United States
Environmental Protection
. Agency
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EPA/600/R-02/028
December 2002
Toxicological Effects of Fine Particulate
Matter Derived from the Destruction of
the World Trade Center
National Health and Environmental Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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Notice
This report has been reviewed and approved for release by the National Health and
Environmental Effects Research Laboratory of the US Environmental Protection Agency. Approval
does not signify that the contents necessarily reflect the views and policies of the Agency, nor does
mention of trade names or commercial products constitute endorsement or recommendation for use.
This report has been audited for quality assurance purposes and a Quality Assurance statement is
included. Supporting documentation and raw data are available from Dr. Stephen H. Gavett,
National Health and Environmental Effects Research Laboratory (MD-82), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711 (telephone 1-919-541-2555, e-mail
gavett.stephen@epa.gov).
Cover photographs courtesy of the Federal Emergency Management Agency
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Contents
Authors, Contributors, and Reviewers v
Executive Summary vi
I. Introduction 1
II. Materials and Methods 3
A. WTC PM Sample Collection and Size Fractionation 3
B. Extraction of PM from Teflon Filters 4
C. Control PM Samples Used in WTC2001 Study 5
D. Physical and chemical analysis of solid (bulk and filter) samples 6
1. Scanning electron microscopy / energy-dispersive x-ray (SEM/EDX) analysis 6
2. X-ray diffraction (XRD) analysis 6
3. X-ray fluorescence (XRF) analysis 7
4. Carbon fraction analysis 7
E. Chemical analysis of liquid extracts of bulk and filter samples 7
1. pH 7
2. Endotoxin 7
3. Inductively coupled plasma - atomic emission spectrometry (ICP-AES) and - mass
spectrometry (ICP-MS) 7
4. Ion chromatography (1C) of deionized water extracts 8
F. Experimental Animals and Weight Randomization 8
G. Toxicological Endpoints: Experimental Design 8
1. Experiment A 9
2. Experiment B 9
3. Experiment C 9
H. Oropharyngeal Aspiration of PM Samples 9
I. Nose-Only Inhalation Exposure 10
J. Respiratory Responses Assessed by Whole Body Plethysmography 10
1. Immediate Airway Responses to PM25 Exposure 10
2. Airway Responsiveness to Methacholine Aerosol 11
K. Diffusing Capacity of the Lung for Carbon Monoxide 11
L. Bronchoalveolar Lavage (BAL) 11
M. Histopathology 12
1. Lung histopathology 12
2. Nasal histopathology 12
N. Statistical Analysis 12
in
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III. Results 14
A. Chemical analysis of solid samples and liquid extracts 14
1. Endotoxin and pH levels 14
2. Elemental and Ion Analysis 14
3. Carbon analysis 16
4. Compound analysis by XRD 16
5. SEM/EDX analysis 17
6. Summary 18
B. Experiment A: Dose-Response Relationships of WTC PM25 19
1. Body weights and immediate airway responses 19
2. DLCO 20
3. BAL parameters 20
4. Responsiveness to methacholine aerosol 22
5. Lung histopathology 22
6. Summary 25
C. Experiment B: Effects of Nose-Only Inhalation Exposure 25
1. Exposure results 25
2. Body weights 26
3. Immediate airway responses to nose-only exposure 26
4. DLCO measurements 27
5. Responsiveness to methacholine aerosol 27
6. BAL parameters 29
7. Nasal histopathology 30
8. Lung histopathology 31
9. Summary 31
D. Experiment C: Effect of Geographical Location of WTC PM Samples on Responses . 31
1. Sub-experiments and body weights 31
2. Responsiveness to methacholine aerosol 32
3. BAL cells 35
4. BAL proteins and enzymes 38
5. Lung histopathology 38
6. Summary 41
IV. Discussion 44
V. Quality Assurance Statement 48
VI. References 50
IV
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Authors, Contributors, and Reviewers
Authors
Stephen H. Gavett, Najwa Haykal-Coates, John K. McGee, Jerry W. Highfill, Allen D. Ledbetter,
and Daniel L. Costa — National Health and Environmental Effects Research Laboratory (MD-
82), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711.
Contributors
John J. Vandenberg, Thomas J. Hughes, Brenda T. Culpepper, M. Ian Gilmour, Judy H. Richards,
Paul A. Evansky, Dock Terrell, James R. Lehmann, Elizabeth H. Boykin, Mette J. Schladweiler,
and Hassell G. Hilliard — National Health and Environmental Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Triangle Park, NC.
Lung Chi Chen, Mitchell D. Cohen, Glenn R. Chee, Colette M. Prophete, and Jessica Duffy — New
York University, Tuxedo, NY (supported by NIEHS Center grant ES00260 and EPA PM Center
grant R827351).
Glen E. Marrs and Staff— Experimental Pathology Laboratories, Research Triangle Park, NC.
Jack R. Harkema and James G. Wagner — Michigan State University, East Lansing, MI.
Shirley J. Wasson — National Risk Management Research Laboratory, U.S. Environmental
Protection Agency, Research Triangle Park, NC.
Teri L. Conner—National Exposure Research Laboratory, U.S. Environmental Protection Agency,
Research Triangle Park, NC.
Annette S. King and A. Glenn Ross — NCCBA / Senior Environmental Employment Program,
Research Triangle Park, NC.
Dennis D. Williams and William D. Ellenson — ManTech Environmental, Research Triangle Park,
NC.
Robert A. Gary and David F. Smith — Sunset Laboratory, Hillsborough, NC.
Reviewers
John B. Morris — University of Connecticut, Storrs, CT.
Michelle M. Schaper — Mine Safety and Health Administration, Pittsburgh, PA.
Michael C. Madden — National Health and Environmental Effects Research Laboratory, U.S.
Environmental Protection Agency, Chapel Hill, NC.
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Executive Summary
The goal of the experiments described in this report was to evaluate the toxicity of fine
particulate matter (PM) derived from the destruction of the World Trade Center (WTC) on the
respiratory tract of mice, and thereby contribute to the short-term health risk assessment of WTC PM
being conducted by the Environmental Protection Agency. The adopted approach allowed a
comparison of the intrinsic acute toxicity of fine WTC PM in the respiratory tract to well-studied
PM reference samples that range in toxicity from essentially inert to quite toxic. The fundamental
question was whether fine WTC PM was uniquely highly toxic.
This toxicological research complements efforts by EPA and other organizations to assess the
extent and level of worker and public exposures to PM derived from the WTC disaster and recovery
efforts. This research is informative, but it is of limited scope, with a focus on the toxicological
effects of the fine fraction of WTC dust from a single exposure. A more complete characterization
of potential health effects would include consideration of other size fractions, repeated exposures,
additional doses and endpoints, and responses in species or strains of differing sensitivity. It was
not possible to assess these other considerations in the present study.
Fallen dust samples were collected on September 12 and 13 from various sites around Ground
Zero, and the fine PM fraction (< 2.5 microns in diameter; PM2 5) was isolated on filters. PM2 5 was
extracted from the filters and extensively analyzed by several chemical and physical techniques. A
dose-response study in mice was conducted comparing aspirated WTC PM25 (pooled from 7
different locations near the WTC site) with low and high toxicity PM2 5 control samples (Mt. St.
Helens and residual oil fly ash (ROFA), respectively). An acute nose-only inhalation exposure study
was conducted on one WTC PM25 sample, since upper airway irritation has been a primary
complaint of those living and working in the WTC area. Finally, a short-term time course study was
conducted comparing aspirated samples from the 7 different locations with each other and with a
standard PM25 sample (NIST 1649a, an ambient air PM sample collected in Washington, DC).
Fine size-fractionated WTC PM25 was composed primarily of calcium-based compounds such
as calcium sulfate (gypsum) and calcium carbonate (calcite, the main component of limestone).
These and other compounds and elements found in the WTC PM2 5 samples are indicative of crushed
building materials such as cement, concrete aggregate, ceiling tiles, and wallboard. Levels of carbon
were relatively low, suggesting that combustion-derived particles did not form a significant fraction
of these samples recovered in the immediate aftermath of the destruction of the towers. Gypsum and
calcite are known to cause irritation of the mucus membranes of the eyes and respiratory tract.
Samples of WTC PM25 induced mild to moderate degrees of inflammation when administered
at a relatively high dose (100 jig) directly into the airways of mice. The pulmonary inflammatory
response was not as great as that caused by the reference PM25 samples (toxic ROFA and ambient
air NIST 1649a). However, this same dose of WTC PM25 caused airway hyperresponsiveness (a
VI
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greater sensitivity to agents which constrict breathing passages) comparable to NIST 1649a and to
a greater degree than ROFA. Doses of 10 and 32 jig administered directly into the airways, or
inhalation at 10 mg/m3, did not induce significant inflammation or hyperresponsiveness. The
significant degree of airway hyperresponsiveness induced by the high dose of WTC PM25 implies
that components of the dust can promote mechanisms of airway obstruction.
The results from these studies indicate that a high dose of WTC dust as PM25 would be
necessary to elicit effects in healthy people. Hypothetical calculations are presented indicating that
a healthy worker at Ground Zero would have to inhale about 425 |ig/m3 WTC PM2 5 for 8 hours to
achieve the same dose per tracheobronchial surface area as occurred with the high dose of WTC
PM25 used in the mouse studies. These high concentrations are conceivable in the aftermath of the
collapse of the towers when rescue and salvage efforts were in effect. Therefore a healthy worker
without respiratory protection could have inhaled enough WTC PM25 to cause pulmonary
inflammation, airway hyperresponsiveness, and manifestations of sensory irritation such as cough.
Species differences in responses to inhalation of WTC PM2 5 are unknown and were not considered
in these calculations. Individuals who are especially sensitive to inhalation of dusts, such as
asthmatics, may experience these effects at lower doses of inhaled WTC PM25. These studies
suggest that most healthy people would not respond to a single exposure to moderately high WTC
PM25 levels (about 130 |ig/m3 or less for 8 hours) with any adverse respiratory responses. However,
it should be emphasized that the effects of chronic (long-term) or repeated exposures to lower levels
of WTC PM25, or the persistence of any respiratory effects are unknown and were not components
of this study. Although only fine PM2 5 was tested in these experiments, its composition was similar
to coarser PM, suggesting that biological responses to both size fractions within the respiratory
system may be similar. The results of these studies will need to be placed within the context of an
overall risk assessment for exposures to pollutants generated by the World Trade Center disaster.
vn
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I. Introduction
The World Trade Center (WTC) disaster sparked
enormous concern about the quality of the environment in
the surrounding neighborhoods. One of the immediate
concerns was the effect of dust from the collapse and
burning of the towers on breathing, especially in more
susceptible individuals. Dust infiltrated indoors into
homes and apartments, in many cases up to several inches
in depth. Fires at the WTC site continued for several
months before finally being extinguished, and emitted
significant quantities of particulate matter (PM).
Recovery and reconstruction efforts have also contributed
to emissions of fine (< 2.5 microns; PM25), coarse (> 2.5
and < 10 microns; PM25_10), and larger (> 10 microns) size
PM fractions. The dust particles from the WTC site
appear to be quite alkaline in nature, probably due to
partial dissolution of concrete, gypsum, and glass fiber
particles (USGS, 2002). As people are trying to move
back, decisions must be made about cleaning procedures
since potential exposure issues are associated with
redispersal and residual dust.
Those moving back to their homes as well as those
who work in the area have reported throat irritation,
cough, and other indications of mucous tissue sensory
irritation (New York Times, 2001; Washington Post,
2002). Nose and throat irritation may be caused by
particles which deposit in the nasal passages and upper
airways and stimulate sensory nerve reflexes (Costa and
Schelegle, 1999). Airborne dust may elicit inflammation,
mucus production, coughing, and sneezing in an effort to
clear the lung of particles (Raabe, 1999). However,
inflammation, mucus production, and airway
hyperresponsiveness may all contribute to airway
obstruction. Since asthma is characterized by all of these
cardinal features (Sears, 1997), it is logical to suspect that
asthmatic individuals may be more sensitive to agents
which further promote airway obstruction.
The National Exposure Research Laboratory (NERL,
USEPA), in coordination with Region 2 of the U.S.
Environmental Protection Agency (USEPA or EPA) and
the New York Department of Environmental Protection
(NYDEP), has been monitoring ambient pollutants
including volatile organic compounds (VOCs), dioxins,
and PM in an effort to ascertain exposures. In addition,
New York University (NYU) and Rutgers University have
collected bulk samples of ash and dust in the immediate
aftermath of the disaster. The National Health and
Environmental Effects Research Laboratory (NHEERL,
USEPA) has collaborated with these organizations to
study health effects of PM from the immediate vicinity of
the WTC site.
The primary goal of the present study was to evaluate
the potential health effects of PM in people working or
living in the vicinity of the WTC and downwind of fires
and dispersed building materials immediately after the
WTC collapse. Toxicologic assessment of entrained
(settled) dusts and combustion-derived PM dispersed in
the areas surrounding the WTC will provide basic hazard
identification information from which a broad health
assessment may be derived. These findings would provide
objective information to EPA, New York State, and local
authorities to communicate to the public about collateral
public health concerns.
In order to begin assessment of the toxicity of dust
derived from the destruction of the WTC towers, scientists
from NYU (led by Drs. Lung Chi Chen and Mitchell
Cohen) went to the area around "Ground Zero" on
September 12 and September 13, 2001. They collected
bulk samples of settled dust from several sites in the
immediate vicinity (<0.5 miles). Back in their laboratories
at NYU, they utilized a procedure to size-fractionate the
dust to obtain both fine and coarse PM fractions which can
be readily inhaled and deposit in the respiratory tract, and
are therefore relevant for study of toxicological effects.
On October 2, 2001, Dr. Chen contacted Dr. Daniel L.
Costaof the U.S. EPA NHEERL in orderto collaborate on
investigations of the toxicity of these size-fractionated
WTC PM samples.
The approach of the present study (code name
WTC2001) was to compare the toxicity of samples of size-
fractionated WTC PM25 with previously tested PM
samples in mice. Mice offer a number of advantages for
toxicity studies: 1) less sample is needed to assess toxicity;
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2) the biology of the mouse has been intensively studied in
the scientific literature; 3) a wide array of mouse-specific
analytical reagents is available; and 4) we have extensive
experience in assessing physiological responses,
inflammation, and respiratory tract injury in mice exposed
to other samples of air pollutants. The WTC PM25
samples were thoroughly characterized by a number of
chemical and physical techniques in order to compare the
composition of the samples with other reference samples.
A dose-response study in mice was conducted comparing
aspirated WTC PM2 5 (pooled from 7 different locations
near the WTC site) with low and high toxicity PM25
control samples. An acute inhalation exposure study was
conducted on one WTC PM2 5 sample, since upper airways
irritation is a primary complaint of those living and
working in the WTC area. Finally, a short-term time
course study was conducted comparing aspirated samples
from 7 different locations with each other and with a
standard PM25 sample.
Several methods were common to all three of these
experiments to determine the toxicological effects of WTC
PM25. The ability of these PM25 samples to affect
respiratory tract responsiveness to aerosolized
methacholine was determined. Since this chemical
triggers airway narrowing, the test is appropriate to
determine sensitivity to agents which induce airway
obstruction. Bronchoalveolar lavage is a common
standard technique which quantifies numbers of
inflammatory cells and levels of proteins and enzymes
indicative of lung injury. Lung pathological effects were
assessed in a semi-quantitative fashion in all studies, and
pathological effects in the nasal region were determined in
the inhalation study. Comparison of the toxicological
effects of dust derived from the destruction of the WTC
with PM25 samples which have been extensively
characterized in the literature will be clearly beneficial and
relevant to the overall assessment of health consequences
of environmental pollutants related to this disaster.
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II. Materials and Methods
A. WTC PM Sample Collection and Size
Fractionation
On 9/12/2001 and 9/13/2001, scientists from New
York University went to the WTC area to collect bulk
samples of fallen dust. Using a paper scoop, bulk samples
were taken from various outdoor locations (e.g. car hood,
window ledge, park bench) as well as one indoor location,
all of which appeared undisturbed since the collapse of the
towers, as judged by the presence of a smooth uniform
layer of dust and the absence of indicators of recent human
activity. Thirteen samples were collected and labeled with
numbers (1 - 13) on 9/12/2001, and six samples were
collected and labeled with letters (A - F) on 9/13/2001.
Samples were stored in 75 ml or 250 ml polystyrene
flasks at room temperature. All samples were collected
before rain fell on 9/14/2001, which certainly altered
chemical and physical characteristics of the dust. Samples
were taken back to NYU for processing to isolate different
size fractions.
Bulk samples of dust were sieved with a 53 jj, mesh
screen (USA Standard Testing Sieves, Fisher Scientific,
Pittsburgh, PA) on a shaker (Portable Sieve Shaker, Tyler
Industrial Products, Mentor, OH). The sieved material
(PM53) was aerosolized through a 10 jo, cut inlet to remove
particles in the 10 - 53 jo, range and isolate the PM10
fraction. The PM10 fraction then passed through a 2.5 (j,
cyclone (made in house) to remove the PM25_10 (coarse)
fraction and isolate the PM25 (fine) fraction. The PM25
fraction was collected on Teflon filters (Pall Gelman
Sciences, Port Washington, NY - Zefluor Supported
PTFE, 2 micron pore size, 47 mm, part # P5PJ047). While
fractionating the PM samples, the filters became loaded
and slowed airflow. Consequently, loaded filters were
replaced with fresh filters periodically, and about 10-40
filters were used to completely size-fractionate each WTC
sample. Analysis of the weights found in the 4 size
fractions showed that roughly half of the sample was in the
PM53 sieved fraction. Of the PM53 fraction, about 80-89%
was in the 10 - 53 jo, size range, which is too large to use in
respiratory toxicology studies since only 45% of 10 jo,
particles are even inhalable in small laboratory animals
(Menache et al., 1995), and deposition of particles greater
than 5 jam is minimal (Raabe et al., 1988). The amount of
the 2.5 -10 n fraction was very small (0.04 -1.14 % of the
PM53 fraction, except 3.23% in sample 13) and was
therefore not feasible to study. The PM25 fraction,
however, was present in large enough amounts (2.29 - 4.06
% of PM53 fraction) to study for potential respiratory
health effects, and is lexicologically relevant since it is
associated with epidemiological findings of health effects
in humans (Dockery et al., 1993). [The sum of the size
fraction percentages does not total 100% of the original
PM53 fraction because of loss of sample during
fractionation steps.] After examination of the available
inventory of filters and the locations where the samples
Figure 1. WTC dust samples were collected by New York
University (NYU) scientists from 13 sites on 9/12/2001
(numbers) and from 6 sites on 9/13/2001 (letters). Collection
sites are shown only for samples used in the WTC2001 study.
Map provided by MapQuest.com, Inc.
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.-: '
Figure 2. WTC bulk dust samples were size-fractionated by
NYU. Filters containing the PM2 5 fraction were received at the
U.S. EPA in Research Triangle Park, NC on 10/26/2001, and
were inspected and photographed 10/29/2001.
were collected, filters containing the PM2 5 fraction were
selected from seven locations (sites 8, 11, 13, B, C, E, and
F) around Ground Zero, in order to assess toxicity of
samples from different geographical locations as well as
overall toxicity of a pooled sample from these locations
(Figure 1). The locations were selected to represent a
distribution surrounding the WTC site, with more
collection sites in the east reflecting the predominant
winds in that direction.
Fourteen Teflon filters containing the PM2 5 fraction
from the 7 different sites collected around the World
Trade Center on 9/12/01 and 9/13/01 were shipped by
overnight express to EPA and received on October 26,
2001, and these were inspected and photographed on
October 29,2001 (Figure 2). [Throughout the WTC2001
study, sample transfers were accompanied by signed
chain-of-custody letters]. A total quantity of about 50 mg
from each site, collected on 1 to 3 filters per site, was
provided. The weight of PM25 on the filters was
determined by NYU, and was separately determined at
EPA after overnight dessication using a Calm
electrobalance. The description of the locations of the 7
samples and the total weight of PM2 5 on the filters from
each site is provided in Table 1. PM25 could not be
efficiently scraped off of one filter, so it was necessary to
isolate the PM25 using an aqueous extraction procedure
(see below).
Throat irritation, cough, nosebleeds, and other mucous
tissue/sensory irritation were reported by residents and
workers in the WTC area (Washington Post, 2002).
Oropharyngeal aspiration of PM bypasses the nose and
therefore potentially relevant effects may go undetected.
