Molecular Pharmacology, Vol 16, 997-1010, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

Specificity of the In Vitro Destruction of Adrenal and Hepatic Microsomal Steroid Hydroxylases by Thiosteroids

¹ Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, Laboratory of Molecular Carcinogenesis, National Cancer Institute, Developmental Pharmacology Branch, National Institute of Child Health and Human Development, and Laboratory of Chemical Physics National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014

Studies are presented to show that thiosteroids such as deacetylspironolactone or 7-thiotestosterone may be used as biochemical probes to correlate the amount of cytochrome P-450 associated with specific steroid hydroxylases. The ability of the thiosteroids to destroy cytochrome P-450 differed markedly among microsomes prepared from liver, adrenal and testicular tissues, and seemed proportional to the magnitude of the spectral interactions of the thiosteroids with cytochrome P-450. At low concentrations (1.0 µM), 7-thiotestosterone caused a NADPH-dependent destruction of hepatic cytochrome P-450 which was associated with a preferential decrease in the activity of testosterone 7-hydroxylase. At 4.5 µM, it also caused a NADPH-dependent decrease in 2, 6, and 16-testosterone hydroxylase, but no NADPH-dependent decrease in benzo(a)pyrene hydroxylation. The destruction of adrenal cytochrome P-450 by deacetylspironolactone in guinea pig microsornes was concurrent with a decrease in the activity of progesterone 17-hydroxylase, but not of progesterone 21-hydroxylase. Studies with radiolabeled deacetylspironolactone suggest that during the loss of cytochrome P-450 by thiosteroids the sulfur atom of the thio group after activation by cytochrome P-450 is eliminated from the steroid moiety and binds covalently to the cytochrome P-450-apoenzymes, thereby resulting in the concomitant loss of the activity and the heme of the cytochrome P-450-dependent steroid hydroxylase.