DNA加合物8-羟基脱氧鸟苷特性研究

Characteristic Study of DNA Adduct 8-Hydroxy-2-Deoxyguanosine

作为DNA氧化损伤的生物学标志物,8-羟基脱氧鸟苷(8-OH-dG)的特性研究,对稳定、灵敏、准确地定量8-OH-dG,进而研究有毒化学物质对生物体内的氧化损伤很重要。该文研究了氧化损伤后DNA中8-OH-dG的分析、水解、储存、稳定性等方面的问题。采用Fenton型产羟自由基系统如螯合剂Fe2+-H2O2为氧化源,与脱氧鸟苷和小牛胸腺DNA反应,生成的8-OH-dG用高压液相色谱-电化学法检测,并对8-OH-dG的分析条件进行优化。该法最低检出限为32fmol,比光学吸收法高2～3个数量级,线性范围可高达4个数量级,从0.32pmol到3.2nmol,相关系数0.9996。并对酶水解DNA的条件、储存酸度、介质环境及其稳定性等进行了探讨,结果表明8-OH-dG储存在中性偏酸的缓冲溶液中损失较少,其形成与所处介质环境有关,在氧化源存在或有氧环境中一定时间内有累积作用,添加抗氧化剂等干预措施后,能灵敏而稳定地对DNA中的8-OH-dG进行测定。

DNA adduct 8-hydroxy-2-deoxyguanosine (8-OH-dG)as a biomarker of oxidative DNA damage, its characteristic study is very important for realization of its stability, sensitivity, accuracy of quantitative determination as well as extensive investigation of the oxidative damage caused by the toxic materials in vivo. Studies on analysis, hydrolysis, storage and stability of 8-OH-dG in oxidatively damaged DNA have been conducted. The oxidative source, taking Fenton type reaction as a hy-droxyl free radical generating system(such as chelator-Fe2+-H2O2 system), reacted with deoxyguanosine and calf thymus DNA to yield mounts of 8-OH-dG. 8-OH-dG was analyzed by HPLC-EC method, its sensitivity was 2-3 orders of magnitude higher than the spectrophoto-metric method, the detection limit was 32 fmol, and its dynamic range was 4 orders of magnitude, from 32pmol to 3.2 nmol, and correlation coefficient was 0.9996. Studies have also been conducted on enzymatic digestion conditions of DNA, storage pH, medium environment and stability etc. The results showed that there was less loss of 8-OH-dG stored in neutral and acidic buffer solution than in alkaline buffer solution and the amount of 8-OH-dG formed in DNA depending on the buffer system. The 8-OH-dG within DNA was accumulated under oxidative source or environment with oxygen, and improved that the method by adding antioxidants to detect 8-OH-dG in DNA were sensitive and accurate.