Diindoles produced from commensal microbiota metabolites function as endogenous CAR/Nr1i3 ligands.

Numerous studies have demonstrated the correlation between human gut bacteria and host physiology, mediated primarily via nuclear receptors (NRs). Despite this body of work, the systematic identification and characterization of microbe-derived ligands that regulate NRs remain a considerable challenge. In this study, we discover a series of diindole molecules produced from commensal bacteria metabolites that act as specific agonists for the orphan constitutive androstane receptor (CAR). Using various biophysical analyses we show that their nanomolar affinities are comparable to those of synthetic CAR agonists, and that they can activate both rodent and human CAR orthologues, which established synthetic agonists cannot. We also find that the diindoles, diindolylmethane (DIM) and diindolylethane (DIE) selectively up-regulate bona fide CAR target genes in primary human hepatocytes and mouse liver without causing significant side effects. These findings provide new insights into the complex interplay between the gut microbiome and host physiology, as well as new tools for disease treatment.

Structural Insights into the Activation of Human Aryl Hydrocarbon Receptor by the Environmental Contaminant Benzo[a]pyrene and Structurally Related Compounds.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor belonging to the bHLH/PAS protein family and responding to hundreds of natural and chemical substances. It is primarily involved in the defense against chemical insults and bacterial infections or in the adaptive immune response, but also in the development of pathological conditions ranging from inflammatory to neoplastic disorders. Despite its prominent roles in many (patho)physiological processes, the lack of high-resolution structural data has precluded for thirty years an in-depth understanding of the structural mechanisms underlying ligand-binding specificity, promiscuity and activation of AHR. We recently reported a cryogenic electron microscopy (cryo-EM) structure of human AHR bound to the natural ligand indirubin, the chaperone Hsp90 and the co-chaperone XAP2 that provided the first experimental visualization of its ligand-binding PAS-B domain. Here, we report a 2.75 Å resolution structure of the AHR complex bound to the environmental pollutant benzo[a]pyrene (B[a]P). The structure substantiates the existence of a bipartite PAS-B ligand-binding pocket with a geometrically constrained primary binding site controlling ligand binding specificity and affinity, and a secondary binding site contributing to the binding promiscuity of AHR. We also report a docking study of B[a]P congeners that validates the B[a]P-bound PAS-B structure as a suitable model for accurate computational ligand binding assessment. Finally, comparison of our agonist-bound complex with the recently reported structures of mouse and fruit fly AHR PAS-B in different activation states suggests a ligand-induced loop conformational change potentially involved in the regulation of AHR function.

Steatosis and Metabolic Disorders Associated with Synergistic Activation of the CAR/RXR Heterodimer by Pesticides.

The nuclear receptor, constitutive androstane receptor (CAR), which forms a heterodimer with the retinoid X receptor (RXR), was initially reported as a transcription factor that regulates hepatic genes involved in detoxication and energy metabolism. Different studies have shown that CAR activation results in metabolic disorders, including non-alcoholic fatty liver disease, by activating lipogenesis in the liver. Our objective was to determine whether synergistic activations of the CAR/RXR heterodimer could occur in vivo as described in vitro by other authors, and to assess the metabolic consequences. For this purpose, six pesticides, ligands of CAR, were selected, and Tri-butyl-tin (TBT) was used as an RXR agonist. In mice, CAR's synergic activation was induced by dieldrin associated with TBT, and combined effects were induced by propiconazole, bifenox, boscalid, and bupirimate. Moreover, a steatosis, characterized by increased triglycerides, was observed when TBT was combined with dieldrin, propiconazole, bifenox, boscalid, and bupirimate. Metabolic disruption appeared in the form of increased cholesterol and lowered free fatty acid plasma levels. An in-depth analysis revealed increased expression of genes involved in lipid synthesis and lipid import. These results contribute to the growing understanding of how environmental contaminants can influence nuclear receptor activity and associated health risks.

