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The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity
Toxicology Letters 144 (2003) 279 /288 www.elsevier.com/locate/toxlet
Short survey
The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity Hartmut Jaeschke a,*, Tamara R. Knight a,b, Mary Lynn Bajt a a
Liver Research Institute, College of Medicine, University of Arizona, 1501 N. Campbell Avenue, Room 6309, Tucson, AZ 85724, USA b Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, USA Received 16 April 2003; received in revised form 7 May 2003; accepted 7 May 2003
Abstract Acetaminophen (AAP) overdose can cause severe hepatotoxicity and even liver failure in experimental animals and humans. Despite substantial efforts over the last 30 years, the mechanism of AAP-induced liver cell injury is still not completely understood. It is widely accepted that the injury process is initiated by the metabolism of AAP to a reactive metabolite, which first depletes glutathione and then binds to cellular proteins including a number of mitochondrial proteins. One consequence of this process may be the observed inhibition of mitochondrial respiration, ATP depletion and mitochondrial oxidant stress. In the presence of sufficient vitamin E, reactive oxygen formation does not induce severe lipid peroxidation but the superoxide reacts with nitric oxide to form peroxynitrite, a powerful oxidant and nitrating agent. Peroxynitrite can modify cellular macromolecules and may aggravate mitochondrial dysfunction and ATP depletion leading to cellular oncotic necrosis in hepatocytes and sinusoidal endothelial cells. Thus, we hypothesize that reactive metabolite formation and protein binding initiate the injury process, which may be then propagated and amplified by mitochondrial dysfunction and peroxynitrite formation. This concept also reconciles many of the controversial findings of the past and provides a viable hypothesis for the mechanism of hepatocellular injury after AAP overdose. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Acetaminophen; Oxidant stress; Peroxynitrite; Liver cell necrosis; Apoptosis
1. 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,
* Corresponding author. Tel.: /1-520-626-1449; fax: /1520-626-5975. E-mail address: [email protected] (H. Jaeschke).
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,b). 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
0378-4274/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0378-4274(03)00239-X
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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.
2. 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,b; 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 al., 1973). The reactive metabolite was later identified as N -acetyl-p-benzoquinone imine (NAPQI) (Dahlin et al., 1984; Nelson, 1990). However, there are arguments that NAPQI may not exist in a free form during AAP metabolism (Smith and Mitchell, 1985). Nevertheless, the basic concept that the covalent binding of a reactive metabolite may interrupt the function of proteins and, therefore, can have detrimental effects on the cell leading eventually to cell death was widely accepted as a general principle of cell toxicity (Mitchell et al., 1981, 1984; Smith et al., 1985; Nelson, 1990). However, in the case of AAP, it was also consistently challenged from the beginning (reviewed in Smith et al., 1985). One of the main concerns was that covalent binding of the reactive AAP metabolite appeared not to be always
correlated with the severity of liver injury. In response to these concerns, Corcoran et al. (1985) demonstrated that the trapping of blood in the liver vasculature during the injury process dilutes the hepatic protein pool. This influx of protein without bound AAP metabolites results in an ‘‘apparent’’ reduction of protein binding after a more severe injury. This effect may be at least in part responsible for some of the observed discrepancies between the extent of covalent binding and severity of injury (Corcoran et al., 1985). Although, in general, no AAP hepatotoxicity is observed without protein binding, there is evidence for covalent binding in female rats without detectable liver injury (Tarloff et al., 1996). In addition, exposure to high doses of 3?-hydroxyacetanilide, a regioisomer of AAP, results in similar overall covalent binding in mouse liver without injury (Tirmenstein and Nelson, 1989; Myers et al., 1995). Because of some of these concerns with the protein binding hypothesis, alternative mechanisms for AAP-induced liver injury were investigated. With the recognition that the P450 system can release reactive oxygen species during its enzyme activity (Kuthan et al., 1978), Wendel and coworkers postulated that AAP metabolism triggers massive LPO, which was responsible for liver injury (Wendel et al., 1979; Wendel and Feuerstein, 1981). Because of the technical difficulties involved in measuring ethane and pentane exhalation as reliable indicators of LPO in mice in vivo, Wendel et al. (1979, 1982) made the animals more susceptible by feeding a diet low in vitamin E and high in polyunsaturated fatty acids. Moreover, the animals were not only fasted but also received inducers of P450 such as benzo-[a]-pyrene (Wendel et al., 1979, 1982). Under these conditions, AAP triggered a rapid loss of GSH and caused massive LPO followed by cell death and liver failure within 4 h (Wendel and Feuerstein, 1981; Wendel et al., 1982). LPO and injury could be dramatically reduced by inhibition of AAP metabolism (Wendel and Feuerstein, 1981), enhancement of liver GSH levels (Wendel et al., 1982) and pretreatment with vitamin E (Wendel et al., 1988). Quantitatively, LPO parameters, such as ethane and pentane exhalation and the hepatic content of thiobarbituric acid-reactive substances, increased 20- to 50-
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fold above baseline. Moreover, there was a substantial and selective loss of polyunsaturated fatty acids in liver cell membranes. These observations, together with the protective effect of vitamin E treatment, strongly supported the hypothesis that massive LPO was the only mechanism of AAPinduced liver injury. However, the reports by Wendel and coworkers led to a vigorous challenge by Mitchell’s group. The hypothesis that P450derived reactive oxygen may be responsible for LPO and cell injury was questioned because of the lack of glutathione disulfide (GSSG) formation as indicator for reactive oxygen generation during the metabolism of AAP in rats and in mice (Lauterburg et al., 1984; Smith and Jaeschke, 1989). Moreover, ethane and pentane exhalation was mainly observed after the injury was established. These findings led to the conclusion that LPO may be a consequence rather than the cause of cell damage (Mitchell et al., 1981, 1984). In addition, animals on a standard diet developed injury much slower and showed a distinct centrilobular necrosis. Treatment with vitamin E, which protected animals on Wendel’s diet (Wendel et al., 1988; Jaeschke et al., 1987, 1992b), had no effect on AAP-induced injury in animals on a standard diet (Jaeschke and Smith, Unpublished). Because of these serious discrepancies, it became obvious that both groups worked on different models with different mechanisms of liver injury. In retrospect, it is clear that the dietary manipulations did not just amplify an existing mechanism but inadvertently switched the mechanisms of injury. It can be concluded from all data available that cell destruction by massive LPO is not a relevant mechanism of AAP-induced liver failure.
