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Estrogens regulate important processes in reproductive, skeletal, cardiovascular, and central nervous systems that impact women's overall health. Understanding endogenous and exogenously administered estrogen metabolism is vital to determining therapeutic estrogen levels. The present review provides an overview of estrogen metabolites formed in non-pregnant and pregnant women, and those resulting from exogenous estrogen administration. There are four principal endogenous estrogens: estrone (E 1), estradiol (E 2), estriol (E 3), and estetrol (E 4). E 4 , which is produced only in pregnancy, has emerged recently as an estrogen with significant therapeutic potential. E 1 , E 2 , and E 3 undergo extensive metabolism primarily through phase I (hydroxylation, oxidation, reduction) and phase II (primarily conjugation) reactions, whereas E 4 undergoes only phase II reactions. Exogenous estrogens commonly used for menopausal treatment and/or contraception, including micronized E 2 , conjugated equine estrogens, and ethinyl estradiol, also undergo phase I and phase II reactions, but differ widely in the types of metabolites formed. The mechanisms by which estrogen metabolites are formed and their excretion in urine, bile, and feces, are still poorly understood. We highlight areas that require further research to foster a better understanding of how estrogen metabolism impacts dosing of oral estrogens for therapeutic use, as well as the physiological regulation of endogenous estrogens. • Estrogens play an important role in health and disease. • Understanding metabolism is vital to determine therapeutic estrogen levels. • E 1 , E 2 , E 3 , conjugated equine estrogens, ethinyl estradiol undergo extensive metabolism. • Estetrol undergoes a distinctive metabolic pathway. • Regulation of these metabolic processes is poorly understood. [ABSTRACT FROM AUTHOR]

• ERα, ERβ1, ERβ2 and GPER are all involved in the regulation of vitellogenesis. • ERα is the central factor, acting interactively with ERβ1, ERβ2 and GPER in zebrafish. • GPER gets involved in vitellogenesis directly and interactively with ERα, ERβ1 and ERβ2 in zebrafish. Estrogens exert their biological functions through the estrogen receptors (ERs). In zebrafish, three nuclear estrogen receptors (nERs) named ERα, ERβ1 and ERβ2 and one membrane-bound G protein-coupled estrogen receptor (GPER) are identified. Vitellogenin (Vtg) is predominantly expressed in liver and strongly response to the stimulation of estrogen. It has been proposed that all three nERs are functionally involved in vitellogenesis and ERα may act as the major mediator in teleost. However, the role of GPER and its interaction with nERs in this process are not yet defined in teleost species. In the present study, we provide genetic evidence for the functional significance of ERα that the expression of Vtg genes (vtg1 , vtg2 , vtg3) and their response to estradiol stimulation were significantly decreased in esr1 mutant zebrafish. Activation of ERβ1 and ERβ2 induced Vtg expression through ERα. Moreover, the involvement of GPER in vitellogenesis and its interaction with nERs in zebrafish were firstly proposed in this work. Activation of GPER induced Vtg genes expression while inhibition of GPER significantly attenuated the estrogenic effect on Vtg. Both treatments altered the expression levels of nERs, suggesting GPER acts interactively with nERs. Collectively, the involvement of both nERs and GPER in regulation of vitellogenesis is demonstrated. ERα is the central factor, acting interactively with ERβ1, ERβ2 and GPER, and GPER regulates vitellogenesis directly and interactively with nERs. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • 17β-estradiol accelerates the process of oocyte meiosis resumption via GPR30. • 17β-estradiol-GPR30 mediated meiosis resumption ascribe downregulation of gap junction permeability in goat COCs. • GPR30-mediated gap junction permeability downregulation resulted from GPR30-MAP kinase-dependent phosphorylation of Cx43. Abstract Estrogen plays a critical role in the regulation of gap junctions between oocytes and granulosa cells in mammalian ovaries. G protein-coupled receptor 30 (GPR30) was identified as a membrane estrogen receptor, mediating rapid, nongenomic signaling events that might be responsible for the regulation of oocyte meiosis resumption and gap junction intercellular communications (GJICs). The present study aimed to determine the expression and localization of GPR30 and its role in oocyte meiotic progression and GJICs in goat cumulus-oocyte complexes (COCs). Immunofluorescence experiments revealed that GPR30 was primarily located in the plasma membrane of cumulus cells and oocytes in goats. 17β-estradiol could promote oocyte meiotic progression, which was blocked by G15 (a selective GPR30 antagonist) but not ICI182780 (a nuclear estrogen receptor inhibitor) in the early stage of in vitro culture. The effect of 17β-estradiol on the GJICs was quantified by lucifer yellow (LY) microinjection and calcein-AM fluorescent dye diffusion. 17β-estradiol treatment of goat COCs resulted in rapid downregulation of GJICs. The transfer of calcein from cumulus cells to oocytes could be significantly inhibited by carbenoxolone (a known gap junction blocker), 17β-estradiol or G1 (a GPR30 agonist), and this inhibition could be reversed by G15 but not ICI182780, indicating that GPR30 mediates the effect of 17β-estradiol on the rapid downregulation of GJICs. 17β-estradiol also stimulated the serine 368 phosphorylation of connexin 43 (Cx43) when COCs were in vitro cultured for 4 h, 6 h, and 8 h. More importantly, 17β-estradiol or G1 could separately promote the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and Cx43 significantly when COCs were cultured for 4 h. Furthermore, both ERK1/2 and Cx43 phosphorylation could be inhibited by G15 and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or by the ERK1/2 inhibitor PD98059, indicating that EGFR-ERK1/2 signaling was involved in these events. These results supported the hypothesis that GPR30 mediated 17β-estradiol-stimulated meiotic resumption and GJIC reduction in goat COCs. Thus, the present study provides novel insights into elucidating the mechanisms for steroid hormone action in the regulation of oocyte maturation. [ABSTRACT FROM AUTHOR]

Cytochrome P450 aromatase (AROM) catalyzes the biosynthesis of estrogen from androgen. Previously crystal structures of human AROM in complex with the substrate androstenedione, and inhibitors exemestane, as well as the newly designed steroidal compounds, have been reported. Here we report the first crystal structure of testosterone complex of human placental AROM. Testosterone binds at the androgen-specific heme distal pocket. The polar and hydrophobic interactions with the surrounding residues resemble the interactions observed for other ligands. The heme proximal region comprises the intermolecular interface in AROM, and also the putative interaction surface of its redox partner cytochrome P450 reductase. Unreported previously, the proximal region is characterized by a large surface cavity, unlike most known P450’s. Using five best X-ray data sets from androstenedione and testosterone complexes of AROM, we now unequivocally show the presence of an unexplained ligand electron density inside the proximal cavity. The density is interpreted as ordered five ethylene glycol units of polyethylene glycols used as a solvent for steroids and also in crystallization. Interestingly, polyethylene glycol exhibits weak inhibition of AROM enzyme activity in a time dependent manner. Besides its critical role in the redox partner coupling and electron transfer process, the proximal cavity possibly serves as the interaction site for other molecules that may have regulatory effects on AROM activity. In addition, the new data also reveal a previously unidentified water channel linking the active site to the lipid interface. The channel could be the predicted passage for water molecules involved in catalysis. [ABSTRACT FROM AUTHOR]

The numerous estrogen functions reported across vertebrates have been classically explained by their binding to specific transcription factors, the nuclear estrogen receptors (ERs). Rapid non-genomic estrogenic responses have also been recently identified in vertebrates including fish, which can be mediated by membrane receptors such as the G protein-coupled estrogen receptor (Gper). In this study, two genes for Gper, namely gpera and gperb , were identified in the genome of a teleost fish, the European sea bass. Phylogenetic analysis indicated they were most likely retained after the 3R teleost-specific whole genome duplication and raises questions about their function in male and female sea bass. Gpera expression was mainly restricted to brain and pituitary in both sexes while gperb had a widespread tissue distribution with higher expression levels in gill filaments, kidney and head kidney. Both receptors were detected in the hypothalamus and pituitary of both sexes and significant changes in gpers expression were observed throughout the annual reproductive season. In female pituitaries, gpera showed an overall increase in expression throughout the reproductive season while gperb levels remained constant. In the hypothalamus, gpera had a higher expression during vitellogenesis and decreased in fish entering the ovary maturation and ovulation stage, while gperb expression increased at the final atresia stage. In males, gpers expression was constant in the hypothalamus and pituitary throughout the reproductive cycle apart from the mid- to late testicular development stage transition when a significant up-regulation of gpera occurred in the pituitary. The differential sex, seasonal and subtype-specific expression patterns detected for the two novel gper genes in sea bass suggests they may have acquired different and/or complementary roles in mediating estrogens actions in fish, namely on the neuroendocrine control of reproduction 2 Protein and gene nomenclature followed that recommended by genenames.org and used for fish at http://zfin.org/ ; in this abbreviation list for each case protein abbreviation is presented first followed by the corresponding gene abbreviation. . [ABSTRACT FROM AUTHOR]

