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.

The NIMH Research Portfolio: An Update.

Objective: To examine the funding priorities of the National Institute of Mental Health (NIMH) since 2016 to assess whether NIMH was continuing to prioritize basic research at the expense of clinical research.Methods: Six psychiatric disorders (schizophrenia, bipolar disorder, depression, anxiety disorders, eating disorders, autism) were assessed using 2 publicly available data sources (ClinicalTrials.gov and the National Institutes of Health Research, Condition, and Disease Categorization [RCDC]) to determine the degree of NIMH support for drug trials and research on these disorders in general since 2016.Results: From 2017 through 2022, ClinicalTrials.gov lists just 1 drug trial each for schizophrenia and bipolar disorder. The RCDC database for 2016 through 2021 shows that NIMH support for research projects on schizophrenia and bipolar disorder decreased by 22% and 20%, respectively. During that time, Congress increased the budget of NIMH by 40%.Conclusions: NIMH has continued to prioritize basic research over clinical trials, resulting in a steep decline in funding for possible treatments for the most serious and costly psychiatric diseases.Prim Care Companion CNS Disord 2023;25(4):23m03486. Author affiliations are listed at the end of this article.

Liquid Application Dosing Alters the Physiology of Air-Liquid Interface Primary Bronchial Epithelial Cultures and In vitro Testing Relevant Endpoints.

Differentiated Primary human bronchial epithelial cell (dpHBEC) cultures grown under air-liquid interface (ALI) conditions exhibit key features of the human respiratory tract and are thus critical for respiratory research as well as efficacy and toxicity testing of inhaled substances (e.g., consumer products, industrial chemicals, and pharmaceuticals). Many inhalable substances (e.g., particles, aerosols, hydrophobic substances, reactive substances) have physiochemical properties that challenge their evaluation under ALI conditions in vitro. Evaluation of the effects of these methodologically challenging chemicals (MCCs) in vitro is typically conducted by "liquid application," involving the direct application of a solution containing the test substance to the apical, air-exposed surface of dpHBEC-ALI cultures. We report that the application of liquid to the apical surface of a dpHBEC-ALI co-culture model results in significant reprogramming of the dpHBEC transcriptome and biological pathway activity, alternative regulation of cellular signaling pathways, increased secretion of pro-inflammatory cytokines and growth factors, and decreased epithelial barrier integrity. Given the prevalence of liquid application in the delivery of test substances to ALI systems, understanding its effects provides critical infrastructure for the use of in vitro systems in respiratory research as well as in the safety and efficacy testing of inhalable substances.

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.

Mucus increases cell iron uptake to impact the release of pro-inflammatory mediators after particle exposure.

We tested the hypothesis that (1) mucus production can be included in the cell response to iron deficiency; (2) mucus binds iron and increases cell metal uptake; and subsequently (3) mucus impacts the inflammatory response to particle exposure. Using quantitative PCR, RNA for both MUC5B and MUC5AC in normal human bronchial epithelial (NHBE) cells decreased following exposures to ferric ammonium citrate (FAC). Incubation of mucus-containing material collected from the apical surface of NHBE cells grown at air-liquid interface (NHBE-MUC) and a commercially available mucin from porcine stomach (PORC-MUC) with iron demonstrated an in vitro capacity to bind metal. Inclusion of either NHBE-MUC or PORC-MUC in incubations of both BEAS-2B cells and THP1 cells increased iron uptake. Exposure to sugar acids (N-acetyl neuraminic acid, sodium alginate, sodium guluronate, and sodium hyaluronate) similarly increased cell iron uptake. Finally, increased metal transport associated with mucus was associated with a decreased release of interleukin-6 and -8, an anti-inflammatory effect, following silica exposure. We conclude that mucus production can be involved in the response to a functional iron deficiency following particle exposure and mucus can bind metal, increase cell uptake to subsequently diminish or reverse a functional iron deficiency and inflammatory response following particle exposure.

Liquid application dosing alters the physiology of air-liquid interface (ALI) primary human bronchial epithelial cell/lung fibroblast co-cultures and in vitro testing relevant endpoints.