Consequently, it was decided that an inhalation exposure
study should be conducted which might reveal important
information about the toxicity and mode of action of WTC
PM25. Since there was not enough PM25 or PM25_10
sample available to conduct an inhalation exposure study
(> 2 g necessary), it was decided to use a PM53 sample
(sieved but not further fractionated) which was available
in large enough quantities to run through the inhalation
exposure system. The EPA inhalation exposure system
has a 2.5 \m\ cut-point cyclone to remove larger particles
(Ledbetter et al., 1998), and therefore measurement of the
PM concentration in the exposure zone of the chamber
represents exposure to PM25. A sample of PM53 from
location #3 (figure 1), 0.3 miles east of Ground Zero (in
the predominant wind direction), was available in large
enough quantities for the nose-only inhalation exposure
study. This sample was sent by overnight express from
NYU and received on November 21, 2001.
B. Extraction of PM from Teflon Filters
Filters were extracted using a modification of a
method by Biran and coworkers (1996). Each filter was
handled with clean sterilized stainless steel forceps.
Filters from each of the 7 individual collection sites (1-3
filters per site) were extracted into a single volume of
distilled water (Gibco BRL ultrapure 10977-015, lot
1063705) in the ratio of 0.5 ml water per mg sample (2 mg
PM / ml water; range 24.96 - 27.14 ml). This volume of
water was pipetted into a 100 ml sterile plastic specimen
cup containing a 3 mm thick Teflon ring at the bottom of
the cup designed to support the filter. Filters were wetted
with 200 \i\ of 70% ethanol on the particle side. The
liquid was gently spread on the filter with the pipet tip,
taking care not to scrape the filter. The filter was then
placed on top of the 3 mm thick Teflon ring in the
specimen cup with the particle side down, and a 6 mm
thick Teflon ring was placed on top of the filter. The cup
with the filter was secured to an orbital shaker (Titer Plate
Shaker, Lab-Line Instruments, Melrose Park, IL). A
cleaned sonicator probe (18 mm diameter, Sonic 300
Dismembrator, Artek Systems Corp., Farmingdale, NY)
was rinsed with 1% Triton X-100 (Sigma Chemical Co.,
St. Louis MO; T8787), and then ultrapure distilled water
(Gibco BRL ultrapure 10977-015, lot 1063705) before and
after each extraction. The probe was then lowered into the
water in the specimen cup to a level just above the filter.
Ice was placed around the specimen cup to prevent rising
temperatures during sonication, and the temperature of the
water was measured before and after sonication. The
shaker was turned to the lowest speed at which it would
mn continuously (setting = 2). The sonicator power was
set to 30, and the filter was sonicated for 30 minutes while
rotating on the orbital shaker. After sonication, the filter
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Table 1. Description of Samples Used in WTC2001 Study
WTC Site Samples
Experiment Sample
Code
A, C WTC 8
A, C WTC 11
A, C WTC 13
A, C WTC B
A, C WTC C
A, C WTC E
A, C WTC F
B WTC 3
Collection
Date
9/12/2001
9/12/2001
9/12/2001
9/13/2001
9/13/2001
9/13/2001
9/13/2001
9/12/2001
Location, Description
Beekman Street - filters #9, #14
0.4 miles E of Ground 0 center
55 Church Street - filters #13, #14, #15
In front of Millenium Hilton Hotel
0. 1 miles E of Ground 0 center
Church & Liberty St. - filters #4, #5
0.1 miles SE of Ground 0 center
Trinity & Rector - filter #4
From a car hood and windshield
0.25 miles S of Ground 0 center
Winter Garden Park - filters #4, #7
From a park bench facing the Hudson
0.2 miles WNW of Ground 0 center
Murray & Greenwich - filters #5, #6
From a window ledge
0.25 miles NNE of Ground 0 center
Inside 120 Broadway - filters #2, #12
From a marble staircase with no footprints
0.25 miles SE of Ground 0 center
23 Park Row - Ground sample in front of
J&R Electronics (across City Hall Park)
0.3 miles E of Ground 0 center
Size
Fraction
PM25
PM2,
PM25
PM2.5
PM2.5
PM2.5
PM2.5
PM<53
Total Weight
on Filters
53.316mg
50.097 mg
51.006mg
52.969 mg
54.285 mg
49.919mg
53.600 mg
21.521 g
sieved material
Extracted Wt,
% Extracted
46.70 mg
87.6%
29.79 mg
59.5%
46.29 mg
90.8%
42.31 mg
79.9%
47.67 mg
87.8%
45.13mg
90.4%
38.73 mg
72.3%
sieved - not
further
fractionated
Control PM Samples
Experiment
C
A
A
Sample
Code
NIST
MSH
ROFA
Collection
Date
1976-1977
1980
1994
Description
NIST Standard Reference Material 1649a
(Urban Dust collected in Washington DC)
Mt. St. Helens ash, Washington State, from
Graham etal., 1985
Residual oil fly ash, ROFA Sample 3 from
Kodavanti etal., 1998
Size
Fraction
PM2.5
PM25
MMAD:
2.665
Weight
Available
47.984 mg
>10g
>2g
Extracted Wt.,
% Extracted
39.33 mg
82.0%
previously size-
separated
milled - not
extracted
was gently removed with forceps and excess liquid was
drained from the filter into the cup. Filters were placed
back in their petri dishes, allowed to dry, and were
dessicated before reweighing to determine quantity
extracted (i.e. removed) from the filters (Table 1). The
suspension of PM was thoroughly mixed, the pH was
determined, and 10 ml was pipetted from each of the 7
samples into a single sterilized 150 ml Erlenmeyer flask on
ice to make a pooled sample (WTCX). The pH of the
pooled sample was also determined. Of the remaining
amount from each individual sample, 1 ml was taken for
endotoxin analysis, and the remainder was pipetted into
sterile 15 ml polystyrene tubes.
The flask containing the pooled sample was covered
with parafilm, and the pooled and individual site samples
were frozen at -80 °C prior to lyophilization. Holes were
poked in the parafilm of the pooled sample, while the caps
on the 15 ml individual site sample tubes were loosened.
Samples were lyophilized for 2 days at -55 °C and 140
mtorr (Virtis Company, Gardiner, NY). After
lyophilization, samples were stored at 4 °C until
resuspension in sterile saline on the day of use in
oropharyngeal aspiration.
C. Control PM Samples Used in WTC2001 Study
In order to assess toxicity of WTC PM25, pooled and
individual site samples were compared with three other
well-characterized PM25 samples. Standard Reference
Materials (SRM) are extensively characterized samples
available from the National Institute of Standards and
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Technology (NIST, Gaithersburg, MD). SRM 1649a is an
urban particulate matter sample which was collected in the
Washington DC area in 1976-1977 over a 12 month period
and represents a time-integrated sample (NIST, 2001).
This material was selected in order to compare toxicity of
WTC PM2 5 with other typical urban air PM2 5 (albeit from
an earlier era when leaded gasoline was still in use). Since
this material was collected as a total suspended particulate
(TSP) sample with a large amount of coarse non-respirable
PM, it was necessary to size-fractionate it in order to
compare it with the WTC PM25 samples. Vials of NIST
1649a were purchased and then sent to NYU for size-
fractionation using the same procedures as outlined above.
The PM2 5 fraction was sent back to EPA, and NIST 1649a
was extracted from Teflon filters as described above.
The toxicity of WTC PM2 5 was also compared to that
of a PM2 5 fraction of ash from Mt. St. Helens (MSH) in
Washington state (Graham et al., 1985). Approximately
half of MSH is crystalline in nature, primarily plagioclase,
a series of compounds beginning with NaAlSi3O8 and
ending with CaAl2Si2O8 which show continuous solid
solution from albite to anorthite, with CaAl replacingNaSi
as the series progresses. The remaining portion of MSH
is amorphous (glass), while there are minor amounts of
cristobalite (3%) and quartz (< 1%). The PM2 5 fraction of
MSH has low toxicity in rats (Raub et al., 1985) and mice
(Hatch et al., 1984). Since the MSH sample had already
been size-fractionated (Graham et al., 1985), it was not
necessary for NYU to further size-fractionate it with their
system.
Residual oil fly ash (ROFA) is a fugitive fine PM
sample with a high content of bioavailable transition
metals including vanadium, nickel, and iron. Numerous
studies by investigators at EPA and other institutions have
demonstrated that these metals are associated with lung
injury in both healthy animals and animal models of
cardiopulmonary injury (Dreher et al., 1997; Gavett et al.,
1999,Kodavantietal., 1998,Watkinsonetal., 1998). For
the WTC2001 study, we chose a sample of ROFA from a
boiler system which is toxic yet not as soluble in water as
previous samples of ROFA (ROFA sample #3 from
Kodavanti et al., 1998), and is therefore more comparable
to WTC PM samples which are not extremely water-
soluble. The ROFA samples in the study by Kodavanti
(1998) were reduced in size by placing each sample with
a stainless-steel ball in a stainless-steel cup and shaking
vigorously in a ball mill shaker for 30-60 minutes, and
then passing the sample through a 100 n mesh nylon
screen. ROFA sample #3 has a mass median aerodynamic
diameter (MMAD) of 2.665 jo,. Although it was slightly
larger in size compared with the other samples used in the
WTC2001 study, it was decided that further size
fractionation at NYU was not necessary. Control PM
samples were stored at room temperature in polystyrene or
polypropylene tubes shielded from light. See Table 1 for
the summary descriptions of control PM samples.
Samples of WTC PM, NIST, MSH, and ROFA were
characterized by scanning electron microscopy / energy
dispersive X-ray (SEM/EDX), X-ray diffraction (XRF), X-
ray fluorescence (XRD), carbon fraction analysis, pH and
endotoxin analysis, inductively coupled plasma-mass
spectrometry / atomic emission spectrometry (ICP-MS /
ICP-AES), and ion chromatography (1C).
D. Physical and chemical analysis of solid (bulk and
filter) samples.
1. Scanning electron microscopy/energy-dispersive
x-ray (SEM/EDX) analysis. SEM/EDX was used to
obtain physical and chemical characteristics of particles
and fibers found in bulk WTC2001 and control dust PM2 5
samples, and on polycarbonate filters taken during an
inhalation exposure using the WTC3 sample. The
Personal SEM® (PSEM) (formerly R. J. Lee Instruments,
Ltd., now Aspex Instruments, Trafford, PA) was used to
conduct the manual, single-particle analyses. The PSEM
is a digital SEM/EDX system equipped with secondary
and backscattered electron detectors for imaging, and a
thin-window EDX detector enabling X-ray detection of
carbon and heavier elements. For bulk samples, a small
amount was applied to an adhesive carbon tab affixed to
an aluminum SEM stub. For filter samples, small pieces
(less than 1 cm2) were affixed to aluminum SEM stubs
using a carbonaceous suspension. Images were created
using the backscattered electron mode, which enhances the
contrast of metals and other heavy elements with the
background carbonaceous medium compared with lighter
element particles. Photomicrographs of the individual
features provide particle morphology and approximate
physical size; an x-ray spectrum displayed below the
image provides information on the elemental composition
of the feature. SEM/EDX analysis was performed by the
National Exposure Research Laboratory, Research
Triangle Park, NC. Since only 15-30 images were
examined from each sample, the results should not be
interpreted as quantitative or comprehensive (Mamane et
al., 2001). Rather, these qualitative results were primarily
used to determine consistency with other analytical
techniques described below.
2. X-ray diffraction (XRD) analysis. XRD was used
to determine qualitatively whether any crystalline
compounds were present in sufficient quantity to be
identified in the WTC3 sample used in the inhalation
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exposure study. The bulk solid PM53 sample was side-
drifted into an aluminum holder and mounted into the
Siemens D-500 Diffractometer (Bruker Analytical X-Ray
Systems, Madison, WI). The generator, set at 45 kilovolts
(kV) and 40 milliamperes (mA), generated x-rays from a
copper-target x-ray tube. The filament current was 3.46
mA. Intensities were collected by a lithium-drifted silicon
detector fitted with a monochromator riding on a
goniometer in the coupled 0/20 mode. Peaks were
collected in the range 20 = 5 to 85 degrees. Collection
software used was Materials Data, Inc. (MDI, Livermore,
CA) Datascan, Version 3.2. Evaluation software was MDI
Jade 5 using the pattern library Powder Diffraction File
(PDF), release 2000 (International Centre for Diffraction
Data). XRD analysis was performed by the National Risk
Management Research Laboratory, Research Triangle
Park, NC.
3. X-ray fluorescence (XRF) analysis. Five
polycarbonate filters (Isopore O.Sum #ATTP04700,
Millipore Corporation, Bedford, MA) loaded with the
WTC3 PM2 5 sample used in the inhalation exposure study,
along with five lot-matched blank filters, were loaded into
liquid-type polyethylene sample cups, placed in a stainless
steel sample holder, and analyzed. No film was used to
cover blanks or samples in the analysis. X-ray intensities
were collected with the Philips PW2404 XRF (Philips
Analytical, Inc., Natick, MA), and the loaded particulate
was analyzed using the "standardless" software, UniQuant
4, after subtraction of the counts due to the blank filter
system (includes filter, polyethylene liquid sample cup and
stainless steel sample holder). The intensities were
averaged in each channel needed for background
subtraction. The blank filter analysis showed slightly
elevated counts due to Fe, Cr, Cu, Ca, Cl, S, and Si. The
constituents of the dust were evaluated as oxides, but are
reported quantitatively as elements with the oxygen
stripped. XRF analysis was performed by the National
Risk Management Research Laboratory, Research
Triangle Park, NC.
4. Carbon fraction analysis. Carbon fraction analysis
was used to speciate the carbon content of samples into
organic, elemental, and carbonate carbon. Analysis was
performed on bulk WTC2001 and control dust PM25
samples, and on quartz filters taken during an inhalation
exposure using WTC3 PM2 5. The thermo-optical method,
based upon sequential pyrolytic vaporization and detection
of the three carbon fractions (Birch and Gary, 1996;
Sunset, 2002), was performed by Sunset Laboratory,
Forest Grove, OR (bulk samples), and Hillsborough, NC
(filter samples).
E. Chemical analysis of liquid extracts of bulk and
filter samples.
1. pH. The pH of samples isolated by aqueous
extraction was determined immediately after the extraction
procedure with an audited calibrated Corning 440 pH
meter (audited by Research Triangle Institute, Research
Triangle Park, NC).
2. Endotoxin. Aliquots of samples isolated by
aqueous extraction were frozen on dry ice and sent by
overnight delivery to Associates of Cape Cod, Inc.
(Falmouth, MA) for analysis of endotoxin content using
the Limulus Amebocyte Lysate (LAL) gel-clot method.
LAL-reagent water (lot # 308-331) was used to
reconstitute or dilute Pyrotell lysate, endotoxin, and
samples, and served as the negative control. Samples were
titered using a twofold dilution scheme against control
standard endotoxin (CSE; lot #85, Escherichia coli Ol 13,
5 EU/ng). Preliminary inhibition tests (positive product
controls) were performed on the undiluted samples spiked
with CSE equivalent to twice the sensitivity (A,; 0.03
EU/ml). The error of the gel-clot method is ± one twofold
dilution.
3. Inductively coupled plasma - atomic emission
spectrometry (ICP-AES) and-mass spectrometry (ICP-
MS). WTC2001 PM25 samples, control dust PM25
samples, and polycarbonate filters taken during an
inhalation exposure using WTC3 PM25 were extracted
with deionized (d.i.) water or 1M HC1, and analyzed for
their elemental content. The two extraction liquids are
used to estimate easily bioavailable and total bioavailable
metal content, respectively. While this speciation scheme
is a rough approximation of bioavailability, it has proved
useful in characterizing inhalation toxicology endpoints
for various source and ambient particulates (Costa and
Dreher, 1997; Kodavanti et al., 1998). Milligram-sized
aliquots of bulk samples were extracted with 1.6 ml of
either liquid. Polycarbonate filters were extracted with 13
ml of either liquid. High-speed centrifugation was used to
separate the liquid and solids (17000 x g for 1.6 ml
samples, 51000 x g for 13 ml samples). After dilution,
extraction solutions were analyzed quantitatively using
ICP-AES (Model P40, PerkinElmer Instruments, Shelton,
CT) operated closely following EPA Method 200.7 (EPA,
2002a), and ICP-MS (ELAN 6000, PerkinElmer
Instruments, Shelton, CT) operated closely following EPA
Method 6020 (EPA, 2002b). Blank Gelman Teflo and
Millipore Isopore filters (used in the inhalation study)
were run through the extraction procedure. Filter blanks
levels for all elements were negligible compared to the
levels in the PM samples. Gelman Zefluor and Teflo
filters and Millipore Isopore filters are of known, similar
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low background levels. These filters are all produced for
air particulate sampling and are commonly used for
chemical analysis since their background chemical levels
are negligible relative to the mass of samples amounts in
this study. ICP-AES and ICP-MS analyses were
performed by the National Health and Environmental
Effects Laboratory, Research Triangle Park, NC.
4. Ion chromatography (1C) of deionized water
extracts. Deionized water extracts from the ICP sample
prep as described above were analyzed quantitatively for
anion and cation content using 1C (DX-500, Dionex,
Sunnyvale, CA). The AS 14 column was used for anion
analysis and the CS12 column was used for cation
analysis. 1C analysis was performed by ManTech
Environmental, an onsite contractor for the National
Exposure Research Laboratory, Research Triangle Park,
NC.
F. Experimental Animals and Weight Randomization.
Young adult (7 week old) female CD-I mice (an
outbred strain) were obtained from Charles River Breeding
Laboratory (Crl:CD-l® (ICR) BR) in Raleigh, NC or
Portage, MI (the latter used in Experiment A5 only). An
outbred strain was chosen because results from any
specific inbred strain might be applicable only to that
strain. CD-I mice were selected since researchers in the
Experimental Toxicology Division of the U.S. EPA have
extensive experience with this strain, while females were
chosen for convenience so that they could be housed
together in groups corresponding to treatment. The health
screening report of mice from the colony accompanied
each shipment of animals and was evaluated to determine
if there were pathogens detected in the colony which could
potentially affect responses. In all shipments, no
pathogens were detected which could affect respiratory
responses. Mice were housed in plastic cages on beta-chip
bedding in groups of 4 per cage in room JJ-4 of the animal
colony of the Environmental Research Center, Research
Triangle Park, NC. Food (Prolab RMH 3000) and water
were provide ad libitum and cages were changed at least
twice a week. Mice were maintained on a 12 hr light/dark
cycle at approximately 22 °C and 50% relative humidity in
our AAALAC-approved facility, and held for a minimum
of 5 days before treatment. Monthly sentinel screens were
negative for sendai, mouse hepatitis virus, mycoplasma
pulmonis, CARbacillus, parvovirus, endo- and ecto-
parasites, and pinworms. Protocols used in this study were
reviewed and approved by the EPA Institutional Animal
Care and Use Committee (Laboratory Animal Project
Review number 02-03-003 with amendments), and were
conducted using national guidelines for the care and
protection of animals.
In all experiments, mice were randomly assigned to
exposure groups based on weights. The weight
randomization program (RandomVB) was developed in-
house, validated, and documented in operating procedure
OP-NHEERL-H/ETD/IEG/97/18/01 (Animal
randomization using a personal computer). The program
takes all animal weights and ranks them from lowest to
highest. A group mean and standard deviation is
calculated for all animals. The number of animals per
group and the number of groups is entered. The numbers
of animals available at 1, 2, or 3 standard deviations (SD)
are calculated. The user then selects the lowest SD which
contains the required number of animals for the study. All
outliers are eliminated. Additional animals are then
eliminated to fit into the required number for the study.
Animals are then randomized by weight into the required
groups. All animals are accounted for and reasons why
they were not selected are displayed. The weight
randomization program was used to identify the groups of
4 mice which are housed together in a single plastic cage.
Within each cage, mice were individually identified by 1
to 4 marks applied to the base of the tail with a Sharpie
permanent ink marker (Sanford, Bellwood IL). Different
experimental groups were identified with different colors
(e.g. saline control mice - green marks, etc.). In addition
the cage cards were marked to identify the experimental
group. Marks remained evident for at least two days
which was long enough to identify mice at 24 hr
termination points. In cases where mice were killed more
than 2 days after the initial marking, the tails were
remarked where necessary because of excessive fading.
Mice were weighed at the time of randomization, again
immediately before exposure if randomization occurred
before the day of exposure, and whenever one group of
mice was killed.
G. Toxicological Endpoints: Experimental Design.
The toxicity of WTC PM25 samples was assessed in
three separate experiments, designated Experiments A, B,
and C. Experiment A was designed to study the dose-
response characteristics of the pooled sample of WTC PM
(WTCX) in comparison with ROFA (toxic control), MSH
(low toxicity control), and saline vehicle control.
Experiment B was designed to study the responses
associated with nose-only inhalation exposure of WTC3
PM25 in comparison with the responses of mice exposed
to air only. Experiment C was designed to compare toxic
responses of WTC PM25 from individual sites with each
other and with NIST 1649a.
In all experiments, a group size of 8 was selected
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based on scientific judgement and experience with the
typical variability of collected data (except the first part of
experiment A, where n = 12; see Results - Experiment A -
BAL parameters for explanation). Endpoints were
analyzed in a total of 388 mice in all 3 experiments. In
general, the endpoints were chosen to assess pulmonary
function impairment, lung injury and inflammation, and
pathological manifestations of respiratory tract injury.