The endocannabinoid system (ECS) is involved in several physiological events that resulted in a growing interest in its modulation. Moreover, the uterine levels of anandamide (AEA), the major endocannabinoid, must be tightly regulated to create proper embryo implantation conditions. However, there are no evidences about the regulation of AEA in uterus by estrogen. Thus, the aim of this study is to elucidate whether estradiol benzoate (EB) and tamoxifen (TAM) administration to ovariectomized (OVX) rats can induce changes in the expression of cannabinoid receptors and AEA-metabolic enzymes in uterus by evaluating gene transcription and protein levels by qPCR, Western blot and immunohistochemistry. Moreover, the plasmatic and uterine levels of AEA and of prostaglandin E 2 (PGE 2 ) and prostaglandin F 2 α (PGF 2α ), the major cyclooxygenase-2 (COX-2) products, were determined by UPLC–MS/MS. The immunohistochemistry showed that cannabinoid receptors, as well as AEA-metabolic enzymes are mainly located in the epithelial cells of both lumen and glands and, to a lesser extent, in the muscle cells. Moreover, EB administration to OVX rats significantly increased CB1, CB2, NAPE-PLD, FAAH and COX-2 expression and transcription. These effects were absent in TAM and TAM + EB treatments showing that this response is estrogen receptor dependent. Additionally, although uterine levels of AEA remained unchanged in EB or TAM treated animals, they showed a rise with EB treatment in plasma. The latter also produced a decrease in uterine PGE 2 levels. In summary, these data collectively indicate that the expression of ECS components, as well as, the AEA and PGE 2 levels in rat uterus is modulated by EB. Thus, estradiol may have a direct regulatory role in the modulation of ECS in female reproductive tissues. [ABSTRACT FROM AUTHOR]

Celecoxib is known to alter the preferred position of SULT2A1-catalyzed sulfonation of 17β-estradiol (17β-E2) and other estrogens from the 3- to the 17-position. Understanding the effects of celecoxib on estrogen sulfonation is of interest in the context of the investigational use of celecoxib to treat breast cancer. This study examined the effects on celecoxib on cytosolic sulfotransferases in human and rat liver and on SULT enzymes known to be expressed in liver. Celecoxib’s effects on the sulfonation of several steroids catalyzed by human liver cytosol were similar but not identical to those observed previously for SULT2A1. Celecoxib was shown to inhibit recombinant SULT1A1-catalyzed sulfonation of 10 nM estrone and 4 μM p -nitrophenol with IC 50 values of 2.6 and 2.1 μM, respectively, but did not inhibit SULT1E1-catalyzed estrone sulfonation. In human liver cytosol, the combined effect of celecoxib and known SULT1A1 and 1E1 inhibitors, quercetin and triclosan, resulted in inhibition of 17β-E2-3-sulfonation such that the 17-sulfate became the major metabolite: this is of interest because the 17-sulfate is not readily hydrolyzed by steroid sulfatase to 17β-E2. Investigation of hepatic cytosolic steroid sulfonation in rat revealed that celecoxib did not stimulate 17β-E2 17-sulfonation in male or female rat liver as it does with human SULT2A1 and human liver cytosol, demonstrating that rat is not a useful model of this effect. In silico studies suggested that the presence of the bulky tryptophan residue in the substrate-binding site of the rat SULT2A homolog instead of glycine as in human SULT2A1 may explain this species difference. [ABSTRACT FROM AUTHOR]

Obesity and ageing are associated with lower serum testosterone levels in men. How fat distribution or adipose tissue metabolism, independent of genetic factors and age, are related to sex steroid metabolism is less clear. We studied the associations between adiposity and serum sex hormone concentrations, and mRNA expression of genes regulating sex hormone metabolism in adipose tissue in young adult male monozygotic (MZ) twin pairs. The subjects [n = 18 pairs; mean age, 32 years; individual body mass indexes (BMIs) 22–36 kg/m 2 ] included 9 male MZ twin pairs discordant for BMI [intra-pair difference (Δ) in BMI ≥3 kg/m 2 ]. Sex steroid concentrations were determined by liquid chromatography–tandem mass spectrometry, body composition by dual-energy X-ray absorptiometry and magnetic resonance imaging, and mRNA expressions from subcutaneous adipose tissue by Affymetrix. In BMI-discordant pairs (mean ΔBMI = 5.9 kg/m 2 ), serum dihydrotestosterone (DHT) was lower [mean 1.9 (SD 0.7) vs . 2.4 (1.0) nmol/l, P = 0.040] and mRNA expressions of DHT-inactivating AKR1C2 ( P = 0.021) and cortisol-producing HSD11B1 ( P = 0.008) higher in the heavier compared to the leaner co-twins. Serum free 17 β -estradiol (E2) was higher [2.3 (0.5) vs . 1.9 (0.5) pmol/l, P = 0.028], and in all twin pairs, serum E2 and estrone concentrations were higher in the heavier than in the leaner co-twins [107 (28) vs . 90 (22) pmol/l, P = 0.006; and 123 (43) vs . 105 (27) pmol/l, P = 0.025]. Within all twin pairs, i.e. independent of genetic effects and age, 1) the amount of subcutaneous fat inversely correlated with serum total and free testosterone, DHT, and sex hormone-binding globulin (SHBG) concentrations ( P < 0.01 for all), 2) intra-abdominal fat with total testosterone and SHBG ( P < 0.05), and 3) liver fat with SHBG ( P = 0.006). Also, 4) general and intra-abdominal adiposity correlated positively with mRNA expressions of AKR1C2 , HSD11B1 , and aromatase in adipose tissue ( P < 0.05). In conclusion, acquired adiposity was associated with decreased serum DHT and increased estrogen concentrations, independent of genetic factors and age. The reduction of DHT could be linked to its increased degradation (by AKR1C2 and HSD11B1) and increased estrogen levels to increased adiposity-related expression of aromatase in adipose tissue. [ABSTRACT FROM AUTHOR]

Steroid hormones, estrogen and androgen, control transcription in various reproductive and non-reproductive tissues. Both hormones are known to be important for control of sperm release from the seminiferous epithelium (spermiation), a process characterized by extensive remodeling of actin filaments and endocytosis. Earlier studies with an estrogen (E2)-induced rat model of spermiation failure revealed genes involved in actin remodeling (Arpc1b and Evl) and endocytosis (Picalm, Eea1, and Stx5a) to be differentially regulated. Further, among these genes, Arpc1b and Evl were found to be estrogen-responsive whereas Eea1 and Stx5a were androgen-responsive and Picalm was responsive to both hormones in seminiferous tubule cultures. Yet, the mechanism by which these genes are regulated by estrogen and androgen in the testis was unclear. Here, we report the presence of a functional estrogen response element (ERE) upstream of Arpc1b and Evl genes and androgen response element (ARE) upstream of Picalm, Eea1, and Stx5a genes. Chromatin immunoprecipitation in control versus E2-treated testes revealed significant changes in estrogen receptor beta (ERβ) recruitment along with coregulators to the EREs upstream of Arpc1b and Evl genes and androgen receptor (AR) at AREs upstream of Picalm, Eea1, and Stx5a genes. Enrichment patterns of these EREs/AREs with coregulators, activating and repressing histone modifications along with RNA polymerase II recruitment, correlated with the observed expression patterns of these genes upon E2 treatment. Taken together, our results reveal direct targets of estrogen and androgen in the testes and provide insights into transcriptional control of sperm release by the two steroid hormones. [ABSTRACT FROM AUTHOR]

As the knowledge on the estrogenic system in the brain grows, the possibilities to modulate it in order to afford further neuroprotection in brain damaging disorders so do it. We have previously demonstrated the ability of the selective estrogen receptor modulator, bazedoxifene (BZA), to reduce experimental ischemic brain damage. The present study has been designed to gain insight into the molecular mechanisms involved in such a neuroprotective action by investigating: 1) stroke-induced apoptotic cell death; 2) expression of estrogen receptors (ER) ERα, ERβ and the G-protein coupled estrogen receptor (GPER); and 3) modulation of MAPK/ERK1/2 and PI3K/Akt signaling pathways. For comparison, a parallel study was done with 17β-estradiol (E2)-treated animals. Male Wistar rats subject to transient right middle cerebral artery occlusion (tMCAO, intraluminal thread technique, 60 min), were distributed in vehicle-, BZA- (20.7 ± 2.1 ng/mL in plasma) and E2- (45.6 ± 7.8 pg/mL in plasma) treated groups. At 24 h from the onset of tMCAO, RT-PCR, Western blot and histochemical analysis were performed on brain tissue samples. Ischemia-reperfusion per se increased apoptosis as assessed by both caspase-3 activity and TUNEL-positive cell counts, which were reversed by both BZA and E2. ERα and ERβ expression, but not that of GPER, was reduced by the ischemic insult. BZA and E2 had different effects: while BZA increased both ERα and ERβ expression, E2 increased ERα expression but did not change that of ERβ. Both MAPK/ERK1/2 and PI3K/Akt pathways were stimulated under ischemic conditions. While BZA strongly reduced the increased p-ERK1/2 levels, E2 did not. Neither BZA nor E2 modified ischemia-induced increase in p-Akt levels. These results show that modulation of ERα and ERβ expression, as well as of the ERK1/2 signaling pathway accounts, at least in part, for the inhibitory effect of BZA on the stroke-induced apoptotic cell death. This lends mechanistic support to the consideration of BZA as a potential neuroprotective drug in acute ischemic stroke treatment. [ABSTRACT FROM AUTHOR]