2,4-Di-tert-butylphenol Induces Adipogenesis in Human Mesenchymal Stem Cells by Activating Retinoid X Receptors.

2,4-Di-tert-butylphenol (2,4-DTBP) is an important commercial antioxidant and a toxic natural secondary metabolite that has been detected in humans. However, there is scant information regarding its toxicological effects. We asked whether 2,4-DTBP is a potential obesogen. Using a human mesenchymal stem cell adipogenesis assay, we found that exposure to 2,4-DTBP led to increased lipid accumulation and expression of adipogenic marker genes. Antagonist assays revealed that 2,4-DTBP increased lipid accumulation by activating the peroxisome proliferator-activated receptor (PPAR) γ-retinoid X receptor (RXR) heterodimer. 2,4-DTBP likely activated the PPARγ/RXRα heterodimer by activating RXRα but not directly binding to PPARγ. We confirmed that 2,4-DTBP directly bound to RXRα by solving the crystal structure of this complex, then predicted and demonstrated that related compounds could also activate RXRα. Our study demonstrated that 2,4-DTBP and related chemicals could act as obesogens and endocrine disruptors via RXRs. These data showed that 2,4-DTBP belongs to a family of compounds whose endocrine-disrupting and obesogenic effects can be strongly modulated by their chemical composition. Structure-activity studies such as the present one could help guide the rational development of safer antioxidants that do not interact with important nuclear receptors having broad effects on human development and physiology.

Interlaboratory prevalidation of a new in vitro transcriptional activation assay for the screening of (anti-)androgenic activity of chemicals using the UALH-hAR cell line.

We report an interlaboratory evaluation of a recently developed androgen receptor (AR) transactivation assay using the UALH-hAR reporter cell line that stably expresses the luciferase gene under the transcriptional control of androgen receptor elements (AREs) with no glucocorticoid receptor (GR) crosstalk. Herein, a two-step prevalidation study involving three laboratories was conducted to assess performance criteria of the method such as transferability as well as robustness, sensitivity, and specificity. The first step consisted in the validation of the transfer of the cell line to participant laboratories through the testing of three reference chemicals: the AR agonist dihydrotestosterone, the AR antagonist hydroxyflutamide and the glucocorticoid dexamethasone. Secondly, a blinded study was conducted by screening a selection of ten chemicals, including four AR agonists, five AR antagonists, and one non-active chemical. All test compounds yielded the same activity profiles in all laboratories. The logEC50 (agonist assay) or logIC50 (antagonist assay) were in the same range, with intra-laboratory coefficients of variation (CVs) of 0.1-3.4% and interlaboratory CVs of 1-4%, indicating very good within- and between-laboratory reproducibility. Our results were consistent with literature and regulatory data (OECD TG458). Overall, this interlaboratory study demonstrated that the UALH-hAR assay is transferable, produces reliable, accurate and specific (anti)androgenic activity of chemicals, and can be considered for further regulatory validation.

Environmental water extracts differentially activate zebrafish and human nuclear progesterone receptors.

Many reports on anti-progestogenic activities in aquatic environments have been published in the past decade. These are monitored mainly by in vitro reporter gene bioassays based upon the human progesterone receptor (PR). However, results obtained by some human in vitro bioassays may not be relevant for aquatic animals, especially fish. The present work aimed to detect fish (anti-)PR activity in waste- and receiving surface waters. In parallel, human (anti-)PR activity was analysed to determine if there was any connection between human and fish (anti-)PR activities. Finally, (anti-)PR activities were linked to the occurrence of progestins in water samples. Human PR agonistic activity was detected in all wastewater and most receiving surface water samples. Nevertheless, zebrafish PR (zfPR) agonistic activity was found in only two influent wastewater samples (max. 117 ng/L 17α,20ß-dihydroxy-4-pregnen-3-one [DHP] equivalents). Analysed synthetic progestins and progesterone accounted for 14 % to 161 % of detected human PR (hPR) agonistic activity in water samples. Progesterone also contributed significantly to zfPR agonistic activity (up to 10 %) in raw wastewater. The anti-hPR activity was detected also in most wastewater and some surface water samples, but synthetic progestins did not trigger anti-zfPR activity in excess of LOQ values. In addition, altrenogest, dienogest, and ulipristal acetate were tested for their potency to zfPR for the first time. The activity analyses of both pure substances and environmental samples showed that human and zebrafish progesterone receptors are differentially activated. Therefore, results based on human PR in vitro bioassays could not predict fish PR activities in the environment.

