Yuchao Xie

Acetaminophen-induced Liver Injury in Rats and Mice: Comparison of Protein Adducts, Mitochondrial Dysfunction, and Oxidative Stress in the Mechanism of Toxicity

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the West. In mice, APAP hepatotoxicity can be rapidly induced with a single dose. Because it is both clinically relevant and experimentally convenient, APAP intoxication has become a popular model of liver injury. Early data demonstrated that rats are resistant to APAP toxicity. As a result, mice are the preferred species for mechanistic studies. Furthermore, recent work has shown that the mechanisms of APAP toxicity in humans are similar to mice. Nevertheless, some investigators still use rats. New mechanistic information from the last forty years invites a reevaluation of the differences between these species. Comparison may provide interesting insights and confirm or exclude the rat as an option for APAP studies. To this end, we treated rats and mice with APAP and measured parameters of liver injury, APAP metabolism, oxidative stress, and activation of the c-Jun N-terminal kinase (JNK). Consistent with earlier data, we found that rats were highly resistant to APAP toxicity. Although overall APAP metabolism was similar in both species, mitochondrial protein adducts were significantly lower in rats. Accordingly, rats also had less oxidative stress. Finally, while mice showed extensive activation and mitochondrial translocation of JNK, this could not be detected in rat livers. These data support the hypothesis that mitochondrial dysfunction is critical for the development of necrosis after APAP treatment. Because mitochondrial damage also occurs in humans, rats are not a clinically relevant species for studies of APAP hepatotoxicity.

Receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice.

Acetaminophen (APAP) overdose is a major cause of hepatotoxicity and acute liver failure in the U.S., but the pathophysiology is incompletely understood. Despite evidence for apoptotic signaling, hepatic cell death after APAP is generally considered necrotic in mice and in humans. Recent findings suggest that the receptor interacting protein kinase 3 (RIP3) acts as a switch from apoptosis to necrosis (programmed necrosis). Thus, the aim of the current investigation was to determine if RIP3 is involved in APAP‐induced liver cell death. APAP (200‐300 mg/kg) caused glutathione depletion and protein adduct formation, oxidant stress, mitochondrial release of apoptosis inducing factor, and nuclear DNA fragmentation resulting in centrilobular necrosis in C57Bl/6J mice. Inhibiting RIP3 protein induction with antisense morpholinos in wild‐type animals or using RIP3‐deficient mice had no effect on protein adduct formation but attenuated all other parameters, including necrotic cell death, at 6 hours after APAP. In addition, cultured hepatocytes from RIP3‐deficient mice showed reduced injury compared to wild‐type cells after 24 hours. Interestingly, APAP‐induced mitochondrial translocation of dynamin‐related protein 1 (Drp1), the initiator of mitochondrial fission, was inhibited by reduced RIP3 protein expression and the Drp1 inhibitor MDIVI reduced APAP‐induced cell death at 24 hours. All of these protective effects were lost after 24 hours in vivo or 48 hours in vitro. Conclusion: RIP3 is an early mediator of APAP hepatotoxicity, involving modulation of mitochondrial dysfunction and oxidant stress. Controlling RIP3 expression could be a promising new approach to reduce APAP‐induced liver injury, but requires complementary strategies to control mitochondrial dysfunction for long‐term protection. (Hepatology 2013; 58:2099–2108)

Plasma and liver acetaminophen-protein adduct levels in mice after acetaminophen treatment: dose-response, mechanisms, and clinical implications.

At therapeutic doses, acetaminophen (APAP) is a safe and effective analgesic. However, overdose of APAP is the principal cause of acute liver failure in the West. Binding of the reactive metabolite of APAP (NAPQI) to proteins is thought to be the initiating event in the mechanism of hepatotoxicity. Early work suggested that APAP-protein binding could not occur without glutathione (GSH) depletion, and likely only at toxic doses. Moreover, it was found that protein-derived APAP-cysteine could only be detected in serum after the onset of liver injury. On this basis, it was recently proposed that serum APAP-cysteine could be used as diagnostic marker of APAP overdose. However, comprehensive dose-response and time course studies have not yet been done. Furthermore, the effects of co-morbidities on this parameter have not been investigated. We treated groups of mice with APAP at multiple doses and measured liver GSH and both liver and plasma APAP-protein adducts at various timepoints. Our results show that protein binding can occur without much loss of GSH. Importantly, the data confirm earlier work that showed that protein-derived APAP-cysteine can appear in plasma without liver injury. Experiments performed in vitro suggest that this may involve multiple mechanisms, including secretion of adducted proteins and diffusion of NAPQI directly into plasma. Induction of liver necrosis through ischemia-reperfusion significantly increased the plasma concentration of protein-derived APAP-cysteine after a subtoxic dose of APAP. While our data generally support the measurement of serum APAP-protein adducts in the clinic, caution is suggested in the interpretation of this parameter.

