OFFICE OF RESEARCH AND DEVELOPMENT
National Health and Environmental Effects Research Laboratory
CRITERIA AIR POLLUTANTS
PARTICULATE MATTER
HEALTH EFFECTS RESEARCH
PROGRESS REPORT
OCTOBER, 1997
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CONTENTS
Introduction 3
Summary of the Particulate Matter Health Effects Research Program 4
FY96-97 Program Highlights 6
Particulate Matter Problem Characterization Research Program 8
Particulate Matter Dosimetry Research Program 11
Particulate Matter Mechanisms of Toxicity Research Program 14
Particulate Matter Host Susceptibility Factors Research Program 18
Conclusions 21
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INTRODUCTION
PURPOSE
The purpose of this report is to communicate results from the Particulate Matter Research
Program of EPA's National Health and Environmental Effects Research Laboratory
(NHEERL).
CONTENT
The report contains
• a summary of the NHEERL Particulate Matter Research Program, including an
explanation of its regulatory and programmatic context, the overall program goal,
the rationale for the program, and the research strategy;
• a section that highlights recent key findings (FY96-97 Program Highlights); and
• a more detailed description of the NHEERL Particulate Matter Research Program,
by program area, including a summary of recent research accomplishments and
anticipated progress for the near future.
COMMENTS WELCOME
The format of this report is still evolving, and we welcome feedback. Readers with
comments, questions, or requests for further information are encouraged to contact:
John Vandenberg, Assistant Laboratory Director
National Health and Environmental Effects Research Laboratory (MD-51A)
U.S. EPA
Research Triangle Park, N.C. 27711
Phone: (919) 541-4527 or FAX: (919) 541-0642
E-mail: vandenberg.john@epamaii.epa.gov
NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997
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PARTICULATE MATTER
RESEARCH PROGRAM SUMMARY
REGULATORY AND PROGRAMMATIC
CONTEXT
The 1970 Clean Air Act (CAA) authorizes
EPA to establish National Ambient Air Quality
Standards (NAAQS) for criteria air pollutants,
including particuiate matter (PM). The Act
states that the scientific basis for the NAAQS
is to be reviewed periodically and revised as
appropriate to reflect new scientific know-
ledge. EPA's Office of Research and
Development (ORD) provides scientific
support for this process. ORD's Particuiate
Matter Research Program, which is based
upon a peer-reviewed research strategy, is
designed to produce scientifically sound data
to help guide decision-makers in their
considerations of the standard. Four areas of
uncertainty have been identified by ORD and
are the focus of its research efforts: health
effects research, ambient monitoring and
exposure research, source characterization
and management research, and risk
assessment. NHEERL is responsible for
health effects research. This document
summarizes NHEERL's health effects
research on particuiate matter and highlights
some of its recent accomplishments in this
area.
PROGRAM GOAL ,,-
To provide credible PM health effects data
that reduce the uncertainties in risk assess-
ment and thereby support evaluation of the
PM NAAQS.
RATIONALE
Recent epidemiological studies of urban
populations, have indicated that current
exposures to particuiate matter may lead to
increased morbidity from pulmonary disorders
and increased mortality from cardiopulmonary
diseases. Age and pre-existing cardio-
pulmonary disease appear to be important
factors in PM susceptibility. PM research is
needed (e.g., on toxicological mechanisms) to
affirm the biological effects of PM and thereby
improve our understanding of these
epidemiological findings. There are also
uncertainties in the association between
particle size/composition, deposition, and
adverse effects. Research in these areas will
help expand the scientific basis for the
assessment-and ultimate reduction-of the
public health effects caused by exposure to
particuiate matter.
RESEARCH STRATEGY
To ensure that the Agency is equipped with
scientific and technical data relevant to the
formulation of sound environmental policy,
ORD operates a research program founded
on principles of risk assessment. In the area
of health effects, the risk assessment
paradigm of the National Academy of
Sciences (NAS) provides the research
context. The risk paradigm consists of 4
fundamental steps that support risk manage-
ment decisions: hazard identification, dose-
response assessment, exposure assessment,
and risk characterization. NHEERL's
research programs emphasize two of these
steps: hazard identification and dose-
response assessment.
PM research is being conducted by NHEERL
in four critical areas:
PROBLEM CHARACTERIZATION, in
which we are integrating field and clinical
studies to evaluate health effects caused by
PM, and we are developing advanced
molecular-based methods to provide more
definitive assessments of target dose and
damage;
DOSIMETRY (exposure-dose
relationships), in which we are measuring and
modeling particle deposition in the lungs while
taking into account factors such as age,
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gender, and disease status;
MECHANISMS OF TOXICITY, in which
we are determining the role of PM
composition, size, and physical properties in
provoking health effects; and
HOST SUSCEPTIBILITY FACTORS, in
which we are examining various host traits,
health conditions, and physiological changes
responsible for enhancing PM susceptibility in
certain population subgroups.
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NHEERL PARTICULATE MATTER RESEARCH
FY96-97 PROGRAM HIGHLIGHTS
PROBLEM .CHARACTERIZATION (pg 8)
The goal ot this research is to improve our understanding of the epidemiological observations of a
relationship between increased mortality/morbidity and PM exposure.
