EPA-600/1-76-021
April 1976
TRACE SUBSTANCES AND TOBACCO SMOKE
IN INTERACTION WITH NITROGEN OXIDES.
Biological Effects
By
Gustave Freeman and Laszlo T. Juhos
Stanford Research Institute
Menlo Park, California 94205
Contract No. 68-02-1243
Project Officer
David L. Coffin
Clinical Studies Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
A T> V
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.
11
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CONTENTS
LIST OF ILLUSTRATIONS AND TABLES iii
INTRODUCTION 1
SPECIALIZED FACILITIES AND EQUIPMENT 2
RESULTS AND DISCUSSION 3
Response of Newborn Rats to N02 3
Long-Term Exposure of Primates to Nitrogen Dioxide
and Ozone 3
The Presence and Role of N02 in Tobacco Smoke and
Its Relationship to Atmospheric NO2 14
Initial Reaction Between NO2 and Tissue Components .... 17
PUBLICATIONS RESULTING FROM THIS RESEARCH 23
APPENDICES
A DELAYED MATURATION OF RAT LUNG IN AN ENVIRON-
MENT CONTAINING NITROGEN DIOXIDE 24
B ADDRESS TO CDC/EPA/WHO INTERNATIONAL SYMPOSIUM .... 31
ill
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ILLUSTRATIONS
1 Mean Body Weights and Mean Respiration Rates of
Monkeys on 0^ 2, and 9 ppm NO2 5
2 Weight Curves of Growing Stump-Tailed Macaques on
0, 2, and 9 ppm N02 6
3 Cigarette Smoking Apparatus for Rats 15
4 Modification of Smoking Apparatus 16
TABLES
1 Review of Repeated Functional Respiratory Studies on
Monkeys Exposed to 0, 2; and 9 ppm N02
2 Observed Functional Residual Capacity of Control and
2 and 9 ppm N02-Exposed Macaques
3 Functional Residual Capacity and Related Respiratory
Parameters for Control Monkey and for One Exposed to
9 ppm N02 10
4 Hematological Parameters for Control and 2 and
9 ppm N02-Exposed Monkeys 11
5 Hematologic Observations of Monkey Exposed Continuously
to 0.91 + 0.08 ppm Ozone 12
6 Hematologic Parameters for Control Monkey and for One
Exposed to 9 ppm N02 13
7 Reported Values for the Oxides of Nitrogen from
Tobacco Smoke 18
8 15N Content of Various Rat Lung Protein Samples
Exposed to 15N02 20
i s*
9 5N Content of Various Rat Lung Protein Samples
\
Exposed to 15N02 21
IV
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INTRODUCTION
During the past several years, we have been observing and reporting
the effects of oxides of nitrogen on rats and on monkeys (Macaca speciosa) .
As a result of exposure to nitrogen dioxide (N02), rats developed a disease
resembling human emphysema. In numerous publications, we have described
morphological, physiological, and hematological changes accompanying
this disease .*
During the past contract year (July 16, 1973, through September 15,
1974), emphasis was on determining the response of newborn animals living
in an environment containing N02. Also, continuing ongoing observations,
we examined:
• Both mature and newborn monkeys exposed continuously to N02 or
to ozone.
• The relative effects of tobacco smoke compared with those of NO2 .
• The binding of NO2 in tissue, based on the use of isotopically
labeled, nonradioactive 15N02.
• The detailed hematologic effects of exposure to NO2 or to ozone.
In addition to the usual parameters for detecting changes in the erythro-
cytic series, we conducted biochemical studies on blood of exposed animals.
This final report describes progress in these areas of investigation.
This report is final only in an administrative sense, since we are con-
tinuing the work in particular unresolved areas. However, the published
paper, "Delayed Maturation of Rat Lung in an Environment Containing
Nitrogen Dioxide," does describe research completed during the contract
year. Thus, this report is in its final form for Contract No. 68-02-1243,
covering the period July 1973 to September 1974.
Refer to "Publications Resulting from This Research" for the most recent
papers .
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SPECIALIZED FACILITIES AND EQUIPMENT
Approximately 3,000 square feet of laboratory space was divided into
an exposure-chamber facility and into physiology, pathology, electron
microscopy and cell biology, biochemistry, and tissue culture laboratories.
A mass spectrometer, scanning electron microscope, and a variety of other
high-precision instruments were available within the Institute for various
phases of the project.
Five 80-cubic-foot exposure chambers, each capable of holding 60
full-grown rats or four adult monkeys, were used. Each chamber was
equipped with the necessary control and safety devices. With an air-
turnover rate of 40 cubic feet per minute, the gas concentrations were
maintained within 10$ of the nominal value so that feedback control tech-
niques were not necessary. An alarm system signaled equipment failures,
and necessary repairs could be made without materially upsetting exposure
schedules.
Four NO2 exposure chambers were equipped with hardware facilities
to permit the addition and monitoring of ozone (03) concentrations in
their atmospheres.
A Bendix Model 8101-BX N02 analyzer provided by EPA was installed
for determining the concentrations of NO2 alone and in mixtures with O3.
NO2 values were confirmed by use of a modified Saltzman method on wet
samples. Ozone determinations were made on a Bendix Model 8002 ozone
analyzer, provided by SRI, and were confirmed with a Mast Meter.
The Bendix N02 analyzer has not worked satisfactorily. The manufac-
turer kept it for six weeks for repair and replacement of a failing
solenoid valve, but the analyzer still performed unsatisfactorily after
reinstallation. Our initial observations suggested a loss of analytical
range and general instrumental instability. Also, ammonia from the
excrement of animals in the chambers interfered with the determination
of N02. Although we have been communicating with the Application Research
Department of the Bendix Corporation in West Virginia to correct these
problems, no solutions appear to be forthcoming. We are considering
returning the analyzer to EPA. For the time being, we will make no
further attempts to automate analysis of N02 concentrations.
Because proper automated control, monitoring, and recording of gas
concentrations requires continuous surveillance, revision, and maintenance,
hand sampling and flow rate control are being used at least twice daily
to monitor desired concentrations. Although this system is satisfactory,
it is time consuming and inefficient. A new analyzer, preferably a Meloy,
is urgently needed.
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RESULTS AND DISCUSSION
Response of Newborn Rats to N02
With pregnancy and birth occurring regularly in smoggy atmospheres,
it is important to assess the relative vulnerability of newborn infants
and their responses to the oxides of nitrogen, especially to NC>2 . We
already were observing monkeys born to mothers exposed continuously to
either 2 or 9 ppm of N02. Offspring were born and reared in each of the
atmospheres, and those born in NO2-free chambers served as controls.
The response of monkeys to the NO2-containing environments is described
in the following sections.
Meanwhile, we initiated a study of neonatal growth, using the rat
because of its relatively shorter growth period and life span and because
of our extensive experience with that species. Results of this study
were just published in the American Review of Respiratory Disease
(Appendix A).
Long-Term Exposure of Primates to Nitrogen Dioxide and Ozone
To evaluate the influence of the oxides of nitrogen on the increasing
prevalence of chronic obstructive pulmonary diseases in man, an experiment
was designed to simulate in an animal model the insidious, long-range
pathogenesis of these diseases. In view of the 25 to 40 years required
by man to develop overt emphysema, primates were chosen for study over
rats because of their extended life span (only three years iu the rat)
and because of their closer biological resemblance to man. For more
than seven years, we have clinically observed the macaques living con-
tinuously in either 2 or 9 ppm of N02. Their growth and respiratory
rates have been monitored regularly, erythropoietic responses have been
followed, and selected studies have been performed periodically. During
the latter part of this contract year, we were developing techniques for
the determination of specific functional states of ventilation of the
exposed monkeys as compared with their controls.
For ten months, we conducted experiments involving exposure of a
monkey to ozone and planned to add NO2 to form a more realistic mixture.
Toward the end of the contract year, we planned to sacrifice the monkeys
and designed procedures for terminal evaluation of the effects of their
chronic exposure to N02 and ozone.
