APAP Overdose In Mice Research Articles

Kaempferol sophoroside glucoside mitigates acetaminophen-induced hepatotoxicity: Role of Nrf2/NF-κB and JNK/ASK-1 signaling pathways

APAP (Acetaminophen)-induced hepatic injury is a major public health threat that requires continuous searching for new effective therapeutics. KSG (Kaempferol-3-sophoroside-7-glucoside) is a kaempferol derivative that was separated from plant species belonging to different genera. This study explored the protective effects of KSG on ALI (acute liver injury) caused by APAP overdose in mice and elucidated its possible mechanisms. The results showed that KSG pretreatment alleviated APAP-induced hepatic damage as it reduced hepatic pathological lesions as well as the serum parameters of liver injury. Moreover, KSG opposed APAP-associated oxidative stress and augmented hepatic antioxidants. KSG suppressed the inflammatory response as it decreased the genetic and protein expression as well as the levels of inflammatory cytokines. Meanwhile, KSG enhanced the mRNA expression and level of anti-inflammatory cytokine, IL-10 (interleukin-10). KSG repressed the activation of NF-κB (nuclear-factor kappa-B), besides it promoted the activation of Nrf2 signaling. Additionally, KSG markedly hindered the elevation of ASK-1 (apoptosis-signal regulating-kinase-1) and JNK (c-Jun-N-terminal kinase). Furthermore, KSG suppressed APAP-induced apoptosis as it decreased the level and expression of Bax (BCL2-associated X-protein), and caspase-3 concurrent with an enhancement of anti-apoptotic protein, Bcl2 in the liver. More thoroughly, Computational studies reveal indispensable binding affinities between KSG and Keap1 (Kelch-like ECH-associated protein-1), ASK1 (apoptosis signal-regulating kinase-1), and JNK1 (c-Jun N-terminal protein kinase-1) with distinctive tendencies for selective inhibition. Taken together, our data showed the hepatoprotective capacity of KSG against APAP-produced ALI via modulation of Nrf2/NF-κB and JNK/ASK-1/caspase-3 signaling. Henceforth, KSG could be a promising hepatoprotective candidate for ALI.

Growth differentiation factor 15 is dispensable for acetaminophen-induced liver injury in mice.

Acetaminophen (APAP)-induced liver injury (AILI) has been recognized as a pivotal contributor to drug-induced liver failure in Western countries, but its molecular mechanism remains poorly understood. Growth differentiation factor 15 (GDF15) is a pleiotropic factor that alleviates non-alcoholic liver steatohepatitis, liver fibrosis and liver injury. The aim of the present study was to examine the possibility whether GDF15 confers protection against AILI. We found that the gene expression of Gdf15 was increased significantly after APAP overdose in mice. Next, the role of Gdf15 in AILI was evaluated by hepatic Gdf15 overexpression (using adeno-associated virus serotype 8), injection with recombinant human GDF15 (rhGDF15) and Gdf15 knockout mice after challenge with APAP. A marked elevation of Gdf15 was observed after AILI. However, there were no significant differences in AILI-related liver injury and JNK phosphorylation after Gdf15 overexpression, rhGDF15 injection or Gdf15 deficiency. Together, we conclude that, despite a noticeable elevation of Gdf15 level after AILI, Gdf15 is dispensable for APAP-induced AILI. Our study further suggests that genomic analysis of mRNA expression after APAP overdose is of limited relevance unless followed up by a functional analysis of candidate genes in vivo.

Schisandra chinensis essential oil attenuates acetaminophen-induced liver injury through alleviating oxidative stress and activating autophagy

