15-acetyldeoxynivalenol and deoxynivalenol

The reactive electrophilic species (RES), typically the molecules bearing α,β-unsaturated carbonyl group, are widespread in living organisms and notoriously known for their damaging effects. Many of the mycotoxins released from phytopathogenic fungi are RES and their contamination to cereals threatens food safety worldwide. However, due to their high reactivity, RES are also used by host organisms to synthesize specific metabolites. The evolutionary conserved glyoxalase (GLX) system scavenges the cytotoxic α-oxoaldehydes that bear RES groups, which cause host disorders and diseases. In cotton, a specialized enzyme derived from glyoxalase I (GLXI) through gene duplications and named as specialized GLXI (SPG), acts as a distinct type of aromatase in the gossypol pathway to transform the RES intermediates into the phenolic products. In this review, we briefly introduce the research progress in understanding the RES, especially the RES-type mycotoxins, the GLX system and SPG, and discuss their application potential in detoxification and synthetic biology.

Topics: Aromatase; Edible Grain; Food Contamination; Food Safety; Fungi; Humans; Lactoylglutathione Lyase; Mycotoxins; Phenol; Signal Transduction; Trichothecenes

This study was aimed to evaluate the fate of D3G, 3-ADON, and 15-ADON during various processing steps (milling, fermentation, baking and cooking with water) of different cereal-based products, as well as the co-occurrence of culmorin (CUL) and its derivatives (15-Hydroxy-CUL and 5-Hydroxy-CUL. Some databases such as Science Direct, PubMed, Scopus, and Embase were screened to collect the relevant published papers between January 1983 to October 2018, and 23 articles with 319 data were included. The baking resulted in reductions in the concentration of all types of investigated masked mycotoxins, i.e., 15-ADON (-25%) > 3-ADON (-15%) > D3G (-6%). Also, rank order of CUL and its derivatives based on occurrence was CUL (70%) > 15-Hydroxy-CUL (47%) > 5-Hydroxy-CUL (15%) and their rank based on their concentration was 5-Hydroxy-CUL (99.21 µg/kg) > CUL (48.84 µg/kg) > 15-Hydroxy-CUL (9.39 µg/kg) > Hydroxy -CUL (0.06 µg/kg) > 12-Hydroxy-CUL (0.05 µg/kg) > 14-Hydroxy-CUL (0.01 µg/kg).

Topics: Chromatography, High Pressure Liquid; Cooking; Databases, Factual; Edible Grain; Food Contamination; Glucosides; Mycotoxins; Sesquiterpenes; Trichothecenes

Mycotoxins are the most frequently occurring natural contaminants in human and animal diet. Among them, deoxynivalenol (DON), produced by Fusarium, is one of the most prevalent and thus represents an important health risk. Recent detection methods revealed new mycotoxins and new molecules derivated from the "native" mycotoxins. The main derivates of DON are the acetylated forms produced by the fungi (3- and 15-acetyl-DON), the biologically "modified" forms produced by the plant (deoxynivalenol-3-β-D-glucopyranoside), or after bacteria transformation (de-epoxy DON, 3-epi-DON and 3-keto-DON) as well as the chemically "modified" forms (norDON A-C and DON-sulfonates). High proportions of acetylated and modified forms of DON co-occur with DON, increasing the exposure and the health risk. DON and its acetylated and modified forms are rapidly absorbed following ingestion. At the molecular level, DON binds to the ribosome, induces a ribotoxic stress leading to the activation of MAP kinases, cellular cell-cycle arrest and apoptosis. The toxic effects of DON include emesis and anorexia, alteration of intestinal and immune functions, reduced absorption of the nutrients as well as increased susceptibility to infection and chronic diseases. In contrast to DON, very little information exists concerning the acetylated and modified forms; some can be converted back to DON, their ability to bind to the ribosome and to induce cellular effects varies according to the toxin. Except for the acetylated forms, their toxicity and impact on human and animal health are poorly documented.

Topics: Acetylation; Animal Feed; Animals; Biological Availability; Biotransformation; Carcinogens, Environmental; Food Contamination; Fusarium; Glucosides; Humans; Intestinal Absorption; Molecular Conformation; Renal Elimination; Tissue Distribution; Toxicokinetics; Trichothecenes

Differences in the geographic distribution of distinct trichothecene mycotoxins in wheat and barley were first recorded two decades ago. The different toxicological properties of deoxynivalenol (DON), nivalenol (NIV) and their acetylated derivatives require careful monitoring of the dynamics of these mycotoxins and their producers. The phylogenetic species concept has become a valuable tool to study the global occurrence of mycotoxin-producing Fusarium species. This has revolutionised our views on the terrestrial distribution of trichothecene-producing Fusaria in the context of agronomics, climatic conditions, and human interference by the global trade and exchange of agricultural commodities. This paper presents an overview of the dynamics of the different trichothecene-producing Fusarium species as well as their chemotypes and genotypes across different continents. Clearly not one global population exists, but separate ones can be distinguished, sometimes even sympatric in combination with different hosts. A population with more pathogenic strains and chemotypes can replace another. Several displacement events appear to find their origin in the inadvertent introduction of new genotypes into new regions: 3-acetyl-DON-producing F. graminearum in Canada; 3-acetyl-DON-producing F. asiaticum in Eastern China; 15-acetyl-DON F. graminearum in Uruguay; and NIV-producing F asiaticum in the southern United States.

Topics: Australia; Canada; China; Europe; Food Contamination; Food Microbiology; Fusarium; Genotype; Hordeum; Iran; Mycotoxins; New Zealand; Phylogeny; Phylogeography; Republic of Korea; Trichothecenes; Triticum; United States; Uruguay

The type B trichothecenes pollute food crops and have been associated to alimentary toxicosis resulted in emetic reaction in human and animal. This group of mycotoxins consists deoxynivalenol (DON) and four structurally related congeners: 3-acetyl-deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON), nivalenol (NIV) and 4-acetyl-nivalenol (fusarenon X, FX). While emesis induced by intraperitoneally dosed to DON in the mink has been related to plasma up-grading of 5-hydroxytryptamine (5-HT) and neurotransmitters peptide YY (PYY), the impact of oral dosing with DON or its four congeners on secretion of these chemical substances have not been established. The aim of this work was to contraste emetic influence to type B trichothecene mycotoxins by orally dosing and involve these influence to PYY and 5-HT. All five toxins attracted marked emetic reaction that are relevant to elevated PYY and 5-HT. The reduction in vomiting induced by the five toxins and PYY was due to blocking of the neuropeptide Y2 receptor. The inhibition of the induced vomiting response by 5-HT and all five toxins is regulated by the 5-HT3 receptor inhibitor granisetron. In a word, our results indicate that PYY and 5-HT take a key role in the emetic reaction evoked by type B trichothecenes.

Topics: Animals; Emetics; Humans; Mink; Mycotoxins; Peptide YY; Serotonin; Trichothecenes; Trichothecenes, Type B; Vomiting

Although

Topics: Edible Grain; Fusarium; Mycotoxins; Plant Diseases

Type B trichothecenes commonly contaminate cereal grains and include five structurally related congeners: deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenon X (FX), and nivalenol (NIV). These toxins are known to have negative effects on human and animal health, particularly affecting food intake. However, the pathophysiological basis for anorexic effect is not fully clarified. The purpose of this study is to explore the potential roles of the brain-gut peptides substance P (SP) and glucagon-like peptide-1

Topics: Amides; Animals; Anorexia; Appetite Depressants; Glucagon-Like Peptide 1; Humans; Substance P; Trichothecenes; Trichothecenes, Type B

Pathogens within Fusarium species are the primary agents of Fusarium head blight (FHB) of wheat, which bring about yield reduction and deoxynivalenol (DON) contamination and are of great concern worldwide. DON-producing Fusarium species can be classified into 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) chemotypes according to the trichothecene metabolites they produce. The detection of these two chemotypes of pathogens is paramount to the successful implementation of disease management strategies and pathogen-related DON forecasting models. In this study, a duplex droplet digital PCR (duplex ddPCR) assay was developed that allowed for the simultaneous quantitation of 3ADON and 15ADON chemotypes of DON-producing Fusarium species. The assay specificity was tested against 30 isolates of target Fusarium species and several non-target Fusarium species that are frequently isolated from wheat in China. Analyzing 90 wheat samples collected from the North China plain and Yangtze River plain demonstrated that the duplex ddPCR assay coupled with magnetic bead-based DNA extraction was competent for investigating composition of 3ADON and 15ADON chemotypes in Chinese wheat. This assay will be useful for monitoring the epidemic and geographic distribution of 3ADON and 15ADON chemotypes of FHB pathogens, which will help with the disease control and DON management.

Topics: China; DNA, Fungal; Fusariosis; Fusarium; Genotype; Plant Diseases; Polymerase Chain Reaction; Trichothecenes; Triticum

Topics: Animal Feed; Biotransformation; Chromatography, Liquid; Food Microbiology; Fusarium; Germany; Glucosides; Mass Spectrometry; Risk Assessment; Trichothecenes; Zea mays; Zearalenone; Zeranol

Deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) are type B trichothecenes; one of the major pollutants in food and feed products. Although the toxicity of DON has been well documented, information on the toxicity of its acetylated derivative remains incomplete. To acquire more detailed insight into 3-ADON and 15-ADON, Caco-2 cells under 0.5 µM DON, 3-ADON and 15-ADON treatment for 24 h were subjected to RNA-seq analysis. In the present study, 2656, 3132 and 2425 differentially expressed genes (DEGs) were selected, respectively, and were enriched utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Gene Ontology (GO) database. The upregulation of ataxia-telangiectasia mutated kinase (ATM), WEE1 homolog 2 (WEE2) and downregulation of proliferating cell nuclear antigen (PCNA), minichromosome maintenance (MCMs), cyclin dependent kinase (CDKs), and E2Fs indicate that the three toxins induced DNA damage, inhibition of DNA replication and cell cycle arrest in Caco-2 cells. Additionally, the upregulation of sestrin (SENEs) and NEIL1 implied that the reason for DNA damage may be attributable to oxidative stress. Our study provides insight into the toxic mechanism of 3-ADON and 15-ADON.

Topics: Acetylation; Caco-2 Cells; Cell Survival; Dose-Response Relationship, Drug; Epithelial Cells; Gene Expression Profiling; Gene Regulatory Networks; Humans; Inhibitory Concentration 50; Intestinal Mucosa; RNA-Seq; Transcriptome; Trichothecenes

Fusarium head blight (FHB) is a major disease in wheat causing severe economic losses globally by reducing yield and contaminating grain with mycotoxins. In Canada,

Topics: Canada; Edible Grain; Food Microbiology; Fusarium; Genetic Variation; Genotype; Minisatellite Repeats; Phenotype; Trichothecenes; Triticum; United States

Among the family of mycotoxins of deoxynivalenol (DON) detected in nature, high proportions of 15-acetyldeoxynivalenol (15ADON) co-occur with the prototype DON and increase the combined exposure and synergistic health risks. The current study aimed to explore the mechanisms underlying the toxicity of 15ADON and compare them with those of DON. As the natural flavonoid compound quercetin (QUE) possesses antioxidant properties, we also aimed to determine the antioxidant effects of QUE on the tested mycotoxins. First, the global metabolomics approach was applied and showed that the metabolites produced from 15ADON or DON were almost identical, while QUE reversed the changes in the levels of key metabolites. Specifically, both DON and 15ADON activated the cell apoptosis pathway mediated by p38 and JNK, but inhibited the cell survival pathway mediated by ERK1/2 in GES-1 cells. Simultaneously, 15ADON induced FOXO3a nuclear translocation, similar to the results described for DON in our recent report. Furthermore, the addition of QUE appeared to counteract the detrimental effects of 15ADON and DON. We observed the effects of QUE treatment on mutant yeast strains with defects in their antioxidant system. More interestingly, QUE also substantially restored the increased ROS levels and the inhibited the growth rate following exposure to the mycotoxins DON and 15ADON. The data reported here support the hypothesis that QUE rescues the toxic effects of DON or 15ADON due to the similar mechanisms of DON and 15ADON toxicity.

