Ovarian C17,20-lyase: changes in intact and hypophysectomized immature rats treated with pregnant mare's serum gonadotropin

The steroid C17,20-lyase activity of immature rat ovarian microsomal (105,000 g pellet), mitochondrial (10,000 g pellet) and combined fractions was measured using progesterone and 17-hydroxyprogesterone as substrates. Steroid 17 alpha-hydroxylase was measured, using progesterone as substrate, in some of the preparations for comparison. With progesterone about 3.5 times more product (acetic acid) was formed than with 17-hydroxyprogesterone as substrate. The half-time for lyase activity following hypophysectomy was 51.8 h, while that for 17 alpha-hydroxylase was 51.3 h. Following an intravenous injection of 20 iu of pregnant mare's serum gonadotropin (PMS) into immature hypophysectomized rats lyase activity decreased for 12 h followed by recovery during the next 12 h with a rapid increase between 24 and 72 h. In contrast, a subcutaneous injection of the same dose produced an initial rise in activity with a decline between 12 and 24 h, followed by a second large increase. In intact animals injection (s.c.) of PMS produced an initial fall in lyase activity followed by an increase beginning 12 h later. A dramatic decrease in activity occurred between 48 and 72 h concomitant with ovulation; hypophysectomy at 48 h not only prevented the decrease, but produced an increase in activity. The changes in ovarian C17,20-lyase activity following administration of PMS mimic those of 17 alpha-hydroxylase.     

No inhibitory effects of gestrinone and medroxyprogesterone acetate on the estrogen production by ovaries of hypophysectomized rats stimulated by gonadotropins.

The in vivo effects of gestrinone (R2323) and medroxyprogesterone acetate (MPA) on the estrogen production by rat ovaries were investigated. Hypophysectomized immature female rats treated with 2.5 or 5 IU of pregnant mare serum gonadotropin (PMS) were daily given vehicle only, gestrinone (0.5 mg/kg body weight) or MPA (10 mg/kg body weight), and the activities of 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, 17, 20-lyase, 17 beta-hydroxysteroid dehydrogenase and aromatase in ovaries of these rats were measured. Gestrinone suppressed the 3 beta-hydroxysteroid dehydrogenase activity and increased activities of 17 alpha-hydroxylase, 17, 20-lyase and aromatase in ovaries stimulated by 5 IU of PMS, while MPA suppressed activities of 17 alpha-hydroxylase and aromatase in these ovaries. On the other hand, the aromatase activity in ovaries stimulated by 2.5 IU of PMS was suppressed by gestrinone and increased by MPA, and neither gestrinone nor MPA affected the production of aromatizable androgens from progesterone by these ovaries. Thus, gestrinone and MPA administrated in vivo showed divergent influences on steroidogenic enzyme activities in ovaries, but they did not affect the serum concentration of estradiol-17 beta. The present results suggest that neither gestrinone nor MPA reduced estrogen production by the rat ovary under the gonadotropin stimulation although they influenced some process of its steroidogenesis.     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of phenyl alkyl imidazole-based compounds as potent inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

