xvEPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA i '"! 001
)79
Research and Development
Effect of Exposure to
PAN and Ozone on
Susceptibility to
Chronic Bacterial
Infection
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6, Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-79-001
January 1979
EFFECT OF EXPOSURE TO PAN AND OZONE ON SUSCEPTIBILITY
TO CHRONIC BACTERIAL INFECTION
by
Gail B. Thomas, James D. Fenters and Richard Ehrlich
IIT Research Institute
Life Sciences Research Division
10 West 35th Street
Chicago, Illinois 60616
Contract No. 68-02-1273
Project Officer
Donald E. Gardner
Clinical Studies Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
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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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 participates in
the development and revision of air quality criteria documents on
pollutants for which national ambient air quality standards exist or
are proposed, provides the data for registration of new pesticides or
proposed suspension of those already in use, conducts research on
hazardous and toxic materials, and is primarily responsible for providing
the health basis for non-ionizing radiation standards. Direct support
to the regulatory function of the Agency is provided in the form of
expert testimony and preparation of affidavits as well as expert advice
to the Administrator to assure the adequacy of health care and surveillance
of persons having suffered imminent and substantial endangerment of
their health.
This report describes the results of an in vivo investigation
designed to determine the effects of peroxyacetyl nitrate and ozone on
susceptibility of mice and guinea pigs to chronic and acute respiratory
infection. The agent used for the acute infectious disease was
Streptococcus pyogenes whereas Mycobacterium tuberculosis served as the
agent for the chronic respiratory infection. In these studies the relative
comparison of the effects of these two pollutants on the resistance to
laboratory induced bacterial infections of the lung could be obtained. As
appropriate to the specific experiments, the parameters measured were
mortality, survival time, retention or growth of inhaled microorganisms
in the lung, histopathology of the respiratory tract tissue, pulmonary
cellular defense system, and immunological response.
F. G. Hueter, Ph. D.
Acting Director,
Health Effects Research Laboratory
iii
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ABSTRACT
The effects of peroxyacetyl nitrate (PAN) and ozone (Oo) on susceptibility
of mice and guinea pigs to chronic and acute respiratory infections were
studied. The agent used for the acute infectious disease was Streptococcus
sp. whereas Mycobacterium tuberculosis served as the agent for chronic
respiratory infection.
A significant increase in mortality due to streptococcal pneumonia was
seen upon a single 3-hr exposure to PAN in concentrations ranging from 14.8
to 28.4 mg/m3. The excess mortalities ranged from 8 to 39% and reduction in
the survival time from 2.4 to 7.9 days. Within this concentration range of
PAN a close relationship was present between the duration of exposure and
concentration. Multiple daily exposures to 4.9 or 7.4 mg/m3 PAN 3 hr/day,
5 days/week for up to 3 weeks had no effect on mortality, survival rates, or
ability to clear inhaled Streptococcus sp. from the lungs.
q
Daily 3-hr exposures to 25.0 mg/m PAN did not produce any marked changes
in the chronic infection as measured by M. tuberculosis titers in their lungs.
The diameter of erythemas, expressing the cutaneous delayed hypersensitivity
reaction were persistently smaller in guinea pigs exposed to PAN than those
exposed to air. Multiple exposures to 19.8 mg/m3 PAN resulted in initial
elevation of antibody titers, but depression of titers during the later (12 to
15 week) observation period. A single exposure to the same concentration of
PAN resulted in a significant increase in total number of cells lavaged from
their lungs but somewhat decreased levels of adenosine triphosphate (ATP).
Exposure to 7.4 mg/m3 PAN 3 hr/day, 5 days/week for 2 weeks resulted in reduced
total cell counts and a significant reduction of ATP levels in alveolar macro-
phages.
Scanning electron microscopic observations of the respiratory tract showed
that the nonciliated cells of the nasal cavities and tracheas of mice exposed
to PAN were raised and sloughing and excess mucus was present. In older mice
lung congestion was enhanced by PAN exposure.
Exposures to ozone resulted in increased titers of M. tuberculosis in
the lungs, depression of hypersensitivity reaction and elevation in serum
antibody titers.
IV
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CONTENTS
Foreword iii
Abstract 1v
Figures vi
Tables vii
Abbreviations viii
1. Introduction 1
2. Conclusions 3
3. Recommendations 5
4. Materials and Methods 6
Animals , ." 6
Infectious Aerosols .... 6
Pollutants 7
Parameters Measured 9
5. Results and Discussion 14
Health Effects of Exposure to PAN 14
Effects of Ozone on Resistance to
Chronic Respiratory Infection 26
References ' 31
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FIGURES
Number Page
1 Relationship between excess mortality due to streptococcal
pneumonia in mice and CT index for PAN exposure
(Mean and 95% CL) 16
2 Serum hemagglutination antibody titers in guinea pigs
challenged with M. tuberculosis and within <3 hr
exposed to 19.8 mg/m3 PAN 3 hr/day for 5 days 19
3 Nonciliated epithelial cells in nasal cavities of mice .... 22
4 Ciliated epithelial cells in nasal cavities of mice 23
5 Nonciliated epithelial cells in nasal cavities of mice .... 24
6 Lungs of mice exposed to 7.4 mg/m PAN 3 hr/day, 5 days/week
for 2 weeks 25
7 Titers of M. tuberculosis in lungs of mice exposed to
1.96 mg/m3 03 3 hr/day, 5 days/week for 6 to 7 weeks .... 27
8 Cutaneous sensitivity to PPD in guinea pigs challenged
with M. tuberculosis and within 3 hr exposed for
3 hr to 2.9 mg/m3 03 28
9 Serum hemagglutination antibody titers in guinea pigs
challenged with M. tuberculosis and within 3 hr exposed
to 0.98 mg/m3 63 3 hr/day for 5 days 30
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TABLES
Number Page
1 Mortality and Survival Rates of Mice Exposed for 2 or 3 Hr
to PAN and Challenged Within 1 Hr With Streptococcus
Aerosol 15
2 Mortality and Survival Rates of Mice Daily Exposed 3 Hr/Day
5 Days/Week for 2 or 3 Weeks to PAN and Challenged Within
1 Hr With Streptococcus Aerosol 15
3 Clearance of Inhaled streptococcus From Lungs of Mice
Exposed to PAN . . . ' 17
4 Effect of Exposure to PAN on Free Cells Lavaged from
the Lungs of Mice 21
VI1
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LIST OF ABBREVIATIONS
AM -- alveolar macrophages
ATP — adenosine triphosphate
BSA — bovine serum albumin
CPU — colony-forming units
C02 -- carbon dioxide
CT -- product of concentration x time
D/T — deaths per total number of animals
IR ~ infrared
LC5Q -- lethal concentration for 50% of the animals
L/min -- liters per minute
mg/m3 — milligrams per cubic meter
N02 — nitrogen dioxide
NOs -- nitrate
03 ~ ozone
OAA — oleic acid albumin
PAN — peroxyacetyl nitrate
PBS — phosphate buffered saline
PPD — purified protein derivative
ppm — parts per mil lion
RH — relative humidity
RMSR — relative mean survival rate
S.D. -- standard deviation
S.E. — standard error
SEM — scanning electron microscopy
S02 -- sulfur dioxide
tso ~ time required for 50% of inhaled bacteria to be
cleared from the lungs
TU -- tuberculin units
y — micron
vm
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SECTION 1
INTRODUCTION
Peroxyacetyl nitrate (PAN) is the first member of a series of peroxy-
acetyl nitrates (R-C"-OON02) that are important constituents of photochemical
air pollution. PAN is a highly oxidized, unstable organic nitrogen compound
that results from ultraviolet irradiation in air of certain unsaturated
hydrocarbons in the presence of oxides of nitrogen. PAN was the first pure
compound recognized as causing plant damage and eye irritation (1). A number
of studies conducted to determine effects on animals indicate that the toxicity
of PAN for mice is greater than that of sulfur dioxide ($03)> similar to
nitrogen dioxide (N02) and lower than that of ozone (03).
