The human estrogen receptor alpha dimer binds a single SRC-1 coactivator molecule with an affinity dictated by agonist structure.

Nuclear receptors act as ligand-inducible transcription factors. Agonist binding leads to interaction with coactivator proteins, and to the assembly of the general transcription machinery. In addition to structural information, a thorough understanding of transcriptional activation by the nuclear receptors requires the characterization of the thermodynamic parameters governing these protein/protein interactions. In this study we have quantitatively characterized the interactions of full-length baculovirus expressed human estrogen receptor alpha (ERalpha), as well as ERalpha hormone binding domain (ERHBD) with a fragment of the coactivator protein SRC-1 (amino acid residues 570 to 780). Fluorescence anisotropy and fluorescence correlation spectroscopy of fluorescently labeled SRC-1(570-780) demonstrate unambiguously that the stoichiometry of the SRC-1/ERalpha/estradiol complex is one coactivator molecule per ERalpha dimer. The affinity of the estradiol or estriol bound ERalpha/SRC-1 complexes was found to be significantly higher than that observed in the presence of estrone. No binding was observed in the absence of ligand or in the presence of antagonists. Distinct anisotropy values for the ERalpha-SRC-1 complexes with different agonists suggest distinct conformations of the complexes depending upon agonist structure

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

New insights on cross-species differences in the modulation of human and zebrafish nuclear receptors by single chemicals and environmental mixtures

In the context of contamination of aquatic ecosystems by endocrine disrupting chemicals (EDCs), this work aims to provide new insights on cross-species differences in the modulation of nuclear receptors (NRs) and the aryl hydrocarbon receptor (AhR) by individual chemicals and environmental mixtures, in order to help further cross-species extrapolation in the frame of the environmental risks of EDCs. To this end, a panel of individual ligands and environmental mixtures from an urban waste water treatment plant (WWTP), were screened on a set of recently developed in vitro reporter cell lines based on both human (h) and zebrafish (zf) NRs and AhR. Our results revealed that for some of the receptors marked cross-species differences occurred (PXR, PPARγ, PR) while for other receptors the differences were lower (ER, AR, GR, MR) or almost absent (AhR, ERRγ). For instance, promegestone acts as a full agonist of the hPR but as partial agonist of the zfPR whereas the dihydroxy-4-pregnen-3-one -reference ligand of the zfPR- antagonizes the hPR. In the same way, none of the reference ligands of the hPXR (T0913117) modulates the zfPXR whereas the clotrimazole -reference ligand of zfPXR- modulates also the hPXR but with lower potency. Then the hAR was more sensitive to the agonist mifepristone and the antagonist OH-flutamide than the zfAR whereas the dexamethasone was a more potent agonist of the zfGR than the hGR. Also significant differences in selectivity were noted among h and zf ER subtypes. Finally, the in vitro profiling of an urban WWTP confirmed these cross-species differences in terms of level, type (agonist vs antagonist), distribution along the WWTP. For instance, h and zf estrogenic activity was differentially detected in the sludge and the suspended material. In the same way, strong zf anti-androgenic activity was detected in the effluent while no human one can be detected. Also, strong zf mineralocorticoidic activity was detected in both influent and effluent whereas only h anti-mineralocorticoid activity was detected. Alogether, our results showed that h and zf NRs are, for some of them, differentially modulated by individual chemicals and environmental mixtures. Also, interaction of EDCs towards NRs cannot always be extrapolated between these species highlighting the need to further document NRs modulation between human and fish and associated responses, to improve human health and environmental risk assessment of EDCs

Parallel biotransformation of tetrabromobisphenol A in Xenopus laevis and mammals: Xenopus as a model for endocrine perturbation studies.

The flame retardant tetrabromobisphenol A (TBBPA) is a high production flame retardant that interferes with thyroid hormone (TH) signaling. Despite its rapid metabolism in mammals, TBBPA is found in significant amounts in different tissues. Such findings highlight first a need to better understand the effects of TBBPA and its metabolites and second the need to develop models to address these questions experimentally. We used Xenopus laevis tadpoles to follow radiolabeled (14)C-TBBPA uptake and metabolism. Extensive and rapid uptake of radioactivity was observed, tadpoles metabolizing > 94% of (14)C-TBBPA within 8 h. Four metabolites were identified in water and tadpole extracts: TBBPA-glucuronide, TBBPA-glucuronide-sulfate, TBBPA-sulfate, and TBBPA-disulfate. These metabolites are identical to the TBBPA conjugates characterized in mammals, including humans. Most radioactivity (> 75%) was associated with sulfated conjugates. The antithyroid effects of TBBPA and the metabolites were compared using two in vivo measures: tadpole morphology and an in vivo tadpole TH reporter gene assay. Only TBBPA, and not the sulfated metabolites, disrupted thyroid signaling. Moreover, TBBPA treatment did not affect expression of phase II enzymes involved in TH metabolism, suggesting that the antithyroid effects of TBBPA are not due to indirect effects on TH metabolism. Finally, we show that only the parent TBBPA inhibits T3-induced transactivation in cells expressing human, zebrafish, or X. laevis TH receptor, TRα. We conclude, first, that perturbation of thyroid signaling by TBBPA is likely due to rapid direct action of the parent compound, and second, that Xenopus is an excellent vertebrate model for biotransformation studies, displaying homologous pathways to mammals