Project 1:
Relative Toxicity of Air Pollution Mixtures
Principal Investigator: John Godleski
Co-Principal Investigator: Petros Koutrakis
ABSTRACT – Project 1
a. EPA-RC2009-STAR-C1
b. Title: Relative Toxicity of Air Pollution Mixtures
c. Investigators: PI: John Godleski, Co-PI: Petros Koutrakis
d. Institution: Harvard School of Public Health, Boston, MA
e. Project Period and Location: 2010-2015, Boston MA
f. Project Cost: $2 Million
g. Project Summary:
1) Objectives and hypotheses: Project 1, an inhalation toxicological animal exposure study, will
investigate the relative toxicity of different component concentration combinations of air
pollution mixtures. These components include both particles and gases that are emitted directly
from sources (primary species) or are formed in the atmosphere through a series of reactions that
are predominantly photochemical (secondary species) Using a novel integration of our ambient
particle concentrator and photochemical chamber technologies to generate realistic mixtures, we
will test these specific hypotheses: (i) secondary gaseous pollutants formed from the
photochemical oxidation of Boston ambient gases can induce biological responses; (ii) aging
Boston concentrated ambient particles (CAPs) in the photochemical chamber enhances their
toxicity; (iii) toxicological effects of photochemically aged CAPs are exacerbated by coexposure to ozone and other secondary gases; and (iv) mixture composition and toxicity exhibit
inter- and intra-seasonal variability due to changes in source emissions and weather conditions.
2) Experimental approach: Toxicity will be assessed in Sprague-Dawley rats by changes in 1) in
vivo oxidant response, 2) blood pressure, 3) measures of inflammation, and 4) vascular blood
flow/resistance. Three concurrent exposures groups (Sham, Control Exposure and Exposure) will
allow us to control for the variability in CAPs composition. With this design, there can always be
a direct comparison between two exposure mixtures on every exposure day, making it possible to
determine which mixture is more toxic. In studies of vascular blood flow/resistance using a
crossover design, each animal will have multiple exposures that will include each of the three
types, including baseline measurements. This will permit for control of inter-subject variability
in the biological response. Exposure atmospheres will be chemically and physically
characterized using a broad array of measurement techniques for CO, NOx, O3, PM, BC, particle
count and size distribution, EC/OC, elemental composition, sulfate, formaldehyde, acetaldehyde
and VOCs. For the biological effects observed during each exposure, inter-group differences will
be assessed using multi-way analysis of variance. To determine the effect of PM composition on
biological response, linear regression models containing exposure concentrations as predictors
will be fitted to each response outcome measure. Multiple pollutant linear regressions will be
used to assess the independent effects of multiple pollution components on biological response.
3) Expected Results: The proposed study will differentiate the health effects of components of
multi-pollutant exposure mixtures. We expect to add to our understanding of the exposureresponse relationship, the interaction between particulate matter and photochemical gases, and
the extent to which the resultant products exert toxicity. The biological outcomes assessed in this
Project focus on responses important in oxidant initiation of pulmonary inflammation, and
important functional measures of vascular and cardiovascular health.
Key words: Air pollution, concentrated ambient particles, vascular blood flow and resistance,
inflammation, blood pressure, reactive oxygen species, oxidant response,
atmospheric photochemistry
D- 2
1. OBJECTIVES/HYPOTHESES:
Components of air pollution mixtures include both particles and gases that are emitted directly
from sources (primary species) or are formed in the atmosphere through a series of reactions that
are predominantly photochemical (secondary species).1-3 The overarching hypothesis of Project
1, and the Center as a whole, is that the toxicity of air pollution mixtures can be related to
specific component concentrations, but some combinations of components in mixtures are more
toxic than others. Testing this overarching hypothesis is a challenging task that will require the
collective efforts of many research groups. Here we propose to advance current understanding by
examining how the addition or subtraction of individual or multiple components influences
mixture toxicity. In particular we focus on toxic effects defined by changes in blood pressure, in
vivo oxidant response, inflammatory parameters, and vascular blood flow/resistance. Using novel
approaches to generate exposures of realistic pollutant mixtures, we will test four specific
hypotheses:
1) Secondary gaseous pollutants formed from the photochemical oxidation of Boston ambient
gases can induce biological responses. These gaseous species include O3, which is the most
abundant atmospheric oxidant, as well as a spectrum of reactive species including nitrogen
compounds, peroxides, organic radicals, polar organic compounds, and others. We further
postulate that such biological effects are not solely O3-related, but can also be induced by the
concomitant reactive gaseous species;
2) Aging of Boston Concentrated Ambient Particles (CAPs) in the photochemical chamber
enhances their toxicity. Many of our Center studies have reported toxic effects of Boston CAPs.416
Now we postulate that further photochemical aging of CAPs renders them more toxic by
altering their organic components and adding secondary PM. Secondary PM includes many
reactive species including mono- and di-carboxylic acids, aldehydes and other polar organic
compounds, sulfuric acid, nitrates, and organic radicals. Photochemical oxidation may alter the
organic component of CAPs, forming various polar derivatives more reactive than their parent
compounds, as well as organic radicals and peroxides, which may enhance CAPs toxicity;
3) Toxicological effects of photochemically aged CAPs are exacerbated by co-exposure to ozone
and other secondary gases. Mixtures of secondary gases, secondary PM, and aged CAPs may
elicit increased toxic effects. More complex exposure mixtures will produce greater responses.
And;
4) Both mixture composition and toxicity exhibit inter- and intra-seasonal variability due to
changes in source emissions and weather conditions. Some variability of mixture toxicity may
be explained in part by changes in mixture composition.
Objectives:
1) Test each of the 4 specific hypotheses by generating different types of multi-pollutant mixtures:
We will combine two exposure-generation technologies developed by our ongoing and previous
Centers including: i) the Harvard Fine Particle Concentrator (HFPC) that is used to generate
CAPs for inhalation studies;17-19 and ii) the Toxicological Evaluation of Realistic Emission
Source Aerosol (TERESA) exposure system that includes a photochemical chamber to simulate
atmospheric aging of source emissions.20-22 For this study, we will use the TERESA
photochemical chamber on the output of the HFPC to form secondary particles and gases from
ambient air and to age the CAPs. Central to this effort is the use of a parallel-plate membrane
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22
denuder to remove gaseous species from the generated pollutant mixtures.
This device will
make it possible to examine the effects of particles and gases combined and separately.
