Estrogenic Activity of Mycoestrogen (3ß,5α,22E)-Ergost-22-en-3-ol via Estrogen Receptor α-Dependent Signaling Pathways in MCF-7 Cells.

Armillariella tabescens (Scop.) Sing., a mushroom of the family Tricholomataceae, has been used in traditional oriental medicine to treat cholecystitis, improve bile secretion, and regulate bile-duct pressure. The present study evaluated the estrogen-like effects of A. tabescens using a cell-proliferation assay in an estrogen-receptor-positive breast cancer cell line (MCF-7). We found that the methanol extract of A. tabescens fruiting bodies promoted cell proliferation in MCF-7 cells. Using bioassay-guided fractionation of the methanol extract and chemical investigation, we isolated and identified four steroids and four fatty acids from the active fraction. All eight compounds were evaluated by E-screen assay for their estrogen-like effects in MCF-7 cells. Among the tested isolates, only (3ß,5α,22E)-ergost-22-en-3-ol promoted cell proliferation in MCF-7 cells; this effect was mitigated by the ER antagonist, ICI 182,780. The mechanism underlying the estrogen-like effect of (3ß,5α,22E)-ergost-22-en-3-ol was evaluated using Western blot analysis to detect the expression of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), Akt, and estrogen receptor α (ERα). We found that (3ß,5α,22E)-ergost-22-en-3-ol induced an increase in phosphorylation of ERK, PI3K, Akt, and ERα. Together, these experimental results suggest that (3ß,5α,22E)-ergost-22-en-3-ol is responsible for the estrogen-like effects of A. tabescens and may potentially aid control of estrogenic activity in menopause.

Screening for Biologically Annotated Drugs That Trigger Triacylglycerol Accumulation in the Diatom Phaeodactylum.

Microalgae are a promising feedstock for the production of triacylglycerol (TAG) for a variety of potential applications, ranging from food and human health to biofuels and green chemistry. However, obtaining high TAG yields is challenging. A phenotypic assay for the accumulation of oil droplets was developed to screen a library of 1,200 drugs, annotated with pharmacology information, to select compounds that trigger TAG accumulation in the diatom Phaeodactylum tricornutum Using this screen, we identified 34 molecules acting in a dose-dependent manner. Previously characterized targets of these compounds include cell division and cell signaling effectors, membrane receptors and transporters, and sterol metabolism. Among the five compounds possibly acting on sterol metabolism, we focused our study on ethynylestradiol, a synthetic form of estrogen that is used in contraceptive pills and known for its ecological impact as an endocrine disruptor. Ethynylestradiol impaired the production of very-long-chain polyunsaturated fatty acids, destabilized the galactolipid versus phospholipid balance, and triggered the recycling of fatty acids from membrane lipids to TAG. The P. tricornutum transcriptomic response to treatment with ethynylestradiol was consistent with the reallocation of carbon from sterols to acetyl-coenzyme A and TAG. The mode of action and catabolism of ethynylestradiol are unknown but might involve several up-regulated cytochrome P450 proteins. A fatty acid elongase, Δ6-ELO-B1, might be involved in the impairment of very-long-chain polyunsaturated fatty acids and fatty acid turnover. This phenotypic screen opens new perspectives for the exploration of novel bioactive molecules, potential target genes, and pathways controlling TAG biosynthesis. It also unraveled the sensitivity of diatoms to endocrine disruptors, highlighting an impact of anthropogenic pollution on phytoplankton.

Schisandrin A and B induce organic anion transporting polypeptide 1B1 transporter activity.

