United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA/600/S1-85/008 July 1985
oEPA Project Summary
Morphometric Studies of the
Effects of Ozone on Rodent
Lungs
J. D. Crapo, B. E. Barry, and R. R. Mercer
The degree of lung injury caused by
prolonged inhalation of low levels of
ozone is relevant because a number of
urban environments periodically reach
levels of 0.2 - 0.3 ppm ozone. Morpho-
metric methods were used to evaluate
the effects of 0.25 ppm ozone on lung
tissue of both young adult and juvenile
rats. Three regions of the lung were
examined, the distal alveolar region, the
proximal alveolar region, and the termi-
nal bronchioles. In addition, the effects
of inhalation of 0.12 ppm O3 on the
proximal alveolar region of young adult
rats were also evaluated.
The results showed that ozone in
these low concentrations reacts mainly
in the terminal bronchioles and the
proximal alveolar region. After 6 weeks
of inhaling 0.25 ppm ozone the number
of type I epithelial cells in the proximal
alveolar region doubled in the animals
exposed from 1 day of age. The mean
surface area of type I epithelial cells
decreased 38% and their mean thick-
ness increased 24%. The number of
alveolar type II epithelial cells also
increased and the number of alveolar
macrophages doubled. Young adult
animals exposed to 0.25 ppm ozone
showed similar changes in the epithe-
lium of the proximal alveolar region,
moreover, these animals showed a
doubling of interstitial macrophages
indicating a mile inflammatory stimulus
in the interstitium. In the terminal bron-
chioles, exposure to 0.25 ppm ozone
produced a significant 14-16% decrease
(p< 0.05) in the average luminal surface
area of clara cells. Inhalation of 0.12
ppm ozone for 6 weeks also caused
measureable changes in the proximal
alveolar region of young adult rats. Type
I cell number increased 36% (p< 0.05)
and the mean surface area of both the
air side and the basement membrane
side of the cell decreased 23%. These
results suggest that 0.25 ppm ozone
causes epithelial injury in the proximal
alveolar region and in the terminal
bronchioles of both juvenile and young
adult rats. Exposure to 0.12 ppm ozone
causes a detectable but less extensive
injury to the type I epithelium in the
proximal alveolar region.
This Project Summary was developed
by EPA's Health Effects Research Lab-
oratory. Research Triangle Park, NC, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Ozone is an atmospheric pollutant
occurring as a photochemical oxidant in
ambient air. The concentration of ozone
in ambient air depends on the concentra-
tion of the primary pollutants as well as
local environmental conditions such as
temperature, air stagnation, and sunlight
intensity. For several years oxidant con-
centrations have been measured by state
and local control agency monitoring sta-
tions. These data are gathered and ana-
lyzed by the U.S. Environmental Protec-
tion Agency (EPA). Most urban centers
have intermittently exceeded the current
national air quality standard for ozone
(0.12 ppm), and several cities (Los
Angeles, Denver, Philadelphia, Houston,
and New York) have had high hourly lev-
els in excess of 0.20 to 0.30 ppm. The
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chronic effects of such ozone concentra-
tions are unknown.
Currently there is a scientific agree-
ment that ozone concentrations in excess
of one ppm are hazardous to human
health. Experiments on laboratory ani-
mals (rats, mice, guinea pigs, rabbits, and
monkeys) at ozone concentrations of 0.2
to 1.0 ppm for short exposures have dem-
onstrated qualitative histopathologic chang-
es in conducting airways and alveolar
spaces. Various investigations have des-
cribed: (1) damage to ciliated cells in the
terminal bronchioles with nonciliated
progenitor cell division for bronchiolar
epithelial cell renewal; '(2) hyperplasia
and hypertrophy of nonciliated bronch-
iolar epithelial cells in respiratory bronch-
ioles and intrabronchial accumulation of
alveolar macrophages; (3) significant
cellular damage at the alveolar duct level;
(4) swelling and desquamation of type I
alveolar lining cells with subsequent
proliferation of type II cells to cover
denuded areas; and (5) ultrastructural
evidence of damage to endothelial cells
and the basement membrane. However,
there are currently no qualitative or
quantitative morphologic studies of an-
imals exposed to ozone concentrations
less than 0.2 ppm. Such experiments are
obviously vital to our understanding of
possible damage to the human lung in
urban areas, where low level exposures
occur intermittently in the range of 0.20
ppm or less.
Materials and Methods
Specific pathogen-free male, Fisher
344 rats that were either 1 day or 6 weeks
old were exposed to 0.12 ppm or 0.25
ppm ozone. Rats that breathed filtered
room air served as controls. After 6 weeks
of exposure, rats were sacrificed and
their lungs fixed by intratracheal instilla-
tion of 2% glutaraldehyde. The procedure
used to isolate terminal bronchioles and
proximal alveolar tissue from slices of the
left lung is illustrated in Figure 1.
