Analysis of 8-oxo-7, 8-dihydroGuanine formation and oxidation mediated by Fenton reaction induced DNA oxidative stress
White, Blánaid (2005) Analysis of 8-oxo-7, 8-dihydroGuanine formation and oxidation mediated by Fenton reaction induced DNA oxidative stress. PhD thesis, Dublin City University.
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DNA undergoes an estimated 10,000 oxidative hits per day. Oxidative DNA damage caused by reactive oxygen species (ROS) can result in multiple base modifications, which have been implicated in mutagenesis, disease and aging. The primary product of G oxidation is 8-oxo-7,8-dihydroguanine (8- oxoG), which is considered by many as a biomarker for oxidative DNA damage. 8-oxoG has an oxidation potential about 0.5 V lower than G, and so can be accurately quantified using electrochemical (EC) detection. EC detection coupled to HPLC resulted in a sensitive and accurate mode of detection for 8- oxoG without requiring any preconcentration or removal of undamaged G, which was simultaneously detected by UV detection.
The aim of this investigation was to measure the rate of 8-oxoG formation in DNA subjected to continuous oxidative attack. The hydroxyl radical, the most aggressive ROS, was generated via the iron-mediated Fenton reaction, and used to generate 8-oxoG in both free G and double stranded DNA. HPLC-UV-EC was utilised for the quantitative analysis of both G and 8-oxoG concentrations with respect to incubation time with the hydroxyl radical. The concentration of 8-oxoG was observed to oscillate with respect to time. After approximately 18 min incubation with Fenton reagents, a maximum 8-oxoG concentration of 0.68 uM was detected in DNA. Thereafter, however, there was an overall decrease in 8-oxoG concentration over time. 8-oxoG concentration was not found to be proportional to the level of oxidative damage which occurred. The concentration of G was also observed to decrease with increasing DNA oxidation, so that as oxidation continued, both G and 8-oxoG were oxidised.
Copper, another important biological metal ion, binds tightly to DNA, inducing significant oxidative DNA damage. It was also investigated as a metal catalyst for the Fenton reaction-mediated DNA oxidation. Again, 8-oxoG concentration was found to oscillate with increasing oxidation of DNA. There were significant differences between the iron- and copper-mediated oxidation of DNA. Oscillation periods for copper-mediated oxidation were shorter, with greater concentration amplitudes. A maximum 8-oxoG concentration of 4.2 fiM was detected after 35 min oxidation. Overall, however, the trend was again towards oxidation of both G and 8-oxoG with increasing oxidation of DNA.
8-oxoG is a hotspot for further oxidation. It was observed in both studies outlined above to be further oxidised during DNA oxidation by the Fenton reaction. The final products of 8-oxoG oxidation were determined using HPLCMS/ MS. Oxidised guanidinohydantoin (oxGh) was identified as the primary product of 8-oxoG oxidation, when both iron and copper catalysts were used. A mechanism for the formation of oxGh by hydroxyl radical attack of 8-oxoG was also proposed.
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