Identification of the Cytochrome P-450 Isozymes Responsible for Testosterone Oxidation in Rat Lung, Kidney, and Testis: Evidence that Cytochrome P-450a (P450IIA1) Is the Physiologically Important Testosterone 7α-Hydroxylase in Rat Testis*

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Abstract
Previous studies have shown that several forms of cytochrome P-450 present in rat liver microsomes oxidize testosterone with a high degree of regio- and stereospecificity. The aim of this study was to characterize the pathways of testosterone oxidation catalyzed by rat extrahepatic microsomes. Lung, kidney, testis, prostate, and brain were isolated from 3- and 14-week-old-male Sprague-Dawley rats. Microsomes from lung, kidney, and testis catalyzed distinctly different pathways of testosterone oxidation, whereas microsomes from prostate and brain failed to hydroxylate testosterone directly in a time- and protein-dependent manner. Lung microsomes from immature and mature rats converted testosterone ot 16.alpha.-hydroxytestosterone, 16.beta.-hydroxytestosterone, and androstenedione. Lung microsomes were shown by Western immunoblot to contain cytochrome P-450b (P450IIB1), which has been shown previously to catalyze these pathways of testosterone oxidation. Antibody against cytochrome P-450b strongly inhibited (> 80%) androstenedione formation and completely inhibited (> 95%) the 16.alpha.- and 16.beta.-hydroxylation of testosterone catalyzed by lung microsomes (as did carbon monoxide and antibody against NADPH-cytochrome P-450 reductase). Kidney microsomes from mature male rats converted testosterone to 2.alpha.-hydroxytestosterone, 16.alpha.-hydroxytestosterone, and androstenedione, whereas only the latter pathway was catalyzed by kidney microsomes from immature rats. Kidney microsomes from mature male rats were shown by Western immunoblot to contain cytochrome P-450h (P450IIC11), which has been shown previously to convert testosterone to 2.alpha.-hydroxytestosterone, 16.alpha.-hydroxytestosterone, and androstenedione. Antibody against cytochrome P-450h completely inhibited (> 95%) the 2.alpha.- and 16.alpha.-hydroxylation of testosterone shown previously to convert testosterone to 2.alpha.-hydroxytestosterone, 16.alpha.-hydroxytestosterone, and androstenedione. Antibody against cytochrome P-450h completely inhibited (> 95%) the 2.alpha.- and 16.alpha.-hydroxylation of testosterone by kidney microsomes, but had little effect on androstenedione formation, which is catalyzed by 17.beta.-hydroxysteroid dehydrogenase. Testicular microsomes from mature, but not immature, rats catalyzed the 7.alpha.-hydroxylation of testosterone. Previous studies have shown that this reaction is catalyzed in liver microsomes by cytochrome P-450a (P450IIA1). Testicular microsomes from mature, but not immature, rats were shown by Western immunoblot to contain cytochrome P-450a. Antibody against cytochrome P-450a or NADPH-cytochrome P-450 reductase completely inhibited (> 95%) the 7 .alpha.-hydroxylation of testosterone by testicular microsomes. A 90:10 atmosphere of carbon monoxide and oxygen did not appreciably block the 7 .alpha.-hydroxylation of testosterone by testicular microsomes, which is consistent with the relative insensitivity of cytochrome P-450a to carbon monoxide inhibition. Whereas the levels of cytochrome P-450a declined with age in liver microsomes from male rats, the levels of cytochrome P-450a and the rate of testosterone 7.alpha.-hydroxylation increased with age in testicular microsomes. These results identify cytochrome P-450a as the developmentally regulated enzyme previously proposed to inactivate androgens in rat testis.