Topics: Antioxidants; Apoptosis; Cell Line; Cell Survival; DNA, Mitochondrial; Humans; Metabolomics; Mitochondria; NAD; Quercetin; Reactive Oxygen Species; Saccharomyces cerevisiae; Trichothecenes

Deoxynivalenol (DON), along with 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON), occur in grains and cereal products and is often hazardous to humans and livestock. In this study, 579 wheat samples and 606 maize samples intended for consumption were collected from China in 2017 and analyzed to determine the co-occurrence of type-B trichothecenes (DON, 3-ADON, and 15-ADON). All the wheat samples tested positive for DON, while 99.83% of the maize samples were DON-positive with mean DON concentrations of 165.87 and 175.30 μg/kg, respectively. Per the Chinese standard limits for DON, 3.63% of wheat and 2.97% of the maize samples were above the maximum limit of 1000 μg/kg. The DON derivatives (3-ADON and 15-ADON) were less frequently found and were present at lower levels than DON in wheat. 3-ADON and 15-ADON had incidences of 13.53% and 76.40%, respectively, in maize. By analyzing the distribution ratio of DON and its derivatives in wheat and maize, DON (95.51%) was the predominant toxin detected in wheat samples, followed by 3.97% for the combination of DON + 3-ADON, while DON + 3-ADON + 15-ADON and DON + 15-ADON were only found in 0.17% and 0.35% of wheat samples, respectively. Additionally, a large amount of the maize samples were contaminated with DON + 15-ADON (64.19%) and DON (22.11%). The samples with a combination of DON + 3-ADON and DON + 3-ADON + 15-ADON accounted for 1.32% and 12.21%, respectively. Only one maize sample did not contain all three mycotoxins. Our study shows the necessity of raising awareness of the co-occurrence of mycotoxin contamination in grains from China to protect consumers from the risk of exposure to DON and its derivatives.

Topics: Acetylation; Chromatography, High Pressure Liquid; Edible Grain; Food Contamination; Limit of Detection; Reproducibility of Results; Tandem Mass Spectrometry; Trichothecenes; Triticum; Zea mays

Deoxynivalenol (DON), a notorious mycotoxin mainly found in Fusarium-contaminated crops, causes great loss in livestock farming and severe safety risks to human health. Here we report the isolation of a Gram-negative bacterial strain with effective biodegrading abilities on DON and its derivatives including 3-acetyl-DON and 15-acetyl-DON. The strain was identified as Devosia insulae A16 on the basis of morphological and physiological characteristics and 16S rRNA-based phylogenetic analysis. D. insulae A16 was able to degrade 88% of 20 mg/l DON within 48 h under aerobic conditions at 35 °C and neutral pH. The major degradation product of DON and its derivatives was 3-keto-DON by the oxidation of the hydroxyl group at C-3. Both 3-acetyl-DON and 15-acetyl-DON underwent a deacetylation reaction to generate DON prior to the degradation to 3-keto-DON. The results provide the potential use of D. insulae A16 as a biodegradation agent to control DON contamination in cereals.

Topics: Chromatography, High Pressure Liquid; Hydrogen-Ion Concentration; Hyphomicrobiaceae; Oxidation-Reduction; Phylogeny; RNA, Ribosomal, 16S; Temperature; Trichothecenes

This work studied the effect of deoxynivalenol (DON) and two of its acetylated analogs (3-ADON, 15-ADON) on first indicators of oxidative stress status, namely production of reactive oxygen species (ROS) and induction of lipid peroxidation (LPO), in HepG2 cells. HepG2 cells were exposed to different concentrations of the three toxins, either alone or in combinations, for 24, 48, and 72 h. Results of cytotoxicity obtained in HepG2 cells were correlated with the detection of ROS and LPO. This effect was inversely correlated with ROS while directly correlated with LPO for the assayed mycotoxins in individual treatment. Combinations of two toxins containing 15-ADON yielded highest values, while for two-toxin combinations with 3-ADON, the effects were minor. A combination of all three mycotoxins alleviated ROS production and the highest levels in LPO were detected, in association to a final breakdown of adaption of ROS early produced by HepG2. In conclusion, parameters of stress evaluation presented in this study (ROS and LPO), revealed increases in HepG2 cells exposed to DON, 3-ADON, and 15-ADON either individually or combined.

Topics: Hep G2 Cells; Hepatocytes; Humans; Lipid Peroxidation; Mycotoxins; Oxidative Stress; Reactive Oxygen Species; Trichothecenes

The mycotoxin deoxynivalenol (DON) is highly prevalent in cereals in moderate climates and therefore pigs are often exposed to a DON-contaminated diet. Pigs are highly susceptible to DON and intake of DON-contaminated feed may lead to an altered immune response and may influence the pathogenesis of specific bacterial diseases. Therefore, the maximum guidance level in feed is lowest in this species and has been set at 900 μg/kg feed by the European Commission. This study aimed to determine the effect of in-feed administration of a moderately high DON concentration (1514 μg/kg) on the severity of an experimental Mycoplasma hyopneumoniae (M. hyopneumoniae) infection in weaned piglets. Fifty M. hyopneumoniae-free piglets were assigned at 30 days of age [study day (D)0] to four different groups: 1) negative control group (NCG; n = 5), 2) DON-contaminated group (DON; n = 15), 3) DON-contaminated and M. hyopneumoniae-inoculated group (DONMHYO; n = 15), 4) M. hyopneumoniae-inoculated group (MHYO; n = 15). The piglets were fed the experimental diets ad libitum for five weeks and were monitored during this period and euthanized at day 35 [27 days post infection (DPI)] or 36 (28 DPI). The main parameters under investigation were macroscopic lung lesions (MLL) at euthanasia, respiratory disease score (RDS) from day 8 until day 35, histopathologic lesions and log copies of M. hyopneumoniae DNA detected by qPCR, determined at the day of euthanasia.. No significant difference was obtained for MLL at euthanasia, RDS (8-35), histopathologic lung lesions and log copies of M. hyopneumoniae DNA in the DONMHYO and MHYO group and consequently, no enhancement of the severity of the M. hyopneumoniae infection could be detected in the DONMHYO compared to the MHYO group.. Under present conditions, the findings imply that feed contaminated with DON (1514 μg/kg) provided to weaned pigs for five weeks did not increase the severity of an experimental M. hyopneumoniae infection. Further research is needed to investigate the impact of DON on M. hyopneumoniae infections in a multi-mycotoxin and multi-pathogen environment.

Topics: Animal Feed; Animals; Bronchoalveolar Lavage; Food Contamination; Lung; Mycoplasma hyopneumoniae; Pneumonia of Swine, Mycoplasmal; Real-Time Polymerase Chain Reaction; Swine; Swine Diseases; Trichothecenes

In this report, a UPLC-ESI-MS/MS method for the simultaneous determination of aflatoxins, ochratoxin A, zearalenone, deoxynivalenol, fumonisins, T-2 and HT-2 toxins, fusarenone X, diacetoxyscirpenol, and 3- and 15-acetyldeoxynivalenol in feedstuffs was developed. A quadrupole-time-of-flight mass spectrometer detector (QTOF-MS) operating in full scan mode was combined with the UPLC-ESI-MS/MS system to confirm the identity of detected mycotoxins and to identify other possible microbial metabolites occurring in samples. Sixty-two feed samples from the Spanish market were analyzed. Extraction of metabolites was carried out with acetonitrile-water-formic acid (80:19:1, v/v/v). Method detection and quantification limits and performance criteria set by Commission Regulation (EC) No 401/2006 were fulfilled. Relatively high levels of the main regulated mycotoxins and presence of non-regulated mycotoxins in feed samples were found. This is the first study in which mycotoxins and other microbial metabolites occurring in feed are studied using a UPLC-QTOF-MS system being therefore a reference report.

Topics: Aflatoxins; Animal Feed; Chromatography, High Pressure Liquid; Fumonisins; Mass Spectrometry; Mycotoxins; Ochratoxins; T-2 Toxin; Trichothecenes; Zearalenone

3-Acetyldeoxynivalenol (3-AcDON) and 15-acetyldeoxynivalenol (15-AcDON) are converted to deoxynivalenol (DON) in vivo and their simultaneous presence may increase DON intake. Mixtures of DON and its derivatives are a public health concern. In this study DON, 3-AcDON and 15-AcDON were evaluated in vitro and in silico. The in vitro cytotoxicity of DON and its derivatives individually and combined was determined by the Neutral Red (NR) assay in human hepatocarcinoma (HepG2) cells. The concentrations tested were from 1.25 to 15 μM (DON) and from 0.937 to 7.5 μM (DON derivatives). The IC

Topics: Cell Survival; Complex Mixtures; Computer Simulation; Cytochrome P-450 CYP3A; Dose-Response Relationship, Drug; Gastrointestinal Absorption; Hep G2 Cells; Humans; In Vitro Techniques; Inhibitory Concentration 50; Mycotoxins; Trichothecenes

Analysis of deoxynivalenol (DON) and its metabolites 3-acetyl and 15-acetyldeoxynivalenol (3-ADON and 15-ADON) in wheat flour samples by liquid chromatography-tandem mass spectrometry (LC/MS/MS) during 2011-2013 was conducted. [(13)C15]-DON was used as the internal standard to accomplish as accurate as possible quantitation. Of all wheat samples (n=672), 91.5% were positive for DON, at levels ranging from 2.4 to 1130 μg/kg, with a median value of 154 μg/kg. The DON derivatives (3-Ac-DON, 15-Ac-DON) were far less frequently found and at lower levels than DON. The probable daily intakes (PDI) of DON (0.49 in 2011; 0.86 in 2012; 0.56 in 2013, expressed as μg/kg body weight/day) were all within the PDI of 1.0 μg/kg of bw/day for DON set by Scientific Committee for Food (SCF) in 2002. Still, persistent monitoring of DON is important.

Topics: Chromatography, Liquid; Flour; Humans; Risk Assessment; Tandem Mass Spectrometry; Trichothecenes; Triticum

The Fusarium graminearum species complex infects several cereals and causes the reduction of grain yield and quality. Many factors influence the extent of Fusarium infection and mycotoxin levels. Such factors include crop rotation. In the present study, we explored the effect of rice or maize as former crops on mycotoxin accumulation in wheat grains.. More than 97% of samples were contaminated with deoxynivalenol (DON). DON concentrations in wheat grains from rice and maize rotation fields were 884.37 and 235.78 µg kg(-1) . Zearalenone (ZEN) was detected in 45% of samples which were mainly collected from maize-wheat rotation systems. Fusarium strains were isolated and more F. graminearum sensu stricto (s. str.) isolates were cultured from wheat samples obtained from maize rotation fields. DON levels produced by Fusarium isolates from rice rotation fields were higher than those of samples from maize rotation fields.. Rice-wheat rotation favours DON accumulation, while more ZEN contamination may occur in maize-wheat rotation models. Appropriate crop rotation may help to reduce toxin levels in wheat grains. © 2016 Society of Chemical Industry.

