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Antimutagenic activity of eggplant (Soranum melongena L.) extract
375
Formation of 8-hydroxydeoxyguanosine in DNA by auto-oxidized unsaturated fatty acids We have previously found the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in DNA by treatment with various oxygen radical-forming agents. As 8-OH-dG in DNA induces misreading during DNA synthesis in vitro, it might be one of the DNA damages related to mutagenesis or carcinogenesis by oxygen radical. In this presentation we show that auto-oxidized unsaturated fatty acids are effective for the formation of 8OH-dG in DNA. Various auto-oxidized unsaturated fatty acids were shaken with a solution of deoxyguanosine or DNA in phosphate buffer (pH 7.4) and reacted in emulsion at 37°C. The formation of 8-OH-dG was monitored with highperformance liquid chromatography coupled with electrochemical detector. After the reaction with auto-oxidized linolenic acid and linoleic acid, a considerable amount of 8-OH-dG was detected. When EDTA or DETAPAC (iron chelator) was added to the reaction mixture the formation of 8-OH-dG was inhibited. It has been reported that unsaturated fatty acids stimulate rat mammary carcinogenesis and that their auto-oxidized products are mutagenic. DNA damages such as 8-OHdG or strand break induced by lipid peroxide may be involved in the mutagenesis and carcinogenesis.
14 Kato, T., and K. Kikugawa, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03 (Japan)
Formation of mutagenic diazoquinone by interaction of phenol with nitrite Reaction of phenol with nitrite under mildly acidic conditions produced p-nitrosophenol, p-diazoquinone (I) and o-diazoquinone (II). Compound I revealed mutagenicity on Salmonella typhimurium TA98 and TA100 strains without metabolic activation. The number of His + revertant colonies on TA98 strain was 85 at the dose of 20 nmoles. The mutagenicity did not increase over the value at the higher dose of the compound owing to its strong bactericidal property. While
the reaction of phenol with an equivalent amount of nitrite at pH 3 produced p-nitrosophenol in a high yield, the reaction with an excess amount of nitrite produced I in a high yield, p-Nitrosophenol was converted into I by reaction with nitrite. Formation of II also increased with the amount of nitrite. Formation of these compounds was not largely affected in the presence of dimethylamine. Nitrosation of dimethylamine with nitrite was stimulated by phenol due to the formation of p-nitrosophenol and its stimulatory effect. Thus, the reaction of phenol with nitrite can produce mutagenic p-diazoquinone (I), and stimulate nitrosation of secondary amines by production of p-nitrosophenol.
15 Kawasaki, Y., H. Tamaki, T. Nunoshiba 1 and H. Nishioka 1, Department of Home Economics, Doshisha Women's College, and 1 Division of Biochemistry, Doshisha University, Kyoto 602 (Japan)
Antimutagenic activity of eggplant (Soranum melongena L.) extract Antimutagens are divided into mutagen inactivators and mutation inhibitors. Many of the known antimutagens in vegetables belong to the former. We found that eggplant extract inhibits UV-induced mutation of E. coli WP2, without affecting the survival rate. Eggplant was homogenized with distilled water, centrifuged, and the supernatant fraction was used. The suspension of E. coli WP2 or WP2uvrA that had been irradiated with UV was incubated with the extract for 30 rain at 37°C, and then plated on SEM. After incubation for 48 h the number of Trp +-revertant colonies was counted. The eggplant extract clearly inhibited mutation in the wild-type, but not in the uvrA strain. The molecular weight of this inhibitory factor obtained by separation using ultrafiltration was estimated to be smaller than 500, and this factor was stable to heat, acid, alkali and some organic solvents. To elucidate the mechanism of mutation inhibition, we examined the effect of the eggplant extract on the induction of SOS responses in UV-irradiated E. coli KY700/
376
pSK1002 and KY706/pSK1002. However, the expression of the umuC gene in these strains was not inhibited by the eggplant extract. These results suggested that antimutagenesis of the eggplant extract is related to neither the combination repair nor to the error-prone repair derived from umuC gene. Possible promotion of error-free repair is now under investigation.
16 Kinae, N., M. Yamashita, N. Daicho, I. Tomita and I. Kimura 1, Shizuoka College of Pharmaceutical Sciences, Shizuoka 422, and 1 Aichi Cancer Center Research Institute, Nagoya 464 (Japan) Mutagenicity of pulp and paper mill effluents, and their tumor-inducing effect on catfish (Plotosus
anguillaris ) It has been reported that pulp and paper mill effluents contain several mutagenic compounds which are formed at the chlorination stage. Effects of these mutagens on fish are not known. We report here identifications of several mutagenic components in a pulp and paper mill effluent, and demonstrate the tumor-inducing activity of the effluents on catfish. The spent chlorination liquor obtained by the treatment of unbleached pulp with C12 or NaC10 in the laboratory and 2 kinds of effluents discharged from the mill into the coastal area were used as water samples. Diethyl ether extracts of these samples showed mutagenic activity toward S. typhimurium TA100 without metabolic activation. 3 dicarbonyl compounds, glyoxal, methylglyoxal and diacetyl, and 2,4,4,4tetrachloro-2-butenal in addition to 4 chloroacetones were identified by GC-MS analysis as mutagens from the above ether extracts. On exposure of catfish to these industrial water samples, pigment cell hyperplasia, and melanosis were induced.
