Gut metabolic changes during pregnancy reveal the importance of gastrointestinal region in sample collection.

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.

Multi-tissue transcriptomic and serum metabolomic assessment reveals systemic implications of acute ozone-induced stress response in male Wistar Kyoto rats.

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.

Prenatal ozone exposure programs a sexually dimorphic susceptibility to high-fat diet in adolescent Long Evans rats.

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.

Intratracheal instillation of respirable particulate matter elicits neuroendocrine activation.

OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

The contribution of the neuroendocrine system to adaption after repeated daily ozone exposure in rats.

Ozone-induced lung injury/inflammation dissipates despite continued exposure for 3 or more days; however, the mechanisms of adaptation/habituation remain unclear. Since ozone effects are mediated through adrenal-derived stress hormones, which also regulate longevity of centrally-mediated stress response, we hypothesized that ozone-adaptation is linked to diminution of neuroendocrine stress-axes activation and glucocorticoid levels. Male Wistar-Kyoto-rats (12-week-old) were injected with vehicle or a therapeutically-relevant dexamethasone dose (0.01-mg/kg/day; intraperitoneal) for 1-month to determine if suppression of glucocorticoid signaling was linked to adaptation. Vehicle- and dexamethasone-treated rats were exposed to air or 0.8-ppm ozone, 4 h/day × 2 or 4 days to assess the impacts of acute exposure and adaptation, respectively. Dexamethasone reduced thymus and spleen weights, circulating lymphocytes, corticosterone and increased insulin. Ozone increased lavage-fluid protein and neutrophils and decreased circulating lymphocytes at day-2 but not day-4. Ozone-induced hyperglycemia, glucose intolerance and inhibition of beta-cell insulin release occurred at day-1 but not day-3. Ozone depleted circulating prolactin, thyroid-stimulating hormone, and luteinizing-hormone at day-2 but not day-4, suggesting central mediation of adaptation. Adrenal epinephrine biosynthesis gene, Pnmt, was up-regulated after ozone exposure at both timepoints. However, genes involved in glucocorticoid biosynthesis were up-regulated after day-2 but not day-4, suggesting that acute 1- or 2-day ozone-mediated glucocorticoid increase elicits feedback inhibition to dampen hypothalamic stimulation of ACTH release in response to repeated subsequent ozone exposures. Although dexamethasone pretreatment affected circulating insulin, lymphocytes and adrenal genes, it had modest effect on ozone adaptation. In conclusion, ozone adaptation likely involves lack of hypothalamic response due to reduced availability of circulating glucocorticoids.

Social isolation exacerbates acute ozone inhalation induced pulmonary and systemic health outcomes.

Psychosocially-stressed individuals might have exacerbated responses to air pollution exposure. Acute ozone exposure activates the neuroendocrine stress response leading to systemic metabolic and lung inflammatory changes. We hypothesized chronic mild stress (CS) and/or social isolation (SI) would cause neuroendocrine, inflammatory, and metabolic phenotypes that would be exacerbated by an acute ozone exposure. Male 5-week-old Wistar-Kyoto rats were randomly assigned into 3 groups: no stress (NS) (pair-housed, regular-handling); SI (single-housed, minimal-handling); CS (single-housed, subjected to mild unpredicted-randomized stressors [restraint-1 h, tilted cage-1 h, shaking-1 h, intermittent noise-6 h, and predator odor-1 h], 1-stressor/day*5-days/week*8-weeks. All animals then 13-week-old were subsequently exposed to filtered-air or ozone (0.8-ppm) for 4 h and immediately necropsied. CS, but not SI animals had increased adrenal weights. However, relative to NS, both CS and SI had lower circulating luteinizing hormone, prolactin, and follicle-stimulating hormone regardless of exposure (SI > CS), and only CS demonstrated lower thyroid-stimulating hormone levels. SI caused more severe systemic inflammation than CS, as evidenced by higher circulating cytokines and cholesterol. Ozone exposure increased urine corticosterone and catecholamine metabolites with no significant stressor effect. Ozone-induced lung injury, and increases in lavage-fluid neutrophils and IL-6, were exacerbated by SI. Ozone severely lowered circulating thyroid-stimulating hormone, prolactin, and luteinizing hormone in all groups and exacerbated systemic inflammation in SI. Ozone-induced increases in serum glucose, leptin, and triglycerides were consistent across stressors; however, increases in cholesterol were exacerbated by SI. Collectively, psychosocial stressors, especially SI, affected the neuroendocrine system and induced adverse metabolic and inflammatory effects that were exacerbated by ozone exposure.

