BRAIN CYTOCHROME-P450 AND TESTOSTERONE-METABOLISM BY RAT-BRAIN SUBCELLULAR-FRACTIONS - PRESENCE OF CYTOCHROME-P450-3A IMMUNOREACTIVE PROTEIN IN RAT-BRAIN MITOCHONDRIA

The hydroxylation of testosterone by rat brain subcellular fractions has been studied using an HPLC method with an enhanced resolution for the separation of testosterone and its monohydroxy derivatives. Although the analysis time is longer than that reported for earlier methods, a baseline separation was obtained between all hydroxytestosterones, excepting 6α-hydroxytestosterone and 15β-hydroxytestosterone, which were separated using a second chromatography system. This separation was important as rat brain microsomes metabolized testosterone to 15α-, 6β-, 15β-, 16β-, 2β-, 1β-hydroxytestosterone and androstenedione. Testosterone metabolism was found to be linear with time and protein concentration. The rat brain mitochondrial fraction metabolized testosterone to androstenedione. Small amounts of immunoreactive bands comigrating with purified cytochromes P450j, P450b, and P450p were detected by Western blot analysis in rat brain microsomes, while only an immunoreactive protein related to cytochrome P450p was found in the mitochondrial fractions. Immunoinhibition studies showed that BEA33, a monoclonal antibody to cytochrome P450b and simultaneously recognizing cytochromes P450e and P450a, was able to inhibit the metabolism of testosterone to the 1β-, 15α-, 2β-, and 6α-hydroxylated metabolites, whereas polyclonal anticytochrome P450p did not inhibit the formation of the 6β-hydroxytestosterone by rat brain microsomes. The metabolism of testosterone by rat brain microsomal or mitochondrial fractions was refractory to induction by 3-methylcholanthrene or pregnenolone-16α-carbonitrile. Thus, in the brain multiple isozymes of cytochrome P450 are constitutively expressed in different subcellular fractions, which suggests that brain cytochrome P450 may play an important role in the metabolism of endogenous compounds. The significance and role of cytochrome P450p-related protein in the rat brain mitochondrial fraction are yet to be determined. © 1992.