生殖細胞および培養細胞を用いた遺伝毒性試験法の開発と国際標準化への貢献

抄録

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.