Inhibition of steroid 5α-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450

Abstract

It has been shown previously that liver microsomal steroid 5α-reductase activity increases with age in female but not male rats, which coincides with a female-specific, age-dependent decline in the cytochrome P-450-dependent oxidation of testosterone to 1β-, 2α-, 2β-, 6α-, 6β-, 7α-, 15β-, 16α-, 16β-, and 18-hydroxytestosterone and androstenedione. To determine whether the increase in steroid 5α-reductase activity is responsible for the decrease in testosterone oxidation, we have examined the effects of the steroid 5α-reductase inhibitor, 4-MA (17β- N,N-diethylcarbamoyl-4-methyl-4-aza-5α-androstan-3-one), on the pathways of testosterone oxidation catalyzed by rat liver microsomes. We have also determined which hydroxytestosterone metabolites are substrates for steroid 5α-reductase. At concentrations of 0.1 to 10 μ m, 4-MA completely inhibited steroid 5α-reductase activity without inhibiting the pathways of testosterone oxidation catalyzed by liver microsomes from rats of different age and sex, and from rats induced with phenobarbital or pregnenolone-16α-carbonitrile. 4-MA (10 μ m) had little or no effect on the oxidation of testosterone catalyzed by liver microsomes from mature male rats (which have low steroid 5α-reductase activity). In contrast, the hydroxylated testosterone metabolites formed by liver microsomes from mature female rats (which have high steroid 5α-reductase activity) accumulated to a much greater extent in the presence of 4-MA. Evidence is presented that 4-MA increases the accumulation of hydroxytestosterones by two mechanisms. First, 4-MA inhibited the 5α-reduction of those metabolites (such as 6β-hydroxytestosterone) that were found to be excellent substrates for steroid 5α-reductase. In the absence of 4-MA, these metabolites eventually disappeared from incubations containing liver microsomes from mature female rats. Second, 4-MA inhibited the formation of 5α-dihydrotestosterone, which otherwise competed with testosterone for oxidation by cytochrome P-450. This second mechanism explains why 4-MA increased the accumulation of metabolites (such as 7α-hydroxytestosterone) that were found to be poor substrates for steroid 5α-reductase. Despite its marked effect on the accumulation of hydroxylated testosterone metabolites, 4-MA had no effect on their initial rate of formation by liver microsomes from either male or female rats. From this we conclude that age- and sex-dependent differences in testosterone hydroxylation are largely due to age- and sex-dependent differences in the profile of cytochrome P-450 isozymes in liver microsomes, and are not an artifact due to differences in steroid 5α-reductase activity. The lack of effect of 4-MA on the initial rate of testosterone hydroxylation by liver microsomes from mature female rats also suggests that rat liver microsomal steroid 5α-reductase and cytochrome P-450 do not compete with each other for electrons from NADPH-cytochrome P-450 reductase.