Formation of intracellular free radicals in guinea pig airway epithelium during in vitro exposure to ozone

In vivo exposure to ozone (O-3) has been shown to cause airway epithelial damage and lipid peroxidation. The oxidation of polyunsaturated fatty acids has been shown to produce hydrogen peroxide and aldehydes with reactive oxygen species (ROS) as intermediates. These products of ozonation may react with other bioorganic molecules and cause cellular damage. To assess the production of ROS, confluent primary cultures of guinea pig airway epithelial cells were grown on Costar membrane with a liquid-air interface and exposed to 0.2, 0.4, and 0.6 ppm O-3. The concentrations of intracellular ROS during the exposure were monitored using the fluorescent dye dihydrorhodamine-123. The intracellular concentration of ROS increased immediately upon the commencement of the O-3 exposure and persisted until the end of the exposure period (up to 1 hr). The concentration of ROS increased with increasing O-3 concentration. To determine the species of ROS produced during O-3 exposure, airway epithelial cells were perfused with dimethyl sulfoxide (DMSO), sodium formate (hydroxyl radical scavengers), NaN3 (catalase inhibitor), or diethyl-dithio carbamate (DEDC, superoxide dismutase inhibitor) prior to and during the exposure period and the fluorescent intensity was monitored continuously. While both DMSO and sodium formate decreased the concentration of ROS, DEDC and NaN3 had no effect. We concluded that hydroxyl radicals instead of H2O2 or superoxide anions were produced immediately following the commencement of O-3 exposure in guinea pig airway epithelial cells in an exposure concentration-dependent fashion. (C) 1997 Academic Press.