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

The modified DNA base 8-hydroxyguanine has been implicated inspontaneous mutagenesis, carcinogenesis and cellular aging.Polyclonal antibodies specific for the 8-hydroxy-2'-deoxyguanosinemoiety in oxidized DNA were used for sensitive detection andquantitation of this biomarker of oxidative damage to cellularDNA. The analysis was performed with immunoslot blot assay (ISB)of oxidized DNA modified in vitro with methylene blue plus lightand upon H₂O₂ treatment of cultured human cells. The level of8-OHdG in DNA exposed to 90 and 120 min light in the presenceof 100 µM methylene blue showed 15.96 ± 2.4 and22.65 ± 3.65 pmol/µg DNA compared to 0.107 ±0.024 pmol/µg in commercial calf thymus DNA control. Inherentdamage, due to cellular endogenous oxidation of DNA, increasedsignificantly upon inhibition of catalase activity in humancells with 10 mM azide. The damage increased further on exposureof azide-treated cells to H₂O₂. The amounts of 8-OHdG followingtreatment of cells with 10 and 100 µM H₂O₂ were determinedto be 205 ± 42 and 333 ± 17.5 pmol/µg DNArespectively. Very low but quantifiable antibody binding wasseen with the ‘control unoxidized’ human nuclearDNA. This DNA, obtained under controlled conditions to restrictthe induction of 8-OHdG during isolation, provides a backgroundlevel of 0.022 ± 0.005 pmol 8-OHdG/µg DNA. Thequantitative assessment of 8-OHdG by ISB assay, with fmol sensitivityand direct analysis using unhydrolyzed DNA, should prove a highlyvaluable alternative to currently used approaches to detecting8-OHdG in enzymatic DNA hydrolysates.