Tracking protein-protein interactions by NMR: conformational selection in human steroidogenic cytochrome P450 CYP17A1 induced by cytochrome b5.

The human steroidogenic cytochrome P450 CYP17A1 catalyzes two types of reactions in the biosynthetic pathway leading from pregnenolone to testosterone and several other steroid hormones. The first is the hydroxylation of pregnenolone or progesterone to the corresponding 17α-hydroxy steroid, followed by a lyase reaction that converts these 17α-hydroxy intermediates to the androgens dehydroepiandrosterone and androstenedione, respectively. cytochrome b5 (cytb5) is known to act as both an effector and electron donor for the lyase oxidations, markedly stimulating the rate of the lyase reaction in its presence relative to the rate in its absence. Extensive sequential backbone 1H,15N and 13C nuclear magnetic resonance assignments have now been made for oxidized CYP17A1 bound to the prostate cancer drug and inhibitor abiraterone. This is the first eukaryotic P450 for which such assignments are now available. These assignments allow more complete interpretation of the structural perturbations observed upon cytb5 addition. Possible mechanism(s) for the effector activity of cytb5 are discussed in light of this new information.

Effect of food and polymorphisms in SLCO2B1, CYP3A4 and UGT1A4 on pharmacokinetics of abiraterone and its metabolites in Chinese volunteers.

AIMS: Abiraterone acetate, a prodrug of abiraterone (ABI), provides an efficient therapeutic option for metastatic castration-resistant prostate cancer patients. ABI undergoes extensive metabolism in vivo and is transformed into active metabolites Δ4 -abiraterone and 3-keto-5α-abiraterone as well as inactive metabolites abiraterone sulfate and abiraterone N-oxide sulfate. We aimed to examine the effect of polymorphisms in SLCO2B1, CYP3A4 and UGT1A4 on the pharmacokinetics of ABI and its metabolites. METHODS: In this study, 81 healthy Chinese subjects were enrolled and divided into 2 groups for fasted (n = 45) and fed (n = 36) studies. Plasma samples were collected after administering a 250 mg abiraterone acetate tablet followed by liquid chromatography-tandem mass spectrometry analysis. Genotyping was performed on a MassARRAY system. The association between SLCO2B1, CYP3A4, UGT1A4 genotype and pharmacokinetic parameters of ABI and its metabolites was assessed. RESULTS: Food effect study demonstrated high fat meal remarkedly increased systemic exposure of ABI and its metabolites. The geometric mean ratio and 90% confidence interval of area under the plasma concentration-time curve from time 0 to the time of the last quantifiable concentration (AUC0-t ) and maximum plasma concentration (Cmax ) of ABI in fed state vs. fasted state were 351.64% (286.86%-431.04%) and 478.45% (390.01%-586.94%), respectively, while the corresponding results were ranging from 145.11% to 269.42% and 150.10% to 478.45% for AUC0-t and Cmax of ABI metabolites in fed state vs. fasted state, respectively. The SLCO2B1 rs1077858 had a significant influence on AUC0-t and Cmax , while 7 other SLCO2B1 variants prolonged half-life of ABI under both fasted and fed conditions. As for ABI metabolites, the systemic exposure of Δ4 -abiraterone, abiraterone sulfate and abiraterone N-oxide sulfate as well as the elimination of 3-keto-5α-abiraterone were significantly affected by SLCO2B1 polymorphisms. Polymorphisms in CYP3A4 and UGT1A4 did not significantly affect pharmacokinetics of ABI and its metabolites. CONCLUSION: Polymorphisms in SLCO2B1 were significantly related to the pharmacokinetic variability of ABI and its metabolites under both fasted and fed conditions.

Maternal 11-Ketoandrostenedione Rises Through Normal Pregnancy and Is the Dominant 11-Oxygenated Androgen in Cord Blood.

