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.     

Inhibition of human and rat 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities by perfluoroalkylated substances

Perfluoroalkylated substances (PFASs) including perfluorooctane acid (PFOA) and perfluorooctane sulfonate (PFOS) have been classified as persistent organic pollutants and are known to cause reduced testosterone production in human males. The objective of the present study was to compare the potencies of five different PFASs including PFOA, PFOS, potassium perfluorooctane sulfonate (PFOSK), potassium perfluorohexane sulfonate (PFHxSK) and potassium perfluorobutane sulfonate (PFBSK) in the inhibition of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) activities in the human and rat testes. Human and rat microsomal enzymes were exposed to various PFASs. PFOS and PFOSK inhibited rat 3β-HSD activity with IC₅₀ of 1.35 ± 0.05 and 1.77 ± 0.04 μM, respectively, whereas PFHxSK and PFBSK had no effect at concentrations up to 250 μM. All chemicals tested weakly inhibited human 3β-HSD activity with IC₅₀s over 250 μM. On the other hand, PFOS, PFOSK and PFOA inhibited human 17β-HSD3 activity with IC₅₀s of 6.02 ± 1.02, 4.39 ± 0.46 and 127.60 ± 28.52 μM, respectively. The potencies for inhibition of 17β-HSD3 activity were determined to be PFOSK > PFOS > PFOA > PFHxSK = PFBSK for human 17β-HSD3 activity. There appears to be a species-dependent sensitivity to PFAS-mediated inhibition of enzyme activity because the IC₅₀s of PFOS(K) for inhibition of rat 17β-HSD3 activity was greater than 250 μM. In conclusion, the present study shows that PFOS and PFOSK are potent inhibitors of rat 3β-HSD and human 17β-HSD3 activity, and implies that inhibition of steroidogenic enzyme activity may be a contributing factor to the effects that PFASs exert on androgen secretion in the testis.     

Immunocytochemical localization of 17β-hydroxysteroid dehydrogenase in porcine testis

Summary The immunocytochemical localization of 17-hydroxysteroid dehydrogenase (17-HSD) in porcine testes was examined by applying an indirect-immunofluorescence method using an antiporcine testicular 17-HSD antibody. Only the Leydig cells located in the interstitial tissue exhibited a positive immunoreaction for 17-HSD: the germ cells and Sertoli cells located in the seminiferous tubules were entirely negative. These results suggest that, in porcine testis, the biosynthesis of testicular testosterone, the final step of which is the conversion of androstenedione to testosterone, takes place in the Leydig cells.Supported by grants from the Ministry of Education, Science, and Culture, Japan     

Inhibition of 3β- and 17β-hydroxysteroid dehydrogenase activities in rat Leydig cells by perfluorooctane acid

Perfluorooctane acid (PFOA) is classified as a persistent organic pollutant and as an endocrine disruptor. The mechanism by which PFOA causes reduced testosterone production in males is not known. We tested our hypothesis that PFOA interferes with Leydig cell steroidogenic enzymes by measuring its effect on 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) activities in rat testis microsomes and Leydig cells. The IC₅₀s of PFOA and mode of inhibition were assayed. PFOA inhibited microsomal 3β-HSD with an IC₅₀ of 53.2 ± 25.9 μM and 17β-HSD3 with an IC₅₀ 17.7 ± 6.8 μM. PFOA inhibited intact Leydig cell 3β-HSD with an IC₅₀ of 146.1 ± 0.9 μM and 17β-HSD3 with an IC₅₀ of 194.8 ± 1.0 μM. The inhibitions of 3β-HSD and 17β-HSD3 by PFOA were competitive for the substrates. In conclusion, PFOA inhibits 3β-HSD and 17β-HSD3 in rat Leydig cells.     

