APAP Administration Research Articles

ORY supplementation mitigates acetaminophen-induced acute liver failure in male mice: role of oxidative stress and apoptotic markers.

The aim of the present study was to assess the possible protective effect of γ-oryzanol (ORY) supplementation in a model of acute liver failure (ALF) induced by acetaminophen (APAP) in mice. Male Swiss strain mice were supplemented with ORY (10 and 50mg/kg, per oral route) daily for 7days. One hour after the last supplementation, animals received APAP (300mg/kg, intraperitoneal). Twenty-four hours after APAP administration, mice were euthanized, and biochemical and histopathological determinations were performed. Histopathological analysis revealed that APAP caused vascular congestion, loss of cellular structure, and cellular infiltration in hepatocytes. Moreover, it caused oxidative damage (enzymatic and non-enzymatic analysis of oxidative stress), with loss of hepatic function leading to cell apoptosis (apoptotic parameters). ORY supplementation (ORY-10 and ORY-50) protected against all changes in ALF model. Thus, the protective effect of ORY supplementation was due to modulation of antioxidant defenses avoiding the apoptotic process.

Improvement of therapeutic potential N-acetylcysteine in acetaminophen hepatotoxicity by encapsulation in PEGylated nano-niosomes

AimsN-Acetylcysteine (NAC) is an effective antidote for the treatment of acetaminophen (APAP) poisoning; however, due to its low stability and bioavailability, repeated dosing of NAC is needed. This study investigated the therapeutic efficacy of NAC by niosomal carriers. Materials and methodsNiosomes were synthesized using surface active agents film hydration method and their physicochemical properties were characterized. In the in vivo study, in addition to control group, male rats were divided in different groups and challenged with an oral dose of APAP (2000 mg/kg); 4 h later, rats were administered normal saline, empty niosome (NIO), NAC (25 mg/kg) and NAC-loaded niosome (NAC-NIO) respectively, and sacrificed 48 h post-APAP overdose. Key findingsThe particle size and zeta potential of NAC-NIO were 242.3 ± 18.5 nm and −23.9 ± 1.6 mV. The loading and encapsulation efficiency of niosomes were 1.22% ± 0.02% and 26.76% ± 6.02%. APAP administration leads to hepatic damage as evidenced by increases in serum hepatic enzyme levels and tissue levels of nitric oxide and lipid peroxidation as well as decreases in hepatic levels of reduced glutathione, catalase, superoxide dismutase, and glutathione peroxidase. Treatment of rats with NIO-NAC was remarkably more effective than NAC in improving biochemical changes such as serum hepatic aminotransferases. These findings were correlated well to the histopathological experiments. SignificanceOur results suggest that NAC when delivered as a niosomal structure, is potentially more effective than NAC standard, in improving APAP-induced hepatotoxicity.

Omega-3 fatty acids protect against acetaminophen-induced hepatic and renal toxicity in rats through HO-1-Nrf2-BACH1 pathway

Although acetaminophen (APAP) is a commonly used analgesic antipyretic drug, hepatotoxicity and nephrotoxicity are common after the overdose. The main mechanism of APAP toxicity is oxidative stress based. Stress may induce the production of heme oxygenase 1 (HO)-1 which is regulated by interleukin (IL)-10 and inhibit the production of tumor necrosis factor-alpha (TNF-α). HO-1 expression is further regulated by nuclear factor erythroid 2–related factor 2 (Nrf2) and the transcription factor BTB and CNC homology 1 (BACH1). Drug-induced toxicity can be relieved by several natural products, which are preferred due to their dietary nature and less adverse reactions. Of these natural products, omega-3 (ω-3) fatty acids are known for anti-inflammatory and antioxidant actions. However, effects of ω-3fatty acids on APAP-induced hepatic and renal toxicity are not well addressed. We designed this study to test the potential protecting actions of ω-3 fatty acids (270 mg/kg Eicosapentaenoic acid and 180 mg/kg docosahexaenoic acid, orally, for 7 days) in hepatotoxicity and nephrotoxicity induced by APAP (2 g/kg, once orally on day 7) in rats. Moreover, we focused on the molecular mechanism underlying APAP hepatotoxicity and nephrotoxicity. Pre-treatment with ω-3 fatty acids enhanced liver and kidney functions indicated by decreased serum aminotransferases activities and serum creatinine and urea concentrations. These results were further confirmed by histopathological examination. Moreover, ω-3 fatty acids showed antioxidant properties confirmed by decreased malondialdehyde level and increased total antioxidant capacity. Antioxidant Nrf2, its regulators (HO-1 and BACH1) and the anti-inflammatory cytokine (IL-10) were up-regulated by APAP administration as a compensatory mechanism and they were normalized by ω-3 fatty acids. ω-3 fatty acids showed anti-inflammatory actions through down-regulating nuclear factor kappa B (NF-ĸB) and its downstream TNF-α. Moreover, Western blot analysis showed that ω-3 fatty acids promoted Nrf2 translocation to the nucleus; BACH1 exit from the nucleus and inhibited NF-ĸB nuclear translocation. These findings suggested the protecting actions of ω-3 fatty acids against APAP-induced hepatic and renal toxicity through regulation of antioxidant Nrf2 and inflammatory NF-ĸB pathways.

