Stability of drug metabolizing enzymes during the incubation conditions of the liver microsomal assay with non-induced and induced mouse liver S-9 fractions

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Abstract
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 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 β -naphthoflavone ( β -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 ∼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 monooxygenase 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 β -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 β -NF + PB mixture, however, showed that induced enzymes were more stable than those obtained by simple induction with either β -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.