Errol M. Thomson

Mapping Acute Systemic Effects of Inhaled Particulate Matter and Ozone: Multiorgan Gene Expression and Glucocorticoid Activity

Recent epidemiological studies have demonstrated associations between air pollution and adverse effects that extend beyond respiratory and cardiovascular disease, including low birth weight, appendicitis, stroke, and neurological/neurobehavioural outcomes (e.g., neurodegenerative disease, cognitive decline, depression, and suicide). To gain insight into mechanisms underlying such effects, we mapped gene profiles in the lungs, heart, liver, kidney, spleen, cerebral hemisphere, and pituitary of male Fischer-344 rats immediately and 24h after a 4-h exposure by inhalation to particulate matter (0, 5, and 50mg/m3 EHC-93 urban particles) and ozone (0, 0.4, and 0.8 ppm). Pollutant exposure provoked differential expression of genes involved in a number of pathways, including antioxidant response, xenobiotic metabolism, inflammatory signalling, and endothelial dysfunction. The mRNA profiles, while exhibiting some interorgan and pollutant-specific differences, were remarkably similar across organs for a set of genes, including increased expression of redox/glucocorticoid-sensitive genes and decreased expression of inflammatory genes, suggesting a possible hormonal effect. Pollutant exposure increased plasma levels of adrenocorticotropic hormone and the glucocorticoid corticosterone, confirming activation of the hypothalamic-pituitary-adrenal axis, and there was a corresponding increase in markers of glucocorticoid activity. Although effects were transient and presumably represent an adaptive response to acute exposure in these healthy animals, chronic activation and inappropriate regulation of the hypothalamic-pituitary-adrenal axis are associated with adverse neurobehavioral, metabolic, immune, developmental, and cardiovascular effects. The experimental data are consistent with epidemiological associations of air pollutants with extrapulmonary health outcomes and suggest a mechanism through which such health effects may be induced.

Cytotoxic and inflammatory potential of size-fractionated particulate matter collected repeatedly within a small urban area.

BackgroundExposure to coarse, fine, and ultrafine particles is associated with adverse population health impacts. We investigated whether size-fractionated particles collected repeatedly in the vicinity of industrial (steel mills and associated coking operations, wastewater treatment), high traffic, and residential areas display systematic differences in biological potency.MethodsParticulate matter (PM<0.1, PM0.1–0.5, PM0.5–2.5, PM2.5–10, PM>10) samples collected at sites within Windsor, Ontario, were screened for biological potency in human A549 lung epithelial and murine J774A.1 macrophage-like cells using cytotoxicity bioassays (cellular ATP, resazurin reduction, lactate dehydrogenase (LDH) release), cytokine production, and transcript profiles. Potency was determined from the slope of each dose-effect relationship.ResultsCytotoxic potency varied across size fractions and within a fraction across sites and sampling periods, suggesting that particle composition, in addition to size and mass, affected particle toxicity. While ATP and LDH profiles showed some similarity, resazurin reduction (a measure of metabolic activity) exhibited a unique pattern of response, indicating that the cytotoxicity assays were sensitive to distinct particle characteristics. Chemical speciation varied in relation to prevailing winds, consistent with enrichment of source emissions (e.g. higher metal and polycyclic aromatic hydrocarbon content downwind of the industrial site). Notwithstanding this variability, site-dependent differences in particle toxicity were evident, including greater potency of coarse fractions at the industrial site and of ultrafine particles at the traffic site (Site × Size interactions, p < 0.05). Regression of potency against particle constituents revealed correlations between resazurin reduction, induction of metal-responsive genes, and metal content, which were particularly strong for the coarse fraction, and between cytokine release and endotoxin, suggesting that these factors were important drivers of biological effects that explain, at least in part, the contrasting potencies of particles compared on an equivalent mass basis.ConclusionsThe data show that 1) particle potency and composition can exhibit significant temporal variation in relation to source contributions; 2) sources may differentially impact the potency of specific size fractions; and 3) particle constituents, notably metals and endotoxin, may elicit distinct biological responses. Together, the data are consistent with the notion that sources and composition, in addition to size and mass concentration, are relevant to particle toxicity.