Organic anion transporting polypeptide 1B1 (OATP1B1) is the most important transporter in the organic anion transporting polypeptide family. OATP1B1 plays an important role in the hepatic uptake of many endogenous compounds and xenobiotics, including many clinical drugs. At present, the combinational usage of Chinese traditional herbal medicines and conventional chemical pharmaceuticals may affect the activity of enzymes and transporters activity and cause absorption of their substrates and metabolic changes. In this study, we aimed to investigate the effect of schisandrin A, schisandrin B and tanshinone IIA, which were extracted from medicinal plants, on OATP1B1 activity. HepG2 cells are used as in vitro models for OATP1B1 activity studies. A combination of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tertazolium bromide (MTT) assays, real-time RT-PCR, and transporter activity analysis were employed. We found that schisandrin A and B increased OATP1B1 mRNA levels by 1.81-fold (p < 0.01) and 1.87-fold (p < 0.01) at concentration of 10 µM, respectively. Schisandrin A of 1 µM and 10 µM and schisandrin B of 10 µM significantly increased the uptake of [3H] estrone-3-sulfate (p < 0.05 or p < 0.01). Tanshinone IIA had no effect on the mRNA expression and transport activity of OATP1B1 at nontoxic concentrations. Our study suggests that schisandrin A and B induced OATP1B1 expression and increased its transporter activity in HepG2 cells.

Improvement of neuronal bioenergetics by neurosteroids: implications for age-related neurodegenerative disorders.

The brain has high energy requirements to maintain neuronal activity. Consequently impaired mitochondrial function will lead to disease. Normal aging is associated with several alterations in neurosteroid production and secretion. Decreases in neurosteroid levels might contribute to brain aging and loss of important nervous functions, such as memory. Up to now, extensive studies only focused on estradiol as a promising neurosteroid compound that is able to ameliorate cellular bioenergetics, while the effects of other steroids on brain mitochondria are poorly understood or not investigated at all. Thus, we aimed to characterize the bioenergetic modulating profile of a panel of seven structurally diverse neurosteroids (progesterone, estradiol, estrone, testosterone, 3α-androstanediol, DHEA and allopregnanolone), known to be involved in brain function regulation. Of note, most of the steroids tested were able to improve bioenergetic activity in neuronal cells by increasing ATP levels, mitochondrial membrane potential and basal mitochondrial respiration. In parallel, they modulated redox homeostasis by increasing antioxidant activity, probably as a compensatory mechanism to a slight enhancement of ROS which might result from the rise in oxygen consumption. Thereby, neurosteroids appeared to act via their corresponding receptors and exhibited specific bioenergetic profiles. Taken together, our results indicate that the ability to boost mitochondria is not unique to estradiol, but seems to be a rather common mechanism of different steroids in the brain. Thus, neurosteroids may act upon neuronal bioenergetics in a delicate balance and an age-related steroid disturbance might be involved in mitochondrial dysfunction underlying neurodegenerative disorders.

Involvement of specific transport system on uptake of lactone form of SN-38 in human intestinal epithelial cell line Caco-2.

The aim of this study was to elucidate the characteristics of the transport of lactone and carboxylate forms of SN-38 (SN-38L and SN-38C, respectively), a metabolite of irinotecan hydrochloride (CPT-11), with the human intestinal epithelial cell line, Caco-2. We examined SN-38L and SN-38C uptake from the apical side into Caco-2, and the effects of various compounds on the uptake of SN-38L. SN-38L and SN-38C in the cells were determined by HPLC with a fluorescence detector. When either SN-38L (0.5 µM) or SN-38C (0.5 µM) was added extracellularly at 37°C, the accumulation of SN-38L into the cells was about 10-fold higher than that of SN-38C, suggesting a dominant role of the lactone form in the uptake of SN-38 into Caco-2. The accumulation of SN-38L in Caco-2 increased time-dependently up to 10 min at 37°C, whereas the accumulation markedly decreased at 4°C. The initial uptake rate of SN-38L approached saturation at high concentrations with Michaelis-Menten constant and 'Hill coefficient,' 2.84±1.00 µM and 2.13±1.14, respectively (mean±S.E.). The accumulation of SN-38L was markedly inhibited by baicalin, an active ingredient of a Chinese herbal medicine, Hange-Shashin-To, as well as CPT-11. The type of inhibition by baicalin was competitive. In contrast, concomitant sulfobromophthalein, taurocholate and estrone 3-sulfate significantly increased SN-38L uptake. These results suggest that apical uptake of SN-38 by Caco-2 is dominantly performed as a lactone form through a specific transporter, which is competitively inhibited by baicalin.

