Ppm O3 Research Articles

Vagal afferents contribute to exacerbated airway responses following ozone and allergen challenge

Brown-Norway rats (n=113) sensitized and challenged with nDer f 1 allergen were used to examine the contribution of lung sensory nerves to ozone (O3) exacerbation of asthma. Prior to their third challenge rats inhaled 1.0ppm O3 for 8h. There were three groups: (1) control; (2) vagus perineural capsaicin treatment (PCT) with or without hexamethonium; and (3) vagotomy. O3 inhalation resulted in a significant increase in lung resistance (RL) and an exaggerated response to subsequent allergen challenge. PCT abolished the O3-induced increase in RL and significantly reduced the increase in RL induced by a subsequent allergen challenge, while hexamethonium treatment reestablished bronchoconstriction induced by allergen challenge. Vagotomy resulted in a significant increase in the bronchoconstriction induced by O3 inhalation and subsequent challenge with allergen. In this model of O3 exacerbation of asthma, vagal C-fibers initiate reflex bronchoconstriction, vagal myelinated fibers initiate reflex bronchodilation, and mediators released within the airway initiate bronchoconstriction.

Abstract P305: Detrimental Effects of Chronic Activation of AKT in Hearts from Ozone-Exposed Healthy Adult Rats

Death of cardiomyocytes results in decreased cardiac function, which correlates with overall morbidity and mortality in many different clinical settings. For these reasons, understanding the intracellular signaling pathways that control cardiomyocyte survival has significant clinical implications. Each year in the US 60,000 cardiovascular deaths have been linked to environmental pollutants such as ozone (O 3 ). Earlier studies from our laboratory have shown that myocardial dysfunction, subsequent to chronic O 3 exposure, in normal adult rats. This attenuation of cardiac function was associated with increased myocardial TNF-alpha (TNF-α) levels. Progressive increases in the expression of myocardial TNF-α in 4 and 8 week O 3 exposed animals were followed by decreases in cardiac caveolin-1 levels. Acute activation of the serine-threonine kinase Akt is cardioprotective and reduces both infarction and dysfunction after ischemia/reperfusion injury (IRI). Akt is activated in samples from patients with chronic heart failure, however, less is known about the chronic effects of Akt activation in the hearts from ozone exposed patients. Although caveolin-1 has been shown to negatively regulate AKT expression; a cell survival pathway, regulatory role of AKT in hearts from ozone exposed subjects has never been studied. Sprague Dawley rats were exposed 8 hr/day for 28 and 56 days to filtered air or 0.8 ppm O 3 . In order to assess the chronic effects to O 3 , expressions of AKT and p-AKT were determined by wetern blotting in hearts extracted from rats, 24 hr after termination of air or O 3 exposure. Compared to rats exposed to filtered air, total AKT levels were significantly increased in all O 3 exposed animals. Similar results were seen with pAKT levels. Since earlier studies have determined detrimental effects of chronic activation of AKT in ischemia reperfusion injury, our novel findings suggest the interesting possibility of a mechanism by which chronic Akt activation can become maladaptive after exposure to chronic levels of ozone. It is possible that PI3K-dependent but Akt-independent effectors are required for full cardioprotection. Studies are currently in progress to prove this hypothesis and complete study will be presented at the meeting in New Orleans in 2011.

Abstract P292: Possible Mechanism and Potential Markers for Ozone-Induced Cardiac Toxicity

