Resistance to Fusarium verticillioides and fumonisin accumulation in maize inbred lines involves an earlier and enhanced expression of lipoxygenase (LOX) genes.

Fusarium verticillioides causes ear rot in maize and contaminates the kernels with the fumonisin myco-toxins. It is known that plant lipoxygenase (LOX)-derived oxylipins regulate defence against pathogensand that the host-pathogen lipid cross-talk influences the pathogenesis. The expression profiles of fif-teen genes of the LOX pathway were studied in kernels of resistant and susceptible maize lines, grownin field condition, at 3, 7 and 14 days post inoculation (dpi) with F. verticillioides. Plant defence responseswere correlated with the pathogen growth, the expression profiles of fungal FUM genes for fumonisinbiosynthesis and fumonisin content in the kernels. The resistant genotype limited fungal growth andfumonisin accumulation between 7 and 14 dpi. Pathogen growth became exponential in the susceptibleline after 7 dpi, in correspondence with massive transcription of FUM genes and fumonisins augmentedexponentially at 14 dpi. LOX pathway genes resulted strongly induced after pathogen inoculation in theresistant line at 3 and 7 dpi, whilst in the susceptible line the induction was reduced or delayed at 14 dpi.In addition, all genes resulted overexpressed before infection in kernels of the resistant genotype alreadyat 3 dpi. The results suggest that resistance in maize may depend on an earlier activation of LOX genesand genes for jasmonic acid biosynthesis

Deoxynivalenol & deoxynivalenol-3-glucoside mitigation through bakery production strategies: Effective experimental design within industrial rusk-making technology

In the scientific field, there is a progressive awareness about the potential implications of food processing on mycotoxins especially concerning thermal treatments. High temperatures may cause, in fact, transformation or degradation of these compounds. This work is aimed to study the fate of mycotoxins during bakery processing, focusing on deoxynivalenol (DON) and deoxynivalenol-3-glucoside (DON3Glc), along the chain of industrial rusk production. Starting from naturally contaminated bran, we studied how concentrations of DON and DON3Glc are influenced by modifying ingredients and operative conditions. The experiments were performed using statistical Design of Experiment (DoE) schemes to synergistically explore the relationship between mycotoxin reduction and the indicated processing transformation parameters. All samples collected during pilot plant experiments were analyzed with an LC-MS/MS multimycotoxin method. The obtained model shows a good fitting, giving back relevant information in terms of optimization of the industrial production process, in particular suggesting that time and temperature inbaking and toasting steps are highly relevant for minimizing mycotoxin level in rusks. A reduction up to 30% for DON and DON3Glc content in the finished product was observed within an acceptable technological range

Extention and interlaboratory comparison of an LC-MS/MS multi-class method for the determination of 15 different classes of veterinary drug residues in milk and poultry feed

An HPLC-MS/MS multi-class method for quantitation of 15 different classes of veterinary drug residues (>140 analytes) in milk and poultry feed was developed and validated. Accuracy criteria for routine laboratories were met for the majority of analytes, > 83 % in milk and between 50 and 60 % in chicken feed, with an apparent recovery of 60-140 %. Extraction efficiency criteria were met for >95 % of the analytes for milk and > 80 % for chicken feed. Intermediate precision meets the SANTE criterion of RSD < 20 % for 80-90 % of the analytes in both matrices. For all analytes with an existing MRL in milk, the LOQ was below the related MRL. Twenty-nine samples of commercial milk and chicken feed were analyzed within the interlaboratory comparison. No residues of veterinary drugs were found in the milk samples. However, the feed samples exhibited high levels of nicarbazin, salinomycin, and decoquinate

Deoxynivalenol-3-β-D-glucoside : in vitro cytotoxicity and in vivo oral bioavailability and hydrolysis in broiler chicken and pig

