MANGANESE MONOGRAPH
ACUTE PULMONARY TOXICITY OF MANGANESE OXIDE
by
Bernard Adkins, Jr.
Health Effects Research
Northrop Services, Inc.
Research Triangle Park, N.C. 27709
and
Donald E. Gardner, Ph.D.
Biomedical Research Branch
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
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MANGANESE MONOGRAPH
ACUTE PULMONARY TOXICITY OF MANGANESE OXIDE
by
Bernard Adkins, Jr.
Health Effects Research
Northrop Services, Inc.
Research Triangle Park, N.C. 27709
and
Donald E. Gardner, Ph.D.
Biomedical Research Branch
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
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MANGANESE MONOGRAPH
ACUTE PULMONARY TOXICITY OF MANGANESE OXIDE
I. Introduction
II. Experimental Procedures
A. Experimental Animals
B. Chemicals
C. Inhalation Exposure Systems
D. Manganese Quantitation Procedures
E. Toxicological Testing: In Vivo Models
1. Infectivity Studies
2. Streptococcal Clearance Studies
3. Septicemia Studies
4. Immunology Studies
F. Toxicological Testing: Iri Vitro Models
1. Cytological Measurements: Endotracheal Saline Lavage
Obtained Pulmonary Cells
a. Total Cell Yield
b. Differential Cell Counts
c. Viability: Dye Exclusion
d. Phagocytic Capability
e. Intracellular ATP
f. Total Protein (Lowry)
g. Acid Phosphatase Activity
h. Lactic Acid Dehydrogenase Activity
2. Inflammation Measurements
a. Wet/Dry Tissue Ratios
b. Lavage Supernatant Total Protein (.Lowry)
III. Results: Post-exposure Data
A. Manganese Lung Deposition Rates
B. Manganese Lung Clearance Rates
C. Manganese Tissue Distribution Data
D. Infectivity Studies
1. Relative Mean Survival Rates
2. Streptococcal Lung Clearance Rates
3. Streptococcal Blood Clearance Rates
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OUTLINE (continued) - Page 2
E. Effects of Manganese Inhalation on Various Murine Immune Mechanisms
F. Biochemical Cytology Studies
1. Lung Edema: Lavage Supernatant Total Protein
2. Cytotoxicity Data
3. Enzyme Assays
IV. Conclusions/Discussion
V. Bibliography
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INTRODUCTION
The American Conference of Governmental Industrial Hygienists (1971) set
the Threshold Limit Value (TLV) for manganese industrial exposure at 5 mg
Mn/m for an 8-hour day. This TLV is considered to represent a low margin of
safety for susceptible individuals in occupational areas (Scientific and
Technical Assessment Report of Manganese, 1975). Case reports by Whitlock
and coworkers (1966) and Tanaka and Leiben (1969) indicated that exposure
levels to lower than 30 mg/m manganese fumes produced chronic poisonings.
Industrial exposures to different forms of manganese have resulted in
two major clinical findings: chronic manganese poisoning with central nervous
system involvement, and a manganic pneumonia. Rodier (1955) documented
cases of chronic manganese poisoning in mining processes of manganese ore.
Most of the toxicological effects resulting from the exposure to manganese
appear to result from prolonged inhalation, and are reversible when the person
is removed from the exposure source. Exposure to manganese dust produces
croupous pneumonia, which primarily affects one lobe of the lung, with a
corresponding high mortality rate (Davies, 1946).
The type of manganese ion present and the corresponding oxidation state
appears to dictate the severity of animal toxicity observed with manganese
compounds. Manganese oxides, such as MnO, MnO2, Mn 0 , and Mn O., have
demonstrated toxicities in rats, but appear to be less toxic than the higher
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1-2
oxide forms. Jonderko and Szczurek (1969) concluded that manganese poisoning
directly affects functional portions of the brain. Similar findings were
reported by Chandra and Srivastava (1970) indicating that the severity of
the degenerated neurons was directly related to the concentration of manganese
in the brain. Borisenkova (1967) reported the inhalation toxicity of various
manganese ore dusts on motor reflex response. The primary effect summarized
in these studies again indicated central nervous system pathology. Additionally,
the rats in this study developed interstitial chronic pneumonia after four
months inhalation exposure to all manganese ore dusts tested.
In a series of reports, the Ethyl Corporation (1972, 1974) suggested
the maximum contribution to airborne manganese from the use of MMT in fuels
would be 0.05 mg/m . A chronic inhalation study involving squirrel monkeys
and rats with an exposure regimen of 22 hours/day, 7 days/week at exposure
levels of 10, 100, and 1000 mg/m was reported by Ulrich and Van Petten (1975).
Data from these studies involving exposure to respirable Mn 0. aerosols (0.2 pm
MMED) indicated there were no exposure-related changes to the hematopoietic
system, central nervous system, respiratory system, and other tissues pathologi-
cally examined. Moore et al. (1975) reported no gross changes in general
condition or appearance were observed in rats and hamsters following chronic
exposure to automotive emission resulting from the combustion of gasoline
with the MMT additive. Additionally, microscopic examination of animal tissues
indicated changes related to exhaust emission and not solely attributable to
the manganese which was maintained at an average manganese aerosol particulate
concentration of 117 pg/m for 56 consecutive days. Similarly, Griffin and
Coulston (1978) reported results from chronic exposure of rats and Rhesus
monkeys daily for 23 hours/day to 100 yg Mn/m combusted from MMT. These studies
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1-3
indicated no signs of toxicity, changes in various clinical parameters, or
changes in morphology, either gross or microscopic, which could be attributed
to the exposure to manganese.
