RESUMO
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.
RESUMO
Liver injury caused by acetaminophen (APAP) overdose is the leading cause of acute liver failure in western countries. The mode of APAP-induced cell death has been controversially discussed with ferroptosis emerging as a more recent hypothesis. Ferroptosis is characterized by ferrous iron-catalyzed lipid peroxidation (LPO) causing cell death, which can be prevented by the lipophilic antioxidants ferrostatin-1 and UAMC-3203. To assess the efficacy of these ferroptosis inhibitors, we used two murine models of APAP hepatotoxicity, APAP overdose alone or in combination with FeSO4 in fasted male C57BL/6J mice. APAP triggered severe liver injury in the absence of LPO measured as hepatic malondialdehyde (MDA) levels. In contrast, ferrous iron co-treatment aggravated APAP-induced liver injury and caused extensive LPO. Standard doses of ferrostatin-1 did not affect MDA levels or the injury in both models. In contrast, UAMC-3203 partially protected in both models and reduced LPO in the presence of ferrous iron. However, UAMC-3203 attenuated the translocation of phospho-JNK through downregulation of the mitochondrial anchor protein Sab resulting in reduced mitochondrial dysfunction and liver injury. Thus, APAP toxicity does not involve ferroptosis under normal conditions. The lack of effects of ferroptosis inhibitors in the pathophysiology indicates that ferroptosis signaling pathways are not relevant therapeutic targets.
Assuntos
Acetaminofen , Doença Hepática Induzida por Substâncias e Drogas , Modelos Animais de Doenças , Ferroptose , Camundongos Endogâmicos C57BL , Animais , Acetaminofen/efeitos adversos , Acetaminofen/toxicidade , Ferroptose/efeitos dos fármacos , Masculino , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Camundongos , Cicloexilaminas/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fenilenodiaminas/farmacologiaRESUMO
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.
Assuntos
Acetaminofen , Doença Hepática Induzida por Substâncias e Drogas , Regeneração Hepática , Fígado , Camundongos Endogâmicos C57BL , Trombopoetina , Animais , Acetaminofen/toxicidade , Masculino , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Trombopoetina/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Regeneração Hepática/efeitos dos fármacos , Camundongos , Acetilcisteína/farmacologia , Pirazóis/farmacologia , Hepatócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Receptores de Trombopoetina/metabolismo , Proliferação de Células/efeitos dos fármacosRESUMO
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.
RESUMO
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.
Assuntos
Acetaminofen , Doença Hepática Induzida por Substâncias e Drogas , Camundongos , Animais , Acetaminofen/toxicidade , Fator 2 Relacionado a NF-E2/metabolismo , Antídotos/farmacologia , Antídotos/uso terapêutico , Antídotos/metabolismo , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controleRESUMO
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.
Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Doença Hepática Induzida por Substâncias e Drogas , Masculino , Feminino , Animais , Camundongos , Neutrófilos , Acetaminofen/toxicidade , Camundongos Endogâmicos C57BL , Fígado , Necrose , Doença Hepática Induzida por Substâncias e Drogas/etiologiaRESUMO
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.
Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Doença Hepática Induzida por Substâncias e Drogas , Acetaminofen/toxicidade , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Doença Hepática Crônica Induzida por Substâncias e Drogas/metabolismo , Desferroxamina/farmacologia , Hepatócitos , Ferro/metabolismo , Peroxidação de Lipídeos , Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Minociclina/farmacologia , Mitocôndrias Hepáticas , Estresse Oxidativo , Ácido Peroxinitroso/farmacologiaRESUMO
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.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Células de Kupffer , Acetaminofen/metabolismo , Acetaminofen/toxicidade , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hepatócitos/metabolismo , Células de Kupffer/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-8BRESUMO
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.
Assuntos
Acetaminofen/intoxicação , Antídotos/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Acetilcisteína/farmacologia , Analgésicos não Narcóticos/intoxicação , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Overdose de Drogas , Fomepizol/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , CamundongosRESUMO
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.
Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Doença Hepática Induzida por Substâncias e Drogas , Células-Tronco Mesenquimais , Geleia de Wharton , Humanos , Camundongos , Animais , Acetaminofen/metabolismo , Acetilcisteína/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Butionina Sulfoximina/metabolismo , Butionina Sulfoximina/farmacologia , Fígado , Hepatócitos , Modelos Animais de Doenças , Fibroblastos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Acetaminofen/intoxicação , Acetilcisteína/farmacologia , Antídotos/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Fomepizol/farmacologia , Acetilcisteína/administração & dosagem , Animais , Antídotos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Overdose de Drogas/tratamento farmacológico , Fomepizol/administração & dosagem , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de TempoRESUMO
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.
Assuntos
Acetaminofen/toxicidade , Analgésicos não Narcóticos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Acetaminofen/administração & dosagem , Analgésicos não Narcóticos/administração & dosagem , Animais , Autofagia/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leupeptinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas/metabolismoRESUMO
Acetaminophen (APAP) hepatotoxicity is the most common cause of acute liver failure in the United States, and while a significant percentage of APAP overdose patients develop kidney injury, molecular mechanisms involved in APAP-induced nephrotoxicity are relatively unknown. We have shown that 4-methylpyrazole (4MP, Fomepizole) protects against APAP-induced liver injury by inhibiting reactive metabolite formation through Cyp2E1, and analysis of data from APAP overdose patients indicated that kidney dysfunction strongly correlated with severe liver injury. Since Cyp2E1 is also expressed in the kidney, this study explored protection by 4MP against APAP-induced nephrotoxicity. Male C57BL/6 J mice were treated with either 300 or 600 mg/kg APAP with or without 4MP for 2, 6 or 24 h, followed by measurement of APAP metabolism and tissue injury. Interestingly, levels of APAP and its non-oxidative metabolites were significantly higher in kidneys when compared to the liver. APAP-protein adducts were present in both tissues within 2 h, but were absent in kidney mitochondria, unlike in the liver. While GSH depletion was seen in both tissues, activation of c-jun N-terminal kinase and its translocation to the mitochondria, which is a critical feature of APAP-induced liver injury, was not detected in the kidney. Treatment with 4MP attenuated APAP oxidative metabolite generation, GSH depletion as well as kidney injury indicating its potential use in protection against APAP-induced nephrotoxicity. In conclusion, since reactive metabolite formation seems to be common in both liver and kidney, 4MP mediated inhibition of Cyp2E1 protects against APAP-induced nephrotoxicity. However, downstream mechanisms of APAP-induced nephrotoxicity seem distinct from the liver.
Assuntos
Acetaminofen/efeitos adversos , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/tratamento farmacológico , Fomepizol/uso terapêutico , Substâncias Protetoras/uso terapêutico , Injúria Renal Aguda/metabolismo , Adolescente , Adulto , Idoso , Animais , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Feminino , Glutationa/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Estudos Prospectivos , Substâncias Protetoras/farmacologia , Adulto JovemRESUMO
Though mitochondrial oxidant stress plays a critical role in the progression of acetaminophen (APAP) overdose-induced liver damage, the influence of mitochondrial bioenergetics on this is not well characterized. This is important, since lifestyle and diet alter hepatic mitochondrial bioenergetics and an understanding of its effects on APAP-induced liver injury is clinically relevant. Pyruvate dehydrogenase (PDH) is critical to mitochondrial bioenergetics, since it controls the rate of generation of reducing equivalents driving respiration, and pyruvate dehydrogenase kinase 4 (PDK4) regulates (inhibits) PDH by phosphorylation. We examined APAP-induced liver injury in PDK4-deficient (PDK4-/-) mice, which would have constitutively active PDH and hence elevated flux through the mitochondrial electron transport chain. PDK4-/- mice showed significant protection against APAP-induced liver injury when compared to wild type (WT) mice as measured by ALT levels and histology. Deficiency of PDK4 did not alter APAP metabolism, with similar APAP-adduct levels in PDK4-/- and WT mice, and no difference in JNK activation and translocation to mitochondria. However, subsequent amplification of mitochondrial dysfunction with release of mitochondrial AIF, peroxynitrite formation and DNA fragmentation were prevented. Interestingly, APAP induced a rapid decline in UCP2 protein levels in PDK4-deficient mice. These data suggest that adaptive changes in mitochondrial bioenergetics induced by enhanced respiratory chain flux in PDK4-/- mice render them highly efficient in handling APAP-induced oxidant stress, probably through modulation of UCP2 levels. Further investigation of these specific adaptive mechanisms would provide better insight into the control exerted by mitochondrial bioenergetics on cellular responses to an APAP overdose.
