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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.
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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.
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Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Enfermedad Hepática Inducida por Sustancias y Drogas , Masculino , Femenino , Animales , Ratones , Neutrófilos , Acetaminofén/toxicidad , Ratones Endogámicos C57BL , Hígado , Necrosis , Enfermedad Hepática Inducida por Sustancias y Drogas/etiologíaRESUMEN
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.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Macrófagos del Hígado , Acetaminofén/metabolismo , Acetaminofén/toxicidad , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hepatocitos/metabolismo , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores de Interleucina-8BRESUMEN
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.
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Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Enfermedad Hepática Inducida por Sustancias y Drogas , Células Madre Mesenquimatosas , Gelatina de Wharton , Humanos , Ratones , Animales , Acetaminofén/metabolismo , Acetilcisteína/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Butionina Sulfoximina/metabolismo , Butionina Sulfoximina/farmacología , Hígado , Hepatocitos , Modelos Animales de Enfermedad , Fibroblastos , Ratones Endogámicos C57BLAsunto(s)
Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Enfermedad Hepática Inducida por Sustancias y Drogas , Acetaminofén/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Humanos , Hígado , Músculos , NecroptosisRESUMEN
INTRODUCTION: Acetaminophen (APAP) overdose is the leading cause of drug-induced liver injury and can cause a rapid progression to acute liver failure (ALF). Therefore, the identification of prognostic biomarkers to determine which patients will require a liver transplant is critical for APAP-induced ALF. AREAS COVERED: We begin by relating the mechanistic investigations in mouse models of APAP hepatotoxicity to the human APAP overdose pathophysiology. We draw insights from the established sequence of molecular events in mice to understand the progression of events in the APAP overdose patient. Through this mechanistic understanding, several new biomarkers, such as CXCL14, have recently been evaluated. We also explore how single-cell RNA sequencing, spatial transcriptomics, and other omics approaches have been leveraged for identifying novel biomarkers and how these approaches will continue to push the field of biomarker discovery forward. EXPERT OPINION: Recent investigations have elucidated several new biomarkers or combination of markers such as CXCL14, a regenerative miRNA signature, a cell death miRNA signature, hepcidin, LDH, CPS1, and FABP1. While these biomarkers are promising, they all require further validation. Larger cohort studies analyzing these new biomarkers in the same patient samples, while adding these candidate biomarkers to prognostic models will further support their clinical utility.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Fallo Hepático Agudo , MicroARNs , Humanos , Ratones , Animales , Acetaminofén/efectos adversos , Fallo Hepático Agudo/inducido químicamente , MicroARNs/genética , Biomarcadores , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Perfilación de la Expresión GénicaRESUMEN
Fifty percent of all acute liver failure (ALF) cases in the United States are due to acetaminophen (APAP) overdose. Assessment of canonical features of liver injury, such as plasma alanine aminotransferase activities are poor predictors of acute liver failure (ALF), suggesting the involvement of additional mechanisms independent of hepatocyte death. Previous work demonstrated a severe overdose of APAP results in impaired regeneration, the induction of senescence by p21, and increased mortality. We hypothesized that a discrete population of p21+ hepatocytes acquired a secretory phenotype that directly impedes liver recovery after a severe APAP overdose. Leveraging in-house human APAP explant liver and publicly available single-nuclei RNAseq data, we identified a subpopulation of p21+ hepatocytes enriched in a unique secretome of factors, such as CXCL14. Spatial transcriptomics in the mouse model of APAP overdose confirmed the presence of a p21+ hepatocyte population that directly surrounded the necrotic areas. In both male and female mice, we found a dose-dependent induction of p21 and persistent circulating levels of the p21-specific constituent, CXCL14, in the plasma after a severe APAP overdose. In parallel experiments, we targeted either the putative senescent hepatocytes with the senolytic drugs, dasatinib and quercetin, or CXCL14 with a neutralizing antibody. We found that targeting CXCL14 greatly enhanced liver recovery after APAP-induced liver injury, while targeting senescent hepatocytes had no effect. These data support the conclusion that the sustained induction of p21 in hepatocytes with persistent CXCL14 secretion are critical mechanistic events leading to ALF in mice and human patients.
