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.     

Inhibition of rat testis microsomal 3beta-hydroxysteroid dehydrogenase activity by tributyltin

In this study, we have examined the effects of a range of organotin compounds (mono-, di-, tributyltin, mono-, di-, trioctyltin) on the activities of rat testis microsomal 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17-hydroxylase (17-OHase) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). 17-OHase activity was inhibited by more than 50% compared with the control rate by 59 microM tributyltin (TBT) but other organotin compounds showed no inhibition. 17beta-HSD activity was unaffected by all organotins tested. 3beta-HSD was inhibited by monooctyltin (81 microM) and by TBT at all concentrations tested in a dose-dependent manner, with almost complete loss of activity at TBT concentrations of 12 microM. The mechanism of inhibition of 3beta-HSD was investigated in kinetic analysis with 0-12 microM TBT. Three rat testis microsomal preparations were incubated with dehydroepiandrosterone as the steroid substrate ranging from 1 to 10,000 nM. Tributyltin was primarily a competitive inhibitor of 3beta-HSD activity, causing an increase in the value of the K(m(app)). However, the mechanism was not entirely competitive as while there was an increase in K(m(app)), a decrease in the V(max(app)) was also observed with increasing concentrations of TBT. Slope and intercept replots demonstrated that the K(i)((app)) from slope replots was around 2.7 microM whereas the K(i)((app)) value from intercept replots was around 30 microM. When compared with the K(m(app)) for 3beta-HSD of around 0.42 microM, TBT could be an effective inhibitor of this enzyme.     

Interferon-gamma inhibits steroidogenesis and accumulation of mRNA of the steroidogenic enzymes P450scc and P450c17 in cultured porcine Leydig cells

The inhibitory effects of recombinant porcine interferon-gamma (IFN gamma) on human CG (hCG)-stimulated testosterone production, and on mRNA concentrations of cholesterol side-chain cleavage (P450scc) and 17 alpha-hydroxylase/C17-20lyase (P450c 17) were investigated using porcine primary Leydig cell culture as a model. After preincubation of Leydig cells for 24 h with 1000 pM IFN gamma, hCG-stimulated (10 ng/ml, 2 h) testosterone production was inhibited by 50%, whereas no significant changes were seen in hCG-stimulated cAMP production. Incubation with 10 microM 5-cholestene-3 beta,22(R)-diol or 10 microM 5-cholestene-3 beta,20 alpha-diol together with hCG (10 ng/ml, 2 h) reversed most of the inhibitory effect of IFN gamma, suggesting that IFN gamma inhibits P450scc activity, possibly by inhibiting the substrate (cholesterol) availability for P450scc. Incubation with IFN gamma also decreased basal concentrations of P450scc (45%) and P450c 17 (35%) mRNA, although these changes probably did not contribute to the decreased testosterone production. Long-term treatment with hCG (100 ng/ml, 24 h) increased P450scc mRNA (3- to 4-fold) and P450c 17 mRNA (4- to 5-fold) concentrations. Simultaneous treatment with IFN gamma attenuated these hCG-induced increases in P450scc mRNA (50%) and P450c 17 mRNA (40-100%) concentrations, as well as in testosterone production (77%). This inhibition of testosterone production could only be partly reversed by the hydroxylated cholesterol derivatives. This suggests that in addition to possible suppression of cholesterol availability, decreased P450scc and/or P450c 17 activities (through decreased mRNA concentrations) were also involved in the IFN gamma suppressed steroidogenic capacity of porcine Leydig cells during long-term hCG stimulation.     

Influence of season and low-level oestradiol immunoneutralization on episodic LH and testosterone secretion and testicular steroidogenic enzymes and steroidogenic acute regulatory protein in the adult ram

