Role of brain cytochrome P450 in regulation of the level of anesthetic steroids in the brain

The role of brain cytochrome P450 (P450) in regulating the levels of the potent anesthetic steroid 3α-hydroxy-5α-pregnan-20-one (3α-OH-DHP) has been investigated. By analogy with the elimination of androgen from its target tissues, we present evidence that it is 3β-hydroxy-5α-pregnan-20-one (3β-OH-DHP) and not 3α-OH-DHP that represents the major pathway for the formation of more polar metabolites and thus the elimination of the 5α-reduced metabolites of progesterone from target tissues. No polar metabolites were formed when 3α-OH-DHP was incubated with microsomal fractions prepared from rat brain, but 3β-OH-DHP was hydroxylated at the 6α- and 7α-positions. These 3β-diols were not formed to any detectable extent in the liver or kidney but were formed in prostate, pituitary, brain, and breast. The highest catalytic activity, 512 nmol of products formed/g of tissue/hr, was found in the prostate. The corresponding rates in the pituitary, brain, and breast were 71.9, 28.1, and 6.7 nmol/g/ hr, respectively. These hydroxylations were confirmed to be P450-catalyzed reactions by solubilization of the P450 from prostate, brain, and breast microsomes and reconstitution of the catalytic activity with NADPH-P450 reductase (EC 1.6.2.4) and lipid. Because 5α-androstane-3β,17β-diol (3β-Adiol) has been shown to be a good substrate for prostate and brain P450, competition experiments were performed to determine whether the same form of P450 is involved in the elimination of 3β-Adiol and 3β-OH-DHP in the brain. These two substrates competed with each other for metabolism in microsomal fractions and in reconstitution experiments with P450 extracted from the brain or prostate. To test the hypothesis that the hydroxylation of 30-OH-DHP represents a pathway for regulation of the level of 3α-OH-DHP in the brain, the effect of inhibition of the hydroxylation of 3β-OH-DHP on the duration of 3α-OH-DHP-induced anesthesia was examined. The nonanesthetic steroid 3β-Adiol was used as a competitive inhibitor of the metabolism of 3β-OH-DHP. The duration of anesthesia upon intravenous administration of 3α-OH-DHP was increased by 33% when 3β-Adiol was coadministered. We conclude that, in the central nervous system, P450-catalyzed hydroxylation of 3β-OH-DHP is a degradative pathway that plays an important role in regulation of the levels of the neuroactive steroid 3α-OH-DHP.