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Acetaminofén , Enfermedad Hepática Inducida por Sustancias y Drogas , Quimiocinas CXC , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Hepatocitos , Ratones Endogámicos C57BL , Acetaminofén/toxicidad , Animales , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Masculino , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Ratones , Quimiocinas CXC/metabolismo , Quimiocinas CXC/genética , Regeneración Hepática/efectos de los fármacos , Sobredosis de Droga , Analgésicos no Narcóticos/toxicidadRESUMEN
The adenosine A2B receptor (A2BAR) is a member of a family of G-protein coupled receptors (GPCRs), which has a low affinity for adenosine and is now implicated in several pathophysiological conditions. We have demonstrated the beneficial effects of A2BAR activation in enhancing recovery after acute liver injury induced by an acetaminophen (APAP) overdose. While receptor trafficking within the cell is recognized to play a role in GPCR signaling, its role in the mediation of A2BAR effects in the context of APAP-induced liver injury is not well understood. This was investigated here, where C57BL/6J mice were subjected to an APAP overdose (300 mg/kg), and the temporal course of A2BAR intracellular localization was examined. The impact of A2BAR activation or inhibition on trafficking was examined by utilizing the A2BAR agonist BAY 60-6583 or antagonist PSB 603. The modulation of A2BAR trafficking via APAP-induced cell signaling was explored by using 4-methylpyrazole (4MP), an inhibitor of Cyp2E1 and JNK activation. Our results indicate that APAP overdose induced the translocation of A2BAR to mitochondria, which was prevented via 4MP treatment. Furthermore, we demonstrated that A2BAR is localized on the mitochondrial outer membrane and interacts with progesterone receptor membrane component 1 (PGRMC1). While the activation of A2BAR enhanced mitochondrial localization, its inhibition decreased PGRMC1 mitochondria levels and blunted mitochondrial Cyp2E1 activity. Thus, our data reveal a hitherto unrecognized consequence of A2BAR trafficking to mitochondria and its interaction with PGRMC1, which regulates mitochondrial Cyp2E1 activity and modulates APAP-induced liver injury.
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Acetaminophen (APAP) is a widely used pain reliever that can cause liver injury or liver failure in response to an overdose. Understanding the mechanisms of APAP-induced cell death is critical for identifying new therapeutic targets. In this respect it was hypothesized that hepatocytes die by oncotic necrosis, apoptosis, necroptosis, ferroptosis and more recently pyroptosis. The latter cell death is characterized by caspase-dependent gasdermin cleavage into a C-terminal and an N-terminal fragment, which forms pores in the plasma membrane. The gasdermin pores can release potassium, interleukin-1ß (IL-1ß), IL-18, and other small molecules in a sublytic phase, which can be the main function of the pores in certain cell types such as inflammatory cells. Alternatively, the process can progress to full lysis of the cell (pyroptosis) with extensive cell contents release. This review discusses the experimental evidence for the involvement of pyroptosis in APAP hepatotoxicity as well as the arguments against pyroptosis as a relevant mechanism of APAP-induced cell death in hepatocytes. Based on the critical evaluation of the currently available literature and understanding of the pathophysiology, it can be concluded that pyroptotic cell death is unlikely to be a relevant contributor to APAP-induced liver injury.
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Peripheral neuropathy (PN), a debilitating complication of diabetes, is associated with obesity and the metabolic syndrome in nondiabetic individuals. Evidence indicates that a high fat diet can induce signs of diabetic peripheral PN in mice but the pathogenesis of high fat diet-induced PN remains unknown. PURPOSE: Determine if neuronal inflammation is associated with the development of mechanical hypersensitivity and nerve fiber changes in high fat fed mice. METHODS: Male C57Bl/6 mice were randomized to a standard (Std, 15% kcal from fat) or high fat diet (HF, 54% kcal from fat) for 2, 4, or 8 weeks (n = 11-12 per group). Lumbar dorsal root ganglia were harvested and inflammatory mediators (IL-1α, IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17, MCP-1, IFN-γ, TNF-α, MIP-1α, GMCSF, RANTES) were quantified. Hindpaw mechanical sensitivity was assessed using the von Frey test. Intraepidermal nerve fiber density (IENFD) and TrkA nerve fiber density were quantified via immunohistochemistry. RESULTS: After 8 weeks, HF had greater body mass (33.3 ± 1.0 vs 26.7 ± 0.5 g, p < 0.001), fasting blood glucose (160.3 ± 9.4 vs 138.5 ± 3.4 mg/dl, p < 0.05) and insulin (3.58 ± 0.46 vs 0.82 ± 0.14 ng/ml, p < 0.001) compared to Std. IL-1α, RANTES and IL-5 were higher in HF compared to Std after 2 and 4 weeks, respectively (IL-1α: 4.8 ± 1.3 vs 2.9 ± 0.6 pg/mg, p < 0.05; RANTES: 19.6 ± 2.2 vs 13.3 ± 1.2 pg/mg p < 0.05; IL-5: 5.8 ± 0.7 vs 3.1 ± 0.5 pg/mg, p < 0.05). IENFD and TrkA fiber density were also higher in HF vs Std after 4 weeks (IENFD: 39.4 ± 1.2 vs 32.2 ± 1.3 fibers/mm, p < 0.001; TrkA: 30.4 ± 1.8 vs 22.4 ± 1.3 fibers/mm). There were no significant differences in hindpaw sensitivity for Std vs HF. CONCLUSION: Increased inflammatory mediators preceded and accompanied an increase in cutaneous pain sensing nerve fibers in high fat fed mice but was not accompanied by significant mechanical allodynia. Diets high in fat may increase neuronal inflammation and lead to increased nociceptive nerve fiber density.
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Acetaminophen (APAP) overdose is the main cause of acute liver failure in Western countries. The mechanism of APAP hepatotoxicity is associated with centrilobular necrosis which initiates infiltration of neutrophils, monocytes, and other leukocytes to the area of necrosis. Although it has been recognized that this infiltration of immune cells plays a critical role in promoting liver repair, mechanism of immune cell clearance that is important for resolution of inflammation and the return to normal homeostasis are not well characterized. CXCR4 is a chemokine receptor expressed on hepatocytes as well as neutrophils, monocytes, and hematopoietic stem cells. CXCR4 function is dependent on its selective expression on different cell types and thus can vary depending on the pathophysiology. This study aimed to investigate the crosstalk between hepatocytes and macrophages through CXCR4 to promote macrophage apoptosis after APAP overdose. C57BL/6J mice were subjected to APAP overdose (300 mg/kg). Flow cytometry and immunohistochemistry were used to determine the mode of cell death of macrophages and expression pattern of CXCR4 during the resolution phase of APAP hepatotoxicity. The impact of CXCR4 in regulation of macrophage apoptosis and liver recovery was assessed after administration of a monoclonal antibody against CXCR4. RNA sequencing analysis was performed on flow cytometry sorted CXCR4+ macrophages at 72 h to confirm the apoptotic cell death of macrophages. Our data indicate that the inflammatory response is resolved by recovering hepatocytes through induction of CXCR4 on macrophages, which triggers their cell death by apoptosis at the end of the recovery phase.
