Human cytochrome P450 3A7 binding four copies of its native substrate dehydroepiandrosterone 3-sulfate.

Human fetal cytochrome P450 3A7 (CYP3A7) is involved in both xenobiotic metabolism and the estriol biosynthetic pathway. Although much is understood about cytochrome P450 3A4 and its role in adult drug metabolism, CYP3A7 is poorly characterized in terms of its interactions with both categories of substrates. Herein, a crystallizable mutated form of CYP3A7 was saturated with its primary endogenous substrate dehydroepiandrosterone 3-sulfate (DHEA-S) to yield a 2.6 Å X-ray structure revealing the unexpected capacity to simultaneously bind four copies of DHEA-S. Two DHEA-S molecules are located in the active site proper, one in a ligand access channel, and one on the hydrophobic F'-G' surface normally embedded in the membrane. While neither DHEA-S binding nor metabolism exhibit cooperative kinetics, the current structure is consistent with cooperativity common to CYP3A enzymes. Overall, this information suggests that mechanism(s) of CYP3A7 interactions with steroidal substrates are complex.

The Sulfated Steroids Pregnenolone Sulfate and Dehydroepiandrosterone Sulfate Inhibit the α1ß3γ2L GABAA Receptor by Stabilizing a Novel Nonconducting State.

The GABAA receptor is inhibited by the endogenous sulfated steroids pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). It has been proposed in previous work that these steroids act by enhancing desensitization of the receptor. Here, we have investigated the modulatory effects of the steroids on the human α1ß3γ2L GABAA receptor. Using electrophysiology and quantitative model-based data analysis, we show that exposure to the steroid promotes occupancy of a nonconducting state that retains high affinity to the transmitter but whose properties differ from those of the classic, transmitter-induced desensitized state. From the analysis of the inhibitory actions of two combined steroids, we infer that PS and DHEAS act through shared or overlapping binding sites. SIGNIFICANCE STATEMENT: Previous work has proposed that sulfated neurosteroids inhibit the GABAA receptor by enhancing the rate of entry into the desensitized state. This study shows that the inhibitory steroids pregnenolone sulfate and dehydroepiandrosterone sulfate act through a common interaction site by stabilizing a distinct nonconducting state.

Derivatization-based quadruplex LC/ESI-MS/MS method for high throughput quantification of serum dehydroepiandrosterone sulfate.

The quantification of the circulating dehydroepiandrosterone sulfate (DHEAS) might be of diagnostic help for several diseases. For the DHEAS quantification, LC/ESI-MS/MS has the advantage of a high specificity compared with immunoassay, whereas LC/ESI-MS/MS has room to improve the analysis throughput. One of the promising solutions to enhance the analysis throughput is sample-multiplexing in the same injection, which can reduce the total LC/ESI-MS/MS run time. In this study, a quadruplex LC/ESI-MS/MS method was developed to quantify DHEAS in four different serum samples in a single run. After the four samples were separately deproteinized and derivatized with one of four Girard reagents (Girard reagent T, P and their isotopologs), the resulting samples were mixed, then injected into the LC/ESI-MS/MS. The applicability and advantage of the developed method were evaluated based on the analysis of nine batches of serum samples from healthy subjects (total 36 samples). The limit of quantitation was 0.050 µg/ml, which was sensitive enough for clinical laboratory use. The method was precise (intra- and inter-assay RSDs ≤ 3.6%), accurate (94.4-108.1%) and robust for the matrix effects. The analysis time was also shortened by about 60% for 36 samples by the introduced method compared with the conventional method.

Partial Agonistic Actions of Sex Hormone Steroids on TRPM3 Function.

