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LETTERS TO THE EDITOR
School of Biomedical and Molecular Sciences, University of Surrey, Guildford, United Kingdom
Received June 5, 2005; accepted June 9, 2005.
; Yuan et al., 2003) that is thought to result from a loss of cellular energy (Leist et al., 1997; Tsujimoto, 1997). Without ATP, cells fail to complete apoptosis and die by necrosis. This paradigm seems applicable to drug-induced liver injury in vivo. For instance, Ledda-Columbano et al. (1991) have shown that the hepatotoxic agent thioacetamide activates an initial apoptotic response in the liver that is followed by necrosis (oncosis). Likewise, high doses of acetaminophen (AAP) undisputedly lead to rapid and massive necrotic (oncotic) liver cell death (Gujral et al., 2002). However, at lower doses of the drug (or in less susceptible species and strains), cytokines and chemokines such as tumor necrosis factor, interferon-
, macrophage inflammatory protein 2, and interleukin 10 have been reported to significantly alter the degree of AAP-induced liver injury and lethality (Laskin et al., 1995; Blazka et al., 1996; Bourdi et al., 2002a,b; Gardner et al., 2002; Ishida et al., 2002, 2004; Liu et al., 2004), and this in the absence of any detectable change in AAP bioactivation and protein adduct formation (e.g., see Gardner et al., 2002; Bourdi et al., 2002a,b; Liu et al., 2004). In addition, there is substantial evidence for the involvement of the apoptosis-inducing system CD95L/CD95 in the liver pathology after AAP administration (Zhang et al., 2000; Fiorucci et al., 2002; Ishida et al., 2002; Liu et al., 2004) and for up-regulation of the expression of a number of apoptosis-related genes (Reilly et al., 2001; Williams et al., 2004). Taking all of this evidence into consideration, it is clear that in the case of submaximal doses, the mechanism of AAP-induced hepatic injury can no longer be reduced to an exclusively oncotic destruction of parenchymal liver cells, and a much more complex biology in the response of the liver cells to the injury needs to be considered.
Yet, even at submaximal doses, the final outcome of AAP-induced liver cell injury is typical of necrosis. However, this does not present proof against a role of apoptosis or of an apoptosis-like pathway of cell death at an early stage of the injury. Evidence for a causative role of apoptosis in AAP-induced hepatic injury was recently published by our laboratory (El-Hassan et al., 2003). In this study, large areas in the livers of AAP-treated mice with many parenchymal cells in the midzonal area showing condensed nuclei that were TUNEL-positive were observed. Here, the TUNEL staining was always nuclear and not cytoplasmic. In addition, inhibitors of caspases protected the livers of mice from injury by AAP, demonstrating that the initiation of apoptosis (or of an apoptosis-like caspase-dependent form of cell death) was critical in the development of toxicity. The protection by caspase inhibitors was found after 500 mg of AAP per kilogram in BALB/c mice but was lost with higher doses of AAP that shifted the mode of cell death initiation to necrosis. Evidence from work on the apoptosis-inducing receptor CD95 has demonstrated that apoptosis is not necessarily a single cell event but can be induced in the liver at such a large scale that massive cell content release and inflammation are observed (Ogasawara et al., 1993); however, unlike CD95, the apoptosis pathway activated by AAP was atypical and incomplete. Chromatin condensation (but not fragmentation), DNA fragmentation, Bid cleavage, Bax translocation, and sensitivity to caspase inhibitors were observed as typical features of apoptosis, yet there was a distinct lack of execution of caspase processing, and cell death rapidly degenerated into necrosis (El-Hassan et al., 2003).
The obvious choice for an in vitro model should be primary hepatocyte cultures. However, cultured hepatocytes are relatively resistant to the induction of apoptosis not only by AAP (Neuman et al., 1999; Nagai et al., 2002) but also by the well established and extremely potent inducer of liver parenchymal cell apoptosis in vivo, the anti-CD95 antibody Jo2 (Ni et al., 1994; Galle et al., 1995). CD95-induced apoptosis of cultured mouse hepatocytes requires at least 12 h to develop, which clearly differs from the occurrence of massive hepatocyte damage within 4 to 6 hours of CD95 activation in vivo. Another fundamental difference between cultured hepatocytes and the liver is that CD95-induced apoptosis is not affected by AAP in cultured hepatocytes (Nagai et al., 2002), whereas the hepatotoxic drug completely prevents CD95-induced liver cell apoptosis in vivo (Lawson et al., 1999). Therefore, primary hepatocyte cultures may be ideal to investigate AAP-induced cytotoxicity as a model for the in vivo hepatotoxicity of very high single doses of AAP but are less suitable for studying the initial apoptotic or apoptotic-like events that occur under less severe injury. To circumvent this problem, we have used a human hepatoma cell line, and this model shares many of the events seen under our in vivo conditions, including the loss of mitochondrial proapoptotic proteins and the sensitivity to caspase inhibitors. HuH7 cells also express significant levels of CYP3A (Phillips et al., 2005), which can efficiently metabolize AAP to N-acetyl-p-benzoquinone imine (Sinclair et al., 1998; Guo et al., 2004), and this is in line with the observed depletion of glutathione in our study (Macanas-Pirard et al., 2005).
In conclusion, although there is no argument that large doses of AAP administered to rodents injure the liver through the activation of massive necrosis, a dose-response window exists where a more complex pattern of toxicity occurs that is initiated by apoptosis or by a caspase-dependent apoptosis-like pathway of cell death. Interestingly, liver damage in humans requires 24 to 48 h before transaminases, bilirubin levels, and prothrombin time begin to increase (Salgia and Kosnik, 1999), which is similar to the temporal development of HuH7 cell death but much slower than the extremely rapid necrotic liver damage produced by high doses of AAP in rodents. This delayed development of toxicity in humans suggests that additional pathways of cell death, including apoptosis and apoptosis-like cell death, may also contribute to the hepatic and renal damage observed in patients that have taken an overdose of AAP.
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ABBREVIATIONS: AAP, acetaminophen; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.
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