••In a pilot study of elderly individuals, we found evidence suggesting that cardiovascular-
compromised individuals may be at higher risk than the general population to the effects of
fine PM.
" In collaboration with the Czech Ministry of Health and the Czech Ministry of the
Environment, we conducted a study of the impact of fine paniculate matter from coke oven
emissions on occupational and urban populations in toe Czech Republic.
DOSIMETRY (pg 11)
The goal of this research is to develop animal and human dosimetric models to better understand
the role of particle size and pre-existing conditions on the health effects of PM and to facilitate
animal-to-human extrapolation.
•• A research physicist in our Experimental Toxicology Division has used supercomputers
to develop two- and three-dimensional models that simulate the movement of inhaled
substances through the human lung. These models were considered so innovative that they
won the Smithsonian Institution's 1997 Computerworid Award for Medicine.
* We have shown that individuals with pre-existing respiratory disease receive higher doses
of fine (PMis) panicles relative to healthy individuals. The dose for patients with chronic
obstructive pulmonary disease was as much as 5 times higher than that for healthy
individuals; for asthmatics, the dose was 59% higher; and for smokers, 49% higher.
> Using our newly developed serial bolus aerosol delivery technique, we conducted the first
systematic investigation of regional deposition in human lungs using inert aerosols. We
found that the site of peak fine particle deposition is in the distal region (toward the alveoli).
With increasing particle size, the peak shifted toward the proximal region (mouth).
MECHANISMS OF TOXICITY (pg 14)
The goal of this research is to identify and evaluate plausible biological mechanisms that evoke the
health effects associated with exposures to PM and its components.
» We have found that bioavailable transition metals present on particles play a significant
role in inflammation and lung damage. The toxic potencies differ from metal to metal, as do
their mechanisms of toxicity.
•• In studies of ambient PM collected in the Utah Valley, our Laboratory has demonstrated
for the first time a correlation between a physiological response (production of inflammatory
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mediators) and epidemiology findings (particle-related mortality), with bioavailable transition
metals present on the PM demonstrating a strong correlation with the physiological
response.
• In studies ofneuroreceptors, we have shown for the first time that neurogenic factors are
associated with PM-induced inflammation. Neuroreceptors are triggered by PM to stimulate
production of inflammatory cytokines.
HOST SUSCEPTIBILITY FACTORS (pg 18)
The goal of this research is to evaluate various host traits and health conditions that may potentiate
the effects associated with PM and to describe the pathophysiology of the susceptibility.
•• When we exposed rodents with pre-existing cardiopulmonary disease to PM, they
exhibited an enhanced mortality rate (50%) compared to healthy animals. We showed that
death was related to cardiac dysfunction and that specific biochemical changes associated
with heart disease may trigger the cardiovascular events.
••We demonstrated that PM enhances pulmonary infections by altering host defense
systems, and we are beginning to explain some of the mechanisms involved in the effects
of PM on host defense functions. For example, we found that PM,0 inhibits the antimicrobial
defenses of macrophages.
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PARTICULATE MATTER PROBLEM CHARACTERIZATION
RESEARCH PROGRAM
NHEERL defines problem characterization
research as research to identify and describe
the health and environmental risks posed by
exposure to environmental contaminants.
ISSUE
Do current exposures to PM produce
increased mortality and morbidity? If so, what
are the specific health effects caused by PM
and its components, and are there segments
of the population who are especially
vulnerable to these effects?
Recent assessments of epidemiological data
have shown significant associations between
various measures of ambient paniculate
matter and excess mortality and morbidity,
raising serious concerns that exposure to PM
may impose a heavy burden on human
health. However, uncertainties in the data
(such as limited characterizations of expo-
sure) have clouded our understanding of
these observations. There is also the issue of
susceptibility. Certain population subgroups,
such as children and the elderly, may be more
vulnerable than others to the adverse effects
ofPM.
PROGRAM DESCRIPTION
This research program is helping EPA
understand the relationship between PM
exposures and health effects through more
accurate exposure estimates and improved
assessments of effect. NHEERL has
combined forces with investigators in EPA's
National Exposure Research Laboratory
(NERL), public institutions, and international
governments to implement a two-pronged
research strategy. First, we are combining
innovative field and clinical approaches to
assess physiological changes in small
populations exposed to PM in real-world
settings. Personal exposure monitors and
particle size discrimination are improving our
exposure characterizations. Secondly, we are
developing molecular and biochemical
methods to strengthen epidemiologic assess-
ments. By studying changes at the molecular
and biochemical level, we can provide more
definitive estimates of target dose and
contribute to a better understanding of the
biological mechanisms that evoke the health
effects associated with PM.
