EPA-600/1-77-007
January 1977
EFFECT OF ATMOSPHERIC POLLUTANTS ON HUMAN PHYSIOLOGIC FUNCTION
By
Jack D. Hackney, M.D.
The Professional Staff Association of the
Rancho Los Amigos Hospital, Inc.
7413 Golondrinas Street
Downey, California 90242
Grant No. R-801396
Project Officer
Dr. George S. Malindzak
Clinical Studies Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U S ' .I;;'...,:. .1 '1 PROTECTION ft£t\&
w, j, 08^17
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 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. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
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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
establishment 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,
epidemiology, 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 develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing 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 endangerment of their health.
The studies reported here were initiated under EPA support in 1971. In
subsequent years, support for the overall effort was also provided by other
organizations -- the National Heart and Lung Institute and the California
Air Resources Board. Since the investigations funded by all three agencies
are closely interrelated and relevant to each other, information from all is
included here. No attempt is made to distinguish sources of support; instead,
the presentation attempts to give maximum scientific coherence to the overall
findings. The bulk of the report (Section B) consists of publication reprints
and preprints on specific effects of air pollution on human health; these are
preceded by a summary and overview (Section A) which attempts to place the
findings in the broader context of current environmental health concerns.
H. Knelson, M.D.
.Director,
Health Effects Research Laboratory
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TABLE OF CONTENTS
PAGE
NO
FOREWORD iii
SUMMARY 1
SECTION A
1. Goals 2
2. Experimental Design and Methods 2
3. Results 4
4. Conclusions 6
5. References - 8
SECTION B
1. Ozone and Human Blood 10
2. Experimental Studies on Human Health Effects
of Air Pollutants I. Design Considerations 14
3. Experimental Studies on Human Health Effects
of Air Pollutants II. Four-Hour Exposure
to Ozone Alone and in Combination With Other
Pollutant Gases 20
4. Experimental Studies on Human Health Effects
of Air Pollutants III. Two-Hour Exposure
to Ozone Alone and in Combination With Other
Pollutant Gases 26
5. Health Effects of Ozone Exposure in Canadians Vs
Southern Californians: Evidence for Adaptation?-- 32
6. Studies in Adaptation to Ambient Oxidant Air
Pollution: Effects of Ozone Exposure in
Los Angeles Residents vs New Arrivals 54
7. Respiratory Effects of Exposure to Ozone-Sulfur
Dioxide Mixtures 70
8. Nitrogen Dioxide Inhalation and Human Blood
Biochemistry 74
9. Experimental Studies on Human Health Effects of
Air Pollutants IV. Short-Term Physiological
and Clinical Effects of Nitrogen Dioxide
Exposure 91
IV
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SUMMARY
Short-term health effects of common ambient air pollutants,
particularly photochemical oxidants, were investigated under controlled
conditions simulating typical ambient exposures. Volunteer subjects
were exposed, in an environmental control chamber providing highly
purified background air, to single pollutants or mixtures under
conditions of realistic secondary stress (heat and intermittent exercise)
Normal men exposed to ozone (Ch) showed respiratory symptoms, pulmonary
function decrement, and alterations in red-cell biochemistry. These
effects were dose-related, with apparent "threshold" for detectable
effect levels as low as 0.2-0.3 ppm in a 2-hr exposure for the most
sensitive subjects. Addition of 0.3 ppm nitrogen dioxide (f^) and
30 ppm carbon monoxide (CO) did not noticeably enhance adverse effects
of 03, but addition of 0.37 ppm sulfur dioxide ($02) to 0.37 ppm 0^
produced slightly greater effects than did 0.37 ppm 03 alone. Subjects
with asthma or clinical airway hyperactivity appeared to experience more
severe effects of 03 than normals, and subjects chronically exposed to
ambient 03 appeared to be less reactive than those living in non-03-
polluted areas. Normal subjects exposed to N02 at 1 ppm or 2 ppm showed
little clinical or physiological response, but did show changes in
red-cell biochemistry at both concentrations. These effects were less
than produced by 0.37 ppm 03, but significantly greater than produced
by heat and exercise stress alone.
Ozone appears to be the most hazardous of common gaseous photo-
chemical pollutants, with respect to short-term human health effects.
Further investigation is needed of additive or synergistic effects of
pollutants in mixtures, of chronic effects of repeated exposures, and
of effects in population groups with preexisting disease or other
high-risk characteristics.
1.
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SECTION A. INTRODUCTION AND OVERVIEW
1. Goals
The basic purpose of this project has been to provide part of
the scientific data base needed for establishment of ambient air quality
standards for the protection of public health, as required by the Clean
Air Act of 1970. Since overly stringent controls on pollutant emissions
are likely to result in economic harm, just as insufficiently stringent
controls are likely to result in harm to human health, it is vitally
important that air quality standards for any given pollutant be based
on a careful and realistic appraisal of the hazards associated with
that pollutant. We sought to determine, for specific pollutants found
in photochemical smog, whether or not doses attainable in severe pollution
episodes produced detectable short-term adverse health effects in normal
adults. A broad range of tests was employed to maximize the probability
of detecting any adverse effects which might occur. Chronic effects
of often-repeated exposures were not considered; therefore, the results
have no direct bearing on questions of public-health risks from chronic
exposures.
2. Experimental Design and Methods
The basic experimental approach, facilities, and physiological
test procedures are described in detail in Section B, Part 2. Biochemical
test procedures are described in Section B, Part 1. In general, a
volunteer subject is exposed to purified air to which a carefully
controlled amount of a given pollutant has been added, under conditions
simulating as closely as possible an ambient exposure to the same
pollutant. Following the exposure, health-effects tests are performed.
Results are compared with those obtained in a control experiment—an
identical exposure to purified air with no pollutant added. Since
inhaled pollutants first come in contact with the respiratory tract,
sensitive respiratory physiological tests are the logical choice as
sensitive indicators of pollutant effects. Pollutant molecules or
their reaction products which successfully pass through the respiratory
tract enter the circulation, thus blood biochemical variables may also
be expected to be sensitive indicators. In studies of oxidant pollutants,
biochemical investigations are logically directed to the pentose-pathway
enzymes of the red cell, which are important in maintaining its correct
redox potential, as well as to the cell membrane—the cell's first line
of defense against toxic substances.
Exposure conditions in most instances were chosen to simulate
weather conditions typically prevailing during photochemical pollution
episodes in Southern California--temperature 31°C (88°F) and relative
humidity 35 percent. These conditions are also realistic with respect
to photochemical episodes in other parts of the United States, except
that humidity may be somewhat higher in other areas. Exposures were
also performed under "normal room temperature" conditions--21°C (70°F)
and 50 percent relative humidity—when appropriate.
2.
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Calibration of pollutant monitors is described here since it is
not covered in detail in Section B. Chemiluminescent ozone (Og)
monitors are calibrated by the EPA 1 percent neutral buffered potassium
iodide method (1) using (^-containing purified air at 35 percent relative
humidity as the sample atmosphere. This calibration is similar to that
previously used by the State of California, which employed 2 percent
buffered potassium iodide (2). The current California standard is based
on an ultraviolet absorption method which gives readings about 20 percent
lower than given by the above methods.(3) A commercial ultraviolet
instrument with calibration traceable to the California standard (DASIBI
Environmental Corp. 1003-AH) is currently used as a calibration check;
however, concentrations are reported in terms of the EPA method.
Nitrogen dioxide calibrations are performed with a National Bureau of
Standards permeation tube whose output is diluted with dry nitrogen,
then with air filtered by silica gel, activated carbon, and molecular
sieves. Sulfur dioxide calibrations are performed similarly with a
Metrom'cs permeation tube. Carbon monoxide calibrations are performed
with commercial calibrating gases (Scott Laboratories); two or more gas
cylinders are compared for consistency as a check on reliability.
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3. Results
Detailed results are given in Section B. The following is a brief
description of work performed, approximately in chronological order.
Initial exposure studies employed 1 ppm N02 (approximating the
highest ambient concentration observed in the Los Angeles area). Five
staff members served as subjects in pilot studies. No symptoms or
adverse clinical findings were produced in this group by exposure for
2 hr or longer. One individual appeared to develop frequency dependence
of dynamic lung compliance with exposure, but this finding could not be
reproduced in later studies. No other meaningful pulmonary function
changes were observed. In further investigation of N02 effects, 15
healthy male pre-professional students were exposed to 1 ppm for 2 hr
on each of two successive days. No meaningful pulmonary function changes
were found, nor were any found in a smaller group exposed to 2 ppm.
Blood biochemical changes suggestive of an oxidant challenge were found
at both concentrations, however. To test the possibility that heat
and/or exercise stress alone could account for these changes, another
group was exposed to pure air only on three successive days. Biochemical
measures were essentially unaffected by this exposure.
Initial studies of 03 employed exposures to 0.50 ppm-experienced
occasionally in Los Angeles and rarely, if at all, elsewhere. The
question whether effects could be shown under the worst possible realistic
exposure conditions was addressed by exposing four staff members for 4 hr
(twice the typical duration of an ambient exposure) on two successive days
in each of three successive weeks. To test whether coexisting pollutants
might modify Oo effects, 0.3 ppm NO? was added to the exposure atmosphere
the second week and 30 ppm CO plus 0.3 ppm N02 the third week. Total
03 dose received in the three-week protocol was estimated to equal that
received in an entire summer season of ambient exposures. The four
initial subjects completed this protocol without showing detectable
clinical or physiological effects, although pronounced oxidative changes
in biochemical parameters were seen beginning with the first exposure
to 03 alone.
The relative lack of response of the initial subjects prompted us to
repeat the same experiment using subjects suspected of being "hyperreactive"
to inhaled irritants. These four people had normal baseline pulmonary
function but had either mild asthma or a history of respiratory symptoms
subjectively associated with smog exposure. All four subjects became ill
and showed substantial pulmonary function changes with the first Oo
exposure, thus it was impossible to complete the planned experimental
protocol. Subsequent studies were conducted at 0.25 ppm 03 and 0.37 ppm
63 for two or four hours and at 0.50 ppm 03 for 2 hrs, and included both
"normal" and "hyperreactive" subjects. No substantial reactions to
0.25 ppm were found, but some "hyperreactors" experienced effects at
0.37 ppm, as did most "hyperreactors" and some "normals" at 0.50 ppm.
4.
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At the highest concentration, effects were clearly cumulative in some
individuals exposed on two successive days, with substantial increases
in symptoms and function decrement occurring on the second day. This
finding is important in that pollution episodes often recur on several
successive days. Biochemical, physiological, and symptom data showed
dose-response relationships with apparent "thresholds" near 0.25 ppm.
In comparing our results with those reported by Bates's group in
Canada (4,5), we observed that the Canadian subjects' response to Oo
appeared about twice as severe at a given exposure concentration, bates
and Hazucha also reported that a mixture of 0.37 ppm Oq plus 0.37 ppm
S02 produced far more severe physiological responses than did 0.37 ppm
63 alone (6). To evaluate the significance of the Canadian findings,
wa undertook a series of comparative studies. When subjects previously
studied in Canada werereexposed to 0^ in this laboratory, their
responses were similar to those previously seen in Canada and the more
reactive individuals showed more severe effects than even the "hyper-
reactors" who were Los Angeles area residents. Similar results were
obtained in a subsequent study of student volunteers--Los Angeles residents
were less reactive on the average than nonresidents, even though resident
and nonresident groups were well matched in other characteristics. Thus
it appears likely that adaptation which modifies short-term 03 effects
develops in Los Angeles residents. In studies of 03/802 mixtures,
Los Angeles residents failed to show the marked synergistic effect seen
by Hazucha and Bates, but those studied did show slightly increased
effects with the mixture relative to 03 alone. Canadian subjects studied
in this laboratory showed responses less severe on the average than they
had previously showed in Canada, but more severe than were seen in Los
Angeles residents. Individual variability was considerable, making
firm conclusions difficult. One possible explanation of these findings
which remains to be tested is that accompanying particulate pollutants
contributed to the Oo/S02 effect in the Canadian studies, in which highly
efficient air filtration such as used in our studies was not available.
5.
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4. Conclusions
a. Ozone
Ozone exposure produces loss in pulmonary function and respiratory
symptoms severe enough to impair performance of normal tasks at levels
easily attainable in Los Angeles area sraog episodes and probably
occasionally attainable elsewhere. Blood biochemical changes also occur;
these cannot as yet be shown to be correlated with physiological or
clinical adverse effects, but must be considered harmful in the absence
of evidence to the contrary. Physiological and clinical effects of
exposure are generally reversed within 24 hrs after exposure ceases,
but biochemical variables appear to require longer to return to
baseline levels. While in the population studied, the average "threshold" of
detectable effect appeared to be 0.25 ppm 03 or higher, effects might
well be found at lower concentrations in high-risk groups such as the
young, the old, and persons with preexisting cardiopulmonary disease.
Further investigation of high-risk groups will be needed to improve the
scientific data base for air-quality standards. While adaptation
probably operates to reduce the acute effects of 03 exposure in
individuals exposed repeatedly, this should not be considered a
justification for relaxing Og air quality standards, since the adaptive
process might have other harmful effects and might not operate satis-
factorily in some individuals.
b. Ozone in Mixtures
When realistic concentrations of NOp and CO were added to 0^, no
additional effects were observed. This experiment was done only with
Oo concentrations which produced little or no clinical or physiological
effect in the individuals studied (0.25 ppm in "hyperreactors", 0.50 ppm
in "normals"). The possibility that coexisting NO? might exacerbate
effects of 03 exposures above the "threshold" level has not been investi-
gated. Adding S02 to Oo concentrations near the "threshold" level did
appear to produce modest increases in exposure effects. Large numbers
of sulfate particles were also formed in the mixture. Further investigation
of the health-effects role of particulates in pollutant mixtures of this
kind will be necessary before health hazards of ambient mixtures can be
assessed reliably.
c. Nitrogen Dioxide
Exposures to 1 ppm or 2 ppm N0£ produced no meaningful physiological
changes in the population studied. A minority of the study population
experienced mild respiratory symptoms possibly, but not certainly,
attributable to exposure. Blood biochemical changes were seen at both
concentrations. These were not clearly dose-related, and a threshold of
detectable effect has not been determined. While the physiological
and clinical findings suggest that N02 is far less hazardous than 03
6.
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at ambient concentrations, the biochemical findings must, in the absence
of contrary evidence, be considered as indicating an adverse health
effect at concentrations of 1 ppm and possibly lower. Furthermore,
Orehek et al. (7), recently reported that NC^ produced an increased
reactivity to bronchoconstrictor challenge in some asthmatics at
concentrations as low as 0.1 ppm--a level commonly experienced in most
urban areas. The report of that study does not make clear whether
control of the experimental environment was sufficiently rigorous to
assure that the observed effects were entirely due to NOo; however, the
work at least raises the possibility that some susceptible individuals
may be adversely affected even by relatively low ambient pollutant levels.
d. General Comments
In the course of this project and concurrent studies elsewhere,
there has developed considerable understanding of the short-term health
effects of individual gaseous pollutants in relatively healthy people.
Most needed now is information on health effects of particulate pollutants
and mixtures of gaseous and particulate pollutants, both in healthy and
in diseased or hypersusceptible populations. Since the possible combinations
of ambient pollutants and resulting health-effects interactions are almost
infinite, control!ed-exposure studies like those reported here can hope
to provide only a part of the necessary health-effects data base. The
rest must be provided by studies of people exposed to actual ambient
pollution, in which both health-effect variables and environmental variables
are quantitated as comprehensively and carefully as possible, in order
to document relationships between health and specific pollutants or
mixtures. In addition to large-scale epidemiologic investigations,
smaller studies should be considered in which panels of clinically well-
characterized individuals are placed in a given well-monitored polluted
environment and examined for short-term health effects attributable to
that environment. This approach should help bridge the gap between
control!ed-environment laboratory studies, in which results may be difficult
to relate to real-life health hazards, and epidemiologic studies, in which
the reliability and sensitivity of the health-effects testing is often
inadequate to detect or quantitate the health hazard of concern. Possible
chronic effects of repeated pollutant exposure are an additional serious
research problem which must be approached mostly by epidemiologic and
animal toxicology studies.
7.
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5. References
1. Selected Methods for Measurement of Air Pollutants. Division of
Air Pollution, Cincinnati, U. S. Public Health Service Publication
No. 999-AP-ll, 1965.
2. Recommended Methods, Air and Industrial Hygiene Laboratory,
California Department of Public Health, Berkeley, 1968.
3. De More, W. B., Calibration report. California Air Resources Board
Bulletin, 5 (11):1> December 1974.
4. Bates, D. V., Bell, G., Burnham, C., Hazucha, M., Mantha, J.,
Pengelly, L. D., Silverman, F.: Short-term effects of ozone on
the lung, J Appl Physiol 32:176-181, 1972.
5. Hazucha, M., Silverman, F., Parent, C., Field, S., Bates, D. V.:
Pulmonary function in man after short-term exposure to ozone.
Arch Environ Health 27:183-188, 1973.
6. Hazucha, M., Bates, D. V.: Combined effect of ozone and sulphur
dioxide on human pulmonary function. Nature 257:50-51, 1975.
7. Orehek, J., Massari, J. P., Gayrard, P., Grimaud, C., Charpin, J.:
Effect of short-term, low-level nitrogen dioxide exposure on
bronchial sensitivity of asthmatic patients. J CHn Invest
57:301-307, 1976.
8.
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SECTION B
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Reprinted from the Archives of Environmental Health
January 1975, Volume 30
Copyright 1975, American Medical Association
Ozone and Human Blood
Ramon D. Buckley, PhD; Jack D. Hackney, MD; Kenneth Clark; Clara Posm
Statistically significant changes
(P <.05) were observed in erythrocytes
(RBC) and sera of young adult human
males following a single short-term expo-
sure to 0.50 ppm ozone (O ) for 2% hours.
The RBC membrane fragility, glucose-
6-phosphate dehydrogenase (G-6-PDH)
and lactate dehydrogenase (LDH) enzyme
activities were increased, while RBC
acetylcholinesterase (AcChase) activity
and reduced glutathione (GSH) levels
were decreased. The RBC glutathione re-
ductase (GSSRase) activities were not
significantly altered. Serum GSSRase ac-
tivity, however, was significantly de-
creased while serum vitamin E, and lipid
peroxidation levels were significantly in-
creased. These alterations tend to dis-
appear gradually, but were still detectable
two weeks following exposure.
The toxicity of inhaled ozone (0,)
is well known and the presence of
the oxidant in many urban and in-
dustrial environments has made the
study of the biological effects of in-
haled 0 important.' Recent studies
have been directed toward the clari-
Submitted for publication Aug 8 1974, ac-
cepted Aug 26
From the Department of Environmental
Health, Rancho Los Amigos Hospital, Downey,
Calif (Dr Buckley, Dr Hackney, Mr, Clark, and
Ms Posm), and the University of Southern Cali-
fornia School of Medicine, Los Angeles (Dr
Buckley and Dr Hackney)
Reprint requests to Room 48, Medical Science
Bldg, Rancho Los Amigo- Hospital, 7601 E Im-
perial Hwy Downey, CA 90242 (Dr Buckley)
fication of the biochemical changes
that occur in tissues following 0, ex-
posure. Radiomimetic changes were
described- as well as the oxidation of
unsaturated fatty acids, and oxida-
tion of biologically active reducing
substances such as reduced sulfhydryl
groups and the cofactors NADH and
NADPH.' Sufficient knowledge is
now available to allow speculation
about the importance of some of the
observed changes in the development
of acute or chronic pulmonary dis-
ease, or in the development of 0, tol-
erance. Ozone is known to produce a
dose-related reaction in in vitro cell
cultures extending from mild meta-
bolic suppression at low le\ els to cell
death at high levels. It is natural
that the target organ for 0 toxicity
studies is the lung, and much of our
knowledge derives from studies of ef-
fects of oxidants in this organ. The
results point to loss of reduced
sulfhydryl groups and reduction of
activities of sulfhydryl-containing
enzymes, while the pentose and gly-
colytic pathway activity levels are in-
creased.' " Oxidant effects of 0 inha-
lation have also been shown to occur
in the blood of rodents following high,
but sublethal levels of the irritant,
and changes in erythrocyte (RBC)
and sera also suggest that oxidant-
induced alterations have taken place
beyond the blood-air barrier'
It is not known if 0, at ambient
levels crosses the air-blood harrier in
humans or if detectable biochemical
changes occur. The present study was
undertaken to answer that question.
SUBJECTS AND METHODS
Seven healthy young adult human male
volunteers were studied. The same cham-
ber was used for sham control exposures
and for exposure to 0,. The chamber con-
tents are monitored for 0,, NC\, CO, hy-
drocarbons, and particles. Pollutant levels
were essentially zero during sham expo-
sures, and chamber background particle
levels (0.5/i to 5/i) were less than 20,000/cu
ft when empty, and less than 120,000/cu
ft during subject exercise. Subjects per-
formed identical exercise and pulmonary
function tests during a sham control period
and while 0 was being administered. Dur-
ing exposure, a 0,5 ppm atmosphere of 0 ,
produced by a silent-arc generator, was
added to the chamber air supply. Chamber
temperature was maintained at 30 C and
relative humidity at 35c/c to simulate a typ-
ical summer day in Los Angeles.
Venous blood samples were collected in
heparinized and unheparimzed tubes im-
mediately after the sham and the 0 expo-
sure Heparinized blood was stored on ice
until the erythrocyte (RBC) studies could
be completed The unhepannized blood was
allowed to remain at 2 to 4 C for four to six
hours until the serum could be removed
Experiments were planned to detect
blood tissue oxidation after 0 inhalation
since this has been shown to be the princi-
pal effect of high levels of the irritant
on lung and blood tissues of experimental
animals The methods are essentially the
same a-' described in the following refer-
ences with only minor modifications The
RBC membrane fragility was measured by
determining the degree of hemolysis in the
presence of H.O. Activities of RBC en-
zymes glueose-6-phosphate dehydrogen-
ase ('it)l'UH), lactate dehydrogenase
(LDH) glutathione reductase (GSSR
asei ' and acftvlcholinestera.se (Ac-
40 Arch Environ Health/Voi 30, Jan 1975
10.
Ozone aric! Human Blood-Bbckley et
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50-
«
"o
cv
* 30-
o^
-^
CO
ut
O
m
rr
10
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0
N-=6
P--; 05
C
E
-T-
c
4-,
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g 20-
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O)
CO
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0)
03
| 1°-
O
m
rr
n
-ti
C
~i"
E
Fig 1 —Human RBC response to inhaled ozone (0.5 ppm x 2 hr 45 mm) Fragility was
measured as percent hemolysis in 2% hydrogen peroxide and incubated for one hour at
pH 7.4 in Krebs-Rmger bicarbonate buffer Acetylcholmesterase was measured at pH
8.0 in 0.1 M phosphate buffer employing acetylthiocholme as substrate Activity is ex-
pressed as mM/ml of blood/mm
Fig 2 —Human RBC response to ozone (0 5 ppm x 2 hr 45 mm) Glutathione assay
detects soluble GSH employing 5, 5' dithiobis (2-mtrobenzoic acid) as coupling reagent
6-
50-
—
E
8
^
q^
C
o
^ -
2
^
o
10-
0
•
4-
~
ii
C
I^
J~l «•
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O)
~~~-
^
X 2"
Q
QL
CO
0
-
n
N = 6
*
C
f*l
E
PS.05
Chase),'1 were measured at- well as EEC
glutsthione ievois.1 Serum levels of vita-
min E,'1 '" and lipid peroxides'" were deter-
mined as well as activity levels of the se-
rum enzyme glutathiore retuctase.-'"
Each experimental subject served as his
own control and paired-group anah ses were
performed The small "Student's" /-test
was used to test the null hypothesis, with
the critical level at F <.05.
RESULTS
Data in Fig 1 to 4 are means and
standard errors of the means, of
paired-group analyses of seven exper-
imental subjects.
The evidence indicating 0,-induced
changes in the RBC membrane is
shown in Fig 1. The single Oj expo-
sure resulted in a significant increase
(P<001) in RBC fragility to H,02,
while the activity of the membrane-
bound enzyme AcChase was decreased
(P <001). Figure 2 shows that the ac-
tivity of G6PDH was significantly in-
creased (P <.001) while RBC levels of
GSH were decreased (P <.01). Ozone
inhalation also stimulated an increase
in LDH activity (P <.001) although
RBC GSSRase activities were not al-
tered (Fig 3). Serum GSSRase activ-
ity levels are shown in Fig 4 to
be significantly decreased (P<.05).
Vitamin E levels were also increased
(P<025) at the same time that in-
creased oxidation of unsaturated
fatty acids (P <.02o) was observed.
COMMENT
The very/ high oxidation potential
of 0, has led research workers from
the beginning to suspect that the ma-
jor damage from inhalation of this ir-
ritant resulted from oxidation of la-
bile components in biological systems
to produce structural or biochemical
lesions.21 More recent work has veri-
fied that inhaled 0, causes oxidation
of components of rodent lung." Other
studies have shown that changes in
the rate of tissue metabolism also ac-
company 0 -induced changes in the
oxidation states of lung tissue compo-
nents.-1
This study arose from our need to
seek evidence of changes in human
tissues due to inhalation of ambient
levels of 0 , and was encouraged by
evidence from past experiments that
showed that significant alterations in
Arch Environ Health/Vol 30, Jan 1975
Ozone and Human Blood/Buckley et al 41
11.
-------
the blood tissue of rodents did occur
ah p. resuil of inhalation of oxidants.
although the levels were generally
much higher than ambient levels an-
ticipated during a smoggy day. Ex-
periments performed bj others,1" and
in our laboratories/' have shown
that inhalation of high levels of 0 by
rodents results in oxidation changes
in blood similar to those detected
in lung. Considerable question arose
about possible metabolic changes in
blood following low-level Oi inhala-
tion, since it is known that the effi-
ciency of the upper airway in the re-
moval of 0, is quite high, and that the
efficiency increases as the levels of in-
haled 0, decrease.-1 The results indi-
cate that under the conditions of the
experiment a single exposure to O.-i
ppm for 234 hours is above the thresh-
old level.
