Mutation Research, 253 (1991) 241-288


© 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1161/91/$03.50

MUTENV 08802

Environmental Mutagen Society of Japan Selected abstracts of the 19th Annual Meeting 29-31 October 1990, Fukuoka (Japan) (Received 23 May 1991) (Accepted 3 June 1991)

Keywords: Japanese Environmental Mutagen Society; Abstracts; Annual Meeting 1990


Akiyama, M., N. Nakamura, S. Kyoizumi, J. Kushiro and Y. Hirai, Department of Radiobiology, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku, Hiroshima 732 (Japan) Detection of somatic mutations in humans

Extensive documentation exists showing that ionizing radiation (X-rays, y-rays, etc.), ultraviolet rays and various chemical agents can induce mutations. The nature of mutations caused by such mutagens has largely been determined through studies using bacteria, Drosophila or cultured mammalian cell lines. However, methods for the quantitative determination of somatic mutations in man are quite limited, and the only practical method available until very recently was the detection of chromosomal aberrations in peripheral blood lymphocytes. The advantage of the search for chromosomal aberrations is that no special equipment is required, but its disadvantage is that examinations must be made by experienced workers, and require much time and effort to do work on an appropriate population scale. Since chemical mutagens induce mainly point muta-

Correspondence: Shigeaki Sato, Toyama Institute of Health, Kosugi-machi, Toyama 939-03 (Japan).

tions, it is possible that the use of chromosomal aberration as an index may result in underestimation of exposure. Therefore, in order to monitor human exposure to environmental mutagens, the development of selective screening methods, by which mutations at various specific loci may be detected using human blood cells, has been anticipated. Recently, we have been involved in developing and putting into practice 4 methods for the detection of somatic mutation at human specific loci. The first method is a modification of the method of Albertini et al. for cloning of lymphocytes to detect mutations of the sex-linked H PRT gene. The second method is a modification of the method developed by Jensen et al. of the Lawrence Livermore National Laboratory using flow cytometry to determine the frequency of erythrocytes lacking the expression of 1 of 2 alleles (M and N) or a somatic recombination at the glycophorin A (GPA) locus on chromosome 4. The other 2 methods, developed by the Radiation Effects Research Foundation (RERF), use flow cytometry to detect mutations of the T-cell antigen receptor (TCR) gene and HLA class I gene in peripheral blood T lymphocytes. This report introduced the methodology, their characteristics and results of our preliminary studies on A-bomb survivors and other individuals known to have been exposed to various mutagens. It can be concluded that the erythrocyte GPA assay appears to be the only assay for lifetime

242 biological dosimetry. However, the donor restriction to MN heterozygotes is the limitation. By contrast, the lymphocyte TCR assay detects effects of recent exposures only, but is simple, quick and requires no specific donor genotype and thus it has a big potential for biological dosimetry of a large-scale survey.

2 Aonuma, S., M. Nakayasu, T. Ushijima, T. Sugimura and M. Nagao, Carcinogenesis Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuoku, Tokyo 104 (Japan) Mutagenicity of okadaic acid on cultured Chinese hamster cells Okadaic acid (OA) has been implicated as one of the causative agents of diarrhetic shellfish poisoning. It was found to be a potent tumor promoter in a 2-stage carcinogenesis experiment on mouse skin. OA does not activate protein kinase C. However, it has been shown to be a strong inhibitor of s e r i n e / t h r e o n i n e protein phosphatases 1 and 2A. We have analyzed the mutagenicity of OA in Chinese hamster lung (CHL) cells using diphtheria toxin resistance (DT r) as a selective marker. The CHL cells (2 × 105) were treated with various concentrations of OA for 24 h. After expression (1 week), the cells were cultured in the presence of 0.1 L f / m l of DT for 1 week, and colonies were counted. 5-17.5 ng/ m l of OA induced DT r cells dose-dependently. At 17.5 ng/ m l of OA, 41 DT r mutant cells were induced per 2.5 × 105 survivors. The specific mutagenic activity of OA calculated from a concentration-response linear curve was 5500 DT r mutants/106 survivors/#g. This value was about 680 times higher than that of EMS, and comparable to that of 2-amino-N6-hydroxyadenine, one of the strongest mutagens. We isolated several DT r mutant clones which were induced by OA. Four independent DT ~ clones were completely resistant to 1 L f / m l of DT. The ADP-ribosylation of elongation factor-2 (EF-2) in the cytoplasmic extracts from 4 DT r clones was almost completely suppressed. In or-

der to clarify whether there is a genomic mutation in the EF-2 gene, EF-2 cDNAs from these clones were synthesized, amplified by PCR and sequenced. However, no nucleotide mutation around the diphthamide residue, which is the target of ADP-ribosylation by DT, was detected. Mutations of histidine-modifying enzymes are being studied.

3 Arimoto, S., N. Inada, H. Rai 1, H. Nakano, T. Negishi and H. Hayatsu, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700, and 1Tama Biochemical Co. Ltd., Tokyo 163 (Japan) Effect of chlorophyllin derivatives on the mutagenicity of 3-hydroxyamino-l-methyl-5H-pyrido[4,3-b]indole, Trp-P-2(NHOH) Recently we reported that chlorophyll and chlorophyllin derivatives can effectively inhibit the mutagenicity of polycyclic aromatic compounds. We have now studied the effectiveness of inhibition of various chlorophyllin derivatives against Trp-P-2(NHOH) by use of the Ames test (TA98, - $ 9 ) , and also investigated the mechanism of the inhibition by analyzing the chemical interactions between Trp-P-2(NHOH) and the chlorophyllins. Among the chlorophyllin-related compounds examined, the most effective inhibition was observed with Fe-chlorin e6-Na 3. Cuchlorin e6-Na 3 and its e4-Na 2 also inhibited the mutagenicity of Trp-P-2(NHOH). Metal-free chlorophyllin was a weak inhibitor, suggesting the importance of the central metal in this action. The absorption spectrum of Trp-P-2(NHOH) at pH 7.4 changed rapidly during incubation with Fe-chlorin e6-Na 3. HPLC analysis of the resulting solution showed that the nitroso compound, 3nitroso- l-methyl-5 H-pyrido[4,3-b ]indole (Trp-P-2 (NO)), was formed during this incubation. These results suggest that the observed effect of Fechlorin e6-Na 3 on the Trp-P-2(NHOH) mutagenicity was due, at least in part, to a rapid oxidation of Trp-P-2(NHOH) to Trp-P-2 (NO).

Environmental Mutagen Society of Japan. 19th annual meeting. 29-31 October 1990, Fukuoka, Japan. Selected abstracts.

Mutation Research, 253 (1991) 241-288 241 © 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1161/91/$03.50 MUTENV 08802 Environment...
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