Cowpeas (Vigna unguiculata (L.) Walp) is an important African indigenous legume crop for the livelihoods of many relatively poor people residing n less developed countries of the tropics. Rural families derive a nutritious food, animal feed and in income from the production of this crop. Storage of seed is certainly the most important post-harvest operation but the losses incurred are great. These losses, due to an inability to effectively control physical and biological factors, result in problems with storage insects, moisture and associated fungi. Seeds are particularly susceptible to fungal contamination when stored at high ambient temperatures and relative humidities. To determine the storage fungi associated with cowpea seeds, surface-sterilised cowpea seeds (200 seeds from each of nine cultivars) were plated out n malt extract agar. After 5-7 days incubation at 25°C, the most dominant and common fungi recorded were Alternaria spp. followed by Penicillium spp., Aspergillus flavus and A. niger. The influence of a three-year cold storage period at ± 5°C on the fungi associated with the seeds was also investigated. Alternaria, Aspergillus and Penicillium spp. appeared to dominate. Some fungal species recorded prior to cold storage were not recorded thereafter. Certain storage fungi are known to produce mycotoxins, which are secondary fungal metabolites that are toxic to both farm animals and humans, under poor storage conditions. The presence of the fusarial mycotoxins, fumonisin BI, B2 and B3 in four cowpea cultivars (Bechwana Whit, Glenda, Iron Grey, Rhino) was investigated. The samples were extracted with methanol/water (70:30 v/v) and cleaned-up on strong anion exchange solid phase extraction cartridges. High performance liquid chromatography with pre¬column derivatisation using o-phthaldialdehyde (OPA) was used for the detection and quantification of fumonisin Bl, B2 and B3. All sampIes were contaminated with FBI, with levels ranging from 81-1002 ng g-I. Fumonisin B2 and B3 were not detected in any samples. This is believed to be the first report of fumonisin BI in cowpea seeds. Since the known fumonisin-producing Fusarium species were not found in the six different Fusarium species isolated from these four cultivars, further investigations are required to determine which fungal species are species are responsible species are responsible for the FBI production. An alternative approach to the prevention and control of fungal contamination and mycotoxin production of seeds by treating cowpea seed with essential plant oils was tested. The inhibitory activity of five essential oils (thyme, clove, peppermint, soybean and peanut) was investigated, in vitro and in vivo, on five fungal species (A. flavus, A. niger, Penicillium chrysogenum, Fusarium oxysporum and F. equiseti) commonly associated with cowpea seeds and on two cowpea cultivars. Thyme and clove oil significantly inhibited the growth of all five fungal species in vitro at 500 and 1000 ppm, while peppermint oil was successful at 2000 ppm. Peanut and soybean oil did not show any significant inhibition of fungal growth. The in vivo effect of thyme, clove and peppermint oils on naturally infected seed revealed that only thyme at 1000 ppm reduced fungal growth of storage fungi in the PAN 325 cultivar. In the PAN 311 cultivar, thyme and clove oils at 1000 ppm and peppermint oil at 2000 ppm significantIy reduced growth of storage fungi. In artificially infected seed, all three oils significantly inhibited the growth of P. chrysogenum. Thyme reduced the growth of F. oxysporum and F. equisetii, whilst peppermint oil inhibited only F. oxyspomm. These oils did not seem to adversely affect the germination nor emergence of cowpea seed. The storage fungi significantly reduced percentag germination and emergence of the white (IT 93K452-1) seed but had little or no effect on the brown (CH 14) seed. Furthermore, all three oils significantly inhibited the storage fungi on the white seed, possibly increasing the percentage germination and emergence. / Dissertation (MSc (Botany))--University of Pretoria, 2006. / Plant Science / unrestricted

