Ppm O3 Exposure Research Articles

Ozone-induced changes in the murine lung extracellular vesicle small RNA landscape.

Inhalation exposure to ozone (O3) causes adverse respiratory health effects that result from airway inflammation, a complex response mediated in part by changes to airway cellular transcriptional programs. These programs may be regulated by microRNAs transferred between cells (e.g., epithelial cells and macrophages) via extracellular vesicles (EV miRNA). To explore this, we exposed female C57BL/6J mice to filtered air (FA), 1, or 2 ppm O3 by inhalation and collected bronchoalveolar lavage fluid (BALF) 21 h later for markers of airway inflammation, EVs, and EV miRNA. Both concentrations of O3 significantly increased markers of inflammation (neutrophils), injury (total protein), and the number of EV‐sized particles in the BALF. Imagestream analysis indicated a substantial portion of particles was positive for canonical EV markers (CD81, CD51), and Siglec‐F, a marker of alveolar macrophages. Using high‐throughput small RNA sequencing, we identified several differentially expressed (DE) BALF EV miRNAs after 1 ppm (16 DE miRNAs) and 2 ppm (99 DE miRNAs) O3 versus FA exposure. O3 concentration‐response patterns in EV miRNA expression were apparent, particularly for miR‐2137, miR‐126‐3p, and miR‐351‐5p. Integrative analysis of EV miRNA expression and airway cellular mRNA expression identified EV miR‐22‐3p as a candidate regulator of transcriptomic responses to O3 in airway macrophages. In contrast, we did not identify candidate miRNA regulators of mRNA expression data from conducting airways (predominantly composed of epithelial cells). In summary, our data show that O3 exposure alters EV release and EV miRNA expression, suggesting that further investigation of EVs may provide insight into their effects on airway macrophage function and other mechanisms of O3‐induced respiratory inflammation.

Quantification of regional murine ozone-induced lung inflammation using [18F]F-FDG microPET/CT imaging

Ozone (O3) is a highly potent and reactive air pollutant. It has been linked to acute and chronic respiratory diseases in humans by inducing inflammation. Our studies have found evidence that 0.05 ppm of O3, within the threshold of air quality standards, is capable of inducing acute lung injury. This study was undertaken to examine O3-induced lung damage using [18F]F-FDG (2-deoxy-2-[18F]fluoro-D-glucose) microPET/CT in wild-type mice. [18F]F-FDG is a known PET tracer for inflammation. Sequential [18F]F-FDG microPET/CT was performed at baseline (i.e. before O3 exposure), immediately (0 h), at 24 h and at 28 h following 2 h of 0.05 ppm O3 exposure. The images were quantified to determine O3 induced spatial standard uptake ratio of [18F]F-FDG in relation to lung tissue density and compared with baseline values. Immediately after O3 exposure, we detected a 72.21 ± 0.79% increase in lung [18F]F-FDG uptake ratio when compared to baseline measures. At 24 h post-O3 exposure, the [18F]F-FDG uptake becomes highly variable (S.D. in [18F]F-FDG = 5.174 × 10–4 units) with a 42.54 ± 0.33% increase in lung [18F]F-FDG compared to baseline. At 28 h time-point, [18F]F-FDG uptake ratio was similar to baseline values. However, the pattern of [18F]F-FDG distribution varied and was interspersed with zones of minimal uptake. Our microPET/CT imaging protocol can quantify and identify atypical regional lung uptake of [18F]F-FDG to understand the lung response to O3 exposure.

