Microsomal cytochrome P450-dependent steroid metabolism in male sheep liver. Quantitative importance of 6β-hydroxylation and evidence for the involvement of a P450 from the IIIA subfamily in the pathway

Abstract

The metabolism of testosterone (TEST), androstenedione (AD) and progesterone (PROG) was assessed in hepatic microsomal fractions from male sheep. Rates of total hydroxylation of each steroid were lower in sheep liver than in microsomes isolated from untreated male rat, guinea pig or human liver. 6β-Hydroxylation was the most important pathway of biotransfromation of each of the three steroids (0.80, 0.89 and 0.43 nmol/min/mg protein for TEST, AD and PROG, respectively). Significant minor metabolites from TEST were the 2β-, 15β- and 15α-alcohols (0.19, 0.22 and 0.17 nmol/min/mg microsomal protein, respectively). Apart from the 6β-hydroxysteroid, only the 21-hydroxy derivative was formed from PROG at a significant rate (0.27 nmol/min/mg protein). The 6β-alcohol was the only metabolite formed from AD at a rate > 0.1 nmol/min/mg protein. Antisera raised in rabbits to several rat hepatic microsomal P450s were assessed for their capacity to modulate sheep microsomal TEST hydroxylation. Anti- P450 IIIA isolated from phenobarbital-induced rat liver effectively inhibited TEST hydroxylation at the 2β-, 6β-, 15α- and 15β-positions (by 31–56% when incubated with microsomes at a ratio of 5 mg IgG/mg protein). IgG raised against rat P450 IIC11 and IIB1 inhibited the formation of some of the minor hydroxysteroid metabolites but did not decrease the rate of TEST 6β-hydroxylation. Western immunoblot analysis confirmed the cross-reactivity of anti-rat P450 IIIA with an antigen in sheep hepatic microsomes; anti-IIC11 and anti-IIB1 exhibited only weak immunoreactivity with proteins in these fractions. Considered together, the present findings indicate that, as is the case in many mammalian species, 6β-hydroxylation is the principal steroid biotransformation pathway of male sheep liver. Evidence from immunoinhibition and Western immunoblot experiments strongly implicate the involvement of a P450 from the IIIA subfamily in ovine steroid 6β-hydroxylation.