A significant number of deaths each year in the US have been linked to environmental pollutants such as ozone (O 3 ). Earlier studies from our laboratory have shown that myocardial dysfunction, subsequent to chronic O 3 exposure, in normal adult rats may be associated with a decrease in antioxidant reserve and with an increased activity of inflammatory mediators. The present study tested the hypothesis that O 3 induced cardiac dysfunction in healthy normal adult rats may be due to changes in caveolin-1 and caveolin-3 levels. Sprague Dawley rats were exposed 8 hr/day for 28 and 56 days to filtered air or 0.8 ppm O 3 . In order to assess the chronic effects to O 3 , in-vivo cardiac function was assessed by measuring left ventricular developed pressure (LVDP), 24 hr after termination of O 3 exposure. Compared to rats exposed to filtered air, LVDP values significantly decreased in all O 3 exposed animals. This attenuation of cardiac function was associated with increased myocardial TNF-alpha (TNF-α) levels and decreased myocardial activities of superoxidase dismutase (SOD). Progressive increases in the expression of myocardial TNF-α in 4 and 8 week O 3 exposed animals were followed by decreases in cardiac caveolin-1 levels. However, differential changes in the expression of caveolin-3 in hearts from 4 and 8 week O 3 exposed animals were independent of intra-cardiac TNF-α levels. These novel findings suggest the interesting possibility that a balance between caveolin-1 and caveolin-3 may be involved in O 3 - mediated cardiac toxicity. Furthermore, differential changes in caveolin-3 content may serve as a marker that predicts moderate and chronic stages of cardiac injury specific to exposure to O 3 in human populations residing in urban areas with unhealthy levels of O 3 . The study is timely and has clinical significance related to environmental causes of cardiovascular disease. This novel study will form a basis for future studies to understand and define the various components of the mechanistic cascade responsible for generation of cell death signaling subsequent to O 3 exposure. The long-term goal of this study is in guiding regulatory policies to the USEPA regarding air quality standards pertaining to O 3 levels.

Abstract P311: Differential Changes in Caveolin Levels in Lung and Heart from Ozone-Exposed Adult Healthy Rats

Acute exposure to ozone (O 3 ) has been reported to possess beneficial effects in the injured myocardium. We have demonstrated that chronic O 3 exposure elicits cardiac dysfunction in rats. These results suggest that acute and chronic exposures to O 3 have different effects on myocardial function. On the contrary exposure to acute ozone levels has shown to cause lung injury. Although effects of chronic exposure to ozone on the lung has not been determined, it is reasonable to suggest that ozone mediated toxicity may be tissue specific. It appears that in chronic cardiac injury subsequent to an ischemic insult, lipid raft proteins such as caveolin-1 and caveolin-3 play vital roles in the generation of death and survival signaling in the heart. In lung injury subsequent to acute O 3 exposure, dramatic reductions in caveolin-1 levels were associated with increases in lung TNF-α. Our previous studies showed increased expressions of myocardial TNF-α after chronic O 3 exposure. In the present study we wanted to determine the regulatory role of caveolin-1 and caveolin-3 in hearts and lungs from rats exposed to chronic O 3 levels. Sprague Dawley rats were exposed 8 hrs/day for 28 and 56 days to filtered air or 0.8 ppm O 3 . In order to assess the chronic effects to O 3 , levels of caveolin-1 and caveolin-3 in the lung and heart tissue were measured 24 hrs after termination of the O 3 exposure. Caveolin-1 levels in hearts from the ozone exposed groups were significantly decreased compared to air exposed rats; while caveolin-1 levels in lungs from the same animals were not different from the air exposed group. On the other hand caveolin-3 levels in hearts from the ozone exposed groups increased and decreased after 4 and 8 week of ozone exposure compared to air exposed rats; while caveolin-3 levels in lungs from the same animals were not different from the air exposed group. These novel findings suggest the interesting possibility that caveolin expressions subsequent to chronic ozone exposure may be tissue specific.

Abstract P299: Gender-Based Differences in Ozone-Induced Cardiac Dysfunction

Sex related differences have been noted in cardiovascular disease where females have a lower incidence of heart failure, and a higher rate of heart failure survival. On-the-other-hand, some studies have reported increased mortality in women compared with men. Recent data also suggest that women have an increased risk of death due to O 3 air pollution, which is in contrast to reports that short-term variations in gaseous pollutants are associated with an increase in hospitalization for cardiac disease that is not modified by gender. It has been speculated that because of the nature of the photochemical equilibrium of O 3 in the ambient environment and due to other confounding factors epidemiological investigations of the health effects of O 3 may be using O 3 as an exposure surrogate for other oxidants or co-pollutants, some of which may be interfering with response to O 3 . The present study in a controlled exposure protocol tested the hypothesis that female rats compared to male have decreased sensitivity to cardiac injury subsequent to O 3 exposure. Age matched male/female rats were exposed 8 hrs/day for 28 and 56 days to filtered air or 0.8 ppm O 3 . In order to assess the chronic effects to O 3 , in-vivo cardiac function was assessed, 24 hrs after termination of the O 3 exposure. Compared to female rats, LVDP values significantly decreased in O 3 exposed male rats. This enhanced attenuation of cardiac function in male animals was associated with increased myocardial TNF-alpha (TNF-α) levels and decreased myocardial activities of superoxidase dismutase (SOD). These novel findings suggest that decreased attenuation of cardiac function in O 3 exposed females compared to males exposed to similar conditions was associated with decreased inflammatory mediator production and decreased oxidative stress. Identifying the underlying factors for gender based variations in ozone response is very important to recognize at-risk groups who would benefit from preventive strategies. In addition identification of those at risk, their degree of sensitivity will assist with the cost-benefit analysis of “safe” exposure levels in the public health setting. The long-term goal of this study is in guiding regulatory policies for reduced environmental related health costs.

