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Mutagens formed from dibenzo-p-dioxin with nitrogen oxides
299 body and the relation between mutagenicity and carcinogenicity were complicated. 94 The Collaborative Study Group for the Micronucleus Test, Organizer-in-chief: Morita, T., Nippon Glaxo, 43 Wadai, Tsukuba-shi, Ibaraki 30042, Japan
Evaluation of the mouse micronucleus assay as a screen for carcinogens: summary report of the 6th collaborative study by CSGMT/JEMS" MMS
The micronucleus (MN) assay was performed on 107 chemicals and chemical groups with few or no data in the MN assay selected from IARC groups 1 (human carcinogen), 2A (probable human carcinogen), and 2B (possible human carcinogen). Seven out of 15 group 1 chemicals, 8 of 23 group 2A chemicals, and 24 of 68 group 2B chemicals showed positive responses. The rest of the chemicals tested were negative or inconclusive under the experimental conditions. One group 3 (not classifiable) chemical was negative. These study results were combined with selected, reliable published results. MN assays were positive for 77% (23/30), 53% (18/34), and 40% (33/83) of IARC groups 1, 2A, and 2B chemicals, respectively. The highest risk group yielded the highest rate of MN induction. Carcinogenic nitrosamines tended to give negative results in the MN assay, along with halogenated compounds and metals. Sex hormones and chemicals with primarily promoting activity also gave negative results. These data should be taken into account if the MN assay is used to predict chemical carcinogenicity in humans. The relationship between the MN assay and other mutagenicity tests, carcinogenicity tests, or chemical structure-activity analyses is now being assessed. 95 Tsutsumi, N., S. Ishimura, T. Kawata, S. Hizatate and M. Mochizuki, Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo 105, Japan
Conversions of N-nitrosodialkylamines by active oxygens (2). Mutagenicity of products of the treatment with iron(II) sulfate and hydrogen peroxide N-Nitrosodialkylamines require oxidative metabolic activation for mutagenicity. We investigated the mutagenicity of products of the treatment of N-nitrosodialkylamines with a chemical model, and isolated the mutagen formed. Eight N-nitrosodialkylamines were treated with iron(II) sulfate and hydrogen peroxide (Fenton reagent) supplemented with copper(II) acetate (Fe z÷Cu2+-H202), and ethyl acetate extracts of the reaction solutions were tested for their mutagenicity in Salmonella typhimurium TA1535 and E. coli WP2 hcr-. Direct mutagenicity was observed in products from N-nitrosodialkylamines having an alkyl group longer than propyl. In all cases, the mutagenicity was stronger in Salmonella than in E. coli. A synergistic effect of Fe 2÷ and Cu 2÷ on the formation of the mutagen was observed in the reaction of N-nitroso-N-methylbutylamine. The mutagen from N-nitroso-Nmethylbutylamine treated with Fe2+-Cu2+-H202 was isolated as a white crystal, which was directly mutagenic, and its activity explained the whole mutagenicity of products. The results suggest that the compound isolated was the sole mutagen formed from the treatment of N-nitroso-N-methylbutylamine with Fe2+-CuZ+-H202. The same mutagen was also isolated from the reaction solution of N-nitroso-N-butylcarboxymethylamine with FeZ+-Cu2+-H202. The structure of the mutagen was estimated to be 5,6-dihydro-6-methyl6-nitrito-4H-1,2,3-oxadiazine based on various spectral data. 96 Watanabe, T., and T. Hirayama, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607, Japan Mutagens formed from dibenzo-p-dioxin with nitrogen oxides Recently, nitroarenes have become of interest for their mutagenicity, carcinogenicity and ubiquity. Most nitroarenes in the atmosphere are
