APAP Metabolism Research Articles

Deciphering Acetaminophen-Induced Hepatotoxicity: The Crucial Role of Transcription Factors like Nuclear Factor Erythroid 2-Related Factor 2 as Genetic Determinants of Susceptibility to Drug-Induced Liver Injury.

Acetaminophen (APAP) is the most commonly used over-the-counter medication throughout the world. At therapeutic doses, APAP has potent analgesic and antipyretic effects. The efficacy and safety of APAP are influenced by multifactorial processes dependent upon dosing, namely frequency and total dose. APAP poisoning by repeated ingestion of supratherapeutic doses, depletes glutathione stores in the liver and other organs capable of metabolic bioactivation, leading to hepatocellular death due to exhausted antioxidant defenses. Numerous genes, encompassing transcription factors and signaling pathways, have been identified as playing pivotal roles in APAP toxicity, with the liver being the primary organ studied due to its central role in APAP metabolism and injury. Nuclear factor erythroid 2-related factor 2 (NRF2) and its array of downstream responsive genes are crucial in counteracting APAP toxicity. NRF2, along with its negative regulator Kelch-like ECH-associated protein 1, plays a vital role in regulating intracellular redox homeostasis. This regulation is significant in modulating the oxidative stress, inflammation, and hepatocellular death induced by APAP. In this review, we provide an updated overview of the mechanisms through which NRF2 activation and signaling critically influence the threshold for developing APAP toxicity. We also describe how genetically modified rodent models for NRF2 and related genes have been pivotal in underscoring the significance of this antioxidant response pathway. While NRF2 is a primary focus, the article comprehensively explores other genetic factors involved in phase I and phase II metabolism of APAP, inflammation, oxidative stress, and related pathways that contribute to APAP toxicity, thereby providing a holistic understanding of the genetic landscape influencing susceptibility to this condition. SIGNIFICANCE STATEMENT: This review summarizes the genetic elements and signaling pathways underlying APAP-induced liver toxicity, focusing on the crucial protective role of the transcription factor NRF2. This review also delves into the genetic intricacies influencing APAP safety and potential liver harm. It also emphasizes the need for deeper insight into the molecular mechanisms of hepatotoxicity, especially the interplay of NRF2 with other pathways.

Identification of acetaminophen degrading microorganisms in mixed microbial communities using 13C-DNA stable isotope probing

The high frequency of acetaminophen (APAP) detection in water bodies around the world increases the risk to the global environment. A lot of work has concentrated on isolating and identifying the culturable APAP degraders for understanding of the microbiota and their ecophysiology, which greatly underrated the diversity of organisms. Here, 13C-DNA stable isotope probing (DNA-SIP) was applied for the first time to identify microorganisms involved in APAP metabolism under conditions close to in situ from three mixed microbial communities collected in lab-scale nitrification systems. Based on DNA-SIP, Achromobacter (oligotype ATAA), Alicycliphilus (oligotype GTTCG) and Thauera (oligotype GAAACTTTCA) were identified as the key APAP degrading bacteria in the floc sludge, granular sludge and biofilm reactors, respectively, with the relative abundance of 43.20%, 10.12% and 12.60%. Moreover, Leucobacter, Achromobacter, Alicycliphilus, Thauera, Microbacterium and Lactobacillus were found, for the first time, to be directly responsible for APAP biodegradation. However, Achromobacter, Lactobacillus and Alicycliphilus were linked to spread resistance genes (intI1, sul2, qacEdta1-02 and qacH-01). The results highlight that Acinetobacter, Leucobacter, Thauera and Microbacterium could be recommended as bioaugmentation strains for the treatment of wastewater contaminated with APAP. Ultimately, the information acquired in this study could add to the current knowledge on microorganisms degrading APAP and accelerate the implementation of biofortification processes to achieve APAP removal.

