DNA Adducts Formed by Dopamine

多巴胺形成的DNA加合物

• 批准号: 6358586
• 负责人: WILLIAM J BODELL
• 金额: $ 28.43万
• 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 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6358586
• 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）：导致帕金森病发展的病理生理过程已被证实 确定为致密部多巴胺能神经元的损失 黑质。尽管进行了深入的研究，但其分子机制 这些神经元的选择性丢失仍然未知。一个有趣的功能 这些神经元的主要特征是神经黑色素的存在。的生物学功能 这些细胞中的神经黑色素尚不清楚，目前对其的了解 合成基于黑色素生物合成。我们实验室的研究 已经证明，神经黑色素的前体多巴胺可以 酶促和非酶促氧化形成DNA加合物和 氧化碱损伤。基于这些观察和独特的关联 我们建议神经黑色素的存在和特定神经元细胞的损失 确定神经黑色素合成过程是否导致产生 DNA 损伤。为了实现这一目标，我们建议： 1a) 优化 用于检测稳定 DNA 加合物的 32P 后标记程序和 HPLC 用于定量不稳定加合物和 多巴胺形成的氧化碱损伤。 1b) 确定结构 结合光谱技术由多巴胺形成 DNA 加合物。 2) 将人类酪氨酸基因插入表达质粒中。所表达的 蛋白质将被亲和纯化并表征。这种人体酶将 用于研究多巴胺的氧化并将提供有关 产生多巴胺诱导 DNA 损伤的酶机制。 3）我们会 改造 PC12 细胞以在转录控制下表达酪氨酸。这些 细胞 PC12/tyr 将用于确定 DNA 损伤是否发生在 神经黑色素合成。对这些细胞的平行研究将调查 在神经黑色素合成过程中诱导细胞毒性。我们相信 这些具体目标将使我们能够检验 DNA 损伤的假设 可能发生在神经黑色素的合成过程中。此外，结果 这些研究将提供未来使用的独特分子标记 研究评估这一过程是否发生在黑质 人类的大脑。