Aflatoxin Bi (AFBi), a highly toxic fungal metabolite (mycotoxin) of certain strains of Aspergillus, has long been known to be carcinogenic in animal species. Accumulation of epidemiological evidence led to its classification, in 1993, by the International Agency for Research on Cancer as a Group I human carcinogen. Aflatoxin Bi contaminates the food supply in most tropical and sub-tropical countries, where it is associated with increased incidence of hepatocellular carcinoma (HCC). In these countries, AFBi is also linked to kwashiorkor, jaundice, and Rey's syndrome. The biological action of AFBi is through its oxidation to AFBi-8,9 epoxide (AFBiO). This epoxide binds to macromolecules like DNA, RNA and proteins as well as amino acids to form AFBi-macromolecular adducts. Quantitation of these adducts is thought to be the most promising approach in the development of methods to measure levels of exposure to aflatoxins. Aflatoxin Bi was produced, isolated and purified using preparative thin layer chromatography (TLC). The toxin was oxidised to AFBiO using dimethyldioxirane and the UV spectra of both the AFBi and AFBiO were determined. Reaction of selected Na-acetyl amino acids (AA) with AFBiO was studied and UV spectrophotometry, TLC, high performance liquid chromatography (FfPLC) and high performance capillary electrophoresis (CE) were used to characterise the reaction products. The epoxide was also reacted with albumin and DNA. Aflatoxin Bi-albumin reaction mixture was hydrolysed and characterised by TLC. Spectrum measurement of the oxidative product of AFBi gave peaks at 266 and 367nm. Qualitative TLC and the epoxide spray reagents confirmed that epoxidation was successful. The in vivo reaction of selected Na-acetyl AA with the epoxide gave peaks between 300 and 400 nm. Naacetyl-arginine, Na-acetyl-lysine and Na-acetyl-histidine showed reaction with AFBiO with maximum wavelengths at 392, 397 and 391 nm respectively. These results strongly suggest that AFBiO is able to covalently bind to lysine, histidine and arginine in albumin. A total of twenty nine blood samples were analysed by HPLC for the presence of AFBilysyl adduct. Of the twenty nine samples, ten were from HCC patients, ten from control patients and nine from kwashiorkor patients. The results show that AFBi-lysine does occur in patients at King Edward VIII Hospital (KEH) and the highest level was detected in HCC patients followed by kwashiorkor patients. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1998.

Adverse effects and increasing cost of therapeutic drugs have renewed an interest in the use of medicinal plant products for the treatment of a variety of chronic disorders. One such bioactive plant-derived compound is a pentacyclic triterpenoid, oleanolic acid (3ß-hydroxy-olea-12-en-28- oic acid, OA) present in herbs. OA possesses a variety of pharmaceutical activities and of interest in this study are the anti-diabetic properties. Diabetes is associated with disorders grouped as microvascular (retinopathy and nephropathy) and macrovascular (atherosclerotic) complications. Accordingly, this study further investigated the potential of OA in diabetes management by studying the effects of this triterpene on kidney function as well as proximal tubular Na+ handling in an effort to identify the site of action of OA. Furthermore, the study evaluated the effects of OA in kidney and liver cell lines to establish whether this triterpene exhibits any toxicity in these organs. OA was extracted using a previously validated protocol in our laboratory. Briefly, dried flower buds of Syzygium aromaticum were soaked in dichloromethane overnight, thereafter in ethyl acetate to obtain ethyl acetate solubles which contained a mixture of OA/ursolic and maslinic acid (MA). OA/MA mixture was subjected to column chromatograph and pure OA was obtained through recrystallization in methanol. The absolute stereostructure of OA was elucidated using 1H and 13C NMR spectroscopy and was comparable to previously reported data. In kidney function studies, various doses of OA (30, 60, 120 mg/kg, p.o.) were administered to male Sprague-Dawley rats twice (8h apart) every third day for five weeks. Rats administered deionised water served as controls. Measurements of body weight, food and water intake, blood pressure, Na+, K+, Cl-, urea and creatinine were taken 24 h from dosing. Renal clearance studies investigated the influence of OA on Na+ handling in the proximal tubule of anaesthetized rats using lithium clearance. Animals were given water with lithium (12mmol/l) for 48 hours following which they were anaesthetized and