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

Abstract

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.

Ginsenoside Rc, as an FXR activator, alleviates acetaminophen-induced hepatotoxicity via relieving inflammation and oxidative stress

“…Mitochondrial proteins undergo nuclear translocation, which results in cell death and nuclear DNA fragmentation. In the current research, Ginsenoside Rc was shown to significantly reduce the level of NAPQI (Geib et al, 2021). Low levels of NAPQI promote the alleviation of liver cell apoptosis, oxidative stress, and the inflammatory response.…”

“…Mitochondrial protein, the key target of NAPQI, appears with excess APAP (Geib et al, 2021). Studies have shown that APAP can cause oxidative stress in the mitochondria and produce ROS through the percolation of electrons from the transport chain (Wang et al, 2021).…”

“…There is an obvious positive relationship between the activity level of complex I and the degree of liver injury. Mitochondrial oxidative stress caused by APAP inevitably triggers cell death, Frontiers in Pharmacology frontiersin.org 13 which in turn produces an inflammatory response (Geib et al, 2021). Oxidative stress in the mitochondria during the metabolic phase increases their permeability while releasing mitochondrial proteins which causing apoptosis of the factor AIF (Shi et al, 2021).…”

Dual roles of drug or its metabolite−protein conjugate: Cutting‐edge strategy of drug discovery using shotgun proteomics

“…169 Using shotgun proteomics with DDA high-resolution MS, APAP covalently modified proteins in rat and mouse livers were detected in highly complex samples. 170 The DDA is the mainstream mode for low-abundance DMPC detection. However, because of the characteristics of the DDA scanning mode, the combination of dynamic exclusion and DDA-based methods still cannot provide full coverage of peptides in complex proteomics samples, resulting in some low-abundance DMPCs that cannot be selected for fragmentation for MS analysis.…”

“…An additional dynamic exclusion technique greatly reduces redundant peptide precursor selection, extends the dynamic range, and maximizes proteome coverage of detection in shotgun proteomics 169 . Using shotgun proteomics with DDA high‐resolution MS, APAP covalently modified proteins in rat and mouse livers were detected in highly complex samples 170 . The DDA is the mainstream mode for low‐abundance DMPC detection.…”