Nucleotide Excision Repair In DNA Damage Of Human Embryonic Kidney 293 Cells Induced By Nephrotoxicant

DNA damage is thought of one mechanism of chemicals-induced nephrotoxicity Cisplatin (Cis-diamminedichloroplatinum, CDDP), one of primary anticancer drugs, hinders its clinical uses for its nephrotoxicity. It has been reported that oxidative stress was one of the mechanisms of CDDP-induced cytotoxicity. Besides these, CDDP can bind to DNA base as the target and form a stable Pt-DNA adducts which will disturb the structure of DNA and inhibit DNA replication. CDDP-induced nephrotoxicity has a linear correlation with Pt-DNA adducts level. Cadmium(Cd) causes oxidative damage, which is closely associated with the nephrotoxicity of Cd. But some studies provide evidences that oxidative damage is not a direct reason of Cd-induced nephrotoxicity. Cd can cause DNA damage by inducing DNA strand breaks, forming 8-hydroxydeoxyguanosine and inhibiting DNA repair. Aristolochic acid (AA) damages DNA and forms DNA adducts, which may be one of the important mechanisms causing its nephrotoxicity and carcinogenesis.Nucleotide excision repair (NER) can recognize a broad spectrum of helix-distorting lesions and is a major repair system of DNA damage. NER is the sole DNA repair activity for removing bulky adducts and a major defense against the carcinogenic effects. NER consists of removal of the damaged nucleotide(s) from DNA by dual incision of the damaged strand on both sides of the lesion, followed by filling of the resulting gap and ligation.To study whether CDDP, Cd and AA -induced DNA damages play the role in the mechanisms of their nephrotoxicities and to provide experimental evidences in live cell, an over-expressing ERCC1/XPF protein model cell strain was established.Methods The human ERCC1 and XPF gene were coexpressed in 293 cells by using biochemistry and molecular biology technique. The colony-like transfected cells were obtained after being selected with Zeocin and G418. The expression of ERCC1 and XPF was detected by western-blot after the clone cells were expanded. Toinvestigate the morphology changes and cytotoxicity after being exposed to CDDP to testify whether the cell strain which can over-express ERCCj/XPF protein was successfully established.The cytotoxicities of CDDP, cadmium chloride(CdCl2) and AA to different cells were measured by MTT assay. IC50 and its 95% confidence limit were calculated by using Bliss method and used to make statistical deducing.Results There were over-expression of ERCC1/XPF in the ERCC1/XPF-293 cells by Western-blot. The morphology changes and cytotoxicity after being exposed to the same concentration of CDDP were markedly alleviated. Those indicated that the cell strain which can over-express ERCCi/XPF protein had been established successfully.Inhibitory effects on transfected cells decreased after CDDP, CdCl2 and AA treatment, respectively. The IC50 of CDDP for 293 and ERCCi/XPF-293 cells were 0.294 and 1.011 mmol/L, respectively (P<0.05). The IC50 of CdCl2 for 293 and ERCC1/XPF-293 cells were 18.61 and 61.91 U mol/L, respectively (P <0.05). The IC50 of AA for 293 and ERCC1/XPF-293 cells were 201.9 and 321.7 umol/L, respectively (P <0.05).Conclusion The human ERCC1 and XPF gene can be coexpressed in 293 cells by using biochemistry and molecular biological technique, over expressing ERCC1/XPF would increase DNA repair capacity which can decrease the CDDP, CdCl2 and AA-induced cytotoxicities. It indicates that DNA damage is a critical role in the genesis of their nephrotoxicities.