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Masters Degrees (Medical Microbiology)

Permanent URI for this collectionhttps://hdl.handle.net/10413/9620

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Browsing Masters Degrees (Medical Microbiology) by Author "Chuturgoon, Anil Amichund."

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    Fumonisin B1 increases the NAD-dependant deacetylase SIRT1 to disrupt cholesterol flux in galactose supplemented HepG2 cells.
    (2020) Adimulam, Theolan.; Chuturgoon, Anil Amichund.
    The Fusarium species are major fungal contaminants of maize, that produce an extremely toxic mycotoxin called Fumonisin B1 (FB1). Fumonisin B1 is a known causative agent of animal-related mycotoxicosis and is implicated in cancer initiation in humans (epidemiological) and animals (experimental). The mechanisms of tumorigenesis have implicated oxidative stress, incomplete apoptosis, and chromatin instability as major contributing factors. While the canonical mechanism of FB1 induced ceramide dysregulation and oxidative stress are the established drivers of cancer initiation, accumulating evidence indicates that changes in cholesterol metabolism may further enhance the tumorigenic potential of FB1. The cholesterolemic effect of FB1 has been shown in experimental models yet molecular evidence elucidating the disturbed cholesterol flux remains vague. This study investigates the molecular mechanism that regulate the effect of FB1 on cholesterol homeostasis, by investigating the role of cholesterol flux proteins and their regulation at the transcriptional and post translational level, in galactose supplemented HepG2 liver cells. Cancer cells like the liver derived HepG2 cell line, differ from normal tissues with respect to their excessive use of aerobic glycolysis, to satisfy the high energy demand. The proven method of circumventing the Crabtree effect exhibited by cancer cells, is to replace glucose with galactose in the culture media, forcing cells to activate the mitochondria and rely on oxidative phosphorylation rather than glycolysis for ATP. SIRT1 is a NAD-dependent deacetylase, that responds to changes in nutrient availability, to induce regulatory effects on cellular metabolism. The toxicity of FB1 was determined (6hr incubation; IC50 = 25μM) on metabolic output, cholesterol regulatory transcription factors and key cholesterol flux proteins using the spectrophotometric MTT assay, ATP luciferase assay, qPCR (SIRT1, SREBP-1C, LXR, LDLR, PCSK9 and ABCA1) and western blots (SIRT1, SREBP-1C, LXR, LDLR, PCSK9 and ABCA1). FB1 induced differential HepG2 cell viability and metabolic output. There was no effect on cell viability for the glucose-supplemented media, while a decrease in cell viability was observed for the galactose-supplement media. Concurrent with the decrease in cell viability of the galactose-supplementation, a decrease in HepG2 ATP output was observed (p=0.0135). FB1 did not compromise membrane integrity, despite the decrease in cell viability. The expression of the SIRT1 gene (p=0.0004) and protein (p=0.0005) was significantly increased by FB1. The expression of the SREBP-1c gene (p=0.0050) was increased while protein expression (p=0.0063) was decreased. The role of LXR and PCSK9 in the regulation of LDLR was further highlighted. FB1 increased the expressions of LXR (p=0.0003) and LDLR (p=0.0004 and p=0.0049 respectively) genes and proteins but decreased the gene (p=0.0017) and protein (p=0.0018) expression of PCSK9. The data provides evidence that SIRT1 reduces the expression of PCSK9 and deacetylates LXR to prevent degradation of LDLR, resulting in dysregulated cholesterol flux in liver cells. The disruption of cholesterol homeostasis by FB1 is beginning to shift away from established ceramide synthase inhibition, changing the perspective to shed light on the diseases caused by dysregulated cholesterol metabolism such as cancer initiation and promotion.
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    Methyl picolinic acid plays a role in epigenetic modifications in Human HepG2 liver cells.
    (2019) Foolchand, Ashmika.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
    Picolinic Acid (PA) is an endogenous catabolite synthesized from L-Tryptophan via the kynurenine pathway. PA has been reported to possess immunological, neuroprotective, and anti-proliferative properties; however, its most widely researched function is its efficient chelating properties. Despite these findings, the physiological function of PA is yet to be discovered. Many PA derivatives exist, with its methyl derivative being of interest in this study. Studies have shown that Picolinic Acid derivatives such as Fusaric Acid are involved in epigenetic regulation by inducing global DNA hypomethylation in HepG2 liver cells. Therefore, the aim of the study was to investigate the epigenetic properties of Picolinic Acid (parent molecule of Fusaric Acid) and Methyl Picolinic Acid in human HepG2 Liver cells. DNA methylation and Histone modifications are epigenetic phenomena that modify accessibility to DNA and chromatin structure, which results in regulation of gene expressions. While DNA methylation results in long-term repression, Histone methylation leads to formation of heterochromatin. The methyl-CpG binding domain 2 (MBD2) binds to methylated CpG dinucleotides and negatively regulates DNA methylation. MBD2 also associates with histone lysine methyltransferases, such as SUV39H1, which is involved in pericentric heterochromatin silencing and is responsible for methylating Histone 3 on lysine 9 (H3K9). The cytotoxicity of Picolinic Acid and Methyl Picolinic Acid in HepG2 liver cells was assessed by the WST-1 assay. The DNA methylation ELISA kit was used to quantify 5-methylcytosine in HepG2 cells. Gene expression for the DNA demethylase MBD2 was determined by qPCR. Protein expression for MBD2, trimethylated H3K9 (H3K9me3) and its methyltransferase, SUV39H1, was assessed by Western Blotting. The IC50 values for Picolinic Acid (7.5 mM) and Methyl Picolinic Acid (13 mM) were obtained from the WST-1 assay. DNA methylation was significantly decreased in Methyl Picolinic Acid, Picolinic Acid and 5-aza-2-DC treated cells (p<0.0001). In Methyl Picolinic Acid and Picolinic Acid treated cells, MBD2 gene expression was downregulated (p<0.0001) followed by an increase in its protein expression (p<0.0001). MPA increased protein expression of SUV39H1 (p<0.0001) leading to an increase in H3K9me3 (p<0.0001), while Picolinic Acid induced a decrease in SUV39H1 and H3K9me3 protein expression (p<0.0001). Methyl Picolinic Acid induced DNA hypomethylation as a result of increased MBD2 protein expression, despite the decrease in MBD2 gene expression. The upregulation of MBD2 protein expression promoted the activity of SUV39H1 which subsequently enhanced expression of H3K9me3. Since DNA hypomethylation can restore expression of aberrantly expressed genes, this study suggests a possible role for MPA in ameliorating carcinogenesis.