2) Expose an animal model to well characterized air pollution mixtures: Our exposure plan
features three concurrent types of exposures (Sham, Control Exposure and Exposure) in a design
that allows us to control for the variability in CAPs composition and for inter-subject variability
in biological response. For example, to test the effects of aging CAPs we will perform three
concurrent exposures: filtered air sham (Sham); CAPs (Control Exposure); and aged CAPs
(Exposure). Using the effect of aging can be examined because we can control the inherent
variability of CAPs toxicity.
3) Characterize the physico-chemical properties of exposures: Particle and gas exposures will be
characterized using continuous measurements of PM, size distribution, BC, Particle Number,
CO, NO, NOx and O3, along with integrated measurement of PM, sulfate, trace elements,
EC/OC, water soluble OC, formaldehyde and acetaldehyde.21,23
4) Assess biological effects: We will test the biological hypothesis that exposures to these air
pollution mixtures will lead to varying degrees of increased in vivo oxidant response in the lungs
that induce systemic inflammation and cardiovascular responses including increased blood
pressure and changes in vascular reactivity. We will employ well-established assessments we
have used with Sprague-Dawley rats in our CAPs and TERESA studies which will allow us to: i)
measure in vivo oxygen radical activity in the heart and lung;4-6 ii) assess markers of pulmonary
and systemic inflammation;7-10 and iii) monitor blood pressure.15 In addition, we will use a novel
assessment of vascular reactivity using fluorescent microspheres to measure vascular flow and
resistance in a panel of vital organs.16, 24, 25 And;
5) Perform statistical analysis: We will estimate associations between exposure and biologic
response parameters using multi-way ANOVA techniques, univariate analyses and, longitudinal
methods. These analyses will allow us to test our hypotheses regarding the toxicity of different
multi-pollutant mixtures and their components. Our analysis will focus on identifying the
characteristics of these mixtures that are mostly responsible for their toxic effects including:
individual components, combinations of components, formation processes or source types.26
1.2 Background and Introductory Information
1.2.1 Northeast air pollution mixture: As discussed in the Exposure Core, most of the air pollution
in the Boston area comes from sources located outside Massachusetts. These include large urban
centers, power plants, industries and biogenic sources most of which are located in the
continental USA.27 Pollution is transported to the Boston area by prevailing southern,
southwestern, western and northwestern winds. The regional component can often account for a
large fraction of ambient pollution locally, but emission from local sources such as
traffic-derived pollution, off-road mobile source emissions, seasonal space heating (distillate oil,
gas, wood), marine aerosol, seasonal biogenic, and local industrial emissions also
contribute.1,3,27,28 Because air pollution is such a complex mixture, with respect to its origin,
composition, and physical properties, it is a long-term challenge to assess what components,
properties, or sources are responsible for air pollution impacts on human health.
1.2.2 Linking health outcomes with air pollution: Laboratory inhalation studies have a critical role
because of their ability to generate specific exposures and directly measure health outcomes. A
significant advance in air pollution toxicology has been to study the effects of exposure to real
ambient particulate pollution. We developed the HFPC that is used to conduct inhalation
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toxicological assessments of responses to particulate matter (PM). The HFPC permits us to
increase the concentrations of northeast urban ambient fine particle mass by a factor of 30-40 for
our studies.17-19 Concentrator studies from our Center in Boston and other research groups worldwide have substantially contributed to the PM health effects field.
Our concentrator studies rely upon sensitive laboratory assessments to define acute responses to
ambient particulates. In vivo oxidant measurements have been used in our CAPs and TERESA
studies measuring oxygen radical activity in the heart and lung.4-6,29,30 We7-10 and many others
(e.g., 31-33) have shown that CAPs increases pulmonary and systemic inflammation. Increases in
15
blood pressure have been shown with Boston CAPs as well as CAPs in other cities in studies of
34-36
our Center.
A novel assessment of vascular reactivity in the heart using fluorescent
microspheres to measure vascular flow and resistance has been developed in our laboratory16, 24, 25
and will be used in this project.
There has been intense interest recently in the effects of exposures to emissions from specific
sources. To date toxicological approaches have focused on exposures to emissions from
combustion sources, such as cigarette smoke,37-43 diesel exhaust,44-49 gasoline vehicles,50-52 and
wood burning smoke.53-56 All these studies have used only primary emissions. Because
particulate and gaseous source emissions undergo many transformations once released into the
atmosphere, it is likely that secondary and primary pollutants from individual sources will
exhibit different toxicities. Thus, it is critical to investigate the relative toxicity of source-specific
primary and secondary particles and gases.
Another important recent advance in exposure studies has been the development of systems to
simulate atmospheric chemical reactions and expose animals to particles resulting from aged
source emission. Our Toxicological Evaluation of Realistic Emission Source Aerosol (TERESA)
studies have investigated the toxicity of primary and secondary emissions from mobile sources
(funded by our ongoing EPA PM Center) and power plants (funded by EPRI and the previous
Center). In our TERESA studies we have developed technologies to sample source emissions,
form secondary particles inside a photochemical chamber, and expose animals to both primary
and secondary particles. The TERESA approach can readily be adapted to investigate other
combustion sources, and the effects of mixtures of primary and photochemically aged gases and
particles. Here, we propose to combine ambient particle concentrator and TERESA exposure
technologies to explore how atmospheric chemical reactions may modify ambient particles.
1.2.3 Toxicity of photochemically aged gases and particles: Recent research has shown that O3 and
other photochemical gases may play an important role in the toxicological response to ambient
pollution. Sexton et al.57-59 have shown that secondary gases produced from photochemical
oxidation of typical ambient VOCs (or hazardous air pollutants such as 1,3-butadiene) can
enhance the toxicity of the parent compounds beyond the effects of comparable concentrations of
O3 and NOx alone. Studies of human respiratory epithelial cells showed that the effects of a
photochemically oxidized mixture persisted even after the O3 was removed by titration with NO,
suggesting contributions from other components.58 The effects exceeded those of a synthetic
mixture of the identified products (including acrolein, acetaldehyde, formaldehyde, furan, and
O3) at the same concentration.57 However, because the photochemical aging in these studies was
performed in the absence of PM, the interactions of such gases and particles in exposure and
toxicity remain to be determined. It is also important to determine the relevance of these in vitro
studies to inhalation exposure in appropriate in vivo subjects, as we propose here.