The cytochrome P450 enzyme, 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), is a potential target in hormone-dependent cancers. We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of azole-based compounds as inhibitors of the two components of P450(17alpha), i.e., 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results suggest that the imidazole-based compounds are highly potent inhibitors of both components, with N-7-phenyl heptyl imidazole (21) (IC(50)=0.32 microM against 17alpha-OHase and IC(50)=0.10 microM against lyase) and N-8-phenyl octyl imidazole (23) (IC(50)=0.25 microM against 17alpha-OHase and IC(50)=0.21 microM against lyase) being the two most potent compounds within the current study, in comparison to ketoconazole (KTZ) (IC(50)=3.76 microM against 17alpha-OHase and IC(50)=1.66 microM against lyase). Furthermore, consideration of the inhibitory activity against the two components show that the compounds tested are less potent towards the 17alpha-OHase component, a desirable property in the development of novel inhibitors of P450(17alpha). Structure-activity relationship determination of the range of compounds synthesised suggests that logP (log of the partition coefficient) is a key physicochemical factor in determining the overall inhibitory activity. In an effort to determine the viability of these compounds becoming potential drug candidates as well as to show specificity of these compounds, we undertook the biochemical evaluation of the synthesised compounds against two isozymes of 17beta-hydroxysteroid dehydrogenase [namely type 1 (17beta-HSD1) and type 3 (17beta-HSD3)] and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Consideration of the inhibitory activity possessed by the compounds considered within the current study against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 shows that there is no clear structure-activity relationship and that the compounds appear to possess similar inhibitory activity against both 3beta-HSD and 17beta-HSD3 whilst against 17beta-HSD1, the compounds appear to possess poor inhibitory activity at [I]=100 microM. Indeed, two of the most potent inhibitors of P450(17alpha), (compounds 21 and 23), were found to possess relatively good levels of inhibition against the three enzymes-compound 21 was found to possess approximately 32%, approximately 21% and approximately 37% inhibition whilst compound 23 was found to possess approximately 38%, approximately 30% and approximately 28% inhibition against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 respectively. We therefore concluded that the azole-based compounds synthesised within the current study are not suitable for further consideration as potential drug candidates due to their lack of specificity.     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of 4-substituted phenyl alkyl imidazole-based inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

We report the preliminary results of the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of phenyl alkyl imidazole-based compounds as inhibitors of the two components of 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), that is, 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results show that N-3-(4-bromophenyl) propyl imidazole (12) (IC50 = 2.95 microM against 17alpha-OHase and IC50 = 0.33 microM against lyase) is the most potent compound within the current study, in comparison to ketoconazole (KTZ) (IC50 = 3.76 microM against 17alpha-OHase and IC50 = 1.66 microM against lyase). Modelling of these compounds suggests that the length of the alkyl chain enhances the interaction between the inhibitor and the area of the active site corresponding to the C3 area of the steroid backbone, thereby increasing potency.     

4-pregnene-3-one-20 beta-carboxaldehyde: a potent inhibitor of 17 alpha-hydroxylase/C17,20-lyase and of 5 alpha-reductase.

The pregnene derivative, 4-pregnene-3-one-20 beta-carboxaldehyde (22-A) was evaluated as an inhibitor of 17 alpha-hydroxylase/C17,20-lyase in rat testicular microsomes and of 5 alpha-reductase in human prostatic homogenates. The effect of the compound in vivo was studied in adult male rats. The 22-A demonstrated potent and competitive inhibition of 17 alpha-hydroxylase and C17,20-lyase with Ki values 8.48 and 0.41 microM, respectively, significantly below the Km values for these two enzymes (33.75 and 4.55 microM). This compound also showed potent inhibition of 5 alpha-reductase with a Ki value of 15.6 nM (Km for this enzyme is 50 nM). By comparison, ketoconazole, a currently studied 17 alpha-hydroxylase/C17,20-lyase inhibitor for the treatment of prostatic cancer, showed less potent inhibition of 17 alpha-hydroxylase (Ki 39.5 microM) and C17,20-lyase (Ki 3.6 microM) and did not inhibit 5 alpha-reductase. Progesterone which has been reported to inhibit the 17 alpha-hydroxylase/C17,20-lyase, did not significantly reduce the production of testosterone by rat testes in vitro in comparison to controls, while the same concentration of 22-A demonstrated a 42% reduction of testosterone biosynthesis. When the adult male rats were injected s.c. with 22-A at 50 mg/day/kg for a 2 week period, the testosterone concentrations in the rat sera were significantly lower than control values (P less than 0.05), whereas serum corticosterone levels did not change. These results suggest that 22-A is a selective potent inhibitor for 17 alpha-hydroxylase and C17,20-lyase, but is more potent for the C17,20-lyase. The compound also inhibits 5 alpha-reductase, and therefore may reduce biosynthesis of testosterone and dihydrotestosterone effectively. Thus, 22-A may be useful in the treatment of problems associated with the androgen excess and prostatic cancer.     