17 Kong, Z.-L., M. Mitsuiki, M. Nonaka and H. Omura, Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Fukuoka 812 (Japan)
Mutagenic activities of fudurals and the effects of Cu 2+
Mutagenicity of 7 aminocarbonyl-reaction intermediates (furan, furfural, 5-methylfurfural, furfuryl alcohol, 2-methylfuran, 2-acetylfuran and 5hydroxymethylfurfural) which are known to be produced by reactions between food constituents during processing of foodstuffs was investigated. The assays were done with the umu test, the SOS chromotest, the Rec assay and the micronucleus test. We found that 5-methylfurfural, furan and 2-methylfuran (dose: 400 mg/kg) were positive (MNPCE% about 1.4) in the micronucleus test, but were negative in the SOS chromotest and the umu test. 2-Acetylfuran, 5-hydroxymethylfurfural and furfuryl alcohol were positive in the Rec assay. 5-Hydroxymethylfurfural was positive in the SOS chromotest and the umu test. The activity of this compound decreased on mixing with Cu z÷ (1/100 in molar ratio). In contrast, 5-hydroxymethylfurfural in the presence of Cu 2÷ showed a stronger activity in the micronucleus test (MNPCE% about 0.94) than in the absence of Cu 2+. We also detected a cytotoxic effect of 5-hydroxymethylfurfural for mouse ascite sarcoma 180, and the toxicity was stronger in the presence of Cu 2+.
18 Kurihara, K., S. Kurashina, Y. Tabuse 1, j. Miwa 1 and T. Kaminuma, Department of Bioinformatics, TIMS, Bunkyo-ku, Tokyo 113, and i NEC, Kawasaki City, Kanagawa 213 (Japan) Evaluation of the tumor promoter-screening system with the nematode C. elegans
Caenorhabditis elegans is a free-living small soil nematode. Due to its short generation time and ease of mass culture, C. elegans is suitable for screening test. Miwa reported that the growth, fertilization, and movement of C. elegans are inhibited by TPA (10-7-10 -6 M), and he found some TPA-resistant mutants. This finding suggests that if a substance has TPA-like inhibition effects to the wild-type of C. elegans, and if it does not have inhibition effects to the mutants, it is a
Formation of 8-hydroxydeoxyguanosine in DNA by auto-oxidized unsaturated fatty acids We have previously found the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in DNA by treatment with various oxygen radical-forming agents. As 8-OH-dG in DNA induces misreading during DNA synthesis in vitro, it might be one of the DNA damages related to mutagenesis or carcinogenesis by oxygen radical. In this presentation we show that auto-oxidized unsaturated fatty acids are effective for the formation of 8OH-dG in DNA. Various auto-oxidized unsaturated fatty acids were shaken with a solution of deoxyguanosine or DNA in phosphate buffer (pH 7.4) and reacted in emulsion at 37°C. The formation of 8-OH-dG was monitored with highperformance liquid chromatography coupled with electrochemical detector. After the reaction with auto-oxidized linolenic acid and linoleic acid, a considerable amount of 8-OH-dG was detected. When EDTA or DETAPAC (iron chelator) was added to the reaction mixture the formation of 8-OH-dG was inhibited. It has been reported that unsaturated fatty acids stimulate rat mammary carcinogenesis and that their auto-oxidized products are mutagenic. DNA damages such as 8-OHdG or strand break induced by lipid peroxide may be involved in the mutagenesis and carcinogenesis.
14 Kato, T., and K. Kikugawa, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03 (Japan)
Formation of mutagenic diazoquinone by interaction of phenol with nitrite Reaction of phenol with nitrite under mildly acidic conditions produced p-nitrosophenol, p-diazoquinone (I) and o-diazoquinone (II). Compound I revealed mutagenicity on Salmonella typhimurium TA98 and TA100 strains without metabolic activation. The number of His + revertant colonies on TA98 strain was 85 at the dose of 20 nmoles. The mutagenicity did not increase over the value at the higher dose of the compound owing to its strong bactericidal property. While
the reaction of phenol with an equivalent amount of nitrite at pH 3 produced p-nitrosophenol in a high yield, the reaction with an excess amount of nitrite produced I in a high yield, p-Nitrosophenol was converted into I by reaction with nitrite. Formation of II also increased with the amount of nitrite. Formation of these compounds was not largely affected in the presence of dimethylamine. Nitrosation of dimethylamine with nitrite was stimulated by phenol due to the formation of p-nitrosophenol and its stimulatory effect. Thus, the reaction of phenol with nitrite can produce mutagenic p-diazoquinone (I), and stimulate nitrosation of secondary amines by production of p-nitrosophenol.