3. 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
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Western blot analyses (Roberts et al., 1987; Bartolone et al., 1988). Over the years, both groups could identify a number of proteins, which contained adducts of AAP (Cohen and Khairallah, 1997; Pumford and Halmes, 1997). More recently, Qiu et al. (1998) could confirm these findings and expand the list of proteins to about 40. Qui et al. used mass spectrometry to identify the proteins that had AAP metabolites bound to them. However, despite these efforts, it became clear that no particular cellular protein was selectively targeted and its function so severely compromised that it could readily explain the rapid cell death. Although Gupta et al. (1997) reported a more than 50% inhibition of hepatic glutamine synthetase activity within 2 h after 400 mg/kg of AAP, other enzyme activities declined only by 20/25%, 1/2 h after AAP treatment (Halmes et al., 1996; Pumford et al., 1997). Therefore, oxidant stress as an alternative or supplementary mechanism of cell injury was further investigated. Laskin and Pilaro (1986) reported first that liver macrophages isolated from AAP-treated rats were activated but no liver injury was observed. In a subsequent paper, Laskin et al. (1995) provided evidence that resident Kupffer cells and accumulating macrophages may contribute to the overall liver injury in rats. Similar experiments with inhibitors of Kupffer cells in mice showed complete protection against AAP-induced liver injury (Michael et al., 1999). However, others and we could not reproduce these data; at best a minor reduction of liver injury was observed (Ju et al., 2002; Knight and Jaeschke, in press; Ito et al., in press). Furthermore, mice, which lack a functional NADPH oxidase in Kupffer cells and, therefore, are unable to generate superoxide, are equally as sensitive to AAP hepatotoxicity as wild-type animals (James et al., in press). These findings do not support a major role of Kupffer cell-derived oxidant stress in pathophysiology. The Kupffer cell hypothesis also has to be questioned just based on the general knowledge of Kupffer cell cytotoxicity in the liver. The most active Kupffer cells in terms of reactive oxygen formation are located in the periportal area (Bautista et al., 1990; Jaeschke et al., 1991a). As a consequence, the injury would
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be expected to be located in close proximity to the most cytotoxic Kupffer cells and not just downstream in the centrilobular area, where less-active Kupffer cells are present. Thus, despite activation, Kupffer cells may be at best a minor source of oxidant stress in the liver after AAP overdose. On the other hand, Kupffer cells may be more important in the pathophysiology by generating anti-inflammatory cytokines such as IL-10 (Bourdi et al., 2002; Ju et al., 2002). Another potent source of reactive oxygen in the liver can be infiltrating neutrophils (Jaeschke et al., 1992a, 1993). However, in all experimental models where neutrophils have been shown to cause injury, e.g. ischemia /reperfusion (Jaeschke et al., 1990), endotoxemia (Jaeschke et al., 1991b) and obstructive cholestasis (Gujral et al., in press), neutrophils are systemically activated and accumulate in the liver before excessive liver injury occurs. In contrast, no systemic activation of neutrophils was found after AAP treatment (Lawson et al., 2000). In addition, neutrophils are recruited into the liver with some minor delay after AAP-induced injury (Welty et al., 1993; Lawson et al., 2000). Furthermore, AAP did not induce the expression of hepatic intercellular adhesion molecule-1 (ICAM-1; Welty et al., 1993), which is a prerequisite of neutrophil-induced liver injury (Essani et al., 1995). Consistent with these data, blocking neutrophil activity with anti-CD18 antibodies did not protect against AAP-induced hepatotoxicity (Lawson et al., 2000). Thus, there is no convincing evidence that neutrophils do anything more than cleanup of necrotic cell debris after AAP overdose. A potential intracellular source of reactive oxygen could be the enzyme xanthine oxidase. Indeed, a conversion of xanthine dehydrogenase to oxidase was observed in the liver after AAP overdose (Jaeschke, 1990; Tirmenstein and Nelson, 1990). However, dose /response experiments with the xanthine oxidase inhibitor, allopurinol, clearly demonstrated that doses, which completely inactivated the enzyme, did not prevent an intracellular oxidant stress and did not protect against AAP-induced toxicity (Jaeschke, 1990). These data argue against xanthine oxidase as a
