| Summary: | 碩士 === 台北醫學院 === 醫學研究所 === 90 === Epidemiological evidence has suggested that exposure of the human to cadmium (Cd) and mercury (Hg) might cause pulmonary damages. However, the mechanism has remained unclear. A normal human fetal lung fibroblast cell line (MRC-5) was used as a cell model to investigate the mechanism of cell death triggered by Cd or Hg. Several methods were employed to elucidate apoptosis and necrosis of MRC-5 cells after treatment of 100μM Cd or Hg, respectively; including cytohistochemistry analysis with Hoechst staining, flow cytometric assay with propidium iodide (PI) single staining as well as annexin V plus PI double staining and 1H NMR to analyze the spectral intensity ratio of methylene (1.3 ppm) to methyl (0.9 ppm) resonances. We have previously demonstrated that H2O2 was elevated three folds after treatment of Cd by using dichlorodihydrofluorescein diacetate (DCFH-DA) as a detection agent. These results suggested that reactive oxygen species (ROS) might play pivotal roles during apoptosis and cytotoxicity induced by Cd. Following this line, the antioxidant compounds, such as N-acetylcysteine (NAC), 4, 5-dihydroxy-1, 3 benzene-disulfonic acid (tiron) and mannitol were used to rescue MRC-5 cells from the deleterious effect of ROS. The results showed that the ROS-generating organelle, mitochondrion, was depolarized and participated in Cd-induced apoptosis as revealed by 5,5’,6,6’-tetrachloro-1,1’3,3’-tetra-ethyl benzimidazolyl carbo -cyanime iodide (JC-1)/flow cytometric analysis. In addition, rotenone and oligomycin A are inhibitors of electron transport chain (ETC), which could decrease the cytotoxicity of Cd and reduce the percentage of apoptotic cells. Pre-treatment with cyclosporin A and aristolochic acid (inhibitor of mitochondrial permeability pore formation) significantly reduced the mitochondrial membrane potential, and attenuated the signs of apoptosis such as DNA fragmentation by detection with PI staining. This result indicated that mitochondria might play an arbitrator for Cd-triggered apoptosis of MRC-5 cells. On the other hand, we used the immunoblot to demonstrate that caspases was not activated and its substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Cd-indicated apoptotic cells. Moreover, the pan-caspase inhibitor, z-Val-Ala-Asp- (OME)- fluoromethylketone (z-VAD- fmk), could not prevent Cd-induced cell death, which implied a caspase-independent apoptotic pathway. Recent reports prove that apoptosis-inducing factor (AIF) is normally confined to the mitochondrial membrane space and translocates through the outer mitochondrial membrane to the nucleus in a caspase-independent apoptotic pathway. By indirect-immunofluorescence, AIF was observed to translocate from mitochondria to the nucleus after Cd treatment. In conclusion, our results suggest that Cd triggered a caspase-independent apoptotic pathway through mitochondria and ROS mechanism in human normal lung cell. In contrast, Hg induced cell death by necrosis.
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