Inhibitor of apoptosis signal-regulating kinase 1 protects against acetaminophen-induced liver injury

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Highlights

  • Two ASK1 inhibitors protected against acetaminophen-induced liver injury.

  • The ASK1 inhibitors protect when used as pre- or post-treatment.

  • Protection by ASK1 inhibitor is not due to inhibition of APAP metabolism.

  • The ASK1 inhibitor prevents JNK activation and translocation to mitochondria.

  • Treatment with ASK1 inhibitors does not impair liver regeneration after APAP.

Abstract

Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun-N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300 mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and liver injury as indicated by plasma ALT activities and area of necrosis over a 24 h observation period. Pretreatment with 30 mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affecting the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5 h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients.

Introduction

Acetaminophen (APAP) is a widely used analgesic and antipyretic. A therapeutic dose of APAP is safe and effective while an overdose can cause severe liver injury in animals and humans (Larson, 2007, McGill et al., 2012). Hepatotoxicity is initiated by formation of a reactive metabolite, N-acetyl-p-benzoquinone imine, which is detoxified by glutathione but also binds to cellular proteins (Cohen et al., 1997). Although formation of protein adducts, especially in mitochondria, is a critical event in the pathophysiology (Tirmenstein and Nelson, 1989), it is not sufficient to cause cell death (Jaeschke and Bajt, 2006). The current hypothesis is that protein binding induces mitochondrial dysfunction including inhibition of mitochondrial respiration (Meyers et al., 1988) and formation of reactive oxygen species (ROS) (Jaeschke, 1990) and peroxynitrite (Hinson et al., 1998, Cover et al., 2005). This oxidant stress together with lysosomal iron taken up into the mitochondria by the Ca2 + uniporter (Kon et al., 2010), leads to the opening of the mitochondrial membrane permeability transition (MPT) pore and collapse of the membrane potential (Kon et al., 2004, Reid et al., 2005, Ramachandran et al., 2011a, LoGuidice and Boelsterli, 2011). The massive loss of mitochondrial function triggers necrotic cell death (Gujral et al., 2002). A number of therapeutic interventions that accelerated the recovery of mitochondrial glutathione and improved the detoxification of mitochondrial ROS and peroxynitrite provided clear evidence for the critical role of the mitochondrial oxidant stress in the mechanism of cell death (Knight et al., 2002, James et al., 2003, Ramachandran et al., 2011b, Saito et al., 2010b). However, it remained unclear how this extensive mitochondrial oxidant stress ultimately can be induced.

Studies during the last decade aimed at elucidating intracellular signaling events have demonstrated a critical role of the mitogen-activated protein kinase (MAPK) c-jun-N-terminal kinase (JNK) in APAP-induced cell death in mice (Gunawan et al., 2006, Henderson et al., 2007, Latchoumycandane et al., 2007) and in human hepatocytes (Xie et al., 2014). Importantly, JNK was not only activated (phosphorylated) in the cytosol, p-JNK also translocated to the mitochondria (Hanawa et al., 2008) and amplified the mitochondrial oxidant stress (Saito et al., 2010a) through binding to an anchor protein in the outer mitochondrial membrane (Win et al., 2011). It was also shown that an early mitochondrial oxidant stress triggered JNK activation (Hanawa et al., 2008, Saito et al., 2010a). However, JNK is not a redox-sensitive kinase. In fact, the upstream kinases, apoptosis signal-regulating kinase 1 (ASK1) (Nakagawa et al., 2008) and mixed-lineage kinase 3 (MLK3) (Sharma et al., 2012) have been identified as critical upstream regulators of JNK activation. Oxidant stress can release ASK1 from its complex with reduced thioredoxin by oxidation of thioredoxin resulting in the liberation of the active kinase (Saitoh et al., 1998). Because ASK1 is thought to be mainly responsible for sustained activation of JNK, the substantial reduction in APAP-induced liver injury in ASK1-deficient mice demonstrated the critical role of ASK1 in the pathophysiology (Nakagawa et al., 2008). Recently a potent and selective small molecule inhibitor of ASK1 with properties amenable for in vivo efficacy testing in rodents was developed (Gerczuk et al., 2012, Toldo et al., 2012). This inhibitor enabled us to test if pharmacologic inhibition of ASK has therapeutic potential for treating APAP hepatotoxicity and to study the mechanisms of protection.

Section snippets

Animals

8 week old male C57Bl/6 mice were acquired from Jackson Laboratories for the experiments. Animals were housed in a controlled environment with a 12 hour light/dark cycle and free access to food and water. Animals were acclimatized for at least 3 days and fasted overnight before experiments. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center.

Inhibitors

GS-459679 and GS-444217 are potent and selective ATP competitive inhibitors

Pharmacological inhibition of ASK1 protects against APAP-induced liver injury

As an initial attempt to investigate the effect of ASK1 inhibition on APAP hepatotoxicity, mice were treated with 10 or 30 mg/kg of GS-459679 (ASK1i), 30 min prior to administration of 300 mg/kg APAP. Liver injury was then examined 6 and 24 h later. APAP induced significant liver injury as indicated by the increase in plasma ALT activities, which were elevated at 6 h and further increased by 24 h (Fig. 1A). This was accompanied by development of centrilobular necrosis (Figs. 1B, C). Pretreatment with

Discussion

The objective of the investigation was to evaluate the protective effect of the ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. Our data showed that pretreatment with the ASK1 inhibitor and treatment during a limited therapeutic window after APAP overdose attenuated liver injury.

Conflict of interest

David Breckenridge and John Liles are employees of Gilead Sciences, Inc. Hartmut Jaeschke received a grant from Gilead Sciences, Inc.

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Acknowledgment

This work was supported in part by a grant from Gilead Science Inc., by the National Institutes of Health Grants AA12916 and DK070195 and by grants from the National Center for Research Resources (5P20RR021940-07) and the National Institute of General Medical Sciences (8 P20 GM103549-07) from the National Institutes of Health.

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    Present address: Wellcome Trust Research Laboratory, Division of Gastrointestinal Sciences, Christian Medical College, Vellore 632004, India.

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