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 for oxidative DNA damage, Reported basal levels determined by P-32-postlabeling (PPL) method mere 10-fold or more higher than those obtained with HPLC/electrochemical detection (ECD), This discrepancy was investigated, In commercial calf thymus DNA, levels of 4 +/- 1 and 64 +/- 14 8-oxo-dG per 10(6) 2'-deoxynucleosides (dN) were measured by the standard HPLC/ECD and PPL methods, respectively, DNA digestion by micrococcal nuclease/spleen phosphodiesterase and nuclease P1 (as used in the standard PPL method), followed by ECD analysis resulted in a level of 8 +/- 3, In calf thymus DNA spiked with chemically synthesized 8-oxo-dGp to give an increment of 9 8-oxo-dG/10(6) dN, the added standard produced a significant increase with HPLC/ECD but not PPL, After spiking the DNA with 90 8-oxo-dG/10(6) dN, the added 8-oxo-dGp was detectable also with PPL, with a labeling efficiency of 65%. In order to investigate the role of ionizing radiation from P-32 for the higher 8-oxo-dG levels in PPL, incubation times and amounts of radioactivity in the phosphorylation reaction with commercial dGp were increased, and external irradiation of commercial dG with P-32 was investigated, All modifications resulted in higher values of 8-oxo-dG measured, but the effect was not large enough to fully explain the discrepancy between PPL and HPLC/ECD, Using [gamma-P-33]ATP instead of [gamma-P-32]ATP or adding [P-33]phosphate to a P-32-PPL assay resulted in even higher levels of 8-oxo-dG measured, The increase in 8-oxo-dG levels during the PPL workup is attributed to the presence and oxidation of unmodified dGp in the reaction mixture. For a determination of true basal levels, the PPL method mill have to be modified, including the removal of dGp prior to the phosphorylation reaction.