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The sodium-dependent organic anion transporter (SOAT, gene symbol SLC10A6) specifically transports 3′- and 17′-monosulfated steroid hormones, such as estrone sulfate and dehydroepiandrosterone sulfate, into specific target cells. These biologically inactive sulfo-conjugated steroids occur in high concentrations in the blood circulation and serve as precursors for the intracrine formation of active estrogens and androgens that contribute to the overall regulation of steroids in many peripheral tissues. Although SOAT expression has been detected in several hormone-responsive peripheral tissues, its quantitative contribution to steroid sulfate uptake in different organs is still not completely clear. Given this fact, the present review provides a comprehensive overview of the current knowledge about the SOAT by summarizing all experimental findings obtained since its first cloning in 2004 and by processing SOAT/SLC10A6-related data from genome-wide protein and mRNA expression databases. In conclusion, despite a significantly increased understanding of the function and physiological significance of the SOAT over the past 20 years, further studies are needed to finally establish it as a potential drug target for endocrine-based therapy of steroid-responsive diseases such as hormone-dependent breast cancer. [ABSTRACT FROM AUTHOR]

• Steroid sulfatase (STS) was examined in male and female mouse tissues. • STS activity was high in livers of both sexes and was also high in testes. • Hepatic and testicular STS activities were lower in younger mice. • STS protein was visualized in hepatocytes, Leydig cells and seminiferous tubules. Steroid hormones often circulate in the plasma as inactive sulfated forms, such as estrone sulfate and dehydroepiandrosterone sulfate. The enzyme steroid sulfatase (STS) converts these steroids into active forms, mainly estrogens, in peripheral tissues. STS is present in most tissues, but it occurs at higher levels in certain organs, notably liver and placenta. In this study, we examined the tissue distribution of STS in a prominent laboratory model, the house mouse (Mus musculus). Tissues included were heart, liver, small intestine, skeletal muscle, and gonads of both sexes. An 3H-estrone-sulfate conversion assay was used to measure STS activity in tissue homogenates and extracts. STS activities were high for hepatic tissue homogenates of both genders. Testicular STS levels were similar to those of liver, while STS activities of ovary, small intestine, heart, and muscle were considerably lower. The specific STS inhibitors, EMATE and STX-64 virtually eliminated STS activity in hepatic microsomes and cytosols, verifying that the observed enzyme activity was due to STS. Enzyme kinetic assays showed Km values of 8.6 µM for liver and 9.1 µM for testis, using E 1 S as substrate. Hepatic and testicular STS activities, measured in CHAPS-extracted microsome, were low up to 5 weeks of age and were higher through 56 weeks. Western blotting, with a specific STS antibody, confirmed the presence of STS protein (65 Da) in both liver and testis. Immunofluorescence of tissue sections detected the presence of STS protein in hepatocytes, in testicular Leydig cells and in seminiferous tubules (Leydig cells and developing germ cells). These results suggest that STS may have a significant role in testicular function. [ABSTRACT FROM AUTHOR]

Current heightened recognition of the importance of sulfated steroids led to the examination of conjugates in media from incubations of estrogens in tissues from the reproductive tract of stallions. Previously, we had reported a 'new' unidentified metabolite of estrone (E1) and [³H]-E1, located between 17β-estradiol (E2) and E1 reference standards on chromatography (HPLC) and identified tentatively as a stable 5α,6α-estrone epoxide. Stallion tissues were minced and incubated for 2 h with [³H]-E1 (1 × 106 cpm). Solid-phase extraction of unconjugated and conjugated steroids from media was followed by liquid scintillation counting (LSC), where radioactivity was mostly in the conjugate fractions (>80%). HPLC of conjugated steroids used an isocratic solvent system of acetonitrile/water (8:92) at 700 µL/min with detection by LSC. A radioactive peak between reference standards of E1 and E2 sulfates was examined, after solvolysis, in a second solvent system. Sulfated steroids yielded largely E1, whereas acid treatment of the unconjugated E1 epoxide had earlier formed 6α-OH-E1 almost exclusively. With sulfatase enzyme, at neutral pH, radioactivity was also seen mostly as E1 with trace amounts of polar material. Reduction with KBH4, however, led also to desulfation; radioactivity had alignment with E2 but even more had low retention times as for 6α/6β-OH-E2. These findings point to a different hydrolysis for desulfation; even more, they reveal an additional oxygen atom at C6 and are supportive of biological formation of 5α,6α-epoxides of E1 and E2. As possible alternatives to catechol estrogens, implicated in cancer, the 'new' estrogen metabolites and their sulfated forms may have special interest. [ABSTRACT FROM AUTHOR]