Overt and Latent Cardiac Effects of Ozone Inhalation in Rats: Evidence for Autonomic Modulation and Increased Myocardial Vulnerability

Background: Ozone (O3) is a well-documented respiratory oxidant, but increasing epidemiological evidence points to extrapulmonary effects, including positive associations between ambient O3 concentrations and cardiovascular morbidity and mortality.Objective: With preliminary reports linking O3 exposure with changes in heart rate (HR), we investigated the hypothesis that a single inhalation exposure to O3 will cause concentration-dependent autonomic modulation of cardiac function in rats.Methods: Rats implanted with telemeters to monitor HR and cardiac electrophysiology [electrocardiography (ECG)] were exposed once by whole-body inhalation for 4 hr to 0.2 or 0.8 ppm O3 or filtered air. A separate cohort was tested for vulnerability to aconitine-induced arrhythmia 24 hr after exposure.Results: Exposure to 0.8 ppm O3 caused bradycardia, PR prolongation, ST depression, and substantial increases in atrial premature beats, sinoatrial block, and atrioventricular block, accompanied by concurrent increases in several HR variability parameters that were suggestive of increased parasympathetic tone. Low-O3 exposure failed to elicit any overt changes in autonomic tone, heart rhythm, or ECG. However, both 0.2 and 0.8 ppm O3 increased sensitivity to aconitine-induced arrhythmia formation, suggesting a latent O3-induced alteration in myocardial excitability.Conclusions: O3 exposure causes several alterations in cardiac electrophysiology that are likely mediated by modulation of autonomic input to the heart. Moreover, exposure to low O3 concentrations may cause subclinical effects that manifest only when triggered by a stressor, suggesting that the adverse health effects of ambient levels of air pollutants may be insidious and potentially underestimated.

Identification of Candidate Genes Downstream of TLR4 Signaling after Ozone Exposure in Mice: A Role for Heat-Shock Protein 70

Background: Toll-like receptor 4 (TLR4) is involved in ozone (O3)-induced pulmonary hyperpermeability and inflammation, although the downstream signaling events are unknown.Objectives: The aims of our study were to determine the mechanism through which TLR4 modulates O3-induced pulmonary responses and to use transcriptomics to determine potential TLR4 effector molecules.Methods: C3H/HeJ (HeJ; Tlr4 mutant) and C3H/HeOuJ (OuJ; Tlr4 normal) mice were exposed continuously to 0.3 ppm O3 or filtered air for 6, 24, 48, or 72 hr. We assessed inflammation using bronchoalveolar lavage and molecular analysis by mRNA microarray, quantitative RT-PCR (real-time polymerase chain reaction), immunoblots, immunostaining, and ELISAs (enzyme-linked immunosorbent assays). B6-Hspa1a/Hspa1btm1Dix/NIEHS (Hsp70–/–) and C57BL/6 (B6; Hsp70+/+ wild-type control) mice were used for candidate gene validation studies.Results: O3-induced TLR4 signaling occurred through myeloid differentiation protein 88 (MyD88)-dependent and -independent pathways in OuJ mice and involved multiple downstream pathways. Genomewide transcript analyses of lungs from air- and O3-exposed HeJ and OuJ mice identified a cluster of genes that were significantly up-regulated in O3-exposed OuJ mice compared with O3-exposed HeJ mice or air-exposed controls of both strains; this cluster included genes for heat-shock proteins (e.g., Hspa1b, Hsp70). Moreover, O3-induced inflammation, MyD88 up-regulation, extracellular-signal–related kinase-1/2 (ERK1/2) and activator protein-1 (AP-1) activation, and kerotinocyte-derived chemokine (KC) protein content were significantly reduced in Hspa1a/Hspa1btm1Dix (Hsp70–/–) compared with Hsp70+/+ mice (p < 0.05).Conclusions: These studies suggest that HSP70 is an effector molecule downstream of TLR4 and is involved in the regulation of O3-induced lung inflammation by triggering similar pathways to TLR4. These novel findings may have therapeutic and preventive implications for inflammatory diseases resulting from environmental exposures.