Antioxidant effects of a grapeseed procyanidin extract and oleoyl-estrone in obese Zucker rats.

OBJECTIVE: To substantiate the relation between obesity and oxidative stress and to assess the potential beneficial properties of a grapeseed proanthocyanidin extract (GSPE), the amelioration of obesity with oleoyl-estrone (OE), and the possible combined effect of GSPE and OE on the hepatic and renal antioxidant enzyme system in obesity-induced oxidative stress. METHODS: Male obese Zucker rats were divided into four groups: GSPE, OE, GSPE + OE, and OC (control). For 30 d they were gavaged with GSPE, OE, GSPE + OE, or sunflower oil as the control vehicle (OC). Lean Zucker rats gavaged with the vehicle comprised the lean control group. Hepatic and renal antioxidant enzymes and oxidative biomarkers were determined at the end of the experimental period. RESULTS: Hepatic antioxidant activities were higher in obese rats than in lean ones. All these activities decreased when obese rats were treated with GSPE, whereas only some of these activities decreased with OE and GSPE + OE treatments. In the kidney, few antioxidant enzymes had higher activities in obese than in lean rats, and OE and GSPE + OE were the treatments that inhibited most enzymes studied. Glutathione S-transferase activity was always lower with the exception of the kidney of obese rats and all treatments used increased the low glutathione levels found in obesity. CONCLUSION: GSPE and OE improve oxidative stress in obese Zucker rats. The effect of GSPE + OE is comparable to GSPE for the liver and to OE for the kidney. Thus the effects of GSPE and OE are not additive and are organ dependent.

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.

Effects of estrogens on striatal damage after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in male and female mice.

Emerging evidence shows a beneficial effect of estrogens for Parkinson's disease, yet the exact potency of these compounds implicated remain obscured. In this study, we investigated the neuroprotective effect of 17beta-estradiol and estrone against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced striatal toxicity in mice. The neuroprotective effects of both compounds were evaluated by HPLC and Western blot analyses 5 days after the last of 4 consecutive injections of MPTP at 1-h intervals to mice. Subacute treatment (10 days) with estrone or 17beta-estradiol at low doses (0.05 and 0.2mg/kg) showed no significant changes against MPTP-induced damage of striatal dopamine terminals in mice. Furthermore, acute treatment with estrone at high doses (0.5 and 2.0mg/kg) showed no significant alterations against MPTP-induced damage of striatal dopamine terminals in mice. In contrast, acute treatment with 17beta-estradiol at high doses exhibited a neuroprotective effect against the damage of striatal dopamine terminals in both male and female mice after MPTP treatments. The results demonstrate that estrogen therapy with high doses may have a neuroprotective effect on the damage of striatal dopamine terminals in the MPTP-induced mice. These findings may lead to be development of estrogen therapy for the prevention and treatment of Parkinson's disease.

Inhibitors of 17beta-hydroxysteroid dehydrogenase type 1.

Carcinogenesis of hormone-related cancers involves hormone-stimulated cell proliferation, which increases the number of cell divisions and the opportunity for random genetic errors. In target tissues, steroid hormones are interconverted between their potent, high affinity forms for their respective receptors and their inactive, low affinity forms. One group of enzymes responsible for these interconversions are the hydroxysteroid dehydrogenases, which regulate ligand access to steroid receptors and thus act at a pre-receptor level. As part of this group, the 17beta-hydroxysteroid dehydrogenases catalyze either oxidation of hydroxyl groups or reduction of keto groups at steroid position C17. The thoroughly characterized 17beta-hydroxysteroid dehydrogenase type 1 activates the less active estrone to estradiol, a potent ligand for estrogen receptors. This isoform is expressed in gonads, where it affects circulating levels of estradiol, and in peripheral tissue, where it regulates ligand occupancy of estrogen receptors. Inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 are thus highly interesting potential therapeutic agents for the control of estrogen-dependent diseases such as endometriosis, as well as breast and ovarian cancers. Here, we present the review on the recent development of inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 published and patented since the previous review of 17beta-hydroxysteroid dehydrogenase inhibitors of Poirier (Curr. Med. Chem., 2003, 10, 453). These inhibitors are divided into two separate groups according to their chemical structures: steroidal and non-steroidal 17beta-hydroxysteroid dehydrogenase type 1 inhibitors. Their estrogenic/ proliferative activities and selectivities over other 17beta-hydroxysteroid dehydrogenases that are involved in local regulation of estrogen action (types 2, 7 and 12) are also presented.

