Thiabendazole (TBZ), a post-harvest fungicide commonly used on imported citrus fruits, exhibited photo-mutagenicity following UVA-irradiation (320-400 nm) in Trp⁺ reverse mutation assay using Escherichia coli WP2uvrA/pKM101 strain. The photo-mutagenicity was not observed in the presence of S9 mix, a rat liver homogenate microsome fraction with co-factors for metabolic activation. We found that NADH and NADPH used as co-factor in the S9 mix efficiently suppressed the photo-mutagenicity of TBZ. This evidence strongly suggested that non-mutagenicity in the presence of S9 mix was not due to the metabolic detoxification of TBZ or the scavenging of UVA-activated TBZ by macromolecules in the S9 mix. Rather quenching effect of NADH and NADPH (λmax=338 nm) may be more responsible for suppression of UVA-activation of TBZ, because oxidized forms of NAD⁺ and NADP⁺ did not show inhibitory effects. Mutagenicity of the UVA-irradiated photo-mutagens such as angelicin and chlorpromazine was also suppressed by the addition of NADH or NADPH. Our present results suggest the possible underestimation in risk evaluation for photomutagenic compounds when they are assayed in the presence of S9 mix.

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Genotoxicity is an important consideration in the safety evaluation of chemicals. It is well known that there are in vitro and in vivo assay systems with different endpoints for evaluating chemical genotoxicity. Bacterial gene mutation test and chromosomal aberrations test using mammalian cultured cells are representative examples. It is apparent that there are limitations of in vitro assay systems for chemical safety evaluation and risk assessment for human health, and in vivo assay systems are becoming more important from the viewpoint of weight of evidence. There are several in vivo assay systems that have been developed and which are based on various endpoints. Among these, the rodent micronucleus test using hematopoietic cells has been most widely and frequently used to detect induction of chromosomal aberration. It is evident that there are chemicals that gave a positive result in the in vitro chromosome aberration test but were negative in the rodent micronucleus test. In such case, as a rule, the in vivo negativity is considered dominant to the in vitro positivity.
It is important and necessary to reduce use of test animals without any loss of evaluation accuracy. In the micronucleus test, development of the method using peripheral blood instead of bone marrow cells succeeded in reducing the total number of animals required for chromosomal aberration evaluation in vivo. Sampling of very small amounts of blood can be done without killing animals, which is one of the most important advantages of the method; it also permits combining with other assays for different endpoints that require different optimal sampling times. Based on this development, in vivo multiple endpoint assay systems will be realized and will lead to further reduction of animal use for the evaluation of chemical genotoxicity. In this manuscript, I describe the history of development and applications of the peripheral blood micronucleus assay.

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Biological effects of genotoxic chemicals are classified into two major categories depending on whether the target organs contain germ cells or just somatic cells. Genotoxicity in germ cells may cause heritable damage in offspring. In the case of somatic cells, genetic damage in a single targeted cell may result in cancer through a multistep process. In this review, I present our data obtained from both areas of germ cell and somatic cell genotoxicity. For studies in male mice, alkylating agents are known to induce dominant lethal mutations in the late spermatids and mature sperm. In studying spermatogenesis and oogenesis in mice, we found that there were stage-specific responses to chemicals. Also, we clarified that such responses to induce heritable translocations closely relate to the types of chromosome aberrations induced in the repair-deficient late spermatid and mature sperm stages. Finally the high incidence of heritable translocations in F1 mice originated from the chromosome type aberrations induced in the first cleavage metaphases of fertilized eggs. Thus, germ cell stages in the mature sperm and oocytes just after copulation were sensitive to mutagens according to the results of chromosome analysis at the first cleavage.
Transformation assays have been used for detection of possible carcinogens, not only initiators but also promoters. In our previous studies, we have tried to use transformation assays such as Syrian hamster embryo (SHE) cells and rat tracheal epithelial (RTE) cells as primary culture cells and also Balb/c 3T3 cells and Bhas42 cells as immortalized cell lines. As an example of transformation assays using primary cells, RTE cell transformation assay system is described. In this study, we could detect a promotion activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by the enhancement of MNNG-induced transformation. This promotional activity may be attributed to a promotional effect, a comutagenic action, or a modulation of cell proliferation and/or differentiation mediated through the TCDD receptor. On the other hand, Balb/c 3T3 assay involves immortalized mouse fibroblast cell lines, in which the number of foci in a monolayer are scored. We have performed an inter-laboratory validation study of the improved transformation assay reported by Drs. Tsuchiya and Umeda with members of NGCS (Non-genotoxic Carcinogen Study Group) under JEMS (Japanese Environmental Mutagen Society). In this study, we gained confidence in the usefulness of the modified two-stage transformation assay with Balb/c 3T3 cells. Bhas42 cells have been established by transfection of v-Ha-ras to Balb/c 3T3 cells by Sasaki et al., and we found that Bhas42 cells were extremely susceptible to TPA-type promoters. Recently, NGCS have started a validation study to evaluate the Bhas42 cell assay system to detect promoters using the modified method reported by Omori et al. This cell transformation assay has advantages over the original Balb/c 3T3 cell assay, particularly regarding sensitivity, test period and simplicity.

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Three areas recently researched on DNA adducts derived from environmental mutagens are described in this paper. (1) A new type of DNA adduct was obtained from 3-nitrobenzanthrone, a powerful mutagen present in the atmospheric environment. N-acetoxy-N-acetyl-3-aminobenzanthrone, a metabolic ultimate form of 3-nitrobenzanthrone, was found to react with 2’-deoxyguanosine to generate N-acetyl-2-(2’-deoxyguanosin-8-yl)-3-aminobenzanthrone, where the C8 position of guanosine is covalently bound to the C2 position of the benzanthrone moiety. This C-C bond formation structure is vastly different from the general C-N bond type DNA adducts formed with amino/nitroarenes. (2) ADP-ribosylation of nucleic acids was reported. Pierisin-1 is a cytotoxic protein present in pupa of cabbage butterflies, and has been shown to have ADP-ribosyltransferase activity, targeting DNA molecules. The N² position of guanosine is ADP-ribosylated to form N²-(ADPribos-1-yl)-2’-deoxyguanosine. (3) The last topic described is the general method for generating so-called dG-C8 adducts derived from amino/nitroarenes. Fully protected 8-amino-2’-deoxyguanosine was able to couple with several bromoarenes to form a protected dG-C8 adduct under the condition of arylamination catalyzed by Pd. This procedure can be used for the preparation of authentic samples of dG-C8 adducts or synthesis of a site- specific adducted oligonucleotide.

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