Modes de perturbation de la stéroïdogenèse testiculaire et de la spermatogenèse chez le poisson zèbre (Danio rerio) par des fongicides azolés

Azole fungicides are detected in the aquatic environment and can inhibit enzymatic activities of cytochrome P450 (CYP). This thesis aims to characterize the mechanism of action of the pharmaceutical clotrimazole on testicular steroidogenesis in zebrafish using a network of functional genes along the brain-pituitary-gonad axis and to evaluate the effect of clotrimazole on spermatogenesis. We show that clotrimazole is able to affect steroidogenesis differently in vitro and in vivo (i) the exposure of testicular explants in vitro leads to inhibition of the synthesis of 11-ketotestosterone, showing a direct action on the testis, and (ii) in vivo exposure increases the transcript levels of genes involved in steroidogenesis. An integrative approach of gene expression measurements along the pituitary-gonad axis highlights a mechanism of biological compensation, with a critical role of the Fsh/FshR pathway in mediating the effects of clotrimazole on testicular steroidogenesis. Finally, effects on spermatogenesis were observed in vivo following chronic exposure to clotrimazole, with an increase in gonado-somatic index as well as in number of Leydig cells. These observations are consistent with measurements at molecular level. Taken together, these data show the interest of an integrative approach. This work raises further concerns, primarily on the study of the functional impact of clotrimazole on reproduction, including male and female studies.Les azoles sont des fongicides présents dans les milieux aquatiques et connus pour inhiber des activités enzymatiques de cytochromes P450 (CYP). L'objectif de ce travail de thèse est de caractériser le mécanisme d'action d'un fongicide pharmaceutique, le clotrimazole sur la stéroïdogenèse testiculaire chez le poisson zèbre au travers l'étude d'un réseau de gènes fonctionnels le long de l'axe cerveau-hypophyse-gonade, et d'évaluer la capacité du clotrimazole à perturber la spermatogenèse. Nous montrons que le clotrimazole est capable d'affecter la stéroïdogenèse de manière différente in vitro et in vivo (i) des expositions d'explants testiculaires in vitro conduisent à l'inhibition de la synthèse de 11-kétotestostérone (11-KT), montrant une action directe de la molécule sur le testicule et (ii) l'exposition in vivo provoque une augmentation de l'expression de gènes impliqués dans le processus de la stéroïdogenèse. Nous avons ainsi mis en évidence un système de compensation biologique au niveau de l'organisme, avec un rôle prépondérant de la voie Fsh/FshR dans la médiation des effets du clotrimazole. Enfin, des effets sur la spermatogenèse ont été observés in vivo suite à une exposition chronique au clotrimazole, avec notamment une augmentation de la masse gonadique et du nombre de cellules de Leydig. Les effets tissulaires observés sont cohérents avec des effets mesurés au niveau moléculaire. L'ensemble de ces données montre l'intérêt de la démarche expérimentale utilisée pour caractériser le mécanisme d'action du clotrimazole. Ce travail ouvre de nombreuses perspectives, en premier lieu sur l'étude de l'impact fonctionnel du clotrimazole sur la reproduction

Modes de perturbation de la stéroïdogenèse testiculaire et de la spermatogenèse chez le poisson zèbre (Danio rerio) par des fongicides azolés

