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Changes in Metabolites Present in Lung Lining Fluid Following Exposure of Humans to Ozone
Abstract
Controlled human exposure to the oxidant air pollutant ozone causes decrements in lung function and increased inflammation as evidenced by neutrophil influx into the lung and increased levels of proinflammatory cytokines in the airways. Here we describe a targeted metabolomics evaluation of human bronchioalveolar lavage fluid (BALF) following controlled in vivo exposure to ozone to gain greater insight into its pulmonary effects. In a two-arm cross-over study, each healthy adult human volunteer was randomly exposed to filtered air (FA) and to 0.3 ppm ozone for 2 hr while undergoing intermittent exercise with a minimum of 4 weeks between exposures. Bronchoscopy was performed and BALF obtained at 1 (n = 9) or 24 (n = 23) h post-exposure. Metabolites were detected using ultrahigh performance liquid chromatography-tandem mass spectroscopy. At 1-hour post-exposure, a total of 28 metabolites were differentially expressed (DE) (p < 0.05) following ozone exposure compared to FA-exposure. These changes were associated with increased glycolysis and antioxidant responses, suggesting a rapid increased energy utilization as part of the cellular response to oxidative stress. At 24-hour post-exposure, 41 metabolites were DE. Many of the changes were in amino acids and linked with enhanced proteolysis. Changes associated with increased lipid membrane turnover were also observed. These later-stage changes were consistent with ongoing repair of airway tissues. There were 1.37 times as many metabolites were differentially expressed at 24 hour compared to 1-hour post-exposure. The changes at 1 hour reflect responses to oxidative stress while the changes at 24 hour indicate a broader set of responses consistent with tissue repair. These results illustrate the ability of metabolomic analysis to identify mechanistic features of ozone toxicity and aspects of the subsequent tissue response.
... The perturbation of amino acid metabolism has been recognized as one of the potential pathophysiologic mechanisms of cardiopulmonary diseases (Atanasova and Reznikov, 2018;Tobias et al., 2018). Air pollution exposure was speculated to be associated with the metabolism of amino acids, which may cause perturbed metabolic signaling pathways and further affect cardiopulmonary health Liang et al., 2019;Mu et al., 2019;Cheng et al., 2018). ...
... Hence, the targeted analysis may be more suitable to provide specific and quantitative detections of amino acids for assessment of their associations with air pollution (Kelly and Fussell, 2020). However, evidence from human studies using targeted analysis is still very limited (Huang et al., 2018;Cheng et al., 2018). ...
The molecular mechanisms underlying the associations between air pollution exposure and adverse cardiopulmonary effects remain to be better understood. Altered amino acid metabolism may plays an important role in the development of cardiopulmonary diseases and may be perturbed by air pollution exposure. To test this hypothesized molecular mechanism, we conducted an association analysis from an existing intervention study to examine the relations of air pollution exposures with amino acids in 43 Chinese healthy adults. Plasma levels of amino acids were measured using a UPLC-QqQ-MS system. Time-weighted personal exposure to O3, PM2.5, NO2, and SO2 over four time windows, i.e., 12 h, 24 h, 1 week, and 2 weeks, were calculated using the measured indoor and outdoor concentrations coupled with the time-activity data for each participant. Linear mixed-effects models were used to estimate the associations between air pollutants at each exposure window and amino acids by controlling for potential confounders. We observed significant associations between exposures and plasma concentrations of amino acids, with the direction of associations varying by amino acid and air pollutant. While there is little evidence of associations for NO2 and SO2, the associations with amino acids were fairly pronounced for exposure to PM2.5 and O3. In particular, independent O3 (12- and 24-hour) associations were observed with changes in the amino acids that were related to the urea cycle, including aspartate, asparagine, glutamate, arginine, citrulline, and ornithine. Our findings indicated that air pollution may cause acute perturbation of amino acid metabolism, and that O3 and PM2.5 may affect the metabolism of amino acids in different pathways.
Main finding: Acute air pollution exposure might affect the perturbation of amino acid metabolism, and in particular, was associated with amino acids in relation to the urea cycle.
... Ozone exposure is associated with significant alterations in lung lipids and in lipid metabolism (Cheng et al., 2018;Shore, 2019). This is thought to be a consequence of increased lipid membrane turnover and altered surfactant recycling secondary to oxidative stress (Cheng et al., 2018). ...
... Ozone exposure is associated with significant alterations in lung lipids and in lipid metabolism (Cheng et al., 2018;Shore, 2019). This is thought to be a consequence of increased lipid membrane turnover and altered surfactant recycling secondary to oxidative stress (Cheng et al., 2018). As FXR regulates the transcription of multiple genes regulating lipid handling and metabolism, we speculated that loss of FXR would alter expression of these genes resulting in dysregulation of lung lipids, which is what we observed. ...
Inflammatory macrophages are known to contribute to ozone toxicity. Farnesoid X receptor (FXR) is a nuclear receptor involved in regulating bile acid and lipid homeostasis; it also exerts anti-inflammatory activity by suppressing macrophage NF-κB. Herein, we analyzed the role of FXR in regulating macrophage activation in the lung following ozone exposure. Treatment of wild-type (WT) mice with ozone (0.8 ppm, 3 h) resulted in increases in proinflammatory (F4/80+CD11c+CD11b+Ly6CHi) and anti-inflammatory (F4/80+CD11c+CD11b+Ly6CLo) macrophages in the lung. The accumulation of proinflammatory macrophages was increased in FXR-/- mice compared with WT mice; however, anti-inflammatory macrophage activation was blunted as reflected by reduced arginase and mannose receptor expression, a response correlated with decreased Nur77. This was associated with prolonged oxidative stress, as measured by 4-hydroxynonenal-modified proteins in the lung. Loss of FXR was accompanied by protracted increases in lung NF-κB activity and its target, inducible nitric oxide synthase in response to ozone. Levels of Tnf-α, Il-1β, Ccr2, Ccl2, Cx3cr1, and Cx3cl1 were also increased in lungs of FXR-/- relative to WT mice; conversely, genes regulating lipid homeostasis including Lxrα, Apoe, Vldlr, Abcg1, and Abca1 were downregulated, irrespective of ozone exposure. In FXR-/- mice, ozone caused an increase in total lung phospholipids, with no effect on SP-B or SP-D. Dyslipidemia was correlated with blunting of ozone-induced increases in positive end-expiratory pressure-dependent quasi-static pressure volume curves indicating a stiffer lung in FXR-/- mice. These findings identify FXR as a regulator of macrophage activation following ozone exposure suggesting that FXR ligands may be useful in mitigating inflammation and oxidative stress induced by pulmonary irritants.
... Metabolomics analysis of bronchoalveolar lavage fluid (BALF) from healthy subjects following a 2 h exposure to ozone (0.3 ppm) whilst undertaking light exercise could detect 28 differentially expressed metabolites after 1 h indicative of increased glycolysis and a feedback antioxidant response (65). In contrast, at 24 h post ozone exposure 41 differentially expressed metabolites were observed. ...
... In contrast, at 24 h post ozone exposure 41 differentially expressed metabolites were observed. These were associated with enhanced proteolysis and lipid membrane turnover indicating repair of airway tissues (65). Interestingly, whilst many human ozone exposure models include light exercise, the effect of ozone on exhaled oxidative stress markers were attenuated by physical activity in healthy adolescents (66). ...
Epidemiological and challenge studies in healthy subjects and in individuals with asthma highlight the health impact of environmental ozone even at levels considered safe. Acute ozone exposure in man results in sputum neutrophilia in 30% of subjects particularly young children, females, and those with ongoing cardiopulmonary disease. This may be associated with systemic inflammation although not in all cases. Chronic exposure amplifies these effects and can result in the formation of asthma-like symptoms and immunopathology. Asthmatic patients who respond to ozone (responders) induce a greater number of genes in bronchoalveolar (BAL) macrophages than healthy responders with up-regulation of inflammatory and immune pathways under the control of cytokines and chemokines and the enhanced expression of remodeling and repair programmes including those associated with protease imbalances and cell-cell adhesion. These pathways are under the control of several key transcription regulatory factors including nuclear factor (NF)-κB, anti-oxidant factors such as nuclear factor (erythroid-derived 2)-like 2 NRF2, the p38 mitogen activated protein kinase (MAPK), and priming of the immune system by up-regulating toll-like receptor (TLR) expression. Murine and cellular models of acute and chronic ozone exposure recapitulate the inflammatory effects seen in humans and enable the elucidation of key transcriptional pathways. These studies emphasize the importance of distinct transcriptional networks in driving the detrimental effects of ozone. Studies indicate the critical role of mediators including IL-1, IL-17, and IL-33 in driving ozone effects on airway inflammation, remodeling and hyperresponsiveness. Transcription analysis and proof of mechanisms studies will enable the development of drugs to ameliorate the effects of ozone exposure in susceptible individuals.
... The concentrations of particulate matter 2.5 (PM 2.5 ) and VOCs in the indoor environment are reportedly several times higher than in outdoor air indicating substantial risks to human health (Cheek et al. 2021;López et al. 2021). The exposure of humans to O 3 decreases pulmonary function acutely, which increases the permeability of the pulmonary epithelium (Gerrity et al. 1993), and the levels of proinflammatory cytokines in the airways (Cheng et al. 2018). According to Diaper et al. (2011), inhaling 7.5% of CO 2 causes anxiety, fear, and tension, as well as increased heart rate and blood pressure. ...
Indoor air quality (IAQ) in the built environment is significantly influenced by particulate matter, volatile organic compounds, and air temperature. Recently, the Internet of Things (IoT) has been integrated to improve IAQ and safeguard human health, comfort, and productivity. This review seeks to highlight the potential of IoT integration for monitoring IAQ. Additionally, the paper details progress by researchers in developing IoT/mobile applications for IAQ monitoring, and their transformative impact in smart building, healthcare, predictive maintenance, and real-time data analysis systems. It also outlines the persistent challenges (e.g., data privacy, security, and user acceptability), hampering effective IoT implementation for IAQ monitoring. Lastly, the global developments and research landscape on IoT for IAQ monitoring were examined through bibliometric analysis (BA) of 106 publications indexed in Web of Science from 2015 to 2022. BA revealed the most significant contributing countries are India and Portugal, while the top productive institutions and researchers are Instituto Politecnico da Guarda (10.37% of TP) and Marques Goncalo (15.09% of TP), respectively. Keyword analysis revealed four major research themes: IoT, pollution, monitoring, and health. Overall, this paper provides significant insights for identifying prospective collaborators, benchmark publications, strategic funding, and institutions for future IoT-IAQ researchers.
Graphical Abstract
... The changes in amino acid and lipid metabolic pathways, deranged energy metabolism, and disturbed cellular turnover are noted. [16][17][18][19][20] The simulated uids with varying pH and biochemical composition have been used to mimic the lung microenvironment under healthy and diseased states. Two of the most common simulated lung uids are Gamble's solution and articial lysosomal uid (ALF). ...
Lung inflammation and injuries are major health problems. The SPA4 peptide (amino acid sequence GDFRYSDGTPVNYTNWYRGE) binds to Toll-like receptor-4 and exerts anti-inflammatory activity. In this study, we have determined the stability of the structure and structure-activity relationship of the SPA4 peptide under ambient and stressed conditions of lung injury. The SPA4 peptide was maintained at different pH and temperatures, in solutions of different ionic strengths, and simulated lung fluids. The primary and secondary structure of the SPA4 peptide was determined by ultraviolet-visible (UV-VIS) and circular dichroism (CD) spectroscopy. The activity of the SPA4 peptide was determined by measurement of secreted levels of chemokine C-X-C motif ligand 1/keratinocyte-derived chemokine (CXCL1/KC) and lactate by primary mouse lung epithelial cells against lipopolysaccharide (LPS) stimuli. Our results demonstrate the stability of the structure of the SPA4 peptide at room temperature and 4 °C over 10 days. The original UV-VIS spectra of the SPA4 peptide followed a typical pattern when incubated in solutions of pH 5.7, 7.0, and 8.0 at different temperatures, simulated lung fluids, and most of the chemical components. Slight shifts in the absorbance peaks, derivative values, and vibrational fine structures were noted in the fourth-derivative spectra of the SPA4 peptide under some conditions. An increased level of lactate is the hallmark of lung injury. The SPA4 peptide on its own and in the presence of lactate exerts anti-inflammatory activity. The primary and secondary structure and the activity of the SPA4 peptide remain intact when pre-incubated in 2 mM sodium lactate solution. The results provide important insights about the stability and structure-activity relationship of the SPA4 peptide.
