To my God son Prenavin Mudaly and my nieces Micosha and Theah Palanee, thank you for the joy you bring. Thank you for the laughter and joy you bring to my life and all the memories.
Figure 6.1
Light micrograph of 50!M FBI-treated N2a cells with DAB-labeled FBI 193 immunolocalized within giant cells (G) with extensive areas of cytoplasmic swelling, lysis, or vacuolization (black arrows) (Haematoxylin, Magn. X 400). Light micrograph of 100!J.M FBI-treated N2a cells with DAB-labeled FBI 194 immunolocalized in rounded dead cells (arrows) and in the cytoplasm (C) of cells that appear less compromised but with some cellular swelling (S) (hematoxylin, mag X 400).
Figure 8.1
Figure 9.1
Figure AU
LIST OF TABLES
- Table 1
- Table 2 .1
- Table 3.1
- Table 5
- Table 6.1
- Table 7.1
- Table 8.1
- Table 9.1a
- Table 10. 1
- Table A2
- Table A3
- Table A4
- Table A8.2
- Table A8.3
- Table A8A
- Table A9 .1
- Table A9.2
- Appendix 10.1 Table A 10 .1
- Appendix 10.3 Table AIO.2
Efficiency of the dual extraction of sphinganine and sphingosine from 246 serum samples spiked with increasing volumes of methanol. Determination of the serum concentrations of sphinganine, sphingosine and 407 Fumonisin BI in the serum of 21 non-cancer subjects.
LIST OF ABBREVIATIONS
UTP UV
UltrastructUl'al and immunochemical analyses of brain carcinomas for 303 fumonisin BI
Introduction
- THE DIET-CANCER RELATIONSHIP
- MYCOTOXINS
- EXPOSURE TO Fusarium FUNGI AND FUMONISINS IN SOUTH AFRICA
- BRAIN CANCER IN SOUTH AFRICA
- POTENTIAL FOR FUNGAL ASSOCIATION IN NEUROLOGICAL DISEASES
- OBJECTIVES
Fusarium mycotoxins have been implicated in food-toxic aleukia, Drov or Kashin-Beck disease, Akakabi-byo or scurvy grain intoxication, and esophageal cancer in humans (Marasas et al., 1979a; 1988b; Rheeder et al., 1992). The significant contribution of maize and maize-based foods to the diet, and therefore their potential interaction between diet and toxicity, justified the present study to determine whether selected Fusarium mycotoxins may be associated with the pathophysiology of brain cancer in the population of KwaZulu-Natal, South Africa.
Literature Review
- STRUCTURE OF THE NERVOUS SYSTEM
- STRUCTURE OF THE BRAIN
- The meninges
- The cerebrum
- The cerebellum
- The brain stem
- THE BLOOD BRAIN BARRIER
- WHA T IS BRAIN CANCER?
- NEUROTOXICOLOGY
- Ganglioneuromas
- Meningiomas
- Pineal tumours
- Pituitary Adenomas
- HYPOTHESES AND THEORIES OF CANCER INDUCTION
- MYCOTOXINS
- FUMONISINS
- Fate and significance of sphingolipids
- SPHINGOLIPIDS AND THEJR METABOLISM
- Biochemical pathways and compartmentalization of de novo sphingolipid biosynthesis
- Functions of sphingolipids and their breakdown products in cellular regulation
- THE SPHINGOMYELIN PATHWAY, APOPTOSIS AND CERAMIDE
- Ceramide in Apoptosis
- Ceramide metabolism and disease
- FUMONISIN INDUCED DISRUPTION OF SPHINGOLIPID METABOLISM
- Fumonisin-induced disruption of sphingolipid metabolism in cell systems
- Lipid peroxidation
- Endothelial cell damage
- The role of fumonisins in mitogenesis
- MOLECULAR MECHANISM OF ACTION OF FUMONISINS, SIGNAL TRANSDUCTION PATHWAYS, SPHINGOLIPIDS AND FUMONISIN Bl
- APOPTOSIS BY SPHINGOID BASES
- FUMONISIN-INDUCED DISRUPTION OF SPHINGOLIPID METABOLISM IN VIVO
- Tissue and species specificity
- Short and long term cancer initiation studies
- ANIMAL DISEASES ASSOCIA TED WITH CONSUMPTION OF FUMONISIN Bl
- Equidae
- Porcine pulmonary oedema syndrome
- Vervet monkeys
- HUMAN STUDIES
- HUMAN EXPOSURE ESTIMATES
- OESOPHAGEAL CANCER IN HUMANS
- Transkei, South Africa
- EFFECTS OF FUMONISINS ON THE BRAIN AND BRAIN CELLS
- Effects of fumonisins on myelin synthesis
- T-2 TOXIN
- CONCLUSION
It is therefore an integral component of the mechanisms involved in the tight regulation of the extracellular homeostasis necessary for normal CNS function (Burkitt et al., 1993). Brainstem gliomas are tumors located in the lower part of the brain that connects to the spinal cord (brainstem). Sphingolipids contain a CER backbone that anchors them in the outer fold of the lipid bilayer.
