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PubMed2004

Production of a calibrant certified reference material for determination of the estrogenic mycotoxin zearalenone.

Several previous interlaboratory studies in the field of mycotoxin analysis have revealed considerable problems, apparent as high between-laboratory standard deviations, or rather non-comparable and non-traceable results. A major reason is lack of proper calibrants for external calibration. Public awareness of substances that mimic or interfere with the activity of natural hormones (endocrine disrupters) has led to increased interest in mycotoxins with estrogenic potential, e.g. zearalenone (ZON). During a large-scale standard measurement and testing (SMT) project of the European Commission (EC) dealing with the preparation and certification of reference materials for determination of the mycotoxin ZON in maize, a ZON calibrant in acetonitrile was prepared and intensively checked for purity, homogeneity, and stability. Preparation of the material, study of its homogeneity and stability, and characterisation of the calibrant on the basis of its preparation, with discussion of the results obtained, are described in this paper. The certified value of 9.95 micro g mL(-1) for ZON in acetonitrile and its corresponding expanded uncertainty of +/-0.30 micro g mL(-1) were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement (GUM).

Analysis of Variance↗

PubMed2004

Production of certified reference materials for mycotoxins: IRMM's view on the assessment of uncertainties.

Analytical difficulty and the economic importance of controlling mycotoxin levels in food and feed led the Community Bureau of Reference (BCR) to prepare a series of certified reference materials (CRM) for various mycotoxins. Because of the wide acceptance of these CRM and the need to ensure the comparability and traceability of measurements in the future it is necessary to prepare and certify new batches of mycotoxin reference materials (RM). In the following text two different approaches for evaluation of the characterisation uncertainty of CRM will be compared using the certification of aflatoxin M(1) (AfM(1)) in milk powder as an example. The conventional approach is based on evaluation of characterisation exercise data; the alternative approach is based on measurement uncertainties of the employed analytical methods. Because laboratories are using totally different approaches to estimate the measurement uncertainties, combination of the uncertainties obtained from the participating laboratories was not recommended. Therefore, a new integrated approach for assessment of the measurement uncertainties of the analytical methods on the basis of additional data collected during the characterisation exercise will be described. The conventional approach was found to be the most appropriate and economical approach to evaluate the characterisation uncertainty as a characterisation exercise must be performed anyway to establish the property values of candidate (C)RM, irrespective of whether or not reliable measurement uncertainties can be provided by the laboratories. An integrated approach for assessment of measurement uncertainties based on additional characterisation data as applied here to enable use of an uncertainty-based approach provides more information but is too time-consuming and cost-intensive to become common practice.

Aflatoxin M1↗

PubMed2001

The state-of-the-art in the analysis of estrogenic mycotoxins in cereals.

The increasing public awareness of chemicals that mimic or otherwise interfere with the activity of natural hormones - so-called endocrine disrupters - has also led to greater study of mycotoxins with estrogenic potential. The purpose of this paper is to introduce the topic of estrogenic mycotoxins and to discuss the state-of-the-art in the analysis of these substances in cereals, with special emphasis on zearalenone (ZON) as its most relevant representative. Because the use of immunoaffinity columns (IAC) followed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and immunoassays are currently the most frequently used methods for the determination of ZON and its metabolites, these techniques are discussed in more detail. Recent papers, which have revealed the great potential of HPLC-MS(MS) for the simultaneous detection and identification of several estrogenic mycotoxins, are discussed. The performances of the state-of-the-art methods are finally compared by study of the results obtained in recent international intercomparison studies. On the one hand, these studies revealed the good performance of both chromatographic and antibody-based methods. On the other hand, the need for better means of external quality assurance measures, especially the availability of certified reference materials and certified standards, has clearly been demonstrated.

Chromatography, High Pressure Liquid↗

PubMed2006

Mycotoxins in poultry feed in Kuwait.