... 24,40,59,63 Creatine was inversely associated with O 3 in serum 26 but positively associated with O 3 in BALF. 19 High reproducibility was present between plasma with serum, BALF, and stratum corneum, but not with urine. Figure 3 showed biological pathways reported in eligible studies, and biological pathways with more than five associations reported were included. ...
Background:
Understanding the mechanistic basis of air pollution toxicity is dependent on accurately characterizing both exposure and biological responses. Untargeted metabolomics, an analysis of small-molecule metabolic phenotypes, may offer improved estimation of exposures and corresponding health responses to complex environmental mixtures such as air pollution. The field remains nascent, however, with questions concerning the coherence and generalizability of findings across studies, study designs and analytical platforms.
Objectives:
We aimed to review the state of air pollution research from studies using untargeted high-resolution metabolomics (HRM), highlight the areas of concordance and dissimilarity in methodological approaches and reported findings, and discuss a path forward for future use of this analytical platform in air pollution research.
Methods:
We conducted a state-of-the-science review to a) summarize recent research of air pollution studies using untargeted metabolomics and b) identify gaps in the peer-reviewed literature and opportunities for addressing these gaps in future designs. We screened articles published within Pubmed and Web of Science between 1 January 2005 and 31 March 2022. Two reviewers independently screened 2,065 abstracts, with discrepancies resolved by a third reviewer.
Results:
We identified 47 articles that applied untargeted metabolomics on serum, plasma, whole blood, urine, saliva, or other biospecimens to investigate the impact of air pollution exposures on the human metabolome. Eight hundred sixteen unique features confirmed with level-1 or -2 evidence were reported to be associated with at least one or more air pollutants. Hypoxanthine, histidine, serine, aspartate, and glutamate were among the 35 metabolites consistently exhibiting associations with multiple air pollutants in at least 5 independent studies. Oxidative stress and inflammation-related pathways-including glycerophospholipid metabolism, pyrimidine metabolism, methionine and cysteine metabolism, tyrosine metabolism, and tryptophan metabolism-were the most commonly perturbed pathways reported in >70% of studies. More than 80% of the reported features were not chemically annotated, limiting the interpretability and generalizability of the findings.
Conclusions:
Numerous investigations have demonstrated the feasibility of using untargeted metabolomics as a platform linking exposure to internal dose and biological response. Our review of the 47 existing untargeted HRM-air pollution studies points to an underlying coherence and consistency across a range of sample analytical quantitation methods, extraction algorithms, and statistical modeling approaches. Future directions should focus on validation of these findings via hypothesis-driven protocols and technical advances in metabolic annotation and quantification. https://doi.org/10.1289/EHP11851.
... Disruption of these lipid signaling pathways can result in inefficient or failure of resolution causing chronic inflammation and disease progression [14,15]. Inhalation exposures can modify pulmonary lipids involved in inflammatory signaling [16][17][18]. Recent studies from our laboratory determined reduced pulmonary eicosapentaenoic acid (EPA), docosapentaenoic acid (DHA), 18-hydroxy eicosapentaenoic acid (18-HEPE), 14-hydroxy docosahexaenoic acid (14-HDHA), 17-hydroxy docosahexaenoic acid (17-HDHA), maresin-1, and resolvin D1 (RvD1) levels as well as other SPMs 24 h after AgNP exposure in the MetS mouse model [11,13]. ...
Background
Metabolic syndrome (MetS) exacerbates susceptibility to inhalation exposures such as particulate air pollution, however, the mechanisms responsible remain unelucidated. Previously, we determined a MetS mouse model exhibited exacerbated pulmonary inflammation 24 h following AgNP exposure compared to a healthy mouse model. This enhanced response corresponded with reduction of distinct resolution mediators. We hypothesized silver nanoparticle (AgNP) exposure in MetS results in sustained pulmonary inflammation. Further, we hypothesized treatment with resolvin D1 (RvD1) will reduce exacerbations in AgNP-induced inflammation due to MetS.
Results
To evaluate these hypotheses, healthy and MetS mouse models were exposed to vehicle (control) or AgNPs and a day later, treated with resolvin D1 (RvD1) or vehicle (control) via oropharyngeal aspiration. Pulmonary lung toxicity was evaluated at 3-, 7-, 14-, and 21-days following AgNP exposure. MetS mice exposed to AgNPs and receiving vehicle treatment, demonstrated exacerbated pulmonary inflammatory responses compared to healthy mice. In the AgNP exposed mice receiving RvD1, pulmonary inflammatory response in MetS was reduced to levels comparable to healthy mice exposed to AgNPs. This included decreases in neutrophil influx and inflammatory cytokines, as well as elevated anti-inflammatory cytokines.
Conclusions
Inefficient resolution may contribute to enhancements in MetS susceptibility to AgNP exposure causing an increased pulmonary inflammatory response. Treatments utilizing specific resolution mediators may be beneficial to individuals suffering MetS following inhalation exposures.
... Epidemiological, controlled chamber studies and toxicological literature have suggested an association between O3 and lung inflammation. Recent human studies on short-term O3 effects on airway inflammation continue to be mixed, with some studies finding an association with lung inflammation markers Modig et al. 2014;Karakatsani et al. 2017;Arjomandi et al. 2017;Cheng et al. 2018). ...
Tropospheric ozone (O3) is the third most important greenhouse gas that is transported from the stratosphere or formed locally by photochemical reactions among precursors such as nitrogen oxides (NOx), carbon monoxide (CO), methane (CH4), volatile organic compounds (VOCs), and peroxyacetyl nitrate (PAN). The lifetime of tropospheric O3 is long enough, i.e. a few days in the boundary layer to a few months in the free troposphere, allowing its (and its precursors) long–range transport from regional to hemispheric scale through trans–Atlantic, trans–Pacific, and trans–Eurasian transport. Therefore, remote areas such as the Arctic region can be affected. The current annual mean background O3 concentrations at surface–layer are 35–50 ppb in North Hemisphere and 15–25 ppb in South Hemisphere. At regional and local scales, the O3 production depends on the VOCs/NOx ratio. Since the 1990s, anthropogenic O3 precursor emissions decreased in North America and Europe, while eastern Asian emissions have increased. Therefore, both mean and peak O3 concentrations decreased in North America and Europe and increased in East Asia. The reduction in O3 mean concentrations and higher percentiles is associated with reductions in NOx and VOCs emissions in the EU and North America since the 1990s. The increase in lower concentrations and percentiles can be attributed to a reduction in local NOx emissions, due to e.g. vehicle emission controls, resulting in lower O3 titration by NO. Generally, the global background O3 increase can be driven by the net impacts of climate change, i.e. increase in stratospheric O3 inputs, higher CH4 emissions, changing lightning NOx emissions, weakened NO titration and impacting reaction rates, through sea surface temperatures and relative humidity changes. (note: the final abstract might slightly differ due to copy editing)
... Also, plasma clara cell protein (CC16) levels, which is a common biomarker used for epithelial cell damage, were observed to be significantly increased after low-level ozone exposure in an ozone concentration-dependent manner. Metabolomics analysis of bronchoalveolar lavage (BAL) samples from volunteers exposed to ozone demonstrated oxidative stress responses and subsequent cellular repair, with metabolomic signals of increased energy usage [50]. Focusing on airway inflammation, sputum neutrophils obtained after exposure showed a small significant increase but in contrast, proinflammatory cytokines (IL-6, IL-8, and tumor necrosis factor-α (TNF-α)) were not significantly affected [51]. in vitro, ozone stimulated bronchial epithelial cells induced IL-6 and IL-8 expression [52]. ...
Since the industrial revolution, air pollution has become a major problem causing several health problems involving the airways as well as the cardiovascular, reproductive, or neurological system. According to the WHO, about 3.6 million deaths every year are related to inhalation of polluted air, specifically due to pulmonary diseases. Polluted air first encounters the airways, which are a major human defense mechanism to reduce the risk of this aggressor. Air pollution consists of a mixture of potentially harmful compounds such as particulate matter, ozone, carbon monoxide, volatile organic compounds, and heavy metals, each having its own effects on the human body. In the last decades, a lot of research investigating the underlying risks and effects of air pollution and/or its specific compounds on the airways, has been performed, involving both in vivo and in vitro experiments. The goal of this review is to give an overview of the recent data on the effects of air pollution on healthy and diseased airways or models of airway disease, such as asthma or chronic obstructive pulmonary disease. Therefore, we focused on studies involving pollution and airway symptoms and/or damage both in mice and humans.
... In 12 studies, the spectra were matched against an authentic chemical standard analyzed on the same analytical platform. To do this, the m/z of the parent ion and MS/MS fragments were compared, as well as the retention time to perform the highest level of confidence in metabolite identification [10,11,14,16,20,23,25,27,30,32,33,35]. ...
Purpose of Review
The purpose of this review is to summarize the application of untargeted metabolomics to identify the perturbation of metabolites or metabolic pathways associated with air pollutant exposures.
Recent Findings
Twenty-three studies were included in this review, in adults, children, or pregnant women. The most commonly measured air pollutant is particulate matter smaller than 2.5 μm. Size-fractioned particles, particle chemical species, gas pollutants, or organic compounds were also investigated. The reviewed studies used a wide range of air pollution measurement techniques and metabolomics analyses. Identified metabolites were primarily related to oxidative stress and inflammatory responses, and a few were related to the alterations of steroid metabolic pathways. The observed metabolic perturbations can differ by disease status, sex, and age. Air pollution-related metabolic changes were also associated with health outcomes in some studies.
Summary
Our review shows that air pollutant exposures are associated with metabolic pathways primarily related to oxidative stress, inflammation, as assessed through untargeted metabolomics in 23 studies. More metabolomic studies with larger sample sizes are needed to identify air pollution components most responsible for adverse health effects, elaborate on mechanisms for subpopulation susceptibility, and link air pollution exposure to specific adverse health effects.
... Macrophage activation is characterized by metabolic shifts and the mitochondria are central hubs in inflammatory macrophage activation [6]. Ozone can accelerate both peroxidative and glycolytic metabolisms in AM [54,55]. ...
Co-enzyme nicotinamide adenine dinucleotide (NAD(H)) redox plays a key role in macrophage function. Surfactant protein (SP-) A modulates the functions of alveolar macrophages (AM) and ozone (O 3) exposure in the presence or absence of SPA and reduces mouse survival in a sex-dependent manner. It is unclear whether and how NAD(H) redox status plays a role in the innate immune response in a sex-dependent manner. We investigated the NAD(H) redox status of AM from SP-A2 and SPA knockout (KO) mice in response to O 3 or filtered air (control) exposure using optical redox imaging technique. We found: (i) In SP-A2 mice, the redox alteration of AM in response to O 3 showed sex-dependence with AM from males being significantly more oxidized and having a higher level of mitochondrial reactive oxygen species than females; (ii) AM from KO mice were more oxidized after O 3 exposure and showed no sex differences; (iii) AM from female KO mice were more oxidized than female SP-A2 mice; and (iv) Two distinct subpopulations characterized by size and redox status were observed in a mouse AM sample. In conclusions, the NAD(H) redox balance in AM responds to O 3 in a sex-dependent manner and the innate immune molecule, SP-A2, contributes to this observed sex-specific redox response. Keywords: optical redox imaging; nicotinamide adenine dinucleotide (NAD(H)); oxidized flavoprotein containing flavin adenine dinucleotide (FAD); redox ratio; surfactant protein A2 (SP-A2); surfactant protein A; macrophage activation; ozone; redox heterogeneity; innate immunity; lung
... Long-chain fatty acids have been previously shown to be responsive to short-term exposure to NO 2 29 . In the same participants of our study, fatty acid metabolism and amino acid metabolism were found to be enriched after ozone exposure compared to before the exposure 30 . Particulate matter-associated amino acids were also strongly associated with markers of inflammation and lung function 31 . ...
