Refinement of an OECD test guideline for evaluating the effects of EDCs on aromatase gene expression and reproduction using novel transgenic cyp19a1a-eGFP zebrafish
Résumé
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.