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Antimutagenic substances in enokitake (Flammulina velutipes)
382 66 Tamakawa, K., Y. Mishima, T. Seki, A. Tsunoda, Y. Hisamatsu I and H. Matsushita 1, Sendai Municipal Institute of Public Health, 2-5-10, Oroshimachi-higashi, Sendal, Miyagi 983, and I National Institute of Public Health, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108 (Japan)
Mutagenicity arising from reaction between asphalt tar and nitrogen dioxide Mutagenicity of the products formed by photochemical reaction of road-coating asphalt with nitrogen dioxide (NO2) was investigated using S. typhimurium strains TA98, TA100 and TA98NR, and T A 9 8 / 1 , 8 - D N P 6 which is a strain resistant to the mutagenic action of 1,8-dinitropyrene. An asphalt material dissolved in benzene was deposited on filter paper and was dried to remove benzene. This material was then exposed to N O 2 (10 ppm) in an air stream during irradiation with a high-pressure mercury lamp. The period of reaction was 0.5-4 h. In the absence of N O 2, no mutagenicity was detected using TA98 and TA100 with or without $9 mix. The product obtained by the reaction with N O 2 showed mutagenicity for TA98 without $9 mix (705 revertants/100 ~g of product mixture). The mutagenicity of the reaction product was much lower for strains T A 9 8 N R and T A 9 8 / 1 , 8 - D N P 6 compared with that for TA98. These results suggest that nitroarenes are responsible for most of the mutagenicity. The mutagenic activity did not increase when the amount of asphalt on the filter was increased. It is possible that the reaction proceeded mainly on the surface of the material on the paper.
67 Tatsumi, K., M. Toyoda, A. Tachibana and H. Takebe, Department of Molecular Oncology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606 (Japan)
Mutagenic activity of cimetidine and nitrosated cimetidine in human lymphoblastoid cells Because cimetidine, a histamine H-2 receptor antagonist, has currently been used in several mil-
lion patients for the treatment of disorders of the esophagus, stomach and duodenum, it is important to determine its mutagenic and carcinogenic potential to humans. However, the genotoxicity of cimetidine in mammalian cell cultures has been examined so far only with rat and hamster cells. We, therefore, measured cytotoxicity and mutagenicity of cimetidine (CM) and nitrosated cimetidine (NCM) in a human lymphoblastoid cell line, TK6, using forward mutation to 6-thioguanine resistance (TG~), trifluorothymidine resistance ( T F T r) and ouabain resistance ( O U N ) . Although the incubation of TK6 cells with CM for 1 h at 3 7 ° C resulted in a dosedependent reduction of surviving fraction, no mutation was induced at any of the 3 loci by CM below 1.2 m M which decreased survival to 0.06. At concentrations above 0.1 mM, NCM, formed by treating CM with nitrite and HC1, induced the appearance of T G r, T F T r and O U N mutants in T K 6 line, as efficiently as M N N G . Extensive survey as to the in vivo nitrosation of CM, especially in gastric juice, is imperative to assess the possible genotoxicity of CM.
68 Uejima, M., T. Kinouchi, T. Ogawa 1 and Y. Ohnishi, Departments of Bacteriology and 1 Nutrition, School of Medicine, University of Tokushima, Tokushima 770 (Japan)
Antimutagenic substances in enokitake (Flammuiina velutipes) The antimutagenic activity of an edible mushroom, enokitake (Flammulina velutipes), was studied in the Ames Salmonella/microsome assay using a preincubation method. An ethanol extract of enokitake was fractionated into diethyl ethersoluble neutral, acidic, basic and water-soluble fractions. The antimutagenic effect of these fractions plus an ethanol-insoluble fraction was determined by measuring decreases in the mutations induced by 2 - a m i n o - 3 - m e t h y l i m i d a z o [ 4 , 5 - f ] quinoline (IQ) in Salmonella typhimurium strain TA98 in the presence of an $9 mix. The diethyl ether-soluble acidic fraction had the highest antimutagenic activity with an extract equivalent to 2
383 g of fresh mushrooms decreasing the mutagenicity of 8 ng of IQ by 84%. This fraction was purified by reversed-phase thin-layer chromatography (TLC) to give two bands with R f values of 0.35-0.47 and 0.72-0.84. These bands inhibited the mutagenicity of 5 ng of IQ by 100% and 83%, respectively. Gas chromatography-mass spectrometry, high-performance liquid chromatography, 1H-nuclear magnetic resonance spectroscopy, and fast atom bombardment mass spectrometry suggested that linolenic acid, linoleic acid and palmitoleic acid were in the TLC band with an R f of 0.35-0.47. The other band contained a lactic acid derivative and two additional substances that have not been identified.
