N-acetyl-p-benzoquinone Imine Research Articles

Electrochemical oxidation of acetaminophen in the presence of diclofenac and piroxicam - Synthesis of new derivatives and kinetic investigation of toxic quinone imine/drugs interactions

In this research, electrochemical oxidation of acetaminophen was investigated in the presence of steroidal anti-inflammatory drugs diclofenac and piroxicam by cyclic voltammetry and controlled-potential coulometry techniques. The results indicated that N-acetyl-p-benzoquinone-imine (NAPQI), produced by electrooxidation of acetaminophen, reacts with diclofenac and piroxicam, via the Michael addition reaction. Corresponding products were electrochemically synthesized in aqueous solutions using a carbon electrode in an undivided cell. These products were identified by spectroscopic methods (FT-IR, 1HNMR, 13CNMR and MS). The homogeneous rate constants (kobs), based on the suggested electrode mechanism (i.e. EC), were estimated by comparing the experimental cyclic voltammetric responses with those digital simulated results. A comparison of the estimated kobs for the reaction of electrochemically generated N-acetyl-p-benzoquinone-imine (NAPQI) with steroidal anti-inflammatory drugs revealed that kobs (piroxicam) > kobs(diclofenac) at biological pH.

A reliable LC-MS/MS method for the quantification of N-acetyl-p-benzoquinoneimine, acetaminophen glutathione and acetaminophen glucuronide in mouse plasma, liver and kidney: Method validation and application to a pharmacokinetic study.

A rapid, specific, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated to simultaneously quantify N-acetyl-p-benzoquinoneimine (NAPQI), acetaminophen-glutathione (acetaminophen-glut) and acetaminophen-glucuronide (acetaminophen-gluc) in mouse plasma, liver and kidney homogenates. Analytes were eluted by a binary gradient mobile phase composed of water (phase A) and methanol containing 0.1% formic acid (phase B) at a flow rate of 0.3 mL/min, which was performed on a CAPCELL PAK C18 MG II column. It took 3.2 min to detect three analytes in a single run. Quantification was carried out in positive mode combined with multiple reaction monitoring. The validation of the LC-MS/MS method consisted of specificity, linearity, precision, accuracy, protein precipitation recovery, matrix effect, dilution integrity and stability. The plasma and tissue homogenate calibration curves were linear over concentration ranges of 0.050-5.00, 0.050-5.00 and 0.100-40.0 μg/mL, with a lower limit of quantification of 0.050, 0.050, and 0.100 μg/mL for NAPQI, acetaminophen-glut and acetaminophen-gluc, respectively. The intra- and inter-run precision values were within 12.47% for NAPQI, 12.11% for acetaminophen-glut and 11.86% for acetaminophen-gluc at their lower limit of quantitation levels. The samples were stable under all tested conditions. This method was successfully applied to study the pharmacokinetics of NAPQI, acetaminophen-glut and acetaminophen-gluc in ICR mice following oral administration of 200 mg/kg of acetaminophen suspension.

An insight into paracetamol and its metabolites using molecular docking and molecular dynamics simulation.

Paracetamol is a relatively safe analgesia/antipyretic drug without the risks of addiction, dependence, tolerance, and withdrawal when used alone. However, when administrated in an opioid/paracetamol combination product, which often contains a large quantity of paracetamol, it can be potentially dangerous due to the risk of hepatotoxicity. Paracetamol is known to be metabolized into N-(4-hydroxyphenyl)-arachidonamide (AM404) via fatty acid amide hydrolase (FAAH) and into N-acetyl-p-benzoquinone imine (NAPQI) via cytochrome P450 (CYP) enzymes. However, the underlying mechanism of paracetamol is still unclear. In addition, paracetamol has the potential to interact with other drugs that are also involved with CYP family enzymes (inducer/inhibitor/substrate), an example being illicit drugs. In our present work, we looked into the relationship between paracetamol and its metabolites (AM404 and NAPQI) using molecular docking and molecular dynamics (MD) simulations. We first carried out a series of molecular docking studies between paracetamol/AM404/NAQPI and their reported targets, including CYP 2E1, FAAH, TRPA1, CB1, and TRPV1. Subsequently, we performed MD simulations and energy decomposition for CB1-AM404, TRPV1-AM404, and TRPV1-NAPQI for further investigation of the dynamics interactions. Finally, we summarized and discussed the reported drug-drug interactions between paracetamol and central nervous system drugs, especially illicit drugs. Overall, we are able to provide new insights into the structural and functional roles of paracetamol and its metabolites that can inform the potential prevention and treatment of paracetamol overdose. Graphical abstract Paracetamol and its metabolites.

