The rate of production of toxic, partially reduced species of oxygen is thought to increase in hyperoxic lungs, eventually overwhelming endogenous cellular defenses and leading to lung cell damage. However, development of oxygen tolerance in adult rats was often observed after exposure to 85% O2 for 5-7 days and which is correlated with an increased activity of both the copper/zinc and manganese superoxide dismutases in lung tissue. The overall goal of this research is to understand the molecular basis for regulation of gene expression of these antioxidant enzymes in rat lungs in response to oxidant stress. In order to perform these studies, a cDNA clone coding for the CuZn SOD and an additional cDNA clone coding for the Mn SOD have been isolated. These cDNA probes will be employed to quantitate the level of CuZn and Mn SOD mRNAs in in vitro pulmonary endothelial cells and alveolar type II cells with or without hyperoxic exposure to define whether expression of individual SOD gene in response to hyperoxia is different in various types of lung cells. In situ hybridization at electron microscope level will be performed to determine if the observation of changing mRNA level in in vitro lung cells correlates closely with the results obtained in in vivo lung cells. Elevation of SOD mRNA level in lung cells after hyperoxic insult should direct to investigating the mechanisms that are involved in mRNA accumulation. We would further measure the effect of hyperoxia on the copy number of the SOD gene, rate of RNA transcription and mRNA transportation and stability of mRNAs. The role of cis and trans acting elements in regulation of SOD gene expression will also be defined by DNA binding assays and gene transfer experiments if we observed transcriptional activation of these genes in response to hyperoxia. Finally, translation efficiency of these mRNAs and stability of these enzymes will also be determined by pulse labelling and immunoprecipitation experiments to define if hyperoxia affects these enzyme activities on translational and/or post-translational levels. Antioxidant enzymes are critical for cellular defense against oxidant stress. Unravelling of the effect of hyperoxia on the modulation of SOD gene expression is essential for us to uncover the signal(s) generated by hyperoxia, and should also provide valuable information to design strategies for augmenting antioxidant defense in the lungs.
Showing the most recent 10 out of 16 publications
