Improvement of chemically-activated luciferase gene expression bioassay for detection of dioxin-like chemicals.
OBJECTIVE
To improve the chemically-activated luciferase expression (CALUX) bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms of DLCs.


METHODS
A recombinant vector was constructed and used to transfect human hepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2 used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor (AhR)-meditated luciferase gene expression. The reliability of luciferase induction in this cell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection time was examined and a comparison was made by using the commonly used ethoxyresoufin-O-deethylase (EROD) activity induction assay.


RESULTS
The results suggested that the luciferase activity in recombinant cells was peaked at about 4 h and then decreased to a stable activity by 14 h after TCDD treatment. The detection limit of this cell line was 0.11 pmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100 pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%.


CONCLUSIONS
The luciferase induction is 30-fold more sensitive than EROD induction, the detection time is 68 h shorter and the detection procedure is also simpler.
Zhang
Zhi-Ren
ZR
Institute of Environmental Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Xu
Shun-Qing
SQ
Zhou
Yi-Kai
YK
Xu
Yong-Jun
YJ
Liu
Zhi-Wei
ZW
Cai
Xiao-Kun
XK
Tan
Xiang-Lin
XL
D001681:Biological Assay / Q000379:methods; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis; D004785:Environmental Pollutants / Q000009:adverse effects* / Q000494:pharmacology; D004790:Enzyme Induction; D005786:Gene Expression Regulation; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis*; D000072317:Polychlorinated Dibenzodioxins / Q000009:adverse effects* / Q000494:pharmacology; D012680:Sensitivity and Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
D004785:Environmental Pollutants; D000072317:Polychlorinated Dibenzodioxins; D008156:Luciferases; D019363:Cytochrome P-450 CYP1A1