The sodium-dependent organic anion transporter (SOAT, gene symbol SLC10A6) specifically transports 3′- and 17′-monosulfated steroid hormones, such as estrone sulfate and dehydroepiandrosterone sulfate, into specific target cells. These biologically inactive sulfo-conjugated steroids occur in high concentrations in the blood circulation and serve as precursors for the intracrine formation of active estrogens and androgens that contribute to the overall regulation of steroids in many peripheral tissues. Although SOAT expression has been detected in several hormone-responsive peripheral tissues, its quantitative contribution to steroid sulfate uptake in different organs is still not completely clear. Given this fact, the present review provides a comprehensive overview of the current knowledge about the SOAT by summarizing all experimental findings obtained since its first cloning in 2004 and by processing SOAT/SLC10A6-related data from genome-wide protein and mRNA expression databases. In conclusion, despite a significantly increased understanding of the function and physiological significance of the SOAT over the past 20 years, further studies are needed to finally establish it as a potential drug target for endocrine-based therapy of steroid-responsive diseases such as hormone-dependent breast cancer.

Objective: Little information is available on the steroid sulfates profile in obese children. Therefore, we examined whether sulfated steroids are linked with weight status and associated comorbidities in obese children. Methods: We analyzed 66 obese children (mean age 10.5 ± 2.5 years, 57.6% female, 53.9% prepubertal, mean BMI 27.0 ± 4.6 kg/m2, 50% with BMI-SDS reduction >0.5, 50% without BMI-SDS reduction) who participated in an outpatient 1-year intervention program based on exercise, behavior and nutrition therapy. We measured intact sulfated steroids (cholesterol sulfate (CS), pregnenolone sulfate (PregS), 17αOH pregnenolone sulfate (17OH-PregS), 16αOH dehydroepiandrosterone sulfate (16OH-DHEAS), DHEAS, androstenediol-3-sulfate, androsterone sulfate and epiandrosterone sulfate) by LC–MS/MS, and insulin resistance index HOMA, lipids, blood pressure at baseline and 1 year later. Results: All sulfated steroids except 17OH-PregS, 16OH-DHEAS, androsterone sulfate and epiandrosterone sulfate were higher in boys compared to girls. Concentrations of CS before intervention were higher in children who lost weight. After 1 year of treatment, both groups showed increased levels of DHEAS, 16OH-DHEAS and androstenediol-3-sulfate, but PregS was only increased in children with weight loss. None of the steroid sulfates was significantly related to cardiovascular risk factors or HOMA except 17OH-PregS, which was associated with systolic blood pressure both in cross-sectional (β-coefficient: 0.09 ± 0.07, P = 0.020) and longitudinal analyses (β-coefficient: 0.06 ± 0.04, P = 0.013) in multiple linear regression analyses. Conclusions: Since higher steroid sulfation capacity was associated with successful weight intervention in children disruption of sulfation may be associated with difficulties to lose weight. Future studies are necessary to prove this hypothesis.

Accurate investigations of adrenal hormone levels play a vital role in pediatric endocrinology for the detection of steroid-related disorders. In this study, we developed and validated a simultaneous assay for eight serum steroids, i.e., DHEA, androstenedione, testosterone, progesterone, 17‑hydroxyprogesterone, DHEA‑sulfate, pregnenolone‑sulfate and cholesterol-sulfate, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Serum samples were prepared by liquid-liquid extraction with methyl t ‑butyl ether. Quantitation by LC-MS/MS was performed in multiple reaction monitoring mode with an electrospray ionization source. The run time was 10 min. Analytical performance was evaluated, including imprecision, linearity, ion suppression, carry over and detection capabilities. Serum specimens from 59 children and 120 adults were analyzed to compare the distribution of steroid levels between the groups. All hormones were effectively extracted and separated using our method. The method was essentially free from potential interference and ion suppression. Within-run and between-run imprecision values were <20%. The lower limits of quantification varied from 0.025 to 10 ng/mL. The results were generally good and correlated with those obtained using immunoassay techniques. We developed the HPLC-MS/MS method for the simultaneous measurement of free and sulfated steroid hormones. The performance of the developed method was generally acceptable. Thus, this method may provide a novel approach to steroid profiling in children of age before adrenarche. • We developed HPLC-MS/MS method for the simultaneous measurement of serum free and sulfated steroids. • A novel assay was developed to detect low concentrations of serum steroids in children. • Satisfactory imprecision, linearity, and detection limits were analyzed. • Lower values were obtained by the LC-MS/MS technique than immunoassay. [ABSTRACT FROM AUTHOR]