Cryo-EM structure of the agonist-bound Hsp90-XAP2-AHR cytosolic complex.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates a broad spectrum of (patho)physiological processes in response to numerous substances including pollutants, natural products and metabolites. However, the scarcity of structural data precludes understanding of how AHR is activated by such diverse compounds. Our 2.85 Å structure of the human indirubin-bound AHR complex with the chaperone Hsp90 and the co-chaperone XAP2, reported herein, reveals a closed conformation Hsp90 dimer with AHR threaded through its lumen and XAP2 serving as a brace. Importantly, we disclose the long-awaited structure of the AHR PAS-B domain revealing a unique organisation of the ligand-binding pocket and the structural determinants of ligand-binding specificity and promiscuity of the receptor. By providing structural details of the molecular initiating event leading to AHR activation, our study rationalises almost forty years of biochemical data and provides a framework for future mechanistic studies and structure-guided drug design.

Bisphenol A analogues induce a feed-forward estrogenic response in zebrafish.

Because exposure to bisphenol A (BPA) has been linked to health problems in humans and wildlife, BPA analogues have been synthesized to be considered as replacement molecules. We here have examined estrogenic activity of BPA and five of its analogues, BPAF, BPE, BPC, BPC-Cl, and BPS by a combination of zebrafish-based in vivo and in vitro assays. We used transgenic estrogen reporter (5xERE:GFP) fish to study agonistic effects of bisphenols. Exposures to BPA, BPAF, BPE, and BPC, induced GFP expression in estrogen reporter fish at low exposure concentrations in the heart valves and at higher concentrations in the liver, whereas BPC-Cl activated GFP expression mainly in the liver, and BPS faintly in the heart only. The in vivo response was compared to in vitro estrogenicity of bisphenol exposure using reporter cells that express the zebrafish estrogen receptors driving expression of an estrogen response element (ERE)-luciferase reporter. In these cells, BPA, BPAF, BPC, BPE and BPS preferentially activated Esr1, whereas BPC-Cl preferentially activated Esr2a. By quantitative PCR we found that exposure to BPAF induced expression of the classical estrogen target genes vtg1, esr1, and cyp19a1b in a concentration response manner, but the most responsive target gene was f13a1a. Exposure to BPC-Cl resulted in a different expression pattern of vtg1 and f13a1a with an activation at low concentrations, followed by a declining expression at higher concentrations. Because expression of f13a1a was strongly activated by all compounds tested, we suggest including this mRNA as a biomarker for estrogenicity in larval fish. We further showed that exposure to BPAF and BPC-Cl increased E2 levels in zebrafish larvae, indicating that bisphenol exposures result in a feed-forward response that can further augment the estrogenic activity of these compounds.

PPARα and PPARγ activation is associated with pleural mesothelioma invasion but therapeutic inhibition is ineffective.

