Examination of DNA lesion by the alkaline elution technique. II. DNA lesion induced by hydrogen peroxide

Examination of DNA lesion by the alkaline elution technique. II. DNA lesion induced by hydrogen peroxide

365 13 Ino, T., T. Sawahata, K. Tanaka and Y. Kitano 1, Toxicology Laboratory, Toray Industries, Inc., Otsu and 1 Toray Research Center, Otsu (Japan) ...

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365 13 Ino, T., T. Sawahata, K. Tanaka and Y. Kitano 1, Toxicology Laboratory, Toray Industries, Inc., Otsu and 1 Toray Research Center, Otsu (Japan)

Mutagenic activity of aromatic amines, benzidine and 4,4'-diaminodiphenyl ether: a quantum chemical approach to structure-activity relationship Benzidine (BZ), a well known bladder carcinogen, has been shown to be metabolized in vivo to N-acetyl-BZ, which exhibits a more potent mutagenic activity than the parent compound toward TA98 as well as TA100 in the Ames Salmonella/microsome test. The epoxy resin hardener, 4,4'-diaminodiphenyl ether (DDE), has a structure related to that of BZ and is also metabolized to its acetyl derivative. However, Nacetyl-DDE was found to be less mutagenic than the parent compound toward TA98 and TA100. Furthermore, using a computer program based on the extended Hi)ckel theory, the electron distribution at the nitrogen atom in the lowest unoccupied molecular orbital (ELuMO) of benzidine was calculated to be greater than that of N-acetyl-BZ. In contrast, ELUMOof DDE was calculated to be smaller than that of N-acetyl-DDE. These results suggested the important aspect of ELUMOin understanding the apparently different roles of metabolic acetylation in modulating the mutagenicity of the above two aromatic amines.

14 Inoue, K., T. Shibata, H. Kosaka, T. Kimura, M. Uozumi, Y. Oda and T. Abe 1, Osaka Prefectural Institute of Public Health, Osaka, and 1 Kyoto Prefectural University of Medicine, Kyoto (Japan)

Induction of sister-chromatid exchanges by Nnitrosocimetidine in cultured human iymphocytes and their inhibition by several chemical compounds The frequency of sister-chromatid exchanges (SCEs) was investigated in human lymphocyte cultures by a 72-h exposure to cimetidine and sodium nitrite and by a 1-h exposure to N-nitrosocimetidine (NC). Cimetidine did not lead to an induc-

tion of SCEs in the concentration range 10-5-10-3 M. Sodium nitrite induced a 2-fold increase in the frequency of SCE at a concentration of 10 -2 M. NC caused a significant and dose-response increase. At a NC concentration of 2.6 × 10 -4 M SCE frequency represented an approx. 4-fold increase relative to the solvent control. Furthermore, we examined whether simultaneous application of cysteine, cysteamine, cystamine, glutathione, dithionite, sodium ascorbate and OL-a-tocopherol with NC has any influence on the SCE rate. Cysteine, cysteamine and cystamine, but not the other chemical compounds, significantly reduced the SCEs induced by NC.

15 Kada, T., M. Kato and S. Kiriyama 1, Department of Induced Mutation, National Institute of Genetics, Misima, and 1 Faculty of Agriculture, Hokkaido University, Sapporo (Japan)

Adsorption of pyrolysate mutagens by vegetable fibres Pyrolysates of amino acids and proteins contain mutagenic and carcinogenic products (Sugimura (1982) Cancer, 49, 1970). We previously showed that extracts prepared from many kinds of vegetables suppress mutagenicities of tryptophan pyrolysates. Many vegetable antimutagens were very resistant to heating. We now found that when fibres of different vegetables, such as burdock, cabbage, carrot, and so on, were mixed with aqueous solutions of Trp-P-1, Trp-P-2, Glu-P-1, or Glu-P-2 at concentrations ranging from 0.1 to 0.3 ppm, they were adsorbed into the fibres. Cellulose was not effective. The adsorbed mutagens were not reextractable by phosphate buffer; only 30% of them were recovered by extraction with ethyl acetate.