relevant intracellular source of reactive oxygen during AAP hepatotoxicity. Mitochondria are a target for covalent binding of the reactive metabolite of AAP (Tirmenstein and Nelson, 1989; Pumford et al., 1990, 1997; Gupta et al., 1997). The importance of mitochondrial protein binding for the toxicity is supported by the observation that AAP and 3?-hydroxyacetanilide show similar overall protein binding but the reactive metabolite of AAP binds to substantially more mitochondrial proteins than the metabolite of its non-hepatotoxic isomer (Tirmenstein and Nelson, 1989; Myers et al., 1995; Qiu et al., 2001). Mitochondrial protein binding may be responsible for the early ultrastructural changes in this cell organelle (Placke et al., 1987) and the inhibition of the mitochondrial respiration after AAP overdose (Meyers et al., 1988; Ramsay et al., 1989). Mitochondrial dysfunction, which may be induced directly by protein binding or indirectly through increased cytosolic Ca2 levels (TsokosKuhn et al., 1988; Tirmenstein and Nelson, 1989), can cause a mitochondrial oxidant stress and ATP depletion (Jaeschke et al., 1990; Tirmenstein and Nelson, 1990). Recent studies suggest that AAPinduced mitochondrial oxidant stress is at least in part a consequence of the mitochondrial membrane permeability transition (MPT) (Jaeschke et al., 2003). Cyclosporin, an inhibitor of the membrane permeability transition, protects against AAP-induced liver injury in mice (Haouzi et al., 2002).
4. Oxidant stress: cause or consequence of liver cell injury? It is well-known that severe hepatocellular injury can lead to an intracellular, mitochondriaderived 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 consequence of cell injury rather than
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the cause of it (Rogers et al., 2000). To address this fundamental problem, we evaluated the time course of reactive oxygen formation with 2,7dichlorodihydrofluoresceine acetate (DCHF) in a hepatocyte cell culture model of AAP-induced injury. The results clearly demonstrated that a drastic increase in DCHF fluorescence preceded cell injury by several hours (Jaeschke et al., 2003). These data are consistent with the early increase in the GSSG-to-GSH ratio within less than 1 h after AAP overdose (Knight et al., 2001) and with the protective effect of various radical scavengers against AAP hepatotoxicity in vivo (Nakae et al., 1990; Ferret et al., 2001). Furthermore, induction of heme oxygenase-1 attenuated AAP-induced liver injury presumably through an antioxidant mechanism (Chiu et al., 2002). Interestingly, loading mitochondria with the antioxidant a-tocopherol in vivo inhibited allyl alcohol/iron-induced LPO and reduced liver injury but had no protective effect against AAP-induced liver injury (Knight et al., 2003). These data suggest that lipids are not relevant targets for the mitochondrial oxidant stress during AAP hepatotoxicity.
5. 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 nitrotyrosine correlated with the presence of AAP protein adducts in cells undergoing necrosis (Hinson et al., 1998). Moreover, time course studies indicated that nitrotyrosine staining slightly preceded or developed parallel to cell injury in both hepatocytes and endothelial cells (Knight et al., 2001). Substantial efforts to address the key question whether peroxynitrite formation is relevant for the cell injury or only an epiphenomenon concentrated on studies with inducible NO
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synthase (iNOS) gene knockout mice and various NOS inhibitors. Unfortunately, the results were conflicting. Laskin and coworkers found a partial protective effect against AAP-induced liver injury with the iNOS inhibitor aminoguanidine in rats (Gardner et al., 1998) and in iNOS gene knockout mice (Gardner et al., 2002). Similar approaches used by Hinson and coworkers did not show beneficial effects against AAP hepatotoxicity (Michael et al., 2001; Hinson et al., 2002). These investigators postulated that reduced peroxynitrite formation under these conditions is compensated by enhanced hydroxyradical generation and LPO (Michael et al., 2001; Hinson et al., 2002). In our own studies, we found peroxynitrite formation without iNOS induction or evidence for excessive NO formation (Knight et al., 2001). These data suggest that NO generation by constitutively expressed NOS may be sufficient to form peroxynitrite under conditions of enhanced superoxide formation. If induced, iNOS can also contribute to AAP-induced peroxynitrite formation. In fact, the protective effect of IL-10 against AAP-induced liver injury may be due to suppressed iNOS induction and reduced peroxynitrite formation (Bourdi et al., 2002). GSH reacts effectively with peroxynitrite and can prevent protein nitration in vitro (Kirsch et al., 2001; Knight et al., 2002). The lack of cytosolic and mitochondrial GSH after AAP overdose may be the main reason for the extensive protein nitration and potential cytotoxic action of peroxynitrite. Therefore, we intravenously injected high doses of GSH at various times after AAP treatment to accelerate the recovery of the depleted hepatic GSH pools (Knight et al., 2002). The goal was to delay the injection of GSH long enough to have no relevant effect on the detoxification of the reactive metabolite but predominantly scavenge peroxynitrite. If GSH was injected at 1.5 or 2.25 h after AAP treatment, the mitochondrial dysfunction was still present but nitrotyrosine staining and the injury were significantly attenuated at 6 h (Knight et al., 2002). Moreover, scavenging peroxynitrite with GSH promoted cell cycle activation and regeneration, and long-term survival of the animals (Bajt et al., 2003). These data suggest that peroxynitrite is indeed a critical mediator in the
