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Despite recent advances in using biologicals that target Th2 pathways, glucocorticoids form the mainstay of asthma treatment. Asthma morbidity and mortality remain high due to the wide variability of treatment responsiveness and complex clinical phenotypes driven by distinct underlying mechanisms. Emerging evidence suggests that inhalation of the toxic air pollutant, ozone, worsens asthma by impairing glucocorticoid responsiveness. This review discusses the role of oxidative stress in glucocorticoid resistance in asthma. The underlying mechanisms point to a central role of oxidative stress pathways. The primary data source for this review consisted of peer-reviewed publications on the impact of ozone on airway inflammation and glucocorticoid responsiveness indexed in PubMed. Our main search strategy focused on cross-referencing "asthma and glucocorticoid resistance" against "ozone, oxidative stress, alarmins, innate lymphoid, NK and γδ T cells, dendritic cells and alveolar type II epithelial cells, glucocorticoid receptor and transcription factors". Recent work was placed in the context from articles in the last 10 years and older seminal research papers and comprehensive reviews. We excluded papers that did not focus on respiratory injury in the setting of oxidative stress. The pathways discussed here have however wide clinical implications to pathologies associated with inflammation and oxidative stress and in which glucocorticoid treatment is essential., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Enweasor, Flayer and Haczku.)

Introduction: The Multicenter Ozone Study of oldEr Subjects (MOSES) was a multi-center study evaluating whether short-term controlled exposure of older, healthy individuals to low levels of ozone (O 3 ) induced acute changes in cardiovascular biomarkers. In MOSES Part 1 (MOSES 1), controlled O 3 exposure caused concentration-related reductions in lung function with evidence of airway inflammation and injury, but without convincing evidence of effects on cardiovascular function. However, subjects' prior exposures to indoor and outdoor air pollution in the few hours and days before each MOSES controlled O 3 exposure may have independently affected the study biomarkers and/or modified biomarker responses to the MOSES controlled O 3 exposures., Methods: MOSES 1 was conducted at three clinical centers (University of California San Francisco, University of North Carolina, and University of Rochester Medical Center) and included healthy volunteers 55 to 70 years of age. Consented participants who successfully completed the screening and training sessions were enrolled in the study. All three clinical centers adhered to common standard operating procedures and used common tracking and data forms. Each subject was scheduled to participate in a total of 11 visits: screening visit, training visit, and three sets of exposure visits consisting of the pre-exposure day, the exposure day, and the post-exposure day. After completing the pre-exposure day, subjects spent the night in a nearby hotel. On exposure days, the subjects were exposed for 3 hours in random order to 0 ppb O 3 (clean air), 70 ppb O 3 , and 120 ppm O 3 . During the exposure period the subjects alternated between 15 minutes of moderate exercise and 15 minutes of rest. A suite of cardiovascular and pulmonary endpoints was measured on the day before, the day of, and up to 22 hours after each exposure., In MOSES Part 2 (MOSES 2), we used a longitudinal panel study design, cardiopulmonary biomarker data from MOSES 1, passive cumulative personal exposure samples (PES) of O 3 and nitrogen dioxide (NO 2 ) in the 72 hours before the pre-exposure visit, and hourly ambient air pollution and weather measurements in the 96 hours before the pre-exposure visit. We used mixed-effects linear regression and evaluated whether PES O 3 and NO 2 and these ambient pollutant concentrations in the 96 hours before the pre-exposure visit confounded the MOSES 1 controlled O 3 exposure effects on the pre- to post-exposure biomarker changes (Aim 1), whether they modified these pre- to post-exposure biomarker responses to the controlled O 3 exposures (Aim 2), whether they were associated with changes in biomarkers measured at the pre-exposure visit or morning of the exposure session (Aim 3), and whether they were associated with differences in the pre- to post-exposure biomarker changes independently of the controlled O 3 exposures (Aim 4)., Results: Ambient pollutant concentrations at each site were low and were regularly below the National Ambient Air Quality Standard levels. In Aim 1, the controlled O 3 exposure effects on the pre- to post-exposure biomarker differences were little changed when PES or ambient pollutant concentrations in the previous 96 hours were included in the model, suggesting these were not confounders of the controlled O 3 exposure/biomarker difference associations. In Aim 2, effects of MOSES controlled O 3 exposures on forced expiratory volume in 1 second (FEV 1 ) and forced vital capacity (FVC) were modified by ambient NO 2 and carbon monoxide (CO), and PES NO 2 , with reductions in FEV 1 and FVC observed only when these concentrations were "Medium" or "High" in the 72 hours before the pre-exposure visit. There was no such effect modification of the effect of controlled O 3 exposure on any other cardiopulmonary biomarker., As hypothesized for Aim 3, increased ambient O 3 concentrations were associated with decreased pre-exposure heart rate variability (HRV). For example, high frequency (HF) HRV decreased in association with increased ambient O 3 concentrations in the 96 hours before the pre-exposure visit (-0.460 ln[ms 2 ]; 95% CI, -0.743 to -0.177 for each 10.35-ppb increase in O 3 ; P = 0.002). However, in Aim 4 these increases in ambient O 3 were also associated with increases in HF and low frequency (LF) HRV from pre- to post-exposure, likely reflecting a "recovery" of HRV during the MOSES O 3 exposure sessions. Similar patterns across Aims 3 and 4 were observed for LF (the other primary HRV marker), and standard deviation of normal-to-normal sinus beat intervals (SDNN) and root mean square of successive differences in normal-to-normal sinus beat intervals (RMSSD) (secondary HRV markers)., Similar Aim 3 and Aim 4 patterns were observed for FEV 1 and FVC in association with increases in ambient PM with an aerodynamic diameter ≤ 2.5 μm (PM 2.5 ), CO, and NO 2 in the 96 hours before the pre-exposure visit. For Aim 3, small decreases in pre-exposure FEV 1 were significantly associated with interquartile range (IQR) increases in PM 2.5 concentrations in the 1 hour before the pre-exposure visit (-0.022 L; 95% CI, -0.037 to -0.006; P = 0.007), CO in the 3 hours before the pre-exposure visit (-0.046 L; 95% CI, -0.076 to -0.016; P = 0.003), and NO 2 in the 72 hours before the pre-exposure visit (-0.030 L; 95% CI, -0.052 to -0.008; P = 0.007). However, FEV 1 was not associated with ambient O 3 or sulfur dioxide (SO 2 ), or PES O 3 or NO 2 (Aim 3). For Aim 4, increased FEV 1 across the exposure session (post-exposure minus pre-exposure) was marginally significantly associated with each 4.1-ppb increase in PES O 3 concentration (0.010 L; 95% CI, 0.004 to 0.026; P = 0.010), as well as ambient PM 2.5 and CO at all lag times. FVC showed similar associations, with patterns of decreased pre-exposure FVC associated with increased PM 2.5 , CO, and NO 2 at most lag times, and increased FVC across the exposure session also associated with increased concentrations of the same pollutants, reflecting a similar recovery. However, increased pollutant concentrations were not associated with adverse changes in pre-exposure levels or pre- to post-exposure changes in biomarkers of cardiac repolarization, ST segment, vascular function, nitrotyrosine as a measure of oxidative stress, prothrombotic state, systemic inflammation, lung injury, or sputum polymorphonuclear leukocyte (PMN) percentage as a measure of airway inflammation., Conclusions: Our previous MOSES 1 findings of controlled O 3 exposure effects on pulmonary function, but not on any cardiovascular biomarker, were not confounded by ambient or personal O 3 or other pollutant exposures in the 96 and 72 hours before the pre-exposure visit. Further, these MOSES 1 O 3 effects were generally not modified, blunted, or lessened by these same ambient and personal pollutant exposures. However, the reductions in markers of pulmonary function by the MOSES 1 controlled O 3 exposure were modified by ambient NO 2 and CO, and PES NO 2 , with reductions observed only when these pollutant concentrations were elevated in the few hours and days before the pre-exposure visit. Increased ambient O 3 concentrations were associated with reduced HRV, with "recovery" during exposure visits. Increased ambient PM 2.5 , NO 2 , and CO were associated with reduced pulmonary function, independent of the MOSES-controlled O 3 exposures. Increased pollutant concentrations were not associated with pre-exposure or pre- to post-exposure changes in other cardiopulmonary biomarkers. Future controlled exposure studies should consider the effect of ambient pollutants on pre-exposure biomarker levels and whether ambient pollutants modify any health response to a controlled pollutant exposure., (© 2020 Health Effects Institute. All rights reserved.)