Acute pulmonary toxicity of manganese dioxide has been reported in other
animal model systems. Inhalation of manganese dust (1 hour/day for 39 days)
in combination with pneumococci types I and II was reported by Jotten (1944) to
cause bronchopneumonia in rabbits. No manganese aerosol concentration was
indicated with this study. Zaidi et al. (1973) suggested that intratracheal
instillations of manganese dioxide (50 mg/1.5 ml saline) in combination with
Candida albicans increased the development of mature lesions and fibrosis
in guinea pig lungs. Bergstron (1977) studied a variety of parameters after
MnO exposure (22 mg/m , 24 hours), including manganese clearance, number of
free lung cells obtained by lavage, and the phagocytic capacity of macrophages
encountering a secondary aerosol challenge with a pathogenic bacterium,
Enterobacter cloacae. Recent studies by Maigetter et al. (1976) indicated
potentiating effects of MnO inhalation at 109 mg/m for 3 hours on experi-
mentally-induced bacterial and viral pneumonias.
Several investigations on manganese toxicity have been reported involving
in vitro model systems. Waters et al. (1975) measured the cytotoxicity of
manganese chloride on rabbit alveolar macrophages which was attributed to
reduction in cell viability and, in some cases, cell lysis. Graham and
coworkers (1975) reported reduction in both viability and phagocytic ability
of rabbit alveolar macrophages resulting from in vitro contact with manganese
chloride. Additionally, Mustafa et al. (1971) reported the inhibitory
effects of numerous divalent cations, including Mn , on ATPase activity in
sheep alveolar macrophages.
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1-4
Based on the above in vitro studies and on information that exposures
of human to manganese produces interstitial pneumonia, whole animal studies
were designed to determine the possible effects of manganese on alterations
of host defenses against infectious microbiological agents. The study
reported here will primarily consider the acute pulmonary toxicity of Mn 0. in a
laboratory animal model.
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EXPERIMENTAL PROCEDURES
EXPERIMENTAL ANIMALS
Female, 4- to 6-weeks old, outbred albino, strain CD-I, mice (Charles River,
Wilmington, Massachusetts) weighing 20-25 g were used throughout this investiga-
tion. The animals were housed in plastic cages on wood shavings and provided
antibiotic-free Wayne Lab-Blox chow and water ad_ libidum.
CHEMICALS
Manganous-manganic oxide (Mn 0., 1-3 ym particle size, 99% purity, Aremco
Products, Inc., Ossining, New York) was used throughout this investigation.
INHALATION EXPOSURE SYSTEM
All inhalation exposures of mice were conducted for 2-hours duration to a
Mn O aerosol. Following the exposure, the animals were used in a variety of
experiments designed to investigate the toxicological effects of this pollutant
on the pulmonary system.
A flow diagram of the inhalation exposure chamber is given in Figure 1. The
chamber consists of a rectangular Plexiglass box having the dimensions of eight
feet long and one foot wide and deep (8 ft ). The 0.3 ft mixing chamber shown
was designed for mixing HEPA-filtered dilution air with the Mn 0. aerosol. Four
exposure ports are located on each of two sides of the chamber which are located
in a staggered arrangement. Plexiglass exposure modules, consisting of banks of
6 cylindrical tubes, individually isolated the animals, providing head-only type
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FIGURE 1
FLOW DIAGRAM
METAL AEROSOL EXPOSURE APPARATUS
CLOVE BOX ROOM
MAGNEHEUC GAUGE AIR
EXHAUST
i
VACUUM
SAMPLE A
DILUTION
HEPA FILTER
INLET
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tHEPA
ILTER
H
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I
AEROSOL
GENERATING
SYSTEM
GLOVE BOX
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\
I1
H
C
METERING
/ORIFICE
BAFFLE
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s
CHAMBER PRESSURE
MAGNEHELIC GAUGE
CHAMBER ORIFICE
MAGNEHELIC GAUGE
VACUUM
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E
E
a_j
>
/ ROYCO
AEROSOL
ANALYZER
>-« >
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/ Fl
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EXPOSURE PORT
EXPOSURE TUBE
LINE
VALVES
FILTER
I
.EXHAUST to
OUTLET
TEMPERATURE/RELATIVE
HUMIDITY SENSOR
T
VACUUM
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II-3
exposure conditions. Each animal at the end opposite that projecting into the
chamber. Animals were allowed to condition for approximately 5 minutes to the
confinement conditions on the chamber while breathing clean filtered air prior
to generation of the Mn 0 aerosol.
The chamber air flow was maintained at approximately 9 ft /minute through-
out the 2-hour exposure period. This condition was controlled by an exhaust
blower located downstream from an absolute, high efficiency particulate aerosol
(HEPA) filter. This filter was placed in-line to remove all Mn 0. prior to
exhausting air to the atmosphere. Chamber pressure was maintained and constantly
monitored at 0.35 inches of water (negative pressure). Airflow into the chamber
was monitored with a Magnehelic gauge (Dwyer Instruments, Inc., Michigan City,
Indiana) across the orifice between the mixing and exposure chambers.
The Mn 0. aerosol was generated within an external glove box (LabConCo Cor-
poration, Kansas City, Missouri) using a modified generation system (Model 7330
generator, Environmental Research Corporation, St. Paul, Minnesota). A flow
diagram of the Mn 0. generation system is shown in Figure 2. Input air for
the system was provided from an oil-less Cast air compressor through a Wilkerson
air line filter, regulated at 15 psi, which was then dried through a Silica gel
column. The air was then filtered through an absolute filter prior to being
split into two streams, one supplying dilution air and the other to the particulate
aerosol generator. A Wright dust-feed mechanism (BGI Corporation, Waltham,
Massachusetts) controlled by a digital programmable motor speed controller was
used to generate the Mn 0. aerosols. The two air streams were metered through
rotameters and recombined in a small mixing cylinder prior to introduction into
a Krypton-85 ionizer, which promotes charge neutralization on the particles generated.
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FIGURE 2
FLOW DIAGRAM
PARTICULATE AEROSOL GENERATING SYSTEM
AIR
INLET
(60 psig)
PRESSURE
GAUGE
(35 psig)
C^
I NEBULIZER
F FLOWMETER
PRESSURE
REGULATOR
LJn
DILUTION
FLOWMETER
PARTICULATE
^
DILUTION
VALVE
AEROSOL
OUTLET
IONIZER
MIXING
CHAMBER
WRIGHT OUST-
FEED MECHANISM
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II - 5
The resulting Mn 0 aerosol was then introduced into the Plexiglas mixing chamber
and exposure chamber previously described.