Genetic Association of Single Nucleotide Polymorphisms with Acetaminophen-Induced Hepatotoxicity

Acetaminophen is commonly used to reduce pain and fever. Unfortunately, overdose of acetaminophen is a leading cause of acute liver injury and failure in many developed countries. The majority of acetaminophen is safely metabolized in the liver and excreted in the urine; however, a small percentage is converted to the highly reactive N-acetyl-p-benzoquinone imine (NAPQI). At therapeutic doses, NAPQI is inactivated by glutathione S-transferases, but at toxic levels, excess NAPQI forms reactive protein adducts that lead to hepatotoxicity. Individual variability in the response to both therapeutic and toxic levels of acetaminophen suggests a genetic component is involved in acetaminophen metabolism. In this review, we evaluate the genetic association studies that have identified 147 single nucleotide polymorphisms linked to acetaminophen-induced hepatotoxicity. The identification of novel genetic markers for acetaminophen-induced hepatotoxicity provides a rich resource for further evaluation and may lead to improved prognosis, prevention, and treatment.

N,N'-Bis(2-mercaptoethyl)isophthalamide Binds Electrophilic Paracetamol Metabolites and Prevents Paracetamol-Induced Liver Toxicity.

Paracetamol overdosing may cause liver injury including fulminant liver failure due to generation of the toxic metabolites, N-acetyl-p-benzoquinone imine (NAPQI) and p-benzoquinone (p-BQ). Herein, the chelating agent, N,N'-Bis(2-mercaptoethyl)isophthalamide (NBMI), was examined for its potential ability to entrap NAPQI and p-BQ and to prevent paracetamol-induced liver injury. Both NBMI and the conventional paracetamol antidote N-acetylcysteine (NAC) were investigated with regard to their abilities to scavenge the NAPQI and p-BQ in a Transient Receptor Potential Ankyrin 1-dependent screening assay. Stoichiometric evaluations indicated that NBMI was able to entrap these metabolites more efficiently than NAC. Furthermore, oral administration of either NBMI (680 mg/kg) or NAC (680 mg/kg) prevented the development of the characteristic liver necrosis and elevation of serum alanine aminotransferase in a mouse model for paracetamol-induced liver injury. In summary, these results show that NBMI is able to entrap the toxic metabolites NAPQI and p-BQ and to prevent paracetamol-induced liver injury in mice.

Mitochondrial-Lysosomal Axis in Acetaminophen Hepatotoxicity.

Acetaminophen (APAP) toxicity is the most common cause of acute liver failure and a major indication for liver transplantion in the United States and Europe. Although significant progress has been made in understanding the molecular mechanisms underlying APAP hepatotoxicity, there is still an urgent need to find novel and effective therapies against APAP-induced acute liver failure. Hepatic APAP metabolism results in the production of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which under physiological conditions is cleared by its conjugation with glutathione (GSH) to prevent its targeting to mitochondria. APAP overdose or GSH limitation leads to mitochondrial NAPQI-protein adducts formation, resulting in oxidative stress, mitochondrial dysfunction, and necrotic cell death. As mitochondria are a major target of APAP hepatotoxicity, mitochondrial quality control and clearance of dysfunctional mitochondria through mitophagy, emerges as an important strategy to limit oxidative stress and the engagement of molecular events leading to cell death. Recent evidence has indicated a lysosomal–mitochondrial cross-talk that regulates APAP hepatotoxicity. Moreover, as lysosomal function is essential for mitophagy, impairment in the fusion of lysosomes with autophagosomes-containing mitochondria may compromise the clearance of dysfunctional mitochondria, resulting in exacerbated APAP hepatotoxicity. This review centers on the role of mitochondria in APAP hepatotoxicity and how the mitochondrial/lysosomal axis can influence APAP-induced liver failure.