cannulated using a previously validated standard protocol that has been reported from our laboratories. After a 3½ h equilibration, animals were challenged with hypotonic saline for 4 h of 1 h control, 1½ h treatment and 1½ h recovery periods. OA was added to the infusate during the treatment period. In vitro effects of various OA concentrations (5, 10, 20, 40, 80 μmol/l) were investigated in HEK293, MDBK and HepG2cell lines. Cells were exposed to OA for 24, 48 and 72 h, thereafter, 3-4,5 dimethylthiazol-2-yl- 2,5diphenyltetrozolium bromide (MTT) and single cell gel electrophoresis (comet) assays were conducted. All data are presented as means ±SEM. OA significantly (p<0.05) increased urinary Na+ output from week 2 until the end of the experimental period in a dose independent manner. However, this OA-evoked natriuresis was not reflected in plasma collected at the end of the experiment as there was no change in plasma Na+ concentrations compared with control animals at the corresponding time. OA administration had no significant influence on K+ and Cl- excretion rates throughout the experiment. However, OA significantly (p<0.05) reduced plasma creatinine concentration with a concomitant increase in glomerular filtration rate (GFR). Furthermore, OA administration significantly (p<0.05) decreased mean arterial pressure from week 2 until the end of the experimental period. Intravenous infusion of OA at 90 ug/h for 1 ½ h induced a marked increase in urinary excretion rates of Na+. This increase was accompanied by concomitant increase in FENa proximal and FENa distal and FELi which persisted until the end of the experiment without any apparent changes in GFR. The cell viabilities of HepG2, HEK293 and MDBK cell lines were significantly increased after 24 h exposure, however, the viabilities of all the three cell lines dropped after 72 h exposure to values that did not achieve statistical significance in comparison to the respective controls. In addition, all OA-treated cells in the comet assay had intact DNA after exposure for 24, 48 and 72 h. Hence, the decrease in viability that was observed in the MTT assay after 72 h exposure could probably be attributed to the depletion of nutrients in the culture medium. The results of the present study, apart from confirming our previous observations of the natriuretic effects of OA in rats, indicate that this effect is in part mediated via the inhibition of proximal tubular Na+ reabsorption and increased Na+ secretion. We speculate that this increased Na+ secretion could have been due to increased tubular function and not to the toxicity of OA as indicated by MTT and comet assays. These findings suggest that OA does not exhibit toxicity in the kidney and the liver. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Westville, 2012.

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.

Fusaric acid (FA) (5-butylpicolinic acid) is a divalent ion chelating agent that has low affinity for Ca2+ and Mg2+ and a high affinity for other essential metal ions such as Fe2+ Mn2+, Zn2+ and Cu2+. Its mode of action therefore may involve its interference with various transition metal ions and thus may be analogous to picolinic acid. Fusaric acid inhibits the proliferation of numerous cell lines in vitro. In the current in vitro study the effects of FA on peripheral blood lymphocytes was studied. Lymphocytes from a healthy volunteer were treated with varying concentrations of FA (3uM, 6uM, 25uM, 50uM 100uM 200uM, 400uM, and 1000uM) to assess the toxins apoptotic inducing potential. The 'Comet Assay' (Single cell gel electrophoresis), DNA fragmentation and Annexin V flous assays were employed to assess apoptosis. These assays proved that FA induces apoptosis in human lymphocytes. Lymphocytes were also incubated with phytohaemagglutinin (PHA) (10ug/ml) and increasing doses of FA (10, 50, 100 and 200uM). After 24, 48 and 72 hours of incubation an aliquot of the cells was stained with propidium iodide and subjected to flow cytometric analysis to assess the DNA configuration. Phytohaemagglutinin stimulation led to a significant increase of the S-phase of the cell cycle after 48 and 72 hours of incubation. All the PHA induced effects were reduced by co-incubation with increasing doses of FA. Lymphocytes were inhibited in the S-phase at 100 and 200uM concentration of FA. The current study shows that the in vitro inhibitory effects of FA can be demonstrated using flow cytometric technology on a cellular level. Fusaric acid leads to an inhibition of cell cycle progression in peripheral blood lymphocytes. / Thesis (M.Med.)-University of Natal, 2003.