We used a randomized crossover experiment to estimate the effects of ozone (vs. clean air) exposure on genome-wide DNA methylation of target bronchial epithelial cells, using 17 volunteers, each randomly exposed on two separated occasions to clean air or 0.3-ppm ozone for two hours. Twenty-four hours after exposure, participants underwent bronchoscopy to collect epithelial cells whose DNA methylation was measured using the Illumina 450 K platform. We performed global and regional tests examining the ozone versus clean air effect on the DNA methylome and calculated Fisher-exact p-values for a series of univariate tests. We found little evidence of an overall effect of ozone on the DNA methylome but some suggestive changes in PLSCR1, HCAR1, and LINC00336 DNA methylation after ozone exposure relative to clean air. We observed some participant-to-participant heterogeneity in ozone responses.
... In mice, O 3 exposure caused lung damage, alterations to surfactant protein activity (32) and neutrophilic inflammation in the lower airway (33). Experimentally, O 3 inhalation in humans has been associated with lung inflammation and increased lower airway neutrophils (34). Increased ambient O 3 exposure has also been associated with human mortality from general (35) and lung-related causes (36), particularly in association with increased ambient temperature (35). ...
Ambient pollution is associated with the development and exacerbation of human asthma, but whether air pollution exposure is associated with lower airway inflammation in horses has not been fully evaluated. The Air Quality Health Index (AQHI) is an online tool used by asthmatic Ontarians to modify their outdoor activity when ambient pollution is high. A single AQHI value, falling on a scale from 1 to 10⁺, is calculated from measurements of fine particulate matter (PM2.5), nitrogen dioxide (NO2), and ozone (O3). Increased AQHI values predict an increased risk for presenting to a health care provider for assessment of asthma exacerbation, with a time lag of 0–9 days after an increase. Whether ambient air pollution is a risk factor for identifying increased lower airway inflammatory cells on cytologic evaluation of bronchoalveolar lavage fluid (BALF) of horses has not yet been explored. To investigate this relationship, case data including BALF cytology preparations from horses across southern Ontario, Canada, were retrieved from the Guelph Animal Health Laboratory's archives. Spanning the years 2007–2017, 154 cases were identified within a 41- by 30-km area surrounding the cities of Guelph and Kitchener. In 78 of 154 cases, cytologic reevaluation identified increased proportions of one or a combination of BALF neutrophils (mean 5%, range 0–15%), eosinophils (mean 2%, range 0–31%), and mast cells (mean 4%, range 0–10%). To assess the effect of lagged pollutant and temperature exposures in these 78 cases, weekly mean values of AQHI, PM2.5, NO2, O3, and temperature were recorded for the 4 weeks prior to the date of the horse's presentation for respiratory tract evaluation. The relationship between ambient exposures and increased proportions of lower airway granulocytes was evaluated using a case-crossover design. Single unit increases in 2-, and 3-week lagged weekly mean PM2.5 and NO2, were associated, respectively, with an 11% (p = 0.04, 95% confidence interval, CI = 1.01–1.22), and 24% (p = 0.03, 95% CI = 1.08–1.43) greater risk of identifying increased lower airway granulocytes. These findings suggest that exposure to increased ambient pollutants is associated with lower airway inflammation in Guelph and Kitchener area horses.
A growing body of literature has attempted to characterize how traffic-related air pollution (TRAP) affects molecular and subclinical biological processes in ways that could lead to cardiorespiratory disease. To provide a streamlined synthesis of what is known about the multiple mechanisms through which TRAP could lead to cardiorespiratory pathology, we conducted a systematic review of the epidemiological literature relating TRAP exposure to methylomic, proteomic, and metabolomic biomarkers in adult populations. Using the 139 papers that met our inclusion criteria, we identified the omic biomarkers significantly associated with short- or long-term TRAP and used these biomarkers to conduct pathway and network analyses. We considered the evidence for TRAP-related associations with biological pathways involving lipid metabolism, cellular energy production, amino acid metabolism, inflammation and immunity, coagulation, endothelial function, and oxidative stress. Our analysis suggests that an integrated multi-omics approach may provide critical new insights into the ways TRAP could lead to adverse clinical outcomes. We advocate for efforts to build a more unified approach for characterizing the dynamic and complex biological processes linking TRAP exposure and subclinical and clinical disease and highlight contemporary challenges and opportunities associated with such efforts.
A growing body of literature has attempted to characterize how traffic-related air pollution (TRAP) affects molecular and subclinical biological processes in ways that could lead to cardiorespiratory disease. To provide a streamlined synthesis of what is known about the multiple mechanisms through which TRAP could lead cardiorespiratory pathology, we conducted a systematic review of the epidemiological literature relating TRAP exposure to methylomic, proteomic, and metabolomic biomarkers in adult populations. Using the 139 papers that met our inclusion criteria, we identified the omic biomarkers significantly associated with short- or long-term TRAP and used these biomarkers to conduct pathway and network analyses. We considered the evidence for TRAP-related associations with biological pathways involving lipid metabolism, cellular energy production, amino acid metabolism, inflammation and immunity, coagulation, endothelial function, and oxidative stress. Our analysis suggests that an integrated multi-omics approach may provide critical new insights into the ways TRAP could lead to adverse clinical outcomes. We advocate for efforts to build a more unified approach for characterizing the dynamic and complex biological processes linking TRAP exposure and subclinical and clinical disease, and highlight contemporary challenges and opportunities associated with such efforts.
Short-term exposure to ozone (O3) has been associated with airway inflammation. Given that high temperature (HT) accelerates O3 production, it is of significance to determine whether co-exposure to HT exacerbates O3-induced airway inflammation. The aim of this study was to examine the possible promotive effect of HT on O3-induced airway inflammation and underlying mechanisms. Forty-eight C57BL/6 N male mice were randomly divided into four groups: filtered air (control), O3, HT, and HT + O3 (co-exposure) groups. Mice in control and O3 groups were exposed to filtered air or 1 ppm O3 at 24 °C, respectively, while mice in HT and co-exposure groups were exposed to filtered air or 1 ppm O3 at 36 °C, respectively. The exposure scenario for four groups was 4 h/d for 5 consecutive days. Bronchoalveolar lavage fluids (BALF) were collected 24 h after the last exposure and subjected to examinations of oxidative stress and inflammation biomarkers, 16S rRNA sequencing, and metabolic profiling. Lung tissues were processed for H&E histological staining. The results showed that O3 inhalation triggered oxidative stress and inflammation in the airways, which was worsen by co-exposure to HT. Further studies revealed that co-exposure to HT strengthened O3-induced decline in Firmicutes and Allobaculum in airways. Moreover, co-exposure to HT promoted O3-induced airway metabolic disorder. Spearman correlation analysis revealed correlations among microbiota dysbiosis, metabolic disorder, oxidative stress and inflammation induced by co-exposure to HT and O3. Taken together, HT exposure aggravates O3-induced airway oxidative stress and inflammation, possibly through modulation of microbiota and metabolism of the airways.
Asthma is a common chronic respiratory disease exacerbated by multiple environmental factors. Acute ozone exposure has previously been implicated in airway inflammation, airway hyperreactivity, and other characteristics of asthma, which may be attributable to altered sphingolipid metabolism. This study tested the hypothesis that acute ozone exposure alters sphingolipid metabolism within the lung, which contributes to exacerbations in characteristics of asthma in allergen-sensitized mice. Adult male and female BALB/c mice were sensitized intranasally to house dust mite allergen (HDM) on days 1, 3, and 5 and challenged on days 12-14. Mice were exposed to ozone following each HDM challenge for 6 hr./day. Bronchoalveolar lavage, lung lobes, and microdissected lung airways were collected for metabolomics analysis (N = 8/sex/group). Another subset of mice underwent methacholine challenge using a forced oscillation technique to measure airway resistance (N = 6/sex/group). Combined HDM and ozone exposure in male mice synergistically increased airway hyperreactivity that was not observed in females and was accompanied by increased airway inflammation and eosinophilia relative to control mice. Importantly, glycosphingolipids were significantly increased following combined HDM and ozone exposure relative to controls in both male and female airways, which was also associated with both airway resistance and eosinophilia. However, 15 glycosphingolipid species were increased in females compared to only 6 in males, which was concomitant with significant associations between glycosphingolipids and airway resistance that ranged from R2= 0.33-0.51 for females and R2= 0.20-0.34 in male mice. These observed sex differences demonstrate that glycosphingolipids potentially serve to mitigate exacerbations in characteristics of allergic asthma.
The cardiometabolic effects of air pollution in the context of mixtures and the underlying mechanisms remain not fully understood. This study aims to investigate the joint effect of air pollutant mixtures on a broad range of cardiometabolic parameters, examine the susceptibility of obese individuals, and determine the role of circulating fatty acids. In this panel study, metabolically healthy normal-weight (MH-NW, n = 49) and obese (MHO, n = 39) adults completed three longitudinal visits (257 person-visits in total). Personal exposure levels of PM2.5, PM10, O3, NO2, SO2, CO and BC were estimated based on fixed-site monitoring data, time-activity logs and infiltration factor method. Blood pressure, glycemic homeostasis, lipid profiles, systematic inflammation and coagulation biomarkers were measured. Targeted metabolomics was used to quantify twenty-eight plasma free fatty acids (FFAs). Bayesian kernel machine regression models were applied to establish the exposure-response relationships and identify key pollutants. Significant joint effects of measured air pollutants on systematic inflammation and coagulation biomarkers were observed in the MHO group, instead of the MH-NW group. Lipid profiles showed the most significant changes in both groups and O3 contributed the most to the total effect. Specific FFA patterns were identified, and de novo lipogenesis (DNL)-related pattern was most closely related to blood lipid profiles. In particular, interaction analysis suggested that DNL-related FFA pattern augmented the effects of O3 on triglyceride (TG, Pinteraction = 0.040), high-density lipoprotein cholesterol (HDL-C, Pinteraction = 0.106) and TG/HDL-C (Pinteraction = 0.020) in the MHO group but not MH-NW group. This modification was further confirmed by interaction analysis with estimated activity of SCD1, a key enzyme in the DNL pathway. Therefore, despite being metabolically healthy, obese subjects have a higher cardiometabolic susceptibility to air pollution, especially O3, and the DNL pathway may represent an intrinsic driver of lipid susceptibility. This study provides new insights into the cardiometabolic susceptibility of obese individuals to air pollution.
Air pollution, especially PM2.5 (particulate matter with an aerodynamic diameter ≤2.5 μm) in China, is severe and related to a variety of diseases while the potential mechanisms have not been clearly clarified yet. This study was conducted using a randomized crossover trial protocol among young and healthy college students. Plasma samples were collected before, during, and post two typical air pollution waves with a washout interval of at least 2 weeks under true and sham air purification treatments, respectively. A total of 144 blood samples from 24 participants were included in the final analysis. Metabolomics analysis for the plasma samples was achieved by Ultrahigh Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). Orthogonal Partial Least Squares Discrimination Analysis (OPLS-DA) and linear mixed-effect models were used to identify the differentially expressed metabolites and their associations with PM2.5 exposure. Further use MetaboAnalyst 5.0 to conduct pathway enrichment analysis and correlation analysis of differential metabolites. A total of 40 metabolites were identified to be differentially expressed between the true and sham air purification treatments, and eleven metabolites showed consistent significant changes upon outdoor, indoor, and time-weighted personal PM2.5 exposures. Short-term exposure to PM2.5 might cause disturbances in metabolic pathways such as linoleic acid metabolism, arachidonic acid metabolism, and tryptophan metabolism.
