Liver injury and acute liver failure caused by acetaminophen (APAP, N-acetyl-p-aminophenol, paracetamol) overdose is a significant clinical problem in most western countries. The only clinically approved antidote is N-acetylcysteine (NAC), which promotes the recovery of hepatic GSH. If administered during the metabolism phase, GSH scavenges the reactive metabolite N-acetyl-p-benzoquinone imine. More recently, it was shown that NAC can also reconstitute mitochondrial GSH levels and scavenge reactive oxygen/peroxynitrite and can support mitochondrial bioenergetics. However, NAC has side effects and may not be efficacious after high overdoses. Repurposing of additional drugs based on their alternate mechanisms of action could be a promising approach. 4-Methylpyrazole (4MP) was shown to be highly effective against APAP toxicity by inhibiting cytochrome P450 enzymes in mice and humans. In addition, 4MP is a potent c-Jun N-terminal kinase inhibitor expanding its therapeutic window. Calmangafodipir (CMFP) is a SOD mimetic, which is well tolerated in patients and has the potential to be effective after severe overdoses. Other drugs approved for humans such as metformin and methylene blue were shown to be protective in mice at high doses or at human therapeutic doses, respectively. Additional protective strategies such as enhancing antioxidant activities, Nrf2-dependent gene induction and autophagy activation by herbal medicine components are being evaluated. However, at this point, their mechanistic insight is limited, and the doses used are high. More rigorous mechanistic studies are needed to advance these herbal compounds. Nevertheless, based on recent studies, 4-methylpyrazole and calmangafodipir have realistic prospects to become complimentary or even alternative antidotes to NAC for APAP overdose.

Although necrosis is recognized as the main mode of cell death induced by acetaminophen (APAP) overdose in animals and humans, more recently an increasing number of publications, especially in the herbal medicine and dietary supplement field, claim an important contribution of apoptotic cell death in the pathophysiology. However, most of these conclusions are based on parameters that are not specific for apoptosis. Therefore, the objective of this review was to re-visit the key signaling events of receptor-mediated apoptosis and APAP-induced programmed necrosis and critically analyze the parameters that are being used as evidence for apoptotic cell death. Both qualitative and quantitative comparisons of parameters such as Bax, Bcl-2, caspase processing and DNA fragmentation in both modes of cell death clearly show fundamental differences between apoptosis and cell death induced by APAP. These observations together with the lack of efficacy of pan-caspase inhibitors in the APAP model strongly supports the conclusion that APAP hepatotoxicity is dominated by necrosis or programmed necrosis and does not involve relevant apoptosis. In order not to create a new controversy, it is important to understand how to use these "apoptosis" parameters and properly interpret the data. These issues are discussed in this review.

Liver injury due to acetaminophen (APAP) overdose is the major cause of acute liver failure in the United States. While treatment with N-acetylcysteine is the current standard of care for APAP overdose, anecdotal evidence suggests that administration of 4-methylpyrazole (4MP) may be beneficial in the clinic. The objective of the current study was to examine the protective effect of 4MP and its mechanism of action. Male C57BL/6J mice were co-treated with 300 mg/kg of APAP and 50 mg/kg of 4MP. The severe liver injury induced by APAP at 6 h as indicated by elevated plasma alanine aminotransferase activities, centrilobular necrosis, and nuclear DNA fragmentation was almost completely eliminated by 4MP. In addition, 4MP largely prevented APAP-induced activation of c-Jun N-terminal kinase (JNK), mitochondrial translocation of phospho-JNK and Bax, and the release of mitochondrial intermembrane proteins. Importantly, 4MP inhibited the generation of APAP protein adducts and formation of APAP-glutathione (GSH) conjugates and attenuated the depletion of the hepatic GSH content. These findings are relevant to humans because 4MP also prevented APAP-induced cell death in primary human hepatocytes. In conclusion, early treatment with 4MP can completely prevent liver injury after APAP overdose by inhibiting cytochrome P450 and preventing generation of the reactive metabolite.

