Thesis (M.Sc.)(Biochemistry)--University of Pretoria, 2006. / Includes summary. Includes bibliographical references. Available on the Internet via the World Wide Web.

The fumonisins are a class of polyketide mycotoxins produced by Fusarium verticilliodes (formerly Fusarium monoliforme) which commonly affects maize. Ingestion of these toxins has been associated with leukoencephalomalacia in equine species, pulmonary oedema in swine, hepatocarcinogenesis in rats and have been linked to oesophageal cancer in humans. The structurally related AAL toxins are host specific mycotoxins produced by Alternaria alternata f. sp. lycopersici, producing stem canker disease in susceptible tomato cultivars. Examination of the C-11-C-20 fragment of the fumonisin B1 backbone [(2S,3S,5R,10R,12S,14S, 15R,16R)-2-amino-3,5,10,14,15-hydroxy-12,16-dimethyleicosane] and the C-10-C-17 fragment of the AAL toxin TA backbone[(2S,4S,5R,11S,13S,14R,15R)-1-amino-2,4,5,13,14-hydroxy-11,15- dimethylheptadecane], reveals four common stereogenic centres, with the only difference between the two fragments being the length of the alkyl chain. It is thought that the position and configuration of these four stereogenic centres is conserved among all members of the fumonisin and AAL classes of toxins. Retrosynthetic analysis of the backbones reveals a common intermediate aldehyde, which can be synthesised from methyl (S)-3-hydroxy-2-methylpropionate. A simple synthetic route to access the C-11-C-20 fragment for the fumonisins and the C-10-C-17 fragment of the AAL toxins was devised utilising Sharpless asymmetric epoxidation and an Evans aldol reaction as key transformations. In practice, it was found that although the Sharpless asymmetric epoxidation produced the desired epoxide in low enantiomeric excess, the two diastereomers produced could be separated by two consecutive flash chromatography silica gel columns. In pursuit of a more efficient method for introduction of the stereogenic centre in the target, other synthetic routes and key transformations were considered. Jacobsen’s kinetic resolution of terminal racemic epoxides was explored, requiring a terminal alkene from which the racemic epoxide was synthesised. An attempt to synthesise the terminal alkene from the appropriate tosylate and vinyl-MgBr, mediated by copper (I) iodide, failed. The synthetic route was redesigned, and the terminal alkene was synthesised by two one-carbon additions: the first a nucleophilic substitution with cyanide, and the second a Wittig olefination. The resolution of the terminal epoxide was also unsuccessful with no significant kinetic resolution occurring. Sharpless asymmetric dihydroxylation was also investigated; however, this reaction too failed to produce products of high diastereomeric excess. As a consequence, it was decided to pursue the asymmetric epoxidation route as the diastereomeric products could at least be separated. The second key transformation, the Evans aldol reaction, also provided an interesting result. When the aldol reaction was attempted with benzaldehyde and enolates derived from (4R,5S)-3-butanoyl-4-methyl-5-phenyl-oxazolidin-2-one and (4R,5S)-3-hexanoyl-4-methyl-5-phenyl-oxazolidin-2-one, the butanoyl derivative was found to give the expected Evans syn product, while the hexanoyl derivative was found to give the non-Evans syn product, with proof provided by single crystal X-ray diffraction analysis. It is proposed that the aldol reaction with the hexanoyl derivative does not proceed through the expected Zimmerman-Traxler-type transition state, but rather through an open chain transition state similar to that seen for asymmetric alkylation reactions. Synthesis of the pentanoyl derivative, and subjecting it to the same aldol reaction gave the expected syn Evans product, as deduced from