Final Report: Pathophysiologic Mechanisms of Mortality Associated with Exposure to Concentrated Particulate Urban Air Toxics

EPA Grant Number: R825242
Title: Pathophysiologic Mechanisms of Mortality Associated with Exposure to Concentrated Particulate Urban Air Toxics
Investigators:
Institution: Harvard University
EPA Project Officer: Chung, Serena
Project Period: October 1, 1996 through September 30, 1999
Project Amount: $520,997
RFA: Air Quality (1996) RFA Text |  Recipients Lists
Research Category: Air Quality and Air Toxics , Air

Objective:

The central hypothesis of this research was that ambient urban air particles are complex mixtures with intrinsic toxicity. In concert with pre-existing inflammation, particulate exposure results in stimulation of pro-inflammatory mediators that lead to local and systemic effects that ultimately account for the epidemiological associations.

The specific aims and objectives of this research were to:

  1. Determine the extent of pulmonary and/or systemic inflammation, as well as the role of cytokines and/or circulating toxic products in morbidity and mortality due to inhaled CAPs.
  2. Determine if hypoxia and/or autonomic neural responses are major pathophysiologic mechanisms in responses to inhaled CAPs.
  3. Relate specific components of urban air particulate to particular mechanisms of responses.

Summary/Accomplishments (Outputs/Outcomes):

A. Urban Air Particulate Inhalation Alters Pulmonary Function and Induces Pulmonary Inflammation in a Rodent Model of Chronic Bronchitis. Epidemiological studies have reported increased morbidity in human populations following inhalation of elevated levels of urban particulate matter. These responses are especially prevalent in populations with chronic obstructive pulmonary diseases, including chronic bronchitis. Toxicological studies have reported altered pulmonary function and increased pulmonary inflammation following particulate inhalation in the laboratory setting. However, most of these studies have utilized artificial particles that may not accurately mimic outdoor air pollutant conditions. Few studies have utilized actual urban air particle samples in inhalation studies. In the present study, the effects of inhaled concentrated urban air particulates (CAPs) on pulmonary function and pulmonary inflammation are addressed.

Normal rats and rats with chronic bronchitis (~ 200 ppm SO2 for 6 weeks) were exposed to filtered air or CAPs (12 rats per group x 4 groups = 48 rats total) for 5 hours/day for 3 consecutive days. The particle aerosol levels were 206, 733, and 607 µg/m3 (MMAD=0.18 µm; g=2.9) on days 1, 2, and 3, respectively. Following the final day of exposure, pulmonary function parameters including peak expiratory flow (PEF), tidal volume (TV), respiratory frequency (RF), and minute volume (MV), were measured and compared to pre-exposure baseline levels. Twenty-four hours following the final day of exposure, bronchoalveolar lavage was performed for total cell counts, differential cell counts, and total lavage protein levels.

Pulmonary responses to CAPs in chronic bronchitic animals indicated a significant increase in tidal volume as well as peak expiratory flow. In CAPs-exposed animals without underlying bronchitis, significantly increased tidal volume was observed. Significant pulmonary inflammation was observed in the CAPs-exposed animals, particularly those with chronic bronchitis. Significant increases in neutrophils, lymphocytes, and total lavage protein were observed (see Figures 1 and 2).

Pulmonary inflammation was modestly increased in the study reported here. Neutrophilia and pulmonary vascular permeability suggested an additive, and potentially a synergistic, response between CAPs and CB, as illustrated in Figures 1 and 2. These results are congruous with our starting hypothesis. Mild inflammatory responses to CAPs were expected in normal animals. However, changes in the animals with preexisting disease were expected to be more severe. Previous studies have shown the induction of inflammation in animals with CB (Farone, et al., American Journal of Respiratory Cell and Molecular Biology 1995;12:345-350; Shore, et al., American Journal of Respiratory and Critical Care Medicine 1995;151:1931-1938). The observed effects in animals with CB provide evidence for the hypothesis that preexisting cardiopulmonary disease predisposes individuals to detrimental biological responses following ambient particle exposure.

