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We would like to thank the HBM4EU team including Petra Apel (UBA), Matthieu Meslin and Christophe Rousselle (Anses) for their scientific contribution, as well as the ANSES Working Group on endocrine disruption, and its scientific Anses coordinators François Pouzaud and Sandrine Charles.; International audience; The close structural analogy of bisphenol (BP) S with BPA, a recognized endocrine-disrupting chemical and a substance of very high concern in the European Union, highlights the need to assess the extent of similarities between the two compounds and carefully scrutinize BPS potential toxicity for human health. This analysis aimed to investigate human health toxicity data regarding BPS, to find a point of departure for the derivation of human guidance values. A systematic and transparent methodology was applied to determine whether European or international reference values have been established for BPS. In the absence of such values, the scientific literature on human health effects was evaluated by focusing on human epidemiological and animal experimental studies. The results were analyzed by target organ/system: male and female reproduction, mammary gland, neurobehavior, and metabolism/obesity. Academic experimental studies were analyzed and compared to regulatory data including subchronic studies and an extended one-generation and reproduction study. In contrast to the regulatory studies, which were performed at dose levels in the mg/kg bw/day range, the academic dataset on specific target organs or systems showed adverse effects for BPS at much lower doses (0.5-10 μg/kg bw/day). A large disparity between the lowest-observed-adverse-effect levels (LOAELs) derived from regulatory and academic studies was observed for BPS, as for BPA. Toxicokinetic data on BPS from animal and human studies were also analyzed and showed a 100-fold higher oral bioavailability compared to BPA in a pig model. The similarities and differences between the two bisphenols, in particular the higher bioavailability of BPS in its active (non-conjugated) form and its potential impact on human health, are discussed. Based on the available experimental data, and for a better human protection, we propose to derive human reference values for exposure to BPS from the N(L)OAELs determined in academic studies.

International audience

International audience; WHAT IS KNOWN ABOUT THE ACTION OF ENDOCRINE DISRUPTORS ON THE IMMUNE SYSTEM? The immune system (innate and adaptive immunity), involves different tissues and cell types to defend the body against external aggressions. This physiological mechanism involves some hormonal systems for its proper functioning. Moreover, new relationships between the immune system and endocrine processes have been recently described. Immunotoxicology is therefore a rapidly expanding field of research. Many environmental pollutants, such as organochlorine pesticides, polycyclic aromatic hydrocarbons or heavy metals, have an impact on the immune response leading either to a deficiency or a hyperactivation (autoimmune disease, allergy). More recently, other endocrine disruptors such as organofluorines are suspected to be immunotoxic. Low-level exposure to pollutants in the general population probably does not explain the development of all the pathologies, but it sentisizes organisms to the development of these pathologies, weakening certain key processes of the immune system.

Development of the central nervous system (CNS) relies on many endogenous factors. Thyroid hormones and sex steroid hormones are among the most studied endocrine systems for their regulation of neural functions, since early embryonic stages of development. Environmental exposure to low doses of chemicals is largely documented. The hypothesis that exposure to some of these molecules with endocrine disrupting activity, likely interfering with the production of these hormones and/or their underlying neural mechanisms is therefore plausible and supported by numerous studies. After having recalled the formation of the hypothalamo-hypophyseal axes, and the importance of thyroid and sexual hormones in neurodevelopment, we will present two examples of substances (BPA, PCBs) and their effects on brain development., Le développement du système nerveux central (SNC) dépend de nombreux facteurs endogènes. Les hormones thyroïdiennes et les hormones stéroïdes sexuelles figurent parmi les systèmes endocriniens les plus étudiés pour leurs effets neuraux, et ce dès les stades embryonnaires de développement. L'exposition environnementale à de faibles doses de substances chimiques est largement documentée. L'hypothèse que certaines de ces substances, appelées perturbateurs endocriniens, puissent interférer avec la production de ces hormones et/ou de leurs mécanismes au niveau central est donc plausible et soutenue par une multitude de travaux. Après avoir rappelé la formation des axes hypothalamo-hypophysaires et l'importance des hormones thyroïdiennes et sexuelles dans le neurodéveloppement, nous présenterons deux exemples de substances (bisphénol A, polychlorobiphényles) et leurs effets sur le développement cérébral.

International audience; WHAT ARE WE TALKING ABOUT AND WHAT NEW MECHANISMS OF TOXICITY DO THEY BRING INTO PLAY? Endocrine disruptors (EDs) are chemicals that can interfere with the functioning of the endocrine system and thereby cause an adverse event. They are suspected of being toxic to the environment and to humans and to increase the risk of developing pathologies such as cancer, metabolic, neurological or immune diseases. These substances are defined by their mechanisms of action which are now described as "Adverse Outcome Pathways" or AOPs. AOPs correspond to a logical chain of events leading to an adverse effect. EDs have properties which have modified our concepts in toxicology, in particular due to the low-dose effects of certain EDs, the possible effects of ED mixtures and finally their delayed effects over time, sometimes with years or decades that separate exposure and impact. Epigenetic mechanisms probably explain these delayed effects.

International audience; Endocrine disrupting chemicals (EDCs) are linked to the worldwide epidemic incidence of metabolic disorders and fatty liver diseases, which affects quality of life and represents a high economic cost to society. Energy homeostasis exhibits strong sexual dimorphic traits, and metabolic organs respond to EDCs depending on sex, such as the liver, which orchestrates both drug elimination and glucose and lipid metabolism. In addition, fatty liver diseases show a strong sexual bias, which in part could also originate from sex differences observed in gut microbiota. The aim of this review is to highlight significant differences in endocrine and metabolic aspects of the liver, between males and females throughout development and into adulthood. It is also to illustrate how the male and female liver differently cope with exposure to various EDCs such as bisphenols, phthalates and persistent organic chemicals in order to draw attention to the need to include both sexes in experimental studies. Interesting data come from analyses of the composition and diversity of the gut microbiota in males exposed to the mentioned EDCs showing significant correlations with hepatic lipid accumulation and metabolic disorders but information on females is lacking or incomplete. As industrialization increases, the list of anthropogenic chemicals to which humans will be exposed will also likely increase. In addition to strengthening existing regulations, encouraging populations to protect themselves and promoting the substitution of harmful chemicals with safe products, innovative strategies based on sex differences in the gut microbiota and in the gut-liver axis could be optimistic outlook.

