United States
Environrrwntal Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600 1-79-026
August 1979
Research and Development
Biochemical
Changes in Humans
Upon Exposure to
Ozone and Exercise
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
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The nine series are:
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This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-79-026
August 1979
BIOCHEMICAL CHANGES IN HUMANS UPON
EXPOSURE TO OZONE AND EXERCISE
Suzanne Chaney
Paulette DeWitt
Wendy Blomquist
Keith Muller
Robert Bruce
George Goldstein
Clinical Pathology Branch
Clinical Studies Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Chapel Hill, North Carolina 27514
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
n
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk of
existing and new man-made environmental hazards is necessary for the estab-
lishment of sound regulatory policy. These regulations serve to enhance the
quality of our environment in order to promote the public health and welfare
and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park, conducts
a coordinated environmental health research program in toxicology, epidemio-
logy, and clinical studies using human volunteer subjects. These studies
address problems in air pollution, non-ionizing radiation, environmental
carcinogenesis and the toxicology of pesticides as well as other chemical
pollutants. The Laboratory participates in the development and revision
of air quality criteria documents on pollutants for which national ambient
air quality standards exist or are proposed, provides the data for registra-
tion of new pesticides or proposed suspension of those already in use,
conducts research on hazardous and toxic materials, and is primarily respon-
sible for providing the health basis for non-ionizing radiation standards.
Direct support to the regulatory function of the Agency is provided in the
form of expert testimony and preparation of affidavits as well as expert
advice to the Administrator to assure the adequacy of health care and
surveillance of persons having suffered imminent and substantial endanger-
ment of their health.
Although animal studies have led to information which may be
pertinent to the question of long-term human health effects, much
more data is needed. With the increase in the atmospheric ozone
levels, it is important to study the effects of ozone exposure
directly to humans. This is one of only a few studies on the
effects of human exposure to ozone that have been reported.
F. G. Hueter, Ph.D.
Director
Health Effects Research Laboratory (MD-51)
iii
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ABSTRACT
A total of 44 human subjects were exposed to 784 ,ug/m3 (0.4 ppm) ozone
and 30 human subjects were exposed to air as controls. A four hour exposure
was given on each of five consecutive days. Half of each group received an
higher level of treadmill exercise than the other half.
Nine biochemical blood parameters were measured pre and post exposure:
red blood cell glutathione reductase, vitamin E, red blood cell cholinesterase,
red blood cell glucose-6-phospnate dehydrogenase, lactic acid dehydrogenase,
complement 03, and IgM. Of these variables only vitamin E, red blood cell
glucose-6-phosphate dehydrogenase, and complement C3 showed significant changes
due to treatment.
This report covers a period from June 13, 1977, to May 19, 1978, and work
was completed as of October 1, 1978.
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INTRODUCTION
Modern manufacturing methods and the enormous increase in the
number of cars and trucks on our streets and highways have lead to
increased levels of atmospheric ozone, a key component of oxidant smog.
Our populations, particularly in urban areas, are exposed to ozone
concentrations up to 0.7 ppm at peak times and average hourly concentra-
tions of 0.2-0.3 ppm. This increase raises many questions with respect
to the effects of ozone on human health. Only a few studies on the
2345
effects of human exposure to ozone have been reported. ' ' ' These
studies involved pulmonary function measurements on subjects from dif-
ferent geographic locations after exposure to ozone. Individuals living
in areas with high ozone levels showed an adaptation in pulmonary function
r
when compared to those subjects from areas of low ozone concentrations.
Species differences make generalizing data from animal studies
somewhat tenuous and, for obvious reasons, the human lung is not directly
available for study. However, animal studies have led to some information
which may be pertinent to the question of long-term human health effects.
Previous studies have shown evidence for lipid peroxidation in red blood
cells exposed to ozone iji vitro and in lungs of mice exposed to 0.7 ppm
fi 7 ft
ozone i_n vivo. ' Stokinger et al. have reported that rabbits exposed
to ozone had detectable amounts of circulating antibodies in their serum
as a result of tyrosine oxidation leading to protein alteration and
Q
denaturation. Goldstein et al. indicated that rats deficient in vitamin
E exhibit a greater susceptibility to lethal levels of ozone. In addition,
rats exposed to sublethal ozone concentrations show an accelerated
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decline in serum vitamin E levels. The importance of cellular sulfhydryl
moieties in maintaining cellular integrity is suggested by the fact that
glutathione (GSH) levels were maintained in ozone-tolerant rats but not
in non-tolerant animals.
The study reported here is a part of a concerted effort to define
biochemical parameters which can be used to measure an ozone pollution
burden on the general population. This study is a clinical evaluation
of controlled human exposure, while most other studies reported have
been based on animal exposure models and on epidemiological models.
METHOD
Subjects
Healthy, Caucasian, non-smoking, male, university students charac-
terized by an average height of 181.4 cm (std. dev. 7.0, range 160.0-194.0
cm), an average weight of 76.0 kg (std. dev. 9.6, range 57.2-109.2 kg)
and an average age of 25.1 years (std. dev. 2.5, range 19.7-30.1 years)
3
were exposed to clean air (controls) or to 784 ug/m (0.4 ppm) ozone.
Each group of subjects was recruited and tested separately and sequentially.
In Group 1, 29 subjects (light exercise, air exposure) were tested from
June 13 through August 26, 1977; in Group 2, 15 subjects (light exercise,
ozone exposure) were tested from October 31 through December 9, 1977; in
Group 3, 15 subjects (heavy exercise, ozone) were tested from February
27 through March 31, 1978; and in Group 4, 15 subjects (heavy exercise,
air exposure) were tested from April 17 through May 19, 1978.
Experimental Design
A total of nine plasma, serum, and red blood cell biochemical
measures were chosen as dependent variables. The analysis techniques
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used incidentally yielded many more. The small number of subjects
required that the number of variables analyzed be kept to a minimum.
