published_or_final_version / Biochemistry / Master / Master of Philosophy

Humans are exposed to arsenicals through many routes with the most common being drinking water. Exposure to arsenic has been associated with an increased incidence of skin, lung, liver, prostate, and bladder cancer. Although the relationship between arsenic exposure and carcinogenesis is well documented, the mechanisms by which arsenic participates in tumorigenesis are not fully elucidated. We evaluated the potential epigenetic component of arsenical action by assessing the histone acetylation and DNA methylation state of 13,000 human gene promoters in a cell line model of arsenical-mediated malignant transformation. We show changes in histone H3 acetylation and DNA methylation occur during arsenical-induced malignant transformation, each of which is linked to the expression state of the associated gene. These epigenetic changes occurred non-randomly and targeted common promoters whether the selection was performed with arsenite [As(III)] or with the As(III) metabolite monomethylarsonous acid [MMA(III)]. The epigenetic alterations of these promoters and associated malignant phenotypes were stable after the removal of the transforming arsenical. One of the affected regions was the promoter of WNT5A. This gene is transcriptionally activated during arsenical induced malignant transformation and its promoter region exhibited alterations in each of the four histone modifications examined which were linked to its transcriptional activation. Experimental reduction of WNT5A transcript levels resulted in abrogated anchorage independent growth, suggesting a participative role for the epigenetic remodeling of this promoter region in arsenical-induced malignant transformation. Taken together, these data suggest that arsenicals may participate in tumorigenesis by stably altering the DNA methylation and histone modifications associated with targeted genes, uncovering a likely set of participative genes and representing a mechanism to potentially explain the latency associated with arsenic-induced malignancy.

Normal tissue structure and function are maintained by a dynamic interaction between epithelial cells and the stroma consisting of fibroblasts, adipose, vasculature and resident immune cells, and a multitude of cytokines and growth factors. Stroma was usually studied in the background of the malignant lesion, only in recent years researchers have started considering its role before carcinogenic lesions appear. Recent studies have shown that stromal cells and their products can cause the transformation of adjacent cells through transient signaling during phenomena like adipogenesis and inflammation by secreting various cytokines and chemokines into the matrix which can lead to apoptosis resistance, proliferation, mutations etc. Research in the last few years has demonstrated a functional role for stroma in the initiation and progression of breast, colon and prostate carcinomas. In this study effect of adipogenesis and/or inflammation on prostaglandin biosynthesis is investigated and the effects that these prostaglandins can have on epithelial cells is highlighted. This work demonstrates that normal colonic fibroblasts CCD18Co can produce anti-tumorigenic and pro-tumorigenic prostaglandins during adipogenesis and that this signaling is mediated via COX-2 activation. Although deoxycholic acid (DCA), a secondary bile acid that is responsible for inflammation in the gastro-intestinal tract, induces COX-2 signaling in the fibroblasts the downstream signaling of prostaglandin synthases is suppressed. Adipogenesis also leads to an increased polyamine catabolism. Effects of the prostaglandins were studied on various epithelial colon cancer cell lines. It was seen that 15d-PGJ2 causes growth inhibition and apoptosis in all cell lines tested and it was demonstrated that an activated K-RAS suppressed this phenomena. It was also seen that 15d-PGJ2 treatment could induce MAPK signaling and that an activated K-RAS suppressed JNK activation via AKT and MKK4. In conclusion this work reports that colonic fibroblasts can produce anti-tumorigenic factors like 15d-PGJ2 which may then induce apoptosis in epithelial cancer cells. This would be suppressed by an activated K-RAS and at the same time 15d-PGJ2 mediated MAPK signaling could confer a growth advantage for these cells and thus aid in tumor progression.

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.

Chemoprevention may be defined as the inhibition, delay or reversal of carcinogenesis by dietary compounds or their derivatives. "Imifino" is a collective name for many wild plants used predominantly by rural people as herbs in cooking. Many of these herbs possess medicinal properties. As the rural population is at higher risk of exposure to dietary carcinogens, such as mycotoxins, this pilot study was undertaken to determine whether the Amaranthus lividus plant held potential for use in chemopreventive strategies. The plant leaves were extracted to obtain individual solvent fractions. Cytotoxic profiling of the fractions using the SNO oesophageal adenocarcinoma cell line and normal human lymphocytes was achieved using the methylthiazol tetrazolium salt bioreduction assay. The SNO cell line, the A549 lung adenocarcinoma cell line and normal human lymphocytes were utilised for the evaluation of the anti-mycotoxigenic potential of the plant fractions in combination with two important dietary carcinogens, aflatoxin B1 and fumonisin B1. A specific biomarker assay (the induction of reduced glutathione) was employed using the SNO cell line. Flow cytometry was also conducted to determine the apoptotic properties of the acetone fraction on normal human lymphocytes. The results of the anti-mycotoxigenic study showed that certain fractions did have protective effects against both of the carcinogens tested. In addition, these effects were noted in the two cancerous cell lines, which were of different tissue origin. None of the fractions tested were toxic towards the normal human lymphocytes. The glutathione assay indicated that certain acetone fraction dilutions were inducive to reduced glutathione production. This plant is a promising candidate for further investigation concerning chemoprevention and the rural community could be educated on the possible benefits of this herb. / Thesis (M.Med.)-University of KwaZulu-Natal, 2005.