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progression of AAP-induced liver injury. The validity of this conclusion is based on the assumption that the bulk of reactive metabolite formation is over by 1/2 h after the intraperitoneal injection of a moderately toxic dose of AAP (Jollow et al., 1973; Tirmenstein and Nelson, 1989; Roberts et al., 1991). However, after high doses administered orally, reactive metabolites may be formed for up to 6 h (Corcoran et al., 1985). Under these circumstances, any delayed administration of N acetylcysteine to restore hepatic GSH levels may still protect by scavenging NAPQI rather than peroxynitrite (Corcoran et al., 1985).
6. 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 significantly after AAP overdose (Gujral et al., 2002). However, the dominant mode (/90%) of cell death was found to be oncotic necrosis (Gujral et al., 2002). Consistent with these morphological observations, there is no evidence of increased caspase enzyme activity or proteolytic activation during AAP hepatotoxicity (Lawson et al., 1999; Adams et al., 2001; Gujral et al., 2002) despite translocation of the pro-apoptotic Bcl-2 family member Bax to the mitochondria (Adams et al., 2001) and mitochondrial cytochrome c release (Knight and Jaeschke, 2002). Furthermore, overexpression of the anti-apoptotic protein Bcl-2 actually aggravated AAP-induced liver injury (Adams et al., 2001). AAP hepatotoxicity involves excessive DNA fragmentation (Shen et al., 1991). However, the reported DNA laddering (Shen et al., 1991), DNA fragmentation as measured by an anti-histone ELISA assay (Lawson et al., 1999) and DNA strand breaks as indicated by TUNEL assay (Lawson et al., 1999) are not specific for
apoptotic cell death (Gujral et al., 2001; Jaeschke and Lemasters, in press). TUNEL staining pattern in AAP-damaged cells is quite different from typical apoptotic cells (Gujral et al., 2002). One of the strongest arguments against AAP-induced apoptosis comes from the observation that AAP treatment actually prevented Fas receptormediated hepatocellular apoptosis (Lawson et al., 1999). Since the Fas receptor signaling pathway in hepatocytes depends on mitochondrial amplification and feedback activation of upstream caspases (Yin et al., 1999; Bajt et al., 2000), AAP interrupts this mechanism by causing mitochondrial dysfunction (Knight and Jaeschke, 2002). Thus, AAP causes cell death almost exclusively through oncotic necrosis.
7. 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 reduced mitochondrial respiration and ATP synthesis. In addition to reduced cellular ATP levels, mitochondria generate increased amounts of superoxide. Although superoxide can dismutate to form hydrogen peroxide, which can enhance protein oxidation or cause LPO, this pathway appears to be of limited relevance. On the other hand, superoxide can react with NO to form the potent oxidant and nitrating agent, peroxynitrite. In the absence of cellular GSH, peroxynitrite can cause extensive protein oxidation and nitration, which may induce further mitochondrial dysfunction and eventually lead to irreversible damage (MPT) and severe loss of cellular ATP. The combination of the detrimental events culminates in oncotic necrosis of hepatocytes. Although more mechanistic work needs to be done to elucidate specific consequences of protein oxidation and nitration, the current literature
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Fig. 1. Mechanism of AAP-induced liver cell injury. See text for details. MPT, mitochondrial membrane permeability transition; NAPQI, N -acetyl-p -benzoquinone imine; NO, nitric oxide; LPO, lipid peroxidation; ONOO, peroxynitrite.
strongly supports the concept that the mechanism of AAP-induced hepatocellular injury is initiated by covalent protein binding of the reactive metabolite. However, this initiating event requires amplification and progression, which may involve mitochondrial dysfunction with oxidant stress and peroxynitrite formation and the consequences of these oxidative events. Thus, protein binding and oxidant stress are not competing hypotheses but sequential events in the mechanism of AAP hepatotoxicity. This concept explains many of the controversial findings and reconciles many of the differences and disputes of the past.
Acknowledgements Work from the authors’ laboratory was supported in part by National Institutes of Health grants ES06091 and AA12916.
