Glucuronidation of deoxynivalenol (DON) by different animal species: identification of iso-DON glucuronides and iso-deepoxy-DON glucuronides as novel DON metabolites in pigs, rats, mice, and cows

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-017-2012-z) contains supplementary material, which is available to authorized users.

Mycotoxin Biomarkers of Exposure: A Comprehensive Review

“…In vitro studies using liver microsomes demonstrated that DON is sensitive to metabolization as a large percentage of free DON (>75%) is converted to DON metabolites. DON metabolism is species‐dependent, and significant differences have been observed among animals (rat, fish, cattle, and swine) and humans (Maul et al., ; Schwartz‐Zimmermann et al., ) (Table ). Deoxynivalenol‐15‐glucuronide (DON‐15‐glucuronide) was the predominant DON metabolite after DON‐incubation in human liver microsomes, while deoxynivalenol‐3‐glucuronide (DON‐3‐glucuronide) was the main metabolite for all other studied animals (fish, rat, cattle, and swine) (Maul et al., ; Schwartz‐Zimmermann et al., ).…”

“…DON metabolism is species‐dependent, and significant differences have been observed among animals (rat, fish, cattle, and swine) and humans (Maul et al., ; Schwartz‐Zimmermann et al., ) (Table ). Deoxynivalenol‐15‐glucuronide (DON‐15‐glucuronide) was the predominant DON metabolite after DON‐incubation in human liver microsomes, while deoxynivalenol‐3‐glucuronide (DON‐3‐glucuronide) was the main metabolite for all other studied animals (fish, rat, cattle, and swine) (Maul et al., ; Schwartz‐Zimmermann et al., ). The second prevailing glucuronide was observed in lower levels (<10% compared to the first prevailing glucuronide) for all species and was species‐dependent: DON‐15‐glucuronide in pigs (Maul et al., ), DON‐3‐glucuronide in humans (Maul et al., ; Schwartz‐Zimmermann et al., ), iso‐deoxynivalenol‐3‐glucuronide (iso‐DON‐3‐glucuronide) in fish and bovine (Maul et al., ), and deoxynivalenol‐8,15‐hemiketal‐8‐glucuronide (DON‐8,15‐hemiketal‐8‐glucuronide) in rodents (Schwartz‐Zimmermann et al., ; Uhlig, Ivanova, and Fæste, ).…”

“…The second prevailing glucuronide was observed in lower levels (<10% compared to the first prevailing glucuronide) for all species and was species‐dependent: DON‐15‐glucuronide in pigs (Maul et al., ), DON‐3‐glucuronide in humans (Maul et al., ; Schwartz‐Zimmermann et al., ), iso‐deoxynivalenol‐3‐glucuronide (iso‐DON‐3‐glucuronide) in fish and bovine (Maul et al., ), and deoxynivalenol‐8,15‐hemiketal‐8‐glucuronide (DON‐8,15‐hemiketal‐8‐glucuronide) in rodents (Schwartz‐Zimmermann et al., ; Uhlig, Ivanova, and Fæste, ). It is worth mentioning that DON‐7‐glucuronide was recently identified as iso‐DON‐3‐glucuronide (Schwartz‐Zimmermann et al., ).…”

“…Schwartz‐Zimmermann et al. () detected 6 different DON‐metabolites after rat microsomal incubation: DON‐3‐glucuronide, DON‐8,15‐hemiketal‐8‐glucuronide, DON‐15‐glucuronide, iso‐DON‐3‐glucuronide, iso‐deoxynivalenol‐8‐glucuronide (iso‐DON‐8‐glucuronide), and an unknown DON‐glucuronide (Table ). An inverted phenomenon was observed in intestinal human cells as a higher amount of DON‐3‐glucuronide was produced than DON‐15‐glucuronide.…”

The role of roughage provision on the absorption and disposition of the mycotoxin deoxynivalenol and its acetylated derivatives in calves: from field observations to toxicokinetics

“…In this study it is suggested that roughage provision stimulate the intestinal microbial DON de-epoxidation capacity by the detection of only traces of DOM-1 in the urine of non-ruminating calves while much higher levels were observed in ruminating calves following IV or PO administration of DON or its ADONs. In addition to DOM-1, also GlcA metabolites of DOM-1 such as DOM-3-GlcA, iso-DOM-3-GlcA, and DOM-15-GlcA have been observed in cow urine samples (Schwartz-Zimmermann et al 2017). Consequently, the low systemic DON bioavailability measured in sheep (Prelusky et al 1985) and in ruminating calves in this study might be associated with significant total rumen degradation.…”