Context Schisandra chinensis (Turcz.) Baill. (Magnoliaceae) essential oil (SCEO) composition is rich in lignans that are believed to perform protective effects in the liver. Objective This study investigates the effects of SCEO in the treatment of acetaminophen (APAP)-induced liver injury in mice. Materials and methods C57BL/6 mice (n = 56) were randomly divided into seven groups: normal; APAP (300 mg/kg); APAP plus bicyclol (200 mg/kg); APAP plus SCEO (0.25, 0.5, 1, 2 g/kg). Serum biochemical parameters for liver function, inflammatory factors, and antioxidant activities were determined. The protein expression levels of Nrf2, GCLC, GCLM, HO-1, p62, and LC3 were assessed by western blotting. Nrf2, GCLC, HO-1, p62, and LC3 mRNA were detected by real-time PCR. Results Compared to APAP overdose, SCEO (2 g/kg) pre-treatment reduced the serum levels of AST (79.4%), ALT (84.6%), TNF-α (57.3%), and IL-6 (53.0%). In addition, SCEO (2 g/kg) markedly suppressed cytochrome P450 2E1 (CYP2E1) (15.4%) and attenuated the exhaustion of GSH (43.6%) and SOD (16.8%), and the accumulation of MDA (22.6%) in the liver, to inhibit the occurrence of oxidative stress. Moreover, hepatic tissues from our experiment revealed that SCEO pre-treatment mitigated liver injury caused by oxidative stress by increasing Nrf2, HO-1, and GCL. Additionally, SCEO activated autophagy, which upregulated hepatic LC3-II and decreased p62 in APAP overdose mice (p < 0.05). Discussion and conclusions Our evidence demonstrated that SCEO protects hepatocytes from APAP-induced liver injury in vivo and the findings will provide a reliable theoretical basis for developing novel therapeutics.

CoQ10 Promotes Resolution of Necrosis and Liver Regeneration After Acetaminophen-Induced Liver Injury.

Coenzyme Q10 (CoQ10) which acts as an electron transporter in the mitochondrial respiratory chain has many beneficial effects on liver diseases. In our previous research, CoQ10 has been found to attenuate acetaminophen (APAP)-induced acute liver injury (ALI). However, whether CoQ10 administration is still effective at the late stage of APAP overdose is still unknown. In this study, we aimed to test CoQ10 efficacy at the late stage of APAP overdose. C57BL/6J mice were intraperitoneally treated with APAP to induce liver injury. CoQ10 (5 mg/kg) was given to mice at 16 h after APAP treatment. The results showed that while CoQ10 treatment at 16 h post-APAP overdose had no effects on the expression of ROS generated genes or scavenged genes, it still significantly decreased necrosis of hepatocytes following APAP-induced ALI. Moreover, CoQ10 increased MerTK+ macrophages accumulation in the APAP-overdose liver and inhibition of MerTK signaling partly abrogated the protective role of CoQ10 treatment on the hepatic necrosis. CoQ10 treatment also significantly enhanced hepatocytes proliferation as shown in the increased 5-bromodeoxyuridine incorporation in the APAP-intoxicated mice liver section. In addition, CoQ10 treatment increased hepatic Proliferating Cell Nuclear Antigen (PCNA) and Cyclin D1 expression and promoted activation of the β-catenin signaling in APAP-overdose mice. To conclude, these data provide evidence that CoQ10 treatment is still effective at the late stage of APAP-induced ALI and promotes resolution of necrosis and liver regeneration following ALI.

Metallothioneins alter macrophage phenotype and represent novel therapeutic targets for acetaminophen-induced liver injury.

Acetaminophen (APAP) intoxication is the foremost cause of drug-induced liver failure in developed countries. The only pharmacologic treatment option, N-acetylcysteine (NAC), is not effective for patients who are admitted too late and/or who have excessive liver damage, emphasizing the need for alternative treatment options. APAP intoxication results in hepatocyte death and release of danger signals, which further contribute to liver injury, in part by hepatic monocyte/macrophage infiltration and activation. Metallothionein (MT) 1 and 2 have important danger signaling functions and might represent novel therapeutic targets in APAP overdose. Therefore, we evaluated hepatic MT expression and the effect of anti-MT antibodies on the transcriptional profile of the hepatic macrophage population and liver injury following APAP overdose in mice. Hepatic MT expression was significantly induced in APAP-intoxicated mice and abundantly present in human livers. APAP intoxication in mice resulted in increased serum transaminase levels, extended necrotic regions on liver histology and induced expression of proinflammatory markers, which was significantly less pronounced in mice treated with anti-MT antibodies. Anti-MT antibody therapy attenuated proinflammatory macrophage polarization, as demonstrated by RNA sequencing analyses of isolated liver macrophages and in LPS-stimulated bone marrow-derived macrophages. Importantly, NAC and anti-MT antibodies were equally effective whereas administration of anti-MT antibody in combination with NAC exceeded the efficiency of both monotherapies in APAP-induced liver injury (AILI). We conclude that the neutralization of secreted MTs using a monoclonal antibody is a novel therapeutic strategy as mono- or add-on therapy for AILI. In addition, we provide evidence suggesting that MTs in the extracellular environment are involved in macrophage polarization.