Lycopene and other carotenoids inhibit estrogenic activity of 17beta-estradiol and genistein in cancer cells.

Epidemiological evidence suggests that carotenoids prevent several types of cancer, including mammary and endometrial cancers. On the other hand, such studies have also shown that estrogens are the most important risk factors for these cancer types. Genistein, the phytoestrogen mainly found in soy, also shows significant estrogenic activity when tested at concentrations found in human blood. The aim of this study was to determine whether carotenoids inhibit signaling of steroidal estrogen and phytoestrogen which could explain their cancer preventive activity. Similar to the known effect of 17beta-estradiol (E(2)), treatment of breast (T47D and MCF-7) and endometrial (ECC-1) cancer cells with phytoestrogens induced cell proliferation, cell-cycle progression and transactivation of the estrogen response element (ERE). However, each of the tested carotenoids (lycopene, phytoene, phytofluene, and beta-carotene) inhibited cancer cell proliferation induced by either E(2) or genistein. The inhibition of cell growth by lycopene was accompanied by slow down of cell-cycle progression from G1 to S phase. Moreover, the carotenoids inhibited estrogen-induced transactivation of ERE that was mediated by both estrogen receptors (ERs) ERalpha and ERbeta. The possibility that this inhibition results from competition of carotenoid-activated transcription systems on a limited pool of shared coactivators with the ERE transcription system was tested. Although cotransfection of breast and endometrial cancer cells with four different coactivators (SRC-1, SRC-2, SRC-3, and DRIP) strongly stimulated ERE reporter gene activity, it did not oppose the inhibitory effect of carotenoids. These results suggest that dietary carotenoids inhibit estrogen signaling of both 17beta-estradiol and genistein, and attenuate their deleterious effect in hormone-dependent malignancies.

Rats treated with oleoyl-oestrone maintain glucidic homeostasis: comparisons with a pair-fed model.

To determine whether or not the weight (and fat) loss induced by oleoyl-oestrone treatment results only as a consequence of decreased food intake, we compared treated animals with a pair-fed model. To this end, Wistar female rats received daily oral gavages of 10 mumol/kg per d oleoyl-oestrone in sunflower oil, or vehicle alone for 10 or 20 d. A second group of rats received the gavage of sunflower oil and the same amount of food ingested as the oleoyl-oestrone-treated animals (pair-fed group). Rats treated with oleoyl-oestrone maintained glucidic metabolism homeostasis despite a marked decrease in adipose tissue weight (P<0.001). Pair-fed rats exhibited a different pattern, comparable to short-term starvation, with greatly decreased glycogen stores (P<0.0001). The most significant effects were detected in the 10 d period groups. Oleoyl-oestrone affected the activity of the ponderostat system not only by decreasing appetite but also by modifying energy partition: treated animals maintained their glucose and energy homeostasis despite decreased food intake and the massive depletion of lipid stores.

Human organic anion transporter 3 (hOAT3) can operate as an exchanger and mediate secretory urate flux.