PROGRAM PROGRESS
Field study and clinical approaches. The
objective of this research is to couple size-
specific PM data with physiological data in our
evaluations of health effects relative to PM
size. Specifically, we are examining the
correlation between PM25 and acute
pulmonary and cardiovascular effects in a
sensitive subpopulation, the elderly. We are
attempting to determine whether daily
variations in particulate air quality are
associated with daily variations in physio-
logical parameters. During FY97, in
collaboration with NERL and scientists from
the University of North Carolina at Chapel Hill,
we completed a pilot study in which we
investigated a variety of non-invasive
techniques for evaluating the response of
elderly individuals to PM exposure. We
secured an elderly cohort (individuals >65
years of age) exposed to regional sources of
PM and administered a battery of
physiological tests measuring lung function,
heart rate, blood pressure, blood oxygen
saturation, and indicators of immunity
(inflammatory response factors). Additionally,
we collected environmental monitoring metrics
intended to improve exposure assessment;
for example, we measured personal
particulate exposures and daily indoor and
outdoor concentrations of fine and coarse
particles. The results of our pilot study
showed that elderly individuals are capable of
withstanding the demands associated with the
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non-invasive techniques. Preliminary analysis
of our health data indicate that cardio-
vascular-compromised individuals may be at
higher risk than the general population to the
effects of fine particulate matter. Future work
in this area will focus on more clearly defining
methods capable of detecting human health
endpoints associated with fine PM and on
correlating physiological responses with
changing concentrations of PM25 (Contacts:
R. Calderon, R. Williams, Human Studies
Division)
During FY97, we finished constructing and
testing an exposure chamber capable of
concentrating airborne fine particles approxi-
mately 10-fold. We plan to use this particle
concentrator in upcoming human studies to
assess the degree of lung damage and
inflammation associated with ambient urban
air particles. Bronchoalveolar lavage samples
will be collected, and biochemical and
physiological responses will be evaluated.
(Contact: R. Devlin, Human Studies Division)
Molecular and biochemical epidemiologic
methods. In cooperation with scientists in the
Czech Republic, NHEERL instituted a multi-
disciplinary research program in 1992 to
document the relationship between human
health effects and PM exposures in a heavily
industrialized region of Eastern Europe. Our
study is being conducted in the city of
Ostrava, an area of exceptionally high levels
of PM. During FY96, we assessed the
exposure of occupational and urban popula-
tions to fine particulate matter (PM25) and
carcinogenic polycyclic aromatic hydro-
carbons (PAHs) from coke oven pollution
sources. We used personal exposure
monitors to collect exposure data, and we
collected blood samples for the analysis of
biomarkers. The biomarker we are studying
is the formation of PAH-related DMA adducts,
which are structural changes to DNA caused
by the binding of a reactive chemical with the
DNA helix. Quantitation of these adducts
provides a good indication of genetic damage
from exposure to carcinogens and mutagens.
Our results showed that coke oven workers
are exposed to extremely high concentrations
(mg/m3) of PM25, while urban populations are
exposed to lower levels (ug/m3). In addition,
we found that PAH-related DNA adducts
increase with increasing exposure to PM at
low to moderate concentration levels. At high
levels of exposure, however, the formation of
adducts becomes nonlinear. This non-
linearity in dose-response has been observed
not only in humans, but in experimental
animals as well. The mechanism for the
nonlinearity is uncertain and is under
investigation by our Laboratory because this
observation has important implications for
dose-response extrapolation in risk
assessment. (Contacts: R. Williams, J.
Lewtas, Human Studies Division)
A study in the district of Xuan Wei, China, of
a population with high rates of lung cancer
(due to exposure to coal combustion
emissions) is providing us with a unique
opportunity for application and evaluation of
biomarkers. Studies are underway in our
Human Studies Division to develop methods
for measuring protein over-expression of the
p53 tumor suppressor gene in sputum-
exfoliated airway epithelial cells and lung
tissues. It is anticipated that this biomarker
can be used for assessing individual lung
cancer risk. As a supplement to this study,
we are isolating DNA from lung tumors
collected from these individuals and
attempting to identify the mutations that occur
along the p53 gene using PCR (polymerase
chain reaction) techniques. This research
should help us explain some of the
mechanisms involved in the development of
PM-related lung cancer. (Contacts: J.
Mumford, Human Studies Division; D.
DeMarini, Environmental Carcinogenesis
Division)
During FY97, we devised a comprehensive,
multidisciplinary approach to address
questions concerning the relationship
between ambient PM exposures, internal
dose (as measured in lung tissue and
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surrogate respiratory tract cells and fluids),
and particle-induced cell injury. Through
scanning electron microscopy analysis, we
are determining particle size and chemistry
(inorganic and carbonaceous) for a wide
variety of air pollution sources. We are then
using cell assays to identify the extent to
which these parameters are responsible for
particle-induced damage. In light of recent
evidence suggesting that particle-bound
PAHs enhance production of human IgE (a
factor that may increase airway allergic
disease), we also are examining the role of
particle-bound organics in modulating cell
loxicity. As a complement to these in vitro
studies, we have obtained human lung tissue
from autopsy victims and are evaluating
possible linkages between chronic PM and
ozone exposure, internal particle dose and
chemistry, and histopathological changes in
the lung. In addition, we are exposing rodents
to particles markedly different in size and
organic content to assess the differential
effects of particle burden, chemistry, and size
on inflammation and tumorigenic response.
(Contact: J. Gallagher, Human Studies
Division)
In a collaboration with NERL, we are
providing data to the National Human
Exposure Assessment Survey (NHEXAS),
whose over-arching goal is to assess the
every-day exposure of American citizens to
potentially hazardous substances. During
FY96, we analyzed respirable PM collected as
part of the survey and determined that it
contained detectable quantities of PAHs,
some of which were human carcinogens.
