Liquid Chromatography-Tandem Mass Spectrometry Analysis of Acetaminophen Covalent Binding to Glutathione S-Transferases.

Timon Geib,Derek J Wilson,Lekha Sleno,Cristina Lento

doi:10.3389/fchem.2019.00558

Timon Geib, Derek J Wilson + Show 2 more

Open Access

https://doi.org/10.3389/fchem.2019.00558

Copy DOI

Abstract

Acetaminophen (APAP)-induced hepatotoxicity is the most common cause of acute liver failure in the Western world. APAP is bioactivated to N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite, which can subsequently covalently bind to glutathione and protein thiols. In this study, we have used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize NAPQI binding to human glutathione S-transferases (GSTs) in vitro. GSTs play a crucial role in the detoxification of reactive metabolites and therefore are interesting target proteins to study in the context of APAP covalent binding. Recombinantly-expressed and purified GSTs were used to assess NAPQI binding in vitro. APAP biotransformation to NAPQI was achieved using rat liver microsomes or human cytochrome P450 Supersomes in the presence of GSTA1, M1, M2, or P1. Resulting adducts were analyzed using bottom-up proteomics, with or without LC fractionation prior to LC-MS/MS analysis on a quadrupole-time-of-flight instrument with data-dependent acquisition (DDA). Targeted methods using multiple reaction monitoring (MRM) on a triple quadrupole platform were also developed by quantitatively labeling all available cysteine residues with a labeling reagent yielding isomerically-modified peptides following enzymatic digestion. Seven modified cysteine sites were confirmed, including Cys112 in GSTA1, Cys78 in GSTM1, Cys115 and 174 in GSTM2, as well as Cys15, 48, and 170 in GSTP1. Most modified peptides could be detected using both untargeted (DDA) and targeted (MRM) approaches, however the latter yielded better detection sensitivity with higher signal-to-noise and two sites were uniquely found by MRM.

Highlights

Hepatotoxicity induced by acetaminophen (APAP), a widely used analgesic and antipyretic, presents major health care challenges in Western societies (Fagan and Wannan, 1996; Lee, 2004; Larson et al, 2005; Sivilotti et al, 2005; James et al, 2013)
In a previous study in rat liver microsomes (RLM), we reported that rat microsomal glutathione S-transferases (GSTs) 1 (MGST1) was a target of N-acetyl p-benzoquinone imine (NAPQI) (Golizeh et al, 2015)
For unbiased analysis of binding to cysteines in recombinant GSTs, each GST was probed for covalent GSH-cysteine adducts, which could have occurred during GST purification over GSH affinity chromatography

Summary

Introduction

Hepatotoxicity induced by acetaminophen (APAP), a widely used analgesic and antipyretic, presents major health care challenges in Western societies (Fagan and Wannan, 1996; Lee, 2004; Larson et al, 2005; Sivilotti et al, 2005; James et al, 2013). LC-MS/MS Analysis of NAPQI-GST by APAP correlates often with overdose or misusage, including chronic overuse (Lee et al, 2008). This often results in difficulties in diagnosis and treatment (Cairney et al, 2016). APAP is mostly detoxified via phase II metabolism; with direct glucuronidation (∼55%) and sulfation (∼40%) (Mitchell et al, 1973a; Mazaleuskaya et al, 2015). The fraction of oxidized APAP can increase up to 15%, due to saturation of the sulfation pathway (Mazaleuskaya et al, 2015). NAPQI can be detoxified by subsequent phase II metabolism via conjugation with glutathione (GSH), partly mediated by different glutathione S-transferase (GST) isozymes (Figure 1) (Mitchell et al, 1973b)

Methods

Results

Conclusion