As part of our studies on the role of different forms of cytochrome P-450 in the metabolism of xenobiotics and steroid hormones, we purified 8 forms of rat liver microsomal cytochrome P-450, one of which (designated P-450a) catalyzes the 7 alpha-hydroxylation of testosterone, androstenedione and 5 alpha-dihydrotestosterone. Antibodies against cytochrome P-450a recognize two other forms of rat liver microsomal cytochrome P-450, neither of which correspond to the forms of cytochrome P-450 purified previously (i.e., P-450b, c, d, e, f, g, h, i, j, k, and p). These new forms of cytochrome P-450 are designated cytochromes P-450m and P-450n. We propose to test the hypothesis that cytochromes P-450a, P-450m and P-450n are members of a subfamily of cytochrome P-450 that catalyze the inactivation of steroid hormones. To test this hypothesis, we propose to purify cytochromes P-450a, P-450m and P-450n, and to prepare antibodies to study their function (e.g., their ability to hydroxylate androgens) and their regulation (by such factors as age, sex, hormonal status and xenobiotic treatment). The antibodies will also be used to screen a rat liver genomic library expressed in E. coli. The goal of these recombinant DNA studies is to deduce the amino acid sequence of cytochromes P-450a, P-450m and P-450n, and to use cDNA probes to study their regulation at the mRNA level. In a related study , we propose to purify the form of cytochrome P-450 responsible for inactivating androgens in rat prostate. It has been proposed that 7 alpha-hydroxylation is a major pathway of androgen inactivation in testis, prostate and brain. By immunochemical techniques, we have established that cytochrome P-450a is responsible for the inactivation of androgens in rat testis but, surprisingly, it is not responsible for the inactivation of androgens in rat prostate or brain. Furthermore, it has been hypothesist that the precancerous condition, benign prostatic hyperplasia (BPH), develops in response to an age-dependent increase in steroid 5 alpha-reductase activity (which activates testosterone to 5 alpha-dihydrotestosterone) and an age-dependent decrease in the cytochrome P-450 that inactivates androgens. The proposed studies to purify and characterize the form of cytochrome P-450 responsible for inactivating androgens in the prostate will be an important step toward testing this hypothesis. Studies on the regulation of prostate cytochrome P-450 may identify a mechanism to stimulate the inactivation of androgens, and hence help correct the imbalance of androgen activation/deactivation underlying the cause of BPH. Stimulating the inactivation of androgens in the prostate would compliment current treatments of BPH, which are aimed at reducing circulating testosterone levels, blocking androgen receptors with anti-androgens and/or blocking the activation of testosterone with inhibitors of steroid 5 alpha-reductase.
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