A comparative study of human and zebrafish glucocorticoid receptor activities of natural and pharmaceutical steroids

IntroductionThe action of environmental steroids on the human glucocorticoid receptor (hGR) has been pointed out with the risk to impair physiological immune and metabolic processes regulated by this nuclear receptor. However, there is still a lack of mechanistic information regarding their ability to interact with GR in aquatic species.MethodsTo investigate ligand activation differences between hGR and zebrafish GR (zfGR), we tested several natural and synthetic steroids using reporter cell lines expressing hGR or zfGR.Results and discussionAlmost all the glucocorticoids tested (dexamethasone, cortisol, bimedrazol, medrol, cortivazol and fluticasone) are agonists of the two receptors with similar potencies. The dissociated glucocorticoids, RU24782 and RU24858 are agonists of both zfGR and hGR but with a better potency for the latter. On the other hand, the synthetic glucocorticoid forbimenol and the mineralocorticoid aldosterone are agonist on hGR but antagonist on zfGR. The other steroids tested, androgens and progestins, are all antagonists of both GRs with equal or lower potency on zfGR than on hGR. Surprisingly, the lower efficacy and potency on zfGR of aldosterone, forbimenol and the dissociated glucocorticoids is not related to their affinity for the receptors which would suggest that it could be related to less efficacious recruitment of coactivators by zfGR compared to hGR

Effets biologiques et mécanismes d'action de ligands environnementaux du récepteur nucléaire de la progestérone chez le poisson zèbre

Among vertebrates, progesterone (P4) is an endogenous progestin acting through the nuclear and molecular progesterone receptor to mediate key processes of development and reproduction. Many molecules are designed to mimic the action of P4. These synthetic progestins and P4 are widely used as pharmaceuticals and some of them are found in this aquatic environment, thus posing risks to aquatic species. However, information is lacking to evaluate the dangers and risks of these substances for the environment. This thesis studied the mechanisms of action and the effects of progestins on zebrafish, using a combination of reporter-gene in vitro and in vivo tools based on the mechanism of action of endocrine disruptors. We characterized the toxicological profile of 26 progestins towards different human and zebrafish nuclear steroid receptors and towards the tissue-specific expression of steroidogenic genes (cyp19a1b in the brain and cyp11c1 in the interrenal cells). Our results show that some progestins can disrupt different signaling pathways in developing larvae at the molecular level, cellular and tissular level, and physiological level. Overall, the results of this thesis allow for a better characterization of the dangers of progestins on several endocrine targets.Chez les vertébrés, la progestérone (P4) est un progestatif endogène agissant via les récepteurs nucléaires et membranaires de la progestérone pour médier des processus clés du développement et de la reproduction. De nombreuses molécules sont conçues pour mimer l’action de la P4. Ces progestatifs synthétiques et la P4 sont très utilisés en tant que substances pharmaceutiques et certains sont retrouvées dans l’environnement aquatique, posant un risque pour les organismes. Cependant, les informations sont insuffisantes pour évaluer les dangers et les risques de ces substances pour l’environnement. Dans ce travail de thèse, les mécanismes d’action et les effets biologiques des progestatifs sur le poisson zèbre ont été étudiés grâce à une combinaison d’outils in vitro et in vivo de type gène-rapporteur basés sur le mécanisme d’action des perturbateurs endocriniens. Nous avons caractérisé le profil toxicologique complexe de 26 progestatifs vis-à-vis de différents récepteurs nucléaires stéroïdiens humain et poisson zèbre et vis-à-vis de l’expression tissu-spécifique de gènes de la stéroïdogenèse (cyp19a1b dans le cerveau et cyp11c1 dans les interrénales). Nos résultats montrent que certains progestatifs peuvent perturber différentes voies de signalisation chez les larves en développement au niveau moléculaire, cellulaire et tissulaire ainsi qu’au niveau physiologique. Dans l’ensemble, les résultats de cette thèse permettent une meilleure caractérisation des dangers des progestatifs sur différentes cibles endocriniennes

Modes of action and biological effects of environmental ligands of the nuclear progesterone receptor in zebrafish