Pengaruh Pemberian Cuka Apel 'A' terhadap Kadar MDA Hepar Tikus Wistar Jantan yang Diinduksi Parasetamol Dosis Toksik


 High dose of paracetamol is metabolized by cytochrome P-450 become free radical N-acetyl-p-benzoquinoneimine (NAPQI) but liver Glutathione (GSH) is not adequate to change it become nonreactive metabolite so that NAPQI bind to unsaturated fatty acid of cell membrane, causing lipid peroxidation and increase liver Malondialdehyde (MDA). 'A' apple vinegar contains anthocyanin with an antioxidant effect by electron donor to NAPQI and acetic acid to improve liver GSH level. The aim of research was to investigate the effect of 'A' apple vinegar on the rat liver MDA induced by toxic dose of paracetamol. Research groups consist of normal control (CMC Na 1% 1 ml for 14 days), negative control (CMC Na 1% 1ml for 14 days + paracetamol 291.6 mg/200gBW on the day 12nd,13rd,14th), and treatment group ('A' apple vinegar 0.4 ml/150gBW for 14 days + paracetamol 291.6 mg/200gBW on the day 12nd,13rd,14th). Liver MDA was measured on the day 15th with competitive ELISA. The average of normal control group was 21.58 ng/ml, negative control group was 70,71 ng/ml, treatment group was 37,67 ng/ml. One way ANOVA and Post hoc LSD test showed significantly differences between all groups (p<0,05). It can be concluded that 'A' apple vinegar had an effect on the liver MDA induced by toxic dose of paracetamol.
 
 Keywords: Paracetamol, NAPQI, MDA, 'A' apple vinegar, antioxidant

Acetaminophen‐Induced Hepatotoxicity in Wistar Rats Treated with Annona Muricata Aqueous Stem Extract

The central role of the liver in biochemical homeostasis cannot be overemphasized and its toxicity is owed greatly to its great involvement in xenobiotic metabolism. This study therefore evaluated the hepatoprotective effect of aqueous stem extract of Anonna muricata S. on Acetaminophen‐induced liver damage. Animals were divided into four groups of five rats. Group1‐ normal saline p.o, Groups 2–4 pretreated with 400mg/kg p.o Acetaminophen, then Groups 3 and 4 treated for 7days with 400mg/kg and 800mg/kg extract p.o respectively. The result revealed an elevated level of serum liver enzymes (ALT 32±1.00, AST 42±2.00) to the untreated Group II rats that received only 400 mg/kg acetaminophen, which at concurrent examination showed a significant decrease in these liver enzymes in Groups III (ALT 29±1.00 and AST 34.67±1.53) and IV (ALT 29±3.00 and AST 34±2.00). The increase seen in the treated groups were not dose dependent but were higher than the control. Group II when compared with the control (ALT 17±1.00 and AST 26±1.04) showed a significant (p≤0.05) increase. The ALP, total protein and bilirubin showed little or no effect in both treated and untreated. Acetaminophen overdose induces hepatotoxicity through its intermediate metabolite N‐acetyl‐p‐benzoquinone imine (NAPQI) which reacts with the natural antioxidant Glutathione reducing its concentration and generating more reactive oxygen species that damage the hepatic cells. These reductions in serum liver enzymes by the extract revealed that the aqueous stem extract of Anonna muricata S. has no toxic effect on the liver and could be hepatoprotective at the tested doses.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of Lipocalin‐2 (LCN2) and High Mobility Box Group 1 (HMGB1) in Acetaminophen‐induced Acute Liver Failure

Acetaminophen (APAP) overdose is the most common cause of Acute Liver Failure (ALF). Following APAP overdose, toxicity is initiated by the conversion of APAP to its reactive metabolic N‐acetyl‐p‐benzoquinone imine (NAPQI) by CYP2E1/CYP3A4. NAPQI binds covalently to mitochondrial proteins forming 3‐(cysteine‐S‐yl)‐acetaminophen (APAP‐Cys). This generates initial hepatotoxicity in the form of centrilobular necrosis and sterile inflammation. Lipocalin‐2 (LCN2), is an acute‐phase innate immune protein, upregulated during tissue injury in various organs including the liver. Recently we have demonstrated that LCN2 is pro‐inflammatory and elevated during APAP overdose in mouse model. Interestingly, LCN2 knockout (LCN2 KO) mice exhibit protection from APAP overdose compared to its wild type (WT) littermates. During APAP overdose there is necrosis of hepatocytes, these hepatocytes release Damage Associated Molecular Patterns (DAMPS) that further exacerbate the inflammatory process. One such DAMP is High Mobility Group Box 1 (HMGB1). However, the role of LCN2 and HMGB1 in APAP overdose has not yet been investigated.Present study investigates the molecular mechanism of LCN2 during the progression of acute liver injury in APAP overdose model. We hypothesize that during APAP induced ALF, LCN2 induces the expression and translocation of HMGB1 which in turn mediates inflammation and progression of injury. An in vitro model of HepG2 cells as well as an in vivo model of WT and LCN2 KO mice will be utilized to test our hypothesis. HepG2 cells will be treated with recombinant LCN2 to investigate whether exogenous LCN2 can cause induction and translocation of HMGB1. Translocation of HMGB1 will be assessed by immunofluorescence staining. Serum and tissues samples of WT and LCN2 KO mice subjected to APAP overdose will be assessed for expression of HMGB1. APAP‐cys formation will be measured in WT and LCN2 KO using the High‐Performance Liquid Chromatography method to ensure comparable bioactivation based injury between the WT and LCN2 KO mice. Lastly, histopathology will be performed on liver samples from both groups to assess liver pathology. It is anticipated that LCN2 mediates progression of liver injury in APAP overdose via HMGB1.Support or Funding InformationPhiladelphia College of Osteopathic Medicine‐Division of ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of food restriction on the expression of genes related to acetaminophen-induced liver toxicity in rats.

