| Cytochrome P450 3A enzymes play a dominant role in the metabolism of a wide spectrum of structurally diverse compounds. This study was aimed to identify isoform-specific probes, determine expression levels in rat tissues, and increase our understanding of structure-function relationship of individual rat P450 3A enzymes. P450 3A9, 3A18, and nine mutants of P450 3A18 were co-expressed with NADPH-cytochrome P450 oxidoreductase using baculovirus expression system for functional characterization, immunochemical studies, and structure-function relationship studies, and compared with those of P450 3A1 and 3A2. Individual rat P450 3A enzymes displayed varied regio- and stereo-selectivity toward the metabolism of testosterone, quinine, alprazolam, and buprenorphine. Although no unique catalytic marker for any individual 3A form was identified, the information on metabolite profiles and turnover rates for each individual 3A form can be used to estimate the relative contribution of individual forms in rat tissues. Several isoform-specific antibodies were identified for P450 3A1, 3A2, 3A9 and 3A18, and used to study the expression levels of individual 3A forms in tissues from vehicle- and pregnenolone 16alpha-carbonitrile (PCN)-treated rats. In vehicle-treated rats, liver was the primary site for P450 3A expression, followed by lung, kidney, and small intestine (GI). In brain, the expression of P450 3A proteins was detected in mitochondria but not in the microsomal fractions. Sex dimorphic expression of P450 3A was observed only in livers from vehicle-treated rats. Rats treated with PCN differentially up-regulated the expression of P450 3A enzymes in liver and GI in a gender- and isoform-dependent manner while no significant effect was found on the expression of P450 3A enzymes in lung, kidney, brain, and olfactory bulb. The major isoform induced by PCN was P450 3A1 and P450 3A9 in rat liver and GI, respectively, and the inducibility of these isoforms was greater in females than in males in both tissues. Testosterone catalyzed by P450 3A18 yielded a metabolite, 16alpha-hydroxytestosterone, not detected in the metabolism catalyzed by the other three isoforms. Using site-directed mutagenesis and homology modeling, we demonstrated that residues 111, 119, 205, 210, 305, and 374 in P450 3A18 were important in producing 16alpha-hydroxytestosterone. |
