Estrogenic effects of two progestins, Levonorgestrel and Norethindrone, in zebrafish

Brain aromatase (cyp19a1b) is a gene that is expressed in radial glial cells of adult zebrafish and that is very sensitive to estrogens; thus, cyp19a1b is commonly used as an endpoint to monitor the potentially estrogenic effects of endocrine disruptors (EDs) found in the environment. The estrogendependent cyp19a1b up-regulation requires the presence of functional estrogen receptors (ERs) and, interestingly, occurs in glial cell context only. Previous studies have shown that, while progesterone has no effects on cyp19a1b expression in the brain of zebrafish some progestins, notably Levonorgestrel and Norethindrone, stimulate cyp19a1b expression in the brain of adult zebrafish, an effect blocked by the anti-estrogen inhibitor ICI 182-780. In order to decipher the mechanisms underlying those effects, i.e. direct binding of progestins to ERs or their metabolization into estrogenic compounds, we an in vitro glial cell-based assay using cyp19a1b as the target gene (Le Page et al. 2006). To this end, the ER-negative glial cell line U251-MG was transfected with the three zebrafish ER subtypes (zfER- α, -β1, and -β2) and the cyp19a1b promoter linked to luciferase reporter gene. Using estradiol (E2) as a positive control, we analyzed the doseresponse (10-9 to 10-6M) effects of natural progesterone (P4) and two synthetic progestins: Levonorgestrel and Norethindrone. As expected, P4 has no effects on the activation of the cyp19a1b promoter at any concentration However, Norethindrone and to a lesser extent Levonorgestrel caused a dose-dependent expression of luciferase. All these effects were suppressed by ICI 182-780. Using ER binding assays, we demonstrated that the affinity of Levonorgestrel and Norethindrone for the three zfERs was very weak, only at doses superior to 10 -6M. This suggests that the activity of progestin in vivo and in vitro is probably due to metabolization through 5α-reductase activity. However, we failed to block the luciferase fold induction of progestins using the 5α-reductase inhibitor finasteride. All together, these data provide evidences that Levonorgestrel and Norethistrone have estrogenic activity in vivo and in vitro. However, the underlying mechanisms ate still not completely clear.