Topics: China; Crop Production; Crops, Agricultural; Environmental Pollutants; Food Contamination; Fusarium; Molecular Typing; Mycological Typing Techniques; Mycotoxins; Oryza; Seeds; Soil Microbiology; Spatio-Temporal Analysis; Trichothecenes; Triticum; Zea mays; Zearalenone

We hereby report the transformation of deoxynivalenol (DON) and its acetylated derivatives (3-ADON and 15-ADON) by spiking targeted mycotoxins to Fusarium mycotoxin-free flour in the process of making Chinese steamed bread (CSB). The impacts of pH, yeast level, and steaming time on the transformation of 3-ADON to DON were investigated. DON, 3-ADON, and 15-ADON were analyzed by UPLC-MS/MS. Spiked DON was stable throughout the CSB making process. Spiked 3-ADON and 15-ADON were partially deacetylated and transformed to DON during kneading (54.1-60.0% and 59.3-77.5%, respectively), fermentation (64.0-76.9% and 78.2-91.6%, respectively), and steaming (47.2-52.7% and 52.4-61.9%, respectively). The ADONs level increased after steaming compared with their level in the previous step. The pH level and steaming duration significantly (P<0.05) affected the conversion of 3-ADON during the CSB making process. Briefly, alkaline conditions and short steaming times favored the deacetylation of 3-ADON. The level of yeast did not remarkably (P<0.05) alter the transformation between ADONs and DON.

Topics: Bread; China; Chromatography, Liquid; Fermentation; Hydrogen-Ion Concentration; Mycotoxins; Saccharomyces cerevisiae; Steam; Tandem Mass Spectrometry; Trichothecenes

The present study was performed to identify prevailing Fusarium species and the environmental factors affecting their frequencies and the contamination of grain with major mycotoxins in Jiangsu province. The precipitation levels were 184.2mm, 156.4mm, and 245.8mm in the years 2013-2015, respectively, and the temperature fluctuated by an average of 10.6±7.2°C in 2013, 10.9±7.2°C in 2014, and 10.6±6.3°C in 2015. Co-occurrence of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3ADON), and 15-acetyldeoxynivalenol (15ADON) were observed in wheat. The average concentrations of DON were 879.3±1127.8, 627.8±640.5, and 1628.6±2,168.0μg/kg in 2013-2015, respectively. The average concentrations of 3ADON were 43.5±59.0, 71.2±102.5, and 33.5±111.9μg/kg in 2013-2015, respectively. We found that the average concentration of DON in wheat was positively correlated with precipitation (r=0.998, p<0.01), and 3ADON was negatively correlated with precipitation (r=-0.887, p<0.05). However, there was no correlation between precipitation and 15ADON or nivalenol (NIV). The differences in temperature were not as significant as the differences in rainfall amount over a short time period. Therefore, there were no correlations between temperature and the concentrations of trichothecenes, excluding 3ADON (r=0.996, p<0.01). Our data indicated that Fusarium asiaticum is the primary pathogenic fungus prevalent in the Fusarium head blight disease nursery. The trichothecene chemotype composition differed between Fusarium graminearum sensu stricto (s. str.) and F. asiaticum isolates. The 3ADON chemotype was found only among strains of F. asiaticum. The NIV chemotype was not observed among strains of F. graminearum, while the 15ADON chemotype represented 100% of the F. graminearum strains collected. The results of this study indicated no correlations between environmental conditions and the species or genetic chemotype composition of pathogens in Jiangsu province in 2013-2015.

Topics: China; Edible Grain; Food Contamination; Fusarium; Mycotoxins; Trichothecenes; Triticum

Over a 4-year period (2010-13), a survey aiming at determining the occurrence of Fusarium spp. and their relations to mycotoxins in mature grains took place in southern Belgium. The most prevalent species were F. graminearum, F. avenaceum, F. poae and F. culmorum, with large variations between years and locations. An even proportion of mating type found for F. avenaceum, F. culmorum, F. cerealis and F. tricinctum is usually a sign of ongoing sexual recombination. In contrast, an unbalanced proportion of mating type was found for F. poae and no MAT1-2 allele was present in the F. langsethiae population. Genetic chemotyping indicates a majority of deoxynivalenol (DON)-producing strains in F. culmorum (78%, all 3-ADON producers) and F. graminearum (95%, mostly 15-ADON producers), while all F. cerealis strains belong to the nivalenol (NIV) chemotype. Between 2011 and 2013, DON, NIV, enniatins (ENNs) and moniliformin (MON) were found in each field in various concentrations. By comparison, beauvericin (BEA) was scarcely detected and T-2 toxin, zearalenone and α- and β-zearalenols were never detected. Principal component analysis revealed correlations of DON with F. graminearum, ENNs and MON with F. avenaceum and NIV with F. culmorum, F. cerealis and F. poae. BEA was associated with the presence of F. tricinctum and, to a lesser extent, with the presence of F. poae. The use of genetic chemotype data revealed that DON concentrations were mostly influenced by DON-producing strains of F. graminearum and F. culmorum, whereas the concentrations of NIV were influenced by the number of NIV-producing strains of both species added to the number of F. cerealis and F. poae strains. This study emphasises the need to pay attention to less-studied Fusarium spp. for future Fusarium head blight management strategies, as they commonly co-occur in the field and are associated with a broad spectrum of mycotoxins.

Topics: Belgium; Depsipeptides; DNA, Fungal; Edible Grain; Food Contamination; Fusarium; Genes, Mating Type, Fungal; Humans; Mycotoxins; Principal Component Analysis; Trichothecenes; Zearalenone

Cereal infection by the broad host range fungal pathogen Fusarium graminearum is a significant global agricultural and food safety issue due to the deposition of mycotoxins within infected grains. Methods to study the intracellular effects of mycotoxins often use the baker's yeast model system (Saccharomyces cerevisiae); however, this organism has an efficient drug export network known as the pleiotropic drug resistance (PDR) network, which consists of a family of multidrug exporters. This study describes the first study that has evaluated the potential involvement of all known or putative ATP-binding cassette (ABC) transporters from the PDR network in exporting the F. graminearum trichothecene mycotoxins deoxynivalenol (DON) and 15-acetyl-deoxynivalenol (15A-DON) from living yeast cells. We found that Pdr5p appears to be the only transporter from the PDR network capable of exporting these mycotoxins. We engineered mutants of Pdr5p at two sites previously identified as important in determining substrate specificity and inhibitor susceptibility. These results indicate that it is possible to alter inhibitor insensitivity while maintaining the ability of Pdr5p to export the mycotoxins DON and 15A-DON, which may enable the development of resistance strategies to generate more Fusarium-tolerant crop plants.

Topics: Amino Acid Substitution; ATP-Binding Cassette Transporters; Edible Grain; Food Safety; Fusarium; Plant Diseases; Protein Engineering; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Trichothecenes

Contamination of wheat grains with Fusarium mycotoxins and their modified forms is an important issue in wheat industry. The objective of this study was to analyse the deoxynivalenol (DON) and deoxynivalenol-3-glucosides (D3G) content in Canadian spring wheat cultivars grown in two locations, inoculated with a mixture of 3-acetyldeoxynivalenol (3-ADON)-producing Fusarium graminearum strains and a mixture of 15-acetlyldeoxynivalenol (15-ADON)-producing F. graminearum strains. According to the analysis of variance, significant differences were observed among the cultivars for Fusarium head blight (FHB) disease index, Fusarium-damaged kernel percentage (%FDK), DON content and D3G content. When the effect of chemotype was considered, significant differences were observed for FHB disease index, FDK percentage and DON content. The D3G content and D3G/DON ratio were not significantly different between the chemotypes, except for D3G content at the Winnipeg location. The Pearson correlation coefficient between DON and D3G was 0.84 and 0.77 at Winnipeg and Carman respectively. The highest D3G/DON ratio was observed in cultivars Carberry (44%) in Carman and CDC Kernen (63.8%) in Winnipeg. The susceptible cultivars showed lower D3G/DON ratio compared with the cultivars rated as moderately resistant and intermediate. The current study indicated that Canadian spring cultivars produce D3G upon Fusarium infection.

Topics: Canada; Disease Resistance; Edible Grain; Food Contamination; Fusarium; Glucosides; Mycotoxins; Plant Diseases; Seasons; Trichothecenes; Triticum

The gastrointestinal tract is the first target after ingestion of the mycotoxin deoxynivalenol (DON) via feed and food. Deoxynivalenol is known to affect the proliferation and viability of animal and human intestinal epithelial cells. In addition to DON, feed and food is often co-contaminated with modified forms of DON, such as 3-acetyldeoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) and deoxynivalenol-3-β-D-glucoside (DON3G). The goal of this study was to determine the in vitro intrinsic cytotoxicity of these modified forms towards differentiated and proliferative porcine intestinal epithelial cells by means of flow cytometry. Cell death was assessed by dual staining with Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), which allows the discrimination of viable (FITC-/PI-), apoptotic (FITC+/PI-) and necrotic cells (FITC+/PI+). Based on the data from the presented pilot in vitro study, it is concluded that cytotoxicity for proliferative cells can be ranked as follows: DON3G ≪ 3ADON < DON≈15ADON.

Topics: Animals; Animals, Newborn; Apoptosis; Chromatography, Liquid; Epithelial Cells; Flow Cytometry; Food Contamination; Glucosides; Humans; In Vitro Techniques; Intestines; Magnetic Resonance Spectroscopy; Swine; Tandem Mass Spectrometry; Trichothecenes

In addition to deoxynivalenol (DON), acetylated derivatives, i.e., 3-acetyl and 15-acetyldexynivalenol (or 3/15ADON), are present in cereals leading to exposure to these mycotoxins. Animal and human studies suggest that 3/15ADON are converted into DON after their ingestion through hydrolysis of the acetyl moiety, the site(s) of such deacetylation being still uncharacterized. We used in vitro and ex vivo approaches to study the deacetylation of 3/15ADON by enzymes and cells/tissues present on their way from the food matrix to the blood in humans. We found that luminal deacetylation by digestive enzymes and bacteria is limited. Using human cells, tissues and S9 fractions, we were able to demonstrate that small intestine and liver possess strong deacetylation capacity compared to colon and kidneys. Interestingly, in most cases, deacetylation was more efficient for 3ADON than 15ADON. Although we initially thought that carboxylesterases (CES) could be responsible for the deacetylation of 3/15ADON, the use of pure human CES1/2 and of CES inhibitor demonstrated that CES are not involved. Taken together, our original model system allowed us to identify the small intestine and the liver as the main site of deacetylation of ingested 3/15ADON in humans.

Topics: Acetylation; Adult; Aged; Bacteria; Caco-2 Cells; Carboxylesterase; Colon; Feces; Female; Gastrointestinal Microbiome; Hep G2 Cells; Humans; Hydrolysis; Intestine, Small; Liver; Male; Middle Aged; Time Factors; Trichothecenes

Fusarium head blight (FHB) of wheat is an important disease causing yield losses and mycotoxin contamination. The aim of the work was to detect and characterise trichothecene producing Fusarium species in durum and soft wheat cultivated in an area of central Italy in 2009 and 2010 and to determine trichothecene contamination by LC-MS/MS in the grain.. F. graminearum s. str. was the most frequent species. In 2009, the occurrence of F. avenaceum and F. poae was higher than in 2010. Among F. graminearum strains, the 15-acetyl deoxynivalenol (15-ADON) chemotype could be found more frequently, followed by nivalenol (NIV) and 3-ADON chemotypes, while all F. culmorum isolates belonged to the 3-ADON chemotype. All F. poae strains were NIV chemotypes. In vitro trichothecene production confirmed molecular characterisation. Durum wheat was characterised by a higher average DON contamination with respect to soft wheat, NIV was always detected at appreciable levels while type-A trichothecenes were mostly found in durum wheat samples in 2009 with 6% of samples exceeding the contamination level recently recommended by the European Union.. Climatic conditions were confirmed to be predominant factors influencing mycotoxigenic species composition and mycotoxin contaminations. However, NIV contamination was found to occur irrespective of climatic conditions, suggesting that it may often represent an under-estimated risk to be further investigated.