BACKGROUND: The assessment of human exposure to fast-elimination endocrine disruptors (ED) such as phthalates, bisphenols or pesticides is usually based on urinary biomarkers. The variability of biomarkers concentration, due to rapid elimination from the body combined with frequent exposure is however pointed out as a major limitation to exposure assessment. Other matrices such as hair, less sensitive to short-term variations in the exposure, have been proposed as possible alternatives. Nevertheless, no study compared the information obtained from hair and urine respectively in a follow-up allowing to assess biomarkers variability over time in these two matrices, and to compare the correlation between them. METHODS: In the present study, hair and urine samples were collected from 16 volunteers over a 6 months follow-up. All in all, 92 hair samples and 805 urines samples were collected and analyzed for the presence of 16 phthalate metabolites, 4 bisphenols and 8 pesticides/metabolites. RESULTS: All the biomarkers analyzed were detected in at least one of the two matrices. 21 biomarkers were more frequently detected in hair, 6 in urine, and 1 was equivalent. Biomarkers intraclass correlation coefficients (ICC) ranged from 0.1 to 0.8 (ten above 0.4) in hair, and from 0.09 to 0.51 in urine (two above 0.4). The concentrations of biomarkers in hair and urine were significantly correlated for only one compound. CONCLUSION: This study highlights the complexity of assessing exposure to fast-elimination ED and suggests considering with caution the specificity of the matrix in data interpretation. The results document the respective advantages and limitations of urine and hair, and provide new insight in the understanding of the information provided by these biological matrices and their relevance for the assessment of human exposure to fast elimination contaminants. CAPSULE: 92 hair and 805 urine samples collected from 16 volunteers over 6 months, tested for phthalate metabolites, bisphenols and pesticides. 19 biomarkers (in hair) and 24 (in urine) were detected in >50% of the samples.

International audience; Since reports published in 2015 and 2016 identified 15 probable exposure-outcome associations, there has been an increase in studies in humans of exposure to endocrine-disrupting chemicals (EDCs) and a deepened understanding of their effects on human health. In this Series paper, we have reviewed subsequent additions to the literature and identified new exposure-outcome associations with substantial human evidence. Evidence is particularly strong for relations between perfluoroalkyl substances and child and adult obesity, impaired glucose tolerance, gestational diabetes, reduced birthweight, reduced semen quality, polycystic ovarian syndrome, endometriosis, and breast cancer. Evidence also exists for relations between bisphenols and adult diabetes, reduced semen quality, and polycystic ovarian syndrome; phthalates and prematurity, reduced anogenital distance in boys, childhood obesity, and impaired glucose tolerance; organophosphate pesticides and reduced semen quality; and occupational exposure to pesticides and prostate cancer. Greater evidence has accumulated than was previously identified for cognitive deficits and attention-deficit disorder in children following prenatal exposure to bisphenol A, organophosphate pesticides, and polybrominated flame retardants. Although systematic evaluation is needed of the probability and strength of these exposure-outcome relations, the growing evidence supports urgent action to reduce exposure to EDCs.

International audience; The purpose of this project report is to introduce the European "GOLIATH" project, a new research project which addresses one of the most urgent regulatory needs in the testing of endocrine-disrupting chemicals (EDCs), namely the lack of methods for testing EDCs that disrupt metabolism and metabolic functions. These chemicals collectively referred to as "metabolism Int. Int. J. Mol. Sci. 2020, 21, 3480 2 of 18 disrupting compounds" (MDCs) are natural and anthropogenic chemicals that can promote metabolic changes that can ultimately result in obesity, diabetes, and/or fatty liver in humans. This project report introduces the main approaches of the project and provides a focused review of the evidence of metabolic disruption for selected EDCs. GOLIATH will generate the world's first integrated approach to testing and assessment (IATA) specifically tailored to MDCs. GOLIATH will focus on the main cellular targets of metabolic disruption-hepatocytes, pancreatic endocrine cells, myocytes and adipocytes-and using an adverse outcome pathway (AOP) framework will provide key information on MDC-related mode of action by incorporating multi-omic analyses and translating results from in silico, in vitro, and in vivo models and assays to adverse metabolic health outcomes in humans at real-life exposures. Given the importance of international acceptance of the developed test methods for regulatory use, GOLIATH will link with ongoing initiatives of the Organisation for Economic Development (OECD) for test method (pre-)validation, IATA, and AOP development.

International audience; Obesity and metabolic-related diseases, among which diabetes, are prominent public health challenges of the 21st century. It is now well acknowledged that pollutants are a part of the equation, especially endocrine-disrupting chemicals (EDCs) that interfere with the hormonal aspect. The aim of the review is to focus on adipose tissue, a central regulator of energy balance and metabolic homeostasis, and to highlight the significant differences in the endocrine and metabolic aspects of adipose tissue between males and females which likely underlie the differences of the response to exposure to EDCs between the sexes. Moreover, the study also presents an overview of several mechanisms of action by which pollutants could cause adipose tissue dysfunction. Indeed, a better understanding of the mechanism by which environmental chemicals target adipose tissue and cause metabolic disturbances, and how these mechanisms interact and sex specificities are essential for developing mitigating and sex-specific strategies against metabolic diseases of chemical origin. In particular, considering that a scenario without pollutant exposure is not a realistic option in our current societies, attenuating the deleterious effects of exposure to pollutants by acting on the gut-adipose tissue axis may constitute a new direction of research.

The research was supported by funding from the European Unions ' Horizon 2020 Research and Innovation Programme under grant agreements No 733032 (HBM4EU) , No. 859891 (PRORISK) , and No. 857560 (CETOCOEN EXCELLENCE Teaming 2 project) . The RECETOX Research infrastructure was further supported by the Czech Ministry of Education, Youth and Sports (LM2018121; CZ02.1.01/0.0/0.0/18_046/0015975) ., Background and aim: Endocrine disrupting chemicals (EDCs) constitute a major public health concern because they can induce a large spectrum of adverse effects by interfering with the hormonal system. Rapid identification of potential EDCs using in vitro screenings is therefore critical, particularly for chemicals of emerging concerns such as replacement flame retardants (FRs). The review aimed at identifying (1) data gaps and research needs regarding endocrine disrupting (ED) properties of replacement FRs and (2) potential EDCs among these emerging chemicals. Methods: A systematic search was performed from open literature and ToxCast/Tox21 programs, and results from in vitro tests on the activities of 52 replacement FRs towards five hormone nuclear receptors (NRs) associated with reproductive outcomes (estrogen, androgen, glucocorticoid, progesterone, and aryl hydrocarbon receptors) were compiled and organized into tables. Findings were complemented with information from structure-based in silico model predictions and in vivo information when relevant. Results: For the majority of the 52 replacement FRs, experimental in vitro data on activities towards these five NRs were either incomplete (15 FRs) or not found (24 FRs). Within the replacement FRs for which effect data were found, some appeared as candidate EDCs, such as triphenyl phosphate (TPhP) and tris(1,3-dichloropropyl) phosphate (TDCIPP). The search also revealed shared ED profiles. For example, anti-androgenic activity was reported for 19 FRs and predicted for another 21 FRs. Discussion: This comprehensive review points to critical gaps in knowledge on ED potential for many replacement FRs, including chemicals to which the general population is likely exposed. Although this review does not cover all possible characteristics of ED, it allowed the identification of potential EDCs associated with reproductive outcomes, calling for deeper evaluation and possibly future regulation of these chemicals. By identifying shared ED profiles, this work also raises concerns for mixture effects since the population is co-exposed to several FRs and other chemicals., European Commission 733032 859891 857560, Ministry of Education, Youth & Sports - Czech Republic LM2018121 CZ02.1.01/0.0/0.0/18_046/0015975