17β-estradiol (E2) and progesterone (P) are neuroprotective factors in the brain preventing neuronal death under different injury paradigms. In previous studies, we demonstrated that both steroids dampen neuronal damage, improve local energy metabolism and attenuate pro-inflammatory responses. MicroRNAs (miRNAs) are small regulators of distinct target genes on the RNA level. Their expression patterns are misbalanced in several neurological disorders. To explore the regulatory mechanisms of steroid hormones on selected miRNAs and their validated targets in ischemia, we used the transient middle cerebral artery occlusion (tMCAO) model. 12-week old male rats were subjected to 2 h tMCAO and expression patterns of miR‐223, miR‐200c, miR‐375, miR‐199 and miR‐214 (all −3p) were determined. Using semi-quantitative real time PCR, we examined the role of E2 or P as regulatory factors for miRNAs and theirs target genes. Besides miR-375, all mentioned miRNAs showed a steady increase with a peak at 72 h post tMCAO, whereas highest levels of miR-375 were detected at 12 h post tMCAO. E2 or P selectively dampened miR-223 and miR-214 but further boosted miR-375 levels. With respect to the miR-223 regulated target genes NR2B and GRIA2 which both decreased after tMCAO, E2 and P application reversed this effect. Further, steroid treatment inhibited the hypoxia-induced increase of the miR-375 target genes Bcl-2 and RAD1. These findings provide new insights into the regulatory role of neuroprotection mediated by sex steroids in the brain. Both hormones are capable of influencing the expression of miRNAs which are relevant during neuropathological processes. Thereby, E2 and P indirectly control pro-apoptotic and −inflammatory gene translation and provide a mechanism to dampen explosive tissue damage. [ABSTRACT FROM AUTHOR]

β-estradiol (β-E2) and α-estradiol (α-E2) act as an endo- and an exon-estrogen in humans, respectively. There is a structural variation in C17-OH configuration of the two estrogens. UDP-glucuronosyltransferases (UGT) are responsible for termination of activities of a variety of endogenous hormones, clinical drugs, and environmental toxicants. The current study was conducted to investigate the effects of the two estrogens towards catalytic activities of UGTs. It was found that β-E2 could decrease activities of UGT1A9, − 2B4 and − 2B7, with K i values of a few micro-molars. β-E2 could additionally accelerate the activity of UGT2B17 via promoting enzyme-substrate binding and increasing the turn over number. Comparatively, α-E2 displayed much stronger inhibitory potentials towards UGT2B7 and − 2B4, but showed little influence to UGT1A9 and − 2B17. The K i values for inhibition of UGT2B7 in glucuronidation of different substrates by α-E2 were in a nanomolar range that is only about 1/100–1/50 of β-E2. UGT2B7 structural model was fatherly constructed to explore the mechanism underlying dramatically different inhibition selectivity of the two estrogens. Compared to β-E2, α-E2 formed more hydrophobic and hydrogen-bonded interactions with the residues in the active pocket. It is concluded that the configuration of E2–17-OH determines the inhibitory potentials towards UGTs. The results are useful in better understanding ligand selectivity of UGTs, as well as in further development of α-E2 in health protection. [Display omitted] • Activities of human UGT1A9, − 2B4, − 2B7, and − 2B17 are sensitive to the configuration of estradiol-17-OH. • The shift from β- to α-configuration decreases inhibition to UGT1A9, while increasing inhibition to UGT2B4 and − 2B7. • α-estradiol acts as a potent inhibitor of UGT2B7 with K i value in the nanomolar concentration. [ABSTRACT FROM AUTHOR]

Previous studies have shown that 17β-estradiol plays a cardioprotective role in the central nervous system (CNS) of male rats. The aim of the present study was to determine the influence of 17β-estradiol on sympathetic vasomotor activity and blood pressure in a renovascular hypertensive Goldblatt two-kidney one-clip (2K-1C) male rat model. We also determined the influence of angiotensin II AT1 receptor on the expression of estrogen receptors (ERα, ERβ, and G protein-coupled ER (GPER)) in the rostral ventrolateral medulla (RVLM) of Goldblatt rats. Experiments were performed in Goldblatt and age-matched control rats six weeks after clipping of renal artery to induce hypertension. Microinjection of 17β-estradiol into the RVLM led to a greater reduction in mean arterial pressure and renal sympathetic nerve activity in controls than in 2K-1C rats. Microinjection of the GPER agonist G-1 into the RVLM led to a significantly greater increase in mean arterial pressure and renal sympathetic nerve activity in 2K-1C rats. Expression levels of estrogen receptors GPER and ERα, but not ERβ, were significantly higher in the RVLM of 2K-1C rats than in that of the control rats. Chronic treatment with losartan significantly reduced the expression levels of estrogen receptors in the RVLM of 2K-1C rats. Taken altogether, the data suggest that the imbalance of actions between ERα and GPER, particularly with the predominance of GPER in the RVLM, contributes to sympathetic overactivation in male rats with Goldblatt hypertension. AT1-Angiotensin II receptor in the RVLM upregulated estrogen receptor expression in male Goldblatt rats. • In Goldblatt rats there is an imbalance between ERα and GPER in the brain with the predominance of GPER in the RVLM. • AT1-Angiotensin II receptor in the RVLM upregulated estrogen receptor expression in male Goldblatt rats. • The crosstalk between AT1 and estrogen receptors in the brain may represent a new therapeutic target to treat hypertension. [ABSTRACT FROM AUTHOR]

Endometriosis is a gynecological disorder affecting about 10% of women and can lead to invalidating painful symptoms and infertility. Since there is no current definitive cure for this disease, new therapeutic options are necessary. 17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is involved in the production of estradiol (E2), the most potent estrogen in women, and of 5-androstene-3β,17β-diol (5-diol), a weaker estrogen than E2, but whose importance increases after menopause. 17β-HSD1 is therefore a pharmacological target of choice for the treatment of estrogen-dependent diseases such as endometriosis. We developed a targeted-covalent (irreversible) and non-estrogenic inhibitor of 17β-HSD1, a molecule named PBRM, and herein evaluated its efficiency for the treatment of endometriosis. In a cell-free assay containing estrone (E1), the natural substrate of 17β-HSD1, PBRM was able to block the formation of E2 in a collection of 50 human endometriosis lesions from a different clinical feature type, location, and phase. When given orally by gavage at 15 mg/kg to baboons, the resulting plasmatic concentration of PBRM was found to be sufficiently high (up to 125 ng/mL) for an efficacy study in a non-human primate (baboon) endometriosis model. After 2 months of treatment, the number of lesions/adhesions decreased in 60% of animals (3/5) in the PBRM-treated group, compared to the placebo group which showed an increase in the number of lesion/adhesions in 60% (3/5) of animals. Indeed, the total number of lesions/adhesions decreased in treated group (−6.5 or −19% when excluding one animal) while it increased in the control group receiving a placebo (+11%). Analysis of specific endometriotic lesions revealed that PBRM decreased the number of red lesions (−67%; 8/12) and white lesions (−35%; 11/31), but not of blue-black lesions. Similarly, PBRM decreased the surface area of dense adhesions and filmy adhesions, as compared to placebo. Also, PBRM treatment did not significantly affect the number of menstrual days. Finally, this targeted covalent inhibitor showed no adverse effects and no apparent toxicity for the duration of the treatment. These data indicate that 17β-HSD1 inhibitor PBRM is a promising candidate for therapy targeting endometriosis and supports the need of additional efforts toward clinical trials. • 17β-HSD1 inhibitor PBRM blocks the estradiol synthesis in human endometriosis lesions. • In a non-human primate endometriosis model, PBRM reduced the number of lesions. • As targeted covalent inhibitor, PBRM showed no adverse effects and no apparent toxicity. • PBRM treatment did not significantly affect the number of menstrual days. • PBRM is a promising candidate for therapy targeting endometriosis. [ABSTRACT FROM AUTHOR]

The chemical synthesis of four stereoisomers (compounds 5a–d ) of 16β-( m -carbamoylbenzyl)-estradiol, a potent reversible inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), and two intermediates (compounds 3a and b ) was performed. Assignment of all nuclear magnetic resonance signals confirmed the stereochemistry at positions 13, 16 and 17. Nuclear overhauser effects showed clear correlations supporting a C-ring chair conformation for 5a and b and a C-ring boat conformation for 5c and d . These compounds were tested as 17β-HSD1 inhibitors and to assess their proliferative activity on estrogen-sensitive breast cancer cells (T-47D) and androgen-sensitive prostate cancer cells (LAPC-4). Steroid derivative 5a showed the best inhibitory activity for the transformation of estrone to estradiol (95, 82 and 27%, at 10, 1 and 0.1 μM, respectively), but like the other isomers 5c and d , it was found to be estrogenic. The intermediate 3a , however, was weakly estrogenic at 1 μM, not at all at 0.1 μM, and showed an interesting inhibitory potency on 17β-HSD1 (90, 59 and 22%, at 10, 1 and 0.1 μM, respectively). As expected, no compound showed an androgenic activity. The binding modes for compounds 3a and b , 5a–d and CC-156 were evaluated from molecular modeling. While the non-polar interactions were conserved for all the inhibitors in their binding to 17β-HSD1, differences in polar interactions and in binding conformational energies correlated to the inhibitory potencies. [ABSTRACT FROM AUTHOR]

Estradiol rapidly activates, within minutes, various physiological functions and behaviors including cognition in rodents. This review describes rapid effects of estradiol on hippocampal dependent learning and memory tasks in rodents. Mechanisms underlying the memory enhancements including the activation of signaling molecules and the enhancement of dendritic spinogenesis are briefly reviewed. In addition, the role of estradiol in the cognitive resilience to chronic stress exhibited only in females is discussed including contributions of ovarian as well as intra-hippocampally derived estrogens to this sex difference. Finally, speculations on possible physiologic functions for rapid mnemonic changes mediated by estrogens are made. Overall, the emergence of a novel and powerful mechanism for regulation of cognition by estradiol is described. [ABSTRACT FROM AUTHOR]

