Josée Guénette

Acute Effects of Inhaled Urban Particles and Ozone : Lung Morphology, Macrophage Activity, and Plasma Endothelin-1

We studied acute responses of rat lungs to inhalation of urban particulate matter and ozone. Exposure to particles (40 mg/m3 for 4 hours; mass median aerodynamic diameter, 4 to 5 microm; Ottawa urban dust, EHC-93), followed by 20 hours in clean air, did not result in acute lung injury. Nevertheless, inhalation of particles resulted in decreased production of nitric oxide (nitrite) and elevated secretion of macrophage inflammatory protein-2 from lung lavage cells. Inhalation of ozone (0.8 parts per million for 4 hours) resulted in increased neutrophils and protein in lung lavage fluid. Ozone alone also decreased phagocytosis and nitric oxide production and stimulated endothelin-1 secretion by lung lavage cells but did not modify secretion of macrophage inflammatory protein-2. Co-exposure to particles potentiated the ozone-induced septal cellularity in the central acinus but without measurable exacerbation of the ozone-related alveolar neutrophilia and permeability to protein detected by lung lavage. The enhanced septal thickening was associated with elevated production of both macrophage inflammatory protein-2 and endothelin-1 by lung lavage cells. Interestingly, inhalation of urban particulate matter increased the plasma levels of endothelin-1, but this response was not influenced by the synergistic effects of ozone and particles on centriacinar septal tissue changes. This suggests an impact of the distally distributed particulate dose on capillary endothelial production or filtration of the vasoconstrictor. Overall, equivalent patterns of effects were observed after a single exposure or three consecutive daily exposures to the pollutants. The experimental data are consistent with epidemiological evidence for acute pulmonary effects of ozone and respirable particulate matter and suggest a possible mechanism whereby cardiovascular effects may be induced by particle exposure. In a broad sense, acute biological effects of respirable particulate matter from ambient air appear related to paracrine/endocrine disruption mechanisms.

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.

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.

SPIN TRAPPING STUDY IN THE LUNGS AND LIVER OF F344 RATS AFTER EXPOSURE TO OZONE

Fischer 344 rats were injected with the spin traps C-phenyl N-tert-butyl nitrone (PBN, 150 mg/kg bw, ip) or 4-pyridine-N-oxide N-tert-butyl nitrone (POBN, 775 mg/kg bw, ip), and exposed to clean air or 2 ppm ozone for two hours. The presence of spin adducts was determined by electron paramagnetic resonance (EPR) spectroscopy of chloroform extracts of lung and liver homogenates. No significant levels of adducts were detected in the lungs of air control animals. Benzoyl N-tert-butyl aminoxyl, attributed to direct reaction of ozone with PBN, and tert-butyl hydroaminoxyl, the scission product of the hydroxyl adduct of PBN, were detected in the lungs of ozone exposed rats. EPR signals for carbon-centred alkoxyl and alkyl adducts were also detected with PBN in the lungs and liver of animals exposed to ozone. With POBN, only carbon-centred alkyl radicals were detected. Senescent, 24 months old rats were found to retain about twice more 14C-PBN in blood, heart and lungs by comparison to juvenile, 2 months old animals. Accordingly, the EPR signals were generally stronger in the lungs of the senescent rats by comparison to juvenile rats. Together, the observations were consistent with the previously proposed notion that a significant flux of hydrogen peroxide produced from the reaction of ozone with lipids of the extracellular lining, or from activated macrophages in the lungs could be a source of biologically relevant amounts of hydroxyl radical.

Alteration in aromatic hydroxylation and lipid oxidation status in the lungs of rats exposed to ozone.

Fischer 344 rats were exposed to ozone by inhalation to identify sensitive indices of acute exposure. 5-Aminosalicylic acid (5-ASA) hydroxylation in bronchoalveolar lavage (BAL), an indicator of hydroxyl radical (*OH) formation, and lipid oxidation in various regions of airways, representing oxidative stress, were measured to verify whether they can function as markers of exposure. BAL cells and supernatants taken from rats that received saline or 5-ASA (ip, 50 mg/kg) prior to ozone exposure (0, 0.4, or 0.8 ppm for 4 h) were analyzed for products of lipid oxidation. *OH formation was assessed by analysis of the BAL supernatant for 5-aminotetrahydroxybenzoic acid (5-ATHBA), a hydroxylation product of 5-ASA. The tetrahydroxy derivative of 5-ASA was higher in the BAL of ozone-treated rats than in air controls, reaching significance (p <. 05) at 0.8 ppm of ozone, The products of lipid oxidation propanal and hexanal were higher in BAL cells taken from rats exposed to ozone, reaching significance (p <. 05) at a 0.8 ppm ozone level, compared to air control animals, irrespective of whether they received saline or 5-ASA prior to ozone exposure. Increases in cholesterol levels were also seen in BAL cells after rats were exposed to ozone. However, there were no significant dose-related changes in the lipid oxidation products in BAL supernatants after exposure to ozone. Lipid oxidation products in BAL cells and 5-ATHBA in lavage exhibited the potential to serve as markers of ozone exposure. This work was supported by Health Canada (#4320105) and Toxic Substances Research Initiatives (TSRI #60).

