Acetaminophen (APAP) hepatotoxicity is characterized by an extensive oxidative stress. However, its source, pathophysiological role and possible therapeutic potential if targeted, have been controversially described. Earlier studies argued for cytochrome P450-generated reactive oxygen species (ROS) during APAP metabolism, which resulted in massive lipid peroxidation and subsequent liver injury. However, subsequent studies convincingly challenged this assumption and the current paradigm suggests that mitochondria are the main source of ROS, which impair mitochondrial function and are responsible for cell signaling resulting in cell death. Although immune cells can be a source of ROS in other models, no reliable evidence exists to support a role for immune cell-derived ROS in APAP hepatotoxicity. Recent studies suggest that mitochondrial targeted antioxidants can be viable therapeutic agents against hepatotoxicity induced by APAP overdose, and re-purposing existing drugs to target oxidative stress and other concurrent signaling events can be a promising strategy to increase its potential application in patients with APAP overdose.

Acetaminophen (APAP) overdose is the clinically most relevant drug hepatotoxicity in western countries, and, because of translational relevance of animal models, APAP is mechanistically the most studied drug. This review covers intracellular signaling events starting with drug metabolism and the central role of mitochondrial dysfunction involving oxidant stress and peroxynitrite. Mitochondria-derived endonucleases trigger nuclear DNA fragmentation, the point of no return for cell death. In addition, adaptive mechanisms that limit cell death are discussed including autophagy, mitochondrial morphology changes, and biogenesis. Extensive evidence supports oncotic necrosis as the mode of cell death; however, a partial overlap with signaling events of apoptosis, ferroptosis, and pyroptosis is the basis for controversial discussions. Furthermore, an update on sterile inflammation in injury and repair with activation of Kupffer cells, monocyte-derived macrophages, and neutrophils is provided. Understanding these mechanisms of cell death led to discovery of N-acetylcysteine and recently fomepizole as effective antidotes against APAP toxicity.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure in Western countries. In the last four decades much progress has been made in our understanding of APAP-induced liver injury through rodent studies. However, some differences exist in the time course of injury between rodents and humans. To study the mechanism of APAP hepatotoxicity in humans, a human-relevant in vitro system is needed. Here we present evidence that the cell line HepaRG is a useful human model for the study of APAP-induced liver injury. Exposure of HepaRG cells to APAP at several concentrations resulted in glutathione depletion, APAP-protein adduct formation, mitochondrial oxidant stress and peroxynitrite formation, mitochondrial dysfunction (assessed by JC-1 fluorescence), and lactate dehydrogenase (LDH) release. Importantly, the time course of LDH release resembled the increase in plasma aminotransferase activity seen in humans following APAP overdose. Based on propidium iodide uptake and cell morphology, the majority of the injury occurred within clusters of hepatocyte-like cells. 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 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.

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States and mechanisms of liver injury induced by APAP overdose have been the focus of extensive investigation. Studies in the mouse model, which closely reproduces the human condition, have shown that hepatotoxicity is initiated by formation of a reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which depletes cellular glutathione and forms protein adducts on mitochondrial proteins. This leads to mitochondrial oxi dative and nitrosative stress, accompanied by activation of c-jun N-terminal kinase (JNK) and its transloca tion to the mitochondria. This then amplifies the mitochondrial oxidant stress, resulting in translocation of Bax and dynamin related protein 1 (Drp1) to the mitochondria, which induces mitochondrial fission, and ultimately induction of the mitochondrial membrane permeability transition (MPT). The induction of MPT triggers release of intermembrane proteins such as apoptosis inducing factor (AIF) and endonuclease G into the cytosol and their translocation to the nucleus, causing nuclear DNA fragmentation and activation of regulated necrosis. Though these cascades of events were primarily identified in the mouse model, studies on human hepatocytes and analysis of circulating biomarkers from patients after APAP overdose, indicate that a number of mechanistic events are identical in mice and humans. Circulating biomarkers also seem to be useful in predicting the course of liver injury after APAP overdose in humans and hold promise for sig nificant clinical use in the near future. Relevance for patients: This review focuses on the mechanisms behind APAP-induced hepatotoxicity and the relevance of these to the human pathophysiology. Current investigations on various biomarkers which may be useful in clinical management of APAP overdose patients are also discussed.