EPA-600/1-78-036
May 1978
RESPIRABLE PARTICLES AND MISTS IN MOUSE
PULMONARY INFECTIVITY MODEL
Effect of Chronic or Intermittent Exposure
by
Jeannie N. Bradof, James D. Fenters and Richard Ehrlich
IIT Research Institute
10 West 35th Street
Chicago, Illinois 60616
Contract No. 68-02-1717
Project Officer
Donald E. Gardner
Clinical Studies Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
LtVi.' ;- ''- -:«L PROTECTION
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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.
n
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FOREWORD
The many benefits of our modern, developing, industrial society
are accompanied by certain hazards. Careful assessment of the relative
risk of existing and new man-made environmental hazards is necessary
for the 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.
The overall objective of this research was to determine if exposure
to respirable sized sulfuric acid mist as well as sulfuric acid mist and
carbon particle mixtures can alter the resistance of animals to bacterial
and viral respiratory infection. The concentration-time relationships
for exposure to these pollutants were examined. The parameters measured
to define health effects were: mortality, mean survival time, lung
edema, lung consolidation, the rate at which viable microbes are cleared
from the lungs, histopathologic and scanning electron miscroscopic
examination of the lungs, trachea and nasal cavities. In addition, the
effects of these exposures on the pulmonary cellular (alveolar macro-
phages) and mucociliary defense system as well as measuring the immuno-
competence of the host was examined in this study.
F. G. Hueter, Ph. D.
Acting Director,
Health Effects Research Laboratory
iii
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ABSTRACT
The effects of respirable-sized sulfuric acid mist or mixtures containing
acid mist and carbon particles (A-C) on the susceptibility to bacterial and
viral respiratory infection were studied in mice and hamsters. Throughout the
short-term exposures, varying concentrations of acid mist were used and com-
bined with 5 mg/m3 carbon particles, Toxicity range-finding studies indicated
that hamsters were more resistant than mice, both species showing mortalities
upon single 3-hr exposure to 600 mg/m3 but not 400 mg/m3 acid mist. A single
3-hr exposure of hamster trachea! organ culture to mixtures containing 0.8 -
1,4 mg/m3 acid and 1.1 - 1.9 mg/m3 carbon resulted in reduced ciliary activity
and damage to the ciliated epithelium. In both in vivo and in vitro exposure
the mixture of the pollutants produced a greater effect than the individual
pollutants.
Three combinations of A-C concentrations and durations of exposure re-
sulting in an acid concentration-time (CT) index of 1000 were examined in mice.
Scanning electron microscopic examination, which proved to be much more sen-
sitive than histopathologic evaluation, demonstrated that concentration of acid
was the most critical factor in producing tissue damage. The most severe
changes, including emphysemic-like areas in alveoli, were found after five
daily 3-hr exposures to 200 tng/m3 A-C, Significantly increased mortality
and decreased bacterial clearance from lungs were also observed in mice chal-
lenged with Streptococcus sp. Significantly increased mortality and
pulmonary consolidation, with concomitant decreased survival time, occurred in
mice challenged with influenza virus aerosol and exposed to 50 mg/m3 A-C,
3 hr/day, 5 days/week for 4 weeks. The effects on formation of antibody and
on preformed antibody were examined in mice exposed for 3 hr/day, 5 days/week
to 100 mg/m3 A-C before or after vaccination with influenza A2/Japan virus.
Significantly increased pulmonary consolidation and depressed secondary immune
responses, as measured by serum antibody levels, were observed in various
groups of vaccinated mice exposed to pollutant compared to vaccinated controls
maintained in clean air.
The effects of long-term exposure of 3 hr/day, 5 days/week for 4, 12, or
20 weeks to mixtures of approximately 1.4 mg/m3 sulfuric acid mist and 1.5 mg/m3
carbon particles as well as carbon only were determined in mice. There were
significant alterations of immunoglobulin concentration, depression of primary
antibody response in spleen cells to antigenic stimulation, and decreased re-
sistance to respiratory infection as measured by mortality, survival time, and
pulmonary consolidation after 20 weeks of exposure. Bactericidal capacity of
lungs was also reduced in mice exposed to either A-C or to carbon alone, and
subtle tissue changes were seen in the respiratory tract upon SEM examination.
These studies were conducted by IIT Research Institute for the Environ-
mental Protection Agency in fulfillment of Contract No. 68-02-1717 from June 3,
1974 to June 2, 1977.
IV
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CONTENTS
Foreword in
Abstract iv
Figures vi
Tables vii
Abbreviations x
1. Introduction 1
2. Conclusions 4
3. Recommendations 8
4. Materials and Methods 9
Generation of Acid Mist and Participate Aerosols 9
Animals 12
Infectious Agents 12
Vaccine 13
Infectious Challenge 13
Pulmonary Consolidation 13
Relative Mean Survival Rate (RMSR) 13
Lung Clearance 14
Serological Methods 14
Hematology Tests 14
Clinical Chemistry Tests 15
Serum Immunoglobulin Concentration 15
Jerne Plaque Assay 16
Alveolar Macrophages 16
Growing Radiolabeled Bacteria 16
Bactericidal Activity 17
Scanning Electron Microscopy and Histopathology 17
Preparation of Trachea! Rings and Cultivation Method 18
5. Results and Discussion 20
Short-Term Exposures to Acid Mist and Carbon
Particulate Mixtures 20
Long-Term Exposure to Carbon and A-C Mixtures 61
References 77
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FIGURES
Number Page
1 Sulfuric acid aerosol generator 10
2 Animal exposure chamber for acid aerosols 11
3 Composite scanning electron micrograph of the nasal cavity of
a normal mouse, 20X 25
4 Nasal cavity of a normal mouse 26
5 Trachea of a normal mouse 27
6 Lung of a normal mouse 27
7 Nasal cavity of a mouse exposed to acid mist-carbon
particles mixture 29
8 Trachea of mouse exposed to acid mist-carbon particles
mixture 30
9 Lung of mouse exposed to acid mist-carbon particles mixture ... 31
3
10 Effects of 3 hr exposure to an aerosol of 100 mg/m of
sulfuric acid and 5 mg/m3 of carbon on alveolar macrophages
of mice obtained by tracheobronchial lavage from controls,
from exposed mice in less than 1 hr after exposure and 24
hr after exposure 47
o
11 Body weights of mice exposed to air, carbon only (1.5 mg/m
carbon), or A-C (1.4 mg/m3 sulfuric acid mist and 1.5
mg/m3 carbon) 3 hr/day, 5 days/week for 20 weeks 62
12 Nasal cavity of control and test mice 64
13 Trachea and lungs of control and test mice 65
VI
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TABLES
Number .Page.
1 Mortality of Mice and Hamsters After a Single 3 Hr Exposure
to Acid Mist ....................... . . , 21
2 Mortality Among Male Mice and Hamsters After a Single
Exposure to Sulfuric Acid-Carbon Particle Mixture ....... 22
3 Mortality After Multiple 3 Hr Exposures to Two Concentrations
of Acid Mist Plus 5 mg/m3 Carbon ................ 23
4 Scanning Electron Microscopic Observations from Mice Exposed
to Sulfuric Acid Mist, Carbon Only, or Sulfuric Acid Mist-
Carbon Particle Mixture .................... 33
3
5 Effects of 3-Hr Exposure to 100 mg/m Acid Mist on Resistance
to Infection of Mice ...................... 35
6 Mortality and Survival Rates of Hamsters Challenged Intra-
nasally with Influenza Virus and 72 Hr Later Exposed for
3 Hr to Acid Mist ....................... 36
7 Mortality of Mice Challenged with Airborne Streptococcus sp.
and Exposed to Air Mist-Carbon Mixtures ............ 37
8. Mortality and Survival Time of Hamsters Challenged Intra-
nasally with Influenza A/PR/8 Virus Before or After a Single
3-Hr Exposure to 300 mg/m3 Acid Mist and 5 mg/m3
Carbon Mixture ......................... 38
9. Response of Mice Exposed 3 Hr to Acid Mist and Carbon Particle
Mixture and Challenged with streptococcus sp .......... 40
10. Response of Mice to 5 Daily 3-Hr Exposures to Acid Mist and
Carbon Particle Mixture and Challenged with Influenza
Taiwan Virus ................
11. Response of Mice to 20 Daily Exposures (3 Hr/Day, 5 Days/Week for
4 Weeks) to 50 mg/m3 Acid Mist and 5 mg/m3 Carbon Particle
Mixture and Challenged with Influenza A2/Taiwan Virus ..... 42
12. Response of Mice Exposed to 5 mg/m Carbon Aerosol and
Challenged with Influenza A£/Taiwan Virus ........... 43
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LIST OF TABLES (cont.)
Number
13. Clearance of Viable Streptococcus From Lungs of Mice Exposed
for 3 Hr to Acid Mist and Carbon Particle Mixture 44
14. Viability, Total Cell Count and Phagocytic Index of
Alveolar Macrophages Lavaged from Mice Exposed for 3 Hr
to Acid-Carbon Mist 46
15. Bactericidal Activity in Lungs of Mice Exposed for 3 Hr
to Sulfuric Acid - Carbon Mist 48
16. Response of Mice Vaccinated with Influenza A2/Japan Virus,
Challenged with Homologous Virus and Immediately Exposed
to Five Daily 3-Hr Doses of 100 mg/m3 Acid Mist and
5 mg/m^ Carbon 50
17. Response of Mice Vaccinated with Influenza A2/Japan Virus,
Immediately Exposed to Five Daily 3-Hr Doses of 100 rng/m3
Acid Mist and 5 mg/m3 Carbon, Then Challenged with
Homologous Virus 51
3
18. Response of Mice Exposed to Five Daily 3-Hr Doses of 100 mg/m
Acid Mist and 5 mg/m3 Carbon, Immediately Vaccinated with
Influenza A2/Japan Virus, and Challenged with Homologous
Virus , 52
19. Ciliary Beating Frequency in Tracheas From Hamsters at <1, 24,
48 and 72 Hr After Termination of a 3-Hr In Vivo Exposure
to Sulfuric Acid Mist and Carbon Particles 54
20. Percentage of Normal Epithelium of Tracheas From Hamsters at
<1, 24, 48 and 72 Hr After Termination of a 3-Hr Exposure
to Sulfuric Acid Mist and Carbon Particles 55
21. Ciliary Beating Frequency in Trachea! Ring Cultures Held
In Vitro at <1, 24, 48 and 72 Hr After Termination of a
3-Hr In Vivo Exposure to Sulfuric Acid Mist and
Carbon Particles 57
vm
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LIST OF TABLES (cont.)
Number Page
22. Percentage of Normal Epithelium of Trachea! Ring Cultures
Held In Vitro At <1, 24, 48 and 72 Hr After Termination
of a 3-Hr in Vivo Exposure to Sulfuric Acid Mist and
Carbon Particles 58
23. Ciliary Beating Frequency in Tracheal Ring Cultures at <1,
24, 48 and 72 Hr After Termination of a 3-Hr in Vitro
Exposure to Sulfuric Acid Mist and Carbon Particles 60
24. Percentage of Normal Epithelium in Tracheal Ring Cultures
at <1, 24, 48 and 72 Hr After Termination of a 3-Hr
In Vitro Exposure to Sulfuric Acid and Carbon Particles ... 60
25. Effect of Long-Term Exposure to Carbon and Acid-Carbon
Mixtures on Selected Hematological Parameters , 67
26. Effect of Long-Term Exposure to Carbon and Acid-Carbon
Mixtures on Selected Clinical Chemistry Parameters 68
27. Long-Term Exposure to Acid Mists:Immunoglobulin Concentrations . 70
28. Long-Term Exposure to Acid Mists:Jerne Plaque Assay for
Primary Antibody Response 71
29. Long-Term Exposure to Acid Mists:Viability and Total Cell
Count of Alveolar Macrophages Lavaged from Mice 73
30. Long-Term Exposure to Acid Mists:Bactericidal Activity in
Lungs of Mice 74
31. Long-Term Exposure to Acid Mists:Response of Mice Challenged
with Influenza A2/Taiwan Virus 74
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LIST OF ABBREVIATIONS
Abbreviation Term
A-C acid mist/carbon particle mixture
AM alveolar macrophages
BSA bovine serum albumin
CMD count median diameter
CT concentration x time (index)
HBSS Hanks' balanced salt solution
HI hemagglutination-inhibition
Ig imrnunoglobulin class
mg/m' milligram per cubic meter
MMD mass median diameter
y micron
yg microgram
NOp nitrogen dioxide
PBS phosphate-buffered saline
PFU plaque-forming unit
ppm parts per million
RMSR relative mean survival rate
SD standard deviation
SEM scanning electron microscopy
SN serum neutralization
SOp sulfur dioxide
SRBC sheep red blood cells
TLV threshold limit value
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SECTION 1
INTRODUCTION
Studies on sulfuric acid mists include animal studies of mortality,
pathology, pulmonary function (1,2,3) and combined-effects studies
involving challenge with bacterial and viral infectious agents. Such studies
show that in addition to the influence of concentration and exposure dura-
tion (4,5,6), toxicity of sulfuric acid mists is enhanced by cold (6) and
humid conditions (7). This, in part, explains enhanced acid concentrations
and toxic effects encountered in urban areas under foggy conditions.
Simultaneous exposure to ammonia neutralizes the toxic effects (6,7). Salem
and Cullumbine (8) and Boren (9) have shown enhancement of toxicity of
certain atmospheric pollutants by kerosene smoke and such carriers as car-
bon particles. Particle size is also an extremely important factor in
determination of the toxicity of an acid mist.
Pulmonary function parameters (10) were used to examine the physiologic
response of guinea pigs to low concentrations of sulfuric acid mists of
several particle sizes (11,12). It was found that the toxicologic response
depends on the particle size as well as the atmospheric concentration since
particle size is an important determinant of the amount of toxic substance
penetrating the upper respiratory tract, as well as the site of deposition
of material reaching the lungs.
Long-term continuous exposure of several species to low concentrations
of sulfuric acid mist produced changes in pulmonary function and in the
histology of the respiratory tract. Pulmonary function measurements on dogs
that received sulfuric acid mist demonstrated loss of functional parenchyma
and the development of obstructive pulmonary defects in the conducting air-
ways (13).
Alarie, j?t_ aj_. (14,15) examined the effects of long-term continuous
exposure to sulfur dioxide, sulfuric acid mist, fly ash, and their mixtures
on histopathology and pulmonary function in monkeys and guinea pigs and
found the effects detected from exposures to mixtures could be attributed to
the presence of acid mist alone.
Relatively few studies have been done to examine the interaction of ex-
posure to acid mists and defense mechanisms against respiratory infections.
Fairchild, ejt a]_. (16) observed that exposure of guinea pigs to sulfuric
acid mist resulted in greater total respiratory deposition of radiolabeled
streptococcus aerosol than control animals and a proximal shift in the
regional pattern of deposition to the nasopharynx. Inhalation of sulfuric
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acid mist aerosol by mice before or after exposure to radioactive aerosol also
impaired the mucociliary defense mechanism of the respiratory tract (17).
The overall objective of this project was to determine if exposure to
respirable-sized sulfuric acid mist as well as sulfuric acid mist and carbon
particle mixtures (A-C) alter the resistance of a laboratory animal host to
bacterial and viral respiratory infection.
During the initial phase of the program, the methodologies required for ex-
posure of animals to the pollutants, including the generation of sulfuric acid
mist and particulate aerosols and monitoring of the aerosols, were established.
Preliminary range-finding experiments were conducted to determine the toxicity
of the pollutants resulting from a single 3- or 6-hr exposure. This infor-
mation was then utilized to design combined-effects studies employing a single
acute pollutant exposure.
A series of experiments was then initiated to determine the effects of
multiple exposures to these pollutants on the host's resistance to infection.
