Case Study Report On The Effects Of Pollution On The O-Zone

Effects on Mice of Exposure to Ozone and Ambient Particle Pollution

EPA Grant Number: R828112C106
Subproject:this is subproject number 106 , established and managed by the Center Director under grant R828112
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center:Health Effects Institute (2000 — 2005)
Center Director: Greenbaum, Daniel S.
Title: Effects on Mice of Exposure to Ozone and Ambient Particle Pollution
Investigators: Kobzik, Lester
Institution:Harvard T.H. Chan School of Public Health
EPA Project Officer: Hunt, Sherri
Project Period: April 1, 2000 through March 31, 2005
RFA: Health Effects Institute (1996) RFA Text |  Recipients Lists
Research Category:Health Effects , Air


Epidemiologic studies have indicated that small shortterm increases in the concentration of particulate matter (PM), the complex and variable mixture of particles in the atmosphere, are associated with short-term increases in human morbidity and mortality. Particularly at risk are elderly people and individuals with compromised cardiac or airway function, such as those with asthma.


The investigators used a mouse model of asthma to evaluate how inhaling pollutants affects the airways. The mice were sensitized to the allergen ovalbumin and later challenged with the same aerosol to induce a lung condition in the mice similar to that found in people with asthma.

The investigators studied concentrated ambient particles (CAPs) of respirable size and ozone, a gaseous pollutant known to cause airway inflammation and to compromise respiratory function. To maximize possible CAPs effects, they used a new version of the Harvard Ambient Particle Concentrator to deliver particles at concentrations that were many times higher than those in ambient Boston air (varying from 12-fold to 67-fold on different days of the study). He exposed mice to ozone at 0.3 ppm, a concentration similar to levels found in urban ambient air.

Supplemental Keywords:

Air, ambient air quality, air toxics, epidemiology, health effects, particulate matter, biochemistry, motor vehicle emissions, diesel exhaust, animal models, ozone., RFA, Health, Scientific Discipline, Air, particulate matter, Toxicology, air toxics, Environmental Chemistry, Health Risk Assessment, Risk Assessments, mobile sources, Biochemistry, Atmospheric Sciences, ambient air quality, lung injury, particulates, air pollutants, cardiopulmonary responses, human health effects, aerosol particles, exposure and effects, acute lung injury, cardiovascular vulnerability, environmental risks, exposure, ozone, pulmonary disease, animal model, air pollution, automotive exhaust, diesel exhaust, chronic health effects, human exposure, lung inflammation, ambient particle health effects, ambient particle pollution, Acute health effects, PM, diesel exhaust particles, indoor air quality, mortality, exposure assessment, human health risk

Progress and Final Reports:

  • 2000
  • 2001
  • 2002
  • 2003
  • Final Report

  • Main Center Abstract and Reports:

    R828112    Health Effects Institute (2000 — 2005)