Experiments utilizing oropharyngeal aspiration (A and C)
were emphasized over inhalation experiments (B) for
several reasons: 1) the quantities of samples available for
study were generally limited, and aspiration requires much
less material (10-100 mg) than inhalation (10 g preferred);
2) aspiration delivers a precise quantity of PM to the lung
at a specific time point, while the inhaled dose is more
difficult to predict or quantify; 3) inhalation exposure
studies are labor intensive and therefore fewer
comparative analyses of the WTC PM25 could be
accomplished in the available time frame compared with
studies utilizing oropharyngeal aspiration, and 4)
oropharyngeal aspiration (equivalent to intratracheal
instillation) is specifically recommended in evaluation of
panels of test materials for their relative potential to
produce toxicity (Driscoll et al., 2000).
1. Experiment A. In 5 sub-experiments, groups of
female CD-I mice were exposed to pooled WTC PM25
sample X (10, 31.6, or 100 ng), MSH (100 ng), ROFA
(10 or 100 |o,g), or saline vehicle control by oropharyngeal
aspiration on day zero. The dose of 31.6 |o,g represents the
half-log difference between 10 and 100 |o,g (i.e. 1015 =
31.6). The high dose of 100 |o,g was selected based on our
experience that at this dose nearly all PM samples will
induce at least a mild inflammatory or physiological
response; any sample that does not induce any response at
all at this dose can be judged to possess low toxicity.
Doses higher than 100 |o,g in the mouse may be of
questionable relevance due to the potential for artifactual
local inflammatory responses in response to bolus
administration (Driscoll et al., 2000). Four mice per
sample group were tested within each sub-experiment (n
= 28 per sub-experiment; total experiment A: n = 140).
In sub-experiments Al, A2, and A5 (total n = 12 mice
per sample group), airway responses to aspiration of the
PM samples was assessed by comparison of breathing
parameters just before and after aspiration (see method
below). On day 1, diffusing capacity of the lung for
carbon monoxide (DLCO) was assessed, and mice were
then killed and bronchoalveolar lavage (BAL) fluid cells,
proteins, and enzymes were recovered and quantified to
assess lung injury and inflammation.
In sub-experiments A3 and A4 (n = 8 mice per sample
group), airway responsiveness to methacholine (Mch)
aerosol was determined on day 1. Mice were then killed,
and lungs were removed and fixed for histopathological
assessment. Airway hyperresponsiveness to nonspecific
bronchoconstrictive agents such as Mch is a primary
feature of asthma (Sears, 1997) as well as reactive airways
dysfunction syndrome (RADS) which develops after high-
level occupational exposure to irritant gases, fumes, or
smoke (Gautrin et al., 1999). Induction of this condition
by PM in nonallergic normal mice can be considered as a
marker of respiratory tract injury.
2. Experiment B. Two groups of female CD-I mice
were exposed in nose-only inhalation exposure tubes one
time to a PM2 5 sample (WTC3) or air only for 5 hr (n = 48
per exposure group; total experiment B: n = 96). This
WTC3 sample was derived from a sieved but not
previously fractionated PM53 sample of WTC3 by
aerodynamic size-separation during exposure. Although
some irritant responses are transitory and therefore would
be best measured during exposure (Costa and Schelegle,
1999), we do not currently possess the recently developed
technology (e.g. Buxco Electronics, Sharon, CT or CH
Technologies, Westwood, NJ) which allows some
respiratory parameters to be measured during nose-only
exposures. Therefore, breathing parameters were
compared just before and after inhalation exposure (n = 12
per group). On days 1,3, and 6 after the exposure, 16
mice from each group were assessed for DLCO and BAL
parameters (n = 8) or responsiveness to Mch aerosol and
lung and nasal histopathology (n = 8).
3. Experiment C. In 2 sub-experiments, groups of
female CD-I mice were exposed by oropharyngeal
aspiration to 100 |o,g of PM from one of 7 individual WTC
sample sites, to 100 jog of NIST 1649a (referred to as
NIST hereafter), or to saline vehicle only. In sub-
experiment Cl, mice were exposed to WTC8, WTC13,
WTCF, NIST, or saline. In sub-experiment C2, mice were
exposed to WTC11, WTCB, WTCC, WTCE, or saline.
On days 1 and 3, mice were assessed for responsiveness to
Mch aerosol, BAL parameters, and lung histopathology (n
= 8 per group per time point, except saline sub-experiment
C2: n = 4 per time point; total sub-experiment C1: n = 80,
total sub-experiment C2: n = 72, total experiment C: n =
152).
H. Oropharyngeal Aspiration of PM Samples.
A Sartorius model AC211S analytical balance
(Edgewood, NY; audited by Research Triangle Institute,
Research Triangle Park, NC) was used to weigh PM
samples for oropharyngeal aspiration. The operation of
the balance was tested by weighing calibrated weights
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before and after weighing samples each day (Class U
calibrated weights, Denver Instrument Company, Arvada,
CO). PM samples were allowed to come to room
temperature from 4 °C before weighing. Sterile 2.5 ml
glass vials or 5 ml polystyrene snap cap vials were used to
weigh and resuspend PM samples. Vials weights were
tared, and a sterilized stainless steel spatula was used to
transfer sample to the weighing vial, and the sample was
weighed. The sample was then resuspended with sterile
saline (Sigma S-8776 single use vials, lot 128H2310)
using a calibrated Rainin Pipetman at a concentration of 2
mg/ml. All mice aspirated a volume of 50 jol. Samples
were vortexed and used straight (100 |o,g dose) or diluted
as necessary (to 0.632 mg/ml or 0.2 mg/ml for 31.6 |o,g or
10 |o,g doses, respectively). All samples were sonicated for
2-4 minutes at 22 °C (Branson model 3210R-DTH,
Danbury, CT) prior to oropharyngeal aspiration.
Mice randomized into different exposure groups were
anesthetized in a 2.7 L plexiglass chamber by passing
house air through an aerator containing methoxyflurane
(Metofane; Mallinckrodt, Mundelein, IL). The vapor
induced rapid anesthesia, at which time the mouse was
taken out of the chamber and placed on an aspiration
platform. The tongue was gently pulled back and held
with a forceps, and 50 jol of PM suspension or saline alone
was pipetted in the back of oropharyngeal region using a
200 (0,1 tip. The tongue was held until the animal was
forced to aspirate the sample, and placed back in its cage.
Mice recovered within 5 or 10 minutes of this procedure.
This technique is equivalent to intratracheal instillation in
deposition efficiency (Foster et al., 2001), and several
publications describe experiments in which it was
successfully used (e.g. Dreher et al., 1997, Gavett et al.,
1999, Kodavanti et al., 1998).
I. Nose-Only Inhalation Exposure.
In order to assess the effects of WTC PM25 on upper
respiratory tract responses, mice were exposed to WTC3
or air only in two separate nose-only inhalation exposure
chambers. The exposures were conducted for 5 hours in
52-port nose-only flow-by inhalation chambers (Lab
Products) on November 27, 2001. The exposure time was
based on practical considerations of the tasks involved on
the exposure day. The WTC3 sample was a tan powder
received in a plastic jar, and was desiccated at room
temperature prior to use. Preliminary exposures were
conducted on several days prior to exposure to set
exposure parameters, which indicated that an aerosol
concentration of 10-15 mg/m3 could be achieved. The
control chamber and the WTC3 chamber had similar flow
rates (~ 12 L/min) and received air from the same source.
The aerosol was generated using a unique exposure system
which conserves sample by using a carpenter's chalk line
to pick up particles from a small Tygon tube dust reservoir
(illustrated in Ledbetter et al., 1998). The dust is carried
out through an orifice and blown off the string in a
discharge head with a high velocity air jet. The particles
are carried through a particle charge neutralizer and 2.5 jo,
cut-point cyclone to remove particles larger than PM2 5,
and finally enter the inlet of the nose-only chamber.
Nose-only exposure tubes were constructed from 50
ml polypropylene centrifuge tubes with the bottom end
removed. Mice were randomized into exposure groups as
described above, and 49 in each group were placed in
exposure tubes (1 extra per group in case any mice died
during the exposure). Mice were not acclimated to the
tubes prior to exposure, since stress may be an important
component of the response to WTC PM. In order to
measure immediate airway responses to air or WTC3
sample and also to handle the large number of mice, the
control air exposure was begun 1 hour before the WTC3
exposure.
Dust concentration was determined gravimetrically on
5 Teflon filters (45 mm diameter with 1 jo, pore size) taken
at a sample flow rate of approximately 0.24 L/min. The
filters were weighed just prior to and after sampling using
a Cahn C-30 balance housed in a controlled temperature
and humidity enclosure. Real-time PM concentration was
achieved with an aerosol monitor (Dust Track, TSI Inc.,
St. Paul, MN) on the chamber exhaust. The particle size
was determined gravimetrically using a Mercer Cascade
Impactor (Intox Products, Albuquerque, NM). On
November 29, 2001, the exposure system was restarted
and 7 Teflon and 3 polycarbonate filters were taken for
chemical analysis. On December 17, 2001, 18
polycarbonate filters (9 WTC3 and 9 blanks) were
collected for chemical analysis and 3 quartz filters were
collected for carbon analysis.
J. Respiratory Responses Assessed by Whole Body
Plethysm ography.
1. Immediate Airway Responses to PM2 5 Exposure.
Exposure to PM25 by oropharyngeal aspiration or by
inhalation may result in immediate changes in breathing
parameters. Breathing parameters in unanesthetized
unrestrained mice were assessed in a 12-chamber whole
body plethysmograph system (Buxco Electronics, Sharon,
CT). The animal chambers have apneumotach on the roof
to measure pressure (which is proportional to air flow)
relative to the pressure in a reference chamber vented to
the atmosphere. The breath by breath signals are taken by
the program software to compute respiratory rate
10
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(frequency, f, breaths / min) and other parameters
including enhanced pause (PenH). Although PenH is at
best an indirect measure of flow resistance, it does
correlate well with lung resistance and reflects changes
occurring during bronchoconstriction (Hamelmann et al.,
1997), although other responses such as mucus production
may increase PenH. The convenience of rapidly
measuring respiratory parameters in twelve mice at once
was a maj or consideration in utilizing this technique rather
than the double plethysmograph or tracheotomized
ventilated methods which allow direct measures of airways
resistance and compliance, but are time and labor
intensive. A protocol was written to record and average
baseline measurements of mice in calibrated chambers for
10 min, pause for oropharyngeal aspiration (or stop during
inhalation exposure), and then resume recording
measurements for one hour. The time between
oropharyngeal aspiration and monitoring of responses was
approximately 6 minutes, while about 20 minutes was
needed after inhalation exposure to remove mice from
exposure tubes, weigh them, and transport them to the
plethysmograph chambers. PenH was automatically
calculated by the software (and confirmed by examination
of random data) using expiration time (Te), relaxation time
(RT), and peak expiratory and inspiratory flows (PEF,
PIF) according to the following expression: PenH = [(Te-
RT)/RT] x [PEF/PIF]. Examination of the data after
exposure showed that utilization of the first 10 or 15
minutes of the data was not more sensitive in detecting
changes in respiratory parameters than the entire hour of
post-exposure monitoring, and therefore responses over
the whole post-exposure hour were utilized and averaged.
The percent change in/and PenH after exposure to PM
was expressed as [(Post-value - Pre-value) / Pre-value] x
100%.
2. Airway Responsiveness to Methacholine Aerosol.
Airway responsiveness to increasing concentrations of
aerosolized methacholine (Mch) was measured in mice in
calibrated chambers. After measurement of baseline PenH
for 5 minutes, saline or Mch in increasing concentrations
(4, 8, 16, 32, and 64 mg/ml) was nebulized through an
inlet of the chamber for 1 min. The aerosol drier was
automatically turned on immediately after the
aerosolization period for 2 min. Measurements of PenH
and other parameters were continued for an additional 1,
2, 3, 4, 8, and 12 minutes after saline or increasing doses
of Mch, for atotal time of 4, 5, 6, 7, 11, and 15 minutes (0,
4, 8, 16, 32, 64 mg/ml Mch, respectively). One minute
pause periods between aerosolizations allowed time to
change solutions for nebulization. After subtracting
baseline values from responses to saline or Mch, the area
under the curve (PenH AUC; PenH - sec) for these
recording intervals was calculated using the trapezoid
method.
K. Diffusing Capacity of the Lung for Carbon
Monoxide.
The ability of the lungs to allow diffusion of gases
(O2, CO2) across the alveolar-capillary barrier is dependent
on physical properties of the gases and the alveolar-
capillary membrane, and may be limited by perfusion or
diffusion (Levitzky, 1995). Diffusion limitation may be
caused by thickening of the alveolar-capillary barrier (e.g.
by interstitial or alveolar edema). Diffusion of carbon
monoxide (CO) is limited only by its diffusivity in the
barrier and by the surface area and thickness of the barrier.
The diffusing capacity of the lung for CO (DLCO) is
therefore a useful test of the integrity of the alveolar-
capillary membrane (Levitzky, 1995).
To determine DLCO rapidly and increase sensitivity
from individual mice, 4 mice were placed together in a
single 7.8 L bell jar associated with a Pharmacokinetic
Uptake System (consisting of an oxygen monitor, flow
meter, pump, pressure gauge and transducer, mass flow
controller, and computerized data collection and control
system). Approximately 6.6 ml of research grade CO
(99.99%) was injected into the system. The initial
concentration of CO in the chamber was approximately
700 ±10 ppm. CO concentrations were taken every 15
seconds (Bendix Model 8501-5CA CO Analyzer), and
continued for approximately 10 minutes. Temperature,
humidity, airflow, pressure, and oxygen were monitored
during the test. The DLCO is expressed as the slope of the
fitted line of [CO] vs. time (ppm/min).
L. Bronchoalveolar Lavage (BAL).
Mice were anesthetized with urethane (1.5 g/kg i.p.)
and killed by exsanguination via severing the renal artery.
The trachea and lungs were exposed and a 20 g catheter
was sutured into the trachea. Mice were lavaged with two
aliquots of Ca2+, Mg2+, and phenol red-free Hanks'
balanced salt solution (HBSS; 35 ml/kg, Life
Technologies,Bethesda,MD). Approximately 85%ofthe
total instilled volume was recovered in all treatment
groups. The BAL fluid was maintained on ice and
centrifuged at 360 x g for 10 minutes at 4 °C.
Supernatants were transferred to a separate tube in order
to prepare aliquots for biochemical analyses. BAL cells
were resuspended in 1 ml of HBSS and counted (Coulter
Zl, Hialeah, FL). Cytospin preparations of BAL cells
were made for each sample and stained with Wright's
Giemsa using an automated slide stainer (Hematek 2000,
11
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Elkhart, IN). Cell differentials were performed by one
person (SHG) counting 500 cells per slide. After lavage,
the lungs were removed and stored at -80° C for future
assays (to be determined).
Assays for total protein, albumin, lactate
dehydrogenase (LDH) andN-acetyl-p-D-glucosaminidase
(NAG) are routine measures of lung injury (Henderson et
al., 1985) and were carried out on an aliquot of BAL
supernatant as previously described using a Cobas Fara II
centrifugal spectrophotometer (Gavett et al., 1997). Four
other BAL supernatant aliquots were prepared from each
sample; one of these was supplemented with 10% fetal
bovine serum to prevent loss of cytokines and other
proteins in low protein concentration fluids, and the 4
aliquots from each sample were stored at -80° C. These
samples are available for analysis of cytokines and other
proteins (to be determined).
M. Histopathology
1. Lung histopathology. In experiments A and B,
mice which were tested for Mch responsiveness were
subsequently assessed for lung histopathology, while in
experiment C, all mice were tested for Mch responsiveness
and were lavaged before assessment of lung
histopathology. Mice were anesthetized with urethane and
killed as described above for BAL. Lungs were removed
and fixed by tracheal perfusion in a fume hood with ice
cold 4% paraformaldehyde at 25 cm pressure for 15
minutes. The trachea was then tied off and placed in a vial
of 4% paraformaldehyde at 4 °C. After 24 hours, the lungs
were drained and placed in phosphate buffered saline at 4
°C.
The lungs were transferred to Experimental Pathology
Laboratories (Research Triangle Park, NC), where fixed
lungs were processed to paraffin blocks, sectioned at an
approximate thickness of 5 jo,, placed on glass slides and
stained with hematoxylin and eosin (H&E). Longitudinal
coronal sections were cut on a lateral plane to include
mainstem bronchi for viewing a maximal amount of lung
area. Two additional unstained lung sections were
prepared for future use. Histopathologic observations for
individual animals in each experiment were tabulated, and
the degree of severity of inflammatory changes and the
presence of PM-related pigment were graded on a scale of
one to five (1 = minimal, 2 = slight/mild, 3 = moderate, 4
= moderately severe, 5 = severe/high). The pathologist
knew which animals comprised a group, which group was
the saline or air-exposed control group, the day after
treatment, and the doses given to the experimental groups,
but did not know the identities of the individual PM
samples other than by a unique number or letter.
2. Nasal histopathology. Dr. James Wagner of
Michigan State University (MSU) instructed EPA
personnel in this procedure, utilized on mice from the
nose-only inhalation exposure (experiment B).
Immediately after death, the head of each animal was
removed from the carcass and both nasal passages were
fixed by slowly flushing retrograde through the
nasopharynx with 1-2 ml 4% paraformaldehyde. The
nasal cavity was then immersed in a large volume of the
fixative for at least 24h until further processing. The fixed
nasal cavities were placed in 0.1 M PBS (pH 7.2, 4 °C)
and shipped overnight to Dr. Wagner at MSU. Nasal
cavities were decalcified in a 13% solution of formic acid
for 5 days, and then rinsed in distilled water for Ih. After
decalcification, three transverse tissue blocks of the nasal
cavity, cut perpendicular to the hard palate, were selected
for light microscopic analysis. The first tissue block was
sectioned from the proximal aspect of the nasal cavity
immediately posterior to the upper incisor tooth (Tl). The
second transverse tissue block was taken at the level of the
incisive papilla (T2) and the third and most distal tissue
block was taken at the level of the second palatial ridge
(T3). The tissue blocks were embedded in paraffin, and 6
|im-thick sections were cut from the anterior surface of
each block. Sections were histochemically stained with
hematoxylin and eosin for morphologic identification of
nasal tissues. Nasal tissues (three sections/mouse) from a
total of 48 mice tested for Mch responsiveness (8
mice/exposure group/time point) were microscopically
examined by Dr. Jack Harkema (MSU). Nasal lesions
were graded on the following scale: 1 = minimal, 2 =
mild, 3 = moderate, and 4 = marked inflammation.
N. Statistical Analysis
All statistical analysis were done using SAS
procedures (Gary, NC). There were generally three types
of responses collected: 1) PenH responses recorded
repeatedly for each animal as area under the curve (AUC)
for various concentration exposures to Mch; 2) responses
to DLCO were analyzed from a single response from 4
animals; and 3) individual measurements measured once
for each animal as a univariate variable. Experimental
designs varied with each experiment and each part of an
experiment. Statistical designs used were replicated
completely random designs for experiment A. Crossed-
designs were used for experiment B and C involving
treatments (TRT) and days (DAY). Randomized block
designs were used for DLCO experiments.
When initial multivariate repeated measures analysis
of variance (MANOVA) test showed significant
interactions between dose of Mch and TRT or DAY in the
12
-------
airway responsiveness studies, univariate linear regression
was used in all subsequent tests. The models used in
these regression studies were analysis of covariance
(COV) with tests for parallelism for each TRT and DAY
combination. In experiment A, natural logarithms were
used for both Mch concentration (C) and PenH AUC
responses. The linear regression Log (PenH AUC) = al +
b(Log C) reduces to a power function of the form PenH =
a2'Cb. No logarithms were used in Experiments B and C
due to several negative values resulting after baseline
adjustments. Techniques similar to ordinary stepwise
regression were used in COV analyses. Overall test of
parallelism of regression lines was done first. Subgroups
of the TRT and DAY combinations were determined to get
subgroups exhibiting a common slope. Within a subgroup
with a single slope, subsequent tests were done to
determine if the means were different (using individual
contrast tests). Body weight was determined
repeatedly for animals. Due to animals being removed and
killed, numbers of mice in each group were different on
different days after exposure. For days when few animals
weights were determined, univariate analysis of variance
(ANOVA) was used to test for TRT effects. For those
days where most of the weight data occurred, MANOVA
techniques were used for statistical tests.