Les azoles sont des fongicides présents dans les milieux aquatiques et connus pour inhiber des activités enzymatiques de cytochromes P450 (CYP). L'objectif de ce travail de thèse est de caractériser le mécanisme d'action d'un fongicide pharmaceutique, le clotrimazole sur la stéroïdogenèse testiculaire chez le poisson zèbre au travers l'étude d'un réseau de gènes fonctionnels le long de l'axe cerveau-hypophyse-gonade, et d'évaluer la capacité du clotrimazole à perturber la spermatogenèse. Nous montrons que le clotrimazole est capable d'affecter la stéroïdogenèse de manière différente in vitro et in vivo (i) des expositions d'explants testiculaires in vitro conduisent à l'inhibition de la synthèse de 11-kétotestostérone (11-KT), montrant une action directe de la molécule sur le testicule et (ii) l'exposition in vivo provoque une augmentation de l'expression de gènes impliqués dans le processus de la stéroïdogenèse. Nous avons ainsi mis en évidence un système de compensation biologique au niveau de l'organisme, avec un rôle prépondérant de la voie Fsh/FshR dans la médiation des effets du clotrimazole. Enfin, des effets sur la spermatogenèse ont été observés in vivo suite à une exposition chronique au clotrimazole, avec notamment une augmentation de la masse gonadique et du nombre de cellules de Leydig. Les effets tissulaires observés sont cohérents avec des effets mesurés au niveau moléculaire. L'ensemble de ces données montre l'intérêt de la démarche expérimentale utilisée pour caractériser le mécanisme d'action du clotrimazole. Ce travail ouvre de nombreuses perspectives, en premier lieu sur l'étude de l'impact fonctionnel du clotrimazole sur la reproduction.Azole fungicides are detected in the aquatic environment and can inhibit enzymatic activities of cytochrome P450 (CYP). This thesis aims to characterize the mechanism of action of the pharmaceutical clotrimazole on testicular steroidogenesis in zebrafish using a network of functional genes along the brain-pituitary-gonad axis and to evaluate the effect of clotrimazole on spermatogenesis. We show that clotrimazole is able to affect steroidogenesis differently in vitro and in vivo (i) the exposure of testicular explants in vitro leads to inhibition of the synthesis of 11-ketotestosterone, showing a direct action on the testis, and (ii) in vivo exposure increases the transcript levels of genes involved in steroidogenesis. An integrative approach of gene expression measurements along the pituitary-gonad axis highlights a mechanism of biological compensation, with a critical role of the Fsh/FshR pathway in mediating the effects of clotrimazole on testicular steroidogenesis. Finally, effects on spermatogenesis were observed in vivo following chronic exposure to clotrimazole, with an increase in gonado-somatic index as well as in number of Leydig cells. These observations are consistent with measurements at molecular level. Taken together, these data show the interest of an integrative approach. This work raises further concerns, primarily on the study of the functional impact of clotrimazole on reproduction, including male and female studies.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Effect of azole fungicides on testicular steroidogenesis in vivo and ex vivo in zebrafish (Danio rerio)

The occurrence of several azole fungicides in aquatic environment has been recently reported but their endocrine effects in fish are poorly studied. The aim of the present study was to assess the effect of clotrimazole, a pharmaceutical fungicide, on expression of key target genes (StaR, cyp17a1, cyp11b2) involved in testicular steroidogenesis as well as on 11- ketotestosterone (11KT) biosynthesis. For that purpose, we first exposed adult male zebrafish to a wide range of clotrimazole concentrations(from 0.01 uM to 0.3 uM) for 7 days. In vivo exposure to clotrimazole resulted in an increase of cyp17a1 and cyp11b2 gene expressions in testis. To gain further information about the mechanism of action of clotrimazole, zebrafish testicular explants were exposed to similar concentrations of clotrimazole, with or without forskolin (FSK), an activator of the cAMP pathway that stimulates steroidogenesis. FSK (1 uM) strongly up-regulated cyp17a1 and cyp11b2 gene expressions and increased 11KT release in the culture medium. Clotrimazole (0.2, 1, and 5 uM), alone or in combination with FSK (1 uM), did not change basal or FSK-induced expression of cyp17a1 and cyp11b2. However, it inhibited both basal and FSK-induced 11KT release ex vivo, suggesting it acted as an inhibitor of steroidogenic enzymes. In order to determine whether other compounds belonging to the azole family are able to elicit similar effect on testicular steroidogenesis as clotrimazole, two other azole fungicides known to contaminate aquatic environment were tested, fenbuconazole and propiconazole. We showed that exposure of male zebrafish for 7 days to fenbuconazole and propiconazole had no significant effects on steroidogenic gene expressions while ex vivo both azoles was able to inhibit the 11KT release. Overall, our results show for the first time that the pharmaceutical fungicide clotrimazole is able to affect key steroidogenic genes expressions in a fish testis. However, the marked differences observed between in vivo and ex vivo experiments suggest that clotrimazole does not act directly on testes to regulate cyp17a1and cyp11b2 transcription. Whatever, using the testis tissue explants model, we demonstrate a direct action of clotrimazole, and to a lesser extent propiconazole and fenbuconazole, on the gonad which results in inhibition of 11KT synthesis. These original data deserve further studies on the effect of these compounds on fish reproductio

Les effets du clotrimazole sur la stéroïdogenèse testiculaire chez le poisson zèbre