    Subprojects under this Center:(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R828112C042 Does Inhalation of Methanol Vapor Affect Human Neurobehavior?
    R828112C043 Human Responses to Nitrogen Dioxide
    R828112C044 The Role of Inflammation in Ozone-Induced Lung Injury
    R828112C045 How Does Exercise Affect the Dose of Inhaled Air Pollutants?
    R828112C046 How Do Chemicals in Diesel Engine Exhaust Damage DNA?
    R828112C047 Effect of Nitrogen Dioxide on Bacterial Respiratory infection in Mice
    R828112C048 Effects of Ozone Exposure on Airway Epithelium
    R828112C049 Inhalation of Aldehydes and Effects on Breathing
    R828112C050 Does Ozone Cause Precancerous Changes in Cells?
    R828112C051 Effects of Formaldehyde on Human Airway Epithelial Cells Exposed in a Novel Culture System
    R828112C052 Carbon Monoxide and Cardiac Arrhythmias
    R828112C053 Effects of Formaldehyde and Particle-Bound Formaldehyde on Lung Macrophage Functions
    R828112C054 Mechanisms for Protecting Lung Epithelial Cells Against Oxidant Injury
    R828112C055 Relationship of Nitropyrene-Derived DNA Adducts to Carcinogenesis
    R828112C056 Particle Trap Effects on Heavy-Duty Diesel Engine Emissions
    R828112C057 Carbon Monoxide and Atherosclerosis
    R828112C058 Nitrogen Dioxide and Respiratory Illness in Children
    R828112C059 Noninvasive Methods for Measuring Ventilation in Mobile Subjects
    R828112C060 Oxidant Air Pollutants and Lung Cancer: An Animal Model
    R828112C061 Detection of Carcinogen-DNA Adducts: Development of New Methods
    R828112C062 Effects of Carbon Monoxide on Heart Muscle Cells
    R828112C063 Development of Personal Ozone Samplers: Three Approaches
    R828112C064 Development of Biomarkers to Monitor Carcinogen Exposure
    R828112C065 Effects of Prolonged Ozone Inhalation on Collagen Structure and Content in Rat Lungs
    R828112C065II Prolonged Ozone Exposure and the Contractile Properties of Isolated Rat Airways
    R828112C065III Changes in Complex Carbohydrate Content and Structure in Rat Lungs Caused by Prolonged Ozone Inhalation
    R828112C065IV Genetic Control of Connective Tissue Protein Synthesis After Prolonged Ozone Inhalation
    R828112C065V Pulmonary Function Alterations in Rats After Chronic Ozone Inhalation
    R828112C065VII Prolonged Ozone Exposure Leads to Functional and Structural Changes in the Rat Nose
    R828112C065VIII - IX Studies of Changes in Lung Structure and Enzyme Activities in Rats After Prolonged Exposure to Ozone
    R828112C065X An Innovative Approach to Analyzing Multiple Experimental Outcomes: A Case Study of Rats Exposed to Ozone
    R828112C065XI The Consequences of Prolonged Inhalation of Ozone on Rats: An Integrative Summary of the Results of Eight Collaborative Studies
    R828112C066 Interactive Effects of Nitropyrenes in Diesel Exhaust
    R828112C067 Detection of Formaldehyde–DNA Adducts: Development of New Methods
    R828112C068I Comparison of the Carcinogenicity of Diesel Exhaust and Carbon Black in Rat Lungs
    R828112C068II An Investigation of DNA Damage in the Lungs of Rats Exposed to Diesel Exhaust
    R828112C068III No Evidence For Genetic Mutations Found In Lung Tumors From Rats Exposed To Diesel Exhaust or Carbon Black
    R828112C069 Noninvasive Determination of Respiratory Ozone Absorption: The Bolus-Response Method
    R828112C070 The Effects of Inhaled Oxidants and Acid Aerosols on Pulmonary Function
    R828112C071 Biochemical Consequences of Ozone Reacting with Membrane Fatty Acids
    R828112C072 DNA Mutations in Rats Treated with a Carcinogen Present in Diesel Exhaust
    R828112C073 Developmental Neurotoxicity of Inhaled Methanol in Rats
    R828112C074 Methanol Distribution in Non Pregnant and Pregnant Rodents
    R828112C075 Is Increased Mortality Associated with Ozone Exposure in Mexico City?
    R828112C076 Effects of Fuel Modification and Emission Control Devices on Heavy-Duty Diesel Engine Emissions
    R828112C077 Metabolic Studies in Monkeys Exposed to Methanol Vapors
    R828112C078 Effects of Ozone on Pulmonary Function and Airway Inflammation in Normal and Potentially Sensitive Human Subjects
    R828112C079 Improvement of a Respiratory Ozone Analyzer
    R828112C080 Mechanism of Oxidative Stress from Low Levels of Carbon Monoxide
    R828112C081 Long-Term Exposure to Ozone: Development of Methods to Estimate Past Exposures and Health Outcomes