3α-Hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase in the ovary of young Mongolian gerbils

Summary The ovaries of sexually mature, pregnant mare serum gonadotropin (PMSG) stimulated, 12 week old Mongolian gerbils were investigated morphologically and enzyme histochemically for the appearance of the 3-hydroxysteroid and the 3-hydroxysteroid dehydrogenase during the estrous cycle. Up to ovulation, on day 3 of the estrous cycle, the number of vesicular follicles increases continuously. Primarily atretic follicles can be seen on day 4. On day 5 corpora lutea appear, but they degenerate already by day 6.During the entire estrous cycle, 3-hydroxysteroid dehydrogenase and 3-hydroxysteroid dehydrogenase activity can be found in the theca of tertiary follicles and in the interstitial cells, whereas the theca of secondary follicles and the granulosa of healthy follicles do not exhibit any enzyme activity. The activity decreases from day 1 till day 6. The granulosa of atretic follicles and the cells of corpora lutea show only weak activity. It may be significant that the intensity of enzyme activity in the ovary and the estrogen level in the plasma are differently correlated to the estrous cycle.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Kinetic mechanism of porcine testicular 17ß-hydroxysteroid dehydrogenase

A study on product inhibition of 17ß-hydroxysteroid dehydrogenase from porcine testes was carried out by measuring the initial velocities of NADPH formation using testosterone as the substrate steroid. Type of inhibition by NADPH against NADP⁺ was competitive in both saturated and unsaturated concentrations of testosterone. In the saturated concentration of NADP⁺, activity of the enzyme was not inhibited by NADPH against testosterone. In the unsaturated concentrations of NADP⁺, however, NADPH brought mixed type inhibition against testosterone. The similar modes of inhibition by the product steroid, androstenedione were observed in the saturated and unsaturated concentrations of NADP⁺ and testosterone.The fluorescence of NADPH was increased in the presence of the enzyme, and fluorometric titration indicated that 1 mol of NADPH was bound to 1 mol of the 17ß-hydroxysteroid dehydrogenase. Addition of testosterone to enzyme-NADPH complex reduced the intensity of fluorescence of NADPH, suggesting formation of testosterone-enzyme-NADPH complex as a ternary dead end complex.From the analyses of product inhibition and spectral changes of NADPH, the kinetic mechanism of the enzyme was revealed as rapid equilibrium random system with two dead end complexes which consisted of the two reduced reactants bound to the enzyme and the two oxidized ones bound to it.     

Electron microscopic localization of 3β-hydroxysteroid dehydrogenase and nadh-ferricyanide reductase activities in amphibian interrenal cells

Summary 3-hydroxysteroid dehydrogenase and NADH-ferricyanide reductase activities were localized at the ultrastructural level in amphibian interrenal (adrenocortical) cells previously fixed in a mixture of formaldehyde and glutaraldehyde. Potassium ferricyanide was used as an electron acceptor.Copper ferrocyanide deposits resulting from 3-HSD activity were seen in close association with the external faces of the membranes of the smooth endoplasmic reticulum. Very rare grains of precipitate appeared in mitochondrial cristae. The addition of phenazine methosulfate to the incubation medium had no effect on these localizations.The interrenal cells showed also a strong NADH-ferricyanide reductase activity. The copper ferrocyanide grains were abundant in the mitochondrial cristae and in the hyaloplasm, where they were not preferentially associated with the smooth endoplasmic reticulum.The author is grateful to Miss Marie-Eve Moritz for skillful technical assistance     

Ultracytochemical demonstration and probable localization of 3β-hydroxysteroid dehydrogenase activity with a ferricyanide technique

Summary In order to localize 3-hydroxysteroid dehydrogenase activity on the ultrastructural level, sections of Newt and Rat adrenocortical tissues, fixed in a mixture of glutaraldehyde (0.25%) and formaldehyde (1%), were incubated in a medium containing namely a 3-hydroxysteroid as substrate, NAD, potassium ferricyanide as final electron acceptor, and copper sulfate. In some experiments, phenazine methosulfate (PMS), an electron carrier which can substitute for the activity of the endogenous NADH-diaphorase, is added at various concentrations to the incubation medium.A final precipitate of copper ferrocyanide is observed in the immediate vicinity of the tubules of the smooth endoplasmic reticulum, or in contact with their external faces. The reaction product can also be seen in mitochondrial cristae. The reaction does not take place in incubation media lacking substrate or containing cyanoketone, a specific inhibitor of 3-hydroxysteroid dehydrogenase. The addition of PMS to the incubation medium increases the intensity of the reaction, but does not modify the localization of the precipitate.     