Cellular RNA is chemically modified by exposure to air pollution mixtures

RNAs are more susceptible to modifications than DNA, and chemical modifications in RNA have an effect on their structure and function. This study aimed to characterize chemical effects on total RNA in human A549 lung cells after exposure to elevated levels of major secondary air pollutants commonly found in urban locations, including ozone (O3), acrolein (ACR) and methacrolein (MACR). Enzyme-linked immunosorbent assays (ELISA) were used to measure levels of interleukin (IL)-8 in the growth media and 8-oxoguanine (8OG) levels in total cellular RNA, and lactate dehydrogenase (LDH) in the growth media was measured by a coupled enzymatic assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure levels of microRNA 10b (miR-10b). The study found that 1-h exposure to all tested pollutant mixtures consistently caused significant increases in the levels of 8OG in total RNA. In the case of 4 ppm O3 exposures, measured levels of IL-8, LDH and miR-10b each showed consistent trends between two independent trials, but varied among these three targets. After 1-h exposures to an ACR+MACR mixture, measured levels of IL-8, LDH and miR-10b showed variable results. For mixtures of O3+ACR+MACR, IL-8 measurements showed no change; miR-10b and LDH showed variable results. The results indicate that short-term high-concentration exposures to air pollution can cause RNA chemical modifications. Chemical modifications in RNAs could represent more consistent markers of cellular stress relative to other inflammation markers, such as IL-8 and LDH, and provide a new biomarker endpoint for mechanistic studies in toxicity of air pollution exposure.

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.

Ozone dose-response effects of varied equivalent minute ventilation rates.

While it is well known that exercise minute ventilation (V(E)) results in greater pulmonary function and subjective symptoms (SS) responses upon exposure to a given ozone (O3) dose, the magnitude of V(E) increase to produce a significant forced expiratory volume in 1 s (FEV1.0) response compared to that observed at a lower exercise V(E) for the same O3 concentration and exposure time is unclear, especially in prolonged (i.e., >2 h) exposures. Further, in prolonged exposures, the relationship of body size to FEV1.0 response to a given O3 exposure dose has not been systematically examined. In the present study, 30 young adults were exposed on four occasions for 6 h (during a 6.6-h period) to constant 03 levels of zero (filtered air, FA) or 0.12 parts per million (ppm). At the latter concentration, exercise V(E) was varied in exposures to 17, 20, and 23 l min(-1) m(-2) of BSA, respectively, for each individual to achieve an equivalent ventilation rate, EVR). In the FA exposure, EVR was 23 l min(-1) m2. Percent changes in FEV1.0 for the three 0.12 ppm O3 exposures were significantly greater than that for FA, but did not differ significantly from each other. For the 6.6-h exposures, exercise EVR at or in excess of 17 l min(-1) m(-2), SS values were significantly greater than those observed for the FA protocol. Further, SS values at 6.6 h of exposure to 0.12 ppm O3 for the exercise EVR of 23 l min(-1) m(-2) protocol were significantly greater than for the 0.12 ppm O3 exercise EVR of 17 l min(-1) m(-2) protocol. To achieve a widened EVR, two 1-h exposures to 0.30 ppm O3 with continuous exercise (CE) at a level necessitating an EVR of 17 and approximately 34 l min(-1) m(-2), respectively, were completed by each subject. All postexposure pulmonary function and SS responses were significantly greater for the higher 1-h EVR protocol. In all exposures with significant O3-induced changes in FEV1.0 and SS, it was found that the smaller subjects who exercised at the lowest absolute V(E) had significantly smaller responses than did the larger subjects. These results strongly suggest that for the O3 concentrations and exposure durations used in this study, the effect of V(E) on O3-induced FEV1.0 and SS responses is not body-size-dependent.

