Cardiovascular health impacts of wildfire smoke exposure.

In recent years, wildland fires have occurred more frequently and with increased intensity in many fire-prone areas. In addition to the direct life and economic losses attributable to wildfires, the emitted smoke is a major contributor to ambient air pollution, leading to significant public health impacts. Wildfire smoke is a complex mixture of particulate matter (PM), gases such as carbon monoxide, nitrogen oxide, and volatile and semi-volatile organic compounds. PM from wildfire smoke has a high content of elemental carbon and organic carbon, with lesser amounts of metal compounds. Epidemiological studies have consistently found an association between exposure to wildfire smoke (typically monitored as the PM concentration) and increased respiratory morbidity and mortality. However, previous reviews of the health effects of wildfire smoke exposure have not established a conclusive link between wildfire smoke exposure and adverse cardiovascular effects. In this review, we systematically evaluate published epidemiological observations, controlled clinical exposure studies, and toxicological studies focusing on evidence of wildfire smoke exposure and cardiovascular effects, and identify knowledge gaps. Improving exposure assessment and identifying sensitive cardiovascular endpoints will serve to better understand the association between exposure to wildfire smoke and cardiovascular effects and the mechanisms involved. Similarly, filling the knowledge gaps identified in this review will better define adverse cardiovascular health effects of exposure to wildfire smoke, thus informing risk assessments and potentially leading to the development of targeted interventional strategies to mitigate the health impacts of wildfire smoke.

Macrophages from the upper and lower human respiratory tract are metabolically distinct.

The function and cell surface phenotype of lung macrophages vary within the respiratory tract. Alterations in the bioenergetic profile of macrophages may also be influenced by their location within the respiratory tract. This study sought to characterize the bioenergetic profile of macrophages sampled from different locations within the respiratory tract at baseline and in response to ex vivo xenobiotic challenge. Surface macrophages recovered from healthy volunteers by induced sputum and by bronchial and bronchoalveolar lavage were profiled using extracellular flux analyses. Oxygen consumption and extracellular acidification rates were measured at rest and after stimulation with lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), or 1,2-naphthoquinone (1,2-NQ). Oxygen consumption and extracellular acidification rates were highly correlated for all macrophage samples. Induced sputum macrophages had relatively higher oxygen consumption and extracellular acidification rates and were largely reliant on glycolysis. In contrast, bronchial fraction and bronchoalveolar macrophages depended more heavily on mitochondrial respiration. Bronchoalveolar macrophages showed elevated LPS-induced cytokine responses. Unlike their autologous peripheral blood monocytes, lung macrophages from any source did not display bioenergetic changes following LPS stimulation. The protein kinase C activator PMA did not affect mitochondrial respiration, whereas the air pollutant 1,2-NQ induced marked mitochondrial dysfunction in bronchoalveolar and bronchial fraction macrophages. The bioenergetic characteristics of macrophages from healthy individuals are dependent on their location within the respiratory tract. These findings establish a regional bioenergetic profile for macrophages from healthy human airways that serves as a reference for changes that occur in disease.

Investigating mitochondrial dysfunction in human lung cells exposed to redox-active PM components.