Exposure varied with respect to location
(urban, suburban, rural), ambient
concentration, and chemical specie.
Generally speaking, individuals living in urban
environments had greater exposures to fine
PM and PM-related carcinogenic PAHs.
(Contact: J. Lewtas, Human Studies Division)
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PARTICULATE MATTER DOSIMETRY
RESEARCH PROGRAM
NHEERL defines dosimetry research as
research that elucidates the relationship
between exposure and dose at the site of
toxicity.
ISSUE
What is the relationship between PM
exposure, dose, and effects, especially with
respect to sensitive subpopulations?
Substantial uncertainties exist regarding the
relationship between PM exposure, dose, and
observed effects. One reason for this
uncertainty is the difference that exists in air
flow in the lungs (differences found, for
example, in children vs. the elderly or in
healthy individuals vs. those with lung
disease). Differences in air flow affect the
pattern of particle deposition in the lung,
which, in turn, affects dose. Understanding
deposition patterns and the retention/
clearance of particles is critical for assessing
the potential risks of PM. Mathematical or
computational models, derived from both
human and experimental animal data, help
explain dose distribution. Of particular
interest are dosimetric models for susceptible
subpopulations.
PROGRAM DESCRIPTION
In an effort to improve our understanding of
the exposure-dose-effect relationships
associated with PM, we are conducting
research on particle deposition in the human
lung. Through the combined use of human
studies, computer modeling, and physical
models, we are relating ambient PM
concentrations to delivered dose. Particles
differing both in size and in size distribution
are being tested. A critical component of our
research is the effect of factors such as age,
gender, and pulmonary disease on the
behavior and fate of particles inhaled and
retained by the lung. This research is
enabling us to describe the distribution and
clearance kinetics of PM in the human
respiratory tract, resulting in more realistic
estimates of dose distribution and reduced
uncertainty in risk assessment.
PROGRAM PROGRESS
Human studies. During FY96, scientists in
our Human Studies Division collaborated with
researchers at the University of North
Carolina at Chapel Hill to examine the
regional deposition of coarse (PM4 5) and fine
(PM25) particles in children. Regional deposi-
tion is important because particles are not
deposited uniformly throughout the lung, and
sites receiving higher doses are more likely to
become triggering points for adverse
biological events. Our results suggest that
children have enhanced upper airway
deposition of coarse particles relative to
adults; the increase is correlated with
decreasing height. In light of recent
epidemiotogical studies showing increased
morbidity in children resulting from airborne
particulate pollution, these findings suggest
that if morbidity is due to upper respiratory
problems, enhanced upper airway deposition
may play a role. We also found that the rate
of deposition of fine particles-normalized to
lung surface area-was greater in children
than adults. (Contacts: W. Bennett, K.
Zeman, Human Studies Division)
During FY96-97, we conducted an
investigation of the deposition of particles in
the lungs of individuals with pre-existing
respiratory disease, including asthmatics,
patients with chronic obstructive pulmonary
disease (COPD), and smokers. Our objective
was to examine differences in particle
deposition in diseased vs. healthy lungs.
Working with researchers at the University of
North Carolina at Chapel Hill, we measured
total lung deposition in individuals with varying
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levels of airway obstruction and found a good
correlation between lung deposition and
spirometric lung function tests. We showed
that COPD patients receive a dose of fine
particles (PM, „) as much as five times higher
than the dose received by healthy subjects.
For asthmatics, fine particle deposition was
increased by 59% over healthy individuals; for
smokers with small airway disease, the
increase was 49%. Our data further
suggested that the increase in deposition was
associated with the obstructed airways.
These dosimetric findings are relevant to
recent epidemiologic data indicating greater
increases in morbidity and mortality in
asthmatics and patients with COPD.
Enhanced dose likely contributes to the
severity of the disease. (Contacts: W.
Bennett, C. Kim, Human Studies Division)
In order to obtain more detailed quantitative
data for inhaled particles, scientists in our
Human Studies Division have developed a
new method for assessing regional deposition
in the human lung. It is called the serial bolus
aerosol delivery technique. This method does
not require radioactive aerosols, and there are
no limitations to the number of lung regions
that may be assessed. Initial studies using
this technique were conducted during FY95-
96, representing the first systematic investiga-
tion ever conducted to determine regional
deposition in humans in situ with inert
aerosols. Our results showed that particle
deposition within the healthy human lung is
highly uneven. The site of peak fine particle
deposition is in the distal region (toward the
alveoli). With increase in particle size (PM30.
so), the site of peak deposition shifts from the
distal to the proximal region (toward the
mouth). These findings suggest that, for
coarse particles, early tissue damage may
occur in the proximal airways. This technique
has the potential for wide application in
toxicological studies of aerosols and in the
fields of biomedical and health sciences
research. (Contact: C. Kim, Human Studies
Division)
In FY97, we used this novel aerosol delivery
technique to study gender differences in
particle deposition. We found a significant
difference in dose distribution in the lungs of
males and females: particle deposition is
skewed toward the mouth in females
compared to males. This enhancement of
deposition becomes more pronounced with
coarse particles (PM?0.50). These results
support our findings in children, discussed
above, which showed that height plays a key
role in deposition patterns. They also indicate
the importance of dose distribution in
interpreting potential gender differences in PM
effects. (Contact: C. Kim, Human Studies
Division)
Computerized models. Using computer
modeling that relies on the speed and power
of supercomputers, a research physicist in our
Experimental Toxicology Division has
developed two sophisticated computer
simulations of the human lung. These models
allow us to visualize the movement of inhaled
substances through the lungs in two- and
three-dimensional views. One model displays
the 20 million airways of the human lung; the
other depicts the paths of particles as they
flow through and are deposited in the lung.
