Perinatal activation of ERα and ERß but not GPER-1 masculinizes female rat caudate-putamen medium spiny neuron electrophysiological properties.

Exposure to steroid sex hormones such as 17ß-estradiol (estradiol) during early life potentially permanently masculinize neuron electrophysiological phenotype. In rodents, one crucial component of this developmental process occurs in males, with estradiol aromatized in the brain from testes-sourced testosterone. However, it is unknown whether most neuron electrophysiological phenotypes are altered by this early masculinization process, including medium spiny neurons (MSNs) of the rat caudate-putamen. MSNs are the predominant and primary output neurons of the caudate-putamen and exhibit increased intrinsic excitability in females compared to males. Here, we hypothesize that since perinatal estradiol exposure occurs in males, then a comparable exposure in females to estradiol or its receptor agonists would be sufficient to induce masculinization. To test this hypothesis, we injected perinatal female rats with estradiol or its receptor agonists and then later assessed MSN electrophysiology. Female and male rats on postnatal day 0 and 1 were systemically injected with either vehicle, estradiol, the estrogen receptor (ER)α agonist PPT, the ERß agonist DPN, or the G-protein-coupled receptor 1 (GPER-1) agonist G1. On postnatal days 19 ± 2, MSN electrophysiological properties were assessed using whole cell patch clamp recordings. Estradiol exposure abolished increased intrinsic excitability in female compared to male MSNs. Exposure to either an ERα or ERß agonist masculinized female MSN evoked action potential firing rate properties, whereas exposure to an ERß agonist masculinized female MSN inward rectification properties. Exposure to ER agonists minimally impacted male MSN electrophysiological properties. These findings indicate that perinatal estradiol exposure masculinizes MSN electrophysiological phenotype via activation of ERα and ERß.NEW & NOTEWORTHY This study is the first to demonstrate that estradiol and estrogen receptor α and ß stimulation during early development sexually differentiates the electrophysiological properties of caudate-putamen medium spiny neurons, the primary output neuron of the striatal regions. Overall, this evidence provides new insight into the neuroendocrine mechanism by which caudate-putamen neuron electrophysiology is sexually differentiated and demonstrates the powerful action of early hormone exposure upon individual neuron electrophysiology.

Estrogen Receptor ß as a Possible Double-Edged Sword Molecule in Breast Cancer: A Mechanism of Alteration of Its Role by Exposure to Endocrine-Disrupting Chemicals.

Estrogen is essential for the growth and development of mammary glands and its signaling is associated with breast cancer growth. Estrogen can exert physiological actions via estrogen receptors α/ß (ERα/ß). There is experimental evidence suggesting that in ERα/ß-positive breast cancer, ERα promotes tumor cell proliferation and ERß inhibits ERα-mediated transcriptional activity, resulting in abrogation of cell growth. Therefore, ERß is attracting attention as a potential tumor suppressor, and as a biomarker and therapeutic target in the ERα/ß-positive breast cancer. Based on this information, we have hypothesized that some endocrine-disrupting chemicals (EDCs) that can perturb the balance between ERα and ERß expression levels in breast cancer cells might have effects on the breast cancer proliferation (i.e., down-regulation of the α-type of ER). We have recently reported that 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), an active metabolite of bisphenol A, in ERα/ß-positive human breast cancer significantly down-regulates ERα expression, yet stimulates cell proliferation through the activation of ERß-mediated transcription. These results support our hypothesis by demonstrating that exposure to MBP altered the functional role of ERß in breast cancer cells from suppressor to promoter. In contrast, some EDCs, such as Δ9-tetrahydrocannabinol and bisphenol AF, can exhibit anti-estrogenic effects through up-regulation of ERß expression without affecting the ERα expression levels. However, there is no consensus on the correlation between ERß expression levels and clinical prognosis, which might be due to differences in exposed chemicals. Therefore, elucidating the exposure effects of EDCs can reveal the reason for inconsistent functional role of ERß in ERα/ß-positive breast cancer.

BPA-induced prostatic hyperplasia in vitro is correlated with the unbalanced gene expression of AR and ER in the epithelium and stroma.