Ammonia is one of the major environmental pollutants in the pig industry that seriously affects the airway health of pigs. In this study, we aimed to investigate the metabolic profiling changes of piglets’ lung tissue after the exposure of 0 ppm (CG), 20 ppm (LG) and 50 ppm (HG) ammonia for 30 days. Compared with the control group, the obvious lung lesions were observed in HG, including interstitial thickening, inflammatory cell infiltration and focal hemorrhage. The significantly increased content of malondialdehyde in HG, combined with the significantly decreased mRNA expression of antioxidase and inflammatory‐regulators in exposure groups, implied that ammonia exposure induced oxidative stress and diminished the anti‐inflammatory response in lung tissues. Metabolomic analyses of lung tissues revealed 15 significantly altered metabolites among the three groups including multiple amino acids, carbohydrates and lipids. The accumulation of succinic acid, linoleic acid and phosphorylethanolamine and consumption of glucose, quinolinic acid and aspartic acid in ammonia exposure groups, indicated that energy supply from glucose aerobic oxidation was suppressed and the glycolysis and lipolysis were activated in lung tissues induced by chronic ammonia exposure.
Exposure to traffic-related pollutants, including diesel exhaust, is associated with increased risk of cardiopulmonary disease and mortality; however, the precise biochemical pathways underlying these effects are not known. To investigate biological response mechanisms underlying exposure to traffic related pollutants, we used an integrated molecular response approach that included high-resolution metabolomic profiling and peripheral blood gene expression to identify biological responses to diesel exhaust exposure. Plasma samples were collected from 73 non-smoking males employed in the US trucking industry between February 2009 and October 2010, and analyzed using untargeted high-resolution metabolomics to characterize metabolite associations with shift- and week-averaged levels of elemental carbon (EC), organic carbon (OC) and particulate matter with diameter ≤ 2.5 μm (PM2.5). Metabolic associations with EC, OC and PM2.5 were evaluated for biochemical processes known to be associated with disease risk. Annotated metabolites associated with exposure were then tested for relationships with the peripheral blood transcriptome using multivariate selection and network correlation. Week-averaged EC and OC levels, which were averaged across multiple shifts during the workweek, resulted in the greatest exposure-associated metabolic alterations compared to shift-averaged exposure levels. Metabolic changes associated with EC exposure suggest increased lipid peroxidation products, biomarkers of oxidative stress, thrombotic signaling lipids, and metabolites associated with endothelial dysfunction from altered nitric oxide metabolism, while OC exposures were associated with antioxidants, oxidative stress biomarkers and critical intermediates in nitric oxide production. Correlation with whole blood RNA gene expression provided additional evidence of changes in processes related to endothelial function, immune response, inflammation, and oxidative stress. We did not detect metabolic associations with PM2.5. This study provides an integrated molecular assessment of human exposure to traffic-related air pollutants that includes diesel exhaust. Metabolite and transcriptomic changes associated with exposure to EC and OC are consistent with increased risk of cardiovascular diseases and the adverse health effects of traffic-related air pollution.
In the past 50 years, the number of publications on air pollution and lung disease has increased considerably, although the number of studies considering sex (a biologic factor), or gender (a social construct), has remained low and stagnant. Accumulating data from studies assessing the effects of the environment on lung health have shown direct associations of air pollution exposures with lung inflammation. Sex-specific disaggregation of data has indicated that substantial – but frequently overlooked – differences exist between men and women, highlighting the importance of sex- and gender-stratified analyses to guide the deployment of safe and effective therapeutics options for males and females. In this chapter, we present an overview of the scientific evidence on differential effects of environmental exposures in men and women. We summarize clinical studies and research using animal models aiming to elucidate sex-specific mechanisms of inflammation and toxicity from a wide range of air pollutants. Understanding these mechanisms can lead to the development of more personalized prevention efforts and better-informed environmental policies accounting for sex, gender, and hormonal status.
The increased incidence of non-communicable human diseases may be attributed, at least partially, to exposures to toxic chemicals such as persistent organic pollutants (POPs), air pollutants and heavy metals. Given the high mortality and morbidity of pollutant exposure associated diseases, a better understanding of the related mechanisms of toxicity and impacts on the endogenous host metabolism are needed. The metabolome represents the collection of the intermediates and end products of cellular processes, and is the most proximal reporter of the body’s response to environmental exposures and pathological processes. Metabolomics is a powerful tool for studying how organisms interact with their environment and how these interactions shape diseases related to pollutant exposure. This mini review discusses potential biological mechanisms that link pollutant exposure to metabolic disturbances and chronic human diseases, with a focus on recent studies that demonstrate the application of metabolomics as a tool to elucidate biochemical modes of actions of various environmental pollutants. In addition, classes of metabolites that have been shown to be modulated by multiple environmental pollutants will be discussed with an emphasis on their use as potential early biomarkers of disease risks. Taken together, metabolomics is a useful and versatile tool for characterizing the disease risks and mechanisms associated with various environmental pollutants.
Background
Air pollutant exposure negatively affects human health; however, the molecular mechanisms causing disease remain largely unclear.
Objectives
To explore the effects of respiratory particulate matter (PM2.5) exposure on the serum metabolome and to identify biomarkers for risk assessment of PM2.5 exposure.
Methods
PM2.5 from Nanjing, China, was collected, and its water-soluble extract was subjected to component analysis. BALB/c mice received acute or prolonged exposure to insoluble PM2.5 particles or its water-soluble extract, and lung tissue was submitted to histopathological analyses. Serum samples were collected pre- and post-PM2.5 exposure and analyzed by liquid chromatography/mass spectrometry.
Results
Component analysis revealed that metals and inorganic ions were the most abundant components in the soluble PM2.5 samples. Acute exposure to insoluble PM2.5 particles and prolonged exposure to the water-soluble PM2.5 extract both induced severe lung injury, and the lung histopathological scores were significantly associated with PM2.5 exposure. Metabolomics analysis showed that prolonged exposure to the water-soluble PM2.5 extract was associated with statistically significant metabolite changes; the serum concentrations of 30 known metabolites, including metabolites of phospholipids, amino acids and sphingolipids, differed significantly between the control and PM2.5 exposure group. Pathway analysis identified an association of the tricarboxylic acid cycle (TCA) and the phospholipase metabolism pathway with PM2.5 exposure. The most influential metabolites for discriminating between the PM2.5-exposure group serum and the control serum were LysoPE, LysoPC, LGPC, citric acid, PAF C-18, NeuAcalpha2–3Galbeta-Cer, Lyso-PAF C-16, ganglioside GA2, 1-sn-glycero-3-phosphocholine, PC and L-tryptophan.
Conclusions
Respiratory exposure to water-soluble PM2.5 extract has developmental consequences affecting not only the respiratory system but also metabolism.
The liver is vital for xenobiotic and endobiotic metabolism. Previously, we demonstrated that a compromised liver worsened toxicity associated with exposure to polychlorinated biphenyls (PCBs), through disruption of energy homeostasis. However, the role of a compromised liver in defining dioxin-like PCB126 toxicity on the peripheral vasculature and associated inflammatory diseases is yet to be studied. The current study investigated the effects of PCB126 on vascular inflammation linked to hepatic dysfunction utilizing a liver injury mouse model. Male C57Bl/6 mice were fed either an amino acid control diet (CD) or a methionine-choline deficient diet (MCD) in this 14-week study. Mice were exposed to PCB126 (0.5 mg/kg) and analyzed for inflammatory, calorimetric and metabolic parameters. MCD diet-fed mice demonstrated steatosis, indicative of a compromised liver. Mice fed the MCD-diet and subsequently exposed to PCB126 manifested lower body fat mass, increased liver to body weight ratio and alterations in hepatic gene expression related to lipid and carbohydrate metabolism, implicating metabolic disturbances. PCB126 induced steatosis irrespective of the diet type, but only the MCD+PCB126 group exhibited steatohepatitis and fibrosis. Furthermore, PCB126 exposure in MCD-fed mice led to increased plasma inflammatory markers such as Icam-1, PAI-1 and pro-atherogenic trimethylamine-N-oxide (TMAO), suggesting inflammation of the peripheral vasculature that is characteristic of atherosclerosis. Taken together, our data provide new evidence of a link between a compromised liver, PCB-mediated hepatic inflammation and vascular inflammatory markers, suggesting that environmental pollutants can promote crosstalk between different organ systems, leading to inflammatory disease pathologies.
Background
Short-term exposure to air pollution is associated with morbidity and mortality. Metabolites are intermediaries in biochemical processes, and associations between air pollution and metabolites can yield unique mechanistic insights.
Methods
We used independent cross-sectional samples with targeted metabolomics (138 metabolites across five metabolite classes) from three cohort studies, each a part of the Cooperative Health Research in the Region of Augsburg (KORA). The KORA cohorts are numbered (1 to 4) according to which survey they belong to, and lettered S or F according to whether the survey was a baseline or follow-up survey. KORA F4 (N = 3044) served as our discovery cohort, with KORA S4 (N = 485) serving as the primary replication cohort. KORA F4 and KORA S4 were primarily fasting cohorts. We used the non-fasting KORA F3 (N = 377) cohort to evaluate replicated associations in non-fasting individuals, and we performed a random effects meta-analysis of all three cohorts. Associations between the 0–4-day lags and the 5-day average of particulate matter (PM)2.5, NO2 and ozone were modelled via generalized additive models. All air pollution exposures were scaled to the interquartile range, and effect estimates presented as percent changes relative to the geometric mean of the metabolite concentration (ΔGM).
Results
There were 10 discovery cohort associations, of which seven were lysophosphatidylcholines (LPCs); NO2 was the most ubiquitous exposure (5/10). The 5-day average NO2-LPC(28:0) association was associated at a Bonferroni corrected P-value threshold (P < 1.2x10⁻⁴) in KORA F4 [ΔGM = 11.5%; 95% confidence interval (CI) = 6.60, 16.3], and replicated (P < 0.05) in KORA S4 (ΔGM = 21.0%; CI = 4.56, 37.5). This association was not observed in the non-fasting KORA F3 cohort (ΔGM = −5.96%; CI = −26.3, 14.3), but remained in the random effects meta-analysis (ΔGM = 10.6%; CI = 0.16, 21).
Conclusions
LPCs are associated with short-term exposure to air pollutants, in particular NO2. Further research is needed to understand the effect of nutritional/fasting status on these associations and the causal mechanisms linking air pollution exposure and metabolite profiles.
Background:
In non-diabetic individuals, higher fasting blood glucose (FBG) independently predicts diabetes risk, cardiovascular disease and dementia. Ambient PM2.5 (particulate matter with aerodynamic diameter ≤ 2.5 μm) is an emerging determinant of glucose dysregulation. PM2.5 effects and mechanisms are understudied among non-diabetic individuals.
Objectives:
Our goals were to investigate whether PM2.5 is associated with increase in FBG and to explore potential mediating roles of epigenetic gene regulation.
Methods:
In 551 non-diabetic participants in the Normative Aging Study, we measured FBG, and DNA methylation of four inflammatory genes (IFN-γ, IL-6, ICAM-1, and TLR-2), up to four times between 2000-2011 (median=2). We estimated short/medium-term (1-, 7-, and 28-day preceding each clinical visit) ambient PM2.5 at each participant's address using a validated hybrid land-use regression/satellite-based model. We fitted covariate-adjusted regression models accounting for repeated measures.
Results:
Mean FBG was 99.8 mg/dL (SD=10.7), 18% of the participants had impaired fasting glucose (IFG, i.e., 100-125 mg/dL FBG) at first visit. Interquartile increases in 1-, 7-, and 28-day PM2.5 were associated with 0.57 mg/dL (95% CI: 0.02; 1.11, p=0.04), 1.02 mg/dL (95% CI: 0.41; 1.63, p=0.001), and 0.89 mg/dL (95% CI: 0.32; 1.47, p=0.003) higher FBG. The same PM2.5 metrics were associated with 13% (95% CI: -3%; 33%; p=0.12), 27% (95% CI: 6%; 52%, p=0.01) and 32% (95% CI: 10%; 58%, p=0.003) higher odds of IFG, respectively. PM2.5 was negatively correlated with ICAM-1 methylation (p=0.01), but not with other genes. Mediation analysis estimated that ICAM-1 methylation mediated 9% of the association of 28-day PM2.5 with FBG.
Conclusions:
In non-diabetics, short/medium-term PM2.5 was associated with higher FBG. Mediation analysis indicated that part of this association was mediated by ICAM-1 promoter methylation.
