Establishment of a cell-free bioassay for detecting dioxin-like compounds

Autor:	Ming Ching Hsu, Bo Jeng Wang, Tzu Ching Tsai, Yi-Chien Lu, Hsinyu Lee, Pei Yi Wu, Chi-Hao Chang, Yueh Chien Lin
Rok vydání:	2013
Předmět:	Aryl hydrocarbon receptor nuclear translocator Recombinant Fusion Proteins Health Toxicology and Mutagenesis Blotting Western Dioxins Transfection Toxicology Limit of Detection Humans Bioassay heterocyclic compounds Receptor Fluorescent Dyes Luciferases Renilla biology Chemistry Aryl Hydrocarbon Receptor Nuclear Translocator HEK 293 cells Aryl hydrocarbon receptor Hsp90 HEK293 Cells Receptors Aryl Hydrocarbon Biochemistry Environmental chemistry biology.protein Biological Assay Environmental Pollutants Signal transduction
Zdroj:	Toxicology Mechanisms and Methods. 23:464-470
ISSN:	1537-6524 1537-6516
DOI:	10.3109/15376516.2013.781254
Popis:	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(-18 )M. These results highlight the potential of AAPAA cell-free extracts to detect dioxin-like pollutants.
Databáze:	OpenAIRE
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