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.
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.
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