Influenza virus infection (IVI) can cause primary viral pneumonia, which may progress to acute lung injury (ALI) and respiratory failure with a potentially fatal outcome. At present, the interactions between host and influenza virus at molecular levels and the underlying mechanisms that give rise to IVI-induced ALI are poorly understood. We conducted a comprehensive mass spectrometry-based metabolic profiling of serum, lung tissue and bronchoalveolar lavage fluid (BALF) from a non-lethal mouse model with influenza A virus at 0, 6, 10, 14, 21 and 28 days post infection (dpi), representing the major stages of IVI. Distinct metabolite signatures were observed in mice sera, lung tissues and BALF, indicating the molecular differences between systematic and localized host responses to IVI. More than 100 differential metabolites were captured in mice sera, lung tissues and BALF, including purines, pyrimidines, acylcarnitines, fatty acids, amino acids, glucocorticoids, sphingolipids, phospholipids, etc. Many of these metabolites belonged to pulmonary surfactants, indicating IVI-induced aberrations of the pulmonary surfactant system might play an important role in the etiology of respiratory failure and repair. Our findings revealed dynamic host responses to IVI and various metabolic pathways linked to disease progression, and provided mechanistic insights into IVI-induced ALI and repair process.
Rationale:
Air pollution has been associated with increased prevalence of type-2 diabetes, however, the mechanisms remain unknown. We have shown that acute ozone exposure in rats induces stress hormone release, hyperglycemia, leptinemia, and glucose intolerance that are associated with global changes in peripheral glucose, lipid, and amino acid metabolism.
Objectives:
To examine ozone-induced metabolic derangement in humans using serum metabolomic assessment, establish human to rodent coherence, and identify novel non-protein biomarkers.
Methods:
Serum samples were obtained from a crossover clinical study which included two clinic visits (n=24 each) where each subject was blindly exposed in the morning to either filtered air or 0.3 ppm ozone for 2 hrs during 15 min on-off exercise. Serum samples collected within 1 hr after exposure were assessed for changes in metabolites using a metabolomic approach.
Measurements and main results:
Metabolomic analysis revealed that ozone exposure markedly increased serum cortisol and corticosterone together with increases in monoacylglycerol, glycerol, and medium- and long-chain free fatty acids, reflective of lipid mobilization and catabolism. Additionally, ozone exposure increased serum lysolipids, potentially originating from membrane lipid breakdown. Ozone exposure also increased circulating mitochondrial β-oxidation-derived metabolites, such as acylcarnitines, together with increases in the ketone body 3-hydroxybutyrate. These changes suggested saturation of β-oxidation by ozone in exercising humans.
Conclusions:
As in rodents, acute ozone exposure increased stress hormones and globally altered peripheral lipid metabolism in humans, likely through activation of neurohormonally-mediated stress response pathway. The metabolomic assessment revealed new biomarkers and allowed for establishment of rodent to human coherence. Clinical trial registration available at www.clinicaltrials.gov, ID NCT01492517.
Background:
Inhalation of ambient levels of ozone causes airway inflammation and epithelial injury.
Methods:
To examine the responses of airway cells to ozone-induced oxidative injury, 19 subjects (7 with asthma) were exposed to clean air (0ppb), medium (100ppb), and high (200ppb) ambient levels of ozone for 4h on three separate occasions in a climate-controlled chamber followed by bronchoscopy with bronchoalveolar lavage (BAL) 24h later. BAL cell mRNA expression was examined using Affymetrix GeneChip Microarray. The role of a differentially expressed gene (DEG) in epithelial injury was evaluated in an in vitro model of injury [16HBE14o- cell line scratch assay].
Results:
Ozone exposure caused a dose-dependent up-regulation of several biologic pathways involved in inflammation and repair including chemokine and cytokine secretion, activity, and receptor binding; metalloproteinase and endopeptidase activity; adhesion, locomotion, and migration; and cell growth and tumorigenesis regulation. Asthmatic subjects had 1.7- to 3.8-fold higher expression of many DEGs suggestive of increased proinflammatory and matrix degradation and remodeling signals. The most highly up-regulated gene was osteopontin, the protein level of which in BAL fluid increased in a dose-dependent manner after ozone exposure. Asthmatic subjects had a disproportionate increase in non-polymerized osteopontin with increasing exposure to ozone. Treatment with polymeric, but not monomeric, osteopontin enhanced the migration of epithelial cells and wound closure in an α9β1 integrin-dependent manner.
Conclusions:
Expression profiling of BAL cells after ozone exposure reveals potential regulatory genes and pathways activated by oxidative stress. One DEG, osteopontin, promotes epithelial wound healing in an in vitro model of injury.
Acute exacerbations in chronic obstructive pulmonary disease may be related to air pollution, of which ozone is an important constituent. In this study, we investigated the protein profiles associated with ozone-induced exacerbations in a smoking-induced emphysema model.
Mice were divided into the following groups: group I, no smoking and no ozone (NS + NO); group II, no smoking and ozone (NS + O); group III, smoking and no ozone (S + NO); and group IV, smoking and ozone (S + O). Bronchoalveolar lavage, the mean linear intercept (MLI) on hematoxylin and eosin staining, nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS), and Western blotting analyses were performed.
The MLIs of groups III (S + NO) and IV (S + O) (45 ± 2 and 44 ± 3 µm, respectively) were significantly higher than those of groups I (NS + NO) and II (NS + O) (26 ± 2 and 23 ± 2 µm, respectively; p < 0.05). Fourteen spots that showed significantly different intensities on image analyses of two-dimensional (2D) protein electrophoresis in group I (NS + NO) were identified by LC-MS/MS. The levels of six proteins were higher in group IV (S + O). The levels of vimentin, lactate dehydrogenase A, and triose phosphate isomerase were decreased by both smoking and ozone treatment in Western blotting and proteomic analyses. In contrast, TBC1 domain family 5 (TBC1D5) and lamin A were increased by both smoking and ozone treatment.
TBC1D5 could be a biomarker of ozone-induced lung injury in emphysema.
Background
Our objective is to investigate the genetic polymorphisms of the glutathione S-transferase M1 and T1 genes (GSTM1 and GSTT1) and evaluate oxidative damage in patients with non-small lung cancer (N-SCLC).Methods
One hundred and ten patients with N-SCLC and 100 controls are included in this case-control study. Multiplex polymerase chain reaction (PCR) analyses were used to identify the genotypes. The activities of malondialdehyde (MDA) and nitric oxide (NO) and total antioxidant capacity (T-AOC) were detected by spectroscopic analysis.ResultsThe frequencies of the GSTM1, T1, and GSTM1/T1 null genotypes in the patient group were significantly higher than that in the control group (OR¿=¿2.071, P¿=¿0.009; OR¿=¿1.900, P¿=¿0.024; OR¿=¿3.258, P¿=¿0.003). The activities of MDA and NO were significantly higher in the patient group than that in the control group (P <0.001), and T-AOC was significantly lower in patient group than that in control group (P <0.001). The activities of MDA, and NO were higher but the T-AOC was lower in patients with the GSTM1, T1 and M1/T1 null genotypes than those in patients with GSTM1, T1 and M1/T1 present genotypes (P <0.001).Conclusions
Our results suggest that oxidative damage may be play a important role in patients with N-SCLC, and that GSTM1 and GSTT1 null genotypes may predispose the cells of patients with N-SCLC to increased oxidative damage.
Chronic obstructive pulmonary disease (COPD), asthma and cystic fibrosis (CF) are characterized by airway obstruction and an inflammatory process. Reaching early diagnosis and discrimination of subtypes of these respiratory diseases are quite a challenging task than other chronic illnesses. Metabolomics is the study of metabolic pathways and the measurement of unique biochemical molecules generated in a living system. In the last decade, metabolomics has already proved to be useful for the characterization of several pathological conditions and offers promises as a clinical tool. In this article, we review the current state of the metabolomics of COPD, asthma and CF with a focus on the different methods and instrumentation being used for the discovery of biomarkers in research and translation into clinic as diagnostic aids for the choice of patient-specific therapies.
The non-essential amino acid, glutamine, exerts pleiotropic effects on cell metabolism, signalling and stress resistance. Here we demonstrate that short-term glutamine restriction triggers an endoplasmic reticulum (ER) stress response that leads to production of the pro-inflammatory chemokine, interleukin-8 (IL-8). Glutamine deprivation-induced ER stress triggers colocalization of autophagosomes, lysosomes and the Golgi into a subcellular structure whose integrity is essential for IL-8 secretion. The stimulatory effect of glutamine restriction on IL-8 production is attributable to depletion of tricarboxylic acid cycle intermediates. The protein kinase, mTOR, is also colocalized with the lysosomal membrane clusters induced by glutamine deprivation, and inhibition of mTORC1 activity abolishes both endomembrane reorganization and IL-8 secretion. Activated mTORC1 elicits IL8 gene expression via the activation of an IRE1-JNK signalling cascade. Treatment of cells with a glutaminase inhibitor phenocopies glutamine restriction, suggesting that these results will be relevant to the clinical development of glutamine metabolism inhibitors as anticancer agents.
Background & aims:
Air pollution is a global challenge to public health. Epidemiological studies have linked exposure to ambient particulate matter with aerodynamic diameters<2.5 μm (PM(2.5)) to the development of metabolic diseases. In this study, we investigated the effect of PM(2.5) exposure on liver pathogenesis and the mechanism by which ambient PM(2.5) modulates hepatic pathways and glucose homeostasis.
Methods:
Using "Ohio's Air Pollution Exposure System for the Interrogation of Systemic Effects (OASIS)-1", we performed whole-body exposure of mice to concentrated ambient PM(2.5) for 3 or 10 weeks. Histological analyses, metabolic studies, as well as gene expression and molecular signal transduction analyses were performed to determine the effects and mechanisms by which PM(2.5) exposure promotes liver pathogenesis.
Results:
Mice exposed to PM(2.5) for 10 weeks developed a non-alcoholic steatohepatitis (NASH)-like phenotype, characterized by hepatic steatosis, inflammation, and fibrosis. After PM(2.5) exposure, mice displayed impaired hepatic glycogen storage, glucose intolerance, and insulin resistance. Further investigation revealed that exposure to PM(2.5) led to activation of inflammatory response pathways mediated through c-Jun N-terminal kinase (JNK), nuclear factor kappa B (NF-κB), and Toll-like receptor 4 (TLR4), but suppression of the insulin receptor substrate 1 (IRS1)-mediated signaling. Moreover, PM(2.5) exposure repressed expression of the peroxisome proliferator-activated receptor (PPAR)γ and PPARα in the liver.
Conclusions:
Our study suggests that PM(2.5) exposure represents a significant "hit" that triggers a NASH-like phenotype and impairs hepatic glucose metabolism. The information from this work has important implications in our understanding of air pollution-associated metabolic disorders.
Mitochondrial metabolism provides precursors to build macromolecules in growing cancer cells. In normally functioning tumour cell mitochondria, oxidative metabolism of glucose- and glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, which is required for tumorigenesis. Yet some tumours harbour mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function, and it is unknown how cells from such tumours generate precursors for macromolecular synthesis. Here we show that tumour cells with defective mitochondria use glutamine-dependent reductive carboxylation rather than oxidative metabolism as the major pathway of citrate formation. This pathway uses mitochondrial and cytosolic isoforms of NADP(+)/NADPH-dependent isocitrate dehydrogenase, and subsequent metabolism of glutamine-derived citrate provides both the acetyl-coenzyme A for lipid synthesis and the four-carbon intermediates needed to produce the remaining CAC metabolites and related macromolecular precursors. This reductive, glutamine-dependent pathway is the dominant mode of metabolism in rapidly growing malignant cells containing mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with mutations in fumarate hydratase, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. Our findings reveal the novel induction of a versatile glutamine-dependent pathway that reverses many of the reactions of the canonical CAC, supports tumour cell growth, and explains how cells generate pools of CAC intermediates in the face of impaired mitochondrial metabolism.