Ultrathin sections of terminal bronchi-
oles and proximal alveolar regions were
obtained and EM micrographs taken from
them. Morphometric analysis was applied
to study structural changes caused by
inhalation of ozone. Randomly selected
blocks of distal alveolar tissue were also
studied.
Results
Effects of Inhalation ofO.25
ppm O3 on the Distal Alveolar
Region of Juvenile and Young
Adult Rats
Morphometric methods were used to
evaluate the effects of 0.25 ppm ozone on
lung tissue randomly selected from the
alveolar region of mature and neonatal
rats. For this study this area is defined as
the distal alveolar region and it includes
all alveoli beyond the terminal bronchi-
oles. The results indicate that few struc-
tural changes occur in the distal alveolar
region as a consequence of this level of
Os exposure. There were reductions in
the body weight gained by mature anim-
als exposed to ozone for 6 weeks (p=0.05)
and by neonates exposed for 1 week
(p=0.06). Previous investigators have
reported this difference in rats exposed to
1 ppm or less of ozone. The arithmetic
mean thicknesses of the epithelium and
endothelium increased 10%(p=0.08)and
16% (p=0.08), respectively, in mature rats.
The volume density of air in the alveolar
region was significantly smaller in neo-
Sharpened Canula
Terminal Bronchioles
In Cross-section
Terminal Bronchiole
In Cross-section
Embed
Oriented
Tissue
Plug In
Capsule,
ii-—v-TWC'-r/1^- ^!
*_>- r*. ' &r*r+*A
I Serial Section To
\ First Duct Bifurcation
, * 1
T/rt rX.
r>-
Figure 1. Schematic of the microdissection procedure used to isolate lung tissue containing terminal bronchiole and proximal
alveolar region.
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natal rats exposed to ozone for 1 week,
but this change may have been caused by
variations in fixed lung volumes of these
small rat lungs. In neonatal rats exposed
to ozone for 6 weeks, the average base-
ment membrane surface area covered by
type I cells decreased 20% (p=0.06). There
were no significant increases in the
arithmetic mean thickness of epithelium
or endothelium. No other significant
changes were found in the morphometric
characteristics of the distal alveolar re-
gion of the adult or neonatal rats.
Effects of Inhalation of 0.25
ppm Oa on the Proximal Alveolar
Region of Juvenile and Young
Adult Rats
Since the alveolar region most adjacent
to terminal bronchioles has been reported
as one of the major sites of injury due to
ozone inhalation, this specific lung region
was studied using ultrastructural mor-
phometric analysis. After 6 weeks of
exposure to 0.25 ppm 03, qualitative
examination of the tissue revealed no
observable damage to the terminal bron-
chioles or the adjacent proximal alveolar
tissues. However, by morphometric anal-
ysis, significant changes occurred in the
alveolar epithelium of the proximal alve-
olar region. In the animals exposed from 1
day of age (juvenile animals) the number
of type I epithelial cells doubled, their
mean surface area decreased 38%, and
their mean thickness increased 24%. The
number of alveolar type II epithelial cells
increased and the number of alveolar
macrophages doubled. Young adult ani-
mals exposed to ozone showed similar
changes in the epithelium of the proximal
alveolar reigon. The changes in type I and
type II cells are illustrated in Figures 2 and
3 respectively. Compared to the juvenile
animals, the young adult, ozone exposed
animals showed more interstitial cell
reaction with a doubling of interstitial-
macrophages suggesting a mile inflam-
matory stimulus in the interstitium. The
change in number and size of type I cells
is consistent with an increased cell turn-
over rate due to prolonged ozone inhala-
tion. These results suggest that 0.25 ppm
ozone causes a chronic epithelial injury in
the proximal alveolar region of both juve-
nile and young adult rats.
Effects of Inhalation of 0.12
ppm Oa on the Proximal Alveolar
Region of Young Adult Rats
Morphometric analysis was carried out
on lung tissue of the proximal alveolar
Proximal Alveolar Region
Type I Epithelial Cells
+ 100
I +5°
Cj
6
I
-50
Cell
Number
Mean Cell
Volume
Mean Cell
Basement Membrane
Surface Area
Mean Cell
Thickness
f urn)
*p < .05
§| Juvenile flats
I Young Adult flats
Figure 2. Effects of inhalation of 0.25 ppm ozone on the characteristics of type I epithelial cells
in the proximal alveolar region of juvenile and young adult rats.
Proximal Alveolar Region
Type II Epithelial Cells
+50
+25
I
OS
O
-25 L
Cell
Number
(>#/mm2)
Mean Cell
Volume
(urn3)
Mean Alveolar
Surface Area
Mean Celt
Thickness
(umj
*p<.05
Juvenile flats
Young Adult Rats
Figure 3. Effects of inhalation of 0.25 ppm ozone on the characteristics of type II epithelial
cells in the proximal alveolar region of juvenile and young adult rats
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region isolated from young adult rats that
were exposed to 0.12 ppm, the current air
quality standard for ozone. This investiga-
tion is therefore of fundamental impor-
tance in evaluating the effectiveness of
present air quality control standards. The
results of morphometric analysis indi-
cated that after 6 weeks of ozone inhala-
tion, significant changes occurred in the
type I epithelium of the proximal alveolar
region. Type I cell number increased 36%
(p < 0.05) and both their mean surface
area and mean basement membranesur-
face area decreased 23% (p < 0.05).
