Orientador: Lucia Maria Valente Soares / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-27T08:21:17Z (GMT). No. of bitstreams: 1 Oliveira_AdrianaQueirozde_M.pdf: 4196208 bytes, checksum: 92d5a9097bf404e01a3739a01e11104f (MD5) Previous issue date: 2001 / Resumo: Os tricotecenos são metabolitos secundarios produzidos por especies de Fusarium, Myrothecium, Cephalosporium, Verticimonosporium, Stachybotrys e trichothecium. Estas micotoxinas podem causar vômitos, angina becrótica, diarreia, anorexia, alterações hematológicas, distúrbios neurológicos, destruição da medula óssea e hemorragias generalizadas, seguidos ou não de morte / Abstract: The trichothecenes are secondary metabolites produced by species of Fusarium, Myrothecium, Cephalosporium, Verticimonosporium, Stachybotrys and Trichothecium. These mycotoxins can cause vomiting, angina becrótica, diarrhea, anorexia, haematological disorders, neurological disorders, destruction of bone marrow and widespread bleeding, followed or not the death / Mestrado / Mestre em Ciência de Alimentos

No description available.

O presente trabalho visou utilizar o perfil polifásico, envolvendo características fenotípicas e genotípicas, na identificação de Fusarium spp. isolados de grãos de trigo como também investigar a presença das principais toxinas: desoxinivalenol e zearalenona nos grãos de trigo de três regiões produtoras de trigo (São Paulo, Paraná e Rio Grande do Sul). Nossos resultados indicaram que os gêneros mais frequentes no trigo foram Alternaria, Fusarium e Epicoccum. A determinação do genótipo por qPCR demonstrou predomínio de 15-ADON seguido por NIV e 3-ADON. A quantificação de DNA demostrou que o perfil 15-ADON foi responsável por 96% de todo DNA quantificado, seguido por NIV com 3.84% e 3-ADON, com somente 0.06%, indicando que 15-ADON, é o principal genótipo de tricoteceno nos grãos de trigo nacional. A toxina desoxinivalenol foi detectada em todas amostras analisadas, com mediana de 323, 466 e 783 µg/kg em SP, PR e RS, respectivamente. A determinação de zearalenona demonstrou contaminação em 100%, 80% e 42% dos grãos dos Estados do RS, PR e SP e medianas de 843, 100 e 14 µg/kg, respectivamente. / The present work aimed to use a polyphasic approach, by phenotypic and genotypic characteristics for the identification of Fusarium strains from wheat grains as well to investigate the presence of deoxynivalenol and zearalenone on wheat grains from three wheat producers States (Parana, Rio Grande do Sul and Sao Paulo). Our results showed that the main genera found were Alternaria, Fusarium and Epicoccum. Genotype detection from qPCR revealed predominance of 15-ADON, followed by NIV and 3-ADON. The qPCR demonstrated that 15-ADON genotype was responsible for 96% of all DNA quantified, followed by NIV with 3.84% and 3-ADON with 0.06%, indicating that 15-ADON is the main trichothecene genotype from Brazilian wheat grains. The toxin deoxynivalenol was detected in all 150 samples analyzed from wheat grains, with medians of 323, 466 and 783 µg/kg in SP, PR and RS, respectively. The zearalenone determination showed contamination on 100%, 80% and 42% of wheat grains from RS, PR and SP State and medians of 843, 100 and 14 µg/kg, respectively.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Two studies were performed in order to evaluate, through meta-analysis, the relationship of mycotoxins with performance and organ weights in pigs and broilers. The databases totaled 13,196 pigs (85 articles published between 1968 and 2010) and 37,371 broilers (98 articles, between 1980 and 2009). Meta-analysis followed three sequential analyses: graphical, correlation and variance-covariance. Pigs challenged by mycotoxins reduced feed intake by 18% and weight gain by 21% in relation to the control group. Challenged broilers presented a reduction of 12% in feed intake and 14% in weight gain. Mycotoxins with the greatest impact on performance were deoxynivalenol and aflatoxins in pigs, and ochratoxins and aflatoxins in broilers. The mycotoxin concentration in diets and the animal age at challenge were the variables that more improved the coefficient of determination in equations for estimating the mycotoxin effect on weight gain. The mycotoxin effect on growth was greater in younger animals. In addition, the residual analysis demonstrated that the greater part of the variation in weight gain was explained by the variation in the feed intake (87% in pigs and 65% in broilers). The variation in weight gain in challenged animals was also influenced by nutrient ingestion, such as protein and methionine. The mycotoxin effect on growth was greater in male pigs (-19%) compared to females (-15%). Mortality rate and some hematological parameters were also influenced by mycotoxins in broilers. Relative weight of organs increased in challenged pigs (liver, kidneys and heart) and broilers (liver, kidneys, lungs and gizzard). Mycotoxins influence on performance, productive indexes and organ weight in pigs and broilers. However, the magnitude of the effects varies with the type and concentration of mycotoxin, sex and age of the animal, as well as nutritional factors. / Dois trabalhos foram desenvolvidos com o objetivo de estudar, através de meta-análise, a relação das micotoxinas com o desempenho e o peso de órgãos de suínos e frangos de corte. As bases de dados totalizaram 13.196 suínos (85 artigos publicados entre 1968 e 2010) e 37.371 frangos de corte (98 artigos, entre 1980 e 2009). A meta-análise foi realizada através de três análises sequenciais: estudos gráficos, de correlação e de variância-covariância. Suínos desafiados por micotoxinas apresentaram redução de 18% no consumo de ração e de 21% no ganho de peso. Frangos de corte desafiados apresentaram redução de 12% no consumo de ração e 14% no ganho de peso. As micotoxinas com maior impacto sobre o desempenho foram deoxinivalenol e aflatoxinas para os suínos, e ocratoxinas e aflatoxinas para as aves. A concentração de micotoxinas nas dietas e a idade dos animais ao desafio foram as variáveis que mais ajustaram o coeficiente de determinação nas equações para estimar o efeito das micotoxinas sobre o ganho de peso. O efeito das micotoxinas sobre o crescimento foi maior nos animais jovens. Além disso, a análise de resíduos mostrou que a maior parte da variação no ganho de peso foi explicada pela variação no consumo de ração (87% nos suínos e 65% nas aves). A variação no ganho de peso em animais desafiados também foi influenciada pela ingestão de nutrientes, como proteína e metionina. O efeito das micotoxinas sobre o ganho de peso foi maior nos suínos machos (-19%) que nas fêmeas (-15%). A taxa de mortalidade e alguns parâmetros hematológicos também foram influenciados pelas micotoxinas nos frangos de corte. O peso relativo dos órgãos aumentou nos suínos (fígado, rins e coração) e nas aves (fígado, rins, pulmões e moela) desafiadas. As micotoxinas influenciam o desempenho, os índices produtivos e o peso dos órgãos em suínos e frangos de corte. No entanto, a magnitude destes efeitos varia com o tipo e a concentração de micotoxinas, o sexo e a idade do animal, bem como com fatores nutricionais.

