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

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.