Following is an account of the effort expended toward the assessment
and monitoring of the influence of the atmospheric oxidants on the
monkeys.
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Growth and Respiratory Rates of Control and NO2- and 03-Exposed
Monkeys
Monitoring of the growth and clinical conditions of control and of
N02- and 03-exposed monkeys continued monthly throughout the contract
year. Body weights and respiratory rates of adult monkeys appear in
Figure 1, and the body weights of juvenile monkeys are presented in
Figure 2.
The adult control and exposed monkeys maintained relatively constant
weights, although all groups appeared to show a moderate sinusoidal
seasonal weight fluctuation.
At age five years, four juveniles (Be ?, Ce ?, In cf, and Dy
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Table 1
REVIEW OF REPEATED FUNCTIONAL RESPIRATORY STUDIES
ON MONKEYS EXPOSED TO 0, 2, and 9 ppm N02
Quarterly
Report
Group (No. /Page)
Control
An 9 15/4
19/6
33/2
Br rf 15/4
33/2
Fy $ 15/4
19/6
33/2
Rh 2 15/4
20/8
33/2
2 ppm NO2
Gy 9 20/8
33/2
Jl 9 15/4
33/2
Ls 9 15/4
19/6
33/2
9 ppm NO2
Cy
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Table 2
OBSERVED FUNCTIONAL RESIDUAL CAPACITY (FRC) OF CONTROL
(UNEXPOSED) AND 2 AND 9 ppm N02-EXPOSED MACAQUES
Weight
I.D. (kg)
Juvenile macaques
Control
In c?
Re 2
Ln d*
Av
2 ppm N02-exposed
Dy 2
Ce 2
Av
9 ppm N02-exposed
Be 2
Qa 2
Av
Adult macaques
Control
Br
-------
To examine further the FRCs of a monkey exposed to 9 ppm NO2 and
of a control, we made measurements repeatedly with a double-indicating
system. The monkeys respired in a closed system containing neon and
oxygen, with selective adsorption of carbon dioxide from the expired air.
Neon and nitrogen are expected to exchange—neon from the spirometer to
the lung and nitrogen from the lung to the spirometer—and attain equi-
librium between the lungs and the spirometer. Although, theoretically,
differences in the results of calculated FRC could be justified, the
values should be close. Table 3 presents the results of such measure-
ments .
Again, the FRC was not enlarged. The apparently greater oxygen
consumption by the monkey exposed to 9 ppm N02, despite its lower minute
volume and frequency, suggests a probable arterial-venous difference
in partial pressures of oxygen.
Assessment of Small-Airway Obstruction
Small-airway disease is indicated when compliance of the lungs
decreases as the frequency of breathing increases. Dr. William R.
Powell* conducted preliminary experiments to measure frequency-dependent
compliance for assessing small-airway obstruction in the rats developing
NO2-induced emphysema. Lungs of these rats readily demonstrated fre-
quency dependence of compliance, whereas normal control lungs showed
no decrease in compliance when respired at the same series of frequencies.
This is strong functional support for the morphologic evidence of small-
airway disease that occurs early in the pathogenesis of NO2-induced
emphysema in the rat (Freeman and Haydon, Arch. Environ. Health, 1964)
and in man with developing emphysema.
Preparatory to measuring frequency-dependent compliance on excised
lungs of monkeys, we are perfecting the procedure using exci;ed rat
lungs. The plethysmographic equipment was modified and recalibrated,
and tidal excursions are now controlled by extrapulmonary pressure changes
rather than by intratracheal positive pressure. Lungs of control and
N02-exposed rats were measured for static compliance at a transpulmonary
pressure ranging from ambient to -30 cm H20; such lungs also were measured
for frequency-dependent dynamic compliance at each cm H2O pressure decre-
ment within the same transpulmonary pressure range decrement, i.e., at
frequencies of 1 to 4 Hz. The analysis of data is in progress.
J"~Dr. Powell, a visiting scientist from the Engineering Department of the
University of West Virginia, performed these tests at no cost to the
contract.
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Table 3
FUNCTIONAL RESIDUAL CAPACITY (FRC) AND RELATED RESPIRATORY
PARAMETERS FOR CONTROL MONKEY AND FOR ONE EXPOSED TO 9 ppm N02*
Exposed to
Control 9 ppm of NO2
(Br
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Hematologic Data on Monkeys Exposed to Environmental Oxidants of
2 and 9 ppm N02 and 0.9 ppm O3
During the six years of this research, we have observed that expo-
sure to both 2 and 9 ppm N02 results initially, within two to three
weeks, in the development of a slightly microcytic polycythemia with
normal mean corpuscular hemoglobin concentrations. This polycythemia
later tended to return to normal levels as the monkeys became acclimated
to the environment.
Hematologic studies were repeated on adult monkeys that have been
exposed continuously to N02 for over six years. Circulating blood cells
were counted and evaluated, and Table 4 compares the results of the two
methods of cell enumeration and characterization. Compared with earlier
hematologic studies in the same animals, the data indicate that the
animals accommodated appreciably to the environment.
Table 4
HEMATOLOGICAL PARAMETERS FOR CONTROL
AND 2 AND 9 ppm NO2-EXPOSED MONKEYS
Numbers in Parentheses Determined
by Coulter Counter, Model S
Concentration of NO,
RBC (X 106)
Hbg (g £)
Hct <#)
MCV (n3)
MCH (nng)
MCHC ($)
WBC (X 103)
Controls
(5 monkeys)
5.6 (5.4)
13 .6* (14.1)+
42.6 (41.3)*
77* (76)
24.3 (25.9)*
-- (34.4)*
12.7 (12.1)
2 ppm
(6 monkeys)
5.5 (5.6)
13.3 (14.2)
43.4 (41.4)
80 (74)
24.2 (25.3)
— (34.3)
16.1 (14.8)
9 ppm
(4 monkeys)
6.0 (6.0)
14.7 (14.8)
48.5 (44.4)
81 (77)
24.5 (25.5)
-- (33.8)
14.0 (13.9)
Wong iron method.
'Coulter colorimetric method.
Calculated .
11
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One macaque (Rh ?) was exposed to 0.91 ± 0.08 ppm O3 for 28 days.
This exposure level was at the upper limit of the analytical capability
of the Bendix chemoluminescence ozone analyzer. This 7.4-lb adult monkey
showed no evidence of difficulty in tolerating this concentration.
Hematologic evaluation of the monkey was made just before exposure,
and 24 and 72 hours and 4 weeks after the introduction of ozone. The
available hematologic results, presented in Table 5, demonstrate early
development of polycythemia. By the third day, accommodation is evident,
although young cells—indicated by their reduced fragility—still pre-
dominate. Complete adaptation was achieved before day 28.
Table 5
HEMATOLOGIC OBSERVATIONS OF MONKEY (Rh ?)
EXPOSED CONTINUOUSLY TO 0.91 ± 0.08 ppm OZONE
Sample
RBC Hct
(X lOVmm3)
Pre-exposure
Post-exposure
24 hr
72 hr
28 days
8 months
4.8
6.2
5.5
4.9
6.7
42
47
46
40.5
42.0
WBC
(X lOVmm3)
14.4
14.0
13.5
12.5
13.2
Hb
(gm/100 ml)
13.21
14.77
13.41
12.06
Osmotic
Fragility*
0.42
0.40
0.38
0.42
0.42
Arbitrary units.
Rh ? now has been exposed continuously to 0.9 ppm ozone for ten
months. Its survival suggests that monkeys are more resistant than rats
to ozone, since this exposure level resulted in an LD50 of about 19 days
for rats. We plan to evaluate the ozone-caused changes after the sacri-
fice of the monkey.
Preparatory to sacrifice of the selected monkeys and assessment of
the damage of atmospheric oxidants to the lungs of monkeys, we measured
hematologic parameters for another monkey exposed to 9 ppm N02 and then
compared them with those of an unexposed monkey of approximately the
same age and weight. Table 6 presents the results.