Lack of mitochondrial Cyp2E1 drives acetaminophen-induced ER stress-mediated apoptosis in mouse and human kidneys: Inhibition by 4-methylpyrazole but not N-acetylcysteine

Acetaminophen (APAP) overdose causes liver injury and acute liver failure, as well as acute kidney injury, which is not prevented by the clinical antidote N-acetyl-L-cysteine (NAC). The absence of therapeutics targeting APAP-induced nephrotoxicity is due to gaps in understanding the mechanisms of renal injury. APAP metabolism through Cyp2E1 drives cell death in both the liver and kidney. We demonstrate that Cyp2E1 is localized to the proximal tubular cells in mouse and human kidneys. Virtually all the Cyp2E1 in kidney cells is in the endoplasmic reticulum (ER), not in mitochondria. By contrast, hepatic Cyp2E1 is in both the ER and mitochondria of hepatocytes. Consistent with this subcellular localization, a dose of 600 mg/kg APAP in fasted C57BL/6J mice induced the formation of APAP protein adducts predominantly in mitochondria of hepatocytes, but the ER of the proximal tubular cells of the kidney. We found that reactive metabolite formation triggered ER stress-mediated activation of caspase-12 and apoptotic cell death in the kidney. While co-treatment with 4-methylpyrazole (4MP; fomepizole) or the caspase inhibitor Ac-DEVD-CHO prevented APAP-induced cleavage of procaspase-12 and apoptosis in the kidney, treatment with NAC had no effect. These mechanisms are clinically relevant because 4MP but not NAC also significantly attenuated APAP-induced apoptotic cell death in primary human kidney cells. We conclude that reactive metabolite formation by Cyp2E1 in the ER results in sustained ER stress that causes activation of procaspase-12, triggering apoptosis of proximal tubular cells, and that 4MP but not NAC may be an effective antidote against APAP-induced kidney 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.

CXCL5 Promotes Acetaminophen-Induced Hepatotoxicity by Activating Kupffer Cells.

Kupffer cells (KCs) play a key part in the pathological process of acetaminophen (APAP)-induced acute liver injury (ALI), the leading cause of acute liver failure in the world. CXC motif chemokine ligand 5 (CXCL5) exerts proinflammatory effects in acute respiratory distress syndrome and arthritis. In the current study, we aim to reveal the effects of CXCL5 on the activation of KCs and the role of CXCL5 in the pathogenesis of APAP-induced hepatotoxicity. The in vivo study, conducted on mice intraperitoneally injected with APAP (300 mg/kg) to establish the ALI model and then treated with Anti-CXCL5 mAb at 30 min and 12 h after the APAP challenge, showed that CXCL5 expression significantly increased in injured livers, and Anti-CXCL5 mAb mitigated the degree of APAP-evoked ALI in mice which was proven through biochemicals and histological examination. Also, neutralization of CXCL5 had no significant effect on APAP metabolism in the liver but exhibited anti-inflammatory effects and ameliorated hepatocellular death in the injured liver. The in vitro data displayed that recombinant mouse CXCL5 treatment promoted APAP-induced cellular toxicity in primary hepatocytes co-cultured with KCs, compared with single-cultured hepatocytes. Consistent with the result, we found that the Anti-CXCL5 mAb gradient decreased LPS-induced expression of inflammatory cytokines in single-cultured KCs. Therefore, CXCL5 could stimulate KCs to produce inflammatory mediators, therefore damaging hepatocytes from APAP toxicity.

Analysis of AT7519 as a pro-resolution compound in an acetaminophen-induced mouse model of acute inflammation by UPLC-MS/MS

BackgroundUncontrolled inflammation contributes to the progression of organ damage in acute conditions, such as acetaminophen-induced acute liver injury (APAP-ALI) and there are limited treatments for this condition. AT7519, a cyclic-dependent kinase inhibitor (CDKI), has been used successfully in several conditions, to resolve inflammation and return tissue homeostatic functions. AT7519 has not been assessed in APAP-ALI and its effect on APAP metabolism is unknown. Targeted chromatography and mass spectrometry can be used to assess multiple compounds simultaneously and this approach has not been applied yet to measure APAP and AT7519 in a mouse model.ResultsWe show an optimised simple and sensitive LC–MS/MS method for determining concentrations of AT7519 and APAP in low volumes of mouse serum. Using positive ion mode electrospray ionisation, separation of AT7519 and APAP and their corresponding isotopically labelled internal standards [2H]8-AT16043M (d8-AT7519) and [2H]8-APAP (d4-APAP), was achieved on an Acquity UPLC BEH C18 column (100 × 2.1 mm; 1.7μm). A gradient mobile phase system of water and methanol was delivered at a flow rate of 0.5 mL/min with a run time of 9 min. Calibration curves were linear, intra-day and inter-day precision and accuracy were acceptable and the covariates of all standards and quality control replicates were less than 15%. The method was successfully applied to evaluate AT7519 and APAP levels 20 h post AT7519 (10 mg/mg) in C57Bl6J wild type mouse serum treated with either vehicle or APAP. Serum AT7519 was significantly higher in mice that had received APAP compared to control, but there was no correlation between APAP and AT7519 quantification. There was also no correlation of AT7519 and hepatic damage or proliferation markers.ConclusionWe optimised an LC–MS/MS method to quantify both AT7519 and APAP in mouse serum (50 µL), using labelled internal standards. Application of this method to a mouse model of APAP toxicity proved effective in accurately measuring APAP and AT7519 concentrations after i.p. dosing. AT7519 was significantly higher in mice with APAP toxicity, indicating hepatic metabolism of this CDKI, but there was no correlation with markers of hepatic damage or proliferation, demonstrating that this dose of AT7519 (10 mg/kg) does not contribute to hepatic damage or repair. This optimised method can be used for future investigations of AT7519 in APAP in mice.

