Acetaminophen Hepatotoxicity: Strong Offense and Weakened Defense
Authors: Neil Kaplowitz, Tin Aung Than, Sanda Win
Journal: Hepatology (2020)

See Article on Page 1787 Potential conflict of interest: Nothing to report. Oxidative stress refers to the consequences of exposure to reactive oxygen species. In many disease conditions, interruption along the mitochondrial electron transport chain (ETC) leads to build‐up of electrons, which are transferred to O2. Reactive oxygen species (ROS) then can oxidize any chemical constituent of cells. This causes thiol‐disulfide redox stress or imbalance, which activates or inhibits many enzymes and signal transduction pathways. Countering oxidative/redox stress is antioxidant defense, which detoxifies reactive oxygen. The transcription factor, nuclear erythroid 2 p45‐related factor 2 (Nrf2), regulates the expression of many enzymes of defense. Nrf2 is regulated by two mechanisms.(1) Keap1 is an ubiquitin ligase adaptor. The Neh2 domain of Nrf2 binds to Keap1 in the cytoplasm, allowing an E3‐ubiquitylation complex to facilitate proteasomal degradation of Nrf2.(1) Following redox stress or binding of toxic metabolites, Keap1 releases Nrf2, which translocates to the nucleus and activates transcription of ARE‐driven genes. However, Nrf2 degradation can occur in a Keap1‐independent fashion through the binding of β‐TrCP. β‐TrCP is redox‐independent and is activated by phosphorylation of Nrf2 in the Neh6 domain (such as by glycogen synthase kinase 3β [GSK‐3β]).(1) β‐TrCP binds an E3‐ubiquitylation complex, which promotes proteasomal degradation. Acetaminophen (APAP) hepatotoxicity is an excellent model for examining the consequences of oxidative stress/redox imbalance. A small portion of an APAP dose is converted to N‐acetyl‐p‐quinone imine (NAPQI), a reactive metabolite that is selectively detoxified by glutathione (GSH). At higher doses, sufficient NAPQI is generated to deplete GSH in cytoplasm and mitochondria; the remaining NAPQI is free to covalently bind to protein thiols. In mitochondria, the combination of GSH depletion and covalent binding exposes mitochondria and the cytoplasm to ROS. This early stage of APAP toxicity occurs very rapidly in the mouse model, and the effect reaches maximum with an hour. ROS species released into the cytoplasm activate the redox‐sensitive mitogen‐activated protein kinase (MAPK) kinase cascade through ASK1 or MLK3, leading to c‐Jun N‐terminal kinase (JNK) activation (P‐JNK). The latter binds to and phosphorylates the mitochondrial outer membrane protein, SAB (Sh3bp5). Analogous to a receptor, SAB then activates an intramitochondrial signal transduction pathway, leading to impairment of the ETC and enhanced ROS production and release, and continued JNK activation in a sustained cycle.(2) The continued production of mitochondrial ROS ultimately leads to complete collapse of mitochondrial function and lytic necrosis.(3) However, there is a time window between APAP metabolism and the onset of necrosis when the situation may be reversible. This is well‐demonstrated in the mouse model by the efficacy of delayed administration of N‐acetylcysteine (NAC) or JNK inhibitor up to 1.5‐2 hours after APAP administration but not beyond.(4,5) NAC is a precursor for de novo synthesis of GSH, which very early on enhances detoxification of NAPQI, but in the window period NAC accelerates GSH recovery and detoxifies ROS, interrupting sustained activation of the MAPK pathway (Fig. 1). Therefore, this raises the question of the potential contribution of activation of antioxidant defense mediated by Nrf2 in dampening oxidative stress in the interval between early APAP metabolism, covalent binding and initial GSH depletion, and the commitment to cell death.Fig. 1: Interplay between JNK and Nrf2 degradation in APAP hepatotoxicity. APAP induces mitochondrial ROS production, activating JNK that further amplifies the ROS production, sustaining JNK activation. Oxidative stress leads to release of Nrf2 from KEAP1 in the cytoplasm and P‐JNK mediates Nrf2 degradation in the nucleus. The latter inhibits dampening of oxidative stress. The key issue is the rapidity of these responses in the interval phase between APAP metabolism and onset and progression of cell death. Abbreviations: KD, knockdown; MPT, mitochondrial permeability transition; NAC, N‐acetylcysteine.Chen et al.