O objetivo deste trabalho foi estudar a transferência de aflatoxinas da ração para peixes lambari (Astyanax altiparanae), avaliando a influência dos níveis de toxina na ração sobre o desempenho dos peixes. As aflatoxinas foram incorporadas à ração extrusada para peixes tropicais, previamente testada para a presença de aflatoxinas, e as concentrações foram confirmadas por cromatografia líquida de alta eficiência (CLAE). Os tratamentos foram constituídos por: Controle - ração sem toxina; A. Ração + 10 µg AFB1/kg; B. Ração + 20 µg AFB1/kg e C. Ração + 50 µg AFB1/kg. Os juvenis de lambari foram alocados em aquários com densidade de 1 peixe por litro. O experimento foi realizado por 120 dias, com três repetições. A cada 30 dias foram realizados levantamentos biométricos e avaliação do desempenho (taxa de crescimento, sobrevivência, ganho de peso). A determinação de aflatoxinas nas amostras foi realizada por CLAE em músculo e fígado. Mensalmente, dez peixes por aquário foram utilizados para compor uma amostra, totalizando 48 amostras de músculo e 48 amostras de fígado analisadas ao final do experimento. As aflatoxinas na dieta prejudicaram o desempenho dos lambaris, observando-se menor peso e tamanho dos peixes nos tratamentos com aflatoxinas em relação ao controle. Houve também menor consumo de ração pelos peixes do tratamento C no período final do experimento. De modo geral, observou-se que, principalmente a partir dos 90 dias de experimento, houve acúmulo de aflatoxinas no fígado e no músculo dos animais. A concentração de aflatoxina B1 observada no músculo dos lambaris ao final do período de exposição foi semelhante aos níveis empregados na dieta. Os resultados mostraram que o lambari é uma espécie sensível, havendo efeito das aflatoxinas sobre o desempenho e acúmulo de aflatoxinas no músculo e fígado. Assim, quando da exposição diária e prolongada de lambaris às aflatoxinas, conclui-se que os limites atuais das legislações para aflatoxinas em ração animal não garantem segurança à produção e à saúde dos consumidores. / The aim of this study was to evaluate the transfer of aflatoxins from feed to lambari fish (Astyanax altiparanae), assessing the influence of toxin levels on fish performance. Aflatoxins were incorporated into extruded feed for tropical fish, previously tested for aflatoxins, and the concentrations were confirmed by high-performance liquid chromatography (HPLC). The treatments were: Control - feed without toxin; A. Feed + 10 µg AFB1/kg; B. Feed + 20 µg AFB1/kg and C. Feed + 50 µg AFB1/kg. Juveniles of lambari fish were placed in aquariums with density of 1 fish per liter. The experiment was conducted for 120 days, with three replications. Every 30 days biometric surveys were performed, including weight and standard length. Feed intake, feed conversion, growth rate and survival were also evaluated. Determination of aflatoxins was carried out by HPLC in muscle and liver. Monthly, a pool of 10 fish were used to make a sample, adding up to 48 samples of muscle and 48 samples of liver analyzed at the end of the experiment. Aflatoxins in the diet impaired the lambari performance, with lower weight and size of fish from aflatoxins treatments compared to control. There was also lower feed intake by fish from treatment C in the final period of the experiment. In general, it was observed that, mainly from 90 days of experiment, there was accumulation of aflatoxins in the liver of lambari fish and consequently in the muscle. Aflatoxin B1 concentration observed in the muscle of lambari fish at the end of the exposure period was similar to the levels used in the diet. Results showed that lambari fish is sensitive specie, with effects of aflatoxins on performance and accumulation of aflatoxins in muscle and liver. Therefore, when daily and prolonged exposure of lambaris to aflatoxins, it is concluded that the current regulation limits for aflatoxins in animal feed do not guarantee safety production and consumer health.

