Nitrogen dioxide induces death in lung epithelial cells in a density-dependent manner

Nitrogen dioxide (*NO2) is commonly known as an indoor and outdoor air pollutant. Inhalation of *NO2 is associated with epithelial cell injury, inflammation, and the aggravation of asthma. *NO2 can also be formed during inflammation, by the metabolism of nitric oxide. We describe a gas-phase exposure system for in vitro exposure of lung epithelial cells to *NO2. Immunofluorescence revealed 3-nitrotyrosine immunoreactivity of rat alveolar type II epithelial cells exposed to 5 parts per million of *NO2 for 4 h. Comparative analysis of log-phase and confluent cultures demonstrated that cell death occurred extensively in log-phase cells, whereas minimal death was observed in confluent cultures. Peroxynitrite (ONOO-) or the ONOO- generator 3-morpholinosydnonimine (SIN-1) caused similar amounts of death. Further, exposure of wounded cell cultures to *NO(2) or SIN-1 revealed that death was restricted to cells repopulating a wounded area. Cycloheximide or actinomycin D, inhibitors or protein and messenger RNA synthesis, respectively, significantly reduced terminal transferase reactivity, suggesting that a new protein(s) may be required for cell death. These results suggest that during restitution after pulmonary injury, epithelium may be sensitive to cell death by reactive nitrogen species.