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

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-X(L) 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-X(L) 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.