As a typical environmental endocrine disruptor (EED), bisphenol A (BPA) can induce pathological hyperplasia of the prostatic epithelium and stroma. This study concentrates mainly on the effect and underlying mechanisms of BPA on prostatic hyperplasia, which is based on the culture of primary human prostate epithelial cells (HPEpiC) and human prostate fibroblasts (HPrF). In an effect to screen the optimal pro-survival BPA levels, HPEpiC and HPrF were, respectively, exposed to concentration gradients of BPA (10-12 M-10-4 M) solution diluted with two corresponding medium and incubated for 72 h at 37°C. CCK-8 assay showed that 10-9 M-10-5 M BPA could facilitate the proliferation of HPEpiC, while similar proliferative effect of HPrF only needed 10-11 M-10-7 M BPA. HPrF were more sensitive to BPA than HPEpiC. The qualification of PCNA gene expression measured using quantitative real-time polymerase chain reaction (qRT-PCR) also mirrored the BPA-induced cell proliferation. Additionally, our results considered that androgen receptor (AR), estrogen receptor (ERα, ERß), and NFKB1 gene expressions exhibited up-regulation in HPEpiC treated with 10-9 M BPA for 72 h. However, in HPrF, the identical BPA treatment could activate ERα, ERß, and NFKB1 gene expressions and down-regulated the expression of AR levels. It is further confirmed that low-dose BPA can indeed promote the proliferation of human prostate cells in vitro, and the mechanisms of BPA for prostatic epithelial and stromal hyperplasia may not be consistent.

Inhibition of brain 17ß-estradiol synthesis by letrozole induces cognitive decline in male and female rats.

BACKGROUND: Hippocampal aromatase is responsible for local synthesis of 17ß-estradiol (E2) that has much higher concentrations than serum levels in males and females. Letrozole, an aromatase inhibitor, passes through the brain barriers, distributes to the brain, and affects local E2 synthesis. Here, the effects of intra-cerebroventricular (ICV) letrozole administration in the presence and absence of gonads were examined on the cognitive abilities of male and female rats. METHOD: Animals received intra-ICV injection of letrozole or vehicle for 14 consecutive days. Spatial working memory, novel object recognition memory, and anxiety-related behavior, were evaluated using Y-maze, object recognition test, and elevated plus maze, respectively. The E2 levels in the serum and hippocampal tissue were measured by the ELISA technique. RT-PCR was performed to assess the hippocampal estrogen receptors (ER) expression. Moreover, letrozole effect on neuronal activity of CA1 pyramidal neurons was studied by in vivo single-unit recording. RESULTS: Letrozole (0.2, 0.4, and 0.8 µg) significantly decreased the hippocampal E2 levels compared to the vehicle group. Letrozole caused cognitive impairments in a dose-dependent manner in male and female rats in the presence or absence of gonads. Dose-response analysis revealed that the minimum effective dose of letrozole on the behavioral measures was 0.4 µg. Letrozole also caused an up-regulation of ERα and ERß and a down-regulation of GPR30 gene expression. The firing rate of pyramidal neurons was reduced by letrozole in gonadal-intact animals. CONCLUSION: The detrimental effects of letrozole treatment on cognitive abilities in the presence and absence of gonads indicate that local E2 synthesis in the hippocampus is a crucial factor in normal cognitive performance. The suppressive effect of letrozole on hippocampal neuronal firing might alter synaptic plasticity that is critical for memory formation. These data potentially suggest that memory deficits following letrozole administration should be monitored.

Distinct Function of Estrogen Receptors in the Rodent Anterior Cingulate Cortex in Pain-related Aversion.