Synthesis and biochemical evaluation of a range of potent benzyl imidazole-based compounds as potential inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

The cytochrome P-450 enzyme, 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), is a potential target in hormone-dependent cancers. Here, we report the synthesis and biochemical evaluation of a range of benzyl imidazole-based compounds which have been targeted against the two components of this enzyme, that is, 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results from the biochemical testing suggest that the compounds synthesised are good inhibitors, with N-4-iodobenzyl imidazole (5) (IC50=10.06 microM against 17alpha-OHase and IC50=1.58 microM against lyase) showing equipotent activity against lyase compared to the standard compound, ketoconazole (KTZ) (IC50=3.76+/-0.01 microM against 17alpha-OHase and IC50=1.66+/-0.15 microM against lyase). Furthermore, the compounds tested are less potent towards the 17alpha-OHase component, a desirable property in the development of novel inhibitors of P450(17alpha).     

Xenopus laevis CYP17 regulates androgen biosynthesis independent of the cofactor cytochrome b5

The enzyme CYP17 primarily regulates androgen production by mediating four reactions: conversion of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively (17alpha-hydroxylase activity), followed by conversion of the 17-hydroxylated steroids to dehydroepiandrosterone and androstenedione, respectively (17,20-lyase activity). Most mammalian CYP17 isoforms have high 17alpha-hydroxylase relative to 17,20-lyase activities and preferentially mediate one of the two 17,20-lyase reactions. In contrast, Xenopus laevis CYP17 potently regulates all four reactions in the frog ovary. CYP17 isoforms generally rely on the cofactor cytochrome b(5) for the 17,20-lyase reaction, suggesting that the high lyase activity of Xenopus CYP17 might be due to a lesser dependence on b(5). The kinetics of Xenopus CYP17 expressed in yeast microsomes were therefore examined in the absence and presence of Xenopus on human b(5). Xenopus CYP17 mediated both 17,20-lyase reactions in the absence of b(5), confirming that the activity did not require b(5). However, both Xenopus and human b(5) slightly enhanced Xenopus CYP17-mediated lyase activity, indicating that the enzyme was still at least partially responsive to b(5). Surprisingly, only the human b(5) cofactor enhanced human CYP17-mediated lyase activity, implying that the human enzyme had more specific cofactor requirements than Xenopus CYP17. Studies using human/Xenopus chimeric b(5) proteins revealed that human b(5) residues 16-41 were important for the specific regulation of the lyase activity of HuCYP17, possibly serving as an interacting domain with the enzyme. CYP17 may therefore have evolved from a general producer of sex steroids in lower vertebrates to a more tightly regulated producer of both sex steroids and glucocorticoids in mammals.     

Neurosteroid biosynthesis in the quail brain: a review

The brain traditionally has been considered to be a target site of peripheral steroid hormones. In contrast to this classical concept, new findings over the past decade have shown that the brain itself also has the capability of forming steroids de novo, the so-called "neurosteroids". De novo neurosteroidogenesis in the brain from cholesterol is a conserved property of vertebrates. Our studies using the quail, as an excellent animal model, have demonstrated that the avian brain possesses cytochrome P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/c17,20-lyase (P450(17alpha,lyase)), 17beta-HSD, etc., and produces pregnenolone, progesterone, 3beta, 5beta-tetrahydroprogesterone, androstenedione, testosterone and estradiol from cholesterol. However, the biosynthetic pathway of neurosteroids in the avian brain from cholesterol may be still incomplete, because we recently found that the quail brain actively produces 7alpha-hydroxypregnenolone, a previously undescribed avian neurosteroid. This paper summarize the advances made in our understanding of biosynthesis of neurosteroids in the avian brain.     