12
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Table 6
HEMATOLOGIC PARAMETERS FOR CONTROL MONKEY
AND FOR ONE EXPOSED TO 9 ppm N02
Control Exposed
(Br cO (Gr cO
RBC (lOVmm3) 5.50 5.25
WBC (lOVmtn3) 11.5 10.4
Hematocrit ($) 44.5 41.5
Hemoglobin (g/100 ml) 13.51 13.48
No significant differences exist within this pair of monkeys.
Plans for sacrificing N02-exposed monkeys (and appropriate controls)
have been formulated for the final evaluation of the changes sustained
by their lungs. Evaluation will include measurements of:
(1) Physiological functional parameters in vivo, mainly carbon
monoxide-diffusing capacity and FRC.
(2) Hematological profile including hematocrit, total white and
red cell counts, differential counts, mean corpuscular volume,
and red cell fragility.
(3) Selected chemical parameters of blood reported to be influenced
by exposure to atmospheric oxidants, such as erythrocytic
cholinesterase, tocopherols, glucose-6-phosphate dthydrogenase,
2,3-diphosphoglycerate, and glutathione reductase.
(4) Pulmonary cell renewal as determined with tritiated thymidine,
(5) Selected chemical constituents of lung tissue such as gluta-
thione reductase and tocopherols.
(6) Static and dynamic compliance and resistance to airflow of
the excised lungs.
The whole monkey will undergo pathological examination, and the
lungs will be studied by electron microscopy.
Sacrifice of the monkeys will commence when plans and procedures,
as well as time tables of processing, are finalized.
13
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The Presence and Role of NO2 in Tobacco Smoke and Its Relationship to
Atmospheric N02
These preliminary studies related to the content of N02 in cigarette
smoke likely to be inhaled and likely to contact pulmonary tissue in vivo.
Although we did examine using rats the apparatus we developed, the chang-
ing interests of the Environmental Protection Agency precluded our con-
ducting detailed experiments. Progress made thus far is as follows.
During the 1972-73 contract year, we had constructed a cigarette
smoking apparatus for rats (Figure 3). Initial tests of moisture reten-
tion of the original glass "trachea" showed that the unit could not be
kept uniformly wet. An improved glass "trachea" was designed using a
porous glass tube filter enclosed in a glass envelope (Figure 4). A
special vessel for humidifying the air was built.
Initial testing of the smoking unit involved burning Camel cigarettes
in the apparatus while simultaneously removing a sample of smoke from
one of the rat "portholes." The sample was to be analyzed continuously
with a Bendix Model 8101-B (chemoluminescent) oxides of nitrogen analyzer
connected to a recorder. Further experiments were planned to determine
the NO/ft02 ratio produced by the machine at various dilutions.
The N02 content of the tobacco smoke reaching the lung, as simulated
in our smoking machine, could not be analyzed by direct sampling with
the Bendix gas analyzer. Removal of the aerosol by a millipore filter
did not provide a sufficiently clean gas sample suitable for analysis
by the chemoluminescence reaction. To solve this problem, we considered
removing all particulate matter from the tobacco smoke by filtering and
cryogenic techniques .
We found a filter that was potentially suitable for removing particu-
late material from fresh cigarette smoke and for producing useful samples
for gas analysis by chemoluminescence. The Teflon filter material removed
all microscopic particulate matter from smoke and retained 99^ of the
gaseous components that were reactive with Saltzman's reagent. However,
the filter proved to have a finite capacity to hold back such components;
once this limit was reached, gases were allowed to pass through.
The reactivity of the retained particulate matter with the Saltzman
reagent could not be ascertained readily for its relationship to NO2 or
other reactive organic or inorganic nitrites.
The cryogenic fractionation of the fresh tobacco smoke, although
potentially effective as a separation technique, would not produce a
continuous stream of sample analyzable by either the Saltzman or the
chemoluminescence methods, so we have not yet attempted using the
technique.
Progress in these evaluations also has been hindered by the unsatis-
factory performance of the Bendix N02 analyzer, a problem compounded by
14
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FIGURE 3 CIGARETTE SMOKING APPARATUS FOR RATS
15
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SMOKE SOURCE CHAMBER
FIGURE 4 MODIFICATION OF SMOKING APPARATUS
16
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the interference of ammonia with the chemoluminescence method of analysis
that we discovered. This problem warrants further analytical work.
We reviewed the literature to gather reported values for the oxides
of nitrogen contained in tobacco smoke. Table 7 lists these values
together with references. Because the wide range of reported values
renders the estimation of exposure of smokers rather difficult, the
design of realistic model experiments is frustrating indeed.
Most of the analytical work on the oxides of nitrogen in cigarette
smoke was reviewed by R. L. Stedman'"' and in a subsequent symposium on
the chemical composition of tobacco and tobacco smoke held in Washington,
B.C., in 1971, summarized by I. Schmeltz in The Chemistry of Tobacco and
Tobacco Smoke. We found no publications after 1971 on the analytical
problems involved in measuring the oxides of nitrogen in tobacco smoke.
Part of the difficulty in achieving accurate analysis in tobacco
smoke is the lack of direct, specific analytical methods for the indi-
vidual oxides and the continuous intermolecular chemical reactions
among them.
Our theoretical calculations indicate that NO would oxidize to
NO2 at a rate of 0.001 ppm/sec, using the rate constant of k = 7.5
X 10~10ppm~2min~1 for the reaction of 2NO + 02 - 2 N02 applied to the
average 50-ml puff of cigarette smoke, containing about 300 ppm of NOX
and diluted to 500 ml in the process of inhalation with 20$ oxygen-
containing air. Since these values are based on in vitro conditions,
extrapolation to in vivo would be difficult.
Initial Reaction Between NO2 and Tissue Components
Not only is the biochemical fate of N02 obscure, but its initial
reactions with tissues also are unknown. As a result, our investigations
were initiated along two lines. One was to observe reactions of thin
films of representative model molecular species with gaseous NO2 in
chambers; the other was to identify reaction products between isotopically
labeled 15N in 15NO2 and exposed tissue, both as a homogenate and as
intact lungs of rats in vivo. The first approach revealed a striking
reactivity of vitamin E (Cf-tocopherol) and also of vitamin A with NO2.
(This avenue is being pursued further under an NIH grant.) The other
area of investigation is directed toward determining whether the nitrogen
in NO2 is covalently bound to tissue elements during exposure.
R. L. Stedman, The chemical composition of tobacco and tobacco smoke,
Chem. Rev. 68, 153-207 (1968).
17
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Table 7
REPORTED VALUES FOR THE OXIDES OF NITROGEN FROM TOBACCO SMOKE
Reference
Haagen-Smit et al. Arch. Ind. Health
20, 53/399-54/400 (1959)
Bokhoven et al. Nature, p. 458,
Nov. 4, 1961
Kensler et al. New Engl. J. Med.
269, 1161 (1963)
Tada et al. Rep. Inst. Sci. Lab.
Kurashiki 60, 7_ (1962)
Wescott et al. 18th Tobacco Chemist
Research Conference, Raleigh, 1964
Norman et al. Nature, p. 915,
Feb. 27, 1965
Newsome et al. Tobacco Science 9_,
102 (1965) ~
P. H. Abelson. Science, Dec. 22,
1967
G. Neurath. Experientia 23_, 400-404,
(1967)
Method of
Analysis
Saltzman*
"Normal
analytical
methods"
Saltzman*
Saltzman*
Saltzman*
Concentration of NOX
Cigarette
NO + N02 145-655 ppm
(v/v)
Cigar
NO + NO2 167-1250 ppm
(v/v)
Pipe tobacco
NO + N02 126-1154 ppm
(v/v)
Cigarette
NO + NO2 170-210 ppm
NO2 80-120 ppm
Cigarette
50 |_Lg/puff of N02
NO 72-271 ppm
NO2 19-118 ppm
NO 96-1120 ppm
Cigarette
NO 698-1008 ppm
NO2 0-25 ppm
Cigarette
NO 500-800 ppm
N02 20-80 ppm
Cigarette
NO2 250 ppm
No quantitative data; the
path of NO and N02 to
nitrosamines is described
Rowlands et al. Environ. Res. 2, 47- Electron
71 (1968) spin
resonance
Implied qualitative pres-
ence of NOX; no quantita-
tive data
*Saltzman, Anal. Chem. 26, 1949 (1954)
18
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The potential relevancy of these studies to lung cancer, as well as
to emphysema, is considerable because nitrous acid, a product of NO2 and
water, forms nitrosamines in the presence of secondary amines, and certain
of these are potent carcinogens.
Several other experiments were conducted, and typical data are
compiled in Table 8. In the first type of experiment, frozen normal
rat lung was powdered and exposed to 0.7 mmol of 15NO2 for 30 minutes.
Chloroform/methanol (1/1) was added to remove lipids. The lipid-free
residue was suspended in an NaNO3-NaNO2 solution for 2 hours, removed
by centrifugation, dialyzed overnight, and then recovered by centrifuga-
tion. A portion was removed for 15N analysis, and the remainder was
hydrolyzed in 6N HC1 at 110°C for 18 hours. The acid was removed by
evaporation in a vacuum desiccator containing KOH pellets. The residue
was taken up in water and applied to a Dowex 50 (acid form) column, which
was then washed with 50 column volumes of H2O . The amino acids were
removed with 5 M NH4OH and recovered by evaporation. A portion was taken
for * 5N assay, and the remainder was reacted with ninhydrin to release
amino nitrogen, which also was assayed for 15N.
Table 9 shows an increased level of 15N in all fractions assayed.
The 15N content also was elevated when the lung homogenate was first
treated with 10$ TCA for 1 hour and then boiled for 2 hours before 15N02
exposure. When 1 ml of 0.05 M phosphate buffer (pH 7.4) is exposed to
the equivalent amount of 15NO2 for 30 minutes, flushed with nitrogen,
and the powdered lung is added, significant amounts of 15N appear in
protein amino nitrogen. Thus, when rats were exposed to 18 ppm 15NO2
for 6 hours, significant amounts of 15N were found in lung protein, and
lesser amounts were found in plasma and red blood cells. These experi-
ments suggest the incorporation of the nitrogen of 15N02 into lung and
blood protein of rats.
Further work was conducted to determine the location of 15N in
protein, lipid, and other cellular constituents, the mechanisms of incor-
poration, and the effect of incorporation on biochemical parameters.
The data in Table 9 reflect incorporation of 15N from 15N02 into the
amino nitrogen of amino acids derived from lung protein of exposed lung
homogenate (Experiments 3 through 8). When homogenate was exposed to
15NO3~ and 15N02~ (prepared by reacting 15NO2 with buffer), the amino
nitrogen of amino acids became labeled also (Experiment 9), suggesting
that one or both of the ions were intermediates in the reaction of N02
with lung homogenate. However, incorporation was equally effective
when lung homogenate was first boiled and then treated with trichloro-
acetic acid (Experiment 10), indicating a nonmetabolic reaction. The
presence of J5N in amino acid nitrogen is surprising because conversion
of NO2 to R-NH2 probably would be metabolic. Our tentative explanation,
therefore, is that the reaction of 15NO2 (and J5N03~ + 15N02~) with
tissue was primarily chemical in nature, but that a portion of the
initial product(s) was metabolized to ammonia during the 30-minute
incubation period and was incorporated into amino acids at that time.
19
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Table 9
15N CONTENT OF VARIOUS RAT LUNG PROTEIN SAMPLES EXPOSED TO 15NO2
Experiment
1 In vitro
2 TCA control
Fraction
Unhydrolyzed protein
Amino acids
Amino nitrogen
Unhydrolyzed protein
Amino acids
Atom
Percent
15N
0.50
0.60
0.85
0.53
0.72
3 Ions Unhydrolyzed protein 0.72
4 In vivo RBC - Unhydrolyzed protein 0.32, 0.67
Plasma - Unhydrolyzed protein 0.45, 0.64
Lung - Unhydrolyzed protein 0.72, 0.85
5 14N control Lung - Unhydrolyzed protein 0.34, 0.41
6 Natural abundance
Literature 0.37
SRI 0.36, 0.38
21
-------
Throughout the experimental work, we have followed the fate of the
nonradioactively labeled NO2 by mass spectrometry; these analyses have
been supported by this contract, and the rest of the endeavor has been
funded by an NIH grant. The results in Tables 8 and 9 depend on an as-
yet-unresolved question about the relative validities of using masses 14
and 15 or 28 and 29 as ratios between the normal and isotopic nitrogens
in the spectrometric analyses. The data presented were based on the
former ratio. When N2 masses are used as the baseline, there is no
clear evidence of incorporation or reaction of the isotopic nitrogen
with any tissue component. This impasse is to be resolved.
22
-------
PUBLICATIONS RESULTING FROM THIS RESEARCH
Published
Changes in Dogs' Lungs After Long-Term Exposure to Ozone. G. Freeman,
R. Stephens, D. Coffin, and J. Stara. Arch. Environ. Health 26,
209 (1973) .
The Effects of Mixed Sodium Chloride Aerosol and Nitrogen Dioxide in
Air on Monkeys and Rats. N. J. Furiosi, S. C. Crane, and G. Freeman.
Arch. Environ. Health 2T_, 405 (1973).
Cytological Changes in Dog Lung Induced by Chronic Exposure to Ozone.
R. Stephens, G. Freeman, J. F. Stara, and D. L. Coffin. Am. J.
Pathol. 73_, 711 (1973).
Delayed Maturation of Rat Lung in N02-Containing Environment. G. Freeman,
L. T. Juhos, N. J. Furiosi, R. Mussenden, and T. A. Weiss. Am. Rev.
Respirat. Disease 110, 754 (1974). (See Appendix A)
In Preparation
The Effects of Long-Range N02 Exposure on Monkey Lungs.
Other
Dr. Freeman presented a paper, "Criteria from Animals Exposed to
Known Concentrations of Nitrogen Dioxide and Ozone, With Potential Use
in Epidemiology," at the CDC/EPA/WHO International Symposium in Paris
(24-28 June 1974). A copy of the manuscript is presented in Appendix B.
Also published was "Pathology of Pulmonary Disease From Exposure to
Ambient Gases (Nitrogen Dioxide and Ozone)," G. Freeman, L. T. Juhos,
N. J. Furiosi, R. Mussenden, R. J. Stephens, and M. J. Evans, Arch.
Environ. Health 29^, 203 (1974).
This research was performed principally with funds provided by the
National Institutes of Health. However, the Environmental Protection
Agency participated in having shared in the support of the central
facility for controlled exposure of animals.
23
-------
Delayed Maturation of Rat Lung in an Environment
Containing Nitrogen Dioxide"
GUSTAVE FREEMAN, LASZLO T. JUHOS, NAZZARENO J. FURIOSI,
ROWENA MUSSENDEN, and THOMAS A. WEISS
SUMMARY.
The maturation of lungs was monitored by morphomctric quantification of the number of alveoli
in rats born and raised for 75 days in "clean" air and in air containing !."> ppm of nitrogen dioxide.
Although numbers of alveoli in control lungs and lungs exposed to nitrogen dioxide were com-
parable at birth, there was a consistent delay in maturation in the exposed lungs from the ages of
3 to 60 days. At 75 days, i.e., at the termination of the experiment, however, the deficit had been
made up. Thus, the delay in maturation is probably not an appreciable factor in the attrition of
alveolar surface area apparent in mature rats exposed similarly for months or years.
Introduction
The number of parenchyma! air spaces and the
related gas-exchanging surface area were re-
duced in emphysematous rats that had been ex-
posed continuously to nitrogen dioxide (NO2)
from weaning through old age (1-4). It was
considered that this deficiency might have re-
flected a failure in early maturation, in part,
and attrition of pulmonary tissue later, due
to the continuous exposure. To test the concept
of early retardation of growth in a preliminary
experiment, the lungs of 90-day-old rats that had
(Received in original form June 28, 1974 and in
revised form September 6,1974)
i From the Department of Medical Sciences, Stan-
ford Research Institute, Menlo Park, California
94025.