Consequently, variables were chosen on three bases. First, variables
with large amounts of missing data were eliminated. Second, a priori
biochemical judgment was used to eliminate inappropriate variables.
Third, evaluation of the control day data, in combination with the
logical relations of certain variables, indicated a small number of
variables which were redundant (and hence eliminated) with some included
in the analysis.
Subjects were exposed in groups of three for five consecutive days
(always Monday through Friday) to four hours of either air or ozone.
Exposures began at approximately 8:00 a.m. On Monday, all subjects
3
received air. Groups 2 and 3 received 784 ug/m (0.4 ppm) ozone on
Tuesday through Friday. Groups 1 and 2 received two 15 minute exercise
sessions (at 1 and 3 hours of exposure) which consisted of walking 6.44
km/hr (4 mph) on a treadmill inclined at 0°, and groups 3 and 4 received
the same exercise regimen except that the treadmill was inclined at 10°.
During the exposures, the temperature was maintained at 20°C with a
relative humidity of 40%, and air flow through the exposure chamber of
493 cubic meters/minute.
The factors in the design include two levels of ozone (0, 784
3
ug/m ), two levels of exercise (0°, 10° treadmill inclination), four
levels of day (Tuesday through Friday) and two levels of pre-post
(before exposure, after exposure). Ozone and exercise are between
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subject factors and days and pre-post are repeated measures. A multi-
variate analysis of variance (MANOVA) approach was used: the eight
repeated measures were taken to be eight dependent variables with a two
factor design. Estimation of day effect and pre-post effect were therefore
contrasts between dependent variables, as were any interactions involving
either or both. This approach completely avoids the restrictive assumption
necessary with a univariate random effects model for repeated measures.
One MANOVA was conducted for each dependent variable. This approach
is justly criticized if the observed p values are reported as "significant"
when compared against noainal values of 0.05 or 0.01. As was done for
this paper, that problea is solved by using the Bonferroni technique to
12
correct the false probability levels. Here that means an observed p
of 0.01/9 = 0.0011 was required for the effect to be declared "significant."
This approach insures that the test of a particular effect will be done
at the 0.01 level for the set of nine variables taken together. Hence,
appropriate oultivariate p levels are used rather than the biased univariate
p levels.
A serum and plasma sample were analyzed daily from each participant
before and after each 4 hour exposure during the week. Samples were
also taken the following Monday (a one week follow-up), at two weeks,
and again at four weeks. For the purposes of significance testing, only
data for actual exposure days were analyzed, which included Tuesday
through Friday. Monday data is reported for control information. The
follow-up data were collected to assure that any effects that occurred
returned to baseline to assure subject safety.
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Blood Analysis
The following enzymes were analyzed on the CentiifiChem 400 Auto-
analyzer: (1) serum glutamic-pyruvic acid transaminase (SGPT) by a
13
modified Wroblewski and LaDue technique, (2) alkaline phosphatase by a
14
modified Bessey-Lowry-Broch technique, (3; lactic acid dehydrogenase
(LDH) was assayed in the direction of conversion of lactate to pyruvate,
(4) red blood cell cholinesterase by a modification of the Ell man method,
and (5) red blood cell glucose-6-phosphate dehydrogenase (G6PDH) by the
procedure of Catalano.
Red blood cell and serum glutathione reductase were assayed by the
18
method of Nichoalds modified such that the enzyme was preincubated at
0° for 30 minutes in the presence of 60 uM flavin adenine dinucleotide
(FAD) and the reaction was initiated by the simultaneous addition of 2
mM oxidized glutathione (GSSG) and 0.24 mM reduced nicotinamide adenine
dinucleotide (NADPH). To measure serum vitamin E, the following technique
was used. In a 15 ml, glass-stopped centrifuge tube, 2 ml of ethanol-L-
ascorbic acid reagent (200 mg L-ascorbic acid in 10 ml 95% ethanol) was
mixed with 0.5 ml of serum and immediately vortexed for 15 seconds.
After standing at room temperature at least 2 minutes, the tubes were
then placed in an 80°C water bath for 3 minutes. The samples were
removed from the water bath and 2 ml of water were added to each and
vortexed. After being allowed to cool, 5 ml of hexane was added to each
and each sample was vortexed 75 seconds. The phases were separated by
centrifuging in a clinical centrifuge at 1200 rpm for 10 minutes at room
temperature. A 4 ml aliquot of the top phase was transferred to a
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conical tube and evaporated to dryness in a stream of nitrogen. The
evaporated sample was reconstituted in 0.1 ml benzene and 0.025 ml was
spotted in duplicate on Bodman LQ6D silica gel plates. The plate was
developed for 20 minutes in hexane:acetone:reagent grade anhydrous ethyl
ether (80:20:10). After the plate was air dried for 5 minutes, it was
sprayed with a solution of acidic cupric acetate (6 gm cupric acetate/
100 ml 15% H3P04). The spots were made visible by charring in a 160°C
oven for 10 minutes. The plate was then scanned at 405 nqj with a Schoffel
densitometer.
19
Serum proteins were determined by nephelometry.
Materials
A CentriflChea Model 400 centrifugal analyzer and CentrifiChem
autapipetter (Union Carbide Corporation, Clinical Diagnostics, Rye, New
York 10580) were used for assaying acetylcholinesterase, G6PDH, LDH,
S6PT, and alkaline phosphatase. Manual assays for glutathione reductase
were run on a Cary 118 spectropnotoneter. LDH (L-»P), SGPT and alkaline
phosphatase reagent kits were purchased from Union Carbide Corporation,
Clinical Diagnostics, Rye, New York 10580. Cholinesterase reagent kits
were purchased from Bio-Dynamics/BMC, Norcross, Georgia 30093.
