DNA Adducts Formed by Dopamine

多巴胺形成的DNA加合物

• 批准号: 6683218
• 负责人: WILLIAM J BODELL
• 金额: $ 28.79万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-15 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6683218
• 关键词: DNA damage; PC12 cells; Parkinson' s disease; adduct; apoptosis; biological products; biomarker; cytotoxicity; disease /disorder etiology; dopamine; electrochemistry; high performance liquid chromatography; mass spectrometry; melanins; mitochondrial DNA; molecular pathology; monophenol monooxygenase; neurons; neurotoxicology; oxidation; plasmids; structural biology; transfection /expression vector; transposon /insertion element; tyrosine 3 monooxygenase; ultraviolet spectrometry

DESCRIPTION (Adapted from applicant's abstract): The pathophysiological processes responsible for the development of Parkinson's disease has been identified as a loss of dopaminergic neurons in the pars compacta of the substantia nigra. Despite intensive study, the molecular mechanisms responsible for the selective loss of these neurons remains unknown. An intriguing feature of these neurons is the presence of neuromelanin. The biological function of neuromelanin in these cells is unknown and the current understanding of its synthesis is based on that of melanin biosynthesis. Studies in our laboratory have demonstrated that the precursor to neuromelanin, dopamine can be enzymatically and non-enzymatically oxidized to form both DNA adducts and oxidative base damage. Based on these observations and the unique association of the presence of neuromelanin and specific neuronal cell loss we propose to determine whether the process of neuromelanin synthesis leads to the production of DNA damage. In order to achieve this goal we propose to: 1a) Optimize the 32P-postlabeling procedure for detection of stable-DNA adducts and HPLC with electrochemical detection for the quantification of both unstable adducts and oxidative base damage formed by dopamine. 1b) Identify the structure(s) of the DNA adducts formed by dopamine using a combination of spectroscopic techniques. 2) Insert the human gene for tyrosine into an expressed plasmid. The expressed protein will be affinity purified and characterized. This human enzyme will be used to study oxidation of dopamine and will provide information as to the enzymatic mechanisms for production of dopamine induced DNA damage. 3) We will engineer PC12 cells to express tyrosine under transcriptional control. These cells, PC12/tyr, will be used to determine if DNA damage occurs during neuromelanin synthesis. Parallel studies with these cells will investigate the induction of cellular cytotoxicity during neuromelanin synthesis. We believe that these Specific Aims will allow us to test our hypothesis that DNA damage can occur during the synthesis of neuromelanin. In addition, the results of these studies will provide unique molecular markers that will be used in future studies to evaluate whether this process is occurring in the substantia nigra of human brain.

描述（改编自申请人的摘要）：导致帕金森氏病发展的病理生理过程是 被确定为在Pars compacta中的多巴胺能神经元丧失 黑质尼格拉。尽管进行了深入研究，但分子机制负责 对于这些神经元的选择性丧失仍然未知。一个有趣的功能 这些神经元的存在是神经苯胺的存在。的生物学功能 这些细胞中的神经丙氨酸是未知的，当前对其细胞的理解 合成基于黑色素生物合成。在我们的实验室研究 已经证明了神经苯胺，多巴胺的前体可以是 酶促和非酶的氧化以形成DNA加合物和 氧化碱损伤。基于这些观察和独特的关联 在存在神经素蛋白和特定神经元细胞损失的情况下，我们提出 确定神经苯胺合成的过程是否导致产生 DNA损伤。为了实现此目标，我们建议：1a）优化 32p postlabeling程序，用于检测稳定DNA加合物和HPLC 量化不稳定加合物的电化学检测和 多巴胺形成的氧化碱损伤。 1b）确定结构 多巴胺与光谱技术组合形成的DNA加合物。 2）将酪氨酸的人基因插入表达的质粒中。表达 蛋白质将具有亲和力纯化和表征。这种人类酶将是 用于研究多巴胺的氧化，并将提供有关 产生多巴胺诱导DNA损伤的酶促机制。 3）我们会的 工程师PC12细胞在转录控制下表达酪氨酸。这些 细胞PC12/Tyr将用于确定在 神经苯胺合成。与这些细胞的平行研究将研究 神经丙烷蛋白合成过程中细胞细胞毒性的诱导。我们相信 这些具体目标将使我们能够检验DNA损害的假设 在合成神经苯胺期间可以发生。另外，结果 这些研究将提供独特的分子标记，以后将使用 评估这一过程是否发生在尼格拉的研究中 人脑。