nivalenol and deoxynivalenol

Deoxynivalenol and nivalenol are major type B trichothecenes and the most frequently occurring mycotoxins worldwide. Their 3-β-d-glucoside forms have recently become a safety management issue. These glucoside conjugates are converted back to the parent toxins during human digestion, but studies to confirm their bioavailability are lacking. In this study, a risk assessment was performed considering the bioavailability of glucoside conjugates. A literature review was conducted to compile the existing bioavailability studies of glucoside conjugates, and three exposure scenarios considering bioavailability were established. As a result of a risk assessment using deterministic and probabilistic methods, both the deoxynivalenol and nivalenol groups had safe levels of tolerable daily intake percentage (TDI%), not exceeding 100%. The TDI% for the nivalenol group was approximately 2-3 times higher than that for the deoxynivalenol group. Notably, infants showed higher TDI% than adults for both toxin groups. By food processing type, the overall TDI% was highest for raw material, followed by simple-processed and then fermented-processed. Since glucoside conjugates can be converted into parent toxins during the digestion process, a risk assessment considering bioavailability allows the more accurate evaluation of the risk level of glucoside conjugates and can direct their safety management in the future.

Topics: Adult; Biological Availability; Eating; Edible Grain; Food Contamination; Glucosides; Humans; Infant; Mycotoxins; Republic of Korea

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

This review summarizes the information regarding the in vivo studies of Fusarium mycotoxins in the last decade. The most common studies are classified as subacute toxicity, subchronic toxicity, acute toxicity, toxicokinetic studies and teratogenicity in order of importance. The most used animals in in vivo studies are pigs, rats, chickens and mice. Fumonisin B1, deoxynivalenol, zearalenone, nivalenol and T-2 toxin are the most studied fusarotoxins. Studies with combinations of mycotoxins are also frequent, deoxynivalenol generally being one of them. The predominant route of administration is oral, administered mostly in the form of naturally contaminated feed. Other administration routes also used are intraperitoneal, intravenous and subcutaneous. In vivo research on Fusarium mycotoxins has increased since 2010 highlighting the need for such studies in the field of food and feed safety.

Topics: Animal Feed; Animals; Chickens; Consumer Product Safety; Disease Models, Animal; Food Contamination; Food Microbiology; Fumonisins; Fusarium; Mice; Mycotoxins; Rats; Swine; T-2 Toxin; Trichothecenes; Zearalenone

Topics: Multigene Family; T-2 Toxin; Trichothecenes

The present paper summarizes toxicity data relevant for hazard characterization for the trichothecene mycotoxins deoxynivalenol (DON), nivalenol (NIV), T-2 and HT-2 from recent opinions prepared by the European Commission Scientific Committee on Food (SCF) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Details on immunotoxicity, cardiovascular toxicity and co-occurrence of different trichothecenes and other mycotoxins and their possible interactions are considered in separate papers in the present issue as well as other aspects such as mould growth, trichothecenes formation, storage, processing, sampling, analytical measurements, exposure assessment and surveillance. The toxicological profiles of DON, NIV, T-2 and HT-2 are similar. The general toxicity and immunotoxicity in experimental animals, and for NIV also haematotoxicity, are considered to be the critical effects. Tolerable Daily Intakes of 1, 0.7 and 0.06 microg/kg b.w. were established for DON, NIV and the sum of T-2 and HT-2, respectively. The TDI's for NIV, T-2 and HT-2 were made temporary because of deficiencies the database.

Topics: Animals; European Union; Female; Fusarium; Male; Mice; Mycotoxins; No-Observed-Adverse-Effect Level; Rats; Swine; T-2 Toxin; Toxicity Tests; Trichothecenes; World Health Organization

Topics: Animal Feed; Animals; Carcinogens; Edible Grain; Food Analysis; Foodborne Diseases; Humans; Mycotoxins; Neoplasms; Trichothecenes; Zearalenone

Topics: Animals; Food Contamination; Mycotoxins; Oryza; Sesquiterpenes; T-2 Toxin; Trichothecenes

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

Topics: Fusarium; Glucosides; Mycotoxins; Plant Diseases; Trichothecenes; Triticum

Fusarium culmorum is a major pathogen of grain crops. Infected plants accumulate deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), or nivalenol (NIV), which are mycotoxins of the trichothecene B group. These toxins are also produced by F. graminearum species complex. New trichothecenes structurally similar to trichothecenes B but lacking the carbonyl group on C-8, designated NX toxins, were recently discovered in atypical isolates of F. graminearum from North America. Only these isolates and a few strains of a yet to be characterized Fusarium species from South Africa are known to produce NX-2 and other NX toxins. Here, we report that among 20 F. culmorum strains isolated from maize, wheat, and oat in Europe and Asia over a period of 70 years, 18 strains produced NX-2 simultaneously with 3-ADON and DON or NIV. Rice cultures of strains producing 3-ADON accumulated NX-2 in amounts corresponding to 2−8% of 3-ADON (1.2−36 mg/kg). A strain producing NIV accumulated NX-2 and NIV at comparable amounts (13.6 and 10.3 mg/kg, respectively). In F. graminearum, producers of NX-2 possess a special variant of cytochrome P450 monooxygenase encoded by TRI1 that is unable to oxidize C-8. In F. culmorum, producers and nonproducers of NX-2 possess identical TRI1; the reason for the production of NX-2 is unknown. Our results indicate that the production of NX-2 simultaneously with trichothecenes B is a common feature of F. culmorum.

Topics: Fusarium; Mycotoxins; Trichothecenes

Topics: Animals; Antibodies, Monoclonal; Fusarium; Mice; Mycotoxins; Trichothecenes

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

The current study investigated the fungal diversity in freshly harvested oat samples from the two largest production regions in Brazil, Paraná (PR) and Rio Grande do Sul (RS), focusing primarily on the

Topics: Avena; Brazil; Edible Grain; Food Contamination; Fusarium; Mycotoxins; Trichothecenes; Trichothecenes, Type B

This study investigated the impact of malting of six wheat cultivars inoculated with

Topics: Biotransformation; Food Contamination; Food Handling; Food Microbiology; Fusarium; Trichothecenes; Triticum; Zearalenone

The transformation of deoxynivalenol (DON), nivalenol (NIV), and their glucosides (DON3G and NIV3G) during the malting of grains of two wheat varieties was studied. The concentration of DON3G and NIV3G started to increase significantly before the concentration of DON and NIV increased. This may reflect the transformation of the parent mycotoxin forms into their glucosides due to xenobiotic detoxification reactions. After a sharp rise during the last 2 days of the process (day 6 and 7), the DON concentration reached 3010 ± 338 µg/kg in the Legenda wheat-based malt and 4678 ± 963 µg/kg in the Pokusa wheat-based malt. The NIV concentration, at 691 ± 65 µg/kg, remained the same as that in the dry grain. The concentration of DON3G in the Legenda and Pokusa wheat-based malt was five and three times higher, respectively, than that in the steeped grain. The concentration of NIV3G in the Legenda wheat-based malt was more than twice as high as that in the steeped grain. The sharp increase in the concentration of DON at the end of the malting process reflected the high pathogen activity. We set aside some samples to study a batch that was left undisturbed without turning and aeration, for the entire period of malting. The concentration of DON in the malt produced from the latter batch was 135% and 337% higher, for Legenda and Pokusa, respectively, than that in the malt produced from the batch that was turned and aerated. The NIV concentration was 22% higher in the latter batch.

Topics: Biotransformation; Edible Grain; Food Handling; Food Microbiology; Fusarium; Glucosides; Time Factors; Trichothecenes; Triticum

Topics: Aflatoxins; Biomarkers; Child; Child, Preschool; Diet; Environmental Exposure; Female; Food Contamination; Humans; Male; Mycotoxins; Ochratoxins; Surveys and Questionnaires; T-2 Toxin; Trichothecenes; United Kingdom; Zearalenone; Zeranol

Major type B trichothecene mycotoxins, including deoxynivalenol (DON), nivalenol (NIV), and their respective glucoside conjugates, deoxynivalenol-3-β-D-glucose (DON3G) and nivalenol-3-β-D-glucose (NIV3G), are present in food products, such as cereals, legumes, and their processed products. Thus, here, DON, NIV, and their 3-β-D-glucosides were monitored in 506 Korean market foods, and exposure to these mycotoxins was estimated in the population consuming these foods. The accuracy and precision of our method, which simultaneously determined four toxins, were 80.1-106.5% and 0.3-12.4%, in four representative food matrices assessed. The incidences of DON, DON3G, NIV, and NIV3G among all food samples tested were 13%, 8%, 12%, and 5%, respectively. The glucoside conjugate with free toxin was found to have the maximum co-occurrence of 49%. The estimated daily intakes of DON, DON3G, NIV, and NIV3G through food intake under four different scenarios were 0.019-0.102, 0.004-0.089, 0.007-0.094, and 0.002-0.095 μg kg

Topics: Adolescent; Adult; Child; Child, Preschool; Dietary Exposure; Environmental Monitoring; Food Contamination; Glucosides; Humans; Infant; Middle Aged; Republic of Korea; Risk Assessment; Trichothecenes; Young Adult

Ruminants are generally considered to be less susceptible to the effects of mycotoxins than monogastric animals as the rumen microbiota are capable of detoxifying some of these toxins. Despite this potential degradation, mycotoxin-associated subclinical health problems are seen in dairy cows. In this research, the disappearance of several mycotoxins was determined in an in vitro rumen model and the effect of realistic concentrations of those mycotoxins on fermentation was assessed by volatile fatty acid production. In addition, two hypotheses were tested: (1) a lower rumen pH leads to a decreased degradation of mycotoxins and (2) rumen fluid of lactating cows degrade mycotoxins better than rumen fluid of non-lactating cows. Maize silage was spiked with a mixture of deoxynivalenol (DON), nivalenol (NIV), enniatin B (ENN B), mycophenolic acid (MPA), roquefortine C (ROQ-C) and zearalenone (ZEN). Fresh rumen fluid of two lactating cows (L) and two non-lactating cows (N) was added to a buffer of normal pH (6.8) and low pH (5.8), leading to four combinations (L6.8, L5.8, N6.8, N5.8), which were added to the spiked maize substrate. In this study, mycotoxins had no effect on volatile fatty acid production. However, not all mycotoxins fully disappeared during incubation. ENN B and ROQ-C disappeared only partially, whereas MPA showed almost no disappearance. The disappearance of DON, NIV, and ENN B was hampered when pH was low, especially when the inoculum of non-lactating cows was used. For ZEN, a limited transformation of ZEN to α-ZEL and β-ZEL was observed, but only at pH 6.8. In conclusion, based on the type of mycotoxin and the ruminal conditions, mycotoxins can stay intact in the rumen.

Topics: Acidosis; Animals; Cattle; Depsipeptides; Fermentation; Hydrogen-Ion Concentration; In Vitro Techniques; Lactation; Microbiota; Models, Biological; Rumen; Trichothecenes

A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3Ac-DON), 15-acetyldeoxynivalenol (15Ac-DON), DON-3-glucoside (DON-3Glc) nivalenol and fusarenone-X in feedstuffs. Different techniques of sample preparation were tested: solid-liquid-extraction, QuEChERS, solid phase extraction with OASIS HLB columns or immunoaffinity columns and a Mycosep 225 Trich column. None of the six immunoaffinity columns tested showed cross-reactivity to all of the mycotoxins. Surprisingly, the results show that if the immunoaffinity columns bound 3Ac-DON, then they did not bind 15Ac-DON. The most efficient sample preparation was achieved with a Mycosep 225 Trich column clean-up. The chromatography was optimised to obtain full separation of all analytes (including 3Ac-DON and 15Ac-DON isomeric form). The validation results show the relative standard deviations for repeatability and reproducibility varied from 4% to 24%. The apparent recovery ranged between 92% and 97%, and the limit of quantification described a 1.30 to 50 µg/kg range. The method trueness was satisfactory, as assessed by a proficiency test and analysis of reference material. A total of 99 feed samples were analysed by the developed method, revealing the presence of DON and DON-3Glc in 85% and 86% of examined animal feeds, respectively at concentrations between 1.70 and 1709 µg/kg. The ratios DON-3Glc to DON in the surveyed feedstuffs were from a low of 3% to high of 59%.

Topics: Animal Feed; Chromatography, Liquid; Food Microbiology; Fungi; Limit of Detection; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Trichothecenes

An analytical method for the simultaneous determination of trichothecenes-namely, nivalenol (NIV), deoxynivalenol (DON) and its acetylated derivatives (3- and 15-acetyl-DON), T-2 and HT-2 toxins-and zearalenone (ZEN) in wheat, wheat flour, and wheat crackers was validated through a collaborative study involving 15 participants from 10 countries. The validation study, performed within the M/520 standardization mandate of the European Commission, was carried out according to the IUPAC (International Union of Pure and Applied Chemistry) International Harmonized Protocol. The method was based on mycotoxin extraction from the homogenized sample material with a mixture of acetonitrile-water followed by purification and concentration on a solid phase extraction column. High-performance liquid chromatography coupled with tandem mass spectrometry was used for mycotoxin detection, using isotopically labelled mycotoxins as internal standards. The tested contamination ranges were from 27.7 to 378 μg/kg for NIV, from 234 to 2420 μg/kg for DON, from 18.5 to 137 μg/kg for 3-acetyl-DON, from 11.4 to 142 μg/kg for 15-acetyl-DON, from 2.1 to 37.6 μg/kg for T-2 toxin, from 6.6 to 134 μg/kg for HT-2 toxin, and from 31.6 to 230 μg/kg for ZEN. Recoveries were in the range 71-97% with the lowest values for NIV, the most polar mycotoxin. The relative standard deviation for repeatability (RSD

Topics: Chromatography, Liquid; Flour; Food Contamination; Humans; Intersectoral Collaboration; Mass Spectrometry; T-2 Toxin; Tandem Mass Spectrometry; Trichothecenes; Triticum; Whole Grains; Zearalenone

In this study, 120 silage samples collected in 2015 from farms in Poland were analysed by a multimycotoxin method based on liquid chromatography coupled with tandem mass spectrometry. The study included toxins which are regulated within the European Union (aflatoxins, deoxynivalenol, fumonisins, T-2/HT-2 toxins, ochratoxin A and zearalenone) and non-regulated mycotoxins (enniatins, beauvericin, 8-ketotrichothecenes, sterigmatocystin, zearalenone derivatives). All silage samples were positive for at least one mycotoxin, and 61% of samples contained five or more mycotoxins simultaneously. The most frequently detected toxins were deoxynivalenol, nivalenol, zearalenone, enniatins and beauvericin, although the levels of these toxins were relatively low. The mean concentration of deoxynivalenol and zearalenon was 406 and 80.6 μg/kg, respectively, and two toxins were positive-correlated. This is the first study that provides information about emerging mycotoxins contaminating silage in Poland.

Topics: Chromatography, Liquid; Fumonisins; Fusarium; Mycotoxins; Poland; Silage; Tandem Mass Spectrometry; Trichothecenes; Zearalenone

Topics: Apoptosis; Biological Assay; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Flow Cytometry; Humans; Inhibitory Concentration 50; Jurkat Cells; Trichothecenes

A simple and reliable method for the simultaneous determination of major type B trichothecene mycotoxins, deoxynivalenol (DON) and nivalenol (NIV), along with their 3-β-d-glucosides (DON-3-glucoside (DON3G) and NIV-3-glucoside (NIV3G)) in baby formula and Korean rice wine was validated in the present study. The method was based on immunoaffinity cleanup followed by analysis using an HPLC-UV technique. The method was validated in-house for two matrices as follows: linearity (R

Topics: Chromatography, Affinity; Chromatography, High Pressure Liquid; Edible Grain; Food Contamination; Glucosides; Infant Formula; Republic of Korea; Spectrophotometry, Ultraviolet; Trichothecenes; Wine

Lymphocytes are involved in the adaptive immune response and are highly sensitive to type B trichothecenes. In grains and their products, deoxynivalenol (DON) is the most widely distributed trichothecene. It usually co-occurs with other type B members, such as nivalenol (NIV) and fusarenon-X (FX), because they are all produced by the same Fusarium fungi. However, the combined effects of mycotoxins are complex and cannot be predicted based on individual toxicity. Thus, the adverse effects of combined toxins are of increasing concern. The aim of this study was to compare the toxicity to lymphoid tissues of mice of DON alone or mixed with NIV or FX. Forty, 3-week-old male ICR mice were given a single oral administration of a vehicle control, one toxin, binary, or ternary mixtures and then sacrificed at 12 h after exposure. Mice treated with FX alone showed marked nuclear condensation and fragmentation of lymphocytes in the cortical thymus and germinal center of Peyer's patches and spleen. Similarly, these animals clearly displayed TUNEL- and Caspase-3-positive cells in the regions. In contrast, minimal changes were noticed in the lymphoid tissues of mice receiving combined toxins when compared to this toxin alone. In addition, oral exposure to FX alone significantly up-regulated the relative expression of Bax, Caspase-3, Caspase-9, and Trp53. These data increase our understanding of the toxic actions of DON, NIV, and FX alone or in combination to lymphocytes and can be used to assess the possible risk associated with their co-occurrences in foodstuffs to human and animal health.

Topics: Administration, Oral; Animals; Apoptosis; In Situ Nick-End Labeling; Lymphocytes; Male; Mice; Mice, Inbred ICR; Mycotoxins; Trichothecenes

Fusarium meridionale and F. boothii cause Gibberella Ear Rot (GER) in maize. This study determined the effects of temperature (5-35 °C) and water activity (0.90-0.995 a

Topics: Argentina; Fusarium; Humidity; Plant Diseases; Seasons; Temperature; Trichothecenes; Triticum; Water; Zea mays

The fungus Fusarium graminearum is the causative agent of economically significant plant diseases such as Fusarium Healed Blight (FHB) of cereals, its mycotoxins as deoxynivalenol (DON), Nivalenol (NIV) and Zearalenone (ZEN) contaminate wheat and other grains. The objectives of the present study were to determine the mechanism by which the bacterium Pseudomonas aeruginosa inhibits the growth of F. graminearum. Our results indicate that P. aeruginosa metabolites as pyocyanin has effective antifungal properties. Pyocyanin was produced by P. aeruginosa when cultured on mineral salt medium and reached a maximum concentration after 72 h. Pyocyanin significantly decreased mycotoxins of F. graminearum, a 25 mg/ml of pyocyanin for 72 h decreased DON by 68.7% and NIV by 57.7%.Real-Time PCR analysis demonstrated that the antifungal effect is mediated by downregulation of the Pleiotropic Drug Resistance (PDR) subfamily FgABC3. 25 mg/ml of pyocyanin decreased FgABC3-mRNA by 60%, inhibited the fungal growth and decreased the area of mycelial growth at 12, 24, 36 and 72 h post incubation by 40-50%. Deletion of FgABC3 led to enhanced accumulation of DON and NIV by 40 and 60%, respectively.The data presented in this report may have significance in understanding mechanism by which certain bacterial metabolites exert a beneficial effect and for developing antifungal drugs.

Topics: Antifungal Agents; Drug Resistance; Edible Grain; Fusarium; Mycotoxins; Pseudomonas aeruginosa; Pyocyanine; Trichothecenes; Triticum; Zearalenone

The presence of mycotoxins in cereal grain is a very important food safety factor. The occurrence of "masked" mycotoxins has been intensively investigated in recent years. In this study, the occurrence of nivalenol, deoxynivalenol-3-glucoside, and deoxynivalenol in 92 samples of winter wheat from Polish cultivars was determined. The frequency of the occurrence of deoxynivalenol and nivalenol in the samples was 83% and 70%, respectively. The average content of the analytes was: for deoxynivalenol 140.2 µg/kg (10.5-1265.4 µg/kg), for nivalenol 35.0 µg/kg (5.1-372.5 µg/kg). Deoxynivalenol-3-glucoside, the formation of which is connected with the biotransformation pathway in plants, was present in 27% of tested wheat samples; its average content was 41.9 µg/kg (15.8-137.5 µg/kg). The relative content of deoxynivalenol-3-glucoside (DON-3G) compared to deoxynivalenol (DON) in positive samples was 4-37%. Despite the high frequency of occurrence of these mycotoxins, the quality of wheat from the 2016 season was good. The maximum content of DON, as defined in EU regulations (1250 µg/kg), was exceeded in only one sample. Nevertheless, the presence of a glycosidic derivative of deoxynivalenol can increase the risk to food safety, as it can be hydrolyzed by intestinal microflora.

Topics: Edible Grain; Environmental Monitoring; Food Contamination; Glucosides; Poland; Trichothecenes; Triticum

In recent years, due to the negative impact of toxigenic mycobiota and of the accumulation of their secondary metabolites in malting barley grains, monitoring the evolution of fungal communities in a certain cultivation area as well as detecting the different mycotoxins present in the raw material prior to malting and brewing processes have become increasingly important. In this study, a survey was carried out on malting barley samples collected after their harvest in the Umbria region (central Italy). Samples were analyzed to determine the composition of the fungal community, to identify the isolated Fusarium species, to quantify fungal secondary metabolites in the grains and to characterize the in vitro mycotoxigenic profile of a subset of the isolated Fusarium strains. The fungal community of barley grains was mainly composed of microorganisms belonging to the genus Alternaria (77%), followed by those belonging to the genus Fusarium (27%). The Fusarium head blight (FHB) complex was represented by nine species with the predominance of Fusarium poae (37%), followed by Fusarium avenaceum (23%), Fusarium graminearum (22%) and Fusarium tricinctum (7%). Secondary metabolites biosynthesized by Alternaria and Fusarium species were present in the analyzed grains. Among those biosynthesized by Fusarium species, nivalenol and enniatins were the most prevalent ones. Type A trichothecenes (T-2 and HT-2 toxins) as well as beauvericin were also present with a high incidence. Conversely, the number of samples contaminated with deoxynivalenol was low. Conjugated forms, such as deoxynivalenol-3-glucoside and HT-2-glucoside, were detected for the first time in malting barley grains cultivated in the surveyed area. In addition, strains of F. avenaceum and F. tricinctum showed the ability to biosynthesize in vitro high concentrations of enniatins. The analysis of fungal secondary metabolites, both in the grains and in vitro, revealed also the presence of other compounds, for which further investigations will be required. The combination of microbiological analyses, of molecular biology assays and of multi-mycotoxin screening shed light on the complexity of the fungal community and its secondary metabolites released in malting barley.

Topics: Alternaria; Depsipeptides; Edible Grain; Food Contamination; Fusarium; Glucosides; Hordeum; Italy; Mycotoxins; Seedlings; T-2 Toxin; Trichothecenes

Fusarium meridionale has been frequently isolated from soybean in Argentina and showed similar pathogenicity as F. graminearum sensu stricto. However, no data on their growth and mycotoxin production under different environmental conditions are yet available. The aims of this study were: to determine the effect of temperature, water activity (a

Topics: Argentina; Food Microbiology; Fusarium; Gene Expression Regulation, Fungal; Glycine max; Temperature; Trichothecenes; Water

Deoxynivalenol (DON), Fumonisin B

Topics: Aflatoxin B1; Biosensing Techniques; Food Analysis; Food Contamination; Fumonisins; Limit of Detection; Ochratoxins; Reagent Strips; T-2 Toxin; Trichothecenes

Fusarium graminearum of the 15-acetyl-deoxynivalenol (15-ADON) chemotype is the main cause of Fusarium head blight (FHB) of wheat in southern Brazil. However, 3-ADON and nivalenol (NIV) chemotypes have been found in other members of the species complex causing FHB in wheat. To improve our understanding of the pathogen biology and ecology, we assessed a range of fitness-related traits in a sample of 30 strains representatives of 15-ADON (F. graminearum), 3-ADON (F. cortaderiae and F. austroamericanum), and NIV (F. meridionale and F. cortaderiae). These included perithecia formation on three cereal-based substrates, mycelial growth at two suboptimal temperatures, sporulation and germination, pathogenicity toward a susceptible and a moderately resistant cultivar, and sensitivity to tebuconazole. The most important trait favoring F. graminearum was a two times higher sexual fertility (>40% perithecial production index [PPI]) than the other species (<30% PPI); PPI varied among substrates (maize > rice > wheat). In addition, sensitivity to tebuconazole appeared lower in F. graminearum, which had the only strain with effective fungicide concentration to reduce 50% of mycelial growth >1 ppm. In the pathogenicity assays, the deoxynivalenol producers were generally more aggressive (1.5 to 2× higher final severity) toward the two cultivars, with 3-ADON or 15-ADON leading to higher area under the severity curve than the NIV strains in the susceptible and moderately resistant cultivars, respectively. There was significant variation among strains of the same species with regards asexual fertility (mycelial growth, macroconidia production, and germination), which suggested a strain- rather than a species-specific difference. These results contribute new knowledge to improve our understanding of the pathogen-related traits that may explain the dominance of certain members of the species complex in specific wheat agroecosystems.