International audience; Many rodent studies and a few non-human primate data report impairments of spatial and non-spatial memory induced by exposure to bisphenol A (BPA), which are associated with neural modifications, particularly in processes involved in synaptic plasticity. BPA-induced alterations involve disruption of the estrogenic pathway as established by reversal of BPA-induced effects with estrogenic receptor antagonist or by interference of BPA with administered estradiol in ovariectomized animals. Sex differences in hormonal impregnation during critical periods of development and their influence on maturation of learning and memory processes may explain the sexual dimorphism observed in BPA-induced effects in some studies. Altogether, these data highly support the plausibility that alteration of learning and memory and synaptic plasticity by BPA is essentially mediated by disturbance of the estrogenic pathways. As memory function in humans involves similar signaling pathways, this mode of action of BPA has the potential to alter human cognitive abilities.

International audience; The extensive database on BPA provides strong evidence of its adverse effects on reproductive, neurobehavioural, metabolic functions and mammary gland. Disruption of estrogenic pathway is central in the mediation of these effects although other modes of action may be involved. BPA has a weak affinity for ERα/β but interaction with extranuclearly located pathways activated by estrogens such as ERRγ and GPER reveals how BPA can act at low doses. The effects are observed later in life after developmental exposure and are associated with pathologies of major societal concern in terms of severity, incidence, impact on quality of life, burden on public health system. The complexity of the dose response raise uncertainties on the possibility to establish safe levels and the scope of ED-mediated effects of BPA may be wider. These concerns fulfill the requirements for ED identification under REACH regulation.

International audience; BPA is one of the most investigated substances for its endocrine disruptor (ED) properties and it is at the same time in the center of many ED-related controversies, the analysis on how BPA fits to the regulatory identification as an ED is a challenge in terms of methodology. It is also a great opportunity to test the regulatory framework with a uniquely data-rich substance and learn valuable lessons for future cases. From this extensive database, it was considered important to engage in a detailed analysis so as to provide specific and strong evidences of ED while reflecting accurately the complexity of the response as well the multiplicity of adverse effects. An appropriate delineation of the scope of the analysis was therefore critical. Four effects namely, alterations of estrous cyclicity, mammary gland development, brain development and memory function, and metabolism, were considered to provide solid evidence of ED-mediated effects of BPA.

International audience; Proper cyclicity is essential to reach successful optimal fertility. In rats and mice, BPA exposure is repeatedly and reliably reported to show an adverse effect on the estrous cycle after exposures at different life stages. In humans, a possible association between modifications of menstrual cycle characteristics (e.g. length of the cycle, duration of menstrual bleeding) and sub-fecundity or spontaneous abortion has been observed. Alterations of ovarian cyclicity can therefore be definitely considered as an adverse health outcome. As a prerequisite for the EU REACH regulation to identify a substance as an endocrine disruptor and a SVHC, the proof has to be established that the substance can have deleterious health effects resulting from an endocrine mode of action. This review provides an overview of the currently available data allowing to conclude that the adverse effects of BPA exposure on ovarian cyclicity is mediated by an endocrine mode of action.

International audience; Endocrine disrupting chemicals and especially oestrogen receptor (ER) agonists have been extensively studied over the years due to their potential effects on sexual development and reproduction in vertebrates, notably fish. As ER agonists can exist as complex mixtures in the aquatic environment, evaluating the impact of combined exposure on oestrogenic effects has become increasingly important. Use of predictive models such as concentration addition (CA) and independent action (IA) has allowed assessment of combined estrogenic effects of complex multi-compound mixtures of ER agonists in various fish in vitro and in vivo experimental models. The present work makes use of a transgenic zebrafish strain, tg(cyp19a1b-GFP), which expresses the green fluorescent protein (GFP) under the control of the cyp19a1b (brain aromatase or aromatase B) gene to determine the oestrogenic potency of ER agonists alone or in mixtures. In these studies, tg(cyp19a1b-GFP) zebrafish embryos were exposed for four days (from one to five days post fertilization) to five different oestrogenic chemicals; 17α-ethinylestradiol (EE2), 17β-estradiol (E2), estrone (E1), bisphenol A (BPA) and 4-tert-octylphenol (OP), and three mixtures of up to four of these compounds. The mixture of BPA, OP and E2 was also tested with primary cultures of rainbow trout hepatocytes by analysing the ER-mediated induction of the oestrogenic biomarker vitellogenin in order to compare the performance of the two methods for assessing oestrogenic effects of complex mixtures. The three tested mixtures were predominantly acting in an additive manner on the expression of GFP. Additivity was indicated by the overlap of the 95% confidence interval of the concentration response curves for the observed data with the CA and IA prediction models, and model deviation ratios within a factor of two for a majority of the mixture concentrations. However, minor deviations determined as more than additive effects for the mixture of EE2, E1 and E2 and less than additive effects for the mixture of BPA, OP, EE2 and E1 were observed at the higher mixture concentrations tested. The successful prediction of additivity by CA and IA in tg(cyp19a1b-GFP) zebrafish embryos and deviations at high mixture concentrations seemed to correspond well to results obtained in the rainbow trout hepatocyte assay. The present results clearly show the usefulness of combining predictive modelling and use of in vitro bioassays for rapid screening of oestrogenic effects of complex mixtures and environmental samples.

Objective To study associations between prenatal exposure to phthalates and fetal and postnatal growth up to age 5 years in male offspring. Methods Eleven phthalate metabolites were quantified in spot maternal urine samples collected during gestation among 520 women of the EDEN mother-child cohort who gave birth to a boy. Fetal growth was assessed from repeated ultrasound measurements and measurements at birth. We used repeated measures of weight and height in the first 5 years of life to model individual postnatal growth trajectories. We estimated adjusted variations in pre and postnatal growth parameters associated with an interquartile range increase in ln-transformed phthalate metabolite concentrations. Results Monocarboxyisononyl phthalate (MCNP) was positively associated with femoral length during gestation and length at birth. High molecular weight phthalate metabolites were negatively associated with estimated fetal weight throughout pregnancy. Monoethyl phthalate (MEP) showed positive association with weight growth velocity from two to five years and with body mass index at five years (β=0.17 kg/m2, 95% confidence interval, 0.04, 0.30). Conclusions We highlighted associations between gestational exposure to some phthalates and growth in boys. The positive association between MEP and postnatal growth in boys was also reported in several previous human studies.