Pulmonary and vascular effects of acute ozone exposure in diabetic rats fed an atherogenic diet.

Air pollutants may increase risk for cardiopulmonary disease, particularly in susceptible populations with metabolic stressors such as diabetes and unhealthy diet. We investigated effects of inhaled ozone exposure and high-cholesterol diet (HCD) in healthy Wistar and Wistar-derived Goto-Kakizaki (GK) rats, a non-obese model of type 2 diabetes. Male rats (4-week old) were fed normal diet (ND) or HCD for 12 weeks and then exposed to filtered air or 1.0 ppm ozone (6 h/day) for 1 or 2 days. We examined pulmonary, vascular, hematology, and inflammatory responses after each exposure plus an 18-h recovery period. In both strains, ozone induced acute bronchiolar epithelial necrosis and inflammation on histopathology and pulmonary protein leakage and neutrophilia; the protein leakage was more rapid and persistent in GK compared to Wistar rats. Ozone also decreased lymphocytes after day 1 in both strains consuming ND (~50%), while HCD increased circulating leukocytes. Ozone increased plasma thrombin/antithrombin complexes and platelet disaggregation in Wistar rats on HCD and exacerbated diet effects on serum IFN-γ, IL-6, KC-GRO, IL-13, and TNF-α, which were higher with HCD (Wistar>GK). Ex vivo aortic contractility to phenylephrine was lower in GK versus Wistar rats at baseline(~30%); ozone enhanced this effect in Wistar rats on ND. GK rats on HCD had higher aortic e-NOS and tPA expression compared to Wistar rats. Ozone increased e-NOS in GK rats on ND (~3-fold) and Wistar rats on HCD (~2-fold). These findings demonstrate ways in which underlying diabetes and HCD may exacerbate pulmonary, systemic, and vascular effects of inhaled pollutants.

Peripheral metabolic effects of ozone exposure in healthy and diabetic rats on normal or high-cholesterol diet.

Epidemiological studies show that individuals with underlying diabetes and diet-associated ailments are more susceptible than healthy individuals to adverse health effects of air pollution. Exposure to air pollutants can induce metabolic stress and increase cardiometabolic disease risk. Using male Wistar and Wistar-derived Goto-Kakizaki (GK) rats, which exhibit a non-obese type-2 diabetes phenotype, we investigated whether two key metabolic stressors, type-2 diabetes and a high-cholesterol atherogenic diet, exacerbate ozone-induced metabolic effects. Rats were fed a normal control diet (ND) or high-cholesterol diet (HCD) for 12 weeks and then exposed to filtered air or 1.0-ppm ozone (6 h/day) for 1 or 2 days. Metabolic responses were analyzed at the end of each day and after an 18-h recovery period following the 2-day exposure. In GK rats, baseline hyperglycemia and glucose intolerance were exacerbated by HCD vs. ND and by ozone vs. air. HCD also resulted in higher insulin in ozone-exposed GK rats and circulating lipase, aspartate transaminase, and alanine transaminase in all groups (Wistar>GK). Histopathological effects induced by HCD in the liver, which included macrovesicular vacuolation and hepatocellular necrosis, were more severe in Wistar vs. GK rats. Liver gene expression in Wistar and GK rats fed ND showed numerous strain differences, including evidence of increased lipid metabolizing activity and ozone-induced alterations in glucose and lipid transporters, specifically in GK rats. Collectively, these findings indicate that peripheral metabolic alterations induced by diabetes and high-cholesterol diet can enhance susceptibility to the metabolic effects of inhaled pollutants.

Diets enriched with coconut, fish, or olive oil modify peripheral metabolic effects of ozone in rats.