Topics: Drug Resistance, Fungal; Fungicides, Industrial; Fusarium; Genotype; Host-Pathogen Interactions; Mycelium; Plant Diseases; Species Specificity; Spores, Fungal; Triazoles; Trichothecenes; Triticum; Virulence

Topics: Cell Line; Cell Survival; Epithelial Cells; Humans; Interleukin-6; Interleukin-8; Respiratory System; Trichothecenes

Family 1 UDP-glycosyltransferases (UGTs) in plants primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, which is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin, and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins because of its ability to glycosylate DON, nivalenol, and hydrolyzed T-2 toxin (HT-2). However, Os79 is unable to modify T-2 toxin (T-2), produced by pathogens such as Fusarium sporotrichioides and Fusarium langsethii. Activity toward T-2 is desirable because it would allow a single UGT to inactivate co-occurring mycotoxins. Here, the structure of Os79 in complex with the products UDP and deoxynivalenol 3-O-glucoside is reported together with a kinetic analysis of a broad range of trichothecene mycotoxins. Residues associated with the trichothecene binding pocket were examined by site-directed mutagenesis that revealed that trichothecenes substituted at the C4 position, which are not glycosylated by wild-type Os79, can be accommodated in the binding pocket by increasing its volume. The H122A/L123A/Q202L triple mutation, which increases the volume of the active site and attenuates polar contacts, led to strong and equivalent activity toward trichothecenes with C4 acetyl groups. This mutant enzyme provides the broad specificity required to control multiple toxins produced by different Fusarium species and chemotypes.

Topics: Fusarium; Glucosides; Glucosyltransferases; Glycogen Debranching Enzyme System; Hordeum; Kinetics; Mutagenesis, Site-Directed; Mycotoxins; Oryza; Plant Diseases; Plant Proteins; Trichothecenes; Triticum

Topics: Cell Line; Epithelial Cells; Food Contamination; Humans; Inflammation; Intestinal Mucosa; Reactive Oxygen Species; Trichothecenes

The global incidence of Fusarium head blight and attendant cereal grains multi-contamination by the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are increasing as a possible result of climate change and inadequate agricultural practices. At the molecular level, these mycotoxins bind to the ribosome, activate the mitogen-activated protein kinase and induce a local and systemic inflammation. DON is of public health concern owing to the narrow margin between exposure and tolerable daily intake. The intestinal inflammatory response to DON, NIV and their mixture was analyzed to determine thresholds for their intestinal pro-inflammatory effects and characterize the type and magnitude of their interaction. Fully differentiated three-dimensional porcine jejunal explants were exposed to increasing doses of DON and NIV alone or in combination; the expression levels of IL-1α, IL-1β, IL-8, IL-17A and IL-22 were measured by RT-PCR. Doses as low as 0.16 µM DON or 0.73 µM NIV significantly increase the intestinal expression levels of the tested inflammation-related genes. These doses are lower than those previously reported for other intestinal toxicity endpoints. The combined pro-inflammatory activity of DON and NIV was synergistic for all the tested genes with combination index value range of 0.23-0.8. Our results indicate that (1) inflammation is a very sensitive endpoint for the intestinal toxicity of the trichothecenes and (2) co-exposure to DON and NIV has a greater inflammatory effect than induced by mycotoxins alone. This synergy should be taken into account considering the frequent co-occurrence of DON and NIV in the diet.

Topics: Animals; Cytokines; Dose-Response Relationship, Drug; Drug Synergism; Enteritis; Food Contamination; Gene Expression Regulation; Jejunal Diseases; Jejunum; Mycotoxins; Organ Culture Techniques; Swine; Toxicity Tests; Trichothecenes

Deoxynivalenol is a food borne mycotoxin belonging to the trichothecenes family that may cause severe injuries in human and animals. The inhibition of protein synthesis via the interaction with the ribosome has been identified as a crucial mechanism underlying toxic action. However, it is not still fully understood how and to what extent compounds belonging to trichothecenes family affect human and animal health. In turn, this scenario causes delay in managing the related health risk. Aimed at supporting the hazard identification process, the in silico analysis may be a straightforward tool to investigate the structure-activity relationship of trichothecenes, finding out molecules of possible concern to carry forth in the risk assessment process. In this framework, this work investigated through a molecular modeling approach the structural basis underlying the interaction with the ribosome under a structure-activity relationship perspective. To identify further forms possibly involved in the total trichothecenes-dependent ribotoxic load, the model was challenged with a set of 16 trichothecene modified forms found in plants, fungi and animals, including also compounds never tested before for the capability to bind and inhibit the ribosome. Among them, only the regiospecific glycosylation in the position 3 of the sesquiterpenoid scaffold (i.e. T-2 toxin-3-glucuronide, α and β isomers of T-2 toxin-3-glucoside and deoxynivalenol-3-glucuronide) was found impairing the interaction with the ribosome, while the other compounds tested (i.e. neosolaniol, nivalenol, fusarenon-X, diacetoxyscirpenol, NT-1 toxin, HT-2 toxin, 19- and 20-hydroxy-T-2 toxin, T-2 toxin triol and tetraol, and 15-deacetyl-T-2 toxin), were found potentially able to inhibit the ribosome. Accordingly, they should be included with high priority in further risk assessment studies in order to better characterize the trichothecenes-related hazard.

Topics: DNA Mismatch Repair; Food Contamination; Food Microbiology; Glucuronides; Mycotoxins; Ribosomes; Structure-Activity Relationship; T-2 Toxin; Trichothecenes

The mycotoxins deoxynivalenol (DON) and nivalenol (NIV), worldwide cereal contaminants, raise concerns for human and animal gut health, following exposure through contaminated food and feed. The aim of this work was to analyze the effects of DON and NIV, alone or associated, on the intestinal pig mucosa. Jejunal loops were used for testing DON and NIV individually and in combination (1:1) after a single exposure, for 24 h. For repeated exposure, piglets received a natural contaminated feed, with DON or with DON + NIV for 28 days. Histological investigations, proliferation and apoptosis assessments were conducted. Both experiments were concordant for the total-cell proliferation decreased at the villus tips after DON or DON + NIV at 10 μM acutely, or repeatedly, by 30-35% and 20-25%, respectively. In loops model, apoptotic enterocytes at villus tips increased dose-dependently after DON, NIV alone or DON + NIV in combination. The combination in loops at 10 μM showed higher effects on proliferation and apoptosis than DON alone, and synergism was shown for villus apoptotic enterocyte. These results are to be considered for NIV consumer risk assessment. Our results demonstrate the in vivo disruption of the intestinal balance proliferation/apoptosis explaining, at least partly, the disruption of intestinal barrier by these mycotoxins.

Topics: Animals; Apoptosis; Cell Proliferation; Enterocytes; Female; Intestinal Mucosa; Jejunum; Male; Swine; Trichothecenes

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

Fusarium mycotoxins deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) were investigated in wheat from the 2009 and 2010 crop years. Samples (n = 745) from commercial fields were collected in four wheat producing regions (WPR) which differed in weather conditions. Analyses were performed using HPLC-DAD. Contamination with ZEN, DON and NIV occurred in 56, 86 and 50%, respectively. Also, mean concentrations were different: DON = 1046 µg kg(-1), NIV < 100 µg kg(-1) and ZEN = 82 µg kg(-1). Co-occurrence of ZEN, DON and NIV was observed in 74% of the samples from 2009 and in 12% from 2010. Wet/cold region WPR I had the highest mycotoxin concentration. Wet/moderately hot region WPR II had the lowest mycotoxin levels. Furthermore, the mean concentration of each mycotoxin was higher in samples from 2009 as compared with those from 2010. Precipitation during flowering or harvest periods may explain these results.

Topics: Brazil; Chromatography, High Pressure Liquid; Climate; Diet; Edible Grain; Food Contamination; Fusarium; Humans; Trichothecenes; Triticum; Weather; Zearalenone

In the present study, the occurrence and contamination levels of eight mycotoxins were investigated in wheat flour samples (n = 359) from Shandong Province of China. Samples were determined using a multi-mycotoxin method based on isotope dilution ultrahigh performance liquid chromatography-tandem mass spectrometry. The results indicated that the most frequently found mycotoxins were deoxynivalenol (DON) (97.2%), nivalenol (40.4%) and deoxynivalenol-3-glucoside (33.4%), and mean contamination levels in positive samples were 86.7, 3.55 and 3.34 µg kg(-1), respectively. The obtained data were further used to estimate the daily intake of the local population, and indicated that wheat flour consumption contributes little to DON exposure. However, with the aim to keep the contamination levels under control and to establish a more precise evaluation of the mycotoxin burden in Shandong Province, more sample data from different harvest years and seasons are needed in the future.

Topics: Adult; Carcinogens, Environmental; China; Chromatography, High Pressure Liquid; Diet; Diet Surveys; Flour; Food Contamination; Food Inspection; Glucosides; Humans; Indicator Dilution Techniques; Limit of Detection; Mycotoxins; Reproducibility of Results; Seeds; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Trichothecenes; Triticum

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

Globally, the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are among the most widely distributed mycotoxins that contaminate small grain cereals. In this study, a bacterial consortium, PGC-3, with de-epoxydation activity was isolated from soil by an in situ soil enrichment method. Screening of 14 soil samples that were sprayed with DON revealed that 4 samples were able to biotransform DON into de-epoxydized DON (dE-DON). Among these, the PGC-3 consortium showed the highest and most stable activity to biotransform DON into dE-DON and NIV into dE-NIV. PGC-3 exhibited de-epoxydation activity at a wide range of pH (5-10) and temperatures (20-37 °C) values under aerobic conditions. Sequential subculturing with a continued exposure to DON substantially reduced the microbial population diversity of this consortium. Analyses of the 16S rDNA sequences indicated that PGC-3 comprised 10 bacterial genera. Among these, one species, Desulfitobacterium, showed a steady increase in relative abundance, from 0.03% to 1.55% (a 52-fold increase), as higher concentrations of DON were used in the subculture media, from 0 to 500 μg/mL. This study establishes the foundation to further develop bioactive agents that can detoxify trichothecene mycotoxins in cereals and enables for the characterization of detoxifying genes and their regulation.

Topics: Aerobiosis; Bacteria; DNA, Bacterial; DNA, Ribosomal; Epoxy Compounds; Soil Microbiology; Trichothecenes

Topics: Animals; Aphids; Behavior, Animal; Fusarium; Host-Pathogen Interactions; Plant Diseases; Trichothecenes; Triticum

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

Deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEA) are mycotoxins commonly produced by Fusarium species. The purpose of the present study was to investigate the effects of DON alone and in combination with NIV and ZEA on several parameters including weight gain and histological aspects of pigs submitted to chronic intoxication. Twenty, 5-week-old piglets received for 28 days one of the following diets: a control diet, a diet mono- contaminated with DON (1.5mg/kg), a diet multi-contaminated with DON (2mg/kg)+NIV (1.3mg/kg)+ZEA (1.5mg/kg) or a diet contaminated with DON (3mg/kg)+NIV (1.3mg/kg)+ZEA (1.5mg/kg). Animals fed the multi-contaminated diets presented a significant decrease in weight gain over the total period. The chronic ingestion of the contaminated diets induced a significant increase on histological changes on the intestine, liver and lymphoid organs. In addition, a significant increase on lymphocyte apoptosis was observed in lymph nodes and spleen in the animals receiving the contaminated diets. These data provide a better understanding of the possible effects of Fusarium toxins, alone or in combinations on the morphology of the intestine and lymphoid organs, which would contribute to the risk assessment of these toxins.

Topics: Animal Feed; Animals; Cell Proliferation; Drug Interactions; Food Contamination; Intestines; Liver; Male; Swine; Trichothecenes; Weight Gain; Zearalenone

A comprehensive quantum chemical study was carried out on 35 type A and B trichothecenes and biosynthetic precursors, including selected derivatives of deoxynivalenol and T-2 toxin. Quantum chemical properties, Natural Bond Orbital (NBO) analysis, and molecular parameters were calculated on structures geometry optimized at the B3LYP/6-311+G** level. Type B trichothecenes possessed significantly larger electrophilicity index compared to the type A trichothecenes studied. Certain hydroxyl groups of deoxynivalenol, nivalenol, and T-2 toxin exhibited considerable rotation during molecular dynamics simulations (5 ps) at the B3LYP/6-31G** level in implicit aqueous solvent. Quantitative structure activity relationship (QSAR) models were developed to evaluate toxicity and detection using genetic algorithm, principal component, and multilinear analyses. The models suggest electronegativity and several 2-dimensional topological descriptors contain important information related to trichothecene cytotoxicity, phytotoxicity, immunochemical detection, and cross-reactivity.

Topics: Electrons; Immunochemistry; Models, Molecular; Quantitative Structure-Activity Relationship; Quantum Theory; T-2 Toxin; Toxins, Biological; Trichothecenes

Mycotoxins are secondary fungal metabolites often found as contaminants in almost all agricultural commodities worldwide, and the consumption of food or feed contaminated by mycotoxins represents a major risk for human and animal health. Reactive oxygen species are normal products of cellular metabolism. However, disproportionate generation of reactive oxygen species poses a serious problem to bodily homeostasis and causes oxidative tissue damage. In this study we analyzed the effect of two trichothecenes mycotoxins: nivalenol and deoxynivalenol, alone and in combination, on oxidative stress in the non-tumorigenic intestinal epithelial cell line IEC-6. Our results indicate the pro-oxidant nivalenol effect in IEC-6, the stronger pro-oxidant effect of nivalenol when compared to deoxynivalenol and, interestingly, that nivalenol increases deoxynivalenol pro-oxidative effects. Mechanistic studies indicate that the observed effects were mediated by NADPH oxidase, calcium homeostasis alteration, NF-kB and Nrf2 pathways activation and by iNOS and nitrotyrosine formation. The toxicological interaction by nivalenol and deoxynivalenol reported in this study in IEC-6, points out the importance of the toxic effect of these mycotoxins, mostly in combination, further highlighting the risk assessment process of these toxins that are of growing concern.

Topics: Animals; Calcium Signaling; Cell Line; Epithelial Cells; Heme Oxygenase-1; Intestinal Mucosa; Intestines; Mitochondria; Mycotoxins; Oxidants; Oxidative Stress; Rats; Reactive Oxygen Species; Superoxides; Trichothecenes; Tyrosine

Red ear rot an important disease of maize cultivated in Europe is caused by toxigenic Fusarium species like Fusarium graminearum and Fusarium culmorum. To get detailed information on the time course of the infection process leading to the accumulation of Fusarium mycotoxins in maize ears, a field study was conducted over 2 years with two maize varieties, which were inoculated with F. culmorum or F. graminearum isolates at the stage of female flowering. Every fortnight after inoculation, infection and contamination progress in the ears was followed by visually evaluating disease signs and analysing Fusarium toxin concentrations in the infected ear tissues. In principle, infection and mycotoxin distribution were similar in respect of pathogens, varieties, and years. External infection symptoms showing some small pale or brown-marbled kernels with dark brown pedicels were mainly seen at the ear tip, whereas internal infection symptoms on the rachis were much more pronounced and spread in the upper half showing greyish brownish or pink discoloration of the pith. Well correlated with disease symptoms, a top-down gradient from high to low toxin levels within the ear with considerably higher concentrations in the rachis compared with the kernels was observed. It is suggested that both Fusarium pathogens primarily infect the rachis from the tip toward the bottom, whereas the kernels are subsequently infected via the rachillae connected to the rachis. A special focus on the pronounced disease symptoms visible in the rachis may be an approach to improve the evaluation of maize-genotype susceptibility against red ear rot pathogens. It has to be underlined that the accumulation of Fusarium mycotoxins in the rachis greatly accelerated 6 weeks after inoculation; therefore, highest contamination risk is indicated for feedstuffs containing large amounts of rachis (e.g., corn cob mix), especially when cut late in growing season.

Topics: Climate; Fusarium; Mycotoxins; Plant Diseases; Trichothecenes; Zea mays

Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A Km of 5.4 mM and a Vmax of 16 μmol min(-1) mg(-1) were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples.

Topics: Bifidobacterium; Cellulases; Edible Grain; Fusarium; Glucosides; Hydrolysis; Kinetics; Levilactobacillus brevis; Mycotoxins; Recombinant Proteins; Substrate Specificity; T-2 Toxin; Trichothecenes

The mycotoxins deoxynivalenol (DON) and nivalenol (NIV), worldwide cereal contaminants, raise concerns for animal and human gut health, following contaminated food or feed ingestion. The impact of DON and NIV on intestinal mucosa was investigated after acute exposure, in vitro and in vivo. The histological changes induced by DON and NIV were analyzed after four-hour exposure on pig jejunum explants and loops, two alternative models. On explants, dose-dependent increases in the histological changes were induced by DON and NIV, with a two-fold increase in lesion severity at 10 µM NIV. On loops, NIV had a greater impact on the mucosa than DON. The overall proliferative cells showed 30% and 13% decrease after NIV and DON exposure, respectively, and NIV increased the proliferative index of crypt enterocytes. NIV also increased apoptosis at the top of villi and reduced by almost half the proliferative/apoptotic cell ratio. Lamina propria cells (mainly immune cells) were more sensitive than enterocytes (epithelial cells) to apoptosis induced by NIV. Our results demonstrate a greater impact of NIV than DON on the intestinal mucosa, both in vitro and in vivo, and highlight the need of a specific hazard characterization for NIV risk assessment.

Topics: Animals; Apoptosis; Cell Proliferation; Enterocytes; Female; In Vitro Techniques; Intestinal Mucosa; Jejunum; Reproducibility of Results; Swine; Trichothecenes

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

Trichothecene mycotoxins such as nivalenol and deoxynivalenol frequently contaminate foodstuffs. Recently, several trichothecene glucosides have been found in trichothecene-contaminated foods, and information about their chemistry, toxicity, and occurrence is required. In this study, a glucoside of nivalenol was isolated from nivalenol-contaminated wheat and was identified as nivalenol-3-O-β-D-glucopyranoside. Analytical methods using a multifunctional column or an immunoaffinity column have been developed for the simultaneous determination of nivalenol, nivalenol-3-O-β-D-glucopyranoside, deoxynivalenol, and deoxynivalenol-3-O-β-D-glucopyranoside in wheat. The methods were validated in a single laboratory, and recovery from wheat samples spiked at four levels ranged between 86.4 and 103.5% for the immunoaffinity column cleanup. These mycotoxins in contaminated wheat samples were quantitated by the validated method. Nivalenol-3-O-β-D-glucopyranoside was detected in the nivalenol-contaminated wheat, and the percentage of nivalenol-3-O-β-D-glucopyranoside to nivalenol ranged from 12 to 27%. This result indicates that the analytical method developed in this study is useful for obtaining data concerning the state and level of food contamination by nivalenol, deoxynivalenol, and their glucosides.

Topics: Chemistry Techniques, Analytical; Food Contamination; Glucosides; Mycotoxins; Trichothecenes; Triticum

Suspensions or solutions with 1% of Chinese galls (Galla chinensis, GC) or 1% of tannic acid (TA), inhibited germination of conidia or mycelium growth of Fusarium graminearum (FG) by 98%-100% or by 75%-80%, respectively, whereas dried bark from buckthorn (Frangula alnus, FA) showed no effect at this concentration. In climate chamber experiments where the wheat variety "Apogee" was artificially inoculated with FG and F. crookwellense (FCr) and treated with 5% suspensions of TA, GC and FA, the deoxynivalenol (DON) content in grains was reduced by 81%, 67% and 33%, respectively. In field experiments with two commercial wheat varieties and artificial or semi-natural inoculations, mean DON reductions of 66% (TA) and 58% (FA), respectively, were obtained. Antifungal toxicity can explain the high efficacies of TA and GC but not those of FA. The Fusarium head blight (FHB) and mycotoxin reducing effect of FA is probably due to elicitation of resistance in wheat plants. With semi-natural inoculation, a single FA application in the first half of the flowering period performed best. However, we assume that applications of FA at the end of ear emergence and a treatment, triggered by an infection period, with TA or GC during flowering, might perform better than synthetic fungicides.

Topics: Antifungal Agents; Fusarium; Mycotoxins; Plant Bark; Plant Diseases; Plant Extracts; Plant Tumors; Rhamnus; Rheum; Spores, Fungal; Tannins; Trichothecenes; Triticum

In this pilot survey human urine samples were analyzed for presence of 15 mycotoxins and some of their metabolites using a novel urinary multi-mycotoxin GC-MS/MS method following salting-out liquid-liquid extraction. Fifty-four urine samples from children and adults residents in Valencia were analyzed for presence of urinary mycotoxin and expressed in gram of creatinine. Three out of 15 mycotoxins were detected namely, HT-2 toxin, nivalenol and deoxynivalenol (DON). 37 samples showed quantifiable values of mycotoxins. Co-occurrence of these contaminants was also observed in 20.4% of assayed samples. DON was the most frequently detected mycotoxin (68.5%) with mean levels of 23.3 μg/g creatinine (range: 2.8-69.1 μg/g creatinine). The levels of urinary DON were used to carry out an exposure assessment approach. 8.1% of total subjects were estimated to exceed the DON provisional maximum tolerable daily intake (PMTDI) (1 μg/kg b.w.). Two out of 9 exposed children exceeded the DON PMTDI thus, making them the most exposed based on the urinary results.

Topics: Adolescent; Adult; Child; Chromatography, Gas; Creatinine; Female; Food Contamination; Food Microbiology; Humans; Limit of Detection; Male; Mycotoxins; Pilot Projects; Reproducibility of Results; T-2 Toxin; Tandem Mass Spectrometry; Trichothecenes; Young Adult

Fusarium spp. cause many diseases in farming systems and can produce diverse mycotoxins that can easily impact humans and animals through the ingestion of food and feed. Among these mycotoxins, deoxynivalenol (DON) and nivalenol (NIV) are considered the most important hazards because they can rapidly diffuse into cells and block eukaryotic ribosomes, leading to inhibition of the translation system. Conversely, the effects of DON and NIV mycotoxins on bacteria remain unclear. We employed RNA-seq technology to obtain information regarding the biological responses of bacteria and putative bacterial mechanisms of resistance to DON and NIV mycotoxins. Most differentially expressed genes down-regulated in response to these mycotoxins were commonly involved in phenylalanine metabolism, glyoxylate cycle, and cytochrome o ubiquinol oxidase systems. In addition, we generated an overall network of 1028 up-regulated genes to identify core genes under DON and NIV conditions. The results of our study provide a snapshot view of the transcriptome of Escherichia coli K-12 under DON and NIV conditions. Furthermore, the information provided herein will be useful for development of methods to detect DON and NIV.

