Monitoring intracellular redox changes in ozone-exposed airway epithelial cells

Abstract

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 intracellular 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 glutathione redox potential (EGSH), was used to monitor EGSH in cultured human airway epithelial cells (BEAS-2B cells) undergoing exposure to 0.15-1.0 ppm ozone (O3). Cells were imaged in real time using a custom-built O3 exposure system coupled to a confocal microscope. Results: O3 exposure induced a dose- and time-dependent increase of the cytosolic EGSH. Additional experiments confirmed that roGFP2 is not directly oxidized, but properly equilibrates with the glutathione redox couple: Inhibition of endogenous glutaredoxin 1 (Grx1) disrupted roGFP2 responses to O3, and a Grx1-roGFP2 fusion protein responded more rapidly to O3 exposure. Selenite-induced up-regulation of GPx (glutathione peroxidase) expression-enhanced roGFP2 responsiveness to O3, suggesting that (hydro)peroxides are intermediates linking O3 exposure to glutathione oxidation. Conclusion: Exposure to O3 induces a profound increase in the cytosolic EGSH of airway epithelial cells that is indicative of an oxidant-dependent impairment of glutathione 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.