REACTIVE SPECIES MEDIATED INJURY OF HUMAN LUNG EPITHELIAL CELLS AFTER HYPOXIA-REOXYGENATION

Abstract

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% CO 2) or hypoxia (< 1% O 2 /5% CO 2) 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-acetylcysteine (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 μ M) and trifluorocarbonylcyanide phenylhydrazone (10 μ M) (anti A + FCCP), a mitochondrial complex III inhibitor and respiratory chain uncoupler, which together increase mitochondrial superoxide (O 2 -) and H 2 O 2 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 more in hypoxic than in normoxic cells, suggesting that mitochondrial electron transport complex I may have been altered by hypoxia preexposure.

• Alveolar
• Apoptosis
• Epithelium
• Hypoxia
• Reoxygenation
• Superoxide