L'intestin est la première barrière de l'organisme contre les contaminants alimentaires, dont les mycotoxines. Le déoxynivalenol (DON) est un contaminant majeur des céréales, souvent retrouvé en association avec d'autres trichothécènes B (TCTs B), le 3- et 15-acétyldéoxynivalénol (3-ADON et 15-ADON), le nivalénol (NIV) et la fusarénone X (FX). Au niveau cellulaire, le DON interagit avec l'ARN ribosomique, bloquant ainsi la synthèse protéique et activant la cascade de la voie de signalisation de MAPKinases impliquée dans des mécanismes de la réponse inflammatoire. Au niveau intestinal, cette mycotoxine pourrait donc perturber le renouvellement continu de l'épithélium, et l'homéostasie de la réponse inflammatoire. On suggère ainsi qu'elle pourrait jouer un rôle dans la pathogénie des maladies inflammatoires chroniques de l'intestin. Si les effets du DON sont relativement connus, ceux du NIV et de leurs dérivés acétylés sont moins bien documentés. De même, peu de données existent quant à la toxicité combinée de ces mycotoxines dont la co-occurrence est avérée. Sur des modèles in vitro de cellules épithéliales intestinales humaines et porcines et sur un modèle ex vivo d'explants de jéjunum de porc, nous avons comparé les toxicités individuelles de cinq TCTs B (DON, 3- et 15-ADON, NIV et FX) et analysé leur toxicité combinée en termes de synergie, additivité ou antagonisme vis-à-vis de l'intestin. Les résultats montrent qu'à des concentrations de l'ordre du micromolaire, les TCTs B inhibent la croissance des cellules épithéliales intestinales par ordre croissant de toxicité 3-ADON, DON, 15-ADON, NIV et FX. Aux faibles doses correspondant à des niveaux d'exposition rencontrés chez le consommateur français ou européen, des synergies d'un facteur 3 à 10 ont été observées. Ces travaux ont également permis de caractériser l'activité pro-inflammatoire au niveau intestinal des TCTs B, et l'analyse benchmark de données de transcriptomique a montré que l'exposition de l'intestin à des doses aussi faibles que 0.04µM de FX, 0.1µM de DON ou 0.1µM de NIV s'accompagne d'une activation significative des mécanismes de l'inflammation. Ces doses sont de l'ordre des concentrations attendues dans le chyle sur la base des valeurs toxicologiques de référence actuelles. En conclusion, ces données montrent que le renouvellement de l'épithélium intestinal et l'activité pro-inflammatoire au niveau intestinal pourraient être des marqueurs très sensibles dans le cadre de l'évaluation de la toxicité individuelle et des interactions entre TCTs B. / As for other food-born contaminants, the gastro-intestinal tract represents the first barrier against deoxynivalenol (DON). This mycotoxin frequently co-occurs with other type B trichothecenes (TCTs B) namely 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX). At the cellular level, DON binding to ribosomal RNA results in the inhibition of protein synthesis and triggers the mitogen-activated protein kinases (MAPKs) pathway that have been linked to immune response mechanisms. Thus, intestinal epithelial cell renewing is considered a putative target in DON toxicity. Moreover, based on the ability of DON to disturb the state of homeostasis of the inflammatory response in the intestine mimicking what is found in inflammatory bowel diseases (IBD), it is proposed that this mycotoxin may play a role in such diseases. However, very few is known about the intestinal toxicity of the other co-occuring TCT B, and their combined effects eventually. By means of in vitro human and porcine intestinal epithelial cells models and an ex vivo porcine jejunal explants model, we assessed the individual toxicity of five TCT B (DON, 3- and 15-ADON, NIV and FX) toward the intestine and we analyzed their combined toxicity in terms of additivity, synergy or antagonism. The tested TCT B significantly impaired the intestinal epithelial cell growth in the micromolar range, in increasing order of potency 3-ADON, DON, 15-ADON, NIV and FX. The toxicity of low doses of TCT B was synergistic. For mycotoxin concentrations corresponding to exposure levels reported for French and European consumers, the amplitude of this synergy ranged between 3 and 10. Benchmark dose analyses of the transcriptional data also showed that the exposure of the intestine to mycotoxin concentrations as low as 0.04µM for FX, 0.1µM for DON and 0.1µM for NIV could be associated to a significant activation of the inflammatory response mechanisms. Taken together, these results suggest that epithelial cell renewing and pro-inflammatory effects at the intestinal level may be consider very sensitive biomarkers for the assessment of the individual toxicity and interactions between the co-occurring TCTs B.

