| Popis: |
Dioxin-responsive element (DRE)-driven luciferase reporter assay is a rapid and cost-effective screening method for detection and semi-quantitation of dioxins and dioxin-like chemicals in samples from a variety of matrices. However, most of such stable cell lines gradually lose their dioxin responsiveness over time, possibly due to cellular degradation and/or silence of the transfected reporter gene. The study aimed to establish a recombinant adenovirus-based DRE-driven luciferase reporter system for easy maintenance and improvement of dioxin sensitivity/responsiveness. Both the Ad-4xDR adenovirus-based and the Huh7-4xDRE-Luc cell-based reporter systems carry a luciferase reporter gene driven by the same 4xDRE-TATA cassette, i.e., 4 copies of DRE and a TATA box. Comparison of the two reporter systems indicated that the adenovirus-based system was much more sensitive and responsive to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) than the cell-based system. Among the four recombinant adenoviruses, Ad-4xDR, Ad-6xDR, Ad-9xDR, and Ad-15xDR carrying 4, 6, 9, and 15 copies of DRE, respectively, the Ad-6xDR-infected H4IIE cells showed the highest TCDD responsiveness. Following optimization of assay conditions, including temperature, dimethyl sulfoxide (DMSO) concentration, and multiplicity of infection (MOI) of adenovirus, the Ad-6xDR-based system exhibited markedly superior performance in TCDD sensitivity (limit of detection (LOD) = 0.02 pM) and responsiveness (the half maximal effective concentration ( EC 50 ) = 10.41 pM and a maximum of 409.34 folds of luciferase induction at 300 pM TCDD). Analysis of soil samples showed a high correlation between the Ad-6xDR-based reporter assay and high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). The study reveals that, as compared with our previous cell-based system, the Ad-6xDR-based reporter system is more reliable and sensitive for high-throughput screening of dioxins. |
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