Ozone (O 3 ) is a major component of air pollution, which has been associated with airway inflammation characterized by the influx of neutrophils in asthmatic subjects. Canonical transient receptor potential 6 (TRPC6) channel is recently identified as a target of oxidative stress which is involved in airway inflammation. However, the regulatory role of TRPC6 in airway epithelial cells and neutrophils has not yet been illuminated in detail. In this study, we investigated the role of TRPC6 in neutrophil adhesion to airway epithelial cells exposed to O 3 in vivo and in vitro approaches. Using transgenic mice, the results showed that TRPC6-deficiency attenuated O 3 -induced neutrophil recruitment to airway epithelial cells and intercellular adhesion molecule-1 (ICAM-1) expression. In vitro, O 3 induced ICAM-1 expression and neutrophil adhesion to 16HBE cells (human airway epithelial cell line) and which were reduced by both TRPC6 silencing short hairpin RNA (shRNA) and TRPC6 inhibitor Larixyl Acetate (LA). We also confirmed that TRPC6-dependent Ca 2+ entry and NF-κB activation in 16HBE cells were required for ICAM-1-mediated neutrophil adhesion exposed to O 3 . In conclusion, this study demonstrated the contribution of TRPC6 to O 3 -induced neutrophil adhesion to airway epithelial cells via NF-κB activation and ICAM-1 expression, which may provide new potential concepts for preventing and treating air pollutant-related inflammatory lung diseases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In recent years, concerns over the issue of air pollution have increased as one of the significant environmental and health problems. Air pollutants can be toxic or harmful to the life of plants, animals, and humans. Contrast to primary pollutants, ozone is a secondary pollutant that is produced by the reaction between primary precursors in the atmosphere. The average of air pollutant data was compiled for the purpose of analyzing their correlation with the pulmonary function of students and the FENO biomarker from the air pollutants of the Environmental Protection Agency. According to the average of 3 days, the concentration of ozone in the (S 3 ) region was higher than the other regions, and this level was significantly different from the ANOVA test (p < 0.05). The results of artificial neural network modeling for three particular combinations in the cold season, two hidden layers with 9 and 12 neurons, with R 2  = 0.859 and in the warm season, layer with 13 neurons, with R 2  = 0.74, showed the best performance.

Increased adverse health effects in older subjects due to exposure to ambient air pollutants may be related to the inflammatory response induced by these contaminants. The aim of this study was to assess airway and systemic inflammatory responses in older healthy subjects to a controlled experimental exposure with spark-generated elemental carbon black ultrafine particles (cbUFPs) and ozone (O 3 ). Twenty healthy subjects, age 52-75 years, were exposed on three occasions separated by at least 8 weeks. The exposures to filtered air (FA), to cbUFP (50 μg/m 3 ), or to cbUFP in combination with 250 ppb ozone (cbUFP + O 3 ) for 3 h with intermittent exercise were performed double blind, and in random order. Sputum and blood samples were collected 3.5 h after each exposure. Exposure to cbUFP + O 3 significantly increased plasma club cell protein 16 (CC16), the number of sputum cells, the number and percent of sputum neutrophils, and sputum interleukin 6 and matrix metalloproteinase 9. Exposure to cbUFP alone exerted no marked effect, except for an elevation in sputum neutrophils in a subgroup of 13 subjects that displayed less than 65% sputum neutrophils after FA exposure. None of the inflammatory markers was correlated with age, and serum cardiovascular risk markers were not markedly affected by cbUFP or cbUFP + O 3 . Exposure to cbUFP+O 3 induced a significant airway and systemic inflammatory response in older healthy volunteer subjects. The effects induced by cbUFP alone suggest that the inflammation was predominantly mediated by O 3 , although one cannot rule out that the interaction of cbUFP and O 3 played a role.

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., (Copyright © 2016. Published by Elsevier B.V.)

Inhaled pollutants produce effects in virtually all organ systems in our body and have been linked to chronic diseases including hypertension, atherosclerosis, Alzheimer's and diabetes. A neurohormonal stress response (referred to here as a systemic response produced by activation of the sympathetic nervous system and hypothalamus-pituitary-adrenal (HPA)-axis) has been implicated in a variety of psychological and physical stresses, which involves immune and metabolic homeostatic mechanisms affecting all organs in the body. In this review, we provide new evidence for the involvement of this well-characterized neurohormonal stress response in mediating systemic and pulmonary effects of a prototypic air pollutant - ozone. A plethora of systemic metabolic and immune effects are induced in animals exposed to inhaled pollutants, which could result from increased circulating stress hormones. The release of adrenal-derived stress hormones in response to ozone exposure not only mediates systemic immune and metabolic responses, but by doing so, also modulates pulmonary injury and inflammation. With recurring pollutant exposures, these effects can contribute to multi-organ chronic conditions associated with air pollution. This review will cover, 1) the potential mechanisms by which air pollutants can initiate the relay of signals from respiratory tract to brain through trigeminal and vagus nerves, and activate stress responsive regions including hypothalamus; and 2) the contribution of sympathetic and HPA-axis activation in mediating systemic homeostatic metabolic and immune effects of ozone in various organs. The potential contribution of chronic environmental stress in cardiovascular, neurological, reproductive and metabolic diseases, and the knowledge gaps are also discussed. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu., (Published by Elsevier B.V.)

Exposure to elevated levels of ozone has been associated with a variety of respiratory-related health endpoints in both epidemiology and controlled human exposure studies, including lung function decrements and airway inflammation. A mode of action (MoA) for these effects has not been established, but it has been proposed that they may occur through ozone-induced activation of neural reflexes. We critically reviewed experimental studies of ozone exposure and neural reflex activation and applied the International Programme on Chemical Safety (IPCS) mode-of-action/human relevance framework to evaluate the biological plausibility and human relevance of this proposed MoA. Based on the currently available experimental data, we found that the proposed MoA of neural reflex activation is biologically plausible for the endpoint of ozone-induced lung function decrements at high ozone exposures, but further studies are needed to fill important data gaps regarding the relevance of this MoA at lower exposures. A role for the proposed MoA in ozone-induced airway inflammation is less plausible, as the evidence is conflicting and is also of unclear relevance given the lack of studies conducted at lower exposures. The evidence suggests a different MoA for ozone-induced inflammation that may still be linked to the key events in the proposed MoA, such that neural reflex activation may have some degree of involvement in modulating ozone-induced neutrophil influx, even if it is not a direct role.

We previously observed greater ozone-induced lung function decrements in obese than non-obese women. Animal models suggest that obesity enhances ozone-induced airway reactivity and inflammation. In a controlled exposure study, we compared the acute effect of randomized 0.4ppm ozone and air exposures (2 h with intermittent light exercise) in obese (N = 20) (30

