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Introduction: Studies of gastrointestinal physiology and the gut microbiome often consider the influence of intestinal region on experimental endpoints. However, this same consideration is not often applied to the gut metabolome. Understanding the contribution of gut regionality may be critically important to the rapidly changing metabolic environments, such as during pregnancy. Objectives: We sought to characterize the difference in the gut metabolome in pregnant mice stratified by region—comparing the small intestine, cecum, and feces. Pre-pregnancy feces were collected to understand the influence of pregnancy on the fecal metabolome. Methods: Feces were collected from CD-1 female mice before breeding. On gestation day (GD) 18, gut contents were collected from the small intestine, cecum, and descending colon. Metabolites were analyzed with LC–MS/MS using the Biocrates MetaboINDICATOR™ MxP® Quant 500 kit. Results: Of the 104 small molecule metabolites meeting analysis criteria, we found that 84 (81%) were differentially abundant based on gut region. The most significant regional comparison observed was between the cecum and small intestines, with 52 (50%) differentially abundant metabolites. Pregnancy itself altered 41 (39.4%) fecal small molecule metabolites. Conclusions: The regional variation observed in the gut metabolome are likely due to the microbial and physiological differences between the different parts of the intestines. Additionally, pregnancy impacts the fecal metabolome, which may be due to evolving needs of both the dam and fetus. [ABSTRACT FROM AUTHOR]

Increased use of renewable energy sources raise concerns about health effects of new emissions. We analyzed relative cardiopulmonary health effects of exhausts from (1) 100% soy biofuel (B100), (2) 20% soy biofuel + 80% low sulfur petroleum diesel (B20), and (3) 100% petroleum diesel (B0) in rats. Normotensive Wistar–Kyoto (WKY) and spontaneously hypertensive rats were exposed to these three exhausts at 0, 50, 150 and 500 μg/m3, 4 h/day for 2 days or 4 weeks (5 days/week). In addition, WKY rats were exposed for 1 day and responses were analyzed 0 h, 1 day or 4 days later for time-course assessment. Hematological parameters,in vitroplatelet aggregation, bronchoalveolar lavage fluid (BALF) markers of pulmonary injury and inflammation,ex vivoaortic ring constriction, heart and aorta mRNA markers of vasoconstriction, thrombosis and atherogenesis were analyzed. The presence of pigmented macrophages in the lung alveoli was clearly evident with all three exhausts without apparent pathology. Overall, exposure to all three exhausts produced only modest effects in most endpoints analyzed in both strains. BALF γ-glutamyl transferase (GGT) activity was the most consistent marker and was increased in both strains, primarily with B0 (B0 > B100 > B20). This increase was associated with only modest increases in BALF neutrophils. Small and very acute increases occurred in aorta mRNA markers of vasoconstriction and thrombosis with B100 but not B0 in WKY rats. Our comparative evaluations show modest cardiovascular and pulmonary effects at low concentrations of all exhausts: B0 causing more pulmonary injury and B100 more acute vascular effects. BALF GGT activity could serve as a sensitive biomarker of inhaled pollutants. [ABSTRACT FROM PUBLISHER]

Diesel exhaust has been associated with adverse cardiovascular and pulmonary health effects. The relative contributions of the gas phase and particulate components of diesel exhaust are less well understood. We exposed telemetered Wistar-Kyoto rats to air or diesel exhaust that was either filtered (F) or unfiltered [gas-phase plus diesel exhaust particles (DEP)], containing ~1.9 mg/m3 of particulate matter for 5 h/day; 5 days/week for 4 consecutive weeks. Blood pressure (BP), core temperature ( Tc), heart rate (HR), and cardiac contractility (CC) estimated by the QA interval were monitored by radiotelemetry during exposure as well as during a 2-week period of recovery. Pulmonary injury and inflammation markers were analysed after 2-day, and 4 weeks of exposure, and 2-week recovery. Exposure to F or DEP was associated with a trend for a reduction in BP during weeks 1, 2 and 4. A reduction in HR in the DEP group was apparent during week 4. Exposure to DEP but not F was associated with significant reduction in CC over weeks 1-4. There was also a slight elevation in Tc during DEP exposure. All telemetry parameters were normal during recovery at night and a 2-week recovery period. Neutrophilic inflammation in bronchoalveolar lavage fluid was evident after 2 days and 4 weeks of exposure to F and DEP. There were no signs of inflammation after 2-week recovery. We found a significant decrease in CC and slight reduction in BP. Exposure to DEP and F is associated with pulmonary inflammation, and mild effects on HR, BP, and Tc but there is a marked effect of DEP on CC. [ABSTRACT FROM AUTHOR]

Increased incidences of asbestosis have been reported in workers from Libby, MT, associated with exposures to amphibole-contaminated vermiculite. In this study pulmonary and histopathological changes were investigated following Libby amphibole (LA) exposure in a rat model. Rat respirable fractions of LA and amosite (aerodynamic diameter <2.5 μm) were prepared by water elutriation. Male F344 rats were exposed to single doses of either saline (SAL), amosite (0.65 mg/rat), or LA (0.65 or 6.5 mg/rat) by intratracheal instillation. At times from 1 d to 3 mo after exposure, bronchoalveolar lavage (BAL) was performed and right and left lungs were removed for reverse-transcription polymerase chain reaction (RT-PCR) and histopathological analysis, respectively. Data indicated that 0.65 mg amosite resulted in a higher degree of pulmonary injury, inflammation, and fibrotic events than LA at the same mass dose. Exposure to either amosite or high dose LA resulted in higher levels of cellular permeability and injury, inflammatory enzymes, and iron binding proteins in both BAL fluid and lung tissue at most time points when compared to SAL controls. However, mRNA expression for some growth factors (e.g., platelet-derived growth factor [PDGF]-A and transforming growth factor [TGF]-1β), which contribute to fibrosis, were downregulated at several time points. Furthermore, histopathological examination showed notable thickening of interstitial areas surrounding the alveolar ducts and terminal bronchioles. On a mass dose basis, amosite produced a greater acute and persistent lung injury for at least 3 mo after exposure. However, further testing and analysis of LA are needed with regard to the dose metric to fully evaluate its potential fibrogenicity and carcinogenicity. [ABSTRACT FROM AUTHOR]

Underlying cardiovascular disease (CVD) is a risk factor for the exacerbation of air pollution health effects. Pulmonary oxidative stress, inflammation, and altered iron (Fe) homeostasis secondary to CVD may influence mammalian susceptibility to air pollutants. Rodent models of CVD are increasingly used to examine mechanisms of variation in susceptibility. Baseline cardiac and pulmonary disease was characterized in healthy normotensive Wistar Kyoto (WKY) rats, cardiovascular compromised spontaneously hypertensive rats (SHR), and spontaneously hypertensive heart failure (SHHF) rats. Blood pressure, heart rate, and breathing frequencies were measured in rats 11 to 12 wk of age, followed by necropsy at 14 to 15 wk of age. Blood pressure and heart rate were increased in SHR and SHHF relative to WKY rats (SHR > SHHF > WKY). Increased breathing frequency in SHHF and SHR (SHR > SHHF > WKY) resulted in greater minute volume relative to WKY. Bronchoalveolar lavage fluid (BALF) protein and neutrophils were higher in SHHF and SHR relative to WKY (SHHF >> SHR > WKY). Lung ascorbate and glutathione levels were low in SHHF rats. BALF Fe-binding capacity was decreased in SHHF relative to WKY rats and was associated with increased transferrin (Trf) and ferritin. However, lung ferritin was lower and Trf was higher in SHHF relative to WKY or SHR rats. mRNA for markers of inflammation and oxidative stress (macrophage inflammatory protein [MIP]-2, interleukin [IL]-1α, and heme oxygenase [HO]-1) were greater in SHHF and SHR relative to WKY rats. Trf mRNA rose in SHR but not SHHF relative to WKY rats, whereas transferrin receptors 1 and 2 mRNA was lower in SHHF rats. Four of 12 WKY rats exhibited cardiac hypertrophy despite normal blood pressure, while demonstrating some of the pulmonary complications noted earlier. This study demonstrates that SHHF rats display greater underlying pulmonary complications such as oxidative stress, inflammation, and impaired Fe homeostasis than WKY or SHR rats, which may play a role in SHHF rats' increased susceptibility to air pollution. [ABSTRACT FROM AUTHOR]

BACKGROUND: Exposure to particulate matter (PM) has been associated with increased cardiovascular morbidity; however, causative components are unknown. Zinc is a major element detected at high levels in urban air. OBJECTIVE: We investigated the role of PM-associated zinc in cardiac injury. METHODS: We repeatedly exposed 12- to 14-week-old male Wistar Kyoto rats intratracheally (1×/week for 8 or 16 weeks) to a) saline (control); b) PM having no soluble zinc (Mount St. Helens ash, MSH); or c) whole-combustion PM suspension containing 14.5 μg/mg of water-soluble zinc at high dose (PM-HD) and d ) low dose (PM-LD), e) the aqueous fraction of this suspension (14.5 μg/mg of soluble zinc) (PM-L), or f ) zinc sulfate (rats exposed for 8 weeks received double the concentration of all PM components of rats exposed for 16 weeks). RESULTS: Pulmonary inflammation was apparent in all exposure groups when compared with saline (8 weeks > 16 weeks). PM with or without zinc, or with zinc alone caused small increases in focal subepicardial inflammation, degeneration, and fibrosis. Lesions were not detected in controls at 8 weeks but were noted at 16 weeks. We analyzed mitochondrial DNA damage using quantitative polymerase chain reaction and found that all groups except MSH caused varying degrees of damage relative to control. Total cardiac aconitase activity was inhibited in rats receiving soluble zinc. Expression array analysis of heart tissue revealed modest changes in mRNA for genes involved in signaling, ion channels function, oxidative stress, mitochondrial fatty acid metabolism, and cell cycle regulation in zinc but not in MSH-exposed rats. CONCLUSION: These results suggest that water-soluble PM-associated zinc may be one of the causal components involved in PM cardiac effects. [ABSTRACT FROM AUTHOR]