Exposure to ambient particulate matter (PM) causes cardiopulmonary morbidity and mortality through mechanisms that involve oxidative stress. 1,2-naphthoquinone (1,2-NQ) is a ubiquitous component of PM and a potent redox-active electrophile. We previously reported that 1,2-NQ increases mitochondrial H2O2 production through an unidentified mechanism. We sought to characterize the effects of 1,2-NQ exposure on mitochondrial respiration as a source of H2O2 in human airway epithelial cells. We measured the effects of acute exposure to 1,2-NQ on oxygen consumption rate (OCR) in the human bronchial epithelial cell line BEAS-2B and mitochondrial preparations using extracellular flux analysis. Complex-specific assays and NADPH depletion by glucose deprivation distinguished between mitochondrial and non-mitochondrial oxygen utilization. 1,2-NQ exposure of BEAS cells caused a rapid, marked dose-dependent increase in OCR that was independent of mitochondrial respiration, exceeded the OCR observed after mitochondrial uncoupling, and remained sensitive to NADPH depletion, implicating extra-mitochondrial redox cycling processes. Similar effects were observed with the environmentally relevant redox-cycling quinones 1,4-naphthoquinone and 9,10-phenanthrenequinone, but not with quinones that do not redox cycle, such as 1,4-benzoquinone. In mitochondrial preparations, 1,2-NQ caused a decrease in Complex I-linked substrate oxidation, suggesting impairment of pyruvate utilization or transport, a novel mechanism of mitochondrial inhibition by an environmental exposure. This study also highlights the methodological utility and challenges in the use of extracellular flux analysis to elucidate the mechanisms of action of redox-active electrophiles present in ambient air.

Mechanisms of the acute effects of inhaled ozone in humans.

Ambient air ozone (O3) is generated photochemically from oxides of nitrogen and volatile hydrocarbons. Inhaled O3 causes remarkably reversible acute lung function changes and inflammation. Approximately 80% of inhaled O3 is deposited on the airways. O3 reacts rapidly with CC double bonds in hydrophobic airway and alveolar surfactant-associated phospholipids and cholesterol. Resultant primary ozonides further react to generate bioactive hydrophilic products that also initiate lipid peroxidation leading to eicosanoids and isoprostanes of varying electrophilicity. Airway surface liquid ascorbate and urate also scavenge O3. Thus, inhaled O3 may not interact directly with epithelial cells. Acute O3-induced lung function changes are dominated by involuntary inhibition of inspiration (rather than bronchoconstriction), mediated by stimulation of intraepithelial nociceptive vagal C-fibers via activation of transient receptor potential (TRP) A1 cation channels by electrophile (e.g., 4-oxo-nonenal) adduction of TRPA1 thiolates enhanced by PGE2-stimulated sensitization. Acute O3-induced neutrophilic airways inflammation develops more slowly than the lung function changes. Surface macrophages and epithelial cells are involved in the activation of epithelial NFkB and generation of proinflammatory mediators such as IL-6, IL-8, TNFa, IL-1b, ICAM-1, E-selectin and PGE2. O3-induced partial depolymerization of hyaluronic acid and the release of peroxiredoxin-1 activate macrophage TLR4 while oxidative epithelial cell release of EGFR ligands such as TGFa or EGFR transactivation by activated Src may also be involved. The ability of lipid ozonation to generate potent electrophiles also provides pathways for Nrf2 activation and inhibition of canonical NFkB activation. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

GAPDH inhibition mediated by thiol oxidation in human airway epithelial cells exposed to an environmental peroxide.

Intracellular redox homeostasis in the airway epithelium is closely regulated through adaptive signaling and metabolic pathways. However, inhalational exposure to xenobiotic stressors such as secondary organic aerosols (SOA) can alter intracellular redox homeostasis. Isoprene hydroxy hydroperoxide (ISOPOOH), a ubiquitous volatile organic compound derived from the atmospheric photooxidation of biogenic isoprene, is a major contributor to SOA. We have previously demonstrated that exposure of human airway epithelial cells (HAEC) to ISOPOOH induces oxidative stress through multiple mechanisms including lipid peroxidation, glutathione oxidation, and alterations of glycolytic metabolism. Using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition to tag intracellular protein thiol oxidation, we demonstrate that exposure of HAEC to micromolar levels of ISOPOOH induces reversible oxidation of cysteinyl thiols in multiple intracellular proteins, including GAPDH, that was accompanied by a dose-dependent loss of GAPDH enzymatic activity. These results demonstrate that ISOPOOH induces an oxidative modification of intracellular proteins that results in loss of GAPDH activity, which ultimately impacts the dynamic regulation of the intracellular redox homeostatic landscape in HAEC.

Sequestration of mitochondrial iron by silica particle initiates a biological effect.