A castanheira (Bertholletia excelsa Humb. & Bonpl.) é uma árvore nativa da região Amazônica muito valorizada por suas sementes, as castanhas-do-Brasil, que apresentam alto valor nutritivo e são uma rica fonte de selênio, um agente antioxidante. O Brasil está entre os países que mais produzem e exportam estas castanhas. As condições climáticas da região Amazônica, assim como as demais etapas da cadeia produtiva, podem favorecer a infecção fúngica neste substrato, principalmente por Aspergillus nomius, espécie extremamente relacionadas à produção de aflatoxinas. Esta micotoxina está associada ao desenvolvimento de tumores, imunossupressão e alterações hepáticas tornando-a um risco para a saúde pública. Sendo assim, a realização de estudos que forneçam informações adequadas sobre o comportamento de A. nomius é de extrema relevância, pois contribuem no conhecimento das condições propícias para a produção de aflatoxinas. Neste contexto, o objetivo deste estudo foi avaliar a resposta ecofisiológica de cepas de A. nomius isoladas de castanhas-do-Brasil (crescimento micelial, expressão gênica e produção de aflatoxinas) em diferentes temperaturas (25, 30 e 35 °C). O crescimento micelial foi mensurado diariamente a partir da inoculação de 8 cepas em ágar coco, mantidas no escuro até 7 dias. A partir destas colônias foi feita análise da expressão dos genes aflR, aflD e aflQ, envolvidos na biossíntese das aflatoxinas, com utilização de PCR em Tempo Real. Com as mesmas colônias também foi feita análise do potencial aflatoxigênico (B1, B2, G1 e G2) qualitativo (Cromatografia em Camada Delgada) e quantitativo (Cromatografia Líquida de Alta Eficiência). A temperatura ideal para crescimento micelial das cepas de A. nomius foi 30 °C. Esta condição foi a melhor para a expressão dos genes aflR, aflD e aflQ. Contudo, o gene aflQ também apresentou alta expressão a 25 °C e foi o gene mais expresso em todas as temperaturas avaliadas. Em relação ao potencial toxigênico das cepas, a maior produção ocorreu a 25 °C. Em todas as temperaturas avaliadas houve maior produção de aflatoxinas do grupo B do que do grupo G. Pôde-se observar que a temperatura que propiciou a maior produção destas toxinas coincide com as da região Amazônica, território nativo das castanheiras. Com base nos resultados reportados, este estudo poderá servir como ferramenta na elaboração de eficientes estratégias para o controle de A. nomius e aflatoxinas em castanhas-doBrasil. / The Brazil nut tree (Bertholletia excelsa Humb. & Bonpl.) is an Amazonian native species that produce seeds with high nutritional value and rich source of selenium, an antioxidant agent. Brazil is one of the major producers and exporters of these nuts. The Amazon weather conditions in the production area and also on the productive chain play a critical role in the fungal infection, specially by Aspergillus nomius, important species associated to the aflatoxin contamination. This mycotoxin is related to the development of tumors, immunosuppression and liver alterations that becomes a risk to public health. Therefore, studies that provides adequate informations about the behavior of A. nomius are extremely relevant, contributing to better understand the favorable conditions to the aflatoxins production. The main goal of the present study was to evaluate the ecophysiological response of A. nomius strains isolated from Brazil nuts (mycelial growth, gene expression and aflatoxin production) at different temperatures (25 °C, 30 °C, and 35 °C). The mycelial growth of 8 strains was measured daily for 7 days in coconut agar. From these colonies, the expression of aflR, aflD, and aflQ genes, that are involved in aflatoxin biosynthesis was analyzed by using Real Time PCR. From the same colonies, the aflatoxigenic potential (B1, B2 , G1 and G2 ) were analyzed qualitative and quantitative by Thin Layer Chromatography and High Performance Liquid Chromatography, respectively. Mycelial growth assessment revealed that the optimal temperature for the radial growth rate and the average of final growth was at 30 °C. This was also the best condition for the expression of aflR, aflD, and aflQ genes. However, the aflQ also showed high expression at 25 °C and was the most expressed gene at all evaluated temperatures. The highest aflatoxin production occurred at 25 °C, with higher toxins production on group B than group G. It was possible to notice that the optimum temperature to aflatoxin production coincides with those in Amazon region, the most important producing area. These results also may contribute to enhance the management strategies of aflatroxin control in Brazil nuts.

