United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-81-051 Sept. 1981
Project Summary
Ozone Exposure and
Pulmonary Metabolic
Effects of Mediators and
Hormones
Ibert C. Wells
The effects of acute ozone exposure
on smooth muscle contracting sub-
stances in lung were studied in male
rats with body weights in the 1 80-250
g range (45-60 days of age). Ozone
concentrations employed were either
0.98 or 1.96 mg/m3andtheexposure
time was four hours. The following
effects were produced: (a) increases in
the amounts of prostaglandin
F2or(PGF2a) and thromboxane A2
(TXA2) in the lumen fluid; (b) an in-
crease in the angiotensin converting
enzyme activity; and (c) a decrease in
the rate of serotonin uptake from the
blood. Histamine and slow reacting
substances of anaphylaxis (SRS-A)
were not released under these experi-
mental conditions, nor was the hista-
mine forming capacity of the lungs
altered. Edema formation was
variable. As indicated in the reports by-
previous investigators, lung succin-
oxidase activity was observed to be
decreased by exposure to 1.96
mg/m3 ozone for four hours, but was
increased after trie-same exposure on
each of four consecutive days.
The exposure of rats to 3.92 mg/m3
ozone for four hours also caused
increased amounts of PGF2a and
TXA2 in their lungs, but this ozone
dose was ineffective in this regard if
15 hours prior to it the animals had
received 0.98 mg/m3 ozone for four
hours. While this low dose of ozone
had a minimal effect on the lung
content of PGF2cr and TXA2 if meas-
ured immediately after its administra-
tion, it apparently did produce an
adaptation to ozone which was
evident 15 hours later.
Indomethacin is a potent inhibitor of
fatty acid cyclo-oxygenase (prosta-
glandin synthetase), the enzyme
required for the biosynthesis of all the
prostaglandins, as well as the
thromboxanes and the prostacyclins.
The administration of this substance
to rats prior to their exposure to 1.96
mg/m3 ozone for four hours prevents
the ozone caused increase of PGF2o
and TXA2 in their lungs. This end result
is the same as that of the apparent
adaptation to ozone produced by ex-
posure of rats to 0.98 mg/m3 ozone
for four hours. It was, therefore, an-
ticipated that "ozone-adapted" and
indomethacin-treated rats would
respond similarly to chronic ozone
exposure and this response would
differ from that of similarly exposed,
untreated controls. This possibility
was studied by measuring the survival
times of treated rats when continu-
ously exposed to 8.82 mg/m3 ozone.
The average survival times of the two
groups of experimental rats were
significantly less than that of the
control group. This result suggests
that the presence of increased
amounts of at least these two medi-
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ators in the lungs may serve to protect
the animals from the deleterious
effects of ozone. Supportive of this
implication is the further observation
that rats treated with Clotrimazole®
(Sobering) survived significantly
longer than control animals under
such conditions. This substance is a
specific inhibitor of TXA2 synthesis,
and it has been proposed that when
TXA2 synthesis is prevented, the
synthesis of other prostaglandins,
such as PGFzff, is enhanced. PGF2a
has the same effect on lung as TXA2
and, though less potent, is much more
stable and therefore longer acting
than TXA2.
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 that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
General Methods
Ozone exposure. Two male rats
having body weights in the 180-250 g
range (45-60 days of age) are placed in a
closed, two compartment, glass
chamber, the volume of which is six
liters (0.21 CF). Air which has been
cooled by passage through a coil of tub-
ing kept at 0°C is pumped through the
chamber at a rate of 12 litersper minute
(C.42 CFM). Prior to its entrance into the
chamber, the air is passed through a
silent, controlled electric discharge to
convert some of the oxygen to ozone It
is introduced into the chamber below
the level of the rats and is exhausted
from the chamberatthe level of the rats'
noses Ozone concentration in the ex-
hausted air is monitored by the spectro-
photometric method of Saltzman [Anal.
Chem. 31, 1914 (1959)] and is ex-
pressed as parts per million (ppm) as
officially specified [Federal Register 35
(No. 84), 8196 (1971)]. Readings are
taken at intervals of 1 5-20 minutes and
the ozone generator adjusted, if re-
quired, to maintain the ozone concen-
tration at the desired level ±010 ppm.
Preparation of lung exudate. The
intact lungs are removed from the de-
capitated rat, blotted, placed onto ice,
and weighed Asmall piece (50-100 mg)
of the tissue is removed for the deter-
mination of the water content by
heating at 110°C to constant weight.
