Kidney DNA Adductomics

项目摘要 人类经常暴露于环境中的危险化学品，这会损害DNA， 导致癌症。然而，在过去的30年里，DNA加合物的测量主要是在 通过监测一个或多个DNA加合物，数据提供了有限的信息 癌症的病原体。有一个关键的未满足的需要，建立一个强大的技术，以筛选广泛的 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。我们应用的目标- 目的是改进我们的质谱（MS）加合物组学方法，以筛选DNA加合物阵列 在一个单一的分析与危险化学品形成，并开发自动化分析算法。我们会- 利用高分辨精确质谱（HRAMS）技术，通过提取DNA加合物的特征， DNA加合物在经历多级（MSn）扫描片段化时的特征光谱特征。这些 MSn扫描方法可以检测人体组织中各种结构的DNA加合物，范围很广， 暴露。在本提案中，我们将优化我们在啮齿动物中的MS技术，在受控暴露于 一系列被认为是导致肾癌的化学物质，我们将创建一个质谱数据库， 用于基因组DNA加合物的全面表征的软件和信息学工具。 在目标1中，啮齿动物暴露于毒物后，将在肾脏中产生DNA加合物谱 在烟草中发现，环境，饮食，从促氧化剂，和亲电体产生内源性。面板 由这些暴露产生的DNA加合物谱的特征将通过非靶向数据依赖（DDA） 和数据独立采集（DIA），采用纳流液相色谱和多级MSn 在目标2中，将构建DNA加合物数据库，并使用生物信息学工具 将创建无偏和自动检测DNA加合物的提取的质谱 DNA加合物的特征。生物信息学工具将识别与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模板来检查不同类别的DNA 加合物在目标3中，我们将测试我们的技术，以筛选肾癌患者的活检，并探索 烟草暴露是这种癌症的少数公认的危险因素之一。 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，并可以帮助确定化学剂和生活方式因素，诱导DNA损伤和癌症风险。 DNA损伤剂的识别可以导致减轻或防止危险暴露的策略 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.