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ToxSci Advance Access published online on April 7, 2004
Toxicological Sciences, doi:10.1093/toxsci/kfh137
Toxicological Sciences © Society of Toxicology 2004; all rights reserved
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Received January 19, 2004; accepted March 26, 2004
© 2004 Toxicological Sciences © Society of Toxicology 2004; all rights reserved.

Environmental Toxicology

Development of an Exonuclease Protection Mediated PCR Bioassay for Sensitive Detection of Ah Receptor Agonists

Xi Sun 1, Fang Li 1, You-jie Wang 1, Yi-rong Li 1, Yan-hua Su 1, Yuan-yuan Li 1, Hong Yan 1, and Shun-qing Xu 1*

1 Institute of Environmental Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

* To whom correspondence should be addressed. E-mail: shunqing{at}public.wh.hb.cn.


  Abstract

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 electrophoreses 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 0.01pM~10nM of TCDD. Minimal detection limit of the assay was below 0.01pM TCDD and whole detection time was less than 5 hours. Comparison of 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 .


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