Sex hormone steroidal drugs were reported to have modulating actions on the ion channel TRPM3. Pregnenolone sulphate (PS) presents the most potent known endogenous chemical agonist of TRPM3 and affects several gating modes of the channel. These includes a synergistic action of PS and high temperatures on channel opening and the PS-induced opening of a noncanonical pore in the presence of other TRPM3 modulators. Moreover, human TRPM3 variants associated with neurodevelopmental disease exhibit an increased sensitivity for PS. However, other steroidal sex hormones were reported to influence TRPM3 functions with activating or inhibiting capacity. Here, we aimed to answer how DHEAS, estradiol, progesterone and testosterone act on the various modes of TRPM3 function in the wild-type channel and two-channel variants associated with human disease. By means of calcium imaging and whole-cell patch clamp experiments, we revealed that all four drugs are weak TRPM3 agonists that share a common steroidal interaction site. Furthermore, they exhibit increased activity on TRPM3 at physiological temperatures and in channels that carry disease-associated mutations. Finally, all steroids are able to open the noncanonical pore in wild-type and DHEAS also in mutant TRPM3. Collectively, our data provide new valuable insights in TRPM3 gating, structure-function relationships and ligand sensitivity.

Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.

The high-energy sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), generated by human PAPS synthase isoforms PAPSS1 and PAPSS2, is required for all human sulfation pathways. Sulfotransferase SULT2A1 uses PAPS for sulfation of the androgen precursor dehydroepiandrosterone (DHEA), thereby reducing downstream activation of DHEA to active androgens. Human PAPSS2 mutations manifest with undetectable DHEA sulfate, androgen excess, and metabolic disease, suggesting that ubiquitous PAPSS1 cannot compensate for deficient PAPSS2 in supporting DHEA sulfation. In knockdown studies in human adrenocortical NCI-H295R1 cells, we found that PAPSS2, but not PAPSS1, is required for efficient DHEA sulfation. Specific APS kinase activity, the rate-limiting step in PAPS biosynthesis, did not differ between PAPSS1 and PAPSS2. Co-expression of cytoplasmic SULT2A1 with a cytoplasmic PAPSS2 variant supported DHEA sulfation more efficiently than co-expression with nuclear PAPSS2 or nuclear/cytosolic PAPSS1. Proximity ligation assays revealed protein-protein interactions between SULT2A1 and PAPSS2 and, to a lesser extent, PAPSS1. Molecular docking studies showed a putative binding site for SULT2A1 within the PAPSS2 APS kinase domain. Energy-dependent scoring of docking solutions identified the interaction as specific for the PAPSS2 and SULT2A1 isoforms. These findings elucidate the mechanistic basis for the selective requirement for PAPSS2 in human DHEA sulfation.

Δ4-3-ketosteroids as a new class of substrates for the cytosolic sulfotransferases.

Cytosolic sulfotransferase (SULT)-mediated sulfation is generally known to involve the transfer of a sulfonate group from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to a hydroxyl group or an amino group of a substrate compound. We report here that human SULT2A1, in addition to being able to sulfate dehydroepiandrosterone (DHEA) and other hydroxysteroids, could also catalyze the sulfation of Δ4-3-ketosteroids, which carry no hydroxyl groups in their chemical structure. Among a panel of Δ4-3-ketosteroids tested as substrates, 4-androstene-3,17-dione and progesterone were found to be sulfated by SULT2A1. Mass spectrometry analysis and structural modeling supported a reaction mechanism which involves the isomerization of Δ4-3-ketosteroids from the keto form to an enol form, prior to being subjected to sulfation. Results derived from this study suggested a potential role of SULT2A1 as a Δ4-3-ketosteroid sulfotransferase in steroid metabolism.

Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts.