Mechanisms of Acetaminophen-induced Cell Death in Primary Human Hepatocytes

UNLABELLED Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5mM, 10mM or 20mM APAP over a period of 48 h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24h and 48h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6h after APAP and a partial protection when given at 15 h. CONCLUSION These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic.

Models of drug-induced liver injury for evaluation of phytotherapeutics and other natural products.

Extracts from medicinal plants, many of which have been used for centuries, are increasingly tested in models of hepatotoxicity. One of the most popular models to evaluate the hepatoprotective potential of natural products is acetaminophen (APAP)-induced liver injury, although other hepatotoxicity models such as carbon tetrachloride, thioacetamide, ethanol and endotoxin are occasionally used. APAP overdose is a clinically relevant model of drug-induced liver injury. Critical mechanisms and signaling pathways, which trigger necrotic cell death and sterile inflammation, are discussed. Although there is increasing understanding of the pathophysiology of APAP-induced liver injury, the mechanism is complex and prone to misinterpretation, especially when unknown chemicals such as plant extracts are tested. This review discusses the fundamental aspects that need to be considered when using this model, such as selection of the animal species or in vitro system, timing and dose-responses of signaling events, metabolic activation and protein adduct formation, the role of lipid peroxidation and apoptotic versus necrotic cell death, and the impact of the ensuing sterile inflammatory response. The goal is to enable researchers to select the appropriate model and experimental conditions for testing of natural products that will yield clinically relevant results and allow valid interpretations of the pharmacological mechanisms.

Acetaminophen-induced Liver Injury: from Animal Models to Humans

Drug-induced liver injury is an important clinical problem and a challenge for drug development. Whereas progress in understanding rare and unpredictable (idiosyncratic) drug hepatotoxicity is severely hampered by the lack of relevant animal models, enormous insight has been gained in the area of predictable hepatotoxins, in particular acetaminophen-induced liver injury, from a broad range of experimental models. Importantly, mechanisms of toxicity obtained with certain experimental systems, such as in vivo mouse models, primary mouse hepatocytes, and metabolically competent cell lines, are being confirmed in translational studies in patients and in primary human hepatocytes. Despite this progress, suboptimal models are still being used and experimental data can be confusing, leading to controversial conclusions. Therefore, this review attempts to discuss mechanisms of drug hepatotoxicity using the most studied drug acetaminophen as an example. We compare the various experimental models that are used to investigate mechanisms of acetaminophen hepatotoxicity, discuss controversial topics in the mechanisms, and assess how these experimental findings can be translated to the clinic. The success with acetaminophen in demonstrating the clinical relevance of experimental findings could serve as an example for the study of other drug toxicities.

Purinergic receptor antagonist A438079 protects against acetaminophen-induced liver injury by inhibiting p450 isoenzymes, not by inflammasome activation.

Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in the western world. Controversy exists regarding the hypothesis that the hepatocyte injury is amplified by a sterile inflammatory response, rather than being the result of intracellular mechanisms alone. A recent study suggested that the purinergic receptor antagonist A438079 protects against APAP-induced liver injury by preventing the activation of the Nalp3 inflammasome in Kupffer cells and thereby preventing inflammatory injury. To test the hypothesis that A438079 actually affects the intracellular signaling events in hepatocytes, C57Bl/6 mice were treated with APAP (300 mg/kg) and A438079 (80 mg/kg) or saline and GSH depletion, protein adduct formation, c-jun-N-terminal kinase (JNK) activation, oxidant stress, and liver cell necrosis were determined between 0 and 6 h after APAP administration. APAP caused rapid GSH depletion, extensive protein adduct formation in liver homogenates and in mitochondria, JNK phosphorylation and mitochondrial translocation of phospho-JNK within 2 h, oxidant stress, and extensive centrilobular necrosis at 6 h. A438079 significantly attenuated GSH depletion, which resulted in a 50% reduction of total liver and mitochondrial protein adducts and substantial reduction of JNK activation, mitochondrial P-JNK translocation, oxidant stress, and liver injury. The same results were obtained using primary mouse hepatocytes. A438079 did not directly affect JNK activation induced by tert-butyl hydroperoxide and GSH depletion. However, A438079 dose-dependently inhibited hepatic P450 enzyme activity. Thus, the protective effect of A438079 against APAP hepatotoxicity in vivo can be explained by its effect on metabolic activation and cell death pathways in hepatocytes without involvement of the Nalp3 inflammasome.

