Acetaminophen (APAP) hepatotoxicity represents a major

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1 phisms and haplotypes in primary biliary cirrhosis. Clin Gastroenterol Hepatol 2007;5: Juran BD, Atkinson EJ, Schlicht EM, et al. Interacting alleles of the coinhibitory immunoreceptor genes cytotoxic T-lymphocyte antigen 4 and programmed cell-death 1 influence risk and features of primary biliary cirrhosis. Hepatology 2008;47: Carlson CS, Eberle MA, Rieder MJ, et al. Selecting a maximally informative set of single-nucleotide polymorphisms for association analyses using linkage disequilibrium. Am J Hum Genet 2004;74: Homer N, Tembe WD, Szelinger S, et al. Multimarker analysis and imputation of multiple platform pooling-based genome-wide association studies. Bioinformatics 2008 Jul 10 [Epub ahead of print]. 35. Nyholt DR. sssnper: identifying statistically similar SNPs to aid interpretation of genetic association studies. Bioinformatics 2006;22: Hirschhorn JN, Lohmueller K, Byrne E, et al. A comprehensive review of genetic association studies. Genet Med 2002;4: Address requests for reprints to: M. Eric Gershwin, MD, Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, TB 192, Davis, CA 95616; megershwin@ucdavis.edu; Fax: Supported by National Institute of Health grant DK by the AGA Institute /08/$34.00 doi: /j.gastro How to Protect Against Acetaminophen: Don t Ask for JUNK See Deletion of ASK1 attenuates acetaminophen-induced liver injury by inhibiting JNK activation, by Nakagawa H, Maeda S, Hikiba Y, et al, on page Acetaminophen (APAP) hepatotoxicity represents a major public health problem in the United States and the United Kingdom, accounting for approximately one third of the cases of acute liver failure that are equally divided between suicidal overdose and inadvertent overdose when it is used with therapeutic intent or in combination with opiates in drug abuse situations. 1 In the majority of these cases, the dose exceeds the recommended limits. However, because use of maximal recommended doses is associated with frequent asymptomatic alanine aminotransferase elevations, 2 the possibility exists that certain susceptible individuals can develop severe liver injury while not exceeding the maximal recommended dose, 3 although this is probably a rare occurrence. In the early 1970s, groundbreaking work by Mitchell et al 4 demonstrated that the toxicity of APAP requires conversion to a reactive metabolite (N-acetyl-P-benzoquinone imine [NAPQI]) that is preferentially detoxified by glutathione (GSH). 4 Once the GSH is depleted, NAPQI binds to protein-sh. 4 Hepatotoxicity occurs when nearly all the GSH has been depleted, especially in zone III hepatocytes. Since then, there have been literally thousands of publications on the mechanisms of APAP hepatotoxicity and our understanding of certain aspects of the mechanism continue to be debated. Although Cyp2e1 is the major enzyme for the conversion to NAPQI, it is a minor pathway for APAP metabolism, which explains why large doses of APAP are required to produce sufficient NAPQI to deplete the GSH stores and cause severe toxicity. However, other Cyp enzymes for example, Cyp3a4 and Cyp1a2 may also contribute. Once the threshold (GSH depletion) for toxicity is reached, considerable controversy has centered on whether the toxicity is directly due to covalent binding, perhaps incapacitating critical target proteins, or whether it is due to the profound depletion of GSH, unleashing oxidative and nitrosative stress. In the early 1990s Nelson s group identified an analog (regioisomer) of APAP, referred to as AMAP, which causes comparable whole liver GSH depletion and covalent binding but no toxicity. Careful comparison of the effects of AMAP and APAP by these investigators identified 1 major difference in the target organelles, namely mitochondria. AMAP spared mitochondria of GSH depletion and covalent binding, whereas APAP did not, indicating the importance of mitochondria as a target of APAP. 5 More recently, the work of Hinson s group identified an important temporal pattern in studies in isolated hepatocytes. 6 They demonstrated that there are 2 phases of APAP toxicity: (1) in the first 2 hours after exposure to APAP, NAPQI production, GSH depletion, and covalent binding occurred but the cells were viable; (2) the cells were then resuspended without APAP and gradually went on to die over the next few hours in the absence of APAP by a process dependent upon oxidative stress and accompanied by the opening of the mitochondrial permeability transition (MPT) pore. Thus, this was an important conceptual advance that identified an APAP metabolism phase and subsequent oxidative stress, mitochondrial permeability phase that was independent of continued exposure to APAP. As 1047

