Physiology

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The cytotoxic effects of aflatoxin B1 and fumonisin B1 on cultured human cells.
(2004) Van der Stok, Mary Elizabeth.; Myburg, Rene Bernadette.
Aflatoxin B1 (AFB1) and Fumonisin B1 (FB1), potentially cytotoxic and carcinogenic mycotoxins are common contaminants of agricultural commodities in South Africa and thus could be detrimental to the human immune system. Many of the cytotoxic effects of AFB1 require its bioactivation to an epoxide, which will bind covalently to macromolecules to form protein and DNA adducts. Fumonisin B1 is a competitive inhibitor of sphingosine and sphinganine N aceyltransferase, which are key components in the pathways for sphingolipid biosynthesis. Accumulation of free sphingoid bases, which are both cytotoxic and mitogenic, could provide a plausible explanation for the toxicity and carcinogenicity of FB1. The cytotoxic effects of AFB1 and FB1 on normal human lymphocytes, individually and in combination were assessed using the methylthiazol tetrazolium (MTT) bioassay. Two different methods of treatment were used, the treatment of isolated normal human lymphocytes for 12, 24, 48, 72 and 96 hours and whole blood treated for 12 hours. Flow cytometry and fluorescent microscopy were used to determine whether AFB1 and FB1 (5uM and 50uM), individually or in combination, were capable of inducing apoptosis, necrosis or nuclear fragmentation in isolated lymphocytes and whole blood treated for 12 hours. DNA damage was evaluated using the comet assay. The results showed that AFB1routinely induced higher levels of cytotoxicity in isolated lymphocytes than FB1. In the combination treatment, the mitogenic properties of FB1 appeared to partially counteract the cytotoxic effect exerted by AFB1. When whole blood was treated with the same concentration and ratio of toxin, FB1 was shown to be more cytotoxic than AFB1. The combination treatment of whole blood was shown to be cytotoxic in a dose dependent manner. The toxins appeared to exert a greater cytotoxic effect, when treated in combination than individually at higher concentrations. Aflatoxin B1 induced increased levels of apoptosis and necrosis in isolated lymphocytes while treatment with the FB1 resulted in increased levels of apoptosis at both concentrations. Treatment with the combination also resulted in increased levels of apoptosis. The levels of apoptosis were reduced in whole blood lymphocytes when compared to isolated lymphocytes. However, treatment with AFB1 and FB1 resulted in increased levels of apoptosis. Both AFB1 and FB1 are capable of inducing nuclear fragmentation. Treatment with FB1 (5uM and 50uM) resulted in greater degree of fragmentation than AFB1. The most nuclear fragmentation was induced by the 5uM combination treatment. The 50uM combination treatment of isolated lymphocytes induced the most DNA damage. As both toxins are common contaminants and have been known to coexist, this could be a potential area of concern for public health.
A cytotoxic evaluation of aflatoxin B1, zearalenone and their epoxide derivatives using human cell lines.
(1996) Pillay, Dharmarai.; Chuturgoon, Anil Amichund.; Dutton, Michael Francis.
Since the discovery of mycotoxins in food, the thrust of biochemical and toxicological research has been carried out on animals which has proven to be uncoordinated and not easily extrapolated to humans. Over the last decade, there have been increasing pressures to review and reduce the use of animals in experimental toxicological studies. Consequently in this study aflatoxin B1 (AFB1), zearalenone (Zea) and their epoxide derivatives have been evaluated using in vitro assays. The HepG2, A549 and Hela cell lines were used for assessing the cytotoxicity, effects on cellular metabolism and sites of action of AFB1, Zea and their derivatives. The cytotoxicity of these mycotoxins was evaluated using the methylthiazol tetrazolium (MTT) reduction assay. Cells, treated with mycotoxins were prepared for transmission electron mlcroscopy (TEM), immunocytochemistry (ICC), scanning electron microscopy (SEM), confocal and light microscopy. From the cytotoxicity assay it was found that the epoxide derivatives were more toxic than the parent toxin when exposed to HepG2 cells with no significant differences in toxicity levels in A549 and Hela treated cells. Both epoxide derivatives displayed a regression of hepatoma cell proliferation at high doses (25ug/ml) while lower concentrations (<12.5ug/ml) enhanced cell growth. Microscopy analyses showed distinct cellular alterations. When exposed to AFB1 (12.5ug/ml) hepatoma cells showed prominent ultrastructural alterations such as areas of cytoplasmic lysis and increased numbers of secondary lysosomes while cells exposed to Zea (l2.5ug/ml) displayed numerous ovoid mitochondria and proliferation of rough endoplasmic reticulum which is indicative of enhanced protein synthesis. The presence of label in toxin treated cells is suggestive of the effects of these mycotoxins. Such cellular changes may lead to altered metabolism and cell function.
