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Activation of acrylonitrile to mutagens: possible contribution of reactive forms of O2
229 original $9 fractions. This is particularly true for the mice which have a much lower endogenous G-6-PDH activity than the rats. At the present time we are studying if the addition of G-6-PDH in the incubation mixtures may increase the sensitivity of the mutagenic assay.
II.2B.5 de Waziers, I., and F. Decloitre, Institut de Recherches Scientifiques sur le Cancer, BP No. 8, 94802 Villejuif Cedex (France)
Comparative study of the mutagenic activation of tryptophan pyrolysis products (Trp-P-1 and Trp-P-2) by rat intestinal and liver $9 fraction It is well known that charred meat contains mutagenic compounds. Analyzing tryptophan pyrolysates, Sugimura et al. isolated 2 derivatives Trp-P-1 and Trp-P-2 which exerted a mutagenic activity after undergoing metabolic activation by liver enzymes contained in the $9 fraction. The intestinal metabolism of orally ingested foreign chemicals could play a crucial role in the toxic and carcinogenic action of these chemicals. Thus, the purpose of our work was to study, in the Ames test, the activity of the intestinal enzymes involved in the formation of mutagenic derivatives from Trp-P-1 and Trp-P-2. Furthermore different inducers (phenobarbital, methylcholanthrene and the 2 inducers given together) were used to compare the inducibility of mutagenic activation of Trp-P-1 and Trp-P-2 in the intestine and in the liver. Our results revealed the formation of mutagenic metabolites of Trp-P-1 and Trp-P-2, after metabolic activation in the presence of the intestinal $9 fraction. Moreover we observed that Trp-P-2 had a much higher mutagenic activity than Trp-P-1. The pretreatment of rats with different inducers confirmed the importance of cytochrome P448 in the formation of mutagenic metabolites of Trp-P-1 and Trp-P-2 in rat liver and intestine. In spite of the high mutagenic activity of Trp-P-1 and Trp-P-2, their car.cinogenic potency in male rats and mice was weak as compared with other chemical carcinogens. In order to evaluate the role of detoxifying pathways in the mutagenicity of Trp-P-1 and Trp-P-2, we also studied the effect of the addition of two endogenous substrates of conjugation, glutathione and uridine 5'-diphosphoglucuronic acid to the Salmonella/intestinal or liver $9 test.
II.2B.6 Duverger-van Bogaert, M., M. Lambotte-Vandepaer and M. Mercier, Laboratory of Toxicology and Bromatology, U.C.L. 7237, Avenue Em. Mounier, 72, 1200 Brussels (Belgium)
Activation of acrylonitrile to mutagens: possible contribution of reactive forms of O 2 Acrylonitrile, a well known monomer used in the manufacture of plastics and synthetic fibres, is recognized as mutagen and suspected as carcinogen for man. The mechanism of its metabolic activation remains unclear: if classical epoxidation of the monomer by the mono-oxygenases occurs, other pathways like radical activation are to be considered. In order to evaluate the formation of reactive intermediate(s), a modified methodology of preincubation in liquid medium of the Ames test, using Salmonella typhimurium TA1530, was used (Duverger-van Bogaert et al., 1981). Acrylonitrile indirect mutagenic activity is decreased by the addition of ethanol (85 raM) or dimethyl sulphoxide (87 raM), two known hydroxyl radical scavengers (Cederbaum et al., 1977). The presence of catalase (32 U/ml) or peroxidase (0.1 U/ml) in the incubation mixture results in a significant decrease of acrylonitrile indirect mutagenic activity, while superoxide dismutase is without effect. Those
230 results suggest the implication of O2-reactive forms in the production of acrylonitrile mutagenic intermediate(s), which, in addition to previously reported data (Duverger-van Bogaert et al., 1981) allow to propose that acrylonitrile activation into mutagenic intermediates results probably from the concomitant action of mono-oxygenases and reactive forms of 02.
References Cederbaum, A.I., E. Dicker, E. Rubin and G. Cohen (1977) Biochem. Biophys. Res. Commun., 78, 1254-1262. Duverger-vanBogaert,M., M. Lambotte-Vandepaer,C. de Meester, B. Rollmann, F. Ponceletand M. Mercier(1981)Toxicol.Lett., 7, 311-318.