Topics: Escherichia coli; Fusarium; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Mycotoxins; RNA, Bacterial; Sequence Analysis, RNA; Trichothecenes

Fusarium mycotoxins occur worldwide in foods such as cereals and animal forages, leading to acute and chronic exposures in human and animals. Intestinal epithelial cells (IECs) are an important first target site for these dietary toxins. This study investigated the cytotoxicity of four common Fusarium mycotoxins, deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEA) and fumonisin B1 (FB1) on a normal porcine jejunal epithelial cell line, IPEC-J2. A dose response relationship between individual mycotoxins and cell viability (MTT assay) was initially investigated, and subsequently cytotoxic and non-cytotoxic concentrations were selected to investigate combinations of two, three and all four of the mycotoxins. For individual mycotoxins, a dose response was observed with cell viability, such that the potency ranking was NIV>DON>ZEA>FB1. At cytotoxic doses of individual mycotoxins, all mixtures gave reduced cell viability compared to control. At noncytotoxic concentrations of individual mycotoxins, all mixtures were cytotoxic with DON-NIV, DON-ZEA, DON-NIV-FB1, DON-ZEA-FB1, NIV-ZEA-FB1 and all four mixed causing the greatest loss of cell viability. The latter observation in particular raises concerns over safety margins based on single toxin species, and suggests that the effects of multiple complex mixtures need to be better understood to assess health risks.

Topics: Animals; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Drug Synergism; Epithelial Cells; Fumonisins; Jejunum; Mycotoxins; Swine; Trichothecenes; Zearalenone

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

Wheat is often infected by Fusarium species producing mycotoxins, which may pose health risks to humans and animals. Deoxynivalenol (DON) is the most important Fusarium toxin in Swedish wheat and has previously been shown to be produced mainly by Fusarium graminearum. However, less is known about the co-occurrence of DON and F. graminearum with other toxins and Fusarium species in Sweden. This study examined the distribution of the most important toxigenic Fusarium species and their toxins in winter wheat (2009 and 2011) and spring wheat (2010 and 2011). DNA from seven species was quantified with qPCR and the toxin levels were quantified with a multitoxin analysis method based on liquid chromatography/electrospray ionisation-tandem mass spectrometry (HPLC/ESI-MS/MS). The method enabled detection of many fungal metabolites, including DON, zearalenone (ZEA), nivalenol (NIV), T-2 toxin, HT-2 toxins, moniliformin (MON), beauvericin (BEA), and enniatins (ENNs). It was found that Fusarium poae and Fusarium avenaceum were present in almost all samples. Other common Fusarium species were F. graminearum and F. culmorum, present in more than 70% of samples. Several species occurred at lower DNA levels in 2011 than in other years, but the reverse was true for F. graminearum and Fusarium langsethiae. The most prevalent toxins were ENNs, present in 100% of samples. DON was also common, especially in spring wheat, whereas ZEA and NIV were common in 2009 and in winter wheat, but less common in 2011 and in spring wheat. Only three samples of spring wheat contained T-2 or HT-2 above LOQ. Annual mean levels of several mycotoxins were significantly lower in 2011 than in other years, but the reverse applied for DON. The strongest correlations between mycotoxin and Fusarium DNA levels were found between F. avenaceum and ENNs (r(2) = 0.67) and MON (r(2) = 0.62), and F. graminearum and DON (r(2) = 0.74). These results show that several Fusarium species and toxins co-occur in wheat. The highest toxin levels were detected in spring wheat and DON and ENNs, the latter belonging to the group of so called "emerging toxins", which were the most prevalent toxins and those occurring at the highest levels.

Topics: Chromatography, High Pressure Liquid; Cyclobutanes; Depsipeptides; DNA, Fungal; Food Contamination; Fusarium; Real-Time Polymerase Chain Reaction; Sweden; T-2 Toxin; Tandem Mass Spectrometry; Trichothecenes; Triticum; Zearalenone

Fusarium moulds frequently contaminate oats and other cereals world-wide, including those grown in Northern Europe. To investigate the presence of toxigenic Fusarium species and their toxins in oats, samples were taken during 2010 and 2011 in three geographical regions of Sweden (east, west, south). The samples were analysed by real-time PCR for the specific infection level of seven Fusarium species associated with oats and other cereals (Fusarium poae, Fusarium graminearum, Fusarium langsethiae, Fusarium culmorum, Fusarium tricinctum, Fusarium sporotrichioides and Fusarium avenaceum) and with a multi-mycotoxin method based on liquid chromatography/electrospray ionisation-tandem mass spectrometry (HPLC/ESI-MS/MS) for the detection of many fungal metabolites, including deoxynivalenol (DON), zearalenone (ZEA), nivalenol (NIV), T-2 toxin, HT-2 toxins, moniliformin (MON), beauvericin (BEA) and enniatins (ENNs). Most samples contained at least four of the seven Fusarium species analysed and F. poae, F. langsethiae and F. avenaceum were present in approximately 90-100% of all samples. The most common toxins detected were DON, NIV, BEA and ENNs, which were present in more than 90% of samples. Most Fusarium species and their toxins occurred in higher concentrations in 2010 than in 2011, with the exception of DON and its main producer F. graminearum. Significant regional differences were detected for some moulds and mycotoxins, with higher levels of F. graminearum, DON and ZEA in western Sweden than in the east (P<0.05) and higher levels of F. tricinctum and MON in the south (P<0.05). Correlation analysis showed significant correlations between many Fusarium species and toxin levels. For example, F. tricinctum was significantly correlated to F. avenaceum (r = 0.72, P<0.001), DON to ZEA (r = 0.52, P<0.001), DON to F. graminearum (r = 0.77, P<0.001) and the sum of T-2 and HT-2 to F. langsethiae (r = 0.77, P<0.001). The multi-toxin approach employed allowed simultaneous detection of many Fusarium mycotoxins in each sample. In combination with real-time PCR analysis of seven toxigenic Fusarium spp., the results gave an overall picture of the presence of Fusarium and their toxins in Swedish oats and revealed significant annual and regional differences. This is the first study of the so-called emerging mycotoxins (e.g., ENNs, MON and BEA) in oats grown in Sweden.

Topics: Avena; Chromatography, High Pressure Liquid; Cyclobutanes; Depsipeptides; DNA, Fungal; Edible Grain; Food Contamination; Fusarium; Geography; Real-Time Polymerase Chain Reaction; Sweden; T-2 Toxin; Tandem Mass Spectrometry; Trichothecenes; Zearalenone

Bio-monitoring of human exposure to mycotoxin has mostly been limited to a few individually measured mycotoxin biomarkers. This study aimed to determine the frequency and level of exposure to multiple mycotoxins in human urine from Cameroonian adults. 175 Urine samples (83% from HIV-positive individuals) and food frequency questionnaire responses were collected from consenting Cameroonians, and analyzed for 15 mycotoxins and relevant metabolites using LC-ESI-MS/MS. In total, eleven analytes were detected individually or in combinations in 110/175 (63%) samples including the biomarkers aflatoxin M1, fumonisin B1, ochratoxin A and total deoxynivalenol. Additionally, important mycotoxins and metabolites thereof, such as fumonisin B2, nivalenol and zearalenone, were determined, some for the first time in urine following dietary exposures. Multi-mycotoxin contamination was common with one HIV-positive individual exposed to five mycotoxins, a severe case of co-exposure that has never been reported in adults before. For the first time in Africa or elsewhere, this study quantified eleven mycotoxin biomarkers and bio-measures in urine from adults. For several mycotoxins estimates indicate that the tolerable daily intake is being exceeded in this study population. Given that many mycotoxins adversely affect the immune system, future studies will examine whether combinations of mycotoxins negatively impact Cameroonian population particularly immune-suppressed individuals.

Topics: Adolescent; Adult; Biomarkers; Cameroon; Environmental Exposure; Environmental Monitoring; Feeding Behavior; Female; Food Contamination; Fumonisins; Glucuronides; Humans; Male; Middle Aged; Mycotoxins; No-Observed-Adverse-Effect Level; Ochratoxins; Surveys and Questionnaires; Trichothecenes; Young Adult; Zearalenone

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

Soybean (Glycine max L.), the main source of protein throughout the world, is used both as a food and a feedstuff. Currently, limited information about the occurrence of Fusarium species and mycotoxins in soybean grain and by-products is available. The aims of the present study were: (1) to identify toxigenic Fusarium species associated with soybean during crop reproductive stages; (2) to determine the occurrence of deoxynivalenol (DON) and nivalenol (NIV) in soybean seeds; (3) to determine the genotype and chemotype of selected Fg complex strains using molecular and chemical analysis, respectively; and (4) to characterize the strains using AFLP(s) markers. One soybean field located at Córdoba Province, Argentina, was monitored and samples of soybean tissue were harvested at three reproductive stages: flowering (R2), full seed (R6) and full maturity (R8). A total of 389 Fusarium strains F. equiseti (40%) was the most frequently species recovered followed by F. semitectum (27%) and F. graminearum (Fg) (11%). From the 40 soybean samples analysed, only two presented detectable DON levels. Based on DON occurrence on soybean seeds at ripening stages, the toxigenic ability of Fg complex strains isolated from soybean seeds, pods and flowers were analysed. The trichothecene genotype was determined by a multiplex PCR using primers based on Tri3, Tri5 and Tri7 toxin genes and then the chemotype was verified by chemical analysis. Most Fg complex strains showed 15-ADON genotype and five strains presented a DON/NIV; these also produced both toxins under in vitro culture. Neither the NIV nor the 3-ADON genotypes were detected among the members of the population evaluated. All the 15-ADON genotype strains were characterized as F. graminearum sensu stricto (lineage 7), while the strains presented a DON/NIV genotype were characterized as F. meridionale (lineage 2). The present study contributes new information on the occurrence of Fusarium species and trichothecenes toxins on soybean at the pre-harvest stages. Also, this is the first report on the chemotype, genotype and lineages among Fg complex isolated from soybean.

Topics: Argentina; Fusarium; Gene Expression Regulation, Fungal; Genotype; Glycine max; Seeds; Trichothecenes

Fusarium graminearum (Gibberella zeae) is an important pathogen of wheat, maize, barley, and rice in South Korea, and harvested grain often is contaminated with trichothecenes such as deoxynivalenol and nivalenol. In this study, we examined 568 isolates of F. graminearum collected from maize at eight locations in South Korea. We used amplified fragment length polymorphisms (AFLPs) to identify four lineages (2, 3, 6, and 7); lineage 7 was the most common (75%), followed by lineage 6 (12%), lineage 3 (12%), and lineage 2 (1%). The genetic identity among populations was high (>0.98), and the effective migration rate between locations was higher than that between lineages. Female fertility varied by lineage: all lineage 7 isolates were fertile, while 70%, 26%, and 14% of the isolates in lineages 6, 3, and 2, respectively, were fertile. All lineage 3 and lineage 7 isolates produced deoxynivalenol, whereas most lineage 2 and 6 isolates produced nivalenol. Genotypic diversity in lineage 3 and lineage 6 populations is similar to that found in previously described Korean rice populations, but genotypic diversity in lineage 7 is much lower, even though similar levels of gene flow occur between lineage 7 populations. We conclude that lineage 7 was relatively recently introduced into South Korea, perhaps accompanying imported maize seeds.

Topics: Amplified Fragment Length Polymorphism Analysis; Fungal Proteins; Fusarium; Gene Flow; Genetic Variation; Genetics, Population; Genotype; Mycotoxins; Plant Diseases; Republic of Korea; Sequence Analysis, DNA; Trichothecenes; Zea mays

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

To investigate the effects of 3 mycotoxins, deoxynivalenol (DON), nivalenol (NIV) and T-2 toxin, in the presence and absence of selenium (Se) on the metabolism of tissue-engineered cartilage to mimic conditions found in Kashin-Beck disease (KBD) environments.. Chondrocytes were seeded onto bone matrix gelatin (BMG) to construct engineered cartilage. The 3 toxins were added to the culture media for 3 weeks followed by immunhistochemical analyses of collagens type II and X, aggrecan, matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3), MMP inhibitors 1 and 3 (TIMP-1 and TIMP-3) and α(2) macroglobulin (α2M).. Type II collagen was decreased while type X collagen was increased in response to DON, NIV and T-2 toxin. Aggrecan was reduced by all 3 mycotoxins. Compared with the control, the 3 toxins decreased the expression of α2M, TIMP-1 and TIMP-3, and increased the expression of MMP-1 and MMP-3. Se could partially inhibit the effects of DON, NIV and T-2 toxins.. Under the low Se condition, the 3 mycotoxins produced procatabolic changes in cartilage resulting in the loss of aggrecan and type II collagen and promoted a hypertrophic phenotype of chondrocytes characterized by increasing type-X-collagen expression, enhancing the expression of MMPs, while weakening the TIMPs. Se could partially block the effects mentioned above. These results support the hypothesis that the combination of mycotoxin stress and Se deficiency would be the causative factors for KBD.

Topics: Aggrecans; Biomarkers; Cells, Cultured; Chondrocytes; Collagen Type II; Dose-Response Relationship, Drug; Drug Therapy, Combination; Humans; Kashin-Beck Disease; Mycotoxins; Selenium; T-2 Toxin; Tissue Engineering; Trichothecenes

This study aimed to investigate mycotoxin contamination of cereal grain commodities for feed and food production in North Western Europe during the last two decades, including trends over time and co-occurrence between toxins, and to assess possible effects of climate on the presence of mycotoxins. For these aims, analytical results related to mycotoxin contamination of cereal grain commodities, collected in the course of national monitoring programmes in Finland, Sweden, Norway and the Netherlands during a 20-year period, were gathered. Historical observational weather data, including daily relative humidity, rainfall and temperature, were obtained from each of these four countries. In total 6382 records, referring to individual sample results for mycotoxin concentrations (one or more toxins) in cereal grains were available. Most records referred to wheat, barley, maize and oats. The most frequently analysed mycotoxins were deoxynivalenol, 3-acetyl-deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin and zearalenone. Deoxynivalenol had the highest overall incidence of 46%, and was mainly found in wheat, maize and oats. Mycotoxins that showed co-occurrence were: deoxynivalenol and 3-acetyl-deoxynivalenol in oats; deoxynivalenol and zearalenone in maize and wheat; and T-2 toxin and HT-2 toxin in oats. The presence of both deoxynivalenol and zearalenone in wheat increased with higher temperatures, relative humidity and rainfall during cultivation, but the presence of nivalenol was negatively associated with most of these climatic factors. The same holds for both nivalenol and deoxynivalenol in oats. This implies that climatic conditions that are conducive for one toxin may have a decreasing effect on the other. The presence of HT-2 toxin in oats showed a slight decreasing trends over time, but significant trends for other toxins showed an increasing presence during the last two decades. It is therefore useful to continue monitoring of mycotoxins. Obtained results can be used for development of predictive models for presence of mycotoxins in cereal grains.

Topics: Acetylation; Agriculture; Animals; Climate Change; Crops, Agricultural; Edible Grain; Food Contamination; Fusarium; Humans; Mycotoxins; Netherlands; Scandinavian and Nordic Countries; Seeds; Spatio-Temporal Analysis; T-2 Toxin; Trichothecenes; Weather; Zearalenone

Mycotoxins are secondary metabolites produced by fungi that can cause adverse health effects. Due to climate change, temperatures are expected to rise and changes in rainfall patterns are foreseen. These developments may increase fungal occurrence and mycotoxin concentrations in maize. It is therefore useful to monitor mycotoxin levels in maize and record the accompanying agronomic factors and weather parameters. This paper describes a field survey in the Netherlands in which information on soil, cultivar, green manure, tillage as well as sowing, emergence, flowering and harvest dates of silage maize were collected from 148 growers. A small number of these growers (42 in total) were visited to collect maize samples revealing that 50% of the samples were contaminated with Fusarium species and mycotoxins were detected in 25% of the samples. The Fusarium species that was most commonly found was F. crookwellense followed by F. graminearum, F. culmorum, F. sporotrichiodes and F. equiseti. In total 31 mycotoxins were analysed. The predominant mycotoxins present were (sum of 3 and 15)-acetyl-DON and nivalenol; other mycotoxins found were alternariol, beauvericin, deoxynivalenol, diacetoxyscirpenol, moniliformin and zearalenone. Nivalenol was present in concentrations up to 1670 µg kg⁻¹ and acetylated DON was usually present at higher concentrations than DON. Statistical analysis of the current data showed no correlation between mycotoxins present and agronomic factors recorded. Field studies as described in this paper are useful and need to be continued in the future in order to observe trends in mycotoxin occurrence.

Topics: Acetylation; Agriculture; Animals; Climate Change; Crops, Agricultural; Environmental Monitoring; Food Safety; Fungi; Humans; Mycotoxins; Netherlands; Seeds; Silage; Soil; Species Specificity; Surveys and Questionnaires; Trichothecenes; Zea mays

To evaluate LC methods with UV or MS detection for simultaneous analysis of deoxynivalenol (DON) and nivalenol (NIV) in wheat, an interlaboratory study was conducted in 11 laboratories. DON and NIV were purified using a multifunctional column, and their concentrations were determined using LC-UV or LC-MS(/MS). No internal standards were used. Three fortified wheat samples (0.1, 0.5 and 1 mg/kg), one naturally contaminated wheat sample, and one blank wheat sample were used. The recoveries ranged from 90% to 110% for DON and from 76% to 83% for NIV. For DON, the relative standard deviations for repeatability (RSDr) ranged from 1.1% to 7.6%. The relative standard deviations for reproducibility (RSDr) ranged from 7.2% to 25.2%. For NIV, the RSDr ranged from 2.0% to 10.7%, and the RSDr ranged from 7.0% to 31.4%. Regardless of sample and detector, the HorRat values for DON and NIV ranged from 0.4 to 1.4. Both LC-UV and LC-MS(/MS) methods were considered to be suitable for application as an official method.

Topics: Chromatography, Liquid; Food Analysis; Food Contamination; Mass Spectrometry; Trichothecenes; Triticum; Ultraviolet Rays

A rapid, sensitive and selective analytical method was developed for the quantitative determination of deoxynivalenol (DON) and nivalenol (NIV) in cereals intended for human and animal consumption. The method, based on liquid chromatography and fluorescence detection, involves an automated 2 channel post-column derivatization, performed with sodium hydroxide, methyl acetoacetate and ammonium acetate. The chromatographic separation was accomplished using a C18 column eluted in isocratic mode with a mixture of 0.01% acetic acid and acetonitrile. Optimal fluorescence detection was obtained by an excitation and emission wavelength of 360 nm and 470 nm, respectively. The sample preparation required a rapid extraction of mycotoxins with water and a purification step by hydrophilic-lipophilic balance column clean-up. Under the optimized experimental conditions, a complete separation of DON and NIV was obtained in less than 20 min. The on-line post-column derivatization ensures excellent results in terms of simplicity and sensitivity, with limits of detection down to 0.014 mg/kg. The proposed method was extensively validated and the analytical performances of linearity (correlation coefficient of 0.9998), selectivity, precision (intra-day precision lower than 8%) and recovery (ranging from 89% to 101%) were evaluated, demonstrating the method feasibility in accurate confirmation analyses.

Topics: Calibration; Chromatography, Reverse-Phase; Edible Grain; Fluorometry; Food Analysis; Humans; Reproducibility of Results; Trichothecenes

Type B trichothecenes, deoxynivalenol (DON) and nivalenol (NIV), are secondary metabolites of Fusarium species and are major pollutants in food and feed products. Recently, the production trend of their derivatives, 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), and 4-acetylnivalenol (4-AcNIV or fusarenon-X), has been changing in various regions worldwide. Although in vivo behavior has been reported, it is necessary to acquire more detailed information about these derivatives. Here, the yeast PDR5 mutant was used for toxicity evaluation, and the growth test revealed that DON, 15-AcDON, and 4-AcNIV had higher toxicity compared to 3-AcDON and NIV. 15-AcDON exerted the most significant gene expression changes, and cellular localization clustering exhibited repression of mitochondrial ribosomal genes. This study suggests that the toxicity trends of both DON products (DON and its derivatives) and NIV products (NIV and its derivatives) are similar to those observed in mammalian cells, with a notable toxic response to 15-AcDON.

Topics: Food Contamination; Fusarium; Gene Expression; Mutation; Mycotoxins; Oligonucleotide Array Sequence Analysis; Saccharomyces cerevisiae; Structure-Activity Relationship; Transcriptome; Trichothecenes

To elucidate the mechanisms underlying the toxicities of the trichothecene mycotoxins deoxynivalenol and nivalenol, their effects on the secretion of anti-hematopoietic chemokines, macrophage inflammatory protein-1α (MIP-1α) and MIP-1β in human promyelocytic leukemia cell line HL60 were investigated. Exposure to deoxynivalenol for 24h significantly induced the secretion of chemokines. The induction of these chemokines may account for the leukopenia after exposure to trichothecene mycotoxins. Treatment with nivalenol decreased the secretion of these chemokines. Our finding that deoxynivalenol induces the secretion of these chemokines, whereas nivalenol has the opposite effect, clearly indicates that the toxicity mechanisms of deoxynivalenol and nivalenol differ.

Topics: HL-60 Cells; Humans; Macrophage Inflammatory Proteins; Mycotoxins; Trichothecenes

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

The integrity of the gastrointestinal tract represents a crucial first level defence against ingested toxins. Among them, Nivalenol is a trichotecenes mycotoxin frequently found on cereals and processed grains; when it contaminates human food and animal feed it is often associated with another widespread contaminant, Deoxynivalenol. Following their ingestion, intestinal epithelial cells are exposed to concentrations of these trichothecenes high enough to cause mycotoxicosis. In this study we have investigated the effects of Nivalenol and Deoxynivalenol on intestinal cells in an in vitro model system utilizing the non-tumorigenic rat intestinal epithelial cell line IEC-6. Both Nivalenol and Deoxynivalenol (5-80 µM) significantly affected IEC-6 viability through a pro-apoptotic process which mainly involved the following steps: (i) Bax induction; (ii) Bcl-2 inhibition, and (iii) caspase-3 activation. Moreover, treatment with Nivalenol produced a significant cell cycle arrest of IEC-6 cells, primarily at the G(0)/G(1) interphase and in the S phase, with a concomitant reduction in the fraction of cells in G(2). Interestingly, when administered at lower concentrations (0.1-2.5 µM), both Nivalenol and Deoxynivalenol affected epithelial cell migration (restitution), representing the initial step in gastrointestinal wound healing in the gut. This reduced motility was associated with significant remodelling of the actin cytoskeleton, and changes in expression of connexin-43 and focal adhesion kinase. The concentration range of Nivalenol or Deoxynivalenol we have tested is comparable with the mean estimated daily intake of consumers eating contaminated food. Thus, our results further highlight the risks associated with intake of even low levels of these toxins.

