Development of novel techniques for quantitative proteomics, and their use to investigate the in vivo effects of long-term ozone exposure on 1-nitronaphthalene toxicity to the rat airway epithelium

Anthropogenic air pollutants are a significant factor in causing lung disease, a leading cause of morbidity and mortality in urban areas worldwide. Recent developments in genomics, proteomics and metabolomics hold substantial promise for understanding cellular responses to multiple toxicants. However, the cellular complexity of the lung presents great challenges for in vivo proteomics, and improved isolation methods for proteins from specific lung cell-phenotypes are required. To address this issue, I have developed a novel method for isolation of rodent airway epithelial cell proteins, which facilitates in vivo proteomics studies of two target-cell phenotypes of the lung, Clara cells and ciliated cells. The airway epithelial cell proteins are reproducibly solubilized, leaving the underlying basement membrane and smooth muscle intact as shown by histopathological analyses. The method yields epithelial cell-specific proteins in 5-fold higher concentrations and reduces the yield of confounding non-epithelial cell proteins 13-fold in comparison to airway microdissection.;Two-dimensional electrophoresis (2DE) remains the most capable method for separation of complex proteomes. However, large variability in the method caused by inter-gel variation limit its use for quantitative proteomics studies. To address this issue, I have developed a fluorescent internal protein standard which facilitates quantitative 2DE studies.;These two novel proteomics tools were used to probe the underlying mechanisms for the synergistic effects of two abundant air pollutants, ozone and 1-nitronaphthalene (1-NN), on injury to rat airway epithelium. Previous pathology studies have shown that rats chronically exposed to ozone are more susceptible to acute exposure to 1-NN than controls. I investigated how long-term ozone exposure alters the protein adduct formation in the rat airway epithelium. Molecular chaperones and proteins involved in antioxidant defense were found to be adducted by 1-NN metabolites. Interestingly, the molecular chaperone calreticulin was found to be adducted exclusively in animals exposed to ozone prior to 1-NN, and not in filtered air exposed controls. This indicates that adduction of calreticulin may be an important factor in the synergistic toxicity of two common air pollutants, ozone and 1-NN. This represents an important step towards understanding differences in the mechanism of toxicity following exposure to single versus multiple air pollutants.