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Background & AimsMultiple mechanisms for the gut microbiome contributing to the pathogenesis of non-alcoholic fatty liver disease (NAFLD) have been implicated. Here, we aim to investigate the contribution and potential application for altered bile acid (BA) metabolizing microbe in NAFLD using whole metagenome sequencing (WMS) data.Methods86 well-characterized biopsy-proven NAFLD patients and 38 healthy controls were included in the discovery cohort. Assembly-based analysis was performed to identify BA-metabolizing microbes. Statistical tests, feature selection and microbial interaction analysis were integrated to identify microbial alterations and markers in NAFLD. An independent validation cohort was subjected to similar analyses.ResultsNAFLD microbiota exhibited decreased diversity and microbial interactions. We established a classifier model with 53 differential species exhibiting a robust diagnostic accuracy (AUC=0.97) for dectecting NAFLD. Next, 8 important differential pathway markers including secondary BA biosynthesis were identified. Specifically, increased abundance of 7α-HSDH, baiA and baiB were detected in NAFLD. Further, 10 of 50 BA-metabolizing metagenome-assembled genomes (MAG)s, from Bacteroides ovatus and Eubacterium biforme, were dominant in NAFLD and interplayed as a synergetic ecological guild. Importantly, two subtypes of NAFLD patients were observed according to secondary BA metabolism potentials. Elevated capability for secondary BA biosynthesis was also observed in the validation cohort.ConclusionsWe identified novel bacterial BA-metabolizing genes and microbes that may contribute to NAFLD pathogenesis and serve as disease markers. Microbial differences in BA-metabolism and strain-specific differences among patients highlight the potential for precision medicine in NAFLD treatment.

3α-Hydroxysteroid dehydrogenase (3α-HSD) is an enzyme that is essential in the regulation of the concentration of 5α-dihydrotestosterone (5α-DHT) in the prostate. It catalyzes the hydride reduction of 5α-DHT to 3α-androstanediol, which activates androgen receptors. Elucidating details about the hydride reduction of 5α-DHT by 3α-HSD and the environment around the active site of the enzyme could lead to the development of effective drugs for the treatment of prostate cancer. In this study, the X-ray crystal structure of human 3α-HSD type 3 was comprehensively evaluated. Moreover, molecular dynamics (MD) simulations and hybrid ONIOM-type quantum mechanics/molecular mechanics (QM/MM) calculations were performed using a large QM region (maximum 232 atoms). It was determined that the reaction proceeded in a single step without the formation of an alkoxide ion owing to the direct hydride reduction of the substrate by nicotinamide adenine dinucleotide phosphate (NADPH) and concerted proton transfer by Tyr55 and Lys84. Noncovalent interaction (NCI) analysis highlighted the roles of Tyr216 and Trp227 in 3α-HSD. Specifically, Tyr216 assisted the reaction by π/π interactions with the neighboring nicotinamide ring of NADP(H), whereas Trp227 played an important role in recognition of the size of the substrate by CH/π interactions.

Enzymatic assays based on bacterial 3α-hydroxysteroid dehydrogenase are the method of choice for quantification of total bile acids (BAs) in serum. Although non-specific, it is generally considered precise and robust. The aim of this study was to investigate how changes in the BA spectrum might affect the reliability of the method. We measured standard solutions of twenty-three human and murine BAs using a commercial enzymatic assay and compared the measured vs. expected concentrations. Additionally, total BA concentrations in rat and human cholestatic samples with an abnormal BA spectrum were measured using an enzymatic assay, and a more specific LC-MS/MS method. We observed a great variability in the response of individual BAs in the enzymatic assay. Relative signal intensities ranged from 100% in glycocholic acid (reference) to only 20% in α-muricholic acid. The enzymatic assay markedly underestimated the BA concentrations in both human and rat cholestatic sera when compared to the LC-MS/MS assay. Our study indicated that the performance of an enzymatic assay largely depends on the BA spectrum, and the total concentration of BAs can be markedly underestimated. Samples with an atypical BA spectrum (viz. in rodents) should preferably be measured by other methods.

In the present study, we analyzed the effects of a short-term environmental enrichment on the mRNA expression and DNA methylation of steroidogenic enzymes in the hippocampus. Thus, young adult (80-day-old) and middle-aged (350-day-old) Wistar female rats were exposed to sensory (SE) or motor (ME) enrichment during 10 days and compared to animals housed under standard conditions. SE was provided by an assortment of objects that included plastic tubes and toys; for ME, rodent wheels were provided. In young adult animals, SE and ME increased the mRNA expression of cytochrome P450 17α-hydroxylase/c17,20-lyase, steroid 5α-reductase type 1 (5αR-1) and 3α-hydroxysteroid dehydrogenase and decreased the methylation levels of 5αR-1 gene. In middle-aged rats, ME and SE upregulated the gene expression of aldosterone synthase and decreased the methylation state of its promoter. These results propose that SE and ME differentially regulate the transcription of neurosteroidogenic enzymes through epigenetic mechanisms in young and aged rats. Fil: Rossetti, María Florencia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Schumacher, Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Varayoud, Jorgelina Guadalupe. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Cátedra de Fisiología Humana; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Ramos, Jorge Guillermo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina

Bile acids level in serum is a useful index for screening and diagnosis of hepatobiliary diseases. As bile acids concentration is closely related to the degree of hepatobiliary diseases, detecting it is a vital factor to understand the stage of the diseases. The prevalent determination for bile acids is the enzymatic cycling method which has low sensitivity while reagent-consuming. It is desirable to develop a new method with lower cost and higher sensitivity. An indirect electrochemical detection (IED) for bile acids in human serum was established using the screen printed carbon electrode (SPCE). Since bile acids do not show electrochemical signals, they were converted to 3-ketosteroids by 3-α-hydroxysteroid dehydrogenase (3α-HSD) in the presence of nicotinamide adenine dinucleotide (NAD(+)), which was reduced to NADH. NADH could then be oxidized on the surface of SPCE, generating a signal that was used to calculate the total bile acids (TBA) concentration. A good linear calibration for TBA was obtained at the concentration range from 5.00μM to 400μM in human serum. Both the precisions and recoveries were sufficient to be used in a clinical setting. The TBA concentrations in 35 human serum samples by our IED method didn't show significant difference with the result by enzymatic cycling method, using the paired t-test. Moreover, our IED method is reagent-saving, sensitive and cost-effective.

Human 3α-HSD3 (3α-hydroxysteroid dehydrogenase type 3) plays an essential role in the inactivation of the most potent androgen 5α-DHT (5α-dihydrotestosterone). The present study attempts to obtain the important structure of 3α-HSD3 in complex with 5α-DHT and to investigate the role of 3α-HSD3 in breast cancer cells. We report the crystal structure of human 3α-HSD3·NADP+·A-dione (5α-androstane-3,17-dione)/epi-ADT (epiandrosterone) complex, which was obtained by co-crystallization with 5α-DHT in the presence of NADP+. Although 5α-DHT was introduced during the crystallization, oxidoreduction of 5α-DHT occurred. The locations of A-dione and epi-ADT were identified in the steroid-binding sites of two 3α-HSD3 molecules per crystal asymmetric unit. An overlay showed that A-dione and epi-ADT were oriented upside-down and flipped relative to each other, providing structural clues for 5α-DHT reverse binding in the enzyme with the generation of different products. Moreover, we report the crystal structure of the 3α-HSD3·NADP+·4-dione (4-androstene-3,17-dione) complex. When a specific siRNA (100 nM) was used to suppress 3α-HSD3 expression without interfering with 3α-HSD4, which shares a highly homologous active site, the 5α-DHT concentration increased, whereas MCF7 cell growth was suppressed. The present study provides structural clues for 5α-DHT reverse binding within 3α-HSD3, and demonstrates for the first time that down-regulation of 3α-HSD3 decreases MCF7 breast cancer cell growth.