The models are the culmination of many years
of research and are so innovative that they
won the Smithsonian Institution's 1997
Computerworld Award for Medicine. They are
assisting predictions of exposure-dose
relationships under a variety of conditions,
such as obstructed airways and uneven
ventilation. Potential uses for the models
include research related to pulmonary
diseases (e.g., cancer, asthma, and Cystic
fibrosis) and research on the effects of air
pollution on children. Other possible applica-
tions are in tracking the movement of
aerosolized drugs to ensure delivery to
diseased areas of the lung. Future research
will emphasize the refinement of the models
and the development of algorithms for 3-D
morphological descriptions and fluid dynamics
patterns of respiratory systems. (Contact: T.
Martonen, Experimental Toxicology Division)
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Airway models. We have developed
branching airway models constructed of glass
tubes that permit us to analyze dose
distribution under realistic breathing condi-
tions. Studies conducted by our Laboratory
during FY96 showed that particle deposition in
the bronchial airways is highly localized.
Surface features, such as rings and ridges,
have profound effects on localized PM
deposition. We also determined that
deposition dose is greater with polydispersed
aerosols relative to monodispersed aerosols.
(Polydispersed aerosols contain particles of
varying sizes and are more representative of
real-world exposures than monodispersed
aerosols, which are composed of particles of
uniform size.) These results are providing
new insights into the dynamics of poly-
dispersed aerosols and are helping us
improve our dose estimates. We are now in
the process of using these data to develop an
empirical mathematical formula for estimating
deposition dose in the bronchial airways.
(Contact: C. Kim, Human Studies Division)
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PARTICULATE MATTER MECHANISMS OF TOXICITY
RESEARCH PROGRAM
NHEERL defines mechanisms of toxicity
research as research to identify and
characterize the physical, chemical and/or
biological mechanisms whereby an
environmental agent may induce health or
environmental effects.
ISSUE
What are the causal mechanisms that can
provide a biologic explanation for the
epidemiologic observations of excess
mortality and morbidity due to PM?
Despite human evidence linking low-level PM
exposures with adverse health effects, there
are uncertainties about the biological
mechanisms responsible for the observed
toxicity. Understanding causal mechanisms
would help explain recent findings that relate
PM toxicity to differences in particle size and
composition.
PROGRAM DESCRIPTION -:. . .
Our research on the mechanisms of PM
toxicity is aimed at determining the effect of
particle size and composition on PM toxicity.
In vitro tests that utilize cultured lung cells
(epithelial cells and alveolar macrophages)
are being developed and used to evaluate
PM-induced injury at the cellular level. These
cell cultures tend to approximate in vivo
responses, and they have the added
advantage of allowing us to study damage to
specific cell types. Our approach is to expose
the cells to a wide range of PM in order to test
specific hypotheses regarding the relationship
between toxicity and particle size/composition.
To complement our in vitro work, we are
conducting in vivo studies to investigate the
role of particles in lung injury and inflam-
mation. This involves exposing experimental
animals to PM with differing physicochemical
properties, and then measuring various
physiological and biochemical indicators of
damage, such as lung inflammation and
oxidant formation.
PROGRAM PROGRESS
Collectively, our data demonstrate that metal
content and bioavailability-as well as particle
size, acidity, and sulfate content-are
important physicochemical properties that
influence PM toxicity.
In vitro studies. Scientists in our Human
Studies Division and Experimental Toxicology
Division have hypothesized that bioavailable
transition metals present on particles (e.g.,
iron, vanadium, and copper) can contribute to
inflammation and lung damage. During FY96-
97, we tested this hypothesis at the cellular
level. We exposed cultured lung cells to a
variety of metals and to residual oil fly ash
(ROFA), which is paniculate matter with a
high metal content emitted by power plants.
When exposed to ROFA, the cells responded
by increasing the expression of inflammatory
cytokine genes and by producing
inflammatory mediators, such as IL-6, IL-8,
TNF, and PGE2. When the cells were
exposed to pure transition metals, we
discovered that different metals have different
potencies. For example, vanadium is more
reactive than zinc or nickel. We then began
to examine some of the molecular
mechanisms by which metals exert their
effects on lung cells and found that metals are
toxic by different means. We found, for
example, that vanadium inhibits tyrosine
phosphate activity, resulting in elevated levels
of phosphorylated proteins. This, in turn,
activates nuclear transcription factor NF-KB,
which is related to the inflammatory response.