8-methoxypsoralen protects against acetaminophen-induced liver injury by antagonising Cyp2e1 in mice

This study aimed to investigate the effect and mechanism of 8-methoxypsoralen (8-MOP) on acetaminophen (APAP)-induced hepatotoxicity in mice. The study found that 1 h after intraperitoneal injection of 300 mg/kg APAP, treatment with 40 mg/kg, 80 mg/kg and 120 mg/kg 8-MOP could reduce serum transaminase level and histopathological liver necrosis area. Elevated mRNA expression of liver inflammatory mediators caused by excessive APAP was also reversed. 8-MOP significantly reduced APAP-induced hepatotoxicity dose-dependently, and the highest therapeutic dose of 8-MOP (120 mg/kg) had no harmful effects on the liver. Cocktail probe assay revealed that 8-MOP can inhibit Cyp2e1 enzymatic activities of mice, thereby reducing the production of acetaminophen-cysteine (APAP-CYS), a toxic metabolite of APAP. 8-MOP had no significant effect on the protein and gene expression of Cyp2e1. The three-dimensional structures of mouse Cyp2e1 were constructed by homologous modeling. Molecular docking showed that 8-MOP had a good binding effect on the enzyme activity site of Cyp2e1. In summary, 8-MOP dose-dependently attenuated APAP-induced hepatotoxicity by binding to Cyp2e1 and occupying the active center of the enzyme, thus competitively inhibiting the oxidative metabolism of APAP, and reducing the generation of toxic product APAP-CYS.

Nuciferine Effectively Protects Mice against Acetaminophen-Induced Liver Injury.

Acetaminophen (APAP) overdose still poses a major clinical challenge and is a leading cause of acute liver injury (ALI). N-acetylcysteine (NAC) is the only approved antidote to treat APAP toxicity while NAC therapy can trigger side effects including severe vomiting and even shock. Thus, new insights in developing novel therapeutic drugs may pave the way for better treatment of APAP poisoning. Previous research has reported that nuciferine (Nuci) possesses anti-inflammatory and antioxidant properties. Therefore, the objective of this study was proposed to investigate the hepatoprotective effects of Nuci and explore its underlying mechanisms. Mice were intraperitoneally (i.p.) administered with APAP (300 mg/kg) and subsequently injected with Nuci (25, 50, and 100 mg/kg, i.p.) at 30 min after APAP overdose. Then, all mice were sacrificed at 12 h after APAP challenge for further analysis. Nuci-treated mice did not show any side effects and our results revealed that treating Nuci significantly attenuated APAP-induced ALI, as confirmed by histopathological examinations, biochemical analysis, and diminished hepatic oxidative stress and inflammation. The in silico prediction and mRNA-sequencing analysis were performed to explore the underlying mechanisms of Nuci. GO and KEGG enrichment of the predicted target proteins of Nuci includes reactive oxygen species, drug metabolism of cytochrome P450 (CYP450) enzymes, and autophagy. Furthermore, the mRNA-sequencing analyses indicated that Nuci can regulate glutathione metabolic processes and anti-inflammatory responses. Consistently, we found that Nuci increased the hepatic glutathione restoration but decreased APAP protein adducts in damaged livers. Western blot analysis further confirmed that Nuci effectively promoted hepatic autophagy in APAP-treated mice. However, Nuci could not affect the expression levels of the main CYP450 enzymes (CYP1A2, CYP2E1, and CYP3A11). These results demonstrated that Nuci may be a potential therapeutic drug for APAP-induced ALI via amelioration of the inflammatory response and oxidative stress, regulation of APAP metabolism, and activation of autophagy.

Absence of Increased Susceptibility to Acetaminophen-Induced Liver Injury in a Diet-Induced NAFLD Mouse Model.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease and its influence on drug-induced liver injury (DILI) is not fully understood. We investigated whether NAFLD can influence acetaminophen (APAP [N-acetyl-p-aminophenol])-induced hepatotoxicity in a diet-induced obese (DIO) mouse model of NAFLD. The male C57BL/6NTac DIO mice, fed a high-fat diet for more than 12 weeks, developed obesity, hyperinsulinemia, impaired glucose tolerance, and hepatomegaly with hepatic steatosis, similar to human NAFLD. In the acute toxicity study after a single dose of APAP (150 mg/kg), compared with control lean mice, the DIO mice had decreased serum transaminase levels and less severe hepatocellular injury. The DIO mice also had altered expression of genes related to APAP metabolism. Chronic APAP exposure for 26 weeks did not predispose the DIO mice with NAFLD to more severe hepatotoxicity compared with the lean mice. These results suggested that the C57BL/6NTac DIO mouse model appears to be more tolerant to APAP-induced hepatotoxicity than lean mice, potentially related to altered xenobiotic metabolizing capacity in the fatty liver. Further mechanistic studies with APAP and other drugs in NAFLD animal models are necessary to investigate the mechanism of altered susceptibility to intrinsic DILI in some human NAFLD patients.

Acetaminophen-induced liver injury: Molecular mechanism and treatments from natural products.

Acetaminophen (APAP) is a widely used analgesic and antipyretic over-the-counter medicine worldwide. Hepatotoxicity caused by APAP overdose is one of the leading causes of acute liver failure (ALF) in the US and in some parts of Europe, limiting its clinical application. Excessive APAP metabolism depletes glutathione and increases N-acetyl-p-benzoquinoneimide (NAPQI) levels, leading to oxidative stress, DNA damage, and cell necrosis in the liver, which in turn leads to liver damage. Studies have shown that natural products such as polyphenols, terpenes, anthraquinones, and sulforaphane can activate the hepatocyte antioxidant defense system with Nrf2 as the core player, reduce oxidative stress damage, and protect the liver. As the key enzyme metabolizing APAP into NAPQI, cytochrome P450 enzymes are also considered to be intriguing target for the treatment of APAP-induced liver injury. Here, we systematically review the hepatoprotective activity and molecular mechanisms of the natural products that are found to counteract the hepatotoxicity caused by APAP, providing reference information for future preclinical and clinical trials of such natural products.

Pectolinarigenin ameliorates acetaminophen-induced acute liver injury via attenuating oxidative stress and inflammatory response in Nrf2 and PPARa dependent manners

BackgroundCirsii Japonici Herba Carbonisata (Dajitan in Chinese) has been used to treat liver disorders in Asian countries. Pectolinarigenin (PEC), an abundant constituent in Dajitan, has been found to possess a wide range of biological benefits, including hepatoprotective effects. However, the effects of PEC on acetaminophen (APAP)-induced liver injury (AILI) and the underlying mechanisms have not been studied. PurposesTo explore the role and mechanisms of PEC in protecting against AILI. Study design and methodsThe hepatoprotective benefits of PEC were studied using a mouse model and HepG2 cells. PEC was tested for its effects by injecting it intraperitoneally before APAP administration. To assess liver damage, histological and biochemical tests were performed. The levels of inflammatory factors in the liver were measured using RT-PCR and ELISA. Western blotting was used to measure the expression of a panel of key proteins involved in APAP metabolism, as well as Nrf2 and PPARα. PEC mechanisms on AILI were investigated using HepG2 cells, while the Nrf2 inhibitor (ML385) and PPARα inhibitor (GW6471) were used to validate the importance of either Nrf2 and PPARα in the hepatoprotective effects of PEC. ResultsPEC treatment decreased serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) levels in the liver. PEC pretreatment increased the activity of superoxide dismutase (SOD) and glutathione (GSH) while decreasing malondialdehyde production (MDA). PEC could also up-regulate two important APAP detoxification enzymes (UGT1A1 and SULT1A1). Further research revealed that PEC reduced hepatic oxidative damage and inflammation, and up-regulated APAP detoxification enzymes in hepatocytes by activating the Nrf2 and PPARα signaling pathways. ConclusionsPEC ameliorates AILI by decreasing hepatic oxidative stress and inflammation while increasing phase Ⅱ detoxification enzymes related to APAP harmless metabolism through activation of Nrf2 and PPARα signaling. Hence, PEC may serve as a promising therapeutic drug against AILI.