Overexpression of Tumor Necrosis Factor-α in the Lungs Alters Immune Response, Matrix Remodeling, and Repair and Maintenance Pathways

Increased production of tumor necrosis factor (TNF)-α and matrix metalloproteinases (MMPs) is a feature of inflammatory lung diseases, including emphysema and fibrosis, but the divergent pathological characteristics that result indicate involvement of other processes in disease pathogenesis. Transgenic mice overexpressing TNF-α in type II alveolar epithelial cells under the control of the surfactant protein (SP)-C promoter develop pulmonary inflammation and emphysema but are resistant to induction of fibrosis by administration of bleomycin or transforming growth factor-β. To study the molecular mechanisms underlying the development of this phenotype, we used a microarray approach to characterize the pulmonary transcriptome of SP-C/TNF-α mice and wild-type littermates. Four-month-old SP-C/TNF-α mice displayed pronounced pulmonary inflammation, airspace enlargement, increased MMP-2 and MMP-9 levels, and altered expression of 2332 probes. The functional assessment of genes with increased expression revealed enrichment of inflammatory/immune responses and proteases, whereas genes involved in protease inhibition, angiogenesis, cross-linking of basement membrane proteins, and myofibroblast differentiation were predominantly decreased. Comparison with multiple lung disease models identified a set of genes unique to the SP-C/TNF-α model and revealed that lack of extracellular matrix production distinguished SP-C/TNF-α mice from fibrosis models. Activation of inflammatory and proteolytic pathways and disruption of maintenance and repair processes are central features of emphysema in this TNF-overexpression model. Impairment of myofibroblast differentiation and extracellular matrix production may underlie resistance to induction of fibrosis.

Effects of ambient air particles on the endothelin system in human pulmonary epithelial cells (A549)

Inhalation of urban particles results in higher circulating levels of the vasoconstrictor peptide endothelin-1 (ET-1), which may account for the adverse cardiovascular impacts associated with air pollution. The objective of this study was to examine the direct effects of urban particles on the production of ET-1 by human epithelial cells (A549). A549 cells were exposed to TiO2, SiO2, Ottawa urban particulate matter EHC-93, and fractions of the urban particles. The levels of ET-1, interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) in the culture medium were detected by ELISA. The mRNA levels of preproET-1, endothelin converting enzyme (ECE-1), ETa receptor and ETb receptor, matrix metalloproteinase (MMP-2), tissue inhibitor of MMP (TIMP-2), and heat shock protein (HSP-70) were determined by quantitative real-time RT-PCR. Cluster analysis of the variables identified similarities in the patterns of effects. Cluster I comprised variables that were primarily inhibited by particles: ET-1 and MMP-2 mRNAs, ET-1 and bigET-1 peptides, and cell viability. Clusters II and III comprised variables that were either inhibited or induced, depending on the test material: HSP-70, ETaR and ECE mRNAs, and IL-8 and VEGF proteins. Cluster IV comprised variables that were mainly induced by particle preparations: ETbR and TIMP-2 mRNAs. The decreased expression of preproET-1 in A549 cells suggests that epithelial cells may not be the source of higher pulmonary ET-1 spillover in the circulation measured in vivo in response to inhaled urban particles. However, higher ECE-1 in A549 cells after exposure to particles suggests an increased ability to process bigET-1 into the mature ET-1 peptide, while increased receptor expression implies higher responsiveness. The increased release of IL-8 and VEGF by epithelial cells in response to particles could possibly upregulate ET-1 production in the adjacent pulmonary capillary endothelial cells, with concomitant increased ET-1 spillover in the systemic circulation.

