Dataset Information

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

ABSTRACT: Acetaminophen (N-acetyl-p-aminophenol; APAP) is a mild analgesic and antipyretic used commonly worldwide. Although APAP is 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 in vivo 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 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) using scheduled multiple reaction monitoring (MRM) acquisition. The targeted 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 forming a positional isomer of the APAP modification on all cysteine residues, in order to build an in-house modified peptide database for targeted analysis. A list of putative in vivo targets of APAP were screened from previous in vitro studies, data-dependent high-resolution MS/MS analyses of liver digests, as well as selected proteins from the target protein database (TPDB), an online resource which references previous reports of proteins found to be modified by acetaminophen. Multiple protein targets of APAP in each species were found, while confirming modification sites.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Rattus Norvegicus (rat) Mus Musculus (mouse)

TISSUE(S): Liver

SUBMITTER: Lekha Sleno

LAB HEAD: Lekha Sleno

PROVIDER: PXD027674 | Pride | 2022-02-17

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	Mouse_11_300mgkg_4h_100ul_SDS_homo_MCX1_10mMAmAc.wiff	Wiff
	Mouse_11_300mgkg_4h_100ul_SDS_homo_MCX1_10mMAmAc.wiff.scan	Wiff
	Mouse_11_300mgkg_4h_100ul_SDS_homo_MCX2_20mMAmAc.wiff	Wiff
	Mouse_11_300mgkg_4h_100ul_SDS_homo_MCX2_20mMAmAc.wiff.scan	Wiff
	Mouse_11_300mgkg_4h_100ul_SDS_homo_MCX3_25mMAmAc.wiff	Wiff

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

Geib Timon T Moghaddam Ghazaleh G Supinski Aimee A Golizeh Makan M Sleno Lekha L

Frontiers in chemistry 20210820

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, <i>N</i>-acetyl <i>p</i>-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 spec ...[more]

PMID: 34490218

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Project description:Sluka2016 - Acetaminophen metabolism Liver metabolism of Acetaminophen: Acetaminophen (APAP) is metabolized in the liver in both Phase I and Phase II reactions. Phase II reactions convert APAP to APAP-glucuronide and APAP-sulfate. Phase I reactions involve Cytochrome P450 mediated (mostly Cyp450-2E1 and -1A2) conversion of APAP to N-acetyle-p-quinoneimine (NAPQI), which goes on to react with cellular nucleophiles such as glutathione (GSH). At high doses of APAP significant GSH depletion in hepatocyte occurs resulting in cell necrosis and and in extreme cases death. This model is described in the article: A Liver-Centric Multiscale Modeling Framework for Xenobiotics. Sluka JP, Fu X, Swat M, Belmonte JM, Cosmanescu A, Clendenon SG, Wambaugh JF, Glazier JA. PLoS ONE 2016; 11(9): e0162428 Abstract: We describe a multi-scale, liver-centric in silico modeling framework for acetaminophen pharmacology and metabolism. We focus on a computational model to characterize whole body uptake and clearance, liver transport and phase I and phase II metabolism. We do this by incorporating sub-models that span three scales; Physiologically Based Pharmacokinetic (PBPK) modeling of acetaminophen uptake and distribution at the whole body level, cell and blood flow modeling at the tissue/organ level and metabolism at the sub-cellular level. We have used standard modeling modalities at each of the three scales. In particular, we have used the Systems Biology Markup Language (SBML) to create both the whole-body and sub-cellular scales. Our modeling approach allows us to run the individual sub-models separately and allows us to easily exchange models at a particular scale without the need to extensively rework the sub-models at other scales. In addition, the use of SBML greatly facilitates the inclusion of biological annotations directly in the model code. The model was calibrated using human in vivo data for acetaminophen and its sulfate and glucuronate metabolites. We then carried out extensive parameter sensitivity studies including the pairwise interaction of parameters. We also simulated population variation of exposure and sensitivity to acetaminophen. Our modeling framework can be extended to the prediction of liver toxicity following acetaminophen overdose, or used as a general purpose pharmacokinetic model for xenobiotics. This model is hosted on BioModels Database and identified by: BIOMD0000000624. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.