;PA-600/1-77-017
March 1977
Environmental Health Effects Research Series
CYTOTOXIC EFFECT OF TRACE METALS
ADSORBED ONTO FLY ASH PARTICLES
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environ-
mental Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application
of environmental technology. Elimination of traditional grouping was con-
sciously planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS
RESEARCH series. This series describes projects and studies relating to the
tolerances of man for unhealthful substances or conditions. This work is gener-
ally assessed from a medical viewpoint, including physiological or psycho-
logical studies. In addition to toxicology and other medical specialities, study
areas include biomedical instrumentation and health research techniques uti-
lizing animals—but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-77-017
March 1977
CYTOTOXIC EFFECT OF TRACE METALS
ADSORBED ONTO FLY ASH PARTICLES
by
Catherine Aranyi
IIT Research Institute
Chicago, Illinois 60616
Contract No. 68-02-0761
Project Officers
Donald E. Gardner
and
Michael D. Waters
Biomedical Research Branch
Clinical Studies Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S.,ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
11
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants. The Laboratory develops and revises 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 preparing the health basis for
non-ionizing radiation standards. Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation of
affidavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.
This report describes the results of an in vitro study designed to
determine the effects of fly ash coated with the oxides of lead, nickel
or manganese on alveolar macrophages which defend the deep lung against
infectious microorganisms. Since particulates of various sizes exist
within the environment, the influence of particle size was also examined
in order to provide data helpful in designing pollutant control strategies.
It was found that as the concentration of pollutant was increased, the
toxicity increased. In addition, the smaller-sized particles were more
toxic than larger particulates.
John H. Knelson, M.D.
Director,
Health Effects Research Laboratory
m
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ABSTRACT
Fly ash particles fractionated into three size ranges (<2,
2-5 and 5-8y) were coated with lead, nickel and manganese oxides
to determine the effects of particle size and surface area on the
in vitro toxicity of these metals for alveolar macrophages. When
uncoated or coated fly ash particles in concentrations ranging
from 50 to 1500 yg/ml were incubated with lO^AM/ml, reduced
viability and increased phagocytosis were observed upon increase
in concentration. This was seen irrespective of the particle
size. However, within a given particle coating and concen-
tration, decrease in particle size resulted in decreased viability
and increased phagocytosis. The percentage of lead, nickel or
manganese adsorbed on the fly ash particles varied within a rel-
atively narrow range, and was not affected by the particle size.
Since the macrophages were exposed to the particles on a weight-
dose-per-cell basis, the concentration of metal oxides to which
they were exposed was constant, irrespective of the particle
size. The greater toxicity of the small particles appeared to
be due to surface interaction between particles and the alveolar
macrophages. Thus, the toxic effect is surface-area as well as
dose related. These observations were further supported by de-
creases in total cellular protein levels and specific activity
of LDH.
This report was submitted in fulfillment of Contract 68-02-
0761 by IIT Research Institute under the sponsorship of the U.S.
Environmental Protection Agency. This report covers the period
from April 1975 to September 1976 and work was completed as of
October 31, 1976.
IV
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CONTENTS
Foreword iii
Abstract iv
Figures vi
Tables vi
1. Introduction 1
2. Conclusions 2
3. Recommendations 3
4. Materials and Methods 4
Animals 4
Preparation of Test Particles 4
Harvesting and Characterization of AM 4
Exposure of AM to Particulates 4
Viability and Phagocytosis 5
Screening of Particles for Soluble
Cytotoxic Components , 5
Enzyme Activity 5
Calculation and Analysis of Data 6
5. Results and Discussion 7
Viability and Phagocytosis 7
Enzyme Activity 13
Effect of Time 16
6. Related Studies 18
References 21
v
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FIGURES
Number Page
1 Effect of particle concentration and size on
viability of macrophages 8
2 Effect of particle size and concentration on
phagocytosis 10
3 Effect of particle exposure on macrophage
viability, total protein content and specific
enzyme activity 14
4 Comparison of the effects of PbO-coated or
NiO-coated and uncoated fly ash on viability,
total protein content and specific enzyme
activity 15
TABLES
1 Estimated Concentration of Particles Required to
Reduce Macrophage Viability to 75% During a 21-
Hr Exposure 7
2 Percent of Metal Elements Adsorbed on Fly Ash
Particles 9
