Wilson V L, Basu A K, Essigmann J M, Smith R A, Harris C C
Division of Cancer Etiology, National Cancer Insitute, NIH, Bethesda, Maryland 20892.
Cancer Res. 1988 Apr 15;48(8):2156-61.
The 32P-postlabeling procedure, developed originally by Randerath and coworkers, has been modified for the detection and analytical quantitation of O6-alkyl-2'-deoxyguanosine residues in DNA. Chromatographic techniques were developed to resolve individually the normal deoxyribonucleotide-3'-monophosphates and the O6-alkyldeoxyguanosine-3'-monophosphates by high-pressure liquid chromatography. Selective deoxyribonucleotide-3'-monophosphates (e.g., O6-alkyldeoxyguanosine-3'-monophosphates) were then converted to labeled deoxyribonucleotide-[5'-32P]monophosphates by 32P-postlabeling and nuclease P1 treatment and separated by two-dimensional thin layer chromatography. The O6-methyl- and O6-ethyl-2'-deoxyguanosine-3'-monophosphate nucleotides, and the respective 5'-monophosphates, were chemically synthesized for standardization of these quantitative procedures. The quantitation of O6-methl- and O6-ethyl-2'-deoxyguanosine was observed to be analytically accurate between one O6-alkyl-2'-deoxyguanosine residue per 10(4) and 10(7) 2'-deoxyguanosines. The limit of detection was less than one O6-alkyl-2'-deoxyguanosine in 10(7) 2'-deoxyguanosine residues in a sample size of 100 micrograms of DNA, i.e., approximately 10 pg of adduct. The quantitation of O6-methyl-2'-deoxyguanosine in the liver DNAs of rats treated with [14C-Me]N-nitrosodimethylamine compared well with values obtained by both 14C and high-pressure liquid chromatography coupled with fluorescence detection. Thus, these 32P-postlabeling and nucleotide chromatographic procedures should be useful in monitoring human exposure to methylating and ethylating carcinogens.
最初由兰德拉斯及其同事开发的³²P后标记程序已被修改，用于检测和分析定量DNA中的O⁶-烷基-2'-脱氧鸟苷残基。已开发出色谱技术，通过高压液相色谱分别分离正常的脱氧核糖核苷酸-3'-单磷酸和O⁶-烷基脱氧鸟苷-3'-单磷酸。然后，通过³²P后标记和核酸酶P1处理，将选择性的脱氧核糖核苷酸-3'-单磷酸（例如，O⁶-烷基脱氧鸟苷-3'-单磷酸）转化为标记的脱氧核糖核苷酸-[5'-³²P]单磷酸，并通过二维薄层色谱进行分离。化学合成了O⁶-甲基-和O⁶-乙基-2'-脱氧鸟苷-3'-单磷酸核苷酸以及相应的5'-单磷酸，用于这些定量程序的标准化。观察到O⁶-甲基-和O⁶-乙基-2'-脱氧鸟苷的定量在每10⁴至10⁷个2'-脱氧鸟苷中有一个O⁶-烷基-2'-脱氧鸟苷残基时分析准确。在100微克DNA的样本量中，检测限小于10⁷个2'-脱氧鸟苷残基中的一个O⁶-烷基-2'-脱氧鸟苷，即约10皮克加合物。用[¹⁴C-甲基]N-亚硝基二甲胺处理的大鼠肝脏DNA中O⁶-甲基-2'-脱氧鸟苷的定量与通过¹⁴C和高压液相色谱结合荧光检测获得的值相比良好。因此，这些³²P后标记和核苷酸色谱程序应有助于监测人类对甲基化和乙基化致癌物的暴露。