BACKGROUND/AIMS: Renal secretion of organic anions is critically dependent on their basolateral uptake against the electrochemical gradient. Due to their localization, two transporters are likely involved, namely OAT1 and OAT3. While OAT1 as an exchanger clearly operates in the secretory direction, OAT3 in its previously supposed mode as a uniporter should move anionic substrates from cell to blood. It would thus dissipate gradients established by OAT1 of common OAT1/OAT3 substrates. In the present study we therefore reinvestigated the driving forces of human OAT3. METHODS: The human OAT3 obtained Xenopus laevis oocyte expression system, hOAT3-mediated transport of estrone sulfate (ES) and dicarboxylates was assayed for cis-inhibition and/or trans-stimulation in both the uptake and efflux direction. RESULTS: hOAT3-mediated efflux of glutarate (GA), can be significantly trans-stimulated by a variety of ions with high cis-inhibitory potency, including GA (282%), alpha-ketoglutarate (476%), p-aminohippurate (179%), and, most notably, urate (167%). Urate cis-inhibited ES uptake with an IC(50) close to normal serum urate concentrations. CONCLUSION: These data indicate that OAT3 does not represent a uniporter but operates as an organic ion%dicarboxylate exchanger similar to OAT1, and may mediate renal urate secretion.

Equine estrogens differentially prevent neuronal cell death induced by glutamate.

OBJECTIVE: In the present study, neuronal PC12 cells and hippocampal HT22 cells maintained in culture were used to test the neuroprotective effect of equine estrogens estrone, 17beta-estradiol, 17alpha-estradiol, equilin, 17beta-dihydroequilin, 17alpha-dihydroequilin, equilenin, 17beta-dihydroequilenin, 17alpha-dihydroequilenin, Delta(8)-estrone(,) and Delta(8),17beta-estradiol against glutamate toxicity. METHODS: The HT22 and PC12 cells were grown in Dulbecco modified Eagle medium supplemented with 5% horse serum, 10% fetal bovine serum, and 10 mM HEPES. The undifferentiated PC12 cells were plated on collagen-coated, 96-well plastic plates at 10,000 cells per well, and the HT22 cells were plated on uncoated 96-well plates at 2500 cells per well. Twenty-four hours after plating, various concentrations of estrogens (0.1-40 microM) and glutamate (1-10 mM) were added in a total volume of 100 microL. After 24 hours, cell viability was determined using the MTS cell proliferation assay. Results were verified in some experiments by using the lactate dehydrogenase cytotoxicity assay. RESULTS: The results indicate that cell toxicity in both cell lines was directly proportional to the concentration of glutamate. The lowest dose of glutamate that reduced cell viability by 50% under these conditions was 1.8 mM for HT22 cells and 3 mM for PC12 cells. All estrogens tested were neuroprotective against glutamate-induced cell death in a typical dose-related manner. However, these estrogens differed extensively with respect to their neuroprotective potencies. In both cell lines, the Delta(8)-ring B unsaturated estrogens were the most neuroprotective, whereas the classic estrogens 17beta-estradiol, estrone, and 17alpha-estradiol were the least potent. The order of potency was Delta(8),17beta-estradiol > Delta(8)-estrone > 17beta-dihydroequilenin > 17alpha-dihydroequilenin > equilenin > 17beta-dihydroequilin = equilin > 17alpha-dihydroequilin > 17beta-estradiol > estrone > 17alpha-estradiol in PC12 cells and Delta(8),17beta-estradiol > Delta(8)-estrone > equilenin = 17beta-dihydroequilenin > 17beta-dihydroequilin > equilin > 17alpha-dihydroequilenin > 17alpha-dihydroequilin > 17alpha-estradiol = 17beta-estradiol > estrone in HT22 cells. CONCLUSIONS: Our data indicate that the neurotoxic effects of glutamate can be inhibited differentially by various equine estrogens. The less estrogenic (uterotropic) Delta(8) estrogens were the most effective neuroprotectors, and further chemical modifications of these estrogens may provide compounds that are useful for preventing neurodegenerative diseases in both women and men.

[Piperazinyl estrone prevents bone loss in ovariectomized rats].