3 Testing Metal Oxide-Coated and Uncoated Particles
for Soluble Cytotoxic Components by Macrophage
Viability 12
4 Estimated Concentration of Particles Required to
Reduce Macrophage Viability, Total Protein Content
and LDH Specific Activity to 50% During 21-Hr
Exposure 16
5 Effect of Duration of Incubation with Particles
On Viability of Macrophages 17
6 Effect of CdO-Coated Fly Ash Particles on
Macrophage Viability 18
7 Testing of CdO-Coated and Uncoated Particles for
Soluble Cytotoxic Components by Macrophage
Viability 19
vi
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SECTION 1
INTRODUCTION
The overall objective of this project was to examine in vivo
and in vitro effects of various air pollutants on pulmonary al-
veolar macrophages, During the first two years the effects of
inhalation of nitrogen dioxide (N02) and ozone (03) on mice were
investigated. The observations included total and differential
cell counts, viability, cell surface morphology, and in vitro
phagocytic function of the alveolar macrophages obtained by
tracheobronchial lavage from mice exposed to the pollutants. The
changes in the bactericidal capacity of alveolar macrophages in
lungs of mice challenged, by the respiratory route, with
Staphyloooocus aureus after exposure to the air pollutants were
determined. In addition, the surface morphologic alterations
observed in alveolar macrophages lavaged from the lungs and the
emphysemateous changes in the alveolar and terminal airway
structure of the lungs of animals exposed to N02 were studied by
scanning electron microscopy. These studies have been summarized
in annual reports IITRI Report L6070-4 (1974) and IITRI Report
L6070-8 (1975). In another phase of the program, studies were
conducted on the exposure of guinea pigs to N02 (IITRI Report
L6070-10P (1975)).
During the year covered by this report, studies were carried
out to correlate the toxicity for normal rabbit alveolar macro-
phages of lead, nickel and manganese oxides with the physical
characteristics of fly ash particles. The toxicity of the metal
oxides as affected by the particle size and surface area of the
carrier fly ash on which they had been adsorbed was investigated.
The parameters of toxicity were changes in alveolar macrophage
viability, cell lysis and activity of selected cellular enzymes.
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SECTION 2
CONCLUSIONS
Various concentrations of fly ash separated into <2, 2 to 5
and 5 to 8y particle size ranges and coated with lead, nickel or
manganese oxide were incubated for 21 hr with normal rabbit al-
veolar macrophages. Changes in viability, total cellular protein
content and specific enzyme activities were monitored. A linear
dose response relationship between concentration and cell viabil-
ity, total cellular protein levels and LDH specific activity was
observed for all particles. The levels of the lysosomal enzymes
acid phosphatase and (3-glucuronidase showed, generally, no change.
The results provided a toxic ranking for the metal oxides, i.e.,
Pb >Ni >Mn and demonstrated that toxicity was not due to solu-
bilization of the metal compounds in the incubating medium. For
a given metal oxide and a given particle concentration, toxic
effects increased with decreasing particle size. Since the macro-
.phages were exposed to the particles on a weight concentration
per cell number basis and, moreover, the percentage of Pb, Ni or
Mn, was approximately the same for all particle sizes, the con-
centration of the metals was not affected by the particle size.
Therefore, the greater toxicity of the smaller particles appeared
to be due to their larger surface area. Consequently, the
particle itself played a role in the toxicity, serving as a
carrier of the metal compounds into the intracellular millieu.
The stability of the specific activity of the lysosomal hydro-
lases (acid phosphatase, B-glucuronidase) as compared to that of
a soluble enzyme in the cytosol (LDH) suggests that the release
of hydrolytic enzymes in the cell may be a secondary event in the
cytotoxic response.
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SECTION 3
RECOMMENDATIONS
Viability of alveolar macrophages appears to be the parameter
of choice in establishing toxic ranking of particulate pollutants.
Although it would be expected that the cellular enzyme activities
should characterize the state of cellular injury more precisely
and with higher sensitivity, the simplicity of the viability
assay is an overriding advantage for screening studies.
The studies provide an experimental evidence that the physical
nature of the particle is extremely relevant to its toxicity by
demonstrating that the size of a carrier particle affects the
toxicity to macrophages of metal oxides adsorbed on its surface.
To explore further how the shape of a particle is involved, fi-
brous material of known toxicity (e.g., asbestos) should be ex-
amined relative to an inert material with simila.r geometric
properties (e.g., glass fibers). An interesting variable to
follow would be the effect on toxicity of simultaneously changing
size and shape of the fibers by varying the length to width
ratio in the fiber axis.