Effect of maternal exposure to ozone on reproductive outcome and immune, inflammatory, and allergic responses in the offspring

There is growing concern that exposure to air pollutants during pregnancy affects health outcomes in the offspring due to alterations in the development of immune and other homeostatic processes. To assess the risks of maternal inhalation exposure to ozone (O3), timed pregnant BALB/c mice were exposed to different concentrations of O3 (0, 0.4, 0.8, and 1.2 ppm) for 4 h/day for 10 days during gestation (GD9–GD18), and pulmonary inflammation and immune responses were assessed in the offspring at 6 weeks-of-age. Maternal O3 exposure reduced the number of productive dams by 25% at the highest O3 concentration (1.2 ppm) and decreased the rate of weight gain in the offspring. Delayed-type hypersensitivity responses to bovine serum albumin were suppressed in the female offspring by maternal exposure to the two highest concentrations of O3, whereas humoral immune responses to sheep red blood cells were not altered in either sex. Maternal exposure to 1.2 ppm O3 increased lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluid (BALF) of the offspring but did not affect the number of inflammatory cells or levels of total protein, IFN-γ, IL-17, and IL-4 cytokines in BALF, or CD4+, CD8+, CD25+, and TCRβ+CD1d+ T-cells in the spleen. Offspring born from air-exposed dams sensitized early in life (postnatal day [PND] 3) to ovalbumin (OVA) antigen and then challenged as adults developed eosinophilia, elevated levels of LDH activity and total protein in BALF, and increased pulmonary responsiveness to methacholine, compared with animals sensitized at PND42. Maternal O3 exposure in the 1.2 ppm O3 group decreased BALF eosinophilia and serum OVA-specific IgE in the female offspring sensitized early in life but did not affect development of allergic airway inflammation by offspring sensitized late in life. In summary, maternal exposure to O3 affected reproductive outcome and produced modest decreases in immune function and indicators of allergic lung disease in surviving offspring.

Ozone Exposure Induces Beta‐Adrenergic Insensitivity

The association between ambient ozone (O3) and adverse cardiopulmonary outcomes has long been recognized. Acute O3 classically induces bradycardia, hypotension, and increases heart rate variability – all of which are affected by the heart's response to sympathetic output. To test whether O3 exposure changes the sensitivity of the heart to beta adrenergic stimulus, C57BL/6 mice (n=10) were exposed to either Room Air (RA) 24 hours of 0.3 ppm O3 and then underwent in‐vivo hemodynamic assessment and isoproterenol (Iso) challenge involving I.V. bolus doses at half log intervals. Mice exposed to O3 displayed decreased left ventricular systolic function as characterized by decreased heart rate 565 bpm vs. 595 bmp in RA; systolic pressure decreased from 106 mmHg in RA mice to 87 mmHg in O3 exposed mice; dP/dT max decreased 10% with O3 exposure. Diastolic function was also depressed by O3 exposure: dP/dT min increased by 25% and Tau (Log) increased by 30% with O3 exposure. O3 exposure resulted in a decreased response to Iso as indicated by a leftward shift in the dose‐response curve. At the maximal dose (6 ng Iso in 10 μL PBS) heart rate increased by 60 bpm in RA controls and 35 bpm in O3 treated mice; dP/dT max increased by 6000 mmHg/s in control and only increased by 3000 mmHg/s in O3 exposed mice. In addition to confirming the systolic and diastolic dysfunction typically associated with O3, this study demonstrates that acute O3 exposure induces beta adrenergic insensitivity – a condition thought to contribute to heart failure in patients with heart disease that may be important in understanding the deleterious cardiopulmonary effects of inhaled O3.NIH R01 HL087033