The observed changes suggest that
oxidation is the initial event, and that
additional changes occur as a result of
the system's attempt to compensate
for the changes in blood tissue redox
potentials. Reduced glutathione, con-
sidered to be an important biological
anti.^ddant, is significantly decreased
by 0, inhalation. The RBC enzvme
Hcetylcholinesterase (AcChase), con-
taining a -SH group essential for its
activity is also depressed. The in-
crease in the presence of peroxidized
lipids and increase in vitamin E in
the sera also suggest that oxidation
is responsible for the priman 0
response. Glutathione red:i cell.
The increases in ;>)ucose-t5-phos-
phate dehydrogenase (GGPDHl and
lactate dehydrogenase (LDH) activi-
ties suggest that RBt' metabolism is
stimulated by 0 . The increase in
G6PDH activity may also IK related
to the adaptation phenomenon de-
scribed by others" One of the pos-
sible pathways by which tissue le>'eU
of GSH are maintained is schemat-
ically represented in Fig •'•>. An in-
crease in G6PUH ac'hity couU'. pro-
vide reduced cot'.idor essential lo- the
1 40-
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Fig 3 —Human RBC response to ozone (0 5 ppm x 2 hr 45 mm). Glutathione reduc-
tase activity was expressed as international units/ml of blood/mm Oxidized glutathione
was used as substrate. Lactate dehydrogenase activity was measured by following the
disappearance of NADH at 340 nm and was expressed as international units/gm hemo-
globin mm
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-------
function of GSSRasc The an,i>unLs of
GSH normally present in human t.s-
sues is small compared to many other
species of higher animals but the
levels of GSSRase were higher in
humans (RBCs) than in any species
tested.^7 The RBC levels of GSSRdse
were not decreased by 0, exposure,
and thus the mechanism for the re-
generation of NADPH appears to be
unaltered. The increase in G6PDH ac-
tivity has been shown in other experi-
ments (unpublished) to persist for a
period of at least two weeks following
0, stimulation. It will be important in
future experiments to follow the time
course of the 0,-induced blood
changes in order to determine the
length of time they persist and if
they are related to the adaptation
phenomenon observed in laboratory
animals.
The increase in serum vitamin E
levels probably results from the adap-
!i\{ rr-Fj' .", jf tht whole i;/g:m.--m
md resi/lt- r. '-lie availability of in-
creased k-vtJs of circuiting anti-
oxidants This mobilization ->f vita-
mir E a grin suggests that 0 , or
oxidizing free-radical, does pass the
air-blood barrier at the comparatively
low levels used in the experiment.
Whether vitamin E acts alone to in-
hibit lipid peroxidation,-' acts in con-
cert with \itamin A or other sub-
stances to protect tissue- from
oxidation,-"1 or functions in some other
way, there is now little doubt of its
protective function-1: " in animals
exposed to a strong oxidant such as
ozone. It would be important in a fu-
ture experiment to determine if in-
gestion of relatively large amounts
of vitamin E would protect humans
against the biochemical effects of 0 .
Future experiments will determine
the level of inhaled 0 below which no
observable changes occur in humans.
Knowledge of these "thresho'd" lev-
els will be useful :n determining the
acceptable and permissible atmospher-
ic standards. It will also :ie important
to establish the length of time re-
quired for changes to disappear, as
well as to determine if successive ex-
posures produce cumulative effects. A
very wide range of subjective reac-
tions to oxidizing air pollution is ex-
pressed by people living in cities
where this type of smog is severe. It
will be important to seek possible cor-
relations between the degree of sub-
jective irritative response and bio-
chemical change, to help determine if
differences exist in people's capacity
to resist 0,.
This study was supported by National Heart
and Lung Institute, grant No HL 15098, En-
vironmental Protection Agency grant No. R-
801396, and California Air Resources Board No
2-372.
1. Stockinger HE- Evaluation of the hazards
of ozone and oxides of nitrogen Arch fnd Health
15-181-190, 1957.
2. Menzel DB: Toxicity of ozone, oxygen and
radiation. Ann Rev Pharmacol 10 379-384, 1970
3 Roehm JN, Hadley JG, Menzel DB. Oxida-
tion of unsaturated fatty acids by ozone and ni-
trogen dioxide A common mechanism of action
Arch Environ Health 23 142-148, 1971
4 Menzel DB: Oxidation of biologically active
reducing substances by ozone. Arch Environ
Health 23-149-153, 1971
5. Pace DM, Landolt PA, Aftonomos BT Ef-
fects of ozone on cells in vitro Arch Environ
Health 18 165-170, 1969
6. DeLucia AJ, Hoque PM, Mustafa MG, et al
Ozone interaction with rodent lung Effect on
sulfhydryls and sulfhydryl-contaming enzyme
activities J Lab Chn Med 80.559-566, 1972."
7 Chow CK, Tappel AL' Activities of pentose
shunt and glycolytic enzymes in lungs of ozone-
exposed rats Arch Environ Health 26205-208,
1973.
8. Chow CK, Tappel AL An enzymatic protec-
tive mechanism against lipid peroxidation dam-
age to lungs of ozone-exposed rats. Lipids 7 518-
524, 1972
9. Goldstein BD, Buckley RD, Cardenas R, et
al. Ozone and vitamin E Science 169:606-616,
1970.
10 Goldstein BD, Pearson B, Lodi C. et al
The effect of ozone on mouse blood in vivo Arch
Environ Health 16-648-650, 1968
11 Younkin S, Oski IA, Barness LA Mecha-
References
nism of the H:02 hemolysis test and its reversal
with phenols Am J Chn Nutr 24.7-13, 1971
12 Lohr GW, Waller HD. In, Bergmeyer HV
(ed) Methods of Enzymatic Analysis New York,
Academic Press Inc, 1963, pp 744-751
13 Bergmeyer HV, Bernt E, Hess B In, Berg-
meyer HV (ed)' Methods of enzymafv Analysis
New York, Academic Press Inc, 1963, pp 736-741
14. Beutler E Effect of flavin compound on
glutathione reductase activity J Chn In ,-est
48 1957-1966, 1969
15 Ellman GL, Courtney KD Andrew V, et
al A new and rapid colonmetnc determina-
tion of acetylchohnesterase activity Biwhem
Pharmacol 788-95, 1961.
16 Beutler E, Duron 0, Kelly BM Improved
method for determination of blood glutathione
,/ Lab Chn Med 61 882-888, 1963
17. Bieri JG, Prival EL Serum vitamin E de-
termined by thin layer chromatography Pror Soc
Exp Bwl Med 120:554-577, 1965.
18. Tsen CC. An improved spectrophotometnc
method for the determination of using 4, 7-di-
phenyl.-l, 10-phenanthroline Anal Chem 33849-
851, 1961.
19 Mengel CE, Kahn HE Jr.. Effects of in vivo
hyperoxia on erythrocytes III. In vivo peroxida-
tion of erythrocyte lipid J Chn Invent 45-1150-
1158, 1966".
20 Horn HD In, Bergmeyer HV (ed) Method*
of Enzymatic Analysis New York, Academic
Press Inc, 1963, pp 875-879
21 Stoki'.ger HE, Coffin DL Biologic effects
uf air pollutants, in Stern AC (ed) An Pollution,
ed 2 New York, Academic Press Inc, 1968, vol 1,
pp 401-546.
22. DeLucia AJ, Hoque PM, Mustafa MG, et
al Ozone interaction with rodent lung Effect on
sulfhydryls and sulfhydryl-contaimng enzyme
activities J Lab din Med 80-539-566. 1972.
23 Chow CK, Tappel AL Activities of pen-
tose shunt and glycolytic enzymes in lungs of
ozone-exposed rats. Arch Environ Health 26.205-
208, 1973
24 Goldstein BD, Lodi C, Collinson C, et al
Ozone and lipid peroxidation Arch Environ
Health 18.631-635, 1969
25 Goldstein BOB, Pearson C, Lodi RD et al-
The effect of ozone on mouse blood in vivo Arch
Environ Health 16:648-650, 1968
26 Yokoyama E, Frank R. Respiratory uptake
of ozone in dogs Arch Em-iron Health 25 132-
138, 1972
27 Lankish PG, Schroeter R, Lege L, et al
Reduced glutathione and glutathione reductase
A comparative study of erythracytes from vari-
ous species Compr Bwchem Physiol 46B 639-641,
1973
28 Kann HE Jr, Mengel CE, Smith W, et al
Oxygen toxicity and vitamin E. Aerospace Med
35:840-844, 1964.
29 Menzel DB Vitamins A and E help main-
tain lung health Chem Engineering A'ew.s- 48 38-
39, 1970
30 Menzel DB, Roehm JN, Duk Lee S. Vita-
min E The biological and environmental anti-
oxidant J Agric Food Chem 20:486-490, 1972
Arch Environ Health/Vol 30, Jan 1975
Ozone and Human Blood/Buckley et al 43
• 3.
fV- '/nitpl 5'-|r-s I,'
-------
Reprinted from the Archives of Environmental Hea/rh
August 1975, Volume 31
Copyright 19/5, American Medical Association
Experimental Studies on Human
Health Effects of Air Pollutants
I. Design Considerations
Jack D. Hackney, MD; William S. Linn, MA; Ramon D. Buckley, PhD;
E. Eugene Pedersen. PhD; Sarunas K. Karuza, PhD; David C. Law, MD; D Armin Fischer, MD
Because of the possible threat to public
health posed by photochemical air pollu-
tion, a need exists for experimental
studies of short-term respiratory effects of
air pollutant exposure in humans. Such
studies require rigorous control and com-
prehensive documentation of the experi-
mental air environment and exposure
conditions to ensure that results are both
reliable and relevant to public health
questions.
In addition to biochemical, behavioral,
and clinical evaluations, comprehensive
pulmonary testing is required to assure
that effects at different levels of the respi-
ratory tract are detected. An experimental
design based on these principles Is
described. Studies using this design have
shown a wide range of sensitivity to the
pollutant ozone and important adverse
hearth effects in sensitive individuals
under exposure conditions similar to
those experienced during ambient pollu-
tion episodes.
Submitted for publication Oct 24. 1974, accept-
ed Jan 2. 1975
From the Specialized Center of Research in
Em iron mental Lung Disease, Environmental
Health Sen-ice, and the Pulmonary service,
Rancho Los Amigos Hospital, Downey, Calif, and
the School of Medicine, UrmrrHtj of Southern
California, Los Angeles
Reprint requests to the Environmental Health
Laboratories, Rm 51, Medical Science Bldg
Raniho Los Amigos Hospital, 7fi(ll E Imporia!
Hwy, Downej, CA 90212 il)r Hackne\)
Photochemical smog is a complex
mixture of substances, including
powerful oxidizing agents such as
ozone (0,), nitrogen dioxide (N02), and
organic peroxides. It is formed by the
action of atmospheric oxygen and
sunlight on effluent gases, particu-
larly hydrocarbons and nitric oxide
(NO), emitted as a result of automo-
tive and industrial fuel combustion.'
The respiratory and other health
effects of exposure of humans to
photochemical smog have not been
well documented. The Los Angeles
area is most often associated with
such exposures, but they are by no
means limited to this region Substan-
tial photochemical oxidant concentra-
tions have been reported in Canada^
and in Europe,1 and can probably
occur in most areas where there are
concentrations of automobile traffic
or fuel-burning industry when sun-
light is strong and \vinds are too light
to disperse effluent gases. Thus,
photochemical smog and other air
pollutants present a widespread po-
tential public health problem. The
appropriate government agencies
have responded by setting air quality
standards intended to protect the
population from dangerous levels of
exposure. Most existing standards,
however, are based on limited scien-
tific information on the health effects
of pollutants.4 Legal challenges to the
standards are also frequent because
the economic and social costs of
conforming may be high. It is thus
apparent that there is a need for
comprehensive experimental studies
on the health effects of exposure to
oxidants and other air pollutants.
Such studies must be controlled and
documented as rigorously as possible
to ensure reproducibility and to with-
stand legal challenges. Although nu-
merous animal studies exist and
others are in progress, at present they
cannot be quantitatively related to
human health. Epidemiologic studies
can be useful, but are limited by cost,
dose-range available, presence of in-
terfering pollutant substances, and
problems caused by many uncon-
trolled variables.
Human experimental studies of the
health effects of oxidant air pollut-
ants can be accomplished at fixed
concentrations in the absence of inter-
fering pollutants, under well-con-
trolled environmental conditions and
with a well-characterized subject
group of limited size. Studies of the
health effects of well-specified am-
bient air are also possible. A survey of
the environmental control and moni-
toring technology used in previous
Arch Environ Health /Vo! 30, Aug 1975
Health and Pollutants i /Hackney et al 373
14.
-------
Measure
Temperature, F
Relative Humidity
Total No4 particles per cubic feet
CO, ppm
NO, ppm
NO2, ppm
03, ppm
S02, ppm
Hydrocarbons, ppm
Table 1.-
Ambient*
25-110
10°,o-100%
> 106
30
2
1
07
1
30
—Chamber Environmental Contiol
Design Specification
Chamber
Within ± 1, range 14-110
Within ± 4%, range 10%-100%
<105
2
.01
.01
.01
.05
5
Factors
Ambient
25-110
10%-100%
> 106
20#
1#
0.7#
0.4#
r*
100**
Actual Performance
Cbambei-t
Within ± 1, range 15-130
200-400§
2 X 103- 10M
2 X 10<- ID5!!
1
002
<001
<001
001**
5**
• Extreme ambient conditions experienced in Los Angeles area
f Over at least a six-hour period.
j Particles with diameter > 0.5 cm.
§ No one in chamber.
j| One to four subjects at rest.
11 One to four subjects exercising.
# Maximum concentration typically found outside of the environment at control facility.
** When deliberately challenged at or above the ambient level.
experimental studies1"8 indicates that
many limitations existed. More rigid-
ly controlled studies are thus required
to extend previous work.
Bates et al and Hazucha et alv* have
discussed some of the problems
encountered in the design and exe-
cution of experimental studies on
humans exposed to air pollutants, and
have described an appropriate experi-
mental protocol. Despite some limita-
tions in environmental assessment
and control, they have verified impair-
ment in pulmonary function in sub-
jects exposed to ozone concentrations
equal to or less than those encoun-
tered in severe photochemical smog
episodes. This communication is in-
tended to report the operational
approach for a new series of studies,
drawing on the experience of Bates
and others, and using current tech-
nology to provide more rigorous expe-
rimental control and more comprehen-
sive information than was obtainable
previously on the effects of human
exposure to pollutants, singly and in
combination. A unique environmental
control facility combined with an
interdisciplinary research team pro-
vides the capability to conduct this
research.
The basic design of studies of pollu-
tant exposure should seek to maximize
information relevant to public health.
The tests for effects of smog must be
reliable and sensitive, the experimen-
tal atmospheric environment must be
rigorously controlled and; equally im-
portant, the manner in which subjects
are exposed to the experimental envi-
ronment must realistically simulate
actual air pollution exposures if
results are to be relevant. These
constraints impose complications in
experimental design and necessitate
focusing attention on several distinct
problems: environmental control, pol-
lutant generation, environmental
monitoring, physiological testing, and
evaluation of symptoms and clinical
observations. The following sections
describe approaches to each of these
problems.
FACILITIES
Environmental Chamber
Studies are performed in the Ran-
cho Los Amigos Clinical Environmen-
tal Stress Testing Laboratory. This
facility consists of a stainless steel-
sheathed controlled environment
chamber, approximately 28 sq m in
area, accessible through a double-door
lock compartment that contains lava-
tory facilities and through which air is
exhausted. The main chamber con-
tains physiological test equipment
and can hold five subjects at the same
time. Data recording and monitoring
equipment are located outside the
chamber in an adjacent laboratory
area. Air flows in an agproximately
laminar manner through the main
chamber from ceiling to floor at a rate
that provides a complete change of air
every five minutes, and is then
exhausted without recirculation. The
air is highly purified and can be
adjusted to simulate a wide range of
meteorological conditions (Table I).
The air purification unit (Mine Safety
Appliances, Inc.) contains high-effi-
ciency particulate filters, a catalytic
oxidation unit for conversion of
carbon monoxide and hydrocarbons to
carbon dioxide, and chemical filters
containing activated charcoal and alu-
minum oxide pellets impregnated
with potassium permanganate (Pura-
fil, Inc.). The air-conditioning unit
consists of refrigerant coils, heaters,
and steam injectors, controlled auto-
matically to maintain desired levels of
temperature and humidity.
Pollutant Generation
Each pollutant gas is introduced
through its own stainless steel inlet
line into the inlet duct for purified air.
Complete mixing occurs before the air
reaches the main chamber, producing
uniform concentrations throughout
the chamber (within 5% of the mean
value). Each inlet line incorporates a
safety device that stops generation if
electric power fails. Carbon monoxide,
nitric oxide, nitrogen dioxide, sulfur
dioxide, and ozone have been studied.
The CO may be introduced from a
cylinder of pure gas with appropriate
flowmeter. The NO and S02 are simi-
larly introduced from cylinders in the
form of nitrogen-diluted mixtures.
374 Arch Environ Health/Vol 30, Aug 1975
Health and Pollutants I 'Hackney et al
-------
The NO, is introduced by bubbling
nitrogen gas through a cylinder of
liquid N204 and metering the result-
ing N..-NO., mixture through a special-
ly c'esigned flow control apparatus.
Ozone is generated from dried and
filtered air using a high-voltage
discharge ozonator (Welsbach T-408).
No contaminating nitrogen oxides are
produced when this technique is
employed. All pollutants can be gener-
ated in concentration ranges realistic-
ally simulating ambient conditions
and concentrations can be controlled
to within 10% of the expected value.
Environmental Monitoring
The chamber atmosphere is moni-
tored continuously utilizing instru-
ments and techniques equivalent to
those used in ambient air monitoring
networks. Instruments are calibrated
as recommended by the California
Department of Public Health and the
California Air Resources Board.''
Cross comparisons of ozone moni-
toring techniques are made with
analytical laboratories of the latter
agency. When it is possible, two
different monitoring instruments are
used for each gaseous pollutant under
study. Ozone and nitrogen oxides are
monitored using chemiluminesctnt
analyzers (Models 612 and 642, REM
Scientific), which provide fast re-
sponse and freedom from interference
by other pollutants. Total oxidants (ie,
ozone) and nitrogen oxides are also
monitored by the neutral buffered
potassium iodide solution and Saltz-
man reagent methods, using contin-
uous-flow colorimetric analyzers
(Beckrnan K-76). Carbon monoxide is
monitored by a nondispersive infrared
analyzer (Mine Safety Appliances)
and by an oxidative electrochemical
analyzer (Model 2100, Energetics
Science, Inc.). Sulfur dioxide is moni-
tored using a flame photometric
analyzer (Meloy SA-1GO). A light-scat-
tering, single-particle cojnu-r (Royco
Instruments, Mode! 225) monitors
part'culates in five subrange; be-
tween 0.5/i and 10/j in diameter.
METHODS
Physiological Testing
An initial target o'' any air pollut-
ant challenge is the respiratory tract,
which i.- thus the center o'" attention
in test; of effects of exposure. Other
aret's of interest include hematology,
blood enzyme biochemistry, and psy-
chomotor performance. Insult by pol-
lutants ran be manifested at various
sHes in the respiratory system. Bron-
choconstriction in the large airways,
maldistribution of ventilation due to
hypersecretion in small airways, con-
striction of alveolar units, and impair-
ment of diffusion as a result of edema
are possible effects. A variety of
pulmonary tests is required to exam-
ine the various possibilities; those
employed in this study are described
below.
Flow-Volume Curves.—These are re-
corded using a low-resistance spirom-
eter (Electro-Med 780). Partial and
maximum forced expiratory maneu-
vers are performed. Partial forced
expirations are initiated at 65'/r of
vital capacity. These tests may be
affected more by mild bronchocon-
striction than are full-vital capacity
forced expirations.10 The functions
measured are forced vital capacity
(FVC), one-second forced expiratory
volume (FEV,), peak expiratory flow
rate (Vmax), and flow rates at 50'?
and 25f! FVC (V,,,, V,,) for partial and
maximum flow-volume curves. These
measurements give an easily ob-
tained, relatively reproducible evalua-
tion of overall pulmonary mechanical
performance, but provide little infor-
mation on the mechanisms respon-
sible for any observed changes.
Airway Resistance (Ra») and Thoracic
Gas Volume (TGV).-These are deter-
mined in a whole-body plethysmo-
graph using the method of DuBois et
;il." 1J The measurement of R,^. is
more sensitive to bronchoconstriction
than maximum-flow measurements,
but the test is more difficult to
perform and less stable. These prob-
lems similarly affect the measure-
ment of TGV which, however, may be
useful for detecting gas trapped as a
consequence of airways dysfunction
(in combination with a gas-dilution
lung-volume determination).
Total Respiratory Resistance (R,).—
This is determined by the forced oscil-
lation technique." The method of
Goldman" is used to eliminate the
need to achieve or simulate resonance.
To eliminate the phase shift intro-
duced by Fleisch pneumotachograph
at higher frequencies,'- phase com-
pensation is used to ensure correct
relationships of the flow and pressure
signals. Resistance is measured at
pressure perturbation frequencies of
3,6, 9, and 12 hertz. This measurement
is affected by changes in upper-
airway configuration, which may com-
plicate detection of changes in pul-
monary airways per se. The method is
believed to be capable of detecting
asynchronous mechanical behavior
(unequal regional ventilatory time
constants) as predicted by Otis et al,"'
which otherwise can be documented
only by the considerably more diffi-
cult measurement of dynamic lung
compliance.
Closing Volume (CV).- This is deter-
mined by the single-breath nitrogen
washout method17 using a linear nitro-
gen analyzer (Med-Science 505). This
test is believed to be sensitive to
changes in small airways in depen-
dent lung regions. It determines the
lung volume at which closure of a
great number of small airways pre-
sumably occurs, and also provides an
estimate of residual volume (RV) and •
total lung capacity (TLQ through the
expired nitrogen concentration1" and
an estimate of the uniformity of
ventilation distribution through the
slope of the alveolar plateau.1"
Static and Dynamic Lung Compliance
(C,, €,,„).-These are measured from
recordings of transpulmonary pres-
sure and respiratory flow and volume.
Transpulmonary pressure is measured
by the esophageal balloon method of
Milic-Emili et al.'" Flow at the mouth
is measured by a pneumotachograph
(Fleisch) and volume by a spirometer
(Electro-Med 780). Adequate dynamic
response of the system has been
verified at frequencies up to 100
breaths/mm. Dynamic compliance in
the tidal range is measured at normal
breathing frequency and at 20, 40, 60,
80, and 100 breaths/min with tidal
volume monitored and kept constant
at 0.75 liter. Static compliance is
measured by closing a rnouth shutter
intermittently during an inspiration
from functional residual capacity
(FRC) t.i TLC. followed by an expira-
tutn to RV Static compliance determi-
Aich Environ Health/voi 30, Aug 1975
Hoalih a''d Pollutants I /Hackney et al 375
16.
-------
nations are preceded by inspiration-:
to TLC to give a consistent volume-
history.
Compliance measurements are in-
dispensable for delineation of changes
in the mechanical characteristics of
the lung, particularly the develop-
ment of unequal time constants.
Unfortunately, the measurements are
somewhat unstable and require con-
siderable effort. In this study these
tests are performed only on a
subgroup of subjects selected for
motivation and performance.
Pulmonary Diffusing Capacity
(DLeo).-This is determined by the
single-breath carbon monoxide meth-
od.21 In the calculation of DL, „ correc-
tion is made for back pressure of CO
due to substantial levels of carbon
monoxide hemoglobin in the blood of
some subjects. Reproducibility of this
test is poor under conditions of this
study (heat and intermittent exer-
cise), but the test offers the possibility
of detecting changes in the blood-air
interface (such as alveolar edema)
that might otherwise go undetected.
Oxygen Consumption.—This is meas-
ured at rest and during exercise on a
constant-load bicycle ergometer (Mod-
el 844, Quinton Instruments) at a level
yielding 65^ of predicted maximum
oxygen consumption.-- Expired air is
collected in meteorological balloons
and emptied into a spirometer (Collins
120-liter) to determine total expired
volume. Gas samples are analyzed for
oxygen using a paramagnetic analyz-
er (Beckman E-2) and for carbon
dioxide using a gas chromatograph
(Beckman GC-M). Oxygen consump-
tions are calculated after the method
of Consolazio et al.J1 A telemetry
system (Spacelabs, Inc.) records an
exercise electrocardiogram during
this test.
Carboxyhemoglobin Concentration
(COHb).-This is estimated from CO
concentration in alveolar air after a
20-second breathhold, using the meth-
od of Jones and co-workers.21 The CO
is measured with an electrochemical
analyzer (Model 2100, Energetics
Science). The test is performed prior
to exposure in the chamber to verify
that the subject has not received an
inordinate ambient pollutant expo-
sure and performed again at the
Table 2. — txuenmental Protocol
Overhall Exposure Schedule
Week 1, O3. weel- 2, 03 J- N02, week 3, 03 + NO2 + CO
Weekly Schedule
Monday, Tuesday
Thursday, Friday
(Wednesday)' Cortrol (clean air)
Pollutant exposure,
4 sjbjects
4 subjects
Daily Schedule
Subject No.
1
2
3
4
Begin Exposure Begin Test Cycle
0700 hr 0845 hr
0800 0945
0900 1045
1000 1145
Individual Subject Schedule
Time, hr, min
-0-01
0.00
1.45
2 00
2:05
2:10
2.15
2:22
2-40
2:58
3:00
3 15
3:20
Procedure
COHb
Enter chamber, exercise first 15 mm of each half hour
Last rest period, psychomotor performance test*
Respiratory resistance (forced oscillation)
Flow volume maneuvers
Closing volume
Body plethysmography*
Lung compliance*
Exercise testing*
COHb
OLCO*
Exit chamber, venous blood sample
Physician interview and examination
* Deleted in abbreviated test protocol.
conclusion of the chamber exposure
period.
Symptomatology.—Immediately af-
ter exposure each subject is ques-
tioned concerning symptoms by the
project physician according to a stan-
dard questionnaire. Subjects also keep
a standard record of symptoms during
and after exposure. Caution is exer-
cised in interpreting symptoms since
these are not blind studies (see Exper-
imental Protocol).
Clinical.—An attending physician is
present in the chamber area during
the study and is able to view its
progress by means of closed-circuit
television. In addition, heart rate, and
ECG are monitored via telemetry.
Equipment necessary for treatment
of an acute cardiovascular or respira-
tory emergency is maintained in the
chamber area.
Development of substantial chest
pain during exercise, cardiac irritabil-
ity, intractable wheezing, or ECG
changes in any subject, constitutes an
indication for termination of the
study for that subject and a thorough
clinical examination, with subsequent
treatment, if indicated. In the event
untoward symptoms or intercurrent
illness occurs and the subject is willing
to continue, the attending physician
makes the ultimate decision as to
whether or not a subject is to persist
in the study.
EXPERIMENTAL PROTOCOL
The exposure protocol has been
designed to simulate as realistically as
possible the ambient exposure of a
person working outdoors on a smoggy
summer day. A two-hour exposure
period is realistic in that high ambient
pollutant concentrations usually per-
sist about that length of time. Inter-
mittent light exercise (sufficient to
approximately double mir,ute volume)
during exposure gives a realistic level
of ventilation (to which pollutant dose
is proportional) during work. Elevated
temperature is an additional stress
factor frequently present during air
pollution episodes, and consequently
introduced into the experimental
situation. The design provides for
exposures on successive days, as dele-
terious effects of exposure may be
cumulative. These requirements are
incorporated into the protocol in a
cost-effective manner that tests sev-
eral subjects on a given day and
requires staggered exposure and test-
ing periods, precluding blind studies
or control measurements on the same
day. Thus, since two or three sham
control runs (exposures to purified
air) precede the pollutant exposure
376 Arch Environ Health/Vol 30, Aug 1975
Health and Pollutants I /Hackney et al
-------
reliable base line values of the meas-
ured functions can be obtained.
A test series may be reasonably
designed to support or reject the null
hvpothcftis that no effects of pollutant
exposure at realistic levels will be
detected in subjects. Results support-
ing or rejecting the null hypothesis
are useful in recommending air pollu-
tion standards. This approach pro-
vides a simple method to test for
combined effects of two or more pol-
lutants: a single pollutant is tested
initially, a second pollutant is added to
the first in the next test cycle. Expo-
sure times may also be increased to
simulate two days of pollutant expo-
sure in one day's testing. If no effects
are found, even under the "worst
case" exposure conditions, valuable
information relevant to standard-
setting may be obtained. If effects are
found at some point, additional stud-
ies will be required to determine if
they are attributable to a single pol-
lutant, to a combined effect, or to
cumulative effects of repeated expo-
sures. If minimal effects are found in
"normal" subjects, "hyperreactive"
?uHtH.-ts are studied as the next step.
These subjects are characterized by a
prestudy history of cough, chest
discomfort, or wheezing, associated
with allergy or exposure to air pollu-
tion.
Table 2 describes the detailed ex-
perimental protocol incorporating the
features described. A shorter protocol,
eliminating certain tests (marked
with an asterisk), is also used. This
protocol retains tests considered rela-
tively simple to perform and likely to
detect effects of exposure. It requires
less training of subjects and thus
uIlovNi study of subject groups more
representative of larger populations.
Statistical Analysis
Experimental data are subjected to
repeated one-way variance analyses.
Post hoc comparisons using "he New-
man-Kuels test-'1 are made when
significant F values are found For
each measurement comparisons are
made among controls, first day of
exposure, and second day of exposure,
for each subject as well as each subject
group A few significant differences
due to random variation mav be found
because of the number of statistical
comparisons being Ti.aue; therefore,
all observed statistically significant
changes must be examined critically
for phjsioiogical importance.
Subjects
Ethical and legal considerations
require that the utmost care be exer-
cised in human experimentation. Risk
is inherent in this work as well as in
other research,-6 but can be mini-
mized by taking all reasonable precau-
tions consistent with satisfactory per-
formance of the study.
The investigators serve as the first
subjects for each exposure study.
Pollutant exposure levels selected nev-
er exceed the highest recorded am-
bient levels. The exposure environ-
ment and the subjects are constantly
monitored and immediate corrective
measures are taken if hazardous
conditions are encountered.
Prospective subjects are screened
by a physician who is not associated
with the study. A standardized psy-
chological evaluation (Minnesota Mul-
tiphasic Personality Inventory)27 is
administered on physician request.
After a full explanation of the proce-
dure, including known risks and
discomfort, informed consent is ob-
tained. All subjects receive a small
reimbursement for their expenses and
RESULTS
In comparison to previous experi-
mental studies,'" more rigorous con-
trol of experimental variables (pollut-
ant concentration, background air
purity, temperature, and humidity)
was possible in the present study.
Also, more comprehensive biological
information was sought through use
of the present experimental design.
Additional measures included detailed
tests of lung mechanics as well as
biochemical and behavioral studies.
During and after exposure, clinical
responses were described according to
a standard format by both the subject
and the project physician. Details of
the following subject variables are
reported: age, sex, number of non-
smokers, number and type of smokers,
health status, socioeconomic level, any
known unusual ambient pollution ex-
posure history during the f-xperi-
ment-this included estimation of
blood carbon monoxide hemoglobin
before each run to help screen for
unknown excessive exposure.
A combination of air pollutants of
known or suspected health importance
was selected. Concentrations were
chosen on the basis of recorded
ambient levels for metropolitan Los
Angeles and adjacent areas. Duration
of pollution exposure and associated
environmental temperature and hu-
midity were also surveyed and consid-
ered in the design. The resulting
protocol as described in this communi-
cation represents an attempt to mimic
"worst case" conditions of summer
exposure in the area. Four-hour expo-
sure periods were chosen to compress
two successive daily episodes of two-
hours each. Thus, four hours exposure
on two successive exposure days were
used to mimic actual pollution epi-
sodes of up to four days' duration.
Although the goal was to use a combi-
nation of pollutant gases, considera-
tions of medical safety directed that
effects of the potentially most toxic
gas be established before adding
others. Because, in the concentrations
chosen, 0, was considered potentially
the most toxic, it was tested the first
week; N02 was added the second week
and CO the third.
We have conducted a series of ex-
perimental studies using this core
protocol. Detailed findings on our first
eight subjects exposed to ozone at
realistic levels, alone and in combina-
tion with other pollutants, are de-
scribed in a second article in this
issue.'" The definite symptoms and
functional decrement found in some
of these subjects indicated that the
exposure time should be shortened.
Therefore the core protocol was mod-
ified to provide a two-hour instead of
a four-hour exposure period. Results
of studies on additional subjects using
the shortened exposure time are pre-
sented in the third article.1' Several
general observations that are docu-
mented in the accompanying articles
are of interest and will be described
here:
1 At least sor>v lightly exercising
individuals develop discomfort and
measurable effects when exposed to
Arcri Environ Health/Vol 30, Aug 1975
Hedkh &nd Pollutants I /Hackney el ai 377
18.
-------
realistic concentrations of ozone.
2. Some individuals are not notice-
ably affected by ozone doses two or
three times greater than those at
which other individuals experience
symptoms and measurable respira-
tor}' dysfunction. Thus, if only group
comparisons are made, risks to more
sensitive individuals may go unde-
tected—a matter of concern in experi-
mental and in epidemiologic studies.
3. A striking result in exposure
studies so far conducted is that, gener-
ally, pulmonary tests that are simplest
to perform are most reliable in
demonstrating changes. "Reliability"
in this sense means that changes
observed after exposure are signifi-
cant compared to the normal test-
to-test variability under control con-
ditions. These tests include FVC,
PEV,, V,,,, Rt, and the slope of the
alveolar plateau of the closing-volume
tracing. Thus, more complex tests
may not be essential to studies
concerned primarily with verifying
exposure effects.
Experimental studies of effects of
air pollutants on human health are by
nature controversial. Results will he
closely examined by proponents of
both lower and higher pollution stan-
dards, as well as by representatives of
various nonmedical disciplines. We
report here in detail the experimental
approach to a comprehensive investi-
gation of air pollutant health effects
and the scientific and practical ration-
ale for this approach. We have, in
addition, filed comprehensive data
tables with the sponsoring agencies in
the form of an "Operational Summa-
ry." We hope this detailed documenta-
tion can help to dispel doubts concern-
ing the reliability of pollutant expo-
sure studies and promote progress in
this field.
This study was supported by a contract from
the California Air Resources Boi.rd, No 2-372, a
National Heart and Lung Institute Specialized
Center of Research grant, HL ES 1509?, and an
Environmental Protection Agency grant, R-
801396.
Charles E. Spier, BS, Leonard Wightman, Julie
Patterson, MS, Howard Greenherg, BS, and
Melvin White gave technical assistance Dale
Hutchinson, PhD (deceased), and other staff
members and consultants of the California Air
Resources Board aided in this study.
1. Chambers LA: Classification and extent of
air pollution. Air Pollution. New York, Academic
Press Inc, 1968, vol 1, pp 1-21
2. Edmonton Air Pollution Surrey, Dei-ember
1969-Norember 1970. Edmonton, Canada, Envi-
ronmental Health Service Division, Alberta
Department of Health, 1971.
3. Atkins DHF, Cox RA, Eggleton AEJ: Photo-
chemical ozone and sulphuric acid aerosol forma-
tion in the atmosphere over Southern England.
Nature 235:372-376, 1972.
4 Air Quality Criteria for Photochemical
Oxidants, NAPCA #AP-63, pp 9-13, section 4A,
col 1. paragraph 1.
5 Bates DV, Bell G, Burnham C, et al Prob-
lems in studies of human exposure to air pollut-
ants, Can Med ASM J 103:833-837, 1970
6 Bates DV. Air pollutants and the human
lung Am Rev Respir Di* 105.1, 1972
7 Bates DV, Bell GM, Burnham CD, et al:
Short-term effects of ozone on the lung J Appl
Phytwl 32:176-181, 1972
8. Hazucha M, Silverman F, Parent C, et al
Pulmonary function in man after short-term
exposure to ozone An:h Ein'iror* Health 27 183-
188, 1973.
9 Recommended Methods Berkeley, Calif, Air
and Industrial Hygiene Laboratory California
Department of Public Health, 1968.
10. Bouhuys A, Hunt VR. Kim BM, et al
Maximum expiratory flow rates in induced bron-
choconstriction in man. J Clin Inre^t 48-1159-
1168, 1969.
11 Du Bois AB, Botelho SY, Bedell GN, et al-
References
Rapid plethysmographic method for measuring
thoracic gas volume ./ OIH Imn,t 35:322-326,
1956
12 Du Bois AB, Botelho SY, et al New method
for measuring airway resistance in man using a
bod} plethysmograph J Clin Im-ei.t 35:327-335,
1956
13 Du Bois AB, Brody AW, Lewis DH, et al
Oscillation mechanics of lungs and chest in man
J Appl Phy-v>l 8:587-594, 1956.
14 Goldman MD, Knudson RJ, Mead J, et al
Simplified measurement of respiratorj resist-
ance by forced oscillation ./ App! Phyt-iol 28 113,
1970
15 Yamashiro SM, Karuza SK, Hackney JD
Phase compensation of Fleisch pneurnotacho-
graphs J Appl Pht/nol 36.493-495, 1974
16 Otis AB, McKerrow CB, Bartlett RA, et al:
Mechanical factors in distribution of pulmonary
ventilation ./ Appl Phyt>u,l 8427-443, 1958
17 Anthonisen NR, Danson J, Robertson PS,
et al Airway closure as a function of age Hi-^pir
P}iy,ml 8 58^65, 1969-1970
18 Buist AS, Ross BB Predicted \alues for
closing volumes using a modified single-breath
nitrogen test Am Rec Re^pir Lh-, 107744-752,
1973
19 Buist AS, Ross BB Quantitatue analysis
of the alveolar plateau in the diagnosis of earlj
airway obstruction A m Rev Re^pi r Dit, 108 1078-
1087, 1973.
20 Milic-Emili J, Mead J, Turner JM, et al
Improved technique for estimating pleural pres-
sure from esophageal balloons J Appl Phy^u/l
19:207-211, 1964
21 Ogilvie CM, Forster RE, Blakemore WS, et
al: A standardized breathholding technique for
clinical measurement of the diffusing capacity of
the lung for carbon monoxide ./ Chn Incest
36.1-17, 1957
22. Astrand PO, Ryhming I. A nomogram for
calculation of aerobic capacity (physical fitness)
from pulse rate during submaximal work •/ Appl
Physiol 7-218-221, 1954-1955.
23. Consolazio CF, Johnson RF, Pecora LJ
Phys'ioloyieai Measurements of MetaboUt Fum-
tionx in Man New York, McGra.v-Hi]l Book Co
Inc, 1963.
24. Jones RH, Ellicott MF, Cadigan JB, et al:
The relationship between alveolar and blood
carbon monoxide concentrations during breath-
holding J Lab Clin Meil 51.553-564, 1958.
25. Winer BJ Stuti-,tii
-------
Reprinted from the Archi /es c-f Environaipntal Health
August 19/5, Volume 31
Copyright 1975, Aniern.an Medical Association
Experimental Studies on
Human Health Effects
of Air Pollutants
II. Four-Hour Exposure to Ozone Alone and in
Combination With Other Pollutant Gases
Jack D. Hackney, MD; William S. Linn, MA; John G Mohler MD;
E. Eugene Pedersen, PhD; Peter Breisaeher, PhD; Anthony Russo, MD
Eight adult male volunteers were ex-
posed to ozone (O ) alone and then in
combination with nitrogen dioxide and
carbon monoxide under conditions simu-
lating ambient air pollution exposures.
Four "normal" men showed few or no
effects from repeated exposures. Four
Ozone (03) is a major component of
photochemical smog. High smog
levels occur frequently in the Los
Angeles region and are reported in
many other urban areas. Other pollu-
tant gases include nitrogen dioxide
(XOj and organic peroxides.' Ozone is
one of the most powerful oxidizing
agents in smog, and its well-known
toxicity in industrial exposures
makes it a pollutant of great concern
in air quality protection. Bates and his
co-workers' and Hazucha et a!' have
reported that normal adults perform-
ing intermittent light exercise devel-
oped marked pulmonary function
decrement from two hours' exposure
to 0 at concentration levels of 0.75
ppm, and measurable decrement from
similar exposures at 0.37 ppm. For
comparison, the highest one-hour av-
Submitted for publication Oct ^4 1974. accept-
ed Jan 2, 1975.
From the Environmental Health Laboratories,
Kancho Los Amigos Hospital, Downey. Calif (Dr
Haikni\. Mr Linn, and Dr Ped< r^en), the
Department of Pulmonan Medicine, Los \n-
gele.-Couatj -T S C Medical Center, Lo., \nxeles
(Dr Moh'en, the Chemical Kinetics Lahorniones,
the Aerospace Corporation, El Se^urdo, Calif
1, Medka! Science Bld^,
Ranho Los Amigos Hospital, 7601 E Imperial
Hw\. Downey, CA 90242 (Dr Hackne> i
male volunteers with a history of "hyper-
reactive" airways, but with normal base
line pulmonary function spirometric stud-
ies, after O exposure developed definite
symptoms and decrement in pulmonary
function.
erage oxidant concentration reported
in the Los Angeles area between 1963
and 1973^ was 0.71 ppm. On the other
hand, subjective experience in the Los
Angeles area suggests that the major-
ity of citizens are not greatly affected
by ambient oxidant concentrations
near 0.5 ppm. Furthermore, other
experimental studies of exposure'17
have not found marked effects or
changes in spirometric measures at
concentrations of 0.5 ppm and below.
Possible explanations for these appar-
ent discrepancies include differences
in exposure time, subject activity, and
subject sensitivity, as well as the
possible presence of interfering sub-
stances in the environment. The pres-
ent study was undertaken to repeat
the previous work under more highly
controlled conditions and to expand
the scope of investigation to include
biochemical tests of blood and tests of
psychomotor performance. The expe-
rimental plan called for studies of
"normal" subjects first, and then
testing suspected "hyperreactive"'
subjects if only minimal effects occur-
red in normals.
METHODS
The detailed experimental protocol and
rationale are given in the preceding arti-
cle." Four normal male volunteer subjects
(with no prestudy history of cough, ihest
discomfort, or wheezing associated with
allergy or exposure to air pollution) were
recruited from the project investigators or
technical staff (designated group 1) and
were tested five days per week for three
successive weeks. The first three days of
each iveek were devoted to control runs
(exposure to purified air); pollutant expo-
sures took place on the fourth and fifth
days. Conditions were designed to simulate
a composite of extreme ambient conditions
experienced during a Los Angeles summer
day. Exposures were: first week, 0.50 ppm
0,; second week, 0.50 ppm 0, plus 0.30 ppm
N0_,; third week, 0.50 ppm 0,, plus 0.30 ppm
NO,, plus 30 ppm carbon monoxide (CO).
The variability of the mean concentration
of each pollutant over different exposure
days was ±5% of the nominal value.
During all exposures maximum variation
of pollutant concentrations was ± 0.06
ppm for 0 and NO,, and ± 3 ppm for CO.
Temperature was held at 31 C and relative
humdity at 35%. Each exposure lasted four
hours before testing was started and
continued during the following hour, or for
the period required to complete testing.
Although similar ambient conditions occur
in the Los Angeles region, the exposure
time was approximately twice as long as
commonly experienced during severe am-
bient pollution episodes. This exposure was
designed to support or reject the null hypo-
thesis that no effects would be found under
the "worst" applied stress. Functions eval-
uated were forced vital capacity (FVC),
one-second forced expiratory volume
(FEV,), partial and maximum forced expir-
atory flow-volume curves, total respiratory
resistance (R,) by the forced oscillation
method, closing volume (CV), alveolar
plateau slope from single-breath nitrogen
test (delta-nitrogen or AN.,), plethysmo-
graphic thoracic gas volume (TGVj and
airway resistance (R,,,), 3-breath rebreath-
ing residual volume (RV),' blood carboxy-
hemoglobin (COHh) concentration esti-
mate, resting and exercise oxygen con-
sumption (VOj, static and dynamic lung
compliance (CSl, C,.,), and pulmonary
diffusing capacity (Du,,). Data were ana-
lyzed by repeated measures one-way anal-
ysis of variance, comparing the individual's
and the group's performance on control
days, first exposure days, and second (.suc-
cessive) exposure days. All lung volumes
are expressed as BTPS. Smokers were not
asked to alter their smoking habits, but no
smoking was permitted in the main
chamber or during testing
Because only minimal effects occurred in
the normal group, four additional male
subjects (designated group 2) were re-
cruited from tbe project investigators or
technical Maff and were tested as de-
Arch Environ Health/Vol 30, Aug 1975
Health and f-ollutants II /Hackney et al 379
20.
-------
bulbed above, extupt I'-jilu.a'jl e\p.;suie.-.
were modified. These <-i.bj«ts had normal
FVC, FEV,, and 0V, but had prestudv
histories of cough, rhoht dimvmfort. ",r
wheezing, associated with allergy or expo-
sure to air pollution Marked effects devel-
oped among some of these subjects during
the first exposure to 0 50 ppm 0, Accord-
ingly, the second 0 50 ppm exposure period
(week one, day five) was shortened to two
hours for three of the four subjects. In light
of the results in week one, the protocol was
further modified to look at dose-response
relations rather than possible effects of
additional pollutants. The subjects were
exposed to 0.25 ppm 0, on two successive
days in week two and to 0.37 ppm 0, on one
day in week three.
RESULTS
Biochemical and behavioral find-
ings are reported elsewhere E. E.
Pedersen, unpublished data.1"
Few important pulmonary function
changes or respiratory symptoms
were detected in group 1 under any
exposure condition. The physiological
importance of the changes in pulmo-
nary function data is in doubt, since
the changes occurred in the less stable
measurements (Rt, Cdyn, DLro), were
small in magnitude, and did not occur
consistently throughout all exposures.
Statistical analyses for this group are
given in Table 1. In group 2, numerous
changes were observed with 0, expo-
sure, as indicated in Table 2 and Fig 1
to 4. At 0.5 ppm, all four subjects
developed marked respiratory symp-
toms and physiological changes, and
felt physically ill to the extent that
they were unable to perform normal
tasks adequately. No such effects
were manifested at 0.25 ppm. At 0.37
ppm, physiological changes were rela-
tively mild but symptoms were more
severe than at 0.5 ppm.
Individual responses to exposures
were variable, but some consistent
patterns emerged. These are dis-
cussed below along with important
individual findings. Prestudy charac-
teristics of individual subjects are
given in Table 3.
FVC, FEV,, and Maximum Expiratory
Flow Rates
These functions were consistently
reduced in sensitive (group 2) sub-
jects. Contributing factors appeared
to be increased airway resistance.
ie ' To?'j|rs of Analysis of '/'Fiance for Physiological Me'jsufemenfs
•"roijp 1 F^posures 0 -= 0 5 ppm 03, N
Measure Exposure
- "jfyTJ" ~Q
ON
ONC
V5o 0
ON
ONC
V25 0
ON
ONC
RV, single breath 0
ON
ONC
TLC, single breath 0
ON
ONC
DLCO 0
ON
ONC
Rt, 3 hz O
ON
ONC
CV O
ON
ONC
Cd,n. normal frequency O
ON
ONC
Cd,n, 60/mm 0
ON
ONC
Cd,n, 100/min O
ON
ONC
C!t.t 0
ON
ONC
TGV (RV) 0
ON
ONC
TGV (TLC) O
ON
ONC
0 3 ppm NO2;
f
204~~
4 17
<1 0
1 .25
<1 0
<1.0
<1 0
<1 0
n/a
1 18
<1.0
7.73
6.79
<1.0
<1.Q
11.1
<1.0
159
<1.0
<1.0
795
<1.0
3 12
<1.0
<1 0
<1.0
1.79
5.62
<1 0
242
1 45
<1.0
<1.0
<1.0
1 06
<1 0
5 67
407
<1 0
547
5 11
206
0 -- 30
dft
2,6
2,6
?,6
2,6
2,5
2,6
2,6
2,6
n/a
2,6
2,6
2,6
2,6
2,6
2,6
2,22
2,22
2,22
2,6
2,6
2,6
2,22
2,22
2,22
2,78
2,72
2,72
2,78
2,72
2,72
2,78
2,78
2,78
2,22
2,22
2,22
2,16
2,16
2,16
2,16
2,16
2,16
ppn CO
Pi
NS
NS
NS
NS
NS
NS
NS
NS
n/a
NS
NS
<05§
<05||
NS
NS
<.oiu
NS
<01||
NS
NS
<.05||,#
NS
NS
NS
NS
NS
NS
-------
Table 2. — Mean Values
and Results of Variance Analysis for Physiological
Measures: Group 2
Measure 0, Exposure Control
FVC 50
25
37
FEV, 50
25
37
V50 50
25
37
V25 50
.25
37
V50, partial 50
25
37
V25, partial 50
25
37
CC 50
25
37
AN2 .50
.25
37
TLC, single br 50
25
37
RV, single br 50
25
37
Rs (FRC) .50
25
.37
Cj -, 50
normal frequency 25
37
C~ ., 100/mm 50
25
37
Dtco .50
25
37
503
508
507
396
407
407
432
409
4 15
1.99
1.72
1 80
455
466
442
1 83
200
1 62
2 35
233
2 21
073
0.67
069
684
691
685
1 69
1 66
1 60
1 26
1 09
1 35
196
.241
224
.179
184
.173
41 8
41 4
40 1
Exposure 1
455§
4 94
5 04
3 54j
4.03
395
342§
424
4 10
1 50
1 87
1..8
3.14§
456
3.52
1.15|'
1.71
1 39
2 27
236
228
1.60§
071
0.84
642
685
683
1 88
1.64
1.74§
1.70
1 24
1.49
.187
227
222
153
.189
127
424
404
45.4I!
Exposure 2
469§
5 13
None
368
4.11
None
327'§
433
None
1 20'§
242
None
3.05*§
460
None
1.19JI
1 72
None
2.47
220
None
1.03
0.63
None
6 18*§
6.77
None
1 76
1 58
None
1 65
1.11
None
None
266
None
None
172
None
38.8
49.8 i
None
F*
5 17
<1 0
<1.0
U
<1 0
2 15
406
201
<1 0
606
226
<1 0
534
<1.0
849
767
1 39
222
308
1 05
207
421
2.07
2 13
396
<1 0
<1.0
1.23
1.51
8.17
2.50
<1.0
275
<1.0
<1.0
<1 0
1 68
<1 0
307
2 18
578
194
dft
2,22
2.22
1,11
2,22
1,10
2,14
2,14
1,8
2,6
2.10
1,7
2,16
2,18
1,9
2,22
2,20
1.11
2,20
2,22
1,11
2,20
2,22
1,11
2,20
2,22
1,11
2,20
2,22
1,11
2,22
2,22
1.11
1,32
2,68
1,36
1,30
2,60
1,39
2,20
2,16
1,11
P*
<05
NS
NS
NS
NS
<05
NS
NS
<05
NS
NS
<.05
NS
<01
<.01
NS
<.01
NS
NS
NS
<.05
NS
NS
<05
NS
NS
NS
NS
<05
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
<.01
<01
* F = statistic for analysis of variance
t df — degrees of freedom for analysis of variance
~ P = probability of control-exposure difference being due to chance
§ Significant change from control, P < 05.
Significant change from control, P <.01.
F Insufficient data, some subjects unable to complete specified number of tests satis-
factorily
changes appear to be due to pain
produced by attempting to reach
extremes of lung volume.
Single-Breath Nitrogen Test
This test determines phase-4 vol-
ume (closing volume), presumably
affected by changes in dependent
peripheral airways; and phase-3 or
alveolar plateau slope (delta-nitro-
gen), presumably affected by substan-
tial changes in ventilatory distribu-
tion anywhere in the lung. No great
changes in closing volume of closing
capacity (CV plus RV) were found in
any subject in this study, whereas in
all sensitive subjects delta-nitrogen
values increased with exposure.