For each TRT and DAY combinations with a
univariate response, a determination was made if the
variances could be considered homogeneous. If the
variance ratios were greater than 10-fold then all of the
responses were ranked from smallest to largest across all
TRT and DAY combinations. Then ranks replaced the
original responses for the univariate ANOVA. Sometimes
the variances of the ranks for TRT and DAY combinations
still indicated heterogeneity. Then additional judgment
was used to help insure that this heterogeneity of variance
did not affect the overall conclusions. In experiment A, a
replicate was called DAY. When replication was shown
to have no significant contribution in the ANOVA results,
DAY was not included in subsequent ANOVAs. For
responses with many "zero" values, the residuals from the
ANOVA were plotted and analyzed by univariate
techniques to determine if the residuals generally met the
assumptions required for ANOVA. When interactions
between TRT and DAY occurred, these were pointed out
and in some cases further ANOVA were done for each
DAY. When ranks were used for the response, the ranks
were regenerated for each day separately. When TRT was
significant, follow-up comparisons of means were done
using Tukey's multiple comparison tests.
The statistical tests examined only whether groups
were significantly different from each other. In the
reporting of the results, for the sake of brevity, groups are
sometimes referred to as having significantly greater
values than other groups. These statements should be read
as groups are significantly different from each other, and
the mean of one group is greater than the mean of another.
13
-------
III. Results
A. Chemical analysis of solid samples and liquid
extracts.
1. Endotoxin and pH levels. The pH of water-
extracted WTC PM25 ranged from 8.88 in WTCEto 10.00
in WTC8 (Table 2). The alkaline pH is consistent with
previous reports of WTC PM (USGS, 2002) and probably
results from the building materials comprising much of the
dust (see below). The pH of lyophilized WTC PM25
reconstituted in unbuffered saline was very close to
neutral, while MSH was very slightly acidic and ROFA
was moderately acidic (average 3.74 at 2 mg/ml). It is not
known why the pH of WTC PM2 5 should be close to
neutral after reconstitution in saline; perhaps the salt
neutralizes a basic component of the extract. Endotoxin
levels in WTC PM2 5 samples were minimal in comparison
with other urban PM samples such as NIST 1649a, which
was also low (Table 2). Several thousand times this level
of endotoxin caused an acute neutrophilic response in the
lungs of CD-I mice (Dhingra et al., 2001). The level of
endotoxin in the samples used in this study would not be
anticipated to contribute directly to any inflammatory
response in the lungs.
2. Elemental and Ion Analysis. The ICP data showed
that water-soluble calcium and sulfate content amounted
to 56-63% of the WTC PM25 samples (Table 3). In
general, the elemental and ion compositions were
consistent among the different samples tested. ICP data
for the IMHCl-soluble extracts of WTC PM2 5 showed an
additional 1-2 weight percent calcium content. This
increase may be attributed to calcite or other water-
insoluble calcium salts which are soluble in 1M HC1 (see
below for data on compound analysis). There was no
evidence of stainless steel contamination from the forceps
used to handle the WTC PM filters or from the stainless
steel balls used to size-fractionate the ROFA sample.
The ICP results for the aerosolized PM2 5 cut fraction
of WTC3 generally agree well with those determined by
XRF (Table 3). Calcium content of acid-extracted WTC3
was somewhat lower by ICP (20-22%) than calcium
content of solid WTC3 by XRF (26.6%). This may reflect
an incomplete extraction in the one hour timeframe of
sample preparation method for ICP, or the presence of
other insoluble forms of calcium in the WTC3. The XRF
values are higher for most other elements, which reflects
the incomplete dissolution of the WTC3 matrix by the
weak (water) and moderate (1M HC1) extraction liquids.
Elements such as magnesium and zinc, which exist in
compounds more amenable to acid dissolution, agree more
closely (Weast, 1985;Budavari, 1996). Elements such as
aluminum, iron, and titanium, which are in the form of
Table 2. Endotox
Extraction and Re
Sample
Code*
Water b
WTC 8-100
WTC 11-100
WTC 13-100
WTC B-100
WTC C-100
WTC E-100
WTC F-100
NIST- 100 c
Saline
WTCX-10
WTCX-31.6
WTCX-100
MSH-100
ROFA-100
in and pH Levels of PM Samples after Water
suspension in Saline.
pHin Endotoxin d pHin
water EU/ml Inhibition Saline e
5.28
10.00 0.50
9.16 0.25
9.47 0.50
9.54 0.25
9.32 0.50
8.88 0.25
9.55 0.50
4.20 25
9.35
none
none
none
none
none
none
none
none
6.67
7.38
7.38
7.36
6.61
3.74
'WTCX indicates pooled sample of WTC8, WTC11,WTC13,
WTCB, WTCC, WTCE, and WTCF. "-100" indicates 100 jig/50 |il
dose = 2 mg/ml. "-31.6" indicates 31.6 jig/50 |il dose = 0.632
mg/ml. "-10" indicates 10 |ig/50 |il dose = 0.2 mg/ml.
Water-extracted PM samples were lyophilized and resuspended in
sterile saline.
Water used to extract filters.
NIST Standard Reference Material 1649a (Washington DC TSP
PM).
Endotoxin levels measured as endotoxin units (EU) per ml water
extract. Samples were tested for inhibition of the endotoxin assay
(none was detected).
Average of 3-4 measurements.
14
-------
Table 3. Elemental and Ion Analysis of WTC2001 Samples :
Sample:
Diluent:
No. Analyses:
Analyte
s
-------
Table 4. Carbon fraction analysis of PM Samples in WTC2001
Study a
Table 5. XRD Analysis of Compounds Present in WTC 3
Sample a
Sample:
% Carbon
Fraction:
Organic
Elemental
Carbonate
Total
WTC3
6.88
0.31
1.39
8.58
WTCX
(pooled)
0.93
0.00
0.60
1.53
WTCB
2.11
0.01
0.35
2.47
MSH
0.06
0.07
0.00
0.13
ROFA
1.31
13.63
1.32
16.26
NIST
SRM
1649a
10.82
15.10
0.00
25.92
ICDD
Number
05-0586
33-0311
41-0224
46-1045
Formula
CaC03
CaSCy2H2O
CaSCy0.5H20
SiO2
Mineral
Name
Calcite
Gypsum
Bassanite
Quartz
Relative
Amount
Major
Major
Minor
Minor
a Organic, elemental, and carbonate carbon fractions were analyzed as
described in text. Results are expressed as percent of total mass of
sample.
nickel, and iron which are important in its toxicity
(Kodavanti et al., 1998). The 1M HCl-soluble metal
content trend is Mt. St. Helens < WTC2001 < ROFA (not
enough NIST sample was available to run the test).
3. Carbon analysis. The WTC2001 samples had low
total carbon content, in the range of 1.5-8.5% (Table 4), in
comparison with control samples such as NIST (26%) and
ROFA (16%). MSH had almost no carbon, as expected
from this crustal PM sample. The WTC3 sample used in
the inhalation study had about 4 times as much carbon as
1000-
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=;
0
~ ti
' 'm 500 -
d
OJ
250-
0
rU.S. EPA
[020118A.MDI] WTC dust, as received
,\
1\^**+J
• 1
. — U.
^CJuLw
[
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10
20
j)Ml»AubkJLlM^^^ — «*™_A-
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oo-Oo 1 1 > Gypium - C3S04!iH2i:i
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41-D224J Bassanite- CaS04!0.5H20
05-0586 > Caloite - CaCOS
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46-1046* QuartE- SI02
1 '
•TO 50 60 70 80
2-Th8laO
IXRDIAJministratorl Wednesday. Feb 06. 2002 04:13p (MDI/JADE
Figure 3. X-ray diffraction (XRD) analysis of WTC 3 sample (PM53) used in nose-only
inhalation exposure study (Experiment B). Peaks were collected in the range, 26 = 5- 85°.
Collection software used was Materials Data, Inc. Datascan, version 3.2
a Analysis showed about half crystalline materials (50.6% above
background), and the remainder was amorphous. After smoothing
and subtracting background, evaluation software (MDI Jade 5) was
used to match patterns with library available from International
Centre for Diffraction Data Powder Diffraction File, release 2000.
the other two WTC samples. This result may be due to
differences in the method by which the samples were
isolated (physical separation vs. aqueous extraction and
lyophilization) or may simply be due to variability in
carbon content of samples from different locations.
Despite the variation in total carbon content of WTC PM
samples, the ratios of elemental, organic, and carbonate
carbon were similar. Elements not listed in Table 3 or 4
(-30% of total mass) are likely O and H from adsorbed
water and O, H, and N from
organic or inorganic compounds.
4. Compound analysis by
XRD. XRD analysis of WTC3
PM53 (before size segregation by
the inhalation exposure system)
showed a complex pattern
containing 25 peaks, indicating the
presence of several crystalline
materials. The peak area above the
background curve was 50.6%. The
49.4% below the curve indicated
mat WTC3 consisted of about half
amorphous materials. Four
patterns were identified as being
consistent with peaks identified in
the dust. Figure 3 shows the XRD
spectra of WTC3 and those of the
matched compounds. Two
compounds were identified as
major constituents (calcium
carbonate (calcite) and calcium
sulfate dihydrate (gypsum)), and
two were identified as minor
constituents (bassanite and quartz,
Table 5). The XRD data are
16
-------
Figure 4. SEM/EDX results from water-extracted lyophilized WTC PM samples. The upper-left
quadrant of each photomicrograph shows a field of view with the particle of interest within the
smaller square in that field. The upper-right quadrant shows a zoomed-in view of the feature (the
area from within the square in the upper-left quadrant), and the lower half shows the elemental
spectrum acquired with the electron beam centered on the small (barely visible) square in the
zoomed-in view. A. Example of Ca-S crystal which dominated the samples. B. Example of fine
particle aggregate which was prominent in the samples. C. Example of fiber found in the samples.
D. Example of metallic particle within fine particle aggregate.
consistent with the TCP data which show water-soluble
calcium and sulfate in the same proportions as gypsum.
Gypsum is completely water-soluble at the solid/liquid
ratio of the extraction conditions used in the ICP analysis,
while calcite is not water-soluble. The sample of MSH
was also analyzed by XRD and the results were consistent
with those previously reported (Graham et al., 1985; data
not shown).
5. SEM/EDX analysis. Water-extracted and
lyophilized WTC PM samples were dominated by
snowflake-like crystals composed of calcium and sulfur
(Figure 4A). Aggregates of fine particles composed of
various combinations of Mg, Al, Si, S, and Ca were also
prominent (Figure 4B). Fibers approximately 1 |im in
diameter were found in most of the samples and had a
composition similar to the fine particle aggregates (Figure
4C). Metallic particles (mostly Ti and Fe, though Zn, Pb,
Ba, and Cu were also found) were found typically as
inclusions in the large fine particle aggregates (Figure 4D).
The crystals and aggregates were likely not original to the
bulk sample but were formed as a result of the aqueous
extraction process.
SEM/EDX analysis of the aerosolized PM25 cut
fraction of WTC3 showed the same overall chemistry as
the extracted and lyophilized WTC PM samples: the
majority of particles were composed of Ca or Ca-S, some
17
-------
Figure 5. Particle types found in the WTC3 sample used in the nose-only inhalation exposure
(Experiment B). A. Example of Ca-S particle which was prominent in the sample. B. Example of
Ca particle which was prominent in the sample.
also containing Si. Some representative particles are
shown in Figures 5 A and 5B. In contrast to the crystals
and aggregates of the bulk solid samples as described
above and shown in Figure 4, the particles of WTC3 are
small, typically about 1 urn, with rough, irregular features.
The different form of the Ca-based particles in WTC3
reflects the dry size segregation of the inhalation exposure
system. Particles with other compositions were found
with far less frequency. These included particles
composed of Fe, C, and Sb-Zn (one example found). One
or more possible asbestos fibers (Mg-Si composition) were
also found, howeverpolarized light microscopy ratherthan
SEM/EDX is the preferred method for identifying
asbestos. SEM/EDX analysis was also performed on
MSH, NIST 1649a, and ROFA and showed results
typically found in previous analyses (data not shown).
6. Summary. WTC PM samples consist primarily of
construction materials from the fallen-down WTC
buildings. The bulk of the WTC PM samples are calcium-
based compounds, specifically calcium sulfate (gypsum)
Group
Saline
MSH- 100
ROFA- 10
ROFA- 100
WTCX-10
WTCX-31.6
WTCX-100
Table 6.
B.Wt. d 0
g
25.8
0.4
25.5
0.4
25.6
0.4
25.2
0.5
25.2
0.6
25.3
0.5
25.6
0.6
Experiment A
B.Wt. d 1
g
24.9
0.4
25.0
0.3
24.3
0.6
25.2
0.4
24.3
0.5
24.7
0.5
25.1
0.4
Body Weights and Immediate
Breathing Frequency (min"1)
Pre- Post- % increase
492.3 348.1 -29.7
11.5 25.6 4.2
474.0 320.6 -31.7
13.5 23.0 5.2
492.0 343.5 -29.7
14.3 18.6 4.1
461.0 307.0 -33.4
11.9 18.7 3.7
467.2 322.4 -31.1
14.7 22.5 4.1
476.8 348.5 -26.3
15.5 18.9 4.3
486.8 325.3 -33.1
14.1 26.6 5.1
Airway
Pre-
0.73
0.08
0.92
0.13
0.74
0.11
0.88
0.10
0.85
0.15
0.86
0.18
0.79
0.08
Responses. a
PenH (unitless)
Post- %
0.96
0.17
1.25
0.18
1.05
0.17
1.51 j~~
0.20 |_
1.14
0.18
1.14
0.34
1.11
0.16
increase
23.9
8.3
40.5
14.8
42.3
13.5
76.4 1
18.6 1
52.4
21.8
26.8
15.6
40.7
11.8
Values shown are means (in bold) and SEM immediately below means (n=12 per group). Body weight (B. Wt.)
was measured in the morning. Respiratory parameters were measured immediately before (Pre-) and after (Post-)
oropharyngeal aspiration of dust samples or saline on day 0. Values within solid-line boxes indicate significantly
greater values in ROFA-100 mice vs. Saline mice (P < 0.05).
18
-------
and calcium carbonate (calcite). Together these salts
compose about two-thirds of WTC PM53 on a weight
percent basis. Given the prevalent use of gypsum in
ceiling tiles and wallboard, and the ease with which these
building materials can be crumbled into dust, the high
gypsum content is reasonable. Elemental analysis
indicates that the other main components of WTC PM are
construction materials such as cement and concrete
aggregate. The elemental composition of WTC PM25 was
consistent with that of sieved unfractionated WTC PM53
(as WTC3, Table 5). Carbon and metal content of the
WTC samples were low, as expected from crustal-derived
building materials (McKetta, 1978). A more complete
chemical and physical analysis of dust samples has
recently been reported by the U.S. Geological Survey
(USGS, 2002). In that study, dust samples were collected
from undisturbed locations within a 1 km radius of the
WTC site on September 17 and 18,2001 (after the rain of
September 14,2001). The present report generally agrees
with the findings of the USGS study, including the
alkaline nature of the WTC PM extracts.
B. Experiment A: Dose-Response Relationships of
WTC PM2 5
1. Body weights and immediate airway responses.
Table 7. Experiment A: Diffusing Capacity of the Lung for
Carbon Monoxide (DLCO)a
Sub-experiment:
Date:
Treatment Group
Saline
MSH-100
ROFA-10
ROFA-100
WTCX-10
WTCX-31.6
WTCX-100
Al
11/6/01
-3.474
-3.734
-3.904
-4.015
-2.759
-4.051
-3.551
A2
11/8/01
-3.610
-3.944
-3.597
-3.089
-3.811
-3.338
-4.433
A5
12/6/01
-4.510
-4.338
-3.928
-4.293
-4.301
-4.014
-4.299
Mean
n=3
-3.865
-4.005
-3.810
-3.799
-3.624
-3.801
-4.094
SEM
0.325
0.177
0.107
0.364
0.455
0.232
0.275
a Diffusing capacity of the lung for carbon monoxide was determined
one day after exposure on four mice from each treatment group, placed
together in a single bell jar, in order to rapidly assess DLCO and
reduce individual variability. Values shown are slopes of chamber
[CO] vs. time (ppm/min), after subtraction of value from empty
chamber. No significant differences among any treatment groups were
detected.
Mice were exposed by oropharyngeal aspiration with
PM25 samples of pooled WTC sample X (10, 31.6, or 100
Hg), MSH (100 ng), ROFA (10 or 100 ng), or saline on
day zero. In sub-experiments Al, A2, and A5, immediate
airway responses were determined on day 0, and DLCO
Table 8. Experiment A: BAL Parameters (Day 1). a
Group
Saline
MSH-100
ROFA-10
ROFA-100
WTCX-10
WTCX-31.6
WTCX-100
BAL Cell Number (x lO'4)
Mac
18.80
4.05
27.78
5.96
27.36
2.70
28.69
3.98
22.28
4.03
31.36
7.73
20.48
1.73
Eos
0.02
0.02
0.07
0.03
0.00
0.00
0.16
0.06
0.00
0.00
0.01
0.01
0.06
0.03
Neut
0.23
0.16
0.78
0.21
0.21
0.05
13.18
2.44
0.09
0.02
0.37
0.14
1.43
_ _Q-24 _
Lym
0.10
0.02
0.18
0.05
0.18
0.07
0.42
0.09
0.14
0.04
0.21
0.03
0.23
0.04
Protein
US/ml
155.2
6.8
168.8
8.8
157.9
5.3
279.5
16.8
153.7
4.3
160.2
6.3
161.4
4.8
LDH
U/L
29.8
2.1
27.8
1.7
32.3
1.2
93.2
10.3
30.4
1.8
33.6
1.6
33.7
2.1
Albumin
US/ml
21.8
1.2
22.3
1.2
20.8
0.9
39.2
2.8
20.8
0.8
21.7
1.3
21.3
1.0
NAG
U/L
2.2
0.4
2.0
0.4
3.1
0.4
7.9
1.2
1.9
0.3
1.8
0.2
2.3
0.3
Values shown are means (in bold) and SEM immediately below means (n= 12 per group). Bronchoalveolar lavage
(BAL) cell numbers and proteins were recovered 1 day after exposure. Cell types shown are macrophages and
monocytes (Mac), eosinophils (Eos), neutrophils (Neut), and lymphocytes (Lym). Total protein, lactate
dehydrogenase (LDH), albumin, and N-acetyl-|3-D-glucosaminidase (NAG) were measured in BAL fluid
supernatant. Values within solid-line boxes indicate significantly greater values in ROFA-100 mice vs. Saline
mice (P < 0.05). Values with dashed-line boxes indicate significantly greater values (P < 0.05) compared with
Saline mice (excluding ROFA-100 data which generally had much larger variances than other groups).
19
-------
50-,
40-
TT
o
30-
0-
Mac
T
T
T
I
16-,
12-
4*
U
4-
0-L
Neut
Saline
MSH-100
ROFA - 10
^H ROFA - 100
EZZZZ2WTCX-10
ESSS3WTCX-31.6
WTCX-100
Eos
Lym
1.0-,
.0.6-
0.4-
0.2-
I Saline
I MSH-100
I ROFA - 10
^H ROFA - 100
EZZZZ2WTCX-10
ESSSS3WTCX-31.6
I WTCX-100
1.0-,
0.8-
,0.6-
0.4-
0.2-
0.0J
I Saline
I MSH-100
I ROFA - 10
^H ROFA - 100
EZZZZ3WTCX-10
Figure 6. Experiment A. Bronchoalveolar lavage cell numbers recovered from mice one day after exposure by intratracheal
instillation to PM samples in saline or saline vehicle alone. Values shown are means and SEM (n=12 per group). Cell types shown
are macrophages and monocytes (Mac), eosinophils (Eos), neutrophils (Neut), and lymphocytes (Lym). a P < 0.05 vs. Saline group.
bP < 0.05 vs. Saline group (comparison of rank values) after exclusion of ROFA-100 data which had much larger variances than other
groups.
and BAL parameters were determined on day 1. There
were no significant differences in body weights of the
seven groups on day 0 or day 1 (Table 6). Ventilatery
parameters were assessed in mice immediately before and
after exposure. There were no differences among groups
in breathing frequency, but mice exposed to the 100 |o,g
dose of ROFA (ROFA-100) had a significant increase in
PenH immediately after exposure in comparison with
saline control mice (Table 6). There were no significant
changes in immediate responses in mice exposed to any
dose of WTCX.
2. DLCO. The diffusing capacity of the lung for
carbon monoxide was determined 24 hr after
oropharyngeal aspiration on groups of 4 mice from the
same exposure group together in the testing chamber.
There were no significant differences in DLCO among any
of the groups of mice, which would be indicated by a
slower uptake of CO and a reduced slope (Table 7). These
data indicate that none of the PM samples caused injury
severe enough to significantly reduce gas exchange at the
alveolar-capillary barrier.
