Previously, we found that inhalation of cigarette smoke by guinea pigs fed a diet deficient in ascorbic acid (AA), induced nitric oxide synthase (iNOS), and increased the respiratory levels of 3-nitrotyrosine (3-NT) and 8-oxodeoxyguanosine (8-oxo-dG). All of these effects, indicative of inflammation and of protein and DNA damage, were significantly decreased in guinea pigs fed a diet high in AA. The work proposed here is a logical extension of these findings. We postulate that cigarette smoke-derived oxidants, probably H2O2 and/or peroxynitrite, activate the transcription factors NF-kappaB and AP-1, and that this is an anti- apoptotic, tumor promoting event. We further propose that the activation of NF-kappaB and AP-1 will be inhibited by anti-oxidants, specifically AA, vitamin E, and the tea polyphenol, (-)-epigallocatechin gallate, given singly or in combination in the diet. To test these hypotheses, we propose short term (approximately 40 day) cigarette smoke inhalation studies in which we determine changes in the levels of DNA 8-oxo-dG, 8- nitroguanine and selected etheno adducts, and the modulation of NF- kappaB, AP-1, c-myc, matrix metalloproteinase-9, iNOS, 3-NT, cell proliferation and apoptosis in the guinea pig respiratory system, and the effects on these factors by dietary antioxidants.
Specific Aim 1 will furnish basic information on the time courses of formation and loss of 8- oxo-dG, ethene adducts and 8-nitroguanine in lung DNA, The studies in Specific Aims 2 and 3 will give information on the degree of activation of inflammation- and tumor promotion-related transcription factors NF- kappaB, AP-1 and c-myc, and on the consequences of such activation, including induction of matrix metalloproteinase-9 and iNOS, the appearance of 3-NT in protein, appearance of 8-oxo-dG, ethano adducts and 8-nitroguanine in lung DNA, and in changes in cell proliferation and apoptosis. Studies will then be undertaken to determine how these molecular marker changes in cell proliferation and apoptosis. Studies will then be undertaken to determine how these molecular markers are affected by dietary antioxidants, and whether the effects of the antioxidants, when given in combination, are either null, additive, or synergistic.
As Specific Aim 2, we will continue the development of a method for the quantitative determination of 3-NT. The extension of this method, which has a sensitivity in the femtomol range for 3-NT (a marker for peroxynitrite-induced damage to proteins), to the array of 8- nitroguanine (a marker of peroxynitrite-induced damage to DNA), and to nitrated benzene metabolites generated in Project 2, will also be examined under this Aim. The results of our studies will read to a better understanding of the relationship of chronic respiratory inflammation to cigarette smoke-induced cancers, and the influence of dietary anti- oxidants. We expect that conclusions drawn into our research will be directly translatable to simple dietary intervention strategies in those smokers unable to quit.
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