CONTEXT: Adrenal-derived 11-oxygenated androgens (11oAs) are known important contributors to human physiology and disease but have not been studied in pregnancy. OBJECTIVE: We characterize 11oAs in normal human pregnancy and neonatal period and assess the ratios between 11oAs and compare with ratios of other steroids that undergo placental metabolism. DESIGN: Prospective cohort study, 2010-2018. SETTING: Academic institution. PATIENTS: Pairs of pregnant women and newborns (n = 120) were studied. Inclusion criteria were maternal age between 18 and 42 years old, spontaneous singleton pregnancies, and intention to deliver at University of Michigan. INTERVENTION: Maternal venous blood was collected during first trimester and at term. Neonatal cord blood was collected following delivery. Steroids were measured via liquid chromatography-tandem mass spectrometry. MAIN OUTCOME MEASURES: Levels of 11ß-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), 11ß-hydroxytestosterone, and 11-ketotestoterone (11KT) in maternal first trimester, maternal term, and neonatal cord blood were compared. 11OHA4-to-11KA4 ratios were correlated with cortisol-to-cortisone ratios. RESULTS: Dominant 11oAs in pregnancy and the cord blood are 11OHA4 and 11KA4, compared to 11OHA4 and 11KT in adult men and nonpregnant women. We found a rise in 11oA concentrations, particularly 11KA4, from first to third trimester. In cord blood, the concentration of 11KA4 exceeded those of both 11OHA4 and 11KT, reflecting placental 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) and 17ß-hydroxysteroid dehydrogenase (17ßHSD2) activities, respectively. 11OHA4-to-11KA4 ratios are concordant with cortisol-to-cortisone ratios across all maternal and fetal compartments, reflecting placental 11ßHSD2 activity. CONCLUSIONS: Placental 17ßHSD2 activity defends the fetus against the androgen 11KT. Our normative values may be used in future studies of 11oAs in complicated pregnancies.

Neuroactive steroids, WIN-compounds and cholesterol share a common binding site on muscarinic acetylcholine receptors.

Endogenous neurosteroids and their synthetic analogues-neuroactive steroids-have been found to bind to muscarinic acetylcholine receptors and allosterically modulate acetylcholine binding and function. Using radioligand binding experiments we investigated their binding mode. We show that neuroactive steroids bind to two binding sites on muscarinic receptors. Their affinity for the high-affinity binding site is about 100 nM. Their affinity for the low-affinity binding site is about 10 µM. The high-affinity binding occurs at the same site as binding of steroid-based WIN-compounds that is different from the common allosteric binding site for alcuronium or gallamine that is located between the second and third extracellular loop of the receptor. This binding site is also different from the allosteric binding site for the structurally related aminosteroid-based myorelaxants pancuronium and rapacuronium. Membrane cholesterol competes with neurosteroids/neuroactive steroids binding to both high- and low-affinity binding site, indicating that both sites are oriented towards the cell membrane..

Bioconversion of antifungal viridin to phytotoxin viridiol by environmental non-viridin producing microorganisms.

Biotransformation of viridin, an antifungal produced by biocontrol agent, with non-viridin producing microorganisms is studied. The results show that some environmental non-targeted microorganisms are able to reduce it in the known phytotoxin viridiol, and its 3-epimer. Consequently, this reduction, which happens in some cases by detoxification mechanism, could be disastrous for the plant in a biocontrol of plant disease. However, a process fermentation/biotransformation could be an efficient approach for the preparation of this phytotoxin.

The interactions of a number of steroid-metabolizing cytochromes P450 with abiraterone D4A metabolite: spectral analysis and molecular docking.

The interactions of pharmacologically active 3-keto-Δ4-metabolite of anticancer drug abiraterone (D4A) with steroid-metabolizing cytochromes P450 (CYP51A1, CYP11A1, CYP19A1) was studied by absorption spectroscopy and molecular docking. Both abiraterone and D4A induce type I spectral changes of CYP51A1, one of the enzymes of cholesterol biosynthesis. We have revealed that D4A did not induce spectral changes of CYP11A1, the key enzyme of pregnenolone biosynthesis, unlike abiraterone (type II ligand of CYP11A1). On the contrary, D4A interacts with the active site of CYP19A1, the key enzyme of estrogen biosynthesis, inducing type II spectral changes, while abiraterone does not. Spectral analysis allowed us to calculate spectral dissociation constant (KS) for each complex of cytochrome P450 with respective ligands. The data were supported by molecular docking. The obtained results broaden understanding of interactions of D4A with some of the key steroid-metabolizing cytochromes P450 and allow one to predict possible disproportions of steroid metabolism.

