Apoptosis and Liver Disease: Its Role in Paracetamol Hepatotoxicity.

Paracetamol (Acetaminophen, N-acetyl-p-aminophenol, AAP) is a popular domestic analgesic and antipyretic for adults and children. It has a high margin of safety when used in therapeutic doses. However, acute paracetamol overdose results in hepatic (and renal) damage. It is believed that such liver damage occurs in two phases. The first phase is due to formation of the highly reactive oxidation product, N-acetyl-p-benzoquinone-imine (NAPQI). After consuming the hepatic reservoir of glutathione, NAPQI oxidises thiol (SH-) groups of key enzymes, which causes cell death, liver failure, and occasionally death. The pathophysiology of the second phase of liver damage is poorly characterised and probably results from the continuing damage inflicted by persistently high levels of serum paracetamol. Although it is generally accepted that centrilobular hepatocyte necrosis is characteristic of paracetamol hapatotoxicity, several sporadic reports have previously presented evidence for the occurrence of some morphological and biochemical (such as DNA fragmentation) changes that are suggestive of apoptosis. Therefore, in this in vivo study, a more in-depth investigation of the role of apoptosis in paracetamol-induced hepatic injury was carried out using different modulators of the induction of parenchymal cell apoptosis by paracetamol. Six hours after paracetamol 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 the apoptosis inducing death receptor CD95. Paracetamol- induced loss of mitochondrial cytochrome c coincided with the appearance in the cytosol of a fragment corresponding to truncated Bid (t-Bid). At the same time, t-Bid became detectable in the mitochondrial fraction, and concomitantly, Bax was found translocated to mitochondria. However, paracetamol 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. On the other hand, pretreating BALB/c mice with the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-fmk) or the caspase-3 family specific inhibitor Z-Asp-Glu-Val-Asp-chloromethylketone (Z-DEVD-cmk) nearly completely abrogated paracetamol-induced damage as reflected by the degree of histopathological lesions (centrilobular necrosis, haemorrhagic damage and congestion), the rise in serum transaminases, and DNA damage (as assessed by the TUNEL method and DNA agarose gel electrophoresis). At the same time, the analogue benzyloxycarbonyl-Phe-Ala-fluoromethylketone (Z-FA-fmk) that does not inhibit caspases did not prevent the development of paracetamol-induced liver injury or the appearance of apoptotic parenchymal cells. This demonstrates that caspase inhibitors protected the liver by pharmacologically targeting caspases. I found that Z-VAD-fmk was protective when administered up to two hours after paracetamol dosing. This protective effect against paracetamol-related liver damage by Z-VAD-fmk also correlated with the inhibition of the processing of Bid to tBid. However, Z-VAD-fmk failed to prevent both the redistribution of Bax to the mitochondria and the loss of cytochrome c. In contrast, pretreatment of mice with gadolinium chloride to block Kupffer cell function did not prevent liver injury but completely blocked paracetamol-induced parenchymal cell DNA fragmentation (as assessed by the TUNEL method). Our findings also suggest that the development of liver injury in paracetamol toxicity is not necessarily a direct consequence to parenchymal cell apoptosis but instead, damage to the sinusoidal endothelial cells may play a major role through haemorrhagic congestion and shut down of hepatic microvascular perfusion. In conclusion, apoptosis is an important causal event in the initiation of the hepatic injury inflicted by paracetamol and is primarily triggered by Kupffer cell-derived factors. As suggested by the lack of activation of the main execution caspases, apoptosis is not properly executed and degenerates into necrosis. However, the causal role of apoptosis in paracetamol-induced hepatic damage meant that an opportunity for pharmacological intervention to protect the liver from drug-induced injury was uncovered