Estrogen signaling through the G protein-coupled estrogen receptor regulates granulocyte activation in fish.

Neutrophils are major participants in innate host responses. It is well known that estrogens have an immune-modulatory role, and some evidence exists that neutrophil physiology can be altered by these molecules. Traditionally, estrogens act via classical nuclear estrogen receptors, but the identification of a G protein-coupled estrogen receptor (GPER), a membrane estrogen receptor that binds estradiol and other estrogens, has opened up the possibility of exploring additional estrogen-mediated effects. However, information on the importance of GPER for immunity, especially, in neutrophils is scant. In this study, we report that gilthead seabream (Sparus aurata L.) acidophilic granulocytes, which are the functional equivalent of mammalian neutrophils, express GPER at both mRNA and protein levels. By using a GPER selective agonist, G1, it was found that GPER activation in vitro slightly reduced the respiratory burst of acidophilic granulocytes and drastically altered the expression profile of several genes encoding major pro- and anti-inflammatory mediators. In addition, GPER signaling in vivo modulated adaptive immunity. Finally, a cAMP analog mimicked the effects of G1 in the induction of the gene coding for PG-endoperoxide synthase 2 and in the induction of CREB phosphorylation, whereas pharmacological inhibition of protein kinase A superinduced PG-endoperoxide synthase 2. Taken together, our results demonstrate for the first time, to our knowledge, that estrogens are able to modulate vertebrate granulocyte functions through a GPER/cAMP/protein kinase A/CREB signaling pathway and could establish therapeutic targets for several immune disorders in which estrogens play a prominent role.

Gal4 turnover and transcription activation.

Growing evidence supports the notion that proteasome-mediated destruction of transcriptional activators can be intimately coupled to their function. Recently, Nalley et al. challenged this view by reporting that the prototypical yeast activator Gal4 does not dynamically associate with chromatin, but rather 'locks in' to stable promoter complexes that are resistant to competition. Here we present evidence that the assay used to reach this conclusion is unsuitable, and that promoter-bound, active Gal4 is indeed susceptible to competition in vivo. Our data challenge the key evidence that Nalley et al. used to reach their conclusion, and indicate that Gal4 functions in vivo within the context of dynamic promoter complexes.

Resolvin D2 supports MCF-7 cell proliferation via activation of estrogen receptor.

Inflammation has been implicated in tumor initiation, angiogenesis, and metastasis, and linked to the development of more aggressive, therapy-resistant estrogen receptor (ER)-positive breast cancer. Resolvin D2 (RvD2) is a potent anti-inflammatory lipid mediator. As RvD2 may be synthesized within breast tumors by both tumor cells and the surrounding stroma cells and is present in plasma at bioactive concentrations, we sought to characterize the impact of RvD2 on cell processes underlying breast tumor growth and spread. Trypan-blue exclusion, transfection with estrogen response element (ERE) reporter, real-time quantitative polymerase chain reaction, competitive radioligand binding assays, Western blotting, and immunofluorescence were the techniques used. Unexpectedly, whereas RvD2 (10-1000 nM) supported the proliferation of the ER-positive breast tumor (MCF-7) cells, it did not affect the ER-negative MDA-MB-231 cell number. The proliferative effect of RvD2 in MCF-7 cells was attenuated by the ER antagonist ICI 182,780 (7α-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17ß-diol). Furthermore, RvD2 increased ERE transcriptional activity in a number of ER-positive breast and ovarian tumor cell lines. This activation was also inhibited by ICI 182,780. RvD2 altered the expression of a subset of estrogen-responsive genes. Although binding experiments showed that RvD2 did not directly compete with [(3)H]17ß-estradiol for ER binding, prior exposure of MCF-7 cells to RvD2 resulted in a significant reduction in the apparent cytosolic ER density. Confocal immunocytochemistry and Western blotting studies showed that RvD2 promoted nuclear localization of ERα. These observations indicate that RvD2 displays significant but indirect estrogenic properties and has the potential to play a role in estrogen-dependent breast cancer progression.

Psoralidin, a coumestan analogue, as a novel potent estrogen receptor signaling molecule isolated from Psoralea corylifolia.