CoQ10 protects against acetaminophen-induced liver injury by enhancing mitophagy

Coenzyme Q10 (CoQ10), which is a key cofactor of the electron transport chain in the mitochondria has shown many beneficial effects on liver diseases. However, the mechanisms of CoQ10 protective role on the acetaminophen (APAP)-induced liver injury are elusive and unclear. In this study, we further investigated the CoQ10 therapeutic effects on APAP-overdose liver injury. C57BL/6 J mice were intraperitoneally treated with APAP to induce liver injury. CoQ10 (5 mg/kg) was given to mice at 1.5 h after APAP treatment. The results showed that hepatic CoQ10 levels were decreased during the APAP-induced hepatotoxicity and preceded serum ALT elevation. Treatment of CoQ10 significantly improved the liver injury induced by APAP. Moreover, CoQ10 treatment decreased the ROS levels and promoted the antioxidative related gene expression in APAP overdose mice. Importantly, results showed that even though CoQ10 had no effects on the mtDNA copy number and the expression of genes related to mitochondrial biogenesis, it significantly improved the mitochondrial complex I and V activities and promoted the mitophagy in APAP-overdose mice. To further authenticate mitophagy role in CoQ10-mediated improved liver injury in vivo, we administrated APAP-overdose mice with chloroquine 1 h prior to APAP treatment and found that chloroquine treatment functionally abrogated the CoQ10 protective role on APAP overdose mice. To conclude, this study provides evidence that CoQ10 activates mitophagy to protect against APAP-induced liver injury. Therefore, CoQ10 may represent a novel therapeutic option for the prevention and treatment of drug-induced liver injury.

The Late-Stage Protective Effect of Mito-TEMPO against Acetaminophen-Induced Hepatotoxicity in Mouse and Three-Dimensional Cell Culture Models.

An overdose of acetaminophen (APAP), the most common cause of acute liver injury, induces oxidative stress that subsequently causes mitochondrial impairment and hepatic necroptosis. N-acetyl-L-cysteine (NAC), the only recognized drug against APAP hepatotoxicity, is less effective the later it is administered. This study evaluated the protective effect of mitochondria-specific Mito-TEMPO (Mito-T) on APAP-induced acute liver injury in C57BL/6J male mice, and a three dimensional (3D)-cell culture model containing the human hepatoblastoma cell line HepG2. The administration of Mito-T (20 mg/kg, i.p.) 1 h after APAP (400 mg/kg, i.p.) injection markedly attenuated the APAP-induced elevated serum transaminase activity and hepatic necrosis. However, Mito-T treatment did not affect key factors in the development of APAP liver injury including the activation of c-jun N-terminal kinases (JNK), and expression of the transcription factor C/EBP homologous protein (CHOP) in the liver. However, Mito-T significantly reduced the APAP-induced increase in the hepatic oxidative stress marker, nitrotyrosine, and DNA fragmentation. Mito-T markedly attenuated cytotoxicity induced by APAP in the HepG2 3D-cell culture model. Moreover, liver regeneration after APAP hepatotoxicity was not affected by Mito-T, demonstrated by no changes in proliferating cell nuclear antigen formation. Therefore, Mito-T was hepatoprotective at the late-stage of APAP overdose in mice.

Absolute measurement of the tissue origins of cell-free DNA in the healthy state and following paracetamol overdose

BackgroundDespite the emergence of cell-free DNA (cfDNA) as a clinical biomarker in cancer, the tissue origins of cfDNA in healthy individuals have to date been inferred only by indirect and relative measurement methods, such as tissue-specific methylation and nucleosomal profiling.MethodsWe performed the first direct, absolute measurement of the tissue origins of cfDNA, using tissue-specific knockout mouse strains, in both healthy mice and following paracetamol (APAP) overdose. We then investigated the utility of total cfDNA and the percentage of liver-specific cfDNA as clinical biomarkers in patients presenting with APAP overdose.ResultsAnalysis of cfDNA from healthy tissue-specific knockout mice showed that cfDNA originates predominantly from white and red blood cell lineages, with minor contribution from hepatocytes, and no detectable contribution from skeletal and cardiac muscle. Following APAP overdose in mice, total plasma cfDNA and the percentage fraction originating from hepatocytes increased by ~ 100 and ~ 19-fold respectively. Total cfDNA increased by an average of more than 236-fold in clinical samples from APAP overdose patients with biochemical evidence of liver injury, and 18-fold in patients without biochemically apparent liver injury. Measurement of liver-specific cfDNA, using droplet digital PCR and methylation analysis, revealed that the contribution of liver to cfDNA was increased by an average of 175-fold in APAP overdose patients with biochemically apparent liver injury compared to healthy subjects, but was not increased in overdose patients with normal liver function tests.ConclusionsWe present a novel method for measurement of the tissue origins of cfDNA in healthy and disease states and demonstrate the potential of cfDNA as a clinical biomarker in APAP overdose.