Narirutin activates TFEB (transcription factor EB) to protect against Acetaminophen-induced liver injury by targeting PPP3/calcineurin

ABSTRACT Acetaminophen (APAP) overdose is the predominant cause of drug-induced liver injury worldwide. The macroautophagy/autophagy-lysosomal pathway (ALP) is involved in the APAP hepatotoxicity. TFEB (transcription factor EB) promotes the expression of genes related to autophagy and lysosomal biogenesis, thus, pharmacological activation of TFEB-mediated ALP may be an effective therapeutic approach for treating APAP-induced liver injury. We aimed to reveal the effects of narirutin (NR), the main bioactive constituents isolated from citrus peels, on APAP hepatotoxicity and to explore its underlying mechanism. Administration of NR enhanced activities of antioxidant enzymes, improved mitochondrial dysfunction and alleviated liver injury in APAP-treated mice, whereas NR did not affect APAP metabolism and MAPK/JNK activation. NR enhanced TFEB transcriptional activity and activated ALP in an MTOR complex 1 (MTORC1)-independent but PPP3/calcineurin-dependent manner. Moreover, knockout of Tfeb or knockdown of PPP3CB/CNA2 (protein phosphatase 3, catalytic subunit, beta isoform) in the liver abolished the beneficial effects of NR on APAP overdose. Mechanistically, NR bound to PPP3CB via PRO31, LYS61 and PRO347 residues and enhanced PPP3/calcineurin activity, thereby eliciting dephosphorylation of TFEB and promoting ALP, which alleviated APAP-induced oxidative stress and liver injury. Together, NR protects against APAP-induced liver injury by activating a PPP3/calcineurin-TFEB-ALP axis, indicating NR may be a potential agent for treating APAP overdose. Abbreviations: ALP: autophagy-lysosomal pathway; APAP: acetaminophen; APAP-AD: APAP-protein adducts; APAP-Cys: acetaminophen-cysteine adducts; CAT: catalase; CETSA: cellular thermal shift assay; CQ: chloroquine; CYP2E1: cytochrome P450, family 2, subfamily e, polypeptide 1; CYCS/Cyt c: cytochrome c, somatic; DARTS: drug affinity responsive target stability assay; ENGASE/NAG: endo-beta-N-acetylglucosaminidase; GOT1/AST: glutamic-oxaloacetic transaminase 1, soluble; GPT/ALT: glutamic pyruvic transaminase, soluble; GSH: glutathione; GPX/GSH-Px: glutathione peroxidase; KD: dissociation constant; Leu: leupeptin; MCOLN1: mucolipin 1; MTORC1: MTOR complex 1; NAC: N-acetylcysteine; NAPQI: N-acetyl-p-benzoquinoneimine; NFAT: nuclear factor of activated T cells; NR: narirutin; OA: okadaic acid; RRAG: Ras related GTP binding; ROS: reactive oxygen species; PPP3CB/CNA2: protein phosphatase 3, catalytic subunit, beta isoform; PPP3R1/CNB1: protein phosphatase 3, regulatory subunit B, alpha isoform (calcineurin B, type I); SOD: superoxide dismutase; SPR: surface plasmon resonance analysis; TFEB: transcription factor EB.

C-Jun-N Terminal Kinase-Mediated Degradation of γ-Glutamylcysteine Ligase Catalytic Subunit Inhibits GSH Recovery After Acetaminophen Treatment: Role in Sustaining JNK Activation and Liver Injury.

