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Final Report: Mechanisms of Age-dependent Ozone Induced Airway Dysfunction
EPA Grant Number: R827447Title: Mechanisms of Age-dependent Ozone Induced Airway Dysfunction
Investigators: Shore, Stephanie , Johnston, Richard , Laporte, Johanne , Murthy, G.G. Krishna , Want, Matt
Institution: Harvard University
EPA Project Officer: Deener, Kacee
Project Period: July 1, 1999 through June 30, 2002 (Extended to July 31, 2003)
Project Amount: $852,937
RFA: Children's Vulnerability to Toxic Substances in the Environment (1999)
Research Category: Health Effects , Children's Health
Description:
Objective:Acute exposure to ozone (O3) causes damage to lung and airway epithelial cells, airway inflammation, decreases in lung function, and airway hyperresponsiveness (AHR) in human subjects. The mechanistic basis for O3-induced AHR appears to be inflammation arising from oxidant injury to the lungs and airways. However, the precise aspect of the inflammatory cascade required for AHR is still not firmly established. O3 may be a particularly important respiratory hazard for children because they spend more time outdoors, where O3 levels are higher, and they are more physically active. Children have a higher minute ventilation (VE) and consequently a higher delivered dose of O3. In addition, animal studies suggest that O3 may be more toxic to the lungs of immature animals compared to mature animals.
The objective of this research project was to address the following questions:
(1) Is O3-induced AHR greater in juvenile than in adult animals, and if so, why?
(2) What is the mechanism of O3-induced AHR?
(3) Is the mechanistic basis for O3-induced AHR the same or different in juvenile
and adult animals? In particular, we proposed to test the following hypotheses:
(a) that age-related differences in ventilation and in the effect of O3 on
the ventilatory pattern contribute to age-related differences in O3-induced
AHR, (b) that TNF
formation induced by O3 is important for O3-induced AHR,
and (c) that O3-induced TNF formation is different in juvenile and adult animals.
Age-Related Differences in the Ventilatory Response to Ozone
The dose of O3 delivered to the lungs is the product of O3 concentration, exposure time, and VE. Immature animals and humans have a higher metabolic rate than adults, in part because they are growing. Consequently, their ventilation also is higher. In adult rats and in some strains of mice, O3 causes a decrease in VE that occurs with a slight delay after the initiation of exposure, and this can be profound. O3-induced decreases in VE are likely to be protective because they reduce the overall inhaled dose of O3, but prior to the studies reported here, there was no information as to whether such changes also occurred in immature animals. To characterize the extent to which age-related differences in the ventilation might impact inhaled O3 dose, we exposed A/J mice to O3 (0.3, 0.5, 1.0, 2.0, or 3.0 ppm for 3 hours) in nose-only exposure plethysmographs, and we measured VE throughout the exposure. There was a consistent reduction in VE normalized for body weight (specific VE) with increasing age. Specific VE ranged from 4.94 ± 0.14 mL/min/g in 2-week-old mice to 1.87 ± 0.16 mL/min/g in 12-week-old mice. This observation is consistent with the greater metabolic rate of younger animals.
In 8-week-old mice, there was a significant effect of O3 concentration on O3-induced changes in VE (p < 0.001 by repeated measures ANOVA). Relative to air exposure, exposure to O3 for 3 hours caused a decrease in VE at all O3 concentrations greater than 0.3 ppm. At 3.0 ppm O3, the decrease in VE was statistically significant within 30 minutes of the onset of O3 exposure, and it declined to as little as 28 percent of the baseline VE by the end of the 3-hour exposure period. At lower O3 concentrations, the effects were progressively smaller in magnitude, but even 1 ppm O3 caused an approximate 50-percent decrease in VE.
Animal age had a significant effect on O3-induced changes in VE. Analysis of variance indicated that the ventilatory responses to O3 were not different among the 4-, 8-, and 12-week-old mice, which all had prominent decreases in VE with exposure to O3. In contrast, decreases in VE induced by O3 were significantly less in 2-week-old mice than in the more mature animals.