Fumonisin BI also induced cell cycle arrest at the G1 phase in CVl cells (Wang et al., 1996). Alteration of blood vessels in the CNS and/or disruption of the BBB may have effects on the transfer of FBI or Sa to brain tissue (Kwon et al., 1997a). In the rat, brain rate is fastest during postnatal development day (PND) 7 (Dobbing and Sands, 1979).
Thus, the greater sensitivity of SM synthesis is probably due to changes in the Km and/or subcellular localization of the respective enzymes (Merrill et al., 1993b).
Cytotoxicity of selected Fusarium mycotoxins and sphingoid bases on the N2a mouse neuroblastoma cell line
INTRODUCTION
This assay depends on the reduction of the yellow-colored MTT salt to purple formazan crystals by the succinate-tetrazolium reductase system belonging to the respiratory chain of mitochondria in viable cells (Altmann, 1976; Slater et al. showed that the reduction is intracellular, and is mainly catalyzed by microsomal enzymes requiring reduced nicotinamide nucleotides, and that succinate can also act as an electron donor in MTT reduction, via mitochondrial succinate dehydrogenase. Hanelt et al. reported the suitability of the MTT assay for the cytotoxic evaluation of the mycotoxins DON, ZEA and ochratoxin The MTT assay has also been used to test other Fusarium mycotoxins on many cell lines (Visconti et al., 1991).
The aim of the present study was to assess the cytotoxicity of selected water-soluble (FBI, MaN and FA) and ethanol-soluble (ZEA, T2 and DON) Fusarium mycotoxins on the mouse N2a neuroblastoma cell line. In addition, the cytotoxic effects of the exogenously added sphingoid bases, Sa and So, were also assessed.
MA TERIALS AND METHODS .1 Materials
- Cell culture media
This suspension was filtered through a 0.45 µm filter to remove insoluble residues of the MTT salt. Aliquots of the cell suspension (10 µl) were dispensed into each of the 96 wells of the microtiter plate, giving a final cell count of 40,000 cells/well. The diluted mycotoxins and sphingoid bases were mixed thoroughly and an aliquot (200 µl) of the test compound at each of the above concentrations was transferred to individual wells of the microtiter plate.
The medium and MTT salt solution were then aspirated and the formazan crystals were dissolved with DMSO (100 µl) for 1 h at 37 °C. Optical density (OD) of cells was measured spectrophotometrically, at dual wavelengths of 595 nm and 610 nm, on a Bio-Rad microplate reader.
RESULTS AND DISCUSSION
The results of the MTT cytotoxicity assays and the comparative effects of FBI, MON, and F A on the N2a neuroblastoma cell line are shown in Figure 3.1. It is likely that the levels of FBI cytotoxicity shown by the MTT assay are related to the FB I-induced inhibition of N2a cell differentiation in culture induced by So. The results of the MTT assay show that Sa and So were extremely cytotoxic at all tested concentrations on the N2a cell line (Figure 3.2).
Lower cell viability may have been detected due to the inhibition of this enzyme by T2 in the N2a cell line. These results are similar to those of the present study, where after 48 hours of exposure to 75~M ZEA, 60% cell death was observed in the N2a cell line.
CONCLUSION
12.5 ~ g.mrl (-50-75 ~ M) ZEA significantly inhibited the mitochondrial division activity of lymphocytes and K562 cells, while in granulocytes none of the tested concentrations were toxic. 1995) reported that when using the brine shrimp bioassay, the concentration of ZEA that caused 50% mortality was -75 ~ M. This colorimetric assay may underestimate cellular damage and detects cell death only in the later stages of apoptosis when the metabolic activity of cells is reduced. Despite this disadvantage, this colorimetric assay was useful for quantifying factor-induced cytotoxicity within a 24- to 48-h period of cell culture and paved the way for further more comprehensive analyzes of the effects of these compounds on the cell line. N2a neuroblastoma cells.
Bioluminescent determination of apoptosis and necrosis using adenylate nucleotide measurements to analyze its effects.
