Inhibition of rainbow trout (Oncorhynchus mykiss) P450 aromatase activities in brain and ovarian microsomes by various environmental substances

International audienceAromatase, a key steroidogenic enzyme that catalyses the conversion of androgens to estrogens, represent a target for endocrine disrupting chemicals. However, little is known about the effect of pollutants on aromatase enzymes in fish. In this study, we first optimized a rainbow trout (Oncorhynchus mykiss) microsomal aromatase assay to measure the effects of 43 substances belonging to diverse chemical classes (steroidal and non steroidal aromatase inhibitors, pesticides, heavy metals, organotin compounds, dioxins, polycyclic aromatic hydrocarbons) on brain and ovarian aromatase activities in vitro. Our results showed that 12 compounds were able to inhibit brain and ovarian aromatase activities in a dose-dependent manner with IC50 values ranging from the low nM to the high uM range depending on the substance: steroidal and non steroidal inhibitors of aromatase (4-hydroxyandrostenedione, androstatrienedione, aminogluthethimide), imidazole fungicides (clotrimazole, imazalil, prochloraz), triazole fungicides (difenoconazole, fenbuconazole, propiconazole, triadimenol), the pyrimidine fungicide fenarimol and methylmercury. Overall, this study demonstrates that rainbow trout brain and ovarian microsomal aromatase assay is suitable for evaluating potential aromatase inhibitors in vitro notably with respect to environmental screening. The results highlight that methylmercury and some pesticides that are currently used throughout the world, have the potential to interfere with the biosynthesis of endogenous estrogens in fish

Expression of Zebra Fish Aromatase cyp19a and cyp19b Genes in Response to the Ligands of Estrogen Receptor and Aryl Hydrocarbon Receptor

Many endocrine-disrupting chemicals act via estrogen receptor (ER) or aryl hydrocarbon receptor (AhR). To investigate the interference between ER and AhR, we studied the effects of 17β-estradiol (E2) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of zebra fish cyp19a (zfcyp19a) and cyp19b (zfcyp19b) genes, encoding aromatase P450, an important steroidogenic enzyme. In vivo (mRNA quantification in exposed zebra fish larvae) and in vitro (activity of zfcyp19-luciferase reporter genes in cell cultures in response to chemicals and zebra fish transcription factors) assays were used. None of the treatments affected zfcyp19a, excluding the slight upregulation by E2 observed in vitro. Strong upregulation of zfcyp19b by E2 in both assays was downregulated by TCDD. This effect could be rescued by the addition of an AhR antagonist. Antiestrogenic effect of TCDD on the zfcyp19b expression in the brain was also observed on the protein level, assessed by immunohistochemistry. TCDD alone did not affect zfcyp19b expression in vivo or promoter activity in the presence of zebra fish AhR2 and AhR nuclear translocator 2b (ARNT2b) in vitro. However, in the presence of zebra fish ERα, AhR2, and ARNT2b, TCDD led to a slight upregulation of promoter activity, which was eliminated by either an ER or AhR antagonist. Studies with mutated reporter gene constructs indicated that both mechanisms of TCDD action in vitro were independent of dioxin-responsive elements (DREs) predicted in the promoter. This study shows the usefulness of in vivo zebra fish larvae and in vitro zfcyp19b reporter gene assays for evaluation of estrogenic chemical actions, provides data on the functionality of DREs predicted in zfcyp19 promoters and shows the effects of cross talk between ER and AhR on zfcyp19b expression. The antiestrogenic effect of TCDD demonstrated raises further concerns about the neuroendocrine effects of AhR ligand

Adverse effects in wild fish living downstream from pharmaceutical manufacture discharges

International audienceA set of biochemical and histological responses was measured in wild gudgeon collected upstream and downstream of urban and pharmaceutical manufacture effluents. These individual end-points were associated to fish assemblage characterisation. Responses of biotransformation enzymes, neurotoxicity and endocrine disruption biomarkers revealed contamination of investigated stream by a mixture of pollutants. Fish from sampled sites downstream of the industrial effluent exhibited also strong signs of endocrine disruption including vitellogenin induction, intersex and male-biased sex-ratio. These individual effects were associated to a decrease of density and a lack of sensitive fish species. This evidence supports the hypothesis that pharmaceutical compounds discharged in stream are involved in recorded endocrine disruption effects and fish population disturbances and threaten disappearance of resident fish species. Overall, this study gives argument for the utilisation of an effect-based monitoring approach to assess impacts of pharmaceutical manufacture discharges on wild fish populations