To examine the implications of the age-related differences in both baseline specific VE and O3-induced changes in VE on inhaled O3 dose, we integrated VE over the 3-hour exposure period to calculate the total volume inhaled during this period. We multiplied this total volume by O3 concentration to calculate O3 dose normalized for body weight (specific O3 dose) (see Figure 1). Animal age had a marked and statistically significant effect on inhaled specific O3 dose. Specific O3 dose was significantly greater in the 2-week-old mice than in adult (8- and 12-week-old) mice at all O3 concentrations except 0.3 ppm. Specific O3 dose also was significantly greater in 4-week-old mice than in adult mice at O3 concentrations greater than 1 ppm. The importance of the decrease in VE that occurs with exposure to O3 on total specific O3 dose is particularly apparent when comparing the O3 dose at O3 concentrations of 2 versus 3 ppm. Despite a 50-percent greater inhaled concentration, there was no difference in O3 dose at 3 ppm versus 2 ppm O3 in any of the age groups. Overall, the net effect of the greater metabolic rate and the failure to decrease VE with O3 in 2-week-old mice is that the inhaled dose of O3, relative to lung size, is about 3-4 times greater in these mice than in adult (8- to 12-week-old) mice.
Essentially, similar results were obtained in Sprague Dawley rats. Baseline VE normalized for body weight decreased with age from 2.1 ± 0.1 mL/min/g in 2-week-old rats to 0.72 ± 0.03 mL/min/g in 12-week-old rats. In adult (8- and 12-week-old) rats, O3 caused 40- to 50-percent decreases in VE. In 6-week-old rats, O3-induced changes in VE were significantly less, and in 2 and 4-week-old rats, no significant changes in VE were observed during O3 exposure.
Figure 1. Effect of O3 Concentration and Age on the Total Inhaled Dose of O3 Normalized for Body Weight During a 3-Hour Exposure. Results are mean ± the standard error (SE) of data from 6 to 12 animals in each group.
The observed age-related differences in the ventilatory response to O3 suggest that the ability of O3 to reduce metabolism may be reduced in immature rodents. We do not know what accounts for this difference. It is unlikely, however, to represent a general inability of immature rodents to lower their metabolic rates, because immature rats are capable of reducing metabolic rate in response to other stimuli, such as hypoxia, and do so to a greater extent than adults. It also is possible that the younger animals, which still lack full coats of fur, are more dependent on activity to maintain their core body temperature than are adult rats. If so, in immature rats and mice, the need to maintain a high metabolic rate may override any signals arising from O3 exposure that would tend to decrease activity, metabolic rate, and consequently, VE. Alternatively, it may be that the neural pathways driving O3-induced changes in activity, similar to much of the central nervous system, are not completely developed in the younger animals.
Age-Related Differences in O3-Induced Lung Injury
Based on the greater inhaled dose of O3 observed in immature mice and rats, we hypothesized that there also would be greater O3-induced lung injury. To test this hypothesis, we measured bronchoalveolar lavage (BAL) protein, a sensitive indicator of O3-induced lung injury. O3 exposure resulted in greater lung injury in 2-week-old than in 8-week-old mice, as evidenced by changes in BAL protein. In 2-week-old mice, O3 exposure (2 ppm for 3 hours) resulted in an approximate 100-percent increase in BAL protein (p < 0.05), whereas in 8-week-old mice, O3 exposure resulted in only a 40-percent increase in BAL protein, which was not statistically significant. Similar results were obtained in rats. BAL protein increased 267 ± 47 percent versus air controls in O3-exposed 2-week-old rats, but only 165 ± 22 percent in 8-week-old rats (p < 0.05). Although the inhaled dose of O3 and the dose delivered to the tissues might not necessarily be the same, it is likely that the increased dose of O3 delivered to the lungs of the younger animals accounts for this increased injury. Relatively few studies have considered age as a factor in any aspect of the response to O3, and the results vary with the species used. In mice, the results of existing studies measuring aspects of lung injury (the lethal dose of O3, O3-induced alveolar damage) are consistent with our results in that they indicate more severe responses to O3 in younger animals (4, 30). Data from rats are less consistent.
