| Objective:To preliminarily explore mitochondria DNA oxidative damage and the repair effect of human8-hydroxyguanine deoxyribonucleic acid glycosidase (hOGG1) in Cr(Ⅵ)-treated hepatocytes in vitro, and to provide novel clues for further elucidating the mechanism of Cr(Ⅵ)-induced mitochondria oxidative damage.Methods:L-02human hepatocyte was used in this study. By applying MTT method, we detected the effect of Cr(Ⅵ) on hepatocytes survival rate and then chose four concentrations (0,2,8,32μmol/L) with the exposure time of24h for the following study. Cellular ROS, ATP levels as well as mitochondrial8-hydroxydeoxy guano sine (8-OHdG) content and superoxide dismutase (SOD) activity were measured by the multifunctional fluorescence microplate reader; the mRNA expression level of hOGG1was detected by real-time quantitative polymerase chain reaction (RT-qPCR); mitochondrial hOGGl protein expression level was evaluated by western blotting; and the activities of catalase (CAT) and glutathione peroxidase (GSH-PX) were determined by spectrophotometer.Results:1. Cr(Ⅵ) induces the inhibition of L-02hepatocytes survival rate. In the concentration range of2-256μmol/L, Cr(Ⅵ) significantly inhibited the hepatocytes survival rate (p<0,05), and a obvious negative correlation was found between the Cr(Ⅵ)-treated concentrations and cell survival rate (r=-0.924, p<0.05). Three suitable Cr(Ⅵ)-treated concentrations32,8, and2μmol/L (high, moderate and low) were chosen for the following studies based on the MTT assay.2. Cr(Ⅵ) induces ROS accumulation in L-02hepatocytes. Compared with the control group, the2μmol/L treatment group showed no significant increase of ROS levels (p>0.05), while the8,32μmol/L treatment group showed great increase of ROS levels (p<0.05) in a dose-response manner. Cr(Ⅵ)-treated concentrations and cellular ROS levels were positively correlated (r=0.942,;?<0.05).3. The effect of Cr(Ⅵ) on energy metabolism of L-02hepatocytes. Compared with control, the2μmol/L treatment group showed the similar ATP levels (p>0.05). The8,32μmol/L treatment group showed decreased ATP levels (P<0.05) in a dose-response manner. Cr(Ⅵ)-treated concentrations and ATP levels were negatively correlated (r=-0.967, p<0.05).4. The effect of Cr(Ⅵ) on mitochondrial DNA (mtDNA). Compared with control, the2μmol/L treatment group showed no significant change of8-OHdG content (p>0.05). The8,32μmol/L treatment group showed increased8-OHdG content (P<0.05) in a dose-response manner. Cr(Ⅵ)-treated concentrations and8-OHdG content were positively correlated (r=0.816,p<0.05).5. The effect of Cr(Ⅵ) on the expression of hOGGl. Compared with that of control, the levels of both mRNA and protein in the2μmol/L treatment group were significantly increased (P<0.05); the levels in the8μmol/L treatment group showed no significant change (p>0.05), and lower than that of the2μmol/L treatment group (P<0.05); the levels in the32μmol/L treatment group were significantly decreased (P<0.05). In the concentration range of2-32μmol/L, the levels of both mRNA and protein were gradually decreased.6. The effect of Cr(Ⅵ) on the enzymes of antioxidant system. Compared with that of control, the activities of SOD, CAT and GSH-PX were all significantly increased in the2μmol/L treatment group (P<0.01); the activities of SOD and CAT were decreased in the8μmol/L treatment group, while the activity of GSH-PX was increased, and higher than that of the2μmol/L treatment group (P<0.05); the activities of all of the enzymes in the32μmol/L treatment group were significantly decreased (P<0.05).Conclusions:Cr(Ⅵ) induces ROS accumulation in the hepatocytes, which further damages mtDNA, increases mitochondrial8-OHdG content, affects ATP production, causes energy metabolism dysfunction. The changes of hOGG1mRNA level and enzymes activities of antioxidant system affect the repair ability of mtDNA. In a word, hOGGl plays an important role in Cr(Ⅵ)-induced mtDNA oxidative damage. |
