Monitoring Intracellular Redox Changes in Ozone-Exposed Airway Epithelial Cells

BACKGROUND: The toxicity of many xenobiotic compounds is believed to involve oxidative injury to cells. Direct assessment of mechanistic events involved in xenobiotic-induced oxidative stress is not easily achievable. Development of genetically encoded probes designed for monitoring intra-cellular redox changes represents a methodological advance with potential applications in toxicological studies. OBJECTIVE: We tested the utility of redox-sensitive green fluorescent protein (roGFP)-based redox sensors for monitoring real-time intracellular redox changes induced by xenobiotics in toxicological studies. METHODS: roGFP2, a reporter of the gluta-thione redox potential (E-GSH), was used to monitor E-GSH in cultured human airway epithelial cells (BEAS-2B cells) undergoing exposure to 0.15-1.0 ppm ozone (O-3). Cells were imaged in real time using a custom-built O-3 exposure system coupled to a confocal microscope. RESULTS: O-3 exposure induced a dose-and time-dependent increase of the cytosolic E-GSH. Additional experiments confirmed that roGFP2 is not directly oxidized, but properly equilibrates with the gluta-thione redox couple: Inhibition of endogenous gluta-redoxin 1 (Grx1) disrupted roGFP2 responses to O-3, and a Grx1-roGFP2 fusion protein responded more rapidly to O-3 exposure. Selenite-induced up-regulation of GPx (gluta-thione peroxidase) expression-enhanced roGFP2 responsiveness to O-3, suggesting that (hydro) peroxides are intermediates linking O-3 exposure to gluta-thione oxidation. CONCLUSION: Exposure to O-3 induces a profound increase in the cytosolic E-GSH of airway epithelial cells that is indicative of an oxidant-dependent impairment of gluta-thione redox homeostasis. These studies demonstrate the utility of using genetically encoded redox reporters in making reliable assessments of cells undergoing exposure to xenobiotics with strong oxidizing properties.