Peripheral intracrine sex steroid synthesis from adrenal precursors dehydroepiandrosterone (DHEA) and DHEA-sulfate has evolved in humans. We sought to establish if there are differences in intracrine, paracrine, and endocrine regulation of sex steroids by primary cultures of human skin epidermal keratinocytes and dermal fibroblasts. Microarray analysis identified multifunctional genes modulated by steroids, quantitative RT-PCR (qRT-PCR) mRNA expression, enzymatic assay aromatase activity, scratch assay cell migration, immunocytochemistry α-smooth muscle actin (α-SMA), and collagen gel fibroblast contraction. All steroidogenic components were present, although only keratinocytes expressed the organic anion organic anion transporter protein (OATP) 2B1 transporter. Both expressed the G-protein-coupled estrogen receptor (GPER1). Steroids modulated multifunctional genes, up-regulating genes important in repair and aging [angiopoietin-like 4 (ANGPTL4), chemokine (C-X-C motif) ligand 1 (CXCL1), lamin B1 (LMNB1), and thioredoxin interacting protein (TXNIP)]. DHEA-sulfate (DHEA-S), DHEA, and 17ß-estradiol stimulated keratinocyte and fibroblast migration at early (4 h) and late (24-48 h) time points, suggesting involvement of genomic and nongenomic signaling. Migration was blocked by aromatase and steroid sulfatase (STS) inhibitors confirming intracrine synthesis to estrogen. Testosterone had little effect, implying it is not an intermediate. Steroids stimulated fibroblast contraction but not α-SMA expression. Mechanical wounding reduced fibroblast aromatase activity but increased keratinocyte activity, amplifying the bioavailability of intracellular estrogen. Cultured fibroblasts and keratinocytes provide a biologically relevant model system to investigate the complex pathways of sex steroid intracrinology in human skin.

What is the optimal bone-preserving strategy for patients with Addison's disease?

Addison's disease is associated with low bone mineral density and increased risk of hip fractures. Causes are multifactorial, contributed by underlying adrenocortical hormonal deficiency, associated autoimmune endocrinopathies, electrolyte disturbances and, in some patients, supraphysiologic glucocorticoid replacement. Recent realization of physiologic cortisol production rate has revised downwards glucocorticoid replacement dosages. Meanwhile, new research has emerged suggesting complex interplay between sodium and calcium homoeostasis under the influence of mineralocorticoid and parathyroid hormone that may impact bone health. As the prevalence of Addison's disease is rising, and osteoporosis and fractures are associated with significant morbidity and increased mortality, attention to bone preservation in Addison's disease is of clinical relevance and importance. We suggest an approach to bone health in Addison's disease integrating physiologic adrenocortical hormonal replacement with electrolyte and mineral homoeostasis optimization.

Insight into binding of endogenous neurosteroid ligands to the sigma-1 receptor.

The sigma-1 receptor (σ1R) is a non-opioid membrane receptor, which responds to a diverse array of synthetic ligands to exert various pharmacological effects. Meanwhile, candidates for endogenous ligands of σ1R have also been identified. However, how endogenous ligands bind to σ1R remains unknown. Here, we present crystal structures of σ1R from Xenopus laevis (xlσ1R) bound to two endogenous neurosteroid ligands, progesterone (a putative antagonist) and dehydroepiandrosterone sulfate (DHEAS) (a putative agonist), at 2.15-3.09 Å resolutions. Both neurosteroids bind to a similar location in xlσ1R mainly through hydrophobic interactions, but surprisingly, with opposite binding orientations. DHEAS also forms hydrogen bonds with xlσ1R, whereas progesterone interacts indirectly with the receptor through water molecules near the binding site. Binding analyses are consistent with the xlσ1R-neurosteroid complex structures. Furthermore, molecular dynamics simulations and structural data reveal a potential water entry pathway. Our results provide insight into binding of two endogenous neurosteroid ligands to σ1R.

Microbial Baeyer-Villiger oxidation of steroidal ketones using Beauveria bassiana: Presence of an 11α-hydroxyl group essential to generation of D-homo lactones.