Ozone induces pulmonary activating transcription factor 3 (ATF3) expression

Ozone, a major component of air pollution, has been linked to detrimental health effects including inflammation, asthma, cardiovascular disease, and increased mortality. While recent evidence suggests that transcription factor ATF3 is a negative regulator of pulmonary inflammation, its role in O3-induced lung injury remains unknown. To address this question, we exposed 8-10 wk male C57BL/6 mice to 2 ppm O3 or filtered air (FA) for 3 hrs and allowed subsequent recovery in room air for up to 24 hrs. Compared to FA, ATF3 protein was up-regulated by 5-fold after exposure to O3 for 3 hrs (p<0.01) while the expression of ATF4 and ATF5 proteins remained unchanged. O3 increased the nuclear accumulation of ATF3, but not ATF4. Quantitative PCR revealed a 3-fold increase in ATF3 after O3 but no change in ATF4 or ATF5. Both ATF3 protein and mRNA levels remained elevated during early recovery (4 hrs post-exposure), but declined by 24 hrs. Induction of ATF3 expression involves multiple overlapping signal transduction pathways known to be sensitive to oxidative stress. We found that O3 increased the phosphorylation of ERK1/2, JNK, and p38 MAPK, but not the phosphorylation of eIF2α. In addition, p38 phosphorylation remained elevated during early recovery. Altogether, these results illustrate that O3 induces expression of ATF3 in the lung. The increase in ATF3 may play a role in the negative regulation of O3-induced inflammation.

Serum biomarkers in young adult and aged Brown Norway (BN) rats following episodic (weekly) ozone exposure

Ozone (O3) is an air pollutant that is associated with cardiovascular and respiratory diseases. Older adults are considered to be particularly susceptible to oxidant air pollutants such as O3. Serum biomarkers are being sought that would lead to better predictions of susceptibility in the aged to air pollutants. We exposed young adult (4 month) and aged (20 month) male BN rats (N=7) to air (control) or 0.8 ppm O3 for 6 hr/d, 1 d/wk for 17 wks. Respiratory parameters assessed by unrestrained plethysmography the day after exposure revealed that O3 led to airway‐flow limitation. Rats were terminated 24 hr after the last exposure. Bronchoalveolar fluid macrophages were reduced in aged rats and neutrophils increased in young rats exposed to O3. A panel of 58 protein analytes was quantified in serum, including cytokines, chemokines, and tissue factors (Rules Based Medicine). Age affected 20 analytes, including C‐reactive protein, interferon, factor VII, and macrophage inflammatory protein‐2 (MIP‐2). O3 affected 6 analytes, including fibroblast growth factor (basic), MIP‐2, leukemia inhibitor factor, and haptoglobin. Age by O3 interactions were obtained for MIP‐2 and myoglobin. The data illustrate that serum analytes may be beneficial as biomarkers to assess aging and susceptibility to criteria air pollutants. This is an abstract of a proposed presentation and does not reflect US EPA policy.

Prostaglandin I2 regulates IL-17 production by invariant natural killer T cells (108.7)

Abstract PGI2 is an immunoregulatory prostanoid that is known to limit allergic inflammation. Our previous studies have shown that the PGI2 receptor, IP, also plays an important role in the development of IL-17-producing γδ T cells. Using mice lacking the PGI2 receptor (IPKO), we have investigated whether PGI2 is required for programming invariant natural killer T cells (iNKT) to produce IL-17. The GalCer response of spleen cells from the IPKO mouse was markedly reduced compared to wild type (WT) control animals. Paradoxically, the IL-17 production elicited by iNKT cells isolated from the lungs of IPKO mice stimulated with GalCer was 1.7-5.7 fold elevated over WT control animals. The elevated GalCer response in IPKO lungs was not associated with an increase in CD1d expression or the number of CD4+NK1.1+ or CD4+DX5+ iNKT cells in the lung. The exposure of IPKO animals to 1.5 ppm O3 resulted in a further increase in IL-17 production by lung mononuclear cells (LMC) in response to GalCer. These observations are consistent with iNKT responses being limited by a suppressive mechanism operative in the WT, but not IPKO lung tissue. The regulatory cells were not γδ T cells since the IL-17 produced by LMC in mice lacking the δ TCR chain remained low. Collectively, these results demonstrate that the GalCer response in normal lungs is depressed by a PGI2 dependent mechanism. The role for PGI2 in promoting the regulation of iNKT cells by regulatory cells, including CD4+ nTreg will be discussed.