Fungos filamentosos com capacidade de produzir micotoxinas podem estar presentes em alimentos, desde o cultivo até o produto após industrialização. Devido a isso, estratégias para controlar o crescimento fúngico devem ser investigadas, a fim de evitar o desenvolvimento desses microrganismos, bem como a produção de suas toxinas nos alimentos. Neste trabalho, duas abordagens para o controle de fungos toxigênicos foram avaliadas. A primeira estratégia foi a utilização de bactérias provenientes de diferentes ambientes aquáticos, sendo que 10 linhagens de Bacillus spp. e a linhagem Pseudomonas sp. 4B foram testadas quanto à influência sobre os parâmetros de crescimento (taxas de crescimento micelial, esporulação e germinação de esporos) de fungos toxigênicos (Aspergillus e Penicillium) e formação de micotoxinas. Todas as bactérias foram capazes de inibir o crescimento dos fungos em meio de cultura, apresentando halos de inibição variando de 1,0 até 15,7 mm. Bacillus sp. P11 apresentou resultados mais expressivos em relação às demais linhagens do gênero Bacillus com maiores valores de redução na maioria dos parâmetros de crescimento. Além disso, Bacillus sp. P11 e Pseudomonas sp. 4B apresentaram efeito sobre as taxas de crescimento micelial, esporulação e germinação de esporos, com níveis de redução acima de 43,3, 32,1 e 84,1% respectivamente. Mesmo assim, as demais linhagens também apresentaram resultados satisfatórios sobre esses parâmetros. Estas bactérias também reduziram a síntese de aflatoxina B1 e ocratoxina A em mais de 94 e 63%, respectivamente, quando cultivadas simultaneamente com os fungos produtores de cada micotoxina. Adicionalmente, a capacidade de Bacillus sp. P11 em produzir os lipopeptídeos iturina A (167,9 mg/mL de extrato butanólico) e surfactina (361,9 mg/mL de extrato butanólico) foi confirmada. Estes compostos podem ter contribuído para a atividade antifúngica desta bactéria. A segunda estratégia investigada neste estudo para controlar o desenvolvimento de fungos toxigênicos foi o emprego de nanofibras de poli-ɛ-caprolactona (PCL) incorporadas com cetoconazol e natamicina como material antimicrobiano. Nesta abordagem, as nanofibras foram produzidas pela técnica de eletrofiação e posteriormente caracterizadas e avaliadas quanto ao seu potencial antifúngico. Nanofibras funcionalizadas com cetoconazol ou natamicina apresentaram atividade antifúngica contra os isolados toxigênicos uma vez que zonas de inibição variando de 6 a 44 mm foram observadas. Além disso, as análises de microscopia eletrônica e espectroscopia demonstraram que a incorporação dos antifúngicos não altera de forma expressiva as principais características das nanofibras. Também foi possível verificar a capacidade de liberação controlada dos antifúngicos durante 72 h de contato das nanofibras com diferentes soluções simulantes. Valores próximos a 80 e 45 μg/mL de cetoconazol e natamicina, respectivamente, foram observados em solução de Tween 20 (5%). Portanto, o processo de eletrofiação foi capaz de agregar propriedades antifúngicas às nanofibras de PCL. Os resultados demonstraram que as bactérias e os nanomateriais investigados neste estudo são promissores para o controle de fungos toxigênicos e produção de micotoxinas. / Filamentous fungi that have the potential to produce mycotoxins may be present in food, from cultivation to after industrialization. Therefore, several strategies to control fungal growth must be investigated in order to avoid the development of these microorganisms and the production of their toxins in food. In this work, two approaches to toxigenic fungi control were evaluated. The first one was the use of bacteria from different aquatic environments as biocontrol agents in which 10 Bacillus spp. strains and the Pseudomonas sp. 4B strain were tested in relation to the effect on growth parameters (mycelial growth, sporulation and spore germination rates) of toxigenic fungi (Aspergillus and Penicillium) and mycotoxin formation. All bacteria were able to inhibit the fungal growth in culture medium with inhibition zones ranging from 1.0 to 15.7 mm. It was also observed that Bacillus sp. P11 had better results compared to other Bacillus strains with larger reduction values in most of growth parameters. Furthermore, Bacillus sp. P11 and Pseudomonas sp. 4B exhibited effect on mycelial growth, sporulation and spore germination rates with reduction values above of 43.3, 32.1 and 84.1%, respectively. Even so, the other strains also showed satisfactory results on these parameters. Finally, these