Humans with underlying cardiovascular disease, including stroke, are more susceptible to ambient particulate matter (PM)-induced morbidity and mortality. We hypothesized that stroke-prone spontaneously hypertensive rats (SHRSP) would be more susceptible than healthy Wistar Kyoto (WKY) rats to PM-induced cardiac oxidative stress and pulmonary injury. We further postulated that PM-induced injury would be greater in SHRSP than in spontaneously hypertensive rats (SHR) based on the greater disease severity in SHRSP than SHR. First, male WKY and SHRSP were intratracheally (IT) instilled with saline or 1.11, 3.33, or 8.33 mg/kg of oil combustion PM and responses were analyzed 4 or 24 h later. Second, SHR and SHRSP were IT instilled with saline or 3.33 or 8.33 mg/kg of the same PM and responses were analyzed 24 h later. Pulmonary injury and inflammation were assessed in bronchoalveolar lavage fluid (BALF) and cardiac markers in cytosolic and mitochondrial fractions. BALF neutrophilic inflammatory response was induced similarly in all strains following PM exposure. BALF protein leakage, γ-glutamyl transferase, and N-acetylglucosaminidase activities, but not lactate dehydrogenase activity, were exacerbated in SHRSP compared to WKY or SHR. Pulmonary cytosolic and cardiac mitochondrial ferritin levels decreased, and cardiac cytosolic superoxide dismutase (SOD) activity increased in SHRSP only. Pulmonary SOD activity decreased in WKY and SHRSP. Cardiac mitochondrial isocitrate dehydrogenase (ICDH) activity decreased in PM-exposed WKY and SHR; control levels were lower in SHRSP than SHR or WKY. In summary, strain-related differences exist in pulmonary protein leakage and oxidative stress markers. PM-induced changes in cardiac oxidative stress sensitive enzymes are small, and appear only slightly exacerbated in SHRSP compared to WKY or SHR. Multiple biological markers may be differentially affected by PM in genetic models of cardiovascular diseases. Preexisting cardiovascular disease may influence susceptibility to PM pulmonary and cardiac health effects in a disease-specific manner. [ABSTRACT FROM AUTHOR]

It was recently demonstrated that particulate matter (PM) containing water-soluble zinc produces cardiac injury following pulmonary exposure. To investigate whether pulmonary zinc exposure produces systemic metal imbalance and direct cardiac effects, male Wistar Kyoto (WKY) rats (12–14 wk age) were intratracheally (IT) instilled with saline or 2 μmol/kg zinc sulfate. Temporal analysis was performed for systemic levels of essential metals (zinc, copper, and selenium), and induction of zinc transporter-2 (ZT-2) and metallothionein-1 (MT-1) mRNA in the lung, heart, and liver. Additionally, cardiac gene expression profile was evaluated using Affymetrix GeneChips (rat 230A) arrays to identify zinc-specific effects. Pulmonary zinc instillation produced an increase in plasma zinc to &nvsim;20% at 1 and 4 h postexposure with concomitant decline in the lung levels. At 24 and 48 h postexposure, zinc levels rose significantly (&nvsim;35%) in the liver. At these time points, plasma and liver levels of copper and selenium also increased significantly, suggesting systemic disturbance in essential metals. Zinc exposure was associated with marked induction of MT-1 and ZT-2 mRNA in lung, heart, and liver, suggesting systemic metal sequestration response. Given the functional role of zinc in hundreds of proteins, the gene expression profiles demonstrated changes that are expected based on its physiological role. Zinc exposure produced an increase in expression of kinases and inhibition of expression of phosphatases; up- or downregulation of genes involved in mitochondrial function; changes in calcium regulatory proteins suggestive of elevated intracellular free calcium and increases in sulfotransferases; upregulation of potassium channel genes; and changes in free radical-sensitive proteins. Some of these expression changes are reflective of a direct effect of zinc on myocardium following pulmonary exposure, which may result in impaired mitochondrial respiration, stimulated cell signaling, altered Ca2+ homeostasis, and increased transcription of sulfotransferases. Cardiotoxicity may be an outcome of acute zinc toxicosis and occupational exposures to metal fumes containing soluble zinc. Imbalance of systemic metal homeostasis as a result of pulmonary zinc exposure may underlie the cause of extrapulmonary effects. [ABSTRACT FROM AUTHOR]

Several studies have reported health effects of concentrated ambient particles (CAP) in rodents and humans; however, toxicity end points in rodents have provided inconsistent results. In 2000 we conducted six 1-day exposure studies where spontaneously hypertensive (SH) rats were exposed to filtered air or CAPs (≤ 2.5 µm, 1,138-1,765 µg/m3) for 4 hr (analyzed 1-3 hr afterward). In seven 2-day exposure studies in 2001, SH and Wistar Kyoto (WKY) rats were exposed to filtered air or CAP (≤ 2.5 µm, 144-2,758 µg/m3) for 4 hr/day 2 days (analyzed 1 day afterward). Despite consistent and high CAP concentrations in the 1-day exposure studies, no biologic effects were noted. The exposure concentrations varied among the seven 2-day exposure studies. Except in the first study when CAP concentration was highest, lavageable total cells and macrophages decreased and neutrophils increased in WKY rats. SH rats demonstrated a consistent increase of lavage fluid -glutamyltransferase activity and plasma fibrinogen. Inspiratory and expiratory times increased in SH but not in WKY rats. Significant correlations were found between CAP mass (microgram per cubic meter) and sulfate, organic carbon, or zinc. No biologic effects correlated with CAP mass. Despite low chamber mass in the last six of seven 2-day exposure studies, the levels of zinc, copper, and aluminum were enriched severalfold, and organic carbon was increased to some extent when expressed per milligram of CAP. Biologic effects were evident in those six studies. These studies demonstrate a pattern of rat strain-specific pulmonary and systemic effects that are not linked to high mass but appear to be dependent on CAP chemical composition. [ABSTRACT FROM AUTHOR]

Toll-like receptor 4 (TLR4) has been shown to play a role in cell signaling that results in neutrophilic inflammation in response to lipopolysaccharide and respiratory syncytial virus infection. TLR4 also interacts with CD14, which upon complex formation triggers TLR4-associated signaling pathways to produce a proinflammatory response. This mechanism results in the activation of NF-κB and subsequent inflammatory gene induction. In order to determine the effect of combustion source particle matter (PM), rich in zinc and nickel but with negligible endotoxin, on a possible activation of TLR4-mediated cell signaling and inflammation, we intratracheally (IT) instilled 3.3 mg/kg of PM into 12-w-old healthy male Wistar Kyoto (WKY) and susceptible spontaneously hypertensive (SH) rats. Inflammation, inflammatory-mediator gene expression, bronchoalveolar lavage fluid (BALF) protein and LDH, TLR4 and CD14 protein, and NF-κB activation in the lung were determined after 24 h. Dose-response data (0.0, 0.83, 3.33, and 8.3 mg/kg PM) for BALF LDH were obtained as a marker of lung cell injury in SH rats. BALF neutrophils, but not macrophages, were significantly increased in the PM-exposed WKY and SH rats. SH rats showed a greater PMN increase than WKY rats. Similarly, BALF protein and LDH levels were also increased following PM exposure but to a significantly greater extent in SH rats. Plasma fibrinogen was increased only in SH rats exposed to PM. The increased inflammation seen in PM-exposed SH rats was accompanied by a significant increase in TLR4 protein in the lung tissue, which was primarily localized in alveolar macrophages and epithelial cells. CD14 was also increased by PM exposure in both SH and WKY rats but was significantly greater in the SH rats. These increases were associated with greater translocation of NF-κB in the lungs of SH rather than WKY rats. This was accompanied by increased macrophage inhibitory protein (MIP)-2 mRNA expression at 24 h of exposure. These data suggest that the increased inflammation in the lungs of PM-exposed SH rats compared to WKY rats is accompanied by an increase in TLR4-mediated cell signaling. Thus, one of the mechanisms for greater susceptibility of SH rats to PM exposure may involve an increased activation of the TLR4 signaling pathway. [ABSTRACT FROM AUTHOR]

Exposure to particulate matter (PM) has been associated with increased morbidity and mortality among individuals with cardiovascular disease. It is hypothesized that systemic alterations occur concurrent to pulmonary injury/inflammation, and contribute to cardiac events in compromised hosts. We explored this hypothesis using a rat model for human hypertension and cardiovascular disease (spontaneously hypertensive, SH), and normotensive Wistar Kyoto (WKY) rats. SH and WKY rats (12-13 wk old) were exposed either intratracheally (IT; 0.0, 1.0, or 5.0 mg/kg in saline) or nose-only (15 mg/m[sup 3] × 6 h/d × 3 d/wk × 1, 2 or 4 wk) to combustion source residual oil fly ash (ROFA) with low metal content, and examined 1, 2 or 4 d later. Bronchoalveolar lavage fluid (BALF) albumin and neutrophils increased (SH ≃ WKY) at d 1 following ROFA IT. With inhalation exposure, both strains experienced progressive histological lung damage and increases in BALF albumin and neutrophils during 1 to 4 wk (SH > WKY). Acute lung injury from ROFA IT was temporally associated with increases in plasma fibrinogen in both strains, but only the SH rats responded to the acute 1-wk ROFA inhalation. Longer term (2 or 4 wk) ROFA caused progressive lung injury (SH > WKY), but did not sustain the increase in fibrinogen. BALF glutathione increased in a temporal fashion similar to fibrinogen; however, only WKY rats demonstrated this response. There was a small but consistent decrease in blood lymphocytes and an increase in blood neutrophils in SH rats exposed to ROFA acutely. In conclusion, acute PM exposure can provoke an acute systemic thrombogenic response associated with pulmonary injury/inflammation and oxidative stress in cardiovascular compromised rats. This evidence is consistent with greater cardiovascular events during acute PM episodes in compromised humans. [ABSTRACT FROM AUTHOR]

We have recently shown that the spontaneously hypertensive (SH) rats with underlying cardiovascular disease exhibited greater pulmonary vascular leakage and oxidative stress than healthy normotensive (Wistar Kyoto, WKY) rats after a 3-day inhalation exposure to residual oil fly ash (ROFA) particles (Kodavanti et al., 2000). Since host responsiveness to a 3-day episodic ROFA inhalation could be different from a single acute exposure, we examined ROFA and its constituent metal (vanadium, V; nickel, Ni)-induced lung injury after a single intratracheal (IT) exposure. Male SH and WKY rats (12-13 wk) were IT instilled with either saline or ROFA (0.0, 0.83 or 3.33 mg/kg). The bronchoalveolar lavage fluid (BALF) was analyzed for lung injury markers at 24 and 96 h post-IT. Rats were also IT instilled with 0.0 or 1.5 μmol/ kg of either VSO[sub 4] or NiSO[sub 4]•6H[sub 2]O in saline (equivalent to a dose of 2-3 mg ROFA), and assessed at 6 and 24 h post-IT. Basal levels of BALF protein, macrophages, and neutrophils, but not lactate dehydrogenase (LDH), were higher in control SH compared to control WKY rats. Lung histology of control SH rats exhibited mild focal alveolitis and perivascular inflammation; these changes were minimal in control WKY rats. ROFA-induced increases in BALF protein, and to a lesser extent in LDH, were greater in SH compared to WKY rats. ROFA IT was associated with the increases in BALF total cells in both strains (SH > WKY). BALF neutrophils increased at 24 h and macrophages at 96 h in a dose-dependent manner (SH > WKY). The increase in BALF neutrophils was largely reversed by 96 h in both rat strains. The V-induced increases in BALF protein and LDH peaked at 6 h post-IT and returned to control by 24 h in WKY rats. In SH rats, BALF protein and LDH were not affected by V. Ni caused BALF protein to increase in both strains at 6 and 24 h; however, the control values at 24 h were high in SH rats, and were not distinguishable from exposed rats. The Ni-induced increase in LDH activity was progressive over a 24-h time period (WKY > SH). The number of macrophages decreased following V and Ni exposure at 6 h, and this decrease was reversed by 24 h in both strains. V caused BALF neutrophils to increase only in WKY rats. The Ni-induced increase in BALF neutrophils was more dramatic and progressive than that of V, but was similar in both strains. Lung histology similarly revealed more severe and persistent edema, perivascular and peribronchiolar inflammation, and hemorrhage in Ni- than in V-exposed rats. This effect of Ni appeared slightly more severe in SH than in WKY rats. In summary, the acute single IT exposure to ROFA resulted in greater pulmonary protein leakage and inflammation in SH rats than in WKY rats. The metallic constituents of ROFA produced these effects in a strain-specific manner such that, at the dose level used, V caused pulmonary injury only in WKY rats, whereas Ni was toxic to both strains. [ABSTRACT FROM AUTHOR]