Iron, on the other hand, stimulates the cellular
production of lactoferritin and ferritin, which
internalize iron inside the cell and render it
inactive. However, iron complexed with
substances such as humic acid, which is
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present on some fine particles, is not
internalized and remains active. We will
continue to examine the involvement of
metals in PM toxicity by studying the
molecular mechanisms responsible for PM-
induced effects. (Contacts: P. Devlin, S.
Becker, A. Ohio, Human Studies Division; J.
Dye, K. Dreher, Experimental Toxicology
Division)
We have used our in vitro methods in several
studies of ambient PM toxicity. During FY96
we showed that PM collected from the Salt
Lake City airshed stimulates the production of
inflammatory cytokines in human lung cells
and that copper ts the most active component
of these particles. (Contacts: R. Devlin, A.
Ohio. C. Kennedy, Human Studies Division)
In a collaborative effort with the National
Institute of Environmental Health Sciences
(NIEHS) and the National Cancer Institute of
Mexico, we examined the toxicity of ambient
PM collected from various locations in Mexico
City. The most toxic samples were from the
industrialized northern region. We showed
that these particles also contained the highest
amount of sulfate and metals. (Contact: K.
Dreher, Experimental Toxicology Division)
Finally, in a study of ambient PM collected
over a three-year period in the Utah Valley,
we found that cultured lung cells produced
significantly more inflammatory mediators
when exposed to PM collected during a time
when a major polluting industry was in full
operation compared to a time when the
industry was on strike. When we compared
these results to epidemiology findings
reported for the same period, we were the first
to demonstrate an association between a
physiological response and epidemiology
results (reduced particle-related mortality for
the year the industry was on strike). We are
currently instilling extracts of these particles
into the lungs of animals and human
volunteers to characterize in vivo response.
Preliminary evidence suggests that the in vivo
toxicity of the Utah Valley samples is
correlated with bioavailable metal content.
(Contacts: R. Devlin, A. Ghio, Human Studies
Division; D. Costa, Experimental Toxicology
Division)
In another project, we are studying the role of
alveolar macrophages in the clearance of
particles and infectious microbes from the
lung. While most of our experiments are
being performed with alveolar macrophages
exposed in vitro, we also plan to obtain
macrophages from volunteers experimentally
exposed to PM,,, in our chambers on the
campus of the University of North Carolina at
Chapel Hill. The primary purpose of these
studies is to understand how PM interferes
with macrophages, which help prevent the
development of infections in the lungs. There
is interest in this area because it has been
shown that high particle pollution levels are
associated with increased incidence of
pneumonia. The project was initiated in 1995
with the identification of particle-induced cell
toxicity and cytokine production in macro-
phages. In FY96, we reported that PM10
modulates macrophage surface receptor
expression and phagocytosis. During FY97,
we found that PM10 can inhibit the anti-
microbial defenses of macrophages. These
studies are beginning to explain some of the
mechanisms involved in the effects of PM1Q on
host defense functions. Our future plans are
to examine the effects of PM on macrophage
antiviral functions, such as virus-induced
production of inflammatory mediators.
(Contact: S. Becker, Human Studies Division)
During FY95, we formulated an artificial lung
lining fluid to simulate fluids in the human
lung. During FY96-97, we used this in vitro
model to investigate a number of mechanisms
involved in PM toxicity. We were especially
interested in the oxidative effects exerted by
PM. We found that particle acidity affects
oxidative reactions: high acidity appears to
increase the oxidative reactions induced by
PM. In tests of the protective effects of
Vitamin C and other antioxidants, we showed
that Vitamin C inhibits effects caused by low
levels of PM, but that the protective effect
disappears at high PM exposures. (Contact:
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G. Hatch, Experimental Toxicology Division)
In a new area of research, we are focusing on
defining the role of neuroreceptors in the
initial events of PM inflammation. Recent
data (FY96-97) produced by our Laboratory
suggest that neuroreceptors, which are
located on the sensory fibers that innervate
the airways, are triggered by PM and
stimulate inflammatory cytokines in bronchial
epithelial cells. Our data are the first to
associate neurogenic factors with PM
inflammation. Based on these data, neuro-
immunological interactions appear to be a
potential mechanism of PM toxicity. We
propose that certain types of PM can activate
capsaicin receptors, located on sensory fibers
and target cells, which in turn causes the
release of neuropeptides that trigger the
cellular events of inflammation. Currently, we
are conducting studies in rodents to
determine whether in vivo results can support
our findings. In the future, we will examine
the role of neuroimmunological interactions in
other airway target cells, such as alveolar
macrophages. (Contacts: B. Veronesi,
Neurotoxicology Division; S. Becker, R.
Devlin, Human Studies Division)
In vivo studies. During FY97, through a
collaboration with ORD's National Risk
Management Research Laboratory (NRMRL),
we exposed rodents by intratracheal
instillation to PM from a variety of sources.
Our samples included stationary combustion
emissions (ROFA and coal fly ash), a
spectrum of inert dusts (such as Mt. St.
Helens ash), and ambient air from urban
locations (e.g., Washington, DC, and St.
Louis. MO). These samples exhibit different
metal and sulfate contents, enabling us to
obtain information on the association between
particle composition and health effects. The
health endpoints we are examining include
acute pulmonary responses, such as
inflammation and airway hyper-reactivity, and
chronic responses associated with pulmonary
fibrosis.
