20α-hydroxysteroid Dehydrogenase Activity Research Articles

Effects of apoptosis by 20α-hydroxysteroid dehydrogenase activity on corpus luteum formation during early pregnancy in cattle

20α-hydroxysteroid dehydrogenase (20α-HSD) is a member of the aldo-keto reductase family. These enzymes play a pivotal role in regulating steroid hormones, such as androgens, oestrogen, and progesterone, and are, therefore, considered vital targets for determining whether a pregnancy is maintained. In this study, we investigated the association between 20α-HSD and apoptosis-related genes in luteal tissues (at 30, 60, and 90 days of gestation) and cells from early gestation in cattle. The corpus luteum altered the number of large lutein cells from 30–90 days. The change in the junction of the connective tissue appeared to affect the density of the corpus luteum. In addition, 20α-HSD was detected in the corpus luteum and cultured cells during early pregnancy, in contrast to the results of previous studies. The overall expression pattern of the 20α-HSD and Casp-3 proteins was lower on day 50 of gestation than on days 30 and 90. However, the 20α-HSD expression gradually increased from 30 to 90 days of gestation. When the 20α-HSD protein was increased to 0.5 μg/ml, 1 μg/ml, and 1.5 μg/ml in the luteal cells collected on day 30 of pregnancy, apoptosis was analysed after 48 hours. 20α-HSD generated in the cells was confirmed, and the concentration gradually decreased as the concentration increased. However, the expression of Casp-3 showed an overall similar expression pattern. Notably, the 20α-HSD and Casp-3 proteins were lowest at 1.5 μg/ml supplemented with 20α-HSD, with higher levels in the cytosol than in the cytoplasm. These results suggest that 20α-HSD plays a role in maintaining normal pregnancy, particularly by regulating the progesterone concentrations during luteal cell development.

Steroid Metabolism in Children and Adolescents With Obesity and Insulin Resistance: Altered SRD5A and 20α/20βHSD Activity.

Alterations in glucocorticoid metabolism may contribute to the development of obesity and insulin resistance (IR). Obesity in turn affects the androgen balance. The peripheral metabolism of steroids is equally an important determinant of their bioavailability and activity. The aim of this study was to evaluate steroid metabolism in obese children and to define which enzyme alterations are associated with IR. Clinical characteristics and anthropometric measurements were determined in 122 obese children and adolescents (72 girls, 50 boys) aged 8 – 18 years. 26 of them (21.3%) were diagnosed with IR (13 boys, 13 girls). Routine laboratory tests were performed and 24h urinary steroid excretion profiles were analyzed by gas chromatography/mass spectrometry. Positive relationship between 5α-reductase (SRD5A) activity and IR was found. According to the androsterone to etiocholanolone (An/Et) ratio the activity of SRD5A was significantly increased in obese children with IR, but the difference remained insignificant once the 5α-dihydrotestosterone to testosterone (5αDHT/T) ratio was considered. Furthermore, this relationship persisted in boys but was not observed in girls. The activity of 20α-hydroxysteroid dehydrogenase (20αHSD) and 20β-hydroxysteroid dehydrogenase (20βHSD) was reduced only in obese girls with IR. Conclude, in the context of obese children and adolescents with IR, we surmise that increased SRD5A represents a compensatory mechanism to reduce local glucocorticoid availability. This phenomenon is probably different in the liver (restriction) and in the adipose tissue (expected increase in activity). We show significant changes in 20αHSD and 20βHSD activity in obese girls with IR, but it is difficult to clearly determine whether the activity of these enzymes is an indicator of the function in their ovaries or adrenal glands.

Inhibition study on anti-type 3 of 3α-hydroxysteroid dehydrogenase activity against 1,2,3-triazolo[4,5-D]pyrimidine derivatives: Molecular modelling approach

Series of havoc posed by breast cancer to women all over the world remain a serious concern to scientist globally. The desire to find an efficient cure to this dreaded disease is still an on-going work amidst researchers; thus, the biological interaction of thirty-four 1,2,3-triazolo[4,5-d]pyrimidine [1,2,3-TPD] derivatives were studied. The studied compounds were optimised using density functional theory on Spartan 14. These compounds were complexed with 3a-Hydroxysteroid dehydrogenases (3 3α-HSD), their binding energies and the non-bonding interactions were examined using molecular docking and molecular dynamics simulation studies. All the studied compounds displayed efficient inhibiting capacity better than the standard used (5FU). In addition, compound 19 possess a greater inhibiting ability than other studied molecules. This study can serve as a lead to discovery of potent and new drug candidates against this women health threat.