Steroids are primarily present in human fluids in their sulfated forms. Profiling of these compounds is important from both diagnostic and physiological points of view. Here, we present a novel method for the quantification of 11 intact steroid sulfates in human serum by LC-MS/MS. The compounds analyzed in our method, some of which are quantified for the first time in blood, include cholesterol sulfate, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-α-hydroxy-dehydroepiandrosterone sulfate, dehydroepiandrosterone sulfate, androstenediol sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and dihydrotestosterone sulfate. The assay was conceived to quantify sulfated steroids in a broad range of concentrations, requiring only 300 μl of serum. The method has been validated and its performance was studied at three quality controls, selected for each compound according to its physiological concentration. The assay showed good linearity (R2 > 0.99) and recovery for all the compounds, with limits of quantification ranging between 1 and 80 ng/ml. Averaged intra-day and between-day precisions (coefficient of variation) and accuracies (relative errors) were below 10%. The method has been successfully applied to study the sulfated steroidome in diseases such as steroid sulfatase deficiency, proving its diagnostic value. This is, to our best knowledge, the most comprehensive method available for the quantification of sulfated steroids in human blood.

Three new sulfated steroid monoglycosides, leptaochotensosides A–C (1–3), and a new sulfated polyhydroxylated steroid (4) were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis. The structures of compounds 1–4 were established by extensive nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESIMS) analyses and chemical transformations. Although the isolated compounds did not show any apparent cytotoxicity against melanoma RPMI-7951 and breast cancer T-47D cell lines, leptaochotensoside A (1) demonstrated inhibition of T-47D cell colony formation in a soft agar clonogenic assay at nontoxic doses. In addition, this compound decreased the epidermal growth factor (EGF)-induced colony formation of mouse epidermal JB6 Cl41 cells. The cancer preventive action of 1 is realized through regulation of mitogen-activated protein kinase (MAPK) signaling pathway.

Steroid sulfatase (STS) deficiency is the underlying cause of the skin condition known as recessive X-linked ichthyosis (RXLI). RXLI patients show scales on their skin caused by high concentrations of cholesterol sulfate (CS), as they are not capable of releasing the sulfate group from its structure to obtain free cholesterol. CS has been reported, so far, as the sole sulfated steroid with increased concentrations in the blood of RXLI patients. A non-targeted LC-MS approach in negative mode detection (LC-MS precursor ion scan mode) was applied to serum samples of 12 RXLI patients and 19 healthy males. We found that CS was not the only sulfated compound consistently elevated in RXLI patients, because a group of compounds with a m/z of 481 was found in high concentrations too. Further LC-MS/MS demonstrated that the main contributor to the m/z 481 signal in RXLI serum is 27-hydroxycholesterol-3-sulfate (27OHC3S). Accordingly, a new method for 27OHC3S quantification in the context of RXLI has been developed and validated. Other hydroxycholesterol sulfate compounds were elevated as well in RXLI patients.

The sodium-dependent organic anion transporter SOAT/Soat shows highly specific transport activity for sulfated steroids. SOAT substrates identified so far include dehydroepiandrosterone sulfate, 16α-hydroxydehydroepiandrosterone sulfate, estrone-3-sulfate, pregnenolone sulfate, 17β-estradiol-3-sulfate, and androstenediol sulfate. Apart from these compounds, many other sulfated steroids occur in mammals. Therefore, we aimed to expand the substrate spectrum of SOAT and analyzed the SOAT-mediated transport of eight different sulfated steroids by combining in vitro transport experiments in SOAT-transfected HEK293 cells with LC–MS/MS analytics of cell lysates. In addition, we aimed to better understand the structural requirements for SOAT substrates and so selected structural pairs varying only at specific positions: 3α/3β-sulfate, 17α/17β-sulfate, mono-sulfate/di-sulfate, and 17α-hydroxylation. We found significant and sodium-dependent SOAT-mediated transport of 17α-hydroxypregnenolone sulfate, 17β-estradiol-17-sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and 5α-dihydrotestosterone sulfate. However, 17β-estradiol-3,17-disulfate was not transported by SOAT. In conclusion SOAT substrates from the group of sulfated steroids are characterized by a planar and lipophilic steroid backbone in trans-trans - trans conformation of the rings and a negatively charged mono-sulfate group at positions 3′ or 17′ with flexibility for α- or β- orientation. Furthermore, 5α-reduction, 16α-hydroxylation, and 17α-hydroxylation are acceptable for SOAT substrate recognition, whereas addition of a second negatively charged sulfate group seems to abolish substrate binding to SOAT, and so 17β-estradiol-3,17-disulfate is not transported by SOAT. [ABSTRACT FROM AUTHOR]

