Abstract
In response to a cytotoxic insult, a cell can die by necrosis or by apoptosis. Therefore, the overall objective of this investigation was to identify the mechanisms of drug-induced liver cell apoptosis in vitro to help with the discovery of novel markers of apoptosis that could be used for predicting hepatocyte apoptosis in vivo. In this investigation, the capability of drugs to induce apoptosis in liver cells was investigated using two cellular models, namely primary mouse hepatocytes and the hepatoma cell line, HuH7. This study was performed using three model compounds, paracetamol, thapsigargin and duroquinone. The hepatotoxin, paracetamol, is a widely used analgesic drug, can induce fatal liver injury through a combination of apoptosis and necrosis, when taken in large doses. Our work has previously shown that apoptosis plays an essential role in paracetamol-induced hepatic injury since inhibiting apoptosis, prevents the development of acute liver failure. The endoplasmic reticulum (ER) stress inducer, thapsigargin, acts by selectively inhibiting the ER Ca2+ -ATPase, which pumps calcium against a concentration gradient into the ER. Both paracetamol and thapsigargin caused marked cytotoxicity in HuH7 cells as evidenced by chromatin condensation and DNA fragmentation. Similarly, the redox-cycler, duroquinone, caused cell death in the HuH7 cells and primary hepatocytes by both the induction of apoptotic and necrotic cell death. All three compounds caused the activation of executioner caspases-3 and -7 and also the cleavage of caspase-3 cellular substrates including fodrin and cytokeratin 18. Furthermore, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-fmk) protected from paracetamol and thapsigargin-induced cytotoxicity. However, Z-VAD-fmk did not afford protection from duroquinone-induced cell death by oxidative stress in the HuH7 cells. Non-caspase proteases including calpains, cathepsins, serine proteases and the proteasome were not involved in cell death by these compounds. The manifestation of apoptosis was preceded by a loss of mitochondrial membrane potential and the release of cytochrome c and Smac/DIABLO. However, the inhibitors of the membrane permeability transition pore (PTP) cyclosporine A and bongkrekic acid failed to prevent apoptosis. In contrast, the Bcl-2 pro-apoptotic protein Bax, was found to translocate to the mitochondria to allow the release of cytochrome c in the HuH7 cells treated with paracetamol, thapsigargin and duroquinone. It was postulated that as a result of the metabolic activation of paracetamol and the concomitant production of reactive oxygen species (ROS), a cellular stress response was induced in HuH7 cells. To investigate the role of this stress response in the initiation of apoptosis by paracetamol and also thapsigargin and duroquinone, stress-activated protein kinases including c-Jun N-terminal kinase (JNK) and p38 and also MEK1/2 and glycogen synthase kinase-3 (GSK-3) were investigated. Although JNK was shown to be activated, the pharmacological inhibition of JNK, p38 and MEK did not afford protection from cell death by these compounds in the HuH7 cells. However, the inhibition of pro-apoptotic GSK-3 protected significantly from paracetamol and thapsigargin-induced cell death by apoptosis.