High-Dose Acetaminophen with Concurrent CYP2E1 Inhibition Has Profound Anticancer Activity without Liver Toxicity.

Acetaminophen (AAP) is metabolized by a variety of pathways such as sulfation, glucuronidation, and fatty acid amide hydrolase-mediated conversion to the active analgesic metabolite AM404. CYP2E1-mediated metabolism to the hepatotoxic reactive metabolite NAPQI (N-acetyl-p-benzoquinone imine) is a minor metabolic pathway that has not been linked to AAP therapeutic benefits yet clearly leads to AAP liver toxicity. N-acetylcysteine (NAC) (an antioxidant) and fomepizole (a CYP2E1 inhibitor) are clinically used for the treatment of AAP toxicity. Mice treated with AAP in combination with fomepizole (plus or minus NAC) were assessed for liver toxicity by histology and serum chemistry. The anticancer activity of AAP with NAC and fomepizole rescue was assessed in vitro and in vivo. Fomepizole with or without NAC completely prevented AAP-induced liver toxicity. In vivo, high-dose AAP with NAC/fomepizole rescue had profound antitumor activity against commonly used 4T1 breast tumor and lewis lung carcinoma lung tumor models, and no liver toxicity was detected. The antitumor efficacy was reduced in immune-compromised NOD-scid IL2Rgammanull mice, suggesting an immune-mediated mechanism of action. In conclusion, using fomepizole-based rescue, we were able to treat mice with 100-fold higher than standard dosing of AAP (650 mg/kg) without any detected liver toxicity and substantial antitumor activity. SIGNIFICANCE STATEMENT: High-dose acetaminophen can be given concurrently with CYP2E1 inhibition to allow for safe dose escalation to levels needed for anticancer activity without detected evidence of toxicity.

Role of HNF4alpha-cMyc interaction in liver regeneration and recovery after acetaminophen-induced acute liver injury.

BACKGROUND AND AIMS: Overdose of acetaminophen (APAP) is the major cause of acute liver failure in the western world. We report a novel signaling interaction between hepatocyte nuclear factor 4 alpha (HNF4α) cMyc and nuclear factor erythroid 2-related factor 2 (Nrf2) during liver injury and regeneration after APAP overdose. APPROACH AND RESULTS: APAP-induced liver injury and regeneration were studied in male C57BL/6J (WT) mice, hepatocyte-specific HNF4α knockout mice (HNF4α-KO), and HNF4α-cMyc double knockout mice (DKO). C57BL/6J mice treated with 300 mg/kg maintained nuclear HNF4α expression and exhibited liver regeneration, resulting in recovery. However, treatment with 600-mg/kg APAP, where liver regeneration was inhibited and recovery was delayed, showed a rapid decline in HNF4α expression. HNF4α-KO mice developed significantly higher liver injury due to delayed glutathione recovery after APAP overdose. HNF4α-KO mice also exhibited significant induction of cMyc, and the deletion of cMyc in HNF4α-KO mice (DKO mice) reduced the APAP-induced liver injury. The DKO mice had significantly faster glutathione replenishment due to rapid induction in Gclc and Gclm genes. Coimmunoprecipitation and ChIP analyses revealed that HNF4α interacts with Nrf2 and affects its DNA binding. Furthermore, DKO mice showed significantly faster initiation of cell proliferation resulting in rapid liver regeneration and recovery. CONCLUSIONS: These data show that HNF4α interacts with Nrf2 and promotes glutathione replenishment aiding in recovery from APAP-induced liver injury, a process inhibited by cMyc. These studies indicate that maintaining the HNF4α function is critical for regeneration and recovery after APAP overdose.

The chemokine CXCL14 is a novel early prognostic biomarker for poor outcome in acetaminophen-induced acute liver failure.