Differentiated primary human bronchial epithelial cell (dpHBEC) cultures grown under air-liquid interface (ALI) conditions exhibit key features of the human respiratory tract and are thus critical for respiratory research as well as efficacy and toxicity testing of inhaled substances (e.g., consumer products, industrial chemicals, and pharmaceuticals). Many inhalable substances (e.g., particles, aerosols, hydrophobic substances, reactive substances) have physiochemical properties that challenge their evaluation under ALI conditions in vitro. Evaluation of the effects of these methodologically challenging chemicals (MCCs) in vitro is typically conducted by "liquid application," involving the direct application of a solution containing the test substance to the apical, air-exposed surface of dpHBEC-ALI cultures. We report that the application of liquid to the apical surface of a dpHBEC-ALI co-culture model results in significant reprogramming of the dpHBEC transcriptome and biological pathway activity, alternative regulation of cellular signaling pathways, increased secretion of pro-inflammatory cytokines and growth factors, and decreased epithelial barrier integrity. Given the prevalence of liquid application in the delivery of test substances to ALI systems, understanding its effects provides critical infrastructure for the use of in vitro systems in respiratory research as well as in the safety and efficacy testing of inhalable substances.

Lactate Production can Function to Increase Human Epithelial Cell Iron Concentration.

Introduction: Under conditions of limited iron availability, plants and microbes have evolved mechanisms to acquire iron. For example, metal deficiency stimulates reprogramming of carbon metabolism, increasing activity of enzymes involved in the Krebs cycle and the glycolytic pathway. Resultant carboxylates/hydroxycarboxylates then function as ligands to complex iron and facilitate solubilization and uptake, reversing the metal deficiency. Similarly, human intestinal epithelial cells may produce lactate, a hydroxycarboxylate, during absolute and functional iron deficiency to import metal to reverse limited availability. Methods: Here we investigate (1) if lactate can increase cell metal import of epithelial cells in vitro, (2) if lactate dehydrogenase (LDH) activity in and lactate production by epithelial cells correspond to metal availability, and (3) if blood concentrations of LDH in a human cohort correlate with indices of iron homeostasis. Results: Results show that exposures of human epithelial cells, Caco-2, to both sodium lactate and ferric ammonium citrate (FAC) increase metal import relative to FAC alone. Similarly, fumaric, isocitric, malic, and succinic acid coincubation with FAC increase iron import relative to FAC alone. Increased iron import following exposures to sodium lactate and FAC elevated both ferritin and metal associated with mitochondria. LDH did not change after exposure to deferoxamine but decreased with 24 h exposure to FAC. Lactate levels revealed decreased levels with FAC incubation. Review of the National Health and Nutrition Examination Survey demonstrated significant negative relationships between LDH concentrations and serum iron in human cohorts. Conclusions: Therefore, we conclude that iron import in human epithelial cells can involve lactate, LDH activity can reflect the availability of this metal, and blood LDH concentrations can correlate with indices of iron homeostasis.

Benchmark Dose Modeling Approaches for Volatile Organic Chemicals Using a Novel Air-Liquid Interface In Vitro Exposure System.

Inhalation is the most relevant route of volatile organic chemical (VOC) exposure; however, due to unique challenges posed by their chemical properties and poor solubility in aqueous solutions, in vitro chemical safety testing is predominantly performed using direct application dosing/submerged exposures. To address the difficulties in screening toxic effects of VOCs, our cell culture exposure system permits cells to be exposed to multiple concentrations at air-liquid interface (ALI) in a 24-well format. ALI exposure methods permit direct chemical-to-cell interaction with the test article at physiological conditions. In the present study, BEAS-2B and primary normal human bronchial epithelial cells (pHBEC) are used to assess gene expression, cytotoxicity, and cell viability responses to a variety of volatile chemicals including acrolein, formaldehyde, 1,3-butadiene, acetaldehyde, 1-bromopropane, carbon tetrachloride, dichloromethane, and trichloroethylene. BEAS-2B cells were exposed to all the test agents, whereas pHBECs were only exposed to the latter 4 listed above. The VOC concentrations tested elicited only slight cell viability changes in both cell types. Gene expression changes were analyzed using benchmark dose (BMD) modeling. The BMD for the most sensitive gene set was within one order of magnitude of the threshold-limit value reported by the American Conference of Governmental Industrial Hygienists, and the most sensitive gene sets impacted by exposure correlate to known adverse health effects recorded in epidemiologic and in vivo exposure studies. Overall, our study outlines a novel in vitro approach for evaluating molecular-based points-of-departure in human airway epithelial cell exposure to volatile chemicals.

