Monitoring redox stress in human airway epithelial cells exposed to woodsmoke at an air-liquid interface.

Wildland fires contribute significantly to the ambient air pollution burden worldwide, causing a range of adverse health effects in exposed populations. The toxicity of woodsmoke, a complex mixture of gases, volatile organic compounds, and particulate matter, is commonly studied in vitro using isolated exposures of conventionally cultured lung cells to either resuspended particulate matter or organic solvent extracts of smoke, leading to incomplete toxicity evaluations. This study aimed to improve our understanding of the effects of woodsmoke inhalation by building an advanced in vitro exposure system that emulates human exposure of the airway epithelium. We report the development and characterization of an innovative system that permits live-cell monitoring of the intracellular redox status of differentiated primary human bronchial epithelial cells cultured at an air-liquid interface (pHBEC-ALI) as they are exposed to unfractionated woodsmoke generated in a tube furnace in real time. pHBEC-ALI exposed to freshly generated woodsmoke showed oxidative changes that were dose-dependent and reversible, and not attributable to carbon monoxide exposure. These findings show the utility of this novel system for studying the molecular initiating events underlying woodsmoke-induced toxicity in a physiologically relevant in vitro model, and its potential to provide biological plausibility for risk assessment and public health measures.

Real-time redox adaptations in human airway epithelial cells exposed to isoprene hydroxy hydroperoxide.

While redox processes play a vital role in maintaining intracellular homeostasis by regulating critical signaling and metabolic pathways, supra-physiological or sustained oxidative stress can lead to adverse responses or cytotoxicity. Inhalation of ambient air pollutants such as particulate matter and secondary organic aerosols (SOA) induces oxidative stress in the respiratory tract through mechanisms that remain poorly understood. We investigated the effect of isoprene hydroxy hydroperoxide (ISOPOOH), an atmospheric oxidation product of vegetation-derived isoprene and a constituent of SOA, on intracellular redox homeostasis in cultured human airway epithelial cells (HAEC). We used high-resolution live cell imaging of HAEC expressing the genetically encoded ratiometric biosensors Grx1-roGFP2, iNAP1, or HyPer, to assess changes in the cytoplasmic ratio of oxidized glutathione to reduced glutathione (GSSG:GSH), and the flux of NADPH and H2O2, respectively. Non-cytotoxic exposure to ISOPOOH resulted in a dose-dependent increase of GSSG:GSH in HAEC that was markedly potentiated by prior glucose deprivation. ISOPOOH-induced increase in glutathione oxidation were accompanied by concomitant decreases in intracellular NADPH. Following ISOPOOH exposure, the introduction of glucose resulted in a rapid restoration of GSH and NADPH, while the glucose analog 2-deoxyglucose resulted in inefficient restoration of baseline GSH and NADPH. To elucidate bioenergetic adaptations involved in combatting ISOPOOH-induced oxidative stress we investigated the regulatory role of glucose-6-phosphate dehydrogenase (G6PD). A knockout of G6PD markedly impaired glucose-mediated recovery of GSSG:GSH but not NADPH. These findings reveal rapid redox adaptations involved in the cellular response to ISOPOOH and provide a live view of the dynamic regulation of redox homeostasis in human airway cells as they are exposed to environmental oxidants.

Improvement in Fitted Filtration Efficiency of N95 Respirators With Escalating Instruction of the Wearer.

Introduction: Fitted filtration performance of an N95 respirator may benefit from differing levels of instructions. Methods: Using a modified Occupational Safety and Health Administration fit test protocol, we measured fitted filtration efficiency for an N95 respirator in 21 screened, healthy participants given 4 levels of escalating instruction: (1) uninstructed (baseline), (2) written/pictorial manufacturer instructions, (3) step-by-step video demonstration, and (4) staff instruction (visual inspection of respirator fit and verbal suggestions to adjust when applicable). Results: Baseline fitted filtration efficiency was not significantly different between participants with or without previous experience of N95 use. Clear improvements in fitted filtration efficiency were observed progressing from baseline (average=86.1%) to manufacturer paper instructions (93.3%), video instructions (97.5%), and post staff intervention (98.3%). Baseline fitted filtration efficiency values were significantly lower than those after video instruction (p<0.037) and staff intervention (p<0.033) sessions. Conclusions: Beyond uninstructed wear or provision of manufacturer instructions, efforts to train and instruct users in proper respirator fit principles with visual feedback are likely to yield benefits to public health outcomes in reducing respiratory exposure during air quality emergencies such as airborne viral outbreaks and wildland fires.

Live cell imaging of oxidative stress in human airway epithelial cells exposed to isoprene hydroxyhydroperoxide.

Exposure to respirable air particulate matter (PM2.5) in ambient air is associated with morbidity and premature deaths. A major source of PM2.5 is the photooxidation of volatile plant-produced organic compounds such as isoprene. Photochemical oxidation of isoprene leads to the formation of hydroperoxides, environmental oxidants that lead to inflammatory (IL-8) and adaptive (HMOX1) gene expression in human airway epithelial cells (HAEC). To examine the mechanism through which these oxidants alter intracellular redox balance, we used live-cell imaging to monitor the effects of isoprene hydroxyhydroperoxides (ISOPOOH) in HAEC expressing roGFP2, a sensor of the glutathione redox potential (EGSH). Non-cytotoxic exposure of HAEC to ISOPOOH resulted in a rapid and robust increase in EGSH that was independent of the generation of intracellular or extracellular hydrogen peroxide. Our results point to oxidation of GSH through the redox relay initiated by glutathione peroxidase 4, directly by ISOPOOH or indirectly by ISOPOOH-generated lipid hydroperoxides. We did not find evidence for involvement of peroxiredoxin 6. Supplementation of HAEC with polyunsaturated fatty acids enhanced ISOPOOH-induced glutathione oxidation, providing additional evidence that ISOPOOH initiates lipid peroxidation of cellular membranes. These findings demonstrate that ISOPOOH is a potent environmental airborne hydroperoxide with the potential to contribute to oxidative burden of human airway posed by inhalation of secondary organic aerosols.

Non-redox cycling mechanisms of oxidative stress induced by PM metals.

Metallic compounds contribute to the oxidative stress of ambient particulate matter (PM) exposure. The toxicity of redox inert ions of cadmium, mercury, lead and zinc, as well as redox-active ions of vanadium and chromium is underlain by dysregulation of mitochondrial function and loss of signaling quiescence. Central to the initiation of these effects is the interaction of metal ions with cysteinyl thiols on glutathione and key regulatory proteins, which leads to impaired mitochondrial electron transport and persistent pan-activation of signal transduction pathways. The mitochondrial and signaling effects are linked by the production of H2O2, generated from mitochondrial superoxide anion or through the activation of NADPH oxidase, which extends the range and amplifies the magnitude of the oxidative effects of the metals. This oxidative burden can be further potentiated by inhibitory effects of the metals on the enzymes of the glutathione and thioredoxin systems. Along with the better-known Fenton-based mechanisms, the non-redox cycling mechanisms of oxidative stress induced by metals constitute significant pathways for cellular injury induced by PM inhalation.