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

Abstract

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-tranfected with zebrafish estrogen receptors (zfERs) and the luciferase gene under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentrationresponse relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures.

Mixture Concentration-Response Modeling Reveals Antagonistic Effects of Estradiol and Genistein in Combination on Brain Aromatase Gene (cyp19a1b) in Zebrafish

“…Fertilized cyp19a1b -GFP transgenic zebrafish eggs were exposed to chemicals (alone or in mixtures) or to solvent control (DMSO, 0.02% v / v ) according to [ 12 ]. Each experimental condition contained 20 embryos in 100 mL of water.…”

“…The mixture experimental design was developed based on relative potency of single compounds according to [ 12 ].…”

“…Several studies have evaluated the combined effects of estrogenic compounds on aquatic organisms [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ], generally concluding on an additive effect in mixtures both in vivo and in vitro. However, most in vivo studies addressed the effects of mixtures of estrogenic compounds on gonads and other peripheral organs, while only few of them studied effects on the central nervous system despite increasing evidences that estrogenic compounds interfere in neuroendocrine regulations.…”

“…Estrogenic compounds are known to highly up-regulate cyp19a1b gene expression by an ER-dependent mechanism [ 3 , 15 ]. By using zebrafish-specific in vitro and in vivo bioassays based on the cyp19a1b gene coupled to a complete modeling approach, we recently demonstrated additive effects of ethynilestradiol and levonorgestrel, a pro-estrogenic compound, on cyp19a1b gene in glial cells [ 12 ].…”

Emerging Estrogenic Pollutants in the Aquatic Environment and Breast Cancer

“…For example, using wild-type and transgenic Tg(cyp19a1b)-GFP embryo zebrafish, we showed that some progestins are able to exert additive effects to the environmental estrogens, therefore acting as estrogenic-like compounds. This resulted in the activation of the estrogen-specific marker, the cyp19a1b aromatase gene, in the developing zebrafish brain through the activation of ERs [ 99 , 117 ]. Furthermore, we have studied the effects of cadmium (Cd, a heavy metal) exposure on estrogen signaling in zebrafish.…”