The clinical use of the steroidal aromatase inhibitor Formestane (4-hydroxandrostenedione, 4-OHA) in the treatment of advanced ER+ breast cancer has been discontinued, and therefore, interest in this remarkable drug has vanished. As a C-19 sterol, 4-OHA can undergo extensive intracellular metabolism depending on the expression of specific enzymes in the corresponding cells. We used the metabolites 4β-hydroxyandrosterone, 4β-hydroxyepiandrosterone and its 17β-reduced derivative as standards for the proof of catalytic activity present in the cell culture medium and expressed by the isolated enzymes. All of the aldo-keto reductases AKR1C1, AKR1C2, AKR1C3 and AKR1C4 catalysed the reduction of the 3-keto-group and the Δ4,5 double bond of 4-OHA at the same time. Molecular docking experiments using microscale thermophoresis and the examination of the kinetic behaviour of the isolated enzymes with the substrate 4-OHA proved that AKR1C3 had the highest affinity for the substrate, whereas AKR1C1 was the most efficient enzyme. Both enzymes (AKR1C1and AKR1C3) are highly expressed in adipose tissue and lungs, exhibiting 3β-HSD activity. The possibility that 4-OHA generates biologically active derivatives such as the androgen 4-hydroxytestosterone or some 17β-hydroxy derivatives of the 5α-reduced metabolites may reawaken interest in Formestane, provided that a suitable method of administration can be developed, avoiding oral or intramuscular depot-injection administration.

One of the reasons it is difficult to analyze protein structural dynamics at atomic resolution using NMR is the molecular size of the protein. The selective amino acid labeling method is one of the effective methods that can solve this problem. In this study, to determine the site-specific conformational change in 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 (Ps3αHSD), which forms a dimer composed of two 26 kDa subunits, we expressed and purified

Gut metagenomic sequences provide a rich source of microbial genes, the majority of which are annotated by homology or unknown. Genes and gene pathways that encode enzymes catalyzing biotransformation of host bile acids are important to identify in gut metagenomic sequences due to the importance of bile acids in gut microbiome structure and host physiology. Hydroxysteroid dehydrogenases (HSDHs) are pyridine nucleotide-dependent enzymes with stereospecificity and regiospecificity for bile acid and steroid hydroxyl groups. HSDHs have been identified in several protein families, including medium-chain and short-chain dehydrogenase/reductase families as well as the aldo-keto reductase family. These protein families are large and contain diverse functionalities, making prediction of HSDH-encoding genes difficult and necessitating biochemical characterization. We located a gene cluster in Eggerthella sp. CAG:298 predicted to encode three HSDHs (CDD59473, CDD59474, and CDD59475) and synthesized the genes for heterologous expression in Escherichia coli . We then screened bile acid substrates against the purified recombinant enzymes. CDD59475 is a novel 12α-HSDH, and we determined that CDD59474 (3α-HSDH) and CDD59473 (3β-HSDH) constitute novel enzymes in an iso-bile acid pathway. Phylogenetic analysis of these HSDHs with other gut bacterial HSDHs and closest homologues in the database revealed predictable clustering of HSDHs by function and identified several likely HSDH sequences from bacteria isolated or sequenced from diverse mammalian and avian gut samples. IMPORTANCE Bacterial HSDHs have the potential to significantly alter the physicochemical properties of bile acids, with implications for increased/decreased toxicity for gut bacteria and the host. The generation of oxo-bile acids is known to inhibit host enzymes involved in glucocorticoid metabolism and may alter signaling through nuclear receptors such as farnesoid X receptor and G-protein-coupled receptor TGR5. Biochemical or similar approaches are required to fill in many gaps in our ability to link a particular enzymatic function with a nucleic acid or amino acid sequence. In this regard, we have identified a novel 12α-HSDH and a novel set of genes encoding an iso-bile acid pathway (3α-HSDH and 3β-HSDH) involved in epimerization and detoxification of harmful secondary bile acids.

3α-Hydroxysteroid dehydrogenase (3α-HSD) catalyzes the oxidation of the 3-hydroxyl group of steroids. The enzymatic conversion is a critical step in the enzymatic assay of urinary sulfated bile acids (SBAs), which is a valuable diagnosis index of hepatobiliary diseases. However, the source of 3α-HSD for clinical applications is limited. In this study, an open reading frame (ORF) encoding a novel 3α-HSD was successfully cloned from Pseudomonas aeruginosa and expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified by immobilized metal ion affinity chromatography. Enzyme characterization studies revealed that the protein has 3α-HSD activity and the Km value for sodium cholate is 1.06 mmol L(-1). More than 60% relative enzyme activity was observed in a wide range of pH and temperature, with an optimum pH at 8.0 and an optimum temperature at 30°C. The enzyme's good thermostability under 40°C would be favorable in clinical applications. Ion interference experiments indicated that Zn(2+) was an activating cofactor which increased the enzyme activity 1.75-fold. With the favorable characteristics mentioned above, the new 3α-HSD is a promising enzyme for clinical applications. More importantly, the present work is the first report on a 3α-HSD from P. aeruginosa.

A bioinformatic and phylogenetic search identifies five enzymes involved in the conversion of DCA to isoDCA in the bacterial bile acid biosynthetic pathway. An investigation of the biological roles of bile acids defines a mutualism between the producer R. gnavus and the nonproducer Bacteroides. The gut bile acid pool is millimolar in concentration, varies widely in composition among individuals and is linked to metabolic disease and cancer. Although these molecules are derived almost exclusively from the microbiota, remarkably little is known about which bacterial species and genes are responsible for their biosynthesis. Here we report a biosynthetic pathway for the second most abundant class in the gut, 3β-hydroxy(iso)-bile acids, whose levels exceed 300 μM in some humans and are absent in others. We show, for the first time, that iso–bile acids are produced by Ruminococcus gnavus, a far more abundant commensal than previously known producers, and that the iso–bile acid pathway detoxifies deoxycholic acid and thus favors the growth of the keystone genus Bacteroides. By revealing the biosynthetic genes for an abundant class of bile acids, our work sets the stage for predicting and rationally altering the composition of the bile acid pool.

The presence of steroidogenic enzymes in the brain suggests de novo synthesis of steroid hormones in the brain. The current study was designed to determine the developmental profiles of cytochrome p450 aromatase (cyp19), 17β-hydroxysteroid dehydrogenase (17β-HSD), 5α-reductase type I and 3α-hydroxysteroid dehydrogenase (3α-HSD) mRNA expression levels in the fetal mouse brain and potential influence of peripheral steroids, and the steroidogenic factor 1 (SF-1) gene on their expression. Brains were collected from WT and SF-1 knockout male and female fetuses at embryonic (E) days E12, E14, E16, and E18. Quantitative PCR analyses revealed age related increases in the expression levels of 17β-HSD and 5α-reductase. Differences between genotypes in the expression levels of 17β-HSD and 5α-reductase were detected on E14, with reduced levels of expression in SF-1 KO males and females for 17β-HSD and only between females for 5α-reductase. Expression of 3α-HSD mRNA did not differ significantly between sexes, age groups or genotypes with the exception of SF-1 KO males, which had an unexplained increase in mRNA for this enzyme on day E18. Expression of cyp19 was at the limit of detection and could not be analyzed effectively. There were no sex differences and, with the exception of small difference on E14 for 17β-HSD and 5α-reductase, no differences between genotypes. The results suggest that gonadal steroids do not influence the production of neurosteroids in the fetal brain, nor does SF-1 play a major role in the regulation of steroidogenic enzyme expression in the brain.

The brain is a steroidogenic tissue. It expresses key molecules involved in the synthesis and metabolism of neuroactive steroids, such as steroidogenic acute regulatory protein (StAR), translocator protein 18 kDa (TSPO), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenases (3β-HSD), 5α-reductases (5α-R) and 3α-hydroxysteroid oxidoreductases (3α-HSOR). Previous studies have shown that the levels of brain steroids are different in male and female rats under basal conditions and after gonadectomy. In the present study, we assessed gene expression of key neurosteroidogenic molecules in the cerebral cortex and cerebellum of gonadally intact and gonadectomised adult male and female rats. In the cerebellum, the basal mRNA levels of StAR and 3α-HSOR were significantly higher in females than in males. By contrast, the mRNA levels of TSPO and 5α-R were significantly higher in males. In the cerebral cortex, all neurosteroidogenic molecules analysed showed similar mRNA levels in males and females. Gonadectomy increased the expression of 5α-R in the brain of both sexes, although it affected the brain expression of StAR, TSPO, P450scc and 3α-HSOR in females only and with regional differences. Although protein levels were not investigated in the present study, our findings indicate that mRNA expression of steroidogenic molecules in the adult rat brain is sexually dimorphic and presents regional specificity, both under basal conditions and after gonadectomy. Thus, local steroidogenesis may contribute to the reported sex and regional differences in the levels of brain neuroactive steroids and may be involved in the generation of sex differences in the adult brain function., This research was supported by grants from MIUR Progetto Eccellenza and Intramural Grant Line‐B from Università degli Studi di Milano to Silvia Giatti. We also thank Agencia Estatal de Investigación, Spain (grant number BFU2017‐82754‐R), Centro de Investigación Biomédica en Red de Fragilidad y Envejecimiento Saludable (CIBERFES), Instituto de Salud Carlos III, Madrid, Spain, and Fondos Feder for financial support to L.M. Garcia‐Segura.