O presente trabalho visou utilizar o perfil polifásico, envolvendo características fenotípicas e genotípicas, na identificação de Fusarium spp. isolados de grãos de trigo como também investigar a presença das principais toxinas: desoxinivalenol e zearalenona nos grãos de trigo de três regiões produtoras de trigo (São Paulo, Paraná e Rio Grande do Sul). Nossos resultados indicaram que os gêneros mais frequentes no trigo foram Alternaria, Fusarium e Epicoccum. A determinação do genótipo por qPCR demonstrou predomínio de 15-ADON seguido por NIV e 3-ADON. A quantificação de DNA demostrou que o perfil 15-ADON foi responsável por 96% de todo DNA quantificado, seguido por NIV com 3.84% e 3-ADON, com somente 0.06%, indicando que 15-ADON, é o principal genótipo de tricoteceno nos grãos de trigo nacional. A toxina desoxinivalenol foi detectada em todas amostras analisadas, com mediana de 323, 466 e 783 µg/kg em SP, PR e RS, respectivamente. A determinação de zearalenona demonstrou contaminação em 100%, 80% e 42% dos grãos dos Estados do RS, PR e SP e medianas de 843, 100 e 14 µg/kg, respectivamente. / The present work aimed to use a polyphasic approach, by phenotypic and genotypic characteristics for the identification of Fusarium strains from wheat grains as well to investigate the presence of deoxynivalenol and zearalenone on wheat grains from three wheat producers States (Parana, Rio Grande do Sul and Sao Paulo). Our results showed that the main genera found were Alternaria, Fusarium and Epicoccum. Genotype detection from qPCR revealed predominance of 15-ADON, followed by NIV and 3-ADON. The qPCR demonstrated that 15-ADON genotype was responsible for 96% of all DNA quantified, followed by NIV with 3.84% and 3-ADON with 0.06%, indicating that 15-ADON is the main trichothecene genotype from Brazilian wheat grains. The toxin deoxynivalenol was detected in all 150 samples analyzed from wheat grains, with medians of 323, 466 and 783 µg/kg in SP, PR and RS, respectively. The zearalenone determination showed contamination on 100%, 80% and 42% of wheat grains from RS, PR and SP State and medians of 843, 100 and 14 µg/kg, respectively.

