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PubMed1995

BCR- and M&T-activities in the area of mycotoxin analysis in food and feedstuffs.

In order to avoid undesirable side effects such as high contents of mycotoxins in food and feed, measurements at critical steps of the food and feed production chain are performed. These measurements need to be accurate and precise to satisfy these aims. The accuracy and precision requirements to measurements form the basis of the Bureau Communautaire de Référence (BCR)-and Measurements and Testing (M&T)-Projects. Such projects aim to improve and/or develop analytical methodology, sampling plans, to harmonise agreement of results between European Union Member States and to prepare suitable certified reference materials (CRMs). The selection of the matrix CRMs and their corresponding level of mycotoxin contamination is made on the basis of requirements of the applied analytical procedures, stability requirements, regulations, norms, and inter-or intra-trade agreements which are in relation to quality specifications of imported and/or exported goods. The availability of appropriate matrix CRMs is not only a prerequisite for the implementation of directives and norms. It allows the validation of new methods and provides a possible solution to trade disputes, means for the statistical control of analytical results, tools for laboratory accreditation, and it is the basis for harmonisation and traceability of proficiency schemes.

Food Analysis↗

PubMed2006

Development of a liquid chromatography/tandem mass spectrometry method for the simultaneous determination of 16 mycotoxins on cellulose filters and in fungal cultures.

The development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of 16 mycotoxins possibly related to the 'Sick Building Syndrome' on filters and in fungal cultures is described. Fungi-surface sampling as regards the 'Sick Building Syndrome' preferably happens by scraping off fungal material and vacuuming onto cellulose filters. Therefore, these two media were used as samples. They were spiked with nivalenol, deoxynivalenol, zearalenone, diacetoxyscirpenol, T-2 toxin, verrucarol, verrucarin A, neosolaniol, sterigmatocystin, roridin A, ochratoxin A, aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2, which can be produced by isolates from fungi-damaged buildings. Deepoxy-deoxynivalenol was used as internal standard. Samples were extracted with organic solvents and the different mycotoxins were separated by high-performance liquid chromatography (HPLC) using a C18 reversed-phase SunFire analytical column and a mobile phase of variable mixtures of ammonium acetate (10 mM) and sodium acetate (20 microM) in water (solvent A) and in methanol (solvent B). The samples were run on-line with a Micromass Quattro Micro triple quadrupole mass spectrometer in positive electrospray ionisation mode using multiple reaction monitoring (MRM). The detection limits of the procedure varied from 50 to 0.009 pg/microL for filter samples and from 75 to 0.04 pg/microL for fungal culture samples. As the method includes few and non-labourious sample treatment steps, it should allow for a high throughput of samples.

Biomass↗

PubMed1997

A rapid bioassay to detect mycotoxins using a melanin precursor overproducer mutant of the ciliate Tetrahymena thermophila.

Four different mycotoxins (patulin, T-2 toxin, diacetoxyscirpenol and roquefortine) were used to study growth inhibitory effects on a melanin precursor overproducer mutant of the ciliate Tetrahymena thermophila. This strain is especially sensitive to diacetoxyscirpenol and T-2 toxin. The secretion capacity of melanin precursors into the culture medium by this mutant and its biosensor capacity are very useful characteristics to elaborate a rapid bioassay to detect some specific mycotoxins.

Animals↗

PubMed1994

Influence of low-level exposure to Fusarium mycotoxins on selected immunological and hematological parameters in young swine.

The effects of low dietary concentrations of Fusarium mycotoxins (deoxynivalenol (DON), 15-acetyl-DON, and zearalenone) on growth, immunological, and hematological parameters were determined in young pigs during a 28-day feeding experiment. Clean and naturally contaminated corn were incorporated into basal diets formulated to contain 0.00, 0.75, 1.50, and 3.00 mg DON/kg diet. A pair-fed control animal was used for comparison with each animal receiving the highest level of contamination (diet 4). Skin temperature, measured during the first week of the experiment, decreased linearly as the dietary mycotoxin concentration increased. Several other linear effects were observed: depressed feed intake throughout the experiment, reduction in thyroid size (absolute/relative), and changes in the appearance of the esophageal region of the stomach (thicker and higher degree of folding with increasing toxin concentration). Serum T4 (thyroxine) levels increased quadratically after 7 and 28 days of exposure compared to control animals. This change coincided with an increase in albumin levels, a decrease in alpha-globulin levels, and an overall increase in albumin/globulin ratio as the level of contamination increased. After immunization with sheep red blood cells (SRBC), animals fed contaminated diets showed a delayed response in peak titers. At the end of the experiment an increase in the segmented neutrophil count was observed. The following observations were made for animals consuming diet 4 as compared to the pair-fed controls: lower skin temperature, better feed efficiency, more corrugated stomachs, reduced alpha-globulin levels, and lower antibody titers to SRBC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Feed↗

PubMed2002

The mycotoxin patulin alters the barrier function of the intestinal epithelium: mechanism of action of the toxin and protective effects of glutathione.

