United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
X-/EPA
Research and Development
EPA-600/S1-81-003 Mar. 1981
Project Summary
Effect of Industrial Paniculate
Samples on Alveolar
Macrophages
Catherine Aranyi
Toxic ranking of the <3 /um size
fraction of particulate samples in in
vitro rabbit alveolar macrophage assay
showed generally low to intermediate
cytotoxicity for samples obtained
from a steel foundry, an electric arc
furnace steel plant and from four coal-
fired power plants. Particulates ob-
tained from an aluminum and from a
copper smelter were highly toxic to
alveolar macrophages as monitored
by viability, total cellular protein and
ATP levels. The two smelter samples
also contained soluble components
that significantly contributed to their
cytotoxicity. None of the particulate
samples tested were true emission or
effluent samples, but were collected
from in-plant control devices.
The copper smelter dust and the
fluidized-bed coal fly ash chosen on
the basis of their respective high and
low in vitro cytotoxicity were used in
aerosol exposures to examine their in
vivo effects on the pulmonary free
cells, bactericidal activity and resist-
ance to respiratory infection in mice.
The results of multiple daily 3-hour
exposures to 2.0,1.0 and 0.5 mg/m3
of the pollutants closely correlated
with the in vitro data. Inhalation of
copper smelter particle aerosols pro-
duced significant changes in more of
the parameters than inhalation of the
coal fly ash. Thus the overall objective
of these studies was realized by dem-
onstrating the validity of prediction of
inhalation hazard on the basis of the in
vitro screening assay.
This Project Summary was develop-
ed by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC, to announce key findings of the
research project which is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction
Alveolar macrophages protect the
lungs principally by phagocytosis of
inhaled particles and infectious agents.
Adverse effects on the activity of the
alveolar macrophages can lead to in-
creased retention of non-biological
particles, as well asto impairment in the
defensive capacity of the lung and
consequent increased susceptibility to
respiratory disease. Since resistance to
infection is known to be lowered by
exposure to pollutants, changes in the
functional characteristics of alveolar
macrophages can be used to monitor
effects of such exposure in the intact
animal. However, since alveolar macro-
phages can be obtained easily by tra-
cheobronchial lavage and maintained in
culture, they are frequently used in in
vitro toxicology to assess the potential
inhalation hazard of various substances.
The advantages of in vitro screening
assay in terms of cost and time efficiency
are well known. The rabbit alveolar
macrophage test, a rapid, in vitro assay
has been used extensively at EPA and
IITRI laboratories to evaluate the relative
cytotoxicity of a variety of soluble com-
pounds and particulates (1, 2, 3,4, 5,6,
-------
7, 8). This bioassay system is capable of
screening and toxicity ranking of a broad
spectrum of materials and thereby
identified not only the potentially haz-
ardous, but also the inert compounds.
Based on results of the in vitro assays
the number of samples which must be
studied further in vivo can be signifi-
cantly reduced.
The objective of the studies was to
determine if in vitro exposure of alveolar
macrophages to a series of complex
industrial particles resulted in the same
relative toxicity ranking as in vivo aerosol
inhalation exposures to these particles
in intact animals. The studies were
designed to examine the relative toxicity
of a number of particulate samples. The
in vitro studies provided initial informa-
tion on the toxicity of the particles and
these data formed the basis for selection
of samples for the subsequent in vivo
investigations. The test particles used in
the studies were collected from various
metal manufacturing plants and coal-
fired power plants directly from in-plant
control devices and were not post-
control device emission samples. All
samples were air classified; only
particles in the 3/um size-fraction were
used.
Conclusions
Particles provided by the EPA were
collected as baghouse samples, or from
electrostatic precipitators, or by cyclone
sampling train from three conventional
and one fluidized-bed coal fired power
plants, and electric arc furnace steel
plant, a steel foundry and an aluminum
and copper smelter. None of the samples
reported here were emission or effluent
samples collected after pollution control
devices and were not necessarily similar
in composition or toxicity to emission
samples. All samples were air classified
and only particles in the <3 /um size-
fraction were used.
The in vitro effects on rabbit alveolar
macrophages were monitored in dose
response experiments using cell viability,
total protein and ATP levels as experi-
mental parameters. Regression analysis
applied to these data showed a signifi-
cant negative linear dose response
relationship for each parameter in all
samples, thus enabling the evaluation
of their relative cytotoxicity. The results
indicated that particles from a steel
foundry and an electric arc furnace steel
plant, and coal fly ash samples from
three conventional combusiton processes
and one fluidized-bed system had a low
to intermediate cytotoxic effect. Samples
from a copper and an aluminum smelter
ranked high in cytotoxicity relative to all
others, with the copper smelter sample
being the most toxic. In addition to the
particles per se soluble components
released from copper and aluminum
smelter samples contributed to their
cytotoxic effect on alveolar macrophages.
