The Thyroid Hormone Receptor Mediated Luciferase Reporter Gene Assays Screening for EDCs
Abstract
This study in order to use report gene assay based on the thyroid hormone receptor (TR) α/β from human origin for screening endocrine disruptors chemicals (EDCs), evaluating the thyroid hormone activity of Bisphenol (BPA), 1-Naphthaleny methyl carbamate and 1-naphthol (1-NAP). Methods Using Rhesus monkey kidney cells (LLC-MK2) as transfection cell to establish the gene report assay system based on pGL-3-promega and pGL4.27 of TRα/β through the method of transient transfection. Using T3 and T4 as positive subjects to evaluation the effectiveness of two detection systems and detect the thyroid hormone activity of BPA, 1-Naphthaleny methyl carbamate, 1-NAP. Results The TRβ LLC-MK2 report gene assay based on pGL3-promega, the minimum detectable limit of T3 is 1.216×10-11 mol/L, the largest induction multiple was shown at 7.482×10-6 mol/L, the expression multiple of induced Lucifrerasewas 5.98-fold that of the vehicle control, the EC50 was 3.327×10-8 mol/L; The minimum detectable limit of T4 was 1.622×10-8 mol/L, the largest induction Luc expression was 3.4-fold of vehicle control, the EC50 was 2.213×10-7 mol/L. The TRβ LLC-MK2 report gene assay based on pGL4.27, the minimum detectable limit of T3 was 9.863×10-12 mol/L, the largest induction Luc expression as shown at 1.671×10-6 mol/L, resulting in 8.57-fold of vehicle control, the EC50 is 3.327×10-8 mol/L. The minimum detectable limit of T4 was 1.349×10-9mol/L, the largest induction Luc expression was 4.6-fold of vehicle control, the EC50 is 4.074×10-7 mol/L. There was no thyroid hormone activity by using TRβ report gene assay to evaluate BPA, 1-Naphthaleny methyl carbamate or 1-NAP, but 1-Naphthaleny methyl carbamate and1-NAP have some degree receptor antagonism. Conclusion The TRβ LLC-MK2 report gene assay based on pGL3-] promega and pGL4.27 show highly sensitive (pGL4.27 relatively higher), can be used to screen for EDCS and test chemical thyroid hormone activity effectively.
Keywords: Thyroid hormone receptors, Endocrine disrupting chemicals, Receptor reporter gene assay, Luciferase report gene
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