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Assessment of urinary deoxynivalenol biomarkers in UK children and adolescents

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Deoxynivalenol (DON), the mycotoxin produced mainly by Fusarium graminearum and found in contaminated cereal-based foodstuff, has been consistently detected in body fluids in adults. Available data in children and adolescents are scarce. This study assessed urinary DON concentrations in children aged 3–9 years (n = 40) and adolescents aged 10–17 years (n = 39) in the UK. Morning urine samples were collected over two consecutive days and analysed for free DON (un-metabolised form), DON-glucuronides (DON-GlcA), deepoxy deoxynivalenol (DOM-1), and total DON (sum of free DON, DON-GlcA, and DOM-1). Total DON was detected in the urine of > 95% of children and adolescents on both days. Mean total DON concentrations (ng/mg creatinine) were 41.6 and 21.0 for children and adolescents, respectively. The greatest total DON levels were obtained in female children on both days (214 and 219 ng/mg creatinine on days 1 and 2, respectively). Free DON and DON-GlcA were detected in most urine specimens, whereas DOM-1 was not present in any sample. Estimation of dietary DON exposure suggested that 33–63% of children and 5–46% of adolescents exceeded current guidance regarding the maximum provisional tolerable daily intake (PMTDI) for DON. Although moderate mean urinary DON concentrations were shown, the high detection frequency of urinary DON, the maximum biomarker concentrations, and estimated dietary DON exposure are concerning

Heterologous screening of hybridomas for the development of broad-specific monoclonal antibodies against deoxynivalenol and its analogues

Hapten heterology was introduced into the steps of hybridoma selection for the development of monoclonal antibodies (MAbs) against deoxynivalenol (DON). Firstly, a novel heterologous DON hapten was synthesised and covalently coupled to proteins (i.e. bovine serum albumin (BSA), ovalbumin and horseradish peroxidase) using the linkage of cyanuric chloride (CC). After immunisation, antisera from different DON immunogens were checked for the presence of useful antibodies. Next, both homologous and heterologous enzyme-linked immunosorbent assays were conducted to screen for hybridomas. It was found that heterologous screening could significantly reduce the proportion of false positives and appeared to be an efficient approach for selecting hybridomas of interest. This strategy resulted in two kinds of broad-selective MAbs against DON and its analogues. They were quite distinct from other reported DON-antibodies in their cross-reactivity profiles. A unique MAb 13H1 derived from DON-CC-BSA immunogen could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyl-DON > DON > nivalenol, with IC50 ranging from 1.14 to 7.69 mu g/ml. Another preferable MAb 10H10 generated from DON-BSA immunogen manifested relatively similar affinity to DON, 3-acetyl-DON and 15-acetyl-DON, with IC50 values of 22, 15 and 34 ng/ml, respectively. This is the first broad-specific MAb against DON and its two acetylated forms and thus it can be used for simultaneous detection of the three mycotoxins

Stability of DON and DON-3-glucoside during baking as affected by the presence of food additives

The mycotoxin deoxynivalenol (DON) is one of the most common mycotoxins of cereals worldwide, and its occurrence has been widely reported in raw wheat. The free mycotoxin form is not the only route of exposure; modified forms can also be present in cereal products. Deoxynivalenol-3-glucoside (DON-3-glucoside) is a common DON plant conjugate. The mycotoxin concentration could be affected by food processing; here, we studied the stability of DON and DON-3-glucoside during baking of small doughs made from white wheat flour and other ingredients. A range of common food additives and ingredients were added to assess possible interference: ascorbic acid (E300), citric acid (E330), sorbic acid (E200), calcium propionate (E282), lecithin (E322), diacetyltartaric acid esters of fatty acid mono- and diglycerides (E472a), calcium phosphate (E341), disodium diphosphate (E450i), xanthan gum (E415), polydextrose (E1200), sorbitol (E420i), sodium bicarbonate (E500i), wheat gluten and malt flour. The DON content was reduced by 40%, and the DON-3-glucoside concentration increased by >100%, after baking for 20 min at 180°C. This confirmed that DON and DON-3-glucoside concentrations can vary during heating, and DON-3-glucoside could even increase after baking. However, DON and DON- 3-glucoside are not affected significantly by the presence of the food additives tested.The authors are grateful to the Spanish government (project AGL2014-55379-P) for providing financial support. A. Vidal thanks the Spanish government (Ministry of Education) for the pre-doctoral grant

The impact of deoxynivalenol on pigeon health : occurrence in feed, toxicokinetics and interaction with salmonellosis

Seed-based pigeon diets could be expected to result in exposure of pigeons to mycotoxins such as deoxynivalenol (DON). Ingestion of low to moderate contamination levels of DON may impair intestinal health, immune function and/or pathogen fitness, resulting in altered host-pathogen interactions and thus different outcome of infections. Here we demonstrate that DON was one of the most frequently detected mycotoxins in seed-based racing pigeons feed, contaminating 5 out of 10 samples (range 177-1,466 mu g/kg). Subsequently, a toxicokinetic analysis revealed a low absolute oral bioavailability (F) of DON in pigeons (30.4%), which is comparable to other avian species. Furthermore, semi-quantitative analysis using high-resolution mass spectrometry revealed that DON-3 alpha-sulphate is the major metabolite of DON in pigeons after intravenous as well as oral administration. Following ingestion of DON contaminated feed, the intestinal epithelial cells are exposed to significant DON concentrations which eventually may affect intestinal translocation and colonization of bacteria. Feeding pigeons a DON contaminated diet resulted in an increased percentage of pigeons shedding Salmonella compared to birds fed control diet, 87 +/- 17% versus 74 +/- 13%, respectively. However, no impact of DON was observed on the Salmonella induced disease signs, organ lesions, faecal and organ Salmonella counts. The presented risk assessment indicates that pigeons are frequently exposed to mycotoxins such as DON, which can affect the outcome of a Salmonella infection. The increasing number of pigeons shedding Salmonella suggests that DON can promote the spread of the bacterium within pigeon populations