In the ovarian follicular membrana granulosa there are morphological and functional differences between cells adjacent to the follicular fluid lumen, or aligning the basal lamina. Amongst the observed functional differences are steroidogenic capacity and expression levels of a novel basal lamina, focimatrix; both of which increase in the later stages of antral follicle growth. A number of different studies have produced apparently inconsistent results as to which cell layers are more steroidogenic. To examine this systematically, individual bovine follicles, confirmed as healthy by post hoc histological examination, were used to isolate populations of apical and basal granulosa cells. Cell counts revealed that the respective groups did not differ in the numbers of cells, thus confirming the separation of these populations. We measured gene expression (quantitative RT-PCR, n=8-10, follicle diameter 14.0±0.5 mm) and protein levels (Western immunoblotting, n=14, follicle diameter 11.9±0.5 mm) and hormone production from granulosa cells (2.5×10(5) viable cells/well in serum-free conditions for 24 h, n=15, diameter 12±0.5 mm). Levels of mRNA of HSD3B1 and CYP19A1 and three focimatrix genes COL4A1, HSPG2 and LAMB2 and LHCGR were significantly lower in apical granulosa cells (P0.05), whereas, expression of CYP11A1 and HSD17B1 were not different (P0.05). The protein levels of steroidogenic enzymes P450scc and P450arom were significantly higher in apical cells (P0.05), whereas those of 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase type 1 were not different (P0.05). Progesterone production was significantly lower and oestradiol production was significantly higher in apical granulosa cells (P0.05). These results confirm that apical and basal cells are functionally different, and the differences might be explained by the location of cells of different ages and maturity within the membrana granulosa. Discrepancies in the literature on their steroidogenic capacity may reflect differences in the steroidogenic parameters measured.

In previous studies, we identified two major Comamonas testosteroni TA441 gene clusters involved in steroid degradation. Because most of the genes included in these clusters were revealed to be involved in degradation of basic steroidal structures and a few were suggested to be involved in the degradation of modified steroid compounds, we investigated the spectrum of steroid compounds degradable for TA441 to better identify the genes involved in steroid degradation. TA441 degraded testosterone, progesterone, epiandrosterone, dehydroepiandrosterone, cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid. The results suggested TA441 having 3alpha-dehydrogenase and Delta4(5)-isomerase, and 3beta-,17beta-dehydrogenase gene, we isolated these genes, all of which had high homology to the corresponding genes of C. testosteroni ATCC11996. Results of gene-disruption experiments indicated that 3beta,17beta-dehydrogenase is a unique 3beta-dehydrogenase which also acts as a 17beta-dehydrogenase in TA441, and there will be at least one more enzyme with 17beta-dehydrogenating activity. The 3alpha-dehydrogenase and Delta4(5)-isomerase genes were found adjacent in the DNA region between the two main steroid degradation gene clusters together with a number of other genes that may be involved in steroid degradation, suggesting the presence of a steroid degradation gene hot spot over 100kb in size in TA441., The Journal of steroid biochemistry and molecular biology, 122(4), pp.253-263; 2010

The enzyme 3alpha-hydroxysteroid oxido-reductase (3alpha-HSOR) catalyzes the synthesis and bioavailability of 3alpha,5alpha-neurosteroids as allopregnanolone (3alpha,5alpha-THP) which activates GABA(A) receptors and blocks T-type calcium channels involved in pain mechanisms. Here, we used a multidisciplinary approach to demonstrate that 3alpha-HSOR is a cellular target the modulation of which in dorsal root ganglia (DRG) may contribute to suppress pain resulting from peripheral nerve injury. Immunohistochemistry and confocal microscope analyses showed 3alpha-HSOR-immunostaining in naive rat DRG sensory neurons and glial cells. Pulse-chase, high performance liquid chromatography and Flo/One characterization of neurosteroids demonstrated 3alpha,5alpha-THP production in DRG. Behavioral methods allowed identification of pain symptoms (thermal and mechanical hyperalgesia and/or allodynia) in rats subjected to sciatic nerve chronic constriction injury (CCI). Reverse transcription and real-time polymerase chain reaction revealed that 3alpha-HSOR mRNA concentration in CCI-rat ipsilateral DRG, 5-fold higher than in contralateral DRG, was also 4- to 6-fold elevated than that in sham-operated or naive rat DRG. Consistently, Western blotting confirmed increased 3alpha-HSOR protein levels in CCI-rat ipsilateral DRG and double immunolabeling showed that 3alpha-HSOR overexpression occurred in DRG neurons but not in glia. Functional plasticity of 3alpha-HSOR leading to increased 3alpha,5alpha-THP production was evidenced in CCI-rat DRG. Interestingly, behavioral and molecular time-course investigations revealed that 3alpha-HSOR gene upregulation was correlated to pain symptom development. Most importantly, in vivo knockdown of 3alpha-HSOR expression in healthy rat DRG using 6-carboxyfluorescein-3alpha-HSOR-siRNA exacerbated thermal and mechanical pain perceptions. This paper is the first to show that siRNA-induced knockdown of a key neurosteroid-synthesizing enzyme directly affects an important function as nociception. Hopefully, these results may be useful for the development of novel analgesics.

Monovalent bile acids, such as taurine- and glycine-conjugated bile acids, are excreted into bile by bile salt export pumps (BSEP, ABCB11). Human BSEP (hBSEP) is physiologically important because it was identified as the gene responsible for the genetic disease: progressive familial intrahepatic cholestasis type 2 (PFIC-2). The evaluation of the inhibitory effect of hBSEP transport activity provides significant information for predicting toxic potential in the early phase of drug development. The role and function of hBSEP have been investigated by the examination of the ATP-dependent transport of radioactive isotopically (RI)-labeled bile acid such as a tritium labeled taurocholic acid, in membrane vesicles obtained from hBSEP-expressing cells. The chemiluminescence detection method using 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) had been developed for a simple analysis of bile acids in human biological fluids. This method is extremely sensitive and it may be applicable for the measurements of bile acid transport activities by hBSEP vesicles without using RI-labeled bile acid. The present paper deals with an application of the chemiluminescence detection method using 3alpha-HSD with enzyme cycling method to the measurement of ATP-dependent transport activities of taurocholic acid (T-CA) in membrane vesicles obtained from hBSEP-expressing Sf9 cells. Calibration curves for T-CA was linear over the range from 10 to 400 pmol/ml. The values of the kinetic parameters for hBSEP vesicles obtained by the chemiluminescence detection method were comparable with the values of that obtained by liquid chromatography-mass spectrometry method. This assay method was highly useful for the measurements of bile acid transport activities.

Cortisol availability is modulated by several enzymes: 11β-HSD2, which transforms cortisol (F) to cortisone (E) and 11β-HSD1 which predominantly converts inactive E to active F. Additionally, the A-ring reductases (5α- and 5β-reductase) inactivate cortisol (together with 3α-HSD) to tetrahydrometabolites: 5αTHF, 5βTHF, and THE. The aim was to assess 11β-HSD2, 11β-HSD1, and 5β-reductase activity in hypertensive patients. Free urinary F, E, THF, and THE were measured by HPLC-MS/MS in 102 essential hypertensive patients and 18 normotensive controls. 11β-HSD2 enzyme activity was estimated by the F/E ratio, the activity of 11β-HSD1 in compare to 11β-HSD2 was inferred by the (5αTHF + 5βTHF)/THE ratio and 5β-reductase activity assessed using the E/THE ratio. Activity was considered altered when respective ratios exceeded the maximum value observed in the normotensive controls. A 15.7% of patients presented high F/E ratio suggesting a deficit of 11β-HSD2 activity. Of the remaining 86 hypertensive patients, two possessed high (5αTHF + 5βTHF)/THE ratios and 12.8% had high E/THE ratios. We observed a high percentage of alterations in cortisol metabolism at pre-receptor level in hypertensive patients, previously misclassified as essential. 11β-HSD2 and 5β-reductase decreased activity and imbalance of 11β-HSDs should be considered in the future management of hypertensive patients.