Discovery of potent and orally bioavailable 17β-hydroxysteroid dehydrogenase type 3 inhibitors

We have previously reported the discovery of a new class of potent inhibitors of 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) derived from benzylidene oxazolidinedione and thiazolidinedione scaffolds. In this study, these analogs were designed, synthesized, and evaluated in a human cell-based assay. The detailed structure-activity relationship (SAR) surrounding this pharmacophore were developed, and consequently a number of compounds from this series demonstrated single-digit nanomolar 17β-HDS3 inhibitory activity in vitro. Subsequent optimization work in pursuit of the improvement of oral bioavailability demonstrated in vivo proof-of-concept by prodrug strategy based on phosphate esters for these 17β-HSD3 inhibitors. When a phosphate ester 16 was administered orally at a high dose of 100mg/kg, 16 showed approximately two times more potent testosterone (T)-lowering effect against a positive control in the luteinizing hormone-releasing hormone (LH-RH)-induced T production assay. The T-lowering effect continued at ca 10% level of control over 4h after administration. The nonsteroidal molecules based on this series have the potential to provide unique and effective clinical opportunities for treatment of prostate cancer.     

A quantitative cytochemical study of glucose-6-phosphate dehydrogenase and Δ 5-3β-hydroxysteroid dehydrogenase activity in the Membrana granulosa of the ovulable type of follicle of the rat

Summary During the last four days of follicular development prior to ovulation, the activities of ⁵-3-hydroxysteroid dehydrogenase (3OHD) and glucose-6-phosphate dehydrogenase (G-6-PD) were quantified in cryostat sections of the rat ovary. The product of the enzyme reactions were measured using a scanning and integrating microdensitometer. The enzyme activity was measured in the peripheral region, the antral region and the cumulus of the membrana granulosa (MG) of these follicles on the morning of each of the four days of the estrous cycle. G-6-PD activity was measured in the presence and absence of an intermediate hydrogen acceptor, phenazine methosulphate, to provide a measure of the quantity of Type I and Type II Hydrogen (H) generated: Type I H is considered to be related to hydroxylating reactions such as those of steroids and Type II H to other general biosynthetic activities of cells.In all three regions of the MG of follicles of the ovulable type, 3OHD activity was lowest in estrus and diestrus-1, increased on diestrus-2 and peaked in proestrus. In estrus and diestrus-1, the level of 3OHD activity in the three regions was comparable. However, by diestrus-2, and even more conspicuously in proestrus, enzyme activity was significantly greater in the peripheral region than in the antral region or in the cumulus. During the same period, the level of enzyme activity remained comparable in the last two regions. Throughout the estrous cycle, both Type I and Type II H generation from G-6-PD was greatest in the peripheral region, less in the antral region and least in the cumulus. In the peripheral region, Type I H generation increased progressively after diestrus-1, to reach a maximum in proestrus. In the antral region, Type I H generation increased between diestrus-1 and diestrus-2 and then remained unchanged through proestrus. In the cumulus, Type I H generation remained at levels seen in estrus throughout the remainder of the cycle. Generation of Type II H, in the peripheral region was constant throughout the estrous cycle. In contrast, in the antral region and cumulus, Type II H generation was greater in diestrus-1 and diestrus-2 than on either proestrus or estrus.This work was supported by research grants from the National Institute of Child Health and Human Development (# HD-12684) and (# HD-09542) and from the Rockefeller Foundation     