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Short survey
The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity Hartmut Jaeschke a,*, Tamara R. Knight a,b, Mary Lynn Bajt a a
Liver Research Institute, College of Medicine, University of Arizona, 1501 N. Campbell Avenue, Room 6309, Tucson, AZ 85724, USA b Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, USA Received 16 April 2003; received in revised form 7 May 2003; accepted 7 May 2003
Abstract Acetaminophen (AAP) overdose can cause severe hepatotoxicity and even liver failure in experimental animals and humans. Despite substantial efforts over the last 30 years, the mechanism of AAP-induced liver cell injury is still not completely understood. It is widely accepted that the injury process is initiated by the metabolism of AAP to a reactive metabolite, which first depletes glutathione and then binds to cellular proteins including a number of mitochondrial proteins. One consequence of this process may be the observed inhibition of mitochondrial respiration, ATP depletion and mitochondrial oxidant stress. In the presence of sufficient vitamin E, reactive oxygen formation does not induce severe lipid peroxidation but the superoxide reacts with nitric oxide to form peroxynitrite, a powerful oxidant and nitrating agent. Peroxynitrite can modify cellular macromolecules and may aggravate mitochondrial dysfunction and ATP depletion leading to cellular oncotic necrosis in hepatocytes and sinusoidal endothelial cells. Thus, we hypothesize that reactive metabolite formation and protein binding initiate the injury process, which may be then propagated and amplified by mitochondrial dysfunction and peroxynitrite formation. This concept also reconciles many of the controversial findings of the past and provides a viable hypothesis for the mechanism of hepatocellular injury after AAP overdose. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Acetaminophen; Oxidant stress; Peroxynitrite; Liver cell necrosis; Apoptosis
1. 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,
* Corresponding author. Tel.: /1-520-626-1449; fax: /1520-626-5975. E-mail address: [email protected] (H. Jaeschke).
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,b). 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
0378-4274/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0378-4274(03)00239-X
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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.
2. 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,b; 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 al., 1973). The reactive metabolite was later identified as N -acetyl-p-benzoquinone imine (NAPQI) (Dahlin et al., 1984; Nelson, 1990). However, there are arguments that NAPQI may not exist in a free form during AAP metabolism (Smith and Mitchell, 1985). Nevertheless, the basic concept that the covalent binding of a reactive metabolite may interrupt the function of proteins and, therefore, can have detrimental effects on the cell leading eventually to cell death was widely accepted as a general principle of cell toxicity (Mitchell et al., 1981, 1984; Smith et al., 1985; Nelson, 1990). However, in the case of AAP, it was also consistently challenged from the beginning (reviewed in Smith et al., 1985). One of the main concerns was that covalent binding of the reactive AAP metabolite appeared not to be always
correlated with the severity of liver injury. In response to these concerns, Corcoran et al. (1985) demonstrated that the trapping of blood in the liver vasculature during the injury process dilutes the hepatic protein pool. This influx of protein without bound AAP metabolites results in an ‘‘apparent’’ reduction of protein binding after a more severe injury. This effect may be at least in part responsible for some of the observed discrepancies between the extent of covalent binding and severity of injury (Corcoran et al., 1985). Although, in general, no AAP hepatotoxicity is observed without protein binding, there is evidence for covalent binding in female rats without detectable liver injury (Tarloff et al., 1996). In addition, exposure to high doses of 3?-hydroxyacetanilide, a regioisomer of AAP, results in similar overall covalent binding in mouse liver without injury (Tirmenstein and Nelson, 1989; Myers et al., 1995). Because of some of these concerns with the protein binding hypothesis, alternative mechanisms for AAP-induced liver injury were investigated. With the recognition that the P450 system can release reactive oxygen species during its enzyme activity (Kuthan et al., 1978), Wendel and coworkers postulated that AAP metabolism triggers massive LPO, which was responsible for liver injury (Wendel et al., 1979; Wendel and Feuerstein, 1981). Because of the technical difficulties involved in measuring ethane and pentane exhalation as reliable indicators of LPO in mice in vivo, Wendel et al. (1979, 1982) made the animals more susceptible by feeding a diet low in vitamin E and high in polyunsaturated fatty acids. Moreover, the animals were not only fasted but also received inducers of P450 such as benzo-[a]-pyrene (Wendel et al., 1979, 1982). Under these conditions, AAP triggered a rapid loss of GSH and caused massive LPO followed by cell death and liver failure within 4 h (Wendel and Feuerstein, 1981; Wendel et al., 1982). LPO and injury could be dramatically reduced by inhibition of AAP metabolism (Wendel and Feuerstein, 1981), enhancement of liver GSH levels (Wendel et al., 1982) and pretreatment with vitamin E (Wendel et al., 1988). Quantitatively, LPO parameters, such as ethane and pentane exhalation and the hepatic content of thiobarbituric acid-reactive substances, increased 20- to 50-
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fold above baseline. Moreover, there was a substantial and selective loss of polyunsaturated fatty acids in liver cell membranes. These observations, together with the protective effect of vitamin E treatment, strongly supported the hypothesis that massive LPO was the only mechanism of AAPinduced liver injury. However, the reports by Wendel and coworkers led to a vigorous challenge by Mitchell’s group. The hypothesis that P450derived reactive oxygen may be responsible for LPO and cell injury was questioned because of the lack of glutathione disulfide (GSSG) formation as indicator for reactive oxygen generation during the metabolism of AAP in rats and in mice (Lauterburg et al., 1984; Smith and Jaeschke, 1989). Moreover, ethane and pentane exhalation was mainly observed after the injury was established. These findings led to the conclusion that LPO may be a consequence rather than the cause of cell damage (Mitchell et al., 1981, 1984). In addition, animals on a standard diet developed injury much slower and showed a distinct centrilobular necrosis. Treatment with vitamin E, which protected animals on Wendel’s diet (Wendel et al., 1988; Jaeschke et al., 1987, 1992b), had no effect on AAP-induced injury in animals on a standard diet (Jaeschke and Smith, Unpublished). Because of these serious discrepancies, it became obvious that both groups worked on different models with different mechanisms of liver injury. In retrospect, it is clear that the dietary manipulations did not just amplify an existing mechanism but inadvertently switched the mechanisms of injury. It can be concluded from all data available that cell destruction by massive LPO is not a relevant mechanism of AAP-induced liver failure.