Bioluminescent determination of apoptosis and necrosis using adenylate nucleotide measurenlents to analyse the effects of
Jurkat cell lines
MA TERIALS AND METHODS
Only the effects of FB I and FA were assessed in this international collaborative study, the reason being that this work was part of the first experiments performed and that the other mycotoxins included in the assessments in Chapters 3, 5 and 6 , were added later. in the timeline of the work presented in this thesis. A Lucy 1 Anthos Luminoskan Labsystems microplate luminometer was used, equipped with reagent dispensers capable of injecting 20I-.t1 volumes, allowing full automation of the assay. The surface of the skin contains significant amounts of ATP. Therefore, gloves were worn when performing the test and ATP-free microtiter plates and tubes were also used to minimize errors.
The N2a cells were grown in 75cm2 culture flasks until confluent, typsinized, and following a cell count, 2.5 x 104 cells were dispensed into each well of the microtiter plates. To minimize dilution of the mycotoxins by the media into the pure cell suspension, cells were concentrated in small volumes of CCM.
RESULTS AND DISCUSSION ·
At the higher range of concentrations (i.e., between 256 and 1000~g.mrl), the ADP:ATP ratios of the FA-treated N2a cells began to approach apoptotic levels. In the (FB 1+ F A) treated N2a cells this effect started at the lower doses of 8~g.mrl and 32~g.mrl, going up to 512 ~g.mrl with the combination of the mycotoxins (FBI+ FA) which may act together to cause apoptosis in the cells after the 24 hour exposure period (Appendix 4). Necrosis was not induced in the N2a cell line at any of the mycotoxin test concentrations according to the results reported for the ApoGlow™ assay (Figure 4.3).
In the N2a cell line, necrosisI and proliferation were not detected at any of the concentrations of FBI or FA evaluated in this study. No distinct trends were observed in the responses of neuroblastoma N2a and the Jurkat suspension cell line to exposure to these mycotoxins.
CHAPTERS
INTRODUCTION
On a flow cytometer this manifests as a reduction in the FSC signal and an initial increase in the SSC signal, partly due to a change in the refractive index of the CelIs cytoplasm. The opening of megachannels results in the collapse of the asymmetric distribution of protons on both sides of the inner mitochondrial membrane, known as the mitochondrial transmembrane potential (TMP) transition (Martinou, 1999) and will be measured using Rhodamine 123 (Rh 123) and the dual emission potential sensitive probe tetrachloro-1,1',3,3'-tetraethylbenzimidazole-carbocyanine iodide (JC-1). The transition of the mitochondrial transmembrane potential is an early and universal event in apoptosis.
In the present investigation, the effects of FBI, MON, FA, ZEA, T2, DON, Sa and So on the N2a cell line were assessed after a 48-hour exposure period, qualitatively using fluorescence. The advantage of this technique is that by using short wavelength light as the illuminating beam, an increase in detail is observed, and the long wavelength light emitted strengthens the label.
MA TERIALS AND METHODS
- Materials
- Precautions
- Preparation of the mycotoxin and sphingoid base stock solutions
- Preparation of N2a cells for fluorescence microscopy
- Acridine orange and ethidium bromide staining of N2a cells for assessment by fluorescence microscopy
- Flow cytometric analysis of cells stained with rhodamine 123/
In the flow cytometer, the value of the photomultiplier tube (PMT) that detected the signal in fluorescence channel 1 (FL-1) was set to approx. 390V, and fluorescence channel 2 (FL-2) PMT for 320V. Propidium iodide uptake was a reflection of the extent of membrane damage and permeability of these unfixed N2a cells. The flow cytometric analyzes of the staining of the treated N2a cells stained with Rh 123 and PI are also summarized in Table 5.
Flow cytometric analyzes of the mycotoxin- and sphingoid base-treated N2a cells after staining with JC-1, rhodamine 123, and propidium iodide. Staining with RhI23/PI indicated that at this concentration the N2a cells were either late apoptotic or necrotic, as indicated by PI uptake.
INTRODUCTION
An immunochemical study on the effects of the fumonisin Bh zearalenone and T-2 toxin on the N2a neuroblastoma cell line. When using light and EM in combination with immunochemical techniques, an investigation in cell culture allows one to construct a possible sequence of events to account for the effects of a mycotoxin in a biological system. This in vitro investigation aimed to monitor the effects of FBI, ZEA and T2 on the structure and ultrastructure of cultured N2a cells and to evaluate the sites of action of these environmental xenobiotics.
Immunohistochemical techniques were performed on N2a neuroblastoma cells treated with LEA, FB I, and T2, as these were the only mycotoxin antibodies available at the time. Immunoelectron microscopy analyzes were also performed on FBI-treated N2a cells, as this was the mycotoxin of primary importance in this study.
MATERIALS AND METHODS .1 Materials