Diacetyl exposure disrupts iron homeostasis in animals and cells.

OBJECTIVE: Several mechanisms have been proposed for the biological effect of diacetyl. We tested the postulate that animal and cell exposures to diacetyl are associated with a disruption in iron homeostasis. MATERIALS AND METHODS: Male, Sprague-Dawley rats were intratracheally-instilled with either distilled water or diacetyl. Seven days after treatment, animals were euthanized and the lungs removed, fixed, and embedded. Sections were cut and stained for iron, collagen, and ferritin. Human epithelial (BEAS-2B) and monocytic (THP-1) cells were exposed in vitro to ferric ammonium citrate (FAC), diacetyl, and both FAC and diacetyl. Cell non-heme iron concentrations and ferritin levels were quantified using inductively coupled plasma optical emission spectroscopy and an immunoassay respectively. RESULTS: After exposure of animals to diacetyl, there were airway polypoid lesions which stained positively for both iron and the intracellular storage protein ferritin. Trichrome stain showed a deposition of collagen immediately adjacent to accumulated metal following diacetyl exposure. In in vitro cell exposures, FAC increased non-heme iron concentration but co-incubations of FAC and diacetyl elevated levels to significantly greater values. Levels of ferritin were increased with exposures of BEAS-2B and THP-1 cells to FAC but were similarly greater after co-exposure with FAC and diacetyl. CONCLUSIONS: Results of animal and cell studies support a disruption of iron homeostasis by diacetyl. It is proposed that, following internalization, diacetyl complexes intracellular sources of iron. The cell recognizes a loss of its requisite iron to diacetyl and imports greater concentrations of the metal.

Ozone Responsive Gene Expression as a Model for Describing Repeat Exposure Response Trajectories and Interindividual Toxicodynamic Variability In Vitro.

Inhaled chemical/material exposures are a ubiquitous part of daily life around the world. There is a need to evaluate potential adverse effects of both single and repeat exposures for thousands of chemicals and an exponentially larger number of exposure scenarios (eg, repeated exposures). Meeting this challenge will require the development and use of in vitro new approach methodologies (NAMs); however, 2 major challenges face the deployment of NAMs in risk assessment are (1) characterizing what apical outcome(s) acute assays inform regarding the trajectory to long-term events, especially under repeated exposure conditions, and (2) capturing interindividual variability as it informs considerations of potentially susceptible and/or vulnerable populations. To address these questions, we used a primary human bronchial epithelial cell air-liquid interface model exposed to ozone (O3), a model oxidant and ubiquitous environmental chemical. Here we report that O3-induced proinflammatory gene induction is attenuated in repeated exposures thus demonstrating that single acute exposure outcomes do not reliably represent the trajectory of responses after repeated or chronic exposures. Further, we observed 10.1-, 10.3-, 14.2-, and 7-fold ranges of induction of interleukin (IL)-8, IL-6, heme oxygenase 1, and cyclooxygenase 2 transcripts, respectively, within in our population of 25 unique donors. Calculation of sample size estimates that indicated that 27, 24, 299, and 13 donors would be required to significantly power similar in vitro studies to identify a 2-fold change in IL-8, IL-6, HMOX1, and cyclooxygenase 2 transcript induction, respectively, to inform considerations of the uncertainty factors to reflect variability within the human population for in vitro studies.

Ozone Reacts With Carbon Black to Produce a Fulvic Acid-Like Substance and Increase an Inflammatory Effect.

Exposure to ambient ozone has been associated with increased human mortality. Ozone exposure can introduce oxygen-containing functional groups in particulate matter (PM) effecting a greater capacity of the particle for metal complexation and inflammatory effect. We tested the postulate that (1) a fulvic acid-like substance can be produced through a reaction of a carbonaceous particle with high concentrations of ozone and (2) such a fulvic acid-like substance included in the PM can initiate inflammatory effects following exposure of respiratory epithelial (BEAS-2B) cells and an animal model (male Wistar Kyoto rats). Carbon black (CB) was exposed for 72 hours to either filtered air (CB-Air) or approximately 100 ppm ozone (CB-O3). Carbon black exposure to high levels of ozone produced water-soluble, fluorescent organic material. Iron import by BEAS-2B cells at 4 and 24 hours was not induced by incubations with CB-Air but was increased following coexposures of CB-O3 with ferric ammonium citrate. In contrast to CB-Air, exposure of BEAS-2B cells and rats to CB-O3 for 24 hours increased expression of pro-inflammatory cytokines and lung injury, respectively. It is concluded that inflammatory effects of carbonaceous particles on cells can potentially result from (1) an inclusion of a fulvic acid-like substance after reaction with ozone and (2) changes in iron homeostasis following such exposure.

Supplementation with omega-3 fatty acids potentiates oxidative stress in human airway epithelial cells exposed to ozone.

BACKGROUND: Dietary intake of the omega-3 family of polyunsaturated fatty acids (ω-3 FA) is associated with anti-inflammatory effects. However, unsaturated fatty acids are susceptible to oxidation, which produces pro-inflammatory mediators. Ozone (O3) is a tropospheric pollutant that reacts rapidly with unsaturated fatty acids to produce electrophilic and oxidative mediators of inflammation. OBJECTIVE: Determine whether supplementation with ω-3 FA alters O3-induced oxidative stress in human airway epithelial cells (HAEC). METHODS: 16-HBE cells expressing a genetically encoded sensor of the reduced to oxidized glutathione ratio (GSH/GSSG, EGSH) were supplemented with saturated, monounsaturated, or ω-3 FA prior to exposure to 0, 0.08, 0.1, or 0.3 ppm O3. Lipid peroxidation was measured in cellular lipid extracts and intact cells following O3 exposure. RESULTS: Relative to cells incubated with the saturated or monounsaturated fatty acids, cells supplemented with ω-3 FA containing 5 or 6 double bonds showed a marked increase in EGSH during exposure to O3 concentrations as low as 0.08 ppm. Consistent with this finding, the concentration of lipid hydroperoxides produced following O3 exposure was significantly elevated in ω-3 FA supplemented cells. DISCUSSION: Supplementation with polyunsaturated ω-3 FA potentiates oxidative responses, as indicated by EGSH, in HAEC exposed to environmentally relevant concentrations of O3. This effect is mediated by the increased formation of lipid hydroperoxides produced by the reaction of O3 with polyunsaturated fatty acids. Given the inflammatory activity of lipid hydroperoxides, these findings have implications for the potential role of ω-3 FA in increasing human susceptibility to the adverse health effects of O3 exposure.

Air pollutants disrupt iron homeostasis to impact oxidant generation, biological effects, and tissue injury.

Air pollutants cause changes in iron homeostasis through: 1) a capacity of the pollutant, or a metabolite(s), to complex/chelate iron from pivotal sites in the cell or 2) an ability of the pollutant to displace iron from pivotal sites in the cell. Through either pathway of disruption in iron homeostasis, metal previously employed in essential cell processes is sequestered after air pollutant exposure. An absolute or functional cell iron deficiency results. If enough iron is lost or is otherwise not available within the cell, cell death ensues. However, prior to death, exposed cells will attempt to reverse the loss of requisite metal. This response of the cell includes increased expression of metal importers (e.g. divalent metal transporter 1). Oxidant generation after exposure to air pollutants includes superoxide production which functions in ferrireduction necessary for cell iron import. Activation of kinases and phosphatases and transcription factors and increased release of pro-inflammatory mediators also result from a cell iron deficiency, absolute or functional, after exposure to air pollutants. Finally, air pollutant exposure culminates in the development of inflammation and fibrosis which is a tissue response to the iron deficiency challenging cell survival. Following the response of increased expression of importers and ferrireduction, activation of kinases and phosphatases and transcription factors, release of pro-inflammatory mediators, and inflammation and fibrosis, cell iron is altered, and a new metal homeostasis is established. This new metal homeostasis includes increased total iron concentrations in cells with metal now at levels sufficient to meet requirements for continued function.

A new cell culture exposure system for studying the toxicity of volatile chemicals at the air-liquid interface.

A cell culture exposure system (CCES) was developed to expose cells established at an air-liquid interface (ALI) to volatile chemicals. We characterized the CCES by exposing indigo dye-impregnated filter inserts inside culture wells to 125 ppb ozone (O3) for 1 h at flow rates of 5 and 25 mL/min/well; the reaction of O3 with an indigo dye produces a fluorescent product. A 5-fold increase in fluorescence at 25 mL/min/well versus 5 mL/min/well was observed, suggesting higher flows were more effective. We then exposed primary human bronchial epithelial cells (HBECs) to 0.3 ppm acrolein for 2 h at 3, 5, and 25 mL/min/well and compared our results against well-established in vitro exposure chambers at the U.S. EPA's Human Studies Facility (HSF Chambers). We measured transcript changes of heme oxygenase-1 (HMOX1) and interleukin-8 (IL-8), as well as lactate dehydrogenase (LDH) release, at 0, 1, and 24 h post-exposure. Comparing responses from HSF Chambers to the CCES, differences were only observed at 1 h post-exposure for HMOX1. Here, the HSF Chamber produced a ∼6-fold increase while the CCES at 3 and 5 mL/min/well produced a ∼1.7-fold increase. Operating the CCES at 25 mL/min/well produced a ∼4.5-fold increase; slightly lower than the HSF Chamber. Our biological results, supported by our comparison against the HSF Chambers, agree with our fluorescence results, suggesting that higher flows through the CCES are more effective at delivering volatile chemicals to cells. This new CCES will be deployed to screen the toxicity of volatile chemicals in EPA's chemical inventories.

ERK1/2 and p38 regulate inter-individual variability in ozone-mediated IL-8 gene expression in primary human bronchial epithelial cells.

Inter-individual variability is observed in all biological responses; however this variability is difficult to model and its underlying mechanisms are often poorly understood. This issue currently impedes understanding the health effects of the air pollutant ozone. Ozone produces pulmonary inflammation that is highly variable between individuals; but reproducible within a single individual, indicating undefined susceptibility factors. Studying inter-individual variability is difficult with common experimental models, thus we used primary human bronchial epithelial cells (phBECs) collected from many different donors. These cells were cultured, exposed to ozone, and the gene expression of the pro-inflammatory cytokine IL-8 was measured. Similar to in vivo observations, we found that ozone-mediated IL-8 expression was variable between donors, but reproducible within a given donor. Recent evidence suggests that the MAP kinases ERK1/2 and p38 mediate ozone-induced IL-8 transcription, thus we hypothesized that differences in their activation may control IL-8 inter-individual variability. We observed a significant correlation between ERK1/2 phosphorylation and IL-8 expression, suggesting that ERK1/2 modulates the ozone-mediated IL-8 response; however, we found that simultaneous inhibition of both kinases was required to achieve the greatest IL-8 inhibition. We proposed a "dimmer switch" model to explain how the coordinate activity of these kinases regulate differential IL-8 induction.

The biological effect of asbestos exposure is dependent on changes in iron homeostasis.

Functional groups on the surface of fibrous silicates can complex iron. We tested the postulate that (1) asbestos complexes and sequesters host cell iron resulting in a disruption of metal homeostasis and (2) this loss of essential metal results in an oxidative stress and biological effect in respiratory epithelial cells. Exposure of BEAS-2B cells to 50 µg/mL chrysotile resulted in diminished concentrations of mitochondrial iron. Preincubation of these cells with 200 µM ferric ammonium citrate (FAC) prevented significant mitochondrial iron loss following the same exposure. The host response to chrysotile included increased expression of the importer divalent metal transporter-1 (DMT1) supporting a functional iron deficiency. Incubation of BEAS-2B cells with both 200 µM FAC and 50 µg/mL chrysotile was associated with a greater cell accumulation of iron relative to either iron or chrysotile alone reflecting increased import to correct metal deficiency immediately following fiber exposure. Cellular oxidant generation was elevated after chrysotile exposure and this signal was diminished by co-incubation with 200 µM FAC. Similarly, exposure of BEAS-2B cells to 50 µg/mL chrysotile was associated with release of the proinflammatory mediators interleukin (IL)-6 and IL-8, and these changes were diminished by co-incubation with 200 µM FAC. We conclude that (1) the biological response following exposure to chrysotile is associated with complexation and sequestration of cell iron and (2) increasing available iron in the cell diminished the effects of asbestos exposure.

Air pollution particles and iron homeostasis.

