Curcumin derivatives inhibit testicular 17β-hydroxysteroid dehydrogenase 3

Non-steroidal compounds that inhibit 17β-hydroxysteroid dehydrogenase isoform 3 (17β-HSD3), an enzyme catalyzing the final step in testosterone biosynthesis in Leydig cells, are under development for male contraceptive or treatment of androgen dependent diseases including prostate cancer. A series of curcumin analogues with more stable chemical structures were compared to curcumin as inhibitors of 17β-HSD3 in rat intact Leydig cells as well as rat and human testis microsomes.     

Effects of phthalates on 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities in human and rat testes

The 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) are involved in the reactions that culminate in androgen biosynthesis in Leydig cells. Human and rat testis microsomes were used to investigate the inhibitory potencies on 3β-HSD and 17β-HSD3 activities of 14 different phthalates with various carbon numbers in the ethanol moiety. The results demonstrated that the half-maximal inhibitory concentrations (IC(50)s) of dipropyl (DPrP), dibutyl (DBP), dipentyl (DPP), bis(2-butoxyethyl) (BBOP) and dicyclohexyl (DCHP) phthalate were 123.0, 24.1, 25.5, 50.3 and 25.5μM for human 3β-HSD activity, and 62.7, 30.3, 33.8, 82.6 and 24.7μM for rat 3β-HSD activity, respectively. However, only BBOP and DCHP potently inhibited human (IC(50)s, 23.3 and 8.2μM) and rat (IC(50)s, 30.24 and 9.1μM) 17β-HSD3 activity. Phthalates with 1-2 or 7-8 carbon atoms in ethanol moieties had no effects on both enzyme activities even at concentrations up to 1mM. The mode of action of DCHP on 3β-HSD activity was competitive with the substrate pregnenolone but noncompetitive with the cofactor NAD+. The mode of action of DCHP on 17β-HSD3 activity was competitive with the substrate androstenedione but noncompetitive with the cofactor NADPH. In summary, our results showed that there are clear structure-activity responses for phthalates in the inhibition of both 3β-HSD and 17β-HSD3 activities. The length of carbon chains in the ethanol moieties of phthalates may determine the potency to inhibit these two enzymes.     

Unusual evolution of 11β- and 17β-hydroxysteroid and retinol dehydrogenases

11β-hydroxysteroid dehydrogenases regulate glucocorticoid concentrations and 17β-hydroxysteroid dehydrogenases regulate estrogen and androgen concentrations in mammals. Phylogenetic analysis of the sequences from two 11β-hydroxysteroid dehydrogenases and four mammalian 17β-hydroxysteroid dehydrogenases indicates unusual evolution in these enzymes. Type 1 11β- and 17β-hydroxysteroid dehydrogenases are on the same branch; Type 2 enzymes cluster on another branch with β-hydroxybutyrate dehydrogenase, 11-cis retinol dehydrogenase and retinol dehydrogenase; Type 3 17β-hydroxysteroid dehydrogenase is on a third branch; while the pig dehydrogenase clusters with a yeast multifunctional enzyme on a fourth branch. Pig 17β-hydroxysteroid dehydrogenase appears to have evolved independently from the other three 17β-hydroxysteroid dehydrogenase dehydrogenases; in which case, the evolution of 17β-hydroxysteroid dehydrogenase activity is an example of functional convergence. The phylogeny also suggests that independent evolution of specificity toward C11 substituents on glucocorticoids and C17 substituents on androgens and estrogens has occurred in Types 1 and 2 11β- and 17β-hydroxysteroid dehydrogenases.     

Steroid-transforming enzymes in fungi

Fungal species are a very important source of many different enzymes, and the ability of fungi to transform steroids has been used for several decades in the production of compounds with a sterane skeleton. Here, we review the characterised and/or purified enzymes for steroid transformations, dividing them into two groups: (i) enzymes of the ergosterol biosynthetic pathway, including data for, e.g. ERG11 (14α-demethylase), ERG6 (C-24 methyltransferase), ERG5 (C-22 desaturase) and ERG4 (C-24 reductase); and (ii) the other steroid-transforming enzymes, including different hydroxylases (7α-, 11α-, 11β-, 14α-hydroxylase), oxidoreductases (5α-reductase, 3β-hydroxysteroid dehydrogenase/isomerase, 17β-hydroxysteroid dehydrogenase, C-1/C-2 dehydrogenase) and C-17-C-20 lyase. The substrate specificities of these enzymes, their cellular localisation, their association with protein super-families, and their potential applications are discussed. Article from a special issue on steroids and microorganisms.     

