Microbiota release of bound mycotoxins contributes to human exposure: in vitro and in vivo evidence

Mycotoxins are toxic fungal metabolites in cereal foods and pose a potential health risk for consumers. In addition to fungal mycotoxins, plant-derived mycotoxin metabolites are frequently found in cereals. These mycotoxin sugar-conjugates may contribute to overall exposure and toxicity in humans. Our
previous work used a range of in vitro model systems to predict the intestinal fate of masked mycotoxin-glucosides in the human gut. We have demonstrated that DON-3-glucoside, the most commonly detected mycotoxin-conjugate in cereals, is stable under small intestinal conditions and is not absorbed through the intestinal epithelium intact [1]. Following transport to the large intestine and contact with the gut microbiota, DON-3-glucoside could be rapidly hydrolysed and free DON released, as demonstrated in human faecal batch cultures. However, the in vivo degradation of DON-3-glucoside and absorption from contaminated food has not been demonstrated in humans. Hence, this study assessed the exposure of human volunteers to DON from barley porridge naturally contaminated with DON-3-glucoside at a low, safe level.
Following 3 days of cereal-restricted diet, 9 healthy fasted volunteers (3 male, 6 female) consumed one portion of barley porridge containing 664 ng DON-3-glucoside. All urine excreted was collected at defined time points (1, 3, 5 and 24 hours post-dosing) and a pooled urine sample was collected overnight. Urinary excretion of free DON was monitored using an established extraction and LC-MS/MS method(2). Results for average urinary DON in early time points (1–5 hours) were compared to overnight samples and 24 hour time points by ANOVA and post-hoc t-test.
From in vitro results we found that DON-3-glucoside is not absorbed in the small intestine while free DON is rapidly transported through the gut epithelium. This finding is confirmed in vivo where only small proportions (average 9–20%) of the DON-3-glucoside dose were excreted as DON in the early urine samples 1–5 hours post-dosing. The majority of urinary DON was detected in the overnight
sample (average 81% of dose, p < 0.05 compared to 1–5 hour time points), suggesting the release of DON from DON-3-glucoside in the lower GI tract. Marked inter-individual differences were observed in the kinetics of DON excretion in human urine in the present study, which also confirms previous in vitro results highlighting different DON-3-glucoside hydrolysis rates between microbiota samples from different donors.
In summary, we demonstrate for the first time that exposure to DON-3-glucoside through contaminated food results in urinary excretion of free DON through microbial release and colonic absorption. This finding confirms that sugar-bound mycotoxin conjugates can contribute to mycotoxin exposure and toxicity in humans and need to be included in future risk assessments.

This work was supported by the Scottish Government Rural and Environment Science and Analytical Services Division (RESAS).