Age-Related Differences in O3-Induced AHR
Because of the importance of AHR for asthma, and because O3 induces airway responsiveness in adult humans and adult animals and is a trigger for asthmatic episodes, we were particularly interested in whether differences in O3 dose between immature and adult animals would result in differences in O3-induced AHR. Therefore, we measured airway responsiveness to inhaled methacholine on the day prior to, and then again 3 hours after cessation of, O3 exposure in mice aged 2, 4, 8, or 12 weeks. Airway responsiveness was assessed by whole-body plethysmography using Penh as the outcome indicator. Note that other investigators have demonstrated that Penh correlates empirically with pulmonary resistance, an index of bronchoconstriction. The effective concentration of methacholine required to increase Penh by 2 units (EC2Penh) was calculated for each animal under each experimental condition. Table 1 shows the change in log EC2Penh induced by exposure to O3 in 2-, 4-, 8-, and 12-week-old mice. Note that a negative value (decrease in EC2Penh) indicates an increase in responsiveness. Data from the 2- and 3-ppm exposures were combined because the O3 dose was not different for these 2 concentrations (see Figure 1). ANOVA indicated a significant effect of age (p < 0.001) on the change in log EC2Penh induced by O3. Two- and 4-week-old mice had no statistically significant change in log EC2Penh after exposure to any concentration of O3. In contrast, in 8- and 12-week-old mice, there was a statistically significant decrease in log EC2Penh at 2.0/3.0 ppm O3 (p < 0.001 in each case), but not at lower O3 concentrations. In addition, the change in log EC2Penh observed at 2.0/3.0 ppm O3 was greater in 8- and 12-week-old mice than in 2- and 4-week-old mice (p < 0.001 in each case). Thus, despite the greater inhaled dose of O3 normalized for body weight delivered to the younger mice and the greater pulmonary injury as evidenced by increases in BAL protein in these mice, they were actually less sensitive to O3-induced AHR than adult mice. To our knowledge, this is the first report of age-related differences in O3-induced AHR in any species.
Table 1. Effect of Age on Changes in Log EC2Penh Induced by O3 (0.3-3.0 ppm) in Mice. Results are mean ± SE of data from 6-18 mice in each group. A decrease in EC2Penh indicates an increase in airway responsiveness.
2 week |
4 week |
8 week |
12 week |
|
| 0.3 ppm | 0.01 ± 0.22 |
-0.24 ± 0.20 |
-0.25 ± 0.20 |
-0.28 ± 0.20 |
| 0.5 ppm | 0.07 ± 0.22 |
-0.14 ± 0.20 |
-0.36 ± 0.20 |
-0.11 ± 0.20 |
| 1.0 ppm | 0.17 ± 0.20 |
-0.34 ± 0.20 |
-0.34 ± 0.20 |
-0.23 ± 0.20 |
| 2.0/3.0 ppm | 0.08 ± 0.13 |
-0.01 ± 0.12 |
-0.78 ± 0.14 *# |
-0.55 ± 0.11*# |
| * p < 0.001 compared to 0 (i.e., no change in responsiveness). | ||||
| # p < 0.001 compared to 2- and 4-week-old mice. | ||||
Mechanism of Ozone-Induced AHR in Mice
To determine why O3-induced AHR was reduced in immature mice despite evidence
of increased O3 dose and increased O3-induced lung injury, we began to explore
the mechanistic basis for O3-induced AHR. Because data in the literature indicated
the release of cytokines, TNF and IL-6, and neutrophil chemotactic factors,
KC and MIP-2, following O3 exposure, we examined O3-induced AHR in wildtype
mice, mice deficient in TNFR1 and TNFR2 (the 2 TNF receptors), mice deficient
in IL-6, and mice deficient in CXCR2, the receptor for MIP-2 and KC.
Wildtype C57BL/6J mice, TNFR1 deficient mice, TNFR2 deficient mice, as well as mice deficient in both TNFR1 and TNFR2, were exposed to O3. Three hours after cessation of O3, airway responses to inhaled methacholine were determined by whole-body plethysmography using changes in Penh as an index of airway narrowing, as described above. In wildtype mice, O3 exposure (0.5 ppm, 3 hours) caused a significant increase in airway responsiveness as indicated by a 1.2 log leftward shift in the methacholine dose response curve. In contrast, in mice deficient in both TNFR1 and TNFR2, O3 caused only a 0.5 log shift in the dose response curve (p < 0.05 compared to wildtype). Similar results were obtained in mice deficient in only TNFR2. In contrast, O3-induced AHR was not different in wildtype mice and mice deficient in only TNFR1.
Airway responsiveness to O3 also was measured by whole-body plethysmography in wildtype C57BL/6J mice and mice deficient in IL-6. Both groups developed AHR following exposure to O3 (0.3 ppm for 3 hours or 0.5 ppm for 3 hours), but there was no difference in the magnitude of this response between the two groups. The results indicate that IL-6 does not contribute to O3-induced AHR.