90-Day Repeated Inhalation Exposure of Surfactant Protein-C/Tumor Necrosis Factor-α (SP-C/TNF-α) Transgenic Mice to Air Pollutants

Tumor necrosis factor (TNF)-α, a cytokine present in inflammed lungs, is known to mediate some of the adverse effects of ozone and inhaled particles. The authors evaluated transgenic mice with constitutive pulmonary expression of TNF-αunder transcriptional regulation of the surfactant protein-C promoter as an animal model of biological susceptibility to air pollutants. To simulate a repeated, episodic exposure to air pollutants, wild-type and TNF mice inhaled air or a mixture of ozone (0.4 ppm) and urban particles (EHC-93, 4.8 mg/m3) for 4 h, once per week, for 12 consecutive weeks and were sacrificed 20 h after last exposure. TNF mice exhibited chronic lung inflammation with septal thickening, alveolar enlargement, and elevated protein and cellularity in bronchoalveolar lavage fluid (genotype main effect, p <.001). Repeated exposure to pollutants did not result in measurable inflammatory changes in wild-type mice and did not exacerbate the inflammation in TNF mice. The pollutants decreased recovery of alveolar macrophages in lavage fluid of both wild-type and TNF mice (exposure main effect, p < .001). Exacerbation of the rate of protein nitration reactions specifically in the lungs of TNF mice was revealed by the high ratio of 3-nitrotyrosine to L-DOPA after exposure to the air pollutants (Genotype × Exposure factor interaction, p = .014). Serum creatine kinase-MM isoform increased in TNF mice exposed to pollutants (Genotype × Exposure factor interaction, p = .043). The marked pollutant-related nitration in the lungs of the TNF mice reveals basic differences in free radical generation and scavenging in the inflamed lungs in response to pollutants. Furthermore, elevation of circulating creatine kinase-MM isoform specifically in TNF mice exposed to pollutants suggests systemic adverse impacts from lung inflamma-tory mediators, possibly on muscles and the cardiovascular system.

STABLE GASEOUS ATMOSPHERES FOR NOSE-ONLY INHALATION USING MASS FLOW CONTROLLERS

The maintenance of stable test atmospheres in small-volume inhalation systems such as a nose-only exposure manifold requires rapid and appropriate responses to fluctuations in a number of parameters. For example, changes in flows or voltages can affect test compound generation efficiency and gas dilution ratios, resulting in excursions of the test concentration outside an acceptable range. A feedback control loop was designed that makes use of mass flow controllers in maintaining the target concentration of a test compound. The system was tested by generating ozone in a nose-only exposure manifold. An ozone analyzer provided updated concentration values every 30 s, which were compared to the user-defined target concentration recorded in the interface readout box. Values of gain, integral, and lead determining an appropriate response of the controller to concentration fluctuations were implemented based on proportional-integral-derivative control algorithms. Using this approach, the flow of ozone mixed wit...

Ozone modifies the metabolic and endocrine response to glucose: Reproduction of effects with the stress hormone corticosterone

ABSTRACT Air pollution is associated with increased incidence of metabolic disease (e.g. metabolic syndrome, obesity, diabetes); however, underlying mechanisms are poorly understood. Air pollutants increase the release of stress hormones (human cortisol, rodent corticosterone), which could contribute to metabolic dysregulation. We assessed acute effects of ozone, and stress axis involvement, on glucose tolerance and on the metabolic (triglyceride), endocrine/energy regulation (insulin, glucagon, GLP‐1, leptin, ghrelin, corticosterone), and inflammatory/endothelial (TNF, IL‐6, VEGF, PAI‐1) response to exogenous glucose. Male Fischer‐344 rats were exposed to clean air or 0.8 ppm ozone for 4 h in whole body chambers. Hypothalamic‐pituitary‐adrenal (HPA) axis involvement in ozone effects was tested through subcutaneous administration of the glucocorticoid synthesis inhibitor metyrapone (50 mg/kg body weight), corticosterone (10 mg/kg body weight), or vehicle (40% propylene glycol) prior to exposure. A glucose tolerance test (2 g/kg body weight glucose) was conducted immediately after exposure, with blood samples collected at 0, 30, 60, 90, and 120 min. Ozone exposure impaired glucose tolerance, an effect accompanied by increased plasma triglycerides but no impairment of insulin release. Ozone diminished glucagon, GLP‐1, and ghrelin responses to glucose, but did not significantly impact inflammatory/endothelial analytes. Metyrapone reduced corticosterone but increased glucose and triglycerides, complicating evaluation of the impact of glucocorticoid inhibition. However, administration of corticosterone reproduced the profile of ozone effects, supporting a role for the HPA axis. The results show that ozone‐dependent changes in glucose tolerance are accompanied by altered metabolic and endocrine responses to glucose challenge that are reproduced by exogenous stress hormone. HighlightsOzone impaired glucose tolerance in Fischer‐344 rats.Ozone also altered the metabolic and endocrine response to glucose challenge.Effects of ozone were reproduced by the stress hormone corticosterone.

