N-acetyl-p-benzoquinone Imine Research Articles

Schisandrin C improves acetaminophen-induced liver injury in mice by regulating Nrf2 signaling pathway

Excess acetaminophen(APAP) can be converted by the cytochrome P450 system to the toxic metabolite N-acetyl-p-benzoquinoneimine(NAPQI), which consumes glutathione(GSH). When GSH is depleted, NAPQI covalently binds with proteins, inducing mitochondrial dysfunction and oxidative stress and thereby leading to hepatotoxicity. Schisandrin C(SinC) is a dibenzocyclooctadiene derivative isolated from Schisandra chinensis. Although there is some evidence showing that SinC has hepatoprotective activity, its protective effect and mechanism on APAP-induced liver injury remain unclear. In this paper, an acute liver injury mouse model was established by intraperitoneal injection of APAP at a dose of 400 mg·kg~(-1) to evaluate the effect of SinC administration on the APAP-induced liver injury and its mechanism through an animal experiment. At the same time, a potential candidate drug was provi-ded for traditional Chinese medicine(TCM) prevention and treatment of overdose APAP-induced liver injury. In the APAP-induced liver injury mouse model, we found that SinC can relieve hepatic histopathological lesions and significantly reduce the activities of alanine aminotransferase(ALT), aspartate aminotransferase(AST) and alkaline phosphatase(ALP). It was also capable of increasing the content of GSH and superoxide dismutase(SOD) and decreasing the levels of total bilirubin(TBIL), direct bilirubin(DBIL), malondialdehyde(MDA), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α). Further analysis showed that SinC decreased the content of CYP2 E1 in liver tissues at protein and mRNA levels and increased nuclear factor erythroid 2-related factor 2(Nrf2) and the expression of its downstream targets(including HO-1, NQO1 and GCLC). Taken together, the above results indicate that SinC can alleviate APAP-induced liver injury by reducing the expression of CYP2 E1, suppressing apoptosis, improving inflammatory response and activating the Nrf2 signaling pathway to inhibit oxidative stress.

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.

Galangin Nanoparticles Protect Acetaminophen-Induced Liver Injury: A Biochemical and Histopathological Approach.

One of the main causes of acute liver failure is overdose with acetaminophen. Excessive consumption of acetaminophen leads to the production of NAPQI (N-acetyl-p-benzoquinone imine) through the activity of the enzyme cytochrome c oxidase. For this purpose, the effect of galangin nanoparticles with antioxidant activities will be evaluated for the treatment of acetaminophen-induced hepatotoxicity. In this study, after the synthesis of galangin nanoparticles and particle size determination, mice were divided into six groups. Before treatment, a single dose (350 mg/kg) of acetaminophen was administered by gavage in all groups. The activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), as well as biochemical factors FRAP and MDA in serum were measured and a histopathological study was performed. The prepared nanoparticles produced in this research were characterized by the SEM, DLS, and ZETA potential, and the average particle size was obtained in the range of 150 nm. Serum levels of liver enzymes (AST and ALT) in the nanoparticle group decreased significantly compared with the control group (P < 0.05). In the group without treatment, the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes increased significantly compared with the treatment groups. Also, galangin nanoparticles, at a dose of 20 mg/kg, improve cell damage in hepatocytes and preserve the tissue structure of the liver. Galangin nanoparticles reduce the acetaminophen-induced hepatotoxicity by reducing the number of liver function indices. According to our findings, the liver-protective effects of the nanoparticle may be due to its antioxidant properties.

Metabolomic Evaluation of N-Acetyl-p-Benzoquinone Imine Protein Adduct Formation with Therapeutic Acetaminophen Administration: Sex-based Physiologic Differences.

