[32P]ATP mediates formation of 8-hydroxy-2′-deoxyguanosine from 2′-deoxyguanosine, a possible problem in the 32P-postlabeling assay

8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a biomarker for oxidative stress on DNA, a common lesion in mammalian cells, A correlation between increased levels of 8-OH-dG and diseases like diabetes, infections and cystic fibrosis has been found in humans, 8-OH-dG levels have been shown to be decreased by antioxidants, an indication of the importance of dietary habits, 8-OH-dG is used as a biomarker for oxidative stress in vivo as well as in vitro and is suggested to be a mutagenic DNA lesion, Different methods are used for the analyses of 8-OH-dG, i.e. GC-MS, HPLC-EC and P-32-postlabeling. The most commonly used method is HPLC-EC. In the analysis of 8-OH-dG, the work-up procedure for DNA, as well as the preparation for analysis, are of critical importance as there is a risk for auto-oxidation of deoxyguanosine (dG), which would result in false high background levels and low sensitivity in analysis, P-32-Postlabeling has recently been applied to the analysis of 8-OH-dG and has shown to be a very sensitive method for the detection of DNA adducts, It is shown here that after extrapolation to normal P-32-postlabeling conditions, [P-32]ATP generated 8-OH-dG to levels of 25 8-OH-dG/10(5) dG. [P-32]ATP mediated the formation of 8-OH-dG from dG in a dose-dependent manner at all dose levels (0.13-12 mu Ci), The reaction occurred immediately and increased with time in a linear dose-response fashion, At high doses (6.0 and 12 mu Ci) the dose-response declined after 24 h, which indicates a possible decomposition or rearrangement of 8-OH-dG, A repeated experiment with 5 mu Ci [P-32]ATP during 2 h resulted in a linear formation of 8-OH-dG and a level of 19 8-OH-dG/10(5) dG, The results indicated that awareness of the autooxidation generated by P-32[ATP] in the postlabeling assay is of utmost importance and that dG must be separated before P-32-postlabeling of 8-OH-dG.