Patulin is a mycotoxin mainly found in apple and apple products. In addition to being toxic for animals, mutagenic, carcinogenic and teratogenic, patulin induces intestinal injuries, including epithelial cell degeneration, inflammation, ulceration, and hemorrhages. In a study of the cellular mechanisms associated with the intestinal toxicity of patulin, two human epithelial intestinal cell lines (HT-29-D4 and Caco-2-14) were exposed to the mycotoxin. Micromolar concentrations of patulin were found to induce a rapid and dramatic decrease of transepithelial resistance (TER) in both cell lines without major signs of toxicity as assessed by the LDH release assay. Since TER reflects the organization of tight junctions, these data indicate that patulin affected the barrier function of the intestinal epithelium. The inhibitory effect of patulin on TER was closely associated with its reactivity for SH groups: (i) cysteine and glutathione prevented the cells from patulin injury; (ii) patulin toxicity was potentiated by buthionine sulfoximine, a specific glutathione-depleting agent; (iii) treatment of the cells with N-ethylmaleimide, a compound known to react with SH groups, resulted in a marked decrease of TER. Moreover, the inhibitory effect of patulin on TER was mimicked and potentiated by phenylarsine oxide, a specific inhibitor of protein tyrosine phosphatase (PTP). This cellular enzyme is a key regulator of intestinal epithelial barrier function. The active site of PTP contains a cysteine residue (Cys215) that is essential for phosphatase activity. Sulfhydryl-reacting compounds such as acetaldehyde decrease TER through covalent modification of Cys215 of PTP. We propose that the toxicity of patulin for intestinal cells involves, among other potential mechanisms, an inactivation of the active site of PTP.

Antidotes↗

PubMed2002

U.S. perspective on mycotoxin regulatory issues.

Control programs set up by the Food and Drug Administration (FDA) for aflatoxin, an unavoidable natural contaminant produced by specific molds that invade a number of feedstuffs and basic foods, provide an example of forces that affect risk assessment and management strategies by a regulatory agency. More recently, on an international scale, efforts to establish international food standards for fumonisin, deoxynivalenol, ochratoxin A, zearalenone, and patulin, as well as for aflatoxin, demonstrate the complexity of developing regulations and/or standards designed to protect consumer health and ensure fair trade practices on a global scale. Current FDA regulations for aflatoxins address public health concerns for potential contamination in basic foods, residues in milk, and animal feeds for numerous commodities and applications. Regulatory limits, sampling and analytical procedures, decontamination and/or diversion to less risk uses for contaminated product are components of mycotoxin control programs. Current efforts by FDA to establish regulatory controls for deoxynivalenol, fumonisin, and patulin add further insight on the role that safety and risk assessment procedures play in the development of action levels and advisories for mycotoxins.

Food Contamination↗

PubMed1999

Effects of Fusarium mycotoxins on levels of serotonin, melatonin, and 5-hydroxytryptophan in pineal cell cultures.

Analysis of melatonin (MEL) in pineal cell cultures by enzyme linked immunosorbent assay showed its concentration was increased by fusaric acid (FA), a mycotoxin produced by Fusarium species and associated with toxic duck and ostrich feeds. Subsequent cell culture studies demonstrated the precursors of MEL, 5-hydroxytryptophan (5HTP) and serotonin (5HT), were also affected by FA as well as other Fusarium mycotoxins. Herein we describe a technique for the analysis of 5HTP and 5HT in pineal cell cultures using HPLC with electrochemical detection (EC), and report on the effects of FA alone and in combination with fumonisin B1 (FB1) and deoxynivalenol (DON) on the levels of these MEL precursors.

5-Hydroxytryptophan↗

PubMed1991

Cytotoxic and immunotoxic effects of Fusarium mycotoxins using a rapid colorimetric bioassay.

