Several synthetic progestins disrupt the glial cell specific-brain aromatase expression in developing zebra fish

Abstract

The effects of some progestins on fish reproduction have been recently reported revealing the hazard of this class of steroidal pharmaceuticals. However, their effects at the central nervous system level have been poorly studied until now. Notwithstanding, progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. Herein, we investigated the effects of a large set of synthetic ligands of the nuclear progesterone receptor on the glial-specific expression of the zebrafish brain aromatase (cyp19a1b) using zebrafish mechanism-based assays. Progesterone and 24 progestins were first screened on transgenic cyp19a1b-GFP zebrafish embryos. We showed that progesterone, dydrogesterone, drospirenone and all the progesterone-derived progestins had no effect on GFP expression. Conversely, all progestins derived from 19-nortesterone induced GFP in a concentration-dependent manner with EC50 ranging from the low nM range to hundreds nM. The 19-nortestosterone derived progestins levonorgestrel (LNG) and norethindrone (NET) were further tested in a radial glial cell context using U251-MG cells co-transfected with zebrafish ER subtypes (zfERα, zfERβ1 or zfERβ2) and cyp19a1b promoter linked to luciferase. Progesterone had no effect on luciferase activity while NET and LNG induced luciferase activity that was blocked by ICI 182,780. Zebrafish-ERs competition assays showed that NET and LNG were unable to bind to ERs, suggesting that the effects of these compounds on cyp19a1b require metabolic activation prior to elicit estrogenic activity. Overall, we demonstrate that 19-nortestosterone derived progestins elicit estrogenic activity by inducing cyp19a1b expression in radial glial cells. Given the crucial role of radial glial cells and neuro-estrogens in early development of brain, the consequences of exposure of fish to these compounds require further investigation.

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase ( cyp19a1b ) in zebrafish specific in vitro and in vivo bioassays

“…The discrepancy between these studies may be attributed to the differences in LNG concentrations used (one order of magnitude higher in roach (3 µg/L) as compared to three-spined stickleback), to the different developmental stages of the fish (adult three-spined stickleback vs pubertal roach), and to different metabolization/biotransformation capabilities between the two species. Nevertheless, in our study, LNG induced GFP expression in the brain of cyp19a1b-GFP transgenic zebrafish after 4 days of exposure with an EC50 of 4.55x10 -8 M (14.2 µg/L), confirming our previous findings on the estrogenic potency of LNG , Cano-Nicolau et al, 2016. In zebrafish, exposure to 231.95 ng/L of LNG from 4 hours post fertilization (hpf) to 144 hpf also led to a slight induction of vtg expression (1.2 fold), supporting this estrogenic effect of LNG in zebrafish larvae (Zucchi et al 2012).…”

“…ERs either (Cano-Nicolau et al, 2016), suggesting its conversion into estrogenic metabolites, especially in the brain. The differences of sensitivity to LNG of the different biological models (in vitro human with hERs or zfERs, in vitro fish, in vivo fish) could thus rely on differences in LNG metabolization capabilities of these different cell lines/organisms and on differences in binding affinity of metabolites to hERs and zfERs.…”

An Approach for Using In Vitro and In Silico Data to Identify Pharmaceuticals with Potential (Anti‐)Estrogenic Activity in Aquatic Vertebrates at Environmentally Relevant Concentrations

“…Tox21 assays. This cross-reactivity has been shown previously in both in vitro (Jordan et al 1993;Ekena et al 1998) and in vivo (Brion et al 2012;Cano-Nicolau et al 2016) studies and is likely related to common structural features shared by all steroid hormones (Huang et al 2010). The interaction of such ligands with ERs has the potential to induce biological effects.…”

Effects of Elevated β-Estradiol Levels on the Functional Morphology of the Testis - New Insights

“…It is of special interest that the only patient who did not show fatty degeneration in the connective tissue took dydrogesterone, a strong acting progestin. Recently, it has been shown that several synthetic progestins are able to disrupt the aromatase expression in the zebra fish brain60. Thus, dydrogesterone might have blocked the increased aromatase activity caused by the elevated estradiol levels.…”