A survey was conducted at a poultry feed production unit in Kuwait for mycotoxin contamination in the samples of yellow maize, soybean meal, wheat bran used as raw material and the poultry feed prepared for broiler starter, broiler finisher, and layer mash. Individual aflatoxins were determined by high-pressure liquid chromatography after immunoaffinity column purification. Repeated analysis revealed average aflatoxin concentration in maize at 0.27 ppb (range 0 to 1.69 ppb), soybean meal at 0.20 ppb (range 0 to 1.27 ppb), wheat bran at 0.15 ppb (range 0 to 1.07 ppb), prepared poultry feed for broiler starter at 0.48 ppb (range 0 to 3.26 ppb), broiler finisher at 0.39 ppb (range 0 to 1.05 ppb), and layer mash at 0.21 ppb (range 0 to 1.30 ppb). Other mycotoxins (ochratoxin, fumonisin, deoxynivalenol (DON), and zearalenone), were detected by quantitative enzyme-linked immunosorbent assay. The average levels of ochratoxin A ranged from 4.6 to 9.6 ppb, fumonisin from 1.4 to 3.2 ppm, DON from 0.17 to 0.29 ppm, and zearalenone from 46.4 to 67.6 ppb in various commodities and prepared feed samples. The study revealed the coexistence of determined mycotoxins, although their concentrations in general were found to be lower than the permissible levels, wherever defined, for the poultry feed.

Animal Feed↗

PubMed2004

Combined effects of selected Penicillium mycotoxins on in vitro proliferation of porcine lymphocytes.

The in vitro effect of combinations of the Penicillium mycotoxins citrinin (CIT), cyclopiazonic acid (CPA), ochratoxin A (OTA), patulin (PAT), penicillic acid (PIA) and roquefortine C (RQC) on mitogen induced lymphocyte proliferation was determined using purified lymphocytes from six piglets. Dose-response curves for each mycotoxin and mycotoxin combinations were generated. The combined effects of toxin pairs based on IC20 were illustrated in isobole diagrams and statistically calculated. OTA and CIT elicited a synergistic effect. Four toxin pairs elicited additive effects, four pairs less-than-additive effects and six pairs independent effects. Thus, the majority of toxin pairs tested produced lower combined effects than an additive effect. The results indicate that the sum effect of all toxins is less than that from the summation of concentrations of the individual compounds, adjusted for differences in potencies.

Animals↗

PubMed1978

Mutagenicity to Salmonella typhimurium of some Aspergillus and Penicillium mycotoxins.

17 mycotoxins produced by various Aspergillus and Penicillium species were screened for their mutagenic activity to Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, both with and without metabolic activation. Austdiol, austocystins A and D, kojic acid and viridicatumtoxin were found to be mutagenic after metabolic activation, while austdiol was also mutagenic per se. Aflatoxin B1, sterigmatocystin and versicolorin A, which were used as positive controls were also mutagenic. No mutagenic activity was evident in the case of citrinin, cyclopiazonic acid, fumitremorgen B, griseofulvin, luteoskyrin, O-methylsterigmatocystin, mycophenolic acid, ochratoxin A, patulin, penicillic acid, secalonic acid D and TR2-toxin. A good relationship was found between the mutagenic activity, or lack of it, of most of the mycotoxins with existing data on carcinogenicity. Inadequate information on the carcinogenicity of austdiol, austocystins A and D, kojic acid and viridicatumtoxin precluded correlations with mutagenicity to S. typhimurium. The relationship between chemical structure and mutagenicity of the mycotoxins is discussed.

Aspergillus↗

PubMed1995

Mycotoxin-producing potential of fungi isolated from amaranth seeds in Argentina.

To evaluate the potential for mycotoxin production by molds in amaranth grains, the mycoflora was determined both before and after surface disinfection on dichloran-chloramphenicol-peptone agar (DCPA) and dichloran-18% glycerol agar (DG18). On both media Aspergillus, Penicillium, Fusarium and Alternaria were the predominant genera. A flaus and A. parasiticus were the Aspergillus species most frequently isolated. P. chrysogenum was the species most common among the penicillia. F. equiseti was the predominant Fusarium species. Isolates of Aspergillus, Penicillium and Fusarium were screened for mycotoxin production on sterile rice substrate and also using a simple screening method. Toxinogenic strains of A. flavus and A. parasiticus (aflatoxins), A. versicolor (sterigmatocystin), P. citrinum (citrinin), P. viridicatum (penicillic acid), F. moniliforme, F. equiseti and F. semitectum (zearalenone), were encountered. The simple screening method for toxinogenic molds showed good performance for the detection of molds producing aflatoxins and zearalenone compared with mycotoxins production on the natural substrate.