BACKGROUND AND AIMS: Patients with acetaminophen-induced acute liver failure are more likely to die while on the liver transplant waiting list than those with other causes of acute liver failure. Therefore, there is an urgent need for prognostic biomarkers that can predict the need for liver transplantation early after an acetaminophen overdose. APPROACH AND RESULTS: We evaluated the prognostic potential of plasma chemokine C-X-C motif ligand 14 (CXCL14) concentrations in patients with acetaminophen (APAP) overdose (n=50) and found that CXCL14 is significantly higher in nonsurviving patients compared to survivors with acute liver failure ( p < 0.001). Logistic regression and AUROC analyses revealed that CXCL14 outperformed the MELD score, better discriminating between nonsurvivors and survivors. We validated these data in a separate cohort of samples obtained from the Acute Liver Failure Study Group (n = 80), where MELD and CXCL14 had similar AUC (0.778), but CXCL14 demonstrated higher specificity (81.2 vs. 52.6) and positive predictive value (82.4 vs. 65.4) for death or need for liver transplantation. Next, combining the patient cohorts and using a machine learning training/testing scheme to mimic the clinical scenario, we found that CXCL14 outperformed MELD based on AUC (0.821 vs. 0.787); however, combining MELD and CXCL14 yielded the best AUC (0.860). CONCLUSIONS: We find in 2 independent cohorts of acetaminophen overdose patients that circulating CXCL14 concentration is a novel early prognostic biomarker for poor outcomes, which may aid in guiding decisions regarding patient management. Moreover, our findings reveal that CXCL14 performs best when measured soon after patient presentation to the clinic, highlighting its importance for early warning of poor prognosis.

The thrombopoietin mimetic JNJ-26366821 reduces the late injury and accelerates the onset of liver recovery after acetaminophen-induced liver injury in mice.

Acetaminophen (APAP)-induced hepatotoxicity is comprised of an injury and recovery phase. While pharmacological interventions, such as N-acetylcysteine (NAC) and 4-methylpyrazole (4-MP), prevent injury there are no therapeutics that promote recovery. JNJ-26366821 (TPOm) is a novel thrombopoietin mimetic peptide with no sequence homology to endogenous thrombopoietin (TPO). Endogenous thrombopoietin is produced by hepatocytes and the TPO receptor is present on liver sinusoidal endothelial cells in addition to megakaryocytes and platelets, and we hypothesize that TPOm activity at the TPO receptor in the liver provides a beneficial effect following liver injury. Therefore, we evaluated the extent to which TPOm, NAC or 4-MP can provide a protective and regenerative effect in the liver when administered 2 h after an APAP overdose of 300 mg/kg in fasted male C57BL/6J mice. TPOm did not affect protein adducts, oxidant stress, DNA fragmentation and hepatic necrosis up to 12 h after APAP. In contrast, TPOm treatment was beneficial at 24 h, i.e., all injury parameters were reduced by 42-48%. Importantly, TPOm enhanced proliferation by 100% as indicated by PCNA-positive hepatocytes around the area of necrosis. When TPOm treatment was delayed by 6 h, there was no effect on the injury, but a proliferative effect was still evident. In contrast, 4MP and NAC treated at 2 h after APAP significantly attenuated all injury parameters at 24 h but failed to enhance hepatocyte proliferation. Thus, TPOm arrests the progression of liver injury by 24 h after APAP and accelerates the onset of the proliferative response which is essential for liver recovery.

Central mechanisms of acetaminophen hepatotoxicity: mitochondrial dysfunction by protein adducts and oxidant stress.

Acetaminophen (APAP) is an analgesic and antipyretic drug used worldwide, which is safe at therapeutic doses. However, an overdose can induce liver injury and even liver failure. Mechanistic studies in mice beginning with the seminal papers published by B.B. Brodie's group in the 1970s have resulted in important insight into the pathophysiology. Although the metabolic activation of APAP with generation of a reactive metabolite, glutathione depletion and protein adduct formation are critical initiating events, more recently the mitochondria came into focus as important target and decision point of cell death. This review provides a comprehensive overview of the induction of mitochondrial superoxide and peroxynitrite formation and its propagation through a mitogen activated protein kinase cascade, the mitochondrial permeability transition pore opening caused by iron-catalyzed protein nitration and the mitochondria-dependent nuclear DNA fragmentation. In addition, the role of adaptive mechanisms that can modulate the pathophysiology including autophagy, mitophagy, Nrf2 activation and mitochondrial biogenesis, are discussed. Importantly, it is outlined how the mechanisms elucidated in mice translate to human hepatocytes and APAP overdose patients, and how this mechanistic insight explains the mechanism of action of the clinically approved antidote N-acetylcysteine and led to the recent discovery of a novel compound, fomepizole, which is currently under clinical development. Significance Statement Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in western countries. Extensive mechanistic research over the last several decades revealed a central role of mitochondria in the pathophysiology of APAP hepatotoxicity. This review article provides a comprehensive discussion of a) mitochondrial protein adducts and oxidative/nitrosative stress, b) mitochondria-regulated nuclear DNA fragmentation, c) adaptive mechanisms to APAP-induced cellular stress, d) translation of cell death mechanisms to overdose patients, and e) mechanism-based antidotes against APAP-induced liver injury.