D,L-ortocopherol, oxidized glutathione (tree acid, grade III), FAD
(disodium salt, grade III), nicotinamide adenine dinucleotide phosphate
reduced form (tetra sodium salt Type 1, NADPH), nicotinamide adenine
dinucleotide phosphate monosodium salt (NADP ) Sigma grade, 6-phospho-
gluconic dehydrogenase Type V from yeast, 50 U/ml, and D-glucose-6-
phosphate monosodium salt were purchased from Sigma Chemical Company,
St. Louis, Missouri 63178.
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RESULTS
The results are organized into two major sections. The first
addresses characteristics of all of the variables simultaneously, while
the second provides a detailed description of the analysis of each
separate variable. Our approach has been to provide sufficient detail
so that the interested reader has enough information available to examine
any particular variable in detail. We have included comments on only
those results of particular interest.
Overall Statistical Description
Table 1 provides summary statistics for each dependent variable,
for each day at both pre and post sampling times. Means, standard
deviations, minima, maxima, and skewnesses are included. Note that not
all lines are based on exactly the same number of observations, and that
the data for all four exposure groups have been combined. Of the approx-
imately 75 subjects who participated in the experiment, complete data
sets were obtained on 50 to 60 subjects for each test. Some subjects
were excluded due to: (1) clinical reasons not related to the experiment,
for example, one subject was excluded when it was discovered that he had
a G6PDH deficiency, or (2) the limited quantity of blood available for
each test prevented repeating the test in the case of equipment failure.
These measures are included to indicate the range of the data observed.
All values observed appear to fall within a "normal" range. Consideration
of Table 1 and associated frequency histograms did not indicate any
serious problems in using analysis of variance on these data.
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Table 2 presents means for each group for pre and post measurements
taken on the control day proceeding the exposure days. A number of com-
ments need to be made. First, these measurements were taken 4 hours
apart, with two 15-minute exercise periods at the beginning of the
second and fourth hours. Second, each group is in the "normal range" of
values. Third, it is instructive to note that the variability pre to
post seems to be much less than the variability among groups. In particular,
Group 3 haptoglobin results seem far higher than the other groups.
These variations may reflect the way the data were collected, i.e., that
all subjects in Group 1 were tested in a contiguous time period, then
all subjects in Group 2, then all subjects in Group 3, and finally, all
subjects in Group 4.
Table 3 presents the intercorrelations of all nine dependent vari-
ables for the control day plus height, weight, and age. Height, weight,
and age were included in order to evaluate their usefulness as covariates.
Since the maximum correlation of any of the three with any dependent
variable is 0.29, and the next largest correlation is 0.17, it is ob-
vious that height, weight, and age are useless as covariates. Since no
dependent variables overlap to any great extent with any other dependent
variable, it is possible to examine each dependent variable separately,
particularly since a few variables which showed excessive overlap were
eliminated from the analysis.
Analysis of Variance Summary
Tables 4.1-4.9 summarize the multivariate analyses of variance
(NANOVA) for the nine dependent variables. In these source tables most
8
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of the tests are multivariate tests. For these tests the likelihood
ratio criterion was used and tested with Rao's F approximation. Where
appropriate, univariate tests were computed, and therefore likelihood
ratios are not presented. The actual alr^ha level for each family of
hypotheses was 0.01. Further, note that some lower order effects which
are "significant" by this standard are not marked as significant. They
should not be because they are subsumed under higher order effects which
were significant. These results indicate at least some effects of
ozone, exercise, and time. However, not all variables show these effects.
Table 4.0 summarizes the significant effects. Results for each variable
will be discussed in detail later.
Tables 5.1-5.9 present means for each dependent variable for each
group based on the MANOVA. The single variable results to be discussed
will lead to simpler tables presented later. One simplification of
these tables is presented in Figures 1.1-1.9. Since the analysis indicates
little, if any, pre to post difference, the averages of each day's pre
and post means were plotted. These will be referred to specifically
when each variable is discussed individually.
Red Blood Cell Glutathione Reductase
Table 4.1 summarizes the MANOVA for RBC glutathione reductase.
Table 5.1 provides, for each treatment condition, the mean response
estimated by the analysis. These results show no indication of any
effect of ozone, exercise, or pre-post factor. Figure 1.1 plots the
mean response of RBC glutathione reductase for each of the groups for
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each day. This figure supports the statistical analysis by also suggesting
that natural variability is at least as large as any of the observed
differences.
Vitamin E
Table 4.2 provides a source table for the MANOVA conducted on serum
vitamin E. Only the ozone by exercise interaction produced a significant
effect. This result indicates that the effect of ozone on serum vitamin
E depends upon the level of exercise. Thus, a greater burden of ozone
per unit time may result with increased exercise. Consideration of
Table 5.2 and Figure 1.2 do not directly address this question.
Note that the effect is averaged across days and pre and post
reasureaents- The four means tested by this hypothesis are: 5.8 ug/ml
for Group 1
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the mean response for each group, for each point in time based on the
analysis of variance. Figure 1.3 presents a plot of the means averaged
across pre-post for each day for each group. Taken together these
results appear to indicate no effect on RBC cholinesterase.
RBC G6PDH
Table 2 indicates that Group 3 subjects (heavy exercise, ozone)
began the experiment with a G6PDH level higher than the other groups.
This observation influences our interpretation of the remaining analyses.
Consider Table 4.4, which summarizes the MANOVA for G6PDH, and Table 5.4
which contains means for each treatment condition. Two interactions are
significant: exercise by day by pre-post and ozone by exercise. Figure
3 plots the means for exercise by day by pre-post. Only one clear
pattern emerges, that of heavy exercise levels showing more variability
than light exercise levels. Results of the interaction trend tests bear
this out. The one degree of freedom contrast for a linear trend yielded
a p value of 0.54, the contrast for a quadradic trend yield a p value of
0.0174 and the contrast for a cubic trend yield a p value of 0.0008. On
this basis the cubic trend was decided to be the significant one. Since
only four days are present, this only indicates that the means do differ
reliably but does not indicate the nature of the relationship other than
indicating that it is not a simple relationship.
