DNA Cross-linking by diepoxybutane

描述(申请人提供)：1，2，3，4-二环氧基丁烷(DEB)是一种遗传毒性中间体，由1，3-丁二烯(BD)代谢产生，1，3-丁二烯(BD)是一种已知的人类致癌物质，工业生产，在汽车尾气和香烟烟雾中发现。DEB是BD最具诱变性和细胞毒性的代谢产物，可能在BD的致癌作用中发挥重要作用。DEB分子结构中的两个环氧基团的存在使其能够通过使DNA双链中的两个相邻的核苷酸碱基连续烷基化而形成DNA-DNA交联链。此外，DEB可以通过烷基化相同DNA碱基的两个位置形成潜在的致突变外环损伤。我们研究的长期目标是建立双官能性烷基化试剂产生生物效应的分子机制。该项目的目标是确定与DEB和BD的遗传毒性效应有关的特定DNA损伤。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BD的致癌和突变特性。我们建议的研究将通过提供有关双功能DEB-DNA加合物的关键信息，包括它们在暴露于BD后在体内的形成，它们对DNA结构的影响，错配特征和细胞修复，来提高对BD致突变和细胞毒性机制的理解。我们将追求以下四个具体目标：1.在体内定量吸入1，3-丁二烯后的双功能DEB-DNA损伤。一种灵敏和特异的基于质谱学的方法将用于分析从接触BD的小鼠和大鼠组织中提取的DNA中的DNA-DNA交联物和外环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每个DEB-DNA加合物的跨病变旁路效率和突变特性。3.分析双功能DEB-DNA加合物的修复。我们将确定负责去除DEB-DNA加合物的主要DNA修复机制，并分析加合物构象与修复效率之间的关系。4.用DEB法表征DNA-蛋白质的交联度。蛋白质组学和免疫学检测的结合将被用来研究DEB对DNA-蛋白质的交联，作为BD细胞毒性和诱变的另一种途径。总的来说，这些研究将确定双功能DNA加合物对BD的生物活性负责，并为其诱变性和细胞毒性的机制提供新的见解，减少人类接触BD的癌症风险评估的不确定性。公共卫生相关性：这项工作将调查1，2，3，4-二环氧基丁烷的生物活性机制，这是一种遗传毒性中间体，由1，3-丁二烯(BD)的代谢激活产生，1，3-丁二烯是一种已知的人类致癌物质，工业生产，在汽车尾气和香烟烟雾中发现。我们将分析暴露于BD的实验动物中二环氧丁烷-DNA加合物的形成，并检测它们诱导DNA突变的能力。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: This work will investigate the mechanisms of biological activity of 1,2,3,4-diepoxybutane, 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. We will analyze the formation of diepoxybutane -DNA adducts in laboratory animals exposed to BD and examine their ability to induce DNA mutations.