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Les fongicides azolés ( e.g. prochloraze, PCZ ; imazalil, IMZ) sont retrouvés dans l’environnement et notamment dans les milieux aquatiques à des concentrations pouvant aller jusqu’à 2000 ng/L pour le PCZ (Kreuger, 1998) et 410 ng/L pour l’IMZ (CCanccapa et al., 2015). Un de leurs mécanismes de toxicité bien connu est leur capacité à inhiber le cytochrome P450 aromatase (aromatase A, codée par le gène cyp19a1a) (Hinfray et al., 2006). L'altération de ce complexe enzymatique au niveau des gonades est un mécanisme d'action de perturbation endocrinienne souvent étudié. C’est d’ailleurs une des rares voies de toxicité pour laquelle une qAOP ( i.e. quantitative Adverse Outcome Pathway, dit voie de toxicité quantitative) est disponible. Celle-ci a été développée chez le vairon à tête-de-boule ( Pimephales promelas) et appliquée au fadrozole, inhibiteur non stéroïdien hautement spécifique de l'aromatase (Conolly et al., 2017). Cependant, de récentes études suggèrent fortement que l’intégration du profil toxicocinétique (TK) des molécules étudiées permettrait d’améliorer les prédictions des relations quantitatives existantes entre chaque évènement clé décrites dans cette qAOP. Ainsi, l’objectif de ce travail est de comprendre et modéliser le processus ADME ( i.e. Absorption, Distribution, Métabolisation, Elimination) du PCZ et de l’IMZ chez le poisson. Pour cela, des mesures de concentrations internes ont été réalisées au niveau des gonades, du foie et de la carcasse de poissons zèbres exposés à trois concentrations d’IMZ (3, 30, 300 µg/L). Ces données en complément de celles disponibles au sein de l’Ineris et issues de la littérature ont permis de paramétrer un modèle de toxicocinétique à fondement physiologique ( i.e. PBTK) (Grech et al., 2019) chez le poisson zèbre appliqué au PCZ et à l’IMZ. De plus, les paramètres de métabolisation ont été estimés à partir de données ex vivo permettant d’améliorer le processus de métabolisation chez le poisson zèbre. Finalement, la modélisation du profil TK de ces substances a permis de prédire les concentrations internes dans les organes cible chez le poisson mâle et femelle. Ainsi, intégrer ce modèle PBTK à une qAOP permettra d’améliorer les prédictions des relations quantitatives qui y sont décrites (Villeneuve et al., 2021).

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (Test Guideline 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 µg/L). In addition to “classical” endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that the highest PCZ concentration significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 µg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. These experiments showed that the physiological responses observed in control and PCZ-exposed cyp19a1aeGFP transgenic zebrafish (E2, VTG, reproduction) are similar to those of wild-type zebrafish, both qualitatively and quantitatively. Besides, the cyp19a1a-eGFP model reliably informs on the time- and concentrationdependent effects of PCZ on ovarian aromatase through in vivo GFP fluorescence of the ovaries, thereby providing novel mechanistic information without increasing the number of animals needed. Overall, the cyp19a1a-eGFP transgenic zebrafish line proved to be a relevant model to study the effects of EDCs in fish, which might also be wisely used to refine mechanism-based assays such as OECD TG 229.