Dietary factors may modulate metabolic effects of air pollutant exposures. We hypothesized that diets enriched with coconut oil (CO), fish oil (FO), or olive oil (OO) would alter ozone-induced metabolic responses. Male Wistar-Kyoto rats (1-month-old) were fed normal diet (ND), or CO-, FO-, or OO-enriched diets. After eight weeks, animals were exposed to air or 0.8 ppm ozone, 4 h/day for 2 days. Relative to ND, CO- and OO-enriched diet increased body fat, serum triglycerides, cholesterols, and leptin, while all supplements increased liver lipid staining (OO > FO > CO). FO increased n-3, OO increased n-6/n-9, and all supplements increased saturated fatty-acids. Ozone increased total cholesterol, low-density lipoprotein, branched-chain amino acids (BCAA), induced hyperglycemia, glucose intolerance, and changed gene expression involved in energy metabolism in adipose and muscle tissue in rats fed ND. Ozone-induced glucose intolerance was exacerbated by OO-enriched diet. Ozone increased leptin in CO- and FO-enriched groups; however, BCAA increases were blunted by FO and OO. Ozone-induced inhibition of liver cholesterol biosynthesis genes in ND-fed rats was not evident in enriched dietary groups; however, genes involved in energy metabolism and glucose transport were increased in rats fed FO and OO-enriched diet. FO- and OO-enriched diets blunted ozone-induced inhibition of genes involved in adipose tissue glucose uptake and cholesterol synthesis, but exacerbated genes involved in adipose lipolysis. Ozone-induced decreases in muscle energy metabolism genes were similar in all dietary groups. In conclusion, CO-, FO-, and OO-enriched diets modified ozone-induced metabolic changes in a diet-specific manner, which could contribute to altered peripheral energy homeostasis.

Iron and zinc homeostases in female rats with physically active and sedentary lifestyles.

To determine the effects of repeated physical activity on iron and zinc homeostases in a living system, we quantified blood and tissue levels of these two metals in sedentary and physically active Long-Evans rats. At post-natal day (PND) 22, female rats were assigned to either a sedentary or an active treatment group (n = 10/group). The physically active rats increased their use of a commercially-constructed stainless steel wire wheel so that, by the end of the study (PND 101), they were running an average of 512.8 ± 31.9 (mean ± standard error) min/night. After euthanization, plasma and aliquots of liver, lung, heart, and gastrocnemius muscle were obtained. Following digestion, non-heme iron and zinc concentrations in plasma and tissues were measured using inductively coupled plasma optical emission spectroscopy. Concentrations of both non-heme iron and zinc in plasma and liver were significantly decreased among the physically active rats relative to the sedentary animals. In the lung, both metals were increased in concentration among the physically active animals but the change in zinc did not reach significance. Similarly, tissue non-heme iron and zinc levels were both increased in heart and muscle from the physically active group. It is concluded that repeated physical activity in an animal model can be associated with a translocation of both iron and zinc from sites of storage (e.g. liver) to tissues with increased metabolism (e.g. the lung, heart, and skeletal muscle).

Ozone-induced acute phase response in lung versus liver: the role of adrenal-derived stress hormones.

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.

Fish oil and olive oil-enriched diets alleviate acute ozone-induced cardiovascular effects in rats.

Fish oil (FO) and olive oil (OO) supplementations attenuate the cardiovascular responses to inhaled concentrated ambient particles in human volunteers. This study was designed to examine the cardiovascular effects of ozone (O3) exposure and the efficacy of FO and OO-enriched diets in attenuating the cardiovascular effects from O3 exposure in rats. Rats were fed either a normal diet (ND), a diet enriched with 6% FO or OO starting at 4 weeks of age. Eight weeks following the start of these diet, animals were exposed to filtered air (FA) or 0.8 ppm O3, 4 h/day for 2 consecutive days. Immediately after exposure, cardiac function was measured as the indices of left-ventricular developed pressure (LVDP) and contractility (dP/dtmax and dP/dtmin) before ischemia. In addition, selective microRNAs (miRNAs) of inflammation, endothelial function, and cardiac function were assessed in cardiac tissues to examine the molecular alterations of diets and O3 exposure. Pre-ischemic LVDP and dP/dtmax were lower after O3 exposure in rats fed ND but not FO and OO. Cardiac miRNAs expressions were altered by both diet and O3 exposure. Specifically, O3-induced up-regulation of miR-150-5p and miR-208a-5p were attenuated by FO and/or OO. miR-21 was up-regulated by both FO and OO after O3 exposure. This study demonstrated that O3-induced cardiovascular responses appear to be blunted by FO and OO diets. O3-induced alterations in miRNAs linked to inflammation, cardiac function, and endothelial dysfunction support these pathways are involved, and dietary supplementation with FO or OO may alleviate these adverse cardiovascular effects in rats.