Design of anti-inflammatory heparan sulfate to protect against acetaminophen-induced acute liver failure.

Acetaminophen/paracetamol (APAP) overdose is the leading cause of drug-induced acute liver failure (ALF) in the United States and Europe. The progression of the disease is attributed to sterile inflammation induced by the release of high mobility group box 1 (HMGB1) and the interaction with receptor for advanced glycation end products (RAGE). A specific, effective, and safe approach to neutralize the proinflammatory activity of HMGB1 is highly desirable. Here, we found that a heparan sulfate (HS) octadecasaccharide (18-mer-HP or hepatoprotective 18-mer) displays potent hepatoprotection by targeting the HMGB1/RAGE axis. Endogenous HS proteoglycan, syndecan-1, is shed in response to APAP overdose in mice and humans. Furthermore, purified syndecan-1, but not syndecan-1 core protein, binds to HMGB1, suggesting that HMGB1 binds to HS polysaccharide side chains of syndecan-1. Last, we compared the protection effect between 18-mer-HP and N-acetyl cysteine, which is the standard of care to treat APAP overdose. We demonstrated that 18-mer-HP administered 3 hours after a lethal dose of APAP is fully protective; however, the treatment of N-acetyl cysteine loses protection. Therefore, 18-mer-HP may offer a potential therapeutic advantage over N-acetyl cysteine for late-presenting patients. Synthetic HS provides a potential approach for the treatment of APAP-induced ALF.

Propagation of Pericentral Necrosis During Acetaminophen-Induced Liver Injury: Evidence for Early Interhepatocyte Communication and Information Exchange.

Acetaminophen (APAP)-induced liver injury is clinically significant, and APAP overdose in mice often serves as a model for drug-induced liver injury in humans. By specifying that APAP metabolism, reactive metabolite formation, glutathione depletion, and mitigation of mitochondrial damage within individual hepatocytes are functions of intralobular location, an earlier virtual model mechanism provided the first concrete multiattribute explanation for how and why early necrosis occurs close to the central vein (CV). However, two characteristic features could not be simulated consistently: necrosis occurring first adjacent to the CV, and subsequent necrosis occurring primarily adjacent to hepatocytes that have already initiated necrosis. We sought parsimonious model mechanism enhancements that would manage spatiotemporal heterogeneity sufficiently to enable meeting two new target attributes and conducted virtual experiments to explore different ideas for model mechanism improvement at intrahepatocyte and multihepatocyte levels. For the latter, evidence supports intercellular communication via exosomes, gap junctions, and connexin hemichannels playing essential roles in the toxic effects of chemicals, including facilitating or counteracting cell death processes. Logic requiring hepatocytes to obtain current information about whether downstream and lateral neighbors have triggered necrosis enabled virtual hepatocytes to achieve both new target attributes. A virtual hepatocyte that is glutathione-depleted uses that information to determine if it will initiate necrosis. When a less-stressed hepatocyte is flanked by at least two neighbors that have triggered necrosis, it too will initiate necrosis. We hypothesize that the resulting intercellular communication-enabled model mechanism is analogous to the actual explanation for APAP-induced hepatotoxicity at comparable levels of granularity.

Dynamic visualization of the recovery of mouse hepatobiliary metabolism to acetaminophen-overdose damage.

Acetaminophen (APAP) overdose is one of the world's leading causes of drug-induced hepatotoxicity. Although traditional methods such as histological imaging and biochemical assays have been successfully applied to evaluate the extent of APAP-induced liver damage, detailed effect of how APAP overdose affect the recovery of hepatobiliary metabolism and is not completely understood. In this work, we used intravital multiphoton microscopy to image and quantify hepatobiliary metabolism of the probe 6-carboxyfluorescein diacetate in APAP-overdose mice. We analyzed hepatobiliary metabolism for up to 7 days following the overdose and found that the excretion of the probe molecule was the most rapid on Day 1 following APAP overdose and slowed down on Days 2 and 3. On Day 7, probe excretion capability has exceeded that of the normal mice, suggesting that newly regenerated hepatocytes have higher metabolic capabilities. Our approach may be further developed applied to studying drug-induced hepatotoxicity in vivo.