Contribution of microsomal cytochrome b5 electron transport system coupled with Δ5-3β-hydroxysteroid dehydrogenase to androgen synthesis from progesterone

Cytochromes P-450 and $\text{b}$₅ were observed in the microsomal fraction of interstitial tissue of rat testes. Microsomal cytochrome $\text{b}$₅ was reduced by the NADH coupled with the activities of Δ⁵-3β-hydroxysteroid dehydrogenase with Δ⁵-Δ⁴ isomerase through conversion of pregnenolone to progesterone. Activities of NADPH-supported 17α-hydroxylase and C-17-C-20 lyase which converted progesterone to androstenedione were stimulated by either the presence of NADH or the oxidative reaction by the dehydrogenase upon Δ⁵-3β-hydroxysteroids. Androstenedione production enhanced by the reaction of the dehydrogenase was decreased by addition of the antibody against NADH-cytochrome $\text{b}$₅ reductase which was purified from rat hepatic microsomes, suggesting the active participation of cytochrome $\text{b}$₅ in the androgen synthesis.     

Identification of phospholipids capable of modulating the activities of some enzymes involved in androgen and 16-androstene biosynthesis in the immature pig testis.

Testicular steroidogenic enzymes in the microsomal fraction from immature pigs were investigated for the effects of phospholipids of known structure on androgen and 16-androstene biosynthesis. Untreated (control) microsomes metabolized pregnenolone to 17-hydroxypregnenolone, DHA and small quantities of progesterone, 17-hydroxyprogesterone, androstenedione and testosterone; and to 5,16-androstadien-3 beta-ol (andien-beta) and 4,16-androstadienone (dienone) in the 16-androstene pathway. Phosphatidyl(P)-serine, P-glycerol, P-ethanolamine, P-inositol, P-choline and phosphatidic acid did not significantly alter the 17-hydroxylase/C-17,20 lyase or "andien-beta-synthetase" activities. Thus, the C21 side-chain cleavage reactions appeared not to be dependent upon phospholipids for optimal activity. The conversion of pregnenolone to 4-ene steroids (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) was inhibited by dilinoleoyl-phosphatidyl-choline, but other phospholipids tested were without effect. On the other hand, the conversion of andien-beta to dienone was inhibited by P-serine, P-inositol and P-cholines with short saturated or long polyunsaturated acyl chains. Therefore, the presence of these phospholipids in pregnenolone incubations had different consequences for 3 beta-hydroxysteroid dehydrogenase-isomerase activities. It is concluded that substrate specific 3 beta-HSD-isomerases exist for androgen and 16-androstene biosynthesis and that phospholipids may play an intrinsic role in their catalytic activity.     

1α,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells

The current study presents data indicating that 1α,25-dihydroxyvitamin D₃ affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1α,25-dihydroxyvitamin D₃ suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1α,25-dihydroxyvitamin D₃ on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3β-hydroxysteroid dehydrogenase (3βHSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1α,25-dihydroxyvitamin D₃. Interestingly, the two CYP17A1-mediated activities were influenced reciprocally — the 17α-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1α,25-dihydroxyvitamin D₃-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1α,25-dihydroxyvitamin D₃-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex.     