3. 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
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Western blot analyses (Roberts et al., 1987; Bartolone et al., 1988). Over the years, both groups could identify a number of proteins, which contained adducts of AAP (Cohen and Khairallah, 1997; Pumford and Halmes, 1997). More recently, Qiu et al. (1998) could confirm these findings and expand the list of proteins to about 40. Qui et al. used mass spectrometry to identify the proteins that had AAP metabolites bound to them. However, despite these efforts, it became clear that no particular cellular protein was selectively targeted and its function so severely compromised that it could readily explain the rapid cell death. Although Gupta et al. (1997) reported a more than 50% inhibition of hepatic glutamine synthetase activity within 2 h after 400 mg/kg of AAP, other enzyme activities declined only by 20/25%, 1/2 h after AAP treatment (Halmes et al., 1996; Pumford et al., 1997). Therefore, oxidant stress as an alternative or supplementary mechanism of cell injury was further investigated. Laskin and Pilaro (1986) reported first that liver macrophages isolated from AAP-treated rats were activated but no liver injury was observed. In a subsequent paper, Laskin et al. (1995) provided evidence that resident Kupffer cells and accumulating macrophages may contribute to the overall liver injury in rats. Similar experiments with inhibitors of Kupffer cells in mice showed complete protection against AAP-induced liver injury (Michael et al., 1999). However, others and we could not reproduce these data; at best a minor reduction of liver injury was observed (Ju et al., 2002; Knight and Jaeschke, in press; Ito et al., in press). Furthermore, mice, which lack a functional NADPH oxidase in Kupffer cells and, therefore, are unable to generate superoxide, are equally as sensitive to AAP hepatotoxicity as wild-type animals (James et al., in press). These findings do not support a major role of Kupffer cell-derived oxidant stress in pathophysiology. The Kupffer cell hypothesis also has to be questioned just based on the general knowledge of Kupffer cell cytotoxicity in the liver. The most active Kupffer cells in terms of reactive oxygen formation are located in the periportal area (Bautista et al., 1990; Jaeschke et al., 1991a). As a consequence, the injury would
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be expected to be located in close proximity to the most cytotoxic Kupffer cells and not just downstream in the centrilobular area, where less-active Kupffer cells are present. Thus, despite activation, Kupffer cells may be at best a minor source of oxidant stress in the liver after AAP overdose. On the other hand, Kupffer cells may be more important in the pathophysiology by generating anti-inflammatory cytokines such as IL-10 (Bourdi et al., 2002; Ju et al., 2002). Another potent source of reactive oxygen in the liver can be infiltrating neutrophils (Jaeschke et al., 1992a, 1993). However, in all experimental models where neutrophils have been shown to cause injury, e.g. ischemia /reperfusion (Jaeschke et al., 1990), endotoxemia (Jaeschke et al., 1991b) and obstructive cholestasis (Gujral et al., in press), neutrophils are systemically activated and accumulate in the liver before excessive liver injury occurs. In contrast, no systemic activation of neutrophils was found after AAP treatment (Lawson et al., 2000). In addition, neutrophils are recruited into the liver with some minor delay after AAP-induced injury (Welty et al., 1993; Lawson et al., 2000). Furthermore, AAP did not induce the expression of hepatic intercellular adhesion molecule-1 (ICAM-1; Welty et al., 1993), which is a prerequisite of neutrophil-induced liver injury (Essani et al., 1995). Consistent with these data, blocking neutrophil activity with anti-CD18 antibodies did not protect against AAP-induced hepatotoxicity (Lawson et al., 2000). Thus, there is no convincing evidence that neutrophils do anything more than cleanup of necrotic cell debris after AAP overdose. A potential intracellular source of reactive oxygen could be the enzyme xanthine oxidase. Indeed, a conversion of xanthine dehydrogenase to oxidase was observed in the liver after AAP overdose (Jaeschke, 1990; Tirmenstein and Nelson, 1990). However, dose /response experiments with the xanthine oxidase inhibitor, allopurinol, clearly demonstrated that doses, which completely inactivated the enzyme, did not prevent an intracellular oxidant stress and did not protect against AAP-induced toxicity (Jaeschke, 1990). These data argue against xanthine oxidase as a