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1.2.4 PM and O3 interaction in biological outcomes: Understanding the interactions of particles
and gases (specifically O3) in generating biological responses remains the subject of much
interest. Many studies have tested the combined effects of O3 and PM (reviewed in 60). Many
important gaps remain, but laboratory exposure studies have shown evidence of synergy between
PM and O3 in humans and animals.34-36,61-63 However, CAPs plus O3 does not have greater
effects on some cardiovascular functions such as heart rate variability compared to CAPs alone.64
The focus of this project is to not only explore the simple combination of CAPs and O3, but to
assess the interactions between CAPs, O3, and the resulting products of photochemistry on
measures of toxicity.
1.2.5 Ties to other Projects and Cores: Our studies will strongly complement the Center’s 3
proposed Cohort Studies (Projects 2, 3, and 4), which focus on cardiovascular and central
nervous system outcomes. A potential mechanism for pollution-related decreases in cognitive
and neurobehavioral functions is decreased vascular perfusion. In this project, we will directly
measure vascular perfusion and vascular resistance in the hearts and brains of animals using
microspheres, a powerful approach for assessing vascular flow. Microspheres distribute to all
organs permitting flow measurements in any vascular bed in relationship to exposure or other
parameters.15,24,25 Also, both our animal toxicology study and the three Cohort Studies will
assess pollution-related pulmonary and systemic inflammation. Our toxicology study will thus
help us to understand the biological mechanisms underlying associations observed within the
Cohorts between outcomes and the exposure mixtures.
1.3. Progress Report and Preliminary Results
1.3.1 TERESA Exposure Studies
1.3.1.1 Coal power plants: Our TERESA technologies have been used successfully in field studies
at three coal fired power plants with distinctly different emissions control, resulting in distinctly
different SO2 and NO concentrations. During these studies, we: 1) optimized stack sampling
methods to prevent condensation and PM loss from the hot and humid emissions; 2) developed a
photochemical chamber to age primary emissions for animal exposures;20,21 3) developed and
validated denuder technology to remove excess gaseous components from the exposure
atmosphere;22 and 4) developed a state-of-the-art mobile toxicology laboratory. During these
field investigations, we tested several exposure scenarios including: primary PM alone (P);
primary PM plus secondary acid sulfate (PO); primary PM plus secondary neutralized sulfate
(PON); primary PM plus secondary organic aerosol and acid sulfate (POS) and; primary PM plus
secondary organic aerosol and neutralized sulfate (PONS); secondary acidic sulfate only with no
primary PM (O) and; secondary organic aerosol with secondary acidic sulfate and no primary
PM (OS). We exposed male Sprague-Dawley rats to these mixtures, and measured biological
responses including continuously monitored pulmonary function, post-exposure measurement of
pulmonary and cardiac oxidative stress, pulmonary inflammation and injury, and blood
chemistry and cytology. To further assess cardiovascular effects, we repeated the most complex
exposure scenarios in a rat model of myocardial infarction65 and monitored cardiac function
using implanted telemeters. Overall, we found that the TERESA aerosols from power plant
emissions were less toxic than CAPs.30 These findings are summarized in Table 1. We found that
the most significant responses occurred with the more complex oxidized scenarios studied,
supporting our Hypothesis 2, that photochemically aged particles are more toxic than primary
particles.
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Table 1: Summary of the statistically significant biological outcomes from the coal power plant TERESA studies.
Scenario
Summary of Statistically Significant Biological Outcomes
PO
Only effect was a breathing pattern change to rapid shallow breathing
POS
Increases in in vivo chemiluminescence of the heart, abrogation of the change in breathing pattern
with PO; Increases in total cell count and macrophage number on broncho-alveolar lavage and;
Increases in premature ventricular heart beats in the myocardial infarction model
PONS
Decreases in peak expiratory air flow and expiratory flow at 50%;
Increases in in vivo chemiluminescence of the lung and; Increases in total cell count,
polymorphonuclear leukocyte number, and macrophage number on broncho-alveolar lavage
1.3.1.2 Mobile Source Emissions: We have successfully adapted the TERESA approach to
investigate vehicular emission sources in a laboratory pilot study and in a northeastern highway
tunnel. Our adaptation of the TERESA approach for mobile source emissions is described in
Section 4 of the Engineering Core. During the laboratory development study for vehicular
emissions, we optimized our technique for formation of secondary particle mass from the dilute
tailpipe emissions of a single car. Because there was very little PM in the car exhaust, we used
an inert seed aerosol particle for the secondary PM to condense on. We then conducted pilot
exposures using normal male Sprague-Dawley rats, and measured pulmonary and cardiac
oxidative stress using in vivo chemiluminescence as well as bronchoalveolar lavage to assess
pulmonary inflammation. For these exposures, a sample of Mount St Helen’s Ash (MSHA) was
used as the seed aerosol, because in our experience it is toxicologically inert.66 Two scenarios
were tested, diluted car exhaust with MSHA (Primary PM), and diluted car exhaust with MSHA
irradiated with UV light to produce secondary PM. The exposure results are summarized below
in Table 2:
Table 2: Pilot exposure study with car exhaust diluted to 5 ppm CO (at 50 min residence time).
Scenario
Seed PM
(μg/m3)
PM
(μg/m3)
EC/OC
(μg/m3)
Primary
PM
298*
298
N/A
Secondar
y PM
142*
212
0.5/6.9
BIOLOGICAL RESPONSES
In Vivo
Chemiluminescence
Total BAL Cells
(x106)
Total BAL PMNs
(x105)
No heart or lung
difference from control
Lung: 2.3±1.3
Significant lung ↑↑↑
from control and
Primary PM
15.7±6.0**
No difference
from control
0.4±0.8
Significant ↑↑
from control and
Primary PM
4.9±1.1**
No difference
from control
0.0±0.1
Significant ↑ from
control and
Primary PM
3.7±1.1**
*MSHA: Mt St Helens Ash (from the Bates Ridge site), a toxicologically and chemically inert particle
** Significant outcome differences from primary PM, p<0.05
These pilot exposures were small scale, using small numbers of exposed and sham rats (n=8), but
show that secondary PM from car exhaust can significantly increase oxidant radicals in the lung,
increase total BAL cells, and increase BAL polymorphonuclear leukocytes (PMNs) over sham
controls (p=0.005, p=0.001, and p=0.05, respectively). Most importantly, there were significant
differences between primary and secondary PM for all of these outcomes (p<0.05) even with
slightly less mass in the secondary PM exposure, thus further supporting our specific hypotheses.