Interaction of an abiraterone with calf thymus DNA: Investigation with spectroscopic technique and modelling studies.

Binding of toxic ligands to DNA could result in undesirable biological processes, such as carcinogenesis or mutagenesis. Binding mode of Abiraterone (ABR), a steroid drug and calf thymus DNA (ctDNA) was investigated in this study using fluorescence and ultraviolet-visible spectroscopy. The probable prediction of binding and the type of interaction forces involved in the arrangement between ABR and ctDNA were explored through spectroscopic and molecular docking studies. The results indicated that ABR binds to the ctDNA in the minor groove. The binding constants were in the range of 1.35 × 106-0.36 × 106 L mol-1 at the studied temperatures. Fluorescence and spectrophotometric data suggested static quenching between ctDNA and ABR. The endothermic values of thermodynamic parameters ΔH°=-82.84 kJ mol-1; ΔS°=-161 J mol-1K-1 suggested that hydrogen bonding is the main force involved in binding of ABR with ctDNA. In experimental studies, the free binding energy at 298 K was -34.9 kJ mol-1 with the relative binding energy ≈ -29.65 kJ mol-1 of docked structure. The Ksv obtained for ABR-KI was similar to that for ABR- ctDNA -KI demonstrating no protection by ctDNA against quenching effect of KI. Thus, suggesting involvement of groove binding between ABR and ctDNA. No change in the fluorescence intensity of ABR-ctDNA was observed in presence of NaCl. Thus, ruling out the involvement of electrostatic interaction. These studies could serve as new insights in understanding the mechanisms of toxicity, resistance and side effects of ABR.

A photochemical approach to 18-nor-17ß-hydroxymethyl-17α-methylandrost-13-ene steroids.

A photochemical approach to 18-nor-17ß-hydroxymethyl-17α-methylandrost-13-ene unit of the long-term metabolites of 17-methylated androgenic anabolic steroids (AAS) is reported. It is based on a visible light-promoted radical decarboxylative alkynylation of steroidal redox-active ester. The developed method was used in synthesis of the long-term metabolite of AAS oxymesterone.

Identification of Potential Biomarkers for Thyroid Cancer Using Bioinformatics Strategy: A Study Based on GEO Datasets.

BACKGROUND: The molecular mechanisms and genetic markers of thyroid cancer are unclear. In this study, we used bioinformatics to screen for key genes and pathways associated with thyroid cancer development and to reveal its potential molecular mechanisms. METHODS: The GSE3467, GSE3678, GSE33630, and GSE53157 expression profiles downloaded from the Gene Expression Omnibus database (GEO) contained a total of 164 tissue samples (64 normal thyroid tissue samples and 100 thyroid cancer samples). The four datasets were integrated and analyzed by the RobustRankAggreg (RRA) method to obtain differentially expressed genes (DEGs). Using these DEGs, we performed gene ontology (GO) functional annotation, pathway analysis, protein-protein interaction (PPI) analysis and survival analysis. Then, CMap was used to identify the candidate small molecules that might reverse thyroid cancer gene expression. RESULTS: By integrating the four datasets, 330 DEGs, including 154 upregulated and 176 downregulated genes, were identified. GO analysis showed that the upregulated genes were mainly involved in extracellular region, extracellular exosome, and heparin binding. The downregulated genes were mainly concentrated in thyroid hormone generation and proteinaceous extracellular matrix. Pathway analysis showed that the upregulated DEGs were mainly attached to ECM-receptor interaction, p53 signaling pathway, and TGF-beta signaling pathway. Downregulation of DEGs was mainly involved in tyrosine metabolism, mineral absorption, and thyroxine biosynthesis. Among the top 30 hub genes obtained in PPI network, the expression levels of FN1, NMU, CHRDL1, GNAI1, ITGA2, GNA14 and AVPR1A were associated with the prognosis of thyroid cancer. Finally, four small molecules that could reverse the gene expression induced by thyroid cancer, namely ikarugamycin, adrenosterone, hexamethonium bromide and clofazimine, were obtained in the CMap database. CONCLUSION: The identification of the key genes and pathways enhances the understanding of the molecular mechanisms for thyroid cancer. In addition, these key genes may be potential therapeutic targets and biomarkers for the treatment of thyroid cancer.