II.2B.7 Elliott, B., and J. Ashby, Central Toxicology Laboratory, Imperial Chemical Industries PLC, Macclesfield (Great Britain)
Correlation between azoreductase activity and carcinogenicity of analogues of p-dimethylaminophenylazobenzene (DAB) in the rat Azoreductase activity towards the rat hepatocarcinogen DAB and 4 analogues has been measured spectrophotometrically in vitro in the liver (9000 × g supernatant) and caecum (caecal contents) of Alderley Park (Wistar-derived) rats fed a standard pelleted diet. Two carcinogenic DAB analogues, 3'-methyl-p-dimethylaminophenylazobenzene (3M) and 6-p-dimethylaminophenylazobenzothiazole (6BT), and two noncarcinogenic analogues, 4-N-pyrrolidinylazobenzene (4N) and 5-p-dimethylaminophenylazoindazole (5I), have been examined. 3M and 4N were reduced by the liver at a rate of about 50% that of DAB, whereas 5I and 6BT were cleaved at a much lower rate (5-20% that of DAB), with all the chemicals being reduced by an oxygen-insensitive enzyme as has previously been reported for DAB. In contrast, DAB, 3M and 6BT were reduced at a similar rate to each other by a fraction containing caecal contents, with 4N and 51 having only 30-50% of their cleavage rate. Anaerobicity was required for maximal activity of the caecal preparation. No marked differences were observed in these relative activities when animals were fed a riboflavin-low diet (2-3 mg/kg) for 2 weeks prior to azoreductase determinations, or when Sprague-Dawley rats were used. Neither the liver nor the caecal azoreductase activity profiles correlated well with the reported carcinogenic potency of the chemicals, and other factors are suggested to be more important in determining this toxicity for the azo chemicals examined.
II.2B.8 EinistiS, P., H. Norppa and K. Falck, Institute of Occupational Health, SF-00290 Helsinki 29 (Finland)
An attempt to activate styrene by erythrocytes in bacterial mutagenieity assays The induction of sister-chromatid exchanges by styrene in human lymphocytes and CHO cells has been shown to depend on the presence of erythrocytes. In bacterial mutagenicity tests styrene has given slightly positive or completely negative results if $9 mix has been used as a metabolic activation system. The ability of erythrocytes to activate styrene in the bacterial assays was investigated by the fluctuation test and liquid incubation assay with Escherichia coli WP2uvrA ( t r p - , detects base-pair substitution
II.2B.5 de Waziers, I., and F. Decloitre, Institut de Recherches Scientifiques sur le Cancer, BP No. 8, 94802 Villejuif Cedex (France)
Comparative study of the mutagenic activation of tryptophan pyrolysis products (Trp-P-1 and Trp-P-2) by rat intestinal and liver $9 fraction It is well known that charred meat contains mutagenic compounds. Analyzing tryptophan pyrolysates, Sugimura et al. isolated 2 derivatives Trp-P-1 and Trp-P-2 which exerted a mutagenic activity after undergoing metabolic activation by liver enzymes contained in the $9 fraction. The intestinal metabolism of orally ingested foreign chemicals could play a crucial role in the toxic and carcinogenic action of these chemicals. Thus, the purpose of our work was to study, in the Ames test, the activity of the intestinal enzymes involved in the formation of mutagenic derivatives from Trp-P-1 and Trp-P-2. Furthermore different inducers (phenobarbital, methylcholanthrene and the 2 inducers given together) were used to compare the inducibility of mutagenic activation of Trp-P-1 and Trp-P-2 in the intestine and in the liver. Our results revealed the formation of mutagenic metabolites of Trp-P-1 and Trp-P-2, after metabolic activation in the presence of the intestinal $9 fraction. Moreover we observed that Trp-P-2 had a much higher mutagenic activity than Trp-P-1. The pretreatment of rats with different inducers confirmed the importance of cytochrome P448 in the formation of mutagenic metabolites of Trp-P-1 and Trp-P-2 in rat liver and intestine. In spite of the high mutagenic activity of Trp-P-1 and Trp-P-2, their car.cinogenic potency in male rats and mice was weak as compared with other chemical carcinogens. In order to evaluate the role of detoxifying pathways in the mutagenicity of Trp-P-1 and Trp-P-2, we also studied the effect of the addition of two endogenous substrates of conjugation, glutathione and uridine 5'-diphosphoglucuronic acid to the Salmonella/intestinal or liver $9 test.