Pulmonary tissue injury and repair processes involve complex and coordinated cellular events such as necrosis, inflammation, cell growth/differentiation, apoptosis, and remodeling of extracellular matrix. These processes are regulated by expression of multiple mediator genes. Commercially available microarray blots and slides allow screening of hundreds to thousands of genes in a given tissue or cell preparation. However, often these blots do not contain cDNAs of one's interest and are difficult to interpret. In order to analyze the tissue expression profile of a large number of genes involved in pulmonary injury and pathology, we developed a rat gene array filter using array technology. This array consisted of 27 genes representing inflammatory and anti-inflammatory cytokines, growth factors, adhesion molecules, stress proteins, transcription factors and antioxidant enzymes; 3 negative controls, and 2 blank spots. Using rat gene-specific polymerase chain reaction (PCR) primer pairs, cDNAs for these genes were amplified and cloned into a TA vector. Plasmids with recombinant cDNA inserts were purified and blotted onto a nylon membrane. Lung total RNA was isolated at 3 or 24 h following intratracheal (IT) exposure of male Sprague Dawley rats to either saline (control), residual oil fly ash (ROFA; 3.3 mg/kg) or metals found in one instillate of ROFA: nickel (NiSO4; 1.3 µmol/ kg) or vanadium (VSO4; 2.2 µmol/kg). 32P-Labeled cDNA was generated from RNA sam [ABSTRACT FROM AUTHOR]

Inhalation of ozone activates central sympathetic-adrenal-medullary and hypothalamic-pituitary-adrenal stress axes. While airway neural networks are known to communicate noxious stimuli to higher brain centers, it is not known to what extent responses generated from pulmonary airways contribute to neuroendocrine activation. Unlike inhalational exposures that involve the entire respiratory tract, we employed intratracheal (IT) instillations to expose only pulmonary airways to either soluble metal-rich residual oil fly ash (ROFA) or compressor-generated diesel exhaust particles (C-DEP). Male Wistar–Kyoto rats (12–13 weeks) were IT instilled with either saline, C-DEP or ROFA (5 mg/kg) and necropsied at 4 or 24 hr to assess temporal effects. IT-instillation of particulate matter (PM) induced hyperglycemia as early as 30-min and glucose intolerance when measured at 2 hr post-exposure. We observed PM- and time-specific effects on markers of pulmonary injury/inflammation (ROFA>C-DEP; 24 hr>4hr) as corroborated by increases in lavage fluid injury markers, neutrophils (ROFA>C-DEP), and lymphocytes (ROFA). Increases in lavage fluid pro-inflammatory cytokines differed between C-DEP and ROFA in that C-DEP caused larger increases in TNF-α whereas ROFA caused larger increases in IL-6. No increases in circulating cytokines occurred. At 4 hr, PM impacts on neuroendocrine activation were observed through depletion of circulating leukocytes, increases in adrenaline (ROFA), and decreases in thyroid-stimulating-hormone, T3, prolactin, luteinizing-hormone, and testosterone. C-DEP and ROFA both increased lung expression of genes involved in acute stress and inflammatory processes. Moreover, small increases occurred in hypothalamic Fkbp5, a glucocorticoid-sensitive gene. Respiratory alterations differed between C-DEP and ROFA, with ROFA inducing greater overall lung injury/inflammation; however, both PM induced a similar degree of neuroendocrine activation. These findings demonstrate neuroendocrine activation after pulmonary-only PM exposure, and suggest the involvement of pituitary- and adrenal-derived hormones. [ABSTRACT FROM AUTHOR]

Recent epidemiological findings link asthma to adverse cardiovascular responses. Yet, the precise cardiovascular impacts of asthma have been challenging to disentangle from the potential cardiovascular effects caused by asthma medication. The purpose of this study was to determine the impacts of allergic airways disease alone on cardiovascular function in an experimental model. Female Wistar rats were intranasally sensitized and then challenged once per week for 5 weeks with saline vehicle or a mixture of environmental allergens (ragweed, house dust mite, and Aspergillus fumigatus). Ventilatory and cardiovascular function, measured using double-chamber plethysmography and implantable blood pressure (BP) telemetry and cardiovascular ultrasound, respectively, were assessed before sensitization and after single and final allergen challenge. Responses to a single 0.5 ppm ozone exposure and to the cardiac arrhythmogenic agent aconitine were also assessed after final challenge. A single allergen challenge in sensitized rats increased tidal volume and specific airways resistance in response to provocation with methacholine and increased bronchoalveolar lavage fluid (BALF) eosinophils, neutrophils, lymphocytes, cytokines interleukin (IL)-4, IL-5, IL-10, IL-1β, tumor necrosis factor-α, and keratinocyte chemoattract-growth-related oncogene characteristic of allergic airways responses. Lung responses after final allergen challenge in sensitized rats were diminished, although ozone exposure increased BALF IL-6, IL-13, IL-1 β, and interferon-γ and modified ventilatory responses only in the allergen group. Final allergen challenge also increased systolic and mean arterial BP, stroke volume, cardiac output, end-diastolic volume, sensitivity to aconitine-induced cardiac arrhythmia, and cardiac gene expression with lesser effects after a single challenge. These findings demonstrate that allergic airways responses may increase cardiovascular risk in part by altering BP and myocardial function and by causing cardiac electrical instability. [ABSTRACT FROM AUTHOR]

Altered fetal growth, which can occur due to environmental stressors during pregnancy, may program a susceptibility to metabolic disease. Gestational exposure to the air pollutant ozone is associated with fetal growth restriction in humans and rodents. However, the impact of this early life ozone exposure on offspring metabolic risk has not yet been investigated. In this study, fetal growth restriction was induced by maternal inhalation of 0.8 ppm ozone on gestation days 5 and 6 (4 hr/day) in Long Evans rats. To uncover any metabolic inflexibility, or an impaired ability to respond to a high‐fat diet (HFD), a subset of peri‐adolescent male and female offspring from filtered air or ozone exposed dams were fed HFD (45% kcal from fat) for 3 days. By 6 weeks of age, male and female offspring from ozone‐exposed dams were heavier than offspring from air controls. Furthermore, offspring from ozone‐exposed dams had greater daily caloric consumption and reduced metabolic rate when fed HFD. In addition to energy imbalance, HFD‐fed male offspring from ozone‐exposed dams had dyslipidemia and increased adiposity, which was not evident in females. HFD consumption in males resulted in the activation of the protective 5'AMP‐activated protein kinase (AMPKα) and sirtuin 1 (SIRT1) pathways in the liver, regardless of maternal exposure. Unlike males, ozone‐exposed female offspring failed to activate these pathways, retaining hepatic triglycerides following HFD consumption that resulted in increased inflammatory gene expression and reduced insulin signaling genes. Taken together, maternal ozone exposure in early pregnancy programs impaired metabolic flexibility in offspring, which may increase susceptibility to obesity in males and hepatic dysfunction in females. [ABSTRACT FROM AUTHOR]

Exposure to wildfire smoke is associated with both acute and chronic cardiopulmonary illnesses, which are of special concern for wildland firefighters who experience repeated exposure to wood smoke. It is necessary to better understand the underlying pathophysiology by which wood smoke exposure increases pulmonary disease burdens in this population. We hypothesize that wood smoke exposure produces pulmonary dysfunction, lung inflammation, and gene expression profiles associated with future pulmonary complications. Male Long-Evans rats were intermittently exposed to smoldering eucalyptus wood smoke at 2 concentrations, low (11.0 ± 1.89 mg/m3) and high (23.7 ± 0.077 mg/m3), over a 2-week period. Whole-body plethysmography was measured intermittently throughout. Lung tissue and lavage fluid were collected 24 h after the final exposure for transcriptomics and metabolomics. Increasing smoke exposure upregulated neutrophils and select cytokines in the bronchoalveolar lavage fluid. In total, 3446 genes were differentially expressed in the lungs of rats in the high smoke exposure and only 1 gene in the low smoke exposure (Cd151). Genes altered in the high smoke group reflected changes to the Eukaryotic Initiation Factor 2 stress and oxidative stress responses, which mirrored metabolomics analyses. xMWAS-integrated analysis revealed that smoke exposure significantly altered pathways associated with oxidative stress, lung morphogenesis, and tumor proliferation pathways. These results indicate that intermittent, 2-week exposure to eucalyptus wood smoke leads to transcriptomic and metabolic changes in the lung that may predict future lung disease development. Collectively, these findings provide insight into cellular signaling pathways that may contribute to the chronic pulmonary conditions observed in wildland firefighters. [ABSTRACT FROM AUTHOR]

Background: The release of catecholamines is preceded by glucocorticoids during a stress response. We have shown that ozone-induced pulmonary responses are mediated through the activation of stress hormone receptors. Objective: To examine the interdependence of beta-adrenergic (βAR) and glucocorticoid receptors (GRs), we inhibited βAR while inducing GR or inhibited GR while inducing βAR and examined ozone-induced stress response. Methods: Twelve-week-old male Wistar-Kyoto rats were pretreated daily with saline or propranolol (PROP; βAR-antagonist; 10 mg/kg-i.p.; starting 7-d prior to exposure) followed-by saline or dexamethasone (DEX) sulfate (GR-agonist; 0.02 mg/kg-i.p.; starting 1-d prior to exposure) and exposed to air or 0.8 ppm ozone (4 h/d × 2-d). In a second experiment, rats were similarly pretreated with corn-oil or mifepristone (MIFE; GR-antagonist, 30 mg/kg-s.c.) followed by saline or clenbuterol (CLEN; β2AR-agonist; 0.02 mg/kg-i.p.) and exposed. Results: DEX and PROP + DEX decreased adrenal, spleen and thymus weights in all rats. DEX and MIFE decreased and increased corticosterone, respectively. Ozone-induced pulmonary protein leakage, inflammation and IL-6 increases were inhibited by PROP or PROP + DEX and exacerbated by CLEN or CLEN + MIFE. DEX and ozone-induced while MIFE reversed lymphopenia (MIFE > CLEN + MIFE). DEX exacerbated while PROP, MIFE, or CLEN + MIFE inhibited ozone-induced hyperglycemia and glucose intolerance. Ozone inhibited glucose-mediated insulin release. Conclusions: In summary, 1) activating βAR, even with GR inhibition, exacerbated and inhibiting βAR, even with GR activation, attenuated ozone-induced pulmonary effects; and 2) activating GR exacerbated ozone systemic effects, but with βAR inhibition, this exacerbation was less remarkable. These data suggest the independent roles of βAR in pulmonary and dependent roles of βAR and GR in systemic effects of ozone. [ABSTRACT FROM AUTHOR]