Dual Role of Epidermal Growth Factor Receptor in Liver Injury and Regeneration after Acetaminophen Overdose in Mice.

Epidermal growth factor receptor (EGFR) plays a crucial role in hepatocyte proliferation. Its role in acetaminophen (APAP)-mediated hepatotoxicity and subsequent liver regeneration is completely unknown. Role of EGFR after APAP-overdose in mice was studied using pharmacological inhibition strategy. Rapid, sustained and dose-dependent activation of EGFR was noted after APAP-treatment in mice, which was triggered by glutathione depletion. EGFR-activation was also observed in primary human hepatocytes after APAP-treatment, preceding elevation of toxicity markers. Treatment of mice with an EGFR-inhibitor (EGFRi), Canertinib, 1h post-APAP resulted in robust inhibition of EGFR-activation and a striking reduction in APAP-induced liver injury. Metabolic activation of APAP, formation of APAP-protein adducts, APAP-mediated JNK-activation and its mitochondrial translocation were not altered by EGFRi. Interestingly, EGFR rapidly translocated to mitochondria after APAP-treatment. EGFRi-treatment abolished mitochondrial EGFR activity, prevented APAP-mediated mitochondrial dysfunction/oxidative-stress and release of endonucleases from mitochondria, which are responsible for DNA-damage/necrosis. Treatment with N-acetylcysteine (NAC), 4h post-APAP in mice did not show any protection but treatment of EGFRi in combination with NAC showed decrease in liver injury. Finally, delayed treatment with EGFRi, 12-h post-APAP, did not alter peak injury but caused impairment of liver regeneration resulting in sustained injury and decreased survival after APAP overdose in mice. Impairment of regeneration was due to inhibition of cyclinD1 induction and cell cycle arrest. Our study has revealed a new dual role of EGFR both in initiation of APAP-injury and in stimulation of subsequent compensatory regeneration after APAP-overdose.

Mitochondria-targeted antioxidant Mito-Tempo protects against acetaminophen hepatotoxicity.

Acetaminophen (APAP) hepatotoxicity is characterized by an extensive mitochondrial oxidant stress. However, its importance as a drug target has not been clarified. To investigate this, fasted C57BL/6J mice were treated with 300mg/kg APAP and the mitochondria-targeted antioxidant Mito-Tempo (MT) was given 1.5h later. APAP caused severe liver injury in mice, as indicated by the increase in plasma ALT activities and centrilobular necrosis. MT dose-dependently reduced the injury. Importantly, MT did not affect APAP-protein adducts formation, glutathione depletion or c-jun N-terminal kinase activation and its mitochondrial translocation. In contrast, hepatic glutathione disulfide and peroxynitrite formation were dose-dependently reduced by MT, indicating its effective mitochondrial oxidant stress scavenging capacity. Consequently, mitochondrial translocation of Bax and release of mitochondrial intermembrane proteins such as apoptosis-inducing factor were prevented, and nuclear DNA fragmentation was eliminated. To demonstrate the importance of mitochondria-specific antioxidant property of MT, we compared its efficacy with Tempo, which has the same pharmacological mode of action as MT but lacks the mitochondria targeting moiety. In contrast to the dramatic protection by MT, the same molar dose of Tempo did not significantly reduce APAP hepatotoxicity. In contrast, even a 3h post-treatment with MT reduced 70% of the injury, and the combination of MT with N-acetylcysteine (NAC) provided superior protection than NAC alone. We conclude that MT protects against APAP overdose in mice by attenuating the mitochondrial oxidant stress and preventing peroxynitrite formation and the subsequent mitochondrial dysfunction. MT is a promising therapeutic agent for APAP overdose patients.

