title: 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 beta-naphthoflavone) and rat (induced with Aroclor 1254) were set out at different pre-incubation times with and without exogenous isocitrate dehydrogenase (IC-DH) in the LMA. Such LMA mixtures contain Mn2+, NADP+, DL-isocitrate (IC) and endogenous IC-DH as NADPH-generating machinery. No changes in mono-oxygenase stability and lipid peroxidation (LP) were observed in the presence of exogenous 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 assays. Exogenous IC-DH in the incubation for LMA did not alter the mitotic crossing-over and the mitotic gene conversion of dimethylnitrosamine (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 during incubations for LMA without additional external NADPH-generating enzyme activity.
mesh_terms: D000818:Animals; D002474:Cell-Free System; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D007700:Kinetics; D008054:Lipid Peroxides / Q000378:metabolism; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009152:Mutagenicity Tests / Q000379:methods*; D009249:NADP / Q000378:metabolism*; D051381:Rats
chemical_list: D008054:Lipid Peroxides; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D007521:Isocitrate Dehydrogenase