Various preparations of the African tree Amphimas pterocarpoides Harms are traditionally used to treat endocrine- related adverse health conditions. In the ovariectomized rat, the enriched in phenolics fraction of the methanol extract of stem bark of A. pterocarpoides acted as vaginotrophic agent of considerably weaker uterotrophic activity compared to estradiol. Evaluation of the fraction and 11 isoflavonoids isolated therefrom using Ishikawa cells and estrogen receptor (ER) isotype-specific reporter cells suggested that the estrogenic activity of the fraction could be attributed primarily to daidzein and dihydroglycitein and secondarily to glycitein. The potency-based selectivity of daidzein, dihydroglycitein and glycitein for gene expression through ERβ versus ERα, expressed relative to estradiol, was 37, 27 and 20, respectively. However, the rank order of relative-to-estradiol potencies of induction of alkaline phosphatase in Ishikawa cells, a reliable marker of estrogenic activity, was daidzein > dihydroglycitein >> glycitein. The considerably higher estrogenic activity of dihydroglycitein compared to glycitein could be attributed to the partial agonist/antagonist activity of dihydroglycitein through ERβ. Calculation of theoretical free energies of binding predicted the partial agonism/antagonism of dihydroglycitein through ERβ. The fraction and the isolated isoflavonoids promoted lactogenic differentiation of HC11 mammary epithelial cells at least as effectively as premenopausal levels of estradiol. This data suggests that the estrogenic activity of the fraction likely depends on the metabolism of glycitein to dihydroglycitein; that the fraction could exert vaginotrophic activity likely without challenging endocrine cancer risk more than estrogen-alone supplementation; and that the fraction's safety for the reproductive track warrants a more detailed evaluation. [ABSTRACT FROM AUTHOR]

Many known estrogens, both natural and synthetic, may act as antioxidants. We designed and synthesized 22 novel estrogen analogues with different ring junctions or substitutions, such as fluorine. We studied the antioxidant capacity in vitro of 35 synthetic estrogen analogues in aqueous lipoprotein solution by monitoring the formation of conjugated dienes. In addition to a free C-3 hydroxyl group, the two most active antioxidants had either a methyl group at C-4 and a six-carbon D-ring, or a fluorine atom at C-2 and an unsaturated B-ring. Extension of the D-ring increased the antioxidant capacity of 6-oxa estrogens. Compounds with a fluorine atom at C-2 were similar or more potent antioxidants compared with the principal endogenous estrogen, 17β-estradiol. In compounds with a substituted C-3 hydroxyl group, the antioxidant capacity could be significantly increased by additional double bonds in the C- or D-rings. In conclusion, we show that the antioxidant capacity of estrogen analogues could be increased by structural changes. [ABSTRACT FROM AUTHOR]

Neuroinflammation in the central nervous system is triggered by toxic stimuli or degenerative events, orchestrates the interplay of brain-intrinsic immune cells and neighboring neural cells, and sequentially allows leukocyte extravasation from the periphery into the brain parenchyma. During the inflammatory cascade, immune-competent cells become activated and secrete a plethora of cytokines and chemokines which form a local inflammatory signaling network important for warding off harmful stimuli to the host but are likewise necessary to preserve damaged brain tissue. Inflammatory responses are initiated by extra- and intra-cellular pathogen and danger-associated receptors. These signals are processed by multi-protein complexes termed inflammasomes which trigger the production of biologically active interleukins-1 and 18 after the cleavage of caspase-1. Estrogens and progesterone are neuroprotective and anti-inflammatory in diverse disease models of the brain in particular under acute inflammatory conditions such as stroke and traumatic brain injury. Both steroids are able to attenuate pro-inflammatory cytokine activity. Recent literature and our own studies provide convincing evidence that the anti-inflammatory potency of these steroids result from a complex interaction with the inflammasome activation and their up-stream regulatory network of miRNAs in brain-intrinsic innate immune cells. This article examines steroid–inflammasome interactions in the brain during brain injury and illuminates the importance of regulation initial upstream events during neuroinflammation. This article is part of a Special Issue entitled ‘Steroid Perspectives’. [ABSTRACT FROM AUTHOR]

17β-amino-1,3,5(10)estratrien-3-ol (17βAE 2 ), is the 17β-aminoestrogens prototype possessing anticoagulant activity, contrasting with the procoagulant effects of 17β-estradiol (17βE 2 ). Its estrogenicity profile has not been reported, and it was evaluated by uterotrophic assay, estrogen receptor binding affinity and its ability to induce gene transcription of the human estrogen receptor (hER)α mediated in a Saccharomyces cerevisiae yeast expression system. Additionally, 17βAE 2 and 17αAE 2 were compared with 17βE 2 in HeLa cells co-transfected with expression vectors for hERα or hERβ subtypes and for an estrogen-responsive reporter gene. Immature female CD1 mice and Wistar rats (21 days old) were treated for three days with 17βAE 2 (10–5000 μg/kg), 17βE 2 (0.001–1000 μg/kg) or vehicle (propylenglycol 10 ml/kg) and uterine weights were estimated. 17βAE 2 increased uterine weight in a dose-dependent manner. The effective dose (ED) 50 uterine weight values: 17βAE 2 = 552 and 764 μg/kg (17βE 2 = 4.8 and 16 μg/kg) and their relative uterotrophic potency were 0.86 and 2.1 (17βE 2 = 100) in mice and rats, respectively. 17βAE 2 competed with [ 3 H]E 2 for the estrogen receptor. The 17βAE 2 relative binding affinities (RBAs) were: 0.074; K i = 2.2 × 10 −6 M (17βE 2 = 100; K i = 1.6 × 10 −9 M); 0.029 and K i = 3.8 × 10 −6 M (17βE 2 = 100; K i = 1.1 × 10 −9 M) for mice and rats uteri respectively. 17βAE 2 activated hERα-mediated β-galactosidase transcription activity in the yeast system co-transfected with hERα gene. 17βAE 2 effective concentration (EC) 50 = 1.82 μM (17βE 2 = 2.14 nM) with a relative potency of 0.12 (17βE 2 = 100). These transactivation effects were abolished by the antagonist fulvestrant (ICI 182,780), similarly to 17βE 2 . 17βAE 2 and 17αAE 2 bind with low relative affinity to hERα and hERβ. Both induced hER-mediated reporter gene transactivation in a dose-response manner. The overall results provide evidence that 17βAE 2 has a weak agonist estrogenic action greatly mediated through the hERβ and to a lesser extent the hERα at genomic level. [ABSTRACT FROM AUTHOR]

Selective estrogen receptor modulators (SERMs) have been reported to enhance synaptic plasticity and improve cognitive performance in adult rats. SERMs have also been shown to induce neuroprotection against cerebral ischemia and other CNS insults. In this study, we sought to determine whether acute regulation of neurogenesis and spine remodeling could be a novel mechanism associated with neuroprotection induced by SERMs following cerebral ischemia. Toward this end, ovariectomized adult female rats were either implanted with pellets of 17β-estradiol (estrogen) or tamoxifen, or injected with raloxifene. After one week, cerebral ischemia was induced by the transient middle-cerebral artery occlusion (MCAO) method. Bromodeoxyuridine (BrdU) was injected to label dividing cells in brain. We analyzed neurogenesis and spine density at day-1 and day-5 post MCAO. In agreement with earlier findings, we observed a robust induction of neurogenesis in the ipsilateral subventricular zone (SVZ) of both the intact as well as ovariectomized female rats following MCAO. Interestingly, neurogenesis in the ipsilateral SVZ following ischemia was significantly higher in estrogen and raloxifene-treated animals compared to placebo-treated rats. In contrast, this enhancing effect on neurogenesis was not observed in tamoxifen-treated rats. Finally, both SERMs, as well as estrogen significantly reversed the spine density loss observed in the ischemic cortex at day-5 post ischemia. Taken, together these results reveal a profound structural remodeling potential of SERMs in the brain following cerebral ischemia. This article is part of a Special Issue entitled “Sex steroids and brain disorders”. [ABSTRACT FROM AUTHOR]

Estradiol (E) has been suggested to have a neuroprotective effect in young animals but has neutral or harmful effects when it is administered to aged animals. In the present study, we determined whether the post-ovariectomy (post-OVX) timeframe elapsed before the initiation of chronic E treatment is critical for the estrogenic induction of neurotrophins (brain-derived neurotrophic factor, BDNF, and synaptophysin, SYN) in the rodent hippocampus. Adult mice were OVX and, a short period (short-term E (STE) animals) or a long period (long-term E (LTE) animals) after the OVX, were daily treated with E. Control animals were treated with sesame oil (short-term control (STC) and long-term control (LTC) animals). Protein expression was determined using an immunohistochemical approach. Transcriptional activity in the hippocampus of individual BDNF promoters was assessed by real-time quantitative RT-PCR, and the methylation levels of regulatory regions were analyzed by methylation-specific PCR and combined bisulfite restriction analysis. STE animals showed increased BDNF and SYN protein expression and a higher activity of BDNF II, IV, and V promoters. In contrast, LTE animals did not show E induction of neurotrophins. In these animals, the methylation levels of regulatory sequences of the BDNF were higher than in the STE animals in a CpG island of promoter V and in the CRE regulatory site located in promoter IV. With this experiment, we determined that a prolonged period of hypoestrogenicity disrupts the E-induction of neurotrophins, and we postulated that DNA methylation is one of the epigenetic mechanisms that could explain the E-insensitivity of the BDNF after a long period post-OVX. [ABSTRACT FROM AUTHOR]

