Monitoring Intracellular Redox Changes in Ozone-Exposed Airway Epithelial Cells

Eugene A. Gibbs-Flournoy,¹ Steven O. Simmons,² Philip A. Bromberg,³ Tobias P. Dick,⁴ and James M. Samet⁵

¹Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; ²Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory (NHEERL), U.S. Environmental Protection Agency (EPA), Research Triangle Park, North Carolina, USA; ³Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; ⁴Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany; ⁵Environmental Public Health Division, NHEERL, U.S. EPA, Chapel Hill, North Carolina, USA

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 (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 (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 E_GSH. 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 E_GSH 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.

Key words: glutathione, human airway epithelial cells, imaging, intracellular, oxidative stress, ozone, NADPH, redox, roGFP.

Environ Health Perspect 121:312–317 (2013). http://dx.doi.org/10.1289/ehp.1206039 [Online 18 December 2012]

Address correspondence to J.M. Samet, 104 Mason Farm Rd., U.S. EPA Human Studies Facility, Chapel Hill, NC 27599-7310 USA. Telephone: (919) 966-0665. E-mail: samet.james@epa.gov

Supplemental Material is available online (http://dx.doi.org/10.1289/ehp.1206039).

The authors gratefully acknowledge the encouragement and support of W.-Y. Cheng, R. Silbajoris, and L. Dailey.

The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. The contents of this article should not be construed to represent agency policy, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

The authors declare they have no actual or potential competing financial interests.

Received 20 September 2012; Accepted 17 December 2012; Online 18 December 2012.