Mesothelioma is a cancer that typically originates in the pleura of the lungs. It rapidly invades the surrounding tissues, causing pain and shortness of breath. We compared cell lines injected either subcutaneously or intrapleurally and found that only the latter resulted in invasive and rapid growth. Pleural tumors displayed a transcriptional signature consistent with increased activity of nuclear receptors PPARα and PPARγ and with an increased abundance of endogenous PPAR-activating ligands. We found that chemical probe GW6471 is a potent, dual PPARα/γ antagonist with anti-invasive and anti-proliferative activity in vitro. However, administration of GW6471 at doses that provided sustained plasma exposure levels sufficient for inhibition of PPARα/γ transcriptional activity did not result in significant anti-mesothelioma activity in mice. Lastly, we demonstrate that the in vitro anti-tumor effect of GW6471 is off-target. We conclude that dual PPARα/γ antagonism alone is not a viable treatment modality for mesothelioma.

Structure-Based and Knowledge-Informed Design of B-Raf Inhibitors Devoid of Deleterious PXR Binding.

Dabrafenib is an anticancer drug currently used in the clinics, alone or in combination. However, dabrafenib was recently shown to potently activate the human nuclear receptor pregnane X receptor (PXR). PXR activation increases the clearance of various chemicals and drugs, including dabrafenib itself. It may also enhance cell proliferation and tumor aggressiveness. Therefore, there is a need for rational design of a potent protein kinase B-Raf inhibitor devoid of binding to the secondary target PXR and resisting rapid metabolism. By determining the crystal structure of dabrafenib bound to PXR and analyzing its mode of binding to both PXR and its primary target, B-Raf-V600E, we were able to derive new compounds with nanomolar activity against B-Raf and no detectable affinity for PXR. The crystal structure of B-Raf in complex with our lead compound revealed a subdomain swapping of the activation loop with potentially important functional implications for a prolonged inhibition of B-Raf-V600E.

Interspecies Differences in Activation of Peroxisome Proliferator-Activated Receptor γ by Pharmaceutical and Environmental Chemicals.

Endocrine disrupting chemicals (EDCs) are able to deregulate the hormone system, notably through interactions with nuclear receptors (NRs). The mechanisms of action and biological effects of many EDCs have mainly been tested on human and mouse but other species such as zebrafish and xenopus are increasingly used as a model to study the effects of EDCs. Among NRs, peroxisome proliferator-activated receptor γ (PPARγ) is a main target of EDCs, for which most experimental data have been obtained from human and mouse models. To assess interspecies differences, we tested known human PPARγ ligands on reporter cell lines expressing either human, mouse, zebrafish, or xenopus PPARγ. Using these cell lines, we were able to highlight major interspecies differences. Known hPPARγ pharmaceutical ligands modulated hPPARγ and mPPARγ activities in a similar manner, while xPPARγ was less responsive and zfPPARγ was not modulated at all by these compounds. On the contrary, human liver X receptor (hLXR) ligands GW 3965 and WAY-252623 were only active on zfPPARγ. Among environmental compounds, several molecules activated the PPARγ of the four species similarly, e.g., phthalates (MEHP), perfluorinated compounds (PFOA, PFOS), and halogenated derivatives of BPA (TBBPA, TCBPA), but some of them like diclofenac and the organophosphorus compounds tri-o-tolyl phosphate and triphenyl phosphate were most active on zfPPARγ. This study confirms or shows for the first time the h, m, x, and zfPPARγ activities of several chemicals and demonstrates the importance of the use of species-specific models to study endocrine and metabolism disruption by environmental chemicals.

A Comparative Study of Human and Zebrafish Pregnane X Receptor Activities of Pesticides and Steroids Using In Vitro Reporter Gene Assays.

The nuclear receptor pregnane X receptor (PXR) is a ligand-dependent transcription factor that regulates genes involved in xenobiotic metabolism in mammals. Many studies suggest that PXR may play a similar role in fish. The interaction of human PXR (hPXR) with a variety of structurally diverse endogenous and exogenous chemicals is well described. In contrast, little is known about the zebrafish PXR (zfPXR). In order to compare the effects of these chemicals on the PXR of these two species, we established reporter cell lines expressing either hPXR or zfPXR. Using these cellular models, we tested the hPXR and zfPXR activity of various steroids and pesticides. We provide evidence that steroids were generally stronger activators of zfPXR while pesticides were more potent on hPXR. In addition, some chemicals (econazole nitrate, mifepristone, cypermethrin) showed an antagonist effect on zfPXR, whereas no antagonist chemical has been identified for hPXR. These results confirm significant differences in the ability of chemicals to modulate zfPXR in comparison to hPXR and point out that zfPXR assays should be used instead of hPXR assays for evaluating the potential risks of chemicals on aquatic species.