Aflatoxins↗

PubMed1987

Effect of the esophageal carcinogen methylbenzylnitrosamine and of a putative potentiating factor, a trichothecene mycotoxin, on O6-methylguanine-dna methyl transferase in rat esophagus and liver.

Epidemiological evidence from China and South Africa has implicated Fusaria mycotoxins in the etiology of esophageal cancer, although treatment of animals with extracts of Fusaria cultures did not cause cancer of the esophagus. Fusaria are the major producers of trichothecenes, and animal experiments have shown that these mycotoxins can damage the esophagus but they have not been shown to cause esophageal cancer. A plausible concept is therefore that esophageal cancer is initiated by the potent environmental esophageal carcinogens, certain nitrosamines, but that the levels of exposure are too low to cause clinical cancer unless their effects are enhanced by additional risk factors. Among the most likely enhancing factors in the regions mentioned above are Fusaria mycotoxins. As trichothecenes are known to inhibit sulphydryl-dependent reactions and to inhibit protein synthesis, experiments were carried out to determine whether potentiation of cancer could be mediated via inhibition of the DNA repair protein O6-methylguanine-DNA methyl transferase (O6MG-MT). The effect of diacetoxyscirpenol (DS) on O6MG-MT was studied. Chronic or acute treatment with DS did not alter the level of O6MG-MT in esophagus, or affect the depletion which occurs after injection of methylbenzylnitrosamine, or alter the rate of reappearance of O6MG-MT. A high dose of DS induced O6MG-MT in liver. These results suggest that if trichothecenes are risk factors for esophageal cancer, the effect is unlikely to be mediated by inhibition of O6MG-MT. Induction of the repair protein in liver may be relevant in the animal toxicoses caused by consumption of trichothecenes, but is unlikely to be implicated in the etiology of liver cancer in man.

Animals↗

PubMed1995

Mycotoxins, general view, chemistry and structure.

Mycotoxins induce diverse and powerful biological effects in test systems; some are carcinogenic, mutagenic, teratogenic, estrogenic, hemorrhagic, immunotoxic, nephrotoxic, hepatotoxic, dermotoxic, and neurotoxic. Mycotoxins have been unambiguously linked to the etiology of several diseases in animals. The discovery of aflatoxins in the early 1960s led to the resurgence of interest in human mycotoxicoses; mycotoxins are now recognized as causal factors of primary liver cancer, ergotism and alimentary toxic aleukia. The fumonisins and ochratoxins are suspected of playing a role in the etiology of esophageal cancer and Balkan endemic nephrotoxicity, respectively.

Aflatoxins↗

PubMed1995

Genetic implications in the metabolism and toxicity of mycotoxins.

In common with many xenobiotics, metabolic activation and detoxification play crucial roles in the determination of a toxic response of animal species, including man, to exposure to mycotoxins. Control of expression of the relevant enzymes, both constitutive and inducible, is therefore a major factor in mycotoxin-induced acute or chronic toxicities. The involvement of these factors in the toxic responses to aflatoxins and ochratoxins will be briefly reviewed. In the case of the aflatoxins, the importance of secondary, conjugating metabolism has become increasingly evident. The specific control of expression of these enzymes, through sequences present in the 5' region of the gene, is evident (e.g. by the changes seen during development and differences between the sexes). The existence of these control mechanisms has made feasible the development of chemoprotective strategies. Although less detailed information is available concerning the metabolic activation and detoxification of the ochratoxins, it appears probable that future studies will reveal a role for the genetic control of expression of enzymes responsible for the target nephrotoxicity of these mycotoxins.

Aflatoxin B1↗

PubMed2006

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

Deoxynivalenol (DON) and its hydroxylated form nivalenol (NIV) are Fusarium mycotoxins that occur in cereal grains alone or in combination. Several studies have shown that these metabolites affect lymphocyte functions. However, the molecular mechanisms underlying their activities are still partially known. To address this issue, we examined the influence of NIV and DON in modulating 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.

Cell Survival↗

PubMed2005

The effect of substrate on mycotoxin production of selected Penicillium strains.

