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

Oxidant stress is arguably a universal feature in toxicology. Research studies on the role of oxidant stress induced by xenobiotic exposures have typically relied on the identification of damaged biomolecules using a variety of conventional biochemical and molecular techniques. However, there is increasing evidence that low-level exposure to a variety of toxicants dysregulates cellular physiology by interfering with redox-dependent processes

Src-Mediated EGF Receptor Activation Regulates Ozone-Induced Interleukin 8 Expression in Human Bronchial Epithelial Cells

Background:Human exposure to ozone (O3) results in pulmonary function decrements and airway inflammation. The mechanisms underlying these adverse effects remain unclear. Epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of lung inflammation.ObjectiveWe examined the role of EGFR activation in O3-induced expression of the chemokine interleukin 8 (IL-8) in human bronchial epithelial cells (HBEC).MethodsWe detected phosphorylated EGFR using immunoblotting. EGFR dimerization was examined through cross-linking reaction and immunoblotting, and levels of IL-8 protein were measured using ELISA.ResultsExposure to O3 (0.25–1.0 ppm) induced rapid and marked increase in EGFR phosphorylation at the autophosphorylation site Y1068 and the transphosphorylation site Y845, implicating the involvement of Src kinase. Further investigation showed that O3 stimulation induced phosphorylation of Src at Y416, indicative of Src activation. Pharmacological inhibition of Src kinase activity abrogated O3-induced EGFR phosphorylation at tyrosines 1068 and 845. Moreover, pretreatment of BEAS-2B cells with inhibitor of either EGFR or Src kinase activities significantly blocked O3-induced IL-8 expression.ConclusionConclusion: O3 exposure increased IL-8 expression through Src-mediated EGFR transactivation in HBEC.Citation>Wu W, Wages PA, Devlin RB, Diaz-Sanchez D, Peden DB, Samet JM. 2015. Src-mediated EGF receptor activation regulates ozone-induced interleukin 8 expression in human bronchial epithelial cells. Environ Health Perspect 123:231–236; http://dx.doi.org/10.1289/ehp.130737

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

Human exposure to particulate matter (PM) is a global environmental health concern. Zinc (Zn2+) is a ubiquitous respiratory toxicant that has been associated with PM health effects. However, the molecular mechanism of Zn2+ toxicity is not fully understood. H2O2 and Zn2+ have been shown to mediate signaling leading to adverse cellular responses in the lung and we have previously demonstrated Zn2+ to cause cellular H2O2 production. To determine the role of Zn2+-induced H2O2 production in the human airway epithelial cell response to Zn2+ exposure. BEAS-2B cells expressing the redox-sensitive fluorogenic sensors HyPer (H2O2) or roGFP2 (EGSH) in the cytosol or mitochondria were exposed to 50 µM Zn2+ for 5 min in the presence of 1 µM of the zinc ionophore pyrithione. Intracellular H2O2 levels were modulated using catalase expression either targeted to the cytosol or ectopically to the mitochondria. HO-1 mRNA expression was measured as a downstream marker of response to oxidative stress induced by Zn2+ exposure. Both cytosolic catalase overexpression and ectopic catalase expression in mitochondria were effective in ablating Zn2+-induced elevations in H2O2. Compartment-directed catalase expression blunted Zn2+-induced elevations in cytosolic EGSH and the increased expression of HO-1 mRNA levels. Zn2+ leads to multiple oxidative effects that are exerted through H2O2-dependent and independent mechanisms

Src-Mediated EGF Receptor Activation Regulates Ozone-Induced Interleukin 8 Expression in Human Bronchial Epithelial Cells

