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
NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997

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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,
NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997

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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.
NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997

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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
NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997

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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.
NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997

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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
NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997
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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)
 NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997
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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
 NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997
                                   11

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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
 NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997
                                    19

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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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