2 was the case with many toxicologic models, APAP metabolism was viewed as causing chemical consequences due to reactive metabolites and reactive oxygen species (ROS) that overwhelmed the cellular defenses leading directly to cell death as a result of profound loss of mitochondrial function. The assumption was that mitochondrial targeted covalent binding and GSH depletion with accompanying oxidative stress directly lead to opening of the MPT pore leading to a bioenergetic catastrophe. The next milestone in our understanding of APAP toxicity was the discovery of the participation of JNK in APAP toxicity. This represents a paradigm shift in that the link between NAPQI and GSH depletion and the destruction of the liver was demonstrated to be indirect and required the participation of JNK. 7 Inhibition of JNK with a potent small molecule inhibitor or the silencing of the expression JNK1 and JNK2 with antisense oligonucleotides in vivo markedly protected against toxicity without changing the APAP metabolism phase (covalent binding and GSH depletion). 7 Therefore, active participation of a cell death promoting kinase was required. These findings were subsequently confirmed by 2 other groups. 8,9 Two important questions arose from this work: (1) How does APAP lead to JNK activation, and (2) How does activated JNK lead to cell death? The paper by Nakagawa et al 10 in this issue of GASTROENTEROLOGY provides important insight into the explanation for JNK activation. Transient early JNK activation is an expected nontoxic response to tumor necrosis factor (TNF) receptor engagement; concurrent activation of nuclear factor (NF)- B followed by rapid up-regulation of several NF- B dependent genes is known to dampen JNK, accounting for transient activation, which usually only lasts minutes after TNF receptor engagement. Sustained JNK activation lasting for 1 hour, however, leads to lethal consequences. The authors looked at upstream kinases that lead to JNK activation and showed that knockout of ASK1, a mitogen-activated (MAP) kinase kinase kinase, blunted JNK activation and APAP toxicity. 10 ASK1 is known to associate with redox-sensitive proteins such as thioredoxin-1, which inhibit kinase activation, 11 When oxidative stress occurs (eg, increased exposure to ROS or peroxynitrite), thioredoxin-1 becomes oxidized and releases ASK1, which then self-activates. 11 However, the authors observed that the protective effect of ASK1 knockout was not as potent in inhibiting JNK activation or liver toxicity as the small molecule JNK inhibitor, SP This suggests that other mechanisms for JNK activation also contribute. One possibility is that JNK itself is released from inhibitory binding to redox-sensitive proteins. The GSH S-transferase Pi monomeric subunit is known to accomplish this function. 12,13 Alternatively, the protein phosphatase that dephosphorylates and inactivates phospho-jnk is known to be inhibited by ROS. Thus, although the authors suggest that inhibition of ASK1 would be an excellent target for protection against APAP, this would seem to be less effective than inhibiting JNK. Furthermore, ASK1 leads to downstream activation of both JNK and p38 kinase. Although the authors confirmed earlier work that p38 does not mediate APAP toxicity, 7 the consequences of preventing p38 maybe complex. In some contexts, p38 is protective. It is noteworthy that the sustained JNK activation observed by Nakagawa et al 10 exhibited 2 components, an early ASK-1 independent phase of sustained JNK activation seen at 1.5 hours and a late ASK-1 dependent phase seen at 3.0 hours. This raises the possibility that direct JNK activation in the early phase may contribute to the subsequent activation of ASK-1 in a self-amplifying loop. For example, JNK could enhance mitochondrial ROS production, augmenting ASK-1 activation and more JNK activation. It would be of interest to test this possibility by assessing the effect of the JNK inhibitor on APAPinduced ASK-1 activation. An associated issue is the mechanism for thioredoxin-1 modification, which permits the release of ASK1. Recent evidence supports the hypothesis that NAPQI s effects on mitochondria lead to increased ROS release from mitochondria as the mechanism for activation of this MAP kinase pathway. 14 However, it also remains possible that NAPQI directly binds to thioredoxin. AMAP, which induces extramitochondrial GSH depletion and covalent binding, does not lead to JNK activation (or toxicity). 14 This strongly suggests that mitochondria are the source of the oxidizing species, which alters thioredoxin and probably other redoxsensitive proteins that inhibit ASK1 and JNK. The second major question concerns how activated JNK exerts its toxic effect that is, what is the target of JNK? Recent evidence in the APAP model suggests that JNK, once activated, translocates to mitochondria where it promotes the MPT. 14 The protein(s) with which JNK associates in the mitochondria and the substrate of this kinase in the mitochondria remain to be identified, so it is not known whether MPT pore proteins or Bcl2 family members are directly phosphorylated by JNK or if other kinases are also involved. The interplay with MAP kinases and crosstalk with PKC, Akt, AMPK, and GSK-3 are of considerable interest because these pathways can promote or inhibit injury and most are activated by oxidative stress. Nakagawa et al 10 also provide evidence that ASK1 / mice are not protected from CCl 4, consistent with earlier work showing that inhibition of JNK is not protective in this model. 7,9 Therefore, how generalizable are the findings in APAP toxicity to drug-induced or other causes of liver injury, or what is so unique about APAP toxicity? Some insight can be gained from recent experiments showing that when pure, activated JNK is added to isolated hepatic mi- 1048

3 Figure 1. Two-hit model of acetaminophen (APAP) hepatotoxicity. The metabolism of APAP exposes mitochondria to a reactive metabolite (NAPQI), which sensitizes mitochondria to JNK. JNK is activated by oxidative stress derived from the mitochondria by oxidation of redox-sensitive inhibitory proteins either upstream in the MAP kinase cascade or downstream at the level of JNK. Activated JNK translocates to mitochondria where it leads to MPT and necrosis. Gst, glutathione S-transferase; Trx, thioredoxin. tochondria from APAP-treated mice that also have been treated with JNK inhibitor, there is profound loss of mitochondrial function, whereas active JNK has no effect on normal mitochondria. 14 The mitochondria from APAP JNK inhibitor treated mice exhibited JNK-independent moderate impairment of respiration, GSH depletion, and covalent binding, which were insufficient to cause loss of viability. However, the addition of JNK provided a second hit, which led to nearly complete loss of function of these mitochondria. Furthermore, cyclosporine blocked the effect of JNK on mitochondrial function in this cell-free system, supporting the hypothesis that APAP, via NAPQI-induced covalent binding and GSH depletion (first hit), sensitizes mitochondria to JNK induced MPT (second hit; Figure 1). Although Nakagawa et al 10 show that CCl 4 activates JNK, it is possible that the reactive metabolite of CCl 4 does not initially target mitochondria as NAPQI does. Further work analyzing the mechanistic differences between CCl 4 and APAP would be of interest. Despite the inability to extend the APAP mechanism to CC1 4, there remains a very important potential link to hepatic ischemia reperfusion injury 15 and other causes of drug-induced liver disease There is growing evidence that many drugs that are associated with delayed idiosyncratic hepatotoxicity target mitochondria and gradually induce the accumulation of sufficient mitochondrial impairment (compared with rapid progression with APAP) to cross a threshold to injury with impairment of electron transport leading to exposure to increased ROS. 16,17 Thus, a scenario similar to APAP (but with slower kinetics) may be set up in which drugs cause mitochondrial impairment, which both enhances ROS production and may sensitize mitochondria to stress kinases activated by ROS. An interesting example is troglitazone, which caused idiosyncratic hepatotoxicity in man. Initial toxicology studies revealed no clear toxic effects of the troglitazone in animals. Ong et al 18 provided novel insight by administrating troglitazone to SOD2 / mice, which have compromised mitochondrial antioxidant defense. Delayed (4 weeks) appearance of hepatotoxicity was observed in SOD2 / but not SOD2 / mice. Mitochondria isolated at 4 weeks, but not at 2 weeks, exhibited impaired function and increased ROS production. Using an immortalized human hepatocyte cell line, they went on to show that the parent troglitazone directly caused inhibition of election transport and depolarization of mitochondria leading to increased ROS, which then activated the MAP kinase cascade followed by cell death. 19 A caveat of this work was that activated phospho-ask1 was localized in mitochondria and was accompanied by oxidation of the mitochondrial form of thioredoxin, thioredoxin-2 (Trx-2). This raises the possibility that Trx-2 ASK1 localized in mitochondria may act as a sensor of oxidative stress and mitochondrial damage. Because JNK does not reside in mitochondria, the pathway from intramitochon- 1049

