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 (E(GSH)), was used to monitor EGSH 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 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 O(3), and a Grx1-roGFP2 fusion protein responded more rapidly to O(3) exposure. Selenite-induced up-regulation of GPx (glutathione peroxidase) expression-enhanced roGFP2 responsiveness to O(3), suggesting that (hydro)peroxides are intermediates linking O(3) exposure to glutathione 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 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.

Figure 1

roGFP2 interactions with the glutathione system (adapted from Meyer and Dick 2010). Glutathione peroxidases (GPx) oxidize GSH to GSSG in response to peroxides, including H₂O₂ and lipid hydroperoxides (LOOHs), thus increasing the glutathione redox potential (E_GSH). Abbreviations: LOH, reduced lipid oxide; Se^–, reduced selenocyteine; SeOH, oxidized selenocyteine; SeSG, glutathionylated selenocysteine. In response to the increase in GSSG, one of the engineered vicinal cysteines of roGFP2 becomes S-glutathionylated by glutaredoxin (Grx). Glutathionylation in turn causes disulfide bond formation and alteration of the spectral properties of the GFP fluorophore. In the reductive pathway, Grx catalyzes the reduction of roGFP2 disulfide bonds through deglutathionylation as GSSG levels decrease and normal levels of GSH are reestablished by glutathione reductase (GR), at the expense of NADPH, causing a renormalization of E_GSH. Glucose and the pentose-phosphate pathway (PPP) create NADPH, which is used by GR to reduce GSSG to GSH.

Figure 2

Exposure to O₃ induces a dose- and time-dependent increase in the cytosolic E_GSH in airway epithelial cells. BEAS-2B cells expressing cytosolic roGFP2 were exposed to clean air for 5 min followed by a 0 (air control), 0.15, 0.25, or 0.50 ppm O₃ exposure for 35 min in a stage-top exposure system maintained at 37°C, > 90% relative humidity, and 5% CO₂. Shown are ratiometric values (404/488) calculated from the fluorescence intensity emitted at 510 nm induced by sequential excitation at 404 and 488 nm, plotted relative to the 5-min baseline. Addition of 0.1 mM H₂O₂ at the end of the O₃ exposure produced a maximal response, which was fully reversible with the addition of 10 mM DTT. The data shown were derived from three or more separate experiments monitoring seven or more cells in real time throughout the exposure period.

Figure 3

Glucose deprivation sensitizes cells to O₃-induced roGFP2 oxidation. Shown are the responses of seven BEAS-2B cells equilibrated in Locke solution containing 1 mg/mL glucose (LS + G: A), or 0 mg/mL (LS – G B). Cells were exposed to 0.5 ppm O₃; addition of 0.1 mM H₂O₂ at the end of the O₃ exposure produced a maximal response, which was fully reversible with the addition of 10 mM DTT.

Figure 4

Manipulation of the glutathione system modulates roGFP2 responses 0.5 ppm O₃ exposure as reported by BEAS-2B cells expressing either roGFP2 or Grx1-roGFP2. (B) BEAS-2B cells were pretreated with 100 µM 2-AAPA, a glutaredoxin inhibitor, before exposure to 0.5 ppm O₃; the responses shown are the normalized 404/488 ratios plotted relative to their established baseline. (C) BEAS-2B cells were pretreated with 1 µM sodium selenite for 48 hr before 0.5 ppm O₃ exposure. To facilitate comparison of the responses in (A) and (C), the normalized ratios were plotted as a percentage of the signals obtained at maximal oxidizing and reducing conditions achieved using 1 mM H₂O₂ and 10 mM DTT, respectively. Other experimental conditions were as described for Figure 2. Values shown are mean ± SE (n ≥ 3).

Figure 5

Comparison between roGFP2 and HyPer responses to O₃. BEAS-2B cells expressing either roGFP2 or the H₂O₂ sensor, HyPer, were exposed to 0.5 ppm O₃ as described for Figure 2. To facilitate comparison, the normalized ratios were plotted as a percentage of the signals at maximal oxidation and reduction achieved using 1 mM H₂O₂ and 10 mM DTT, respectively. Values shown are mean ± SE (n ≥ 3).

Figure 6

O₃-induced E_GSH changes affect the cytosol more rapidly than the mitochondrial matrix. BEAS-2B cells expressing roGFP2 targeted to either the cytosol or mitochondria were exposed to either 0.5 ppm (roGFP2-cyto) or 1.0 ppm (roGFP2-mito). For direct comparison, the normalized ratios were plotted as a percentage of the maximal oxidation achieved using 1 mM H₂O₂ and 10 mM DTT. Other experimental conditions were as described in Figure 2. Values shown are mean ± SE (n ≥ 3).