It is well known that fasting substantially affects the metabolism of drugs and chemicals. Food restriction also affects drug kinetics, such as absorption, metabolism, and excretion, and therefore, it can potentially modulate the onset of chemical toxicity or drug-induced adverse reactions. In the present study, the expression of drug-metabolizing enzyme genes and total glutathione content in the liver, which are related to toxicity induced by overdose of the hepatotoxic drug acetaminophen (N-acetyl-p-aminophenol; APAP), were examined in rats reared under different feeding conditions: ad libitum feeding, 16-h fasting, and food restriction (fed 70% of the average intake of ad libitum feeding for 10 days) conditions. The rats under food restriction conditions as well as fasted rats showed significantly higher expression of Cyp2e1, the gene encoding the enzyme that metabolizes APAP to its toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). They also had lower levels of liver total glutathione, which detoxifies NAPQI. In contrast, the gene expression of UDP-glucuronosyltransferase 1A6 (Ugt1a6), sulfotransferase 1A1 (Sult1a1), and glutathione S-transferase M1 (Gstm1) was not affected by food restriction or fasting. When APAP was administered (800 mg/kg), histopathological changes were not observed in rats fed ad libitum, while hepatocellular necrosis was observed in most of the rats treated with APAP after fasting or food restriction. Taken together, these results suggest that not only fasting but also food restriction exacerbate APAP-induced acute liver injury, probably by the induction of CYP2E1 and the reduction of liver glutathione contents, in rodents.

Novel approach for evaluating pharmaceuticals toxicity using Daphnia model: analysis of the mode of cytochrome P450-generated metabolite action after acetaminophen exposure

Because of its widespread use, the pharmaceutical acetaminophen (APAP) is frequently detected in aquatic environments. APAP can have serious physiological effects, such as reduced reproduction, low growth rates, and abnormal behavior, in aquatic organisms. However, the methods available for evaluation of the aquatic toxicity of APAP are of limited usefulness. The present study aimed to develop reliable and sensitive markers for evaluation of APAP toxicity using Daphnia as a model organism. We focused on N-acetyl-p-benzoquinoneimine (NAPQI) production from APAP via cytochrome P450 metabolism because NAPQI causes APAP toxicity. Daphnia magna were exposed to APAP (0, 50, or 100 mg/L for 12 h or 24 h), and the total metabolites were extracted and analyzed for NAPQI. Direct detection of NAPQI was difficult because of its high reactivity, and its peak was close to that for APAP. Therefore, we tried to identify molecular and biochemical indicators associated with NAPQI generation, elimination, and its interactions with macromolecules. We identified changes in CYP370A13 gene expression, glutathione depletion, inhibition of thioredoxin reductase activity, and production of reactive oxygen species as indicators of D. magna exposure to APAP. These indicators could be used to develop sensitive and accurate techniques to evaluate the environmental toxicity of APAP.