This diploma thesis is focused on the analysis of toxins and their extraction from sandyloamy soil. Particularly, saxitoxin and trichothecene mycotoxins deoxynivalenol, HT-2 and T-2 toxins are in the centre of interest of this work. Their occurrence, toxic properties and influence on living organisms are described in theoretical part of this thesis. In next chapters, currently published extraction methods for individual toxins and analytical approaches for their quantitative evaluation are summarized. In experimental part of this thesis, optimized process of sample pretreatment based on extraction of toxins from soil using 1mM HCOONH4 in 84% acetonitrile was proposed as the best option. Simultaneous determination of toxins was performed by liquid chromatography on a CN column (3.0 x 150 mm, 3 Îm, 100 ) in gradient elution mode. Mass spectrometer with electrospray as ion source and linear ion trap as analyzer was used as detector. Recovery of designed method was over 80% for trichothecene mycotoxins and 51% for saxitoxin.

No description available.

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.

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.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In the first part of the thesis, the spontaneous occurrence of an outbreak of Baccharis megapotamica var. weirii poisoning in buffalo in the Central region of the State of Rio Grande do Sul is reported. Ten out of 50 buffalo died 24 48 hr after being introduced into a pasture containing abundant amounts of the plant. Factors influencing the ingestion of the plant and consequent toxicosis included hunger, stress caused by shipment, and unfamiliarity with the plant. Clinical signs included serous ocular discharge, incoordination, mild bloat, and muscle trembling. One buffalo was necropsied. Gross findings included dehydration, abundant liquid in the rumen, reddening of the mucosa of forestomachs, abomasum, and intestine, and edema of the wall of the rumen. The main histologic lesions were superficial to full thickness degeneration and necrosis of the stratified epithelium lining the forestomachs, necrosis of the intestinal mucosa, and widespread lymphoid necrosis. A calf (Bos taurus) was fed a single dose of 5 g/kg/body weight of B. megapotamica var. weirii harvested from the same site where the buffalo died. Twenty hours after the administration of the plant this calf died with clinical signs and lesions similar to those observed in the naturally poisoned buffalo.sido consumida pelos búfalos. In the second part of the thesis, Five male 6-8 month-old Murrah buffalo calves were orally dosed with the fresh aerial parts of Baccharis megapotamica var. weirii at doses of 1, 3, 4, 5 and 10 g/kg body weight (bw) (~1-10 mg macrocyclic trichothecenes/kg/bw). The B. megapotamica used for the experiment was harvested on a farm where a recent spontaneous outbreak of poisoning caused by such plant had occurred. Clinical signs appeared 4-20 hours and 4 buffalo died 18-49 hours after the ingestion of the plant. Clinical signs were apathy, anorexia, and watery diarrhea, fever, colic, drooling, muscle tremors, restlessness, laborious breathing and ruminal atony, and dehydration. The most consistent gross findings were restricted to the gastrointestinal (GI) tract consisted of varying degrees of edema and reddening of the mucosa of the fore-stomachs. Histopathological findings consisted of varying degrees of necrosis of the epithelial lining of the fore-stomachs and of lymphocytes within lymphoid organs and aggregates. Fibrin thrombi were consistently found in sub-mucosal vessels of the fore stomachs and in the lumen of hepatic sinusoids. It is suggested that dehydration, septicemia and disseminated intravascular coagulation participate in the pathogenesis of the intoxication and play a role as a cause of death. A subsample of the B. megapotamica var. weirii was frozen-dried and ground and analyzed using UHPLC (Ultra High Performance Liquid Chromatography) with high resolution Time of Flight mass spectrometry and tandem mass spectrometry, it was shown that the plant material contained