Intact female rats and gonadectomized rats of both sexes were administered 0.5 mg 5 alpha-dihydrotestosterone s.c. daily for 15 days. On the 16th day the activities of hepatic microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase were determined. Androgen treatment increased the activity of both hydroxysteroid dehydrogenases and decreased the activity of the 5 alpha-reductase in all animal models. Simultaneous administration of a steroidal antiandrogen, cyproterone (5 mg/day; 15 days s.c.) did not prevent this androgenic effect, whereas oestradiol (5 micrograms/day; 15 days s.c.) completely reversed the action of 5 alpha-dihydrotestosterone. The nonsteroidal antiandrogen, flutamide, also exhibited a potent antiandrogenic influence on these enzyme activities in female rats, whereas its antagonizing action in males was limited. The effects of cyproterone, oestradiol and flutamide on enzyme activities of hepatic steroid metabolism do not reflect the biological activity of these compounds on the weights of target organs (uterus, seminal vesicle, levator ani).

The influence of cyproterone acetate (CA) upon the behaviour of hydroxysteroid dehydrogenases (HSDH) in the Wistar rat Leydig cells was investigated during the perinatal phase with the help of enzymhistochemical cum morphometrical techniques. The pregnant rats as well as their offsprings were injected with CA (dosage: 35 mg/kg body wt) sc, daily from 14 fetal day upto 31 postnatal day (p.n.d.). The animals were killed on 5, 20, and 32 p.n.d.; the enzymhistochemical reactions for 3-beta-HSDH, 11-beta-HSDH, 17-HSDH and 3-alpha-HSDH were performed in the cryostat sections of the testis, and the morphometric evaluation of HSDH positive Leydig cells was carried out. On 5 p.n.d. the activity of 17-beta-HSDH was slightly impaired in the intertubular Leydig cells of the CA treated animals. On 20 p.n.d., CA prevented nearly completely the HSDH activity in the newsly built peritubular Leydig cells; the activities of 3-beta-HSDH, 17-beta-HSDH, and 3-alpha-HSDH resided mainly in the intertubular Leydig cells. On 32 p.n.d. the HSDH activities in the Leydig cells were observed in the control as well as in treated animals. It seems that the differentiation of peritubular Leydig cells, and thereby the steroid production, is delayed by CA, but not entirely blocked.

The aldo-keto reductase (AKR) human type 3 3alpha-hydroxysteroid dehydrogenase (h3alpha-HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3alpha-HSD3-NADP(H) at 1.9 A resolution. During the refinement process, we found acetate and citrate molecules deeply engulfed in the steroid-binding cavity. Superimposition of this structure with the h3alpha-HSD3-NADP(H)-testosterone/acetate ternary complex structure reveals that one of the mobile loops forming the binding cavity operates a slight contraction movement against the citrate molecule while the side chains of many residues undergo numerous conformational changes, probably to create an optimal binding site for the citrate. These structural changes, which altogether cause a reduction of the substrate-binding cavity volume (from 776 A(3) in the presence of testosterone/acetate to 704 A(3) in the acetate/citrate complex), are reminiscent of the "induced-fit" mechanism previously proposed for the aldose reductase, another member of the AKR superfamily. We also found that the replacement of residues Arg(301) and Arg(304), localized near the steroid-binding cavity, significantly affects the 3alpha-HSD activity of this enzyme toward 5alpha-DHT and completely abolishes its 17beta-HSD activity on 4-dione. All these results have thus been used to reevaluate the binding mode of this enzyme for androgens.

Developing methods that result in targeting of therapeutic molecules in gene therapies to target tissues has importance, as targeting can increase efficacy and decrease off target-side-effects. Work from my laboratory previously showed that the extracellular matrix protein Del1 is organized in the extracellular matrix (ECM) via the Del1 deposition domain (DDD). In this work, a fusion protein with DDD was made to assay the ability to immobilize an enzyme without disrupting enzymatic function. A prostatic cancer-derived cell line LNCap that grows in an androgen-dependent manner was used with 3alpha-hydroxysteroid dehydrogenase (3 alphaHD), which catalyzes dihydrotestosterone (DHT). Plasmids encoding a 3alphaHD:DDD fusion were generated and transfected into cultured cells. The effects of 3alphaHD immobilized in the ECM by the DDD were evaluated by monitoring growth of LNCap cells and DHT concentrations. It was demonstrated that the DDD could immobilize an enzyme in the ECM without interfering with function.

Triclosan (TCS) is an antimicrobial chemical widely used in different commercial preparations. The present study demonstrated the mechanism of action of TCS-induced anti-androgenicity in rat Leydig cells. Treatment of purified cells with increasing concentrations of TCS (0.001, 0.01, 0.1, 1 and 10 microM) resulted in a significantly decreased activity of adenylyl cyclase enzyme which was followed by a decreased synthesis of cAMP. This decreased cAMP level resulted in the disruption of entire steroidogenic cascade causing a depressed synthesis of testosterone. However, TCS-induced decrease in the production of testosterone returned to normalcy when cells were treated with forskolin (an adenylyl cyclase activator). Transcription followed by translational of four prominent steroidogenic enzyme/proteins, cytochrome P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and steroidogenic acute regulatory (StAR) protein, also decreased in a dose-dependent manner in TCS-treated Leydig cells as determined by RT-PCR, enzyme assay and Western blot. These results suggested that the disruption of the activity of adenylyl cyclase enzyme by TCS in turn leads to the disruption of intermediate steroidogenic cascade causing a depressed testosterone production. The study further confirmed the anti-androgenic activity of TCS in Leydig cells with highest effective concentration at 1 microM.

Context: Dihydrotestosterone (DHT), the primary active androgen in peripheral target tissues, is metabolized by 3α-hydroxysteroid dehydrogenase type III (3α-HSD), encoded by the AKR1C2 gene, forming 5α-androstane-3α,17β-diol (3α-diol). 3α-HSD may play a role in the pathogenesis of hirsutism.Objectives: Our objective was to evaluate the role of 3α-HSD in hirsutism by comparing 1) tissue levels of active androgens, 2) relative gene expression of AKR1C2, and 3) activity of 3α-HSD in genital skin from normal and hirsute women.Design: Genital skin was obtained from normal and hirsute women. After homogenization, testosterone (T) and DHT levels were quantified by conventional RIA. From isolated RNA, relative expression of AKR1C2 was determined by real-time PCR. In addition, minced genital skin was incubated with [3H]DHT, and the product, [3H]3α-diol, was quantified by radio-HPLC.Setting: The study took place at an inner-city hospital.Patients: Patients included women undergoing posterior colporrhaphy.Main Outcome Measures: We assessed 1) tissue levels of T, DHT, and 3α-diol; 2) relative expression of AKR1C2; and 3) conversion ratio of [3H]3α-diol to [3H]DHT.Results: In genital skin, tissue DHT and T concentrations in hirsute women were 1.90-fold and 1.84-fold higher than in normal women (P =0 .002 and 0.03), and relative expression of AKR1C2 mRNA was reduced approximately 7-fold (P = 0.04). Genital skin from hirsute women showed less metabolism of [3H]DHT to [3H]3α-diol (conversion ratio, 0.24 ± 0.19 vs. 0.85 ± 0.55, P = 0.01).Conclusions: In genital skin of hirsute women, reduced AKR1C2 gene expression and 3α-HSD activity results in decreased DHT metabolism and elevated tissue levels of DHT. Diminished DHT metabolism may play an important role in the pathogenesis of hirsutism.

Identification of cellular targets pertinent for the development of effective therapies against pathological pain constitutes a difficult challenge. We combined several approaches to show that 3alpha-hydroxysteroid oxido-reductase (3alpha-HSOR), abundantly expressed in the spinal cord (SC), is a key target, the modulation of which markedly affects nociception. 3alpha-HSOR catalyzes the biosynthesis and oxidation of 3alpha,5alpha-reduced neurosteroids as allopregnanolone (3alpha,5alpha-THP), which stimulates GABA(A) receptors. Intrathecal injection of Provera (pharmacological inhibitor of 3alpha-HSOR activity) in naive rat SC decreased thermal and mechanical nociceptive thresholds assessed with behavioral methods. In contrast, pain thresholds were dose-dependently increased by 3alpha,5alpha-THP. In animals subjected to sciatic nerve injury-evoked neuropathic pain, molecular and biochemical experiments revealed an up-regulation of 3alpha-HSOR reductive activity in the SC. Enhancement of 3alpha,5alpha-THP concentration in the SC induced analgesia in neuropathic rats while Provera exacerbated their pathological state. Possibilities are opened for chronic pain control with drugs modulating 3alpha-HSOR activity in nerve cells.