Mechanistic insights into the synergistic activation of the RXR-PXR heterodimer by endocrine disruptor mixtures.

Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures may alter physiology through interaction with RXR-PXR and potentially several other nuclear receptor heterodimers.

A Novel N-Substituted Valine Derivative with Unique Peroxisome Proliferator-Activated Receptor γ Binding Properties and Biological Activities.

A proprietary library of novel N-aryl-substituted amino acid derivatives bearing a hydroxamate head group allowed the identification of compound 3a that possesses weak proadipogenic and peroxisome proliferator-activated receptor γ (PPARγ) activating properties. The systematic optimization of 3a, in order to improve its PPARγ agonist activity, led to the synthesis of compound 7j (N-aryl-substituted valine derivative) that possesses dual PPARγ/PPARα agonistic activity. Structural and kinetic analyses reveal that 7j occupies the typical ligand binding domain of the PPARγ agonists with, however, a unique high-affinity binding mode. Furthermore, 7j is highly effective in preventing cyclin-dependent kinase 5-mediated phosphorylation of PPARγ serine 273. Although less proadipogenic than rosiglitazone, 7j significantly increases adipocyte insulin-stimulated glucose uptake and efficiently promotes white-to-brown adipocyte conversion. In addition, 7j prevents oleic acid-induced lipid accumulation in hepatoma cells. The unique biochemical properties and biological activities of compound 7j suggest that it would be a promising candidate for the development of compounds to reduce insulin resistance, obesity, and nonalcoholic fatty liver disease.

Assessing the Selectivity of FXR, LXRs, CAR, and RORγ Pharmaceutical Ligands With Reporter Cell Lines.

To characterize human nuclear receptor (NR) specificity of synthetic pharmaceutical chemicals we established stable cell lines expressing the ligand binding domains (LBDs) of human FXR, LXRα, LXRß, CAR, and RORγ fused to the yeast GAL4 DNA binding domain (DBD). As we have already done for human PXR, a two-step transfection procedure was used. HeLa cells stably expressing a Gal4 responsive gene (HG5LN cell line) were transfected by Gal4-NRs expressing plasmids. At first, using these cell lines as well as the HG5LN PXR cells, we demonstrated that the basal activities varied from weak (FXR and LXRs), intermediate (PXR), to strong (CAR and RORγ), reflecting the recruitment of HeLa co-regulators in absence of ligand. Secondly, we finely characterized the activities of commercially available FXR, LXRα, LXRß, CAR, RORγ, and PXR agonists/antagonists GW4064, feraxamine, DY268, T0901317, GW3965, WAY252623, SR9238, SR9243, GSK2033, CITCO, CINPA1, PK11195, S07662, SR1078, SR0987, SR1001, SR2211, XY018, clotrimazole, dabrafenib, SR12813, and SPA70, respectively. Among these compounds we revealed both, receptor specific agonists/antagonists, as well as less selective ligands, activating or inhibiting several nuclear receptors. FXR ligands manifested high receptor selectivity. Vice versa, LXR ligands behaved in non-selective manner, all activating at least PXR. CAR was selectively influenced by their ligands, while it also responded to several LXR ligands. Finally, although PXR was quite selectively activated or antagonized by its own ligands, it responded to several NRs ligands as well. Thus, using these reporter cell lines enabled us to precisely characterize the selectivity of pharmaceutical ligands for different nuclear receptors.

Assessing species-specific differences for nuclear receptor activation for environmental water extracts.