This paper demonstrates for the first time transformation of a series of 17-oxo steroidal substrates (epiandrosterone, dehydroepiandrosterone, androstenedione) by the most frequently used whole cell biocatalyst, Beauveria bassiana, to 11α-hydroxy-17a-oxa-d-homo-androst-17-one products, in the following sequence of reactions: 11α-hydroxylation and subsequent Baeyer-Villiger oxidation to a ring-D lactone. 11α-Hydroxyprogesterone, the product of the first stage of the progesterone metabolism, was further converted along two routes: hydroxylation to 6ß,11α-dihydroxyprogesterone or 17ß-acetyl chain degradation leading to 11α-hydroxytestosterone, the main metabolite of the substrate. Part of 11α-hydroxytestosterone underwent a rare reduction to 11α-hydroxy-5ß-dihydrotestosterone. The experiments have demonstrated that the Baeyer-Villiger monooxygenase produced by the strain catalyzes solely oxidation of C-20 or C-17 ketones with 11α-hydroxyl group. 17-Oxo steroids, beside the 11α-hydroxylation and Baeyer-Villiger oxidation, also underwent reduction to 17ß-alcohols; activity of 17ß-hydroxysteroid dehydrogenase (17ß-HSD) has significant impact on the amount of the formed ring-D δ-lactone.

Organic anion transporter 3 mediates the efflux transport of an amphipathic organic anion, dehydroepiandrosterone sulfate, across the blood-brain barrier in mice.

The present study investigated the efflux transport systems of organic anions across the blood-brain barrier (BBB) using dehydroepiandrosterone sulfate (DHEAS) as a probe. The elimination of DHEAS from the brain after microinjection into the cerebral cortex was characterized in wild-type mice and mice with deficiency of well characterized organic anion transporters, organic anion-transporting polypeptide 1a4 (Oatp1a4)/Slco1a4 and organic anion transporter 3 (Oat3)/Slc22a8, at the BBB. The saturable efflux of DHEAS from the brain was completely inhibited by probenecid, benzylpenicillin, and estrone-3-sulfate and moderately inhibited by taurocholate and p-aminohippurate (50-57%). Uptake of DHEAS and estrone-3-sulfate was greater in murine Oat3 cRNA-injected oocytes than that in water-injected oocytes. Efflux of these compounds from the brain was significantly delayed in Oat3(-/-) mice compared with that in wild-type mice, indicating that indeed Oat3 is functionally important in vivo. Furthermore, probenecid and taurocholate inhibited DHEAS efflux completely in Oat3(-/-) mice. Contrary to the past report in rats that suggested involvement of Oatp1a4, specific uptake of DHEAS and estrone-3-sulfate by murine Oatp1a4 was not detected in vitro, and efflux of both compounds from the brain was not altered in Oatp1a4(-/-) mice. There was no significant difference in the uptake of DHEAS by brain slices prepared from wild-type, Oatp1a4(-/-), and Oat3(-/-) mice. Taken together, these results suggest that Oat3 plays a significant role in the efflux of steroid conjugates across the BBB in mice and that the BBB also expresses other unknown organic anion transporters for the efflux of DHEAS. Transport mechanisms of organic anions at the BBB are far more diverse than they were assumed to be.

Validation of steroid sulfates deconjugation for metabolic studies. Application to human urine samples.

BACKGROUND: Urinary sulfate fraction of the anabolic androgenic steroids is not analyzed routinely in anti-doping analyses but has demonstrated in the last years an increasing interest among the anti-doping community. Sulfate conjugates are linked to plasma proteins increasing the residence time in the body compared to glucuro-conjugated metabolites, and then their analyses may allow improving the detection time window of specific metabolites. Hydrolysis of sulfates can be made enzymatically or chemically and can be challenging, depending on the strategy selected. METHODS: Hydrolysis by solvolysis was validated for metabolic studies, focusing on setting a quality control able to assess the hydrolytic step. To the internal standards mixture, androsterone-D4 and etiocholanolone-D5 sulfate were added. The proposed protocol was applied over samples collected after dehydroepiandrosterone (DHEA) administrations. RESULTS: The stability of the structures showed good results, and no evident formation of degradation products was observed. Internal standard to monitor the efficiency of hydrolysis, recovery, and retention time was successfully introduced. Additional analytes (4ß-hydroxy-DHEA, 5-androstene-3ß,17ß-diol and 5α-androstane-3ß,17ß-diol) were found to be affected besides of DHEA and epiandrosterone (epiA) as previously described. CONCLUSIONS: Results in terms of linearity, precision, and accuracy, showed that the method is suitable to quantify seven analytes in urine in the sulfated fraction. The validated method was successfully applied to urine samples after administration of DHEA to detect this compound in the sulfate fraction and preliminarily to negative samples from athletes of both sexes, to determine Q1 and Q3 inter-quartiles. A quality control assessment for the hydrolysis efficiency was established for every individual sample.

