Abstract
The Fusarium mycotoxin deoxynivalenol (DON) is a frequent contaminant of cereal-based food and feed. Mammals metabolize DON by conjugation to glucuronic acid (GlcAc), the extent and regioselectivity of which is species-dependent. So far, only DON-3-glucuronide (DON-3-GlcAc) and DON-15-GlcAc have been unequivocally identified as mammalian DON glucuronides, and DON-7-GlcAc has been proposed as further DON metabolite. In the present work, qualitative HPLC–MS/MS analysis of urine samples of animals treated with DON (rats: 2 mg/kg bw, single bolus, gavage; mice: 1 mg/kg bw, single i.p. injection; pigs: 74 µg/kg bw, single bolus, gavage; cows: 5.2 mg DON/kg dry mass, oral for 13 weeks) revealed additional DON and deepoxy-DON (DOM) glucuronides. To elucidate their structures, DON and DOM were incubated with human (HLM) and rat liver microsomes (RLM). Besides the expected DON/DOM-3- and 15-GlcAc, minor amounts of four DON- and four DOM glucuronides were formed. Isolation and enzymatic hydrolysis of four of these compounds yielded iso-DON and iso-DOM, the identities of which were eventually confirmed by NMR. Incubation of iso-DON and iso-DOM with RLM and HLM yielded two main glucuronides for each parent compound, which were isolated and identified as iso-DON/DOM-3-GlcAc and iso-DON/DOM-8-GlcAc by NMR. Iso-DON-3-GlcAc, most likely misidentified as DON-7-GlcAc in the literature, proved to be a major DON metabolite in rats and a minor metabolite in pigs. In addition, iso-DON-8-GlcAc turned out to be one of the major DON metabolites in mice. DOM-3-GlcAc was the dominant DON metabolite in urine of cows and an important DON metabolite in rat urine. Iso-DOM-3-GlcAc was detected in urine of DON-treated rats and cows. Finally, DON-8,15-hemiketal-8-glucuronide, a previously described by-product of DON-3-GlcAc production by RLM, was identified in urine of DON-exposed mice and rats. The discovery of several novel DON-derived glucuronides in animal urine requires adaptation of the currently used methods for DON-biomarker analysis.
Highlights
Formed pre-harvest by Fusarium species, the mycotoxin deoxynivalenol (DON) is one of the most frequent fungal contaminants of food and feed worldwide
As isolation of several hundred microgram amounts of these compounds—which is required for structure elucidation by nuclear magnetic resonance spectroscopy (NMR)—is not practicable, we decided to incubate DON and DOM with rat- and human liver microsomes, to isolate the formed glucuronides and compare them with the unknown glucuronides detected in animal urine samples
The only DON glucuronides identified beyond doubt in human and animal urine samples had been DON-3-glucuronic acid (GlcAc) and DON-15-GlcAc
Summary
Formed pre-harvest by Fusarium species, the mycotoxin deoxynivalenol (DON) is one of the most frequent fungal contaminants of food and feed worldwide. DON affects eukaryotic cells by inhibition of protein-, DNA-, and RNA synthesis, resulting in toxic effects in animals and plants (Rocha et al 2005). These effects include feed refusal and emesis, growth retardation, and modulation of immune response in animals (Pestka 2010). Depending on the animal species, animals conjugate DON to glucuronic acid (GlcAc) (reviewed by Payros et al 2016) or sulfate (Schwartz-Zimmermann et al 2015; Wan et al 2014), which are both phase II metabolism reactions. The most prominent microbial metabolite of DON is deepoxy-DON (DOM)(Fuchs et al 2002) which can, in turn, be subject to glucuronidation (Nagl et al 2014), sulfation (SchwartzZimmermann et al 2015), or sulfonation (Schwartz-Zimmermann et al 2014)
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