A colorimetric MTT (tetrazolium salt) cleavage test was used to evaluate cytotoxicity of twenty-three Fusarium mycotoxins on two cultured human cell lines (K-562 and MIN-GL1) as well as their inhibitory effect on proliferation of phytohemagglutinin-stimulated human peripheral blood lymphocytes. The values of 50% inhibition of lymphocyte blastogenesis were very close to the 50% cytotoxic doses observed with the more sensitive cell line (MIN-GL1). T-2 toxin was the most cytotoxic with CD50 and ID50 values less than 1 ng/ml. Type A trichothecenes were the most cytotoxic followed by the type B trichothecenes; the non-trichothecenes were the least cytotoxic. The MTT cleavage test, in conjunction with cell culture, is a simple and rapid bioassay to evaluate cytotoxicity and immunotoxicity of Fusarium mycotoxins.

Cell Line, Transformed↗

PubMed1979

[Determination of the antibacterial activity of various mycotoxins using Bacillus thuringiensis (Berliner)].

Antibacterial activity of pure preparations of mycotoxins, was studied against strains of Bacillus thuringiensis (Berliner). Most of fungal metabolites used provokes: 1) Growth bacterial inhibition similar to lethal effect of antibiotics 2) Enlargment of cell volume; this cell abnormality induction resembles those obtained with mitomycin C. The bioassay of fungal toxins with B. thuringiensis can be recommended as a sensitive technique to supplement chemical characterization. By using specific cell lines to such a mycotoxin and specific inactivators as coumarin for aflatoxin, or sulfhydryl compounds for penicillic acid and patulin.

Aflatoxins↗

PubMed1991

Mycotoxin formation by different geographic isolates of Fusarium crookwellense.

Eighteen Fusarium crookwellense isolates from the continents of Australia, Europe, and North America were compared for their ability to produce mycotoxins on corn at 25 degrees C after 2 weeks. Extracts from corn fermented with each Fusarium isolate were analyzed by thin-layer chromatography (TLC) and gas chromatography/mass spectroscopy (GS/MS) for mycotoxins. Toxins detected were zearalenone (13 isolates), fusarin C (11 isolates), nivalenol (4 isolates), and diacetoxyscirpenol (2 isolates). Zearalenone and fusarin C were produced by isolates from each continent, while nivalenol was detected in the Fusarium isolates originating from Australia and one isolate from the United States.

Australia↗

PubMed1991

Differential cytotoxicity and mycotoxin content among isolates of Fusarium moniliforme.

Ten different isolates of the common corn fungus, Fusarium moniliforme, were cultured on corn, and the production by the isolates of two important mycotoxins, fusarin C and fumonisin B1, was compared. Additionally, both aqueous and organic extracts of the cultures were tested for cytotoxicity to rat primary hepatocytes by measuring the effects of three dose levels on the ability of the cells to take up valine and to cause the release of the cytoplasmic enzyme, lactate dehydrogenase. The fungal isolates differed drastically in their ability to produce the two mycotoxins and in their cytotoxicity. However the toxic effects could not be accounted for by the content of the two toxins measured. Therefore it appears that there are other toxins, both organic and aqueous soluble compounds, that are toxic to liver cells.

Animals↗

PubMed1996

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat.

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 micrograms/kg (DON) and 17 micrograms/kg (ZEA), maximum concentrations were 1800 micrograms kg-1 and 120 micrograms kg-1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1% and 0.7% of the samples, at at maximum level of about 100 micrograms kg-1. Only one sample was positive for T-2 (55 micrograms/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.

Bulgaria↗

PubMed1989

Production of trichothecene and non-trichothecene mycotoxins by Fusarium species isolated from maize in Minnesota.

Eighty-two cultures of Fusarium species isolated in 1986 from moldy maize in Minnesota were each cultured on rice for 4 weeks and found to produce the following mycotoxins: F. graminearum isolates, deoxynivalenol (DON, 4-225 micrograms/g), 3-acetyldeoxynivalenol (3-ADON, 2-4 micrograms/g), 15-acetyldeoxynivalenol (15-ADON, 1-35 micrograms/g) and zearalenone (ZEA, 5-4350 micrograms/g); F. moniliforme, fusarin C (detectable amounts to 1000 micrograms/g); F. moniliforme, F. oxysporum, F. proliferatum and F. subglutinans isolates, moniliformin (15-6775 micrograms/g); F. moniliforme, F. proliferatum, and F. subglutinans isolates, fusaric acid (detectable amounts). Other mycotoxins screened for in each rice sample and not detected were T-2 toxin, HT-2 toxin, neosolaniol, T-2 tetraol, nivalenol, fusarenon-X, scirpenols, alpha and beta trans-zearalenols, wortmannin, and fusarochromanone. The rat feeding bioassay indicated that other, unidentified toxins may be present.

