DNA cross-linking by diepoxybutane

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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.