Cardiovascular and inflammatory mechanisms in healthy humans exposed to air pollution in the vicinity of a steel mill

BackgroundThere is a paucity of mechanistic information that is central to the understanding of the adverse health effects of source emission exposures. To identify source emission-related effects, blood and saliva samples from healthy volunteers who spent five days near a steel plant (Bayview site, with and without a mask that filtered many criteria pollutants) and at a well-removed College site were tested for oxidative stress, inflammation and endothelial dysfunction markers.MethodsBiomarker analyses were done using multiplexed protein-array, HPLC-Fluorescence, EIA and ELISA methods. Mixed effects models were used to test for associations between exposure, biological markers and physiological outcomes. Heat map with hierarchical clustering and Ingenuity Pathway Analysis (IPA) were used for mechanistic analyses.ResultsMean CO, SO2 and ultrafine particles (UFP) levels on the day of biological sampling were higher at the Bayview site compared to College site. Bayview site exposures “without” mask were associated with increased (p < 0.05) pro-inflammatory cytokines (e.g IL-4, IL-6) and endothelins (ETs) compared to College site. Plasma IL-1β, IL-2 were increased (p < 0.05) after Bayview site “without” compared to “with” mask exposures. Interquartile range (IQR) increases in CO, UFP and SO2 were associated with increased (p < 0.05) plasma pro-inflammatory cytokines (e.g. IL-6, IL-8) and ET-1(1–21) levels. Plasma/saliva BET-1 levels were positively associated (p < 0.05) with increased systolic BP. C-reactive protein (CRP) was positively associated (p < 0.05) with increased heart rate. Protein network analyses exhibited activation of distinct inflammatory mechanisms after “with” and “without” mask exposures at the Bayview site relative to College site exposures.ConclusionsThese findings suggest that air pollutants in the proximity of steel mill site can influence inflammatory and vascular mechanisms. Use of mask and multiple biomarker data can be valuable in gaining insight into source emission-related health impacts.

Stress axis variability is associated with differential ozone-induced lung inflammatory signaling and injury biomarker response

ABSTRACT Ozone (O3), a ubiquitous urban air pollutant, causes adverse pulmonary and extrapulmonary effects. A large variability in acute O3‐induced effects has been observed; however, the basis for interindividual differences in susceptibility is unclear. We previously demonstrated a role for the hypothalamic‐pituitary‐adrenal (HPA) stress axis and glucocorticoid response in acute O3 toxicity. Glucocorticoids have important anti‐inflammatory actions, and have been shown to regulate lung inflammatory responses. We hypothesised that a hyporesponsive HPA axis would be associated with greater O3‐dependent lung inflammatory signaling. Two genetically‐related rat strains with known differences in stress axis reactivity, highly‐stress responsive Fischer (F344) and less responsive Lewis (LEW), were exposed for 4 h by nose‐only inhalation to clean air or 0.8 ppm O3, and euthanized immediately after exposure. As expected, baseline (air‐exposed) plasma corticosterone was significantly lower in the hypo‐stress responsive LEW. Although O3 exposure increased plasma corticosterone in both strains, corticosterone remained significantly lower in LEW when compared to F334. LEW exhibited greater O3‐induced inflammatory cytokine/chemokine signaling compared to F344, consistent with the lower corticosterone levels. Since we observed strain‐specific differences in inflammatory signaling, we further investigated injury biomarkers (total protein, albumin and lactate dehydrogenase). Although the hyper‐responsive F344 exhibited lower inflammatory signaling in response to O3 compared with LEW, they had greater levels of lung injury biomarkers. Our results indicate that stress axis variability is associated with differential O3‐induced lung toxicity. Given the large variability in stress axis reactivity among humans, stress axis regulation could potentially be a determining factor underlying O3 sensitivity. HighlightsFischer and Lewis rats exhibited distinct stress axis responses to ozone inhalation.Hypo‐stress responsive Lewis rats had a greater lung inflammatory response.Hyper‐stress responsive Fischer rats had a greater lung injury response.Innate stress axis differences may play a role in ozone‐induced lung toxicity.