Activation of the capsaicin-receptor TRPV1 by the acetaminophen metabolite N-arachidonoylaminophenol results in cytotoxicity

AimsThe anandamide reuptake inhibitor N-arachidonoylaminophenol (AM404) and the reactive substance N-acetyl-p-benzoquinone imine (NAPQI) are both metabolites of acetaminophen and may contribute to acetaminophen-induced analgesia by acting at TRPV1 expressed in the peripheral or central nervous system. While NAPQI slowly sensitizes and activates TRPV1 by interacting with distinct intracellular cysteine residues, detailed properties of AM404 as an agonist of TRPV1 have not yet been reported on. We explored the effects of AM404 on recombinant human TRPV1 and in rodent dorsal root ganglion (DRG) neurons. Materials and methodsHEK 293 cells expressing different isoforms of recombinant TRPV1 and rodent DRG neurons were employed for patch clamp and calcium imaging experiments. Cytotoxicity was assessed by propidium iodide and Annexin V staining on TRPV1-HEK 293 cells and with trypan blue staining on DRG neurons. Key findingsAM404 activates hTRPV1 at concentrations >1μM and in a concentration-dependent manner. AM404 also potentiates TRPV1-mediated currents evoked by heat and anandamide. Moreover, AM404-evoked currents are potentiated by NAPQI. While the partly capsaicin-insensitive rabbit (o) TRPV1 fails to respond to AM404, AM404-sensitivity is restored by insertion of the capsaicin binding-domain of rat TRPV1 into oTRPV1. In DRG neurons, AM404-evoked calcium influx as well as cell death is mediated by TRPV1. SignificanceAM404 gates TRPV1 by interacting with the vanilloid-binding site, and TRPV1 is the main receptor for AM404 in DRG neurons. While direct activation of TRPV1 requires high concentrations of AM404, it is possible that synergistic effects of AM404 with further TRPV1-agonists may occur at clinically relevant concentrations.

Glutathione peroxidase 3 is a protective factor against acetaminophen‑induced hepatotoxicity in vivo and in vitro.

Acetaminophen (APAP) is a widely available antipyretic and analgesic; however, overdose of the drug inflicts severe damage to the liver. It is well established that the hepatotoxicity of APAP is initiated by formation of a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which can be detoxified by conjugation with reduced glutathione (GSH), a typical antioxidant. We recently found that the blood mRNA expression level of glutathione peroxidase 3 (Gpx3), which catalyzes the oxidation of GSH, is associated with the extent of APAP-induced hepatotoxicity in mice. The present study was carried out to determine the in vivo and in vitro role of GPx3 in APAP-induced hepatotoxicity. In in vivo experiments, oral administration of APAP to mice induced liver injury. Such liver injury was greater in males than in females, although no gender difference in the plasma concentration of APAP was found. Female mice had a 2-fold higher expression of Gpx3 mRNA and higher plasma GPx activity than male mice. 17β-estradiol, a major female hormone, decreased APAP-induced hepatotoxicity and increased both the expression of blood Gpx3 mRNA and plasma GPx activity, suggesting that the cytoprotective action of this hormone is mediated by the increase in GPx3. To further clarify the role of GPx3 in APAP-induced hepatotoxicity, we evaluated the effect of a change in cellular GPx3 expression resulting from transfection of either siRNA-GPx3 or a GPx3 expression vector on NAPQI-induced cellular injury (as assessed by a tetrazolium assay) in in vitro experiments using heterogeneous cultured human cell lines (Huh-7 or K562). NAPQI-induced cell death was reduced by increased GPx3 and was enhanced by decreased GPx3. These results suggest that GPx3 is an important factor for inhibition of APAP-induced hepatotoxicity both in vivo and in vitro. To our knowledge, this is the first report to show a hepatoprotective role of cellular GPx3 against APAP-induced liver damage.

Pharmacokinetic modelling of modified acetylcysteine infusion regimens used in the treatment of paracetamol poisoning.