Pulmonary Expression of PreproET-1 and PreproET-3 mRNAs Is Altered Reciprocally in Rats After Inhalation of Air Pollutants

Perturbation of vascular homeostasis is an important mechanism related to the acute health effects of inhaled pollutants. Inhalation of urban particulate matter and ozone by rats has been shown to result in increased synthesis of the potent vasoactive peptide endothelin (ET)-1 in the lungs, with spillover into the circulation. In the present work, we have analyzed the interrelationships between responses of the three major endothelin isoforms, ET-1[1–21], ET-2[1–21], and ET-3[1–21], to inhaled pollutants at the peptide and gene expression levels. Fisher-344 rats were exposed for 4 hrs by nose-only route to clean air, urban particles EHC-93 (0, 50 mg/m3), ozone (0, 0.8 ppm), or ozone and particles together. Circulating levels of both the ET-1 [1–21] and ET-3[1–21] peptides were increased immediately after exposure to particulate matter or ozone. While expression of preproET-1 mRNA in the lungs increased, expression of preproET-3 mRNA decreased immediately after exposure. PreproET-2 mRNA was not detected in the lungs, and exposure to either pollutant did not affect plasma ET-2 levels. Co-exposure to ozone and particles, while altering lung preproET-1 and preproET-3 mRNA levels in a fashion similar to ozone alone, did not cause changes in the circulating levels of the two corresponding peptides. Thus, de novo synthesis of ET-3 in the lungs is not responsible for the increase of circulating plasma ET-3 after inhalation of pollutants, which implies regulation of preproET-3 at a remote site and, hence, systemic impacts of the pollutants. Upregulation of preproET-1 coupled with down-regulation of preproET-3 in the lungs of animals exposed to air pollutants implies a mismatch of local ET-1/ETA receptor–mediated vasoconstriction and ET-3/ETB receptor–mediated vasodilation.

Nitrogen Dioxide and Ultrafine Particles Dominate the Biological Effects of Inhaled Diesel Exhaust Treated by a Catalyzed Diesel Particulate Filter

We studied the impact of a catalyzed diesel particulate filter (DPF) on the toxicity of diesel exhaust. Rats inhaled exhaust from a Cummins ISM heavy-duty diesel engine, with and without DPF after-treatment, or HEPA-filtered air for 4h, on 1 day (single exposure) and 3 days (repeated exposures). Biological effects were assessed after 2h (single exposure) and 20h (single and repeated exposures) recovery in clean air. Concentrations of pollutants were (1) untreated exhaust (-DPF), nitric oxide (NO), 43 ppm; nitrogen dioxide (NO2), 4 ppm; carbon monoxide (CO), 6 ppm; hydrocarbons, 11 ppm; particles, 3.2×10(5)/cm(3), 60-70nm mode, 269 μg/m(3); (2) treated exhaust (+DPF), NO, 20 ppm; NO2, 16 ppm; CO, 1 ppm; hydrocarbons, 3 ppm; and particles, 4.4×10(5)/cm(3), 7-8nm mode, 2 μg/m(3). Single exposures to -DPF exhaust resulted in increased neutrophils, total protein and the cytokines, growth-related oncogene/keratinocyte chemoattractant, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1 in lung lavage fluid, as well as increased gene expression of interleukin-6, prostaglandin-endoperoxide synthase 2, metallothionein 2A, tumor necrosis factor-α, inducible nitric oxide synthase, glutathione S-transferase A1, heme oxygenase-1, superoxide dismutase 2, endothelin-1 (ET-1), and endothelin-converting enzyme-1 in the lung, and ET- 1 in the heart. Ratio of bigET-1 to ET-1 peptide increased in plasma in conjunction with a decrease in endothelial nitric oxide synthase gene expression in the lungs after exposure to diesel exhaust, suggesting endothelial dysfunction. Rather than reducing toxicity, +DPF exhaust resulted in heightened injury and inflammation, consistent with the 4-fold increase in NO2 concentration. The ratio of bigET-1 to ET-1 was similarly elevated after -DPF and +DPF exhaust exposures. Endothelial dysfunction, thus, appeared related to particle number deposited, rather than particle mass or NO2 concentration. The potential benefits of particulate matter reduction using a catalyzed DPF may be confounded by increase in NO2 emission and release of reactive ultrafine particles.

