Use of several zebrafish-based bioassays to study the mechanisms of action and tissue-specific effects of ligands of the progesterone receptor
Résumé
Background and Objectives : To date, most studies on endocrine disrupting compounds have focused on estrogenic compounds with an attention to natural (estradiol, estrone, estriol) and synthetic estrogens (17α- ethinylestradiol). However, far fewer studies focused on natural and synthetic progestins, another class of endocrine disrupting steroids. Yet, progestins are widely used as pharmaceuticals, notably in human medicine as oral contraceptives (often in combination with estrogens) and hormone replacement therapy. This results in a lack of data regarding their toxicological effects and their mechanisms of action. To fulfill these gaps, this work aimed at investigating the modes of action of a broad range of progestins and their effects in fish using a combination of several zebrafish-based bioassays. Methods and Results : A broad range (26) of currently used progestins were screened using in vitro and in vivo reporter gene assays for their capacities to interact with human (h) or zebrafish (zf) nuclear progesterone receptor (PR) and to deregulate the tissue-specific expression of steroidogenic genes in zebrafish embryos. By using human reporter cell lines stably expressing either hPR or zfPR, we revealed marked interspecies differences on the ability of progestins to activate/antagonize PRs. We further documented the interaction of progestins with multiple nuclear steroid receptors and showed that they were able to interact with zfARs, zfERs and zfGRs, potentially disrupting the signaling pathways regulated by these receptors. The estrogenic effects have been revealed in vivo using transgenic cyp19a1b-GFP zebrafish eleuthero-embryos. In this model, the progestins derived from 19-nor-testosterone induced brain aromatase in radial glial cells through an ER-dependent mechanism. Using both GRE-luc zebrafish eleuthero-embryos and the newly developped cyp11c1-GFP zebrafish eleuthero-embryos, the effects of several progestins on the glucocorticoid signaling pathway were investigated and we showed that progestins can negatively affect the hypothalamo-pituitary-interrenal axis. This disruption appeared to cause a downregulation of cortisol levels in larvae. Discussion and Conclusion : This work provides an extensive toxicological characterization of a large number of currently used (anti-)progestin pharmaceuticals on key molecular targets of the endocrine system. We showed the multiplicity and complexity of the modes of actions of progestins, which are able to act on different signaling pathways through their interaction with nuclear receptors. We highlighted major differences between hPR and zfPR in vitro. Furthermore, we showed tissue-specific effects at early developmental stages in brain and interrenal cells of zebrafish embryos exposed to progestins. Overall, this study demonstrates the usefulness of combining different mechanismbased bioassays to characterize the endocrine potency of pharmaceuticals, raising further questions regarding their developmental and reproductive effects.