International audience; The number of environmental chemical contaminants suspected to act as endocrine disruptor compounds by interacting with estrogen receptor (ER) signaling pathway has been continuously increasing. To study such interaction, the use of stable reporter gene assays is relevant, but species-specific in vitro screening assays are still lacking to address hazard assessment of estrogenic chemicals in aquatic vertebrates. Here, we describe the development of stable reporter gene assays based on stable expression of subtypes of zebrafish ER (zfERα, zfERβ1, and zfERβ2) coupled to estrogen response element-driven luciferase in a zebrafish liver (ZFL) cell line. The three established cell models, named ZELH-zfERα, ZELH-zfERβ1, and ZELH-zfERβ2, expressed stable and significant basal luciferase signal, which was induced by 17β-estradiol (E2) in a sensitive and dose-response manner at EC(50)s of 0.2, 0.03, and 0.05 nM, respectively. In addition, E2 significantly altered cell proliferation in ZELH-zfERα and ZELH-zfERβ2 cells, but not in parental ZFL and ZELH-zfERβ1 cells, suggesting a functionality of these two receptors to modulate endogenous gene expression in the transfected clones. The screening of various xenoestrogens from different classes in the three models resulted in different luciferase response patterns. Natural and synthetic estrogens and 1,1,1-trichloro-2-(2 chlorophenyl)-2-(4-chlorophenyl)ethane were active at lower concentrations in ZELH-zfERβ1 and ZELH-zfERβ2 than in ZELH-zfERα cells, whereas genistein and zearalenone metabolites as well as three benzophenone derivatives preferentially activated zfERα. Altogether, the newly established models provide specific and convenient in vitro tool for comparative assessment of zfERs selective activation by chemicals within ZFL cell context.

International audience; Benzophenone (BP) derivatives, BP1 (2,4-dihydroxybenzophenone), BP2 (2,2',4,4'-tetrahydroxybenzophenone), BP3 (2-hydroxy-4-methoxybenzophenone), and THB (2,4,4'-trihydroxybenzophenone) are UV-absorbing chemicals widely used in pharmaceutical, cosmetics, and industrial applications, such as topical sunscreens in lotions and hair sprays to protect skin and hair from UV irradiation. Studies on their endocrine disrupting properties have mostly focused on their interaction with human estrogen receptor alpha (hERalpha), and there has been no comprehensive analysis of their potency in a system allowing comparison between hERalpha and hERbeta activities. The objective of this study was to provide a comprehensive ER activation profile of BP derivatives using ER from human and fish origin in a battery of in vitro tests, i.e., competitive binding, reporter gene based assays, vitellogenin (Vtg) induction in isolated rainbow trout hepatocytes, and proliferation based assays. The ability to induce human androgen receptor (hAR)-mediated reporter gene expression was also examined. All BP derivatives tested except BP3 were full hERalpha and hERbeta agonists (BP2>THB>BP1) and displayed a stronger activation of hERbeta compared with hERalpha, the opposite effect to that of estradiol (E2). Unlike E2, BPs were more active in rainbow trout ERalpha (rtERalpha) than in hERalpha assay. All four BP derivatives showed anti-androgenic activity (THB>BP2>BP1>BP3). Overall, the observed anti-androgenic potencies of BP derivatives, together with their proposed greater effect on ERbeta versus ERalpha activation, support further investigation of their role as endocrine disrupters in humans and wildlife.

Many endocrine disruptors are found in our environment. They act on hormonal receptors, on their action and synthesis. They may alter neuronal transmission and neuronal formation. A lot of neurodevelopmental diseases have a growing prevalence, raising many questions about a possible association with endocrine disruptors. One of their action mechanisms may be an effect on DNA methylation of the developing brain.Objectives: To study neurodevelopmental characteristics and the epigenetic signature of patients exposed in utero to diethylstilbestrol. To identify differentially methylated regions in neurodevelopmental related genes, which could lead to a psychiatric vulnerability.Materials and Methods: We recruited 75 siblings from 31 families, in which at least one member was exposed in utero to diethylstilbestrol. A psychiatric evaluation was assessed with standardized questionnaires. We used the Infinium HumanMethylation450 BeadChip and analyzed the methylation variations of 411 947 CpG loci.Results: We found clinical differences in non-specific psychiatric symptoms and in neurological soft signs, such as lateralization, associated with the exposure. We found several biological pathways in which methylation modifications were associated to diethylstilbestrol exposure. These pathways are implicated in neurodevelopmental pathways, general metabolism and oncogenesis mostly.Conclusion : Prenatal diethylstilbestrol exposure seems associated with non-specific psychiatric symptoms, lateralization abnormalities and methylation alterations in genes that participate to pathways known to be involved in psychiatric diseases, notably in neurotransmitters signalization.; De nombreux perturbateurs endocriniens sont retrouvés dans notre environnement. Ils interfèrent sur l’action des récepteurs hormonaux. Plusieurs travaux argumentent qu’ils altéreraient la formation des réseaux neuronaux. Un parallèle a été évoqué avec l’augmentation de la prévalence des troubles neurodéveloppementaux. Un mécanisme d’action évoqué est un effet sur la méthylation de l’ADN dans le cerveau en maturation.Objectifs : Étudier les caractéristiques neurodéveloppementales et la signature épigénétique des patients exposés in utero au diéthylstilbestrol. Identifier des régions de méthylation différentielles sur des gènes impliqués dans le neurodéveloppement, pouvant mener à une vulnérabilité psychiatrique.Matériels et méthodes: Nous avons recruté 75 frères et sœurs issus de 31 familles dont au moins un membre fut exposé in utero au diéthylstilbestrol. Une évaluation neuropsychiatrique a été réalisée. Nous avons utilisé une puce de méthylation et avons analysé les variations de méthylation de 411 947 CpG.Résultats: Nous avons observé des différences significatives au niveau de symptômes psychiatriques aspécifiques et de signes neurologiques mineurs, tels que la latéralisation, associées à l’exposition. Plusieurs réseaux biologiques présentant des modifications de méthylation sont associés à l’exposition. Ils concernent les fonctions neurodéveloppementales, le métabolisme et l’oncogenèse.Conclusion : L’exposition au diéthylstilbestrol paraît associée à des profils cliniques aspécifiques, à des troubles de la latéralisation, et à des altérations de méthylation au sein de réseaux biologiques spécifiques.