Mice were exposed 5, 10, and 20 times to 5 mg/nH carbon only (3 hr/day,
5 days/week) immediately before or after challenge with influenza virus. We
then determined the lowest concentration of A-C mixtures and number of repeated
exposures to the mixture that produced increased mortality in conjunction with
either a bacterial or viral challenge. The concentration-time (CT) relation-
ship for exposure to A-C and infectious challenge was examined. The parameters
measured to define the health effects were: mortality, mean survival time,
lung edema, lung consolidation, the rate at which viable bacteria are cleared
from the lungs of mice, histopathologic examination of appropriate tissues,
and scanning electron microscopic observation of lung, trachea, and nasal
cavities. In limited experiments, the effects of exposures on the pulmonary
cellular defense system of mice were determined. These included the examination
of total and differential cell counts, viability, cell surface morphology and
in vitro phagocytic function of alveolar macrophages lavaged from lungs, as
well as studies of the in vivo bactericidal capacity in the lungs. These
studies were summarized in Annual IITRI Report L6080-4 (1975) and I1TRI
Report L6080-8 (1976) and the data reported in the open literature (18,19).
Studies of the effect of exposure to A-C mixtures on vaccine-induced
immunity to influenza virus were completed. Mortality, mean survival time,
lung consolidation, serum neutralization (SN) and hemagglutination-inhibition
(HI) antibody titers were measured. In addition, preliminary studies were
initiated to determine the effects of a single 3-hr exposure to 1.4 +^ 0.4
mg/m3 sulfuric acid mist and/or 1.5 +_ 0.4 mg/m3 carbon on tracheal epithelium
in organ culture. Changes in ciliary activity were determined and alter-
ations in tracheal epithelium following exposure to A-C were examined by con-
ventional and scanning electron microscopic techniques.
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The effects of long-term (4,12 and 20 week) exposures (3 hr/day, 5 days/
week) to 1.4 +_ 0.4 SD mg/m3 acid mist and 1.5 +_ 0.4 mg/m3 carbon as well as car-
bon only were examined in normal mice and in combined-effects studies with in-
fluenza virus challenge. The effects of the exposure on normal (uninfected)
mice were examined using the following parameters: hematology, blood chemistry,
immunoglobulin concentration, Jerne plaque assay for number of plaque-forming
cells in the spleen, bactericidal assay in lungs, examination of the respiratory
tract by SEM and conventional microscopy, and body weight and temperature
measurements. Parameters for examining the effects of exposure on the response
to challenge with infectious influenza virus included mortality, mean survival
time, and lung consolidation.
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SECTION 2
CONCLUSIONS
Data were accumulated on single, 3-hr exposures to aerosols of sulfuric
acid mists and A-C. Mortalities were found at 600 mg/m3 acid mist, whereas
the animals tolerated 400 mg/m3 and below. Hamsters were more resistant to acid
mist per se than mice. The effects of single and multiple 3-hr inhalation
exposures to varying concentrations of sulfuric acid mist in combination with
a constant concentration of 5 mg/m3 of carbon particles were examined. As was
true with acid mist alone, mice were more sensitive to A-C than hamsters.
Only minimal mortality occurred among CFi mice after a single 3-hr exposure to
300 mg/m3 or five daily 3-hr exposures to 200 mg/m3 A-C.
Three combinations of exposure duration and varying acid concentration
combined with 5 mg/m3 of carbon particles resulting in the same concentration-
exposure time index (CT) were examined: 5 daily 3-hr exposures to 200 mg/m3,
10 daily exposures to 100 mg/m3, and 20 daily exposures to 50 mg/m3 A-C.
Scanning electron microscopic (SEN) examination of nasal cavities, trachea,
and lungs showed that the initial concentration of A-C is a more critical factor
in tissue damage than the total number of exposures. Exposure to acid mist
alone caused damage near the top and middle of the trachea, while the lower
portion of the trachea was relatively free of damage and the lungs were normal.
Addition of the 5 mg/m3 carbon particulates to the acid mist extended the damage
to the lower trachea and the bronchus of the lungs. The most severe changes,
including emphysemic-1ike areas in the alveoli, were found in mice following
five daily 3-hr exposures to 200 mg/m3 A-C. Histopathologic examination of
these tissues did not disclose any marked changes, indicating the sensitivity
of SEM in detecting tissue damage.
Infectivity studies conducted with various concentrations of acid in con-
junction with a constant concentration of 5 mg/m3 of carbon particles demon-
strated the following.
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Increased mortalities occurred in hamsters after a single 6-hr
exposure to 300 mg/m3 A-C 24 hr or 48 hr after intranasal
challenge with influenza virus.
Five daily 3-hr exposures to 200 mg/m A-C immediately before
or after challenge with a viral or bacterial agent signifi-
cantly increased mortality of mice. A concurrent marked
decrease in clearance rate of inhaled bacteria from the lungs
was observed.
Five as well as ten daily 3-hr exposures 5 days/week to
100 mg/m3 A-C immediately after challenge with a bacterial
agent also resulted in markedly increased mortality and
decreased survival time. Significantly increased mortality
and decreased survival time were seen in mice exposed daily
for 3 hr for 5 days to 100 mg/m3 A-C 24 hr before or after
challenge with influenza virus.
-%
t Twenty 3-hr exposures, 5 days/week, to 50 mg/m° A-C followed
immediately by challenge with influenza virus resulted in
significantly increased mortality and decreased survival time
in experimental mice compared to controls.
Studies of the cellular defense system indicated a decreased capacity
to clear bacteria in lungs of mice infected immediately after a single 3-hr
exposure to 100 mg/m3 acid and 5 mg/m3 carbon mists. Parallel extensive
structural changes were seen upon scanning electron microscopic examination
of alveolar macrophages lavaged from their lungs immediately after A-C
exposure.
Studies of the effects of multiple exposures (3 hr/day, 5 days/week) to
5 mg/m3 of carbon aerosols showed that 5 or 10 exposures either immediately
before or after challenge with airborne influenza virus caused no enhancement
of mortality or alteration of mean survival time of male CDi mice. However,
20 daily exposures immediately before virus challenge resulted in significant
increase in mortality and pulmonary consolidation. Upon SEM examination of
the respiratory tract,slightly increased sloughing of squamous cells in the ex-
ternal nares was seen, whereas the remaining respiratory epithelium aopeared
normal.
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The effects of inhalation of A--C on formation of antibody and on pre-
formed antibody were examined in mice exposed for 3 hr/day, 5 days/week to
100 mg/m3 A-C before or after vaccination with influenza A2/Japan virus. At
various time intervals after the vaccination, the mice were challenged with
homologous influenza virus and mortality, survival rate, pulmonary consoli-
dation and immune response were measured. In terms of mortality and survival
time, the vaccine protected A-C and air-exposed animals equally well. How-
ever, significantly increased pulmonary consolidation and depressed secondary
immune responses, as measured by HI and SN antibody levels, occurred in many
groups of vaccinated mice exposed to pollutant compared to vaccinated con-
trols maintained in clean air. In general, there was little effect of ex-
posure to A-C on preformed antibody in studies in which A-C exposure occurred
immediately after the infectious challenge which followed vaccination at
various intervals. In studies in which A-C exposure occurred immediately
before or after vaccination, pulmonary consolidation scores measured after
subsequent infectious challenge were generally significantly higher- in the
vaccinated mice which had been exposed to A-C. The primary SN antibody re-
sponse was significantly increased when the interval between vaccination and
infectious challenge was 8 weeks. Secondary HI and SN antibody responses
were generally depressed when challenge occurred 2, 8, or 16 weeks after
vaccination.
Examination of the hamster tracheal epithelium in organ culture showed
that exposure to A-C reduced ciliary activity with damage to the ciliated
epithelium immediately after a single 3-hr exposure to 0.8 to 1.4 mg/m3 acid and
1.1 to 1.9 mg/m3 carbon. In both in vivo and in vitro exposure the acid-
carbon mixture produced a greater effect than the two pollutants singly.
Recovery from the acid-carbon mixture was similar to explants exposed to acid
alone. Carbon-exposed explants recovered to an activity index similar to
the controls. There was a clear correlation between initial damage to
tracheal cells in organ culture and their recovery, and the ability to
reproduce these effects in hamsters,
Long-term exposures of mice 3 hr/day, 5 days/week for 4, 12 or 20 weeks
to 1.4 +_ 0.4 (SD) mg/m3 sulfuric acid mist and 1.5 + 0.4 mg/m3 carbon aerosol
as well as carbon only demonstrated the following:
Subtle changes were detected by SEM examination of the respiratory
tract. After 12 and 20 weeks exposure to either carbon or A-C, in-
creased sloughing of squamous cells in the nasal cavity and areas
of thickened alveoli in the lungs were observed. The changes were
more widespread in animals exposed to A-C.
Mice exposed to A-C showed markedly slower weight gain than air con-
trols during the period of rapid growth in the first 8 weeks of
exposure to the pollutant.
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t Serum immunoglobulin concentrations varied in response to exposure
to pollutants, with the changes more marked in mice exposed to A-C
than to carbon. Concentrations of IgA did not change until 20 weeks
exposure when they were slightly depressed. Levels of IgG] were
depressed during the first 4 weeks of the A-C exposure. Concen-
trations of IgG2a slightly increased initially in the A-C exposure
group, then depressed from 4 to 12 weeks of exposure. IgG2b levels
were elevated initially and again after 20 weeks of exposure to both
carbon and A-C. Concentration of IgM was elevated after one week
of exposure to A-C but depressed throughout the remainder of the
20 week exposure.
Primary antibody response of spleen cells derived from mice exposed
to A-C or carbon assayed by the Jerne plaque technique followed the
pattern typical of other stress exposures. The response was stimu-
lated after 4 weeks of exposure, did not differ from the controls
after 12 weeks exposure, and a marked depression of the response,
especially in mice exposed to A-C, was observed after 20 weeks
exposure.
Bactericidal capacity in the lungs of mice exposed for 4 or 12 weeks
to carbon or A-C was significantly reduced compared to air controls.
Loading of the macrophages with carbon was apparently the most
significant factor in the decrease. A slight reduction in bacter-
icidal activity remained after 20 weeks of exposure.
t Marked increases in mortality and pulmonary consolidation and de-
creased survival time were seen in mice exposed to A-C for 20 weeks
and challenged with influenza A2/Taiwan virus.
The studies indicated that the effects of long-term exposure to mixtures
of concentrations of 1.4 +_ 0.4 mg/nr sulfuric acid and 1.5 +_ 0.4 mg/m^ carbon
(0.4 yMMD) as well as carbon only (0.3 yMMD) have been detected using sensitive
parameters. The immunologic state of the animal was examined directly in
response to specific antigens by the primary response of spleen cells, and in-
directly by infectivity studies. A quantitative measure of the effects on the
immune system without antigenic stimulation was obtained by determination of
serum immunoglobulin concentrations. These parameters have shown significant
alterations of immunoglobulin titer5depression of primary antibody response in
spleen cells to antigenic stimulation, and decreased resistance to respiratory
infection as measured by mortality, survival time, and pulmonary consolidation
after 20 weeks of exposure to A-C. In addition, bactericidal capacity of lungs
was reduced in mice exposed to either A-C ov^ to carbon alone, and subtle mor-
phological changes in the respiratory tract were detected by SEM.
The alterations of the pulmonary defense system suggest that prolonged ex-
posure to low concentrations of sulfuric acid and carbon particle mixtures
reduce the ability of mice to resist the secondary stress of respiratory
infection.
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SECTION 3
RECOMMENDATIONS
To further explore the effects of exposure to low concentrations of
sulfuric acid mists on the defense mechanisms of a host, research efforts
should be extended to life-tine studies on an animal host. Cellular and
humoral immunity (B and T cell stimulation), viability and enzyme activity of
alveolar macrophages, examination of effects on interferon production and
lung clearance mechanisms should be examined in addition to the parameters
used in the present study. The effects of the exposure stress on the acti-
vation of latent infections should also be examined. In terms of the present
model system, regular serologic monitoring of animals in long-term exposure
studies is recommended in order that such periodic measurements will detect
effects of the exposure stress on the activation of unexpected latent
bacterial and viral infections.
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SECTION 4
MATERIALS AND METHODS
GENERATION 0^ ACID MIST AND PARTICIPATE AEROSOLS
The apparatus used for generation of the sulfuric acid mist. (96^ purity,
Baker Co.. Phil 1 ipsburg, NJ) arid carbon particle (Sterling MT CT-6729, carbon
black 9?. 5'; purity, Cabot Corp. Boston, MA) aerosol is shown in Figure 1. Dry
filtered compressed arr was regulated by a low pressure regulator. A portion
of the air, controlled by valve V3 and monitored by gauge P2> was bubbled
through distilled water maintained at room temperature while another portion
of the air (controlled by valve V2 and monitored by the flowmeter) mixed
with the air stream from the bubbler to provide a 60% RH and a flow rate of
100 "liters/ruin. The third stream of air (controlled by valve Vl and monitored
by P]) passed through a DeVilbiss No. 40 nebulizer containing 40% sulfuric
acid solution (or a 0.4% sulfuric acid solution for the 1-2 mg/m3 acid
studies). The aerosol produced by the nebulizer was diluted with the 60/' RH
air in a flask and passed into the animal exposure chamber.
To prevent changes in particle size due to evaporation and condensation
the humidity in the chamber was maintained fairly constant. Since the size
change is most significant when humidity is below 20% or above 80% RH, a
humidity of 60% RH was chosen for these experiments.
The carbon aerosol was generated by a Wright dust feeder. A slowly
rotating scraper blade continuously released a small quanity of carbon
powder. The powder carried by the air stream was impacted on a elate to break
up the agglomerates and disperse the particles uniformly in the air. In the
short term, multiple-exposure experiments, a mean carbon particle size of
0.3 ym and a concentration of 5 mg/m3 were used.
The A-C aerosol was produced by mixing the sulfuric acid mist and the
carbon aerosol stream and passing the mixture through a tube maintained at
3100C. The sulfuric acid vaporized in this tube and, as the mixture cooled
in the section following the heated tube, the acid vapor condensed on the
solid carbon nuclei. The resulting aerosol was then diluted with the wet
and dry air in the three-neck flask to adjust the humidity to 60% RH. The
aerosol was then passed into the animal exposure chamber.
The chamber used for exposure of animals to the pollutants is shown
in Figure 2. The acid mist was introduced near the bottom at one end of
the chamber and exhausted from the top diagonally across from the inlet.
-------
I. COARSE FILTER
2. REGULATOR
3. ABS FILTER
4. GLASS WOOL
5. VARIAC
6. HUMIDIFIER
7. FLOWMETER
8. NEBULIZER
9. WRIGHT DUST FEEDER
10. HEATING COIL
II. MIXING FLASK
TEST
AEROSOL
Figure 1. Sulfuric acid aerosol generator
10
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ISOLATION
CHAMBER
H2 S04
AEROSOL
ANIMALTREATMENT
CHAMBER
SAMPLING PORTS
EXHAUST
Figure 2. Animal exposure chamber for acid aerosols,
11
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For particle size analysis of all concentrations of acid mists produced
by hot generation and for acid coated carbon aerosols with mean particle
diameters of 0.12ym and 0.4ym, respectively, the aerosol in the chamber
was sampled from Port No. 2 using an electric mobility analyzer. The
sampling system used for the quantitative assay consisted of two Greenberg
impingers in series, each containing 50 ml of isopropanol-water mixture
(80:20 v/v). The sample was collected for a known period of time (at least
10 min) at a flow rate of 1 liter/min held constant by a stainless steel
critical orifice. The contents of the impingers were transferred to a 200-ml
volumetric flask and titrated with 0.001 N barium perchlorate using thorin
as an indicator. The acid concentrations used in the experiments ranged from
50 to 200 mg/m^ and were obtained by varying the pressure of the air passing
through the nebulizer. The carbon concentration was monitored by a con-
densation nuclei counter and was maintained at 5 mg/m3 throughout the short-
term experiments.
o
Long-term exposure mean concentrations were 1.4 +_ 0.4 (SD) mg/m sulfuric
acid mist and 1.5 +_ 0.4 mg/m3 carbon. As in the short term studies, mean
particle diameters were 0.12 ym, 0.3 ym, and 0.4 ym for acid only, carbon, and
A-C mists respectively. The acid mist generation system was the same as out-
lined above using 0.4% acid solution instead of 40%. Quantitative assay of
acid concentration was performed by pulling a 200 liter sample from the chamber
through a type A-E glass fiber filter (Gelman Instrument Company). The acid
was dissolved from the filter into 15 ml of 5% isopropanol. Acid concentration
was determined by measuring the resistance of this solution using a 1650-B
impedance bridge (General Radio Company, Concord, MA) and comparing to standard
curve values. Carbon concentration was monitored as above using a condensation
nuclei counter.
ANIMALS
The experimental animals used included 3- to 6-week-old COBS mice from
Charles River Laboratory (male CDi strain) and ARS/Sprague-Dawley (male and
female ARS2-CF] strain), conventional 3- to 6-week-old BDF-] strain mice from
Murphy Breeding Labs, and 12- to 14-week-old male CrrRGH (SYR) Syrian golden
hamsters obtained from ARS/Sprague-Dawley. The animals were quarantined for
2 weeks and were provided with food and water ad libitivn throughout the
experiments. For exposure to pollutants and airborne infectious agents the
animals were placed into specially constructed stainless steel wire cages,
keeping each animal in a separate compartment. In a few selected experiments,
two to five mice were groups per compartment for exposure to pollutant only.
INFECTIOUS AGENTS
Influenza virus was prepared by rapid passage through mice or hamsters
and identified by a NIH reference reagent antiserum. Influenza A2/Taiwan/64
and A2/Japan/170 viruses were used for mice and A/PR/8 for hamsters. Within
24 to 48 hr after intranasal inoculation, lungs were removed aseptically and
a 20% suspension was prepared in phosphate-buffered saline (PBS). For intra-
nasal inoculation dilutions were made in PBS; for aerosolization the diluent
was PBS containing 0.2% bovine serum albumin. For the vaccine study A2/Japan/
170 influenza virus was prepared by rapid passage through mice followed by
growth in 10-day-old embryonated chicken eggs and identification by NIH ref-
erence reagent antiserum.
12
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streptosoccus sp. , Lancefield's group C, was grown in Todd Hewitt broth for
18 hr at 37°C. Suspensions of the bacteria were prepared in 0.1% peptone water
for aerosol ization.
Klebs-iella pneumonias, type 1, passaged in mice and isolated from the heart,
was grown in trypticase soy broth for 18 hr at 37°C. Suspensions of the bac-
teria were prepared in 0.1% peptone water for aerosol ization.
VACCINE
Influenza A2/Japari/l 70/62 virus vaccine (Lot 2JG63, Eli Lilly and
Company, Indianapolis, IN) was used. This commercial preparation had been
zonal centrifuged and contained 3696 CCA units per ml
INFECTIOUS CHALLENGE
The experimental animals were infected by exposure to an aerosol or by
intranasal inoculation of the agent. Infectious aerosol challenge was con-
ducted in a 350-liter plastic aerosol chamber (60x60x95 cm) installed within
a microbiological safety cabinet. A continuous flow DeVilbiss atomizer
(Model 84) was used to disseminate the infectious agent with particles of
1 to 5 ym MMD by means of filtered compressed air. For the challenge, mice
were placed in the aerosol chamber and exposed for 5 to 10 min to the air-
borne infectious agent. Control mice were exposed to the sterile diluent for
a comparable interval. After challenge, the animals were removed from the
chamber and held for 14 days in an isolated clean air animal room.
PULMONARY CONSOLIDATION
Lung consolidation in mice was scored on a scale ranging from 0 to 5.
0 = no consolidation
1 = 1 to 25% consolidation
2 = 26 to 50?»
3 = 51 to 75%
4 = 76 to 100%
A value of 5 was assigned to mice that died during the experiment (20).
RELATIVE MEAN SURVIVAL RATE (RMSR)
The RMSR was calculated according to the equation:
where A is the last day on which any individual mouse is alive; B is the
number of mice surviving A d?;-:: d is the last day of observation; L is the
number of mice alive on the c.\ d; and n is the original number of mice in
the experimental group (21).
13
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LUNG CLEARANCE
Groups of four or five mice were killed immediately after challenge with
Streptococcus sp. and at hourly intervals for up to 6 hr and at 24, 48 and 72
hr. Each lung was aseptically removed, weighed and homogenized in 1.8 ml of
sterile peptone water. The homogenates were diluted in 0.1% peptone water
and plated in duplicate on blood agar base media (BBL) with 5 to 10% defibrin-
ated sheep blood. The number of viable streptococcus per gram wet weight of
lung determined at 0 hr was considered 100% recovery. The microorganisms in
lungs at hourly intervals were assayed by the same procedure and the number
of viable microorganisms/gram wet weight of lung was compared to the 0-hr
value and plotted on semi logarithmic paper.
The rate of bacterial clearance was calculated and expressed as the t^
value representing the time required for 50% of the original bacterial popu-
lation to be cleared from the lungs. The \h was determined on a semilogarith-
mic regression after converting the percent recovery to logio values.
SEROLOGICAL METHODS
HI antibody tests were performed in duplicate by the microtiter method
in disposable V-plates. In all tests, 1% chicken red-blood cells and fpur
hemagglutinating units of egg-adapted influenza A2/Japan/170 virus were used.
Type A2 influenza (Japan/170/62) antiserum, received from the National
Institutes of Health, served as the positive control.
SN tests were performed in 10-day-old embryonated chicken eggs. Serially
diluted serum samples were mixed with equal volumes of 10 to 320 LDso of in-
fluenza virus and incubated for 1 hr at 4°C. The mixtures were then inoculated
into the allantoic cavity and the eggs incubated for 48 hr. The hemagglutina-
tion test was then performed on the harvested fluids by using 0.5% chick
red-blood cells. The SN titer:; were calculated by the Reed-Muench method
(22).
HEMATOLOGY TESTS
Erythrocyte and Leukocyte
A Coulter Electronic Particle Counter with 100 aperature was used. Each
blood sample was counted in duplicate. Reference blood samples (Coulter
Electronics, Inc) were counted for standardization (23).
Hematocrit
Packed cell volume was determined in capillary tubes using a micro-
capillary head centrifuge (International Equipment Company, Model MB).
Hemoglobin
Hemoglobin was measured as cyanomethemoglobin, with a reference solution
as standard (24).
14
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Differential Leukocyte Count
Wright's stain was used to stain the leukocytes for examination.
Reticulocyte Count
These cells were counted by the new methylene blue N method (25).
Platelet Count
Platelets were counted visually in a hemocytometer with a phase
microscope.
CLINICAL CHEMISTRY TESTS
The following tests were performed on a centrifugal analyzer (CentrifiChem,
Union Carbide, Tarrytown, NY) using microassays. Tests were run the day the
blood was collected, except in a few instances where technical difficulties
required storage of sera at -20°C. Previous work had shown that such storage
had no significant adverse effect.
Alkaline Phosphatase
A modified Bessey-Lowry-Brock technique utilizing p-nitrophenyl phosphate
as substrate for AP was used (26).
Lactic Dehydrogenase-L
The microassay developed by Wacker, _et jil_. (27) was used.
gHydroxybutyrate Dehydrogenase
The microassay developed by Rosalki, et al_. (28) was used.
Isocitric Dehydrogenase
The microassay developed by Wolfson, et al_. (29) was used.
Lactic Dehydrogenase Isoenzymes
A Beckman Microzone Electrophoresis system was adapted from the method
developed by Dade Division (Amer. Hosp. Supply Diy.).
SERUM IMMUNOGLOBULIN CONCENTRATION
Quantitative radial immunodiffusion plates for mouse immunoglobulins
IgA, IgG-j, IgG;?a,IgG2b and I9M were procured from Meloy Laboratories, Inc.,
Springfield, Virginia. Reference standards obtained from pooled sera of nor-
mal mice were assayed daily, in duplicate, to provide quality control.
Mouse immunoglobulin standards (Meloy Laboratories) were assayed concurrently
to quantitate the experimental samples. Duplicate serum samples were placed
in preformed wells, the plates incubated at 220C for 18 hr, and the radial
diffusion diameters measured using a Bausch & Lomb 7x lens. Duplicate radial
diffusion diameters were recorded for each of the 6 to 9 serum samples.
15
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JERNE PLAQUE ASSAY
The plaque assay i.e., hemolysis-in-gel first described by Jerne, et
a1_. (30) and modified by Kaliss (31) was used to determine humoral antibody
response to sheep red blood cells (SRBC). Six mice from each exposure group
were injected intraperitoneally with 0.5 ml of a 2% suspension of washed
SRBC in 0.85% saline. After 4 days the spleens were removed and a single-
cell suspension prepared from each.
For the plaque assay, 0.6 ml of 1.5% Seaplaque agarose (Marine Colloids)
in Hanks' balanced salt solution (HBSS), 0.3 ml of 2% SRBC in 0,85% saline,
and 0.3 ml of the appropriate spleen cell suspension were added in sequence
to plastic tubes (17 x 100 mm, Falcon) maintained at 42°C. The contents were
mixed and poured into petri plates containing a preformed agarose layer (3 ml
of Seakem agarose, Marine Colloids, Rockland, Maine at. a 1.5% solution in
HBSS in 60 x 15 mm petri dishes). Each spleen cell suspension was plated in
triplicate. The plates were gelled at 24°C for 10 min and incubated for
1 hr at 370C in a 5% C02 incubator. One milliliter of guinea pig complement
(Microbiological Associates, Bethesda, MD) at a 20% solution in HBSS was
carefully added to each plate and incubated 60 min at 37°C. The plates were
stored overnight at 4°C and p" aques were counted the following day. Prior work
had shown that such storage produced no alterations.
ALVEOLAR MACROPHAGES
Alveolar macrophages obtained from mice by tracheobronchial lavage were
examined for total and differential cell count and viability by conventional
methods.
GROWING RADIOLABELED BACTERIA
Cultures of 35S-labeled K. pnewnoniae were grown in Anderson's medium
by an adaptation of the method of Berlin and Rylander (32) used for labeling
of E. Goli. In the medium composed of 0.1 g MgS04, 3.0 g Na2HP04, 5.0 g
NaCl, 1.0 g NH4C1 and 4.0 g glucose per 1000 ml, the MgS04 was replaced by
MgCl2 and the sulfate requirement of the bacteria was then provided by
addition of 35$-labeled NaaS04. For each experiment, a culture of K.
pnewnoniae was first grown in conventional Anderson's medium for 24 hr from
a bacterial stock maintained in trypticase soy broth. The concentration of
this bacterial suspension was then adjusted to 108/ml after counting in a
Petroff-Hauser chamber, and a 0.1 ml aliquot was used to inoculate 10 ml of
Anderson's medium containing ^180 yCi of 35s-NaaS04 (specific activity
>-100 mCi/mM). After 16 hr of incubation,the 35s-labeled K. pnewnoniae was
harvested and the bacteria were washed and centrifuged at 12,000 xg 10 times
for removal of unattached radiolabel. All bacterial counts were first deter-
mined by dark field microscopy in a Petroff-Hauser bacterial counting chamber
and subsequently also by culture plate technique.
16
-------
BACTERICIDAL ACTIVITY
Intrapulmonary bacterial inactivation was determined in the lungs of
individual animals by the method of Green and Goldstein (33). Mice are
exposed to radiolabeled live bacteria and the ratio of the viable bacterial
count to the radioactive count in each animal's lung provides a measure of
bacteria which are destroyed by the lung at a given time after infection.
After exposure to A-C mist, groups of mice were infected by intranasal
inoculation with 35s-labeled K. pneumoni-ae. Four hr after infection they
were killed, and their lungs were aseptically removed and homogenized. From
the radioactive counts made on aliquots of the homogenates and the radio-
active labeling ratio determined for each bacterial culture, the total number
of bacteria deposited in each lung could be calculated immediately. On the
basis of this information, another aliquot of each lung homogenate was appro-
priately diluted for determination by culture-plate technique of the bacteria
that remained live in the lungs.
SCANNING ELECTRON MICROSCOPY AND HISTOPATHOLOGY
Nasal cavities, trachea, and lungs were taken for SEM examination from
mice killed immediately after exposure to air, carbon, and A-C mist. The
animals 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 opened, and
the lungs and trachea were removed ->. tote. The trachea was cannulated to
the level of the first cartilaginous ring and the lungs expanded with
Karnovsky's paraformaldehyde-glutaraldehyde phosphate-buffered fixative (34)
at 20 cm water pressure. Perfusion continued for at least 2 hr with the lungs
completely immersed in fixative. Upon completion of airway perfusion, the
trachea was ligated and the lungs floated in fixative.
After cannulation of the trachea, the head was removed, the skin was 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.
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. All tissues were washed in distilled water and de-
hydrated with increasing concentrations of alcohol. Pentyl acetate was then
substituted for the alcohol and the tissues dried by the critical point
method in carbon dioxide. The dried trachea was sectioned longitudinally,
and all tissues were cemented to stubs, gold-coated in a Denton vacuum evapor-
ator equipped with a rotating turntable, and examined in a Kent-Cambridge
Mark II Stereoscan scanning electron microscope at 20 kV.
Samples of lung, trachea and nasal cavities were also processed for
light microscopy by conventional histologic methods. Sections were cut at
and stained with hematoxylin and eosin.
17
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PREPARATION OF TRACHEAL RINGS AND CULTIVATION METHOD
Four-week-old Syrian Golden Hamsters were killed with C02 and immediately
opened from the middle of the sternum to the larynx. The trachea was exposed
and the surrounding membranes aseptically teased away with fine forceps. The
trachea was gently lifted,and a forceps was slid underneath to free it from
the esophagus and to facilitate cleaning. After removing remaining membrane,
the trachea was excised, cutting just above the larynx and just above the
bifurcation. The trachea was briefly rinsed in HBSS and cleaned of remaining
membrane. The larynx-end of the trachea was grasped with a sterile curved
hemostat which was laid against the edge of the dish at an acute angle with
the trachea held over the dish. Each trachea was opened from the larynx to
the corina along the membranous dorsal wall. After grasping the loose end
with sterile forceps, the trachea was gently stretched to slightly separate
the white cartilage rings. With a sterile scapel the trachea was cut into
rings approximately 1 mm thick. Approximately 10 to 12 trachea! rings were
obtained from each animal.
Tracheal rings were washed in Eagle's basal medium and placed on a section
cross-hatched with a scapel blade in 35 x 10 mm unwettable plastic petri dishes
containing 0.7 ml of HEPES buffered CMRL 1066 medium supplemented with 0.2%
bovine serum albumin, penicillin (250 U/ml), and streptomycin (250 yg/ml). The
rings were incubated for 24 hr at 37°C in 5% C02 atmosphere and 90% relative
humidity. This initial incubation period allows the rings to adjust to the
external environment and ensures that normal beating frequency is restored
before the test substance is applied.
Immediately following removal from the hamster, all trachea! rings were
examined in inverted Nikon microscope at 200X for normal ciliary activity or
evidence of cytopathology. The vigor of ciliary activity at the periphery of
the explants was determined using an electronic strobescope (General Radio,
Type 1531-AB) as the light source. A Sage air curtain (Sage Instruments, Model
279) was placed adjacent to the microscope stage to maintain the temperature
at 37°C while determining the ciliary beat frequency. When the flash rate of
the strobescope is set to the same speed as the ciliary beat, ciliary move-
ment appears to stop. Ciliary beat frequency of ring explants was measured
at four separate areas on the lumen and their average was recorded as the
initial beats per minute. The ciliary activity of whole trachea explants was
observed by focusing on the mucosa through the explant. After the initial
24 hr incubation period , the baseline cilia beating frequency for each ex-
plant was determined. Cilia beating frequency was determined every 24 hr
for 3 days in the A-C mixture studies.
18
-------
For light microscopy, ring explants were washed in Hank's salt solution
to remove mucus, fixed in 10% neutral-buffered formaldehyde solution, dehydrated
and infiltrated. Paraffin-embedded tissue was cut into 6-ym thick sections
and stained with hematoxylin and eosin for histologic examination. Paraffin
sections were also stained with an alcian blue at pH 2.5-periodic acid Schiff
(PAS) sequence which distinguished blue- or purple-stained acidic mucosubstance
from red-colored neutral mucosaccharide.
Trachea specimens for scanning electron microscopy were fixed with
Karnovsky's paraformaldehyde-glutaraldehyde phosphate-buffered fixative. The
tissues were washed in distilled water, and dehydrated with increasing con-
centration of alcohol. Pentyl acetate was then substituted for the alcohol,
and the tissues were critical point dried in carbon dioxide. The dried
specimens were cemented to a stub, gold coated by vacuum evaporation and
examined in a Mark II Stereoscan scanning electron microscope at 20 kV.
DATA ANALYSIS
Results of the experiments were subjected to statistical analyses and
the significance of the observed differences reported at the 5% probability
level. As appropriate, linear regression analysis, analysis of variance,
Chi square test, and Student t test were used. Reciprocal antibody titers
were converted to logio for statistical analysis (35).
In order to eliminate animal-to-animal variability in statistical analysis
of tracheal explant data, each treatment group and control were assigned two
ring explants from each hamster. Experimental error was further reduced by
using the same ring to determine the differences between the baseline cilia
beating frequency and the beating frequency at different times following ex-
pcsure. Two-way analysis of variance was used to test the hypothesis of
no treatment differences for the time periods studied. Duncan's new multiple-
range test and the Chi square distribution test were used to elucidate patterns
of significant treatment differences.
19
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SECTION 5
RESULTS AND DISCUSSION
SHORT-TERM EXPOSURES TO ACID MIST AND CARBON PARTICULATE MIXTURES
Toxici'ty
The first phase of this investigation consisted of range finding experi-
ments to provide the concentrations; of acid mist which did not result in
mortalities and which could be used in the infectivity studies. Table 1 shows
mortality of mice after a single 3-hr exposure to acid mist. Mortalities were
observed at 600 mg/m^ while at 400 mg/m^ and below only one out of 360 animals
died. It is interesting to note that the population (density) of mice per cage
during the acid mist exposure had an effect on mortality. Whenever two mice
were housed per cage the mortalities were markedly higher than when five
animals were housed per cage. Similarly, the data suggest that, irrespective
of the housing density, male mice were more sensitive to the acid mist than
female. The mortality data for hamsters indicate that this species is more
resistant to the acid mist exposure than mice.
Initial experiments were also conducted to determine the concentration
and duration of exposure to sulfuric acid mist-carbon particle mixture (A-C)
that could be used in the infectivity studies without resulting in mortalities
due to pollutant per se. Mice and hamsters were exposed once for 3 or 6 hr
to a mixture of sulfuric acid mist ranging from approximately 100 to 700 mg/m
combined with a constant concentration of 5 mg/m3 carbon aerosol. The results
summarized in Table 2 indicate that CF] mice were more sensitive to the ex-
posure than the BDF'| strain of mice or hamsters. The maximum concentrations-
of A-C that did not result in deaths in CF] mice were approximately 300 mg/m
for the 3 hr or 200 mg/m3 for the 6 hr exposures.
The mortalities due to multiple exposures to A-C in mice of different
ages and strains are shown in Table 3, Groups of 6- to 7-week-old and 9- to
10-week-old BDF] and CFi mice were exposed at 24-hr intervals five times for
3 hr to 100 or 200 mg/m3 A-C; only minimal mortalities were observed as the
result of the multiple exposures irrespective of the concentration of acid.
Upon exposure to 200 mg/m3 A-C, the BDF-] strain mice again appeared to be
somewhat more resistant than the CFi mice. In all cases, the mortalities
were low, with the older mice perhaps somewhat more susceptible to the toxic
effects of the A-C mixture exposure.
20
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TABLE 2. MORTALITY AMONG MALE MICE AND HAMSTERS
AFTER A SINGLE EXPOSURE TO SULFURIC
ACID-CARBON PARTICLE MIXTURE
3
Mortality
Concn.. mg/m Mice CFl Mice BDF1 Hamsters
Acid Carbon D/T %__ D/T %_ D/T %_
3 hr-Exposure
100 5 1/53 2 - 0/24 0
200 5 1/25 4 - 0/12 0
300 5 0/24 0 0/48 0 0/12 0
400 5 4/24 17 0/36 0 0/12 0
500 5 12/50 24 0/44 0 0/12 0
700 5 5/24 21 - 0/12 0
6 hr-Exposure
100 5 0/24 0 - 0/12 0
200 5 0/24 0 0/12 0
300 5 7/72 10 0/48 0 0/12 0
400 5 5/24 21 0/36 0 1/12 8
500 5 22/48 46 2/44 5
22
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TABLE 3. MORTALITY AFTER MULTIPLE 3-HR EXPOSURES TO TWO
CONCENTRATIONS OF ACID MIST PLUS 5 mg/m3 CARBON
Species
CF, mice (M)
1
CF, mice (M)
1
BDF, mice (M)
1
BDF, mice (M)
1
Number
Age of
(weeks) Exposures
7 1
2
3
4
5
9 1
2
3
4
5
6 1
2
3
4
5
9 1
2
3
4
5
Acid + Carbon
100+5
D/T
1/353
0/357
1/357
2/356
6/354
0/138
0/138
0/138
0/138
2/138
0/48
0/48
0/48
1/48
0/47
0/48
0/48
0/43
0/48
0/48
Concn. , mg/m3
200 + 5
D/T
0/192
0/192
0/192
2/96
2/94
1/152
11/151
4/95
2/91
5/89
0/96
0/96
0/96
0/dS
0/43
0/48
0/48
0/48
0/48
2/48
23
-------
3
The 7-week-old CFi mice exposed five times to 200 mg/m A-C showed an
approximate 20% loss in body weight, while 9-week-old CFi mice lost 25%
weight. The 7-week-old CFi mice exposed five times to luO mg/m3 A-C showed
a 16% weight loss, whereas control mice held in the exposure chamber for
3 hr/day for 5 days at ambient condition lost only 5%. Thus, in terms of
body weight loss, exposure to the pollutant mixture appears to be a con-
siderably greater stress than the mere manipulation of the animals or depri-
vation of food and water during exposures.
To complete the studies of the effects of short-term multiple exposures
to mixtures of acid and carbon, experiments were conducted in which 3- to 4-
week-old CDl male mice were exposed for 3 hr/day, 5 days/week to aerosols
containing 5 mg/m3 of carbon particles only. This concentration was the
same as that used in combination with all of the various acid concentrations
in single or short-term multiple exposure studies. Mice were exposed 5, 10,
or 20 times (1, 2 or 4 weeks) to the carbon aerosol or to ambient air with
no mortality resulting in any of the groups.
Scanning Electron Microscopy
The mortality studies were accompanied by histologic and scanning electron
microscopic examination of the respiratory tract to further define the effects
of acid mist and acid mist-carbon mixtures on the animals.
Tissues from Control Mice --
Nasal cavity -- As seen in the composite scanning electron micrograph
(Figure 3), the nasal cavity is composed of squamous, ciliated, and nonciliated
respiratory epithelium. Starting with the external nares, the surface is
covered with squamous cells, which are characterized by their rough invaginated
surface and well-defined cell walls (Figure 4a). The anterior portion of
the septum shows a general transition from the squamous cells of the external
nares to a rough irregular surface of nonciliated cells (Figure 4b). The
microvilli on these cells are generally very short and compact; however, each
cell varies in number of and length of microvilli (Figure 4c). Toward mid-
septum a few ciliated cells can be seen (Figure 4d), while microvilli on the
nonciliated cells are becoming longer and the cell surface is becoming
smoother. At the posterior end, the cell surface is smooth with a heavy
population of ciliated cells (Figure 4e). Cilia are longest here, although
a few nonciliated cells can be seen.
Trachea -- Both ciliated and nonciliated cell types are found in the
trachea. Cilia tend to be uniform in length and microvilli tend to be short
and compact (Figure 5).
Lung, bronchus and alveoli -- The cells in the bronchus are similar to
those found in the trachea (Figure 6a). Alveoli have a honeycomb appearance,
each alveolus being divided by a thin septum (Figure 6b). Pores are also
visible as small openings in each alveolus.
24
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25
-------
Figure 4. Nasal cavity of a normal mouse: (a) squamous cells
in the external nares, 5100X; (b) nonciliated cells,
2200X; (c) nonciliated cells showing varying lengths
of microvilli, 1050X; (d) nonciliated and ciliated
cells of mid septum, 2100X; (e) ciliated cells at
posterior septum,2100X.
26
-------
Figure 5. Trachea of a normal mouse:
cells,2000X.
nonciliated and ciliated
Figure 6. Lung of a normal mouse: (a) nonciliated and ciliated
cells in the bronchus, 2000X; (b) alveoli,110X.
27
-------
Tissues from Mice Exposed to Acid Only and A-C --
The toxicity studies were extended to include scanning electron micro-
scopic examination of the nasal cavity, trachea, and lung to further define
the effects of exposure to A-C mixtures. Lungs from mice and hamsters exposed
to 100 mg/m3 acid mist for 3 hr and examined at 1, 3, 24, 48 and 72 hr past
exposure did not reveal any change. The same was found true of those animals
exposed to mixtures of 100 mg/m3 acid mist and 5 mg/m3 carbon. In animals
exposed to the A-C mixtures damage was distributed throughout the entire
trachea as well as down into the main stem bronchus, while in those exposed
to acid mist only, the main destruction was found in the upper portion of the
trachea. SEM examination of the respiratory tract following 5, 10 or 20 3-hr
exposures to 5 mg/m3 carbon only revealed only slightly increased sloughing
of squamous cells in the external nares of animals exposed to carbon compared
to the controls. The rest of the respiratory epithelium was normal.
The type of damage observed in mice exposed to various concentrations of
A-C was similar in all samples, with variations in the degree of severity and
the tissues involved. In general, starting at the external nares of the
nasal cavity, small holes could be seen in the cell surface in the squamous
cells (Figure 7a). On the anterior septum and midseptum, nonciliated cells
had holes, tears, and/or missing microvilli (Figure 7b). Some dead cells
began sloughing (Figure 7c) and areas could be found where a dead cell had
totally sloughed off and left a hole in the surface (Figure 7d). Some ciliated
cells were seen tearing away at their cell edges (Figure 7e), On the posterior
septum, ciliated cells showed little damage except for a heavy mucous coating.
3
In mice exposed five times to 200 mg/m A-C, or three, five and ten times
to 100 mg/m3 A-C, macrophages were found in the nasal cavity, especially on
the anterior septum and midseptum. The highest number of macrophages was
present in the group exposed to 200 mg/m13 A-C where the damage was most severe
and extensive, and a lesser number was found in the latter groups where the
damage was less severe. These are believed to be alveolar macrophages brought
up in the mucus into the nasal cavity. A mucous coating was found throughout
damaged areas, being most prevalent in high damage areas and probably a re-
sult of the acid-carbon treatment.
In the trachea, a mucous coating and matted cilia were present in mice
exposed to the various A-C concentrations (Figure 8a). In the trachea of some
mice exposed five times to 200 mg/m0 A-C, the mucous coating on the cells was
so heavy that it was not possible to estimate the damage to the cell surface,
while in tracheas of other mice holes, tears and dying cells were seen
(Figure 8b and c), and some regeneration of cilia could be found 2 weeks after
exposure (Figure 8d). In the bronchus of the lung, results were similar, i.e.,
mucus, matted cilia, holes, tears, and dying cells were noted.
The alveoli showed damage only when mice were exposed five times to
200 mg/m3 A-C. Many of the septa dividing each alveolus were thickened and yet.
many emphysemic-like areas were also present where the septa were thin and
filamentous (Figure 9).
28
-------
Figure 7. Nasal cavity of mouse exposed to acid mist-carbon
particles mixture: (a) small holes in squamous cells,
1000X; (b) missing microvilli in nonciliated cells,
1050X; (c) damaged nonciliated cells beginning to
slough off, cell shows small holes and missing micro-
villi, 2300X; (d) holes in cell surface where dead
cells have sloughed off,500X; (e) ciliated cell
tearing away at its border on mid septum,2000X.
29
-------
Figure 8.
Trachea of mouse exposed to acid mist-carbon particles
mixture: (a) mucous coating and matted cilia 1800X-
9nnnv° ,^ the Cel1 surface of nonciliated cells, '
2009X; (c) dying ciliated cell, 5200X; (d) regenerating
ciliated cell at 2 weeks after exposure to the
pollutants,10,050X.
30
-------
Figure 9. Lung of mouse exposed to acid mist-carbon particles
mixture: thickened alveolar walls as well as thin
filamentous septa (a) 200X; (b) 550X.
31
-------
CT indices were calculated (Table 4) for all A-C studies so that a com-
parison of comparable concentrations could be evaluated. In the first four
groups the amount of damage to the tissues increased as the CT for acid and
the CT for carbon particles increased.
o
After a single exposure to 100 mg/m of acid mist (Group I), damage was
found near the top and middle of the trachea. The lower portion of the trachea
was relatively free of damage and the lung was normal. When the same concen-
tration of acid was combined with 5 mg/m3 of carbon (Group II), damage was
more extensive and extended into the bronchus of the lungs.
3
In mice exposed three or five times to 100 mg/m A-C (Groups III and
IV), the damage became more severe deeper into the bronchus. After 2 weeks,
however, some signs of healing were noted.
Mice in Groups V, VII and IX were exposed to a total acid CT dose of
1000 mg/m3 with carbon. From the results shown in the table it is apparent
that although the total acid exposure dose was equivalent, the tissue damage
differed markedly. In mice exposed ten times to 100 mg/m3 A-C (Group VII) 3
the damage was similar to that seen in mice exposed 3 or 5 times to 100 mg/m
A-C (Groups III and IV) and was much less severe than in mice exposed five
times to 200 mg/m3 A-C (Group \l). Mice in Group V showed the most severe
damage, which extended into the alveoli, and remained essentially the same
at 2 weeks after the exposure. Mice exposed 20 times to 50 mg/m3 A-C
(Group IX), the most exposures with the lowest concentration nevertheless still
equaling the CT of 1000 for acid, showed a total damage similar to those ex-
posed to acid only (Group I). Thus, the damage here was slight despite the
total exposure concentration.
By comparing the CT values for groups IV, V and VI, an increase in damage
could only be seen where the acid concentration was the highest, the least
damage where no acid was present, and minimal damage at 500 mg/m3. Since the
CT for carbon was the same in all three groups, it appeared that the concen-
tration of acid was more important. By comparing groups VII and VIII as well
as IX and X, it could also be seen that even though the CT values were equal
for carbon and acid within the groups, the most damage was found in the A-C
groups, the damage being more extensive in group VII where the acid concen-
tration was the higher of the two. These observations suggest that although
the carbon caused some damage, its presence served to enhance the toxicity
of the acid mist. Moreover, the results demonstrated that the frequency of
exposure to A-C was not as important a factor in inducing tissue damage as the
initial concentration of sulfuric acid mist,
Histopathologic Examination of Lung and Trachea Tissues
In general, tissues from experimental mice were similar to those of the
controls. At the highest A-C concentration (5 daily exposures to 200 mg/m3
and 5 mg/m3 carbon) a slight trend toward squamous metaplasia in a few small
bronchioles was apparent. Mild bronchiolar epithelial hyperplasia and bronch-
iolites were also present. However, very similar lesions were found in con-
trol mice and the general conclusion was that the control and experimental
tissues could not be differentiated.
32
-------
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Infectious Challenge
In the infectivity studies, the combined effects of exposure to acid mists
or acid-carbon mists with viral or bacterial challenge were examined. The
pathogens used were influenza virus, Klebsiella pneumonlae and streptoccccus
sp. Lancefield's Group C.
Single Exposure to Acid only
Table 5 shows the results of experiments where mice were exposed for 3-hr
to 100 mg/m^ acid mist and then challenged by the respiratory route with air-
borne Streptococcus or influenza virus. The interval between the acid mist
exposure and the infectious challenge varied from 1 to 24-hrs. As can be seen,
mortality and mean survival time did not appear to be affected by the exposure
and challenge with Streptococcus. The extent of lung consolidation observed
after the challenge with influenza virus was higher in mice exposed to acid
mist than those challenged with the influenza virus only. This was especially
noticable in animals challenged with the infectious agent within 1 hr after
the termination of acid mist exposure, where two-fold increase in mortality
and a significant increase in lung consolidation were seen.
In a similar group of experiments not shown in the table, mice were
challenged with K, pneumoniae first and 1 to 24-hr later were exposed to
100 mg/m3 of acid mist. This experimental condition did not result in any
enhancement of mortality.
TABLE 5. EFFECTS OF 3-HR EXPOSURE TO 100 mg/nT
RESISTANCE TO INFECTION OF MICE
ACID MIST ON
Agent
Streptococcus
Influenza
Interval Between
Mortality
Acid and Infect, (.hr) D/T %
O3
1
3
24
Oa
1
3
24
12/53
9/45
9/45
11/45
9/60
16/58
10/60
13/55
23
20
20
24
15
28
17
24
RMSR
(days)
12.2
12.1
12.4
11.8
13.2
12.6
13.2
12.3
Lung
Consol .
mmf
-
-
-
2.05
2.74*
2.34
2.57
Significant difference (p <0.05) compared to influenza virus
infected control.
Animals infected and not exposed to the pollutant.
35
-------
In another study, hamsters were first intranasally infected with in-
fluenza virus and 72 hr later exposed to acid mist concentrations of 500 and
700 mg/m3 for 3 hr. Control hamsters were given saline intranasally and then
exposed to the same concentration of acid mist. As can be seen in Table 6
the exposure to acid mist per se had no effect on mortality or survival rate
of the hamsters. The mortality due to infection only was 12% and increased to
27% upon exposure to 500 mg/tn3 and 46% upon exposure to 700 mg/m3. Similarly,
the survival rates were reduced from 13.1 to 84. days. A Chi square analysis
performed on the data suggests a high significance of the differences
(X2 = 6.87). Based on this exploratory experiment, additional studies were
conducted using the 72-hr interval to determine whether the mortality of mice
challenged with airborne Streptococcus is altered by high concentration acid
mist-carbon mixture exposures.
TABLE 6. MORTALITY AND SURVIVAL RATES OF
HAMSTERS CHALLENGED INTRANASALLY
WITH INFLUENZA VIRUS AND 72 HR
LATER EXPOSED FOR 3 HR TO ACID
MIST
Agent
Virus
Saline
Acid
mg/m3
0
500
700
0
500
700
Mortality
D/T
3/25
3/11
11/24
0/4
0/5
0/4
%
12
27
46*
0
0
0
RMSR
(days)
13.1
11.0
8.4*
14
14
14
Significant difference (p <0.05)
compared to influenza virus infected
control.
Single Exposure to A-C Mixtures --
Table 7 shows the mortality of mice challenged with airborne streptococcus
and 72 hr later exposed to A-C mixtures for 3 hr. The data show very little
if any effect on mortality after the combined exposures to the pollutant and
infectious challenge when compared to mortality observed in animals challenged
with the Streptococcus only. As is seen, the exposure to peptone alone did
not produce any mortalities but a few animals died after exposure to peptone
followed by 3-hr exposure to the pollutant.
36
-------
TABLE 7. MORTALITY OF MICE CHALLENGED WITH
AIRBORNE STREPTOCOCCUS SP. AND
EXPOSED TO ACID MIST-CARBON
MIXTURES
Agent
Streptococcus
Peptone
Acid
mg/fn
0
100
100
500
500
0
100
100
500
500
Carbon
mg/m3
0
0
5
0
5
0
0
5
0
5
Mortality
D/T
19/102
13/50
9/48
14/50
8/48
0/30
1/15
1/15
3/15
0/14
%
19
26
19
28
17
0
7
7
20
0
In another experiment, hamsters were used in a single acute exposure to
A-C to investigate the importance of the interval between exposure to the
pollutants and infectious challenge. At <1, 24, 48 or 72 hr before or after
intranasal challenge with influenza A/PR/8 virus or PBS control, hamsters were
exposed to 300 mg/m3 and 5 mg/m3 carbon for 6 hr. Results shown in Table 8
indicate that when exposure to A-C preceded the infectious challenge by <1,
24 and 72 hr, although not statistically significant, some increases in
mortality and decreased survival times were noted. However, when the sequence
was reversed, i.e., challenge followed by A-C exposure, significantly in-
creased mortality was observed at the 24-hr interval and survival times de-
creased significantly at the 24- or 48-hr intervals.
Short-Term Multiple Exposures to A-C Mixtures --
To determine the lowest concentration and the number of repeated exposures
that produce increased mortality in conjunction with either a bacterial or
viral challenge, studies were initiated with infectious challenge before or
after five or ten repeated 3-hr exposures to either 100 or 200 mg/m3 acid mist
and 5 mg/m3 carbon particle mixture. Male CF-| or CD] mice were used for all
of these studies.
Streptococcus sp. -- Mice were exposed daily for 3 hr, 5 days/week for 1
and 2 weeks, to 100 mg/m3 acid mist and 5 mg/m3 carbon particle mixture either
immediately before or after challenge with airborne streptococcus sp. When the
A-C exposures preceded the infectious challenges, no differences in mortality and
37
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RMSR were noted (Table 9). Reversing the sequence (infection followed by A~C
exposure) both five and ten exposures produced an increase in mortality which
was more marked when the number of A-C exposures increased. When the concen-
tration of pollutant was increased to 200 mg/m3 acid mist and 5 mg/m3 carbon
particle mixture, a significant increase in mortality and decreased survival
time compared to infected controls occurred with both pollutant exposure/
infection sequences. These data correlate with the SEM observations indicating
more severe lung damage in mice exposed five times to 200 mg/m3 than ten times
to 100 mg/m3 A-C,
Out of a total of 188 mice serving as controls challenged with 0.1% pep-
tone water aerosol and exposed to either A-C or air, 11 died (6%). In the
reverse experimental sequence no death occurred among mice challenged with
peptone water after exposure to A-C or air.
Influenza virus -- Studies were initiated in which five daily 3-hr exposures
to 100 mg/m3 acid mist and 5 mg/m3 carbon particle mixture were combined with
challenge with influenza A2/Taiwan virus aerosol at <1 , 24, 48 or 72 hr before
or after the A-C exposure. The 24- and 48-hr periods were included because these
intervals resulted in most marked mortality increases when a single 6-hr
exposure to 300 mg/m3 acid mist and 5 mg/m3 carbon particle mixture was combined
with influenza virus challenge in hamsters. The results shown in Table 10 in-
dicate a significant increase in mortality and decrease in mean survival time of
mice exposed to the pollutants in the 24-hr interval experiments. Slightly in-
creased mortalities were also seen when the infectious challenge occurred 1 or
72 hr after the final A-C exposure, but these increases were not statistically
significant. Among control mice used in the various sequence studies, 3 out
of 287 died (1%).
A similar series of experiments was conducted using airborne influenza
Taiwan virus as the challenge agent immediately before or after five daily 3-hr
exposures to 200 mg/m3 acid mist and 5 mg/m3 carbon particle mixture. In both
sequences of challenge and pollutant exposures, a significant decrease in sur-
vival time and significantly increased mortality in A-C exposed infected groups
was observed when compared with ambient controls (Table 10).
Another condition which produced a CT value of 1000 was twenty daily ex-
posures to 50 mg/m3 acid mist. Mice were exposed 3 hr/day, 5 days/week, for
4 weeks to 50 mg/m3 acid mist and 5 mg/m3 carbon particle mixture and chal-
lenged by the respiratory route with influenza A?/Taiwan virus aerosol within
1 or 24 hr after the final pollutant exposure. The results (Table 11) show
that, in both time- interval groups, the mortality increased and mean
survival time decreased compared with ambient controls. The control mortality
in the 24-hr interval study was quite high, but some increase was never-
theless detected in the experimental mice. The increase in mortality in the
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Short-Term Multiple Exposures to Carbon Only --
Limited experiments were also conducted in which 3- to 4-week-old CD! male
mice were exposed to aerosols containing 5 mg/m3 of carbon particles only.
This concentration was the same as that used in combination with all of the
various acid concentrations in single or short-term multiple exposures.
Mice were challenged with airborne influenza A2/Taiwan virus within 1 hr
before or after 5, 10 or 20 daily exposures to 5 mg/m3 carbon, 3 hr/day, 5
days/week. The results in Table 12 show that there were no changes in mortality,
survival time or pulmonary consolidation in mice exposed 5 or 10 times to the
carbon aerosol either before or after virus challenge. However, the mice
challenged with virus after 20 exposures to the carbon aerosol had significantly
increased mortality and pulmonary consolidation and decreased mean survival
time compared to the controls.
TABLE 12. RESPONSE OF MICE EXPOSED TO 5 mg/rri CARBON AEROSOL
AND CHALLENGED WITH INFLUENZA A2/TAIWAN VIRUS
Challenge Number of 3-hr
Mortality
Sequence Carbon Exposures D/T
Pre- carbon
exposure
Post-carbon
exposure
5
10
Air-Control
10
20
Air-Control
0/48
1/48
2/48
6/48
11/48
3/48
of
0
2
4
12.5
23*
6.25
RMSR
Pulmonary
(day_sj^ Consolidation
14. G
13.9
13.9
12.4
12.3*
12.8
1.44
1.56
1.40
2.58
3.02*
2.38
Significant difference (p <0.05) compared to air control.
Bacterial Clearance Rate
The clearance rates of viable Streptococcus sp. from lungs of mice exposed
to several different concentrations of A-C were examined. The t% values repre-
senting the time in hours required to clear 50% of viable bacteria from the
lungs were based on results of two replicate experiments. As seen in Table 13,
control mice held in air cleared bacteria more rapidly than any of the A-C
groups. Moreover, the increase of t% values appeared to be related to increase
in A-C concentration or the number of exposures.
43
-------
TABLE 13. CLEARANCE OF" VIABLE STREPTOCOCCUS FROM
LUNGS OF MICE EXPOSED FOR 3 HR TO ACID
MIST AND CARBON PARTICLE MIXTURE
Exposure t^
Concn. ,
Acid
100
300
200
mg/m3
Carbon
5
5
5
Number of
Exposures
1 1.35
1 1,56
5 1 . 58
i (hr)
Control
1.31
1.26
1,04
o o
A single 3-hr exposure to 100 mg/m acid mist and 5 mq/m carbon particle mix-
ture did not alter the rate of clearance of viable bacteria from the lungs.
This paralleled with absence of changes in mortality due to bacterial respira-
tory infection in response to a single 3-hr exposure to 100 mg/m3 A-C. Upon
a single 3-hr exposure to 300 mg/m3 acid mist and 5 mg/m3 carbon particle mix-
ture, the highest A-C concentration that did not result in toxicity deaths,
the t?2 was somewhat increased. Five daily 3-hr exposures to 200 mg/m^ A-C
produced a much greater increase in the time required to clear 50% of viable
bacteria from the lungs. For this exposure condition significant increases
in mortality due to bacterial"respiratory infection have been found, demon-
strating a relationship between the efficacy of clearance of viable bacteria
from the lung and the ability of mice to survive an infectious challenge. It
should be noted that markedly fewer bacteria were initially recovered (0 hr)
from lungs of mice exposed 5 times to 200 A-C than from control mice. Hence,
it is apparent that mice exposed to A-C inhaled fewer bacteria, but were not
able to clear them as efficiently as control mice which initially inhaled more
viable bacteria.
Alveolar Macrpphages
The specific capacity of the AM to handle inhaled bacteria is the result
of many factors including their metabolic activity, immunologic experience,
and the presence of pulmonary or other disease, In addition, certain environ-
mental factors have been shown experimentally to impair the functional state
of the cells, thus pulmonary antibacterial activity. Among these are hypoxia,
ethanol, acute starvation, corticosteroids, progressive renal disease, and
gaseous air pollutants (36).
44
-------
Since phagocytosis by AM is considered to be an important defense mechanism,
the number and the activity of these cells in lung washings have been used as
an indication of the capability of lungs to deal with foreign particles.
Mechanisms of clearance of viable bacteria from the lung include physical
removal of the bacterial material by the mucociliary apparatus and lymphatic
drainage system, and the phagocytosis which destroys the viability of the
organism, with or without removal from the lung, A number of studies (32,
37-42) have shown that physical removal of bacteria deposited in the lung
accounts for only a small fraction of the total reduction in viable counts re-
covered over a given period. Therefore the loss of viable microorganisms is
primarily a result of in situ bactericidal activity.
Green and Goldstein (32) described a method to measure in vivo inactiyatipn
of staphylococci in the lungs of mice by simultaneous determination of physical
removal and bactericidal activity. They quantitated intrapulmonary bacterial
inactivation in individual animals, rather than by the conventional method
determining group mean bacterial clearance. Animals are exposed to radiolabeled
live bacteria and the ratio of the viable bacterial count to the radioactive
count in each animal's lung provides the rate at which bacteria are destroyed
by the lung in a given time after infection. Decline in the radioactive count
in the lungs gives the rate of physical removal of bacteria by the mucociliary
system.
Rylander (42) used this technique to investigate the effects of exposure to
coal dust, sulfur dioxide (S02), and a combination of both on the pulmonary
bactericidal activity and on physical removal of inhaled bacteria in guinea
pigs. Exposure to 26.2 mg/m3 (10 ppm) S02 for four weeks did not affect the
bacterial elimination mechanisms, whereas 15 mg/m3 carbon black reduced the
bactericidal capacity with the implication that the capacity of the mechanisms
of phagocytosis had decreased. When the two agents were given simultaneously
they produced a synergistic effect decreasing the mucous flow, In studies in-
volving other air pollutants, it was found that exposure to ozone and NQ2
alone, and in combination, resulted in significant decreases in pulmonary
bactericidal activity (43-45) and no synergistic effect.
For this program, a limited number of experiments was conducted to examine
the effect of sulfuric acid-carbon mixtures on the cellular defense system in
the lungs of mice. In all of these experiments single exposures of 3 hr dur-
ation were used at concentrations of 100 mg/m3 of sulfuric acid and 5 mg/m3
carbon. Two approaches were taken; in the first, after exposure to A-C mist
the mice were killed and alveolar macrophages lavaged from their lungs were
examined for total and differential cell counts, viability, in vitro phagocytic
activity, and cell surface morphology. In the second approach, after A-C ex-
posure the mice were infected with 5. aureus by intranasal inoculation and
bacterial inactivation was determined in the lungs.
45
-------
Results on alveolar macrophage examinations are summarized in Table 14,
The table lists the viability, total cell count and phagocytic index values
determined on alveolar macrophages lavaged less than 1 hr and 24 hr after the
3 hr exposure to A-C mixtures. There are no changes in viability but a slight
trend toward decreased cell counts and increased phagocytic indices in macro-
phages of mice could be observed immediately as well as 24 hr after the ex-
posure. Differential counts, not shown in the table, were in the range of
97-98% alveolar macrophages with polymorphonuclear leukocytes and lymphocytes
under 2% and were not affected by the exposure.
Examination of the effect of A-C exposure on the surface structure of
alveolar macrophages showed that compared with air controls there were pro-
nounced changes in macrophages lavaged in less than 1 hr after A-C exposure,
as shown in Figures lOa through f..
TABLE 14. VIABILITY, TOTAL CELL COUNT AND PHAGOCYTIC INDEX OF ALVEOLAR
MACROPHAGES LAVAGED FROM MICE EXPOSED FOR 3 HR TO ACID-
CARBON MIST a
Interval Between
Acid-Carbon Mist Viability,% Total Cell Count x 105 Phagocytic Index, %
and Lavage Air A-C Air A-C Air A-C
<1
24
hr
hr
93.
93.
9
9
S3. 3
53.7
9.
9.
1
6
8.
8.
2
5
12.
10.
4
1
14.0
13.5
100 mg/m H2SO. and 5 mg/m carbon.
Figure lOa and b show macrophages typical of those obtained from control
mice. The intricate surface structure with numerous surface processes of the
cells extending in all directions can be seen especially clearly at higher
magnification. In the contrcl samples between 90 and 100% of the cells had
similar surface structure. In Figure lOc macrophages lavaged from mice less
than 1 hr after 3 hr exposure to 100 mg/m3 acid and 5 mg/m3 carbon are seen.
