DNA Cross-linking by diepoxybutane

描述（由申请人提供）：1,2,3,4-二氧丁烷（DEB）是一种基因毒性中间体，由1,3-丁二烯（BD）的代谢激活产生，BD是一种已知的工业生产的人类致癌物质，存在于汽车尾气和香烟烟雾中。DEB是BD最具诱变性和细胞毒性的代谢物，可能在BD诱导的癌变中发挥重要作用。在DEB的分子结构中，两个氧环基团的存在允许它通过连续烷基化DNA双链中相邻的两个核碱基形成DNA-DNA交联。此外，DEB可以通过烷基化同一DNA碱基的两个位点形成潜在的促生外环病变。我们的长期研究目标是建立双功能烷基化剂引发其生物效应的分子机制。该项目的目的是确定导致DEB和BD基因毒性作用的特定DNA病变。本研究的中心假设是DEB形成DNA-DNA交联和外环加合物，并在靶组织中积累。我们提出的研究将通过提供双功能BD -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，从而提高目前对BD暴露导致的突变和细胞毒性机制的理解。我们将努力实现以下四个具体目标：定量吸入1,3-丁二烯后体内双功能DEB-DNA损伤。一种基于质谱的灵敏和特异的方法将被用于分析从暴露于BD的小鼠和大鼠组织中提取的DNA中的DNA-DNA交联和外环DEB加合物。确定双功能脱氧核糖核酸加合物对DNA双工结构和复制的影响。核磁共振结构分析将用于分析双链DNA中的加合物构象，而位点特异性诱变实验将确定每个DEB-DNA加合物的翻译绕过效率和突变特性。3. 分析双功能脱氧核糖核酸加合物的修复。我们将确定主要的DNA修复机制负责去除DEB-DNA加合物，并分析加合物构象与修复效率之间的关系。4. 用DEB表征dna -蛋白交联。蛋白质组学和免疫学检测的结合将用于研究DNA-蛋白质交联作为双相障碍细胞毒性和诱变的额外途径。总的来说，这些研究将确定双相障碍生物活性的双功能DNA加合物，并为其致突变性和细胞毒性的机制提供新的见解，减少人类暴露于双相障碍的癌症风险评估的不确定性。这项工作将研究1,2,3,4-二氧丁烷的生物活性机制，这是一种基因毒性中间体，由1,3-丁二烯（BD）的代谢激活产生，BD是一种已知的工业生产的人类致癌物质，存在于汽车尾气和香烟烟雾中。我们将分析二氧丁烷-DNA加合物在暴露于BD的实验动物中的形成，并检查它们诱导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.