Figure 4 plots the ozone by exercise means, averaged across days.
The significance of this effect appears to be due to Group 3 (ozone,
heavy exercise) being different from the other groups. However, it is
important to note again that Group 3 began the experiment near the same
11
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level as illustrated in Figure 4. Furthermore, the other three groups
also began the experiment near the same levels as shown in Figure 4.
Refer to Table 2 to compare this plot against control day measures.
Lactic Acid Dehydrogenase (LDH)
A normal range of 0-110 IU/1 for LDH has been reported for the
20
method used. In contrast, our data show a range of 32-232 IU/1. Note
that these two extreme values are not unique in our data. A possible
factor may be the uncontrolled exercise level inherent in a subject
getting from home to the facilities preceding the initial blood draw for
the day.
Table 4>.5 summarizes the MANOVA for LDH. The analysis indicates no
effects of any of the treatments either individually or in combination.
Means for each combination for LDH are presented in Table 5.5. Figure
1.5 plots mean LDH activity for each group for each day. No consistent
pattern is present and the differences are small in light of the standard
deviation of 20 IU/1.
otj-Antitrypsin
Table 4.6 summarizes the MANOVA for a-,-antitrypsin. No effects
were declared significant according to the decision rule discussed
earlier, although some observers might argue that the ozone by exercise
by pre-post or the ozone by days factors should be significant. In a
situation like this, it is important to consider the means for each
treatment condition reported in Table 5.6. They do not appear to indicate
any particular pattern of results.
12
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Figure 1.6 plots mean ot..-antitrypsin for each day for each group.
In fact, it does appear to show a consistent pattern of an ozone-by-days
interaction. Although these results appear more regular, the amount of
variability present in a,-antitrypsin cauces the differences to be
declared not significant.
Haptoglobin
Table 2 lists mean response of haptoglobin for each group on the
control day. These results indicate two things. First, little difference
is seen pre to post exposure. Second, Group 3 began the experiment with
haptoglobin levels approximately 3 standard deviations higher than the
other three groups, which are approximately the same.
Table 4.7 summarizes the MANOVA for haptoglobin. No results were
declared significant. Given the apparent initial deviation of Group 3,
it is useful to also consider Table 5.7 which lists the means for each
group for each measurement point, and Figure 1.7 which plots the mean
for each group for each day's haptoglobin data. Figure 1.7 and Table 2
provide a consistent picture. Group 3 began at a much higher level and
remained there. Consequently, even if any result had been declared
"significant," its interpretation would have had to have been strongly
modified in light of the initial differences.
Complement C^
Table 4.8, which summarizes the MANOVA for complement C~, indicates
a significant interaction of ozone by exercise by days. No other effects
were declared significant. Table 5.8 provides mean response for each
treatment condition. Figure 1.8 plots the means for the significant
13
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ozone by exercise by day interaction. Tests for trends in this inter-
action clearly indicate a linear trend, p < 0.00004. In particular,
Group 3 means increase linearly across days. However, the importance of
this result is diminished in light of the change seen in Figure 1.8 in
the other three groups.
IgM
Table 2 gives mean response of IgM for each group on the control
day. Although the groups appear to vary, the differences are not large
in comparison to the standard deviation of IgM. The standard deviation
for IgM is much larger than complement C-, haptog1 obin, and a,-antitrypsin,
which are all measured on the same scale and by the same technique.
Table 4.9 indicates no significant results from the MANOVA for IgM.
Table 5.9 provides mean levels of IgM for each treatment combination,
while Figure 1.9 plots mean response across days for each group. Neither
the table nor the figure indicate any pattern to the results.
Summary of Results
Although ozone is a highly reactive oxidizing agent, it showed no
effect on several biochemical parameters, chosen for their sensitivity
to an oxidative insult, as well as a pulmonary insult. These parameters
included RBC glutathione reductase, RBC cholinesterase, LDH, a,-anti-
trypsin, haptoglobin, and IgM. Three variables, however, serum vitamin
E, RBC G6PDH, and complement C,, appear to have shown some effect.
For serum vitamin E, the ozone by exercise interaction was signif-
icant. This was attributed to Group 2 (light exercise, ozone) being
slightly higher than the other three groups in its response.
14
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For RBC G6PDH, the ozone by exercise effect and the ozone by day by
pre-post were significant. The ozone by exercise effect appears to be
due to Group 3 being higher than the other three groups. It was noted
that Group 3 also began the experiment at a higher level. The ozone by
day by pre-post effect indicates that heavy exercise causes G6PDH levels
to vary more than light exercise does.
Complement C3 showed a significant ozone by exercise by day effect.
The level for Group 3 was seen to increase linearly across time; however,
Group 1 levels also increased across time. As with G6PDH, no clear
pattern emerges.
DISCUSSION
In light of the highly oxidative effect of ozone, it would seem
reasonable to expect a response in G6PDH, serum vitamin E and complement
C- levels in volunteers exposed to ozone. The results, however, are not
borne out by this simple relation, and in fact may be due to the variability
in the data. This variability may result from the data collection
scheme being sequential rather than counterbalanced. Results from a
subsequent unpublished blood study, unrelated to the ozone exposure,
support this hypothesis. The protocol of that study provided counter-
balancing for day of the week and week of the year. Consequently,
subjects were uniformly distributed across time and group. Analysis of
data, including some of the same variables, indicate less variability
between subjects and smaller initial differences between groups.
