DNA Adducts Formed by Dopamine

描述（改编自申请人摘要）： 帕金森病的发展过程一直是 被鉴定为多巴胺能神经元的损失， 黑质尽管进行了深入的研究， 对于这些神经元的选择性损失仍然未知。一个有趣的特征 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是未知的，目前对其的理解是， 合成是基于黑色素生物合成的。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 酶促和非酶促氧化以形成DNA加合物， 氧化性碱损伤根据这些观察和独特的关联 神经黑色素的存在和特定的神经元细胞损失，我们建议， 确定神经黑色素合成的过程是否导致产生 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32 P-后标记程序和HPLC 用于定量不稳定加合物和 由多巴胺形成的氧化性碱损伤。1b）确定 使用光谱技术的组合由多巴胺形成的DNA加合物。 2)将人酪氨酸基因插入表达质粒中。表达的 蛋白质将被亲和纯化和表征。这种人体酶将 用于研究多巴胺的氧化，并将提供有关 多巴胺诱导的DNA损伤的酶促机制。3)我们将 工程化PC 12细胞以在转录控制下表达酪氨酸。这些 细胞，PC 12/tyr，将被用来确定是否发生DNA损伤， 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。我们认为 这些特定目标将使我们能够测试我们的假设， 在神经黑色素合成过程中发生。此外， 这些研究将提供独特的分子标记， 研究以评估这一过程是否发生在黑质 人类的大脑。

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.