DAMAGE TO MITOCHONDRIAL DNA IN AGING

工作概述：有丝分裂后组织衰老机制的一种理论认为，线粒体DNA （mt-DNA）氧化损伤的积累导致线粒体生物发生受损，从而无法维持细胞能量稳态。我们正在测试这个假设的几个要素。本项目采用高效液相色谱法分离核苷，电化学阵列检测器测定不同年龄大鼠肝脏mt-DNA中脱氧鸟苷氧化产物8-OHdeoxyguanosine （8OHdG）的含量。我们发现8-OHdG在mt-DNA中比在核DNA中更普遍。此外，随着年龄的增长，有显著的增加；从6月龄时的每10万个脱氧鸟苷残基中有8个到12月龄时的14个和23月龄时的22个。核DNA中没有这种与年龄相关的变化。正如先前使用GC/MS分离/检测8-OHdG的工作所显示的那样，mt- DNA中8-OHdG没有随着年龄的增长而增加。我们花了一些功夫来验证HPLC/EC技术。用8-OHdG特异性甲酰胺嘧啶糖基酶（fpg酶）处理来自组织的mt-DNA和商业小牛胸腺DNA，导致8-OHdG峰的化学计量去除。fpg酶和HPLC技术同样可以识别亚甲蓝存在下光诱导的氧化损伤，这鼓励我们在当前和未来的工作中使用fpg处理和Southern blotting相结合的方法来研究非常小的组织样本（例如用FACS技术分类的t淋巴细胞）中mt-DNA的8-OHdG含量。这对我们来说很重要，因为我们之前的大部分工作都涉及到衰老的大鼠心脏，而从一只大鼠心脏中分离出的mt-DNA的产量太小，无法支持HPLC分析。我们还计划将这项工作扩展到mt-DNA的另一种氧化产物5- ohcyto嘧啶的测量。我们还使用了另一种方法，我们可以用一种特定的抗体直接检测体内线粒体中8-OHdG的存在。该抗体还可以检测到核DNA中存在这种DNA损伤，并允许我们在体内直接比较各种细胞类型和各种条件下核DNA和线粒体DNA的DNA损伤。- 8-羟基脱氧鸟苷的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