In vitro bioassays are increasingly applied to detect endocrine disrupting chemicals (EDCs) in environmental waters. Most studies use human nuclear receptor assays, but this raises questions about their relevance for evaluating ecosystem health. The current study aimed to assess species-specific differences in the activation or inhibition of a range of human and zebrafish nuclear receptors by different water extracts. Wastewater and surface water extracts were run in transactivation assays indicative of the estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), mineralocorticoid receptor (MR), pregnane X receptor (PXR) and peroxisome proliferator-activated receptor gamma (PPARγ). The transactivation assays were complemented with competitive binding assays for human AR, GR, PR and MR. In most cases, both human and zebrafish nuclear receptor activity were detected in the water extracts. Only some species-specific differences in potency and activity were observed. Water extracts were more active in zebrafish PXR compared to human PXR whereas the opposite was observed for PPARγ. Further, all water extracts inhibited zebrafish PR, while only one extract showed weak anti-progestagenic activity for human PR. Due to these observed differences, zebrafish nuclear receptor assays may be preferable over human nuclear receptor assays to assess the potential risks of EDCs to aquatic organisms. However, recognizing issues with availability of zebrafish nuclear receptor assays and the relatively small differences in responsiveness for many of the human and zebrafish nuclear receptors, including the widely studied ER, the current study supports the continued use of human nuclear receptor assays for water quality monitoring.

The Anti-Cancer Drug Dabrafenib Is a Potent Activator of the Human Pregnane X Receptor.

The human pregnane X receptor (hPXR) is activated by a large set of endogenous and exogenous compounds and plays a critical role in the control of detoxifying enzymes and transporters regulating liver and gastrointestinal drug metabolism and clearance. hPXR is also involved in both the development of multidrug resistance and enhanced cancer cells aggressiveness. Moreover, its unintentional activation by pharmaceutical drugs can mediate drug-drug interactions and cause severe adverse events. In that context, the potential of the anticancer BRAF inhibitor dabrafenib suspected to activate hPXR and the human constitutive androstane receptor (hCAR) has not been thoroughly investigated yet. Using different reporter cellular assays, we demonstrate that dabrafenib can activate hPXR as efficiently as its reference agonist SR12813, whereas it does not activate mouse or zebrafish PXR nor hCAR. We also showed that dabrafenib binds to recombinant hPXR, induces the expression of hPXR responsive genes in colon LS174T-hPXR cancer cells and human hepatocytes and finally increases the proliferation in LS174T-hPXR cells. Our study reveals that by using a panel of different cellular techniques it is possible to improve the assessment of hPXR agonist activity for new developed drugs.

High Content Screening Using New U2OS Reporter Cell Models Identifies Harmol Hydrochloride as a Selective and Competitive Antagonist of the Androgen Receptor.

Prostate cancer is the most commonly diagnosed malignancy in men. Its growth mainly relies on the activity of the androgen receptor (AR), justifying the use of androgen deprivation therapy as a gold standard treatment for the metastatic disease. Inhibition of the androgen axis using second generation antagonists has improved patients' survival, but is systematically confronted to resistance mechanisms, leading to a median survival that does not exceed 5 years. Counteracting this resistance has been the object of a large number of investigations, with a particular emphasis towards the identification of new AR inhibitors, whether they antagonize the receptor by a competitive or a non-competitive binding. To this end, many high content screens have been performed, to identify new non-steroidal AR antagonists, using a variety of approaches, but reported somewhat controversial results, depending on the approach and on the cell model that was used for screening. In our study, we used the U2OS osteosarcoma cells stably transfected with AR or ARv7 and a luciferase reporter as a previously validated model to screen the Prestwick Phytochemical library. The results of our screen identified ellipticine, harmol, and harmine hydrochloride as confirmed hits. Surprisingly, we could demonstrate that harmol hydrochloride, previously identified as a non-competitive inhibitor of AR or a weak inhibitor of androgen signaling, was actually a competitive antagonist of AR, which inhibits the growth of VCaP prostate cancer line, at concentrations for which it did not affect the growth of the AR negative DU145 and PC3 cells. Interestingly, we also report for the first time that harmol hydrochloride was selective for AR, as it could not alter the activity of other nuclear receptors, such as the glucocorticoid receptor (GR), the progesterone receptor (PR), or the mineralocorticoid receptor (MR). Additionally, we demonstrate that, conversely to enzalutamide, harmol hydrochloride did not show any agonistic activity towards the pregnane X receptor (PXR), a master regulator of drug metabolism. Together, our results shed light on the importance of the cellular context for the screening of new AR antagonists. They further indicate that some of the potential hits that were previously identified may have been overlooked.