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1.3.2.3 TERESA approach with ambient gases and particles: When the TERESA technology is
integrated with the ambient fine particle concentrator, we will use ambient air rather than source
emissions in the photochemical chamber. We performed feasibility testing with Boston ambient
air to confirm that the concentration of oxidizable gaseous species present in ambient air is still
sufficient to produce O3 and secondary PM when photochemically aged in our reaction chamber.
Results from a typical day (evolution of the particle size distribution and accumulation of O3 –
see figures below) support the validity of this approach.
Evolution of ozone from photochemical aging of ambient air
Residence Time = 100min
Evolution of secondary PM from photochemical aging of ambient air
Residence Time = 100min
200
0 min
180
30 min
12000
Ozone Generation (ppb)
Particle Number Concentration (#/cc)
14000
60 min
120 min
10000
180 min
240min
8000
300 min
6000
4000
160
140
120
100
80
60
40
20
2000
0
0
10
100
1000
0
30
60
90
120
150
180
210
240
270
300
330
360
Elapsed Irradiation Time (min)
Mobility Diameter (nm)
1.3.3 Microsphere studies in dogs: Microspheres have been used as measures of organ vascular
perfusion in numerous studies of both large animals16,24,25,67 and rodents.68-71 Our laboratory has
pioneered the use of this approach to study cardiac blood flow and coronary vascular resistance
in relationship to CAPs exposures. Our results show that this method is sensitive enough to
detect changes in response to single 5-hr exposures. We found that acute exposure to CAPs
decreases myocardial blood flow during subsequent coronary artery occlusion in chronically
instrumented dogs. The decrease in myocardial blood flow was most pronounced in tissue within
or near the ischemic zone and was accompanied by increases in coronary vascular resistance.16
This finding has important, direct relevance to the potential influence of pollutant exposure on
the size, severity, and chances of survival of an acute myocardial infarction. Our observation that
CAPs exposure increases coronary vascular resistance during ischemia suggests an important
role for PM-induced coronary vasoconstriction. This is consistent with the findings by Brook et
al.72 that a 2-hr exposure to CAPs and O3 in healthy volunteers decreased brachial artery
diameter, but not endothelium-dependent vascular reactivity. Also, we have previously shown by
histology that increased vasoconstriction occurs acutely in pulmonary vessels of CAPs-exposed
rats11 and that chronic ambient exposure of mice results in both pulmonary and coronary
vasoconstriction.73 Thus, we now propose to further test the hypothesis that PM-induced
alterations in vascular structures is associated with decreased blood flow and increased vascular
resistance in the heart and potentially in the vasculature of all major organs including the brain.
1.3.4 Microspheres in rats: As presented above, we have considerable experience with
microsphere methodology in the dog,16,24 but we needed to adapt the surgical techniques for
chronic rat experiments. In a number of preliminary studies, we have been able to chronically
implant catheters in the left ventricle and thoracic aorta for perfusion and sampling, respectively,
and connect these to two subcutaneous vascular access ports implanted subcutaneously in the
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posterior
intrascapular
area. Data Sciences blood
pressure telemeters will
be
implanted
▼
▼
abdominally. We have
▼
found that rats with
abdominally implanted
telemeters routinely live
for a year or more
without any adverse
effect of the telemeter.
▼
During this time, the
telemeter will remain
operational if it is off
A
B
between
experiments.
After implantation of the
vascular access ports, the A. Picture at surgery of cathers implanted in the heart ▼ and aorta▼ and
rats fully recover within attached to vascular access ports ▼. B. After tunneling the catheters
through the chest wall and subcutaneously to the intrascapular area, the
two weeks. The cardiac picture shows the skin partially closed over the vascular access ports ▼.
port is used to inject
microspheres. The aortic port is used to obtain a blood sample while the microspheres are
injected into the heart. Both the injection and the sample are done with a calibrated pump so that
both the injection rate and the sample rate are constant. We have routinely used 7 different colors
obtained from IMT Stason laboratories. Microspheres can be injected at weekly intervals to
measure vascular perfusion and vascular resistance in every vascular bed of the animal. The
blood pressure measurement is used to calculate vascular resistance.
2. APPROACH/ACTIVITIES
2.1 Overall study design: The figure below presents our exposure generation plan. We will first
produce CAPs using our HFPC and introduce them into our photochemical chamber to simulate
the reactions that occur naturally in the atmosphere. These reactions will result in the formation
of O3 and other photochemical gases, secondary PM, and photochemically aged CAPs.
P ro je c t 1 : E x p e rim e n ta l C o n c e p tu a l S c h e m a tic
A m b ie n t A ir
P o rt 1
L o c a l S o u r c e E m is s io n s a n d R e g io n a l P o llu ta n ts
F in e
P a r t ic le
C o n c e n tra to r
CAPs
F ilte r
P o rt 2
P h o to c h e m ic a l
C ham ber
Y e s /N o
P o rt 3
S ham E xp osure
D enuder
Y e s /N o
F ilte r
Y e s /N o
P o rt 4 -C E
D enuder
Y e s /N o
A d d O zon e
Y e s /N o
P o rt 4 -E
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C o n tr o l E x p o s u r e
E xp osure
Secondary gases can be removed or reduced using our non-selective denuder, or alternatively
they can remain components of the exposure mixture. Likewise, the photochemically aged CAPs
and secondary PM can also be removed, leaving only the secondary gases. The exposure
scenarios that we will use to test our hypotheses are detailed below in Section 2.4.3.
Our exposure plan, described in detail in section 2.4.4 below, features three concurrent exposures
(e.g., Exposure Mixture, Control Exposure Mixture, Filtered Air Sham). We will use a crossover
design that allows us to control for: 1) variability in CAPs composition and concentration; and 2)
inter-subject variability in biological response. Subsequently, these exposures will be repeated
with co-exposure to controlled concentrations of O3 and other photochemical gases. Because
northeastern aerosol has significant differences in seasonal composition, a full crossover study
will be done during winter and summer to test each of the study hypotheses. Animals used in the
crossover studies will have implanted hardware for: 1) telemetric monitoring of blood pressure
and heart rate; and 2) injection of fluorescent microspheres for vascular perfusion assessment. In
addition, normal rats will be exposed during each experiment for measurement of in vivo
chemiluminescence and other acute biological outcomes.
2.2 Exposure system: We will integrate our HFPC with the exposure technology developed for
the TERESA studies. The TERESA exposure technology is a complex system designed to
sample source emissions, simulate atmospheric photochemistry, generate an exposure, expose
laboratory animals, characterize the exposure, and determine the biological outcomes of
exposure. Key elements of this exposure system include the photochemical reaction simulation
system, and the parallel plate membrane denuder, detailed in the Engineering Core, Section 3.2.
2.3 Planned Exposures: The HFPC and photochemical chamber will be interfaced to produce
exposures, consisting of the mixtures of components described below and summarized in Table 3
below. The scenarios are designed to represent four components of ambient air pollution
(ambient PM, ambient gases, secondary PM (SPM), secondary gases (SG) including O3)
separately and in combination. Comparisons of biological outcomes among these exposure
scenarios will enable us to ascertain the relative contribution of each component to the toxicity of
ambient air pollution. To facilitate these comparisons, we have developed the following novel
exposure plan that will control for the inherent variability of CAPs composition. Our exposure
plan features three concurrent exposures, including two exposed groups and a filtered air
exposure sham group. The responses are thus always directly comparable for two scenarios
(mixtures) and sham control for every experiment. This means, for example, that the response to
photochemically aged CAPs is assessed concurrently with the response to CAPs alone.
Table 3: Exposure Study Plan for CAPs/TERESA Project
Year/Seasons
Exposure Mixture
Control Exposure Mixture
Sham/Exposure
Year 1 Winter/Summer
CAPs + SPM* (no Gases)
CAPs Only (no gases or SPM)
Filtered clean air
Year 2 Winter/Summer
CAPs + SPM+ SG**
CAPs + SPM (no gases)
Filtered clean air
Year 3 Winter/Summer
CAPs + SPM+ SG
SG Only (no CAPs or SPM)
Filtered clean air
Year 4 Summer
CAPs + SPM+ O3
CAPs + SPM (no gases)
Filtered clean air
*SPM=Secondary Particles (including photochemical aging of CAPs); **SG=Secondary Gases
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In general, significant variability in ambient pollution concentrations and composition occur due
to the contributions of sources and weather conditions. It is expected that there will be day-today variability in composition and concentration, as we have observed in many of our CAPs
studies. We also expect seasonal variability as discussed previously (biogenic emissions, space
heating, regional scale photochemistry, etc.). Because of these seasonal differences in source
contributions, each of the scenarios will be conducted during warm and cold seasons (Table 3).
Based on our previous concentrator studies and preliminary studies using secondary PM formed
from photochemical oxidation of ambient air, we expect that CAPs concentrations will be 300350 μg/m3 on average. We estimate that photochemically aged CAPs + SPM will add an average
of 40 μg/m3 to the CAPs concentration, varying according to the concentrations of VOCs and
NOx in ambient air. Likewise, the amount of O3 formed will vary depending on the VOCs and
NOx concentrations in the ambient air. In our preliminary studies with ambient air, we found O3
concentrations of 120-350 ppb downstream of the photochemical chamber. Because the effects of
O3 at high concentrations have already been studied extensively, we will dilute the chamber
output, if necessary, to keep the O3 exposure concentration to < 120 ppb. The series of exposures
described above will enable us to test our 4 specific hypotheses (Section 1) by comparing
responses as indicated below in Table 4.
Table 4: Comparisons to address specific hypotheses (presented above in Section 1)
Specific Hypothesis
Comparison(s) to Evaluate Hypothesis
1
SG* and O3 from oxidation of AG** have toxic effects
Year 3, Control Exposure Mixture vs. Sham
2.a
CAPs have toxic effects
Year 1, Control Exposure Mixture vs. Sham
2.b
SPM*** has toxic effects
Year 1, Exposure Mixture vs. Sham
Year 2, Control Exposure Mixture vs. Sham
2.c
CAPs toxicity is enhanced by photochemical oxidation
Year 1, Exposure vs. Control Exposure Mixture
3.a
SPM effects are exacerbated by O3
Year 4, Exposure vs. Control Exposure Mixture
3.b
SPM effects are exacerbated by SG including O3
Year 2, Exposure vs. Control Exposure Mixture
3.c
SG/O3 exacerbate effects of SPM more than O3 only
Year 2 Exposure vs. Control Exposure Mixture
compared to Year 4 Exposure vs. Control Exposure
3.d
Effects: SPM/SG/O3 > SPM/O3 > SG ≥ SPM > CAPs
All Exposures, Longitudinal Analysis
4.a
SPM effects differ in summer vs winter
Year 1, Exposure Mixture Summer vs. Winter
Year 2, Control Exposure Summer vs. Winter
4.b
SPM toxicity depends on composition of CAPs and AG
All Summer and Winter Exposures
*SG=Secondary Gases;
** AG=Ambient Gases;
***SPM=Secondary Particles (includes photochemically aged CAPs)
Although our hypotheses follow a step-wise logical plan of increasing complexity, our strategic
plan takes these hypotheses out of turn for several reasons. First, we need to establish the relative
toxicity of CAPs and SPM in order to effectively compare scenarios that may generate greater or
lesser responses. Secondly, we can apply the results of some studies to more than one hypothesis.
For example, we will test Hypothesis 3 (toxicological effects of photochemically aged CAPs are
exacerbated by co-exposure to O3 and other secondary gases) during Year 2 by comparing the
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Exposure Mixture (photochemically aged CAPs with secondary gases) to the Control Exposure
Mixture (photochemically aged CAPs with secondary gases removed). We will test Subhypothesis 3a, that effects of photochemically aged CAPs may be exacerbated by O3, during
Year 4, by comparing the Exposure Mixture (photochemically aged CAPs with the secondary
gases removed and O3 added) with the Control Exposure Mixture (photochemically aged CAPs
with the gases removed). Sub-hypothesis 3b, that secondary gases including O3 will increase the
effects of exposure to photochemically aged CAPs more than will O3 without the other
secondary gases, will be tested by comparing responses in Year 2 to those in Year 4. We will
evaluate the extension of hypothesis 3, that mixtures containing greater numbers of oxidized
components (i.e., more complex mixtures) are more toxic than those containing fewer oxidized
components. Within the successive years covering this relatively short span, a given summer or
winter should be comparable to other summers of the study, respectively.