Pregnenolonyl-α-glucoside exhibits marked anti-cancer and CYP17A1 enzymatic inhibitory activities.

We report here that pregnenolonyl-α-glucoside (2), a steryl glycoside synthesized directly from pregnenolone and glucose via a consecutive multienzyme-catalyzed process, exhibits marked dose-dependent cytotoxic activity against HT29, AGS, and ES-2 cells with IC50 values of 23.5 to 50.9 µM. An in vitro CYP17A1 binding pattern assay and protein-ligand docking model support that 2, like abiraterone, binds in the active site heme iron pocket of CYP17A1.

Glucuronidation of Abiraterone and Its Pharmacologically Active Metabolites by UGT1A4, Influence of Polymorphic Variants and Their Potential as Inhibitors of Steroid Glucuronidation.

Abiraterone (Abi) acetate (AA) is a prodrug of Abi, a CYP17A1 inhibitor used to treat patients with advanced prostate cancer. Abi is a selective steroidal inhibitor that blocks the biosynthesis of androgens. It undergoes extensive biotransformation by steroid pathways, leading to the formation of pharmacologically active Δ4-Abi (D4A) and 5α-Abi. This study aimed to characterize the glucuronidation pathway of Abi and its two active metabolites. We show that Abi, its metabolites, and another steroidal inhibitor galeterone (Gal) undergo secondary metabolism to form glucuronides (G) in human liver microsomes with minor formation by intestine and kidney microsomal preparations. The potential clinical relevance of this pathway is supported by the detection by liquid chromatography-tandem mass spectrometry of Abi-G, D4A-G, and 5α-Abi-G in patients under AA therapy. A screening of UGT enzymes reveals that UGT1A4 is the main enzyme involved. This is supported by inhibition experiments using a selective UGT1A4 inhibitor hecogenin. A number of common and rare nonsynonymous variants significantly abrogate the UGT1A4-mediated formation of Abi-G, D4A-G, and 5α-Abi-G in vitro. We also identify Gal, Abi, and its metabolites as highly potent inhibitors of steroid inactivation by the UGT pathway with submicromolar inhibitor constant values. They reduce the glucuronidation of both the adrenal precursors and potent androgens in human liver, prostate cancer cells, and by recombinant UGTs involved in their inactivation. In conclusion, tested CYP17A1 inhibitors are metabolized through UGT1A4, and germline variations affecting this metabolic pathway may also influence drug metabolism. SIGNIFICANCE STATEMENT: The antiandrogen abiraterone (Abi) is a selective steroidal inhibitor of the cytochrome P450 17α-hydroxy/17,20-lyase, an enzyme involved in the biosynthesis of androgens. Abi is metabolized to pharmacologically active metabolites by steroidogenic enzymes. We demonstrate that Abi and its metabolites are glucuronidated in the liver and that their glucuronide derivatives are detected at variable levels in circulation of treated prostate cancer patients. UDP-glucuronosyltransferase (UGT)1A4 is the primary enzyme involved, and nonsynonymous germline variations affect this metabolic pathway in vitro, suggesting a potential influence of drug metabolism and action in patients. Their inhibitory effect on drug and steroid glucuronidation raises the possibility that these pharmacological compounds might affect the UGT-associated drug-metabolizing system and pre-receptor control of androgen metabolism in patients.

A Novel 3-Phytosterone-9α-Hydroxylase Oxygenation Component and Its Application in Bioconversion of 4-Androstene-3,17-Dione to 9α-Hydroxy-4-Androstene-3,17-Dione Coupling with A NADH Regeneration Formate Dehydrogenase.