The analysis of acetaminophen (paracetamol) and seven metabolites in rat, pig and human plasma by U(H)PLC-MS.

A U(H)PLC-MS/MS method is described for the analysis of acetaminophen and its sulphate, glucuronide, glutathione, cysteinyl and N-acetylcysteinyl metabolites in plasma using stable isotope-labeled internal standards. P-Aminophenol glucuronide and 3-methoxyacetaminophen were monitored and semi-quantified using external standards. The assay takes 7.5min/sample, requires only 5μl of plasma and involves minimal sample preparation. The method was validated for rat plasma and cross validated for human and pig plasma and mouse serum. LOQ in plasma for these analytes were 0.44μg/ml (APAP-C), 0.58μg/ml (APAP-SG), 0.84μg/ml (APAP-NAC), 2.75μg/ml (APAP-S), 3.00μg/ml (APAP-G) and 16μg/ml (APAP). Application of the method is illustrated by the analysis of plasma following oral administration of APAP to male Han Wistar rats.

Aldehyde dehydrogenase-2 activation decreases acetaminophen hepatotoxicity by prevention of mitochondrial depolarization

Oxidative stress contributes to acetaminophen (APAP) hepatotoxicity. Since lipid peroxidation produces reactive aldehydes, we investigated whether activation of mitochondrial aldehyde dehydrogenase-2 (ALDH2) with Alda-1 decreases liver injury after APAP. Male C57BL/6 mice fasted overnight received Alda-1 (20 mg/kg, i.p.) or vehicle 30 min before APAP (300 mg/kg, i.p.). Blood and livers were collected 2 or 24 h after APAP. Intravital multiphoton microscopy of rhodamine 123 (Rh123) and propidium iodide (PI) fluorescence was conducted 6 h after APAP administration to detect mitochondrial polarization status and cell death. 4-Hydroxynonenal protein adducts were present in 0.1% of tissue area without APAP treatment but increased to 7% 2 h after APAP treatment, which Alda-1 blunted to 1%. Serum alanine and aspartate aminotransferases increased to 7594 and 9768 U/L at 24 h respectively, which decreased ≥72% by Alda-1. Alda-1 also decreased centrilobular necrosis at 24 h after APAP from 47% of lobular areas to 21%. N-acetyl-p-benzoquinone imine protein adduct formation and c-Jun-N-terminal kinase phosphorylation increased after APAP as expected, but Alda-1 did not alter these changes. Without APAP, no mitochondrial depolarization was detected by intravital microscopy. At 6 h after APAP, 62% of tissue area showed depolarization, which decreased to 33.5% with Alda-1. Cell death as detected by PI labeling increased from 0 to 6.8 cells per 30× field 6 h after APAP, which decreased to 0.6 cells by Alda-1. In conclusion, aldehydes are important mediators of APAP hepatotoxicity. Accelerated aldehyde degradation by ALDH2 activation with Alda-1 decreases APAP hepatotoxicity by protection against mitochondrial dysfunction.

Peroxiredoxin 6 mediates acetaminophen-induced hepatocyte death through JNK activation

Acetaminophen (APAP) is one of the most frequently used drugs; however, its overdose leads to acute liver injury. Recently, studies have reported that the adduction of peroxiredoxin 6 (PRDX6), a member of the PRDX family of antioxidant enzymes, is associated with liver diseases. However, the role of PRDX6 in APAP-induced liver injury remains unclear. Here, we assessed both age-matched (about 12 weeks) PRDX6-overexpressing transgenic mice (PRDX6 mice) and wild type (WT) mice presenting acute liver injury induced by the intraperitoneal injection of APAP (500 mg/kg). Although PRDX6 is known as an antioxidant enzyme, PRDX6 mice unexpectedly demonstrated severe liver injury following APAP injection compared with WT mice. We observed that PRDX6 was hyperoxidized after APAP administration. Additionally, calcium-independent phospholipase A2 (iPLA2) activity and lysophosphatidylcholine (LPC) levels were markedly elevated in PRDX6 mice following APAP administration. Moreover, APAP-induced JNK phosphorylation was considerably increased in the liver of PRDX6 mice. MJ33, an inhibitor of PRDX6, attenuated APAP-induced liver injury both in WT and PRDX6 mice. Notably, MJ33 reduced the APAP-induced increase in JNK activation, iPLA2 activity, and LPC levels. Although SP600125, a JNK inhibitor, abolished APAP-induced liver injury, it failed to affect the APAP-induced hyperoxidation of PRDX6, iPLA2 activity, and LPC levels. These results suggested that PRDX6 was converted to the hyperoxidized form by the APAP-induced high concentration of hydrogen peroxides. In the liver, hyperoxidized PRDX6 induced cellular toxicity via JNK activation by enhancing iPLA2 activity and LPC levels; this mechanism appears to be a one-way cascade.

Inhibitory activity of black mulberry (Morus nigra) extract against testicular, liver and kidney toxicity induced by paracetamol in mice.