A novel biological activity of psoralidin as an agonist for both estrogen receptor (ER)α and ERß agonist has been demonstrated in our study. Psoralidin has been characterized as a full ER agonist, which activates the classical ER-signaling pathway in both ER-positive human breast and endometrial cell lines as well as non-human cultured cells transiently expressing either ERα or ERß. The estrogenic activity was determined using the relative expression levels of either reporter or the endogenous genes dependent on the agonist-bound ER to the estrogen response element (ERE). Psoralidin at 10 µM was able to induce the maximum reporter gene expression corresponding to that of E2-treated cells and such activation of the ERE-reporter gene by psoralidin was completely abolished by the cotreatment of a pure ER antagonist, implying that the biological activities of psoralidin are mediated by ER. Psoralidin was also able to induce the endogenous estrogen-responsive gene, pS2, in human breast cancer cells MCF-7. It was observed that activation of the classical ER-signaling pathway by psoralidin is mediated via induction of ER conformation by psoralidin and direct binding of the psoralidin-ER complex to the EREs present in the promoter region of estrogen-responsive genes, as shown by chromatin immunoprecipitation assay results. Finally, molecular docking of psoralidin to the ligand binding pocket of the ERα showed that psoralidin is able to mimic the binding interactions of E2, and thus, it could act as an ER agonist in the cellular environment.

Estrogenic chemicals often leach from BPA-free plastic products that are replacements for BPA-containing polycarbonate products.

BACKGROUND: Xenobiotic chemicals with estrogenic activity (EA), such as bisphenol A (BPA), have been reported to have potential adverse health effects in mammals, including humans, especially in fetal and infant stages. Concerns about safety have caused many manufacturers to use alternatives to polycarbonate (PC) resins to make hard and clear, reusable, plastic products that do not leach BPA. However, no study has focused on whether such BPA-free PC-replacement products, chosen for their perceived higher safety, especially for babies, also release other chemicals that have EA. METHODS: We used two, well-established, mammalian cell-based, assays (MCF-7 and BG1Luc) to assess the EA of chemicals that leached into over 1000 saline or ethanol extracts of 50 unstressed or stressed (autoclaving, microwaving, and UV radiation) BPA-free PC-replacement products. An EA antagonist, ICI 182,780, was used to confirm that agonist activity in leachates was due to chemicals that activated the mammalian estrogen receptor. RESULTS: Many unstressed and stressed, PC-replacement-products made from acrylic, polystyrene, polyethersulfone, and Tritan™ resins leached chemicals with EA, including products made for use by babies. Exposure to various forms of UV radiation often increased the leaching of chemicals with EA. In contrast, some BPA-free PC-replacement products made from glycol-modified polyethylene terephthalate or cyclic olefin polymer or co-polymer resins did not release chemicals with detectable EA under any conditions tested. CONCLUSIONS: This hazard assessment survey showed that many BPA-free PC- replacement products still leached chemicals having significant levels of EA, as did BPA-containing PC counterparts they were meant to replace. That is, BPA-free did not mean EA-free. However, this study also showed that some PC-replacement products did not leach chemicals having significant levels of EA. That is, EA-free PC-replacement products could be made in commercial quantities at prices that compete with PC-replacement products that were not BPA-free. Since plastic products often have advantages (price, weight, shatter-resistance, etc.) compared to other materials such as steel or glass, it is not necessary to forgo those advantages to avoid release into foodstuffs or the environment of chemicals having EA that may have potential adverse effects on our health or the health of future generations.

Cooperative activation of gene expression by agonists and antagonists mediated by estrogen receptor heteroligand dimer complexes.

Estrogen receptor (ER) antagonists are generally thought to inhibit estrogen action through competitive inhibition, resulting in receptor binding to antagonist rather than agonist. However, microarray analyses reveal a group of genes for which ER agonist and antagonist cooperatively regulate expression, suggesting additional models of combined agonist/antagonist action must exist. In conjunction with a chimeric reporter gene and two modified ERs, one [ERα(GSCKV)] with a mutation in the DNA-binding domain and the other (ERα-G521R) with a ligand-binding specificity mutation, we herein demonstrate that ER agonist and antagonist cooperatively activate gene expression through an ER heteroligand dimer complex (ER-HLD) consisting of one subunit of the receptor dimer bound to agonist and another occupied by antagonist. Coimmunoprecipitation experiments confirmed interaction between the agonist-bound and antagonist-bound receptors. This cooperative activation of gene expression was enhanced by steroid receptor coactivator 3 coactivator, and required each ligand-bound subunit of the dimer to bind to DNA, as well as both activation function 1 domains for maximal transcriptional activity. Ligand combinations able to induce ER-HLD transcriptional activity include the agonists 17ß-estradiol or conjugated estrogens with the antagonists tamoxifen, raloxifene, bazedoxifene, or fulvestrant. Moreover, ER-HLD can activate transcription in the context of a natural promoter. Taken together, these findings broaden our understanding of the complex relationship between ER agonist and antagonist, and suggest a novel model by which cell and tissue selective effects of antiestrogens may be achieved.

