Development Of A Screening System For DNA Damage And Repair Of Potential Carcinogens Based On Dual Luciferase Assay In Human HepG2Cell

Objective：Different methods are presently used for the detection of earlybiological effects of DNA-damagingagents in environment. Because DNAdamage and repair play important roles in maintaining geneticstability, DNAdamage and repair have been good biomarkers of early biological effects ofDNAdamagingagents and measurement of individual DNA repair capacityshould be a valued tool inmolecular epidemiology studies. Some sensitivetesting methods using different inducing genes whichincluded RAD51,RAD54and gadd153gene are used to detect the DNA damage. The HCRassay is afunctional assay which bases on the independent transfection of cellswith either damaged or undamagedplasmid DNA and allows the identificationof the genes responsible for DNA repair-deficient syndromes.Here wecombined the Gadd153-luc test system and HCR assay to measure the DNAdamage and DNArepair by dual luciferase assay.Medthods:1Cell lineThe HepG2cell line was derived from China Center for Type CultureCollection (CCTCC). Cells were grown in Dulbecco’s modified Eagle’smedium (DMEM) with10%heat inactivated fetal calf serum (FCS),penicillin-streptomycin (100IU/ml and100ug/ml respectively) andL-glutamine (2mmol/l). The XRCC1(-) cell with XRCC1knockdown genewas constructed by ShRNA transfection. All the cells were incubated inDMEM medium with5%CO2at37°C.2Construction of vector and transfectionpGadd153-Luc containing hamster gadd153promoter and luciferencereporter gene was reconstructed in our lab，pRL-TK was purchased from Clontech. The vectors were transfected into HepG2cells, grown to95%confluence, by lipofectamine2000reagentaccording to the manufacturer’sinstructions in6-well plates with a final concentration of50nM.3Host cell reactivation assayThe reporter plasmid pRL-TK containing the RL-luciferase gene drivenby the SV40promoter was treated withCdCl2in TE buffer at a DNAconcentration of100μg/ml.The damaged plasmid was recovered byprecipitation with ethanol, and after it was dissolved in TE buffer.The cellswere treated with different carcinogenics for12h and then were transientlytransfected with damaged reporter plasmid and pGL3-luciferase reporterplasmid. Luciferase activity values were quantified with a luminometerandnormalized protein concentrationwas calculated as the average of triplicatewells for each chemical dose.4Dectection of Dual-Luciferase activityAfter transfection, cells were harvested and lysed. Cell extracts were usedto determine dual-luciferase actixity by Dual-Lucy Assay Kit according to theprotocol described previously and the manufacturer’s instructions. Luciferaseactivity values were quantified with a luminometer and normalized proteinconcentration was calculated as the average of triplicate wells for eachchemical dose. Values of firefly-andRL-luciferase expression were normalizedto the luciferase activities of the untreated.5Comet assayComet assay is a traditional approach for the assessment of DNA damageand repair. We compared the sensitivity of the Gadd153-Luc test[1]and HCRassay responded to the genotoxic agents with that of the comet assay.6Extraction of organic compounds from Soil samplesThe samples collection and preparation methods employed for threedifferent e-waste contaminated study sites. The DNA damage and repair whichstimulate by the soils extractive were detected by the dualluciferase reportertest system and comet assay. Results:1DNA repair was detected by the host-cell reactivationIn the present study,2hours were used as treatment time point before pRL-TKwas transfected because most of the plasmid can be repaired in wild typeHepG2cells at that time point.5μmol/L CdCl2treated pRL-TK for2hours and90%plasmid DNA can be repaired in the HepG2cells.Therefore, in thefollowing HCR assay and the dual luciferase assay,5μmol/L CdCl2was usedto damage the pRL-TK for2hours before transfection.2HCR assaySeven different kinds of DNA-damaging agents were assessed the DNArepair by HCR assay and compared with that of the comet assay.Comparingwith comet assay, the HCR assay had a high sensitivity to detect the most ofenvironmental chemicals in DNA repaired research.The threshold of HCR is1000fold lower than that of comet assay after benzopyrene treatment and10fold lower than that of comet assay after CdCl2treatment.3Dual luciferase assayNineteen different chemical were assessed the DNA damages and repaircapabilities by dual luciferase assay. The renilla luciferase activity indicatesthe DNA repair capability and the firefly luciferase activity indicates the DNAdamage. There is no different that the thresholds of16kinds of chemicals withgenetic toxicity we tested in the present study by dual luciferase assay and thatof gadd153-luc assay or HCR assay. And nongenotoxic agents could notinduce DNA damage and repair.4Soil samples were detected by dual luciferase assaySoil samples increased firefly luciferase activity and decrease the renillaluciferase activity.The DNA strand breakwas evaluated by comet assay. Cometassay had the coincident results with that of the dual luciferase assay.Amongof the three e-waste contaminated soil samples, the DNA damage increasingand repair decreasing were at the following order: resident’s area> garbagedump> farmland.Conclusions:1The screening system for DNA damage and repair of potential carcinogens based on dual luciferase assay in human HepG2cell had beenconstructed.2The levels of DNA damages and repair were quantified by luciferaseactivity following drug exposure.3The dual luciferase assay screening system could sensitively andcorrectly detect the DNA damages and repair induced by carcinogens andenvironmental samples. This method had a broad range of detection and a highdetection rate.