Stability of drug metabolizing enzymes during the incubation conditions of the liver microsomal assay with non-induced and induced mouse liver S-9 fractions.
The purpose of this work was to study the relative activities and stabilities of phase-I and phase-II drug metabolizing enzymes in incubation mixtures used in vitro genotoxicity testing in order to optimize the conditions of the assay, increase sensitivity and eliminate false negative results. Cytochrome P-450, NADPH-cytochrome P-450 (cytochrome c) reductase activity and various phase-I and phase-II enzyme activities of the drug-metabolizing system were determined in incubation mixtures used in liver microsomal assays. The behaviour of aminopyrine N-demethylase and p-nitroanisole O-demethylase activities as phase-I markers have been reported previously. Other activities measured were glutathione S-transferase, glutathione S-epoxide transferase and epoxide hydrase, and lipid peroxidation (LP) was determined. The experiments were carried out on liver S9 fractions derived from non-induced mice or mice induced with sodium phenobarbital (PB), and/or beta-naphthoflavone (beta-NF). The phase-II enzymes were much more stable (70-90% residual activity) than phase-I enzyme activities (35-60%) in all conditions tested. The residual cytochrome P-450 was approximately 70% stable and the remaining activity of NADPH-cytochrome c-reductase about 80%, indicating that this latter enzyme does not limit the rate of the monoxygenase system in these conditions. Phase-II enzymes were induced to a smaller extent (about 2 times) than in phase-I enzymes (5-6 times) by beta-NF + PB. NADPH-cytochrome c-reductase behaved as phase-II enzymes in this respect as well as for stability. LP was appreciably higher in non-induced than in induced animals. Treatment with the beta-NF + PB mixture, however, showed that induced enzymes were more stable than those obtained by simple induction with either beta-NF or PB alone. These results lead to the conclusion that prolonged incubation times in mutagenicity assays are unnecessary when considering the relative stabilities of the various phase-I and phase-II enzyme activities in the drug-metabolizing system.
D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D004790:Enzyme Induction; D004851:Epoxide Hydrolases / Q000032:analysis; D005260:Female; D005982:Glutathione Transferase / Q000032:analysis; D008054:Lipid Peroxides / Q000378:metabolism; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D009152:Mutagenicity Tests; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D008054:Lipid Peroxides; D004364:Pharmaceutical Preparations; D019324:beta-Naphthoflavone; D009251:NADPH-Ferrihemoprotein Reductase; D005982:Glutathione Transferase; D004851:Epoxide Hydrolases; D010634:Phenobarbital