Fusarium graminearum est un champignon pathogène des plantes, responsable de la fusariose de l'épi (plus connue sous le nom de Fusarium Head Blight : FHB) sur céréales, notamment sur le blé et le maïs. En interaction avec la plante, le champignon produit des mycotoxines, parmi lesquellse le déoxynivalénol (DON), dont la finalité pour le champignon producteur est méconnue mais qui sont toxiques pour les humains et les animaux. Ainsi la qualité des grains contribue fortement aux pertes de rendement observées et les résidus contaminés restent au champ. Une première revue bibliographique (Leplat et al 2012) a mis en évidence l'importance des résidus de culture (habitat écologique) pour la survie saprophyte du champignon, pour sa reproduction sexuée et pour l'établissement de l'inoculum primaire susceptible d'infecter la prochaine culture. Une seconde revue bibliographique a souligné les lacunes en ce qui concerne le rôle que les mycotoxines pourraient jouer dans la survie de F. graminearum dans un cet habitat. L'objectif principal de cette thèse était donc de vérifier si la présence de mycotoxines dans les résidus de récolte donne un avantage compétitif à F. graminearum vis-à-vis des composantes biotiques du sol et des résidus et notamment les champignons, les bactéries, les protozoaires, les nématodes et les vers de terre. L'impact du DON sur ces différentes communautés a été évalué dans des résidus de maïs et de blé, au champ et en microcosmes, en condition de labour et de travail superficiel du sol. Le développement de la maladie et ses conséquences sur le rendement ont été observés dans l'expérience de terrain à l'Unité Expérimentale de l'INRA de Dijon.Au cours de cette étude, la survie et les dynamiques de développement de la souche modèle d'étude F. graminearum MIAE00376 et des communautés fongiques et bactériennes ont été mesurées en utilisant la réaction de polymérisation en chaîne en temps réel (Q-PCR) ainsi que par comptage sur boîtes. Dans le même temps, l'évolution des structures des communautés microbiennes a été déterminée par analyse du polymorphisme de longueur des fragments de restriction terminaux (T-RFLP). Les nématodes et les vers de terre ont été quantifiés par extraction et observations à l'œil ou a la loupe binoculaire. Le DON introduit dans le sol et les résidus a été extrait et quantifié au cours du temps par chromatographie liquide haute performance (CLHP). Des dynamiques de population de la souche MIAE00376 associée à différents microorganismes isolés de paille en décomposition et sélectionnés pour leur résistance au DON, à des bactéries fixatrice d'azote et à des Fusarium sp. appartenant au complexe fongique du FHB ont été mesurées en microcosmes de paille en présence ou non de DONLes résultats suggèrent que le DON dans les résidus de culture a une incidence sur les composantes biotiques du sol, mais l'impact dépend des communautés et de la localisation des résidus (en surface ou incorporés dans le sol). La biomasse moléculaire montre que les densités bactériennes et fongiques ont été significativement affectées par la présence de DON. La présence de DON a joué un rôle significatif sur la structure des communautés bactériennes et protozoaires, plus faible sur les communautés fongiques et nul sur les nématodes voire positif sur les vers de terre.Il est conclu que le DON est rapidement inaccessible en profondeur et un peu moins rapidement en surface (immobilisation ou dégradation), qu'il ne confère pas d'avantage compétitif au champignon producteur et que la gestion de l'habitat privilégié que constituent les résidus de culture pour F. graminearum peut être envisagée par le travail du sol en favorisant la décomposition rapide des résidus, par le labour ou l'utilisation d'organismes décomposeurs indigènes ou introduits. / Fusarium graminearum is a plant pathogenic fungus, causing devastating disease “Fusarium head blight” (FHB) in cereals including wheat and maize. It also contaminates the grains with mycotoxins including deoxynivalenol (DON) which are toxic to human and animals. This disease has resulted in the serious losses in grain yield and quality. We established through a first bibliographic review that during off season fungus survives saprophytically on the crop residues (ecological habitat) and serves as primary inoculum for the next season crop. However, we noticed also that the literature was poor about the role mycotoxins could play in the establishment of F. graminearum in such a habitat. The main aim of this thesis was therefore to test whether the presence of mycotoxins in the crop residues gives an advantage to F. graminearum to survive and develop a primary inoculum in the presence of the whole soil biota including fungi, bacteria, protozoa, nematodes and earthworms. We studied the impact of DON on the soil communities in the field as well as in microcosms, in wheat as well as in maize residues under tillage and no-tillage conditions. The disease development and the yield were noted in the field experiment. Some DON resistant active fungal decomposers and nitrogen fixing bacteria were picked and the dynamics of F. graminearum was observed by accelerating decomposition of crop residue in their presence, in the presence or absence of DON.During this study, the dynamic and survival of F. graminearum and total fungal and bacterial communities were examined by using quantitative real time polymerase chain reaction (qPCR) as well as by plate counting. At the same time, the structures of microbial communities were determined by using terminal restriction fragment length polymorphism analysis (T-RFLP). The DON resistance of isolated fungal decomposers and nitrogen fixers was tested by using minimal inhibitory concentration test (MIC). Nematodes and earthworms were quantified through binocular observations. The fate of DON was determined by quantifying the mycotoxin by high performance liquid chromatography (HPLC).The results suggested that DON in crop residues showed an impact on the biotic components of the soil but the impact depended on the communities and on the location of the residues (on surface or incorporated in the soil). The molecular biomass shows that the fungal and bacterial densities were significantly affected by the presence of DON. The presence of DON played significant role on the structure of bacterial and protozoan community while the nematodes and fungal communities remained unaffected. MIC results showed that the susceptibility of some competitive fungal strains towards DON was dependent on the dose of mycotoxin. The earthworms (Lumbricus terrestris) were not affected by the presence of mycotoxin. The degradation of DON in the residues was dependent on the time, the location of residues and the soil biota. The quantification of F. graminearum suggested that the presence of DON gave no advantage in the survival and development of primary inoculum during the decomposition of crop residues in the soil. We conclude that fungal decomposers can be selected on their enzymatic potential towards organic matter more than on the DON resistance to increase the degradation of the straw left at the surface and limit the subsequent development of F. graminearum.