9α-Hydroxy-4-androstene-3,17-dione (9-OH-AD) is one of the significant intermediates for the preparation of ß-methasone, dexamethasone, and other steroids. In general, the key enzyme that enables the biotransformation of 4-androstene-3,17-dione (AD) to 9-OH-AD is 3-phytosterone-9α-hydroxylase (KSH), which consists of two components: a terminal oxygenase (KshA) and ferredoxin reductase (KshB). The reaction is carried out with the concomitant oxidation of NADH to NAD+. In this study, the more efficient 3-phytosterone-9α-hydroxylase oxygenase (KshC) from the Mycobacterium sp. strain VKM Ac-1817D was confirmed and compared with reported KshA. To evaluate the function of KshC on the bioconversion of AD to 9-OH-AD, the characterization of KshC and the compounded system of KshB, KshC, and NADH was constructed. The optimum ratio of KSH oxygenase to reductase content was 1.5:1. An NADH regeneration system was designed by introducing a formate dehydrogenase, further confirming that a more economical process for biological transformation from AD to 9-OH-AD was established. A total of 7.78 g of 9-OH-AD per liter was achieved through a fed-batch process with a 92.11% conversion rate (mol/mol). This enzyme-mediated hydroxylation method provides an environmentally friendly and economical strategy for the production of 9-OH-AD.

Structural basis for human sterol isomerase in cholesterol biosynthesis and multidrug recognition.

3-ß-hydroxysteroid-Δ8, Δ7-isomerase, known as Emopamil-Binding Protein (EBP), is an endoplasmic reticulum membrane protein involved in cholesterol biosynthesis, autophagy, oligodendrocyte formation. The mutation on EBP can cause Conradi-Hunermann syndrome, an inborn error. Interestingly, EBP binds an abundance of structurally diverse pharmacologically active compounds, causing drug resistance. Here, we report two crystal structures of human EBP, one in complex with the anti-breast cancer drug tamoxifen and the other in complex with the cholesterol biosynthesis inhibitor U18666A. EBP adopts an unreported fold involving five transmembrane-helices (TMs) that creates a membrane cavity presenting a pharmacological binding site that accommodates multiple different ligands. The compounds exploit their positively-charged amine group to mimic the carbocationic sterol intermediate. Mutagenesis studies on specific residues abolish the isomerase activity and decrease the multidrug binding capacity. This work reveals the catalytic mechanism of EBP-mediated isomerization in cholesterol biosynthesis and how this protein may act as a multi-drug binder.

Conformational selection dominates binding of steroids to human cytochrome P450 17A1.

Cytochrome P450 (P450, CYP) enzymes are the major catalysts involved in the oxidation of steroids as well as many other compounds. Their versatility has been explained in part by flexibility of the proteins and complexity of the binding mechanisms. However, whether these proteins bind their substrates via induced fit or conformational selection is not understood. P450 17A1 has a major role in steroidogenesis, catalyzing the two-step oxidations of progesterone and pregnenolone to androstenedione and dehydroepiandrosterone, respectively, via 17α-hydroxy (OH) intermediates. We examined the interaction of P450 17A1 with its steroid substrates by analyzing progress curves (UV-visible spectroscopy), revealing that the rates of binding of any of these substrates decreased with increasing substrate concentration, a hallmark of conformational selection. Further, when the concentration of 17α-OH pregnenolone was held constant and the P450 concentration increased, the binding rate increased, and such opposite patterns are also diagnostic of conformational selection. Kinetic simulation modeling was also more consistent with conformational selection than with an induced-fit mechanism. Cytochrome b5 partially enhances P450 17A1 lyase activity by altering the P450 17A1 conformation but did not measurably alter the binding of 17α-OH pregnenolone or 17α-OH progesterone, as judged by the apparent Kd and binding kinetics. The P450 17A1 inhibitor abiraterone also bound to P450 17A1 in a multistep manner, and modeling indicated that the selective inhibition of the two P450 17A1 steps by the drug orteronel can be rationalized only by a multiple-conformation model. In conclusion, P450 17A1 binds its steroid substrates via conformational selection.

Monitoring of behavior, sex hormones and boar taint compounds during the vaccination program for immunocastration in three sire lines.