Topics: Chromatography, Liquid; DNA, Fungal; Food Contamination; Food Microbiology; Fusarium; Genotype; Humans; Italy; Plant Diseases; Polymerase Chain Reaction; Seeds; Species Specificity; Tandem Mass Spectrometry; Trichothecenes; Triticum

In case of mycotoxin contaminations, food and feedstuff are usually contaminated by more than one toxin. However toxicological data concerning the effects of mycotoxin combinations are sparse. The intestinal epithelium is the first barrier against food contaminants and this constantly renewing organ is particularly sensitive to mycotoxins. The aim of this study was to investigate the effects of deoxynivalenol (DON) and four other type B trichothecenes (TCTB), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX) alone or in combination on intestinal epithelial cells. Proliferating, non-transformed IPEC-1 cells were exposed to increasing doses of TCTB, alone or in binary mixtures and mycotoxin-induced cytotoxicity was measured with MTT test. The toxicological interactions were assessed using the isobologram-Combination index method. The five tested mycotoxins and their mixtures had a dose-dependent effect on the proliferating enterocytes. DON-NIV, DON-15-ADON and 15-ADON-3-ADON combinations were synergistic, with magnitude of synergy for 10 % cytotoxicity ranging from 2 to 7. The association between DON and 3-ADON also demonstrated a synergy but only at high doses, at lower doses antagonism was noted. Additivity was observed between NIV and FX, and antagonism between DON and FX. These results indicate that the simultaneous presence of mycotoxins in food commodities and diet may be more toxic than predicted from the mycotoxins alone. This synergy should be taken into account considering the frequent co-occurrence of TCTB in the diet.

Topics: Animals; Cell Culture Techniques; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Drug Synergism; Epithelial Cells; Intestinal Mucosa; Swine; Trichothecenes

A QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method was optimized and validated for the simultaneous extraction of 12 trichothecenes (type A and type B) from baby foods, followed by gas chromatography-mass spectrometry (GC-MS) analysis. Using this methodology, limits of detection and quantification ranging from 0.37 to 19.19 μg/kg and 1.24 to 63.33 μg/kg, respectively, were achieved. Mean recoveries between 44% and 135% were obtained and repeatability, expressed as relative standard deviation, was always lower than 29%. A comparison between the developed method and two alternative cleanup procedures (MultiSep and IAC--immunoaffinity columns) was performed, being the advantages and drawbacks of each one presented. The screening of nine commercially available cereal-based baby foods revealed the presence of 4 out of 12 studied trichothecenes: DON (deoxynivalenol), 15AcDON (15-acetyl-deoxynivalenol), T2-Tetrol and NEO (Neosolaniol). DON was the most commonly found, being detected in 4 samples in significant levels (29-270 μg/kg), sometimes exceeding the maximum permitted level. 15AcDON, T2-Tetrol and NEO were found only in one sample each.

Topics: Edible Grain; Food Contamination; Gas Chromatography-Mass Spectrometry; Infant Food; Liquid-Liquid Extraction; Reproducibility of Results; Sensitivity and Specificity; Trichothecenes

The present study investigated the changes and conversion profiles of DON, its conjugations 3-ADON, and 15-ADON during bread making process, by spiking targeted mycotoxin standards to Fusarium mycotoxins-free wheat flour. No significant (p < 0.05) changes of DON levels were observed during dough preparation stages, including kneading, fermentation, and proofing. A reduction of DON level ranged from 4% to 14% was observed during baking process. The main thermal degradation products of DON, namely norDON A, B, C, and F were detected in the bread crust. Regarding ADONs, decreases of 20-40% for 3-ADON and 28-60% for 15-ADON were found during fermentation stage, and further losses of ADONs were observed after proofing process. Although ADONs levels gained an increase after baking. This study demonstrated that ADONs were converted to DON, while no ADONs were detectable in DON spiked samples during bread making process. The mechanism that ADONs could be converted into DON is unclear so far.

Topics: Bread; Chromatography, Liquid; Fermentation; Flour; Food Analysis; Food Handling; Fusarium; Mycotoxins; Tandem Mass Spectrometry; Trichothecenes

A critical assessment of three previously published indirect methods based on acidic hydrolysis using superacids for the determination of "free" and "total" deoxynivalenol (DON) was carried out. The modified mycotoxins DON-3-glucoside (D3G), 3-acetyl-DON (3ADON), and 15-acetyl-DON (15ADON) were chosen as model analytes. The initial experiments focused on the stability/degradation of DON under hydrolytic conditions and the ability to release DON from the modified forms. Acidic conditions that were capable of cleaving D3G, 3ADON, and 15ADON to DON were not found, raising doubts over the efficacy of previously published indirect methods for total DON determination. Validation of these indirect methods for wheat, maize, and barley using UHPLC-MS/MS was performed in order to test the accuracy of the generated results. Validation data for DON, D3G, 3ADON, and 15ADON in nonhydrolyzed and hydrolyzed matrices were obtained. Under the tested conditions, DON was not released from D3G, 3ADON, or 15ADON after hydrolysis and thus none of the published methods were able to cleave the modified forms of DON. In addition to acids, alkaline hydrolysis with KOH for an extended time and at elevated temperatures was also tested. 3ADON and 15ADON were cleaved under the alkaline pH caused by the addition of KOH or aqueous K2CO3 to "neutralize" the acidic sample extracts in the published studies. The published additional DON increase after hydrolysis may have been caused by huge differences in matrix effects and the recovery of DON in nonhydrolyzed and hydrolyzed matrices as well as by the alkaline cleavage of 3ADON or 15ADON after the neutralization of hydrolyzed extracts.

Topics: Chromatography, Liquid; Edible Grain; Glucosides; Hordeum; Hydrolysis; Mycotoxins; Tandem Mass Spectrometry; Trichothecenes; Triticum; Zea mays

TRI6 is a positive regulator of the trichothecene gene cluster and the production of trichothecene mycotoxins [deoxynivalenol (DON)] and acetylated forms such as 15-Acetyl-DON) in the cereal pathogen Fusarium graminearum. As a global transcriptional regulator, TRI6 expression is modulated by nitrogen-limiting conditions, sources of nitrogen and carbon, pH and light. However, the mechanism by which these diverse environmental factors affect TRI6 expression remains underexplored. In our effort to understand how nutrients affect TRI6 regulation, comparative digital expression profiling was performed with a wild-type F. graminearum and a Δtri6 mutant strain, grown in nutrient-rich conditions. Analysis showed that TRI6 negatively regulates genes of the branched-chain amino acid (BCAA) metabolic pathway. Feeding studies with deletion mutants of MCC, encoding methylcrotonyl-CoA-carboxylase, one of the key enzymes of leucine metabolism, showed that addition of leucine specifically down-regulated TRI6 expression and reduced 15-ADON accumulation. Constitutive expression of TRI6 in the Δmcc mutant strain restored 15-ADON production. A combination of cellophane breach assays and pathogenicity experiments on wheat demonstrated that disrupting the leucine metabolic pathway significantly reduced disease. These findings suggest a complex interaction between one of the primary metabolic pathways with a global regulator of mycotoxin biosynthesis and virulence in F. graminearum.

Topics: Amino Acids, Branched-Chain; Carbon-Carbon Ligases; Fungal Proteins; Fusarium; Gene Expression Regulation, Fungal; Genotype; Leucine; Metabolic Networks and Pathways; Multigene Family; Mutation; Transcription Factors; Trichothecenes; Triticum

The goal of this study was to determine the absolute oral bioavailability, (presystemic) hydrolysis and toxicokinetic characteristics of deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol in broiler chickens and pigs. Crossover animal trials were performed with intravenous and oral administration of deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol to broilers and pigs. Plasma concentrations were analyzed by using liquid chromatography-tandem mass spectrometry, and data were processed via a tailor-made compartmental toxicokinetic analysis. The results in broiler chickens showed that the absorbed fraction after oral deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol administration was 10.6, 18.2, and 42.2%, respectively. This fraction was completely hydrolyzed presystemically for 3-acetyldeoxynivalenol to deoxynivalenol and to a lesser extent (75.4%) for 15-acetyldeoxynivalenol. In pigs, the absorbed fractions were 100% for deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol, and both 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol were completely hydrolyzed presystemically. The disposition properties of 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol demonstrate their toxicological relevance and consequently the possible need to establish a tolerable daily intake.

Topics: Administration, Oral; Animals; Chickens; Hydrolysis; Mycotoxins; Swine; Toxicokinetics; Trichothecenes

To assess the dietary exposure of Shanghai residents to a compound of deoxynivalenol (DON) and its acetylated derivatives, 3-acetyl-deoxynivalenol (3-Ac-DON) and 15-acetyl-deoxynivalenol (15-Ac-DON) through wheat flour.. DON, 3-Ac-DON and 15-Ac-DON in wheat flour collected from 2011 to 2013 were respectively analyzed by high performance liquid chromatography tandem mass spectrometry. (HPLC-MS/MS) method and the total content of the compound was calculated. Dietary intake assessments of the compound through wheat flour were carried out in combination of wheat flour consumption data of Shanghai residents with the compound content data using both the point estimate method and the probabilistic assessment method. Group provisional maximum tolerable daily intake(PMTDI, 1 jg/(kg BW- d) )of the compound was used to assess the risk of the exposure.. (1) At the mean and 50th percentile consumption level of wheat flour, the dietary exposure of people to the compound accounted for 18%-83% of PMTDI based on different content levels (mean and the 50th, 75th, 90th and 95th percentiles) of the compound. At the 95th percentile consumption level of wheat flour, the exposure was 1.08-2.55 times higher than PMTDI based on different toxin levels (mean and the 50th, 75th, 90th and 95th percentiles). (2) 89.99% of total Shanghai residents, 87.00% of Shanghai wheat flour consumers, and 76.55% of Shanghai residents under 15 years old had a daily exposure to the compound lower than PMTDI.. The dietary exposure of Shanghai residents to the compound of DON, 3-Ac-DON and 15-Ac-DON increased along with the increase of wheat flour consumption. Most of the residents were safe through wheat flour consumption, but still 10.01% of the population was at risk, and residents under 15 years old were a high-risk group for the compound exposure.

Topics: Acetylation; Adult; China; Chromatography, High Pressure Liquid; Diet; Edible Grain; Flour; Food Contamination; Humans; Mycotoxins; Nutrition Surveys; Risk Assessment; Tandem Mass Spectrometry; Trichothecenes; Triticum

At least 20 epidemics of Fusarium head blight (FHB) of wheat have been registered in the last 50 years in Argentina, with variable intensity. Damage induced by the disease is further aggravated by the presence of mycotoxins in affected grains that may cause health problems to humans and animals. The trichothecene chemotype was analyzed for 112 isolates of Fusarium graminearum from Argentina by polymerase chain reaction and two field trials were conducted to study the aggressiveness of a subsample of 14 representative isolates and to analyze deoxynivalenol (DON) production in planta and in vitro. All isolates belonged to the 15-acetyl-DON chemotype. Significant differences were observed in both the symptom severity induced in wheat spikes and the in vivo DON production, and a close correlation was found between these two variables. However, in vitro toxigenic potential was not correlated with the capacity of F. graminearum isolates to produce DON under natural conditions. The progress of infection in the rachis of inoculated wheat spikes was analyzed and the pathogen presence verified in both symptomatic and symptomless spikes. Even isolates with a limited capacity to induce symptoms were able to colonize the vascular tissue and to produce considerable amounts of DON in planta.

