Bronchopulmonary dysplasia, a chronic debillitating lung disease, is a serious consequence of modern neonatal care resulting from the use of high oxygen concentrations in premature infants. Exposure to hyperoxia results in increased production of potentially damaging, partially reduced oxygen species. Protection against these oxygen metabolites requires adequate antioxidant enzyme activity, and the balance between oxygen radical production and antioxidant capacity determines whether pulmonary oxygen injury occurs. This proposal is based on the premise that copper-zinc superoxide dismutase is an important antioxidant because it eliminates superoxide anion and prevents the generation of other damaging oxygen metabolites that arise from superoxide anion. Two hypotheses will be tested: 1) CuZn SOD is temporally and hormonally regulated during the perinatal period: 2) Oxygen increases pulmonary CuZn SOD activity by increasing CuZn SOD mRNA. To test these hypotheses, a cDNA encoding human CuZn SOD will be isolated and characterized. Temporal and hormonal regulation of CuZn SOD expression during development will be evaluated in explant cultures of human and rabbit fetal lung. Ontogenic regulation will be analyzed at the protein level by ELISA and at an activity level using the nitroblue tetrazolium assay. Hybridization assays with the cDNA in both Northern and slot blot will be performed to quantitate changes in specific mRNA. Runoff transcription experiments will be done to determine whether increases in CuZn SOD RNA are related to changes in transcription rates or in message stability. To evaluate oxygen-induced increases in CuZn SOD activity, Type II cells isolated from oxygen treated adult rats will be used. The mechanisms of oxygen induction will be evaluated by measuring CuZn SOD protein, enzyme activity, CuZn SOD RNA, and specific mRNA transcription rates. The final goals of this project are to isolate human genomic DNA encoding CuZn SOD. To understand regulation of CuZn gene expression, promoter and 5' flanking regions of the gene will be subcloned in a CAT vector and used in transfection experiments in continuous cell cultures or primary type II cells. These studies will improve the understanding of control of CuZn SOD expression during development and hyperoxic exposure. In the future, these studies may suggest ways to increase SOD activity in newborns who require oxygen therapy.
