Isolation, characterization, and chromosomal mapping of mouse P450 17 alpha-hydroxylase/C17-20 lyase.

Cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P45017 alpha) catalyzes the conversion of C-21 steroids to C-19 steroids in gonads. A full-length mouse cDNA encoding P450 17 alpha was isolated from a mouse Leydig cell library and characterized by restriction mapping and sequencing. The predicted amino acid sequence has 83% homology to rat, 66% homology to human, and 62% homology to bovine P45017 alpha amino acid sequences. The protein is 507 amino acids in length, which is 1 amino acid shorter than the human protein and 2 amino acids shorter than the bovine protein. The structural gene encoding P450 17 alpha (Cyp17) was localized utilizing an interspecific testcross to mouse chromosome 19, distal to Got-1. Another cytochrome P450, P4502c (Cyp2c), also is located at the distal end of chromosome 19. CYP17, CYP2c, and GOT1 have been mapped to human chromosome 10, with CYP2C and GOT1 mapped to the distal region, q24.3 and q25.3, respectively. The data in the present study indicate conserved syntenic loci on mouse chromosome 19 and human chromosome 10 and predict that the structural gene encoding P45017 alpha will be found distal to GOT1 on human chromosome 10.     

Inhibition of 17 alpha-hydroxylase/C17-C20 lyase by bifluranol and its analogues

A simple assay for the measurement of the activities of both 17 alpha-hydroxylase and C17-C20 lyase is described. No extraction procedures are required. The separation of substrate and products is achieved using HPLC which allows the collection of the components of interest and the monitoring of the recovery of various steroids. Using this assay, bifluranol (known to show anti-prostatic activity in vivo) and some analogues were tested for inhibitory activity towards these enzyme activities. Each compound was active, although less potent than ketoconazole, and this activity may contribute towards the in vivo action.     

Novel steroidal vinyl fluorides as inhibitors of steroid C17(20) lyase

20-fluoro-17(20)-pregnenolone derivatives were designed as enol mimics of pregnenolone. All of the targeted, novel fluoroolefins were potent inhibitors of C17(20) lyase.     

Hormonal regulation of cytochrome P450 enzymes, cholesterol side-chain cleavage and 17 alpha-hydroxylase/C17-20 lyase in Leydig cells

Testosterone biosynthesis in Leydig cells is dependent on two cytochrome P450 enzymes, cholesterol side-chain cleavage (P450scc) and 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha]. The expression of these two enzymes is differentially regulated by LH acting via its second messenger, cyclic adenosine 3',5'-monophosphate (cAMP), and by specific steroid hormones. P450scc is constitutively expressed in normal mouse Leydig cells and in MA-10 tumor Leydig cells. Chronic cAMP stimulation increases the steady state levels of P450scc mRNA and de novo P450scc protein synthesis. In contrast, cAMP is obligatory for de novo synthesis of P450(17 alpha) in normal mouse Leydig cells; P450(17 alpha) synthesis ceases in the absence of luteinizing hormone or cAMP. MA-10 tumor Leydig cells do not express P450(17 alpha) even after treatment with cAMP. The amount of P450(17 alpha) in Leydig cells is negatively regulated by testosterone acting by two distinct mechanisms. At low concentrations, testosterone acts via the androgen receptor to repress cAMP-induced synthesis of P450(17 alpha), whereas at high concentrations this steroid increases the rate of degradation of the enzyme by an oxygen-mediated mechanism. Both constitutive and cAMP-induced synthesis of P450scc protein and steady state levels of mRNA are modulated by glucocorticoids. In normal mouse Leydig cells, glucocorticoids repress P450scc synthesis and steady state levels of P450scc mRNA, whereas glucocorticoids stimulate P450scc synthesis and levels of P450scc mRNA in the tumor Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human chorionic gonadotropin treatment prevents depressed 17 alpha-hydroxylase/C17-20 lyase activities and serum testosterone concentrations in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats

It is known that administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes decreased serum testosterone concentrations in the rat. Previous studies in this laboratory have shown that in rats TCDD exposure results in decreased 17 alpha-hydroxylase and C17-20 lyase activities. The decreases in these activities paralleled decreases in testicular microsomal heme and cytochrome P450 contents. As reported herein, neither testicular mitochondrial cytochrome P450 content nor the activity of cholesterol side-chain cleavage was altered in rats exposed to TCDD. Since the production of testosterone in the testis is dependent on LH, it is important to determine the early effects of TCDD on serum LH concentrations in the rat. Male Sprague-Dawley rats were given a single, oral dose of TCDD (50 micrograms/kg). Serum LH concentrations were determined by RIA on Days 1, 2, 3, 5, and 7 following TCDD treatment. Rat serum LH concentrations were decreased to 60% of controls as early as Day 1 and continued to be depressed on Days 2 and 3 at 53% and 59% of control values, respectively. Rat serum LH returned to control values by Day 5 in spite of continued depression of serum testosterone concentrations. The early depression in serum LH levels caused by TCDD may be related to the subsequent androgenic deficiency in the rat. Treatment of rats with hCG was found to be able to prevent the depression of the activities of testicular microsomal 17 alpha-hydroxylase and C17-20 lyase and serum testosterone concentrations caused by TCDD. These data indicate that TCDD decreases serum testosterone by decreasing P450(17 alpha) and C17-20 but not P450sec activities and that hCG treatment prevents the TCDD-induced decrease.     

