DNA Cross-linking by diepoxybutane

描述：参见说明。说明申请的广泛、长期目标和具体目标，并提及 该项目（即，与原子能机构的使命相关）。简要描述研究设计和实现这些目标的方法。描述 你将用来追求这些目标的基本原理和技术。 此外，用两三句话，用通俗的语言描述这项研究与公共卫生的相关性。如果申请得到资助， 这样的描述将成为公开信息。因此，不包括专有/机密信息。不要超过空间 提供了 1，2，3，4-二环氧丁烷（DEB）是一种遗传毒性中间体，在1，3- 丁二烯（BD）是一种已知的人类致癌物质，在工业上生产，并在汽车尾气和 香烟烟雾。DEB是BD最具致突变性和细胞毒性的代谢产物， BD诱发的致癌作用。分子结构中存在两个环氧乙烷基团， DEB允许它通过连续烷基化DNA中两个相邻的核碱基来形成DNA-DNA交联 复式。此外，DEB可通过烷基化两个位点形成潜在的促突变性环外病变， 相同的DNA碱基我们研究的长期目标是建立分子机制， 双官能烷基化剂引起它们的生物学效应。该项目的目标是确定具体的 DNA损伤导致DEB和BD的遗传毒性作用。这项研究的核心假设是 DEB形成DNA-DNA交联和在靶组织中积累的环外加合物， 观察到的BD的致癌性和致突变性。我们建议的研究将改善目前的 了解BD暴露导致的诱变和细胞毒性机制， 关于双功能DEB-DNA加合物的关键信息，包括它们在体内暴露于 BD，它们对DNA结构、错配特征和细胞修复的影响。我们将继续努力， 四个具体目标： 1.定量吸入暴露于1，3-丁二烯后体内双功能DEB-DNA损伤。 一种灵敏和特异的基于质谱的方法将用于分析DNA-DNA交叉， 从暴露于BD的小鼠和大鼠的组织中提取的DNA中的环外DEB加合物。 2.确定双功能DEB-DNA加合物对DNA双链体结构和复制的影响。 将通过NMR进行结构分析以分析双链DNA中的加合物构象， 虽然位点特异性诱变实验将确定跨损伤旁路效率和突变 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.通过DEB表征DNA-蛋白质交联。结合蛋白质组学和免疫学 检测将被用来研究DNA-蛋白质交联的DEB作为一个额外的途径， BD的细胞毒性和致突变性。 总之，这些研究将确定负责BD生物活性的双功能DNA加合物 并提供新的见解，其致突变性和细胞毒性的机制，减少不确定性， 人类暴露于BD的癌症风险评估。 履约地点（组织、城市、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