BACKGROUND: The mechanism underlying biological effects, including pro-inflammatory outcomes, of particles deposited in the lung has not been defined. MAJOR CONCLUSIONS: A disruption in iron homeostasis follows exposure of cells to all particulate matter including air pollution particles. Following endocytosis, functional groups at the surface of retained particle complex iron available in the cell. In response to a reduction in concentrations of requisite iron, a functional deficiency can result intracellularly. Superoxide production by the cell exposed to a particle increases ferrireduction which facilitates import of iron with the objective being the reversal of the metal deficiency. Failure to resolve the functional iron deficiency following cell exposure to particles activates kinases and transcription factors resulting in a release of inflammatory mediators and inflammation. Tissue injury is the end product of this disruption in iron homeostasis initiated by the particle exposure. Elevation of available iron to the cell precludes deficiency of the metal and either diminishes or eliminates biological effects. GENERAL SIGNIFICANCE: Recognition of the pathway for biological effects after particle exposure to involve a functional deficiency of iron suggests novel therapies such as metal supplementation (e.g. inhaled and oral). In addition, the demonstration of a shared mechanism of biological effects allows understanding the common clinical, physiological, and pathological presentation following exposure to disparate particles. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

Differential expression of pro-inflammatory and oxidative stress mediators induced by nitrogen dioxide and ozone in primary human bronchial epithelial cells.

CONTEXT: NO2 and O3 are ubiquitous air toxicants capable of inducing lung damage to the respiratory epithelium. Due to their oxidizing capabilities, these pollutants have been proposed to target specific biological pathways, but few publications have compared the pathways activated. OBJECTIVE: This work will test the premise that NO2 and O3 induce toxicity by activating similar cellular pathways. METHODS: Primary human bronchial epithelial cells (HBECs, n = 3 donors) were exposed for 2 h at an air-liquid interface to 3 ppm NO2, 0.75 ppm O3, or filtered air and harvested 1 h post-exposure. To give an overview of pathways that may be influenced by each exposure, gene expression was measured using PCR arrays for toxicity and oxidative stress. Based on the results, genes were selected to quantify whether expression changes were changed in a dose- and time-response manner using NO2 (1, 2, 3, or 5 ppm), O3 (0.25, 0.50, 0.75, or 1.00 ppm), or filtered air and harvesting 0, 1, 4 and 24 h post-exposure. RESULTS: Using the arrays, genes related to oxidative stress were highly induced with NO2 while expression of pro-inflammatory and vascular function genes was found subsequent to O3. NO2 elicited the greatest HMOX1 response, whereas O3 more greatly induced IL-6, IL-8 and PTGS2 expression. Additionally, O3 elicited a greater response 1 h post-exposure and NO2 produced a maximal response after 4 h. CONCLUSION: We have demonstrated that these two oxidant gases stimulate differing mechanistic responses in vitro and these responses occur at dissimilar times.

Baseline Chromatin Modification Levels May Predict Interindividual Variability in Ozone-Induced Gene Expression.

Traditional toxicological paradigms have relied on factors such as age, genotype, and disease status to explain variability in responsiveness to toxicant exposure; however, these are neither sufficient to faithfully identify differentially responsive individuals nor are they modifiable factors that can be leveraged to mitigate the exposure effects. Unlike these factors, the epigenome is dynamic and shaped by an individual's environment. We sought to determine whether baseline levels of specific chromatin modifications correlated with the interindividual variability in their ozone (O3)-mediated induction in an air-liquid interface model using primary human bronchial epithelial cells from a panel of 11 donors. We characterized the relationship between the baseline abundance of 6 epigenetic markers with established roles as key regulators of gene expression-histone H3 lysine 4 trimethylation (H3K4me3), H3K27 acetylation (H3K27ac), pan-acetyl H4 (H4ac), histone H3K27 di/trimethylation (H3K27me2/3), unmodified H3, and 5-hydroxymethylcytosine (5-hmC)-and the variability in the O3-induced expression of IL-8, IL-6, COX2, and HMOX1. Baseline levels of H3K4me3, H3K27me2/3, and 5-hmC, but not H3K27ac, H4ac, and total H3, correlated with the interindividual variability in O3-mediated induction of HMOX1 and COX2. In contrast, none of the chromatin modifications that we examined correlated with the induction of IL-8 and IL-6. From these findings, we propose an "epigenetic seed and soil" model in which chromatin modification states between individuals differ in the relative abundance of specific modifications (the "soil") that govern how receptive the gene is to toxicant-mediated cellular signals (the "seed") and thus regulate the magnitude of exposure-related gene induction.