NADPH as rate-limiting factor for microsomal metabolism an alternative and economic NADPH-generating system for microsomal mono-oxygenase in in vitro genotoxicity studies

Abstract

The effect of NADPH supply on enzymatic activity and its stability were investigated with respect to the mono-oxygenase activities of 7-ethoxyresorufin O-deethylase (ERD), dinemorphan N-demethylase (DND), aminopyrine N-demethylase (APD), 7-ethoxycoumarin O-deethylase (ECD) and p-nitroanisole O-demethylase ( p-NAD) under incubation conditions for the liver microsomal assay (LMA). Experiments with S9 liver fractions of mouse (induced with Na-phenobarbital and β-naphthoflavone) and rat (induced with Aroclor 1254) were set out at different pre-incubation time with and without exogenous isocitrate dehydrogenase (IC-DH) in the LMA. Such LMA mixtures Mn 2+, NADP +, DL-isocitrate (IC) and endogeneous IC-DH as NADPH-generating machinery. No changes in mono-exygenase stability and lipid peroxidation (LP) were observed in the presence of exogeneous IC-DH. The metabolizing capability at the considered times was the maximal one, as shown by no stability changes after the direct addition of IC-DH to the enzymatic assay. Exogenous IC-DH in the incubation for LMA did not alter the mitotic crossing-over and the mitotic gene conversion of dimetylnitrosamine (DMNA) and AR2MNFN (a nitroimidazo[2,1- b]thiazole) in the tester D7 strain of Saccharomyces cerevisiae. It was concluded that endogenous IC-DH seems to be sufficient to provide a saturating level of NADPH for mono-oxygenase activities incubations for LMA without additional external NADPH-generating enzyme activity.