Morphologic injury and lipid peroxidation in monolayer cultures of rabbit tracheal epithelium exposed in vitro to ozone

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Abstract
Numerous reports have documented airway epithelial damage and lipid peroxidation in the lungs of animals exposed to ozone. However, the response of isolated tracheal epithelial (TE) cells to ozone has not been extensively studied. To assess ozone‐induced injury in cultured TE cells, an in vitro exposure system was developed in which cells were maintained at gas‐fluid interface analogous to in vivo conditions. Confluent monolayer cultures of rabbit TE cells were exposed for 30 min to atmospheres of 5% CO2/air containing 0.05, 0.1, 0.5, 1, 2, 4, 6, or 8 ppm ozone. Morphologic injury was assessed by phase‐contrast microscopy and by determination of TE cell number and viability (trypan blue dye exclusion) pre‐ and postexposure, and the lipid peroxide content of TE cells was measured as thiobarbituric acid (TBA) reactive substances. Exposure to 5% CO2/air alone did not affect monolayer morphology, cell number or viability. Cultures exposed to 0.05 or 0.1 ppm ozone demonstrated no consistent light microscopic changes, whereas exposure to 0.5 ppm and higher ozone concentrations caused distortion of monolayer morphology, cytoplasmic vacuolization, and decreased viability. Exposure to 0.5 or 1 ppm resulted primarily in cytoplasmic vacuolization while exposure to 2, 4, 6, or 8 ppm induced more pronounced cellular injury associated with cell necrosis (viability post 8 ppm ozone 75.0 ± 7.0%, vs. 95.9 ± 2.6% for 5% CO2/air controls). Ozone exposure also caused changes in cell shape, which on occasion resulted in loss of cell‐to‐cell contact. Increased production of TBA‐reactive substances was detected in TE cells following ozone exposure, including exposure to 0.05 and 0.1 ppm. The morphologic changes induced by in vitro ozone exposure in the cultured TE cells were similar to those described in the tracheal epithelium of ozone‐exposed animals and occurred independent of recruited inflammatory cells or extravasated circulating mediators.