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microsome test
255
Studies on the weak mutagenic effect of coffee in the Salmonella/microsome assay: cytotoxic effects and antimutagenic effects of rat-liver enzymes Aqueous extracts of instant coffee powder were examined in the Salmonella/microsome assay using strain TA100. A strong bactericidal effect (50% survival with l0 mg coffee per plate) was found if a diluted cell suspension was plated in the presence of the coffee extract. The toxic component seems to act preferentially on dividing cells. The weak mutagenic effects of coffee per se (Aeschbacher et al., Food Cosmet. Toxicol., 18 (1980) 605- 613; Nagao et al., Mutation Res., 68 (1979) 101-106) could be reproduced. In the preincubation assay a dose-related mutagenic effect was found between 4 and 20 mg coffee per plate. This effect is not a secondary consequence of cytotoxic effects (histidine release) as was shown by the addition of ethanol or by treatment with ultrasound. As a result of the addition of active $9 (9000 × g supernatant) or S 105 (105 000 x g supernatant) fractions of rat-liver homogenate, the mutagenic effect disappeared. This inactivation seems to be a process catalyzed by enzymes since after the addition of inactivated $9 (20 min at 60°C) the weak mutagenic effect could not be prevented. These results suggest that the constituents of roasted coffee beans showing weak direct-acting mutagenic activity towards S. typhimurium TA100 will be inactivated by mammalian metabolism (as already mentioned by Aeschbacher and Wiirzner, Toxicol. Lett., 5 (1980) 139-145). The presented results clearly demonstrate that results from standard bacterial mutagenicity tests cannot be used for human risk estimation without additional experiments.
56 Ftille, I., M. Scheutwinkel-Reich, A.M. Preiss and H.J. Stan, Institut for Lebensmittelchemie, Technische Universit~t Berlin, Mtiller-Breslau-Strasse 10, 1000 Berlin 12 (F.R.G.)
Mutagenicity of waste composts in the Salmonella/microsome test Waste compost is commonly used in plant cultivation for soil improvement, especially in viticulture. In order to estimate the potential risk for human health it seemed to be necessary to investigate the possible mutagenicity of these composts. In our study we subjected two kinds of waste composts to the Salmonella/microsome test according to B.N. Ames et al.: (1) composts produced only of domestic refuse and (2) composts containing an addition of sewage sludge. Concentrates were obtained by Soxhlet extraction using a mixture of hexane, acetone and ethanol (120 + 60 + 20). The results demonstrated clear dose-response curves for all investigated waste composts using the tester strains TA98 and TA 1538 indicating frameshift mutagenicity. Microsomal activation led to a decrease of mutagenic potential. However, one compost showed mutagenic activity using all standard tester strains. In this case an
256 increase in mutagenicity was observed using metabolic activation effected by rat-liver homogenate. As a consequence of our results we recommend a standard mutagenicity control of waste composts to be used in agriculture. For this control we suggest as a minimum the Ames Salmonella/microsome test system using the tester strains TA98 and TA 100.
57 Gatehouse, D., Genetic Toxicology Unit, Glaxo Group Research Ltd., Ware, Herts (Great Britain) Procarbazine: a bacterial mutagen in vitro using the microtitre fluctuation test
The immunosuppressant procarbazine is genetically active in a variety of systems and possesses carcinogenic and teratogenic potential (Lee, I.P. et al., Mutation Res., 55 (1978) 1). The compound has given false negative results in in vitro microbial tests in the presence and absence of mammalian metabolic activation (Adler, I.D., Mutation Res., 74 (1980) 77). The microtitre fluctuation test is a sensitive technique for the detection of bacterial mutagens. Using this assay, procarbazine proved to be a bacterial mutagen after metabolic activation in vitro at concentrations as low as 300/~g/ml. The activity was dependent upon higher concentrations of liver $9 than those usually attained in agar assays. Mutagenic activity was only detectable when $9 fractions were derived from Aroclor-treated or phenobarbitone + flnaphthoflavone-treated rat livers. The excision-repair-proficient strains E. coli WP2 and S. typhimurium G46 were considerably more sensitive than repair-deficient strains, E. coli WP2 uvrA and S. typhimurium TA1535. Furthermore, the differential mutagenic effects obtained using S. typhimurium TA1535, TA1530 and G46 indicated that the absence of an rfa mutation was essential for procarbazine mutagenesis. Procarbazine was not mutagenic in standard Ames tests using high $9 concentrations (up to 500/~1 S9/plate). The results suggest the presence of low concentrations of activating enzyme(s) either initially in the $9 fraction or as a result of rapid breakdown during storage or incubation. Inducing agents appear to increase this level. The low concentrations of mutagenic species formed in vitro, may only be detectable using a highly sensitive assay such as the microtitre fluctuation test.
58 Gebhart, E., J. L6sing, R.L. Mueller and B. Windolph, Institut ftir Humangenetik der Universit~it, Bismarckstrasse 10, D-8520 Erlangen (F.R.G.)