4 drial ASK1 to extramitochondrial JNK and whether this is relevant to the APAP model remains uncertain. Alternatively, it is possible that the entire cytoplasmic activated kinase module, including phospho-ask1 and phospho- JNK, translocates to mitochondria. More work is needed on the compartmentation of ASK-1. Nevertheless, either rapidly or slowly developing impairment of mitochondrial function can lead to increased ROS production, which activates the MAP kinase pathway leading to MPT. The mitochondrial electron transport chain can be inhibited by covalent binding or GSH depletion (APAP) or by inhibitory effects of a parent drug (troglitazone) or its metabolites. Therefore, the mechanistic insights derived from the APAP model may be widely applicable to idiosyncratic drug-induced liver injury. Two isoforms of JNK are expressed in the liver. There is evidence that each may have unique roles in liver disease. JNK1 has been suggested to play a major role in insulin resistance and fatty liver disease, 20 whereas JNK2 has been shown to participate in TNF-induced apoptosis 21 and ischemia reperfusion injury. 15 In the APAP model, when APAP is given dissolved in dimethyl sulfoxide (DMSO), a slower and less severe injury is seen as a result of the protective effect of DMSO. 22 Under these conditions, a predominant role for JNK2 in toxicity is seen. 7 However, when APAP is administered without DMSO, inhibition of both JNK1 and JNK2 is required for protection. 14 Nakagawa et al 10 demonstrated a slight protective effect of deletion of JNK2. Overall, it seems that maximal protection is afforded by inhibiting both JNK1 and JNK2. Another area of controversy concerns the mode of cell death in APAP toxicity. Nakagawa et al 10 provide some evidence that apoptosis (TUNEL stain) may contribute but the combination of cytoplasmic and nuclear staining shown in their studies has been found to be more consistent with necrosis. 23 Indeed, the bulk of evidence shows that APAP overdose causes a necrotic cell death. The fact that MAP kinases, particularly JNK, mediate necrosis in this model underscores the emerging evidence that toxin-induced necrosis, like apoptosis, is a programmed process in which built-in pathways for active cell demise can be recruited to kill hepatocytes. This knowledge provides therapeutic targets to prevent necrosis downstream of drug metabolism and oxidative stress. NEIL KAPLOWITZ MIE SHINOHARA ZHANG-XU LIU DERICK HAN USC Research Center for Liver Disease Keck School of Medicine University of Southern California Los Angeles, California References 1. Larson AM, Polson J, Fontana RJ, et al ;Acute Liver Failure Study Group. 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Glutathione S-transferase p elicits protection against H2O2-induced cell death via coordinated regulation of stress kinases. Cancer Res 2000;60: Hanawa N, Shinohara M, Saberi B, et al. Role of JNK translocation to mitochondria leading to inhibition of mitochondria bioenergetics in acetaminophen-induced liver injury. J Biol Chem 2008; 283: Theruvath TP, Czerny C, Ramshesh VK, et al. C-Jun N-terminal kinase 2 promotes graft injury via the mitochondrial permeability transition after mouse liver transplantation. Am J Transplant 2008;8: Dykens JA, Jamieson JD, Marroquin LD, et al. In vitro assessment of mitochondrial dysfunction and cytotoxicity of nefazodone, trazodone, and buspirone. Toxicol Sci 2008;103: Wallace KB, Starkov AA. Mitochondrial targets of drug toxicity. Annu Rev Pharmacol Toxicol 2000;40: Ong MM, Latchoumycandane C, Boelsterli UA. Troglitazone-induced hepatic necrosis in an animal model of silent genetic mitochondrial abnormalities. Toxicol Sci 2007;97: Lim PL, Liu J, Go ML, et al. The mitochondrial superoxide/ thioredoxin-2/ask1 signaling pathway is critically involved in tro- 1050

5 glitazone-induced cell injury to human hepatocytes. Toxicol Sci 2008;101: Schattenberg JM, Singh R, Wang Y, et al. JNK1 but not JNK2 promotes the development of steatohepatitis in mice. Hepatology 2006;43: Wang Y, Singh R, Lefkowitch JH, et al. Tumor necrosis factorinduced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway. J Biol Chem 2006;281: Jaeschke H, Cover C, Bajt ML. Role of caspases in acetaminophen-induced liver injury. Life Sci 2006;78: Gujral JS, Bucci TJ, Farhood A, et al. Mechanism of cell death during warm hepatic ischemia-reperfusion in rats: apoptosis or necrosis? Hepatology 2001;33: Address requests for reprints to: Neil Kaplowitz, MD, Keck School of Medicine, 2011 Zonal Avenue, HMR 101, Los Angeles, California 90033; kaplowit@usc.edu by the AGA Institute /08/$34.00 doi: /j.gastro