bacteria reduced the synthesis of aflatoxin B1 and ochratoxin A at levels above 94 and 63%, respectively, when co-cultivated with each mycotoxin producing fungi. Additionally, the ability of Bacillus sp. P11 to produce lipopeptides such as iturin A (167.9 mg/ml of butanolic extract) and surfactin (361.9 mg/ml of butanolic extract) was confirmed. These compounds may have contributed to antifungal activity of this bacterium. The second investigation of this work in order to control the growth of toxigenic fungi was the use of poly-ε-caprolactone nanofibers incorporated with ketoconazole and natamycin as antimicrobial material. In this approach, nanofibers were produced by the electrospinning technique and subsequently characterized and evaluated for their antifungal potential. Both nanofibers functionalized with ketoconazole and natamycin showed antifungal activity against toxigenic isolates since inhibitory zones ranging from 6 to 44 mm were observed. In addition, scanning electron microscopy and infrared spectroscopy analysis showed that the antifungals incorporation does not change the characteristics of nanofibers. It was also possible to verify the ability of controlled drug release during 72 h of nanofibers contact with different simulants solutions. Values near 80 and 45 μg/ml of ketoconazole and natamycin, respectively, were observed in the solution containing 5% Tween 20. Therefore, the electrospinning process was able to provide antifungal properties to the nanofibers. The results showed that bacteria and nanomaterials investigated in this study are promising for developing control strategies of toxigenic fungi and mycotoxin production.

<p>Unwanted fungal growth is one of the most common causes of food spoilage throughout the world, and is causing health risks for both humans and animals and economical losses for the food- and agricultural industries. In Europe the mycotoxin producing Fusarium species F. sporotrichioides, F. culmorum, F. poae and F. graminearum is of greatest importance, due to their production of the trichothecene deoxynivalenol (DON) among other mycotoxins. Today’s conventional determination methods of these Fusarium species is time-consuming and quicker screening methods directly on cereals is therefore of interest to develop. In this project a species-specific PCR-protocol targeting the trichodiene synthase (tri5) gene in F. sporotrichioides, F. culmorum, F. poae and F. graminearum was used to evaluate two different DNA-extraction methods for cereals. The PCR-protocol was first verified with pure fungal cultures and optimized with a PCR-gradient before it was applied on cereals. The PCR-gradient resulted in a background reduction in the PCR-analysis of F. sporotrichioides infected cereals.</p><p>The two methods, called the Hammer-method (cereals was crushed with a hammer) and the Nitrogen-method (cereals were crushed in a mortar together with liquid nitrogen), is both combinations of a published DNA-extraction method (CTAB-based) for cereals and a DNA-purifying kit (chaotropic agent-based). Within these two methods some modifications were made and a comparison of the results showed that the Nitrogen-method indicated to be more stable than the Hammer-method. Too few analyses have though been made for a definite conclusion. A verification of the Nitrogen-method showed that the PCR-protocol can be considered as stable and reliable also on cereals but the DNA-extraction method for cereals is still to be optimized and stabilized. Sonification of the cereals is under consideration for further tests and studies.</p> / <p>Mögelsvampsinfektioner av spannmålsprodukter är ett av de vanligaste problemen inom mat- och jordbruksindustrierna runt om i världen. Enligt FNs Food and Agriculture Organization beräknas att cirka 25 procent av världens spannmålsgrödor är infekterade med mykotoxinbildande mögelsvampar vilket kan leda till stora hälsorisker för konsumenterna och ekonomiska förluster för mat- och jord-bruksindustrierna. Mykotoxiner är sekundära produkter från svampens ämnesomsättning som troligen har betydelse för svampens överlevnad, men kan ge toxiska effekter hos människa och djur. I Europa är mögelsvampsläktet Fusarium den vanligaste och viktigaste av mykotoxinbildande svampar och producerar de för jordbruksnäringen två viktigaste mykotoxingrupperna trichotechener och fumonisi-ner.