Protein phosphatase 2A and phosphoprotein SET regulate androgen production by P450c17

Cytochrome P450c17 catalyzes 17 alpha-hydroxylation needed for cortisol synthesis and 17,20 lyase activity needed to produce sex steroids. Serine phosphorylation of P450c17 specifically increases 17,20 lyase activity, but the physiological factors regulating this effect remain unknown. Treating human adrenal NCI-H295A cells with the phosphatase inhibitors okadaic acid, fostriecin, and cantharidin increased 17,20 lyase activity, suggesting involvement of protein phosphatase 2A (PP2A) or 4 (PP4). PP2A but not PP4 inhibited 17,20 lyase activity in microsomes from cultured cells, but neither affected 17 alpha-hydroxylation. Inhibition of 17,20 lyase activity by PP2A was concentration-dependent, could be inhibited by okadaic acid, and was restored by endogenous protein kinases. PP2A but not PP4 coimmunoprecipitated with P450c17, and suppression of PP2A by small interfering RNA increased 17,20 lyase activity. Phosphoprotein SET found in adrenals inhibited PP2A, but not PP4, and fostered 17,20 lyase activity. The identification of PP2A and SET as post-translational regulators of androgen biosynthesis suggests potential additional mechanisms contributing to adrenarche and hyperandrogenic disorders such as polycystic ovary syndrome.     

Location of enzymes of androgen and estrogen biosynthesis in the testis of the ground squirrel (Citellus lateralis)

The intratesticular localization of enzymes of androgen and estrogen biosynthesis was studied in the ground squirrel (Citellus lateralis). In mature animals, interstitium and tubules were isolated by manual dissection. Microsomes were prepared and enzymes assayed by analysis of product formation after incubation with appropriate 3H-labeled substrates. In the immature testis, tubules and interstitium are not readily separable; thus, distribution was inferred after analysis of whole testicular microsomes from control, follicle-stimulating hormone (FSH)-treated, and luteinizing hormone (LH)-treated animals. To verify the cellular composition of tissues and the status of steroidogenic organelles in Leydig and Sertoli cells, samples were also analyzed by light and electron microscopy. In mature squirrels, enzymes of androgen biosynthesis were concentrated in the interstitium; however, levels present in the tubules were sufficient to account for a substantial fraction of whole testicular activity (1/3 to 1/5). By contrast, virtually all of the testicular aromatase was accounted for by that in the seminiferous tubules. The purity of these fractions was checked by light microscopy; they showed little cross-contamination. In whole testicular microsomes of immature squirrels, androgen biosynthetic enzymes had a much lower specific activity than in mature animals; however, the opposite was true for aromatase, its activity being approximately 5-fold higher in prepubertal animals. Luteinizing hormone treatment markedly stimulated hydroxylase and lyase but not aromatase. Luteinizing hormone also induced an increase in Leydig cell size and a dramatic proliferation of smooth endoplasmic reticulum. These changes were correlated with increased serum testosterone. As shown previously in rats, 3 beta-hydroxysteroid dehydrogenase was independent of LH control. Follicle-stimulating hormone had no effect on any of the enzymes studied, but induced some increase of agranular reticulum in Sertoli cells. Results from immature squirrels thus corroborate data from mature animals, showing a predominant interstitial location of androgen biosynthetic enzymes. While we cannot explain the absence of FSH stimulation of aromatase activity, the data do not refute the findings in mature animals showing a predominant tubular location of this enzyme. We conclude that the distribution of steroidogenic enzymes in the testis of squirrels differs in several important respects from rats, although both are members of the order Rodentia.     

A histochemical study of the circumtesticular Leydig cells of a teiid lizard, Cnemidophorus tigris

Several oxidative enzymes in the testis of the teiid lizard Cnemidophorus tigris were studied histochemically. The cells of the circumtesticular sheath (Leydig cell tunic) are functionally equivalent to Leydig cells of the interstitium on the basis of similar histochemical reactions for the five enzyme systems studied. Both groups of cells were positive for 3β-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase, NADH diaphorase, NADPH diaphorase, and glucose-6-phosphate dehydrogenase. These results support the hypothesis that the circumtesticular sheath has endocrine function as indicated by its vascularity and its ability to catalyze histochemical reactions involving steroid biosynthesis.     