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Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Enfermedad Hepática Inducida por Sustancias y Drogas , Acetaminofén/metabolismo , Acetaminofén/toxicidad , Animales , Apoptosis , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Necrosis/metabolismoRESUMEN
BACKGROUND & AIMS: The liver has a unique capacity to regenerate after injury in a highly orchestrated and regulated manner. Here, we report that O-GlcNAcylation, an intracellular post-translational modification regulated by 2 enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a critical termination signal for liver regeneration following partial hepatectomy (PHX). METHODS: We studied liver regeneration after PHX on hepatocyte specific OGT and OGA knockout mice (OGT-KO and OGA-KO), which caused a significant decrease (OGT-KO) and increase (OGA-KO) in hepatic O-GlcNAcylation, respectively. RESULTS: OGA-KO mice had normal regeneration, but the OGT-KO mice exhibited substantial defects in termination of liver regeneration with increased liver injury, sustained cell proliferation resulting in significant hepatomegaly, hepatic dysplasia, and appearance of small nodules at 28 days after PHX. This was accompanied by a sustained increase in expression of cyclins along with significant induction in pro-inflammatory and pro-fibrotic gene expression in the OGT-KO livers. RNA-sequencing studies revealed inactivation of hepatocyte nuclear 4 alpha (HNF4α), the master regulator of hepatic differentiation and a known termination signal, in OGT-KO mice at 28 days after PHX, which was confirmed by both Western blot and immunohistochemistry analysis. Furthermore, a significant decrease in HNFα target genes was observed in OGT-KO mice, indicating a lack of hepatocyte differentiation following decreased hepatic O-GlcNAcylation. Immunoprecipitation experiments revealed HNF4α is O-GlcNAcylated in normal differentiated hepatocytes. CONCLUSIONS: These studies show that O-GlcNAcylation plays a critical role in the termination of liver regeneration via regulation of HNF4α in hepatocytes.
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Hepatocitos , Regeneración Hepática , Animales , Hepatectomía , Hepatocitos/metabolismo , Hígado/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Extracellular vesicles (EV) are defined as nanosized particles, with a lipid bilayer, that are unable to replicate. There has been an exponential increase of research investigating these particles in a wide array of diseases and deleterious states (inflammation, oxidative stress, drug-induced liver injury) in large part due to increasing recognition of the functional capacity of EVs. Cells can package lipids, proteins, miRNAs, DNA, and RNA into EVs and send these discrete packages of molecular information to distant, recipient cells to alter the physiological state of that cell. EVs are innately heterogeneous as a result of the diverse molecular pathways that are used to generate them. However, this innate heterogeneity of EVs is amplified due to the diversity in isolation techniques and lack of standardized nomenclature in the literature making it unclear if one scientist's "exosome" is another scientist's "microvesicle." One goal of this chapter is to provide the contextual understanding of EV origin so one can discern between divergent nomenclature. Further, the chapter will explore the potential protective and harmful roles that EVs play in DILI, and the potential of EVs and their cargo as a biomarker. The use of EVs as a therapeutic as well as a vector for therapeutic delivery will be discussed.
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Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Vesículas Extracelulares/metabolismo , Biomarcadores/metabolismo , HumanosRESUMEN
Introduction: Liver injury induced by drugs is a serious clinical problem. Many circulating biomarkers for identifying and predicting drug-induced liver injury (DILI) have been proposed.Areas covered: Biomarkers are mainly predicated on the mechanistic understanding of the underlying DILI, often in the context of acetaminophen overdose. New panels of biomarkers have emerged that are related to recovery/regeneration rather than injury following DILI. We explore the clinical relevance and limitations of these new biomarkers including recent controversies. Extracellular vesicles have also emerged as a promising vector of biomarkers, although the biological role for EVs may limit their clinical usefulness. New technological approaches for biomarker discovery are also explored.Expert opinion: Recent clinical studies have validated the efficacy of some of these new biomarkers, cytokeratin-18, macrophage colony-stimulating factor receptor, and osteopontin for DILI prognosis. Low prevalence of DILI is an inherent limitation to DILI biomarker development. Furthering mechanistic understanding of DILI and leveraging technological advances (e.g. machine learning/omics) is necessary to improve upon the newest generation of biomarkers. The integration of omics approaches with machine learning has led to novel insights in cancer research and DILI research is poised to leverage these technologies for biomarker discovery and development.