Sensory effects in eyes and airways are common symptoms reported by aircraft crew and office workers. Neurological symptoms, such as headache, have also been reported. To assess the commonality and differences in exposures and health symptoms, a literature search of aircraft cabin and office air concentrations of non-reactive volatile organic compounds (VOCs) and ozone-initiated terpene reaction products were compiled and assessed. Data for tricresyl phosphates, in particular tri-ortho-cresyl phosphate (ToCP), were also compiled, as well as information on other risk factors such as low relative humidity. A conservative health risk assessment for eye, airway and neurological effects was undertaken based on a "worst-case scenario" which assumed a simultaneous constant exposure for 8h to identified maximum concentrations in aircraft and offices. This used guidelines and reference values for sensory irritation for eyes and upper airways and airflow limitation; a tolerable daily intake value was used for ToCP. The assessment involved the use of hazard quotients or indexes, defined as the summed ratio(s) (%) of compound concentration(s) divided by their guideline value(s). The concentration data suggest that, under the assumption of a conservative "worst-case scenario", aircraft air and office concentrations of the compounds in question are not likely to be associated with sensory symptoms in eyes and airways. This is supported by the fact that maximum concentrations are, in general, associated with infrequent incidents and brief exposures. Sensory symptoms, in particular in eyes, appear to be exacerbated by environmental and occupational conditions that differ in aircraft and offices, e.g., ozone incidents, low relative humidity, low cabin pressure, and visual display unit work. The data do not support airflow limitation effects. For ToCP, in view of the conservative approach adopted here and the rareness of reported incidents, the health risk of exposure to this compound in aircraft is considered negligible., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The ozone challenge model can be used to assess the efficacy of anti-inflammatory compounds in early phases of clinical drug development. PUR118, a calcium salt based formulation engineered in the iSPERSE(TM) dry powder delivery technology, is a novel anti-inflammatory drug for COPD. Here we evaluated the efficacy and safety of three doses of PUR118 in attenuating ozone-induced airway inflammation in healthy volunteers., Methods: In a single-blind, phase 1B proof of concept study, 24 subjects were enrolled to sequentially receive three doses of PUR118 (5.5 mg, n = 18; 11.0 mg, n = 18; 2.8 mg, n = 16). Each dose was inhaled 3 times (1, 13, 25 h, preceded by 2 puffs salbutamol) before the ozone exposure (250 ppb, 3 h intermittent exercise). Sputum was induced 3 h after the end of exposure., Results: Sputum neutrophils, sputum CD14+ cells, as well as concentrations of IL1B, IL6, IL8, MMP9, and TNFA in sputum supernatant significantly increased after ozone exposure (n = 24). The percentage of sputum neutrophils (n = 12 who completed all treatments) did not change following treatment with different doses of PUR118. The high dose treatment group (n = 16) showed a decrease in the percentage and number of sputum macrophages (p ≤ 0.05) as well as a decrease in blood neutrophils (p = 0.04), and an increase in blood CD14 + cells (p = 0.04) compared to baseline. All dosages of PUR118 were safe and well tolerated., Conclusion: Ozone challenge resulted in the expected and significant increase of sputum inflammatory parameters. Treatment with multiple rising doses of PUR118 was safe and three applications within 25 h prior to the ozone challenge had small effects on ozone-induced airway inflammation., Trial Registration: ClinicalTrials.gov: NCT01690949. Registered 12 September 2012.

Previous studies demonstrated that interleukin-1β (IL-1β) and nerve growth factor (NGF) increase synthesis of substance P (SP) in airway neurons both after ozone (O3) exposure and by direct application. It was postulated that NGF mediates O3-induced IL-1β effects on SP. The current study specifically focused on the influence of O3 on IL-1β, NGF, and SP levels in mice bronchoalveolar lavage fluid (BALF) and whether these mediators may be linked in an inflammatory-neuronal cascade in vivo. The findings showed that in vivo O3 exposure induced an increase of all three proteins in mouse BALF and that O3-induced elevations in both NGF and SP are mediated by the inflammatory cytokine IL-1β. Further, inhibition of NGF reduced O3 induced increases of SP in both the lung BALF and lung tissue, demonstrating NGF serves as a mediator of IL-1β effects on SP. These data indicate that IL-1β is an early mediator of O3-induced rise in NGF and subsequent SP release in mice in vivo.

Air pollution has been associated with many different diseases, such as cancer, and respiratory, cardiovascular, and cutaneous chronic diseases. These effects are enhanced in people exposed to combined air pollutants, such as ozone and cigarette smoke. Chronic exposure to these pollutants causes an increase in oxidative stress and inflammation and has been associated with an increase in pulmonary diseases and mortality. Clinical and epidemiological studies reported interindividual variability in the adverse health effects of air pollutants, suggesting a genetic predisposition. The identification of subgroups of the population who are particularly vulnerable to air pollution is, therefore, of importance. Mouse models are a useful tool for studying the mechanisms underlying different susceptibility, as they show differences in strain responses to both ozone and cigarette smoke. This review analyses the role of inflammation and the influence of genetic factors on the mechanisms of lung injury caused by ozone and cigarette smoke., (© 2012 New York Academy of Sciences.)

Repeated low-level indoor air exposure to volatile organic compounds (VOCs) may influence the reporting of sensory irritation in the eyes and airways. The ozone-initiated reaction products of limonene, an abundant VOC, were used as a model of indoor air mixtures to study upper airway (sensory) irritation, bronchoconstrictive and alveolar level effects after repeated exposures. Mice were exposed 1h/day for 10 consecutive days to: air, limonene (52 ppm/289 mg/m(3)); ozone (0.1 ppm/0.2mg/m(3)); a reaction mixture of limonene (52±8 ppm) and ozone (0.5, 2.5 and 3.9 ppm) resulting in ~0.05 ppm residual ozone. Neither the limonene nor the ozone exposures alone showed consistent effects on the respiratory parameters. In the limonene/ozone groups, the respiratory rate decreased concentration-dependently with an extrapolated no-effect-level of ~0.3 ppm admixed ozone. Both sensory irritation and airflow limitation were conspicuous effects of the mixtures; sensory irritation appeared rapidly and airflow limitation developed slowly during each exposure. The effects of these parameters did not change with increasing number of exposures. No firm conclusion could be drawn about alveolar level effects. Cells in bronchoalveolar lavage were unchanged irrespective of exposure to air, ozone, and limonene with and without ozone. In conclusion, the study indicated that repeated exposures to ozone-initiated limonene mixtures did not cause sensitization of sensory irritation and airflow limitation. Bronchoalveolar lavage after exposures to ozone, and limonene with and without ozone, respectively, did not show airway inflammation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Selectins, a family of cell adhesion molecules, are involved in the activation and extravasation of leukocytes in inflammatory diseases. Inhalation of ozone induces an inflammation of the airways, which is dominated by neutrophils. We investigated the effect of repeated inhalations of the pan-selectin antagonist Bimosiamose on ozone-induced airway inflammation in healthy volunteers. In a double-blind, placebo-controlled, randomized, cross-over study Bimosiamose (10 mg bid) was inhaled via a breath actuated nebulizer (AKITA2 APIXNEB(®)) for 4 days. Treatment was followed by inhalation of ozone (250 ppb) for 3 h with intermittent exercise. Induced sputum was collected 3 h post ozone challenge for analysis of cellular and non-cellular composition. 18 subjects were randomized and completed the study. All treatments were safe and well tolerated. Compared to placebo Bimosiamose reduced the numbers of sputum neutrophils by 40% (p = 0.068) and concentrations of interleukin-8 and matrix-metalloproteinase-9 in sputum supernatant by 35% (p = 0.004) and 46% (p = 0.022), respectively. Inhalation of Bimosiamose showed favourable anti-inflammatory effects on ozone-induced airway inflammation in healthy volunteers. Further studies have to proof and translate this anti-inflammatory effect of Bimosiamose into a clinical benefit in patients with chronic obstructive pulmonary disease. (ClinTrialgov Ident: NCT01108913)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Airway neural plasticity contributes to the process of airway remodeling in response to airway irritants. However, the mechanisms of neural remodeling in the airways during the early postnatal period, when responses to airway irritation may be most sensitive, have not been characterized. This study used a rat model to examine a possible mechanism of ozone (O(3))-induced neural hyperresponsiveness during a critical period of developmental, postnatal day (PD) 6, that may be mediated by the neurotrophin nerve growth factor (NGF), resulting in an enhanced release of inflammatory neuropeptide substance P (SP) from airway nerves. Rat pups between PD6-PD28 were killed 24 hours after exposure to O(3) (2 ppm, 3 hours) or filtered air (FA), to establish a timeline of NGF synthesis, or else they were exposed to O(3) or NGF on PD6 or PD21 and re-exposed to O(3) on PD28, and killed on PD29. Measurement endpoints included NGF mRNA in tracheal epithelial cells, NGF protein in bronchoalveolar lavage fluid, airway SP-nerve fiber density (NFD), and SP-positive airway neurons in vagal ganglia. Acute exposure to O(3) increased NGF in bronchoalveolar lavage fluid on PD10 and PD15, and mRNA expression in epithelial cells on PD6, compared with FA controls. NGF protein and mRNA expression in the O(3)-PD6/O(3)-PD28 groups were significantly higher than in the O(3)-PD21/O(3)-PD28 and O(3)-PD6/FA-PD28 groups. NGF-PD6/O(3)-PD28 increased the SP innervation of airway smooth muscle and SP-positive sensory neurons, compared with the NGF-PD21/O(3)-PD28 or NGF-PD6/FA-PD28 groups. NGF enhanced sensory innervation, which may mediate acute responses or prolong sensitivity to O(3) during early life. The model may be relevant in O(3) responses during early childhood.

