title: Establishment of a cell-free bioassay for detecting dioxin-like compounds.
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(-18 )M. These results highlight the potential of AAPAA cell-free extracts to detect dioxin-like pollutants.
authors.affiliation: Institute of Zoology, National Taiwan University, Taipei, Taiwan, ROC.
authors.forename: Bo-Jeng
authors.initials: BJ
authors.lastname: Wang
authors.forename: Pei-Yi
authors.initials: PY
authors.lastname: Wu
authors.forename: Yi-Chien
authors.initials: YC
authors.lastname: Lu
authors.forename: Chi-Hao
authors.initials: CH
authors.lastname: Chang
authors.forename: Yueh-Chien
authors.initials: YC
authors.lastname: Lin
authors.forename: Tzu-Ching
authors.initials: TC
authors.lastname: Tsai
authors.forename: Ming-Ching
authors.initials: MC
authors.lastname: Hsu
authors.forename: Hsinyu
authors.initials: H
authors.lastname: Lee
mesh_terms: D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D015153:Blotting, Western; D004147:Dioxins / Q000032:analysis*; D004785:Environmental Pollutants / Q000032:analysis*; D005456:Fluorescent Dyes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D057230:Limit of Detection; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D004147:Dioxins; D004785:Environmental Pollutants; D005456:Fluorescent Dyes; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D049410:Luciferases, Renilla