Mechanism for the differential induction of mutation by S9 activated benzo[a]pyrene employing either a glucose-6-phosphate-dependent NADPH-regenerating system or an isocitrate-dependent system.
A significant difference in mutation frequency has been observed in CHO cells exposed to benzo[a]pyrene with alternative activation systems. Each system employed rat-liver S9 homogenate with one using isocitrate dehydrogenase to provide reduced NADP, while the other method uses glucose-6-phosphate dehydrogenase. Total aryl hydrocarbon hydroxylase (AHH) activity was greater for the isocitrate dependent system, however, this yielded a lower level of HGPRT mutants. It was ascertained that this reduced mutation frequency may result from sequestering of B[a]P substrate by crystals in the medium, possibly calcium phosphate, which decreased the effective substrate concentration. This sequestration enhances B[a]P internalization, which would explain the dichotomy between the AHH values and the mutation frequency data. The production of specific B[a]P metabolites was also examined by reverse phase HPLC quantitation of extracts of solutions in which the two activation systems were used. The levels of 7,8 dihydroxybenzo[a]pyrene produced by the glucose-6-phosphate protocol were consistently greater than with isocitrate. This may also be a contributing mechanism for elevating the mutation frequency with this procedure. These results demonstrate several interactions between test compound, cells, and metabolizing system which must be considered with in vitro activation systems.
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001564:Benzo(a)pyrene; D001580:Benzopyrenes / Q000378:metabolism* / Q000494:pharmacology; D001711:Biotransformation; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D019298:Glucose-6-Phosphate; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D005958:Glucosephosphates / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism*; D009152:Mutagenicity Tests; D009153:Mutagens / Q000494:pharmacology*; D009154:Mutation; D009249:NADP / Q000378:metabolism*; D010053:Ovary; D010084:Oxidation-Reduction; D051381:Rats
D001580:Benzopyrenes; D005958:Glucosephosphates; D007523:Isocitrates; D009153:Mutagens; D001564:Benzo(a)pyrene; D009249:NADP; D019298:Glucose-6-Phosphate; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D001189:Aryl Hydrocarbon Hydroxylases