Cumene hydroperoxide-supported microsomal hydroxylations of warfarin—A probe of cytochrome P-450 multiplicity and specificity

Abstract

The hepatic microsomal metabolism of R and S warfarin, supported by NADPH or cumene hydroperoxide, has been investigated to probe the multiplicity and specificity of cytochromes P-450. Microsomes were uninduccd, and phenobarbital (PB)-, 3-methylcholanthrene (MC)- or 3β-hydroxy-20-oxopregn-5-ene-16-α-carbonitrile (PCN)-induced from rat liver. Cumene hydroperoxide supported the formation of all the NADPH-supported warfarin metabolites (4′-, 6-, 7- and benzylic hydroxywarfarin and dehydrowarfarin). except 8-hvdroxywarfarin. Comparisons of the rates of formation of the metabolites supported by NADPH or cumene hydroperoxide (with uninduced and induced microsomes) revealed that cumene hydroperoxide had the following effects: (1) rates of hydroxylation of the phenyl substituent of warfarin (4′-hydroxywarfarin) were increased; (2) rates of metabolism of the aliphatic portion of warfarin (benzylic hydroxywarfarin and dehydrowarfarin) were increased, except with S warfarin and uninduced microsomes; and (3) rates of hydroxylation of the phenyl ring of the coumarin group of warfarin were (a) decreased (7-. 8-hydroxywarfarin) or (b) decreased (6-hydroxywarfarin) with MC-induced microsomes and increased or unchanged with uninduced and PB- or PC'N-induced microsomes. We concluded from these studies that multiple cytochromes P-450 are implicated in the metabolism of warfarin: that the cytochromes P-450 catalyzing the formation of 7- and 8-hydroxywarfarin differ from those catalyzing the other metabolites. except foro-hydroxylation by MC-induced microsomes: that the cytochromes catalyzing 7- and 8- hydroxywarfarin formation differ from one another; that for each metabolite of warfarin, the cytochrome P-450 type predominantly responsible for its formation is the same. irrespective of the mode of induction of the microsomes: and that 6-hydroxylase activity is the exception to the previous point, and is predominantly associated with different cytochromes P-450 in differently induced microsomes. The effects of cumene hydroperoxide have been ascribed to differences in cumene hydroperoxide affinities, differences in cumene hydroperoxide-induced destruction, and differences in cumene hydroperoxidc inhibitions of warfarin binding to different cytochromes P-450. together with differences in the situation of cytochromes P-450 in the microsomal membrane.