INHIBITION OF ARACHIDONIC ACID ESTERIFICATION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO OZONE IN VITRO

There is evidence that some lung responses to ozone (O3) exposure are mediated through the altered metabolism of arachidonic acid (AA). Increased concentrations of some AA metabolites such as prostaglandin E2 (PGE2) and prostaglandin F2 (PGF2) are observed in the bronchoalveolar lavage fluid recovered from human subjects exposed to O3. Airway epithelial cells may contribute to this increase in eicosanoid formation. Previous reports have shown increased PGE2 and PGF2 production by O3-exposed air way epithelial cells, believed to be mediated at least in part by increased phospholipase activity. We examined other potential biochemical mechanisms for the increased formation of these two prostaglandins by O3-exposed epithelial cells. Cultured normal human bronchial epithelial (NHBE) cells, prelabeled with 3H-AA, were exposed to air or to 0.1 ppm or1.0 ppm O3 for 60 min in a Transwell cell culture system. NHBE cells produced increased amounts of 3H-PGE2 and 3H-PGF2 in response to 0.1 ppm O3 exposure but not with 1.0 ppm exposure as measured in the conditioned media by high-performance liquidchromatography. Other 3H-AA products, including [3H]-15-hydroxyeicosatetraenoic acid, [3H]-12-heptadecatrienoic, and [3H]aldehydic substances derived from the ozonation of 3H-AA, also were observed in increased amounts. Pulsing of cells with 15 n 3H-AA after 0.1 ppm and 1.0 ppm O3 exposure revealed a decrease in 3H-AA esterification into cellular phospholipids, resulting in an increase in free 3H-AA available for metabolism to prostaglandins. No O3-induced alteration in NHBE cell cyclooxyge nase activity was observed with the 0.1 ppm exposures. Impaired esterification of free AA into cellular phospholipids appears to be a very sensitive target for O3-induced effects on AA metabolism, and may play a role in the increased prostaglandin production observed upon O3 inhalation in vivo.

OZONE-INDUCED DNA SINGLE STRAND BREAKS IN HUMAN AND GUINEA PIG LUNG CELLS IN VIVO

Ozone O3 has been postulated to induce DNA damage and has been shown to be mildly tumorigenic in some studies utilizing long-term rodent exposures. We investigated lung DNA damage induced by controlled O3 exposure in vivo in guinea pigs and human subjects. We specifically examined DNA single-strand breaks SSB using the single-cell gel electrophoresis assay. Guinea pigs were exposed for 2 h to air, 0.4 ppm O3, or 1.0 ppm O3, and lung cells were collected by bronchoalveolar lavage BAL and bronchial scraping within 1 h after exposure. Both the 0.4 and 1.0 ppm O3 exposures induced significant increases in SSB in both the BAL cells and tracheal cells as indicated by an increased cell DNA length in electrophoresized agarose gel. The increase in DNA SSB was a more sensitive biomarker of exposure compared to more traditional biomarkers BAL total protein and lactate dehydrogenase, alterations in BAL cell differential, which changed only at the 1.0 ppm exposure. In an initial study with human volunteers, BAL and bronchial epithelial cells were collected from human volunteers 1-2 h after an air or 0.4 ppm O3 exposure in vivo without exercise. BAL cells primarily macrophages and bronchial epithelial cells showed no change in DNA SSB compared to the air-exposed controls. In a second study, DNA SSB in bronchial epithelial cells and BAL cells collected from exercising subjects exposed to 0.4 ppm O3 were not altered by steroid prednisone, beclomethasone pre- treatment compared to placebo treatment. However the bronchial epithelial cell DNA SSB values in the O3-exposed, placebo-pretreated group were significantly increased compared to values air or O3 exposed, no exercise in the first study. The dosimetry of O3 deposi- tion in the guinea pig and human subjects appeared similar based on the amount of 18O derived from 18O-labeled O3 found in the BAL cell fraction. These data suggest that O3 exposure at 0.4 ppm induces DNA SSB in rodent and human lung cells, although the effect of exercise on the increase of human lung cell SSB is unclear. Formation of DNA SSB may be an indicator of the tumorigenic potential of O3. Additionally, DNA SSB can potentially be a good biomarker of O3 exposure in humans and animal model systems. 3

Effects of acute ozone exposure on the electrophysiological properties of guinea pig trachea