In studies conducted by Campbell and coworkers (2) the LC5Q for mice for
a 2-hr exposure was 105 to 150 ppm PAN. The LC$Q was closely related to the
age of the animals and the environmental temperature maintained during the
exposure. Mice, 4 to 6 month old were more susceptible than those
2 to 3 month old and increased susceptibility to PAN was seen at 9QOF as
compared to 80°F. The same investigators,reported a depression of voluntary
motor activity in mice during exposure to approximately 8 ppm PAN (3). Daily
6 hr exposure to 15 ppm PAN for 6 months caused 18% mortality, weight loss,
and damage to the respiratory tract tissue in mice (4). The damage included
tracheobronchitis, broncholotis, pneumonitis, mild emphysema, squamous meta-
plasia, and epithelial hyperplasia. In a study involving 21-year-old males
exposed for 5 min to 0.3 ppm PAN and then exercised for 5 min a significant
increase in oxygen uptake was reported when compared to those breathing
normal air (5).
Tuberculosis infection in rodents has been described by various investi-
gators (6,7,8,9). Many of the studies have been conducted to determine the
effects of vaccination on the growth of the virulent human Mycobacteriian
tuberculosis strain H37Rv in the lungs and other organs of mice and guinea
pigs (6,9). The attenuated M. tuberculosis strain RlRv used in our studies
has been grown in liver and spleen of rats (6). It is the most
virulent of the attenuated strains and causes disease in silicotic animals
(Personal communications, Mr. Logy, Trudeau Institute, Saranac Lake, NY).
Immunological responses, especially cutaneous delayed hypersensitivity, to
M. tuberculosis have been measured extensively in guinea pigs (10).
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Studies of the effects of inhalation of air pollutants on the resistance
to chronic respiratory infection, such as tuberculosis are very limited.
Thienes and coworkers (11) investigated the effects of ozone on M. tuberculosis
infection in mice. Exposure to 1.5 ppm 03 4 hr/day, 5 days/week for 2 months
did not alter the resistance to M. tuberculosis H37Rv nor the BCG vaccine
strain. The authors noted that this infectious model was less sensitive to
ozone than the acute disease model which used Klebsiella pnewnoniae as the
infectious agent.
The overall purpose of this program was to study the effects of PAN and
ozone on the susceptibility of animals to acute and chronic respiratory in-
fections. The acute disease was established in mice by respiratory challenge
with Streptococcus sp., (Lancefield group C) aerosols. The chronic infection
was induced in mice and guinea pigs by challenge with an aerosol of Af.
tuberculosis attenuated human strain RlRv.
The various parameters measured in mice during the chronic disease
studies were the Af. tuberculosis titers in the lungs and spleens, number of
tubercules in the lungs and histopathologic changes in lungs. The parameters
determined in guinea pigs were serum hemagglutination antibody titers and
cutaneous delayed hypersensitivity reactions to purified protein derivative
(FDD). The parameters assayed in conjunction with the acute infection included
mortality and survival time, clearance rate of inhaled bacteria from the lungs,
and histopathologic alterations in the lungs, tracheas, and nasal cavities as
observed by light and scanning electron microscopy. In a limited number of
experiments the effects of exposure to the pollutants on the pulmonary cellular
defense system were determined by examining the total and differential cell
counts, viability, cell surface morphology and the phagocytic function of
alveolar macrophages lavaged from lungs.
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SECTION 2
CONCLUSIONS
Effects of single and multiple exposures to PAN on resistance to
streptococcal pneumonia were determined. Single 2 to 3 hr exposures to PAN
in concentrations ranging from 14.8 to 28.4 mg/m3 significantly enhanced the
susceptibility of mice to streptococcal pneumonia. A linear relationship was
observed between CT index and percent increase in mortality. The average
excess mortality among mice exposed to PAN was 20%. Multiple daily 3 hr ex-
posures to 7.4 mg/m3 PAN for 2 weeks or 4.9 mg/m3 for 3 weeks had no effect
on mortality, survival time or the rate of clearance of inhaled bacteria from
the lungs.
Studies to determine the effects of PAN on the susceptibility of mice to
M. tuberculosis were limited by the difficulty to produce large quantities
of pure PAN. The highest concentration used was 25 mg/m3 PAN given 3 hr daily
for 6 days beginning 1 week after the challenge. This exposure caused only
a minimal increase in the bacterial titers in the lungs, seen in the later
stages of the infection.
3 3
A 3 hr exposure of guinea pigs to 29 mg/m PAN or 2.9 mg/m 03 initiated
within less than 3 hr after challenge with M. tuberculosis aerosol indicated
that both pollutants had some effect on delayed hypersensitivity. The diameters
of erythema were persistently smaller in animals exposed to PAN or ozone than
those exposed to air. Exposure of guinea pigs to 19.8 mg/m3 PAN or 0.98 mg/m3
03 3 hr/day for 5 days, immediately after challenge resulted in antibody titers
that were generally higher in guinea pigs exposed to ozone than those exposed
to air. Titers in guinea pigs exposed to PAN were initially higher but later
lower than those in guinea pigs exposed to air. Exposure to 0.98 or 1.96 mg/m3
03 3 hr/day 5 days/week for 6 to 8 weeks beginning 1 to 3 weeks after challenge
enhanced susceptibility of mice to tuberculosis. The effect was seen as marked
increases in bacterial titers in the lungs.
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Other parameters measured in conjunction with the exposure to PAN in-
cluded damage to lungs, tracheas and nasal cavities observed by scanning electron
microscopy. The single 3-hr exposure to 25 mg/m3 PAN caused the nonciliated
epithelial cells of the nasal cavity to rise up from the basal membrane and
slough. Excess mucus was also present. A similar damage was seen upon daily
3 hr exposures for 2 weeks to 7.4 mg/m-5 PAN in young, 6 week old mice. In
older mice (4 month old) exposed to PAN, congestion in the lungs was greater than
in air controls. Daily 3 hr exposure for 3 weeks to 4.9 mg/m3 PAN caused damage
to nonciliated cells of the trachea similar to that observed in the nasal
cavities.
A single 3-hr exposure to 19.8 mg/m PAN did not alter the differential
cell counts or the activity and viability of alveolar macrophages. However,
there was a significant increase in total cell counts (macrophages and lympho-
cytes). Daily 3 hr exposure for 2 weeks to 7.4 mg/nP PAN caused a decrease in
total cell counts and reduced cellular ATP levels in alveolar macrophages.
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SECTION 3
RECOMMENDATIONS
The availability of sufficiently large quantities of pure PAN is
mandatory to enable the continuation of health effects studies of this
pollutant. This is esepcially important for the extension of multiple and
chronic exposure studies to determine their effect on the resistance to
respiratory infections.
Short-term exposures to PAN alone or in combination with other pollutants
such as ozone can be investigated since the effects of acute exposures to PAN
were in part determined in this study. Based on results obtained, such studies
should include the determination of the effects of PAN on immune response.
Specifically, the activity and viability of alveolar macrophages, serum
antibody formation, macrophage migration inhibition and mitogen stimulation
of lymphocytes in animals exposed to PAN should be defined.
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SECTION 4
MATERIALS AND METHODS
ANIMALS
Sprague-Dawley CF, outbred female albino mice, 6 to 16 weeks old C57BL/6
black mice 6 weeks old and Murphy Breeding Laboratory CD2F] hybrid brown mice,
7 to 10 weeks old, were used in these studies. The mice were housed in groups
of 10 in stainless steel shoebox cages and provided food and water ad libitum.
Hartley strain female guinea pigs (Murphy Breeding Laboratory) weighing
300-500 g were housed in groups of 2 or 3 and provided food and water ad
libitum.
