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

生物氧化应激是由氧自由基与DNA反应引发的，氧自由基首先诱导DNA自由基。DNA自由基然后与氧反应形成过氧自由基，过氧自由基继续被证明是多种DNA损伤。因此，已经开发了几种方法作为定性和定量测量DNA终产物的工具。由于DNA自由基的种类比最终产物的种类少，在自由基阶段检测和鉴定DNA损伤似乎更好。在本研究中，自旋捕获方法适用于这一目的。自旋捕获是一种利用自旋捕获剂捕获不稳定、短寿命自由基的方法。随后通过电子自旋共振光谱法（ESR）分析自旋捕获的化合物，即自旋加合物。使用1-甲基-2-亚硝基丙烷（MNP）作为自旋捕获试剂。利用X射线照射产生的氧自由基与DNA反应，检测和鉴定了DNA链断裂和8-羟基脱氧腺苷（oh^8dG）的前体自由基，并分别着重于染色体畸变和点突变的前体自由基的检测。结果，在糖部分的C4'位点和鸟嘌呤碱基部分的N7位点分别观察到自由基的形成，作为链断裂和oh^8dG的前体自由基。用两个10聚体寡核苷酸oligo（dC）_和oligo（dT）_作为DNA模型，进一步证实了C4'糖自由基是链断裂前体的证据<10><10>。寡聚体<32>在5'末端被13 P标记，并且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目。观察到MNP对C4'糖自由基的捕获确实抑制了两种寡聚体中链断裂的数目。

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.