Removal of acetaminophen protein adducts by autophagy protects against acetaminophen-induced liver injury in mice

BACKGROUND & AIMS Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in the US and many other countries. Metabolism of APAP results in formation of APAP protein adducts (APAP-AD) in hepatocytes and triggers mitochondrial dysfunction and necrosis. However, the mechanisms for how APAP-AD are removed from hepatocytes remain unknown. METHODS Mice or primary hepatocytes were treated with APAP. APAP-AD were determined by immunoblot, immunostaining and high pressure liquid chomatography with electrochemical detection analysis. RESULTS We found that APAP-AD were detected at 1h, peaked at approximately 2h, declined at 6h and almost full removed at 24h post treatment with APAP in mouse livers and in primary mouse hepatocytes. APAP-AD displayed a punctate pattern and were colocalized with GFP-LC3 positive autophagosomes and Lamp1 positive lysosomes in APAP-treated primary hepatocytes. Moreover, isolated autophagosomes and autolysosomes from APAP-treated mouse livers contained APAP-AD, suggesting autophagy may selectively remove APAP-AD. APAP-AD were detected in both detergent soluble and insoluble pools in APAP-treated mouse livers and hepatocytes. More importantly, pharmacological inhibition of autophagy by leupeptin or chloroquine increased whereas induction of autophagy by Torin 1 decreased serum APAP-AD levels in APAP-treated mice, which correlated with alanine aminotransferase levels and liver necrosis. Furthermore, SQSTM1/p62, an autophagy receptor protein, was recruited to APAP-AD. Adenovirus-mediated shRNA knockdown of SQSTM1/p62 led to increased APAP-AD and necrosis in primary hepatocytes. CONCLUSIONS Our data indicate that APAP-AD are removed though selective autophagy. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury. LAY SUMMARY Acetaminophen overdose can form acetaminophen protein adducts and mitochondria damage in hepatocytes resulting in liver injury. Activation of autophagy-lysosomal degradation pathway can help to remove acetaminophen protein adducts. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury.

Inhibitor of apoptosis signal-regulating kinase 1 protects against acetaminophen-induced liver injury

Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun-N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300 mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and liver injury as indicated by plasma ALT activities and area of necrosis over a 24h observation period. Pretreatment with 30 mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affecting the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients.

Pathophysiological significance of c-jun N-terminal kinase in acetaminophen hepatotoxicity

Background: Acetaminophen (APAP) overdose is the leading cause of acute liver failure in the US. Although substantial progress regarding the mechanisms of APAP hepatotoxicity has been made in the past several decades, therapeutic options are still limited and novel treatments are clearly needed. c-jun N-terminal Kinase (JNK) has emerged as a promising therapeutic target in recent years. Areas covered: Early studies established the critical role of JNK activation and mitochondrial translocation in APAP hepatotoxicity. However, this concept has also been challenged. Initial studies failed to reproduce the protection of JNK deficiency in APAP toxicity and concerns over off-target effects of JNK inhibitors and even in knock-out mice are increasing. Interestingly, recent studies have even shown that liver injury can be altered with or without effects on JNK activation. The current review addresses these discrepancies and tries to explain or reconcile some of the conflicting results. Expert opinion: JNK is a potential therapeutic target for APAP poisoning. However, controversies still exist regarding its actual role in APAP hepatotoxicity. Future studies are warranted for more in-depth testing of specific inhibitors in well-defined preclinical models and human hepatocytes before JNK can be considered a relevant therapeutic target for APAP poisoning.