The mitochondrial calcium uniporter mediates mitochondrial Fe2+ uptake and hepatotoxicity after acetaminophen.

Acetaminophen (APAP) overdose disrupts hepatocellular lysosomes, which release ferrous iron (Fe2+) that translocates into mitochondria putatively via the mitochondrial calcium uniporter (MCU) to induce oxidative/nitrative stress, the mitochondrial permeability transition (MPT), and hepatotoxicity. To investigate how MCU deficiency affects mitochondrial Fe2+ uptake and hepatotoxicity after APAP overdose, global MCU knockout (KO), hepatocyte specific (hs) MCU KO, and wildtype (WT) mice were treated with an overdose of APAP both in vivo and in vitro. Compared to strain-specific WT mice, serum ALT decreased by 88 and 56%, respectively, in global and hsMCU KO mice at 24 h after APAP (300 mg/kg). Hepatic necrosis also decreased by 84 and 56%. By contrast, when MCU was knocked out in Kupffer cells, ALT release and necrosis were unchanged after overdose APAP. Intravital multiphoton microscopy confirmed loss of viability and mitochondrial depolarization in pericentral hepatocytes of WT mice, which was decreased in MCU KO mice. CYP2E1 expression, hepatic APAP-protein adduct formation, and JNK activation revealed that APAP metabolism was equivalent between WT and MCU KO mice. In cultured hepatocytes after APAP, loss of cell viability decreased in hsMCU KO compared to WT hepatocytes. Using fructose plus glycine to prevent cell killing, mitochondrial Fe2+ increased progressively after APAP, as revealed with mitoferrofluor (MFF), a mitochondrial Fe2+ indicator. By contrast in hsMCU KO hepatocytes, mitochondrial Fe2+ uptake after APAP was suppressed. Rhod-2 measurements showed that Ca2+ did not increase in mitochondria after APAP in either WT or KO hepatocytes. In conclusion, MCU mediates uptake of Fe2+ into mitochondria after APAP and plays a central role in mitochondrial depolarization and cell death during APAP-induced hepatotoxicity.

Nrf2 as a therapeutic target in acetaminophen hepatotoxicity: A case study with sulforaphane.

Acetaminophen (APAP) overdose can cause severe liver injury and acute liver failure. The only clinically approved antidote, N-acetylcysteine (NAC), is highly effective but has a narrow therapeutic window. In the last 2 decades, activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates acute phase proteins and antioxidant defense genes, has emerged as a putative new therapeutic target against APAP hepatotoxicity. However, virtually all studies that propose Nrf2 activation as mechanism of protection used prolonged pretreatment, which is not a clinically feasible approach to treat a drug overdose. Therefore, the objective of this study was to assess if therapeutic activation of Nrf2 is a viable approach to treat liver injury after APAP overdose. We used the water-soluble Nrf2 activator sulforaphane (SFN; 5 mg/kg) in a murine model of APAP hepatotoxicity (300 mg/kg). Our results indicate that short-term treatment (≤3 h) with SFN alone did not activate Nrf2 or its target genes. However, posttreatment with SFN after APAP partially protected at 6 h likely due to more rapid activation of the Nrf2-target gene heme oxygenase-1. A direct comparison of SFN with NAC given at 1 h after APAP showed a superior protection with NAC, which was maintained at 24 h unlike with SFN. Thus, Nrf2 activators have inherent problems like the need to create a cellular stress to activate Nrf2 and delayed adaptive responses which may hamper sustained protection against APAP hepatotoxicity. Thus, compared to the more direct acting antidote NAC, Nrf2 activators are less suitable for this indication.

