Sensitive detection of 8-hydroxy-2'-deoxyguanosine in DNA by P-32-postlabeling assay and the basal levels in rat tissues

Oxidative damage from reactive oxygen species including free radicals has been considered to play a vital role in many degenerative diseases and measurement of 8-hydroxy-2'-deoxyguanosine (Oh(8)dG) in tissue DNA has been used as a benchmark for oxidative DNA damage, We report here an ultrasensitive P-32-postlabeling method to detect and quantitate Oh(8)dG in DNA and have determined basal levels of Oh(8)dG in rat tissues, The method is comprised of DNA digestion to 3'-monophosphates, 5'-P-32-labeling, conversion to 5'-monophosphates and separation by a 2-directional PEI- cellulose TLC (D1 = 1.5 M formic acid; and D2 = 0.6 M ammonium formate, pH 6.0), Under these conditions, all radioactive contaminants were either removed from the chromatogram (normal nucleotides and (32)Pi) or remained at the origin (ATP and other contaminants), while Oh(8)dG migrated in the middle of the chromatogram, Calf thymus DNA incubated with ascorbic acid and H2O2 produced predominantly one spot under the chromatography conditions used; a chromatographically identical spot was also detected in untreated DNA, but at a much lower level (125 +/- 40 Oh(8)dG/10(6) nucleotides), A chromatographically identical spot was also found in dGp incubated with ascorbic acid and H2O2, but not with dAp, dCp or dTp, When applied to rat tissue DNA, the assay readily permitted detection of Oh(8)dG in the liver, lung, kidney, heart, brain, spleen, intestines and mammary epithelial cells of 3-month old female Sprague-Dawley rats, The tissue Oh(8)dG levels were found in the range of 87 +/- 29 to 133 +/- 49 per 10(6) nucleotides, with liver and heart being the highest and kidney and brain the lowest. These values are in the vicinity to those found by gas chromatography/mass spectrometry but 10-50 times higher than those reported by HPLC-electrochemical detection, Because of its high sensitivity (<1 Oh(8)dG per 10(5-6) nucleotides) to detect Oh(8)dG using nanogram quantity of DNA digest, the P-32-postlabeling method is likely to be valuable in quantitating Oh(8)dG in human tissue biopsies.