BACKGROUND: Brain-derived estrogen is implicated in pain-related aversion; however, which estrogen receptors mediate this effect remains unclear. This study hypothesized that the different estrogen receptors in the rostral anterior cingulate cortex play distinct roles in pain-related aversion. METHODS: Formalin-induced conditioned place avoidance and place escape/avoidance paradigms were used to evaluate pain-related aversion in rodents. Immunohistochemistry and Western blotting were used to detect estrogen receptor expression. Patch-clamp recordings were used to examine N-methyl-D-aspartate-mediated excitatory postsynaptic currents in rostral anterior cingulate cortex slices. RESULTS: The administration of the estrogen receptor-ß antagonist 4-(2-phenyl-5,7-bis [trifluoromethyl] pyrazolo [1,5-a] pyrimidin-3-yl) phenol (PHTPP) or the G protein-coupled estrogen receptor-1 antagonist (3aS*,4R*,9bR*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta [c] quinolone (G15) but not the estrogen receptor-α antagonist 1,3-bis (4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy) phenol]-1H-pyrazole dihydrochloride (MPP) into the rostral anterior cingulate cortex blocked pain-related aversion in rats (avoidance score, mean ± SD: 1,3-bis [4-hydroxyphenyl]-4-methyl-5-(4-[2-piperidinylethoxy] phenol)-1H-pyrazole dihydrochloride (MPP): 47.0 ± 18.9%, 4-(2-phenyl-5,7-bis [trifluoromethyl] pyrazolo [1,5-a] pyrimidin-3-yl) phenol (PHTPP): -7.4 ± 20.6%, and [3aS*,4R*,9bR*]-4-[6-bromo-1,3-benzodioxol-5-yl]-3a,4,5,9b-3H-cyclopenta [c] quinolone (G15): -4.6 ± 17.0% vs. vehicle: 46.5 ± 12.2%; n = 7 to 9; P < 0.0001). Consistently, estrogen receptor-ß knockdown but not estrogen receptor-α knockdown by short-hairpin RNA also inhibited pain-related aversion in mice (avoidance score, mean ± SD: estrogen receptor-α-short-hairpin RNA: 26.0 ± 7.1% and estrogen receptor-ß-short-hairpin RNA: 6.3 ± 13.4% vs. control short-hairpin RNA: 29.1 ± 9.1%; n = 7 to 10; P < 0.0001). Furthermore, the direct administration of the estrogen receptor-ß agonist 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN) or the G protein-coupled estrogen receptor-1 agonist (±)-1-([3aR*,4S*,9bS*]-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta [c]quinolin-8-yl)-ethanone (G1) into the rostral anterior cingulate cortex resulted in conditioned place avoidance (avoidance score, mean ± SD: 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN): 35.3 ± 9.5% and (±)-1-([3aR*,4S*,9bS*]-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta [c]quinolin-8-yl)-ethanone (G1): 43.5 ± 22.8% vs. vehicle: 0.3 ± 14.9%; n = 8; P < 0.0001) but did not affect mechanical or thermal sensitivity. The activation of the estrogen receptor-ß/protein kinase A or G protein-coupled estrogen receptor-1/protein kinase B pathway elicited the long-term potentiation of N-methyl-D-aspartate-mediated excitatory postsynaptic currents. CONCLUSIONS: These findings indicate that estrogen receptor-ß and G protein-coupled estrogen receptor-1 but not estrogen receptor-α in the rostral anterior cingulate cortex contribute to pain-related aversion by modulating N-methyl-D-aspartate receptor-mediated excitatory synaptic transmission.

BRCA1-associated mammary tumorigenesis is dependent on estrogen rather than progesterone signaling.

Hereditary breast cancers in BRCA1 mutation carriers are mostly estrogen receptor α (ERα)-negative and progesterone receptor (PR)-negative; however, hormone depletion via bilateral oophorectomy does result in a marked reduction in breast cancer risk, suggesting that BRCA1-associated breast tumorigenesis is dependent on hormone signaling. We used geneticaly engineered mouse models to determine the individual influences of ERα and PR signaling on the development of BRCA1-deficient breast cancer. In line with the human data, BRCA1-deficient mouse mammary tumors are ERα-negative, and bilateral ovariectomy leads to abrogation of mammary tumor development. Hormonal replacement experiments in ovariectomized mice showed that BRCA1-deficient mammary tumor formation is promoted by estrogen but not by progesterone. In line with these data, mammary tumorigenesis was significantly delayed by the selective ERα downregulator fulvestrant, but not by the selective PR antagonist Org33628. Together, our results illustrate that BRCA1-associated tumorigenesis is dependent on estrogen signaling rather than on progesterone signaling, and call into question the utility of PR antagonists as a tumor prevention strategy for BRCA1 mutation carriers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tamoxifen attenuates development of lithium-induced nephrogenic diabetes insipidus in rats.

Lithium is widely used in treatment of bipolar affective disorders but often causes nephrogenic diabetes insipidus (NDI), a disorder characterized by severe urinary-concentrating defects. Lithium-induced NDI is caused by lithium uptake by collecting duct principal cells and altered expression of aquaporin-2 (AQP2), which are essential for water reabsorption of tubular fluid in the collecting duct. Sex hormones have previously been shown to affect the regulation of AQP2, so we tested whether tamoxifen (TAM), a selective estrogen receptor modulator, would attenuate lithium-induced alterations on renal water homeostasis. Rats were treated for 14 days with lithium, and TAM treatment was initiated 1 wk after onset of lithium administration. Lithium treatment resulted in severe polyuria and reduced AQP2 expression, which were ameliorated by TAM. Consistent with this, TAM attenuated downregulation of AQP2 and increased phosphorylation of the cAMP-responsive element-binding protein, which induced AQP2 expression in freshly isolated inner-medullary collecting duct suspension prepared from lithium-treated rats. In conclusion, TAM attenuated polyuria dose dependently and impaired urine concentration and downregulation of AQP2 protein expression in rats with lithium-induced NDI. These findings suggest that TAM is likely to be a novel therapeutic option for lithium-induced NDI.