Acute ozone (O3) exposures produce an increase in the apparent permeability of the tracheal epithelium, but the mechanism of this response is poorly understood. Comparison of previous studies suggests that qualitative differences may exist between measurements made in vivo or in vitro. To test this possibility we used both in vitro and in vivo electrophysiological techniques to investigate the effects of O3 exposure on guinea pig tracheal epithelium. Male Hartley guinea pigs were exposed to either 1 or 2 ppm O3 or to filtered air for 3 h and were studied 0, 6, or 24 h after exposure. Air-exposed animals had in vitro mean tracheal potential (VT) -32.0 +/- 1.5 mV, conductance (GTL) 2.18 +/- 0.22 mS/cm, short-circuit current (ISCL) 62.6 +/- 3.7 microA/cm, and diameter (D) 2.44 +/- 0.10 mm. In vitro properties after 1 ppm O3 exposure did not differ at any time point from control. Two parts per million O3 increased ISCL, but only at 6 h postexposure. The effect of O3 on ISCL was abolished by amiloride. There were no significant changes in VT, GTL, or D. In vivo tracheal potential under pentobarbital anesthesia was -19.7 +/- 1.7 mV. At 6 h postexposure to 2 ppm O3, but not at 0 or 24 h, in vivo VT was increased. Thus, acute exposure of guinea pigs to a high concentration of O3 caused a delayed increase in Na+ absorption by the trachea with no change in conductance. This indicates that paracellular permeability of guinea pig tracheal epithelium was not substantially increased by acute O3 and suggests that enhanced macromolecular uptake in this species probably occurs transcellularly. Furthermore, the increase of in vivo VT following O3 exposure is consistent with the in vitro response, indicating that in vivo/in vitro differences are not responsible for the discrepancies between previous electrophysiological and "permeability" studies.

Susceptibility to ozone-induced inflammation. II. Separate loci control responses to acute and subacute exposures.

We demonstrated previously that inbred strains of mice are differentially susceptible to acute (3 h) and subacute (48 h) exposures to 2 parts per million (ppm) ozone (O3) and 0.30 ppm O3, respectively. Genetic studies with O3-resistant C3H/HeJ and O3-susceptible C57BL/6J strains have indicated that susceptibility to each of these O3 exposures is under Mendelian (single gene) control. In the present study, we hypothesized that the same gene controls susceptibility to the airway inflammatory responses to 2 ppm and 0.30 ppm O3 exposures. To test this hypothesis, airway inflammation was induced in 10 BXH and 16 BXD recombinant inbred (RI) strains of mice by acute as well as subacute O3 exposures. Airway inflammation was assessed by counting the number of polymorphonuclear leukocytes (PMNs) in bronchoalveolar lavage (BAL) returns obtained immediately after 48-h subacute exposure to 0.30 ppm O3, or 6 h after 3 h acute exposure to 2 ppm O3. Each RI strain was classified as susceptible or resistant to each exposure, based on a comparison of mean numbers of PMNs with those of the respective progenitor strains. For each RI set, a phenotypic strain distribution pattern (SDP) was thus derived for each exposure regimen, and the SDPs were then compared for concordance. Among the BXH RI strains, 4 of 10 responded discordantly to the two exposures: 3 were susceptible to acute exposure and resistant to subacute exposure, whereas 1 was conversely susceptible. Among the BXD RI strains, 4 of 16 were discordant: 1 was susceptible to acute exposure, and resistant to subacute exposure, whereas 3 were conversely susceptible.(ABSTRACT TRUNCATED AT 250 WORDS)

Ozone inhalation effects in females varying widely in lung size: comparison with males

It has been suggested that lung size accounts for observed gender differences in responsiveness to the same total inhaled dose of O3. To test the hypothesis that lung size is a determinant of magnitude of response within a gender, two groups of 14 healthy young adult females differing significantly in forced vital capacity [FVC; i.e., small-lung group mean = 3.74 liters (range 3.2-4.0) and large-lung group mean = 5.11 liters (range 4.5-6.2] were exposed for 1 h to filtered air (FA) and to 0.18 and 0.30 ppm O3. On each occasion, subjects exercised continuously on a cycle ergometer at a work rate that elicited a mean minute ventilation of approximately 47 l/min. For the small-lung group [mean total lung capacity (TLC) = 4.52 liters] exercise O2 uptake was 67% of maximal O2 uptake (VO2max), and that for the large-lung group (TLC 6.37 liters) was 61% of VO2max. Statistical analysis revealed significant decrements for both groups in FVC, forced expiratory volume in 1 s (FEV1.0), and forced expiratory flow rate in the middle half of FVC on exposure to 0.18 and 0.30 ppm O3. Exercise respiratory frequency increased, and tidal volume decreased significantly in both groups in response to 0.18 and 0.30 ppm O3 exposure. On exposure to 0.30 ppm O3, the number of individual subjective symptoms reported and their severity were significantly greater for both groups than those reported for the FA and 0.18 ppm O3 exposures. Both groups evidenced similar percent changes in pulmonary function and exercise ventilation response, and in subjective symptom response.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of short- and long-term exposure to ozone on heart rate and blood pressure of emphysematous rats

