The goal of this proposal is to gain an understanding of the molecular mechanisms controlling the biosynthesis of antioxidant enzymes in lung cells using """"""""state-of-the-art"""""""" recombinant DNA techniques. Three diploid cell types (tracheobronchial and type II epithelial, fibroblasts, alveolar macrophages) isolated from Fischer 344 rat lung will be used under normal culture conditions and after exposure to oxidant stresses (hyperoxia, addition of asbestos) to determine the molecular mechanisms for regulation of copper-zinc superoxide dismutase (CuZn, SOD), manganese superoxide dismutase (Mn SOD) and glutathione peroxidases (GPX). The objectives over a 5-year period of funding are to: 1) construct a lambda gt11 expression cDNA library from a rat tracheobronchial epithelial cell line (2C5); 2) isolate cDNA clones for the rat lung antioxidant enzymes, CuZn COD, Mn SOD and GPX; 3) examine the expression of the mRNA for these enzymes in epithelial cells, fibroblasts and alveolar macrophages in response to stimuli (hyperoxia, crocidolite asbestos) which reportedly induce these enzymes; 4) isolate the complete genes for CuZn SOD, Mn SOD and GPX; 5) delineate the structural organization of the CuZn SOD, Mn SOD and GPX genes; and 6) amplify transfected CuZn, SOD, Mn SOD and GPX genes in cells of the lung determine if high levels of enzymes will afford protection from hyperoxic and asbestos induced injury in vitro. Results should determine whether different types of oxidant stresses cause selective activation of antioxidant enzyme genes and/or synthesis of enzymes in various cell types of the respiratory tract. In addition, information on whether regulation of antioxidant enzyme synthesis occurs at the transcriptional, translational and/or post translational level will be obtained.
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