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Acetaminofén/efectos adversos , Analgésicos no Narcóticos/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Hígado/metabolismo , Animales , Biomarcadores/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Pruebas de Función Hepática , Regeneración Hepática , Aprendizaje Automático , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/patología , Valor Predictivo de las PruebasRESUMEN
An acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States. A hallmark characteristic of APAP hepatotoxicity is centrilobular necrosis. General, innate mechanisms such as lower amounts of GSH and higher cytochrome P450 2e1 expression in pericentral (PC) hepatocytes are known to contribute to the differences in susceptibility to cell injury between periportal (PP) hepatocytes and PC hepatocytes. Although a sequence of molecular events involving formation of the reactive metabolite N-acetyl-p-benzoquinone imine, GSH depletion, oxidative stress, and c-Jun N-terminal kinase activation define the early cell stress trajectory following APAP exposure, their activation in PC versus PP hepatocytes is not well characterized. By using single-cell RNA-sequencing, we provide the first reconstruction of the early transcriptomic APAP liver lobule after validation of our methodology using human liver single-cell RNA-sequencing data. Two hours after APAP treatment, we find that PP hepatocytes progress along the APAP stress axis to oxidative stress, before resolving injury due to innate and adaptive mechanisms. However, PC hepatocytes continue along this stress axis as indicated by activation of mitogen-activated protein kinase genes, which is absent in PP hepatocytes. We also identify a population of glutamine synthetase enriched PC hepatocytes in close proximity to the central vein, where a stepwise induction of a stress program culminated in cell death. Collectively, these findings elucidate a molecular sequence of events distinguishing the differential response to APAP exposure between PP and PC hepatocytes and identify a subset of uniquely susceptible PC hepatocytes.
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Acetaminofén , Enfermedad Hepática Inducida por Sustancias y Drogas , Acetaminofén/metabolismo , Acetaminofén/toxicidad , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Glutatión/metabolismo , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , ARN/metabolismo , TranscriptomaRESUMEN
Mitochondria have been studied for decades from the standpoint of metabolism and ATP generation. However, in recent years mitochondrial dynamics and its influence on bioenergetics and cellular homeostasis is also being appreciated. Mitochondria undergo regular cycles of fusion and fission regulated by various cues including cellular energy requirements and pathophysiological stimuli, and the network of critical proteins and membrane lipids involved in mitochondrial dynamics is being revealed. Hepatocytes are highly metabolic cells which have abundant mitochondria suggesting a biologically relevant role for mitochondrial dynamics in hepatocyte injury and recovery. Here we review information on molecular mediators of mitochondrial dynamics and their alteration in drug-induced liver injury. Based on current information, it is evident that changes in mitochondrial fusion and fission are hallmarks of liver pathophysiology ranging from acetaminophen-induced or cholestatic liver injury to chronic liver diseases. These alterations in mitochondrial dynamics influence multiple related mitochondrial responses such as mitophagy and mitochondrial biogenesis, which are important adaptive responses facilitating liver recovery in several contexts, including drug-induced liver injury. The current focus on characterization of molecular mechanisms of mitochondrial dynamics is of immense relevance to liver pathophysiology and have the potential to provide significant insight into mechanisms of liver recovery and regeneration after injury.
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Mitochondrial morphology plays a critical role in regulating mitochondrial and cellular function. It is well established that oxidative stress and mitochondrial injury are central to acetaminophen (APAP) hepatotoxicity. However, the role of mitochondrial dynamics, namely the remodeling of mitochondrial morphology through fusion and fission, has largely gone unexplored. To investigate this, we used primary mouse hepatocytes treated with APAP which allowed for real-time visualization of mitochondrial morphology using mitotracker green. We found that alterations in mitochondrial morphology were dose dependent, with a biphasic response in mitochondrial shape at higher APAP doses. Importantly, these two distinct mitochondrial morphologies corresponded with differences in mitochondrial respiratory function and polarization. The early change in mitochondrial morphology can be reversible and appears to be an adaptive response caused by alterations in membrane potential, which ultimately help preserve mitochondrial function. The later delayed change in mitochondrial morphology is irreversible and is driven by loss of mitochondrial membrane potential, decreased canonical fusion proteins, and alterations in mitochondrial lipid composition. Collectively, these later changes tilt the scales toward mitochondrial fission resulting in fragmented mitochondria with reduced functionality. This work provides evidence of adaptive early changes in mitochondrial morphology, which results in functional consequences that are dictated by the severity of APAP overdose.