The residual fresh lung tissue is re-
weighed and placed in a volume (ml) of
cold Tyrode's solution equal to 2.5 times
its weight (grams) and cut with scissors
into about 10 pieces of roughly equal
size. After the mixture has remained on
ice for 3-5 minutes, the lung tissue is
removed and frozen. The residual
Tyrode's solution is the "lung exudate"
and is used immediately or is stored at
-20°C until used
Quantitation of prostaglandm F2a
(PGF2a) and thromboxane A2(TXA2)
PGF2a and thromboxane B2 (TXB2), the
mole-for-mole inactivation product of
TXA2, in the lung exudates were quanti-
tated with the use of radioimmuno-
assays by Dr H H Tai at the Texas
College of Osteopathic Medicine,
Denton, Texas [Anal Chem 87, 343-
349 (1978)]. In these procedures,
aliquots of the lung exudates are acidi-
fied and the prostaglandins and TXB2
are extracted into ethylacetate and then
separated chromatographically using
columns of silica gel PGF2a and TXB2,
as well as PGE2, are then assayed by
means of competitive binding measure-
ments using specific rabbit antibodies
for each constituent. In these studies,
no differences in the concentrations of
PGE2 were observed
Angiotensin converting enzyme activ-
ity. Lung tissue samples remaining from
the preparation of lung exudates are
frozen and thawed twice and homogen-
ized in 0 05 M phosphate buffer, pH 7 0,
2 0 ml per gram of tissue The homog-
enates are strained through gauze,
frozen and thawed twice, and centri-
fuged for two hours at 2,000 x g and
4°C. The supernatant solutions are
diluted with 40 volumes of buffer [0 5 M
NaCI, 0.05 M sodium phosphate (pH
8 0), 0.05 percent Bnj 35] to establish
optimal assay conditions. The angio-
tensin converting enzyme activity radio-
assay system obtained from Ventrex
Labs, Inc (217 Read Street, Portland,
ME 04103), is used to determine the
enzyme activities m these diluted
solutions This assay system uses [3H]-
hippuryl-glycyl-glycine for enzyme sub-
strate and the enzyme activity is ex-
pressed as nanomoles of [3H]-hippunc
acid generated per minute per gram of
lung.
Uptake of blood serotonin by lung
Rats which have been fasted overnight
are used. Both control and exposed
animals are anesthetized with
Nembutal (5 mg/100g body weight,
I.P ) The abdominal aorta is cannulated
and the inferior vena cava is exposed A
tourniquet is applied to the neck of the
animal and immediately thereafter a
saline solution of serotonin [010
ml/200 g body weight, containing 10 fjg
(14C) serotonin (3-5 /uCi/yumole)] is in-
jected into the vena cava Blood flowing
from the aortic cannula is then collected
for the next minute m a heparin-
contammg tube. Each blood sample is
hemolyzed by freezing and thawing, is
diluted to 8 0 ml with distilled water and
the total protein is precipitated by the
addition of 2 Oml of 40 percent (w/v)tn-
chloroacetic acid One ml of the
supernate is added to 15 ml of scintilla-
tion cocktail and the tola I dpm are deter-
mined with a liquid scintillation
spectrometer (Beckman LS-1 50) A0.1
ml aliquot of the serotonin solution used
for injection is treated similarly and the
percentage of the injected dose not
taken up by the lung is calculated for
each animal
Experimental Results
Prostaglandin F2a (PGF2a) andthrom-
boxane A^TXAz) Male rats were
exposed to 1 96 mg/m3 ozone for four
hours and the lung exudates were pre-
pared as described above The results of
PGF2a and TXB2 assays are reported in
Table 1
Conclusions and
Recommendations
These studies have revealed three
effects on the lung of acute ozone
exposure Each of these effects involves
a smooth muscle contracting sub-
stance(s)- (a) the increased content of
PGF2a and TXB2 in lung, (b) the in-
creased activity of angiotensm
converting enzyme, and (c) the
decreased ability of the lung to remove
serotonin from the blood The first effect
would seem to be concerned only with
the lung itself, whereas the other two
effects would seem to be concerned
with the interaction of the lungs and
other systems of the organism
The effort m these studies was largely
directed at identifying the effects pro-
duced by acute ozone exposure and
ascertaining the minimum dose of
ozone required to produce each effect
These objectives were essentially
achieved in the case of the increases of
PGF2a and TXB2 in the lung. The mini-
mal doses of ozone necessary to
produce the two remaining observed
effects remain to be determined How-
ever, because of the potential impor-
tance of these two effects, the investi-
gations of them should be completed
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Table 1. Effect of Ozone Exposure on Lung Content of Prostaglandms and
Thromboxane
Rats
Control
Exposed^
Body
Weight
(9)
282 ±
20 (8)b
249 +
3 (20)
Lung
Weight
(9)
0.92 ±
079 (8)
J 23 ±
029 (18)
Ozone
Concen-
tration
(mg/m3)
0
1 96
Lung Exudates
TXB2a
(ng/g)
15.3 ±
211 (6)
250 ±
2 82 (8)
PGE2a
(ng/g)
15.4 ±
2 63 (8)
147 +
1.06 (8)
PGF2cra
(ng/g)
8.4 ±
1.67 (8)
13.0 +
1 17(9)
Difference
NS
NS
''TXBi thromboxane Bz PCE? - prostaglandin Ez PGFid prostaglandin F2a Determined by Dr H H Jai
(Texas College of Osteopathic Medicine, Denton, Texas) using rad/o/mmunoassay
bMean t 5 E Number of animals is in parentheses
C7ime four hours
"Significant difference, P 002, student's "t" test
From a consideration of the overall
project, one is intrigued by the recurring
question of "adaptation to ozone" or
"ozone tolerance," its mechanism or
mechanisms and its significance rela-
tive to health effects on the exposed
animal It would appear that this basic
question must be answered before a
decision can be reached as to which of
the acute effects of ozone a re significant
from the standpoint of injury Further,
the mechanism of the phenomenon
must be understood in order to make
effects of chronic ozone exposure
Ibert C Wells is with Creighton University, Omaha, NE 68178
Donald E. Gardner is the EPA Project Officer (see below)
The complete report, entitled "Ozone Exposure and Pulmonary Metabolic Effects
of Mediators and Hormones," (Order No PB 81 -222 408; Cost. $5 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 2771 1
ft U S GOVERNMENT PRINTING OFFICE 1981 757012 7325
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