relevant intracellular source of reactive oxygen during AAP hepatotoxicity. Mitochondria are a target for covalent binding of the reactive metabolite of AAP (Tirmenstein and Nelson, 1989; Pumford et al., 1990, 1997; Gupta et al., 1997). The importance of mitochondrial protein binding for the toxicity is supported by the observation that AAP and 3?-hydroxyacetanilide show similar overall protein binding but the reactive metabolite of AAP binds to substantially more mitochondrial proteins than the metabolite of its non-hepatotoxic isomer (Tirmenstein and Nelson, 1989; Myers et al., 1995; Qiu et al., 2001). Mitochondrial protein binding may be responsible for the early ultrastructural changes in this cell organelle (Placke et al., 1987) and the inhibition of the mitochondrial respiration after AAP overdose (Meyers et al., 1988; Ramsay et al., 1989). Mitochondrial dysfunction, which may be induced directly by protein binding or indirectly through increased cytosolic Ca2 levels (TsokosKuhn et al., 1988; Tirmenstein and Nelson, 1989), can cause a mitochondrial oxidant stress and ATP depletion (Jaeschke et al., 1990; Tirmenstein and Nelson, 1990). Recent studies suggest that AAPinduced mitochondrial oxidant stress is at least in part a consequence of the mitochondrial membrane permeability transition (MPT) (Jaeschke et al., 2003). Cyclosporin, an inhibitor of the membrane permeability transition, protects against AAP-induced liver injury in mice (Haouzi et al., 2002).
4. Oxidant stress: cause or consequence of liver cell injury? It is well-known that severe hepatocellular injury can lead to an intracellular, mitochondriaderived 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 consequence of cell injury rather than
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the cause of it (Rogers et al., 2000). To address this fundamental problem, we evaluated the time course of reactive oxygen formation with 2,7dichlorodihydrofluoresceine acetate (DCHF) in a hepatocyte cell culture model of AAP-induced injury. The results clearly demonstrated that a drastic increase in DCHF fluorescence preceded cell injury by several hours (Jaeschke et al., 2003). These data are consistent with the early increase in the GSSG-to-GSH ratio within less than 1 h after AAP overdose (Knight et al., 2001) and with the protective effect of various radical scavengers against AAP hepatotoxicity in vivo (Nakae et al., 1990; Ferret et al., 2001). Furthermore, induction of heme oxygenase-1 attenuated AAP-induced liver injury presumably through an antioxidant mechanism (Chiu et al., 2002). Interestingly, loading mitochondria with the antioxidant a-tocopherol in vivo inhibited allyl alcohol/iron-induced LPO and reduced liver injury but had no protective effect against AAP-induced liver injury (Knight et al., 2003). These data suggest that lipids are not relevant targets for the mitochondrial oxidant stress during AAP hepatotoxicity.
5. 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 nitrotyrosine correlated with the presence of AAP protein adducts in cells undergoing necrosis (Hinson et al., 1998). Moreover, time course studies indicated that nitrotyrosine staining slightly preceded or developed parallel to cell injury in both hepatocytes and endothelial cells (Knight et al., 2001). Substantial efforts to address the key question whether peroxynitrite formation is relevant for the cell injury or only an epiphenomenon concentrated on studies with inducible NO
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synthase (iNOS) gene knockout mice and various NOS inhibitors. Unfortunately, the results were conflicting. Laskin and coworkers found a partial protective effect against AAP-induced liver injury with the iNOS inhibitor aminoguanidine in rats (Gardner et al., 1998) and in iNOS gene knockout mice (Gardner et al., 2002). Similar approaches used by Hinson and coworkers did not show beneficial effects against AAP hepatotoxicity (Michael et al., 2001; Hinson et al., 2002). These investigators postulated that reduced peroxynitrite formation under these conditions is compensated by enhanced hydroxyradical generation and LPO (Michael et al., 2001; Hinson et al., 2002). In our own studies, we found peroxynitrite formation without iNOS induction or evidence for excessive NO formation (Knight et al., 2001). These data suggest that NO generation by constitutively expressed NOS may be sufficient to form peroxynitrite under conditions of enhanced superoxide formation. If induced, iNOS can also contribute to AAP-induced peroxynitrite formation. In fact, the protective effect of IL-10 against AAP-induced liver injury may be due to suppressed iNOS induction and reduced peroxynitrite formation (Bourdi et al., 2002). GSH reacts effectively with peroxynitrite and can prevent protein nitration in vitro (Kirsch et al., 2001; Knight et al., 2002). The lack of cytosolic and mitochondrial GSH after AAP overdose may be the main reason for the extensive protein nitration and potential cytotoxic action of peroxynitrite. Therefore, we intravenously injected high doses of GSH at various times after AAP treatment to accelerate the recovery of the depleted hepatic GSH pools (Knight et al., 2002). The goal was to delay the injection of GSH long enough to have no relevant effect on the detoxification of the reactive metabolite but predominantly scavenge peroxynitrite. If GSH was injected at 1.5 or 2.25 h after AAP treatment, the mitochondrial dysfunction was still present but nitrotyrosine staining and the injury were significantly attenuated at 6 h (Knight et al., 2002). Moreover, scavenging peroxynitrite with GSH promoted cell cycle activation and regeneration, and long-term survival of the animals (Bajt et al., 2003). These data suggest that peroxynitrite is indeed a critical mediator in the