Purification and properties of 17 alpha-hydroxylase from microsomes of pig adrenal: a second C21 side-chain cleavage system

We have purified a cytochrome P-450 from microsomes of pig adrenal glands to homogeneity (11.1 n moles heme/mg protein) as demonstrated by electrophoresis on polyacrylamide gels with sodium dodecyl SO4 and by line of identity with an antibody using double diffusion. The enzyme shows both 17 alpha-hydroxylase and C17,20-lyase activities and therefore constitutes a C21 side-chain cleavage system like that previously purified in this laboratory from neonatal pig testis. Antibody to the testicular enzyme cross-reacts (line of identity) with both enzymes. It is concluded that the adrenal enzyme is the same or very similar to the testicular enzyme, that each enzyme possesses two enzymatic activities, and that microsomes provide some regulatory device to limit the lyase activity of the enzyme in vivo. No evidence was found for the usually accepted existence of an adrenal steroid 17 alpha-hydroxylase without lyase activity.     

Distribution, metabolism and excretion of a synthetic androgen 7alpha-methyl-19-nortestosterone, a potential male-contraceptive

A synthetic androgen 7α-Methyl-19-nortestosterone (MENT) has a potential for therapeutic use in ‘androgen replacement therapy’ for hypogonadal men or as a hormonal male-contraceptive in normal men. Its tissue distribution, excretion and metabolic enzyme(s) have not been reported. Therefore, the present study tested the distribution and excretion of MENT in Sprague-Dawley rats castrated 24 h prior to the injection of tritium-labeled MENT (³H-MENT). Rats were euthanized at different time intervals after dosing, and the amount of radioactivity in various tissues/organs was measured following combustion in a Packard oxidizer. The radioactivity (% injected dose) was highest in the duodenal contents in the first 30 min of injection. Specific uptake of the steroid was observed in target tissues such as ventral prostate and seminal vesicles at 6 h, while in other tissues radioactivity equilibrated with blood. Liver and duodenum maintained high radioactivity throughout, as these organs were actively involved in the metabolism and excretion of most drugs. The excretion of ³H-MENT was investigated after subcutaneous injection of ³H-MENT into male rats housed in metabolic cages. Urine and feces were collected at different time intervals (up to 72 h) following injection. Results showed that the radioactivity was excreted via feces and urine in equal amounts by 30 h.Aiming to identify enzyme(s) involved in the MENT metabolism, we performed in vitro metabolism of ³H-MENT using rat and human liver microsomes, cytosol and recombinant cytochrome P₄₅₀ (CYP) isozymes. The metabolites were separated by thin-layer chromatography (TLC). Three putative metabolites (in accordance with the report of Agarwal and Monder [Agarwal AK, Monder C. In vitro metabolism of 7α-methyl-19-nortestosterone by rat liver, prostate, and epididymis. Endocrinology 1988;123:2187-93]), [i] 3-hydroxylated MENT by both rat and human liver cytosol; [ii] 16α-hydroxylated MENT (a polar metabolite) by both rat and human hepatic microsomes; and [iii] 7α-methyl-19-norandrostenedione (a non-polar metabolite) by human hepatic microsomes, were obtained. By employing chemical inhibitors and specific anti-CYP antibodies, ³H-MENT was found to be metabolized specifically by rat CYP 2C11 and 3-hydroxysteroid dehydrogenase (3-HSD) enzymes whereas in humans it was accomplished by CYP 3A4, 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3-HSD enzymes.     

Ontogenesis of Oxidative Reaction of 17β-hydroxysteroid Dehydrogenase and 11β-hydroxysteroid Dehydrogenase in Rat Leydig Cells, a Histochemical Study

