Student Research: Seth Stuebbe

MS, , 1993
Faculty Advisor: Daniel L. Luchtel

Flow Cytometric Measurement of Glutathione Content in Respiratory Epithelial Cells Exposed In Vitro to Ozone


Ozone is a ubiquitous secondary atmospheric air pollutant frequently measured in many urban areas at levels exceeding the National Ambient Air Quality Standard, currently 120 ppb averaged over a one hour period. Many adverse effects are known to occur in humans as a consequence of exposure to elevated concentrations of ozone, and a growing body of evidence suggests that exposure to ozone at levels below the standard can also have adverse effects on the lung. However, the mechanism(s) of ozone's toxicity have not been fully elucidated. Therefore, this project investigated the hypothesis that exposing human nasal (HNE) and monkey bronchial (PBE) epithelial cells to ozone in vitro would cause depletion of their intracellular glutathione content ([GSH]i), presumably as a consequence of liquid peroxidation of cellular membranes. Cells were grown on collagen-coated, microporous membrane inserts until confluents, and then exposed to either air or 250 ppb ozone for 3 hours, followed by a 3 hour incubation period at 37 C. Air and ozone-exposed cells were analyzed on a flow cytometer for changes in [GSH]i. Cells were stained for 30 minutes post-incubation with the nonfluorescent dye monochlorobimane (MCB). Reduced glutathione (GSH) is congugated to MCB by intracelular glutathione-S-transferases. This conjugate fluoresces at a characteristic wavelength (475 nm) when illuminated with the 354 nm line of an argon laser. The intensity of fluorescence is thus indicative of the amount of GSH present within the cell. The viability of the cells at the time of the flow cytometric measurements was also determined by flow cytometry by adding propidium iodide to cell suspensions immediately prior to analysis. Analysis showed that ozone did not significantly alter [GSH]i of HNE or PBE cells exposed to ozone as compared to air-exposed cells. However, significant differences were observed between cell types with respect to [GSH]i, and with respect to cell count in a defined range of fluorescence intensities. Viability was identical between the cell types, and no significant change in viability was observed between ozone and air-exposed within either cell type. Analysis of the magnitude of forward light scatter, an indicator of relative cell size, showed that PBE cells are significantly smaller overall than HNE cells. These results indicate that HNE and PBE ceell populations differ with respect to their overall glutathione content, and with respect to the proportion of cells within defined ranges of glutathione content. These differences cannot be explained by either viability or size differences between cell types.