Particle size analysis of each exposure aerosol was conducted continuously
using a Royco Aerosol Particle Monitor (Model 203, Royco Instruments, Inc., Menlo
Park, California) equipped with a Model 508 Module. Frequency counts were deter-
mined in each of ten size channels, including: >_ 0.2-10, >_ 0.3-10, >_ 0.4-10,
>_ 0.6-10, >_ 0.8-10, >_ 1.0-10, >^ 1.5-10, >_ 2.0-10, >_ 3.0-10, >_ 5.0-10 ym diameter.
Particle size analysis indicated an average mass median diameter of 1.40 ± 0.09 ym
for all Mn 0 exposures.
Inert particulate aerosols were also generated using the system shown in
Figures 1 and 2. These included iron Oxide (Fe 0 , Pressure Chemical Company,
Pittsburg, Pennsylvania) and carbon black (Pressure Chemical Company). Aerosols of
these particles were generating from pressure packaged (1000 psi) pellets using the
Wright dust-feed mechanism previously described.
The temperature and relative humidity of the exposure atmospheres in the
chamber were monitored continuously with a Rustrak Temperature and Relative
Humidity Sensor (Model 225, Gulton Industries, Inc., Manchester, New Hampshire).
The average chamber temperature was 23.8°C with a standard error of 0.2°C, while
the relative humidity was 30.4 ± 0.7%.
MANGANESE QUANTITATION PROCEDURE
Filter samples (0.22 ym, Millipore Corporation, Bedford, Massachusetts), for
aerosol mass quantitation of manganese, were routinely performed at regular intervals
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II - 6
during the exposure period for seven minutes at 2.65 I/minute using the sampling
ports indicated (Figure 1). Filters were treated with 5.0 ml concentrated H SO
(Fisher Scientific Co., Fairlawn, New Jersey) for 15 minutes on a platform
shaker (120 oscillations/minute, Eberback Corporation, Ann Arbor, Michigan) prior
to the addition of 15.0 deionized water. Filters were allowed an additional
15-minute shaking period prior to decanting the eluate which was stored at 4°C
prior to analysis.
Lungs were excised at the carina immediately, and at various time intervals
post-exposure, from randomly selected animals sacrificed by cervical dislocation.
The lungs were placed in individually tared Pyrex boats and immediately weighed,
prior to mincing, to determine wet weight. Samples were then oven-dried under
vacuum (> 90°C, 24 inches Hg) for 24 to 72 hours, prior to low temperature ashing
(Model LTA-505, LFE Corporation, low temperature asher) at 400 RF Watts and
200 ml/minute oxygen flow. The dried residue was dissolved with 10 ml 0.6 N
hydrochloric acid (reagent grade, Fisher Scientific Company) and stored at -20°C
in polyethylene vials until analyzed.
o
Manganese quantitaton was performed at 2795 A using a Jarrell-Ash Atomic
Absorption Spectrophotometer (Model 850-2, Fisher Scientific Company) equipped
with a heated graphite furnace (Model FLA-10). The following conditions were
used during analysis: slit size, 3A; drying for 25 seconds, 400°C, ashing for
10 seconds, 900°C; and atomizing for 10 seconds, 2500°C; with background correc-
tion. Sample manganese quantitation was determined by interpolation on a multipoint
standard curve at 2795A with a Fisher certified manganese atomic absorption
standard (1000 yg/ml, Fisher Scientific Company).
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II-7
TOXICOLOGICAL TESTING: IN VIVO MODELS
Streptococcal Infectivity. . Manganese-exposed mice were randomized and com-
bined with control animals in stainless steel mesh exposure racks which individ-
ually partition animals collectively in groups of 40 each. The infectivity expo-
sure racks were then placed on a single tier in a stainless steel chamber (Young
and Bertke Company, Cincinnati, Ohio). Exposure to an aerosol of a viable 24-hour
9
culture of Streptococcus pyogenes (Group C, approximately 2 x 10 bacteria/exposure)
in Todd Hewitt broth (THE, Baltimore Biological Laboratories (BBL), Cockeysville,
Maryland) was conducted for a 20-minute period. The streptococci were generated
using a DeVilbiss atomizer (No. 40, the DeVilbiss Company, Somerset, Pennsylvania).
Animals were separated into their appropriate treatment groups and were
maintained in clean filtered room air for fifteen (15) days duration. Total
cumulative mortality rates were determined for Mn 0 -exposed and infected control
mice. The mortality and relative mean survival rates are presented as difference
from control response.
Streptococcal Deposition and Clearance Studies. Immediately following the
Streptococcal infectivity, either 2 or 4 control animals were sacrificed for
total lung Streptococcal deposition determinations. All aseptically excised
lungs were homogenized in 2.8 ml sterile THE using a Sorvall homogenizer
(Model Omni-Mixer, DuPont Instruments, Newtown, Connecticut) and sterile individual
10 ml stainless steel micro-chamber homogenizer cups. The lung-broth homogenate
total volume was 3.0 ml. All homogenizations were performed in an ice bath for
two three-minute periods separated by a 15-minute cooling period on ice. Complete
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II-8
homogenization of lungs was verified by transferring the entire homogenate into a
clear polystyrene culture tube prior to culturing. A 48-hour culture of homogenate
aliquots at 37°C on triplicate sheep blood supplemented (5%) Trypticase Soy Agar
plates (TSA, BBL) provided direct quantitation of viable streptococci.
Clearance of viable in vivo streptococci following the laboratory-induced
infection was determined on excised lungs at various time intervals using the
above procedure.
Septicemia Studies. Septicemia determinations were performed from blood
cultures obtained by asceptically transverse sectioning the distal ends of mouse
tails with scissors. The first drop of blood obtained in all cases was removed
by wiping with surgical gauze saturated with 70% ethanol. Duplicate 0.05 ml
aliquots were applied to TSA plates containing 5% sheep blood and streaked
according to standard microbiological procedures. Following a 48-hour incubation
period at 37°C, the percent positive blood cultures were determined.