This master thesis deals with a monitoring of a content of the trichothecene mycotoxins deoxynivalenol, nivalenol, T-2 toxin and HT-2 toxin in malting barley using the LC-MS/MS method. The theoretical part describes general characteristics of mycotoxins and their significant producer filamentous fungus of Fusarium species. Further, important trichothecene mycotoxins and mycotoxins generally which are commonly found in malting barley were also characterized. In the theoretical part of the thesis possibilities for a determination of the mycotoxins by the chromatographic methods were presented too; the immunochemical methods were also mentioned. In the experimental section an analysis of the B type trichothecenes was optimized by LC/APCI-MS/MS and of the A type trichothecenes by LC/ESI-MS/MS. When analyzing 57 samples of different barley varieties the deoxynivalenol reached the highest values (up to 945,2 µg.kg-1), namely in the case of the Sebastian variety with corn as the fore-crop. The highest values of nivalenol, T-2 toxin and HT-2 toxin (138,4 µg.kg-1; 21,8 µg.kg-1 and 68,7 µg.kg-1 respectively) were found in the Prestige variety of barley with winter wheat as the fore-crop. Subsequently a second set of four experimental samples of the Sebastian variety of barley and malt produced from the variety with corn as the fore-crop were analysed. In this group three samples were artificially infected with the filamentous fungi of Fusarium species; the fourth sample was not artificially infected and served as a control sample. Even in the case of the artificially infected samples the deoxynivalenol reached the highest values. The master thesis was implemented in the Research Institute of Brewing and Malting, Plc. in Brno.

Fungi represent one of the most diverse and abundant eukaryotes on earth. The interplay between mold exposure and the host immune system is still not fully elucidated. Literature research focusing on up-to-date publications is providing a heterogenous picture of evidence and opinions regarding the role of mold and mycotoxins in the development of immune diseases. While the induction of allergic immune responses by molds is generally acknowledged, other direct health effects like the toxic mold syndrome are controversially discussed. However, recent observations indicate a particular importance of mold/mycotoxin exposure in individuals with pre-existing dysregulation of the immune system, due to exacerbation of underlying pathophysiology including allergic and non-allergic chronic inflammatory diseases, autoimmune disorders, and even human immunodeficiency virus (HIV) disease progression. In this review, we focus on the impact of mycotoxins regarding their impact on disease progression in pre-existing immune dysregulation. This is complemented by experimental in vivo and in vitro findings to present cellular and molecular modes of action. Furthermore, we discuss hypothetical mechanisms of action, where evidence is missing since much remains to be discovered.