The immunocytochemical and electrophoretic localisation of aflatoxin B1-binding proteins in isolated liver mitochondria.
(1998) Raman, Gareth.; Chuturgoon, Anil Amichund.; Dutton, Michael Francis.
Mitochondria perform functions which are central to the life of most eukaryotic cells. These organelles can be considered the ultimate energy power house of a living cell. The role of mitochondria in cancer phenotype remains a fertile area of research. Several carcinogens are known to enter the mitochondria, resulting in impaired functioning and altered structure. Aflatoxin BI (AFB1) a primary type I mycotoxin elaborated by Aspergillus flavus and Aspergillus parasiticus, is carcinogenic for a wide species range. The epoxide is capable of binding to nucleic acids and proteins, resulting in induced mutations, cellular toxicity, and eventually carcinogenesis. Approximately 250 000 deaths occur annually in both China and Africa due to patients presenting with Hepatocellular Carcinoma (HCC). The causative agents being AFB1-ingestion via contaminated foods and feeds, and the Hepatitis B Virus infection. The toxin has a multifaceted mode of attack, capable of being activated to a highly reactive and carcinogenic derivative, the AFB1-8,9-epoxide, via the cytochrome P450 enzyme system of the microsomes, endoplasmic reticulum and also the mitochondria. The epoxide is capable of binding to nucleic acids and proteins, resulting in the formation of covalent adducts. The repeated occurrence of gold labelled toxin within mitochondria from hepatomas of patients presenting with HCC suggested that these organelles were direct sites of toxin binding. Despite observations that mitochondria appear as direct and perhaps preferential targets for attack by AFB1, the actual in vivo immunolocalisation and characterisation of bound AFB1 within liver mitochondria has not been reported previously. In addition the role of AFB1-protein binding within mitochondria was investigated to determine the mode of action of the toxin, within the mitochondrial system. Liver sections from rats treated with a single lethal dose of AFB1, showed distinct ultrastructural abnormalities viz. large nuclei, increased heterochromatin, and swollen mitochondria. Immunocytochemistry revealed for the first time, the selective localisation of conjugated gold labelled toxin within the mitochondria. Toxin was found in the intracristal and peripheral spaces and frequently within the mitochondrial matrix. The mitochondria isolated from treated rats revealed significant alterations and damage to the mitochondrial membranes. The cristae were also markedly swollen with the associated clearing of the mitochondrial matrix. Western blot immunoassays revealed the presence of five AFB1-bound proteins (150kDa, 50kDa, 25kDa, 18kDa, 14kDa) in the inner mitochondrial fraction of isolated mitochondria. High pressure liquid chromatography also revealed that a significant proportion (84%) of an initial dose of toxin, was absorbed by mitochondrial protein. This study is the first to show the presence of specific mitochondrial proteins involved in toxin binding. In addition, the presence of toxin within the mitochondria and the specific binding to inner mitochondrial proteins suggest that the toxin specifically targets the electron transport chain and hence effects ATP production. This study conclusively indicates that mitochondria are direct targets for attack by AFB1 during experimental carcinogenesis. Mitochondria therefore play an important role in AFB1-mediated carcinogenesis.
The occurrence and detection of aflatoxin-macromolecular conjugates in humans.
(1998) Myeni, Sibongiseni Selby.; Chuturgoon, Anil Amichund.; Dutton, Michael Francis.
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.
The synthesis of xanthone derivatives and their enzymatic conversion and inhibition of aflatoxin biosynthesis.
(1996) Gengan, Robert Moonsamy.; Mulholland, Dulcie Aca.; Dutton, Michael Francis.
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.

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