16 Kananishi, N., M. Watanabe 1, y. Kinjo i and N. Tanaka 2, Tokyo Metropolitan Research Laboratory of Public Health, Tokyo, I Tokyo Metropoli-

366 tan Isotope Research Institute, Tokyo, and 2 Teikyo University, Tokyo (Japan) Examination of DNA lesion by the alkaline elution technique. II. DNA lesion induced by hydrogen peroxide DNA single-strand breaks (SSBs) induced by hydrogen peroxide (H202) in the chromatin of mouse leukemic cells (L5178Y) were examined employing an 'alkaline elution' technique. An obvious relationship was obtained between the concentration of H202 and the amount of single-stranded DNA fragments. The amount of fragmented DNA increased along the time course of H202 treatment at low concentration (0.5 /~g/ml). However, this relation was reversed when raising the concentration of H202 up to 10 #g/ml. HzOz-induced DNA SSB was inhibited after pretreatment of cells with catalase within 5 min at 0 °C or 25 °C. DNA SSB repair was evident after removal of H202 and cell incubation for 5 min at 37 o C although not evident after incubation for 20 min at 0°C. The repair was not inhibited by 2 mM caffeine or 0.2 mM cyclohexamide.

17 Kanaya, H., and Y. Takeda, Hatano Research Institute, Food and Drug Safety Center, Hadano, Kanagawa (Japan)

18 Kasai, H., H. Hayami and S. Nishimura, National Cancer Center Research Institute, Tokyo (Japan) Hydroxylation of deoxyguanosine at the C-8 position by mutagens and carcinogens The C-8 position of deoxyguanosine (dGuo) was hydroxylated by ascorbic acid in the presence of oxygen (02) in 0.1 M phosphate buffer (pH 6.8) at 37°C. Addition of hydrogen peroxide (H202) remarkably enhanced this hydroxylation. The Udenfriend system (ascorbic acid, Fe n, ethylenediaminetetraacetic acid (EDTA) and O 2) was also effective for hydroxylation of dGuo in high yield. Guanine residues in DNA were also hydroxylated by ascorbic acid. Other reducing agents, such as hydroxylamine, hydrazine, dihydroxymaleic acid, sodium bisulfite and acetol, were also effective for the hydroxylation reaction, as were metal-EDTA complexes (Fe ll-, Snn-, Ti m-, CuLEDTA). Dihydroxymaleic acid showed considerable mutagenic activity to TA100 (about 3000 revertants/mg). An OH radical seemed to be involved in this hydroxylation reaction in most of the above hydroxylating systems, but another reaction mechanism may also be involved, particularly when dGuo is hydroxylated by ascorbic acid alone or ascorbic acid plus H202. The possible biological significance of the hydroxylation of guanine residues in DNA in relation to mutagenesis and carcinogenesis is discussed.

Formation of mutagens and nitroso compounds from cardiovascular drugs by reaction with nitrite The formation of mutagens and nitroso compounds from 18 cardiovascular drugs by drugnitrite interaction was examined by the screening procedure developed in our laboratory (Takeda and Kanaya (1982) Chem. Pharm. Bull., 30, 3399-3404). On the reaction of 50 mM drug and 500 mM nitrite at pH 3.0-3.4 and 37 o C for 4 h, formation of bacterial mutagens (S. typhimurium TA98 and TA100) was observed for clonidine, bamethan, hydralazine and bethanidine. The formation of nitroso compounds was observed for ajmaline, alprenolol, dilazep, trimetazidine, bamethan and propranolol.

19 Katoh, M., and S. Iwahara, Hatano Research Institute, Food and Drug Safety Center (FDSC), Kanagawa (Japan) Formation of chromatid-type aberrations with procarbazine hydrochloride in postmeiotic male germ cells of mice To study the relationship between the types of chromosome aberrations and the rates of heritable translocations induced in postmeiotic male germ cells of mice after treatment with mutagens, we investigated whether procarbazine hydrochloride