Ozone Reacts With Carbon Black to Produce a Fulvic Acid-Like Substance and Increase an Inflammatory Effect.

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.

Independent roles of beta-adrenergic and glucocorticoid receptors in systemic and pulmonary effects of ozone.

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 (ßAR) and glucocorticoid receptors (GRs), we inhibited ßAR while inducing GR or inhibited GR while inducing ßAR and examined ozone-induced stress response.Methods: Twelve-week-old male Wistar-Kyoto rats were pretreated daily with saline or propranolol (PROP; ßAR-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; ß2AR-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 ßAR, even with GR inhibition, exacerbated and inhibiting ßAR, even with GR activation, attenuated ozone-induced pulmonary effects; and 2) activating GR exacerbated ozone systemic effects, but with ßAR inhibition, this exacerbation was less remarkable. These data suggest the independent roles of ßAR in pulmonary and dependent roles of ßAR and GR in systemic effects of ozone.

A single exposure to eucalyptus smoke sensitizes rats to the postprandial cardiovascular effects of a high carbohydrate oral load.

OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. DISCUSSION AND CONCLUSIONS: Exposure to a model wildfire air pollution source modifies cardiovascular responses to HC challenge, suggesting air pollution sensitizes the body to systemic triggers.

The influence of maternal and perinatal high-fat diet on ozone-induced pulmonary responses in offspring.

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.

Adrenergic and glucocorticoid receptor antagonists reduce ozone-induced lung injury and inflammation.

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 7days with propranolol (PROP; a non-selective ß adrenergic receptor [AR] antagonist, 10mg/kg, i.p.), mifepristone (MIFE; a glucocorticoid receptor [GR] antagonist, 30mg/kg, s.c.), both drugs (PROP+MIFE), or respective vehicles, and then exposed to air or ozone (0.8ppm), 4h/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.

Acute inhalation of ozone induces DNA methylation of apelin in lungs of Long-Evans rats.

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.

Adrenal-derived stress hormones modulate ozone-induced lung injury and inflammation.

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 (1ppm), 4h/day for 1 or 2days. 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.

Systemic metabolic derangement, pulmonary effects, and insulin insufficiency following subchronic ozone exposure in rats.

Acute ozone exposure induces a classical stress response with elevated circulating stress hormones along with changes in glucose, protein and lipid metabolism in rats, with similar alterations in ozone-exposed humans. These stress-mediated changes over time have been linked to insulin resistance. We hypothesized that acute ozone-induced stress response and metabolic impairment would persist during subchronic episodic exposure and induce peripheral insulin resistance. Male Wistar Kyoto rats were exposed to air or 0.25ppm or 1.00ppm ozone, 5h/day, 3 consecutive days/week (wk) for 13wks. Pulmonary, metabolic, insulin signaling and stress endpoints were determined immediately after 13wk or following a 1wk recovery period (13wk+1wk recovery). We show that episodic ozone exposure is associated with persistent pulmonary injury and inflammation, fasting hyperglycemia, glucose intolerance, as well as, elevated circulating adrenaline and cholesterol when measured at 13wk, however, these responses were largely reversible following a 1wk recovery. Moreover, the increases noted acutely after ozone exposure in non-esterified fatty acids and branched chain amino acid levels were not apparent following a subchronic exposure. Neither peripheral or tissue specific insulin resistance nor increased hepatic gluconeogenesis were present after subchronic ozone exposure. Instead, long-term ozone exposure lowered circulating insulin and severely impaired glucose-stimulated beta-cell insulin secretion. Thus, our findings in young-adult rats provide potential insights into epidemiological studies that show a positive association between ozone exposures and type 1 diabetes. Ozone-induced beta-cell dysfunction may secondarily contribute to other tissue-specific metabolic alterations following chronic exposure due to impaired regulation of glucose, lipid, and protein metabolism.