v>EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600 1-78-057
August 1978
Research and Development
interactions of
Various
Pollutants on
Causation of
Pulmonary Disease
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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-600/1-78-057
August 1978
INTERACTIONS OF VARIOUS POLLUTANTS ON
CAUSATION OF PULMONARY DISEASE
by
Richard Ehrlich
I IT Research Institute
Chicago, Illinois 60616
Contract No. 68-02-2274
Project Officer
Donald E. Gardner
Biomedical Research Branch
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.
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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.
Studies of health effects of air pollutants have traditionally been
concerned with causa.! association between a .single pollutant and a disease
state. However,..sinee it is well established that multiple factors are
frequently responsible for the occurrence of natural diseases it is important
to consider such multiple causality in the evaluation of the biological
effects of air pollutants. One of the interactions is depicted by the
experimental animal model system which reflects an enhancement of severity
of bacterial or viral infections by an exposure to air pollutants. The
animal model represents an overall toxic response of the respiratory system,
which includes edema, inflammation, cellular necrosis, altered macrophage
function and ciliostatis. Thus, this model indicates impairment of the
basic defense mechanism in the lung by the combination of exposure to air
pollutants and superimposed challenge with airborne infectious micro-
organisms. The objective of the project was to determine the effects of
single or multiple short-term exposures to various gaseous and particulate
pollutants on the resistance to respiratory infections.
F. G. Hueter, Ph. D.
Acting Director,
Health Effects Research Laboratory
iii
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ABSTRACT
Studies were conducted to determine the effects of various pollutants and
exposure regimens to the pollutants on the resistance to respiratory infections
caused by inhalation of Streptoaoecue pyogenee or influenza virus aerosols.
The individual pollutants or their mixtures included nitrogen dioxide (N02)>
ozone (03), sulfur dioxide (SQ2). zinc sulfate (ZnS04),zinc ammonium sulfate
(Zn(NH4)2(S04)2) and ammonium sulfate
A single 3 hr exposure to 3760 yg/m (2.0 ppm) N02 or to 196 yg/m3
(0.1 ppm) 03 increased the susceptibility of mice to streptococcal pneumonia.
This was demonstrated by excess deaths and reduced survival time compared to
infected mice exposed to filtered air. Within the N02 concentration range of
2820 (1.5 ppm) to 9400 yg/m3 (5.0 ppm) and 03 ranging from 98 (0.05 ppm) to
980 yg/m3 (0.5 ppm) the response was linear whereby Increase in concentration
Of tne pollutant resulted in Increased excess mortalities.
The effect of a single 3 hr exposure to a mixture containing N02 and Oq
was additive. At most concentrations the total excess mortality was equivalent
to the sum of excess deaths resulting from exposure to each individual pollu-
tant. At higher concentrations of the pollutant mixture (I.e. 6580 yg/m3
(3.5 ppm) N02 and 980 yg/m3 (0.5 ppm) Oo a 1 hr exposure was equally effective
as the 3 hr exposure. However, at the Tower concentrations the 1 hr exposure
was less effective in inducing excess mortality.
The ability to clear inhaled bacteria from lungs was impaired by exposure
to the N02 and 03 mixture. A marked increase in the time required to clear 50%
of viable bacteria within 4 to 5 hr after the infectious challenge was seen
upon exposure to 6580 yg/m3 (3,5 ppm) N02 and 196 yg/m3 (0.1 ppm; 03 mixture,
At the same time a larger percentage of mice exposed to the pollutants had
S. pyogenes present in their lungs 6 days after the respiratory challenge than
mice exposed to filtered air.
3 3
A single 3 hr exposure to either 6580 yg/m (3.5 ppm) N02 or 980 yg/m
(0.5 ppm) 03 but not to a mixture of the two enhanced the susceptibility of mice
to influenza infection. Exposure to these pollutants after a respiratory
challenge with influenza virus aerosol resulted in excess mortality, reduced
survival time and increased lung consolidation.
IV
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Daily 3 hr exposures, 5 days/week for 1, 2 or 4 weeks to mixtures con-
sisting of 3760 yg/m3 (2.0 ppm) N02 and 98 yS/m3 (0,05 ppm) 03 resulted in
significant excess mortalities from streptococcal pneumonia, Inasmuch as the
exposure to the individual pollutants had no effect on the susceptibility to
infection, a syngeristic interaction between the two pollutants appeared to
be present. An identical exposure regimen but to 2820 yg/m3 (1.5 ppm) N02
and 196 yg/m3 (0.1 ppm) 03 mixture induced significant excess mortalities only
after 4 week exposure. Susceptibility to influenza infection was not
affected by the multiple exposure to either of the pollutants or pollutant
mixtures.
3
Daily 3 hr exposures, 5 days/week for 4 weeks to 3760 yg/m (2.0 ppm)
N02 and 98 yg/m3 (0.05 ppm) 03 did not diminish the effectiveness of influenza
vaccine in protecting mice from a respiratory challenge with infectious influenza
virus. However, in general more severe lung lesions, increased hemagglutination
inhibition (HI) antibody titers and increased rate of HI seroconversion was
seen in vaccinated mice exposed to the pollutants.
To more closely simulate ambient environmental exposure of human popu-
lations, studies were conducted using five different exposure regimens, three
of which were of major importance.
3
• daily 3 hr exposure, 5 days/week to 940 yg/m (0.5 ppm) N02
• daily 3 hr exposure, 5 days/week to a mixture of 940 yg/m (0.5 ppm)
N02 and 196 yg/m3 (0.1 ppm) 03
o
• continuous exposure 24 hr/day, 7 days/week to 188 yg/m (0.1 ppm) N0«
with superimposed daily 3 hr peaks, 5 days/week of 940 yg/m3 (0.5 ppffl)
N02 and 196 yg/m3 (0.1 ppm) 03
The 6 month exposure to any of the pollutant combinations resulted in a
marked increase in the susceptibility to streptococcal pneumonia. This was
manifested by excess mortality and reduced survival time. The increase in
susceptibility was much more pronounced after 1, 2 or 3 months when the animals
continued to be exposed for 14 days after the infectious challenge to the
pollutants instead to filtered air.
After a 3 month exposure to the N02 and 03 mixtures there was a marked
decrease in the ability of the animals to clear inhaled 5. pyogenes from
their lungs. This impairment was not present in mice exposed dally for 3 hr
to 940 yg/m3 (0.5 ppm) N02 only. Pulmonary cellular defenses were somewhat
affected by the 3 month exposure to the pollutant mixture but not by the
daily exposure to N02 only. The total cell count in the fluid lavaged from
the lungs was markedly reduced as was the viability and phagocytic activity
of the alveolar macrophages.
In non-infected mice the 6 month continuous exposure to 188 yg/m3
(0.1 ppm) N02 with the superimposed dally 3 hr NO? and 03 mixture peaks resulted
in significant decrease in serum cholinesterase, increase in serum glutamic
pyruvic transaminase and serum glutamic oxalic transaminease and shift to
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lactate dehydrogenise (LDH-1) isoenzyme. In the same treatment group a de-
crease in hematocrit and an increase in platelet counts was seen, A similar
trend in changes was seen in mice daily exposed for 3 hr to the N02 and 03
mixture only. The changes in blood serum enzyme activity were in close agree-
ment with those reported for guinea pigs exposed for 4 months to 94C yg/mJ
(0.5 ppm) N02-
o
Single or up to 15 multiple daily 3 hr exposures to 13.1 mg/m (5.0 ppm)
S02 alone or in mixture with either, carbon particles, zinc sulfate particles
or N02 and 63 had no effect on the susceptibility of mice to streptococcal
pneumonia. Continuous exposure 24 hr/day for up to 3 month to the same S02
concentration also had little effect on the susceptibility to respiratory
infections as well as on other health effect parameters.
Inhalation of zinc sulfate (Vi.2 mg/m ) or zinc ammonium sulfate
(>2.1 mg/m3) followed by a respiratory challenge with 5". pyogenes aerosol
resulted in significant excess mortality and reduced survival time in mice.
For the 3 hr inhalation exposure the estimated concentration of zinc sulfate
which induced 20% excess mortality (ED2o) was 1.45 mg/m3 whereas that of
zinc ammonium sulfate was 2,40 mg/m3. Exposure to ammonium sulfate aerosol.
in concentrations ranging from 1.1 to 5.3 mg/m3 had no effect on susceptibility
to streptococcal pneumonia.
vi
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CONTENTS
Foreword , iii
Abstract iv
Figures , . , . , viii
Tables , . , . . . viii
Abbreviations , x
1. Introduction ., 1
2. Conclusions , 3
3. Recommendations 5
4. Materials and Methods . . , . 6
Animals 6
Exposure Chambers 6
Pollutants 7
Infectious Agents , , 9
Infectious Challenge 9
Health Effects Parameters 9
Statistical Analyses 12
5. Results and Discussion 13
Single 3-Hr Exposure to N02 and 03 Mixtures 13
Multiple 3-Hr Exposure to N02 and 03 Mixtures 19
Long-Term Exposure to N02 end 03 Mixtures 24
Exposure to S02 - 37
Exposure to Sulfates 40
References 47
vii
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FIGURES
Number Page
1 Percent mortality in mice exposed for 3 hr to various
concentrations of N02 or 03 and challenged with
Streptococcus aerosol ,,.,..,.,., 14
2 Excess mortality in mice after multiple 3 hr exposures to
N02 and 03 mixtures and respiratory challenge with
Streptococcus aerosol ... 20
3 Changes in mortality from streptococcal pneumonia after
various regimens of exposure to N02 and 03 mixtures .... 28
4 Effect of various regimens of exposure to N02 and 03
mixtures on blood serum enzymes 34
5 Excess mortality in mice exposed for 3 hr to sulfates and
challenged with Streptococcus aerosol 43
6 Effect of 3 hr exposure to sulfates on the survival time
of mice infected with Streptococcus . 44
TABLES
Number
1 Excess Mortality in Mice Challenged with Streptococcus
Aerosol After Single 3-Hr Exposure to N02 and 03 Mixtures . 15
2 Mortality and Survival Rates of Mice Exposed for 3 or 1 Hr
to N02 and 03 Mixtures and Challenged with Streptococcus
Aerosol 16
3 Changes in Resistance in Mice Challenged with A2/Taiwan
Influenza Virus Followed 24 Hr Later with a 3 Hr N02-03
Exposure 17
4 Retention of Inhaled Viable Streptococcus in Lungs of Mice
Exposed for 3 Hr to N02 and 03 Mixtures 18
5 Effect of Daily 3 Hr Exposure, 5 Days/Week for 4 Weeks to
N02 and 03 Mixtures on Resistance tc Influenza Infection . 21
vi ii
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LIST OF TABLES (cent.)
Number Page
6 Lung Consolidation and Geometric Mean Reciprocal HI
Antibody Titers in Mice Exposed 3 Hr/Day, 5 Days/Week
for 4 Weeks to N02 and Os Mixtures, Vaccinated
Challenged with Infectious Influenza Virus Aerosol , . , , 23
7 Effect of Exposure to N02 and Os Mixtures on Mortality
and Survival Time of Mice Challenged with streptococcus
Aerosol , 26
8 Changes in Mortality and Survival Rates of Mice Exposed
to N02 and 03 Mixtures, Challenged with streptococcus
Aerosol and Maintained for 14 Days Either in Filtered
Air or Pollutant Mixtures 29
9 Clearance of Inhaled Viable Streptococcus From Lungs of
Mice Exposed to N02 and 03 Mixtures 30
10 Effect of Exposure to N02 and 03 Mixtures on Alveolar
Macrophages 32
11 Summary of Health Effects in Mice Exposed for 6 Months
to N02 or N02 and 03 Mixtures 36
12 Mortality and Survival Time of Mice Daily Exposed for
3 Hr to 13100 yg/m3 S02 and Challenged with Streptococcus
Aerosol 38
13 Mortality and Mean Survival Time of Mice Exposed to
13100 yg/m3 S02 and Challenged with Streptococcus or
Influenza Virus Aerosol .... 39
14 Mortality and Survival Rate of Mice Exposed for 3 Hr to
Sulfates and Challenged with Streptococcus Aerosol .... 41
15 Mortality of Mice Exposed for 3 Hr to ZnS04, and N02 and 03
Mixtures and Challenged with Streptococcus Aerosol .... 45
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ABBREVIATIONS
A1P — alkaline phosphatase
AM -- alveolar macrophages
AP -- acid phosphatase
CHE -- cholinesterase
D/T -- dead/total
ED -- effective dose
HBDH — hydroxybutyrate dehydrogenase
HI -- hemagglutination-inhibitiun antibody
hr — hour
ICDH -- isocitric dehydrogenase
LD -- lethal dose
LDH -- lactate dehydrogenase
MMD -- mass median diameter
MST -- mean survival time, days
m3 . -- cubic meter g
yg -- microgram, 1 yg = 10" g
N02 -- nitrogen dioxide (ppm x 1880 = yg/m3)
(NH4)2S04 -- ammonium sulfate
03 — ozone (ppm x 1960 = yg/m3)
ppm -- parts per million
RH, -- relative humidity
SGOT -- serum glutamic oxaloacetic transaminase
SGPT -- serum glutamic pyruvic transaminase
S02 — sulfur dioxide (ppm x 2620 = yg/m3)
t5g — time (hr) to clear 50% of inhaled bacteria from lungs
Zn(NH4)2(S04)2 -- zinc ammonium sulfate
-- zinc sulfate
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SECTION 1
INTRODUCTION
Studies of health effects of air pollutants have traditionally been con-
cerned with causal association between a single pollutant and a disease
state. However, since it is well established that multiple factors are fre-
quently responsible for the occurrence of natural diseases It is important to
consider such multiple causality in the evaluation of the biological effects
of air pollutants. One of the interactions 1s depicted by the experimental
animal model system which reflects an enhancement of severity of bacterial
or viral infections by an exposure to air pollutants. The animal model repre-
sents an overall toxic response of the respiratory system, which includes
edema, inflammation, cellular necrosis, altered macrophage function and dlio-
statis. Thus, this model Indicates impairment of the basic defense mechanism
in the lung by the combination of exposure to air pollutants and superimposed
challenge with airborne infectious microorganisms.
