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 7 alpha-hydroxylase in rat testis.
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 to 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 three pathways of testosterone oxidation. Antibody against cytochrome P-450b strongly inhibited (greater than 80%) androstenedione formation and completely inhibited (greater than 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 (greater than 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 (greater than 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, wh
Sonderfan
A J
AJ
Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City 66103.
Arlotto
M P
MP
Parkinson
A
A
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism* / Q000502:physiology; D000072467:Cytochrome P450 Family 2; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000378:metabolism* / Q000502:physiology; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008861:Microsomes / Q000032:analysis / Q000378:metabolism; D010084:Oxidation-Reduction; D011467:Prostate / Q000166:cytology / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D039181:Steroid 16-alpha-Hydroxylase; D013737:Testis / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D013739:Testosterone / Q000378:metabolism* / Q000502:physiology*
D007527:Isoenzymes; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase