DNA Cross-linking by diepoxybutane

说明：请参阅说明。说明应用程序的广泛、长期目标和具体目标，并提及与健康相关的 项目(即与该机构使命的相关性)。简明扼要地描述实现这些目标的研究设计和方法。描述 你将用来追求这些目标的基本原理和技术。 此外，用两三句简单明了的话描述这项研究与公共卫生的相关性。如果应用程序得到资助，则此 原样的描述将成为公开信息。因此，不包括专有/机密信息。不要超过空格 但前提是。 1，2，3，4-二环氧基丁烷(DEB)是1，3-二环氧基丁烷代谢活化后产生的一种遗传毒性中间体。 丁二烯(BD)，一种已知的人类致癌物质，工业生产，在汽车尾气和 香烟味。Deb是BD最具诱变性和细胞毒性的代谢物，很可能在BD的 在BD致癌中的作用。在分子结构中存在两个环氧基团 Deb使DNA中相邻的两个碱基连续烷基化，从而形成DNA-DNA交联链 双面打印。此外，DEB可以通过烷基化两个位点形成潜在的致突变外环损伤 相同的DNA碱基。我们研究的长期目标是建立 双官能性烷基化试剂可产生生物效应。该项目的目标是确定特定的 DNA损伤与DEB和BD的遗传毒性效应有关。这项研究的中心假设是 DEB形成DNA-DNA交联物和外环加合物，积聚在靶组织中，有助于 观察到的BD的致癌和致突变特性。我们建议的研究将改善目前的 了解接触BD致突变和细胞毒性的机制 关于双功能DEB-DNA加合物的关键信息，包括它们在暴露于 BD，它们对DNA结构、错配特征和细胞修复的影响。我们将致力于 以下是四个具体目标： 1.对吸入1，3-丁二烯后体内双功能的DEB-DNA损伤进行定量。 将使用一种灵敏和特异的基于质谱学的方法来分析DNA-DNA交叉 从接触BD的小鼠和大鼠组织中提取的DNA中的连接和外环DEB加合物。 2.确定双功能DEB-DNA加合物对DNA双链结构和复制的影响。 核磁共振结构分析将用于分析双链DNA中的加合物构象， 而定点突变实验将确定跨病变搭桥效率和突变 每个DEB-DNA加合物的性质。 3.分析双功能DEB-DNA加合物的修复。我们将确定主要的DNA修复机制 负责DEB-DNA加合物的去除，并分析加合物之间的关系 构象和修复效率。 4.用DEB法表征DNA-蛋白质的交联度。蛋白质组学和免疫学的结合 DEB的检测将用于研究DNA-蛋白质的交联性，作为一种额外的途径 BD的细胞毒性和致突变性。 总的来说，这些研究将确定与BD生物活性有关的双功能DNA加合物 并对其致突变性和细胞毒性的机制提供了新的见解，减少了 人类接触BD的癌症风险评估。 演出现场(S)(组织、市、州) 明尼苏达大学癌症中心 明尼苏达州明尼阿波利斯

DESCRIPTION: See instructions. State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project (i.e., relevance to the mission of the agency). Describe concisely the research design and methods for achieving these goals. Describe the rationale and techniques you will use to pursue these goals. In addition, in two or three sentences, describe in plain, lay language the relevance of this research to public health. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE PROVIDED. 1,2,3,4-diepoxybutane (DEB) is a genotoxic intermediate produced upon the metabolic activation of 1,3- butadiene (BD), a known human carcinogen produced industrially and found in automobile exhaust and in cigarette smoke. DEB is the most mutagenic and cytotoxic metabolite of BD and is likely to play an important role in BD-induced carcinogenesis. The presence of two oxirane groups within the molecular structure of DEB allows it to form DNA-DNA cross-links by consecutively alkylating two adjacent nucleobases in a DNA duplex. In addition, DEB can form potentially promutagenic exocyclic lesions by alkylating two sites of the same DNA base. The long-range goal of our research is to establish the molecular mechanisms by which bifunctional alkylating agents elicit their biological effects. The objective of this project is to identify specific DNA lesions responsible for the genotoxic effects of DEB and BD. The central hypothesis of this research is that DEB forms DNA-DNA cross-links and exocyclic adducts that accumulate in target tissues, contributing to the observed carcinogenic and mutagenic properties of BD. Our proposed studies will improve the current understanding of the mechanisms of mutagenesis and cytotoxicity resulting from BD exposure by providing key information about bifunctional DEB-DNA adducts, including their formation in vivo following exposure to BD, their effects on DNA structure, mispairing characteristics, and cellular repair. We will be pursuing the following four Specific Aims: 1. Quantify bifunctional DEB-DNA lesions in vivo following inhalation exposure to 1,3-butadiene. A sensitive and specific mass spectrometry-based methodology will be used to analyze DNA-DNA cross- links and exocyclic DEB adducts in DNA extracted from tissues of mice and rats exposed to BD. 2. Determine the effects of bifunctional DEB-DNA adducts on DNA duplex structure and replication. Structural analyses by NMR will be performed to analyze adduct conformations in double stranded DNA, while site specific mutagenesis experiments will determine translesion bypass efficiencies and mutational properties of each DEB-DNA adduct. 3. Analyze the repair of bifunctional DEB-DNA adducts. We will identify the major DNA repair mechanisms responsible for the removal of DEB-DNA adducts and analyze the relationships between adduct conformations and repair efficiency. 4. Characterize DNA-protein cross-linking by DEB. A combination of proteomics and immunological detection will be used to investigate DNA-protein cross-linking by DEB as an additional pathway to cytotoxicity and mutagenesis of BD. Collectively, these studies will identify bifunctional DNA adducts responsible for the biological activity of BD and afford new insights into the mechanisms of its mutagenicity and cytotoxicity, reducing the uncertainty in cancer risk assessment for human exposure to BD. PERFORMANCE SITE(S) (organization, city, state) The Cancer Center, University of Minnesota Minneapolis, Minnesota