Mechanisms of formation and protection against oxidative DNA damage in vitro and in vivo

University dissertation from Stockholm : Stockholm University

Abstract: Oxidative DNA damage has been linked to cancer and aging as well as other diseases. The aim of this thesis was to study the mechanisms for DNA damage generated in cells or in vitro systems by ionizing radiation or transition metals like iron ions. The damage primarily studied, was 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG), formed by the attack of hydroxyl radical on DNA. Due to its incorrect base pairing with deoxyadenosine during cell replication, 8-oxo-dG is potentially mutagenic. This base damage was analysed with the method of high performance liquid chromatography and electrochemical detection. Also other endpoints of DNA damage were measured, such as the formation of DNA strand breaks with the alkali unwinding assay.Oxidation of iron ions was shown to be dependent on chelation to phosphate ions in solution as well as phosphate bound to DNA. Increasing the ratio of phosphate to ferrous iron (Fe2+) was correlated with the increased formation of 8-oxo-dG. Quin2, a metal chelator, was shown to redox cycle normally unreactive ferric iron (Fe3+) to its reduced form, Fe2+ in the presence of hydrogen peroxide, thereby producing hydroxyl radicals and damage such as 8-oxo-dG. Compounds with these properties would thus be considered as potentially mutagenic.In vitro, it was seen that the yield of 8-oxo-dG was severalfold higher upon irradiation of DNA as compared to irradiation of deoxyguanosine in solution, due to properties of the DNA structure itself. Adding scavengers to DNA, highest protection against damage could be seen using thiols with positive net charge, due to the formation of a counterion condensation complex with the negatively charged DNA, thus protecting more efficiantly against oxygen radical damage. The novel finding was that scavengers with reducing properties, like thiols or ascorbate, added to the nucleoside deoxyguanosine in solution, increased the radiation induced formation of 8-oxo-dG severalfold.Further, the protective effect of chromatin conformation was studied. The yield of radiation induced DNA damage increased when removing low molecular weight nuclear components upon permeabilisation of cells, demonstrating a larger role of hydroxyl radicals for the formation of 8-oxo-dG relative to DNA strand breaks. The removal of histones produced the highest levels of DNA damage, in the form of DNA strand breaks and 8-oxo-dG. In histone free DNA, thiol with positive net charge provided the best protection against formation of 8-oxo-dG.In vivo formation of 8-oxo-dG was studied in rats fed diets supplemented with iron and/or alcohol. In response to iron overload there was only a minor, insignificant, increase of 8-oxo-dG in rat liver cells, and a synergistic effect of iron and alcohol on cytotoxicity but with no genotoxicity.The excretion of 8-oxo-dG in urine was followed in response to radiotherapy of patients treated for breast cancer. The levels of 8-oxo-dG in the urine, before and in response to radiotherapy, correlated with the patients radiosensitivity. Thus, patients with higher radiosensitivity were considered to have a lower repair capacity for this damage.