These results suggest two distinct mechanistic responses to inhaled particles:  a stress-type pulmonary function response marked by increases in flow and volume (i.e., deeper breathing); and acute pulmonary inflammation marked by cellular influx, particularly neutrophils. From these data, it is concluded that inhaled urban air particles alter pulmonary breathing parameters and increase pulmonary inflammation.

B. Age-Related Responses in Rats to CAPs. Since epidemiological studies have reported that elderly individuals have a higher risk of detrimental responses following exposure to elevated levels of ambient particulate matter, we investigated this finding in a toxicological model.

Aged Fisher rats were exposed for 3 days to CAPs from Boston. The hypothesis tested was that older animals would exhibit more severe pulmonary inflammation and hematological changes following the CAPs exposure when compared to young, normal animals. Aged Fisher rats (> 17 months) and juvenile Fisher rats (4-6 weeks) were maintained in a virus-antigen free facility for 3 months prior to exposure. Following this period, aged and young rats were exposed to CAPs or sham-exposed to filtered air (FA) for 5 hours/day for 3 consecutive days (10 rats/group x 4 groups total = 40 rats). Daily integrated CAPs concentrations were 80, 170, and 50 mµg/m3 on days 1, 2, and 3, respectively.

None of the animals died throughout the duration of exposure. Twenty-four hours following the last day of exposure, blood was collected by cardiac puncture, and bronchoalveolar lavage (BAL) was performed. Young rats had significantly higher total BAL cell counts compared to old rats as well as a significant increase in BAL polymorphonuclear leukocytes (PMN) following CAPs exposure compared to sham.

 

Figure 1. Percent of total
and whole number differential
cell counts from normal and
chronic bronchitic rats exposed
to filtered air or CAPs. a,
significant effect of SO2 exposure
(p < .05); asterisk, significant
effect of CAPs exposure (p < .05).
Two-way ANOVA was employed
in the present analyses. Each value
represents the mean ? SE of at
least eight determinations.

 

 

 

 

 

Figure 2. Total BAL protein from normal and chronic bronchitic rats exposed to filtered air or CAPs. a, significant effect of SO2 exposure (p < .05); asterisk, significant effect of CAPs exposure (p < .05). Two-way ANOVA was employed in the present analyses. Each value represents the mean ? SE of at least eight determinations.

 

Figure 3. Mean of (A) BAL polymorphonuclear leukocyte (PMN), (B) lymphocyte (LYM), and (C) macrophage (MAC) cell counts following 3 days of filtered air or CAPs exposure in young and aged rats. Each value represents the mean ? SE for each treatment group (6-10 animals per group). Asterisk indicates p < .05 versus CAPs; a indicates p < .05 versus age.

 

Old rats also exhibited a small, but significant, increase in BAL PMN following exposure to CAPs compared to sham. There were no CAPs-related significant changes in aged animals in total BAL cell counts, BAL lactate dehydrogenase, total white blood cell (WBC) counts, or the percent of WBC PMN, lymphocytes, and monocytes. When comparing aged versus young (CAPs- or filtered air-exposed) animals, advanced age was associated with significant decrements in the total BAL cell counts, total WBC counts, percent of blood lymphocytes, and blood hemoglobin; a significant increase in the percent of blood PMN was also observed.

The results suggest that:  (1) young Fisher rats may represent a sensitive model for the examination of pulmonary inflammatory responses following CAPs exposure, and (2) the lack of a pulmonary inflammatory response in the aged rats, despite the presence of a higher percentage of circulating neutrophils, may reflect decreased sensitivity to inhaled particles.