The regulation of LH-dependent and -independent increases in testosterone secretion by key proteins in the testes of adult rams was investigated. Serial blood samples were collected from groups of four control and passively immunized (oestradiol antiserum for 3 weeks) rams and the animals were gonadectomized in either the non-breeding season (April) or the breeding season (September). LH pulse frequency and basal (interpulse) concentrations were several times greater (P < 0.01) in the breeding season than in the non-breeding season. Neither of these parameters nor LH pulse amplitude were affected by oestradiol immunization. Parameters of testosterone episodic secretion and response to an injection (i.v.) of 15 micrograms NIH-LH-S25 were also greater (P < 0.05) in the breeding season and, with the exception of pulse frequency, in immunized rams versus controls. Substrate utilization established that testosterone biosynthesis was predominantly via the 5-ene pathway. Increases in blood testosterone concentration in the breeding season were associated with a fivefold higher (P < 0.01) activity of cytochrome P450 17alpha-hydroxylase/C-17,20 lyase (P450(17alpha)) and a 65% higher (P < 0.05) relative amount of mRNA for cytochrome P450 cholesterol side-chain cleavage enzyme complex (P450scc) in the testis. Of the steroidogenic enzyme activities examined, only that for 17beta-hydroxysteroid dehydrogenase (17beta-HSD) tended to be increased by oestradiol immunization. Blood concentrations of cholesterol lipoproteins and expression of the testicular low density lipoprotein receptor were not affected by season or immunization. The amount of steroidogenic acute regulatory protein (StAR) mRNA was 65% higher (P < 0.01) in the breeding season and 20% higher (P < 0.01) in immunized rams versus controls. These results indicate that greater LH stimulation may increase testosterone biosynthesis in the breeding season by increasing StAR mRNA (and presumably delivery of cholesterol to P450scc) and the activity of P450(17alpha), and possibly that of P450scc (activity not measured). More moderate increases in StAR mRNA and 17beta-HSD activity may explain, in part, the increases in testosterone secretion with oestradiol immunization.     

Inhibition of human cytochrome P450 aromatase activity by butyltins

Organotin compounds are widely used as antifouling agents and bioaccumulate in the food chain. Tributyltin chloride (TBT) has been shown to induce imposex in female gastropods. On the basis of this observation it has been suggested that TBT acts as an endocrine disrupter inhibiting the conversion of androgens to estrogens mediated by the aromatase cytochrome P450 enzyme. However, to date, the molecular basis of TBT-induced imposex and in particular its putative inhibitory effects on human aromatase cytochrome P450 activity have not been investigated. Therefore, we examined the effects of the organotin compounds tetrabutyltin (TTBT), TBT, dibutyltin dichloride (DBT) and monobutyltin trichloride (MBT) on human placental aromatase activity. TBT was found to be a partial competitive inhibitor of aromatase activity with an IC(50) value of 6.2 microM with 0.1 microM androstenedione as substrate. TBT impaired the affinity of the aromatase to androstenedione but did not affect electron transfer from NADPH to aromatase via inhibiting the NADPH reductase. DBT acted as a partial but less potent inhibitor of human aromatase activity (65% residual activity), whereas TTBT and MBT had no effect. The residual activity of TBT-saturated aromatase was 37%. In contrast, human 3beta-HSD type I activity was only moderately inhibited by TBT (80% residual activity). Moreover, neither TTBT or DBT nor MBT inhibited the 3beta-HSD type I activity. Together, these results suggest that the environmental pollutants TBT and DBT, both present in marine organisms, textile and plastic products, may have specific impacts on the metabolism of sex hormones in humans.     

Effects of corticosterone deficiency and its replacement on Leydig cell steroidogenesis

Clinical and experimental studies have shown the adverse effects of glucocorticoid deficiency/metyrapone treatment on testicular Leydig cell testosterone production. However, molecular mechanisms that underlie the effects of glucocorticoid deficiency on Leydig cell steroidogenesis are not yet determined. Therefore, the present study was designed to assess the mechanism of this phenomenon. Following metyrapone-induced corticosterone deficiency, serum testosterone, and Leydig cell 14C-glucose oxidation were decreased. StAR mRNA and protein levels were significantly increased in Leydig cells of corticosterone-deficient animals. mRNA levels and the specific activities of P(450)scc and 17beta-HSD were decreased by corticosterone deficiency, whereas the activity and mRNA of 3beta-HSD were increased. Simultaneous administration of corticosterone prevented its deficiency-induced changes in Leydig cells. Our results show that metyrapone-induced corticosterone deficiency impairs Leydig cell testosterone production by decreasing the activities of steroidogenic enzymes and their mRNA expression and glucose oxidation.     

Studies on cytochrome P-450-dependent microsomal enzymes of testicular androgen and estrogen biosynthesis in a urodele amphibian, Necturus

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.     

