[Show abstract][Hide abstract] ABSTRACT: Clotrimazole is an azole fungicide used as a human pharmaceutical that is known to inhibit cytochrome P450 (CYP) enzymatic activities, including several steroidogenic CYP. In a previous report, we showed that a 7-day exposure to clotrimazole induced the expression of genes related to steroidogenesis in the testes as a compensatory response, involving the activation of the Fsh/Fshr pathway. In this context, the aim of the present study was to assess the effect of an in vivo 21-day chronic exposure to clotrimazole (30-197 μg/L) on zebrafish testis function, i.e., spermatogenesis and androgen release. The experimental design combined (1) gene transcript levels measurements along the brain-pituitary-gonad axis, (2) 11-ketotestosterone (11-KT) quantification in the blood, and (3) histology of the testes, including morphometric analysis. The chronic exposure led to an induction of steroidogenesis-related genes and fshr in the testes as well as fshβ in the pituitary. Moreover, increases of the gonadosomatic index and of the volume proportion of interstitial Leydig cells were observed in clotrimazole-exposed fish. In accordance with these histological observations, the circulating concentration of 11-KT had increased. Morphometric analysis of the testes did not show an effect of clotrimazole on meiotic (spermatocytes) or postmeiotic (spermatids and spermatozoa) stages, but we observed an increase in the number of type A spermatogonia, in agreement with an increase in mRNA levels of piwil1, a specific molecular marker of type A spermatogonia. Our study demonstrated that clotrimazole is able to affect testicular physiology and raised further concern about the impact of clotrimazole on reproduction.
Full-text · Article · Jan 2013 · Environmental Science and Pollution Research
[Show abstract][Hide abstract] ABSTRACT: Estrogens 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 analyzed 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 (17β-œstradiol, 10 nM) on the localization of these enzymes after 7 and 14 days of in vivo exposure in order to study how œstrogen-mediated modulation of their synthesis is linked to œstrogen 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, 17β-œstradiol exposure specifically affects only Leydig cell Cyp17a1 synthesis, preceding the disruption of spermatogenesis. The œstrogen-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 the testicular physiology.
Full-text · Article · Jan 2013 · Journal of Endocrinology
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Clotrimazole is a pharmaceutical fungicide known to inhibit several cytochrome P450 enzyme activities, including several steroidogenic enzymes. This study aimed to assess short-term in vivo effects of clotrimazole exposure on blood 11-ketotestosterone (11-KT) levels and on the transcriptional activity of genes in pituitary and testis tissue that are functionally relevant for androgen production with the view to further characterize the mode of action of clotrimazole on the hypothalamus-pituitary-gonad axis in zebrafish, a model vertebrate in toxicology. Adult male zebrafish were exposed to measured concentrations in water of 71, 159 and 258μg/L of clotrimazole for 7 days. Expression of pituitary gonadotropins β subunit (lhb, fshb), testicular gonadotropins receptors (lhcgr, fshr) and testicular steroidogenesis-related genes (e.g., star, cyp17a1, cyp11c1) were assessed. Blood concentrations of 11-KT were measured. Short-term exposure to clotrimazole induced a concentration-dependent increase of star, cyp17a1, and cyp11c1 gene expression and Cyp17a1 and Cy11c1 protein synthesis in Leydig cells, but androgen levels in blood remained unchanged. fshb, but not lhb mRNA levels in the pituitary tended to increase in clotrimazole-exposed zebrafish. Testicular expression of the Fsh receptor gene was significantly up-regulated following exposure, when expression of this receptor was significantly correlated to the expression of steroidogenesis-related genes. Moreover, the Fsh-regulated insulin-like growth factor 3 (igf3) gene, a fish-specific Igf peptide expressed in Sertoli cells, was induced in testes. By using a network of genes functioning in pituitary and testis tissue, our study demonstrated that clotrimazole induced a cascade of molecular and cellular events which are in agreement with a role for Fsh (1) in stimulating Leydig cell steroidogenesis to compensate the inhibitory action of clotrimazole on 11-KT synthesis and (2) in inducing the expression of Fsh-regulated igf3 in Sertoli cells.
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to characterize P450 17α-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.
Full-text · Article · Sep 2011 · General and Comparative Endocrinology
[Show abstract][Hide abstract] ABSTRACT: 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 biosynthesis
[Show abstract][Hide abstract] ABSTRACT: 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 reproduction