Impaired Effect Of Pb On HL-7702 Cells

Objective: To observe the injured effect of Pb on human liver cells HL-7702, and to investigate the possible mechanisms from the aspect of oxidative damage and apoptosis.Methods: HL-7702 cells were cultured in vitro, and exposed to 0, 50, 100, 200 or 400μmol/L lead acetate respectively for 24 hours for revealing the dose-, and time-effect relation. HL-7702 cells were treated with 100μmol/L lead acetate for 0, 6, 12, 24 or 48 hours respectively. The control was exposed to D-Hank's solution instead of lead acetate. The morphological and ultrastructural changes of cells were observed by HE staining and transmission electron microscope (TEM). The antioxygen ability of the cells was revealed by detecting activity of SOD and GSH-Px in culture supernatant. The state of membrane integrity was denoted by detecting the content of LDH in culture supernatant. The cell cycle proportion and apoptotic ratio were detected by Flow Cytometer (FCM) with propidium iodide(PI) staining. The expression of P53 in cytoplasm was observed by immunocytochemical staining.Results:①HE staining showed that after treated with Pb, cellular swelling and vacuolar degeneration of cytoplasm and nucleus could be seen in 50μmol/L group. As the dose increased, the cell body shrinked, and vacuolar degeneration of cytoplasm and nucleus became more severe,and the trachychromatic chromatin and chromatin margination became obvious. In 400μmol/L group, the cell density significantly decreased, and shrinkage of cell body was more severe, and pyknosis or disaggregation of nucleus appeared, with the acting time of Pb prolonged. There were no changes of cell size and density, but vacuolar degeneration of cytoplasm and nucleus became severe, and trachychromatic chromatin and chromatin margination became more severe.②TEM showed that after treated with 50μmol/L Pb for 24 hours, the ultrastracture of most of the cells were basically normal, only a few of them had regional membrane damage and vacuolar degeneration of mitochondrium, with some loss of crista mitochondriales. After treated with 400μmol/L Pb for 24 hours, almost all of the cells underwent degeneration at some extent, charactered by incompletement of cell membrane, naked nucleus, and severely injured mitochondriums.③After treated with Pb, the activity of SOD and GSH-Px in culture supernatant decreased and the content of LDH in culture supernatant increased in a dose- and time-depended pattern.④The results of FCM with PI staining denoted that the apoptotic ratio increased as the dose and action time of Pb extended. The cell circle analysis showed that there were no obvious changes of the percentage of cells in each stage comparing 50 and 400μmol/L groups with control. The percentage of cells in G2/M stage decreased while that in S stage increased in 100μmol/L group. The percentage of cells at G1 stage increased in 200μmol/L group while that in S stage decreased. There were no obvious changes of the percentage of cells in each stage comparing 6h and 48h groups with control. The percentage of cells in G1 and S stages decreased while that in G2/M stage increased in 12h group. The percentage of cells in G2/M stages decreased while that in S stage increased in 24h group.⑤P53 immunocytochemistry revealed an increase of P53 expression in cytoplasm as the dose of Pb increased and action time prolonged, but the expression of P53 in 400μmol/L group was less than that in 200μmol/L group.Conclusion: Pb has a dose- and time-related damage effect on liver cells. The effect may be mediated by oxidative damage through inhibiting activity of SOD and GSH-Px, and by induction of apoptosis via up-regulation of P53 expression.