spectroscopic properties. When the aldol reaction was attempted with the appropriate aldehyde intermediate, it was found that the dibutylboron triflate in the reaction medium caused the cleavage of the O-TBS ether protection, resulting in the formation of (3S,5R)-3-(4-methoxybenzyloxy)-5-methyl-tetrahydropyran-2-ol, before the aldehyde could undergo the aldol reaction. In order to avoid this problem, it is suggested that an alternative protecting group strategy using a more robust protecting group, such as a benzyl group which is stable to Lewis acids, could be substituted for the O-TBS group. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Chemistry / unrestricted

Fusarium verticillioides (=Fusarium moniliforme) a common fungal contaminant of maize throughout the world has been associated with diseases in both man and animals. The structure of the fumonisins, a family of structurally related mycotoxins isolated from cultures associated with the high incidence of human oesophageal cancer in the Transkei region in South Africa and with equine leucoencephalomalacia, a neurological disorder in horses and donkeys, has been established. The main mycotoxin, fumonisin B₁ consists of the diester formed by the C(14) and C(15) hydroxy groups of (2S,3S,5R,10R,12S,14S,15R,16R)-2-amino-12,16-dimethyleicosane-3,10,14,15-pentaol with the Si carboxy group of propane-1,2,3-tricarboxylic acid. A comparison of the structures of the 28 known fumonisins isolated since 1988 reveals that they share a common structural motif for the C(11)–C(20) unit, and probably also the same stereochemistry for the 4 stereogenic centres present in this part of the C20 backbone. Disconnection of the C(9)–C(10) bond in a retrosynthetic analysis of the fumonisins identifies (3S,5S,6R,7R)-3,7-dimethylundecane-1,5,6-triol as a common building block for the synthesis of any of the fumonisins. In the dissertation the retrosynhetic analysis of this 3,7-dimethylundecane-1,5,6-triol building block identifies a simple precursor, ethyl 2-heptenoate, as the starting material for the proposed synthetic route toward this target. The Sharpless asymmetric epoxidation reaction plays a pivotal role in this synthetic route as all 4 stereogenic centres present in the 3,7-dimethylundecane-1,5,6-triol target are generated by this methodology at three different stages of the proposed synthesis. The epoxy alcohol formed at each stage was subjected to regioselective ring opening followed by a protective group strategy which allowed for the protection of the secondary hydroxyl group as the benzyl ether and left the primary hydroxyl group, available after oxidation to the aldehyde, for a two-carbon chain extension to an α,β-unsaturated ester. This ester in turn was reduced to an allylic alcohol which formed the starting material for a second cycle of reactions. In this manner a synthetic route towards the target compound was developed and problems associated with the route investigated. The dissertation shows that a viable route was developed with complete stereochemical control in the formation of the stereogenic centres, even though the final product, the protected 3,7-dimethylundecane-1,5,6-triol was not obtained due to time constraints and material shortages. / Dissertation (MSc (Chemistry))--University of Pretoria, 2007. / Chemistry / MSc / unrestricted