Exposure to air pollution near a steel plant and effects on cardiovascular physiology: A randomized crossover study

BACKGROUND Iron and steel industry is an important source of air pollution emissions. Few studies have investigated cardiovascular effects of air pollutants emitted from steel plants. OBJECTIVE We examined the influence of outdoor air pollution in the vicinity of a steel plant on cardiovascular physiology in Sault Ste. Marie, Canada. METHODS Sixty-one healthy, non-smoking subjects (females/males=33/28, median age 22 years) spent 5 consecutive 8-hour days outdoors in a residential area neighbouring a steel plant, or on a college campus approximately 5 kilometres away from the plant, and then crossed over to the other site with a 9-day washout. Mid day, subjects underwent daily 30-minute moderate intensity exercise. Blood pressure (BP) and pulse rate were determined daily and post exercise at both sites. Flow-mediated vasodilation (FMD) was determined at the site near the plant. Air pollution was monitored at both sites. Mixed-effects regressions were run for statistical associations, adjusting for weather variables. RESULTS Concentrations of ultrafine particles, sulphur dioxide (SO2), nitrogen dioxide (NO2) and carbon monoxide (CO) were 50-100% higher at the site near the plant than at the college site, with minor differences in temperature, humidity, and concentrations of particulate matter ≤2.5 μm in size (PM2.5) and ozone (O3). Resting pulse rate [mean (95% confidence interval)] was moderately higher near the steel plant [+1.53 bpm (0.31, 2.78)] than at the college site, male subjects having the highest pulse rate elevation [+2.77 bpm (0.78, 4.76)]. Resting systolic and diastolic BP and pulse pressure, and post-exercise BP and pulse rate were not significantly different between two sites. Interquartile range concentrations of SO2 (2.9 ppb), NO2 (5.0 ppb) and CO (0.2 ppm) were associated with increased pulse rate [0.19 bpm (-0.00, 0.38), 0.86 bpm (0.03, 1.68), and 0.11 bpm (0.00, 0.22), respectively], ultrafine particles (10,256 count/cm(3)) associated with increased pulse pressure [0.85 mmHg (0.23, 1.48)], and NO2 and CO inversely associated with FMD [-0.14% (-0.31, 0.02), -0.02% (-0.03, -0.00), respectively]. SO2 during exercise was associated with increased pulse rate [0.26 bpm (0.01, 0.51)]. CONCLUSION Air quality in residential areas near steel plants may influence cardiovascular physiology.

Ozone Inhalation Provokes Glucocorticoid-Dependent and -Independent Effects on Inflammatory and Metabolic Pathways

Growing evidence implicates air pollutants in adverse health effects beyond respiratory and cardiovascular disease, including metabolic impacts (diabetes, metabolic syndrome, obesity) and neurological/neurobehavioral outcomes (neurodegenerative disease, cognitive decline, perceived stress, depression, suicide). We have shown that inhalation of particulate matter or ozone activates the hypothalamic-pituitary-adrenal axis in rats and increases plasma levels of the glucocorticoid corticosterone. To investigate the role of corticosterone in mediating inflammatory and metabolic effects of pollutant exposure, in this study male Fischer-344 rats were administered the 11β-hydroxylase inhibitor metyrapone (0, 50, 150 mg/kg body weight) and exposed by nose-only inhalation for 4 h to air or 0.8 ppm ozone. Ozone inhalation provoked a 2-fold increase in plasma corticosterone, an effect blocked by metyrapone, but did not alter epinephrine levels. Inhibition of corticosterone production was associated with increased inflammatory signaling in the lungs and plasma in response to ozone, consistent with a role for glucocorticoids in limiting local and systemic inflammatory responses. Effects of ozone on insulin and glucagon, but not ghrelin or plasminogen activator inhibitor-1, were modified by metyrapone, revealing glucocorticoid-dependent and -independent effects on circulating metabolic and hemostatic factors. Several immunosuppressive and metabolic impacts of ozone in the lungs, heart, liver, kidney, and spleen were blocked by metyrapone and reproduced through exogenous administration of corticosterone (10 mg/kg body weight), demonstrating glucocorticoid-dependent effects in target tissues. Our results support involvement of endogenous glucocorticoids in ozone-induced inflammatory and metabolic effects, providing insight into potential biological mechanisms underlying health impacts and susceptibility.

