Short surveyThe role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity
Introduction
Acetaminophen (AAP) is a safe and effective analgesic when used at therapeutic levels. However, an overdose can induce severe hepatotoxicity in experimental animals and in humans (Thomas, 1993). In fact, AAP overdose is the leading cause of drug-induced liver failure requiring transplantation in US (Ostapowicz et al., 2002). Early investigations into the mechanism identified the critical role of glutathione (GSH) in detoxifying the reactive metabolite of AAP (Mitchell et al., 1973a, Mitchell et al., 1973b). These studies led to the clinical use of N-acetylcysteine as standard treatment for patients with AAP overdose. However, despite this initial breakthrough, where the new insight into the mechanism of AAP-induced toxicity in animal studies resulted in the rapid translation of this knowledge into novel therapeutic strategies in the clinic, the mechanism of cell injury and liver failure is still not completely understood. In particular, the role of reactive oxygen species in the pathophysiology remains controversial despite three decades of research. In the present review, we will summarize this historic discussion and, based on more recent findings, we will attempt to reconcile these data and provide a unified hypothesis where protein binding of AAP is the initiating event, which is followed by mitochondrial dysfunction, oxidant stress and peroxynitrite formation, as critical events of an amplification phase of the cell injury mechanism.
Section snippets
Historical perspective: protein binding versus lipid peroxidation (LPO)
The first comprehensive mechanism for AAP hepatotoxicity was published in a series of landmark papers in 1973 (Mitchell et al., 1973a, Mitchell et al., 1973b, Potter et al., 1973, Jollow et al., 1973). These investigators demonstrated that the toxicity involved the activation of AAP to a reactive metabolite (Mitchell et al., 1973a, Jollow et al., 1973), which initially depletes GSH (Mitchell et al., 1973b) and subsequently covalently binds to cellular proteins (Jollow et al., 1973, Potter et
Oxidant stress during AAP-induced liver injury
Criticism of the protein binding theory led to the hypothesis that the overall protein binding of AAP was not as important as the covalent modification of specific vital protein targets in the cell. The groups of Cohen and Khairallah, and Hinson and Pumford developed antibodies against AAP adducts, which were used for immunohistochemistry to localize protein adducts in tissue and also for Western blot analyses (Roberts et al., 1987, Bartolone et al., 1988). Over the years, both groups could
Oxidant stress: cause or consequence of liver cell injury?
It is well-known that severe hepatocellular injury can lead to an intracellular, mitochondria-derived oxidant stress in other experimental models (Jaeschke and Mitchell, 1989). After AAP overdose, significant increases of the hepatic and mitochondrial GSSG levels, indicators of mitochondrial reactive oxygen formation, were only observed after 4–6 h, i.e. after the onset of cell injury (Jaeschke, 1990, Knight et al., 2001). Thus, it is feasible that the oxidant stress could be primarily a
Peroxynitrite formation and protein nitration
Enhanced generation of superoxide in the presence of equimolar concentrations of nitric oxide (NO) will lead to the formation of the potent oxidant and nitrating agent peroxynitrite (Squadrito and Pryor, 1998). The immunohistochemical detection of nitrotyrosine protein adducts in hepatocytes (Hinson et al., 1998) and in sinusoidal endothelial cells (Knight et al., 2001, Knight and Jaeschke, in press) suggested the formation of peroxynitrite after AAP overdose. The staining pattern for
Apoptosis versus oncotic necrosis
Although the mode of AAP-induced cell death was generally considered to be hemorrhagic necrosis (oncosis), several more recent reports suggest that apoptosis may play a significant role in the overall pathophysiology (Ray et al., 1996, Ferret et al., 2001). Oxidant stress and peroxynitrite can induce apoptosis under certain conditions (Hampton and Orrenius, 1998). A careful quantitative analysis of morphological changes in hepatocytes confirmed that the number of apoptotic cells increased
Summary and conclusions
AAP hepatotoxicity is initiated by the metabolic activation of AAP to a reactive metabolite, which first depletes cellular GSH pools (Fig. 1). When the liver GSH levels are exhausted, the reactive metabolite will covalently bind to cellular proteins including proteins of the plasma membrane and mitochondria. This may result in reduced Ca2+ ATPase activities and increased cytosolic Ca2+ levels. The direct effects of modified mitochondrial proteins or increased uptake of Ca2+ can both lead to
Acknowledgements
Work from the authors' laboratory was supported in part by National Institutes of Health grants ES06091 and AA12916.
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