To learn more about the toxic mechanism it would be important
to determine the time course of the processes. Macrophages
should be incubated with the particles in concentrations that
produce a 50% viability decrease in a given period and the changes
in viability, activity of selected enzymes and levels of ATP for
monitoring functional impairment should be determined at various
time intervals. Particles containing nonsoluble toxic compounds
and particles from which a toxic compound is partially solubil-
ized during incubation should be included. The time course
studies would demonstrate differences in the nature of the toxic
effects on macrophages of different compounds. Most importantly
the studies would provide means of comparison between the mech-
anism of action of a soluble and nonsoluble toxic substance on
macrophages. If solubilized the compound can directly interfere
with metabolic processes in the cytoplasm by permeating the
plasma membrane; if nonsoluble and bound to the particle by
acting after engulfment of the toxic particle in the phagolyso-
somes.
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SECTION 4
MATERIALS AND METHODS
ANIMALS
Male albino New Zealand rabbits weighing 1.5 to 2 kg were used
to obtain high yields of alveolar macrophages (AM). The rabbits
were quarantined for 2 weeks before use and were provided food and
water ad libitum.
PREPARATION OF TEST PARTICLES
The particles used in the experiments were prepared by the
Fine Particles Section of IITRI from a fly ash sample provided by
EPA. The fly ash was fractionated by air classification (Bahco
Microparticle Classifier) into three particle size ranges, namely
<2, 2 to 5 and 5 to 8y. The metals in form of their hydroxides
were then precipitated onto the surface of the fly ash particles
dispersed in an aqueous phase. After filtration and washing, the
particles were heated to 675°C to convert the metal hydroxides
into the respective oxides (1).
HARVESTING AND CHARACTERIZATION OF AM
The rabbits were killed by injection of sodium pentobarbital
into the marginal ear vein and the lungs were lavaged in situ
with warm (37°C) sterile saline through a catheter inserted into
an incision in the trachea (2-3). The AM were isolated from the
lavage fluid by centrifugation at 365xg for 10 min and washing
in Hanks salt solution. Total cell counts were made in a hemo-
cytometer (3), and viability was determined by the trypan-blue
dye exclusion technique (4). For differential counts, smears of
air-dried cells were fixed in methanol and stained with Wright's
stain.
EXPOSURE OF AM TO PARTICULATES
Medium 199 in Hanks balanced salt solution was used for incu-
bation and exposure of the AM to the particulates. Supplements
in the maintenance medium included 0.25 M Hepes buffer to further
stabilize the pH (7.2 - 7.4), 100 yg/ml Gentamicin (Schering
Corp.) and 10% heat-inactivated fetal calf serum. Separate AM
and particle suspensions were prepared in the medium at twice the
projected exposure concentrations and then equal volumes of the
-------
two suspensions were mixed (Serum was omitted from the particle
suspension to prevent coating of the particles and was added at
20% concentration to the cell suspension.) The final AM concen-
tration in the suspensions was maintained constant at lO^AM/ml,
whereas the concentration of particles ranged from 50 to 1500
yg/lO^AM/ml medium. The suspensions were incubated in wells of
disposable plastic cluster dishes placed on a rocker platform for
continuous agitation for 21 hr at 37°C in a humidified 470 C02
atmosphere.
VIABILITY AND PHAGOCYTOSIS
After incubation, the suspensions were withdrawn from the
cluster dishes with Pasteur pipettes and transferred into sili-
conized test tubes. The AM attached to the cluster dishes were
removed by treatment with 0.2570 trypsin or by use of rubber
policemen, and were combined with the corresponding suspensions
in the test tubes and chilled. Viability was determined by micro-
scopic counting of 400 to 500 AM and the percentage of AM that
had phagocytized was determined on the same slides.
SCREENING OF PARTICLES FOR SOLUBLE CYTOTOXIC COMPONENTS
The fly ash particles coated with the various metal oxides
were incubated in the culture medium under similar conditions to
those used in the experiments, with the omission of AM and serum.
After incubation, the particles were removed from the media by
centrifugation and were resuspended in fresh medium. The super-
natant was filtered through a 0.22y Millipore filter. The fil-
tered supernatant (S) and the resuspended particles (P) and the
original suspension (P + S) were then separately tested for cyto-
toxicity using the AM viability assay.
