SEPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/D-84-111 May 1 984
ENVIRONMENTAL
RESEARCH BRIEF
Health Effects of Fine- and Coarse-Mode Particulate Matter:
Exposures by Inhalation and Intratracheal Instillation
Catherine Aranyi1, John Drummond2, Richard Ehrlich1, James D. Renters1,
Jean Graf1, Leonard J. Schiff1, and Peter T. Thomas1
Abstract
The effects of fine- and coarse-mode particles on the
respiratory defense system were studied in a two-phase
investigation. Aerosols of fine-mode particles were used in
inhalation exposures, whereas coarse mode particles were
administered by intratracheal instillation.
Rangefinding acute and repeated exposures to ammonium
sulfate or ammonium nitrate aerosols were followed by
subchronic exposures to ammonium sulfate, sulfur dioxide
and ozone mixtures to elucidate the potential impact on
health of interactions of these particles and gases. The
effects of exposure to 0.2 mg/m3 ozone, or to a mixture
containing ozone, 13.2 mg/m3 sulfur dioxide and 1.04
mg/m3 ammonium sulfate for 5 hr/day, 5 days/week for
up to 103 days were evaluated in mice in a series of in vivo
and in vitro health effects assays following the exposures.
Statistically significant changes were observed in suscep-
tibility to streptococcal pneumonia, in the bactericidal
activity of alveolar macrophages, in in vitro cytostasis in
tumor target cells cocultured with peritoneal macrophages
and in splenic T-lymphocyte function measured by blasto-
genesis to mitogens and alloantigens.
The effects of coarse-mode particles of quartz, ferric oxide,
calcium carbonate and sodium feldspar, on host defenses
against pulmonary bacterial infection were also investi-
gated. Mice receiving intratracheal instillations of 10, 33 or
100 A/g/mouse were challenged with Streptococcus sp
aerosols within 1 or 24 hr after the exposure. Mortality from
streptococcal pneumonia was significantly increased by
'NT Research Institute, Life Sciences Research Division, Chicago, IL 6061 6.
international Research and Development Corporation, Inhalation Toxicology
Mattawan, Ml 49071.
exposure to all particles at the 33 or 100 A/g/mouse
concentration. Delaying the infectious challenge to 24 hr
caused partial recovery in mice exposed to sodium feldspar
but not to those exposed to the other particles.
The following publications resulted from the studies: Effects
of Subchronic Exposure to a Mixture of Oa, S02, and
(NhUJsSCU on Host Defenses of Mice. Catherine Aranyi,
Stanley C. Vana, Peter T. Thomas, Jeannie N. Bradof,
James D. Renters, Judith A. Graham, and Frederick J.
Miller; J. of Toxic. Environ. Health 12:55-71, 1 983.
The Effects of Intratracheally Administered Coarse Mode
Particles on Respiratory Tract Infection in Mice. Catherine
Aranyi, Jean L. Graf, William J. O'Shea, Judith A. Graham,
and Frederick J. Miller; Toxicol. Lett. 19:63-72, 1983.
Introduction and Summary Text
Atmospheric aerosols are classified as fine- and coarse-
mode according to particle size. Sulfate, nitrate, and
ammonium salts and organic compounds are generally
found as fine-mode particles produced by condensation,
growth and agglomeration. Coarse-mode particlesoriginate
from mechanical abrasion of earth crustal materials and
typically contain iron, aluminum, titanium, silica and
calcium. The demarcation between fine- and coarse-mode
urban aerosols is at approximately 2.5 pm aerodynamic
diameter with the greatest mass of particles occurring at.
0.4 to 0.5 and 5 to 7 ^m for fine- and coarse-mode particles
respectively. Although a significant amount of health effects
information is available, many uncertainties exist, particu-
larly with respect to effects on host defenses against
pulmonary infection of these particles. Epidemiological
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studies have shown associations between exposure to
mixtures of sulfur dioxide (SOa) and particles and increased
incidence of pulmonary infections. However, little informa-
tion is available to define the chemical species of the
particles which were responsible for these observations.
Initial studies were conducted using single and/or repeated
exposures to ammonium sulfate [(NH4)2SO4] and ammon-
ium nitrate (NH4NOa) to obtain rangefinding data which
form the basis for the design for the subchronic study. The
purpose of the subchronic study was to evaluate the influ-
ence of SO2 and particles in the presence of ozone (O3) on
essential host defense functions. Sulfur dioxide (NH4)2S04,
and Qa were chosen because of (a) our interest in S02-
particulate interactions; (b) because (NH4)aSO4 is a major
component of fine-mode urban aerosols; and (c) because of
the ubiquitous presence of O3 and its effects on host
defenses against infection. The 5-hr experimental exposure
period corresponds to the ambient Oa peak patterns usually
observed in urban environments. The panicle-size distribu-
tion of (NH4)zS04 was chosen to correspond to that of this
pollutant in fine-mode urban aerosols. Since SO2 and
(NH4)2SO4 given singly at the same or at higher concentra-
tions had no effect on the major endpoints to be examined,
(Ehrlich 1979;Ehrlichetal. 1978) there was an insufficient
rationale for further study of these pollutants alone. Thus, a
mixture of Oa, SO: and (NH4)2S04 aerosol, was decided
upon, especially since Oa co-exists with ambient aerosols.
To evaluate the contribution of Oa to any effects observed
with the mixture, an Oa group was included. The control
group was exposed to filtered air.
Several health effect parameters were assessed to evaluate
.effects of the exposures. Changes in susceptibility to
respiratory infection in which experimental and control
mice are simultaneously challenged with streptococcal
aerosol and mortality rates are compared, was selected
because of the sensitivity of this model in determining the
impact of pollutants on several host defense mechanisms.
Pulmonary bactericidal activity was measured to determine
if the exposures affected the function of alveolar macro-
phages in situ. The activity of alveolar macrophages and
other free lung cells obtained by tracheobronchial lavage
was also examined. In vitro peritoneal macrophage cyto-
statis to tumor target cells was measured to assess whether
the inhalation exposures affected extrapulmonary macro-
phage activation, a potential defensive capacity against
tumor growth. The effect of the exposures on the systemic
immune system was studied whereby the blastogenic
response of splenic lymphocytes to mitogens and alloanti-
gens, and plaque-forming cell response to sheep red blood
cells were used to measure the cell-mediated and humoral
immunity, respectively. Trachea! ciliary beating frequen-
cies and alterations in cytology in the trachea! epithelium
from exposed animals were assessed.
Intratracheal instillation was used for exposures to various
coarse-mode particles to obtain a toxicity ranking that
would provide guidance for future inhalation studies. The
use of this exposure method circumvented the extreme
difficulty in generating stable coarse-mode polydisperse
aerosols at sufficient mass concentrations and for adequate
length of time. Iron oxide, calcium carbonate and kaolin
(clay) were chosen for evaluation, as relatively common
coarse-mode particles in the atmosphere. Silicon doxide
was included since silica in fine-mode form is known to be
toxic and it isfound in ambient aerosols in the coarse-mode.
In selecting the actual compounds to be used, the primary
consideration was to obtain materials as close as possible
in crystal structure, crystal habit, and phase purity to those
encountered in ambient atmospheric aerosols. Therefore,
hematite (FezOa) was selected as the typical iron oxide
compound, calcite (CaCOa) as the calcium carbonate, and
quartz (SiOz) was selected as the free silica compound.
Because coarse-mode clay particles in the atmosphere are
actually agglomerates and would therefore disperse when
suspended in the saline required for intratracheal instilla-
tion, its geological precursor, the sodium feldspar albite
(NaAISi308), was used.
1. Fine-Mode Particles
a. Acute and repeated exposures to ammonium
sulfate and ammonium nitrate aerosols
Single 3-hr exposures to approximately 2.5 mg/m3
(NH4)2S04 aerosol did not affect susceptibility to respiratory
infection and pulmonary bactericidal activity in mice and
did not produce any significant alterations intracheal
explants in mice and hamsters. Similarly, only minor
changes were noted in the various health effects param-
eters examined after 5, 10 or 20 daily 3-hr inhalation
exposures to approximately 1 mg/m3 (NH4)2S04 aerosol
(59% of the particles had <1.0//m MM AD). After 5 daily
exposures to the aerosol, ATP levels of pulmonary free cells
were decreased in both female and male mice, but no
consistent changes in these parameters were observed
after 10 or 20 daily exposures. Altered trachea! epithelial
cytology was noted only in hamsters after 5 exposures.
