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 free radicals has been considered to play a vital role in many degenerative diseases and measurement of 8-hydroxy-2'-deoxyguanosine (Oh8dG) in tissue DNA has been used as a benchmark for oxidative DNA damage. We report here an ultrasensitive 32P-postlabeling method to detect and quantitate Oh8dG in DNA and have determined basal levels of Oh8dG in rat tissues. The method is comprised of DNA digestion to 3'-monophosphates, 5'-32P-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 32Pi) or remained at the origin (ATP and other contaminants), while Oh8dG migrated in the middle of the chromatogram. Calf thymus DNA incubated with ascorbic acid and H202 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 Oh8dG/10(6) nucleotides). A chromatographically identical spot was also found in dGp incubated with ascorbic acid and H202, but not with dAp, dCp or dTp. When applied to rat tissue DNA, the assay readily permitted detection of Oh8dG in the liver, lung, kidney, heart, brain, spleen, intestines and mammary epithelial cells of 3-month old female Sprague-Dawley rats. The tissue Oh8dG 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 Oh8dG per 10(5-6) nucleotides) to detect Oh8dG using nanogram quantity of DNA digest, the 32P-postlabeling method is likely to be valuable in quantitating Oh8dG in human tissue biopsies.
Devanaboyina
U
U
Graduate Center for Toxicology, University of Kentucky Medical Center, Lexington 40536-0305, USA.
Gupta
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RC
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002855:Chromatography, Thin Layer; D004247:DNA / Q000032:analysis*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D005260:Female; D006801:Humans; D010761:Phosphorus Radioisotopes; D051381:Rats; D017207:Rats, Sprague-Dawley
D010761:Phosphorus Radioisotopes; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine