Selective Activation of Zebrafish Estrogen Receptor Subtypes by Chemicals by Using Stable Reporter Gene Assay Developed in a Zebrafish Liver Cell Line

The number of environmental chemical contaminants suspected to act as endocrine disruptor compounds by interacting with estrogen receptor (ER) signaling pathway has been continuously increasing. To study such interaction, the use of stable reporter gene assays is relevant, but species-specific in vitro screening assays are still lacking to address hazard assessment of estrogenic chemicals in aquatic vertebrates. Here, we describe the development of stable reporter gene assays based on stable expression of subtypes of zebrafish ER (zfER alpha, zfER beta 1, and zfER beta 2) coupled to estrogen response element-driven luciferase in a zebrafish liver (ZFL) cell line. The three established cell models, named ZELH-zfER alpha, ZELH-zfER beta 1, and ZELH-zfER beta 2, expressed stable and significant basal luciferase signal, which was induced by 17 beta-estradiol (E2) in a sensitive and dose-response manner at EC(50)s of 0.2, 0.03, and 0.05nM, respectively. In addition, E2 significantly altered cell proliferation in ZELH-zfER alpha and ZELH-zfER beta 2 cells, but not in parental ZFL and ZELH-zfER beta 1 cells, suggesting a functionality of these two receptors to modulate endogenous gene expression in the transfected clones. The screening of various xenoestrogens from different classes in the three models resulted in different luciferase response patterns. Natural and synthetic estrogens and 1,1,1-trichloro-2-(2 chlorophenyl)-2-(4-chlorophenyl)ethane were active at lower concentrations in ZELH-zfER beta 1 and ZELH-zfER beta 2 than in ZELH-zfER alpha cells, whereas genistein and zearalenone metabolites as well as three benzophenone derivatives preferentially activated zfER alpha. Altogether, the newly established models provide specific and convenient in vitro tool for comparative assessment of zfERs selective activation by chemicals within ZFL cell context.