AIM: To determine the effect of piperazinyl estrone, a new estrogen derivative, on bone turnover, bone mass and uteri in ovariectomized rats. METHODS: Female Sprague-Dawley rats were ovariectomized (OVX) or sham operated (sham) at the age of 3 months and treated with estrone (E) at 0.75 mg.kg-1.d-1, or with piperazinyl estrone (P-E) at 1 or 10 mg.kg-1.d-1, orally, for 3 months. At the time of death, the uterine weight was measured. Bone histomorphometric analysis of proximal tibial metaphyses (PTM) was performed in undecalcified sections. RESULTS: Bone histomorphometric data showed that the percent trabecular area (% Tb.Ar) of OVX rats with bone high turnover was significantly decreased. The uteri were atrophied. The percent trabecular area (% Tb.Ar) of estrone treated group was increased in decreasing bone turnover manner. But the size and weight of uteri in this group were increased vs OVX group. The bone loss induced by OVX was preserved by P-E treatment, but the mechanism of maintaining bone is different from that of E-treated rats. P-E treatment in low dose did not decrease any bone formation indices, such as percent labeling perimeter, bone formation rate per bone volume (BFR/BV), except bone mineral apposition rate (MAR) compared with E-treated group, and maintained them at OVX level. The uteri were found to be in atrophy compared with the match dose (0.75 mg) of E-treated OVX rats. But rats treated with high dose of P-E showed the same change like E-treated group. CONCLUSION: The finding of this study shows that lower dosage of piperazinyl estrone has effect on preventing the bone losses in OVX rats, while the bone formation and the uterus are not affected, thus supporting the hypothesis that piperazinyl estrone has the potential to prevent postmenopausal bone loss in women with less side effects.

Steroidal oxathiazine inhibitors of estrone sulfatase.

The presence of estrone sulfatase in breast tumors and the high levels of circulating estrone sulfate may contribute the major portion of estrogen synthesized locally in breast tissues through conversion of estrone sulfate to estrone by the enzyme. Using inhibitors of estrone sulfatase for the treatment of estrogen-dependent (estrogen receptor positive, ER(+)) breast cancer could be a very effective therapeutic strategy for the treatment of estrogen-dependent breast tumors in postmenopausal women. Therefore, we designed and synthesized several steroidal 2',3'-oxathiazines that inhibit estrone sulfatase and have greatly reduced estrogenic side effects. Our in vitro studies indicate that the oxathiazine compounds have inhibitory activity on estrone sulfatase in MCF-7 human breast cancer cells. These estrone sulfatase inhibitors (ESIs) also inhibit the growth of MCF-7 cells induced by estrone sulfate. In addition, our in vivo experiments demonstrate that our ESIs have moderate antitumor activity against MCF-7 breast cancer xenografts in Balb/c athymic nude mice. The synthesis and biological activity of a number of these unique steroidal ESIs are described.

Conjugated equine estrogens, estrone sulphate and estradiol valerate oral administration in ovariectomized rats: effects on central and peripheral allopregnanolone and beta-endorphin.

OBJECTIVES: Several natural or synthetic estrogenic molecules are commonly used in oral hormone replacement therapy for the relief of menopausal complaints and for the primary prevention of cardiovascular disease and osteoporosis. Little information is available concerning the comparative efficacy of different compounds on neuroendocrine function. The opioid peptide beta-endorphin (beta-EP), and the neurosteroid allopregnanolone are considered markers of neuroendocrine function and their synthesis and action is regulated by gonadal steroids. The present study aimed to investigate the effects of a 2-week oral treatment with estradiol valerate (EV), estrone sulphate (ES), or conjugated equine estrogen (CEE) on central and peripheral beta-EP and allopregnanolone levels in ovariectomized (OVX) female rats. METHODS: Twelve groups of Wistar OVX rats received oral EV (0.05, 0.1, 0.5 and 1 mg/Kg/day) or ES (0.1, 0.5, 1 and 2 mg/Kg/day), or CEE (0.1, 0.5, 1 and 2 mg/Kg/day) for 14 days. One group of fertile and one group of OVX rats were used as controls. beta-EP content was assessed in hypothalamus, hippocampus, anterior and neurointermediate pituitary, and plasma, while allopregnanolone content was assessed in hypothalamus, hippocampus, anterior pituitary, adrenals and serum. RESULTS: Ovariectomy induced a significant decrease in beta-EP and allopregnanolone content in hypothalamus, hippocampus, pituitary, and serum, while it increased allopregnanolone content in the adrenals. In OVX rats, the administration of each molecule reversed the ovariectomy-induced beta-EP and allopregnanolone changes in a dose-dependent fashion, therefore completely restoring their concentration. At higher doses, the estrogenic compounds induced significantly higher levels of allopregnanolone and beta-EP than in fertile rats. CEE induced higher allopregnanolone levels in hypothalamus, anterior pituitary and serum than the other estrogenic molecules, and in the hippocampus with respect to EV alone. CEE produced higher beta-EP levels in the hippocampus and hypothalamus with respect to EV and ES. CONCLUSION: In the examined tissue and serum estrogens restore the ovariectomy induced changes in allopregnanolone and beta-EP content in a dose-dependent manner; the magnitude of these effects is not uniform and it is related to the different tissues and the employed compounds.

