United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 2771 1
Research and Development
EPA-600-S1-81-018 July 1981
Project Summary
Ultrastructure and X-Ray
Microanalysis of Macrophages
Exposed to Non-Criteria
Pollutants, with Emphasis on
Certain Metals
John D Shelburne
It is well known that the alveolar
macrophage represents one of the
important first lines of defense of man
and other species against inhaled
environmental pollutants. The study
was undertaken to increase under-
standing of the effects of a wide
variety of environmental pollutants on
alveolar macrophages.
The objectives of this study were as
follows: 1) to investigate the ultra-
structural effects of certain soluble
metals on alveolar macrophages, 2) to
investigate the ultrastructural effects
of realistic and quasirealistic airborne
respirable particles on alveolar macro-
phages, 3) to develop new techniques
not currently available in the literature
that would permit study of the sub-
cellular movement (metabolism) of
ions which either enter cells from
solution or which enter cells from
surfaces, of particles be they outside
cells or within phagolysosomes, and
4) to use these new techniques to
study as many soluble ions and respi-
rable particles as possible.
The study resulted in the develop-
ment of a variety of new techniques
which permit investigators to freeze-
fix alveolar macrophages and thus to
trap normal ions and xenobiotics in
their in vivo location. Using these
techniques, we have studied the sub-
cellular metabolism of numerous solu-
ble ions such as cadmium, nickel,
vanadium, copper, cobalt, and mag-
anese as well as a number of particles
including 2-5 micron coal fly ash
particles with or without coatings of
PbO or NiO, and particles of PbO,
PbaO/t, and NiO. These studies have
shown that this work is possible and
have pointed clearly to future studies
involving new, more sensitive imaging
techniques such as secondary ion
microscopy. These new techniques
will be explored in the future.
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC, to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back}.
Introduction
Interest in this area began with a
series of experiments using NiO and
MnC>2 particles Rabbit alveolar macro-
phages (RAMs) phagocytized these
particles and then fixed them with
conventional techniques. These cells
were then embedded in Epon and stud-
ied by SEM/EDX. Several new mapping
techniques were developed which
worked quite well in the STEM mode
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and which have permitted clear illustra-
tion of the location of these particles
within cells
Next, further study sought to demon-
strate location of a soluble metal within
the cytoplasm of the cell exposed in
vitro—not to particles, but to a metal in
solution. For the initial experiments,
manganese was selected. Manganese
was demonstrated within the cytoplasm
of cells but it was demonstrable only in
electron dense cytoplasmic precipitates
In addition, a wide variety of ultrastruc-
tural effects of manganese was de-
scribed. During these studies, manganese
could not be detected elsewhere in the
cytoplasm of these cells even though
the cells had been incubated with rather
high levels Also, atthistimepreliminary
experiments with soluble cadmium in
vitro were initiated. Again, a large
number of ultrastructural effects of
cadmium using conventional chemical
fixation with glutaraldehyde and con-
ventional embedding in Epon were
described and published However,
even though a galaxy of ultrastructural
changes were present, including fas-
cinating nuclear inclusions not previously
described in the literature, using this
conventionally prepared material it was
not possible to detect the location of
cadmium even though its presence was
very likely High levels had been used for
the exposure and subcellular changes
were very pronounced.
Physiologists have shown that phys-
iologic ions such as sodium and potas-
sium are redistributed tremendously
during chemical fixation and that the
only way to preserve their true in vivo
location is to use freezing techniques.
Experiments with manganese and cad-
mium seem to support that observation
for physiologic ions as well as xenobiotics
No published papers were available,
however, describing how to snap-freeze
mammalian cells in suspension so that
they could be serially sectioned using
cryoultramicrotomy. It was important to
cut sections of these cells since this is
the only way to get inside of them to map
their ultrastructure. Therefore, the first
requirement was to develop new tech-
niques for the freezing of macrophages
either in suspension or as monolayers.
These techniques were used throughout
these experiments.
This study shows that cadmium pro-
duces a lamellar nuclear inclusion
which we have proven to contain cad-
mium by microprobe analysis Inter-
estingly, similar lamellar nuclear inclu-
sions were observed in RAMs exposed
to nickel, either in soluble or particulate
form, as discussed below and in RAMs
exposed in vitro to soluble copper. These
inclusions were not observed in RAMs
exposed to manganese, lead, cobalt, or
iron. In addition to the experiments
noted in this manuscript, the following
new experiments with cadmium were
performed.
Since Chinese hamster ovary (CHO)
cells possess fewer lysosomes and
more mitochondria than do RAMs,
these cells were exposed to soluble
cadmium as had been done previously
for RAMs. The results to date of this
work are that cytoplasmic densities
similar to those noted in RAMs are still
present and these are positive by micro-
probe analysis for calcium, magnesium
and cadmium. The presence of mag-
nesium is strong evidence that these
inclusions are in mitochondria. Freeze
substituted images are consistent with
but do not absolutely unambiguously
prove that hypothesis since the mito-
chondria appeared damaged by the
cadmium and thus are difficult to recog-
nize as mitochondria. The striking
finding was that cadmium did not pro-
duce nuclear inclusions in CHO cells
even though it did at the same doses and
times produce nuclear inclusions in
RAMs. Thus, CHO cellsare nota suitable
model for studying the pathophysiology
of cadmium with respect to itseffectson
nuclei. Cadmium did produce clumping
of heterochromatin in treated cells but
this did not appear to be a specific
finding in the same sense that cadmium
nuclear inclusions are.
Since the cytoplasmic densities ob-
served in previous experiments with
RAMs exposed to cadmium also gave
peaks for phosphorus when studied by
EDX, and because of the presence of
extracellular cadmium phosphate pre-
cipitates, these experiments were re-
peated using special phosphate and
sulfate free medium. When compared to
cells exposed to regular media, control
cells exposed to this special media
without cadmium exhibited more num-
erous homogeneous nuclear bodies at4
hours, swelling of ER cisternae, and
peculiar flocculant mitochondria! matrix
densities. Cells in this special media
treated with cadmium did exhibit char-
acteristic lamellar nuclear inclusions
but fewer than observed in previous
experiments with regular medium. No
cytoplasmic densities at all were ob-
served. Homogeneous nuclear bodies
were extremely common. No extracellu-
lar precipitation of any sort was seen in
frozen sections Thus, this special
media did prevent the problem of extra-
cellular precipitation noted previously
but the lack of phosphate (probably)
seems to have blocked the formation of
mitochondnal densities containing cad-
mium and blocking partially the forma-
tion of cadmium specific nuclear bodies
Microprobe analysis of frozen sections
revealed - in marked contrast to previous
experiments with regular media - a
surprising lack of detectable cadmium.
Nuclear inclusions were magnesium,
calcium and phosphorus positive, but
only occasionally exhibited a trace of
cadmium No other cadmium localization
was detected. Perhaps the absence of
phosphate prevented cadmium transport
into cells (phosphate is, in some in-
stances, necessary for calcium trans-
port). Or, perhaps the phagocytosis of
cadmium phosphate precipitates is
necessary to get large amounts of
cadmium into cells in a hurry (4 hours),
bypassing normal membrane barriers.
Experiments to test these hypotheses
are underway
A series of experiments were con-
ducted on a mutant strain of CHO cells
developed by Dr. Ken McCarty, Sr ,
Department of Biochemistry, Dukej
University. These CHO cells are resistant!
to cadmium and can successfully bind
large amounts of cadmium mtracellu-
larly without visible toxic effects An
induced metallothionem is responsible
for the remarkable behavior of these
cells. Four different variables were
studied "Wild" CHO cells with and
without cadmium and mutant CHO cells
with and without cadmium were incu-
bated on glass andThermonoxcoverslips
for 4 hours (wild) and 48 hours (mutant)
in 0.1 mM cadmium chloride. At the end
of the incubation, these coverslips were
snap frozen in liquid nitrogen and
freeze-dned on an Edwards freeze-
drying apparatus or in a rotary vacuum
pump chamber. After a light carbon
coating, these cells were studied by
SEM-EDX Some coverslips were rinsed
in medium without cadmium before
snap freezing. The principal findings to
date are' 1) The glass coverslip is a poor
substrate because it contributes to high
background radiation and to numerous
spurious peaks including silicon, zinc,
titanium, and chromium On the other
hand, Thermonox coverslips are an
excellent substrate as there is no in-
creased background radiation. 2) The
mutant cells exposed to cadmium dc
exhibit cadmium peaks and there are
differences between cells. No cadmiurr
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peaks were seen in "wild" cells with or
without cadmium exposure. These stud-
ies were done using the spot probe
mode since cell size varies significantly.
3) "Wild" cells treated with cadmium
were rounded. "Wild" cells not treated
with cadmium and most mutant cells
th or without cadmium were relatively
flat 4) Sulfur peaks were consistently
higher in treated mutant cells than in
any of these other groups, possibly due
to the presence of the induced metal-
lothionem. 5) The results with rinsed vs
unrmsed cells were the same.
In addition, perhaps the most inter-
esting results of this work with CHO
cells concerned viral particles. Experi-
ments with CHO cells in two different
laboratories have shown that treatment
with cadmium causes the production of
a type RNA virus, probably a tumor
virus In view of the fact that cadmium is
a carcinogen, we feel this hitherto un-
reported finding is a significant discovery
John D. Shelburne is with the Department of Pathology, Duke University Medi-
cal Center, Durham, NC 27710.
Michael D. Waters is the EPA Project Officer (see below).
The complete report, entitled "Ultrastructure andX-Ray Microanalysis ofMacro-
phages Exposed to Non-Criteria Pollutants, with Emphasis on Certain Metals,"
{Order No PB 81 -157 935, Cost $14 00, subject to change) will be available
only from.
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone. 703-487-4650
The EPA Project Officer can be contacted at
Health Effects Research Laboratory
U S Environmental Protection Agency
Research Triangle Park, NC 27711
',- US GOVERNMENT PRINTING OFFICE 1981 -757-012/7234
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Environmental Protection
Agency
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Information
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