There are distinct changes in surface structure. The cells are excessively
spread out on the glass substrate, the surface processes have atrophied and
distinct holes can be seen in one of them. The damage is shown in more detail
in Figure lOd at higher magnification, while in Figure lOe a macrophage in
the transitional stage is shown: some of the normal surface structure is
still retained in the center, but it is increasingly spread and flattened out
near the periphery.
46
-------
Figure 10. Effects of 3 hr exposure to an aerosol of 100 mg/m of sulfuric acid
and 5 mg/m3 of carbon on alveolar macrophages of mice obtained by
tracheobronchial lavage from controls (a,b), from exposed mice in
less than 1 hr after exposure (c,d,e) and 24 hr after exposure (f).
The bar in the lower right hand corner represents 2 y.
47
-------
Counts made to obtain an estimate of the altered cell population in the
A-C exposed samples compared to the controls showed that 78% of the macrophages
had these distinctly different surface structural features.
When macrophages were lavaged from mice 24 hr after A-C exposure these
changes were no longer present and the morphology (shown in Figure lOf) as
well as the percent distribution (96%) was similar to the unexposed controls.
Bactericidal activity in the lungs of mice challenged with s. aureus less
than 1 hr and 24 hr after A-C exposure is shown in Table 15. The number of
mice used ranged from 25 to 30 per exposure group. The intranasally administered
dose of S. aureus was 1 x 10° bacteria in 0.05 ml of 0.1% peptone suspension;
the number of bacteria actually deposited in the lungs, as determined by radio-
active counts, ranged from 3.5 to 7.0 x 105. The results show a significant
increase in the percent of bacteria remaining, reflecting decrease in bac-
tericidal activity, in lungs of mice infected with S. aureus immediately after
exposure to A-C. However, mice infected 24 hr after the exposure cleared the
bacteria at a rate equal to that of the corresponding controls. These data
support the scanning electron microscopic observation of morphologic alterations
in the macrophages which were seen immediately after A-C exposure but not
24 hr later.
TABLE 15. BACTERICIDAL ACTIVITY IN LUNGS OF MICE EXPOSED FOR
3 HR TO SULFURIC ACID - CARBONa MIST
Bacteria Remaining (%) in Lungs
Interval Between
A-C Mist and
S. aureus Challenge
<1 hr
24 hr
of Mice Exposed
Geom.
25
30
Air
Mean
.7
.9
SE
3.8
3.8
Geom.
33
30
to:
A-C
Mean
.1*
.9
SE
4.0
3.8
3 100 mg/m H?SO» and 5 mg/m carbon.
Significant difference (<0.05) compared to control.
48
-------
Vaccine-Induced Immunity
The effects of exposure on preformed antibody as well as formation of
antibody during A-C exposure were examined using vaccine-induced immunity.
Briefly, 5- to 6-week-old male CDi mice were vaccinated subcutaneously with
approximately 370 CCA units of influenza A2/Oapan/170 virus vaccine. Control
mice received subcutaneous injections of PBS. Before or after vaccination the
mice were exposed daily for 3 hr, 5 days/week to 100 mg/m^ acid mist and
5 mg/m3 carbon particle mixture or ambient air. At 2, 8 and 16 weeks after
vaccination, one group of mice, consisting of 20 animals/group,was killed to
determine HI and SN antibody levels. The remaining mice were challenged by
the respiratory route with infectious influenza A2/Japan/170 virus. Two weeks
after challenge, the surviving animals were killed, lung consolidation scored,
and sera collected for serological assay to allow comparison of primary and
secondary response. All sera were pooled by groups of two.
At 2 weeks after vaccination (Table 16),exposure to A-C had no effect
on the secondary SN antibody response obtained following infectious challenge.
This is consistent with the mortality, survival time, and pulmonary consoli-
dation data which similarly showed no difference between vaccinated mice ex-
posed to air or A-C. However, the secondary HI antibody response was sig-
nificantly depressed. At 8 weeks after vaccination, exposure to A-C caused
a marked depression of the secondary SN antibody response following in-
fectious challenge,although the HI antibody response was unchanged. Simi-
larly, although the mortality and survival time were not affected, there was
a significant increase in pulmonary consolidation in mice exposed to A-C
compared to the ambient controls. At 16 weeks after vaccination,A-C exposure
resulted in slightly higher secondary HI and SN antibody titers, but no
change in the other parameters.
The effects of A-C mists on antibody formation are shown in Tables 17
and 18. Exposure to A-C or ambient air occurred immediately before or after
vaccination. At 2, 8, and 16 weeks after vaccination, sera were collected
and all remaining mice challenged with infectious homologous virus. In
both studies, there was generally no effect on mortality nor change in
the primary SN or HI antibody response when measured 2 weeks after vaccination
or in the secondary SN antibody titers following infectious challenge. How-
ever, a significant decrease in lesions and significantly depressed secondary
HI titers were seen when vaccination was followed by A-C exposure (Table 17),
while significantly increased pulmonary consolidation was observed when
vaccination was preceded by A-C exposure (Table 18), The primary SN antibody
titers measured 8 weeks after vaccination were, in both cases, significantly
higher in mice exposed to A-C. However, this pattern was not paralleled by
the primary HI antibody response which did not change significantly. Follow-
ing infectious challenge,the secondary SN antibody response was significantly
depressed in mice which had been exposed to A-C immediately before vaccina-
tion (Table 18), although no changes were observed in the other parameters. In
49
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addition, the HI antibody response was somewhat depressed. Both HI and SN
secondary responses were similar when the A-C exposure followed immediately
the vaccination. Mortality and survival times were unchanged, but lung
consolidation scores were significantly higher in the A-C exposure group.
At 16 weeks after vaccination,there were no differences in the primary SN or
HI antibody responses between the A-C or ambient exposure groups. Secondary
antibody titers following infectious challenge were slightly depressed in all
A-C exposure groups, the HI response significantly so in mice exposed to
A-C immediately before vaccination. Lung consolidation was also significantly
increased in both experimental groups.
In summary, 2 weeks after vaccination, the primary immune response
ranged from <8 to 8. Nevertheless, the vaccinated mice, whether exposed to
air or A-C mists, were protected to the same extent against infectious
challenge and showed good HI and SN secondary responses. Only the lung con-
solidation and secondary HI antibody data indicated differences between ex-
perimental and control mice in this time period. No pattern was noted with
lung consolidation. However, the secondary HI response was signifcantly
depressed when A-C exposure followed immediately after vaccination. This was
the same study in which lung consolidation was significantly depressed. When
A-C exposure preceded vaccination, the significant increase in pulmonary con-
solidation was accompanied by a slight increase in secondary HI antibody
response.
At 8 weeks after vaccination,a primary immune response was noted with
considerably higher SN titers in mice exposed to A-C mists. The secondary
HI and SN response in the experimental mice were in general similar to
the vaccinated air controls. Only when A-C preceded vaccination was the SN
secondary response significantly depressed compared to the vaccinated air
control (Table 18).
By 16 weeks after vaccination,no effects were noted on preformed anti-
body (Table 16). However some slight depression in the secondary response
was still present, as was a significantly increased pulmonary consolidation
and a significant increase in mortality in mice vaccinated, immediately ex-
posed to A-C mists, and 16 weeks later challenged with infectious virus.
Significantly increased pulmonary consolidation and marked decreases in both
HI and SN secondary antibody responses were noted in mice exposed to A-C
mists immediately before vaccination (Table 18),
In general, both the HI and SN antibody levels showed changes in the
same direction.
53
-------
Hamster Tracheal Organ Culture
Hamster trachea! organ culture was used to assess the effects of sul-
furic acid mist, carbon particles and mixtures of A-C on ciliary activity
and alterations in tracheal epithelium. Two approaches were used, namely
in vivo and in vitro exposure to the pollutants. Different combinations of
in vivo and in vitro exposure and/or maintenance were also examined to
determine the correlation between in vivo exposure damage and recovery and
the effects observed in organ culture.
In vivo Exposure and Maintenance
Tracheas from hamsters exposed for 3 hr to a range of 0.8-1.4 mg/m
sulfuric acid mist alone, mist combined with 1.1-1.9 mg/m3 carbon, and
1.1-1.9 mg/m3 carbon only were removed and cut transversely into a series
of rings immediately after and 24, 48, and 72 hr after exposure. Alterations
in cilia beating frequency were observed,and the rings were processed for
histological and scanning electron microscopic examinations. Each experiment
was performed in duplicate.
The data shown in Table 19 indicate that the means for the carbon treatment
group did not differ significantly from controls throughout the study. The
mean ciliary beating frequency for sulfuric acid mist alone and acid mist-carbon
measured immediately and 24 hr after exposure was significantly lower than
controls. At 48 and 72 hr the means of the acid mist treatment group were sig-
nificantly lower than the controls, carbon alone,and acid mist-carbon.
TABLE 19. CILIARY BEATING FREQUENCY IN TRACHEAS FROM
HAMSTERS AT <1, 24, 48 AND 72 HR AFTER
TERMINATION OF A 3-HR IN VIVO EXPOSURE TO
SULFURIC ACID HIST AND CARBON PARTICLES
0 Ciliary Beat Frequency
Concn.
H2S04
0
0
1.1
1.1
, , mg/m Time after Exposure,
Carbon < 1
0 1190a*
1.5 11903
0 1101b
1.5 1122&
24
11863
11553b
1107b
1106b
48
11773
11663
1027b
1133a
hour,
72
11903
11803
1090b
11473
Identical superscripts do not differ significantly
from each other (Duncan's New Multiple-Range Test),
54
-------
Immediately after exposure to sulfuric acid mist-carbon the trachea!
epithelium showed significantly greater cytological alteration than when exposed
to air, carbon,and air mist alone (Table 20). The pattern of damage to the
ciliated epithelium seen in the acid mist group was similar but less severe than
that seen after exposure to acid mist-carbon. The general appearance of the
epithelium no longer had its sharp outline; it was uneven and had a swollen
appearance. A number of epithelial cells appeared in clusters protruding into
the lumen. Immediately after exposure to carbon some ciliary disorganization
and, in some cases, loss of cilia and rounding and sloughing of epithelial
cells were seen. Tracheal epithelium of hamsters exposed to carbon and acid-
mist alone showed continuous recovery throughout the 72-hr period. By 72 hr
after exposure, only tracheal epithelium from the A-C treatment group still
showed significant damage as compared to the ambient air control.
TABLE 20. PERCENTAGE OF NORMAL EPITHELIUM OF TRACHEAS FROM
HAMSTERS AT <1, 24, 48 AND 72 HR AFTER TERMINATION
OF A 3-HR EXPOSURE TO SULFURIC ACID MIST AND
CARBON PARTICLES
3 Percentage of Normal Epithelium*
Concn., mg/m Time in Culture, hour
H2
1
1
:S04
0
0
.1
.1
Carbon <1
0
1.
0
1.
5
5
91
74
63
43
a*
b
b
c
24
94
84
70
46
a
ab
b
c
48
97
94
75
50
a
a
b
c
72
97
97
84
72
a
a
ab
b
**
Normal epithelium is defined as a smooth luminal surface
with beating cilia.
t
Identical superscripts do not differ significantly from
each other (Chi square Distribution Test).
55
-------
Histopathological examinations of normal hamster trachea immediately after
removal from the animal revealed a layer of pseudostratified, ciliated, columnar
epithelium, with a single layer of basal cells over a relatively thtn lamina
propria and submucosa. The epithelium of specimens exposed to carbon exhibited
moderate pathological alterations during the 72 hr observation period. The
surface of the tracheal epithelium 24 hr after exposure showed focal loss of
ciliated cells which became more diffuse over the next 48 hr. Exfoliation
resulting from dying cells resulted in some flattening of the epithelium. With
the alcian blue-PAS sequence, a moderate PAS reactivity demonstrative of a
high concentration of mucosubstance was observed. In tracheas removed <1 hr
after exposure to acid mist alone and A-C, severe loss of ciliated cells in
focal areas was observed. By 72 hr after exposure, the epithelium was composed
of one to three layers of basal cells, goblet cells and a severe loss of
ciliated cells in focal areas. A marked increase in PAS positive (acid) mucp-
substance was observed at 72 hr.
SEM examinations of tracheas of animals exposed in vivo to carbon or acid
mist alone, or acid mist-carbon showed similar epithelial surface alterations
as tracheas exposed in vivo and maintained in vitro. Description of these
alterations will be described in the following section (In vivo Exposure;
In vitro Maintenance),
These data indicate that acid mist alone and acid mist in combination
with carbon participates produce a significant cytotoxic effect in hamster
tracheal epithelium. The overall damage, i.e., depression of cilia beat
frequency and damage to the normal columnar orientation of the cells in the
epithelium, produced by the A-C mixture was greater than that produced by the
acid mist or carbon alone. This again indicates ciliary beating frequency
alone is an insufficient indicator of assessing toxicity and should be com-
bined with other observations and measurements,
In vivo Exposure; In vitro Maintenance --
3
The effects of sulfuric acid mist (0,8 to 1.8 mg/m ) combined with carbon
(1.1 to 1.9 mg/m3) as well as acid mist (0.8 to 1.8 mg/m3) or carbon (1-1
to 1.9 mg/m3) only were studied in hamster tracheal epithelium maintained
in vitro. Tracheas were removed immediately after a single 3-hr exposure to
the pollutants and processed as ring organ .cultures.
Immediately after removal the decline in cilia beating frequency for
all pollutant treatment groups (carbon and acid mist alone, and A-C mixture)
was significant when compared to ambient air control cultures (Table 21).
Within 24 hr after exposure, the ciliary beat frequency of the carbon anc
A-C treatment groups returned to control levels. At 24 hr only the mean
of the acid mist group was significantly different from the control. The
acid mist group showed a significant recovery at 48 hr following exposure.
56
-------
TABLE 21. CILIARY BEATING FREQUENCY IN TRACHEAL RING CULTURES
HELD IN VITRO AT <1 , 24, 48 AND 72 HR AFTER
TERMINATION OF A 3 HR IN VIVO EXPOSURE TO SULFURIC
_ ACID MIST AND CARBON PARTICLES
Ciliary Beat Frequency
______
Concn. , mg/m Time after Exposure, hour
H2S04
0
0
1.1
1.3
Carbon
0
1.5
0
1.5
<1
1204a*
1137b
1075b
1119b
24
1180a
1129ab
1094b
1116ab
48
11559
11343
1103a
1119a
72
1168a
1144a
1129a
1132a
Identical superscripts do not differ significantly from each
other (Duncan's New Multiple-Range Test).
The amount of damage to the epithelial layer immediately after exposure
to carbon and acid mist alone, or A-C combination was significantly different
from the ambient air controls (Table 22), Significant cytological alterations
continued in the acid mist and A-C groups throughout the 72 hr observation
period. Twenty-four hr after exposure, the cytological alterations in both
acid mist and A-C exposed groups were significantly different from either the
control or carbon treated groups. Morphological changes observed by light
microscopy resembled those described in the section on in vivo exposure and
maintenance.
Tracheal ring organ cultures also proved applicable to the examination
of the recovery from in vivo exposure to sulfuric acid mist and/or carbon.
Tracheas maintained in organ culture following exposure to acid mist or A-C
mixtures showed similar recovery patterns during the 72 hr observation period.
Organ cultures exposed to carbon only had significant alterations in the
ciliated epithelium compared to the controls, but were normal after 24 hr.
With the exception of one or two observations, this trend of recovery was
similar to that observed in the in vivo experiments previously described.
Pathological changes in the control and pollutant treatment groups during
the 72 hr observation period were also similar to those described in the
in vivo test system. There was a moderate loss of ciliated cells in focal
areas of the epithelium of tracheas exposed to acid mist and A-C mixture.
After 72 hr in culture, the cellular composition of trachea! epithelium was
the same as described for in vitro maintenance. The histochemical properties
indicated moderate increase in PAS reactivity (acidic mucosubstances).