</p><p>På grund av den breda förekomsten av dessa Fusarium-svampar finns det idag ett behov av att utveckla snabba och pålitliga metoder för att detektera och identifiera mögelsvamparna redan direkt på de färska spannmålen. Under hösten 2002 startades projektet Bestämning av potentiella mykotoxin-producerande Fusariumsvampar med PCR-metodik vid Mikrobiologiska enheten på Livsmedelsver-ket i Uppsala. Projektet syftar till att utveckla en molekylärbiologisk bestämningsmetod där identifie-ringen av svamparna sker med hjälp av dess DNA istället för via mikroskopiska undersökningar. Det-ta examensarbete har varit en del av det projektet och har i huvudsak inriktats på att utveckla en stabil metod för själva utvinningen av svamp-DNA från de färska spannmålen. De slutsatser som nåtts är att de utvinningsmetoder som examensarbetet omfattade inte kan anses stabila i nuläget utan behöver utvecklas och stabiliseras ytterligare. Vad gäller de molekylärbiologiska metoderna har de kunnat visas stabila även på färskt spannmål.</p>

Unwanted fungal growth is one of the most common causes of food spoilage throughout the world, and is causing health risks for both humans and animals and economical losses for the food- and agricultural industries. In Europe the mycotoxin producing Fusarium species F. sporotrichioides, F. culmorum, F. poae and F. graminearum is of greatest importance, due to their production of the trichothecene deoxynivalenol (DON) among other mycotoxins. Today’s conventional determination methods of these Fusarium species is time-consuming and quicker screening methods directly on cereals is therefore of interest to develop. In this project a species-specific PCR-protocol targeting the trichodiene synthase (tri5) gene in F. sporotrichioides, F. culmorum, F. poae and F. graminearum was used to evaluate two different DNA-extraction methods for cereals. The PCR-protocol was first verified with pure fungal cultures and optimized with a PCR-gradient before it was applied on cereals. The PCR-gradient resulted in a background reduction in the PCR-analysis of F. sporotrichioides infected cereals. The two methods, called the Hammer-method (cereals was crushed with a hammer) and the Nitrogen-method (cereals were crushed in a mortar together with liquid nitrogen), is both combinations of a published DNA-extraction method (CTAB-based) for cereals and a DNA-purifying kit (chaotropic agent-based). Within these two methods some modifications were made and a comparison of the results showed that the Nitrogen-method indicated to be more stable than the Hammer-method. Too few analyses have though been made for a definite conclusion. A verification of the Nitrogen-method showed that the PCR-protocol can be considered as stable and reliable also on cereals but the DNA-extraction method for cereals is still to be optimized and stabilized. Sonification of the cereals is under consideration for further tests and studies. / Mögelsvampsinfektioner av spannmålsprodukter är ett av de vanligaste problemen inom mat- och jordbruksindustrierna runt om i världen. Enligt FNs Food and Agriculture Organization beräknas att cirka 25 procent av världens spannmålsgrödor är infekterade med mykotoxinbildande mögelsvampar vilket kan leda till stora hälsorisker för konsumenterna och ekonomiska förluster för mat- och jord-bruksindustrierna. Mykotoxiner är sekundära produkter från svampens ämnesomsättning som troligen har betydelse för svampens överlevnad, men kan ge toxiska effekter hos människa och djur. I Europa är mögelsvampsläktet Fusarium den vanligaste och viktigaste av mykotoxinbildande svampar och producerar de för jordbruksnäringen två viktigaste mykotoxingrupperna trichotechener och fumonisi-ner. På grund av den breda förekomsten av dessa Fusarium-svampar finns det idag ett behov av att utveckla snabba och pålitliga metoder för att detektera och identifiera mögelsvamparna redan direkt på de färska spannmålen. Under hösten 2002 startades projektet Bestämning av potentiella mykotoxin-producerande Fusariumsvampar med PCR-metodik vid Mikrobiologiska enheten på Livsmedelsver-ket i Uppsala. Projektet syftar till att utveckla en molekylärbiologisk bestämningsmetod där identifie-ringen av svamparna sker med hjälp av dess DNA istället för via mikroskopiska undersökningar. Det-ta examensarbete har varit en del av det projektet och har i huvudsak inriktats på att utveckla en stabil metod för själva utvinningen av svamp-DNA från de färska spannmålen. De slutsatser som nåtts är att de utvinningsmetoder som examensarbetet omfattade inte kan anses stabila i nuläget utan behöver utvecklas och stabiliseras ytterligare. Vad gäller de molekylärbiologiska metoderna har de kunnat visas stabila även på färskt spannmål.