C. Hematologic and Inflammatory Cytokine Responses to Inhaled CAPs. In another experiment, mature male rats were exposed to CAPs using the Harvard Ambient Particle Concentrator to determine if hematological responses were affected. Chronic bronchitic rats (CB; 250 ppm SO2 x 5 days/week for 6 weeks) were employed as a model of pulmonary disease. Age-matched, room air-exposed rats were used as a normal group. For urban particle exposures, normal or CB animals were inhalation exposed for 6 hours/day for 3 days to CAPs or filtered air followed by collection of blood for cell analyses as well as assessment of circulating cytokines, bronchoalveolar lavage (BAL), and lung tissue for morphology 24 hours post-final exposure. Hematological markers including total white blood cell counts (WBC) and differentials, red blood cell (RBC) counts, and hemoglobin as well as plasma levels of TNF and MIP-2, were measured. Pulmonary inflammatory markers assessed included BAL total and differential cell counts, BAL lactate dehydrogenase (LDH), and total BAL protein. CAPs exposures were 374.6, 254.2, and 173.3 mµg/m3 on day 1, 2, and 3, respectively (mass median aerodynamic diameter + geometric standard deviation = 0.28 mm + 2.7). Increases in RBC counts, blood monocytes (MONO), and blood eosinophils (EOS) were observed in normal, CAPs-exposed animals whereas CB, CAPs-exposed animals had increased RBC counts, blood MONO, and blood PMN. In this study, BAL parameters showed no significant changes in total cell counts (TCC), BAL lymphocytes (LYM), or BAL LDH. CB, CAPs-exposed animals exhibited increased BAL PMN (p = 0.07) while CAPs exposure in all animals actually caused a significant decrease in total BAL protein. Morphometry of the lung tissue showed significant increases in neutrophils in all groups compared to normal sham-treated animals. No differences in circulating MIP-2 and TNF were detected in any treatment group, but MIP-2 was detected by Northern Blot analyses in BAL cells and in lung tissues.

Figure 4. Mean of peripheral blood percentages of (A) monocytes, (B) PMN, (C) LYM and (D) eosinophils following 3 days of filtered air or CAPs exposure in young and aged rats. Each value represents the mean ? SE for each treatment group (6-10 animals per group). Asterisk indicates p < .05 versus CAPs; a indicates p < .05 versus age.


D. Rat Cardiovascular Dysfunction Prior to Death During Exposure to CAPs. Groups of 12 rats with or without monocrotaline treatment (MT)-induced pulmonary inflammation were exposed by inhalation to either filtered air (FA) or CAPs for 5 hours per day on 3 consecutive days. Continuous electrocardiograms (EKGs) were recorded on three members of each group during exposures. CAPs concentrations were approximately 200, 600, and 150 µg/m3, respectively, on days 1, 2 and 3 of exposure. During the exposure period, one MT, FA-exposed (8.3%) and three MT, CAPs-exposed (25%) animals expired. Continuous EKG was recorded on one of the MT rats which succumbed on the third day of CAPs exposure. Death occurred 90 minutes into exposure and was preceded by an initially steady heart rate (HR) for 15 minutes, followed by a linear reduction in HR from 340 to 290 beats per minute over the next 60 minutes. Concomitant increases in PR interval, QRS duration, HR standard deviation, low frequency HR variability power, and low-to-high frequency ratio were observed. QT interval contemporaneously decreased accompanied by a decrease in T wave height, deepening of the S wave, and emergence of possible U waves. Numerous premature ventricular contractions and other dysrhythmias were noted prior to demise. To our knowledge, this is the first continuous recording of cardiac death apparently induced by inhalation of CAPs. A similar mode of death (bradycardia and dysrhythmia followed by asystole) was reported in MT animals instilled with residual oil fly-ash particles (Watkinson, et al., Toxicological Sciences 41:209). Canines exposed to CAPs in our laboratory have also exhibited HR slowing. Furthermore, analysis of human data obtained from implantable cardiac defibrillators has associated bradycardic events with PM2.5 elevation. Cardiac conduction system dysfunction may be an important mechanism of death associated with fine particle exposure in the MT rat.

E. Pulmonary Inflammation is Associated with the Metal Content of CAPs. Data from six different rat chronic bronchitis/CAPs studies, as described in sections A and C above, were assessed. The relationship of morphometric measurements of pulmonary neutrophils was ranked versus the total metal content of the CAPs exposures. The association is illustrated in Figure 5, below.

