Study On The Mechanisms Of Ochratoxin A-induced Cell Cycle Arrest In Human Embryonic Kidney Cells(HEK 293)

Ochratoxin A(OTA)is a ubiquitous mycotoxin contaminating the grain crop and food,becoming one of the risk factors in the realm of food safety in recent years.OTA has been shown to be nephrotoxic,hepatotoxic,immunotoxic,and teratogenic.As kidney is the main target of OTA,many studies focusing on the mechanisms of nephrotoxicity induced by OTA have been reported.However,there were no reports about the influence of OTA on the cell cycle distribution and DNA damage in human embryonic kidney cells(HEK 293).In this study,the OTA-induced cell cycle arrest and DNA damage has been investigated,providing new insights into the mechanisms of nephrotoxicity induced by OTA.The main results and conclusions were as follows:1.OTA induced the decrease of cell viability,the overproduction of reactive oxygen species(ROS),and the decrease of membrane potential in a dose-dependent manner,indicating that OTA induced oxidative damage in HEK 293 cells.In comet assay,the value of comet length,olive tail moment,and the percentage of tail DNA after treatment with OTA for 24 h were significantly higher than the control group,suggesting the OTA induced breakage of DNA in HEK 293 cells.Through the flow cytometry,HEK 293 cells were arrested at the S phase by OTA.The S-phase related key proteins including cyclin A2,cyclin E1,and CDK2 were down-regulated.2.Pretreatment with N-acetylcysteine(NAC)partly reversed the adverse effects of OTA on the cell proliferation and oxidative damage in HEK 293 cells.Compared with the OTA treatment group,NAC pretreatment reduced the ROS production,elevated the membrane potential and SOD activity.The results of comet assay indicated that NAC pretreatment alleviated the OTA-induced DNA damage in HEK 293 cells.NAC pretreatment reversed the level of 8-hydroxy-2'-deoxyguanosine(8-OHdG)and global DNA hypomethylation indicated in the results of HPLC/MS/MS.NAC pretreatment ameliorated the cell cycle arrest and up-regulated the expression of cyclin A2,cyclin E1,and CDK2 at both mRNA and protein level.3.Co-incubation of ZnSO4 and OTA alleviated the OTA-induced inhibition of cell proliferation.The addition of ZnSO4 inhibited the OTA-induced production of ROS and reversed the decrease of membrane potential and SOD activity,indicating the antioxidative effects of ZnSO4.OTA induced-DNA strand break,formation of 8-OHdG and the reduction of global DNA methylation were ameliorated by addition of ZnSO4.Zinc helped to keep the integrity and stability of DNA.4.High expression vector of Chkl(pcDNA3.1(+)-Chk1)were successfully established in HEK 293 cells.The expression of Chkl in trasnsfection cell line was higher than the control.RNA interfering vector of Chkl(pEGFP-H1-Chk1-shRNA)were successfully established in HEK 293 cells.The expression of Chkl in trasnsfection cell line was lower than the control,providing in vitro model for the study of Chkl in the OTA-induced cell cycle arrest.