On July 10, 2010, the beautiful City of Rouen (France) hosted what we believe was the First International Symposium on the “Neuroendocrine Effects of Endocrine Disruptors (NEED).” Several laborator...

International audience; Tributyltin (TBT) and triphenyltin (TPT) are emblematic endocrine disruptors, which have been mostly studied in gonochoric prosobranchs. Although both compounds can simultaneously occur in the environment, they have mainly been tested separately for their effects on snail reproduction. Because large discrepancies in experimental conditions occurred in these tests, the present study aimed to compare the relative toxicity of TBT and TPT under similar laboratory conditions in the range of 0 ng Sn/L to 600 ng Sn/L. Tests were performed on the simultaneous hermaphrodite Lymnaea stagnalis, a freshwater snail in which effects of TPT were unknown. Survival, shell length, and reproduction were monitored in a 21-d semistatic test. Frequency of abnormal eggs was assessed as an additional endpoint. Triphenyltin hampered survival while TBT did not. Major effects on shell solidity and reproduction were observed for both compounds, reproductive outputs being more severely hampered by TBT than by TPT. Considering the frequency of abnormal eggs allowed increasing test sensitivity, because snail responses to TBT could be detected at concentrations as low as 19 ng Sn/L. However, the putative mode of action of the 2 compounds could not be deduced from the structure of the molecules or from the response of apical endpoints. Sensitivity of L. stagnalis to TBT and TPT was compared with the sensitivity of prosobranch mollusks with different habitats and different reproductive strategies.

International audience; The purpose of this study was to investigate the impact of temperature on the release of PET-bottle constituents into water and to assess the potential health hazard using in vitro bioassays with bacteria and human cell lines. Aldehydes, trace metals and other compounds found in plastic packaging were analysed in PET-bottled water stored at different temperatures: 40, 50, and 60 °C. In this study, temperature and the presence of CO2 increased the release of formaldehyde, acetaldehyde and antimony (Sb). In parallel, genotoxicity assays (Ames and micronucleus assays) and transcriptional-reporter gene assays for estrogenic and anti-androgenic activity were performed on bottled water extracts at relevant consumer exposure levels. As expected, and in accordance with the chemical formulations specified for PET bottles, neither phthalates nor UV stabilisers were present in the water extracts. However, 2,4-di-tert-butylphenol, a degradation compound of phenolic antioxidants, was detected. In addition, an intermediary monomer, bis(2-hydroxyethyl)terephthalate, was found but only in PET-bottled waters. None of the compounds are on the positive list of EU Regulation No. 10/2011. However, the PET-bottled water extracts did not induce any cytotoxic, genotoxic or endocrine-disruption activity in the bioassays after exposure.

International audience; Bisphenol A (BPA) is an endocrine-disrupting chemical that leaches from polycarbonate plastics that consequently leads to low-dose human exposure. In addition to its known xenoendocrine action, BPA exerts a wide variety of metabolic effects, but no data are available on its actions on the functions of liver mitochondrial. To assess these effects, HepG2 cells were exposed to BPA (10(-4)-10(-12)M) and physiological parameters were measured by flow cytometry. We demonstrated a significant mitochondrial dysfunction including ROS production, ΔΨ(M) hyperpolarization, lipid accumulation, lipoperoxidation and the release of pro-inflammatory cytokines. In conclusion, we showed that low concentrations of BPA promote lipid accumulation in hepatic cells triggered by disturbances in mitochondrial function, alterations in lipid metabolism and by inflammation that can therefore contribute to steatosis.

International audience; More than half the pregnant women in the Western world report taking mild analgesics. These pharmaceutical compounds have been associated with congenital cryptorchidism in humans, the best-known risk factor for low semen quality and testicular germ cell cancer. Furthermore, some of these mild analgesics exert potent anti-androgenic effects in the male rat and several endocrine-disrupting compounds, known to alter masculinization, have also been shown to be potent inhibitors of prostaglandin (PG) synthesis similar to mild analgesics. Using a 3-day ex vivo organotypic model system based on gestational day 14.5 rat testes, we herein show that testosterone production was inhibited by paracetamol, at doses of 0.1 μm to 100 μm. Similar results were obtained for aspirin (1-100 μm) and indomethacin (10 μm). The production of the other Leydig cell hormone, Insl3, was not disrupted by exposure to paracetamol. Investigations of the gross anatomy of the testis as well as Leydig cells number and rate of gonocyte apoptosis after the 3 days of ex vivo differentiation showed no significant effect of the analgesics tested compared with controls. These data indicate therefore that mild analgesics specifically inhibit testosterone production in rat foetal testes in vitro and that these compounds had no effect on gonocyte survival. Parallel determinations of prostaglandin D2 (PGD2) production indicated that the effects of paracetamol and aspirin on PGD2 and testosterone were not connected, whereas the effects of indomethacin were correlated. We conclude that mild analgesics exert direct and specific anti-androgenic effects in rat foetal testis in our experimental setup and that the mechanism of action is probably uncoupled from the inhibition of PG synthesis.

International audience; The presence of dyes in wastewater effluent of textile industry is well documented. In contrast, the endocrine disrupting effects of these dyes and wastewater effluent have been poorly investigated. Herein, we studied twenty-three commercial dyes, usually used in the textile industry, and extracts of blue jean textile wastewater samples were evaluated for their agonistic and antagonistic estrogen activity. Total estrogenic and anti-estrogenic activities were measured using the Yeast Estrogen Screen bioassay (YES) that evaluates estrogen receptor binding-dependent transcriptional and translational activities. The estrogenic potencies of the dyes and wastewater samples were evaluated by dose-response curves and compared to the dose-response curve of 17β-estradiol (E2), the reference compound. The dose-dependent anti-estrogenic activities of the dyes and wastewater samples were normalized to the known antagonistic effect of 4-hydroxytamoxifen (4-OHT) on the induction of the lac Z reporter gene by E2. About half azo textile dyes have anti-estrogenic activity with the most active being Blue HFRL. Most azo dyes however have no or weak estrogenic activity. E2/dye or E2/waste water ER competitive binding assays show activity of Blue HFRL, benzopurpurine 4B, Everzol Navy Blue FBN, direct red 89 BNL 200% and waste water samples indicating a mechanism of action common to E2. Our results indicate that several textile dyes are potential endocrine disrupting agents. The presence of some of these dyes in textile industry wastewater may thus impact the aquatic ecosystem.