Immunocastration (vaccination against boar taint) is an alternative method to prevent boar taint without the need for surgical castration. This study investigates the evolution of boar taint compounds in serum and fat, serum steroid compounds as well as behavior in immunocastrated pigs from 3 sire lines: 15 stress positive Belgian Piétrain (BP), 20 stress negative French Piétrain (FP), and 20 stress negative Canadian Duroc (CD). Hormone and boar taint compounds in serum were determined at 4 time points; boar taint compounds in fat were determined at 3 time points. Behavior, skin lesions, animal and pen fouling were also recorded before the first vaccination (V2). Aggressiveness, eating and drinking and general activity behavior declined from  V2 for all sire lines. Pigs from BP were cleaner than FP and CD pigs. Even though immunocastration was effective in general (reduced testosterone, estradiol as well as androstenone in serum) for all sire lines, some individual pigs showed either androstenone or skatole levels in fat above cutoff values. While the immunocastration mechanism works as intended for androstenone, and also for skatole for the three sire lines, the risk of carcasses with boar taint compounds above cutoff levels (respectively 1.9 and 3.7%) still remains to some extent.

The 11ß-hydroxysteroid dehydrogenase isoforms: pivotal catalytic activities yield potent C11-oxy C19 steroids with 11ßHSD2 favouring 11-ketotestosterone, 11-ketoandrostenedione and 11-ketoprogesterone biosynthesis.

The 11ß-hydroxysteroid dehydrogenase (11ßHSD) types 1 and 2 are primarily associated with glucocorticoid inactivation and reactivation. Several adrenal C11-oxy C19 and C11-oxy C21 steroids, which have been identified in prostate cancer, 21-hydroxylase deficiency and polycystic ovary syndrome, are substrates for these isozymes. This study describes the kinetic parameters of 11ßHSD1 and 11ßHSD2 towards the C11-keto and C11-hydroxy derivatives of the C19 and C21 steroids. The apparent Km and Vmax values indicate the more prominent 11ßHSD2 activity towards 11ß-hydroxy androstenedione, 11ß-hydroxytestosterone and 11ß-hydroxyprogesterone in contrast to the 11ßHSD1 reduction of the C11-keto steroids, as was demonstrated in the LNCaP cell model in the production of 11-ketotestosterone and 11-ketodihydrotestosterone. Data highlighted the role of 11ßHSD2 and cytochrome P450 17A1 in the contribution of C11-oxy C21 steroids to the C11-oxy C19 steroid pool in the C11-oxy backdoor pathway. In addition, 11ßHSD2 activity, catalysing 11-ketotestosterone biosynthesis, was shown to be key in the production of prostate specific antigen and in the progression of prostate cancer to castration resistant prostate cancer. The study at hand thus provides evidence that 11ßHSD isozymes play key roles in pathophysiological states, more so than was previously put forward.

The synthesis of 16-androstene sulfoconjugates from primary porcine Leydig cell culture.

Increased public interest in the welfare of pigs reared for pork production has led to an enhanced effort in finding alternatives to castration for controlling the unpleasant odour and flavour from heated pork products known as boar taint. The purpose of this study was to investigate the testicular metabolism of androstenone, one of the major components of boar taint. Leydig cells were isolated from mature boars and incubated with radiolabeled androstenone for 10 min, 1 h, 4 h, 8 h, and 12 h. Steroid profiles were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS/MS). Sulfoconjugated, but not glucuronidated steroids were produced by Leydig cells. Approximately 85% of androstenone was converted into sulfoconjugated metabolites in Leydig cell incubations after 8 h. This sulfoconjugate fraction included androstenol-3-sulfate and two major sulfated forms of androstenone. Following removal of the sulfate group, these two sulfated forms of androstenone returned the parent compound androstenone, and not a hydroxylated metabolite. These findings provided direct evidence for the testicular production of sulfoconjugated forms of androstenone and androstenol in the boar. The high proportion of sulfoconjugates produced by the Leydig cells emphasizes the importance of steroid conjugation, which serves to regulate the amount of unconjugated steroid hormones available for accumulation in adipose tissue.

11-Oxygenated C19 Steroids Do Not Decline With Age in Women.

