Mitochondrial Damage Induced By Hexavalent Chromium Under The Condition Of P53 Closed In L-02 Hepatocytes

Objective:To explore the mitochondria damage induced by hexavalent chromium(Cr(Ⅵ)under the condition of p53 closed in L-02 hepatocytes,and to further investigate the pathway of apoptosis induced independently by mitochondria damage in L-02 hepatocytes treated with Cr(Ⅵ).Methods:L-02 hepatocytes were cultured,and MTT test was adopted to test the viability of L-02 hepatocytes treated with PFT-αalone and/or with Cr(Ⅵ).The detectation of p53 protein level in nuclei of L-02 hepatocytes was performed to verify the inhibitive effect of PFT-αon p53, in order to select the exposure concentrations of PFT-αand Cr(Ⅵ).The concentrations of PFT-αand Cr(Ⅵ)were both designed at 20μmol/L. After cells were treated with PFT-αfor 4hrs,and then treated with Cr(Ⅵ) for 24hrs.The cultured L-02 hepatocytes were randomly divided into six groups,namely control group,Cr(Ⅵ)alone,PFT-αalone,PFT-α+Cr(Ⅵ), PFT-α+Z-VAD-fmk+Cr(Ⅵ),and PFT-α+NAC+Cr(Ⅵ).The activity of SOD and GST and the levels of MDA and GSH were detected by the colorimetric method.The mitochondrial permeability transition pore(PTP) and the mitochondrial transmembrane potential(△ψm)were determined by the fluorospectrophotometric method.The protein expression levels of p53,cytochrome C and apoptosis-inducing factor(AIF)were assayed with the western blotting.The gene expressions of Bax and Bcl-2 were detected with the reverse transcription polymerase chain reaction(RT-PCR).Genomic DNA damage ladder was analyzed with gel electrophoresis.Results:1.The level of p53 protein was significantly inhibited in L-02 hepatocytes pretreated with 20μmol/L PFT-α.2.Compared with control group,the level of GSH and the acticity of SOD and GST in groups treated with Cr(Ⅵ)alone,PFT-α+Cr(Ⅵ)and PFT-α+Z-VAD-fmk+Cr(Ⅵ)were significantly decreased,and the level of MDA was significantly increased(P＜0.05).While the above changes were protected by NAC(P＜0.05).3.The mitochondrial transmembrane potential(△ψm)of L-02 hepatocytes in the groups treated with Cr(Ⅵ)alone,PFT-α+Cr(Ⅵ),and PFT-α+Z-VAD-fmk+Cr(Ⅵ)was significantly decreased,and the open rate of PTP significantly increased(P＜0.05),compared with control group,.The mitochondria damage was protected by NAC(P＜0.05).4.The levels of cytC and AIF in cytoplasm in the groups of Cr(Ⅵ), PFT-α+Cr(Ⅵ),and PFT-α+Z-VAD-fmk+Cr(Ⅵ)significantly increased(P＜0.05),compared with control group,and these changes were inhibited by NAC(P＜0.05).5.Compared with control group,the level of gene expressions of Bcl-2 of cells in the groups exposed to Cr(Ⅵ)alone,PFT-α+Cr(Ⅵ),and PFT-α+Z-VAD-fmk+Cr(Ⅵ)was decreased significantly,and the level of Bax was increased significantly,which were inhibited by NAC.6.The genomic DNA in cells treated with Cr(Ⅵ),PFT-α+Cr(Ⅵ) and PFT-α+Z-VAD-fmk+Cr(Ⅵ)showed ladder degradation,and this effect was not be protected by the addition of NAC.Conclusions:Under the condition of p53 inhibited,Cr(Ⅵ)at the concentration of 20μmol/L could significantly cause oxidation stress of L-02 hepatocytes,damage to mitochondiral membrane and genomic DNA. The results show that Cr(Ⅵ)could cause the mitochondria damage,and the apoptosis pathway in L-02 hepatocytes with Cr(Ⅵ)can induce by mitochondira-dependent without p53 and caspase.NAC is able to protect L-02 hepatocytes from mitochondria damage.