 

Figure 5.

 

 

F. Summary. Inhalation of concentrated urban air particles by rats results in modest pulmonary inflammation in normal animals and model populations in most, but not all, experiments. Increases in circulating neutrophils as evidence of systemic inflammation were also variable in these experiments. However, when circulating neutrophils were increased, there was not a measurable increase in circulating cytokines. In studies with rats treated with monocrotaline, an agent with endothelial toxicity, exposure to concentrated urban air particles resulted in the deaths of some animals with electrocardiographic evidence of both sympathetic nervous system enhancement and arrhythmia. The assessment of particle composition in relationship to exposure outcomes requires many repetitions of experiments. Preliminary analyses suggest that the metal content of particles is associated with pulmonary inflammatory responses. Research supported by this grant has contributed to the beginnings of our understanding of the biologic mechanisms by which inhaled ambient particles cause health effects.


Journal Articles on this Report : 4 Displayed | Download in RIS Format

Other project views: All 14 publications 5 publications in selected types All 4 journal articles
Type Citation Project Document Sources
Journal Article Batalha JRF, Saldiva PHN, Clarke RW, Coull BA, Stearns RC, Lawrence J, Krishna Murthy GG, Koutrakis P, Godleski JJ. Concentrated ambient air particles induce vasoconstriction of small pulmonary arteries in rats. Environmental Health Perspectives 2002;110(12):1191-1197. R825242 (Final)
R827353 (Final)
R827353C014 (Final)
R832416 (2008)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Journal Article Clarke RW, Catalano PJ, Koutrakis P, Krishna Murthy GG, Sioutas C, Paulauskis J, Coull B, Ferguson S, Godleski JJ. Urban air particulate inhalation alters pulmonary function and induces pulmonary inflammation in a rodent model of chronic bronchitis. Inhalation Toxicology 1999;11(8):637-656. R825242 (Final)
  • Abstract from PubMed
  • Abstract: Taylor and Francis-Abstract
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  • Journal Article Clarke RW, Catalano P, Coull B, Koutrakis P, Krishna Murthy GG, Rice T, Godleski JJ. Age-related responses in rats to concentrated urban air particles (CAPs). Inhalation Toxicology 2000;12(Suppl 1):73-84. R825242 (Final)
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  • Journal Article Saldiva PHN, Clarke RW, Coull BA, Stearns RC, Lawrence J, Krishna Murthy GG, Diaz E, Koutrakis P, Suh H, Tsuda A, Godleski JJ. Lung inflammation induced by concentrated ambient air particles is related to particle composition. American Journal of Respiratory and Critical Care Medicine 2002;165(12):1610-1617. R825242 (Final)
    R827353 (Final)
    R827353C014 (Final)
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  • Full-text: AJRCCM-Full Text HTML
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  • Abstract: AJRCCM-Abstract
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  • Other: AJRCCM-Full Text PDF
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  • Supplemental Keywords:

    ambient air, health effects, particulates, pathology, northeast, air, atmosphere, animal, toxics, biology, Massachusetts, MA, Region 1, RFA, Health, Scientific Discipline, Air, Geographic Area, particulate matter, air toxics, Epidemiology, State, Disease & Cumulative Effects, Atmospheric Sciences, EPA Region, ambient air quality, health effects, particle size, particulates, urban air, air pollutants, cardiopulmonary responses, human health effects, pathophysiologic mechanisms, exposure and effects, ambient air, cardiovascular vulnerability, exposure, respiratory problems, Massachusetts (MA), biologically plausible mechanisms, atmospheric transport, human exposure, Acute health effects, coronary artery disease, environmental toxicant, epidemiological studies, harmful environmental agents, inhaled, ambient particulates, cardiotoxicity, mortality, atmospheric chemistry, exposure assessment, heart rate , Region 1

    Progress and Final Reports:

    Original Abstract
  • 1997
  • 1998