Aims: Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States. Liver glutathione (GSH) depletion and sustained P-JNK (c-Jun-N-terminal kinase) activation are key modulators in the mechanism leading to hepatic necrosis. GSH depletion is directly related to the consumption of GSH by APAP metabolites N-acetyl-p-benzoquinone imine (NAPQI). We previously noticed that the glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in GSH synthesis, rapidly decreased at the same time P-JNK increased. Our aims were to determine if JNK was directly responsible for decreased GCLC causing impaired recovery of GSH and if this was an important factor in determining APAP hepatotoxicity. Results: Immunoprecipitation of JNK after APAP identified binding to GCLC. Expression of a site-directed mutated canonical JNK docking site in GCLC was resistant to degradation and led to rapid restoration of GSH and inhibited sustained JNK activation. The JNK-resistant GCLC markedly protected against necrosis and alanine aminotransferase (ALT) elevation. The proteolytic loss of GCLC was abrogated by inhibition of the proteasome, ubiquitination, or calpain. Innovation: Using mutated-GCLC resistant to JNK-induced degradation, the results allowed us to identify impaired GSH recovery as an important contributor to early progression of APAP toxicity after the metabolism of APAP and initial GSH depletion had occurred. Conclusion: Activated JNK interacts directly with GCLC and leads to proteolytic degradation of GCLC. Degradation of GCLC impairs GSH recovery after APAP allowing the continued activation of JNK. Conversely, rapid recovery of GSH inhibits the sustained activation of the mitogen-activated protein (MAP) kinase cascade and dampens APAP toxicity by suppressing the continued activation of JNK. Antioxid. Redox Signal. 38, 1071-1081.

Oridonin ameliorates acetaminophen-induced acute liver injury through ATF4/PGC-1α pathway.

Acetaminophen (APAP) overdose-induced acute liver injury (ALI) causes hepatocyte cell death, oxidative stress, and inflammation. Oridonin (Ori), a covalent NLRP3-inflammasome inhibitor, ameliorates APAP-induced ALI through an unclear molecular mechanism. This study found that Ori decreased hepatic cytochrome P450 2E1 level and increased glutathione content to prevent APAP metabolism, and then reduced the necrotic area, improved liver function, and inhibited APAP-induced proinflammatory cytokines and oxidative stress. Ori also decreased activating transcription factor 4 (ATF4) protein levels and increased peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) to reduce APAP-induced endoplasmic reticulum stress activation and mitochondrial dysfunction. Furthermore, western blot and luciferase assay found that ATF4 inhibited transcription in the PGC-1α promoter -507 to -495 region to reduce PGC-1α levels, while ATF4 knockdown neutralized the hepatoprotective effect of Ori. Molecular docking showed that Ori bound to ATF4's amino acid residue glutamate 302 through 6, 7, and 18 hydroxyl bands. Our findings demonstrated that Ori prevented metabolic activation of APAP and further inhibited the ATF4/PGC-1α pathway to alleviate APAP overdose-induced hepatic toxicity, which illuminated its potential therapeutic effects on ALI.

Desorption Electrospray Ionization Mass Spectrometry Imaging Allows Spatial Localization of Changes in Acetaminophen Metabolism in the Liver after Intervention with 4-Methylpyrazole.

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the US, and hepatotoxicity is initiated by a reactive metabolite which induces characteristic centrilobular necrosis. The only clinically available antidote is N-acetylcysteine, which has limited efficacy, and we have identified 4-methylpyrazole (4MP, Fomepizole) as a strong alternate therapeutic option, protecting against generation and downstream effects of the cytotoxic reactive metabolite in the clinically relevant C57BL/6J mouse model and in humans. However, despite the regionally restricted necrosis after APAP, our earlier studies on APAP metabolites in biofluids or whole tissue homogenate lack the spatial information needed to understand region-specific consequences of reactive metabolite formation after APAP overdose. Thus, to gain insight into the regional variation in APAP metabolism and study the influence of 4MP, we established a desorption electrospray ionization mass spectrometry imaging (DESI-MSI) platform for generation of ion images for APAP and its metabolites under ambient air, without chemical labeling or a prior coating of tissue which reduces chemical interference and perturbation of small molecule tissue localization. The spatial intensity and distribution of both oxidative and nonoxidative APAP metabolites were determined from mouse liver sections after a range of APAP overdoses. Importantly, exclusive differential signal intensities in metabolite abundance were noted in the tissue microenvironment, and 4MP treatment substantially influenced this topographical distribution.

Ginsenoside Rc, as an FXR activator, alleviates acetaminophen-induced hepatotoxicity via relieving inflammation and oxidative stress.

