ORIGINAL RESEARCH published: XX XX 2021 doi: 10.3389/fchem.2021.736788 1 58 2 59 3 60 4 61 5 62 6 63 7 64 8 65 9 66 10 Protein Targets of Acetaminophen 67 11 68 12 Covalent Binding in Rat and Mouse 69 13 70 14 Q2 Liver Studied by LC-MS/MS 71 15 Q3 72 Q1 16 Timon Geib, Ghazaleh Moghaddam, Aimee Supinski, Makan Golizeh† and Lekha Sleno* Q4 73 17 Q5 74 18 Chemistry Department, Université du Québec à Montréal, Montréal, QC, Canada Q6 75 19 76 20 Acetaminophen (APAP) is a mild analgesic and antipyretic used commonly worldwide. 77 21 78 Although considered a safe and effective over-the-counter medication, it is also the leading 22 79 23 cause of drug-induced acute liver failure. Its hepatotoxicity has been linked to the covalent 80 24 binding of its reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), to proteins. The 81 Edited by: 25 aim of this study was to identify APAP-protein targets in both rat and mouse liver, and to 82 26 Marcus S Cooke, 83 University of South Florida, compare the results from both species, using bottom-up proteomics with data-dependent 27 United States 84 28 high resolution mass spectrometry and targeted multiple reaction monitoring (MRM) 85 Reviewed by: 29 experiments. Livers from rats and mice, treated with APAP, were homogenized and 86 Hartmut Jaeschke, 30 University of Kansas Medical Center digested by trypsin. Digests were then fractionated by mixed-mode solid-phase extraction 87 31 Research Institute, United States prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Targeted LC- 88 32 Anthony P DeCaprio, 89 MRM assays were optimized based on high-resolution MS/MS data from information- 33 Florida International University, 90 34 United States dependent acquisition (IDA) using control liver homogenates treated with a custom 91 35 Q7 *Correspondence: alkylating reagent yielding a isomeric modification to APAP on cysteine residues, to 92 36 Lekha Sleno build a modified peptide database. A list of putative in vivo targets of APAP were 93 [email protected] 37 screened from data-dependent high-resolution MS/MS analyses of liver digests, 94 38 †Present address: 95 previous in vitro studies, as well as selected proteins from the target protein database 39 Department of Mathematical and 96 40 Physical Sciences, (TPDB), an online resource compiling previous reports of APAP targets proteins. Multiple 97 Concordia University of Edmonton, 41 protein targets in each species were found, while confirming modification sites. Several 98 Edmonton, AB, Canada 42 proteins were modified in both species, including ATP-citrate synthase, betaine- 99 43 100 Specialty section: homocysteine S-methyltransferase 1, cytochrome P450 2C6/29, mitochondrial 44 101 This article was submitted to glutamine amidotransferase-like protein/ES1 protein homolog, glutamine synthetase, 45 Analytical Chemistry, 102 46 a section of the journal microsomal glutathione S-transferase 1, mitochondrial-processing peptidase, 103 47 Frontiers in Chemistry methanethiol oxidase, protein/nucleic acid deglycase DJ-1, triosephosphate isomerase 104 48 Received: 05 July 2021 and thioredoxin. The targeted method afforded better reproducibility for analysing these 105 49 Accepted: 04 August 2021 106 50 Published: XX XX 2021 107 51 Citation: Abbreviations: ACN, acetonitrile; APAP, acetaminophen; BHMT, betaine—homocysteine S-methyltransferase 1; CA3, 108 52 Geib T, Moghaddam G, Supinski A, carbonic anhydrase 3; CAM, carbamidomethylation; CPS1, mitochondrial carbamoyl-phosphate synthase [ammonia]; DTT, 109 dithiothreitol; ER, endoplasmic reticulum; EST, estrogen sulfotransferase; GS, glutamine synthetase; HDAg, hepatitis delta Golizeh M and Sleno L (2021) Protein 53 antigen; HP-CAM, N-(4-hydroxyphenyl)-2-carbamidomethylation; HP-IAM, N-(4-hydroxyphenyl)-2-iodoacetamide; 3α- 110 Targets of Acetaminophen Covalent 54 HSD, 3-alpha-hydroxysteroid dehydrogenase; IAM, iodoacetamide; IDA, information-dependent acquisition; IP, intraperi- 111 55 Binding in Rat and Mouse Liver toneal; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MeOH, methanol; MGST1, microsomal glutathione 112 Studied by LC-MS/MS. 56 S-transferase 1; MRM, multiple reaction monitoring; MTO, methanethiol oxidase; NAPQI, N-acetyl p-benzoquinone imine; 113 Front. Chem. 9:736788. PMPCB, mitochondrial-processing peptidase subunit beta; SDS, n-dodecyl sulfate; S/N, signal-to-noise; SPE, solid-phase 57 doi: 10.3389/fchem.2021.736788 extraction; TPDB, Target Protein Database; TPI, triosephosphate isomerase; TXN, thioredoxin. 114 Frontiers in Chemistry | www.frontiersin.org 1 August 2021 | Volume 9 | Article 736788 Geib et al. Acetaminophen Protein Targets by LC-MS/MS 115 low-abundant modified peptides in highly complex samples compared to traditional data- 172 116 dependent experiments. 173 117 174 118 Keywords: acetaminophen, rodent model, reactive metabolites, liver proteins, LC-MS/MS, LC-MRM, protein 175 119 Q8 modification, NAPQI, N-acetyl-p-benzoquinone, mouse, rat, covalent binding 176 120 177 121 178 122 Q9 INTRODUCTION homogenates. This comparative analysis in rat and mouse 179 123 could potentially provide novel mechanistic insight into 180 124 Drugs are generally metabolized by the liver into biologically APAP-related hepatotoxicity. 