MtDNA damage as a biomarker for environmental mitochondrial toxicity

描述（由申请人提供）：Greenamyre实验室（匹兹堡大学）最近获得资助，研究线粒体DNA （mtDNA）损伤作为暴露于环境线粒体毒素的潜在生物标志物。所采用的基于pcr的检测方法非常敏感、定量和高度可重复性，它可以检测到任何类型的损害，减缓聚合酶的进展。当前申请的基础是父母资助，它是实验性的，研究暴露于线粒体毒素的大鼠mtDNA损伤的各个方面。基本前提是，线粒体毒素通过产生活性氧（ROS），会在线粒体中引起各种形式的DNA损伤。帕金森氏症（PD）在美国影响着大约100万人，它似乎与遗传和环境因素都有关系，其中系统性线粒体缺陷与之密切相关。帕金森病最典型的环境风险因素是职业性农药暴露，其中许多农药是线粒体抑制剂。在这方面，目前的申请涉及两位流行病学家，他们在确定特定农药作为PD的真正危险因素方面发挥了重要作用。新的合作者Caroline Tanner和Sam Goldman（加州森尼维尔帕金森研究所）表明，使用鱼藤酮或百草枯的农民患帕金森病的风险增加了一倍以上，这两种物质都是产生ros的线粒体抑制剂。此外，他们报告说，接触溶剂三氯乙烯（TCE）也会增加患帕金森病的风险。在大鼠中，TCE暴露与线粒体复合体i的抑制有关。重要的是，现有的基因组DNA样本可从Tanner和Goldman的流行病学研究中获得。因为(i)帕金森病与线粒体缺陷和暴露于已知线粒体毒素有关，（ii）即使是亚临床暴露于线粒体毒素的大鼠也会在血液中产生强大的mtDNA损伤，他们提出了具体的目标来解决以下问题：百草枯和/或TCE暴露会导致mtDNA损伤吗？人类暴露于鱼藤酮、百草枯或TCE与mtDNA损伤有关吗？散发性PD是否与mtDNA损伤有关，无论是否暴露于鱼藤酮、百草枯或TCE ？目前的提案建立在母体应用程序的优势之上，其创新和极其敏感的mtDNA损伤检测，并建立了从啮齿动物实验工作（Greenamyre）到帕金森病人类流行病学转化研究（Tanner & Goldman）的桥梁。他们将利用现有的技术和人员，以及储存的DNA样本，利用现有的资产，努力开发一种生物标志物，用于人类暴露于某些环境毒物（例如鱼藤酮、百草枯和TCE），并可能用于帕金森病。因此，与一项新研究相比，拟议的工作可以显著节省成本和效率。此外，主要研究人员有一个独特的机会，在两个独立的流行病学队列中测试我们的分析作为PD的生物标志物。鉴于他们的综合经验和专业知识，他们相信他们在评估这种潜在的生物标志物方面处于独特的地位。

DESCRIPTION (provided by applicant): The Greenamyre laboratory (University of Pittsburgh) was recently funded to examine mitochondrial DNA (mtDNA) damage as a potential biomarker of exposure to environmental mitochondrial toxins. The PCR-based assay employed is extremely sensitive, quantitative and highly reproducible - and it detects any type of damage that slows polymerase progression. The parent grant, upon which the current application is based, is experimental and examines various aspects of mtDNA damage in rats exposed to mitochondrial toxins. The basic premise is that mitochondrial toxins, by generating reactive oxygen species (ROS), will cause various forms of DNA lesions in the mitochondria. Parkinson's disease (PD), affecting about 1 million people in the United States, appears to involve both genetic and environmental factors - and systemic mitochondrial defects have been strongly implicated. The best characterized environmental risk factor for PD is occupational exposure to pesticides, many of which are mitochondrial inhibitors. In this regard, the current application engages two of the epidemiologists who were instrumental in identifying specific pesticides as bona fide risk factors for PD. The new collaborators, Caroline Tanner and Sam Goldman (Parkinson's Institute, Sunnyvale, CA), showed that the risk of PD more than doubled in farmers using rotenone or paraquat, which are both ROS-generating mitochondrial inhibitors. Additionally, they reported that exposure to the solvent, trichloroethylene (TCE), also increases the risk of PD. In rats, TCE exposure is associated with inhibition of mitochondrial complex I. Importantly, existing genomic DNA samples are available from the epidemiological studies of Tanner and Goldman. Because (i) PD is associated with mitochondrial defects and exposures to known mitochondrial toxins, and (ii) even subclinical exposure of rats to a mitochondrial toxin produces robust mtDNA damage in blood, they propose specific aims to address the following questions: Does paraquat and/or TCE exposure cause mtDNA damage? Is human exposure to rotenone, paraquat or TCE associated with mtDNA damage? Is sporadic PD, with or without exposure to rotenone, paraquat or TCE associated with mtDNA damage? The current proposal builds upon the strengths of the parent application, with its innovative and extremely sensitive assay for mtDNA damage, and establishes a bridge from experimental work in rodents (Greenamyre) to translational research in the human epidemiology of Parkinson's disease (Tanner & Goldman). Using existing technology and staff, and banked DNA specimens, they will leverage current assets in an effort to develop a biomarker for human exposures to certain environmental toxicants (e.g., rotenone, paraquat & TCE) and, possibly, for Parkinson's disease. As a result, the proposed work can be done with dramatic cost savings and efficiency compared to a new study. Moreover, the principal investigators have a unique opportunity to test our assay as a biomarker for PD in two independent epidemiological cohorts. Given their combined experience and expertise, they believe they are uniquely positioned to assess this potential biomarker.