    R828112C082 Effects of Ambient Ozone on Healthy, Wheezy, and Asthmatic Children
    R828112C083 Daily Changes in Oxygen Saturation and Pulse Rate Associated with Particulate Air Pollution and Barometric Pressure
    R828112C084 Evaluation of The Potential Health Effects of the Atmospheric Reaction Products of Polycyclic Aromatic Hydrocarbons
    R828112C085 Mechanisms of Response to Ozone Exposure: The Role of Mast Cells in Mice
    R828112C086 Statistical Methods for Epidemiologic Studies of the Health Effects of Air Pollution
    R828112C087 Development of New Methods to Measure Benzene Biomarkers
    R828112C088 Alveolar Changes in Rat Lungs After Long-Term Exposure to Nitric Oxide
    R828112C089 Effects of Prenatal Exposure to Inhaled Methanol on Nonhuman Primates and Their Infant Offspring
    R828112C090 A Pilot Study of Potential Biomarkers of Ozone Exposure
    R828112C091 Effects of Concentrated Ambient Particles on the Cardiac and Pulmonary Systems of Dogs
    R828112C092 Cancer, Mutations, and Adducts in Rats and Mice Exposed to Butadiene and Its Metabolites
    R828112C093 Effects of Concentrated Ambient Particles in Rats and Hamsters: An Exploratory Study
    R828112C094I The National Morbidity, Mortality, and Air Pollution Study: Methods and Methodologic Issues
    R828112C094II The National Morbidity, Mortality, and Air Pollution Study: Morbidity and Mortality from Air Pollution in the United States
    R828112C095 Association of Particulate Matter Components with Daily Mortality and Morbidity in Urban Populations
    R828112C096 Acute Pulmonary Effects of Ultrafine Particles in Rats and Mice
    R828112C097 Identifying Subgroups of the General Population That May Be Susceptible to Short-Term Increases in Particulate Air Pollution
    R828112C098 Daily Mortality and Fine and Ultrafine Particles in Erfurt, Germany
    R828112C099 A Case-Crossover Analysis of Fine Particulate Matter Air Pollution and Out-of-Hospital Sudden Cardiac Arrest
    R828112C100 Effects of Mexico City Air on Rat Nose
    R828112C101 Penetration of Lung Lining and Clearance of Particles Containing Benzo[a]pyrene
    R828112C102 Metabolism of Ether Oxygenates Added to Gasoline
    R828112C103 Characterization and Mechanisms of Chromosomal Alterations Induced by Benzene in Mice and Humans
    R828112C104 Acute Cardiovascular Effects in Rats from Exposure to Urban Ambient Particles
    R828112C105 Genetic Differences in Induction of Acute Lung Injury and Inflammation in Mice
    R828112C106 Effects on Mice of Exposure to Ozone and Ambient Particle Pollution
    R828112C107 Emissions from Diesel and Gasoline Engines Measured in Highway Tunnels
    R828112C108 Case-Cohort Study of Styrene Exposure and Ischemic Heart Disease Investigators
    R828112C110 Effects of Metals Bound to Particulate Matter on Human Lung Epithelial Cells
    R828112C111 Effect of Concentrated Ambient Particulate Matter on Blood Coagulation Parameters in Rats
    R828112C112 Health Effects of Acute Exposure to Air Pollution
    R828112C113 Benzene Metabolism in Rodents at Doses Relevant to Human Exposure from Urban Air
    R828112C114 A Personal Particle Speciation Sampler
    R828112C115 Validation and Evaluation of Biomarkers in Workers Exposed to Benzene in China
    R828112C116 Biomarkers in Czech Workers Exposed to 1,3-Butadiene: A Transitional Epidemiologic Study
    R828112C117 Peroxides and Macrophages in the Toxicity of Fine Particulate Matter in Rats
    R828112C118 Controlled Exposures of Healthy and Asthmatic Volunteers to Concentrated Ambient Particles in Metropolitan Los Angeles
    R828112C119 Manganese Toxicokinetics at the Blood-Brain Barrier
    R828112C120 Effects of Exposure to Concentrated Ambient Particles from Detroit Air on Healthy Rats and Rats with Features of Asthma or Mild Bronchitis
    R828112C121 Field Evaluation of Nanofilm Detectors for Measuring Acidic Particles in Indoor and Outdoor Air
    R828112C123 Time-Series Analysis of Air Pollution and Mortality: A Statistical Review
    R828112C126 Effects of Exposure to Ultrafine Carbon Particles in Healthy Subjects and Subjects with Asthma
    R828112C128 Neurogenic Responses of Rat Lung to Diesel Exhaust
    R828112C130-I Relationships of Indoor, Outdoor, and Personal Air (RIOPA). Part I. Collection Methods and Descriptive Analyses
    R828112C132 An Updated Study of Mortality Among North American Synthetic Rubber Industry Workers