Extensive research has been compiled on mycotoxin production by mesophilic fungi. However, toxin production by the thermophilic fungi has only slightly been explored. In 1970 Sister Donovan of this laboratory hinted at the possibility of mycotoxin production in thermophiles when she demonstrated that extracts of Fenicillium duponti were toxic towards brine shrimp (Artemia,salina).Thermophilic fungi from natural composting substrates and from existing laboratory stock cultures were investigated to determine the possibility of mycotoxin production by thermophilic fungi. Three bioassay organisms were used to test the toxicity of the thermophilic fungi extracts.Sixteen of the 16 thermophiles reduced brine shrimp viability by more than 25% over controls. Ten of the 16 thermophiles produced toxic substances at each of three fungal incubation temperatures.Germinating Bacillus megatherium spores were found sensitive to extracts of 9 of the 16 thermophiles. Five of the nine thermophiles produced bacterial toxic substances at each of three fungal incubation temperatures.All embryos exposed to the thermophilic extracts lived and developed normally showing no sign of somatic deformities upon opening the eggs. One-day-old hatched chicks were then investigated. Sixteen of the 48 chicks injected with thermophilic extracts manifested viscerallesions of the heart, liver and stomach on autopsy seven days after injection.Results of three bioassays indicate that the toxic secondary metabolites were found to be present in crude extracts of 16 thermophilic fungi. Data indicated that many of the thermophiles produced toxic substances at each of three incubation temperatures.Ball State UniversityMuncie, IN 47306

In questa tesi di dottorato sono stati indagati i punti critici legati ai fattori biotici e abiotici che possono influenzare la crescita del fungo Fusarium verticillioides, produttore di fumonisine in mais. le fumonisine sono metaboliti secondari prodotte da funghi appartenenti al genere Fusarium e sono state classificate come possibili cancerogene per l’uomo e per gli animali. Gli argomenti trattati nei vari capitoli sono stati: parametri ecologici che condizionano la crescita e l’accumulo di fumonisine nelle prime fasi post raccolta e durante lo stoccaggio; relazione che intercorre tra aw, umidita’ relativa e tipo di ibrido; controllo con mezzi chimici e biologici in campo e in vitro su F. verticillioides e A. flavus. / The aim of this work was to collect missing information about critical point related to abiotic and biotic factors that can influence the growth of Fusarium verticillioides in maize and the consequent production of fumonisins in kernels. Fumonisins are secondary metabolites reported as toxigenic in humans and animals. Issues treated are: variables influencing growth and toxin accumulation during post-harvest and storage; the relationship between aw, relative humidity and type of hybrids; chemical and biological control of F. verticillioides e A. flavus in field and in vitro.

Mycotoxins are poisonous secondary metabolites commonly produced by fungi and are involved in human disease conditions known as mycotoxicoses. There is evidence to show that food eaten by the rural Black population of Southern Africa is contaminated with mycotoxins. A tenuous relationship exists between the occurrence of mycotoxins in foods and certain disease conditions in humans. In order to verify this relationship, efforts have, in the past, been made to detect mycotoxins and their metabolites in physiological fluids and tissues. The difficulty with this approach is that mycotoxins in the body have short half lives, being rapidly excreted or metabolised to other forms. More recently it has been shown that aflatoxin B1, as its activated epoxide, can conjugate with macromolecules such as nucleic acids and proteins. These survive for much longer than the free toxins and by suitable methods can be isolated and measured. This allows for a much better estimate of exposure of the individual to aflatoxin. This study reviews and evaluates screening methods for the detection and analysis of mycotoxin contamination in rural foodstuffs such as maize and groundnuts. Methods for the production of aflatoxin-lysine and protein adducts are motivated and developed then used in the identification of naturally occurring adducts in humans. Isolation and quantitative analysis techniques are proposed to routinely screen patients for evidence of aflatoxin exposure. / Thesis (M.Med.)-University of Natal, Durban, 1995.

