xvEPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-81-031 May 1981
Project Summary
Effects of Ozone on
Leukocyte DNA
Stephen G. Chaney
This research program was initiated
with the overall objective of deter-
mining whether exposure to ozone
could damage the DNA of peripheral
blood cells.
An animal model system was de-
signed in which glycogen was used to
stimulate the production of peritoneal
exudate cells (PECs) in mice. These
PECs were labeled by repeated i.v.
injection of 3H-thymidine. The labeled
PECs circulated briefly through the
peripheral blood and eventually ac-
cumulated in the peritoneal cavity.
The experimental animals were either
exposed to ozone or ambient atmo-
sphere (sham treated) during the
period of time when the PECs were
circulating through the peripheral
blood. These PECs were then harvest-
ed and their DNA analyzed for single
strand breaks on alkaline sucrose
gradients.
At very high levels of ozone exposure
(5 ppm 9800 A
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Another unresolved question is the
manner in which ozone caused systemic
damage. Ozone itself is very reactive
and might be expected to react primarily
with the lung tissue without penetrating
any further. It is widely assumed, there-
fore, that ozone does not cause systemic
damage directly, but via production of
peroxides andozonides which migrate
to the target cell and generate free
radicals. For the most part, this mecha-
nism is speculative, although it has
been shown that ozonides and peroxides
are produced in response to ozone
exposure and that they can cause some
of the observed effects in cell culture.
One method of assessing ozone re-
lated damage to the DNA is to measure
the extent of DNA repair synthesis.
Cultured lymphocytes have previously
been shown to carry out repair of
damage to their DNA caused by UV
radiation, ionizing radiation, or alkylating
agents. This repair DNA synthesis is
quite specific and easy to measure. In
addition, the magnitude of the repair
synthesis is proportional to the extent of
the damage. Lymphocytes, in fact would
appear to provide an ideal system for
studying DNA repair, since normal
replicative DNA synthesis is quite low in
unstimulated lymphocytes.
Since most examples of systemic
ozone damage do seem to mimic the
effects of ionizing radiation and since
ozone apparently does damage lympho-
cyte DNA, one would predict that ex-
posure to ozone should set off a round of
repair synthesis in the circulating blood
lymphocytes of the exposed animal. If
this prediction held true, it would provide
a valuable new probe for measuring the
extent and mechanism of ozone damage.
In the first place, the magnitude of the
repair synthesis should be proportional
to the extent of damage. Secondly, by
examining the repair process closely,
one should be able to gather some
information as to the mechanism by
which ozone damaged the DNA. In this
project, the extent of DNA repair syn-
thesis in cultured lymphocytes was
measured following exposure of rabbits,
guinea pigs, and hamsters to ozone.
Of course, since ozone damage should
be radiomimetic, one would expect the
damaged DNA to contain an increased
number of single strand breaks. Thus, it
should also be possible to measure the
effects of ozone on the DNA directly by
looking for single strand breaks in the
DNA. In this project the number of
single strand breaks in the DNA of
peritoneal exudate cells was measured
by alkaline elution profile or by alkaline
sucrose gradient following exposure of
mice to ozone.
Conclusions
This assay technique does not appear
to be sensitive enough to detect DNA
damage by the exposure of the whole
animal to ozone. Clearly, exposure of
the mice to 5 ppm (9800 /ug/m3) ozone
for 24 hours causes a significant increase
in single strand breaks in the DNA of the
leukocytes which are formed and which
circulate through the peripheral blood
during the ozone exposure. While this
experimental result establishes the
validity of the experimental approach, it
does not provide any information on the
chances of DNA damage occurring upon
exposure to levels of ozone which are
likely to be found in the environment. In
the first place, exposure to 5 ppm ozone
for a 24-hour period is extremely unlikely.
Secondly, previous experiments have
shown that normal defense mechanisms
begin to be overwhelmed at 5 ppm.
While exposure to 1 ppm ozone for a
24-hour period is unlikely, exposure to
levels approaching 1 ppm for shorter
times does occur. In these experiments
a 24-hour exposure to 1 ppm did appear
to cause a slight decrease in the weight
average molecular weight of the DNA in
each of the four separate experimental
trials. This suggests that some DNA
damage in the form of single strand
breaks was occurring. However, the
difference in size distribution was rarely
statistically significant. Thus, these
experiments suggest that some DNA
damage may occur at lower levels of
ozone exposure, but the technique is not
sensitive enough to accurately deter-
mine whether damage occurs at environ-
mentally significant doses.
Finally, the effect of 48-hours exposure
to 1 ppm (1960/yg/m3) was measured in
a single experiment. In this case, no
decrease in weight average molecular
weight of the DNA was noted following
ozone exposure. The reasons for this
could be twofold: 1) Most of the leukocyte
accumulation in the peritoneal cavity
will have occurred by 24 hours. By 48
hours, a repair synthesis may have
ligated the damaged DNA. 2) By 48
hours, the cell population which accu-
mulates in the peritoneal cavity is more
heterogeneous. The other cell types
which accumulate may be less sensitive
to ozone-caused DNA damage.
Recommendation
This study still leaves open the possi-
bility that ozone may damagethe DNA of
peripheral blood cells. As more sensitive
techniques for assaying DNA damage
become available, this question should
be reinvestigated.
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Stephen G. Chaney is with the School of Medicine, University of North Carolina,
Chape I Hill, NC 27514.
Gary Hatch is the EPA Project Officer (see below).
The complete report, entitled "Effects of Ozone on Leukocyte DNS," (Order No.
PB 81-179 277; 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. 1S61 -757-012/7122
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Environmental Protection
Agency
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