Culture Media↗

PubMed1991

Mycotoxins and mycotoxigenic moulds in nuts and sunflower seeds for human consumption.

A survey was carried out to obtain data on the occurrence of mycotoxins and the mycotoxin-producing potential of fungi isolated from nuts (almonds, peanuts, hazelnuts, pistachio nuts) and sunflower seeds in Spain. Thin-layer chromatography was used to separate the toxins. Aflatoxins were detected in one sample of almonds (95 ppb aflatoxin B1 and 15 ppb aflatoxin B2) and in one sample of peanuts at a level below 10 ppb of aflatoxin B1. 100% of samples showed variable incidence of fungal contamination. The predominant fungi present in samples were Penicillium spp, Aspergillus niger, A. flavus, A. glaucus and Rhizopus spp. The results showed that isolates of different species were able to produce aflatoxins B1, B2, G1, and G2, sterigmatocystin, ochratoxin A, patulin, citrinin, penicillic acid, zearalenone, and griseofulvin.

Analysis of Variance↗

PubMed1988

Mycotoxin-producing potential of fungi isolated from red kidney beans.

The predominant fungi present in samples of reject and retail red kidney beans were Aspergillus glaucus, Penicillium spp. and Alternaria spp. Together with A. ochraceus, A. flavus, Fusarium spp., and Trichoderma, these isolates from the reject beans were screened for numerous mycotoxins by TLC. The most consistently produced mycotoxins were penicillic acid (from A. ochraceus and Penicillium spp.) and Alternaria toxins (tenuazonic acid and alternariol). A. glaucus strains were tested for cytotoxicity in three tissue culture cell lines with positive results.

Aspergillus↗

PubMed1990

A survey of the natural occurrence of Fusarium mycotoxins, deoxynivalenol, nivalenol and zearalenone, in cereals harvested in the Netherlands.

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.

Edible Grain↗

PubMed1981

Fungal contamination and mycotoxin-producing potential of dried beans.

A total of 604 samples of about 7 different types of beans was examined to determine their mycological profiles, and suitability for use as solid substrates for mycotoxin production. All of the samples were collected from bean jam makers in Tokyo by the official food examiners. Genera Penicillium and Aspergillus were predominant, and genus Wallemia was also found commonly in all types of beans. Mycotoxin-producing Aspergillus strains were isolated from 52 samples of beans, approximately 9% of the total. The highest incidence of toxigenic Aspergillus (14.1%) was found in kidney beans. Red beans and peas inoculated with Aspergillus ochraceus were found to produce about 7 to 8 times more toxin than was obtained in a liquid medium. and red beans inoculated with A. versicolor produced more toxin than was obtained in yeast extract sucrose broth. Green peas inoculated with Fusarium graminearum produced about 8 times more T-2 toxin than was obtained in 1% peptone containing Czapek solution under comparable culture conditions.

Aspergillus↗

PubMed1989

Immunochemical assay applied to mycotoxin biosynthesis: ELISA comparison of sterigmatocystin production by Aspergillus versicolor and Aspergillus nidulans.

Conventional thin layer and instrumental methods for analyzing mycotoxins and their precursors are time-consuming and make the investigation of mycotoxin biosynthesis particularly difficult. As an alternative, sensitive enzyme-liked immunosorbent assays (ELISAs) can be utilized to analyze for these compounds. In this report, sterigmatocystin production in test tube cultures of Aspergillus versicolor ATCC 18643 and Aspergillus nidulans ATCC 32610 were compared using competitive ELISA. Polyclonal antiserum that was prepared against a sterigmatocystin hemiacetal-bovine serum albumin conjugate exhibited greatest specificity for sterigmatocystin hemiacetal and sterigmatocystin with less reactivity for O-methylsterigmatocystin. The antiserum could be used to detect as little as 50 ng/ml sterigmatocystin in ELISA. Direct ELISA could be performed on diluted culture broth and on mycelial extracts solubilized with N,N-dimethylformamide. Aspergillus versicolor ATCC 18643 produced more sterigmatocystin in SLS medium than in YES medium, and showed maximal levels at between 9 to 12 days incubation. Approximately 75% of sterigmatocystin was detectable in mycelium (254 micrograms/ml culture) compared to the extracellular fraction (87 micrograms/ml culture). Aspergillus nidulans exhibited qualitatively similar patterns of growth and toxigenesis in SLS medium but accumulated maximal levels of only 15 micrograms mycelial sterigmatocystin/ml culture and 5 micrograms extracellular sterigmatocystin/ml broth, respectively.

Aspergillus↗