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Acetaminofén , Enfermedad Hepática Inducida por Sustancias y Drogas , Acetaminofén/metabolismo , Acetaminofén/toxicidad , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hepatocitos/metabolismo , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Mitocondrias , Mitocondrias Hepáticas/metabolismo , Estrés OxidativoRESUMEN
Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States and formation of APAP-protein adducts, mitochondrial oxidant stress and activation of the mitogen activated protein (MAP) kinase c-jun N-terminal kinase (JNK) are critical for APAP-induced cell death. However, direct evidence linking these mechanistic features are lacking and were investigated by examining the early temporal course of these changes in mice after 300 mg/kg APAP. Protein adducts were detectable in the liver (0.05-0.1 nmol/mg protein) by 15 and 30 min after APAP, which increased (>500 %) selectively in mitochondria by 60 min. Cytosolic JNK activation was only evident at 60 min, and was significantly attenuated by scavenging superoxide specifically in the cytosol by TEMPO treatment. Treatment of mouse hepatocytes with APAP revealed mitochondrial superoxide generation within 15 min, accompanied by hydrogen peroxide production without change in mitochondrial respiratory function. The oxidant stress preceded JNK activation and its mitochondrial translocation. Inhibitor studies identified the putative source of mitochondrial superoxide as complex III, which released superoxide towards the intermembrane space after APAP resulting in activation of JNK in the cytosol. Our studies provide direct evidence of mechanisms involved in mitochondrial superoxide generation after NAPQI-adduct formation and its activation of the MAP kinase cascade in the cytosol, which are critical features of APAP hepatotoxicity.
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Acetaminofén , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Citosol/enzimología , Sobredosis de Droga , Hepatocitos/enzimología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Mitocondrias Hepáticas/enzimología , Proteínas Mitocondriales/metabolismo , Superóxidos/metabolismo , Animales , Benzoquinonas/metabolismo , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Modelos Animales de Enfermedad , Activación Enzimática , Hepatocitos/patología , Iminas/metabolismo , Masculino , Ratones Endogámicos C57BL , Mitocondrias Hepáticas/patología , Estrés Oxidativo , Transporte de Proteínas , Factores de TiempoRESUMEN
Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses. The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology. As a result, this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions. However, the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic. However, many studies using this model are flawed, which jeopardizes the scientific and clinical relevance. The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained. The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism, mitochondrial dysfunction, necrotic cell death, autophagy and the sterile inflammatory response. In addition, the most frequently made mistakes when using this model are discussed. Thus, considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions.
RESUMEN
Liver injury and acute liver failure caused by acetaminophen (APAP, N-acetyl-p-aminophenol, paracetamol) overdose is a significant clinical problem in most western countries. The only clinically approved antidote is N-acetylcysteine (NAC), which promotes the recovery of hepatic GSH. If administered during the metabolism phase, GSH scavenges the reactive metabolite N-acetyl-p-benzoquinone imine. More recently, it was shown that NAC can also reconstitute mitochondrial GSH levels and scavenge reactive oxygen/peroxynitrite and can support mitochondrial bioenergetics. However, NAC has side effects and may not be efficacious after high overdoses. Repurposing of additional drugs based on their alternate mechanisms of action could be a promising approach. 4-Methylpyrazole (4MP) was shown to be highly effective against APAP toxicity by inhibiting cytochrome P450 enzymes in mice and humans. In addition, 4MP is a potent c-Jun N-terminal kinase inhibitor expanding its therapeutic window. Calmangafodipir (CMFP) is a SOD mimetic, which is well tolerated in patients and has the potential to be effective after severe overdoses. Other drugs approved for humans such as metformin and methylene blue were shown to be protective in mice at high doses or at human therapeutic doses, respectively. Additional protective strategies such as enhancing antioxidant activities, Nrf2-dependent gene induction and autophagy activation by herbal medicine components are being evaluated. However, at this point, their mechanistic insight is limited, and the doses used are high. More rigorous mechanistic studies are needed to advance these herbal compounds. Nevertheless, based on recent studies, 4-methylpyrazole and calmangafodipir have realistic prospects to become complimentary or even alternative antidotes to NAC for APAP overdose.