Inhalation of particulate matter has presented a challenge to human health for thousands of years. The underlying mechanism for biological effect following particle exposure is incompletely understood. We tested the postulate that particle sequestration of cell and mitochondrial iron is a pivotal event mediating oxidant generation and biological effect. In vitro exposure of human bronchial epithelial cells to silica reduced intracellular iron, which resulted in increases in both the importer divalent metal transporter 1 expression and metal uptake. Diminished mitochondrial (57)Fe concentrations following silica exposure confirmed particle sequestration of cell iron. Preincubation of cells with excess ferric ammonium citrate increased cell, nuclear, and mitochondrial metal concentrations and prevented significant iron loss from mitochondria following silica exposure. Cell and mitochondrial oxidant generation increased after silica incubation, but pretreatment with iron diminished this generation of reactive oxygen species. Silica exposure activated MAP kinases (ERK and p38) and altered the expression of transcription factors (nF-κB and NF-E2-related factor 2), proinflammatory cytokines (interleukin-8 and -6), and apoptotic proteins. All of these changes in indexes of biological effect were either diminished or inhibited by cell pretreatment with iron. Finally, percentage of neutrophils and total protein concentrations in an animal model instilled with silica were decreased by concurrent exposure to iron. We conclude that an initiating event in the response to particulate matter is a sequestration of cell and mitochondrial iron by endocytosed particle. The resultant oxidative stress and biological response after particle exposure are either diminished or inhibited by increasing the cell iron concentration.

Pulmonary responses in current smokers and ex-smokers following a two hour exposure at rest to clean air and fine ambient air particles.

BACKGROUND: Increased susceptibility of smokers to ambient PM may potentially promote development of COPD and accelerate already present disease. OBJECTIVES: To characterize the acute and subacute lung function response and inflammatory effects of controlled chamber exposure to concentrated ambient fine particles (CAFP) with MMAD ≤ 2.5 microns in ex-smokers and lifetime smokers. METHODS: Eleven subjects, aged 35-74 years, came to the laboratory 5 times; a training day and two exposure days separated by at least 3 weeks, each with a post-exposure visit 22 h later. Double-blind and counterbalanced exposures to "clean air" (mean 1.5 ± 0.6 µg/m3) or CAFP (mean 108.7 ± 24.8 µg/m3 ) lasted 2 h with subjects at rest. RESULTS: At 3 h post-exposure subjects' DTPA clearance half-time significantly increased by 6.3 min per 100 µg/m3 of CAFP relative to "clean air". At 22 h post-exposure they showed significant reduction of 4.3% per 100 µg/m3 in FEV1 and a significant DLCO decrease by 11.1% per 100 µg/m3 of CAFP relative to "clean air". At both 3 h and 22 h the HDL cholesterol level significantly decreased by 4.5% and 4.1%, respectively. Other blood chemistries and markers of lung injury, inflammation and procoagulant activity were within the normal range of values at any condition. CONCLUSIONS: The results suggest that an acute 2 h resting exposure of smokers and ex-smokers to fine ambient particulate matter may transiently affect pulmonary function (spirometry and DLCO) and increase DTPA clearance half-time. Except for a post exposure decrease in HDL no other markers of pulmonary inflammation, prothrombotic activity and lung injury were significantly affected under the conditions of exposure.

Correlative ultrastructural investigations of airway epithelium following experimental exposure to defined air pollutants and lifestyle exposure to tobacco smoke.

CONTEXT: Investigations of cell/molecular level effects of in vivo exposure of airway mucosa of experimental animals to common irritant gases have demonstrated structural and physiological changes reflective of breaches in epithelial barrier function, presence of inflammatory cell infiltrate and compromised ciliary function. These experimental animal studies provided useful perspectives of plausible, but more subtle pathologic outcomes having relevance to lifestyle exposure to gaseous environmental irritants including tobacco smoke. METHODS: Freeze-fracture technology was applied to ultrastructural examination of large airway epithelium, with appropriate controls, from guinea pigs exposed to ozone and of nasal mucosa of human subjects exposed to ozone or sulfur dioxide, and nasal mucosa of active smokers. RESULTS: We documented substantive membrane structural changes to tight junctional complexes and cilia as well as an infiltrate of neutrophils into the surface mucosal layer in exposed animals. These patterns also were evident but not as pervasive among human subjects acutely exposed experimentally to irritant gases and those chronically exposed by their lifestyle to tobacco smoke. DISCUSSION: Our intent was to characterize respiratory tract mucosal membrane disorganization associated with high level acute irritant exposures in an experimental animal model and to evaluate evidence of similar but perhaps more subtle pathologic change associated with lower level experimental or lifestyle exposures. Our studies demonstrate continuity, albeit subtle, of pathologic change from high dosage experimental animal exposure to low dosage human exposures. CONCLUSIONS: This study represents the first report of ultrastructural airway epithelial membrane anomalies associated with lifestyle exposure to tobacco smoke irritants.

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.

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.

Fish oil blunts lung function decrements induced by acute exposure to ozone in young healthy adults: A randomized trial.

BACKGROUND: Over one-third of the U.S. population is exposed to unsafe levels of ozone (O3). Dietary supplementation with fish oil (FO) or olive oil (OO) has shown protection against other air pollutants. This study evaluates potential cardiopulmonary benefits of FO or OO supplementation against acute O3 exposure in young healthy adults. METHODS: Forty-three participants (26 ± 4 years old; 47% female) were randomized to receive 3 g/day of FO, 3 g/day OO, or no supplementation (CTL) for 4 weeks prior to undergoing 2-hour exposures to filtered air and 300 ppb O3 with intermittent exercise on two consecutive days. Outcome measurements included spirometry, sputum neutrophil percentage, blood markers of inflammation, tissue injury and coagulation, vascular function, and heart rate variability. The effects of dietary supplementation and O3 on these outcomes were evaluated with linear mixed-effect models. RESULTS: Compared with filtered air, O3 exposure decreased FVC, FEV1, and FEV1/FVC immediately post exposure regardless of supplementation status. Relative to that in the CTL group, the lung function response to O3 exposure in the FO group was blunted, as evidenced by O3-induced decreases in FEV1 (Normalized CTL -0.40 ± 0.34 L, Normalized FO -0.21 ± 0.27 L) and FEV1/FVC (Normalized CTL -4.67 ± 5.0 %, Normalized FO -1.4 ± 3.18 %) values that were on average 48% and 70% smaller, respectively. Inflammatory responses measured in the sputum immediately post O3 exposure were not different among the three supplementation groups. Systolic blood pressure elevations 20-h post O3 exposure were blunted by OO supplementation. CONCLUSION: FO supplementation appears to offer protective effects against lung function decrements caused by acute O3 exposure in healthy adults.

Enhancement of systemic and sputum granulocyte response to inhaled endotoxin in people with the GSTM1 null genotype.

OBJECTIVE: To determine if the GSTM1 null genotype is a risk factor for increased inflammatory response to inhaled endotoxin. METHODS: 35 volunteers who had undergone inhalation challenge with a 20 000 endotoxin unit dose of Clinical Center Reference Endotoxin (CCRE) were genotyped for the GSTM1 null polymorphism. Parameters of airway and systemic inflammation observed before and after challenge were compared in GSTM1 null (n=17) and GSTM1 (n=18) sufficient volunteers. RESULTS: GSTM1 null volunteers had significantly increased circulating white blood cells (WBCs), polymorphonuclear neutrophils (PMNs), platelets and sputum PMNs (% sputum PMNs and PMNs/mg sputum) after CCRE challenge. GSTM1 sufficient volunteers had significant, but lower increases in circulating WBCs, PMNs and % sputum PMNs, and no increase in platelets or PMNs/mg sputum. Linear regression analysis adjusted for baseline values of the entire cohort revealed that the GSTM1 null genotype significantly increased circulating WBCs, platelets and % sputum PMNs after challenge. CONCLUSION: These data support the hypothesis that the GSTM1 null genotype is a risk factor for increased acute respiratory and systemic inflammatory response to inhaled CCRE. These data are consistent with other observations that the GSTM1 null genotype is associated with increased respiratory, systemic and cardiovascular effects linked to ambient air particulate matter exposure and indicate that the GSTM1 null genotype should be considered a risk factor for adverse health effects associated with exposure to environmental endotoxin.