Divergent responsiveness of two isoforms of the estrogen receptor to mixtures of contaminants of emerging concern in four vertebrates.

Contaminants of emerging concern (CECs) are ubiquitous in aquatic environments with well-established endocrine-disrupting effects. A data matrix of 559 water samples was queried to identify two commonly occurring CECs mixtures in Great Lakes tributaries. One mixture consisted of eight agricultural CECs (AG), while another contained 11 urban CECs (UB). The known estrogenic compounds bisphenol A, estrone and nonylphenol were present in both mixtures. According to the EPA Tox21 in ToxCast database, AG and UB mixture at an environmentally relevant concentration were estimated to account for 6.5% and 3.4% estrogenicity of the model endocrine disruptor estradiol-17ß, respectively. Two isoforms of the estrogen receptor (Esr1 and -2, former Erα and Erß) cloned from fathead minnow, bluegill sunfish, American alligator and human, responded differently to AG and UB mixtures. Human and bluegill Esr1 were the most sensitive to AG and UB mixtures, respectively. Fathead minnow Esr1 and Esr2b were the least sensitive to 10× AG and UB in estrogen dose equivalents, respectively. Even at environmentally documented concentrations, UB significantly activated bluegill Esr1. Moreover, 100× concentrated UB hyperstimulated fathead minnow Esr1 beyond the maximum induction of estradiol-17ß. These results indicate that efficacious receptors and species differ in their response to CEC mixtures. Furthermore, estrogenicity may be present in some CECs not previously considered estrogenic, or, alternatively, estrogenicity of a mixture may be enhanced through chemical interactions. Our study highlights the need for further studies of CECs utilizing a variety of receptors cloned from diverse species.

Estrogen Receptors α and ß Play Major Roles in Ethanol-Evoked Myocardial Oxidative Stress and Dysfunction in Conscious Ovariectomized Rats.

BACKGROUND: We documented the dependence of ethanol (EtOH)-evoked myocardial dysfunction on estrogen (E2 ), and our recent estrogen receptor (ER) blockade study, in proestrus rats, implicated ERα signaling in this phenomenon. However, a limitation of selective pharmacological loss-of-function approach is the potential contribution of the other 2 ERs to the observed effects because crosstalk exists between the 3 ERs. Here, we adopted a "regain"-of-function approach (using selective ER subtype agonists) to identify the ER subtype(s) required for unraveling the E2 -dependent myocardial oxidative stress/dysfunction caused by EtOH in conscious ovariectomized (OVX) rats. METHODS: OVX rats received a selective ERα (PPT), ERß (DPN), or GPER (G1) agonist (10 µg/kg; i.v.) or vehicle 30 minutes before EtOH (1.0 g/kg; infused i.v. over 30 minutes) or saline, and the hemodynamic recording continued for additional 60 minutes. Thereafter, left ventricular tissue was collected for conducting ex vivo molecular/biochemical studies. RESULTS: EtOH had no hemodynamic effects in OVX rats, but reduced the left ventricular contractility index, dP/dtmax , and MAP after acute ERα (PPT) or ERß (DPN) activation. These responses were associated with increases in the phosphorylation of ERK1/2 and eNOS, and in reactive oxygen species (ROS) and malondialdehyde (MDA) levels in the myocardium. GPER activation (G1) only unraveled a modest EtOH-evoked hypotension and elevation in myocardial ROS. PPT enhanced catalase, DPN reduced ALDH2, while G1 had no effect on the activity of either enzyme, and none of the agonists influenced alcohol dehydrogenase or CYP2E1 activities in the myocardium. Blood EtOH concentration (96.0 mg/dl) was significantly reduced following ERα (59.8 mg/dl) or ERß (62.9 mg/dl), but not GPER (100.3 mg/dl), activation in EtOH-treated OVX rats. CONCLUSIONS: ERα and ERß play major roles in the E2 -dependent myocardial dysfunction caused by EtOH by promoting combined accumulation of cardiotoxic (ROS and MDA) and cardiodepressant (NOS-derived NO) molecules in female myocardium.