Topics: Argentina; Edible Grain; Fusarium; Genotype; Inflorescence; Mycotoxins; Plant Diseases; Regression Analysis; Trichothecenes; Triticum

Cereal grain contamination by trichothecene mycotoxins is known to negatively impact human and animal health with adverse effects on food intake and growth being of particular concern. The head blight fungus Fusarium graminearum elaborates five closely related 8-ketotrichothecene congeners: (1) deoxynivalenol (DON), (2) 3-acetyldeoxynivalenol (3-ADON), (3) 15-acetyldeoxynivalenol (15-ADON), (4) fusarenon X (FX), and (5) nivalenol (NIV). While anorexia induction in mice exposed intraperitoneally to DON has been linked to plasma elevation of the satiety hormones cholecystokinin (CCK) and peptide YY₃₋₃₆ (PYY₃₋₃₆), the effects of oral gavage of DON or of other 8-keotrichothecenes on release of these gut peptides have not been established. The purpose of this study was to (1) compare the anorectic responses to the aforementioned 8-ketotrichothecenes following oral gavage at a common dose (2.5 mg/kg bw) and (2) relate these effects to changes plasma CCK and PYY₃₋₃₆ concentrations. Elevation of plasma CCK markedly corresponded to anorexia induction by DON and all other 8-ketotrichothecenes tested. Furthermore, the CCK1 receptor antagonist SR 27897 and the CCK2 receptor antagonist L-365,260 dose-dependently attenuated both CCK- and DON-induced anorexia, which was consistent with this gut satiety hormone being an important mediator of 8-ketotrichothecene-induced food refusal. In contrast to CCK, PYY₃₋₃₆ was moderately elevated by oral gavage with DON and NIV but not by 3-ADON, 15-ADON, or FX. Taken together, the results suggest that CCK plays a major role in anorexia induction following oral exposure to 8-ketotrichothecenes, whereas PYY₃₋₃₆ might play a lesser, congener-dependent role in this response.

Topics: Administration, Oral; Animals; Anorexia; Chemokines, CC; Cholecystokinin; Female; Mice; Mycotoxins; Peptide Fragments; Peptide YY; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Trichothecenes

This study aims to develop an LC-MS/MS method allowing the determination of 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, deoxynivalenol and its main in vivo metabolite, deepoxy-deoxynivalenol, in broiler chickens and pigs. These species have a high exposure to these toxins, given their mainly cereal based diet. Several sample cleanup strategies were tested and further optimized by means of fractional factorial designs. A simple and straightforward sample preparation method was developed consisting out of a deproteinisation step with acetonitrile, followed by evaporation of the supernatant and reconstitution in water. The method was single laboratory validated according to European guidelines and found to be applicable for the intended purpose, with a linear response up to 200ngml(-1) and limits of quantification of 0.1-2ngml(-1). As a proof of concept, biological samples from a broiler chicken that received either deoxynivalenol, 3- or 15-acetyl-deoxynivalenol were analyzed. Preliminary results indicate nearly complete hydrolysis of 3-acetyl-deoxynivalenol to deoxynivalenol; and to a lesser extent of 15-acetyl-deoxynivalenol to deoxynivalenol. No deepoxy-deoxynivalenol was detected in any of the plasma samples. The method will be applied to study full toxicokinetic properties of deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol in broiler chickens and pigs.

Topics: Animals; Chickens; Chromatography, High Pressure Liquid; Male; Pilot Projects; Sensitivity and Specificity; Sus scrofa; Tandem Mass Spectrometry; Toxicokinetics; Trichothecenes

To validate an LC-MS/MS method for simultaneous determination of deoxynivalenol (DON) and its acetylated derivatives, 3-acetyl-deoxynivalenol (3ADON) and 15-acetyl-deoxynivalenol (15ADON), in wheat using a multifunctional column, an inter-laboratory study was performed in 9 laboratories using one blank wheat sample, three spiked wheat samples (10, 50, 150 µg/kg) and one naturally contaminated wheat sample. The recoveries ranged from 98.8 to 102.6% for DON, 89.3 to 98.7% for 3ADON, and from 84.9 to 90.0% for 15ADON. The relative standard deviations for repeatability (RSDR) and reproducibility (RSDR) of DON were in the ranges of 7.2-11.3% and 9.5-22.6%, respectively. For 3ADON, the RSDR ranged from 5.3 to 9.5% and the RSDR ranged from 16.1 to 18.0%, while for 15ADON, the RSDR ranged from 6.2 to 11.2% and the RSDR ranged from 17.0 to 27.2%. The HorRat values for the three analytes ranged from 0.4 to 1.2. These results validate this method for the simultaneous determination of DON and its acetylated derivatives, 3ADON and 15ADON.

Topics: Chromatography, Liquid; Food Analysis; Japan; Laboratory Proficiency Testing; Reproducibility of Results; Taiwan; Tandem Mass Spectrometry; Trichothecenes; Triticum

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. DON is often present with other type B trichothecenes such as 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX). Although the cytotoxicity of individual mycotoxins has been widely studied, data on the toxicity of mycotoxin mixtures are limited. The aim of this study was to assess interactions caused by co-exposure to Type B trichothecenes on intestinal epithelial cells. Proliferating Caco-2 cells were exposed to increasing doses of Type B trichothecenes, alone or in binary or ternary mixtures. The MTT test and neutral red uptake, respectively linked to mitochondrial and lysosomal functions, were used to measure intestinal epithelial cytotoxicity. The five tested mycotoxins had a dose-dependent effect on proliferating enterocytes and could be classified in increasing order of toxicity: 3-ADON<15-ADON≈DON

Topics: Algorithms; Caco-2 Cells; Cell Survival; Coloring Agents; Dose-Response Relationship, Drug; Drug Synergism; Epithelial Cells; Humans; Intestinal Mucosa; Mycotoxins; Tetrazolium Salts; Thiazoles; Trichothecenes

The effects of the trichothecene mycotoxin deoxynivalenol (DON) and its acetylated derivatives, 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) on human intestinal cell Caco-2 were investigated by the studies of transepithelial transport, gene expression, and cytokine secretion. Permeability across a Caco-2 cell monolayer was evaluated by transport study. Transport rates were ranked as DON, 3ADON<15ADON in apical-basolateral direction. 15ADON showed the highest permeability, induced the highest decrease in transepithelial electrical resistance (TEER), and prompted significant Lucifer Yellow permeability. These results showed that 15ADON affect paracellular barrier function extremely. In addition, gene expressions induced by toxins were screened by DNA microarray for investigating cellular effect on Caco-2 cell. The most remarkable gene induced by DON and 15ADON was inflammatory chemokine IL-8 and thus mRNA expression and secretion of IL-8 were analyzed by PCR and ELISA. Both DON and acetylated DONs could induce mRNA expression and production of IL-8. In particular, ELISA assay showed that the ability to produce IL-8 was ranked as 3ADON

Topics: Biological Transport; Caco-2 Cells; Cell Survival; Gene Expression Profiling; Humans; Interleukin-8; Intestinal Absorption; Intestinal Mucosa; Oligonucleotide Array Sequence Analysis; Trichothecenes

Although the acute toxic effects of trichothecene mycotoxin deoxynivalenol (DON or vomitoxin), a known cause of human food poisoning, have been well characterized in several animal species, much less is known about closely related 8-ketotrichothecenes that similarly occur in cereal grains colonized by toxigenic fusaria. To address this, we compared potencies of DON, 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), fusarenon X (FX), and nivalenol (NIV) in the mink emesis model following intraperitoneal (ip) and oral administration. All five congeners dose-dependently induced emesis by both administration methods. With increasing doses, there were marked decreases in latency to emesis with corresponding increases in emesis duration and number of emetic events. The effective doses resulting in emetic events in 50% of the animals for ip exposure to DON, 15-ADON, 3-ADON, FX, and NIV were 80, 170, 180, 70, and 60 µg/kg bw, respectively, and for oral exposure, they were 30, 40, 290, 30, and 250 µg/kg bw, respectively. The emetic potency of DON determined here was comparable to that reported in analogous studies conducted in pigs and dogs, suggesting that the mink is a suitable small animal model for investigating acute trichothecene toxicity. The use of a mouse pica model, based on the consumption of kaolin, was also evaluated as a possible surrogate for studying emesis but was found unsuitable. From a public health perspective, comparative emetic potency data derived from small animal models such as the mink should be useful for establishing toxic equivalency factors for DON and other trichothecenes.

Topics: Administration, Oral; Animals; Dose-Response Relationship, Drug; Female; Injections, Intraperitoneal; Male; Mice; Mink; Mycotoxins; Pica; Toxicity Tests; Trichothecenes; Vomiting

While induction of food refusal by the trichothecene mycotoxin deoxynivalenol (DON) has been described in several animal models, much less is known about the anorectic effects of structurally related 8-ketotrichothecenes, 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenon X (FX) and nivalenol (NIV). Here, we compared the capacities of these congeners to induce anorexia in the mouse. As previously observed for DON, anorectic responses to 3-ADON and 15-ADON in the B6C3F1 female mouse following both intraperitoneal (IP) and oral exposure were transient, lasting only a few hours, with food intake recovering to control levels within 16 h. For both ADONs, the no observed adverse effect levels (NOAEL) and lowest observed adverse effect levels (LOAEL) were 0.5 and 1mg/kg bw following IP exposure, respectively, and 1 and 2.5mg/kg bw after oral exposure, respectively. In contrast, food refusal persisted from 48 to 96 h following IP and oral exposure to FX and NIV. For both IP and oral FX exposure, the NOAEL was 0.025 mg/kg bw and LOAEL was 0.25mg/kg bw, whereas the NOAELs and LOAELs for NIV were 0.01 and 0.1mg/kg bw, respectively, after IP exposure and 0.1 and 1mg/kg bw, respectively, following oral exposure. Both these data and a prior DON study suggest that anorectic responses to 8-ketotrichothecenes were always greater when administered IP as compared to oral exposure and follow an approximate rank order of NIV>FX>DON≈3-ADON≈15-ADON for IP exposure and FX>NIV>DON≈3-ADON≈15-ADON for oral exposure. Toxic potency data such as is described here will be applicable to future comparative risk assessments for this important group of trichothecene mycotoxins.

