DNA cross-linking by diepoxybutane

描述（申请人提供）：1,2,3,4-二氧丁烷（DEB）是1,3-丁二烯的重要代谢物，而1,3-丁二烯是一种主要的工业化学品和环境污染物，存在于汽车尾气和香烟烟雾中。DEB是一种双功能烷基化剂，被美国卫生与公众服务部列为“合理预期的人类致癌物”。到目前为止，DEB是1,3- bd最具遗传毒性的代谢物，其致突变效力比单环氧丁二烯高两个数量级。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可以通过同时烷基化DNA双链内的两个核碱基形成DNA-DNA交联。根据其结构不同，交联病变可诱导细胞毒性或促生作用。虽然N7-G-N7-G DEB交联在40多年前首次从DNA中分离出来，但没有这些病变的详细结构信息。凝胶电泳研究提供了DEB在腺嘌呤核碱基交联的证据，但没有允许这些病变的结构鉴定。提出的研究的总体目标是评估DNA-DNA交联在二氧丁烷和1,3-丁二烯的遗传毒性作用中的作用。我们将通过质谱、分子生物学和分子模型的结合来研究deb诱导的DNA交联。首先，我们将在结构上表征DEB-DNA交联，并确定其形成的序列偏好。接下来，我们将评估DEB-DNA交联的水解稳定性及其在大肠杆菌UvrABC修复复合体中的识别能力。最后，采用毛细管HPLC-ESI-MS/MS方法定量测定DEB暴露后啮齿动物组织中DEB- dna交联的形成。本研究结果将为二氧丁烷基因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试剂，以解释其生物活性的观察差异。

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.