In Utero Tobacco Smoke Exposure Alters Pulmonary Responses of Newborn Rats to Ozone

Prenatal tobacco smoke (TS) exposure has been implicated in various adverse health outcomes in the offspring, including poor development of lung and immune system, which in turn can alter the response of neonates to environmental challenges. This study was performed to determine whether in utero exposure to TS influences the pulmonary response of newborn rat pups to ozone (O3). Timed pregnant Sprague-Dawley (SD) rats were exposed to TS or air for 3 h/d from gestation d 7 through 21. The pulmonary response of pups was assessed following a single 3-h exposure to air or 0.6 ppm O3 on d 13 after birth. In all, 4 exposure groups were evaluated: (1) Air/Air (in utero air and postnatal air), (2) Air/O3 (in utero air and postnatal O3), (3) TS/Air (in utero TS and postnatal air), and (4) TS/O3 (in utero TS and postnatal O3). Bronchoalveolar lavage (BAL) was performed, and BAL cells and fluid were analyzed. Data revealed a significant increase in polymorphonuclear leukocytes (PMN) and total BALF protein in the Air/O3 group compared to the Air/Air control, reflecting the inflammatory and cytotoxic effects of O3. However, in utero exposure to TS attenuated PMN infiltration into the air spaces for recovery in the BAL of TS/O3 pups. Lung tissue myeloperoxidase activity significantly increased only in the TS/O3 group but not in Air/O3 pups, thus suggesting that PMN are sequestered in the lung tissue and that the in utero TS likely inhibits O3-mediated influx of PMN into the air spaces. Lung tissue analyses further showed a significant rise in manganese superoxide dismutase (SOD) protein and a decrease in extracellular SOD protein in the Air/O3 group, suggesting oxidative effects of O3. Interestingly, in utero TS exposure again suppressed these effects in the TS/O3 group. Overall, results suggest that in utero exposure to TS alone produced minimal acute pulmonary effects in newborn rats, but modulated adverse effects of postnatal O3 exposure. The results are contrary to the interactive toxic responses predicted for sequential exposures to TS and O3, and may represent the development of “cross-tolerance.”

Protective Role of Interleukin-10 in Ozone-Induced Pulmonary Inflammation

BackgroundThe mechanisms underlying ozone (O3)-induced pulmonary inflammation remain unclear. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is known to inhibit inflammatory mediators.ObjectivesWe investigated the molecular mechanisms underlying interleuken-10 (IL-10)–mediated attenuation of O3-induced pulmonary inflammation in mice.MethodsIl10-deficient (Il10−/−) and wild-type (Il10+/+) mice were exposed to 0.3 ppm O3 or filtered air for 24, 48, or 72 hr. Immediately after exposure, differential cell counts and total protein (a marker of lung permeability) were assessed from bronchoalveolar lavage fluid (BALF). mRNA and protein levels of cellular mediators were determined from lung homogenates. We also used global mRNA expression analyses of lung tissue with Ingenuity Pathway Analysis to identify patterns of gene expression through which IL-10 modifies O3-induced inflammation.ResultsMean numbers of BALF polymorphonuclear leukocytes (PMNs) were significantly greater in Il10−/− mice than in Il10+/+ mice after exposure to O3 at all time points tested. O3-enhanced nuclear NF-κB translocation was elevated in the lungs of Il10−/− compared with Il10+/+ mice. Gene expression analyses revealed several IL-10–dependent and O3-dependent mediators, including macrophage inflammatory protein 2, cathepsin E, and serum amyloid A3.ConclusionsResults indicate that IL-10 protects against O3-induced pulmonary neutrophilic inflammation and cell proliferation. Moreover, gene expression analyses identified three response pathways and several genetic targets through which IL-10 may modulate the innate and adaptive immune response. These novel mechanisms of protection against the pathogenesis of O3-induced pulmonary inflammation may also provide potential therapeutic targets to protect susceptible individuals.