Metabolomics is an emerging component of systems biology that may be a viable strategy for the identification and validation of physiologically relevant biomarkers. Nuclear magnetic resonance (NMR) spectroscopy allows for establishing quantitative data sets for multiple endogenous metabolites without preconception. Sepsis-induced acute lung injury (ALI) is a complex and serious illness associated with high morbidity and mortality for which there is presently no effective pharmacotherapy. The goal of this study was to apply ¹H-NMR based quantitative metabolomics with subsequent computational analysis to begin working towards elucidating the plasma metabolic changes associated with sepsis-induced ALI. To this end, this pilot study generated quantitative data sets that revealed differences between patients with ALI and healthy subjects in the level of the following metabolites: total glutathione, adenosine, phosphatidylserine, and sphingomyelin. Moreover, myoinositol levels were associated with acute physiology scores (APS) (ρ = -0.53, P = 0.05, q = 0.25) and ventilator-free days (ρ = -0.73, P = 0.005, q = 0.01). There was also an association between total glutathione and APS (ρ = 0.56, P = 0.04, q = 0.25). Computational network analysis revealed a distinct metabolic pathway for each metabolite. In summary, this pilot study demonstrated the feasibility of plasma ¹H-NMR quantitative metabolomics because it yielded a physiologically relevant metabolite data set that distinguished sepsis-induced ALI from health. In addition, it justifies the continued study of this approach to determine whether sepsis-induced ALI has a distinct metabolic phenotype and whether there are predictive biomarkers of severity and outcome in these patients.
Although ozone (O3) has been shown to induce inflammation in the lungs of animals, very little is known about its inflammatory effects on humans. In this study, 11 healthy nonsmoking men, 18 to 35 yr of age (mean, 25.4 +/- 3.5), were exposed once to 0.4 ppm O3 and once to filtered air for 2 h with intermittent exercise. Eighteen hours later, bronchoalveolar lavage (BAL) was performed and the cells and fluid were analyzed for various indicators of inflammation. There was an 8.2-fold increase in the percentage of polymorphonuclear leukocytes (PMN) in the total cell population, and a small but significant decrease in the percentage of macrophages after exposure to O3. Immunoreactive neutrophil elastase often associated with inflammation and lung damage increased by 3.8-fold in the fluid while its activity increased 20.6-fold in the lavaged cells. A 2-fold increase in the levels of protein, albumin, and IgG suggested increased vascular permeability of the lung. Several biochemical markers that could act as chemotactic or regulatory factors in an inflammatory response were examined in the BAL fluid (BALF). The level of complement fragment C3 alpha was increased by 1.7-fold. The chemotactic leukotriene B4 was unchanged while prostaglandin E2 increased 2-fold. In contrast, three enzyme systems of phagocytes with potentially damaging effects on tissues and microbes, namely, NADPH-oxidase and the lysosomal enzymes acid phosphatase and beta-glucuronidase, were increased neither in the lavaged fluid nor cells. In addition, the amounts of fibrogenic-related molecules were assessed in BALF.(ABSTRACT TRUNCATED AT 250 WORDS)
Although acute ozone (O3) exposure injures tracheal epithelium, the response of the tracheal epithelium to prolonged O3 exposure, and the degree of repair following cessation of exposure have not been previously reported. The purpose of this experiment was to characterize the morphologic response of rat tracheal epithelium to acute (3 days) and prolonged (60 days) exposure to 0.96 ppm O3 as well as to evaluate repair in a 7- and 42-day post-60-day exposure period. Quantitative light- and electron-microscopic evaluation and thymidine labeling indices showed that after 3 days of O3 exposure there was ciliary damage, cell necrosis, an increased density of intermediate cells, and an elevated thymidine labeling index. Following 60 days of exposure, the only major change from controls was the presence of ciliated cells with uniformly short cilia. Tracheal superoxide dismutase levels did not differ between control and 60-day exposure groups. Our findings suggest that the tracheal epithelium adapts to prolonged ozone exposure with the exception of cilia formation in ciliated cells. Complete epithelial recovery occurred by 42 days after exposure.
The airway epithelial cells is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products--hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines--macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretory leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease.
Antioxidants present within lung epithelial lining fluids (ELFs) constitute an initial line of defense against inhaled environmental oxidants such as ozone, nitrogen oxides, and tobacco smoke, but the antioxidant composition of human ELFs is still incompletely characterized. We analyzed ELF concentrations of the low-molecular-mass antioxidants ascorbate, urate, glutathione (GSH), and alpha-tocopherol by obtaining bronchoalveolar lavage (BAL) and nasal lavage fluids from healthy nonsmoking volunteers and compared two different BAL procedures. ELF dilution by the lavage procedures was estimated by measurement of urea in recovered BAL fluids in comparison with those in blood plasma from the same subjects. The results indicated that a recently developed single-cycle BAL procedure minimizes influx of non-ELF urea into the instilled fluid and thus allows for a more accurate determination of ELF antioxidant concentrations. Using this procedure, we determined that bronchoalveolar ELF contains 40 +/- 18 (SD) microM ascorbate, 207 +/- 167 microM urate, 109 +/- 64 microM GSH, and 0.7 +/- 0.3 microM alpha-tocopherol (n = 12 subjects). Similar analysis of nasal lavage fluid yielded nasal ELF levels of 28 +/- 19 microM ascorbate and 225 +/- 105 microM urate (n = 12 subjects), whereas GSH was undetectable (<0.5 microM). Our results demonstrate that ascorbate and urate are major low-molecular-mass ELF antioxidants in both the upper and lower respiratory tract, whereas GSH is present at significant concentrations only in bronchoalveolar ELF.
We tested the hypothesis that exposure of healthy volunteers to concentrated ambient particles (CAPS) is associated with an influx of inflammatory cells into the lower respiratory tract. Thirty-eight volunteers were exposed to either filtered air or particles concentrated from the immediate environment of the Environmental Protection Agency (EPA) Human Studies Facility in Chapel Hill, North Carolina. Particle concentrations in the chamber during the exposures ranged from 23.1 to 311.1 microgram/m(3). While in the exposure chamber, volunteers alternated between moderate exercise (15 min) and rest (15 min) for a total exposure time of 2 h. There were no symptoms noted by volunteers after the exposure. Similarly, there were no decrements in pulmonary function. Eighteen hours after exposure, analysis of cells and fluid obtained by bronchoalveolar lavage showed a mild increase in neutrophils in both the bronchial and alveolar fractions in those individuals exposed to CAPS (8.44 +/- 1.99 and 4.20 +/- 1.69%, respectively, in those with the greatest exposure) relative to filtered air (2.69 +/- 0.55 and 0.75 +/- 0.28%, respectively). Blood obtained 18 h after exposure to CAPS contained significantly more fibrinogen relative to samples obtained before exposure. We conclude that ambient air particles are capable of inducing a mild inflammation in the lower respiratory tract, as well as an increased concentration of blood fibrinogen.
Glutamine is an important mitochondrial substrate implicated in the protection of cells from oxidant injury, but the mechanisms of its action are incompletely understood. Human pulmonary epithelial-like (A549) cells were exposed to 95% O2 for 4 days in the absence and presence of glutamine. Cell proliferation in normoxia was dependent on glutamine, and glutamine deprivation markedly accelerated cell death in hyperoxia. Glutamine significantly increased cellular ATP levels in normoxia and prevented the loss of ATP in hyperoxia seen in glutamine-deprived cells. Mitochondrial membrane potential as assessed by flow cytometry with chloromethyltetramethylrosamine was increased by glutamine in hyperoxia-exposed A549 cells, and a glutamine dose-dependent increase in mitochondrial membrane potential was detected. Glutamine-supplemented, hyperoxia-exposed cells had a higher O2 consumption rate and GSH content. Electron and fluorescence microscopy revealed that, in hyperoxia, glutamine protected cellular structures, especially mitochondria, from damage. In hyperoxia, activity of the tricarboxylic acid cycle enzyme alpha-ketoglutarate dehydrogenase was partially protected by its indirect substrate, glutamine, indicating a mechanism of mitochondrial protection.
To determine whether antioxidants can influence human susceptibility to ozone (O(3))-induced changes in lung function and airway inflammation, we placed 31 healthy nonsmoking adults (18 to 35 yr old) on a diet low in ascorbate for 3 wk. At 1 wk, subjects were exposed to filtered air for 2 h while exercising (20 L/min/m(2)), and then underwent bronchoalveolar lavage (BAL) and were randomly assigned to receive either a placebo or 250 mg of vitamin C, 50 IU of alpha-tocopherol, and 12 oz of vegetable cocktail daily for 2 wk. Subjects were then exposed to 0.4 ppm O(3) for 2 h and underwent a second BAL. On the day of the O(3) exposure, supplemented subjects were found to have significantly increased levels of plasma ascorbate, tocopherols, and carotenoids as compared with those of the placebo group. Pulmonary function testing showed that O(3)-induced reductions in FEV(1) and FVC were 30% and 24% smaller, respectively, in the supplemented cohort. In contrast, the inflammatory response to O(3) inhalation, as represented by the percent neutrophils and the concentration of interleukin-6 recovered in the BAL fluid at 1 h after O(3) exposure was not different for the two groups. These data suggest that dietary antioxidants protect against O(3)-induced pulmonary function decrements in humans.
“Sola dosis facit venenum.” These words of Paracelsus, “the dose makes the poison”, can lead to a cavalier attitude concerning potential toxicities of the vast array of low abundance environmental chemicals to which humans are exposed. Exposome research teaches that 80-85% of human disease is linked to environmental exposures. The human exposome is estimated to include >400,000 environmental chemicals, most of which are uncharacterized with regard to human health. In fact, mass spectrometry measures >200,000 m/z features (ions) in microliter volumes derived from human samples; most are unidentified. This crystalizes a grand challenge for chemical research in toxicology: to develop reliable and affordable analytical methods to understand health impacts of the extensive human chemical experience. To this end, there appears to be no choice but to abandon the limitations of measuring one chemical at a time. The present article reviews progress in computational metabolomics to provide probability-based annotation linking ions to known chemicals and serve as a foundation for unambiguous designation of unidentified ions for toxicologic study. We review methods to characterize ions in terms of accurate mass m/z, chromatographic retention time, correlation of adduct, isotopic and fragment forms, association with metabolic pathways and measurement of collision-induced dissociation products, collision cross section and chirality. Such information can support a largely unambiguous system for documenting unidentified ions in environmental surveillance and human biomonitoring. Assembly of this data would provide a resource to characterize and understand health risks of the array of low-abundance chemicals to which humans are exposed.
The resurgence of research into cancer metabolism has recently broadened interests beyond glucose and the Warburg effect to other nutrients, including glutamine. Because oncogenic alterations of metabolism render cancer cells addicted to nutrients, pathways involved in glycolysis or glutaminolysis could be exploited for therapeutic purposes. In this Review, we provide an updated overview of glutamine metabolism and its involvement in tumorigenesis in vitro and in vivo, and explore the recent potential applications of basic science discoveries in the clinical setting.
Ozone is a highly reactive environmental toxicant that can react with the double bonds of lipids in pulmonary surfactant. This study was undertaken to investigate the proinflammatory properties of the major lipid-ozone product in pulmonary surfactant, 1-palmitoyl-2-(9’-oxo-nonanoyl)-glycerophosphocholine (16:0/9al-PC), with respect to eicosanoid production. A dose-dependent increase in the formation of 5-lipoxygenase (5-LO) products was observed in murine resident peritoneal macrophages (RPM) and alveolar macrophages (AM) upon treatment with 16:0/9al-PC. In contrast, the production of cyclooxygenase (COX) derived eicosanoids did not change from basal levels in the presence of 16:0/9al-PC. When 16:0/9al-PC and the TLR2 ligand, zymosan, were added to RPM or AM, an enhancement of 5-LO product formation along with a concomitant decrease in COX product formation was observed. Neither intracellular calcium levels or arachidonic acid release were influenced by the addition of 16:0/9al-PC to RPM. Results from mitogen-activated protein kinase (MAPK) inhibitor studies and direct measurement of phosphorylation of MAPKs revealed that 16:0/9al-PC activates the p38 MAPK pathway in RPM, which results in activation of 5-LO. Our results indicate that 16:0/9al-PC has a profound effect on the eicosanoid pathway, which may have implications in inflammatory pulmonary disease states where eicosanoids have been shown to play 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.
