Characterization of Deoxynivalenol-Glutathione Conjugates Using Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography-High-Resolution Mass Spectrometry Ana Stanic, †,§ Silvio Uhlig, † Morten Sandvik, † Frode Rise, § Alistair L. Wilkins, †,⊥ and Christopher O. Miles* ,† † Norwegian Veterinary Institute, P.O. Box 750, Sentrum, NO-0106 Oslo, Norway § Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315 Oslo, Norway ⊥ Chemistry Department, University of Waikato, Private Bag 3105, 3240 Hamilton, New Zealand * S Supporting Information ABSTRACT: Glutathione (GSH) conjugates of the mycotoxin 4-deoxynivalenol (DON), 1, have been detected in plants by LC-MS, but their identities were not confirmed due to a lack of standards. We have synthesized DON-GSH conjugates in alkaline solution. The major products 2 and 5 were isolated and their structures determined by mass spectrometry and NMR spectroscopy as GSH adducts at C-13 and C-10 (via epoxide and Michael addition, respectively) of 1. Other Michael addition products were also tentatively identified by LC-MS. Concentrations of 2 and 5 were determined by quantitative NMR and are suitable for use as quantitative standards for LC-MS studies of plant and animal metabolism of 1. LC-MS showed that in the presence of human glutathione S-transferases of the alpha and mu classes, the reaction of DON and GSH proceeded with a half- life of 17 h, identical with the rate of the uncatalyzed reaction rate, indicating an absence of catalysis. KEYWORDS: thiol, mycotoxin, HRMS, LC-MS, NMR, ERETIC, conjugation, GSH, GST ■ INTRODUCTION Fungi of the genus Fusarium infect grain and other crops worldwide and are responsible for contamination of grain products with a range of mycotoxins, including trichothecenes, zearalenone, and fumonisins. 1 Among the trichothecene mycotoxins, 4-deoxynivalenol (DON), 1 (Figure 1), is one of the most widespread and economically important, causing feed refusal, reduced weight gain, and emesis in livestock consuming heavily contaminated grain products. 2 Several detoxification mechanisms for 1 have been identified in plants infected with Fusarium, including sulfation, 3 glycosylation, 4 and conjugation with the tripeptide L-glutathione (GSH). 5 The presence of DON-GSH, as well as its presumed degradation products DON-cysteinylglycine and DON-cysteine (DON-Cys), was recently demonstrated by LC-MS in DON-treated wheat lines, 6 although the actual structures of the conjugates were not determined. This, together with the observed up-regulation of glutathione transferase (GST) and related pathways in contaminated plants, 7 suggests a role for GST-catalyzed conjugation in this detoxification process. GSTs are a widely distributed superfamily of multifunctional enzymes found in invertebrates and vertebrates, plants, yeasts, and bacteria. 8 Their main role is catalysis of the nucleophilic conjugation of GSH to electrophilic substrates, but they also have less well characterized roles in endogenous metabolism including functioning as GSH-dependent peroxidases or GSH-dependent isomerases or have noncatalytic functions including binding of nonsubstrate ligands and modulation of signaling processes. 9 Thus, a wide array of glutathione conjugates can be formed with endogenous and xenobiotic electrophilic species. 10,11 Characteristic features of glutathione are its high cellular concentration relative to other thiols, with glutathione accumulating to millimolar concentrations in tissues, well in excess of the concentration of free cysteine, and the high reduction state of the cellular glutathione pool. 12 Recent studies with 1 suggested that it reacts with a wide range of thiols, including Cys and GSH, and that the reactive species is the thiolate. 13 The reaction of 1 with Cys leads to reversible Michael addition of the thiolate to C-10 to form multiple diastereoisomers and to irreversible nucleophilic addition of the thiolate to C-13 of the epoxy group. 14 Thus, the Cys and GSH conjugates of 1 formed naturally in plants could plausibly have a number of structures. Here we report the base-catalyzed synthesis and isolation of GSH adducts of 1 and their identification by LC-MS and NMR. The adducts were then used as standards to study the possible role of GST catalysis in the conjugation of 1 with GSH in vitro. ■ MATERIALS AND METHODS Chemicals. DON, 1 (≥98%), GSH (≥98%), 5.0 mm Norell Standard Series NMR tubes, D 2 O (99.9 atom % D), glutathione S- transferase (GST) assay kit (CS0410, human GSTA4 GSTM1), and sodium carbonate (pro analysis) were from Sigma-Aldrich (Steinheim, Germany). LC-MS grade water and acetonitrile were from Fisher Scientific (Oslo, Norway), whereas ammonium formate (puriss p.a. for HPLC) and dioxane were from Fluka (Steinheim, Germany). Sodium Received: June 24, 2016 Revised: August 18, 2016 Accepted: August 22, 2016 Published: August 22, 2016 Article pubs.acs.org/JAFC © 2016 American Chemical Society 6903 DOI: 10.1021/acs.jafc.6b02853 J. Agric. Food Chem. 2016, 64, 6903-6910