2 This work was supported mainly by the Division
of Health Effects Research, National Environmen-
tal Research Center, Environmental Protection Agen-
cy, Research Triangle Park, North Carolina, Con-
tract No. 68-02-1243, and partly by the National
Institute of Environmental Health Sciences, Nation-
al Institutes of Health, Bethesda, Maryland, Grant
No. ES-00842-02.
3 Requests for reprints should be addressed to
Gustave Freeman, M.D., Stanford Research Institute,
333 Ravenswood Avenue, Mcnlo Park, California
94025.
been exposed from birth to approximately 10
ppm of NO2 were studied. No deficiency in the
number of alveoli was revealed. Subsequently,
the effects of 15 ppm of NO2 on the lung were
explored more definitively during growth from
birth to 75 days. Evidence indicating a tem-
porary lag in "septation" of air spaces during
the first 6 weeks after birth is presented here.
Material* and Methods
Experiment A: Ten rats, approximately 14 days'
pregnant, were placed in chambers containing 10 ±
2 ppm of NO2 in air, where they subsequently de-
livered their progeny. A comparable number deliv-
ered their offspring in a controlled environment of
ambient air without added pollutants. Progeny were
weighed at 62 days ± 1 day, and their skeletal length
was measured from the tip of the nose to the end of
the outstretched tail. At age 90 days, randomly se-
lected male and female control rats and rats exposed
to NO2 were sacrificed, and their lungs were pre-
pared for quantitative assessment of alveolation, as
described earlier (1).
Experiment B: Hilltop rats, 18 days' pregnant,
were placed either in exposure chambers contain-
ing approximately 15 ppm of NO£ or in an environ-
ment without added contaminants. The concentra-
tion of NO2 was monitored by frequent sampling
and use of the Saltzman reaction. Three rats from
each of 3 different litters born in environments
754
AMERICAN RF.VIKW Oh IO SI'lR A I ORV DISK-VSt, VOLl'MK 110, 197^
25
-------
DELAYED LUNG MATURATION ON EXPOSURE TO NITROGEN DIOXIDE
755
TABLE 1
RELATIVE ALVEOLAR MATURATION AT 90 DAYS OF AGE IN NEONATAL
CONTROL RATS AND RATS EXPOSED TO 10 PPM OF NITROGEN DIOXIDE
Lungs
Mean ±SD Grid Count'
(alveoli/equivalent
square)
Mean + SD Lineart
(points on equivalent
length of line)
Control
Exposed to NC>2
50.5 ±11.5
49.0 ± 14.2
22.1 ±3.0
23.5 ±4.9
'Approximately 2,300 alveoli per lung were counted.
^Approximately 400 line Intercepts per lung were counted.
with and without NO2 were removed at 1,3, 5, 10,
15,20,30,40,50,60, and 75 days.
After anesthesia was induced with sodium pento-
barital, the lungs were exposed, and the trachea was
cannulated with a shortened hypodermic needle. The
lungs were left in the opened thorax of rats younger
than 10 days, but were excised from older ones. An
apparatus was constructed for fixing the lungs with
formaldehyde steam, according to the method of
VVeibel and Vidone (5), and was modified to accept
a hypodermic needle-cannula. The degree of disten-
tion of the lungs was controlled by holding the pres-
sure of the fixing chamber at —15 cm H2O and
keeping the pressure of formaldehyde slightly higher
than atmospheric, sufficient to overcome the icsis-
tancc of the cannula, which was considerable in
the small cannulas used in very young rats. This re-
sulted in uncertain pressure control of the formal-
dehyde steam, as reported also by Weibel (6).
Fixed lung volume was determined by water dis-
placement. Lungs of animals of adjacent ages (hav-
ing similar volumes) were grouped, and an average
age was assigned. Histologic slides were prepared by
cutting 4-fi sections uniformly through the great-
est diameter of the right lung from apex to base.
Adjacent sections were stained with hematoxylin-
eosin and with acid orccin. Counts on 10 randomh
selected parcnchymal fields of each lung were made
at a magnification of 200X. The 10 fields covered ex-
tensive portions of the parenchyma at all levels of
the relative!) small neonatal lungs. Equivalent poi
tions of all lobes were counted in all lungs.
The proportion of space to tissue in the paren-
chyma was determined by the point-counting
method (7, 8), without differentiating between al-
veoli and ducts, and by using a 100-point integrat-
ing eyepiece (Zeiss). The transections of alveolai
and ductal airspace units also were counted (7,
8). Transections extending beyond the upper hor-
izontal and the righthand vertical limits of the in-
tegrating eyepiece field were included, and those that
extended beyond the lower horizontal and lefthand
veitical borders were excluded. The parenchymal
portion of a lung was assumed to represent 90 per
cent of the whole lung volume, as estimated by Wei-
bel (8), Dunnill (9), and Angus and Thurlbeck
(10).
Result*
The relative number of alveoli in the lungs
from Experiment A was determined as described
earlier (1). The relative numbers of alveolar
air spaces per unit volume of lung, adjusted for
differences in distention, were proportionally
equivalent to absolute alveolar counts in Exper-
iment B (table 1). No significant difference was
found between the degrees of alveola tion in
control lungs and lungs exposed to NO2 at age
90 days.
TABLE 2
EFFECT OF EXPOSURE SINCE BIRTH TO APPROXIMATELY 10 PPM OF
NITROGEN DIOXIDE ON WEIGHT AND LENGTH OF RATS DURING
62 DAYS OF GROWTH
NC-2
Exposure
(ppm)
0
10 + 2
P value
0
10±2
P value
Average Weight
(g>
No.
12
31
5
10
Sax
M
M
F
F
Mean
313.8
275.7
< 0.001
210.8
190.8
< 0.025
SD
28.1
30.6
10.9
15.4
Average Height
(cm)
Mean
40.9
38.6
< 0.001
37.9
35.5
< 0.005
SD
1.5
1.4
0.9
1.2
26
-------
756
FREEMAN, JUHOS, FURIOSI, MUSSENDKX, AND WEISS
The body weights and skeletal lengths for con-
trol rats and those exposed to 10 ppm of NO2
at 62 days of age are presented in table 2.
In calculating the number of parenchymal
air spaces for Experiment B, the method of Wei-
bel (7, 8) was used, as expressed by the for-
mula:
NL = 0.9 x VLF X
where NL = total number of parenchymal (al-
veolar plus ductal) air spaces in the whole lung;
0.9 = the assumption that 90 per cent of the
whole lung is parenchyma; VLF = volume of
whole lung (in milliliters) after fixation; n =
number of transectional units in 1 cm2 of histo-
logic preparation of parenchyma; 3/2 — con-
version factor from 2-dimensional planar to 3-
dimensional volumetric units; f)= 1.92, a shape
coefficient (8) based on the assumption that
two thirds of the parenchymal air space consists
of spherical alveoli, and one-third consists of
cylindrical alveolar ducts; 8 = proportion of
space to tissue in the parenchyma, determined
by point counting.
Lung volumes and transectional and point
counts are presented in table 3. At the termina-
tion of the study (75 days), lung volumes had
not readied a plateau and presumably were con-
tinuing to increase in porportion to body weight
until maturity; however, from approximately 25
days of age, the lung volumes of rats exposed to
NO2 were considerably larger than those of con-
trol rats. The proportion of space to tissue fluc-
tuated with age more in the parenchyma of con-
trol lungs than in lungs exposed to NO2, and
the mean value was slightly higher in control
lungs. The number of parenchymal air spaces
per unit volume (1 cm3) of parenchyma and
per whole lung (figure 1) are presented in ta-
ble 4. Also listed are the computed rates of for-
mation of individual parenchymal airspace units
between the sampling periods and the average
volumes.