Acetaminophen (APAP)-associated transaminase elevation, induced by N-acetyl-p-benzoquinone imine (NAPQI) protein adduction, remains an area of research interest. Distinct from known genetic, physiologic, and dosage associations dictating severity of hepatic injury, no known factors predict an absence of protein adduct formation at therapeutic APAP dosing. Sex-based physiology is predictive of APAP-induced protein adduct formation and differential metabolite expression at therapeutic doses. This retrospective study interrogated serum samples collected for a prior study investigating fluctuations of alanine aminotransferase (ALT) over time with 4G daily APAP dosing for ≥ 16days in subjects from Denver, Colorado. Subjects were grouped by adduct formation (n = 184) vs no adducts (n = 20). Samples were run on ultra-high-performance liquid chromatography mass spectrometry from study days 0, 7, 16, and 31. Significant metabolite expressions were identified using t-tests with false discovery rate correction (FDR), partial least squares discriminant, and ANOVA simultaneous comparison analyses. Demographic and clinical data were explored using t-tests with FDR (age, weight, BMI, ALT) and Chi-square (sex, ethnicity, race) analyses. In pre-treatment samples, relative quantitation caprylic acid was expressed ninefold higher and 6-carboxyhexanoate was expressed threefold lower in subjects who did not develop adducts. Lactate had greater expression in the no adducts group (p = 0.001). Using absolute quantitation, glutathione was expressed 2.6-fold greater among no adduct subjects. Odds of males developing NAPQI protein adducts at therapeutic APAP dosing were 5.91 times lower than females (95% CI = 2.3-14.9; p = 0.0001). Multiple metabolites were differentially expressed based on adduct group and sex. Metabolites were identified unique to adduct development independent of sex. At therapeutic APAP dosing, males were less likely to develop APAP protein adducts. Further research into lipid biosynthesis and metabolism may provide further insight into physiology associated with adduct production.

Protective effect of probiotics against acetaminophen induced nephrotoxicity.

Acetaminophen (APAP) is commonly prescribed as an antipyretic and analgesic agent in the practical field. Like every other drug(s), APAP also undergo metabolism by oxidation or conjugation by glucuronate and sulphate to form the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). Moreover, the NAPQI is detoxified by conjugation with reduced glutathione (GSH). Interestingly, APAP is also metabolized in the kidney by deacetylation reaction in the presence of N-deacetylase enzyme into another severely toxic but minor metabolite, p-aminophenol. Both NAPQI and p-aminophenol shows nephrotoxicity as well as hepatotoxicity. Hence, the long-term therapeutic dose use and unnecessary overdose of APAP are of great concern as prolonged negligence may cost the nephrotoxicity that may lead to uremia and finally to kidney failure. It has recently been investigated that probiotic supplementation inhibits the sequential events associated with APAP-induced nephrotoxicity. This review emphasizes the role of different probiotics that have already been investigated in nephrotoxicity or uremia caused by APAP overdose.

Mitochondrial versus microsomal bioactivation of paracetamol by human liver and kidney tissues

Mitochondria appeared to be a major target for paracetamol (PAR)-induced hepatotoxicity. Studies suggested that microsomal CYPs catalyse bioactivation of PAR to N-acetyl-p-benzoquinone imine (NAPQI), which alkylates mitochondrial proteins, and causes transmission of death signal from mitochondria to nucleus. We hypothesised that local formation of NAPQI within mitochondria seems more likely compared to the translocation of NAPQI. We therefore tested whether the formation of NAPQI may be catalysed by mitochondrial CYPs. Cellular fractions were isolated from human liver and kidney to compare the metabolic capacities. Liver and kidney mitochondria are capable to generate NAPQI. Mitochondrial CYP2E1 and CYP3A4 activities were comparable to the microsomal counterparts in both organs. Previously reported higher kidney microsomal CYP2E1 activity in men compared women were observed in mitochondrial CYP2E1 as well in the present study. On the other hand, no correlation between kidney CYP2E1 activity and quantity of NAPQI formation, as well as no induction on mitochondrial permeability transition pore (mPTP) opening by PAR in kidney mitochondria strongly suggested a different toxicity mechanism in this organ.

Fenton-reaction-triggered metabolism of acetaminophen for enhanced cancer therapy

Acetaminophen (APAP), a classic nonsteroidal anti-inflammatory drug (NSAID), has attracted much attention due to the overdose-induced hepatotoxicity in the past several decades. N-Acetyl-p-benzoquinone imine (NAPQI), the P450-dependent metabolism of APAP, leads to GSH depletion, protein binding, mitochondrial oxidative stress, and eventually the liver injury. Herein, we develop a Fe-based metal-organic framework (MOF) to deliver and transform acetaminophen into toxic “chemo” drug through the cascade reaction for enhanced cancer therapy. In the acidic tumor microenvironment, the Fe-based MOF collapses and releases abundant Fe ions to generate hydroxyl radicals (•OH) via Fenton reaction, subsequently catalyzing nontoxic APAP into toxic NAPQI. Meanwhile, NAPQI depletes intracellular glutathione (GSH) rapidly, leading to alleviating the antioxidant ability of cancer cells and amplifying Fenton activity. The intracellular oxidative stress and the toxic metabolite of APAP can provide a synergistic effect on antitumor activity.

