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
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Environmental Protection
Agency
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Information
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