DETECTION OF ENDOG AND EXOGEN DERIVED ETHYLENE OXIDE DNA ADDUCTS BY 3H & 14C AM

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ethylene oxide is a widely used intermediate in the chemical industry and is also formed endogenously from the metabolic oxidation of ethylene, which is generated during normal physiological processes. Although ethylene oxide is classed as a human carcinogen, epidemiological studies provide conflicting evidence regarding its ability to induce human cancers. Consequently, there is a need to assess the risks associated with low dose occupational exposures to this chemical. Ethylene oxide reacts with DNA, primarily forming N7-(2-hydroxyethyl)-2¿-deoxyguanosine adducts (7HEG), which can be used as a biomarker of exposure and potential cancer risk. The ultimate goal of this project is to identify sources of endogenous adduct formation and determine the relative contribution of low dose ethylene oxide exposures to the overall level of 7HEG adducts formed in vivo. This will be achieved through the administration of [3H]-labeled biological precursors of ethylene (unsaturated fatty acids and methionine) and [14C]-ethylene oxide, coupled with accelerator mass spectrometry analysis. Specifically, this project will involve first establishing the sources and level of endogenous 7HEG adducts formed in rat tissues through oral administration of [3H]-labeled unsaturated fatty acids or methionine. The level of exogenously derived 7HEG adducts formed in rat tissues following acute administration of [14C]-ethylene oxide over a range of doses, including occupational exposure levels will also be determined in parallel studies. In order to determine the relative contribution of adducts from endogenous and exogenous sources, a [3H]-labeled fatty acid or [3H]-methionine will be co-administered with [14C]-ethylene oxide. Analysis of the DNA adducts formed using dual-isotope AMS will demonstrate whether adduct formation by different routes is additive and whether ethylene oxide is able to influence endogenous adduct levels. Through this work we will identify sources of endogenous 7HEG adduct formation and quantify the effect of occupational levels of ethylene oxide on adduct formation, which will aid in assessing the risk to humans exposed to this chemical. Furthermore, the methods developed could be applied to the study of other chemical carcinogens capable of generating DNA adducts also formed endogenously. This will lead to a better appreciation of the relative roles of endogenous and exogenous pathways of DNA adduct formation and the risks associated with exposure to industrial chemicals. This past year several abstracts were published for presentations from this work: Marsden, D. A.; Jones, D. J. L.; Lamb, J. H.; Tompkins, E. M.; Crookston, R. J. R.; Farmer, P. B.; Brown, K. Development of an LC-MS/MS method for quantifying N7-(2-hydroxyethyl)-guanine adducts. 35th Annual Meeting of the European Environmental Mutagen Society, Kos, Greece. European Journal of Genetic and Molecular Toxicology (2005) 155, #P183. Marsden, D. A.; Farmer, P. B.; Jones, D. J. L.; Lamb, J. H.; Tompkins, E. M.; Crookston, R. J. R.; Brown, K. Determination of N7HEG adducts in ethylene oxide treated rats using LC-MS/MS. AACR 97th Annual meeting (April 2006). Proceedings of the American Association for Cancer Research, V47 #703. Marsden, D.; Farmer, P.B.; Jones, D.J.L.; Lamb, J.H.; Tompkins, E.M.; Crookston, R.J.R.; Brown. K. Measurement of endogenous and exogenously derived N7-HEG adducts in ethylene oxide treated rats using LC-MS/MS. Joint congress of the United Kingdom Environmental Mutagen Society and the British Toxicology Society Warwick, UK In press, Mutagenesis (2006).