The sodium-dependent organic anion transporter SOAT (gene name SLC10A6 in man and Slc10a6 in mice) is a plasma membrane transporter for sulfated steroids, which is highly expressed in germ cells of the testis. SOAT can transport biologically inactive sulfated steroids into specific target cells, where they can be reactivated by the steroid sulfatase (STS) to biologically active, unconjugated steroids known to regulate spermatogenesis. Significantly reduced SOAT mRNA expression was previously found in different forms of impaired spermatogenesis in man. It was supposed that SOAT plays a role for the local supply of steroids in the testis and consequently for spermatogenesis and fertility. Thus, an Slc10a6 −/− Soat knockout mouse model was established by recombination-based target deletion of the Slc10a6 gene to elucidate the role of Soat in reproduction. However, the Slc10a6 −/− knockout mice were fertile, produced normal litter sizes, and had normal spermatogenesis and sperm vitality. This phenotype suggests that the loss of Soat can be compensated in the knockout mice or that Soat function is not essential for reproduction. In addition to reproductive phenotyping, a comprehensive targeted steroid analysis including a set of 9 un-conjugated and 12 sulfo-conjugated steroids was performed in serum of Slc10a6 −/− knockout and Slc10a6 +/+ wildtype mice. Only cholesterol sulfate, corticosterone, and testosterone (only in the males) could be detected in considerable amounts. Interestingly, male Slc10a6 −/− knockout mice showed significantly higher serum levels for cholesterol sulfate compared to their wildtype controls. As cholesterol sulfate has a broader impact apart from the testis, further analysis of this phenotype will include other organs such as skin and lung, which also show high Soat expression in the mouse. [ABSTRACT FROM AUTHOR]

Sulfo-conjugated steroid hormones, such as dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate or estrone-3-sulfate are abundant in the body, but are biologically inactive at classical androgen and estrogen steroid receptors. However, after carrier-mediated import and de-conjugation by the steroid sulfatase, these compounds participate in the overall steroid regulation of reproductive organs. The sodium-dependent organic anion transporter SOAT, coded by the SLC10A6 gene, is specific for the transport of steroid sulfates and is highly expressed in testicular germ cells, including pachytene spermatocytes, secondary spermatocytes, and round spermatids. Therefore, SOAT is supposed to be involved in the regulation of spermatogenesis and male fertility. In the present study, the SLC10A6 gene was analyzed for rare genetic variants, which might affect transport function or membrane expression of SOAT. Among the 31 SOAT variants analyzed, L44P, Q75R, P107L, G109S, S112F, N113K, S133F, G241D, G263E, G294R, and Y308N showed no transport activity for DHEAS at all. In the case of P107L, G241D, G263E, and Y308N, this was most likely due to significantly reduced expression in the plasma membrane. Other variants are located directly at (Q75R, S112F, N113K) or close to (G109S, S133F, and G263E) the supposed SOAT Na + binding sites and thus could disable the sodium-coupled transport cycle. If these loss-of-function SOAT variants are more frequent in men with impaired spermatogenesis or infertility needs further investigation. [ABSTRACT FROM AUTHOR]