Ozone (O(3)) and endotoxin are common environmental contaminants that cause asthma exacerbation. These pollutants have similar phenotype response characteristics, including induction of neutrophilic inflammation, changes in airway macrophage immunophenotypes, and ability to enhance response to inhaled allergen. Evoked phenotyping studies of volunteers exposed to O(3) and endotoxin were used to identify the response characteristics of volunteers to these pollutants. New studies support the hypotheses that similar innate immune and oxidant processes modulate response to these agents. These include TLR4 and inflammasome-mediated signaling and cytokine production. Innate immune responses are also impacted by oxidative stress. It is likely that continued discovery of common molecular processes which modulate response to these pollutants will occur. Understanding the pathways that modulate response to pollutants will also allow for discovery of genetic and epigenetic factors that regulate response to these pollutants and determine risk of disease exacerbation. Additionally, defining the mechanisms of response will allow rational selection of interventions to examine. Interventions focused on inhibition of Toll-like receptor 4 and inflammasome represent promising new approaches to preventing pollutant-induced asthma exacerbations. Such interventions include specific inhibitors of innate immunity and antioxidant therapies designed to counter the effects of pollutants on cell signaling., (© 2011 John Wiley & Sons A/S.)

Nonsteroidal anti-inflammatory drugs ameliorate pain and fever by inhibiting cyclooxygenase (COX) and suppressing prostanoid formation. Microsomal prostaglandin E synthase-1 (mPGES-1) catalyzes formation of PGE(2) from the COX product PGH(2) and has emerged as a therapeutic target. Inhibition of mPGES-1, however, renders the PGH(2) substrate available for diversion to other PG synthases. To address the possibility that substrate diversion augments formation of PGs that might modulate bronchial tone, we assessed the impact of mPGES-1 deletion in a mouse model of ozone-induced airway hyper-responsiveness. Ozone exposure increased total lung resistance to inhaled methacholine in wild-type mice. Deletion of mPGES-1 had little effect on total lung resistance in either naive or ozone-exposed animals. The carbachol-induced narrowing of luminal diameter in intrapulmonary airways of lung slices from acute ozone-exposed mice was also unaltered by mPGES-1 deletion. Likewise, although concentrations of PGE(2) were reduced in bronchoalveolar lavage fluid, whereas 6-keto-PGF(1alpha), PGD(2), and PGF(2alpha), all were increased, deletion of mPGES-1 failed to influence cell trafficking into the airways of either naive or ozone-exposed animals. Despite biochemical evidence of PGH(2) substrate diversion to potential bronchomodulator PGs, deletion of mPGES-1 had little effect on ozone-induced airway inflammation or airway hyper-responsiveness. Pharmacologically targeting mPGES-1 may not predispose patients at risk to airway dysfunction.

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.

Two physiotherapeutic complexes are proposed for the improvement of functional characteristics of the respiratory and cardiovascular systems that can be used for the non-medicamentous treatment of patients with cardiovascular diseases or concurrent broncho-pulmonary and cardiovascular pathologies. One complex is designed for the treatment of patients with asbestosis including that complicated by chronic obstructive bronchitis in the phase of remission with the well-apparent signs of myocardial electric instability. The use of the other complex is indicated to patients with chronic obstructive bronchitis in the phase of exacerbation and the signs of chronic pulmonary heart associated with cardiac dysrhythmia.

There are concerns about ozone-initiated chemistry, because the formation of gaseous oxidation products and ultrafine particles may increase complaints, morbidity and mortality. Here we address the question whether the gaseous products or the ultrafine particles from the ozone-initiated chemistry of limonene, a common and abundant indoor pollutant, cause acute airway effects. The effects on the airways by d-limonene, a ca. 16s old ozone/d-limonene mixture, and clean air were evaluated by a mice bioassay, from which sensory irritation of the upper airways, airflow limitation, and pulmonary irritation can be obtained. A denuder was inserted to separate the ultrafine particles from the gaseous products prior to the exposure chamber. Reduction of mean respiratory frequency (>30%) and 230% increase of time of brake were observed without denuder, during 30min exposure, to the ozonolyzed d-limonene mixture, which are indicative of prominent sensory effects. The initial concentrations (ppm) were 40 d-limonene and 4 ozone. The exposure concentrations (ppm) were about 35 d-limonene and 0.05 ozone. Formaldehyde and residual d-limonene, the salient sensory irritants, accounted for up to three-fourth of the sensory irritation. The upper airway effects reversed to baseline upon cessation of exposure. An effect on the conducting airways was also significant, which did not reverse completely upon cessation. Airway effects were absent with the denuder inserted, which did not alter the size distribution of ultrafine particles ( approximately 10mg/m(3)), significantly. The result was statistically indistinguishable from clean dry air. It is concluded that ultrafine particles that are generated from ozone-initiated d-limonene chemistry and denuded are not causative of sensory effects in the airways.

Objective: To investigate the potential effects of lifetime cumulative ozone (O3) exposure on acute pulmonary responses to O3., Methods: Fifteen healthy subjects from a larger cohort of young adults were exposed to 200 ppb O3 for 4 hours followed by bronchoscopy and bronchoalveolar lavage 18 hours later. Lung function, symptom questionnaires, and blood samples were obtained before and after each exposure. Subjects' lifetime cumulative O3 exposures were estimated from residential histories and air-quality monitoring data., Results: Acute exposure to O3 caused decrements in forced expiratory volume in 1 second (FEV1), maximal mid-expiratory flow rate (FEF25-75), and forced expiratory flow rate at 75% of forced vital capacity (FEF75), and an increase in plasma clara cell protein (CC16) level. Changes in CC16 and lower respiratory symptoms, but not in lung function, were positively correlated with lifetime cumulative O3 exposure., Conclusion: Higher lifetime cumulative O3 exposure was associated with airway injury and respiratory symptom responses, but not with airway inflammatory or lung function responses, to acute O3 exposure.

Objective: Recent studies indicate that the U.S. Environmental Protection Agency (EPA) ozone standards may not protect sensitive individuals. In this study we examined respiratory effects of ozone in infants who may be vulnerable, particularly if they are children of asthmatic mothers., Design: Women delivering babies at one of five hospitals in southwestern Virginia between 1994 and 1996 were invited to participate in a cohort study; 780 women enrolled. Ambient air quality data (ozone and particulate matter) were collected at a central monitoring site., Participants: This analysis is of 691 infants followed for approximately 83 days between 10 June and 31 August 1995 and/or 1996 ; they contributed a total of 52,421 infant-days of follow-up. Mothers were interviewed at enrollment and approximately biweekly to report infants' daily symptoms. Repeated measures logistic regression models were run separately for wheeze, difficulty breathing, and cough. Ozone metrics included 24-hr average, peak 1-hr, and maximum 8-hr average. Analyses were repeated for the 61 infants whose mothers had asthma., Results: For every interquartile-range increase in same-day 24-hr average ozone, likelihood of wheeze increased 37% [95% confidence interval (CI), 2-84%]. Among infants of asthmatic mothers, same-day 24-hr average ozone increased likelihood of wheeze 59% (95% CI, 1-154%) and of difficulty breathing 83% (95% CI, 42-136%). Maximum 8-hr ozone and peak 1-hr ozone were associated with difficulty breathing, but not wheeze, in infants of asthmatic mothers. Ozone was not associated with cough., Conclusions: At levels of ozone exposure near or below current U.S. EPA standards, infants are at increased risk of respiratory symptoms, particularly infants whose mothers have physician-diagnosed asthma.