Glucocorticoid metabolism and the action of 11 beta-hydroxysteroid dehydrogenase 2 in canine congestive heart failure

The enzyme 11-beta-hydroxysteroid dehydrogenase isoenzyme 2 (11BHSD2) is responsible for converting the active glucocorticoid cortisol to inactive cortisone and in the renal medulla protects the mineralocorticoid receptor (MR) from activation by cortisol. Derangements in 11BHSD2 activity can result in reduced conversion of cortisol to cortisone, activation of the MR by cortisol and, consequently, sodium and water retention. The objective of this study was to examine glucocorticoid metabolism in canine congestive heart failure (CHF), specifically to evaluate whether renal 11BHSD2 activity and expression were altered. Dogs were prospectively recruited into one of two phases; the first phase (n=56) utilized gas chromatography-tandem mass spectrometry to examine steroid hormone metabolites normalised to creatinine in home-caught urine samples. Total serum cortisol was also evaluated. The second phase consisted of dogs (n=18) euthanased for refractory CHF or for behavioural reasons. Tissue was collected from the renal medulla for examination by quantitative reverse transcription polymerase chain reaction, immunohistochemistry and protein immune-blotting. Heart failure did not change urinary cortisol:cortisone ratio (P=0.388), or modify renal expression (P=0.303), translation (P=0.427) or distribution of 11BHSD2 (P=0.325). However, CHF did increase excretion of 5α-tetrahydrocortisone (P=0.004), α-cortol (P=0.002) and α-cortolone (P=0.009). Congestive heart failure modifies glucocorticoid metabolism in dogs by increasing 5α-reductase and 20α-hydroxysteroid dehydrogenase activity. Differences between groups in age, sex and underlying disease processes may have influenced these results. However, 11BHSD2 does not appear to be a potential therapeutic target in canine CHF.

PTSD in women is associated with a block in conversion of progesterone to the GABAergic neurosteroids allopregnanolone and pregnanolone measured in plasma

There is a need to identify new and more effective treatments for posttraumatic stress disorder (PTSD). Allopregnanolone and its stereoisomer pregnanolone (together termed ALLO) are metabolites of progesterone that positively and allosterically modulate GABA effects at GABAA receptors, thereby reducing anxiety and depression. Previous research revealed that women with PTSD had low cerebrospinal fluid (CSF) ALLO levels and a low ratio of ALLO to the allopregnanolone precursor 5α-DHP, consistent with deficient activity of the ALLO synthetic enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD). The current study examined ALLO and the ratio of ALLO to 5α-DHP in plasma at rest and in response to psychophysiological stressors in trauma-exposed, medication-free women with and without PTSD. Participants were examined twice in random order during the early follicular phase (eFP) and mid-luteal phase (mLP) of the menstrual cycle. Plasma neurosteroids were measured using gas chromatography-mass spectrometry. Results indicate that the ALLO to 5α-DHP ratio in plasma increases between the eFP and mLP. In addition, women with PTSD have a lower ratio of ALLO to 5α-DHP than trauma-exposed healthy women, as well as blunted increases in this ratio in response to a moderately stressful laboratory procedure, i.e., differential fear conditioning, across the menstrual cycle. Clinically feasible testing for 3α-HSD dysfunction is critical to translating this line of research into clinical care. Measurement of this ratio in plasma could facilitate patient stratification in clinical treatment trials, as well as precision medicine targeting of treatments that address ALLO synthesis deficits in women with PTSD.

НАРУШЕНИЕ АКТИВНОСТИ 20α-ГИДРОКСИСТЕРОИДДЕГИДРОГЕНАЗЫ В СИНЦИТИОТРОФОБЛАСТЕ ВОРСИНЧАТОГО ХОРИОНА ПРИ ОБОСТРЕНИИ ЦИТОМЕГАЛОВИРУСНОЙ ИНФЕКЦИИ ВО ВРЕМЯ БЕРЕМЕННОСТИ

