The objective of this proposal is to understand the nature of the phenomenon of elevated ozone resistance of conducting airway epithelium. Previous in vivo animal studies have demonstrated the phenomenon of """"""""elevated ozone resistance"""""""" in conducting airway epithelium after a repeating ozone exposure. Several years ago, we developed a biphasic culture system in which conducting airway epithelial cells are able to grow between air phase and liquid medium. Under the basal feeding, epithelial cells differentiate and form polarized epithelium resembling that in vivo. Since ozone is such a highly reactive chemical, this biphasic culture system allows a direct exposure of airway epithelial cells to ozone. In the Preliminary Study, the toxicity of ozone on airway epithelial cells can be demonstrated. However, we observed different toxicity among different airway epithelial cultures. for most of human and non-human primate primary airway epithelial cells, the LT50 is more than 30 hrs; in contrast, the immortalized human tracheobronchial epithelial (TBE) cell lines and established monkey cell lines have LT50 of 4-6 hrs. Numerous explanations can be postulated to address this difference, such as different levels in antioxidant/oxidant enzymes, different toxicity response, different membrane structures, and other mechanisms still undefined. We will test two hypotheses in this proposal, 1) antioxidant enzymes are involved in the elevation of ozone resistance; 2) differential gene expressions are involved in the regulation of different ozone sensitivity in different cells. To address the former hypothesis, we need to demonstrate the existence of different levels of antioxidant enzymes in these primary TBE cells and the cell lines. Most of all, we need to demonstrate that the resistance of cell lines to ozone can be elevated upon the transfection with antioxidant enzyme expressional constructs. To address the latter hypothesis, we will construct the cDNA expressional library from the resistance TBE cells and use in transfection for selecting cDNA clones that can convert the sensitive cell lines to be resistant to ozone. This approach will allow us to identify genes which are previously not recognized in a role of ozone resistance. Currently, we have obtained expressional cDNA constructs of manganese-superoxide dismutase (SOD), extracellular form of SOD, and catalase, and full length cDNA clones of gluthionine peroxidase and gluthionine-S-transferase. The activity of these enzymes in transfected cells will be determined biochemically. We will construct the latter two cDNA constructs in the eukaryotic expressional vectors. We have considerable experience in cDNA construction. This genetic approach will help to elucidate the genes underlined the mechanism of the phenomenon of elevated ozone resistance.
