A new bioluminescent cellular assay to measure the transcriptional effects of chemicals that modulate the alpha-1 thyroid hormone receptor.
Interactions of environmental pollutants with the thyroid endocrine axis have received much attention especially because thyroid hormones (THs) play a major role in mammalian brain development. In order to screen for compounds that act on the triiodothyronine (T3) signaling pathway, we developed a new reporter gene assay expressing luciferase under the control of the TH receptor (TR). PC12 cells expressing the alpha1-isoform of TR of avian origin were stably transfected with a luciferase gene controlled by the SV40 promoter, and enhanced by a four-spaced direct repeat (DR4) thyroid response element (TRE). The resulting PC-DR-LUC cells were used to optimize a T3 assay in multiwell microplates. This assay was highly sensitive (30 pM T3) and reproducible, and responded as expected to TH analogues. Several halogenated phenolic (3,3',5,5'-tetrabromobisphenol A, 3,3',5,5'-tetrachlorobisphenol A, 4-hydroxy-2',3,4',5,6'-pentachlorobiphenyl) and phenol (pentachlorophenol, 2,4,6-triiodophenol) compounds suspected of being thyroid-disrupting environmental chemicals induced partial agonistic and/or complex competitive/uncompetitive antagonistic responses in PC-DR-LUC cells at micromolar concentrations. A cell viability test indicated that these effects were not related to cytotoxicity of the chemicals. These results suggest that the PC-DR-LUC assay could be a valuable tool for the large-scale screening for thyroid receptor agonists and antagonists in vitro, and for detecting thyroid disruptors in the environment.
Jugan
M L
ML
Univ Paris-Sud, Faculté de Pharmacie, Laboratoire Santé Publique-Environnement, F-92296 Châtenay-Malabry, France.
Lévy-Bimbot
M
M
Pomérance
M
M
Tamisier-Karolak
S
S
Blondeau
J P
JP
Lévi
Y
Y
D000818:Animals; D001681:Biological Assay; D052244:Endocrine Disruptors / Q000633:toxicity*; D004785:Environmental Pollutants / Q000633:toxicity; D005411:Flame Retardants / Q000633:toxicity; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D049449:Luminescence; D016716:PC12 Cells; D010636:Phenols / Q000633:toxicity; D011075:Polybrominated Biphenyls / Q000633:toxicity; D011078:Polychlorinated Biphenyls / Q000633:toxicity; D051381:Rats; D037021:Thyroid Hormone Receptors alpha / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D052244:Endocrine Disruptors; D004785:Environmental Pollutants; D005411:Flame Retardants; D010636:Phenols; D011075:Polybrominated Biphenyls; D037021:Thyroid Hormone Receptors alpha; D011078:Polychlorinated Biphenyls; D008156:Luciferases; C020806:tetrabromobisphenol A