Information-dependent enrichment analysis reveals time-dependent transcriptional regulation of the estrogen pathway of toxicity.

The twenty-first century vision for toxicology involves a transition away from high-dose animal studies to in vitro and computational models (NRC in Toxicity testing in the 21st century: a vision and a strategy, The National Academies Press, Washington, DC, 2007). This transition requires mapping pathways of toxicity by understanding how in vitro systems respond to chemical perturbation. Uncovering transcription factors/signaling networks responsible for gene expression patterns is essential for defining pathways of toxicity, and ultimately, for determining the chemical modes of action through which a toxicant acts. Traditionally, transcription factor identification is achieved via chromatin immunoprecipitation studies and summarized by calculating which transcription factors are statistically associated with up- and downregulated genes. These lists are commonly determined via statistical or fold-change cutoffs, a procedure that is sensitive to statistical power and may not be as useful for determining transcription factor associations. To move away from an arbitrary statistical or fold-change-based cutoff, we developed, in the context of the Mapping the Human Toxome project, an enrichment paradigm called information-dependent enrichment analysis (IDEA) to guide identification of the transcription factor network. We used a test case of activation in MCF-7 cells by 17ß estradiol (E2). Using this new approach, we established a time course for transcriptional and functional responses to E2. ERα and ERß were associated with short-term transcriptional changes in response to E2. Sustained exposure led to recruitment of additional transcription factors and alteration of cell cycle machinery. TFAP2C and SOX2 were the transcription factors most highly correlated with dose. E2F7, E2F1, and Foxm1, which are involved in cell proliferation, were enriched only at 24 h. IDEA should be useful for identifying candidate pathways of toxicity. IDEA outperforms gene set enrichment analysis (GSEA) and provides similar results to weighted gene correlation network analysis, a platform that helps to identify genes not annotated to pathways.

Bisphenol A suppresses glucocorticoid target gene (ENaCγ) expression via a novel ERß/NF-κB/GR signalling pathway in lung epithelial cells.

We previously demonstrated that prenatal exposure to Bisphenol A (BPA) disrupts fetal lung maturation likely through the glucocorticoid signalling pathway, but the precise molecular mechanisms remain obscure. Given that BPA diminished the expression of epithelial sodium channel-γ (ENaCγ), a well-known glucocorticoid receptor (GR) target gene, in fetal lungs, we used this GR target gene to delineate the molecular pathway through which BPA exerts its effects on lung cells. The A549 lung epithelial cell line was used as an in vitro model system. As a first step, we validated our in vitro cell model by demonstrating a robust concentration-dependent suppression of ENaCγ expression following BPA exposure. We also showed that both dexamethasone and siRNA-mediated knockdown of GR expression blocked/abrogated the inhibitory effects of BPA on ENaCγ expression, suggesting that BPA repressed ENaCγ expression via inhibition of GR activity. Given the well-known antagonistic interactions between the pro-inflammatory transcriptional factor NF-κB and GR, we then showed that BPA inhibited GR activity through the activation of NF-κB. Lastly, since BPA is known to function as a pro-inflammatory factor via the estrogen receptor ß (ERß), we provided evidence that BPA signals through ERß to activate the NF-κB signalling pathway. Taken together, these findings demonstrate that BPA acts on ERß to activate the NF-κB signalling pathway, which in turn leads to diminished GR activity and consequent repression of ENaCγ expression in lung epithelial cells. Thus, our present study reveals a novel BPA signalling pathway that involves ERß, NF-κB and GR.

2-Methoxyestradiol, an endogenous 17ß-estradiol metabolite, inhibits microglial proliferation and activation via an estrogen receptor-independent mechanism.