Comparative study on the metabolism of the androgen precursor androstenedione in two gastropod species: in vitro alterations by TBT and TPT

A comparative study was performed to assess the metabolism of the androgen precursor androstenedione (AD) in two gastropod species from the Muricidae family: Bolinus brandaris and Hexaplex trunculus. AD was mainly converted to 5alpha-dihydrotestosterone by microsomal fractions isolated from Bolinus brandaris, whereas it was primarily metabolized to testosterone by Hexaplex trunculus. Sex differences in the metabolism of AD were only detected in Bolinus brandaris and attributed to higher 5alpha-reductase activity in males. Thereafter, the effect of the organotin compounds, tributyltin (TBT) and triphenyltin (TPT), on the metabolism of AD was investigated. A significant interference was only detected in females, and differences between the modes of action of the two compounds were observed: TPT was a strong inhibitor of 5alpha-reductase activity in B. brandaris at a concentration as low as 100 nM whereas only TBT (10 microM) altered the metabolism of AD in H. trunculus by increasing the activity 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Thus, this work shows that the metabolism of the androgen precursor AD strongly differs among gastropod species, both in terms of activity and metabolic profile, and further demonstrates the ability of TBT and TPT to interfere with key enzymatic pathways involved in androgen synthesis.     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of 4-hydroxyphenyl ketones as potent and specific inhibitors of the type 3 of 17beta-hydroxysteroid dehydrogenase (17beta-HSD3)

We report the synthesis and biochemical evaluation of a number of 4-hydroxyphenyl ketones as potential inhibitors of the enzyme 17beta-hydroxysteroid dehydrogenase (17beta-HSD). In particular, we evaluated compounds against the catalysis of the conversion of androstenedione (AD) to testosterone (T) [17beta-HSD type 3 (17beta-HSD3)], furthermore, in an effort to determine the specificity of our compounds, we evaluated the ability of the compounds to inhibit the catalysis of the conversion of estrone (E1) to estradiol (E2) [17beta-HSD type 1 (17beta-HSD1)] as well as the conversion of dehydroepiandrosterone (DHEA) to AD [by 3beta-hydroxysteroid dehydrogenase (3beta-HSD)]. The results of our study suggest that the synthesised compounds are, in general, able to inhibit 17beta-HSD3 whilst being weak inhibitors of 17beta-HSD1. Against 3beta-HSD, we discovered that all of the synthesised compounds were weak inhibitors (all were found to possess less than 50% inhibition at [I]=500 microM). More specifically, we discovered that 1-(4-hydroxy-phenyl)-nonan-1-one (15) was the most potent against 17beta-HSD3 (IC(50)=2.9 microM) whilst possessing poor inhibitory activity against 17beta-HSD1 ( approximately 36% inhibitory activity against this reaction at [I]=100 microM) and less than 10% inhibition for the conversion of DHEA to AD. We have therefore provided good lead compounds in the design and synthesis of novel non-steroidal inhibitors of 17beta-HSD3.     

Tetrahydroisoquinoline alkaloids mimic direct but not receptor-mediated inhibitory effects of estrogens and phytoestrogens on testicular endocrine function. Possible significance for Leydig cell insufficiency in alcohol addiction

Possible effects of various tetrahydroisoquinolines (TIQs) on rat testicular endocrine function were tested in vitro in order to prove whether these compounds, some of which have been claimed to accumulate in alcoholics, may be mediators of the development of Leydig cell insufficiency, a well-known side-effect of ethanol ingestion. TIQ effects on different levels of regulation of testis function were compared in vitro with estrogen effects, since both classes of compounds have structural similarities. Gonadotropin-stimulated testosterone production by testicular Leydig cells was inhibited by tetrahydropapaveroline and isosalsoline, the IC50 values (30 microM) being comparable to those of estradiol (3 microM), 2-hydroxyestradiol (10 microM), and the phytoestrogens, coumestrol (15 microM) and genistein (7 microM); salsolinol (85 microM) and salsoline (240 microM) were less effective, and salsolidine was ineffective. None of these TIQs interacted significantly with testicular estrogen receptor as analyzed by estradiol displacement. However, tetrahydropapaveroline, isosalsoline and salsolinol competitively inhibited (Ki 130-150 microM) substrate binding to cytochrome P450XVII, one key enzyme of androgen biosynthesis, with similar efficiency as the estrogens did (Ki 50-110 microM); salsoline and salsolidine were again much less effective. Since the efficient TIQ concentrations in this system are identical with those reported to generate central-nervous effects, it is concluded that certain TIQs may amplify peripheral inhibitory effects of ethanol on testicular endocrine function by their interaction with at least one enzyme of the androgen biosynthetic pathway.     

Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure

Depriving rats of luteinizing hormone (LH) causes Leydig cells to lose smooth endoplasmic reticulum and diminishes their P450 C17-hydroxylase/C17,20-lyase activity (Wing et al., 1984). LH administration to hypophysectomized rats prevents these changes in Leydig cell structure and function (Ewing and Zirkin, 1983). We adopted a multistep procedure of rat Leydig cell isolation to study the trophic effects of LH on steroidogenesis in the Leydig cell. Our method employs vascular perfusion, enzymatic dissociation, centrifugal elutriation, and Percoll gradient centrifugation. The purified Leydig cell fraction obtained after Percoll density-gradient centrifugation contains 95% well-preserved 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)-staining cells with ultrastructural characteristics of Leydig cells. These Leydig cells produced 248 and 29 ng of testosterone/10(6) Leydig cells when incubated for 3 h with and without a maximally stimulating concentration of ovine LH. Purified Leydig cells obtained from control rats and rats treated with testosterone-estradiol (T-E) implants for 4 days to inhibit LH production were incubated with a saturating concentration (2 microns) of pregnenolone. Leydig cells from control and T-E-implanted rats produced 537 and 200 ng of testosterone/10(6) Leydig cells X 3 h, respectively, suggesting a defect in the steroidogenic reactions converting pregnenolone to testosterone in Leydig cells from T-E-implanted rats. By using rabbit antibodies to the P450 C17-hydroxylase/C17,20-lyase pig microsomal enzyme, immunoblots of one-dimensional sodium dodecyl sulfate polyacrylamide gels of Leydig cell microsomal protein from control and 4- and 12-day T-E implanted rats revealed a continued loss of enzyme as the period of LH withdrawal continues. These results show that Leydig cells from animals deprived of LH had diminished capacity to convert pregnenolone to testosterone and reduced P450 C17-hydroxylase/C17,20-lyase content.     

Resveratrol is a selective human cytochrome P450 1A1 inhibitor.

Resveratrol (trans-3,4',5-trihydroxystilbene) is a phytoalexin compound found in juice and wine produced from dark-skinned grape cultivars and reported to have anti-inflammatory and anticarcinogenic activities. To investigate the mechanism of anticarcinogenic activities of resveratrol, the effects on cytochrome P450 (P450) were determined in human liver microsomes and Escherichia coli membranes coexpressing human P450 1A1 or P450 1A2 with human NADPH-P450 reductase (bicistronic expression system). Resveratrol slightly inhibited ethoxyresorufin O-deethylation (EROD) activity in human liver microsomes with an IC(50) of 1.1 mM. Interestingly, resveratrol exhibited potent inhibition of human P450 1A1 in a dose-dependent manner with IC(50) of 23 microM for EROD and IC(50) of 11 microM for methoxyresorufin O-demethylation (MROD). However, the inhibition of human P450 1A2 by resveratrol was not so strong (IC(50) 1.2 mM for EROD and 580 microM for MROD). Resveratrol showed over 50-fold selectivity for P450 1A1 over P450 1A2. The activities of human NADPH-P450 reductase were not significantly changed by resveratrol. In a human P450 1A1/reductase bicistronic expression system, resveratrol inhibited human P450 1A1 activity in a mixed-type inhibition (competitive-noncompetitive) with a K(i) values of 9 and 89 microM. These results suggest that resveratrol is a selective human P450 1A1 inhibitor, and may be considered for use as a strong cancer chemopreventive agent in humans.     

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.     

