Original Articles

Apoptosis initiated by carbon tetrachloride in mitochondria of rat primary cultured hepatocytes

Yan Cai, Li-kun Gong, Xin-ming Qi, Xiang-hong Li, Jin Ren


Aim: To investigate the mitochondria-initiated apoptosis pathway involved in Carbon tetrachloride (CCl4) hepatotoxicity in vitro.
Methods: Several cytotoxicity endpoints, including WST-8 metabolism, lactate dehydrogenase leakage and morphological changes, were examined. The 5,5'-dithio-bis(2-nitrobenzoic acid) reaction was used to measure reduced glutathione level, and the malondialdehyde level was determined using the thiobarbituric acid assay. The release of cytochrome c and Bcl-XL was detected by Western blot. Caspase-3 activity was measured using the fluorogenic substrate Ac-DEVD-AMC. DNA fragmentation was used to evaluate cell apoptosis.
Results: A time-and dose-dependent decrease in cellular glutathione content was observed, along with a concomitant increase in malondialdehyde levels following the application of CCl4. Caspase 3 activity was stimulated at all doses of CCl4, with the most significant activation at 3 mmol/L. Cytochrome c was released obviously after CCl4 treatment. A time-dependent decrease in Bcl-XL expression was observed. DNA fragmentation results revealed apoptosis and necrosis following CCl4 treatment.
Conclusion: Oxidative damage is one of the essential mechanisms of CCl4 hepatotoxicity, which triggers apoptosis via the mitochondria-initiated pathway.