Paracetamol overdose is common and is treated with acetylcysteine to prevent the development of hepatotoxicity. N-acetyl-p-benzoquinone imine (NAPQI) is the toxic metabolite of paracetamol overdose. We aimed to assess the expected acetylcysteine concentration time profiles following delivery of modified acetylcysteine regimens proposed for those at high and low risk of hepatotoxicity. In addition, we will determine acetylcysteine concentrations post-cessation of abbreviated infusions. We performed pharmacokinetic simulations using Berkeley Madonna (version 8.3.23.0) comparing the time course of acetylcysteine concentration during and after the cessation of an abbreviated 12-h regimen (250mg/kg) using a two-bag infusion and compared this to the standard 21-h three-bag (300mg/kg) regimen. We also simulated extended duration acetylcysteine regimens and other increased dosing strategies that have been recommended in specific paracetamol poisoning scenarios. A more sustained serum concentration is achieved when the acetylcysteine loading dose is delivered over 4h using the two-bag compared to the 1-h loading dose of the three-bag regimen. When administering an abbreviated 12-h acetylcysteine regimen, circulating acetylcysteine is detectable for 8h after cessation of the infusion. This may provide a continued hepatoprotective effect if NAPQI is still being generated after the infusion is ceased. This pharmacokinetic simulation study is an important step in determining plasma acetylcysteine concentrations that are likely to be achieved using various modified treatment regimens. Importantly, for patients at low risk of liver injury after acute overdose, acetylcysteine is likely to be detectable many hours post-cessation of a 12-h regimen. This should provide a safety factor against development of hepatotoxicity for any ongoing paracetamol metabolism after cessation of the acetylcysteine infusion.

Nontargeted Identification of Reactive Metabolite Protein Adducts.

Metabolic bioactivation of many different chemicals results in the formation of highly reactive compounds (chemically reactive metabolites, CRMs) that can lead to toxicity via binding to macromolecular targets (e.g., proteins or DNA). There is a need to develop robust, rapid, and nontargeted analytical techniques to determine the identity of the protein targets of CRMs and their sites of modification. Here, we introduce a nontargeted methodology capable of determining both the identity of a CRM formed from an administered compound as well as the protein targets modified by the reactive metabolite in a single experiment without prior information. Acetaminophen (N-acetyl-p-aminophenol, APAP) and 13C6-APAP were incubated with rat liver microsomes, which are known to bioactivate APAP to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). Global tryptic digestion followed by liquid chromatographic/mass spectrometric (LC/MS) analysis was used to locate "twin" ion peaks of peptides adducted by NAPQI and for shotgun proteomics via tandem mass spectrometry (MS/MS). By the development of blended data analytics software called Xenophile, the identity of the amino acid residue that was adducted can be established, which eliminates the need for specific parametrization of protein database search algorithms. This combination of experimental design and data analysis software allows the identity of a CRM, the protein target, and the amino acid residues that are modified to be rapidly established directly from experimental data. Xenophile is freely available from https://github.com/mgleeming/Xenophile .

Acetaminophen analog N-acetyl-m-aminophenol, but not its reactive metabolite, N-acetyl-p-benzoquinone imine induces CYP3A activity via inhibition of protein degradation

Cytochrome P450 (CYP) 3A subfamily members are known to metabolize various types of drugs, highlighting the importance of understanding drug–drug interactions (DDI) depending on CYP3A induction or inhibition. While transcriptional regulation of CYP3A members is widely understood, post-translational regulation needs to be elucidated. We previously reported that acetaminophen (APAP) induces CYP3A activity via inhibition of protein degradation and proposed a novel DDI concept. N-Acetyl-p-benzoquinone imine (NAPQI), the reactive metabolite of APAP formed by CYP, is known to cause adverse events related to depletion of intracellular reduced glutathione (GSH). We aimed to inspect whether NAPQI rather than APAP itself could cause the inhibitory effects on protein degradation. We found that N-acetyl-l-cysteine, the precursor of GSH, and 1-aminobenzotriazole, a nonselective CYP inhibitor, had no effect on CYP3A1/23 protein levels affected by APAP. Thus, we used APAP analogs to test CYP3A1/23 mRNA levels, protein levels, and CYP3A activity. We found N-acetyl-m-aminophenol (AMAP), a regioisomer of APAP, has the same inhibitory effects of CYP3A1/23 protein degradation, while p-acetamidobenzoic acid (PAcBA), a carboxy-substituted form of APAP, shows no inhibitory effects. AMAP and PAcBA cannot be oxidized to quinone imine forms such as NAPQI, so the inhibitory effects could depend on the specific chemical structure of APAP.

The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity.

Long-term acetaminophen treatment induced liver fibrosis in mice and the involvement of Egr-1.

