Quantitative immunoanalysis of promutagenic 8-hydroxy-2'-deoxyguanosine in oxidized DNA

The modified DNA base 8-hydroxyguanine has been implicated in spontaneous mutagenesis, carcinogenesis and cellular aging. Polyclonal antibodies specific for the 8-hydroxy-2'-deoxyguanosine moiety in oxidized DNA were used for sensitive detection and quantitation of this biomarker of oxidative damage to cellular DNA. The analysis was performed with immunoslot blot assay (ISB) of oxidized DNA modified in vitro with methylene blue plus light and upon H2O2 treatment of cultured human cells. The level of 8-OHdG in DNA exposed to 90 and 120 min light in the presence of 100 microM methylene blue showed 15.96 +/- 2.4 and 22.65 +/- 3.65 pmol/micrograms DNA compared to 0.107 +/- 0.024 pmol/micrograms in commercial calf thymus DNA control. Inherent damage, due to cellular endogenous oxidation of DNA, increased significantly upon inhibition of catalase activity in human cells with 10 mM azide. The damage increased further on exposure of azide-treated cells to H2O2. The amounts of 8-OHdG following treatment of cells with 10 and 100 microM H2O2 were determined to be 205 +/- 42 and 333 +/- 17.5 pmol/micrograms DNA respectively. Very low but quantifiable antibody binding was seen with the 'control unoxidized' human nuclear DNA. This DNA, obtained under controlled conditions to restrict the induction of 8-OHdG during isolation, provides a background level of 0.022 +/- 0.005 pmol 8-OHdG/micrograms DNA. The quantitative assessment of 8-OHdG by ISB assay, with fmol sensitivity and direct analysis using unhydrolyzed DNA, should prove a highly valuable alternative to currently used approaches to detecting 8-OHdG in enzymatic DNA hydrolysates.