Relationship Between Cr (â…¥)-induced Apoptosis And Mitochondrial Electron Transport Chain Dysfunction

Objective:To explore the action of electron transport chain dysfunction on the L-02 hepatocytes apoptosis induced by hexavalent chromium Cr(Ⅵ) and its possible mechanisms.Methods:The effects of Cr(Ⅵ) or Cr(Ⅵ) and inhibitors (DPI, ROT, AA) or protective agent (Vit C) on cell survival rate were assessed by the reductions of tetrazolium dye (MTT) in cultured L-02 hepatocytes. L-02 hepatocytes in all tests were incubated with 25μmol/L concentrations of Cr(VI), and 2.5μmol/L DPI,5μmol/L ROT,1μg/mL AA and 2mmol/L Vit C for 12h according to cell survival rate. The reduction rate of Cr(Ⅵ) in mitochondria was measured by the diphenylcarbazid (DPC) method with 756P ultraviolet-visible pectrophotometer. The respiratory function of mitochondria was detected by oxygen electrode. The levels of ATP and ROS in L-02 hepatocytes were respectively detected by Luminometer method and fluorometry method with Varioskan Flash 4.00.52 multifunctional microplate reader. The activity of caspase-3 was measured with the Microplate Reader. AnnexinV-FITC/PI double staining with FCM detection showed the cellular apoptosis and necrosis ratios.Results:1. The concentration-dependent decrease in cell survival rate of Cr(Ⅵ)-treated L-02 cells was observed. The relationship between concentration and survival rate was significantly negative correlation (r12h=-0.980, P<0.05), and 2.5μmol/L DPI,5μmol/L ROT,lμg/mL AA and 2mmol/L Vit C was chosen for follow-up experiments.2. The reduction rate of Cr(Ⅵ) in mitochondria:different substrates had different effects on the reduction of Cr(Ⅵ) in mitochondria. Compared to the same concentration of Cr(Ⅵ) control group, the reduction rates of Cr(Ⅵ) in the mitochondria complex I respiration substrate malate plus glutamate treated groups increased markedly. While the reduction rates of Cr(Ⅵ) in the complex IVrespiration substrate Vit C treated groups decreased significantly (P<0.05). Different respiratory inhibitors had different impacts on the reduction of Cr(Ⅵ) (P<0.05). When treated with NaN3, the inhibitor of complex IV, the reduction of Cr(Ⅵ) in mitochondria got larger than other inhibitor treated group.3. Effects of Cr(Ⅵ) on the L-02 hepatocytes respiratory function: Compared with the control group, the hepatocyte'oxygen consumption rate of Cr(Ⅵ) single treated group decreased significantly(P<0.05). Compared to the same concentration of Cr(Ⅵ) single treated group, the hepatocyte oxygen consumption rate of Cr(Ⅵ) plus respiratory chain inhibitor (include DPI, ROT, AA) combined groups decreased significantly(P＜0.05). The oxygen consumption rate of the Cr(Ⅵ) plus Vit C combined group increased markedly(P<0.05).4. Effects of Cr(Ⅵ) on the content of ATP in L-02 hepatocytes: compared with the control group, the content of ATP in Cr(Ⅵ) single treated group decreased significantly(P<0.05). Compared to the same concentration of Cr(Ⅵ) single treated group, the content of ATP in Cr(Ⅵ) plus respiratory chain inhibitors (include DPI, ROT, AA) combined groups decreased significantly(P<0.05). The content of ATP of the Cr(Ⅵ) plus Vit C combined group increased markedly(P<0.05).5. Effect of Cr(Ⅵ) on the level of ROS in L-02 hepatocytes: compared with the control group, the level of ROS in Cr(Ⅵ) single treated group increased significantly(P<0.05). Compared to the same concentration of Cr(Ⅵ) single treated group, the level of ROS in Cr(Ⅵ) plus respiratory chain inhibitors (include DPI, ROT, AA) combined groups increased significantly(P<0.05). The level of ROS in the Cr(Ⅵ) plus Vit C combined group decreased markedly(P<0.05).6. Effect of Cr(Ⅵ) on the expression of caspase-3:Compared to the control group, the caspase-3 activity increased remarkably in the Cr(Ⅵ) single treated group (P<0.05), Compared to the same concentration of Cr(Ⅵ) single treated group, the caspase-3 activity in the Cr(Ⅵ) plus respiratory chain inhibitors (include DPI, ROT, AA) combined groups increased significantly(P<0.05). The caspase-3 activity in the Cr(Ⅵ) plus Vit C combined group decreased markedly (P<0.05). 7. Effect on the L-02 hepatocytes apoptosis rate of Cr(Ⅵ):compared with the control group, the hepatocytes apoptosis rate of Cr(Ⅵ) single treated group increased significantly(P<0.05). Compared to the same concentration of Cr(Ⅵ) single treated group, the hepatocytes apoptosis rate of Cr(Ⅵ) plus respiratory chain inhibitors (include DPI, ROT, AA) combined groups increased significantly(P<0.05). The apoptosis rate of the Cr(Ⅵ) plus Vit C combined group decreased markedly (P<0.05).Conclusions:1. Cr(Ⅵ) can be reduced by getting electrons from mitochondrial complex I, and ROS which generated from the reduction process of Cr(Ⅵ), could damage mitochondria and cause the release of the apoptosis inducing factor caspase-3, and then induce L-02 hepatocyte apoptosis.2. The main respiratory chain inhibitors (especially ROT) can induce the electron leak from the respiratory chain complex I, the electron leaked from complex I can be used for the reduction of Cr(Ⅵ). The increased electron leakage promote the reduction of Cr(VI), so that the L-02 hepatocytes apoptosis induced by Cr(Ⅵ) increase.3. Vit C is able to improve the ATP level in cells, repairs the function of mitochondrial respiration partly, decreases the caspase-3 activity, and debases the damage of mitochondria and protects hepatocytes from Cr(Ⅵ)-induced apoptosis.4. Electron transport dysfunction plays an important role in the apoptosis process.