O projeto teve por finalidade isolar e identificar fungos, avaliar o potencial toxigênico dos isolados de Aspergillus spp. e Fusarium spp., detectar e quantificar aflatoxinas B1, B2, G1, G2 e fumonisinas B1 e B2 em amostras de misturas de cereais. Foram analisadas 15 marcas de misturas de cereais, prontas para o consumo, adquiridas de supermercados e de empresas que comercializam o produto nacionalmente via internet. Foram adquiridas amostras por sete meses, totalizando 105 amostras ao final do experimento. A contagem de bolores nas amostras variou de 1,0 x 101 a 2 x 105 unidades formadoras de colônias (UFC)/g, com isolamento de sete cepas de Aspergillus flavus. As aflatoxinas B1 e G1 foram detectadas em poucas amostras e em baixos níveis, sendo que estes resultados podem ser devidos à baixa atividade de água nos produtos avaliados, a qual foi inferior a 0,63. A fumonisina B1 foi detectada em 84,8% das amostras, no entanto, a ingestão diária provável calculada para as fumonisinas esteve abaixo da recomendação do JECFA. Apenas uma amostra apresentou níveis de fumonisinas acima do limite esperado para 2016. Adicionalmente, foi observado que 21% das amostras apresentaram mais de um tipo de micotoxina, o que poderia conduzir à potencialização de efeitos tóxicos. / The project aimed to isolate and identify fungi, evaluate the toxigenic potential of isolates of Aspergillus spp. and Fusarium spp. detect and quantify aflatoxins B1, B2, G1, G2 and fumonisins B1 and B2 in samples of cereal mixtures. We analyzed 15 brands of cereal mixtures ready to eat adding up to 105 samples at the end of the experiment. Samples were acquired in supermarkets and from companies that market the product nationally by internet. The mold count in the samples ranged from 1.0 x 101 to 2 × 105 colonies forming units (CFU)/ g, with isolation of seven strains of Aspergillus flavus. Aflatoxin B1 and G1 were detected in a few samples and at low levels, what might be due to the low water activity in the product reviews, which was less than 0.63. Fumonisin B1 was detected in 84.8% of the samples, however, the probable daily intake calculated for fumonisin was bellow the JECFA recommendation. Only one sample showed fumonisin levels above the expected limit for 2016. Additionally, it was observed that 21% of the samples presented more than one type of mycotoxin, which could lead to enhancement of toxic effects.

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.

The mycotoxin, fumonisin B1 is produced by the mould Fusarium moniliforme, a common contaminant of maize and maize products. Small doses (mg/kg) of ingested fumonisin B1 have been shown to cause diseases and even death in animals, including non-human primates. Thus highly sensitive methods have been employed to detect fumonisin B1 presence in foods, feeds and in animals. This study comprised two parts.The initial part focused on establishing reliable extraction, purification and quantitation of fumonisin B1 using high-performance liquid chromatography (HPLC) on culture extracts. The second part was to analyse sera of Black African women with pre-eclampsia for the presence of fumonisin B1 using HPLC. Maize patty cultures and broth cultures were inoculated with Fusarium moniliforme PPRI 1059 and incubated. Fumonisin B1 was extracted and purified by centrifugation strong anion exchange chromatography (SAX). Eluents from SAX cartridges were analysed using Thin-layer chromatography (TLC) and fluorescence HPLC after o-phythadialdehyde (OPA) derivatisation. Fumonisin B1 standards on HPLC gave a retention time of 7.5 minutes using methanol/0.1 M sodium dihydrogen phosphate (68 + 32, pH 3.3) as mobile phase and a 25 cm C8 column. Patty cultures produced the highest yields of fumonisin B1. In the case of serum samples, a double-blind study was carried out using women attending the obstetric clinic at a large city teaching hospital. The population comprised normal, pre-eclamptic and eclamptic women. On HPLC analysis a significantly higher mean concentration of fumonisin B1 concentration was found in the eclamptic group (P<0,005) as compared to the other two groups.Thus fumonisin B1 may have a role to play in eclampsia for which the aetiology is still unknown. / Thesis (M.Med.Sc.)-University of Natal, 1999.