International audience; Some environmental contaminants interact with hormones and may exert adverse consequences as a result of their actions as endocrine disrupting chemicals (EDCs). Exposure in people is typically a result of contamination of the food chain, inhalation of contaminated house dust or occupational exposure. EDCs include pesticides and herbicides (such as dichlorodiphenyl trichloroethane or its metabolites), methoxychlor, biocides, heat stabilisers and chemical catalysts (such as tributyltin), plastic contaminants (e.g. bisphenol A), pharmaceuticals (i.e. diethylstilbestrol; 17α-ethinylestradiol) or dietary components (such as phytoestrogens). The goal of this review is to address the sources, effects and actions of EDCs, with an emphasis on topics discussed at the International Congress on Steroids and the Nervous System. EDCs may alter reproductively-relevant or nonreproductive, sexually-dimorphic behaviours. In addition, EDCs may have significant effects on neurodevelopmental processes, influencing the morphology of sexually-dimorphic cerebral circuits. Exposure to EDCs is more dangerous if it occurs during specific 'critical periods' of life, such as intrauterine, perinatal, juvenile or puberty periods, when organisms are more sensitive to hormonal disruption, compared to other periods. However, exposure to EDCs in adulthood can also alter physiology. Several EDCs are xenoestrogens, which can alter serum lipid concentrations or metabolism enzymes that are necessary for converting cholesterol to steroid hormones. This can ultimately alter the production of oestradiol and/or other steroids. Finally, many EDCs may have actions via (or independent of) classic actions at cognate steroid receptors. EDCs may have effects through numerous other substrates, such as the aryl hydrocarbon receptor, the peroxisome proliferator-activated receptor and the retinoid X receptor, signal transduction pathways, calcium influx and/or neurotransmitter receptors. Thus, EDCs, from varied sources, may have organisational effects during development and/or activational effects in adulthood that influence sexually-dimorphic, reproductively-relevant processes or other functions, by mimicking, antagonising or altering steroidal actions.

Background Environmental epidemiology and biomonitoring studies typically rely on biological samples to assay the concentration of non-persistent exposure biomarkers. Between-participant variations in sampling conditions of these biological samples constitute a potential source of exposure misclassification. Few studies attempted to correct biomarker levels for this error. We aimed to assess the influence of sampling conditions on concentrations of urinary biomarkers of select phenols and phthalates, two widely-produced families of chemicals, and to standardize biomarker concentrations on sampling conditions. Methods Urine samples were collected between 2002 and 2006 among 287 pregnant women from Eden and Pélagie cohorts, from which phthalates and phenols metabolites levels were assayed. We applied a 2-step standardization method based on regression residuals. First, the influence of sampling conditions (including sampling hour, duration of storage before freezing) and of creatinine levels on biomarker concentrations were characterized using adjusted linear regression models. In the second step, the model estimates were used to remove the variability in biomarker concentrations due to sampling conditions and to standardize concentrations as if all samples had been collected under the same conditions (e.g., same hour of urine collection). Results Sampling hour was associated with concentrations of several exposure biomarkers. After standardization for sampling conditions, median concentrations differed by ‒ 38 % for 2,5-dichlorophenol to +80 % for a metabolite of diisodecyl phthalate. However, at the individual level, standardized biomarker levels were strongly correlated (correlation coefficients above 0.80) with unstandardized measures. Conclusions Sampling conditions, such as sampling hour, should be systematically collected in biomarker-based studies, in particular when the biomarker half-life is short. The 2-step standardization method based on regression residuals that we proposed in order to limit the impact of heterogeneity in sampling conditions could be further tested in studies describing levels of biomarkers or their influence on health.

International audience; Phthalates are abundantly produced plasticizers, and di(ethylhexyl) phthalate (DEHP) is the most widely used derivative in various consumer products and medical devices. Animal studies show that DEHP and various other phthalates cause reproductive and developmental toxicity. Although the evidences are limited, it seems reasonable that DEHP may have a potential for similar adverse effects in humans. Such concerns are increasing, particularly for the developing reproductive system of male infants and children. By taking into account the essentiality of selenium (Se) in testicular structure and functions and the high prevalence of inadequate Se intake in various part of the world, this study was designed to investigate the testicular toxicity of DEHP in Se-deficient male rats and to examine the possible preventive effects of Se supplementation on phthalate toxicity. Se deficiency was generated by feeding 3-week-old Sprague-Dawley rats with a ≤0.05 Se mg/kg diet for 5 weeks. Supplementation groups were on a 1 mg Se/kg diet, and DEHP-treated groups received a 1,000 mg/kg dose by gavage during the last 10 days of the feeding period. Testicular histopathology, sperm count and motility, and sperm morphology were examined, and plasma levels of sex hormones were measured. Toxicity and antiandrogenic effects of DEHP were evidenced by disturbed testicular histology and spermatogenesis, diminished testosterone, leutinizing hormone (LH) and follicle stimulating hormone (FSH) levels, and sperm motility. The effects of DEHP were much more pronounced in Se-deficient rats, whereas Se supplementation was found to be protective, reflecting its regulating role in cellular redox equilibrium.

Background The plastic monomer and plasticizer bisphenol A (BPA), used for manufacturing polycarbonate plastic and epoxy resins, is produced at over 2.5 million metric tons per year. Concerns have been raised that BPA acts as an endocrine disruptor on both developmental and reproductive processes and a large body of evidence suggests that BPA interferes with estrogen and thyroid hormone signaling. Here, we investigated BPA effects during embryonic development using the zebrafish and Xenopus models. Results We report that BPA exposure leads to severe malformations of the otic vesicle. In zebrafish and in Xenopus embryos, exposure to BPA during the first developmental day resulted in dose-dependent defects in otolith formation. Defects included aggregation, multiplication and occasionally failure to form otoliths. As no effects on otolith development were seen with exposure to micromolar concentrations of thyroid hormone, 17-ß-estradiol or of the estrogen receptor antagonist ICI 182,780 we conclude that the effects of BPA are independent of estrogen receptors or thyroid-hormone receptors. Na+/K+ ATPases are crucial for otolith formation in zebrafish. Pharmacological inhibition of the major Na+/K+ ATPase with ouabain can rescue the BPA-induced otolith phenotype. Conclusions The data suggest that the spectrum of BPA action is wider than previously expected and argue for a systematic survey of the developmental effects of this endocrine disruptor.

International audience; Estrogens and estrogen receptors (ER) are key actors in the control of differentiation and survival and act on extrareproductive tissues such as brain. Thus, estrogens may display neuritogenic effects during development and neuroprotective effects in the pathophysiological context of brain ischemia and neurodegenerative pathologies like Alzheimer's disease or Parkinson's disease. Some of these effects require classical transcriptional "genomic" mechanisms through ER, whereas other effects appear to rely clearly on "membrane-initiated mechanisms" through cytoplasmic signal transduction pathways. Disturbances of these mechanisms by endocrine-disrupting chemicals (EDC) may exert adverse effects on brain. Some EDC may act via ER-independent mechanisms but might cross-react with endogenous estrogen. Other EDC may act through ER-dependent mechanisms and display agonistic/antagonistic estrogenic properties. Because of these potential effects of EDC, it is necessary to establish sensitive cell-based assays to determine EDC effects on brain. In the present review, some effects of estrogens and EDC are described with focus on ER-mediated effects in neuronal cells. Particular attention is given to PC12 cells, an interesting model to study the mechanisms underlying ER-mediated differentiating and neuroprotective effects of estrogens.