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.

Malaria disease poses a serious global health burden as recent reports have indicated that nearly half of the world’s population is at risk (WHO 2008). The World Health Organization (WHO) Expert Consultative Team has reported that 90% of all malaria deaths occur in Sub Saharan Africa. (WHO, 2008). Despite the numerous global efforts to control and manage the disease, through various ways, use of chemotherapeutic agents continues to be the major intervention strategy in the control of malaria. The WHO recommended use of Artermisinin combination therapy (ACT) has been hampered by an imbalance between demand and supply in the poor socioeconomically challenged rural populations of Sub Saharan Africa, the epicenter of malaria infection. Chloroquine (CHQ), therefore, continues to be used in most malaria endemic areas in developing countries despite development of P. falciparum resistance to the drug (WHO, 2006). Oral administration is the major delivery route for CHQ. However, CHQ is a bitter drug, with an inconvenient dosing schedule leading to incomplete courses of therapy by most malaria patients. Oral CHQ administration is also associated with adverse effects in various organ systems resulting from deposition of CHQ in these organs to elicit impairment of glucose homeostasis, renal and cardiovascular function. Alternative methods of CHQ administration such as transdermal delivery have, therefore, been suggested, in an effort not only to avoid the bitter taste, but also to modify the dosing schedule, which may improve patient comfort and compliance. Transdermal delivery of CHQ via an amidated matrix patch, which is envisaged to ensure a slow, controlled and sustained release of therapeutic concentrations of CHQ, may circumvent the previously reported adverse effects of oral CHQ. It is against this background that the current study compared the effects of transdermal CHQ patch and oral chloroquine in the management of malaria as assessed by the ability to clear parasites of P. berghei infected rats. The other aims were to investigate and distinguish between the patho physiological effects of malaria and CHQ treatments on blood glucose and plasma insulin concentration, renal and cardiovascular function in male Sprague-Dawley rats. To investigate and distinguish between the pathophysiological effects malaria infection and CHQ treatments on blood glucose homeostasis, renal and cardiovascular function markers, separate groups of non infected and P. berghei infected male Sprague Dawley rats (90g-150g) were used. The animals were treated twice daily with oral CHQ (60mg/kg) and a once off transdermal delivery of CHQ via topical application of pectin CHQ matrix patch (53mg/kg) in a 21 day study divided into pre treatment, (days 0-7) treatment (days 8-12) and post treatment (days 13-21) periods. The animals were housed individually in metabolic cages for the duration of the study. Treatment was for 5 consecutive days. Measurements of body weight, food and water intake, mean arterial pressure (MAP), blood glucose concentration, % parasitaemia, haematocrit, and 24 hour urine volume, Na+, K+, urea and creatinine outputs were done every day during the treatment period, and every third day during the pre and post treatment periods. Separate groups of non fasted conscious animals (n=6) were sacrificed on days 0, 7, 8, 9, 10, 12, 14 and 21, at 24 hours after the last treatment for oral CHQ administration and after a once off patch application on the first day of treatment. The plasma obtained was assayed for plasma insulin, lipid profile parameters and plasma Na+, K+, urea and creatinine. The harvested liver and gastrocnemius muscle were used for determination of glycogen concentration. The current study has demonstrated the sustained controlled release of CHQ from the pectin matrix patch, demonstrating the therapeutic ability to clear P. berghei malaria parasites from systemic circulation. Malaria infection and oral CHQ treatment exhibited blood glucose lowering effects which were circumvented by topical application of the pectin CHQ matrix patch. Oral CHQ elevated hepatic glycogen concentration through mechanisms that are still to be elucidated. Topical application of CHQ via pectin matrix patch did not alter hepatic and gastrocnemius muscle glycogen concentrations. Malaria infection and oral CHQ delivery reduced food intake, water intake and % body weight changes of the animals as well as inducing natriuresis, reduced urine output and increased urinary creatinine outputs. Malaria infection was also shown to elicit hyperkalaemia and kaliuresis in experimental animals. Hypotensive effects of malaria infection and oral CHQ delivery were also demonstrated in the current study. Malaria infection and oral CHQ delivery elevated plasma total cholesterol and LDL-c as well as reduction in the cardio protective particle, plasma HDL-c, concentrations. Topically delivered CHQ via pectin CHQ matrix patch did not evoke any such alterations, suggestive of its ability to circumvent the observed adverse effects of oral CHQ delivery due to sustained, controlled release of therapeutic concentrations of CHQ from the transdermal formulation. To the best of our knowledge, the results of the present study provides the first evidence of the release of therapeautic CHQ concentrations from pectin CHQ matrix patch that cleared the malaria parasites from systemic circulation as well as demonstrating the ability of the transdermal formulation to circumvent the adverse effects of oral CHQ delivery in glucose homeostasis, renal and cardiovascular function markers. This is clinically relevant as it provides a feasible and novel alternative method of CHQ delivery that could play a major role in the effective management of malaria. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

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.