Goblet cell hyperplasia was observed in. mouse tracheas
after 10 and 20 exposures. Slight alterations of alveolar
shape and wall thickness were observed by scanning
electron microscopy after all three exposure regimens. No
changes were seen in susceptibility to respiratory infection,
pulmonary bactericidal activity, type 01 viability of free
pulmonary cell, trachea! ciliary beating frequency or
pulmonary histology.
The effects of a single 3 hr exposure to 2 mg/m3 of NH4NO3
and 5 and 20 3-hr exposures to 1 mg/m3 NH4N03 aerosol
(MMAD = 0.9 fjm, erg = 1.8) were evaluated in mice or
hamsters. In general, only a few minor changes were found
in the health effects parameters examined. After the single
exposure, a decrease in trachea! cilia beating frequency,
reduced percent of normal tracheal epithelium, and minor
histologic changes in tracheal epithelium were seen in
hamsters. After 5 exposures, significant decreases in cilia
beating frequency and percent of normal epithelium were
seen only in the hamster tracheal organ culture. After 20
exposures, slight congestion and enlarged alveolar pores
were noted in the lungs of mice. Total pulmonary free cell
counts tended to be greater than control value after 1 or 5
exposures, but lower than control values after 20 exposures
and the ATP levels were lower in pulmonary free cells
lavaged from exposed mice. No changes were seen at any
time in susceptibility to respiratory streptococcus infection,
in pulmonary bactericidal activity and in differential counts
or viability of pulmonary free cells.
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b. Subchronic exposure to a mixture of ozone,
sulfur dioxide and ammonium sulfate
Mice exposed 5 hr/day, 5 days/week for up to 103 days, to
0.2 mg/m3 O3 or to a mixture of 0.2 mg/m3 of O3, 13.2
mg/m3 SO2and 1.04 mg/m3 (NH4)2SC>4showed significant
increase in susceptibility to streptococcal infection when
compared to filtered air controls. Bactericidal activity was
significantly enhanced in the lungs of mice exposed to the
mixture as compared to those exposed to filtered air or to O3
alone. The total number and distribution of the free cells
lavaged from the lungs, as well as cellular ATP levels were
not affected. At a target-to-effector-cell ratio of 1:10, in
vitro cytostasis in tumor target cells cocultured with peri-
toneal macrophages from the exposed mice was signifi-
cantly enhanced by exposure to Os or the pollutant mixture
group relative to controls. The magnitude of the effect of the
mixture was greater than that of O3 alone. No such effects
were observed when the target-to-effector cell ratio was
1:20. Splenic T-lymphocyte function, as measured by
blastogenesis to mitogens and alloantigens, was decreased
by exposure to O3 alone and increased by exposure to the
mixture. Splenic B-cell function and macrophage antigen
processing, as measured by the generation of antibody
plaque-forming cells, was not affected.
2. Coarse-Mode Particles
The effects of coarse-mode quartz, ferric oxide, calcium
carbonate, and sodium feldspar particles on host defenses
against bacterial pulmonary infection were investigated.
Intratracheal instillations of 33 and 100//g/mouse, resulted
in significantly increased mortality from streptococcal pneu-
monia. At 10//g/mouse, only ferric oxide caused a signifi-
cant increase. To evaluate potential delayed effects, mice
were challenged with the bacterial aerosol 24 hr after
instillation of 100/yg/mouse. Delay of the challenge did not
significantly alter the response, except for the sodium
feldspar where a partial recovery was observed. Intratra-
cheal instillation of 100/ug/mouse had no significant effect
on pulmonary bactericidal activity. For the model system
used, it appears that ferric oxide, calcium carbonate and
sodium feldspar have effects roughly equivalent to quartz.