Therapeutic Potential of Justicia secundaon Oxidative Stress Indices in N‐Acetyl‐P‐Benzoquinone Imine Induced‐ Hepatotoxic Rats

The therapeutic potential of Hot Water Leaf Extract of Justicia secunda(HWEJS) on oxidative stress indices in paracetamol (N‐Acetyl‐p‐Benzoquinone Imine) induced hepatotoxic rats was investigated. Justicia secunda is a blood boosting plant used traditionally with so many medicinal activities. Five groups of 10 rats each were used for the study, GpD was the control given n‐saline and the test grps A,B and C were administered with 2000mg/kg N‐Acetyl‐p‐Benzoquinone Imine (NAPQI) and after 24hrs, groupsB and C were administered with 200 and 400mg/kg of HWEJS respectively for 7 days. The body weights were also measured. Results of the study showed a relative weight loss of all the groups except the control in the first 2 days thereafter a relative increase was observed throughout the duration of the experiment. This could be as a result of the ameliorative effect of the extract against NAPQI overdose. Oxidative stress indices in the animals treated with NAPQI (as shown in the figure) increased the levels of the oxidative enzymes while the 200 and 400mg/kg HWEJS reduced them except in Malondealdehyde which showed a reduced level with NAPQI with increased levels on the treated groups. Gpx showed a significant (p&lt;0.05) decrease at 200mg/kg and 400mg/kg. CAT and SOD decreased significantly (p&lt;0.05) at 400mg/kg and non‐significant (p&gt;0.05) at 200mg/kg respectively relative to control. MDA, an indicator for lipid peroxidation recorded non‐significant (p.&gt;0.05) levels for all the test groups when compared to the control. These results suggest that the oral administration (p.o) of hot water extract of the leaves of J.S have antioxidant activities at the doses of 200 and 400mg/kg in the hepatotoxic rats. The combination of SOD, CAT and GPx enzymes constitute a first line antioxidant defense system that plays a crucial role in total defense mechanisms during metabolic processes. In the extract treated animals, SOD showed that it may have removed superoxide anions from oxidative insult by helping in breaking down potentially cellular harmful oxygen molecules while the CAT and GPX protected the cells from damage due to free radicals like the peroxides. This result could suggest that the hot water extract of Justicia secunda can be used in conditions where harmful oxygen molecules are believed to play a role in a disease or in oxidative stress‐related diseases as a therapeutic agent.

Molecular pathogenesis of acetaminophen-induced liver injury and its treatment options.

Acetaminophen, also known as N-acetyl-p-aminophenol (APAP), is commonly used as an antipyretic and analgesic agent. APAP overdose can induce hepatic toxicity, known as acetaminophen-induced liver injury (AILI). However, therapeutic doses of APAP can also induce AILI in patients with excessive alcohol intake or who are fasting. Hence, there is a need to understand the potential pathological mechanisms underlying AILI. In this review, we summarize three main mechanisms involved in the pathogenesis of AILI: hepatocyte necrosis, sterile inflammation, and hepatocyte regeneration. The relevant factors are elucidated and discussed. For instance, N-acetyl-p-benzoquinone imine (NAPQI) protein adducts trigger mitochondrial oxidative/nitrosative stress during hepatocyte necrosis, danger-associated molecular patterns (DAMPs) are released to elicit sterile inflammation, and certain growth factors contribute to liver regeneration. Finally, we describe the current potential treatment options for AILI patients and promising novel strategies available to researchers and pharmacists. This review provides a clearer understanding of AILI-related mechanisms to guide drug screening and selection for the clinical treatment of AILI patients in the future.

Metabolite alteration analysis of acetaminophen-induced liver injury using a mass microscope.