Evaluation of 7-hydroxy-flavones as inhibitors of oestrone and oestradiol biosynthesis

A series of 4-aryl substituted 7-hydroxy-flavones were prepared using the three-step Baker-Venkataraman synthesis in good overall yields. The flavones were all evaluated in vitro for inhibitory activity against aromatase (P450AROM, CYP19), using human placental microsomes, and for inhibitory activity against 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD-1) using human placental cytosol. The phenyl, 4-fluoro-phenyl and 4-bromo-phenyl derivatives displayed moderate inhibitory activity against P450AROM (IC50 17.2, 13.5 and 10.1 microM, respectively), none of the flavones, including the standard genistein, displayed any inhibitory activity against 17beta-HSD type 1 at 100 microM concentration.     

Effect of nonylphenol on serum testosterone levels and testicular steroidogenic enzyme activity in neonatal, pubertal, and adult rats.

Previous dose range-finding studies with nonylphenol (NP) administered to rats in a soy- and alfalfa-free diet showed apparent feminization of several endpoints in male rats at doses of 25 ppm and above. One possible mechanism contributing to these effects is a reduction of testosterone at critical developmental periods. The present study was conducted as an adjunct to a multigeneration study and was designed to examine the effect of NP on testosterone production. Male rats in the F1 and F2 generations were exposed through their dams or directly to various dietary doses of NP (0, 25, 200 and 750 ppm) throughout gestation and until sacrifice at either postnatal day 2 (PND2), PND50, or PND140. Male pups in the F3 generation were examined only on PND2. At PND2, serum testosterone levels were significantly decreased in all groups exposed to NP in the F1 generation, but not in the F2 or F3 generations. The activity of 17alpha-hydroxylase/C17, 20 lyase (P450c17) in PND2 testicular homogenates was not affected by NP treatment. In F1 and F2 PND50 and PND140 rats, NP treatment did not affect serum testosterone levels. The absolute dorsolateral prostate weight was increased in the 200 and 750 ppm dose groups only in the F1 PND50 rats, however, no significant effects were observed in other male reproductive organs. NP treatment did not affect P450c17 activity in microsomes prepared from testes of F1 PND50 or PND140 rats. However, P450c17 activity was significantly decreased in testicular microsomes of F(2) PND50 (200 and 750 ppm dose groups) and PND140 (25, 200, and 750 ppm dose groups) rats. A decrease in testicular beta-nicotinamide adenine dinucleotide phosphate (NADPH) P450 reductase was also observed in all PND50 and PND140 NP-exposed rats of the F1 and F2 generations. The ability of NP to directly inhibit P450c17 activity in vitro at concentrations of 1-100 microM was also demonstrated. These results indicate that NP can inhibit the activity of enzymes involved in testosterone synthesis, but suggest minimal effects on testosterone or testosterone-dependent endpoints via this mechanism.     

Effects of flavonoid phytochemicals on cortisol production and on activities of steroidogenic enzymes in human adrenocortical H295R cells

Inhibitory effects of flavonoid phytochemicals, flavones, flavonols and isoflavones on cortisol production were examined in human adrenal H295R cells stimulated with di-buthylyl cAMP. In addition, the inhibitory effects of these chemicals on the activity of P450scc, 3beta-HSD type II (3beta-HSD II), P450c17, P450c21 and P45011beta, steroidogenic enzymes involved in cortisol biosynthesis, were examined in the same cells. Exposure to 12.5 microM of the flavonoids 6-hydroxyflavone, 4'-hydroxyflavone, apigenin, daidzein, genistein and formononetin significantly decreased cortisol production (by 6.3, 69.6, 47.5, 26.6, 13.8 and 11.3%, respectively), and biochanin A significantly decreased cortisol production (by 47.3%) at a concentration of 25 microM without any significant cytotoxic effects or changes in cell number. Daidzin, the 7-glucoside of daidzein, did not alter cortisol production by H295R cells at concentrations over 10 microg/ml (24 microM). Daidzein-induced reduction of cortisol production by H295R cells was not inhibited by the estrogen receptor antagonist ICI 182,780. The flavonoids 6-hydroxyflavone, daidzein, genistein, biochanin A and formononetin strongly and significantly inhibited microsomal 3beta-HSD II activity at concentrations from 1 to 25 microM, and I(50) values were estimated to be 1.3, 2, 1, 0.5 and 2.7 microM, respectively. In addition, these flavonoids significantly inhibited microsomal P450c21 activity at 12.5 and/or 25 microM. In addition, 6-hydroxyflavone inhibited activity of microsomal P450c17 and mitochondrial P45011beta at 12.5 and/or 25 microM. Results of Lineweaver-Burk's plot analysis indicate that daidzein is a competitive inhibitor of the activity of 3beta-HSD II and P450c21. K(m) and V(max) values of 3beta-HSD II for DHEA were estimated to be 6.6 microM and 328pmol/minmg protein, respectively. K(m) and V(max) values of P450c21 for progesterone were estimated to be 2.8 microM and 16pmol/minmg protein, respectively. K(i) values of 3beta-HSD II and P450c21 for daidzein were estimated to be 2.9 and 33.3 microM, respectively.     

