Detection Of Oxidative Dna Base Damage

Electron leak from the ETC results in the formation of ROS, which may cause oxidative damage to mtDNA. To date, 24 major products of oxidative damage to DNA bases have been identified (Evans et al., 2004), of which 8-oxodG has received the most attention as marker of oxidative DNA damage because it can readily be detected by various techniques. However, wide discrepancies in the reported basal levels (over two orders of magnitude) of 8-oxodG in DNA recently have prompted the establishment of the European Standards Committee on Oxidative DNA Damage (ESCODD), which consists of 27 member laboratories who will examine critically the different approaches to measuring base oxidation in DNA, in particular 8-oxodG. It is now widely recognized that the guanine in DNA is readily oxidized during sample preparation. Techniques that utilize gas chromatography followed by mass spectro-metry (GC-MS) are particularly susceptible to this artifact if the derivatization step is carried out at high temperature in the presence of oxygen. Therefore, protocols currently in use address this problem by the inclusion of anti-oxidants and metal chelators during sample preparation. Several techniques were evaluated by ESCODD: HPLC with electrochemical detection (HPLC-ECD), GC-MS, and HPLC followed by tandem mass spectrometry-mass spectrometry (HPLC-MS/MS). Laboratories that employ HPLC-ECD were able to detect induced 8-oxodG with similar efficiencies with very similar dose response profiles whereas GC-MS and HPLC-MS/MS, employed in three laboratories, failed to detect a dose response. Median values for 8-oxodG in untreated cells were determined to be 4.01 per 106 guanines. However, approximately a 10-fold difference was observed between the highest and the lowest values of background mtDNA damage (ESCODD 2003). Overall, it can be concluded that because of this high variability in the background damage determined by different labs using the same techniques and starting material, any published quantitative data on the 8-oxodG content in mtDNA should be interpreted with caution.

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Your heart pumps blood throughout your body using a network of tubing called arteries and capillaries which return the blood back to your heart via your veins. Blood pressure is the force of the blood pushing against the walls of your arteries as your heart beats.Learn more...

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