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In vitro metabolic activation by Drosophila melanogaster microsomes
66 base-pair substitutions with a b o u t 0.03 revertants/nmole. Various limitations imposed by the composition of the minimal medium and possible variations of the test procedure as applied to inorganic c o m p o u n d s will be discussed.
2 T.K. Rao, A.A. Hardigree, J.A. Young, W. Winton and J.L. Epler, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (U.S.A.)
Mutagenicity o f N-nitrosopiperidines: typbimurium system
in vitro studies with the Salmonella
Carcinogenic p o t e n c y of various substituted nitrosopiperidines was investigated by Lijinsky et al. (Proc. Am. Assoc. Cancer Res., 16, 61, 1975). We have examined the mutagenic potential of these c o m p o u n d s with Salmonella tester strains. Nitrosopiperidines show a specificity for TA1535, a missense mutant, and require metabolic activation with phenobarbital-induced rat liver microsomal preparation. A quantitative relationship was observed between mutagenicity and carcinogenicity of methyl-substituted nitrosopiperidines. A block in the alpha position (2,6-dimethyl; 2,2,6,6-tetramethyl) abolished the mutagenic activity. Substitution by a single methyl group did not inhibit mutagenic activity. A good correlation was also noted between mutagenicity and carcinogenicity with oxygenated and halogenated derivatives of nitrosopiperidines. Substitution with a carboxyl group eliminated both mutagenic and carcinogenic activity. Nitroso guvacoline was the only non-carcinogen that gave a positive effect with metabolic activation using Aroclor-induced rat liver microsomal preparation. These results support the hypothesis (Lijinsky, Int. J. Cancer, 16, 318--322, 1975) that activation at the alpha position is necessary for both mutagenic and carcinogenic activity. The results also support the predictive value of a bacterial test system for the detection of mutagenic or carcinogenic compounds. Research jointly supported by EPA and Energy Research and Development Administration under contract with Union Carbide Corporation.
3 C.E. Nix, Bobbie Brewen, R u b y Wilkerson and J.L. Epler, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830 {U.S.A.)
Mutagenesis of nitrosopiperidines in Drosophila melanogaster Nitrosopiperidine and many of its derivatives were fed to Drosophila melanogaster males in a solution of 2% sucrose and 4% DMSO. Treatment time was 24--48 hours except with those c o m p o u n d s such as the halogenated and methyl derivatives which were extremely toxic. Each c o m p o u n d was fed at several concentrations up to a maximum of 30 mM, except where toxicity
67 precluded doses greater than 10 mM. Treated males were mated individually to three Muller-5 females for a period of four days and the F, females then checked for the presence of sex-linked recessive lethals. All suspected lethals were confirmed in the F3 generation. Nitrosopiperidine was a weak mutagen, giving a maximum frequency of a b o u t 2.50% sex-linked recessive lethals in mature sperm and spermatids. All of the methyl derivatives except (2,6-dimethyl) were also weak mutagens. The halogenated derivative (3,4-dichloro) was the most mutagenic nitrosopiperidine tested in terms of the lowest effective dose. The h y d r o x y (nitroso-4-piperidinol) and carboxy (nitrosopipecolic acid) derivatives were n o t mutagenic for mature sperm and spermatids in Drosophila. All of the nitrosopiperidines tested seemed to be independent of concentration fed, although the increase in percent lethals was linear with time up to a maximum of 48 hours. Results with DMN, using the feeding technique, reveal a linear increase in percent lethals with concentration fed. Correlation between these results and those of Lijinsky et al. (Proc. Am. Assoc. Cancer Res., 16, 61, 1975) on the carcinogenic p o t e n c y of the nitrosopiperidines will be discussed. This research jointly supported by EPA and Energy Research and Developm e n t Administration under contract with Union Carbide Corporation.
4 Bobbie Brewen and C.E. Nix, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830 (U.S.A.) In vitro metabolic activation b y
Drosopbila melanogaster microsomes
Our laboratory has developed procedures for the isolation of Drosophila microsomes in an a t t e m p t to characterize the Drosophila activation system. Oregon-R males and females were collected b y etherization and immediately placed on ice. The flies were then homogenized b y mortar and pestle in icecold 0.1 M potassium phosphate buffer, pH 7.5 until a smooth brei was formed. The homogenate was filtered through four layers of cheesecloth and centrifuged at 750 × g f o r 10 min. After t w o 10 000 X g centrifugations, the supernatant was dispensed into 2 ml aliquots, immediately frozen and stored at --70°C. Microsomes were assayed for activity using the standard Salmonella assay. Benzo(a)pyrene and dibenzanthracene are n o t activated by Drosophila microsomes. This agrees with our in vivo results in which these c o m p o u n d s are ineffective in producing sex-linked recessive lethals and Minutes in our Oregon-R stock. Aromatic amines such as 2-acetylaminofluorene (AAF) and 2-aminobenzanthracene are also ineffective mutagens in vivo, b u t isolated microsomes can activate these t w o c o m p o u n d s very effectively. On the basis of the number of revertants/mg microsomal protein, the Drosophila microsomes are more active than those isolated from phenobarbitol-induced rat liver. Possible explanations for the discrepancy between the in vivo and in vitro results as well as activation of other classes of c o m p o u n d s will be discussed. Research jointly supported b y Environmental Protection Agency and Energy
2 T.K. Rao, A.A. Hardigree, J.A. Young, W. Winton and J.L. Epler, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (U.S.A.)
