Stereochemical biotransformation of warfarin as a probe of the homogeneity and mechanism of microsomal hydroxylases

The hepatic microsomal metabolism of R and S warfarin by normal Wistar and Sprague-Dawley rats was compared with that of phenobarbital (PB) and 3-methylcholanthrene (3-MC)-pretreated animals. In all the microsomal systems examined, R warfarin was metabolized faster than the S enantiomer. Induction of microsomal mixed-function oxidase activity with PB, and especially 3-MC, caused significant alterations in the normal stereochemical pattern of R and S warfarin hydroxylation which were independent of the method of microsomal preparation and the technique employed in the quantitation of hydroxylated products. PB pretreatment of Sprague-Dawley rats resulted in an increase in all hydroxylated products but to differing extents. Similar results were obtained from Wistar rats except for the processes of 4' and benzylic hydroxylation of (S) warfarin. 3-MC pretreatment resulted in the selective induction of 6- and 8-hydroxylation in both species. These results suggest that liver microsomes from normal animals contain at least two major, A and B, and two minor, C and D, mono-oxygenases which differ in their stereoselectivity (as measured by the rates for the formation of two enantiomeric products), regioselectivity (as measured by the ratio of any two isomeric products from the same substrate), and inducibility. In this model, normal animals have hydroxylase activity enzyme A which is not inducible by PB or 3-MC and which is stereoselective for the R enantiomer of warfarin in 7- and benzylic hydroxylation and for the S enantiomer in 4'-hydroxylation. Microsomes from normal and PB-induced animals contain additional hydroxylase activity, enzyme B, which catalyzes both the 6- and 8-hydroxylation of warfarin and which has low stereoselectivity but is regioselective for 6-hydroxylation. Enzyme B may also be responsible for some 4'-hydroxylation. PB-induced animals have additional mono-oxygenase activity, enzyme C, which displays the opposite stereoselectivity compared to enzyme A for benzylic and less stereoselectivity for 7-hydroxylation. 3-MC-induced animals have greatly enhanced levels of 6- and 8-hydroxylase activity, enzyme D, which is stereoselective for the R enantiomer and regioselective for 8-hydroxylation of R warfarin and 6-hydroxylation of S warfarin.