II.2B.6 Duverger-van Bogaert, M., M. Lambotte-Vandepaer and M. Mercier, Laboratory of Toxicology and Bromatology, U.C.L. 7237, Avenue Em. Mounier, 72, 1200 Brussels (Belgium)
Activation of acrylonitrile to mutagens: possible contribution of reactive forms of O 2 Acrylonitrile, a well known monomer used in the manufacture of plastics and synthetic fibres, is recognized as mutagen and suspected as carcinogen for man. The mechanism of its metabolic activation remains unclear: if classical epoxidation of the monomer by the mono-oxygenases occurs, other pathways like radical activation are to be considered. In order to evaluate the formation of reactive intermediate(s), a modified methodology of preincubation in liquid medium of the Ames test, using Salmonella typhimurium TA1530, was used (Duverger-van Bogaert et al., 1981). Acrylonitrile indirect mutagenic activity is decreased by the addition of ethanol (85 raM) or dimethyl sulphoxide (87 raM), two known hydroxyl radical scavengers (Cederbaum et al., 1977). The presence of catalase (32 U/ml) or peroxidase (0.1 U/ml) in the incubation mixture results in a significant decrease of acrylonitrile indirect mutagenic activity, while superoxide dismutase is without effect. Those
230 results suggest the implication of O2-reactive forms in the production of acrylonitrile mutagenic intermediate(s), which, in addition to previously reported data (Duverger-van Bogaert et al., 1981) allow to propose that acrylonitrile activation into mutagenic intermediates results probably from the concomitant action of mono-oxygenases and reactive forms of 02.
References Cederbaum, A.I., E. Dicker, E. Rubin and G. Cohen (1977) Biochem. Biophys. Res. Commun., 78, 1254-1262. Duverger-vanBogaert,M., M. Lambotte-Vandepaer,C. de Meester, B. Rollmann, F. Ponceletand M. Mercier(1981)Toxicol.Lett., 7, 311-318.
II.2B.7 Elliott, B., and J. Ashby, Central Toxicology Laboratory, Imperial Chemical Industries PLC, Macclesfield (Great Britain)
Correlation between azoreductase activity and carcinogenicity of analogues of p-dimethylaminophenylazobenzene (DAB) in the rat Azoreductase activity towards the rat hepatocarcinogen DAB and 4 analogues has been measured spectrophotometrically in vitro in the liver (9000 × g supernatant) and caecum (caecal contents) of Alderley Park (Wistar-derived) rats fed a standard pelleted diet. Two carcinogenic DAB analogues, 3'-methyl-p-dimethylaminophenylazobenzene (3M) and 6-p-dimethylaminophenylazobenzothiazole (6BT), and two noncarcinogenic analogues, 4-N-pyrrolidinylazobenzene (4N) and 5-p-dimethylaminophenylazoindazole (5I), have been examined. 3M and 4N were reduced by the liver at a rate of about 50% that of DAB, whereas 5I and 6BT were cleaved at a much lower rate (5-20% that of DAB), with all the chemicals being reduced by an oxygen-insensitive enzyme as has previously been reported for DAB. In contrast, DAB, 3M and 6BT were reduced at a similar rate to each other by a fraction containing caecal contents, with 4N and 51 having only 30-50% of their cleavage rate. Anaerobicity was required for maximal activity of the caecal preparation. No marked differences were observed in these relative activities when animals were fed a riboflavin-low diet (2-3 mg/kg) for 2 weeks prior to azoreductase determinations, or when Sprague-Dawley rats were used. Neither the liver nor the caecal azoreductase activity profiles correlated well with the reported carcinogenic potency of the chemicals, and other factors are suggested to be more important in determining this toxicity for the azo chemicals examined.
II.2B.8 EinistiS, P., H. Norppa and K. Falck, Institute of Occupational Health, SF-00290 Helsinki 29 (Finland)
An attempt to activate styrene by erythrocytes in bacterial mutagenieity assays The induction of sister-chromatid exchanges by styrene in human lymphocytes and CHO cells has been shown to depend on the presence of erythrocytes. In bacterial mutagenicity tests styrene has given slightly positive or completely negative results if $9 mix has been used as a metabolic activation system. The ability of erythrocytes to activate styrene in the bacterial assays was investigated by the fluctuation test and liquid incubation assay with Escherichia coli WP2uvrA ( t r p - , detects base-pair substitution