Mutagenicity o f N-nitrosopiperidines: typbimurium system
in vitro studies with the Salmonella
Carcinogenic p o t e n c y of various substituted nitrosopiperidines was investigated by Lijinsky et al. (Proc. Am. Assoc. Cancer Res., 16, 61, 1975). We have examined the mutagenic potential of these c o m p o u n d s with Salmonella tester strains. Nitrosopiperidines show a specificity for TA1535, a missense mutant, and require metabolic activation with phenobarbital-induced rat liver microsomal preparation. A quantitative relationship was observed between mutagenicity and carcinogenicity of methyl-substituted nitrosopiperidines. A block in the alpha position (2,6-dimethyl; 2,2,6,6-tetramethyl) abolished the mutagenic activity. Substitution by a single methyl group did not inhibit mutagenic activity. A good correlation was also noted between mutagenicity and carcinogenicity with oxygenated and halogenated derivatives of nitrosopiperidines. Substitution with a carboxyl group eliminated both mutagenic and carcinogenic activity. Nitroso guvacoline was the only non-carcinogen that gave a positive effect with metabolic activation using Aroclor-induced rat liver microsomal preparation. These results support the hypothesis (Lijinsky, Int. J. Cancer, 16, 318--322, 1975) that activation at the alpha position is necessary for both mutagenic and carcinogenic activity. The results also support the predictive value of a bacterial test system for the detection of mutagenic or carcinogenic compounds. Research jointly supported by EPA and Energy Research and Development Administration under contract with Union Carbide Corporation.
3 C.E. Nix, Bobbie Brewen, R u b y Wilkerson and J.L. Epler, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830 {U.S.A.)
Mutagenesis of nitrosopiperidines in Drosophila melanogaster Nitrosopiperidine and many of its derivatives were fed to Drosophila melanogaster males in a solution of 2% sucrose and 4% DMSO. Treatment time was 24--48 hours except with those c o m p o u n d s such as the halogenated and methyl derivatives which were extremely toxic. Each c o m p o u n d was fed at several concentrations up to a maximum of 30 mM, except where toxicity
67 precluded doses greater than 10 mM. Treated males were mated individually to three Muller-5 females for a period of four days and the F, females then checked for the presence of sex-linked recessive lethals. All suspected lethals were confirmed in the F3 generation. Nitrosopiperidine was a weak mutagen, giving a maximum frequency of a b o u t 2.50% sex-linked recessive lethals in mature sperm and spermatids. All of the methyl derivatives except (2,6-dimethyl) were also weak mutagens. The halogenated derivative (3,4-dichloro) was the most mutagenic nitrosopiperidine tested in terms of the lowest effective dose. The h y d r o x y (nitroso-4-piperidinol) and carboxy (nitrosopipecolic acid) derivatives were n o t mutagenic for mature sperm and spermatids in Drosophila. All of the nitrosopiperidines tested seemed to be independent of concentration fed, although the increase in percent lethals was linear with time up to a maximum of 48 hours. Results with DMN, using the feeding technique, reveal a linear increase in percent lethals with concentration fed. Correlation between these results and those of Lijinsky et al. (Proc. Am. Assoc. Cancer Res., 16, 61, 1975) on the carcinogenic p o t e n c y of the nitrosopiperidines will be discussed. This research jointly supported by EPA and Energy Research and Developm e n t Administration under contract with Union Carbide Corporation.
4 Bobbie Brewen and C.E. Nix, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830 (U.S.A.) In vitro metabolic activation b y
Drosopbila melanogaster microsomes
Our laboratory has developed procedures for the isolation of Drosophila microsomes in an a t t e m p t to characterize the Drosophila activation system. Oregon-R males and females were collected b y etherization and immediately placed on ice. The flies were then homogenized b y mortar and pestle in icecold 0.1 M potassium phosphate buffer, pH 7.5 until a smooth brei was formed. The homogenate was filtered through four layers of cheesecloth and centrifuged at 750 × g f o r 10 min. After t w o 10 000 X g centrifugations, the supernatant was dispensed into 2 ml aliquots, immediately frozen and stored at --70°C. Microsomes were assayed for activity using the standard Salmonella assay. Benzo(a)pyrene and dibenzanthracene are n o t activated by Drosophila microsomes. This agrees with our in vivo results in which these c o m p o u n d s are ineffective in producing sex-linked recessive lethals and Minutes in our Oregon-R stock. Aromatic amines such as 2-acetylaminofluorene (AAF) and 2-aminobenzanthracene are also ineffective mutagens in vivo, b u t isolated microsomes can activate these t w o c o m p o u n d s very effectively. On the basis of the number of revertants/mg microsomal protein, the Drosophila microsomes are more active than those isolated from phenobarbitol-induced rat liver. Possible explanations for the discrepancy between the in vivo and in vitro results as well as activation of other classes of c o m p o u n d s will be discussed. Research jointly supported b y Environmental Protection Agency and Energy