There is growing interest in understanding how maternal diet might affect the sensitivity of offspring to environmental exposures. Previous studies demonstrated that adult rat offspring (approximately 6-months-old) from dams given a high-fat diet (HFD) prior to, during, and after pregnancy displayed elevated pulmonary responses to an acute ozone (O3) exposure. The aim of this study was to examine the influence of maternal and perinatal HFD on pulmonary and metabolic responses to O3 in male and female young-adult offspring (approximately 3-month old). One-month-old F0 female Long-Evans rats commenced HFD (60% kcal from fat) or control diet (CD; 10.5% kcal from fat) and were bred on PND 72. Offspring were maintained on respective HFD or CD until PND 29 when all groups were switched to CD. The 3-months-old female and male offspring (n = 10/group) were exposed to air or 0.8 ppm O3 for 5hr/day for 2 consecutive days. Maternal and perinatal HFD significantly increased body weight and body fat % in offspring regardless of gender. Ozone exposure, but not maternal and perinatal diet, induced hyperglycemia and glucose intolerance in the offspring. Ozone-induced alterations in pulmonary function were exacerbated by maternal and perinatal HFD in both offspring genders. Pulmonary injury/inflammation markers in response to O3 exposure such as bronchoalveolar lavage fluid total protein, lactate dehydrogenase, total cells, and neutrophils were further augmented in offspring (males>females) from dams fed the HFD. Data suggest that maternal and perinatal HFD may enhance the susceptibility of offspring to O3-induced pulmonary injury and that these effects may be sex-specific. [ABSTRACT FROM AUTHOR]

Acrolein, a respiratory irritant, induces systemic neuroendocrine stress. However, peripheral metabolic effects have not been examined. Male and female WKY rats were exposed to air (0 ppm) or acrolein (3.16 ppm) for 4 h, followed by immediate serum and liver tissue collection. Serum metabolomics in both sexes and liver transcriptomics in males were evaluated to characterize the systemic metabolic response. Of 887 identified metabolites, > 400 differed between sexes at baseline. An acrolein biomarker, 3-hydroxypropyl mercapturic acid, increased 18-fold in males and 33-fold in females, indicating greater metabolic detoxification in females than males. Acrolein exposure changed 174 metabolites in males but only 50 in females. Metabolic process assessment identified higher circulating free-fatty acids, glycerols, and other lipids in male but not female rats exposed to acrolein. In males, acrolein also increased branched-chain amino acids, which was linked with metabolites of nitrogen imbalance within the gut microbiome. The contribution of neuroendocrine stress was evident by increased corticosterone in males but not females. Male liver transcriptomics revealed acrolein-induced over-representation of lipid and protein metabolic processes, and pathway alterations including Sirtuin, insulin-receptor, acute-phase, and glucocorticoid signaling. In sum, acute acrolein inhalation resulted in sex-specific serum metabolomic and liver transcriptomic derangement, which may have connections to chronic metabolic-related diseases. [ABSTRACT FROM AUTHOR]

We have shown that acute ozone inhalation activates sympathetic-adrenal-medullary and hypothalamus-pituitary-adrenal stress axes, and adrenalectomy (AD) inhibits ozone-induced lung injury and inflammation. Therefore, we hypothesized that stress hormone receptor agonists (β2 adrenergic-β2AR and glucocorticoid-GR) will restore the ozone injury phenotype in AD, while exacerbating effects in sham-surgery (SH) rats. Male Wistar Kyoto rats that underwent SH or AD were treated with vehicles (saline + corn oil) or β2AR agonist clenbuterol (CLEN, 0.2 mg/kg, i.p.) + GR agonist dexamethasone (DEX, 2 mg/kg, s.c.) for 1 day and immediately prior to each day of exposure to filtered air or ozone (0.8 ppm, 4 h/day for 1 or 2 days). Ozone-induced increases in PenH and peak-expiratory flow were exacerbated in CLEN+DEX-treated SH and AD rats. CLEN+DEX affected breath waveform in all rats. Ozone exposure in vehicle-treated SH rats increased bronchoalveolar lavage fluid (BALF) protein, N -acetyl glucosaminidase activity (macrophage activation), neutrophils, and lung cytokine expression while reducing circulating lymphocyte subpopulations. AD reduced these ozone effects in vehicle-treated rats. At the doses used herein, CLEN+DEX treatment reversed the protection offered by AD and exacerbated most ozone-induced lung effects while diminishing circulating lymphocytes. CLEN+DEX in air-exposed SH rats also induced marked protein leakage and reduced circulating lymphocytes but did not increase BALF neutrophils. In conclusion, circulating stress hormones and their receptors mediate ozone-induced vascular leakage and inflammatory cell trafficking to the lung. Those receiving β2AR and GR agonists for chronic pulmonary diseases, or with increased circulating stress hormones due to psychosocial stresses, might have altered sensitivity to air pollution. [ABSTRACT FROM AUTHOR]

Apelin has cardiopulmonary protective properties that promote vasodilation and maintenance of the endothelial barrier. While reductions in apelin have been identified as a contributor to various lung diseases, including pulmonary edema, its role in the effect of air pollutants has not been examined. Thus, in the current study, we sought to investigate if apelin is a downstream target of inhaled ozone and if such change in expression is related to altered DNA methylation in the lung. Male, Long-Evans rats were exposed to filtered air or 1.0 ppm ozone for 4 h. Ventilation changes were assessed using whole-body plethysmography immediately following exposure, and markers of pulmonary edema and inflammation were assessed in the bronchoaveolar lavage (BAL) fluid. The enzymatic regulators of DNA methylation were measured in the lung, along with methylation and hydroxymethylation of the apelin promoter. Data showed that ozone exposure was associated with increased enhanced pause and protein leakage in the BAL fluid. Ozone exposure reduced DNA cytosine-5-methyltransferase (DNMT) activity and Dnmt3a/b gene expression. Exposure-induced upregulation of proliferating cell nuclear antigen, indicative of DNA damage, repair, and maintenance methylation. Increased methylation and reduced hydroxymethylation were measured on the apelin promoter. These epigenetic modifications accompanied ozone-induced reduction of apelin expression and development of pulmonary edema. In conclusion, epigenetic regulation, specifically increased methylation of the apelin promoter downstream of DNA damage, may lead to reductions in protective signaling of the apelinergic system, contributing to the pulmonary edema observed following the exposure to oxidant air pollution. [ABSTRACT FROM AUTHOR]

Recent studies showed that the circulating stress hormones, epinephrine and corticosterone/cortisol, are involved in mediating ozone-induced pulmonary effects through the activation of the sympathetic-adrenal-medullary (SAM) and hypothalamus-pituitary-adrenal (HPA) axes. Hence, we examined the role of adrenergic and glucocorticoid receptor inhibition in ozone-induced pulmonary injury and inflammation. Male 12-week old Wistar-Kyoto rats were pretreated daily for 7 days with propranolol (PROP; a non-selective β adrenergic receptor [AR] antagonist, 10 mg/kg, i.p.), mifepristone (MIFE; a glucocorticoid receptor [GR] antagonist, 30 mg/kg, s.c.), both drugs (PROP + MIFE), or respective vehicles, and then exposed to air or ozone (0.8 ppm), 4 h/d for 1 or 2 consecutive days while continuing drug treatment. Ozone exposure alone led to increased peak expiratory flow rates and enhanced pause (Penh); with greater increases by day 2. Receptors blockade minimally affected ventilation in either air- or ozone-exposed rats. Ozone exposure alone was also associated with marked increases in pulmonary vascular leakage, macrophage activation, neutrophilic inflammation and lymphopenia. Notably, PROP, MIFE and PROP + MIFE pretreatments significantly reduced ozone-induced pulmonary vascular leakage; whereas PROP or PROP + MIFE reduced neutrophilic inflammation. PROP also reduced ozone-induced increases in bronchoalveolar lavage fluid (BALF) IL-6 and TNF-α proteins and/or lung Il6 and Tnfα mRNA. MIFE and PROP + MIFE pretreatments reduced ozone-induced increases in BALF N -acetyl glucosaminidase activity, and lymphopenia. We conclude that stress hormones released after ozone exposure modulate pulmonary injury and inflammatory effects through AR and GR in a receptor-specific manner. Individuals with pulmonary diseases receiving AR and GR-related therapy might experience changed sensitivity to air pollution. [ABSTRACT FROM AUTHOR]

Air pollutant exposures have been linked to systemic disease; however, the underlying mechanisms between responses of the target tissue and systemic effects are poorly understood. A prototypic inducer of stress, ozone causes respiratory and systemic multiorgan effects through activation of a neuroendocrine stress response. The goal of this study was to assess transcriptomic signatures of multiple tissues and serum metabolomics to understand how neuroendocrine and adrenal-derived stress hormones contribute to multiorgan health outcomes. Male Wistar Kyoto rats (12–13 weeks old) were exposed to filtered air or 0.8 ppm ozone for 4-hours, and blood/tissues were collected immediately post-exposure. Each tissue had distinct expression profiles at baseline. Ozone changed 1,640 genes in lung, 274 in hypothalamus, 2,516 in adrenals, 1,333 in liver, 1,242 in adipose, and 5,102 in muscle (adjusted p-value < 0.1, absolute fold-change > 50%). Serum metabolomic analysis identified 863 metabolites, of which 447 were significantly altered in ozone-exposed rats (adjusted p-value < 0.1, absolute fold change > 20%). A total of 6 genes were differentially expressed in all 6 tissues. Glucocorticoid signaling, hypoxia, and GPCR signaling were commonly changed, but ozone induced tissue-specific changes in oxidative stress, immune processes, and metabolic pathways. Genes upregulated by TNF-mediated NFkB signaling were differentially expressed in all ozone-exposed tissues, but those defining inflammatory response were tissue-specific. Upstream predictor analysis identified common mediators of effects including glucocorticoids, although the specific genes responsible for these predictors varied by tissue. Metabolomic analysis showed major changes in lipids, amino acids, and metabolites linked to the gut microbiome, concordant with transcriptional changes identified through pathway analysis within liver, muscle, and adipose tissues. The distribution of receptors and transcriptional mechanisms underlying the ozone-induced stress response are tissue-specific and involve induction of unique gene networks and metabolic phenotypes, but the shared initiating triggers converge into shared pathway-level responses. This multi-tissue transcriptomic analysis, combined with circulating metabolomic assessment, allows characterization of the systemic inhaled pollutant-induced stress response. [ABSTRACT FROM AUTHOR]