INFECTIOUS AEROSOLS
Mycobaaterium tubefoulosis
Mycobacterium tuberculosis RlRv (Trudeau Institute #205) was grown in
Proskauer and Beck broth in prescription bottles incubated horizontally for
4 to 8 weeks at 37°C until confluent pellicle growth was obtained. A loopful
of the pellicle was transferred to 50 ml of Middlebrook 7H9 broth (Difco)
containing 0.1% Tween 80 and incubated 7 to 10 days until the culture was
turbid. The unwashed culture was homogenized using a teflon pestle and
50-ml homogenizing vessel. The titer was determined by serially diluting in
0.1% phosphate-gelatin (Bacto hemagglutination buffer (Difco) with 0.1%
gelatin), plating 0.1 ml aliquots onto Dubos oleic acid albumin (OAA) agar
containing 50 y/ml penicillin G and 0.1% cycloheximide, and counting the
colonies after 3 weeks incubation at 37°C. The cultures were frozen in 8-ml
aliquots at -70°C.
For aerosol dissemination, 6 ml of thawed culture was diluted approxi-
mately 1:10 in Middlebrook 7H9 broth to which a 10% solution of antifoam A
(Dow Chemical, Midland, MI) was added. The bacterial suspension was dissem-
inated at a rate of 0.75 ml/min from a DeVilbiss Model 841 continuous flow
nebulizer using a primary filtered air flow of 8 liter/min into a 30x30x90-cm
plexiglass chamber installed within a microbiological safety hood.
Groups of six guinea pigs were exposed to the aerosol for 2 min. Groups
of 48 to 60 mice, housed in individual compartments of specially designed
aluminum wire cages, were exposed for 20 min to the aerosol. The inhaled dose
was determined by homogenizing lungs from three mice immediately after the
challenge in 2 ml of 2% albumin and plating 0.2 ml of ten-fold phosphate-
gelatin dilutions onto OAA agar. The plates were incubated for 4 weeks at
37°C. The number of bacteria in a total volume of 2.2 ml was considered as
the number of M. tuberculosis per lung.
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Streptococcus pyogenes
Streptococcus pyogenes group C (Lancefield strain) was passaged in mice,
isolated from hearts, incubated for 18 hr at 37°C in Todd Hewitt broth and
1-ml aliquots frozen at -70°C. For aerosol dissemination, 1 ml of thawed
culture was added to 100 ml of Todd Hewitt broth and incubated for 18 hr at
37°C. The culture was diluted approximately 1:10 in 0.1% peptone water and
adjusted to 60% transmittance at 440 nm in a spectrophotometer (Bausch and
Lomb Spectronic 20, Rochester, NY).
The bacteria were disseminated using a DeVilbiss Model 841 nebulizer by
using a primary filtered air flow of 8 liter/min and a secondary humidified
air flow of 28.3 liter/min into a 60x70x90-cm plexiglass chamber placed inside
a microbiological safety hood.
Mice were exposed to the aerosol for 10 min. The inhaled dose was
determined by homogenizing lungs from three mice in 0.1% peptone water and
plating tenfold dilutions onto blood agar. The colonies were counted after
48-hr incubation at 37°C. The titer of the culture was determined by plating
tenfold dilutions onto blood agar.
POLLUTANTS
Ozone (03)
Animals were exposed to 03 in a 120x60x60-cm plexiglass chamber maintained
at 24 +_ 2°C and <40% relative humidity (RH). To prevent build-up of ammonia,
deotized cage board (Upjohn Co., Kalamazoo, MI) was placed on the bottom of
the chamber. A high voltage generator (IITRI) was used to convert filtered
air to 03. To provide the desired concentration, 03 was mixed with filtered
air in a glass mixing chamber and the mixture passed into the exposure chamber
at a rate of 60 +_ 5 liters/min. Concentration was monitored continuously
with an 03 Chemiluminescent Analyzer (Model OA 310, Meloy Laboratories,
Springfield, VA), and was expressed in mg/m3 (ppm x 1.95 = mg/m3).
Peroxyacetyl Nitrate
Synthesis—
PAN was synthesized using procedures described by Kacmarek, et al. (12).
Briefly, biacetyl was photolyzed in the presence of NOX and oxygen. Biacetyl
(1-1/2 torr) and N02 (4 torr) were loaded into a 3 liter bulb and then oxygen
was introduced. The bulb was floated in an ice bath and cold water from the
bath was circulated over the bulb. The chemicals were irradiated for 4 hr by
UV light from an A-H6 lamp (Illumination Industries, Sunnyvale, CA) placed
35 cm from the bulb. Over 5 ml of the gas (equivalent at STP) produced from
this photolysis yielded 80%.
(CH3C(0)C(0)CH3+N02 ^> CH3C(0)OON02)
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Crude PAN was initially purified by vacuum line fractionation to remove methyl
nitrate, carbondioxide, and excess N02i followed by gas chromatographic puri-
fication. The 152 cm, 0.64 cm O.D. pyrex tube column contained Fluoropak 80
support with a KEL-F grease (10£) stationary phase. The purity of the PAN was
determined by IR analysis. PAN was allowed to decompose in a pyrex infrared
cell at 10 torr pressure. The decomposition products in order of decreasing
quantities were: COg, CH30H02, CH2N02, CO, 02 and Cfy. The half life of PAN
under these conditions was 100 hr.
Exposure to PAN—
Pure PAN was introduced into a 60x70x40 cm plexiglass exposure chamber
by passing N2 at a rate of 300-700 ml/min over liquid PAN held at -30°C in
Freon 12 in a modified cold trap. A speedaire filter and hydropurge removed
moisture and oil from the nitrogen line. The chamber temperature was main-
tained at 26.5° HH l.QOC. The relative concentration of PAN in the chamber
was monitored continuously using a Mast oxidant analyzer containing KI solu-
tion, with an attached strip chart recorder (Model 725-3CS Mast Development
Co., Davenport, IA). The concentrations were maintained by adjusting N2 and
air flow rates. All PAN concentrations (ppm x 4.95 = mg/rn^) were based on
an NOX Chemiluminescent Analyzer (Model 7101-B, Bendix Corp., Ronceverte,
WV), which was used to calibrate the Mast Analyzer.
To confirm the chemiluminescent measurements, the concentration and
purity of PAN in the chamber were analyzed in an infrared spectrophotometer
(Perkin-Elmer Model 283) equipped with a wave number marker and a repetitive
scan device. PAN was monitored at 1165 cm-1 and methyl nitrate at 1019 cnH.
Samples were passed into a glass cell with Csl windows having a pathlength
of 11 cm using inlet and outlet ports. A calibration spectrum was obtained
by bubbling argon at 200 to 700 ml/min through PAN contained in the cold trap
held at -30°C and passing the saturated gas through the cell. The efficiency
of delivery of PAN from the vessel into the IR cell was calculated by measuring
the vapor pressure of the existing PAN and comparing it to the vapor pressure
of the saturated gas.
To determine PAN concentration in the animal exposure chamber a slight
positive pressure was maintained in the chamber and exhausted through the IR
cell. After a large volume of gas (compared to the cell volume) passed
through the cell, the cell was valved-off and placed into the spectrophotometer.
The concentration of PAN determined by IR was compared to that measured by
the Mast Analyzer-Chemiluminescent Analyzer method.
The concentrations of PAN in the exposure chamber determined by the IR
analysis were in the range of concentrations of NOX determined by the Mast
Analyzer-NOx Chemiluminescent Analyzer measurements. Based on the qualitiatve
and quantitative IR analyses it was concluded that:
8
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• The PAN was >90% pure.
• The gaseous PAN did not contain break-down products,
such as, methyl nitrate.
• The efficiency of generation of gaseous PAN from the
liquid state into the chamber was
• The concentration of PAN in the chamber determined with
50% accuracy by IR was within the range determined
by chemiluminescent analysis (4.5 ppm versus 3.0 ppiu).
• Methyl nitrate was detected when animals were present in
the chamber, suggesting that breakdown of PAN upon
contact with animals, cages, and in the presence of
exhaled C02 is inevitable. While breakdown products
could have contributed to NOX levels assumed to be PAN
by chemiluminescent and oxidant analysis, the flow
rates of N2 and air required to maintain the desired
PAN concentrations in the chamber were similar with
and without animals. Moreover, the IR analysis specific
for PAN indicated higher rather than lower concentrations
compared to chemiluminescent measurements.
PARAMETERS MEASURED
Chronic Infection
Tubercles on Lungs—
At weekly intervals lungs were removed from five mice per group and
placed in buffered formaldehyde. Tubercles present on the surface were counted
and average counts in pollutant-exposed and control mice were compared.
Lung Tissue Histology-
Lungs from three mice per group were cannulated and perfused with
buffered formaldehyde. Tissues were processed for light microscopy by con-
ventional histologic methods and sections were cut at 5y and stained with
hematoxylin and eosin.
M. tuberculosis Titers in Lung and Spleen—
M. tuberculosis present in the lungs and/or spleens was determined at
weekly intervals for 8 weeks. At each interval 5 mice were killed and the
lung or spleen tissue homogenized in 2 ml of 2% albumin. Approximate tenfold
dilutions were made in 0.1% phosphate-gelatin and 0.2 ml aliquots from two
dilutions were plated in triplicate onto OAA agar using an Oxford Sampler
micropipettor (Fisher). Thirty plates were sealed in a CO?- impermeable 29x46 cm
supervac bag (VacPac Mfg. Co., Balitmore, MD) and incubated at 37°C. Plates
containing 1 to 350 colonies were counted after 3 and 4 weeks incubation. The
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colony-forming units (CPU) from all plates were averaged and multiplied by
the dilution factor (11) to give the total M. tuberculosis in 2.2 ml of homo-
genized tissue suspension.
The log^o of the CPU from each lung or spleen was averaged for each time
period and the concentrations of M. tuberculosis in the experimental groups
were compared with those in the controls using the Student t test.
Skin Sensitivity—
At weekly intervals, guinea pigs infected with M. tuberculosis were given
a 0.1 ml (25 TU) intradermal injection of a 5.0 yg/ml buffered solution of
tuberculin PPD (Parke, Davis and Co., Detroit, MI or Connaught Labs., Willowdal,
Ontario, Canada) and 24 hr later the diameter of erythema was measured (mm).
The difference of diameters between polutant-exposed and control animals were
compared by Student t test.
Passive Hemaggluti nation Test--
Guinea pigs were bled weekly by cardiac puncture, approximately 1.5 ml
of blood was drawn and the sera stored at -70°C. Before use, the sera were
heat- inactivated at 56°C for 30 min and diluted 1:2 in isotonic saline. The
procedures for tanning and sensitizing the red cells were those of Boyden (13)
and Middlebrook and Dubos (14). The tests were performed using modifications
of the Indirect Hemagglutination Test (Conrath (15) and the method of Daniel (16).
Briefly, citrated sheep red blood cells (Colorado Serum Co., Denver, CO)
were washed three times in isotonic saline and packed by centrifuging at
2000 rpm for 5 min. A 5% red cell suspension in buffered saline at pH 7.2
was treated with a 1:40,000 (wt/vol) tannic acid solution for 10 min in a
37°C water bath. The cells were then washed twice and resuspended to half the
original volume in buffered saline (PBS) providing a 10% red cell suspension.
One half of the tanned cells were sensitized by adding one volume of a PPD
solution (2.5 mg/ml in PBS) and incubated in a 37°C water bath for 15 min.
The cells were washed in 0.1% PBS and resuspended to twice the original volume
(2.5%) in the same diluent. Each serum was tested with both sensitized and
unsensitized cells to assure absence of nonspecific agglutination.
Sera were adsorbed twice to the sheep red cells to remove nonspecific
agglutination factors. One-tenth volume of packed, washed red cells was added
to one volume of serum. After frequent shaking at room temperature for 20 min
the suspension was centrifuged, the supernatant removed, and treated again
in the same manner. The tests were performed in microtiter disposotrays (V)
(Linbro Scientific Co., Hamden, CT). A 0.025 ml volume of 0.1% BSA was added
to all wells except the first ones where 0.05 ml of the 1:2 dilution of each
serum was added. Twofold dilutions were made using 0.025 ml microdiluters.
A second 0.025 ml volume of 0.1% BSA was added to each well.
10
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To the first row of every serum sample, 0.05 ml of 2.5% tanned, un-
sensitized red cells was added; to the second row, 0.05 ml of 2.5% tanned,
sensitized red cells was added. A saline control was used for each type of
cells. The plates were incubated in a 37°C water bath for 45 min, the results
read, and reread after overnight storage at 4°C. The reciprocal of the highest
dilution causing >^50% specific hemagglutination was considered the end point.
Because of nonspecific agglutination of normal guinea pig serum to sheep
erythrocytes, prevaccination serum for each guinea pig was tested along with
each postvaccination serum.
Acute Infection
Mortality and Mean Survival Rate--
During the experiments with S. pyogenes the mice were observed over a
28-day period after the infectious challenge. The percent mortality and
relative mean survival rate (RMSR) were determined. The RMSR were calculated
according to the following equation:
RMSR = (AxB)+(dxl)t
where A is the last day on which any individual mouse was alive; B is the
number of mice surviving A days; d is the last day of observation; L is the
number of mice alive on day d; and, n is the original number of mice in the
experimental group.
The significance of the observed differences in mortality rates was
determined by the chi-square test with a 2x2 contingency table. Analysis of
variance, the Student t test and linear regression analysis were used when
appropriate.
Bacterial Clearance Rates from Lungs--
The clearance rate of inhaled viable S. pyogenes from the lungs was
determined by sacrificing groups of 4 mice at 0,1,2,3 and 4 hr after challenge,
aseptically removing, weighing and homogenizing the lungs in 2.0 ml peptone
water, and plating 0.1 ml aliquots in duplicate onto blood agar plates. The
colonies were counted after 48 hr incubation at 37°C. The number of S. pyogenes
per gram of lung was determined for each exposure condition at 0 time and
this number was designated as 100% recovery. At each time interval the number
of S. pyogenes per gram of lung was expressed as percent of the 0 hr. The
rate of clearance, i.e., the time required for 50% of the original bacterial
population to be cleared from the lungs (tso)» was determined on a semilog-
arithmic regression after converting the percent recoveries to logio values.
The slopes of the regression lines for the experimental and control groups
were compared using the Student t test.
11
-------
Pulmonary Cell Population--
The free cells in the lungs were obtained by tracheobronchial lavage.
Mice were killed by intraperitoneal injection of sodium pentobarbital and
the lung-heart-trachea complex removed in toto. A blunted hypodermic needle
inserted into the trachea and fastened with surgical suture was used to lavage
the lungs by repeated infusions of warm saline.
The cells were isolated from the lavage fluids by centrifuging at 365 xg
and washing in Hanks' medium. Total cell counts were made using a white blood
cell diluting pipette and a henrocytometer. To determine the cellular distri-
bution (i.e., the proportion of alveolar macrophage, polymorphonuclear
leukocytes and lymphocytes), differential counts were made on air-dried smears
of cells fixed in methanol and stained with Wright's stain. Viability of
alveolar macrophages (AM) was determined by dye exclusion using \% trypan
blue. At least 200 cells selected at random were counted microscopically for
determination of differential counts and viability.
Cellular adenosine triphosphate (ATP) concentration in the lavaged cells
was determined using a DuPont 760 Luminescence Biometer employing procedures
recommended for the instrument. The assay is based on the principal that
when a microsample containing ATP is injected into a suitably buffered reaction
mixture of luciferase and luciferin, the peak intensity of the resulting light
flash is directly proportional to the concentration of ATP. ATP was extracted
from the cells by dimethyl sulfoxide (DMSO) from aliquots of the lavaged and
washed cellular suspension. The DMSO extracts were diluted with 0.01 M
morpholinopropane sulfonic acid (MOPS) buffer to overcome the quench effect
of the high concentration of DMSO in the aqueous extract. ATP levels were
expressed as femtograms (10-15g) of ATP per 105AM (calculated from initial
cell counts).
In vitro phagocytic activity was evaluated by light microscopy. Macro-
phages obtained from individual mice were resuspended to provide 4xlObAM/ml in
Medium 199 in Hanks' salt solution supplemented with Hepes buffer, Gentamicin
(Schering Corp.) and heat-inactivated fetal calf serum. A saline suspension
of polystyrene latex particles (l.ly) was prepared to contain 2xlOs particles/ml.
To determine the phagocytic activity 0.5 ml aliquots of AM suspension were mixed
with 0.1 ml of the particle suspension resulting in a final concentration of
3.33x10? particles and 3.33x105 AM/ml,(100 to 1 ratio of particles to AM).
After 1 hr incubation at 37°C the AM were washed in Hanks' salt solution,
centrifuged at 365 xg for 5 min, smeared onto glass slides, fixed in formalin
vapor overnight and stained with methylene blue. The non-engulfed latex spheres
were removed from the slides by a 3-hr immersion in xylene. The percentage of
phagocytizing cells was determined for each lavage sample by counting 200 cells
on duplicate slides.
12
-------
For scanning electron microscopy examination isolated AM were resuspended
in Hanks' medium supplemented with heat-inactivated fetal calf serum and
Gentamicin. One-mi aliquots of this suspension containing between 3.5 and
7.5x105 were incubated at 37°C in a 5% C02 humidified atmosphere for 1 3/4 hr
in plastic petri dishes containing glass coverslips as substrates to allow
the cells to attach. Subsequently the coverslips were removed, rinsed with
warm PBS and placed into 1% glutaraldehyde fixative-solution prepared in PBS.
After fixation, the coverslips with the attached cells were washed in a gentle
stream of distilled water, immediately frozen by immersion into liquid Freon-22,
and freeze-dried in vacuum.
For scanning electron microscopy (SEM) examination the coverslips were
glued to metal specimen holders. To render them conductive, the macrophages
were covered with a thin layer of carbon followed by gold evaporated onto
their surface in high vacuum. Micrographs were taken at 20 kV in a Kent-
Cambridge Mark II Stereoscan scanning electron microscope.
Scanning Electron Microscopy--
Tissues from the nasal cavities, tracheas, and lungs were taken for SEM
examination from mice exposed to PAN or filtered air and challenged <1 hr
later with S. pyogenes. The mice were anesthetized by an intraperitoneal
injection of pentobarbital, exsanguinated from the ventral aorta, the chest
opened, and the lungs and trachea were removed in toto. The trachea was
cannulated to the level of the first cartilaginous ring and the lungs expanded
with Karnovsky's paraformaldehyde-glutaraldehyde phosphate-buffered fixative
at 20 cm water pressure. Perfusion continued for at least 2 hr with the lungs
completely immersed in fixative. Upon completion of airway perfusion, the
trachea was ligated and the lungs floated in fixative.
After cannulation of the trachea, the head was removed, the skin was
retracted and the lower jaw removed by sectioning through the ramus. The
cranium was cut off immediately posterior to the orbits, leaving only the
nasal cavity. The nasal bones were reflected utilizing small forceps and the
cavity halved by inserting a razor blade on one side of the medium septum and
severing the hard palate.
The trachea and main stem bronchi were isolated from the lungs. The
lungs were sectioned with a razor blade in order to reveal the bronchus and
alveoli of each lobe. All tissues were washed in distilled water and
dehydrated with increasing concentrations of alcohol. Pentyl acetate was
then substituted for the alcohol and the tissues were dried by the critical
point method in carbon dioxide. The dried trachea was sectioned longitudinally.
The tissues were cemented to stubs, gold-coated in a Denton vacuum evaporator
equipped with a rotating turntable, and examined in a Kent-Cambridge Mark II
Stereoscan scanning electron microscope at 20kV.
Samples of lung, trachea and nasal cavities were also processed for
light microscopy examination. Sections were cut at 5y and stained with
hematoxylin and eosin.
13
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SECTION 5
RESULTS AND DISCUSSION
HEALTH EFFECTS OF EXPOSURE TO PAN
The effects of PAN on the resistance to acute and chronic respiratory
infections were studied in mice and guinea pigs. In some of the acute in-
fection experiments, initiated by inhalation of Streptococcus sp. aerosol,
mice exposed to ozone were included in parallel with those exposed to PAN.
Thus relative comparison of the effects of these two pollutants on the
resistance to streptococcal pneumonia could be obtained. As appropriate to
the specific experiments the parameters measured were mortality, survival time,
retention or growth of inhaled bacteria in the lungs, histopathology of
respiratory tract tissues, pulmonary cellular defense system, and immunological
response.
Effect on Resistance to Streptococcal Pneumonia
Mortality and Survival Rates-
Mortality rates and mean survival time of mice resulting from single
2 or 3 hr exposures to various concentrations of PAN and challenged within
1 hr with Streptococcus sp. aerosol are shown in Table 1. Significant increases
in mortality and decreases in mean survival time were seen in mice exposed to
PAN in concentrations ranging from 14.8 to 28.4 mg/m3. The mean mortality
rate for the six exposure conditions was 31.7% (+7.5% S.E.) and excess mortality
ranged from 8 to 39%. The reduction in the survival time ranged from 2.4
to 7.9 days. The mean mortality of mice exposed for 2 or 3 hr to 0.98 mg/m^ 03
and challenged with the infectious aerosol, which were included in the
experiments as positive controls, was 37.9% (+6.5% S.E.).
To further assess the effects of the short term exposure to PAN the CT
index, representing the products of PAN concentration and the duration of
exposure was calculated. The percent changes in mortality observed at each
exposure condition were subjected to regression analysis and the resulting
least square lines were plotted against the CT index (Figure 1). The relation-
ship between excess mortality due to streptococcal pneumonia and the CT index
can be clearly seen (correlation coefficient r = 0.95, p^O.Ol).
14
-------
TABLE 1. MORTALITY AND SURVIVAL RATES OF MICE EXPOSED FOR
2 OR 3 HR TO PAN AND CHALLENGED WITHIN 1 HR WITH
STREPTOCOCCUS AEROSOL
PAN Exposure
Concn
mg/m3
14.8
19.8
22.3
24.7
27.2
28.4
Time
hr
3
3
2
3
2
3
Air
D/T
6/90
4/147
6/49
40/1 50
9/100
30/201
Mortality
%
7
3
12
27
9
1-5
PAN
D/T
14/91
23/142
13/50
84/150
23/100
105/196
%
15
16
26
56
23
54
Change
+ 8**
+13*
+14**
+29*
+14*
+39*
RMSR (day)
Air
26.5
27.4
26.0
22.3
27.2
23.2
PAN
24.1
24.6
22.5
14.7
23.0
15.3
Change
-2.4
-2.8*
-3.5*
-7.6*
-4.2*
-7.9*
Significant change from corresponding infected mice exposed to
filtered air.
*
p ,<0.05.
**
Multiple daily exposures to PAN did not affect the resistance of mice to
the streptococcal infection. Daily exposure 3 hr/day, 5 days/week to 4.9
PAN for 2 and 3 weeks (10 and 15 exposures) or to 7.4 mg/m3 for 2 weeks (10
exposures) had no appreciable effect on mortality or mean survival time
(Table 2).
TABLE 2. MORTALITY AND SURVIVAL RATES OF MICE DAILY EXPOSED
3 HR/DAY 5 DAYS/WEEK FOR 2 OR 3 WEEKS TO PAN AND
CHALLENGED WITHIN 1 HR WITH STREPTOCOCCUS AEROSOL
PAN Exposure
Concn
mg/m3
4.9
4.9
7.4
Duration Air
(days)
10
15
10
D/T
31/160
2/30
17/75
%
19
7
23
Mortality
PAN
D/T
41/159
2/27
12/77
%
26
.8
16
Change
%
+7
+1
-7
RMSR (days)
Air
23.8
26.6
23.2
PAN
22.6
26.4
25.0
Change
-1.2
-0.2
+1.8
15
-------
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tr
lOOr
90
i son
o
5 70
38 60
UJ
50 -
UJ
40
>-
t 30
£
Q 20
10
10 20 30
40 50
CXT
60 70 80 90 (00
(PAN, mg/m x Exposure, hr)
Figure 1. Relationship between excess mortality due to
streptococcal pneumonia in mice and CT index
for PAN exposure (Mean and 95% CL).
16
-------
Lung Clearance of Inhaled Bacteria—•
The rates of clearance of inhaled viable Streptococcus sp. (tsg) from
lungs of mice after single and multiple 3-hr exposures to PAN were not sig-
nificantly different from those seen in mice exposed to air (Table 3). After
single 3-hr exposures to 14.8 or 19.8 mg/m3 PAN clearance rates fluctuated
with respect to air controls, although mortality rates were significantly
higher in mice exposed to the same concentrations of PAN. Upon repeated daily
exposures 3 hr/day, 5 days/week for 2 or 3 weeks to 4.9 or 7.4 mg/m3 PAN the
clearance rates were similar to those seen in mice exposed to air.
TABLE 3. CLEARANCE OF INHALED STREPTOCOCCUS FROM LUNGS
OF MICE EXPOSED TO PAN
PAN Ejcposure
Concn Number of
mg/m3 3-hr Exposures
14.8 1
19.8 1
4.9 10
7.4 10
Mice
Strain Age
(wks)
CDF! 9-12
CDF-, 5
CFi 16
CFn 6-9
Clearance Rate
t50 (hr)a
Air PAN
1.13 1.00
0.66 0.84
1.10 1.25
0.96 1.09
4.9
15
8
1.20 1.00
a Time in hours required to clear 50% of inhaled viable
bacteria.
Effect on Resistance to Tuberculosis
To study the effects on the resistance to chronic mycobacterial infection
mice and guinea pigs were exposed to various concentrations of PAN. Inasmuch
as inhalation of M. tuberculosis aerosols per se did not result in mortalities,
other indicators of changes in the resistance to the infection were used.
Initially these parameters included the determination of the numbers of
tubercles present on the lung surface and histopathological changes in the
respiratory tract.
Neither parameter provided satisfactory information as to the course
or the severity of the infection. Formation of tubercles on the lung surface
did not occur before the 4th week and the tubercles were difficult to count.
Typical focal granulomas appeared at 3 weeks and became confluent during the
later stages of the infection so that the differences between lungs were
difficult to quantitate.
17
-------
Mycobacterial liters in Lungs--
The changes in resistance to the infection, as determined by M. tuberculosis
titers in lungs were studied in mice exposed to PAN and compared to those in
mice exposed to filtered air. In all experiments the mice were challenged
first with M. tuberculosis aerosol and exposed to various PAN regimens after
the infectious challenge.
The inhaled dose of M. tuberculosis ranged from 45 to 125 CFU/lung and
there was no apparent increase in the titers at 1 week after the infectious
challenge. In control mice exposed to filtered air the titer of mycobacteria
showed a continuous increase reaching a peak concentration at 4 to 5 weeks
after the infectious challenge. In general the titers remained approximately
constant for an additional 2 to 3 weeks and showed a slow decrease thereafter.
o
Daily 3 hr exposure to 25.0 mg/m PAN for 6 days beginning one week after
the respiratory challenge with M. tuberculosis aerosol did not produce any changes
in the infection in mice, as measured by bacterial lung titers. In mice exposed
to PAN and those exposed to filtered air the titer of M. tuberculosis in lungs
reached a peak at approximately 4 weeks after the infectious challenge and a
slight decrease during an additional 6 week holding period was seen.
Immune Response--
The cutaneous sensitivity to PPD was determined in guinea pigs challenged
with M. tuberculosis aerosol and within less than 3 hr exposed for 3 hr to
29 mg/m3 PAN or exposed daily 3 hr/day for 5 days to 19.8 mg/m3 PAN. After the
single 3-hr exposure to 29 mg/m3 PAN the diameter of erythema increased gradually
until a peak response was seen at 6 weeks after the infectious challenge.
Between the 7th and 18th week after the challenge a slow decrease of the
erythema was seen. Guinea pigs exposed to filtered air responded in a similar
manner. After five daily 3-hr exposures to 19.8 mg/m3 PAN a very slow increase
in diameter of erythema was seen and a peak occurred at 11 weeks. In guinea
pigs exposed to filtered air a more rapid increase in diameter was seen whereas
the peak diameter was reached at 8 weeks after the infectious challenge. In
general during the 16 weeks observation period the diameters of erythemas, ex-
pressing the cutaneous delayed hypersensitivity reaction were persistently
smaller in guinea pigs exposed to PAN than those exposed to air. The differences
in diameter ranged from approximately 0.5 to 2.0 mm, however because of the wide
variations in responses of individual guinea pigs the statistical significance
of the differences could not be ascertained.
The serum hemagglutination antibody titers were determined at approximately
3-week intervals in guinea pigs exposed to 19.8 mg/m3 PAN 3 hr/day for 5 days.
The multiple exposures resulted in elevated antibody titers at 3, 6 and 9 weeks
after the challenge but lower titers at 12 and 15 weeks when compared to those
in guinea pigs exposed to filtered air (Figure 2). At 26 weeks after the
challenge the serum antibody titers were identical in both groups of animals.
18
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o.
32
16
6 9 12
WEEKS AFTER CHALLENGE
15
Fioure 2
Serum hemagglutination antibody titers in guinea pigs
challenged with M. tuberculosis and within <3 hr
exposed to 19.8 mg/m3 PAN 3 hr/day for 5 days.
-------
Effects on Free Pulmonary Cells
Limited experiments were conducted to determine the effect of exposure
to PAN on free cells lavaged from lungs of mice. The treatment groups in-
cluded mice exposed for 3 hr to 19.8 mg/m3 PAN and those exposed daily,
3 hr/day, 5 days/week for 2 weeks to 7.4 mg/m3 PAN. Mice maintained in
filtered air for corresponding periods of time were included with each ex-
posure regimen (Table 4).
3
The single 3-hr exposure to 19.8 mg/m PAN resulted in a significant
(p>£0.05) increase in total cell counts, some decrease in ATP levels but
did not affect the differential counts nor the viability or phagocytic activity
of the alveolar macrophages. The multiple exposures to 7.4 mg/m3 PAN resulted
in a decrease although not significant in total cell counts and significant
decrease in the ATP levels in the alveolar macrophages. The differential
counts and the viability and phagocytic activity of the alveolar macrophages
were not affected. The decrease in ATP activity indicates some impairment
to the alveolar macrophages, since utilization of ATP in the cells provides
the energy requirement for the phagocytic process (17).
Cells lavaged from the lungs of mice in the various treatment groups
were incubated to determine their ability to attach to a glass substrate.
Scanning electron microscopy examination indicated that after a single e-hr
exposure to 19.8 mg/m3 PAN a large number of alveolar macrophages attached to
the glass; no surface structural changes compared to cells obtained from
control mice exposed to filtered air were apparent. After repeated 10 ex-
posures to 7.4 mg/m3 PAN practically no cells were found on the glass substrates
and therefore the determination of morphologic changes could not be made.
Thus, it appears that the ability of alveolar macrophages to attach to a
suitable substrate was impaired.
Respiratory Tract Tissue Morphology
Changes in the respiratory tract tissues of mice after single and
multiple exposures to PAN were examined by scanning electron microscopy.
An abnormal raised appearance of nonciliated epithelial cells of the nasal
cavity was the major change seen in mice exposed for 3 hr to 25 mg/m-3 PAN
(Figure 3). The lungs and trachea appeared normal upon both light and
scanning electron microscopic examination. A similar raised appearance of
nonciliated epithelial cells with some sloughing and excess mucus was apparent
in the nasal cavities of mice exposed daily for 3 hr, 5 days/week for 2
weeks of 5-week-old mice to 7.4 mg/m3 PAN resulted in a similar damage in the
nonciliated epithelial cells of the trachea. The same exposure regimen of
16-week-old mice caused congestion in the lungs evidenced by thickened
alveolar walls, however to some extent this thickening was also seen in
mice exposed to filtered air (Figure 6). In both groups of animals the nasal
cavity tissues and tracheas appeared normal.
20
-------
TABLE 4. EFFECT OF EXPOSURE TO PAN ON FREE CELLS LAVAGED FROM THE LUNGS OF MICE3
Exposure
Cell Count
Concn Number of Total
mg/m3 3 hr Exposures xlO^
ro
0 1
19.8 1
0 10
7.4 10
Mean
6.92
9.95*
8.86
6.11
SE
0.72
1.03
0,86
1.10
Macrophage
Mean
97.0
97.7
97.7
96.0
SE
1.5
0.9
0.7
1.2
Differential
Alveolar Macrophage
Viability,
Lymphocytes Neutrophils %
Mean
3.0
2.3
2.3
4.0
SE Mean SE
1.5 <1
0.9 <1
0.7 <1
1.2 <1
Mean
95.2
96.7
95.2
95.6
SE
1.3
0.5
0.9
0.5
Phagocytosis, & ATP
6
1
Mean
91.0
88.0
89.9
90.6
I
SE
2.0
1.0
1.8
1.1
Mean
1.03
0.89
0.98
0.81*
SE
0.05
0.05
0.05
0.05
a Cells lavaged from 6 mice, except for 4 mice used for total cell count after 10 exposures to PAN.
Percent of alveolar macrophages engulfing at least one latex sphere after 1 hr incubation at 37 C.
*
Significant change from corresponding mice exposed to air (p^0.05).
-------
ro
ro
Figure 3 Nonciliated epithelial cells in nasal cavities of mice, (a) normal;
(b) and (c) raised and clumped cells in mice exposed for hr to
25 mg/m3 PAN (5 ym).
-------
f.)
Figure 4.
Ciliated epithelial cells in nasal
mucus in mice exposed to 4.9 mg/m3
(5 ym).
cavities of mice
PAN, 3 hr/day, 5
(a) normal;
days/week for
(b) excess
3 weeks
-------
Figure 5.
Nonciliated epithelial cells in nasal cavities of mice, (a) normal; (b) raised
and sloughing cells in mice exposed to 4.9 mg/m3 PAN, 3 hr/day, 5 days/week
for 3 weeks (5 ym).
-------
I
•1
Figure 6. Lungs of mice exposed to 7.4 mg/m PAN 3 hr/day, 5 days/week for 2 weeks.
(a) normal tissue in 6 week-old-mice exposed to air; (b) slight thickening
of alveolar walls in 16 week-old-mice exposed to air; (c) pronounced
thickening of alveolar walls in 16 week-old mice exposed to PAN (20 um).
-------
EFFECTS OF OZONE ON RESISTANCE TO CHRONIC RESPIRATORY INFECTION
The effects of ozone on the resistance to chronic mycobacterial infection
were studied in mice and guinea pigs. Although CF] mice served as the experi-
mental host in the majority of the experiments, similar results were obtained
when CDF or C57BL/6 mice were used.
Kycobacterial Titers in Lungs
The changes in resistance to the infection, as determined by M.
tuberculosis titers in lungs and, in some experiments, in spleens were studied
in mice exposed to ozone and compared to those in mice exposed to filtered
air. In all experiments mice were challenged first with M. tuberculosis
aerosol and exposed to various ozone regimens after the infectious challenge.
3
A single 3 hr exposure to 2.9 mg/m 03 initiated within less than 3 hr
or 1 days after the challenge with M. tuberculosis aerosol resulted in bacterial
lung titers that did not differ from those seen in mice exposed to air.
Similarily no marked changes in titers were seen in lungs or spleens in mice
challenged with M. tuberculosis aerosol and within «:3 hr, 7 days or 14 days
exposed 3 hr/day for up to 7 weeks to 0.98 mg/m3 03.
Multiple daily exposures to 1.96 mg/m3 03, 3 hr/day, 5 days/week for
up to 8 weeks initiated at 7 and 14 days after the challenge with M. tuberculosis
aerosol resulted in increased bacterial titers in lungs when compared to those
of mice exposed to filtered air. In all groups, irrespective whether or not
exposed to ozone, a lag in bacterial growth was seen during the first week
after the challenge. In lungs of mice exposed to air a peak M. tuberculosis
titer was attained at 4 weeks after the challenge and a slow decrease was
seen thereafter. A similar initial lag and growth pattern was seen in mice
exposed to ozone and a peak bacterial titer in lungs also appeared at 4 weeks
after the challenge. However, the titers were markedly higher than those
seen in lungs of mice exposed to filtered air. A decrease in titers occurred
during the ensuing 4-week period with the titer being persistently higher
than in mice exposed to air (Figure 7). At most time intervals the differ-
ences in lung titers were significant (p<:0.05).
Immune Response
The effects of ozone on cell mediated and humoral immune responses to
tuberculosis infection were studied in guinea pigs by determining the cutaneous
delayed hypersensitivity reaction and serum antibody titers. Guinea pigs
were challenged with M, tuberculosis aerosol and 3 hr or 7 days later exposed
to various concentrations of ozone for single or multiple 3 hr periods.
Continuous sensitivity to PPD, expressed as diameter of erythemas in
guinea pigs infected with M. tuberculosis, was significantly affected by a
single 3 hr exposure to 2.9 mg/m3 03 (Figure 8). The diameters of the
erythemas in guinea pigs exposed to ozone were significantly smaller during
the 4 to 7 week period, but did not differ from those in guinea pigs exposed
to air during the 8 to 18 week period after the infectious challenge.
26
-------
o
c
3
0>
O.
FORMINK
0
1 2345678
INITIATION OF
03 EXPOSURE
3
O
o
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«•
<
ui 12.0
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Figure 8.
I
I
I
I
I I
4 6 8 10 12 14
WEEKS AFTER CHALLENGE
16
Cutaneous sensitivity to PPD in guinea pigs
challenged with M. tuberculosis and within
3 hr exposed for 3 hr to 2.9 mg/m3 03.
28
-------
The peak response (i.e., the largest diameter) in guinea pigs exposed to ozone
occurred at 10 weeks instead of 5 weeks as in those exposed to air. The
hemagglutination antibody titers determined in the same guinea pigs exposed
to ozone ranged from 16 to 128 over a 14 week observation period. The titers
did not differ from those seen in animals exposed to filtered air.
3
Exposure to 0.98 mg/m 03, 3 hr/day for 5 days initiated within 3 hr
after the challenge with M. tuberculosis aerosol had no effect on the hyper-
sensitivity response in guinea pigs. The diameters of erythemas measured at
weekly intervals during a 36 week observation period did not differ from those
seen in guinea pigs exposed to filtered air.
The hemagglutination antibody titers were determined at approximately
3 week intervals during the 15 weeks. The titers were markedly higher in
guinea pigs exposed to ozone than in those exposed to air during the initial
12 weeks (Figure 9). The peak antibody titer appeared to be attained at
9 weeks after the challenge and the titers decreased slowly during the
remaining 7-week period. In guinea pigs exposed to air, the peak antibody
titer appeared at 15 weeks after the challenge.