Results of experimental studies which utilized this animal model clearly
demonstrated that Inhalation of either ozone or nitrogen dioxide significantly
enhances the susceptibility to bacterial pneumonias. On the other hand, only
sparse data are available on the effect of exposure to mixture of these two
pollutants or to other pollutants on the resistance to respiratory infection.
It is well recognized that effects of air pollutants may be additive or
synergistic with each other. Such combined effects can be ascribed to a
variety of factors. For example, one pollutant can affect the site of the
deposition of another, a pollutant can affect the lung clearance mechanism so
that upon inhalation the second pollutant cannot be removed, or a pollutant
can produce an effect in the lung which makes it more vulnerable to the effects
of the second pollutant. Occasionally the type of interaction can be pre-
dicted on the basis of chemical composition of each of the components of the
pollutant mixture. More often, however, 1t is not possible to forecast
effects of the mixture and therefore empirical studies must be carried out.
The objective of the project was to determine the effects of single or
multiple short-term exposures to various pollutants on the resistance to
respiratory infections. The studies were conducted using mice as the experi-
mental animal host, exposed for 3 hours to either ozone, nitrogen dioxide,
sulfur dioxide, various sulfates as well as mixtures containing various con-
centrations of these pollutants, and then challenged with Streptococcus pyogenes
aerosol. Extensive studies were also carried out to define the effects of
daily 3-hr peak exposure tc nitrogen dioxide and ozone mixtures on the re-
sistance to infection and other health parameters.
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Results of these studies were sumamrized in a series of quarterly progress
reports (IITRI Report No. L6089-1 through L6089-7), Phases of the studies were
reported at the International Conference on Photochemical Qxidant Pollution
and Its Control, September 12-17, 1976, Raleigh, NC and several Environmental
Protection Agency workshops. Publications resulting from the studies are:
Effects of Short-Term Inhalation of Nitrogen Dioxide and Ozone Mixture
R. Ehrlich, J. C. Findlay, J. D. Renters and D. E. Gardner.
Environmental Research J4_, 223-231, 1977,
Susceptibility to Bacterial Pneumonia of Animals Exposed to Sulfates
R. Ehrlich, J. C. Findlay and D. E. Gardner.
Toxicology Letters, (accepted for publication).
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SECTION 2
CONCLUSIONS
Studies were conducted to determine the effects of single and multiple
3 hr exposures to individual air pollutants and pollutant mixtures on the
resistance to respiratory infections. The results reconfirm the utility and
applicability of this experimental animal model system to studies of the
effects of air pollutants in concentrations approximating those found 1n
ambient environment.
Results of nitrogen dioxide (N02) studies point out to the greater Im-
portance of short-term exposures to peak concentrations than prolonged exposures
to low concentrations of N02 in increasing the susceptibility to streptococcal
pneumonia, A single 3 hr exposure to 3760 yg/m3 (2.0 ppm) NO? or a daily
3 hr exposure, 5 days/week for 6 months to 940 yg/m3 (0.5 ppm] N02 resulted
in significant excess mortality and reduced survival time 1n mice Infected with
streptococcus pyogenes aerosol. The data reemphaslze the necessity for the
establishment of primary air quality standard for short-term exposure to N02«
The current air quality standard of 100 yg/m3 (0.05 ppm) NOg represents an
annual arithmetic average which does not take into consideration the health
effects of short-term exposures to elevated concentrations of this pollutant.
Single 3-hr exposure to mixtures containing various concentrations of
N02 and ozone (03) had an additive effect. The excess mortalities due to
streptococcal pneumonia in mice exposed to such mixtures were equivalent to
the sum of excess death resulting from exposure to each Individual pollutant.
Repeated dally 3-hr exposures for up to 4 weeks to mixtures consisting of
3760 yg/m3 (2,0 ppm) N02 and 98 yg/m3 (0.05 ppm) Oo suggested the presence of
a synerglstic interaction between the two pollutants in Increasing the sus-
ceptibility to respiratory infection.
3
Daily 3 hr exposures,q5 days/week for 6 months to mixtures of 940 yg/m
(0.5 ppm) N0£ and 196 yg/m (0.1 ppm) 0? resulted in significant excess mortal-
ity, and reduced mean survival time in infected animals. Continuation of
exposure to the pollutants for 14 days after the Infectious challenge resulted
in a pronounced increased susceptibility to the respiratory infection, after 1»
2 or 3 month exposure. In non-infected mice this exposure regimen induced
changes in the activity of several blood serum enzymes.
Single or multiple 3 hr exposures as well as^continuous exposures to
sulfur dioxide (SOo) In concentration ofl3.1mg/m3 (5.0 ppm) had no marked
effect on the susceptibility to bacterial pneumonia.
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A single 3 hr exposure tg sulfates indicated that, 1n decreasing order
of effectiveness zinc sulfate, zinc ammonium sulfate and ammonium sulfate
reduced resistance to streptococcal pneumonia. The decrease in resistance to
the respiratory infection appeared to be related to the zinc ion present in the
sulfate complex. The lesser effect of zinc ammonium sulfate than zinc sulfate
in enhancing the severity of the infection and the absences of any effect due
to inhalation of ammonium sulfate might be due to the presence of the ammonium
ion which could possibly neutralize the effectiveness of the metallic cation.
These studies indicate that resistance to respiratory infection can be used as
a sensitive parameter for evaluation and ranking of health effects of sulfates.
Moreover, the differences in response to inhalation of the three sulfates
suggest the advisability of identification of the molecular composition of sul-
fate particles during air monitoring. This is of special importance when the
air monitoring is conducted as part of epidemiological studies of health effects
of environmental pollution.
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SECTION 3
RECOMMENDATIONS
Studies of the effects of exposure to air pollutants on the resistance
to respiratory infections represent a sensitive and realistic approach to
defining the health effects of pollutants. Thus, studies should continue to
more precisely define the causal relationship between inhalation of air
pollutants and acute respiratory infections in laboratory animals. This
animal model should be used to establish a toxic ranking of various pollutants
especially those within a given group of related pollutants.
To simulate environmental pollution conditions studies should include the
use of mixture of gaseous as well as particulate pollutants. Exposures to
additional stresses, such as exercise or extremes of temperature and humidity
should also be included in such investigations.
Since man is usually exposed to low concentrations of pollutants with
superimposed cyclical higher concentrations, similar exposure regimens should
be used during long-term exposure studies. This 1s especially Important 1n
studies of pollutants such as nitrogen dioxide, where within a given exposure
regimen the concentration is of greater importance than the duration of
exposure.
Additional studies should also be carried out to elucidate the mechanisms
by which the defense systems against respiratory Infections are affected by
exposure to the pollutants. Thus, the cellular and humoral Immunity as well
as the activity of alveolar macrophages in several species of laboratory
animals must be Investigated,
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SECTION 4
MATERIALS AND METHODS
ANIMALS
The strains of mice used in the various experiments included female CM
(ARS, Madison, WI) and CD2F1 (Murphy Laboratory, Plainfield, IN), and male
and female BDFi (Laboratory Supply, Indianapolis, IN; Murphy Laboratory).
The 5-to-8 week old mice were quarantined for 7-14 days before being used in
the studies. During the quarantine period and throughout the experiments the
mice were housed in groups of 8 in stainless steel shoe box cages. Food and
water were provided, ad libitum.
EXPOSURE CHAMBERS
The 432-liter capacity (120 x 60 x 60 cm) chambers used for short-term
exposure to afr pollutants were constructed from plexiglass. During the short-
term exposures the mice were housed individually in separate compartments of
specially designed stainless steel wire cages. The pollutants entered the
chambers from the top of one side at the approximate rate of 60 ^ 5 liters/min
and were exhausted at the top of the opposite side. To assure a homogeneous
distribution of the pollutants a small fan was operated continuously in each
chamber. To prevent a build-up of ammonia during the exposure of animals to
the pollutants, deotized cage board (Upjohn Co., Kalamazoo, MI) was placed on
the floor of each chamber.
Temperature in the exposure chambers was maintained at 24 t 2°C at ambient
humidity ("o40% RH). During experiments designed to study the eTfects of
elevated humidity, pressurized steam was introduced into the chambers after it
passed through condensation traps. Continuous reading of the relative humidity
was provided by Airguide Humidity Indicator (Model 605) located in each
chamber. An electro-hygometer (Lab-line Instruments, Inc., Melrose Park, IL)
was used to check and calibrate the humidity indicators.
The compressed air supplied to the exposure chambers was passed first
through an Alemite filter (Model 7620, Steward Warner, Chicago, IL) to dry
the air and to remove traces of oil. Before entering the individual exposure
chambers the air was further purified by passage through disposable air puri-
fier and flow equalizer (Koby Inc., Marlboro, MA).
For long-term exposures to the pollutants, five identical 4420-liter
aluminum-lined chambers 1.2 x 1.8 x 2.0 m were used. Randomly selected mice
were housed in suspended wire cages, which were rotated 2 to 3 times/week
to various positions on the cage racks to assure unbiased exposure to the
experimental environment. The pollutants diluted in charcoal-filtered air
entered the chambers at a rate of approximately 20 changes per hr. The same
air flow pattern was maintained in the control chamber, where ambient air
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passed through charcoal filters, was used. The mean temperature In the
chambers was 24° + 2°C at ambient humidity (%30% RH). To verify the con-
centration and homogeneity of the pollutants, air samples were taken from
different sections of the chambers. All exposure chambers were connected to
a manifold system capable of manual or automatic sampling. When the auto-
matic arrangement was used, the sampling time for each chamber was 15 min.
POLLUTANTS
Nitrogen Dioxide
A 1 or 5% N02 gas mixture in balanced air, >99,5% pure, (Matteson, Joliet,
IL) was diluted with the filtered air in a mixing chamber and then passed into
the animal exposure chambers. The N02 concentration was monitored continuously
by a N0-N02-N0x chemi luminescent analyzer (Model 8101 B. Bendix Corp.,
Ronceverte, WV) and was expressed in ppm and yg/m3 (ppm x 1880 = yg/m3).
Ozone (03)
A high-voltage generator (IITRI) was used to convert filtered air or oxygen
to 03. To provide the desired concentration, 03 was diluted with filtered air
in a glass mixing chamber and then passed into the animal exposure chambers.
The 03 concentration was monitored continuously with an 03 chemi luminescent
analyzer (Model OA 310, Meloy Laboratories, Springfield, VA) and was expressed
in ppm and yg/m3 (ppm x 1960 = yg/m3).
N02-03 Mixture
Each gas was introduced into separate glass vessel and then combined in
a glass mixing chamber. The N02 and 03 mixture was then passed into the
animal exposure chambers. The concentration of each gas in the exposure chamber
was monitored continuously with the NOx and 03 chemi luminescent analyzers.
Sulfur Dioxide ($02)
A 1 or 5% C02 gas mixture in balanced air, >99.98% pure, (Matteson,
Joliet, IL) was diluted with filtered air in a glass mixing chamber and then
passed into the animal exposure chambers. The S02 concentration was monitored
continuously with a total sulfur analyzer (Model 8300, Bendix Corp.,
Ronceverte, WV). The analyzer was connected to a dilution panel (Bendix Corp.)
equipped with a stainless steel capillary, metering regulator, pull-to-test
diverter valve and diluting manifold. The gas sample was diluted 10-fold
with filtered air before being analyzed. Periodically the S02 concentration
in the chamber was monitored by the pararosaline method (1). Air samples
for the assays were collected in a all -glass impinger (AGI) operated for
30 min at a rate of 1 liter/min. The concentration of S02 was expressed in
ppm and yg/m3 (ppm x 2620 = yg/m3).
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Carbon Particles
The carbon (Carbon black, 99.5% pure,Sterling MT CT-6729, Cabot Corp.,
Boston, MA) aerosol was generated by a Wright Dust Feeder (L. Adams Ltd.,
London, England). A slowly rotating scraper blade continuously released
a small quantity of carbon powder into an air stream passed through a
nozzle with an impaction plate to break the agglomerates. Particles larger
than 5y were removed by sedimentation during passage of the aerosol through
a 30 cm long settling chamber before entering the mixing chamber.
The average mass concentration of carbon in the aerosol was estimated by
gravimetric analysis. The particles were collected on a HA Q.45y Millipore
filter backed by a 0.22u Millipore filter, both 47 mm in diameter. The total
air collected for assay during the 3 hr animal exposures ranged from 210 to
350 liters. The concentration of carbon particles per cubic meter of air was
estimated by dividing the weight of the particles collected on the membrane
filters by the total volume of collected air.
Sulfates
Aqueous dilutions of zinc sulfate (ZnS04, 99.4% pure, Baker Chem.,
Phillisburg, NJ), ammonium sulfate ((NH4)2S04, 99.7% pure. Fisher, Chicago,
IL), and zinc ammonium sulfate (Zn(NH4)2(S04), 95.0% pure, ICN Pharmaceuticals,
Plainview, NY) were used to generate the aerosol by using a DeVilbiss Model
40 nebulizer. Depending on the desired concentration of the sulfate, the
nebulizer was operated at pressures ranging from 2 to 6 psi. The sulfate
aerosol was passed through a heated glass tube into a glass mixing vessel
where the particles were diluted with dried filtered air and then introduced
into the animal exposure chamber.
The mass median size of droplets produced by the DeVilbiss 40 was 3 ym (2),
The estimated mass median diameter of the evaporated sulfate aerosol particles
was 0.63 ym and the count median diameter approximately 0.31 ym. For particle
size analysis aerosol samples were collected through a 7 ym single-stage
preimpactor on a methanol-cleaned 25 mm glass discs coated with a 50% glycerol-
water solution. Microscopic examination indicated that approximately 90% of
the airborne particles were <3 ym in diameter.
For quantitative assay, the airborne sulfate particles in the chamber were
collected on membrane filter (0.22 yM) at a flow rate of 1.5 to 3.0 liters/nrin.
After collection the filteres were refluxed with 20 ml of distilled water for
90 min. The extract was treated with barium chloride to form barium sulfate
and the turbidity was measured against a standard curve. The concentration
of sulfate was expressed as mg/m^ of S04 (3).
8
-------
INFECTIOUS AGENTS
Bacteria
Streptococcus pyogenes (Lancefield Group C) was used for the respiratory
infectious challenge. To maintain the stock culture, mice were injected
intraperitoneally with a suspension of S. pyog&nes and killed 24 hr later.
Heart blood was incubated on blood agar for 48 hr at 370C and s. pyogenes
colonies isolated from the agar were inoculated in Todd Hewitt broth (BBL).
After 18 hr incubation at 37°C the bacteria were harvested in approximately
1 ml aliquots and frozen at -70°C. For aerosol dissemination, 0,8 ml of the
thawed bacteria was grown in Todd Hewitt broth for 18 hr at 37°C. The optical
density of the culture was adjusted in 0,1% peptone-water to the appropriate
density as measured at 440 My in Spectronic 20 densitometer.
Virus
Mouse-adapted influenza virus was passaged several times in mice and a 20%
lung suspension of the virus was prepared and frozen at -70°C. The virus was
identified by use of specific antiserum obtained from the National Institutes
of Health. For aerosol dissemination, the virus was appropriately diluted in
0.2% bovine albumin containing 100 units/ml of Polymyxin and 100 units/ml of
Neomycin.
INFECTIOUS CHALLENGE
Infectious respiratory challenge was performed in a 400-liter plexiglass
aerosol chamber (71 x 61 x 92 cm) contained within a microbiological safety
cabinet. Temperature and humidity in the aerosol chamber were maintained at
24 +. 20C and 65 +_ 5% RH. A continuous flow DeVilbiss nebulizer (Model 841)
was used to produce the bacterial aerosol. Filtered air was supplied to the
inlet of the nebulizer at a flow rate of approximately 8 liters/nun. For the
infectious challenge, mice were housed in individual compartments of a stain-
less steel wire cage and exposed to the aerosol for 10 to 20 min. After the
challenge, the mice were removed from the chamber and housed in conventional
cages in a clean-air, isolated animal room.
The inhaled dose of bacteria was determined by killing three mice
immediately after the infectious challenge. The lungs were removed, weighed,
and homogenized in 1.8 ml of sterile 0.1% peptone water. The suspension was
diluted, plated on blood agar and the colonies counted after 48 hr incubation
at 37°C. The inhaled dose generally ranged from 5-25 x 103 viable bacteria
per gram of lung tissue.
HEALTH EFFECTS PARAMETERS
Mortality and Survival Time
Deaths were recorded daily during the 14-day observation period following
the infectious challenge. The mortality rates were, reported as either number
of dead out of the total mice used (D/T), percent mortality (%) or percent
excess mortality (% deaths pollutants - % deaths filtered-air controls).
-------
The mean survival tine was calculated according to the following
equation
MST =
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 total number of mice used in the
experiment.
Lung Clearance of Inhaled Bacteria
Groups of five mice were killed immediately after the respiratory
challenge with s. pyogenes aerosol. The lungs were removed aseptically,
weighed, homogenized and cultured quantitatively by using the procedure
described for the determination of the inhaled dose. These initial counts
(0 hr), calculated as the number of viable bacteria per gram of lung, were
considered as unity (100%). Thereafter groups of five mice were killed at
1,2,3,4 and 5 hr and, as appropriate, at 1,2,3 and 6 days after the respira-
tory challenge and their lungs assayed in an identical manner. The counts
were calculated as percent recovery of these present at the 0 hr.
The rate of clearance was expressed as tso i.e., time in hours required
for 50% of the inhaled viable bacteria to be cleared from the lungs. The
was determined on a semilogarithmic regression after converting the percent
recovery to logarithmic values.
Phagocytic Activity
Mice were killed by intraperitoneal injection of sodium pentobarbital and
the lung-heart-trachea complex was removed from the chest cavity in tot-o. A
blunted hypodermic needle inserted into the trachea and fastened with surgical
suture was used to lavage the lungs by repeated infusions of warm (37°C)
saline. Alveolar macrophages were isolated from the lavage fluids by centri-
fugation at 365 xg and washing in Hanks medium.
Total cell counts were made in a hemocytometer. Differential counts
were made on air-dried smears of cells fixed in methanol and stained with
Wright's stain to determine the cellular distribution (i.e., percent of
alveolar macrophages, polymorphonuclear leucocytes and lymphocytes). Viability
of the macrophages was determined by dye exclusion using 1% trypan blue.
Lung Edema
To detect the extent of edema, groups of 8 to 10 mice were killed and
individual lungs weighed immediately after removal. The lungs were then dried
in a vacuum dessicator and reweighed at 24 hr intervals until no additional
weight loss was apparent. The extent of lung edema was expressed as the wet-
to-dry weight ratio.
10
-------
Lung Consolidation
The extent of pulmonary lesions after challenge with influenza virus was
expressed as the percentage of the total lung consolidated (4). A score of
1 represented 25% lung consolidation, 2 = 50%, 3 - 75%, 4 - 100%, and a score
of 5 was assigned to dead animals that succumbed to the infection.
Serological Assays
The hemagglutination inhibition (HI) antibody levels were determined in
duplicate by the microtiter method in disposable "V" plates. In all HI tests,
1% chicken red blood cells and four hemagglutinating units of egg-adapted
influenza A2/Taiwan virus were used. Type A2 influenza (Taiwan/1/64, NIH)
antiserum served as the positive control. To remove nonspecific inhibitors
of hemagglutination the sera were heat inactivated at 56°C for 30 min and
treated with trypsin-periodate. The primary HI antibody titer in all mice
was <8.
Scanning Electron Microscopy (.SEM) and Histopathology
Mice were anesthetized by an intraperitoneal injection of pentobarbital
and the abdomen was opened on the midline, permitting access to the ventral
aorta. The aorta was severed, the animal exsanguinated, the chest was opened,
and the lungs and trachea removed in toto. The trachea was cannulated to the
level of the first cartilaginous ring and the lungs were expanded with
Karnovsky's paraformaldehyde-glutaraldehyde phosphate-buffered fixative at
20 cm water pressure. After a 2 hr perfusion with the lungs completely
immersed in the fixative, the trachea was ligated and the lungs floated in
fixative. The trachea and main stem bronchi were Isolated from the lungs.
The lungs were sectioned with a razor blade so as to reveal the bronchus and
alveoli of each lobe.
After cannulation of the trachea, the head was removed, the skin was re-
tracted 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 sectioned in half by inserting a razor blade on one side of the median
septum and severing the hard palate.
All tissues were washed in distilled water, and dehydrated with, increasing
concentration of alcohol, Pentyl acetate was then substituted for the alcohol,
and the tissues were critical point dried in carbon dioxide. The dried
trachea was sectioned longitudinally, and all tissues were cemented to stubs,
gold coated in a Denton vacuum evaporator and examined in a Kent-Cambridge
Mark II Stereoscan scanning electron microscope at 20 kV.
Lung and trachea tissues were also processed for conventional histo-
pathological examination. Sections were cut at 5y, stained with hematoxylin
and eosin and examined microscopically.
11
-------
Hematology Tests
Erythrocytes and leukocytes were counted in duplicate in 9. Coulter
Electronic Particle Counter with 100 aperature. Reference blood samples were
counted for standardization (5). Hematocrit was determined in capillary tubes
using a microcapillary head centrifuge. Hemoglobin was measured as cyano-
methemoglobin, with a reference solution as standard (6). Wright's stain was
used for differential leukocyte counts. Retlculocytes were counted by the new
methylene blue N method (7). Platelets were counted in a hemocytometer with
a phase microscope.
Clinical Chemistry Tests
The clinical chemistry tests were run on the day of blood collection,
except in a few instances where storage of serum at -20 C was necessary. The
tests on the sera were performed on a centrifugal analyzer (Union Carbide,
Tarrytown, NY) and Included alkaline phosphatase (A1P) (8). lactic dehydro-
genase-L (LDH) (9), hydroxybutyrate dehydrogenase (HBDH) (10), 1soc1tric
dehydrogenase (ICDH) (11), glutamic oxalic transaminase (SG0T) (12), glutamic
pyruvic transaminase (SGPT) (12), cholinesterase (CHE) (13) and acid phos-
phatase (AP) (14), The lactic dehydrogenase isoenzytnes were determined in a
Beckman microzone electrophoresis system.
STATISTICAL ANALYSES
The significance of the-differences in mortality rates was determined by
the Chi-square (X2) test with a 2 x 2 contingency table, The Student t test,
analysis of variance, and least square linear regression were applied to the
data as appropriate.
12
-------
SECTION 5
RESULTS AND DISCUSSION
SINGLE 3-HR EXPOSURE TO NQ2 AND 03 MIXTURES
Mortality and Survival Time
Results of previous studies conducted in our laboratories (15) indicated
that a single 2 hr exposure of mice to 6580 yg/m3 (3,5 ppm} NQ2 significantly
enhanced the mortality resulting from a superimposed bacterial pneumonia
initiated by inhalation of airborne Klebsiella pneumoniae. Coffin and
Gardner (16) reported a similar enhancement of mortality after a 3 hr exposure
to 157 yg/m3 (0.08 ppm) 03 and challenge with Streptococcus sp, aerosol. To
provide unified data in terms of duration of exposure and the Infectious agent,
mice were exposed for 3 hr to various concentrations of N02 and 03 and then
challenged with s. pyogenes aerosol. The death rates obtained during numerous
replicate exposures to the pollutants are summarized in Figure 1. A sig-
nificant increase in mortality rates over those in control mice was seen upon
the 3 hr exposure to 196 yg/m3 (0,1 ppm) Os or 3760 yg/m3 (2,0 ppm) N02-
Moreover, a linear relationship was present between the concentration of the
pollutants and mortality rates with a correlation coefficient (r) of 0.969 for
N02 and 0.996 for 03.
To determine the effects of inhalation of air pollutant mixtures, groups
of mice were exposed to various concentrations of N02, 03, N02 and 03 mixture
or to filtered air. The four treatment groups, usually consisting of 24 mice
each, were then simultaneously challenged with s. pyogenes aerosol. The
mortality rates were Initially compared on the basis of individual exposure
experiments and results of replicate experiments were pooled for further
statistical analysis. The excess mortalities based on a minimum of four
replicate experiments at each concentration of the pollutants are summarized
in Table 1. In most instances the excess mortality was equivalent to the sum
of those induced by inhalation of each individual pollutant. Thus, the effect
of the 3-hr inhalation of N02 and 03 mixtures was additive.
13
-------
100
90
80
>, 70
"5
5 60
S
_ 50
c
fly
fc 40
a.
30
20
10
0
K\N Significant Mortality Change (p<0.05)
( ) Number Of Mice
0 2820 3760 658O 94OO
N Og LL g / f
98 196 98O
.3
Figure I. Percent mortality in mice exposed for 3 hr to various concentrations
of NO2 or 03 and challenged with Streptococcus aerosol.
-------
TABLE 1. EXCESS MORTALITY IN MICE CHALLENGED WITH
STREPTOCOCCUS AEROSOL AFTER SINGLE 3-HR
EXPOSURE TO NQ2 AND 03 MIXTURES
o
Concn. (yg/m ) Excess Mortality (%)
N02 \ 03
0
2820
3760
6580
9400
0
0
-1.7
14.3*
28.2*
35.7*
(Pollutant
98
5.4
4.6
22.0*
-
-
- Control)
196
7.2
4.2
9.1*
38.5*
-
980
28.6*
23.9*
56.2*
68,7*
65.3*
*
Significant change from corresponding infected mice
exposed to filtered air (p^O.05).
The mean survival time was also affected by the exposures to the in-
dividual pollutants as well as the pollutant mixtures. Noninfected mice ex-
posed to only the pollutants survived the 14 day observation period, whereas
the mean survival time of infected mice, not exposed to the pollutants, was
12 days. Significant shortening of survival time was observed upon exposure
to 6580 (3.5 ppm) or 9400 yg/m3 (5 ppm) N02 or 980 yg/m3 (0.5 ppm) 03. Al-
though the single 3 hr exposure to 3760 yg/m3 (2.0 ppm) N02 or 196 yg/m3
(0.1 ppm) 03 significantly enhanced the mortality rates no apparent effect on
the course of the infection was noted. Shortened survival time was also seen
upon exposures to all mixtures containing 980 yg/m3 (0.5 ppm) 03 irrespective
of concentration of N0?, and to the mixture containing 6580 mg/m3 (3.5 ppm)
N02 and 196 yg/m3 (0.1 ppm) 03.
An exploratory experiment was conducted to determine whether a 1-hr ex-
posure to the pollutants induces a change in resistance to streptococcal
pneumonia similar to that observed upon a 3-hr exposure. As seen in Table 2
the 3-hr exposure to 6580 yg/m3 (3.5 ppm) NC^ resulted in a significant in-
crease in mortality and decrease in the survival time. However, a 1 hr ex-
posure to the same N02 concentration failed to produce any marked changes.
Inhalation of 980 yg/m3 (0.5 ppm) 03 or a mixture consisting of 6580 yg/m3
(3.5 ppm) N0£ and 980 yg/m3 (0.5 ppm) 03 resulted in significant excess mor-
tality and decrease in survival time, irrespective of the duration of the
exposure. The 3-hr exposure to the N02 and 03 mixtures at the two lower con-
centrations also resulted in a marked enhancement of mortalities. However,
this was not seen upon 1 hr exposure to the mixture of the pollutants.
15
-------
TABLE 2. MORTALITY AND SURVIVAL RATES OF MICE EXPOSED FOR 3 OR 1 HR TO
N02 AND 03 MIXTURES AND CHALLENGED WITH STREPTOCOCCUS AEROSOL
•
Duration of
Exposure, hr
3
Concn.,
N02
0
6580
0
6580
0
3760
0
3760
0
2820
0
2820
3
tjg/m
03
0
0
980
980
0
0
196
196
0
0
98
98
Mortal i ty
D/T
3/36
8/36
19/36
26/36
1/44
1/44
1/44
6/44
4/24
3/24
3/24
8/24
•*
8.3
22.2
52.8
72.2
2.3
2.3
2.3
13.6
16.7
12.5
12.5
33.3
Change
%
—
+13.9**
+44.5*
+63.9*
_
0
0
+10.7*
*»
- 4.2
- 4.2
+16.6
MST,
day
13.5
12.2*
10.1*
8.2*
13.8
13.9
13.8
12.8
12.5
13.1
13.0
12.0
1
Mortality
D/T
10/72
8/72
18/72
27/72
4/68
5/96
6/96
10/96
7/48
7/48
4/48
3/48
%
13.9
11.1
25.0
37.5
5.9
5.2
6.3
10.4
14.6
14.6
8.3
6.3
Change
%
_
- 2.8
+11.1**
+23.6*
_
- 0.7
+ 0.4
+ 4.5
M
0
- 6.3
- 6.3
MST,
day
12.9
13.1
12.0**
11.1*
13.7
13.6
13.5
13.1
12.8
12.9
13.4
13.5
Significant change from corresponding infected mice exposed to filtered air.
**,
(p ^0.05)
-------
The inability to establish the statistical significance of the increased mortal-
ity upon exposure to 2820 yg/m3 (1.5 ppm) N02 and 98 yg/m3 (0.05 ppm) 03 mix-
ture appears to be due to the small number of animals used in this experiment.
The data suggest that a single 1-hr exposure to the individual pollutants as
well as a mixture of the pollutants is less effective in altering the resistance
to streptococcal pneumonia than a 3 hr exposure.
A single experiment was conducted to determine the susceptibility of mice
challenged with A2/Taiwan influenza virus followed 24 hr later by a single
3 hr exposure to 6580 yg/m3 (3.5 ppm) N02, 980 yg/m3 (0.5 ppm) 03 or a mixture
containing the same N02 and 63 concentrations. Only exposure to N02 resulted
in significant excess mortality, increase in lung consolidation and reduced
survival time (Table 3). Although mortality was almost twice as high among
mice exposed to 03 than infected mice exposed to filtered air, the difference
was not significant.
TABLE 3. CHANGES IN RESISTANCE IN MICE CHALLENGED WITH A2/TAIWAN
INFLUENZA VIRUS FOLLOWED 24 HR LATER WITH A 3 HR NO?-Q3 EXPOSURE
Concn. ,
N02
0
6580
0
6580
yq/m
_Q3_
0
0
980
980
D/T
7/47
23/48
13/48
6/48
Mortality
% Change, %
14.8
47.8* +33.0
27.0 +12.2
12.5 - 2.3
Lung Lesions
in Survivors
3.00
3.90*
3.15
2.69
MST,
day
13.6
12.5*
13.3
13.6
Significant change from corresponding infected mice exposed to
filtered air (p<:0.05).
17
-------
Clearance of Inhaled Bacteria from Lungs
The effects on clearance of viable bacteria from lungs were investigated
in mice exposed for 3 hr to mixtures of 6580 yg/m3 [3,5 ppm) N02 and 196
yg/m3 (0.1 ppm) Os, or 3760 yg/m3 (2.0 ppm) NQ2 and 98 yg/m3 [0,05 ppm) 03.
Groups of 5 mice, either exposed to filtered air or the pollutants were killed
at hourly intervals up to 5 hr and at 1,2,3, and 6 days after the respiratory
challenge and the concentration of 51, pyogenes in their lungs was assayed.
The clearance rate of viable bacteria from the lungs was markedly delayed
after exposure to the 6580 yg/m3 (3.5 ppm) N02 and 196 yg/m3 (0.1 ppm) 03
mixture. The time required to clear 50% of inhaled bacteria in mice exposed
to filtered air was approximately!. 35 hr but 2.18 hr in those exposed to
the pollutant mixture. No differences were seen in bacterial clearance upon
exposure to 3760 yg/m3 (2 ppm) N02 and 98 yg/m3 (0.05 ppm) 03 mixture.
The clearance rates over an extended period were calculated as the
number of mice out of the total number of mice killed on a given day having
viable s. pyogenes present in their lungs. The effects of exposure to mixtures
of the pollutants were much more pronounced over this 6 day assay period
(Table 4). Among the mice exposed to 6580 yg/m3 (3.5 ppm) N02 and 196 yg/m3
(0.1 ppm) 03 mixture, 15/19 (79%) and 3760 yg/m3 (2.0 ppm) N02 and 98 yg/m3
(0.05 ppm) 03 mixture 16/18 (89%) showed viable S. pyogenes in the lungs,
whereas among the corresponding controls 7/20 (35%) and 8/20 (40%) were
positive. Thus, it appears that the ability to clear inhaled bacteria is
markedly impaired by a single 3 hr inhalation of the pollutant mixtures.
TABLE 4. RETENTION OF INHALED VIABLE
STREPTOCOCCUS IN LUNGS OF MICE
EXPOSED FOR 3 HR TO N02 AND 03
MIXTURES
Concentration, yg/m
Day of
Lung
Assay
1
2
3
6
6580/N02
Positive
Control
2/5
3/5
0/5
2/5
+196/03
/Total*
Expt
5/5
3/5
3/4
4/5
3760/NQ2
Positive
Control
0/5
2/5
4/5
2/5
H-98/Q3
.'/Total*.
Expt
4/4
4/5
3/4
5/5
*
Number of mice showing viable s,- pyogenes
out of total number of mice assayed.
18
-------
SUMMARY
A single 3 hr exposure to 3760 yg/m (2,0 ppm) NOg or 196 yg/m3 (0.1 ppm)
03 increased the susceptibility of mice to streptococcal pneumonia, This was
demonstrated by excess deaths and reduced survival time compared to infected
mice exposed to filtered air. Within the N02 concentration range of 2820
(1.5 ppm) to 9400 yg/m3 (5.0 ppm) and 03 ranging from 98 (0.05 ppm) to 980 yg/m3
(0.5 ppm) the response was linear whereby an increase in concentration of the
pollutant resulted in increased excess mortalities.
The effect of a single 3 hr exposure to the N02 and 03 mixtures was additive.
At most concentrations the excess mortality was equivalent to the sum of excess
deaths resulting from exposure to each individual pollutant. At the higher con-
centrations of the pollutant mixture (i.e., 6580 yg/m3 (3,5 ppm) N02 and 980
yg/m3 (0.5 ppm) 03) a 1 hr exposure was equally effective in inducing excess
mortality as was the 3 hr exposure. However, at the lower concentrations of
the pollutants the 3 hr exposure was more effective.
The ability to clear inhaled bacteria from lungs was impaired by the exposure
to the N02 and 03 mixture. A marked increase in the time required to clear 50%
of viable bacteria within 4 to 5 hr after the infectious challenge was seen
upon exposure to 6580 yg/m3 (3.5 ppm) N02 and 196 yg/m3 (0.1 ppm) 03 mixture.
Moreover, a large percentage of mice had 5. pyogenes present in their lungs
6 days after the respiratory challenge.
A 3 hr exposure to either 6580 yg/m3 (3.5 ppm) N02 or 980 yg/m3 (0.5 ppm) 03
but not to a mixture of the two markedly enhanced the susceptibility of mice to
influenza infection. Exposure to these pollutants within 24 hr after a respiratory
challenge with influenza virus resulted in excess mortality, reduced survival
time and increase in lung consolidation.
MULTIPLE 3-HR EXPOSURE TO N02 AND Q3 MIXTURES
Mortality and Survival Time
In studies of the effects of multiple exposures, mice were exposed daily for
3 hr, 5 days/week, for 1, 2 and 4 weeks to mixtures consisting of 3760 yg/m3
(2.0 ppm) N02 and 98 yg/m3 (0.5 ppm) 03, or 2820 yg/m3 (1.5 ppm) N02 and
196 yg/m3 (0.1 ppm) 03. Within 1 hr after termination of the final exposure
the mice, along with control mice exposed to filtered air, were challenged
with S. pyogenes aerosol. The excess mortalities, based on exposure of 48 mice
to the 2820 yg/m3 (1.5 ppm) N02 and 196 yg/m3 (0.1 ppm) 03 mixture, and 104
mice to the 3760 yg/m3 (2.0 ppm) N02 and 98 yg/m3 (0.05 ppm) 03 mixture are
shown in Figure 2.
Repeated daily exposure to the mixture consisting of 3760 yg/m3 (2.0 ppm)
N02 and 98 yg/m3 (0.05 ppm) 03 followed by the infectious challenge resulted
in significant excess deaths over those observed in control mice. The excess
mortalities were present irrespective of the number.of the daily 3-hr exposures.
19
-------
NO,
N0
20
15
10
5
8 0
o
C3 ^ ^)
o
~ -10
S5
^ 20
O
(A
in
x
UJ
15
10
5
0
-5
-10
Figure 2
* p<0.05
** p<0.l
3760>zg/m3 N02 + 98
10
20
m m
2820 /ig/m2 N02 + 196 /ig/m3 03
10
Number Of 3 hr Exposures
20
Excess mortality in mice after multiple daily 3 hr exposures
to N02 and 03 mixtures and respiratory challenge with
Streptococcus aerosol.
20
-------
Among the mice exposed to the individual pollutants, only those exposed 10
times to 3760 yg/m3 NC>2 showed significant excess mortality when compared to
infected mice exposed to filtered air. Thus, daily 3-hr exposure to the
individual pollutants had no major effect on the mortality rates. On the other
hand, daily exposures to the N02 and 03 mixture containing the same concen-
tration of each pollutant resulted in significant excess mortality. This
suggests a synergistic relation between the two pollutants that makes them
more effective in reducing resistance to respiratory infection. At the same
time the absence of effect of N02 after 20 daily exposure coul-J suggest a
possible development of tolerance to this pollutant.
Results of daily 3-hr exposures to a mixture consisting of 2820
(1.5 ppm) NO;? and 196 yg/m3 (0.1 ppm) 03 indicate that there were no remarkable
differences in mortalities at any of the exposure conditions after five daily
exposures. However, marked excess deaths were present after 10 or 20 exposures
to 03 or the N02 and 03 mixture. The fact that excess mortalities seen upon
inhalation of the mixture approximated those observed in mice exposed to 03
only, suggests that they were due to the presence of 03 in the mixture.
In a single experiment the effects of daily 3-hr exposures 5 days/week for
4 weeks to 3760 ug/m3 (2.0 ppm) N02 and 98 yg/m3 (0.05 ppm) 03 mixtures on the
resistance to influenza infection were studied. The mice were exposed to the
pollutants and within 1 hr after termination of the final exposure were
challenged with A2/Taiwan influenza virus aerosol. The mortality and survival
rates and lung consolidation seen in Table 5 indicate that inhalation of these
pollutants had no pronounced effect on the resistance to influenza.
TABLE 5. EFFECT OF DAILY 3 HR EXPOSURE, 5 DAYS/WEEK
FOR 4 WEEKS TO N02 AND 03 MIXTURES ON
RESISTANCE TO INFLUENZA INFECTION
Concn,
N02
0
3760
0
3760
yg/m3
03_
0
0
98
98
D/T
18/48
23/48
18/48
21/48
Mortal
%
37.5
47.9
37.5
43.8
ity
Change, %
-
-t-10.4
0
+ 6.3
Lung
Lesions
3.21
3.42
2.94
3.15
MST,
day
16.5
16.2
16.8
16.2
21
-------
Immune Response
3
Mice were exposed 3 hr/day, 5 days/week, for 4 weeks to 3760 yg/m (2.0 ppm)
NQ2, 98 yg/m3 (0.05 ppm) 03, or to a mixture containing the same concentrations
of the two pollutants, Within 1 hr after termination of the final exposure
to the pollutants, the mice were vaccinated with 0.1 ml of chick embryo A2/
Taiwan/1/64 influenza vaccine (Lot No. B80549 Zonomune, Eli Lilly and Company,
Indianapolis, IN). A single subcutaneous injection of approximately 300 CCA
units of the vaccine was used. Control mice received subcutaneous injection
of phosphate-buffered saline. After the vaccination all mice were kept in
isolated animal room in clean ambient air.
At 2, 12 and 22 weeks after the vaccination groups of mice were exsanguinated
and blood serum was collected to determine the hemagejlutination-inhibition
(HI) antibody levels. The primary HI antibody titer in all groups of mice was
<8. At the same time intervals (.2, 12 and 22 weeks) groups of mice representing
all exposures to the pollutants were challenged by the respiratory route with
infectious Az/Taiwan influenza virus and mortality and survival time were re-
corded. The surviving mice were exsanguinated 16 days after the infectious
challenge to determine the secondary HI antibody formation and lung consoli-
dation.
Because of the inadvertent use of lew challenge dose and the ensuing
low mortality among the nonvaccinated mice it was difficult to determine the
protection provided by the vaccine at 2 and 12 weeks after the vaccination.
The mortality among the nonvaccinated mice was 12.5% (3/24) and 8.3% (2/24)
whereas that among vaccinated mice exposed to filtered air was 0% (Q/24) and
4.2% (1/24), The mortality rates in mice exposed to the pollutants ranged
from 0% (0/24) to 8.3% (2/24). At 22 weeks the vaccine afforded significant
protection against the Infectious challenge. Mortality in nonvaccinated mice
was .. 31..6S5 (12/38), in vaccinated mice exposed to filtered air 5.7% (2/35) and
in those exposed to the pollutants ranged from 6.1 to 12.8%. Thus the
mortality of mice vaccinated with influenza virus vaccine and challenged with
infectious virus was not affected by exposure to the pollutants.
The lung consolidation, HI titers and percent seroconversion in surviving
mice exposed to the pollutants for 4 weeks, vaccinated and challenged with
infectious influenza virus at various time periods after the vaccination are
shown in Table 6. At 4 weeks after vaccination, mice exposed to either
3760 yg/m3 (2.0 ppm) N02> 98 yg/m3 (0.05 ppm) 03 or to the mixture consisting
of the two pollutants, showed significant increase in lung consolidation com-
pared to the vaccinated mice exposed to filtered air. At 14 weeks after
vaccination only mice exposed to NOg showed significantly higher lung lesion
scores than vaccinated mice held in filtered air. At 24 weeks more severe lung
consolidation were seen in all mice exposed to the pollutants, but the statis-
tical significance of the differences could be ascertained only for the mice
exposed to 98 yg/m3 (0.05 ppm) 03. The lung lesion scores in nonvaccinated
mice challenged with infectious virus were 2.61, 2.50, and 2.57 at 4, 14 and
24 weeks, respectively.
22
-------
TABLE 6. LUNG CONSOLIDATION AND GEOMETRIC MEAN RECIPROCAL HI ANTIBODY TITERS IN MICE EXPOSED
3 HR/DAY.5 DAYS/WEEK FOR 4 WEEKS TO N02 AND 03 MIXTURES, VACCINATED AND CHALLENGED
WITH INFECTIOUS INFLUENZA VIRUS AEROSOL
3
Concn., yg/m Week after Vaccination
4 14 24
Lung Consol HI Seroconv Lung_"Consp1 HI Seroconv Lung Consol HI Seroconv
N02 03 Mean SE Titer % Mean SE Titer % Mean SE Titer %
^ 0 0 0.48 0.12 19.5 78 0.88 0.20 12.9 42 1.06 0.20 34.7 67
co
3760 0 0.92* 0.15 9.1* 44 1.96* 0.29 49,0* 85 1,31 0.25 36,3 71
0 98 1.00* 0.22 7.6* 20 0,79 0,12 19.5 75 1.79* 0,27 93,3 86
3760 98 1.05* 0.20 20.9 63 1.00 0.17 30.2 75 1.24 0.21 72.4 79
Significant change from corresponding infected mice exposed to filtered air (p 40.05).
-------
At 4 weeks after vaccination, exposure to 3760 yg/m3 (2,0 ppm) NQ2 or to
98 yg/m3 (0.05 ppm) Os caused a significant depression of the secondary HI
antibody response (Table 6). At 14 weeks a 4-fold increase in HI antibody titer
was seen in mice exposed to N02 and a 2-fold increase in those exposed to the
N02 and 03 mixture. This was in agreement with the lung consolidation data,
suggesting extensive virus replication. At the same time period, marked increase
in HI seroconversion (i.e., the percentage of mice showing titers of ^8} was
seen in all groups of mice exposed to the individual or mixture of the pollu-
tants. The generally higher HI antibody titers seen In all mice at 24 weeks
reflect the higher infectious virus dose used in the challenge. Exposure to
98 yg/m3 (0.5 ppm) 03 or the N0£ and 03 mixture resulted in an approximately 3-
and 2-fold increase of HI antibody titers. The higher HI antibody titer was
consistent with pulmonary consolidation especially in mice exposed to 98 yq/m^
(0.05 ppm) 03.
SUMMARY
Daily 3 hr exposures, 5 days/week for 1, 2 or 4 weeks to mixtures con-
sisting of 3760 yg/m3 (2.0 ppm) N02 and 98 yg/m3 (0.05 ppm) 03 resulted in
significant excess mortalities from streptococcal pneumonia. Inhalation of
the individual pollutants had no effect on the susceptibility to Infection,
thus suggesting a possible synergistic interaction upon exposure to the pollu-
tant mixture. An identical exposure regimen to 2820 yg/m3 (1.5 ppm) N02 and
196 yg/m3 (0.1 ppm) Os mixture induced significant excess mortalities only
after 4 week exposure.
Susceptibility to influenza infection was not affected by the multiple
exposure to either of the pollutants or pollutant mixture,
o
The daily 3 hr exposure, 5 days/week for 4 weeks to 3760 yg/m (2.0 ppm)
N02 and 98 yg/m3 (0.05 ppm) 63 did not diminish the effectiveness of influenza
vaccine in protecting mice from a respiratory challenge with infectious in-
fluenza virus. However, vaccinated mice exposed to the pollutants in general
had more severe lung lesions, increased HI antibody titers and increased rate
of seroconversion,
LONG-TERM EXPOSURE TO N02 AND 03 MIXTURES
Studies were conducted to determine the effect of long-term exposure to
various concentrations of N02> 03 and N02 and Oo mixtures on the resistance
to streptococcal pneumonia as measured by mortality and survival rates,
clearance of inhaled bacteria from the lungs and phagocytic activity of al-
veolar macrophages. In addition, body weights, lung edema and changes in
various hematological and clinical pathology parameters were determined.
Selected tissues from the respiratory tract were examined by conventional and
scanning electron microscopy. The exposure conditions used in the experiments
were:
24
-------
Continuous Exposure Peak Exposure
24 hr/day, 7 days/week 3 hr/day, 5 days/week
Filtered Air Filtered Air
3
Filtered Air 940 yg/m (0.5 ppm) NO?
and 196 yg/m3 (0.1 ppm) Os
188 yg/m3 (0.1 ppm) N0? 940 yg/m3 (0.5 ppm) NO?
and 196 yg/m3 (0,1 ppm) 03
Filtered Air 940 yg/m (0.5 ppm) N02
188 yg/m3 (0.1 ppm) N02 940 yg/m3 (0.5 ppm) N02
Filtered Air 196 yg/m3 (0.1 ppm) 03
Mortality and Survival Time
Mice exposed to the pollutants for 1,2,3 or 6 months were challenged with
the s. pyogenes aerosol and held in clean air environment for a 14-day obser-
vation period. Mortality rates and mean survival time for each exposure con-
dition are shown in Table 7.
The 1 or 2 month exposure did not induce any marked changes in mortality
rates. The exception was a significant decrease in mortality among mice ex-
posed for 1 month to filtered air with the superimposed daily 3 hr peaks of
940 yg/m3 N02 and 196 yg/m3 03 mixture. In this treatment group the mortality
was 14.0% below and the survival time 1.3 day above that of infected mice
exposed to filtered air.
The 3 month exposure resulted in significant changes in mortality rates.
Excess mortality was seen in mice exposed to filtered air with superimposed
NO? and 03 peaks (+21.0%) and those exposed to filtered air with superimposed
daily 3 hr peak of 940 yg/m3 N02 (+11.2%). The corresponding mean survival
time decreased by 2.5 and 1.8 days. On the other hand, significant decrease
in mortality was seen among mice continuously exposed to 188 yg/m3 N0? with
daily 3 hr peaks of N02 and 03 mixture (-15.3%) or N02 (-17.0%). The decrease
was not accompanied by significant changes in mean survival time, however.
The 6 month exposure resulted in excess mortalities in all treatment
groups. The significance of the differences in mortalities could be ascertained
for the group of mice exposed to filtered air with the N02 and 03 peaks (+27.2%)
and those continuously exposed to 188 yg/m3 N02 with the superimposed N02 and
03 peaks (+14.8%). The inability to demonstrate the statistical significance
of the excess mortalities in the other two NO? treatment groups can be ascribed
to the small number of mice used in the experiments.
25
-------
ro
o>
TABLE 7. EFFECT OF EXPOSURE TO NO? AND 03 MIXTURES ON MORTALITY AND SURVIVAL TIME OF MICE CHALLENGED
WITH STREPTOCOCCUS AEROSOL
Exposure Concn. ,
yg/m3
24 hr/day 3 hr/day
N02 N02
0 0
0 940
188 940
0 940
188 940
0 0
03
0
196
196
0
0
196
1
Mortality
D/T
24/126
5/99
21/127
28/127
18/127
19/127
%
19.1
5.1*
16.5
22.1
14.2
15.0
MST
day
12.3
13.6*
12.5
11.7
12.7
12.9
Duration of Exposure,
2
Mortality
D/T
21/87
20/72
14/80
26/196
28/117
26/107
%
24.1
27.8
17,5
27.1
23.9
24.3
MST
day
12.2
12.0
12.9
12,0
12.1
12.0
Month
3
Mortality
D/T
62/156
94/1 55
38/156
86/169
39/172
70/158
%
39,7
60.7*
24.4*
50.9*
22.7*
44.3
MST
day
11.3
8,8*
11.9
9.5*
12.4
11.1
6
Mortality
D/T
9/44
21/44
18/51
13/46
14/42
12/47
%
20.5
47.7*
35,3**
28,3
33,3
25.5
MST
day
12.8
n.o*
11,2*
11.8*
11.5*
12.3
**
Significant change from corresponding infected mice exposed to filtered air.
p <0.05.
t
p «0.01
-------
The differences in percent mortality for the various exposure groups are
shown in Figure 3. The data indicate that the mode of exposure to the pollu-
tants is important in altering the host defense against respiratory infection.
Most effective in reducing the resistance to streptococcal pneumonia appeared
to be the daily 3 hr exposure to 940 yg/m3 N02 and 196 yg/m3 03 mixture in mice
otherwise kept in filtered air. During the first month of exposure there was
an increase in resistance to infection. This was followed by a gradual decrease
in resistance demonstrated by significant excess mortalities after the 3 and 6
month exposures.
A gradual increase in resistance to the respiratory infection was seen
during the first 3 months of continuous exposure to 188 yg/m3 NQ2 with the
superimposed daily 3 hr peak of 940 yg/m3 N02 and 196 yg/m3 03 mixture or upon
continuous exposure to 188 yg/m3 N02 with 940 yg/m3 N02 peak. However, after
6 month exposure the effect was reversed and marked increase in susceptibility
to the respiratory infection was observed. Daily 3 hr exposure to 940 yg/m3
N02 also resulted in increased susceptibility to the infection after 3 and 6
month. However, the daily 3 hr exposure to 196yg/m3 03 for up to 6 months had
no effect on the susceptibility to the streptococcal pneumonia.
The effect of inhalation of the pollutants before and after the infectious
challenge was investigated in conjunction with the 1, 2 and 3 month exposures.
In these studies, instead of being maintained in filtered air for the 14 day
observation period after the infectious challenge, mice were kept in the same
environment to which they were exposed before the challenge. The results
summarized in Table 8 indicate that inhalation of the pollutants during the
14 day post-infection period had a pronounced effect on mortality rates and
survival time. After 1 month exposure the changes were small and not sig-
nificant. Nevertheless, in contrast to mice maintained in filtered air after
the infectious challenge, all exposures resulted in excess mortalities and
slight decrease in the survival time. After 2 or 3 months exposure, when the
mice were held for 14 days in polluted atmosphere after the challenge with
S. pyogenes aerosol, marked excess mortality and decreased survival time was
seen.
27
-------
I
c
3O
20
,o
o
S
I ] I Month
* p<0.05
2 Months ^\S^j 3 Months
6 Months
Exposure Regimen
A: 24 Hr/Doy. 7 Days/Week
B: 3 Hr/Oay, 5 Days/Week
-IO
-20
- A:
B:
Air
m
!88Mg/msN02
,-H96Mg/msOs
188 fig/m3
94O/ig/m*
Air
94Oftg/ms
Air
l96/tg/msO9
Figure 3. Changes in mortality from streptococcal pneumonia after various exposure regimens to N02 and 03
mixtures.
-------
TABLE 8: CHANGES IN MORTALITY AND SURVIVAL RATES OF MICE EXPOSED TO N02 AN 03 MIXTURES, CHALLENGED
WITH STREPTOCOCCUS AEROSOL AND MAINTAINED FOR 14 DAYS EITHER IN FILTERED AIR OR
POLLUTANT MIXTURES
Exposure Concn. ,
24 hr/day
N02
1 month
0
0
188
0
188
2 month
0
0
188
0
188
3 month
0
0
188
0
188
3 hr/day
N02 03
0 0
940 196
940 196
940 0
940 0
0 0
940 196
940 196
940 0
940 0
0 0
940 196
940 196
940 0
940 0
P
- 1C - .1
Mortality
D/T
24/126
5/99
21/127
28/127
18/127
21/87
20/72
14/80
26/96
28/117
62/156
94/155
38/1 56
86/169
39/172
»
19.1
5.1
16.5
22.1
14.2
24.1
27.8
17.5
27.1
23.9
39.7
60.7
24.4
50.9
22.7
Change
.
-14.0*
- 2.6
+ 3.0
- 4.9
.
+ 3.7
- 6.6
+ 3.0
- 0.2
-
+21.0*
-15.3*
+11.2*
-17.0*
Exposure
Condition
i(a) P + 1C -»P
Survival
Day C
12.3
13.6 +
12.5 +
11.7 -
12.7 +
12.2
12.0 -
12.9 +
12.0 -
12.1
11.3
8.8 -
11.9 +
9.5 -
12.4 +
Time
hange
.
1.3
0.2
0.6
0.4
.
0.2
0.7
0.2
0.1
.
2.5
0.6
1.8
1.1
Mortality
D/T
0/78
2/78
3/77
2/78
1/84
5/50
18/52
11/45
12/52
14/67
4/60
24/60
16/60
8/60
9/60
%
0
2.6
3.9
2.6
1.2
10.0
34.6
24.4
23.1
20.9
6.7
40.0
26.7
13.3
15.0
Change
.
+ 2.6
+ 3.9*
+ 2.6
+ 1.2
'.
+24.6*
+14.4**
+13.1**
+10.9**
.
+33.3*
+20.0*
+ 6.6
+ 8.3
(a)
Survival Time
Day
14.0
13.9
13.8
13.8
13.9
13.2
12.0
12.1
12.5
12.5
13.4
10.8
11.9
13.2
12.7
Change
.
- 0.1
- 0.2
- 0.2
- 0.1
.
- 1.2
- 1.1
- 0.7
- 0.7
.
- 2.6
- 1.5
- 0.2
- 0.7
a Pollutant
•* Infectious Challenge -+
Filtered
Air (from
Table
7).
Significant change from corresponding infected mice exposed to filtered air.
*p $0.05.
-------
Clearance of Bacteria from Lungs
To study the effect of N02 and Os mixture on the ability to clear viable
inhaled bacteria from the lungs, mice were challenged with 5. gyogenes aerosol
after 1, 2 and 3 months exposure to the pollutants. Groups of four mice from
each treatment group were killed immediately after and at 1,2,3 and 4 hours
after the infectious challenge. Clearance rates of bacteria from lungs of mice
exposed to the various treatments are shown in Table 9. Daily 3~hr exposure
to a mixture of 940 yg/m3 N0£ and 196 pg/m3 03 and the continuous exposure to
188 yg/m3 N02 with superimposed 3-hr daily peaks of either the NOz and Ch
mixture or 940 yg/m3 N0£ were most effective in delaying the clearance or in-
haled bacteria. The other two exposure conditions, namely N02 and Os peaks only,
were not effective and in some instances the clearance rate of bacteria from
the lungs was faster than that in the control mice.
TABLE 9. CLEARANCE OF INHALED VIABLE STREPTOCOCCUS
FROM LUNGS OF MICE EXPOSED TO N02 AND 03
MIXTURES
Exposure Concn., yg/m3 Estimated tSQ Rate3
24 hr/day 3 hr/day" Duration of Exposure CMonthj
N02
0
0
188
0
188
0
N02
0
940
940
940
940
0
03
0
196
196
0
0
196
1
1.34
1.57
1.46
1.10
1,59
0.90
2
1.03
1.26
1,11
1 .00
1.35
1.30
3
1.36
1,88
1,63
1.28
1.82
1.19
a Time in hours required to clear 50% of inhaled
bacteria from lungs.
30
-------
Pulmonary Cellular Defense
Limited experiments were conducted to determine the effect of 1,2,
3 month exposure to the pollutants on alveolar macrophages. The treatment
groups included in these studies were control mice, those continuously ex-
posed to 188 yg/m3 N02 with 3 hr peaks of 940 yg/m3 NQ2 and 196 yg/m3 03
mixture, and mice exposed to daily peaks of either NO? and 03 mixture or
940 yg/m3 N02- After each exposure,groups of 5 to 7 mice were killed and their
lungs lavaged. The total cell count, the percentage of alveolar macrophages
and their viability and phagocytic activity were determined in the lavage fluid
(Table 10).
The total cell count, which contained 96.7 to 99.3% alveolar macrophages,
appeared to be influenced by the pollutants, although there was no apparent
relationship to the duration of the exposure. After 1 month all three treat-
ment groups showed a total cell count which was lower than that of the controls
and the decrease was significant for mice continuously exposed to 188 yg/m3 N0£
with the N02 and 03 mixture peaks and those exposed to the 3 hr daily peaks of
940 yg/m3 N02- The 2 month exposure dfd not have any effect on the total cell
count. At this exposure period mice exposed daily to peaks of N02 and 03
mixture showed statistically significant decrease in the percentage of al-
veolar macrophages, this difference, however, is not considered to be bio~
logically significant. After 3 month exposure reduced total cell counts were
seen in mice exposed to the peaks of N02 and 03 mixture and somewhat reduced
counts in those exposed continuously to 188 yg/m3 with daily N02 and 03
mixture peaks.
The viability of the alveolar macrophages was not affected by a 1 or 2
months exposure to the pollutants. However, after 3 month a significant de-
crease in viability was seen in macrophages isolated from mice exposed to the
N02 and 03 peaks. Some decrease in viability was also seen 1n macrophages of
mice exposed continuously to 188 yg/m3 N02 with the superimposed dally peaks
of N02 and 03 mixture.
The phagocytic activity of alveolar macrophages, expressed as the percent
of macrophages which engulf at least one latex sphere after 1 hr Incubation
at 37°C, was not affected by 1 month exposure to the pollutants. After 2 and
3 months, however, a marked decrease was seen in activity of macrophages from
mice exposed to the NO? and 03 mixture peaks and those continuously exposed
to 188 yg/m3 N02 and the superimposed N02 and 03 peaks. After 2 months re-
duced phagocytosis was also seen in mice exposed to the 3 hr daily peaks of
940 yg/m3 N02.
31
-------
TABLE 10. EFFECT OF EXPOSURE TO NQ2 AND 03 MIXTURES ON ALVEOLAR MACROPHAGES
3
Exposure Concn.,yg/m
24 hr/day
NO?
1 month
0
0
188
0
2 month
0
0
188
0
3 month
0
a
188
0
3 hr/day
N02
0
940
940
940
0
940
940
940
0
940-
940
940
03_
0
196
196
0
0
196
196
0
0
196
196
0
Total
Mean
1.68
1.50
1.04*
1.02*
1.37
1.54
1.53
1.30
0.94
0.60*
0.78
1.04
Cell Count
x!06
SE
0.21
0.30
0.27
0.10
0.23
0.21
0.17
0.09
0,11
0.04
0.09
0.11
Macroph %
Mean
98.8
99.3
98.8
98.6
98.6
96.8*
97.5
97.8
96.7
97.4
97.1
97.4
SE
0.8
0.5
0.6
0.2
0.4
0.9
0.7
0.6
0.7
0,6
0.4
0.8
Macrophage
Viability, %
Mean
93.2
92.3
94.9
89.6
93.8
95.2
95.0
93.8
87.3
78.9*
84.9
88.1
SE
1.6
1.2
0.7
1.7
1.0
1.1
1.1
1.1
2.2
1.1
2.5
1.4
Phagocyt, %a
Mean
87.2
87.2
87.8
89.5
89.1
82.1*
86.5*
84.0*
85.8
83.0*
83.7
86.2
SE
0.6
0.7
0.6
0.7
0.9
0.6
0.8
1.9
0.7
1.2
1.0
1.0
a Percent of alveolar macrophages engulfing one or more latex sphere after 1 hr
incubation at 37°C (Ratio Maerophages to Latex - 1:200).
*
Significant change from corresponding control mice exposed to filtered air
(p^O.05).
32
-------
Clinical Pathology
Limited experiments were conducted to determine the effects of exposure
to the pollutants on activity of selected blood serum enzymes. Mice were ex-
sanguinated after 1, 3 and 6 months exposure and serum was assayed for the
activity of acid phosphatase (AP), cholinesterase (CHE), lactate dehydrogenase
(LDH), serum glutamic oxalic transaminase (SGOT) and serum glutamic pyruvic
transaminase (SGPT). In addition, gel electrophoresis was used to separate
the isoenzyme forms of LDH. The geometric means, standard error, statistical
significance of the differences between the means and the number of mice ex-
amined in each assay are shown in Figure 4.
In general elevation of SGPT, SGOT and LDH, and depression of AP and CHE
activity were observed. Moreover, a significant increase of the LDH-1 iso-
enzyme with a corresponding decrease in the other four isoenzymes was observed
after 6 month exposure. The continuous exposure to 188 yg/m3 N02 with the
superimposed 3 hr daily peaks of 940 yg/m3 N02 and 196 yg/m3 03 mixture
appeared to be most effective in inducing these changes. Because of the small
numbers of mice available for these determinations and the variability of the
enzyme levels the statistical significance of the differences could be
ascertained in only a limited number of exposure conditions.
The changes in serum enzyme activity were in a close agreement with those
reported by Kosmider (unpublished data) and Menzel, et al. (17) for guinea
pigs exposed to 940 yg/m3 N0£ 8 hr/day, 7 days/week for 4 month. In spite of
the differences in animal species (mice vs guinea pigs) and the duration and mode
of exposure to the pollutants the trends of the changes closely resembled those
seen in mice after 6 month continuous exposure to 188 yg/m with superimposed
daily 3-hr peaks of 940 yg/m3 N02 and 196 yg/m3 03.
Kosmider Menzel IITRI
AP 4* +* +
LDH t* t
CHE 4* 4-* 4*
SGOT t t*
(t increase;4 decrease)
(* significant change from control values)
Elevation of SGOT, SGPT and LDH is usually seen in many pathologic con-
ditions whereas these enzymes are released as the result of damage to heart,
liver, muscle and other tissue such as lung. An increase in levels of these
enzymes could be indicative of damage to lung resulting from exposure to air
pollutants. Acid phosphatase is present in lung lysosomal fractions capable
of tissue digestion. Depressed plasma AP in presence of increase in lung
tissue AP levels could suggest continuing elevation of phagocytosis in the
lung due to inhalation of the pollutants (17).
33
-------
80
70
60
5O
40
30
20
10
*~ Serum Glutamic Pyruvic Trontminote ^
-
-
_
;
i
\
s
•
i, S
s S
s s
1
!
140
120
r _ • 100
•
T
W///////////A
I80
to
60
40
20
~ Strum Glutamic Oxalic Trantamina**
T
_
-
•>'
S /
y
/ j
T
I
1
SI
,jv
^L
X
y//////////////A
/
/
<
/
/
/
/
/
/
/
/
/
/
/
136 1
12
10
8
6
4
2
O
~ Acid Photphatase T i
~
-
A
il
5
^
X
X
1
h
^
\
X
X
X
\
\
•> •)
' S
y,
\
\
12
,
10
T 8
\
\
UJ
z 6
0
4
2
— O
~ Cholinetterate
m
\
\\
•
'
t
t
/
/
/
/
/
/
/
/
/
•
S
S
s
s
m Concn.^g/m3
s
s
N
S
vH
Y///////////////,
3
n
\
rt
i
^s,
^\
X
'//////////////A
[
24Hr/Dav 3 Hr/Dov
^ Air
Air
Air 940N02+I96 0,
^ 188 N02 940NOz-»-i96 09
• p
2
K
t\l O
~ Lactate Dehydrogenose
-
•ft
~ ^ X
y x
y ^
i
- *
^
- y/
^ ^
ft
\////////s
• •
1
I
s
X
\
X
\
\
I 3 6
Duration Of Exposure, Month
I 3 6
Duration Of Exposure, Month
I 3 6
Duration Of Exposure, Month-
Figure 4. Effect of various regimens of exposure to N02 and 03 mixtures on blood serum enzymes
-------
Clinical Hematology
After 1, 3 and 6 months groups of 4 mice continuously exposed to air or
188 yg/m3 NC>2 with superimposed 3 hr daily peaks of 940 yg/nP NQ2 and 196 yg/m3
03 mixture and groups of 4 control mice exposed to air only were exsanguinated
and various hematological parameters were determined. Because of the small
number of mice used in these exploratory studies it was difficult to ascertain
the statistical significance of the observed differences. Moreover, the paucity
of information available in the literature on blood characteristics of mice in
general and specifically as related to age made the interpretation of the data
difficult. Exposure to the pollutants did not have any apparent effect on
hemoglobin content or erythrocyte count, whereas the hematocrit values were
reduced. White blood cell, reticulocyte, platelet and differential counts
showed variations which, however, did not appear to be related to the exposure
to the pollutants.
Pulmonary Edema
The wet-to-dry lung weight ratios expressing the extent of pulmonary edema
was determined after exposure to the pollutants. After 1 month the lung water
content did not differ between control mice and those exposed to the pollutants.
