Development of an exonuclease protection mediated PCR bioassay for sensitive detection of Ah receptor agonists.
The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. Here we developed a novel method to detect the presence of AhR ligands using Exonuclease Protection Mediated PCR bioassay (EPM-PCR). This assay measures the ability of a chemical to activate AhR DNA binding in vitro. In the presence of AhR ligand, an expected length PCR product was observed on electrophoresis, but no signal was detected in the absence of ligand. Real-time quantitative PCR was performed to quantify DNA bound to ligand:AhR complex. We obtained a standard curve with TCDD concentration to bound DNA copies in the range of 0.01 pM-10 nM of TCDD. Minimal detection limit of the assay was below 0.01 pM TCDD, and the whole detection time was less than 5 h. In comparison to the chemical-activated luciferase gene expression (CALUX) bioassay, EPM-PCR bioassay is more sensitive and easier to perform. These results suggest that this assay is useful for detection and quantification of TCDD and related AhR ligands in a cell-free system without the use of radioactivity.
Sun
Xi
X
Institute of Environmental Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Li
Fang
F
Wang
You-Jie
YJ
Li
Yi-Rong
YR
Su
Yan-Hua
YH
Li
Yuan-Yuan
YY
Yan
Hong
H
Xu
Shun-Qing
SQ
D000818:Animals; D001681:Biological Assay / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D004785:Environmental Pollutants / Q000494:pharmacology; D005092:Exonucleases / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008156:Luciferases / Q000235:genetics; D008297:Male; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists* / Q000737:chemistry
D004785:Environmental Pollutants; D008024:Ligands; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D008156:Luciferases; D005092:Exonucleases