Chez les vertébrés, la progestérone (P4) est un progestatif endogène agissant via les récepteurs nucléaires et membranaires de la progestérone pour médier des processus clés du développement et de la reproduction. De nombreuses molécules sont conçues pour mimer l’action de la P4. Ces progestatifs synthétiques et la P4 sont très utilisés en tant que substances pharmaceutiques et certains sont retrouvées dans l’environnement aquatique, posant un risque pour les organismes. Cependant, les informations sont insuffisantes pour évaluer les dangers et les risques de ces substances pour l’environnement. Dans ce travail de thèse, les mécanismes d’action et les effets biologiques des progestatifs sur le poisson zèbre ont été étudiés grâce à une combinaison d’outils in vitro et in vivo de type gène-rapporteur basés sur le mécanisme d’action des perturbateurs endocriniens. Nous avons caractérisé le profil toxicologique complexe de 26 progestatifs vis-à-vis de différents récepteurs nucléaires stéroïdiens humain et poisson zèbre et vis-à-vis de l’expression tissu-spécifique de gènes de la stéroïdogenèse (cyp19a1b dans le cerveau et cyp11c1 dans les interrénales). Nos résultats montrent que certains progestatifs peuvent perturber différentes voies de signalisation chez les larves en développement au niveau moléculaire, cellulaire et tissulaire ainsi qu’au niveau physiologique. Dans l’ensemble, les résultats de cette thèse permettent une meilleure caractérisation des dangers des progestatifs sur différentes cibles endocriniennes.Among vertebrates, progesterone (P4) is an endogenous progestin acting through the nuclear and molecular progesterone receptor to mediate key processes of development and reproduction. Many molecules are designed to mimic the action of P4. These synthetic progestins and P4 are widely used as pharmaceuticals and some of them are found in this aquatic environment, thus posing risks to aquatic species. However, information is lacking to evaluate the dangers and risks of these substances for the environment. This thesis studied the mechanisms of action and the effects of progestins on zebrafish, using a combination of reporter-gene in vitro and in vivo tools based on the mechanism of action of endocrine disruptors. We characterized the toxicological profile of 26 progestins towards different human and zebrafish nuclear steroid receptors and towards the tissue-specific expression of steroidogenic genes (cyp19a1b in the brain and cyp11c1 in the interrenal cells). Our results show that some progestins can disrupt different signaling pathways in developing larvae at the molecular level, cellular and tissular level, and physiological level. Overall, the results of this thesis allow for a better characterization of the dangers of progestins on several endocrine targets

Les progestatifs, les poissons et les milieux aquatiques : apports du projet ANR « proofs »

During the past twenty years, numerous studies have examined the effects of estrogenic compounds, notably 17α-ethinylestradiol a potent synthetic steroidal estrogen used as pharmaceuticals in contraceptive pills, because these substances were among the first identified as endocrine disrupting compounds responsible for the feminization of wild populations of fish observed worldwide. There is now evidence of the occurrence of other natural and synthetic steroids in aquatic environment. Among steroidal pharmaceuticals, the risks encountered by synthetic progestins on aquatic species have been recently pointed out but data on their occurrence and effects on fish endocrinology and physiology are missing to properly assess their hazard and risk to aquatic species. The national project ANR “PROOFS” aimed at investigating the effect of progestins on key molecular and cellular targets of the endocrine system using zebrafish in vitro and in vivo reporter gene mechanismbased assays. It also aimed at providing data on the occurrence of progestagenic activity of environmental samples and to identify substances responsible for these activities through an effectdirected analysis (EDA) approach. Among the 26 pharmaceutical progestins investigated, our data revealed that they can act on several steroidal nuclear receptors. By using novel transgenic zebrafish models, we demonstrated for the first time their estrogenic activity in the developing brain and their capacity to disrupt corticosteroigenesis in interrenal cells. Finally, we reported strong progestagenic activity in waste waters and identified several pharmaceuticals as potential candidate responsible for these activities. Overall, the ANR project “PROOFS” brings novel and relevant data on occurrence and endocrine potency of progestins stressing the need to further study these emerging contaminantsDurant ces vingt dernières années, la question des effets des perturbateurs endocriniens (PE) sur des fonctions physiologiques clés telles que la reproduction a fait l’objet de nombreux travaux en raison des risques encourus pour les organismes et les milieux aquatiques. À cet égard, de nombreux travaux ont porté sur les risques associés aux composés oestrogéniques et, en particulier, les oestrogènes utilisés dans les pilules contraceptives [1 ; 2]. Pourtant, la présence d’autres pharmaceutiques stéroïdiens dans les milieux aquatiques est aujourd’hui avérée sans que l’on puisse en appréhender les conséquences. Parmi ces stéroïdes, les progestatifs de synthèse représentent un ensemble de substances largement utilisées en médecine humaine et vétérinaire. Certains progestatifs ont pu être quantifiés dans des effluents de stations d’épuration et des eaux de surface, à des concentrations faibles (ng/L-μg/L) mais capables d’affecter la reproduction des poissons exposés en laboratoire. Cependant, il existe un manque d’information sur les mécanismes, les effets et l’exposition des organismes aux progestatifs qu’il est nécessaire de combler dans l’optique d’évaluer les dangers et les risques que ces substances soulèvent pour les espèces aquatiques et l’environnemen