Background: A single, acute exposure to ozone has been shown to modify the antioxidant defense mechanism in the respiratory tract., Objective: The aim of this study was to evaluate the effects of ozone exposure on antioxidant response in BALB/c mice., Methods: We measured enhanced pause of breathing (Penh) as a marker of airway obstruction using barometric whole-body plethysmography before and after ozone exposure [groups (n = 6): filtered air, 0.12 ppm, 0.5 ppm, 1 ppm, 2 ppm] for 3 h. Antioxidant levels were measured using high-performance liquid chromatography with electrochemical detection in bronchoalveolar lavage (BAL) fluid and lung tissue homogenates., Results: Malondialdehyde concentrations in lung tissue homogenates were significantly increased in the group exposed to 2-ppm ozone compared to the filtered air group. Uric acid and gamma-tocopherol concentrations in BAL fluid were significantly increased in the ozone exposure group compared to the filtered air group (p < 0.01). Uric acid concentrations were increased in a concentration-dependent manner according to ozone concentration to which the animals were exposed. Increases in Penh after ozone exposure were significantly higher in an ozone concentration-dependent manner. The proportion of neutrophils in BAL fluid was significantly higher in the group exposed to 2 ppm than in the filtered air and the group exposed to 0.12 ppm (p < 0.01, respectively). The level of ascorbate correlated with the level of gamma-tocopherol., Conclusion: These findings suggest that antioxidant responses may serve as a protective mechanism against a range of oxidants in BALB/c mice exposed to ozone., (Copyright (c) 2005 S. Karger AG, Basel.)

Ozone (O(3)), a common air pollutant, induces airway inflammation and airway hyperresponsiveness. In mice, the neutrophil chemokines KC and macrophage inflammatory protein-2 (MIP-2) are expressed in the lungs following O(3) exposure. The purpose of this study was to determine whether CXCR2, the receptor for these chemokines, is essential to O(3)-induced neutrophil recruitment, injury to lungs, and increases in respiratory system responsiveness to methacholine (MCh). O(3) exposure (1 ppm for 3 h) increased the number of neutrophils in the bronchoalveolar lavage fluid (BALF) of wild-type (BALB/c) and CXCR2-deficient mice. However, CXCR2-deficient mice had significantly fewer emigrated neutrophils than did wild-type mice. The numbers of neutrophils in the blood and concentrations of BALF KC and MIP-2 did not differ between genotypes. Together, these data suggest CXCR2 is essential for maximal chemokine-directed migration of neutrophils to the air spaces. In wild-type mice, O(3) exposure increased BALF epithelial cell numbers and total protein levels, two indirect measures of lung injury. In contrast, in CXCR2-deficient mice, the number of BALF epithelial cells was not increased by O(3) exposure. Responses to inhaled MCh were measured by whole body plethysmography using enhanced pause as the outcome indicator. O(3) exposure increased responses to inhaled MCh in both wild-type and CXCR2-deficient mice 3 h after O(3) exposure. However, at 24 h after exposure, responses to inhaled MCh were elevated in wild-type but not CXCR2-deficient mice. These results indicate CXCR2 is essential for maximal neutrophil recruitment, epithelial cell sloughing, and persistent increases in MCh responsiveness after an acute O(3) exposure.

Recently, we reported the formation of unidentified strong upper airway irritants in reaction mixtures of terpenes and ozone. The identified products included aldehydes, ketones and carboxylic acids. Here we report the effects of variation of reaction time, relative humidity and initial ozone concentration on irritant formation in a flow reaction system using R-(+)-limonene and isoprene. Upper airway irritation was measured in mice as reduction of the respiratory rate. For both substances maximum irritation was observed for low humidity (<2% RH)/short time (16-30 s) reaction mixtures, and both moderate humidity ( approximately 32% RH) and longer reaction times (60-90 s) resulted in significantly less irritation. These results suggest that some unidentified intermediates react with water vapor to give less irritating products. Irritation measured at four ozone concentrations (0.5, 1, 2 and 3.5 ppm) using low humidity/short time reaction conditions for limonene (50 ppm) and isoprene (500 ppm) revealed that at 0.5 ppm, irritation was at the same level as that for the pure terpenes, indicating that at 0.5 ppm ozone the combined irritant effect was near the no effect level for the product mixture.

Exercise during exposure to inhaled toxicants increases inhaled dose rate and alters dose distribution within the respiratory tract, and exercise has long been recognized as a critical exposure variable. While modern inhalation exposure studies with human subjects routinely use an exercise protocol, most investigations with animal models are performed with resting subjects. Animal inhalation exposures under exercising conditions frequently induce respiratory toxic effects well beyond that expected from the simple increase in ventilation dose rate. Different classes of irritants can induce different reflex breathing patterns, and small mammals have different capacities for hypothermic or hyperthermic responses to rest or exercise exposure. Respiratory tract injury and breathing pattern responses of rats to upper and lower airway irritants ozone and formaldehyde were measured under conditions of rest and exercise to illustrate the complexities of comparison to human exposures. In addition to the exercise effects on dose and dose distribution and the possibility of hypo- or hyperthermic responses, exercise enhancement of inhalation injury may also be related to a combination of toxicant-induced disruption of the pulmonary epithelial permeability barrier and exercise-induced pulmonary capillary stress failure.

The air pollutant ozone induces both airway inflammation and restrictions in lung function. These responses have been proposed to arise as a consequence of the oxidizing nature of ozone, depleting endogenous antioxidant defenses with ensuing tissue injury. In this study we examined the impact of an environmentally relevant ozone challenge on the antioxidant defenses present at the surface of the lung in two groups known to have profound differences in their antioxidant defense network: healthy control (HC) and mild asthmatic (MA) subjects. We hypothesized that baseline differences in antioxidant concentrations within the respiratory tract lining fluid (RTLF), as well as induced responses, would predict the magnitude of individual responsiveness. We observed a significant loss of ascorbate (ASC) from proximal (-45.1%, p <.01) and distal RTLFs (-11.7%, p <.05) in healthy subjects 6 h after the end of the ozone challenge. This was associated (Rs, -0.71, p <.01) with increased glutathione disulphide (GSSG) in these compartments (p =.01 and p <.05). Corresponding responses were not seen in asthmatics, where basal ASC concentrations were significantly lower (p <.01) and associated with elevated concentrations of GSSG (p <.05). In neither group was any evidence of lipid oxidation seen following ozone. Despite differences in antioxidant levels and response, the magnitude of ozone-induced neutrophilia (+20.6%, p <.01 [HC] vs. +15.2%, p =.01 [MA]) and decrements in FEV(1) (-8.0%, p <.01 [HC] vs. -3.2%, p <.05 [MA]) did not differ between the two groups. These data demonstrate significant differences between the interaction of ozone with RTLF antioxidants in MA and HC subjects. These responses and variations in basal antioxidant defense were not, however, useful predictive markers of group or individual responsiveness to ozone.

Tropospheric ozone is the major oxidizing component in photochemical smog and is one of the most pervasive problems to human health of the criteria air pollutants for which the National Ambient Air Quality Standards have been designated by the Clean Air Act. Although many adverse health effects of ozone exposure have been documented in both humans and laboratory animals, controversy surrounds the establishment and implementation of ozone standards set forth by the U.S. Environmental Protection Agency. Because people are commonly exposed to more than one air pollutant at a time, studies that examine coexposures to airborne materials may be more relevant for assessing their risks to human health. Airborne biogenic substances such as pollens, spores, and bacterial products are ubiquitous in the environment, and when inhaled can cause adverse respiratory symptoms. One such biogenic agent, bacterial endotoxin, is a potent stimulus of airway inflammation and is a ubiquitous airborne contaminant commonly found in domestic, agricultural, and industrial settings. Little is known about the interaction of exposures to biogenic substances and criteria air pollutants such as ozone. In the last few years we have performed a series of studies in rodents that examined the biologic responses of the respiratory epithelium after airway exposures to both endotoxin and ozone. When exposed to ozone (0.5 ppm 8 hr/day for 3 days), Fischer rats develop lesions in the nasal transitional epithelium, whereas intranasal instillation of endotoxin (20 microg) elicits epithelial lesions in the respiratory epithelium of the nose and conducting airways. Our studies were designed to examine how exposure to one toxicant may affect the airway epithelial lesions induced by the other toxicant. We investigated the potential role of acute inflammation in the enhancement of airway epithelial lesions after exposure of these two toxicants in neutrophil-sufficient and neutrophil-deficient rodents. A summary of these results indicates that epithelial and inflammatory responses to coexposure of these two pollutants are greater than those elicited by either agent alone. Interestingly, each toxicant enhances the epithelial alterations induced by the other. Furthermore, the synergistic effects elicited by coexposure to ozone and endotoxin are mediated partly by neutrophils. These studies provided some new insights into how inhaled co-pollutants interact to initiate and promote alterations of airway epithelium. Further studies with these and other air pollutants will help define their true risk to human health.