The positive responses that were observed are descriptive of the
body's response to an oxidative challenge. G6PDH is an important enzyme
15
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in the metabolism of glucose via the hexose nonophosphate shunt. The
hexose nonophosphate shunt is responsible for maintaining levels of the
reduced coenzyrae NADPH. The NAOPH is necessary to maintain sufficient
levels of reduced glutathione which acts to protect cellular components
fron oxidation. In the presence of an oxidant such as ozone, the body
may respond by increasing G6PDH activity or by increasing the levels of
G6PDH. The mobilization of vitamin E, a known antioxidant, may also
indicate a metabolic response to increased oxidant exposure, as demonstrated
by the elevated levels of serum vitamin E.
The elevated level of complement C^ is indicative of a mild inflam-
matory response. In turn, elevation of C3 will result in elevated C-
which is one of the anaphylatoxins of the complement system. This
Increase may be one of the reasons for the increased coughing and short-
ness of breath experienced by the subjects.
Taken together, these results Indicate biochemical responses to
ozone and exercise. The data at hand do not allow elucidation of the
mechanism of such an effect. This study indicates directions which such
research might profitably follow.
16
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18
-------
TABLE 1
SUMMARY STATISTICS FOR ALL FOUR GROUPS COMBINED FOR ALL NINE VARIABLES
FOR THE CONTROL DAY
Variable
GSH Reductase
Serum Vitamin E
RBC Cholinesterase
G6PDH
LDH
Oj-Antitrypsin
Haptoglobin
Complement C3
IgM
Units
umoles/gm Hgb/min
ug/ml serum
mU/109 RBC
U/gm Hgb
IU/1
mg/dl
mg/dl
mg/dl
mg/dl
Time
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Mean
7.59
7.59
6.11
6.27
1069
1087
1.92
1.97
87.6
90.3
175
178
64
65
90
92
112
119
Std. Dev.
1.75
1.95
1.28
1.25
221
234
0.36
0.42
20.7
18.4
29
28
31
34
17
15
47
53
Min.
4.29
4.06
2.42
3.20
373
393
1.05
1.06
51.0
48.5
90
97
13
13
51
61
22
31
Max.
11.45
13.20
9.89
9.99
1595
1814
2.85
3.64
155.0
148.
238
242
154
168
129
134
213
255
Skewness
0.13
0.46
0.1
0.0
-0.2
0.1
-0.4
0.8
0.9
0.6
-0.7
-0.5
0.6
0.8
0.3
0.8
0.3
0.7
N
54
V5
61
50
54
57
57
57
57
-------
TABLE 2
GROUP MEANS ON CONTROL DAY
Pre Exposure
Group 1 Group 2 Group 3 Group 4
Air 03 03 Air
Variable Light Exercise Light Exercise Heavy Exercise Heavy Exercise
BBC Glut. Reduct.
inoles/ag Hgb/oin
Sena Vitamin E ug/«l
BBC Cholinesterase
*UA09 RBC
6SHJH U/ga Hgb
UDH IU/1
aj-ftatitrypsin ag/rfl
ttaptoglobin og/dl
CooplaBent £3 ag/dl
Ig* ag/dl
7.69
5.45
1086
1.72
92.0
170
56
88
105
7.30
6.34
1061
1.92
85.6
177
58
92
117
7.59
6.29
908
2.17
77.6
180
89
88
100
7.81
6.36
1202
1.93
92.0
175
57
90
125
Post Exposure
Group 1 Group 2 Group 3 Group 4
Air 03 03 Air
Variable Light Exercise Light Exercise Heavy Exercise Heavy Exercise
fiBC Glut. Reduct.
IMoles/ng Hgb/ain
Serum Vitamin E ug/al
RBC Cholinesterase
aU/10» RBC
G6POH U/ga Hgb
LDH IU/1
ai-Antitrypsin ag/dl
Haptoglobin ag/dl
Coapleaent €3 ag/dl
Igttag/dl
8.08
5.62
1075
1.75
94.2
173
54
91
116
7.49
6.63
1077
1.95
87.0
178
60
97
127
7.50
6.15
975
2.31
83.5
182
86
88
105
7.22
6.60
1214
1.96
92.9
179
69
89
126
20
-------
TABLE 3
INTERCORRELATIONS OF CONTROL DAY MEASURES
RBC
Glutathione
Reductase
RBC
Glutathione 1.0
Serum Vit. E .04
RBC Cholin-
esterase -.15
G6PHD -.04
LDH
tti-Antitrypsin
Haptoglobin
Complement C3
IgM
Ht
Wt
Age
.11
.01
.13
.21
.00
-.01
.01
-.01
Serum
Vit. E
1.0
.13
.12
.16
.07
.05
.15
.20
.11
-.02
.03
RBC
Cholin-
esterase
1.0
-.21
-.07
-.03
-.10
.00
.17
-.01
-.09
.03
G6PDH
1.0
.06
.30
.00
.00
-.18
.12
.17
-.18
LDH
1.0
.12
-.05
.30
.13
.02
.08
-.07
<*i
anti-
trypsin
1.0
.07
-.12
-.29
.29
.11
.05
Hapto-
globin
1.0
.41
-.01
.12
.01
.13
Comple-
ment C3
1.0
.15
.10
-.19
-.13
IgM
1.0
-.17
- 'J7
Ht Wt
r
1.0
.43 1.0
- 14 -.24 -.09
Age
1.0
-------
ro
ro
Ozone (0)
Exercise (E)
Days (D)
Pre-post (P)
0 x E
TABLE 4.0
SUMMARY OF SIGNIFICANT EFFECTS FOR ALL DEPENDENT VARIABLES
Variable
RBC RBC a i
GlutatMone Serum Choi In- antl- Hapto- Comple-
Reductase Vlt. E esterase G6PDH LDH trypsln globln ment C3 IgM
0
E
E
D
0
0
0
E
0
X
X
X
X
X
X
X
X
X
P
D
P
P
E
E
D
D
'E
X
X
X
X
X
D
P
P
P
D x
P
-------
TABLE 4.1
MANOVA FOR RBC GLUTATHIONE REDUCTASE
Source
Ozone (0)
Exercise (E)
Days (D)
Pre-Post (P)
0 x E
0 x D
0 x P
E x D
E x P
D x P
0 x E x D
0 x E x P
0 x D x P
E x D x P
0 x E x D x P
Likelihood
Ratio
-
-
.991
-
-
.874
-
.918
-
.892
.965
-
.904
.945
.939
F
.01
0.0
.15
.52
2.92
2.30
.27
1.42
.68
1.94
.59
2.94
1.69
.94
1.03
Num. d.f.