3. BAL parameters. Bronchoalveolar lavage
parameters were determined immediately after testing for
DLCO. Originally, we planned to do just two sub-
experiments for this part of Experiment A. However, we
noted that there was significant variation in the total cell
numbers recovered from the 4 saline control mice in each
of sub-experiments Al and A2 (average of 7 x 104 vs. 33
x 104, respectively). There was no evidence of any
infection (in both cases 99% of BAL cells recovered from
control mice were alveolar macrophages (AMs)), mice
came from the same shipment in the same week, and no
20
-------
Protein
300 n
^ 250 -_
a :
~ei 200 -j
^ 150~
"S
g looq
a.
so ^
I Saline
I MSH - 100
I ROFA - 10
^H ROFA - 100
EZZZZ3WTCX-10
ESSS3WTCX-31.6
IWTCX-100
Albumin
50-,
25-
.a
0J
Saline
MSH-100
ROFA - 10
^H ROFA - 100
EZZZZ2WTCX- 10
WTCX-31.6
WTCX-100
LDH
NAG
120-,
80-
40-
I Saline
I MSH - 100
I ROFA - 10
^H ROFA - 100
ZZZZZaWTCX-10
WTCX-100
10-,
Q
-J
4-
0J
I Saline
I MSH-100
I ROFA - 10
^H ROFA - 100
EZZZZ2WTCX-10
ESSS3WTCX-31.6
IWTCX-100
Figure 7. Experiment A. Values for total protein, lactate dehydrogenase (LDH), albumin, and N-acetyl-b-D-glucosaminidase (NAG)
were measured inbronchoalveolar lavage fluid supernatants recovered from mice one day after exposure by intratracheal instillation
to PM samples in saline or saline vehicle alone. Values shown are means and SEM (n=12 per group). a P < 0.001 vs. Saline group.
other reason could be deduced for the difference.
Consequently, we performed a third sub-experiment to
examine these endpoints and increase the number of mice
per group (sub-experiment A5). The average total cell
number recovered from saline control mice in A5 was 18
x 104 (97% AMs) - about in the middle between Al and
A2. All data shown is combined from the 3 sub-
experiments. Due to the high variance of data in the
ROFA-100 group, we judged that it was necessary to
compare ROFA-100 data alone vs. saline control data.
Other comparisons were made between the saline control
group and the other groups after excluding ROFA-100
data. Significant increases in neutrophils, eosinophils, and
lymphocytes were found in ROFA-100 mice compared
with saline control mice (Table 8). Neutrophils comprised
31% of total BAL cells in ROFA-100 mice (Figure 6).
After excluding the ROFA-100 data, significant
differences in neutrophil numbers were found between the
saline control group and both the MSH-100 group and the
WTCX-100 group (Table 8, Figure 6; P < 0.05).
Neutrophils comprised about 7% of total BAL cells in the
WTCX-100 group, but only about 1% or less in the
WTCX-31.6 and WTCX-10 groups.
Levels of proteins and enzymes were measured in the
BAL supernatantto assess lung damage. Both total protein
and albumin are increased after damage to the alveolar
epithelial barrier (Henderson et al., 1985). Lactate
dehydrogenase (LDH) is a cytoplasmic enzyme which is
released by dead or dying cells, while N-acetyl-p-D-
glucosaminidase (NAG) is indicative of lysosomal enzyme
release (Henderson et al., 1985). All of these parameters
were significantly increased in the ROFA-100 group in
comparison to saline control mice (Table 8). Total protein
and albumin were both increased about 80% compared to
saline, while LDH was increased 3-fold and NAG almost
4-fold (Figure 7). No significant changes in BAL proteins
21
-------
Table 9.
Group
Saline
MSH-100
ROFA-10
ROFA-100
WTCX-10
WTCX-31.6
WTCX-100
Experiment A: Body
B.Wt. dO B.Wt. dl
(g) (g)
24.78
0.42
24.82
0.51
24.99
0.43
24.86
0.44
24.96
0.38
24.56
0.46
24.68
0.44
23.30
0.65
23.80
0.45
24.55
0.45
24.60
0.45
24.44
0.28
23.73
0.51
24.14
0.42
Weights,
Baseline
PenH
0.97
0.13
1.10
0.12
0.81
0.10
0.91
0.11
0.92
0.11
0.85
0.11
1.04
0.12
Baseline
0
0.5
0.5
2.4
0.3
15.5
3.2
6.1
6.3
18.5
6.4
15.3
3.4
9.5
8.2
PenH, and Responsiveness to Methacholine Aerosol
Dose Mch (nig/ml) and PenH AUC (PenH - sec)
4 8 16
39.1 54.0 142.3
8.4 8.9 28.5
36.4 99.2 177.9
5.5 22.5 23.7
42.4 114.6 205.5
8.9 11.3 56.1
36.7 118.2 242.1
4.1 34.1 37.2
43.9 88.3 169.1
11.6 16.2 52.3
38.3 55.2 114.4
4.8 8.8 7.0
67.4 208.3 397.3
11.3 47.6 61.4
32
309.0
30.6
437.9
54.3
432.4
88.5
642.2
94.0
281.1
35.5
249.2
39.8
2265.0
260.7
64
1249.9
360.7
1173.3
358.3
1182.5
294.2
2190.9
706.4
968.4
129.9
923.3
174.2
4009.1
580.1
Values shown are means (in bold) and SEM immediately below means (n=8 per group). Body weight (B. Wt.) was measured
in the morning (no significant differences were found). No significant differences were found in baseline PenH (enhanced
pause; unitless) on day 1. Methacholine aerosol (Mch) was then administered (see Methods for details) at the indicated doses,
and the airway response was calculated as the area under the curve (AUC) of the PenH response over time in seconds. See
Figure 8 for description of statistical analysis of PenH AUC data.
and enzymes were found in any of the other PM exposure
groups relative to saline controls. In general, the
inflammatory response in the WTCX-100 group can be
considered to be quite mild considering the fairly high
dose.
4. Responsiveness to methacholine aerosol. In sub-
experiments A3 and A4, the same groups of mice were
exposed and the same time points were examined as
described for sub-experiments Al, A2, and A5, but
different endpoints were examined. There were no
differences in body weights on day 0 or day 1 among the
7 groups of mice (Table 9). On day 1, there was no
difference in baseline PenH values (immediately before
Mch aerosol) among the 7 groups. Responsiveness to
increasing concentrations of Mch aerosol was assessed and
quantified by integrating the area under the PenH - time
curve (PenH AUC; Table 9). In order to assess overall
responsiveness and account for variability, power function
equations were fit to the PenH AUC vs. [Mch] data for
each group (Figure 8). The analysis showed that the
Saline, MSH, ROFA-10, WTCX-10, and WTCX-31.6
groups could all be modeled with a common power
function exponent (1.157). It is important to note that
once the lines were determined to come from groups with
a common exponent, the lines for these 5 groups were fit
simultaneously, resulting in fitted equations that did not fit
as well as an individual line would fit the group-specific
data. The responses to saline or individual doses of Mch
aerosol are not as important as the fitted line describing
the groups. Among these 5 groups, ROFA-10 mice had a
small but significant increase in the coefficient of the
equation vs. the Saline group CP = 0.03). The ROFA-100
and WTCX-100 groups could be modeled with a power
function with a significantly different exponent (1.471; P
= 0.001) vs. the common exponent of the other 5 groups,
indicating that these 2 groups are hyperresponsive
compared with the other 5 groups. In addition, the
coefficient for the WTCX-100 group was significantly
different from and greater than that of the ROFA-100
group (P = 0.0001), showing that mice exposed to the 100
|o,g dose of WTCX were more reactive to Mch than the
ROFA-100 group.
5. Lung histopathology. Following tests for airway
responsiveness to Mch aerosol, mice from sub-
experiments A3 and A4 were killed and assessed for
pathological changes in the lungs. No remarkable findings
were observed in the lungs of the saline control group
(Table 10). In both the MSH-100 and ROFA-100 groups,
focal subacute bronchiolar inflammation was found at
similar incidences and average severity, which was
minimal (average score: MSH-100 = 0.8; ROFA-100 =
1.0). The ROFA-10 group had a lower average severity
22
-------
C/3
U
4000-
3000-
2000-
1000-
Saline
Saline
PenHAUC = 5.726*[Mch]A1.157
4000-
3000-
2000-
1000-
WTCX10
WTCX10
- PenHAUC = 5.726*[Mch]A1.157
4000-
3000-
2000-
1000-
WTCX31.6,WTCX100
WTCX31.6
PenHAUC = 5.726* [Mchf 1.157
WTCX100 c a
•PenHAUC = 8.482* [Mchf 1.471
• MSH
PenHAUC = 7.178*[Mch]A1.157
ROFA10
ROFA10 jj
PenHAUC = 7.538* [Mchfl. 157
ROFA100
• ROFA100
a
- PenHAUC = 3.975*[Mch]A1.471
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
[Mch] (mg/ml)
Figure 8. Experiment A: Airway responsiveness to methacholine aerosol challenge in mice exposed to PM samples or saline vehicle
and tested one day later (n = 8/group; data shown are mean + SEM). Power function equations were fit to the data. Saline, MSH,
ROFA10, WTCX10, and WTCX31.6 equation exponents were not significantly different. a Significantly different exponent vs.
common Saline, MSH, ROFA10, WTCX10, and WTCX31.6 exponent (P = 0.001). b Significantly different coefficient vs. Saline
coefficient (P = 0.03). c Significantly different coefficient vs. ROFA100 coefficient (P = 0.0001).
23
-------
Table 10. Experiment A: Summary of Treatment-Related Histopathologic Findings in Mice One Day after
Intratracheal Instillation of Paniculate Matter Samplesa
Treatment
Group
Saline
MSH-100
ROFA-10
ROFA-100
WTCX-10
WTCX-31.6
WTCX-100
Bronchiole,
Inflammation,
Subacute, Focal
Incidence Severity
0/8 0.0
6/8 0.8
2/8 0.3
6/8 1.0
1/8 0.1
0/8 0.0
0/8 0.0
Bronchiole,
Pigment,
Free, Focal
Incidence Severity
0/8 0.0
0/8 0.0
6/8 0.8
8/8 1.5
0/8 0.0
1/8 0.1
0/8 0.0
Bronchiole,
Pigment,
Macrophage, Focal
Incidence Severity
0/8 0.0
2/8 0.3
0/8 0.0
0/8 0.0
0/8 0.0
0/8 0.0
0/8 0.0
Peribronchiolar,
Inflammation,
Acute, Focal
Incidence Severity
0/8 0.0
0/8 0.0
1/8 0.1
0/8 0.0
0/8 0.0
0/8 0.0
0/8 0.0
Incidence denotes number of mice in group with finding / total number of mice examined. Average severity score for
the group is shown based on the following scoring system: 0 = not present, 1 = minimal, 2 = slight/mild, 3 = moderate,
4 = moderately severe, 5 = severe/high.
score (0.3) than the MSH-100 and ROFA-100 groups, and
also had one mouse with minimal focal acute
peribronchiolar inflammation. Although 1 mouse in the
^ /"* ~-r- **• i "\' •••
'./
^
r*'
r •/ ^ i. •-
-^ ' ^
WTC-X group had a finding of minimal focal subacute
bronchiolar inflammation, for an average group score of
0.1, this lesion was not found in any of the mice in the
£il
Figure 9. Experiment A: Representative micrographs of lesions occuring in lungs of mice one day after
intratracheal instillation of PM samples or saline vehicle (all panels same magnification: bar length =100 um).
A. Saline-instilled control mouse (#69) with no remarkable findings. B. Mouse #84 instilled with 100 ug
MSH showing minimal degree of focal subacute bronchiolar inflammation. C. Mouse #57 instilled with 100
ug pooled WTCX sample with no remarkable findings. D. Mouse #73 instilled with 100 ug ROFA showing
slight/mild degree of focal subacute bronchiolar inflammation.
24
-------
WTCX-31.6 orWTCX-100 groups (Figure 9), suggesting
that the lesion in the one WTCX-10 mouse was not
treatment-related. Free bronchiolar pigment (presumably
corresponding to PM) was identified in all ROFA-100
mice at an average severity of 1.5 (Table 10), and in 6 of
8 mice in the ROFA-10 group at an average severity of
0.8. One mouse in the WTCX-31.6 group (but none in the
WTCX-100 group) had minimal free bronchiolar pigment;
again suggesting that this finding is not treatment-
dependent. However, it may be more difficult to see the
WTC PM which is lighter in color than the ROFA or MSH
PM. Focal bronchiolar macrophage pigment was found in
2 of 8 mice in the MSH-100 group at an average severity
of 0.3. These findings indicate that both ROFA-100 and
MSH-100, but not the pooled WTCX-100 or any lower
dose, caused focal subacute bronchiolar inflammation.
6. Summary. Results from investigation of the dose-
response relationships of pooled WTCX PM showed that
the two lower doses of WTCX (10 jog and 31.6 |o,g) did not
have any significant effects on inflammatory parameters,
lung histopathologic findings, or respiratory responses.
The 100 |o,g dose of WTCX caused a slight but significant
increase in BAL neutrophils (7% of total cells) as
determined by BAL parameters, and no inflammation as
determined by histopathologic examination, while the
toxic PM control, ROFA, caused minimal inflammation by
histopathologic examination, significant increases in BAL
neutrophils and other cell types, and significant increases
in biochemical indicators of lung injury. Despite the lack
of effect of WTCX on lung injury and the relatively low
level of neutrophilic inflammation, mice in the WTCX-
100 group were significantly more responsive to Mch
aerosol challenge than all other groups. A lack of
correlation between lung inflammation and airway
hyperresponsiveness is not uncommon (e.g. Alvarez et al.,
2000; Smith and McFadden Jr., 1995). The significant
degree of airway hyperresponsiveness induced by WTC
PM25 implies that components of the dust can promote
mechanisms of airway obstruction.
C. Experiment B: Effects of Nose-Only Inhalation
Exposure
1. Exposure results. The gravimetric concentration
for the WTC3 exposure chamber was 10.64± 3.10mg/m3.
The mass median aerodynamic diameter (MMAD) was
1.05 |om, and the geometric standard deviation (ag) was
2.67. Chamber temperature and relative humidity was 74
°F and 11% in the control chamber and 75 °F and 11% in
the WTC3 chamber. The low humidity was required to
prevent the PM from sticking to the string in the aerosol
generation system; the humidity within the exposure tubes
was significantly higher due to body heat from the mice in
a confined environment. At the end of the exposure, two
control mice (#131 and #146) and one WTC3-exposed
mouse (#203) were found dead in the exposure tubes,
apparently from attempting to turn around in the exposure
tubes and suffocating. The incidence of this problem was
not unusual considering the large number of mice exposed
simultaneously (AD Ledbetter, personal communication).
The two spare mice (designated #146a and #203a) were
used to replace the dead ones, and were killed on day 6
(December 3, 2001). An additional control mouse
(designated #13la) was exposed to air for 5 hours on
November 30, 2001 to replace the second dead control
mouse, and was killed on day 3 (December 3, 2001).
Therefore all groups of mice had the full number of 8 per
group per time point.
Table 11. Experiment B: Body Weights of Mice in Nose-Only
Inhalation Exposure Studya
Day:
Group
Air
WTC 3
Body Weight (g)
-1
23.91
0.16
23.93
0.15
0
(Pre-)
23.81
0.18
23.47
0.18
0.25
(Post-)
21.83
0.18
21.82
0.16
1
23.00
0.18
23.05
0.18
3
24.04
0.23
23.89
0.15
6
24.78
0.35
24.66
0.34
Values shown are means (in bold) and SEM immediately below
means (n=48 days -1 through 1, n=32 day 3, n = 16 day 6). Body
weight was measured in the morning except on day 0.25
(immediately after nose-only exposure). There was no significant
difference between the two groups.
Nose-only exposure
20
-2 -1
1234
Days after Exposure
Figure 10. Experiment B: Body weights in nose-only inhalation
exposure experiment. Values shown are means and SEM
(numbers of mice shown in Table 11). Nose-only inhalation
exposure caused a significant drop in body weight but there was
no significant difference between groups.
25
-------
Table 12.
Group
Air
WTC3
Experiment
B: Immediate Airway Responses a
Breathing Frequency (min1)
Pre-
549.2
9.1
560.3
10.3
Post-
357.7
24.4
389.3
19.0
% increase
-35.0
4.1
-30.1
3.9
PenH (unitless)
Pre-
0.88
0.05
0.94
0.06
Post-
1.09
0.09
1.47
0.16
% increase
29.5
13.8
60.1
18.2
Values shown are means (in bold) and SEM immediately below means (n=12). Respiratory
parameters were measured immediately before (Pre-) and after (Post-) nose-only inhalation
exposure on day 0. No significant differences in percent change in frequency or PenH between
groups were found.
2. Body weights. Animal weights were monitored on
days -1 (before exposure), 0 (both before and after
exposure), 1, 3, and 6 (Table 11). Body weight was
measured between 7:00 and 8:00 each morning, except
immediately after exposure. There were no significant
differences between the two groups at any time point. The
nose-only exposure caused a significant 2 g drop in body
weight in both groups of mice (Figure 10).
3. Immediate airway responses to nose-only
exposure. Ventilatory parameters were measured in 12
mice from each group before and after the nose-only
exposure. Ventilatory rate decreased after exposure in
both groups but there was no significant difference
between them (Table 12). It should be noted that many
physiological responses are readily reversible, and the time
required to unload the mice from the exposure tubes and
C/5
C/5
2.5n
2.0-
Air
£ 1.5-
« 1.0-
0.5-
0.0-
Pre-
Post-
C/5
C/5
2.5n
2.0-
WTC3
£ 1.5-
« 1.0-
0.5-
0.0-
Pre-
Post-
Figure 11. Experiment B. PenH values measured immediately before and after nose-only exposure to WTC 3 PM or Air only.
Legends refer to individual mouse numbers. Immediate response (calculated as [(Post-value -Pre-value) /Pre-value x 100%] was not
significantly different between the two groups but data indicate the possibility of individual sensitivity to dust exposure.
26
-------
Table 13. Experiment B: Diffusing Capacity of the Lung for
Carbon Monoxide a
Treatment
Group
Air
WTC3
Air
WTC3
Air
WTC3
Day after
Treatment
1
1
3
3
6
6
Subjects
1-4
-3.761
-3.715
-4.102
-3.818
-3.528
-3.487
Subjects
5-8
-4.185
-3.981
-3.826
-4.078
-4.162
-3.809
Average
n = 2
-3.973
-3.848
-3.964
-3.948
-3.845
-3.648
Diffusing capacity of the lung for carbon monoxide was determined
1, 3, or 6 days after exposure on four mice from each treatment
group placed together in a single bell jar. Values shown are slopes
of chamber [CO] vs. time (ppm/min), after subtraction of value from
empty chamber.
begin the measurement of breathing parameters (~20
minutes) may have caused us to miss some changes. PenH
was increased by an average of 30% after exposure to air
and by an average of 60% after exposure to WTC3 (P =
0.20). Although this difference was not significant,
examination of the changes in individual mice showed that
PenH increased in all 12 mice exposed to WTC3, but only
8 of 12 mice exposed to Air (Figure 11). Furthermore
some of the increases in WTC3-exposed mice were quite
large. These data indicate the possibility that individual
mice in this outbred strain may be susceptible to
bronchoconstrictive effects of WTC PM.
4. DLCO measurements. DLCO was determined 1,
3, and 6 days after exposure on 4 mice from each group
placed together in the test chamber. Since there were 8
mice per group per time point, only two tests of DLCO
were conducted within each group, and no statistical
comparison was possible between Air and WTC3 mice.
Examination of the data showed little apparent difference
in DLCO at different times in the two groups (Table 13).
5. Responsiveness to methacholine aerosol. Analysis
of baseline PenH values (immediately before Mch aerosol)
between the two groups showed a significant difference
depending on day, but not due to treatment (day 6
baselines were lower in both groups; P = 0.0007; Table
14). Responsiveness to increasing concentrations of Mch
aerosol was assessed and quantified as described in the
Methods section (Table 14). Unlike Experiment A, the
results could be modeled with linear equations (Figure 12).