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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 study of retroviruses has been of pivotal significance to the field of biomedical science, where it has provided fundamental insights into the processes underlying both viral and non-viral carcinogenesis. Ovine pulmonary adenocarcinoma (OP A), a contagious lung cancer of sheep and goats, has emerged over the past three decades as an invaluable model of human epithelial cancers. It is one of the very few animal models of retrovirus induced neoplasia of epithelial tissues, whereas most other such animal models of human cancers pertain to the haematopoietic system. OP A represents a unique, naturally occurring, inducible, outbred animal model of peripheral lung carcinomas, and is caused by a betaretrovirus - jaagsiekte sheep retrovirus (JSRV) - that is receiving increasing attention in the fields of retrovirology and lung cancer research. JSRV exists in two highly homologous, yet molecularly distinct forms. The first is an exogenous form of the virus that is transmitted horizontally from one animal to another. This form is infectious and the direct cause of OP A. The other is an endogenous form, 15 to 20 proviral copies of which reside benignly in the genome of sheep and are transmitted vertically from one generation to the next. At the time this study commenced, no knowledge existed regarding the underlying pathogenic mechanism by which JSRV causes OPA. Even though the nucleotide sequence of exogenous JSRV had been elucidated seven years earlier, only limited sequence information was available on endogenous JSRVs. With a view towards identifying genetic regions or elements within exogenous JSRV that could potentially be implicated in its pathogenic function, this study began with the cloning of the first three full-length endogenous JSRV loci ever isolated from sheep. The DNA sequences of these full-length endogenous JSRV loci were determined and comprehensively analysed. Comparison with exogenous JSRV isolates revealed that the two forms of the virus are highly homologous, yet can be consistently distinguished in three short regions within the coding genes. Two of these reside in the gag gene, and one at the end of the env gene. These regions were named the variable regions (VRs) of sheep betaretroviruses. The JSRV VR3 in env was linked by our collaborators to the virus's ability to transform cells in tissue culture. The effects and biological significance of VRI and VR2 in gag are subtler and more difficult to determine. After identifying these regions, it became the objective of this study to develop relevant molecular tools that could be used to discern the significance of these variable regions in vivo, and to characterise these tools in vitro to assess their suitability for in vivo studies. The development of these tools entailed the design of a novel strategy that was implemented to precisely substitute the endogenous VRI and VR2 (individually and in combination) into an infectious molecular clone of exogenous JSRV. These chimeric constructs were shown to support retroviral particle release into the supernatant of transiently transfected cells in tissue culture. These particles were confirmed by independent experiments to have arisen specifically from transfection with the chimeric clones. Finally, the particles were shown to be capable of infecting cultured cells and of productively integrating their genomes into those of their host cells, rendering these particles fully competent retroviruses that can be used in the context of in vivo studies to determine the biological significance of VRI and VR2. This study has made a significant contribution to the further development of the OP A / JSRV model system of human epithelial lung cancers. It has also led to the design of a molecular substitution strategy that can be adapted to introduce any genetic region into a cloned DNA construct, regardless of the degree - or lack of interrelation - of the two DNA sequences, thereby creating a highly versatile molecular biological tool. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2007.

Experiments were conducted to examine the role of dietary selenium (Se) and changes in antioxidant capacity during DMBA-induced mammary carcinogenesis. Weanling rats were fed graded amounts of Se in an AIN-76 diet, modified to approximate the amount and type of dietary fat consumed in Canada. Animals fed 3-4 ppm Se had a reduced tumor incidence but there was evidence of chronic Se toxicity. Low Se did not elevate tumor incidence. Thus, supplemental dietary Se did not offer any protection in reducing the incidence of cancer when rats were fed a diet representative of North American intakes of fat. Blood GSHPx activity was elevated in rats that remained free of tumors (NT group) compared to animals that would eventually develop tumors (WT group). SOD activity was reduced in WT and NT rats, but appeared to be related to carcinogen administration. These differences were observed before tumor development and over a wide range of Se intakes. WT rats excreted more $ sp{75}$Se compared to both control and NT rats. These changes, however, were not reflected in elevated lipid peroxidation. Thus, one of the protective mechanisms during carcinogenesis may be the capacity of the animal to elevate GSHPx activity.

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