2.4 Animal exposures design: Our planned design, including Exposure, Control-Exposure, and
Sham-Exposure will enable us to control for the inherent variability in CAPs concentration and
composition. During the two seasons tested (summer, winter) we will use a crossover exposure
design for blood pressure and vascular reactivity outcomes. Repeated exposures of individual
animals will allow us to control for variability between subjects, as each animal will be included
at least once in the Exposed, Control Exposed, or Sham groups. The crossover design for each
exposure (Year 1: summer, winter, etc.) is summarized below in Table 5. Three groups of 6 rats,
each implanted with Telemeters and hardware for injecting microspheres and sampling cardiac
output (Section 2.4.6) will be exposed in the rotating pattern indicated (Table 5). Each group will
have a total of 5 exposures. This will result in 15 Exposures, 15 Control Exposures, and 10 Sham
exposures per group, and animal exposure day totals of 45 Exposure, 45 Control Exposure, and
30 Sham per study.
Table 5: Treatment Plan: Implanted rats for crossover study and normal rats for acute exposure
Group
1
2
3
CL
Rat
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
1
2
3
4
A
Day
1
S
CE
E
S
CE
E
Day
2
Day
3
Day
4
E
S
CE
E
S
CE
S
CE
E
S
CE
E
E
E
CE
S
E
CE
CE
S
Microsphere Color
C
B
Day
5
Day
6
Day
7
CE
E
S
CE
E
S
E
S
CE
E
S
CE
S
CE
E
S
CE
E
E
CE
S
S
E
E
CE
S
E
CE
CE
S
Day
8
Day
9
D
Day
10
S
CE
E
S
CE
E
CE
E
S
CE
E
S
E
S
CE
E
S
CE
E
CE
S
S
E
E
CE
S
E
CE
CE
S
Day
11
E
Day
12
Day
13
E
S
CE
E
S
CE
S
CE
E
S
CE
E
CE
E
S
CE
E
S
E
CE
S
S
E
E
CE
S
E
CE
CE
S
Day
15
E
S
CE
E
S
CE
S
CE
E
S
CE
E
E
CE
S
S
E
E
CE
S
E: Exposure Mixture; CE: Control Exposure Mixture; S: Filtered Air Sham Exposure
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Day
14
E
CE
CE
S
E
S
CE
E
S
CE
E
CE
S
S
Only 5 microsphere colors (A, B, C, D, and E) are shown in Table 5. Not included in Table 5 are
the initial and final baseline color determinations which are needed in order to internally control
all flow measurements. Therefore, a total of 7 colors will be used in each animal. In addition, to
those animals in the crossover studies, 4 acute normal rats will be exposed each day, and used in
the studies of acute outcomes listed below in Table 6, including in vivo oxidant response and
measures of inflammation. All animals will be exposed in individual whole body
plethysmograph chambers.
2.5 Toxicological Assessments: Toxicological assessments to be performed are summarized in
Table 6 for the exposure studies listed in Table 4. Methods are detailed in sections 2.4.5.1-7.
Table 6: Toxicology plan – Normal Rats (each scenario from Table 3)
Exposure
Type
One Day
(Acute)
All
Exposures
Crossover
Studies
(Chronic)
Evaluation
Method
Effect Assessed
Specific Parameters
Organ
Chemiluminescence
Pulmonary, Cardiac
Oxidative Stress
Blood Chemistry and
Blood Cytology
Systemic Markers
of Inflammation
Histopathology and
Morphometry
Heart, Lung,
Brain, &
Other Organs
Non-invasive
Plethysmography
BUXCO
Pulmonary Function
In vivo whole organ chemiluminescence of
heart and lung
Total while cell counts, differential
profiles, platelets, cbc, cytokines
(interlekin-6), atrial naturetic peptide,
vasoactive mediator endothelin-1
3 Randomly selected slices of fixed heart,
lung, brain, and other tissue assessed for
inflammation,
injury, and vasoconstriction
Peak inspiratory flow, peak expiratory
flow, tidal volume, minute volume,
respiratory frequency, end inspiratory
pause, end expiratory pause, derived
parameter PenH
Microspheres
Vascular blood
Flow/Resistance
Blood Pressure
Telemetry (DSI)
Blood Pressure and
Cardiac Function
Measureme
nt Period
Immediately
post exposure
Immediately
post exposure
Immediately
post in vivo
chemiluminescence
Continuously
during exposure
Microsphere quantification in organs,
vascular perfusion rates; with BP vascular
resistance
Post exposure
injections
Systolic and diastolic pressure;
Heart rate, heart rate variability
Continuously
during exposure
and injections
2.5.1 Pulmonary function testing: We will measure breathing parameters on 6 animals from each
group each day to assess acute pulmonary irritant responses in calibrated whole body
plethysmography chambers with an automated software system (Buxco Electronics, Sharon,
CT). After 10 min of animal acclimatization, respiratory parameters will be measured
continuously throughout exposure. Measurements will be calculated from flow changes in
pressure transducers connected to the plethysmographs. During exposures, breathing patterns are
continuously measured and analyzed breath-by-breath to derive breathing rate, tidal volume,
minute ventilation, as well as inspiration and expiration times, peak inspiratory and expiratory
flows, and end inspiratory and expiratory pauses. These parameters are used to estimate the
parameters Penh (Penh = te/RT –1; where RT is the relaxation time derived from the expiratory
cycle), Inspiratory Ventilatory Drive (IVD = TV/ti), and Inspiratory Duty Cycle (IDC = ti/ti+te),
measures indicative of bronchoconstriction and respiratory effort. Changes in respiratory
parameters have provided important evidence of pollution-induced health effects.23,26,74
2.5.2 Blood pressure and pulse rate: These will be monitored with our DSI telemetry system.
Physiotel PA-C40 transmitters will be surgically implanted in the abdomen of the rat at least 2
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weeks before thoracic hardware implantation. The pressure transmission catheter will be secured
in the iliac/femoral artery. Systolic, diastolic, mean blood pressure, heart rate, and heart rate
variability (HRV) will be obtained from the continuous arterial pressure signal.15,75 Increases in
blood pressure and changes in heart rate and HRV indicate significant cardiovascular health
effects.13 Rats with these implanted devices will be used in multiple exposure experiments in a
crossover design with at least one week between exposures for any animal.
2.5.3 Regional blood flow and vascular resistance in multiple organ systems: Regional blood flow
and vascular resistance will be evaluated with a fluorescent microsphere technique.16,67,69 Briefly,
polystyrene microspheres containing one of seven distinct fluorescent dyes are injected into the
left ventricle of the heart. Injected microspheres become lodged within a given capillary bed.