Assuntos
Acetaminofen/intoxicação , Doença Hepática Induzida por Substâncias e Drogas/patologia , Overdose de Drogas/complicações , Fígado/patologia , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Modelos Animais de Doenças , Overdose de Drogas/etiologia , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Proteína Desacopladora 2/metabolismoRESUMO
Acetaminophen (APAP) is one of the most popular and safe pain medications worldwide. However, due to its wide availability, it is frequently implicated in intentional or unintentional overdoses where it can cause severe liver injury and even acute liver failure (ALF). In fact, APAP toxicity is responsible for 46% of all ALF cases in the United States. Early mechanistic studies in mice demonstrated the formation of a reactive metabolite, which is responsible for hepatic glutathione depletion and initiation of the toxicity. This insight led to the rapid introduction of N-acetylcysteine as a clinical antidote. However, more recently, substantial progress was made in further elucidating the detailed mechanisms of APAP-induced cell death. Mitochondrial protein adducts trigger a mitochondrial oxidant stress, which requires amplification through a MAPK cascade that ultimately results in activation of c-jun N-terminal kinase (JNK) in the cytosol and translocation of phospho-JNK to the mitochondria. The enhanced oxidant stress is responsible for the membrane permeability transition pore opening and the membrane potential breakdown. The ensuing matrix swelling causes the release of intermembrane proteins such as endonuclease G, which translocate to the nucleus and induce DNA fragmentation. These pathophysiological signaling mechanisms can be additionally modulated by removing damaged mitochondria by autophagy and replacing them by mitochondrial biogenesis. Importantly, most of the mechanisms have been confirmed in human hepatocytes and indirectly through biomarkers in plasma of APAP overdose patients. The extensive necrosis caused by APAP overdose leads to a sterile inflammatory response. Although recruitment of inflammatory cells is necessary for removal of cell debris in preparation for regeneration, these cells have the potential to aggravate the injury. This review touches on the newest insight into the intracellular mechanisms of APAP-induced cells death and the resulting inflammatory response. Furthermore, it discusses the translation of these findings to humans and the emergence of new therapeutic interventions.
Assuntos
Acetaminofen , Analgésicos não Narcóticos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fígado/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Acetaminofen/farmacocinética , Acetaminofen/intoxicação , Analgésicos não Narcóticos/farmacocinética , Analgésicos não Narcóticos/intoxicação , Animais , Apoptose/efeitos dos fármacos , Humanos , Estresse Oxidativo/efeitos dos fármacosRESUMO
Acetaminophen (APAP)-induced liver injury is an important clinical and toxicological problem. Understanding the mechanisms and modes of cell death are vital for the development of therapeutic interventions. The histological and clinical features of APAP hepatotoxicity including cell and organelle swelling, karyolysis, and extensive cell contents release lead to the characterization of the cell death as oncotic necrosis. However, the more recent identification of detailed signaling mechanisms of mitochondrial dysfunction, the amplification mechanisms of mitochondrial oxidant stress and peroxynitrite formation by a mitogen-activated protein kinase cascade, mechanisms of the mitochondrial permeability transition pore opening and nuclear DNA fragmentation as well as the characterization of the sterile inflammatory response suggested that the mode of cell death is better termed programmed necrosis. Additional features like mitochondrial Bax translocation and cytochrome c release, mobilization of lysosomal iron and the activation of receptor-interacting protein kinases and the inflammasome raised the question whether other emerging modes of cell death such as apoptosis, necroptosis, ferroptosis and pyroptosis could also play a role. The current review summarizes the key mechanisms of APAP-induced liver injury and compares these with key features of the newly described modes of cell death. Based on the preponderance of experimental and clinical evidence, the mode of APAP-induced cell death should be termed programmed necrosis; despite some overlap with other modes of cell death, APAP hepatotoxicity does not fulfill the characteristics of either apoptosis, necroptosis, ferroptosis, pyroptosis or autophagic cell death.