The central nervous system is a potential site of action for the Fusarium mycotoxin Fumonisin B1 (FB1), and is exemplified in horses by the disease equine leukoencephalomalacia. Structurally resembling sphingoid bases, FB1 inhibits ceramide synthase, an enzyme involved in sphingolipid metabolism, leading to accumulation of free sphinganine (Sa) and sphingosine (So). This investigation focused on FB1, Sa, So and the Fusarium mycotoxins fusaric acid (FA), moniliformin (MaN), zearalenone (ZEA), deoxynivalenol (DON), and T-2 toxin (T2). Effects of the Fusarium mycotoxins and sphingoid bases on the N2a neuroblastoma cell line were assessed using the methylthiazol tetrazolium (MIT) and ApoGlow™ assays. The MIT assay revealed significant differences between the viability of N28 control cells and the cytotoxic effects of FB1 (p=0.001), So (p=1.1 x10-6 ), Sa (p=1.9x10-6 ), MON (p=0.002), DON (p=0.04) and ZEA (p=0.003) on N28 cells between 5-250µM. The cytotoxic effects of FA did not differ significantly from controls (p=0.1). The ApoGlow™ assay revealed that in N28 cells, FB1 at 8µg.ml-1, FA at 128µg.ml-1, and (FBI+FA) combined induced growth arrest at 2 and 4µg·ml-1. Assessment of the effects of FBI and FA on the Jurkat leukaemic suspension cell line revealed that FB1 induced apoptosis at 1.56,12.5 and 50µ.ml-1, growth arrest at 100, 200 and 800µg.ml-1 and proliferation at 400µg.mg-1. Fusaric acid induced proliferation at 1. 56µg.ml-1, apoptosis at 3.15µg.mrl, growth arrest at 100 and 200µg.mrl, and necrosis at 800µg.ml-1. Combined, (FB1+FA) induced apoptosis at 1.56, 3.15,12.5 and 800µg.ml-1. Flow cytometry and fluorescence microscopy revealed that mycotoxins, Sa and So induced varying levels of apoptosis and necrosis in N28 cells. Acridine orange and ethidium bromide staining facilitated discrimination between viable, apoptotic and necrotic cells. Transition of the mitochondrial transmembrane potential was measured using Rhodamine 123 with propidium iodide, and the dual emission potential sensitive stain JC-1. Changes in mitochondrial membrane potential and plasma membrane integrity were expressed as increases or decreases in fluorescence intensity. An increase in mycotoxin concentration from 50 to 200µM was usually paralleled by a decrease in J-aggregate formation, suggesting a decrease in the ?¦¥m. Staining with Rh 123/PI indicated at specific concentrations whether N28 cells were either late apoptotic or necrotic reflected by the levels of PI uptake. No dose dependant mechanism of cell death was established using either method, as fluctuations were evident. Immunolocalisation of T2, ZEA and FB1 within cellular organelles that exhibited ultrastructural pathology provided correlation between mycotoxin exposure and effects. Multinucleate giant cells and retraction of cellular processes were observed. At the electron microscope (EM) level, FB1 was immunolocalised within microsegregated and peripherally condensed nucleoli, the nucleoplasm, distorted mitochondria and dilated endoplasmic reticulum (ER). The capacity of cells to incorporate mycotoxins and effect cytological changes represents a major factor in the potential for initiation of malignant transformation. Exposure of N2a cells to FB1 for 72 hours increased intracellular free Sa and depleted complex sphingolipids. Using High Performance Liquid chromatography (HPLC), acid hydrolysis revealed reduction in Sa from a level of O.6±0.12µM in control cells, to 02±0.lµM in cells exposed to 50µM and lOOµM FB1. Base hydrolyses revealed increase in free Sa: So ratios from 0.52±0.2 in control cells, to 1.14±0.2 and 1.4±0.3 in cells exposed to 50 and l00µM FB1 respectively. The Sa: So ratio in the complete culture media (CCM) increased from 1. 7±0. 