Because the number of occluded capillaries is small, local vascular resistance is not altered.
Regional blood flow is proportional to the number of microspheres trapped in the region of
interest.67 A reference blood sample withdrawn at a known rate allows calculation of regional
blood flow in ml/min. A chronic indwelling injection catheter will be positioned in the left
ventricle, and a chronic indwelling withdrawal catheter will be place in the aorta at the time of
surgery. Both are connected to subcutaneous vascular access ports as shown in section 1.2.3. For
injections, microspheres (15 µm, IMT Stason Labs, CA) will be diluted to 1x105 in 0.5 ml of
saline solution and injected over a 30-second period. Up to 100,000 microspheres can be used
per injection in rats. Reference blood samples will be withdrawn using a dual-piston syringe
pump at a constant rate of 0.3 ml per 30 seconds through the aortic vascular access port. After all
experiments are completed, the rats will be sacrificed and the organs excised and sectioned. We
will dissect the brain into its major anatomic areas and separately analyze the cerebral cortex,
midbrain and thalamus, cerebellum, and medulla and brain stem. The heart will be dissected into
the left ventricle, right ventricle, and septum. The kidneys, adrenals, a lobe of the liver, the
spleen, pancreas, and right lower lobe of the lung will be collected. All tissues will be sent to
IMT Stason laboratories for analyses. All other tissues from the animal will be saved in 10%
formaldehyde. Each sample will be weighed (W in mg) and digested in a strong base.
Microspheres are recovered and quantified. Regional blood flow (Q in ml/min/g) will be
calculated from fluorescence measurements of each tissue sample (flsample) and the reference
blood samples (flref). Since arterial pressure will be monitored throughout the procedure, we will
also be able to calculate regional resistance (flow/mean arterial pressure) as we have
reported.16,24,25
2.5.4 In vivo chemiluminescence: Spontaneous chemiluminescence of the lung and heart surface is
a sensitive measure of reactive oxygen species (ROS) in tissues. We will monitor ROS using a
photon counter with a photomultiplier responsive in the range 300-900 nm.4-6 Rats will be
anesthetized (Nembutal 50 mg/kg body weight), connected to an animal ventilator, and the chest
opened to expose the lungs and heart. Measurements will be performed within 10-20 min in a
light-tight chamber kept at 32-36°C. We will shield the heart or lung using aluminum foil while
measuring the other organ in order to record both measurements in the same animal. Emission
will be expressed as counts per second per unit of lung or heart surface (cps/cm2) as we have
previously published.4-6,76,77
2.5.5 Blood collection: One day prior to exposure, 0.2-0.5 ml of blood will be drawn under sterile
conditions from the tail vein of each animal. Serum and plasma will be prepared, stored at -20°C,
and used for comparisons with post-exposure levels of circulating mediators (e.g., endothelin and
IL-6). Immediately after 6 hr exposure and completing the chemiluminescence measurements, 3
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ml blood will be collected by cardiac puncture, and a portion used for complete blood count and
peripheral blood cell differential. Half of the remaining sample will be saved as plasma and half
as serum for analyses of circulating mediators. After blood collection, the animals will be given
an overdose of pentobarbital to harvest tissues for morphological studies.
2.5.6 Mediator analyses in blood: Blood Endothelin-1, atrial naturetic peptide (ANP), and IL-6
will be analyzed. All standard peptides, antibodies, and assay reagents will be obtained from a
commercial radio immunoassay kit for rat ANP (Peninsula). For endothelin-1 and plasma IL-6
assays we will use commercial kits (rat endothelin-1 ELISA kit, Assay Designs; Quantikine rat
IL-6 ELISA, R&D Systems).
2.5.7 Histopathologic and morphometric assessments: Morphometric assessments will quantify
vasoconstriction of vessels in heart and lung tissues. The trachea will be tied off and the lung
removed. The lungs will be fixed by intratracheal fixation in situ at a constant pressure of 20 cm
H2O with 2% glutaraldehyde in 0.085 M cacodylate buffer, 0.05% CaCl2, pH 7.4. The lungs and
heart will be removed and stored in fixative at 4oC. Morphometry will be carried out as described
in our previous studies.9
2.6 Characterization of exposures: We will use a variety of continuous and integrated methods to
characterize the exposures generated. Many of these methods are described in detail in the
Engineering Core, and are briefly listed here. Particle and gas exposures will be characterized
using continuous measurements of PM (TEOM), size distribution (APS, SMPS), BC
(Aethalometer), particle count (CPC), CO, NO, NOx and O3, along with integrated measurement
of PM (gravimetric), sulfate (Ion Chromatography), trace elements (X-Ray Fluorescence),
EC/OC (Thermal/Optical Reflectance), water soluble OC78 and formaldehyde and acetaldehyde
(HPLC). Sampling will be conducted at four locations indicated on the schematic diagram
(Section 2.4.1): 1) concentrator inlet (ambient air); 2) concentrator outlet/photochemical chamber
inlet; 3) outlet of the photochemical chamber (aged emissions) and; 4) at the point of exposure.
The first two locations will be used to monitor the effectiveness of the HFPC, looking
specifically at the enrichment of PM2.5 mass to verify that the concentrator performance. The
second and third locations will provide information on the effectiveness of the photochemical
chamber, specifically by monitoring changes in gas and particle concentrations and size
distribution. The fourth sampling location will be used for exposure characterization, with the
sampling location and conditions as comparable to the exposure conditions as possible.
2.7 Statistical methods and data analysis: The exposure experiments use a partial factorial design.
Outcomes of interest on each animal will include chemiluminescence, histological outcomes
assessing inflamation, pulmonary breathing rate, volumes, and flow measures, and blood
parameters. In these studies, subjects are randomized to receive the Exposure Mixture, Control
Exposure Mixture, or Sham Exposure either within a straightforward factorial structure or a more
complicated cross-over design. To analyze these data, we will conduct multiple analyses that use
exposure metrics of increasing sensitivity. First, we begin by using (potentially multi-way)
ANOVA techniques that treat exposure as a treatment. That is, we will assess overall differences
between Exposure, Control Exposure, or Sham Exposure responses (i.e., a binary exposure
covariate). Second, to assess univariate associations between mixture mass and component levels
and health, we will conduct single-pollutant analyses in which a separate regression model is
fitted using biologic response as the dependent variable and either PM mass, number, single
particle component or single gas as the exposure metric. Third, to confirm univariate findings,
we will conduct multiple pollutant analyses to investigate joint effects of multiple mixture
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components. For the studies of blood flow and resistance, we will employ longitudinal methods
to estimate associations between exposure and biologic parameters in the proposed cross-over
design. Briefly, data from each animal is collected during two baselines (one at the beginning
and the end of the experiment) and 5 exposures. Each animal will be exposed to each of filtered
air (Sham), control exposures (e.g., CAPs), and exposures (e.g., aged CAPs).