Streptococcus suis est un agent pathogène bactérien important qui affecte les porcelets post-sevrés et qui cause des septicémies, des méningites et des arthrites. L’infection se transmet principalement par voie respiratoire, cependant il existe une hypothèse que la voie intestinale pourrait aussi représenter une porte d’entrée pour le pathogène. Le déoxynivalénol (DON) est une mycotoxine soupçonnée depuis longtemps d’être un facteur prédisposant dans le développement de la maladie causée par S. suis, mais cela n’a encore jamais été étudié. L’objectif de cette étude est donc d’évaluer l’effet du DON sur les interactions de S. suis avec les cellules dendritiques (DCs) porcines, les macrophages alvéolaires porcins (PAMs) et les cellules épithéliales intestinales du porc (IPEC-J2). L’induction de médiateurs inflammatoires mesurée par RT-PCR ont révélé que le DON cause une diminution significative de l’expression des cytokines pro-inflammatoires (IL-6, IL-8, TNF-α) des DCs porcines et des PAMs favorisant ainsi un environnement immunosuppresseur. L’étude de l’expression du CMH-II par FACS a révélé que de faibles concentrations (≤ 1 μM) de DON augmentent significativement l’expression du CMH-II alors que de fortes concentrations (≥ 2 μM) causent plutôt une diminution significative de son expression influençant ainsi la mise en place de l’immunité adaptative. De plus, les tests de phagocytose ont révélé que de fortes concentrations (≥ 1 μM) de DON diminuent la capacité de phagocytose de S. suis par les PAMs favorisant ainsi la dissémination de la bactérie. Des tests d’adhésion et d’invasion ont révélé que le DON n’a pas d’impact sur l’adhésion de S. suis aux IPEC- J2 et que de fortes concentrations (≥ 4 μM) de DON favorisent l’invasion de S. suis dans ces cellules. Finalement, des tests ELISA ont montré que le DON (0,5, 1 et 4 μM) diminue significativement la production de cytokines pro-inflammatoires (IL-8) des IPEC-J2. / Streptococcus suis is an important bacterial pathogen that affects post-weaner piglets and causes septicemia, meningitis and arthritis. The infection is mainly transmitted by the respiratory route; however, there is a hypothesis that the intestinal tract could also represent an entry point for the pathogen. Deoxynivalenol (DON) is a mycotoxin that has long been suspected of being a predisposing factor in the development of disease caused by S. suis, but has never been studied. The objective of this study is therefore to evaluate the effect of DON on the interactions of S. suis with porcine dendritic cells (DCs), porcine alveolar macrophages (PAMs) and porcine intestinal epithelial cells (IPEC-J2). The induction of inflammatory mediators measured by RT-PCR revealed that DON causes a significant decrease in the expression of pro-inflammatory cytokines (IL-6, IL- 8, TNF-α) in porcine DCs and PAMs, thus promoting an immunosuppressive environment. The study of MHC-II expression by FACS revealed that low concentrations (≤ 1 μM) of DON significantly increase the expression of MHC-II while high concentrations (≥ 2 μM) rather cause a significant decrease in its expression, thus influencing the establishment of adaptive immunity. In addition, phagocytosis assays revealed that high concentrations (≥ 1 μM) of DON decrease the phagocytosis capacity of S. suis by PAMs, thus promoting the dissemination of the bacteria. Adhesion and invasion assays revealed that DON does not impact S. suis adhesion to IPEC-J2 and that high concentrations (≥ 4 μM) of DON promote S. suis invasion into IPEC-J2. Finally, ELISA have shown that DON (0,5, 1 et 4 μM) significantly reduce IPEC-J2’s production of pro-inflammatory cytokines (IL-8).

Day care centers and schools are important environments for children, but few epidemiological studies exist from these environments. Mould, dampness, fungal DNA and allergens levels in these environments and respiratory health effects in school children were investigated in this thesis. In the day care centers studies, Allergen Avoidance Day care Centers (AADCs) and Ordinary Day care Centers were included. One third of the Swedish day care centers had a history of dampness or mould growth. Total fungal DNA levels were positively associated with risk construction buildings, reported dampness/moulds, rotating heat exchangers, linoleum floors and allergens (cat, dog, horse allergen) levels. The two school studies included secondary schools in Johor Bahru, Malaysia and elementary schools from five European countries (Italy, Denmark, Sweden, Norway, and France) (HESE-study). In Malaysia, 13 % of the pupils reported doctor-diagnosed asthma but only 4 % had asthma medication. The prevalence of wheeze in the last 12 months was 10 % in Malaysia and 13 % in the HESE-study. Cough and rhinitis were common among children in the HESE-study. There were associations between fungal DNA and reported dampness or mould growth. Fungal DNA levels and viable mould (VM) concentration in the classrooms were associated with respiratory symptoms (wheeze, rhinitis, cough, daytime breathlessness) in school children. In the HESE-study, associations were found between total fungal DNA, Aspergillus/Penicillium DNA and respiratory symptoms among children. Moreover, Aspergillus versicolor DNA and Streptomyces DNA were associated with respiratory symptoms in Malaysia and the HESE-study, as well as reduced lung function [forced vitality capacity (FVC) and forced expiratory volume in 1 second (FEV1)] among children in the HESE-study. In conclusion, fungal DNA and pet allergens were common in day care centers and schools and respiratory symptoms in school children were common. The associations between VM concentration and fungal DNA levels in the schools and respiratory health effects in school children indicated a need for improvement of these environments. Moreover, risk constructions should be avoided and buildings should be maintained to avoid dampness and microbial growth. Health relevance of microbial exposure and biodiversity needs to be further studied using molecular methods.