While the average type I cell volume
decreased 11%, this change was not sta-
tistically significant. The volume of type I
epithelium increased 13% and the thick-
ness of type II epithelium increased 29%.
No significant changes were found in the
mean cell volume or surface area of type II
cells. The changes observed with the
inhalation of 0.12 ppm ozone were found
to be similar to, but of lesser magnitude
than, those occurring with exposure to
0.25 ppm ozone (see Figure 4).
The higher concentration of ozone pro-
duced both a greater and a broader degree
of injury. In addition to increases in type I
epithelium, the volumes of type II epithe-
lium, alveolar macrophage, and cellular
interstitium were found to increase after
6 weeks of exposure to 0.25 ppm ozone.
This study demonstrates that the expo-
sures to ozone concentrations near cur-
rent air quality standard of 0.12 ppm
ozone can cause measurable structural
changes in the alveolar epithelium. The
degree of tissue injury caused by ozone
can be rigorously quantified and in-
creased with exposure to higher concen-
trations of ozone.
Effects of Inhalation of 0.25
ppm Oa on the Terminal
Bronchioles of Juvenile and
Young Adult Rats
Terminal bronchioles comprise the final
portion of the conducting airway in the
lung. It has been shown to be particularly
sensitive to the effects of ozone exposure.
Morphometric methods were developed
to evaluate the effects of inhalation of
0.25 ppm ozone on the terminal bronchi-
oles of rats. This concentration of ozone is
near the current air quality standard of
0.12 ppm and is below the level at which
changes can be reliably documented
qualitatively. The results of the morpho-
metric analysis indicated that ciliated
cells composed 48% (juvenile) or 53%
(young adult) of the total population of
terminal bronchiolar cells and clara cells
were 33% (juvenile) or 29% (young adult).
The average terminal bronchiolar diam-
eter (210 fjm) and the average thickness
of the bronchiolar epithelium (7.5 fjm)
was not significantly changed by the
exposure to ozone. Exposure to 0.25 ppm
ozone for 6 weeks produced a significant
14-16% decrease (p < 0.05) in the aver-
age luminal surface area of clara cells in
the terminal bronchioles of both juvenile
and young adult rats. No changes oc-
curred in mean clara cell volume or in
average clara cell basement membrane
surface area. The changes found in cil-
iated cells are illustrated in Figure 5.
The effect of ozone on terminal bron-
chiolar cells are subtle, especially when
compared with changes induced in the
epithelium of the proximal alveolar region
after exposure to the same concentra-
tions of ozone. These differences may be
due to the terminal bronchiolar epithelial
cells being less susceptible to this con-
centration of ozone than are the cells of
the alveolar epithelium. Alternatively, it
may be due to the fact that the epithelium
Total Volumes of Alveolar Tissue Compartments
+50
£+25
§
o
I
I
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in the terminal bronchiole is covered by a
mucous/serous coat which may react
with the low concentrations of Oa and
thereby prevent the Oa from reaching the
underlying cells.
Conclusions and
Recommendations
EM morphometry is extremely sensi-
tive and particularly suited as a tool to
evaluate health effects of air pollutants
such as ozone at low concentrations. By
the combined use of microdissection and
EM morphometry, we demonstrated in
this study that subchronic exposure to
0.25 ppm ozone produced significant
structural changes in the cells of the ter-
minal bronchioles and their adjacent
alveolar tissues. In addition, inhalation of
0.12 ppm 03, the current ambient air
quality standard, caused structural chang-
es to occur in the alveolar epithelium
immediately peripheral to the terminal
bronchioles.
A wide array of effects have previously
been reported as a result of subchronic
exposure to ozone. These investigations,
although mostly at higher concentrations
of Oa, were essentially in agreement with
the results of the present study in the
location and nature of the injury invoked
by ozone. The major impact of the current
study is that by using quantitative tech-
niques subchronic exposure to ozone at
extremely low levels can be shown to
cause structural changes in lung cells
which are suggestive of injury. These
changes can be detected at the current air
quality standard of 0.12 ppm Oa. Because
the long-term effects of lung cell injury
caused by low levels of 03 are not known,
chronic exposure to Oa at near ambient
levels should be performed and adverse
health effects carefully defined.
J. D. Crapo, B. E. Barry, andR. R. Mercer are with Duke University Medical Center,
Durham, NC 27710.
Jean Wiester is the EPA Project Officer (see below).
The complete report, entitled "Morphometric Studies of the Effects of Ozone on
Rodent Lungs, "(Order No. PB 85-207 470/AS; Cost: $8.50, subject to change)
will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA2216J
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC27711
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