Acrolein and trichloroethylene (TCE) are priority hazardous air pollutants due to environmental prevalence and adverse health effects; however, neuroendocrine stress-related systemic effects are not characterized. Comparing acrolein, an airway irritant, and TCE with low irritancy, we hypothesized that airway injury would be linked to neuroendocrine-mediated systemic alterations. Male and female Wistar-Kyoto rats were exposed nose-only to air, acrolein or TCE in incremental concentrations over 30 min, followed by 3.5-hr exposure to the highest concentration (acrolein - 0.0, 0.1, 0.316, 1, 3.16 ppm; TCE - 0.0, 3.16, 10, 31.6, 100 ppm). Real-time head-out plethysmography revealed acrolein decreased minute volume and increased inspiratory-time (males>females), while TCE reduced tidal-volume. Acrolein, but not TCE, inhalation increased nasal-lavage-fluid protein, lactate-dehydrogenase activity, and inflammatory cell influx (males>females). Neither acrolein nor TCE increased bronchoalveolar-lavage-fluid injury markers, although macrophages and neutrophils increased in acrolein-exposed males and females. Systemic neuroendocrine stress response assessment indicated acrolein, but not TCE, increased circulating adrenocorticotrophic hormone, and consequently corticosterone, and caused lymphopenia, but only in males. Acrolein also reduced circulating thyroid-stimulating hormone, prolactin, and testosterone in males. In conclusion, acute acrolein inhalation resulted in sex-specific upper respiratory irritation/inflammation and systemic neuroendocrine alterations linked to hypothalamic-pituitary-adrenal axes activation, which is critical in mediating extra-respiratory effects. • Acrolein but not TCE elicits acute respiratory irritancy and systemic stress. • Acrolein-induced respiratory injury prompts sex-specific SAM/HPA axes activation. • Acute acrolein inhalation increases HPA and decreases HPT and HPG neurohormones. • Integrating neuroendocrine assessment could inform etiology of HAPs stress effects. [ABSTRACT FROM AUTHOR]

Acute ozone exposure increases circulating stress hormones and induces metabolic alterations in animals. We hypothesized that the increase of adrenal-derived stress hormones is necessary for both ozone-induced metabolic effects and lung injury. Male Wistar-Kyoto rats underwent bilateral adrenal demedullation (DEMED), total bilateral adrenalectomy (ADREX), or sham surgery (SHAM). After a 4 day recovery, rats were exposed to air or ozone (1 ppm), 4 h/day for 1 or 2 days and responses assessed immediately postexposure. Circulating adrenaline levels dropped to nearly zero in DEMED and ADREX rats relative to SHAM. Corticosterone tended to be low in DEMED rats and dropped to nearly zero in ADREX rats. Adrenalectomy in air-exposed rats caused modest changes in metabolites and lung toxicity parameters. Ozone-induced hyperglycemia and glucose intolerance were markedly attenuated in DEMED rats with nearly complete reversal in ADREX rats. Ozone increased circulating epinephrine and corticosterone in SHAM but not in DEMED or ADREX rats. Free fatty acids (P = .15) and branched-chain amino acids increased after ozone exposure in SHAM but not in DEMED or ADREX rats. Lung minute volume was not affected by surgery or ozone but ozone-induced labored breathing was less pronounced in ADREX rats. Ozone-induced increases in lung protein leakage and neutrophilic inflammation were markedly reduced in DEMED and ADREX rats (ADREX>DEMED). Ozone-mediated decreases in circulating white blood cells in SHAM were not observed in DEMED and ADREX rats. We demonstrate that ozone-induced peripheral metabolic effects and lung injury/inflammation are mediated through adrenal-derived stress hormones likely via the activation of stress response pathway. [ABSTRACT FROM AUTHOR]

BACKGROUND: Inhaled irritant air pollutants may trigger stress-related metabolic dysfunction associated with altered circulating adrenal-derived hormones. OBJECTIVES: We used implantable telemetry in rats to assess real-time changes in circulating glucose during and after exposure to ozone and mechanistically linked responses to neuroendocrine stress hormones. METHODS: First, using a cross-over design, we monitored glucose during ozone exposures (0.0, 0.2, 0.4, and 0:8 ppm) and nonexposure periods in male Wistar Kyoto rats implanted with glucose telemeters. A second cohort of unimplanted rats was exposed to ozone (0.0, 0.4 or 0.8 ppm) for 30 min, 1 h, 2 h, or 4 h with hormones measured immediately post exposure. We assessed glucose metabolism in sham and adrenalectomized rats, with or without supplementation of adrenergic/glucocorticoid receptor agonists, and in a separate cohort, antagonists. RESULTS: Ozone (0.8 ppm) was associated with significantly higher blood glucose and lower core body temperature beginning 90 min into exposure, with reversal of effects 4–6 h post exposure. Glucose monitoring during four daily 4-h ozone exposures revealed duration of glucose increases, adaptation, and diurnal variations. Ozone-induced glucose changes were preceded by higher levels of adrenocorticotropic hormone, corticosterone, and epinephrine but lower levels of thyroid-stimulating hormone, prolactin, and luteinizing hormones. Higher glucose and glucose intolerance were inhibited in rats that were adrenalectomized or treated with adrenergic plus glucocorticoid receptor antagonists but exacerbated by agonists. DISCUSSION: We demonstrated the temporality of neuroendocrine-stress-mediated biological sequalae responsible for ozone-induced glucose metabolic dysfunction and mechanism in a rodent model. Stress hormones assessment with real-time glucose monitoring may be useful in identifying interactions among irritant pollutants and stress-related illnesses. [ABSTRACT FROM AUTHOR]

Acute ozone-induced pulmonary injury and inflammation are well characterized in rats; however, mechanistic understanding of the pathways involved is limited. We hypothesized that acute exposure of healthy rats to ozone will cause transcriptional alterations, and comprehensive analysis of these changes will allow us to better understand the mechanism of pulmonary injury and inflammation. Male Wistar Kyoto rats (10–12 week) were exposed to air, or ozone (0.25, 0.5 or 1.0 ppm) for 4 h and pulmonary injury and inflammation were assessed at 0-h or 20-h (n = 8/group). Lung gene expression profiling was assessed at 0-h (air and 1.0 ppm ozone,n = 3–4/group). At 20-h bronchoalveolar lavage, fluid protein and neutrophils increased at 1 ppm ozone. Numerous genes involved in acute inflammatory response were up-regulated along with changes in genes involved in cell adhesion and migration, steroid metabolism, apoptosis, cell cycle control and cell growth. A number of NRF2 target genes were also induced after ozone exposure. Based on expression changes, Rela, SP1 and TP3-mediated signaling were identified to be mediating downstream changes. Remarkable changes in the processes of endocytosis provide the insight that ozone-induced lung injury and inflammation are likely initiated by changes in cell membrane components and receptors likely from oxidatively modified lung lining lipids and proteins. In conclusion, ozone-induced injury and inflammation are preceded by changes in gene targets for cell adhesion/migration, apoptosis, cell cycle control and growth regulated by Rela, SP1 and TP53, likely mediated by the process of endocytosis and altered steroid receptor signaling. [ABSTRACT FROM AUTHOR]

To elucidate key factors of host susceptibility to air pollution, healthy and cardiovascular (CV)-compromised rats were exposed to air or ozone (O3) at 0.25, 0.5, or 1.0 ppm for 4 h. We hypothesized that rat strains with the least cardiac reserve would be most prone to develop significant health effects. Using flow whole body plethysmography (FWBP), ventilatory responses in healthy 3-month-old male rats [i.e. Wistar–Kyoto (WKY), Wistar (WIS), and Sprague–Dawley (SD) strains] were compared with hypertensive [i.e. spontaneously hypertensive (SH), fawn-hooded-hypertensive (FHH), and SH-stroke-prone (SHSP)] strains and obese [i.e. SH-heart failure-prone (SHHF) and JCR:LA-cp, atherosclerosis-prone (JCR)] strains. SH were slower to acclimate to the FWBP chambers. At 0-h post-air-exposure, SHSP and SHHF exhibited hyperpnea, indicative of cardiopulmonary insufficiency. At 0-h-post-O3, all but one strain showed significant concentration-dependent decreases in minute volume [MV = tidal volume (TV) × breathing frequency]. Comparing air with 1.0 ppm responses, MV declined 20–27% in healthy, 21–42% in hypertensive, and 33% in JCR rats, but was unchanged in SHHF rats. Penh increased significantly in all strains, with disproportionate increases in “responder” WKY and FHH strains. By 20 h, most changes had resolved, although Penh remained elevated in WKY, SH, and SHSP. Based on theeffectivedose estimates (O3 ppm × h × MV), the most CV-compromised (SHSP and SHHF) strains received significantly greater O3lung deposition (25% and 40%, respectively). Data support epidemiologic associations that individuals with cardiopulmonary insufficiency are at greater risk for urban pollutant exposure due, in part, to enhanced lung deposition and exacerbation of hypoxia and pathophysiologic processes of heart failure. [ABSTRACT FROM AUTHOR]

Ozone-induced lung injury, inflammation, and pulmonary/hypothalamus gene expression changes are diminished in adrenalectomized (AD) rats. Acute ozone exposure induces metabolic alterations concomitant with increases in epinephrine and corticosterone. We hypothesized that adrenal hormones are responsible for observed hepatic ozone effects, and in AD rats, these changes would be diminished. In total, 5–7 days after sham (SH) or AD surgeries, male Wistar-Kyoto rats were exposed to air or 0.8-ppm ozone for 4 h. Serum samples were analyzed for metabolites and liver for transcriptional changes immediately post-exposure. Ozone increased circulating triglycerides, cholesterol, free fatty-acids, and leptin in SH but not AD rats. Ozone-induced inhibition of glucose-mediated insulin release was absent in AD rats. Unlike diminution of ozone-induced hypothalamus and lung mRNA expression changes, AD in air-exposed rats (AD-air/SH-air) caused differential hepatic expression of ∼1000 genes. Likewise, ozone in AD rats caused differential expression of ∼1000 genes (AD-ozone/AD-air). Ozone-induced hepatic changes in SH rats reflected enrichment for pathways involving metabolic processes, including acetyl-CoA biosynthesis, TCA cycle, and sirtuins. Upstream predictor analysis identified similarity to responses produced by glucocorticoids and pathways involving forskolin. These changes were absent in AD rats exposed to ozone. However, ozone caused unique changes in AD liver mRNA reflecting activation of synaptogenesis, neurovascular coupling, neuroinflammation, and insulin signaling with inhibition of senescence pathways. In these rats, upstream predictor analysis identified numerous microRNAs likely involved in glucocorticoid insufficiency. These data demonstrate the critical role of adrenal stress hormones in ozone-induced hepatic homeostasis and necessitate further research elucidating their role in propagating environmentally driven diseases. [ABSTRACT FROM AUTHOR]