The enzyme 17β-hydroxysteroid dehydrogenase is required for the synthesis and 11β-hydroxysteroid dehydrogenase for the regulation of androgens in rat Leydig cells. This histochemical study describes ontogenetic changes in distribution and intensity of these enzymes in Leydig cells from postnatal day (pnd) 1–90. Using NAD or NADP as the cofactor, 17β-hydroxysteroid dehydrogenase (substrate: 5-androstene-3β, 17β-diol) peaks were observed on pnd 16 for fetal Leydig cells and on pnd 19 and 37 for adult Leydig cells. Between pnd 13 and 25 the fetal cells showed a higher intensity for the 17β-enzyme than the adult cells; more fetal Leydig cells were stained with NADP, whereas more adult cells were positive with NAD on pnd 13 and 16. A nearly identical distribution of 11β-hydroxysteroid dehydrogenase (substrate: corticosterone) was observed with NAD or NADP as the cofactor; the reaction was present from pnd 31 onwards, first in a few adult Leydig cells and later in almost all these cells homogeneously. The ontogenetic curves of the two enzymes show an inverse relationship. To conclude: (1) Generally, a stronger reaction for 17β-hydroxysteroid dehydrogenase is shown with NAD as cofactor than with NADP; using NADP, fetal Leydig cells show a stronger staining than adult Leydig cells. (2) The data possibly support the notion of a new isoform of 11β -hydroxysteroid dehydrogenase in addition to types 1 and 2.     

Androgen regulation of progestin biosynthetic enzymes in FSH-treated rat granulosa cells in vitro

The influence of androgens on the FSH modulation of progestin biosynthetic enzymes was studied $\text{in vitro}$. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 3 days in a serum-free medium containing FSH (20 ng/ml) with or without increasing concentrations (10^?9?10^?6 M) of 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone; DHT), 5α-androstane-3α, 17β-diol (3α-diol), or the synthetic androgen 17β-hydroxy-17-methyl-4,9,11-estratrien-3-one (methyltrienolone; R1881). FSH treatment increased progesterone and 20α-hydroxy-4-pregnen-3-one(20α-OH-P) production by 10.2- and 11-fold, respectively. Concurrent androgen treatment augmented FSH-stimulated progesterone and 20α-OH-P production in a dose-related manner (R1881 > 3α-diol > DHT). In the presence of an inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), the FSH-stimulated pregnenolone (3β-hydroxy-5-pregnen-20-one) production (a 20-fold increase) was further enhanced by co-treatment with R1881, 3α-diol or DHT. Furthermore, FSH treatment increased 4.4-fold the activity of 3β-HSD, which converts pregnenolone to progesterone. This stimulatory action of FSH was further augmented by concurrent androgen treatment. In contrast, androgen treatment did not affect FSH-stimulated activity of a progesterone breakdown enzyme, 20α-hydroxysteroid dehydrogenase(20α-HSD). These results demonstrate that the augmenting effect of androgens upon FSH-stimulated progesterone biosynthesis is not due to changes in the conversion of progesterone to 20α-OH-P, but involves an enhancing action upon 3β-HSDΔ⁵, Δ⁴-isomerase complexes and additional enzymes prior to pregnenolone biosynthesis.     

Phospholipases modulate the rat testicular androgen biosynthetic pathway in vitro

The role of membrane phospholipids in testicular androgen biosynthesis was investigated by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Androgen biosynthesis in untreated rat testicular microsomes was examined by monitoring the temporal appearance of pregnenolone metabolites and was found to proceed through the 4-ene route. When phospholipase A2 was included, the 5-ene steroids 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) were formed in greater quantities, and the production of 4-ene steroids was reduced indicating that the conversion of 5-ene steroids to the 4-ene configuration was inhibited by phospholipase A2 treatment. Phospholipase C, in addition to inhibiting this step, also inhibited the conversion of C21 steroids to C19 steroids. When the enzymatic steps were measured individually, phospholipase A2 inhibited 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-Isomerase) with an ED50 of 73 mU/ml but had no effect on the activities of 17-hydroxylase, C-17, 20 lyase, or 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). However, though phospholipase C treatment inhibited 3 beta-HSD-Isomerase, it caused less inhibition (the ED50 value was 149 mU/ml). Furthermore, 17-hydroxylase and C-17, 20 lyase activities were also inhibited by phospholipase C treatment (ED50 values were 410 and 343 mU/ml, respectively), but no effect on 17 beta-HSD was observed. The differences in the apparent phospholipid requirements of the steroidogenic enzymes provides the possibility that the metabolic fate of pregnenolone may be regulated by changes in the phospholipid composition of the microenvironment.     