The aim of the work is to assess the effects of cytomegalovirus infection on the 20α-hydroxysteroid dehydrogenase activity in the villous chorion syncytiotrophoblast in the first trimester of pregnancy. The materials for the study included 48 villous chorions samples taken during spontaneous abortions at 8th–10th weeks of gestation from women with reactivation of cytomegalovirus infection (the main group). The control group included 35 villous chorions samples from pregnant women with chronic cytomegalovirus infection in the latent stage taken at medical abortions at the same stage of gestation. The results of women’s examination were studied taken into account the activity of cytomegalovirus infection by immunoassay by detection of IgM antibodies or by 4 times and more increase of IgG antibody titers in the paired serums in dynamics in 10 days. The activity of 20α-hydroxysteroid dehydrogenase was assessed by the histochemical method. Quantitative evaluation of the reaction products was carried out on sections under the microscope MT (Japan) connected to software-hardware complex «SCION Corporation» (USA). At histochemical specimen of villous chorion of pregnant women who had a reactivation of cytomegalovirus infection during pregnancy (main group), there was a decrease of cytophotometric parameter of 20α-hydroxysteroid dehydrogenase activity till 30.1±2.12 pixels/μm2 (p<0.001) compared with the control group. The decrease in the reaction activity in syncytiotrophoblast indicated a decrease in the contents of 20α-dihydroprogesterone in placenta, which contributed, in our opinion, to spontaneous abortion.

A single dose of allopregnanolone affects the ovarian morphology and steroidogenesis.

Allopregnanolone, a progesterone metabolite, is one of the best characterized neurosteroids. In a dose that mimics serum levels during stress, allopregnanolone inhibits sexual receptivity and ovulation and induces a decrease in luteinizing hormone levels. The aim of this work was to examine the effect of an intracerebroventricular administration of allopregnanolone on ovarian morphophysiology, serum and tissue levels of progesterone and estrogen, and enzymatic activity of 3β-hydroxysteroid dehydrogenase, 20α-hydroxysteroid dehydrogenase and 3α-hydroxysteroid oxido-reductase in the ovary and in the medial basal hypothalamus on the morning of estrus. Ovarian morphology was analyzed under light microscopy. The hormone assays were performed by radioimmunoassay. The enzymatic activities were measured by spectrophotometric analysis. The morphometric analysis revealed that, in allopregnanolone-treated animals, the number of secondary and Graafian follicles was decreased while that of atretic follicles and cysts was significantly increased. Some cysts showed luteinized unruptured follicles. There were no differences in the number of tertiary follicles or corpora lutea in comparison with the corresponding control groups. In allopregnanolone-treated animals, progesterone serum levels were increased, while ovarian progesterone levels were decreased. Moreover, 3β-HSD and 3α-HSOR enzymatic activities were increased in the medial basal hypothalamus while ovarian levels were decreased. The enzyme 20α-hydroxysteroid dehydrogenase showed the opposite profile. The results of this study showed that allopregnanolone interferes on ovarian steroidogenesis and ovarian morphophysiology in rats, providing a clear evidence for the role of this neurosteroid in the control of reproductive function under stress situations.

Parturition failure in mice lacking Mamld1.

In mice, the onset of parturition is triggered by a rapid decline in circulating progesterone. Progesterone withdrawal occurs as a result of functional luteolysis, which is characterized by an increase in the enzymatic activity of 20α-hydroxysteroid dehydrogenase (20α-HSD) in the corpus luteum and is mediated by the prostaglandin F2α (PGF2α) signaling. Here, we report that the genetic knockout (KO) of Mamld1, which encodes a putative non-DNA-binding regulator of testicular steroidogenesis, caused defective functional luteolysis and subsequent parturition failure and neonatal deaths. Progesterone receptor inhibition induced the onset of parturition in pregnant KO mice, and MAMLD1 regulated the expression of Akr1c18, the gene encoding 20α-HSD, in cultured cells. Ovaries of KO mice at late gestation were morphologically unremarkable; however, Akr1c18 expression was reduced and expression of its suppressor Stat5b was markedly increased. Several other genes including Prlr, Cyp19a1, Oxtr, and Lgals3 were also dysregulated in the KO ovaries, whereas PGF2α signaling genes remained unaffected. These results highlight the role of MAMLD1 in labour initiation. MAMLD1 likely participates in functional luteolysis by regulating Stat5b and other genes, independent of the PGF2α signaling pathway.

Neuromodulation of the luteal regression: presence of progesterone receptors in coeliac ganglion.

