The broad, long-term objective of this application is to establish chemical markers of internal exposure and genetic damage through biomonitoring DNA adducts to assess the risk of carcinogens. Human risk assessment has been severely impeded by the paucity of analytical methods that unambiguously identify and quantitate DNA adducts in vivo, and by the inability to easily obtain relevant tissues to assess cancer development. We hypothesize that the recent advances in sensitivity of liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) instrumentation allow for the identification and quantification of DNA adducts in exfoliated buccal cells of individuals exposed to tobacco smoke and diet-related carcinogens. The successful use of buccal cells as a reliable tissue to measure DNA adducts requires optimization of the analytical MS methods and the establishment of standardized methods to isolate human buccal cell DNA. To address this hypothesis, the specific aims are: 1) To establish ultra-trace LC/ESI-MS methods to measure DNA adducts of suspected human carcinogens found in tobacco smoke that include: 4-methylnitrosamino-1-(3-pyridyl)-1-butanone, benzo(a)pyrene, 2-amino-a-carboline, 2-amino-3-methyl-a-carboline, 4-aminobiphenyl; and abundant heterocyclic aromatic amine (HAAs) carcinogens formed in grilled meats; 2) Cytobrush and mouthwash rinsing techniques will be compared to isolate buccal cells that maximize the recovery of human DNA and minimize contaminating bacterial DNA. The standardization of the buccal cell DNA isolation method will permit reliable assessment of genotoxin exposure and DNA damage; and 3) Recruit volunteers that include: smokers, non-smokers, and meat-eaters and probe for buccal cell DNA adducts by LC/ESI-MS. Adduct formation will be correlated to salivary nicotine levels for tobacco exposure and HAAs in the diet. The results of this research are expected to show that quantitative LC/ESI-MS analysis of buccal cell DNA adducts may be used to assess damage posed by genotoxins, and reduce the invasiveness of risk assessment procedures by validation of exfoliated cell specimens. The impact of this research on public health is the development of new analytical tools that may be used for biomonitoring and risk assessment, which examine exposure to genotoxicants, assess gene-environment interactions and determinants of cancer, or chemopreventive efficacy. ? ? ?