(6) have now shown convincingly that Nrf2 can be added to the large number of proteins that are phosphorylated by JNK. In an elegant series of experiments in A549 cells that lack functional Keap1, they identified the JNK phosphorylation site (serine335) in the Neh6 region of Nrf2, distinct from the Keap1 binding region in Neh2,(1) permitting β‐TrCP‐E3 ubiquitylation complex to bind to Nrf2 followed by proteasomal degradation. Thus, two counteracting mechanisms may occur in toxic stress: redox modification of Keap1, which favors Nrf2 activation of the gene programs for defense, and P‐JNK‐mediated degradation of Nrf2 that impairs defense. Chen et al.(6) demonstrated that inhibitors or knockdown of JNK prevented APAP hepatotoxicity and stabilized Nrf2. The key question is whether these two outcomes of JNK inhibition are causally linked. In approaching this question, it is very important to distinguish pre‐existent effects of Nrf2, as exemplified by the previously identified effect of Nrf2 knockout versus the effect of APAP on the response of Nrf2 from its basal condition, leading to increased transcripts and increased proteins of the antioxidant gene repertoire with sufficient rapidity to dampen the oxidative stress in the window period after APAP treatment. Chen et al.(6) showed that while the Nrf2 level actually increased, ARE‐dependent gene messenger RNAs (mRNAs) were suppressed by 6 hours, although actually up‐regulated in the first 3 hours after APAP. At 6 hours, the authors observed that despite the decline in mRNAs, the respective proteins they examined in the Nrf2 repertoire remained unaffected. However, nontranscriptional effects of P‐JNK on many targets are known to occur very rapidly and promote necrosis, which is already well‐established in the first 6 hours. By the time peak necrosis had occurred (24 hours), the expression of antioxidant proteins had declined. Therefore, it is conceivable that in the interval between 6 hours after APAP, when injury was approximately half maximum until peak injury, the declining expression of Nrf2‐responsive genes might contribute to the progression to peak injury. However, this remains unproven. Alternatively, because the antioxidant response usually accompanies cell proliferation, the late decline in Nrf2‐regulated genes might impair recovery. It should be noted that Nrf2 regulates expression of many antioxidant and detoxification genes, only some of which were examined by Chen et al.(5) Thus, it remains possible that selective decrease of antioxidative proteins due to faster protein turnover in the absence of continued synthesis may contribute in the early phase. Proteins of potential importance include GSH peroxidase, peroxiredoxins, thioredoxin, thioredoxin reductase, sulforedoxin, ferritin, heme oxygenase, MKP‐1, and GSH synthetic enzymes. The authors did examine the GCLC protein, which was not significantly changed at 6 hours after APAP. This is a key enzyme in de novo GSH synthesis and mediates GSH recovery in this model. Others have found increased GCLC mRNA up to 6 hours after APAP, but rapid decline in GCLC protein after APAP,(7) presumably contributing to delayed GSH recovery, which impairs GSH dampening of oxidative stress. Chen et al.(6) demonstrated that JNK inhibitor did not protect Nrf2−/− mice from APAP. However, Nrf2−/− mice are highly susceptible to APAP toxicity and have altered basal defense, which may overwhelm the ability of JNK inhibitors to protect.(8) Strikingly, Nrf2−/− mice expressing mutated JNK phosphorylation site were markedly resistant to APAP toxicity. It should be noted that 70% mortality at 6 hours was seen after APAP (300 mg/kg) in the knockout mice, but no mortality at 6 hours after expression of either wild‐type or JNK‐resistant Nrf2. As proof of principle, the results of these experiments are quite striking. However, Nrf2−/− mice are very different from wild‐type mice, so extrapolation is not straightforward. In summary, the paper by Chen et al.(6) provides an important advance, namely the demonstration that Nrf2 is a target substrate of JNK, which leads to degradation of Nrf2. The impact of this on acute APAP toxicity requires more work with respect to its contribution to various stages of injury onset and progression in the rapid sequence of events during the time course of acute APAP injury. Nevertheless, the implications of JNK‐mediated Nrf2 degradation have much broader implications in the pathophysiology of diseases. The determinants of the balance between Nrf2 activation by release from Keap1‐mediated degradation versus activation of JNK‐mediated Nrf2 degradation appear to be a pivotal aspect of the response to oxidative stress.