300 considered to be emitted from combustion sources or formed from polycyclic aromatic hydrocarbons with nitrogen oxides (NOx) during transport through the atmosphere. In the present study, we investigated the formation of mutagens from dibenzo-p-dioxin (DD) with various concentrations of NOx, and the structures of mutagens were elucidated. DD (0.2 mmole, 36.8 mg) was coated on the inner surface of a 1-1 gas collection tube made of glass. Nitrogen monoxide (0.2-3.6 mmole) was injected into the tube after removal of the air inside the tube. Then air was admitted into the tube to convert nitrogen monoxide into NOx. The sample was allowed to stand at 25-30°C for 3 h in the dark or under light irradiation. The light was radiated from a xenon lamp (1000 W) equipped with a filter to cut off light of less than 300 nm wavelength. DD-NOx (molar ratios 1 : 3, 1 : 6 and 1 : 18) reaction mixtures, in spite of the light irradiation, exhibited mutagenicity in S. typhimurium TA98 and TA98/1,8-DNP6 without $9 mix. DDNOx in the dark reaction mixture was a little more mutagenic than under light irradiation. 2Nitrodibenzo-p-dioxin (2-NDD) was isolated from DD-NOx (1 : 6, in the dark) reaction mixture. In a gas chromatography/mass spectrometry study, a peak ( m / z 274, M ÷) of dinitrodibenzo-p-dioxin (DNDD) was detected in DD-NOx (1 : 18, in the dark) reaction mixture. The reaction mixture was reduced by NaSH and 2,7-DNDD and 2,8-DNDD were identified as corresponding diamino-DDs in the reduced mixture by HPLC. 2-NDD contributed more than 85% of the mutagenicity of the DD-NOx (1:3) and (1:6) reaction mixtures, in spite of light irradiation. 90% of the mutagenic potency of DD-NOx (1 : 18, with or without light irradiation) reaction mixtures was accounted for by DNDDs. 97 Watanabe, T., H. Kaji and T. Hirayama, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misa-sagi, Yamashina-ku, Kyoto 607, Japan Mutagenicity of nitrodibenzofurans in Salmonella
typhimurium strains Nitroarenes have become of interest for their mutagenicity and carcinogenicity. They were re-
ported not only to be emitted from combustion sources, but to be produced by the reaction of aromatic hydrocarbons with nitrogen oxides in the environment. Dibenzofurans (DF), which are environmental pollutants, were identified in air, groundwater and diesel exhaust gas particulates, so that the existence of nitrodibenzofurans was expected in the air. Accordingly, 4 kinds of nitrodibenzofurans and 2 kinds of dinitrobenzofurans were synthesized and their mutagenicity was tested by the Ames method in S. typhimurium strains. Authentic 1-, 2-, 3-, 4-NDF, 2,7- and 2,8-DNDF were synthesized by the nitration of DF. Their structures and purities were confirmed by melting point measurement, I H-NMR, GC and HPLC. The mutagenicity of synthesized NDFs and DNDFs, whose purities were above 99%, were tested using S. typhimurium TA98, TA100, TA98NR, TA98/1,8-DNP6 in the presence or absence of $9. $9 was prepared from the livers of male SD rats induced with PCB, phenobarbital, 3-methylcholanthrene and /3-naphthoflavone. In strain TA98, all NDFs and DNDFs except 1-NDF exhibited mutagenicity without $9 mix. The order of mutagenicity was 2,7- > 2,8- > 3- > 2-> 4-nitro substituted dibenzofuran and especially 2,7-DNDF exhibited potent mutagenicity, 900 rev.//xg. Their mutagenicity in strains TA98NR and TA98/1,8-DNP6 without $9 mix was lower than in strain TA98, but the activities of 2-NDF, 2,7- and 2,8-DNDF in strain TA98NR were markedly enhanced by the addition of $9 induced by 3-methylcholanthrene. 98 Watanabe, M., A. Matsuoka, M. Yamada, N. Yamazaki, M. Hayashi, T. Nohmi and T. Sofuni, Division of Genetics and Mutagenesis, National Institute of Hygienic Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158, Japan Expression of genes of nitroreductase and Oacetyltransferase of Salmonella typhimurium in Chinese hamster lung (CHL) cells
