title: Selective activation of zebrafish estrogen receptor subtypes by chemicals by using stable reporter gene assay developed in a zebrafish liver cell line.
abstract: 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 (zfERα, zfERβ1, and zfERβ2) coupled to estrogen response element-driven luciferase in a zebrafish liver (ZFL) cell line. The three established cell models, named ZELH-zfERα, ZELH-zfERβ1, and ZELH-zfERβ2, expressed stable and significant basal luciferase signal, which was induced by 17β-estradiol (E2) in a sensitive and dose-response manner at EC(50)s of 0.2, 0.03, and 0.05 nM, respectively. In addition, E2 significantly altered cell proliferation in ZELH-zfERα and ZELH-zfERβ2 cells, but not in parental ZFL and ZELH-zfERβ1 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-zfERβ1 and ZELH-zfERβ2 than in ZELH-zfERα cells, whereas genistein and zearalenone metabolites as well as three benzophenone derivatives preferentially activated zfERα. 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.
authors: Institut National de l'Environnement Industriel et des Risques (INERIS), Unité d'Ecotoxicologie in vitro et in vivo, Parc Technologique Alata, BP2, f-60550 Verneuil-en-Halatte, France.
authors: Anne
authors: A
authors: Cosnefroy
authors: François
authors: F
authors: Brion
authors: Emmanuelle
authors: E
authors: Maillot-Maréchal
authors: Jean-Marc
authors: JM
authors: Porcher
authors: Farzad
authors: F
authors: Pakdel
authors: Patrick
authors: P
authors: Balaguer
authors: Selim
authors: S
authors: Aït-Aïssa
mesh_terms: D000826:Animal Testing Alternatives; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D052244:Endocrine Disruptors / Q000633:toxicity*; D004958:Estradiol / Q000633:toxicity; D047628:Estrogen Receptor alpha / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D004967:Estrogens / Q000633:toxicity*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D008024:Ligands; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011960:Receptors, Estrogen / Q000819:agonists / Q000235:genetics; D018675:Toxicity Tests; D014162:Transfection; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000819:agonists* / Q000235:genetics / Q000378:metabolism
chemical_list: D052244:Endocrine Disruptors; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D004967:Estrogens; D008024:Ligands; D011960:Receptors, Estrogen; D029961:Zebrafish Proteins; C516067:estrogen receptor 2a, zebrafish; D004958:Estradiol; D008156:Luciferases