A novel transgenic zebrafish line (cyp19a1a-GFP) : refinement of an OECD test guideline for evaluating the effects of prochloraz on aromatase gene expression and reproduction
Résumé
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.