181 125 inactive forms and eliminated from the body through bile and 182 126 urine. However during these processes, they can also be 183 127 bioactivated by hepatic enzymes into reactive electrophilic MATERIALS AND METHODS 184 128 intermediates and subsequently react with nucleophilic sites of 185 129 proteins to form covalent adducts (Xu et al., 2005; Park et al., Chemicals and Materials 186 130 2011). Reactive metabolites can in turn cause oxidative stress, Urea was purchased from BioRad (Mississauga, ON, Canada). 187 131 depleting intracellular glutathione, and affect energy metabolism APAP, trypsin (TPCK-treated, from bovine pancreas), sodium 188 132 by binding to mitochondrial protein complexes (Pessayre, 1995; n-dodecyl sulfate (SDS), dithiothreitol (DTT), iodoacetamide 189 133 Srivastava et al., 2010). (IAM), thiourea, ammonium bicarbonate, ammonium acetate, 190 134 Acetaminophen (N-acetyl p-aminophenol, APAP) is one of ammonium hydroxide, formic acid, acetic acid, acetonitrile 191 135 the most commonly used analgesic and antipyretic, and is known (ACN), methanol (MeOH) and all other reagents were from 192 136 to be the number one cause of acute liver failure in North America Sigma-Aldrich (St. Louis, MO, United States). Labeling agent N- 193 137 (Bateman, 2016). APAP is deemed safe as long as recommended (4-hydroxyphenyl)-2-iodoacetamide (HP-IAM) was synthesized 194 138 doses are not exceeded, but very high doses can cause severe in-house as previously described (LeBlanc et al., 2014). Ultra-pure 195 139 hepatotoxicity, liver injury and even death in both children and water was produced using a Millipore Synergy UV system 196 140 adults (Tittarelli et al., 2017). APAP metabolism primarily occurs (Billerica, MA, United States). 197 141 in the liver, where major biotransformations include sulfate and 198 142 glucuronide conjugation forming readily excretable phase II In Vivo Experiments 199 143 metabolites. A smaller proportion, between 10 and 15%, is Four Sprague-Dawley male rats (450−550 g) were dosed with 200 144 converted to the reactive electrophile N-acetyl p-benzoquinone 600 mg/kg APAP (IP; solubilized in 60% PEG 200) or two 201 145 imine (NAPQI), by multiple cytochrome P450 enzymes (Laine animals were treated with vehicle for control samples. Rat 202 146 et al., 2009). NAPQI can generally be detoxified via glutathione livers were collected after 24 h post dosing. Male C57BL/6 203 147 conjugation and further excreted via the mercapturic acid mice (27−35 g) were treated (IP, in saline) with 150 mg/kg (2 204 148 pathway. However, excess NAPQI can bind to nucleophilic and 6 h) and 300 mg/kg (2 and 6 h), as well as with vehicle for 205 149 sulfhydryl groups in cysteines of cellular proteins, leading to control samples. Two mice were treated at each dose and 206 150 possible protein dysfunction due to conformational changes, timepoint. All experiments were performed at INRS Centre 207 151 potentially leading to acute liver failure (James et al., 2003; National de Biologie Expérimentale (Laval, QC, Canada). The 208 152 Graham and Scott, 2005). Mitochondrial protein adducts have protocol was approved by the Ethics Committee of the INRS 209 153 been shown to directly correlate to the initiation of hepatotoxicity Centre National de Biologie Expérimentale under the ethical 210 154 through mitochondrial dysfunction and DNA fragmentation practices of the Canadian Council on Animal Care (project 211 155 (Tirmenstein and Nelson, 1989; McGill et al., 2012; McGill UQLK.14.02). 212 156 et al., 2014; Xie et al., 2015; Ramachandran and Jaeschke, 213 157 2020). Identifying and monitoring modified proteins from in Sample Preparation 214 158 vivo samples could help better understand the metabolic Frozen liver samples, stored at −80°C, were homogenized in 215 159 processes involved in APAP-related hepatotoxicity and 100 mM ammonium bicarbonate (ABC buffer, pH 8–8.5) at 216 160 potentially lead to the detection of important biomarkers 5 ml/g tissue weight using a hand-held homogenizer 217 161 (Cohen et al., 1997; Bissell et al., 2001). (Tissuemiser; Thermo Fisher Scientific, Waltham, MA, 218 162 In this study, liquid chromatography coupled to tandem mass United States), and 100 μl aliquots were combined with 25 μl 219 163 spectrometry (LC-MS/MS) was employed to identify in vivo of 0.1% SDS, and heated for 5 min at 95°C. After cooling samples, 220 164 protein targets of acetaminophen, via the formation of 50 μl of a solution of 7 M urea and 2 M thiourea in water was 221 165 NAPQI, in mouse and rat. Liver homogenates were digested added, and a probe sonicator was used ((XL-2000; Qsonica, 222 166 by trypsin and fractionated by mixed-mode solid-phase Newtown, CT, United States) for three cycles of 10 s, followed 223 167 extraction (SPE) prior to LC-MS/MS analysis.