Mitochondria are critical cellular organelles for energy generation and are now also recognized as playing important roles in cellular signaling. Their central role in energy metabolism, as well as their high abundance in hepatocytes, make them important targets for drug-induced hepatotoxicity. This review summarizes the current mechanistic understanding of the role of mitochondria in drug-induced hepatotoxicity caused by acetaminophen, diclofenac, anti-tuberculosis drugs such as rifampin and isoniazid, anti-epileptic drugs such as valproic acid and constituents of herbal supplements such as pyrrolizidine alkaloids. The utilization of circulating mitochondrial-specific biomarkers in understanding mechanisms of toxicity in humans will also be examined. In summary, it is well-established that mitochondria are central to acetaminophen-induced cell death. However, the most promising areas for clinically useful therapeutic interventions after acetaminophen toxicity may involve the promotion of adaptive responses and repair processes including mitophagy and mitochondrial biogenesis, In contrast, the limited understanding of the role of mitochondria in various aspects of hepatotoxicity by most other drugs and herbs requires more detailed mechanistic investigations in both animals and humans. Development of clinically relevant animal models and more translational studies using mechanistic biomarkers are critical for progress in this area.

Acetaminophen (APAP) overdose is the most common cause of hepatotoxicity and acute liver failure in the United States and many western countries. However, the only clinically approved antidote, N-acetylcysteine, has a limited therapeutic window. 4-Methylpyrazole (4MP) is an antidote for methanol and ethylene glycol poisoning, and we have recently shown that cotreatment of 4MP with APAP effectively prevents toxicity by inhibiting Cyp2E1. To evaluate if 4MP can be used therapeutically, C57BL/6J mice were treated with 300 mg/kg APAP followed by 50 mg/kg 4MP 90 min later (after the metabolism phase). In these experiments, 4MP significantly attenuated liver injury at 3, 6, and 24 h after APAP as shown by 80%-90% reduction in plasma alanine aminotransferase activities and reduced areas of necrosis. 4MP prevented c-Jun c-Jun N-terminal kinase (JNK) activation and its mitochondrial translocation, and reduced mitochondrial oxidant stress and nuclear DNA fragmentation. 4MP also prevented JNK activation in other liver injury models. Molecular docking experiments showed that 4MP can bind to the ATP binding site of JNK. These data suggest that treatment with 4MP after the metabolism phase effectively prevents APAP-induced liver injury in the clinically relevant mouse model in vivo mainly through the inhibition of JNK activation. 4MP, a drug approved for human use, is as effective as N-acetylcysteine or can be even more effective in cases of severe overdoses with prolonged metabolism (600 mg/kg). 4MP acts on alternative therapeutic targets and thus may be a novel approach to treatment of APAP overdose in patients that complements N-acetylcysteine.

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Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses. The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology. As a result, this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions. However, the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic. However, many studies using this model are flawed, which jeopardizes the scientific and clinical relevance. The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained. The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism, mitochondrial dysfunction, necrotic cell death, autophagy and the sterile inflammatory response. In addition, the most frequently made mistakes when using this model are discussed. Thus, considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions.

p53 is the major cellular gatekeeper involved in proliferation, cell death, migration, and homeostasis. The role of p53 in pathogenesis of drug-induced liver injury is unknown. We investigated the role of p53 in liver injury and regeneration after acetaminophen (APAP) overdose, the most common cause of acute liver failure in the Western world. Eight-week-old male wild-type (WT) and p53 knockout (p53KO) mice were treated with 300 mg/kg APAP, and the dynamics of liver injury and regeneration were studied over a time course of 0 to 96 hours. Deletion of p53 resulted in a threefold higher liver injury than in WT mice. Interestingly, despite higher liver injury, p53KO mice recovered similarly as the WT mice because of faster liver regeneration. Deletion of p53 did not affect APAP bioactivation and initiation of injury. Microarray analysis revealed that p53KO mice had disrupted metabolic homeostasis and induced inflammatory and proliferative signaling. p53KO mice showed prolonged steatosis correlating with prolonged liver injury. Initiation of liver regeneration in p53KO mice was delayed, but once initiated, cell cycle was significantly faster than WT mice because of sustained AKT, extracellular signal-regulated kinase, and mammalian target of rapamycin signaling. These studies show that p53 plays a pleotropic role after APAP overdose, where it prevents progression of liver injury by maintaining metabolic homeostasis and also regulates initiation of liver regeneration through proliferative signaling.