Dose-dependent pleiotropic role of neutrophils during acetaminophen-induced liver injury in male and female mice.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure in western countries. APAP can cause extensive hepatocellular necrosis, which triggers an inflammatory response involving neutrophil and monocyte recruitment. Particularly the role of neutrophils in the injury mechanism of APAP hepatotoxicity has been highly controversial. Thus, the objective of the current study was to assess whether a potential contribution of neutrophils was dependent on the APAP dose and the sex of the animals. Male and female C57BL/6 J mice were treated with 300 or 600 mg/kg APAP and the injury and inflammatory cell recruitment was evaluated between 6 and 48 h. In both male and female mice, ALT plasma levels and the areas of necrosis peaked at 12-24 h after both doses with more severe injury at the higher dose. In addition, Ly6g-positive neutrophils started to accumulate in the liver at 6 h and peaked at 6-12 h after 300 mg/kg and 12-24 h after 600 mg/kg for both sexes; however, the absolute numbers of hepatic neutrophils in the liver were significantly higher after the 600 mg/kg dose. Neutrophil infiltration correlated with mRNA levels of the neutrophil chemoattractant Cxcl2 in the liver. Treating mice with an anti-Cxcl2 antibody at 2 h after APAP significantly reduced neutrophil accumulation at 24 h after both doses and in both sexes. However, the injury was significantly reduced only after the high overdose. Thus, neutrophils, recruited through Cxcl2, have no effect on APAP-induced liver injury after 300 mg/kg but aggravate the injury only after severe overdoses.

Interruption of bile acid uptake by hepatocytes after acetaminophen overdose ameliorates hepatotoxicity.

BACKGROUND & AIMS: Acetaminophen (APAP) overdose remains a frequent cause of acute liver failure, which is generally accompanied by increased levels of serum bile acids (BAs). However, the pathophysiological role of BAs remains elusive. Herein, we investigated the role of BAs in APAP-induced hepatotoxicity. METHODS: We performed intravital imaging to investigate BA transport in mice, quantified endogenous BA concentrations in the serum of mice and patients with APAP overdose, analyzed liver tissue and bile by mass spectrometry and MALDI-mass spectrometry imaging, assessed the integrity of the blood-bile barrier and the role of oxidative stress by immunostaining of tight junction proteins and intravital imaging of fluorescent markers, identified the intracellular cytotoxic concentrations of BAs, and performed interventions to block BA uptake from blood into hepatocytes. RESULTS: Prior to the onset of cell death, APAP overdose causes massive oxidative stress in the pericentral lobular zone, which coincided with a breach of the blood-bile barrier. Consequently, BAs leak from the bile canaliculi into the sinusoidal blood, which is then followed by their uptake into hepatocytes via the basolateral membrane, their secretion into canaliculi and repeated cycling. This, what we termed 'futile cycling' of BAs, led to increased intracellular BA concentrations that were high enough to cause hepatocyte death. Importantly, however, the interruption of BA re-uptake by pharmacological NTCP blockage using Myrcludex B and Oatp knockout strongly reduced APAP-induced hepatotoxicity. CONCLUSIONS: APAP overdose induces a breach of the blood-bile barrier which leads to futile BA cycling that causes hepatocyte death. Prevention of BA cycling may represent a therapeutic option after APAP intoxication. LAY SUMMARY: Only one drug, N-acetylcysteine, is approved for the treatment of acetaminophen overdose and it is only effective when given within ∼8 hours after ingestion. We identified a mechanism by which acetaminophen overdose causes an increase in bile acid concentrations (to above toxic thresholds) in hepatocytes. Blocking this mechanism prevented acetaminophen-induced hepatotoxicity in mice and evidence from patients suggests that this therapy may be effective for longer periods after ingestion compared to N-acetylcysteine.

The role of Iron in lipid peroxidation and protein nitration during acetaminophen-induced liver injury in mice.