Acetaminophen (APAP)-induced liver injury (APAP-ILI), which occurs during APAP overdose, has been extensively studied. The production of N-acetyl-p-benzoquinone imine (NAPQI), the reactive metabolite of APAP, primarily contributes to liver injury. However, the mechanism underlying APAP-ILI has not been fully characterized. For further clarification, it is important to consider drug localization and endogenous substances in the injured liver. Herein, we show the localization of NAPQI metabolites and the injury site-specific changes in endogenous substances in the rat liver following APAP overdose using a mass microscope. Our results of on-tissue derivatization matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) showed that the glutathione metabolite of APAP, a detoxified metabolite of NAPQI, localized in the damaged central vein region in the rat liver following APAP administration. Moreover, in the conventional MALDI-MSI, the intensities of some phospholipids, phosphocreatine, and ceramides decreased or increased in the damaged regions compared with those in non-damaged regions. Phosphocreatine was localized in the damaged cells, whereas its related mitochondrial creatine kinase was localized in the non-damaged cells. These results are expected to contribute to further elucidation of the mechanisms underlying APAP-ILI. Our findings illustrate the localization of NAPQI-related metabolites and endogenous molecules associated with APAP-ILI, which may be related to apoptosis or metabolic adaptation ultimately protecting the cells. As MALDI-MSI can analyze and differentiate regions with tissue damage, it is a valuable tool for analyzing the mechanism underlying drug-induced liver injury to identify novel biomarkers.

Tanshinone IIA prevents acetaminophen-induced nephrotoxicity through the activation of the Nrf2-Mrp2/4 pathway in mice.

It's known that APAP overdose often leads to hepatotoxicity and nephrotoxicity. In the present study, we investigated the preventative effect of Tan IIA on APAP-induced nephrotoxicity. Mice were orally administrated with Tan IIA (10 or 30 mg/kg/day) for 1 week and subsequently gavaged with 200 mg/kg of APAP. Tan IIA reduced APAP-induced nephrotoxicity as evidenced by histopathological evaluation and serum creatinine levels. Tan IIA pretreatment promoted the efflux of the toxic intermediate metabolite N-acetyl-p-benzoquinone imine (NAPQI), thus reduced its injury to mouse kidney. After Tan IIA pretreatment, a remarkable increase in mRNA and protein expression of Nrf2 and its target genes Mrp2 and Mrp4 was observed in Nrf2+/+ mice kidneys, however, no obvious change of Mrp2 and Mrp4 mRNA and protein expression was detected in Nrf2-/- mice kidneys. HK-2 cells were used for exploring the roles of Tan IIA in the Nrf2-MRPs pathway in vitro. Consistently, Tan IIA up-regulated the Nrf2-MRPs pathway and promoted the nuclear Nrf2 accumulation in HK-2 cells. Collectively, our findings suggested that Tan IIA facilitated the clearance of toxic intermediate metabolite NAPQI from the kidney through upregulation of the Nrf2-MRP2/4 pathway, thereby, performing preventive effects against APAP-induced nephrotoxicity.

Propacetamol in dogs: First description of its pharmacokinetics after intravenous and oral administration

Propacetamol is a prodrug form of paracetamol (APAP) licensed for human use as a pain reliever in postoperative care. It is prescribed if APAP cannot be administered orally or rectally to a patient and for patients in whom nonsteroidal anti-inflammatory drugs are contraindicated. In this study, we aimed to quantify the pharmacokinetics of APAP and its metabolites, paracetamol sulfate (PS), paracetamol glucuronide (PG), and N-acetyl-p-benzoquinone imine (NAPQI), after a single oral and intravenous (IV) administration of 30 mg/kg of propacetamol to six healthy adult Labrador dogs according to a 2 × 2 crossover study. The analyses were performed using a validated HPLC-MS/MS method.PS and PG exposures were higher than that of APAP, while NAPQI concentrations were constantly below the detection limit of the analytical method. IV propacetamol administration produced 30% more APAP than oral administration. However, propacetamol released a significantly lower amount of active moiety in dogs than in humans. The propacetamol dose administered in this study did not produce plasma APAP concentrations above the threshold sufficient to provide analgesia in adult humans (4 μg/mL). In conclusion, direct IV injection of APAP instead of propacetamol might be a better clinical option for pain relief in dogs.