15 Kawasaki, Y., H. Tamaki, T. Nunoshiba 1 and H. Nishioka 1, Department of Home Economics, Doshisha Women's College, and 1 Division of Biochemistry, Doshisha University, Kyoto 602 (Japan)
Antimutagenic activity of eggplant (Soranum melongena L.) extract Antimutagens are divided into mutagen inactivators and mutation inhibitors. Many of the known antimutagens in vegetables belong to the former. We found that eggplant extract inhibits UV-induced mutation of E. coli WP2, without affecting the survival rate. Eggplant was homogenized with distilled water, centrifuged, and the supernatant fraction was used. The suspension of E. coli WP2 or WP2uvrA that had been irradiated with UV was incubated with the extract for 30 rain at 37°C, and then plated on SEM. After incubation for 48 h the number of Trp +-revertant colonies was counted. The eggplant extract clearly inhibited mutation in the wild-type, but not in the uvrA strain. The molecular weight of this inhibitory factor obtained by separation using ultrafiltration was estimated to be smaller than 500, and this factor was stable to heat, acid, alkali and some organic solvents. To elucidate the mechanism of mutation inhibition, we examined the effect of the eggplant extract on the induction of SOS responses in UV-irradiated E. coli KY700/
376
pSK1002 and KY706/pSK1002. However, the expression of the umuC gene in these strains was not inhibited by the eggplant extract. These results suggested that antimutagenesis of the eggplant extract is related to neither the combination repair nor to the error-prone repair derived from umuC gene. Possible promotion of error-free repair is now under investigation.
16 Kinae, N., M. Yamashita, N. Daicho, I. Tomita and I. Kimura 1, Shizuoka College of Pharmaceutical Sciences, Shizuoka 422, and 1 Aichi Cancer Center Research Institute, Nagoya 464 (Japan) Mutagenicity of pulp and paper mill effluents, and their tumor-inducing effect on catfish (Plotosus
anguillaris ) It has been reported that pulp and paper mill effluents contain several mutagenic compounds which are formed at the chlorination stage. Effects of these mutagens on fish are not known. We report here identifications of several mutagenic components in a pulp and paper mill effluent, and demonstrate the tumor-inducing activity of the effluents on catfish. The spent chlorination liquor obtained by the treatment of unbleached pulp with C12 or NaC10 in the laboratory and 2 kinds of effluents discharged from the mill into the coastal area were used as water samples. Diethyl ether extracts of these samples showed mutagenic activity toward S. typhimurium TA100 without metabolic activation. 3 dicarbonyl compounds, glyoxal, methylglyoxal and diacetyl, and 2,4,4,4tetrachloro-2-butenal in addition to 4 chloroacetones were identified by GC-MS analysis as mutagens from the above ether extracts. On exposure of catfish to these industrial water samples, pigment cell hyperplasia, and melanosis were induced.
17 Kong, Z.-L., M. Mitsuiki, M. Nonaka and H. Omura, Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Fukuoka 812 (Japan)
Mutagenic activities of fudurals and the effects of Cu 2+
Mutagenicity of 7 aminocarbonyl-reaction intermediates (furan, furfural, 5-methylfurfural, furfuryl alcohol, 2-methylfuran, 2-acetylfuran and 5hydroxymethylfurfural) which are known to be produced by reactions between food constituents during processing of foodstuffs was investigated. The assays were done with the umu test, the SOS chromotest, the Rec assay and the micronucleus test. We found that 5-methylfurfural, furan and 2-methylfuran (dose: 400 mg/kg) were positive (MNPCE% about 1.4) in the micronucleus test, but were negative in the SOS chromotest and the umu test. 2-Acetylfuran, 5-hydroxymethylfurfural and furfuryl alcohol were positive in the Rec assay. 5-Hydroxymethylfurfural was positive in the SOS chromotest and the umu test. The activity of this compound decreased on mixing with Cu z÷ (1/100 in molar ratio). In contrast, 5-hydroxymethylfurfural in the presence of Cu 2÷ showed a stronger activity in the micronucleus test (MNPCE% about 0.94) than in the absence of Cu 2+. We also detected a cytotoxic effect of 5-hydroxymethylfurfural for mouse ascite sarcoma 180, and the toxicity was stronger in the presence of Cu 2+.
18 Kurihara, K., S. Kurashina, Y. Tabuse 1, j. Miwa 1 and T. Kaminuma, Department of Bioinformatics, TIMS, Bunkyo-ku, Tokyo 113, and i NEC, Kawasaki City, Kanagawa 213 (Japan) Evaluation of the tumor promoter-screening system with the nematode C. elegans
Caenorhabditis elegans is a free-living small soil nematode. Due to its short generation time and ease of mass culture, C. elegans is suitable for screening test. Miwa reported that the growth, fertilization, and movement of C. elegans are inhibited by TPA (10-7-10 -6 M), and he found some TPA-resistant mutants. This finding suggests that if a substance has TPA-like inhibition effects to the wild-type of C. elegans, and if it does not have inhibition effects to the mutants, it is a