Genetically manipulated mice exhibiting altered innervation of the airways were used to examine the role of sensory nerves in ozone-induced lung inflammation. Transgenic mice expressing nerve growth factor (NGF) from the lung-specific Clara cell secretory protein (CCSP) promoter exhibit hyperinnervation of the airways by sympathetic and tachykinin-containing sensory nerve fibers. Mice carrying a mutation in the low-affinity NGF receptor (NGFR) gene possess deficits in sensory innervation. CCSP-NGF transgenic mice exhibited a twofold increase in the number of lung lavage neutrophil level whereas NGFR knockout mice exhibited a nearly 50% decrease in neutrophilic inflammation compared with wild-type mice 18 h after ozone inhalation. Treatment with neurokinin receptor antagonists reduced the level of neutrophilic inflammation in both wild-type and CCSP-NGF mice. Examination of lavage fluid cytokine concentrations revealed that 4 h after ozone exposure CCSP-NGF mice produced significantly higher amounts of the chemokine KC than wild-type mice exposed to ozone. The results of this study indicate that sensory nerves are important mediators of ozone-induced inflammation in mice.

A complex mixture of air pollutants is present in the ambient air in urban areas. People, animals, and vegetation are chronically and sequentially exposed to outdoor pollutants. The objective of this first of 2 studies is to evaluate by light and electron microscopy the lungs of Mexico City dogs and compare the results to those of 3 less polluted cities in MEXICO: One hundred fifty-two clinically healthy stray mongrel dogs (91 males/61 females), including 43 dogs from 3 less polluted cities, and 109 from southwest and northeast metropolitian Mexico City (SWMMC, NEMMC) were studied. Lungs of dogs living in Mexico City and Cuernavaca exhibited patchy chronic mononuclear cell infiltrates along with macrophages loaded with particulate matter (PM) surrounding the bronchiolar walls and extending into adjacent vascular structures; bronchiolar epithelial and smooth muscle hyperplasia, peribronchiolar fibrosis, microthrombi, and capillary and venule polymorphonuclear leukocytes (PMN) margination. Ultrafine PM was seen in alveolar type I and II cells, endothelial cells, interstitial macrophages (Mtheta), and intravascular Mtheta-like cells. Bronchoalveolar lavage showed significant numbers of alveolar macrophages undergoing proliferation. Exposure to complex mixtures of pollutants-predominantly particulate matter and ozone-is causing lung structural changes induced by the sustained inflammatory process and resulting in airway and vascular remodeling and altered repair. Cytokines released from both, circulating inflammatory and resident lung cells in response to endothelial and epithelial injury may be playing a role in the pathology described here. Deep concern exists for the potential of an increasing rise in lung diseases in child populations exposed to Mexico City's environment.

We examined the effect of tetracaine aerosol inhalation, a local anesthetic, on lung volume decrements, rapid shallow breathing, and subjective symptoms of breathing discomfort induced by the acute inhalation of 0.30 ppm ozone for 65 min in 22 ozone-sensitive healthy human subjects. After 50 min of ozone inhalation FEV(1) was reduced 24%, breathing frequency was increased 40%, tidal volume was decreased 31%, and total subjective symptom score was increased (71.2, compared with 3.8 for filtered air exposure). Inhalation of tetracaine aerosol resulted in marked reductions in ozone-induced subjective symptoms of throat tickle and/or irritation (92.1%), cough (78.5%), shortness of breath (72.5%), and pain on deep inspiration (69.4%). In contrast, inhalation of tetracaine aerosol (mass median aerodynamic diameter of 3.52 microm with a geometric standard deviation of 1.92) resulted in only minor and inconsistent rectification of FEV(1) decrements (5.0%) and breathing frequency (-3.8%) that was not significantly different from that produced by saline aerosol alone (FEV(1), 5.1% and breathing frequency, -2.7%). Our data are consistent with afferent endings located within the large conducting airways of the tracheobronchial tree being primarily responsible for ozone-induced subjective symptoms and provides strong evidence that ozone-induced inhibition of maximal inspiratory effort is not dependent on conscious sensations of inspiratory discomfort.

The rat has been used extensively as a health sentinel, indicator, or monitor of environmental health hazards, but this model has not been directly validated against human exposures. Humans in Mexico City show upper respiratory tract lesions and evidence of pulmonary damage related to their environmental inhalation exposure. In this study, male and female F344 rats were exposed (23 hr/day) in Mexico City to local Mexico City air (MCA)* for up to seven weeks. Controls were maintained at the same location under filtered air. Prior to these exposures, several steps were taken. First, the nasal passages of normal male rats shipped from the United States and housed in Mexico City were examined for mycoplasma infection; no evidence of infection was found. In addition, a mobile exposure and monitoring system was assembled and, with an ozone (O3) exposure atmosphere, was tested along with supporting histopathology techniques and analysis of rat nasal and lung tissues. Last, the entire exposure model (equipment and animals) was transported to Mexico City and validated for a three-week period. During the seven-week study there were 18 one-hour intervals during which the average O3 concentration of MCA in the exposure chamber exceeded the US National Ambient Air Quality Standard (NAAQS) of 0.120 ppm 03 (hourly average, not to be exceeded more than once per year). This prolonged exposure of healthy F344 rats to MCA containing episodically low to moderate concentrations of 03 (as well as other urban air pollutants) did not induce inflammatory or epithelial lesions in the nasal airways or lung as measured by qualitative histologic techniques or quantitative morphometric techniques. These findings agree with those of previous controlled O3 inhalation studies, but they are in contrast to reports indicating that O3-polluted MCA causes significant nasal mucosal injury in adults and children living in southwestern Mexico City. Taken together, these findings may suggest that human airways are markedly more susceptible to the toxic effects of MCA than are the airways of the F344 rat.

We examined the effects of ozone (O(3)) and endogenous antioxidant transport on canine peripheral airway function, central airway function, epithelial integrity, and inflammation. Dogs were either untreated or pretreated with probenecid (an anion-transport inhibitor) and exposed for 6 h to 0.2 parts/million O(3). Peripheral airway resistance (Rpa) and reactivity (DeltaRpa) were monitored in three sublobar locations before and after exposure to either air or O(3). Pulmonary resistance and transepithelial potential difference in trachea and bronchus were also recorded. Bronchoalveolar lavage fluid (BALF) was collected before, during, and after exposure. O(3) increased Rpa and DeltaRpa only in probenecid-treated dogs and in a location-dependent fashion. Pulmonary resistance and potential difference in bronchus increased after O(3) exposure regardless of treatment. O(3) markedly increased BALF neutrophils only in untreated dogs. With the exception of hexanal, O(3) did not alter any BALF constituent examined. Probenecid reduced BALF ascorbate, BALF protein, and plasma urate. We conclude that 1) a 6-h exposure to 0.2 parts/million O(3) represents a subthreshold stimulus in relation to its effects on peripheral airway function in dogs, 2) antioxidant transport contributes to the maintenance of normal airway tone and reactivity under conditions of oxidant stress, 3) O(3)-induced changes in Rpa and DeltaRpa are dependent on location, and 4) peripheral airway hyperreactivity and inflammation reflect independent responses to O(3) exposure. Finally, although anion transport mitigates the effect of O(3) on peripheral airway function, it contributes to the development of airway inflammation and may represent a possible target for anti-inflammatory prevention or therapy.