Paracetamol Overdose in the Postoperative Postpartum Period

Paracetamol is the most commonly used medication for pain and fever worldwide and is readily available over the counter. The recommended maximum daily dose for an adult is three to four-gram. Paracetamol poisoning is the foremost cause of acute liver failure in the Western world and accounts for the highest percentage of drug overdose worldwide. Paracetamol administered in therapeutic doses is mainly conjugated with sulfate and glucuronide to form non-toxic metabolites via phase II metabolism. A small portion is metabolized to N-acetyl-p-benzoquinoneimine (NAPQI), a toxic metabolite. NAPQI is conjugated with glutathione and detoxified to form cysteine and mercapturic acid conjugates in normal circumstances. However, under certain conditions, NAPQI is not effectively detoxified due to depleting glutathione, causing hepatocellular necrosis and subsequently, liver failure. We present a case involving paracetamol overdose in a 35-year-old female who self-medicated on a large number of paracetamol pills daily for two weeks because of postoperative pain. This resulted in deranged liver enzymes. N-acetylcysteine was administered and led to clinical improvement and normalized LFTs. The case highlights patients’ ignorance of the dangers of paracetamol overdose and the importance of N-acetylcysteine in its management.

The Cytotoxicity and Nephroprotective Activity of the Ethanol Extracts of Angelica keiskei Koidzumi Stems and Leaves against the NAPQI-Induced Human Embryonic Kidney (HEK293) Cell Line.

Materials and Methods A. keiskei Koidzumi plant was collected from Mount Rinjani, Lombok, Indonesia, and was identified at the School of Biology Sciences and Technology, Bandung Institute of Technology, Indonesia. Extraction of the stems (ASE) and leaves (ALE) was performed by employing ethanol 70% for 3 × 24 h at 26°C. The cytotoxicity study of the extracts was assessed using the water-soluble tetrazolium salt-8 (WST-8) reagent on the HEK293 cell line, while the nephroprotective activity assay was determined on the NAPQI-induced HEK293 cell line. Results The WST-8 assay showed that the cytotoxicity IC50 of ASE = 2322 μg/mL and IC50 of ALE = 2283 μg/mL. The nephroprotective activity assay revealed that ASE possesses nephroprotective activity against the NAPQI-induced HEK293 cell line at 1161 μg/mL, while ALE does not show the nephroprotective activity. Conclusion Taken together, lower concentrations of ASE and ALE (<2000 μg/mL) are not toxic to the HEK293 cell line, and only ASE indicates the activity to protect the HEK293 cell line against NAPQI damage. This Japanese celery could be further explored for its potential as a plant-based nephroprotective drug.

The Timing and Effects of Low-Dose Ethanol Treatment on Acetaminophen-Induced Liver Injury.

Acetaminophen (APAP) overdose is the major cause of drug-induced liver injury and acute liver failure. Approximately 10% of APAP is metabolized by cytochrome P450 (CYP2E1) into toxic N-acetyl-p-benzoquinone imine (NAPQI). CYP2E1 also contributes to ethanol metabolism, especially during conditions of high blood ethanol concentration. Acute and chronic ethanol consumption appears to have opposite effects on APAP-induced liver injury. We determined the effects of different doses, pre- and post-treatment, and various schedules of ethanol exposure in APAP-induced liver injury. Treatment with ethanol (0.5 g/kg) after 1 h of APAP (300 mg/kg) administration decreased serum ALT levels, histopathological features, and inflammatory cell infiltration. Moreover, ethanol treatment 1 h after APAP treatment reduced APAP-induced liver injury compared with later administration. Interestingly, ethanol pretreatment did not provide any protective effect. Furthermore, ethanol treatment was associated with a significant decrease in ERK and AKT phosphorylation during the acute injury phase. Ethanol exposure also increased CYP2E1 expression and decreased PCNA expression during the liver regeneration phase.