Effects of mirtazapine on dehydroepiandrosterone-sulfate and cortisol plasma concentrations in depressed patients.

BACKGROUND: Among the neuroactive steroids, dehydroepiandrosterone sulfate (DHEA-S) is at least in part produced in the adrenal gland and is therefore under the control of the hypothalamic-pituitary-adrenocortical (HPA)-system. In the present study, the impact of mirtazapine on DHEA-S and cortisol (COR) levels was investigated in relation to clinical response in depressed patients. METHODS: A total of 23 inpatients 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 h and quantified for COR and DHEA-S levels. RESULTS: Mirtazapine significantly reduced both COR and DHEA-S concentrations, but had no impact on the COR/DHEA-S ratio. The percentage decrease of DHEA-S, but not that of COR was significantly and positively correlated with the percentage reduction in the sum score of the Hamilton Depression Rating Scale at week 5, suggesting a relationship between DHEA-S reduction and clinical efficacy of mirtazapine. There was a significant positive correlation between the decline in COR and DHEA-S levels. CONCLUSIONS: Apparently, the decrease in COR and DHEA-S concentrations conjointly reflects an attenuating impact of mirtazapine on HPA axis activity, thereby decreasing the adrenal secretion of COR and DHEA-S.

Modulation of neurotransmitter systems by dehydroepiandrosterone and dehydroepiandrosterone sulfate: mechanism of action and relevance to psychiatric disorders.

Dehydroepiandrosterone (DHEA) is synthesized in the brain and several studies have shown that this steroid is a modulator of synaptic transmission. The effect of DHEA, and its sulfate ester DHEAS, on glutamate and GABA neurotransmission has been extensively studied but some effects on other neurotransmitter systems, such as dopamine, serotonin and nitric oxide, have also been reported. This review summarizes studies showing the effect of DHEA and DHEAS on neurotransmitter systems at different levels (metabolism, release, reuptake, receptor activation), as well as the activation of voltage-gated ion channels and calcium homeostasis, showing the variety of effects that these steroids exert on those systems, allowing the discussion of its mechanisms of action and its relevance to psychiatric disorders.

[The applications of chiroptical spectroscopy for the determination and the detection of steroids and for the examination of their cyclodextrin-mediated enantioselective solubility]. / A kiroptikai spektroszkópia alkalmazása szteroidok meghatározására es elválasztásuk detektálására, valamint ciklodextrinekkel történo enantioszelektív oldhatóságuk tanulmányozására.

Chiroptical spectroscopy is one of the most developing areas of chiral analysis which role has been enhanced in recent years. In the thesis new applications of circular dichroism (CD) and optical rotatory dispersion (ORD) methods are presented via some examples. A quick and accurate CD spectroscopic method was developed for the simultaneous determination of ethisterone and its delta5-isomer (delta5-ethisterone). The method is based on the selective negative Cotton effect of the delta4-3-oxo group of ethisterone. The concentrations of the two isomers can be easily determined by ellipticity measurements at two different wavelengths without the application of any separation technique. CD detection for the separation of ethisterone isomers by high performance liquid chromatography (HPLC) is suitable even for the determination of the delta5-ethisterone, that contains isolated oxo groups. The separation of dehydroepiandrosterone (DHEA), dehydroepi-androsterone sulfate (DHEA-S) and related steroids was also elaborated by a CD detected HPLC technique. CD detection is a good tool for the determination of saturated ketones. Enantioselectivity was observed when the solubility of raceme norgestrel was examined in aqueous solutions of gamma-cyclodextrin and hydroxypropyl-gamma-cyclodextrin. A CD spectroscopic method, based on the measurement of g-factor was applied for the determination of the enantiomer ratio obtained. Levonorgestrel, the effective enantiomer was dissolved in greater extent using either cyclodextrins. The obtained solubility of norgestrel was greater using gamma-cyclodextrin, although the enantioselectivity was more significant when hydroxypropyl-gamma-cyclodextrin was applied. Phase-solubility examinations were appropriate for the verification of 1:1 stoichiometry and that the enantiomer ratios were independent of the cyclodextrin concentrations. A new calculation method was also elaborated, suitable for the direct determination of the concentrations of norgestrel enantiomers in aqueous cyclodextrin solutions.

Dehydroepiandrostenedione sulphate interferes in many direct immunoassays for testosterone.

BACKGROUND: In response to a report that the Abbott Architect direct testosterone immunoassay was affected by dehydroepiandrostenedione sulphate (DHEAS) cross-reactivity, this study investigated the effect of DHEAS on other testosterone methods using a UK National External Quality Assessment Scheme (UK NEQAS) for steroid hormones special distribution. METHODS: Separate male and female matrix pools of normal human serum were prepared and divided into three portions. Two portions from each matrix were spiked with 10.07 and 20.14. micromol/L DHEAS, respectively. Aliquots of these pools were distributed to participants in UK NEQAS for male and female testosterone at distribution 319 (July 2006). Median testosterone results for each method group were examined for an increase in testosterone concentration. RESULTS: In the female matrix, the effect on Abbott Architect was confirmed and shown also for Roche Elecsys, Roche E170 Modular and Beckman Access (includes some Beckman DxI users). In the male matrix, the effect on Abbott Architect, Roche Elecsys and Roche E170 Modular was similar in magnitude to that in the female matrix, but was smaller for Beckman Access/DxI. However, the effect on Immulite 2000 (includes some Immulite 2500 users) was greater in the male matrix. Indications of interference in other methods, used by only a few laboratories (notably Tosoh AIA and Wallac Delfia), were also observed. CONCLUSIONS: DHEAS interference in testosterone assays is a feature of many routine testosterone methods, and occurs in both male and female matrices.

Determination of salivary 17-ketosteroid sulfates using liquid chromatography-electrospray ionization-mass spectrometry.

A simple and sensitive liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) method for the simultaneous quantification of representative 17-ketosteroid sulfates, dehydroepiandrosterone sulfate (DHEAS), androsterone sulfate and epiandrosterone sulfate, in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a solid-phase extraction cartridge and subjected to LC-MS. Deuterium-labeled DHEAS was used as the internal standard and quantification was based on the selected ion monitoring (SIM) mode of each deprotonated molecule. This method allowed the reproducible and accurate quantification of the salivary sulfates using a 100-microl sample; the intra- and inter-assay coefficients of variation were below 6.8 and 6.3%, respectively, and the % accuracy values were quantitative for all the sulfates. The limits of quantitation for all the sulfates were 100 pg/ml. No significant matrix effect or change in the measured value by freeze/thaw repetitions was observed. The developed method was applied to clinical studies, and produced satisfactory results.

High-Affinity Nucleic-Acid-Based Receptors for Steroids.

Artificial receptors for hydrophobic molecules usually have moderate affinities and limited selectivities. We describe three new classes of high affinity hydrophobic receptors for nonaromatic steroids based on deoxyribonucleotides, obtained through five high stringency selections coupled with tailored counter-selections. The isolation of multiple classes of high affinity steroid receptors demonstrates the surprising breadth of moderately sized hydrophobic binding motifs (<40 nucleotides) available to natural nucleic acids. Studies of interactions with analogs indicate that two classes, four-way junctions and 4XGN motifs, comprise receptors with shapes that prevent binding of specific steroid conjugates used in counter-selections. Furthermore, they strongly prefer nonhydroxylated steroid cores, which is typical for hydrophobic receptors. The third new class accommodates hydroxyl groups in high-affinity, high-selectivity binding pockets, thus reversing the preferences of the first two classes. The high-affinity binding of aptamers to targets efficiently inhibits double-helix formation in the presence of the complementary oligonucleotides. The high affinity of some of these receptors and tailored elimination of binding through counter-selections ensures that these new aptamers will enable clinical chemistry applications.

