DNA cross-linking by diepoxybutane

描述(申请人提供)：1，2，3，4-二氧基丁烷(DEB)是1，3-丁二烯的重要代谢物，1，3-丁二烯是汽车尾气和香烟烟雾中发现的主要工业化学品和环境污染物。DEB是一种双官能性烷基化试剂，被归类为“合理预期为人类致癌物质”(美国卫生与公众服务部)。DEB是迄今为止1，3-BD的遗传毒性最强的代谢物，其致突变性比丁二烯环氧化物高两个数量级。DEB的细胞毒性和遗传毒性被认为是其双功能性质的结果。DEB可以通过同时使DNA双链中的两个碱基烷基化来形成DNA-DNA交联链。根据结构的不同，交联损伤可能会引起细胞毒性或致突变效应。虽然N7-G-N7-G DEB交联链最早是在40多年前从DNA中分离出来的，但这些损伤的详细结构信息尚不清楚。凝胶电泳法研究已经提供了腺嘌呤碱基上的DEB交联物的证据，但不能对这些损伤进行结构鉴定。这项拟议研究的总体目标是评估DNA-DNA交联在二环氧丁烷和1，3-丁二烯遗传毒性效应中的作用。我们将通过质谱学、分子生物学和分子模拟相结合的方法研究DEB诱导的DNA交联。首先，我们将从结构上表征DEB-DNA交联链，并确定其形成的序列偏好。接下来，我们将评估DEB-DNA交联物的水解性及其被大肠杆菌UvrABC修复复合体识别的情况。最后，将用毛细管高效液相色谱-电喷雾质谱质谱联用法对暴露于DEB后的啮齿动物组织中DEB-DNA交联物的形成进行定量。这项研究的结果将为二环氧丁烷遗传毒性活性的分子机制提供有价值的信息。这些发现将扩展到其他双功能亲电体，以解释观察到的生物活性差异。

DESCRIPTION (provided by applicant): 1,2,3,4-Diepoxybutane (DEB) is an important metabolite of 1,3-butadiene, a major industrial chemical and environmental pollutant found in automobile exhaust and cigarette smoke. DEB is a bifunctional alkylating agent classified as "reasonably anticipated to be a human carcinogen" (U.S. Department of Health and Human Services). DEB is by far the most genotoxic metabolite of 1,3-BD, with mutagenic potency two orders of magnitude higher than that of butadiene monoepoxide. The cytotoxicity and genotoxicity of DEB is thought to be a result of its bifunctional nature. DEB can form DNA-DNA cross-links by simultaneously alkylating two nucleobases within the DNA duplex. Depending on their structure, the cross-linked lesions can induce cytotoxic or promutagenic effects. Although N7-G-N7-G DEB cross-links were first isolated from DNA over 40 years ago, no detailed structural information for these lesions is available. Gel electrophoresis studies have provided evidence for DEB cross-linking at adenine nucleobases, but did not allow structural identification of these lesions. The overall goal of the proposed research is to evaluate the role of DNA-DNA cross-linking in the genotoxic effects of diepoxybutane and 1,3-butadiene. We will investigate DEB-induced DNA cross-linking by a combination of mass spectrometry, molecular biology, and molecular modeling. First, we will structurally characterize DEB-DNA cross-links and determine sequence preferences for their formation. Next, we will evaluate the hydrolytic stability of DEB-DNA cross-links and their recognition by the E. coli UvrABC repair complex. Finally, the formation of DEB-DNA cross-links in rodent tissues following DEB exposure will be quantified by capillary HPLC-ESI-MS/MS methods. The results of this research will provide valuable information on the molecular mechanisms underlying the genotoxic activity of diepoxybutane. These findings will be extended to other bifunctional electrophiles to explain the observed differences in their biological activity.