T -2 toxin is an immunosuppressive mycotoxin that has been conjoined with several symptoms and diseases as early as the turn of the century, but whose mechanisms of action are still being investigated. Accordingly, this study was an attempt to determine the cytotoxic effects of T -2 toxin on normal human lymphocytes in vitro, with particular emphasis on mitochondrial viability, cellular and nuclear morphology as well as the localisation of the subcellular sites of toxin interaction. The cytotoxicity of T -2 toxin was assessed with the use of a methylthiazol tetrazolium (MTT) assay. This assay targeted the succinate dehydrogenase activity of the lymphocytic mitochondria, over a range of concentrations of T-2 toxin at various incubation times. The morphology of treated lymphocytes was analysed with the use of transmission electron microscopy and the localisation of the toxin was accomplished via immunocytochemistry. DNA fragmentation studies formed an integral part of the analyses. The cytotoxicity assay indicated that not only was cell viability inversely proportional to both the dose and exposure time, but that the eftects of the different doses were only evident at prolonged incubation times (12-24 hours). The electron microscopy studies showed that T-2 toxin (1,56 ug/ml) induced apoptosis (cell suicide) in normal human lymphocytes. This was determined by the observation of chromatin condensation and nuclear disintegration within the toxin treated lymphocytes. Apoptosis seemed to occur independently of mitochondrial damage at 6 hours of exposure to T-2 toxin. The presence of polyribosomes within the treated lymphocytes indicated that protein synthesis was not inhibited. Anti-T-2 toxin conjugated gold label was present in all areas of damage, particularly within the nuclei of the T-2 toxin treated lymphocytes. The DNA fragmentation results showed that T-2 toxin induced fragmentation in lymphocytes, the extent of which was directly proportional to the exposure time. It appears that the early signs of T-2 toxin induced apoptosis in normal human lymphocytes can be determined by damage to the nucleus. / Thesis (M.Med.)-University of Natal, Durban, 1998.

The human population can be considered as a subject of combined exposure to chemicals against which the gastrointestinal tract represents the first barrier. The most relevant are those compounds that occur in plants which are used as foods, medicines and beverages. Of special interest are the mycotoxins deoxynivalenol (DON) and fumonisin B1 (FB1), two of the most commonly encountered food-borne mycotoxins and curcumin, a popular spice and pigment reported to have antineoplastic properties. In this study, the HT-29 cell line was used to assess the toxicity of the mycotoxins DON and FB1 (5uM and 50uM) as well as the possible cytoprotective effects of curcumin (50uM) on colonic cells. Mixtures of both mycotoxins were also assessed to determine any possible interaction. Cytotoxicity, DNA fragmentation, cellular morphology and cell surface alterations were evaluated using the methylthiazol tetrazolium (MTT) bioassay, the single cell gel electrophoresis (SCGE) assay, fluorescence microscopy and scanning electron microscopy respectively. Deoxynivalenol displayed cytotoxic and genotoxic effects as well as induced morphological features of apoptosis and cell surface alterations that worsened with increasing concentration. Fumonisin B1 exhibited a proliferative effect at the high concentration however DNA damage and cell surface alterations worsened with decreasing concentration. Mixtures of DON and FB1 displayed similar effects to those exhibited by DON in terms of cytotoxicity, DNA fragmentation, morphology and cell surface alterations indicating that DON is able to antagonise the effects of FB1 at the concentrations tested. Curcumin appeared to exhibit a protective effect that was prominent when co-administered with the 50uM toxin concentration. Low concentrations of DON and FB1 (5uM) were sufficient to induce apoptosis in this cell line and suggest a danger from natural contamination by these toxins. Curcumin, however, warrants further investigation with regards to its cytoprotective activities in the presence of these mycotoxins as it could present a promising candidate for a natural chemoprotective agent in the armamentarium against mycotoxin induced cancers. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, 2005.