Differential activity of BPA, BPAF and BPC on zebrafish estrogen receptors in vitro and in vivo.

The high volume production compound bisphenol A (BPA) is of environmental concern largely because of its estrogenic activity. Consequently, BPA analogues have been synthesized to be considered as replacement molecules for BPA. These analogues need to be thoroughly evaluated for their estrogenic activity. Here, we combined mechanism zebrafish-based assays to examine estrogenic and anti-estrogenic activities of BPA and two of its analogues, bisphenol AF (BPAF) and bisphenol C (BPC) in vitro and in vivo. In vitro reporter cell lines were used to investigate agonistic and antagonistic effects of the three bisphenols on the three zebrafish estrogen receptors. The transgenic Tg(5 × ERE:GFP) and Cyp19a1b-GFP zebrafish lines were then used to analyze estrogenic and anti-estrogenic responses of the three bisphenols in vivo. BPA, BPAF and BPC were agonists with different potencies for the three zebrafish estrogen receptors in vitro. The potent zfERα-mediated activity of BPA and BPAF in vitro resulted in vivo by activation of GFP expression in zebrafish larvae in the heart (zfERα-dependent) at lower concentrations, and in the liver (zfERß-dependent) at higher concentrations. BPC induced zfERß-mediated luciferase expression in vitro, and the zfERß agonism led to activation of GFP expression in the liver and the brain in vivo. In addition, BPC acted as a full antagonist on zfERα, and completely inhibited estrogen-induced GFP expression in the heart of the zebrafish larvae. To summarize, applying a combination of zebrafish-based in vitro and in vivo methods to evaluate bisphenol analogues for estrogenic activity will facilitate the prioritization of these chemicals for further analysis in higher vertebrates as well as the risk assessment in humans.

Insights into the activation mechanism of human estrogen-related receptor γ by environmental endocrine disruptors.

The estrogen-related receptor γ (ERRγ, NR3B3) is a constitutively active nuclear receptor which has been proposed to act as a mediator of the low-dose effects of a number of environmental endocrine-disrupting chemicals (EDCs) such as the xenoestrogen bisphenol-A (BPA). To better characterize the ability of exogenous compounds to bind and activate ERRγ, we used a combination of cell-based, biochemical, structural and computational approaches. A purposely created stable cell line allowed for the determination of the EC50s for over 30 environmental ERRγ ligands, including previously unknown ones. Interestingly, affinity constants (Kds) of the most potent compounds measured by isothermal titration calorimetry were in the 50-500 nM range, in agreement with their receptor activation potencies. Crystallographic analysis of the interaction between the ERRγ ligand-binding domain (LBD) and compounds of the bisphenol, alkylphenol and naphthol families revealed a partially shared binding mode and minimal alterations of the receptor conformation upon ligand binding. Further biophysical characterizations coupled to molecular dynamics simulations suggested a mechanism through which ERRγ ligands would exhibit their agonistic properties by preserving the transcriptionally active form of the receptor while rigidifying some loop regions with associated functions. This unique mechanism contrasts with the classical one involving a ligand-induced repositioning and stabilization of the C-terminal activation helix H12.