Studies recently showed that intratracheal (IT) instillation of Libby amphibole (LA) increases circulating acute-phase proteins (APP; α-2 macroglobulin, A2M; and α-1 acid glycoprotein, AGP) and inflammatory biomarkers (osteopontin and lipocalin) in rats. In this study, objectives were to (1) compare changes in biomarkers of rats after instillation of different naturally occurring asbestos (NOA) minerals including LA, Sumas Mountain chrysotile (SM), El Dorado Hills tremolite (ED), and Ontario ferroactinolite cleavage fragments (ON), and (2) examine biomarkers after subchronic LA or amosite inhalation exposure. Rat-respirable fractions (aerodynamic diameter approximately 2.5 μm) prepared by water elutriation were delivered via a single IT instillation at doses of 0, 0.5, and 1.5 mg/rat in male F344 rats. Nose-only inhalation exposures were performed at 0, 1, 3.3, and 10 mg/m3for LA and at 3.3 mg /m3for amosite, 6h/d, 5 d/wk for 13 wk. Inflammation, metabolic syndrome, and cancer biomarkers were analyzed in the serum for up to 18 mo. IT instillation of some asbestos materials significantly increased serum AGP and A2M but to a varying degree (SM = LA > ON = ED). Numerical increases in interleukin (IL)-6 and osteopontin occurred in rats instilled with SM. SM and ED also elevated leptin and insulin at 15 mo, suggesting potential metabolic effects. LA inhalation tended to raise A2M at d 1 but not cytokines. Serum mesothelin appeared to elevate after 18 mo of LA inhalation. These results suggest that the lung injury induced by high levels of asbestos materials may be associated with systemic inflammatory changes and predisposition to insulin resistance. [ABSTRACT FROM AUTHOR]

Epidemiological evidence suggests the potential for air pollutants to induce male reproductive toxicity. In experimental studies, exposure to ozone during sensitive windows in the sperm lifecycle has been associated with impaired sperm motility. Subsequently, we sought to investigate the effects of episodic exposure to ozone during sperm maturation in the rat. Long-Evans rats were exposed to either filtered air or ozone (0.4 or 0.8 ppm) for five non-consecutive days over two weeks. Ozone exposure did not impact male reproductive organ weights or sperm motility ∼24 hours following the final exposure. Furthermore, circulating sex hormones remained unchanged despite increased T 3 and T 4 in the 0.8 ppm group. While there was indication of altered adrenergic signaling attributable to ozone exposure in the testis, there were minimal impacts on small non-coding RNAs detected in cauda sperm. Only two piwi-interacting RNAs (piRNAs) were altered in the mature sperm of ozone-exposed rats (piR-rno-346434 and piR-rno-227431). Data across all rats were next analyzed to identify any non-coding RNAs that may be correlated with reduced sperm motility. A total of 7 microRNAs (miRNAs), 8 RNA fragments, and 1682 piRNAs correlated well with sperm motility. Utilizing our exposure paradigm herein, we were unable to substantiate the relationship between ozone exposure during maturation with sperm motility. However, these approaches served to identify a suite of non-coding RNAs that were associated with sperm motility in rats. With additional investigation, these RNAs may prove to have functional roles in the acquisition of motility or be unique biomarkers for male reproductive toxicity. • Ozone exposure during sperm maturation does not affect sperm motility in rats. • Ozone exposure has minimal effects on mature sperm non-coding RNA populations but alters piR-rno-346434 and piR-rno-227431. • Increased piRNA expression was associated with reduced sperm quality in rats. [ABSTRACT FROM AUTHOR]

Complexation of host iron (Fe) on the surface of inhaled asbestos fibers has been postulated to cause oxidative stress contributing to in vivo pulmonary injury and inflammation. We examined the role of Fe in Libby amphibole (LA; mean length 4.99 µµm ±± 4.53 and width 0.28 µµm ±± 0.19) asbestos-induced inflammogenic effects in vitro and in vivo. LA contained acid-leachable Fe and silicon. In a cell-free media containing FeCl3, LA bound #17 µµg of Fe/mg of fiber and increased reactive oxygen species generation #3.5 fold, which was reduced by deferoxamine (DEF) treatment. In BEAS-2B cells exposure to LA, LA loaded with Fe (FeLA), or LA with DEF did not increase HO-1 or ferritin mRNA expression. LA increased IL-8 expression, which was reduced by Fe loading but increased by DEF. To determine the role of Fe in LA-induced lung injury in vivo, spontaneously hypertensive rats were exposed intratracheally to either saline (300 µµL), DEF (1 mg), FeCl3 (21 µµg), LA (0.5 mg), FeLA (0.5 mg), or LA ++ DEF (0.5 mg). LA caused BALF neutrophils to increase 24 h post-exposure. Loading of Fe on LA but not chelation slightly decreased neutrophilic influx (LA ++ DEF > LA > FeLA). At 4 h post-exposure, LA-induced lung expression of MIP-2 was reduced in rats exposed to FeLA but increased by LA ++ DEF (LA ++ DEF > LA > FeLA). Ferritin mRNA was elevated in rats exposed to FeLA compared to LA. In conclusion, the acute inflammatory response to respirable fibers and particles may be inhibited in the presence of surface-complexed or cellular bioavailable Fe. Cell and tissue Fe-overload conditions may influence the pulmonary injury and inflammation caused by fibers. [ABSTRACT FROM AUTHOR]

Spontaneously hypertensive (SH) and normotensive Wistar Kyoto (WKY) rats have been used for understanding the mechanisms of variations in susceptibility to airborne pollutants. We examined the lung burden of diesel exhaust particles (DEP) following inhalation of diesel engine exhaust (DEE) in both strains. The kinetics of clearance was also examined after single intratracheal (IT) instillation of DEP. Lungs were analyzed for DEP elemental carbon (EC) after exposure to DEE (0, 500, or 2000 μg/m3 4 h/day, 5 days/week × 4 weeks). SH rats had 16% less DEP-EC at 500 and 32% less at 2000 μg/m3 in the lungs, despite having 50% higher than the average minute volume. No strain-related differences were noted in number of alveolar macrophages or their average DEP load as evident from examining cells in bronchoalveolar lavage fluid (BALF). The kinetics of DEP clearance from lungs of male WKY and SH rats was studied following a single instillation at 0.0 or 8.33 mg/kg of DEP standard reference material (SRM 2975) from the National Institute of Standards Technology. SH rats cleared 60% DEP over 112 days while minimal clearance occurred from the lungs of WKY. The pattern of DEP-induced inflammatory response assessed by BALF analysis was similar in both strains, although the overall protein leak was slightly greater in SH rats. A time-dependent accumulation of DEP occurred in tracheal lymph nodes of both strains (SH > WKY). Thus, SH rats may clear DEP more efficiently from their lungs than normotensive WKY rats, with a small contribution of more effective lymphatic drainage. [ABSTRACT FROM PUBLISHER]

Tire and brake wear particles contain transition metals, and contribute to near-road PM. We hypothesized that acute cardiopulmonary injury from respirable tire particles (TP) will depend on the amount of soluble metals. Respirable fractions of two types of TP (TP1 and TP2) were analyzed for water and acid-leachable metals using ICP-AES. Both TP types contained a variety of transition metals, including zinc (Zn), copper (Cu), aluminum, and iron. Zn and Cu were detected at high levels in water-soluble fractions (TP2 > TP1). Male Wistar Kyoto rats (12-14 wk) were intratracheally instilled, in the first study, with saline, TP1 or TP2 (5 mg/kg), and in the second study, with soluble Zn, Cu (0.5 μ mol/kg), or both. Pulmonary toxicity and cardiac mitochondrial enzymes were analyzed 1 d, 1 wk, or 4 wk later for TP and 4 or 24 h later for metals. Increases in lavage fluid markers of inflammation and injury were observed at d 1 (TP2 > TP1), but these changes reversed by wk 1. No effects on cardiac enzymes were noted with either TP. Exposure of rats to soluble Zn and Cu caused marked pulmonary inflammation and injury but temporal differences were apparent (Cu effects peaked at 4 h and Zn at 24 h). Instillation of Zn, Cu, and Zn+ Cu decreased the activity of cardiac aconitase, isocitrate dehydrogenase, succinate dehydrogenase, cytochrome-c-oxidase and superoxide dismutase suggesting mitochondrial oxidative stress. The observed acute pulmonary toxicity of TP could be due to the presence of water soluble Zn and Cu. At high concentrations these metals may induce cardiac oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract: Cardiovascular damage induced by pulmonary exposure to environmental chemicals can result from direct action or, secondarily from pulmonary injury. We have developed a rat model of pulmonary exposure to zinc to demonstrate cardiac, coagulative, and fibrinolytic alterations. Male Wistar Kyoto rats were instilled intratracheally with saline or zinc sulfate, 131 μg/kg (2 μmol/kg); the alterations were determined at 1, 4, 24, and 48 h postexposure. High-dose zinc enabled us to show changes in circulating levels of zinc above normal and induce significant pulmonary inflammation/injury such that cardiac impairments were likely. At 1–24 h postexposure, plasma levels of zinc increased to nearly 20% above the base line. Significant pulmonary inflammation and injury were determined by analysis of bronchoalveolar lavage fluid and histopathology in zinc-exposed rats at all time points. Starting at 4 h postexposure, pulmonary damage was accompanied by persistently increased gene expressions of tissue factor (TF) and plasminogen activator-inhibitor-1 (PAI-1), but not thrombomodulin (TM). Cardiac tissues demonstrated similar temporal increases in expressions of TF, PAI-1, and TM mRNA following pulmonary instillation of zinc. In contrast to extensive pulmonary edema and inflammation, only mild, and focal acute, myocardial lesions developed in a few zinc-exposed rats; no histological evidence showed increased deposition of fibrin or disappearance of troponin. At 24 and 48 h postexposure to zinc, increases occurred in levels of systemic fibrinogen and the activated partial thromboplastin time. These data suggest that cardiovascular blood coagulation impairments are likely following pulmonary zinc exposure and associated pulmonary injury and inflammation. [Copyright &y& Elsevier]

Particulate matter air pollution has been associated with cardiopulmonary morbidity and mortality in many recent epidemiological studies. Previous toxicological research has demonstrated profound cardiac and thermoregulatory changes in rats following exposure to residual oil fly ash (ROFA), a combustion-derived particulate. The response to ROFA appeared biphasic, consisting of both immediate (0-6 h) and delayed (24-96 h) bradycardia and hypothermia. Other studies have demonstrated that much of the pulmonary toxicity of ROFA was caused by its constitutive transition metals, namely, Fe, Ni, and V. This study examined the contributions of these metals to the observed cardiac and thermoregulatory changes caused by ROFA in conscious, unrestrained rats. Prior to exposure, each animal was surgically implanted with a radiotelemetry device capable of continuously monitoring heart rate, electrocardiographic, and core temperature data. Individual metals were intratracheally instilled in healthy rats (n = 4 per metal species) and in rats with monocrotaline (MCT; 60 mg/kg)-induced pulmonary hypertension (n = 10 per metal species); combinations of metals were instilled in MCT-treated rats only (n = 6 per combination of metal species). Metals were administered in doses equivalent to those found in the highest dose of ROFA used in previous studies, that is, 105 μg Fe[sub 2](SO[sub 4])[sub 3], 263 μg NiSO[sub 4], and 245 μg VSO[sub 4]. Healthy and MCT-treated rats demonstrated similar responses to metals. Fe caused little response, whereas V caused marked bradycardia, arrhythmogenesis, and hypothermia immediately following instillation and lasting ~6 h. Ni caused no immediate response, but induced a delayed bradycardia, arrhythmogenesis, and hypothermia that began ~24 h after instillation and lasted for several days. When instilled in combination, Ni appeared to exacerbate the immediate effects of V, whereas Fe attenuated them. These data suggest that the biphasic response to instilled ROFA may result from a summation of the temporally different effects of V and Ni. [ABSTRACT FROM AUTHOR]