Experimentally determined protein tertiary structures are rapidly accumulating in a database, partly due to the structural genomics projects. Included are proteins of unknown function, whose function has not been investigated by experiments and was not able to be predicted by conventional sequence-based search. Those uncharacterized protein structures highlight the urgent need of computational methods for annotating proteins from tertiary structures, which include function annotation methods through characterizing protein local surfaces. Toward structure-based protein annotation, we have developed VisGrid algorithm that uses the visibility criterion to characterize local geometric features of protein surfaces. Unlike existing methods, which only concerns identifying pockets that could be potential ligand-binding sites in proteins, VisGrid is also aimed to identify large protrusions, hollows, and flat regions, which can characterize geometric features of a protein structure. The visibility used in VisGrid is defined as the fraction of visible directions from a target position on a protein surface. A pocket or a hollow is recognized as a cluster of positions with a small visibility. A large protrusion in a protein structure is recognized as a pocket in the negative image of the structure. VisGrid correctly identified 95.0% of ligand-binding sites as one of the three largest pockets in 5616 benchmark proteins. To examine how natural flexibility of proteins affects pocket identification, VisGrid was tested on distorted structures by molecular dynamics simulation. Sensitivity decreased approximately 20% for structures of a root mean square deviation of 2.0 A to the original crystal structure, but specificity was not much affected. Because of its intuitiveness and simplicity, the visibility criterion will lay the foundation for characterization and function annotation of local shape of proteins.

A dramatic difference exists in the timing of development of cardiovascular disease in men vs. women. The primary candidates underlying the cause of this gender difference are the sex steroids, estrogen and testosterone. The vasculature is considered to be a site of action of these steroids. In spite of these concepts there is little data on the direct effects of estrogen and testosterone on gene expression in the vasculature. In this study, ovariectomized Sprague Dawley rats were treated for 4 days with vehicle (sesame oil), estradiol benzoate (0.15 mg/kg/day), or testosterone (1 mg/kg/day). The mesenteric arteries were obtained, total RNA was extracted, and CodeLink Uniset Rat I DNA microarrays were used to identify differential gene expression. Seven genes were identified as differentially expressed from the DNA microarray data and confirmed by real time RT-PCR. The expression of D site albumin promoter binding protein and fatty acid synthase were increased in response to both estrogen and testosterone. 3 alpha-hydroxysteroid dehydrogenase, interleukin 4 receptor, JunB and c-Fos expression were increased by estrogen but not by testosterone. Aryl hydrocarbon nuclear translocator-like gene was reduced by testosterone. These data identify genes not previously known to be responsive to estrogen and testosterone in the vasculature.

Allopregnanolone (ALLO) and androsterone (ADT) are naturally occurring 3alpha-hydroxysteroids that act as positive allosteric regulators of gamma-aminobutyric acid type A receptors. In addition, ADT activates nuclear farnesoid X receptor and ALLO activates pregnane X receptor. At least with respect to gamma-aminobutyric acid type A receptors, the biological activity of ALLO and ADT depends on the 3alpha-hydroxyl group and is lost upon its conversion to either 3-ketosteroid or 3beta-hydroxyl epimer. Such strict structure-activity relationships suggest that the oxidation or epimerization of 3alpha-hydroxysteroids may serve as physiologically relevant mechanisms for the control of the local concentrations of bioactive 3alpha-hydroxysteroids. The exact enzymes responsible for the oxidation and epimerization of 3alpha-hydroxysteroids in vivo have not yet been identified, but our previous studies showed that microsomal nicotinamide adenine dinucleotide-dependent short-chain dehydrogenases/reductases (SDRs) with dual retinol/sterol dehydrogenase substrate specificity (RoDH-like group of SDRs) can oxidize and epimerize 3alpha-hydroxysteroids in vitro. Here, we present the first evidence that microsomal nicotinamide adenine dinucleotide-dependent 3alpha-hydroxysteroid dehydrogenase/epimerase activities are widely distributed in human tissues with the highest activity levels found in liver and testis and lower levels in lung, spleen, brain, kidney, and ovary. We demonstrate that RoDH-like SDRs contribute to the oxidation and epimerization of ALLO and ADT in living cells, and show that RoDH enzymes are expressed in tissues that have microsomal 3alpha-hydroxysteroid dehydrogenase/epimerase activities. Together, these results provide further support for the role of RoDH-like SDRs in human metabolism of 3alpha-hydroxysteroids and offer a new insight into the enzymology of ALLO and ADT inactivation.

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 17beta-hydroxysteroid dehydrogenase, 20alpha-hydroxysteroid dehydrogenase and 3alpha-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 17beta-HSD possesses 95.9% amino acid sequence identity with human type 5 17beta-HSD. It catalyzes the transformation of 4-androstenedione into testosterone, but it lacks 20alpha-hydroxysteroid dehydrogenase activity that is present in the human enzyme. This activity seems to be specific to human, since mouse type 5 17beta-HSD does not show significant 20alpha-HSD activity. In addition, monkey and mouse 20alpha-HSD possess relatively high 20alpha-, 3alpha-, and 17beta-HSD activities, while their human counterpart is confined to 20alpha-HSD activity. The monkey 3alpha-HSD possesses relatively high 3alpha-, 17beta-, and 20alpha-HSD activities; human type 1 3alpha-HSD exerts 3alpha- and 20alpha-HSD activities; the mouse 3alpha-HSD displays a unique 3alpha-HSD activity. Quantification of mRNA expression shows that the monkey 3alpha-HSD is exclusively expressed in the liver, while the type 5 17beta-HSD is predominately found in the kidney, with lower levels observed in the stomach, liver, and colon. Monkey 20alpha-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.

Background Koumine is an alkaloid monomer found abundantly in Gelsemium plants. It has been shown to reverse thermal hyperalgesia and mechanical allodynia induced by sciatic nerve chronic constriction injury (CCI) in rats in a dose-dependent manner. Interestingly, this effect is mediated by elevated allopregnanolone levels in the spinal cord (SC). Since 3α-hydroxysteroid oxidoreductase (3α-HSOR), the key synthetase of allopregnanolone, is responsible for allopregnanolone upregulation in the SC, the objective of the present study was to investigate the role of its expression in the SC in koumine-induced analgesia using a rat model of neuropathic pain following peripheral nerve injury. Results Time-course investigations of immunohistochemistry and real-time polymerase chain reaction revealed that the immunoreactivity and mRNA expression of 3α-HSOR markedly increased in a time-dependent manner in the SC of koumine-treated CCI rats. Furthermore, 3α-HSOR activity in the SC of koumine-treated CCI rats increased by 15.8% compared to the activity in untreated CCI rats. Intrathecal injection of medroxyprogesterone acetate, a selective 3α-HSOR inhibitor, reversed the analgesic effect of koumine on CCI-induced mechanical pain perception. Our results confirm that koumine alleviates neuropathic pain in rats with CCI by enhancing 3α-HSOR mRNA expression and bioactivity in the SC. Conclusion This study demonstrates that 3α-HSOR is an important molecular target of koumine for alleviating neuropathic pain. Koumine may prove a promising compound for the development of novel analgesic agents effective against intractable neuropathic pain.

To explore the effects of AKR1C3 on kidney damage of preeclamptic rats and elucidate the possible therapeutic mechanism of glyburide.The rat model of preeclampsia was established by an intraperitoneal injection of Nw-nitro-L-arginine methyl ester hydrochloride (L-NAME). A total of 40 Wistar rats were randomly divided into 4 groups of preeclampsia (pregnant rats and L-NAME), treatment (pregnant rats, L-NAME and glyburide), non-pregnancy (L-NAME) and control (pregnant rats and NS) (n = 10 each). The rats in treatment group received an intragastric dose of glyburide. Successful modeling was confirmed by measuring blood pressure and 24 h urine protein content and observing the structure of kidney under transmission electron microscopy (TEM). The methods of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were employed to detect the expression levels of AKR1C3, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA).The blood pressures of all rats had no significant difference prior to modeling. After modeling, the blood pressure of preeclampsia group was significantly higher than those of control and treatment groups [(143.83 ± 9.62), (120.83 ± 4.31), (129.43 ± 14.4) mmHg, both P0.05]. The 24 h urine protein content of all rats had no significant difference prior to modeling. After modeling, the 24 h urine protein content of preeclampsia group was higher than those of control and treatment groups. The TEM observation of kidney slices verified the success of modeling. In preeclampsia group, the expression levels of AKR1C3 in protein and mRNA were significantly lower than control group [(0.48 ± 0.09) vs (0.98 ± 0.27), (0.05 ± 0.02) vs (0.87 ± 0.45), both P0.05]. Compared with preeclampsia group, the expression levels of AKR1C3 in protein and mRNA significantly increased in treatment group [(0.48 ± 0.09) vs (1.05 ± 0.20), (0.05 ± 0.02) vs (0.22 ± 0.06), both P0.05]. As compared with control group, the levels of SOD, CAT and GSH-Px were significantly lower while MDA was higher. Compared with preeclampsia group, the levels of SOD, CAT and GSH-Px in treatment group were significantly higher while MDA was lower.The expression level of AKR1C3 decreases in kidney of preeclamptic rats and the mechanism is related with oxidative stress. Glyburide increases the expression of AKR1C3 and have therapeutic effect for preeclampsia.