Let Yij be a biologic endpoint recorded for animal i at time j. We will use models of the form:
Yij = β 0 + β1shamij + β 2 expoij + β 3c expoij + β 4 j + ui +εij
(1)
where shamij , expoij and c expoij are indicator variables equal to 1 if subject i receives sham,
exposure or controlled exposure, respectively, at time j, and β 3 j is a linear term corresponding to
the time (order) of measurement. The model compares the mean outcome under each exposure
condition (sham, controlled exposure, exposure) to that measured under baseline conditions. We
will consider interaction terms that allow the effect of exposure to vary over time. If necessary
we will also consider more general forms for the effect of time and more flexible correlations
structures than implied by random subject-specific intercepts. We have used this class of models
to analyze similar microsphere data in a study of CAPs effects in dogs.16
We will continue to use a multi-tiered strategy that successively incorporates more refined
exposure measures to assess the relative contributions of different pollutants to observed health
effects.26,79 Furthermore, we will extend the above model to use concentrations of individual or
multiple pollutants, depending on the specific hypothesis of interest. For a given pollutant, let
conc ij denote the concentration of that pollutant for animal i at exposure occasion j. The model
becomes:
(2)
Yij = β 0 + β1shamij + β 2conc ij expoij + β 3conc ij c expoij + β 4 j + ui +εij ,
Fitting this model yields estimates of a exposure-response slope under both exposure and
controlled exposure conditions, and yields a test of whether these slopes are equal to one another.
This test will assess the evidence that exposure increases the toxicity of particular air pollution
components. Extension to non-linear forms for the exposure-response function or multicomponent models is straight forward.
2.7.1 Power calculations: We estimated the power we will have to detect realistic effect sizes for
four primary outcomes using the study design presented in Table 5. The primary outcomes used
were organ chemiluminescence (CL) and three main respiratory parameters: respiratory
frequency (F), peak expiratory flow (PEF) and expiratory flow at 50% (EF50). We assumed
effect sizes observed in our previous studies:4,74,79 1) 1.7 fold increase for CL; 2) 28 breaths/min
for F; 3) -0.37 ml/s for EF50; and 4) -4.6 ml/s for PEF. We assumed residual variances equal to
estimates from these earlier analyses. The estimated power to detect these effects for any one
exposure setting versus sham is 93, 84, 89 and 95% for CL, F, EF50, and PEF, respectively,
suggesting the proposed sample sizes will provide adequate power to detect exposure scenario
effects.
2.8 Animal Subjects: Animals will be maintained and studied in accordance with the National
Institutes of Health (NIH) guidelines for animal care and use in research. The proposed protocols
have already been approved by the Harvard Medical Area Standing Committee on Animals. Rat
strain selected for this project is Sprague-Dawley, used most successfully in our prior CAPs
studies. Our School is accredited by the American Association for the Accreditation of
F- 31
Laboratory Animals Care and meets the NIH standards as set forth in the “Guide for the Care
and Use of Laboratory Animals,” DHEW Publication No. [NIH] 78-23 Revised.
2.9 Timeline: Table 3 shows the timeline for the proposed project, which will begin during Year
1 of our Center, and conclude after the summer of Year 4. Two Exposure studies will be
conducted each year, one during the winter and the other during the summer. Each study will
consist of 15 exposure days, leaving sufficient time between exposure campaigns for analysis.
The second half of Years 4 and 5 will be used to complete data analysis and integrate the results.
3. EXPECTED RESULTS: These studies will differentiate the effects of different components of
multi-pollutant mixtures on toxicological outcomes, using a novel and sophisticated controlledexposure plan that controls both variability in exposure to ambient particles and variability of
response between subjects. Using ambient particles and gases will allow us to generate realistic
exposure mixtures that are toxicologically relevant to public health. Exposure mixtures include
CAPs, photochemically aged CAPs, O3 and other photochemical gases, separately and in
combination. The sequence of exposures, progressively adding or subtracting components of the
mixture, will provide insight into the role of each component in producing biological responses.
Inherent variability in the concentration and composition of CAPs and other components of the
exposure mixture will provide insight into the shape of exposure-response relationships. We
expect to advance understanding of interactions between PM and photochemical gases in health
outcomes, addressing a critical knowledge gap. The biological outcomes assessed in this Project
include responses important in oxidant induced pulmonary inflammation, and functional
vascular/cardiovascular health responses that can also contribute to neurocognitive toxicities and
impairments. Our Project strongly complements the other Center Projects, which comprise
human epidemiological studies that are focused on many of the same outcomes but cannot
directly assess the underlying molecular or morphological outcomes critical to understanding
pollutant toxicity and mechanisms of such vascular and cardiovascular responses.
4. GENERAL PROJECT INFORMATION: Dr. Godleski will be the Project PI. He is an
experimental Pathologist with extensive experience in inhalation toxicology studies of CAPs and
TERESA exposures. He will be responsible for aspects of this study that pertain to the biological
outcomes. Dr. Godleski will be assisted by Co-PI Dr. Koutrakis, who will be responsible for
design of the exposure scenarios. Drs. Lawrence and Wolfson will assist Dr. Koutrakis with the
technical and administrative aspects of this project. Dr. Lawrence is an atmospheric chemist who
will be responsible for the HFPC and the reaction chamber, generation and characterization of
exposures. Dr. Edgar Diaz will be responsible for animal exposures and biological assessments.
Finally, Dr Brent Coull, Center Biostatistician, will participate in the data analysis. Facilities:
The HFPC and Inhalation Toxicology laboratory will be available for use in this study, including
all the instrumentation and equipment for generating and characterizing CAPs exposures. The
photochemical chamber and denuders designed, built, and used in the TERESA studies funded
by the existing PM Center, will also be available for this study.
F- 32
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