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.

A redox mechanism underlying nucleolar stress sensing by nucleophosmin

“…Glutaredoxin 1 (Grx1) is a thiol oxidoreductase that selectively interacts with GSH31. In previous reports, a Grx1-roGFP2 fusion protein was constructed to monitor the dynamics of reduced GSH versus oxidized GSH (GSSG)323334. We therefore constructed an EGFP-Grx1-NPM1 fusion protein and observed its translocation dynamics in a live-cell imaging system.…”

Live-cell imaging approaches for the investigation of xenobiotic-induced oxidant stress

“…As an example, we may consider the case of a hypothetical dithiocarbamate derivative that inhibits Grx, the proximal enzyme in the relay through which roGFP senses the oxidant effect of H 2 O 2 on E GSH (Fig. 1, [101,212]). Since the effect of Grx inhibition would be to sever the ability of roGFP to sense E GSH , any oxidant effect that this dithiocarbamate derivative may have on E GSH, either through the production of H 2 O 2 or by direct reactivity with GSH, could not be reported by the sensor, resulting in a false negative result.…”

“…1). Our laboratory used this approach in a recent study [212] that investigated the basis for roGFP2-reported increases in E GSH induced in human airway epithelial cells undergoing real time exposure to O 3 , a highly reactive environmental electrophile. The requirement for Grx in the O 3 -induced E GSH change was tested two different ways.…”

“…Our experiments showed that glucose deprivation effectively sensitized the cells to increases in E GSH induced by a subsequent exposure to O 3 [212], without a detectable alteration in basal E GSH . Depletion of intracellular levels of GSH using an inhibitor of glutathione synthesis, such as buthionine sulfoximine (BSO), is also an effective tool that can be brought to bear in studies aimed at scrutinizing the integrity of the roGFP redox relay in cells exposed to xenobiotics [199].…”

Role of H2O2 in the oxidative effects of zinc exposure in human airway epithelial cells

“…In order to determine the dependence of Zn 2+ -induced responses on H 2 O 2 , the present study expanded our previous live-cell imaging approach to monitor oxidative changes in the cytosol and mitochondria of HAEC exposed to Zn 2+ [28] , [29] . We utilized cytosolic overexpression or ectopic mitochondrial expression of the H 2 O 2 scavenging enzyme catalase in BEAS-2B cells bearing the genetically-encoded fluorogenic ratiometric sensors HyPer or roGFP2, which report on H 2 O 2 and the glutathione redox potential ( E GSH ), respectively [30] , [31] , [32] .…”