DNA adducts are believed to be a major source of the mutations involved in carcinogenesis. The availability of innovative technologies for investigating the presence of these adducts will greatly aid basic research epidemiological and chemoprevention studies, risk assessment and occupational health. We propose to develop technologies for the routine analysis of DNA adducts that arise from medical, environmental and occupational exposures, as well as from endogenous processes. We have previously developed highly sensitive gas chromatography/mass spectroscopy (GC-MS) methods for DNA adducts, but these methods are time consuming and labor intensive. The primary focus of this Phase I application will be the development of ultrasensitive techniques for monitoring DNA adduct formation using liquid- chromatography/electrospray mass spectroscopy (LC-MS). Specifically, we will develop LC-MS methods for 7-hydroxyethylguanine, 7- methylguanine, O6- methylguanine, N2,3-ethenodeoxyguanosine, l ,N2- ethenodeoxyguanosine, l ,N6-ethenodeoxyadenosine, 3,N4-ethenodeoxy- cytosine, 7-(2-hydroxy-2-phenylethyl)guanine, and 7-(2-hydroxy- l - phenyl-ethyl)guanine. These adducts are formed in DNA from animals and humans exposed to vinyl chloride, simple alkylating agents and styrene oxide, and most can be demonstrated in DNA of unexposed animals and humans. Following LC-MS methods development, the methods will be compared with our GC-MS methods to determine the sensitivity, specificity and feasibility of LC-MS for routine use. Triangle Laboratories, with three LC-MS/MS instruments, will be the primary site for the development of the new technology. This will be facilitated by the expertise and stable isotope internal standards that exist in Dr. Swenberg's laboratory. Since the equipment, standards and expertise required for these analyses are extensive, it will be more cost and time effective for many investigators in academia, industry and government to have such assays run by a specialized biotechnology laboratory. PROPOSED COMMERCIAL APPLICATIONS: The availability of innovative technologies for investigating the presence of these adducts will greatly aid basic research, epidemiological and chemoprevention studies, risk assessment and occupational health. Since the equipment, standards and expertise required for these analyses are extensive, it will be more cost and time effective for many investigators in academia, industry and government to have such assays run by a specialized biotechnology laboratory.
Thesaurus Terms: DNA damage, adduct, liquid chromatography mass spectrometry, method development guanine analog, nucleoside, technology /technique development gas chromatography mass spectrometry
