title: Novel cell-based assay for detection of thyroid receptor beta-interacting environmental contaminants.
abstract: Even though the presence of endocrine disrupting chemicals (EDCs) with thyroid hormone (TH)-like activities in the environment is a major health concern, the methods for their efficient detection and monitoring are still limited. Here we describe a novel cell assay, based on the translocation of a green fluorescent protein (GFP)-tagged chimeric molecule of glucocorticoid receptor (GR) and the thyroid receptor beta (TRβ) from the cytoplasm to the nucleus in the presence of TR ligands. Unlike the constitutively nuclear TRβ, this GFP-GR-TRβ chimera is cytoplasmic in the absence of hormone while translocating to the nucleus in a time- and concentration-dependent manner upon stimulation with triiodothyronine (T3) and thyroid hormone analogue, TRIAC, while the reverse triiodothyronine (3,3',5'-triiodothyronine, or rT3) was inactive. Moreover, GFP-GR-TRβ chimera does not show any cross-reactivity with the GR-activating hormones, thus providing a clean system for the screening of TR beta-interacting EDCs. Using this assay, we demonstrated that Bisphenol A (BPA) and 3,3',5,5'-Tetrabromobisphenol (TBBPA) induced GFP-GR-TRβ translocation at micro molar concentrations. We screened over 100 concentrated water samples from different geographic locations in the United States and detected a low, but reproducible contamination in 53% of the samples. This system provides a novel high-throughput approach for screening for endocrine disrupting chemicals (EDCs) interacting with TR beta.
authors.affiliation: Laboratory of Receptor Biology and Gene Expression, Building 41, B602, 41 Library Dr., National Cancer Institute, NIH, Bethesda, MD 20892-5055, United States. Electronic address: stavrevd@mail.nih.gov.
authors.forename: Diana A
authors.initials: DA
authors.lastname: Stavreva
authors.affiliation: Laboratory of Receptor Biology and Gene Expression, Building 41, B602, 41 Library Dr., National Cancer Institute, NIH, Bethesda, MD 20892-5055, United States.
authors.forename: Lyuba
authors.initials: L
authors.lastname: Varticovski
authors.affiliation: Department of Physics and Astronomy, Physics and Astronomy, University of Utah, Salt Lake City, UT, United States.
authors.forename: Ludmila
authors.initials: L
authors.lastname: Levkova
authors.affiliation: Laboratory of Receptor Biology and Gene Expression, Building 41, B602, 41 Library Dr., National Cancer Institute, NIH, Bethesda, MD 20892-5055, United States.
authors.forename: Anuja A
authors.initials: AA
authors.lastname: George
authors.affiliation: Laboratory of Receptor Biology and Gene Expression, Building 41, B602, 41 Library Dr., National Cancer Institute, NIH, Bethesda, MD 20892-5055, United States.
authors.forename: Luke
authors.initials: L
authors.lastname: Davis
authors.affiliation: Laboratory of Receptor Biology and Gene Expression, Building 41, B602, 41 Library Dr., National Cancer Institute, NIH, Bethesda, MD 20892-5055, United States.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Pegoraro
authors.affiliation: U.S. Geological Survey, Leetown Science Center, National Fish Health Research Laboratory, 11649 Leetown Road, Kearneysville, WV 25430, United States.
authors.forename: Vicki
authors.initials: V
authors.lastname: Blazer
authors.affiliation: U.S. Geological Survey, Leetown Science Center, National Fish Health Research Laboratory, 11649 Leetown Road, Kearneysville, WV 25430, United States.
authors.forename: Luke
authors.initials: L
authors.lastname: Iwanowicz
authors.affiliation: Laboratory of Receptor Biology and Gene Expression, Building 41, B602, 41 Library Dr., National Cancer Institute, NIH, Bethesda, MD 20892-5055, United States. Electronic address: hagerg@exchange.nih.gov.
authors.forename: Gordon L
authors.initials: GL
authors.lastname: Hager
mesh_terms: D001559:Benzhydryl Compounds / Q000032:analysis; D001681:Biological Assay; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D052244:Endocrine Disruptors / Q000032:analysis*; D004785:Environmental Pollutants / Q000032:analysis*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D061986:MCF-7 Cells; D010636:Phenols / Q000032:analysis; D011075:Polybrominated Biphenyls / Q000032:analysis; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D037042:Thyroid Hormone Receptors beta / Q000378:metabolism*; D014158:Transcription, Genetic; D014178:Translocation, Genetic; D014284:Triiodothyronine / Q000031:analogs & derivatives / Q000378:metabolism; D000098826:Bisphenol A Compounds
chemical_list: D001559:Benzhydryl Compounds; D052244:Endocrine Disruptors; D004785:Environmental Pollutants; D049452:Green Fluorescent Proteins; D010636:Phenols; D011075:Polybrominated Biphenyls; D011965:Receptors, Glucocorticoid; D037042:Thyroid Hormone Receptors beta; D014284:Triiodothyronine; C010642:3,3',5-triiodothyroacetic acid; C020806:tetrabromobisphenol A; C006780:bisphenol A; D000098826:Bisphenol A Compounds
keywords: BPA; EDCs; High-throughput cell assay; TBBPA; TRβ