Air pollution has been associated with metabolic diseases and hepatic steatosis-like changes. We have shown that ozone alters liver gene expression for metabolic processes through neuroendocrine activation. This study aimed to further characterize ozone-induced changes and to determine the impact of hepatic vagotomy (HV) which reduces parasympathetic influence. Twelve-week-old male Wistar-Kyoto rats underwent HV or sham surgery 5–6 days before air or ozone exposure (0 or 1 ppm; 4 h/day for 1 or 2 days). Ozone-induced lung injury, hyperglycemia, glucose intolerance, and increases in circulating cholesterol, triglycerides, and leptin were similar in rats with HV and sham surgery. However, decreases in circulating insulin and increased HDL and LDL were observed only in ozone-exposed HV rats. Ozone exposure resulted in changed liver gene expression in both sham and HV rats (sham > HV), however, HV did not change expression in air-exposed rats. Upstream target analysis revealed that ozone-induced transcriptomic changes were similar to responses induced by glucocorticoid-mediated processes in both sham and HV rats. The directionality of ozone-induced changes reflecting cellular response to stress, metabolic pathways, and immune surveillance was similar in sham and HV rats. However, pathways regulating cell-cycle, regeneration, proliferation, cell growth, and survival were enriched by ozone in a directionally opposing manner between sham and HV rats. In conclusion, parasympathetic innervation modulated ozone-induced liver transcriptional responses for cell growth and regeneration without affecting stress-mediated metabolic changes. Thus, impaired neuroendocrine axes and parasympathetic innervation could collectively contribute to adverse effects of air pollutants on the liver. [ABSTRACT FROM AUTHOR]

Acute-phase response (APR) is an innate stress reaction to tissue trauma or injury, infection, and environmental insults like ozone (O3). Regardless of the location of stress, the liver has been considered the primary contributor to circulating acute-phase proteins (APPs); however, the mechanisms underlying APR induction are unknown. Male Wistar–Kyoto rats were exposed to air or O3 (1 ppm, 6-hr/day, 1 or 2 days) and examined immediately after each exposure and after 18-hr recovery for APR proteins and gene expression. To assess the contribution of adrenal-derived stress hormones, lung and liver global gene expression data from sham and adrenalectomized rats exposed to air or O3 were compared for APR transcriptional changes. Data demonstrated serum protein alterations for selected circulating positive and negative APPs following 2 days of O3 exposure and during recovery. At baseline, APP gene expression was several folds higher in the liver relative to the lung. O3-induced increases were significant for lung but not liver for some genes including orosomucoid-1. Further, comparative assessment of mRNA seq data for known APPs in sham rats exhibited marked elevation in the lung but not liver, and a near-complete abolishment of APP mRNA levels in lung tissue of adrenalectomized rats. Thus, the lung appears to play a critical role in O3-induced APP synthesis and requires the presence of circulating adrenal-derived stress hormones. The relative contribution of lung versus liver and the role of neuroendocrine stress hormones need to be considered in future APR studies involving inhaled pollutants. [ABSTRACT FROM AUTHOR]

Exposure to ambient ozone has been associated with increased human mortality. Ozone exposure can introduce oxygen-containing functional groups in particulate matter (PM) effecting a greater capacity of the particle for metal complexation and inflammatory effect. We tested the postulate that (1) a fulvic acid-like substance can be produced through a reaction of a carbonaceous particle with high concentrations of ozone and (2) such a fulvic acid-like substance included in the PM can initiate inflammatory effects following exposure of respiratory epithelial (BEAS-2B) cells and an animal model (male Wistar Kyoto rats). Carbon black (CB) was exposed for 72 hours to either filtered air (CB-Air) or approximately 100 ppm ozone (CB-O3). Carbon black exposure to high levels of ozone produced water-soluble, fluorescent organic material. Iron import by BEAS-2B cells at 4 and 24 hours was not induced by incubations with CB-Air but was increased following coexposures of CB-O3 with ferric ammonium citrate. In contrast to CB-Air, exposure of BEAS-2B cells and rats to CB-O3 for 24 hours increased expression of pro-inflammatory cytokines and lung injury, respectively. It is concluded that inflammatory effects of carbonaceous particles on cells can potentially result from (1) an inclusion of a fulvic acid-like substance after reaction with ozone and (2) changes in iron homeostasis following such exposure. [ABSTRACT FROM AUTHOR]

Previous studies have shown that air pollution exposure primes the body to heightened responses to everyday stressors of the cardiovascular system. The purpose of this study was to examine the utility of postprandial responses to a high carbohydrate oral load, a cardiometabolic stressor long used to predict cardiovascular risk, in assessing the impacts of exposure to eucalyptus smoke (ES), a contributor to wildland fire air pollution in the Western coast of the United States. Three-month-old male Sprague Dawley rats were exposed once (1 h) to filtered air (FA) or ES (700 µg/m3 fine particulate matter), generated by burning eucalyptus in a tube furnace. Rats were then fasted for six hours the following morning, and subsequently administered an oral gavage of either water or a HC suspension (70 kcal% from carbohydrate), mimicking a HC meal. Two hours post gavage, cardiovascular ultrasound, cardiac pressure–volume (PV), and baroreceptor sensitivity assessments were made, and pulmonary and systemic markers assessed. ES inhalation alone increased serum interleukin (IL)-4 and nasal airway levels of gamma glutamyl transferase. HC gavage alone increased blood glucose, blood pressure, and serum IL-6 and IL-13 compared to water vehicle. By contrast, only ES-exposed and HC-challenged animals had increased PV loop measures of cardiac output, ejection fraction %, dP/dtmax, dP/dtmin, and stroke work compared to ES exposure alone and/or HC challenge alone. Exposure to a model wildfire air pollution source modifies cardiovascular responses to HC challenge, suggesting air pollution sensitizes the body to systemic triggers. [ABSTRACT FROM AUTHOR]

Agonists of β2 adrenergic receptors (β2AR) and glucocorticoid receptors (GR) are prescribed to treat pulmonary diseases. Since ozone effects are mediated through the activation of AR and GR, we hypothesized that the treatment of rats with relevant therapeutic doses of long acting β2AR agonist (LABA; clenbuterol; CLEN) and/or GR agonist (dexamethasone; DEX) would exacerbate ozone-induced pulmonary and systemic changes. In the first study, male 12-week-old Wistar-Kyoto rats were injected intraperitoneally with vehicle (saline), CLEN (0.004 or 0.02 mg/kg), or DEX (0.02 or 0.1 mg/kg). Since dual therapy is commonly used, in the second study, rats received either saline or combined CLEN + DEX (each at 0.005 or 0.02 mg/kg) one day prior to and on both days of exposure (air or 0.8ppm ozone, 4 hr/day x 2-days). In air-exposed rats CLEN, DEX or CLEN + DEX did not induce lung injury or inflammation, however DEX and CLEN + DEX decreased circulating lymphocytes, spleen and thymus weights, increased free fatty acids (FFA) and produced hyperglycemia and glucose intolerance. Ozone exposure of vehicle-treated rats increased bronchoalveolar lavage fluid protein, albumin, neutrophils, IL-6 and TNF-α. Ozone decreased circulating lymphocytes, increased FFA, and induced hypeerglycemia and glucose intolerance. Drug treatment did not reverse ozone-induced ventillatory changes, however, lung effects (protein and albumin leakage, inflammation, and IL-6 increase) were exacerbated by CLEN and CLEN + DEX pre-treatment in a dose-dependent manner (CLEN > CLEN + DEX). Systemic effects induced by DEX and CLEN + DEX but not CLEN in air-exposed rats were analogous to and more pronounced than those induced by ozone. These data suggest that adverse air pollution effects might be exacerbated in people receiving LABA or LABA plus glucocorticoids. [ABSTRACT FROM AUTHOR]

Acute ozone inhalation increases circulating stress hormones through activation of the sympathetic-adrenal-medullary and hypothalamic-pituitary-adrenal axes. Rats with adrenalectomy (AD) have attenuated ozone-induced lung responses. We hypothesized that ozone exposure will induce changes in circulating pituitary-derived hormones and global gene expression in the brainstem and hypothalamus, and that AD will ameliorate these effects. Male Wistar-Kyoto rats (13 weeks) that underwent sham surgery (SHAM) or AD were exposed to ozone (0.8 ppm) or filtered-air for 4 h. In SHAM rats, ozone exposure decreased circulating thyroid-stimulating hormone (TSH), prolactin (PRL), and luteinizing hormone (LH). AD prevented reductions in TSH and PRL, but not LH. AD increased adrenocorticotropic hormone approximately 5-fold in both air- and ozone-exposed rats. AD in air-exposed rats resulted in few significant transcriptional differences in the brainstem and hypothalamus (approximately 20 genes per tissue). In contrast, ozone-exposure in SHAM rats resulted in either increases or decreases in expression of hundreds of genes in the brainstem and hypothalamus relative to air-exposed SHAM rats (303 and 568 genes, respectively). Differentially expressed genes from ozone exposure were enriched for pathways involving hedgehog signaling, responses to alpha-interferon, hypoxia, and mTORC1, among others. Gene changes in both brain areas were analogous to those altered by corticosteroids and L-3,4-dihydroxyphenylalanine, suggesting a role for endogenous glucocorticoids and catecholamines. AD completely prevented this ozone-induced transcriptional response. These findings show that short-term ozone inhalation promotes a shift in brainstem and hypothalamic gene expression that is dependent upon the presence of circulating adrenal-derived stress hormones. This is likely to have profound downstream influence on systemic effects of ozone. [ABSTRACT FROM AUTHOR]

Implantation is a sensitive window in reproductive development during which disruptions may increase the risk of adverse pregnancy outcomes including intrauterine growth restriction. Ozone exposure during implantation in rats reduces fetal weight near the end of gestation, potentially though impaired trophoblast migration and invasion and altered implantation. The current study characterized changes in ventilation, pulmonary injury, and circulating factors including hormonal, inflammatory, and metabolic markers related to exposure to ozone (0.4–1.2 ppm) for 4-h on gestation days 5 and 6 (window of implantation) in Long-Evans dams. To determine the effects of this exposure on trophoblast function, placental-derived, first trimester, HTR-8/SVneo cells were exposed to serum from air- or ozone (0.8 ppm×4 h)-exposed dams and examined for impacts on metabolic capacity, wound-closure, and invasion. Peri-implantation exposure to ozone induced ventilatory dysfunction and lung vascular leakage in pregnant rats, with little effect on most of the circulating markers measured. However, ozone inhalation induced a significant reduction in several serum cytokines (interferon-γ, interleukin-6, and interleukin-13). Treatment of HTR-8/SVneo trophoblasts with serum from ozone-exposed dams for 16-h downregulated metabolic capacity, wound-closure, and invasion through a Matrigel membrane compared with both air-serum and fetal bovine serum-treated cells. Ozone-serum treated cells increased the release of a critical inhibitor of invasion and angiogenesis (soluble fms-like receptor 1; sFlt1) compared with air-serum treatment. Together, our data suggest that circulating factors in the serum of pregnant rats exposed to ozone during implantation receptivity can hinder critical processes of implantation (eg, invasion and migration) and impair trophoblast metabolic capacity. [ABSTRACT FROM AUTHOR]