Total Pulmonary Resistance
Further investigation is required
before results of this test can be fully
evaluated, since normal limits and
test-to-test variability have not been
fully worked out. Substantial changes
occurred with 0.5 ppm 0, exposure,
however. Resistance determined by
forced oscillation increased at all oscil-
lation frequencies in three of four
sensitive subjects and in one "normal"
subject (6) in whom no other impor-
tant physiological changes were
found. The increases in oscillatory
resistance in the sensitive subjects
generally correlated with increases in
airway resistance measured plethys-
mographically. In the fourth sensitive
subject (10), total resistance, which
was already elevated in comparison
with other subjects, did not increase
overall but became greatly frequency-
dependent, as did dynamic lung com-
pliance (Fig 5), suggesting exposure-
related small airways dysfunction.
The frequency dependence of resist-
ance persisted through the second and
third weeks of the study; this was the
only physiological change in any
subject that appeared not to return to
base line values between the time of
exposure and the next following
control study.
Lung Compliance
Subject 7 showed a reduction in
static compliance with 0.5 ppm expo-
sure, and a corresponding drop in
dynamic compliance at all frequencies
without substantial frequency-depen-
dence. Subject 10 developed frequency
dependence as indicated previously.
No other statistically significant
changes in compliance were found.
Exercise Testing
No consistent changes in resting or
exercise oxygen consumption were
found with exposure. One normal
subject (6) developed possible angina
and minimal electrocardiogram
changes during exercise while ex-
posed to CO plus oxidants.
Pulmonary Diffusing Capacity
In both groups 1 and 2, slight
decreases in diffusing capacity oc-
curred on the second day of exposure
Arch Environ Health /Vol 30, Aug 1975
Health and Pollutants II /Hackney et ai 381
22.
-------
Table 3. — Subject Characteristics
Subject
No.
Group 1 1
3
4
6
Mean
(SD)
Group 2 7
8
9
10
Mean
(SD)
Age, yr
49
36
44
42
43
(54)
36
29
41
30
34
(5.6)
Height,
cm (Inches)
178 (70)
178 (70)
188 (74)
175 (695)
180 (71)
(5) (21)
173 (685)
172 (68)
188 (74)
172 (68)
176 (695)
7 (29)
Weight,
kg (Ib)
84 (185)
84 (185)
88 (195)
84 (185)
85 (188)
(2) (5)
70 (155)
71 (157)
80 (177)
78 (172)
74 (165)
5 (11)
Smoking
History
Pack-Years*
17
34
4|l
50fl#
0
0
20U
30H
Prestudy His-
tory of Smog
Sensitivityt
0
0
0
0
+
+
+
+
History of
Asthmat
0
0
0
0
0
0
+
-f
History of Allergy§
Personal Family
0 0
0 0
+ 0
+ 0
+ -t-
0 0
+ +
~f~ "^
* One pack year = one pack of cigarettes per day for one year.
t Defined by symptoms of cough, wheezing, or chest discomfort when outside on high pollution days (Los Angeles area) '
+ Defined by spontaneous attacks of bronchospasm requiring bronchodilator therapy separated by asymptomatic intervals
§ Defined by symptoms of wheezing, gastroenteritis, or dermatitis when in contact with specific antigens or a history of ch-onic rhinitis,
postnasal drip, or hayfever.
11 Now smokes pipe.
tl Still smoking- all others have been nonsmokers for at least four years prior to study.
# Also smokes and inhales cigars.
as compared with the first day of
exposure, when results were essen-
tially unchanged from control values.
Test results appeared less reproduc-
ible in this study than expected under
normal conditions (resting subjects,
normal room temperature).
Symptomatology
Symptoms were absent in group 1
during control runs, but some subjects
reported mild pharyngitis or subster-
nal discomfort during exposures. A
low frequency of symptoms in group 2
during control runs increased dramat-
ically with 0, exposure at 0.5 and 0.37
ppm. All four subjects experienced
cough, substernal pain, wheezing, and
malaise. Cough was generally nonpro-
ductive, but subject 7 produced a small
amount of blood-streaked sputum dur-
ing the second exposure at 0.5 ppm. Of
the sensitive subjects, only subject 9
was able to complete two successive
four-hour exposures; his symptoms
and physiological changes were only
slightly increased on the second expo-
sure day. The sensitive subjects'
symptoms generally moderated soon
after exposure was completed but
some persisted throughout the re-
mainder of the exposure day, and
sometimes throughout the next day.
The project physician assigned
symptom scores for each subject on
6-
5-
I 4H
3-
Week 1, .50 PPM
FVC
FEV
,H
Week 2, .25 PPM
I I
1—9
Week 3, .37 PPM
S EX EX S S EX EX
S EX
Fig 1 —Daily group means ( ± 1 SE) for FVC and FEV,, group 2 Open circles,control,
and black circles, O, exposure *Significant change from control, P < 05; fSigmficant
change from control, P < .01 Arrow, exposure time decreased from four to two hours in
three of the four subjects S = sham exposure, and EX, O, exposure.
each day following an interview using
a standard questionnaire. Symptoms
of cough, wheezing, sputum produc-
tion, substernal pain, dyspnea, fa-
tigue, headache, laryngitis, and nasal
discharge were scored. Each was rated
from 0 to 4 points, based on severity,
during each of the three time peri-
ods—during exposure, remainder of
the exposure day, and the following
morning. Less specific symptoms such
as malaise and muscular aches were
not scored. In group 2, symptom
scores were strongly correlated with
observed physiological changes except
in the 0.37 ppm 0, exposure, in which
symptoms were more severe than
would be expected, and out of propor-
tion to the physiological findings. The
0.37 ppm exposure was during the
third week and the severity of symp-
toms at that time was greater than for
the 0.5 ppm exposure of the first
week.
382 Arch Environ Health/Vol 30, Aug 1975
Health and Pollutants II /Hackney et al
-------
3-
Z 2-
Week 2, 25 PPM | Week 3, 37 PPM
S S S EX EX S S EX EX S S EX
6-
~ 4H
2-
Week 1, 50 PPM
| Week 2, 25 PPM | Week 3, 37 PM
S EX EX S S EX EX
S EX
Fig 3.— Daily group means ( ± 1 SE) for lung volumes calculated from single-breath
nitrogen tracings. Open circles, control, and black circles O( exposure *Sigmficant
change from control, P < 05. Arrow, exposure time decreased from four to two hours in
three of the four subjects S = sham exposure, and EX, O, exposure
30-
20-
Week 1, .50 PPM
O
Week 2, 25 PPM (Week 3, .37 PPM
EX EX S S EX EX
Symptom Scores
T
S
EX
Fig 2.—Daily group means ( ± 1 SE) for
closing capacity (CC) and delta-nitrogen
(AN2), group 2. Open circles, control, and
black circles, O, exposure. *Significant
change from control, P < 05. Arrow,
exposure time decreased from four to two
hours in three of the four subjects.
S = sham exposure, and EX, Oj expo-
sure
COMMENT
A relatively broad range of sensi-
tivity to 0, has been demonstrated,
with observed effects of exposure
correlating well with subjects' own
opinions concerning their sensitivity.
Although the number of subjects
tested is small, the results support the
hypothesis that individuals with pre-
existing pulmonary hyperreactivity
are more severely affected by expo-
sure and thus more at risk in polluted
environments. All four subjects with
any history of pulmonary hyperreac-
tivity were significantly affected by
0.37 ppm 0;, while all four subjects
without such a history were affected
minimally or not at all by 0.50 ppm Ov
In the latter subjects, addition of a
second oxidant pollutant, NO, at 0.3
ppm, did not produce additional de-
tectable effects. This does not, how-
ever, preclude the possibility that
additive or synergistic effects of
exposure to 0, and NO., in combina-
tion may occur at higher NOj concen-
trations, at higher relative humidity,
or in more sensitive subjects. Addition
of CO to the pollutant mixture also
failed to produce detectable effects
other than increases in blood carboxy-
hemoglobin levels.
A comparison of the present results
with the findings of Bates et al' and
Huzucha et al' is in order. As shown in
Table 4, the symptoms experienced
and most of the changes in pulmonary
function observed were similar in the
two studies. Discrepancies may be
related to differences in methodology
rather than in physiological responses.
One might infer that Bates' Canadian
subjects, although they had no history
Fig 4 —Daily mean symptom scores
related to O exposure for group 1 (dotted
line, week 1 only) and group 2 (solid lines)
Open circles, control, and black circles, O
exposure Arrow, exposure time decreas-
ed from four to two hours in three of four
subjects of group 2 S = sham exposure,
and EX, O exposure
Arch Environ Health/Vol 30, Aug 1975
Health and Pollutants II 'Hackney et al 383
24.
-------
02-
O
d b\ a contract from
the California Air Resources Board. No 2-372, a
National Heart and Lung Institue Specialized
Center of Research grant, HL ES 15098. and an
Environmental Protection Agency grant, R-
801396.
C E Spier, L Wightman. H Greenberg,
J Patterson, and I)r D C Lau aided with
technical assistance and consultat on
References
1 Chamhers LA Classification and extent of
air pollution, in Air Pollution New York, Aca-
demic Press Tnc, 1968, vol 1, p 1
2. Stokinger HE: E\aluation of the hazards of
ozone and oxides of nitrogen Aril Intl Health
15 181-190, 1957
3 Bates DV, Bell C, Burnham C, et al Short-
term effects of ozone on the lung / Ap/il Pky.nol
32176-181, 1972
4. Hazucha M, Silverman F. Parent C, et al
Pulmonary function in man after short-term
exposure to ozone Ar736 1949
10 Buckley RD, Hatkne.% .ID, (.lark K, et al
Ozone and Human Blood Ar<}< Eriiroii. Health
30 40-43, 1975
11. Buist AS, Ross BB Predicted values for
closing volumes using a modified -ingle-breath
nitrogen test Am Re< Rt-^pir />>- 107 "44-752,
1973
12. Du Bois AB, Botehlo SY, Comroe J H New
method for measuring airwaj res «tance in man
using a l>ody plethysmograph / Clm Inreit
35.322, 1956
13. Stokinger HE, Wagner WD, Wright HG
Studies on ozone toxicity I Potentiating effects
of exercise and toleram e dev elopment ,4 ri h hid
Health 14.158-160, 1956
14 Hazucha M, Parent C, Bates DV Combina-
tion effect of ozone and sulfur dioxide on
puirnonarv function in man F^l Pro< 33 350.
1974
384 Arch Environ Health/Vol 30, Aug 1975
25.
Health and Pollutants II /Hackney et al
nteJ 3,-jo' f'ui/.
-------
Reprinted from the Archives of Environmental Health
August 1975, Volume 31
Copyright 1975, American Medical Association
Experimental Studies on
Human Health Effects
of Air Pollutants
III. Two-Hour Exposure to Ozone Alone and in
Combination With Other Pollutant Gases
Jack D. Hackney, MD; William S. Linn, MA; David C. Law, MD; Sarunas K. Karuza, PhD;
Howard Greenberg; Ramon D. Buckley, PhD; E. Eugene Pedersen, PhD
Adult male volunteers were exposed to
ozone (O3) at 0.25, 0.37, or 0.50 ppm, and
to O3 in combination with nitrogen dioxide
(NO2) and carbon monoxide (CO), with
secondary stresses of heat, intermittent
light exercise, and repeated exposure.
Few important physiological changes,
and only mild symptoms, were found with
0.25 ppm O3, with 0.25 ppm Og plus 0.30
ppm NO2, or when 30 ppm CO was added
to the latter mixture. With 0.37 ppm O3,
Ozone (0 ) is a common photochem-
ical oxidant air pollutant that
may be an important health hazard.
Bates and co-workers' and Hazucha et
al- found that volunteers having no
history of respiratory disease and
living in an area with little oxidant
Submitted for publication Oct 24, 1974, accept-
ed Jan 1. 1975
From the Environmental Health Laboratories,,
RarH'ho Lo^ .\migub Hospital, Downey, Calif
Reprint requests to the Environmental Health
Laboratories. Rm 51, Medical Science Bldg,
Rancho Lo> Amigos Hospital, 7601 E Imperial
Hv.y, Downej, CA 90242 (Dr Hackney)
Arch Environ Health/Vol 30, Aug 1975
more symptoms were present and some
subjects developed definite decreases in
pulmonary function. With 0.50 ppm O3,
most subjects had symptoms and about
half showed substantial pulmonary func-
tion decrement.
In reactive subjects exposed on two
successive days, changes were usually
greater the second day, indicating that
effects of successive exposures were
cumulative.
pollution developed substantial respi-
ratory symptoms and function decre-
ment when exposed for two hours to
0, at concentrations as low as 0.37
ppm, with intermittent light exercise.
In previous work in this laboratory,1'
similar studies were conducted on
volunteers living in the Los Angeles
area, where ambient Oi concentra-
tions of 0.5 ppm or higher are possible.
Four "normal" subjects (without a
prestudy history of cough, chest
discomfort, or wheezing associated
with allergy or air pollution exposure)
failed to react to 0, exposure at 0.50
ppm, even when exposed for four-hour
periods on two successive days. Four
"reactive" subjects (with a prestudy
history of cough, chest discomfort, or
wheezing associated with allergy or
air pollution exposure, but with nor-
mal base line pulmonary function
studies) during one four-hour expo-
sure to 0.50 ppm 0, developed
substantial decrease in pulmonary
function and symptoms severe enough
to restrict normal activity. The same
subjects did not react appreciably to
0.25 ppm 0, but did react somewhat to
0.37 ppm 0,. The degree of pulmonary
function decrement found in reactive
subjects led us to decrease the expo-
sure time to two hours. The present
studies use a two-hour exposure time
and were undertaken to better define
the range of sensitivity and dose-
response characteristics in "normal"
and "reactive" populations. Such in-
formation is required as a basis for
setting realistic air-quality standards
for the protection of public health.
Health and Pollutants III /Hackney et al 385
26.
-------
Table 1. — Subject Characteristics
Subject
No.
3
7
8
9
10
11
12
13
14
15
16
17
18
Exposure
Group (s)
3
4
5
4,5
4,5
3,4
3
3
3
3,4
3,4
4,5
5
Age, yr
36
36
29
41
30
30
28
28
34
22
30
41
27
Height.
cm (in)
178 (70)
173 (685)
173 (68)
188 (74)
173 (68)
183 (72)
183 (72)
188 (74 5)
175 (69 5)
165 (65)
183 (72)
167 (56)
178 (70)
Weight,
kg (Ib)
84 (185)
70 (155)
71 (157)
80 (177)
78 (172)
70 (155)
75 (166)
70 (155)
68 (150)
58 (128)
79 (175)
64 (142)
68 (150)
Smoking
Hislory
Pack-Years'
34
0
0
?0"
3011
0
0
0
0
1?
75fl
0
0
Prestudy
History of
Smog
Sensitivityt
0
+
+
_L
+
0#
0
0
0
0#
0#
0
0
History of
Asthma!
0
0
0
-j-
4
0
0
0
0
0
0
0
0
History
Personal
0
—
0
+
_!_
0
0
+
0
+
0
0
0
of Allergy§
Family
0
+
0
+
+
0
+
+
0
+
0
0
0
* One pack year = one pack of cigarettes per day for one year
T Defined by symptoms of cough, wheezing, or chest discomfort when outside on high pollution days (Los Angeles area)
i Defined by spontaneous attacks of bronchospasm requiring bronchodilator therapy separated by asymptomatic intervals.
§ Defined by symptoms of wheezing, gastroenteritis, or dermatitis when in contact with specific antigens or a history of chronic rhinitis,
postnasal drip, or hay fever.
Now smokes pipe
H Still smoking all others have been nonsmokers for at least four years prior to study
# Following the chamber study a close revaluation of their history suggested that these subjects should be classified as smog sensitive
Table 2. — Mean Values and Results of Variance
Measurements, Group 3 (0.50 ppm
Measure
FVC
FEV,
V,.,
Vso
V25
VMP
VUP
cc
AN2
TLC, single-breath
RV, single-breath
RV, rebreathing
R(, 6 hz
DLCO
Control
5 15
446
120
595
269
642
268
1 85
068
664
1 41
1 66
339
392
Exposure 1
503
425
11 7
573
239J
606
2 54
1 81
075
6.44
1 32
1 66
352
388
Exposure 2
4.75f
3.86t
1091:
484t
220|
5.12t
221t
1.86
1 04$
597t
1 38
1 70
477f
Not available
Analysis for Physiological
03 Exposure)
F
646
7.58
3.52
733
686
135
6.68
<1 0
4.64
529
242
<1 0
5.39
df*
2,38
2,38
2,38
2.34
2,34
2,34
2.38
2,38
2,38
2,38
2,38
2,40
2,98
P
<01
<01
<05
<01
<01
<01
<01
NS§
<05
<.01
NS§
NS§
<.01
* df = degrees of freedom
t Significant change from control, P < 01
4 Significant change from control, P<05.
§ NS = change not significant at 05 level.
METHODS
The controlled-environment exposure fa-
cility and detailed experimental protocol
used for these studies have been described
previously.1 Thirteen volunteer subjects,
recruited from the project professional and
technical staff, participated in one or more
exposure groups (Table 1). Group 3, con-
sisting of seven "normal" subjects (without
a prestudy history of cough, chest discom-
fort, or wheezing associated with allergy or
air pollution exposure), was exposed to 0.50
ppm 0, in a one-week study. (Groups 1 and
2 are described in the previous report').
Groups 4 and 5 included both "normal" and
"reactive" subjects. In a three-week study,
group 4 was exposed to 0.25 ppm 0 the
first week, to 0 25 ppm 0 plus 0 30 ppm
NO, the second week, and to 025 ppm O,
plus 0 30 ppm NO, plus 30 ppm CO the third
week. Group 5 was exposed to 0.37 ppm 0
in a one-week study During each week of
studies, the first two or three days were
devoted to sham exposures (exposures to
purified air) to establish base line mea-
sures of functions, and the final two days
were devoted to pollutant exposures (the
given concentration of pollutant added to
purified air)
Each subject was exposed in the con-
trolled-environment chamber for two
hours, and then performed the battery of
physiological tests while still under expo-
sure. During the two-hour period, the first
15 minutes of every 30 was spent in exer-
cise at a level sufficient approximately to
double the volume of resting ventilation
per minute Exercise consisted of walking
briskly or riding a bicycle ergometer Each
subject also recorded symptoms on a stan-
dard form. After completing the physiolog-
ical tests, the subject left the exposure
chamber and was immediately examined
by the project physician. A venous blood
sample was taken for biochemical analysis
and the subject was interviewed for symp-
toms according to a standard question-
naire. Symptoms experienced during the
remainder of the day were also recorded on
a standard form.
Physiological results evaluated were
forced vital capacity (FVC); one-second
forced expiratory volume (FEV,); partial
and maximum forced expiratory flow-
volume curves, total respiratory resistance
by forced oscillation at 3, 6, 9, and 12 hertz
(R,); closing volume (C\), lung volume
estimates, and slope of the alveolar plateau
(AN.,/liter) from the single-breath nitrogen
test; carboxyhemoglobin (COHb) concen-
tration estimate by brea.h analysis, and
single-breath carbon monoxide diffusing
capacity (T)L,,,). Supplementary physiolog-
ical tests included plethysmographic tho-
racic gas volume (TGV) and airway resist-
386 Arch Environ Health/Vol 30, Aug 1975
Health and Pollutants Hi /Hackney et al
27.
-------
Fig 1 —Daily variation of pulmonary
functions in group 3, mean ± 1 SE S,
sham exposure, and EX = 0.5 ppm O3
exposure TLC, RV, CC, and AN2 calcu-
lated from single-breath nitrogen trac-
ings
ance (R,J (groups 4 and 5), static (CS1) and
dynamic (C,lln) lung compliance (group 5),
and residual volume (RV) by rebreathing
(group 3). Psychomotor performance was
evaluated in group 4 through measurement
of reaction time, heart rate variability, and
tracking performance, in a combined
central and peripheral tracking task (E. K.
Pedersen et al, unpublished data). Data for
each exposure group were analyzed using a
repeated measures, one-way analysis of
variance as described previously '
RESULTS
Group 3
This "normal" group showed de-
creases, after 0.5 ppm 0, exposure, in
forced expiratory measurements in-
cluding FVC, FEV,, flow rate at 50f>
FVC on maximum and partial forced
expiratory flow-volume curves (V-,,
and V,0 P, respectively), and flow-
rates on flow-volume curves at 25^
FVC (V,-, and V,, P). Closing volume
and closing capacity as measured by
the single-breath nitrogen test did not
change, but the alveolar nitrogen
plateau slope (AN,) increased, indi-
cating less uniform distribution of
ventilation as a result of exposure.
Residual volume measurement by
rebreathing nitrogen dilution' pro-
duced larger values than were ob-
tained from the single-breath nitro-
gen RV estimate, but neither meas-
urement indicated significant
changes in RV with exposure. Total
lung capacity (TLC) decreased due to
the decrease in vital capacity. Expo-
sure increased total pulmonary resis-
: snee at all oscillation frequencies.
The group changes in physiological
measurements, in many cases, did not
achieve statistical significance until
the second exposure day, and in most
cases the decrements in function were
markedly worse on the second day.
The group changes were primarily due
to subjects 11, 13, and 16. The re-
maining four subjects (including one
Fig 3 —Daily variation of lung volumes,
calculated from single-breath nitrogen
tracings, in group 4, mean ± 1 SE error
(see Fig 2 for explanation)
Liters
5-
4-1
3-
Liters
8-1
FVC
FEV
2-
Liters or
% N2/Liter
TLC
RV
B 5—-S Z I
2-
1-
CC
AN
S S S EX EX
S S S EX EX
Exposure
—I 1 1 1 1—
S S S EX EX
Liters
6-1
5-
4-
3-
1
0
FVC 5-5x2
rvu 2 :2 J £
rrv 7L -^- X B
1 LV( g- j. £ *
;
j
T .._-$— X X
2 I S I
T S- T I
^ 5— 3— -f— i
SS11 SS22 SS33
Exposure
Fig 2 —Daily variation of forced expiratory measurements in group 4, mean -t 1 SE
Exposure conditions: S = sham; 1 = 0 25 ppm O3, 2 = 0 25 ppm O3 + 0 30 ppm NO2, 3
= 0 25 ppm O + 0 30 ppm NO2 + 30 ppm CO Successive weeks of study are
separated by broken lines Subject 9 absent week three, subject 11 absent week two
Liters
6-
4-
2-
TLC
MV T£ —
1
S
— " — I *
1 1 1
S 1 1
1
S
1
S
1
2
1
1
1
2
B—
1
S
— It—
1
S
— 3f —
1
3
>
F
3
Exposure
Arch Environ Health/Voi 30, Aug 1975
Health and Pollutants III./ Hackney et al 387
28.
-------
Table 3. — Mean Values and Results of Variance Analysis for Physiologies!
Measurements, Group 4
Measure
FVC
FEV,
50
25
50
25
cc
2
RV, single-breath
TLC, single-breath
R,, 6 hz
DLco
Exposure
Condi-
tions*
0
ON
ONC
0
ON
ONC
O
ON
ONC
0
ON
ONC
O
ON
ONC
0
ON
ONC
0
ON
ONC
0
ON
ONC
O
ON
ONC
0
ON
ONC
0
ON
ONC
O
ON
ONC
Control
482
4.88
466
3.94
394
3 77
466
463
458
201
1 91
1 86
5 12
467
491
2 11
1 91
1 87
1.88
1 98
1 63
0.70
075
072
1.30
1 40
1.15
6.27
650
6 16
3.25
3 33
3.35
40.4
45.0
36.2
Exposure
1
4.80
4 88
4 64
3 95
3 96
374
4 69
447
458
207
1 85
1 77
4 91
462
493
207
1.85
1 78
1 85
1 93
1 64
0.68
074
0.75
1 37
1 38
1.11
625
653
6 14
3 83§
350
3 65
42.0
36 6§
384
Exposure
2
4.79
501§
4 68
396
4 03
379
484
448
463
2 10
1 87
1 92
5 10
492
483
2 10
1.93
1 92
1 77§
1 93
1 65
0.66
072
0.72
1 32
1 36
1 11
633
666§
6 12
307
328
354
34 2-
39 5|
38.0
F
<1.0
685
<-1 0
<1 0
409
1 67
1 50
<1 0
<1 0
<1 0
<1 0
<\ 0
1 45
2 55
<1 0
<1 0
<1 0
<1 0
10.7
<1 0
<1 0
<1 0
<1 0
<1 0
2 13
1 74
2.01
1 34
791
<1 0
7.11
<1 0
<1 0
461
6.27
<1 0
dft
2,40
2,32
2,32
2,38
2,30
2,32
2,12
2,10
2.10
2,12
2,10
2,10
2,12
2,10
2,10
2,12
2,10
2,10
2,40
2,34
2,34
2,40
2,34
2,32
2,40
2,34
2,34
2,40
2,34
2,34
2,94
2,82
2,80
2,40
2,34
2,32
P
NSt
NSt
NSt
<05
NSt
NSt
NSt
NSt
NSt
NSt
NSt
NSt
NSt
NSt
NSt
NSt
NSt
<01
NSt
NSt
NSt
NSt
NSt
NSi
NSt
NSt
NSt
< 01
NSt
<01
NSt
NSt
<05
<01
NSt
* Exposure conditions: 0 = 0.25 ppm 03; N = 0 3 ppm N02; C = 30 ppm CO
t df = degrees of freedom.
t NS = change not significant at 05 level.
§ Significant change from control, P<.01.
II Significant change from control, P<.05.
who had been exposed previously and
found nonreactive) showed few or no
changes or symptoms. Predominant
symptoms in the reactive subjects
were cough, substernal discomfort,
and malaise. Biochemical analysis re-
vealed oxidative changes in erythro-
cyte and plasma enzymes and in-
creased erythrocyte fragility."
Physiological results for group 3 are
summarized in Table 2 and Fig 1.
