DAMAGE TO MITOCHONDRIAL DNA IN AGING

工作总结：有丝分裂后组织衰老机制的一种理论提出，线粒体DNA（mt-DNA）的氧化损伤的积累导致线粒体的生物合成受损，从而无法维持细胞能量稳态。我们正在检验这一假设的几个要素。本研究采用高效液相色谱法分离核苷，电化学阵列检测器检测脱氧鸟苷的氧化产物8-OHdG在不同年龄大鼠肝线粒体DNA中的含量。我们发现8-OHdG在线粒体DNA中比在核DNA中更普遍。此外，有一个显着的增加与老化，从8每100000脱氧鸟苷残基在6个月至14在12个月和22在23个月的年龄。核DNA中没有这种与年龄相关的变化。由于先前的工作使用GC/MS分离/检测8-OHdG显示mt-DNA中没有随着老化而增加。我们已经努力验证HPLC/EC技术。用8-OHdG特异性甲酰胺基嘧啶糖基化酶（fpg酶）处理来自组织的线粒体DNA和商业小牛胸腺DNA，导致化学计量消除了归因于8-OHdG的峰。光诱导的氧化损伤，在亚甲蓝的存在下，同样认识到的fpg酶和HPLC技术，鼓励我们使用的fpg处理和Southern印迹相结合，以调查8-OHdG含量的线粒体DNA从非常小的组织样本，如T淋巴细胞分选的流式细胞仪技术，在当前和未来的工作。这对我们来说很重要，因为我们以前的工作涉及衰老的大鼠心脏，从一个大鼠心脏中分离的mtDNA的产量太小，不支持HPLC分析。我们还计划将这项工作扩展到测量5-OH胞嘧啶作为另一种线粒体DNA的氧化产物。我们还使用另一种方法，其中我们可以用特异性抗体直接检测体内线粒体中8-OHdG的存在。该抗体还检测核DNA中这种DNA损伤的存在，并使我们能够在各种条件下直接在体内比较各种细胞类型中核和线粒体DNA中的DNA损伤。- 8-羟基脱氧鸟苷HPLC电化学检测

Summary of work: One theory of the mechanism of aging of post-mitotic tissues proposes that an accumulation of oxidative damage to mitochondrial DNA (mt-DNA) leads to impaired biogenesis of mitochondria and thus a failure to maintain cellular energy homeostasis. We are testing several elements of this hypothesis. In this project, we have quantitated the occurrence of 8-OHdeoxyguanosine (8OHdG), an oxidative product of deoxyguanosine, in mt-DNA from liver of rats of various ages, using separation of nucleosides by HPLC and measurement by an electrochemical array detector. We found that 8-OHdG is more prevalent in mt-DNA than in nuclear DNA. Further, there is a significant increase with aging; from 8 per 100000 deoxyguanosine residues at 6 months to 14 at 12 months and 22 at 23 months of age. There was no such age-linked change in nuclear DNA. As previous work using GC/MS for separation/detection of 8-OHdG had shown no increase with aging in mt- DNA. we have taken some pains to validate the HPLC/EC technique. Treatment both of mt-DNA from tissues and of commercial calf thymus DNA with the 8-OHdG-specific formamidopyrimidine glycosylase (fpg enzyme) resulted in stoichiometric removal of the peak attributed to 8-OHdG. Oxidative damage induced by light in the presence of methylene blue was equally recognized by the fpg enzyme and HPLC techniques, encouraging us to use a combination of fpg treatment and Southern blotting to investigate the 8-OHdG content of mt-DNA from very small tissue samples, eg T-lymphocytes sorted by FACS technology, in current and future work. This is important to us since much of our previous work has involved the aging rat heart and the yield of mt-DNA isolated from one rat heart is too small to support the HPLC analysis. We also plan to extend this work to the measurement of 5-OHcytosine as another oxidative product of mt-DNA. We are also using another approach in which we can directly detect the presence of 8-OHdG in mitochondria in vivo with a specific antibody. This antoibody also detects the presence of this DNA lesion in nuclear DNA and permits us to compare the DNA damage in nuclear and mitochondrial DNA directly in vivo in various cell types under various conditions. - 8-hydroxydeoxyguanosine HPLC electrochemical detection