IMMUNOLOGY STUDIES
Formalinized S. pyogenes were used for the immunization of mice. Colony-
forming units were determined as previously described on an 18-hour 5. pyogenes
THE culture prior to harvesting by centrifugation (Sorvall RC-2B, DuPont Instru-
ments, Newtown, Connecticut) at 2500 rpm for 10 minutes at 4-8°C. Harvested
cells were washed 3X with sterile physiological saline (0.85%, pH 7.4). The
final cell pellet was resuspended to the original volume with sterile 1.0%
formalinized (Fisher Scientific Company) saline and allowed to stand at room
temperature for 24 hours. Complete formalin killing of the cells was verified
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II-9
prior to use for immunization by innoculating duplicate 0.1 ml aliquots in THE
and incubated at 37°C for 72 hours. Formalinized cells were then harvested by
centrifugation as previously described and washed 2X with saline prior to storage
in sterile injection vials at 4°C.
Immunization by the subcutaneious and intramuscular route employed inocular
9
containing approximately 1 x 10 streptococci/ml. Water-in-oil emulsions were
prepared using the formalinized streptococci in saline and Freund's complete
adjuvant (1:1, Difco Laboratories, Detroit, Michigan). The first immunization
was performed using 0.1 ml of the emulsion injected subcutaneously (SQ) on the
back of each mouse at the base of the tail. Two subsequent intramuscular
injections were performed at one-week intervals seven days after the SQ injection
in the calf of separate hind legs.
Immunization by the aerosol route involved aerosolization of 5.0 ml of the
formalinized streptococci in saline. This suspension was generated as previously
described for the infectivity studies for a 30-minute exposure period. Mice
were individualized in the partition stainless steel exppsure cages as described
previously for the streptococcal infectivity. Following the first aerosol immuniza-
tion, mice were similarly immunized on two or more occasions separated by one-week
intervals.
Both groups of immunicated animals and untreated control mice were randomized
in in the exposure modules, and exposure for two hours to the Mn 0 atmosphere, as
described previously. Immediately following this treatment all animals were
separated into their respective treatment groups and again randomized with untreated
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11-10
control animals. These animals were then infected with viable S. pyogenes
using the infectivity system as previously mentioned.
All animals were again segregated into their respective treatment groups
and maintained in clean filtered room air for fifteen (15) days duration.
Total cumulative mortality rates were monitored and determined for both
immunized and non-immunized animals that were subsequently exposed to Mn 0.
or only infected as described. The mortality and relative mean survival
rates are presented as percent difference from control response.
TOXICOLOGY TESTING: IN VITRO MODELS
Cytological Measurements. Free cells were obtained from the lungs
of both manganese-exposed and control mice. Both groups were handled identi-
cally with the mice being sacrificed with 50 mg sodium pentobarbital
(Nembutal, Abbot Laboratories, Chicago, Illinois). The free pulmonary cells
were isolated and quantitated according to the procedure previously described
(Coffin et al., 1968), with modifications for the smaller animal. The in_
situ lung lavage was performed using prewarmed sterile 0.85% physiological
saline at 37°C. The first 0.5 ml volume instilled into the mouse lungs
remained approximately 5 minutes; three to four subsequent 0.5 ml
instillations were aspirated, withdrawn immediately, and pooled. The pooled
cellular fraction was washed three times by centrifugation (365 x g) and
resuspended with 1.0 ml sterile physiological saline. The pulmonary cell
suspension was maintained in an ice bath during all subsequent manipulations.
The cellular composition of the harvested cells from the pooled lavage fluid
was determined from Wright-stained smears. Viability determinations were
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performed using the trypan blue-dye exclusion method (0.4% trypan blue,
Grand Island Biological Company, Grand Island New York; GIBCO; Hanks and
Wallace, 1958). Total cell count yields were determined by hemocytometer
counting.
Phagocytic Capability. Phagocytic acitvity of pulmonary cells obtained from
mice following two-hours exposure to a Mn.,0. aerosol were compared with an untreated
animal response. Polystyrene-latex spheres (1.1 ym, Dow Diagnostics, Indianapolis,
Indiana) were used with the pulmonary cells cultured in culture tubes (12 x 75 mm,
Falcon Plastics, Oxnard, California). Pulmonary cells in 0.5 ml saline were diluted
equally with 2X Medium 199 (Earle's Base, with L-glutamine; GIBCO) supplemented
with 30% fetal calf serum (GIBCO). The incubation mixture contained approximately
2.4 x 10 cells/ml and 0.1 ml styrene particles containing approximately 1.3 to
Q
2.5 x 10 particles/ml. This system maintained approximately 50 to 100 particles/cell
in 1.1 ml volumes of the IX supplemented medium. All subsequent manipulations,
including incubation conditions, and determinations of phagocytic activity, were
performed according to procedures previously reported (Waters et al., 1975; and
Gardner et al., 1973).
The suspension cultures were harvested by centrifugation (365 x g). The
styrene particle containing supernatant medium was decanted and replaced with
200 ul fresh IX supplemented medium. Smear slides were prepared from this
suspension and air-dried prior to Wright-staining. All stained slides were
then emersed totally in xylene (Fisher Scientific Company) for one (1) hour
to remove extracellular latex spheres (Gardner et al., 1973). Slides were then
microscopically examined by counting 100 consecutive monocytic cells for the
presence or absence of styrene beads.
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Intracellular ATP. Pulmonary cells were harvested by centrifugation (365 x g)
from the saline suspension cultures and resuspended to original volume with sterile
Tris buffer ([Tris(hydroxymethyl)aminomethaneJhydrochloride; 0.1 M, pH 7.7). The
resuspended cell suspensions were then transferred to sterile glass culture tubes
(12 x 75 mm) and placed in a boiling water bath (> 100°C) for 10 minutes. The
culture tubes were allowed to cool at room temperature before storing at -20°C
prior to assay.
The ATP assay was performed using the Tris buffer lysate above. Frozen
samples were thawed at 37°C and maintained on ice throughout these manipulations.
A 200 yl aliquot of each lysate was mixed equally in glass scintillation vials
(Fisher Scientific Company) with freshly rehydrated firefly lantern extract
(Grade B, Calbiochem, San Diego, California) containing 5 mg/ml dried lanterns
in 0.025 M potassium arsenate and 0.01 M magnesium sulfate at pH 7.4. Following
a 15-second delay, the light emitted was integrated for 60 seconds in an ATP
photometer (Model 3000, SAI Technology Company, San Diego, California). The
ATP concentration/ml was calculated from the integrated counts interpolated
from a multipoint standard curve of ATP standard solutions (adenosine triphos-
phate-magnesium sulfate, Calbiochem) in Tris buffer. The ATP concentrations were
then normalized by dividing the concentration/ml by the total cell count for each
sample.
Preparation of Cell Sonicates. The saline suspensions of the pulmonary
cell were harvested by centrifugation as previously described. The cell pellets
were resuspended with sterile deionized water and stored at -20°C in sterile
plastic culture tubes (12 x 75 mm). Fresh extracts were prepared by thawing the
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frozen cell suspensions at 37°C and sonicating for 20 seconds at approximately
135 Watts in an ice bath using a titanium microprobe (Model 300, Sonic Dismem-
brator, Artek Systems Corporation, Farmingdale, New York). Sonicated samples
were maintained in an ice bath for all subsequent manipulations.
Total Protein. Total protein was determined in the cell sonicates above
following the procedure of Lowry et al. (1951) with slight modifications. A
100 yl aliquot of the sonicate was mixed with 1.0 ml of fresh alkaline copper
reagent containing NaOH (0.1 M; Sigma Chemical Corporation, St. Louis, Missouri),
Na CO (0.19 M, Sigma), NaKC H O 4H O (0.7 mM, Sigma), and CuSO 5H 0 (0.02 M,
2. 3 4 4 D «( 4 ^
Fisher) and allowed to stand for 10-30 minutes. Following this incubation period
at room temperature, a 100 yl volume of freshly diluted Folin phenol reagent
(0.1 N, Fisher) was then added, mixed, and allowed to stand for an additional 20-120
minutes. The absorbance was determined on each sample at 750 nm in a double-beam
spectrophotometer (Model 25, Beckman Instruments, Inc., Irvine, California) against
a reagent blank containing c'eionized water. Total protein was calculated as mg
protein/ml by interpolation of all sample absorbance values on a bovine serum
albumin (Miles Laboratories, Inc., Kankakee, Illinois) multi-point standard
curve (750 nm). The total protein concentration was then normalized by dividing
by the total cell count.
Acid Phosphatase Activity. The acid phosphatase activity was determined
according to the procedures of Brandenberger and Hanson (1953) and Hofster (1954).
A 250 yl aliquot of each cell sonicate was mixed individually prior to assay with
1.25 ml reaction mixture containing 1.0 ml acetate buffer (0.15 M, pH 5.0) and
0.25 ml o-carboxyphenyl-phosphate (3.65 mM, Sigma). The increase in absorbance
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II - 14
at 300 nm resulting from the liberation of salicylic acid was measured in the
double-beam Beckman spectrophotometer against an acetate buf fer/o-carboxyphenyl
phosphate reagent blank containing 250 yl deionized water. The enzyme reaction
was followed for 2-3 minutes using a Recordall recorder (Model 5000, Fisher Scien-
tific Company) attached to the spectrophotometer. The change in absorbance
was determined from the initial linear portion of the reaction curve. Acid phos-
phatase activity was calculated as units/mg protein according to the following:
AA /minute x 1000 divided by the salicylic acid molar extinction coefficient
(3500) x mg protein/ml reaction mixture.
Lactic Acid Dehydrogenase Activity. The lactic acid dehydrogenase activity
was determined according to the procedure of Wacker et al. (1956) with slight
modifications. A 100 yl aliquot of each cell sonicate was mixed individually
prior to assay with 2.9 ml of reaction mixture containing 2.7 ml Tris-HCl buffer
(0.2 M, pH 7.3), 0.1 ml nicotinamideadenine dinucleotide (reduced form, NADH,
6.6 mM in Tris-HCl buffer, Sigma), and 0.1 ml sodium pyruvate (30 mM in Tris-HCl
buffer, Sigma) . The decrease in absorbance at 340 nm resulting from the
oxidation of NADH was measured in the double-beam Beckman spectrophotometer
against a reagent blank containing 100 yl deionized water. The enzyme reaction
was followed for 3-4 minutes on the Recordall recorder as previously described.
The change in absorbance (AA ) was determined from the initial linear portion
of the reaction curve. Lactate dehydrogenase activity was calculated as units/mg
protein according to the following: AA /minute divided by the lactate molar
extinction coefficient (6.2) x mg protein/ml reaction mixture.
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Inflammation Measurements.
Wet-Dry Tissue Ratios. All wet tissues were weighed immediately fol-
lowing excision on an analytical balance (Model H64, Mettler Instruments
Corporation, Hightstown, New Jersey) in individually tared Pyrex ashing boats.
Following 24-48 hours drying time in an evacuated oven at 90°C, the same tissue
samples were again weighed. The dry:wet tissue weight ratio was then calculated
for each tissue sample as an index of edema.
Lavage Supernatant Total Protein. The saline lavage supernatant fluid was
decanted from the pulmonary cell pellet, volumetrically determined by pipette,
and transferred individually to culture tubes (12 x 75 mm). All supernatant
samples were maintained in an ice bath and assayed for total protein according
to the procedure previously described. The total protein was normalized by
multiplying the protein concentration/ml by the total volume of the lavage
supernatant fluid.
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RESULTS
MANGANESE LUNG DEPOSITION RATES
The initial pulmonary deposition of manganese following 2-hours inhalation
of aerosol concentrations ranging from 0 to 3.0 mg/m is shown in Figure 3. Re-
gression analysis yielded a statistically significant increasing linear relation-
2
ship (p < .0001 with an R of 81%) between yg Mn/g dry lung and manganese aerosol
concentration for mice sacrificed immediately after exposure.
MANGANESE LUNG CLEARANCE RATES
The retention/clearance of manganese following 2-hours inhalation of a
1.798 mg Mn/m aerosol over a 24-hour period is given in Figure 4. Regression
analysis yielded a statistically significant decreasing non-linear relationship
(p < .001) between yg Mn/g dry lung and the post-exposure assay interval. These
data indicated approximately 47%, 27%, and 14% inhaled manganese remained four,
six, and twenty-four hours, respectively, after exposure.
MANGANESE TISSUE DISTRIBUTION
The distribution of manganese in various murine tissues was assayed between 0
and 48-hours post-exposure following inhalation of 1.837 mg Mn/m (Table 1). Sig-
nificant levels of manganese were observed in the lungs immediately after exposure
and in the kidney and spleen 48-hours post-exposure when compared to control levels.
The systemic distribution of manganese observed agrees with the molecular exclusion
for biliary excretion. The retention/clearance levels in the 48-hour lungs parallel
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INI I IAL pdlMoNARY DEPOSITION OF MANGANESE FOLLOWING TWO HOURS INHALATJON IN MICE
60
50
40
D
CC
Q
O)
a.
30
20
10
I
I
I
I
1
0.4 0.8 1.2 1.6 2.0 2.4
MANGANESE AEROSOL CONCENTRATION, mg/m3
m
2.8
-------
III-3
FIGURE 4
LUNG RETENTION OF INHALED MANGANESE FOLLOWING TWO-HOURS
INHALATION OF A MANGANESE OXIDE AEROSOL
(1.798mg/m3)
DC
Q
o>
c
IE
O)
28
24
20
16
12
8
I I I I I I I I I I I I
= 2.17 + 29.15e -217
O _
I I I I I I I I I I II
8 10 12 14 16 18 20 22 24 26
POST EXPOSURE INTERVAL, hours
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Ill - 4
TABLE 1. MANGANESE DISTRIBUTION IN VARIOUS MURINE TISSUES FOLLOWING INHALATION
OF 1.837 mg Mn/m3.
POST-EXPOSURE
INTERVAL
(hours)
0
24
48
CONTROL
TISSUE SAMPLED, yg Mn/g DRY LUNG
LUNG LIVER KIDNEY SPLEEN
21.70 ± 2.19* 7.37 ± 0.39 5.77 ± 0.63 2.08 ± 0.28
2.98 ± 0.20 6.12 ± 0.38 '6.53 ± 0.65 1.48 ± 0.11
2.69 ± 0.17 7.22 ± 0.23 10.29 ± 0.89* 2.57 ± 0.29*
2.00 ± 0.07 6.14 ± 0.65 6.53 ± 0.82 1.61 ± 0.28
* p < .05 using Dunnett's test for significance between treatment and control
mean values.
data presented previously (Figure 4) with 12% of the manganese remaining The
manganese levels observed in both kidneys and spleen represented 1.6-fold increases
over control levels at 48-hours post-exposure.
INFECTIVITY STUDIES
Relative Mean Survival Rates. The mortality difference between Mn 0 -exposed
and control mice indicated a statistically significant (p < .001 with R = 71%)
positive linear relationship with the manganese aerosol concentration (Figure 5).
Similarly, the increasing toxicity of Mn O with increasing aerosol concentration
2
is reflected in a significant (p < .001 with R = 69%) negative linear relationship
expressed as relative mean survival time (difference from control). Figure 5
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, MABS. oscoa
en
-------
Ill - 6
demonstrates the 95% confidence limits for these two response parameters which
excludes zero for Mn 0 concentrations in excess of 0.5 mg/m . Using inverse
prediction techniques, these data indicate 1.55 mg Mn/m as the estimated manganese
aerosol concentration for which not less than 10% mortality difference from
control will result with 95% confidence, and 1.323 mg Mn/m for 7.5% mortality
difference from control with 95% confidence.
Inert particulates, namely iron oxide and carbon black, were tested in the
infectivity system as particulate controls to test the hypothesis of possible
physical irritation toxicity exerted by the particulate aerosols per se.
Iron oxide exposure at 2.5 mg/m for 2-hours resulted in < 2% mortality rate
(difference from control) following subsequent infection. Carbon black
exposure at 5.0 mg/m for 2-hours resulted in < 8% mortality rate (difference
.from control) following subsequent infection. These data indicated the failure
of these inert particulates to ellicite toxic effects in the infectivity
system and further substatiate true metal toxicity rather than physical irritation
by the manganese oxide particles.
Streptococcal Lung Clearance Rates. Manganese inhalation toxicity data
presented previously is supported by data presented in Figure 6. These data
indicate the delayed clearance and subsequent inhanced growth of inhaled
streptococci in mouse lungs following exposure to 2.122 +_ 0.314 mg Mn/m as
compared to infected control animals. An average deposition of 1000 viable
organisms/lung were deposited in the lungs of both control and Mn 0 -exposed
mice. Growth of in vivo streptococci continued in Mn 0 -exposed mice
throughout the 96-hour testing period. However, the number of viable
microorganisms in the control group did not follow this trend.
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III-7
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I
-J
cc
in
o.
5
O
o
o
1
01
cc
(O
UJ
s!
u.
O
CC
UJ
CO
I
EFFECT OF MANGANESE INHALATION ON THE
GROWTH AND CLEARANCE OF STREPTOCOCCI
IN MOUSE LUNGS FOLLOWING INFECTION
I
Mn-TREATED
(Mn304;2.|2ZmgMn/m3)
INFECTED
CONTROL
24
48
72
96
POST - INFECTION INTERVAL, hours
W-* . ». X_ 1* *,-».,. ^. >
-------
i ' i i ! i ; : . i i i ' ' ' i : i : ! I I ' ! ' ' I ' I »
OF MlM^O^. XN)\AAuATiOfO OM N\oRrrAuvfvj RAres
OF
i
CO
-------
Ill - 9
Streptococcal Blood Clearance Rates. The time course of the pathogenesis
of the laboratory-induced Streptococcal infection was demonstrated in Figure 7.
The data correlate the occurrence of septicemia with the demonstrated mortality
rate for both Mn 0.-exposed and infected control animals. Two different levels
of manganese were tested as indicated. Significant differences between exposure
groups and the infected control mice were not observed with either occurrence
of septicemia or mortality rate. Peak mortality and septicemia rates with
both exposed and control animals occurred at day 5 and day 4, respectively.
These data support the invasiveness of the organism in the infection and indicate
the early occurrence of the septicemia in relation to the ensuing death.
EFFECTS OF MANGANESE INHALATION ON VARIOUS MURINE IMMUNE MECHANISMS
Two routes of immunization of Mn 0 -exposed and control mice were compared
with regards to protection against the Streptococcal infection (Figure 8).
Successful immunization was observed in control groups not exposed to Mn 0
with both routes of immunization tested. The immunization provided some
protection against the effects of Mn 0 exposure (1.854 _+ 0.175 mg/m ) and
subsequent Streptococcal infection. Immunization by the SQ/IM route afforded
greater protection than the aerosol route, based on the parameters considered,
but in neither case provided total immunity against the Streptococcal infection
following the deleterous effects of Mn 0 inhalation.
BIOCHEMICAL CYTOLOGY STUDIES
Several parameters relating to pulmonary toxicity resulting from the
inhalation of a Mn 0 atmosphere were investigated. Table 2 summarized data at
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cc
O
LU
O
cc
LU
O.
COMPARISON OF TWO ROUTES OF IMMUNIZATION WITH FORMALINIZED
Streptococcus pyogenes ON THE TOXICITY OF MANGANESE BY
INHALATION USING THE INFECTIVITY SYSTEM IN MICE
70
60
50 -
MANGANESE - TREATMENT
(l.
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TABLE £. EFFECTS OF
INHALATION ON VARIOUS CYTOLOGICAL PARAMETERS IN MURINE PULMONARY CELLS
Mn(jjg/m )
0
897.0133.9
AEROSOL
MASS MEDIAN
DIAMETER
(pm)
-
2.4 ± 0.6
DIFFERENTIAL CELLS COUNTS (%)
MACROPHAGES
91. 3 ±2. 6
(22)
93.3 ± 1 .0
(44)
PMNs
0.0
(22)
0.07 ±0.04
(44)
LYMPHOCYTES
9.1 ± 2.7
(22)
6.7 ±0.9
(44)
TOTAL CELL
COUNT
(xl05/ml)
5.41 ± 0.20
(132)
H*
4.651 0.14
(195)
TOTAL PROTEIN
SUPERNATANT
FLUID(mg)
1.152 ±0.042
(132)
U33± 0.036**
(191)
VIABILITY(%)
91.0 ±0.4
(100)
89.8 ±0.5*
(135)
H
H
H
I
a Mean values± one Standard Error' numbers in parentheses represent numbers of animals tested
*» Mean values significantly different (p <.OI) from control
-------
Ill - 12
897.0 yg Mn/M and compares it with the appropriate control animals.
Differential pulmonary cell counts indicated normal cellular populations
between animals used as controls and Mn 0 -exposure. However, there was a
significant decrease, from control animals, in the number of total cells
isolated from the mice lungs following manganese treatment (p<.01). Decreases
in cellular viability was also observed with the pulmonary cells following
Mn 0 exposure. This response was not statistically different as determined
by student's t comparison of exposure and control groups. Increases in
extracellular protein in the lavage supernatant fluid were not observed
following this level of Mn 0 exposure.
5 ^t
Inflammatory responses were measured in lungs and other tissues excised
at various time intervals following exposure at 1.837 mg Mn/m . A significant
(p<.0001) increase in spleen weight was observed immediately after exposure,
which did not persist through 48-hours post-exposure. Lungs, kidneys, and
livers did not exhibit any increase in tissue weight through the same assay
period.
Mouse lungs were assayed for increases in tissue weight immediately after
exposure throughout the entire aerosol dose range (0.1 to 2.8 mg Mn/m ).
Dry:wet tissue ratios did not show consistently significant increases over
control animal lungs at every aerosol dose concentration studied.
Several biochemical parameters were also studied with the pulmonary cells
obtained in these studies. Table 3 summarizes data at the same exposure level.
No effect was observed in phagocytic capability of monocytic cells following
Mn 0 exposure. Intracellular ATP determinations indicated a significant
-------
TABLE 3. EFFECTS OF
INHALATION ON SPECIFIC BIOCHEMICAL PARAMETERS IN MURINE PULMONARY CELLS
Mn (jjg/m3)
0
897. 0± 33.9
PHAGOCYTIC
CAPABILITY
(%)
90.8 ±0.7
(44)
89.2 ± 0.7+ +
(62)
_o ATP 5
(x 10 9 g/IO° cells)
0.52 ±0.06
(40)
0.82 ± 0.11*
(58)
PULMONARY CELL SONICATE
TOTAL PROTEI N
(mg/ml/105 cells)
0.016 ± 0.001
(55)
0.013 ± 0.001*
(71)
LDH
(units/mg protein)
0.004+0.0004
(27)
0.004 ±0.0002
(32)
ACID PHOSPHATES
(units/mg protein)
0.025 ± 0.001
(46)
0.032 ±0.003*
(60)
H
a Mean .values ± one Standard Error; numbers in parenthesis represent number of animals tested
* Mean values significantly different (p < .05) from control
-------
Ill - 14
(p<0.05) increase following Mn 0 exposure as compared to control pulmonary
cell sonicates.
Other biochemical parameters measured included enzymatic assays of the
pulmonary cells. Total protein measurements in alveolar macrophages indicated
a statistically significant decrease following Mn 0 exposure. Lactic acid
dehydrogenase (LDH) activity was analyzed to parallel the viability determinations
as well as indicate the general metabolic state of the pulmonary cells. No
difference in LDH activity was observed following exposure. Acid phosphatase
activity was also analyzed in the pulmonary alveolar macrophages to assess
hydrolytic enzyme activity. A statistical increase in acid phosphatase
acticity was observed following manganese treatment which did not necessarily
parallel the phagocytic response reported previously (Table 2).
Similiarly, cytological and biochemical parameters were also measured in
pulmonary cells from mice exposed to a lower Mn 0 aerosol concentration
(532.2 +_ 10.7 yg Mn/m ). The aerosol mass median diameter in these studies
was different than the previous study, and was 1.9 +_ 0.3 ym. Differential
characterization of the isolated pulmonary cells from both the Mn 0 -exposed
and control mice indicated approximately 50% macrophages and 50% lymphocytes.
From past experience, these data indicated that both groups of animals, control
and treatment, were not healthy according to this pulmonary parameter and
thus the data will not be reported in this monograph. Pulmonary cells from
these sick animals, however, indicated a similar response as reported at
897.0 yg Mn/m for healthy animals. That is, there was a significant decrease
in the total number of lavaged cells, viability, and phagocytic activity; and
a statistical increase in total protein in lavage fluid. All other parameters
measured did not statistically differ from animals exposed to clean air.
-------
CONCLUSIONS/DISCUSSION
Manganese was found in measurable amounts in the majority of suspended
particulate matter samples collected by the National Air Surveillance Networks
(NASN). Data from this survey indicated the highest ambient concentrations
of manganese to be in areas adjacent to ferromanganese alloy processing
plants or related activities. The NASN urban average manganese concentra-
tion was determined to be less than 0.2 yg/m , with annual average ranges of
0.5 to 3.3 yg/m in several cities. Lee et al. (1972) reported that at least
50% of the mass of suspended manganese was associated with _<_ 2 ym (Stokes
equivalent diameter) particles and that 80% of the manganese is found in the
respirable (< 5 ym) particle size range. The greater occurrence of manganese
in suspended particulate matter in the respirable range favors the widespread
distribution of this pollutant. The prevalence of manganese in air was
confirmed by the analysis of precipitation samples collected at several remote
locations in the United States (Scientific and Technical Assessment Report
on Manganese, EPA-600/6-75-002, 1975).
Because of the abundance of manganese in nature, exposure via the inhala-
tion route does not significantly contribute to the total dose, as compared to
other routes (Schroeder et al., 1970). However, the toxicity via the inhala-
tion route can easily be quite different and may be substantially a greater
risk than by other routes. Currently, there is a scarcity of information
identifying adverse health effects resulting from inhalation exposures to
ambient levels of manganese. The widespread use of manganese fuel additives
has been speculated to make resulting emissions more ubiquitous. The manganese
tested in these studies was at concentrations below the threshold limit value
-------
IV-2
(TLV), which was set at 5.0 mg/m for an 8-hour day. Exposure to manganese
aerosol concentrations ranging from 0.22 to 2.65 mg Mn/m for 2-hours caused
enhancement in subsequent respiratory infection. Toxicological consequences
of the manganese oxide inhalation were indicated additionally by a reduced
clearance initially of inhaled streptococci with subsequent enhanced growth
of the streptococci over that observed with control animals. Streptococcal
septicemia occurred earlier in manganese-exposed animals, paralleling the increase
in subsequent mortality. Protective immunity against streptococci did not
surmount the deleterous effects of manganese inhalation and Streptococcal
infection; again supporting the complexity of the toxicity as measured in
relation to respiratory infection.
Several biochemical parameters were measured in pulmonary cells obtained
from mice acutely exposed to Mn 0. aerosols. These efforts were designed to
support information obtained on the suppression of pulmonary defense mechanisms
against respiratory infections following acute manganese inhalation. Cytological
information from these studies, although not statistically significant,
indicated a reduction in total cell yield, cellular, viability, and phagocytic
ability of pulmonary cells following Mn O. exposure. Total protein in pulmonary
cells after sonication was significantly decreased compared to control cell
sonicates. Enzymatic assays indicated no change in lactate dehydrogenase
activity following exposure; whereas, acid phosphatase activity increased
over that observed with control cells. Additionally, the Mn 0. aerosol did
not produce an inflammatory reaction as determined by the total protein in
the lavage supernatant fluid.
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IV-3
Other investigations have shown a variety of toxicity effects of manganese
on components of the host defense system. Waters et al. (1975) correlated
manganese cytotoxicity on rabbit alveolar macrophages with reduction in cell
viability in an in_ vitro system. Decreases in cell number to 50% of control
levels and reduction in acid phosphatase specific activity were observed with
4-5 mM Mn , which paralleled reduction in cell viability. Graham et al.
(1975) reported the toxicity of manganese on the in vitro phagocytic activity of
rabbit alveolar macrophages. Similarly, Mustafa and coworkers (1971) reported
manganese to be slightly toxic to pulmonary alveolar macrophage ATPase activity
2+ 2+ 2+ 2+ 2+
compared with other divalent cations such as Ca , Ba , Cd , Zn , and Hg
2+
These findings suggested other interactions of divalent cations, such as Mn ,
on other cellular functions; namely, labilize the lysosomal enzymes of alveolar
macrophages causing additional damage to the lung parenchyma (Allison et al.,
1966). Depression in the protective role of pulmonary alveolar macrophages
against inanimate and microbial agents caused by exposure to such divalent
cations was also suggested.
Maigetter and coworkers (1976) reported single and multiple 3 hour
exposures of mice to respirable MnO aerosols altered the resitance to bacterial
and viral penumonias. Bergstron (1977) investigated a variety of parameters
associated with pulmonary toxicity of Mn02 aerosols in guinea pigs. These
studies indicated that acute MnO. exposure produced an inflammatory reaction
in the guinea pig respiratory tract without the presence of pathogenic bacteria
(Enterobacter Cloacae). This reaction was more pronounced in lungs challenged
with bacteria simultaneously with the MnO_ particles. Additonally, the decrease
in bacterial clearance reported in a similar study involving another bacteriaum,
Escherichia coli (Rylander, 1968) , was supported by Bergstrom (1977) in the
studies previously cited.
-------
IV-4
The results reported in this investigation substantiate many of these
findings in the literature. Additional studies involving chronic exposure
to manganese aerosols using appropriate animal models seems warranted.
-------
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V - 2
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V - 3
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