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

Oxidative damage from reactive oxygen species including freeradicals has been considered to play a vital role in many degenerativediseases and measurement of 8-hydroxy-2'-deoxyguanosine (Oh⁸dG)in tissue DNA has been used as a benchmark for oxidative DNAdamage. We report here an ultrasensitive ³²P-postlabeling methodto detect and quantitate Oh⁸dG in DNA and have determined basallevels of Oh⁸dG in rat tissues. The method is comprised of DNAdigestion to 3'-monophosphates, 5'-³²P-labeling, conversionto 5'-monophosphates and separation by a 2-directional PEI-cellulose TLC (Dl = 1.5 M formic acid; and D2 = 0.6 M ammoniumformate, pH 6.0). Under these conditions, all radioactive contaminantswere either removed from the chromatogram (normal nucleotidesand ³²Pi) or remained at the origin (ATP and other contaminants),while Oh⁸dG migrated in the middle of the chromatogram. Calfthymus DNA incubated with ascorbic acid and H₂O₂ produced predominantlyone spot under the chromatography conditions used; a chromatographicallyidentical spot was also detected in untreated DNA, but at amuch lower level (125 ± 40 Oh⁸dG/10⁶ nucleotides). Achromatographically identical spot was also found in dGp incubatedwith ascorbic acid and H₂O₂, but not with dAp, dCp or dTp. Whenapplied to rat tissue DNA, the assay readily permitted detectionof Oh⁸dG in the liver, lung, kidney, heart, brain, spleen, intestinesand mammary epithelial cells of 3-month old female Sprague-Dawleyrats. The tissue Oh⁸dG levels were found in the range of 87± 29 to 133 ± 49 per 10⁶ nucleotides, with liverand heart being the highest and kidney and brain the lowestThese values are in the vicinity to those found by gas chromatography/massspectrometry but 10–50 times higher than those reportedby HPLC-electrochemical detection. Because of its high sensitivity(<1 Oh⁸dG per 10^5–6 nucleotides) to detect Oh⁸dG usingnanogram quantity of DNA digest, the ³²P-postlabeling methodis likely to be valuable in quantitating Oh⁸dG in human tissuebiopsies.