title: Third-generation Ah receptor-responsive luciferase reporter plasmids: amplification of dioxin-responsive elements dramatically increases CALUX bioassay sensitivity and responsiveness.
abstract: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like chemicals are widespread and persistent environmental contaminants that produce diverse toxic and biological effects through their ability to bind to and activate the Ah receptor (AhR) and AhR-dependent gene expression. The chemically activated luciferase expression (CALUX) system is an AhR-responsive recombinant luciferase reporter gene-based cell bioassay that has been used in combination with chemical extraction and cleanup methods for the relatively rapid and inexpensive detection and relative quantitation of dioxin and dioxin-like chemicals in a wide variety of sample matrices. Although the CALUX bioassay has been validated and used extensively for screening purposes, it has some limitations when screening samples with very low levels of dioxin-like chemicals or when there is only a small amount of sample matrix for analysis. Here, we describe the development of third-generation (G3) CALUX plasmids with increased numbers of dioxin-responsive elements, and stable transfection of these new plasmids into mouse hepatoma (Hepa1c1c7) cells has produced novel amplified G3 CALUX cell bioassays that respond to TCDD with a dramatically increased magnitude of luciferase induction and significantly lower minimal detection limit than existing CALUX-type cell lines. The new G3 CALUX cell lines provide a highly responsive and sensitive bioassay system for the detection and relative quantitation of very low levels of dioxin-like chemicals in sample extracts.
authors.affiliation: Department of Environmental Toxicology, University of California, Davis, California 95616, USA.
authors.forename: Guochun
authors.initials: G
authors.lastname: He
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Tsutsumi
authors.forename: Bin
authors.initials: B
authors.lastname: Zhao
authors.forename: David S
authors.initials: DS
authors.lastname: Baston
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.forename: Sharon
authors.initials: S
authors.lastname: Heath-Pagliuso
authors.forename: Michael S
authors.initials: MS
authors.lastname: Denison
mesh_terms: D000818:Animals; D001681:Biological Assay; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism; D045744:Cell Line, Tumor; D002999:Clone Cells; D004784:Environmental Monitoring / Q000379:methods; D004790:Enzyme Induction; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D011237:Predictive Value of Tests; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D020218:Response Elements / Q000187:drug effects / Q000235:genetics*; D013045:Species Specificity; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014874:Water Pollutants, Chemical / Q000032:analysis / Q000633:toxicity
chemical_list: D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D014874:Water Pollutants, Chemical; D008156:Luciferases