at least 51 different macrocyclic trichothecenes at a total level of 1.1-1.2 mg/g. About 15-20% of the total trichothecenes contents was found to be monosaccharide conjugates, with two thirds of these being glucose conjugates and one third constituted by six aldopentose conjugates (probably xylose), which has never been reported in the literature. / Na primeira parte dessa tese, relatamos a ocorrência natural de um surto de intoxicação por Baccharis megapotamica var. weirii em búfalos na Região Central do Rio Grande do Sul. Os animais haviam sido transportados de uma propriedade onde a planta não ocorria para uma propriedade infestada pela planta. Durante o transporte, os animais foram submetidos a um longo período de jejum e estresse. Como resultado, após o desembarque dos 50 búfalos transportados, dez morreram com doença de evolução aguda (24-48 horas). A maioria dos búfalos foi encontrada morta, mas os sinais clínicos observados em um búfalo incluíam lacrimejamento, incoordenação e fraqueza dos membros posteriores, desorientação, decúbito esternal, lateral e morte. Na necropsia de um animal foi observado acentuada desidratação, avermelhamento e edema da mucosa dos pré-estômagos e intestino. Na microscopia, as áreas vermelhas dos pré-estômagos e intestino correspondiam à necrose acentuada do epitélio. Em visita à propriedade foi observada grande quantidade de B. megapotamica (identificada posteriormente como B. megapotamica var. weirii) com sinais de ter sido consumida pelos búfalos. Na segunda parte da tese, reproduzimos experimentalmente a intoxicação por B. megapotamica var. weirii em búfalos para melhor caracterizar o quadro clínico-patológico da intoxicação na espécie, assim como determinar a dose tóxica e avançar no estudo da patogênese da intoxicação. Para tal, utilizamos cinco búfalos da raça Murrah com 6 a 8 meses de idade e peso variando entre 122 e 143 kg. Esses animais receberam em uma única administração por via oral, 1, 3, 4, 5 e 10 g/Kg das partes aéreas de Baccharis megapotamica var. weirii. A planta usada no experimento foi colhida na fazenda onde ocorreu o surto de intoxicação espontânea descrito acima. Os sinais clínicos apareceram 4-20 horas e quatro búfalos morreram 18-49 horas após a ingestão da planta. Os sinais clínicos consistiram de apatia, anorexia, diarreia aquosa, febre, cólica, salivação, tremores musculares, inquietação, respiração laboriosa, atonia ruminal e desidratação. Os achados macroscópicos mais consistentes estavam restritos ao trato gastrointestinal (GI) e consistiram de graus variados de edema e avermelhamento da mucosa dos pré-estômagos. Os achados histopatológicos consistiam de vários graus de necrose do epitélio de revestimento dos pré-estômagos e de linfócitos em agregados e órgãos linfoides. Trombos de fibrina foram consistentemente encontrados nos vasos da submucosa dos pré-estômagos e na luz dos sinusoides hepáticos. Uma subamostra de B. megapotamica var. weirii foi congelada a seco, moída e analisada usando UHPLC (Cromatografia Líquida de Ultra Alta Performance) com espectrometria de tempo-de-vôo de alta resolução e espectrometria de massa em tandem. Foi demonstrado que o material de planta analisado continha pelo menos 51 tricotecenos macrocíclicos diferentes num nível total de 1,1-1,2 mg/g. Cerca de 15-20% do conteúdo total de tricotecenos eram conjugados de monossacarídeos, sendo dois terços desses, conjugados de glicose e um terço constituídos por seis conjugados de aldopentose (provavelmente xilose). Em conclusão, o presente estudo descreveu pela primeira vez a intoxicação em búfalos por plantas do gênero Baccharis. A reprodução experimental demostrou que búfalos são um pouco mais resistentes a intoxicação por B. megapotamica var. weirii do que bovinos. Quanto à patogênese, foi sugerido que desidratação, septicemia e coagulação intravascular disseminada sejam fatores responsáveis pela morte dos animais afetados. Adicionalmente, foi descrito presença de tricotecenos constituídos por seis conjugados de aldopentose (provavelmente xilose) no B. megapotamica var. weirii, o que nunca tinha sido antes relatado na literatura.