X15695 is a 2-phenylimidazo[1,2-a] pyridine derivative previously described as an orally active, selective estrogen receptor (ER) degrader that inhibits the proliferation of ER+ breast cancer cells. Here, we show that X15695 and derivatives are aryl hydrocarbon receptor (AHR) ligands. Knockout of AHR abolishes the anti-proliferative property of the imidazopyridine derivatives. In the presence of estradiol, X15695 and derivatives outperform the standard of care drug fulvestrant in suppressing the growth of ER+ breast cancer cells, expressing either the wild-type or clinically relevant ER mutant forms (Y537S and D538G) and of patient-derived organoids established from ER+ tumors. Using computational techniques, we discovered that a low pKa value resulting from electron-withdrawing substituents in the 2-phenylimidazo[1,2-a] pyridine compounds is a key feature that identifies them as potent AHR ligands, leading to the potential discovery of additional derivatives for future therapeutic development.

Most in vitro reporter gene bioassays for detecting biological activities in aquatic environments rely on mammalian receptors and may not accurately assess risk for aquatic organisms. This study explored species-specific differences in activation or inhibition of six human and zebrafish nuclear receptors by environmental water extracts from the Czech Republic. Active sampling was conducted at wastewater treatment plants (WWTP) influents and effluents, receiving waters, and ponds; passive sampling in rivers across the Czech Republic, spanning the Bohemian and Moravian regions. A battery of bioassays including human (h) and zebrafish (zf) estrogen receptor-alpha (ERα), androgen (AR), progesterone (PR), glucocorticoid (GR), mineralocorticoid (MR), and pregnane X (PXR) receptors were employed. hERα activity was detected more frequently than zfERα. Detection and responsiveness of AR activity were similar in both human and zebrafish bioassays. hPR activity appeared only in influent of WWTP Protivín, whereas zfPR activity was found in influents of both WWTP Protivín and Vodňany. zfMR activity was detected in both WWTP influents; no hMR activity was observed. zfPXR activity occurred in influents and surface waters, whereas hPXR activity was restricted to WWTP influents. No (anti-)glucocorticoid activity was detected. Our findings indicate that both human and zebrafish nuclear receptor-based bioassays are suitable for monitoring ERα and AR activities, while zebrafish bioassays are better for PR, MR, and PXR activities in fish habitats. These results may guide researchers in selecting the most appropriate bioassay for assessing endocrine activity in waters.

The pregnane X receptor (PXR) is a key chemosensory protein that helps the organism adapt to its chemical environment. Indeed, humans are continuously exposed to a wide range of external chemicals, known as xenobiotics, which include environmental pollutants, food components, cosmetics, and pharmaceuticals. PXR has the unique property to sense a large variety of xenobiotics and regulate the expression of detoxifying enzymes and transporters, facilitating the clearance of these chemicals. However, prolonged activation of this pathway can lead to negative effects, such as drug-drug interactions, chemoresistance, endocrine disruption, a heightened risk of metabolic diseases, or enhanced cell growth and tumor aggressiveness. Understanding how PXR interacts with xenobiotics is crucial for predicting, assessing, and preventing the potential impacts of these chemicals on human health. Here, we present the structural and functional analysis of the interaction of PXR with 2 approved drugs (nimodipine and liranaftate), a natural flavor commonly used in cosmetics and the food industry (sclareol), and an environmentally relevant halogenated derivative of the emblematic endocrine disruptor bisphenol A (2,2'-dichlorobisphenol A). Cell-based assays show that these 4 compounds display different PXR binding potencies, stimulate the expression of key target genes related to drug metabolism and cell proliferation, and promote colon cancer cell proliferation to varying degrees. Crystallographic analysis reveals their distinct mechanisms of binding to PXR. The integrated functional and structural characterization pipeline we have established yields critical insights for both environmental risk assessment and the rational development of industrial and pharmaceutical compounds with minimal harmful PXR activity. Significance Statement The pregnane X receptor is an off-target of many pharmaceuticals and industrial chemicals whose activation is associated with clinically relevant drug-drug interactions. This study describes a pipeline for the identification and characterization of pregnane X receptor ligands, designed to support environmental risk assessment and the informed design of safer substitutes.