The significant difference (P < 0.05) between
the over-all means of the transectional counts of
TABLE 3
VOLUMES AND TRANSECTIONAL AND POINT COUNTS OF PARENCHYMAL AIR SPACES
IN MATURING CONTROL LUNGS AND LUNGS EXPOSED TO NITROGEN DIOXIDE (NO?)
Afle
(days) No.
Fixed Lung
Volume
(cm*)
Mean SE
No. of Tran»ection»/Grld of 0.0016 cm2
Per cm2
Mean SE (X 103)
Proportion of Space
to Tissue in
Parenchyma bv
"oint Counts
<%)
Mean SE
Rats exposed to air
1
4
10
15
25
40
50
60
75
Mean
0.30
0.90
1.23
1.59
2.58
2.60
3.48
5.70
7.05
0.08
0.09
0.08
0.42
0.51
0.03
0.65
0.35
0.70
16.8
16.2
30.9
34.0
31.1
41.8
34.8
26.7
23.4
28.4
Rat* exposed 19 approximately 15 ppm of NO? in air
1 2
3 3
10 9
20 3
35 5
55 4
75 3
Mean
P value (control
versus experimental)
0.35
0.60
1.02
2.55
3.90
7.64
7.95
0.15
0.10
0.16
0.00
0.68
1.53
2.30
19.5
18.9
21.6
23.0
19.8
20.6
21.7
20.7
<0.05
0.80
0.95
1.61
1.67
1.50
2.60
2.56
2.00
1.51
1.52
1.20
0.92
1.99
1.59
1.32
2.35
10.5
10.1
30.9
21.2
19.4
26.1
21.7
16.7
14.6
19.0
12.2
11.8
13.5
14.4
12.4
12.9
13.6
13.0
< 0.05
71.8
69.9
65.4
66.7
66.9
49.8
58.8
66.2
67.2
61.4
69.4
69.2
69.9
73.9
71.1
69.9
1.67
1.48
1.15
1.09
1.73
2.48
5.05
1.90
2.01
9 79
3.73
0.90
3.00
1.41
3.14
1.80
27
-------
DEl.AYKD ITNC; MVILRV1ION ON KXI'OSLRK TO NITROGEN' DIOXIDE
757
the lungs from control rats and rats exposed to
NO2 (table 3) generated the significant differ-
ence between the means of the number of par-
enchymal air spaces per unit volume (1
cm8) in table 4. In lungs from both 1- and 75-
day-old control rats and rats exposed to NO2,
the total numbers of parenchymal airspace units
were approximately equal. Between the ages of
10 and 45 days, however, there was approxi-
mately a 36 per cent deficit in the number of
parenchymal airspace units among the rats ex-
posed to NO2 and, presumably, in "over-all
pulmonary maturation" (figure 1). Compari-
son of the slopes of the growth curves between
these ages for both groups revealed a significant-
ly reduced rate of "septation" for the rats ex-
posed to NO2 (P < 0.05).
The calculated formation rates of new paren-
chymal airspace units varied between lungs
from control rats and rats exposed to NO2. Be-
tween the ages of 4 and 10 days, the rate of ap-
proximately 7,400 units per day in the control
group was 32 per cent higher than that in the
group exposed to NO2; however, during the lat-
ter part of the experimental period, the growth
rate of new air spaces in the lungs exposed to
NO2 was somewhat higher than that in con-
trol lungs.
Variations in the average size of individual
parenchym.il airspace units in the control
group indicate that normal growth of lungs in-
volves periodic expanding and stretching of the
parenchyma in addition to formation of new
units. In contrast, the relative homogeneity in
average size of individual parenchymal airspace
units in the lungs exposed to NO2 suggests per-
sistent distention with increased volume of in-
dividual air spaces of approximately 30 per
cent.
Discussion
The total alveolar count in lungs was used as a
measure of pulmonary maturation by Dunnill
(9) and Davies and Reid (11) in children
from birth to 8 years of age, and by Angus and
Thurlbeck (10) in adults from 12 through 85
years of age. In rats, \Veibel (12) and Burri
and associates (13) used parameters other than
direct alveolar counts. Bartlett (14-16) studied
the effects of altitude and endocrine constitu-
ents on pulmonary development in terms of
numbers of alveoli.
It was apparent that Weibel's formula (8)
for enumerating parench>mal air spaces in the
normal lung might not be appropriate when
applied wholly to lungs altered by disease. Dis-
eased lungs with changes in cellularity, connec-
tive tissue, fluid content, types of cells, thickness
of basement membranes, and alterations of al-
veolar septa and of distensibility under stan-
dard pressure (—15 cm H2O) might not meet
the criteria for shapes of air spaces (/3) or pro-
portions of space to tissue (S) or both applied
to normal lungs. Fortuitously, the over-all aver-
age ratio of space to tissue (table 3) revealed
little or no difference between the control group
and the group exposed to NO2, although there
was greater homogeneity of this ratio among the
latter. During growth of diseased animals, there-
fore, maturation in terms of total number of
parenchymal air spaces can only be expressed
as an estimate.
Maximal growth rate of airspace units (table
4) occurred between 4 and 10 days of age in the
control group. This is consistent with "Phase
II" of pulmonary alveolation described by
Burri and associates (13). The lack of statisti-
280
260
•S
I 240
o
.c
I 220
C/>
5 200
13
LU
< 180
Q_
E 160
< 140
5 120
< 100
80
O
cc
CO
i 60
<
O
40
20
I I I I 1 I
- SLOPE
NORMAL = 5.4
15 ppm NO2 EXPOSED = 3.4
p = < 0.05
• Normal (Control)
o 15 ppm NOj Exposed
I
10
20
30 40 50 60 70 80
AGE — days
Fig. 1. Comparative growth of parenchymal airspace
units in lungs from maturing control rats and rats
exposed to 15 ppm of nitrogen dioxide (NO2).
28
-------
758
1REEMAN, JL'HOS, KURIOSI, MLSSEMJEN, AM) WEISS
TABLE 4
NUMBER OF PARENCHYMAL AIRSPACE UNITS (ALVEOLAR AND
DUCTAL), RATES OF FORMATION, AND INDIVIDUAL VOLUMES
IN LUNGS OF MATURING CONTROL RATS AND RATS EXPOSED
TO NITROGEN DIOXIDE (N02)
Age
(days)
No.
No. of Parenchymal Airspace Units
Per Unit Formed
Volume Per Whole Lung per Day
(X 703,) (x 703; fx
Volume of
I ndividual
Airspace Units
(ml X 10-s)
Exposed to air
1
4
10
15
25
40
50
60
75
Mean
20.9
20.0
54.6
62.2
54.4
98.3
68.3
43.7
35.3
50 9
5.6
16.2
60.4
89.1
126.3
230.0
214.8
224.0
224.9
Exposed to approximately 15 ppm of NO2 In air
1
3
10
20
35
55
75
Mean
P value (control
versus experimental)
28.3
25.3
31.0
34.1
26.5
28.6
31.2
29.3
<0.05
8.9
13.7
28.5
78.2
93.0
196.8
223.3
3.2
7.4
5.7
3.7
1.9
2.4
2 1
5.0
2.6
4.8
5.0
1.8
1.6
1.8
1.0
1.5
2.3
2 8
3.5
3.9
3.2
2.9
3.8
3.5
3.2
cal distinction in number of air spaces beyond
the age of 60 days supports the earlier observa-
tion in 90-day-old rats exposed to approximately
10 ppm of NO2; however, the difference in
slope of the two "alveolar growth" curves (fig-
ure 1) between 10 and 45 days of age indicates
a temporary lag, or delay, in pulmonary matura-
tion in rats exposed to NO2. This effect may re-
flect an error in assuming identical coefficients
for alveolar shape for both control lungs and
lungs exposed to NO2. Bignon and associates
(17) also did not use an altered value in their
detailed study of human chronic, obstructive,
bronchopulmonary disease.