Effects of ozone exposure on the oxidative capacity for drug biotransformation in Wistar rats

The aim of the present study was to determine if ozone (O3) exposure modifies the oxidative capacity of cytochrome P450 (CYP450) using pharmacokinetic analysis of metronidazole. A total of 84 male Wistar rats were placed in two groups. Group I was subdivided into two subgroups of 30 and 12 rats. The metronidazole pharmacokinetic study was performed on the group of 30 rats, which was in turn divided into two subgroups of 15 rats each, one exposed to 0.15 ppm O3, the other not exposed (control group). The group of 12 rats was subdivided into two subgroups of 6 rats each; one was exposed to O3 (0.15 ppm), the other was not exposed (control). Liver microsomes were obtained from these two subgroups for immunodetection of CYP450 families. The same procedure was applied to group II, except for an O3 concentration of 0.45 ppm. Plasma metronidazole was determined by high performance liquid chromatography. The maximum plasma concentration (Cpmax), area under the curve (AUCo → ∞), volume of distribution (Vd), and clearance (CL) showed a significant increase in the group exposed to 0.15 ppm O3 compared to the group exposed to 0.45 ppm. Further, data were significantly increased compared to control. The elimination half-life time (t1/2el) in the 0.15 ppm O3 group was similar to the 0.45 ppm rats, however, these values were significantly decreased compared controls. No statistically significant differences were found among rats in the electrophoretic band pattern for any of the CYP450 families evaluated, 1A, 3A, 2B and 2E. The expression of these subfamilies in hepatic and renal tissue is in agreement with other reports.

Responses measured in the exhaled breath of human volunteers acutely exposed to ozone and diesel exhaust

Exhaled breath collection is used to identify and monitor inflammatory or oxidative components in breath. Exhaled breath sample collection is noninvasive and would greatly benefit human pollutant exposure research. We demonstrate the efficacy of exhaled breath collection and analysis in two human exposure studies to ozone (O3) and diesel exhaust, respectively. O3 study: we collected exhaled breath (gas phase) from healthy human volunteers (age 18–35 years, 12 subjects) immediately before and after exposure to filtered air or 0.4 ppm O3 for 2 h with and without intermittent exercise. Six subjects received antioxidant supplementation for 2 weeks before their O3 exposure, while the remaining six subjects received placebo treatments. We demonstrate increased amounts of non-polar carbonyls exhaled immediately post O3 exposure. The O3-induced increase in exhaled carbonyl concentrations was attenuated in the group receiving antioxidants. Our data demonstrate that exhaled exposure biomarkers can be measured in the breath gas phase in humans exposed to O3. Diesel study: we collected exhaled breath condensate (EBC; liquid phase) from healthy human volunteers (age 18–40 years; 10 subjects) immediately before, immediately after and 20 h post filtered air or diesel exhaust (106 ± 9 µg m−3) exposure. Clean air and diesel exposures were separated by 3 weeks to 6 months. We obtained reproducible intra-subject EBC volumes and total protein concentrations across our six collection time points. Diesel exposure did not affect either EBC volume or total protein concentrations. Our data demonstrated EBC volume and total protein reproducibility over several months. Volume and total protein concentration may serve as normalizing factors for other EBC constituents.

Impact of Pollution, Heat and Humidity on Athletic Performance

The 2008 Olympic Games will take place in Beijing, a city that presents high concentration of different pollutants. One of the primary pollutants that is of concern to athletes competing in Beijing is the oxidant gas ozone, especially when combined with the heat and humidity (H) of the summer months. PURPOSE: To investigate the impact of a hot, humid and ozone polluted environment during an 8 km time trial run on (a) inflammatory response in the lungs, (b) changes in lung function and (c) performance. METHODS: Using a single blinded randomized design, 8 male endurance athletes (mean ± SD, 25 ± 5 years; 73 ± 8 kg; 182 ± 9 cm; 64 ± 3 ml.kg-1.min-1) took part in a time trial run in four different environmental conditions: 20°C + 50% H (Control); 20°C + 50% H + 0.10 ppm O3 (Control + O3); 31°C + 70% H (Heat); 31°C + 70% H + 0.10 ppm O3 (Heat + O3). Heart rate (HR), RPE and expired air were collected during the exercise. Nasal lavage samples were collected post-exercise to analyse the neutrophil count in the upper respiratory airways. Lung function was measured pre and post-exercise. Respiratory symptoms were also recorded. Data was analysed using Repeated Measures ANOVA with Tukey's post-hoc test. RESULTS: The mean speed attained on the Heat + O3 trial (14.8 km·h-1) was significantly lower when compared with the Control (16.1 km·h-1). The completion time of both Heat (32m03s) and Heat + O3 (32m26s) was significantly higher when compared with the Control (29m53s). Even though there is no statistical difference between the Heat and the Heat + O3 trials, the time to complete the latter was, on average, 23 sec longer when compared to the former. In a competitive 8 km race environment, this time difference could be ecologically very significant. Mean and peak HR did not differ between trials, whereas mean and peak RPE (16; 18) was significantly higher in the Heat + O3 when compared with the Control (15; 17). Respiratory symptoms reported by the athletes increased following Heat + O3 and all of them reported not being able to perform maximally if it were a competition. No significant differences in the mean lung function or neutrophil counts were found. CONCLUSION: Ozone alone does not impair performance. However, combined with heat and humidity then significant performance decrements may be seen. Supported by Napier University and UK Sport.