Clotrimazole (CLO) is a pharmaceutical fungicide that has been recently detected in aquatic environment but little is known about its endocrine disrupting potency in fish. In vivo exposures of male zebrafish to CLO led to inductions of steroidogenic gene and protein expressions. Using zebrafish testicular explant culture, no effect of CLO was observed on transcript levels of steroidogenic enzymes but it inhibited 11 ketotestorone (11-KT) release in the medium. It suggests that CLO does not act directly on testis. Induction of these genes could be interpreted as a compensatory biological response to inhibition of cytochrome P-450 dependent steroidogenic enzymes. To support this hypothesis, a network of functional genes of the pituitary-gonad axis was used. We showed that CLO induce a cascade of molecular events in pituitary and testes. Transcript levels of genes encoding for folliculo- stimulating hormone (FSH), testicular FSH receptor and steroidogenic enzymes were induced. These molecular events are consistent with the involvement of FSH in inducing steroidogenic gene expressions to compensate the inhibitory action of CLO on 11-KT synthesis. Our study highlights the relevance of studying a network of genes of the pituitary-gonad axis to investigate the mode of action of compounds acting as inhibitor of P450-steroidogenic enzymes.Il est aujourd’hui clairement admis que des composés chimiques, d’origine naturelle ou de synthèse, présents dans les milieux aquatiques sont capables de perturber l’endocrinologie et l’homéostasie des organismes. Ces substances, appelées perturbateurs endocriniens (PE), représentent un large éventail de contaminants de l’environnement, qui interfèrent avec le système endocrinien via de multiples modes d’action. Au cours des dernières décennies, de nombreux travaux ont permis de montrer que ces produits chimiques peuvent agir comme des agonistes des récepteurs des hormones stéroïdiennes et induire des effets biologiques similaires à ceux des hormones endogènes. C’est le cas de nombreux produits chimiques comme, par exemple, les alkylphénols ou le bisphénol A, qui miment l’action des oestrogènes et induisent des effets féminisants. Toutefois, d’autres mécanismes peuvent rendre compte de la perturbation endocrinienne chez les organismes aquatiques. En particulier, on sait que les PE peuvent agir sur les cytochromes P450 impliqués dans la stéroïdogenèse. La stéroïdogenèse est le processus responsable de la biosynthèse des hormones stéroïdiennes telles que les oestrogènes et les androgènes, qui jouent un rôle prépondérant dans le développement et la reproduction. Parmi les substances chimiques connues pour agir sur les cytochromes P450, impliqués dans la biosynthèse hormonale, figurent les azoles [A]. Cette famille de molécules regroupe des composés qui sont utilisés dans diverses applications comme agents antifongiques en agriculture, en médecine humaine et vétérinaire. Bien que l’occurrence environnementale de ces composés soit peu renseignée, des études récentes montrent que des azoles tels que le propiconazole, le tebuconazole, le prochloraz ou encore le clotrimazole sont présents dans les effluents de station d’épuration ainsi que dans les eaux de surface. Cependant, les mécanismes et effets in vivo de ces composés sur le système endocrinien et la reproduction des poissons sont peu renseignés. Dans ce contexte, nous nous sommes spécifiquement intéressés aux effets du clotrimazole, une substance pharmaceutique utilisée dans le traitement des mycoses vaginales, sur la stéroïdogenèse testiculaire et la biosynthèse d’androgènes. Pour ce faire, une série d’expérimentations combinant des modèles in vivo et in vitro a été menée chez une espèce modèle largement utilisée en toxicologie et en écotoxicologie, le poisson zèbre (Danio rerio)

Characterization of the mode of action of the pharmaceutical clotrimazole on testicular steroidogenesis in zebrafish

Clotrimazole is a pharmaceutical fungicide that has been recently detected in aquatic environment. This substance is known to inhibit CYP enzymatic activities, including several steroidogenic CYP but little is known about its in vivo endocrine disrupting potency in fish. In vertebrates gonadal steroidogenesis is under the control of the hypothalamus-pituitary-gonad (HPG) axis. The aim of the present study was (i) to assess the effect of clotrimazole on zebrafish testicular steroidogenesis by conducting in vivo and in vitro experiments and (ii) to characterize its mode of action by studying a network of functional target genes of the pituitary-gonad axis by means of Q-PCR. In vivo exposures of male zebrafish to clotrimazole were conducted for 7 (70- 150 micro g/L) or 21 days (20-200 micro g/L) and led to concentration-dependent inductions of steroidogenic gene and protein expressions. In vitro testicular explants were exposed to similar concentrations of clotrimazole, and no effect was observed on transcript levels of steroidogenic enzymes. However, clotrimazole inhibited 11-KT release in the culture medium. This result suggests that clotrimazole does not act directly on testis to regulate the transcriptional activity of these genes. Induction of steroidogenic genes could be interpreted as a compensatory biological response to inhibition of cytochrome P-450dependent steroidogenic enzymes. To support this hypothesis, a network of functional genes of the pituitary-gonad axis was used. We showed that clotrimazole induce a cascade of molecular events in pituitary and testes. Transcript levels of genes encoding for pituitary Gonadotropin releasing hormone receptors (GnRH-R) and folliculo - stimulating hormone (FSH) - subunit, as well as testicular FSH receptor and steroidogenic enzymes were induced. All together, these molecular events are consistent with the involvement of FSH in inducing steroidogenic gene expressions to compensate the inhibitory action of clotrimazole on 11-KT synthesis. Our study highlights the relevance of studying a network of relevant genes of the pituitary-gonad axis to investigate the mode of action of clotrimazole on the endocrine system of fish. Such approach could be extended to other compounds acting as inhibitor of P450-steroidogenic enzymes. The disruption of testicular steroidogenesis raises further concerns about the impact of clotrimazole on reproduction