Topics: Animals; Appetite Depressants; Dose-Response Relationship, Drug; Eating; Female; Fusarium; Mice; Mice, Inbred Strains; Mycotoxins; No-Observed-Adverse-Effect Level; Trichothecenes

The intestinal epithelium is the first barrier against food contaminants and is highly sensitive to mycotoxins, especially de oxynivalenol (DON). Consumption of DON-contaminated food is associated with outbreaks of gastroenteritis. In cereals and their byproducts, DON is present together with two acetylated derivatives, 3-ADON and 15-ADON. The aim of this study was to compare the intestinal toxicity of DON and A-DONs, using noncytotoxic doses. The toxicity was assessed using in vitro (intestinal epithelial cell line), ex vivo (intestinal explants), and in vivo (animals exposed to mycotoxin-contaminated diets) models. The effects were studied on cell proliferation, barrier function, and intestinal structure. The mechanism of toxicity was investigated by measuring the expression of the tight junction proteins and of phosphorylated ERK1/2, p38, and JNK, which are effectors of signaling pathway involved in cellular programs including embryogenesis, proliferation, differentiation, and apoptosis. On proliferating cells, 3-ADON was less toxic than DON, which was less toxic than 15-ADON. On differentiated cells, 15-ADON impaired the barrier function, whereas DON and 3-ADON did not have a significant effect. Similarly, ex vivo and in vivo, 15-ADON caused more histological lesions than DON or 3-ADON. At the molecular level, the 15-ADON activated the mitogen-activated protein kinases (MAPK) ERK1/2, p38, and JNK in the intestinal cell line, explants, and the jejunum from exposed animals at lower dose than DON and 3-ADON. Our results show that the higher toxicity of 15-DON is due to its ability to activate the MAPK. Given that cereal-based foods are contaminated with DON and acetylated-DON, the higher toxicity of 15-ADON should be taken into account.

Topics: Animals; Animals, Newborn; Cell Line; Cell Proliferation; Electrophysiological Phenomena; Food Contamination; Intestinal Mucosa; Intestine, Small; Mitogen-Activated Protein Kinases; Patch-Clamp Techniques; Swine; Tight Junction Proteins; Tight Junctions; Trichothecenes

We developed a purification method based on liquid chromatography-tandem mass spectrometry for the identification of deoxynivalenol (DON), its acetylated derivatives (3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol), and a glycosylated derivative (deoxynivalenol-3-glucoside [D3G]) in corn-based products. The analytes were extracted from samples with acetonitrile-water (85:15, vol/vol) and then purified with multifunctional columns. Evaluation of five kinds of multifunctional columns revealed that DON and its acetylated derivatives were recovered well (96 to 120%) by all columns, but D3G was recovered adequately (93.5%) by only one column, InertSep VRA-3. Samples of corn grits and corn flour were analyzed using the purification method with InertSep VRA-3. DON, D3G, and 15-acetyl-deoxynivalenol were the major contaminants in the samples harvested in 2009, but only DON was detected in the samples harvested in 2010. These results suggest that the purification method using InertSep VRA-3 is effective for identification of DON and its derivatives in corn-based products.

Topics: Acetylation; Chromatography, Liquid; Consumer Product Safety; Edible Grain; Flour; Food Contamination; Glucosides; Glycosylation; Humans; Tandem Mass Spectrometry; Trichothecenes; Zea mays

The objective of the present study was to monitor the occurrence and distribution of a spectrum of trichothecene toxins in different parts of maize plants. Therefore maize plants were sampled randomly from 13 fields in southwest Germany and the fractions kernels, cobs, husks, stalks, leaves and rudimentary ears were analyzed for eight A-type and five B-type trichothecenes. Each of the toxins was found in at least three of the total of 78 samples. The study revealed that both A-type and B-type trichothecenes may be present in all parts of the maize plant but may be unevenly distributed. For the contents of deoxynivalenol, 3- and 15-acetyldeoxynivalenol, nivalenol, scirpentriol, 15-monoacetoxyscirpenol, HT-2 and T-2 toxin significant differences (p < 0.05) were found between different parts of the maize plants whereas no significant differences were observed for fusarenon-X, 4,15-diacetoxyscirpenol, neosolaniol, T-2 triol and T-2 tetraol. Up to twelve toxins co-occurring in one sample were detected. As a group B-type trichothecenes dominated over A-type trichothecenes concerning incidences and levels. Contamination was strongest with rudimentary ears based on incidence and mean and maximum contents; mean contents with few exceptions tended towards a higher level than in other fractions with significant (p < 0.05) differences compared to leaves for seven toxins.

Topics: Food Contamination; Food Microbiology; Germany; T-2 Toxin; Trichothecenes; Zea mays

Argentina is the fourth largest exporter of wheat in the world. The main pathogen associated with Fusarium Head Blight (FHB) of wheat in Argentina is Fusarium graminearum lineage 7 also termed F. graminearum sensu stricto in the F. graminearum species complex, which can produce the Type B trichothecenes, usually deoxynivalenol (DON) and its acetylated forms (3-ADON and 15-ADON) or nivalenol (NIV). We used a multiplex PCR assay of Tri3, Tri7, and Tri13 to determine the trichothecene genotype of 116 strains F. graminearum collected from three locations in Argentina and then verified the chemotype by chemical analysis. PCR assays and chemical analyses gave the same results for all strains that produced trichothecenes. Most strains (> 92%) had the 15-ADON genotype, with the remaining strains having the DON/NIV genotype. We observed neither the NIV nor the 3-ADON genotypes amongst the strains evaluated. The nine strains with the DON/NIV genotype produced DON when analyzed chemically. Thus, the Argentinean populations of F. graminearum are similar to those from wheat elsewhere in the world, in that all the strains produced DON/15-ADON and belong to lineage 7. However approximately 8% of the strains tested were incorrectly diagnosed as DON/NIV producers with the current multiplex PCR and were only DON producers by chemical analysis.

Topics: Argentina; DNA, Fungal; Fusarium; Genotype; Polymerase Chain Reaction; Trichothecenes; Triticum

Fusarium head blight is a disease of primary concern to small-grain cereals of Brazil, including barley. Its main causal agent, Fusarium graminearum species complex (Fg complex)¸ is able to produce mycotoxins, especially deoxynivalenol (DON) and nivalenol (NIV), that usually contaminate grain. Strains that produce DON may also produce its acetylated derivatives: 3-acetyl-DON (3-ADON) and 15-acetyl-DON (15-ADON). Ninety two isolates were obtained from samplings of barley grain during three years (2007, 2008 and 2009) from several fields in both southern and northern production regions of Rio Grande do Sul state, Brazil. These isolates were examined for polymerase chain-reaction-based (PCR) trichothecene genotype based on the amplification of portions of Tri3 and Tri12. There was no effect of year or region on the proportion of trichothecene genotypes. Overall, 66% of the strains (61/92) were 15-ADON, 4.4% (4/92) were 3-ADON and 29.3% (27/92) were NIV. The overall NIV/DON ratio estimated (0.41) was five times higher than that found in previous studies with strains from wheat grown in the same region. Species identification of nine strains representing the trichothecene genotypes, based on comparisons of DNA sequences of portions of the PHO, RED and URA genes with sequences from curated reference isolates of Fusarium from GenBank, revealed that they belong to F. graminearum sensu stricto (four 15-ADON and one 3-ADON strain), F. meridionale (three NIV strains) and F. austroamericanum (one 3-ADON strain). These results add to the current regional knowledge of trichothecene genotypes and species within the Fg complex affecting barley in the region.

Topics: Bacterial Typing Techniques; Brazil; DNA, Fungal; Fusarium; Genotype; Hordeum; Mycotoxins; Polymerase Chain Reaction; Trichothecenes

Fusarium graminearum is the most important pathogen causing Fusarium head blight (FHB) of small cereal grains worldwide responsible for quantitative and qualitative yield losses. The presence in crops is often associated with mycotoxin contamination of foodstuff limiting its use for human and animal consumption. A collection of isolates of F. graminearum from Germany was characterized genetically and chemically for their potential to produce the B trichothecenes deoxynivalenol (DON) and nivalenol (NIV). Molecular methods with eight PCR assays were implemented based on functional Tri7 and Tri13 genes and on the tri5-tri6 intergenic region to differentiate between chemotaxonomic groups DON and NIV, resulting in a marked majority (61/63) of DON chemotypes. Mycotoxins produced on rice kernels were quantified by means of LC-MSMS including DON, NIV, 3-acetyl-DON (3-ADON), 15-acetyl-DON (15-ADON), DON-3-glucoside, fusarenon X, as well as zearalenone; all of them proving to be present in high concentration among the isolates. All DON-chemotype isolates also produced lower amounts of NIV with the amount being positively correlated (R²=0.89) to the DON amount. 15-ADON and 3-ADON are reported to be produced simultaneously by the isolates, the former dominating over the latter in all but one isolate. Fungal biomass, was quantified via ergosterol amount on rice. It was used to calculate specific mycotoxin production per biomass of isolates, ranging from 0.104 to 1.815mg DON mg-1 ergosterol, presenting a Gaussian distribution. Genotype and phenotype characterization revealed discrepancies with respect to mycotoxin production potential of the fungi, i.e. isolates from one chemotype were able to produce mycotoxins from other chemotypes in considerable amounts.

Topics: DNA, Fungal; Ergosterol; Fusarium; Genotype; Germany; Glucosides; Oryza; Phenotype; Polymerase Chain Reaction; Trichothecenes; Zearalenone

A new analytical method for the rapid and simultaneous determination of five mycotoxins (zearelenone, deoxynivalenol, Fusarenon X, 15-acetyldeoxynivalenol and nivalenol) in breakfast cereals and flours by heart-cutting GC-MS has been developed and validated. Extraction was performed with MeCN, applying a modified QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) procedure, and the extracts were analyzed after a silylation of the analytes under study. Careful optimization of the parameters of Deans Switch device and GC-MS was achieved in order to attain a fast separation in SIM mode, allowing a total run time of only 8 min. Acceptable recoveries for all mycotoxins at two different spiking levels (20 and 100 microg/kg) were achieved with good repeatability (from 9 to 21%). LOD ranged from 2 to 15 microg/kg and LOQ ranged from 5 to 50 microg/kg, which were lower than the maximum limit legal established by the European Union (EU). The method developed was applied to commercial breakfast cereals and flours; among the mycotoxins studied, deoxynivalenol and zearalenone were the most predominant.

Topics: Edible Grain; Flour; Gas Chromatography-Mass Spectrometry; Molecular Structure; Mycotoxins; Reproducibility of Results; Safety; Time Factors; Trichothecenes; Zearalenone

Reducing production of type B trichothecenes by Fusarium graminearum on cereals is necessary to control contamination, prevent yield reduction and protect human and animal health. Thus, an understanding of how trichothecene biosynthesis is induced is essential. The effect of ambient pH on fungal growth, toxin biosynthesis and expression of TRI genes was studied during in vitro liquid culture of F. graminearum on minimal medium. Fungal development stopped at day 3 after a sharp pH drop in the medium. At the same time, induction of TRI gene expression was observed and toxin began accumulating 1 day later. Acidification seems a determinant of induction, as neither the toxin nor the TRI genes were detected when the pH was maintained neutral. Shifting from neutral to acidic pH by mycelium transfer induced TRI gene expression and toxin accumulation. The regulation of toxin production by ambient pH appears to be specific to some TRI genes since TRI5, located in the core FgTRI5 cluster, showed an immediate induction while TRI101, located elsewhere in the genome, showed a more progressive response. The regulation of trichothecene biosynthesis by the ambient pH appears to be a general mechanism, independent of strain or chemotype, as all tested strains, including F. graminearum and F. culmorum species, showed a regulation of toxin production in response to the ambient pH. We conclude that, in vitro, external acidification is required for induction of TRI gene expression.