The biosynthesis of Aflatoxin B1 (AFB1) has been the subject of conflicting speculation and numerous reviews. The currently accepted scheme for the aflatoxin pathway is based on data obtained from feeding studies using isotopically labelled precursors. In these studies the conversion of possible intermediate metabolites to AFBl by mutants of Aspergillus parasiticus illustrated their role as biogenetic precursors. Currently there is now agreement on the identity of most of the intermediate Illetabolites involved in the biosynthesis of AFB1. However, there is a lack of clarity on the details of AFB1 biosynthesis including the conversion of sterigmatocystin (ST) to AFB1 via the metabolite O-methylsterigmatocystin (OMST). There is no clear cut evidence of the metabolic role of OMST, i.e., either it is a compulsory intermediate or a shunt metabolite and hence part of a metabolic grid. In order to investigate this step in AFBl biosynthesis, ST was isolated from surface cultures of A. versicolor (M1101) and purified by silica gel column chromatography and repeated recrystallisation. Sterigmatocystin was characterised by thin layer chromatography (t.1.c.), low resolution mass spectrometry (M.S) and nuclear magnetic resonance spectroscopy (N.M.R). A series of seven derivatives of the free hydroxyl group of ST were synthesised by known chemical reactions, purified by silica gel column chromatography and characterised by high resolution mass spectrometry and proton nuclear magnetic resonance spectroscopy. A high pressure liquid chromatography (HPLC) method was developed using a fluorescence detector. The optimum parameters for the separation of the four major aflatoxins, namely AFBl, AFB2, AFGl and AFG2, using trifluoroacetic acid as the derivatising reagent, were obtained for a reversed phase Prodigy C18 column with a mobile phase of water: acetonitrile: isopropanol: acetic acid (8: 1: 0.5: 0.5, v/v). Feeding studies, using whole cells of A. parasiticus (WhI-11-105), showed that ST and the ST derivatives were converted to AFB1. A time courser study for the conversion of ST and selected ST derivatives to AFB1 indicated a decrease in the rate of conversion in the order: a-propyl sterigmatocystin (OPROST) > a-ethyl sterigmatocystin > a-methylsterigmatocystin > Sterigmatocystin> a-benzoyl sterigmatocystin (OBzST). It was apparent that the "enzyme" responsible for the conversion of the derivatives to AFB1 did not display a high degree of substrate specificity, since it was unable to recognize the difference between the various alkyl groups, either as ether or ester functional groups. An HPLC method was developed using a diode array detector. The optimum parameters for the separation of aflatoxin metabolites and the synthesised derivatives were obtained for a reversed phase Lichrosphere RP-I8 column with a 30 minute gradient elution program with water and acetonitrile as the mobile phase. Crude cell-free extracts were prepared by lyophilisation of the mycelia of A. parasiticus (Whl-11l-105) with phosphate buffer. The temperature and pH for the conversion of ST to AFB1, were found to be optimum at 28°C and 7.2, respectively. The addition of SAM (1.5 mM) and NADPH (1.5 mM) increased the conversion of ST to AFBl from 11.21 % to 27.10 %. A time course study with ST, OMST and OPROST showed that the rate of conversion to AFBl was close to linear for an incubation time of up to 60 minutes. Approximation of the reaction rate indicated a decrease in the order: OMST > ST > OPROST. This indicated that the time course reaction using whole cells was in part a measure of membrane permeability rather than substrate specificity. Molecular exclusion chromatography was used to separate enzymatic protein from primary and secondary metabolites, small biomolecules and indigenous co-factors (MW < 10 000) and the partially purified "enzyme" was concentrated by dialysis against solid sucrose. The "enzyme" was subjected to non-denaturing polyacrylamide gel electrophoresis and was found to be made of sub-units ranging from 58 kDa to over 200 kDa. Enzymatic investigations with ST, as substrate, indicated that OMST is a compulsory intermediate