Topics: Actin Cytoskeleton; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Cycle Checkpoints; Cell Line; Cell Movement; Cell Survival; Connexin 43; Epithelial Cells; Focal Adhesion Protein-Tyrosine Kinases; Gap Junctions; Intestinal Mucosa; Intestines; Mycotoxins; Proto-Oncogene Proteins c-bcl-2; Rats; Trichothecenes

To clarify the underlying mechanisms of IgA nephropathy (IgAN) induced by nivalenol (NIV), a trichothecene mycotoxin, we examined the time and dose relationships of glomerular deposition of IgA by NIV in BALB/c mice (Experiment 1), and also evaluated the modification of NIV on spontaneous IgAN in an inbred murine model, a high IgA strain (HIGA), during its early stage of pathogenesis (Experiment 2). In Experiment 1, female BALB/c mice were given a diet containing 0, 12, or 24 ppm concentration of NIV for 4 or 8 weeks. An increase in serum IgA levels was found at 24 ppm from 4 weeks. At week 8 of treatment, dose-dependent increases in serum IgA levels and glomerular deposition of IgA and IgG were observed without accompanying histopathological glomerular changes. On the other hand, in Experiment 2, control HIGA mice exhibited rather high levels of serum IgA as compared with BALB/c mice from 4 weeks of experiment as well as glomerular deposition of IgA and IgG and mesangial proliferation as revealed at week 8. NIV at 24ppm further increased serum IgA in this strain; however, it did not enhance glomerular immunoglobulin deposition or histopathological lesion. These results suggest that NIV-induced increase of serum IgA levels may be primarily responsible for glomerular immunoglobulin deposition; however, NIV does not enhance glomerular IgA deposition that may lead to exacerbation of predisposed IgAN in the short term, irrespective of the further elevation of serum IgA from the high basal levels.

Topics: Animals; Dose-Response Relationship, Drug; Female; Fluorescent Antibody Technique, Indirect; Glomerular Mesangium; Glomerulonephritis, IGA; Immunoglobulin A; Immunoglobulin G; Mice; Mice, Inbred BALB C; Trichothecenes

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

The occurrence of mycotoxins in small grain cereals and their retention in final products are serious concerns for food safety. Previously, we investigated the fate of deoxynivalenol and nivalenol in a Japanese soft red winter wheat cultivar during milling and we found that deoxynivalenol and/or nivalenol was readily distributed among flours for human consumption. In the present study, we analyzed the ergosterol concentrations in the milling fractions as an index of fungal biomass to elucidate the relationship between deoxynivalenol/nivalenol accumulation and fungal invasion into the grain, after the in-house validation of an analytical method for quantifying ergosterol in the resulting milling fractions (patent flour, low-grade flour, bran, and shorts). Using three samples with different levels of deoxynivalenol and/or nivalenol contamination, the contents of deoxynivalenol/nivalenol and ergosterol in the resulting milling fractions were evaluated. The concentration of ergosterol was always lowest in patent flour and highest in bran or shorts, indicating that most of the fungi is retained in the outer layers of grain (bran and shorts) even in highly contaminated grain. On the other hand, the concentrations of deoxynivalenol and nivalenol were similar in the low-grade and patent flours and only slightly lower than in the medium-level and high-level contaminated grains. Moreover, the percentage distribution of ergosterol was higher in bran than in other fractions in all cases, which differed from that of deoxynivalenol/nivalenol. This result indicates the diffusion of deoxynivalenol/nivalenol inside the grain that is independent of fungal invasion.

Topics: Ergosterol; Food Microbiology; Fusarium; Trichothecenes; Triticum

Crop rotations with putative non-host crops such as sugar beet are often recommended to reduce Fusarium head blight (FHB) in cereals. However, recent observations have shown pathogenic, endophytic, and saprotrophic colonization of sugar beet with various Fusarium spp. Therefore, strains of seven species frequently isolated from sugar beet were tested for pathogenicity on wheat. Species-specific symptoms on heads and kernels were evaluated and the grains were analyzed for 20 mycotoxins with liquid chromatography-tandem mass spectrometry. Fusarium graminearum, F. culmorum, and F. cerealis from sugar beet caused typical FHB symptoms and mycotoxin contamination with deoxynivalenol and nivalenol, while a high incidence of black point was observed in heads inoculated with F. tricinctum or F. equiseti. Black point kernels revealed 3.4 to 14.5 times higher mycotoxin concentrations than symptomless grains, containing enniatin B1 at 38,000 μg/kg, moniliformin at 4,900 μg/kg, and 2-amino-14,16-dimethyloctadecan-3-ol at 5,500 μg/kg, as well as monoacetoxyscirpenol at 2,600 μg/kg and nivalenol at 3,800 μg/kg. Monitoring of these latter two species in the field is hampered by the lack of typical head symptoms after infection. In further experiments, the impact of sugar beet residues on FHB severity and the correlation between mycotoxin contamination of cereal lots and the amount of black point have to be evaluated.

Topics: Beta vulgaris; Cyclobutanes; Depsipeptides; Edible Grain; Fusarium; Mycotoxins; Plant Diseases; Plant Leaves; Plant Roots; Species Specificity; Sphingolipids; Trichothecenes; Triticum

Rapid and sensitive methods to detect Fusarium culmorum and trichothecene and zearalenone producing strains in food and feed are valuable in predicting potential contamination. In this study the effectiveness of primers, recommended in the literature, for species identification of F. culmorum and basic genes encoding for mycotoxin production was tested. A total of 68 isolates of F. culmorum were collected from cereals and potato between 2005 and 2008 from different Polish provinces. It was shown that from among the four primer pairs enabling the identification of F. culmorum, and therefore also to establish its presence in the material, only primers Fc01F/Fc01R seem to be fully effective in the case of Polish strains. Determination of material contamination by F. culmorum, however, is only a first step in determining food safety. It is also extremely important to identify genes encoding the potential ability to produce mycotoxins. It was shown that three pairs of primers (tox5-1/tox5-2, HATriF/HATriR and Tri5F/Tri5R) enable a fully effective identification of the presence of the Tri5 gene responsible for producing trichothecenes. Determination of the DON-chemotype, and thus identification of the strains of F. culmorum potentially producing deoxynivalenol, is enabled equally by MinusTri7F/MinusTri7F, Tri7F/Tri7DON and Tri13F/Tri13DONR. However, a determination of the NIV-chemotype, and thus identification of the strains potentially producing nivalenol, is enabled by Tri7F/Tri7R, Tri7F/Tri7NIV and Tri13NIVF/Tri13R. The potential ability of isolates to produce ZEA can be determined to the same degree in assay with PKS4-PS.1/PKS4-PS.2 and F1/R1.

Topics: Base Sequence; DNA Primers; DNA, Fungal; Food Contamination; Food Microbiology; Fusarium; Genes, Fungal; Mycotoxins; Polymerase Chain Reaction; Species Specificity; Trichothecenes; Zearalenone

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

A total of 201 samples of brown rice, polished rice, and two types of by-products, blue-tinged rice and discolored rice, were collected from rice stores maintained at 51 rice processing complexes in Korea. These samples were analyzed for the presence of Fusarium mycotoxins such as deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEA). Contaminants (and their ranges) found in discolored rice samples were DON (59 to 1,355 ng g(-1)), NIV (66 to 4,180 ng g(-1)), and ZEA (25 to 3,305 ng g(-1)); those found in blue-tinged (less-ripe) rice were DON (86 to 630 ng g(-1)), NIV (50 to 3,607 ng g(-1)), and ZEA (26 to 3,156 ng g(-1)). Brown rice samples were contaminated mostly with NIV and ZEA (52 to 569 ng g(-1) and 47 to 235 ng g(-1), respectively). Polished rice samples were largely free from mycotoxins, although one sample was contaminated with NIV (77 ng g(-1)). When the fungal flora associated with each rice sample was investigated, blue-tinged rice was the most often contaminated with Fusarium graminearum (3.8%), followed by the discolored rice (2.4%) and brown rice (1.6%) samples. Using PCR, toxin genotyping of 266 isolates of F. graminearum revealed that most isolates (96%) were NIV producers. In conclusion, this survey is the first report of the cocontamination of Korean rice and its by-products with trichothecenes and ZEA. Importantly, it also provides new information on the natural contamination of rice by Fusarium mycotoxins.

Topics: Consumer Product Safety; Food Contamination; Food Microbiology; Fusarium; Humans; Korea; Mycotoxins; Oryza; Prevalence; Trichothecenes; Zearalenone

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

Maize is frequently infected by the Fusarium species producing mycotoxins. Numerous investigations have focused on grain maize, but little is known about the Fusarium species in the entire plant used for silage. Furthermore, mycotoxins persist during the ensiling process and thus endanger feed safety. In the current study, we analyzed 20 Swiss silage maize samples from growers' fields for the incidence of Fusarium species and mycotoxins. The species spectrum was analyzed morphologically and mycotoxins were measured by LC-MS/MS. A pre-harvest visual disease rating showed few disease symptoms. In contrast, the infection rate of two-thirds of the harvest samples ranged from 25 to 75% and twelve different Fusarium species were isolated. The prevailing species were F. sporotrichioides, F. verticillioides and F. graminearum. No infection specificity for certain plant parts was observed. The trichothecene deoxynivalenol (DON) was found in each sample (ranging from 780 to 2990 µg kg(-1)). Other toxins detected in descending order were zearalenone, further trichothecenes (nivalenol, HT-2 and T-2 toxin, acetylated DON) and fumonisins. A generalized linear regression model containing the three cropping factors harvest date, pre-precrop and seed treatment was established, to explain DON contamination of silage maize. Based on these findings, we suggest a European-wide survey on silage maize.

Topics: Chromatography, Liquid; Food Contamination; Fumonisins; Fusarium; Mycotoxins; Silage; T-2 Toxin; Tandem Mass Spectrometry; Trichothecenes; Zea mays; Zearalenone

Nivalenol (NIV) and Deoxynivalenol (DON), mycotoxins of the trichothecene family are considered very common food contaminants. In this work, we investigated whether the immunotoxic effects ascribed to these trichothecenes may be mediated by perturbations in the activity of dendritic cells (DCs). Murine bone marrow-derived DCs were used to evaluate the effects of NIV and DON on the LPS-induced maturation process. We found that the expression of the class II MHC and of the accessory CD11c molecules, but not of the costimulatory CD86 marker, was down-regulated by NIV and DON exposure in LPS-treated DCs, as well as nitric oxide (NO) production. Interestingly, NIV, but not DON, induced DC necrosis. Moreover, the analysis of the cytokine pattern showed that IL-12 and IL-10 expressions induced by LPS exposure were suppressed by both trichothecenes in a dose-dependent fashion. On the other hand, the secretion of the proinflammatory cytokine TNF-alpha was increased as a direct consequence of DON and NIV exposure. Taken together, our data indicated that the immunotoxicity of NIV and DON was related to the capacity of both trichothecenes to interfere with phenotypic and functional features of maturing DCs.

Topics: Animals; Bone Marrow Cells; CD11c Antigen; Cell Differentiation; Cell Survival; Cells, Cultured; Cytokines; Dendritic Cells; Dose-Response Relationship, Drug; Flow Cytometry; Genes, MHC Class II; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mycotoxins; Necrosis; Nitric Oxide; Trichothecenes

Deoxynivalenol (DON) and nivalenol (NIV), trichothecene mycotoxins, are secondary metabolites produced by Fusarium fungi. Trichothecene mycotoxins cause immune dysfunction, thus leading to diverse responses to infection. The present study evaluated the effect of DON and NIV on nitric oxide (NO) production by RAW264 cells stimulated with lipopolysaccharide (LPS). LPS-induced NO production was reduced in the presence of these toxins. The transcriptional activation and expression of inducible NO synthase (iNOS) by LPS were also repressed by these toxins. DON or NIV inhibited LPS-induced expression of interferon-beta (IFN-beta), which plays an indispensable role in LPS-induced iNOS expression. These results indicate that DON and NIV inhibit the LPS-induced NO and IFN-beta production, which both play an important role for host protection against invading pathogens, and suggests that the inhibition of these factors may be involved in the immunotoxic effects of these mycotoxins.

Topics: Animals; Cell Line; Fusarium; Interferon-beta; Lipopolysaccharides; Macrophages; Mice; Mycotoxins; Nitric Oxide; Nitric Oxide Synthase; Trichothecenes

Fusarium graminearum, a known producer of trichothecene mycotoxins in cereal hosts, has been recently documented as a cause of dry rot of potato tubers in the United States. Due to the uncertainty of trichothecene production in these tubers, a study was conducted to determine the accumulation and diffusion of trichothecenes in potato tubers affected with dry rot caused by F. graminearum. Potato tubers of cv. Russet Burbank were inoculated with 14 F. graminearum isolates from potato, sugar beet, and wheat and incubated at 10 to 12 degrees C for 5 weeks to determine accumulation of trichothecenes in potato tubers during storage. Twelve of the isolates were classified as deoxynivalenol (DON) genotype and two isolates were as nivalenol (NIV) genotype. Trichothecenes were detected only in rotted tissue. DON was detected in all F. graminearum DON genotype isolates up to 39.68 microg/ml in rotted potato tissue. Similarly, both NIV genotype isolates accumulated NIV in rotted potato tissue up to 18.28 microg/ml. Interestingly, isolates classified as genotype DON accumulated both DON and NIV in the dry rot lesion. Potato tubers were then inoculated with two isolates of F. graminearum chemotype DON and incubated up to 7 weeks at 10 to 12 degrees C and assayed for DON diffusion. F. graminearum was recovered from >53% of the isolations from inoculated tubers at 3 cm distal to the rotted tissue after 7 weeks of incubation but DON was not detected in the surrounding tissue. Based in this data, the accumulation of trichothecenes in the asymptomatic tissue surrounding dry rot lesions caused by F. graminearum is minimal in cv. Russet Burbank potato tubers stored for 7 weeks at customary processing storage temperatures.

Topics: Fusarium; Genotype; Mycotoxins; Plant Diseases; Solanum tuberosum; Trichothecenes

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

A surface plasmon resonance (SPR) immunoassay using a monoclonal antibody was developed to measure nivalenol (NIV) and deoxynivalenol (DON) contamination in wheat. A highly sensitive and stable DON-immobilized sensor chip was prepared, and an SPR detection procedure was developed. The competitive inhibition assay used a monoclonal antibody that cross-reacts with NIV and DON. The half maximal inhibitory concentration (IC(50)) values of the SPR assay were 28.8 and 14.9 ng mL(-1) for NIV and DON, respectively. The combined responses of NIV and DON in wheat were obtained using a simultaneous detection assay in a one-step cleanup procedure. NIV and DON were separated using a commercial DON-specific immunoaffinity column (IAC) and their responses were obtained using an independent detection assay. Spiked tests using these toxins revealed that recoveries were in the range 91.5-107% with good relative standard deviations (RSDs) (0.40-4.1%) and that detection limits were 0.1 and 0.05 mg kg(-1) for NIV and DON, respectively. The independent detection using IAC showed detection limits of 0.2 and 0.1 mg kg(-1) for NIV and DON, respectively. SPR analysis results were correlated with those obtained using a conventional LC/MS/MS method for wheat co-contaminated with NIV and DON. These results suggested that the developed SPR assay is a practical method to rapidly screen the NIV and DON co-contamination of wheat and one of a very few immunoassays to detect NIV directly.

Topics: Animals; Antibodies, Monoclonal; Cattle; Immunoassay; Serum Albumin, Bovine; Surface Plasmon Resonance; Trichothecenes; Triticum

The manner in which deoxynivalenol (DON) and nivalenol (NIV) accumulation progresses in wheat grain infected with Fusarium graminearum and the influence of the time of infection on the accumulation of toxins were investigated. Four cultivars were tested in a greenhouse environment, where the plants were spray inoculated at three different stages with a mixture of DON and NIV chemotypes of F. graminearum. The results indicate that high levels of DON and NIV can be produced beyond 20 days after anthesis (DAA), even by early infection. The results of field experiments performed on seven cultivars, where inoculation was conducted using colonized maize kernel inoculum, were consistent with the greenhouse results. In addition, in the greenhouse experiments, late infection, at least as late as 20 DAA, caused grain contamination with these toxins even without clear disease symptoms on the spike. These results indicate the importance of the late stage in grain development in DON and NIV contamination, suggesting that control strategies that cover the late as well as the early stage of grain development should be established to effectively reduce the risk of these toxins' contaminating wheat.

Topics: Fusarium; Host-Pathogen Interactions; Mycotoxins; Plant Diseases; Seeds; Trichothecenes; Triticum

The fate of the Fusarium mycotoxins deoxynivalenol and nivalenol during the milling of Japanese wheat cultivars artificially infected with Fusarium was investigated. Grain samples with different mycotoxin concentrations were milled using a laboratory-scale test mill to produce eight fractions: three breaking flours (1B, 2B, and 3B), three reduction flours (1M, 2M, and 3M), wheat bran, and wheat shorts. Patent flour for human consumption was made from the 1B, 2B, 1M, and 2M flours, and low-grade flour was made from 3B and 3M flours. The four resulting samples (patent flour, low-grade flour, bran, and shorts) were analyzed for deoxynivalenol and/or nivalenol with an in-house validated analytical method using high-performance liquid chromatography with UV absorbance detection. In samples with different mycotoxin concentrations, the distribution of those toxins differed among the milling fractions. Grains with a lower level of contamination produced bran and shorts samples with a high relative concentration of nivalenol. A high percentage of nivalenol was found in patent flour, followed by bran. Contrary to the less-contaminated sample, the concentration of nivalenol in moderately contaminated grain was high only in the shorts sample. The highest percentage of deoxynivalenol and nivalenol was observed in the patent flour. The results of this study indicate that the distribution of deoxynivalenol and nivalenol in milled Japanese wheat could be influenced by the contamination level of the original grain, and the milling process is not always effective for removal of toxins from wheat grains.

Topics: Chromatography, High Pressure Liquid; Consumer Product Safety; Flour; Food Contamination; Food Handling; Fusarium; Humans; Trichothecenes; Triticum

To elucidate the dietary exposure of Chinese populations to deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN).. Ten Fusarium toxins including DON, NIV, ZEN in domestic wheat flour and corn-based products harvested and collected in 2009 were analyzed by UPLC-MS/MS. Dietary intake assessments of human exposure to DON, NIV and ZEN were carried out in combination of national food consumption data with toxin concentration data by deterministic estimate method.. (1) There are 2.5% adults and 10% children with the dietary exposure to DON exceeding the tolerable daily intake (TDI) on the basis of the average food consumption. At the 75th percentile food consumption level, the dietary exposure of populations to DON was higher than its TDI, 1.72 and 2.02 times (adults) as well as 1.19 and 1.09 times higher than TDI (children), respectively, based on the higher DON exposure (adults : P90 for wheat flour and P97. 5 for corn-based products, children: P50 for wheat flour and P75 for corn-based products). At the average toxin concentration, children with the high consumption level (90th, 97. 5th and 99th percentile) of either wheat flour or corn-based products, the dietary exposure to DON exceed the TDI, 1.81 to 3.17 times (wheat flour) and 1.47 - 3.97 times (corn-based products) higher than TDI, respectively. The dietary exposure of adults to ZEN exceed the TDI, based on the average food consumption data and higher ZEN level (P99), the 75th consumption data combined with P97. 5th toxin concentration, as well as 90th percentile of both food consumption data and ZEN concentration, respectively. There are 1%, 2.5%, 25%, 25% and 50% populations of children exposed ZEN higher than TDI, respectively at the 50th, 75th, 90th, 97. 5th and 99th percentile of food consumption data. (2) The level of concern (LOC) of DON in wheat flour calculated with high consumption data (90th, 97. 5th and 99th percentile) for all populations as well as DON and ZEN in corn-based products calculated with high consumption data (adults: 99th percentile, children: 97. 5th and 99th percentile) were lower than their average concentrations in above cereals. (3) No matter adults or children, the maximum daily safe intake of both wheat flour and corn-based products with high concentration of DON (97. 5th and 99th percentile, and 90th else for children) were lower than their own average food consumption data. For adults, the maximum daily safe intake of corn-based products with high ZEN contamination level (99th percentile) was lower than the average amount of corn-based products consumption. While, the maximum daily safe intake of both wheat flour and corn-based products derived from high ZEN concentration (97.5th and 99th percentile) for children were lower than their own average food consumption.. Children dietary exposure to DON, NIV and ZEN was higher than adults. Children are the populations at the high risk of dietary exposure to these three mycotoxins. The risk to health caused by long-term consumption of wheat flour and corn-based products heavily contaminated with such high concentration of DON and ZEN was relatively high. The present tolerance limit for DON and ZEN in foods implemented in China should be revised on the basis of assessment results.

Topics: Adult; Child; China; Diet; Edible Grain; Environmental Exposure; Food Contamination; Food Microbiology; Fusarium; Humans; Mycotoxins; T-2 Toxin; Trichothecenes; Zearalenone

Fusarium graminearum is an important fungal pathogen of cereal crops and produces mycotoxins, such as the trichothecenes nivalenol and deoxynivalenol. This species may be subdivided into a series of genetic lineages or phylogenetic species. We identified strains of F. graminearum from the Republic of Korea to lineage, tested their ability to produce nivalenol and deoxynivalenol, and determined the genetic composition and structure of the populations from which they were recovered. Based on amplified fragment length polymorphism (AFLP), PCR genotyping, and chemical analyses of trichothecenes, all 249 isolates from southern provinces belonged to lineage 6, with 241 having the nivalenol genotype and 8 having the deoxynivalenol genotype. In the eastern Korea province, we recovered 84 lineage 6 isolates with the nivalenol genotype and 23 lineage 7 isolates with the deoxynivalenol genotype. Among 333 lineage 6 isolates, 36% of the AFLP bands were polymorphic, and there were 270 multilocus haplotypes. Genetic identity among populations was high (>0.972), and genotype diversity was low (30 to 58%). To test the adaptation of lineage 6 to rice, conidial mixtures of strains from lineages 3, 6, and 7 were inoculated onto rice plants and then recovered from the rice grains produced. Strains representing lineages 6 and 7 were recovered from inoculated spikelets at similar frequencies that were much higher than those for the strain representing lineage 3. Abundant perithecia were produced on rice straw, and 247 single-ascospore isolates were recovered from 247 perithecia. Perithecia representing lineage 6 (87%) were the most common, followed by those representing lineage 7 (13%), with perithecia representing lineage 3 not detected. These results suggest that F. graminearum lineage 6 may have a host preference for rice and that it may be more fit in a rice agroecosystem than are the other lineages present in Korea.

Topics: Amplified Fragment Length Polymorphism Analysis; Cluster Analysis; DNA Fingerprinting; DNA, Fungal; Fusarium; Genetic Variation; Genotype; Korea; Mycotoxins; Oryza; Trichothecenes

Nivalenol (NIV) and deoxynivalenol (DON) are trichothecenes mycotoxins produced by Fusarium fungi that occur in cereal grains alone or in combination. Several studies have shown that exposure to high concentrations of these mycotoxins resulted in decreased cell proliferation; however, the molecular mechanism underlying their activities are still partially known. In this study, we evaluated the effects of NIV and DON, alone and in combination, on J7741.A macrophages viability. The results of the current study show that both NIV and DON (10-100 microM) significantly stimulate apoptosis in J774A.1 macrophages in a concentration-dependent manner; in particular, NIV results a stronger pro-apoptotic effect than DON on cultured J774A.1 murine macrophages. No interactive effects were observed by exposing J774A.1 cells to both NIV and DON simultaneously. Pro-apoptotic activity induced by both mycotoxins seems to be essentially mediated by caspase-3 and is associated with a cell cycle blocking in G0/G1 phase. Moreover, our results show that NIV and DON are able to influence apoptotic pathway by ERK, pro-apoptotic protein Bax, caspase-3 and poly-ADP-ribose synthase (PARP), DNA repairing enzyme.