What is the central question of this study? The processes involved in luteal involution have not yet been clarified and, in general, have been studied only from a hormonal point of view. We investigated whether progesterone, from the coeliac ganglion through the superior ovarian nerve, is able to modify the luteal regression of late pregnancy in the rat. What is the main finding and its importance? We showed that the luteal regression might be reversed by the neural effect of progesterone and demonstrated the presence of its receptors in the coeliac ganglion. This suggests that the peripheral neural pathway, through neuron-hormone interaction, represents an additional mechanism to control luteal function in addition to the classical endocrine regulation. The corpus luteum (CL) is a transitory endocrine gland that produces progesterone (P). At the end of its useful life, it suffers a process of functional and structural regression until its complete disappearance from the ovary. To investigate whether P is able to regulate the process of luteal regression through the peripheral neural pathway, we used the coeliac ganglion (CG)-superior ovarian nerve-ovary system from rats on day21 of pregnancy. We stimulated the CG with P and analysed the functional regression through ovarian P release measured by radioimmunoassay, expression by RT-PCR and activity of luteal 3β- and 20α-hydroxysteroid dehydrogenase (anabolic and catabolic P enzymes, respectively). The luteal structural regression was evaluated through a study of apoptosis measured by TUNEL assay and the expression of apoptotic factors, such as Bcl-2, Bax, Fas and Fas ligand (FasL) by RT-PCR. To explore whether the effects mediated by P on the CL may be associated with P receptors, their presence in the CG was investigated by immunohistochemistry. In the group stimulated with P in the CG, the ovarian P release and the 3β-hydroxysteroid dehydrogenase activity increased, whereas the expression and activity of 20α-hydroxysteroid dehydrogenase decreased. In addition, a decrease in the number of apoptotic nuclei and a decrease of the expression of FasL were observed. We demonstrated the presence of P receptors in the CG. Overall, our results suggest that the regression of the CL of late pregnancy may be reprogrammed through the peripheral neural pathway, and this effect might be mediated by P bound to its receptor in the CG.

Comparison of cell types in the rat Leydig cell lineage after ethane dimethanesulfonate treatment

The objective of this study was to purify cells in the Leydig cell lineage following regeneration after ethane dimethanesulfonate (EDS) treatment and compare their steroidogenic capacity. Regenerated progenitor (RPLCs), immature (RILCs), and adult Leydig cells (RALCs) were isolated from testes 21, 28 and 56 days after EDS treatment respectively. Production rates for androgens including androsterone and 5α-androstane-17β, 3α-diol (DIOL), testosterone and androstenedione were measured in RPLCs, RILCs and RALCs in media after 3-h in vitro culture with 100 ng/ml LH. Steady-state mRNA levels of steroidogenic enzymes and their activities were measured in freshly isolated cells. Compared to adult Leydig cells (ALCs) isolated from normal 90-day-old rat testes, which primarily produce testosterone (69.73%), RPLCs and RILCs primarily produced androsterone (70.21%) and DIOL (69.79%) respectively. Leydig cells isolated from testes 56 days post-EDS showed equivalent capacity of steroidogenesis to ALCs and primarily produced testosterone (72.90%). RPLCs had cholesterol side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1 and 17α-hydroxylase but had almost no detectable 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities, while RILCs had increased 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities. Because RPLCs and RILCs had higher 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase activities they produced mainly 5α-reduced androgens. Real-time PCR confirmed the similar trends for the expressions of these steroidogenic enzymes. In conclusion, the purified RPLCs, RILCs and RALCs are similar to those of their counterparts during rat pubertal development.