Some pregnant rats died from the NO2 expo-
sure before delivering their progeny, and lit-
ters generally were smaller than those of control
rats. The mortality of the exposed progeny re-
mained high for 15 days, indicating that ani-
mals used in our experiments were survivors
of an environment that was only marginally com-
patible with life.
The extent ol the observed lung enlargement
in these experiments is similar to that reported
by Bartlett (14) in studies of altitude-imposed
hypoxia, but it is unlikely that the basic mecha-
nisms are the same.
It was anticipated that continuous exposure
of newboin animals to a high "subacute" con-
centration of NO., would result in injury and
disease to their lungs, as it did in older animals
(1-4). Although the pulmonary tissue fixed
with volatilized formaldehyde did not provide
optimal visualization of pathologic detail, mi-
29
-------
DELAYED LUNG MATURATION ON EXPOSURE TO NITROGEN DIOXIDE
759
croscopy revealed a lack of both fixed tissue re-
action and macrophage aggregation in these im-
mature lungs exposed to NO2.
The objective of this study was to explore fur-
ther the basis for the reduced number of par-
enchymal air spaces in the lungs of rats ren-
dered emphysematous during long-term, inter-
mittent exposure to approximately 15 ppm of
NO2 from the age of 1 month (1). The data
support evidence for loss of parenchyma! tis-
sue essentially by a destructive process during
the latter part of the animals' lives, although
there also may well have been a transient neo-
natal delay in maturation. Such an effect was
unlikely, however, because the emphysematous
rats (1) had not been exposed to NO2 until
they were at least 1 month old.
References
I. Kieeinun, C., Crane, S. C., F'uriosi, N.J., Stephens,
R. J., Evans, M. J.. and Mooic, W. D.: Covert
leduction in vcntilatory surface in rats duiing
prolonged cxposuic to subacute nitrogen diox-
ide. Am Rev Rcspir Dis, 1972,106, 563.
2. Havdon, G. H., Freeman, C., and Kuriosi, N.
J.: Coveit pathogencsis of NO2-induccd em-
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12 Weibel. E. R.: Postnatal giowth of the lung and
pulmonary gas-exchange capacity, in Develop-
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ter, ed., Churchill, London, 1967, p. 131.
13. Buni, P. H., Dbaly, J., and Weibel, E R.: Post-
natal giowth in the rat lung, Experientia, 1972,
2,V. 727.
14 Baitlett, D., Jr.: Postnatal growth of the mam-
malian lung: Influence of low and high oxy-
gen tensions, Resp Physiol, 1970, 9, 58.
I:' Barllett. D., Jr.: Postnatal growth of the mam
malian lung: Influence of exercise and thyroid
activity, Resp Phvsiol, 1970,9,50.
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malian lung: Influence of excess growth hor-
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RespirDis, 1969,99,669.
30
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CRITERIA FROM ANIMALS EXPOSED TO KNOWN CONCENTRATIONS OF NITROGEN
DIOXIDE AND OZONE, WITH POTENTIAL USE IN EPIDEMIOLOGY
G. FREEMAN, L. T. JUHOS, N. J. FURIOSI,
W. R. POWELL, AND R. MUSSENDEN
Stanford Research Institute
Menlo Park, California, USA
DR. POCKIANELLA, LADIES AND GENTLEMEN:
Much has been learned about the responses of laboratory animals to
atmospheres polluted by either nitrogen dioxide (NO2) (Freeman et al.
[1-4]) or ozone (O3) (Stokinger et al. [5], Werthamer et al. [6], Freeman
et al. [?]). But applying this knowledge to the epidemiology of disease
is difficult because of the variability of man's activities, the complex-
ity of photochemical reactions [8,9], and the difficulties in devising
effective epidemiologic designs.
At least four criteria are required to group individuals for a
meaningful epidemiologic study of the effects of oxides of nitrogen (NOX)
and oxidants. The first concerns residence in crowded, traffic-laden,
and industrialized areas where the toxic, interdependent, photochemically
induced atmospheric pollutants N02 and O3 cycle simultaneously during
half of each day in varying ratios and in similar average concentrations
[8,9], Thus, epidemiologic observations are related to the impact of
mixtures. (Our experience with rats reveals that in short-term experi-
ments, 03 is about 10- to 20-fold more injurious to the lungs than NO2
(Freeman et al. [10])) .
Whereas one may limit atmospheric components to NO2 and 03 under
laboratory conditions, in smoggy atmospheres peroxyacetyl nitrates also
exist as photochemical products [8], and independent pollutants such
as sulfur dioxide (SO2) I'll] appear frequently to complicate interpre-
tation .
Cigarette smoking is a second factor [12], Among the injurious
components of tobacco smoke, oxides of nitrogen are found in hundreds
of parts per million (Haagen-Smit et al. [13]), of which an undetermined
portion is N02. Almost all the NOX carried into the lungs with each
bolus of inhaled smoke is retained (Freeman et al. [14]). The close
correlation between the prevalence of human emphysema and chronic
bronchitis and the amount of tobacco smoke inhaled over a period of
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years [12 ] is especially noteworthy. Pulmonary disease is readily
induced in rats by NO2 in concentrations similar to those in tobacco
smoke [4]. As a corollary, nonsmokers are largely spared from chronic
obstructive pulmonary diseases, regardless of their environments.
A third factor is occupation. Hazardous conditions are produced
by confined concentrations of NO2 or O3, or both, independent of photo-
chemical activity. Moreover, certain activities create high concentra-
tions of N02 or 03 not achieved in photochemical reactions (Stokinger
[15]). NOX also is produced naturally by bacterial action on crops
stored in silos and is the cause of silo-filler's disease in farmers.
Industrial processes such as welding and the use of explosives in mines
also can be hazardous when high temperatures accelerate the oxidation
of nitrogen to form toxic oxides [9].
The fourth factor is individual susceptibility, which includes age
at initial exposure. Generally, the very young are poorly defended
against exogenously induced disease compared with more mature individuals,
but this may not be a universal truth, as we shall see. The elderly
also are vulnerable because of their susceptibility to cardiopulmonary
disease and possibly because of aging. Individuals prone to respiratory
allergy are particularly responsive to atmospheric irritants. With
regard to chronologic age, the type or response to exogenous insults
may depend on whether an individual was born in a polluted environment
or moved into it later in life.
What is the evidence for this? We exposed four-week-old rats con-
tinuously to N02. Their rates of red cell formation increased rapidly,
and the rats developed polycythemia [3]. [See Slide 1.] In contrast,
animals exposed from birth generated red cells less rapidly than rats
born in clean air. As a result, such exposed rats were relatively
anemic at seven weeks of age and contrasted sharply with polycythemic
rats of the same age that were exposed only during the last two of the
seven weeks [14],
Also, in rats exposed from birth to high subacute concentrations
of N02 (—15 ppm), the mechanical compliance of lungs was not affected
by increasing the frequency of respiration. Lungs of animals of the
same age, exposed after they were a month old, developed frequency
dependence of compliance, because of changes in the small airways [14]
[Slide 2]. Thus, the failure of animals exposed from birth to have
developed small airway disease, as judged by frequency dependence of
compliance, may have been determined by neonatal contact with NO2 .
Both the hematologic and respiratory observations, then, suggest altered
responsiveness. Incidentally, growth of the lung defined as the rate
of formation of new alveoli—in animals exposed continuously to N02
from birth—was less than normal for the first ten weeks [14]. [See
Slide 3.] The significance of very early exposure relative to subsequent
pathogenesis appears to deserve further study.
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Whereas smoking clearly is associated with chronic pulmonary disease,
ambient ozone has not yet been so identified epidemiologically [8].
What is ozone's impact on the nonsmoking population? The odor of ozone
is known to be detectable at a concentration of about 0.02 ppm; increased
susceptibility to infection in animals begins at about 0.08 ppm; and
eye irritation to peroxyacetyl nitrates is noticeable at about 0.1 ppm
[8], At the 0.1-ppm level, increased airway resistance also becomes
evident in some individuals and is readily apparent in subjects breath-
ing a concentration of about 0.5 ppm for three hours on five consecutive
days. Exposure to about 1.0 ppm rapidly becomes intolerable to man [8].