in the biosynthesis of AFBl. Also, enzymatic investigations of selected ST derivatives showed that the partially purified "enzyme" displayed relative specificity for these substrates, viz., OMST, OPROST and OBzST. Three xanthones, namely, 1-hydroxy-,6-dimethylxanthone, I-methoxy-3,6-dimethylxanthone and l-acetyl-3,6-dimethylxanthone were synthesised, purified and characterised spectroscopically. Whole cell studies of A. parasiticus (CMI 91019b) and A. parasiticus (Wh1-11-105) showed that these xanthones inhibited AFBl production to varying extents. Kinetic studies of cell-free extracts revealed that the 1-methoxy-3,6-dimethylxanthone derivative was a non-competitive inhibitor. The Michaelis Menten constant (Km) of approximately 5.60 uM (for OMST) was determined for a cell-free reaction at pH 7.2 and 28 QC. A Clark oxygen electrode was used to carry out oxygen consumption studies in a partially purified "enzyme" preparation. A calibration system was designed and the enzymatic conversion of OMST to AFB1 and NADPH consumption were monitored by HPLC and UV spectroscopy, respectively. From the results of these enzymatic reactions, the following stoichiometric relationship was determined: 2 mole oxygen consumed = 1 mole NADPH consumed = 1 mole AFB1 produced A tentative mechanism is discussed for the conversion of OMST to AFB1 which utilizes a monooxygenase and a dioxygenase. / Thesis (Ph.D.)-University of Natal, Durban, 1996.

Methamphetamine is a highly addictive stimulant drug whose illicit use and resultant addiction has become an alarming global phenomenon. The mesolimbic dopaminergic system in the brain, originating in the ventral tegmental area and terminating in the nucleus accumbens, has been shown to be central to the neurobiology of addiction and the establishment of addictive behaviour. This pathway, as part of the reward system of the brain, has also been shown to be important in classical conditioning, which is a learnt response. This common pathway has supported theories suggesting addiction as a case of maladaptive associative learning. Within the modulation of learning and memory, the neurohypophyseal hormones vasopressin and oxytocin have been seen to play a vital role. Vasopressin exerts a long- term facilitatory effect on learning and memory processes. Studies have shown that the stress responsive AVP V1b receptor systems are a critical component of the neural circuitry underlying emotional consequences of drug reward. Oxytocin, on the other hand, has an effect on learning and memory opposite to that of vasopressin. Previous studies have shown that oxytocin caused a decrease in heroin self-administration, as well as attenuated the appearance of cocaine-induced hyperactivity and stereotyped behaviour. Therefore, we adopted a reinstatement conditioned place preference model to investigate whether a V1b antagonist or oxytocin treatment would cause a decrease in methamphetamine seeking behaviour. Behavioural findings indicated that methamphetamine induced a change in the place preference in the majority of our animals. This change in preference was not seen after vasopressin administration in the extinction phase. On the other hand, the change in place preference was enhanced during the reinstatement phase in the animals treated with oxytocin. Striatal dopamine levels were determined, as methamphetamine is known to increase dopamine transmission in this area. Results showed that rats that received both methamphetamine and oxytocin had significantly higher striatal dopamine than those that received oxytocin alone. Western blot analysis for hippocampal cyclic AMP response element binding protein (CREB) was also conducted as a possible indicator of glutamatergic NMDA receptor activity, a pathway that is important for learning and memory. The Western blot analysis showed no changes in hippocampal pCREB expression. Overall our data led us to conclude that methamphetamine treatment can change place preference behaviour in rats and that this change may be partially restored by vasopressin antagonism, but exaggerated by oxytocin. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Westville, 2012.