Topics: Acetylation; Acetyltransferases; Carbon-Carbon Lyases; Edible Grain; Food Contamination; Fungal Proteins; Fusarium; Gene Expression Regulation, Fungal; Genes, Fungal; Hydrogen-Ion Concentration; Kinetics; Microbiological Phenomena; Multigene Family; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Trichothecenes

A speedy and selective ultra-HPLC-MS/MS method for simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-ADON, nivalenol and fusarenon X in traditional Chinese medicines (TCMs) was developed. The method was based on one-step sample cleanup using reliable homemade cleanup cartridges. A linear gradient mobile-phase system, consisting of water containing 0.2% aqueous ammonia and acetonitrile/methanol (90:10, v/v) at a flow rate of 0.4 mL/min, and an Acquity UPLC HSS T3 column (100 mm x 2.1 mm, 1.8 microm) were employed to obtain the best resolution of the target analytes. [(13)C(15)]-DON was used as the internal standard to accomplish as accurate as possible quantitation. The established method was further validated by determining the linearity (R(2) > or = 0.9990), sensitivity (LOQ, 0.29-0.99 microg/kg), recovery (88.5-119.5%) and precision (RSD < or = 15.8%). It was shown to be a suitable method for simultaneous determination of DON, 3-ADON, 15-ADON, nivalenol and fusarenon X in various TCM matrices. The utility and practical impact of the method was demonstrated using different TCM samples.

Topics: Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Medicine, Chinese Traditional; Molecular Conformation; Stereoisomerism; Tandem Mass Spectrometry; Trichothecenes

Fusarium head blight (FHB) is primarily caused by Fusarium graminearum in North America. Isolates of F. graminearum can be identified as one of three chemotypes: 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON), and nivalenol (NIV). In this study, we characterized F. graminearum isolates collected in 1980 to 2000 (old collection) and in 2008 (new collection) from North Dakota and found a 15-fold increase of 3ADON isolates in the new collection. Evaluation of randomly selected 3ADON isolates and 15ADON isolates on three spring wheat genotypes (Grandin, Steele-ND, and ND 2710) by single-floret inoculation indicated that the 3ADON population caused a higher disease severity and produced more DON at a significant level than the 15ADON population on Grandin (susceptible to FHB) and ND 2710 (with FHB resistance from Sumai 3). However, no significant differences in disease severity and DON production were observed between the two populations on Steele-ND (with moderate resistance from Triticum dicoccoides). The 3ADON isolates also exhibited a higher DON production in rice culture and produced more spores on agar media than the 15ADON isolates, suggesting a fitness advantage of the newly emerging 3ADON population over the prevalent 15ADON population. Population genetic analyses using DNA markers revealed a significant genetic differentiation between the two populations. The information obtained in this study could have an impact on development of FHB-resistant wheat cultivars and disease management.

Topics: Fusarium; Genetic Variation; Genotype; Host-Pathogen Interactions; Oryza; Plant Diseases; Trichothecenes; Triticum

CCAAT/enhancer-binding protein homologous protein (CHOP) is a crucial stress-responsive factor in various mucosal injuries, including cellular translational stress conditions. In this study, chemical ribosome-inactivating stresses were assessed for their effects on stress-inducible CHOP expression and its association with epithelial inflammatory cytokine production. Several representative ribotoxic agents (deoxynivalenol, anisomycin, and 15-acetyldeoxynivalenol) enhanced CHOP expression and its nuclear translocation in human intestinal epithelial cells. Moreover, CHOP was a strong positive regulator of IL-8 production, but CHOP-mediated IL-8 production was inversely associated with expression of the mucosal regulatory factor peroxisome proliferator-activated receptor γ (PPARγ). Based on our recent report that PPARγ is a negative regulator of mRNA stability of IL-8, PPARγ was linked to a notable mRNA stabilizing protein, HuR, since ribotoxin-induced IL-8 mRNA is stabilized by HuR protein. Expression of exogenous PPARγ suppressed ribotoxin-triggered cytoplasmic translocation of HuR. In contrast, PPARγ-regulating CHOP was a positive modulator of HuR protein export from nuclei. Taken together, the results indicate that ribotoxin-induced CHOP protein is positively associated with production of proinflammatory cytokine IL-8, but it downregulates PPARγ action, subsequently allowing the cytosolic translocation of HuR protein and stabilization of IL-8 mRNA in gut epithelial cells. CHOP and PPARγ may represent critical mechanistic links between ribotoxic stress and proinflammatory cytokine production, and they may have a broader functional significance with regard to gastrointestinal stresses by toxic mucosal insults.

Topics: Anisomycin; Antigens, Surface; Blotting, Western; Cell Line; ELAV Proteins; ELAV-Like Protein 1; Enzyme-Linked Immunosorbent Assay; Gene Expression; Gene Expression Regulation; Humans; Immunity, Mucosal; Interleukin-8; Intestinal Mucosa; Microscopy, Confocal; PPAR gamma; Protein Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; Transcription Factor CHOP; Transfection; Trichothecenes

A total of 120 freshly harvested wheat samples from the 2004 season in nine locations from Northern Buenos Aires Province, Argentina, were analysed for trichothecene natural occurrence and associated mycoflora, and for determining the influence of commonly used fungicide field treatment and the cultivar type on trichothecene contamination. The trichothecenes T-2 tetraol, T-2 triol, HT-2 and T-2 toxin (HT-2, T-2), diacetoxyscirpenol (DAS), nivalenol (NIV), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) were analysed by gas chromatography and electron capture detection. Detection limits ranged from 4 to 20 microg/kg. The isolation frequencies of species were calculated. Alternaria alternata, Fusarium graminearum, Fusarium poae and Fusarium semitectum were the predominant fungal species identified as endogenous mycoflora. The type of cultivar and the fungicide field treatment did not affect significantly the trichothecene contamination. The trichothecenes type A detected were HT-2 and T-2 triol toxins and the type B were DON, NIV and 3-ADON. Based on 120 samples the incidences were 21.7% for 3-ADON, 22.5% for HT-2, 27.5% for T-2 triol and 85% for DON. NIV was confirmed in one sample. Mean levels of trichothecene positive samples were between 7 and 2788 microg/kg.

Topics: Alternaria; Argentina; Chromatography, Gas; Fungi; Fusarium; Species Specificity; Trichothecenes; Triticum

Effect of exogenous H(2)O(2) and catalase was tested in liquid cultures of the deoxynivalenol and 15-acetyldeoxynivalenol-producing fungus Fusarium graminearum. Accordingly to previous results, H(2)O(2) supplementation of the culture medium leads to increased toxin production. This study indicates that this event seems to be linked to a general up regulation of genes involved in the deoxynivalenol and 15-acetyldeoxynivalenol biosynthesis pathway, commonly named Tri genes. In catalase-treated cultures, toxin accumulation is reduced, and Tri genes expression is significantly down regulated. Furthermore, kinetics of expression of several Tri genes is proposed in relation to toxin accumulation. Biological meanings of these findings are discussed.

Topics: Base Sequence; Catalase; DNA, Fungal; Fungal Proteins; Fusarium; Gene Expression; Genes, Fungal; Hydrogen Peroxide; Kinetics; Models, Biological; Oxidative Stress; Trichothecenes

A large number of isolates from the Fusarium graminearum clade representing all regions in China with a known history of Fusarium head blight (FHB) epidemics in wheat were assayed using PCR to ascertain their trichothecene mycotoxin chemotypes and associated phylogenetic species and geographical distribution. Of the 299 isolates assayed, 231 are from F. asiaticum species lineage 6, which produce deoxynivalenol and 3-acetyldeoxynivalenol (3-AcDON); deoxynivalenol and 15-acetyldeoxynivalenol (15-AcDON); and nivalenol and 4-acetylnivalenol (NIV) mycotoxins, with 3-AcDON being the predominant chemotype. Ninety-five percent of this species originated from the warmer regions where the annual average temperatures were above 15 degrees C, based on the climate data of 30 y during 1970-1999. However, 68 isolates within F. graminearum species lineage 7 consisted only of 15-AcDON producers, 59% of which were from the cooler regions where the annual average temperatures were 15 degrees C or lower. Identification of a new subpopulation of 15-AcDON producers revealed a molecular distinction between F. graminearum and F. asiaticum that produce 15-AcDON. An 11-bp repeat is present in F. graminearum within their Tri7 gene sequences but is absent in F. asiaticum, which could be directly used for differentiating the two phylogenetic species of the F. graminearum clade.

Topics: China; Fusarium; Mycological Typing Techniques; Mycotoxins; Phylogeny; Plant Diseases; Polymerase Chain Reaction; Species Specificity; Trichothecenes; Triticum

Liquid cultures of Fusarium graminearum were supplemented with H2O2 or other oxidative compounds. The accumulation kinetics of the resulting trichothecenes were monitored. At non-lethal concentrations, the H2O2 treatments modulated toxin accumulation, dependent on the method of supplementation. When H2O2 was added at the same time as the inoculation, higher levels of toxins accumulated 30 days later. Conversely, adding H2O2 2 or 7 days after inoculation had little effect. When H2O2 was added daily over the course of the culture, the accumulation of trichothecenes was rapidly and strongly enhanced. The fungus may adapt to oxidative stress when the first exposure to H2O2 occurs at the beginning of the culture course. The highest toxin levels were measured when the H2O2 was added daily. The importance of the first hours of culture was confirmed: pre-treating conidia with H2O2 does not affect their germination kinetics but leads to a reduction in the yield of trichothecenes 40 days later. The H2O2 regulation of this trichothecene accumulation may be specific, as paraquat, another pro-oxidant compound, inhibits their production. Since H2O2 is a major component of the oxidative burst occurring in pathogen/host interactions, these data support the theory that trichothecenes may act as virulence factors.

Topics: Diamide; Fusarium; Hydrogen Peroxide; Oxidative Stress; Paraquat; Trichothecenes

A new, rapid and sensitive method is reported for the multiresidual determination of type A (diacetoxyscirpenol, HT-2 toxin, T-2 toxin) and type B (nivalenol, deoxynivalenol, fusarenon X, 15-O-acetyl-4-deoxynivalenol) trichothecenes in wheat flour samples. Sample extraction was performed with acetonitrile/water mixtures. Mycosep columns were used for a fast and effective clean-up procedure. The analytes were separated by HPLC with a RP C18 column by means of a gradient elution and detected in an ESI-interfaced single quadrupole mass spectrometer. Type B and type A trichothecenes were monitored in the negative and in the positive ion mode, respectively. The method performance is reported in terms of linearity (r2 = 0.999), specificity, accuracy (recoveries from 70-120%) and precision (CV% = 5), the LOQs are in the range 10-20 microg/Kg.

Topics: Chromatography, High Pressure Liquid; Drug Residues; Flour; Food Contamination; Mycotoxins; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; T-2 Toxin; Trichothecenes; Triticum

The purpose of this study was to develop an LC/MS assay to accurately detect three mycotoxins produced by Fusarium graminearum in various matrices. Using different LC conditions, deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), and zearalenone (ZEN) were detected in four different matrices (fungal liquid cultures, maize grain, insect larvae and pig serum). The sensitivity of MS detection allowed us to detect concentrations as low as 8 ppb of DON and 12 ppb of ZEN. A very small quantity of matrix was therefore necessary for successful analysis of these toxins and a variety of experimental situations were successfully investigated using this technique. Production of 15-ADON and butenolide was monitored in a liquid culture of F. graminearum under controlled conditions. Using simple extraction procedures, the differential accumulation of DON and 15-ADON was followed in inoculated maize genotypes varying in susceptibility to F. graminearum. Toxicokinetic studies were carried out with maize insect pests reared continually on artificial diets containing ZEN and suggested that larvae may possess the ability to degrade ZEN. Finally, persistence of DON was assessed in pigs fed diet supplemented with DON, results indicated that DON accumulates quickly in pig blood and then levels decline progressively for 12 hours thereafter. The LC/MS study reported here is very useful and flexible for the detection of these mycotoxins in different media and at very low concentrations.

