Glutathione-Conjugates of Deoxynivalenol in Naturally Contaminated Grain Are Primarily Linked via the Epoxide Group

Abstract

A glutathione (GSH) adduct of the mycotoxin 4-deoxynivalenol (DON), together with a range of related conjugates, has recently been tentatively identified by LC-MS of DON-treated wheat spikelets. In this study, we prepared samples of DON conjugated at the 10- and 13-positions with GSH, Cys, CysGly, γ-GluCys and N-acetylcysteine (NAC). The mixtures of conjugates were used as standards for LC-HRMS analysis of one of the DON-treated wheat spikelet samples, as well as 19 Norwegian grain samples of spring wheat and 16 grain samples of oats that were naturally-contaminated with DON at concentrations higher than 1 mg/kg. The artificially-contaminated wheat spikelets contained conjugates of GSH, CysGly and Cys coupled at the olefinic 10-position of DON, whereas the naturally-contaminated harvest-ripe grain samples contained GSH, CysGly, Cys, and NAC coupled mainly at the 13-position on the epoxy group. The identities of the conjugates were confirmed by LC-HRMS comparison with authentic standards, oxidation to the sulfoxides with hydrogen peroxide, and examination of product-ion spectra from LC-HRMS/MS analysis. No γ-GluCys adducts of DON were detected in any of the samples. The presence of 15-O-acetyl-DON was demonstrated for the first time in Norwegian grain. The results indicate that a small but significant proportion of DON is metabolized via the GSH-conjugation pathway in plants. To our knowledge, this is the first report of in vivo conjugation of trichothecenes via their epoxy group, which has generally been viewed as unreactive. Because conjugation at the 13-position of DON and other trichothecenes has been shown to be irreversible, this type of conjugate may prove useful as a biomarker of exposure to DON and other 12,13-epoxytrichothecenes.

Stable Isotope-Assisted Metabolomics for Deciphering Xenobiotic Metabolism in Mammalian Cell Culture

“…(Stanic et al 2016a;Stanic et al 2016b). In addition, a comparison of the MS2 spectrum and the retention time with a wheat sample in which the presence of DON-10-GSH was confirmed before (Uhlig et al 2016) further supported the identification. The remaining conjugates were annotated at level 2b (probable structure/diagnostic).…”

“…Both activities are needed to generate DON-Cys from DON-GSH. Cysteine might directly react non-enzymatically as well (Uhlig et al 2016) in an alkaline cell compartment, but this seems unlikely.…”

“…For all masses of interest, agreement (retention time, accurate mass, MS2) with the available reference standards was checked. Known metabolites included in the multi-component standard and other metabolites described in literature like the plant metabolites DON-GSH, DON-Cys and DON-CysGly(Kluger et al 2015;Stanic et al 2016a;Uhlig et al 2016) were annotated automatically by MetExtract II in case they were present. According to a study bySchwartz et al (2013) other potential metabolites, namely the DON-sulfonates, were included in the analysis.…”

Do Plant-Bound Masked Mycotoxins Contribute to Toxicity?

“…In addition to these single-sugar conjugates, di-, tri-, and tetra-glucosides as well as mixed disaccharides and malonyl-glucosides have been described for DON, T2, HT2, and ZEN [18,19]. Glutathione-conjugates of DON have been identified in barley [20], wheat, and oats [21]. Sulfo-conjugates have been described for ZEN (ZEN14S) in naturally contaminated animal feed [22] and for DON (DON3S, DON15S) in artificially contaminated wheat [23,24].…”

Yesterday masked, today modified; what do mycotoxins bring next?

“…The most powerful technique among them is liquid chromatography coupled to orbitrap high resolution mass spectrometry (LC-HRMS), which has pointed to a whole new range of possibly masked mycotoxins (19) that have later been confirmed by triple quadrupole (QqQ) mass spectrometry analyser in real wheat (20) and human urine samples (21). Beside these, liquid chromatography/tandem mass spectrometry (LC-MS/ MS) has confirmed the existence of theoretical molecules such as DON-glutathione conjugates in cereals, first synthesised and characterised by nuclear magnetic resonance spectroscopy (22,23).…”