International audience; Regulatory assays available from the OECD for the testing of chemicals, such as endocrine disrupting chemicals (EDC) testing (TG229, TG230), can be performed with zebrafish without any recommendation regarding the strain that should be used. In addition to endpoints defined in the TG, transgenic fish could inform on mechanisms underlying EDC effects. By avoiding interference with pigmentation during imaging, use of casper background may improve tests sensitivity. In the present study, we compared 4 zebrafish lines (wild type AB, casper, cyp19a1a-eGFP and cyp19a1a-eGFP-casper) to determine whether transgene insertion and mutations could modify their basal physiology and their response to pollutant, thereby impeding their use for EDC testing. We thus compared the behavior (anxiety, sociability) and reproductive parameters (circulating vitellogenin (VTG) concentrations, female fecundity, egg viability) of control fish of the 4 lines and evaluated their responses to a model pollutant, e.g. clotrimazole. Sociability was evaluated by monitoring the time spent near congeners in a shuttle box while anxiety was evaluated using the novel tank diving test. No difference was observed between lines for either sociability or anxiety level. Concerning reproduction, no significant difference in the number of eggs laid per female, in the viability of eggs or in the female circulating VTG concentrations was noted between the 4 lines studied. Clotrimazole exposure (28 days; 250 µg/L) had similar effects in the 4 zebrafish lines despite a slightly higher sensitivity in wild type AB. Clotrimazole led to a masculinization of one part of the females, an inhibition of VTG, an inhibition of aromatase A expression (GFP measurement and qPCR) and an induction of 11-ketotestosterone. In summary, no critical difference in the behavior, reproductive parameters and response to pollutant was found between the 4 zebrafish lines, indicating that the transgene insertion and the mutations did not modify basal physiology of the fish. Therefore, their use in OECD tests for the study of EDC appears to be relevant. Further work will be conducted on strains sensitivity by evaluating dose-response effects of pollutants.

Concern about the effects of endocrine disrupting chemicals (EDCs) on reproductive health has stimulated the development of mechanism-based models and assays. In this regard, transgenic fish are powerful biological models that can provide mechanistic information regarding the endocrine activity of test chemicals. The objective of this study is to assess the feasibility of using a new biological model in an OECD fish screening assay to provide additional mechanistic information, as compared to wild-type models. In the present study, we used a transgenic zebrafish line cyp19a1a-GFP expressing GFP under the control of the “ovarian” aromatase promoter. Based on the OECD Fish Short Term Reproduction Assay (TG229), the effect of a prochloraz (PCZ; 3, 30 and 300µg/L), was assessed on “classical” endpoints. Additionally, fluorescence of ovarian GFP was monitored in vivo at 4 times from T0, to determine the baseline level of fluorescence, to T21 days of exposure to determine the kinetic of perturbation. After 21 days of exposure, a significant decrease in the number of eggs laid per female per day was observed at 300 µg/L. PCZ led to a concentration-dependent inhibition of circulating vitellogenin concentrations in females most probably reflecting the decreasing E2 synthesis due to inhibition of ovarian aromatase activity. GFP intensities were similar over treatment groups at 7 and 14 days of exposure but significantly increased after 21 days of exposure at 30 and 300 µg/L. A similar profile was observed on the endogenous cyp19a1a gene expression analyzed by qPCR. The overexpression of the aromatase gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities consistent with the literature on PCZ. Thus, the cyp19a1a-GFP transgenic zebrafish line allows in vivo non-invasive monitoring over time of the GFP fluorescence of the ovary, providing information on aromatase expression and its perturbation. The protocol developed for GFP measures has no observable effects on reproduction and survival of fish. Moreover, it allows evaluation of EDCs effects over time and concentrations. Taken together, our findings highlight that the transgenic cyp19a1a-GFP model is a sensitive and relevant tool, which can bring complementary data without increasing the number of individuals or costs of the experiments.

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study our objective was to refine the OECD Fish Short Term Reproduction Assay (TG229) using a new transgenic zebrafish line expressing GFP under the control of the cyp19a1a promoter, to provide additional mechanistic information. For this purpose, we exposed this zebrafish line using prochloraz (PCZ), one of the TG229 reference molecule, at 3, 30 and 300 µg/L. In addition to “classical” endpoints, the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. At the end of the experiment, we found that PCZ significantly decreased the number of eggs laid per female per day at 300 µg/L and the concentrations of vitellogenin in females, most probably reflecting the decreasing E2 synthesis due to inhibition of the ovarian aromatase activities. Additional information on the mode of action of PCZ were provided through the measurement of GFP intensities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 30 and 300 µg/L. A similar profile was observed for cyp19a1a expression (qPCR) thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-GFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information of the time- and concentration-dependent effect on a key target gene through in vivo GFP imaging without increasing the number of individuals or costs of the experiments.