Mitochondrial protection by the JNK inhibitor leflunomide rescues mice from acetaminophen-induced liver injury

Abstract

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses but which can precipitate liver injury at high doses. We have previously found that the antirheumatic drug leflunomide is a potent inhibitor of APAP toxicity in cultured human hepatocytes, protecting them from mitochondria-mediated cell death by inhibiting the mitochondrial permeability transition. The purpose of this study was to explore whether leflunomide protects against APAP hepatotoxicity in vivo and to define the molecular pathways of cytoprotection. Male C57BL/6 mice were treated with a hepatotoxic dose of APAP (750 mg/kg, ip) followed by a single injection of leflunomide (30 mg/kg, ip). Lefluno-mide (4 hours after APAP dose) afforded significant protection from liver necrosis as assessed by serum ALT activity and histopathology after 8 and 24 hours. The mechanism of protection by leflunomide was not through inhibition of cytochrome P450 (CYP)-catalyzed APAP bioactivation or an apparent suppression of the innate immune system. Instead, leflunomide inhibited APAP-induced activation (phosphorylation) of c-jun NH 2-terminal protein kinase (JNK), thus preventing downstream Bcl-2 and Bcl-X L inactivation and protecting from mitochondrial permeabilization and cytochrome c release. Furthermore, leflunomide inhibited the APAP-mediated increased expression of inducible nitric oxide synthase and prevented the formation of peroxynitrite, as judged from the absence of hepatic nitrotyrosine adducts. Even when given 8 hours after APAP dose, leflunomide still protected from massive liver necrosis. Conclusion: Leflunomide afforded protection against APAP-induced hepato-toxicity in mice through inhibition of JNK-mediated activation of mitochondrial permeabi-lization. (HEPATOLOGY 2007;45:412-421.) A cetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses. However, when taken at high doses or, rarely in particularly susceptible people at therapeutic doses, APAP can precipitate severe liver injury that can develop into fulminant liver failure. 1 The clinical significance of this adverse effect is underscored by APAP being, among all drugs, the single major cause of drug-induced hepatotoxicity in the United States and the United Kingdom. 2 The mechanisms underlying APAP-induced liver injury have been studied for several decades, and excellent recent reviews have summarized the cellular and molecular pathways of this toxic response. 3-5 Although the initial steps in the sequence of events leading to hepatocyte ne-crosis (bioactivation of APAP and glutathione depletion) have been well known for many years, the more distal events (signaling pathways that lead to the precipitation of cell death) are less clear. However, recently, the mito-chondrial permeability transition (mPT) has been identified as a pivotal mechanism mediating APAP-induced cell death. 6,7 According to this concept, a combination of mi-tochondrial oxidant stress, increased Ca 2 levels, and other factors may favor ...

Protective role of c-Jun N-terminal kinase 2 in acetaminophen-induced liver injury

“…The role of JNKs in AILI has also been studied in WT mice in vivo with the use of pharmacological inhibitors [14,21,22,35] and anti-sense oligonucleotides [21 ]. Treatment of mice with pharmacological inhibitors that blocked the activity of both JNK1 and 2 reduced AILI and mortality, while administration of JNK2 but not JNK1 anti-sense oligonucleotides partially inhibited AILI when administered individually.…”

“…Treatment of mice with pharmacological inhibitors that blocked the activity of both JNK1 and 2 reduced AILI and mortality, while administration of JNK2 but not JNK1 anti-sense oligonucleotides partially inhibited AILI when administered individually. It was proposed that JNK had a pathological role in AILI in part by contributing to mitochondrial injury [14,21,35]. Potential limitations of these studies, as acknowledged by the investigators [21,22] is that JNK inhibitors may not be specific and as reported can affect other signaling events that could lead to the misinterpretation of results [36][37][38][39].…”

“…Potential limitations of these studies, as acknowledged by the investigators [21,22] is that JNK inhibitors may not be specific and as reported can affect other signaling events that could lead to the misinterpretation of results [36][37][38][39]. Further, some of the JNK inhibitors were dissolved in vehicles containing DMSO [14,21] and polyethylene glycol [22,35], which may affect various parameters at the cellular and molecular level [34,[40][41][42][43].In conclusion, our findings suggest that JNK2 plays a protective role against AILI in mice at least, in part, by modulating hepatocellular regeneration and repair. Although other research in mice provides evidence for JNK having a pathologic role in AILI, we feel that it is prudent to reconsider the use of JNK inhibitors as a therapeutic intervention in AILI [21,22,35,44].…”

“…Further, some of the JNK inhibitors were dissolved in vehicles containing DMSO [14,21] and polyethylene glycol [22,35], which may affect various parameters at the cellular and molecular level [34,[40][41][42][43].…”

Chronic Deletion and Acute Knockdown of Parkin Have Differential Responses to Acetaminophen-induced Mitophagy and Liver Injury in Mice

“…JNK activation, particularly in the mitochondria, has been shown to exacerbate APAP-induced liver injury (24,25,27,47).…”