Darkfield-confocal microscopy detection of nanoscale particle internalization by human lung cells.

BACKGROUND: Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm). Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM) for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane. RESULTS: Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal) and scattered transmitted light (Darkfield) along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO2 particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependent internalization process. CONCLUSIONS: DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron density. DF-CLSM is especially well suited to the task of establishing the spatial localization of nanoparticles within cells, a critical topic in nanotoxicology. This technique has advantages to 2D darkfield microscopy as it visualizes nanoparticles in 3D using confocal microscopy. Use of this technique should aid toxicological studies related to observation of NP interactions with biological endpoints at cellular and subcellular levels.

Atopic asthmatic subjects but not atopic subjects without asthma have enhanced inflammatory response to ozone.

BACKGROUND: Asthma is a known risk factor for acute ozone-associated respiratory disease. Ozone causes an immediate decrease in lung function and increased airway inflammation. The role of atopy and asthma in modulation of ozone-induced inflammation has not been determined. OBJECTIVE: We sought to determine whether atopic status modulates ozone response phenotypes in human subjects. METHODS: Fifty volunteers (25 healthy volunteers, 14 atopic nonasthmatic subjects, and 11 atopic asthmatic subjects not requiring maintenance therapy) underwent a 0.4-ppm ozone exposure protocol. Ozone response was determined based on changes in lung function and induced sputum composition, including airway inflammatory cell concentration, cell-surface markers, and cytokine and hyaluronic acid concentrations. RESULTS: All cohorts experienced similar decreases in lung function after ozone. Atopic and atopic asthmatic subjects had increased sputum neutrophil numbers and IL-8 levels after ozone exposure; values did not significantly change in healthy volunteers. After ozone exposure, atopic asthmatic subjects had significantly increased sputum IL-6 and IL-1beta levels and airway macrophage Toll-like receptor 4, Fc(epsilon)RI, and CD23 expression; values in healthy volunteers and atopic nonasthmatic subjects showed no significant change. Atopic asthmatic subjects had significantly decreased IL-10 levels at baseline compared with healthy volunteers; IL-10 levels did not significantly change in any group with ozone. All groups had similar levels of hyaluronic acid at baseline, with increased levels after ozone exposure in atopic and atopic asthmatic subjects. CONCLUSION: Atopic asthmatic subjects have increased airway inflammatory responses to ozone. Increased Toll-like receptor 4 expression suggests a potential pathway through which ozone generates the inflammatory response in allergic asthmatic subjects but not in atopic subjects without asthma.

Diesel particle-induced transcriptional expression of p21 involves activation of EGFR, Src, and Stat3.

Exposure to diesel exhaust particles (DEP) has been associated with adverse health outcomes such as inflammation, adjuvancy, and mutagenesis. However, the molecular mechanisms by which DEP inhalation exerts these effects are still largely unknown. We previously reported that exposure to DEP activates the transcription factor Stat3 in airway epithelial cells, a primary target cell of inhaled DEP. To elucidate the functional role of Stat3 activation in these cells, we investigated the function of Stat3 in DEP-induced expression of the p21 gene in the human bronchial epithelial cell line BEAS-2B. We report that DEP exposure induces increased levels of p21 mRNA and protein in a manner that is independent of p53 and Sp1 expression or DNA binding to the p21 gene. Using chromatin immunoprecipitation assays and expression of a dominant-negative Stat3 mutant, we show that activation of Stat3 and its binding to the p21 promoter are required for DEP-induced expression of p21, suggesting that Stat3 plays an essential role in the induction of p21 by DEP. Additional experiments demonstrated that activation of p21 gene expression is dependent on the activation of epidermal growth factor receptor and Src kinase activities. Finally, we provide evidence suggesting that DEP exposure can inhibit the proliferation of human bronchial epithelial cells, suggesting a functional role of p21 activation airway epithelial cells exposed to DEP.