Inhibitory effect of synthetic progestins, 4-MA and cyanoketone on human placental 3 beta-hydroxysteroid dehydrogenase/5----4-ene-isomerase activity

Human placental 3 beta-hydroxysteroid dehydrogenase/5----4-ene isomerase (3 beta-HSD) purified from human placenta transforms C-21 (pregnenolone and 17 alpha-hydroxy pregnenolone) as well as C-19 (dehydroepiandrosterone and androst-5-ene-3 beta, 17 beta-diol) steroids into the corresponding 3-keto-4-ene-steroids and is thus involved in the biosynthesis of all classes of hormonal steroids. Trilostane, epostane and cyanoketone are potent inhibitors of 3 beta-HSD with Ki values of approximately 50 nM. 4-MA, a well known 5 alpha-reductase inhibitor, is also a potent inhibitor of 3 beta-HSD with a Ki value of 56 nM. Synthetic progestin compounds such as promegestone and RU2323 show relatively strong inhibitory effects with Ki values of 110 and 190 nM, respectively. Cyproterone acetate, a progestin used in the treatment of hirsutism, acne and prostate cancer as well as norgestrel and norethindrone that are widely used as oral contraceptives also inhibit 3 beta-HSD activity at Ki values of 1.5, 1.7 and 2.5 microM, respectively.     

Synthesis,X-ray crystal structures and biological activity of 16-amino-17-substituted-D-homo steroid derivatives

D-Homo derivatives in the androstane and estrane series, 12-19, were synthesized by a fragmentation-cyclization reaction of 16-oximino-17-hydroxy-17-substituted derivatives 3-9, or by cyclization of the corresponding D-seco derivatives 20-26. The structures were confirmed by X-ray analysis of compounds 12 and 16. Preliminary assessment of inhibitory effects of D-homo derivatives from androstane series towards aromatase, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase/C17-20 lyase (P450c17) and 17 beta-HSD indicated much lower inhibitory potential compared to previously tested activity of another type of D-modified steroids, namely D-seco derivatives. Also, assessment of potential antiestrogenic activity of derivatives from estrane series showed absence of such an activity.     

Synthesis, biochemical evaluation of a range of potent 4-substituted phenyl alkyl imidazole-based inhibitors of the enzyme complex 17alpha-Hydroxylase/17,20-Lyase (P45017alpha)

We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of azole-based compounds as inhibitors of the two components of the cytochrome P-450 enzyme 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), i.e. 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results suggest that the compounds synthesised are potent inhibitors, with 7-phenyl heptyl imidazole (11) (IC(50)=320 nM against 17alpha-OHase and IC(50)=100 nM against lyase); 1-[7-(4-fluorophenyl) heptyl] imidazole (14) (IC(50)=170 nM against 17alpha-OHase and IC(50)=57 nM against lyase); 1-[5-(4-bromophenyl) pentyl] imidazole (19) (IC(50)=500 nM against 17alpha-OHase and IC(50)=58 nM against lyase) being the most potent inhibitors within the current study, in comparison to ketoconazole (KTZ) (IC(50)=3.76 microM against 17alpha-OHase and IC(50)=1.66 microM against lyase). Furthermore, consideration of the inhibitory activity against the two components shows that all of the compounds tested are less potent towards the 17alpha-OHase in comparison to the lyase component, a desirable property in the development of novel inhibitors of P450(17alpha). From the modelling of these compounds onto the novel substrate heme complex (SHC) for the overall enzyme complex, the length of the compound, along with its ability to undergo interaction with the active site corresponding to the C(3) area of the steroidal backbone, are suggested to play a key role in determining the overall inhibitory activity.     

The effects of prolactin on rat testicular steroidogenic enzyme activities

To investigate whether hyperprolactinemia directly affects rat testicular steroidogenesis, we examined the effects of prolactin (PRL) on microsomal 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) 17-hydroxylase (17-OH), 17,20-desmolase (17,20-D), 17-ketosteroid reductase (17-KSR) and aromatase enzyme activities. Adult hypophysectomized, gonadotropin-treated Fisher rats were rendered hyperprolactinemic by isografting pituitaries under the kidney capsule. The controls received skeletal muscle. All rats were sacrificed 7 days later and serum PRL was measured in each animal. PRL levels were 198 +/- 14 ng/ml in the hyperprolactinemic rats and 4.3 +/- 0.6 ng/ml in the controls (P less than 0.001). The testes were resected, pooled according to PRL levels, and microsomes were prepared from each pool. The activities of the 3 beta-HSD, 17-OH, 17,20-D, 17-KSR and aromatase were measured using as substrates 14C dehydroepiandrosterone, progesterone, 17-hydroxyprogesterone, androstenedione and testosterone, respectively. Hyperprolactinemia was associated with significant decreases in 3 beta-HSD, 17-OH, 17,20-D, 17-KSR and aromatase activities when compared to controls (P less than 0.005). We conclude that prolactin may have a direct effect on rat testicular steroidogenesis which appears to be independent of changes in gonadotropin secretion.     