Inhibitory effect of cadmium on estrogen signaling in zebrafish brain and protection by zinc

International audienceThe present study was conducted to assess the effects of Cd exposure on estrogen signaling in the zebrafish brain, as well as the potential protective role of Zn against Cd-induced toxicity. For this purpose, the effects on transcriptional activation of the estrogen receptors (ERs), aromatase B (Aro-B) protein expression and molecular expression of related genes were examined in vivo using wild-type and transgenic zebrafish embryos. For in vitro studies, an ER-negative glial cell line (U251MG) transfected with different zebrafish ER subtypes (ERα, ERβ1 and ERβ2) was also used. Embryos were exposed either to estradiol (E2), Cd, E2+Cd or E2+Cd+Zn for 72 h and cells were exposed to the same treatments for 30 h. Our results show that E2 treatment promoted the transcriptional activation of ERs and increased Aro-B expression, at both the protein and mRNA levels. Although exposure to Cd, does not affect the studied parameters when administered alone, it significantly abolished the E2-stimulated transcriptional response of the reporter gene for the three ER subtypes in U251-MG cells, and clearly inhibited the E2 induction of Aro-B in radial glial cells of zebrafish embryos. These inhibitory effects were accompanied by a significant downregulation of the expression of esr1, esr2a, esr2b and cyp19a1b genes compared to the E2-treated group used as a positive control. Zn administration during simultaneous exposure to E2 and Cd strongly stimulated zebrafish ERs transactivation and increased Aro-B protein expression, whereas mRNA levels of the three ERs as well as the cyp19a1b remained unchanged in comparison with Cd-treated embryos. In conclusion, our results clearly demonstrate that Cd acts as a potent anti-estrogen in vivo and in vitro, and that Cd-induced E2 antagonism can be reversed, at the protein level, by Zn supplement

The Herbicide Atrazine Activates Endocrine Gene Networks via Non-Steroidal NR5A Nuclear Receptors in Fish and Mammalian Cells

Atrazine (ATR) remains a widely used broadleaf herbicide in the United States despite the fact that this s-chlorotriazine has been linked to reproductive abnormalities in fish and amphibians. Here, using zebrafish we report that environmentally relevant ATR concentrations elevated zcyp19a1 expression encoding aromatase (2.2 µg/L), and increased the ratio of female to male fish (22 µg/L). ATR selectively increased zcyp19a1, a known gene target of the nuclear receptor SF-1 (NR5A1), whereas zcyp19a2, which is estrogen responsive, remained unchanged. Remarkably, in mammalian cells ATR functions in a cell-specific manner to upregulate SF-1 targets and other genes critical for steroid synthesis and reproduction, including Cyp19A1, StAR, Cyp11A1, hCG, FSTL3, LHß, INHα, αGSU, and 11ß-HSD2. Our data appear to eliminate the possibility that ATR directly affects SF-1 DNA- or ligand-binding. Instead, we suggest that the stimulatory effects of ATR on the NR5A receptor subfamily (SF-1, LRH-1, and zff1d) are likely mediated by receptor phosphorylation, amplification of cAMP and PI3K signaling, and possibly an increase in the cAMP-responsive cellular kinase SGK-1, which is known to be upregulated in infertile women. Taken together, we propose that this pervasive and persistent environmental chemical alters hormone networks via convergence of NR5A activity and cAMP signaling, to potentially disrupt normal endocrine development and function in lower and higher vertebrates

Glucocorticoid deficiency causes transcriptional and post-transcriptional reprogramming of glutamine metabolism

