An investigation of DNA damage induced by Oxidative stress in neuronal cells

This project aimed to investigate the hypothesis that oxidative DNA damage has a role in neuronal dysfunction. Reactive oxygen species (ROS) are known to damage important cellular macromolecules, including DNA. They have been implicated in many pathological conditions, including common neurodegenerative disorders, but the mechanisms of cellular dysfunction and death involved remain unclear. Oxidative DNA damage was therefore studied in an in vitro model system and pathological tissue. A novel, sensitive assay based on the fortuitous finding that avidin binds directly to the DNA lesion 8-oxodeoxyguanosine (8-oxodG) was used in these investigations. Pre-lethal DNA damage was assessed in terminally differentiated human neuroblastoma IMR32 cultures after exposure to various forms of oxidative stress (hydrogen peroxide, UVA irradiation and paraquat). Avidin binding was significantly increased in these cells, indicating the presence of oxidative DNA damage; repairable and non-repairable damage were both detected. Binding was decreased by pre-incubation with the antioxidant -tocopherol or iron chelator desferrioxamine, demonstrating the involvement of ROS in the mechanism of DNA damage induced during oxidative stress. Avidin binding was also assessed visually in cells exposed to hydrogen peroxide via fluorescent microscopy, and was shown to be located primarily within the nucleus. Once again, levels of damage were decreased after incubation with -tocopherol. Finally preliminary studies were carried out to assess levels of oxidatively-damaged DNA in spinal cord sections from patients suffering from MND compared to age-matched control subjects. Binding was once again detected directly in situ using immunofluorescence microscopy. The novel methodology used allows the demonstration of oxidative DNA damage directly in situ. The direct demonstration of damage may help elucidate the mechanisms involved in cellular dysfunction produced by oxidative stress, and so might provide a greater understanding of the causes of the most common neurodegenerative diseases.