Analytical methods are presented for detecting simultaneously 11 fungal metabolites (aflatoxins B1, B2, G1 and G2, citrinin, cyclopiazonic acid, mycophenolic acid, ochratoxin A, penicillic acid, penitrem A and roquefortine C) on different matrices. The methods were applied to determine the mycotoxins produced by different Penicillium crustosum, Penicillium nordicum and Penicillium verrucosum strains on yeast extract sucrose (YES) agar and cheese and bread analogues and are based on high-performance liquid chromatography (HPLC) and photodiode array detection (PDA). The growth substrate had a distinctive effect on the mycotoxin production ability of the fungi examined. The P. crustosum strains produced roquefortine C on all the substrates, with the highest amounts being detected on the cheese analogue. Penitrem A was synthesised on the cheese analogue only. The strains of P. verrucosum produced exclusively citrinin on YES, but both ochratoxin A and citrinin were detected in considerable amounts on the bread analogue. On the bread, toxin profiles varied significantly between the individual P. verrucosum strains. The cheese analogue was not favourable for the mycotoxin production of this species. The growth substrate had the least effect on the toxin production of the P. nordicum strains, which synthesised ochratoxin A in moderate amounts on all three media.

Bread↗

PubMed2005

Occurrence of mycotoxin producing fungi in bee pollen.

The natural mycobiota occurring in bee pollen is studied in the present report with special attention to analyze the incidence of fungal species that are potential producers of mycotoxins. A total of 90 ready-to-eat bee pollen samples were analyzed. Eighty-seven samples were collected in stores placed in different Spanish areas and three were from Buenos Aires (Argentina). The statistical results (ANOVA) showed that yeasts and Penicillium spp. were the predominant fungi. With regard to the potential mycotoxin producing species, Penicillium verrucosum, Aspergillus niger aggregate, Aspergillus carbonarius, Aspergillus ochraceus, Aspergillus flavus, Aspergillus parasiticus and Alternaria spp. were found. The last genus was isolated very frequently. The potential ability for producing ochratoxin A (OTA) and aflatoxins B(1), B(2), G(1) and G(2) was studied by culturing in vitro the isolates followed by analysis of these mycotoxins in culture extracts by HPLC with fluorescent detection. It was found that 100%, 53.3%, 33.3% and 25% of the isolates of A. carbonarius, A. ochraceus, P. verrucosum and A. niger aggregate, respectively, produced OTA. Moreover, 28.6% of the isolates from the A. flavus plus A. parasiticus group were able to produce aflatoxin B(1). Aflatoxin B(2) was detected in only 10% of the cultures. Aflatoxins G(1) and G(2) were not detected in cultures under the assayed conditions. This is the first report carried out on the natural mycobiota occurring in bee pollen in general and on the toxigenic capability of these isolates in particular.

Aflatoxins↗

PubMed2004

Cytotoxic effects of the mycotoxin beauvericin to human cell lines of myeloid origin.

Beauvericin, a cyclic hexadepsipeptide of potential importance to the health of humans and domestic animals, has been reported to exert cytotoxic effects on several mammalian cell types and to induce apoptosis. We investigated the cytotoxicity of this compound to two human cell lines of myeloid origin: the monocytic lymphoma cells U-937 and the promyelocytic leukemia cells HL-60. In some experiments HL-60 cells partially differentiated towards the eosinophilic phenotype were also used. Cultures of U-937 cells and HL-60 cells in stationary phase were exposed to beauvericin at concentrations ranging from 100 nM to 300 microM for periods of time of 4 and 24h, respectively. The effects of beauvericin on cell viability were assessed by the Trypan blue exclusion method. In another set of experiments, performed with U-937 cells, the mycotoxin was included in the culture medium at passaging, in order to assess its possible effects on cell growth. Viability of both U-937 cells and HL-60 cells was not affected by beauvericin at concentrations up to 3 microM, after 4h exposure, whereas a steady decline was seen at higher concentrations. Similarly, after an exposure time of 24h, a decline in viability was observed in cultures exposed to beauvericin at a concentration of 10 microM or higher. Thus, 50% cytotoxic concentrations at 24h of congruent with 30 microM and congruent with 15 microM were estimated for U-937 cells and HL-60 cells, respectively.Similar experiments were performed with cultures of HL-60 cells partially differentiated towards the eosinophilic phenotype, revealing that, in 4h exposure experiments (but not in 24h experiments), the viability of these cultures underwent a significantly less pronounced decline, in comparison to undifferentiated HL-60 cultures. Interestingly, when U-937 cells were allowed to proliferate in the presence of the mycotoxin, included in the culture medium at passaging, a substantial cytotoxicity was observed at lower concentrations, compared with prevalently resting, stationary phase cultures. Accordingly, a definite inhibition of the proliferative capability of the cells was detected. The information provided by this work may be useful in selecting appropriate myeloid cell models for the development of biossays aimed at detecting beauvericin (and, possibly, other mycotoxins) in foods and other commodities.

