Glycogen synthase kinase-3 mediates acetaminophen-induced apoptosis in human hepatoma cells.

The mild analgesic drug acetaminophen (AAP) induces severe hepatic injury when taken at excessive doses. Recent evidence shows that the initial form of damage is through apoptosis, but this fails to go to completion and degenerates into necrosis. The aim of this study was to elucidate the mechanism through which AAP induces apoptosis using human HuH7 hepatoma cells as an in vitro model system to investigate the initial phase of AAP-induced hepatic injury. AAP-induced apoptosis in HuH7 cells as evidenced by chromatin condensation was preceded by the translocation of Bax to mitochondria and the cytoplasmic release of the proapoptotic factors cytochrome c and Smac/DIABLO. A concomitant loss of mitochondrial membrane potential occurred. Activation of the mitochondrial pathway of apoptosis led to the activation of execution caspases-3 and -7. AAP-induced apoptosis and cell death was blocked by inhibitors of caspases but not by inhibitors of calpains, cathepsins, and serine proteases. Apoptosis was unaffected by inhibitors of the mitochondrial permeability transition pore and by inhibitors of Jun NH(2)-terminal kinases, p38 mitogen-activated protein kinase, or mitogen-activated protein kinase kinase 1/2. However, pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) delayed and decreased the extent of AAP-induced apoptosis. In comparison, endoplasmic reticulum stress-induced but not prooxidant-induced apoptosis of HuH7 cells was sensitive to GSK-3 inhibition. It is concluded that AAP-induced apoptosis involves the mitochondrial pathway of apoptosis that is mediated by GSK-3 and most likely initiated through an endoplasmic reticulum stress response.

Involvement of mitochondria in acetaminophen-induced apoptosis and hepatic injury: roles of cytochrome c, Bax, Bid, and caspases.

The role of apoptosis in acetaminophen (AAP)-induced hepatic injury was investigated. Six hours after AAP administration to BALB/c mice, a significant loss of hepatic mitochondrial cytochrome c was observed that was similar in extent to the loss observed after in vivo activation of CD95 by antibody treatment. AAP-induced loss of mitochondrial cytochrome c coincided with the appearance in the cytosol of a fragment corresponding to truncated Bid (tBid). At the same time, tBid became detectable in the mitochondrial fraction, and concomitantly, Bax was found translocated to mitochondria. However, AAP failed to activate the execution caspases 3 and 7 as evidenced by a lack of procaspase processing and the absence of an increase in caspase-3-like activity. In contrast, the administration of the pan-inhibitor of caspases, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (but not its analogue benzyloxycarbonyl-Phe-Ala-fluoromethylketone) prevented the development of liver injury by AAP and the appearance of apoptotic parenchymal cells. This correlated with the inhibition of the processing of Bid to tBid. The caspase inhibitor failed to prevent both the redistribution of Bax to the mitochondria and the loss of cytochrome c. In conclusion, apoptosis is an important causal event in the initiation of the hepatic injury inflicted by AAP. However, as suggested by the lack of activation of the main execution caspases, apoptosis is not properly executed and degenerates into necrosis.

The role of apoptosis in acetaminophen-induced injury.

Apoptosis plays a critical role in acetaminophen (AAP)-induced hepatic injury, since inhibiting apoptosis also prevents the development of acute liver failure. In this study, the mechanism of apoptosis induction by AAP was investigated in the human hepatoblastoma cell line HuH7. AAP caused marked cytotoxicity in HuH7 cells as a result of apoptosis. Processing of execution caspases to their corresponding active fragments and cleavage of cytokeratin-18 were observed, supporting a role of caspases in AAP-induced apoptosis. The manifestation of apoptosis was preceded by a translocation of cytochrome c from mitochondria to the cytosol. In conclusion, AAP induces apoptosis in human hepatoblastoma HuH7 cells through mitochondrial cytochrome c release and caspase activation.