COMBINATION THERAPY WITH N-ACETYLCYSTEINE AND 4-METHYLPYRAZOLE FOR ACETAMINOPHEN OVERDOSE

TOPIC: Critical Care TYPE: Medical Student/Resident Case Reports INTRODUCTION: Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States. We report a case of a patient who presented with acetaminophen overdose and was successfully treated with N-acetylcysteine (NAC) and 4-methylpyrazole (4-MP). CASE PRESENTATION: A 58-year-old female with history of alcohol abuse, hypertension, and hyperlipidemia presented with abdominal pain, nausea, and vomiting. Patient had fallen several days prior, resulting in rib fractures, and had been taking APAP for pain relief. Vital signs were within normal limits. Physical exam showed a lethargic, encephalopathic patient who only followed simple commands and had right upper quadrant abdominal tenderness. Labs revealed total bilirubin 6.4, AST 19816, ALT 5544, INR 3.7, acetaminophen level 58.3, lactic acid 12.2, creatinine 3.1, lipase 1187, and ammonia 82. Imaging was consistent with hepatic steatosis. Patient was treated with aggressive fluid resuscitation for pancreatitis; NAC and 4-MP for acetaminophen toxicity; lactulose and rifaximin for hepatic encephalopathy; vitamin K for coagulopathy; and albumin, octreotide, and midodrine for hepatorenal syndrome. Mental status and lab abnormalities gradually improved and acetaminophen level became undetectable. DISCUSSION: Our patient presented with unintentional APAP overdose. She likely had underlying chronic liver disease, as evidenced by hepatic steatosis on imaging, due to alcohol abuse. This likely made her more susceptible to liver injury, even with therapeutic doses of APAP.The pathogenesis APAP-induced hepatotoxicity starts with the conversion of APAP to N-acetyl-p-benzoquinone imine (NAPQI) by cytochrome P450 enzymes, which is detoxified by conjunction with glutathione.[1] When excessive amounts of APAP are ingested, hepatic glutathione levels are depleted, resulting in oxidant stress. NAC, the current standard of care and only clinically approved treatment for APAP overdose, attempts to limit this by accelerating glutathione synthesis and subsequent scavenging of NAPQI.[2]Recent literature has suggested that 4-MP may also be beneficial for attenuating liver injury in APAP overdose. 4-MP is classically known for its inhibition of alcohol dehydrogenase in methanol or ethylene glycol poisoning. However, it is unique in that it also directly inhibits CYP2E1, one of the major enzymes involved in the generation of NAPQI. 4-MP has been shown to be effective at preventing APAP-induced liver injury in mice and human hepatocytes and limiting oxidative metabolism in five human volunteers who ingested supratherapeutic APAP doses.[1,2] It may also protect against APAP-induced nephrotoxicity via a similar mechanism.[3] CONCLUSIONS: In patients presenting with APAP overdose, combination therapy with NAC and 4-MP may provide complementary protective effects through targeting of different therapeutic pathways. REFERENCE #1: Kang AM, Padilla-Jones A, Fisher ES, et al. The effect of 4-methylpyrazole on oxidative metabolism of acetaminophen in human volunteers. J Med Toxicol. 2020;16(2):169-176. REFERENCE #2: Akakpo JY, Ramachandran A, Kandel SE, et al. 4-Methylpyrazole protects against acetaminophen hepatotoxicity in mice and in primary human hepatocytes. Hum Exp Toxicol. 2018;37(12):1310-1322. REFERENCE #3: Akakpo JY, Ramachandran A, Orhan H, et al. 4-methylpyrazole protects against acetaminophen-induced acute kidney injury. Toxicol Appl Pharmacol. 2020;409:115317. DISCLOSURES: No relevant relationships by Travis Homan, source=Web Response No relevant relationships by Brittany Wichman, source=Web Response

Protein Targets of Acetaminophen Covalent Binding in Rat and Mouse Liver Studied by LC-MS/MS.

Acetaminophen (APAP) is a mild analgesic and antipyretic used commonly worldwide. Although considered a safe and effective over-the-counter medication, it is also the leading cause of drug-induced acute liver failure. Its hepatotoxicity has been linked to the covalent binding of its reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), to proteins. The aim of this study was to identify APAP-protein targets in both rat and mouse liver, and to compare the results from both species, using bottom-up proteomics with data-dependent high resolution mass spectrometry and targeted multiple reaction monitoring (MRM) experiments. Livers from rats and mice, treated with APAP, were homogenized and digested by trypsin. Digests were then fractionated by mixed-mode solid-phase extraction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Targeted LC-MRM assays were optimized based on high-resolution MS/MS data from information-dependent acquisition (IDA) using control liver homogenates treated with a custom alkylating reagent yielding an isomeric modification to APAP on cysteine residues, to build a modified peptide database. A list of putative in vivo targets of APAP were screened from data-dependent high-resolution MS/MS analyses of liver digests, previous in vitro studies, as well as selected proteins from the target protein database (TPDB), an online resource compiling previous reports of APAP targets. Multiple protein targets in each species were found, while confirming modification sites. Several proteins were modified in both species, including ATP-citrate synthase, betaine-homocysteine S-methyltransferase 1, cytochrome P450 2C6/29, mitochondrial glutamine amidotransferase-like protein/ES1 protein homolog, glutamine synthetase, microsomal glutathione S-transferase 1, mitochondrial-processing peptidase, methanethiol oxidase, protein/nucleic acid deglycase DJ-1, triosephosphate isomerase and thioredoxin. The targeted method afforded better reproducibility for analysing these low-abundant modified peptides in highly complex samples compared to traditional data-dependent experiments.