1
1
3
1
1
3
1
3
1
3
3
1
3
3
3
Den. d.f.
50
50
48
50
50
48
50
48
50
48
48
50
48
48
48
P
value
.9322
.9772
.9324
.4758
.0940
.0889
.6044
.2483
.4142
.1352
.6277
.0925
.1814
.4289
.3870
23
-------
TABLE 4.2
MANOVA FOR VITAMIN E
Source
Ozone (0)
Exercise (E)
Days (D)
Pre-Post (P)
0 x E
0 x D
0 x P
E x D
E x P
D x P
0 x E x D
0 x E x P
0 x D x P
E x D x P
0 x E x D x P
Likelihood
Ratio
-
-
.993
-
-
.812
-
.887
-
.945
.947
-
.807
.935
.859
F
3.77
4.31
.12
1.23
12.46
3.79
1.87
2.08
1.39
.95
.44
.04
3.90
1.13
2.69
Num. d.f.
1
1
3
1
1
3
1
3
1
3
3
1
3
3
3
Den. d.f.
51
51
49
51
51
49
51
49
51
49
49
51
49
49
49
P
value
.0578
.0429
.9494
.2733
. 0009*
.0160
.1773
.1149
.2434
.4223
.7249
.8401
.0141
.3446
.0567
^Significant at .01 level (Bonferroni).
24
-------
TABLE 4.3
MANOVA FOR RBC CHOLINESTERASE
Source
Ozone (0)
Exercise (E)
Days (D)
Pre-Post (P)
0 x E
0x0
0 x P
E x 0
E x P
D x P
0 x E x 0
0 x E x P
0 x 0 x P
E x D x P
0 x E x D x P
Li kel 1 hood
Ratio
-
-
.958
-
-
.967
-
.937
-
.882
.923
-
.904
.882
.899
F
.68
6.77
.81
.56
3.82
.63
1.06
1.23
.24
2.45
1.53
1.47
1.95
2.44
2.05
Num. d.f.
1
1
3
1
1
3
1
3
1
3
3
1
3
3
3
Den. d.f.
57
57
55
57
57
55
57
55
57
55
55
57
55
55
55
P
value
.4121
.0118
.4959
.4561
.0554
.6022
.3068
.3074
.6262
.0724
.2152
.2304
.1308
.0727
.1157
25
-------
TABLE 4.4
MANOVA FOR RBC G6PDH
Source
Ozone (0)
Exercise (E)
Days (D)
Pre-Post (P)
0 x E
0 x D
0 x P
E x D
E x P
D x P
0 x E x D
0 x E x P
0 x D x P
E x D x P
0 x E x D x P
Likelihood
Ratio
-
.873
-
-
.955
-
.938
-
.482
.979
-
.797
.628
.931
F
3.25
2.74
2.13
1.61
26.66
0.70
1.13
.96
.97
15.77
.31
.53
3.73
8.67
1.09
Num. d.f.
1
1
3
1
1
3
1
3
1
3
3
1
3
3
3
Den. d.f.
46
46
44
46
46
44
46
44
46
44
44
46
44
44
44
P
value
.0779
.1046
.1098
.2112
4.0 x 10~6
.5589
.2926
.4189
.3287
4.1818 x 10~7*
.8146
.4719
.0178
. 0001*
.3614
*Significant at the .01 level (Bonferroni corrected).
26
-------
TABLE 4.5
MANOVA FOR LDH
Source
Ozone (0)
Exercise (E)
Days (D)
Pre-Post (P)
0 x E
0x0
0 x P
E x 0
E x P
D x P
0 x E x 0
0 x E x P
0 x D x P
E x 0 x P
0 x E x 0 x P
Likelihood
Ratio
-
-
.830
-
-
.927
-
.880
-
.942
.938
-
.863
.958
.913
F
.03
.76
3.27
.39
3.14
1.25
2.92
2.19
.01
.99
1.05
1.03
2.54
0.70
1.53
Num. d.f.
1
1
3
1
1
3
1
3
1
3
3
1
3
3
3
Den. d.f.
50
50
48
50
50
48
50
48
50
48
48
50
48
48
48
P
value
.8626
.3882
.0292
.5370
.0825
.3009
.0938
.1019
.9329
.4043
.3770
.3155
.0675
.5538
.2200
27
-------
TABLE 4.6
MANOVA FOR c^-ANTITRYPSIN
Source
Ozone (0)
Exercise (E)
Days (D)
Pre-Post (P)
0 x E
0 x D
0 x P
E x D
E x P
D x P
0 x E x D
0 x E x P
0 x D x P
E x D x P
0 x E x D x P
Likelihood
Ratio
-
-
.798
-
-
.801
-
.968
-
.885
.925
-
.921
.930
.892
F
2.34
1.03
4.30
6.67
.11
4.21
2.75
.57
1.41
2.19
1.38
7.16
1.46
1.27
2.05
Num. d.f.
1
1
3
1
1
3
1
3
1
3
3
1
3
3
3
Den. d.f.