17ß-Estradiol (estradiol) inhibits microglia proliferation. 2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol with little affinity for estrogen receptors (ERs). We hypothesize that 2-ME inhibits microglial proliferation and activation and contributes to estradiol's inhibitory effects on microglia. We compared the effects of estradiol, 2-hydroxyestradiol [2-OE; estradiol metabolite produced by cytochrome P450 (CYP450)], and 2-ME [formed by catechol-O-methyltransferase (COMT) acting upon 2-OE] on microglial (BV2 cells) DNA synthesis, cell proliferation, activation, and phagocytosis. 2-ME and 2-OE were approximately three- and 10-fold, respectively, more potent than estradiol in inhibiting microglia DNA synthesis. The antimitogenic effects of estradiol were reduced by pharmacological inhibitors of CYP450 and COMT. Inhibition of COMT blocked the conversion of 2-OE to 2-ME and the antimitogenic effects of 2-OE but not 2-ME. Microglia expressed ERß and GPR30 but not ERα. 2,3-Bis(4-hydroxyphenyl)-propionitrile (ERß agonist), but not 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (ERα agonist) or G1 (GPR30 agonist), inhibited microglial proliferation. The antiproliferative effects of estradiol, but not 2-OE or 2-ME, were partially reversed by ICI-182,780 (ERα/ß antagonist) but not by 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole (ERα antagonist) or G15 (GPR30 antagonist). Lipopolysaccharide increased microglia iNOS and COX-2 expression and phagocytosing activity of microglia; these effects were inhibited by 2-ME. We conclude that in microglia, 2-ME inhibits proliferation, proinflammatory responses, and phagocytosis. 2-ME partially mediates the effects of estradiol via ER-independent mechanisms involving sequential metabolism of estradiol to 2-OE and 2-ME. 2-ME could be of potential therapeutic use in postischemic stroke injuries. Interindividual differences in estradiol metabolism might affect the individual's ability to recover from stroke.

Divergent effects of ERα and ERß on fluid intake by female rats are not dependent on concomitant changes in AT1R expression or body weight.

Estradiol (E2) decreases both water and saline intakes by female rats. The ERα and ERß subtypes are expressed in areas of the brain that control fluid intake; however, the role that these receptors play in E2's antidipsogenic and antinatriorexigenic effects have not been examined. Accordingly, we tested the hypothesis that activation of ERα and ERß decreases water and saline intakes by female rats. We found a divergence in E2's inhibitory effect on intake: activation of ERα decreased water intake, whereas activation of ERß decreased saline intake. E2 decreases expression of the angiotensin II type 1 receptor (AT1R), a receptor with known relevance to water and salt intakes, in multiple areas of the brain where ERα and ERß are differentially expressed. Therefore, we tested for agonist-induced changes in AT1R mRNA expression by RT-PCR and protein expression by analyzing receptor binding to test the hypothesis that the divergent effects of these ER subtypes are mediated by region-specific changes in AT1R expression. Although we found no changes in AT1R mRNA or binding in areas of the brain known to control fluid intake associated with agonist treatment, the experimental results replicate and extend previous findings that body weight changes mediate alterations in AT1R expression in distinct brain regions. Together, the results reveal selective effects of ER subtypes on ingestive behaviors, advancing our understanding of E2's inhibitory role in the controls of fluid intake by female rats.

Hormone therapy and large bowel cancer revisited.

Many studies have investigated potential links between postmenopausal hormone therapy (HT) and the risk for colorectal cancer. Most of these studies showed some reduction in risk, but association with the exact grade of the tumor or with cancer mortality is less consistent. Activation of estrogen receptor beta (ERß) related cellular pathways is probably the pathophysiological basis for this HT effect, since ERßs exert antiproliferative and pro-apoptotic signaling, inhibit inflammatory signaling and modulate the tumor microenvironment. To note, the impact on health may not be substantial as the absolute numbers point at the order of only a few saved cases per 10 000 women per year of hormone use.

Different mechanisms of action of 2, 2', 4, 4'-tetrabromodiphenyl ether (BDE-47) and its metabolites (5-OH-BDE-47 and 6-OH-BDE-47) on cell proliferation in OVCAR-3 ovarian cancer cells and MCF-7 breast cancer cells.

Data concerning the possible action of polybrominated diphenyl ethers (PBDEs) in hormone-dependent cancer are scarce. Some data showed that PBDEs may directly affect breast cancer cells formation and only one research showed increased proliferation of the OVCAR-3 cells, but the results are ambiguous and the mechanisms are not clear. There is growing evidence that not only parent compounds but also its metabolites may be involved in cancer development. The present study was, therefore, designed to determine the effect of BDE-47 and its metabolites (2.5 to 50 ng ml-1 ) on proliferation (BrdU), cell-cycle genes (real-time PCR) and protein expression (Western blot), protein expression of oestrogen receptors (α ß), extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase Cα (PKCα) in OVCAR-3 ovarian and MCF-7 breast cancer cells. In OVCAR-3 cells, the parent compound stimulated cell proliferation by activating CDK1, CDK7, E2F1 and E2F2. Independent of time of exposure, BDE-47 had no effect on ERα and ERß protein expression and ERK1/2 and PKCα phosphorylation. Metabolites had no effect on cell proliferation but increased both ERs protein expression and ERK1/2 and PKCα phosphorylation. In MCF-7 cells, the parent compound displayed no effect on cell proliferation but decreased ERα and increased ERß protein expression with concomitant induction of PKCα phosphorylation. Both metabolites increased MCF-7 cell proliferation, ERK1/2 and PKCα phosphorylation and decreased ERα and ERß protein expression.We suggest that studies concerning PBDEs with fewer bromine atoms should be continued to understand environmental links to different hormone-dependent cancers. Copyright © 2016 John Wiley & Sons, Ltd.