Significant interactions of treatment, day, and Mch
concentration were detected (P = 0.01), implying that the
results depended upon a combination of factors. Slopes of
the Day and Treatment combinations were significantly
different (P = 0.0001). Analysis of the data showed that
one equation could be used to describe the data for Air
Day 6 and for WTC3 Day 1 (Figure 12). The slope of this
line was significantly different from and less than that of
Table
Treatment
Group
Air
WTC 3
Air
WTC 3
Air
WTC 3
14. Experims
Day after
Treatment
1
1
3
3
6
6
;nt B: Baseline PenH
Baseline
PenH
0.80
0.04
0.68
0.05
0.76
0.05
0.86
0.07
0.61
0.05
0.58
0.04
0
-1.5
10.8
21.8
7.9
-13.7
9.8
-11.4
9.7
-3.5
3.8
2.0
2.9
and Responsiveness to Methacholine Aerosol a
Dose Mch (me/ml) and PenH AUC (PenH - sec)
4
20.0
15.3
18.8
20.8
50.4
20.9
15.2
8.3
-7.6
12.4
31.1
9.0
8
61.5
41.7
88.9
12.7
238.4
68.7
37.8
12.6
35.5
11.7
72.0
20.4
16
262.0
71.4
227.2
50.0
384.9
77.4
198.6
108.8
106.5
43.6
276.7
97.1
32
945.9
227.1
697.8
175.4
1188.1
172.5
679.1
317.2
70.1
59.5
1030.4
252.8
64
1940.7
597.3
492.3
104.0
2031.4
178.8
1293.3
356.6
786.6
269.9
1935.6
385.1
Values shown are means (in bold) and SEM immediately below means (n=8 per group). Baseline PenH (enhanced
pause; unitless) was measured immediately before methacholine aerosol challenge. No significant differences were
found between treatment groups, but there was a significant difference in day, with day 6 values being significantly
lower than other days (solid line box; P = 0.0007). Methacholine aerosol (Mch) was administered (see Methods
for details) at the indicated doses, and the airway response was calculated as the area under the curve (AUC) of
the PenH response over time in seconds. See Figure 12 for description of statistical analysis of PenH AUC data.
27
-------
o
CD
t/3
CD
CD
O
1
o3
I
CD
PH
2500:
2000-j
1500:
Air Day 1
Air Day 1
- PenH AUC = 30.53* [Mch] -122.6
2500n
2000-
1500-j
1000-j
500-
AirDay 3
• Air Day 3
PenH AUC = 30.53* [Mch] -122.6
2500-]
2000-
1500-
1000-
AirDay 6
Air Day 6
a
• PenH AUC = 10.72* [Mch] - 17.76
WTC3 Day 1
• WTC Day 1
a
PenH AUC = 10.72* [Mch] -17.76
WTC3 Day 3
• WTC Day 3
PenH AUC = 30.53* [Mch] -122.6
WTC3 Day 6
WTC Day 6
- PenH AUC = 30.53* [Mch] -122.6
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
[Mch] (mg/ml)
Figure 12. Experiment B: Airway responsiveness to metnacholine aerosol challenge in mice exposed nose-only to Air or aerosolized
WTC (sample 3) and tested 1, 3, or 6 days later (n = 8/group; data shown are mean + SEM). Linear dose-response relationships were
found. a Slope of Air Day 6 and WTC3 Day 1 were significantly different from and lower than the 4 other groups.
the equation used to fit the other four groups. It should be lines were fit simultaneously. This resulted in an equation
noted that as in Experiment A, once the lines were for the common groups (e.g. Air Day 6andWTC3 Day 1)
determined to come from groups with equal slopes, the that did not fit as well as lines fit to the individual group
28
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Table 15. Experiment B: BAL Cell Numbers after Nose-Only Exposure '
Group
Air
WTC3
Air
WTC3
Air
WTC3
Day
1
1
3
3
6
6
BAL Cell Number Cx 10'4)
Mac
14.80
3.11
17.48
3.13
16.56
1.13
26.72
3.11
22.24
1.13
129.86
2.58
Neut
0.012
0.004
0.006
0.003
0.008
0.004
0.034
0.017
0.000
0.000
0.019
0.008
Eos
0.003
0.002
0.000
0.000
0.000
0.000
0.016
0.009
0.000
0.000
0.005
0.004
Lym
0.046
0.010
0.067
0.013
0.125
0.041
| 0.197 |
|_ 0.036 _|
0.140
0.026
| 0.281 |
|_ 0.056 _|
Values shown are means (in bold) and SEM immediately below means (n=8 per
group). Solid-line box: Significant difference (P = 0.01) between Air and WTC3,
Day 6 different from Day 1. Dashed-line boxes: Significant difference (P = 0.02)
between Air and WTC3, Day 3 and Day 6 both different from Day 1.
data. These results could be interpreted as saying that
mice exposed to WTC3 became more responsive to Mch
in the days following exposure, while Air-exposed mice
became less responsive. However, close
examination of the data from experiment B
showed it was more variable than that from
experiment A. Therefore, although the Air Day 6
and WTC3 Day 1 groups were less responsive to
Mch aerosol challenge than the other four groups,
the biological significance of this finding is
unclear.
6. BAL parameters. Numbers of BAL cells
were quantified 1,3, and 6 days after nose-only
exposure to Air or WTC3 (Table 15). Analysis of
the data showed that mice exposed to WTC3 had
significantly greater numbers of macrophages (P
= 0.01) and lymphocytes (P = 0.02) compared
with Air-exposed mice. Macrophage numbers
were significantly greater on Day 6 vs. Day 1, and
lymphocyte numbers were significantly greater on
both Day 3 and Day 6 vs. Day 1. Over all time
points, WTC3 mice had 38% more macrophages,
and 75% more lymphocytes (Figure 13).
However, macrophages comprised 99% of all recovered
cells in both groups at all time points. Lymphocytes
constituted about 1% or less of total BAL cells, while both
Mac
0.6-,
0.4-
0.2-
Day 6
IZZlAir
0.6-1
0.4-
0.2-
Neut
Air
WTC3
Day 1 Day 3 Day 6 Day 1 Day 3 Day 6
Figure 13. Experiment B: Bronchoalveolar lavage cell numbers recovered from mice 1, 3, or 6 days after 5 hr
nose-only inhalation exposure to WTC sample 3 or Air only. Values shown are means and SEM (n=8 per group).
Cell types shown are macrophages and monocytes (Mac), eosinophils (Eos), neutrophils (Neut), and lymphocytes
(Lym). a Significant difference (P = 0.01) between Air and WTC3, Day 6 different from Day 1. b Significant
difference (P = 0.02) between Air and WTC3, Day 3 and Day 6 different from Day 1.
29
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Table 16. Experiment B:
Nose-Only Exposure a
BAL Supernatant Biochemical Values after
Group
Air
WTC3
Air
WTC3
Air
WTC3
Day Protein Albumin LDH
|ig/ml |-ig/ml U/L
1 165.2 21.0 29.0
6.1 1.1 3.4
1
147.1
6.7
16.9
1.2
23.9
3.3
3 136.6 16.2 33.0
10.4 1.2 6.4
3
138.1
7.8
15.8
1.5
28.9
3.4
6 172.6 22.4 30.2
8.5 1.3 2.4
6
146.5
6.8
17.5
1.1
27.1
3.1
NAG
U/L
1.5
0.1
1.6
0.1
1.8
0.0
1.6
0.1
1.4
0.2
1.4
0.1
Values shown are means (in bold) and SEM immediately below means (n=8 per
group). Heavy solid-line boxes: Significant overall treatment effect (WTC3 < Air;
no significant day effect); P = 0.05 (Protein) or P = 0.007 (Albumin).
neutrophils and eosinophils were about 0.1% or less of
total BAL cells, indicating that WTC3 did not induce a
significant acute inflammatory reaction. The increase in
macrophages and lymphocytes is probably a
nonspecific reaction to inhalation of large
amounts of dust which induces macrophage
recruitment for phagocytosis and clearance of
the particles (Adamson and Bowden, 1981).
Levels of proteins and enzymes in the BAL
supernatant were assessed in the two groups of
mice at 3 time points (Table 16). Significant
differences between Air and WTC3 groups were
found for total protein (P = 0.05) and albumin (P
= 0.007), but there was no significant day effect.
Surprisingly, the levels of protein and albumin
were higher in the Air group. However, the
overall levels of all proteins and enzymes was
low in both groups and at all time points (Figure
14), in comparison with Experiment A. The
results indicate that at this exposure
concentration and duration, WTC3 PM25 does
not induce severe acute lung injury.
7. Nasal histopathology. No exposure-
related nasal lesions were found in mice exposed
to air alone (controls). Similarly no nasal lesions were
found in the mice exposed to WTC3 and killed 3 or 6 days
post-exposure. None of the mice in any group had
Protein
LDH
250-1
lO-i
s 6"
14
Day 6
Air
WTC3
Day 1 Day 3 Day 6 Day 1 Day 3 Day 6
Figure 14. Experiment B: BAL supernatant biochemical values in mice 1, 3, or 6 days after 5 hr nose-only
inhalation exposure to WTC3 or Air only. Values shown are means and SEM (n=8 per group). a Significant overall
treatment effect (WTC3 < Air; no significant day effect); P = 0.05 (Protein) or P = 0.007 (Albumin).
30
-------
exposure-related alterations in the mucosal tissues lined by
respiratory or olfactory epithelium in the more distal tissue
sections examined (T2 and T3).
The only nasal alterations observed by light
microscopic examination was minimal to mild acute, focal
inflammation (rhinitis) in four of the eight mice exposed
to WTC3 PM2 5 and killed 24 h post-exposure (animal #
161, 162, 163, 164). This minimal inflammatory response
was bilateral and restricted to the most proximal tissue
section examined (Tl). It was characterized by a slight
increase in the number of neutrophils in the mucosal
tissues lining the lateral meatus, especially in the ventral
lateral meatus, the dorsomedial aspect of the proximal
maxilloturbinate, and the ventral aspect of the proximal
nasoturbinate in both nasal passages. It must be
emphasized, however, that the severity of this focal rhinitis
was minimal to mild (i.e., severity score of 1 or 2 out of 4).
In addition, there were no associated histologic alterations
in the surface epithelium or in the subepithelial tissues in
the affected areas. In mouse #162 there was a small
accumulation of mucus and fiber-like material in the
lateral meatus of one nasal passage in Tl.
In summary, some but not all mice exposed to WTC3
and killed 1 day after exposure had a minimal acute
rhinitis that was restricted to the proximal nasal airways.
This minimal inflammatory response was probably due to
stimulation by the WTC3 exposure. This stimulation,
however, did not result in any apparent epithelial cell
inj ury that is often observed with many inhaled agents. No
nasal lesions were observed in mice exposed to WTC3 and
killed 3 or 6 days post-exposure. This suggests that any
acute inflammation that may have been induced by the
dust exposure quickly resolved and did not result in any
persistent injury to the nasal mucosathat could be detected
by light microscopy.
8. Lung histopathology. No remarkable findings
were observed in any of the mice exposed to Air or to
WTC3 at any time point. Since nasal lesions as described
above were restricted to the proximal Tl region and were
not found in the more distal T2 and T3 regions, the lack of
any findings in the lung suggests that the proximal region
of the nose effectively scrubbed out enough of the
particulate matter during the exposure to WTC3 to limit
deposition further down the respiratory tract. It should be
noted that mice are obligate nose-breathers, while humans
have significant oral breathing, and therefore significantly
more PM can bypass the nasal passages in humans
(Schlesinger, 1985). Studies have shown considerably less
deposition efficiency in the alveolar region of rodents
compared with humans (Asgharian et al., 1995).
9. Summary. Results from investigation of the
effects of nose-only exposure to the WTC3 sample
indicate that WTC3 PM25 induced mild transitory
neutrophilic inflammation in proximal nasal airways of
some mice, but WTC3 PM2 5 did not induce neutrophilic
inflammation in the lungs of any mice. However, numbers
of macrophages were significantly increased after
exposure, suggesting that some WTC3 PM2 5 penetrated
into the lower respiratory tract, which stimulated
recruitment of macrophages to phagocytize and clear the
particulate matter. Biochemical parameters of lung injury
were not increased at all by WTC3. The data suggested
that individual mice in this outbred strain may be sensitive
to the immediate effects of WTC3 exposure and respond
with increased airway obstruction, although this effect was
not significant for the group as a whole. Groups of mice
exposed to Air or WTC3 PM25 differed in their
responsiveness to Mch aerosol at different times after
exposure, but the biological significance of these results
was unclear. The dose deposited in the respiratory tract
following nose-only inhalation may be estimated as
follows: 18.8 ml/min (mouse minute ventilation based on
weight; Costa et al., 1992) x 300 min (exposure time) x
0.001 L/ml x 0.001 m3/L x 10.64 mg/m3 (exposure
concentration) x 1000 |o,g/mg x 0.23 (deposition efficiency
estimate in total respiratory tract) ~ 14 jog. Thus, the
significant difference in dose deposited into the airways
between oropharyngeal aspiration (100 |o,g) and nose-only
inhalation probably accounts for the lack of effect in many
of the endpoints examined following nose-only inhalation
exposure.
D. Experiment C: Effect of Geographical Location of
WTC PM Samples on Responses
1. Sub-experiments and body weights. WTC PM2 5
samples from 7 different sites comprised the pooled
WTCX sample (Figure 1, Table 1). The effects of the
pooled sample may have been dominated by one or more
site samples which were toxic in comparison with other
site samples. Experiment C was designed to address this
possibility and to examine the variability of pulmonary
responses associated with WTC PM25 samples collected
from different geographical locations. The 7 sites were
located east (WTC11 - 0.1 miles, WTC8 - 0.4 miles),
southeast (WTC13 - 0.1 miles, WTCF - 0.25 miles), south
(WTCB - 0.25 miles), west-northwest (WTCC - 0.2 miles),
and north-northeast (WTCE - 0.25 miles) from the center
point of Ground Zero. Sub-experiment Cl examined
responses to WTC8, WTC13, WTCF, NIST, and Saline
control mice. Sub-experiment C2 examined responses to
WTC 11, WTCB, WTCC, WTCE, and Saline control mice.
31
-------
Table 17.
Group
Saline
WTC8
WTC13
WTCF
NIST
Saline
WTC11
WTCB
WTCC
WTCE
Sub-
Experiment
Cl
Cl
Cl
Cl
Cl
C2
C2
C2
C2
C2
Experiment
Day-1
24.69
0.39
24.76
0.49
24.69
0.40
24.72
0.43
24.75
0.50
ndb
nd
nd
nd
nd
C: Body
Body
DayO
24.76
0.42
24.54
0.52
24.26
0.42
24.46
0.42
24.32
0.53
22.55
0.27
23.72
0.39
23.66
0.33
23.67
0.27
23.69
0.29
Weights a
Weight (g)
Day 1
24.72
0.45
24.16
0.58
23.91
0.38
24.17
0.38
24.15
0.50
22.23
0.25
23.20
0.43
23.11
0.33
23.12
0.24
23.27
0.36
Day 3
24.91
0.42
25.35
0.90
24.14
0.43
24.41
0.49
24.31
0.71
22.85
0.55
23.72
0.68
23.29
0.61
23.96
0.41
23.44
0.41
a Values shown are means (in bold) and SEM immediately below means (on days -1,0, and
1, n=16 per group, except Saline sub-experiment C2: n=8; on day 3, n=8 per group, except
Saline sub-experiment C2: n=4). b nd - Not determined. No treatment-related differences
in body weight among groups within each sub-experiment were detected.
Responses were examined in 8 mice per group at 1 and 3
days after oropharyngeal aspiration of 100 |o,g of each PM
sample or saline alone (n = 4 per time point in sub-
experiment C2 Saline mice). Responses were examined at
both 1 and 3 daytime points in order to begin examination
of persistence of exposure effects. Statistical analysis of
the data was performed within each sub-experiment.
Body weights were determined on days -1 (before
oropharyngeal aspiration), 0, 1, and 3 in sub-experiment
Cl, and on days 0, 1, and 3 in sub-experiment C2 (Table
17). No treatment-related differences in body weight
among groups within each sub-experiment were detected,
although there were differences on the day the animals
were weighed (P = 0.0001).
2. Responsiveness to methacholine aerosol. In sub-
experiment Cl, the WTC8 group had significantly greater
baseline PenH values 1 day after exposure compared with
the WTC13 group (Table 18). No other significant
differences in baseline PenH values in sub-experiments C1
or C2 were found. Responsiveness to methacholine
aerosol was quantified as PenH AUC (Table 18). Analysis
of the data in both sub-experiments showed that linear
regression equations could be fit to the PenH AUC vs.
[Mch] data (Figure 15). In both sub-experiments, tests for
equal slopes on days 1 and 3 after exposure showed that
day was not a significant factor. Therefore, a single
equation was fit to the data for each group, and day does
not appear in the equations.
In sub-experiment Cl, the WTC8, WTCF, and NIST
groups could be described with a common slope and
intercept. The common slope of these 3 groups was
significantly different from and greater than that of the
WTC13 or Saline Cl groups (P < 0.0005), indicating that
WTC8, WTCF, and NIST were hyperresponsive to
methacholine aerosol. The slope of the WTC13 group was
significantly greater than that of the Saline C1 group,
showing that WTC 13 mice were hyperresponsive
compared with control mice, though less so than WTC8,
32
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Table 18.
Group
WTC8
WTC13
WTCF
NIST
Saline
WTC8
WTC13
WTCF
NIST
Saline
WTC11
WTCB
WTCC
WTCE
Saline
WTC11
WTCB
WTCC
WTCE
Saline
Sub-
Experiment
Day
Experiment
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
1
1
1
1
1
3
3
3
3
3
1
1
1
1
1
3
3
3
3
3
C: Baseline
Baseline
PenH
0.68 1
0.05 1
0.45
0.02
0.55
0.05
0.59
0.03
0.57
0.02
0.66
0.04
0.78
0.08
0.77
0.07
0.76
0.05
0.71
0.03
0.69
0.06
0.67
0.05
0.62
0.06
0.62
0.02
0.61
0.08
0.59
0.05
0.59
0.05
0.56
0.04
0.62
0.05
0.62
0.05
PenH
0
35.2
10.1
35.5
5.1
25.9
2.1
31.6
3.1
24.2
1.5
14.5
3.0
27.9
9.0
18.8
6.4
21.1
8.0
-2.8
4.6
15.9
6.5
7.3
9.4
5.9
5.9
8.3
3.1
3.2
4.0
3.8
6.3
22.2
4.3
10.0
7.1
5.7
6.3
3.5
3.5
and Responsiveness to
Dose Mch
4
69.6
19.2
50.5
4.4
62.3
14.4
52.7
2.9
40.4
4.0
41.9
10.9
51.2
9.7
29.3
22.3
47.1
14.8
30.6
17.2
66.9
23.3
46.4
10.0
27.5
9.9
28.7
12.6
52.5
7.5
24.7
12.5
31.9
14.6
22.8
6.6
42.3
11.7
27.5
27.0
Methacholine
Aerosol a
(mg/ml) and PenH AUC (PenH - sec)
8
197.0
92.8
87.9
9.1
164.4
29.0
97.3
11.5
61.4
6.6
62.2
25.1
207.0
62.6
87.5
34.4
86.1
19.4
99.2
16.8
238.8
90.7
205.4
53.8
118.7
30.6
155.6
86.8
111.7
19.3
45.0
19.2
98.0
34.7
118.9
37.4
138.6
33.2
84.0
65.1
16
429.4
87.0
264.7
44.2
369.1
47.3
279.3
54.0
171.7
19.9
182.5
49.7
271.9
68.3
280.8
130.7
212.3
72.2
280.4
67.0
811.0
223.6
695.5
193.0
737.6
249.0
390.0
177.1
167.7
29.5
256.8
112.0
238.6
59.2
351.7
144.1
412.4
117.6
114.7
17.2
32
1115.0
126.6
674.8
135.4
1145.3
155.8
811.1
149.2
534.5
74.2
690.3
206.4
1000.8
204.3
994.4
368.1
962.5
271.0
481.0
40.8
1496.5
437.7
1561.4
404.0
1826.9
466.3
1112.5
360.2
308.7
26.0
1003.0
325.5
738.5
216.6
1282.4
365.5
1257.9
297.5
341.1
65.2
64
1953.5
203.5
1151.3
206.6
1966.5
293.0
1882.2
324.6
1077.0
163.2
1907.0
370.0
1554.5
190.8
2160.8
563.2
1886.2
437.2
1287.9
79.5
1835.9
306.0
2333.1
611.8
2800.2
672.8
1722.8
452.6
825.5
101.6
2304.1
559.8
2609.2
585.5
2669.0
520.9
2088.7
472.9
1058.7
45.7
Values shown are means (in bold) and SEM immediately below means (n=8 per group, except Saline sub-experiment
C2: n=4). A significant difference in baseline PenH (enhanced pause; unitless) was found between the WTC8 group
and the WTC13 group on day 1 (heavy solid line box). No other significant differences in baseline values on day 1 or
day 3 were detected. Methacholine aerosol (Mch) was administered at the indicated doses, and the airway response was
calculated as the area under the curve (AUC) of the PenH response over time in seconds. See Figure 15 for description
of statistical analysis of PenH AUC data.