Sodium-dependent organic anion transporter (SOAT) represents a membrane transporter specific for sulfated steroid hormones, which are supposed to participate in the regulation of reproductive processes. In man, SOAT shows predominant mRNA expression in the testis and here was localized to primary spermatocytes. SOAT mRNA expression is significantly downregulated in different disorders of spermatogenesis, including hypospermatogenesis. The resulting decline of SOAT-mediated transport of sulfated steroids may participate in the impairment of functional spermatogenesis. Apart from downregulation of SOAT mRNA expression, genetic polymorphisms affecting the transport function of SOAT may have the same negative effect on spermatogenesis. Therefore, in the present study we searched for functionally relevant SOAT polymorphisms, aiming to comparatively analyze their occurrence in patients with impaired spermatogenesis vs. patients with intact spermatogenesis. We found that the SOAT polymorphism L204F showed a significantly reduced transport function for DHEAS when expressed in HEK293 cells. Although the K m value was identical with that of the SOAT wildtype, the V max value dramatically declined for the SOAT-L204F variant (942.5 vs. 313.6 pmol × mg protein −1 × min −1 ). Although the same amount of total SOAT-L204F protein was detected in transfected HEK293 cells compared to the SOAT wildtype, plasma membrane expression was significantly reduced, which points to a plasma membrane sorting defect of the SOAT-L204F variant. Groups of 20 subjects with normal spermatogenesis and 26 subjects with hypospermatogenesis were genotyped for this polymorphism. Both groups showed nearly identical distributions of the SOAT-L204F polymorphism (∼10% heterozygous and ∼5% homozygous), indicating that this polymorphism seems not be causative for hypospermatogenesis. [ABSTRACT FROM AUTHOR]

The impact of steroid sulfatase (STS) activity in the circulating levels of both sulfated and unconjugated steroids is only partially known. In addition, the sulfated steroid pathway, a parallel pathway to the one for unconjugated steroids, which uses the same enzymes, has never been characterized in detail before. Patients with steroid sulfatase deficiency (STSD) are unable to enzymatically convert sulfated steroids into their unconjugated forms, and are a good model to elucidate how STS affects steroid biosynthesis and to study the metabolism of sulfated steroids. We quantified unconjugated and sulfated steroids in STSD serum, and compared these results with data obtained from serum of healthy controls. Most sulfated steroids were increased in STSD. However, androstenediol-3-sulfate and epiandrosterone sulfate showed similar levels in both groups, and the concentrations of androsterone sulfate were notably lower. Hydroxylated forms of DHEAS and of pregnenolone sulfate were found to be increased in STSD, suggesting a mechanism to improve the excretion of sulfated steroids. STSD testosterone concentrations were normal, but cholesterol and DHEA were significantly decreased. Additionally, serum bile acids were three-fold higher in STSD. Correlations between concentrations of steroids in each group indicate that 17α-hydroxy-pregnenolone-3-sulfate in men is mainly biosynthesized from the precursor pregnenolone sulfate and androstenediol-3-sulfate from DHEAS. These findings confirm the coexistence of two steroidogenic pathways: one for unconjugated steroids and another one for sulfated steroids. Each pathway is responsible for the synthesis of specific steroids. The equal levels of testosterone, and the reduced level of unconjugated precursors in STSD, support that testosterone is primarily synthesized from sulfated steroids. In consequence, testosterone synthesis in STSD relies on an enzyme with sulfatase activity other than STS. This study reveals that STS is a key player of steroid biosynthesis regulating the availability of circulating cholesterol. [ABSTRACT FROM AUTHOR]

Within the human testis, large amounts of sulfated steroid hormones are produced. As shown in breast tissue and placenta, these might not only be excretion intermediates, but re-activated in target cells by steroid sulfatase (STS). This process is called sulfatase pathway and may play a pivotal role in para- and/or intracrine regulation by creating a local supply for steroid hormones. This requires a facilitated transport via uptake carriers and efflux transporters as these hydrophilic molecules cannot pass the cell membrane. Moreover, blood-testis barrier formation in the testis requires a transport through Sertoli cells (SCs) to reach germ cells (GCs). Sertoli cells are therefore expected to play a key role as gate-keepers for sulfatase pathway in human seminiferous epithelium. We analyzed the mRNA and protein expression of uptake carriers and efflux transporters like organic anion-transporting polypeptides (OATP2B1, OATP3A1) and multidrug resistance-related proteins (MRP1, MRP4) in testicular tissue and cultured Sertoli cells (FS1, HSEC). Additionally, expression pattern of STS as well as sulfonating enzymes (SULTs) were assessed. OATP2B1, OATP3A1 and STS were detected in SCs as well as GCs, whereas MRP1 is only expressed in SCs, and SULT1E1 only in Leydig cells, respectively. By transcellular transport of [H]DHEAS in HSEC, we showed a functional transport of sulfated steroids in vitro. Our data indicate that steroid synthesis via sulfatase pathway in Sertoli cells in vivo and in vitro is possible and may contribute to paracrine and intracrine regulation employing the local supply of sulfated and free steroid hormones inside seminiferous tubules. [ABSTRACT FROM AUTHOR]

The mouse vomeronasal organ expresses chemosensory receptors that detect intra-species as well as inter-species cues. The vomeronasal neurons are thought to be highly selective in their responses. The tuning properties of individual receptors remain difficult to characterize due to the lack of a robust heterologous expression system. Here, we take a transgenic approach to ectopically express two Type 1 vomeronasal receptors in the mouse vomeronasal organ and characterize their responses to steroid compounds. We find that V1rj2 and V1rj3 are sensitive to two sulfated estrogens and can be activated by a broad variety of sulfated and glucuronidated steroids at high concentrations. Individual neurons exhibit narrow range of concentration-dependent activation. Collectively, a neuronal population expressing the same receptor covers a wide dynamic range in their responses to sulfated estrogens. These properties recapitulate the response profiles of endogenous neurons to sulfated estrogens.

Three new sulfated steroid monoglycosides, leptaochotensosides A-C (1-3), and a new sulfated polyhydroxylated steroid (4) were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis. The structures of compounds 1-4 were established by extensive nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESIMS) analyses and chemical transformations. Although the isolated compounds did not show any apparent cytotoxicity against melanoma RPMI-7951 and breast cancer T-47D cell lines, leptaochotensoside A (1) demonstrated inhibition of T-47D cell colony formation in a soft agar clonogenic assay at nontoxic doses. In addition, this compound decreased the epidermal growth factor (EGF)-induced colony formation of mouse epidermal JB6 Cl41 cells. The cancer preventive action of 1 is realized through regulation of mitogen-activated protein kinase (MAPK) signaling pathway. [ABSTRACT FROM AUTHOR]

The mouse vomeronasal organ (VNO) expresses chemosensory receptors that detect intra-species as well as inter-species cues. The vomeronasal neurons are thought to be highly selective in their responses. The tuning properties of individual receptors remain difficult to characterize due to the lack of a robust heterologous expression system. Here, we take a transgenic approach to ectopically express two type 1 vomeronasal receptors in the mouse VNO and characterize their responses to steroid compounds. We find that V1rj2 and V1rj3 are sensitive to two sulfated estrogens (SEs) and can be activated by a broad variety of sulfated and glucuronidated steroids at high concentrations. Individual neurons exhibit narrow range of concentration-dependent activation. Collectively, a neuronal population expressing the same receptor covers a wide dynamic range in their responses to SEs. These properties recapitulate the response profiles of endogenous neurons to SEs. [ABSTRACT FROM AUTHOR]

Highlights: [•] 6E-hydroximinosteroids were synthesized from the corresponding ketones. [•] Cytotoxic activity of the compounds was evaluated against PC-3 and LNCaP. [•] The biological activity was compared to that of sulfated polyhydroxysteroid analogs. [•] 2β,3α,6β-sulfated steroid showed the highest cytotoxicity against LNCaP. [Copyright &y& Elsevier]

Highlights: [•] Disulfated and trisulfated steroids were synthesized starting from cholesterol. [•] Acetylcholinesterase inhibitory activity of the compounds was evaluated. [•] 2β,3α,6α-Sulfated steroid showed the highest inhibitory activity. [•] Diols and triols were inactive. [ABSTRACT FROM AUTHOR]

Abstract: Disodium 2β,3α-dihydroxy-5α-cholestan-6-one disulfate (8) has been synthesized using cholesterol (1) as starting material. Sulfation was performed using trimethylamine–sulfur trioxide complex in dimethylformamide as the sulfating agent. The acetylcholinesterase inhibitory activity of compound 8 was evaluated and compared to that of disodium 2β,3α-dihydroxy-5α-cholestane disulfate (10) and diols 7 and 9. Compounds 8 and 10 were active with IC50 values of 14.59 and 59.65μM, respectively. Diols 7 and 9 showed no inhibitory activity (IC50 >500μM). [Copyright &y& Elsevier]