    Materials and methods

    Population studied

    We performed a 5-year (2007–2011) case-crossover analysis in Nice, France. We retrospectively enrolled consecutive patients with stroke admitted at the University Hospital of Nice between January 2007 and December 2011. Querying French DRG-based database (PMSI: Programme de Médicalisation des Systèmes d'Information) with I60–I69 codes from the International Classification of Diseases (10th revision), we screened all patients hospitalised for stroke. We filtered the sample to patients living in Nice (geographical area defined by zip codes: 06000, 06100, 06200 and 06300). The diagnosis of ischaemic stroke was reviewed and confirmed by a panel of neurologists using clinical and radiological data of medical records. Patients with another diagnosis than stroke were excluded. Demographic data, vascular risk factors (WHO definitions), clinical and radiological characteristics of stroke were also collected from medical records.

    Outdoor air pollution and meteorological data

    Nice is an urban city situated in the south-eastern part of France on the Mediterranean coast. According to the latest census, Nice has a population of 340 735 in 2009. Its climate is temperate and qualified as Mediterranean type. Surrounded by hills and mountains (south Alps), the city of Nice is sheltered from continuous violent winds. Outdoor air pollution comes mainly from traffic due to high density of roads and an international airport (first one in France after Paris airports).

    Air pollution data were obtained from the regional agency for air quality monitoring (AirPACA). Exposure measurements during the study period were carried out in 2 of 13 permanent monitoring stations in the study area. Measures (µg/m3) were performed in an urban station (Cagnes Ladoumègue) for following atmospheric pollutants: particulate matter (PM10; tapered element oscillating microbalance), nitrogen dioxide (NO2) (chemiluminescence), sulfur dioxide (SO2; ultraviolet photometry) and ozone (O3; ultraviolet photometry). Missing values were replaced by measures performed by the observational monitoring station located at Nice Airport. We computed for each pollutant during 24 h average and specifically for O3 during 8 h daytime periods.

    Daily meteorological data were obtained from the National Meteorological Office of Nice, including temperature (°C) and humidity (%). Moreover, data on influenza epidemics (weekly count) in the region of Provence-Alpes-Cote-d'Azur were obtained from the Sentiweb network.

    Statistical analysis

    Continuous variables were expressed as mean (SD) or median (IQR), and categorical variables as percentages. Spearman correlation coefficients (r) between air pollutants and atmospheric parameters were calculated. The time-stratified case-crossover design was used to examine the relationship between short-term effects of outdoor air pollutants and stroke. In this design, each participant enrolled was his own control. Case days were defined as the day of stroke. Control days were defined as the same day of the same stratum as the case day. Study time was stratified by months. Therefore, explicative variable levels at the case day were compared with levels of the same variables at control days. This method has the main advantage to control individual factors, the day of the week, season and time trend.24 Conditional logistic regression was performed to estimate the association between short-term effects of each air pollutant measured and stroke onset. OR and 95% CI for a 10 µg/m3 increase of pollutant level were adjusted for temperature and humidity with a 1-day lag, influenza epidemics and holidays without day lag. The pollutant exposure was tested in models for 1-day, 2-day or 3-day lag. Stratified analyses by subgroups were performed according to age, gender, risk vascular factors (tobacco use, diabetes mellitus, hypercholesterolaemia and hypertension) and stroke aetiological subtypes according to the Trial of ORG 10 172 in acute stroke treatment (TOAST). We evaluated dose–response relationships across four exposure levels of pollutants studied, and the first quartile was used as the reference group. A p value less than 0.05 was considered as significant. The data were analysed using Stata V.10.0 SE software.