ENZYME ACTIVITY
For determination of enzyme activity, the AM were first mixed
with the particles in siliconized Erlenmeyer flasks, then 10 ml
of the test suspension containing 10? AM was distributed in four
wells of a disposable plastic cluster dish. This sample volume
was used to provide a sufficient number of AM required for the
assays. After 21 hr incubation at 37°C, the viability of AM was
determined and, after transfer into siliconized tubes, they were
centrifuged at 365xg and washed in Hanks salt solution three
times. The cell pellet was resuspended in distilled water for
osmotic shock, the volume adjusted to 2.0 ml and the suspension
cooled in ice was sonicated for a total of 45 sec in two bursts
with the microtip of an ultrasonic generator using maximum
setting. A portion of the cell suspension was used for enzyme
assays after centrifugation at 63xg to remove the metal particles
and a second portion was treated with sodium deoxycholate, the
resulting lysate centrifuged at 10,000xg and the supernatant used
for protein assay.
-------
Enzyme activity was determined in triplicate using commercial
assay kits (Boehringer Mannheim Corporation, Sigma Chemical
Company). Acid phosphatase activity was determined by using the
enzyme to hydrolyze p-nitro-phenylphosphate at 37°C, and measuring
the liberated p-nitrophenol colorimetrically at 405 my. Beta-
glucuronidase activity was measured by using phenolphtalein-
glucuronic acid as a substrate and monitoring the liberated
phenolphtalein colorimetrically at 550 my. LDH activity was
analyzed by measuring the rate of oxidation of NADH at 366 my.
This oxidation is proportional to the conversion by the enzyme
of pyruvate to lactate. Total protein content was analyzed by
the Lowry method (5) using a bovine serum albumin standard.
CALCULATION AND ANALYSIS OF DATA
Specific enzyme activities were calculated in mU/mg cellular
protein. Changes in enzyme activity and in total cellular pro-
tein content were expressed as percentage of the corresponding
controls. Since viability of the control AM was not affected by
the 21 hr incubation, the viability values were reported directly
as a percentage of total AM in the experimental sample. The data
on AM viability, specific enzyme activity and total protein
levels obtained in the dose response experiments were subjected
to linear regression analysis.
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SECTION 5
RESULTS AND DISCUSSION
VIABILITY AND PHAGOCYTOSIS
Viability of the macrophages was studied to establish the
relative toxicity of the coated and uncoated fly ash in the three
particle size ranges. When the cells were incubated in the
presence of 50 to 1500 yg of particles per lO^AM/ml, a close dose
response relationship between the concentration of the particles
and viability was observed. Within each particle size, decreased
viability of AM was seen upon increase in concentration of the
particles. This response appeared to be linear, generally pro-
viding a significant correlation coefficient (r) ranging from
0.906 to 0.995 for nine out of the twelve particles tested.
Figure 1 shows the least square lines of AM viability plotted
for the various particles as well as the actual mean percent
viability. The data indicate that, at a given concentration, AM
viability decreased consistently with decrease in particle size.
Although untreated fly ash reduced AM viability to a much lesser
extent, nevertheless the particle size effect was clearly evident,
The estimated concentrations of particles that reduced the
AM viability to 75% (LCys) during a 21-hr incubation are shown in
Table 1. The LC75 rather than the LC5Q values were chosen to
TABLE 1. ESTIMATED CONCENTRATION OF PARTICLES REQUIRED TO
REDUCE MACROPHAGE VIABILITY TO 75% DURING A
21-HR EXPOSURE
Particle Particle Concentration, yg/106AM/ml
Coating\Size, y <22-55-8
PbO 180 270 470
NiO 320 550 870
Mn02 375 510 860
None 870 1160 2700*
Extrapolated value.
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00
100
eo
60
m
<40
20
PbO-COATED FLY ASH
2-5/i
100
NiO-COATEDFLYASH
200 400 600 800
CONCENTRATION./ig/ml
1000
20O 400
600 80O 1000
CONCENTRATION ,/ig/ml
1200
I4OO 1600
100
Mn 0 2 - COATED FLY ASH
200 400 600 800
CONCENTRATION./ig/ml
1000
2OO 40O
600 80O 1000
CONCENTRATION ,/ig/ml
1200 1400 1600
Figure 1. Effect of particle concentration and size on viability of macrophages
Each point represents the mean of multiple experiments.
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establish the toxic ranking because in most instances the concen-
trations of particles that reduced AM viability to 50% were mark-
edly higher than those actually included in the exposure experi-
ments, thus would have to be determined by extrapolation. The
results indicate that, irrespective of the particle size, PbO-
coated fly ash was the most toxic with the NiO- and Mn02-coated
fly ash particles following close to each other. The uncoated
fly ash particles reduced viability at markedly higher concen-
trations than the coated particles.
The effect of particle concentration and size on phagocytic
activity was determined. The data indicate that the percentage of
macrophages that had engulfed particles increased with increasing
particle concentration and decreasing particle size. Moreover,
in presence, of the smaller particles not only more macrophages
have phagocytized but also more particles were found within each
cell. The data also show that, as expected, for a given particle
size the percentage of phagocytizing AM was independent from the
test compounds adsorbed on their surface. This is demonstrated
in Figure 2 where the means of pooled observations of the four
particle types within each size range are shown. In presence of
the <2y particles, more than 9070 of the AM phagocytized at the
lowest concentration (250 vig/ml) and saturation was partially
attained at the next highest concentration (500 yg/ml). For the
intermediate and the largest particles the process was more grad-
ual and more strongly concentration dependent. Also, for the 5-8y
particles, even at the highest concentration, only 85% of the
AM phagocytized.
The amounts of metal elements deposited on the various fly
ash particles were analyzed by atomic absorption spectrophotometry
and the results are shown in Table 2.
TABLE 2. PERCENT OF METAL ELEMENTS
ADSORBED ON FLY ASH PARTICLES
Metal Element,
Particle Size,
Coating
PbO
NiO
Mn02
None
<2
3
2
3
.85
.63
,22
a
2-5
3
2
3
.84
.91
.67
a
5-8
3
2
2
.15
.77
.91
a
a Uncoated fly ash contained <0.03% Pb, <0.03%
Ni, and <0.01% Mn.
-------
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-------
The percentage of adsorbed test metal varied within a rela-
tively narrow range for all particles, and the variability within
each particle type was even smaller. Therefore, on a weight/cell
basis, the AM were exposed to fairly constant concentration of
the test metals irrespective of particle size. The increased
toxicity observed with the decreasing particle size, suggests that
the toxic effect is due to concentration as well as to surface
interaction between particles and alveolar macrophages. This is
in agreement with the observations that, within a given concen-
tration, the percentage of phagocytizing macrophages and the
apparent number of particles engulfed by the AM increases with
decreasing particle size. Thus, as smaller particles can be
phagocytized easier in larger numbers, they can provide more sur-
face area for interaction with the lysosomal membrane or release
of cytotoxic components within the phagolysosomes. Large parti-
cles do not affect AM viability as readily because they cannot be
engulfed in similar numbers and, after engulfment, a smaller sur-
face area is available to interact with the lysosomal hydrolases.
Separate studies were conducted to determine whether the ob-
served toxic effects were due to the solubilization of the metal
element from the particles during the exposure. The test parti-
cles were incubated at 37°C for 21 hr and the particle suspension
(P + S) , the filtered particles resuspended in fresh maintenance
medium (P), and the maintenance medium filtered through a 0.22 y
Millipore filter (S) were subsequently incubated with AM suspen-
sions, and after 21 hr AM viabilities were determined. The re-
sults summarized in Table 3 indicate that viability of AM was
equally affected by exposure to the original particle suspension
or the incubated particles resuspended in fresh maintenance
medium. The filtered culture media did not produce decreases in
macrophage viability. This clearly demonstrates that the tox-
icity was not the result of solubilization of the test compounds
in the medium.
11
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TABLE 3. TESTING METAL OXIDE-COATED AND UNCOATED
PARTICLES FOR SOLUBLE CYTOTOXIC COMPONENTS
BY MACROPHAGE VIABILITY
Particle
Size, y Coating
<2 PbO
NiO
Mn02
None
2-5 PbO
NiO
Mn02
None
5-8 PbO
NiO
Mn02
None
AM Viability,
P + Sb
24.3
33.3
31.7
35.2
22.5
52.6
37.3
52.0
26.7
66.2
44.7
80.1
pb
25.1
31.2
31.4
35.8
26.7
41.9
33.2
49.5
26.2
58.6
53.5
81.3
cycl
/o
Sb
92.2
91.5
93.4
92.6
90.9
91.7
92.7
93.0
92.6
92.0
94.0
92.0
3 Viability of control AM = 94.0%
Viability determined after exposure at 37°C for 21 hr
at concentrations of 1000 yg/lO^AM/ml to
P + S Original particle suspension
P Particles separated from medium
S Particle-free filtered medium
12
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ENZYME ACTIVITY
Activity of LDH, acid phosphatase, and B-glucuronidase and
the levels of total cellular protein in AM after 21-hr incu-
bation with the particles were studied to determine if a corre-
lation with viability could be demonstrated or if a more sensi-
tive indicator of cell dysfunction could be established. The
PbO- and Ni.0-coated <2y and 2 to 5y particles were used with ex-
posures to five concentrations for each particle type that re-
sulted in viabilities ranging from 30 to 8070. The results
(Figure 3) indicate that the decrease in viability was generally
paralleled by decreases in cellular protein levels (representing
cell lysis) and specific activity of LDH. A inconsistent de-
creasing trend could be observed for acid phosphatase and there
appeared to be no change in the activity of B-glucuronidase.
The values shown in Figure 3 represent the means and standard
error of three replicate experiments with triplicate assay deter-
minations at each point. The considerably wider standard error
in enzyme and occasionally in protein determinations compared to
those of viability are the result of the complexity of the assay
procedures. In addition, errors due to procedural factors in-
troduced in the unexposed controls, which serve to calculate the
percentage changes, could skew considerably the final specific
enzyme activity levels calculated for the exposed samples. This
is the reason for obtaining specific enzyme values exceeding the
control levels, as we found to be the case frequently for g-
glucuronidase and occasionally at lower concentrations for acid
phosphatase and LDH.
The relationship between the concentration of the particles
and decreases in viability, LDH activity and protein levels was
linear (correlation coefficient ranging from 0.906 to 0.995). As
can be seen from Table 4, the estimated concentrations of the
particles that reduced these experimental parameters to 5070
generally confirmed the toxic ranking determined in the viability-
dose response experiments.
The PbO- and NiO-coated <2y and 2 to 5y particles at concen-
trations that resulted in 50% reduction of viability were com-
pared with uncoated fly ash particles in the same particle size
ranges and concentrations. Although all parameters were affected
by the uncoated fly ash particles, the decreases produced were
generally markedly smaller than upon exposure to the coated
particles (Figure 4). The results of six replicate experiments
demonstrated that the differences in viability and total protein
levels were significant (P <0.001); however, for enzyme activi-
ties this was not always the case.
13
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9-O.UCUROMDASE
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il
4
1
ft
§ § 1§
rt|
^
CONCENTRATION ,/ig/ml OF PbO-COATED FLY ASH, 2-5/u.
IOO
eo
t
ffi
S 40
20
h
*
•yr
^
i
-. ACTIVITY, % OF CONTROL
i I
I
Jit. 0
i
%
CONCENTRATION ,/j.q / m 1
LOH
1
t
^
ACID »<
PHOSPHATASE
1
J
.
i
>UXXIRONIDASE
1
i
3F NiO- COATED FLY ASH . 2-5/U
CONCENTRATION ,/ag /ml OF NIO - COATED FLY ASH , < 2/11
Figure 3. Effect of particle exposure on macrophage viability, total protein
content and specific enzyme activity. Means + SE of three experiments
are shown.
-------
400 500 700 1400
«2) (2-5) «2) (2-5)
400 500 700 1400
«2) (2-5) «2) (2-5)
400 500 700 1400 400 50O 700 1400 400 5OO 70O 1400
«2) (2-5) «2) (2-5) «2) (2-5) «2) (2-5) «2) (2-5) «2) (2-5)
CONCENTRATION , ttg/ml
(SIZE./i)
Figure 4. Comparison of the effects of PbO-coated (•) or NiO-coated (H) and
uncoated (Q) fly ash on viability, total protein content and specific
enzyme activity. Means + SE of six experiments are shown. Significance
for the comparisons: * P<:0.001; ** P^O.Ol; + P
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TABLE 4. ESTIMATED CONCENTRATION OF PARTICLES REQUIRED TO
REDUCE MACROPHAGE VIABILITY, TOTAL PROTEIN CONTENT
AND LDH SPECIFIC ACTIVITY TO 50% DURING 21-HR EXPOSURE
Particle Conc,yg/10 AM/ml, that Reduce to 50%
Particle
Coating\Size, y
PbO
NiO
Viability
2
450
685
2-5
560
1370
Protein Content
2
670
760
2-5
480
1100
LDH Activity
2
485
1345*
2-5
700
3960*
Above the tested concentration range.
Another experiment with these PbO- and NiO-coated and uncoated
fly ash particles was conducted to determine if the presence of
particles affected the enzyme assay system. The particles at the
concentrations used in the experiments were added to AM preincu-
bated for 21 hr at 37°C. The enzyme assay procedure, including all
preparative steps, was immediately performed. The results showed
no differences in the enzyme levels of these samples compared to
control cells. Thus, there appeared to be no interference by the
particles with any of the enzyme assays.
EFFECT OF TIME
Preliminary experiments were also conducted to investigate the
effect on AM viability of length of incubation in presence of
particles in the <2y size range. As shown in Table 5, no changes
were observed during the first 5 hr of the incubation. After 14
hr, gradual decrease in viability was seen for all particles with
the most rapid decrease observed in AM exposed to PbO-coated fly
ash. In control AM, the viability remained constant throughout
the 21-hr period. These observations suggest that the mechanism
of action of these particles is similar to that described for
silica where cell destruction starts after a lag time that permits
sufficient hydrogen bonding of the lysosomal membranes to the
silica in the phagolysosomes.
16
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TABLE 5. EFFECT OF DURATION OF INCUBATION WITH PARTICLESa
ON VIABILITY OF MACROPHAGES
Viability, %
Exposure
Time, hr
1
3
5
14
15.5
18.5
20
21
Particle Coating
Control
97
97
98
97
96
95
97
96
None
94
94
96
88
84
71
60
70
PbO
94
96
97
76
62
46
32
40
NiO
95
95
94
74
80
56
48
55
Mn02
96
97
96
87
83
66
57
53
ft
Estimated concentration of particles producing 50% reduction
in AM viability in 21 hr. Particle size range: <2y.
17
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SECTION 6
RELATED STUDIES
Preliminary studies were conducted with cadmium oxide-(CdO)
coated fly ash. The particles were prepared in<2, 2 to 5 and 5 to 8y
size ranges with respectively 12.1, il.9 and 12.370 cadmium ad-
sorbed on their surface. Cadmium was not detected by atomic
adsorption spectrophotometry on the uncoated control fly ash
particles.
In presence of 100 to 1000 yg particles/106AM/ml, viability
of macrophages decreased between 50 to 60% even after exposure to
the lowest concentrations. Results summarized in Table 6 indicate
that this abrupt decrease in viability occurred in all three
particle size ranges, with relatively little further change.
TABLE 6. EFFECT OF CdO-COATED FLY ASH PARTICLES ON
MACROPHAGE VIABILITY*
Particle
Concentratior
yg/106AM/ml
100
250
300
500
600
750
900
1000
Viability, %
i,
CdO-Coated
Uncoated
<2y 2-5y 5-8y <2y
58
49
53
54
49
51
37
36
.4
.3
.4
.3
,6
.6
.5
.8
59
46
47
40
48
33
36
33
.9
.6
.1
.0
.7
.4
.9
.5
50
44
55
42
42
34
28
27
.1
.0
.0
.9
,7
.0
,1
.9
90
89
80
85
73
81
65
77
.2
.5
.5
.3
.9
.1
.8
.5
2-5y 5-ay
92
95
85
89
74
87
63
82
.7
.2
.7
.0
.8
.7
.1
.8
95.4
92.7
91.8
94.0
90.9
91.5
88.3
90.5
a Determined after 21 hr incubation at 37°C. Viability of control
AM=96.7%.
18
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Comparison with the uncoated fly ash showed that a further de-
crease in viability was generally observed at higher concentra-
tions where the fly ash particles themselves produced a decrease
in AM viability. These results strongly suggested that the
toxicity of the test particles was partially due to solubiliza-
tion of the CdO coating in the incubating medium. This was
supported by visual observations in the light microscope of pro-
fuse bleb formation in the CdO-exposed AM, a phenomenon also
seen under the scanning electron microscope on AM exposed -in
vitro to soluble cadmium salts (6).
To determine whether solubilization of the CdO coating was
indeed taking place, CdO-coated test particles at the highest
exposure concentration were incubated and AM were exposed to the
filtered (0.22y Millipore) medium. A viability decrease to
approximately 50% was seen when AM were exposed to the filtered
medium (S) confirming that solubilization has occurred (Table 7),
TABLE 7. TESTING OF CdO-COATED AND UNCOATED PARTICLES
FOR SOLUBLE CYTOTOXIC COMPONENTS BY MACROPHAGE
VIABILITY
Particle AM Viability. %a
Size, y
<2
2-5
5-8
Coating
None
CdO
None
CdO
None
CdO
P +
49.
9.
69.
20.
77.
19.
o f*\
9
7
9
9
1
4
Pb
67.
22.
63.
37.
67.
25.
6
1
1
6
8
2
Sb
94.8
49.8
90.8
50.2
82.7
49.5
a
Viability of control AM = 91.2%
Viability determined after exposure at 37°C for 21 hr at
concentrations of 1000 yg/106AM/ml to
P + S Original particle suspension
P Particles separated from medium
S Particle-free filtered medium
19
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The viability of AM exposed to the original suspensions (P 4- S)
was considerably lower since, in addition to the CdO that was
released into the medium in the "presolubilization" period, the
cells were also exposed to the whole system for an additional
21 hr. Solubilization of the CdO was also continuing since
the resulting viability values were markedly lower than those
shown in Table 6 for the corresponding exposure concentrations.
According to this hypothesis, AM exposed to the preincubated
washed particles (P) only should have intermediate viability
values, which indeed was noted.
A preliminary experiment was conducted to monitor release
of enzymes after incubation of the AM for 21 hr at 37°C with
CdO-coated and uncoated fly ash particles (<2jj) at 500 yg/106AM/
ml. The results indicated a marked decrease in acid phosphatase
and $-glucuronidase activity in the CdO-exposed AM compared to
the unexposed controls whereas LDH activity could no longer be
detected after the CdO exposure. The AM exposed to uncoated fly
ash showed little or no change in the activity of the three
enzymes compared to the unexposed AM controls.
20
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REFERENCES
1. Yamate, G., and H. Ashley. Preparation and Characterization
of Finely Divided Particulate Environmental Contaminants
for Biological Experiments. IITRI Report No. C6321-5, IIT
Research Institute, Chicago, Illinois, September 1975.
2. Myrvik, 0. N., E. S. Leake, and B. Fariss. Studies on
Pulmonary Alveolar Macrophages from the Normal Rabbit: A
Technique to Procure Them in a High State of Purity. J.
Immun., 86:128-132, 1961.
3. Coffin, D. L., D. E. Gardner, R. S. Holzman and F. J.
Wolock. Influence of Ozone on Pulmonary Cells. Arch.
Environ. Health, 16:633-636, 1968.
4. Weissbecker, L., R. D. Carpenter, P. C. Luchsinger and T. S.
Osdene. In Vitro Alveolar Macrophage Viability. Arch.
Environ. Health, 18:756-759, 1969.
5. Lowry, 0. H. , N. J. Rosebrough, A. L. Farr and R. J.
Randall. Protein Measurement with Folin Phenol Reagent.
J. Biol. Chem., 193:265-275, 1951.
6. Waters, M. D., D. E. Gardner, C. Aranyi and D. L. Coffin.
Metal Toxicity for Rabbit Alveolar Macrophages In Vitro.
Environ. Res., 9:32-47, 1975.
21
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-77-017
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
CYTOTOXIC EFFECT OF TRACE METALS ADSORBED ONTO
FLY ASH PARTICLES
5. REPORT DATE
March 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Catherine Aranyi
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
10. PROGRAM ELEMENT NO.
IIT Research Institute
10 West 35th Street
Chicago, Illinois 60616
1AA601
11. CONTRACT/GRANT NO.
68-02-0761
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory RTP, NC
Office of Research and Development
U..S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final - 4/75 to 9/76
14. SPONSORING AGENCY CODE
EPA/600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Fly ash particles fractionated into three size ranges (<2, 2-5 and 5-8y) were
coated with lead, nickel and manganese oxides to determine the effects of particle
size and surface area on the in vitro toxicity of these metals for alveolar macropha-
ges. When uncoated or coated fly ash particles in concentrations ranging from 50 to
1500 ng/ml were incubated with 10 AM/ml, reduced viability and increased phagocytosis
were observed upon increase in concentration. This was seen irrespective of the
particle size. However, within a given particle coating and concentration, decrease
in particle size resulted in decreased viability and increased phagocytosis. The
percentage of lead, nickel or manganese adsorbed on the fly ash particles varied
within a relatively narrow range, and was not affected by the particle size. Since
the macrophages were exposed to the particles on a weight-dose-per-cell basis, the
concentration of metal oxides to which they were exposed was constant, irrespective
of the particle size. The greater toxicity of the small particles appeared to be due
to surface interaction between particles and the alveolar macrophages. Thus, the
toxic effect is surface-area as well as dose related. These observations were
further supported by decreases in total cellular protein levels and specific
activity of LDH.
17.
a.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COS AT I Field/Group
Toxicity
Phagocytes
Fly Ash
Particles
Lead Oxides
Nickel Oxides
Manganese Oxides
06 T, F
1!3. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
27
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA F:orm 2220-1 (9-73)
22
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