CONTEXT: The ovaries and adrenals are sources of androgens in women. Although dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and testosterone (T) all decline with age, these C19 steroids correlate poorly with parameters of androgen action in postmenopausal women. OBJECTIVE: To comprehensively compare the androgen profiles of pre- and postmenopausal women. METHODS: We quantified 19 steroids-including DHEA; DHEAS; T; androstenedione (A4); and the following adrenal-specific 11-oxygenated C19 steroids (11oxyandrogens): 11ß-hydroxytestosterone (11OHT), 11-ketotestosterone (11KT), 11ß-hydroxyandrostenedione (11OHA4), and 11-ketoandrostenedione (11KA4)-using liquid chromatography-tandem mass spectrometry in morning serum obtained from 100 premenopausal (age 20 to 40 years) and 100 postmenopausal (age ≥ 60 years) women. Double immunofluorescence of 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2) with cytochrome b5 (CYB5A) or sulfotransferase 2A1 (SULT2A1) was performed in normal adrenal glands obtained from eight premenopausal and eight postmenopausal women. RESULTS: DHEA, DHEAS, A4, and T were significantly higher in pre- than in postmenopausal women (2.9, 2.8, 2.9, and 1.6-fold, respectively; P < 0.0001). In contrast, the 11-oxyandrogens did not decrease with aging, and the 11OHT/T and 11OHA4/A4 ratios showed strong positive correlations with age (r = 0.5 and 0.8, respectively; P < 0.0001). Double immunofluorescence analysis showed that with the involution of the zona reticularis in the old adrenals, the sharp zonal segregation of HSD3B2 and CYB5A becomes less distinct, and areas of HSD3B2 and CYB5A overlap are observed. CONCLUSIONS: Unlike DHEA, DHEAS, A4, and T, the 11oxyandrogens do not decline in aging women. Structural changes within the adrenal cortex might explain the evolution of androgen profiles in aging women.

The characterization of androstenone transport in boar plasma.

The transport of steroids by plasma proteins influences the amount of steroid available for uptake by the target tissue. In the boar, androstenone is transported to the adipose tissue where it accumulates to cause an off-odour or off-flavour in pork, known as boar taint. The mechanism of the transport of androstenone in the boar remains unclear, and the plasma protein responsible for binding androstenone has yet to be identified. Therefore, the purpose of the present study was to characterize the binding of androstenone to plasma proteins in the boar. The binding specificity of androstenone to plasma proteins was first investigated using a HPLC gel filtration method. [3H]-androstenone was incubated with plasma in the presence or absence of unlabeled competitors and the displacement of androstenone from plasma proteins was measured. In the presence of excess unlabeled competitors, [3H]-androstenone was only partially displaced from plasma proteins, indicating it binds to a low affinity high capacity plasma protein. Binding kinetics studies were also conducted to characterize the binding of androstenone and dehydroepiandrosterone (DHEA) to plasma proteins. The Bmax of androstenone and DHEA was approximately the same (89.1% and 92.3%, respectively). However, the binding affinity (K) of androstenone was 6.5 fold greater than DHEA (0.39 nmol/ml and 0.06 nmol/ml, respectively). Affinity chromatography was used to remove albumin from the plasma proteins. Following incubations with androstenone and DHEA, the binding observed in the albumin free protein fraction was reduced 2.6 and 2.1 fold, respectively relative to the binding in the albumin protein fractions. These results provide direct evidence that androstenone is transported non-specifically by albumin in the plasma of the boar.

Identification of 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid as a metabolite of steroid degradation in Comamonas testosteroni TA441 and the genes involved in the conversion.

Comamonas testosteroni TA441 degrades steroid compounds via aromatization of the A-ring to produce 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (a metabolite with C- and D-rings), which is presumed to be further degraded via ß-oxidation. In elucidating the complete steroid degradation process in C. testosteroni, we isolated 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid and several other metabolites containing only C-ring. For conversion of the CoA-ester of this compound, a two-subunit ß -ketoacyl-CoA-transferase encoded by ORF1 and ORF2 was shown to be indispensable. ORF1 and ORF2 are located just after tesB, the meta-cleavage enzyme gene in one of the two major steroid degradation gene clusters of strain TA441. Conversion by the CoA-transferase leads to cleavage of the remaining C-ring, and the product was suggested to be further degraded by ß-oxidation involving other genes in the cluster. ORF1 and ORF2 are considered orthologues of ipdAB gene in Mycobacterium tuberculosis H37Rv, which is recently reported as the CoA-transferase of 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid (Crowe AM, Casabon I, Brown KL, Liu J, Lian J, Rogalski JC, Hurst TE, Snieckus V, Foster LJ, Eltis LD. 2017. MBio 8).