Effects of transgene expression of superoxide dismutase and glutathione peroxidase on pulmonary epithelial cell growth in hyperoxia

1 CardioPulmonary Research Institute and the Departments of 2 Pediatrics, 3 Medicine, and 4 Biostatistics, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Mineola, New York; 5 Department of Computer Information Systems and Decision Sciences, St. John's University, Jamaica, New York; 6 Department of Pathology, Thomas Jefferson University Medical Center, and 7 Joseph Stokes Jr. Research Institute, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Submitted 31 December 2003 ; accepted in final form 29 November 2004 Prolonged exposure to supraphysiological oxygen concentrations results in the generation of reactive oxygen species, which can cause significant lung injury in critically ill patients. Supplementation with human recombinant antioxidant enzymes (AOE) may mitigate hyperoxic lung injury, but it is unclear which combination and concentration will optimally protect pulmonary epithelial cells. First, stable cell lines were generated in alveolar epithelial cells (MLE12) overexpressing one or more of the following AOE: Mn superoxide dismutase (MnSOD), CuZnSOD, or glutathione peroxidase 1. Next, A549 cells were transduced with 50–300 particles/cell of recombinant adenovirus containing either LacZ or each of the three AOE (alone or in combination). Cells were then exposed to 95% O 2 for up to 3 days, with cell number and viability determined daily. Overexpression of either MnSOD (primarily mitochondrial) or CuZnSOD (primarily cytosolic) reversed the growth inhibitory effects of hyperoxia within the first 48 h of exposure, resulting in a significant increase in viable cells ( P < 0.05), with 1.5- to 3-fold increases in activity providing optimal protection. Protection from mitochondrial oxidation was confirmed by assessing aconitase activity, which was significantly improved in cells overexpressing MnSOD ( P < 0.05). Data indicate that optimal protection from hyperoxic injury occurs in cells coexpressing MnSOD and glutathione peroxidase 1, with prevention of mitochondrial oxidation being a critical factor. This has important implications for clinical trials in preterm infants receiving SOD supplementation to prevent acute and chronic lung injury. viral transduction; gene therapy; adenovirus; antioxidants Address for reprint requests and other correspondence: J. A. Kazzaz, CardioPulmonary Research Institute, Winthrop Univ. Hospital, 222 Station Plaza North, Suite 604, Mineola, NY 1