69 Wakabayashi, K., M. Yano, M. Nagao and T. Sugimura, Carcinogenesis Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104 (Japan) Presence of nitrosable mutagen precursors in heated foods
Various kinds of foods, such as soybean fermentation products, vegetables and pickled vegetables, show direct-acting mutagenicity after nitrite treatment, and tyramine and indole compounds are identified as mutagen precursors in these foods. Broiled sun-dried fish, beef, chicken, mutton and pork were also found to yield mutagenicity to S. typhimurium TA100 without $9 mix after nitrite treatment. Their mutagenic activities were 430028800 revertants/g heated food. However, unheated materials showed very weak or no mutagenicity after nitrite treatment. These results suggested that mutagen precursors were produced in the process of heating protein food. Next, we tested the mutagenicity of heated various food components such as amino acids, guanine, glucose and cholesterol after nitrite treatment. Heated tryptophan and a mixture of tryptophan and glucose were found to show directacting mutagenicity inducing 730 and 2600 revertants/mg original tryptophan after nitrite treatment, respectively. Therefore, some mutagen
precursors in heated foods are presumably derived from tryptophan.
70 Watanabe, M., T. Nohmi and M. Ishidate Jr., Division of Mutagenesis, National Institute of Hygienic Sciences, Setegaya-ku, Tokyo 158 (Japan) Cloning of Salmonella typhimurium genes for nitroreductase and acetyltransferase, enzymes known to activate some nitroarenes
Efforts to clone Salmonella typhimurium genes for nitroreductase and acetyltransferase produced some strains which had high levels of these enzymes which convert some nitroarenes to active mutagens. An S. typhimurium TA1538 gene library was constructed in the plasmid pBR322. S. typhimurium TA1538NR and T A 1 5 3 8 / 1 , 8 - D N P were transformed with this library, and the transformants were selected in terms of sensitivity to the toxicity of 2-nitrofluorene. Using the transformants, the mutagenicity of 2-nitrofluorene was tested. The transformants were found to be more sensitive than the parent strain (TA1538) and the deficient strains (TA1538NR, TA1538/1,8-DNP) in the mutagenicity assay. These transformants of TA1538NR were also tested for nitrofurazone nitroreductase activity, and one of these transformants had about 50 times as much activity as that of the parent strain. Selected transformants of TA1538/1,8-DNP were tested for isoniazid and 2-aminofluorene N-acetyltransferase activities, and found to have activities 10-100 times higher than the parent strain. It is suggested that plasmids of these transformants contain nitroreductase or acetyltransferase genes and that these transformants will be useful in the detection of mutagenic nitroarenes.
71 Watanabe, M., T. Ohta, Y. Shirasu, T. Inoue 1 and T. Kada 1, Institute of Environmental Toxicology, Kodaira, Tokyo 187, and 1 National Institute of Genetics, Mishima, Shizuoka 411 (Japan)
Mutagenicity arising from reaction between asphalt tar and nitrogen dioxide Mutagenicity of the products formed by photochemical reaction of road-coating asphalt with nitrogen dioxide (NO2) was investigated using S. typhimurium strains TA98, TA100 and TA98NR, and T A 9 8 / 1 , 8 - D N P 6 which is a strain resistant to the mutagenic action of 1,8-dinitropyrene. An asphalt material dissolved in benzene was deposited on filter paper and was dried to remove benzene. This material was then exposed to N O 2 (10 ppm) in an air stream during irradiation with a high-pressure mercury lamp. The period of reaction was 0.5-4 h. In the absence of N O 2, no mutagenicity was detected using TA98 and TA100 with or without $9 mix. The product obtained by the reaction with N O 2 showed mutagenicity for TA98 without $9 mix (705 revertants/100 ~g of product mixture). The mutagenicity of the reaction product was much lower for strains T A 9 8 N R and T A 9 8 / 1 , 8 - D N P 6 compared with that for TA98. These results suggest that nitroarenes are responsible for most of the mutagenicity. The mutagenic activity did not increase when the amount of asphalt on the filter was increased. It is possible that the reaction proceeded mainly on the surface of the material on the paper.
67 Tatsumi, K., M. Toyoda, A. Tachibana and H. Takebe, Department of Molecular Oncology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606 (Japan)
Mutagenic activity of cimetidine and nitrosated cimetidine in human lymphoblastoid cells Because cimetidine, a histamine H-2 receptor antagonist, has currently been used in several mil-
lion patients for the treatment of disorders of the esophagus, stomach and duodenum, it is important to determine its mutagenic and carcinogenic potential to humans. However, the genotoxicity of cimetidine in mammalian cell cultures has been examined so far only with rat and hamster cells. We, therefore, measured cytotoxicity and mutagenicity of cimetidine (CM) and nitrosated cimetidine (NCM) in a human lymphoblastoid cell line, TK6, using forward mutation to 6-thioguanine resistance (TG~), trifluorothymidine resistance ( T F T r) and ouabain resistance ( O U N ) . Although the incubation of TK6 cells with CM for 1 h at 3 7 ° C resulted in a dosedependent reduction of surviving fraction, no mutation was induced at any of the 3 loci by CM below 1.2 m M which decreased survival to 0.06. At concentrations above 0.1 mM, NCM, formed by treating CM with nitrite and HC1, induced the appearance of T G r, T F T r and O U N mutants in T K 6 line, as efficiently as M N N G . Extensive survey as to the in vivo nitrosation of CM, especially in gastric juice, is imperative to assess the possible genotoxicity of CM.