Topics: Animals; Apoptosis; Blotting, Western; Cell Cycle; Cell Line; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Interactions; Macrophages; Mice; Mycotoxins; Trichothecenes

This study examined a total of 82 consignments of French and Argentinean raw maize as received at maize mills in the UK between 2004 and 2007. Samples were analysed for deoxynivalenol (DON), nivalenol (NIV), other trichothecenes, zearalenone (ZON), and fumonisins B(1), B(2), and B(3) (FB(1), FB(2), and FB(3)) using fully validated analytical methods with limits of quantification of 10 microg kg(-1) for DON, NIV, and each fumonisin mycotoxin and 3 microg kg(-1) for ZON. All samples except two containing fumonisins met the European Commission statutory maximum permissible levels for DON, ZON, and FB(1) + FB(2) as operating in 2007. The maximum concentrations found for DON, NIV, ZON, and FB(1) + FB(2) were 444, 496, 165 and 5002 microg kg(-1), respectively. Fumonisins were detected in almost every sample with 65% of Argentinean maize containing more than 1000 microg kg(-1) of FB(1) + FB(2). In contrast, ZON was not detectable in almost 50% of consignments. During this period there was a distinct difference in mycotoxin concentrations between harvests and geographic origin. Flint maize from Argentina usually contained lower concentrations of DON and related trichothecenes and higher levels of fumonisins than maize from France, although concentrations of fumonisins up to 2000 microg kg(-1) or greater occurred in samples from both regions. The incidence and concentrations of fumonisins were similar to those in a similar previous survey, while zearalenone concentrations were lower. The distribution of mycotoxins in multi-hold ships was also investigated showing that fumonisins were much more evenly distributed than DON, thus indicating their general level in the ship as a whole. The effect of cleaning regimes was found to be very variable, especially for DON, ranging from no removal of mycotoxins to greater than 50% in some instances, but was not related to concentration. Evidence here suggests that while cleaning is essential for removing foreign bodies before milling, it cannot be used as a reliable tool for reducing mycotoxins.

Topics: Animal Feed; Animals; Argentina; Food Contamination; Food Handling; France; Fumonisins; Fusarium; Humans; Mycotoxins; Trichothecenes; United Kingdom; Zea mays; Zearalenone

Every year between 2002 and 2005 approximately 100 samples of oats from fields of known agronomy were analysed by GC/MS for 10 trichothecenes: deoxynivalenol (DON), nivalenol, 3-acetylDON, 15-acetylDON, fusarenone X, T-2 toxin (T2), HT-2 toxin (HT2), diacetoxyscirpenol, neosolaniol and T-2 triol. Samples were also analysed for moniliformin and zearalenone by HPLC. Of the 10 trichothecenes analysed from 458 harvest samples of oat only three, 15-acetylDON, fusarenone X and diacetoxyscirpenol, were not detected. Moniliformin and zearalenone were absent or rarely detected, respectively. HT2 and T2 were the most frequently detected fusarium mycotoxins, present above the limit of quantification (10 microg kg(-1)) in 92 and 84% of samples, respectively, and were usually present at the highest concentrations. The combined mean and median for HT2 and T2 (HT2 + T2) was 570 and 213 microg kg(-1), respectively. There were good correlations between concentrations of HT2 and all other type A trichothecenes detected (T2, T2 triol and neosolaniol). Year and region had a significant effect on HT2 + T2 concentration. There was also a highly significant difference between HT2 + T2 content in organic and conventional samples, with the predicted mean for organic samples five times lower than that of conventional samples. This is the largest difference reported for any mycotoxin level in organic and conventional cereals. No samples exceeded the legal limits for DON or zearalenone in oats intended for human consumption. Legislative limits for HT2 and T2 are currently under consideration by the European Commission. Depending on the limits set for unprocessed oats intended for human consumption, the levels detected here could have serious consequences for the UK oat-processing industry.

Topics: Agriculture; Analysis of Variance; Avena; Food Contamination; Fusarium; Humans; Mycotoxins; Seeds; T-2 Toxin; Trichothecenes; United Kingdom

Fusarium species, a plant pathogenic fungus of wheat and other cereals, produces toxic metabolites such as nivalenol (NIV) and deoxynivalenol (DON). Control of contamination by these toxins is very difficult, and a continuous survey of the occurrence is necessary for these toxins. Thus, the accurate and convenient determination of the cereals contaminated with these toxins is important for the supply of safe foods. A selective analytical method based on high-performance liquid chromatography, combined with atmospheric pressure photoionization (APPI) mass spectrometry, has been developed for simultaneous determination of NIV and DON. The parameters investigated for the optimization of APPI were the ion source parameters fragmentor voltage, capillary voltage, and vaporizer temperature, and also mobile phase composition and flow rate. Furthermore, chemical noise and signal suppression of analyte signals due to sample matrix interference were investigated for APPI. The results indicated that APPI provides lower matrix effect and the correlation coefficient of NIV and DON in the range 0.2-100 ng x mL(-1) was above 0.999. Recoveries of NIV and DON in wheat ranged from 86 to 107% and limits of detection of NIV and DON were 0.20 ng x g(-1) and 0.39 ng x g(-1), respectively. In addition, the proposed method was applied for the analysis of naturally contaminated wheat samples. APPI was found to offer lower matrix effect and was a convenient technique for routine analysis of NIV and DON residues in wheat at trace levels.

Topics: Chromatography, High Pressure Liquid; Mass Spectrometry; Mycotoxins; Trichothecenes; Triticum

Fusarium graminearum trichothecene producing isolates can be broadly divided into two chemotypes based on the production of the 8- ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). Functional Tri13 gene required for the production of NIV and 4- acetyl NIV, whereas in the isolates producing DON and its acetylated derivates, this gene is nonfunctional. In this study, a total of 57 isolates from different fields of Mazandaran province, Iran were identified as F. graminearum using classical methods and species specific primers. In order to assess the potential of isolates to produce NIV or DON, we used PCR to determine whether isolates carried a functional or nonfunctional Tri13 gene. Out of the 57 tested F. graminearum isolates with Tri13 PCR assays, 46 yielded an amplicon similar to the size predicted for nivalenol production, while 11 yielded an amplicon similar to the size predicted for deoxynivalenol production. From regions where more than one F. graminearum isolate was obtained, isolates were not exclusively of a single chemotype. It seems that genetic diversity among the isolates has relation with geographical region and wheat cultivar. The assay can provide information about the distribution of Tri13 haplotype that can be used in tracing of trichothecene contaminated samples.

Topics: Agriculture; Fusarium; Genes, Fungal; Iran; Plant Diseases; Polymerase Chain Reaction; Trichothecenes; Triticum

The fungus Fusarium graminearum (sexual stage Gibberella zeae) causes ear rot of maize (Zea mays) and contamination with the 8-ketotrichothecenes nivalenol (1) or 4-deoxynivalenol (2), depending on diversity of the fungal population for the 4-oxygenase gene (TRI13). To determine the importance of 1 and 2 in maize ear rot, a survey of naturally contaminated maize in Nepal was combined with experiments in the field and in a plant growth room. In the survey, 1 contamination was 4-fold more frequent than 2 contamination and 1-producers (TRI13) were isolated more than twice as frequently as 2-producers (Psi TRI13). In maize ear rot experiments, genetically diverse 1-producers and 2-producers caused ear rot and trichothecene contamination. Among strains with the same genetic background, however, 1-producers caused less ear rot and trichothecene contamination than did 2-producers. The high frequency of 1 contamination and the high virulence of many 1-producers are of concern because maize is a staple food of rural populations in Nepal and because 1 has proven to be more toxic than 2 to animals.

Topics: Fruit; Fungal Proteins; Fusarium; Mycotoxins; Nepal; Oxygenases; Phylogeny; Plant Diseases; Trichothecenes; United States; Zea mays

The in vitro effects of four Fusarium toxins, fumonisin B(1) (FB(1)), alpha-zearalenol (alpha-ZEA), nivalenol (NIV) and deoxynivalenol (DON), on mitogen-induced cell proliferation were determined in swine whole-blood cultures. Considering the lack of sufficient toxicological data both on single and in combination effects, in vitro studies may contribute to risk assessment of these toxins. Incubation with increasing concentrations of FB(1) did not produce any consequence on proliferation; in contrast alpha-ZEA, NIV and DON showed an inhibitory effect. Dose-response curves for each mycotoxin were generated. NIV was found to be the most potent toxin followed by DON and alpha-ZEA. The effects of both FB(1)+alpha-ZEA and NIV+DON mixtures were also analysed to investigate possible interactions. The results indicated that combination of FB(1)+alpha-ZEA produces a synergistic inhibition of porcine cell proliferation; whereas there is no interaction between DON and NIV on porcine whole-blood proliferation, at tested concentrations.

Topics: Animals; Dose-Response Relationship, Drug; Drug Synergism; Fumonisins; Lymphocyte Activation; Male; Swine; Trichothecenes; Zeranol

To investigate possible co-occurrences of type B trichothecenes and zearalenone within a Fusarium culmorum-infected wheat harvest lot, kernels were fractionated into six groups by visual criteria. The Fusarium-damaged kernels were subdivided into white, shrunken, and red kernel groups, and the remaining kernels were sorted into healthy, black spotted, and nonspecific groups. The distribution patterns of nivalenol, deoxynivalenol, zearalenone, and ergosterol were determined for possible correlations. Significant correlations between the distribution patterns were found for the mycotoxins and ergosterol for the grouped kernels (r = 0.997-0.999, p < 0.0001). Additionally, remarkably outstanding levels of nivalenol (24-fold more than the mean at 1.16 mg/kg), deoxynivalenol (27-fold more than the mean at 0.16 mg/kg), zearalenone (25-fold more than the mean at 77 microg/kg), and ergosterol (17-fold more than the mean at 13.4 mg/kg) were found in the red kernel group. Further, detailed mycotoxin and ergosterol analyses were carried out on various segments (kernel surface, conidia, bran, and flour) of the red kernels. However, the mycotoxin and ergosterol distribution profiles revealed nonsignificant correlations for these kernel segments, with the exception of deoxynivalenol and nivalenol, which were moderately correlated (r = 0.948, p = 0.035).

Topics: Ergosterol; Fusarium; Mycotoxins; Plant Diseases; Seeds; Trichothecenes; Triticum; Zearalenone

Topics: Animals; Fumonisins; Humans; Jurkat Cells; Lymphocytes; Mycotoxins; Respiration; Swine; Trichothecenes; Zeranol

Three Fusarium species: F. graminearum, F. culmorum and F. cerealis were identified in laboratory cultures and in sporodochia from spikelets of scabby wheat. SCAR (sequence characterized amplified region) primers were used to identify Fusarium species and nivalenol (NIV) and deoxynivalenol (DON) chemotypes within species in laboratory cultures and field collected heads harvested in 2006. Results from PCR analyses confirmed preliminary identifications of species on the basis of examination of macroconidia under a light microscope and identification of cultures on agar media. NIV and DON (3Ac-DON and 15Ac-DON) chemotypes were identified using PCR assay. Among samples and isolates of F. graminearum, the 15Ac-DON chemotype dominated, and among those where F. culmorum was identified, the 3Ac-DON chemotype prevailed. Only 5 of the 41 isolates of F. graminearum tested, displayed the NIV chemotype. An increase in the frequency of F. graminearum and a decrease in the frequency of F. culmorum were found during 1998 to 2006.

Topics: Fusarium; Mycoses; Plant Diseases; Poland; Polymerase Chain Reaction; Trichothecenes; Triticum

A sensitive, accurate and precise method for the simultaneous determination of nivalenol (NIV), deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in different food matrices, including wheat, maize, barley, cereal-based infant foods, snacks, biscuits and wafers, has been developed. The method, using liquid chromatography coupled with atmospheric pressure chemical ionization triple quadrupole mass spectrometry (LC-APCI-MS/MS), allowed unambiguous identification of the selected trichothecenes at low microg per kg levels in such complex food matrices. A clean-up procedure, based on reversed phase SPE Oasis HLB columns, was used, allowing good recoveries for all studied trichothecenes. In particular, NIV recoveries significantly improved compared to those obtained by using Mycosep #227 columns for clean-up of the extracts. Limits of detection in the various investigated matrices ranged 2.5-4.0 microg kg(-1) for NIV, 2.8-5.3 microg kg(-1) for DON, 0.4-1.7 microg kg(-1) for HT-2 and 0.4-1.0 microg kg(-1) for T-2. Mean recovery values, obtained from cereals and cereal products spiked with NIV, DON, HT-2 and T-2 toxins at levels from 10 to 1000 microg kg(-1), ranged from 72 to 110% with mean relative standard deviation lower than 10%. A systematic investigation of matrix effects in different cereals and cereal products was also carried out by statistically comparing the slopes of standard calibration curve with matrix-matched calibration curve for each of the four toxins and the eight matrices tested. For seven of the eight matrices tested, statistically significant matrix effects were observed, indicating that, for accurate quantitative analysis, matrix-matched calibration was necessary. The method was applied to the analysis of 57 samples of ground wheat originated from South Italy and nine cereal food samples collected from retail markets.

Topics: Bread; Chromatography, Liquid; Edible Grain; Food Contamination; Fusarium; Hordeum; Humans; Infant; Infant Food; T-2 Toxin; Tandem Mass Spectrometry; Trichothecenes; Triticum; Zea mays

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

In the European Union, deoxynivalenol in cereals and cereal products is controlled by recent legislation with the objective of minimizing consumer exposure to this mycotoxin. Relatively few studies have examined the loss of Fusarium mycotoxins during processing and whether this is accurately reflected by the processing factors. The behaviour of deoxynivalenol, nivalenol and zearalenone during extrusion of naturally contaminated wholemeal wheat flour has been examined using pilot-scale equipment. Factors examined were temperature and moisture content. Concentrations of the three mycotoxins were little changed by extrusion although the amount of deoxynivalenol decreased at the lowest moisture content. However, this effect did not appear to be temperature-dependent, suggesting that the apparent loss is either due to binding or inability to extract the residue. Under some conditions, concentrations of the mycotoxins, particularly nivalenol, were higher after extrusion.

Topics: Edible Grain; Flour; Food Contamination; Food Industry; Fusarium; Humidity; Mycotoxins; Temperature; Trichothecenes; Triticum; Zearalenone

One hundred and fifty-six samples of breakfast cereals were collected from the Canadian retail marketplace over a 3-year period. The samples were analysed for the mycotoxins deoxynivalenol, nivalenol, HT-2 toxin, zearalenone, ochratoxin A, and fumonisins B1 and B2 to contribute to dietary exposure estimates in support of the development of Canadian guidelines for selected mycotoxins in foods. The samples included corn-, oat-, wheat- and rice-based cereals, as well as mixed-grain cereals, and were primarily from North American processors. Overall, deoxynivalenol was the most frequently detected mycotoxin--it was detected in over 40% of all samples analysed. Fumonisins and ochratoxin A were each detected in over 30% of all samples. Zearalenone was detected in over 20% of all samples. Nivalenol and HT-2 toxin were each detected in only one sample. The survey clearly demonstrated regular occurrence of low levels of multiple mycotoxins in breakfast cereals on the Canadian market.

Topics: Canada; Edible Grain; Environmental Monitoring; Food Contamination; Fumonisins; Fusarium; Mycotoxins; Ochratoxins; T-2 Toxin; Trichothecenes; Zearalenone

Cell transformation assays using BALB/3T3 cells can mimic the two-stage process of chemical carcinogenesis in experimental animals. A short-term transformation assay using v-Ha-ras-transfected BALB/3T3 cells (Bhas 42 cells), which was developed by Ohmori et al. and modified by Asada et al., has been reported to detect both tumor initiators and promoters as transformation initiators and promoters, respectively, with their differences based on their protocols. In this new short-term assay, we examined mycotoxins derived from Fusarium and related substances for the initiation and promotion activities of the transformation. The tested substances included deoxynivalenol, nivalenol, fusarenon-X, T-2 toxin, fumonisin B(1), fumonisin B(2), zearalenone, alpha-zearalanol, beta-zearalanol, alpha-zearalenol and beta-zearalenol. Fumonisin B(1) and T-2 toxin were positive for promoting activity in the assay. Especially, T-2 toxin was active at concentrations as low as 0.001-0.002microg/mL in the culture medium. From a comparison between the results of this study and published carcinogenicity assay data, it was expected that the Bhas 42 cell transformation assay had a good correlation with the two-stage carcinogenicity tests using experimental animals for estimation of the tumor-promoting activity.

Topics: Animals; BALB 3T3 Cells; Carcinogenicity Tests; Cell Survival; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Fumonisins; Fusarium; Genes, ras; Mice; Mycotoxins; T-2 Toxin; Transfection; Trichothecenes; Zearalenone

This paper presents results from the European Commission-funded project Doncalibrant, the objective of which was to produce calibrators with certified mass fractions of the Fusarium toxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-Ac-DON), 15-acetyldeoxynivalenol (15-Ac-DON), and nivalenol (NIV), in acetonitrile. The calibrators, available in ampoules, were sufficiently homogeneous, with between-bottle variations (s (bb)) of less than 2%. Long-term stability studies performed at four different temperatures between -18 and 40 degrees C revealed no significant negative trends (at a confidence level of 95%). Molar absorptivity coefficients (in L mol(-1) cm(-1)) were determined for all four toxins (DON: 6805 +/- 126, NIV: 6955 +/- 205, 3-Ac-DON: 6983 +/- 141, 15-Ac-DON: 6935 +/- 142) on the basis of a mini-interlaboratory exercise. The overall uncertainty of the calibrators' target values for DON and NIV were evaluated on the basis of gravimetric preparation data and include uncertainty contributions from possible heterogeneity, storage, and transport. The Doncalibrant project resulted in the production of calibrators for DON (IRMM-315) and NIV (IRMM-316) in acetonitrile with certified mass fractions of 25.1 +/- 1.2 microg g(-1) and 24.0 +/- 1.1 microg g(-1), respectively. Both CRMs became commercially available from the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium) at the beginning of 2007.

Topics: Automation; Calibration; Chemistry Techniques, Analytical; Chromatography, Liquid; Fusarium; Models, Chemical; Models, Statistical; Reproducibility of Results; Spectrophotometry, Ultraviolet; Temperature; Trichothecenes

Pearl millet is increasingly being grown as a premium-value grain for the recreational wildlife and poultry industries in the southern US. We conducted three experiments to assess grain mold development in storage conditions typically encountered in the region of production. Variables included production year, temperature, relative humidity, atmosphere, and grain moisture content. In the first experiment, grain was stored for 9 weeks at 20 or 25 degrees C and maintained at 86% or 91% relative humidity (r.h.). In the second experiment, grain was stored for 9 weeks at 20 or 25 degrees C in either air (aerobic) or N2 (anaerobic), and maintained at 100% r.h. In the third experiment, high-moisture grain was stored for 3 weeks at 20 or 25 degrees C and maintained at 100% r.h. Grain was sampled at weekly intervals and plated to determine changes in fungal frequency. Fungi isolated included Fusarium chlamydosporum (19% of grain), Curvularia spp. (14%), F. semitectum (16%), Alternaria spp. (9%), Aspergillus flavus (8%), "Helminthosporium"-type spp. (6%), and F. moniliforme sensu lato (3%). Year of grain production significantly affected isolation frequency of fungi. Isolation frequencies from low-moisture grain were rarely affected by temperature, relative humidity, or atmosphere treatments, but was affected by storage duration for some fungi. Changes in isolation of toxigenic fungi occurred in high-moisture grain. Isolation frequency of F. chlamydosporum increased in grain stored at 86% and 91% r.h. Incidence of A. flavus increased in high-moisture grain treatments, particularly at 25 degrees C. Incidence of deoxynivalenol was not affected by storage treatment. Low concentrations of nivalenol were detected in most grain incubated at 100% r.h. Zearalenone was detected only when grain moisture content was 20-22%. Aflatoxin contamination averaged 174 ng g(-1) over all treatments, and increased up to 798 ng g(-1) in high-moisture grain at stored at 25 degrees C.

Topics: Aerobiosis; Aflatoxins; Anaerobiosis; Ascomycota; Aspergillus; Food Microbiology; Fungi; Fusarium; Humidity; Mycotoxins; Pennisetum; Temperature; Time Factors; Trichothecenes; Zearalenone

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

Several studies have shown that the mycotoxins T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON) and nivalenol (NIV) affect lymphocyte functioning. However, the molecular mechanisms underlying the immunomodulatory effects of these trichothecenes are not defined yet. In this study, the potency of the type A trichothecenes T-2 toxin and DAS, and the type B trichothecenes DON (and its metabolite de-epoxy-deoxynivalenol; DOM-1) and NIV to reduce mitochondrial activity and to induce apoptosis of Jurkat T cells (human T lymphocytes) were examined. T-2 toxin and DAS are much more cytotoxic at low concentrations than DON and NIV as shown by the AlamarBlue cytotoxicity assay. In addition, the mechanism whereby DON and NIV induced cytotoxicity is mainly via apoptosis as we observed phosphatidylserine externalization, mitochondrial release of cytochrome c, procaspase-3 degradation and Bcl-2 degradation. In contrast, type A trichothecenes reduce the mitochondrial activity at approximately 1000-fold lower concentrations than the type B trichothecenes, resulting in necrosis. These data suggest that the mechanisms resulting in cytotoxic effects are different for type A and type B trichothecenes.

Topics: Apoptosis; Blotting, Western; Flow Cytometry; Humans; Jurkat Cells; Mitochondria; Trichothecenes

The ability to rapidly distinguish trichothecene chemotypes in a given species/population of the genus Fusarium is important due to significant differences in the toxicity of these secondary metabolites. A multiplex PCR assay, based on primer pairs derived from the Tri3, Tri5 and Tri7 genes of the trichothecene gene cluster was established for the identification of the different chemotypes among Fusarium graminearum, F. culmorum and F. cerealis. Using the selected primers, specific amplification products of 625, 354 and 708 bp were obtained from Fusarium isolates producing nivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, respectively. Moreover, the multiplex PCR was successfully used to identify the chemotype of the Fusarium species contaminating wheat kernels. Four picograms of fungal DNA were found to be necessary to obtain a visible amplification product.

Topics: DNA, Fungal; Fungal Proteins; Fusarium; Mycological Typing Techniques; Plant Diseases; Polymerase Chain Reaction; Trichothecenes; Triticum

Nivalenol is a mycotoxin produced by certain fungi that are pathogenic to important cereal crops, in particular maize, wheat, and barley. This toxin, 3alpha,4beta,7alpha,15-tetrahydroxy-12,13-epoxytrichothec-9-en-8-one, is found worldwide and is closely related to 4-deoxynivalenol (DON or vomitoxin), a mycotoxin associated with outbreaks of Fusarium head blight in North America. The literature on the toxicity of nivalenol suggests it is similar, if not more toxic, than DON. Despite the development of rapid immunologically based assays for detecting DON, such assays have not existed for detecting nivalenol without chemical modification of the analyte. This paper describes the development of a monoclonal antibody using a nivalenol-glycine protein conjugate. The monoclonal antibody was most specific for an acetylated form of DON (3-Ac-DON), but it exhibited sensitivity and cross-reactivity that were useful for detecting nivalenol and DON at relevant levels without the need to modify either toxin chemically. In an competitive indirect ELISA format, the concentrations of toxins able to inhibit colour development by 50% (IC50) were 1.7, 15.8, 27.5, 68.9, and 1740 ng ml(-1) for the mycotoxins 3-Ac-DON, DON, nivalenol, 15-Ac-DON, and fusarenon-X, respectively. The antibody was also used to develop a competitive direct ELISA for DON and nivalenol, with IC50's of 16.5 ng ml(-1) (DON) and 33.4 ng ml(-1) (nivalenol). These assays are capable of detecting both DON and nivalenol simultaneously, a property that may be useful in regions where these toxins co-occur or in formats, such as immunoaffinity columns, where co-isolation of both toxins is desirable.