Bisphenol a exposure promotes the migration of NCM460 cells via estrogen receptor-mediated integrin ß1/MMP-9 pathway.

Bisphenol A (BPA) is a widely used industrial chemical and also an environmental endocrine disruptor (EED), which serves as a monomer in the manufacture of polycarbonate plastics. BPA enters human body mainly through oral intake, and has been reported as being linked to oncogenesis in many tissues. However, the association of BPA intake with gastrointestinal cancer, such as colon cancer, has received less attention. The present study was conducted to investigate the effects of BPA on the migration of normal colon epithelial cells (NCM460 cells) and further elucidate the underlying mechanisms. Our data showed that 1 × 10(-8) M (equivalent to environmental concentration) of BPA potently promoted the migration of NCM460 cells. Interestingly, BPA treatment induced an increase of integrin ß1 expression, and the functional blocking of integrin ß1 abolished the migration-promoting effects of BPA. Moreover, the results showed that it was estrogen receptor ß but not estrogen receptor α that was involved in this migration promotion. In addition, cellular exposure of BPA stimulated the expression and activity of MMP-9, a well-known factor of cell migration. Taken together, these results indicate that environmental concentration of BPA exposure promotes cell migration through activating ERß-mediated integrin ß1/MMP-9 pathway, suggesting exposure to BPA in the colon may present a potential cancer risk. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 799-807, 2016.

Estradiol Elicits Proapoptotic and Antiproliferative Effects in Human Trophoblast Cells.

During the first trimester of pregnancy, appropriate regulation of estradiol (E2) is essential for normal placental development. Previous studies demonstrate that premature elevation in E2 concentrations can lead to abnormal placentation, but have not fully elaborated the mechanism of this effect in the first-trimester trophoblast. Our aim was to determine whether E2 elicits trophoblast cell death or inhibits proliferation. The first-trimester human cytotrophoblast cell line HTR-8/SVneo was cultured in phenol red-free medium containing charcoal-stripped serum and treated with 17beta-E2 at concentrations between 0 and 100 nM. TUNEL and invasion assays indicated that E2 significantly increased cell death and reduced cell invasion at 10 nM, and nuclear Ki67 expression revealed that it decreased cell proliferation at 1 nM. A similar effect on cell death was observed in first-trimester placental explants. The E2 antagonist fulvestrant blocked all effects of E2. Immunohistochemistry showed that protein expression of proapoptotic caspases 3, 8, and 9 increased at E2 concentrations of 25 nM and greater, whereas expression of antiapoptotic BCL2-alpha decreased at E2 concentrations of 10 nM and greater. Additionally, treatments with estrogen receptor (ER) alpha-specific and ERbeta-specific agonists at concentrations between 0 and 1000 nM indicated that only ERalpha mediates E2's effects, although immunohistochemistry and Western immunoblotting showed that HTR-8/SVneo cells and placental explants express both ERalpha and ERbeta. Taken together, these findings reveal the interplay between elevated serum E2 and apoptosis in the first trimester of pregnancy. These factors could be associated with pregnancy complications including infertility and uteroplacental insufficiency.

Estrogen receptor-α is a key mediator and therapeutic target for bladder complications of benign prostatic hyperplasia.