Acetaminophen (APAP) intake leads to excessive NAPQI deposition, stimulating inflammatory and oxidative stress and causing fatal liver injury. However, the detailed molecular mechanism involved is unknown, and effective therapeutic approaches remain insufficient. In this study, we discovered that treatment with ginsenoside Rc can prevent the inflammatory response caused by APAP and oxidative stress in mouse primary hepatocytes (MPHs), along with the corresponding changes in related genes. Additionally, Ginsenoside Rc effectively alleviates APAP-induced cellular apoptosis and NAPQI accumulation in MPHs. In vivo, Ginsenoside Rc administration remarkably attenuates APAP-induced hepatotoxicity, repairing liver damage and improving survival. Moreover, Ginsenoside Rc treatment modulates genes involved in APAP metabolism, leading to a decrease in NAPQI and resulting in the alleviation of fatal oxidative stress and inflammatory response after APAP exposure, along with the expression of their related indicators. Furthermore, our RNA-seq and molecular docking analysis implies that FXR expression and FXR transcriptional activity are stimulated by Ginsenoside Rc treatment. Notably, due to the lack of FXR in mice and MPHs, ginsenoside Rc can no longer play its original protective role against hepatotoxicity and cell damage caused by APAP, and it is difficult to improve the corresponding survival rate and prevent hepatic apoptosis, NAPQI generation, fatal oxidative stress, and the inflammatory response induced by APAP and the expression of related genes. In summary, our results indicate that Ginsenoside Rc could act as an effective FXR activator and effectively regulate FXR-induced antioxidant stress and eliminate inflammation while also having an anti-apoptotic function.

The Relationship between Prohibitin 1 Expression, Hepatotoxicity Induced by Acetaminophen, and Hepatoprotection by S-Adenosylmethionine in AML12 Cells.

Prohibitin 1 (Phb1) is a pleiotropic protein, located mainly in the mitochondrial inner membrane and involved in the regulation of cell proliferation and the stabilization of mitochondrial protein. Acetaminophen (APAP) is one of the most commonly used over-the-counter analgesics worldwide. However, at high dose, the accumulation of N-acetyl-p-benzoquinone imine (NAPQI) can lead to APAP-induced hepatotoxicity. In this study, we sought to understand the regulation of mRNA expression in relation to APAP and GSH metabolism by Phb1 in normal mouse AML12 hepatocytes. We used two different Phb1 silencing levels: high-efficiency (HE, >90%) and low-efficiency (LE, 50-60%). In addition, the siRNA-transfected cells were further pretreated with 0.5 mM of S-adenosylmethionine (SAMe) for 24 h before treatment with APAP at different doses (1-2 mM) for 24 h. The expression of APAP metabolism-related and antioxidant genes such as Cyp2e1 and Ugt1a1 were increased during SAMe pretreatment. Moreover, SAMe increased intracellular GSH concentration and it was maintained after APAP treatment. To sum up, Phb1 silencing and APAP treatment impaired the metabolism of APAP in hepatocytes, and SAMe exerted a protective effect against hepatotoxicity by upregulating antioxidant genes.

Metabolic Competency of Larval Zebrafish in Drug-Induced Liver Injury: A Case Study of Acetaminophen Poisoning.

Larval zebrafish is emerging as a new model organism for studying drug-induced liver injury (DILI) with superiorities in visual assessment, genetic engineering as well as high throughput. Metabolic bioactivation to form reactive intermediates is a common event that triggers DILI. This study first addressed the correlation between acetaminophen metabolism and hepatotoxicity in zebrafish larvae (3-day postfertilization) and demonstrated the occurrence of cytochrome P450 enzymes-mediated acetaminophen (APAP) bioactivation at early developmental stage through characterizing the dose-effect (0-1.6 mg/ml) and the time course (0-72 h) of liver injury and metabolism in the AB strain and LiPan transgenic line Tg(lfabp10a: DsRed; elaA:egfp) expressing the liver-specific fluorescent protein. APAP caused multiorgan developmental retardation and elicited dose- and time-dependent hepatotoxicity. Liver imaging revealed significant changes earlier than histological and biochemical measurements. APAP bioactivation in larval zebrafish was first confirmed by the detection of the glutathione conjugate of the reactive intermediate NAPQI (NAPQI-GSH) and subsequent mercapturate derivatives NAPQI-cysteine and NAPQI-N-acetylcysteine after even short (0.5-h postexposure) or low (0.2 mg/ml) APAP exposure. APAP overdose impaired metabolic function, in particular sulfation, whereas facilitated GSH depletion and APAP sulfate excretion. Meanwhile, APAP displayed triphasic accumulation in the larvae, agreeing with fluctuating metabolic capabilities with sulfation dominating the early larval developmental stage. Most importantly, the dose-response effects and time course of APAP accumulation and metabolism agree well with those of the liver injury development. Overall, larval zebrafish has developed mammalian-like metabolic function, enabling it an ideal model organism for high-throughput screening hepatotoxicity and mechanistic study of bioactivation-based DILI.