The involvement of estrogens, which influence many physiologic processes, has been shown in the development or progression of several diseases including some cancers, most notably breast cancer, and autoimmune disorders. Estrogenic signal is transferred via estrogen receptors (ER) which have dual localization, predominantly intracellular but also in plasma membrane. The discovery of membrane-associated ER (mER) has greatly expanded our understanding of estrogen action; upon ligand binding, mER rapidly activate different signaling pathways inducing downstream transcription factors. Some target genes of the mER pathway may be activated independently of the intracellular ER. Additionally, intracellular ER action can be modulated by mER-initiated signaling. Most notably, the identification of autoantibodies reacting with ER (ERAB) and their possible pathogenic role in autoimmunity and cancer have opened a new path for the research in the estrogen-related receptor activity. In this review, we briefly recapitulate the localization and function of ER and mostly discuss the possible role of ERAB as novel potential prognostic and/or predictive tools in autoimmunity and cancer. [ABSTRACT FROM AUTHOR]

Steroids were first analyzed by immunoassay-based methods followed by gas chromatography mass spectrometry (GC–MS or GC–MS/MS) with derivatization techniques since steroids are neutral and do not ionize at a high level using the electrospray ionization technique. We now report a validated liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of seven steroidal compounds, i.e., estradiol (E 2 ), estrone (E 1 ), testosterone (Testo), dihydrotestosterone (DHT), androst-5-ene-3β, 17β-diol (5-diol), dehydroepiandrosterone (DHEA) and androstenedione (4-dione). The system used is a UPLC–MS/MS (Qtrap 6500) system. With this method, the sample preparation is the combination of liquid–liquid extraction and a simple selective derivatization for only E 1 and E 2 . This assay method is simple and practically eliminates potential contamination. Low quantification limits of 1 pg/mL, 4 pg/mL, 50 pg/mL, 10 pg/mL, 100 pg/mL, 500 pg/mL and 100 pg/mL have been found, respectively for the steroids mentioned above. Without derivatization, DHT sensitivity can be as low as 4 pg/mL with S/N ≥ 5. A full validation has been performed for the seven compounds in compliance with GLP and FDA guidelines for bioanalytical method development and validation. Recovery of all seven compounds in unstripped serum is similar to that in stripped serum: 72.1–84.7% for E 2 , 83.6–94.5% for E 1 , 88.2–90.3% for Testo, 82.0–90.6% for DHT, 84.9–92.0% for 5-diol, 88.1–93.8% for DHEA and 86.2–90.3% for 4-dione, respectively. A good linearity is obtained with R > 0.99 for each compound within its calibration range. Accuracies of all levels of QC are within the range of 15% for all seven compounds. The between day variation coefficients are 6.1–8.9% for the low limits of quantification of all seven compounds with 0.7–6.1% for higher levels of QCs for all seven compounds. All results of other test parameters similarly meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines. By comparison of GC–MS/MS and LC–MS/MS data for six derivatized and nonderivatized free steroids, the present data show the crucial importance to use validated assays according to the FDA guidelines to increase specificity, precision and reliability of the absolute values associated with MS/MS-based assays. This method has already been applied to series of samples from clinical trials and is ready for wide clinical use. [ABSTRACT FROM AUTHOR]

Estrogen is involved in inflammation/pain of temporomandibular joint (TMJ), but the underlying mechanisms are largely unknown. Cadherin-11 plays an essential role in synovial inflammation. This study examined whether estrogen could potentiate cadherin-11 in synoviocytes and contribute to TMJ inflammatory pain. Female rats were ovariectomized, treated with increasing doses of 17β-estradiol for 10 days, and injected intra-articularly with complete Freund’s adjuvant to induce TMJ inflammation. The expression of cadherin-11 in synovial membrane was evaluated. TMJ pain was blocked with intra-articular injection of anti-cadherin-11 antibody and evaluated by head withdrawal threshold. Primary TMJ synoviocytes were treated with estradiol and tumor necrosis factor (TNF)-α or blocked with anti-cadherin-11 antibody to assess the expression of cadherin-11, interleukin (IL)-6, cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS). We observed that estradiol potentiated the inflammation-induced expression of cadherin-11 in the synoviocytes of synovial membrane from inflamed TMJ. Estradiol induced cadherin-11 expression in a dose- and time-dependent manner in primary synoviocytes and further potentiated the induction of cadherin-11 by TNF-α in synoviocytes. Furthermore, an estrogen receptor antagonist or a NF-κB inhibitor partially blocked the effects of estradiol on cadherin-11 induction in the synovial membrane. Blocking cadherin-11 partially reversed the TMJ inflammatory pain and estradiol-potentiated proliferation of synovial lining cells accompanied with iNOS expression. In addition, blocking cadherin-11 reversed TNF-α-induced and estradiol-potentiated transcription of IL-6, COX-2, and iNOS in primary synoviocytes. These results suggest that estrogen aggravated TMJ inflammatory pain partially through cadherin-11-mediated release of proinflammatory cytokines and enzymes in the synoviocytes. [ABSTRACT FROM AUTHOR]

Until recently, synonymous mutations (which do not change amino acids) have been much neglected. Some evidence suggests that this kind of mutations could affect mRNA secondary structure or stability, translation kinetics and protein structure. To explore deeper the role of synonymous mutations, we studied their consequence on the functional activity of the estrogen receptor alpha (ERα). The ERα is a ligand-inducible transcription factor that orchestrates pleiotropic cellular effects, at both genomic and non-genomic levels in response to estrogens. In this work we analyzed in transient transfection experiments, the activity of ERα carrying the synonymous mutation Ala87, a polymorphism involving about 5–10% of the population. In comparison to the wild type receptor, our results show that ERαA87 mutation reduces the transactivation efficiency of ERα on an ERE reporter gene while its expression level remains similar. This mutation enhances 4-OHT-induced transactivation of ERα on an AP1 reporter gene. Finally, the mutation affects the subcellular localization of ERα in a cell type specific manner. It enhances the cytoplasmic location of ERα without significant changes in non-genomic effects of E2. The functional alteration of the ERαA87 determined in this work highlights the relevance of synonymous mutations for biomedical and pharmacological points of view. [ABSTRACT FROM AUTHOR]

Highlights: [•] Brain SRC-1 has multifunctional potentials in the mediating of steroids action. [•] Hippocampal estradiol, AR, ERα and ERβ were downregulated by letrozole. [•] Letrozole decreased SRC-1 in specific limbic, diencephalic and brainstem nuclei. [•] SRC-1 my mediate letrozole regulation of memory, neuroendocrine and integration. [•] The side effects of letrozole on CNS should be taken to serious consideration. [Copyright &y& Elsevier]

Highlights: [•] ERβ is upregulated in the hippocampus of the aromatase-KO-mouse. [•] In hippocampal neurons, ERβ is upregulated after aromatase inhibition (letrozole). [•] In hippocampal neurons, agonists of ERβ downregulate synaptopodin. [•] Synaptopodin is downregulated by estradiol and letrozole in neurons and in the ArKO. [•] Balance of ER subtype and ligand levels controls hippocampal synaptic plasticity. [ABSTRACT FROM AUTHOR]

Highlights: [•] Serum estradiol (E2) should remain subthreshold during menopause management. [•] Intravaginal prasterone (DHEA) permits strictly local intracellular formation of E2. [•] Serum sex steroids remain within the normal postmenopausal range with DHEA. [ABSTRACT FROM AUTHOR]

Highlights: [•] Apurinic endonuclease (Ape1) is required to maintain protein and DNA integrity. [•] 17β-Estradiol (E2) increases Ape1 protein expression in cerebral cortical neurons. [•] E2 reduces hypoxia-induced oxidative DNA damage in cortical neurons. [•] The E2-induced expression of Ape1 helps to reduce hypoxia-induced neuronal damage. [•] These studies identify a mechanism by which E2 neuroprotection may be conferred. [ABSTRACT FROM AUTHOR]

Abstract: The neuroactive steroids 17β-estradiol and progesterone control a broad spectrum of neural functions. Besides their roles in the regulation of classical neuroendocrine loops, they strongly influence motor and cognitive systems, behavior, and modulate brain performance at almost every level. Such a statement is underpinned by the widespread and lifelong expression pattern of all types of classical and non-classical estrogen and progesterone receptors in the CNS. The life-sustaining power of neurosteroids for tattered or seriously damaged neurons aroused interest in the scientific community in the past years to study their ability for therapeutic use under neuropathological challenges. Documented by excellent studies either performed in vitro or in adequate animal models mimicking acute toxic or chronic neurodegenerative brain disorders, both hormones revealed a high potency to protect neurons from damage and saved neural systems from collapse. Unfortunately, neurons, astroglia, microglia, and oligodendrocytes are comparably target cells for both steroid hormones. This hampers the precise assignment and understanding of neuroprotective cellular mechanisms activated by both steroids. In this article, we strive for a better comprehension of the mutual reaction between these steroid hormones and the two major glial cell types involved in the maintenance of brain homeostasis, astroglia and microglia, during acute traumatic brain injuries such as stroke and hypoxia. In particular, we attempt to summarize steroid-activated cellular signaling pathways and molecular responses in these cells and their contribution to dampening neuroinflammation and neural destruction. This article is part of a Special Issue entitled ‘CSR 2013’. [Copyright &y& Elsevier]

Abstract: The aims of this work were to investigate if oestradiol 10−8 M in the incubation media of either the ovary alone (OV) or the ganglion compartment of an ex vivo coeliac ganglion–superior ovarian nerve–ovary system (a) modifies the release of ovarian progesterone (P4) and oestradiol (E2) on dioestrus II, and (b) modifies the ovarian gene expression of 3β-HSD and 20α-HSD enzymes and markers of apoptosis. The concentration of ovarian P4 release was measured in both experimental schemes, and ovarian P4 and E2 in the ex vivo system by RIA at different times. The expression of 3β-hydroxysteroid dehydrogenase, 20α-hydroxysteroid dehydrogenase and antiapoptotic bcl-2 and proapoptotic bax by RT-PCR were determined. E2 added in the coeliac ganglion caused an increase in the ovarian release of the P4, E2 and 3β-HSD, while in the ovary incubation alone it decreased P4 and 3β-HSD but increased and 20α-HSD and bax/bcl-2 ratio. It is concluded that through a direct effect on the ovary, E2 promotes luteal regression in DII rats, but the addition of E2 in the coeliac ganglion does not have the same effect. The peripheral nervous system, through the superior ovarian nerve, has a protective effect against the apoptotic mechanism on DII. [Copyright &y& Elsevier]