Impact of nose-only exposure system on pulmonary gene expression

Nose-only exposure is used to study the distribution and toxicity of airborne contaminants. Restraint of animals in nose-only tubes causes stress, but the impact on pulmonary mRNA levels is unknown. Since stress and xenobiotics activate common pathways, we assessed whether nose-only exposure would alter expression of toxicologically relevant genes in the lungs. To identify candidate genes for further analysis, we first interrogated microarray data to examine time-dependent changes in gene expression in air-control animals from a nose-only inhalation study involving male wild-type C57BL/6 mice and transgenic tumor necrosis factor (TNF)-α over-expressing littermates. Comparison of transcript levels immediately and 24 h after a single 4-h nose-only exposure to air revealed differential expression of 280 genes (false discovery rate-adjusted, p < .05). Functional analysis revealed enrichment of immune response, apoptosis, and signalling terms, consistent with effects of restraint stress. We then selected a subset of target genes for comparison of naive animals and air-exposed animals from the inhalation study by real-time polymerase chain reaction (PCR). Expression of genes involved in stress (BNIP, sestrin-1, CDKN1A [p21], GADD45γ), glucocorticoid-response (GILZ, Sgk), and signal transduction (MAP3K6, C/EBP-δ) was increased as a result of nose-only exposure (p < .05). In contrast, proinflammatory factors (lymphotoxin-β, chemokine receptor CXCR5) were decreased (p < .05). Immune gene responses observed in wild-type animals were reduced in animals with lung inflammation, indicating that pathological states can modify the response to nose-only exposure. Observed responses may warrant consideration in the evaluation of materials delivered by nose-only inhalation, and suggest that incorporation of naive animals into nose-only studies should be considered as a best practice.

Acute changes in lung function associated with proximity to a steel plant: a randomized study.

BACKGROUND Steel production is a major industry worldwide yet there is relatively little information on the pulmonary effects of air quality near steel manufacturing plants. OBJECTIVES The aim of this study was to examine how lung function changes acutely when healthy subjects are situated near a steel plant which is adjacent to a residential area. METHODS Sixty-one subjects were randomly assigned to spend 5 consecutive, 8-hour days in a residential neighborhood approximately 0.9km from a steel plant, or approximately 4.5km away at a college campus. Subjects crossed-over between sites after a nine-day washout period. Lung function was measured daily at both sites along with air pollutants including SO2, NO2, O3, PM2.5, and ultrafine particles. Diffusion capacity and pulse oximetry were also examined. RESULTS Compared with the college site, the forced expiratory volume in 1-second/forced vital capacity, forced expiratory flow between 25% and 75% of the FVC, total lung capacity, functional residual capacity, and residual volume were lower near the steel plant by 0.67% (95% CI: 0.28, 1.06),1.62% (95% CI: 0.50, 2.75), 1.54% (95% CI: 0.68, 2.39), 3.54% (95% CI: 1.95, 5.13) and 11.3% (95% CI: 4.92, 17.75), respectively. Diffusion capacity, forced expiratory volume in 1s, and pulse oximetry were also lower near the plant but these effects were not statistically significant. Sulfur dioxide, ultrafine particulates, and oxides of nitrogen were greater near the steel plant site compared to the college site. CONCLUSIONS Spending short periods of time near a steel plant is associated with a decrease in lung function.