International audience; Because a large proportion of potential endocrine disruptors (EDC) end up in surface waters, aquatic species are particularly vulnerable to their potential adverse effects. Recent studies identified a number of brain targets for EDC commonly present in environmentally relevant concentrations in surface waters. Among those neuronal systems disrupted by EDC are the gonadotropin-releasing hormone (GnRH) neurons, the dopaminergic and serotoninergic circuits, and more recently the Kiss/GPR54 system, which regulates gonadotropin release. However, one of the most striking effects of EDC, notably estrogen mimics, is their impact on the cyp19a1b gene that encodes the brain aromatase isoform in fish. Moreover, this is the only example in which the molecular basis of endocrine disruption is fully understood. The aims of this review were to (1) synthesize the most recent discoveries concerning the EDC effects upon neuroendocrine systems of fish and (2) provide, when possible, the underlying molecular basis of disruption for each system concerned. The potential adverse effects of EDC on neurogenesis, puberty, and brain sexualization are also described. It is important to point out the future environmental, social, and economical issues arising from endocrine disruption studies in the context of risk assessment.

International audience; The presence of pharmaceuticals and endocrine disruptors in the environment raises many questions about risk to the environment and human health. Environmental exposure has been largely studied, providing to date a realistic picture of the degree of contamination of the environment by pharmaceuticals and hormones. Conversely, little information is available regarding human exposure. NSAIDS, carbamazepine, iodinated contrast media, β-blockers, antibiotics have been detected in drinking water, mostly in the range of ng/L. it is questioned if such concentrations may affect human health. Currently, no consensus among the scientific community exists on what risk, if any, pharmaceuticals and endocrine disruptors pose to human health. Future European research will focus, on one hand, on genotoxic and cytotoxic anti-cancer drugs and, on the other hand, on the induction of genetic resistance by antibiotics. This review does not aim to give a comprehensive overview of human health risk of drug residues and endocrine disruptors in drinking water but rather highlight important topics of discussion.

International audience; There is growing evidence that neuroendocrine circuits controlling development and reproduction are targeted by EDCs. We have previously demonstrated that low concentrations of 17α-ethinylestradiol (EE2) disrupt the development of forebrain GnRH neurons during zebrafish development. The objectives of the present study were to determine whether the weak estrogenic compound, nonylphenol (NP), could elicit similar effects to EE2 and to what extent the estrogen receptors are involved in mediating these effects. Using immunohistochemistry, we confirmed that EE2 exposure induces an increase in the number of GnRH-ir neurons and we demonstrated that NP is able to produce similar effects in a concentration-dependent manner. The effects of both NP and EE2 were shown to be blocked by the estrogen receptors (ERs) antagonist ICI 182-780, demonstrating the involvement of functional ERs in mediating their effects. Altogether, these results highlight the need to consider neuroendocrine networks as critical endpoints in the field of endocrine disruption.

International audience; Until now, studies dedicated to the actions of endocrine disrupting chemicals (EDCs) on the reproductive axis have been concerned with their effects at the gonadal level leaving their actions on neuroendocrine circuits controlling reproduction virtually unexplored. In vertebrates, gonadotropin-releasing hormone (GnRH) is the key factor controlling the activity of the reproductive axis. The development and functioning of GnRH neurons are finely tuned by a series of factors, notably sex steroids, making these neurons potential targets of EDCs, notably in aquatic species. By means of immunohistochemistry, we examined the effects of low levels of ethinylestradiol (EE2 0.02 nM, 0.1 nM, 0.5 nM), a potent synthetic estrogen, on early development (at 5, 10, 20, 30 days post-fertilization) of the forebrain GnRH neurons in a model fish species, the zebrafish (Danio rerio). In parallel, the ER-regulated expression of cytochrome P450 aromatase B (AroB) protein, which is encoded by the cyp19a1b gene, was precisely mapped at the brain and pituitary levels in developing control and EE2-exposed zebrafish. We show that EE2 disrupts the ontogeny of GnRH system by inducing an increase in the number of GnRH-ir neurons and GnRH fibers based on their immunoreactivity as well as a decrease in the size of the GnRH-ir soma and a modification of the migration profile of GnRH-ir neurons. Furthermore, we report a spectacular dose and time-dependent induction of AroB expression in radial glial cells of the developing brain further illustrating the extreme sensitivity of AroB to xenoestrogen and the relevance of AroB as biomarker of xenoestrogen effects on the central nervous system. Collectively, these original and relevant observations highlight the sensitivity of GnRH and AroB to a synthetic estrogen during embryogenesis. These data reinforce the need to further study the mechanisms underlying EDC effects on key neuroendocrine circuits involved in reproduction and brain development of vertebrates.

International audience; BACKGROUND: Prostaglandins (PGs) play key roles in development and maintenance of homeostasis of the adult body. Despite these important roles, it remains unclear whether the PG pathway is a target for endocrine disruption. However, several known endocrine-disrupting compounds (EDCs) share a high degree of structural similarity with mild analgesics. OBJECTIVES AND METHODS: Using cell-based transfection and transduction experiments, mass spectrometry, and organotypic assays together with molecular modeling, we investigated whether inhibition of the PG pathway by known EDCs could be a novel point of endocrine disruption. RESULTS: We found that many known EDCs inhibit the PG pathway in a mouse Sertoli cell line and in human primary mast cells. The EDCs also reduced PG synthesis in ex vivo rat testis, and this reduction was correlated with a reduced testosterone production. The inhibition of PG synthesis occurred without involvement of canonical PG receptors or the peroxisome proliferator-activated receptors (PPARs), which have previously been described as targets of EDCs. Instead, our results suggest that the compounds may bind directly into the active site of the cyclooxygenase (COX) enzymes, thereby obstructing the conversion of arachidonic acid to PG precursors without interfering with the expression of the COX enzymes. A common feature of the PG inhibitory EDCs is the presence of aromatic groups that may stabilize binding in the hydrophobic active site of the COX enzymes. CONCLUSION: Our findings suggest a hitherto unknown mode of action by EDCs through inhibition of the PG pathway and suggest new avenues to investigate effects of EDCs on reproductive and immunological disorders that have become increasingly common in recent decades.