Platelets and protease-activated receptor-4 contribute to acetaminophen-induced liver injury in mice

AbstractAcetaminophen (APAP)-induced liver injury in humans is associated with robust coagulation cascade activation and thrombocytopenia. However, it is not known whether coagulation-driven platelet activation participates in APAP hepatotoxicity. Here, we found that APAP overdose in mice caused liver damage accompanied by significant thrombocytopenia and accumulation of platelets in the liver. These changes were attenuated by administration of the direct thrombin inhibitor lepirudin. Platelet depletion with an anti-CD41 antibody also significantly reduced APAP-mediated liver injury and thrombin generation, indicated by the concentration of thrombin-antithrombin (TAT) complexes in plasma. Compared with APAP-treated wild-type mice, biomarkers of hepatocellular and endothelial damage, plasma TAT concentration, and hepatic platelet accumulation were reduced in mice lacking protease-activated receptor (PAR)-4, which mediates thrombin signaling in mouse platelets. However, selective hematopoietic cell PAR-4 deficiency did not affect APAP-induced liver injury or plasma TAT levels. These results suggest that interconnections between coagulation and hepatic platelet accumulation promote APAP-induced liver injury, independent of platelet PAR-4 signaling. Moreover, the results highlight a potential contribution of nonhematopoietic cell PAR-4 signaling to APAP hepatotoxicity.

Resveratrol prevents protein nitration and release of endonucleases from mitochondria during acetaminophen hepatotoxicity

Overdose of acetaminophen (APAP) is a common cause of acute liver injury and liver failure. The mechanism involves formation of a reactive metabolite, protein binding, oxidative stress and activation of c-Jun N-terminal kinase (JNK), mitochondrial dysfunction, and nuclear DNA fragmentation caused by endonucleases released from damaged mitochondria. Previous work has shown that the natural product resveratrol (RSV) can protect against APAP hepatotoxicity in mice through prevention of lipid peroxidation and anti-inflammatory effects. However, these earlier studies did not take into consideration several fundamental aspects of the pathophysiology. To address this, we treated C57Bl/6 mice with 300 mg/kg APAP followed by 50 mg/kg RSV 1.5 h later. Our results confirmed that RSV reduced liver injury after APAP overdose in mice. Importantly, RSV did not inhibit reactive metabolite formation and protein bindings, nor did it reduce activation of JNK. However, RSV decreased protein nitration after APAP treatment, possibly through direct scavenging of peroxynitrite. Interestingly, RSV also inhibited release of apoptosis-inducing factor and endonuclease G from mitochondria independent of Bax pore formation and prevented the downstream nuclear DNA fragmentation. Our data show that RSV protects against APAP hepatotoxicity both through antioxidant effects and by preventing mitochondrial release of endonucleases and nuclear DNA damage.

Lower susceptibility of female mice to acetaminophen hepatotoxicity: Role of mitochondrial glutathione, oxidant stress and c-jun N-terminal kinase

Acetaminophen (APAP) overdose causes severe hepatotoxicity in animals and humans. However, the mechanisms underlying the gender differences in susceptibility to APAP overdose in mice have not been clarified. In our study, APAP (300mg/kg) caused severe liver injury in male mice but 69–77% lower injury in females. No gender difference in metabolic activation of APAP was found. Hepatic glutathione (GSH) was rapidly depleted in both genders, while GSH recovery in female mice was 2.6 fold higher in the mitochondria at 4h, and 2.5 and 3.3 fold higher in the total liver at 4h and 6h, respectively. This faster recovery of GSH, which correlated with greater induction of glutamate-cysteine ligase, attenuated mitochondrial oxidative stress in female mice, as suggested by a lower GSSG/GSH ratio at 6h (3.8% in males vs. 1.4% in females) and minimal centrilobular nitrotyrosine staining. While c-jun N-terminal kinase (JNK) activation was similar at 2 and 4h post-APAP, it was 3.1 fold lower at 6h in female mice. However, female mice were still protected by the JNK inhibitor SP600125. 17β-Estradiol pretreatment moderately decreased liver injury and oxidative stress in male mice without affecting GSH recovery. Conclusion: The lower susceptibility of female mice is achieved by the improved detoxification of reactive oxygen due to accelerated recovery of mitochondrial GSH levels, which attenuates late JNK activation and liver injury. However, even the reduced injury in female mice was still dependent on JNK. While 17β-estradiol partially protects male mice, it does not affect hepatic GSH recovery.