331 ANDROGEN DEPRIVATION PROMOTES INTRATUMORAL SYNTHESIS OF DIHYDROTESTOSTERONE FROM ANDROGEN METABOLITES IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research (II)1 Apr 2013331 ANDROGEN DEPRIVATION PROMOTES INTRATUMORAL SYNTHESIS OF DIHYDROTESTOSTERONE FROM ANDROGEN METABOLITES IN PROSTATE CANCER Fumio Ishizaki, Tsutomu Nishiyama, Takashi Kawasaki, Yoshimichi Miyashiro, Noboru Hara, Itsuhiro Takizawa, Makoto Naito, and Kota Takahashi Fumio IshizakiFumio Ishizaki Nagaoka, Japan More articles by this author , Tsutomu NishiyamaTsutomu Nishiyama Niigata, Japan More articles by this author , Takashi KawasakiTakashi Kawasaki Niigata, Japan More articles by this author , Yoshimichi MiyashiroYoshimichi Miyashiro Kawasaki, Japan More articles by this author , Noboru HaraNoboru Hara Niigata, Japan More articles by this author , Itsuhiro TakizawaItsuhiro Takizawa Niigata, Japan More articles by this author , Makoto NaitoMakoto Naito Niigata, Japan More articles by this author , and Kota TakahashiKota Takahashi Niigata, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1716AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Dihydrotestosterone (DHT) is reduced by aldo-keto reductase 1C2 and 1C1 (AKR1C2 and AKR1C1) and is metabolized to 5α-androstane-3α/β,17β-diol (3α/β-diol), respectively. 3α-diol can be converted back to DHT via oxidative 3α-hydroxysteroid dehydrogenase (HSD) activity. On the other hand, it has been reported that synthesis of DHT from 3β-diol is prevented. The significance of the back conversion in humans during androgen deprivation therapy (ADT) has barely been studied, despite evidence that the balance between DHT synthesis and metabolization determines intratumoral DHT concentrations. METHODS Intracellular androgen levels under incubation with the addition of 3α- or 3β-diol into LNCaP and VCaP cells were measured by the liquid chromatography tandem mass spectrometry (LC/MS). LNCaP was cultured in steroid hormone-depleted medium with 1 nM dehydroepiandrosterone (DHEA) for 70 passages (LNCaP-ADT). Transcriptome expression were analyzed by real-time PCR. Expression scores for 17β-HSD type 6 (HSD17B6) in radical prostatectomy specimens treated with or without ADT were evaluated using immunohistochemistry. We prospectively analyzed blood samples of 72 patients receiving ADT to reveal the in vivo kinetics of 3α-diol. RESULTS Our studies showed the synthesis of DHT from 3β-diol in both prostate cancer cell lines and culture medium. Different from the results of 3α-diol, we found a lot of other C19 androgens such as A-dione, A-diol, and DHEA in prostate cancer (PCa) cells and the culture medium. Additionally, long-term culture in androgen-deprived media increased mRNA expression of HSD17B6 in PCa cells. Conversely, AKR1C1·2 was down-regulated at the beginning of androgen deprivation. The expression score for HSD17B6 in prostatectomy specimens of 42 PCa patients undergoing neoadjuvant ADT was about 2-fold higher than that in tissues of 100 untreated individuals (p<0.001). In men receiving ADT, patients showing biochemical progression had a higher HSD17B6 score than those without progression (p=0.038). Transfection with HSD17B6 siRNA significantly reduced PSA by 20.6% in LNCaP-ADT cells treated with 3α-diol (P=0.014). Serum 3α-diol glucuronide levels correlated with serum adrenal androgen such as DHEA sulfate (DHEA-S) and A-dione before and after ADT. CONCLUSIONS Our study revealed that 3α- and 3β-diol has a much more significant role in intratumoral androgen metabolism during ADT than previously thought, and the back conversion of DHT from 3α- and 3β-diol can be a promising target for advanced combination hormone therapy. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e134 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Fumio Ishizaki Nagaoka, Japan More articles by this author Tsutomu Nishiyama Niigata, Japan More articles by this author Takashi Kawasaki Niigata, Japan More articles by this author Yoshimichi Miyashiro Kawasaki, Japan More articles by this author Noboru Hara Niigata, Japan More articles by this author Itsuhiro Takizawa Niigata, Japan More articles by this author Makoto Naito Niigata, Japan More articles by this author Kota Takahashi Niigata, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Syndrome of 17,20 Lyase Deficiency

Disorders of steroidogenesis have been instrumental in delineating human steroidogenic pathways. Each genetic disorder seemed to correspond to a different steroidogenic activity, helping to identify several enzymes. Beginning in 1972, several patients have been reported as having "17,20 lyase deficiency," but there have been inconsistent genetic findings. This manuscript reviews the biochemistry, genetics, and clinical disorders of 17,20 lyase activity, which converts 21-carbon precursors of glucocorticoids to 19-carbon precursors of sex steroids. A single enzyme, cytochrome P450c17, catalyzes both 17α-hydroxylase activity and 17,20 lyase activity. The 17,20 lyase activity is especially sensitive to the activities of the accessory proteins P450 oxidoreductase and cytochrome b(5). The first cases of genetically and biochemically proven 17,20 lyase deficiency were reported in 1997, in which specific P450c17 mutations were identified that lost 17,20 lyase activity but not 17α-hydroxylase activity when assayed in vitro. Subsequent work identified other P450c17 mutations and mutations in the genes encoding P450 oxidoreductase and cytochrome b(5). Recently, the initially reported cases from 1972 were found to carry mutations in two aldo-keto reductases, AKR1C2 and AKR1C4. These AKR1C isozymes catalyze 3α-hydroxysteroid dehydrogenase activity in the so-called "backdoor pathway" by which the fetal testis produces dihydrotestosterone without the intermediacy of testosterone. 17,20 Lyase deficiency should be considered a syndrome with multiple causes, and not a single disease. Study of this very rare disorder has substantially advanced our understanding of the pathways, mechanisms, and control of androgen synthesis. Mutations in other, as-yet unidentified genes may also cause this phenotype.