We have found that particle surface chemistry
plays a key role in the pulmonary toxicity of
PM. The most toxic combustion PM samples
are fine (PM^) acidic particles with a high
content of sulfate and bioavailable transition
metals. Metals are largely, if not entirely,
responsible for the initial acute inflammatory
response to PM, and specific metal composi-
tion is very important in determining the
severity of lung injury. For example, we found
that a significantly greater degree of lung
damage is induced by fly ash containing high
levels of zinc, and nickel poses a greater risk
of lung injury than vanadium or iron. Although
these in vivo findings do not seem to
corroborate our in vitro results using cultured
cells (discussed above), which showed
vanadium to be more reactive than nickel or
zinc, the contrast in response is related to
differential cell sensitivity. In vivo, nickel
causes more of a fibrotic than inflammatory
response, which is probably related to effects
on alveolar macrophages rather than
epithelial cells. Epithelial cells, which
measure inflammatory response, indicate
vanadium is more reactive than nickel. Thus,
vanadium seems to drive the inflammatory
component of pulmonary response. While
reinforcing the hypothesis that the
composition of soluble metals and sulfate is
critical to the development of airway hyper-
reactivity and lung injury, these results also
suggest that differential cell sensitivities may
be critical to the overall injury caused by PM.
Our findings emphasize that in vitro studies
must be conducted using "relevant" cell types
in order to approximate in vivo responses.
We are currently testing nickel in vitro with
alveolar macrophages to see whether we can
mimic the in vivo (fibrotic) effects with this cell
type. (Contacts: S. Gavett, U. Kodavanti, K.
Dreher, Experimental Toxicology Division; A.
Ghio, Human Studies Division)
We also fractionated ambient air PM from
Washington, DC, into a coarse (PM37.20) and
two fine (PM<17 and PM17.37) fractions and
found that all size fractions induced the same
modest increase in airway hyper-reactivity.
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However, PM0 7 induced the greatest degree
of injury in terms of pulmonary inflammation,
edema, and hemorrhage. Furthermore, we
found that the chemistry of ambient air PM
changes with particle size. For example, fine
ambient particles (PM^.y) are chemically
similar to combustion emissions (i.e., they are
acidic and have a high content of sulfate and
bioavailable metals). (Contact: K. Dreher,
Experimental Toxicology Division).
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PARTICULATE MATTER HOST SUSCEPTIBILITY FACTORS
RESEARCH PROGRAM
NHEERL defines host susceptibility factors
research as research to characterize traits
and pre-existing health conditions that may
potentiate the health effects caused by an
environmental contaminant.
ISSUE
What are the factors responsible for the
enhanced susceptibility to PM exhibited by
certain segments of the population?
Certain subpopulations, including individuals
with pre-existing disease or weakened
physical condition, appear to be more
vulnerable to the effects of PM and may be
predisposed to unusual response, injury, or
death when exposed to PM. For example,
there is evidence of an association between
pre-existing cardiopulmonary disease and
excess mortality due to PM. Because risk
assessment strategies must consider the
spectrum of population responses, it is
important to understand variability in
susceptibility to PM.
PROGRAM DESCRIPTION
The issue of PM susceptibility is a recurrent
theme throughout this document (for example,
the elderly are being studied under
PROBLEM CHARACTERIZATION, and
DOSIMETRY research is being conducted on
children and asthmatics). The objectives of
the HOST SUSCEPTIBILITY FACTORS
research program are to study various traits or
health conditions that make an individual
more susceptible to PM and determine how
these pre-existing conditions potentiate the
toxicity of PM. To accomplish these
objectives, we are developing animal models
of cardiopulmonary disease, asthma, and
infectious disease to represent sensitive
human subpopulations. These models then
enable us to study the pathophysiology of PM
susceptibility. Another facet of our research
involves the study of animals and humans to
help explain genetic predisposition to the
effects of PM.
PROGRAM PROGRESS
Cardiopulmonary disease. Using a variety
of rat models of human cardiopulmonary
disease, we have begun to explore
susceptibility to PM by examining effects on
cardiopulmonary function, pulmonary
pathology, and lung damage. In our
pulmonary hypertension model, PM exposure
resulted in more severe inflammation, greater
lung impairment, and higher mortality when
responses were compared to normal animals.
The mortality rate (50%) was of special
interest and prompted us to investigate its
cause. We used radiotelemetry to examine
cardiac function, and during FY96 we found
that both the number and severity of cardiac
arrhythmias were significantly increased
following ROFA exposure in hypertensive
animals compared to controls. These animals
also exhibited gas uptake abnormalities,
possibly causing a transient hypoxic condition,
and fibrinogen levels and leukocyte counts in
the blood were elevated, both of which are
risk factors for ischemic heart disease. These
physiological changes may constitute
mechanisms whereby cardiopulmonary
events are triggered in susceptible individuals.
Collectively, these findings (effects of ROFA
on lethality, arrhythmia, and biochemical risk
factors) strongly support epidemiology studies
that show an association between PM and
cardiovascular morbidity and mortality
(Contacts: U. Kodavanti, P. Watkinson,
Experimental Toxicology Division).