Immunocytochemical and biochemical studies on the localization and changes of 17 alpha-hydroxylase/C17-C20 lyase activity in immature rat ovary treated with PMSG and hCG

The localization of cytochrome P-450 of 17 alpha-hydroxylase/C17-C20 lyase (P-450(17 alpha, lyase] and the changes of the enzyme activity were studied immunocytochemically and biochemically in the ovaries of immature rats treated with PMSG (pregnant mare serum gonadotropin) and hCG (human chorionic gonadotropin). Immunocytochemically, P-450(17 alpha, lyase) was localized in both the theca interna cells and interstitial gland cells of the ovaries of immature rats treated with PMSG for 48 h. After hCG administration, the immunoreactive cells rapidly decreased in number in the PMSG-pretreated rat ovary. Namely, 6 h after the hCG injection, positive staining for P-450(17 alpha, lyase) was recognized only in a few theca interna cells, while 12 h after the injection to immunostained cells were detected in the ovary. Forty-eight hours after the hGC treatment (96 h after the PMSG injection), most of the theca interna cells and the interstitial gland cells became immunopositive for P-450(17 alpha, lyase) again. The 17 alpha-hydroxylating activity of P-450(17 alpha, lyase) was 0.5, 0.22 and 0.03 nmol/min/mg protein in the ovarian microsomes of PMSG-treated, PMSG + hCG(3 h)-treated and PMSG + hCG(6 h)-treated rats, respectively. Changes of the hydroxylase activities in all the experimental groups are almost parallel to those of P-450 contents in the microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active site proton delivery and the lyase activity of human CYP17A1

Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon–carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17’s physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.     

A normal phase high-performance liquid chromatography system for steroid 17 alpha-hydroxylase/C17-20 lyase (cytochrome P-450 21scc) assays

A normal phase HPLC system has been developed which is applicable to all of the steroid separations involved in the assay of steroid 17 alpha-hydroxylase (EC 1.14.99.9) and C17-20 lyase activities, in both the delta 4 (progesterone) and delta 5 (pregnenolone) pathways. A hexane-tetrahydrofuran (THF) gradient system is used with silica stationary phase and a flow cell radioactivity detector having a high efficiency for tritium. Folch extraction produces uniform extraction of substrates and products from the microsomal incubates, and this uniformity is maintained through HPLC separation and measurement. The hexane-THF mobile phase is convenient for product isolation and substrate purification and should be adaptable to other steroid separations. The system is especially useful for steroid enzyme assays utilizing radiolabeled substrates, since internal standards are not required for measuring recoveries of substrates and products.     

Reasons for reduced activities of 17 alpha-hydroxylase and C17-C20 lyase in spite of increased contents of cytochrome P-450 in mature rat testis fetally irradiated with 60Co

Pregnant rats received whole body irradiation with 2.6 Gy gamma-ray from a 60Co source at Day 20 of gestation. When pups were 4 months old, activities of electron transport system and steroid monooxygenase in tests were assayed. The content of total cytochrome P-450 in the irradiated testes had increased to 170% of that in non-irradiated rats, but NADPH-cytochrome P-450 reductase activity had reduced to 36% of the control. Also, amounts of cytochrome b5 in testicular microsomal fraction were decreased markedly after irradiation, but no significant change of NADH-cytochrome b5 reductase activity was observed in the treated pups. Because both 17 alpha-hydroxylase and C17-C20 lyase activities tended to be decreased by fetal irradiation, testosterone production from progesterone and 17 alpha-hydroxyprogesterone was reduced to about 30% of the control. From these results, it has been suggested that the testicular cytochrome P-450 is radioresistant but steroid monooxygenase activities are reduced after the fetal irradiation. We propose that the discrepancy arises from the marked decrement of NADPH-cytochrome P-450 reductase activity.     

Studies on the activity of 17 beta-hydroxysteroid dehydrogenase in human adipose tissue

The activity of 17 beta-hydroxysteroid dehydrogenase has been investigated in human subcutaneous adipose tissue. Using oestrone as substrate, oestradiol formation was linear with time and the concentration of protein in the tissue homogenate. The optimum pH was 8.0 and the Km for oestrone was 2.5 x 10(-6) M. With NADH, the production of oestradiol was about 30% of that with NADPH. Oestradiol was also a substrate for the enzyme although under the experimental conditions used reduction of oestrone appeared to be favoured in adipose tissue. In the presence of progesterone (31.8 x 10(-6) M) the Km for oestrone was increased fivefold.     