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Antibody Specificity; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Female; Glycine; Immunotoxins; Mice; Mice, Inbred BALB C; Mycotoxins; Solvents; Trichothecenes

The occurrence of mycotoxins in barley, sorghum, teff (Eragrostis tef) and wheat from Ethiopia has been studied. Samples were analyzed for aflatoxin B(1) (AFB1), ochratoxin A (OTA), deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) using high performance liquid chromatography (HPLC) and for fumonisins (FUM) using enzyme linked immunosorbent assay (ELISA). AFB1 and OTA were detected in samples of all the four crops. AFB1 was detected in 8.8% of the 352 samples analyzed at concentrations ranging from trace to 26 microg kg(-1). OTA occurred in 24.3% of 321 samples at a mean concentration of 54.1 microg kg(-1) and a maximum of 2106 microg kg(-1). DON occurred in barley, sorghum and wheat at 40-2340 microg kg(-1) with an overall incidence of 48.8% among the 84 mainly 'suspect' samples analyzed; NIV was co-analyzed with DON and was detected at 40 microg kg(-1) in a wheat sample and at 50, 380, and 490 microg kg(-1) in three sorghum samples. FUM and ZEN occurred only in sorghum samples with low frequencies at concentrations reaching 2117 and 32 microg kg(-1), respectively. The analytical results indicate higher mycotoxin contamination in sorghum, which could be related to the widespread storage of sorghum grain in underground pits leading to elevated seed moisture contents. This is the first report on the occurrence of OTA in teff.

Topics: Aflatoxin B1; Chromatography, High Pressure Liquid; Edible Grain; Ethiopia; Fumonisins; Mycotoxins; Ochratoxins; Trichothecenes; Zearalenone

The occurrence of mycotoxins in agricultural commodities is a major health concern for livestock, humans, and the environment. Barley and subsequently malt quality is of fundamental importance to obtain good quality beer. Classical methods of analysis often require tedious, laborious, and expensive processes. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) instrumentation enables highly sensitive and fast analysis and/or detection using a very small sample. The possibilities of MALDI-TOF MS for he identification and/or detection of trichothecene mycotoxins (deoxynivalenol (DON) and nivalenol (NIV), respectively) in barley malt were examined, and it was found that almost all classical MALDI matrices failed to ionize the compounds being studied. This detailed study of the ionization conditions and the search for unconventional matrices led to the discovery of suitable MALDI matrices, which enable ionization of trichothecene mycotoxins. These were: fine powdered synthetic diamond, sodium azide, or hydrazine hydrate. It is possible to detect 8.5 x 10(-12) mol (2.5 ng) of deoxynivalenol or 64 x 10(-12) mol (20 ng) of nivalenol in just 1 microL of barley malt extract (equivalent to 600 microg of DON in 1 kg of barley malt). The procedure developed enables fast determination of DON and NIV in barley, malt, or similar products.

Topics: Beer; Hordeum; Mycotoxins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trichothecenes

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

Topics: Cell Survival; Cytokines; Gene Expression Regulation; Humans; Jurkat Cells; Mycotoxins; RNA, Messenger; Trichothecenes

Based on the intergenic sequences of Tri5-Tri6 genes involved in the mycotoxin pathways of Fusarium species, a generic PCR assay was developed to detect a 300 bp fragment of deoxynivalenol (DON)-chemotypes and a 360 bp sequence of nivalenol (NIV)- chemotypes of Fusarium graminearum. Mycotoxin chemotypes identified by the PCR assays were confirmed by the chemical analyses of HPLC or GC/MS. Further analysis of 364 F. graminearum isolates from 12 provinces of China showed that 310 were DON-chemotypes and 54 were NIV-chemotypes. Sequence analyses revealed that DON-chemotypes display more variations than NIV-chemotypes. This PCR assay could be used to detect mycotoxin-producing Fusarium-species and may thus help to develop strategies to avoid or reduce mycotoxin contamination of cereals. Also this assay may provide useful alternatives to antibody-based mycotoxin tests.

Topics: Base Sequence; DNA, Ribosomal Spacer; Edible Grain; Fungal Proteins; Fusarium; Mycotoxins; Phylogeny; Polymerase Chain Reaction; Sequence Analysis, DNA; 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

This study reports estimates on dietary exposure from the first French Total Diet Study (FTDS) and compares these estimates with both existing tolerable daily intakes for these toxins and the intakes calculated during previous French studies. To estimate the dietary exposure of the French population to the principal mycotoxins in the French diet (as consumed), 456 composite samples were prepared from 2280 individual samples and analysed for aflatoxins, ochratoxin A, trichothecenes, zearalenone, fumonisins and patulin. Average and high percentile intakes were calculated taking account of different eating patterns for adults, children and vegetarians. The results showed that contaminant levels observed in the foods examined 'as consumed' complied fully with current European legislation. However, particular attention needs to be paid to the exposure of specific population groups, such as children and vegans/macrobiotics, who could be exposed to certain mycotoxins in quantities that exceed the tolerable or weekly daily intake levels. This observation is particularly relevant with respect to ochratoxin A, deoxynivalenol and zearalenone. For these mycotoxins, cereals and cereal products were the main contributors to high exposure.

Topics: Adult; Aflatoxins; Child; Diet; Diet, Vegetarian; Environmental Exposure; Feeding Behavior; Food Contamination; Food Microbiology; France; Fumonisins; Humans; Mycotoxins; Ochratoxins; Patulin; Trichothecenes; Zearalenone

A reliable, sensitive and selective method was developed to determine different Fusarium mycotoxins (trichothecenes Type A and B, zearalenone) simultaneously in cereals and cereal-based samples using liquid chromatography with tandem mass spectrometry (LC-ESI-MS/MS). Sample preparation is based on a standard solvent extraction step followed by two different kinds of solid-phase clean-up procedures: using a multifunctional MycoSep material for trichothecenes and zearalenone. The average recoveries for trichothecenes ranged from 65% for nivalenol (NIV) up to 96% for deoxynivalenol (DON) and 89% for zearalenone (ZON). The limit of quantification varied between 0.02 and 10 ppb for each substance. In addition, a screening survey with 685 samples was carried out to compare contents of T-2 toxin and deoxynivalenol and to investigate potential coherence in contamination pattern.

Topics: Chromatography, Liquid; Edible Grain; Flour; Food, Fortified; Fusarium; Mass Spectrometry; T-2 Toxin; Trichothecenes; Triticum; Zearalenone

Deoxynivalenol (DON) and nivalenol (NIV) are toxic Fusarium secondary trichothecene metabolites that often co-occur regularly in cereal grains. These compounds were compared for their toxicity towards C57BL/6 mice on several parameters including alteration in plasma biochemistry, immune system reactivity and hepatic drug metabolism capacity. Mice received individual or combined oral doses of each toxin: 0.071 or 0.355 mg/kg of body weight, administrated three days a week for 4 weeks. Food consumption was altered by the single administration of 0.355 mg/kg of NIV, although no noticeable change of body and organ weights or liver protein contents was detected. NIV administration did cause also significant changes in total CO2 and uric acid concentrations in plasma. Individual toxin exposures led to increases in plasma IgA without no detectable change in the ex vivo production of cytokine by splenocytes. The liver ethoxyresorufin O-deealkylase, pentoxyresorufin O-depenthylase and glutathione S-transferase activities were increased in concert with cytochrome P4501a and P4502b subfamily expression. Administration of combinations of DON and NIV resulted in responses similar to that observed using individual doses of each toxin. However, depending on the ratio of toxin doses and biochemical parameters, some responses could be also additive (plasma IgA and hepatic DCNB conjugation) or synergistic (plasma uric acid).

Topics: Administration, Oral; Animals; Mice; Mice, Inbred C57BL; Trichothecenes

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON) and fumonisin B1 (FB1) were studied in the K562 human erythroleukemia cell line using the Trypan Blue, MTT and BrdU uptake analyses of cytotoxicity, cell metabolism and cell proliferation, respectively. Nuclear staining with propidium iodide and DNA analysis by flow cytometry were used to identify apoptosis and cell cycle distribution. By the MTT and BrdU tests, both NIV and DON were significantly more toxic than FB1 by at least one order of magnitude, with ID50s ranging from 0.5 microM for NIV to 70 microM for FB1. The MTT test indicated that NIV was significantly (approximately four times) more toxic than DON. In contrast, the Trypan Blue test did not reveal any effects of mycotoxin exposure suggesting that, at the concentrations tested, NIV, DON and FB1 did not induce cytotoxicity through plasma membrane damage. Cell cycle analysis suggested apoptotic cytotoxicity, revealing 100% cellular debris at the highest concentrations of NIV and DON and approximately 2.9 times more debris than control at the highest FB1 concentration. Morphological evidence of apoptosis was related to the toxicity of the substances, such that the more toxic NIV and DON resulted in more late stage apoptotic events than FB1. This study suggests that human blood cells are sensitive to mycotoxin exposure, that NIV is more toxic than DON which is more toxic than FB1, and that DNA damage and apoptosis rather than plasma membrane damage and necrosis may be responsible for the observed cytotoxicity.

Topics: Apoptosis; Bromodeoxyuridine; Cell Cycle; Cell Differentiation; Cell Division; Cell Membrane; Cell Physiological Phenomena; Cell Survival; Cycloheximide; DNA Replication; Dose-Response Relationship, Drug; Flow Cytometry; Fumonisins; Humans; K562 Cells; Leukemia, Erythroblastic, Acute; Mitochondria; Mycotoxins; Tetrazolium Salts; Thiazoles; Trichothecenes; Trypan Blue

Many Fusarium species produce toxic sesquiterpenoids known as trichothecenes, including deoxynivalenol and nivalenol by Fusarium graminearum and T-2 toxin by Fusarium sporotrichioides. These toxins are potent inhibitors of protein synthesis and are a significant agricultural problem due to their adverse affect on human, animal, and plant health. Previously, 10-12 co-regulated orthologous genes within a 26-kb region were identified in F. graminearum and F. sporotrichioides, respectively. A majority of these clustered genes have been shown to be involved in different aspects of trichothecene metabolism including 7 of 15 biosynthetic steps. Three other biosynthetic steps are carried out by genes located elsewhere in the genome. In this study, we sequenced 14-16 kb of DNA on both sides of the core clusters and identified 12 new ORFs in both Fusarium species. Although the predicted functions of some of the new ORFs are consistent with some unassigned biochemical reactions, gene expression and gene deletion studies indicate that none are required for trichothecene biosynthesis. These results provide evidence to demarcate both ends of the core trichothecene gene cluster. Index descriptors: Fungal secondary metabolite, Pathogenic fungi, Gene cluster, Fusarium, Trichothecene, DON

Topics: DNA, Fungal; Fungal Proteins; Fusarium; Gene Deletion; Gene Expression; Gene Order; Genes, Fungal; Molecular Sequence Data; Multigene Family; Mycotoxins; Open Reading Frames; Sequence Analysis, DNA; Sequence Homology; T-2 Toxin; Trichothecenes

The cytotoxicity of the de-epoxy metabolites of trichothecenes nivalenol (NIV) and deoxynivalenol (DON) was determined and compared with the cytotoxicity of the respective toxin with an intact epoxy group and their acetylated derivatives. The cytotoxic effects was determined by using the 5-bromo-2'-deoxyuridine (BrdU) incorporation assay assessing DNA-synthesis. The toxicity of NIV and DON expressed as the concentration inhibiting 50% of the DNA synthesis (IC(50)), was occurring at similar micromolar concentrations (1.19+/-0.06 and 1.50+/-0.34 microM). The toxicity of fusarenon X (4-acetyl NIV) in the assay was similar to the toxicity of NIV, and the toxicity of 15-AcDON was equal to the toxicity of DON. 3-AcDON was less toxic than DON and 15-AcDON. The IC(50) value for de-epoxy DON was 54 times higher in the assay than the IC(50) for DON, while the IC(50) of de-epoxy NIV was 55 times higher than the IC(50) for NIV. The results verify previous findings that the de-epoxidation is a detoxification reaction.

Topics: Acetylation; Animals; Biological Assay; Bromodeoxyuridine; Chromatography, Gas; DNA; Dose-Response Relationship, Drug; Inhibitory Concentration 50; Mice; Mycotoxins; Swiss 3T3 Cells; Trichothecenes

In vitro screening of 14 adsorbent materials, including some commercial products used to detoxify Fusarium-mycotoxins, were tested in the pH range of 3-8 for deoxynivalenol (DON)- and nivalenol (NIV)-binding ability. Only activated carbon showed to be effective with binding capacities of 35.1 micromol and 8.8 micromol DON and NIV/g adsorbent, respectively, calculated from the adsorption isotherms. A dynamic laboratory model simulating the gastrointestinal (GI) tract of healthy pigs (TIM system) was used to evaluate the small-intestinal absorption of DON and NIV and the efficacy of activated carbon in reducing the relevant absorption. The in vitro intestinal absorptions of DON and NIV were 51% and 21%, respectively, as referred to 170 microg DON and 230 microg NIV ingested through contaminated (spiked) wheat. Most absorption occurred in the jejunal compartment for both mycotoxins. The inclusion of activated carbon produced a significant reduction in the intestinal mycotoxin absorption. At 2% inclusion level the absorption with respect to the intake was lowered from 51% to 28% for DON and from 21% to 12% for NIV. The binding activity of activated carbon for these trichothecenes was lower than that observed for zearalenone, a mycotoxin frequently co-occurring with them in naturally contaminated cereals.

Topics: Adsorption; Animals; Charcoal; Food Contamination; Hydrogen-Ion Concentration; In Vitro Techniques; Intestinal Absorption; Intestine, Small; Models, Biological; Swine; Trichothecenes

Various species of Fusarium can produce trichothecene mycotoxins that contaminate food commodities and can represent a risk for human and animal health. In this paper, a full factorial design was applied to study the influence of incubation temperature, water activity (a(w)) and type of isolate on the production of deoxynivalenol (DON), nivalenol (NIV) and 3-acetyldeoxynivalenol (3-AcDON) in corn kernel cultures by three isolates of Fusarium graminearum and three isolates of Fusarium culmorum from crops grown in Spain. The tested temperatures were 15, 20, 28 and 32 degrees C. The a(w)-values were 0.960, 0.970 and 0.980. Moisture of cultures (within the studied range) did not affect significantly production of trichothecenes; however, the temperature affected significantly mycotoxin production and the optimal values were 28, 20 and 15 degrees C for DON, NIV and 3-AcDON, respectively. Four additional isolates of F. graminearum and two additional isolates of F. culmorum were examined for production of these mycotoxins at the optimal temperatures. Of the seven isolates of F. graminearum, four produced DON (0.88-3.97 microg/g), seven produced NIV (1.53-124 microg/g), and three produced 3-AcDON (0.65-10.6 microg/g). Of the five isolates of F. culmorum, four produced DON (1.20-4.93 microg/g), four produced NIV (6.94-701 microg/g), and four produced 3-AcDON (0.83-7.70 microg/g). Practically all isolates seem to belong to the NIV-chemotype. This is the first study done with regard to interaction between strain and ecological variables on type B trichothecene production by isolates of these two species from crops grown in Spain.

Topics: Food Contamination; Food Microbiology; Fusarium; Trichothecenes; Water; Zea mays

This is the first report that a Fusarium toxin nivalenol (NIV) naturally existing at high levels in dietary food in high-risk areas of cancer of esophagus and gastric cardia in China induced benign and malignant tumors in mice. The levels of two Fusarium toxins, nivalenol and deoxynivalenol (DON) were quantitated using high performance liquid chromatography (HPLC) in a total of 97 samples of dietary wheat flour, barley and corn collected from families in two areas with high mortality rate of cancer of esophagus and gastric cardia (132/100,000), Linxian, Henan province and Cixiang, Hepei province, China. The mean level of NIV and DON in three dietary foods was 830+/-927 microg/kg (range 584-1,780 microg/kg) and 4,281+/-6,114 microg/kg (range 732-10,980 microg/kg) respectively. The highest mean level of NIV was 1,780+/-1,705 microg/kg found in barley from Linxian, that of DON was 10,980+/-10,139 microg/kg found in corn from Cixiang. NIV was undetectable in 2 samples of rice from USA. The mean levels of NIV in three main dietary foods in those two high-risk areas were estimated at 400 to 800-fold higher than that in the USA, where NIV was undetectable in dietary food, and the mortality rate of esophageal cancer is <5/100,000 in white Caucasians in the USA, (odds ratio was estimated at 17-34, p<0.000005). These data suggest that Linxian and Cixiang peasants who consumed a diet with high NIV had significantly higher risk for developing esophageal cancer than the US residents who consumed food without or with negligible amounts of NIV. Three repeated experiments were performed using Balb/C mice with inter-mittent application of NIV, alternate with 12-Tetradeconoyl-phorbol-13-acetate (TPA) application on skin. Papillomas and carcinomas developed in a total of 23/49 (47%) mice that survived 11-60 weeks of experiments. Among all the tumors, 4 carcinomas in 3 mice were identified. No tumors were found in the 60 control mice applying either TPA or acetone (solvent) only on skin.

Topics: Animal Feed; Animals; Carcinoma; Cardia; Chromatography, High Pressure Liquid; Chromosome Aberrations; Diet; Esophageal Neoplasms; Female; Flour; Fusarium; Hordeum; Humans; Male; Mice; Mice, Inbred BALB C; Mycotoxins; Neoplasms, Experimental; Odds Ratio; Papilloma; Stomach Neoplasms; Temperature; Trichothecenes; Zea mays

In 1998, a typhoon struck before rice harvesting in Japan, and the unpolished rice was found to be stained brown. Samples were collected and analyzed for the presence of trichothecenes using GC/MS. The mycotoxins deoxynivalenol (DON), fusarenon-X (Fus.-X) and nivalenol (NIV) were detected and confirmed with GC/MS. The quantity of trichothecenes was determined using GC-ECD. To our knowledge, this is the first report of the presence of the trichothecene Fus.-X in rice.

Topics: Gas Chromatography-Mass Spectrometry; Japan; Oryza; Trichothecenes

Fusarium mycotoxins are increasingly studied agronomically, chemically, and pathologically in the context of food safety, as a means of preventing new major health crises. Reliable mycotoxin techniques and sampling procedures are required for assessment of the effects of different sources of variation on grain mycotoxin content in agronomic experiments. Analyses were performed with the aim of formulating guidelines for grain sampling to increase the reliability of grain mycotoxin measurement in agronomic experiments. Two toxins in wheat samples, deoxynivalenol and nivalenol, were targeted. With a nested linear mixed model, it was estimated that the uncertainty of nivalenol determination was low (+/-15 microg/kg), whereas that for deoxynivalenol determination was higher (+/-38 microg/kg). It was also found that grinding of the grain decreased the variability of the results. Moreover, despite the heterogeneity in grain mycotoxin content across a given field, it was shown that heads can be harvested manually for agronomic experiments provided that sampling is representative (evenly distributed over the entire plot area). Finally, delaying the assay until after harvest was found to affect the results obtained and should therefore be avoided.

Topics: Edible Grain; Food Contamination; Food Handling; Fusarium; Sensitivity and Specificity; Trichothecenes

We have developed and tested an enzyme-linked immunosorbent assay system for individual measurement of deoxynivalenol, nivalenol, and T-2 + HT-2 toxin using monoclonal antibodies for 3,4,15-triacetyl-nivalenol, for both 3,4,15-triacetyl-nivalenol and 3,15-diacetyl-deoxynivalenol, and for acetyl-T-2 toxin. The assay system comprised three kits (desinated the DON + NIV kit, the NIV kit, and the T-2 + HT-2 kit). The practical performance of the enzyme-linked immunosorbent assay system was assessed by assaying trichothecene mycotoxins in wheat kernels. The enzyme-linked immunosorbent assay system meets all the requirements for use in a routine assay in terms of sensitivity (detection limit: deoxynivalenol 80 ng/g, nivalenol 80 ng/g, T-2 toxin 30 ng/g), reproducibility (total coefficient of variation: 1.9-6.2%), accuracy (recovery: 93.8-112.0%), simplicity and rapidity (time required: <2 h), mass handling (>42 samples/assay), and a good correlation with gas chromatography-mass spectrometry (r=0.9146-0.9991). Components derived from the wheat extract did not interfere with the assay kits. The enzyme-linked immunosorbent assay system is a useful alternative method to gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, or liquid chromatography-ultraviolet absorption for screening cereals and foods for trichothecene mycotoxin contamination.

Topics: Antibodies, Monoclonal; Enzyme-Linked Immunosorbent Assay; Flour; T-2 Toxin; Trichothecenes; Triticum

Diversity in trichothecene mycotoxin production by 167 isolates of Fusarium graminearum was examined by chemical and molecular methods. Isolates from barley, corn, and wheat grown in Korea produced either deoxynivalenol (DON) or nivalenol (NIV), whereas isolates from corn grown in the United States produced DON only. Southern blotting of MseI-digested genomic DNA's from these isolates was performed using a 0.6-kb fragment of Tri5, a key enzyme for trichothecene production, as a probe. This technique revealed a single-band polymorphism between these isolates, with 1.8- and 2.2-kb bands arising from DON and NIV producers, respectively. The same set of isolates was subjected to previously developed PCR assays using primers derived from Tri7 or Tri13. These assays also revealed a single-band polymorphism between NIV- and DON-producing chemotypes. The polymorphisms at Tri5, Tri7, or Tri13 in all of the US isolates were consistent with their chemotypes as identified by GC-MS. However, for seven Korean isolates, chemical and molecular analyses yielded seemingly inconsistent results. This issue was resolved by Southern blot analysis with the Tri5 probe using two other restriction enzymes and sequence comparison of a 3.8-kb region spanning Tri5. In addition, one of these exceptional isolates was found to carry both DON and NIV chemotype-specific regions, possibly resulting from recombination between the two chemotypes.

Topics: Base Sequence; Fungal Proteins; Fusarium; Hordeum; Molecular Sequence Data; Multigene Family; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Sequence Analysis, DNA; Trichothecenes; Triticum; Zea mays

Information on the contamination of Danish cereals and cereal products with Fusarium toxins is limited and the last survey is from 1984/1985. In the present study, the occurrence of deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin, T-2 toxin and zearalenone (ZON) was investigated in flour of common wheat, durum wheat and rye. The samples were collected from 1998 to 2001 from both mills and the retail market in Denmark. A total of 190 flour samples were analysed for DON and NIV and about 60 samples for HT-2, T-2 toxin and ZON. DON was most frequently detected with an incidence rate of 78% over all samples for all years. The contamination level varied considerably from year to year, and for wheat and rye the highest incidence and DON concentrations were found in samples from the 1998 harvest. There were regular and heavy rainfalls in Denmark during the flowering period of the crops that year, and DON was found in all samples, with mean concentrations in wheat and rye flour of 191 microg kg(-1) (n=14) and 99 microg kg(-1) (n=16), respectively. Comparison of data from each harvest year showed higher contents of DON in samples of wheat (range 20-527 microg kg(-1)) than in rye (20-257 microg kg(-1)). Contents of NIV, HT-2 toxin and ZON in samples of wheat and rye were generally low, and even in positive samples the contents were close to the detection limit of the methods. The T-2 toxin was detected in only a few of the wheat samples and in low amounts. However, the toxin was found in about 50% of the rye samples collected during 1998-2000, with a mean content of 49 microg kg(-1) (n=25). Durum wheat flour showed the highest DON contamination level, and all samples (n=33) collected during 2000 and 2001 contained DON with means and medians above 1100 microg kg(-1). Over 70% of the samples contained more than 500 microg kg(-1) DON, and the highest observed concentration was 2591 microg kg(-1). The concentration of T-2 toxin in durum wheat flour was also high with five of the 10 analysed samples containing more than 100 g kg(-1).

