title: Establishment of a bioluminescence-based bioassay for the detection of dioxin-like compounds.
abstract: Dioxin and dioxin-like compounds are among the most prevalent and toxic environmental pollutants. At present, analytical chemical techniques are considered the gold standard for detection of dioxins. Here, we describe a highly sensitive and cost-effective alternative, based on bioluminescence and bioluminescence resonance energy transfer (BRET). Upon binding to dioxin, aryl hydrocarbon receptor (AHR) dissociates from HSP90 and subsequently translocates to the nucleus, where it interacts with AHR nuclear translocator (ARNT). We generated cell lines that stably co-express a fusion protein of AHR and Renilla luciferase (AHR-RL) and either HSP90 or ARNT tagged with yellow fluorescent protein (HSP90-YFP or ARNT-YFP). The fluorescent signals of YFP are activated by the emission of RL while the interactions between AHR and HSP90 (or ARNT) were monitored. Application of 3-methylcholanthrene, the AHR agonist, enhances BRET signals in cells co-expressing AHR-RL, AIP-His, P23-His and ARNT-YFP (AAPA cells), while suppressing BRET signals in cells co-expressing AHR-RL, AIP-His, P23-His and HSP90-YFP (AAPH cells). In addition, dioxin treatment reduced Renilla luminescence in AAPH cells in a concentration-dependent manner, due to the degradation of AHR. Intriguingly, the detection limit for dioxin in our AHR degradation assay was as low as 10(-17) M. This work highlights the potential of AHR-RL degradation assays 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: Yung-Feng
authors.initials: YF
authors.lastname: Liao
authors.forename: Ying-Tsen
authors.initials: YT
authors.lastname: Tung
authors.forename: Ling-Huei
authors.initials: LH
authors.lastname: Yih
authors.forename: Cho-Chun
authors.initials: CC
authors.lastname: Hu
authors.forename: Hsinyu
authors.initials: H
authors.lastname: Lee
mesh_terms: D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D015153:Blotting, Western; D004147:Dioxins / Q000032:analysis*; D004785:Environmental Pollutants / Q000032:analysis*; D057809:HEK293 Cells; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057230:Limit of Detection; D049410:Luciferases, Renilla / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D004147:Dioxins; D004785:Environmental Pollutants; D018841:HSP90 Heat-Shock Proteins; D008164:Luminescent Proteins; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D049410:Luciferases, Renilla