Bisphenol A (BPA) is a widely used plastic monomer with well-established endocrine-disrupting properties and emerging evidence for metabolism-disrupting effects. Following regulatory restrictions, structurally modified alternatives, including halogenated derivatives, have been introduced, but information on their metabolic effects remains scarce. Hence, we analyzed proteomic and metabolomic responses in SGBS human adipocytes after exposure to BPA and five analogues (TBBPA, TCBPA, BPS, TBBPS, and TCBPS), including environmentally relevant concentrations of 10 nM. Intracellular target proteins were identified using thermal proteome profiling (TPP). All tested alternatives significantly increased intracellular triglyceride levels, indicating adipogenic potential. Proteomic and metabolomic analyses revealed alterations in lipid and energy metabolism and the central carbon cycle. TPP identified protein targets within steroid hormone pathways, fatty acid, central carbon, and amino acid metabolism, which were confirmed by nano differential scanning fluorimetry. Among these, AKR1C1 showed strong binding interactions with the tested bisphenols, resulting in reduced enzymatic activity. Pharmacological inhibition of AKR1C1 induced metabolic changes resembling those caused by BPS and its halogenated derivates. Overall, these findings identify AKR1C1 as a potential molecular target of halogenated bisphenols and demonstrate that these compounds disrupt adipocyte metabolism highlighting the importance of mechanistic data of chemical risk assessment.

The obesity epidemic is increasingly linked to environmental factors like endocrine disrupting chemicals (EDCs). Bisphenol A (BPA), a known EDC, has been suspected to be linked to adiposity through activation of peroxisome proliferator activated receptor gamma (PPARγ), a key regulator of adipogenesis. Though many BPA alternatives have been introduced as substitutes, their effects on metabolic health remain unclear. This study aimed to investigate the mechanistic interactions of 11 BPA alternatives with PPARγ and their adipogenic potential. Using a PPARγ reporter assay, we assessed the binding affinity and activation potential of BPA alternatives, followed by X-ray crystallography of two potent activators, 4-benzyloxyphenyl 4-hydroxyphenyl sulfone (BPS4BE) and bisphenol PH (BPPH). Additionally, adipogenesis was assessed via a human mesenchymal stem cells (hMSCs) differentiation assay. Results revealed that the alternatives BPPH and BPS4BE potently activated PPARγ (BMD20 (μM): 0.23 and 0.34 respectively). Both significantly induced adipogenesis and a positive correlation was found between PPARγ activation and adipogenic differentiation. Crystallography revealed distinct binding modes for BPPH and BPS4BE compared to rosiglitazone, indicating partial agonism. These findings raise significant concerns about the safety of BPA alternatives and underscore the need for structure-based risk assessment to ensure safer substitutes.

Pesticides are essential in modern agriculture but raise concerns about long-term metabolic effects, particularly through nuclear receptor activation. This study examines the impact of chronic exposure to a cocktail of five pesticides (dieldrin, propiconazole, boscalid, bupirimate, and pendimethalin) with or without tributyltin (TBT) on glucose and lipid metabolism in mice, focusing on the role of the constitutive androstane receptor (CAR) and pregnane X receptor (PXR). TBT was included for its strong affinity for RXR, a key heterodimerization partner of CAR and PXR, to assess whether RXR activation modulates or amplifies the metabolic effects of pesticide exposure. Results showed that pesticide exposure at NOAEL levels altered CAR and PXR dependent metabolic pathways. CAR knockout mice exhibited reduced free fatty acids and fasting glycemia, while PXR activation led to lower peak glycemia in oral glucose tolerance tests. Transcriptomic analysis identified disrupted pathways linked to glucose uptake and insulin sensitivity. These findings suggest that low-dose pesticide exposure can subtly affect metabolism via nuclear receptor interactions. The inclusion of TBT emphases RXR’s role in metabolic regulation. Overall, the study underscores the need to consider cocktail effects in risk assessment.

