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• 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]

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]