Acetaminophen (APAP) hepatotoxicity, a leading cause of acute liver failure in western countries, is characterized by mitochondrial superoxide and peroxynitrite formation. However, the role of iron, especially as facilitator of lipid peroxidation (LPO), has been controversial. Our aim was to determine the mechanism by which iron promotes cell death in this context. Fasted male C57BL/6J mice were treated with the iron chelator deferoxamine, minocycline (inhibitor of the mitochondrial calcium uniporter) or vehicle 1 h before 300 mg/kg APAP. Deferoxamine and minocycline significantly attenuated APAP-induced elevations in serum alanine amino transferase levels and hepatic necrosis at 6 h. This protection correlated with reduced 3-nitro-tyrosine protein adducts; LPO (malondialdehyde, 4-hydroxynonenal) was not detected. Activation of c-jun N-terminal kinase (JNK) was not affected but mitochondrial release of intermembrane proteins was reduced suggesting that the effect of iron was at the level of mitochondria. Co-treatment of APAP with FeSO4 exacerbated liver injury and protein nitration and triggered significant LPO; all effects were reversed by deferoxamine. Thus, after APAP overdose, iron imported into mitochondria facilitates protein nitration by peroxynitrite triggering mitochondrial dysfunction and cell death. Under these conditions, endogenous defense mechanisms largely prevent LPO. However, after iron overload, protein nitration and LPO contribute to APAP hepatotoxicity.

Platanosides, a Potential Botanical Drug Combination, Decrease Liver Injury Caused by Acetaminophen Overdose in Mice.

Oxidative stress plays an important role in acetaminophen (APAP)-induced hepatotoxicity. Platanosides (PTSs) isolated from the American sycamore tree (Platanus occidentalis) represent a potential new four-molecule botanical drug class of antibiotics active against drug-resistant infectious disease. Preliminary studies have suggested that PTSs are safe and well tolerated and have antioxidant properties. The potential utility of PTSs in decreasing APAP hepatotoxicity in mice in addition to an assessment of their potential with APAP for the control of infectious diseases along with pain and pyrexia associated with a bacterial infection was investigated. On PTS treatment in mice, serum alanine aminotransferase (ALT) release, hepatic centrilobular necrosis, and 4-hydroxynonenal (4-HNE) were markedly decreased. In addition, inducible nitric oxide synthase (iNOS) expression and c-Jun-N-terminal kinase (JNK) activation decreased when mice overdosed with APAP were treated with PTSs. Computational studies suggested that PTSs may act as JNK-1/2 and Keap1-Nrf2 inhibitors and that the isomeric mixture could provide greater efficacy than the individual molecules. Overall, PTSs represent promising botanical drugs for hepatoprotection and drug-resistant bacterial infections and are effective in protecting against APAP-related hepatotoxicity, which decreases liver necrosis and inflammation, iNOS expression, and oxidative and nitrative stresses, possibly by preventing persistent JNK activation.

Comparing N-acetylcysteine and 4-methylpyrazole as antidotes for acetaminophen overdose.

Acetaminophen (APAP) overdose can cause hepatotoxicity and even liver failure. N-acetylcysteine (NAC) is still the only FDA-approved antidote against APAP overdose 40 years after its introduction. The standard oral or intravenous dosing regimen of NAC is highly effective for patients with moderate overdoses who present within 8 h of APAP ingestion. However, for late-presenting patients or after ingestion of very large overdoses, the efficacy of NAC is diminished. Thus, additional antidotes with an extended therapeutic window may be needed for these patients. Fomepizole (4-methylpyrazole), a clinically approved antidote against methanol and ethylene glycol poisoning, recently emerged as a promising candidate. In animal studies, fomepizole effectively prevented APAP-induced liver injury by inhibiting Cyp2E1 when treated early, and by inhibiting c-jun N-terminal kinase (JNK) and oxidant stress when treated after the metabolism phase. In addition, fomepizole treatment, unlike NAC, prevented APAP-induced kidney damage and promoted hepatic regeneration in mice. These mechanisms of protection (inhibition of Cyp2E1 and JNK) and an extended efficacy compared to NAC could be verified in primary human hepatocytes. Furthermore, the formation of oxidative metabolites was eliminated in healthy volunteers using the established treatment protocol for fomepizole in toxic alcohol and ethylene glycol poisoning. These mechanistic findings, together with the excellent safety profile after methanol and ethylene glycol poisoning and after an APAP overdose, suggest that fomepizole may be a promising antidote against APAP overdose that could be useful as adjunct treatment to NAC. Clinical trials to support this hypothesis are warranted.

