Reactive species mediated injury of human lung epithelial cells after hypoxia-reoxygenation

This study tested the hypothesis that hypoxia exposure predisposed lung epithelial cells to reactive oxygen species-(ROS) mediated cellular injury. Human lung carcinoma cells (ATCC line H441) having epithelial characteristics (including lamellar bodies, surfactant protein [SP]-A, and SP-B) were cultured in air (air/5% CO2) or hypoxia (<1% O-2/5% CO2) for 0 to 24 hours before imposition of oxidant stress. Cellular manganese superoxide dismutase (MnSOD) activity (units/mg protein) decreased significantly after 24 hours of hypoxia. In normoxic culture after hypoxia, the cells produced increased ROS, detected as dichlorofluorescein (DCF) fluorescence and H(2)o(2) accumulation in medium. Antioxidants N-acetylosteine (N-Ac) and ebselen inhibited increased DCF fluorescence after hypoxia. To test their ability to tolerate oxidant stress, some cells were incubated with antimycin A (100 muM) and trifluorocarbonylcyanide phenylhydrazone (10muM) (anti A + FCCP), a mitochondrial complex HI inhibitor and respiratory chain uncoupler which together increase mitochondrial superoxide (O-2(-)) and H2O2 production. Lung epithelial cells preexposed to hypoxia released more lactate dehydrogenase (LDH) than normoxic controls in response to increased O-2(-) production. Increased LDH release from hypoxia-preexposed cells treated with anti A + FCCP was inhibited by 1 mM N-Ac. Rotenone and myxothiazole increased DCF oxidation mare in hypoxic than in normoxic cells, suggesting that mitochondrial electron transport complex I may have been altered by hypoxia preexposure.