We analyzed the effects of aging and environmental enrichment on the mRNA expression and DNA methylation state of steroidogenic enzymes in the hippocampus. The effects of agingwere evaluated by comparing young adult (90-day-old) and middle-aged (450-day-old) female Wistar rats. To elucidate the effects of environmental enrichment, a subgroup of middle-aged rats exposed to sensory and social stimulation for 105 days was compared to rats housed under standard laboratory conditions. Aging decreased the transcription of neurosteroidogenic-related genes and increased the promoter methylation state of cytochrome P450 side chain cleavage, 3α-hydroxysteroid dehydrogenase (3α-HSD) and 5α-reductase-1. Exposure of middle-aged rats to environmental enrichment increased mRNA levels of 5α-reductase-1, 3α-HSD and cytochrome P450 17α-hydroxylase/c17,20-lyase and decreased the methylation state of the 5α-reductase-1 gene. Thus, sensory and social stimulation attenuate the age-related decline in the mRNA expression of hippocampal steroidogenic enzymes. Epigenetic mechanisms associated with differential promoter methylation could be involved. Fil: Rossetti, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Varayoud, Jorgelina Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Moreno Piovano, Guillermo Samuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Luque, Enrique Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Ramos, Jorge Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina

Maternal social stress during late pregnancy programs hypothalamo-pituitary-adrenal (HPA) axis hyper-responsiveness to stressors, such that adult prenatally stressed (PNS) offspring display exaggerated HPA axis responses to a physical stressor (systemic interleukin-1β; IL-1β) in adulthood, compared with controls. IL-1β acts via a noradrenergic relay from the nucleus tractus solitarii (NTS) to corticotropin releasing hormone neurons in the paraventricular nucleus (PVN). Neurosteroids can reduce HPA axis responses, so allopregnanolone and 3β-androstanediol (3β-diol; 5α-reduced metabolites of progesterone and testosterone, respectively) were given subacutely (over 24 h) to PNS rats to seek reversal of the “programmed” hyper-responsive HPA phenotype. Allopregnanolone attenuated ACTH responses to IL-1β (500 ng/kg, i.v.) in PNS females, but not in PNS males. However, 3β-diol normalized HPA axis responses to IL-1β in PNS males. Impaired testosterone and progesterone metabolism or increased secretion in PNS rats was indicated by greater plasma testosterone and progesterone concentrations in male and female PNS rats, respectively. Deficits in central neurosteroid production were indicated by reduced 5α-reductase mRNA levels in both male and female PNS offspring in the NTS, and in the PVN in males. In PNS females, adenovirus-mediated gene transfer was used to upregulate expression of 5α-reductase and 3α-hydroxysteroid dehydrogenase mRNAs in the NTS, and this normalized hyperactive HPA axis responses to IL-1β. Thus, downregulation of neurosteroid production in the brain may underlie HPA axis hyper-responsiveness in prenatally programmed offspring, and administration of 5α-reduced steroids acutely to PNS rats overrides programming of hyperactive HPA axis responses to immune challenge in a sex-dependent manner.

Fast inhibitory neurotransmission in the brain is largely mediated by the γ-aminobutyric acid-type A (GABAA) receptor. The 3α,5α-reduced neurosteroids (e.g., allopregnanolone) are the most potent endogenous modulators of the GABAA receptor. Although it is known that 3α,5α-reduced neurosteroid levels change during stress or depression and over the estrus cycle, a basic physiological role consistent with their pharmacological action remains elusive. We used the unique architecture of the auditory midbrain to reveal a role for 3α,5α-reduced neurosteroids in regulating inhibitory efficacy. After blocking the massive GABAergic projection from the dorsal nucleus of the lateral lemniscus (DNLL) to the contralateral central nucleus of the inferior colliculus (ICC) in anesthetized rats, a reactive increase in the efficacy of other inhibitory circuits in the ICC (separable because of the dominant ear that drives each circuit) was demonstrated with physiological measures—single-neuron activity and a neural-population-evoked response. This effect was prevented by blocking 3α,5α-reduced neurosteroid synthesis with a 5α-reductase inhibitor: finasteride. Immunohistochemistry confirmed that the DNLL blockade induced an increase in 3α,5α-reduced neurosteroids in the contralateral ICC. This study shows that when GABAergic inhibition is reduced, the brain compensates within minutes by locally increasing synthesis of neurosteroids, thereby balancing excitatory and inhibitory inputs in complex neural circuits.

A novel quinone derivative, Griseusin C (1), along with a known quinone, Naphthoquinone C (2), was isolated from the lyophilized culture broth of the marine-derived fungus Penicillium sp. The structures were elucidated on the basis of extensive 1 D- and 2D-NMR, as well as HRESI-MS, spectroscopic analysis. The relative stereochemistries of the compounds were assessed by NOESY analysis.

Human type 3 3α-hydroxysteroid dehydrogenase, or aldo-keto reductase (AKR) 1C2, eliminates the androgen signal in human prostate by reducing 5α-dihydrotestosterone (DHT, potent androgen) to form 3α-androstanediol (inactive androgen), thereby depriving the androgen receptor of its ligand. The kcat for the NADPH-dependent reduction of DHT catalyzed by AKR1C2 is 0.033 s-1. We employed transient kinetics and kinetic isotope effects to dissect the contribution of discrete steps to this low kcat value. Stopped-flow experiments to measure the formation of the AKR1C2·NADP(H) binary complex indicated that two slow isomerization events occur to yield a tight complex. A small primary deuterium isotope effect on kcat (1.5) and a slightly larger effect on kcat/Km (2.1) were observed in the steady state. In the transient state, the maximum rate constant for the single turnover of DHT (ktrans) was determined to be 0.11 s-1 for the NADPH-dependent reaction, which was ∼4-fold greater than the corresponding kcat. ktrans wa...

Allopregnanolone (ALLO) and tetrahydrodeoxycorticosterone (THDOC) are potent positive allosteric modulators of GABA action at GABA A receptors. ALLO and THDOC are synthesized in the brain from progesterone or deoxycorticosterone, respectively, by the sequential action of two enzymes: 5α-reductase (5α-R) type I and 3α-hydroxysteroid dehydrogenase (3α-HSD). This study evaluates 5α-R type I and 3α-HSD mRNA expression level in mouse brain by using in situ hybridization combined with glutamic acid decarboxylase 67/65, vesicular glutamate transporter 2, glial fibrillary acidic protein, and S100β immunohistochemistry. We demonstrate that 5α-R type I and 3α-HSD colocalize in cortical, hippocampal, and olfactory bulb glutamatergic principal neurons and in some output neurons of the amygdala and thalamus. Neither 5α-R type I nor 3α-HSD mRNAs are expressed in S100β- or glial fibrillary acidic protein-positive glial cells. Using glutamic acid decarboxylase 67/65 antibodies to mark GABAergic neurons, we failed to detect 5α-R type I and 3α-HSD in cortical and hippocampal GABAergic interneurons. However, 5α-R type I and 3α-HSD are significantly expressed in principal GABAergic output neurons, such as striatal medium spiny, reticular thalamic nucleus, and cerebellar Purkinje neurons. A similar distribution and cellular location of neurosteroidogenic enzymes was observed in rat brain. Taken together, these data suggest that ALLO and THDOC, which can be synthesized in principal output neurons, modulate GABA action at GABA A receptors, either with an autocrine or a paracrine mechanism or by reaching GABA A receptor intracellular sites through lateral membrane diffusion.