Kupffer cells regulate liver recovery through induction of chemokine receptor CXCR2 on hepatocytes after acetaminophen overdose in mice.

Acetaminophen (APAP) is a widely used analgesic, but also a main cause of acute liver injury in the United States and many western countries. APAP hepatotoxicity is associated with a sterile inflammatory response as shown by the infiltration of neutrophils and monocytes. While the contribution of the immune cells to promote liver repair have been demonstrated, the direct interactions between macrophages or neutrophils with hepatocytes to help facilitate hepatocyte proliferation and tissue repair remain unclear. The purpose of this study was to investigate the relationship between resident macrophages (Kupffer cells) and hepatocytes with a focus on the chemokine receptor CXCR2. C57BL/6J mice were subjected to an APAP overdose (300 mg/kg) and the role of CXCR2 on hepatocytes was investigated using a selective antagonist, SB225002. In addition, clodronate liposomes were used to deplete Kupffer cells to assess changes in CXCR2 expression. Our data showed that CXCR2 was mainly expressed on hepatocytes and it was induced specifically in hepatocytes around the necrotic area 24 h after APAP treatment. Targeting this receptor using an inhibitor caused a delayed liver recovery. Depletion of Kupffer cells significantly prevented CXCR2 induction on hepatocytes. In vitro and in vivo experiments also demonstrated that Kupffer cells regulate CXCR2 expression and pro-regenerative gene expression in surviving hepatocytes through production of IL-10. Thus, Kupffer cells support the transition of hepatocytes around the area of necrosis to a proliferative state through CXCR2 expression.

Human Wharton's Jelly-derived mesenchymal stem cells prevent acetaminophen-induced liver injury in a mouse model unlike human dermal fibroblasts.

The persistence of hepatotoxicity induced by N-acetyl-para-aminophenol (Acetaminophen or Paracetamol, abbreviated as APAP) as the most common cause of acute liver failure in the United States, despite the availability of N-acetylcysteine, illustrates the clinical relevance of additional therapeutic approaches. While human mesenchymal stem cells (MSCs) have shown protection in mouse models of liver injury, the MSCs used are generally not cleared for human use and it is unclear whether these effects are due to xenotransplantation. Here we evaluated GMP manufactured clinical grade human Wharton's Jelly mesenchymal stem cells (WJMSCs), which are currently being investigated in human clinical trials, in a mouse model of APAP hepatotoxicity in comparison to human dermal fibroblasts (HDFs) to address these issues. C57BL6J mice were treated with a moderate APAP overdose (300 mg/kg) and WJMSCs were administered 90 min later. Liver injury was evaluated at 6 and 24 h after APAP. WJMSCs treatment reduced APAP-induced liver injury at both time points unlike HDFs, which showed no protection. APAP-induced JNK activation as well as AIF and Smac release from mitochondria were prevented by WJMSCs treatment without influencing APAP bioactivation. Mechanistically, WJMSCs treatment upregulated expression of Gclc and Gclm to enhance recovery of liver GSH levels to attenuate mitochondrial dysfunction and accelerated recovery of pericentral hepatocytes to re-establish liver zonation and promote liver homeostasis. Notably, preventing GSH resynthesis with buthionine sulfoximine prevented the protective effects of WJMSCs. These data indicate that these GMP-manufactured WJMCs could be a clinically relevant therapeutic approach in the management of APAP hepatotoxicity in humans.

Contrasting model mechanisms of alanine aminotransferase (ALT) release from damaged and necrotic hepatocytes as an example of general biomarker mechanisms.

