ToxSci Advance Access originally published online on April 7, 2004
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Toxicological Sciences 80, 49-53 (2004)
Toxicological Sciences vol. 80 no. 1 © Society of Toxicology 2004; all rights reserved.
Development of an Exonuclease Protection Mediated PCR Bioassay for Sensitive Detection of Ah Receptor Agonists
Institute of Environmental Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
Received January 19, 2004; accepted March 26, 2004
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.
Key Words: Ah receptor; 2,3,7,8- TCDD; exonuclease III; S1 nuclase; real-time PCR.