68 Uejima, M., T. Kinouchi, T. Ogawa 1 and Y. Ohnishi, Departments of Bacteriology and 1 Nutrition, School of Medicine, University of Tokushima, Tokushima 770 (Japan)
Antimutagenic substances in enokitake (Flammuiina velutipes) The antimutagenic activity of an edible mushroom, enokitake (Flammulina velutipes), was studied in the Ames Salmonella/microsome assay using a preincubation method. An ethanol extract of enokitake was fractionated into diethyl ethersoluble neutral, acidic, basic and water-soluble fractions. The antimutagenic effect of these fractions plus an ethanol-insoluble fraction was determined by measuring decreases in the mutations induced by 2 - a m i n o - 3 - m e t h y l i m i d a z o [ 4 , 5 - f ] quinoline (IQ) in Salmonella typhimurium strain TA98 in the presence of an $9 mix. The diethyl ether-soluble acidic fraction had the highest antimutagenic activity with an extract equivalent to 2
383 g of fresh mushrooms decreasing the mutagenicity of 8 ng of IQ by 84%. This fraction was purified by reversed-phase thin-layer chromatography (TLC) to give two bands with R f values of 0.35-0.47 and 0.72-0.84. These bands inhibited the mutagenicity of 5 ng of IQ by 100% and 83%, respectively. Gas chromatography-mass spectrometry, high-performance liquid chromatography, 1H-nuclear magnetic resonance spectroscopy, and fast atom bombardment mass spectrometry suggested that linolenic acid, linoleic acid and palmitoleic acid were in the TLC band with an R f of 0.35-0.47. The other band contained a lactic acid derivative and two additional substances that have not been identified.
69 Wakabayashi, K., M. Yano, M. Nagao and T. Sugimura, Carcinogenesis Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104 (Japan) Presence of nitrosable mutagen precursors in heated foods
Various kinds of foods, such as soybean fermentation products, vegetables and pickled vegetables, show direct-acting mutagenicity after nitrite treatment, and tyramine and indole compounds are identified as mutagen precursors in these foods. Broiled sun-dried fish, beef, chicken, mutton and pork were also found to yield mutagenicity to S. typhimurium TA100 without $9 mix after nitrite treatment. Their mutagenic activities were 430028800 revertants/g heated food. However, unheated materials showed very weak or no mutagenicity after nitrite treatment. These results suggested that mutagen precursors were produced in the process of heating protein food. Next, we tested the mutagenicity of heated various food components such as amino acids, guanine, glucose and cholesterol after nitrite treatment. Heated tryptophan and a mixture of tryptophan and glucose were found to show directacting mutagenicity inducing 730 and 2600 revertants/mg original tryptophan after nitrite treatment, respectively. Therefore, some mutagen
precursors in heated foods are presumably derived from tryptophan.
70 Watanabe, M., T. Nohmi and M. Ishidate Jr., Division of Mutagenesis, National Institute of Hygienic Sciences, Setegaya-ku, Tokyo 158 (Japan) Cloning of Salmonella typhimurium genes for nitroreductase and acetyltransferase, enzymes known to activate some nitroarenes
Efforts to clone Salmonella typhimurium genes for nitroreductase and acetyltransferase produced some strains which had high levels of these enzymes which convert some nitroarenes to active mutagens. An S. typhimurium TA1538 gene library was constructed in the plasmid pBR322. S. typhimurium TA1538NR and T A 1 5 3 8 / 1 , 8 - D N P were transformed with this library, and the transformants were selected in terms of sensitivity to the toxicity of 2-nitrofluorene. Using the transformants, the mutagenicity of 2-nitrofluorene was tested. The transformants were found to be more sensitive than the parent strain (TA1538) and the deficient strains (TA1538NR, TA1538/1,8-DNP) in the mutagenicity assay. These transformants of TA1538NR were also tested for nitrofurazone nitroreductase activity, and one of these transformants had about 50 times as much activity as that of the parent strain. Selected transformants of TA1538/1,8-DNP were tested for isoniazid and 2-aminofluorene N-acetyltransferase activities, and found to have activities 10-100 times higher than the parent strain. It is suggested that plasmids of these transformants contain nitroreductase or acetyltransferase genes and that these transformants will be useful in the detection of mutagenic nitroarenes.
71 Watanabe, M., T. Ohta, Y. Shirasu, T. Inoue 1 and T. Kada 1, Institute of Environmental Toxicology, Kodaira, Tokyo 187, and 1 National Institute of Genetics, Mishima, Shizuoka 411 (Japan)