Interpretations of elevated blood levels of alanine aminotransferase (ALT) for drug-induced liver injury often assume that the biomarker is released passively from dying cells. However, the mechanisms driving that release have not been explored experimentally. The usefulness of ALT and related biomarkers will improve by developing mechanism-based explanations of elevated levels that can be expanded and elaborated incrementally. We provide the means to challenge the ability of closely related model mechanisms to generate patterns of simulated hepatic injury and ALT release that scale (or not) to be quantitatively similar to the wet-lab validation targets, which are elevated plasma ALT values following acetaminophen (APAP) exposure in mice. We build on a published model mechanism that helps explain the generation of characteristic spatiotemporal features of APAP hepatotoxicity within hepatic lobules. Discrete event and agent-oriented software methods are most prominent. We instantiate and leverage a small constellation of concrete model mechanisms. Their details during execution help bring into focus ways in which particular sources of uncertainty become entangled with cause-effect details within and across several levels. We scale ALT amounts in virtual mice directly to target plasma ALT values in individual mice. A virtual experiment comprises a set of Monte Carlo simulations. We challenge the sufficiency of four potentially explanatory theories for ALT release. The first of the tested model theories failed to achieve the initial validation target, but each of the three others succeeded. Results for one of the three model mechanisms matched all target ALT values quantitatively. It explains how ALT externalization is the combined consequence of lobular-location-dependent drug-induced cellular damage and hepatocyte death. Falsification of one (or more) of the model mechanisms provides new knowledge and incrementally shrinks the constellation of model mechanisms. The modularity and biomimicry of our explanatory models enable seamless transition from mice to humans.

Impaired protein adduct removal following repeat administration of subtoxic doses of acetaminophen enhances liver injury in fed mice.

Acetaminophen (APAP) is a widely used analgesic and is safe at therapeutic doses. However, an overdose of APAP is hepatotoxic and accidental overdoses are increasingly common due to the presence of APAP in several combination medications. Formation of protein adducts (APAP-CYS) is central to APAP-induced liver injury and their removal by autophagy is an essential adaptive response after an acute overdose. Since the typical treatment for conditions such as chronic pain involves multiple doses of APAP over time, this study investigated APAP-induced liver injury after multiple subtoxic doses and examined the role of autophagy in responding to this regimen. Fed male C57BL/6J mice were administered repeated doses (75 mg/kg and 150 mg/kg) of APAP, followed by measurement of adducts within the liver, mitochondria, and in plasma, activation of the MAP kinase JNK, and markers of liver injury. The role of autophagy was investigated by treatment of mice with the autophagy inhibitor, leupeptin. Our data show that multiple treatments at the 150 mg/kg dose of APAP resulted in protein adduct formation in the liver and mitochondria, activation of JNK, and hepatocyte cell death, which was significantly exacerbated by inhibition of autophagy. While repeated dosing with the milder 75 mg/kg dose did not cause mitochondrial protein adduct formation, JNK activation, or liver injury, autophagy inhibition resulted in hepatocyte death even at this lower dose. These data illustrate the importance of adaptive responses such as autophagy in removing protein adducts and preventing liver injury, especially in clinically relevant situations involving repeated dosing with APAP.

Delayed administration of N-acetylcysteine blunts recovery after an acetaminophen overdose unlike 4-methylpyrazole.

N-acetylcysteine (NAC) is the only clinically approved antidote against acetaminophen (APAP) hepatotoxicity. Despite its efficacy in patients treated early after APAP overdose, NAC has been implicated in impairing liver recovery in mice. More recently, 4-methylpyrazole (4MP, Fomepizole) emerged as a potential antidote in the mouse APAP hepatotoxicity model. The objective of this manuscript was to verify the detrimental effect of NAC and its potential mechanism and assess whether 4MP has the same liability. C57BL/6J mice were treated with 300 mg/kg APAP; 9 h after APAP and every 12 h after that, the animals received either 100 mg/kg NAC or 184.5 mg/kg 4MP. At 24 or 48 h after APAP, parameters of liver injury, mitochondrial biogenesis and cell proliferation were evaluated. Delayed NAC treatment had no effect on APAP-induced liver injury at 24 h but reduced the decline of plasma ALT activities and prevented the shrinkage of the areas of necrosis at 48 h. This effect correlated with down-regulation of key activators of mitochondrial biogenesis (AMPK, PGC-1α, Nrf1/2, TFAM) and reduced expression of Tom 20 (mitochondrial mass) and PCNA (cell proliferation). In contrast, 4MP attenuated liver injury at 24 h and promoted recovery at 48 h, which correlated with enhanced mitochondrial biogenesis and hepatocyte proliferation. In human hepatocytes, 4MP demonstrated higher efficacy in preventing cell death compared to NAC when treated at 18 h after APAP. Thus, due to the wider treatment window and lack of detrimental effects on recovery, it appears that at least in preclinical models, 4MP is superior to NAC as an antidote against APAP overdose.