The food contaminant deoxynivalenol, decreases intestinal barrier permeability and reduces claudin expression

The gastrointestinal tract represents the first barrier against food contaminants as well as the first target for these toxicants. Deoxynivalenol (DON) is a mycotoxin that commonly contaminates cereals and causes various toxicological effects. Through consumption of contaminated cereals and cereal products, human and pigs are exposed to this mycotoxin. Using in vitro, ex vivo, and in vivo approaches, we investigated the effects of DON on the intestinal epithelium. We demonstrated that, in intestinal epithelial cell lines from porcine (IPEC-1) or human (Caco-2) origin, DON decreases trans-epithelial electric resistance (TEER) and increases in a time and dose-dependent manner the paracellular permeability to 4 kDa dextran and to pathogenic Escherichia Coli across intestinal cell monolayers. In pig explants treated with DON, we also observed an increased permeability of intestinal tissue. These alterations of barrier function were associated with a specific reduction in the expression of claudins, which was also seen in vivo in the jejunum of piglets exposed to DON-contaminated feed. In conclusion, DON alters claudin expression and decreases the barrier function of the intestinal epithelium. Considering that high levels of DON may be present in food or feed, consumption of DON-contaminated food/feed may induce intestinal damage and has consequences for human and animal health

The mycotoxin deoxynivalenol predisposes for the development of Clostridium perfringens-induced necrotic enteritis in broiler chickens

Both mycotoxin contamination of feed and Clostridium perfringens-induced necrotic enteritis have an increasing global economic impact on poultry production. Especially the Fusarium mycotoxin deoxynivalenol (DON) is a common feed contaminant. This study aimed at examining the predisposing effect of DON on the development of necrotic enteritis in broiler chickens. An experimental Clostridium perfringens infection study revealed that DON, at a contamination level of 3,000 to 4,000 mg/kg feed, increased the percentage of birds with subclinical necrotic enteritis from 2062.6% to 4763.0% (P<0.001). DON significantly reduced the transepithelial electrical resistance in duodenal segments (P<0.001) and decreased duodenal villus height (P = 0.014) indicating intestinal barrier disruption and intestinal epithelial damage, respectively. This may lead to an increased permeability of the intestinal epithelium and decreased absorption of dietary proteins. Protein analysis of duodenal content indeed showed that DON contamination resulted in a significant increase in total protein concentration (P = 0.023). Furthermore, DON had no effect on in vitro growth, alpha toxin production and netB toxin transcription of Clostridium perfringens. In conclusion, feed contamination with DON at concentrations below the European maximum guidance level of 5,000 mg/kg feed, is a predisposing factor for the development of necrotic enteritis in broilers. These results are associated with a negative effect of DON on the intestinal barrier function and increased intestinal protein availability, which may stimulate growth and toxin production of Clostridium perfringens

Mycotoxins nivalenol and deoxynivalenol differently modulate cytokine mRNA expression in Jurkat T cells.

Deoxynivalenol (DON) and its hydroxylated form nivalenol (NIV) are Fusarium mycotoxins that occur in cereal grains alone or in combination. Several studies have shown that these metabolites affect lymphocyte functions. However, the molecular mechanisms underlying their activities are still partially known. To address this issue, we examined the influence of NIV and DON in modulating IFNc, IL-2 and IL-8 mRNA levels in Jurkat T cells. In PMA/ionomycin stimulated cells, pre-incubated with increasing concentrations of NIV, transcription was induced in the range 0.06–2 lM; higher concentrations of NIV were found non-stimulating (4 lM) or inhibitory (8 lM) for IFNc and IL-2 whereas IL-8 was still induced. DON administration elicited a similar profile for IL-8 and IFNc, whilst IL-2 mRNA was induced in a broader range of concentrations. Combination of NIV and DON at 1:1 and 1:10 ratios essentially restored the cytokine transcriptional pattern observed with NIV alone but the level of transcripts, with the exception of IL-8, peaked at lower concentrations suggesting interactive effects. Moreover both mycotoxins caused inhibition of cell proliferation, mediated by induction of apoptosis, confirming previous results and highlighting the usefulness of Jurkat as a T-cell model to study the effects of mycotoxins on the immune functions in humans

Leaching of organic nitrogen and carbon after cultivating grass-clover pastures

Leaching of organic nitrogen (DON) and carbon (DOC) was measured after cultivating grass-clover of different age. It was found that DON and escpecially DOC was lost in considerable amounts, and that the leaching depends upon crop and management. The highest concentrations of DON were measured in the bare soil treatment, whereas concen-trations in catch crop treatments were between 1.2 and 3.2 mg N L-1. The leaching of DOC showed opposite trends compared to leaching of DON with higher values in the catch crop treatments (296 - 310 kg DOC ha-1) than in bare soil treatments (174 - 217 kg DOC ha-1)