33
-------
0)
U
3
3000-
2000-
1000-
WTC8
a
PenH AUC = 32.8* [Mch} - 139.4
8 Day 1
WTC11
PenH AUC = 38.5* [Mch] - 103.9
O 8 Day 3
3000-
2000-
1000-
WTCF
a
PenH AUC = 32.8* [Mch] - 139.4
FDay 1
WTCB
PenH AUC = 38.5* [Mch] - 103.9
O F Day 3
3000-
2000-
1000-
MST 1649a
a
- PenH AUC = 32.8* [Mch] - 139.4
NIST Day 1
O NIST Day 3
WTCC
c
PenH AUC = 38.5* [Mch] - 103.9
CDay 1
3000-
2000-
1000-
WTC 13, Saline Cl
b
PenH AUC = 22.3* [Mch] - 22.4
• 13 Day 1
O 13 Day 3
PenH AUC =19.3* [Mch]-72.1
A Saline Cl Day 1
A Saline Cl Day 3 T
WTC E, Saline C2
c
PenH AUC = 38.5* [Mch] - 103.9
• EDayl
O EDay3
- - PenH AUC =14.9* [Mch]-61.1
A Saline C2 Day 1
A Saline C2 Day 3
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
[Mch] (mg/ml)
Figure 15. Experiment C: Airway responsiveness. See next page for figure legend.
34
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Figure 15. (previous page.) Experiment C: Airway
responsiveness to methacholine aerosol challenge in mice
exposed to saline vehicle, NIST 1649a, or WTC PM samples
from individual collection sites and tested 1 or 3 days later (n
= 8 per group except Saline sub-experiment C2: n = 4). A
single regression equation was fit to the data for both days in
each group. a In sub-experiment Cl (left panels), a common
equation could be fit to the WTC8, WTCF, and NIST data,
and the slope of the line was significantly different from and
greater than the slopes of the WTC13 and Saline Cl
equations. b In sub-experiment Cl (left panels), the slope of
the equation for the WTC 13 group was significantly different
from and greater than the slope for the Saline Cl group. ° In
sub-experiment C2 (right panels), a common equation could
be fit to the WTC11, WTCB, WTCC, and WTCE data, and
the slope of the line was significantly different from and
greater than the slope of the Saline C2 equation.
WTCF, and NIST mice.
In sub-experiment C2, the WTC11, WTCB, WTCC,
and WTCE groups could all be described with a common
slope and intercept, which was similar to that found for
WTC8, WTCF, and NIST groups in sub-experiment Cl.
The common slope of the 4 WTC groups was significantly
different from and greater than that of the Saline C2 group
(P = 0.001), indicating that WTC 11, WTCB, WTCC, and
WTCE were hyperresponsive to methacholine aerosol.
In general, these results are consistent with those from
Experiment A, where the 100 |o,g dose of the pooled
WTCX sample induced significant hyperresponsiveness to
methacholine aerosol compared with control PM samples
and saline. All but one of the WTC PM samples, as well
as the NIST control PM, appeared to cause similar degrees
of hyperresponsiveness. However, the WTC13 sample,
located just 0.1 miles southeast of Ground Zero, caused a
lower degree of hyperresponsiveness compared with
WTC8, WTCF, and NIST.
3. BAL cells. After assessment of responsiveness to
Mch aerosol, mice were killed and numbers of BAL cells
were quantified (Table 19; Figure 16). In sub-experiment
C1, significant increases in numbers of neutrophils on Day
1 were found in all PM-exposed groups compared with
Saline Cl mice. An average of 14.7 x 104 neutrophils was
recovered from NIST mice (45% of total BAL cells).
Significantly lower numbers of neutrophils were found in
WTC13 (6.1 x 104) and WTCF (6.9 x 104) mice, while
numbers of neutrophils were lower still in WTC8 mice
(3.2 x 104). The neutrophilic response abated by Day 3,
and there were no significant differences among the 5
groups. Numbers of lymphocytes were significantly
increased in WTC8, WTC13, WTCF, and NIST mice in
comparison with Saline C1 mice on both Day 1 and Day
3 after oropharyngeal aspiration (P = 0.0001).
Lymphocyte numbers significantly increased in all groups
from Day 1 to Day 3 (P = 0.0001). Since there were
significant interactions between day and treatment with
respect to eosinophil numbers (P = 0.01), no significant
differences among groups could be discerned. Although
a significant difference in macrophage numbers was
detected in the WTC 13 group compared with saline, it was
very marginal and not considered biologically significant.
In sub-experiment C2, significant increases in
neutrophils and eosinophils were found in WTC 11 and
WTCE mice compared with Saline C2 mice. The average
number of neutrophils in these 2 WTC groups was
comparable to those found in the WTC 13 and WTCF
groups in sub-experiment Cl. In addition, numbers of
neutrophils and eosinophils were significantly greater in
WTCE mice compared with WTCB mice. Neutrophils
numbers declined from Day 1 to Day 3 (P = 0.0001). It
should be noted that of the four mice in the Saline C2 Day
1 group, two had unusually high neutrophil numbers
(individual numbers: 0.15, 0.26, 3.15, and 5.79 x 104
neutrophils), which limited the ability to determine
significant increases in neutrophils in mice exposed to
WTC PM samples in sub-experiment C2. The reasons for
this significant inflammatory response in two control mice
are not apparent, but this finding does not detract from the
overall conclusion that PM collected from specific
locations near the WTC site caused significant
inflammation of neutrophils and eosinophils.
These results differ substantially from those found in
Experiment A, where 100 |o,g of pooled WTCX induced
only a mild neutrophilic response in the lung one day after
oropharyngeal aspiration (average 1.43 x 104). Some
WTC individual site samples (WTCF, WTC13, WTC11,
WTCE) caused about 4 times the amount of neutrophil
recruitment as WTCX, while the others (WTC8, WTCB,
WTCC) caused about twice as much recruitment. It is not
clear how the individual site samples could all cause more
lung inflammation than the pooled WTCX sample which
was composed of the individual site samples. This finding
may be a result of significant differences in responsiveness
of different lots of mice sent on different weeks, which we
have found with some studies of other toxic inhalants.
Additionally, although these mice were lavaged after Mch
challenge, our experience has shown that the challenge
itself does not induce cellular inflammation that might
account for the observations made here. To adequately
address this question, pooled WTCX and individual site
samples would need to be tested together in the same
experiment.
35
-------
Table 19. Experiment C: BAL Cell Numbers'
Group
Saline
WTC8
WTC13
WTCF
NIST
Saline
WTC8
WTC13
WTCF
NIST
Saline
WTC11
WTCB
WTCC
WTCE
Saline
WTC11
WTCB
WTCC
WTCE
Sub-
Experiment
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
C2
C2
BAL Cell Number fx 10"4)
Day
1
1
1
1
1
3
3
3
3
3
1
1
Mac
16.41
1.92
21.43
2.38
24.19
2.24
18.47
1.45
17.36
3.41
24.03
6.56
29.79
2.70
25.96
2.32
24.60
2.23
29.09
4.06
16.25
1.21
23.38
Neut
0.34
0.20
3.23
0.52
6.10
0.88
6.85
0.85
14.67
1.17
0.48
0.45
0.18
0.06
0.46
0.15
0.25
0.07
1.74
0.26
2.34
1_34
5.79
3.84 | L42
C2
C2
C2
C2
C2
1
1
1
3
3
25.91
3.90
27.19
4.02
24.19
2.94
20.87
0.69
25.50
2.98
0.95
2.53
0.42
5.12
0.80
0.03
0_02
1.13
3.80 | (199
C2
C2
C2
3
3
3
29.60
2.87
24.76
2.24
30.11
2.62
0.16
0.05
0.34
0.08
0.21
0.05
Eos Lym
0.02 0.14
0.01 0.02
0.27
0.08
1.33
0.43
0.84
0.22
0.43
0.17
0.38
0.05
0.70
0.14
0.58
0.10
0.48
0.13
0.19 0.36
0.07 0.09
1.48
0.90
0.39
0.10
1.46
0.49
0.67
0.36
0.75
0.15
0.88
0.22
1.16
0.26
1.97
0.41
0.22 0.16
OJ_9 0.06
| 0.75 0.98
| 0^39 | 0.26
0.17 0.49
0.05 0.05
0.28 0.29
0.10 0.08
0.37
0.12
0.38
0.10
0.02 0.27
0_01 0.11
| 0.49 0.75
| OJ.6 | 0.31
0.30 0.46
0.12 0.11
0.39 0.79
0.14 0.19
1.59
0.43
1.45
0.50
Values shown are means (in bold) and SEM immediately below means (n=8 per group, except Saline sub-experiment
C2: n=4). Significant differences shown are within sub-experiments only. Heavy solid-line boxes: Within
sub-experiment Cl day 1 neutrophils, NIST > WTC13 and WTCF > WTC8 > Saline. Solid-line boxes, underlined
values: NIST, WTC13, WTCF, and WTC8 all significantly different from Saline. Dashed-line boxes: WTC11
significantly different from Saline. Solid-line shaded boxes: WTCE significantly different from Saline and WTCB.
36
-------
40n
30-
Saline (Cl)
WTC8
WTC13
WTCF
NIST
Mac
Day 1
Day 3
Neut
Day 1
Day 3
5-,
Eos
I I Saline (Cl)
WTC8
I WTC13
I WTCF
I NIST
Day 1
Day 3
-------
Figure 16. (previous page.) Experiment C: BAL cell
numbers recovered from mice exposed to saline vehicle, NIST
1649a, or WTC PM samples from individual collection sites
and tested 1 or 3 days later (n = 8 per group except Saline
sub-experiment C2: n = 4). Cell types shown are
macrophages (Mac), neutrophils (Neut), eosinophils (Eos),
and lymphocytes (Lym).a NIST significantly greater than all
othergroups. bWTC13 and WTCF significantly greater than
WTC8 and Saline Cl groups. ° WTC8 significantly greater
than Saline Cl group. d Lymphocyte numbers significantly
greater in WTC8, WTC13, WTCF, and NIST groups
compared with Saline Cl group. e Significantly greater
numbers of neutrophils and eosinophils in WTC11 group
compared with Saline C2 group. f Significantly greater
numbers of neutrophils and eosinophils in WTCE group vs.
WTCB and Saline C2 groups.
4. BAL proteins and enzymes. As for other
parameters, BAL protein and enzyme data for sub-
experiments C1 and C2 were analyzed separately (Table
20, Figure 17). In sub-experiment Cl, significant
increases in BAL total protein levels were found in the
NIST group compared with the WTC8 group (P = 0.05).
No significant differences due to Treatment were found
with respect to albumin or LDH levels. There were
significant interactions between Day and Treatment in
NAG values (P = 0.02), indicating the results depended on
the day animals were killed.
In sub-experiment C2, there were significant effects of
Day after treatment for total protein and LDH, but there
were no effects of Treatment group. There were
significant interactions between Day and Treatment in
NAG values (P = 0.005), indicating the results depended
on the day after treatment.
In both sub-experiment Cl and C2, one of the saline
group mice killed on Day 1 had very high values for total
protein, albumin, and LDH, which increased the mean
values and variability in these groups. Although this result
may have limited the ability to detect some statistical
differences, overall the biochemical values were not
greatly different among the treatment groups, and any
additional differences with more consistent control data
would likely have been minimal. Therefore the results for
the individual site WTC PM samples are comparable to
those found with the pooled WTCX sample in Experiment
A, where no differences from control saline mice were
found.
5. Lung histopathology. Following tests for airway
responsiveness to Mch aerosol and lung lavage, lungs were
removed and fixed with 4% paraformaldehyde, and
pathological changes were assessed. Although the lungs
of all mice in Experiment C were lavaged (they were not
lavaged in Experiments A or B), the pattern and the
morphology of the PM induced findings were relatively
consistent in all treated groups.
Focal subacute bronchiolar inflammation and focal
bronchiolar pigmented macrophages (presumably PM)
were consistently observed in all groups of mice dosed
with each of the different PM samples, and both findings
are considered to be PM-induced in all groups (Table 21).
Some groups also had findings of focal free bronchiolar
pigment, consistent with the pigment in macrophages. No
remarkable findings were observed in the lungs of the
saline control group (Figure 18A), except for one mouse
which had a minimal degree of focal subacute bronchiolar
inflammation which was not considered to be treatment-
related. Table 21 shows the rankings of the treatment-
related histopathologic findings in mice 1 or 3 days after
exposure. The degree of focal subacute bronchiolar
inflammation was greatest in the NIST (Figure 18C),
WTCE, and WTC 13 (Figure 18D) groups on Day 1
(average severity scores of 1.9,2.0, and 2.1, respectively).
The scores in the WTCC (Figure 18B), WTCB, WTC8,
WTCF, and WTC 11 groups were lower (average severity
scores of 0.8, 1.1, 1.1, 1.3, and 1.3, respectively). By Day
3, the focal subacute bronchiolar inflammation was
greatest in the NIST group (average severity score 2.1;
Figure 18E), while the scores were reduced in all of the
WTC PM groups relative to their scores on Day 1 (Figure
18F).
The histopathologic scoring system is semi-
quantitative, and much larger numbers of mice per group
would be necessary to determine statistically significant
differences among groups. Nevertheless, these results also
show substantial differences from those found in
Experiment A with the pooled WTCX sample.
Oropharyngeal aspiration of 100 |o,g of WTCX did not
cause any treatment-related histopathologic findings. In
contrast, all individual site samples of WTC PM induced
at least minimal focal subacute bronchiolar inflammation,
and some samples caused slight/mild and even moderate
degrees of inflammation. In addition, pigment associated
with PM was visible in macrophages from all WTC PM-
exposed mice, but none was visible in mice exposed to
pooled WTCX in Experiment A. Re-examination of the
slides by a different observer will be necessary to confirm
this finding. The findings of pulmonary inflammation in
WTC PM groups by histopathologic examination are
consistent with the results from the quantification of BAL
cell numbers.
38
-------
Table 20.
Group
Saline
WTC8
WTC13
WTCF
NIST
Saline
WTC8
WTC13
WTCF
NIST
Saline
WTC11
WTCB
WTCC
WTCE
Saline
WTC11
WTCB
WTCC
WTCE
Sub-
Experiment
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
Experiment C:
Day
1
1
1
1
3
3
3
3
1
1
1
1
1
3
3
3
3
3
BAL Supernatant Biochemical
Protein
US/ml
240.0
73.9
191.9
9.5
201.8
16.3
200.4
11.9
257.1
16.3
274.6
65.4
156.1
13.3
203.8
26.4
197.5
15.9
220.4
17.3
307.8
95.7
202.0
30.8
242.5
56.4
194.8
13.5
194.5
11.7
180.8
21.1
213.0
64.7
131.8
12.2
167.2
16.4
190.2
23.4
Albumin
US/ml
52.5
15.2
40.5
3.1
40.6
4.5
42.9
3.4
152.6
4.1
55.9
10.0
34.9
4.6
47.1
8.1
45.4
4.5
145.2
4.6
75.5
28.0
44.8
7.7
45.2
6.6
42.2
4.6
43.5
3.6
46.8
6.1
49.2
11.5
32.5
4.6
44.8
5.8
51.0
8.1
Values a
LDH
U/L
43.1
5.3
46.1
3.6
40.6
4.4
44.6
3.3
56.8
5.0
37.5
11.1
35.2
1.8
36.0
2.2
37.2
2.8
40.2
5.2
44.5
18.9
48.1
6.2
55.2
13.0
34.6
2.6
39.5
2.7
29.5
4.9
50.1
15.0
32.4
3.6
30.0
1.7
29.4
2.1
NAG
U/L
1.7
0.2
2.7
0.3
2.9
0.2
1.7
0.2
4.5
0.4
3.1
1.3
2.3
0.1
2.7
0.2
2.9
0.4
4.3
0.6
2.9
0.2
2.4
0.7
2.9
0.8
1.7
0.1
3.0
0.4
2.3
0.3
4.4
2.3
2.2
0.1
2.9
0.2
2.5
0.2
Values shown are means (in bold) and SEM immediately below means (n=8 per group, except Saline sub-experiment
C2: n=4). Significant differences shown are within sub-experiments only. Heavy solid-line boxes: NIST significantly
different from WTC8. Significant overall Day effects were found for Protein (sub-experiment C2), and LDH (both
sub-experiments).
39
-------
400 n
Protein
I Saline (Cl)
Day 1
Day 3
400-,
300-
200-
100-
Protein l=1 Saline (C2)
i::::::::::::::::j \VTC11
EZZZZaWTCB
rm-n WTCC
-r
JL
j
c_
i
T
I
I
SS3
J
Wl
L
^CE
V^
\\\
sV\
^
$8
Day 1
Day 3
Albumin
Day 1
Saline (Cl)
WTC8
WTC13
WTCF
NIST
Day 3
100-,
f BOH
•efc
5 60-
40
A
^
Albumin
T
X
I 1 Saline (C2)
WTC11
ZZZZZIWTCB
nun WTCC
ESS3 WTCE
m
I
Day 1
Day 3
LDH
Saline (Cl)
WTC8
WTC13
WTCF
NIST
LDH
p
J
80-
60-
40-
20-
| i Saline
T -r EZ^WTCB
^
i
i
\
Inrm WTCC
1 ESSWTCE
^5n
J
EL
!
X
I
J
-
1
Day 1
Day 3
Day 1
Day 3
I 1 Saline (Cl)
WTPS
I WTC13
I WTCF
INIST
NAG
Day 1
Day 3
6"
4-
2-
Saline (C2)
I WTC11
vzzza WTCB
WTPP
WTCE
JL
NAG
Day 1
I
Day 3
Figure 17. Experiment C: BAL supernatant biochemistry. See next page for figure legend.
40
-------
Figure 17. (previous page.) Experiment C: Bronchoalveolar
lavage supernatant proteins and enzymes recovered from mice
exposed to saline vehicle, NIST 1649a, or WTC PM samples
from individual collection sites and tested 1 or 3 days later (n
= 8 per group except Saline sub-experiment C2: n = 4). a
Significantly greater protein values in NIST group vs. WTC8
group. Other significant differences were found due to Day
of sacrifice or interactions between Day and Treatment, but
there were no other effects due to Treatment alone.
6. Summary. Examination of the effects of WTC
PM collected from different locations surrounding the
WTC site showed that all samples were capable of
inducing pulmonary inflammation and
hyperresponsiveness to Mch aerosol, although overt lung
damage as determined by biochemical parameters of lung
injury was minimal. The neutrophilic response was
substantially greater for all individual site WTC PM
samples compared with the response induced by the
pooled WTCX sample in Experiment A, although differing
responsiveness of different shipments of mice could
account for this finding, and a direct comparison would be
necessary to determine if there is a difference. Numbers
of neutrophils declined from Day 1 to Day 3 after
oropharyngeal aspiration, as determined by both BAL and
histopathologic examination. Other cell types appeared to
be more persistent or increase from Day 1 to Day 3
(especially lymphocytes in sub-experiment Cl), but these
were not large changes. Respiratory responsiveness to
Mch aerosol was significantly increased in all WTC
groups compared with saline controls, although mice
exposed to WTC 13 were less responsive than other WTC
groups. The degree of Mch hyperresponsiveness in the
WTC groups of Experiment C appeared to be comparable
to that from the WTCX group in Experiment A.
No particular geographical significance could be
deduced from the patterns of responses induced by the
individual WTC PM samples. The one group which had
Table 21. Experiment C - Summary of Treatment-Related Histopathologic Findings inMice 1 or 3 Days after
Intratracheal
Treatment
Group
WTC 13
WTCE
NIST
WTC 11
WTCF
WTC8
WTCB
WTCC
Saline
NIST
WTC 11
WTCE
WTC8
WTC 13
WTCB
WTCF
WTCC
Saline
Instillation of Paniculate Matter Samples a
Sub-
Experiment
Cl
C2
Cl
C2
Cl
Cl
C2
C2
Cl
Cl
C2
C2
Cl
Cl
C2
Cl
C2
Cl
Day
1
1
1
1
1
1
1
1
1
3
3
3
3
3
3
3
3
3
Bronchiole,
Inflammation,
Subacute, Focal
Incidence Severity
8/8 2.1
8/8 2.0
8/8 1.9
8/8 1.3
8/8 1.3
6/8 1.1
6/8 1.1
6/8 0.8
1/8 0.1
8/8 2.1
6/8 1.1
6/8 0.8
4/8 0.8
4/8 0.6
3/8 0.4
3/8 0.4
2/8 0.3
0/7 0.0
Bronchiole,
Pigmenl
Macrophage,
Focal
Incidence Severity
8/8
8/8
7/8
7/8
6/8
6/8
6/8
4/8
0/8
8/8
2/8
6/8
1/8
3/8
2/8
1/8
1/8
0/7
2.0
1.9
2.0
0.9
0.8
0.8
0.8
0.5
0.0
2.0
0.3
0.8
0.1
0.4
0.3
0.1
0.1
0.0
Bronchiole,
Pig
ment,
Free, Focal
Incidence
4/8
2/8
7/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/7
Severity
0.6
0.3
0.9
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Saline-instilled control mice in sub-experiment C2 were not examined. Incidence denotes number of mice in group with
finding / total number of mice examined. Average severity score for the group is shown based on the following scoring
system: 0 = not present, 1 = minimal, 2 = slight/mild, 3 = moderate, 4 = moderately severe, 5 = severe/high. Groups
are arranged in descending order of severity within each post-exposure day, first by severity of focal subacute
bronchiolar inflammation, and then by severity of focal bronchiolar pigmented macrophages.