A comprehensive weight of evidence assessment of published acetaminophen genotoxicity data: Implications for its carcinogenic hazard potential.

In 2019, the California Office of Environmental Health Hazard Assessment initiated a review of the carcinogenic hazard potential of acetaminophen, including an assessment of its genotoxicity. The objective of this analysis was to inform this review process with a weight-of-evidence assessment of more than 65 acetaminophen genetic toxicology studies that are of widely varying quality and conformance to accepted standards and relevance to humans. In these studies, acetaminophen showed no evidence of induction of point or gene mutations in bacterial and mammalian cell systems or in in vivo studies. In reliable, well-controlled test systems, clastogenic effects were only observed in unstable, p53-deficient cell systems or at toxic and/or excessively high concentrations that adversely affect cellular processes (e.g., mitochondrial respiration) and cause cytotoxicity. Across the studies, there was no clear evidence that acetaminophen causes DNA damage in the absence of toxicity. In well-controlled clinical studies, there was no meaningful evidence of chromosomal damage. Based on this weight-of-evidence assessment, acetaminophen overwhelmingly produces negative results (i.e., is not a genotoxic hazard) in reliable, robust high-weight studies. Its mode of action produces cytotoxic effects before it can induce the stable, genetic damage that would be indicative of a genotoxic or carcinogenic hazard.

Assessment of the biochemical pathways for acetaminophen toxicity: Implications for its carcinogenic hazard potential.

In 2019 California's Office of Environmental Health Hazard Assessment (OEHHA) initiated a review of the carcinogenic hazard potential of acetaminophen. In parallel with this review, herein we evaluated the mechanistic data related to the steps and timing of cellular events following therapeutic recommended (≤4 g/day) and higher doses of acetaminophen that may cause hepatotoxicity to evaluate whether these changes indicate that acetaminophen is a carcinogenic hazard. At therapeutic recommended doses, acetaminophen forms limited amounts of N-acetyl-p-benzoquinone-imine (NAPQI) without adverse cellular effects. Following overdoses of acetaminophen, there is potential for more extensive formation of NAPQI and depletion of glutathione, which may result in mitochondrial dysfunction and DNA damage, but only at doses that result in cell death - thus making it implausible for acetaminophen to induce the kind of stable, genetic damage in the nucleus indicative of a genotoxic or carcinogenic hazard in humans. The collective data demonstrate a lack of a plausible mechanism related to carcinogenicity and are consistent with rodent cancer bioassays, epidemiological results reviewed in companion manuscripts in this issue, as well as conclusions of multiple international health authorities.

Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)-Mixed Lineage Kinase Domain-Like Protein (MLKL)-Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers.

Increased hepatic ischemia-reperfusion (IR) injury in steatotic livers is a major reason for rejecting the use of fatty livers for liver transplantation. Necroptosis is implicated in the pathogenesis of fatty liver diseases. Necroptosis is regulated by three key proteins: receptor-interacting serine/threonine-protein kinase (RIPK)-1, RIPK3, and mixed-lineage kinase domain-like protein (MLKL). Here, we found that marked steatosis of the liver was induced when a Western diet was given in mice; steatosis was associated with the inhibition of hepatic proteasome activities and with increased levels of key necroptosis-related proteins. Mice fed a Western diet had more severe liver injury, as demonstrated by increases in serum alanine aminotransferase and necrotic areas of liver, after IR than did mice fed a control diet. Although hepatic steatosis was not different between Mlkl knockout mice and wild-type mice, Mlkl knockout mice had decreased hepatic neutrophil infiltration and inflammation and were protected from hepatic IR injury, irrespective of diet. Intriguingly, Ripk3 knockout or Ripk3 kinase-dead knock-in mice were protected against IR injury at the late phase but not the early phase, irrespective of diet. Overall, our findings indicate that liver steatosis exacerbates hepatic IR injury via increased MLKL-mediated necroptosis. Targeting MLKL-mediated necroptosis may help to improve outcomes in steatotic liver transplantation.