Measurements of airway responsiveness also were made in wildtype (Balb/c) mice and mice deficient in CXCR2, the receptor through which KC and MIP-2 signal. Four hours after the cessation of O3 (1 ppm for 3 hours) exposure, airway responsiveness was increased in both wildtype and CXCR2 knockout mice. Twenty-four hours after O3 exposure, AHR was still present in the wildtype mice, but absent in the CXCR2 knockout mice. The results indicate that CXCR2 contributes to the sustained increase in AHR that is observed in Balb/c mice following O3 exposure.
To determine the effect of TNFR, IL-6, or CXCR2 deficiency on O3-induced inflammation, we also examined neutrophil influx into the lungs. Following acute O3 exposure, neutrophil influx was reduced in IL-6 deficient compared to wildtype mice and in CXCR2 deficient compared to wildtype mice, but not in TNFR1/TNFR2 deficient compared to wildtype mice. The results indicate that IL-6 and KC/MIP-2, but not TNF, contribute to O3-induced pulmonary neutrophilia. We also have examined the role of IL-6 in the neutrophil influx that occurs in response to more sustained O3 exposure (72 hours at 0.3 ppm). IL-6 deficient mice also had reduced BAL neutrophils compared to wildtype mice after this type of exposure. It is possible that neutrophils recruited to the lungs following O3 exposure contribute to O3-induced AHR. This could potentially explain the fact that O3-induced AHR was not sustained in CXCR2-deficient mice. We think that this is unlikely because others have reported that in mice in which neutrophils are depleted prior to O3 exposure, O3-induced AHR is as great as in mice with a full complement of neutrophils. Furthermore, IL-6 deficiency reduced O3-induced neutrophil influx, but did not affect O3-induced AHR. CXCR2 receptors have been reported to be present and to induce signaling in cells other than neutrophils, for example airway epithelial cells, and it is possible that activation of these receptors leads to changes in airway responsiveness.
Age-Related Differences in O3-Induced Airway Inflammation
Because we determined that both TNF and MIP-2/KC appeared to be important
for O3-induced AHR, we performed BAL after exposure of 2- and 8-week-old mice
to air or O3 (2 ppm for 3 hours) and measured BAL TNF and MIP-2 (see Figure
2) to determine whether differences in release of these cytokines and chemokines
might explain the observed age-related differences in O3-induced AHR. We also
measured IL-6. In control air-exposed mice, BAL IL-6 and TNFa were higher in
8-week-old than in 2-week-old mice (see Figure 2). This is likely the result
of age-related differences in the dilution of the BAL fluid, because the same
volume was used for the BAL in both ages, but the lungs of the 2-week-old mice
are much smaller. MIP-2 was undetectable after exposure to air. O3 resulted
in greater airway inflammation in 8-week-old than in 2-week-old mice. For example,
BAL MIP-2 was not significantly increased by O3 in 2-week-old mice, but O3
caused a statistically significant increase in MIP-2 in 8-week-old mice. Similarly,
BAL IL-6 levels increased following O3 exposure in both 8- and 2-week-old mice,
but the effect was greater in the 8-week-old mice (325 ± 50-percent increase)
than in the 2-week-old mice (135 ± 50-percent increase) (p < 0.01). BAL
TNF levels increased in both 2- and 8-week-old mice, but the magnitude of
the change was similar in both groups. The results indicate that age-related
differences in the release/expression of MIP-2 or related cytokines may account
for age-related differences in AHR: O3-induced MIP-2 release was greater in
adult than in immature mice, O3-induced AHR was greater in adult than in immature
mice, and in the absence of the receptor for MIP-2, CXCR2 reduced O3-induced
AHR indicating a requirement for this or related cytokines for O3-induced AHR.
Figure 2. BAL Protein (A), TNF (B), IL-6 (C), and MIP-2 (D)
in Air and O3-Exposed Mice Aged 2 or 8 Weeks. Mice were exposed to 2 ppm O3
for 3 hours, and BAL
was performed 4 hours after the cessation of exposure. Results are mean ± SE
of data from 9-19 mice in each group. *p < 0.05 compared to air-exposed
mice of the same age.