Topics: Denmark; Flour; Food Contamination; Fusarium; Humans; Mycotoxins; Secale; Sweden; T-2 Toxin; Trichothecenes; Triticum; Zearalenone

To determine the effect of interacting conditions of water activity (aw, 0.99-0.85), temperature (15, 25 degrees C) and time (40 days) on growth and production of the mycotoxins deoxynivalenol (DON) and nivalenol (NIV) by Fusarium culmorum on a wheat-based agar medium.. Fusarium culmorum grew optimally at 0.995aw and minimally at 0.90 at both 15 and 25 degrees C. No growth was observed at <0.90aw. Overall, temperature, aw and their interaction had a statistically significant effect on the growth rate of F. culmorum. Production of both DON and NIV were over a much narrower range (0.995-0.95aw) than that for growth. The highest concentrations of DON and NIV levels were produced at 0.995aw and 0.981aw at 25 degrees C, respectively, after 40 days of incubation. Statistically, aw, temperature and incubation time, and aw x temperature and temperature x incubation time had a statistically significant effect on DON/NIV production.. This is the first detailed report on the two-dimensional environmental profiles for DON/NIV production by F. culmorum in the UK.. As part of a hazard analysis critical control point (HACCP) approach, this type of information is critical in monitoring critical control points for prevention of DON/NIV entering the wheat production chain.

Topics: Culture Media; Food Microbiology; Fusarium; Humidity; Mycotoxins; Temperature; Time Factors; Trichothecenes; Triticum

The influence of heating temperature and time on deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEA) contents in naturally co-contaminated barley and wheat was investigated intending to establish the basis for a decontamination model of Fusarium mycotoxins in cereals. The standard toxins and whole barley powder samples were heated in a convection oven at 140, 160, 180, 200, or 220 degrees C, and kernel subsamples (200 g each) were roasted in an experimental rotary gas-fired roaster at 150, 180 or 220 degrees C. All treatments resulted in a time-temperature-dependent decomposition of the toxins; the logarithm of the toxin remaining % presented a linear relationship with heating time. The lines equations were used to estimate the half (H) and decimal (D) decomposition times (time required to destroy 50 or 90% of the toxin, respectively). DON and NIV H and D decomposition times were similar and 50% shorter for heated standards than for whole barley powder. ZEA standard values were considerably longer, while whole barley powder values were comparable with those of DON and NIV. At 220 degrees C, D decomposition times of DON, NIV and ZEA heated standards were 11, 10 and 85 min, respectively, while the values obtained in whole barley powder were the same for the three toxins (25 min). The determination of H and D decomposition values constitutes a basis to understand the heating stability nature of each toxin.

Topics: Decontamination; Drug Stability; Food Contamination; Food Handling; Fusarium; Heating; Hordeum; Humans; Mycotoxins; Temperature; Trichothecenes; Triticum; Zearalenone

The capacity of pig gastrointestinal microflora to metabolise the trichothecenes 3-acetyl-deoxynivalenol (3-acDON) and nivalenol (NIV) was investigated. 3-acDON was deacetylated to DON in anaerobic incubations with pig faeces collected at different pig farms. Furthermore, both 3-acDON and NIV were metabolised to the corresponding deepoxy metabolite in these incubates. Five pigs, in which the gastrointestinal microflora lacked the ability to transform 3-acDON and NIV to their corresponding de-epoxidated metabolites, were given low levels of DON in the feed for seven weeks. The gastrointestinal micro-organisms did not acquire the de-epoxidation ability during the seven week long exposure period. At the end of the exposure period, faeces from pigs with a known de-epoxidation ability was spread out in the pens and left for 24 hours. One week after the faeces had been spread out in the pens, the de-epoxidation ability was found in faecal incubations from four out of five experimental pigs. This change in metabolic ability of the intestinal de-epoxidation ability was not accompanied by any detectable changes in the DNA-profiles of the bacterial community composition. The results show that the intestinal de-epoxidation ability is common at pig farms in the Uppsala area, and that the ability may be transferred between pigs in a stock.

Topics: Animal Feed; Animals; Biotransformation; Chromatography; DNA, Bacterial; Feces; Food Contamination; Gastrointestinal Contents; Ileum; Male; Specific Pathogen-Free Organisms; Swine; Trichothecenes

A total of 60 samples of wheat flour were collected during the first 6 months of 1999 from mills and food stores in an area in southwest Germany. Samples included whole-grain and two types of white flour with these three groups characterized by a high, medium and low ash content. The contents of deoxynivalenol (DON), nivalenol (NIV), 3- and 15-acetyldeoxynivalenol, HT-2 toxin (HT-2), T-2 toxin (T-2) and fusarenon-X (FUS-X) were determined by gas chromatography/mass spectrometry, and those of zearalenone (ZEA), alpha- and beta-zearalenol (alpha- and beta-ZOL) by high performance liquid chromatography with fluorescence detection. FUS-X, alpha- and beta-ZOL were not detected in any sample. Based on incidence and level, DON was the predominant toxin followed by NIV and ZEA for all three flour types. The overall degree of toxin contamination was lower with decreasing ash content. This suggests a localization of the toxins analyzed primarily in the outer parts of the original wheat kernels. The median DON content was significantly (P<0.05) higher for wheat flour originating from wheat of conventional than of organic production.

Topics: Chromatography, High Pressure Liquid; Flour; Food Analysis; Food Microbiology; Fusarium; Gas Chromatography-Mass Spectrometry; Germany; Mycotoxins; T-2 Toxin; Trichothecenes; Triticum; Zearalenone; Zeranol

Gibberella zeae, a major cause of cereal scab, can be divided into two chemotypes based on production of the 8-ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). We cloned and sequenced a Tri13 homolog from each chemotype. The Tri13 from a NIV chemotype strain (88-1) is located in the trichothecene gene cluster and carries an open reading frame similar to that of Fusarium sporotrichioides, whereas the Tri13 from a DON chemotype strain (H-11) carries several mutations. To confirm the roles of the Tri13 and Tri7 genes in trichothecene production by G. zeae, we genetically altered toxin production in 88-1 and H-11. In transgenic strains, the targeted deletion of Tri13 from the genome of 88-1 caused production of DON rather than NIV. Heterologous expression of the 88-1 Tri13 gene alone or in combination with the 88-1 Tri7 gene conferred on H-11 the ability to synthesize NIV; in the latter case, 4-acetylnivalenol (4-ANIV) also was produced. These results suggest that Tri13 and Tri7 are required for oxygenation and acetylation of the oxygen at C-4 during synthesis of NIV and 4-ANIV in G. zeae. These functional analyses of the Tri13 and Tri7 genes provide the first clear evidence for the genetic basis of the DON and NIV chemotypes in G. zeae.

Topics: Genes, Fungal; Gibberella; Organisms, Genetically Modified; Trichothecenes

Topics: Animal Feed; Animals; Chromatography; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Food Contamination; Gas Chromatography-Mass Spectrometry; Humans; T-2 Toxin; Trichothecenes

Bioassays were used to determine the production of the trichothecene mycotoxin, deoxynivalenol (DON), by two isolates of Fusarium graminearum when grown in association with potentially competitive fungi and an antifungal chemical, 6-pentyl-alpha-pyrone (6PAP). The presence of 6PAP in the culture medium reduced DON production by as much as 80%, but this effect was reduced for the F. graminearum isolate that most efficiently metabolized the added 6PAP. A 6PAP-producing Trichoderma isolate grown in a competition assay system with the F. graminearum isolates was also able to substantially reduce DON production. When Fusarium isolates (F. crookwellense, F. culmorum, F. subglutinans, F. poae, F. equiseti, F. avenaceum, and F. sambucinum), which co-occur with F. graminearum in New Zealand maize plants (Zea mays), were grown in competition assays, the effect on DON production was variable. However, all isolates of F. subglutinans tested were shown to cause reductions in DON production (by 13-76%, mean = 62%). F. subglutinans frequently co-occurs with F. graminearum, but its presence can vary with location and time of the season. When the competitive fungus tested was also a trichothecene producer (e.g., of nivalenol), both toxins were produced in the assay medium. The results indicate that mycotoxin production by F. graminearum can be affected by the presence of particular competitive fungi. These results have implications for an ecological understanding of pathogenicity and of mycotoxin accumulation in plants. Early establishment of F. subglutinans, for example, may act as a biological control mechanism providing a temporary protection against invasion by more commonly toxigenic fusaria such as F. graminearum.

Topics: Fungicides, Industrial; Fusarium; Mycotoxins; Pyrones; Trichothecenes; Zea mays

In South-Eastern region of Poland (near Lublin), where frequency of scab (fusariosis) is much higher than in other parts of the country, during harvest of 1993 kernels of 25 winter wheat cultivars were collected. On the basis of morphological studies Fusarium graminearum was found in 42% of investigated samples while other fungi appeared less frequently: F. nivale and F. poae (35%), F. avenaceum (31%) and F. culmorum (12%). Chemical analysis (by HPLC) revealed that the tested cultivars were contaminated with deoxynivalenol (96% of investigated samples), its acetyl derivatives (48%), nivalenol (76%) and moniliformin (28%). The average levels of the metabolite concentrations were as follows: 104; 16; 97; and 63 micrograms/kg, respectively. Co-occurrence of 2 toxic metabolites was found in the following percentage of the positive samples: deoxynivalenol and nivalenol (72%), deoxynivalenol and moniliformin, as well as nivalenol and moniliformin (24%). Usually (71-83% of contaminated samples) mycotoxins were accumulated in the concentration range > or = 10, < 100 micrograms/kg.

Topics: Chromatography, High Pressure Liquid; Cyclobutanes; Food Contamination; Food Microbiology; Fusarium; Humans; Mycoses; Mycotoxins; Poland; T-2 Toxin; Trichothecenes; Triticum

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

Gibberella zeae, a major cause of cereal scab, may be divided into two chemotypes based on production of the trichothecenes deoxynivalenol (DON) and nivalenol (NIV). We cloned and sequenced the gene cluster for trichothecene biosynthesis from each chemotype. G. zeae H-11 is a DON producer isolated from corn, and G. zeae 88-1 is a NIV producer from barley. We sequenced a 23-kb gene cluster from H-11 and a 26-kb cluster from 88-1, along with the unlinked Tri101 genes. Each gene cluster contained 10 Tri gene homologues in the same order and transcriptional directions as those of Fusarium sporotrichioides. Between H-11 and 88-1 all of the Tri homologues except Tri7 were conserved, with identities ranging from 88 to 98% and 82 to 99% at the nucleotide and amino acid levels, respectively. The Tri7 sequences were only 80% identical at the nucleotide level. We aligned the Tri7 genes and found that the Tri7 open reading frame of H-11 carried several mutations and an insertion containing 10 copies of an 11-bp tandem repeat. The Tri7 gene from 88-1 carried neither the repeat nor the mutations. We assayed 100 G. zeae isolates of both chemotypes by PCR amplification with a primer pair derived from the Tri7 gene and could differentiate the chemotypes by polyacrylamide gel electrophoresis. The PCR-based method developed in this study should provide a simple and reliable diagnostic tool for differentiating the two chemotypes of G. zeae.

Topics: Cloning, Molecular; Fungal Proteins; Gibberella; Hordeum; Molecular Sequence Data; Multigene Family; Plant Diseases; Polymerase Chain Reaction; Sequence Analysis, DNA; Trichothecenes; Zea mays

The occurrences and concentrations of trichothecenes, ochratoxin A and zearalenone in Finnish cereal samples are presented in this study. Furthermore, injections by moulds, especially Fusarium contamination of grains in the same samples, are reported. In total 68 cereal samples, including 43 rye, 4 wheat, 15 barley and 6 oats samples, were collected after a cool and very rainy growing season in 1998. A gas chromatograph combined with a mass spectrometric detector was used for determination of seven different trichothecenes. A high performance liquid chromatograph with a fluorescence detector was used for ochratoxin A and zearalenone determination. For the identification of moulds, the grain samples were incubated and the moulds were isolated and identified by microscopy. The analytical methods were validated for mycotoxin analysis and they were found to be adequately reliable and sensitive. Heavy rainfalls in the summer and autumn of 1998 caused abundant Fusarium mould infection in Finnish cereals, particularly in rye. Fusarium avenaceum was the most common Fusarium species found in cereals. However, the mycotoxin concentrations were very low and only deoxynivalenol, nivalenol and HT-2 toxin were detected. Deoxynivalenol was detected in 54 samples in the concentration range 5-111 microg/kg. Nivalenol and HT-2 toxin were detected in three and two samples, respectively, in the concentration range 10-20 microg/kg.

Topics: Chromatography, Gas; Chromatography, High Pressure Liquid; Edible Grain; Food Microbiology; Fusarium; Humans; Mass Spectrometry; Ochratoxins; Rain; Reproducibility of Results; Sensitivity and Specificity; T-2 Toxin; Trichothecenes; Zearalenone

The effects of moisture, pH and heat on the stability of nivalenol (NIV), deoxtnivalenol (DON) and zearalenone (ZEN) present as natural contaminants of ground maize were measured for different periods. Standard solution tests were also performed to measure pH, salt and temperature effects on NIV and DON. The solution tests showed NIV and DON to be relatively stable in buffer solutions over the pH range 1-10. Quite harsh conditions (pH 12, high salt concentration, 80 degrees C, prolonged exposure) were needed to give substantial breakdown. In the ground maize substrate, these toxins were further stabilized relative to the solution tests. NIV and DON were both reduced (range 60-100%) by treatment with aqueous bicarbonate solution at 10, 20 or 50% of the ground maize dry weight, and subsequent heating at 80 or 110 degrees C for 2 and 12 days. There was no measurable reduction at lower test temperatures (20, 40 degrees C). NIV (but not DON) also showed some reduction following addition of water and heating at 80 or 110 degrees C for 12 days. ZEN content was not reduced even by 12 days of heating at 110 degrees C after treatment with a sodium bicarbonate solution.

Topics: Analysis of Variance; Bicarbonates; Chromatography, High Pressure Liquid; Fusarium; Hot Temperature; Humans; Hydrogen-Ion Concentration; Mycotoxins; Sodium Chloride; Time Factors; Trichothecenes; Water; Zea mays; Zearalenone

The influence of solvent, storage time and temperature on the stability of the trichothecene mycotoxins T-2 toxin (T-2), HT-2 toxin (HT-2), deoxynivalenol (DON) and nivalenol (NIV) was investigated. Toxins in acetonitrile, ethyl acetate or as thin film were stored in sealed glass ampoules at -18, 4, 25 and 40 degrees C for up to 24 months. Samples were analysed by HPLC with UV detection. The results should that acetonitrile was the preferred solvent and no significant (t0.95-test) decomposition occurred for any of the four trichothecenes when stored for 24 months at 25 degrees C or 3 months at 40 degrees C. T-2 and HT-2 in ethyl acetate or as thin film were also stable under the same conditions. DON and NIV in ethyl acetate or as thin film were stable for up to 24 months at -18 degrees C, but a significant decomposition of DON and NIV in ethyl acetate was observed for both toxins after 24 months of storage at 4 degrees C and after 12 months at 25 degrees C. When stored as thin film, a significant trend of decomposition of DON occurred after 24 months of storage at 4 degrees C and after 6 months of storage at 25 degrees C. A significant decrease of NIV stored as thin filmn was observed after 9 months at 25 degrees C. In conclusion, acetonitrile was the most suitable solvent for long-term storage of T-2, HT-2, DON and NIV.

Topics: Acetates; Acetonitriles; Chromatography, High Pressure Liquid; Humans; Linear Models; Mycotoxins; Solvents; T-2 Toxin; Temperature; Time Factors; Trichothecenes

Twenty-three Fusarium culmorum and 21 F. graminearum isolates were studied for their ability to produce mycotoxins and other secondary metabolites. The strains were cultivated on rice, and the extracts analysed by gas chromatography mass spectrometry (GC-MS) after derivatization with pentafluoropropionic (PFP) reagent. Two F. culmorum strains formed nivalenol and its acetylated derivatives (chemotype II), while all F. graminearum and the other F. culmorum isolates produced deoxynivalenol (DON) via 3-acetyldeoxynivalenol (3-acetyl-DON) (chemotype IA). 15-hydroxy-culmorin, followed by 5-hydroxy-culmorin were the main other metabolites produced F. culmorum, while 5-, 12- and an unidentified hydroxy-culmorin, suggested to be 14-hydroxy-culmorin, were the main metabolites of F. graminearum. The hydroxy-culmorin profile was found to be significantly different for the two Fusarium species. Minor amounts of about ten other hydroxy-culmorins, four hydroxy-culmorones and 3,13-dihydroxy- epiapotrichothecene were also detected in most cultures. Traces of sambucinol seemed to be present in some of the isolates, but were not detected in any significant amounts. The precursors in the biosynthetic sequence to 3-acetyldeoxynivalenol, 7,8-dihydroxycalonectrin and 15-deacetyl-7,8-dihydroxycalonectrin, were detected in most cultures. We also report the assignment of both the 1H and 13C NMR data of 15-deacetyl-7,8-dihydroxycalonectrin, which has only been reported incorrectly before.

Topics: Fermentation; Fusarium; Gas Chromatography-Mass Spectrometry; Norway; Oryza; Sesquiterpenes; Trichothecenes

Ten samples of stored wheat grain and 10 samples of settled grain dust released during machine threshing of wheat grain were collected on 10 farms located in Lublin province (eastern Poland). The samples were examined for the concentration of total microfungi, Fusarium species, fusariotoxins (moniliformin, deoxynivalenol, nivalenol), and ochratoxin. Microfungi able to grow on malt agar were present in 30% of grain samples (median for all examined samples = 0, range 0-227.5 x 10(3) cfu/g) and in all samples of grain dust (median = 977.5 x 10(3) cfu/g, range 115.0-16,700.0 x 10(3) cfu/g). Fusarium species (F. avenaceum) were found only in 10% of grain samples (median = 0, range 0-800.0 x 10(3) cfu/g), but in 90% of grain dust samples (median = 1,150 x 10(3) cfu/g, range 5.5-10,060.0 x 10(3) cfu/g). The species F. avenaceum, F. culmorum, F. graminearum, F. poae and F. sporotrichioides were isolated respectively from 50%, 10%, 20%, 40% and 20% of examined grain dust samples. The presence of the mycotoxins produced by Fusarium (moniliformin, deoxynivalenol, and nivalenol) was found altogether in 70% of wheat grain samples (median = 0.1275 microg/g, range 0-1.480 microg/g) and in 90% of grain dust samples (median = 0.350 microg/g, range 0-1.090 microg/g). Moniliformin (MON), deoxynivalenol (DON), and nivalenol (NIV) were each detected in 40% of grain samples, and respectively in 80%, 40%, and 40% of grain dust samples. Ochratoxin A (OTA) was detected in 60% of grain samples and in 60% of grain dust samples (median in both cases was 0.0005 microg/g). The concentrations of F. poae (p<0.05) and of total Fusarium species (p<0.01) in grain samples, and the concentrations of F. culmorum and F. graminearum (p<0.05) in grain dust samples were significantly correlated with the concentration of deoxynivalenol. The concentrations of F. poae (p<0.05) and of total Fusarium species (p<0.01) in grain dust samples were significantly correlated with the concentration of total fusariotoxins. Moreover, the concentration of total Fusarium species in grain dust samples was significantly correlated with the concentration of nivalenol (p<0.05). In conclusion, the majority of samples of wheat grain and grain threshing dust collected on farms in eastern Poland contained notable quantities of fusaria and/or fusariotoxins. This fact poses a potential risk of mycotoxicoses to agricultural workers exposed to grain dust when handling wheat during threshing, unloading, shuffling, and other f

Topics: Agricultural Workers' Diseases; Air Microbiology; Colony Count, Microbial; Cyclobutanes; Dust; Food Contamination; Food Microbiology; Fungi; Fusarium; Humans; Mycoses; Mycotoxins; Occupational Exposure; Poland; Trichothecenes; Triticum

Infection of cereal grains with Fusarium species can cause contamination with mycotoxins that affect human and animal health. To determine the potential for mycotoxin contamination, we isolated Fusarium species from samples of rice seeds that were collected in 1997 on farms in the foothills of the Nepal Himalaya. The predominant Fusarium species in surface-disinfested seeds with husks were species of the Gibberella fujikuroi complex, including G. fujikuroi mating population A (anamorph, Fusarium verticillioides), G. fujikuroi mating population C (anamorph, Fusarium fujikuroi), and G. fujikuroi mating population D (anamorph, Fusarium proliferatum). The widespread occurrence of mating population D suggests that its role in the complex symptoms of bakanae disease of rice may be significant. Other common species were Gibberella zeae (anamorph, Fusarium graminearum) and Fusarium semitectum, with Fusarium acuminatum, Fusarium anguioides, Fusarium avenaceum, Fusarium chlamydosporum, Fusarium equiseti, and Fusarium oxysporum occasionally present. Strains of mating population C produced beauvericin, moniliformin, and gibberellic acid, but little or no fumonisin, whereas strains of mating population D produced beauvericin, fumonisin, and, usually, moniliformin, but no gibberellic acid. Some strains of G. zeae produced the 8-ketotrichothecene nivalenol, whereas others produced deoxynivalenol. Despite the occurrence of fumonisin-producing strains of mating population D, and of 8-ketotrichothecene-producing strains of G. zeae, Nepalese rice showed no detectable contamination with these mycotoxins. Effective traditional practices for grain drying and storage may prevent contamination of Nepalese rice with Fusarium mycotoxins.

Topics: Carboxylic Acids; Food Contamination; Fumonisins; Fusarium; Gibberellins; Mycotoxins; Nepal; Oryza; Reproduction; Seeds; Trichothecenes

The trichothecenes are mycotoxins produced by fungi of the genus Fusarium, which are commonly present in foods and feed of cereal origin. Owing to the lack of sufficient toxicological data for most of the trichothecenes, in vitro studies may contribute to risk assessments of these toxins. In the present report, human lymphocyte cultures were used to study the individual variation in sensitivity among humans and the effects on in vitro Ig production. Furthermore, proliferative responses of cells exposed to combinations of two of the toxins were studied. Four toxins, T-2 toxin, diacetoxyscirpenol (DAS), nivalenol (NIV) and deoxynivalenol (DON) were included in the study. All four of the tested trichothecenes effectively inhibited mitogen-induced lymphocyte proliferation. There were no statistically significant differences in sensitivity to the toxins between lymphocytes from female and male blood donors. The individual variation in sensitivity, evaluated as the range of IC50 values, was rather limited (within a factor of 3 to 4). Immunoglobulin production by pokeweed-stimulated human lymphocytes was also effectively inhibited with IC50 values similar to the IC50 values in the proliferation tests for DON and NIV. However, IC50 values for Ig synthesis in cultures exposed to T2 were approximately two to three times higher than the corresponding IC50 values found in the proliferation tests. At low levels of exposure, elevated Ig production was observed in lymphocyte cultures from four out of the five blood donors tested. This effect was most pronounced on IgA synthesis. Combinations of NIV with T2, DAS or DON resulted in additive toxicity in the lymphocyte proliferation test, while combinations of DON with T2 or DAS resulted in an inhibition that was slightly lower than what could have been expected from the inhibition produced by the individual toxins. In conclusion, the tested trichothecenes inhibited both proliferation and Ig production in human lymphocytes in a dose-dependent manner with limited variation in sensitivity between individuals. Enhanced Ig production was observed in cell cultures exposed to the lower doses of the toxins. Combined exposure to two of the toxins resulted mainly in additive or antagonistic effects, although synergistic effects cannot be excluded and should be further investigated. These findings indicate that the total intake of type A and B trichothecenes should be taken into account in risk assessments.