Inhibition of steryl sulfatase activity in LNCaP human prostate cancer cells.

The enzyme steryl sulfatase may help support the growth of hormone-dependent tumors, including prostate cancers, by facilitating the conversion of circulating precursor steroids to active hormones. We sought to determine the presence of steryl sulfatase activity in the androgen-dependent human prostate cancer cell line LNCaP, and to determine if this activity was inhibited by known steryl sulfatase inhibitors. Intact LNCaP cultures had steryl sulfatase activity, as determined by conversion of [3H]estrone sulfate (E(1)S) to unconjugated steroids. The level of steryl sulfatase activity was relatively low (4.6 pmol/18 h/million cells) compared to MDA-MB-231 breast cancer cells (284.0 pmol/18 h/million cells). The observed activity in both cell lines was blocked by addition of 1 microM estrone sulfamate (EMATE), an active-site-directed, steroidal inhibitor of steryl sulfatase. Steryl sulfatase activity was also inhibited by Danazol, and by (p-O-sulfamoyl)-tetradecanoyl tyramine (C2-14), a non-steroidal inhibitor. Microsomes prepared from LNCaP cultures also showed steryl sulfatase activity, as determined by hydrolysis of [3H]E(1)S and [3H]dehydroepiandrosterone sulfate (DHEAS) to unconjugated forms. LNCaP and MDA-MB-231 microsomes both hydrolyzed E(1)S about two times faster than DHEAS. Hydrolysis of E(1)S in LNCaP and MDA-MB-231 microsomes was blocked by steryl sulfatase inhibitors with the following relative potencies: EMATE>C2-14>Danazol. These data demonstrate that LNCaP prostate cancer cells contain a steryl sulfatase with properties similar to that found in human breast cancer cells, and that the activity of this enzyme can be blocked by known steryl sulfatase inhibitors. Steryl sulfatase inhibitors may be useful as an adjuvant to androgen deprivation therapy for prostate cancer.

Oral, water-soluble combined estrogen/calcium preparation for postmenopausal therapy.

OBJECTIVES: Estrogen is often prescribed for symptoms and sequelae of ovarian estrogen loss after menopause. METHODS: To assess efficacy and acceptability of a new, highly soluble estrogen-calcium preparation, we formulated a water-soluble powdered combination of estrogen (0.625 mg estrone piperazine sulfate) and calcium (1 g, ions) as the highly soluble glycerophosphate salt (Estrosol). Effects of once-daily administration on bone mineral turnover of Estrosol dissolved in water (n = 11) was compared with 0.625 mg conjugated estrogens (Premarin) + 1 g calcium (Tums 500 Calcium Supplement) (n = 8). All women had had a previous hysterectomy, were between the ages 40 and 75, within 25% of ideal body weight, and had not taken hormonal preparations for at least 3 months. Assessment of bone mineral turnover was by monitoring N-telopeptides and bone specific alkaline phosphatase (BSAP) on 5 occasions: pretreatment and once during each of the 4 months of treatment. RESULTS: Mean N-telopeptide values decreased (p = .005) in both groups: Estrosol, 29.2% (40 --> 29 mmol bone collagen equivalents (BCE)/mmol creatinine), and Premarin(R) + calcium, 44.8% (33 --> 18 mmol). Mean BSAP values also decreased (p = 0.007) in both groups: Estrosol, 12.6% (12.06 --> 10.54 mg/l), Premarin(R) + calcium, 19.1% (11.57 --> 9.36 mg/l). The difference between groups for both N-telopeptides and BSAP was not significant, although sample size was small. Symptoms (hot flashes, vaginal dryness) improved similarly in both groups. Symptoms during treatment (breast or nipple tenderness, bloating) also were similar in both groups. Both preparations were well-tolerated. There were no changes in CBC, liver function tests, electrolytes or urinalyses in either group . CONCLUSIONS: This pilot study indicates that the combined, highly water-soluble preparation of estrogen and calcium is effective in reducing bone mineral turnover, acceptable and well-tolerated. Use of this single aqueous preparation may lead to better compliance than using two separate pills.