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome p450 17alpha-hydroxylase/17,20-lyase and cytochrome p450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.     

Effect of the progestogens, gestodene, 3-keto desogestrel, levonorgestrel, norethisterone and norgestimate on the oxidation of ethinyloestradiol and other substrates by human liver microsomes

A number of different progestogens, levonorgestrel (LNG), norethisterone (NET), gestodene (GSD), desogestrel (DG) and norgestimate (NORG) are used in combination with the oestrogen ethinyloestradiol (EE2) in oral contraceptive steroid preparations. All the progestogens are acetylenic steroids and previous studies have indicated the potential of acetylenic steroids to cause mechanism-based or "suicide" inactivation of cytochrome P-450. We have compared the effects of the different progestogens on EE2 2-hydroxylation (a reaction catalyzed by enzymes from the P-450IIC, P-450IIIA and P-450IIE gene families) and also the oxidative metabolism of other drug substrates (cyclosporin, diazepam, tolbutamide) by human liver microsomes. On coincubation with EE2 as substrate, GSD, 3-keto desogestrel (3-KD, the active metabolite of desogestrel) and LNG produced some concentration-dependent inhibition of EE2 2-hydroxylation (maximum 32% inhibition at 100 microM 3-keto desogestrel). Ki values determined for GSD and 3-KD were 98.5 +/- 12.3 and 93.2 +/- 10.3 microM (mean +/- SD; n = 4), respectively. Preincubation of progestogens in a small volume (50 microliters) incubation for 30 min in the presence of an NADPH-generating system enhanced the inhibitory potential of all the steroids (at 100 microM, inhibition was for GSD 39%, 3-KD 46%, LNG 46%, NET 51% and NORG 43%). Inhibitory effects were therefore comparable and also similar to the macrolide antibiotic troleandomycin. The most marked inhibition seen was of diazepam N-demethylation and hydroxylation by GSD (71 and 57%, respectively) and 3-KD (62 and 50%, respectively). In preincubation studies involving cyclosporin as the substrate, the order of inhibitory potency was GSD greater than 3-KD greater than NET greater than LNG for production of both metabolite M17 and M21. The results of the study indicate that all the progestogens in common use have the propensity to inhibit a number of oxidative pathways but there is little evidence for one progestogen being more markedly inhibitory than others.     

Inhibition of rat testicular 17 alpha-hydroxylase and 17,20-lyase activities by anti-androgens (flutamide, hydroxyflutamide, RU23908, cyproterone acetate) in vitro

Flutamide, hydroxyflutamide, RU23908 and cyproterone acetate (CPA) inhibited rat testicular microsomal 17 alpha-hydroxylase and 17,20-lyase activities in vitro. The Km of [3H] progesterone for 17 alpha-hydroxylase was 45 +/- 0.62 nmol/l (+/- SEM, n = 12) and the Km of [3H] 17 alpha-hydroxyprogesterone for 17,20-lyase was 192 +/- 0.42 nmol/l (+/- SEM, n = 12). The Ki values for 17 alpha-hydroxylase, determined from Lineweaver-Burk plots were 102 +/- 3.2 mumol/l (+/- SEM, n = 6), 363 +/- 3.8 mumol/l (+/- SEM, n = 6), 118 +/- 1.4 mumol/l (+/- SEM, n = 6) and 123 +/- 2.1 mumol/l (+/- SEM, n = 6) for flutamide, hydroxyflutamide, RU23908 and CPA respectively. Flutamide and CPA were mixed-type inhibitors, whereas hydroxyflutamide and RU23908 were competitive inhibitors of 17 alpha-hydroxylase activity. Ki values for 17,20-lyase were 33 +/- 3.1 mumol/l (+/- SEM, n = 6), 112 +/- 3.1 mumol/l (+/- SEM, n = 6), 69 +/- 4.4 mumol/l (+/- SEM, n = 6) and 71 +/- 3.2 mumol/l (+/- SEM, n = 6) for flutamide, hydroxyflutamide, RU23908 and CPA, respectively. Inhibition was found to be competitive in each case. Although the characteristic action of anti-androgens is at the receptor level, these results demonstrate that anti-androgens may also have inhibitory effects on androgen biosynthesis which could prove to be of clinical significance.     

Side-chain cleavage of C21 steroids by testicular microsomal cytochrome P-450 (17 alpha-hydroxylase/lyase): involvement of heme

The two steps in the side-chain cleavage of C21 steroids to give C19 steroids (i.e. 17 alpha-hydroxylation and C17,20 lyase activity) were examined using a highly purified cytochrome P-450 from microsomes of neonatal pig testis to determine the photochemical action spectra for the two reactions. Photochemical action spectra, using either 4-ene (progesterone) or 5-ene (pregnenolone) substrates, showed maximal reversal of inhibition by CO with light of 451 nm. Evidently the heme of cytochrome P-450 is involved in both 17 alpha-hydroxylation and in C17,20-lyase activity as in the case of the side-chain cleavage of cholesterol. Mechanisms proposed to account for enzymatic cleavage of the alpha-ketol side-chain of C21 steroids (C17,20 lyase activity) must be consistent with these findings.     

Characterization of two new enzymatic activities of the rat ventral prostate: 5 alpha-androstane-3 beta, 17 beta-diol 6 alpha-hydroxylase and 5 alpha-androstane-3 beta, 17 beta-diol 7 alpha-hydroxylase.

This study has characterized two new enzymatic hydroxylase activities specific for 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) in the rat ventral prostate: 5 alpha-androstane-3 beta, 17 beta-diol 6 alpha-hydroxylase (6 alpha-hydroxylase) and 5 alpha-androstane-3 beta, 17 beta-diol 7 alpha-hydroxylase (7 alpha-hydroxylase). Both of these irreversible hydroxylase activities require NADPH and are localized in the microsomal fraction of the prostate. The apparent Km for 3 beta-diol is 2.5 microM for both the 6 alpha- and 7 alpha-hydroxylase activities. The apparent Km for NADPH is 7.6 microM for the 6 alpha-hydroxylase and 7.0 microM for the 7 alpha-hydroxylase. The pH optimum for both activities is 7.4. Several steroid inhibitors of these hydroxylase activities in vitro were identified including cholesterol, progesterone, and estradiol. Estradiol was found in vitro to be a noncompetitive inhibitor (Ki = 5 microM). Injection of estradiol into intact male rats, simultaneously receiving exogenous testosterone, also produced a significant lowering of the 6 alpha-plus 7 alpha-hydroxylase activities. Both the 6 alpha- and 7 alpha-hydroxylase were found to be androgen sensitive. Following castration there is a rapid decrease in both activities.     

Inhibition of testicular 17 alpha-hydroxylase and 17,20-lyase but not 3 beta-hydroxysteroid dehydrogenase-isomerase or 17 beta-hydroxysteroid oxidoreductase by ketoconazole and other imidazole drugs

Ketoconazole, an orally active antifungal drug, is known to inhibit testicular androgen production both in vitro and in vivo. The aim of the present study was to examine the effect of ketoconazole and 13 other imidazole drugs on rat testicular microsomal 17 alpha-hydroxylase, 17,20-lyase, 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-I) and 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR). The order of decreasing inhibitory effect (determined from Ki values) on 17 alpha-hydroxylase (substrate [3H]progesterone; Km = 89 +/- 0.65 nmol/l; SEM, n = 8) was bifonazole (Ki = 86 +/- 3.3 nmol/l; SEM, n = 4) greater than ketoconazole (160 +/- 4.92) greater than clotrimazole (170 +/- 5.81) greater than miconazole (599 +/- 7.22) greater than econazole (688 +/- 6.98) greater than tioconazole (901 +/- 1.71) greater than isoconazole (1090 +/- 6.96) and on 17,20-lyase (substrate, [3H]17 alpha-hydroxyprogesterone; Km = 250 +/- 0.75 nmol/l; SEM, n = 8) was bifonazole (56.5 +/- 3.4) greater than clotrimazole (81.5 +/- 3.1) greater than ketoconazole (84 +/- 3.5) greater than miconazole (243 +/- 6.3) greater than econazole (325 +/- 5.1) greater than tioconazole (505 +/- 5.2) greater than isoconazole (610 +/- 6.34). However, these imidazole drugs did not inhibit the 3 beta-HSD-I or 17 beta-HSOR activities. A common structural feature of the imidazole drugs having an inhibitory effect was the presence of one or more aromatic rings on the imidazole side chain. In contrast, the imidazole drugs having the imidazole ring fused to a benezene ring, i.e. benzimidazoles (astemizole, mebendazole, thiabendazole) and those having an aliphatic side chain on the N-1 of the imidazole ring (carbimazole, metronidazole, nimorazole, tinidazole) did not inhibit 17 alpha-hydroxylase, 3 beta-HSD-I or 17 beta-HSOR enzyme activities. However some did inhibit 17,20-lyase activity but only at high concentrations. The results of the present study suggest that some imidazole drugs may be useful in clinical situations requiring the suppression of androgen production, for example in the treatment of hormone-dependent prostatic cancer.     

The C17,20-lyase, 17 alpha-hydroxylase and aromatase activity in rat ovarian granulosa cells stimulated in vivo by gonadotropins

Immature hypophysectomized rats were injected with PMS; some groups received hCG 48h later. The C17,20-lyase activity in the granulosa cells removed from the large preovulatory follicles was estimated by the amount of labelled acetic acid produced from 21 (14C) progesterone or 17-hydroxyprogesterone. 17 alpha-hydroxylase and aromatase activity were measured by the tritium exchange method. Although the granulosa cells contained lyase, it was considerably less than their hydroxylase activity. The remaining tissue, consisting of small follicles and hypertrophied thecal and interstitial tissue, had a great deal more lyase and hydroxylase activity than did the granulosa cells. The results are consistent with the view that granulosa cells can produce estrogen from progesterone and do not require androgen precursors from the theca and/or interstitium.     

5alpha-reduced C21 steroids are substrates for human cytochrome P450c17

The 5alpha-reduction of testosterone in target tissues is a key step in androgen physiology; however, 5alpha-reduced C(19) steroids are sometimes synthesized in testis via a pathway that does not involve testosterone as an intermediate. We studied the metabolism of 5alpha-reduced C(21) steroids by human cytochrome P450c17 (hCYP17), the enzyme responsible for conversion of C(21) steroids to C(19) steroids via its 17alpha-hydroxylase and 17,20-lyase activities. hCYP17 17alpha-hydroxylates 5alpha-pregnan-3,20-dione, but little androstanedione is formed by 17,20-lyase activity. hCYP17 also 17alpha-hydroxylates 5alpha-pregnan-3alpha-ol-20-one and the 5alpha-pregnan-3alpha,17alpha-diol-20-one intermediate is rapidly converted to androsterone by 17,20-lyase activity. Furthermore, 5alpha-pregnan-3alpha,17alpha-diol-20-one is a better substrate for the 17,20-lyase reaction than the preferred substrate 17alpha-hydroxypregnenolone and cytochrome b(5) stimulates androsterone formation only 3-fold. Both 5alpha-pregnan-3alpha-ol-20-one and 5alpha-pregnan-3alpha,17alpha-diol-20-one bind to hCYP17 with higher affinity than does progesterone. We conclude that 5alpha-reduced, 3alpha-hydroxy-C(21) steroids are excellent, high-affinity substrates for hCYP17. The brisk metabolism of 5alpha-pregnan-3alpha,17alpha-diol-20-one to androsterone by CYP17 explains how, when 5alpha-reductases are present, the testis can produce C(19) steroids androsterone and androstanediol from 17alpha-hydroxyprogesterone without the intermediacy of androstenedione and testosterone.