3 for control cells to 2.0±0.2 and 2.50±0.4 for cells exposed to 50 and lOOµM FB1 respectively. Correlation coefficients between Sa: So ratios to FB1 exposure in CCM (R=0.75) and within cells (R=0.85), imply that the free Sa: So ratio within cells appears to be a better biomarker for FB1-induced disruption of sphingolipid metabolism in vitro, than the Sa: So ratio in CCM. Optimisation of HPLC analytical procedures improved recovery of FB I from spiked human sera to 95.8% (n=15) and detection limits to -5ng.ml-1 at a signal to noise ratio of 5:1. Optimisation of methods for recovery of Sa and So from spiked sera, led to recoveries of 77.9% and 85.0%, for So and Sa respectively at levels of spiking with lOng per 500µl of serum. Matched sera Sa:So ratios and FB1 levels in brain cancer and non-cancer subjects in KwaZulu-Natal were determined using these optimised methods. Fumonisin B1 was detected in sera of non-cancer (76.7±62.2nM) and brain cancer subjects (l07.38±116nM). Mean serum Sa:So ratios of 21 non-cancer subjects was 1.7±0.7. There was no correlation (R=0.26) between these variables in non-cancer subjects. The mean serum FB1 level in brain cancer subjects was 107.4±116nM (range 10.5-298nM) (n=50) and the mean Sa:So ratio (n=50) was 1.9±1.7 (range 0.40-8.16). No correlation was found between these variables in the brain cancer subjects either (R = -0.23). Fumonisin B1 was irnmunolocalised in 49 of 76 brain tumour tissue samples analysed using immunohistochemistry (IHC). Thirty-eight of the 76 specimens had matched serum FBI levels and Sa: So ratios, and 23 of these were positive for FB1 presence. Although not significantly different (p=0.ll), the FBI sera levels in the cancer group with FBI within the tumour tissue had higher levels of FB1 in sera than the IHC FB1 negative group. Fumonisin B1 was localised within irregular profiles of nuclei, elongated and swollen mitochondria and ER. Immunolocalisation of FB1 within organelles in the brain showing ultrastructural cellular pathology suggests FBI may be implicated in the aetiology of human brain carcinogenesis. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2004.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 131-144). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 131-144). Also available on the Internet.

O projeto teve por finalidade isolar e identificar fungos, avaliar o potencial toxigênico dos isolados de Aspergillus spp. e Fusarium spp., detectar e quantificar aflatoxinas B1, B2, G1, G2 e fumonisinas B1 e B2 em amostras de misturas de cereais. Foram analisadas 15 marcas de misturas de cereais, prontas para o consumo, adquiridas de supermercados e de empresas que comercializam o produto nacionalmente via internet. Foram adquiridas amostras por sete meses, totalizando 105 amostras ao final do experimento. A contagem de bolores nas amostras variou de 1,0 x 101 a 2 x 105 unidades formadoras de colônias (UFC)/g, com isolamento de sete cepas de Aspergillus flavus. As aflatoxinas B1 e G1 foram detectadas em poucas amostras e em baixos níveis, sendo que estes resultados podem ser devidos à baixa atividade de água nos produtos avaliados, a qual foi inferior a 0,63. A fumonisina B1 foi detectada em 84,8% das amostras, no entanto, a ingestão diária provável calculada para as fumonisinas esteve abaixo da recomendação do JECFA. Apenas uma amostra apresentou níveis de fumonisinas acima do limite esperado para 2016. Adicionalmente, foi observado que 21% das amostras apresentaram mais de um tipo de micotoxina, o que poderia conduzir à potencialização de efeitos tóxicos. / The project aimed to isolate and identify fungi, evaluate the toxigenic potential of isolates of Aspergillus spp. and Fusarium spp. detect and quantify aflatoxins B1, B2, G1, G2 and fumonisins B1 and B2 in samples of cereal mixtures. We analyzed 15 brands of cereal mixtures ready to eat adding up to 105 samples at the end of the experiment. Samples were acquired in supermarkets and from companies that market the product nationally by internet. The mold count in the samples ranged from 1.0 x 101 to 2 × 105 colonies forming units (CFU)/ g, with isolation of seven strains of Aspergillus flavus. Aflatoxin B1 and G1 were detected in a few samples and at low levels, what might be due to the low water activity in the product reviews, which was less than 0.63. Fumonisin B1 was detected in 84.8% of the samples, however, the probable daily intake calculated for fumonisin was bellow the JECFA recommendation. Only one sample showed fumonisin levels above the expected limit for 2016. Additionally, it was observed that 21% of the samples presented more than one type of mycotoxin, which could lead to enhancement of toxic effects.