Ovine steroid 17alpha-hydroxylase cytochrome P450: characteristics of the hydroxylase and lyase activities of the adrenal cortex enzyme

The steroid 17-hydroxylase cytochrome P450 (CYP17) found in mammalian adrenal and gonadal tissues typically exhibits not only steroid 17-hydroxylase activity but also C-17,20-lyase activity. These two reactions, catalyzed by CYP17, allow for the biosynthesis of the glucocorticoids in the adrenal cortex, as a result of the 17-hydroxylase activity, and for the biosynthesis of androgenic C(19) steroids in the adrenal cortex and gonads as a result of the additional lyase activity. A major difference between species with regard to adrenal steroidogenesis resides in the lyase activity of CYP17 toward the hydroxylated intermediates and in the fact that the secretion of C(19) steroids takes place, in some species, exclusively in the gonads. Ovine CYP17 expressed in HEK 293 cells converts progesterone to 17-hydroxyprogesterone and pregnenolone to dehydroepiandrosterone via 17-hydroxypregnenolone. In ovine adrenal microsomes, minimal if any lyase activity was observed toward either progesterone or pregnenolone. Others have demonstrated the involvement of cytochrome b(5) in the augmentation of CYP17 lyase activity. Although the presence of cytochrome b(5) in ovine adrenocortical microsomes was established, ovine adrenal microsomes did not convert pregnenolone or 17-hydroxypregnenolone to dehydroepiandrosterone. Furthermore the addition of purified ovine cytochrome b(5) to ovine adrenal microsomes did not promote lyase activity. We conclude that, in the ovine adrenal cortex, factors other than cytochrome b(5) influence the lyase activity of ovine CYP17.     

Testicular steroidogenic enzymes in the lizard Podarcis sicula during the spermatogenic cycle

Steroidogenic Acute Regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), 5α-Reductase (5α-Red), P450 aromatase are key enzymes involved in steroidogenesis. Recently, we showed the expression and the localization of P450 aromatase in Podarcis sicula testis during the different phases of the reproductive cycle, showing its involvement in the control of steroidogenesis, particularly in 17β-estradiol synthesis. Now, we have investigated the presence and distribution of the other enzymes involved in steroidogenesis, i.e. StAR, 3β-HSD, 17β-HSD and 5α-Red, during three significant periods of the reproductive cycle: summer stasis (July–August), autumnal resumption (November) and reproductive period (May–June). We demonstrated for the first time that all these enzymes are always present in somatic cells (Leydig and Sertoli) and germ cells (spermatogonia, spermatocytes I and II, spermatids and spermatozoa) of Podarcis testis, mainly in spermatids and spermatozoa. The present results strongly suggest that in Podarcis testis both somatic and germ cells could be involved in local sex hormone synthesis and that 5α-Red and P450 could carry out a pivot role.     

Cytochrome P-450(17alpha) in beta-cells of rat pancreas and its local steroidogenesis

We have found cytochrome P-450(17alpha) in the islets of Langerhans of rat pancreas. Its existence coincided with that of insulin and demarcated those of glucagon and somatostatin, demonstrating the localization in beta-cells. The enzyme has not only 17alpha-hydroxylase activity but also lyase one, which is a prerequisite for androgen biosynthesis. The pancreatic microsomes converted progesterone mainly to androstenedione with a minor production of 17alpha-hydroxyprogesterone. Due to a low activity of the built-in lyase, cytochrome P-450(17alpha) requires a sufficient electron-transfer from P-450 reductase or presence of an activator to promote the C-C bond cleavage. In beta-cells, P-450 reductase was abundant and could efficiently transfer electrons to P-450(17alpha). Actually, inhibition with anti-P-450 reductase or limitation of NADPH preferentially reduced the lyase activity. Androstenedione was accumulated when its further metabolism was suppressed. We also found localization of cytochrome P-450scc and 3beta-hydroxysteroid dehydrogenase in beta-cells. These results indicate that the immediate substrate for androgen formation, progesterone, is intracellularly produced and is converted mainly to androstenedione with support by an efficient electron supply from P-450 reductase. The product was supposed to be further metabolized to the reduced derivatives such as testosterone, 5alpha-androstanedione, and dihydrotestosterone, which would act as local steroids in the islets of Langerhans.     

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.