Acute and subacute exposure to ozone (O3) induces lung inflammation and hyperpermeability and causes epithelial injury of both upper (nasal) and lower airways. Mast cells are important regulatory cells in mice for each of these effects. Subacute and chronic O3 exposures cause epithelial injury and inflammation in terminal bronchioles and proximal alveoli. Little is known, however, about the mechanisms of injury. Because inflammatory processes may be linked to the pathogenesis of many airway diseases, it is critical to understand the underlying mechanisms that initiate and propagate these processes. We tested the hypothesis that mast cells mediate airway injury induced by chronic O3 exposure by comparing regional airway inflammation and epithelial injury as well as ventilatory responses in genetically mast cell-deficient mice (WBB6F1-KitW/KitW-v [KitW/KitW-v]) with those in (1) normal, mast cell-sufficient, congenic littermates (WBB6F1(-)+/+ [+/+]) and those in (2) KitW/KitW-v mice that were repleted with mast cells by bone marrow transplantation (BMT) from +/+ donors (KitW/KitW-v-BMT). Thus, three (different) groups of mice were used. The following experimental protocol was utilized to test this hypothesis. Animals from each treatment group (n = 4-6/group) were exposed to 0.26 parts per million (ppm) O3 8 hours/day and 5 days/week for durations of 1, 3, 14, 30, and 90 days. Between 8-hour exposures, mice were exposed continuously to 0.06 ppm O3. Age-matched mice were simultaneously exposed to filtered air (0.0 ppm O3) to serve as O3 controls. To evaluate reversibility of exposure-induced lesions, a set of mice from each genotypic group was exposed to air or O3 for 90 days and then placed in HEPA-filtered air for 35 days. After each period of exposure and after 35-day recovery, the nasal cavity and lungs of O3- and air-exposed mice from each group were evaluated for regional inflammation and permeability, epithelial proliferation, and ventilation pattern. Estimates of airway inflammation and hyperpermeability were obtained by analysis of cell differentials and total protein concentrations, respectively, in fluids obtained through use of bronchoalveolar lavage (BAL). Ozone exposure caused significantly greater increases in lung macrophages, epithelial cells, and polymorphonuclear leukocytes (PMNs) in mast cell-sufficient +/+ and KitW/KitW-v-BMT mice than in mast cell-deficient KitW/KitW-v mice. Comparable ozone exposure also elicited increases in lung lymphocytes and in total protein, but there were no significant differences in these two genotypic groups. Cell and total-protein responses in BAL fluid returned to control levels (that is, air exposure only) in all three groups of mice after a 35-day recovery period. The effects of O3 exposure on cell proliferation in the nose and lung were evaluated in the genotypic groups by counting the number of cells that incorporated bromodeoxyuridine (BrdU, a thymidine analog) into DNA. In the centriacinar region of the lung, DNA synthesis was increased significantly in O3-exposed +/+ and KitW/KitW-v-BMT mice, but not in KitW/KitW-v mice, compared with DNA synthesis in air controls. Epithelial proliferation remained significantly elevated or even increased in +/+ and KitW/KitW-v-BMT mice after O3 exposure. Nasal responses to O3 were also evaluated in these three genotypic groups of mice, and there were slight, although statistically significant, O3-exposure effects on the transitional epithelium. However, there were no differences among the groups up to an exposure of 90 days in duration. After a 35-day recovery period, epithelial cell proliferation in +/+ and KitW/KitW-v-BMT mice was greater than that in KitW/KitW-v mice. There were no significant exposure, genotype, or duration effects on baseline ventilation or responses to hypercapnic hypoxia in the three groups of mice exposed to air or O3. (ABSTRACT TRUNCATED)

The influence of local exposure to ozone (O3) on respiratory epithelial permeability of sublobar lung segments was studied by using aerosolized 99mTc-diethylenetriamine pentaacetic acid (DTPA; mol wt, 492). Two bronchoscopes were inserted through an endotracheal tube in anesthetized, mechanically ventilated, mixed breed dogs and were wedged into sublobar bronchi located in the right and left lower lobes, respectively. Segments were ventilated via the bronchoscope with 5% CO2 in air delivered at 200 ml/min, and an aerosol of 99mTc-DTPA was generated and delivered through the scope and into the sublobar segment over a 30-s period. Clearance of 99mTc-DTPA was measured simultaneously from right and left lower lung segments at baseline and 1, 7, and 14 days after a 6-h sublobar exposure to filtered air or 400 parts per billion O3. O3 treatment significantly decreased the clearance halftime (t50) of 99mTc-DTPA by 50% from the baseline mean of 32.3 to 16.0 min at 1 day postexposure. After 7 days of recovery, t50 was still reduced by 28. 8%; however, by 14 days postexposure, clearance of 99mTc-DTPA had recovered, and the t50 had a mean value of 30.0 min. 99mTc-DTPA clearance was not altered by exposure to filtered air, and t50 values were comparable to baseline at 1, 7, and 14 days postexposure. These results reveal that a single local exposure to O3 increases transepithelial clearance, but only for epithelia directly exposed to O3, and that 7-14 days of recovery are required before permeability to small-molecular-weight solutes returns to normal.

Previous studies that used neonatal capsaicin (Cap) treatment to ablate C fibers indicate that C fibers act to inhibit lung damage and airway hyperresponsiveness after ozone (O3) exposure in rats. The purpose of this study was to determine 1) the role of tachykinins in these protective effects and 2) whether differences in minute ventilation (VE) during O3 exposure might account for the effect of Cap. In the first study, male Sprague-Dawley rats were exposed to 1 part/million O3 or air for 3 h. Four hours later, a bronchoalveolar lavage (BAL) was performed or airway responsiveness was measured. Rats were treated with CP-99994 and SR-48968, selective neurokinin-1- and -2-receptor antagonists, respectively, or with vehicle (Veh). O3 caused an increase in the number of neutrophils recovered from BAL fluid in both the Veh-treated and tachykinin-receptor antagonist (TKRA)-treated rats, but the number of neutrophils was approximately twofold greater in the TKRA-treated rats. In contrast, TKRA treatment had no effect on baseline pulmonary mechanics or airway responsiveness. After O3 exposure, the number of neutrophils in BAL fluid was also greater in Cap- than in Veh-treated rats. O3 reduced VE in both Veh- and Cap-treated rats, but the response was greater (reduction of 44.7 +/- 3.7 vs. 27.8 +/- 6.8%) and occurred earlier (10 vs. 70 min) in Cap- than in Veh-treated rats (P < 0.02). These results suggest that tachykinins mediate protective effects of C fibers against O3-induced lung inflammation. The results also indicate that the more pronounced effect of O3 on BAL neutrophils in Cap-treated rats is not the result of a greater inhaled dose of O3 resulting from greater VE.

Effects of low concentration of O3 on the respiratory system in mice were studied in the present study. Mice were exposed to 0.1, 0.25 and 1.0 mg/m3 of O3, respectively for 8 h per day for 3 days. Cilia in brochia, Lipid peroxide (LPO) in lung tissue and phagocytic function of macrophage in mice were examined. The results showed that there were cilia damage in the 1.0 mg/m3 group and swelling of mitochondrias as well as endoplasmic reticulums in the 0.25 mg/m3 group. But no damage was found in the group of 0.1 mg/m3. Furthermore, phagocytic rates in the 0.25 and 1.0 mg/m3 groups were significantly lower than that of control group (P < 0.01). No differences in LPO leads were found.

Although several lines of evidence have suggested that oxidizing agents can induce heat shock proteins (HSPs) in vitro, little is known about the induction of HSPs during in vivo exposure to oxidants. Guinea pigs were exposed to ozone for 6 h and euthanized up to 72 h later. Proteins from lavage cells and lung tissue were characterized by immunoblotting with 72- and 73/72-kDa HSP monoclonal antibodies. Although 73-kDa HSP was expressed constituitively in lung tissue, it was not affected by ozone. In contrast, 72-kDa HSP was significantly increased in lavage cells and lung tissue of animals exposed to 0.4 and 0.66 parts/million of ozone. Both heat treatment and arsenite induced 72-kDa HSP in cultured alveolar macrophages. The increase in 72-kDa HSP in the lavage cell pellet peaked at 24 h after ozone, whereas the influx of polymorphonuclear leukocytes peaked at 4 h. Examination of the induction of HSPs by ozone may provide clues to the development of ozone tolerance in humans and animals.

People are exposed to a mixture of chemicals, noise, bad smells and other factors. Little is known about the possible interactions between these various contaminants. Although research on the effects of chemical mixtures has been initiated, the combination of chemical and non-chemical factors has received little attention. This paper summarizes the possible effects of combined exposure to noise and the air pollutant ozone, which are both factors with a high prevalence in the Western society. A series of animal experiments are presented to evaluate the possible combined effects of noise and air pollution.

Common air pollutants (O3, SO2) exert their deleterious effects in several ways, mostly on the respiratory functions. Ozone causes formation of peroxides and aldehydes with subsequent release of inflammatory lipids and cytokines. Changes in the activity of neutral endopeptidase and release of neuropeptides may occur. As a result, bronchial hyperreactivity can be detected. In the mechanisms of the disturbances due to sulfur dioxide, cholinergic mechanisms and release of neuropeptides may be involved.

We investigated the time course and regional distribution of the expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells and the polymorphonuclear leukocyte (PMN) inflammatory response in the lung after acute exposure to ozone (O3). C57BL/6J mice were exposed to air or 2 ppm O3 for 3 h and killed immediately or 3, 6, 9, or 21 h after exposure. Expression of ICAM-1 was examined by immunohistochemical staining of frozen sections. PMN influx was evaluated by lavage and by histochemical staining of myeloperoxidase (MPO) and measurement of tissue MPO activity. ICAM-1 expression exhibited regional selectivity and temporal patterns that were unique to each region. Upregulation of ICAM-1 expression on the epithelial cells in the trachea, and to a lesser extent in the lobar and segmental bronchi, was observed 3-9 h after exposure and remained present at 21 h. Enhanced ICAM-1 expression in bronchioles and terminal bronchiole/alveolar duct regions was evident earlier (immediately to 3 h after exposure) but returned to baseline levels by 21 and 9 h, respectively. Maximal ICAM-1 expression and PMN influx in the lung parenchyma were concurrently observed at 3 h, followed by transepithelial migration of PMNs to the airway lumen. These results demonstrate regional variations in airway inflammatory activity and are supportive of the notion that upregulation of ICAM-1 on the airway epithelium may play a role in local regulation of PMN influx to the airways after acute O3 exposure.

In the present paper, we have reviewed experimental animal studies on the effects of the two most important oxidant airborne pollutants, nitrogen dioxide and ozone, on the respiratory system. The toxic effects depend on concentration and length of exposure, and are generally similar for both oxidants, with ozone operative at lower concentrations. High doses of both oxidants cause death due to lung oedema. Exposure to sublethal levels causes functional alterations such as airflow limitation and airway hyperresponsiveness to bronchoconstrictor stimuli. These effects, which are generally reversible, are associated with epithelial injury, oedema and airway and parenchymal infiltration by inflammatory cells. Loss of cilia of airway epithelium and necrosis of type I alveolar epithelial cells are the most prominent consequences at the epithelial level. Inflammation is characterized by early neutrophilic infiltration, followed by an increased number of mononuclear cells, predominantly alveolar macrophages. After long-term exposure, whilst nitrogen dioxide causes predominantly emphysema, ozone produces mainly pulmonary fibrosis. Biochemical effects include lipid peroxidation, increased antioxidant metabolism, and alteration of enzyme activity. Nitrogen dioxide and ozone may also alter the immunological response and reduce the defence against infections, increasing the susceptibility of exposed animals to infections.

Individuals with heightened airways reactivity, such as asthmatics, may be at risk to inflammatory effects of oxidant air pollutants. In the inbred mouse, significant interstrain variation in airways reactivity to acelycholine (ACh) and differential susceptibility to ozone (O3)-induced airways inflammation has been described previously. This study used these murine models to test hypotheses that (1) O3-induced hyperreactivity to ACh is a function of inherent baseline ACh reactivity, and (2) susceptibility to O3-induced inflammation is associated with O3-induced hyperreactivity. Strains (15-25 g, 6-8 weeks) with HYPERREACTIVE (DBA/2J, AKR/J, A/J), HYPOREACTIVE (C3H/HeJ, C57BL/6J, SJL/H), or INTERMEDIATE (129/J) phenotypes for ACh reactivity were exposed for 3 h to 2.0 ppm O3 or air (control). ACh reactivity (25 and 50 micrograms/kg, IV) was assessed 0 and 24 h after exposure. Relative to air controls, mean airways responses to 25 and 50 micrograms/kg ACh 24 h post-O3 increased significantly in the HYPERREACTIVE A/J strain (p < .05). Among HYPOREACTIVE strains, O3 significantly (p < .05) increased the response to 50 micrograms/kg ACh in C57BL/6J and SJL/J strains 24 h postexposure. A/J, C57BL/6J, and SJL/J mice are susceptible to O3-induced lung injury. O3 did not alter ACh reactivity in the other strains. O3 also did not affect airways reactivity to methacholine or carbachol, observations consistent with the hypothesis that O3-induced hyperreactivity to ACh may be due, in part, to O3 effects on cholinesterase function. Treatment of C57BL/6J and A/J mice with an immunosuppressant (cyclophosphamide) or an anti-PMN antibody significantly (p < .05) attenuated circulating and infiltrating polymorphonuclear leukocytes (PMNs), but did not affect O3-induced hyperreactivity. Therefore, O3-induced ACh hyperreactivity was not a function of baseline reactivity, but correlated with susceptibility to acute O3-induced airways injury and inflammation. Pharmacologic studies suggest that although PMNs were associated with O3-induced hyperreactivity, these cells were not the cause of the effect, and that these two events are not codependent.

In the present study, we asked whether platelet-activating factor (PAF) mediates the ozone-induced increase in airway microvascular leakage. To answer this question, we examined the effect of a PAF receptor antagonist on the ozone-induced increase in airway microvascular leakage quantified by the extravasation of Evans blue dye in the guinea pig trachea and main bronchi. Guinea pigs were pretreated with the PAF receptor antagonist, E6123 ((S)-(+)-6-(2-chlorophenyl)-3-cyclopropane-carbonyl-8,11-dimethyl-2,3,4, 5- tetrahydro-8H-pyrido[4',3':4,5]thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepine) (0.01, 0.1 and 1.0 mg/kg i.v.) and then exposed to 3 ppm ozone for 30 min. The PAF receptor antagonist significantly reduced the ozone-induced increase in microvascular leakage in a dose-dependent manner in both the trachea and main bronchi. Our results indicate that PAF mediates the ozone-induced increase in airway microvascular leakage. We therefore suggest that PAF may be involved in ozone-induced airway inflammation.

The purpose of this study was to evaluate the toxicity and potential carcinogenicity or cocarcinogenicity of ozone exposure in rats. Fischer-344/N (F-344/N) rats were exposed 6 hr/day, 5 days/wk, to 0, 0.12, 0.5, or 1.0 ppm ozone by inhalation for 2-yr and lifetime exposures. The cocarcinogenicity study included subcutaneous administration of 0, 0.1, or 1.0 mg/kg body weight of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and inhalation of 0 or 0.5 ppm ozone to male rats. NNK was administered by subcutaneous injections 3 times per week for the first 20 wk with ozone inhalation exposure. The ozone inhalation exposure was for 2 yr (104 wk), including the first 20 wk of NNK treatment and continuing for 84 wk after the last NNK injection. Ozone exposure caused a concentration-related increase in inflammation of the centriacinar region of the lung. There was also increased fibrosis and an extension of the bronchiolar epithelium in these centriacinar regions to involve the proximal alveoli. There was no increased incidence of neoplasms at any site, including the lung, that was associated with ozone exposure. Rats administered 1.0 mg/kg body weight NNK alone had an increased incidence of bronchiolar/alveolar neoplasms, but this effect was not enhanced by ozone exposure. Ozone exposure for 2 yr and lifetime was associated with site-specific toxic alterations in the nasal passage and lung similar to those previously described for short-term exposures. While there was significant attenuation of the pulmonary lesions as compared to short-term exposures, lesions persisted in the lifetime study and there was evidence of a mild progressive fibrosis. We conclude that under the conditions of these studies: (a) ozone exposure is not carcinogenic to either male or female F-344/N rats, (b) ozone does not enhance the incidence of pulmonary neoplasms in F-344/N rats exposed to a known pulmonary carcinogen (NNK), and (c) mild site-specific toxic lesions characteristic of ozone exposure persist in the nasal passage and lung throughout the lifetime of the rat with continued ozone exposure.