    During the study period (January 2007 to December 2011), there were 2067 patients living in Nice and were admitted to the University Hospital Center for ischaemic stroke based on the DRG database. After neurologists review of medical records, 1729 patients with ischaemic stroke were enrolled for final analysis. Six hundred and twenty (35.9%) of these patients were hospitalised in the stroke unit. According to the last population census of 2009, annual ischaemic stroke incidence rates (by 100 000) in the studied area were, respectively, from 2007 to 2011: 100, 100, 98, 96 and 112. The mean age was 76.1±14.0 years, and 46.7% were men (table 1).

    The distribution of air pollutants and meteorological variables is shown in table 2. Spearman correlation coefficients (r) were ranged from 0.01 to 0.25 between each studied pollutants, except between O3 and NO2 (r=−0.54). Correlation coefficient between minimal temperature and O3 was r=0.67 (see online supplementary table I).

    No significant association was found between stroke occurrence and short-term effects of all pollutants tested. In addition, we performed stratified subgroup analysis according to gender, age by decade, incident/recurrent stroke status, vascular risk factors, presence of atrial fibrillation and stroke aetiological subgroups. We measured only significant associations between stroke and short-term effect of O3 in following both groups: recurrent (n=280) and large artery stroke (n=578) (table 3). In recurrent stroke subgroup, for an increase of 10 µg/m3 of O3 level (mean D-1, D-2 and D-3 lag), stroke risk was significantly increased by 12.1% (95% CI 1.5% to 23.9%). Adjusted OR between O3 exposure (mean D-3 and D-4) and large artery stroke subgroup was 1.080 (95% CI 1.002 to 1.166). No significant association was observed with other pollutants than O3. Adjusted in two-pollutant models, OR was not affected. Using O3 quartiles (1st quartile as the reference group), linear dose–response relationship for both subgroups was observed (figure 1). Baseline characteristics in recurrent stroke and large artery stroke subgroups are shown in figure 2.

    Figure 1

    Dose relationship between ozone and ischaemic stroke events ((A), recurrent ischaemic stroke subgroup and (B), large artery ischaemic stroke subgroup).

    Figure 2

    Baseline characteristics according to recurrent stroke subgroup (A) and large artery stroke subgroup (B) (*p<0.05).

    Table 1

    Baseline characteristics of patients with ischaemic stroke (incident and recurrent) hospitalised in Nice University Hospital from 2007 to 2011

    Table 2

    Distribution of air pollution concentrations and meteorological parameters in Nice (France) between 2007 and 2012

    Table 3

    Adjusted ORs between ischaemic stroke and outdoor pollutants exposure for an increase of 10 µg/m3 in Nice (France) between 2007 and 2011


    Our study assessed the short-term effect of O3 exposure on a selected population of ischaemic stroke in a city especially polluted by O3. An elevation of 10 µg/m3 of O3 concentration increases stroke risk with few days lag in recurrent (≈12%) and large artery stroke (≈8%) subgroups only. Linear dose–response relationship was observed systematically in both groups. In these groups, the common feature of the patients was that they cumulate vascular risk factors. No significant association was found between all ischaemic stroke groups and atmospheric pollutants studied (O3, NO2, SO2 and PM10).