Five new steroid sulfates, sodium 2β,3α-dihydroxy-5α-cholestane 3-sulfate (6), sodium 2β,3α-dihydroxy-5α-cholestane 2-sulfate (7), disodium 2β,3α-dihydroxy-5α-cholestane disulfate (8), sodium 3α-acetoxy-2β-hydroxy-5α-cholestane 2-sulfate (12), and sodium 2β-acetoxy-3α-hydroxy-5α-cholestane 3-sulfate (13), have been synthesized starting from 3β-hydroxy-5α-cholestane (1). The synthetic steroids were completely characterized by one-dimensional and two-dimensional NMR and FABMS spectra. Sulfation was performed using triethylamine–sulfur trioxide complex in dimethylformamide as the sulfating agent. The sulfated steroids were comparatively evaluated for their inhibitory effect on the replication of herpes simplex virus type 2 (HSV-2). Compounds 7 and 8 were the most effective in their inhibitory action against HSV-2. The disulfated steroid 8 also proved to be active against DEN-2 and JV. [Copyright &y& Elsevier]

• Steroid sulfatase (STS) converts inactive steroids to active ones in peripheral tissues. • STS activity is present in mouse NIH-3T3 fibroblasts and in mouse ear and tail fibroblasts. • Glucocorticoids decrease STS activity in NIH-3T3 cells, not due to substrate inhibition. • STS in fibroblasts may increase estrogens and cortisol may decrease them, affecting inflammation. Steroid hormones often circulate in the blood as inactive sulfated forms, such as estrone sulfate and dehydroepiandrosterone sulfate. The enzyme steroid sulfatase (STS) converts these steroids into active forms, mainly estrogens, in peripheral tissues. We have previously characterized STS activity in human and mouse breast and bone tissues, and we have shown that STS can provide estrogens to these tissues from circulating sulfated precursors. This study was designed to characterize STS activity in a mouse fibroblast cell line (NIH-3T3). Using a radioactive estrone sulfate (E 1 S) conversion assay, we detected STS activity in cultured NIH-3T3 cells. This activity was blocked by the STS inhibitors EMATE and STX-64, indicating authentic STS activity. We also found that microsomes prepared from NIH-3T3 cells had relatively high STS activity and that cytosols had low activity, consistent with the known distribution of this enzyme to the endoplasmic reticulum. Michaelis-Menten analysis of the NIH-3T3 microsomes indicated a Km of 10.9 µM using E 1 S as substrate. Primary fibroblasts prepared from mouse ears and tails also had measurable STS activity, as indicated by 3H-E 1 S conversion assay, further supporting the conclusion that fibroblasts possess STS. Furthermore, Western blotting confirmed the presence of immunoreactive STS in NIH-3T3 microsomes. With regard to regulation, treatments of cultured NIH-3T3 cells revealed that cortisol and the synthetic glucocorticoids dexamethasone and prednisolone decreased STS activity, as we have found for cell lines from other tissues. The effect of cortisol was seen at both 10 µM and 1.0 µM but not at 0.1 µM. Western blotting also indicated a decrease in STS immunoreactivity in cortisol-treated microsomes. The reduction in STS activity by dexamethasone in whole cells was reversed by the glucocorticoid receptor antagonist RU-486, indicating that glucocorticoid downregulation of STS activity is receptor mediated. An inhibition assay on NIH-3T3 microsomes revealed that STS activity was inhibited significantly by 10 µM estradiol-17β, a known substrate inhibitor of E 1 S for STS, but not by 10 µM cortisol. This is consistent with the idea that cortisol inhibits STS in NIH-3T3 cells through a regulatory mechanism rather than by substrate inhibition. Our results could have important implications regarding local estrogen production by STS in fibroblasts, which are the most common connective tissue cells in the body, and on possible regulation of local estrogen levels by cortisol. [ABSTRACT FROM AUTHOR]