Abstract: The hippocampus synthesizes estrogen and androgen in addition to the circulating sex steroids. Synaptic modulation by hippocampus-derived estrogen or androgen is essential to maintain healthy memory processes. Rapid actions (1–2h) of 17β-estradiol (17β-E2) occur via synapse-localized receptors (ERα or ERβ), while slow genomic E2 actions (6–48h) occur via classical nuclear receptors (ERα or ERβ). The long-term potentiation (LTP), induced by strong tetanus or theta-burst stimulation, is not further enhanced by E2 perfusion in adult rats. Interestingly, E2 perfusion can rescue corticosterone (stress hormone)-induced suppression of LTP. The long-term depression is modulated rapidly by E2 perfusion. Elevation of the E2 concentration changes rapidly the density and head structure of spines in neurons. ERα, but not ERβ, drives this enhancement of spinogenesis. Kinase networks are involved downstream of ERα. Testosterone (T) or dihydrotestosterone (DHT) also rapidly modulates spinogenesis. Newly developed Spiso-3D mathematical analysis is used to distinguish these complex effects by sex steroids and kinases. It has been doubted that the level of hippocampus-derived estrogen and androgen may not be high enough to modulate synaptic plasticity. Determination of the accurate concentration of E2, T or DHT in the hippocampus is enabled by mass-spectrometric analysis in combination with new steroid-derivatization methods. The E2 level in the hippocampus is approximately 8nM for the male and 0.5–2nM for the female, which is much higher than that in circulation. The level of T and DHT is also higher than that in circulation. Taken together, hippocampus-derived E2, T, and DHT play a major role in modulation of synaptic plasticity. [Copyright &y& Elsevier]

Abstract: Breast cancer is one of several tumors, including prostate, thyroid and kidney, which display a remarkable predilection for metastasis to bone. The preference to metastasize to bone by tumor cells relies on specific interactions among tumor cells, bone marrow microenvironment and bone cells. Osteomimicry is postulated to enable the survival of tumor cells in the bone tissue. Using gene profiling array and RT-PCR we demonstrated the message expression of few bone matrix proteins in mammary adenocarcinoma cells as well as that of cell adhesion molecules (CAMs). A CAM molecule, named SVEP1, was previously shown to be expressed in osteoblastic cells both in vivo and in vitro mediating cell adhesion in the bone-marrow niches. Both estradiol (17βE2) and TNFα regulate the expression of adhesion molecules and act in bone-cancer-crosstalk. We focused on differential regulation of SVEP1 gene comparing pre-osteoblastic MBA-15 and mammary adenocarcinoma DA3 cells. 17βE2 and TNFα activated SVEP1 promoter, increased its message and protein levels in both cell types. Using chromatin immunoprecipitation assay, we quantified SVEP1 promoter occupancy by transcription factors; TFIIB, ERα, NF-κB, Sp1 and their binding was also regulated by both factors. By comparing pre-osteoblastic with mammary adenocarcinoma cells, the study expands our understanding of SVEP1 gene expression regulation and it sheds light on its involvement in bone-cancer-microenvironment interactions. [Copyright &y& Elsevier]

Abstract: A number of studies have demonstrated that 17β-estradiol (E2) protects the brain from ischemia and yet the mechanism by which this hormone brings about its protective effect is unclear. Interestingly, like E2, overexpression of the oxidative stress response protein Cu/Zn superoxide dismutase (SOD1), which plays a critical role in regulating reactive oxygen species, also protects the brain from ischemia. Because we previously showed that E2 treatment of cultured mammary cells increases SOD1 expression, we hypothesized that E2 might increase SOD1 expression in the brain and that this E2-mediated increase in SOD1 expression might help to protect the brain from ischemia. We now show that SOD1 is expressed in cortical neurons, that SOD1 expression is increased by exposure of brain slice cultures to E2, and that the E2-mediated increase in SOD1 expression is further augmented by exposure of brain slice cultures to increased superoxide levels or oxygen and glucose deprivation. Importantly, when cortical neurons are exposed to increased superoxide levels and markers of protein and DNA damage, nitrotyrosine and 8-oxoguanine, respectively, are measured, both protein and DNA damage are reduced. In fact, E2 reduces nitrotyrosine and 8-oxoguanine levels in brain slice cultures regardless of whether they have or have not been exposed to increased superoxide levels. Likewise, when brain slice cultures are treated with E2 and deprived of oxygen and glucose, 8-oxoguanine levels are reduced. Taken together, these studies provide a critical link between E2 treatment, SOD1 expression, and neuroprotection and help to define a mechanism through which E2-mediated neuroprotection may be conferred. [Copyright &y& Elsevier]

Abstract: In the brain, the synthesis of neurosteroids and receptor activation during gonadal sex differentiation in teleosts are poorly understood. In the present study, the protogynous orange-spotted grouper (Epinephelus coioides) was selected as a model fish, and we hypothesized that de novo synthesis of neural estrogen may play a role in the female grouper brain during gonadal sex differentiation. We investigated the temporal expression of the genes StAR, cyp19a1b and pcna and the sex steroid nuclear receptors for estrogen (ERα, ERβ1 and ERβ2), androgen (AR) and the plasma membrane-associated estrogen receptor (GPR30) in the brain during early developmental ages from 90 days after hatching (dah) to 180dah after gonadal sex differentiation. Our results revealed that mRNA for ERs and GPR30 but not AR was significantly increased at 110dah (a time close to gonadal sex differentiation) in the forebrain and midbrain and for cyp19a1b at 110dah in the forebrain. Brain aromatase activity and estradiol (E2) levels, but not testosterone (T), were increased in the forebrain at 110 and 120dah, respectively. Furthermore, exogenous E2 stimulated cyp19a1b transcripts in the forebrain and hypothalamus and immunoreactive (ir)Cyp19a1b (aromatase enzyme) in the forebrain. irCyp19a1b localized in the glial cells of the forebrain regions. Therefore, we identified a peak of functional aromatase activity and estrogen signaling in the early grouper brain during gonadal sex differentiation. Moreover, pcna transcripts (a marker for cell proliferation activity) were higher in the early brain at 110–150dah. Thus, a peak time of development in the brain is suggested to occur during gonadal sex differentiation in the grouper. [Copyright &y& Elsevier]

Abstract: Three sex steroid hormones, estradiol-17β (E2), 11-ketotestosterone (11-KT), and 17α,20β-dihydroxy-4-pregnen-3-one (DHP), are well established as primary estrogen, androgen, and progestin, respectively, in teleost fish. Japanese eel, Anguilla japonica, would be a suitable candidate to study ovarian steroid physiology of fish because the ovarian growth and steroidogenesis is dormant under laboratory condition but can be induced by administration of exogenous gonadotropic reagents. In this review, we summarized our work on the function and production of sex steroid hormones in the ovary of the Japanese eel during ovarian growth and oocyte maturation artificially induced by treatment with extract of salmon pituitary. In vitro and in vivo assays suggest that 11-KT and E2 play primary roles in previtellogenic and vitellogenic growth of oocytes, respectively, whereas DHP is essential for induction of final oocyte maturation. We also reviewed the correlation between ovarian steroidogenesis to produce these sex steroid hormones, serum titers and gene expression. [Copyright &y& Elsevier]

Abstract: DNA-intercalating molecules can impair DNA replication, DNA repair, and gene transcription. We previously demonstrated that XR5944, a DNA bis-intercalator, specifically blocks binding of estrogen receptor-α (ERα) to the consensus estrogen response element (ERE). The consensus ERE sequence is AGGTCAnnnTGACCT, where nnn is known as the tri-nucleotide spacer. Recent work has shown that the tri-nucleotide spacer can modulate ERα–ERE binding affinity and ligand-mediated transcriptional responses. To further understand the mechanism by which XR5944 inhibits ERα–ERE binding, we tested its ability to interact with consensus EREs with variable tri-nucleotide spacer sequences and with natural but non-consensus ERE sequences using one dimensional nuclear magnetic resonance (1D 1H NMR) titration studies. We found that the tri-nucleotide spacer sequence significantly modulates the binding of XR5944 to EREs. Of the sequences that were tested, EREs with CGG and AGG spacers showed the best binding specificity with XR5944, while those spaced with TTT demonstrated the least specific binding. The binding stoichiometry of XR5944 with EREs was 2:1, which can explain why the spacer influences the drug–DNA interaction; each XR5944 spans four nucleotides (including portions of the spacer) when intercalating with DNA. To validate our NMR results, we conducted functional studies using reporter constructs containing consensus EREs with tri-nucleotide spacers CGG, CTG, and TTT. Results of reporter assays in MCF-7 cells indicated that XR5944 was significantly more potent in inhibiting the activity of CGG- than TTT-spaced EREs, consistent with our NMR results. Taken together, these findings predict that the anti-estrogenic effects of XR5944 will depend not only on ERE half-site composition but also on the tri-nucleotide spacer sequence of EREs located in the promoters of estrogen-responsive genes. [Copyright &y& Elsevier]