The Protective Potential of Metformin against Liver Acetaminophen Toxicity

The inadequacy to replace acetaminophen (APAP) with a more effective analgesic continues its use in therapeutic interventions, upholding the risk of hepatotoxicity. Depletion of glutathione reserves by a metabolic intermediate of acetaminophen, N-acetyl-p-benzoquinone imine (NAPQI), is the major reason. The current study presents the combinatorial effect of metformin, a biguanide, in ameliorating the APAP toxicity. HepG2 cells were used for in vitro studies and MTT and LDH leakage assays were used for viability assessment. 10 ?M of metformin improved the cell viability and membrane integrity of cells treated with a high antioxidant enzymes and reduced glutathione were significantly increased in cells co-administrated with metformin and acetaminophen

Glabridin attenuates paracetamol-induced liver injury in mice via CYP2E1-mediated inhibition of oxidative stress

CYP2E1 plays a crucial role in the bio-activation of toxic substances leading to liver damage. In this context, CYP2E1 converts paracetamol (PCM) to N-acetyl-p-benzoquinone imine (NAPQI), which is prone to cause hepatotoxicity. Hence, we aimed to explore the protective effect of glabridin on widely used PCM-induced liver injury model in the present study and, after that, correlated with the role of CYP2E1 toward its efficacy. Glabridin was isolated from Glycyrrhiza glabra and characterized before the investigation in an in-vivo mice model of PCM-induced liver injury. Glabridin after oral treatment at 5–20 mg/kg showed a considerable improvement in serum biochemical parameters (ALT and AST) and oxidative stress markers (MDA, GSH, SOD, and catalase) in comparison to only PCM-treatment. Histopathological examination of the liver depicted that glabridin exhibited substantial protection from PCM-induced liver injury compared to the disease control group. Significant down-regulation of CYP2E1 protein and its mRNA expression levels were observed in the glabridin-treated groups compared to PCM-induced respective elevation of CYP2E1. Moreover, activation of NF-κB was significantly inhibited by glabridin. Therefore, glabridin has the potential to protect PCM-induced liver injury through CYP2E1 inhibition-mediated normalization of oxidative stress. Further research is warranted to establish glabridin as a phytotherapeutics for liver protection for which no effective and safe oral drug is available to date.

The in silico mechanism of hVKOR interaction with acetaminophen and its metabolite, as well as N-acetyl cysteine: caution on application in COVID-19 patients

Acetaminophen and N-acetyl cysteine (NAC) are being used as supportive care in patients suffering from coronavirus disease 2019 (COVID-19). The coagulopathy and cerebral hemorrhage have been recently reported in these patients. Prolonged acetaminophen use increases the international normalized ratio (INR) and the risk of bleeding among patients taking anti-coagulants. Inhibition of vitamin K epoxide reductase (VKOR) by acetaminophen and NAC in chronic applications has been reported, however, detailed knowledge of the molecular mechanism and binding sites are not clear. Herein, we built the homology model of human VKOR (hVKOR) using ITASSER server, confirmed, and applied it for docking analysis of its interaction with acetaminophen and its metabolite, N-acetyl-p-benzoquinone imine (NAPQI), and NAC. We also calculated the lipophilicity and predicted the blood-brain-barrier (BBB) permeation of NAPQI by Swiss ADME. Our analysis showed that NAPQI and NAC, but not acetaminophen, bind strongly to the similar sites in hVKOR via both hydrogen and van der Waals bonding; particularly with Cys135. Thus, it interrupted the vitamin K reducing electron transfer pathway. Further, molecular dynamic (MD) simulation study revealed that the interactions of the ligands with hVKOR are stable. In conclusion, our analysis shed a light on the molecular mechanism of acetaminophen-induced coagulopathy previously reported in some clinical cases with chronic acetaminophen use. Furthermore, considering the anti-coagulopathy of NAPQI and NAC but not acetaminophen, the BBB permeation potency of these agents, and the risk of coagulopathy in COVID-19, we suggest a regular prothrombin time (PT) and INR monitoring of these patients taking acetaminophen and/or NAC. Communicated by Ramaswamy H. Sarma