Contamination of the environment with endocrine-disrupting chemicals (EDC) has raised concerns about potential health hazards for humans and wildlife. Human and wildlife exposure to one such ubiquitous chemical, p-tert-octylphenol (OP), are likely, due to its persistence in the environment and its presence in food, water, and items of daily use. OP is reported to bind to the estrogen receptor (ER) and alter expression of estrogen-responsive genes. Detrimental effects of OP exposures on the reproductive system have been observed in most, but not all, in vivo experiments. This study examined estrogenic effects of oral exposures of adult female rats to OP. In vitro, OP bound weakly to human ER and a co-activator protein, and accelerated proliferation of MCF-7 cells. Adult Sprague-Dawley rats were given OP by gavage daily for 35 d (25, 50, or 125 mg/kg/d). Body and organ weights and ovarian follicle populations were not significantly altered in OP-exposed adult rats, despite detectable levels of OP in reproductive organs. The estrous cycle of rats was slightly altered, but there were no significant estrogen-like changes in histomorphology or gene expression of the uterus. Prepubertal rats given 125 or 250 mg/kg OP by gavage for 3 d had reduced body weight compared to vehicle-exposed rats but failed to show any uterotrophic response, although 17alpha-ethinyl estradiol (EE, 10 microg/kg/d, ip) induced a threefold increase in uterine weight. Overall, results suggest that toxicity will occur before estrogenic effects with oral exposures to OP. Relevant environmental exposures likely pose little risk for estrogenic effects.

International audience; A comprehensive evaluation of organic contamination was performed in sediments sampled in two reference and three impacted small streams where endocrine disruptive (ED) effects in fish have been evidenced. The approach combined quantitative chemical analyses of more than 50 ED chemicals (EDCs) and a battery of in vitro bioassays allowing the quantification of receptor-mediated activities, namely estrogen (ER), androgen (AR), dioxin (AhR) and pregnane X (PXR) receptors. At the most impacted sites, chemical analyses showed the presence of natural estrogens, organochlorine pesticides, parabens, polycyclic aromatic hydrocarbons (16 PAHs), bisphenol A and alkylphenols, while synthetic steroids, myco-estrogens and phyto-estrogens were not detected. Determination of toxic-equivalent amounts showed that 28-96% of estrogenic activities in bioassays (0.2-6.3 ng/g 17beta-estradiol equivalents) were explained by 17beta-estradiol and estrone. PAHs were major contributors (20-60%) to the total dioxin-like activities. Interestingly, high PXR and (anti)AR activities were detected; however, the targeted analysed compounds could not explain the measured biological activities. This study highlighted the presence of multiple organic EDCs in French river sediments subjected to mixed diffuse pollution, and argues for the need to further identify AR and PXR active compounds in the aquatic environment.

International audience; Many drugs and chemicals exert their biological effect by modulating protein-protein interactions. In vitro approaches to characterize these mechanisms are often based on indirect measurements (e.g., fluorescence). Here, we used mass spectrometry (MS) to directly monitor the effect of small-molecule ligands on the binding of a coactivator peptide (SRC1) by the human estrogen receptor alpha ligand binding domain (hERalpha LBD). Nanoelectrospray mass spectrometry (nanoESI-MS) and high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking were employed to follow these processes. The chemical cross-linking protocol used prior to high-mass MALDI analysis allows detection of intact noncovalent complexes. The binding of intact hERalpha LBD homodimer with two coactivator peptides was detected with nanoESI-MS and high-mass MALDI-MS only in the presence of an agonist ligand. Furthermore, high-mass MALDI-MS revealed an increase of the homodimer abundance after incubating the receptor with a ligand, independent of the ligand character (i.e., agonist, antagonist). The binding characteristics of the compounds tested by MS correlate very well with their biological activity reported by cell-based assays. High-mass MALDI appears to be an efficient and simple tool for directly monitoring ligand regulation mechanisms involved in protein-protein interactions. Furthermore, the combination of both MS methods allows identifying and characterizing endocrine-disrupting compounds or new drug compounds in an efficient way.

In the present report, a method based on chip-based nanoelectrospray mass spectrometry (nanoESI-MS) is described to detect noncovalent ligand binding to the human estrogen receptor alpha ligand-binding domain (hERalpha LBD). This system represents an important environmental interest, because a wide variety of molecules, known as endocrine disruptors, can bind to the estrogen receptor (ER) and induce adverse health effects in wildlife and humans. Using proper experimental conditions, the nanoESI-MS approach allowed for the detection of specific ligand interactions with hERalpha LBD. The relative gas-phase stability of selected hERalpha LBD-ligand complexes did not mirror the binding affinity in solution, a result that demonstrates the prominent role of hydrophobic contacts for stabilizing ER-ligand complexes in solution. The best approach to evaluate relative solution-binding affinity by nanoESI-MS was to perform competitive binding experiments with 17beta-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hERalpha LBD measured by nanoESI-MS was 4-hydroxtamoxifen approximately diethylstilbestrol > E2 >> genistein >> bisphenol A, consistent with the order of the binding affinities in solution. The limited reproducibility of the bound to free protein ratio measured by nanoESI-MS for this system only allowed the binding constants (K(d)) to be estimated (low nanomolar range for E2). The specificity of nanoESI-MS combined with its speed (1 min/ligand), low sample consumption (90 pmol protein/ligand), and its sensitivity for ligand (30 ng/mL) demonstrates that this technique is a promising method for screening suspected endocrine disrupting compounds and to qualitatively evaluate their binding affinity.

International audience; Experimental studies investigating the effects of endocrine disruptors frequently identify potential unconventional dose-response relationships called non-monotonic dose-response (NMDR) relationships. Standardized approaches for investigating NMDR relationships in a risk assessment context are missing. The aim of this work was to develop criteria for assessing the strength of NMDR relationships. A literature search was conducted to identify published studies that report NMDR relationships with endocrine disruptors. Fifty-one experimental studies that investigated various effects associated with endocrine disruption elicited by many substances were selected. Scoring criteria were applied by adaptation of an approach previously used for identification of hormesis-type dose-response relationships. Out of the 148 NMDR relationships analyzed, 82 were categorized with this method as having a " moderate " to " high " level of plausibility for various effects. Numerous modes of action described in the literature can explain such phenomena. NMDR can arise from numerous molecular mechanisms such as opposing effects induced by multiple receptors differing by their affinity, receptor desensitization, negative feedback with increasing dose, or dose-dependent metabolism modulation. A stepwise decision tree was developed as a tool to standardize the analysis of NMDR relationships observed in the literature with the final aim to use these results in a Risk Assessment purpose. This decision tree was finally applied to studies focused on the effects of bisphenol A.