Asthma. Our Experimental Toxicology
Division has developed two asthma models in
rodents. These models are permitting us to
examine the effects of co-exposure to
allergens and PM. The mouse model, whose
development began in 1995, incorporates key
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features of the human asthmatic condition,
including elevated levels of serum IgE,
pulmonary inflammation highlighted by
increased eosinophil numbers, and, most
importantly, airway hyper-reactivity.
To create the model, we sensitized normal
mice with an allergen and then challenged the
mice with an aerosol of the foreign protein.
During FY96, we used our "asthmatic" mouse
to study the impact of PM exposure on
physiological and inflammatory responses. A
great advantage of our model is that it allows
us to investigate mechanisms through widely
available immunological and genetic tools.
Initial studies were performed using several
different particle types, including ROFA and
ambient air PM samples from St. Louis, MO,
and Washington, DC. Following trachea!
instillation of the particles, we studied the time
course of responses and assessed possible
mechanisms for the observed responses.
Mechanisms were examined in detail by
measuring levels of allergy-related cytokines
in- bronchoalveolar lavage fluid and then
relating these levels to inflammatory and
physiological responses, such as airway
hyper-reactivity.
During FY96, we found that ROFA particles
increase airway reactivity and that there is a
synergistic interaction between allergen and
ROFA in elevating eosinophil numbers. Upon
further investigation of this phenomenon in
FY97, we found that ROFA increases levels
of the allergy-related cytokine lnterieukin-4,
and that the increase is associated with
enhanced pulmonary response. However,
IgE antibodies do not increase. In an
important discovery in FY97, we found that
airway hyper-reactivity following ROFA
exposure may be divided into early (1 day)
and very late (8 day) phases that are
mediated by different mechanisms. In the
future, we will study PM-modulated
mechanisms of allergy by utilizing various
gene knock-out and transgenic mice. Study
of these mutant mice will give us the
opportunity to examine the role of specific
gene products in the pathogenesis of PM
effects. (Contact: S. Gavett, Experimental
Toxicology Division)
In our other allergy model, house dust mite
allergen was used to create an asthmatic rat.
Using this model in FY96-97, we showed that
ROFA treatment enhances the development
of immune responses during sensitization,
and it augments antigen-induced inflammation
during the challenge period. (Contact: I.
Gilmour, Experimental Toxicology Division)
Infectious disease. Other NHEERL studies
are examining the ability of particles to
enhance pulmonary infections. These studies
demonstrate that particulate matter enhances
infectivity by altering host defense systems.
For example, studies conducted by our
Laboratory prior to FY96 showed that several
combustion and ambient air PM samples
enhanced susceptibility to pulmonary
streptococcal infection in mice. In rats, we
showed that dual exposure to ROFA and the
influenza virus produces more severe
respiratory symptoms and lung damage than
either stimulus alone. And in FY97, in
collaboration with investigators at the New
Jersey Medical School, we demonstrated that
ROFA exposure compromises one of the
lung's innate immune responses: it inhibits the
production of antimicrobial peptides.
(Contacts: M. Belgrade, G. Hatch, I. Gilmour,
L Ryan, Experimental Toxicology Division)
Genetic susceptibility. Another avenue of
research is genetic susceptibility. In animal
studies conducted during FY96, we demon-
strated genetic variability in rats with respect
to ROFA-induced pulmonary inflammation,
airway hyper-reactivity, and pulmonary
fibrosis. Sprague-Dawley rats were shown to
be more sensitive than Wistar rats, which
were more sensitive than Fischer rats.
(Contacts: U. Kodavanti, K. Dreher, D. Costa,
Experimental Toxicology Division)
Differences in human response to some
pollutants have been attributed to genetic
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differences between individuals, including
differences in the way in which contaminants
are metabolized. For example, humans have
multiple enzyme-mediated pathways for the
detoxification or activation of DNA-reactive
chemicals. The inter-individual variation in
metabolic enzyme activities is likely to be
associated with variations in susceptibility to
pollutants. As part of the China lung cancer
project (discussed on page 9), we are
evaluating genetic differences in the GSTM1
gene, which is the gene coding for the
enzyme important in the detoxification of
carcinogens present in PM, Determination of
GSTM1 in a lung cancer case-control study is
in progress. The results should help us
understand the causal mechanisms
associated with differences in susceptibility to
PM-related lung cancer (Contact: J. Mumford,
Human Studies Division).
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CONCLUSIONS
As demonstrated clearly in this Progress Report, a variety of methods and models, are being
developed by NHEERL scientists in human, animal, and in vitro systems to significantly improve and
expand the scientific basis for future evaluation of the PM NAAQS. It is quite unlikely that any
research institution in the world is more focused than NHEERL in understanding the biological
effects of PM exposures on human health. NHEERL researchers benefit substantially from
expertise committed to PM research by NERL and NRMRL, with ORD's National Center for
Environmental Assessment poised to incorporate, in the year 2000, the latest research findings in
the next PM Criteria Document. We anticipate that critically important results of the research
conducted by the laboratories of the Office of Research and Development will be available for
inclusion in the next Criteria Document. These results, in combination with extramural studies
conducted through the EPA Science to Achieve Results (STAR) grants program and by other
institutions, will be used to inform risk management decisions with economic consequences
measured in the billions of dollars.
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