We also examined the effect of more sustained exposure to lower concentrations of O3 on aspects of airway inflammation and injury. Mice aged 2 weeks or 8 weeks were exposed to O3 (0, 0.2, 0.3, or 0.5 ppm) for 48 hours. O3-induced pulmonary inflammation and injury were assessed by measuring BAL cytokines protein and inflammatory cells, as described above. In contrast to our previous results, which indicated greater BAL protein following acute exposure to high-dose O3 in immature compared to mature mice, we observed that following more chronic exposure to lower concentrations of O3, immature mice had less protein in BAL fluid compared to adults (see Figure 3). In fact, in the 2-week-old mice, BAL protein was not significantly different from values obtained in air-exposed control mice at any of the concentrations of O3 studied. In contrast, in 8-week-old mice, BAL protein was significantly greater than in air-exposed mice at all O3 concentrations examined. The soluble TNFR receptors, sTNFR1 and sTNFR2, also increased to a greater extent in 8-week-old than in 2-week-old mice; however, sTNFR1 and sTNFR2 were increased in BAL of 2-week-old versus 8-week-old mice, even at baseline. This was true, despite lower BAL protein in the 2-week-old, air-exposed mice. In concert with our data indicating a role for TNF in O3-induced AHR, these data suggest that it is possible that the lack of O3-induced AHR in 2-week-old mice may be related to increased levels of sTNFR's neutralization of TNF.
We also noted substantial differences in BAL neutrophils induced by O3 between 2- and 8-week-old mice. In the 2-week-old mice, O3 exposure did not result in a significant increase in neutrophils at any of the concentrations studied, whereas in adult mice, even the lowest concentration of O3 (0.2 ppm) increased BAL neutrophils (see Figure 3). Surprisingly, eosinophils increased in the 2-week-old mice even at the lowest concentration of O3 studied, whereas they were undetectable in 8-week-old mice. We do not yet know the mechanistic basis for this difference. However, the results suggest that the immature animals may be predisposed to enhanced airway eosinophilia. Given that airway eosinophils are a major component of the inflammation of asthma, this may result in more severe episodes of asthma during periods of high O3.
Figure 3. BAL Protein, sTNFR1, sTNFR2, Neutrophils, and Eosinophils in 2- and 8-Week-Old Mice Exposed to Air or to O3 (0.2, 0.3, or 0.5 ppm) for 48 Hours. Results are mean ± SE of data from 5-16 mice in each group.
Our results indicate that immature mice and rats have a higher specific VE, but a reduced ventilatory response to acute high dose O3 compared to adult animals. These changes in ventilation result in a marked increase in the inhaled dose of O3 in the immature animals. Such age-related differences would be expected to be amplified under conditions of exercise, when ventilation increases. Consistent with the increased dose, immature mice and rats are more susceptible to pulmonary injury than adult animals when exposed acutely to high concentrations of O3. In contrast, immature mice are less susceptible to the induction of AHR. The results indicate that factors other than dose and injury are responsible for the reduced susceptibility to O3-induced AHR observed in immature mice: dose and injury are greater in younger than older animals, but younger animals do not develop AHR, whereas older animals do. Instead, differences in O3-induced inflammation may play a role in the differential susceptibility of immature versus mature mice to O3-induced AHR. For example, immature mice do not demonstrate release of MIP-2 into the airways following acute ozone exposure, and our results demonstrate that MIP-2 or some other ligand for the CXCR2 receptor is required for O3-induced AHR in this species.
Our data also indicate that there are differences between acute high-dose exposure and more sustained low-dose exposure with respect to age-dependent effects on O3-induced lung injury. When they are exposed acutely to high concentrations of O3 (2 ppm for 3 hours), immature rats and mice have greater lung injury than mature rats and mice, as indicated by BAL protein. In contrast, immature mice develop less lung injury than mature mice when they are exposed to lower concentrations of O3 (0.2 to 0.5 ppm), but for longer periods of time (48 hours). As discussed above, it is likely that differences in O3 dose due to differences in ventilation account for the greater lung injury in the young mice that are acutely exposed to ozone. However, it is clear that these younger mice are better able to adapt to continued O3 exposure than older mice. One potential explanation is that they have a greater capacity to produce antioxidants in response to O3. One important difference in the responses of immature versus mature mice to sustained exposure to low concentrations of O3 is that the immature, not the mature, mice develop airway eosinophilia, particularly at lower concentrations of O3. This may make the younger animals more susceptible to O3 as an asthma trigger, because airway eosinophilia is one of the hallmarks of asthma. Understanding the mechanistic basis for this increased susceptibility to eosinophilia might provide new avenues for the treatment of asthmatic children on days of high pollution, and reinforces recently published results indicating that asthmatic children have increased asthma symptoms in response to O3, even when O3 is below the U.S. Environmental Protection Agency (EPA) standard.