In contrast, there is little clinical evidence of an adverse effect
on the respiratory tract during experimental exposure to levels of N02
below 1.0 ppm, although the odor of 0.1 ppm is readily detectable. To
simulate maximum human exposure to the mixture of N02 and ozone on a
cyclic photochemical basis, we exposed rats four hours per day and
observed pathologic effects in the lungs that could relate to chronic
pulmonary disease [10], At a concentration of about 0.9 ppm of each
gas, a lesion developed at the narrow junction of the respiratory bron-
chiole and alveolar duct through which air is conducted to and from the
alveoli. [Slide 4] Epithelial cells were injured and replaced (Evans
et al. [17]), often by hypertrophic cells. The hypertrophic epithelium,
together with fibroblasts and chronic inflammatory cells, proliferated
at the level of the smallest airways to form denser tissue. The effects
of these changes on lung function have not been explored. As with high
subacute concentrations of N02 [l,4], continuous exposure of rats to a
mixture of 0.9 ppm of ozone and of NO2 led to the development of an
emphysema-like disease. [Slide 5] Also, continuous exposure to a mix-
ture of 2.5 ppm of N02 and 0.25 ppm of ozone resulted in disease of
the alveolar ducts characteristic of the response to ozone rather than
to N02 . [Slides 6 and 7] Thus, ozone, clearly, is the primary cause
of deep-seated pulmonary disease in the experimental animal when mixtures
of the agents are present on a schedule approximating man's experience
in photochemically smoggy atmospheres.
Let us shift our attention to animal studies with higher subacute
concentrations of NO2—about 14 to 17 ppm [3,4]. The relationship of
smoking to the epidemiology of chronic obstructive pulmonary diseases
[12], to which N02 may contribute, now comes into focus. Extrapolation
to chronic human disease from the rat, which has a life span of about
4 percent that of man, is hazardous. Nevertheless, the fortuitous
similarity in the relative time required for the development of emphysema
in the rat compared with that in man warrants attention [l,3,4]. The
clinical and morphological features associated with habitual smoking
of cigarettes are consistent with the changes in the lungs of rats
exposed to high subacute concentrations of N02 for long periods [l,3,4].
It is noteworthy, also, that ozone, which is absent from tobacco smoke,
induces a similar disease in the rat at approximately one-twentieth
the concentration of N02 when mixed with it in equal amounts [10].
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In summary, it would not seem feasible to distinguish between the
specific effects of NO2 and O3, epidemiologically, except in the cases
of high occupational concentrations and of habitual smoking. As photo-
chemically interdependent gases, they coexist and act almost simulta-
neously .
This research was supported by Contract NO. 68-02-1243 of the
Environmental Protection Agency (USA), Division of Health Effects Research,
Research Triangle Park, North Carolina, and by Grant ES00842-02 of the
National Institutes of Health.
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References
1. FREEMAN, G., HAYDON, G.B., "Emphysema after low-level exposure to
N02," Arch. Environ. Health 8_, 125 (1964).
2. FREEMAN, G., STEPHENS, R.J., CRANE, S.C., FURIOSI, N . J . , "Lesion
of the lung in rats continuously exposed to two parts per million
of nitrogen dioxide," Arch. Environ. Health 17, 181 (1968).
3. FREEMAN, G., CRANE, S .C ., STEPHENS, R . J ., FURIOSI, N . J ., "Patho-
genesis of the nitrogen dioxide-induced lesion in the rat lung:
A review and presentation of new observations," Am. Rev. Respirat.
Disease 98_, 429 (1968).
4. FREEMAN, G., CRANE, S .C., FURIOSI, N.J., STEPHENS, R.J., EVANS, M.J.,
MOORE, W.D., "Covert reduction of ventilatory surface in rats during
prolonged exposure to subacute nitrogen dioxide," Am. Rev. Respirat.
Disease 106, 536 (1972).
5. STOKINGER, H .E ., WAGNER, W.D., DOBROGORSKI, O.J., "Ozone toxicity
studies: III. Chronic injury to lungs of animals following exposure
at low level,"Arch. Industr. Health 16_, 514 (1957).
6. WERTHAMER, S., et al., "ozone-induced pulmonary lesions: Severe
epithelial changes following sublethal doses," Arch. Environ.
Health 20_, 16 (1970).
7. FREEMAN, G., STEPHENS, R.J., COFFIN, D.L., STARA, J.F., "Changes
in dogs' lungs after long-term exposure to ozone," Arch. Environ.
Health 2£, 209 (1973).
8. U.S. Public Health Service, Air Quality Criteria for Photochemical
Oxidants, National Air Pollution Control Administration Publication
No. AP-63, Washington, D.C. (1970).
9. U.S. Environmental Protection Agency, Air Quality Criteria for
Nitrogen Oxides, Air Pollution Control Office Publication No. AP-84,
Washington, D.C. (1971)
10. FREEMAN, G., JUHOS, L.T., FURIOSI, N.J., MUSSENDEN, R., STEPHENS,
R.J., EVANS, M.J., "Pathology of pulmonary disease from exposure to
ambient gases (nitrogen dioxide and ozone)," Arch. Environ. Health
29, 203 (1974).
11. U.S. Public Health Service, Air Quality Criteria for Sulfur Oxides,
National Air Pollution Control Administration Publication No. AP-50,
Washington, D.C. (1969).
12. U.S. Department of Health, Education and Welfare, The Health Con-
sequences of Smoking, Chapter 2, Public Health Service, January
1973, pp. 31-62.
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13. HAAGEN-SMIT, A.J., BRUNELLE, M.F., HARA, J., "Nitrogen oxide content
of smoke from different types of tobacco," Arch. Industr. Health 20,
399 (1959).
14. FREEMAN, G., et al. Unpublished data.
15. STOKINGER, H .E ., "Ozone toxicology. A review of research and
industrial experience: 1954-1964," Arch. Environ. Health 10, 719
(1965).
16. SCHWARTZ, R., DAMESHEK, W., "Drug induced immunologic tolerance,"
Nature 183, 1682 (1959).
17. EVANS, M.J., STEPHENS, R.J., CABRAL, L.J., FREEMAN, G., "Cell
renewal in the lungs of rats exposed to low levels of N02," Arch.
Environ. Health 24_, 180 (1972).
18. BUCKLEY, R .D., HACKNEY, J.D., CLARK, K., POSIN, C., "Some effects
of ozone inhalation on human erythrocyte metabolism" (Abstract),
Fed. Proc. Abstracts 33, 335 (1974).
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 REPORT NO.
EPA-600/1-76-021
4. TITLE AND SUBTITLE
TRACE SUBSTANCES AND TOBACCO SMOKE IN INTERACTION
WITH NITROGEN OXIDES. Biological Effects.
3. RECIPIENT'S ACCESSION" NO.
5. REPORT DATE
April 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Gustave Freeman and Laszlo T. Juhos
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Stanford Research Institute
Menlo Park, California 94025
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
68-02-1243
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
Final. July 1973/Sept. 1974
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The emphasis of this study is on determination of the response of newborn
animals living in an environment containing nitrogen dioxide.
The following are also examined:
a. Both mature and newborn monkeys (Macaca speciosa) exposed continuously
to N02 or to ozone.
b. The relative effects of tobacco smoke compared with those of N02-
c. The binding of N02 in tissue, based on the use of isotopically labeled,
nonradioactibe N02-
d. The detailed hematologic effects of exposure to N02 or to ozone.
In addition to the usual parameters for detecting changes in the erythrocytic
series, biochemical studies were conducted on the blood of exposed animals.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Nitrogen Oxide
Nitrogen Dioxide
Tobacco
Ozone
Laboratory animals
Respiration
Hematology
Tobacco smoke
06, F, T
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
41
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
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