Electrocardiogram and arterial blood pressure of elastase-treated emphysematous rats (E rats) and saline-treated control rats (S rats) were recorded continuously during exposure to either 1 ppm ozone (O3) for 3 hr or 0.5 ppm O3 for 6 hr. The heart rates (HRs) of both groups decreased to about 50 and 65% of the initial levels at the end of 1 ppm and 0.5 ppm O3 exposure, respectively. Mean arterial blood pressures (MAPs) also decreased to about 76 and 82%, respectively. There was no significant difference in these responses between E and S rats, although the levels of HRs and MAPs of the E rats were always a little lower than those of the S rats. Another group of E and S rats was continuously exposed to 0.2 ppm O3 for 4 weeks. The HRs of both E and S groups decreased to about 81 and 88% of the initial levels on the first day, respectively, although they recovered completely by the third day. No significant difference in the variation of HRs during exposure was noted between E and S rats. However, the HR responses of these rats to a challenge exposure of 0.8 ppm O3 for 1.5 hr appeared to be different. That is, S rats were more tolerant of the challenge exposure to O3 for 1.5 hr than the E rats.

Evaluation of Effects of Ozone Exposure on Influenza Infection in Mice Using Several Indicator of Susjceptibility

Evaluation of Effects of Ozone Exposure on Influenza Infection in Mice Using Several Indicators of Susceptibility. Selgrade, M. J. K., Illing, J. W., Starnes, D. M., Stead, A. G., Menache, M. G., and Stevens, M. A. (1988). Fundam. Appl. Toxicoi. 11, 169–180. Mice were exposed to 1 ppm O3, 3 hr/day, for 5 consecutive days. Separate groups of mice were infected with influenza following each of the individual exposures. A twofold increase in the incidence of mortality and a 3-day decrease in mean survival time were observed in mice infected after the second exposure. There were no effects on percentage mortality or mean survival time due to exposure to 1 ppm O3 in mice infected after the first, third, fourth, or fifth exposure. When the exposure concentration was lowered to 0.5 ppm, there were no effects on mortality in mice infected after the second exposure. Five, daily, 3-hr exposures to 1 ppm O3 had no effect on virus titers in the lungs of mice infected after either the second or fifth exposure. In contrast, wet lung weights were significantly enhanced over infected air controls in mice infected after the second O 3 exposure at both 1 and 0.5 ppm but not at 0.25 ppm exposure concentrations. This effect on lung wet weight was observed in mice infected with a dose of virus which produced 7-33% mortality in controls as well as in mice infected with a sublethal dose of virus. Histopatho-logjc changes due to sublethal influenza infection, including nonsuppurative pneumonitis and necrosis, squamous metaplasia and hyperplasia of the epithelium lining the bronchi and bronchioles, were more severe in mice infected after the second of five, 1 ppm O3 exposure than in comparable air controls. Sublethal infection caused a loss of lung volume with secondary reduction in diffusing capability and homogenity of ventilation distribution. These latter two effects were also exacerbated in mice infected after the second of five, 1 ppm O 3 exposures as compared to air controls. When mice were infected after the fifth, 1 ppm O 3 exposure, there was no effect due to ozone on either lung wet weight or histopathology. The data indicate that O 3 has little if any effect on antiviral defense mechanisms since virus titers in the lungs were not affected by O3 exposure. However, mortality and morbidity, as indicated by lung wet weights, histopathology, and pulmonary function changes, were enhanced by O 3 exposure in mice infected after the second of five exposures suggesting that symptoms due to infection can be enhanced in the absence of enhanced virus replication, possibly due to synergistic effects of O 3 and virus in production of lung pathology. The fact that effects due to daily repeated exposures were only observed when infection occurred after the second exposure may be due to adaptation or a similar phenomenon.