Topics: Adult; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Female; Humans; Immunoglobulins; Lymphocyte Activation; Lymphocytes; Male; Protein Synthesis Inhibitors; T-2 Toxin; Trichothecenes

A new, rapid and sensitive method has been developed for the determination of nivalenol (NIV) and deoxynivalenol (DON) by using HPLC in combination with an atmospheric pressure chemical ionization (APCI)-interface and a single quadrupole mass spectrometer. Different LC and MS parameters have been optimized prior to this in order to obtain better results and sensitivity. The effect of nebulizing temperature on the sensitivity and fragmentation of NIV and DON in an APCI interface was investigated. Also, the influence of the cone voltage on the fragmentation pattern was studied, which was shown to have a tremendous effect. Furthermore, the effect of modifiers such as ammonium acetate, acetic acid and ammonia on the ionisation yield of the above substances have been investigated. The extraction was carried out using acetonitrile-water. A two step purification was then applied on two different Mycosep clean up columns. We have used a modified, rapid and isocratic HPLC method combined with a negative ion APCI-MS for the separation and quantitative determination of NIV and DON in wheat extract. An RP C18 column was used for the separation of selected compounds in wheat extract with water-acetonitrile-methanol (82:9:9, v/v/v) at a flow-rate of 1 ml/min without a split. Calibration curves show good linearity and reproducibility. The detection limit and precision were determined for NIV and DON. Both compounds could be detected down to microg/kg level in wheat using selected ion monitoring of the [M-H]- ions and the main fragments.

Topics: Atmospheric Pressure; Chromatography, High Pressure Liquid; Mass Spectrometry; Mycotoxins; Reference Standards; Reproducibility of Results; Trichothecenes; Triticum

Four trichothecene mycotoxins--the type A trichothecenes T2-toxin and diacetoxyscirpenol and the type B trichothecenes nivalenol and deoxynivalenol--were studied. The effects of these mycotoxins on the expression of the sequentially expressed activation markers CD69, CD25, and CD71 and on proliferation of human lymphocytes were studied in culture with a duration of up to 72 h. All the examined toxins affected activation marker expression in a similar way. After 6 h, the CD69 expression was lower in exposed cultures compared to controls. After 24 and 48 h of exposure, an increased frequency of cells expressing CD69 was found in exposed cultures, indicating a delay in downregulation of CD69 expression. Stimulation of CD25 expression was observed for doses below the IC50 value, while suppression was found for higher doses. The pattern was different from that detected for CD69 expression, in that an increased expression of CD25 never occurred after exposure to the highest concentration of the toxin, and in that no stimulatory effects were found after 48 h of exposure, indicating that the response was inhibited and not delayed. The effects of toxin exposure on CD71 expression were in many respects similar to the effects on CD25 expression. We conclude that the trichothecene mycotoxins investigated in this study inhibited the cell cycle in a similar way and exert their main antiproliferative action rather early in the cell cycle, before or in conjunction with CD25 expression.

Topics: Adult; Antigens, CD; Antigens, Differentiation, B-Lymphocyte; Antigens, Differentiation, T-Lymphocyte; Biomarkers; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cells, Cultured; Female; Humans; Kinetics; Lectins, C-Type; Lymphocyte Activation; Lymphocytes; Mycotoxins; Receptors, Interleukin-2; Receptors, Transferrin; T-2 Toxin; Trichothecenes

A total of 237 commercially available samples of cereal-based foods including bread and related products, noodles, breakfast cereals, baby and infant foods, rice and other foods were randomly collected in southwest Germany during the first six months of 1998. The trichothecenes deoxynivalenol (DON), 3- and 15-acetyl-deoxynivalenol (3-,15-ADON), nivalenol (NIV), fusarenon-X (FUS-X), T-2 toxin (T-2) and HT-2 toxin (HT-2) were determined by gas chromatography/mass spectrometry following clean-up by a two stage solid-phase extraction. Detection limits ranged between 2 and 12 micrograms/kg. Based on all samples, the incidence of DON, HT-2, T-2, 3-ADON, 15-ADON, and NIV was at 71, 18, 4, 4, 4 and 2%, respectively; the average contents in positive samples were at 103, 16, 14, 17, 24 and 109 micrograms/kg, respectively. Fus-X was not detected in any sample. A lower (P < 0.05) DON content was found in baby and infant foods as well as in cookies and cakes compared to bread. Overall, based on the incidence and level of all six toxins, the degree of contamination was lowest in baby and infant foods. Foods produced from either white or whole grain flour did not differ (P > 0.05) with regard to the incidence and level of DON. In foods produced from cereals of organic production both the incidence and median content of DON was lower compared to conventional production. Zearalenone, alpha- and beta-zearalenol were determined by high performance liquid chromatography in 20 selected samples, mostly baby and infant foods. These toxins were not present in excess of the detection limit in any sample.

Topics: Bread; Chromatography, Affinity; Chromatography, High Pressure Liquid; Edible Grain; Food Microbiology; Fusarium; Gas Chromatography-Mass Spectrometry; Germany; Humans; Infant Food; Mycoses; Mycotoxins; Oryza; Secale; Spectrometry, Fluorescence; T-2 Toxin; Trichothecenes; Triticum; Zea mays; Zearalenone; Zeranol

A total of 449 grain samples, 102 barley, 169 wheat and 178 oat samples were collected from different regions of Norway from 1996-1998 crops, mainly from grain loads and silos. The samples were analysed for type A and B trichothecenes, the largest groups of mycotoxins produced by the Fusarium species, by gas chromatography with mass spectrometric detection (GC-MS). Factors affecting the presence of the different trichothecenes are discussed. Deoxynivalenol (DON) and HT-2 toxin were the trichothecenes most frequently detected, followed by T-2 toxin, nivalenol, and scirpentriol, scirpentriol being detected only in seven samples (> 20 micrograms/kg). Oats were the grain species most heavily contaminated with an incidence (% > 20 micrograms/kg) and mean concentration of positive samples of 70% (115 micrograms/kg) for HT-2 toxin, 30% (60 micrograms/kg) for T-2 toxin, 57% (104 micrograms/kg) for DON, and 10% (56 micrograms/kg) for nivalenol. The corresponding values for barley were 22% (73 micrograms/kg), 5% (85 micrograms/kg), 17% (155 micrograms/kg) and 6% (30 micrograms/kg), and for wheat 1.2% (20 micrograms/kg), 0.6% (20 micrograms/kg), 14% (53 micrograms/kg) and 0% for HT-2, T-2, DON and nivalenol, respectively. Norwegian oats were found to contain HT-2 and T-2 toxin in concentrations that might be at threat to human health for high consumers of oats. The amount of DON was significantly lower than in the crop from previous years.

Topics: Chromatography, Gas; Edible Grain; Food Contamination; Food Microbiology; Fusarium; Humans; Mass Spectrometry; Norway; T-2 Toxin; Trichothecenes

The application of cell culture technique for screening of low concentrations of Fusarium mycotoxins was examined. Three colorimetric bioassays were used to determine the cytotoxicity of the trichothecenes T-2 toxin (T-2), HT-2 toxin (HT-2), deoxynivalenol (DON) and nivalenol (NIV) to 3T3 mouse fibroblasts (3T3 cells). The bioassays assess DNA synthesis (incorporation of 5-bromo-2'-deoxyuridine; BrdU), metabolic activity (cleavage of 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT) and cell membrane damage (release of lactate dehydrogenase; LDH), respectively. The BrdU bioassay was the most sensitive and the IC50 values (50% response compared to untreated cells) of T-2, HT-2, DON and NIV were 4.6, 13, 263 and 365 ng/ml, respectively. At the same toxin concentrations used in the BrdU bioassay, only T-2 and HT-2 were toxic enough to obtain IC50 values using the MTT bioassay. The IC50 values for T-2 and HT-2 were 12 and 68 ng/ml, respectively. When determined by the LDH bioassay, the IC50 values of T-2 and HT-2 were 18 and 42 ng/ml, respectively. At the tested concentrations, DON and NIV had a minor effect on the 3T3 cells when evaluated by the MTT and LDH bioassays. The BrdU bioassay in combination with 3T3 cells was found to be a suitable method for determination of trichothecene-induced toxicity at low concentrations.

Topics: 3T3 Cells; Animals; Bromodeoxyuridine; Colorimetry; Dose-Response Relationship, Drug; L-Lactate Dehydrogenase; Mice; T-2 Toxin; Tetrazolium Salts; Toxicity Tests; Trichothecenes

In order to survey the natural occurrence of Fusarium mycotoxins including deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN) in beers consumed in Korea, a total of 54 extracts of Korean and imported beers were derivatized with heptafluorobutyric anhydride and analysed by gas chromatography-mass spectrometry with selected ion monitoring mode. DON was detected in 14 samples (26%) and NIV was detected in 43 samples (80%). ZEN was not detected in any of the samples tested. The incidence of DON was 19% for Korean beers and 50% for imported beers, whereas the incidence of NIV was 85% for Korean beers and 58% for imported beers. This is the first report that the beers consumed in Korea are contaminated with DON and NIV.

Topics: Beer; Food Contamination; Fusarium; Gas Chromatography-Mass Spectrometry; Humans; Korea; Mycotoxins; Trichothecenes; Zearalenone

The capacity of the gastrointestinal microflora of pig, cow, and chicken to metabolize nivalenol (NIV) and deoxynivalenol (DON) was studied both in vivo and in vitro. Before feeding NIV to pigs, no metabolites of NIV or DON were formed in anaerobic incubates of the toxins with the pigs feces. However, after one week on a diet containing 2.5 or 5 ppm NIV, nearly all excreted NIV in feces had been de-epoxidated in five of six pigs. After three weeks on the NIV diet also the sixth pig had acquired this ability. Deoxynivalenol was also de-epoxidated when incubated in vitro with the microorganisms that formed de-epoxy-NIV in vivo. Anaerobic incubation of NIV and DON with cow rumen fluid produced de-epoxides of both toxins in a high proportion. No de-epoxide of NIV, but another unidentified metabolite was found in feces from chicken fed 2.5 or 5 ppm NIV for three weeks.

Topics: Animals; Biotransformation; Cattle; Chickens; Diet; Digestive System; Feces; In Vitro Techniques; Mycotoxins; Rumen; Swine; Time Factors; Trichothecenes

Topics: Adult; Aged; Carcinogens, Environmental; Colombia; Female; Fumonisins; Human T-lymphotropic virus 1; Humans; Male; Middle Aged; Mycotoxins; Paraparesis, Tropical Spastic; Trichothecenes

The effect of trichothecene mycotoxins, deoxynivalenol (DON), fusarenon-X (FX) and nivalenol (NIV), on plaque formation of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) in HEp-2 cells was examined. The 50% effective concentrations (EC50) of DON, FX, and NIV for HSV-1 plaque formation were 160, 56, and 120 ng/ml, respectively. Those for HSV-2 plaque formation were 94, 26, and 50 ng/ml, respectively. These three mycotoxins showed about 2-fold higher selectivity to HSV-2 than to HSV-1. Plaque formation of HSV-1 was not inhibited with trichothecenes at concentrations completely inhibiting plaque formation when cells were treated during virus adsorption period or 15 hr before infection. These results indicate that trichothecenes affect replication of HSV-1 after virus adsorption, but not before or during virus adsorption to the host cells.

Topics: Cell Line; Herpesvirus 1, Human; Herpesvirus 2, Human; Humans; Mycotoxins; Trichothecenes; Viral Plaque Assay; Virus Replication

100 samples of rye and 101 samples of wheat coming out of both conventional and alternative or ecological production were investigated for contamination with mycotoxins with interest for our degree of latitude. Deoxynivalenol (DON) was found with thin-layer-chromatography in 131 of 201 samples altogether. A top level of 1250 micrograms DON kg-1 in rye of alternative offspring was detected. The average burden in contaminated rye coming from ecological production was 427 micrograms kg-1 and a mean level of 160 micrograms kg-1 resulting in rye out of conventional growth conditions. In wheat, conventionally grown yield showed slightly lower contamination (mean levels of 420 micrograms DON kg-1 towards 486 micrograms kg-1). The toxins 3-acetyl-deoxynivalenol, nivalenol and fusarenone X were detected in some samples by thin-layer-chromatography. This results could not be confirmed by gas chromatography -mass spectrometry. Zearalenone was found in 40 out of the number of 201 samples of grain by HPLC with fluorescence detection. An average of 6 micrograms and 24 micrograms zearalenone kg-1 in conventionally and alternatively grown wheat and 4 micrograms and 51 micrograms zearalenone kg-1 in conventionally and alternatively produced rye was detected. The highest finding of zearalenone was 199 micrograms kg-1 in alternatively grown rye. Skin toxicity testing did not show any reference of contamination with type-A-trichothecenes. No correlation between contamination of zearalenone or deoxynivalenol and thousand-kernel-weight was detected.

Topics: Agriculture; Chromatography, Thin Layer; Food Microbiology; Gas Chromatography-Mass Spectrometry; Humans; Mycotoxins; Secale; Skin; Skin Tests; Trichothecenes; Triticum; Zearalenone

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

A preliminary survey for the natural occurrence of Fusarium mycotoxins--nivalenol (NIV), deoxynivalenol (DON) and zearalenone (ZEN)--in cereals imported mostly from Australia to Papua, New Guinea (PNG) in 1991 was carried out by TLC, GLC, and HPLC analysis. The data have revealed various kinds of cereals and cereal flours, such as wholemeal flour, millet meal, corn flour, brown trukai rice, and others, were significantly contaminated with these mycotoxins in various concentrations. Among these Fusarium mycotoxins, the level of ZEN was surprisingly high. This is the first report on the natural occurrence of Fusarium mycotoxins in PNG cereals.

Topics: Chromatography, Gas; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Edible Grain; Food Contamination; Food Microbiology; Fusarium; Mycotoxins; New Guinea; Trichothecenes; Zearalenone

Antibody against nivalenol tetraacetate (tetra-Ac-NIV) was prepared by immunization of rabbits with triacetyl-15-pimelate-NIV conjugated to bovine serum albumin. By using tritiated tetra-Ac-NIV as the test ligand, antibody titers were demonstrated as early as 4 weeks after immunization. Useful antibody for radioimmunoassay (RIA) of tetra-Ac-NIV was obtained 7 weeks after immunization, with one booster injection. Results of competitive RIA revealed that the antibody was most specific to tetra-Ac-NIV. The relative cross-reactivity of this antibody with tetra-Ac-NIV, deoxynivalenol triacetate, and neosolaniol triacetate was found to be 100, 2.2, and less than 1, respectively. Practically no cross-reaction was found with deoxynivalenol, fusarenon X, and NIV. The detection limit for tetra-Ac-NIV by RIA was about 5.0 ng/ml (0.5 ng per assay). The use of this antibody for quantitation of NIV in cereals after acetylation of sample extracts is proposed.

Topics: Acetylation; Animals; Antibodies, Fungal; Antibody Specificity; Fusarium; Molecular Structure; Mycotoxins; Rabbits; Radioimmunoassay; Trichothecenes

A survey for the natural occurrence of Fusarium mycotoxins, deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN), in Dutch cereals (totaling 29 samples) harvested in 1984/1985, showed that 90%, 79% and 62% of samples were contaminated with DON, NIV and ZEN, respectively. Average contents (ng/g) in the total of positive samples were 221 (DON), 123 (NIV) and 61 (ZEN). Among the cereals examined, the highest concentrations (ng/g) was 3198 (DON), 1875 (NIV) and 677 (ZEN) in a yellow corn sample for animal feed. The results of this survey show that Dutch cereals were relatively significantly contaminated with Fusarium mycotoxins.

Topics: Edible Grain; Food Contamination; Fusarium; Hordeum; Mycotoxins; Netherlands; Resorcinols; Secale; Sesquiterpenes; Trichothecenes; Triticum; Zea mays; Zearalenone

By single ascospore isolation, several sets of asci containing eight ascospores were isolated from perithecia of Gibberella zeae. Of these sets, seven were investigated for their ability to produce 8-ketotrichothecene mycotoxins on rice grains. Analyses were made with gas chromatography-mass spectrometry and gas chromatography with 63Ni electron capture detection. Of 56 total isolates, 11 produced nivalenol, 4-acetylnivalenol, and deoxynivalenol, 1 produced nivalenol and deoxynivalenol, 7 produced deoxynivalenol and 3-acetyldeoxynivalenol, 19 produced deoxynivalenol and 15-acetyldeoxynivalenol, and 6 produced deoxynivalenol and both 15- and 3-acetyldeoxynivalenol. The remaining 12 isolates produced nivalenol and 4-acetylnivalenol. All isolates of G. zeae that we examined could produce 8-ketotrichothecenes in this investigation. This report is the first to demonstrate the presence of G. zeae isolates producing both nivalenol and deoxynivalenol. In addition, differences in the production between 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol are discussed in relation to culture conditions.

Topics: Gibberella; Species Specificity; Trichothecenes

Bavarian cereals and wheat flour from the 1987 harvest were analysed for nivalenol (NIV) and deoxynivalenol (DON) using high performance liquid chromatography (HPLC) and for T-2 toxin and zearalenone (ZEA) by enzyme-linked-immunosorbent assay (ELISA). The study included 190 field samples of wheat, barley, rye and oat with visibly damaged ears, 45 samples of wheat intended for feed production and two series of wheat flour (type 550) and whole wheat flour collected in October 1987 and June 1988. The field samples examined showed a high DON contamination of wheat (87%) with an average of 3.96 mg/kg and a maximum of 43.8 mg/kg. Mean levels between 0.33 mg/kg and 0.27 mg/kg DON could be detected in barley, rye and oat. Of the wheat samples, 58% contained ZEA with a maximum of 1.560 mg/kg. The highest levels of ZEA were detected in samples which also showed high concentrations of DON. The NIV and T-2 toxin levels were comparatively low. Thirty percent of the samples showed NIV concentrations between 0.04 mg/kg and 0.29 mg/kg and 38% contained between 0.005 and 0.60 mg/kg of T-2. In the wheat samples for feed production, only DON was detected with an average of 0.190 mg/kg and a maximum of 0.75 mg/kg. The highest DON levels (0.58 mg/kg) from October 1987 were found in the wheat flour samples which were lower than the highest DON concentration (3.24 mg/kg) detected in the samples collected during June 1988. This fact was probably due to a substantial amount of non-contaminated wheat from 1986. The toxin concentrations in the whole wheat flour were not higher than in the type 550 flour.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animal Feed; Animals; Edible Grain; Flour; Food Contamination; Food Microbiology; Fusarium; Germany, West; Humans; Mycotoxins; T-2 Toxin; Trichothecenes; Zearalenone

Nivalenol is a toxic trichothecene metabolite which is produced by a number of different Fusarium species. However, the nature of its immediate biosynthetic precursor is not known. Oxidation of 7-deoxynivalenol(3 alpha,4 beta,15-trihydroxy-12,13- epoxytrichothec-9-ene-8-one) to nivalenol occurred with reagents known to react by a free radical pathway, such as hydrogen peroxide-ferrous ion-ascorbic acid or lead tetracetate, but not with electrophilic reagents requiring prior formation of the enol. These results suggest that 7-deoxynivalenol or an acetylated derivative could be the biosynthetic precursor of nivalenol.

Topics: Fusarium; Hydroxylation; Sesquiterpenes; Trichothecenes

DON and NIV were extracted with methanol-water (3:1) and purified by Florisol column. The mycotoxins in the column eluates were derived by TMS reagent and then analysed by gas chromatography with electron capture detector. The average recoveries of DON and NIV, as counted into wheat, at 100, 500, 1,000 and 2000 ppb were 81.2% and 74.5% respectively. The limits of detection of toxins of wheat were 10 ppb for both DON and NIV.

Topics: Chromatography, Gas; Food Contamination; Mycotoxins; Sesquiterpenes; Trichothecenes; Triticum

An investigation for the occurrence of nivalenol (NIV), deoxynivalenol (DON) and zearalenone (ZEN) in cereals (ten wheat, one rye and one corn) harvested in Canada have been carried out using a procedure, which is rapid and sensitive for Fusarium mycotoxins. NIV, DON and ZEN were detected in 4, 9 and 9 out of ten wheat samples, and their average levels in the positives were 23 ng/g, 1257 ng/g and 9 ng/g, respectively. One rye and one corn were also contaminated with a minor amount of NIV. This is the first evidence for the natural occurrence of NIV in cereals grown in Canada, though its level was far less than DON.

Topics: Canada; Edible Grain; Food Contamination; Fusarium; Resorcinols; Secale; Sesquiterpenes; Trichothecenes; Triticum; Zea mays; Zearalenone

Cereals, foods and feeds sampled in Taiwan, China and the U.S.S.R. were contaminated with nivalenol, deoxynivalenol and zearalenone. The frequencies and levels of contamination are similar to those observed in the cereals of Japan and Korea. This is the first report on the natural occurrence of nivalenol, deoxynivalenol and zearalenone in Chinese and U.S.S.R. cereals, foods and feeds.

Topics: China; Edible Grain; Flour; Food Contamination; Hordeum; Resorcinols; Sesquiterpenes; Taiwan; Trichothecenes; Triticum; USSR; Zearalenone

Topics: Beverages; Citrus; Edible Grain; Food Contamination; Infant Food; Sesquiterpenes; Trichothecenes

Topics: Food Contamination; Food Handling; Hot Temperature; Sesquiterpenes; Trichothecenes; Triticum

Zearalenone, an estrogenic mycotoxin of Fusarium species, in cereals can be extracted with acetonitrile-water (3:1), purified on a Florisil column, resolved by high-performance liquid chromatography (HPLC) with a Nucleosil 50-10 column using 90% water-saturated chloroform-cyclohexane-acetonitrile-ethanol (50:15:2:1) and quantitated by fluorescence measurement. This method is rapid, simple and reproducible, and detects zearalenone in wheat, barley, corn and other cereals with picogram sensitivity. A combination of this HPLC method with a gas-liquid chromatographic method for trichothecenes may be applied to the simultaneous detection of Fusarium mycotoxins (zearalenone, nivalenol and deoxynivalenol) in cereals.

Topics: Chromatography, High Pressure Liquid; Edible Grain; Food Microbiology; Hordeum; Resorcinols; Spectrometry, Fluorescence; Trichothecenes; Triticum; Zea mays; Zearalenone

Topics: Chromatography, Liquid; Edible Grain; Gas Chromatography-Mass Spectrometry; Mass Spectrometry; Mycotoxins; Sesquiterpenes; Trichothecenes

A method for the analysis of deoxynivalenol (DON) und nivalenol (NV) in cereals is described. This method is suitable for routine use for control purposes. The extract is purified on a florisil column. Derivatization with heptafluorobutyrylimidazole is followed by quantitative determination by means of capillary gas chromatography and electron capture detection (ECD). For verification purposes two-dimensional thin layer chromatography is applied to gc-positive extracts. 67 cereal samples and cereal products from the Berlin market place have been assayed. Depending on the type of sample the limit of detection was 20 to 30 micrograms/kg for DON, and 100 to 300 micrograms/kg for NV.

Topics: Chemical Phenomena; Chemistry; Chromatography, Gas; Edible Grain; Fluorocarbons; Food Contamination; Sesquiterpenes; Trichothecenes

A method is described for the determination of Deoxynivalenol (DON) and Nivalenol (NV). A liquid chromatographic clean-up step is followed by silylation of the extract and the trimethylsilylether are determined by gas-chromatography-mass-spectrometry (GC-MS), monitoring the characteristic ions in the negative chemical ionisation mode. 71% of 42 food-samples were contaminated with DON and 38% with NV. The highest contamination detected was 2000 micrograms/kg DON and 120 micrograms/kg NV in a wheat bran sample. Deoxynivalenol and Nivalenol in concentrations of 10 micrograms/kg can be detected easily by this method.

Topics: Chromatography, Thin Layer; Edible Grain; Food Contamination; Gas Chromatography-Mass Spectrometry; Molecular Weight; Mycotoxins; Sesquiterpenes; Trichothecenes