Protection afforded by pre- or post-treatment with 4-phenylbutyrate against liver injury induced by acetaminophen overdose in mice

Acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) is a widely used analgesic/antipyretic drug with few adverse effects at therapeutic doses; suicidal or unintentional overdose of APAP frequently induces severe hepatotoxicity. To explore a new and effective antidote for APAP hepatotoxicity, this study examined the effects of sodium 4-phenylbutyrate (4-PBA) on liver injury induced by APAP overdose in mice. Liver injury was induced in C57BL/6 male mice by intraperitoneal injection of APAP (400mg/kg). The effects of 4-PBA (100–200mg/kg) treatment at 1h before the APAP injection were evaluated with serum alanine aminotransferase (ALT) and blood ammonia levels, hepatic pathological changes, including histopathology, DNA damage, nitrotyrosine formation, and mRNA or protein expression involved in the development of hepatotoxicity, such as X-box binding protein-1 (XBP1), c-Jun N-terminal kinase (JNK), C/EBP homologous protein (CHOP) and B-cell lymphoma 2 interacting mediator of cell death (Bim). In addition, glutathione depletion and CYP2E1 protein expression, which are measures of the metabolic conversion of APAP to a toxic metabolite, were examined. Furthermore, we examined the effects of post-treatment with 4-PBA against APAP-induced hepatotoxicity in mice. When administered at 1h before APAP injection, 4-PBA significantly prevented the increase in serum ALT and blood ammonia levels, centrilobular necrosis of hepatocytes, DNA fragmentation, and nitrotyrosine formation induced by APAP in mice. 4-PBA also inhibited hepatic Xbp1 mRNA splicing and JNK phosphorylation induced by APAP, but did not suppress CHOP and Bim mRNA and protein expression. In addition, 4-PBA had little effect on hepatic glutathione depletion and CYP2E1 expression, parameters of toxic APAP metabolite production. Post-treatment with 4-PBA administration at 1 or 2h after APAP injection also attenuated the increase in serum ALT and blood ammonia levels and hepatic pathological changes in APAP-induced hepatotoxicity in mice. Although post-treatment with 4-PBA did not show any effects on hepatic Xbp1 mRNA splicing and JNK phosphorylation, it drastically attenuated the DNA fragmentation induced by APAP. The precise molecular mechanisms of the protection afforded by 4-PBA against APAP hepatotoxicity in mice are unclear, but they seem to involve inhibition of hepatocellular DNA fragmentation. We suggest that 4-PBA is a promising candidate as an antidote against APAP-induced liver injury.

Ozagrel hydrochloride, a selective thromboxane A2 synthase inhibitor, alleviates liver injury induced by acetaminophen overdose in mice

BackgroundOverdosed acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) causes severe liver injury. We examined the effects of ozagrel, a selective thromboxane A2 (TXA2) synthase inhibitor, on liver injury induced by APAP overdose in mice.MethodsHepatotoxicity was induced to ICR male mice by an intraperitoneal injection with APAP (330 mg/kg). The effects of ozagrel (200 mg/kg) treatment 30 min after the APAP injection were evaluated with mortality, serum alanine aminotransferase (ALT) levels and hepatic changes, including histopathology, DNA fragmentation, mRNA expression and total glutathione contents. The impact of ozagrel (0.001-1 mg/mL) on cytochrome P450 2E1 (CYP2E1) activity in mouse hepatic microsome was examined. RLC-16 cells, a rat hepatocytes cell line, were exposed to 0.25 mM N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic metabolite of APAP. In this model, the cytoprotective effects of ozagrel (1–100 muM) were evaluated by the WST-1 cell viability assay.ResultsOzagel treatment significantly attenuated higher mortality, elevated serum alanine aminotransferase levels, excessive hepatic centrilobular necrosis, hemorrhaging and DNA fragmentation, as well as increase in plasma 2,3-dinor thromboxane B2 levels induced by APAP injection. Ozagrel also inhibited the hepatic expression of cell death-related mRNAs induced by APAP, such as jun oncogene, FBJ osteosarcoma oncogene (fos) and C/EBP homologous protein (chop), but did not suppress B-cell lymphoma 2-like protein11 (bim) expression and hepatic total glutathione depletion. These results show ozagrel can inhibit not all hepatic changes but can reduce the hepatic necrosis. Ozagrel had little impact on CYP2E1 activity involving the NAPQI production. In addition, ozagrel significantly attenuated cell injury induced by NAPQI in RLC-16.ConclusionsWe demonstrate that the TXA2 synthase inhibitor, ozagrel, dramatically alleviates liver injury induced by APAP in mice, and suggest that it is a promising therapeutic candidate for the treatment of APAP-induced liver injury.