    Several studies have investigated the association between outdoor air pollution and stroke.3,5–24 Results of these studies are conflicting and hamper generalisation of conclusions. Heterogeneous methodological considerations are the main explanation of this conflict. Methodological differences are observed in patient selection, study design, outcomes choice (incidence, hospital admission, mortality) and assessment of individual exposure to selected pollutants.20 Few published studies investigated especially the association between occurrence of ischaemic stroke and O3 exposure using the case-crossover design8,15,17,19,21 or the time series analysis method.3,12,16,22 Consistent with our results, the majority of these studies do not observe the relationship between O3 exposure and occurrence of ischaemic stroke.3,8,17,19,21,22 Whenever a relationship was revealed, the association was borderline significant16 or was not confirmed by a second study on the same area of investigation.15,19 Despite the fact that the link between ischaemic stroke and O3 exposure is not obvious, results in subgroup analyses seem to identify a population at risk for O3 exposure. In a recurrent ischaemic stroke subgroup, a significant increase of 12.1% (95% CI 1.5% to 23.9%) in stroke risk was observed for each increase of 10 µg/m3 of O3 concentration during previous days (mean D-1, D-2 and D-3 lag). Consistent with this result, a population-based study in Dijon (France) revealed the same association (OR 1.150; 95% CI 1.027 to 1.209) with 3 days lag.19 Similarly, a significant association was observed in a large artery stroke subgroup (mean D-3, D-4, OR 1.080; 95% CI 1.002 to 1.166). This link was observed in the previous study (Dijon) especially in this stroke aetiological subgroup.15 Associations in other ischaemic stroke subgroups are not systematically confirmed (age, gender, vascular risk factors and season).3,15,19,21,22 Our study confirms the short-term effects of O3 exposure on patients with stroke with high vascular risk.15,19

    Our findings suggest that exposure to O3, the main photochemical pollutant, could increase the risk of ischaemic stroke in population subgroups (recurrent stroke, large arteries stroke) particularly exposed to vascular risk factors inducing atherosclerosis. Physiopathological pathways linking ischaemic stroke and O3 exposure still remain largely unclear and probably complex. Some studies support a delayed effect (1–3 days lag) between acute exposure of O3 pollution and stroke onset.15,19 O3 urban pollution effects on healthy participants are associated with systemic inflammatory responses, oxidative stress and blood coagulation.25,26 These acute phenomena induced by even low levels of O3 could be the trigger of ischaemic event consecutively to atherosclerotic plaque instability, alterations in endothelial function, and increased coagulation and thrombosis.27 As suggested by Henrotin et al,19 we hypothesised that short-term effect of O3 exposure could be involved especially among participants with high vascular risk.

    In order to establish a causal relationship between O3 exposure and stroke onset, we studied the exposure–response relationship, the main criteria identified by Hill.28 Consistent with previous reports, we show a linear exposure–response relationship between O3 concentration and ischaemic stroke in subgroups identified in previous reports.15,19

    Our study has several limitations. The question of completeness of patients with stroke living in Nice in this hospital-based study was discussed. In Nice, patients with suspicion of stroke are admitted in priority in the University Hospital Center. Likewise, incidence of ischaemic stroke was consistent with epidemiological data in France. The question of individual exposure measurement is generally discussed. The main limitation is that we used air pollution levels from air monitoring station to represent individual exposure. However, we limited our investigations to a small geographical area (72 km2), not considered as a polluted town except for O3 (median 53.3 (32.6–69.2) µg/m3). Moreover, in the stroke population studied, elderly patients are mostly retired and have daily activity in the study area. Since O3 concentration is correlated with meteorological parameters, temperature and humidity were incorporated into our models. Association between O3 pollution and stroke can be confounded by other pollutants studied, especially particles. Effects of O3 alone are not modified using adjusted models for each of the other pollutants (NO2, SO2 and PM10). PM2.5 was not studied because it was not monitored in Nice.


    The consequences of O3 pollution on the respiratory system and mortality are well documented.1 Our results confirm the relationship between low-level O3 exposure and ischaemic stroke in high vascular risk subgroup with linear exposure–response relationship, independently of other pollutants and meteorological parameters. Reproducibility of previous results is one of the main Hill’s criterion to induce causality of O3 exposure. Even if the individual's risk is low, to identify an association between O3 and ischaemic stroke incidence is important from a public health point of view, since a large population is concerned. The physiopathological processes underlying this association between ischaemic stroke and O3 exposure remain to be investigated.


    The authors would like to thank AirPACA association and Sentiweb network who provided, respectively, daily measures of outdoor air pollution and data on influenza epidemics in the region of Provence-Alpes-Cote-d'Azur.


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