Free radical induced oxidative DNA damage

Oxidative DNA damage has been implicated in processes such as carcinogenesis, mutagenesis, ageing and cell death. Reactive oxygen species (ROS) such as superoxide (O2), hydrogen peroxide (H2O 2) and hydroxyl radical (OH*) are produced in mammalian cells as a result of aerobic metabolism. However excess generation of these species by endogenous or exogenous sources can result in damage to DNA, producing a large number of sugar and base lesions. In order to understand the biological consequences of such free radical induced damage it is essential to characterise and quantitate this damage. This study describes the establishment of sensitive and specific techniques to chemically characterise and quantitate three markers of oxidative DNA damage, namely: cis-thymine glycol (Tg), 5-hydroxymethyluracil (5-OHMeU) and 8-hydroxyguanine (8-OHG). Techniques using gas chromatography/mass spectrometry (GC/MS) were established for Tg and 5-OHMeU, following their derivatisation with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA). Standards of Tg and 5-OHMeU were synthesised, and stable isotopically labelled analogues were prepared as internal standards. Analysis of the DNA was carried out at the base level and therefore required acidic hydrolysis of the DNA in order to release the modified and intact bases. For the quantitation of 8-OHG a novel procedure using high performance liquid chromatography (HPLC) - electrochemical detection (ECD) with guanase incubation of DNA hydrolysates was established. The established assays were used to quantitate DNA lesions in vitro and in vivo. In vitro dose response curves were established for the three markers upon gamma-irradiation of DNA. In vivo results of an animal inhalation study indicated there was not a significant increase in oxidative damage upon exposure to crocidolite. An antioxidant supplementation study in humans placental DNA also did not show a significant reduction in levels of the three markers upon supplementation. Comparable background levels of Tg and 5-OHMeU were observed in human and calf thymus DNA, while 8-OHG levels were found to be significantly higher.