Context:
Evidence from recent decades supports a causal association between air pollution (particulate matter <10 μ m in diameter [PM10] and PM <2.5 μ m in diameter [PM2.5]) and oxidative stress, possibly involving impaired metabolism of glucose and lipids.
Objective:
Using a satellite based model to assess PM exposure at 1-km spatial resolution, we examined the associations between PM and glucose, hemoglobin A1c (HbA1c), and lipids.
Design:
Population-based retrospective cohort study of a 10-year period.
Setting:
Members of the largest health care provider in Southern Israel.
Participants:
We included all serum glucose, HbA1c, and lipids tests of subjects with known cardiovascular diseases and risk factors. Subjects' glycemic status was defined as normal or diabetes.
Main outcome:
Log-transformed glucose, HbA1c, and lipid values were explored by mixed models, with adjustment for personal and seasonal confounders.
Results:
We assessed 73 117 subjects with over 600 000 samples. Three-month average concentration of PM10, but not 1- to 7-d exposure, was associated with increases of serum glucose, HbA1c, low-density lipoprotein and triglycerides, and decrease of high-density lipoprotein. The strongest associations were observed among subjects with diabetes (percent increase [95% confidence interval], for interquartile range increase of PM10 and PM2.5): 3.58% (1.03%; 6.20%) and 2.93% (0.35%; 5.59%) increase in HbA1c and 2.37% (2.11%; 2.63%) and 1.54% (1.26%; 1.83%) increase in low-density lipoprotein. Antidiabetic medications (other than insulin) attenuated the air pollution effect on serum glucose.
Conclusions:
Intermediate-term, but not short term, exposure to PM is associated with alterations in glucose, HbA1c, and lipids, especially among people with diabetes.
Air pollutants have been associated with increased diabetes in humans. We hypothesized that ozone would impair glucose homeostasis by altering insulin signaling and/or endoplasmic reticular (ER) stress in young and aged rats. One, 4, 12, and 24month old Brown Norway (BN) rats were exposed to air or ozone, 0.25 or 1.0ppm, 6h/day for 2days (acute) or 2d/week for 13weeks (subchronic). Additionally, 4month old rats were exposed to air or 1.0ppm ozone, 6h/day for 1 or 2days (time-course). Glucose tolerance tests (GTT) were performed immediately after exposure. Serum and tissue biomarkers were analyzed 18h after final ozone for acute and subchronic studies, and immediately after each day of exposure in the time-course study. Age-related glucose intolerance and increases in metabolic biomarkers were apparent at baseline. Acute ozone caused hyperglycemia and glucose intolerance in rats of all ages. Ozone-induced glucose intolerance was reduced in rats exposed for 13weeks. Acute, but not subchronic ozone increased α2-macroglobulin, adiponectin and osteopontin. Time-course analysis indicated glucose intolerance at days 1 and 2 (2>1), and a recovery 18h post ozone. Leptin increased day 1 and epinephrine at all times after ozone. Ozone tended to decrease phosphorylated insulin receptor substrate-1 in liver and adipose tissues. ER stress appeared to be the consequence of ozone induced acute metabolic impairment since transcriptional markers of ER stress increased only after 2days of ozone. In conclusion, acute ozone exposure induces marked systemic metabolic impairments in BN rats of all ages, likely through sympathetic stimulation.
The kinetics and products of the reaction of ozone with specific amino acids, peptides, and proteins are reviewed based on studies reported in the literature. Ozone reacts mainly with the unprotonated amino group of the acids and the second-order ozone rate constants for these reactions, except for cysteine, methionine, and tryptophan, vary by about two-orders from 2.6 × 10 to 4.4 × 10 Ms. The site of attack on cysteine and methionine by O3 is at the sulfhydryl rather than the amino group to give sequential O-atom addition products. The order of reactivity for the oxidation of amino acids by O3 at pH 8 is cysteine > tryptophan ≈ methionine > phenylalanine ≈ histidine > others, with half-lives mostly in the range of milliseconds to tens of seconds (1 mg L O3 dose). Reactions of O3 with aliphatic amino acids form nitrate, ammonia, and one or two carbon atom-containing carbonyl and carboxylic byproducts. In the ozonolysis of peptides and proteins, oxidation by O3 occurs at the tyrosine, tryptophan, histidine, cysteine, and methionine residues. Oxidation of proteins results in changes in their folding ability and tertiary structures.
Recent epidemiology studies have reported associations between short-term ozone exposure and mortality. Such studies have previously reported associations between airborne particulate matter pollution and mortality, and support for a causal relationship has come from controlled-exposure studies that describe pathophysiological mechanisms by which particulate matter could induce acute mortality. In contrast, for ozone, almost no controlled-human-exposure studies have tested whether ozone exposure can modulate the cardiovascular system.
Twenty-three young healthy individuals were exposed in a randomized crossover fashion to clean air and to 0.3-ppm ozone for 2 hours while intermittently exercising. Blood was obtained immediately before exposure, immediately afterward, and the next morning. Continuous Holter monitoring began immediately before exposure and continued for 24 hours. Lung function was performed immediately before and immediately after exposure, and bronchoalveolar lavage was performed 24 hours after exposure. Immediately after ozone exposure, we observed a 98.9% increase in interleukin-8, a 21.4% decrease in plasminogen activator inhibitor-1, a 51.3% decrease in the high-frequency component of heart rate variability, and a 1.2% increase in QT duration. Changes in interleukin-1B and plasminogen activator inhibitor-1 were apparent 24 hours after exposure. In agreement with previous studies, we also observed ozone-induced drops in lung function and an increase in pulmonary inflammation.
This controlled-human-exposure study shows that ozone can cause an increase in vascular markers of inflammation and changes in markers of fibrinolysis and markers that affect autonomic control of heart rate and repolarization. We believe that these findings provide biological plausibility for the epidemiology studies that associate ozone exposure with mortality.
URL: http://www.clinicaltrials.gov. Unique identifier: NCT01492517.
Lung inflammation resulting from oxidant/antioxidant imbalance is a common feature of many lung diseases. In particular, the role of enzymes regulated by the NF-E2-related factor 2 transcription factor has recently received increased attention. Among these antioxidant genes, glutathione S-transferase Mu 1 (GSTM1) has been most extensively characterized because it has a null polymorphism that is highly prevalent in the population and associated with increased risk of inflammatory lung diseases. Present evidence suggests that GSTM1 acts through interactions with other genes and environmental factors, especially air pollutants. Here, we review GSTM1 gene expression and regulation and summarize the findings from epidemiological, clinical, animal, and in vitro studies on the role played by GSTM1 in lung inflammation. We discuss limitations in the existing knowledge base and future perspectives and evaluate the potential of pharmacologic and genetic manipulation of the GSTM1 gene to modulate pulmonary inflammatory responses.
Plasmalogens are a unique class of membrane glycerophospholipids containing a fatty alcohol with a vinyl-ether bond at the sn-1 position, and enriched in polyunsaturated fatty acids at the sn-2 position of the glycerol backbone. These two features provide novel properties to these compounds. Although plasmalogens represent up to 20% of the total phospholipid mass in humans their physiological roles have been challenging to identify, and are likely to be particular to different tissues, metabolic processes and developmental stages. Their biosynthesis starts in peroxisomes, and defects at these steps cause the malformation syndrome, Rhizomelic Chondrodysplasia Punctata (RCDP). The RCDP phenotype predicts developmental roles for plasmalogens in bone, brain, lens, lung, kidney and heart. Recent studies have revealed secondary plasmalogen deficiencies associated with more common disorders and allow us to tease out additional pathways dependent on plasmalogen functions. In this review, we present current knowledge of plasmalogen biology in health and disease. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of peroxisomes in Health and Disease.
Adverse health effects from air pollutants remain important, despite improvement in air quality in the past few decades. The exact mechanisms of lung injury from exposure to air pollutants are not yet fully understood. Studying the genome (e.g. single-nucleotide polymorphisms (SNP) ), epigenome (e.g. methylation of genes), transcriptome (mRNA expression) and microRNAome (microRNA expression) has the potential to improve our understanding of the adverse effects of air pollutants. Genome-wide association studies of SNP have detected SNP associated with respiratory phenotypes; however, to date, only candidate gene studies of air pollution exposure have been performed. Changes in epigenetic processes, such DNA methylation that leads to gene silencing without altering the DNA sequence, occur with air pollutant exposure, especially global and gene-specific methylation changes. Respiratory cell line and animal models demonstrate distinct gene expression signatures in the transcriptome, arising from exposure to particulate matter or ozone. Particulate matter and other environmental toxins alter expression of microRNA, which are short non-coding RNA that regulate gene expression. While it is clearly important to contain rising levels of air pollution, strategies also need to be developed to minimize the damaging effects of air pollutant exposure on the lung, especially for patients with chronic lung disease and for people at risk of future lung disease. Careful study of genomic responses will improve our understanding of mechanisms of lung injury from air pollution and enable future clinical testing of interventions against the toxic effects of air pollutants.
Lung cancer is one of the most common cancers in the world, but no good clinical markers that can be used to diagnose the disease at an early stage and predict its prognosis have been found. Therefore, the discovery of novel clinical markers is required. In this study, metabolomic analysis of lung cancer patients was performed using gas chromatography mass spectrometry. Serum samples from 29 healthy volunteers and 33 lung cancer patients with adenocarcinoma (n=12), squamous cell carcinoma (n=11), or small cell carcinoma (n=10) ranging from stage I to stage IV disease and lung tissue samples from 7 lung cancer patients including the tumor tissue and its surrounding normal tissue were used. A total of 58 metabolites (57 individual metabolites) were detected in serum, and 71 metabolites were detected in the lung tissue. The levels of 23 of the 58 serum metabolites were significantly changed in all lung cancer patients compared with healthy volunteers, and the levels of 48 of the 71 metabolites were significantly changed in the tumor tissue compared with the non-tumor tissue. Partial least squares discriminant analysis, which is a form of multiple classification analysis, was performed using the serum sample data, and metabolites that had characteristic alterations in each histological subtype and disease stage were determined. Our results demonstrate that changes in metabolite pattern are useful for assessing the clinical characteristics of lung cancer. Our results will hopefully lead to the establishment of novel diagnostic tools.
Ozone is a common environmental toxicant to which individuals are exposed to on a daily basis. While biochemical end points such as increased mortality, decrements in pulmonary function, and initiation of inflammatory processes are known, little is actually understood regarding the chemical mechanisms underlying changes in pulmonary health, especially for low concentrations of ozone. This study was undertaken to investigate ozone-induced oxidation of endogenous lipids that are potentially exposed to environmental ozone within lung, specifically focusing on plasmalogen glycerophospholipids present in pulmonary surfactant. Sensitive liquid chromatography-mass spectrometry methods were developed to follow oxidation of diacyl and plasmalogen phosphatidylethanolamine (PE) phospholipids and to identify and quantitate products generated by ozonolysis. Using a unilamellar vesicle system containing a 1:1 molar mixture of 1-O-octadec-1'-enyl-2-octadecenoyl-PE and 1,2-dihexadecanoyl-PC, these studies revealed that the vinyl ether bond of plasmalogens was oxidized preferentially at low concentrations of ozone (100 ppb), when compared to olefinic bond oxidation on omega-9 of the fatty acyl chain in the same phospholipids. Major phospholipid products generated were identified as 1-formyl-2-octadecenoyl-PE and 1-hydroxy-2-octadecenoyl-PE. Heptadecanal and heptadecanoic acid production was also quantitated using gas chromatography-mass spectrometry, and production was consistent with oxidation of the vinyl ether, at low concentrations of ozone. Analysis of murine lung surfactant from C57Bl/6 mice revealed several plasmalogen PE lipid species, encompassing approximately 38% of total PE species. Upon exposure of ozone (0 and 100 ppb) to murine surfactant, plasmalogen PE molecular species preferentially reacted, as compared to diacyl PE molecular species. Lysophospholipids, pentadecanal, and nonanal were found to be the primary products of surfactant ozone oxidation.
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.
