7-ethenyloxycoumarin as a new substrate for fluorophotometric assay of hepatic microsomal epoxidizing activities.

7-Ethenyloxycoumarin (7-vinyloxycoumarin, VOC) was metabolized by rat liver microsomes in the presence of a reduced nicotinamide adenine dinucleotide phosphate-generating system to 7-hydroxycoumarin (HOC) and glycolaldehyde via the unstable epoxide, 7-(epoxyethoxy)coumarin, as an obligatory intermediate which had a half life of 5.4 min in 0.1 M phosphate buffer, pH 7.4, at 37 degrees C. The epoxide of VOC accumulated in the microsomal incubation mixture in the presence of the epoxide hydrolase inhibitor, 3,3,3-trichloropropene 1,2-oxide, was isolated and identified. HOC and glycolaldehyde were auto-decomposition products of the putative highly unstable intermediate, 7-(1',2'-dihydroxyethoxy)coumarin, mostly formed by microsomal epoxide hydrolase from the epoxide. Direct fluorophotometry of HOC made it possible to determine epoxidizing activities of very small quantities of the microsomes from untreated rat liver (greater than or equal to 5 micrograms protein). VOC was epoxidizied by rat liver microsomal cytochrome P-450, inducible by 3-methylcholanthrene (3-MC) and phenobarbital (PB), and the microsomal epoxidation reactions were inhibited by IgG preparations raised against the major cytochrome P-450 components isolated from 3-MC- and PB-pretreated rat liver microsomes. In the untreated, 3-MC- and PB-pretreated rat liver microsomes, at least two monooxygenase components with different affinity were strongly suggested by a kinetic study, carried out using the antibodies, to be involved in the epoxidation of VOC.