Molecular mechanism(s) of endocrine-disrupting chemicals and their potent oestrogenicity in diverse cells and tissues that express oestrogen receptors.

Endocrine-disrupting chemicals (EDCs) are natural or synthetic compounds present in the environment which can interfere with hormone synthesis and normal physiological functions of male and female reproductive organs. Most EDCs tend to bind to steroid hormone receptors including the oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR). As EDCs disrupt the actions of endogenous hormones, they may induce abnormal reproduction, stimulation of cancer growth, dysfunction of neuronal and immune system. Although EDCs represent a significant public health concern, there are no standard methods to determine effect of EDCs on human beings. The mechanisms underlying adverse actions of EDC exposure are not clearly understood. In this review, we highlighted the toxicology of EDCs and its effect on human health, including reproductive development in males and females as shown in in vitro and in vivo models. In addition, this review brings attention to the toxicity of EDCs via interaction of genomic and non-genomic signalling pathways through hormone receptors.

Evolution of the tissue selective estrogen complex (TSEC).

Estrogens mediate gene transcription and signaling of numerous cellular processes in a variety of tissues, including the bone, breast, and endometrium, through binding and activation of estrogen receptors (ERs). Estrogen-mediated ER agonist activity has shown benefit in conditions related to estrogen deficiency in women; however, these effects have been associated with stimulation of breast and uterine tissues. Due to the complexity of ER signaling, selective estrogen receptor modulators (SERMs) can exhibit ER agonist or antagonist activity depending on the target tissue. A newer approach to menopausal therapy, the tissue selective estrogen complex (TSEC), pairs a SERM with one or more estrogens with the goal of maintaining the benefits of estrogens without the stimulatory effects on the breast and uterus. Preclinically, different TSECs have been associated with distinct gene expression profiles compared with each other and with their individual SERM/estrogen components. Studies in cultured breast cancer cells and animal models have demonstrated a lack of estrogen-induced stimulation with TSECs in the mammary gland and endometrium. In the breast, biochemical analyses indicate that degradation of the ER is an important mechanism by which TSECs exert their antagonistic effects. TSECs have also shown positive effects similar to estrogens in other tissue types, including bone and the central nervous system, although mechanisms underlying these activities are less clear. Overall, preclinical studies have shown that estrogens, SERMs, and TSECs each exert distinct and tissue specific molecular and pharmacologic effects.

Antiestrogens and their therapeutic applications in breast cancer and other diseases.

The identification of the link between breast cancer and estrogens has led to the development of antiestrogens, in particular tamoxifen, to inhibit the activities of estrogen receptors (ERs) in breast cancer cells. The clinical use of tamoxifen has played a major part in decreasing breast cancer mortality over the past 30 years. Though antiestrogenic in the breast, some antiestrogens have estrogen-like actions in other tissues, acting to promote bone density and protect against cardiovascular disease, thus raising the possibility of their use in counteracting the effects of estrogen loss following menopause. Moreover, antiestrogens show efficacy as chemopreventive agents in women at high risk of developing breast cancer. Thus, antiestrogens define an important and well-understood class of cancer drug, which continue to be a mainstay in breast cancer treatment.

Estrogen receptor-ß ligand treatment after disease onset is neuroprotective in the multiple sclerosis model.

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammation and neurodegeneration. Current MS treatments were designed to reduce inflammation in MS rather than directly to prevent neurodegeneration. Estrogen has well-documented neuroprotective effects in a variety of disorders of the CNS, including experimental autoimmune encephalomyelitis (EAE), the most widely used mouse model of MS. Treatment with an estrogen receptor-ß (ERß) ligand is known to ameliorate clinical disease effectively and provide neuroprotection in EAE. However, the protective effects of this ERß ligand have been demonstrated only when administered prior to disease (prophylactically). Here we tested whether ERß ligand treatment could provide clinical protection when treatment was initiated after onset of disease (therapeutically). We found that therapeutic treatment effectively ameliorated clinical disease in EAE. Specifically, ERß ligand-treated animals exhibited preserved axons and myelin compared with vehicle-treated animals. We observed no difference in the number of T lymphocytes, macrophages, or microglia in the CNS of vehicle- vs. ERß ligand-treated animals. Our findings show that therapeutically administered ERß ligand successfully treats clinical EAE, bearing translational relevance to MS as a candidate neuroprotective agent.

