Kidney DNA Adductomics

项目总结 人类经常暴露在环境中的危险化学物质中，这会破坏DNA和 会导致癌症。然而，在过去的30年里，DNA加合物的测量在很大程度上是通过 通过监测单个或最多几个DNA加合物，数据提供的信息有限 癌症的致病因素。有一个关键的未得到满足的需求是建立一种强大的技术来筛查广泛的 一系列DNA加合物，其中一些可能与癌症驱动基因的突变有关。我们申请的目标是- 我们的目标是改进我们的质谱学(MS)加合物方法，以筛选一系列DNA加合物 在一次化验中使用危险化学品，并开发自动分析的算法。我们会- 采用高分辨精确质谱仪(HRAMS)提取DNA加合物的特征，用于DNA加合物的检测 DNA加合物通过多阶段(MSN)扫描进行碎裂时的空间光谱特征。这些 MSN扫描方法可以在广泛的范围内检测人体组织中各种结构的DNA加合物 曝光。在这项提案中，我们将优化我们在啮齿动物身上的多发性硬化症技术，在受控的情况下暴露于 一系列被认为会导致肾癌的化学物质，我们将创建一个质谱库并开发 用于全面描述基因组DNA加合物的软件和信息学工具。 在目标1中，在暴露于毒物之后，将在啮齿动物的肾脏中产生dna加合物图谱。 在烟草中发现，环境、饮食、促氧化剂和亲电体都是内源性产生的。嵌板 由这些暴露产生的DNA加合物图谱的特征是非靶向数据依赖(DDA) 和数据独立采集(DIA)，使用纳米流液相色谱和多级MSN 在AIM 2中，用Orbitrap MS扫描，将建立DNA加合物数据库，并使用生物信息学工具 将通过提取质谱图来创建用于DNA加合物的无偏自动检测 DNA加合物的特性。生物信息学工具将识别不同类别形成的加合物的模式 并对不同类型的DNA加合物进行系统的比较和鉴定 以及DNA损伤的机制。DIA和DDA扫描方法，DNA加合物 数据库和生物信息学技术将作为检查不同类别DNA的模板 人类体内的加合物。在目标3中，我们将测试我们的技术来筛选肾癌患者的活检组织，并探索 烟草暴露是为数不多的被认为是这种癌症的危险因素之一。 所提出的dna加合物分子分析技术适用于其他靶器官和细胞。 类型，并有助于识别导致DNA损伤和癌症风险的化学制剂和生活方式因素。 识别DNA损伤剂可以导致减轻或防止暴露在危险环境中的策略 对公众健康有重大影响的化学品。

PROJECT SUMMARY Humans are frequently exposed to hazardous chemicals in the environment, which can damage DNA and lead to cancer. However, over the past 30 years, the measurements of DNA adducts have been largely carried out by monitoring a single or up to several DNA adducts, and the data have provided limited information about causative agents of cancer. There is a crucial unmet need to establish a robust technology to screen for a broad range of DNA adducts, some of which can be linked to mutations in cancer driver genes. The goal of our applica- tion is to advance our mass spectrometry (MS) adductomics methods to screen for an array of DNA adducts formed with hazardous chemicals in a single assay and develop algorithms for automated analysis. We will em- ploy high-resolution accurate mass spectrometry (HRAMS) to detect DNA adducts by extracting the charac- teristic spectral features of DNA adducts as they undergo fragmentation by the multistage (MSn) scanning. These MSn scanning approaches can detect DNA adducts of diverse structures in human tissues over a wide range of exposures. In this proposal, we will optimize our MS technology in rodents, under controlled exposures to an array of chemicals thought to contribute to renal cancer, and we will create a mass spectral database and develop software and informatics tools for comprehensive characterization of DNA adducts of the genome. In Aim 1, DNA adduct profiles will be generated in the kidney of rodents, following exposures to toxicants found in tobacco, the environment, diet, from pro-oxidants, and electrophiles produced endogenously. Panels of DNA adduct profiles resulting from these exposures will be characterized by untargeted data-dependent (DDA) and data-independent acquisition (DIA), employing nanoflow liquid chromatography and multistage MSn scanning with the Orbitrap MS. In Aim 2, a DNA adduct database will be constructed, and bioinformatics tools will be created for the unbiased and automated detection of DNA adducts by extraction of the mass spectral features of DNA adducts. The bioinformatics tools will identify patterns of adducts formed with different classes of renal toxicants and conduct systematic comparison and identification of DNA adducts based on different types of exposures and the mechanism of DNA damage. The DIA and DDA scanning methods, the DNA adduct database, and bioinformatics technologies will serve as templates to examine for different classes of DNA adducts in humans. In Aim 3, we will test our technologies to screen biopsies of renal cancer patients and explore tobacco exposure, one of the few recognized risk factors for this cancer. The proposed technology for molecular analysis of DNA adducts is amenable to other target organs and cell types and can help identify chemical agents and lifestyle factors that induce DNA damage and cancer risk. Identification of DNA-damaging agents can lead to strategies to mitigate or prevent exposures to hazardous chemicals with a dramatic impact on public health.