Cell Division↗

PubMed2004

Solutions to Penicillium taxonomy crucial to mycotoxin research and health.

The stability of taxonomy within Penicillium is reviewed with respect to mycotoxin production. Identification remains difficult despite the availability of modern methods. Proficiency testing is rare and conventional identifications do not inform reliably as to whether mycotoxins were detected/produced. A solution which consists of identifying a Penicillium strain as terverticillate and then undertaking mycotoxin analysis is described.

Health↗

PubMed2005

In vitro investigation of individual and combined cytotoxic effects of ochratoxin A and other selected mycotoxins on renal cells.

Hundreds of mycotoxins are known to date and many of them are of great interest with regard to human and animal health since they are detected frequently in plant-derived products. Various mycotoxins may occur simultaneously, depending on the environmental and substrate conditions. Considering this coincident production, it is very likely, that humans and animals are always exposed to mixtures rather than to individual compounds. Therefore, future risk assessments should consider mixture toxicity data. This is particularly true for ochratoxin A (OTA), ochratoxin B (OTB), citrinin (CIT) and occasionally for patulin (PAT) as they are all produced by a number of Penicillium and Aspergillus species. Therefore, these four toxins were chosen to study the interactive effects in vitro, using the well-established porcine renal cell line LLC-PK1 and the MTT reduction test as a cytotoxicity endpoint. By application of a step-wise approach to test combination toxicity, using various full factorial as well as a central composite experimental designs, the interactive (synergistic) cytotoxic effects of the these four toxins were assessed. The results obtained in this study confirm a potential for interactive (synergistic) effects of CIT and OTA and possibly other mycotoxins in cells of renal origin.

Algorithms↗

PubMed2004

Novel sampling methods for the analysis of mycotoxins and the combination with spectroscopic methods for the rapid evaluation of deoxynivalenol contamination.

A novel non-destructive sampling approach is described for the identification of food matrices contaminated with deoxynivalenol and other mycotoxins. This technique is different from currently applied sampling procedures for this purpose and is based on the principle that surface material from the tested goods is collected on a filter and brought to chemical or spectroscopic analysis. This approach has been applied to several matrices and mycotoxins, with a focus on those mycotoxin-matrix combinations that are of main relevance due to current or future legislation. Tests were carried out with a facility that has been shown to be suitable to process large quantities of materials at points of transaction, such as harbour. Further experiments with a small sampling lance prototype showed that analytical results from the chemical analysis of the tested goods can be correlated with the results obtained with this novel sampling procedure.

Chemistry Techniques, Analytical↗

PubMed1985

Thin-layer chromatography of mycotoxins.

TLC has become an extremely powerful, rapid and in most instances inexpensive separation technique in mycotoxicology. This review presents achievements of its applications in this field. General technical aspects of the TLC of mycotoxins that are discussed include extraction and clean-up procedures, adsorbents and solvent systems, detection methods, two-dimensional TLC, high-performance TLC (HPTLC), quantitation and preparative TLC (PLC). Special applications of TLC deal with multi-mycotoxin analyses and with structurally related or individual mycotoxins (aflatoxins, sterigmatocystins, versicolorins, ochratoxins, rubratoxins, patulin, penicillic acid, mycophenolic acid, butenolide, citreoviridin, trichothecenes, cytochalasans, tremorgenic toxins, epipolythiopiperazine-3,6-diones, hydroxyanthraquinones, zearalenone, citrinin, secalonic acids, cyclopiazonic acid, PR toxin, roquefortine, xanthomegnin, viomellein and naphtho-gamma-pyrones).

Aflatoxins↗