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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 ...

Troglitazone, a first-generation thiazolidinedione antidiabetic drug, was withdrawn from the market due to an unacceptable risk of idiosyncratic hepatotoxicity. Troglitazone does not cause hepatotoxicity in normal healthy rodents, but it produces mitochondrial injury in vitro at high concentrations. The aim of this study was to explore whether genetic mitochondrial abnormalities might sensitize mice to hepatic adverse effects of troglitazone. We used heterozygous superoxide dismutase 2 (Sod2(+/-)) mice as a model of clinically silent mitochondrial stress. Troglitazone was daily administered for 4 weeks (0, 10 or 30 mg/kg/day, ip). We found that troglitazone caused overt liver injury in the high-dose group, manifested by increased serum alanine aminotransferase activity (> twofold) and midzonal areas of hepatic necrosis, in Sod2(+/-) but not in wild-type mice. No signs of hepatotoxicity were apparent at 2 weeks of treatment. Hepatic mitochondria isolated from troglitazone-treated mice exhibited decreased activities of aconitase (by 45%) and complex I (by 46%) and increased (by 58%) protein carbonyls, indicative of enhanced mitochondrial oxidant stress. This was paralleled by compensatory increases in mitochondrial glutathione levels. Finally, in hepatocytes isolated from untreated Sod2(+/-), but not wild-type mice, troglitazone caused a concentration-dependent increase in superoxide anion levels as demonstrated with a selective mitochondria-targeting fluorescent probe. In conclusion, prolonged administration of troglitazone can superimpose oxidant stress, potentiate mitochondrial damage, and induce delayed hepatic necrosis in mice with genetically compromised mitochondrial function. These data are consistent with our hypothesis that inherited or acquired mitochondrial abnormalities may be one of the contributing determinants of susceptibility to troglitazone-induced idiosyncratic liver injury.

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