Dehydroepiandrosterone Sulfate Stimulates Expression of Blood-Testis-Barrier Proteins Claudin-3 and -5 and Tight Junction Formation via a Gnα11-Coupled Receptor in Sertoli Cells.

Dehydroepiandrosterone sulfate (DHEAS) is a circulating sulfated steroid considered to be a pro-androgen in mammalian physiology. Here we show that at a physiological concentration (1 µM), DHEAS induces the phosphorylation of the kinase Erk1/2 and of the transcription factors CREB and ATF-1 in the murine Sertoli cell line TM4. This signaling cascade stimulates the expression of the tight junction (TJ) proteins claudin-3 and claudin-5. As a consequence of the increased expression, tight junction connections between neighboring Sertoli cells are augmented, as demonstrated by measurements of transepithelial resistance. Phosphorylation of Erk1/2, CREB, or ATF-1 is not affected by the presence of the steroid sulfatase inhibitor STX64. Erk1/2 phosphorylation was not observed when dehydroepiandrosterone (DHEA) was used instead of DHEAS. Abrogation of androgen receptor (AR) expression by siRNA did not affect DHEAS-stimulated Erk1/2 phosphorylation, nor did it change DHEAS-induced stimulation of claudin-3 and claudin-5 expression. All of the above indicate that desulfation and conversion of DHEAS into a different steroid hormone is not required to trigger the DHEAS-induced signaling cascade. All activating effects of DHEAS, however, are abolished when the expression of the G-protein Gnα11 is suppressed by siRNA, including claudin-3 and -5 expression and TJ formation between neighboring Sertoli cells as indicated by reduced transepithelial resistance. Taken together, these results are consistent with the effects of DHEAS being mediated through a membrane-bound G-protein-coupled receptor interacting with Gnα11 in a signaling pathway that resembles the non-classical signaling pathways of steroid hormones. Considering the fact that DHEAS is produced in reproductive organs, these findings also suggest that DHEAS, by acting as an autonomous steroid hormone and influencing the formation and dynamics of the TJ at the blood-testis barrier, might play a crucial role for the regulation and maintenance of male fertility.

Estrone sulfate and dehydroepiandrosterone sulfate: Transactivation of the estrogen and androgen receptor.

Dehydroepiandrosterone sulfate (DHEAS) and estrone sulfate (E1S) are two of the most abundant steroids in the human circulation. The enzyme steroid sulfatase (STS) cleaves the sulfate group of DHEAS and E1S leading to biosynthesis of endogenous hormones such as testosterone and estrone. In the current study we aimed at determining the effect of E1S and DHEAS on estrogen receptor (ER) and androgen receptor (AR) transactivation. Using luciferase reporter gene assays, the ER and AR transactivities of E1S and DHEAS were determined by direct cell exposure; as well as upon extraction from human serum using a method to extract perfluorinated alkyl acids (PFAAs). By direct cell exposure, both E1S and DHEAS transactivated the ER and the AR in dose-dependent manners. The DHEAS-induced AR transactivity could be abolished by the STS inhibitor STX64. Immunoassay analysis confirmed the presence of E1S and DHEAS in the serum PFAA extracts with mean recoveries below 2.5%. For the PFAA extracts of human male and female serum, only the AR was significantly transactivated. The AR transactivity of the sulfated steroids in the extracts was abolished by STX64 to obtain the net PFAA induced xenohormone transactivity, but further cleanup might be needed at high concentrations of E1S.