Group 4
This was a mixed group including
one subject unreactive by history and
previously tested (No. 17), two sub-
jects unreactive by history but reac-
tive by previous testing (No. 11 and
16), and three subjects reactive by
history and previous testing (Nos. 7, 9,
and 10). Ozone exposure concentration
was 0.25 ppm. No consistent physio-
logical changes attributable to expo-
sure and few or no important bio-
chemical changes were detected in
this exposure series, even when NO.
and CO were added to ozone. Slight
increases in R, and decreases in DL,.,,
were observed in some exposures but
not in others.
Physiological results for group 4 are
summarized in Tab'e 3 and in Pig 2 to
4.
Group 5
This group included three subjects
reactive by history and previous
testing (No. 8, 9, and 10) and two
subjects (No. 17 and 18) unreactive by
history but not tested previously at or
above the 0, concentration of 0.37
ppm. In this group important changes
with exposure in most physiological
measures were not found. However,
subject 8 showed substantial de-
creases in FVC and FEV, and
increases in R, and airway resistance
(RaJ at functional residaal capacity
(FRC) measured plelhysmographical-
ly. These changes became worse on
the second exposure da.y. For the
group, Rin> at FRC did not change
substantially with exposure, but R,
was increased above control on both
exposure days. Symptoms reported by
subject 8 were similar to those of the
reactive subjects in group 3. The
remaining subjects most frequently
reported upper-airway irritation and
coughing. Oxidative biochemical
changes were detected in blood of this
group, but were not as severe as in
groups exposed to 0.5 pprn 0,.
Physiological results for group 5 are
summarized in Table 4 and Fig 5.
COMMENT
The foregoing results, together
with those reported previoush from
this laboratory,1" allow formulation of
dose-response curves for effects of 0,
exposure (Fig 6 to 8) based on less
variable physiological and biochemical
measurements. Equation of these ob-
served responses with "important del-
eterious effects on health" is not
completely straightforward, since the
underlying mechanisms are not fully
understood. However, from a practical
standpoint the observed physiological
changes may reasonably be consid-
ered to represent important health
effects, since (a) the changes are qual-
itatively similar to those observed in
certain pulmonary diseases and (b) in
studies to date, substantial physiolog-
ical changes have always been accom-
panied by marked clinical illness (res-
piratory symptoms severe enough to
388 Arch Environ Health/Vol 30, Aug 1975
Health and Pollutants III /Hackney et al
-------
F
ty a
Fig
F
func
sha
exp
late
mgs
Lite
5-
3-
;
o
g 4 —Daily variation of closing
nd AN2m group 4, mean ± 1
2 for explanation).
g 5 —Daily variation of pu
;tions in group 5, mean ± 1 S
TI exposure, and EX = 0 37
osure. TLC, RV, CC, and AN
d from single-breath nitroge
rs Lite
8-
FVC
6-
FEV, 4-
2-
:
Liters or
% N2/Liter
31
capaci-
SE (see
2" cr I__-J— I - ? 4 1 i
1-
* X T T T I
TIT! T^3~~~l I ^_J — I— I
AN2 $ 2— JIY--J.JT. X J. -L -L
n
u I
monary S
E. S =
ppm O3
2 calcu-
n trac-
;rs Lite
% N
TLC
3-
2-
RV 1.
x -^
a — 5 — i — i
0
"T II 1 1 1 1 1 1 1 I
S11 SS22 SS33
Exposure
rs or PPM 03
20 40 60
-10- "\ ^\Mean
^N Change
X
— 20- \Maximum
Change
CC -30-
^ ^ J * -40
% Change
From Sham
AN,
Fig 6.— Dose-response behavior of FEV,
is T I T ln subjects exposed to O3 Mean and
i 1 * maximum changes from control values
observed in all subjects tested at given
concentrations
1 i I i I I I I I i i I
S S EX EX S S EX EX S S EX EX
Exposure
inhibit normal activity). While bio-
chemical changes have been observed
in asymptomatic as well as sympto-
matic subjects, the dose-response
curves for those biochemical measure-
ments expected to result in an imme-
diate response to oxidant exposure
are remarkably similar to the curves
for the physiological measurements.
The given dose-response curves are
to be considered first approximations
only, since they are based on small
samples, neglect differences in expo-
sure time, and do not distinguish
between initial and cumulative expo-
sures Responses are expressed in
units of percent change from the
appropriate control values. The mean
of individual responses and the larg-
Table 4. — Mean Values and Results
Measurements, Group
Measure
FVC
FEV,
V50
V25
V50P
VaP
CC
AN2
TLC. single-breath
R.«, FRC
R,, 6 hz
Cd»n, 20/min
Cd,n. 100/min
Control
537
4.29
494
2 15
549
2 18
1 97
080
6.81
1.40
324
0 282
0 136
Exposure 1
529
4.19
474
1 9
466
1 86
204
079
666
1 55
401±
0 288
0 130
of Variance
5 (0.37 ppm
Exposure 2
535
4.24
4.68
1 9
450
1.86
2.02
081
C.75
1 48
3.88t
0 290
0 129
Analysis for Physiological
O3 Exposure)
F
<1 0
1.11
<1 0
3 03
103
<1 0
<1 0
<\ 0
<1 0
725
<1 0
<1.0
df
2,8
2,8
2,8
(Not available)
2.8
2,8
2,8
2,28
2,26
2,28
2,26
2,80
2,98
f
NSt
NSt
NSt
NSt
NSt
NSt
NSt
NSt
NSt
<01
NSt
NSt
* df — degrees of freedom.
"t NS — not significant at .05 level.
\ Significant change from control, P < 01.
Arcn Environ Health/Vol 30, Aug 1975
Health and Pollutants III /HacKney et al 389
30.
-------
Maximum
Change
% Change
From Sham
200-
150
100-
50-
Fig 7 —Dose-response behavior of AN,
in subjects exposed to O3 Mean and
maximum changes from control values ob-
served in all subjects tested at given con-
centrations
est individual response observed at
each concentration are plotted as a
function of concentration and smooth
curves drawn through the resulting
points to generate the "mean re-
sponse" and "maximum response"
curves for the study population. Best-
fit straight lines have also been gener-
ated from the dose-response data by
least-squares calculations; these indi-
cate comparable no-effect levels but
appear to overestimate the response
at the intermediate concentration.
The mean dose-response curves sug-
gest a "no-detectable-effect" level of
0.25 to 0.30 ppm. Ambient oxidant
concentrations exceed 0.30 ppm for
one hour or more at least 20 days per
% Change
From Sham
30
Cell Fragility
Mean
Change
Acetylchohnesterase
Fig 8 —Dose-response behavior of se-
lected erythrocyte measurements in sub-
jects exposed to 03 Mean and maximum
changes from control values observed in
all subjects tested at given concentrations
Black circles, fragility of cells, and open
circles, cell membrane acetylcholinester-
ase level
year in parts of the Los Angeles area,"
and such levels are possible in many
other urban areas. Since the exposure
conditions that were studied simulate
light, outdoor, physical work, an
important and widespread public-
health risk related to 0, pollution is
implied, although the precise compa-
rability of exposures to 0 in purified
air and to complex ambient oxidant
mixtures is not known. Furthermore,
many individuals are considerably
more sensitive than the average, and
thus mav be at risk at considerably
lower levels of pollution. The degree of
risk to types of populations not
studied, such as children, the elderly,
or chronic pulmonary disease patients
remains to be determined.
This study was supported by a contract from
the California Air Resources Board, No :i-372, a
National Heart and Lung Institute Specialized
Center of Research grant, HL ES 15098, and an
Environmental Protection Agency grant, R-
801396
References
1 Bates DV, Bell G, Burnham C, et al Short-
term effects of ozone on the lung .' Ajipl Plitf.ni/
32 176-181, 1972
2. Hazucha M. Silverman F, Ps.rent C, et al
Pulmonary function in man after short-term
exposure to ozone Aril: En'inn, Hml'li 27183,
1973
3 Hackney JD, Linn % S. Buckley RD, et al
Experimental studies on hjman health effects of
air pollutants I Design considerations Arch
Environ Health 30373-378, 1975
4. Hackney JD, Linn WS, Mohler JG, et al
Experimental studies on human health effects of
air pollutants II Four-hour exposure to ozone
alone and in combination with other pollutant
gases. Arch Etu-irun Health 30-379-384, 1975
5 Rahn H, Fenn WO, Otis AB DaiK varia-
tions of vital capacity, residual air, and expira-
tory reserve, including a study of the residual air
method. ,/ Art,l I'l, ,,*,;! 1.725-736. 1949
6 Buckley RD, Hackney JD. Clark K, et al
Some effects of ozone inhalation or human eryth-
rocyte metabolism Fed 1'rix 33 334, 1974
7 Buckley RD, Hackney JD, Clark K, et al
Ozone and human blood An I' En n nm Hfultl*
30-40-43, 1975
8 California Air Resources Board. Ti,i-,f<'iir
Sti n> mo rij o1 ('n/1/1 Aml i' ij Datn. Sacra-
mento, Calif, 1974
390 Arch Environ Health/Vol 30, Aug 1975
31.
Health and Pollutants III /Hackney et al
Printed and Published in the United States ol America
-------
HEALTH EFFECTS OF OZONE EXPOSURE IN CANADIANS VS. SOUTHERN CALIFORNIANS:
EVIDENCE FOR ADAPTATION?.
RUNNING TITLE: HEALTH EFFECTS OF OZONE
J. D. Hackney, MD, W. S. Linn, MA, S. K. Karuza, PhD,
R. D. Buckley, PhD, D. C. Law, MD, D. V. Bates, MD,
M. Hazucha, MD, PhD, L. D. Pengelly, PhD, F. Silverman, PhD
SEND CORRESPONDENCE TO: Jack D. Hackney, MD
Medical Science Bldg., Room 51
Rancho Los Amigos Hospital
7601 East Imperial Highway
Downey, CA 90242
From the Specialized Center of Research in Environmental Lung Disease,
Rancho Los Amigos Hospital, Downey, California (Hackney, Linn, Karuza,
Buckley, Law); Faculty of Medicine, University of Briti sh Coiurabia,
Vancouver (Bates); Department of Physiology, McGiI I University, Montreal,
Quebec (Hazucha); Medical School, McMaster University, Hami!ton, Ontario
(Pengelly); and Gage Research Institute, Toronto, Ontario (Silverman).
32.
-------
ABSTRACT
Comparison of published reports on health effects of exposure
to ozone (03) suggests that Canadians are more reactive than
Southern Caliform'ans. Subject responses and experimental methods
were compared further in a cooperative investigation of this apparent
reactivity difference. Four Canadians and four Californians were
exposed to 0.37 ppm Og in purified air at 21°C and 50 percent
relative humidity for two hours with intermittent light exercise.
Exposures to purified air alone served as controls. Subject responses
were similar to those observed previously—Canadians on the average
showed greater clinical and physiological reactivity to exposure than
did Californians, who were no more than minimally reactive. Canadians
also showed larger increases in erythrocyte fragility following
exposure. Methodological differences sufficient to explain different
results of previous studies were not found. Although other possible
explanations have not been entirely ruled out, adaptation of Southern
Californians to chronic ambient 0-j exposure is a rational hypothesis
which can explain these results.
33.
-------
HEALTH EFFECTS OF OZONE LXPOSbRE IN CANADIANS VS. SOUTHERN CALIFORNIANS:
EVIDENCE FOR ADAPTATION?
Introduction
Previous work in our respective separate laboratories has shown
that significant adverse physiological, clinical, and biochemical
changes occur in humans exposed to ozone (03) under conditions
realistically simulating ambient exposures experienced during photo-
chemical smog episodes. Dose-response information obtained from these
studies Indicates that typical Montreal or Toronto residents show
at least twice as great a response to a given 03 dose as do typical
Los Angeles residents studied under presumably similar conditions,
when response is expressed as decrement in a pulmonary function measure-
ment such as one-second forced expiratory volume (FEV^). It was
hypothesized that differences in reactivity might be due to development
of biological adaptation in Los Angeles subjects, somewhat analogous
o
to tolerance observed in experimental animals. (We use the term
"adaptation" in relation to human studies at near-ambient 63 concentra-
tions, since "tolerance" is generally used in reference to higher
concentration animal mortality studies. "Adaptation" in this sense
implies a beneficial effect only in the short term, since its long-
term effects are unknown.) Metropolitan Los Angeles experiences more
frequent 0, pollution episodes and considerably higher 03 concentrations
than Canadian cities, although the latter are not entirely free of 03
pollution (Table 1).
Other possible explanations for the different results of Los
Angeles and Canadian studies include modification of effects of 03 by
coexisting unknown pollutants, random variation in reactivity among
individuals, selective migration of hyperreactive individuals, nutri-
tional differences (known to be relevant in animals9), genetic differences,
-------
and differences in 0., monitoring leading to higher actual doses in
the Canadian studies relative to the Los Angeles studies. Significant
differences among "standard" 03 monitoring techniques have recently
been documented, pointing up the importance of investigating the
last possibility. The hypothesis of adaptation was tested by con-
ducting a cooperative study in which human subjects, monitoring
techniques, and physiological measurement techniques used in the
different laboratories were directly compared. Goals were (a) to
confirm the difference in reactivity between Canadians and Southern
Californians by replicating previous results from the separate
laboratories, and (b) to support the adaptation hypothesis by ruling
out as completely as possible any alternative explanations for the
difference in reactivity.
Methods
Subjects were exposed to purified air (control) and to 0.37 parts
per million (ppm) 0^. Exposures lasted two hours, during which subjects
exercised for 15 minutes in every 30 at a level sufficient to increase
minute ventilation to 2-2 1/2 times the resting level. Exposure
temperature was 21 C and relative humidity was 50% ± 4%. Forced expira-
tory measurements were made at 30-minute intervals during exposure;
other physiological tests were performed at the end of the 2-hour period,
the exposure continuing during testing. After testing was complete,
exposure was stopped. Clinical examinations of the subjects were
immediately performed and venous blood drawn for biochemical studies.
Details of the physiological and biochemical testing have been described
previously. '
35.
-------
Of the exposure facilities available, the Los Angeles facility
(Rancho Los Amigos Hospital Clinical Environmental Stress Testing
Laboratory) had the most elaborate environmental control and monitoring
capability, thus it was used for the cooperative exposure study.
Four volunteer subjects, residents of Toronto and Hamilton, Ontario,
traveled to Los Angeles for the study. During their stay the subjects
remained in the coastal areas of the Los Angeles Basin to minimize
their ambient Oj exposures. A representative Canadian 03 monitoring
instrument (Mast coulometric analyzer from McMaster University) was
also brought to Los Angeles and used to monitor the exposure atmosphere
in parallel with the local instruments (REM chemiluminescent analyzer
and Beckman continuous colorimetric analyzer). All instruments were
originally calibrated to read equivalent to manual samples determined
12
using one percent neutral phosphate-buffered potassium iodide solution.
Four Los Angeles subjects, matched as closely as practical to the
Canadian subjects, were exposed separately under comparable conditions.
Both groups of subjects included smokers, nonsmokers, individuals
previously found to be reactive to 03 exposure, and individuals not
previously studied but with histories of respiratory hypersensitivity,
4
which is associated with hyperreactivity to 0_. Subject characteristics
are summarized in Table 2.
Data were analyzed by t tests or analyses of variance comparing
control and post-exposure measurements. In the physiological tests,
repeated measures on each individual were possible, thus each individual
Jb.
-------
as well as each group could be tested for significant changes. In the
biochemical analyses, only one value was available for each individual
for each condition, thus only group statistical comparisons were made.
Results
Ozone Monitoring. During the exposure of the Canadian subjects,
the mean readings of the 63 monitors were 0.37 ppm for the Los Angeles
chemiluminescent instrument, 0.34 ppm for the Los Angeles colorimetric
instrument, and 0.30 ppm for the Canadian electrochemical instrument.
In a separate experiment with no subjects in the exposure chamber, the
three instruments read respectively 0.52, 0.47, and 0.44 ppm. Standard
deviations of data recorded at fixed intervals from all instruments
did not exceed 0.02 ppm. Relative deviation of the Canadian instrument
from the Los Angeles instruments thus ranged from -6 percent to -19
percent. If these data are typical of differences in monitoring between
the Canadian and Los Angeles laboratories, monitoring differences can
explain some, but certainly not all, of the difference in reactivity
between the locations.
Clinical Response. Canadian subjects 19 and 20 developed definite
clinical illness during 03 exposure. Both experienced nonproductive
cough, substernal discomfort, and upper-airway irritation (laryngitis or
pharyngitis). The symptoms moderated after exposure was terminated, but
did not disappear for several hours. These responses were similar to
•3 c
those exhibited by these individuals when previously studied in Canada. '
Canadian subjects 21 and 22 developed upper-airway irritation during
exposure, and subject 22 also reported slight substernal discomfort.
Los Angeles subjects 7 and 23 reported upper-airway irritation during
exposure; no other exposure-related symptoms were reported by the Los
-------
Angeles subjects. The relative lack of response was consistent with
previous findings in Los Angeles subjects exposed at similar con-
centrations.
Physiological Response. Group mean physiological data are given
in Table 3 . Neither the Canadian nor the Los Angeles group
showed significant differences between control and exposure studies.
The clinically reactive Canadian subjects 19 and 20 showed significant
(P<.05) losses in FEVj, forced vital capacity (FVC), and total lung
capacity (TLC) as estimated from the single-breath nitrogen washout.
Subject 19 also exhibited a significant increase in delta nitrogen,
representing a decrease in uniformity of ventilation distribution.
FEYj -changes were partly reversed within six hours after exposure
and fully reversed after 24 hours. Los Angeles subject 7 showed a
small but significant loss in FEV^ with exposure. The other subjects
showed no statistically significant physiological changes except in
isolated cases among the less stable measurements—thoracic gas
volume and total respiratory resistance. These changes were not
considered physiologically meaningful. Significant individual changes
are summarized in Table 4.
Blood Biochemical Response. Control and post-exposure values
\
for biochemical measures expected to show immediate responses to 03
or other oxidant challenge are given in Table 5 for the groups.
Erythrocyte fragility showed the most striking difference between
Canadian and Los Angeles subjects. While both groups showed similar
decreases in activity of erythrocyte membrane acetylcholinesterase
(a typical response to 03 exposure1), only the Canadians showed a
significant increase in cell fragility with exposure. This response
-------
was fairly consistent among the four Canadians; no obvious differences
in biochemical response between the two highly reactive and the two
relatively unreactive subjects were seen (Table 6). The Canadians
also showed small but significant increases in serum vitamin E levels,
presumably the result of mobilization from body stores in response
to the oxidant challenge. A similar increase in the Los Angeles
subjects did not attain statistical significance since these
individuals were less consistent in baseline values and in response
to exposure.
-------
DISCUSSION
These results support the existence of a real difference in re-
activity to 03 between Southern Californians and Canadians studied to
date. While the number of subjects tested in the present study is small,
the good agreement between present results and previous results in the
separate laboratories allows considerably increased confidence in
direct comparison of the previous results from Los Angeles and Canada,
providing a considerably larger data base from which to judge relative
reactivity. Such comparisons are given in Table 7 and in Figure 1.
Table 7 gives the mean reactivity in 0.37 ppm 03 exposures (expressed
as loss in FEV^) of the present subject groups, the entire group of
Los Angeles subjects studied to date, and a representative larger group
of Montreal subjects. While reactivity is highly variable among different
individuals, both the Canadians studied in Montreal and the Canadians in
the present study show greater mean reactivity than any Los Angeles group,
even a group of relatively sensitive subjects exposed for twice as long
as the other groups. Furthermore, if only the studies done in Los Angeles
are considered, without reference to the Canadian studies, the two highly
reactive Canadian subjects in the present study were far
more reactive than any Los Angeles subject studied under similar
conditions—about as reactive at 0.37 ppm as the most sensitive Los Angeles
subjects exposed at 0.50 ppm, and with FEV^ changes exceeding by three
standard deviations the mean for all Los Angeles subjects in equivalent
exposures. Comparative dose-response curves for Los Angeles and Montreal
subjects, derived by second-order polynomial regression analysis from the
-------
mean response values for each concentration studied, are given in
Figure 1. If these curves are adjusted by assuming a consistent
difference in 03 monitoring between the laboratories equal to the
maximum observed difference described in the preceding section,
they approach each other more closely but still indicate sub-
stantially less reactivity in Los Angeles subjects.
If the difference in response to 63 between Canadians and Southern
Californians studied is accepted as real, the hypothesis of adaptation
in Southern Californians is supported. Further support of the hypothesis
requires demonstration that the subjects tested are meaningfully
representative of larger population groups residing in their respective
areas, and that identifiable factors other than adaptation are not
sufficient to explain the observations. (Positive evidence for 03
adaptation in humans appears to be lacking at present.)
Subjects in both the Los Angeles and the Canadian studies were pre-
dominantly white, middle-class professional and technical workers and pre-
professional students, young to middle-aged adults in normal health or
with mild respiratory hypersensitivity. While they represent highly
selected groups, there appears to be no reason to expect them to be
atypical with respect to pulmonary physiological or biochemical status.
Neither is there any obvious indication of differences between the
Californians and the Canadians in genetic constitution, nutrition, or
environmental stresses other than air pollution exposure which could
41.
-------
be the source of their differences in reactivity. Selective migration
of O^-sensitive individuals away from Los Angeles might be of
importance, but no reliable evidence relevant to this possibility
exists. Adaptation thus remains the most plausible explanation for
the existing data. Since controlled studies designed to demonstrate
adaptation in humans are probably impractical, future investigations
should seek to define more fully the relative reactivity of populations
exposed to different,levels of 03 and the changes in reactivity, if
any, exhibited by individuals with little previous exposure who move
to O^-polluted environments.
This study was supported by National Heart and Lung Institute
Specialized Center of Research grant HL ES 15098.^
We thank Charles Spier, Leonard Wightman, Howard Greenberg,
Julie Patterson, Johnnie Foy, Kenneth Clark, and Clara Posin for
assitance in this study.
42.
-------
REFERENCES
1. Buckley RD, Hackney JD, Clark K, et al.: Ozone and human blood
Arch Environ Health, 30: 40-43, 1975.
2. Bates DV, Bell G, Burnham C, et al.: Short-term effects of
ozone on the lung. J Appl Physiol, 32: 176-181, 1972.
3. Hazucha M, Silverman F, Parent C, et al.: Pulmonary function in
man after short-term exposure to ozone, Arch Environ Health, 2.1 \
183-188, 1973.
4. Hackney JD, Linn WS, Mohler JG, et al.: Experimental studies on
human health effects of air pollutants. II. Four-hour exposure
to ozone alone and in combination with other pollutant gases.
Arch Environ Health, 30: 379-384, 1975.
5. Hackney JD, Linn WS, Law DC, et al.: Experimental studies on
human health effects of air pollutants. III. Two-hour exposure
to ozone alone and in combination with other pollutant gases.
Arch Environ Health, 30: 385-390, 1975.
6. Silverman F: Effects of ozone on pulmonary function in man.
Presented, Canadian Conference on Research in Atmospheric Pollution,
Gravenhurst,Ontario, 6 June 1974.
7. Hazucha M: Effects of ozone and sulfur dioxide on pulmonary
function in man. Thesis, McGill University, October 1973.
8. Stokinger HE, Wagner WD, Wright PG: Studies on ozone toxicity.
I. Potentiating effects of exercise and tolerance development.
Arch Ind Health, 14: 158-160, 1956.
9. Shakman RV: Nutritional influences on the toxicity of environmental
pollutants. Arch Environ Health, 28: 105-113, 1974.
10. DeMore WB: Calibration report. California Air Resources Board
Bulletin, Vol. 5, No. 11, p 1, December 1974.
11. Hackney JD, Linn WS, Buckley RD, et al.: Experimental studies on
human health effects of air pollutants. I. Design considerations.
Aroh Environ Health, 30: 373-378, 1975.
12. Selected methods for measurement of air pollutants. Division of Air
Pollution, Cincinnati, Ohio. PHS Publication No. 999-AP-ll May 1965.
13. Buist AS, Ross BB: Predicted values for closing volumes using a
modified single-breath nitrogen test. Am Rev Respir Disease, 107:
744-752, 1973.
43.
-------
TABLE 1. COMPARATIVE TOTAL OXIDANT (PRIMARILY OZONE) LEVELS FOR
SOUTHERN CALIFORNIA AND ONTARIO URBAN AREAS, 1971 (a)
NO. OF MONTHS CONCENTRATIONS (ONE-HOUR AVERAGE), PPM
LOCATION MONITORED MAXIMUM 99TH PERCENTILE(b) 9QTH PEKCENTILE(c) MEAN
Los Angeles area
Central City 12 .31 .18 .08 .032
Pasadena (d) 12 .68 .31 .14 .052
Toronto area
Central City 11 .07 .037 .006 .010
Etobicoke (d) 10 .35 .131 .064 .034
Hamilton 10 .25 .124 .070 .042
NOTES: (a) Los Angeles area values have been multiplied by 1.3 to
allow for probable differences in instrument calibration (Reference 10).
(b) Level exceeded dun'ng 1 percent of total hours monitored.
(c) Level exceeded during 10 percent of total hours monitored.
(d) Monitoring station showing highest concentration in area
during 1971.
SOURCES: California Air Resources Board Ten-Year Summary of Air Quality
Data, 1963-1972; Ontario Ministry of the Environment Air Quality
Monitoring Report, Vol. 1, 1971.
44.
-------
TABLE 2. SUBJECT CHARACTERISTICS
HISTORY CF:
(Canadian Group)
19 M 39
20 J 33
21 M 35
22 ? 32
HT, 13
73
63
69
63
WT,L3
2CO
140
212
123
SMOKING ALLERGY
N 0
3(1)* 0
3(15) +
3(4) +
WHEEZING C^ REACTIVITY
0
0
+
0
-M-
++
unknown
unknown
(Los Aageles Group)
7 M 38 69
lo « 30 72
11 M 30 72
23 y 22 64
155
175
155
10?
H
S(7)
N
s(3)
+
0
0
+
+
0
0
+
-H+
4-
-H-
xinknown
* Very light sasoker
N = never sacked
3c = current cigarette sisoker (number in parentheses = approximate
lifetime constcaption in packs— per-day x years)
0 = none
+ = sold
-M- = moderate to severe
0 reactivity previously documented at 0.75
0.50 ppn in Los Angeles subjects.
in Canadian subjects,
45.