Effects of Exposure to 0.06 ppm Ozone on FEV 1 in Humans: A Secondary Analysis of Existing Data

BackgroundOzone is a potent photochemical oxidant that produces transient, reversible decrements in the lung function of acutely exposed individuals. A recent study provided previously unavailable clinical data for 30 healthy young adults exposed to O3 at 0.06 ppm. That study showed significant effects of 0.08 ppm on lung function, confirming the findings of others. However, exposure to 0.06 ppm O3 was not reported to significantly affect lung function.ObjectivesWe conducted this analysis to reevaluate the existing lung function data of the volunteers previously exposed to 0.06 ppm O3.MethodsWe obtained pre- and postexposure data on forced expiratory volume in 1 sec (FEV1) for all subjects who were previously exposed for 6.6 hr to filtered air or to 0.06 ppm or 0.08 ppm O3. We used standard statistical methods appropriate for paired comparisons to reanalyze FEV1 responses after exposure to 0.06 ppm O3 relative to filtered air.ResultsControlling for filtered air responses, 24 of the 30 subjects experienced an O3-induced decrement in FEV1. On average, 0.06 ppm O3 exposure caused a 2.85% reduction in FEV1 (p < 0.002), which was consistent with the predicted FEV1 response from existing models. Although the average response was small, two subjects had > 10% FEV1 decrements.ConclusionsExposure to 0.06 ppm O3 causes a biologically small but highly statistically significant decrease in mean FEV1 responses of young healthy adults.

Effect of ozone on nitric oxide synthases gene expression in endothelial cells

The goal was to explore the effects of ozone (O3) on gene expression of nitric oxide synthases (NOSs) in endothelial cells (EC) isolated from rat aorta. EC grew until confluence and then they were exposed (1h) to 1ppm of O3 or to 0.05 ppm of O3 (to assess the effect of separating the cells from the culture environment) under sterile conditions. After O3 exposure EC were incubated within normal culture environment during 3, 6 or 9 hs. mRNA was determined by means of real time RT‐PCR. O3 at 0.05 ppm significantly decreased the expression of NOSs after 6 h (p&lt;0.05, compared to basal expression) with a partial recovery after 9 h. O3 exposure at 1ppm also decreased NOSs expression but it happened earlier (3 h, p&lt;0.001 compared to basal), was more significant at 6 h (p&lt;0.001) and greater than the effect produced by 0.05 ppm of O3 (p&lt;0.01), and it had also a partial recovery after 9 h. It is concluded that O3 inhibits NOSs gene expression in isolated EC, which is different from the effect on the whole animal where O3 decreases the vascular endothelial and neuronal NOS while increases the inducible isoform, probably because of the systemic induction of inflammatory mediators by O3

Ultrastructural Alterations During Embryonic Rats' Lung Development Caused by Ozone

Ozone (O3) is an oxidizing agent that acts on phospholipids, proteins and sugars of cellular membranes producing free radicals, which cause oxidative damages. The O3 exposure has been used as a model to study oxidative stress, in which the respiratory airways represent the entrance to the organism. In this study, ultrastructural alterations were identified at the bronchiolar level during the intra-uterine lung development, using an O3 exposure model in pregnant rats during 18, 20 and 21 days of gestation. Twelve pregnant Wistar rats, six controls and six exposed to 1 ppm O3 inhalation during 12 h per day, were used. The rats were sacrificed at gestational days 18, 20 and 21; the fetuses were obtained and their lungs dissected. The ultrastructural analysis evidenced swollen mitochondria, cytoplasmic vacuolization of the epithelial cells and structural disorder caused by the oxidative stress. At gestation day 20, flake-off epithelial cells and laminar bodies in the bronchiolar lumen were observed. In the 21-gestation-day group, the mitochondria were edematous and their cristae were disrupted by the damage caused in mitochondrial membranes.