53
53
51
53
53
51
53
51
53
51
51
53
51
51
51
P
value
.1317
.3149
.0089
.0126
.7439
.0098
.1031
.6433
.2410
.0992
.2579
.0099
.2364
.2941
.1172
28
-------
TABLE 4.7
MANOVA FOR HAPTOGLOBIN
Source
Ozone (0)
Exercise (E)
Days (D)
Pre-Post (P)
0 x E
0x0
0 x P
E x D
E x P
D x P
0 x E x 0
0 x E x P
0 x D x P
E x D x P
0 x E x D x P
Likelihood
Ratio
-
-
.935
-
-
.839
-
.868
-
.866
.972
-
.965
.964
.980
P
11.29
5.17
1.13
.24
1.85
3.25
.01
2.57
.44
2.63
.49
.14
.61
.62
.34
Num. d.f.
1
1
3
1
1
3
1
3
1
3
3
1
3
3
3
Den. d.f.
53
53
51
53
53
51
53
51
53
51
51
53
51
51
51
P
value
.0014
.0270
.3465
.6247
.1789
.0287
.9270
.0630
.5109
.0587
.6980
.7051
.6143
.6077
.7958
29
-------
TABLE 4.8
MANOVA FOR COMPLEMENT C,
Source
Ozone (0)
Exercise (E)
Days (D)
Pre-Post (P)
0 x E
0 x D
0 x P
E x D
E x P
D x P
0 x E x D
0 x E x P
0 x D x P
E x D x P
0 x E x D x P
Likelihood
Ratio
-
-
.964
-
-
.953
-
.916
-
.974
.725
-
.908
.915
.863
F
1.85
6.00
.63
9.68
.02
.84
1.48
1.56
.61
.45
6.43
1.83
1.72
1.58
2.69
Num. d.f.
1
1
3
1
1
3
1
3
1
3
3
1
3
3
3
Den. d.f.
53
53
51
53
53
51
53
51
53
51
51
53
51
51
51
P
value
.1796
.0176
.6038
.0030
.8866
.4792
.2284
.2096
.4368
.7207
. 0001*
.1812
.1736
.2035
.0547
*Significant at the .01 level (Bonferroni corrected).
30
-------
TABLE 4.9
MANOVA FOR IgM
Source
Ozone (0)
Exercise (E)
Days (D)
Pre-Post (P)
0 x E
0 x D
0 x P
E x D
E x P
D x P
0 x E x D
0 x E x P
0 x D x P
E x D x P
0 x E x D x P
Likelihood
Ratio
-
-
.686
-
-
.875
-
.934
-
.993
.994
-
.968
.878
.963
F
.04
.30
7.27
.11
.99
2.42
.14
1.20
1.28
.13
.11
0.0
.56
2.36
.65
Num. d.f.
1
1
3
1
1
3
1
3
1
3
3
1
3
3
3
Den. d.f.
53
53
51
53
53
51
53
51
53
51
51
53
51
51
51
P
value
.8506
.5880
.0003
.7429
.3249
.0752
.7112
.3173
.2638
.9391
.9506
.9832
.6492
.0811
.5913
31
-------
TABLE 5.1
MEAN RESPONSE FROM MANOVA FOR
RBC GLUTATHIONE REDUCTASE |jmoles/mg Hgb/min
Group 1 • Group 2 Group 3 Group 4
Air 03 03 Air
Day
2
3
4
5
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Light Exercise
7.82
7.37
7.93
8.38
7.78
7.85
8.40
8.06
Light Exercise
7.31
7.95
7.17
7.73
7.64
7.53
7.31
7.65
Heavy Exercise
8.68
8.35
7.88
8.28
8.11
7.19
7.32
7.69
Heavy Exercise
7.38
7.65
7.42
7.31
7.78
7.77
7.46
7.73
TABLE 5.2
MEAN RESPONSE MANOVA
FOR VITAMIN E ug/ml serum
Group 1 Group 2 Group 3 Group 4
Air 03 03 Air
Day
2
3
4
5
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Light Exercise
6.08
5.56
5.82
6.02
5.52
6.55
5.62
5.27
Light Exercise
7.45
8.19
6.78
6.48
6.41
6.60
6.89
7.35
Heavy Exercise
5.60
5.59
5.72
5.80
5.77
5.68
5.78
6.27
Heavy Exercise
5.68
5.62
6.85
6.53
6.10
6.08
6.13
6.02
32
-------
TABLE 5.3
MEAN RESPONSE FROM MANOVA FOR
RBC CHOLINESTERASE mU/109 RBC
Day
2
3
4
5
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Group 1
Air
Light Exercise
1077
1106
1060
1034
1058
1048
1105
1107
Group 2
03
Light Exercise
1039
1078
1118
1200
1147
1076
1186
1105
Group 3
03
Heavy Exercise
1049
1321
1125
1021
1180
1065
1188
1203
Group 4
Air
Heavy Exercise
1288
1272
1320
1200
1252
1235
1268
1185
TABLE 5.4
MEAN RESPONSE FROM MANOVA FOR
RBC G6PDH U/gm Hgb
Group 1 Group 2 Group 3 Group 4
Air 03 03 Air
Day
2
3
4
5
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Light Exercise
2.01
2.04
1.94
1.98
1.99
2.03
2.04 '
2.07
Light Exercise
1.96
1.84
1.86
1.86
1.78
1.89
1.94
1.76
Heavy Exercise
2.43
2.04
1.95
2.29
2.20
2.16
2.23
2.07
Heavy Exercise
1.94
1.74
1.75
1.89
1.94
1.89
1.87
1.81
33
-------
TABLE 5.5
MEAN RESPONSE FROM MANOVA FOR LDH IU/1
Group 1 Group 2 Group 3 Group 4
Air 03 03 Air
Day
2
3
4
5
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Light Exercise
96.3
89.7
87.8
89.2
89.6
89.7
91.6
95.3
Light Exercise
86.6
84.6
85.2
91.4
80.1
75.4
72.6
84.3
Heavy Exercise
81.5
101.8
78.4
89.0
92.2
90.6
78.5
80.2
Heavy Exercise
98.9
81.4
76.1
75.7
76.0
76.9
75.8
74.6
TABLE 5.6
MEAN RESPONSE FROM MANOVA
FOR a,-ANTITRYPSIN mg/dl
Group 1 Group 2 Group 3 Group 4
Air 03 03 Air
Day
2
3
4
5
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Light Exercise
165
171
168
173
168
173
168
178
Light Exercise
177
178
183
167
179
186
178
184
Heavy Exercise
186
185
182
183
191
196
190
193
Heavy Exercise
175
179
177
175
175
177
174
172
34
-------
TABLE 5.7
MEAN RESPONSE FROM MANOVA FOR HAPTOGLOBIN mg/dl