-------
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47.
-------
TABLE 4. SIGNIFICANT INDIVIDUAL CHANGES IN FORCED-EXPIRATORY
AND SINGLE-BREATH NITROGEN MEASURES WITH EXPOSURE
SUBJECT 7 (LOS ANGELES)
FEV (best of 3 trials)
FEY., (mean)
SUBJECT 19 (CANADIAN)
FVC (best of 3 trials)
FVC (mean)
FSV (best of 3 trials)
FS7 (mean)
TLC (mean)
(mean)
SUBJSCT 20 (CANADIAN)
FVC (best of 3 trials)
FVC (mean)
F57 (best of 3 trials)
FEY (mean)
TLC (mean)
CONTROL
3.82
3.80 - .02
3.26
- .03
2.52 - .
EXPOSURE
3.70
3.65 - ,
05
6.07
6.00 - .05
k.7k - .06
7.62 - .05
0.53 - .06
4.90
4.85 i .08*
3.80 i .56
6.4o - .^9
1.25 - .35
- .03
3.00
2.92 - .09*
2.25
2.17 - .07
3.98 i .05*
* Mean change significant t P < .01, by unpaired t test. All other
mean changes significant, P < .05. Three measures obtained under
each condition.
48.
-------
TABLE 5
BLOOD BIOCHEMICAL RESPONSE OF CANADIANS
VS. SOUTHERN CALIFORNIA—GROUP DATA, MEAN ± S.D.
ANALYSIS, GROUP CONTROL EXPOSURE
Erythrocyte Fragility (a)
Canadian (b) 15.3±1.2
Los Angeles (b) 18.6±1.9
Total L.A. (c) 16.8+2,4
Erythrocyte Acetylcholinesterase
Canadian 22.3±1.4
Los Angeles 21.0±1.0
Total L.A. 22.3±2.9
Erythrocyte Glucose-6-Phosphate
Canadian 6.42±0.47
Los Angeles 7.02±0.30
Total L.A. 4.50±1.02
Erythrocyte Glutathione (e)
Canadian 30.9±5.5
Los Angeles 31.4±3.9
-Total L.A. 31.6+4.7
Serum Lipid Peroxidation (F)
Canadian .219±.022
Los Angeles .300±.032
Total L.A. v .191±.060
Serum Vitam E (g)
Canadian 1.71±.34
Los Angeles 2.36±1.65
Total L.A. 2.05±.98
22.1±2.2
17.5±1.3
17.3±3.2
Activity (d)
19.9+1.9
19.4±1.0
20.6±2.4
Dehydrogenase Activity
6.84±0.80
7.13±0.77
4.81±1.21
30.4±5.4
33.2±2.0
28.2±3.4
.242±.030
.307±.015
.199±.030
1.83±.33
2.50±1.61
2.15+.98
4
4
13
4
4
13
(d)
4
4
13
4
4
12
4
4
13
4
4
13
9.53 <.005
0.48 N.S.
0.97 N.S.
8.72 <.005
7.83 <.005
5.49 <.01
2.46 N.S.
0.32 N.S.
2.18 <.05
2.96 N.S.
2.00 N.S.
2.72 <.025
1.00 N.S.
1.80 N.S.
0.62 N.S.
4.10 <.025
1.28 N.S.
0.83 N.S.
NOTES: (a) Percent hemolysis in 2 percent hydrogen peroxide, (b) Present
study, (c) All Los Angeles subjects under initial exposure to
0.37 ppm Oj studied to date, including present Los Angeles subjects.
(d) pmol substrate consumed/g hemoglobin/min. (e) mg/100 ml.
(f) Equivalent yg malonaldehyde/ml. (g) yg/ml.
49.
-------
TABLE 6
INDIVIDUAL BLOOD BIOCHEMICAL
RESPONSES OF CANADIAN SUBJECTS
ANALYSIS, SUBJECT CONTROL EXPOSURE % CHANGE
Erythrocyte Fragility
19 (R)
20 (R)
21
22
19 (R)
20 (R)
21
; 22
Serum Vitamin E
19 (R)
x 20 (R)
21
22
NOTE: (R) = Clinically and physiologically reactive subject. Units are
given in Table 5.
14.2
14.4
16.7
15.8
rase Activity
20.8
21.7
24.0
22.9
1.73
1.94
1.94
1.22
21.9
19.3
24.9
22.5
18.2
18.5
22.3
20.7
1.90
1.98
2.09
1.34
+53
+34
+49
+42
-12
-15
- 7
-10
+10
+ 2
+ 8
+10
50.
-------
TABLE 7. MEAN REACTIVITY OF SUBJECT GROUPS UNDER INITIAL EXPOSURE
TO 0.37 PPM 03 FOR 2 HOURS WITH INTERMITTENT LIGHT EXERCISE
PERCENT CHANGE
GROUP (NUMBER OF SUBJECTS) IN FEVj, MEAN±S.D.
Montreal, nonsmokers (6)* -7.4±5.6**
Montreal, smokers (6)* -3.9±1.7
Los Angeles, total group, nonsmokers (7) -0.6±3.4**
Los Angeles, total group, smokers (6) -2.0+3.0
Los Angeles, total group (13) -1.2±3.2
Toronto/Hamilton, present study (4) -5.8±7.3
Los Angeles, present study (4) 0.0+2.4
f_Los Angeles, cumulative 2-hr exposure (8)3 [-2.5+3.2U
0-OS Angeles, cumulative 4-hr exposure (4)] [-4.0±8.33
* M. Hazucha, Effects of Ozone and Sulfur Dioxide on Pulmonary
Function in Man, dissertation, McGill University, Montreal, 1973.
** Response significantly different in Montreal and Los Angeles
nonsmoker groups (P<.05 by t test).
51.
-------
FIGURE i;-*
COMPARATIVE DOSE-RESPONSE BEHAVIOR
PPM
-20
X
AFEV
52-
-------
LEGENDS
FIGURE 1. COMPARATIVE DOSE-RESPONSE BEHAVIOR. Curve A—mean
response (percent loss in FEV-) of previously-studied
Southern Californians exposed at 0.25-0.50 ppm Oo.
Curve B—mean response of previously-studied Canadians
exposed at 0.25-0.75 ppm (reference 7). Point C--mean
response of Southern Californians in present study.
Point D--mean response of Canadians in present study.
Curves determined by second-order regression analysis.
All exposures for 2 hours with intermittent light
exercise.
53.
-------
STUDIES IN ADAPTATION TO A'iJluiT OXIDANI AIR POLLUTION:
Effects of Ozone Exposure in Los Angeles Residents vs.
New Arrivals
Jack D. Hackney, William S. Linn, Ramon D. Buckley and Helen J. Hi si op
Environmental Health Service, Rancho Los Amigos Hospital,
Downey, California 90242
From the Specialized Center of Research in Environmental Lung Disease (SCOR),
National Heart and Lung Institute, Grant No. HL-15098-05.
Presented in part at the Conference on Recent Developments in Toxicity of
Environmental Oxidants, National Institute of Environmental Health Sciences,
Bethesda, Maryland, March 4-5, 1976.
b4.
-------
•STUDIES IN ADAPTATION TO AMBIENT OXIDANT AIR POLLUTION:
Effects of Ozone Exposure in Los Angeles Residents vs.
.New Arrivals
ABSTRACT
To test the hypothesis that adaptation protecting against acute
effects of ambient ozone (0^) exposures develops in Los Angeles
residents, human volunteers were exposed to 0.4 ppm Og under conditions
simulating ambient pollution exposures. Blood biochemical., pulmonary
physiological, and clinical responses were assessed, Los Angeles
residents (N=6) showed only minimal clinical or physiological response
to 03, while new arrivals (N=9) showed significant losses in pulmonary
function and a tendency toward increased symptoms. Most biochemical
responses did not differ significantly between residents and new
arrivals. These results agree with others in suggesting that exposures
*-*-
to elevated ambient concentrations of 63 produce adaptation in at
least some residents of photochemical pollution areas/- The under-
lying mechanisms and long-term consequences of such adaptation are
unknown.
55.
-------
INTRODUCTION
Development of tolerance to ozone (0^) and other irritant gases
in experimental animals was first described by Stokinger and co-
workers approximately 20 years ago (1) and lias been studied exten-
sively since. The subject has been reviewed by Fairchild (2) and
Morrow (3). Salient features of animal tolerance include the
following: (a) Pretreatment with a relatively low 0^ dose will pre-
vent death or severe lung injury which would otherwise occur with a
higher dose, (b) This tolerance gradually disappears after cessation
of Oq exposure, (c) Cross tolerance exists among 03 and other irri-
tant gases, including some which, like 03, are powerful oxidizing
agents and others which are not. (d) Tolerance does not prevent
the development of chronic lung lesions following repeated exposures.
(e) Tolerance results in decreased edema formation in response to
03 challenge, but no diminution of cytotoxic effects of 03 is
observable (4). (f) The biological mechanisms responsible for
tolerance are largely unknown.
The observation that animals can respond to a toxic inhalation
challenge in a manner which prevents some of the short-term adverse
effects of further exposures suggests the possibility that an analogous
response might occur in humans exposed to community air pollution. We
use the term "adaptation" to describe this hypothetical response in
humans, since the doses of toxicants being considered are much less than
in animal "tolerance" studies, and since responses are less severe and
-------
perhaps depend on different biological phenomena. Metropolitan Los
Angeles experiences uncommonly high ambient levels of 0^ and other
oxidants during photochemical smog episodes, thus residents of this
area constitute an attractive group in which to investigate the
possibility of adaptation. That Los Angeles residents suffer less
deleterious effects of ambient exposures than visitors to the area.
has been previously suggested (5), but the hypothesis has never been
tested extensively.
Previous work in our laboratory (6,7) showed that some healthy
Los Angeles residents develop respiratory symptoms and function
changes when exposed to 03 concentrations of 0.37-0.50 ppm—less than
maximum ambient concentrations in the area. Similar studies in
Canadians not frequently exposed to ambient oxidants (8,9) appeared
to show a greater mean effect of a given dose, suggesting that
responses in Los Angeles residents might 'nave been reduced by adapta-
tion. Methodological differences between studies might have explained
the apparently different responses, however. To test this possibility,
a cooperative investigation was undertaken to compare experimental
methods and responses of a small sample of subjects to 0.4 ppm Oo (10).
The results reproduced to a great extent the previous finding of less
reactivity in Los Angeles residents as compared to Canadians and failed
to reveal any methodological factors which could account for this
difference. The hypothesis of adaptation was thus supported. To test
the hypothesis more rigorously, the present study was undertaken in
-------
order to compare the effects of 0.4 ppm CU in somewhat larger and
more carefully matched groups of Los Angeles residents and non-
residents.
METHODS
The null hypothesis tested was as follows: Healthy Los Angeles
residents (three years or more in area) and new arrivals (five days
or less in area) will not differ in mean clinical, physiological or
biochemical response to 0.4 ppm 0^ exposure under conditions
simulating ambient pollution episodes. Rejection of the null
hypothesis with new arrivals showing significantly greater mean
response would be the necessary result if the hypothesis of 03
adaptation in residents were to be supported.
The exposure facility and basic experimental design have been
described in detail previously (11). Volunteer subjects were studied
on two successive days. The first day's exposure v/as to purified air
only; the second day's exposure was to 0.40 ppm 0^ in purified air.
Exposures lasted 2 hr 15 min. During the first two hours, each sub-
ject exercised at a workload sufficient to increase minute volume
to approximately twice the resting level (150-200 kg-m/min) for
15 min in every half hour. During the last 15 min pulmonary function
tests were performed; these included forced vital capacity (FVC), one-
second forced expiratory volume (FEVi), maximum midexpiratory flow
rate (MMF), total respiratory resistance by forced oscillation (R^),
and indices of the single-breath nitrogen test: closing volume as a
53.
-------
percent of vital capacity (CV/VC), delta nitrogen or slope of the
alveolar plateau (A^) . Each subject's test results were expressed
as control values (those obtained after purified-air exposure) and
as Oo responses (differences between post-O-j exposure and control values).
Subjects' symptoms during and following exposure were recorded and
scored semi quantitatively according to severity and duration using a
standard interview questionnaire administered by the project medical
officer. The symptom response to 0^ was expressed as the difference
in symptom score between 63 exposure and control days. Venous blood
samples were drawn immediately following exposure, and erythrocyte (RBC)
and serum analyses were performed to detect changes expected to result
from an oxidant challenge, as described previously (12).
Paired statistical tests with each subject serving as his own
control were applied to detect differences between control and 03 condi-
tions for the resident group and for the new-arrival group. Unpaired
tests were applied to compare between groups. For physiological measures,
only 03 responses were compared between groups, as control values were
expected to depend mostly on body size and not on adaptation. For bio-
chemical measures, control values could have differed between groups as
a consequence of adaptation, therefore both control values and (k responses
were compared statistically. In addition to the commonly employed t tests,
analogous nonparametric tests—the Wilcoxon signed-rank test for paired
analyses and the Mann-Whitney U test for between-group analyses—were
applied to the pulmonary function data. The nonparametric tests were
expected to be possibly more powerful in analyzing these data since the
data were expected to be skewed, whereas t tests require a normal
59.
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distribution for greatest reliability. Skev/ness is inherent in data of
this nature since there is considerable variability between individuals
in reactivity to exposure, and since function measures remain similar
to control values in relatively unreactive subjects but deviate from
control values in only one direction in more reactive subjects.
Symptom data, which were not rigorously quantitative and not necessarily
expected to show a normal distribution even under control conditions,
were analyzed only with the nonparametric tests.
Subjects were recruited within the incoming 1975 class of the
USC School of Physical Therapy. Fifteen of a possible 44 individuals
volunteered to be studied; six of these were metropolitan Los Angeles
residents and nine were nonresidents. Studies were conducted during
September, i.e. late in the summer smog season when residents should have
had ample time to develop adaptation. Nonresidents were studied within
five days of their arrival in Los Angeles; they were instructed to
minimize intercurrent ambient oxidant exposures by remaining in coastal
areas of metropolitan Los Angeles and/or remaining indoors and at rest
during peak oxidant hours.
Individual subject characteristics are given in Table 1. Since
the nonresidents included two males, while the residents were all female»
the possible effect of sex differences on the overall results was examined.
The males' data were compared individually with the female nonresidents'
for the three measures which showed significant (P<.05) group differences.
Both males' values fell within the females' range, except that one male
had the largest control and post-exposure FEV-j. When statistical analyses
were repeated excluding the males' data, mean group responses were actually
60.
-------
larger than when the males were included; however, due to the reduction
in sample size the level of significance of the group differences
decreased--.05
-------
TABLE 1. INDIVIDUAL SUBJECT CHARACTERISTICS
AGE, HT.
WT.
YEARS IN LOS
°
SEX YEARS INCHES POUNDS SMplONG_ ANGELES__ARE_A RESPONSE*
LOS ANGELES RESIDENTS
52
59=-
60
65
66
69
F
F
F
F
F
F
22
25
25
21
25
22
69
68
68
67
63
65
158
118
118
138
94
125
current
former
18
3
18
10
3
14
p
s
p
NONRESIDENTS (NEW ARRIVALS)
47
49
50
51
53
55
56
57
58
F
M
F
F
F
F
F
F
M
22
22
21
21
22
22
23
21
24
68
71
66
62
73
68
62
64
72
140
160
115
125
155
125
120
121
170
P,S
current
P,S
s
P,S
p
p
* P = physiological response—significant (P<.05) loss in FVC and/or FEV-,
with 03 exposure relative to control, determined by t test, 3 measurements
under each condition. S = symptom response--!ncrease in symptom score
of ^ 4 units (arbitrary definition of "clinically significant" response).
t Spent previous summer in area.
62.
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TABLE 2. COMPARATIVE PULMONARY FUNCTION AND SYMPTOM MEASURES--
CONTROL VALUES AND CHANGE WITH 03 EXPOSURE, MEAN ± S.D.
INTERGROUP COMPARISON
RESIDENTS NEW ARRIVALS t U
Control FVC, liters 4.01±.40 4.57+.89
FVC change, liters -.093+.155(a) -.164+.202(b) 0.72(a) 20(a)
Control FEVp liters 3.49±.22 3.84±.49
FEVj change, liters -.018+.098(a) -.171±.174(b) 1.93(a) 9(P<.05)
Control MMF, I/sec 4.06±.70 4.23±.85
MMF change, I/sec +.175±.336(a) -.252±.320(b) 2.48(P<.05) 9.5(p<.05)
Control CV/VC% 7.6+5.5 6.8±5.8
CV/VC% change +0.4±2.8(a) +0.5±3.2(a) 0.12(a) 24(a)
Control AN2, %H2/1 0.95±.15 0.93±.23
AN2 change, %N2/1 -.117±.094(c) -,050±.206(a) 0.73(a) 21.5(a)
Control Rf, 4.02+.99 3.25±.90
cmH20/(l/sec)
Rt change, +0.13±.98(a) +0.20+.45(a) 0.18(a) 17(a)
cmH20/(l/sec)
Control symptom score 4.9±5.1 3.6±3.5
Symptom score change +0.2±5.5(a) +2.7±4.8(a) - 19(a)
NOTES: (a) Not significant
(b) Significant decrement after exposure, P<.05 by paired t test
and by Wilcoxon signed-rank test.
(c) Change not significant by signed-rank test; apparent
"improvement" after exposure according to paired t test
(P<.05).
63.
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Increases in Al^ ar^ normally u::pected in chronic pulmonary dys-
function and in acute responses to 03 exposure (6). Decreased
values represent increased uniformity of ventilation distribution
and thus could be considered an improvement in function. On the
other hand, more uniform distribution could be the result of adverse
physiological changes, such as complete "closure" of a few small
airways previously only partially obstructed. Nonresidents showed
a smaller, nonsignificant decrease in ANo, but showed significant
03 responses in FVC, FEV^, and M,MF. The MMF response was significantly
more severe than in the residents according to the intergroup com-
parison, but the FVC responses did not differ significantly between
the groups. The FEV^ loss was significantly more severe in non-
residents than in residents according to the U test (P=.03)s but not
according to the t test (P=.06). Since the distributions of FEVj
responses appear skewed (Figure 1), the results of the U test may be
more reliable. Neither group showed significant responses of CV/VC,
Rt, or symptom score, but the nonresidents showed a trend toward
increased symptom score with 0- exposure.
Group mean biochemical measurements and significant changes
related to exposure are summarized in Table 3. None of the analyses
showed significant differences in control values between residents
and new arrivals, although residents showed trends toward less
fragility of RBCs as determined by hydrogen peroxide challenge, and
higher serum concentrations of Vitamin E. Both groups showed 03
-------
TABLE 3. COMPARATIVE BIOCHEMICAL MEASURES-CONTROL VALUES AND CHANGE WITH 0. EXPOSURE,
MEAN ± S.D.
Intergroup Comparison
R^sjjJerrts_ New Arrivals t_
RBC fragility Control 23.0 ± 15.2 31.4 ± 8.0 1.39 (a)
(% hemolysis in H202) Change +6.2 ± 5.5 (b) +3.1 ± 3.4 (b) 1.35 (a)
RBC acetylcholinesterase Control 17.5 ± 1.8 18.8 ± 1.9 1.31 (a)
mM/ml/min) Change -0.6 ± 0.6 (c) -0.8 ± 0.5 (d) 0.83 (a)
RBC glutathione, Control 33.3 ± 6.2 35.3 ± 5.0 0.67 (a)
(mg %) Change -1.6 ± 1.9 (a) -2.0 ± 3.0 (a) 0.28 (a)
RBC 2,3-diphospho- Control 14.9 ± 1.6 14.7 ± 4.1 0.12 (a)
glycerate (yM/g Hb) Change +1.7 ± 0.7 (d) +1.0 ± 3.3 (a) 0.50 (a)
RBC glucose-6-phosphate Control 5.22 ± 1.14 5.05 ± 0.86 0.33 (a)
dehydrogenase (U/g Hb/min) Change +0.23 ± 0.43 (a) +0.21 ± 0.32 (a) 0.09 (a)
RBC lactate dehydrogenase Control 107 ± 16 112 ± 15 0.59 (a)
(U/g Hb/min) Change -6.6 ± 12.4 (a) +8.9 ± 9.5 (d) 2.74 (e)
RdC glutathione peroxidase Control 8.6 ± 1.8 8.8 ± 1.7 0.23 (a)
(U/ml/min) Change +0.8 ±1.1 (a) +0.3 ± 0.8 (a) 1.11 (a)
Serum Vitamin E Control 2.77 ± 0.79 2.64 ± 0.38 0.44 (a)
(mg %) Change +0.09 ± 0.15 (a) +0.03 ± 0.14 (a) 0.66 (a)
Serum glutathione Control 23.7 ± 4.2 22.6 ± 3.0 0.60 (a)
reductase (mU/ml/min) . Change +1.5 ± 1.7 (a) +2.8 ± 3.0 (d) 0.98 (a)
NOTES: (a) Not significant
(b) Significant (P<.05) change after exposure by t test;
not significant (.05
-------
responses generally similar to those seen previously (12)--increased
RBC fragility, reduced RBC acetylcholinesterase activity, and
tendencies toward increased activity of pentose pathv/ay enzymes
(which would tend to protect against excessive oxidation of cellular
components). Lactate dehydrogenase (LDH) activity was the only
biochemical measure to show a significant difference between groups
in response to O,. New arrivals showed the expected increase in LDH
activity, while residents showed a decrease, in contrast to previous
findings (12). The biological significance of this observation, if
indeed it represents other than a chance occurrence, is unclear.
DISCUSSION
These results support the hypothesis of adaptation to 0^ in Los
Angeles residents. Statistical differences found between residents
and new arrivals are relatively small, as should be expected given
the unavoidably small sample sizes and the typically large individual
variability in 0^ responses. Control!ed-exposure studies cannot be
done on a large enough scale to conclusively establish differenes in
response between populations, but the essential agreement of present
and previous results in small-scale studies considerably strengthens
the case for the existence of such differences. Various factors
unrelated to inherent adaptive biological responses could explain
these results--selective migration or diet, for example (10). No
such factor has yet been identified, leaving adaptation as the most
plausible explanation for the experimental observations. No biochemical
66.
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index of the adapted state has yet been found in animals or in man,
nor are the physiological and biochemical mechanisms of 02 toxicity
well understood. Further investigations in these areas will be
necessary before the biological mechanisms of the adaptive response
(if it exists) can be elucidated. Of particular interest is the
possibility that adaptive mechanisms may be inoperative in certain
individuals, who might-then be at increased risk of developing chronic
respiratory disease.
The phenomenon of adaptation may ultimately, but should not
presently, be taken into account in setting ambient or occupational
air-quality standards. By analogy with animal studies, it appears
that human adaptation to acute 03 effects might not protect against
the possible development of chronic lung damage after many exposures.
Unless this possibility and the possibility of failure of adaptation
are conclusively ruled out, air quality standards should continue to
be set to protect the susceptible, least well-adapted individuals in
the exposed population.
67.
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REFERENCES
1. Stokinger, H. E., Wagner, !•;. [)., arid Wr. gnt, P. G.: Studios
on ozone toxicity. I. Potentiating effect of exorcise and
to'l era nee development. Amh. jnd Hualth 14: 158-162, 1956.
2. Fairchild, E. J.: Tolerance mechanisms.Determinants of lung
responses to injurious agents. Arch Environ Health 14: 111-
126, 1967.
3. Morrow, P. E.: Adaptations of the respiratory tract to air
pollutants. Arch Environ Health 14: 127-136, 1967.
4. Gardner, D. E., et al.: Role of tolerance in pulmonary defense
mechanisms. Arch Environ Health 25: 432-438, 1972.
5. Falk, H. L.: Chemical definitions of inhalation hazards. In:
Inliala.t'ion Carcinogenssis: Ah'C SywposivM Series No. 18.
Division of Technical Information, US Atomic Energy Commission,
Oak Ridge, Tenn., 1970.
6. Hackney, J. D., et al.: Experimental studies on human health
effects of air pollutants. II. Four-hour exposure to ozone
alone and in combination with other pollutant gases. Arch
Environ Health 30: 379-384, August, 1975.
7. Hackney, J. D., et al.: Experimental studies on human health
effects of air pollutants. III. Two-hour exposure to ozone
alone and in combination with other pollutant gases. Arch
Environ Health 30: 385-390, August, 1975.
8. Bates, D. V., et al.: Short-term effects of ozone on the lung.
J Appl Physiol 32: 176-181, 1972.
9. Hazucha, M., et al.: Pulmonary function in man after short-
term exposure to ozone. Arch Environ Health 27: 183-188, 1973.
10. Hackney, J. D,, et al.: Health effects of ozone exposure in
Canadians vs. Southern Californians: Evidence for adaptation?
Arch Environ Health,., in press.
11. Hackney, J. D., et al.: Experimental studies on health effects
of air pollutants. I. Design considerations. Arch Environ.
Health 30: 373-378, August, 1975.
12. Buckley, R. D., et al.: Ozone and human blood. Arch Environ
Health 30: 40-43, January 1975. Air Pollution Abstracts., pg. 110,
Abs. No. 50966; September, 1975.
08.
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NO. OF
SUBJECTS
3-
P—
0J
2H
NONRESIDENTS
RESIDENTS
0^--, r ,-
-.6 -A
r i
-.2
+.2
CHANGE IN FEVr LITERS
FIGURE 1. Histograms of 03 responses in FEV-j (change betv;een post-
exposure and control measurements) for non-residents and
residents -- number of subjects showing a given response
(within a 0.5-liter interval) vs. magnitude of response.
69.
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I • RESPIRATORY EFFECTS OF EXPOSURE TO OZONE-SULFUR DIOXIDE MIXTURES
SCOR-Environmental Health Service
Summary
Four normal Loe Angeles residents were exposed to 0, plus SO., each gas
at 0.37 part per million, in purified air at 31°C and 35# relative humidity
in the Rancho Los Amigos enTironmental chamber. The exposure was preceded
by a sham exposure (purified air only) and by an exposure to 0, alone.
