Relationship Between Cr(Ⅵ)-induced Apoptosis And Energy Metabolism Dysfunction In Hepatocyte

Objective:To explore the relationship between the energy metabolism dysfunction and t apoptosis induced by Cr(Ⅵ) he L-02 hepatocytes and provide clues for the further research in the mechanism of Cr (Ⅵ)-induced hepatocyte apoptosis.Methods:The effects of Cr(Ⅵ) single treated or ATP single treated 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 respectively incubated with 2.5μmol/L, 10μmol/L,40μmol/L concentrations of Cr(Ⅵ) or/and 1μmol/L ATP for 24h according to cell survival rate.The cellular apoptosis and necrosis ratios.AnnexinV-FITC/ PI double stained analysed by FCM; DNA damage was observed by using the DNA-Ladder. The mitochondrial permeability transition pore (PTP) and the mitochondrial transmembrane potential (△ψm) were determined by the fluorospectrophotometric method ;the activity of caspase-3 was tested by the Microplate Reader. The levels of cellular ATP, ADP, AMP content as well as the changes of ATP/ADP ratio and the energy charge (Ec) were detected by high performance liquid chromatography (HPLC), The respiratory function of mitochondria was detected by oxygen electrode.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 (r=-0.909, P＜0.05). The 2.5μmol/L,10μmol/L,40μmol/LCr(Ⅵ) concentration and 1μmol/L ATP were chosen.2. Compared to the control group, the apoptosis ratio of all the Cr(Ⅵ)-treated groups increased markedly (P<0.05). Compared to the same concentration of Cr(Ⅵ), the apoptosis ratio in the 10μmol/L Cr(Ⅵ)+ATP group decreased, while the apoptosis ratio in the 4μmol/L Cr(Ⅵ)+ATP group increased but the necrosis ratio decreased significantly (P＜0.05).3. DNA damage was observed in the intermediate dose group[10μmol/L Cr(Ⅵ)] and maximum group[40μmol/L Cr(VI)], but in the intermediate combined group[10μmol/L Cr(Ⅵ)+ATP] and the maximum combined group[40μmol/L Cr(Ⅵ)+ATP] the DNA breakage reduced compared to the same concentration of Cr(Ⅵ) single group (P＜0.05).4. Compared to the control group, the caspase-3 activity increased remarkably in other groups (P＜0.05), and after treated with ATP together, the caspase-3 activity of the maximum group decreased significantly (P＜0.05).5. The ROS in all groups except ATP treated group rose significantly compared to the control group (P＜0.05).6. The mitochondrial openness of permeability transition pore (PTP) and the rate of mitochondrial membrane potential (△ψm) declining significantly increased in all groups compared to the control group(P＜0.05). ATP markedly improved them in the intermediate combined group and maximum combined group compared to the same concentration of Cr(VI) groups(P＜0.05).7. The contents of ATP,ADP and total adenine nucleotide (TAN) significantly increased in the minimum group but declined in the intermediate dose group and the maximum group compared to the control group(P＜0.05).While the energy charge (Ec) significantly increased in the intermediate dose group but decreased in the maximum compared with the control group (p＜0.05).8. In all the Cr(VI) single treated groups, RCR had significantly declined compared to the control group(P＜0.05), ATP apparently improved the RCR in the maximum group compared to the same concerntration of the Cr(VI) group, and the P/O declined in all the Cr(VI)-treated groups compared to the control group(P＜0.05),.Conclusions:Cr(VI) can induce L-02 hepatocyte apoptosis and mitochondrial energy metabolism dysfunction. Energy metabolism dysfunction has a close relationship with the Cr(Ⅵ)-induced hepatocyte apoptosis process. ATP can repair the damage of mitochondrial respiration function, depress the caspase-3 activity, and alleviate the mitochondria and DNA damage in Cr(Ⅵ)-treated hepatocyte. ATP can protect hepatocytes from Cr(Ⅵ)-induced apoptosis at the lower dose(2.5μmol/L-10μmol/L),but increase the apoptosis and decrease the necrosis in the 40μmol/L group.