Tobacco smoke contains high concentrations of oxidants and free radicals and increases the oxidative stress on the lung. This proposal outlines studies of the molecular mechanisms involved in chemical reactions caused by aqueous extracts of cigarette smoke and tar. Extracts of whole (i.e., unfiltered) smoke, of cigarette tar, and of gas-phase smoke contain different compounds and react differently with bio-targets; therefore, this proposal includes studies of all three. In addition, studies of extracts of both mainstream smoke and environmental tobacco smoke are proposed. Extracts of cigarette tar contain high concentrations of a free radical, identified by electron spin resonance (ESR) methods as a semiquinone (QH.). This QH. radical can reduce dioxygen to give superoxide, leading to hydrogen peroxide and the hydroxyl radical. In addition, smoke extracts contain nitric oxide (NO), nitrogen dioxide (NO2), peroxynitrous (HO-ON=O) and peroxynitric acids (HO-ONO2) and their esters, organic peroxides, and aldehydes. Any or all of these species could be involved in smoking-induced pathology, and studies of these compounds also are proposed. Two bio-target molecules are chosen as models for study. (i) The antiprotease alpha-1-proteinase inhibitor (a1PI) is thought to be involved in protecting lung tissue from changes leading to emphysema. The effects of smoke extracts on a1PI and on Met-10, a decapeptide that models the active site of a1PI, will be studied. (ii) The species responsible for smoke extract-induced nicking of DNA in rat alveolar macrophages, spleen cells and thymocytes will be determined. The possibility of base-sequence specificity of the nicking will be examined using plasmid DNA fragments. The radical chemistry of smoke extracts is duplicated by aged solutions of catechol. Both smoke extracts and these aged catechol solutions are used to determine whether the tar radical binds to DNA prior to causing nicks.
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