Non-stereo-selective cytosolic human brain tissue 3-ketosteroid reductase is refractory to inhibition by AKR1C inhibitors

Cerebral 3α-hydroxysteroid dehydrogenase (3α-HSD) activity was suggested to be responsible for the local directed formation of neuroactive 5α,3α-tetrahydrosteroids (5α,3α-THSs) from 5α-dihydrosteroids. We show for the first time that within human brain tissue 5α-dihydroprogesterone and 5α-dihydrotestosterone are converted via non-stereo-selective 3-ketosteroid reductase activity to produce the respective 5α,3α-THSs and 5α,3β-THSs. Apart from this, we prove that within the human temporal lobe and limbic system cytochrome P450c17 and 3β-HSD/Δ 5–4 ketosteroid isomerase are not expressed. Thus, it appears that these brain regions are unable to conduct de novo biosynthesis of Δ 4-3-ketosteroids from Δ 5-3β-hydroxysteroids. Consequently, the local formation of THSs will depend on the uptake of circulating Δ 4-3-ketosteroids such as progesterone and testosterone. 3α- and 3β-HSD activity were (i) equally enriched in the cytosol, (ii) showed equal distribution between cerebral neocortex and subcortical white matter without sex- or age-dependency, (iii) demonstrated a strong and significant positive correlation when comparing 46 different specimens and (iv) exhibited similar sensitivities to different inhibitors of enzyme activity. These findings led to the assumption that cerebral 3-ketosteroid reductase activity might be catalyzed by a single enzyme and is possibly attributed to the expression of a soluble AKR1C aldo-keto reductase. AKR1Cs are known to act as non-stereo-selective 3-ketosteroid reductases; low AKR1C mRNA expression was detected. However, the cerebral 3-ketosteroid reductase was clearly refractory to inhibition by AKR1C inhibitors indicating the expression of a currently unidentified enzyme. Its lack of stereo-selectivity is of physiological significance, since only 5α,3α-THSs enhance the effect of GABA on the GABA A receptor, whereas 5α,3β-THSs are antagonists.

The role of aryl hydrocarbon receptor in regulation of enzymes involved in metabolic activation of polycyclic aromatic hydrocarbons in a model of rat liver progenitor cells

In contrast to hepatocytes, there is only limited information about the expression and activities of enzymes participating in metabolic activation of environmental mutagens, including polycyclic aromatic hydrocarbons (PAHs), in liver progenitor cells. In rat liver “stem-like” WB-F344 cell line, sharing many characteristics with rat liver progenitor cells, PAHs are efficiently activated to their ultimate genotoxic metabolites forming DNA adducts. The present study aimed to characterize expression/activities of enzymes of two major pathways involved in the metabolism of benzo[ a]pyrene (BaP): cytochrome P450 (CYP) family 1 enzymes and cytosolic aldo–keto reductases (AKRs). We report here that, apart from induction of CYP1A1 and CYP1B1 expression and the corresponding enzymatic activity, both BaP and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) induced rat 3α-hydroxysteroid dehydrogenase (AKR1C9) expression and activity. In contrast, the aldehyde reductase AKR1A1 was not induced by either treatment. Thus, both CYP1 and AKR metabolic pathways were inducible in the model of liver progenitor cells. BaP and TCDD were efficient inducers of NAD(P)H:quinone oxidoreductase 1 (NQO1) expression and activity in WB-F344 cells, a principal enzyme of cellular antioxidant defense. Both compounds also induced expression of transcription factor NRF2, involved in control of enzymes protecting cells from oxidative stress. However, although BaP induced a significant formation of reactive oxygen species, it did not induce expression of heme oxygenase-1, suggesting that induction of oxidative stress by BaP was limited. Using shRNA against the aryl hydrocarbon receptor (AhR), we found that similar to CYP1A1 and CYP1B1, the AKR1C9 induction was AhR-dependent. Moreover, constitutive AKR1C9 levels in AhR-deficient rat BP8 hepatoma cells were significantly lower than in their AhR-positive 5L variant, thus supporting possible role of AhR in regulation of AKR1C9 expression. Taken together, both CYP1 and AKR1C9 appear to be AhR-regulated metabolic pathways, which may contribute to formation of pro-carcinogenic PAH metabolites in liver progenitor cells.

