Establishment of a cell-free bioassay for detecting dioxin-like compounds
Journal
Toxicology Mechanisms and Methods
Journal Volume
23
Journal Issue
6
Pages
464-470
Date Issued
2013
Author(s)
Abstract
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 cells that stably co-express Renilla luciferase tagged-AhR (AhR-RL), Ah receptor-interacting protein (AIP), p23 and yellow fluorescent protein-tagged Arnt (Arnt-YFP) (AAPA cells) for detection of dioxin-like compounds. Treatment with 3-methylcholanthrene (3MC), AhR agonist, enhanced the interaction between AhR and Arnt and avoided proteosomal degradation. In addition, treatment with 3MC or TCDD stabilized Renilla luminescence from 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 10-18M. These results highlight the potential of AAPAA cell-free extracts to detect dioxin-like pollutants. © 2013 Informa Healthcare USA, Inc. All rights reserved: reproduction in whole or part not permitted.
Subjects
AhR; Arnt; Bioassay; Dioxin
Other Subjects
2,3,7,8 tetrachlorodibenzo para dioxin; 3 methylcholanthrene; aromatic hydrocarbon receptor; dioxin; Renilla luciferin 2 monooxygenase; yellow fluorescent protein; article; bioassay; bioluminescence; cell free system; cost effectiveness analysis; human; human cell; limit of detection; nucleotide sequence; priority journal; protein degradation; Aryl Hydrocarbon Receptor Nuclear Translocator; Biological Assay; Blotting, Western; Dioxins; Environmental Pollutants; Fluorescent Dyes; HEK293 Cells; Humans; Limit of Detection; Luciferases, Renilla; Receptors, Aryl Hydrocarbon; Recombinant Fusion Proteins; Transfection; Renilla; Renilla luciferase
Type
journal article