57
-------
TABLE 22. PERCENTAGE OF NORMAL EPITHELIUM OF TRACHEAL RING
CULTURES HELD IN VITRO AT <1, 24, 48 AND 72 HR
AFTER TERMINATION OF A 3 HR IN VIVO EXPOSURE TO
SULFURIC ACID MIST AND CARBON PARTICLES
o Percentage of Normal Epithelium*
Concn.. mg/m Time in Culture, hour
H2S04
0
0
1.3
1.3
Carbon
0
1.5
0
1.5
<1
-,**
94a
73b
56C
50C
24
86a
75a
56b
52b
48
88a
87a
62b
62b
72
90a
87ab
74b
75b
Normal epithelium is defined as a smooth luminal surface
with beating cilia.
**
Identical superscripts do not differ significantly from
each other (Chi Square Distribution Test).
Hamster trachea! epithelial surface consists of three distinct cell types,
ciliated, microvillous and goblet cells, in that order of frequency, when
examined by scanning electron microscopy. Ambient air controls fixed immediate-
ly after removal from the animal showed evidence of cilia growth. In control
explants fixed at 24 and 48 hr in culture, cilia buds were being formed and
fully formed ciliated cells were present. In controls fixed after 72 hr in
culture, only fully developed ciliated cells were observed. There were areas,
however, where ciliated cells were almost completely absent. In these areas
microvillous cells were the predominant cell type. Hamster trachea! epithelium
removed and fixed immediately after exposure to carbon showed a network-like
structure with some mucus secretions covering the surface. At 24 hr, cilia
buds were present. The network-like structure was still visible along with
areas of sparsely ciliated cells. In cultures removed 48 hr after exposure
to carbon, good growth of cilia similar to that observed in the ambient air
controls could be found. At 72 hr, nonciliated cells were seen interspersed
within a layer of ciliated cells. Tracheal epithelium observed immediately
after exposure to sulfuric acid mist alone or with carbon showed a pattern
similar to that from carbon exposed animals except the damage was more severe.
Large areas of the network-like structures could be seen in conjunction with
only a few ciliated cells. In trachea! explants 24 hr after exposure, the
network-like structure was still present but ciliary buds were visible. The
acid mist and A-C groups showed new growth of ciliated cells along with heavily
ciliated areas 48 hr following exposure. Areas of dead cells with missing
microvilli were also visible. In trachea! explants 72 hr after exposure,
5
o
-------
new growth of ell fa was present but ft was not as extensive as that observed
in ambient air control or carbon treatment groups. The observations of
epithelial damage by scanning techniques are comparable to those described
above in the histological and cytological studies.
The results indicate that inhalation of acid mist in combination with
carbon, or acid mist or carbon alone, significantly depresses the cilia beating
frequency and damages normal ciliated epithelium. The damage produced by
A-C mixtures did not differ significantly from that produced by acid mist
only, but it was greater than that observed after exposure to carbon alone.
In vitro Exposure and Maintenance --
Studies were conducted to determine the concentration of sulfuric acid
that produces epithelial damage in vitro similar to that obtained from
in vivo exposure. Such microscopic alterations in the trachea! epithelium
were observed with a 1:10^ dilution of concentrated sulfuric acid. Tracheal
ring explants were exposed for 3 hr to this dilution of sulfuric acid,
150 yg/ml of carbon and an A-C combination. Dishes containing the pollutants
were incubated in a 5% C02 atmosphere chamber, placed on a rocker platform
rocking 10 cycles/min, Explants were exposed in 2 ml of simple medium (saline
and 0.05% glucose). A pH of 6.05 + 0.1 was maintained throughout the 3 hr
exposure.
A decline in ciliary activity was observed at <1, 24, 48 and 72 hr after
exposure (Table 23). The ciliary beating frequency of A-C group immediately
after exposure was significantly reduced from the control (no pollutant) and
acid and carbon alone. At 24 hr the means of both acid alone and A-C mixture
groups were significantly lower. By 48 hr after exposure to acid and A-C,
recovery from damage was evident and persisted through 72 hr.
The pattern of damage to the epithelium was similar to that in tracheal
tissue observed after in vivo exposure. Epithelial cells were swollen and
appeared in clumps, giving the luminal surface a moth-eaten appearance.
Damage resulting from exposure to carbon, acid and A-C could be detected <1 hr
after the 3-hr exposure period (Table 24). This persisted for 24 hr. At
48 hr after exposure to A-C or acid only, damage to the organ culture was
still evident. Recovery was seen 72 hr after exposure in all treatment
groups.
Histopathological and scanning electron microscopic alterations similar
to those seen and described in the section titled "in Vivo Exposure; In Vitro
Maintenance" were also present in the tracheal organ cultures exposed to
sulfuric acid and carbon alone, or A-C mixture. As in the in vivo exposure,
sulfuric acid alone and the A-C mixture produced similar effects. Recovery
of the epithelium from the A-C mixture again was similar to that observed in
explants exposed to acid alone. Carbon-exposed explants recovered to a level
similar to the controls. Statistically, there was a clear correlation between
initial damage to tracheal cells in organ culture and their recovery, and the
ability to reproduce the effects observed in hamsters after in vivo exposure.
59
-------
TABLE 23, CILIARY BEATING FREQUENCY IN TRACHEAL RING CULTURES
AT <1, 24, 48 AND 72 HR AFTER TERMINATION OF A 3-HR
IN VITRO EXPOSURE TO SULFURIC ACID MIST AND
CARBON PARTICLES
H2S04
(dilution)
0
0
1:106
1:106
Carbon
0
150
0
150
Cil
Time
<1
1195a*
1140ab
1146ab
noib
iary Beat Frequency
after
24
1191a
1135ab
1112b
1091b
Exposure, hour
48
1143a 1
1144a 1
1115a
1107a I
72
I128a
I131a
!106a
mia
Identical superscripts do not differ significantly from each
other (Duncan's New Multiple-Range Test).
TABLE 24. PERCENTAGE OF NORMAL EPITHELIUM IN TRACHEAL RING
CULTURES AT <1, 24, 48 AND 72 HR AFTER TERMINATION
OF A 3-HR IK VITRO EXPOSURE TO SULFURIC ACID AMD
CARBON PARTICLES
H2S04
(dilution)
0
0
1:106
1:106
Carbon
(yg/ml)
0
150
0
150
Percent
Time
<1
38a**
72b
57b
59b
of Normal
Epithel
ium*
in Culture, hour
24
84a
69ab
61 b
63b
48
89a
74ab
71b
70b
72
90a
80a
78a
75a
Normal epithelium is defined as a smooth luminal surface with
beating cilia.
**
Identical superscripts do not differ significantly from each
other (Chi Square Distribution Test).
60
-------
The question was raised as to whether the damage to the trachea] ciliated
epithelium was due tp sulfun'c acid itself or whether the alterations were
pH related. To answer this question, hydrochloric acid in sufficient dilution
to adjust the pH of a saline and a 0.05% glucose medium to 5.0 was made.
Explants were exposed to this medium for 3 hr and observed with the phase
microscope for ciliostasis or gross exfoliation. Hydrochloric acid sufficient
to reduce the.pH to 5.0 caused approximately 50 to 75% ciliary arrest but did not
alter normal cellular morphology. When explants were transferred to fresh
culture medium (CMRL 1066), the cilia resumed beating at a normal frequency.
These results indicated that pH 5.0 produced no lasting morphological effects
and that other factors are responsible for toxicity produced by sulfuric acid.
LONG-TERM EXPOSURE TO CARBON AND A-C MIXTURE
Long-term low concentration studies were conducted to determine the
effect of exposure to carbon or A-C mixtures on normal (uninfected) mice and
on the response to challenge with infectious influenza virus. Male CDi
mice, 4 to 5 weeks old at the initiation of the study were exposed 3 hr/day,
5 days/week to air, carbon only (mean concentration + standard deviation,
1.5 i_ 0.4 mg/m3 carbon) or A-C (1.4 +_ 0.4 mg/m3 acid mixed with 1.5 +_
0.4 mg/m3 carbon). Body weight and temperature data were collected weekly
from the same mice from each exposure condition throughout the study. To
determine immunoglobulin concentrations a separate group of mice was bled
orbitally after 1, 4, 12, and 20 weeks of exposure. Additional parameters
studied after 4, 12, and 20 weeks exposure of uninfected mice were Jerne
plaque assay for number of plaque-forming cells in the spleen; examination
of the respiratory tract by SEM and conventional microscopy; examination of
total cell counts and viability of alveolar macrophages CAM) lavaged from the
lungs; and examination of in vivo bactericidal capacity in the lungs. Exam-
ination of the effects of 4, 12, and 20 weeks exposure to the pollutant on
the host's response to challenge with infectious influenza virus included
observation of mortality, mean survival time and lung consolidation.
Body Weight and Temperature
During the 20 weeks of pollutant exposure 24 individually marked mice
in each exposure group (air, carbon, and A-C) were weighed weekly and rectal
temperatures recorded. All three groups were similarly caged and deprived
of food and water during the 3-hr exposures. The mean body temperatures
ranged between 36.9 and 38.10C and those of experimental mice were not sig-
nificantly different from the air controls. All three groups of mice gained
weight consistently throughout the 20 weeks. However, the average weight
of mice exposed to A-C was consistently lower than the air controls, with
mice exposed to carbon intermediate (Figure 11). All three groups were
essentially the same weight at the beginning of the study. The mice exposed
to the pollutant showed an initial lag in weight gain, but eventually approxi-
mated weights of control mice. The 20 week exposure interval can be divided
into two phases related to the age of the mice: the interval up to 8 weeks
representing a rapid growth and from 9 to 20 weeks during which growth rate
of control mice decreased and average body weights reached a plateau. Re-
gression analysis shows a markedly lower rate of growth in both the carbon
and A-C groups during the initial rapid growth period.
61
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Tissue Toxjcity
Nasal cavities (Figure 12), tracheas (Figure 13a,b) and lungs (figure 13c,
d,e,f) from experimental and control animals were prepared for SEM and conven-
tional histopathologic examinations. Su&tle changes in the respiratory epithelium
were detected by SEM examination of tissues collected after the final pollutant
exposure. The normal respiratory tract surface as seen by SEM was described
previously in this report and by others (46).
At 12 weeks exposure to carbon only, more squamous cells than normal
were seen sloughing at the external nares (Figure 12b), but the remaining
nasal epithelium was normal. Tracheas showed many mucous cells and some
dying cells with loss of microvilli. Cells in the bronchus appeared normal.
However, some areas of congestion were found in the alveoli. These appeared
as uneven raised areas at low magnification (Figure 13d). At higher magni-
fication the areas appeared to be thickened and fused alveoli with enlarged
alveolar pores (Figure 13f). Such areas were never found in the control
animals (Figures 13c,e).
Although similar, the damage was somewhat more severe in animals ex-
posed to A-C for 12 weeks. As in the carbon group, an increased number of
sloughing cells was seen at the external nares. Some non-ciliated cells were
dying (Figurel2d) or had small holes around their edges. Dying cells in the
trachea had small holes around the cell borders (Figure 13b), Bronchus cells
were normal, but alveoli were fused as in samples from mice exposed to carbon
only. Such areas were more extensive in animals exposed to A-C than in
carbon-exposed animals.
At 20 weeks the damage was similar to that seen at 12 weeks, but to a
lesser degree. In both A-C and carbon-exposed animals, squamous cells were
sloughing more than usual, but the remaining respiratory epithelium was
normal. Tracheas showed some mucus, but no damage. Lung bronchus cells
were normal. Lung alveoli showed the same fusing as before, but to a lesser
degree.
Thus, subtle changes were detected by SEM in the epithelium of the
respiratory tract of mice after long-term exposure to carbon or A-C. In
general, no changes were seen at 4 weeks. At 12 weeks, increased sloughing
of squamous cells, some dying cells in the nasal cavity, and fused and
thickened lung alveoli were seen in both A-C and carbon-exposed animals.
In addition, holes were seen around the borders of the dying cells in the
nasal cavities of mice exposed to A-C. Some dying cells with holes were
also seen in tracheas from these animals. At 20 weeks, sloughing cells in
the nasal cavity and fused and thickened alveoli were similar in both ex-
perimental groups, The changes were less extensive than at 12 weeks.
63
-------
Figure 12. Nasal cavity of contro" and test mice, (a) sloughing of
squamous cells at external nares in normal mice; (b) increased
sloughing of squamous cells after 12 weeks exposure to carbon;
(c) midseptum area showing ciliated cells and microvilli of
normal non-ciliated cells; (d) dying non-ciliated cells showing
loss of microvilli after 12 weeks exposure to A-C.
64
-------
Figure 13. Trachea and lungs of control and test mice, (a) ciliated and non-
ciliated cells of normal trachea; (b) dying non-ciliated cell shows
holes and missing microvilli after 12 weeks exposure to A-C; (c)
normal cross-section of lung; (d) uneven raised areas in lung after
12 weeks exposure to carbon; (e) normal alveoli showing thin alveolar
walls and small pores; (f) fused and thickened alveoli and enlarged
pores in lungs exposed to carbon for 12 weeks.
65
-------
Conventional histopathologic examination of tissues from the respiratory
tract indicated the presence of carbon-loaded macrophages throughout the
lung parenchyma in all samples exposed to carbon or A-C, No other changes
were found, again demonstrating the greater sensitivity of SEM examination for
detection of subtle changes in the respiratory epithelium.
Hematology and Clinical Chemistry
Ten individually marked mice from air, carbon only, and A-C exposure
groups were designated for repeated orbital bleeding after 0,1,4,8,12,16 and
20 weeks exposure (3 hr/day, 5 days/week). Hematological parameters were
measured and individual sera were saved for assay of immunoglobulin concen-
trations. Additional mice were sacrificed after the same exposure intervals
and blood chemistry assays were done on the individually collected sera.
Data for initial and 20 week values of selected hematological parameters
are shown in Table 25. Clinical chemistry data are summarized in Table 26.
Although the levels fluctuated during the exposure they did not vary sig-
nificantly from normal values reported for mice. With our necessarily small
sample size, the variation observed was too great to allow detection of trends
in the experimental data.
Immunoglobulin Quantitation
Among the five main classes of immunoglobulins IgM, IgG, IgA, IgD and
IgE, the first three are involved in defense against infection. IgM is the
first antibody to appear in the serum of an animal after stimulation with a
protein antigen. Each molecule contains 10 antigen binding sites and is an
efficient antibody in certain in vitro tests and in certain aspects of anti-
microbial immunity. IgM makes up 5 to 10% of the total antibody protein in
serum and is usually supplemented by IgG antibody early in the response to
antigen stimulation. At least 80% of the serum antibodies directed against
the antigen belong to the class IgG which has several subclasses. In addition,
IgG is the only immunoglobulin to cross the placenta. IgA is found in the
serum as well as in saliva, colostrum, tears, and nasal and bronchial secre-
tions. Secretory IgA is the predominant immunoglobulin in external secretions
which play an important role in the protection of mucous membranes and conjunctiva.
Serum immunoglobulin concentrations have been quantified to determine which
of the immunoglobulin classes are affected by the exposure to pollutant. The
major site of air pollutant action is the lungs, making the secretory immuno-
globul ins which protect the respiratory membrane a target of interest. Un-
fortunately, there are many technical difficulties involved in obtaining
nasal and bronchial washings from mice. However, study of serum immunoglobulin
levels in experimental animals was still of interest since changes in con-
centration of serum immunoglobulins in man have been reported in studies
of several chronic lung disorders. Elevated levels of serum IgA with nor-
mal IgM and IgG concentrations were observed by Biegel and Krumholz (47)
in adults with chronic obstructive pulmonary emphysema. The mean
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IgA and IgG levels in the sera of patients with fanner's lung disease were
found to be significantly higher than those of normal individuals (43).
Similarly,occupational exposure of workers to 0.5-2.7 ppm N02 for 6 to 8
hours/day for up to 6 years resulted in elevated levels of serum IgA and
IgM but decreased IgG concentrations in most cases (49).
In the present study, individual sera from 8 to 10 marked mice exposed to
either air, carbon only, or A-C mixtures were collected at selected intervals and
quantitated for immunoglobulin concentrations by radial immunodiffusion.
After 1 week exposure, serum levels of IgG] were significantly decreased
in both the carbon and A-C groups compared to controls held at ambient
conditions (Table 27). Levels of IgG2b and ^M were significantly increased
compared to the controls. Serum IgA levels were not affected.
After 4 weeks exposure to A-C, serum levels of IgG], IgG2a> and IgM
were all significantly depressed compared to the controls. Again, IgA levels
were not affected. Immunoglobulin levels in mice exposed to carbon only were
not significantly different from the controls.
After 12 weeks, serum levels of IgG2a and IgM remained significantly de-
pressed in mice exposed to A-C compared to air controls. The other immuno-
globulin classes were not signficantly affected.
At the end of 20 weeks exposure to the pollutants, IgM levels continued
to be suppressed in mice exposed to A-C. IgG2b was significantly increased
in both A-C and carbon mice and IgA was reduced for the first time in mice
exposed to both A-C and carbon, with the suppression being greatest in the
mice exposed to carbon only.
In summary, concentrations of serum immunoglobulins can be measured
and represent a sensitive parameter which is altered by exposure to low con-
centrations of carbon and A-C. In general, the changes observed were more
marked in mice exposed to A-C than in those exposed to carbon alone. No
changes were seen in serum levels of IgA until after 20 weeks of pollutant
exposure when some depression was seen. An immediate depression in IgG]
levels was seen which persisted in the A-C exposure group through 4 weeks of
pollutant exposure. There was an initial slight increase in Ig2a levels at
1 week followed by depressed levels in A-C exposed mice through 12 weeks
of pollutant exposure. Serum levels of IgG?b were significantly elevated
after 1 week exposure to the pollutants (in contrast to depressed IgG]
levels), were similar to controls during the intermediate intervals of ex-
posure and were significantly elevated over the air controls at 20 weeks.
IgM levels were initially increased at 1 week in both experimental groups
after which they were significantly depressed only in the A-C exposed pice
and remained so for the duration of exposure.
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Because a direct relationship between the levels, of immunoglohulins and
chronic pulmonary diseases is not established, it cannot be stated whether
increases in concentrations of immunoglobulins reflect cause or effect. There
have been no other results reported on the effects of exposure to sulfuric
acid mists, with or without particulates, on serum immunoglobulin concen-
trations. Disruption of the elastin and collagen framework of the lung was
reported for guinea pigs exposed to N02 (50). Such tissue damage could alter
lung proteins so that they become antigenie and thereby stimulate the pro-
duction of serum antibodies. An increase in titer of serum antibodies to
normal lung proteins was observed in guinea pigs exposed to N02- Studies at
I IT Research Institute (51) indicate that mice exposed to N02 for 12 weeks
showed a marked decrease in concentrations (mg/ml) of serum IgA, and an in-
crease in serum IgM, IgGi and IgG£ immunoglobulins.
Primary Antibody Response of Spleen Cells
The primary response to SRBC, as measured by the Jerne claque assay, is
the production of specific IgM antibody (52) and is a quantitative reflection
of the ability of the animal to respond to an antigenic stimulus. In the
Jerne plaque assay, lymphoid cells are incorporated together with a dense
population of red blood cells in an agar layer. During incubation, each
lymphoid cell that releases antibody causes the red cells near the lymphoid
cell to become sensitized; the sensitized blood cells are then lysed, in the
presence of complement, forming a "clear area" or plaque against a background
of red cells. The effect on the primary response of spleen cells from mice
exposed to the pollutants was determined after 4, 12, and 20 weeks of pollu-
tant exposure. The SRBC were injected after the final pollutant exposure and
the individual spleens were removed and tested four days later. The results
(Table 28) are reported as plaque-forming units (PFU) per total spleen cell
population.
TABLE 28. LONG-TERM EXPOSURE TO ACID MISTS:
JERNE PLAQUE ASSAY FOR PRIMARY ANTIBODY RESPONSE
Plaque-forming cells/spleen x
following exposure to:
Duration of
Ai
exposure, wk Mean
4
12
20
2
13
32
.14
.15
.64
r
S
1.
3.
6.
-E
01
09
36
Carbon
Mean
7,
13.
16.
41*
00
63*
SE
1.
2.
6.
22
57
76
Acid-Carbon
Mean
4.
18.
4.
72
21
* a
36 'a
SE
1.41
4.44
1.62
Significant difference (p<0.05) compared to air control.
a Significant difference (p^O.05) compared to carbon
exposure.
71
-------
As expected, during the interval of exposure the number of PFU/spleen in-
creased in normal (air-exposed) mice from 2.14 x 10^ PFU/spleen at 4 weeks
to 32.64 x 104 PFU/spleen after 20 weeks. After 4 weeks of pollutant ex-
posure, an early stimulation of the response to SRBC was seen in mice ex-
posed to either carbon alone or the A-C mixture. The increase in PFU/spleen
in carbon-exposed mice was significant compared to the control mice, but not
statistically different from the mice exposed to A-C. At 12 weeks after the
beginning of pollutant exposure there were no differences between the experi-
mental groups and controls. After 20 weeks of exposure there was a signifi-
cant depression of PFU/spleen in mice exposed to carbon and even greater
depression following exposure to A-C.
This appears to follow a classic pattern in which a short exposure to a
stress causes an increased immune response and long-term exposure results
in depression of that response. Thomas, Holt and Keast (53-57) have ob-
served such biphasic changes in systemic spleen and regional lymph node
responsiveness following exposure to tobacco smoke, with moderate exposure
periods producing marked enhancement and prolonged exposure producing
severe depression. Zarkower (58-59) noted a decrease in the overall ability
of animals to form antibody after chronic exposures to carbon alone or in
combination with S02- He observed an enhancement of antibody production in
mediastinal lymph nodes after 102 and 135 days of exposure, but the effect
was reversed by 195 days. A similar pattern was found in our studies with
carbon and A-C exposures. Initially, there was an enhanced antibody response.
After 20 weeks exposure, A-C exposure resulted in the greatest decrease in
PFU/spleen, significantly less than both the control and carbon exposures.
Serum immunoglobulin levels and the plaque assay provide quantitative
measurements of IgM production. In this study, the plaque assay measures
response of spleen cells to antigenic stimulation while the other method
records serum IgM levels in the absence of antigenic stimulation. There is
some correlation between the results obtained by these two methods. It
appears that the ability to respond to antigenic stimuli is not depressed
as rapidly as background levels of serum IgM. The number of PFU/spleen was
still increased after 4 weeks of exposure to pollutant when background IgM
levels were depressed. The response of spleen cells to antigenic stimulation
diminished from increased PFU/spleen after 4 weeks to marked depression after
20 weeks exposure. For both parameters, the more marked initial en-
hancement and subsequent depression was observed for the more severe stress
of A-C exposure.
Alveolar Macrophage
A limited number pf experiments to examine the effect of A-C mixtures on
the cellular defense system in the lungs of mice was conducted. Two appro-
aches were taken; in the first, the mice were killed after pollutant exposure
and alveolar macrophages lavaged from their lungs were examined for total
cell counts and viabilities. In the second approach, the mice were infected
with radioactively labeled K. vneumoniae by intranasal inoculation after the
final pollutant exposure and bacterial inactivatlon was determined in the
lungs.
72
-------
After 4 weeks of exposure,viabilities and cell counts were done only on
macrophages from one mouse from each group, The viability in the three ex-
posure groups ranged from 93.5 to 95.5%. Total cell counts ranged from 5,7
x 10^ to 7.1 x 105 cells. Table 29 shows the viability and total cell count
values determined on alveolar macrophages lavaged from five to nine mice
less than 1 hr after the last of a series of 12 or 20 weeks of exposure to
carbon or A-C. There were no changes in viability at any of the exposure
intervals. A trend toward decreased cell counts was observed in both the
carbon and A-C groups after 12 weeks of exposure. No differences were ob-
served after 20 weeks of pollutant exposure.
TABLE 29. LONG-TERM EXPOSURE TO ACID MISTS;
VIABILITY AND TOTAL CELL COUNT OF ALVEOLAR MACROPHAGES
LAVAGED FROM MICE
Duration of
exposure, wk
12
20
Number of
mice/group
9
5
Viability^?
Air
93.5
94.4
Carbon
93.7
92.4
A-C
92.8
93.8
Total
Air
8.9
11.1
Cell Count xlO5
Carbon
7.0
11.4
A-C
7.8
12.8
The effect of long-term carbon and A-C exposure on the bactericidal
activity in the lungs of mice challenged with K. pnewnon-iae less than 1 hr
after 4, 12, or 20 weeks of pollutant exposure is shown in Table 30. The
number of mice used ranged from 5 to 21 per exposure group. The intranasally
administered dose of K. pneumoniae was 2.5 x 105 bacteria in 0.05 ml of 0.1%
peptone water; the number of bacteria actually deposited in the lungs, as
determined by radioactive counts, ranged from 1.9 x 103 to 5.1 x 105. The
results show a significant increase in the percent of viable bacteria re-
maining, reflecting decrease in bactericidal activity, compared to the air
controls in lungs of mice infected with K. pneimoniae immediately after 4
or 12 weeks exposure to either carbon alone or to A-C. At these exposure
durations the percent bacteria remaining in the lungs was greater in mice
exposed to A-C than to carbon alone, but not significantly so. The most
significant factor in the decreased bactericidal activity in the lungs of
these animals is apparently the loading of the macrophages with carbon. The
slightly increased percent bacteria remaining in the lungs of mice in the ex-
perimental groups infected with K. pnewnoniae immediately after 20 weeks of
pollutant exposure was not statistically significant.
73
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TABLE 30. LONG-TERM EXPOSURE TO ACID MISTS;
BACTERICIDAL ACTIVITY IN LUNGS OF MICE
Bacteria Remaining (%} in
Lungs of Mice Exposed to:
Duration of
exposure, wk
4
12
20
Ai
Mean
40.1
15.5
51.0
r
SE
2.9
2.5
4.7
Carbon
Mean SE
53.8* 3.0
24.0* 1.9
58.1 6.2
A-C
Mean SE
55.3* 4.7
29.7* 2.3
53.4 5.1
*
Significant difference (p^0.05) compared to
air control.
Infectious Challenge
After 4 and 20 weeks of exposure to the pollutant, mice were challenged
within 1 hr of the final exposure with airborne influenza A2/Taiwan virus.
The lung lesions of surviving mice were scored 14 days later.
Although the infectious challenge dose was low, there was no enhancement
of mortality in mice exposed for 4 weeks to either carbon alone or to the
A-C mixture compared to air controls (Table 31). Pulmonary consolidation did
not increase nor was there a decrease in mean survival time.
TABLE 31. LONG-TERM EXPOSURE TO ACID MISTS:
RESPONSE OF MICE CHALLENGED WITH INFLUENZA
A2/TAIWAN VIRUS
Duration of
exposure, wk
4
20
Experimental
Condition
Air
Carbon
A-C
Air
Carbon
A-C
Mortal
D/T
16/164
10/164
13/166
48/133
46/137
61/136
ity
%
10
6
8
37
34
45
RMSR
(days)
13.5
13.7
13.7
12.2
12.3
11.7*
Pulmonary
Consolidation
2.05
1.62
1.70
3.11
3.07
3.43*
Significant difference (p <0.10) compared to both air control
and carbon,
74
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Increased mortality, markedly decreased (p <0.10) mean survival time,
and increased pulmonary consolidation compared to both the air controls
to the mice exposed to carbon only was seen in mice exposed to A-C for 20
weeks. Hence, changes in the response to infectious challenge were detected
following 20 weeks exposure to low concentrations of A-C mixtures.
Results from the 12-week exposure study suggest additional experiments
which might yield information on the response of mice with a low-grade or
latent infection to pollutant stress or to subsequent infectious challenge.
Mice were exposed as in the other experiments. However, difficulties were
encountered in controlling the relative humidity (RH) in the exposure chamber.
Despite our efforts, which included increasing the dry air flow through the
chamber and using trays of calcium chloride in the bottom of the chamber to
lower the atmospheric RH to that which should be in equilibrium with a satu-
rated solution of calcium chloride, the RH frequently reached 80 to 85% in the
chamber by the end of a 3-hr exposure. This was undoubtedly an additional
stress on the animals which appeared very ruffled and damp when they were re-
moved after the exposure. This was especially noticed in the group exposed
to carbon alone. Some of the mice in this group began to appear ill, and random
deaths were recorded although they had received no infectious challenge. As
a check of whether this simply represented the effect of exposure to pollutant
or the activation of some latent infection, a few "sick" animals were sacri-
ficed and the sera tested for antibody to various murine viruses. All three
mice sacrificed had significant antibody titers against Sendai virus (1:10,
1:20, and 1:40). Latent infection with Sendai virus is known to cause
problems in some mouse colonies and stressful conditions may activate the virus
and kill the mice. Our control mice appeared healthy during these studies
and thus no control mice were sacrificed for determination of murine antibody.
Twenty-one animals died in the carbon group during the last 3 weeks of ex-
posure while no deaths occurred in the A-C or air groups. The mice were
challenged with airborne influenza virus after exposure to the pollutant for
12 weeks. The challenge dose was very high. However, the results are none-
theless of some interest.
Experimental
Condition
Mortality
D/T %
RMSR
(days)
Pulmonary
Consolidation
Air
Carbon
A-C
133/163
144/148
147/162
82
97
90
9.3
7.8
8.7
4.56
4.95
4.81
75
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Even with the high virus challenge, the increase in mortality and pulmonary
consolidation in mice exposed to carbon or A-C was statistically significant.
The increase in the carbon group (the "sick" group) is also significant over
the A-C group. Apparently, the presence of a low grade or latent infection
in animals exposed to the pollutant stress leaves them especially susceptible
to a subseguent infectious challenge. Other studies have shown that con-
ventional (not SPF) animals exhibit a lower tolerance for air contaminants
than SPF animals (56).
76
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
FPA-finn/i-7a-n3fi
2.
3. RECIPIENT'S ACCESSI Of* NO.
4. TITLE AND SUBTITLE
RESPIRABLE PARTICLES AND MISTS IN MOUSE PULMONARY
INFECTIVITY MODEL. Effect of Chronic or Intermittent
Exposure
5. REPORT DATE
1Q76
|V|gy
6. PERFC
ORMING ORGANIZATION CODE
7. AUTHOR(S)
J.N. Bradof, J.D. Renters and R. Ehrlich
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
10. PROGRAM ELEMENT NO.
IIT Research Institute
10 West 35th Street
Chicago, IL 60616
1AA601
11. CONTRACT/GRANT NO.
68-02-1717
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park. N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
RTP, NC
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The effects of respirable-sized sulfuric acid mist or mixtures containing acid
mist and carbon particles (A-C) on the susceptibility to bacterial and viral
respiratory infection were studied in mice and hamsters. Both species showed
mortalities upon single 3-hour exposure to 600 mg/m3 but not 400 mg/m3 acid mist.
Scanning electron microscopic examination indicated that the most severe changes,
including emphysemic-like areas in alveoli, were found after five daily 3-hour
exposures to 200 mg/m3 A-C. Significantly increased mortality and decreased bacterial
clearance from lungs were also observed in mice challenged with Streptococcus sp..
Significantly increased mortality and pulmonary consolidation, with concomitant
decreased survival time, occurred in mice challenged with influenza virus aerosol
exposed to 50 mg/m3 A-C, 3 hr/day. 5 days/week for 4 weeks. Depressed secondary
immune responses, as measured by serum antibody levels, were observed in various
groups of vaccinated mice exposed to pollutant. The effects of long-term exposure
mixtures of approximately 1.4 mg/m3 sulfuric acid mist and 1.5 mg/m3 carbon particles
well as carbon only were determined. Significant alterations of immunoglobulin
concentration, depression of primary antibody response in spleen cells and decreased
resistance to respiratory infection as measured by mortality, survival time, and
pulmonary consolidation after 20 weeks of exposure were evident.
and
to
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
sulfuric acid
particles
immunity
respiratory infections
infectivity model
06 F, T
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
92
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
82
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