Testosterone metabolism in rat brain is differentially enhanced by phenytoin-inducible cytochrome P450 isoforms

Abstract

Many cytochrome P450 (P450) isoforms are selectively inducible by xenobiotics, e.g. pharmaceuticals like the anti-epileptic drug phenytoin. Some of these P450 enzymes are involved in the metabolism of gonadal hormones and are of great importance, especially in early brain development. In this study, the hydroxylation of testosterone by rat brain microsomes from control and phenytoin-induced animals was examined by use of high performance liquid chromatography (HPLC) provided with a photodiode array detector (PDA). In control rats, testosterone is converted by cytochrome(s) P450 to 6alpha-hydroxytestosterone (OHT) as the main metabolite and 6beta-OHT as well as androstenedione as minor metabolites. After phenytoin treatment, brain microsomes showed a strong increase of testosterone metabolism to 2alpha-, 6beta-, 16alpha-, 16beta-OHT and androstenedione, whereby 16alpha-OHT was the main degradation product. These metabolites indicated the action of isoforms of the P450 subfamilies CYP2B, CYP2C and CYP3A. Inhibition experiments with antibodies against CYP2B1/2 and with the CYP2B specific inhibitor orphenadrine indicated the occurrence of members of this subfamily which are known to catalyse the oxidation of testosterone to 16alpha-OHT, 16beta-OHT and androstenedione. Western blots revealed the phenytoin-inducible expression of CYP2B1 and the constitutive expression of CYP3A. The latter is involved in the 6beta-hydroxylation of testosterone which was found correspondingly in control microsomes. Distinct CYP2C isoforms involved in the hydroxylation of testosterone in phenytoin-induced microsomes are not yet identified. The highly increased testosterone metabolism by phenytoin-dependent induction of specific cytochrome P450 isoforms in adult rat brain illustrates the potential influence of exogenous substances on internal regulative and metabolic pathways in the brain.