Topics: Animals; Biological Assay; Fusarium; Gas Chromatography-Mass Spectrometry; Insecta; Male; Mycotoxins; Swine; Trichothecenes; Zea mays; Zearalenone

An experiment was conducted to investigate the effects of feeding grains naturally contaminated with Fusarium mycotoxins on growth and immunological parameters of broiler chickens. Three hundred sixty, 1-d-old male broiler chicks were fed 1 of 4 diets containing grains naturally contaminated with Fusarium mycotoxins for 56 d. The diets included (1) control; (2) low level of contaminated grains (5.9 mg/kg deoxynivalenol (DON), 19.1 mg/kg fusaric acid (FA), 0.4 mg/kg zearalenone, and 0.3 mg/kg 15-acetyldeoxynivalenol; (3) high level of contaminated grains (9.5 mg/kg DON, 21.4 mg/kg FA, 0.7 mg/kg zearalenone, and 0.5 mg/kg 15-acetyldeoxynivalenol); and (4) high level of contaminated grains + 0.2% polymeric glucomannan mycotoxin adsorbent (GM polymer). Body weight gains and feed consumption of chickens fed contaminated grains decreased linearly with the inclusion of contaminated grains during the grower phase (d 21 to 42). Efficiency of feed utilization, however, was not affected by diet. Production parameters were not significantly affected by the supplementation of GM polymer to the contaminated grains. Peripheral blood monocytes decreased linearly in birds fed contaminated grains. The feeding of contaminated diets linearly reduced the B-cell count at the end of the experiment, whereas the T-cell count on d 28 responded quadratically to the contaminated diets. The feeding of contaminated diets did not significantly alter serum or bile immunoglobulin concentrations, contact hypersensitivity to dinitrochlorobenzene, or antibody response to SRBC. Supplementation with GM polymer in the contaminated diet nonspecifically increased white blood cell count and lymphocyte count, while preventing mycotoxin-induced decreases in B-cell counts. It was concluded that broiler chickens are susceptible during extended feeding of grains naturally contaminated with Fusarium mycotoxins.

Topics: Animal Feed; Animals; Chickens; Dermatitis, Contact; Energy Intake; Food Contamination; Fusarium; Immunophenotyping; Lymphocytes; Male; Meat; Mycotoxins; Organ Size; Poultry Diseases; Trichothecenes; Weight Gain; Zearalenone

Various analytical methods used in the analysis of type B trichothecenes (deoxynivalenol, nivalenol, 3- and 15-acetyldeoxynivalenol) in cereals were compared and optimised in this work. These methods use either GC-electron-capture detection (ECD) of trimethylsilyl, trifluoroacetyl and heptafluorobutyryl derivatives or HPLC with UV or photodiode array detection of analytes. A new HPLC procedure using fluorescence detection prior derivatisation with coumarin-3-carbonyl chloride has been also tested. Five extraction solvents and two solid-phase extraction cartridges (silica, Florisil) plus a especial clean-up column (MycoSep 225) were compared in order to obtain the best recovery of the mycotoxins with minimal presence of coextractives in the chromatograms. The chosen extraction solvent was a mixture of acetonitrile-water (84:16, v/v). The MycoSep 225 column was chosen as the best alternative for clean-up of grain samples. For GC-ECD analysis, derivatisation of analytes with heptafluorobutyric anhydride prior the final determination was chosen as the most suitable procedure. HPLC-photodiode array (at 221 nm) analysis was more suitable for determination of type B trichothecenes than HPLC of the fluorescent coumarin-3-carbonyl derivatives. Recoveries obtained in spiked corn, rice and wheat are reported. The utility of the proposed methodology was assayed in cereal cultures of various Fusarium strains.

Topics: Calibration; Chromatography, High Pressure Liquid; Edible Grain; Fusarium; Sensitivity and Specificity; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Trichothecenes

The effects of trichothecene structure on cytokine secretion and gene expression were assessed in primary CD4+ T-cells from murine spleen. CD4+ T-cells were stimulated with concanavalin A (Con A) for 2 or 7 days in the presence of various concentrations of the trichothecenes, vomitoxin (VT or deoxynivalenol), nivalenol (NIV), 15-acetyl deoxynivalenol (15-ADON), 3-acetyl deoxynivalenol (3-ADON), T-2 toxin (T-2) and verrucarin A (Ver A). Culture supernatants were subsequently analyzed for interleukin (IL)-2, IL-4 and IL-5 by ELISA. At day 2, all trichothecenes were found to have inhibited production of IL-2, IL-4, and IL-5. However, at day 7, supernatant IL-2 was significantly increased (2-5.5-fold) in cultures containing VT, NIV, 3-ADON, and 15-ADON at 250, 250, 2500, and 1000 ng/ml doses, respectively, when compared to control Con A-stimulated cultures; significant increases in IL-2 were not observed with T-2 and Ver-A. Similarly, at day 7, IL-4 and IL-5 were significantly increased in the presence of VT (100 ng/ml), NIV (100 ng/ml), 3-ADON (1000 ng/ml), 15-ADON (500 ng/ml), T-2 (1 ng/ml), and Ver A (50 pg/ml, only IL-5) when compared to control cultures. IL production was inhibited at trichothecene concentrations exceeding the aforementioned optima. When total RNA of 2-day cultures was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) in conjunction with Southern analysis, IL-2 mRNA was also found to be superinduced by VT (50 and 100 ng/ml), NIV (50, 100 and 250 ng/ml), 3-ADON (1500 ng/ml), 15-ADON (100 ng/ml), T-2 (0.5 ng/ml) and Ver A (25, 50 and 100 pg/ml); IL-4 mRNA by VT (50 ng/ml), NIV (50 ng/ml), and Ver A (25, 50 and 100 pg/ml); IL-5 mRNA by VT (50 ng/ml); and IL-6 mRNA by 15-ADON (100 ng/ml) and Ver A (50 pg/ml). As the trichothecene concentration increased from these levels, inhibition of mRNA transcript levels was also observed for many of the interleukins. Taken together, the results suggest that trichothecenes as a group can either inhibit or superinduce both IL secretion and mRNA levels in CD4+ T-cells. Superinduction exhibited a rank order of macrocyclic > type A > type B trichothecenes and was dependent on acylation of the trichothecene nucleus.

Topics: Acylation; Animals; Antibiotics, Antineoplastic; Blotting, Southern; CD4-Positive T-Lymphocytes; Cells, Cultured; Concanavalin A; Cytokines; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Interleukin-2; Interleukin-4; Interleukin-5; Mice; RNA, Messenger; Structure-Activity Relationship; T-2 Toxin; Transcription, Genetic; Trichothecenes

Wheat cultivars (Stoa, MN87150, SuMai-3, YMI-6, Wheaton) and barley cultivars (Robust, Excel, Chevron, M69) were inoculated in the field with isolates of Fusarium graminearum and F. culmorum. The disease (Fusarium head blight) kernels were analyzed for deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON) and nivalenol (NIV). F. culmorum produced all three trichothecenes on all cultivars tested whereas F. graminearum only produced DON and 15-ADON. There was no well defined correlation between DON production in the host and resistance although the data tended to favor SuMai-3 as having definitive resistance to both F. graminearum and F. culmorum.

Topics: Animal Feed; Food Contamination; Food Microbiology; Fusarium; Hordeum; Trichothecenes; Triticum

Five Fusarium species were isolated from the grain of dent corn (Zea mays) selected from 20 of 32 damaged fields in 10 counties in Minnesota on the basis of hyphal growth visible on kernels in the field. Three mycotoxins were identified in the infected ears: zearalenone, deoxynivalenol, and 15-acetyl-deoxynivalenol. This is the first report of the presence of 15-acetyl-deoxynivalenol on corn ears in the field prior to harvest and in combination with deoxynivalenol and zearalenone. Ninety-nine cultures were selected from colonies growing from kernels on an agar medium; 30% of the cultures were F. graminearum, 23% were F. subglutinans, 20% were F. moniliforme, 14% were F. oxysporum, and 12% were F. proliferatum.

Topics: Fusarium; Minnesota; Mycotoxins; Trichothecenes; Zea mays; Zearalenone

The acute toxic effects of deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-ADON) were compared in the B6C3F1 female mouse after oral and intraperitoneal exposure. Using the abbreviated procedure of Lorke (Archs Toxicol. 1983, 54, 275), LD50 values for DON were estimated to be 78 mg/kg (oral) and 49 mg/kg (ip) whereas the LD50 values for 15-ADON were 34 mg/kg (oral) and 113 mg/kg (ip). Acute doses of these toxins resulted in extensive necrosis of the gastro-intestinal tract, bone marrow and lymphoid tissues, and focal lesions in kidney and cardiac tissue. The minimum doses required for these histopathological effects were consistent with LD50 estimations. The results indicate that 15-ADON was more or less toxic than DON depending on the route of administration. Risk assessments for DON should therefore consider the potential for 15-ADON occurrence and toxicity in food and feed.

Topics: Animals; Bone Marrow; Digestive System; Female; Kidney; Lethal Dose 50; Lymphoid Tissue; Mice; Mice, Inbred Strains; Sesquiterpenes; Trichothecenes

The emetic activity of 15-acetyldeoxynivalenol (15-ADON), a deoxynivalenol (DON) precursor, was evaluated in swine over a dose range of 25-200 micrograms/kg body weight and found to be very similar to that of DON. The minimum effective oral doses for 15-ADON and DON were 75 and 50 micrograms/kg, respectively, with 3/15 of the 15-ADON- and 4/15 of the DON-treated pigs exhibiting emesis, over the total dose range. The minimum effective ip doses for 15-ADON and DON were also 75 and 50 micrograms/kg, respectively, with 9/15 pigs in each group exhibiting emesis, over the total dose range. For pigs receiving 15-ADON and DON ip, increased dosage was associated with decreased average time to vomition, increased duration of emesis and increased average number of vomitions.

Topics: Animals; Dose-Response Relationship, Drug; Regression Analysis; Sesquiterpenes; Swine; Trichothecenes; Vomiting

Growth and toxigenesis by Fusarium graminearum R6576, were compared in four liquid media. Parameters monitored during the fermentation were deoxynivalenol (DON) and 15-acetyl deoxynivalenol (15-ADON) production, fungal mass, carbohydrate utilization, and pH. Factors which were varied included basal medium composition, corn steep liquor (CSL) concentration, sucrose concentration and ammonium tartrate concentration. Growth in modified Fries medium resulted in only low levels of DON (0.25 mg/L) and 15-ADON (0.25 mg/L) after 20 days. Addition of 4% CSL to modified Fries medium raised the 20 day DON yield to 16.5 mg/l. Increasing the sucrose concentration in modified Fries medium amended with 4% CSL resulted in increased mycelial dry weight but decreased levels of DON. Concentrations of ammonium tartrate greater than 1% in modified Fries amended with 4% CSL greatly reduced DON yield. Use of glucose-yeast extract-peptone (GYEP) for toxin production resulted in higher yields of 15-ADON (14.0 mg/L) than DON (5.5 mg/L) after 20 days. However, supplementation of GYEP with 4% CSL resulted primarily in DON production (4.5 mg/L) after 20 days. In general, qualitative and quantitative production of DON and 15-ADON by Fusarium graminearum R6576 were dependent on the composition of the complex liquid medium.

Topics: Carbohydrate Metabolism; Culture Media; Fusarium; Hydrogen-Ion Concentration; Sesquiterpenes; Species Specificity; Trichothecenes