An analysis of 8-oxodeoxyguanosine as a marker of oxidative stress: Involvement in UV-mediated DNA damage in cells.
thesisposted on 19.11.2015, 08:52 by Monica T. V. Finnegan
Oxidative DNA damage is thought to play a role in the aetiology of ageing and a number of diseases including cancer, chronic inflammation, ischemia, degenerative arterial and autoimmune diseases. 8-oxodeoxyguanosine (8-oxodG), an oxidative DNA adduct, has gained much popularity as a biomarker of damage to DNA. In this thesis the reliability of 8-oxodG as a marker for oxidative stress and its involvement in ultraviolet (UV)-mediated DNA damage in cells was investigated. Because of our concern and those expressed in the literature over the possible induction of artefactual 8-oxodG during phenol extraction procedures, a comparative study on DNA extraction methods was undertaken. It was found that phenol isolation of DNA yielded higher levels of 8-oxodG when directly compared to pronase E isolation, irrespective of the model of oxidative stress used to treat DNA or cells. Furthermore results from peripheral blood mononuclear cells from patients under oxidative stress (systemic lupus erythematosus and rheumatoid arthritis) showed a high degree of variability leading to inconclusive results. This lead to investigation of alternative methods of analysis of the 8-oxodG lesion. 8-oxoguanine is measured conventionally as the deoxynucleoside by high performance liquid chromatography with electrochemical detection (HPLC-ECD) or as the free base by gas chromatography combined with mass spectrometry. A 'hybrid' analysis of the 8-oxoG base by HPLC was established. The new procedure combined formic acid hydrolysis of DNA with guanase treatment of the resultant base mixture. Guanase was found to specifically degrade guanine thus allowing reversed-phase HPLC quantitation of 8-oxoG. This procedure avoided problems with enzymes used to degrade DNA to deoxynucleosides which may lack specificity toward oxidatively damaged DNA. A reliable method to measure oxidative DNA damage, including 8-oxodG, at ultra-low levels, without artefactual generation of 8-oxoG which is suggested to be unavoidable when extracting and/or derivatising DNA, is clearly of great importance. Therefore, as a model, a polyclonal antibody to UV-induced DNA damage was developed and utilised in flow cytometric and immunocytochemical techniques to detect UV-mediated DNA damage in cells. Ultraviolet radiation has been reported to produce a number of potentially mutagenic photoproducts and has consequently been implicated in skin tumourigenesis. The polyclonal antibody utilised was successful at detecting UVB-induced DNA damage and UVA-induced single strand breaks. The levels of DNA damage and p53 expression following UVA irradiation of keratinocytes were found to be cell-cycle and dose dependent. UVA was found to cause a dose-dependent increase in 8-oxoG formation also as determined by HPLC. Therefore it is likely that oxidative DNA damage has a role in UVA-induced DNA damage in cells. It is concluded that the use of specific antibodies may represent an accurate and reliable measure of oxidative lesions directly m cellular DNA as an alternative to procedures that require DNA extraction.