Black mulberry (Morus nigra) leaves is broadly used in traditional medicine worldwide. However, there are no scientific reports regarding testicular protection, hepato-and nephroprotective activities of M. nigra leaves. The present investigation wasassessed the protective mechanism by which methanol extract from M. nigra leaves suppressed the damaging effects induced by paracetamol (APAP) in different mouse tissues. Male mice were orally given APAP (500mg/kg) with or without M. nigra extract (150, 300, and 500mg/kg) for four consecutive days. The results showed that crude extract possessed potent antioxidant activity (EC50 = 42.97µg extract/mL) due to the presence of a high amount of polyphenol and flavonoid compounds. Gallic acid, chlorogenic acid, catechin, and rutin were isolated from the n-butanol fraction of M. nigra extract. Unexpectedly, oral administration of APAP did not induce chromosomal aberrations in mouse bone marrow; however, it produced damaging effects on testis, liver, and kidney tissues. Interestingly, M. nigra extract suppressed APAP-induced genotoxicity by lowering meiotic chromosomal aberrations in spermatocytes, morphological sperm abnormalities, and % DNA damage in comet tail in the liver and kidney tissues. The altered levels of glutathione S transferase activity, lipid peroxidation, liver, and kidney functions were significantly reversed when M. nigra was given to APAP group. The restoring of the histo-architectural distortions and decreasing over-expression of p53 protein as determined by immunohistochemistry in the liver, kidney, and testis sectionswere strengthened the protective activity of M. nigra extract. Conclusion, the bioactive components in the leaves of black mulberry appear to be a good candidate for genetic protection, treatment of oxidative stress-induced organotoxicity.

Lophenol and lathosterol from resin of Commiphora kua possess hepatoprotective effects in vivo

Ethnopharmacological relevanceDrug induced liver damage remains a prevalent concern in healthcare and may reduce the effectiveness of therapy by compromising therapeutic regimens. Many Commiphora species are known for their medicinal properties, and some of them are used traditionally for hepatoprotective effect. In the course of our drugs discovery from natural sources, phytosterols (lophenol (Lop) and lathosterol (Lat)), isolated from Commiphora kua were studied to evaluate their hepatoprotective effects in acetaminophen (APAP) induced hepatotoxicity in mice. Aims and objectiveTo evaluate the hepatoprotective effects of phytosterols isolated from C. kua using in vivo experimental model. Materials and methodsMice of either sex were divided into 7 groups: Vehicle, silymarin (SLY), acetaminophen (APAP), Lop 25, Lop 50, Lat 25, Lat 50 (n = 5). Vehicle group received only vehicle (0.1% DMSO solution) for 7 days, APAP group received single dose of acetaminophen on day 7 and SLY group received silymarin for 7 days. Lop 25 and Lop 50 received low and high doses of Lop (25 μg/kg BW and 50 μg/kg BW), respectively, for 7 days, while Lat 25 and Lat 50 received low and high doses of Lat (25 μg/kg BW and 50 μg/kg BW) for 7 days. On day 7, all animals except Vehicle group kept fasted for 18 h and received APAP i. p. 400 mg/kg BW. After 20 h of APAP administration, the animals anesthetized with light chloroform and scarified by cervical decapitation. The blood serum and liver tissue samples were collected for biochemical and histopathological analysis. Liver function tests (LFTs) including lactate deydrogenase (LDH), alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST) and direct bilirubin) were used as biochemical parameters. While catalase (CAT), superoxide dismutase (SOD) and reduced glutathione (GSH) were taken as anti-oxidant enzymes. ResultsSignificant increase in levels of ALT, AST, ALP, LDH and direct bilirubin, and significant decrease in concentration of anti-oxidant enzymes (SOD, CAT and GSH) was observed in APAP-treated group. Similarly, histological slides showed obvious signs of damage to liver cells, reflecting acetaminophen induced hepatotoxicity. Treatment of test animals with phytosterols resulted in significant recovery of LFTs profile and concentration of anti-oxidant enzymes. Similarly, significant improvement of liver tissues was noted in histological analysis. ConclusionsBoth phytosterols possessed hepatoprotective potential and should be further evaluated for acute toxicity studies and pharmacokinetics/pharmacodynamics profile.

Hepatoprotective Effects of the Leaves of Agauria salicifolia against Acetaminophen-Induced Liver Injury in Mice

Agauria salicifolia (Ericaceae) is a medicinal plant traditionally used for the treatment of liver ailments. The present study investigates the hepatoprotective effect of the residual aqueous fraction (RAF) of Agauria salicifolia on acetaminophen (APAP)-induced liver damage. The ethanol extract obtained as maceration of the dried leaves, was fractionated into hexane, ethyl acetate and residual aqueous fractions. Adult mice of both sexes were pre-treated with the reference drug silymarin (50 mg/kg) or RAF (100 and 200 mg/kg) during 6 days followed by a single administration of APAP (500 mg/kg) on day 7. The hepatoprotective effect and the contribution of antioxidant activities were evaluated by determining the level of transaminases in serum samples, the levels of proteins, nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase in liver homogenates. Histological analyses of liver slides were also performed. RAF at the doses of 100 and 200 mg/kg and silymarin significantly (p increase in proteins (p p p p

Folic acid as preventive therapy for hearing loss: effect of ototoxic drug consumption

AbstractIntroductionAge-related hearing loss (ARHL) is a sensory impairment, with a dramatic increase in its incidence, which is caused by genetic and environmental factors such as noise and ototoxic drugs. Recent studies correlated ARHL to elevated plasma homocysteine (Hcy) by folate deficiency, suggesting that reduction of Hcy levels by folate supplementation could potentially ameliorate ARHL.Hyperhomocysteinemia (HHcy), a status that contributes to ARHL, may also arise from malfunction of Hcy remethylation by betaine homocysteine S-methyltransferases (BHMTs) and methionine synthase in the methionine cycle. The expression and/or activity of these enzymes may be altered by ototoxic drugs, including paracetamol (APAP).ObjectiveTo determine the effect of APAP in cochlear morphology and function of control and Bhmt-/- mice, and to analyze putative preventive effects of folic acid (FA) supplementation.Materials and MethodsTwo-month-old Bhmt-/- mice (n = 47), with greater dependence on folate metabolism for Hcy remethylation, and Bhmt + / + mice (n = 42) were fed control or FA supplemented diets for 30 days. The last day APAP (250 mg/kg) or placebo were injected intraperitoneally.Hearing was evaluated by recording auditory brainstem responses (ABR) at the beginning of the experiment and after treatments. Picrosirius red staining was used for evaluation of the cochlear lateral wall cytoarchitecture. Plasma and hepatic metabolite levels were determined by HPLC or on Spinlab 100® autoanalyzer.ResultsLoss of Bhmt expression induced HHcy, but an impact on hearing acuity was not observed. Acute APAP administration did not induce ABR threshold shifts. However, following ototoxic treatment, changes of 5–17% in the areas of the stria vascularis and spiral ligament were detected between Bhmt-/- mice under different dietary treatments; cochlear structures of Bhmt-/- mice receiving APAP plus FA supplementation resemble those of the control group. APAP increases susceptibility to ototoxic damage in the presence of HHcy.DiscussionBHMT plays a central role in cochlear methionine metabolism. FA supplementation modulates Hcy levels, contributing to a proper remethylation status that prevents ARHL.

Intrabody against prolyl hydroxylase 2 ameliorates acetaminophen-induced acute liver injury in mice via concomitant promotion of angiogenesis and redox homeostasis

Acetaminophen (APAP) overdose has become the most common cause of drug-induced acute liver failure. Angiogenesis and redox homeostasis play an important role in liver protection and repair of APAP-induced acute liver injury (AILI). Hypoxia inducible factor-1 (HIF-1) is a transcription factor that plays a crucial role in regulating the expression of genes associated with angiogenesis, redox homeostasis and energy balance. Prolyl hydroxylase 2 (PHD2) predominantly hydroxylates proline residues in HIF-1α to promote its degradation. In our previous study, we reported an intrabody against PHD2 (ER-INP) that enhances angiogenesis by blocking PHD2 activity to increase HIF-1α abundance and activity. The present study was designed to explore the role and possible mechanisms of ER-INP in AILI in mice. Mice were pretreated intravenously with ER-INP before intraperitoneal injection of APAP to induce AILI. The results showed that pretreatment with ER-INP dramatically decreased the high ALT and AST activities and significantly ameliorated the centrilobular necrosis induced by APAP administration. ER-INP expression promoted angiogenesis in vivo by upregulating the mRNA and protein levels of HIF-1α target genes. Meanwhile, ER-INP pretreatment restored redox homeostasis, verified by reinforcement of PRDX4 activity and suppression of GSH depletion. This study demonstrated that ER-INP protects against AILI in part by increasing angiogenesis and maintaining redox homeostasis. These results indicate that ER-INP may provide a potential liver protection strategy against AILI in the future.

High Molecular Weight Kininogen but Not Factor XII Deficiency Attenuates Acetaminophen-Induced Liver Injury in Mice

Acetaminophen (APAP) toxicity is a common cause of acute liver failure (ALF). Dysregulation of coagulation is well documented in ALF patients. Recent reports indicate that small subset of ALF patients (~10%) exhibit spontaneous bleeding, while thrombotic complications are more frequent. Animal studies demonstrated that the inhibition of tissue factor-initiated thrombin generation attenuates APAP-induced liver injury in mice via both fibrin- and protease activated receptor 1-dependent mechanisms. Activation of FXII leads to two events: propagation of coagulation by activation of FXI (intrinsic pathway) and activation of plasma prekallikrein to kallikrein, with subsequent cleavage of high molecular weight kininogen (HK) into bradykinin (BK) and cleaved HK fragments (contact pathway). There is a growing interest in therapeutic targeting of FXII or FXI to prevent thrombosis, primarily because this goal may be attainable without incurring a significant bleeding risk. In this study, we evaluated if the intrinsic coagulation pathway contributes to the activation of coagulation and liver injury in a mouse model of APAP-induced ALF. We postulated that targeting FXII would attenuate coagulation and reduce liver injury without affecting hemostasis. In addition, FXII deficiency could provide a further protection by preventing activation of contact pathway and subsequent reduction of inflammatory response. We used 12 weeks old FXII, FXI, prekallkrein and HK deficient male mice and their respective WT controls. Mice were fasted for 16 hours followed by a single intraperitoneal injection of APAP (400 mg/kg) or sterile saline. Livers and plasma samples were collected 6 and 24 hours after injections. Hepatocellular necrosis was determined on liver sections stained with H&E. Plasma levels of alanine transaminase (ALT- marker of liver injury), thrombin-antithrombin (TAT) complexes, plasmin-α2 antiplasmin (PAP) complexes and interleukin-6 were analyzed using commercially available assays. HK cleavage was determined using BK ELISA, Western blot, and mass spectrometry analysis. Consistently with previously published data, plasma levels of ALT, TAT and IL-6, as well as liver injury scores were significantly elevated in APAP-challenged mice compared to saline-injected WT mice at both 6 and 24 hours. Surprisingly, neither FXII, FXI, nor prekallikrein deficiency had statistically significant effects on any of these parameters in APAP-challenged mice at either time point. Interestingly, however, plasma levels of ALT were significantly attenuated in HK-/- mice (n=23-34) compared to HK+/+ (n=19-31) controls at both 6 (1278 ± 184 vs. 1850 ± 235 U/L; p<0.05) and 24 hours (1546 ± 302 vs. 3857 ± 493 U/L; p<0.01) after injection of APAP. The attenuation of liver injury was observed despite the lack of a significant effect on APAP-induced coagulation activation (plasma TAT levels [mean±SEM]: 72.9 ± 8.2 μg/L in HK+/+ vs. 70.8 ± 4.3 μg/L in HK-/-). HK deficiency also reduced plasma levels of IL-6 (75.8%; p<0.001), number of infiltrating neutrophils in liver (59.8%; p<0.001) and liver necrotic area (28.1%; p<0.001) 24 hours after APAP challenge. Importantly, the protective effects of HK deficiency were completely reversed by the injection of HK protein into APAP-treated HK-/- mice. It has been previously shown that plasminogen deficiency protects against APAP-induced liver injury. Indeed, APAP administration activated the fibrinolytic system in WT mice as demonstrated by increased plasma levels of active tissue plasminogen activator (19.5 fold; p<0.01) and PAP complexes (64.4 fold; p<0.01). Using Western blotting analysis, BK ELISA, and mass spectrometry analysis we demonstrated that plasmin efficiently cleaves HK in a buffer system as well as in mouse and human plasma. Importantly, plasmin mediated cleavage of HK resulted in the release of BK and generation of cleaved HK fragments. In summary, our data indicate that the extrinsic but not the intrinsic coagulation pathway drives the coagulation-mediated pathologies associated with APAP-induced liver injury in mice. HK contributes to the liver injury independently of thrombin generation. We propose that in APAP-challenged mice, HK cleavage and downstream hepatotoxicity, are mediated by plasmin rather than FXIIa-dependent generation of kallikrein. Disclosures McCrae: Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees. Key:Uniqure BV: Research Funding.

Intestinal Epithelial Chemokine (C-C Motif) Ligand 7 Overexpression Enhances Acetaminophen-Induced Hepatotoxicity in Mice

Acetaminophen (APAP) overdose-induced hepatotoxicity is the leading cause of drug-induced liver injury worldwide. The related injury pathogenesis is mainly focused on the liver. Here, the authors report that gut barrier disruption may also be involved in APAP hepatotoxicity. APAP administration led to gut leakiness and colonic epithelial chemokine (C-C motif) ligand 7 (CCL7) up-regulation. Intestinal epithelial cell (IEC)-specific CCL7 transgenic mice (CCL7tgIEC mice) showed markedly increased myosin light chain kinase phosphorylation, and elevated gut permeability and bacterial translocation into the liver compared to wild-type mice. Global transcriptome analysis revealed that the expression of hepatic proinflammatory genes was enhanced in CCL7tgIEC mice compared with wild-type animals. Moreover, CCL7 overexpression in intestinal epithelial cells significantly augmented APAP-induced acute liver injury. These data provide new evidence that dysfunction of CCL7-mediated gut barrier integrity may be an important contributor to APAP-induced hepatotoxicity.

A TYLENOL A DAY BRINGS THE PATIENT TO THE BAY: A CASE OF USUAL DOSING ACETAMINOPHEN TOXICITY

SESSION TITLE: Wednesday Medical Student/Resident Case Report Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/23/2019 09:45 AM - 10:45 AM INTRODUCTION: Acetaminophen (APAP) toxicity is responsible for nearly 50% of acute liver failure (ALF) in the United States (1). Most cases are not deliberate overdoses (2). Chronic administration of APAP is associated with additional toxic pathways, leading to organ dysfunction. Presented is a case of ALF due to therapeutic doses of APAP with toxicity from the metabolite 5-oxoproline (pyroglutamic acid). CASE PRESENTATION: An 80 year old man with hypertension, diabetes, and chronic lower back pain presented with nausea and vomiting found to have fulminant liver failure with AST 15,000 and ALT 4,037 along with an anion gap metabolic acidosis. APAP levels ranged between 29 and 34 (10-30 ug/ml). The patient took only two to three APAP tablets daily for chronic lower back pain for the past 2 years as confirmed by family. He denied taking any other medications or herbal supplements. Ethanol, salicylate and CPK levels were normal. IR-guided liver biopsy confirmed APAP toxicity as culprit for ALF. His liver failure progressed with encephalopathy requiring intubation and rapidly worsening renal failure. The diagnosis of 5-oxoproline intoxication was made and after a week of continuous N-acetylcysteine infusion, his ALF resolved. DISCUSSION: Over 10,000 hospitalizations in the United States are due to APAP toxicity, and what is most concerning is that more than 50% of these cases are unintentional and only in the setting of therapeutic use (2, 5). Typically, in acute APAP toxicity the hepatic injury is due to the accumulation of hepatoxic N-acetyl-para-benzoquinoeimine (NAPQI) via depletion of hepatic glutathione stores. In these cases N-acetylcysteine (NAC) increases and repletes glutathione storage by being a glutathione synthesis precursor. However, in chronic APAP use even when taken in recommended doses a different toxic metabolite can accumulate. This metabolite is known as 5-oxoproline is overproduced due to the activation of an ATP depleting cycle (Figure 1). The condition generally resolves with discontinuation of acetaminophen and general supportive measures. Although many reports suggest that administration of N-acetylcysteine may accelerate recovery, this has not been proven. Here NAC increases the negative feedback of γ-glutamylcysteine synthetase, resulting in reduced generation of pyroglutamic acid from γ-glutamylcysteine (7). Other factors lower the dose threshold for APAP toxicity, including alcohol use, malnutrition, use of CYP 450-inducers, and delayed gastric emptying (3). In our patient, malnutrition and ileus may have increased his risk for APAP toxicity, which were both diagnosed during the hospitalization. We present a case of APAP toxicity with likely contribution from the rarely described 5-oxoproline. CONCLUSIONS: ALF from APAP toxicity is not only associated with acute overdose, but also with usual administration. One of these processes is due to the accumulation of 5-oxoproline. Reference #1: Ostapowicz G, et al. “Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States.” Annals of Internal Medicine 2002; 137:947-954. Reference #2: Ghanem CI. “Acetaminophen from liver to brain: new insights into drug pharmacological action and toxicity.” Pharmacological Research. 2016; 109:119–131. Reference #3: Bunchorntavakul C, Reddy KR. “Acetaminophen (APAP or N-Acetyl-p-Aminophenol) and acute liver failure.” Clinical Liver Disease. 2018; 22:325-246. DISCLOSURES: no disclosure on file for Alfredo Astua; No relevant relationships by Dawn Maldonado, source=Web Response No relevant relationships by Michael Megally, source=Web Response

JNK Signaling Pathway Mediates Acetaminophen-Induced Hepatotoxicity Accompanied by Changes of Glutathione S-Transferase A1 Content and Expression.

Acetaminophen (APAP) is an analgesic–antipyretic drug and widely used in clinics. Its overdose may cause serious liver damage. Here, we examined the mechanistic role of c-Jun N-terminal kinase (JNK) signaling pathway in liver injury induced by different doses of APAP. Male mice were treated with APAP (150 and 175 mg·kg−1), and meanwhile JNK inhibitor SP600125 was used to interfere APAP-induced liver damage. The results showed that JNK signaling pathway was activated by APAP in a dose-dependent manner. C-Jun N-terminal kinase inhibitor decreased JNK and c-Jun activation significantly (P < 0.01) at 175 mg·kg−1 APAP dose, and phosphorylation levels of upstream proteins of JNK were also decreased markedly (P < 0.05). In addition, serum aminotransferases activities and hepatic oxidative stress increased in a dose-dependent manner with APAP treatment, but the levels of aminotransferases and oxidative stress decreased in mice treated with JNK inhibitor, which implied that JNK inhibition ameliorated APAP-induced liver damage. It was observed that apoptosis was increased in APAP-induced liver injury, and SP600125 can attenuate apoptosis through the inhibition of JNK phosphorylation. Meanwhile, glutathione S-transferases A1 (GSTA1) content in serum was enhanced, while GSTA1 content and expression in liver reduced significantly with administration of APAP (150 and 175 mg·kg−1). After inhibiting JNK, GSTA1 content in serum decreased significantly (P < 0.01); meanwhile, GSTA1 content and expression in liver enhanced. These findings suggested that JNK signaling pathway mediated APAP-induced hepatic injury, which was accompanied by varying GSTA1 content and expression in liver and serum.

Acetaminophen induces liver injury and depletes glutathione in mice brain: Prevention by Moringa oleifera extract

Acetaminophen (APAP) overdose is among the leading causes of acute liver failure worldwide. Moringa oleifera is used as food additive and, as phytomedicine for its antioxidant, analgesic, anti-pyretic, anti-inflammatory, immuno-modulatory and hepatoprotective properties. The present study investigated the effect of Moringa oleifera aqueous extract on acetaminophen-induced acute liver injury and glutathione depletion in mice brain. Moringa oleifera leaf aqueous extract (100 and 200 mg/kg, p.o.), distilled water and ascorbic acid (50 mg/kg, p.o.) were administered to mice 1 and 12 h before acetaminophen (500 mg/kg, p.o.). Mice were sacrificed 6 h after APAP treatment. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) serum activities; malondialdehyde (MDA) and nitrite liver level, tumor necrosis factor alpha (TNF-α or TNF) and interleukin-1β (IL-1β) serum levels were assessed. Reduced glutathione (GSH) was evaluated in brain and liver tissues. Histological analyses of liver section were performed by hematoxylin/eosin (H&E) staining. APAP administration induced liver injury with significant (P < .01) depletion of liver and brain GSH level. ALT, ALP and AST serum activities (P < .05), MDA and nitrite liver content, TNF and IL-1β in the serum were increased (P < .05). Moringa oleifera extract significantly (P < .05) reduced liver injury, inhibiting ALT, AST and ALP serum activities. TNF and IL-1β serum levels, nitrite, and MDA liver content were significantly (P < .05) reduced by M. oleifera extract, compared to APAP-treated mice. GSH depletion in brain and liver was inhibited by M. oleifera extract. Histological injuries (vacuolization and inflammation) by acetaminophen were remarkably prevented by M. oleifera administration. Results of the present study revealed that M. oleifera leaf extract strongly protects mice against acetaminophen hepatotoxicity, exhibiting antioxidant and anti-inflammatory effects. The present results support ethnopharmacological uses of M. oleifera for the treatment of liver diseases.

Comparison of toxic responses to acetaminophen challenge in ICR mice originating from different sources

Acetaminophen (APAP) is the most common antipyretic analgesic worldwide. However, APAP overdose causes severe liver injury, especially centrilobular necrosis, in humans and experimental animals. At therapeutic dosage, APAP is mainly metabolized by sulfation and glucuronidation, and partly by cytochrome P450–mediated oxidation. However, APAP overdose results in production of excess reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), by cytochromes P450; NAPQI overwhelms the level of glutathione (GSH), which could otherwise detoxify it. NAPQI binds covalently to proteins, leading to cell death. A number of studies aimed at the prevention and treatment of APAP-induced toxicity are underway. Rats are more resistant than mice to APAP hepatotoxicity, and thus mouse models are mainly used. In the present study, we compared the toxic responses induced by APAP overdose in the liver of ICR mice obtained from three different sources and evaluated the usability of the Korl:ICR stock established by the National Institute of Food and Drug Safety Evaluation in Korea. Administration of APAP (300 mg/kg) by intraperitoneal injection into male ICR mice enhanced CYP2E1 protein expression and depleted hepatic GSH level 2 h after treatment accompanied with significantly increased level of hepatic malondialdehyde, a product of lipid peroxidation. Regardless of the source of the mice, hepatotoxicity, as evidenced by activity of serum alanine aminotransferase, increased from 8 h and peaked at 24 h after APAP treatment. In summary, hepatotoxicity was induced after the onset of oxidative stress by overdose of APAP, and the response was the same over time among mice of different origins.

Augmenter of Liver Regeneration Protects Against Acetaminophen-Induced Acute Liver Injury in Mice by Promoting Autophagy.

Most cases of acute liver failure are caused by acetaminophen (APAP) overdose. Oxidative stress is a key factor in APAP toxicity. Although augmenter of liver regeneration (ALR) has both antioxidative and antiapoptotic effects, its therapeutic potential in APAP hepatotoxicity remains unknown. The current study assessed the protective effects and associated mechanisms of ALR against APAP-induced acute liver injury in female BALB/c mice. We found that serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, intrahepatic hemorrhage and necrosis were increased at 3, 6, 12, 24, 48, and 72 h after 600 mg/kg APAP i.p. injection. During the early stages (before 12 h) of acute liver injury, ALR levels increased significantly, followed by a decrease to control level at 24 h after APAP administration. ALR treatment increased autophagosomes, promoted the conversion of LC3 I to LC3 II, and the degradation of p62. ALR attenuated APAP-stimulated increases in ALT, AST, myeloperoxidase (MPO), malondialdehyde (MDA), and reactive oxidative species (ROS) levels; intrahepatic hemorrhage; and necrosis as well as superoxide dismutase (SOD) and Glutathione (GSH) depletion. We found that APAP caused release of the mitochondrial intermembrane proteins apoptosis-inducing factor (AIF) and cytochrome c and that ALR inhibited this change. Meanwhile, ALR decreased expression of cleaved-caspase 3 and apoptotic cells. Subsequently, we investigated the significance of autophagy in APAP-induced acute liver injury by treatment with 3-methyladenine (3-MA), which were classical pharmaceuticals for suppressing autophagy. ALR directly induced autophagy flux; and the inhibition of autophagy reversed the beneficial effects of ALR on hepatotoxicity. Our findings suggest that ALR protects against APAP hepatotoxicity by activating the autophagy pathway.

SUN-153 ACCUMULATION OF ACETAMINOPHEN METABOLITES EXERTS KIDNEY PROTECTIVE EFFECT IN RENAL FAILURE RAT MODEL

Acetaminophen (APAP) is recommended preferential use in pain management of chronic kidney disease (CKD) patients owing to fewer side effects in clinical dose. Although it is highly safe drug, usefulness of APAP is not consistent during long-term dosing in renal failure. So far, we have obtained the results that repeated administration of APAP does not worsen renal function in the adenine-induced renal failure model rats, suggesting that APAP might have antioxidant effect. In this study, we focused on the antioxidant effect of APAP and its metabolites, evaluated association between long-term APAP administration and renal function or oxidative stress in vivo and in vitro. first, we carried out daily oral administration of APAP (150 or 750 mg / kg) for 12 weeks against 5/6 nephrectomy (Nx) renal failure model rat, and evaluated the biochemical parameters, oxidative stress markers, as well as the drug concentration of APAP and its metabolites. We also examined the antioxidant capacity of APAP using HUVEC and EPR in vitro. Long-term administration of APAP did not induce further renal dysfunction. Furthermore, the APAP treated group significant inhibited the production of plasma 8-OHdG. Also, results of DHE staining of the renal tissue indicated that an oxidative stress was suppressed by APAP administration. Interestingly, APAP metabolites, normally excreted into urine, accumulated in the plasma and renal tissue. The antioxidant effect of these metabolites was evaluated in vascular endothelial cells (HUVEC), APAP metabolites showed a significant ROS inhibitory effect of ROS induced by uremic toxin indoxyl sulfate. From these results, it is suggested that APAP metabolites accumulated by renal failure might exhibit renal protective effect due to a part its antioxidant capacity. in the Future by monitoring of APAP metabolites by TDM, it is possible to establish a more effective method of APAP administration, the efficacy of a new antioxidant effect of APAP metabolites can be expected.

Vitamin D protects against oxidative stress, inflammation and hepatorenal damage induced by acute paracetamol toxicity in rat

Acute paracetamol (APAP) toxicity is a leading cause of liver, and less commonly renal, injuries through oxidative stress and inflammation. Albeit vitamin D (VD) is a well-known anti-oxidant and anti-inflammatory hormone, there is no report on its potential protective/therapeutic actions against APAP acute toxicity. This study, therefore, measured the interplay between APAP toxicity and the hepatorenal expressions of the VD-metabolising enzymes (Cyp2R1, Cyp27b1 & cyp24a1), receptor (VDR) and binding protein (VDBP) alongside the effects of VD treatment on APAP-induced hepatorenal injuries. Thirty-two male rats were distributed equally into negative (NC) and positive (PC) controls besides VD prophylactic (P-VD) and therapeutic (T-VD) groups. All groups, except the NC, received a single oral dose of APAP (1200 mg/kg). The P-VD also received by intraperitoneal injection two cycles of VD3 (1000 IU/Kg/day; 5 days/week) prior to, and a third round after, APAP administration. Similarly, the T-VD group received VD3 (3000 IU/Kg/day) for five successive days post-APAP intoxication. Euthanasia was on the sixth day post-APAP toxicity. The PC group had marked alterations in the hepatorenal biochemical parameters, upregulation in cellular cleaved caspase-3 as well as pronounced increase in the numbers of apoptotic/necrotic cells by TUNEL technique. The PC group plasma levels of 25-hydroxyvitamin D (25-OH VD) also declined markedly and coincided with significant inhibitions in the expression of Cyp2R1 and Cyp27b1 enzymes and VDR, whereas the VDBP and Cyp24a1 increased substantially, in the hepatorenal tissues at the gene and protein levels compared with the NC group. Coherently, the lipid peroxidation marker (MDA) and pro-inflammatory cytokines (IL1β, IL6, IL17A, IFN-γ & TNF-α) augmented significantly, while the anti-oxidative markers (GSH, GPx & CAT) and anti-inflammatory cytokines (IL10 & IL22) diminished substantially, in the PC hepatorenal tissues. Both VD regimens alleviated the APAP-induced hepatorenal damages and restored the 25-OH VD levels together with the hepatorenal expression of Cyp2R1, Cyp27b1, Cyp24a1, VDR and VDBP. Additionally, MDA and all the targeted pro-inflammatory cytokines declined, whereas all the anti-oxidative and anti-inflammatory markers increased, in both VD groups hepatorenal tissues and the results were significantly different than the PC group. Although the P-VD anti-inflammatory and anti-oxidative stress actions were more pronounced than the T-VD group, the results remained markedly abnormal than the NC group. In conclusion, this report is the first to reveal that the circulatory VD levels alongside the hepatorenal VD-metabolising enzymes and VDR are pathologically altered following acute APAP toxicity. Moreover, the prophylactic protocol showed better anti-oxidative and anti-inflammatory effects than the therapeutic regimen against APAP-induced hepatorenal injuries.