Estrogen and androgen influence hypothalamic AVP and CRF concentrations in fetal and adult sheep.

The present study tested the hypothesis that action of sex steroids on the hypothalamus-pituitary-adrenal (HPA) axis is measurable in the hypothalamus. Late-gestation fetal sheep were treated (5 mg/21 days) with either estradiol, androstenedione, or tamoxifen and compared to age-matched control fetuses. Tamoxifen significantly increased hypothalamic corticotropin releasing factor (CRF) and arginine vasopressin (AVP) concentrations, and androstenedione significantly decreased hypothalamic CRF concentration. Adult sheep were treated with estrone (10 mg/21 days), and responded with significant increases in hypothalamic AVP concentration, but not in immunoreactive ACTH concentration or processing within the pituitary. The results demonstrate that the effect of estrogen on the HPA axis is measurable in the hypothalamus, and is therefore not primarily at the anterior pituitary.

Oral oleoyl-estrone induces the rapid loss of body fat in Zucker lean rats fed a hyperlipidic diet.

OBJECTIVE: To test whether oleoyl-estrone affects body weight when given orally, which may help curtail the secondary growth-boosting effects of derived estrone. DESIGN: The rats were fed for 15 days with a powdered hyperlipidic diet (16.97 MJ/kg metabolizable energy) in which 46.6% was lipid-derived and 16.1% protein-derived energy (HL group), containing 1.23+/-0.39micromol/kg of fatty-acyl esters of estrone. This diet was supplemented with additional oleoyl-estrone to produce diets with 2.5 micromol/kg (diet OE2.5), 4.4 micromol/kg (diet OE4.4), and 33.3 micromol/kg content in fatty-acyl estrone (diet OE33). SUBJECTS: Twelve-week old female Zucker lean (Fa/?) rats initially weighing 200-235g. MEASUREMENTS: Food intake and body weight changes; urine and droppings production and nitrogen content. Body composition (water, lipid, protein) and total energy. Energy and nitrogen balances. Plasma chemistry including free amino acids. RESULTS: Oral administration of oleoyl-estrone in a hyperlipidic diet resulted in significant losses of fat, energy and, ultimately, weight, which were dependent on the dose of oleoyl-estrone ingested. Treatment induced the maintenance of energy expenditure combined with lower food intake, creating an energy gap that was filled with internal fat stores whilst preserving body protein. The decrease in food intake was not a consequence of food aversion but of diminished appetite. Energy expenditure was practically constant for all groups except for the OE33, which showed values about 25% lower than the controls. In most of the groups studied, there was a net protein deposition in spite of severe lipid and energy drainage. Amino acid levels agreed with this N-sparing shift. In spite of lowered energy intake, the N balance was positive or near zero in all groups, with a sizeable N-gap in controls and in lower-dose groups that disappeared in the OE33 group. CONCLUSION: Treatment of rats with a hyperlipidic diet containing added oleoyl-estrone resulted in the dose-related loss of fat reserves with scant modification of other metabolic parameters and preservation of body protein. The results agree with the postulated role of oleoyl-estrone as a ponderostat signal and open the way for its development as anti-obesity drug.