Characteristics and application of established luciferase hepatoma cell line that responds to dioxin-like chemicals

摘要

AIM: To establish a luciferase reporter cell line that responds dioxin-like chemicals (DLCs) and on this basis to evaluate its characteristics and application in the determination of DLCs. METHODS: A recombinant luciferase reporter plasmid was constructed by inserting dioxin-responsive element (DREs) and MMTV promoter segments into the pGL_3-promoter plasmid immediately upstream of the luciferase gene, which was structurally demonstrated by fragment mapping analysis in gel electrophoresis and transfected into the human hepatoma cell line HepG_2, both transiently and stably, to identify the inducible expression of luciferase by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The time course, responsive period, sensitivity, structure-inducibility and dose-effect relationships of inducible luciferase expression to DLCs was dynamically observed in HepG_2 cells stably transfected by the recombinant vector (HepG_2-Luc) and compared with that assayed by ethoxyresorufin-Odeethylase (EROD) in non-transfected HepG_2 cells (HepG_2-wt). RESULTS: The inducible luciferase expression of HepG_2-Luc cells was noted in a time-, dose-, and AhR-dependent manner, which peaked at 4 h and then decreased to a stable level at 14 h after TCDD treatment. The responsiveness of HepG_2-Luc cells to TCDD induction was decreased with culture time and became undetectable at 10th month of HepG_2-Luc cell formation. The fact that luciferase activity induced by 3, 3′, 4, 4′-PCB in HepG_2-Luc cells was much less than that induced by TCDD suggests a structure-inducibility relationship existing among DLCs. Within the concentrations from 3.5 x 10~(-12) to 5 x 10~(-9) mol/L, significant correlations between TCDD doses and EROD activities were observed in both HepG_2-luc and HepG_2-wt cells. The correlation between TCDD doses from 1.1 x 10~(-13) to 1 x 10~(-8) mol/L and luciferase activities was also found to be significant in HepG_2-luc cells (r=0.997, p<0.001), but not in their HepG_2-wt counterparts. For the comparison of the enzyme responsiveness between cell lines to TCDD, the luciferase sensitivity and reproducibility in HepG_2-luc cells were both better than that of EROD in HepG_2-wt cells, the former was at 1.1 x 10~(-13) mol/L and 3.5 x 10~(-12) mol/L, and the coefficients of variation (CV) of the latter was 15-30 % and 22-38 %, respectively. CONCLUSION: The luciferase expression of HepG_2-luc cells established in the present study could sensitively respond to the DLCs stimulation and might be a prospective tool for the determination of DLCs.