Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013Cells are continuously exposed to reactive oxygen species (ROS) causing oxidative stress. Cells can withstand and counteract ROS using defence mechanisms which range from free radical scavengers, antioxidant enzymes and DNA repair systems. Prolonged exposure of cells to oxidant species leads to the accumulation of genetic as well as epigenetic alterations. Exposure of cells to the non-radical hydrogen peroxide (H2O2) leads to the generation of hydroxyl radicals (˙OH) by Fenton reactions when H2O2 reacts with a metal iron in the vicinity of DNA. These OH are very reactive and attack DNA giving rise to lesions such as single stand breaks and base modifications, which could influence DNA methylation. DNA methylation is the post synthetic addition of methyl groups to the carbon 5 position of cytosine when cytosines are in the CpG dinucleotide context and is involved in gene expression. DNA methylation is considered to be very stable. Aberrant DNA methylation influences cancer related gene expression and genomic stability. The aim of this study was to investigate early changes in the global DNA -and gene specific methylation patterns of cultured mammalian cells when cells were exposed to H2O2. The term early refers to how soon following exposure to H2O2 over a six hour period changes in the DNA methylation pattern can be observed when exposing cells to H2O2 concentrations that causes oxidative DNA damage. Changes in the hOGG1 promoter methylation status and gene expression were evaluated as this gene plays a crucial role in the initiation of the base excision repair pathway for the repair of oxidative DNA damage caused by H2O2 exposure. Results obtained with the
alkaline comet assay showed that H2O2 exposure led to oxidative DNA damage and decreased DNA repair capacity when cells were exposed to H2O2 in fully supplemented medium (DMEM + 10% FBS). A change in the global DNA methylation pattern was evaluated with the cytosine extension assay and an enzyme based methylation sensitive PCR was used to evaluate the change in the promoter methylation status of hOGG1. Changes in the global DNA- and gene (promoter) specific methylation patterns could be observed where; a degree of global DNA hypomethylation and hypermethylation of the hOGG1 promoter could be observed within the six hour period of exposure to a
concentration of H2O2 that was also associated with a high level of oxidative DNA damage. Finally, a decrease in the expression of the hOGG1 gene was also observed following exposure to this concentration of H2O2 within the six hour exposure period. These findings
suggests that oxidative DNA damage influences DNA methylation (both globally and gene specific) and that the expression of the hOGG1 gene is possibly influenced by promoter hypermethylation which is associated with oxidative DNA damage. Results were generated in human osteosarcoma (143B) cells. This cell line was used in order to investigate the effect of oxidative stress on the global DNA methylation pattern as well as the promoter methylation- and expression of the hOGG1 gene in wild type 143B cells (uncompromised complex III). Previous studies reported that in 143B cells in which complex III of the respiratory chain was compromised, by a knockdown system, deviations in the global DNA methylation pattern as well as the promoter methylation and expression of genes involved in DNA repair pathways could be observed.Master