The glucocorticoid receptor (GR) belongs to the family of steroid receptors (SRs). These receptors regulate a vast selection of cell-, tissue-, and organism biology, and are also targets of endocrine disrupting chemicals warranting design and validation of in vitro assays. Here we report a "blinded" ring trial of an in vitro cell-based GR transactivation assay with four involved laboratories. The laboratories set up the assay and tested 34 selected chemicals with remarkably good concordance. There was agreement between all laboratories for the classification of activity in 97 % of the cases, and three or more laboratories were always in agreement. The within laboratory concordance was very high (99.6 %) with only one of all 272 triplicates deviating. The assay was, thus, deemed easily transferable and reproducible within and between laboratories, since they would arrive at the same qualitative results. Furthermore, for the chemicals with solid data regarding GR activation or inhibition, the laboratories arrived at the expected conclusion in all cases. Overall, the transfer and validation were successful, and the method is under evaluation to become an OECD test guideline. The method is expected to become valuable in tiered approaches for assessing chemicals or environmental samples together with other similar methods.

The action of environmental chemicals (ECs) on the mineralocorticoid receptor (MR) has been suggested to impair physiological processes regulated by this nuclear receptor. However, it remains understudied both as a target of ECs and with respect to potential species-specific differences. In this regard, we have developed reporter cell lines to identify the response to different steroids, ECs, and urban wastewater (WW) sample extracts of human MR (hMR) and zebrafish MR (zfMR). Most of the steroids had a higher efficacy on zfMR than hMR, while the ECs were antagonists to both hMR and zfMR, with a lower potency on the latter. Interestingly, WW sample extracts revealed the presence of MR activity with a greater activity on zfMR compared to hMR, suggesting the presence of steroids in WW. These screening tools have proven to be powerful tools for characterizing the interaction of chemicals with MRs and revealing their presence in environmental samples.

X15695 is a 2-phenyl-imidazo[1, 2α] pyridine derivative identified as an orally active, selective oestrogen receptor (ER) degrader that inhibits the proliferation of ER+ breast cancer cells. Here, we show that X15695 is an aryl hydrocarbon receptor (AHR) ligand that stabilises the AHR more efficiently than its classical ligand, indirubin. X15695 enables AHR to form a complex with the ER, promoting its proteasomal degradation. In the presence of oestradiol, X15695 outperforms the standard of care drug fulvestrant in suppressing the growth of ER+ breast cancer cells, either expressing the wild-type or clinically relevant ER mutant forms (Y537S and D538G), and of patient-derived xenograft organoids established from ER+ tumours. Using computational techniques, we discovered that a low pKa value resulting from electron-withdrawing substituents in the 2-phenyl-imidazo[1, 2α] pyridine compounds is a key feature that identify them as potent AHR ligands, leading to the potential discovery of additional derivatives for future therapeutic development. ### Competing Interest Statement J.S., M.P., S.B., N.J., S.B. and A.C.B.C. report patent application on the compounds (pending). The other authors declare no competing interests China Scholarship Council, 201807090113 Ministry of Health, RF-2021-12371961 AIRC, IG20061 Deutsche Forschungsgemeinschaft, 284178167

The literature documenting the value of drug-like molecules found in natural products is vast. Although many dietary and herbal remedies have been found to be effective for treating intestinal inflammation, the identification of their active components has lagged behind. In this study, we find that a major ginger component, furanodienone (FDN), is a selective pregnane X receptor (PXR) ligand with agonistic transcriptional outcomes. We show that FDN binds within a sub-pocket of the PXR ligand binding domain (LBD), with subsequent alterations in LBD structure. Using male mice, we show that orally provided FDN has potent PXR-dependant anti-inflammatory outcomes that are colon-specific. Increased affinity and target gene activation in the presence of synergistically acting agonists indicates further opportunities for augmenting FDN activity, efficacy and safety. Collectively, these results support the translational potential of FDN as a therapeutic agent for the treatment and prevention of colonic diseases. Here the authors discover that furanodienone (FDN), a small molecule derived from ginger, exhibits PXR agonist activity selective for the intestine and show that when FDN is administered orally in pharmacological doses ameliorates induced colitis in male mice.