After 3 and 6 month exposure the water content was lower 1n mice exposed to
the pollutants then those exposed to filtered air. The differences, although
being only 2%, were statistically significant. One possible explanation for
the lower water content in lungs of mice exposed to the pollutants could be
the development of tolerance to 03 present in the pollutant mixture.
Body Weights
Groups of 100 mice selected at random from each exposure treatment were
weighed weekly to determine body weight changes which may have occurred as the
result of inhalation of the pollutants. In general the weight gains were not
affected by the exposure.
Histopathology
Lung tissues from control mice and those exposed to the pollutant mixtures
for 1 and 3 month were examined by conventional and scanning electron microscopy.
In addition trachea and nasal cavity tissues were subjected to SEM examination.
In general there were no remarkable pathological changes which could be attri-
buted to the exposure to the pollutants.
SUMMARY
Table 11 represents a summary of the health effects observed upon exposure
to the various N02 and 03 mixture environments, The three experimental treat-
ments shown in the table are daily 3 hr exposure 5 days/week to either 940
yg/m3 N02 or a mixture containing 940 yg/m3 NO;? and 196 yg/m3 03, and continuous
exposure to 188 yg/m3 N02 with superimposed daily 3 hr peaks (5 days/week) of
940 yg/m3 N02 and 196 yg/m3 03 mixture. The data represent changes from control
mice exposed to filtered air only. All changes of approximately 10% are shown
and, as appropriate, the statistical significance of the differences is indicated.
35
-------
TABLE 11. SUMMARY OF HEALTH EFFECTS IN MICE EXPOSED FOR 6 MONTHS TO N02 OR N02 AND 0^ MIXTURES
00
Exposure £
Concn. , ng/i" *«
24 hr/day
1
3
6
N02
month
0
0
188
month
0
0
188
month
0
0
188
3 hr/day t
N02
940
940
940
940
940
940
940
940
940
Ol_. £
0 +
196 (-)
196 -
0 (+)
196 (+)
196 (-)
0 +
196 (+)
196 (+)
*.
O)
OJ o
£ 5 2 J
i_ s- ••-> T- >> .5:
•0 C i— 0 ^
•5 ° § S §> ^ o
> 0 0 * * _ ei. 0>
^g>^>D- a:-ujfc-fez=n+»i
55355.'SSSSS.S-S^I
000+ (+) +o o + .+ - n n
+ - - 0 0 + 0 (+) 0 +.0 0 - 0
0 - (-) 00+ (•») + + + 0 0 (-) 0
(-) o + oo o - + + + o o n n
(-) - (-) (-) (-) o o o + - ' o o (-) o
( + ) 0 - - 0 0 ( + ) + 0 0 0 0
(-)nnnnnnnnnnnnn
(-) nnnnooo + o (+) (-) - o
(-) n n n - + (-) (+) (+) (+) (+) (-) (-) o
l/l •!—
-------
The 6 month exposure to any of the pollutant combinations resulted in a
marked increase in the susceptibility to streptococcal pneumonia. This was
manifested by excess mortality and reduced mean survival time. The increase in
susceptibility was much more pronounced after 1, 2 or 3 months when the animals
continued to be exposed to the pollutants for 14 days after the infectious
challenge. After a 3 month exposure to the N02 and 03 mixtures there was a
marked decrease in the ability of the animals to clear inhaled s. pyogenes
from their lungs. This impairment, however, was not present in mice exposed
daily for 3 hr to 940 yg/m3 N02.
Pulmonary cellular defenses were somewhat affected by the 3 month exposure
to the mixture of the pollutants but not by the dally exposure to N02 only.
The total cell count in the fluid lavaged from the lungs was markedly reduced
as was the viability and phagocytic activity of the alceolar macrophages.
In non-infected mice the 6 month exposure to 188 yg/m NO? with the super-
imposed daily 3 hr N02 and 03 mixture peaks resulted in significant decrease in
serum cholinesterase, increase in S6PT and SGPT and shift to LDH 1 isoenzyme.
In the same treatment group a decrease in hematocrit and an increase in platelet
counts was seen. A similar trend in changes was seen in mice daily exposed for
3 hr to the N02 and Oo mixture.
EXPOSURE TO S02
The effects of $03 on the resistance to respiratory infection was studied
in a series of experiments. The studies included single and multiple 3-hr
exposures as well as continuous exposures for up to 3 months to 13100 yg/m3
(5.0 ppm) S02- Exploratory studies were also conducted to determine the effects
of S02 in high humidity environment (85% RH), and 1n presence of respirable
size carbon particles, zinc sulfate particles, and mixture of N02 and 03.
Short-term 3-hr Exposure
The mortality and mean survival time resulting from single and multiple
3-hr exposures to S02 followed by respiratory challenge with S, pyogenes
aerosol are shown in Table 12. It is apparent from these data that inhalation
of 13100 yg/m3 (5.0 ppm) S02 had no effect on the susceptibility to streptococcal
pneumonia. Similar lack of effect was seen upon a single 3 hr exposure to $02
at 85% RH (46.2% vs 39.5% mortality in control mice), in presence of 2200 yg/m3
carbon particles (35.6% vs 38.2% mortality in con-rol mice) or in presence of
800 yg/m3 of zinc sulfate particles (29,1% mortality in S02 exposed mice vs
27.1% mortality in mice exposed to SO? and zinc sulfate mixture). Exposure
to 13100 yg/m3 S02 in mixture with 3760 yg/m3 (2.0 ppm) N02 and 196 yg/m3
(0.1 ppm) 63 resulted in a significant excess mortality (26.3%) which, how-
ever, was probably due to the presence of N02 and 03 in the mixture.
Thus it appears that exposure to 13100 yg/m (5.0 ppm) S02 alone or in mixture with
other pollutants has no effect on the susceptibility to streptococcal pneumonia.
37
-------
TABLE 12. MORTALITY AND SURVIVAL TIME OF MICE DAILY EXPOSED FOR
3 HR TO 13100 pg/ffl3 $02 AND CHALLENGED WITH
STREPTOCOCCUS AEROSOL
Number of Filtered Air
Daily 3-hr Mortality
Exposures
1.
5
10
15
D/T
90/373
13/48
19/48
15/47
%
24,1
27.1
39.6
31.9
MST
day
12.1
11.6
10.4
11.5
D/T
97/372
13/48
18/47
14/46
13100 ug/m S02
Mortality
MST
% Change (%) day
26,1 +2.0
27.1 0
38,3 -1.3
30.4 -1.5
12.0
12.0
10.5
11.5
Continuous Exposure
3
In studies of effects of continuous exposures to 13100 yg/m (5.0 ppm) S02
for up to 3 months various physiological response parameters were measured.
They included resistance to streptococcal and viral pneumonias, clearance of
inhaled bacteria from the lungsj phagocytic activity of alveolar macrophages,
lung edema, hematology, clinical pathology and conventional and scanning
electron microscopic examination of the respiratory tract tissue.
After 1, 2,.or.3 months of continuous exposure to SOg, mice were challenged
by the respiratory route with either 5. pycgenes or influenza virus aerosols.
The summary shown in Table 13 indicate that in general the S02 exposure had
only a slight effect on resistance to streptoccccal pneumonia. A significant
excess mortality was seen after 2 month but not after 1 or 3 month exposure.
In mice challenged with influenza virus mortality rates were significantly
higher after 1 month, but significantly lower after 3 month exposure to S02-
It was interesting to note that lung lesions in mice surviving the influenza
infection were consistently higher in those exposed to S02 than control mice
exposed to filtered air, suggesting that the Infection was more severe in mice
exposed to S02-
After 1 and 2 months exposure to S02, viable inhaled s. pyogenes was
cleared slightly faster from lungs of mice exposed to S02 then from control
mice exposed to filtered air. The t$Q in mice exposed for either 1 or 2 months
to S02 was 1.05 hr while for corresponding control mice were 1.40 and 1.27 hr.
After 3 months the tso for mice exposed to S02 was 1.08 hr and for the
corresponding control mice 0,70 hr. Inasmuch as the 1.08 hr value was in close
agreement with the t5Q values seen after 1 and 2 months S02 exposure, it is
assumed that the tso obtained in control mice is in error, Thus, it can be
concluded that exposure to S02 had no effect on clearance rate of inhaled
bacteria from the lungs.
38
-------
TABLE 13. MORTALITY AND MEAN SURVIVAL TIME OF MICE EXPOSED TO 13100 yg/m S02
AND CHALLENGED WITH STREPTOCOCCUS OR INFLUENZA VIRUS AEROSOL
Duration of
S02 Exposure,
Month
Streptococcus
1
2
3
Influenza Virus
1
2
3
Mortality
Control
D/T
13/131
6/120
4/48
35/50
23/48
33/48
%
9.9
5.0
8.3
70.0
47.9
68.8
SO?
D/T
10/129
15/120
5/48
48/55
30/48
23/48
%
7.8
12.5
10.4
87.3
62.5
47.9
Survival Time, day
Change Control
%
- 2.
+ 7.
+ 2.
+17.
+14.
-20.
1
5*
1
3*
6
9*
13.4
13.7
13.4
11.4
12.0
9.7
S02 Change
%
13
13
13
11
11
12
.5
.1
.3
.3
.5
.1
+ 0.
-0.
-0.
-0.
-0.
+ 2.
1
6
1
1
5
4
Significant change from corresponding infected mice exposed to filtered
air (p <:0.05).
Examination of fluid lavaged from lungs of mice exposed to S02 did not
demonstrate any significant changes in total and differential cell counts, nor
the percent viability of alveolar macrophages or their ability to phagocytize
polystyrene latex spheres.
The wet-to-dry lung weight ratios examined after 1, 2 and 3 month exposure
to S02 showed no significant differences.
During the first month of exposure an increase in body weight was seen
in mice exposed to S02 as well as those exposed to filtered air. Thereafter
the weights leveled off without showing any remarkable differences between the
control and experimental animals during the remaining exposure period.
Rectal temperatures of randomly selected mice from the exposure and control
group were determined during the 3 month period. Temperatures decreased somewhat
in both groups after 1 week and remained depressed for 2 to 3 weeks, at which
time, recovery to normal body temperatures followed. Irrespective of the
exposure treatment marked decrease in body temperature was seen during the first
24 hr after the infectious challenge with s. pyogenes* and a 3°C decrease in
temperature was seen in mice which died due to the streptococcal pneumonia.
39
-------
For clinical pathology and hematologi'c examination blood samples were ob-
tained from orbital bleeding using heparinized micro-hematocrit capillary
tubes. The hematological assay included hematocrit hemoglobin, red, white and
differential blood cell counts, reticulocytes, platelets and sedimentation rate.
Clinical pathology included activity in serum of alkaline phosphatase, lactic
dehydrogenase (LDH) and LDH Isoenzymes, ahydroxybutyrate alkaline dehydrogenase
(HBDH) and isocitric dehydrogenase (ICDH), The variations observed in the
various measurements were within the expected normal range and were not related
to the exposure.
Lungs, trachea, and nasal cavities from mice exposed for 1, 2, and 3 months
to S02 were examined by scanning electron microscopy. Thickened alveolar septa
and enlarged pores were seen in the lungs of control as well as the mice ex-
posed to S02 with slightly more damage in those exposed to the pollutant. Nasal
cavities and trachea appeared normal. Histopathological examination of lung
tissues disclosed that several mice in each group had moderately sized focal
areas of parabronchial lymphocytic aggregations, comparable to those seen in
chronic respiratory disease. These aggregations were considered as background
lesions and were not attributed to the S02 exposure.
SUMMARY
Single or multiple 3 hr exposures of mice to 13100 yg/m (5.0 ppm) SO? alone
or in combination with either, carbon particles, zinc sulfate particles or N02
and 03 mixture had no effect on, their susceptibility to streptococcal pneumonia.
Continuous exposure for up to 3*month to the same $02 concentration also had
little effect on the susceptibility to respiratory infections as well as other
health effect parameters.
EXPOSURE TO SULFATES
The major thrust of these studies was to determine the effect of a single
3-hr exposure to zinc sulfate (ZnS04). ammonium sulfate ((NH4)2$04) and zinc
ammonium sulfate (Zn(NH4)2(S04)2 on the susceptibility to streptococcal
pneumonia. Exploratory experiments were also conducted to study the effects
of air pollutant mixtures containing ZnS04, N02 and 03 on susceptibility to
respiratory infection.
Exposure to Individual Sulfates
Mean mortality rates and survival time of mice exposed for 3 hr to various
concentrations of the sulfates and subsequently challenged by the respiratory
route with S. pyogenes are summarized in Table 14. The concentrations of the
various sulfates were expressed as mg/m^ of $04. Although not shown in the
table, throughout the replicate experiments, there were no deaths among mice
exposed to the sulfates only,
40
-------
TABLE 14. MORTALITY AND SURVIVAL RATE OF MICE EXPOSED FOR 3 MR TO SULFATES
AND CHALLENGED WITH STREPTOCOCCUS AEROSOL
m
<1
1.
2.