Nitroreductase and O-acetyltransferase of S. are enzymes involved in the metabolic activation of nitroarenes and aromatic
typhimurium
Evaluation of the mouse micronucleus assay as a screen for carcinogens: summary report of the 6th collaborative study by CSGMT/JEMS" MMS
The micronucleus (MN) assay was performed on 107 chemicals and chemical groups with few or no data in the MN assay selected from IARC groups 1 (human carcinogen), 2A (probable human carcinogen), and 2B (possible human carcinogen). Seven out of 15 group 1 chemicals, 8 of 23 group 2A chemicals, and 24 of 68 group 2B chemicals showed positive responses. The rest of the chemicals tested were negative or inconclusive under the experimental conditions. One group 3 (not classifiable) chemical was negative. These study results were combined with selected, reliable published results. MN assays were positive for 77% (23/30), 53% (18/34), and 40% (33/83) of IARC groups 1, 2A, and 2B chemicals, respectively. The highest risk group yielded the highest rate of MN induction. Carcinogenic nitrosamines tended to give negative results in the MN assay, along with halogenated compounds and metals. Sex hormones and chemicals with primarily promoting activity also gave negative results. These data should be taken into account if the MN assay is used to predict chemical carcinogenicity in humans. The relationship between the MN assay and other mutagenicity tests, carcinogenicity tests, or chemical structure-activity analyses is now being assessed. 95 Tsutsumi, N., S. Ishimura, T. Kawata, S. Hizatate and M. Mochizuki, Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo 105, Japan
Conversions of N-nitrosodialkylamines by active oxygens (2). Mutagenicity of products of the treatment with iron(II) sulfate and hydrogen peroxide N-Nitrosodialkylamines require oxidative metabolic activation for mutagenicity. We investigated the mutagenicity of products of the treatment of N-nitrosodialkylamines with a chemical model, and isolated the mutagen formed. Eight N-nitrosodialkylamines were treated with iron(II) sulfate and hydrogen peroxide (Fenton reagent) supplemented with copper(II) acetate (Fe z÷Cu2+-H202), and ethyl acetate extracts of the reaction solutions were tested for their mutagenicity in Salmonella typhimurium TA1535 and E. coli WP2 hcr-. Direct mutagenicity was observed in products from N-nitrosodialkylamines having an alkyl group longer than propyl. In all cases, the mutagenicity was stronger in Salmonella than in E. coli. A synergistic effect of Fe 2÷ and Cu 2÷ on the formation of the mutagen was observed in the reaction of N-nitroso-N-methylbutylamine. The mutagen from N-nitroso-Nmethylbutylamine treated with Fe2+-Cu2+-H202 was isolated as a white crystal, which was directly mutagenic, and its activity explained the whole mutagenicity of products. The results suggest that the compound isolated was the sole mutagen formed from the treatment of N-nitroso-N-methylbutylamine with Fe2+-CuZ+-H202. The same mutagen was also isolated from the reaction solution of N-nitroso-N-butylcarboxymethylamine with FeZ+-Cu2+-H202. The structure of the mutagen was estimated to be 5,6-dihydro-6-methyl6-nitrito-4H-1,2,3-oxadiazine based on various spectral data. 96 Watanabe, T., and T. Hirayama, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607, Japan Mutagens formed from dibenzo-p-dioxin with nitrogen oxides Recently, nitroarenes have become of interest for their mutagenicity, carcinogenicity and ubiquity. Most nitroarenes in the atmosphere are
