Effect Of Hexavalent Chromium On Electron Leak From Respiratory Chain Of Mitochondria In Rat Hepatocytes

Objective:To establish mitochondria isolation method from hepatocytes that could achieve mitochondria that were functional intact and relatively pure and to explore mechanisms of electron leak from the respiratory chain of mitochondria treated with hexavalent chromium in rat hepatocyte and provide clues for the further research in mechanism of Cr(Ⅵ) induced hepatocyte oxidative damage.Methods:The intact mitochondria were isolated from rat liver tissues by sucrose gradient centrifugation and commercially available kit-based methods. Mitochondrial morphology and ultra-structure were observed by OPTEC light microscope and H-600 electron microscope respectively. The mitochondrial membrane potential, respiratory control rate and marker enzymes activities were measured by 960MC fluorescence spectrometer, Clark oxygen electrode and SP-756P spectrophotometer respectively. Mitochondrial ROS production from NADH and FADH2 respiratory chain induced by Cr(Ⅵ) at the levels of different concentrations were detected, when mitochondria were energized by glutamate/malate and succinate respectively. Basal respiratory rates of mitochondria NADH and FADH2 respiratory chain were measured by Clark oxygen electrode. Mitochondria were treated with Cr(Ⅵ), GSH and several complex inhibitors. Electron transport of NADH respiratory chain were separately or jointly intervened with Rot, DPI and Ant, while that of FADH2 respiratory chain were separately or jointly intervened with Rot, DPI,TTFA and Ant. ROS and O2·- production were measured with VarioskanFlash 4.00.52 multifunctional microplate reader.Results:1. The electron micrograph of mitochondria isolated by two methods showed that mitochondria were morphologically dense and intact with no signs of swelling or damage. Mitochondrial membrane potential and respiratory control rate of mitochondria isolated by sucrose gradient centrifugation were significantly higher than that of mitochondria isolated by kit-based method (P<0.05), but the purity of mitochondria between them was not statistically different (P> 0.05).2. Cr(Ⅵ) induced concentration-dependent increase of ROS production of NADH and FADH2 respiratory chain of liver mitochondria(P<0.05). Basal respiratory rate of mitochondrial FADH2 respiratory chain was significantly higher than that of NADH respiratory chain (P<0.05).3. For glutamate/maltate-energized mitochondria, ROS content and electron leak rate in Rot, Rot+Ant and DPI+Ant groups increased, while ROS content in DPI group decreased significantly compared with control group. Compared with Cr(Ⅵ) group, ROS content and electron leak rate in Cr(Ⅵ)+Rot, Cr(Ⅵ)+Ant, Cr(Ⅵ)+Rot+DPI, Cr(Ⅵ)+Rot+Ant and Cr(Ⅵ)+DPI+Ant groups increased significantly(P<0.05).4. For succinate-energized mitochondria, ROS content and electron leak rate in Rot, DPI, Rot+DPI, Rot+TTFA and DPI+TTFA group decreased, while that in TTFA, Ant, Rot+Ant, Rot+DPI and TTFA+Ant groups increased significantly(P<0.05). When compared with Cr(Ⅵ) group, ROS content and electron leak rate in Cr(Ⅵ)+TTFA, Cr(VIⅥ)+Rot+TTFA, Cr(Ⅵ)+Ant, Cr(Ⅵ)+Rot+Ant, Cr(Ⅵ)+DPI+Ant and Cr(Ⅵ)+TTFA+Ant groups increased, but that in Cr(Ⅵ)+DPI+TTFA groups decreased significantly (P<0.05).Conclusions:1. Compared with mitochondrial isolated kit method, mitochondria isolated by sucrose gradient centrifugation are functionally and morphologically intact and relatively pure, which can meet the requirement of mitochondrial relevant tests.2.50uM Cr(Ⅵ) induced an increase of liver mitochondrial electron leak from NADH respiratory chain, mainly from ubiquinone binding sites of complexⅠand complexⅢ. For FADH2 respiratory chain,50uM Cr(Ⅵ) promotes electrons pass forward through FADH2 respiratory chain and 硕士毕业论文英文摘要leak through ubiquinone binding site of complexⅢ.3.The protective effect of 200uM GSH for liver mitochondria electron leak induced by 50uM Cr(Ⅵ)is to remove excess H2O2 generated and reduce total ROS content,so as to decrease oxidative damage.