DEVELOPMENT OF A P-32 POSTLABELING METHOD FOR THE ANALYSIS OF 2'-DEOXYGUANOSINE-3'-MONOPHOSPHATE AND DNA-ADDUCTS OF METHYLGLYOXAL
DEVELOPMENT OF A P-32 POSTLABELING METHOD FOR THE ANALYSIS OF 2'-DEOXYGUANOSINE-3'-MONOPHOSPHATE AND DNA-ADDUCTS OF METHYLGLYOXAL
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DOI：
10.1093/carcin/15.9.1887
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发表时间：
1994-09-01
期刊：
影响因子：
4.7
通讯作者：
HOU, SM
中科院分区：
文献类型：
--
作者：
VACA, CE;FANG, JL;HOU, SM
开发了 P-32 后标记测定法，用于分析 2'-脱氧鸟苷-3'-单磷酸与 1,2-二羰基化合物甲基乙二醛（多种食品（特别是速溶咖啡和冲泡咖啡）中的主要诱变剂）反应产生的加合物。通过测试各种参数（例如 T-4 多核苷酸激酶的磷酸化动力学、底物浓度依赖性标记效率以及磷酸化反应的各种成分的浓度）来优化 P-32 后标记反应。还研究了核酸酶 P1 的 3'-单磷酸去磷酸化活性的敏感性。通过HPLC分离四种异构体反应产物，进行结构表征并鉴定为3-(2'-脱氧-β-D-赤式-五呋喃糖基)-6,7-二氢-6,7-二羟基-6-甲基咪唑并[2,3-b]嘌呤-9(8H)酮。在体外与甲基乙二醛反应的小牛胸腺 DNA 中可以检测到相同的加合物。 DNA加合物在酶消化成单核苷酸之后分离，然后用核酸酶pi消化正常核苷酸。获得的甲基乙二醛-DNA 加合物的总水平为 5.7 +/- 1.7 (n = 15) 加合物/10(6) 个核苷酸。 P-32 后标记方法通过检测新鲜分离并刺激的体外暴露的人淋巴细胞 DNA 中的甲基乙二醛加合物得到进一步验证。在用 1.5 和 3.0 mM 甲基乙二醛处理后，这些样品中检测到的加合物浓度分别为 8.2 +/- 0.9 (n = 3) 加合物/10(7) 个核苷酸和 1.5 +/- 0.1(n = 3) 加合物/10(6) 个核苷酸。
A P-32-postlabelling assay was developed for the analysis of adducts arising from the reaction of 2'-deoxyguanosine-3'-monophosphate with the 1,2-dicarbonyl compound methylglyoxal, a major mutagen in several foodstuffs, in particular, instant and brewed coffee. The P-32-postlabelling reaction was optimized by testing various parameters such as the kinetics of phosphorylation by T-4 polynucleotide kinase, substrate concentration-dependent labelling efficiency and the concentration of the various ingredients of the phosphorylation reaction. The sensitivity to the 3'-monophosphate dephosphorylation activity of nuclease P1 was also studied. Four isomeric reaction products were separated by HPLC, structurally characterized and identified as 3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-6,7-dihydro-6,7-dihydroxy-6-methylimidazo[2,3-b]purine-9(8H)one. The same adducts could be detected from calf thymus DNA that had been reacted in vitro with methylglyoxal. DNA adducts were isolated after enzymatic digestion to mononucleotides followed by nuclease pi digestion of normal nucleotides. The total level of methylglyoxal-DNA adducts obtained was 5.7 +/- 1.7 (n = 15) adducts/10(6) nucleotides. The P-32-postlabelling method was further validated by the detection of adducts of methylglyoxal in DNA from freshly isolated and stimulated human lymphocytes exposed in vitro. The concentrations of the adducts detected in these samples were 8.2 +/- 0.9 (n = 3) adducts/10(7) nucleotides and 1.5 +/- 0.1(n = 3) adducts/10(6) nucleotides after treatment with 1.5 and 3.0 mM methylglyoxal respectively.