Estrogen responsiveness of the TFIID subunit TAF4B in the normal mouse ovary and in ovarian tumors.

Estrogen signaling in the ovary is a fundamental component of normal ovarian function, and evidence also indicates that excessive estrogen is a risk factor for ovarian cancer. We have previously demonstrated that the gonadally enriched TFIID subunit TAF4B, a paralog of the general transcription factor TAF4A, is required for fertility in mice and for the proliferation of ovarian granulosa cells following hormonal stimulation. However, the relationship between TAF4B and estrogen signaling in the normal ovary or during ovarian tumor initiation and progression has yet to be defined. Herein, we show that Taf4b mRNA and TAF4B protein, but not Taf4a mRNA or TAF4A protein, are increased in whole ovaries and granulosa cells of the ovary after exposure to 17beta-estradiol or the synthetic estrogen diethylstilbestrol and that this response occurs within hours after stimulation. Furthermore, this increase occurs via nuclear estrogen receptors both in vivo and in a mouse granulosa cancer cell line, NT-1. We observe a significant increase in Taf4b mRNA in estrogen-supplemented mouse ovarian tumors, which correlates with diminished survival of these mice. These data highlight the novel response of the general transcription factor TAF4B to estrogen in the normal ovary and during ovarian tumor progression in the mouse, suggesting its potential role in regulating actions downstream of estrogen stimulation.

Heparanase procoagulant activity is elevated in women using oral contraceptives.

STUDY QUESTION: What is the effect of estrogen on heparanase procogulant activity? SUMMARY ANSWER: Estrogen increases heparanase procoagulant activity. WHAT IS KNOWN ALREADY: Estrogen therapy increases the risk of thrombosis and was previously found to up-regulate heparanase expression. Heparanase is involved in angiogenesis and metastasis, and has been shown to form a complex with tissue factor (TF) and also shown to enhance the generation of factor Xa. STUDY DESIGN, SIZE, DURATION: A case-control study. Thirty-four healthy women using oral contraceptives (OC) and 41 women not using hormonal therapy and not pregnant per history were enrolled, over a 5-month period, at the Rambam Medical Center, Haifa, Israel. In vitro, estrogen receptor-positive (MCF-7) and -negative (MDA-231) cell lines were incubated with estrogen, tamoxifen and ICI-182.780 a pure estrogen receptor antagonist. The cell medium was evaluated for TF/heparanase complex activity, TF activity and heparanase procoagulant activity by chromogenic substrate. PARTICIPANTS/MATERIALS, SETTING, METHODS: Exclusion criteria included age <18 years, post-menopausal women, concomitant medications other than supplement minerals and vitamins, acute or chronic illness. MAIN RESULTS AND THE ROLE OF CHANCE: The study demonstrates increased risk of high heparanase procoagulant activity in OC users. When a cutoff level of 0.25 (absorbance 405-490 nm) was set, the odds ratio was 131 (P < 0.0001). When all results were studied by quartiles, in quartiles 3 and 4 the results were almost exclusively of the OC users (P < 0.0001). In cell cultures, estrogen and tamoxifen increased heparanase procoagulant activity in the medium of estrogen receptor-positive (MCF-7) cells. LIMITATIONS, REASONS FOR CAUTION: The main limitation of the current study is that the two estrogens given to the women and cell cultures, ethinyl estradiol (EE) and 17-ß-estradiol (E2), respectively, may have different effects on the coagulation system, although an increase in heparanase procoagulant activity was demonstrated in both of them. Although the sample size of the study group was limited, significant differences in the activation of the extrinsic coagulation pathway were demonstrated. WIDER IMPLICATIONS OF THE FINDINGS: The clinical relevance of the heparanase procoagulant activity assay as a screening tool in thrombophilia work-up should further be elucidated.

Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches.

Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERß ligands was assembled (546 for ERα and 137 for ERß). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure-activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERß. High predictive accuracy was achieved for ERα binding affinity (MTL R(2)=0.71, STL R(2)=0.73). For ERß binding affinity, MTL models were significantly more predictive (R(2)=0.53, p<0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERß, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERß agonist (PDB ID: 2NV7), and ERß antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation.

Bradycardic effects mediated by activation of G protein-coupled estrogen receptor in rat nucleus ambiguus.

The G protein-coupled estrogen receptor (GPER) has been identified in several brain regions, including cholinergic neurons of the nucleus ambiguus, which are critical for parasympathetic cardiac regulation. Using calcium imaging and electrophysiological techniques, microinjection into the nucleus ambiguus and blood pressure measurement, we examined the in vitro and in vivo effects of GPER activation in nucleus ambiguus neurons. A GPER selective agonist, G-1, produced a sustained increase in cytosolic Ca(2+) concentration in a concentration-dependent manner in retrogradely labelled cardiac vagal neurons of nucleus ambiguus. The increase in cytosolic Ca(2+) produced by G-1 was abolished by pretreatment with G36, a GPER antagonist. G-1 depolarized cultured cardiac vagal neurons of the nucleus ambiguus. The excitatory effect of G-1 was also identified by whole-cell visual patch-clamp recordings in nucleus ambiguus neurons, in medullary slices. To validate the physiological relevance of our in vitro studies, we carried out in vivo experiments. Microinjection of G-1 into the nucleus ambiguus elicited a decrease in heart rate; the effect was blocked by prior microinjection of G36. Systemic injection of G-1, in addition to a previously reported decrease in blood pressure, also reduced the heart rate. The G-1-induced bradycardia was prevented by systemic injection of atropine, a muscarinic antagonist, or by bilateral microinjection of G36 into the nucleus ambiguus. Our results indicate that GPER-mediated bradycardia occurs via activation of cardiac parasympathetic neurons of the nucleus ambiguus and support the involvement of the GPER in the modulation of cardiac vagal tone.

A Novel Membrane Estrogen Receptor Activated by STX Induces Female Sexual Receptivity through an Interaction with mGluR1a.

Membrane initiated estradiol signaling has been shown to be vital for multiple physiological processes. Several receptors have been proposed to mediate the actions of estradiol at the membrane. Here, we examined the ability of STX, an agonist of a novel putative membrane estrogen receptor, to activate sexually receptive behavior in the female rat. Infusions of STX into the arcuate nucleus of the hypothalamus resulted in the activation and internalization of µ-opioid receptors in the medial preoptic nucleus, an action that is required for lordosis behavior. Indeed, STX was able to augment sexual receptivity in female rats given a sub-behavioral dose of estradiol. However, if the mGluR1a antagonist, LY367,385, was administered prior to STX, its circuit-activating effects, the internalization of µ-opioid receptors, were lost. This suggests that the receptor stimulated by STX activates rapid membrane-initiated signaling through an interaction with mGluR1a - an effect previously described for estrogen receptor-α at the membrane.

Design, synthesis and biological evaluation of novel estrogen-derived steroid metal complexes.

A new series of estrogen-derived metal complexes were synthesized and characterized. The functionalized estrogen receptor ligands were prepared by a four-step synthetic strategy, and then three transition metal Pd, Ni, Zn were introduced readily to give the title metal complexes, in which the squaramide was introduced as ion acceptor for the first time in the development of estrogen-derived metal complexes for estrogen receptor. Upon binding to estrogen receptors, all of the estrogen conjugates exhibited acceptable binding affinity (up to 4.04% relative to estradiol), and in transcription assays, all the compounds are agonists on ERα. Molecular modeling studies suggest a structural basis for the agonist activity of these compounds.

Total synthesis of eryvarin H and its derivatives and their biological activity as ERRγ inverse agonist.

Total synthesis of eryvarin H and a biological investigation of its analogues as a potential inverse agonist of ERRγ are described here. Among the 13 analogues prepared by the modular synthetic route, eryvarin H and compound showed meaningful ERRγ inverse agonistic activities along with moderate selectivity over ERα and other nuclear receptors in the cell-based reporter gene assay.

Binary classification of a large collection of environmental chemicals from estrogen receptor assays by quantitative structure-activity relationship and machine learning methods.

