strong candidate for a TPA-type tumor promoter. Based on this assumption we have examined several substances that were reported to have tumor promotion effects by some other methods. It was found that sodium ascorbate, sodium erysorbate, saccharin sodium, L-leucine, L-iso- leucine and L-valine do not affect the brood size at a concentration of 10 -2 M. Cholic acid (5 × 10 -4 M) and oL-tryptophan (10-3 M) slightly reduced the brood size, but did not induce abnormal movement. These results suggest that the above substances are not TPA-like tumor promoters.

19 Kuroda, Y., Laboratory of Phenogenetics, Na- tional Institute of Genetics, Mishima, Shizuoka 411 (Japan)

Mutagen-modifying effects of vitamin A and vita- min E in mammalian cells in culture

Effects of vitamin A and vitamin E on the cytotoxicity and mutagenicity of ethyl methane- sulfonate (EMS) in Chinese hamster V79 cells were examined. Vitamin A had a strong cytotoxic effect on the cells, showing an LDs0 of 302 pg/ml. In the presence of vitamin A at a concentration of 100 #g/ml, which had no cytotoxicity, the cyto- toxicity of EMS was slightly reduced. Vitamin E had a moderate cytotoxicity, showing the LDs0 of 710 /~g/rnl. A mixture of vitamin E and EMS showed a cytotoxicity greater than the sum of the individual cytotoxicity. Vitamin A alone did not induce 6-thioguanine-resistant mutations in V79 cells at concentrations up to 100 #g/ml. In the presence of vitamin A, the EMS-induced mutation was reduced to 1/2-2/3, depending on the dose of the vitamin A (200-600 /~g/ml). Vitamin E alone also had no mutagenic effect on the cells. However, in the presence of vitamin E, the EMS- induced mutation increased markedly, depending on concentrations of vitamin E. Vitamin E at a concentration of 100 #g/rrd enhanced 3 times the frequency of EMS-induced mutations. This en- hancing effect of vitamin E was detected only when vitamin E coexisted with EMS: a pretreat- ment with vitamin E had no effect on EMS-in- duced mutations.

377

2O Matsui, M., K. Matsui, T. Nohmi, H. Mizusawa and M. Ishidate Jr., Division of Mutagenesis, Na- tional Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan)

Mutagenicity of steviol: An analytical approach using a Southern blotting system

Stevioside, a natural sweetener, is decomposed by cecal flora in the body to steviol, the aglycone of stevioside. It has been reported that steviol is negative in a reversion assay (Ames test), but positive in a forward mutation with Salmonella typhimurium TM677 after metabolic activation. This discrepancy could be explained if it is as- sumed that steviol preferentially induces deletions or insertions of more than 1 base pair, which can be detected by TM677 but not by strains used in the standard Ames test. The 8-azaguanine resis- tance of the TM677 mutants appears to reside in the chromosomal gpt gene, since it can be com- pleted by the gpt gene of E. coli (Ecogpt). In this study, the chromosomal DNA of TM677 was di- gested by several restriction enzymes: BamHI, Sau3AI, AluI, TaqI, HaelII, HpalI, and RsaI, and analyzed by a Southern blot hybridization technique with a probe to the gpt gene DNA of E. coli. No significant differences in DNA fragment length were found, however, between the wild-type and spontaneous or steviol-induced mutants. Fur- ther studies on direct sequencing of the gpt gene at the mutated region on the TM677 (gpt-) are in progress.

21 Matsuoka, A., M. Sawada, T. Sofuni and M. Ishidate Jr., Division of Mutagenesis, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan)

Chromosome aberration test on 25 chemicals - - Cooperative study with NTP of U.S.A.

25 chemicals supplied by NTP (National Toxicology Program, U.S.A.) and consisting of tri- or tetra-chlorophenols, 9 phenylenediamines, 2 naphthylamines, 4 diphenylamines and 4 other