X-/EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-81-067 Feb. 1982
Project Summary
Determination of the Effects of
Material from Alternate Energy
Sources on the Upper
Respiratory Tract Clearance
Mechanism: Part I and Part II
Dorothy Adalis
Studies were conducted to measure
the toxic effects of a variety of sub-
stances from the environment on the
clearance mechanism of the upper
respiratory tract using an in vitro
hamster model system.
Studies using hamsters for in vivo
exposures to ozone were also con-
ducted to determine the effects of
ozone on the cilia beat frequency and
cytopathology of the trachea! epithe-
lium.
Organ cultures of hamster trachea!
tissue were exposed to graded con-
centrations of pollutants to determine
effects on the respiratory cilia. Param-
eters studied were beat frequency,
ciliostasis, cytopathology, and ATP
concentration.
A preliminary survey to determine
the "lethal level" and "no measurable
effect level" on ciliary beating for 15
trace metals found in the environment
and in the effluents from coal gasifica-
tion was conducted. The metals toxic
at the 100 Aig/ml level following 24
and 48 hours of in vitro exposure were
cadmium, zinc, nickel, manganese,
mercury and vanadium. Cobalt was
toxic at 10 /ug/ml following 48 hours
of in vitro exposure. Copper was lethal
at 10 jug/ml and chromium was lethal
at 1 /jg/m\ after 24 hours of exposure.
Extensive and/or complete data for
time and dose levels of all the metals
was not determined because of the
preliminary nature of this survey.
Material from an electrostatic pre-
cipitator of a coal-fired power plant
was not toxic at levels as high as 1075
jug/ml in suspension following 48
hours of exposure. Exposure of rings
for 48 hours to a filtrate of 2477
/ug/ml also showed no observable
toxic effect on the epithelial cells and
there was no effect on the beat fre-
quency due to treatment.
In vitro exposures to nickel at 0.63,
6.3 and 63.5/ug/mlfor 24,48, and 72
hours resulted in a dose related pattern
of cytopathological changes in the
trachea! ring explants. ATP content of
nickel treated rings decreased as
concentration of nickel increased
(P=0.01).
Treatment with VO3 in concentra-
tions of 0.63 /ug/ml to 12.5 fjg/m\ did
not produce statistically significant
differences in ATP content of rings.
However, dose related histological
changes resulted from exposure to
vanadium in concentrations of 0.63
fjg/m\ to 12.5 Aig/ml for 24, 48 and
72 hours. Ciliostatis occurred at levels
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as low as 0.63 //g/ml and the results
were statistically significant from the
controls at the P < 0.005 confidence
level.
In vitro exposure studies using zinc,
zinc and ammonium and ammonium
ion showed dose response changes in
ciliostasis and ATP concentration at
levels of 50, 75, 100 /ug/ml zinc and
28, 41, 55 /ug/ml NH4. Differences
from the controls were statistically
significant at P < 0.05. A.combination
of zinc plus ammonium ions was
significantly more toxic than either ion
alone. All treatment levels of zinc or
zinc plus ammonium caused a signifi-
cant decrease in ATP content as com-
pared to the controls (P < 0.001).
Results of in vivo exposures to .25
ppm and .5 ppm ozone indicated a
dose related reduction in cilia beat
frequency that was statistically signif-
icant at the P < 0.01 level exposed
versus control hamsters.
The histological assessment also
indicated dose related, adverse patho-
logical effects in the tracheas of
hamsters exposed to ozone.
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC. to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Part I. In Vitro Exposure to Participate Pollutants
Introduction
Airborne particles emitted into the
environment from the utilization of
materials from all types of power plants
constitute a major source of pollution
directly related to the problem of air
quality for the human population. A
number of new substances from alter-
nate and conventional energy sources
have not been lexicologically evaluated
for their potential adverse health effects
in humans Some of these substances,
including the ones in this study, are
materials derived from shale oil, coal
gasification and liquefaction and par-
ticulate effluents from both mobile and
stationary sources.
Once these substartces are inhaled
and deposited within the lung, they may
exert a toxic effect on the host via one or
more of these mechanisms: (1) the
substance may be intrinsically toxic due
to its inherent chemical or physical
properties, (2) the substance may inter-
fere with one or more of the clearance
mechanisms in the respiratory tract,
and (3) the substance may act as a
carrier of an absorbed toxic substance.