Imidazole substituted biphenyls: a new class of highly potent and in vivo active inhibitors of P450 17 as potential therapeutics for treatment of prostate cancer.

3- And 4-imidazol-1-yl-methyl substituted biphenyl compounds (named as meta- and para-substituted compounds) were synthesized bearing additional substituents in 3'-/4'-position as inhibitors of P450 17 (17alpha-hydroxylase-C17,20-lyase). P450 17 is the key enzyme of androgen biosynthesis. Its inhibition is a novel therapeutic strategy for treatment of prostate cancer (PC). Twenty-nine compounds were synthesized by Ar-Mg-Br, Negishi or Suzuki aryl-aryl cross coupling and tested toward human and rat enzyme. Most of the compounds showed moderate to excellent activity against one of the enzymes (0.087 microM < or = IC50 < or = 7.7 microM (ketoconazole: 0.74 microM) for the human enzyme, 0.63 microM < or = IC50 < or = 32 microM (ketoconazole: 67 microM) for the rat enzyme). Interestingly, strong species differences were observed. In addition compounds were tested for inhibition toward P450 arom. The 3-imidazol-1-yl-methyl substituted compounds showed good inhibitory activity of P450 arom, while for the 4-substituted compounds negligible inhibition was found. For the most active group of P450 17 inhibitors, (i.e. the 4-imidazol-1-yl-methyl substituted compounds) a QSAR study was performed for inhibition of the human enzyme leading to the result that a hydrophilic substituent in 3'-/4'-position is very important. The most promising compounds (with respect to activity toward both enzymes) were tested in vivo using SD-rats for reduction of plasma testosterone concentrations 2 and 6 h after single i.p. application. The fluorine substituted compound 8c decreased the testosterone plasma concentration to castration level (after 2 h; 5 mg/kg) showing a biological half live of about 6 h.     

Rapid mechanisms of glucocorticoid signaling in the Leydig cell

Stress-mediated elevations in circulating glucocorticoid levels lead to corresponding rapid declines in testosterone production by Leydig cells in the testis. In previous studies we have established that glucocorticoids act on Leydig cells directly, through the classic glucocorticoid receptor (GR), and that access to the GR is controlled prior to the GR by a metabolizing pathway mediated by the type 1 isoform of 11beta-hydroxysteroid dehydrogenase (11betaHSD1). This enzyme is bidirectional (with both oxidase and reductase activities) and in the rat testis is exclusively localized in Leydig cells where it is abundantly expressed and may catalyze the oxidative inactivation of glucocorticoids. The predominant reductase direction of 11betaHSD1 activity in liver cells is determined by an enzyme, hexose-6-phosphate dehydrogenase (H6PDH), on the luminal side of the smooth endoplasmic reticulum (SER). Generation of the pyridine nucleotide cofactor NADPH by H6PDH stimulates the reductase direction of 11betaHSD1 resulting in increased levels of active glucocorticoids in liver cells. Unlike liver cells, steroidogenic enzymes including 17beta-hydroxysteroid dehydrogenase 3 (17betaHSD3) forms the coupling with 11betaHSD1. Thus the physiological concentrations of androstenedione serve as a substrate for 17betaHSD3 utilizing NADPH to generate NADP+, which drives 11betaHSD1 in Leydig cells primarily as an oxidase; thus eliminating the adverse effects of glucocorticoids on testosterone production. At the same time 11betaHSD1 generates NADPH which promotes testosterone biosynthesis by stimulating 17betaHSD3 in a cooperative cycle. This enzymatic coupling constitutes a rapid mechanism for modulating glucocorticoid control of testosterone biosynthesis. Under stress conditions, glucocorticoids also have rapid actions to suppress cAMP formation thus to lower testosterone production.