300 considered to be emitted from combustion sources or formed from polycyclic aromatic hydrocarbons with nitrogen oxides (NOx) during transport through the atmosphere. In the present study, we investigated the formation of mutagens from dibenzo-p-dioxin (DD) with various concentrations of NOx, and the structures of mutagens were elucidated. DD (0.2 mmole, 36.8 mg) was coated on the inner surface of a 1-1 gas collection tube made of glass. Nitrogen monoxide (0.2-3.6 mmole) was injected into the tube after removal of the air inside the tube. Then air was admitted into the tube to convert nitrogen monoxide into NOx. The sample was allowed to stand at 25-30°C for 3 h in the dark or under light irradiation. The light was radiated from a xenon lamp (1000 W) equipped with a filter to cut off light of less than 300 nm wavelength. DD-NOx (molar ratios 1 : 3, 1 : 6 and 1 : 18) reaction mixtures, in spite of the light irradiation, exhibited mutagenicity in S. typhimurium TA98 and TA98/1,8-DNP6 without $9 mix. DDNOx in the dark reaction mixture was a little more mutagenic than under light irradiation. 2Nitrodibenzo-p-dioxin (2-NDD) was isolated from DD-NOx (1 : 6, in the dark) reaction mixture. In a gas chromatography/mass spectrometry study, a peak ( m / z 274, M ÷) of dinitrodibenzo-p-dioxin (DNDD) was detected in DD-NOx (1 : 18, in the dark) reaction mixture. The reaction mixture was reduced by NaSH and 2,7-DNDD and 2,8-DNDD were identified as corresponding diamino-DDs in the reduced mixture by HPLC. 2-NDD contributed more than 85% of the mutagenicity of the DD-NOx (1:3) and (1:6) reaction mixtures, in spite of light irradiation. 90% of the mutagenic potency of DD-NOx (1 : 18, with or without light irradiation) reaction mixtures was accounted for by DNDDs. 97 Watanabe, T., H. Kaji and T. Hirayama, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misa-sagi, Yamashina-ku, Kyoto 607, Japan Mutagenicity of nitrodibenzofurans in Salmonella
typhimurium strains Nitroarenes have become of interest for their mutagenicity and carcinogenicity. They were re-
ported not only to be emitted from combustion sources, but to be produced by the reaction of aromatic hydrocarbons with nitrogen oxides in the environment. Dibenzofurans (DF), which are environmental pollutants, were identified in air, groundwater and diesel exhaust gas particulates, so that the existence of nitrodibenzofurans was expected in the air. Accordingly, 4 kinds of nitrodibenzofurans and 2 kinds of dinitrobenzofurans were synthesized and their mutagenicity was tested by the Ames method in S. typhimurium strains. Authentic 1-, 2-, 3-, 4-NDF, 2,7- and 2,8-DNDF were synthesized by the nitration of DF. Their structures and purities were confirmed by melting point measurement, I H-NMR, GC and HPLC. The mutagenicity of synthesized NDFs and DNDFs, whose purities were above 99%, were tested using S. typhimurium TA98, TA100, TA98NR, TA98/1,8-DNP6 in the presence or absence of $9. $9 was prepared from the livers of male SD rats induced with PCB, phenobarbital, 3-methylcholanthrene and /3-naphthoflavone. In strain TA98, all NDFs and DNDFs except 1-NDF exhibited mutagenicity without $9 mix. The order of mutagenicity was 2,7- > 2,8- > 3- > 2-> 4-nitro substituted dibenzofuran and especially 2,7-DNDF exhibited potent mutagenicity, 900 rev.//xg. Their mutagenicity in strains TA98NR and TA98/1,8-DNP6 without $9 mix was lower than in strain TA98, but the activities of 2-NDF, 2,7- and 2,8-DNDF in strain TA98NR were markedly enhanced by the addition of $9 induced by 3-methylcholanthrene. 98 Watanabe, M., A. Matsuoka, M. Yamada, N. Yamazaki, M. Hayashi, T. Nohmi and T. Sofuni, Division of Genetics and Mutagenesis, National Institute of Hygienic Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158, Japan Expression of genes of nitroreductase and Oacetyltransferase of Salmonella typhimurium in Chinese hamster lung (CHL) cells
Nitroreductase and O-acetyltransferase of S. are enzymes involved in the metabolic activation of nitroarenes and aromatic
typhimurium