Group 1 Group 2 Group 3 Group 4
Air 03 03 Air
Day
2
3
4
5
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Light Exercise
53
54
50
52
48
49
53
52
Light Exercise
59
60
64
70
70
67
67
67
Heavy Exercise
89
89
89
90
95
95
94
91
Heavy Exercise
60
59
53
56
59
60
58
57
TABLE 5.8
Day
2
3
4
5
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Group 1
Air
Light Exercise
87
89
88
90
87
92
90
93
11-JL. i ixuri riruiwin I ui\
Group 2
03
Light Exercise
95
97
98
98
97
91
89
96
v»unr i_i_rii_ii i \»— '"y/ u i
Group 3
03
Heavy Exercise
81
82
84
84
85
88
89
91
Group 4
Air
Heavy Exercise
83
84
80
81
78
80
81
81
35
-------
TABLE 5.9
MEAN RESPONSE FROM MANOVA FOR IgM mg/dl
Group
Air
Group 2
Group
03
Group 4
Air
Day
2
3
4
5
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Light Exercise
108
109
105
110
114
105
97
102
Light Exercise
120
120
121
117
125
128
116
124
Heavy Exercise
107
106
98
98
103
103
101
97
Heavy Exercise
119
119
116
112
110
111
108
101
36
-------
8.6
O GROUP 1
O GROUP2
A GROUPS -
O GROUP4
TIME, day
Figure la. Ozone by exercise by day Interaction means for RBC
glutathi one reductase.
37
-------
O GROUP1
D GROUP2
A GROUPS
O GROUP4
TIME, day
Figure Ib. Ozone by exercise by day interaction means for
serum vitamin E.
38
-------
1300,
5 1200 -
E
ui
1100 -
1000
4
O GROUP 1
O GROUP 2
A GROUP3
O GROUP4
TIME, day
Figure Ic.. Ozone by exercise by day Interaction means for RBC
cholinesterase.
-------
OGROUP1
DGROUP2
AGROUP3
OGROUP4
3 4
TIME, day
Figure Id. Ozone by exercise by day interaction means for LDH.
40
-------
OGROUP 1
0 GROUP 2
GROUP 3
OGROUP4
TOE, fey
Figure le. Ozone by exercise by day interaction means
for a-j-*antitrypsin.
41
-------
100
90
80
S 70
O GROUP 1
DGROUP 2
A GROUPS
O GROUP4
TIME, day
Figure If. Ozone by exercise by day interaction means for
haptoglobin.
42
-------
70
OGROUP1
DGROUP 2
AGROUP3
OGROUP4
TIME, day
Figure Ig. Ozone by exercise by day interaction means for
complement C.
43
-------
oGROUPI
a GROUP 2
AGROUP3
OGROUP4
B 110-
Figure Ih. Ozone by exercise by day interaction means for IgM.
44
-------
2.20
2.15
2.10
I 2.05
Q
ZOO
1.90
1.85.
AIR
Figure 2. Ozone by exercise means for serwn
vitamin E.
45
-------
S 1.8
1.6
I I i
o PRE LIGHT
a POST LIGHT
A PRE HEAVY
O POST HEAVY
24 32 40 48 56 64 72 80 88 96
TIME, hours
Figure 3. Pre-post by exercise by days means for 66PDH.
46
-------
7.1
6.9
6.7
6.5
in
> 6.3
5
cc
Ul
vt
6.1 • —
5.9
5.7
AIR
mm.
•I
mm
Figure 4. Ozone by exercise means for 66PDH.
47
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing}
1. REPORT NO.
EPA-600/1-79-026
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
BIOCHEMICAL CHANGES IN HUMANS UPON EXPOSURE TO OZONE
AND EXERCISE
5. REPORT DATE
August 1979
6. PERFORMING ORGANIZATION CODE
, AUTHOR(S)
Suzanne Chaney, Paulette DeWitt, Wendy Blomquist,
Keith Muller, Robert Bruce, George Goldstein
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, N. C. 27711
10. PROGRAM ELEMENT NO.
1AA816
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
In ho'use
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency, Research Pk. NC
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A total of 44 human subjects were exposed to 784 ug/m3 (0.4 ppm) ozone
and 30 human subjects were exposed to air as controls. A four hour exposure
was given on each of five consecutive days. Half of each group received an
higher level of treadmill exercise than the other half.
Nine biochemical blood parameters were measured pre and post exposure:
red blood cell glutathione reductase, vitamin E, red blood cell cholinesterase,
red blood cell glucose-6-phosphate dehydrogenase, lactic acid dehydrogenase,
complement C3, and IgM. Of these variables only vitamin E, red blood cell
glucose-6-phosphate dehydrogenase, and complement C3 showed significant changes
due to treatment.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Blood biochemistry
Human exposure
Oxidizing pollutant
Ozone
Ozadapt 1-4
Pollutant insult screening
Reductive detoxification
06,A
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGO*
51
20. SECURITY CLASS (This page)
UNPI ASSTFTFD
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
48
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