Immunolocalization of cytochrome P450 17alpha-hydroxylase/c17-20 lyase in the ovary of pregnant pigs and fetal gonads

The study was designed to localize P450 17alpha-hydroxylase/c17-20 lyase (P450c17) in the ovaries of pregnant pigs and fetal gonads. Immunoexpression of P450c17 was investigated in the porcine ovaries (follicles and corpora lutea; CL) collected on days 10, 18, 32, 50, 70 and 90 post coitum (p.c.), and fetal gonads (testes and ovaries) on days 50, 70 and 90 p.c. The presence of P450c17 in ovarian follicles was demonstrated on all examined days of pregnancy but was restricted to theca interna cells. In CL, P450c17 was detected on all examined days of pregnancy but only in small luteal cells. In the female porcine fetuses, P450c17 immunostaining was found in oocyte nests and granulosa cells of primary ovarian follicles, while in the male fetuses in fetal Leydig cells. In conclusion, the immunolocalization of P450c17, detected in the ovaries of pregnant pigs and fetal porcine gonads, indicates the potential sites of androgen synthesis. We suggest that androgens may play a role in the maintenance of pregnancy and in the development of prenatal gonads in pigs.     

17 beta-(cyclopropylamino)-androst-5-en-3 beta-ol, a selective mechanism-based inhibitor of cytochrome P450(17 alpha) (steroid 17 alpha-hydroxylase/C17-20 lyase)

A new compound, 17 beta-(cyclopropylamino)-androst-5-en-3 beta-ol, MDL 27,302, has been designed and synthesized as a mechanism-based inhibitor of cytochrome P450(17 alpha). The time-dependent inactivation of human testicular P450(17 alpha) is irreversible by dialysis and requires the cofactor, NADPH; Kiapp. 90 nM (determined on cynomolgous monkey testis enzyme). Inactivation was not affected by the nucleophile DTT, suggesting retention of the inhibitor in the enzyme active site during the inactivation process. Inhibition is specific to the cyclopropylamino compound, since the isopropylamino- and cyclobutylamino-analogs were not inhibitory. Enzymatic specificity of MDL 27,302 for P450(17 alpha) was demonstrated by its failure to inhibit steroid 21-hydroxylase and the cholesterol side chain cleavage enzyme (P450scc). Both the 17 alpha-hydroxylase and C17-20 lyase activities of cytochrome P450(17 alpha) of human testis microsomes were inhibited by MDL 27,302.     

Dehydroepiandrosterone formation is independent of cytochrome P450 17α-hydroxylase/17, 20 lyase activity in the mouse brain

Cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) is a microsomal enzyme reported to have two distinct catalytic activities, 17α-hydroxylase and 17, 20 lyase, that are essential for the biosynthesis of peripheral androgens such as dehydroepiandrosterone (DHEA). Paradoxically, DHEA is present and plays a role in learning and memory in the adult rodent brain, while CYP17 activity and protein are undetectable. To determine if CYP17 is required for DHEA formation and function in the adult rodent brain, we generated CYP17 chimeric mice that had reduced circulating testosterone levels. There were no detectable differences in cognitive spatial learning between CYP17 chimeric and wild-type mice. In addition, while CYP17 mRNA levels were reduced in CYP17 chimeric compared to wild-type mouse brain, the levels of brain DHEA levels were comparable. To determine if adult brain DHEA is formed by an alternative Fe²⁺-dependent pathway, brain microsomes were isolated from wild-type and CYP17 chimeric mice and treated with FeSO₄. Fe²⁺ caused comparable levels of DHEA production by both wild-type and CYP17 chimeric mouse brain microsomes; DHEA production was not reduced by a CYP17 inhibitor. Taken together these in vivo studies suggest that in the adult mouse brain DHEA is formed via a Fe²⁺-sensitive CYP17-independent pathway.     

Acute effect of human chorionic gonadotropin (hCG) on 17 alpha-hydroxylase and C17,20-lyase activity in neonatal or prepubertal rat testis

In contrast to the situation in adults, desensitization of androgen production, secondary to loss of enzyme activity, was not found in testes of neonatal rats exposed to human Chorionic Gonadotropin (hCG). In the present study attention was given to the acute effects of a single injection of hCG upon the activity of testicular 17 alpha-hydroxylase, C17,20-lyase and the concentration of testosterone in the serum of 5, 10 or 28-30 day old rats was investigated. Tritiated H2O from 17 alpha-[3H]progesterone and 14CH3COOH from 21-[14C]progesterone were the products measured to evaluate hydroxylase and lyase activities respectively. Large increases in hCG in the serum were detected within 2 h of a subcutaneous injection. Testosterone, which was highest in 5 day animals, increased quickly in all animals given hCG. In 28-30-day old animals, the concentration of this steroid began to fall 24 h after injection of hCG. 17 alpha-Hydroxylase activity decreased in the testes of all animals given hCG, but only after a brief increase. Activity returned to the starting level, or above, within 24 h in 5 or 10-day old animals. In 28-30-day old rats the activity of both enzymes decreased dramatically to a nadir at 24 h, but increased thereafter. The results indicate that desensitization of testicular androgen synthesizing enzymes occurs in neonatal as well as older testes stimulated with hCG, but the desensitization was very brief in neonatal animals and no desensitization of lyase was found in 5-day old rat testes.