Assuntos
Acetaminofen/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Ferroptose/efeitos dos fármacos , Ferroptose/fisiologia , Humanos , Necroptose/efeitos dos fármacos , Necroptose/fisiologiaRESUMO
We previously reported that delayed treatment with Mito-tempo (MT), a mitochondria-targeted superoxide dismutase mimetic, protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation. However, whether this protection is sustained to the late phase of toxicity is unknown. To investigate the late protection, C57Bl/6J mice were treated with 300 mg/kg APAP followed by 20 mg/kg MT 1.5 h or 3 h later. We found that both MT treatments protected against the late phase of APAP hepatotoxicity at 12 and 24 h. Surprisingly, MT-treated mice demonstrated a significant increase in apoptotic hepatocytes, while the necrotic phenotype was observed almost exclusively in mice treated with APAP alone. In addition, there was a significant increase in caspase-3 activity and cleavage in the livers of MT-treated mice. Immunostaining for active caspase-3 revealed that the positively stained hepatocytes were exclusively in centrilobular areas. Treatment with the pan-caspase inhibitor ZVD-fmk (10 mg/kg) 2 h post-APAP neutralized this caspase activation and provided additional protection against APAP hepatotoxicity. Treatment with N-acetylcysteine, the current standard of care for APAP poisoning, protected but did not induce this apoptotic phenotype. Mechanistically, MT treatment inhibited APAP-induced RIP3 kinase expression, and RIP3-deficient mice showed caspase activation and apoptotic morphology in hepatocytes analogous to MT treatment. These data suggest that while necrosis is the primary cause of cell death after APAP hepatotoxicity, treatment with the antioxidant MT may switch the mode of cell death to secondary apoptosis in some cells. Modulation of mitochondrial oxidative stress and RIP3 kinase expression play critical roles in this switch.
Assuntos
Acetaminofen/intoxicação , Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Compostos Organofosforados/farmacologia , Piperidinas/farmacologia , Acetilcisteína , Animais , Antioxidantes/farmacologia , Caspase 3/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Necrose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Cleistanthus collinus is a poisonous shrub used for deliberate self-harm in rural areas of South India and intake of boiled decoction of leaves is a common method of self-harm. Distal renal tubular acidosis (dRTA) is an important clinical symptom observed in C. collinus poisoning, and renal V-ATPases may be potential targets of damage. However, a lack of understanding of molecular mediators involved hampers medical management, which is mainly supportive. We hypothesized that C. collinus poisoning induces renal oxidative stress; probably by inducing mitochondrial uncoupling, which compromises V-ATPase activity to ultimately produce dRTA. This was tested by exposing renal BBMV, kidney cells in culture, and Wistar rats to C. collinus poisoning. Exposure to C. collinus aqueous extract resulted in significant elevations in the lipid peroxidation marker, conjugated dienes, in cell culture and in vivo. A significant decrease in mitochondrial respiratory control ratio was observed in kidneys from C. collinus-treated animals suggesting that mitochondrial oxidative phosphorylation is uncoupled. This was accompanied by significant increase in ADP levels and a decrease in proton pump activity. Thus, these results demonstrate that C. collinus poisoning induces oxidative stress which influences proton pump activity, probably due to feedback inhibition by elevated ADP levels because of mitochondrial dysfunction in the rat kidney.
Assuntos
Acidose Tubular Renal/induzido quimicamente , Euphorbiaceae/intoxicação , Rim/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/metabolismo , Acidose Tubular Renal/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Mitocôndrias Musculares/metabolismo , Fosforilação Oxidativa , Extratos Vegetais/intoxicação , Ratos WistarRESUMO
Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the US, and decades of intense study of its pathogenesis resulted in the development of the antidote N-acetylcysteine, which facilitates scavenging of the reactive metabolite and is the only treatment in clinical use. However, the narrow therapeutic window of this intervention necessitates a better understanding of the intricacies of APAP-induced liver injury for the development of additional therapeutic approaches that can benefit late-presenting patients. More recent investigations into APAP hepatotoxicity have established the critical role of mitochondrial dysfunction in mediating liver injury as well as clarified mechanisms of APAP-induced hepatocyte cell death. Thus, it is now established that mitochondrial oxidative and nitrosative stress is a key mechanistic feature involved in downstream signaling after APAP overdose. The identification of specific mediators of necrotic cell death further establishes the regulated nature of APAP-induced hepatocyte cell death. In addition, the discovery of the role of mitochondrial dynamics and autophagy in APAP-induced liver injury provides additional insight into the elaborate cell signaling mechanisms involved in the pathogenesis of this important clinical problem. In spite of these new insights into the mechanisms of liver injury, significant controversy still exists on the role of innate immunity in APAP-induced hepatotoxicity.