The progression of injury in these cells involved mitochondrial reactive oxygen and reactive nitrogen formation. APAP did not increase caspase activity above untreated control values and a pancaspase inhibitor did not protect against APAP-induced cell injury. Conclusion: These data suggest that key mechanistic features

of APAP-induced cell death are the same in human HepaRG cells, rodent in vivo models, and primary cultured mouse hepatocytes. Thus, HepaRG cells are a useful model to study mechanisms of APAP hepatotoxicity in humans. (HEPATOLOGY 2011) Acetaminophen (APAP) is a widely used over-the-counter HCS assay analgesic and antipyretic drug and is a common component of opioid-containing prescription formulations. Although safe at therapeutic levels, overdose of APAP causes liver injury and is the foremost cause of acute liver failure in the US and the UK.1 At therapeutic doses, >90% of the drug is glucuronidated or sulfated in the liver and subsequently excreted. The remainder is metabolized by cytochromes P450 (CYP450) to the electrophilic intermediate N-acetyl-p-benzoquinoneimine (NAPQI), which can be neutralized by conjugation with glutathione.2 However, after an overdose of APAP, formation of NAPQI exceeds the detoxification capacity of glutathione, resulting in covalent

binding to cellular proteins.3 Although the overall protein binding caused by an overdose of APAP or its isomer 3′-hydroxyacetanilide is similar and many adducted proteins have been identified, toxicity only occurred with APAP, which shows greater binding to mitochondrial proteins.3-6 The subsequent mitochondrial dysfunction leads Metabolism inhibitor to inhibition of mitochondrial respiration,7 ATP depletion,8 and formation of reactive oxygen8 and peroxynitrite9 (ROS and RNS) inside mitochondria. The oxidant stress is involved in activation of the c-jun-N-terminal kinase (JNK) pathway10 and eventually triggers the opening of the mitochondrial membrane permeability transition (MPT) pore,11 resulting in collapse of the mitochondrial membrane potential.11,

12 Mitochondrial matrix swelling and rupture of the outer membrane causes the Sclareol release of intermembrane proteins including cytochrome c, endonuclease G, and apoptosis-inducing factor (AIF).13 Only endonuclease G and AIF translocate to the nucleus and induce DNA fragmentation.14 The severe impairment of aerobic energy metabolism, massive ATP depletion, and nuclear DNA damage result in necrotic cell death.15 Despite the release of cytochrome c from mitochondria, no significant activation of caspases has been detected and apoptosis contributes less than 5% to the overall injury in mice.15-17 Most of our present knowledge of APAP hepatotoxicity has been learned from rodent studies in vivo and in primary culture.2, 13 However, notable differences exist in the time course of injury between rodents and humans.