PURPOSE: Estrogens are important in prostate growth and have a role in benign prostatic hyperplasia. However, to our knowledge no current therapy directly targets estrogen action. Estrogens act primarily via estrogen receptors α and ß. In a mouse model we evaluated the relative contribution of these receptors to bladder complications of benign prostatic hyperplasia. We also evaluated the prevention of these bladder complications using the selective estrogen receptor modulators raloxifene and tamoxifen (estrogen receptor-α selective antagonists), and R,R-THC (estrogen receptor-ß selective antagonist). MATERIALS AND METHODS: Adult male C57bl/6 mice received implants of 25 mg testosterone and 2.5 mg 17ß-estradiol slow release pellets. Untreated controls underwent sham surgery. We evaluated the contributions of the estrogen receptor subtypes in ERαKO and ERßKO mice compared to their respective wild-type litter mates. Wild-type mice treated with testosterone plus 17ß-estradiol were compared to mice treated with testosterone plus 17ß-estradiol and 25 mg selective estrogen receptor modulators to evaluate the prevention of benign prostatic hyperplasia complications by selective estrogen receptor modulators. RESULTS: Large bladders with urinary retention developed in ERαWT and ERßWT litter mates treated with testosterone plus 17ß-estradiol but such bladders did not develop in ERαKO mice treated with testosterone plus 17ß-estradiol. ERßKO mice treated with testosterone plus 17ß-estradiol had large bladders with urinary retention and increased bladder mass. Cotreatment with the estrogen receptor-α antagonist raloxifene resulted in decreased bladder mass compared to that in wild-type mice treated with testosterone plus 17ß-estradiol. Bladders in mice treated with the estrogen receptor-ß antagonist R,R-THC were similar to those in testosterone plus 17ß-estradiol treated mice. CONCLUSIONS: Estrogen receptor-α but not ß is a key mediator of bladder complications of benign prostatic hyperplasia and a potential target for future therapies.

Effect of diosgenin on metabolic dysfunction: Role of ERß in the regulation of PPARγ.

The present study was designed to investigate the effect of diosgenin (DSG) on metabolic dysfunction and to elucidate the possible molecular mechanisms. High fat (HF) diet-fed mice and 3T3-L1 preadipocytes was used to evaluate the effect of DSG. We showed that DSG attenuated metabolic dysfunction in HF diet-fed mice, as evidenced by reduction of blood glucose level and improvement of glucose and insulin intolerance. DSG ameliorated oxidative stress, reduced body weight, fat pads, and systematic lipid profiles and attenuated lipid accumulation. DSG inhibited 3T3-L1 adipocyte differentiation and reduced adipocyte size through regulating key factors. DSG inhibited PPARγ and its target gene expression both in differentiated 3T3-L1 adipocytes and fat tissues in HF diet-fed mice. Overexpression of PPARγ suppressed DSG-inhibited adipocyte differentiation. DSG significantly increased nuclear expression of ERß. Inhibition of ERß significantly suppressed DSG-exerted suppression of adipocyte differentiation and PPARγ expression. In response to DSG stimulation, ERß bound with RXRα and dissociated RXRα from PPARγ, leading to the reduction of transcriptional activity of PPARγ. These data provide new insight into the mechanisms underlying the inhibitory effect of DSG on adipocyte differentiation and demonstrate that ERß-exerted regulation of PPARγ expression and activity is critical for DSG-inhibited adipocyte differentiation.

Protective effect of porcine placenta in a menopausal ovariectomized mouse.

Menopause is a significant physiological phase that occurs as women's ovaries stop producing ovum and the production of estrogen declines. Human placenta and some amino acids are known to improve menopausal symptoms. In this study, we investigated that porcine placenta extract (PPE) and arginine (Arg), a main amino acid of PPE, would have estrogenic activities in ovariectomized (OVX) mice as a menopause mouse model, human breast cancer cell line (MCF-7) cells, and human osteoblast cell line (MG-63) cells. PPE or Arg significantly inhibited the body weight and increased the vagina weight compared to the OVX mice. PPE or Arg ameliorated the vaginal atrophy in the OVX mice. The levels of 17ß-estradiol and the activities of alkaline phosphatase (ALP) were significantly increased by PPE or Arg in the serum of OVX mice. Trabecular bone parameters such as bone mineral density and porosity were also improved by PPE or Arg in the OVX mice. In the MCF-7 and MG-63 cells, PPE or Arg significantly increased the cell proliferation, estrogen receptor ß mRNA expression, and estrogen-response elements luciferase activity. Finally, PPE or Arg increased the activations of ALP and extracellular signal-regulated kinase 1/2 in the MG-63 cells. These results indicate that PPE or Arg would have estrogenic and osteoblastic activity. Therefore, PPE or Arg may be useful as new pharmacological tools for treating menopausal symptoms including osteoporosis. Free Korean abstract: A Korean translation of this abstract is freely available at http://www.reproduction-online.org/content/150/3/173/suppl/DC1.