Comparative analysis of 8-oxo-2' -deoxyguanosine in DNA by 32P- and 33P- postlabeling and electrochemical detection

8-Oxo-2'-deoxyguanosine (8-oxo-dG) is emerging as a useful marker foroxidative DNA damage. Reported basal levels determined by 32P- postlabeling(PPL) method were 10-fold or more higher than those obtained withHPLC/electrochemical detection (ECD). This discrepancy was investigated. Incommercial calf thymus DNA, levels of 4 +/- 1 and 64 +/- 14 8-oxo-dG per10(6) 2'-deoxynucleosides (dN) were measured by the standard HPLC/ECD andPPL methods, respectively. DNA digestion by micrococcal nuclease/spleenphosphodiesterase and nuclease P1 (as used in the standard PPL method),followed by ECD analysis resulted in a level of 8 +/- 3. In calf thymus DNAspiked with chemically synthesized 8-oxo-dGp to give an increment of 98-oxo-dG/10(6) dN, the added standard produced a significant increase withHPLC/ECD but not PPL. After spiking the DNA with 90 8-oxo-dG/10(6) dN, theadded 8-oxo-dGp was detectable also with PPL, with a labeling efficiency of65%. In order to investigate the role of ionizing radiation from 32P forthe higher 8-oxo-dG levels in PPL, incubation times and amounts ofradioactivity in the phosphorylation reaction with commercial dGp wereincreased, and external irradiation of commercial dG with 32P wasinvestigated. All modifications resulted in higher values of 8-oxo-dGmeasured, but the effect was not large enough to fully explain thediscrepancy between PPL and HPLC/ECD. Using [gamma-33P]ATP instead of[gamma-32P]ATP or adding [33P]phosphate to a 32P-PPL assay resulted in evenhigher levels of 8-oxo-dG measured. The increase in 8-oxo-dG levels duringthe PPL workup is attributed to the presence and oxidation of unmodifieddGp in the reaction mixture. For a determination of true basal levels, thePPL method will have to be modified, including the removal of dGp prior tothe phosphorylation reaction.