Abstract: We re-examined mouse ERα mRNA variants using rapid amplification of cDNA ends (RACE) and RT-PCR. Our analysis showed the presence of several mRNA variants containing unique 5′- or 3′-nucleotide sequences. We mapped the cDNA sequences on the mouse genome, and identified four novel 3′-terminal and 5′-leader exons in the intronic region between exons 4 and 5. RT-PCR analysis revealed that the expression patterns of the C-terminally truncated ERα products (CTERPs) were similar to that of Wild-type ERα and that the N-terminally truncated ERα products (NTERPs) appeared to have different expression profiles. Moreover, we constructed expression vectors and analyzed the subcellular localization and the transcriptional activation abilities of the variant proteins in transfected HEK293 cells using immunocytochemistry and luciferase reporter assay. The CTERP variants localized in the nuclei and constitutively activated estrogen response element (ERE)-driven promoters, while the NTERP variant was located in the extra-nuclear regions and had no ability to activate the ERE promoters in the presence or absence of 10nM estradiol. Our results indicate that the mouse ERα gene is more complex than previously thought in terms of genomic organization and that alternative splicing and alternative usage of intronic promoters contribute to the remarkable diversity of ERα mRNAs and proteins. [Copyright &y& Elsevier]

Abstract: Endogenous estrogen plays an integral role in the etiology of breast and endometrial cancer, and conceivably ovarian cancer. However, the underlying mechanisms and the importance of patterns of estrogen metabolism and specific estrogen metabolites have not been adequately explored. Long-standing hypotheses, derived from laboratory experiments, have not been tested in epidemiologic research because of the lack of robust, rapid, accurate measurement techniques appropriate for large-scale studies. We have developed a stable isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS2) method that can measure concurrently all 15 estrogens and estrogen metabolites (EM) in urine and serum with high sensitivity (level of detection=2.5–3.0fmol EM/mL serum), specificity, accuracy, and precision [laboratory coefficients of variation (CV''s) ≤5% for nearly all EM]. The assay requires only extraction, a single chemical derivatization, and less than 0.5mL of serum or urine. By incorporating enzymatic hydrolysis, the assay measures total (glucuronidated+sulfated+unconjugated) EM. If the hydrolysis step is omitted, the assay measures unconjugated EM. Interindividual differences in urinary EM concentrations (pg/mL creatinine), which reflect total EM production, were consistently large, with a range of 10–100-fold for nearly all EM in premenopausal and postmenopausal women and men. Correlational analyses indicated that urinary estrone and estradiol, the most commonly measured EM, do not accurately represent levels of total urinary EM or of the other EM. In serum, all 15 EM were detected as conjugates, but only 5 were detected in unconjugated form. When we compared our assay methods with indirect radioimmunoassays for estrone, estradiol, and estriol and enzyme-linked immunosorbent assays for 2-hydroxyestrone and 16α-hydroxyestrone, ranking of individuals agreed well for premenopausal women [Spearman r (r s)=0.8–0.9], but only moderately for postmenopausal women (r s =0.4–0.8). Our absolute readings were consistently lower, especially at the low concentrations characteristic of postmenopausal women, possibly because of improved specificity. We are currently applying our EM measurement techniques in several epidemiologic studies of premenopausal and postmenopausal breast cancer. [Copyright &y& Elsevier]

Abstract: The age-related decline in ovarian sex hormone production following the onset of menopause alters skeletal muscle metabolic, structural and functional characteristics. The myosin heavy chain (MHC) expression pattern defines skeletal muscle contraction velocity and is therefore an important factor in skeletal muscle function. The present study was designed to examine the effects of 17β estradiol (E2), estrogen receptor (ER) subtype selective agonists (ERα, ERβ) or genistein (Gen) following ovary removal (OVX) in female Wistar rats in combination with a high intensity treadmill-based exercise protocol (Ex) or normal cage-based activity (NoEx) on MHC protein expression patterns in the slow fiber type m.Soleus (Sol) and the fast fiber type m.Gastrocnemius (Gas). Gen and E2 in the Sol significantly stimulated MHC-I expression relative to OVX only in the absence of exercise (NoEx). MHC-IIb expression in the Gas was significantly increased relative to OVX in Gen Ex and E2 Ex and NoEx groups. The estrogenic effects in the Sol and Gas were both predominantly mediated via ERβ pathways, since the ERβ agonist induced greater MHC increases than OVX or ERα. We therefore propose that high intensity exercise in combination with exposure to E2, Gen, ERα or ERβ agonists in OVX rats exerts differential effects on MHC expression in skeletal muscles composed of mainly slow type I MHC (Sol) or fast type II MHC (Gas). In summary, the data shows that MHC composition is affected by estrogens and exercise in a fiber type specific manner and that these effects are mainly mediated by ER-β. This is of great importance with respect to skeletal muscle health and potential treatment with ER selective agonists. [Copyright &y& Elsevier]

Abstract: As breast cancer cells develop secondary resistance to estrogen deprivation therapy, they increase their utilization of non-genomic signaling pathways. Our prior work demonstrated that estradiol causes an association of ERα with Shc, Src and the IGF-1-R. In cells developing resistance to estrogen deprivation (surrogate for aromatase inhibition) and to the anti-estrogens tamoxifen, 4-OH-tamoxifen, and fulvestrant, an increased association of ERα with c-Src and the EGF-R occurs. At the same time, there is a translocation of ERα out of the nucleus and into the cytoplasm and cell membrane. Blockade of c-Src with the Src kinase inhibitor, PP-2 causes relocation of ERα into the nucleus. While these changes are not identical in response to each anti-estrogen, ERα binding to the EGF-R is increased in response to 4-OH-tamoxifen when compared with tamoxifen. The changes in EGF-R interactions with ERα impart an enhanced sensitivity of tamoxifen-resistant cells to the inhibitory properties of the specific EGF-R tyrosine kinase inhibitor, AG 1478. However, with long term exposure of tamoxifen-resistant cells to AG 1478, the cells begin to re-grow but can now be inhibited by the IGF-R tyrosine kinase inhibitor, AG 1024. These data suggest that the IGF-R system becomes the predominant signaling mechanism as an adaptive response to the EGF-R inhibitor. Taken together, this information suggests that both the EGF-R and IGF-R pathways can mediate ERα signaling. To further examine the effects of fulvestrant on ERα function, we examined the acute effects of fulvestrant, on non-genomic functionality. Fulvestrant enhanced ERα association with the membrane IGF-1-receptor (IGF-1-R). Using siRNA or expression vectors to knock-down or knock-in selective proteins, we further demonstrated that the ERα/IGF-1-R association is Src-dependent. Fulvestrant rapidly induced IGF-1-R and MAPK phosphorylation. The Src inhibitor PP2 and IGF-1-R inhibitor AG1024 greatly blocked fulvestrant-induced ERα/IGF-1-R interaction leading to a further depletion of total cellular ERα induced by fulvestrant and further enhanced fulvestrant-induced cell growth arrest. More dramatic was the translocation of ERα to the plasma membrane in combination with the IGF-1-R as shown by confocal microscopy. Taken in aggregate, these studies suggest that secondary resistance to hormonal therapy results in usage of both IGF-R and EGF-R for non-genomic signaling. [Copyright &y& Elsevier]

Abstract: A 17β-estradiol (E2) is hydrolyzed to 2-hydroxy-E2 (2-OHE2) and 4-hydroxy-E2 (4-OHE2) via cytochrome P450 (CYP) 1A1 and 1B1, respectively. In estrogen target tissues including the mammary gland, ovaries, and uterus, CYP1B1 is highly expressed, and 4-OHE2 is predominantly formed in cancerous tissues. In this study, we investigated the inhibitory effects of chrysoeriol (luteorin-3′-methoxy ether), which is a natural methoxyflavonoid, against activity of CYP1A1 and 1B1 using in vitro and cultured cell techniques. Chrysoeriol selectively inhibited human recombinant CYP1B1-mediated 7-ethoxyresorufin-O-deethylation (EROD) activity 5-fold more than that of CYP1A1-mediated activity in a competitive manner. Additionally, chrysoeriol inhibited E2 hydroxylation was catalyzed by CYP1B1, but not by CYP1A1. Methylation of 4-OHE2, which is thought to be a detoxification process, was not affected by the presence of chrysoeriol. In human breast cancer MCF-7 cells, chrysoeriol did not affect the gene expression of CYP1A1 and 1B1, but significantly inhibited the formation of 4-methoxy E2 without any effects on the formation of 2-methoxy E2. In conclusion, we present the first report to show that chrysoeriol is a chemopreventive natural ingredient that can selectively inhibit CYP1B1 activity and prevent the formation of carcinogenic 4-OHE2 from E2. [Copyright &y& Elsevier]

Abstract: Objective: This study discussed the role of estrogen as an antioxidant in the damage of vascular endothelial cells. Design: We treated bovine aortic endothelial cells (bAEC) either with 1mM of H2O2 alone or with 1μM of 17β-estradiol (E2) for 24h followed by 1mM of H2O2 for 3h. The cell survival was evaluated by MTT assay, cellular apoptosis by fluorescence activated cell sorter (FACS) and Hoechst 33342 staining, oxidative stress by intracellular reactive oxygen species (ROS) and apoptosis after oxidative stress by western blotting for phospho-p38, p38, and Bcl-2. Results: MTT assay showed that bAEC viability was reduced to 55.7±3.0% and 39.1±3.7% after 30 and 60min of H2O2 treatment, respectively. E2 and H2O2 treated cells did not show significant decrease in the cell survival. Similarly the FACS analysis and Hoechst 33342 stain showed that the latter decreased cellular apoptosis induced by H2O2. Intracellular ROS increased by 181.6±68.9% in the former and by 37.0±3.9% in the latter (P <0.05). The expression of phospho-p38 mitogen-activated protein kinase (MAPK) was higher in the latter. Conclusions: E2 mediates antioxidant effects on the oxidative stress induced by H2O2. This antioxidant effect on bAEC may elucidate the scientific basis of hormone therapy for maintaining cardiovascular integrity in postmenopausal women. [Copyright &y& Elsevier]