Objective Little information is available on the steroid sulfates profile in obese children. Therefore, we examined whether sulfated steroids are linked with weight status and associated comorbidities in obese children. Methods We analyzed 66 obese children (mean age 10.5 ± 2.5 years, 57.6% female, 53.9% prepubertal, mean BMI 27.0 ± 4.6 kg/m2, 50% with BMI-SDS reduction >0.5, 50% without BMI-SDS reduction) who participated in an outpatient 1-year intervention program based on exercise, behavior and nutrition therapy. We measured intact sulfated steroids (cholesterol sulfate (CS), pregnenolone sulfate (PregS), 17αOH pregnenolone sulfate (17OH-PregS), 16αOH dehydroepiandrosterone sulfate (16OH-DHEAS), DHEAS, androstenediol-3-sulfate, androsterone sulfate and epiandrosterone sulfate) by LC-MS/MS, and insulin resistance index HOMA, lipids, blood pressure at baseline and 1 year later. Results All sulfated steroids except 17OH-PregS, 16OH-DHEAS, androsterone sulfate and epiandrosterone sulfate were higher in boys compared to girls. Concentrations of CS before intervention were higher in children who lost weight. After 1 year of treatment, both groups showed increased levels of DHEAS, 16OH-DHEAS and androstenediol-3-sulfate, but PregS was only increased in children with weight loss. None of the steroid sulfates was significantly related to cardiovascular risk factors or HOMA except 17OH-PregS, which was associated with systolic blood pressure both in cross-sectional (β-coefficient: 0.09 ± 0.07, P = 0.020) and longitudinal analyses (β-coefficient: 0.06 ± 0.04, P = 0.013) in multiple linear regression analyses. Conclusions Since higher steroid sulfation capacity was associated with successful weight intervention in children disruption of sulfation may be associated with difficulties to lose weight. Future studies are necessary to prove this hypothesis.

Steroids are primarily present in human fluids in their sulfated forms. Profiling of these compounds is important from both diagnostic and physiological points of view. Here, we present a novel method for the quantification of 11 intact steroid sulfates in human serum by LC-MS/MS. The compounds analyzed in our method, some of which are quantified for the first time in blood, include cholesterol sulfate, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-α-hydroxy-dehydroepiandrosterone sulfate, dehydroepiandrosterone sulfate, androstenediol sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and dihydrotestosterone sulfate. The assay was conceived to quantify sulfated steroids in a broad range of concentrations, requiring only 300 μl of serum. The method has been validated and its performance was studied at three quality controls, selected for each compound according to its physiological concentration. The assay showed good linearity (R(2) > 0.99) and recovery for all the compounds, with limits of quantification ranging between 1 and 80 ng/ml. Averaged intra-day and between-day precisions (coefficient of variation) and accuracies (relative errors) were below 10%. The method has been successfully applied to study the sulfated steroidome in diseases such as steroid sulfatase deficiency, proving its diagnostic value. This is, to our best knowledge, the most comprehensive method available for the quantification of sulfated steroids in human blood., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

The mouse vomeronasal organ (VNO) expresses chemosensory receptors that detect intra-species as well as inter-species cues. The vomeronasal neurons are thought to be highly selective in their responses. The tuning properties of individual receptors remain difficult to characterize due to the lack of a robust heterologous expression system. Here, we take a transgenic approach to ectopically express two type 1 vomeronasal receptors in the mouse VNO and characterize their responses to steroid compounds. We find that V1rj2 and V1rj3 are sensitive to two sulfated estrogens (SEs) and can be activated by a broad variety of sulfated and glucuronidated steroids at high concentrations. Individual neurons exhibit narrow range of concentration-dependent activation. Collectively, a neuronal population expressing the same receptor covers a wide dynamic range in their responses to SEs. These properties recapitulate the response profiles of endogenous neurons to SEs.

Steroid sulfatase (STS) deficiency is the underlying cause of the skin condition known as recessive X-linked ichthyosis (RXLI). RXLI patients show scales on their skin caused by high concentrations of cholesterol sulfate (CS), as they are not capable of releasing the sulfate group from its structure to obtain free cholesterol. CS has been reported, so far, as the sole sulfated steroid with increased concentrations in the blood of RXLI patients. A non-targeted LC-MS approach in negative mode detection (LC-MS precursor ion scan mode) was applied to serum samples of 12 RXLI patients and 19 healthy males. We found that CS was not the only sulfated compound consistently elevated in RXLI patients, because a group of compounds with a m/z of 481 was found in high concentrations too. Further LC-MS/MS demonstrated that the main contributor to the m/z 481 signal in RXLI serum is 27-hydroxycholesterol-3-sulfate (27OHC3S). Accordingly, a new method for 27OHC3S quantification in the context of RXLI has been developed and validated. Other hydroxycholesterol sulfate compounds were elevated as well in RXLI patients., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)