Studies on the weak mutagenic effect of coffee in the Salmonella/microsome assay: cytotoxic effects and antimutagenic effects of rat-liver enzymes Aqueous extracts of instant coffee powder were examined in the Salmonella/microsome assay using strain TA100. A strong bactericidal effect (50% survival with l0 mg coffee per plate) was found if a diluted cell suspension was plated in the presence of the coffee extract. The toxic component seems to act preferentially on dividing cells. The weak mutagenic effects of coffee per se (Aeschbacher et al., Food Cosmet. Toxicol., 18 (1980) 605- 613; Nagao et al., Mutation Res., 68 (1979) 101-106) could be reproduced. In the preincubation assay a dose-related mutagenic effect was found between 4 and 20 mg coffee per plate. This effect is not a secondary consequence of cytotoxic effects (histidine release) as was shown by the addition of ethanol or by treatment with ultrasound. As a result of the addition of active $9 (9000 × g supernatant) or S 105 (105 000 x g supernatant) fractions of rat-liver homogenate, the mutagenic effect disappeared. This inactivation seems to be a process catalyzed by enzymes since after the addition of inactivated $9 (20 min at 60°C) the weak mutagenic effect could not be prevented. These results suggest that the constituents of roasted coffee beans showing weak direct-acting mutagenic activity towards S. typhimurium TA100 will be inactivated by mammalian metabolism (as already mentioned by Aeschbacher and Wiirzner, Toxicol. Lett., 5 (1980) 139-145). The presented results clearly demonstrate that results from standard bacterial mutagenicity tests cannot be used for human risk estimation without additional experiments.
56 Ftille, I., M. Scheutwinkel-Reich, A.M. Preiss and H.J. Stan, Institut for Lebensmittelchemie, Technische Universit~t Berlin, Mtiller-Breslau-Strasse 10, 1000 Berlin 12 (F.R.G.)
Mutagenicity of waste composts in the Salmonella/microsome test Waste compost is commonly used in plant cultivation for soil improvement, especially in viticulture. In order to estimate the potential risk for human health it seemed to be necessary to investigate the possible mutagenicity of these composts. In our study we subjected two kinds of waste composts to the Salmonella/microsome test according to B.N. Ames et al.: (1) composts produced only of domestic refuse and (2) composts containing an addition of sewage sludge. Concentrates were obtained by Soxhlet extraction using a mixture of hexane, acetone and ethanol (120 + 60 + 20). The results demonstrated clear dose-response curves for all investigated waste composts using the tester strains TA98 and TA 1538 indicating frameshift mutagenicity. Microsomal activation led to a decrease of mutagenic potential. However, one compost showed mutagenic activity using all standard tester strains. In this case an
256 increase in mutagenicity was observed using metabolic activation effected by rat-liver homogenate. As a consequence of our results we recommend a standard mutagenicity control of waste composts to be used in agriculture. For this control we suggest as a minimum the Ames Salmonella/microsome test system using the tester strains TA98 and TA 100.
57 Gatehouse, D., Genetic Toxicology Unit, Glaxo Group Research Ltd., Ware, Herts (Great Britain) Procarbazine: a bacterial mutagen in vitro using the microtitre fluctuation test
The immunosuppressant procarbazine is genetically active in a variety of systems and possesses carcinogenic and teratogenic potential (Lee, I.P. et al., Mutation Res., 55 (1978) 1). The compound has given false negative results in in vitro microbial tests in the presence and absence of mammalian metabolic activation (Adler, I.D., Mutation Res., 74 (1980) 77). The microtitre fluctuation test is a sensitive technique for the detection of bacterial mutagens. Using this assay, procarbazine proved to be a bacterial mutagen after metabolic activation in vitro at concentrations as low as 300/~g/ml. The activity was dependent upon higher concentrations of liver $9 than those usually attained in agar assays. Mutagenic activity was only detectable when $9 fractions were derived from Aroclor-treated or phenobarbitone + flnaphthoflavone-treated rat livers. The excision-repair-proficient strains E. coli WP2 and S. typhimurium G46 were considerably more sensitive than repair-deficient strains, E. coli WP2 uvrA and S. typhimurium TA1535. Furthermore, the differential mutagenic effects obtained using S. typhimurium TA1535, TA1530 and G46 indicated that the absence of an rfa mutation was essential for procarbazine mutagenesis. Procarbazine was not mutagenic in standard Ames tests using high $9 concentrations (up to 500/~1 S9/plate). The results suggest the presence of low concentrations of activating enzyme(s) either initially in the $9 fraction or as a result of rapid breakdown during storage or incubation. Inducing agents appear to increase this level. The low concentrations of mutagenic species formed in vitro, may only be detectable using a highly sensitive assay such as the microtitre fluctuation test.
58 Gebhart, E., J. L6sing, R.L. Mueller and B. Windolph, Institut ftir Humangenetik der Universit~it, Bismarckstrasse 10, D-8520 Erlangen (F.R.G.)