3.
so^
0
.1
2-2.0
1-3.0
1-4.0
-1
ZnS04
Mortality
D/T
373/1689
125/599
369/813
186/278
-
-
%
22.1
20.9
45.4*
66.9*
-
-
Zn(NH4)2lS04)2
MST
day
12.1
12.2
9.1*
6.2*
-
-
Mortality
D/T
165/756
29/120
112/445
67/192
-
45/48
21
24
25
34
93
%
.8
.2
.2
.9*
-
.8*
MST
day
12.3
11.9
12.0
10.9*
-
4.0*
(NH4)2S04
Mortal
D/T
233/588
22/48
76/191
52/96
47/144
46/110
ity
%
39.6
45.8
39.8
54.2*
32.6
41.8
MST
day
10.1
10.1
10.7
9.2
11.2
10.3
Significant change from infected mice exposed to filtered air (p<:0.05).
Mortality and survival rates were not affected by a 3-hr exposure to
1.1 mg/m3 or lower concentrations of ZnS04- However, inhalation of 1.3 mg/nr
or higher concentrations resulted in significant excess mortality and reduced
survival time. The death rates of control mice challenged with S. pyogenes
and exposed to filtered air was 22.1% (2.6%S.E.) and among those exposed to
0.5 to 1.1 mg/m3 ZnS04 20.9%. Exposure to 1.3 to 1.9 mg/m3 resulted in 23.3%
excess mortality and a decrease in survival time of 3 days. Exposure to 2.3
to 2.9 mg/m3 induced 44.8% excess mortality and an approximately 6 day re-
duction in the mean survival time. The Chi-square (X2) analysis of the results
of individual exposure experiments confirmed this dose effect. Within the
concentration range of 0.5 to 1.1 mg/m2 ZnSO* 6 individual test X2 values did
not show any significance, whereas 9 out of 10 test X2 values were significant
for death rates resulting from exposures to 1.3 to 2.9 mg/m3.
The persistence of ZnS04 effect on the susceptibility to respiratory in-
fection was studied in a single experiment. Groups of mice were exposed for
3 hr to 1.65 mg/m3 ZnS04 and 1, 24, and 48 hr later challenged with 5. pyogenes
aerosol. Significant excess mortality (25%) and reduced survival time (2.4
days) was seen at the 1 hr challenge. However, when the infectious challenge
was delayed to 24 or 48 hr no differences in mortality or survival time were
observed when compared to infected mice exposed to filtered air, thus indicating
a recovery from the effect of ZnS04 inhalation. Scanning electron micro-
scopic observation of tissues of mice exposed to Zn-S04 aerosols and sacrificed
1 and 24-hr later indicated some thickening of the alveolar walls in the lungs
and presence of particles in the nasal cavity.
41
-------
Inhalation of Zn(NH4)2(S04)2 was less effective in altering the resistance
to streptococcal pneumonia. The 3-hr exposure to up to 2,0 mg/m3 had no effect
on the resistance to infection. On the other hand, exposures to concentrations
above 2.1 mg/m3 significantly enhanced mortality and reduced the survival time.
The mean mortality for the control group of mice was 21,8% (2,4% S,E.) and for
those exposed to 1.0 to 2.0 mg/m3 Zn(NH4)2(S04)2 was 25,0%; the corresponding
survival times were 12.3 and 12.0 days. The inhalation of 2.1 to 3.0 mg/m3
Zn(NH4)2 followed by the infectious challenge resulted in 13.1% excess mortality
and approximately 1.5 day decrease in survival time. Exposure to 4.1 mg/m3
resulted in 72.0% excess mortality and 8.3 day decrease in mean survival time.
The X2 analysis of ten individual exposure expriments confirmed this dose
relation, whereas none of the X2 values were significant at concentrations below
2.0 mg/m3, but all four at or above 2.1 mg/m3 were significant.
In general, the exposure to 1.1 to 5.3 mg/m3 (1^4)2804 was not effective in
altering the resistance to streptococcal pneumonia. Although in the concentra-
tion range of 2.1 to 3.0 mg/m3 the excess mortality was significant, there was
otherwise no apparent relationship between death or survival time and concen-
trations of the sul fate. The overall mortality among the infected control mice
was 39.6 (3.5% S.E.) whereas that of mice exposed to (1^4)2804 was 41.3%, the
corresponding mean survival rates were 10.1 and 10.4 days.
To further assess the effect of exposure to sulfates on the resistance to
streptococcal pneumonia the excess mortality and survival time data were sub-
jected to regression analysis. The resulting least square lines are shown in
Figures 5 and 6. The effectiveness and dose relation of ZnS04 1n enhancing
the mortality caused by the infection is clearly seen. The correlation co-
efficient was significant for mortality as well as survival time (r(!4) -
0.899 and r(i4) - 0,891). The estimated concentration of ZnS04 required to
induce 20% excess mortality (ED2o) was 1,45 mg/m3. The dose response relation
for. Zn.(NH4)2(804)2 can also be seen, where the correlation coefficients for
mortality and for the survival time were significant (rQ2) = °-883 and r(12) B
0.744), The dose which, produced a 20% excess mortality was estimated at
2.4 mg/m3.
Exposure to ZnSQ4. NO? and 03 Mixtures
Changes in mortality among mice exposed for 3 hr to various concentrations
of N02, 03 and ZnS04 and challenged with s. pyogeneQ aerosol are shown 1n
Table 15. The table also includes estimated excess mortalities, which could
be expected to result from a single 3-hr exposure to the Individual pollutants
in concentration present in the mixtures. These estimates were based on excess
mortality data obtained during other studies conducted during the project.
With some exceptions the observed excess mortalities were in close agreement
with the expected values, suggesting that the inhalation of mixture on air
pollutants has an additive effect. However, at the higher concentrations of
the pollutants a potentiation appeared to be present resulting in excess
mortalities approximately twice as high as those expected.
42
-------
80
70
60
50
40
30
20
10
S 0
£ -10
a*
3? 80
70
c
o
o
o
8 60
u
X
UJ
50
40
30
20
10
0
-10
Zinc Sulfatote
ZnS04
1.0
2.0
3.0
Zinc Ammonium Sulfate
Zn(NH4)2 (
4.0
1.0 2.0 3.0 4.0
S04 mg/m3
5.0
5.0
Figure 5. Excess mortality in mice exposed for 3 hr to sulfates
and challenged with Streptococcus aerosol.
-------
12
10
-------
TABLE 15. MORTALITY OF MICE EXPOSED FOR 3 HR TO ZnS04 AND N02
AND 03 MIXTURES AND CHALLENGED WITH STREPTOCOCCUS
AEROSOL
Concn. , yg/m
Air
ZnS04
400
800
1100
1200
1400
1400
1500
N02
940
2820
3760
2820
940
2820
3760
P_3_
98
98
196
98
98
98
198
D/T
26/72
34/120
12/144
8/144
13/72
8/96
8/96
%"
36.1
28.3
8.3
5.6
18.1
8.3
8.3
Mortality
Pollutant
D/T
31/72
41/120
38/144
55/144
40/72
79/96
81/96
%
43,1
34.2
26,4
38.2
55,6
82.3
84,4
Change,%
Actual
7.0
5.9
18,1*
32,6*
37,5*
74.0*
76.1*
Expected5
8.0
0.6
20,5
18.7
36.6
34,9
32,1
Significant change from corresponding infected mice exposed to
filtered air (p «0.05),
Expected change in mortality based on exposure to. the individual
pollutants in concentrations present in the mixture.
45
-------
SUMMARY
Inhalation of ZnSCty C> 1.2 tng/tri ) or ZnCNH^CSQ^ 0>2.1 mg/m ) followed
by a respiratory challenge with airborne S, py-ogenes- resulted in significant
excess mortality and reduced survival time in mice. For the 3 hr inhalation
exposure the estimated concentration of ZnS04 which induced 20% excess
mortality (ED2o) was 1-45 rag/m^ whereas that of Zn(NH4)2 was 2.40 mg/m^.
Exposure to (N^^SO^ aerosol in concentrations ranging from 1.1 to 5,3
had no effect on the susceptibility to the respiratory infection.
46
-------
REFERENCES
1. Federal Register. Reference Method for the Determination of Sulfur
Dioxide in the Atmosphere (Pararosaniline Method), Vol. 36, No. 228,
November 25, 1971.
2. Mercer, T. T., M. J. TUlery, and H. Y. Chow. Operating Characteristics
of Some Compressed Air Nebulizers. Am. Ind. Hyg. J. 29:66-78, 1968.
3. Public Health Service, Selected Methods for the Measurement of Air
Pollutants. PHS Publ. No. 999-AP-ll, 1965.
4. Horsfall, F. L. Jr. Neutralization of Epidemic Influenza Virus, 0,
Expt. Med. 70:209-222, 1939.
5. Mattern, C. F., F. S. Brackett, and B. 0. Olson. Determination of Number
and Size of Particles by Electrical GatlngiBlood Cells. 0. Appl. Physiol.
10:56-70, 1957.
6. Sunderman, F. W. Further Modifications in Measurement of Blood Glucose,
Am. J. CUn. Path. 23:193-196, 1953.
7. Schalm, 0. W, A Simple and Rapid Method for Staining Blood Films with
New Methylene Blue. J. Am. Vet, Med, Assoc. 145:1184, 1964,
8. Wilkinson, 0. H., 0. H. Boutwell, and S. Winsten. Evaluation of a New
System for the Kinetic Measurement of Serum Alkaline Phosphatase. Clin.
Chem, 15:487-495, 1969.
9. Hacker, W. E. C., D. D. Ulmer and B. L. Vallee. Metalloenzymes and
Myocardlal Infarction; Malic and Lactic Dehydrogenase Activities and
Zinc Concentrations 1n Serum. New Eng. J. Med. 255:449-456, 1956.
10. Rosalki, S. B., and J. H, Wilkinson. Reduction of a-Ketabutyrate by
Human Serum. Nature 188:1110-1111, 1960.
11. Wolfson, S. K., and S. Ellis. Effects of Glucagon on Plasma Potassium.
Proc. Soc. Exptl. Biol. Med. 91:226-228, 1957,
12. Henry, R. J., N. Chiamori, 0. J. Golub, and 5, Berkman, Revised Spectro-
photometric Methods for the Determination of Glutamic-Oxalacetic
Transaminase, Glutamic-Pyruvlc Transaminase, and Lactic Acid Dehydrogenase.
Am. J. Clin. Path. 34:381-98, 1960.
47
-------
REFERENCES (cont,)
13. Ellman, G. L., D. Courtney, V. Andres, and R, Featherstcm, A New
and Rapid Colorimetric Determination of Acetylcholinesterase Activity.
Biochem. Pharmacol. 7:88-95, 1961.
14. Hillman, G. Fortlaufende Photometrische Messung der Sauren
Prostataphosphatase-Aktlvitat. Z, Klin. Chem. Klin. Biochem. 9:273-274,
1971.
15. Ehrlich, R. Effect of Nitrogen Dioxide on Resistance to Respiratory
Infection. Bacteriol. Rev. 30:604-614, 1966.
16. Coffin, D. L., and D. E. Gardner. Interaction of Biological Agents and
Chemical Pollutants. Ann. Occup. Hyg. 15:219-234, 1972.
17. Menzel, D. B., M. D. Abou-Donia, C. R, Roe, R. Ehrlich, D. E. Gardner,
and D. L. Coffin. Biochemical Indices of Nitrogen Dioxide Intoxication
of Guinea Pigs Following Low Level Long Term Exposure. In: Proc. Int'l.
Conf. on Photochemical Oxldant Pollution and Its Control. Raleigh, NC,
Sept. 1976. EPA Publication No. EPA-60Q/3-77-001b, Vol. II. pp. 577-587,
1977.
48
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-78-057
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
INTERACTIONS OF VARIOUS POLLUTANTS ON CAUSATION OF
PULMONARY DISEASE
5. REPORT DATE
August 1978
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Richard Ehrlich
8. PERFORMING ORGANIZATION REPORT NO.
. PERFORMING ORGANIZATION NAME AND ADDRESS
IIT Research- Institute
10 West 35th Street
Chicago, IL 60616
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
68-02-2274
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research a'nd Development
U.S. Environmental Protection Agency
Research Triangle Park. N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
RTP,NC
«. SPONSORING AGENCY CODE"
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Studies were conducted to determine the effects of single and multiple exposures
to individual and pollutant mixtures on resistance to respiratory infections. Results
of N02 studies indicate the greater importance of short-term peak concentrations than
prolonged exposures to lower concentrations in increasing the susceptibility to
infections. Single 3-hr exposure to mixtures containing various concentrations of
N02 and 03 had an additive effect. Repeated 3-hr exposures for 4 weeks to mixtures
consisting of 3760 yg/m3 N02 and 98 yg/m3 03 suggested a synergistic interaction
between the two pollutants. Daily 3-hr exposures for 6 months to mixtures of 940
yg/m3 N02 and 196 yg/m3 03 resulted in significant excess mortality, and reduced mean
survival time in infected animals. Continuation of exposure to the pollutants for
14 days after the infectious challenge resulted in a pronounced increased susceptibi-
lity to the respiratory infection, after 1, 2 or 3 month exposure. In non-infected
mice this exposure regimen induced changes in the activity of several serum enzymes.
Single, multiple or continuous exposures to S02 in concentration of 13.1 mg/m3 had
no effect on susceptibility. A single 3-hour exposure to sulfates indicated that, in
decreasing order of effectiveness zinc sulfate, zinc ammonium sulfate and ammonium
sulfate reduced resistance to streptococcal pneumonia. The decrease in resistance
to the respiratory infection appeared to be related to the zinc ion present in the
sulfate complex.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
respiratory infections
interactions
nitrogen oxide
ozone
sulfur dioxide
ammonium sulfate
zinc sulfates
air pollution
sulfates
06 F, T
18. DISTRIBUTION STATEMEN1
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
59
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
49
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