Mutation Research, 253 (1991) 241-288 241

© 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

1 tions, it is possible that the use of chromosomal

Akiyama, M., N. Nakamura, S. Kyoizumi, J.
Kushiro and Y. Hirai, Department of Radiobiol-
ogy, 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 mu-
tations. 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 de-
tection 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).
aberration as an index may result in underestima-
tion 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 antic-
ipated. Recently, we have been involved in devel-
oping 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 lympho-
cytes 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 al-
leles (M and N) or a somatic recombination at
the glycophorin A (GPA) locus on chromosome
4. The other 2 methods, developed by the Radia-
tion 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 charac-
teristics 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 restric-
tion to MN heterozygotes is the limitation. By
contrast, the lymphocyte TCR assay detects ef-
fects 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. Sugi-
mura 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 poi-
soning. It was found to be a potent tumor pro-
moter 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 phos-
phatases 1 and 2A. We have analyzed the muta-
genicity 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 in-
duced 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 activ-
ity of OA calculated from a concentration-re-
sponse 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 muta-
tion 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 be-
ing studied.
3
Arimoto, S., N. Inada, H. Rai 1, H. Nakano, T.
Negishi and H. Hayatsu, Faculty of Pharmaceuti-
cal Sciences, Okayama University, Okayama 700,
and 1Tama Biochemical Co. Ltd., Tokyo 163
(Japan)
Effect of chlorophyllin derivatives on the muta-
genicity 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 com-
pounds. 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 mecha-
nism 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 inhibi-
tion was observed with Fe-chlorin e6-Na 3. Cu-
chlorin 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, 3-
nitroso- 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 Fe-
chlorin e6-Na 3 on the Trp-P-2(NHOH) muta-
genicity was due, at least in part, to a rapid
oxidation of Trp-P-2(NHOH) to Trp-P-2 (NO).