Abstract: Previous studies have suggested elevated estrogen production in tumour-bearing breast quadrants as well as in breast cancers versus benign tissue. Using highly sensitive assays, we determined breast cancer tissue estrogen concentrations together with plasma and benign tissue estrogen concentrations in each quadrant obtained from mastectomy specimens (34 postmenopausal and 13 premenopausal women). We detected similar concentrations of each of the three major estrogens estradiol (E2), estrone (E1) and E1S in tumour-bearing versus non-tumour-bearing quadrants. Considering malignant tumours, intratumour E1 levels were reduced in cancer tissue obtained from pre- as well as postmenopausal women independent of tumour ER status (average ratio E1 cancer: benign tissue of 0.2 and 0.3, respectively; p <0.001 for both groups), suggesting intratumour aromatization to be of minor importance. The most striking finding was a significant (4.1–8.6-fold) increased E2 concentration in ER positive tumours versus normal tissue (p <0.05 and <0.001 for pre- and postmenopausal patients, respectively), contrasting low E2 concentrations in ER− tumours (p <0.01 and <0.001 comparing E2 levels between ER+ and ER− tumours in pre- and postmenopausals, respectively). A possible explanation to our finding is increased ligand receptor binding capacity for E2 in receptor positive tumours but alternative factors influencing intratumour estrogen disposition cannot be excluded. [Copyright &y& Elsevier]

Abstract: Estradiol (E2) is produced locally in adipose tissue and could play an important role in fat distribution and accumulation, especially in women. It is well recognized that aromatase is expressed in adipose tissue; however the identity of its estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD) partner is not identified. To gain a better knowledge about the enzyme responsible for the conversion of estrone into estradiol, we determined the activity and expression levels of known estrogenic 17β-HSDs, namely types 1, 7 and 12 17β-HSD in preadipocytes before and after differentiation into mature adipocytes using an adipogenic media. Estrogenic 17β-HSD activity was assessed using [14C]-labelled estrone, while mRNA expression levels of types 1, 7 and 12 17β-HSD were quantified using real-time PCR and protein expression levels of type 12 17β-HSD was determined using immunoblot analysis. The data indicate that there is a low conversion of E1 into E2 in preadipocytes; however this activity is increased ∼5-fold (p <0.0001) in differentiated adipocytes. The increased estrogenic 17β-HSD activity is consistent with the increase in protein expression levels of 17β-HSD12. [Copyright &y& Elsevier]

Abstract: Cystic fibrosis (CF) is a major genetic disease in Caucasians affecting 1 in 2500 newborns. Hepatobiliary pathology is a major cause of morbidity and mortality in CF second only to pulmonary disease. SULT1E1 activity is significantly elevated, generally 20–30-fold, in hepatocytes of mouse models of CF. SULT1E1 is responsible for the inactivation of β-estradiol (E2) at physiological concentrations via conjugation with sulfonate. The increase in SULT1E1 activity results in the alteration of E2-regulated protein expression in CF mouse liver. To investigate the mechanism by which the absence of CFTR in human cholangiocytes induces SULT1E1 expression in hepatocytes, a membrane-separated human MMNK-1 cholangiocyte and human HepG2 hepatocyte co-culture system was developed. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in bile duct cholangiocytes but not hepatocytes, whereas SULT1E1 is expressed in hepatocytes but not cholangiocytes. CFTR expression in MMNK-1 cells was inhibited with siRNA by >90% as determined by immunoblot and immunohistochemical analysis. Control and CFTR-siRNA-MMNK-1 cells were co-cultured with HepG2 cells in a Transwell membrane-separated system. After 8h of co-culture, HepG2 cells were removed from exposure to MMNK-1 cells and placed in fresh medium. After 24–48h, expression of SULT1E1 and selected E2-regulated proteins was analyzed in the HepG2 cells. Results demonstrated that SULT1E1 message and activity were selectively induced in HepG2 cells co-cultured with CFTR-deficient MMNK-1 cells. The expression of E2-regulated proteins (IGF-1, GST-P1 and carbonic anhydrase II) was also altered in response to decreased E2 levels. Thus, the loss of CFTR activity in cholangiocytes stimulates the expression of SULT1E1 in hepatocytes by a paracrine mechanism. SULT1E1 expression in HepG2 cells is inducible by sterol mediated liver-X-receptor (LXR) activation although not by progestins that induce SULT1E1 in the endometrium. SULT1E1 induction in the human cholangiocyte/hepatocyte co-culture system is consistent with and supports the results observed in CF mice. The changes in hepatocyte gene expression affect liver biochemistry and may facilitate the development of CF liver disease. [Copyright &y& Elsevier]

Abstract: Sex steroids, including those through intratumoral production in an intracrine manner, play important roles in the development of invasive ductal carcinoma (IDC) of human breast, but biological and/or clinical significance of intratumoral production and metabolism of sex steroids, have remained largely unknown in the ductal carcinoma in situ (DCIS), an important precursor lesion of IDC. We recently examined tissue concentration of estradiol and 5α-dihydrotestosterone using liquid chromatography/electrospray tandem mass spectrometry in non-neoplastic breast, DCIS, and IDC tissues. Results of our study suggest that intratumoral concentrations of both estradiol and 5α-dihydrotestosterone are increased in DCIS, which is considered due to intratumoral production of these sex steroids. Therefore, both estradiol and 5α-dehydrotestosterone are considered to play important roles in the development of DCIS as well as IDC through an intracrine manner. Intratumoral metabolism and synthesis of estrogens and androgens as a result of the interactions of various enzymes are therefore also considered to play important roles in hormone dependent DCIS. Aromatase, which is one of the estrogen synthesis enzymes, plays an important role in intratumoral production of estrogen but other enzymes also play pivotal roles in intratumoral estrogen and androgen productions in human breast carcinoma. Therefore, in this review, we also focused on the importance of key intracrine enzymes such as 17β-hydroxysteroid dehydrogenases, steroid sulfatase, estrogen sulfotransferase, 5α-reductases in both IDC and DCIS. [Copyright &y& Elsevier]

Abstract: Sex steroids play an important role in the development and differentiation in several tissues. Biologically active hormones that are locally converted in endocrine organs in the tissue where they exert their effects without release into extracellular space is a field of endocrinology that has been called intracrinology. In pre-menopausal women the ovary is the main source of estrogens, but in post-menopausal women the estrogen production as main site of synthesis moves to peripheral tissues and almost all of the sex steroids are synthesised from precursors of adrenal origin. In breast cancer 60–80% of the tumors express high levels of oestrogen receptor (ER) α which gives estrogen a proliferative effect. Breast tumors tend to have a higher intratumoral estrogen concentration than normal breast tissue and plasma, and in situ synthesis and the metabolism of estrogens is believed to be of great importance for the development and progression of the disease. The activity of estrogen metabolizing enzymes in breast are mainly aromatase, estrone sulfatases and 17HSD enzymes. 17HSD1 and 17HSD2 are the family members known to be of main importance in breast cancer. High expression of 17HSD1 has been associated to poor prognosis in breast cancer and late relapse among patients with ER-positive tumors. One of the mechanisms behind high 17HSD1 expression is gene amplification. Low or absent expression of 17HSD2 is associated to decreased survival in ER-positive breast cancer. 17HSD14 is one of the latest discovered 17HSD enzymes, transfection of 17HSD14 in human breast cancer cells significantly decreased the levels of estradiol in the culture medium. Low expression of 17HSD14 mRNA expression in breast cancer was correlated to decreased survival. The understanding of intratumoral synthesis of sex steroids in breast cancer is crucial to understand the disease both in pre- and post-menopausal women. Further studies are desirable to state the direct role of these enzymes in breast cancer and which patients that may benefit from new therapeutic strategies targeting 17HSD enzymes. The new inhibitors targeting 17HSD1 have shown promising results in pre-clinical studies to have clinical potential in the future. [Copyright &y& Elsevier]

Abstract: SERMs bind to both estrogen receptor (ER)α and β, resulting in tissue dependent estrogen agonist or antagonist responses. Both raloxifene and tamoxifen are most frequently used SERMs and exert estrogen agonistic effects on human bone tissues, but the details of their possible direct effects on human bone cells have remained largely unknown. In our present study, we examined the comparative effects of raloxifene, tamoxifen, and native estrogen, estradiol on human osteoblast cell line, hFOB in vitro. Both the cell numbers and the ratio of the cells in S phase fraction were significantly increased by the treatment of raloxifene or tamoxifen as well as estradiol treatments in hFOB. Gene profile patterns following treatment with raloxifene, tamoxifen, and estradiol demonstrated similar patterns in a microarray/hierarchal clustering analysis. We also examined the expression levels of these genes detected by this analysis using quantitative RT-PCR. MAF gene was induced by raloxifene treatment alone. GAS6 gene was induced by raloxifene and tamoxifen as well as estradiol. An estrogen receptor blocker, ICI 18, 286, inhibited an increase of GAS6 gene expression but not the levels of MAF gene mRNA expression. Results of our present study demonstrated that raloxifene exerted direct protective effects on human osteoblasts in both estrogen receptor dependent and independent manners. [Copyright &y& Elsevier]