Nitrate-induced photodegradation of chlorotoluron: ecotoxicological effects in aquatic mesocosms

National audienc

Characterization of testicular expression of P450 17alpha-hydroxylase, 17,20-lyase in zebrafish and its perturbation by the pharmaceutical fungicide clotrimazole

International audienceThe aim of the present study was to characterize P450 17alpha-hydroxylase/17,20-lyase (cyp17a1) expression in zebrafish and to assess the effect of the pharmaceutical clotrimazole, a known inhibitor of various cytochrome P450 enzyme activities, on testicular gene and protein expression of this enzyme as well as on the testicular release of 11-ketotestosterone (11-KT), a potent androgen in fish. We first showed that cyp17a1 is predominantly expressed in gonads of zebrafish, notably in male. In vivo, clotrimazole induced a concentration-dependent increase of cyp17a1 gene expression and Cyp17-I protein synthesis in zebrafish testis. Using zebrafish testicular explants, we further showed that clotrimazole did not directly affect cyp17a1 expression but that it did inhibit 11-KT release. These novel data deserve further studies on the effect of azole fungicides on gonadal steroidogenesis

Cyp17a1 and Cyp19a1 in the zebrafish testis are differentially affected by oestradiol

International audienceOestrogens can affect expression of genes encoding steroidogenic enzymes in fish gonads. However, little information is available on their effects at the protein level. In this context, we first analysed the expression of key steroidogenic enzyme genes and proteins in zebrafish testis, paying attention also to other cell types than Leydig cells. Gene expression was analysed by quantitative PCR on fluorescence-activated cell-sorting fractions coupled or not to differential plating, while protein synthesis was studied by immunohistochemistry using specific antibodies against zebrafish Cyp17a1, Cyp19a1a and Cyp19a1b. Furthermore, we have evaluated the effect of oestrogen treatment (17 beta-oestradiol (E-2), 10 nM) on the localization of these enzymes after 7 and 14 days of in vivo exposure in order to study how oestrogen-mediated modulation of their expression is linked to oestrogen effects on spermatogenesis. The major outcomes of this study are that Leydig cells express Cyp17a1 and Cyp19a1a, while testicular germ cells express Cyp17a1 and both, Cyp19a1a and Cyp19a1b. As regards Cyp17a1, both protein and mRNA seem to be quantitatively dominating in Leydig cells. Moreover, E-2 exposure specifically affects only Leydig cell Cyp17a1 synthesis, preceding the disruption of spermatogenesis. The oestrogen-induced suppression of the androgen production capacity in Leydig cells is a major event in altering spermatogenesis, while germ cell steroidogenesis may have to be fuelled by precursors from Leydig cells. Further studies are needed to elucidate the functionality of steroidogenic enzymes in germ cells and their potential role in testicular physiology

Effect of several azole fungicides on biosynthesis of androgen using an <em>ex vivo</em> zebrafish testis explants culture

International audienc

Effect of several azole fungicides on biosynthesis of androgen using an ex vivo Zebrafish Testis explants Culture

It is known that several azole fungicides can affect the expression and activities of some steroidogenic P450 enzymes in vertebrates, including fish. Among them, propiconazole (PPI), fenbuconazole (FB), clotrimazole (CLO) and ketoconazole (KTZ) have been shown to strongly inhibit ovarían aromatase activities but little is known about their potential effect on hormonal biosynthesis by male gonads. In this study, a recently developped zebrafish testicular explant culture system was used to assess their effect on biosynthesis of 11- ketotestosterone (11KT), a potent androgen in fish as well as on expression of some key steroidogenic genes (star, cyp17a1, cyp11b). In this assay, fungicides were tested alone or in combination with forskolin (FSK, 1uM), an activator of the cAMP pathway that stimulates steroidogenesis. After 6 days of ex vivo exposure to FSK, steroidogenic genes expression was strongly induced as well as 11KT release in the culture medium. Interestingly, azole fungicides (0.2 to 5uM), alone or in combination with FSK did not changed basal or FSK- induced steroidogenic genes expression. However, they inhibited both the basal and FSK-induced 11KT release. The ability of CLO and KTZ to inhibit 11KT release occured at 1uM while for FB and PPI significant inhibitions were observed only at 5uM suggesting that these compounds were less active compared to CLO and KTZ. To conclude, the zebrafish testicular explant culture system was usefull to demonstrate the inhibitory action of azoles on 11KT biosynthesis. Inhibition of 11KT may rely on their ability to interact directly with steroidogenic enzymatic complex as inhibitors. The testicular explant culture system can serve as a tool to identify chemicals that disrupt sex steroid biosynthesi