Background Deficient glucocorticoid biosynthesis leading to adrenal insufficiency is life-threatening and is associated with significant co-morbidities. The affected pathways underlying the pathophysiology of co-morbidities due to glucocorticoid deficiency remain poorly understood and require further investigation. Methods To explore the pathophysiological processes related to glucocorticoid deficiency, we have performed global transcriptional, post-transcriptional and metabolic profiling of a cortisol-deficient zebrafish mutant with a disrupted ferredoxin (fdx1b) system. Findings fdx1b−/− mutants show pervasive reprogramming of metabolism, in particular of glutamine-dependent pathways such as glutathione metabolism, and exhibit changes of oxidative stress markers. The glucocorticoid-dependent post-transcriptional regulation of key enzymes involved in de novo purine synthesis was also affected in this mutant. Moreover, fdx1b−/− mutants exhibit crucial features of primary adrenal insufficiency, and mirror metabolic changes detected in primary adrenal insufficiency patients. Interpretation Our study provides a detailed map of metabolic changes induced by glucocorticoid deficiency as a consequence of a disrupted ferredoxin system in an animal model of adrenal insufficiency. This improved pathophysiological understanding of global glucocorticoid deficiency informs on more targeted translational studies in humans suffering from conditions associated with glucocorticoid deficiency. Fund Marie Curie Intra-European Fellowships for Career Development, HGF-programme BIFTM, Deutsche Forschungsgemeinschaft, BBSRC

Efficacité de l'oxytocine chez la femme enceinte déclenchée en fonction de l’indice de masse corporel

Analyser l'efficacité de l'oxytocine en comparant la dose nécessaire à l'obtention d'une dynamique utérine efficace, entre les patientes de poids normal, les patientes en surpoids et les patientes obèses. Méthodologie : Étude cas-témoin, rétrospective, au CHU de Rouen, de janvier 2013 à novembre 2015. Ont été incluses les patientes ayant eu une grossesse singleton menée à terme, avec présentation fœtale céphalique et déclenchement artificiel du travail par Syntocinon®. Résultats et discussion : Les dossiers de 95 femmes de poids normal, 59 femmes en surpoids et 65 femmes obèses ont été étudiés . La quantité moyenne d'oxytocine permettant d'obtenir une dynamique utérine efficace (4-5 CU/10`) était significativement différente entre les groupes (p=0,017) et variait de manière proportionnelle à l'I.M.C. (0,62±0,57 UI vs 0,87±0,98 UI vs 1,08±1,28). Une corrélation significative à également été prouvée pour les items suivants : la prise de poids pendant la grossesse (p=0,000), le mode d'accouchement (p=0,034) et le poids néonatal (p=0,004). Soulignons qu'aucune différence significative n'a été prouvée en ce qui concerne les anomalies de dynamique utérine, le taux d'hémorragie de la délivrance ainsi que l'adaptation à la vie extra utérine du nouveau-né. Conclusion : Pour obtenir une dynamique utérine efficace lors d'un déclenchement artificiel du travail, il est nécessaire d'adapter la quantité d'oxytocine en fonction de l'I.M.C. de la patiente

Cross-border regions, new territories of project, new poles of development within a polycentric Europe ?

La thèse a pour sujet une réflexion générale sur l'aménagement des espaces transfrontaliers, qui présentent la particularité de s'étendre de part et d'autre d'une frontière nationale. Suite à l'intensification des échanges et aux mouvements d'intégration socio-économique, l'ouverture des frontières européennes a notamment pour conséquences des processus de recomposition territoriale au sein des espaces transfrontaliers. A partir d'une recherche théorique et empirique, il s'agit de s'interroger sur les potentialités de développement territorial des espaces transfrontaliers européens. Dans cette perspective, nous proposons un modèle d'analyse global et systémique traitant de la construction des territoires transfrontaliers, qui permet de mesurer l'intensification et l'interaction des liens de différentes natures (structurel, institutionnel, idéel) qui se mettent en place au travers des frontières. La démarche est appliquée à quatre études de cas, à savoir l'eurorégion Hàjdu-Bihar-Bihor, située sur la frontière hungaro-roumaine, l'eurorégion Norte-Galice entre l'Espagne et le Portugal, l'agglomération transfrontalière franco-valdo-genevoise et le projet urbain de Belval localisé sur la frontière Luxembourg-France.The thesis deals with a general reflection about planning in cross-borders regions. Following the increasing exchanges and dynamics of socio-economical integration, the opening of the European borders contributes to process of territorial rescaling within cross-border regions. The territorial development potentialities of European cross-border regions are interrogated through both theoretical and empirical research. In this purpose, we propose a global and systemic model of analysis dealing with the construction of cross-border territories, making to measure increasing and interacting links of different natures (structural, institutional, ideal) taking place across national borders. Then the approach is applied to four case studies : Euroregion Hàjdu-Bihar-Bihor on the Hungarian-Romanian border, Euroregion Norte-Galice between Spain and Portugal, the crossborder agglomeration of Geneva and the urban project of Belval located on the Luxembourg-France border