Les mycotoxines sont des métabolites secondaires des moisissures qui contaminentnaturellement de nombreuses denrées alimentaires, notamment les céréales. Certainesmycotoxines présentent des effets toxiques potentiellement dangereux pour l’homme à desdoses extrêmement faibles. Les objectifs de ce travail étaient d’évaluer in vitro : (1) la toxicitéde trois mycotoxines qualifiées d’émergentes : la beauvericine, l’enniatine B et lamoniliformine ; (2) les effets de mycotoxines testées simultanément : déoxynivalénol etbeauvericine, déoxynivalénol et fumonisine B1, déoxynivalénol et zéaralénone,déoxynivalénol et toxine T-2, toxine T-2 et zéaralénone, beauvericine et enniatine B. Lesétudes ont été réalisées sur des cellules humaines d’origine hématopoïétique, en raison de leurforte sensibilité connue à certaines mycotoxines majeures. La myélotoxicité a été évaluée surles progéniteurs hématopoïétiques granulo-monocytaires, érythrocytaires et plaquettaires.L’immunotoxicité a été évaluée sur les cellules dendritiques et les macrophages.Un effet cytotoxique a été observé pour les progéniteurs hématopoïétiques exposésaux toxines émergentes. Une inhibition de la différenciation des progéniteurs érythroïdes a éténotée en présence d’enniatine B ou de moniliformine. Une cytotoxicité a été mise en évidencepour les cellules immunitaires exposées à la beauvericine ou à l’enniatine B. L’exposition à lamoniliformine a provoqué une inhibition de la différenciation des monocytes en cellulesdendritiques ou en macrophages. Une inhibition de la capacité de maturation des cellulesdendritiques a été mise en évidence en présence de beauvericine ou d’enniatine B. Les travauxmenés en co-exposition ont montré des effets cytotoxiques principalement additifs sur lesprogéniteurs hématopoïétiques et sur les cellules dendritiques. Une synergie a été observéepour les progéniteurs hématopoïétiques exposés au déoxynivalénol et à la beauvericine. Unantagonisme a été noté pour les progéniteurs et pour les cellules dendritiques exposéssimultanément au déoxynivalénol et à la fumonisine B1.Ces travaux réalisés in vitro suggèrent que les mycotoxines émergentes pourraientprovoquer chez l’homme une diminution du nombre et un dysfonctionnement des celluleshématopoïétiques, avec pour conséquence l’induction de troubles hématologiques etimmunologiques. La présence concomitante de mycotoxines pourrait augmenter le risque demyélotoxicité et d’immunotoxicité chez l’homme. Des études in vivo devraient être menéesafin de compléter ces travaux. / Mycotoxins are secondary metabolites of fungi that are natural contaminants ofseveral commodities, in particular cereals. The main objectives of the thesis were to assess thein vitro toxicity of: (1) emerging mycotoxins beauvericin, enniatin b and moniliformin; (2)combinations of fusariotoxins known to co-contaminate cereals or food commodities:deoxynivalenol and beauvericin, deoxynivalenol and fumonisin B1, deoxynivalenol andzearalenone, deoxynivalenol and T-2, T-2 and zearalenone, beauvericin and enniatin B.Experiences have been done using human hematopoietic cells. Myelotoxicity has beenevaluated using clonogenic assays on granulo-monocytic, erythroid and megakaryocyticprogenitors. Immunotoxicity has been assessed on dendritic cells and macrophages.Emerging mycotoxins were cytotoxic for hematopoietic progenitors. Inhibition oferythroid differentiation has been observed in the presence of enniatin b or moniliformin.Beauvericin and enniatin B were cytotoxic for dendritic cells and macrophages. Thedifferentiation process of monocytes into immature dendritic cells or into macrophages wasdisturbed in the presence of moniliformin. Beauvericin and enniatin B have been shown tointeract with dendritic cells maturation process. Additive myelotoxic and immunotoxic effectswere observed for combination of deoxynivalenol and zearalenone, deoxynivalenol and T-2,T-2 and zearalenone, beauvericin and enniatin B. Co-exposure of granulo-monocyticprogenitors to deoxynivalenol and beauvericin resulted in synergic effects. Combination ofdeoxynivalenol and fumonisin B1 showed antagonist myelotoxic and immunotoxic effects.These in vitro results suggested that emerging mycotoxins could be responsible forhematological and immunological troubles on human in case of consumption of contaminatedcommodities. Simultaneous presence of mycotoxins in food commodities and diet may bemore toxic than the presence of one mycotoxin alone. In vivo studies should be performed totest these hypotheses.

