The overall goals of this proposal are to determine whether mutagens present in urban air induce a significant number of mutations in the genome of bronchoepithelial cells and whether these induced mutations include the oncogene mutations and/or the tumor suppressor gene mutations that are frequently detected inhuman lung carcinomas. In order to address this question, the frequency and distribution of these oncomutations will be measured in normal lung tissue of healthy non-smoking individuals, smokers and lung cancer patients.
The specific aims of this project are as follows: Lungs from adults deceased from causes not related to pulmonary disease will be dissected to reveal the airways and divided into about 126 sectors. Epithelial cells will be isolated separately from each sector. The highly sensitive and specific Mismatch Amplification Mutation Assay (MAMA) will then be used to measure the frequency of oncogenic mutations in the K-ras and p53 genes within the individual sectors. A random distribution among organ sectors will imply that the mutations occurred after lung development. A clustering of mutations within organ sectors (jackpots) would be consistent with their origin during lung development. In order to determine whether the most important mutagens identified in urban air contribute to the induction of gene mutations, the frequency of mutations, and more importantly the number of organ sector containing mutations will be measured as a function of age, smoking, rural versus urban dwelling and indoor air quality. If mutations are induced by environmental exposure, the number of mutant sectors will increase above the fraction of jackpot sectors as a function of age or exposure. In order to permit the utilization of smaller cell numbers to measure mutations, and to explore the possible role of mitochondrial mutagenesis in carcinogenesis, a newly developed assay for mutations affecting """"""""6 cuter"""""""" restriction sites will be applied to all of the sectors' DNA analyzed for oncomutations. If cancer gene mutations of the type induced by the most important mutagens present in urban and/or indoor air are found to increase with age in humans, and if these induced mutations are clustered within human lung sectors that are known to have the highest exposure to particulate or mutagens present in urban air based on position and adduct analysis, then it may be inferred that airborne mutagens contribute significantly to the induction of lung tumors by inducing cancer gene mutations.
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