Acetaminophen (APAP)-induced acute liver injury has already been well studied. However, whether long-term administration of APAP will cause liver fibrosis is still not very clear. This study aims to investigate the liver fibrosis in mice induced by long-term APAP treatment and the involvement of early growth response 1 (Egr-1). C57BL/6 mice were orally given with APAP (200, 300mg/kg) for 2, 6 or 10 weeks, respectively. Liver hydroxyproline content, collagen deposition and inflammatory cells infiltration were increased in mice treated with APAP (200, 300mg/kg) for 6 or 10 weeks. Liver mRNA expression of collagen (COL)1a1, Col3a1, transforming growth factor-β (TGF-β) and serum contents of COL1, COL3, TGF-β were all increased in APAP-treated mice. Liver expression of α-smooth muscle actin (α-SMA) and phosphorylated ERK1/2 and Smad2/3 were all increased in APAP-treated mice. Furthermore, increased liver mRNA expression of Egr-1 and its subsequent nuclear translocation were found in APAP-treated mice. Egr-1 knock-out mice were further applied. APAP-induced liver fibrosis was found to be more serious in Egr-1 knock-out mice. N-acetyl-p-benzoquinoneimine (NAPQI), the APAP hepatotoxic metabolite, increased cellular mRNA expression of α-SMA, Col1a1, Col3a1, TGF-β, induced ERK1/2 and Smad2/3 phosphorylation and Egr-1 nuclear translocation in hepatic stellate LX2 cells. In conclusion, long-term administration of APAP induced liver fibrosis in mice, and Egr-1 was critically involved in this process. This study points out a warning and reference for patients with long-term APAP ingestion in clinic.

Mechanisms of acetaminophen hepatotoxicity and their translation to the human pathophysiology.

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States and mechanisms of liver injury induced by APAP overdose have been the focus of extensive investigation. Studies in the mouse model, which closely reproduces the human condition, have shown that hepatotoxicity is initiated by formation of a reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which depletes cellular glutathione and forms protein adducts on mitochondrial proteins. This leads to mitochondrial oxidative and nitrosative stress, accompanied by activation of c-jun N-terminal kinase (JNK) and its translocation to the mitochondria. This then amplifies the mitochondrial oxidant stress, resulting in translocation of Bax and dynamin related protein 1 (Drp1) to the mitochondria, which induces mitochondrial fission, and ultimately induction of the mitochondrial membrane permeability transition (MPT). The induction of MPT triggers release of intermembrane proteins such as apoptosis inducing factor (AIF) and endonuclease G into the cytosol and their translocation to the nucleus, causing nuclear DNA fragmentation and activation of regulated necrosis. Though these cascades of events were primarily identified in the mouse model, studies on human hepatocytes and analysis of circulating biomarkers from patients after APAP overdose, indicate that a number of mechanistic events are identical in mice and humans. Circulating biomarkers also seem to be useful in predicting the course of liver injury after APAP overdose in humans and hold promise for significant clinical use in the near future.

436 - Susceptibility of Human Platelets, Monocytes, and Neutrophils Bioenergetics to Acetaminophen

Acetaminophen (APAP) is a common over-the-counter pain reliever with high hepatotoxic potential. Although APAP hepatotoxicity has been recognized for years, the mechanisms remain elusive. APAP is metabolized to N-acetyl-p-benzoquinone imine (NAPQI) by cytochrome p450 and conjugated with GSH for detoxification, causing oxidative stress that impairs mitochondrial function. In a previous study, we found that APAP decreases rat hepatocyte respiration, and this effect is exacerbated in alcohol-treated rats. Recently, it has been reported that following APAP overdose, platelets and monocytes accumulate in the liver contributing to hepatotoxicity. In the present study, we report the inhibition of mitochondrial function of platelets and monocytes, as well as the oxidative burst capacity of monocytes and neutrophils treated with APAP. Platelets, monocytes, and neutrophils were isolated from blood of healthy human volunteers. The cells were seeded into XF96 microplates and treated with APAP in a dose-response manner for 30 min prior to mitochondrial stress test (MST). In platelets and monocytes, APAP decreases mitochondrial function in a dose-dependent manner revealed by decreased basal oxygen consumption rates (OCR), as well as decreased maximal OCR, reserve capacity, and ATP-linked OCR. In addition to MST, we tested the platelets activation with 0.2 U/ml of thombin, and monocytes and neutrophiles oxidative burst measured following activation with phorbol 12-myristate 13-acetate (PMA). APAP (5 mM) completly abolished thrombin-activation of platelets. Similarly, APAP inhibited the oxidative burst in neutrophils in a dose-dependent manner. Taken together, these data show that within an hour, APAP causes significant bioenergetic changes of platelets, monocytes and decreases the oxidative burst magnitude of neutrophils and monocytes. This work was supported by National Institute of Health grants R21AA023273 (to VDU and AS).