O presente estudo analisou os efeitos da radiação gama na sobrevivência de fungos em plantas medicinais embaladas, adquiridas do comércio atacadista e varejista, em diferentes períodos (0 e 30 dias) após o tratamento por irradiação. Cinco tipos de plantas medicinais [Peumus boldus, Cameilia sinensis, Maytenus ilicifolia, Pauilinia cupana and Cássia angustifolia), foram coletadas de diferentes municípios do Estado de São Paulo e submetidas ao tratamento por irradiação, utilizando-se uma fonte 60Co (tipo Gammacell 220), com doses de 5,0 kGy e 10 kGy e taxa de dose de 3,0 kGy/h. Amostras não irradiadas (grupo controle) foram usadas na contagem de fungos e diluições seriadas de 10-1 a 10-6 das amostras foram semeadas em duplicata e plaqueadas usando o método de cultura em superfície, em ágar Dicloran Glicerol 18% (DG18) e contadas após cinco dias a 25°C. O grupo controle revelou a presença dos gêneros Aspergilius e Penicillium, os quais são conhecidos como fungos toxigênicos e poucas amostras do grupo controle estavam dentro dos limites seguros, estabelecidos pela Organização Mundial de Saúde (OMS, 1998) para plantas medicinais. Em resposta a resistência do tratamento por ionização, na dose de 5 kGy, foi obsen/ado que os gêneros Aspergilius, Piioma e Syncephalastrum foram radiorresistentes, após o processo (dia 0 e 30° dia). O tratamento por radiação gama foi efetivo na descontaminação de todas as amostras de plantas medicinais, após 30 dias, com a dose de 10 kGy e mantidas em condições de vedação. Não foram detectadas aflatoxinas nas amostras do grupo controle, ainda que estas amostras estivessem intensamente contaminadas com Aspergilius flavus. / This present study evaluated the effect of gamma radiation on the fungal survival in packed medicinal plants, purchased from wholesale and retail market, in different period (0 and 30 days) after the treatment. Five kind of medicinal plants (Peurnus boldus, Camellia sinensis, Maytenus ilicifolia, Pauilinia cupana and Cassia angustifolia), were collected from different cities of São Paulo State, and submitted to irradiation treatment using a 60Co source (type Gammacell 220) with doses of 5,0 kGy and 10 kGy and at dose rate of 3.0 kGy/h. Non-irradiated samples (control group) were used for fungal counts and serial dilutions from 10-1 to 10-6 of the samples were seeded in duplicates and plated using the surface culture method in Dichloran 18% Glycerol Agar (DG 18) and were counted after five days at 25°C. The control group revealed the presence of genera Aspergillus and Penicillium, which are known as toxigenic fungi and a few samples of control group were within the safety limits of World Health Organization (WHO, 1998) to medicinal plants. In response to resistance of ionizing treatment, in the dose of 5 kGy, it was obsen/ed that the genera Aspergillus, Phoma and Syncephalastrum were radio-resistant after the process (day 0 and 30° day). The treatment by gamma radiation was effective in decontamination of all irradiated samples of medicinal plants, after 30 days, with the dose of 10 kGy and kept of veiled conditions. It was not detected aflatoxins in samples of control group, even though these samples were heavily contaminated with Aspergillus flavus.