These are the first data providing evidence for age-related differences in the ventilatory response to O3, and the first data providing evidence for age-related differences in O3-induced AHR. These data also provide the first evidence for a role for the CXCR2 receptor in O3-induced AHR. We do not know to what extent the results reported here can be extrapolated to humans. Of importance, humans do not have robust declines in VE in response to O3. However, because this response reduces the inhaled dose of O3 in rodents, the toxic effects of O3 might be expected to be higher in humans than in rodents for the same inhaled concentration. In contrast, the lower specific ventilation observed in mature versus immature rodents also is observed in humans, and it is likely to contribute to an increased inhaled dose of O3 for the same inhaled concentration in children versus adults. There also may be differences between mice and humans in the degree of postparturition development of systems that are involved in sensing or responding to inhaled irritants such as O3. Such species differences in development further complicate the extrapolation of our results to humans. Nevertheless, the results emphasize the likelihood of age-related differences in response to O3 between human children and adults, and suggest that not all responses to O3 may be affected in the same way by age. As such, data from adult humans regarding sensitivity of pulmonary responses to O3 likely cannot be extrapolated to children.
References:
Guzelian PS, Henry CJ, Olin SS, eds. Similarities and Differences Between Children and Adults: Implications for Risk Assessment. Washington, DC: International Life Sciences Institute, 1992.
Klepeis NE , Tsang AM, Behar JV. Analysis of the National Human Activity Patter Survey (NHAPS) respondents from a standpoint of exposure assessment. EPA/600-R-96/074, 1996.
Mukerjee D. Assessment of risk from multimedia exposures of children to environmental chemicals. Journal of Air and Waste Management Association 1998;48:483-501.
National Academy of Sciences. Pesticides in the Diets of Infants and Children. Washington, DC: National Academy Press, 1993.
Nigg HH, Beier RC, Carter O, et al. Exposure to pesticides. In: Proceedings Baker SR, Wilkinson CS, eds. The Effect of Pesticides on Human Health. Volume 17: Advances in Modern Environmental Toxicology. New York, NY: Princeton Scientific, 1990, pp. 35-130.
Journal Articles on this Report: 3 Displayed | Download in RIS Format
| Other project views: | All 5 publications | 4 publications in selected types | All 4 journal articles |
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Johnston RA, Schwartzman IN, Flynt L, Shore SA. Role of interleukin-6 in murine airway responses to ozone. American Journal of Physiology-Lung Cellular and Molecular Physiology 2005;288(2):L390-L397. |
R827447 (Final) |
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Shore SA, Abraham JH, Schwartzman IN, Krishna Murthy GG, Laporte JD. Ventilatory responses to ozone are reduced in immature rats. Journal of Applied Physiology 2000;88(6):2023-2030. |
R827447 (1999) R827447 (2000) R827447 (Final) |
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Shore SA, Johnston RA, Schwartzman IN, Chism D, Krishna Murthy GG. Ozone-induced airway hyperresponsiveness is reduced in immature mice. Journal of Applied Physiology 2002;92(3):1019-1028. |
R827447 (2000) R827447 (2001) R827447 (Final) |
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air, ambient air, ozone, health effects, sensitive populations, dose-response, animal, mammalian, cellular, children, age, susceptibility, oxidants, biology, pathology, air toxics, tropospheric ozone, acute exposure, age-dependent response, age-related differences, air pollution, airway disease, assessment of exposure, asthma, biomedical research, children's vulnerability, environmental hazard exposures, enzyme systems, exposure, exposure and effects, human exposure, inhalation, lung injury, ozone-induced airway dysfunction, ozone-induced inflammation, respiratory problems, stratospheric ozone. , Air, Scientific Discipline, Health, RFA, PHYSICAL ASPECTS, Susceptibility/Sensitive Population/Genetic Susceptibility, Molecular Biology/Genetics, Risk Assessments, genetic susceptability, Health Risk Assessment, Physical Processes, air toxics, Children's Health, Biochemistry, tropospheric ozone, exposure and effects, environmental hazard exposures, acute exposure, enzyme systems, inhalation, respiratory problems, ozone, ozone induced airway dysfunction, assessment of exposure, age-related differences, lung injury, toxics, sensitive populations, ozone induced inflammation, air pollution, airway disease, children, stratospheric ozone, age dependent response, biomedical research, exposure, children's vulnerablity, dose response model, asthma
Relevant Websites:
http://www.hsph.harvard.edu/facres/shr.html
Progress and Final Reports:
1999 Progress Report
2000 Progress Report
2001 Progress Report
Original Abstract