Use of transgenic zebrafish models to study the endocrine effects of natural and synthetic progestins

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Synthetic progestins disrupt the glial cell specific-brain aromatase expression in the developing brain of fish

Few studies have addressed the biological effects of natural and synthetic ligands of the nuclear progesterone receptor (nPR) on fish development and reproduction, resulting in a significant lack of data to assess the hazard and risk posed by these compounds on aquatic organisms. Yet progesterone plays key roles in many vertebrates and recent data showed that some progestins are present in the aquatic environment at concentrations that can impair reproduction. In light of this context, we studied the effects of progesterone and a panel of synthetic nPR ligands on the estrogen-receptor (ER)-signaling pathway by investigating the expression of the zebrafish brain aromatase cyp19a1b gene using zebrafish-specific mechanism-based in vitro and in vivo assays. Twenty-four nPR ligands were screened on transgenic cyp19a1b-GFP zebrafish embryos to assess their potential estrogenic effect. Progesterone and its isomer dydrogesterone as well as drospirenone and all the progesterone-derived progestins had no effect on GFP expression. However, all progestins derived from 19-nortesterone induced the cyp19a1b expression in an ER- and concentration-dependent manner with EC50 ranging from the low nM to the µM range. These results show that progestins are estrogenic to fish and that early-life exposure of fish to 19-nortestosterone disrupts the ER-signaling pathway within the developing brain. The 19-nortestosterone derived progestins levonorgestrel and norethindrone were further tested in U251-MG cells transfected with any of the three zebrafish ER subtypes zfERα, zfERβ1, or zfERβ2. Progesterone had no effect on luciferase activity with either of the zfER subtypes. Norethindrone and levonorgestrel both induced luciferase activity that was blocked by co-exposing the cells with ICI 182,780. An ER competition assay showed that both progestins were unable to bind to and activate ER except at very high concentrations (10-5M), suggesting they require metabolic activation prior to elicit estrogenic activity. This study highlights the relevance of using zebrafish-specific screening assays to characterize endocrine disrupting properties of emerging contaminants and demonstrates that 19-nortestosterone derived progestins are pro-estrogenic compounds targeting radial glial cells inducing cyp19a1b expression in the developing brain of fish. Given that ER signaling is one of the very first to emerge during early-life stage development, the consequences should be further investigated

A newly cyp11c1-GFP transgenic zebrafish model to study corticosteroidogenesis and its perturbation by endocrine active substances at early developmental stages

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Synthetic progestins activate human and zebrafish nuclear progesterone receptor in vitro

As compared to (xeno-)estrogens, natural and synthetic ligands of the progesterone receptor (PR) have been scarcely studied on aquatic organisms while the potential risk posed by environmental progestins has been recently pointed out. However, there is still a lack of data to accurately characterize the hazards posed by these compounds. In that respect, the capacity of synthetic progestins to interact with fish nuclear progesterone receptor was poorly investigated. In the present work we aimed at assessing potential endocrine disruption of these compounds towards zebrafish nPR and to compare their effects with that of human nPR to identify possible interspecies differences. Two human cell lines co-expressing either human PR (hPR) or the zebrafish PR (ZfPR) and luciferase gene, namely HELN-hPRB and U2OS-zfPR cells, were developed and characterized using promegestone (R5020) as a reference agonist ligand. R5020 induced luciferase activity in both cell lines in a concentration-dependent manner. These effects were completely blocked by co-exposing the cells with mifepristone (RU486), a potent PR antagonist. A large set of natural and synthetic progestins (25) was screened in the two cell lines. All of the compounds except the natural zebrafish progestin 17α,20β-dihydroxy-4-pregnen-3-one (DHP) activated the hPR. About half of the progestins induced a maximum effect of 100% when compared to R5020 while the other half partially induced luciferase activity. In contrast to cells expressing hPR, progestins behaved very differently with the zfPR since only five of them were active on zfPR. Interestingly, we found that DHP strongly activated zfPR but not hPR. To assess whether some progestins were partial agonists, they were coexposed with R5020. Half of them antagonized the activity induced by R5020 on hPR and are thus partial hPR agonists. All the active progestins on zfPR antagonized R5020 and are therefore partial zfPR agonists. Two new luciferase reporter cell lines were developed and characterized, providing novel information regarding the activity of a large set of progestins on the zfPR. These models allowed us to highlight major interspecies differences. These results support the need to further determine the effects of zfPR agonist progestins on PR-dependent physiological processes in order to characterize the hazards posed by progestins in fish