In summary, single 3-hr exposures to 2.9 mg/m 03 caused a suppression
of delayed hypersensitivity in guinea pigs, especially during the peak
response at 5 and 6 weeks after infectious challenge with M. tuberculosis
aerosol. Serum antibody titers were not altered. Daily 3-hr exposures to
0.98 mg/nr 63 for 5 days did not suppress delayed hypersensitivity, but
enhanced the serum antibody titer. Since the host response to tuberculosis
involves primarily cell-mediated immunity it is reasonable to suggest that
a suppression of the delayed hypersensitivity response, which is associated
with cell-mediated immunity, enhanced the local lung infection in mice
exposed to ozone. Although not conclusively demonstrated, a suppression of
delayed hypersensitivity appeared to be present in most of the guinea pigs
exposed to ozone.
29
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12
15
WEEKS AFTER CHALLENGE
Figure 9.
Serum hemagglutination antibody titers in guinea pigs
challenged with M. tuberculosis and within 3 hr
exposed to 0.98 mg/m3 03 3 hr/day for 5 days.
30
-------
REFERENCES
1. Stephens, E. R., E. F. Darley, 0. C. Taylor, and W. E. Scott. Photochemical
Reaction Products in Air Pollution. Int. J. Air and Water Pollut.
4:79-100, 1961.
2. Campbell, K. I., G. L. Clarke, L. 0. Emik, and R. L. Plata. The
Atmospheric Contaminant Peroxyacetyl Nitrate. Arch. Environ. Health
15:739-744, 1967.
3. Campbell, K. I., L. 0. Emik, 6. L. Clarke, and R. L. Plata. Inhalation
Toxixity of Peroxyacetyl Nitrate:Depression of Voluntary Activity in
Mice. Arch. Environ. Health 20:22-27, 1970.
4. Dungworth, D. L., G. L. Clarke, and R. L. Plata. Pulmonary Lesions
Produced in A-strain Mice by Long-term Exposure to Peroxyacetyl Nitrate.
Amer. Rev. Resp. Dis. 99:565-574, 1969.
5. National Air Pollution Control Administration (NAPCA). U.S.D.H.E.W. Air
Quality Criteria for Photochemical Oxidants. AP-63 March, 1970. pp. 8-35.
6. Lefford, M. J., D. D. McGregor, and G. B. Macjcaness. Immune Response to
Mycobacterium tuberculosis in Rats. Inf. and Immun. 8:182-189, 1973.
7. Collins, F. M., and M. M. Smith. A Comparative Study of the Virulence of
Mycobacterium tuberculosis Measured in Mice and Guinea Pigs. Amer. Rev.
Resp. Dis. 100:631-639, 1969.
8. Smith, D. W., D. N. McMurray, E. H. Wiegeshaus, A. A. Grover, and G. E.
Harding. Host-Parasite Relationships in Experimental Airborne Tuberculosis.
Am. Rev. Resp. Dis. 102:973-949, 1970.
9. Schell, R. F., W. F. Ealey, G. E. Harding, and D. W. Smith. The Influence
of Vaccination on the Course of Experimental Airborne Tuberculosis in
Mice. J. Reticuloendothelial Soc. 16(3):131-138, 1974.
10. Kostiala, A. A. I. Delayed Hypersensitivity in the Guinea Pig Immunized
with Killed Tubercle Bacilli in Adjuvant. 1. Development of Peritoneal
Cell Migration Inhibition, Skin Reactions, and Antibodies to Tuberculin
Purified Protein Derivative. Acta. Path. Microbiol. Scand. Section B
79:275-280, 1971.
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REFERENCES (cont.)
11. Thienes, C. H., R. G. Skillen, A. Hoyt, and E. Bogen. Effects of Ozone on
Experimental Tuberculosis and on Natural Pulmonary Infections in Mice.
Am. Ind. Hygiene Assoc. J. 26:255-260, 1965.
12. Kacmarek, A. J., I. J. Solomon and M. Lustig. Preparation and Properties
of Peroxyacetyl Nitrate. J. Inorg. Nucl. Chem. 40:574-576, 1978.
13. Boyden, S. V. The Adsorption of Proteins on Erythrocytes Treated with
Tannic Acid and Subsequent Hemagglutination by Antiprotein Sera. J.
Exp. Med. 93:107-120, 1951.
14. Middlebrook, G., and R. J. Dubos. Specific Serum Agglutination of
Erythrocytes Sensitized with Extracts of Tubercle Bacilli. J. Exp.
Med. 88:521-528, 1948.
15. Indirect Hemagglutination Test for Serodiagnosis of Mycoplasmas, in:
Handbook of Microtiter Procedures, T. B. Conrath, ed. Dynatech. Corp.,
Cambridge, MA 1972. pp. 231-254.
16. Daniel, M., J. G. M. Weyland, Jr., and A. B. Stavitsky. Micromethods
for the Study of Proteins and Antibodies. IV. Factors Involved in
the Preparation and Use of Stable Preparation of Formalinized, Tannic
Acid-Treated, Protein-Sensitized Erythrocytes for Detection of Antigen
and Antibody. J. Imnunol. 90:741-750, 1963.
17. Waters, M. D., T. 0. Vaughan, D. J. Abernethy, H. R. Garland, C. C. Cox,
and, D. L. Coffin. Toxixity of Platinum (IV) Salts for Cells of
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32
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing}
1. REPORT NO.
EPA-600/1-79-001
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
EFFECT OF EXPOSURE TO PAN AND OZONE ON SUSCEPTIBILITY
TO CHRONIC BACTERIAL INFECTION
5. REPORT DATE
January 1979
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Gail B. Thomas, James D. Fenters and Richard Ehrlich
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
IIT Research Institute
Life Sciences Research Division
10 West 35th Street
Chicago, IL 60616
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
68-02-1273
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
RTP.NC
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The effects of peroxyacetyl nitrate (PAN) and ozone (Oj) on susceptibility of mice
and guinea pigs to chronic and acute respiratory infections were studied. The
agent used for the acute infectious disease was Streptococcus sp. whereas Hycobac-
terium tuberculosis served as the agent for chronic respiratory infection. A sign-
ificant increase in mortality due to streptococcal pneumonia was seen upon a single
3-hr exposure to PAN in concentrations ranging from 14.8 to 28.4 mg/m3. Multiple
daily exposures to 4.9 or 7.4 mg/nr PAN 3 hr/day, 5 days/week for up to 3 weeks
had no effect on mortality, survival rates, or ability to clear inhaled Streptococ-
cus sp. from the lungs. Daily 3-hr exposures to 25.0 mg/nr PAN did not produce any
marked changes in the chronic infection as measured by H. tuberculosis titers in the
lungs. The diameter of erythemas, expressing the cutaneous delayed hypersensitivity
reaction were persistently smaller in guinea pigs exposed to PAN than those exposed
to air. Multiple exposures to 19.8 mg/m3 PAN resulted ininitial elevation of anti-
body titers, but depression of titers during the later (12 to 15 week) observation
period. A single exposure to the same concentration of PAN resulted in a significant
increase in total number of cells lavaged from their lungs but somewhat decreased
levels of adenosine triphosphate (ATP). Exposure to 7.4 mg/m3 PAN 3 hr/day, 5 days/
week for 2 weeks resulted in reduced total cell counts and a significant reduction of
ATP levels in alveolar macrophages. Scanning electron microscopic observations of the
respiratory tract showed that the nonciliated cells of the nasal cavities and tracheas
of mice exposed to PAN were raised and sloughing and excess mucus was present. In
older mice lung congestion was enhanced by PAN exposure. Exposures to ozone resulted
in increased titers of M^ tuberculosis in the lungs, depression of hypersensitivity
reaction and elevation in serum antibody titers.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. COS AT I Field/Group
peroxyacetic acid
ozone
respiratory infection
toxicity
sensitivity
06 F, T
18. DISTRIBUTION STATEM-ENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
40
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION IS OBSOLETE
33
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