tate the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project (i.e., relevance to the mission of the agency). Describe concisely the research design and methods for achieving these goals. Describe the rationale and techniques you will use to pursue these goals. In addition, in two or three sentences, describe in plain, lay language the relevance of this research to public health. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE(S) (organization, city, state) The Cancer Center, University of Minnesota Minneapolis, Minnesota

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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA100670-09
Application #
8390506
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Johnson, Ronald L
Project Start
2003-04-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2014-11-30
Support Year
9
Fiscal Year
2013
Total Cost
$207,749
Indirect Cost
$62,158
Name
University of Minnesota Twin Cities
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Wickramaratne, Susith; Banda, Douglas M; Ji, Shaofei et al. (2016) Base Excision Repair of N(6)-Deoxyadenosine Adducts of 1,3-Butadiene. Biochemistry 55:6070-6081
Groehler 4th, Arnold; Villalta, Peter W; Campbell, Colin et al. (2016) Covalent DNA-Protein Cross-Linking by Phosphoramide Mustard and Nornitrogen Mustard in Human Cells. Chem Res Toxicol 29:190-202
Wickramaratne, Susith; Ji, Shaofei; Mukherjee, Shivam et al. (2016) Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases. J Biol Chem 291:23589-23603
Wickramaratne, Susith; Seiler, Christopher L; Tretyakova, Natalia Y (2015) Synthesis of DNA Oligodeoxynucleotides Containing Site-Specific 1,3-Butadiene-Deoxyadenosine Lesions. Curr Protoc Nucleic Acid Chem 61:4.61.1-22
Kotapati, Srikanth; Wickramaratne, Susith; Esades, Amanda et al. (2015) Polymerase Bypass of N(6)-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene. Chem Res Toxicol 28:1496-507
Wickramaratne, Susith; Boldry, Emily J; Buehler, Charles et al. (2015) Error-prone translesion synthesis past DNA-peptide cross-links conjugated to the major groove of DNA via C5 of thymidine. J Biol Chem 290:775-87
Tretyakova, Natalia Y; Groehler 4th, Arnold; Ji, Shaofei (2015) DNA-Protein Cross-Links: Formation, Structural Identities, and Biological Outcomes. Acc Chem Res 48:1631-44
Yeo, Jung Eun; Wickramaratne, Susith; Khatwani, Santoshkumar et al. (2014) Synthesis of site-specific DNA-protein conjugates and their effects on DNA replication. ACS Chem Biol 9:1860-8
Wickramaratne, Susith; Tretyakova, Natalia Y (2014) Structure elucidation of DNA-protein crosslinks by using reductive desulfurization and liquid chromatography-tandem mass spectrometry. Chembiochem 15:353-5
Kowal, Ewa A; Wickramaratne, Susith; Kotapati, Srikanth et al. (2014) Major groove orientation of the (2S)-N(6)-(2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine DNA adduct induced by 1,2-epoxy-3-butene. Chem Res Toxicol 27:1675-86

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