Furthermore, B[a]P and DMBA were evaluated by alkaline comet assay using liver cells after both 3 and 24h. The mean percent of DNA (%DNA) in tail in the 0.05-0.2% MNNG and 0.1-0.25% 4NQO treatment groups was markedly higher than in the control group at 3h post-application. Although the mean %DNA values in the tail in the B[a]P and DMBA groups were the same as the controls at 3h post-application, the 2% B[a]P and 1% DMBA groups showed significantly higher values versus controls 24h after application. No significant increases in the mean %DNA in the tail were observed in the MMC group. No clear increases in %DNA in the tail were observed in the B[a]P and DMBA groups at 3 or 24h after application in the liver. These results suggest that the in vivo skin comet assay is able to accurately identify DNA-damaging potential with a skin-specific response and is a useful method to detect the DNA-damaging potential of genotoxic chemicals on the skin.

In vivo comet assay of acrylonitrile, 9-aminoacridine hydrochloride monohydrate and ethanol in rats.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay, we examined the ability of acrylonitrile, 9-aminoacridine hydrochloride monohydrate (9-AA), and ethanol to induce DNA damage in the liver and glandular stomach of male rats. Acrylonitrile is a genotoxic carcinogen, 9-AA is a genotoxic non-carcinogen, and ethanol is a non-genotoxic carcinogen. Positive results were obtained in the liver cells of male rats treated with known genotoxic compounds, acrylonitrile and 9-AA.

JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: I. Summary of pre-validation study results.

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this validation effort was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The purpose of the pre-validation studies (i.e., Phase 1 through 3), conducted in four or five laboratories with extensive comet assay experience, was to optimize the protocol to be used during the definitive validation study.

JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for detection of genotoxic carcinogens: II. Summary of definitive validation study results.

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity.

Relevance of chemical structure and cytotoxicity to the induction of chromosome aberrations based on the testing results of 98 high production volume industrial chemicals.

Over a 6-year period (1991-1996), the chromosomal aberration testing of high production volume (HPV) industrial chemicals had been conducted using Chinese hamster lung (CHL/IU) cells according to OECD HPV testing program and the national program in Japan. A total of 98 chemicals were tested for the induction of chromosome aberration (CA), consisting of structural CA and polyploidy. Of the 98 chemicals, structural CA and/or polyploidy were induced by 39 chemicals (40%). Anilines and phenols tended to induce only structural CA. p-tert-Butylphenol had a peculiar feature in inducing not only structural CA but also polyploidy at considerably high frequency (93.2%) after continuous treatment for 48 h, posing an aneugenic potential. Not all, but six of 11 carboxylic acids or esters also showed the simultaneous induction of structural CA and polyploidy. The majority of organic phosphates, alcohols or ethers, alkyl benzenes and non-cyclic alkanes had no CA induction activity. For chemicals which were negative in the bacterial reverse mutation assay (Ames test), the proportion of the chemicals that induced CA at a severely cytotoxic dose (doses manifesting more than 50% cytotoxicity) was similar to that of the CA-negative chemicals manifesting severe cytotoxicity, suggesting that severely cytotoxic chemicals do not always induce CA.

Evaluation of effect during cell isolation process in alkaline comet assay using epidermal skin cells.

The aim of the present study was to evaluate the effect of the cell isolation process in the alkaline comet assay using epidermal skin cells. When we explored the cell isolation method for the alkaline comet assay using the 3-dimensional (3D) human epidermal skin model, we found that DNA damage and cytotoxicity were induced during the cell isolation process. In particular, trypsin 5 min treatment with ethylene diamine tetraacetic acid (EDTA) showed about 5 times %DNA in the tail value compared to without EDTA treatment. In general, EDTA is commonly used for cell isolation, but it is known to induce genotoxicity due to secondary effects. We therefore evaluated the effect of EDTA and pH in the alkaline comet assay on a monolayer culture of rat keratinocytes. As a result, there was a significant increase of %DNA in tail values by treatment with 0.1 w/v% EDTA for 60 min; however, there was no difference in the %DNA in tail values between 0.1 w/v% EDTA/PBS(-) (pH 6.8) and 0.1 w/v% EDTA/PBS(-) (pH 7.4). These data imply that there is a need to control the EDTA conditions for cell isolation in the epidermal skin cells.