3-Ketosteroid Reductase Activity and Expression by Fetal Rat Osteoblasts

In addition to reproductive tissue, sex hormones induce transcriptional events in many connective tissue cells, including osteoblasts. Some sex hormone receptor modulators with bone sparing effects selectively target estrogen or androgen receptors, whereas others appear more promiscuous, in part through enzymatic metabolism. Rat osteoblasts express significant oxidative 3alpha-hydroxysteroid dehydrogenase activity, which can convert precursor substrates to potent androgen receptor agonists. Here we show that they also express 3-ketosteroid reductase activity, exemplified by 7-methyl-17-ethynyl-19-norandrostan-5 (10)en-3-one (tibolone) conversion to potent estrogen receptor alpha agonists. Conversion was rapid and quantitative, with 3alpha-hydroxytibolone as the primary metabolite. Consistently, tibolone induced estrogen receptor alpha-dependent gene promoter activity through cis-acting estrogen response elements, increased the stimulatory effect of TGF-beta on Smad-dependent gene promoter activity, and enhanced prostaglandin E2-induced activity of transcription factor Runx2. Rat osteoblasts express the 3-ketosteroid reductase AKR1C9, an aldo-keto reductase gene family member. Exposure to prostaglandin E2 increased AKR1C9 gene promoter activity and mRNA expression. AKR1C9 promoter activity was also enhanced by overexpression of protein kinase A catalytic subunit or transcription factor C/EBPdelta, and the effect of PGE2 was reduced by dominant negative C/EBPdelta competition or C/EBPdelta antisense expression. Moreover, prostaglandin E2 increased the amount of functional endogenous nuclear C/EBPdelta that could bind specifically to a distinct domain approximately 1.8-kb upstream from the start site of AKR1C9 transcription. In summary, in addition to 3alpha-hydroxysteroid dehydrogenase, rat osteoblasts express significant and regulatable 3-ketosteroid reductase activity. Through these enzymes, they may selectively metabolize precursor compounds into potent steroid receptor agonists locally within bone.

Several distinct enzymes catalyze 20α-hydroxysteroid dehydrogenase activity in mouse liver and kidney

The effects of flavonoids and quinones on NADPH- and NADH-dependent 20α-hydroxysteroid dehydrogenase (20α-HSD) activities were examined in cytosolic fractions from the liver and kidney of mice. Judging from the data for the inhibition of NADPH- and NADH-dependent 20α-HSD activities by flavonoids and quinones, enzyme catalyzing renal NADPH-dependent 20α-HSD activity was found to be distinct from enzyme(s) catalyzing hepatic NADPH- and NADH-dependent 20α-HSD activities. Sulfobromophthalein (SBP) had little ability to inhibit hepatic NADPH-dependent 20α-HSD activity and bromophenol blue (BPB) exhibited a weak activation against the enzyme activity, whereas SBP and BPB were potent and moderate inhibitors, respectively, of hepatic NADH-dependent 20α-HSD activity. Thus, enzyme catalyzing hepatic NADPH-dependent 20α-HSD activity appeared to be distinct from enzyme catalyzing hepatic NADH-dependent 20α-HSD activity. The data for the pH profiles of hepatic NADPH- and NADH-dependent 20α-HSD activities also led us to the conclusion. Based on these results, we propose the possibility that several distinct enzymes catalyze NADPH- and NADH-dependent 20α-HSD activities in cytosolic fractions from the liver and kidney of mice.

Molecular characterization of the cynomolgus monkey Macaca fascicularis steroidogenic enzymes belonging to the aldo-keto reductase family

Steroidogenic enzymes belonging to the aldo-keto reductase family (AKR) possess highly homologous sequences while having different activities. To gain further knowledge about the function as well as the regulation of these enzymes in the monkey, we have isolated cDNA sequences encoding monkey type 5 17β-hydroxysteroid dehydrogenase, 20α-hydroxysteroid dehydrogenase and 3α-hydroxysteroid dehydrogenase, and characterized their enzymatic activity and mRNA tissue distribution. Sequence analysis indicates that these enzymes share approximately 94 and 76% amino acid identity with human and mouse homologs, respectively. Monkey type 5 17β-HSD possesses 95.9% amino acid sequence identity with human type 5 17β-HSD. It catalyzes the transformation of 4-androstenedione into testosterone, but it lacks 20α-hydroxysteroid dehydrogenase activity that is present in the human enzyme. This activity seems to be specific to human, since mouse type 5 17β-HSD does not show significant 20α-HSD activity. In addition, monkey and mouse 20α-HSD possess relatively high 20α-, 3α-, and 17β-HSD activities, while their human counterpart is confined to 20α-HSD activity. The monkey 3α-HSD possesses relatively high 3α-, 17β-, and 20α-HSD activities; human type 1 3α-HSD exerts 3α- and 20α-HSD activities; the mouse 3α-HSD displays a unique 3α-HSD activity. Quantification of mRNA expression shows that the monkey 3α-HSD is exclusively expressed in the liver, while the type 5 17β-HSD is predominately found in the kidney, with lower levels observed in the stomach, liver, and colon. Monkey 20α-HSD mRNA is highly expressed in the kidney, stomach, and liver. Our study provides the basis for future investigations on the regulation and function of these enzymes in the monkey.

