The Characterization Of Mitochondrial Toxicity Indued By Hepatotoxic Drugs

Objective:To evaluate the hepatotoxicity and to characterize the mitochondrial toxicity mechanism and dose-effect relationship of drug-induced liver injury caused by tamoxifen,flutamide and troglitazone in vitro using HepG2 cell lines as a model.To provide the experimental basis for the elucidation of the mechanism of mitochondrial toxicity and the screening of mitochondrial toxicity in vitro.Methods:The cytotoxicity of drug was evaluated by detecting cell survival rate and membrane integrity after drug action for 12 h and 24 h and morphological changes after drug action for 24 h.The method of CCK-8was used to detect the survival rate of cells and calculate the survival rate of IC50;Lactate dehydrogenase(LDH)leakage assay was for membrane integrity;The morphological changes of cells were observed by microscope.Mitochondrial toxicity of drug was evaluated by detecting the changes of mitochondrial membrane potential(MMP)after drug action for 3h and reactive oxygen species(ROS)production,cytochrome C(Cyt C)release,adenosine triphosphate(ATP)content changes after drug action for 3h.Application of high content imaging technology,Cell ROX Green probe detected the production of ROS,Mito Tracker Red CMXRos probe was used to detect the changes of MMP,Cytochrome C primary antibody and Alexa Flour 647 fluorescent secondary antibody were used to detect the release of Cyt C.The content of ATP was detected by luciferase chemiluminescence and measured by using the Multiscan Spectrum.The activity of superoxide dismutase(SOD),catalase(CAT)and production of malondialdehyde(MDA)were measured by Multiscan Spectrum after 12 h treament of drugs to evaluate the degree of oxidative damage of HepG2 cells.Western blot was used to detect the changes of Nrf2,SOD2 and HO-1protein expression after 12 h treament of drugs,and to evaluate the effects of drugs on the antioxidant pathway of Nrf2.Results:the IC50 value after the effect of tamoxifen,flutamide and troglitazone for 12 h measured by CCK-8were(33.855 + 4.316),(187.674 + 13.643),(166.821 + 12.127).the IC50 value for 24 h were(19.092±1.385)μM、(126.114±21.682)μM、(88.357±8.672)μM respectively;Compared with the control group,the LDH leakage rate of the drug administration group increased significantly with the increase of dose and time;Three drugs were observed under microscope that HepG2 cells can significantly change the morphology and even die in the medium high concentration.Compared with the control group,a,the MMP of the drug administration group decreased with the increase of dose after administration of 3h,and the MMP of the Tam group decreased first and then increased;After 12 h,the ROS production and Cyt C release of the drug administration group increased with the increase of dose,and the ATP production decreased with the increase of dose.After 12 h,compared with the control group,the drug administration group showed different degrees of decreased SOD and CAT and increased MDA production,and the activity change of SOD was more sensitive than that of CAT.Compared with the control group,after 12 h,the antioxidant protein expression of Nrf2 pathway in low dose group had no obvious change,the antioxidant protein expression of Nrf2 pathway in medium dose group were increased,the antioxidant protein expression of Nrf2 pathway in the high dose group decreased significantly.The antioxidant protein expression of Nrf2 pathway generally increased first and then decreased with the dose.Conclusion:Tamoxifen,flutamide and troglitazone has certain toxic effects on HepG2 cells;Tamoxifen,flutamide and troglitazone can increase the ROS of HepG2 ROS cells,cause mitochondrial membrane dysfunction and produce mitochondrial toxicity.Mitochondrial toxicity may be one of the important mechanisms of hepatotoxicity;Oxidative stress of Hep G2 cells induced by tamoxifen,flutamide and troglitazone can decrease the activity of Oxidase SOD and CAT and increase lipid peroxidation products MDA;The Nrf2 pathway play an important role in the oxidative damage of HepG2 cells aused by tamoxifen,flutamide and troglitazone.Low levels of oxidative stress induce defense mechanisms to activate adaptive responses.The defense mechanism of HepG2 cells will overrun and cause cell injury and death,when the level of oxidative stress is too high to repair.