41
-------
• •.
* /•
;-•»< !• < \ ^
>-W, l-frlt*--'C" -. '*•
:/(c;^11-^^
^V*.. ,'*^ w
rv;^? ?. *.
i..' »«»'* «
V , >&.
Figure 18. Experiment C. Representative micrographs of lesions occuring in lungs of mice 1 or
3 days after intratracheal instillation of 100 (j,g PM sample or saline vehicle (all panels same
magnification: bar length = 100 (j,m). A. Saline-instilled control mouse (#301), Day 1, with no
remarkable findings. B. Mouse #349 instilled with WTCC, Day 1, showing minimal degree of focal
subacute bronchiolar inflammation (FSBI). C. Mouse #291 instilled with NIST, Day 1, with
moderate degree of FSBI. D. Mouse #270 instilled with WTC13, Day 1, with moderate degree of
FSBI. E. Mouse #300 instilled withNIST, Day 3, with slight/mild degree of FSBI. F. Mouse #280
instilled with WTC13, Day 3, with minimal degree of FSBI.
lower Mch responsiveness (WTC13) was centrally located
only 0.1 mile southeast of the center of Ground Zero. The
WTCF sample was blown into a building at 120 Broadway
and collected on an undisturbed marble staircase. The
responses caused by this "indoor" sample were quite
similar to those caused by the other "outdoor" WTC PM
samples.
In general, responsiveness to Mch aerosol and
pulmonary inflammation were not well correlated. Mice
in the WTC 13 group had one of the largest neutrophilic
and eosinophilic responses, yet had a significantly lower
degree of Mch responsiveness. Mice in the WTCC group
had perhaps the greatest response to Mch challenge (not
significantly different from WTCB, WTCE, or WTC11),
42
-------
yet their neutrophil and eosinophil responses were low hyperresponsiveness is not uncommon (Alvarez et al.
relative to the other WTC groups. As noted previously, a 2000; Smith and McFadden Jr., 1995).
lack of correlation between inflammation and airway
43
-------
IV. Discussion
Samples of fallen dust were collected at various
locations in the immediate vicinity of the WTC site one
and two days after the WTC disaster, and were examined
by several physical and chemical techniques. Both coarse
unfractionated and fine size-fractionated WTC PM
samples were composed primarily of calcium-based
compounds such as calcium sulfate (gypsum) and calcium
carbonate (calcite; the main component of limestone).
These and other compounds and elements found in the
WTC PM samples are indicative of crustal material-
derived building materials such as cement, concrete
aggregate, ceiling tiles, and wallboard. Both gypsum and
calcite irritate the mucus membranes of the eyes, nose,
throat, and upper airways (Stellman, 1998). Calcium
carbonate dust causes coughing, sneezing, and nasal
irritation (NLM, 2002). These minerals are often
contaminated with small amounts of silica, which is the
main concern for occupational health hazards (Stellman,
1998). Minor amounts of silica (quartz) were detected in
the WTC PM samples
Our chemical analysis generally agrees with the
extensive analysis of WTC PM performed by the USGS
(USGS, 2002). Levels of carbon were relatively low,
suggesting that combustion-derived particles did not form
a significant fraction of these samples recovered in the
immediate aftermath of the destruction of the towers.
Lastly, there was no evidence of significant asbestos
contamination of the samples used in these studies,
although the physical analyses conducted were not
specifically focused on definitive asbestos quantitation.
As of May 23, 2002, the U.S. EPA had analyzed 9,544 air
samples in Lower Manhattan since September 11, and
found elevated levels of asbestos in only 21 samples (EPA,
2002c).
The effects of exposure to samples of WTC PM25 on
respiratory parameters, pulmonary inflammation, and lung
injury were investigated in young adult female CD-I mice,
an outbred strain expected to have significant variability
in biological responses, in three separate experiments. A
pooled sample of WTC PM25 composed of roughly
equivalent amounts of samples from 7 different locations
around the WTC site caused a mild degree of pulmonary
inflammation in mice (7% neutrophils in BAL fluid), and
had no effect on parameters of acute lung injury at a dose
of 100 |o,g instilled directly into the lungs. ROFA, a toxic
positive control fine PM sample, caused a much higher
degree of lung inflammation and lung injury at the same
dose. However, mice instilled with 100 |o,g pooled WTC
PM25 had highly significant increases in airway
responsiveness to methacholine (Mch) aerosol challenge,
which were significantly greater than that of ROFA. Mice
exposed to lower doses of pooled WTC PM25 (10 |o,g and
31.6 |o,g) and mice exposed by nose-only inhalation
(estimated to have about 14 |o,g WTC PM25 deposited in
the respiratory tract) did not have any biologically
significant changes in methacholine responsiveness or
neutrophilic inflammation. These dose-response
relationships and the lack of effect in nose-only exposure
suggest that inhalation of relatively high doses of WTC
PM2 5 are necessary to elicit respiratory effects in people.
Mice exposed to samples of WTC PM25 from the 7
individual sites around Ground Zero had greater lung
inflammation (2 to 4-fold) than mice exposed to the WTC
PM25 sample pooled from these sites. These findings
occurred in separate experiments and would need to be
confirmed by a direct comparison, but nonetheless all
groups of mice exposed to the individual site samples
developed hyperresponsiveness to Mch aerosol challenge,
similar to mice exposed to the pooled sample. No
particular pattern of responses was found corresponding to
the geographical location where the samples were taken.
Pulmonary inflammation in mice exposed to individual
site WTC PM2 5 samples diminished from 1 day to 3 days
after exposure, although hyperresponsiveness to Mch
aerosol did not diminish significantly. Further
experiments would be necessary to determine the
persistence of pulmonary responses in mice, which may
lead to insights into whether any WTC PM-associated
effects which may exist in people are persistent.
The results of these studies should be examined in the
context of previous studies of the effects of
44
-------
environmentally relevant PM samples in rodents. Rats
were intratracheally instilled with 2.5 mg (~8.3 mg/kg) of
various emission source and urban ambient air PM
samples (Costa and Dreher, 1997), a dose about twice as
high, based on body weight, as the 100 |o,g WTC PM25
dose in mice (~4 mg/kg). Oil fly ashes and urban ambient
air PM samples (including a ROFA similar to the one used
in the present study and NIST1649a) induced strong
neutrophilic responses 24 hr after exposure, while
biochemical markers of lung injury were lower in the
urban air PM samples compared with the oil fly ash
samples. ROFA at this dose induced airway
hyperresponsiveness in rats which persisted at least 4 days,
and was greater than that observed in an urban ambient air
PM sample (Pritchard et al., 1996). The fact that WTC
PM2 5 induced a significantly greater degree of airway
hyperresponsiveness in mice than ROFA, which is used as
atoxic positive control particle in many studies, suggests
a very significant respiratory effect of a relatively high
dose exposure to WTC PM25.
Some people were exposed acutely to high
concentrations of dust in the WTC disaster, and
subsequently developed wheezing or symptoms of sensory
irritation, such as cough and irritation of the nose and
throat. These effects resemble, in some respects, the
reactive airways dysfunction syndrome (RADS). RADS
can occur after single or multiple high-level occupational
exposures to an irritating vapor, fume, or smoke (Gautrin
et al., 1999). Effects can occur within minutes or hours
after exposure, and include cough, dyspnea, and wheezing.
Clinical tests can show airways obstruction, persistent
airway hyperresponsiveness, and inflammation. The
recovery process appears to be dependent on the initial
degree of injury. The effects of a high dose exposure to
WTC PM25 in mice (100 |o,g) appear to mimic at least
some ofthese responses, especially the significant increase
in airway hyperresponsiveness to Mch. It is important to
note that WTC PM25-induced pulmonary inflammation,
although significantly greater than in control mice, was not
as robust as one might expect in a realistic animal model
of RADS. However, the degree to which inflammation
and airway hyperresponsiveness are associated in RADS
is not clear (Gautrin et al., 1999). Examination of other
time points would be necessary to determine the
persistence of WTC PM-induced airway
hyperresponsiveness in mice and its similarity to RADS.
Close examination of the data suggested that
individual mice within the outbred CD-I strain vary in
sensitivity to the effects of WTC PM25. Certain
individuals within the human population may also have
particular susceptibility to the hazards posed by exposure
to WTC PM25. It is known that some asthmatic
individuals are hyperresponsive to nonspecific irritants
such as cold dry air (Anderson and Daviskas, 2000) or
cigarette smoke (Bonham et al., 2001). This
subpopulation is likely to be at high risk for development
of dust-induced airways obstruction (Donaldson et al.,
2000; Peden, 2001; Nel et al., 2001). Very few studies
have been published regarding the effects of alkaline
aerosols on pulmonary function in asthma. One study
reported that inhalation of high concentrations of an
alkaline aerosol (pH 9.8 to 10.3) had no significant effect
on irritant symptoms or specific airways resistance in mild
asthmatic patients (Eschenbacher, 1991). However, this
aerosol was composed of a simple mixture of sodium
carbonate, sodium bicarbonate, and sodium hydroxide.
The chemical composition of the alkaline (pH 8.88 to
10.00) WTC PM2 5 is much more complex and interactions
of numerous chemical species may be associated with
development of airway hyperresponsiveness to
methacholine or other bronchoconstrictors.
How does the dose of 100 jig WTC PM25, which
caused bronchiolar inflammation and airway
hyperresponsiveness in mice, relate to exposure of people
at the WTC site? Because inflammation was observed
mainly in the airways, and airway hyperresponsiveness is
mainly due to dysfunction of airway smooth muscle
(Fredberg, 2000), the dose metric which is probably most
relevant is dose per surface area of the tracheobronchial
(TB) region of the respiratory tract. The TB region is
defined as the airways (excluding the nasal (head) region)
from the trachea down to the terminal bronchioles
(Overton et al., 2001). Therefore, to assess the risks of
exposure in people, the concentrations of WTC PM2 5 in
air which could produce doses per TB surface area in
humans equivalent to that in mice should be calculated.
These WTC PM2 5 concentrations may be estimated (Table
22) using the following assumptions: 1) The mouse
alveolar pulmonary surface area can be estimated from an
allometric equation based on body weight (Jones and
Longworth, 1992), and the TB surface area is very small
in comparison to the alveolar surface area (Overton et al.,
2001); 2) Oropharyngeal aspiration bypasses the mouse
nose and spreads the dose of WTC PM25 evenly over the
TB and pulmonary alveolar surface areas of the mouse
lung; 3) The human TB dose per surface area, selected to
match the mouse dose per surface area, does not clear from
the lung in the time frame of exposure to WTC PM2 5 (an
8-hour work shift was selected); and 4) The model of the
fraction of inhaled PM25 (model particles with MMAD =
1, ag = 2.5, and density = 1 g/cc) deposited in the TB
region (Freijer et al., 1999) assumes a reference 30 year-
45
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Table 22 Estimation of WTC PM2 5 Concentrations Required to Produce Human Doses Equivalent to Mouse
Doses Used in WTC2001 Study
Dose deposited in mouse tracheobronchial and pulmonary regions (jig)
Mouse alveolar pulmonary surface area (m2) a
Mouse dose per tracheobronchial (TB) or pulmonary surface area (mg/m2) b
Human TB surface area (m2) c
Total human TB dose equivalent to mouse TB dose (mg/m2 x m2) d
Deposition fraction in human TB region e
Total inhaled dose in mg (total human TB dose / TB deposition fraction)
Quantity of air breathed in 8 hr workshift at ventilation of 30 L/min (m3) f
WTC PM25 concentrations required to produce human doses equivalent to
mouse doses used in WTC2001 Study (|ig/m3)
10
0.103
0.097
0.415
0.040
0.066
0.612
14.4
42
31.6
0.103
0.307
0.415
0.128
0.066
1.932
14.4
134
100
0.103
0.973
0.415
0.404
0.066
6.115
14.4
425
a From Jones and Longworth (1992) calculated allometric equation: Mammalian alveolar pulmonary surface
area in m2 = 3.36 x (Wt, kg)0935, where weight = 0.024 kg (average mouse weight in all studies).
Tracheobronchial surface area is minimal in comparison to alveolar surface area and can be ignored in
calculation.
b Assumes dose is spread out evenly over tracheobronchial and pulmonary alveolar regions.
0 Based on 30 year old, 5' 10" male with functional residual capacity (FRC) of 3300 ml (Overton et al,
2001).
d Calculations assume no clearance of particles after deposition in human respiratory tract.
e Calculations made with Multiple Path Particle Deposition model version 1.11 (Freijer et al., 1999) which
assume human Yeh-Schum 5-lobe model, FRC = 3300 ml (appropriate for 30 year old, 5'10" male), upper
respiratory tract volume = 50 ml, density of particles = 1 gm/ cc, diameter = 1 umMMAD, inhalability
adjustment on, erg = 2.5, breathing frequency = 15 min', tidal volume = 2000 ml, minute volume = 30
L/min, inspiratory: expiratory ratio = 1, and oronasal mouth breathing.
f Estimate of minute ventilation during moderate to heavy sustained work (Astrand and Rodahl, 1986).
old 5' 10" male breathing oronasally with a minute
ventilation of 30 L/min (estimate during moderate to heavy
sustained work; Astrand and Rodahl, 1986). The total
human TB dose and the fraction deposited in the TB
region are used to back-calculate the total inhaled dose of
PM25. The total inhaled dose divided by the quantity of
air breathed in a typical 8-hour work shift yields the
concentration of PM2 5 in the WTC work or neighborhood
environment required to produce human doses equivalent
to the mouse doses used in the WTC2001 study (Table
22). These calculations show that under these conditions,
concentrations of 42, 134, and 425 |o,g/m3 WTC PM25
would produce human doses per TB surface area
equivalent to the mouse doses of 10, 31.6, and 100 |o,g,
respectively. Obviously many factors may cause wide
variations in the calculation of dose, and extrapolation of
responses from the mouse to the human involves another
dimension of uncertainty which was not considered, but it
seems reasonable to say that a healthy worker breathing
heavily in the dusty environment generated after the
collapse of the towers could have inhaled enough PM2 5 to
approximate the 100 jog dose in the mouse. Therefore,
inhalation of a very high concentration of WTC PM2 5 (e .g.
-425 ng/m3) over a short period of time (8 hr) could have
contributed to development of pulmonary inflammation,
airway hyperresponsiveness, and manifestations of sensory
irritation such as cough. Individuals who are especially
sensitive to inhalation of dusts, such as asthmatics, may
experience these effects at lower doses of inhaled WTC
PM25. However, most healthy people would not be
expected to respond to moderately high WTC PM2 5 levels
(130 |ig/m3 or less for 8 hours) with any adverse
respiratory responses. The effects of chronic or repeated
exposures to lower levels of WTC PM2 5, or the persistence
of any respiratory effects are unknown and were not
components of this study. The persistence of any effects
of inhaled WTC PM25, if similar to RADS, would be
expected to depend on the dose initially deposited in the
respiratory tract.
It is important to consider several limitations of these
studies. First, most of the experiments used oropharyngeal
aspiration to deliver PM samples to the respiratory tract
46
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rather than more physiologically relevant inhalation
exposure methodology. We believe that utilizing
oropharyngeal aspiration, as described in the Experimental
Design section, had many advantages and was necessary
in these circumstances. However, this report indicates that
future studies may be needed to more closely examine
bronchoconstriction and sensory irritation during
inhalation exposure to WTC PM in mice and in guinea
pigs, a species known to be especially sensitive to sensory
irritants (Costa and Schelegle, 1999). Secondly, these
studies only evaluated short-term toxicological effects
(endpoints were examined 1, 3, or 6 days after exposure)
after acute exposure and no direct information is provided
on the long term effects of acute or chronic exposures to
WTC PM25. Thirdly, gaseous and vapor-phase toxicants
(e.g. dioxin and volatile organic compounds such as
benzene) were certainly released, especially during the
fires which continued for months after September 11
(EPA, 2002c). The collection and processing techniques
described in this report do not allow investigation of these
important toxic species, nor are the interactions of
particles with gases or organic vapors considered (Mautz
et al., 2001). Finally, these studies only examined fine
PM2 5, while the toxicity of coarse mode and larger size
PM fractions were not investigated. However, it is
important to remember that the size-fractionation
techniques employed in this report are not absolute, and
significant quantities of PM > 2.5 jam are present in the
samples. Furthermore, analysis of the WTC PM25 and
PM53 samples showed that they were similar in
composition (Tables 3 and 5), suggesting that only
differences in respiratory tract deposition patterns of fine
and coarse WTC PM would affect biological responses.
Coarse mode PM may be more relevant for upper airways
sensory irritation because larger particles will mainly
deposit in the upper airways where sensory innervations
are predominant (Costa and Schelegle, 1999). However,
chronic effects of fine PM may be greater than coarse PM
since it can be inhaled more deeply and deposit in
peripheral regions of the lungs, and is more slowly
cleared. Coarse PM is much less inhalable in small
rodents than in humans, and less is deposited in the
respiratory tract (Menache et al., 1995). Consequently,
interpretation of results derived from exposure of mice to
coarse PM is problematic, and small rodents are probably
not the ideal species to study effects of coarse PM.
Nevertheless, because upper airways irritant responses
seem to be so important in people exposed to WTC-
derived dust, future studies should examine the specific
toxicity of coarse WTC PM on respiratory responses in
appropriate animal models.
47
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V. Quality Assurance Statement
U.S. EPA World Trade Center Research Project:
"Toxicological Effects of Fine Particulate Matter Derived
from the Destruction of the World Trade Center"
Page 1 of2
The study "Toxicological Effects of Fine Particulate Matter Derived from the Destruction of the World Trade Center"
was conducted by the Pulmonary Toxicology Branch, Experimental Toxicology Division (ETD), National Health and
Environmental Effects Research Laboratory (NHEERL), Office of Research and Development, U.S. Environmental
Protection Agency, Research Triangle Park, NC, in compliance with NHEERL QA Guidelines. Results of these
inspections were reported directly to the Principal Investigator (PI) of the Study, Dr. Stephen Gavett. Critical phases in
the study were audited.
Date of Inspection
October 29, 2001
October 31,2001
November 2, 2001
November 2, 2001
Novembers, 2001
November 5-14,2001
November 15, 2001
November 19, 2001
November 26, 2001
November 27,2001
Decembers, 2001
Item Inspected
Particulate Matter (PM) filters delivered to EPA.
Attempted scraping of PM from filters.
Approval of study protocol.
Extraction of PM from filters.
Shipment of WTC dust samples for endotoxin testing.
Conduct of Experiment A1 -A3: weighing of samples and mice, randomization of mice,
dosing of mice, BUXCO, DLCO, BAL, cell counts, methacholine responses, lung
samples.
Delivery of NIST samples and blank filters.
Delivery of #3 Cortland Sample. Shipment of NIST samples for endotoxin testing.
Delivery of Experiment B inhalation sample (WTC 3).
Conduct of Experiment B (Day 0): placing and removal of mice in inhalation chambers,
operation of inhalation pump. Shipment of #3 Cortland Sample Back to NYU.
Completion of Experiment B (Day 6): Nasal fixation.
Receipt of endotoxin results on WTC dust samples.
48
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Quality Assurance Statement
Page 2 of 2
Date of Inspection Item Inspected
December 4, 2001 XRF/XRD laboratory tour. Shipment of 96 mice heads to Michigan State University.
December 11, 2001 Conduct of Experiment C (Day 0): Dosing of Mice.
XRF/XRD technical meeting. Receipt of NIST endotoxin test results.
December 27, 2001 Shipment of 184 lung tissues for histopathological analysis.
January 15-17, 2002 Technical Systems Review of project. Interviews with study personnel and inspection
of project data and records.
January 16, 2002 Shipment of six PM samples, and 12 PM samples and 10 filter samples for chemical
analysis.
January 16-25, 2002 Data audit of spreadsheets against notebooks.
January 30, 2002 Shipment of 10 liquid samples for chemical analysis.
March 15, 2002 Transfer of custody of 12 PM Samples from the EPA Chemist to the PI.
March 4-19, 2002 Data audit of Draft Final Report.
The Quality Assurance Manager of ETD and the Director of Quality Assurance for NHEERL have determined by
the above review process that the conduct of this project was in compliance with EPA quality requirements and the
operating procedures and study protocol (Intramural Research Protocol No.: IRP-NHEERL-H/ETD/PTB/SHG/01-01-
000). Furthermore, the results accurately reflect the raw data obtained during the course of the study.
/s/ 03/22/2002
Thomas J. Hughes, ETD QA Manager Date
/s/ 03/22/2002
Brenda T. Culpepper, NHEERL Director of QA Date
49
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