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2000 Progress 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 School of Public Health
Current Institution: Harvard University
EPA Project Officer: Deener, Kacee
Project Period: July 1, 1999 through June 30, 2002 (Extended to July 31, 2003)
Project Period Covered by this Report: July 1, 2000 through June 30, 2001
Project Amount: $852,937
RFA: Children's Vulnerability to Toxic Substances in the Environment (1999)
Research Category: Children's Health , Health Effects
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 because they are more physically active, and hence 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 than mature animals.
The questions that this research project seeks to address are: (1) Is O3 induced AHR greater in juvenile than adult animals and if so, why? (2) What is the mechanism of O3 induced AHR? and (3) Is the mechanistic basis for O3-induced AHR the same or different in juvenile and adult animals? In particular, we propose to test the following hypotheses: (1) 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; (2) that TNFa formation induced by O3 is important for O3-induced AHR; and (3) that O3-induced TNFa formation is different in juvenile and adult animals.
Progress Summary:Age Related Differences in the Ventilatory Response to O3 in Mice. In the previous reporting period, we reported on age related differences in ventilation in rats aged 2 to 12 weeks and in the effect of 2 ppm O3 on the ventilatory pattern. We now have extended these observations to mice and have investigated a range of O3 concentrations from 0.3 to 3.0 ppm. 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. There was a consistent reduction in VE normalized for body weight (specific VE) with increasing age (p<0.001). 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. Age-related differences in specific VE were primarily the result of age-related differences in specific tidal volume (VT), which also decreased significantly with age (p< 0.001) until 8 weeks of age.
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 significant (p<0.001) within 30
minutes of the onset of O3 exposure and 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. The decreases in VE occurred as
the result of decreases in both VT and f. The change in breathing frequency was
primarily the result of an increase in end expiratory pause (EEP), whereas
inspiratory time was not significantly altered.
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 (p<0.001).
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 specific VE over the 3-hour exposure period to calculate the total volume inhaled during this period, and multiplied this total volume by O3 concentration to calculate O3 dose normalized for body weight. Animal age had a marked and statistically significant effect on inhaled O3 dose (p<0.001 by ANOVA). O3 dose normalized for body weight 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. 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 O3 dose is particularly apparent when comparing the O3 dose at O3 concentrations of 2 ppm 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 three to four times greater in these mice than in adult (8-12 week old) mice.
Age Related Differences in O3-Induced Airway Responsiveness. To determine the functional consequences of the increase in O3 dose in the younger animals, we measured airway responsiveness to inhaled methacholine on the day prior to, and then again 3 hours after cessation of O3 exposure in mice in each age group. AHR is a defining feature of asthma and is observed following O3 exposure in adults of most species. 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. Data from the 2 and 3 ppm exposures were combined because the O3 dose was not different for these two concentrations. 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 logEC2Penh 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).
Age-Related Differences in O3-Induced Pulmonary Injury and Inflammation. To determine whether there were age-related differences in the magnitude of O3-induced injury or inflammation that might account for the differences in 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 protein, TNFa, IL-6, and MIP-2. O3 exposure resulted in greater lung injury in 2 week old than in 8 week old mice. In 2 week old mice, O3 exposure 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. In contrast, O3 resulted in greater airway inflammation in 8 week old than in 2 week old mice. BAL MIP-2 was not significantly increased by O3 in 2 week old mice, but O3 did cause a statistically significant increase in MIP-2 in 8 week old mice. Similarly, BAL IL-6 levels were 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 TNFa levels increased in both 2 and 8 week old mice, but the effect did not reach statistical significance in either age group. Following O3 exposure, BAL PMN increased to approximately the same extent in 2 week and 8 week old mice.
In summary, our results indicate that immature mice have a higher specific VE, but a reduced ventilatory response to O3 compared to adult mice. These changes in ventilation result in a marked increase in the inhaled dose of O3 in the immature mice. Such age-related differences would be expected to be amplified under conditions of exercise, when ventilation increases. Consistent with the increased dose, immature mice are more susceptible to pulmonary injury than adult mice. In contrast, immature mice are less susceptible to the induction of AHR and cytokine formation by O3 than adult mice. The results indicate that factors other than dose and injury are responsible for the reduced susceptibility to O3-induced AHR observed in immature mice.
Future Activities:In the coming year, a study similar to that reported above will be performed using longer exposures to lower concentrations of O3. Such exposure should more accurately reflect the types of exposures that occur in humans. Mice aged 2 weeks through 12 weeks will be exposed to O3 (0.1, 0.2, 0.3, or 0.5 ppm) for 72 hours. O3-induced changes in airway responsiveness to inhaled methacholine, and O3-induced pulmonary inflammation and injury will be assessed as described above. In addition, because we and others have reported that AHR induced by acute exposure to O3 is attenuated in TNF receptor deficient mice, we will perform further studies of age-related differences in the induction of TNF by O3. Our studies using ELISAs of BAL fluid revealed a statistically insignificant increase in TNF following acute O3 exposure. However, because TNF can be associated with cell membranes, it is possible that it is indeed induced by O3, but not released into BAL fluid in quantities sufficient to detect by ELISA. Consequently, TNFa mRNA will be assessed by Northern blotting of RNA derived from immature and adult mice exposed to air or O3. We also will examine the expression of TNF receptors, because others have reported that expression of TNFR2 is altered by O3 exposure.
Journal Articles on this Report: 2 Displayed | Download in RIS Format
| Other project views: | All 5 publications | 4 publications in selected types | All 4 journal articles |
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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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ozone, exposure, health effects, age, biology, sensitive populations, mammalian. , 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
Original Abstract
2001 Progress Report
Final Report