Abstract Wildland fire emissions cause adverse cardiopulmonary outcomes, yet controlled exposure studies to characterize health impacts of specific biomass sources have been complicated by the often latent effects of air pollution. The aim of this study was to determine if postprandial responses after a high fat challenge, long used clinically to predict cardiovascular risk, would unmask latent cardiometabolic responses in rats exposed to peat smoke, a key wildland fire air pollution source. Male Wistar Kyoto rats were exposed once (1 h) to filtered air (FA), or low (0.36 mg/m3 particulate matter) or high concentrations (3.30 mg/m3) of peat smoke, generated by burning peat from an Irish bog. Rats were then fasted overnight, and then administered an oral gavage of a HF suspension (60 kcal% from fat), mimicking a HF meal, 24 h post-exposure. In one cohort, cardiac and superior mesenteric artery function were assessed using high frequency ultrasound 2 h post gavage. In a second cohort, circulating lipids and hormones, pulmonary and systemic inflammatory markers, and circulating monocyte phenotype using flow cytometry were assessed before or 2 or 6 h after gavage. HF gavage alone elicited increases in circulating lipids characteristic of postprandial responses to a HF meal. Few effects were evident after peat exposure in un-gavaged rats. By contrast, exposure to low or high peat caused several changes relative to FA-exposed rats 2 and 6 h post HF gavage including increased heart isovolumic relaxation time, decreased serum glucose and insulin, increased CD11 b/c-expressing blood monocytes, increased serum total cholesterol, alpha-1 acid glycoprotein, and alpha-2 macroglobulin (p = 0.063), decreased serum corticosterone, and increased lung gamma-glutamyl transferase. In summary, these findings demonstrate that a HF challenge reveals effects of air pollution that may otherwise be imperceptible, particularly at low exposure levels, and suggest exposure may sensitize the body to mild inflammatory triggers. Graphical abstract Unlabelled Image Highlights • Peat smoke inhalation modified cardiac functional responses to a high fat oral load. • Peat smoke inhalation altered metabolic responses to a high fat oral load. • Peat smoke worsened lung & systemic inflammatory responses to a high fat oral load. • Peat smoke increased proinflammatory blood monocytes after a high fat oral load. [ABSTRACT FROM AUTHOR]

Fish, olive and coconut oil dietary supplementation have several cardioprotective benefits, but it is not established if they protect against air pollution-induced adverse effects. We hypothesized that these dietary supplements would attenuate ozone-induced systemic and pulmonary effects. Male Wistar Kyoto rats were fed either a normal diet, or a diet supplemented with fish, olive, or coconut oil for 8weeks. Animals were then exposed to air or ozone (0.8 ppm), 4 h/day for 2 days. Ozone exposure increased phenylephrine-induced aortic vasocontraction, which was completely abolished in rats fed the fish oil diet. Despite this cardioprotective effect, the fish oil diet increased baseline levels of bronchoalveolar lavage fluid (BALF) markers of lung injury and inflammation. Ozone-induced pulmonary injury/ inflammation were comparable in rats on normal, coconut oil and olive oil diets with altered expression of markers in animals fed the fish oil diet. Fish oil, regardless of exposure, led to enlarged, foamy macrophages in the BALF that coincided with decreased pulmonary mRNA expression of cholesterol transporters, cholesterol receptors and nuclear receptors. Serum microRNA profile was assessed and demonstrated marked depletion of a variety of microRNAs in animals fed the fish oil diet, several of which were of splenic origin. No ozone-specific changes were noted. Collectively, these data indicate that although fish oil offered vascular protection from ozone exposure, it increased pulmonary injury/inflammation and impaired lipid transport mechanisms resulting in foamy macrophage accumulation, demonstrating the need to be cognizant of potential off-target pulmonary effects that might offset the overall benefit of this vasoprotective supplement. [ABSTRACT FROM AUTHOR]

BACKGROUND: Epidemiological studies suggest that increased ozone exposure during gestation may compromise fetal growth. In particular, the implantation stage of pregnancy is considered a key window of susceptibility for this outcome. OBJECTIVES: The main goals of this study were to investigate the effects of short-term ozone inhalation during implantation on fetal growth outcomes and to explore the potential for alterations in uterine arterial flow as a contributing mechanism. METHODS: Pregnant Long-Evans rats were exposed to filtered air, 0:4 ppm ozone, or 0:8 ppm ozone for 4 h/d during implantation, on gestation days (GD) 5 and 6. Tail cuff blood pressure and uterine artery Doppler ultrasound were measured on GD 15, 19, and 21. To assess whether periimplantation ozone exposure resulted in sustained pulmonary or systemic health effects, bronchoalveolar lavage fluid, serum metabolic and inflammatory end points, and kidney histopathology were evaluated in dams at GD 21. Growth parameters assessed in GD 21 offspring included fetal weight, length, and body composition. RESULTS: Measures of maternal uterine arterial flow, including resistance index and mean velocity, indicated that resistance increased between GD 15 and GD 21 in 0:8 ppm dams but decreased in controls, although absolute values were similar in both groups on GD 21. Ozone-exposed dams also had lower serum glucose and higher free fatty acid concentrations than controls on GD 21. On GD 21, both male and female offspring had lower body weight than controls, and pooled subsets of 3 male and 3 female fetuses from litters exposed to 0:8 ppm ozone had lower lean mass and fat mass than pooled control offspring. CONCLUSIONS: Findings from our experimental model suggest that the offspring of dams exposed to ozone during implantation had reduced growth compared with controls, possibly as a consequence of ozone-induced vascular dysfunction. [ABSTRACT FROM AUTHOR]

Ozone-induced systemic effects are modulated through activation of the neuro-hormonal stress response pathway. Adrenal demedullation (DEMED) or bilateral total adrenalectomy (ADREX) inhibits systemic and pulmonary effects of acute ozone exposure. To understand the influence of adrenal-derived stress hormones in mediating ozone-induced lung injury/inflammation, we assessed global gene expression (mRNA sequencing) and selected proteins in lung tissues from male Wistar-Kyoto rats that underwent DEMED, ADREX, or sham surgery (SHAM) prior to their exposure to air or ozone (1 ppm), 4 h/day for 1 or 2 days. Ozone exposure significantly changed the expression of over 2300 genes in lungs of SHAM rats, and these changes were markedly reduced in DEMED and ADREX rats. SHAM surgery but not DEMED or ADREX resulted in activation of multiple ozone-responsive pathways, including glucocorticoid, acute phase response, NRF2, and PI3K-AKT. Predicted targets from sequencing data showed a similarity between transcriptional changes induced by ozone and adrenergic and steroidal modulation of effects in SHAM but not ADREX rats. Ozone-induced increases in lung Il6 in SHAM rats coincided with neutrophilic inflammation, but were diminished in DEMED and ADREX rats. Although ozone exposure in SHAM rats did not significantly alter mRNA expression of Ifnγ and Il-4 , the IL-4 protein and ratio of IL-4 to IFNγ (IL-4/IFNγ) proteins increased suggesting a tendency for a Th2 response. This did not occur in ADREX and DEMED rats. We demonstrate that ozone-induced lung injury and neutrophilic inflammation require the presence of circulating epinephrine and corticosterone, which transcriptionally regulates signaling mechanisms involved in this response. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that exposure to the pulmonary irritant ozone causes myriad systemic metabolic and pulmonary effects attributed to sympathetic and hypothalamus-pituitary-adrenal (HPA) axis activation, which are exacerbated in metabolically impaired models. We examined respiratory and systemic effects following exposure to a sensory irritant acrolein to elucidate the systemic and pulmonary consequences in healthy and diabetic rat models. Male Wistar and Goto Kakizaki (GK) rats, a nonobese type II diabetic Wistar-derived model, were exposed by inhalation to 0, 2, or 4ppm acrolein, 4 h/d for 1 or 2 days. Exposure at 4ppm significantly increased pulmonary and nasal inflammation in both strains with vascular protein leakage occurring only in the nose. Acrolein exposure (4 ppm) also caused metabolic impairment by inducing hyperglycemia and glucose intolerance (GK > Wistar). Serum total cholesterol (GKs only), lowdensity lipoprotein (LDL) cholesterol (both strains) and free fatty acids (GK > Wistar) levels increased; however, no acroleininduced changes were noted in branched-chain amino acid or insulin levels. These responses corresponded with a significant increase in corticosterone and modest but insignificant increases in adrenaline in both strains, suggesting activation of the HPA axis. Collectively, these data demonstrate that acrolein exposure has a profound effect on nasal and pulmonary inflammation, as well as glucose and lipid metabolism, with the systemic effects exacerbated in the metabolically impaired GKs. These results are similar to ozone-induced responses with the exception of lung protein leakage and ability to alter branched-chain amino acid and insulin levels, suggesting some differences in neuroendocrine regulation of these two air pollutants. [ABSTRACT FROM AUTHOR]

Photochemical smog is a complex mixture of primary and secondary air pollutants including secondary organic aerosols (SOA), ozone and reactive aldehydes which has been linked to increased risk of adverse cardiovascular and pulmonary responses. The components and related health effects of smog are thought to be determined both by the precursor chemicals and reaction conditions. Here we examined the difference between isoprene- (IS-) and toluene- (TL) generated smog in causing cardiopulmonary effects in rats. Wistar-Kyoto rats were exposed to either filtered air (FA) or smog for 4 h. IS-smog contained 660 ppb NO 2 , 335 ppb O 3 , and 178 μg/m3 SOA while TL-smog had 380 ppb NO 2 , 250 ppb O 3 , and 1,250 μg/m3 SOA. Whole-body plethysmography was performed at baseline and after each exposure and left ventricular function was measured using a Millar probe and arrhythmic sensitivity to aconitine. Tidal volume decreased only after IS-smog while inspiratory time and nasal injury increased following both smog atmospheres. Only TL-smog caused an increase in PenH, which is an indicator of airway irritation. On the other hand, only IS-smog caused left ventricular pressure to decrease, while only TL-smog increased sensitivity to cardiac arrhythmia. These data indicate that the source of smog atmosphere plays a large role in the types of responses it elicits and that its relative SOA and gaseous composition determine the extent of cardiovascular versus respiratory effects. [ABSTRACT FROM AUTHOR]