Differential activity of BPA, BPAF and BPC on zebrafish estrogen receptors in vitro and in vivo

The high volume production compound bisphenol A (BPA) is of environmental concern largely because of its estrogenic activity. Consequently, BPA analogues have been synthesized to be considered as replacement molecules for BPA. These analogues need to be thoroughly evaluated for their estrogenic activity. Here, we combined mechanism zebrafish-based assays to examine estrogenic and anti-estrogenic activities of BPA and two of its analogues, bisphenol AF (BPAF) and bisphenol C (BPC) in vitro and in vivo. In vitro reporter cell lines were used to investigate agonistic and antagonistic effects of the three bisphenols on the three zebrafish estrogen receptors. The transgenic Tg(5 x ERE:GFP) and Cyp19a1b-GFP zebrafish lines were then used to analyze estrogenic and anti-estrogenic responses of the three bisphenols in vivo. BPA, BPAF and BPC were agonists with different potencies for the three zebrafish estrogen receptors in vitro. The potent zfER alpha-mediated activity of BPA and BPAF in vitro resulted in vivo by activation of GFP expression in zebrafish larvae in the heart (zfER alpha-dependent) at lower concentrations, and in the liver (zfER beta-dependent) at higher concentrations. BPC induced zfER beta-mediated luciferase expression in vitro, and the zfER beta agonism led to activation of GFP expression in the liver and the brain in vivo. In addition, BPC acted as a full antagonist on zfER alpha, and completely inhibited estrogen-induced GFP expression in the heart of the zebrafish larvae. To summarize, applying a combination of zebrafish-based in vitro and in vivo methods to evaluate bisphenol analogues for estrogenic activity will facilitate the prioritization of these chemicals for further analysis in higher vertebrates as well as the risk assessment in humans.