A quantitative cytochemical method for the demonstration of Β5,3β-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary

Summary A method for the quantitative measurement of ⁵, 3-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary has been described. The method depends on the oxidation of dehydroepiandrosterone (DHEA) and uses nitroblue tetrazolium as the final electron acceptor. Although the dehydrogenase is not a soluble enzyme, polyvinyl alcohol is included in the reaction medium to allow the use of a high substrate concentration whilst employing a low concentration (5%) of dimethyl formamide. The enzyme is equally dependent on NAD⁺ or NADP⁺ for its activity and this activity is significantly enhanced by the presence of cyanide. The NADP⁺ dependence is not abolished by inhibiting nonspecific alkaline phosphomonoesterase. The activity of ⁵, 3-hydroxysteroid dehydrogenase is completely dependent on a functional sulphydryl group. Furthermore, the enzyme activity is totally inhibited in the presence of a steroid substrate analogue at 10^–4 M.     

Steroid-induced regulation of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase activity in wild type and mutants of Streptomyces hydrogenans

The effect of estradiol, hydrocortisone and progesterone on 3,20-and 3,17-hydroxysteroid dehydrogenase (HSD) in mutants of Streptomyces hydrogenans was compared to the steroid response of the wild type. Mutants were defective in arginine biosynthesis and/or aerial mycelial formation and lacked both enzymes or only 17-HSD. Some 17-HSD mutants had lost the ability to be induced by estradiol, by progesterone or by both. Some 20-HSD mutants had lost the ability to be induced by hydrocortisone, by progesterone or by both. Non-inducibility of 17-and 20-HSD by progesterone was not co-ordinate. An additional study of the growth phase-dependent enzyme activity of the wild type after induction with estradiol, hydrocortisone and progesterone was performed.Non-standard abbreviations 17-HSD 3,17-Hydroxysteroid dehydrogenase (EC 1.1.1.51) - 20-HSD 3,20-hydroxysteroid dehydrogenase (EC 1.1.1.53) - AO acridine orange - EBr ethidium bromide - EMS ethyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Identification of oxazolidinediones and thiazolidinediones as potent 17β-hydroxysteroid dehydrogenase type 3 inhibitors

Novel and potent inhibitors of 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) were identified based on oxazolidinedione and thiazolidinedione derivatives, starting from a high-throughput screening hit, 5-(3-bromo-4-hydroxybenzyl)-3-(4-methoxyphenyl)-1,3-thiazol-2-one. 5-(3-Bromo-4-hydroxybenzylidene)-3-(4-methoxyphenyl)-2-thioxo-1,3-thiazolidin-4-one exhibited a promising activity profile and demonstrated significant selectivity over the related 17β-HSD isoenzymes and nuclear receptors.     

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.     

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.     

Histochemical localization of 3β-hydroxysteroid dehydrogenase in marmoset ovaries during pro- and diestrus,with special reference to substrate specificity

Summary We applied qualitative cytochemical procedures to investigate and compare the distribution of 3-hydroxysteroid dehydrogenases (HSDH) in pro- and diestrus ovaries of sexually mature marmosets (Callithrix jacchus) using dehydroepiandrosterone or etiocholane-3-ol-17-one as the substrate. During proestrus dehydroepiandrosterone dehydrogenase (3-5-HSDH) activity was found in the theca of tertiary follicles and in atretic granulosa cells. In granulosa cells at advanced stages of degeneration, HSDH activity was distinctly higher than in thecal cells. The activity of etiocholane-3-ol-17-one dehydrogenase (3-5-HSDH) exhibited a gradient in preovulatory follicles, ranging from high levels in granulosa cells adjacent to the basement membrane to low levels in cells bordering on the antrum and in cumulus oophorus cells. During diestrus 3-5-HSDH activity was only detected in the corpora lutea; the level of 3-5-HSDH activity was unchanged in the theca of tertiary follicles and high in the cells of the corpora lutea. HSDH activity was no longer detectable in atretic granulosa cells using either dehydroepiandrosterone or etiocholane-3-ol-17-one as the substrate. comparison of the distribution of HSDH during proestrus and diestrus revealed that steroidogenesis in marmoset ovaries occurs in follicular elements during diestrus and almost exclusively in the corpora lutea during diestrus. From this phase-dependent localization, it is possible to detemine the stage of the estrous cycle. Furthermore, our findings indicate that the localization of HSDH is dependent on the conformational structure of the substrate used.     