Inhibitory effects of diesel exhaust components and flavonoids on 20α-hydroxysteroid dehydrogenase activity in mouse tissues

The inhibitory effects of diesel exhaust components and flavonoids on 20α-hydroxysteroid dehydrogenase (20α-HSD) activity were examined in cytosolic fractions from the liver, kidney and lung of male mice. 9,10-Phenanthrenequinone (9,10-PQ) and 1,2-naphthoquinone (1,2-NQ), which are contained in diesel exhaust particles (DEPs), potently inhibited 20α-HSD activity in liver cytosol. 9,10-PQ also inhibited the enzyme activity in lung cytosol. However, 20α-HSD activity in kidney cytosol was little inhibited by 9,10-PQ or 1,2-NQ. Flavonoids such as quercetin, fisetin and kaempferol exhibited high inhibitory potencies for 20α-HSD activity in liver cytosol, whereas these flavonoids were poor inhibitors for the enzyme activity in kidney cytosol. It is likely that several diesel exhaust components and flavonoids augment the signaling of progesterone in the liver cells, by potently inhibiting 20α-HSD activity in mouse liver cytosol. The possibility that there are distinct enzymes catalyzing 20α-HSD activity in the non-reproductive tissues of male mice is also discussed.

Enhanced activity of 3α-hydroxysteroid dehydrogenase by addition of the co-solvent 1-butyl-3-methylimidazolium (l)-lactate in aqueous phase of biphasic systems for reductive production of steroids

The enzyme activity of 3alpha-hydrosteroid dehydrogenase (HSDH) was enhanced by the addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate ([Bmim][lactate]) to 50 mM Tris-HCl buffer. When utilizing [Bmim][lactate], the reaction velocity of HSDH increased. Also, reductive production of androsterone was investigated in an aqueous-organic solvent biphasic system containing 5% [Bmim][lactate] as the co-solvent of aqueous phase. In a coupled-enzyme system comprising HSDH and formate dehydrogenase (FDH), a two-fold increase in production rate of androsterone was obtained when utilizing [Bmim][lactate] with NADH regeneration.

Leydig cell function in mice lacking connexin43

Connexin43 (Cx43) is the most abundantly expressed member of the connexin (gap junction protein) family and the only one so far identified in mouse Leydig cell gap junctions. Mice lacking Cx43 were used to investigate its role in testicular androgen production and regulation. Testes from term fetuses were grafted under the kidney capsules of castrated adult males. After 3 weeks, serum from host mice was analyzed for androgens. In order to test their response to stimulation, the grafted testes were incubated in vitro with varying concentrations of LH and their androgen end products analyzed. Incubation with radiolabeled progesterone was followed by high performance liquid chromatography to quantify the androgen-intermediate metabolites. Radiolabeled testosterone in the presence of NADPH was used to determine the activity of testosterone-metabolizing enzymes 17beta-hydroxysteroid dehydrogenase (17betaHSD), 5alpha-reductase (5alphaR), and 3alpha-hydroxysteroid dehydrogenase (3alpha HSD). Serum androgen levels did not differ between hosts carrying wild-type versus null mutant grafts although Cx43-deficient testes had more 17betaHSD and 5alphaR activity than wild-type controls. Furthermore, the genotype of grafted testes did not influence LH-stimulated androgen production in vitro. These results indicate that the steroidogenic function of Leydig cells is not compromised by the absence of Cx43, perhaps because other gap junction proteins are present. Dye transfer experiments demonstrated that Cx43-deficient Leydig cells retain intercellular coupling, indicating that Cx43 is not the only protein contributing to their gap junctions. Thus, despite their prominence in Leydig cells, Cx43 gap junctions are not essential for androgen production.