Conclusions
During subchronic exposures to mixtures consisting of O3,
S02 and (NH4)zS04 it appeared that the presence of 0.2
mg/m3 of O3 was the significant cause of the increase in
susceptibility to streptococcal infection.
Total and differential cell counts, viability, and ATP levels in
the free cells lavaged from lungs of mice exposed to the
pollutants did not differ significantly from those in control
mice exposed to filtered air. This is in agreement with
results of 20 3-hr exposures to 1 mg/m3 (NH<)2SO< and
with studies of Ehrlich (1980), showing that exposure to
similar concentrations of SO2 for up to 3 months did not
change the phagocytic activity of macrophages lavaged
from the lung.
Pulmonary bactericidal activity was significantly higher in
the mixture-exposed group compared to the air controls or
to the Os-exposed group. This increase might be due to
activation of the alveolar macrophages after having phago-
cytized the inhaled particles. In previous studies (Aranyi,
1981; Aranyi et al., 1981), increased bactericidal activity
was observed in lungs of mice exposed to aerosols that did
not have any significant effects on host defenses; inhalation
of more toxic particles appeared to overcome this initial
increase, and depressed the bactericidal capacity of the
macrophages.
The increased in vitro cytostasis to tumor target cells
observed for peritoneal macrophages lavaged from the
exposed mice, indicate a possible activation of these
macrophages by the exposures. The pollutant mixture had a
significantly greater effect than O3 alone. The studies
demonstrated that exposure to O3 alone or to the mixture
altered splenic T-lymphocyte function as measured by
blastogenesis to mitogens and alloantigens. The extent of
the change was related to the pollutants treatment. Splenic
B-cell and T-helper cell function and macrophage antigen
processing, as measured by the generation of antibody
plaque-forming cells, was not affected by exposures. Thus,
these exposures caused extrapulmonary effects, hitherto
unrecognized.
Intratracheal instillation was used to examine specific
coarse-mode particles common in the atmosphere.. The
studies demonstrated that intratracheal exposure to Fe203,
CaC03, Si02 and sodium feldspar particles of 5 to 7 /ym
MM AD increased streptococcal-induced mortality in mice.
This mortality has been correlated primarily, but not solely,
to alveolar macrophage function, suggesting that the
presence of the particles depressed alveolar macrophage
function. Such depression was expected following exposure
to Si02 (Allison et al., 1979) and possibly sodium feldspar,
insofar as it is similar to kaolinite (White and Kuhn, 1980).
However, bactericidal activity, which is a more direct
measurement of alveolar macrophage function than the
infectivity model was not affected. Thus it appears that
decrements in host defense mechanisms (in addition to
alveolar macrophages) such as the mucociliary escalator
and edema, may have been also involved. The approximate
equivalency of the effects of silica to the other coarse-mode
particles indicates that coarse-mode particles cannot be
assumed to be nontoxic.
Recommendations
Results of subchronic inhalation studies indicate that
exposure to 0.2 mg/m3 (0.1 ppm) O3 causes alterations in
some pulmonary defense systems. For some parameters/
exposure to mixture containing O3, S02 and (NH<)2S04 was
more effective than exposure to Oa alone. Given the known
low toxicity of SO2 and (NH^SCU for host defense
mechanisms, their role in admixture with O3 should be
further investigated. Since humans are usually exposed to
complex mixtures of pollutants it is important to elucidate
the causative agents in the mixture or to define a
synergistic/antagonistic relationship.
The immunological studies indicated that the mixture and
03 caused effects to the spleen and cells in the peritoneum.
Examination of such extrapulmonary effects is rare,
requiring more research toenable improved understanding
of the full array of effects of these pollutants. In addition.
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since the systemic immune response was affected, it would
be of interest to investigate the pulmonary immune
response since this system would receive more direct
exposure to higher doses.
Intratracheal instillation of coarse-mode particles of SiO2,
FeaOa, CaCOs and sodium feldspar, caused decreased
resistance to respiratory infection. Therefore it is important
to confirm and characterize these responses further using
inhalation exposures.
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