An investigation of genotoxicity and antioxidant treatment of DNA repair in human cells

Understanding the mechanisms defying endogenous and exogenous processes that threaten DNA integrity remains critical to the elucidation of cancer, disease processes and ageing. The research presented in this thesis examines the formation and the removal of two distinctly different DNA lesions in human cell culture systems. Significant differences in the formation of cyclobutane pyrimidine dimers following monochromatic ultraviolet irradiations at 305nm and 315nm in cultured keratinocytes and DNA were demonstrated immunochemically, offering a non-invasive model to gauge wavelength-specific damage and their potencies. The formation of the oxidative lesion, 7, 8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), was also demonstrated immunochemically and by HPLC with electrochemical detection. In addition the measurement of intracellular reactive oxygen species generation was monitored by a fluorimetric assay. All three approaches clearly illustrated that sub-lethal doses of hydrogen peroxide caused a dose-dependent increase in the genotoxic potential in cultured human cells. Further to the confirmation of this prooxidant condition, the generation of a potentially antioxidant environment was achieved through the uptake and/or regeneration of ascorbic acid, confirmed by capillary electrophoresis measurements of intracellular extracts. The modulation of two base excision repair enzymes, human 8-oxoguanine glycosylase (hOGG1) and human apurinic/apyrimidinic endonuclease (hAPE), important in the removal of 8-oxodG, was investigated in response to prooxidant and antioxidant treatments. No significant change in mRNA and protein expression of hOGG1 and hAPE was observed under either experimental condition. In conclusion however, there may be modulation of these repair enzymes at the post-translational level possibly in response to the changes in cellular redox status and the formation of 8-oxodG. Therefore measuring repair activities alongside genotoxicity may provide useful indications of perturbations that can lead to potential mutational events and cellular dysfunction. Appreciating the interplay of multiple DNA repair pathways presents an interesting challenge for future studies.