Changes in delayed hypersensitivity reaction in mice exposed to O 3

BALB/c mice were continuously exposed to 0.8 ppm O3 for 1, 3, 7, and 14 days. Ozone exposure suppressed the delayed hypersensitivity (DH) reaction to sheep red blood cells (SRBC). The maximum effect was seen after 7 days of exposure. To estimate the suppression of the DH reaction by O3 exposure, the numbers of lymphocytes in thymus and blood of exposed mice were compared with those of control mice. A decrease in the numbers of lymphocytes in both thymus and blood was observed in O3-exposed mice. The percentage of T and B lymphocytes in blood of exposed mice was the same as that in blood of control mice. These results suggest that 0.8 ppm O3 exposure affects the T lymphocytes required for DH reactions.

Reduced exercise time in competitive simulations consequent to low level ozone exposure

Ten highly trained endurance athletes were studied to determine the effects of exposure to low ozone (O3) concentrations on simulated competitive endurance performance and associated physiological and subjective symptom responses. Each subject was randomly exposed to filtered air (FA), and to 0.12, 0.18, and 0.24 ppm O3 while performing a 1 h competitive simulation protocol on a bicycle ergometer. Endurance performance was evaluated by the number of subjects unable to complete rides (last 30 min at an intense work load of approximately 86% VO2max). All subjects completed the FA exposure, whereas one, five, and seven subjects did not complete the 0.12, 0.18, and 0.24 ppm O3 exposures, respectively. Statistical analysis indicated a significant (P less than 0.05) increase in the inability of subjects to complete the competitive simulations with increasing O3 concentration, including a significant difference between the 0.24 ppm O3 and FA exposure. Significant decreases (P less than 0.05) were also observed following the 0.18 and 0.24 ppm O3 exposures, respectively, in forced vital capacity (-7.8 and -9.9%), and forced expiratory volume in 1 s (-5.8 and -10.5%). No significant O3 effect was observed for exercise respiratory metabolism or ventilatory pattern responses. However, the number of reported subjective symptoms increased significantly following the 0.18 and 0.24 ppm O3 protocols. These data demonstrate significant decrements in simulated competitive endurance performance and in pulmonary function, with accompanying enhanced subjective symptoms, following exposure to low O3 levels commonly observed in numerous metropolitan environments during the summer months.

Combined effects of ozone exposure and ambient heat on exercising females.

Ten aerobically trained young adult females exercised continuously at 66% of maximum O2 uptake for 1 h while exposed orally to filtered air and 0.15 and 0.30 parts per million (ppm) ozone (O3) in both moderate (24 degrees C) and hot (35 degrees C) ambient conditions. Exposure to 0.30 ppm O3 induced significant impairment in forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1.0), and other pulmonary function variables. Exercise respiratory frequency (fR) increased, whereas tidal volume and alveolar volume (VA) decreased with 0.30 ppm O3 exposure. Significant interactions of O3 and ambient heat were obtained for fR and VA, whereas FVC and FEV1.0 displayed a trend toward an O3-temperature interaction. Although expired ventilation increased, the interactions could not be ascribed to a greater O3 effective dose in the 35 degrees C exposures. However, subjective discomfort increased with both O3 and heat exposure such that three subjects ceased exercise prematurely when O3 and ambient heat were combined. We conclude that accentuation of subjective limitations and certain physiological alterations by ambient heat coinciding with photochemical oxidant episodes is likely to result in more severe impairment of exercise performance, although the mechanisms remain unclear.