Depending on their site of deposition,
these substances come into intimate
contact with very specialized host cells,
i.e., cilia and alveolar macrophages,
whose function is to rid or clear the body
of inhaled substances. If these defense
systems of the host are reduced m
activity then an accumulation of both
viable and nonviable substances would
occur, which in turn might jeopardize
the health of the host.
The overall purpose of this project
was to screen a variety of substances
from the environment for their toxic
effect on the clearance mechanism of
the upper respiratory tract using an in
vitro hamster model system. In testing
for cytotoxicity, an in vitro organ culture
system was used employing hamster
tracheal rings to determine specific
effects on normal ciliary activity and
ciliated epithelium. This isolated pseudo-
stratified ciliated epithelium provided a.
controlled means for assessing some
direct effects of the pollutants
Samples of pollutants tested were
supplied by the contractor or the EPA
Project Officer. Materials tested and
studies completed in this project include
the following: a screening study using
15 trace elements from the environment,
cytohistological and ATP effects of
nickel, a beat frequency study of a
sample from an electrostatic precipitator
and two in depth studies of the toxicity
of vanadium, and zinc/ammonium sul-
fate ion.
Dose response experiments were
designed using various concentrations
and lengths of exposures. The various
parameters assayed in the hamster
tracheal model included histopatholog-
ical alterations, ciliary beat frequency,
ciliostasis, and ATP determinations.
Conclusions
The purpose of this project was to
evaluate the effects of various materials
on the ciliary function of the upper
respiratory tract. Experiments com-
pleted in this project demonstrate that
alterations in ciliary function seem to be
indicative of an impairment of the
normal clearance mechanism. The
results from the data showed statistically
significant differences between control
and exposed tracheal tissue in an in
vitro organ culture model.
Data from the screening study of 15
trace metal pollutants indicated toxicity
to tracheal ciliary beating activity for
mercury, zinc, cadmium, vanadium,
nickel, cobalt, copper and chromium.
Molydenum, manganese, barium, lead,
chromium (in the +3 valence state),
sulfite, and magnesium were not toxic
after 48 hours of in vitro exposure to
concentrations of at least 100 /ug/ml.
Histologically, there was evidence of
damage to the ciliated epithelium of the
tracheal rings following exposure to
graded concentrations of nickel and
vanadium. Denudation, tufting of cilia,
sloughing, blebbing, vascularization of
cytoplasm, notched nuclei, eosinophilic
inclusions and flattened cells were
observed in a dose related pattern
tracheal rings exposed to either pol-
lutant
Treatment with both nickel and zinc
sulfate or zinc ammonium sulfate re-
sulted in statistically significant (P <
0.05) dose related decreases in the ATP
content of tracheal ring explants. In vitro
exposure to vanadium did not produce
correlating changes in the ATP content
of rings.
In terms of tracheal clearance, the
significant differences in % ciliostasis
with both zinc and vanadium indicate
impairment of normal ciliary activity. If
the effectiveness of the mucociliary
activity depends on normal functioning
of the ciliated epithelium of the upper
respiratory tract, it follows from the data
presented that inhaled environmental
pollutants can inhibit the normal muco-
ciliary function needed to clear possible
harmful and infectious agents from the
respiratory tract.
Recommendations
The in vitro hamster tracheal ring
model used in this study is excellent for
preliminary screening of environmental
chemicals. This in vitro system is a
convenient and inexpensive means of
examining the effect of acute and short
term exposure to potentially toxic sub-
stances. Such substances, if present in
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sufficient quantities to allow for testing,
should be examined by preliminary
screening in such an in vitro system.
Previous studies which compare this in
vitro method with in vivo exposures
support the use of the in vitro model for
testing the effects of pollutants on the
living animal.
In addition, the short term, acute
exposures of the materials evaluated in
this study should be extended to include
multiple exposures and recovery
projects. Pollutants should be evaluated
in combination with a stress factor such
as exercise. Further detailed studies
should include in vivo exposures for
validation of in vitro results.
Our study of ATP concentration was
an attempt to elucidate the site of pol-
lutant effect on ciliary activity. Further
studies might examine oxidative and
enzymatic responses to pollutants.