All exposures lasted 2 hr, during which subjects exercised lightly (approx-
imately 200 kg-m/min) for 15 min in every $0. Pulmonary physiological testa
were performed at the end of the 2-hr exposure period and compared among
different exposure conditions. No significant physiological changes
attributable to 0,/SO_ exposure were found, and only minimal and transient
respiratory symptoms were experienced by the subjects. Atmospheric mon-
itoring of the exposure chamber showed that many condensation nuclei were
formed when 0, and S0_ were mixed (100,000 or more particles/ml with gases
present vs. less than 1000/ml in purified background air). These particles
were too small to be detected by light-scattering particle counters, i.e.
of the order of 0.1 um or smaller in diameter. Filter samples showed
approximately 1 ug/m of aulfate aerosol in the mixed-gas atmosphere; no
sulfate was detectable in the purified background air.
Four Los Angeles residents previously found to be highly reactive to
0, exposure were exposed in the same manner as the normal subjects. Of
these, two showed no more than minimal pulmonary function changes with
0_,/SOp exposure and two showed mild but significant losses in forced-
expiratory measures. In the most reactive subject, these changes were
greater than previously observed in a comparable exposure to 0, alone on
two successive days.
As the above results suggested much less marked short-terra toxicity of
0,/SO- than previously reported by Hazucha et al. (Fed. Proc. 33: 350,
197*0 in normal Canadians, a cooperative study was initiated to investigate
the source of the difference between the two studies. Four previously-
studied Canadian subjects were brought to this laboratory and exposed for
2 hr with intermittent light exercise to purified air on one day and to
0.37 ppm 0, plus 0.37 ppm SO on the following day. Temperature was 21°C
and relative humidity 50#, giving comparability to the previous Canadian
70.
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exposure conditions. One subject experienced a reaction of severity compar-
able to or greater than that previously experienced in the Canadian study,
Two ethers reacted much less severely in this laboratory than in Canada, and
the remaining subject showed a relatively mild reaction in both places.
Pulmonary-function changes for the group in the present study were not
statistically significant due to the large individual variation. Particulate
formation from the 0-./SO_ mixture occurred in this study in essentially the
same manner as described for the previous studies.
The four reactive Los Angeles residents previously mentioned, plus one
additional 0.,-reactive Los Angeles subject, were exposed under the conditions
employed for the Canadian subjects. Results were similar to those previously
obtained in these individuals. Pulmonary function changes seen were slight,
but were more consistent than in the Canadians, allowing the mean loss in
one-second forced expiratory volume to attain marginal statistical significance.
Mean function changes were smaller in these subjects than in the Canadians
but larger than observed previously in these subjects under exposure to
0-, alone. Symptoms were also less severe than in the Canadians but more
severe than in the same subjects when exposed to 0 . Typical symptoms in both
groups were cough and substernal discomfort; some subjects also reported
a burning sensation on the skin, not noticed in 0, exposures.
Conclusions
The Canadians studied in this laboratory appeared to react less than
they had previously in Canada in presumably similar 0,/SO_ exposures.
They also appeared to react more severely than Los Angeles residents.
These findings are consistent with the possibility of biological adaptation
to chronic ambient pollution exposure in Los Angeles residents. More study
of interactions of 0 , SO , and participates is required, since such
interactions are apparently inevitable in controlled exposures as well as
in polluted ambient air and may grossly change the manner in which the
pollutants impinge upon the respiratory tract. Different background
particulate concentrations in the present studies and the Canadian studies
may be the source of the difference in subject responses. Particulate levels
were probably higher in the Canadian studies, since less elaborate air
filtration was available.
71.
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Data
PULMONARY FUNCTION DATA FOR LOS ANGELES GROUPS EXPOSED TO 0,/SO, AT J1°C
MEAN, (STANDARD DEVIATION)
EXPOSURE: Shag
"Normal" Group (N =~4T~
FVC
4.80 (.56)
3.85 (-54)
5.5 (0.7)
13.2 (4.3)
.70 (.18)
4.73 (.5^)
3.91 (.59)
5.6 (1.1)
13.8 (4.2)
.71 (.24)
4.66 (.59)
3.76 (.64)
5.1 (1.0)
13.4 (5.3)
.69 (.24)
FVC
CV (% VC)
Delta N2
"Reactive" Group (N = 4)
FVC 5.11 (.89) 5.06 (1.02) 4.90 (1.05)
FEV1 4.19 (.60) 4.11 (.70) 3.96 (.74) (P < .05)
Vfflax50* FVC 5.0 (0.8) 4.8 (0.7) 4.6 (0.6)
CV (% VC) 11.6 (4.3) 10.6 (5.2) 11.4 (4.0)
Delta N2 .61 (.06) .65 (.04) .76 (.13) (P < .05)
Note: Volumes in liters, flows in liters/second, delta nitrogen in percent
N- concentration increase per liter expired. Changes not significant
at .05 level unless indicated otherwise.
PULMONARY FUNCTION DATA FOR CANADIAN & LOS ANGELES GROUPS EXPOSED AT 21°C
MEAN, (STANDARD DEVIATION)
Sham
Canadian Group (N =4)
FVC
FEV1
Vfflax50* FVC
CV (% VC)
Delta N2
"Reactive" Los
FVC
FE^
i/Bax50^ FVC
CV (% VC)
Delta N_
5.83 (.6?)
4.60 (.91)
5.6 (2.4)
6.0 (0.6)
.78 (.27)
Angeles Group
4.62 (.99)
3.72 (.80)
4.3 (1.1)
9.^ (5.2)
.93 (.38)
5.64 (.75)
4.35 (1.02)
4.7 (2.4)
5.5 (1.8)
.85 (.10)
(N = 5)
4.50 (.98)
3.59 (.79)
4.0 (0.8)
9.8 (3.5)
1.02 (.50)
(P .05)
(See notes following preceding table)
72.
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INDIVIDUALS' RESPONSES IN PREVIOUS CANADIAN STUDY VS. PRESENT
(LOS ANGELES) STUDY—PERCENT CHANGE FROM CONTROL VALUES
MEASURE: FVC
j.
STUDY: Can. L.A. Can. L.A.
Subject No. 31 -2 -6 -9 -13
32 -51 0 -58 -2
33 -5 -2 -1 -6
34 -29 -3 -30 -2
(Mean) (-22) (-3) (-24) (-6)
73.
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NITROGEN DIOXIDE INHALATION AND HUMAN BLOOD BIOCHEMISTRY
Ramon D. Buckley
Clara Posin
Kenneth Clark
Jack D. Hackney
Julie Patterson
Environmental Health Service
Rancho Los Amigos Hospital
7601.East Imperial Highway
Downey, California 90242
Department of Biochemistry
University of Southern California,
School of Medicine
2025 Zonal Avenue
Los Angeles, CA 90033
Supported by Grant No. R-801396-4, Environmental Protection Agency,
and by SCOR Grant No. HL 15098, National Heart and Lung Institute
74.
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NITROGEN DIOXIDE INHALATION AND HUMAN BLOOD BIOCHEMISTRY
ABSTRACT
Blood from ten young adult male humans, exposed to 1.0 ppm or
2 ppm nitrogen dioxide (NO^) for 2.5-3.0 hours, was examined for
evidence of biochemical changes. The experiments lasted three days.
The subjects entered an environmental chamber, performed mild
exercise and completed a series of pulmonary physiology measurements
while breathing filtered air. Blood samples were then taken and
analyzed. This regimen v/as repeated on the second and third day
except, the chamber atmosphere now contained 1.0 ppm or 2 ppm NOo.
Paired group analyses were performed on the data. A statistically
significant decrease was observed in the activity of the erythrocyte
(RBC) membrane enzyme acetylcholinesterase (AcChase) at both N02
levels. Levels of thiobarbituric acid reactive substances were
elevated following inhalation of both levels of the irritant gas
but the differences were statistically significant only at the higher
concentration. Other biochemical values were variable but suggested
the possibility of a transitory response to oxidation damage resulting
from the N02 inhalation.
75.
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INTRODUCTION
The toxic nature of very high levels of nitrogen dioxide (N02)
has been known at least as long as man has been using silos for wet
storage of livestock food. This knowledge has stimulated Man's
curiosity about the mechanism of toxicity and many experiments have
been performed with laboratory animals in an attempt to elucidate
the structural and functional changes resulting from acute and
chronic exposure to the gas. Early experiments were relatively crude
because methods for generating and monitoring the gas were not
1
sufficiently developed. With better methods the experiments have
become more sophisticated and much more useful information began to
2
emerge; • Nitrogen dioxide is commonly found in urban and industrial
atmospheres but at much lower levels than were employed in many early
experiments. More recently, experiments have been designed in which
laboratory animals were exposed to levels of N02 found commonly in
outside atmospheres. Biological effects have been noted at these
levels. Typical experiments, for example, have shown hypertrophy and
hyperplasia of alveolar cells after three weeks continuous exposure
3
to 2 ppm N02, a proteinitria in guinea pigs following 14 days exposure
to 0.5 pprn N02, and a decrease in the animal's resistance to bacterial
5
infection. Reports of significant biochemical changes in blood after
in vivo exposures to low levels of N02 have not come to our attention.
We have exposed humans to low levels of N02 in our environmental
6
chamber, and the results of biochemical tests on the blood of human
subjects are presented here.
76.
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METHODS AND MATERIALS
Healthy young adults volunteered for the studies. Subjects
entered the environmental chamber in groups of four or five and
underwent a regimen of intermittent light exercise and pulmonary
function tests over a two and one-half hour period. Subjects
alternated 15 minutes exercise and 15 minutes rest at a level
sufficient to double the minute ventilation. All pulmonary function
tests were performed during the last 15 minute period. Chamber
temperature and relative humidity were maintained at 31 C and 35
percent respectively. These conditions simulate a "typical" smoggy
day in Los Angeles. Blood was drawn from each subject immediately
after the test period. Each subject was studied in the chamber on
three successive days. The environmental chamber atmosphere con-
tained no N02 the first day. This constituted a "sham control".
The atmosphere contained 1.0 ppm or 2 ppm N02 on the second and third
days. These two days were designated "exposure days". When all bio-
chemical assays were completed the values obtained for each subject
at each concentration during exposure periods were compared to the
control values for the same individual. Thus, each subject served
as his own control and the statistical method of paired group analysis
was utilized. Additional experiments were performed in which subjects
followed the three day experimental protocol but breathed only
filtered air. These "sham" experiments were conducted to detect any
biochemical changes resulting from the chamber procedures per ss.
77.
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7 8
Hemoglobin and Hematocrit values were determined on each blood
sample as a routine hematological procedure. Other observations were
chosen because they had been shown to undergo significant changes
following ozone (Oo) exposure which may have implications about the
subject's response and adaptation to the pollutants. These bio-
chemical assays included measurements of erythrocyte (RBC) membrane
9 10
fragility and acetylene!inesterase (AcChase), Erythrocyte levels
11
of reduced glutathione (GSH)S and the enzymes glucose-6-phosphate
12 13
dehydrogenase (G6PDH), lactate dehydrogenase (LDH), glutathione
14 15
reductase (GSSGase), and glutathione peroxidase (GSHPy). Lipid
16
peroxidation was determined in whole blood and measurements
17 18
of serum components include vitamin E, and GSSGase.
RESULTS
The paired t test was used to statistically compare the values
of each parameter for the first (sharn) day, and the two days of NO2
exposure. Results of experiments in which 1 ppm NOo was employed are
presented in Table 1. The values are means and standard deviations
of results of biochemical measurements on the bloods of 10 subjects.
All possible combinations of pairs were tested and statistically
significant differences are indicated by the lines and arrows.
Results of studies in which 2 ppm NOg was employed are shown on
Table 2. Table 3 contains data from the three days of sham exposure.
All data were treated in a similar way.
78.
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DISCUSSION
Previous work has shown that several significant biochemical
changes occur in the blood of humans breathing 0.5 ppm 03 for 2.5
19
hours. There is much evidence that the primary mechanism for the
toxicity of 0^ may involve the formation of free radicals and
ozonides, primarily via peroxidation of tissue unsaturated fatty
16,20
acids. Other changes have been found in lung tissue, including
21
oxidation of sulfhydryl-containing substances in tissues, and
22
stimulation of pentose shunt and glycolytic enzymes. Roehm, et
23
at. found that trace amounts of Oo and N02 caused the rapid oxida-
tion of unsaturated fatty acids .in vitro and concluded that the oxida-
tion of-essential tissue polyunsaturated fatty acids in vivo by the
air pollutants was likely. These changes are detected as thiobarbituric
acid reactive substances in this study. A variety of biochemical changes
24
are brought about in lung by NOp inhalation and it now appears that
alterations also occur in blood.
Erythrocyte membrane AcChase activity was significantly decreased
following the first and second day of exposure in both N09 atmospheres.
26,27
The newly formed RBC has a full compliment of AcChase ' and cannot
produce any more by enzyme induction since nuclei and ribosomes are
lacking. It is normal for the level of AcChase activity to decrease
as RBCs age and N02 appears to accelerate this process. The erythrocyte
membranes did not show an increase in fragility related to sham at
either N0? concentration in contrast to the response seen with 0.37 ppm
20
or higher levels of 03. Such antioxidants as vitamin L are thought to
79.
-------
provide protection against oxidant-induced biochemical changes in
blood tissue, and the increase detected in serum vitamin L levels
follov/ing the first NC^ exposure may have provided some protection.
The vitamin E levels were elevated at the end of the first 1 ppm NOp
exposure but not the second. Evidence exists that mobilization of
vitamin E from body depots could possibly result in an increase in
membrane-associated vitamin E which would increase the resistance to
oxidant induced damage. However, the unsustained increase seen at
1 ppm N02 and the failure of a graded response to result from the
2 ppm NO £ exposure do not suggest that serum vitamin E levels are
affected by NC^ inhalation.
The activity of the enzyme G6PDH showed some increase but the
change v;as not statistically significant. This enzyme participates
in the pentose shunt pathway and serves both as an important energy
source for lung celts and also provides reduced NADPH required for
the reduction of glutathione. Increases in activity would suggest.
a cellular biochemical response to the injurious effects of N02»
which includes an increase in the rate of cell metabolism. These
data suggest that the respiratory rate of red cells was not
significantly stimulated by NC^ but that some increase in the rate
at which cellular reducing substances (NADPH) were produced might
have occurred.
The levels of both RBC-GSSGase and RBC-GSKPX showed statistically
significant increases following 1 ppm NCL inhalation but not at the
-------
2 ppm level. These data suggest a stimulatory response to the pre-
sence of increased levels of oxidizing free radicals. A similar
response is not seen, however, following exposure to the 2 ppm level,
possibly because the increased toxicity of the higher NO2 level
inactivates the enzymes. Although somewhat lov/er than control values,
the GSH levels were not significantly different following exposure
periods. Glutathione is very sensitive to the presence of oxidants
28
and serves as a cellular antioxidant. Glutathione was significantly
reduced in RBCs of humans following exposure to 0.5 ppm Q~ while
20 6
GSSGase activity was not changed. The increase in GSSGase and
GSHPV could conceivably have restored any GSH lost through oxidation,
A
and suggests that the mechanisms of RBC cellular damage and response
to pollutants may be different for 0^ and M02-
Both N02 and 03 elicit in vivo changes in human blood which
suggests oxidation .of susceptible cell components followed by compen-
satory responses which may serve to adapt the subject to inhaled
oxidants, resulting in enhanced resistance to subsequent exposures.
23
Roehm, et al. found that the in vitro oxidation of polyunsaturated
fatty acids is similar with 03 and N02 but that the process is much
more rapid with 03. They speculated that the reaction with N02 in-
volved free-radical formation at the double bonds which initiate an
autocatalytic chain reaction. Ozone on the other hand was thought to
attack double bonds directly and produce more stable, though very
toxic, ozonides. One of the antioxidant functions of vitamin E is
81.
-------
believed to be to terminate free radical chain reactions in tissues,
so it would be expected to provide more protection against NO-
toxicity than Oq. The results of this experiment suggest this
because biochemical changes seen after the first exposure day tend
to be greater than after the second. Experiments requiring multiple
exposures of humans to 03 are in progress and preliminary data
suggests that biochemical changes are additive and fail to show the
attenuated response seen on the second day of N02 exposure.
23
Roehm, et al. also found that Oq produced much more peroxidation
than NOg when the same concentrations were employed. The -In vivo
toxicity of the two oxidants is also quite different. The biochemical
changes detected in this study are comparable to those seen when
human subjects were exposed for about the same length of time to
30
0.37 ppm 03 or less. Biochemical changes in blood are similar at
these two concentrations of the respective gases and there is no
evidence from these experiments that the irritants produce toxic
effects other than by oxidation. The very high oxidation potential
of Oq compared to NO? could account for much of the greater toxicity.
The lung lesions induced by these two gases occur at the terminal
24,31
bronchioles of the respiratory tree so their absorption rates are
probably similar. Much more evidence is needed before the mechanism
of NOo-induced biochemical changes in vivo are understood but the
results of this experiment suggest that N0? attacks cell membrane
lipids, forming free radicals which not only cause damage to the
82.
-------
membrane but also elicit biochemical responses from within the cell.
The "no measurable effect", or "threshold" level for M02 induced
blood biochemical changes has pot been verified or determined in
humans. The effects of repeated exposure to low NOo levels over
extended periods of time are also unknown and should be investigated
before the possible chronic effects of the air pollutant in human health
can be assessed.
83.
-------
REFERENCES
1. Gray, E. Le B.: Oxides of nitrogen: Their occurrence, toxicity
hazard. Arch Industr Health, 19: 479-486, 1959.
2. Cooper, W.C., Tabershaw, I.R.: Biologic effects of nitrogen dioxide
in relation to air quality standards. Arch Environ Health, 12:
522-530, 1966.
3. Sherwin, R.P., Margolick, J.B., and Azen, S.P.: Hypertrophy of
alveolar wall cells. Secondary to an air pollutant.. Arch Environ
Health, 26: 297-299, 1973.
4. Sherwin, R.P. and Layfield, L.J.: Proteinuria in guinea pigs exposed
to 0.5 ppm nitrogen dioxide. Arch Environ Health,, 28: 336-341, 1974.
5. Goldstein, E., Warshauer, D., Lippert, W., and Tarkington, B.: Ozone
and nitrogen dioxide exposure. Arch Environ Health., 28: 85-90, 1974.
6. Hackney, J.D., Linn, W.S., Buckley, R.D., Pedersen, E.E., Karuza, S.K.,
Lav;, D.C., and Fischer, A.: Experimental studies on human health
effects of air pollutants. I. Design considerations. Arch Environ
Health, 30: 373-378, 1975.
7. Drabkin, D.L. and Austin, J.H.: Spectrophotornetric studies; technique
for analysis of undiluted blood and concentrated hemoglobin. J Biol
Chem, 112: 105-115, 1935.
8. International Micro Capillary Reader. International Equipment Company,
Needham Heights, Massachusetts.
9. Younkin, S., Oski, I.A., and Barness, L.A.: Mechanisms of the hydrogen
peroxide hemolysis test and its reversal with phenols. Am J Nutrition.,
24: 7-13, 1971.
84.
-------
10. Ell man, G. L., Courtney, K. D., et al.: A new and rapid
colorimetric determination of acetylcholinesterase activity.
Biochem Pharmacol, 7: 88-95, 1961.
11. Beutler, E., Duron, 0., and Kelly, B. M.: Improved method for
determination of blood glutathione. J Lab Clin Vied., 61: 882-888,
1963.
12. Lohr, G. W. and Waller, H. D.: In: Methods of Enzymatic Analysis,
Bergmeyer, H. V. (ed). New York: Academic Press, Inc., 1963,
pp. 744-751.
13. Bergmeyer, H. V., Bernt, E., and Hess, B.: In: Methods of
Enzymatic Analysis., Bergmeyer, H. V. (ed). New York: Academic
Press, Inc., 1963, pp 736-741.
14. Beutler, E.: Effect of flavin compounds on glutathione reductase
. activity, j Clin Invest, 48: 1957-1959, 1969.
15. Paglia, D. E. and'Valentine, W. N.: Studies on the quantitative
and qualitative characterization of erythrocyte glutathione
peroxidase. J Lab Clin Med, 70(1): 158-160, 1967.
16. Mengel, C. E., and Kahn, H. E., Jr.: Effects of in vivo hyperoxia
on erythrocyte: III. In vivo peroxidation of erythrocyte lipid.
J Clin Invest, 45: 1150-1158, 1966.
17. Tsen, C. C.: An improved spectrophotometric method for the
determination of vitamin E using 4, 7-diphenyl-l, 10-phenanthroline.
Anal Chem, 33: 849-851, 1961.
85.
-------
18. Horn, H. D.: In: Methods of Enzymatic Analysis, Bergmeyer,
H. V. (ed). New York: Academic Press, Inc., 1963, pp. 875-
879.
19. Buckley, R. D., Hackney, J. D., Clark, K., and Posin, C.:
Ozone and human blood. Arch Environ Health, 30: 40-43, 1975.
20. Menzel, D. B.: Oxidation of biologically active reducing sub-
stances by ozone. Arch Environ Health, 23: 149-153, 1971.
21. De Lucia, A. J., Hogue, P. M., Mustafa, M. G., et a].: Ozone
interaction with rodent lung. Effect on sulfhydryls and
sulfhydryl-containing enzyme activities. J Lab Clin Ned, 80:
559-566, 1972.
22. Chort, C. K. and Tappel, A. L.: Activities of pentose shunt and
glycolytic enzymes in lungs of ozone-exposed rats. Arch Environ
Health, 26: 205-208, 1973.
23. Roehm, J. N.s Hadley, J. G., and Menzel, D. B.: Oxidation of
unsaturated fatty acids by ozone and nitrogen dioxide. Arch
Environ Health, 23: 142-148, 1971.
24. Buckley, R. D. and Loosli, C. G.: Effects of nitrogen dioxide
inhalation on germfree mouse lung. Arch Environ Health, 18:
588-595, 1969.
25. Goldstein, B. D., Buckley, R. D., and Cardenas, R,, et al.:
Ozone and vitamin E. Soienoe, 169: 606-616, 1970.
26. Beutler, E. Red Cell Metabolism. New York, Grune and
Stratton, 1975, pp. 87-88.
-------
27. Wintrobe, M. M.: Clinical Usmabology, 7th ed. Philadelphia:
Lea and Febiger, 1974, pp. 201-204.
28. Menzel, D. B.: Oxidation of biologically active reducing
substances by ozone. Arch Environ Health., 23: 149-153, 1971.
29. Beutler, E.: Red Cell Metabolism. New York: Grune and
Stratton, 1975, p.111.
30. Hackney, J. D., Linn, W. S., Mohler, J. G., et a!.: Experi-
mental studies on human health effects of air pollutants.
III. Two-hour exposure to ozone alone and in combination with
Other pollutant gases. Arch Environ Health, 30(8): 385-390, 1975.
31.- Loosli, C. G., Buckley, R. D., et al.: Pulmonary response in
mice exposed to synthetic smog. Ann Occup Hyg3 15: 251-260, 1972.
87.
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TABLE 1
BIOCHEMICAL CHANGES IN HUMAN BLOOD FOLLOWING 1 PPM N02 INHALATION
MEAN ± S.D.
OBSERVATION
RBC Frag.
(% hern )
AcChase
\ / I )
GSH
(mg %)
G6PDH
(U/gmHb/min)
LDH
(U/gmHb/min)
RBC GSSGase
(U/ml/min)
RBC GSHPX
(U/ml/min)
Vit. E
Lipid Perox.
(ygm/rnl blood)
Serum GSSGase
(mU/ml/min)
RBC Frag. - sensit
SHAM
20.6±3.11
20.7+1.72
f
L
28.4±2.29
5.03±0.86
111.0±17.7
2 96+0.267
1
8.35±2.44
0.99±0.41
0.204±0.070
24.4+3.54
[_
ivity to f.h n2u2
EXPOSURE 1
22.9±3.76
t
20.2±1.58
f t
27.U2.70
5.18+0.70
106.0±22.6
3.690+0.762
t
10.0±3.44
1.08+0.42
0.249+0.069
26.5+3.88
t
EXPOSURE 2|
17.8+3.93
i
19.3+1.16
|
28.3±2.74
5.11+0.64
103.0±17.7
4.020+0.576
1
9.52+3.48
0.98±0.45
0.249+0.046
27.8±3.33
J
Pn ^
.UD
n m
GSH - reduced glutathione n = 10
G6PDH - glucose-6-phosphate dehydrogenase
LDH - lactate dehydrogenase
RBC GSSGase - erythrocyte glutathione reductase
RBC GSHPX - erythrocyte glutathione peroxidase
Vit. E - a-tocopherol
Lipid Perox. - thiobarbituric acid reactive substance
88.
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TABLE 2
BIOCHEMICAL CHANGES IN HUMAN BLOOD
FOLLOWING 2 PPM N0£ INHALATION
MEAN ± S.D.
OBSERVATION
RBC Frag.
(% hem.)
AcChase
(mM/ml/min)
GSH
(mg %)
G6PDH
(U/amHb/min)
LDH
(U/gmHb/min)
RBC GSSGase
(U/ml/min)
RBC GSHP
(U/ml/min)
Vit E
{mg %)
Lipid Perox.
/unm/ml ^
\ My111/ |p| ' 1
Serum GSSGase
( ml I/ml /mi n )
n = 8
n--n cm
_n
-------
TABU: 3
BLOOD BIOCHEMISTRY FROM HUMAN SHAM EXPOSURE
OBSERVATION
RBC Fraq.
(% hem.)
AcChase
(mM/ml/min)
RBC - GSH
(mg %}
RBC-Lipid Perox.
(y gin/ml blood)
RBC-GSSGase
(U/ml/min)
RBC-GSHP
(U/ml/min)
G6PDH
(U/gm Hb/min)
LDH
(U/gm Hb/min)
Hb
(gm %)
Ht
f o/ \
SHAM DAY #
1
2
3
1
2
3.
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
X ± S.