Polymerase chain reaction (PCR) is a powerful tool for qualitative evaluation of nucleic acid expression. PCR has been widely applied to measure DNA and RNA messages expression. Neurosteroids synthesized in the nervous system are potent modulators of synaptic activity and have been implicated in several neuropsychiatric disorders. To examine the possibility of an altered expression of the neurosteroidogenic metabolic enzymes in neurological diseases (like Parkinson's disease, PD) we developed a comparative non-radioactive RT-PCR assay to detect the mRNA levels of the peripheral benzodiazepine receptor, the 5 alpha-reductase type 1 and 3 alpha-hydroxysteroid-oxidoreductase type 1 and 2 in lymphocytes obtained from PD patients. The results were compared with that obtained from simultaneous quantification of progesterone, 5 alpha-dihydroprogesterone and 3 alpha,5 alpha-tetrahydroprogesterone in the plasma and cerebro-spinal fluid of the same individuals using a gas chromatography mass spectrometry (GC/MS) technique. We found a significant decrease of the rate-limiting enzyme 5 alpha-R1 along with a significant decrease in plasma and CSF of the 3 alpha,5 alpha-tetrahydroprogesterone and of the 5 alpha-dihydroprogesterone. Comparative RT-PCR assay, along with complimentary techniques (i.e. GUMS), has the sensitivity, selectivity and dynamic range to allow specific and reliable quantization of the enzymes involved in the neurosteroids pathway and represent a valuable tool to assess their expression in human neuropsychiatric conditions. (c) 2005 Elsevier B.V. All rights reserved.

Zearalenone (ZEA) is a mycoestrogen found in diverse food and feed materials, particularly in corn and small grains. Following ingestion, the parent zearalenone is converted predominantly into alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) by hepatic hydroxy steroid dehydrogenases (HSD). The present study demonstrated by standard RT-PCR the expression of 3alpha- and 3beta-HSD also in porcine cumulus oocyte complexes (COCs) and granulosa cells isolated form cumulus oocyte complexes. Analysis of the rate of bioconversion of zearalenone (ZEA) by the cultured granulose cells showed the extra-hepatic production of both hydroxy metabolites of ZEA with alpha-ZOL being the dominating metabolites as previously observed in incubations with liver microsomes. The endogenous steroids 5alpha-dihydrotestosterone (5alpha-DHT), and progesterone (PGTN), both known substrates for 3alpha-HSD inhibited the conversion of ZEA into alpha-ZOL. In the presence of pregnelonone (PGN), a major substrate for 3beta-HSD only a slight inhibitory effect on the apparent beta-ZOL formation could be observed. In conclusion, these data indicate that both 3alpha- and 3beta-HSDs are expressed in porcine COCs and GCs, whereas the biotransformation experiments confirm the involvement of these enzymes in the extra-hepatic biotransformation of ZEA.

Androgen-dependent prostate diseases initially require 5alpha-dihydrotestosterone (DHT) for growth. The DHT product 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), is inactive at the androgen receptor (AR), but induces prostate growth, suggesting that an oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) exists. Candidate enzymes that posses 3alpha-HSD activity are type 3 3alpha-HSD (AKR1C2), 11-cis retinol dehydrogenase (RODH 5), L-3-hydroxyacyl coenzyme A dehydrogenase , RODH like 3alpha-HSD (RL-HSD), novel type of human microsomal 3alpha-HSD, and retinol dehydrogenase 4 (RODH 4). In mammalian transfection studies all enzymes except AKR1C2 oxidized 3alpha-diol back to DHT where RODH 5, RODH 4, and RL-HSD were the most efficient. AKR1C2 catalyzed the reduction of DHT to 3alpha-diol, suggesting that its role is to eliminate DHT. Steady-state kinetic parameters indicated that RODH 4 and RL-HSD were high-affinity, low-capacity enzymes whereas RODH 5 was a low-affinity, high-capacity enzyme. AR-dependent reporter gene assays showed that RL-HSD, RODH 5, and RODH 4 shifted the dose-response curve for 3alpha-diol a 100-fold, yielding EC(50) values of 2.5 x 10(-9) M, 1.5 x 10(-9) M, and 1.0 x 10(-9) M, respectively, when compared with the empty vector (EC(50) = 1.9 x 10(-7) M). Real-time RT-PCR indicated that L-3-hydroxyacyl coenzyme A dehydrogenase and RL-HSD were expressed more than 15-fold higher compared with the other candidate oxidative enzymes in human prostate and that RL-HSD and AR were colocalized in primary prostate stromal cells. The data show that the major oxidative 3alpha-HSD in normal human prostate is RL-HSD and may be a new therapeutic target for treating prostate diseases.

RS-8359, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine is a selective and reversible MAO-A inhibitor. The (S)-enantiomer of RS-8359 has been demonstrated to be inverted to the (R)-enantiomer after oral administration to rats. In the current study, we investigated the chiral inversion mechanism and the properties of involved enzymes using rat liver subcellular fractions. The 7-hydroxy function of RS-8359 was oxidized at least by the two different enzymes. The cytosolic enzyme oxidized enantiospecifically the (S)-enantiomer with NADP as a cofactor. On the other hand, the microsomal enzyme catalyzed more preferentially the oxidation of the (S)-enantiomer than the (R)-enantiomer with NAD as a cofactor. With to product enantioselectivity of reduction of the 7-keto derivative, it was found that only the alcohol bearing (R)-configuration was formed by the cytosolic enzyme with NADPH and the microsomal enzyme with NADH at almost equal rate. The reduction rate was much larger than the oxidation rate of 7-hydroxy group. The results suggest that the chiral inversion might occur via an enantioselectivity of consecutive two opposing reactions, oxidation and reduction of keto-alcohol group. In this case, the direction of chiral inversion from the (S)-enantiomer to the (R)-enantiomer is governed by the enantiospecific reduction of intermediate 7-keto group to the alcohol with (R)-configuration. The enzyme responsible for the enantiospecific reduction of the 7-keto group was purified from rat liver cytosolic fractions and identified as 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) via database search of peptide mass data obtained by nano-LC/MS/MS.

AKR1C20, a member of the aldo-keto reductase (AKR) superfamily, found by mouse genomic analysis, exhibits the highest sequence identity (89%) with mouse liver 17beta-hydroxysteroid dehydrogenase (HSD) type 5, but its function remains unknown. In this report, we have expressed the recombinant AKR1C20 from its cDNA, and examined its properties. The purified enzyme was a 36-kDa monomer, and showed both 17beta-HSD and 3alpha-HSD activities in the presence of NADP(H) as the coenzymes. While the Km values for testosterone and 5alpha-dihydrotestosterone were high (0.2 mM), those for 3alpha-hydroxy- and 3-keto-steroids were low (0.3-5 microM), resulting in high catalytic efficiency for the substrates. Although no significant dehydrogenase activity towards non-steroidal alcohols was observed, the enzyme highly reduced alpha-dicarbonyl compounds such as 16-ketoestrone, 9,10-phenanthrenequinone, acenaphthenequinone, 1-phenylisatin and camphorquinone. The pH optima of the dehydrogenase and reductase activities were 10.5 and 6.5-7.5, respectively. The enzyme was inhibited by sulfobromophthalein, hexestrol, indomethacin and flufenamic acid. The properties of AKR1C20 are distinct from those of previously known mouse 17beta-HSD type 5 (AKR1C6), 3alpha-HSD (AKR1C14) and other members of the AKR1C subfamily. Thus, AKR1C20 is a novel 3alpha(17beta)-HSD, which may also function as a reductase for xenobiotic alpha-dicarbonyl compounds.

Concentrations of 3alpha-reduced neuroactive steroids are altered in depression and normalize after antidepressant pharmacotherapy with selective serotonin re-uptake inhibitors (SSRIs). We investigated the impact of mirtazapine on the activity of a key neurosteroidogenic enzyme, the 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), and on the levels of neuroactive steroids in relation to clinical response. A total of 23 drug-free in-patients suffering from a major depressive episode (DSM-IV criteria) underwent 5-week treatment with mirtazapine (45 mg/day). Plasma samples were taken weekly at 0800 and quantified for neuroactive steroids by means of combined gas chromatography/mass spectrometry analysis. Enzyme activity was determined by assessment of steroid conversion rates. Irrespective of clinical outcome, there were significant increases in 3alpha,5alpha-tetrahydroprogesterone, 3alpha,5beta-tetrahydroprogesterone, 5alpha-dihydroprogesterone, and 5beta-dihydroprogesterone after mirtazapine treatment, whereas 3beta,5alpha-tetrahydroprogesterone levels were significantly decreased. In vitro investigations demonstrated a dose-dependent inhibitory effect of mirtazapine on the activity of the microsomal 3alpha-HSD in the oxidative direction (conversion of 3alpha,5alpha-tetrahydroprogesterone to 5alpha-dihydroprogesterone). Mirtazapine affects neuroactive steroid composition similarly as do SSRIs. The inhibition of the oxidative pathway catalyzed by the microsomal 3alpha-HSD is compatible with an enhanced formation of 3alpha-reduced neuroactive steroids. However, the changes in neuroactive steroid concentrations more likely reflect direct pharmacological effects of this antidepressant rather than clinical improvement in general.

Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (approximately 37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (approximately 35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-alpha-hydroxysteroid dehydrogenase (3alphaHSD). Silver staining indicated that this 3alphaHSD form selectively bound to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3alphaHSD demonstrated immunostaining of four 3alphaHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3alphaHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3alphaHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles.

The aim of the present study was to investigate the hepatic biotransformation of the mycotoxin zearalenone (ZEA) in vitro using subcellular fractions of pig livers. The dependencies of the enzymatic reactions involved on the enzyme velocity, on the cofactor and on pH were analysed in both the microsomal fraction and the post-mitochondrial cell fraction. Finally, the inhibitory effects of various endogenous substrates on the enzymes involved (3alpha- and 3beta-hydroxysteroid dehydrogenase) were examined. Significant differences were observed between the individual subcellular fractions in terms of prevailing metabolites and absolute amounts of the metabolites produced. Moreover, this study also demonstrated that the reactions for both subcellular fractions of porcine liver are dependent on the cofactor, as alpha-zearalenol (alpha-ZOL) formation increased in the presence of NADPH, whereas beta-zearalenol (beta-ZOL) production only increased in the presence of NADH (P0.001). The optimal pH for alpha-ZOL production was pH 5.6 and that for beta-ZOL formation pH 7.4. Subsequent inhibition studies showed significant inhibitory effects for 5alpha-androstanedioneandrostanedionepregnenolone on alpha-ZOL formation, whereas beta-ZOL production was only inhibited by pregnenolone. Finally, the contributions of 3alpha- and 3beta-hydroxysteroid dehydrogenase during the bioconversion of ZEA are discussed in the context of these experiments.

Acetylpyridines (1–3) are known as aroma components of foods, perfumes, and smoking suppressants, showing several biological activities and constituting part of the structure of some important biologically active compounds. We purified and characterized an enzyme that catalyzes the stereoselective reduction of acetylpyridines so that we could clarify its function. The enzyme participating in the reductive metabolism of 4-acetylpyridine (1) in the rat liver was purified by successively applying ammonium sulfate fractionation, anion-exchange, gel filtration, and affinity chromatography, and it was definitively identified as 3α-HSD. It preferentially reduced acetylpyridines (1–3) and acetophenone (7) to their corresponding (S)-alcohols, with high enantioselectivity. Kinetic analyses of the compounds were performed, and the Vmax/Km values decreased in the order of 4-, 2-, and 3-acetylpyridine (1, 3, 2), while acetophenone (7) showed almost the same value as 3-acetylpyridine (2). These results suggested that the reduction of the substrates by 3α-HSD is affected by the nitrogen atom in the aromatic ring. © 2005 Wiley-Liss, Inc. Chirality 17:494–500, 2005.

A highly sensitive enzymatic cycling method was developed for the serum total bile acids assay. We constructed a prokaryotic expression system to prepare the recombinant 3alpha-hydroxysteroid dehydrogenase in place of the natural enzyme and for the first time used it in the total bile acids assay. The production rate of thio-NADH correlated with the bile acids concentration and was measured by the change of absorbance at 405/660 nm. The enzymatic cycling method could detect 0.22 micromol/L total bile acids in serum. Within-run and between-run imprecisions were 1.2-3.7% and 2.3-4.8%, respectively. The calibration curve for total bile acids in serum was linear between 0.5 and 180 micromol/L. This method was free from interference by bilirubin, hemoglobin, ascorbate, and lactate dehydrogenase. In conclusion, serum total bile acids could be measured by the enzymatic cycling method with recombinant 3alpha-hydroxysteroid dehydrogenase as the tool enzyme.

3Alpha-hydroxysteroid dehydrogenase (3-HSD) from Pseudomonas testosteroni and diaphorase (lipoyl dehydrogenase) from Clostridium spp. have been immobilized individually onto arylamine glass beads through diazotization. A cost-effective enzymic colorimetric method for determination of bile acid in serum and bile employing a mixture of these immobilized enzymes was developed. The method is based on measurement of reduced nicotinamide adenine dinucleotide generated from bile acid in serum/bile by immobilized 3alpha-HSD with a color reagent consisting of nitro blue tetrazolium chloride salt, oxidized nicotinamide adenine dinucleotide, and immobilized lipoyl dehydrogenase in 0.065 M sodium phosphate buffer, pH 7.0. Analytical recovery of added bile acid (50 and 200 micromol/L) was 95.57 and 85.46% in serum and 97.6 and 91.6% in bile, respectively. Within- and between-batch coefficients of variation (CV) for bile acid determination were1.2 and0.2% in serum and0.1 and0.1% in bile, respectively. Good correlations for bile acid in serum (r1=0.92) and in bile (r2=0.97) were obtained by use of a standard chemical method and the present method. The mixture of immobilized 3alpha-HSD dehydrogenase and lipoyl dehydrogenase lost 50% of its initial activity after 6 months of regular use. The cost of bile acid determination in 100 serum and bile samples by the present method has been compared with that of the Sigma kit method.

In late sepsis, it has been established that the liver plays a major role in the initiation of multiorgan failure, which is the most lethal complication in hospitals. The molecular mechanism underlying liver failure that results from sepsis remains elusive. This study was undertaken to identify the bona fide differentially expressed genes in the 18-h septic liver by suppression subtractive hybridization, and the data were corroborated by Northern blot analysis. The differential gene expression profile renders a clue as to the genes involved in septic liver failure. The cecal ligation and puncture (CLP) model of a polymicrobial septic rat was used, with the late sepsis referring to animals sacrificed at 18 h after CLP. We have identified three upregulated genes (TII-kininogen, serine protease inhibitor 2.2 [Spi2.2], and alpha 2 macroglobulin [alpha M]) and six down-regulated genes (hydroxysteroid dehydrogenase [3 alpha HSD], EST189895/mouse RNase4, bile acid-CoA-amino acid N-acyltransferase [kan-1/rBAT], IF1, albumin, and alpha 2u-globulins [alpha 2u-G PGCL1]). Among these genes, the 3 alpha HSD and kan-1/rBAT are involved in bile acid metabolism. The IF1 plays a crucial role in any disease that involves ATP hydrolysis by F1F0-ATPase. The alpha 2M, TII-kininogen, and Spi2.2 are protease inhibitors. The functions of the alpha 2u-G PGCL1 and EST189895/mouse RNase4 genes are unknown. The present results suggest that the roles of disturbance of bile acid metabolism/synthesis and the abolishment of ATP production may contribute to liver failure during late sepsis.

Aldo-keto reductase (AKR1C) isoforms can regulate ligand access to nuclear receptors by acting as hydroxysteroid dehydrogenases. The principles that govern steroid hormone binding and steroid turnover by these enzymes were analyzed using rat 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD, AKR1C9) as the protein model. Systematic alanine scanning mutagenesis was performed on the substrate-binding pocket as defined by the crystal structure of the 3alpha-HSD.NADP(+).testosterone ternary complex. T24, L54, F118, F129, T226, W227, N306, and Y310 were individually mutated to alanine, while catalytic residues Y55 and H117 were unaltered. The effects of these mutations on the ordered bi-bi mechanism were examined. No mutations changed the affinity for NADPH by more than 2-3-fold. Fluorescence titrations of the energy transfer band of the E.NADPH complex with competitive inhibitors testosterone and progesterone showed that the largest effect was a 23-fold decrease in the affinity for progesterone in the W227A mutant. By contrast, changes in the K(d) for testosterone were negligible. Examination of the k(cat)/K(m) data for these mutants indicated that, irrespective of steroid substrate, the bimolecular rate constant was more adversely affected when alanine replaced an aromatic hydrophobic residue. By far, the greatest effects were on k(cat) (decreases of more than 2 log units), suggesting that the rate-determining step was either altered or slowed significantly. Single- and multiple-turnover experiments for androsterone oxidation showed that while the wild-type enzyme demonstrated a k(lim) and burst kinetics consistent with slow product release, the W227A and F118A mutants eliminated this kinetic profile. Instead, single- and multiple-turnover experiments gave k(lim) and k(max) values identical with k(cat) values, respectively, indicating that chemistry was now rate-limiting overall. Thus, conserved residues within the steroid-binding pocket affect k(cat) more than K(d) by influencing the rate-determining step of steroid oxidation. These findings support the concept of enzyme catalysis in which the correct positioning of reactants is essential; otherwise, k(cat) will be limited by the chemical event.