Mykotoxine sind giftige Naturstoffe, die im Rahmen des Sekundärstoffwechsels von Schimmelpilzen beim Wachstum auf pflanzlichen Substraten gebildet werden. Zu den bekanntesten Mykotoxinen zählt das Ochratoxin A (OTA), welches überwiegend in Getreide und davon abgeleiteten Erzeugnissen, aber auch in Wein und Kaffee sowie Gewürzen und Bier nachgewiesen wurde. OTA ist unter anderem immunotoxisch, nephrotoxisch und besitzt teratogene sowie kanzerogene Eigenschaften. Darüber hinaus wird OTA eine hormonelle Wirkung zugesprochen. Aufgrund der Toxizität und des relativ häufigen Vorkommens in Lebensmitteln wurden für OTA Grenzwerte festgelegt. Diese liegen für Wein, Röstkaffee und diätetische Produkte zwischen 0,5 mikrogramm /kg und 10 mikrogramm /kg. Grenzwerte für Gewürze und Bier sind geplant. Im Rahmen der Methodenentwicklung zur Bestimmung von OTA in Lebensmitteln wurden verschiedene clean-up-Techniken miteinander verglichen. Dabei wurde vor allem den kombinierten Ionentauscher/reversed phase-Säulen und der LC-MS/MS wesentliche Bedeutung beigemessen. Innerhalb der Methodenvalidierung wurden die Ergebnisse mit dem entsprechenden Standardverfahren verglichen. Es konnte gezeigt werden, dass mit oben genanntem Material in Kombination mit der LC-MS/MS sehr gute Ergebnisse erzielt werden können. Diese wurden im Rahmen der Validierung durch statistische Auswertung bestätigt. Ferner wurden im Rahmen der Methodenentwicklung, speziell bei der Bestimmung von OTA in Kaffee, Extraktionstechniken miteinander verglichen. Dabei wurde die bislang kaum beachtete ASE (Accelerated Solvent Extraction, Dionex) sowie Ultraschallextraktion mit der Schüttelextraktion aus der gültigen Norm verglichen und ihre Anwendbarkeit auf weitere Lebensmittel geprüft. In diesem Zusammenhang waren die ASE und Ultraschallextraktion der konventionellen Schüttelextraktion überlegen. Darüber hinaus wurde die Ultraschallextraktion in Kombination mit dem Ionentauscher/reversed phase clean-up erfolgreich auf Weizen und Chili angewandt. Parallel zur Methodenentwicklung wurde die Stabilität und Homogenität von OTA in Wein und Röstkaffee untersucht. Sowohl die Homogenität als auch die Stabilität des Analyten sind wichtige Faktoren bei der Entwicklung neuer Verfahren, da zusammen mit Ergebnissen der Validierung, die Messunsicherheit innerhalb neuer Verfahren eingegrenzt werden. / Mycotoxins are naturally occurring toxic substances and produced as secondary metabolites by fungi which are growing on plants predominantly. One of the most interesting mycotoxins is ochratoxin A (OTA), which was found in several foods and feed stuff like wheat and its related products, wine, coffee, spices and beer. Among other things OTA is immunotoxic, nephrotoxic and has teratogenic and cancerogenic properties. Further more OTA shows hormonal effects. Because of its toxicity and common appearance in food and feed the OTA content had to be regulated in European and or national directives. The maximum values are determined between 0.5 micrograms/kg and 10 micrograms/kg for wine, roasted coffee and dietetic products. Other maximum levels for beer and spices are intended. During the investigation of analytical methods for the determination of OTA in food stuff different clean-up techniques were compared. Thereby the mixed anion exchange/reversed phase clean-up in combination with LC-MS/MS was researched intensively. Within method validation the results were compared with the corresponding standard methods. This work shows that the mixed mode clean-up in combination with LC-MS/MS gives excellent results. These results were statistically confirmed during method validation. Further more different extraction techniques were compared during the development of analytical methods, especially for the determination of OTA in coffee. Thereby the ASE (accelerated solvent extraction, Dionex) and the ultrasonic extraction were compared with the shaking technique from current standard methods. The adaptability for other food and feed products was researched as well. In this context the new extraction techniques were superior to conventional shaking techniques. In addition the ultrasonic extraction with mixed mode clean-up and LC-MS/MS detection was applied to chilli and wheat effectively. Besides the investigation of analytical methods the stability and homogeneity of OTA in wine and roasted coffee was researched. The homogeneity and stability of OTA in matrix are very important factors during the investigation of analytical methods. In combination with the relevant results of the method validation these results can be used to determine the uncertainty of the new analytical methods.