Editorial trend: adverse outcome pathway (AOP) and computational strategy - towards new perspectives in ecotoxicology.

The adverse outcome pathway (AOP) has been conceptualized in 2010 as an analytical construct to describe a sequential chain of causal links between key events, from a molecular initiating event leading to an adverse outcome (AO), considering several levels of biological organization. An AOP aims to identify and organize available knowledge about toxic effects of chemicals and drugs, either in ecotoxicology or toxicology, and it can be helpful in both basic and applied research and serve as a decision-making tool in support of regulatory risk assessment. The AOP concept has evolved since its introduction, and recent research in toxicology, based on integrative systems biology and artificial intelligence, gave it a new dimension. This innovative in silico strategy can help to decipher mechanisms of action and AOP and offers new perspectives in AOP development. However, to date, this strategy has not yet been applied to ecotoxicology. In this context, the main objective of this short article is to discuss the relevance and feasibility of transferring this strategy to ecotoxicology. One of the challenges to be discussed is the level of organisation that is relevant to address for the AO (population/community). This strategy also offers many advantages that could be fruitful in ecotoxicology and overcome the lack of time, such as the rapid identification of data available at a time t, or the identification of "data gaps". Finally, this article proposes a step forward with suggested priority topics in ecotoxicology that could benefit from this strategy.

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

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.

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

Oestrogens 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 (17ß-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 in vivo chronic exposure to clotrimazole on zebrafish testis function.

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 fshß 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.

A critical role of follicle-stimulating hormone (Fsh) in mediating the effect of clotrimazole on testicular steroidogenesis in adult zebrafish.

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 ß 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.

Ecotoxicological effects of diuron and chlorotoluron nitrate-induced photodegradation products: monospecific and aquatic mesocosm-integrated studies.

The ecotoxicological impact of nitrate-induced photodegradation products of diuron and chlorotoluron was studied through monospecific biotests conducted in conjunction with experiments in outdoor aquatic mesocosms. Organisms representing three trophic levels were used: two heterotrophic microorganisms, the luminescent bacterium Vibrio fischeri and the ciliated protozoa Tetrahymena pyriformis, and one metazoa, the gastropod Lymnaea stagnalis. Among the variety of the phenylurea photoproducts, the N-formylated ones appeared clearly more toxic than the parent compounds towards the microorganisms, whereas the nitroderivatives showed a similar toxicity. Using photodegraded solutions of diuron, toxicity was maintained or even increased during disappearance of the initial herbicide, demonstrating that some of the photoproducts may have an impact additively or in synergy. Enzymatic biomarker assays performed on Lymnaea stagnalis exposed under monospecific conditions showed significant effects, due to the combination of nitrate with the pesticide and its photoproducts. A positive impact on snail fecundity was observed with chlorotoluron both under monospecific laboratory and integrated mesocosm conditions. Oviposition stimulation took place when first- and second-generation photoproducts were predominant.