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


CONCLUSIONS
Oxidative damage is one of the essential mechanisms of CCl4 hepatotoxicity, which triggers apoptosis via the mitochondria-initiated pathway.
Cai
Yan
Y
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Gong
Li-kun
LK
Qi
Xin-ming
XM
Li
Xiang-hong
XH
Ren
Jin
J
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002251:Carbon Tetrachloride / Q000633:toxicity*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D051020:bcl-X Protein / Q000378:metabolism
D051020:bcl-X Protein; D008315:Malondialdehyde; D045304:Cytochromes c; D002251:Carbon Tetrachloride; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione