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Establishment of a cell‐free bioassay for detecting dioxin‐like compounds
Author(s) -
Tsai TzuChing,
Wang BoJeng,
Lee Hsinyu
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.729.1
Subject(s) - aryl hydrocarbon receptor nuclear translocator , aryl hydrocarbon receptor , luciferase , chemistry , bioluminescence , bioassay , receptor , pollutant , cell , laboratory flask , transfection , microbiology and biotechnology , environmental chemistry , biophysics , biochemistry , biology , transcription factor , gene , genetics , organic chemistry
Dioxins are byproducts from incomplete combustion processes and belong to a group of mostly toxic chemicals known as persistent organic pollutants, and 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is considered to be the most toxic species of all dioxin‐like compounds. Analytical chemical processes are employed to determine the specific dioxin content in environmental samples. However, cost‐ineffectiveness and excess time consumption limit their routine utilization. The aryl hydrocarbon receptor (AhR) is the major TCDD receptor. Upon binding to dioxin, the AhR dissociates from Hsp90 and other cofactors. TCDD‐bound AhR subsequently translocates to the nucleus and interacts with the AhR nuclear translocator (Arnt) to induce signal transduction. Here, we describe a highly sensitive and cost‐effective alternative assay based on detecting stability of bioluminescence signals. We generated AAPA cells that stably coexpress Renilla luciferase tagged‐AhR (AhR‐RL), Ah receptor‐interacting protein (AIP), p23 and yellow fluorescent protein‐tagged Arnt (Arnt‐YFP). Treatment with 3‐methylcholanthrene (3MC), a dioxin agonist, enhanced the interaction between AhR and Arnt and avoided proteosomal degradation. In addition, treatment with 3MC and TCDD stabilized Renilla luminescence in AhR‐RL of AAPA cell‐free extracts in a concentration‐dependent manner. The TCDD detection limit in this cell‐free system was as low as 10 −18 M. These results highlight the potential of AAPA cell‐free extracts to detect dioxin‐like pollutants.