Differential transcriptional regulation of IL-8 expression by human airway epithelial cells exposed to diesel exhaust particles.

Exposure to diesel exhaust particles (DEP) induces inflammatory signaling characterized by MAP kinase-mediated activation of NFkB and AP-1 in vitro and in bronchial biopsies obtained from human subjects exposed to DEP. NFkB and AP-1 activation results in the upregulation of genes involved in promoting inflammation in airway epithelial cells, a principal target of inhaled DEP. IL-8 is a proinflammatory chemokine expressed by the airway epithelium in response to environmental pollutants. The mechanism by which DEP exposure induces IL-8 expression is not well understood. In the current study, we sought to determine whether DEP with varying organic content induces IL-8 expression in lung epithelial cells, as well as, to develop a method to rapidly evaluate the upstream mechanism(s) by which DEP induces IL-8 expression. Exposure to DEP with varying organic content differentially induced IL-8 expression and IL-8 promoter activity human airway epithelial cells. Mutational analysis of the IL-8 promoter was also performed using recombinant human cell lines expressing reporters linked to the mutated promoters. Treatment with a low organic-containing DEP stimulated IL-8 expression by a mechanism that is predominantly NFkB-dependent. In contrast, exposure to high organic-containing DEP induced IL-8 expression independently of NFkB through a mechanism that requires AP-1 activity. Our study reveals that exposure to DEP of varying organic content induces proinflammatory gene expression through multiple specific mechanisms in human airway epithelial cells. The approaches used in the present study demonstrate the utility of a promoter-reporter assay ensemble for identifying transcriptional pathways activated by pollutant exposure.

Concentrated ambient ultrafine particle exposure induces cardiac changes in young healthy volunteers.

RATIONALE: Exposure to ambient ultrafine particles has been associated with cardiopulmonary toxicity and mortality. Adverse effects specifically linked to ultrafine particles include loss of sympathovagal balance and altered hemostasis. OBJECTIVES: To characterize the effects of acute exposure to ambient ultrafine particles in young healthy humans. METHODS: Nineteen healthy nonsmoking male and female subjects between the ages of 18 and 35 were exposed to filtered air or to an atmosphere in which captured ultrafine (<0.16 microm) particles were concentrated by a factor of up to 20-fold over ambient levels with the use of particle concentrators fitted with size-selective outlets (ultrafine concentrated ambient particles [UFCAPs]). Subjects underwent bronchoalveolar lavage 18 hours after each exposure. Cardiovascular endpoints measured included pulmonary function, clinical chemistry, and hematological parameters, as well as heart rate variability and repolarization indices. MEASUREMENTS AND MAIN RESULTS: Exposure to UFCAPs was statistically associated with an increase in frequency domain markers of heart rate variability, specifically indicative of elevated vagal input to the heart. Consistent with this finding were increases in the variance associated with the duration of the QT interval. In addition, UFCAP exposure resulted in a significant increase in blood levels of the fibrin degradation product D-dimer as well as a modest elevation in the inflammatory chemokine IL-8 recovered in the lavage fluid. CONCLUSIONS: These findings show mild inflammatory and prothrombic responses and are suggestive of alterations in cardiac repolarization induced by UFCAP inhalation.

Comparative airway inflammatory response of normal volunteers to ozone and lipopolysaccharide challenge.

Ozone and lipopolysaccharide (LPS) are environmental pollutants with adverse health effects noted in both healthy and asthmatic individuals. The authors and others have shown that inhalation of ozone and LPS both induce airway neutrophilia. Based on these similarities, the authors tested the hypothesis that common biological factors determine response to these two different agents. Fifteen healthy, nonasthmatic volunteers underwent a 0.4 part per million ozone exposure for 2 h while performing intermittent moderate exercise. These same subjects underwent an inhaled LPS challenge with 20,000 LPS units of Clinical Center Reference LPS, with a minimum of 1 month separating these two challenge sessions. Induced sputum was obtained 24 h before and 4-6 h after each exposure session. Sputum was assessed for total and differential cell counts and expression of cell surface proteins as measured by flow cytometry. Sputum supernatants were assayed for cytokine concentration. Both ozone and LPS challenge augmented sputum neutrophils and subjects' responses were significantly correlated (R = .73) with each other. Ozone had greater overall influence on cell surface proteins by modifying both monocytes (CD14, human leukocyte antigen [HLA]-DR, CD11b) and macrophages (CD11b, HLA-DR) versus LPS where CD14 and HLA-DR were modified only on monocytes. However, LPS significantly increased interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, with no significant increases seen after ozone challenge. Ozone and LPS exposure in healthy volunteers induce similar neutrophil responses in the airways; however, downstream activation of innate immune responses differ, suggesting that oxidant versus bacterial air pollutants may be mediated by different mechanisms.

Low-level ozone exposure induces airways inflammation and modifies cell surface phenotypes in healthy humans.

The effects of low-level ozone exposure (0.08 ppm) on pulmonary function in healthy young adults are well known; however, much less is known about the inflammatory and immunomodulatory effects of low-level ozone in the airways. Techniques such as induced sputum and flow cytometry make it possible to examine airways inflammatory responses and changes in immune cell surface phenotypes following low-level ozone exposure. The purpose of this study was to determine if exposure to 0.08 parts per million ozone for 6.6 h induces inflammation and modifies immune cell surface phenotypes in the airways of healthy adult subjects. Fifteen normal volunteers underwent an established 0.08 part per million ozone exposure protocol to characterize the effect of ozone on airways inflammation and immune cell surface phenotypes. Induced sputum and flow cytometry were used to assess these endpoints 24 h before and 18 h after exposure. The results showed that exposure to 0.08 ppm ozone for 6.6 h induced increased airway neutrophils, monocytes, and dendritic cells and modified the expression of CD14, HLA-DR, CD80, and CD86 on monocytes 18 h following exposure. Exposure to 0.08 parts per million ozone is associated with increased airways inflammation and promotion of antigen-presenting cell phenotypes 18 hours following exposure. These findings need to be replicated in a similar experiment that includes a control air exposure.

The glutathione-S-transferase Mu 1 null genotype modulates ozone-induced airway inflammation in human subjects.

BACKGROUND: The glutathione-S-transferase Mu 1 (GSTM1) null genotype has been reported to be a risk factor for acute respiratory disease associated with increases in ambient air ozone levels. Ozone is known to cause an immediate decrease in lung function and increased airway inflammation. However, it is not known whether GSTM1 modulates these ozone responses in vivo in human subjects. OBJECTIVE: The purpose of this study was to determine whether the GSTM1 null genotype modulates ozone responses in human subjects. METHODS: Thirty-five healthy volunteers were genotyped for the GSTM1 null mutation and underwent a standard ozone exposure protocol to determine whether lung function and inflammatory responses to ozone were different between the 19 GSTM1 wild type and 16 GSTM1 null volunteers. RESULTS: GSTM1 did not modulate lung function responses to acute ozone. Granulocyte influx 4 hours after challenge was similar between GSTM1 normal and null volunteers. However, GSTM1 null volunteers had significantly increased airway neutrophils 24 hours after challenge, as well as increased expression of HLA-DR on airway macrophages and dendritic cells. CONCLUSION: The GSTM1 null genotype is associated with increased airways inflammation 24 hours after ozone exposure, which is consistent with the lag time observed between increased ambient air ozone exposure and exacerbations of lung disease.