The purpose of this study was to determine whether the GSTM1 null genotype modulates ozone responses in human subjects.
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.
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.
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.
Based on available knowledge, this study shows that alpha-1-proteinase inhibitor (alpha 1-PI) plays an important role in protecting lung elastin from elastolytic proteinases, particularly human neutrophil elastase (HNE). Studies previous to this one showed that alpha 1-PI was very susceptible to inactivation by oxidants. We sought to use this oxidant sensitivity as an in vivo marker for ozone (O3) and nitrogen dioxide (NO2) exposure. The mechanism of alpha 1-PI inactivation by O3 and NO2 was examined to provide insight concerning the pathogenesis of oxidant-mediated lung damage. Attention also was focused on the bronchial leukocyte proteinase inhibitor (BLPI), which inhibits HNE in the bronchial secretions. Careful examination of blood plasma samples from individuals exposed to 0.5 ppm O3 for four hours on two consecutive days failed to detect any inactivation of alpha 1-PI. This result showed that blood alpha 1-PI was not a satisfactory marker for O3 exposure, but, more importantly, demonstrated that inhaling O3 for short periods does not grossly inactivate this important protein. Studies on BLPI showed that it is a significant inhibitor of HNE and probably plays a more important role in protecting the lung than previously thought. BLPI, like alpha 1-PI, was found to be inactivated by oxidants, including O3 and NO2. The mechanism of O3 inactivation of leukocyte proteinase inhibitors was studied using alpha 1-PI, alpha-1-antichymotrypsin (alpha 1-Achy), BLPI, and Eglin C. While all these inhibitors differed in structure, the concentrations of O3 required for inactivation were essentially the same, except for alpha 1-Achy, which only lost half of its inhibitory activity. It would seem from these results that O3 can damage proteins via the oxidation of any of the following: tryptophan (Trp), methionine (Met), tyrosine (Tyr), or histidine (His) residues. Interestingly, Eglin C, which does not have oxidizable amino acids in its inhibitory active site, was inactivated by the same amount of O3 as BLPI, BLPI was easily inactivated by a methionine-specific oxidant, suggesting an important role for methionine in this inhibitor. In vitro exposure of alpha 1-PI and BLPI to 800 moles of NO2 per mole of inhibitor resulted in 35% and 50% losses of HNE inhibitory activity, respectively. Tryptophan was destroyed by NO2 and studies are in progress to examine effects on other amino acids.
Ozone effects on lung mitochondrial oxidative metabolism were examined after short-term exposure of rats and monkeys to O3. Exposure of animals to 2 ppm O3 for 8 hr or to 4 ppm O3 for 4 hr caused a 15–27% (P < 0.05) depression of lung mitochondrial O2 consumption, using 2-oxoglutarate, succinate, and glycerol-1-phosphate. but not ascorbate plus Wurster's blue as substrates. Under these exposure conditions (4 ppm 4 hr) the ADP:O ratios dropped 25–36% (P < 0.05) and the respiratory control indices decreased 27–33% (P < 0.02) for oxidation of all substrates examined. Lung mitochondria from control animals were relatively impermeable to added NADH, but those from O3-exposed animals showed an increased permeability as judged from NADH oxidation at a rate 3-fold higher than the control. Likewise, added cytochrome c caused a 22% (P < 0.01) stimulation of succinate oxidation in exposed lung mitochondria as against 5% (nonsignificant) in controls. Ozone exposure also caused a 20% (P < 0.01) oxidation of thiol groups in lung mitochondria, but no lipid peroxidation products were detectable in O3-exposed lung tissue. The depression of substrate utilization, coupled phosphorylation and respiratory control observed in lung mitochondria of O3-exposed animals might be related to alteration of membrane permeability, and inhibition of respiratory enzymes (dehydrogenases) due to oxidation of functional thiol groups.
Circular dichroism and potentiometric titration studies of leucine random copolymers in aqueous solutions, as well as a comparison of the conformational stability in poly-α-amino acids, indicate that leucine may possibly be the amino acid with the highest propensity for forming α-helical structures. This suggests that leucine might be found most frequently in the helical regions of proteins. A survey was made on 15 different proteins containing 2473 residues with known sequence and conformation determined by X-ray crystallography: carboxy-peptidase A, α-chymotrypsin, cytochrome b5, elastase, ferricytochrome c, α- and β-hemoglobin, insulin, lysozyme, myogen, myoglobin, papain, ribonuclease A, staphylococcal nuclease, and subtilisin BPN′. It was found that 888 residues in these proteins are in helices, and 422 of them reside in the internal turns of helical regions. While Glu, Ala, Leu and His were found to be present with the highest percentages in helical regions, Leu was clearly the most abundant residue in the inner helical cores of proteins. Polar residues are found preferentially at the helix-coil boundary regions; Asp and Glu at the N-terminal and His, Lys and Arg at the C-terminal helical ends. These findings agree with Ptitsyn's (1969) analysis on seven proteins containing 1132 residues. A more comprehensive analysis in the present survey showed that Ile, Met and Val occur with the greatest frequency in the β-regions of proteins. Leu was also found as the strongest structure-forming residue in proteins (total helical and β-regions). The functional-structural role of leucine was established by showing that it occurs most frequently among residues surrounding the heme in five of the heme proteins. In addition, the greater abundance of leucine as neighbors to active-site residues in enzymes provides strong evidence that hydrophobic residues create a non-aqueous environment, aiding the polar residues in substrate binding and enzymic catalysis. Examples of conservative and non-conservative mutations of leucine in heme proteins are given to illustrate the structure—function relation of proteins, and explain why most leucine residues in the insulin, hemoglobin, and cytochrome c homologs are invariant. Finally, the strong helical-forming power of leucine, as demonstrated experimentally in synthetic copolypeptides and its high occurrence in the inner helical cores of proteins, suggests that it could have a major role as nucleation centers in the folding and evolution of large protein molecules.
Aminoguanidine produces a time-dependent inactivation of the citrulline forming activity of all three nitric oxide synthase isoforms that is blocked by arginine. Aminoguanidine inactivates both the NADPH oxidase and citrulline forming activities of GH3 pituitary constitutive nitric oxide synthase (cNOS) but does not alter its cytochrome c reductase activity. GH3 pituitary cells contain an NOS isoform identical physically, kinetically, and immunologically to cerebellar neuronal NOS (Wolff and Datto, Biochemical J. (1992) 285, 201-206). The inactivation of GH3 cNOS NADPH oxidase activity, as measured without added tetrahydrobiopterin cofactor, is saturable, is inhibited by arginine, and follows pseudo-first-order kinetics with an inactivation rate constant of 0.25 min-1 and a Ki value of 0.83 mM aminoguanidine. The inactivation of the citrulline forming activity of GH3 cNOS by aminoguanidine was not saturable by aminoguanidine. Aminoguanidine, at concentrations in the millimolar range, inhibited the citrulline forming activity of endothelial cNOS by an apparently nonsaturable mechanism. Aminoguanidine inactivates the citrulline forming activity of murine macrophage iNOS. The inactivation is saturable and follows pseudo-first-order kinetics with an inactivation rate constant of 0.46 min-1 and a Ki value of 16 microM. The inactivation of the constitutive isoforms of nitric oxide synthase by aminoguanidine required the concurrent presence of Ca2+, calmodulin, NADPH, tetrahydrobiopterin, and oxygen in preincubations and was not reversed either by dilution or dialysis. These observations support the assertion that aminoguanidine is a mechanism-based inactivator of the nitric oxide synthase isoforms and exhibits marked specificity for the inactivation of the inducible isoform.
Nitric oxide (NO) is synthesized from arginine by nitric-oxide synthase (NOS), and citrulline that is generated can be recycled to arginine by argininosuccinate synthase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS) and expression of the inducible isoform of NOS (iNOS), AS and AL was analysed. In RNA blot analysis, iNOS mRNA was induced by LPS in the lung, heart, liver and spleen, and less strongly in the skeletal muscle and testis. AS and AL mRNAs were induced in the lung and spleen. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2–5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6–12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high upto 24 h. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS and AL were co-induced in rat tissues and cells, citrulline–arginine recycling seems to be important in NO synthesis under the conditions of stimulation.
Arginine is a common substrate of NOS and arginase. Rat peritoneal macrophages were cultured in the presence of LPS and expression of iNOS and liver-type arginase (arginase I) was analysed. mRNAs for iNOS and arginase I were induced by LPS in a dose-dependent manner. iNOS mRNA appeared 2 h after LPS treatment and increased up to a near-maximum at 8–12 h. On the other hand, arginase I mRNA began to increase after 4 h with a lag time and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and arginase I proteins were also induced. Induction of iNOS and arginase I mRNAs were also observed in LPS-injected rats in vivo. Thus, arginase I appears to have an important role in downregulating NO synthesis in murine macrophages by decreasing the availability of arginine.
A cDNA for human arginase II, an arginase isozyme, was isolated. A polypeptide of 354 amino acid residues including the putative NH2-terminal presequence for mitochondrial import was predicted. It was 59% identical with arginase I. mRNA for human arginase II was present in the kidney and other tissues but was not detected in the liver. Arginase II mRNA was co-induced with iNOS mRNA in murine macrophage-like RAW 264.7 cells by LPS. This induction was enhanced by dexamethasone and dibutyrul cAMP, and was prevented by interferon-γ.
These results indicate that NO synthesis is regulated by arginine-synthesizing and -degrading enzymes in a complicated manner.
We have proposed that exposure of epithelial cell membrane lipids in the lung (mainly phospholipids) to ozone will generate lipid ozonation products (LOP), which could be responsible for the proinflammatory effects of ozone. The ozonation of phosphocholine, the principal membrane phospholipid, produces a limited number of LOP, including hydroxyhydroperoxides and aldehydes. We now report that exposure of cultured human bronchial epithelial cells to the ozonized 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) product, 1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphocholine (PC-ALD), a phospholipase A(2) (PLA(2))-stimulatory LOP, resulted in a 113 +/- 11% increase in the amounts of tritiated platelet-activating factor ((3)H-PAF) released apically. (3)H-PAF release was also induced by 1-hydroxy-1-hydroperoxynonane of ozonized POPC (HHP-C9), a phospholipase C (PLC)- stimulatory LOP (134 +/- 40% increase in (3)H-PAF). PC-ALD at 10 microM, but not HHP-C9, induced a 127 +/- 24% increase in prostaglandin E(2) (PGE(2)) release (n = 6, p < 0.05). In contrast, HHP-C9, but not PC-ALD, induced interleukin (IL)-6 release (178 +/- 23% increase, n = 6, p < 0.05) and IL-8 release (101 +/- 23% increase, n = 8, p < 0. 05). These results suggest that LOP-dependent release of proinflammatory mediators may play an important role in the early inflammatory response seen during exposure to ozone.
Nitric oxide (NO) is increased in exhaled air of asthmatics. We hypothesized that endogenous NO contributes to airway inflammation and hyperresponsiveness, and that interleukin-8 (IL-8) might be involved in this mechanism. In human transformed bronchial epithelial cells in vitro, NO donors increased IL-8 production dose-dependently. In addition, tumor necrosis factor-alpha (TNF-alpha) plus IL-1beta plus interferon-gamma (IFN-gamma) increased IL-8 in culture supernatant of epithelial cells; the combination of NO synthase (NOS) inhibitors, aminoguanidine (AG) plus N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the cytokine-induced IL-8 production in epithelial cells. In guinea pigs in vivo, ozone exposure induced airway hyperresponsiveness to acetylcholine and increased neutrophils in bronchoalveolar lavage fluid (BALF), and these changes persisted for at least 5 h. Pretreatment with NOS inhibitors had no effect on airway hyperresponsiveness or neutrophil accumulation immediately after ozone, but significantly inhibited the changes 5 h after ozone. NOS inhibitors also attenuated the increases of nitrite/nitrate levels in BALF and the IL-8 mRNA expression in epithelial cells and in neutrophils in guinea pig airways 5 h after ozone. These results suggest that endogenous NO may play an important role in the persistent airway inflammation and hyperresponsiveness after ozone exposure, presumably partly through the upregulation of IL-8.