The Dual Role of Innate Immune Response in Acetaminophen-Induced Liver Injury.

Simple SummaryAn injury to the liver caused by a drug or its metabolites is referred to as drug-induced liver injury (DILI). The clinical manifestations of DILI are varied, and it may even result in acute liver failure under certain circumstances. Based on findings in the United States and China, Acetyl-para-aminophenol (APAP) may not be the most prevalent cause of DILI, but it is the leading cause of acute liver failure. The study of immune responses in APAP-induced liver injury (AILI) has been making significant progress in recent years. It should be noted, however, that different studies have reported differing results regarding the role played by immune cells in AILI, with some studies indicating they are proinflammatory, while others have showed no effect, or even pro-repair effects. Therefore, we here discuss the mechanisms of the dual role of immune cells in AILI.Acetyl-para-aminophenol (APAP), a commonly used antipyretic analgesic, is becoming increasingly toxic to the liver, resulting in a high rate of acute hepatic failure in Europe and the United States. Excessive APAP metabolism in the liver develops an APAP–protein adduct, which causes oxidative stress, MPTP opening, and hepatic necrosis. HMGB-1, HSP, nDNA, mtDNA, uric acid, and ATP are DMAPs released during hepatic necrosis. DMAPs attach to TLR4-expressing immune cells such KCs, macrophages, and NK cells, activating them and causing them to secrete cytokines. Immune cells and their secreted cytokines have been demonstrated to have a dual function in acetaminophen-induced liver injury (AILI), with a role in either proinflammation or pro-regeneration, resulting in contradicting findings and some research confusion. Neutrophils, KCs, MoMFs, NK/NKT cells, γδT cells, DCs, and inflammasomes have pivotal roles in AILI. In this review, we summarize the dual role of innate immune cells involved in AILI and illustrate how these cells initiate innate immune responses that lead to persistent inflammation and liver damage. We also discuss the contradictory findings in the literature and possible protocols for better understanding the molecular regulatory mechanisms of AILI.

Hepatocyte Specific gp130 Signalling Underlies APAP Induced Liver Injury.

N-acetyl-p-aminophenol (APAP)-induced liver damage is associated with upregulation of Interleukin-11 (IL11), which is thought to stimulate IL6ST (gp130)-mediated STAT3 activity in hepatocytes, as a compensatory response. However, recent studies have found IL11/IL11RA/gp130 signaling to be hepatotoxic. To investigate further the role of IL11 and gp130 in APAP liver injury, we generated two new mouse strains with conditional knockout (CKO) of either Il11 (CKOIl11) or gp130 (CKOgp130) in adult hepatocytes. Following APAP, as compared to controls, CKOgp130 mice had lesser liver damage with lower serum Alanine Transaminase (ALT) and Aspartate Aminotransferase (AST), greatly reduced serum IL11 levels (90% lower), and lesser centrilobular necrosis. Livers from APAP-injured CKOgp130 mice had lesser ERK, JNK, NOX4 activation and increased markers of regeneration (PCNA, Cyclin D1, Ki67). Experiments were repeated in CKOIl11 mice that, as compared to wild-type mice, had lower APAP-induced ALT/AST, reduced centrilobular necrosis and undetectable IL11 in serum. As seen with CKOgp130 mice, APAP-treated CKOIl11 mice had lesser ERK/JNK/NOX4 activation and greater features of regeneration. Both CKOgp130 and CKOIl11 mice had normal APAP metabolism. After APAP, CKOgp130 and CKOIl11 mice had reduced Il6, Ccl2, Ccl5, Il1β, and Tnfα expression. These studies exclude IL11 upregulation as compensatory and establish autocrine, self-amplifying, gp130-dependent IL11 secretion from damaged hepatocytes as toxic and anti-regenerative.