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.

This study describes the production of a new biobased epoxy thermoset and its use with long hemp fibres to produce high-performance composites that are totally biobased. The synthesis of BioIgenox, an epoxy resin derived from a lignin biorefinery, and its curing process have been optimised to decrease their environmental impact. The main objective of this study is to characterise the rheology and kinetics of the epoxy system with a view to optimising the composite manufacturing process. Thus, the epoxy resin/hardener system was chosen considering the constraints imposed by the implementation of composites reinforced with plant fibres. The viscosity of the chosen mixture shows the compatibility of the formulation with the traditional implementation processes of the composites. In addition, unlike BPA—a precursor of diglycidyl ether of bisphenol A (DGEBA) epoxy resin—BioIgenox and its precursor do not have endocrine disrupting activities. The neat polymer and its unidirectional hemp fibre composite are characterised using three-point bending tests. Results measured for the fully biobased epoxy polymer show a bending modulus, a bending strength, a maximum strain at failure and a Tg of, respectively, 3.1 GPa, 55 MPa, 1.82% and 120 °C. These values are slightly weaker than those of the DGEBA-based epoxy material. It was also observed that the incorporation of fibres into the fully biobased epoxy system induces a decrease in the damping peak and a shift towards higher temperatures. These results point out the effective stress transfers between the hemp fibres and the fully biobased epoxy system. The high mechanical properties and softening temperature measured in this work with a fully biobased epoxy system make this type of composite a very promising sustainable material for transport and lightweight engineering applications.

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 ligandbinding PAS-B domain. Here, we report a 2.75 A ⠁ 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 (c) 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://crea

With an increasing demand for safe and natural products from both industries and consumers, paired with the recent ban of decried molecules (i.e. octinoxate, avobenzone or octocrylene) due to their high negative impact on humans and the environment (i.e. endocrine disruption, coral bleaching), safe bio-based alternatives are a necessary and promising surrogate to substitute current commercialized petroleum-based UV filters. In this context, a class of bio-based molecules, displaying interesting UV-B filtering properties and great photostability were developed from furfural and 5-hydroxymethylfurfural (HMF), using the Knoevenagel condensation with a set of green conditions to minimize the impact on environment. Furthermore, those furfural- and HMF-based molecules demonstrated antimicrobial properties as secondary activity, highly sought by industries. Some furan derivatives being recognized to exhibit toxicological risks, in silico and in vitro assays were conducted and demonstrated the absence of endocrine disruption activity for these new molecules.

ABSTRACT Ligand binding to estrogen receptors (ER), ERα and ERβ, controls the physiology of estrogen-responsive tissues through nuclear and extranuclear initiated pathways. We have found that ligands activate the extranuclear pathway by a mechanism involving proton transfer. The low affinity ligand and widespread endocrine disruptor Bisphenol-A (BPA) initiated nuclear and extranuclear actions. Concentrations similar to the receptor affinity initiated the nuclear pathway, whereas much lower concentrations initiated the extranuclear pathway. Experiments in different cell types using deuterated molecules of BPA and the ERβ agonist, diarylpropionitrile (DPN), indicated that a proton transfer from the hydroxyl groups to an amino acid acceptor within the ligand binding domain switches the extranuclear pathway. Activation of this pathway elicited a pattern of protein interactions with ERβ that regulated most of the important cellular functions. Thus, the extranuclear actions of ERβ are initiated by a molecular mechanism different from that of nuclear actions. This mechanism may work for other ligands and nuclear receptors.

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.

Abstract2,4-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.

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.

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,20β-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.