Part II. In Vivo Exposure to Ozone
Introduction
An increase in ozone concentrations
of ambient air has become one of the
prime concerns of air quality organiza-
tions. Ozone, a gas, which has its
chemical origin as a product of photo-
chemical-oxidant formation, is con-
sidered a secondary pollutant since it is
formed as a result of chemical reactions
in the atmosphere rather than being
directly emitted by a pollution source.
Primary pollutants (these directly emitted
by a pollution source) most responsible
for ozone and other oxidant formation in
the air are the nitrogen oxides, hydro-
carbons, and carbon monoxide. Ozone
and other oxidants formation is related
to time of day, meteorologic conditions,
and the amount of primary pollutants in
ambient air. Ozone is used by the U.S.
Environmental Protection Agency as an
indicator of photochemical smog.
The overall purpose of Part II of this
study was to determine the effects of in
vivo exposure to three concentrations of
ozone on the ciliated epithelium of
hamster tracheal rings. Specifically,
two parameters were studied following
in vivo exposures to ozone1
1) a cytopathological assessment
was made to determine if ozone
alters or interferes with the struc-
tural integrity of the ciliated epi-
thelium, and
2) quantitative measurements of the
cilia beat frequency were deter-
mined using a stroboscope and
statistically analyzed for signifi-
cant differences between control
and exposed rings.
A variety of times and doses were
studied to include 0.25, 0.5, and 1.0
ppm for 1, 3, or 5 days.
Conclusions
In vivo exposure to 0.25 and 0.5 ppm
ozone 3 hrs x 3 days resulted in a
significant reduction.in ciliary beat
frequency. This reduction in beat fre-
quency increased as the concentration
of ozone increased. The mean beating
frequency in beats per minute for 94
control rings (8 animals, 4 of each sex)
was 117.4.6. The mean beating fre-
quency for 93 rings from 8 animals
exposed to 0.25 ppm ozone 3 hours x 3
days was 1089.2. Mean beat frequency
for 8 animals exposed to 0.5 ppm ozone
was 1009.0 (89 rings). An analysis of
variance indicated a significant decrease
(P<0.005) in beat frequency in the
ozone treated rings when compared
with controls. There was no significant
difference in beat frequency between
sexes or due to interaction of sex and
treatment.
The results indicate an altered func-
tioning of the tracheal ciliated epithe-
lium which would possibly interfere
with the host's normal pulmonaryclear-
ance mechanism. This in turn would
make the host more susceptible to
respiratory infection.
Effects of in vivo exposure to ozone
produced pathological effects which
were dose related. Increasing the con-
centration of ozone results in increasing
disorganization of the pseudostratified
ciliated epithelium lining of the tracheal
rings. The degree of disorganization
was also directly related to the length of
time or exposure at any one concentra-
tion level.
The resulting damage to tissue mor-
phology due either to concentration or
length of exposure to ozone would alter
the normal functioning of the mucociliary
escalator. This impairment could in-
crease the susceptibility of the host to
respiratory infections.
Recommendations
Because of,the widespread and in-
creasing occurrences of ozone pollution,
further studies should be conducted.
The short term acute exposures to
ozone in this study should be extended
to include ozone in combination with
other pollutants. Also needed are studies
to determine the effects of ozone in
combination with stress factors such as
exercise or in relation to diet.
Other studies recommended include
the following'
1. Recovery of ciliary activity follow-
ing in vivo exposures to ozone.
2. Measurement of ATP production
in hamster heart, liver and gastro-
cnemius muscle following in vivo
exposure to ozone.
3. The effect of ozone on the oxidative
capacity of hamster heart, liver
and muscle.
4. Effect of ozone on the oxidative
capacity of hamster tissues fol-
lowing exercise.
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Dorothy Ada/is is with the Department of Biology, Ball State University, M uncle,
IN 47306.
Donald E. Gardner is the EPA Project Officer (see below).
The complete report, entitled "Determination of the Effects of Material from
Alternate Energy Sources on the Upper Respiratory Tract Clearance
Mechanism: Part I. In Vitro Exposure to Paniculate Pollutants. Part II. In Vivo
Exposure to Ozone." (Order No. PB 82-117 037; Cost: $9.00. 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. 1982 — 559-017/7447
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
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