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progression of AAP-induced liver injury. The validity of this conclusion is based on the assumption that the bulk of reactive metabolite formation is over by 1/2 h after the intraperitoneal injection of a moderately toxic dose of AAP (Jollow et al., 1973; Tirmenstein and Nelson, 1989; Roberts et al., 1991). However, after high doses administered orally, reactive metabolites may be formed for up to 6 h (Corcoran et al., 1985). Under these circumstances, any delayed administration of N acetylcysteine to restore hepatic GSH levels may still protect by scavenging NAPQI rather than peroxynitrite (Corcoran et al., 1985).
6. 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 significantly after AAP overdose (Gujral et al., 2002). However, the dominant mode (/90%) of cell death was found to be oncotic necrosis (Gujral et al., 2002). Consistent with these morphological observations, there is no evidence of increased caspase enzyme activity or proteolytic activation during AAP hepatotoxicity (Lawson et al., 1999; Adams et al., 2001; Gujral et al., 2002) despite translocation of the pro-apoptotic Bcl-2 family member Bax to the mitochondria (Adams et al., 2001) and mitochondrial cytochrome c release (Knight and Jaeschke, 2002). Furthermore, overexpression of the anti-apoptotic protein Bcl-2 actually aggravated AAP-induced liver injury (Adams et al., 2001). AAP hepatotoxicity involves excessive DNA fragmentation (Shen et al., 1991). However, the reported DNA laddering (Shen et al., 1991), DNA fragmentation as measured by an anti-histone ELISA assay (Lawson et al., 1999) and DNA strand breaks as indicated by TUNEL assay (Lawson et al., 1999) are not specific for
apoptotic cell death (Gujral et al., 2001; Jaeschke and Lemasters, in press). TUNEL staining pattern in AAP-damaged cells is quite different from typical apoptotic cells (Gujral et al., 2002). One of the strongest arguments against AAP-induced apoptosis comes from the observation that AAP treatment actually prevented Fas receptormediated hepatocellular apoptosis (Lawson et al., 1999). Since the Fas receptor signaling pathway in hepatocytes depends on mitochondrial amplification and feedback activation of upstream caspases (Yin et al., 1999; Bajt et al., 2000), AAP interrupts this mechanism by causing mitochondrial dysfunction (Knight and Jaeschke, 2002). Thus, AAP causes cell death almost exclusively through oncotic necrosis.
7. 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 reduced mitochondrial respiration and ATP synthesis. In addition to reduced cellular ATP levels, mitochondria generate increased amounts of superoxide. Although superoxide can dismutate to form hydrogen peroxide, which can enhance protein oxidation or cause LPO, this pathway appears to be of limited relevance. On the other hand, superoxide can react with NO to form the potent oxidant and nitrating agent, peroxynitrite. In the absence of cellular GSH, peroxynitrite can cause extensive protein oxidation and nitration, which may induce further mitochondrial dysfunction and eventually lead to irreversible damage (MPT) and severe loss of cellular ATP. The combination of the detrimental events culminates in oncotic necrosis of hepatocytes. Although more mechanistic work needs to be done to elucidate specific consequences of protein oxidation and nitration, the current literature
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Fig. 1. Mechanism of AAP-induced liver cell injury. See text for details. MPT, mitochondrial membrane permeability transition; NAPQI, N -acetyl-p -benzoquinone imine; NO, nitric oxide; LPO, lipid peroxidation; ONOO, peroxynitrite.
strongly supports the concept that the mechanism of AAP-induced hepatocellular injury is initiated by covalent protein binding of the reactive metabolite. However, this initiating event requires amplification and progression, which may involve mitochondrial dysfunction with oxidant stress and peroxynitrite formation and the consequences of these oxidative events. Thus, protein binding and oxidant stress are not competing hypotheses but sequential events in the mechanism of AAP hepatotoxicity. This concept explains many of the controversial findings and reconciles many of the differences and disputes of the past.
Acknowledgements Work from the authors’ laboratory was supported in part by National Institutes of Health grants ES06091 and AA12916.
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