Molecular cloning and expression of 17β-hydroxysteroid dehydrogenase type 2 gene in Hu sheep

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyzes the NADP+-dependent oxidation of the most potent estrogen 17β-estradiol into the weak estrogen estrone, and the conversion of testosterone to androstenedione. It has been reported that 17β-HSD2 was expressed in many tissues in human, rats, however, the full-length sequence of 17β-HSD2 gene and its expression in ewe were still unknown. In this study, we cloned the full-length cDNA sequence and investigated mRNA differential expression in 28 tissues of 12 adult Hu-Sheep which were fed with high- and low- dietary intake. The 1,317 bp full-length cDNA sequence was first cloned. The coding region was 1,167 bp in length, and the monomer was estimated to contain 389 amino acid residues. It shares high AA sequence identity with that of bos Taurus (96.13 %), sus scrofa (77.06 %), canis lupus familiaris (70.44 %), Callithrix jacchus (65.72 %), Nomascus leucogenys (65.46 %), pan troglodytes (65.21 %), human (64.69 %), mus musculus (58.35 %), and a comparatively lower identity to danio rerio (37.85 %). 17β-HSD2 gene was high expressed in gastrointestinal (GI) tract, liver, but weakly expressed in other tissues. No detected expression was examined in lung. 17β-HSD2 gene expression was significantly difference in rumen, omasum, duodenum, cecum, hypophysis after high- and low- dietary intake. Results from the present study suggested that 17β-HSD2 plays a crucial role in almost all tissues protecting against excessive levels of active steroid hormone, and GI tract maybe an important steroid hormone metabolizing organ in Hu-Sheep. This present study is the first to provide the primary foundation for further insight into this ovine gene.     

Expression and NNK reducing activities of carbonyl reductase and 11β-hydroxysteroid dehydrogenase type 1 in human lung

The tobacco specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), which is found in high amounts in tobacco products, is believed to play an important role in lung cancer induction in smokers. NNK requires metabolic activation by cytochrome P450 mediated α-hydroxylation to exhibit its carcinogenic properties. On the other hand, NNK is inactivated by carbonyl reduction to its alcohol-equivalent 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronidation and final excretion into urine or bile. Carbonyl reduction and α-hydroxylation are the predominant pathways in man, and it has been postulated that the extent of these competing pathways determines the individual susceptibility to lung cancer. Moreover, only a minor part of all habitual smokers develop lung cancer, suggesting the existence of susceptibility genes. Microsomal 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD 1) (EC 1.1.1.146) and cytosolic carbonyl reductase (CR) (EC 1.1.1.184) have been shown to be mainly responsible for NNAL formation in liver and lung. In the present study, we performed comparative investigations of human lung tissue samples from several patients with respect to the expression and activity of 11β-HSD 1 and carbonyl reductase. We observed varying levels in 11β-HSD 1 and carbonyl reductase expression in these patients, as revealed by RT-PCR and ELISA. Also, the tissue samples showed a different activity and inhibitor profile for both enzymes. According to our results, variations in the expression and activity of NNK carbonyl reducing enzymes may constitute a major determinant in the overall NNK detoxification capacity and thus may be linked to the great differences observed in the individual susceptibility of tobacco-smoke related lung cancer.