There are thousands of environmental chemicals subject to regulatory decisions for endocrine disrupting potential. The ToxCast and Tox21 programs have tested ∼8200 chemicals in a broad screening panel of in vitro high-throughput screening (HTS) assays for estrogen receptor (ER) agonist and antagonist activity. The present work uses this large data set to develop in silico quantitative structure-activity relationship (QSAR) models using machine learning (ML) methods and a novel approach to manage the imbalanced data distribution. Training compounds from the ToxCast project were categorized as active or inactive (binding or nonbinding) classes based on a composite ER Interaction Score derived from a collection of 13 ER in vitro assays. A total of 1537 chemicals from ToxCast were used to derive and optimize the binary classification models while 5073 additional chemicals from the Tox21 project, evaluated in 2 of the 13 in vitro assays, were used to externally validate the model performance. In order to handle the imbalanced distribution of active and inactive chemicals, we developed a cluster-selection strategy to minimize information loss and increase predictive performance and compared this strategy to three currently popular techniques: cost-sensitive learning, oversampling of the minority class, and undersampling of the majority class. QSAR classification models were built to relate the molecular structures of chemicals to their ER activities using linear discriminant analysis (LDA), classification and regression trees (CART), and support vector machines (SVM) with 51 molecular descriptors from QikProp and 4328 bits of structural fingerprints as explanatory variables. A random forest (RF) feature selection method was employed to extract the structural features most relevant to the ER activity. The best model was obtained using SVM in combination with a subset of descriptors identified from a large set via the RF algorithm, which recognized the active and inactive compounds at the accuracies of 76.1% and 82.8% with a total accuracy of 81.6% on the internal test set and 70.8% on the external test set. These results demonstrate that a combination of high-quality experimental data and ML methods can lead to robust models that achieve excellent predictive accuracy, which are potentially useful for facilitating the virtual screening of chemicals for environmental risk assessment.

Effect-directed analysis of endocrine-disrupting compounds in multi-contaminated sediment: identification of novel ligands of estrogen and pregnane X receptors.

Effect-directed analysis (EDA)-based strategies have been increasingly used in order to identify the causative link between adverse (eco-)toxic effects and chemical contaminants. In this study, we report the development and use of an EDA approach to identify endocrine-disrupting chemicals (EDCs) in a multi-contaminated river sediment. The battery of in vitro reporter cell-based bioassays, measuring estrogenic, (anti)androgenic, dioxin-like, and pregnane X receptor (PXR)-like activities, revealed multi-contamination profiles. To isolate active compounds of a wide polarity range, we established a multi-step fractionation procedure combining: (1) a primary fractionation step using normal phase-based solid-phase extraction (SPE), validated with a mixture of 12 non-polar to polar standard EDCs; (2) a secondary fractionation using reversed-phase-based high-performance liquid chromatography (RP-HPLC) calibrated with 33 standard EDCs; and (3) a purification step using a recombinant estrogen receptor (ER) affinity column. In vitro SPE and HPLC profiles revealed that ER and PXR activities were mainly due to polar to mid-polar compounds, while dioxin-like and anti-androgenic activities were in the less polar fractions. The overall procedure allowed final isolation and identification of new environmental PXR (e.g., di-iso-octylphthalate) and ER (e.g., 2,4-di-tert-butylphenol and 2,6-di-tert-butyl-α-methoxy-p-cresol) ligands by using gas chromatography coupled with mass spectrometry with full-scan mode acquisition in mid-polar fractions. In vitro biological activity of these chemicals was further confirmed using commercial standards, with di-iso-octylphthalate identified for the first time as a potent hPXR environmental agonist.

Selective estrogen receptor modulator BC-1 activates antioxidant signaling pathway in vitro via formation of reactive metabolites.

AIM: Benzothiophene compounds are selective estrogen receptor modulators (SERMs), which are recently found to activate antioxidant signaling. In this study the molecular mechanisms of antioxidant signaling activation by benzothiophene compound BC-1 were investigated. METHODS: HepG2 cells were stably transfected with antioxidant response element (ARE)-luciferase reporter (HepG2-ARE cells). The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) in HepG2-ARE cells was suppressed using siRNA. The metabolites of BC-1 in rat liver microsome incubation were analyzed using LC-UV and LC-MS. RESULTS: Addition of BC-1 (5 µmol/L) in HepG2-ARE cells resulted in a 17-fold increase of ARE-luciferase activity. Pretreatment with the estrogen receptor agonist E2 (5 µmol/L) or antagonist ICI 182,780 (5 µmol/L) did not affect BC-1-induced ARE-luciferase activity. However, transfection of the cells with anti-Nrf2 siRNA suppressed this effect by 79%. Addition of BC-1 in rat microsome incubation resulted in formation of di-quinone methides and o-quinones, followed by formation of GSH conjugates. BC-1 analogues with hydrogen (BC-2) or fluorine (BC-3) at the 4' position did not form the di-quinone methides. Both BC-2 and BC-3 showed comparable estrogenic activity with BC-1, but did not induce ARE-luciferase activity in HepG2-ARE cells. CONCLUSION: Benzothiophene compound BC-1 activates ARE signaling via reactive metabolite formation that is independent of estrogen receptors.