28.54 ±
24.00 ±
31.41 ±
22.90 ±
21.98 ±
21.91 ±
27.16 ±
27.87 ±
27.95 ±
0.289 ±
0.247 ±
0.268 ±
3.387 ±
3.453 ±
3.405 ±
8.275 ±
9.250 ±
9.837 ±
4.219 ±
4.798 ±
4.433 ±
105.62 ±
93.36 ±
105.58 ±
15.70 ±
15.66 ±
15.56 ±
45.02 ±
45.65 ±
D.
3.86
1.83
2.93
4.68
4.52
4.34
2.97
3.55
3.16
0.02 '
OH AA «o — .
.009 -£—
0.006^J
0.319
0.398
0.342
1.172
1.006
1.467
0.392
0.806
0.968
9.28
15.87
7.46
0.669
0.747
0.715
3.14
3.25
n =8
= p<0.05
___ = p<0.01
RBC Frag = erythrocyte fragility to 2% H202
AcChase = acetylcholinesterase
RBC-GSH = erythrocyte reduced glutathione
RBC-lipid perox. = T thiobarbituric acid reactive substance
RBC-GSSGase = erythrocyte glutathione reductase
RBC-GSHPX = erythrocyte glutathione peroxidase
G6PDH = glucose-6-phosphate dehydrogenase
LDH = lactate dehydrogenase
Hb = hemoglobin
Ht = heraatocrit
90.
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EXPERIMENTAL STUDIES ON HUMAN HEALTH EFFECTS OF AIR POLLUTANTS
IV. SHORT-TERM PHYSIOLOGICAL AND CLINICAL EFFECTS OF NITROGEN
DIOXIDE EXPOSURE
RUNNING TITLE: PHYSIOLOGICAL EFFECTS OF NITROGEN DIOXIDE
Jack D. Hackney, M. D.
Frederick C. Thiede, Ph.D.
William S. Linn, M. A.
E. Eugene Pedersen, Ph.D.
Charles E. Spier
David C. Law, M. D.
D. Armin Fischer, M. D.
Supported by Grant No. R-801396-4, Environmental Protection Agency, and
by SCOR Grant No. HL 15098, National Heart and Lung Institute
91.
-------
EXPERIMENTAL STUDIES ON HUMAN HEALTH EFFECTS OF AIR POLLUTANTS
IV. SHORT-TERM PHYSIOLOGICAL AND CLINICAL EFFECTS OF NITROGEN
DIOXIDE EXPOSURE
ABSTRACT
Adult male volunteers were exposed to nitrogen dioxide
at 1.0 ppm in purified air under conditions simulating ambient
photochemical smog exposures (two-hour exposure with intermittent
light exercise at 31°C and 35 percent relative humidity). Sham
exposures (to purified air alone) served as controls. Exposure
effects were assessed by pulmonary physiological tests and by a
standardized clinical evaluation. No statistically significant
physiological changes attributable to NO^ exposure were found except
for a marginal loss in forced vital capacity after two successive
days' exposure (1.5 percent mean decrease, P<.05). Reported respi-
ratory and other symptoms were slightly increased with exposure as
compared to control, but the change was not significant. Short-
term toxicity of N02 at peak ambient concentrations appears to be
substantially less than that of ozone, but adverse health effects
due to NOp or its atmospheric reaction products cannot be ruled out
at present.
-------
INTRODUCTION
Photochemical smog as experienced in many urban areas is a
complex mixture including many oxidizing substances, of which the
two typically present in highest concentration are nitrogen dioxide
(N02) and ozone (03). Short-term toxic effects of exposure to Oo
at concentrations attainable in ambient smog are well documented,
but comparable information on N02 exposure is lacking. In the Los
Angeles Basin, an area of high N02 pollution, peak ambient con-
centrations may exceed 1.0 ppm and one-hour average concentrations
o
of 0.8 ppm have been recorded. Adverse respiratory effects have
been reported in humans exposed briefly to concentrations of 5 ppm
g
and less. This evidence and a variety of findings from animal
studies suggest that N02 in polluted air may constitute a significant
health hazard. This possibility has been investigated in a compre-
hensive study of short-term health effects in adult male volunteers
exposed to N02 in purified air under controlled conditions realis-
tically simulating ambient exposures. Results of blood biochemical
studies are given in the preceding paper; pulmonary physiological
and clinical findings are the subject of this report.
93.
-------
METHODS
Exposure and Physiological Testing. The exposure facility,
test protocol, and design rationale have been described in detail
12
previously. Subjects (N=16) were exposed in groups of five or
six for two hours to 1.0 ppm NOo in purified air on two successive
days. (During one exposure a failure of the N0£ generator occurred,
requiring use of an alternate generator which produced a mixture of
N02 and nitric oxide. No differences in effects were found between
the group thus exposed and the groups exposed normally.) A control
study (exposure to purified air alone) was performed on the day pre-
ceding the first exposure to establish baseline values for the various
measures. Exposures were performed in a staggered manner for most
efficient use of the exposure chamber, with subjects entering and leaving
at 45 minute intervals. During exposure each subject exercised for the
first 15 minutes in every 30 at approximately 200 kgm/min--sufficient to
about double the minute volume of ventilation compared to the resting
level. Physiological testing was started after two hours and lasted for
approximately 20 minutes, during which exposure continued. The subject
then left the exposure chamber and immediately received a clinical
examination, during which venous blood was drawn for biochemical analysis,
Exposure temperature was 31±1 C and relative humidity 35 percent ± 4
percent, simulating a smoggy Los Angeles summer day. Subjects were not
told the nature of the exposure they were to receive on any given test
day, but most were able to distinguish control and exposure conditions
on the basis of the characteristic odor of N02-
94.
-------
Physiological tests performed included partial and maximum
expiratory flow-volume curves, single-breath nitrogen tests,
determination of total respiratory resistance and frequency dependence
of resistance by forced oscillation applied at the mouth, and deter-
mination of thoracic gas volume and airway resistance using a constant-
volume body plethysmograph. Flow-volume data collected included forced
vital capacity (FVC), one-second forced expiratory volume (FEV^), peak
expiratory flow rate (Vmax), and maximum expiratory flow rates at 50
• *
percent and 25 percent FVC from maximum flow-volume curves (Vrn V25)
• •
and partial flow-volume curves (V5QP> ^P). Closing volume (CV),
closing capacity (CC), residual volume (RV), total lung capacity (TLC),
and delta nitrogen (A^) were determined from single-breath nitrogen
tests. Total respiratory resistance (R^) was determined at oscillation
frequencies of 3, 6, 9, and 12 Hz. Thoracic gas volume (TGV) and airway
resistance (Raw) were determined in the body plethysmograph at functional
residual capacity (FRC) and expressed as specific airway conductance
(SGaw), equal to (l/Raw)/(TGV). Other lung volumes were determined from
plethysmographic FRC and inspiratory capacity and expiratory reserve
determined by spirometry.
Clinical Assessment. Specific respiratory and other symptoms
relatable to exposure were evaluated via a standard interview questionnaire
administered by the project physician and were expressed as a semi-
12
quantitative score, as described previously. A total symptom score was
determined for each subject on each test day, and scores on control and
exposure days were compared to test for clinical effects of exposure.
95.
-------
Subjects. Volunteer subjects were recruited from the project
investigators and staff, other hospital workers, and preprofessional
students. All were in good general health and had baseline pulmonary
function tests within normal limits, although a few had some history
of respiratory allergy. Informed consent was obtained from all subjects
prior to exposure. Individual characteristics are given in Table 1.
Data Analysis. Individual and group data were compared among control,
exposure 1 (initial), and exposure 2 (cumulative) conditions by repeated-
measures one-way analysis of variance. As effects found were small and
trends were similar in separate groups, a separate analysis was performed
in which data for three groups exposed similarly (16 subjects total) were
combined to provide greater sensitivity in detecting changes. An additional
analysis was performed excluding the data from the subjects who received
mixed oxides of nitrogen during exposure 1. No substantial changes in
results were produced by excluding these data.
Since symptom-score data were not strictly quantitative, changes in
scores between control and exposure were tested by a nonparametric method,
the Wilcoxon signed-ranks test.
Pilot Studies. Initial studies, in which the project investigators
served as the subjects (N=5), used a different protocol in that single
three-hour exposures at 22°C and 45-52 percent relative humidity were
employed, only one individual was exposed at a time, and some different
pulmonary tests were performed. In particular, esophageal balloons were
placed for measurement of transpulmonary pressure and determinations
made of static and dynamic lung compliance. Subjects were exposed to
0.5, 1, and in some cases 2 ppm .
96.
-------
RESULTS
Pilot Studies. These results have been previously reported in
brief form. None of the five subjects developed respiratory or other
symptoms attributable to exposure or changes in forced-expiratory,
single-breath nitrogen, or plethysmographic measures. Mean values for
these are given in Table 2A. Subject 7 appeared to develop frequency
dependence of dynamic compliance (i.e., reduction of compliance compared
to control measurements at frequencies of 60 to 100 breaths/min) during
two separate exposures to 1 ppm, but failed to show this effect in one
additional exposure at 1 ppm or in another at 2 ppm. None of the other
pilot-study subjects showed substantial changes in compliance under any
test condition. Measurement of compliance was omitted from subsequent
test series because of its instability and technical difficulty, and
because other, simpler techniques proved at least equally sensitive in
detecting effects of 03 exposure in related studies.
Pulmonary Physiology. Test results for the three combined subject
groups and results of analyses of variance performed on these data are
given in Table 2B. As indicated, no changes with exposure were apparent
except for a mean loss in FVC of 1.5 percent after the second exposure
as compared to control. That this change represents other than a random
variation is doubtful due to its small size, its marginal statistical
significance, and the relatively large number of statistical comparisons
being made. Similarly, data for individuals and for single groups failed
to show any marked .changes with exposure.
97.
-------
Clinical Assessment. Mean symptom scores for the three combined
groups are given in Table 3. While symptoms were increased with
exposure, particularly on the second exposure day, the increases were
not statistically significant in comparison to control symptom levels.
Only five subjects--27, 35, 36, 37, and 39—showed substantially
increased symptoms (score increased by three or more points above
control) on the second exposure day. Of these five clinically most-
sensitive subjects, only one (35) reported substernal pain and coughing
reminiscent of typical clinical effects of 03. ' These symptoms were
present only to a mild degree. Two others among the five (27 and 36)
reported lower-respiratory symptoms (cough or feeling of chest
restriction), while all five reported upper-respiratory symptoms
(nasal discharge or laryngitis).
DISCUSSION
The results of this study by themselves fail to show convincing
evidence of adverse short-term health effects-of N0£ exposure under
conditions simulating the worst expected ambient exposure in polluted
urban areas. The simultaneous blood biochemical studies on the same
subjects and others did, however, show statistically significant adverse
changes in red cells under these exposure conditions. The biochemical
findings taken together with the clinical findings in the apparently
most reactive five subjects suggest that N02 exposure at 1 ppm may not
be considered completely innocuous, even in young healthy adults.
98.
-------
Preliminary evidence suggests that individuals with pulmonary disease
are more reactive to Oo than normals; if this were true for N09,
«J L.
pulmonary disease patients might be at substantial risk at exposure
levels experienced in Los Angeles and other urban areas. Other possible
hazards of NOg relate to effects of long-term exposure, synergistic
effects with other pollutants, and effects in other high-risk groups
such as the very young, the very old, and those with preexisting
disorders of red-cell metabolism. Little information relevant to these
possibilities is available, although one study"'' investigated effects
of 0.3 ppm N02 in combination with 0.25 or 0.5 ppm 03 in a small subject
sample and found no evidence of enhanced toxicity of the mixture relative
to 03 alone.
Overall results from the series of studies performed in our
laboratory allow comparison of the short-term toxicity of N0£ and
0^ in relatively healthy adult male Los Angeles residents. Marginal
physiological effects of 03 were found at 0.37 ppm and marked effects
were found at 0.50 ppm. No effects were detectable at 0.25 ppm.
Biochemical effects followed a similar dose-response relationship except
that effects at 0.37 ppm were more pronounced relative to 0.25 ppm
or to control. Biochemical effects of 1.0 ppm N02 were generally less
than those of 0.37 ppm 03 but greater than those of 0.25 ppm 03.
As indicated in Table 4, peak N02 concentrations in photochemical smog
areas of Southern California tend to be similar to, or less than,
peak oxidant (primarily 03) concentrations, thus the apparent markedly
higher toxicity of 03 makes it of greater concern as a health hazard
99.
-------
in acute exposures. Peak levels of NCk in low-oxidant areas tend to
be higher than in high-oxidant areas, thus N02 may be of greater
relative health importance in low-oxidant areas. While Og and NO-
coexist in polluted atmospheres, the peak concentrations of each tend
to occur at different times of day and different seasons, so their
potential for additive or synergistic effects is somewhat less than
suggested by the peak concentrations in Table 4.
These studies do not negate the need for adequate controls on
emissions of oxides of nitrogen. While the short-term toxic effects
of N02 at ambient concentrations may be small in healthy subjects,
respiratory disease patients are likely to be more sensitive. Further-
more, various products of N02 atmospheric reactions may be substantially
more toxic than NCL per se> These include 03, peroxyacetyl nitrate
(PAN) and related compounds, and inorganic nitrate particulates. A
reduction of pollution by these substances is dependent on a reduction
of N02 pollution.
100.
-------
REFERENCES
1. Bates, D. V., Bell, G., Burnham, C., et al.: Short-term effects
of ozone on the lung. J Appl Physiol, 32: 176-181, 1972.
2. Hazucha, M., Silverman, F., Parent, C., et al.: Pulmonary
function in man after short-term exposure to ozone. Arch
Environ Health, 27: 183-188, 1973.
3. Buckley, R. D., Hackney, J. D., Clark, K., Posin, C.: Ozone
and human blood. Arch Environ Health,, 30: 40-43, 1975.
4. Kerr, H. D., Kulle, T. J., Mcllhany, M. L., Swidersky, P.:
Effects of ozone on pulmonary function in normal subjects.
Am Rev Respir Dis, 3: 763-773, 1975.
5. Folinsbee, L. J., Silverman, F., Shephard, R. J.: Exercise
responses following ozone exposure. J Appl Physiol,, 38:
996-1001, 1975.
6. Hackney, J. D., Linn, W. S., Mohler, J. G., et al.: Experimental
studies on human health effects of air pollutants. II. Four-
hour exposure to ozone alone and in combination with other
pollutant gases. Arch Environ Health^* 379-384, Aug. 1975.
7. Hackney, J. D., Linn, W. S., Law, D. C., et al.: Experimental
studies on human health effects of air pollutants. III. Two-
hour exposure to ozone alone and in combination with other
pollutant gases. Arch Environ Health, 30: 385-390, Aug. 1975.
8. Ten-Year Summary of California Air Quality Data 1963-1972.
California Air Resources Board, Sacramento, 1974.
101.
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9. von Nieding, G., Krekeler, H., Fuchs, R., et al.: Studies of
the acute effects of NC>2 on lung function: Influence on
diffusion, perfusion, and ventilation in the lungs, Int Arch.
Arbeitsmed, 31: 61-72, 1973.
10. Morrow, P. E.: An evaluation of recent NOX toxicity data and
an attempt to derive an ambient air standard for NOX by
established toxicological procedures. Environ Research,
10: 92-112, 1975.
11. Buckley, R. D., Clark, K., Posin, C., Hackney, J. D.: Nitrogen
dioxide inhalation and human blood biochemistry. Arch. Environ
Health, Submitted.
12. Hackney, J. D., Linn, W. S., Buckley, R. D., et al.: Experimental
studies on human health effects of air pollutants. I. Design
considerations. Arch Environ Health, 30: 373-378, Augusts 1975.
13. Hackney, J. D., Thiede, F. C., Linn, W. S., et al.: Effect of
short-term NOp exposure on lung function in normal human subjects.
Chest, 64: 395, 1973.
14. De More, W. B.: Calibration report. California Air Resources
Board Bulletin, 5(11): 1 December 1974.
102.
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TABLE 1 SUBJECT CHARACTERISTICS
SUBJECT NO. (a)
1
3
4
7
12
13- -
. 15
17
18
26
27
28
29
30
35
36
37
/->
38
39
40
GROUP (a,b)
pilot
pilot, 10
pilot
pilot
10
pilot
10
10
10
13
13
13
13
13
15
15
15
15
15
15
HEIGHT, CM
172
180
188
175
183
190
165
167
178
170
186
178
170
182
170
167
167
193
178
186
'WEIGHT, KG
84
84
88
70
75
70
60
65
66
62
77
72
66
77
70
63
66
79
67
99
AGE
48
36
43
35
28
28
23
41
27
28
23
26
24
25
24
23
. 27
27
25
23
SMOKER HISTORY
former
former
current (c)
(c,d)
(c,e)
current
(e)
(f)
(c)
former
(c,g
NOTES: (a) Studies were interspersed with others in a continuing series,
accounting for discrepancies in numbering, (b) During exposure 1 for group 13,
the N0£ generator failed, requiring use of an alternate system produced 1.0 ppm
total oxides of nitrogen of which 20-25 percent was N0£ and 75-80 percent was NO.
(c) Hay fever, (d) Rare wheezing episodes, (e) Eczema, (f) Urticaria.
(g)Childhood asthma. 103.
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TABLE 2A PHYSIOLOGICAL TEST RESULTS IN PILOT
GROUP (N-5) EXPOSED TO 1 PPM N02 FOR 3 HR--MEAN±S.D.
MEASURE
FVC
vmax
V50
CV (% VC)
CC (% TLC) (a)
TLC (b)
RV (b)
MOTES: (a) CC = CV from single-breath nitrogen test plus
RV from plethysmographic measurement.
(b) Plethysmographic measurement.
CONTROL
5.28±0.63
12.2±1.7
5.9±1.1
15.4±4.7
41.5±3.4
7.68±1.38
2.4U0.80
EXPOSURE
5.35±0.70
12.6±2.0
5.9±0.8
16.5±5.1
41.1+2.6
7.64±1.42
2.29±0.76
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TABLE 2B PHYSIOLOGICAL TEST RESULTS IN 3
COMBINED SUBJECT GROUPS (N=16) EXPOSED TO 1 PPM N0£
FOR 2 HR--MEAN±S.D. AND ANALYSIS-OF-VARIANCE RESULTS
MEASURE (a)
FVC
Vmax
V50
V25
V P
V~ P"
FEVX
cv (% vc)
CC (% TLC)
AN2
TLC (e)
RV (e)
TLC (f)
RV (f)
FRC (f)
Raw
SGaw
Rt (3 Hz)
Rt (6 Hz)
Rt (9 Hz)
Rt (12 Hz)
•CONTROL
5.44±0.92
12.1±1.68
5.51+1.49
2.31+0.62
5.67+1.49
2.22+0.69
4.44±0.74
6.0±4.5
24.4±5.2
0.66±0.17
6.58±0.96
1.13±0.30
7.12+1.02
2.05+1.43
3.58+0.61
1.20±0.26
2.36±0.80
4.26+0.96
3.36±1.10
3.09±1.15
2.95±1.11
EXPOSURE 1
5.40+0.90
12.0±1.61
5.43+1.57
2.28+0.58
5.97±1.68
2.44±0.70
4.41+0.71
5.8+3.8
24.2+3.6
0.67±0.13
6.52±0.98
1.15+0.34
7.32+1.11
1.85±0.72
3.66±0.79
1.1U0.35
2.37±0.58
4.07+1.20
3.28+0.92
2.99±0.91
2.88±0.88
EXPOSURE 2
5.36±0.90
12.0+1.52
5.39+1.46
2.46±0.82
5.76±1.23
2.48±0.74
4.42±0.70
5.3+4.8
22.7+4.3
0.65±0.15
6.56±0.96
1.13±0.28
7.30±1.04
1.90±0.77
3.69±0.80
1.12±0.25
2.42±0.64
4.09±1.19
3.62±1.45
3.16±1.19
2.90±0.88
F df (b)
3.36 2,86
<1.0 2,30
<1.0 2,30
1.66 2,30
1.08 2,8
3.12 2,8
<1~0 2,76
<1.0 2,30
2.30 2,18
<1.0 2,92
2.82 2,90
<1.0 2,90
1.42 2,30
<1.0 2,30
-<1.0 2,94
2.56 2,94
<1.0 2,36
<1.0 2,340
1.09 2,328
<1.0 2,314
<1.0 2,316
-P
<.05 (c)
NS (d)
NS
NS
NS
NS
NS
NS
MS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
105.
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TABLE 2B PHYSIOLOGICAL TEST RESULTS IN 3
COMBINED SUBJECT GROUPS (M=16) EXPOSED TO 1 PPM N02
FOR 2 HR--MEAN±S.D. AND ANALYSIS-OF-VARIANCE RESULTS
(continued)
NOTES: (a) Volumes in liters, flows in liters/sec, pressures in cm
F^O, AN£ in % N£ increase per liter expired.
(b) Degrees of freedom
(c) Exposure 2 value significantly different from control value.
(d) Not significant at .05 level.
(e) Single-breath method.
(f) Plethysmographic method.
106.
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TABLE 3 SYMPTOM SCORES FOR 3
COMBINED SUBJECT GROUPS (n=16)--CHANGE FROM CONTROL VALUE (a)
EXPOSURE 1 EXPOSURE 2
Mean change in score +0.2 +1.4
Scores increased 7 8
Scores unchanged 4 4
Scores decreased 5 4
T (b) 34.0 (NS) 22.5 (NS)
(Mean control score = 1.6)
NOTES: (a) Symptoms scored: cough, sputum production, dyspnea,
substernal discomfort, chest restriction, wheezing, laryngitis,
nasal discharge, headache, fatigue. Scoring periods: during
exposure, remainder of day after exposure completed, following
morning. Score for each symptom for each period: 0.5 = minimal,
1 = mild, 2 = moderate, 3 = severe, 4 = incapacitating.
(b) T = smaller sum of ranks for Wilcoxon signed-rank test.
T values are not significant (T <17.5 required for significant
increase of score from control, P<.05, 12 pairs of values tested,
excluding those with score unchanged.)
107.
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TABLE 4 MAXIMUM HOURLY AVERAGE N02 AND TOTAL OXIDANT LEVELS
PREVALENT IN REPRESENTATIVE AREAS OF THE LOS ANGELES BASIN, 19/I8 (a)
LOCATION POLLUTANT LEVEL EXCEEDED GIVEN PERCENT OF DAYS
West Los Angeles (b)
Central Los Angeles (b)
Azusa (b)
Riverside
N02
Oxidant
N02
Oxidant
N02
Oxidant
N02
Oxidant
1%
.46
.23
.56
.29
.30
.51
.25
.40
10%
.27
.14
.31
.17
.19
.35
.15
.27
501
.12
.08
.14
.08
.10
.14
.08
.12
MOTES: (a) Levels (in ppm) exceeded for one hour or more on the indicated
percent of days monitored. Ozone is the major contributor to total
oxidant concentrations.
(b) Oxidant levels reported for these monitoring stations have been
multiplied by 1.3 to account for differences among agencies in
calibration techniques.* Values tabulated may thus be directly
compared with those given elsewhere in this paper.
108.
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TECHNICAL REPORT DATA
iPicas,, read iKitiuctions on Hie rc\ cnc bcfoif completing)
1 REPORT NC
EPA-600/1-77-007
~'7LE ANO S
3 RECIPIENT'S ACCESSIOt*NO
Effects of Atmospheric Pollutants on Human Physiologic
Function
b REPORT DATE
January 1977
7 AUTHOR(S)
Jack D. Hackney, M.D.
8. PERFORMING ORGANIZATION REPORT NO
9 PERFORMING ORGANIZATION NAVI& AND ADDRESS
The Professional Staff Association of the Rancho Los
Amigos Hospital, Inc.
7413 Golondrinas Street
Downey, CA 90242
6. PERFORMING ORGANIZATION CODE
10. PROGRAM ELEMENT NO.
1AA601
11 CONTRACT/GRANT NO.
R-801396
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park. N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
fll .1i.no 1 1Q71 Al.mo 1Q7
FINAL
PONSORI
14. SPONSORING AGENCYCODE
EPA-ORD
15. SUPPLEMENTARY NOTES
Short-term health effects of common ambient air pollutants, particularly
photochemical oxidants, were investigated under controlled conditions simulating typica
ambient exposures. Volunteer subjects were exposed, in an environmental control
chamber providing highly purified background air, to single pollutants or mixtures
under conditions of realistic secondary stress (heat and intermittent exercise).Normal
men exposed to ozone (03) showed respiratory symptoms, pulmonary function decrement,
and alterations in red-cell biochemistry. These effects were dose-related, with appar-
ent "threshold" for detectable effect levels as low as 0.2-0.3 ppm in a 2-hr exposure
for the most sensitive subjects. Addition of 0.3 ppm nitrogen dioxide (N02) and 30
ppm carbon monoxide (CO) did not noticeably enhance adverse effects of 63, but addition
of 0.37 ppm sulfur dioxide (S02) to 0.37 ppm 03 produced slightly greater effects than
did 0.37 ppm 03 alone. Subjects with asthma of clinical airway hyperactivity appeared
to experience more severe effects of 03 than normals, and subjects chronically exposed
to ambient 03 appeared to be less reactive than those living in non-03-polluted areas.
Normal subjects exposed to N02 at 1 ppm or 2 ppm showed little clinical or physiologi-
cal response, but did show changes in red-cell biochemistry at both concentrations.
jT^ese effects we^e less than produced by 0.37 ppm 03, but significantly greater than
'produced by heat and exercise stress alone.
DESCRIPTORS
KEV WORDS AND DOCUMENT ANALYSIS
b.IDENTIFIERS/OPEN ENDED TERMS
Air Pollution
Physiological Effects
Ozone
. 1 ield'Group
06F
13 Z.ISTPIB jT c\ ST f-TEMENI
RELEASE TO PUBLIC
r B-I >=o-m :23Q-1 (9-73)
19 SECURITY CLASS • /hf, Repi-.r
_UNCLAS_SIF_I_ED _
20 SECURITY CI-AS3 '"tin page/
]_ UNCLASSIFIED
21 ',O
109
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