To confirm the relevance of the in vitro
assay the inhalation hazard of these
particulate samples was studied in
intact animals. Mice were exposed 3
hr/day, 5 days/week for up to 4 weeks
to aerosols of the copper smelter dust
and of the fluidized-bed coal fly ash, i.e.,
particles that had shown high and low
cytotoxicity in vitro. Aerosol mass con-
centrations used were 2040 and 1010
fjg/m3 for the coal fly ash and 2050,
1020 and 540 /ag/M3 for the more toxic
copper smelter dust. The effects of
inhalation on the pulmonary defense
system were determined after 5,10 and
20 exposures by examination of the
pulmonary cellular lavage, bactericidal
activity in the lungs and the resistance
to experimentally induced respiratory
bacterial infection. Results of these
studies substantiated the in vitro obser-
vations by demonstrating that inhalation
of copper smelter dust was significantly
more deleterious (i.e., increased sus-
ceptibility to streptococcus infection
and decreased pulmonary bactericidal
activity) than of the fluidized-bed coal fly
ash. The major objective of these studies
was accomplished by demonstrating
the feasibility of predicting the potential
inhalation hazard of a particulate sub-
stance on the basis of the in vitro alveolar
macrophage screening assay.
Recommendations
Copper smelter dust which contained
13% arsenic in addition to such other
major trace metal constituents as lead,
copper, iron, antimony and zinc showed
high in vitro cytotoxicity in the rabbit
alveolar macrophage assay. These in
vitro studies also indicated that much of
the arsenic can be solubilized from the
particles and the leachate per se is toxic
to alveolar macrophages. Inhalation of
copper smelter dust «3/jm aerodynamic
diameter) significantly reduced the
pulmonary bactericidal activity and the
resistance to respiratory bacterial infec-
tion in mice. If the arsenic content is
used as one basis of assessment of the
health hazard of the copper smelter
dust, the experimental results suggest
that five daily 3-hr aerosol exposures to
an equivalent of 266 Aig/m3 of arsenic M
resulted in significantly increased mor- ™
tality rates from streptococcal pneumo-
nia. Moreover, a significant depression
of pulmonary bactericidal activity was
seen after five daily exposures to 133
fjg/m3 of arsenic. These concentrations
are considerably lower than the TLV of
500 //g/m3 of arsenic.
Most the arsenic in the atmosphere is
the consequence of emissions from
copper, lead and zinc smelters. At
smelting operating temperatures arsenic
trioxide is formed that, upon cooling,
condenses on the surface of small parti-
culate effluents. Since soluble arsenic
trioxide adsorbed on particulates of <3
fjm may easily penetrate into the gas-
exchange region of the lung, it can be
released there. Larger aerosol particles
containing arsenic in soluble and/or
non-soluble form can partially be adsorb-
ed in the upper respiratory tract and the
conducting airways.
Thus, studies should be conducted to
determine if particulate aerosols that
contain components potentially soluble
in the respiratory tract a priori studies
are necessary to explain how the respira-
tory defense systems are affected by the
form of arsenic or by the particle size of
the inhaled aerosol. Since the copper A
smelter dusts also contain other metals *
such as lead, copper, zinc and antimony
that can potentially contribute to their
toxicity, studies should be undertaken to
compare the effects of inhalation of
smelter dusts with those of aerosol
exposures to such trace metals in a
soluble form and at chemically equiva-
lent concentrations.
The in vivo studies indicated increased
susceptibility to respiratory infection in
mice exposed to copper smelter dust
particles. Further studies are necessary
to determine the effects of inhalation of
such toxic particulate aerosols on the
cellular and humoral immune systems
and thereby elucidate the changes in
the immune mechanisms due to particu-
late pollutant-induced immunotoxicity.
-------
Catherine Aranyi is with IIT Research Institute, Chicago, IL 60616.
Donald £. Gardner is the EPA Project Officer (see below).
The complete report, entitled "Effect of Industrial Paniculate Samples on
Alveolar Macrophages," (Order No. PB 81-150 963; Cost: $6.50, subject to
change} will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
, US GOVERNMENT PRINTING OFFICE 1981-757-012/7013
-------
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45266
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use 8300
Region 5 Library
US EPA
230 S. Darborn St,
Chicago, IL 60604
L
------- |