Acta pharmacologica Sinica · 2005
Apoptosis initiated by carbon tetrachloride in mitochondria of rat primary cultured hepatocytes.
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Cai Yan, Gong Li-kun, Qi Xin-ming, Li Xiang-hong, Ren Jin
Аннотация
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.
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.