Utilization of Spin-Trapping Reactions for Effective Detection of DNA Damage

利用自旋捕获反应有效检测 DNA 损伤

• 批准号: 04680207
• 负责人: KUWABARA Mikinori
• 金额: $ 1.6万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04680207/
• 关键词: DNA Damage; DNA Radicals; X Irradiation; Oxygen Radicals; Spin Trapping; Electron Spin Resonance; DNA Strand Breaks; 8-Hydroxydeoxyguanosine; 放射線; スピントラップ反応; 高速液体クロマトグラフィー

Biological oxidative stress is triggered by the reactions of oxygen radicals with DNA.Oxygen radicals first induced DNA radicals. The DNA radicals then react with oxygens to form peroxy radicals which proceed to turn out to be many kinds of DNA damage. Therefore, several methods have been developed as tools to qualitatively and quantitatively measure DNA final products. Since the kinds of DNA radicals are fewer than those of final products in mumber, it seems better to detect and identify DNA damage at the stage of radicals. In the present study the spin-trapping method was applied for this purpose. The spin trapping is a method to trap unstable, short-lived free radicals by spin trapping reagent. The spin-trapped raticals, the spin adducts, are subsequently analyzed by electron spin resonance spectrometry (ESR). i-Methyl-2-nitrosopropane (MNP) was used as a spin trapping reagent. X irradiation was employed to produce oxygen radicals which reacted with DNA.An effort to detect and identify the precursor radicals of both DNA strand breaks and 8-hydroxydeoxyadenosinn (oh^8dG) was made with spcial emphasis on the detection of precursors of chromosomal aberrations and point mutations, respectively. As a result, the formation of radicals at the C4' site of the sugar moiety and at the N7 site of the guanine base moiety were observed as precursor radicals of strand breaks and oh^8dG, respectively. Evidence for the C4' sugar radical as a precursor of strand breaks was further confirmed by experiments using two 10mers of oligonucleotides, oligo(dC)_<10> and oligo(dT)_<10>, as DNA models. The oligomers were ^<32>P-labeled at the 5' terminus and the numbers of oxygen-radical-induced strand breaks in the presence or absence of MNP were measured by bio-image analyzer BAS 2000. It was observed that the trapping of the C4' sugar radical by MNP surely suppressed the numbers of strand breaks in both oligomers.

生物氧化应激是由氧自由基与DNA反应引发的。氧自由基首先诱发DNA自由基。然后DNA自由基与氧反应形成过氧自由基，进而导致多种DNA损伤。因此，已经开发了多种方法作为定性和定量测量 DNA 最终产品的工具。由于DNA自由基的种类比人类最终产物的种类少，因此在自由基阶段检测和识别DNA损伤似乎更好。在本研究中，自旋捕获方法用于此目的。自旋捕获是一种利用自旋捕获试剂捕获不稳定、短命自由基的方法。随后通过电子自旋共振光谱 (ESR) 分析自旋捕获的自由基，即自旋加合物。 i-甲基-2-亚硝基丙烷 (MNP) 用作自旋捕获试剂。采用X射线照射产生与DNA反应的氧自由基。对DNA链断裂和8-羟基脱氧腺苷(oh^8dG)的前体自由基进行了检测和鉴定，特别重点分别检测了染色体畸变和点突变的前体自由基。结果，观察到在糖部分的C4'位点和鸟嘌呤碱基部分的N7位点处自由基的形成分别作为链断裂和oh^8dG的前体自由基。使用两个10聚体寡核苷酸oligo(dC)_<10>和oligo(dT)_<10>作为DNA模型的实验进一步证实了C4'糖基作为链断裂前体的证据。寡聚物在5'末端进行^ 32 P-标记，并通过生物图像分析仪BAS 2000测量在存在或不存在MNP的情况下氧自由基诱导的链断裂的数量。观察到MNP对C4'糖自由基的捕获确实抑制了两种寡聚物中的链断裂的数量。

项目成果

Kuwabara, M., et al.: "Spin-trapping detection of precursors of hydroxyl-radical-induced DNA damage : Identification of precursor radicals of DNA strand breaks in oligo(dC)_<10> and oligo(dT)_<10>" Biochemistry. 32. 10599-10606 (1993)

Kuwabara, M., 等人：“羟基自由基诱导的 DNA 损伤前体的自旋捕获检测：鉴定oligo(dC)_<10> 和oligo(dT)_<10 中 DNA 链断裂的前体自由基

Kuwabara,M.,et al.: "Spin-trapping detection of precursors of hydroxyl-radical-induced DNA damage:Iden-tification of precursor radicals of DNA strand breaks in oligo(dC)_<10> and oligo(dT)_<10>" Biochemistry. 32. 10599-10606 (1993)

Kuwabara,M.,et al.：“羟基自由基诱导的 DNA 损伤前体的自旋捕获检测：寡聚 (dC)_<10> 和寡聚 (dT)_ 中 DNA 链断裂前体自由基的鉴定

Kuwabara,M.,et al.: "Spin-trapping detection of precursors of hydroxyl-radical-induced DNA damage:Identification of precursor radicals of DNA strand breaks in oligo(dc)_<10> and oligo(dT)_<10>" Biochemistry. 32. 10599-606 (1993)

Kuwabara,M.,et al.：“羟基自由基诱导的 DNA 损伤前体的自旋捕获检测：寡核苷酸 (dc)_<10> 和寡核苷酸 (dT)_<10 中 DNA 链断裂前体自由基的识别