In addition to DON, cereals are often co-contaminated with modified forms of DON, such as deoxynivalenol-3-β-D-glucoside (DON3G), 3-acetyl-deoxynivalenol (3ADON) or 15-acetyl-deoxynivalenol (15ADON). Due to the lack of information on toxicity and bioavailability of modified mycotoxins, current risk assessment assumes that these modified forms are equally toxic to their respective unmodified counterparts. The goal of the in vitro study was to determine the intrinsic cytotoxicity of modified DON forms towards porcine intestinal epithelial cells by means of a flow cytometric technique. Quantification of viable cells, apoptotic and necrotic cells indicate following toxicity ranking: DON3G << 3ADON < DON ≈ 15ADON. For the in vivo part, cross-over animal trials were performed with intravenous and oral administration of DON3G and DON to broiler chickens and pigs. Systemic and portal plasma concentrations of DON and DON3G were quantified using liquid chromatography-tandem mass spectrometry. Liquid chromatography coupled to high-resolution mass spectrometry was used to unravel phase II metabolism of DON. Data were processed via tailor-made compartmental toxicokinetic models. The results in broiler chickens indicate that DON3G is not hydrolysed to DON in vivo. Furthermore, the absolute oral bioavailability of DON3G in broiler chickens was low (3.79±2.68%) and comparable to that of DON (5.56±2.05%). After oral DON3G administration to pigs, only DON was detected in plasma, indicating a complete presystemic hydrolysis. However, the absorbed fraction of DON3G, recovered as DON, was approximately 5 times lower than after oral DON administration, 16.1±5.4% compared to 81.3±17.4%. Additionally, analysis of phase II metabolites revealed that biotransformation of DON in pigs mainly consists of limited glucuronidation, whereas in chickens extensive conjugation with sulfate occurs, these observations might explain the differences in sensitivity of these species to DON. Although in vitro studies demonstrate a decreased toxicity of DON3G compared to DON, the species dependent toxicokinetic data and in vivo hydrolysis to DON illustrate the toxicological relevance of DON3G

Biotransformation of the Mycotoxin Deoxynivalenol in Fusarium Resistant and Susceptible Near Isogenic Wheat Lines

<div><p>In this study, a total of nine different biotransformation products of the <i>Fusarium mycotoxin</i> deoxynivalenol (DON) formed in wheat during detoxification of the toxin are characterized by liquid chromatography—high resolution mass spectrometry (LC-HRMS). The detected metabolites suggest that DON is conjugated to endogenous metabolites via two major metabolism routes, namely 1) glucosylation (DON-3-glucoside, DON-di-hexoside, 15-acetyl-DON-3-glucoside, DON-malonylglucoside) and 2) glutathione conjugation (DON-S-glutathione, “DON-2H”-S-glutathione, DON-S-cysteinyl-glycine and DON-S-cysteine). Furthermore, conjugation of DON to a putative sugar alcohol (hexitol) was found. A molar mass balance for the cultivar ‘Remus’ treated with 1 mg DON revealed that under the test conditions approximately 15% of the added DON were transformed into DON-3-glucoside and another 19% were transformed to the remaining eight biotransformation products or irreversibly bound to the plant matrix. Additionally, metabolite abundance was monitored as a function of time for each DON derivative and was established for six DON treated wheat lines (1 mg/ear) differing in resistance quantitative trait loci (QTL) <i>Fhb1</i> and/or <i>Qfhs.ifa-5A</i>. All cultivars carrying QTL <i>Fhb1</i> showed similar metabolism kinetics: Formation of DON-Glc was faster, while DON-GSH production was less efficient compared to cultivars which lacked the resistance QTL <i>Fhb1</i>. Moreover, all wheat lines harboring <i>Fhb1</i> showed significantly elevated D3G/DON abundance ratios.</p></div

EICs of DON and its corresponding biotransformation products.

<p>EICs of accurate mass traces (± 3 ppm) of DON and its corresponding biotransformation products in a wheat sample harvested 96 hours post treatment with 1 mg DON. Due to low abundance, EIC intensities of DON-di-hexoside were multiplied by a factor of 10.</p

Overview on DON degradation.

<p>Time course for the degradation of DON (1 mg) of wheat lines ‘CM-82036’, C1, C2, C3, C4 and ‘Remus’. Wheat ears were sampled 0, 12, 24, 48, and 96 hours after treatment (n = 5 biological replicates per time point and wheat line). a) Degradation rate of DON. b) boxplot of relative concentrations 96 h after DON treatment. c) DON-glucoside/DON ratio 96 h after DON treatment. * Significantly differing DON levels between wheat lines with and without resistance QTL <i>Fhb1</i> based on a non-paired t-test (5% global significance threshold).</p