Devanaboyina U, Gupta R C
Graduate Center for Toxicology, University of Kentucky Medical Center, Lexington 40536-0305, USA.
Carcinogenesis. 1996 May;17(5):917-24. doi: 10.1093/carcin/17.5.917.
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.
包括自由基在内的活性氧物种所造成的氧化损伤，被认为在许多退行性疾病中起着至关重要的作用，而组织DNA中8-羟基-2'-脱氧鸟苷（Oh8dG）的测量已被用作氧化DNA损伤的基准。我们在此报告一种超灵敏的32P后标记法，用于检测和定量DNA中的Oh8dG，并已测定大鼠组织中Oh8dG的基础水平。该方法包括将DNA消化为3'-单磷酸、5'-32P标记、转化为5'-单磷酸以及通过双向PEI-纤维素TLC分离（D1 = 1.5 M甲酸；D2 = 0.6 M甲酸铵，pH 6.0）。在这些条件下，所有放射性污染物要么从色谱图中去除（正常核苷酸和32Pi），要么留在原点（ATP和其他污染物），而Oh8dG在色谱图中间迁移。在所用的色谱条件下，用抗坏血酸和H2O2孵育的小牛胸腺DNA主要产生一个斑点；在未处理的DNA中也检测到一个色谱上相同的斑点，但水平要低得多（125 +/- 40 Oh8dG/10(6)核苷酸）。在用抗坏血酸和H2O2孵育的dGp中也发现了一个色谱上相同的斑点，但在dAp、dCp或dTp中未发现。当应用于大鼠组织DNA时，该测定法能够轻松检测3个月大的雌性Sprague-Dawley大鼠的肝脏、肺、肾、心脏、大脑、脾脏、肠道和乳腺上皮细胞中的Oh8dG。发现组织中Oh8dG水平在每10(6)核苷酸87 +/- 29至133 +/- 49范围内，肝脏和心脏中的水平最高，肾脏和大脑中的水平最低。这些值与气相色谱/质谱法测得的值相近，但比高效液相色谱-电化学检测法报告的值高10 - 50倍。由于使用纳克量的DNA消化物检测Oh8dG具有高灵敏度（每10(5 - 6)核苷酸<1 Oh8dG），32P后标记法在定量人体组织活检中的Oh8dG方面可能很有价值。