BACKGROUND: Human exposure to ozone (O(3)) results in pulmonary function decrements and airway inflammation. The mechanisms underlying these adverse effects remain unclear. Epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of lung inflammation. OBJECTIVE: We examined the role of EGFR activation in O(3)-induced expression of the chemokine interleukin 8 (IL-8) in human bronchial epithelial cells (HBEC). METHODS: We detected phosphorylated EGFR using immunoblotting. EGFR dimerization was examined through cross-linking reaction and immunoblotting, and levels of IL-8 protein were measured using ELISA. RESULTS: Exposure to O(3) (0.25–1.0 ppm) induced rapid and marked increase in EGFR phosphorylation at the autophosphorylation site Y1068 and the transphosphorylation site Y845, implicating the involvement of Src kinase. Further investigation showed that O(3) stimulation induced phosphorylation of Src at Y416, indicative of Src activation. Pharmacological inhibition of Src kinase activity abrogated O(3)-induced EGFR phosphorylation at tyrosines 1068 and 845. Moreover, pretreatment of BEAS-2B cells with inhibitor of either EGFR or Src kinase activities significantly blocked O(3)-induced IL-8 expression. CONCLUSION: Conclusion: O(3) exposure increased IL-8 expression through Src-mediated EGFR transactivation in HBEC. CITATION>: Wu W, Wages PA, Devlin RB, Diaz-Sanchez D, Peden DB, Samet JM. 2015. Src-mediated EGF receptor activation regulates ozone-induced interleukin 8 expression in human bronchial epithelial cells. Environ Health Perspect 123:231–236; http://dx.doi.org/10.1289/ehp.130737

Protein Sulfenylation: A Novel Readout of Environmental Oxidant Stress

Oxidative stress is a commonly cited mechanism of toxicity of environmental agents. Ubiquitous environmental chemicals such as the diesel exhaust component 1,2-naphthoquinone (1,2-NQ) induce oxidative stress by redox cycling, which generates hydrogen peroxide (H₂O₂). Cysteinyl thiolate residues on regulatory proteins are subjected to oxidative modification by H₂O₂ in physiological contexts and are also toxicological targets of oxidant stress induced by environmental contaminants. We investigated whether exposure to environmentally relevant concentrations of 1,2-NQ can induce H₂O₂-dependent oxidation of cysteinyl thiols in regulatory proteins as a readout of oxidant stress in human airway epithelial cells. BEAS-2B cells were exposed to 0–1000 μM 1,2-NQ for 0–30 min, and levels of H₂O₂ were measured by ratiometric spectrofluorometry of HyPer. H₂O₂-dependent protein sulfenylation was measured using immunohistochemistry, immunoblotting, and isotopic mass spectrometry. Catalase overexpression was used to investigate the relationship between H₂O₂ generation and protein sulfenylation in cells exposed to 1,2-NQ. Multiple experimental approaches showed that exposure to 1,2-NQ at concentrations as low as 3 μM induces H₂O₂-dependent protein sulfenylation in BEAS-2B cells. Moreover, the time of onset and duration of 1,2-NQ-induced sulfenylation of the regulatory proteins GAPDH and PTP1B showed significant differences. Oxidative modification of regulatory cysteinyl thiols in human lung cells exposed to relevant concentrations of an ambient air contaminant represents a novel marker of oxidative environmental stress

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

BACKGROUND: Oxidant stress is arguably a universal feature in toxicology. Research studies on the role of oxidant stress induced by xenobiotic exposures have typically relied on the identification of damaged biomolecules using a variety of conventional biochemical and molecular techniques. However, there is increasing evidence that low-level exposure to a variety of toxicants dysregulates cellular physiology by interfering with redox-dependent processes. SCOPE OF REVIEW: The study of events involved in redox toxicology requires methodology capable of detecting transient modifications at relatively low signal strength. This article reviews the advantages of live-cell imaging for redox toxicology studies. MAJOR CONCLUSIONS: Toxicological studies with xenobiotics of supra-physiological reactivity require careful consideration when using fluorogenic sensors in order to avoid potential artifacts and false negatives. Fortunately, experiments conducted for the purpose of validating the use of these sensors in toxicological applications often yield unexpected insights into the mechanisms through which xenobiotic exposure induces oxidant stress. GENERAL SIGNIFICANCE: Live-cell imaging using a new generation of small molecule and genetically encoded fluorophores with excellent sensitivity and specificity affords unprecedented spatiotemporal resolution that is optimal for redox toxicology studies. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu