[CANCER RESEARCH 42, 2729-2735,
0008-5472/82/0042-OOOOS02.00
July 1982]
Video Time-Lapse Microscopy of Phagocytosis and Intracellular Fate of
Crystalline Nickel Sulfide Particles in Cultured Mammalian Cells1
R. Mark Evans,2 Peter J. A. Davies, and Max Costa
Department of Pharmacology,
The University ot Texas Medical School at Houston, Houston. Texas 77025
ABSTRACT
The endocytosis and intracellular distribution of carcinogenic
crystalline nickel sulfide (NiS) particles in Chinese hamster
ovary cells were studied using time-lapse video recording with
phase-contrast and bright-field optics. Crystalline NiS particles
were phagocytosed by Chinese hamster ovary cells in regions
of membrane ruffling. While these particles may remain bound
to the cell surface for variable time intervals (min to hr), their
internalization generally required only 7 to 10 min. Endocytosed crystalline NiS particles exhibited saltatory motion, and
lysosomes were observed to interact repeatedly with the par
ticles in a manner similar to that observed during the digestion
of macropinosomes. Particles were never observed to be exocytosed from the cell, and with time, most of the internalized
particles aggregated in the region around the nucleus. After
24 to 48 hr, particle saltation decreased to a point where the
particle position became relatively fixed in the perinuclear
region, and in some instances, this was associated with a
conspicuous vacuole formation around the particles. It is con
cluded that the uptake and distribution of crystalline NiS par
ticles occur by normal endocytic and saltatory processes as
occur during the formation and breakdown of macropino
somes. The observed lysosomal interaction with phagocytosed
cytoplasmic NiS may accelerate particulate nickel dissolution
allowing entry of ionic nickel into the nucleus.
INTRODUCTION
The crystalline nickel sulfide compounds (NiS and Ni3S2)
represent a class of extremely potent inducers of cancer in
experimental animals (17, 26, 27, 30) and of neoplastic trans
formation in primary cell cultures (9-12, 23). In contrast,
amorphous NiS exhibited no carcinogenic activity in experi
mental animals (27) and has been repeatedly shown to possess
very weak cell-transforming activity (9-12). Recently, Costa
and Mollenhauer (9,10) related the potent carcinogenic activity
of the crystalline nickel sulfide compounds to their selective
phagocytosis by cell cultures, since amorphous NiS particles
of similar respirable size (< 5 jum) were not endocytosed.
Further studies by Abbracchio et al. (1 -3) demonstrated that
crystalline NiS particles had a negative surface potential ( —
28
mV), while amorphous NiS particles exhibited an overall posi-
tive surface charge (+9 mV). Since the interation of the NiS
particles with the cell membrane involved the particle surface,
it was proposed that the negative surface charge was directly
related to their endocytosis. The level of phagocytosis of crys
talline NiS particles is not influenced by the components of
tissue culture media or serum, since similar endocytosis of
crystalline NiS occurs in a salts/glucose medium as in com
plete culture growth medium (1 -3,16). Recent studies by Heck
and Costa (16) lend further support to this hypothesis, since
chemical reduction of amorphous NiS particle surfaces with
LiAlHi resulted in their avid endocytosis by cultured cells, and
these chemically reduced amorphous NiS particles caused an
incidence of transformation equalling that of the crystalline NiS
in keeping with the direct relationship between phagocytosis
and transforming activity.
It has been hypothesized that the carcinogenic potency of
crystalline NiS particles is derived from the release of ionic
Ni2+ inside the cell following their avid phagocytosis by facul
tative phagocytes (potential target cells for transformation ca
pable of phagocytosis but not performing this function exclu
sively). Thus, particle dissolution is rate limiting in the delivery
of oncogenically active ionic nickel across the nuclear mem
brane and into the nucleus, since this membrane represents a
barrier to the interaction of particulate crystalline NiS with DNA
(9). Studies by Abbracchio ef al. (3) suggested that the disso
lution of phagocytosed crystalline NiS particles was acceler
ated by several possible cytoplasmic events of which lysosomal
interaction was implicated as the most probable, since the
acidic pH of the lysosomes could enhance the dissolution of
crystalline NiS particles. The purpose of the present study was
3-fold: (a) to further characterize the process by which crys
talline NiS particles are phagocytosed; (b) to observe cytoplas
mic lysosome-particle interactions; and (c) to determine particle
movement and distribution inside the cell. For this study, we
have used the technique of video time-lapse microscopy to
obtain a continuous record of the interaction of crystalline NiS
with the cell. Our results describe the phagocytosis process
and lend support to the proposed lysosomal interaction with
cytoplasmic crystalline NiS particles.
MATERIALS
AND METHODS
Materials
' Primary funding for this research
project was provided
by the Office of
Research and Development. Environmental Protection Agency, under Grant R808048. Additional funding was provided by Research Training Grant ES07090
from the National Institute of Environmental Health Sciences and by Research
Contract DE-AS05-81 ER 60016 from the United States Department of Energy.
The Environmental Protection Agency does not necessarily endorse any com
mercial products used in this study, and the conclusions represent the views of
the authors and do not necessarily represent the opinions, policies, or recom
mendations of the Environmental Protection Agency. Additional support was
provided by American Cancer Society Grant BC 334, and National Institute of
Health Grant AM27078.
2 To whom requests for reprints should be addressed.
Received December 1, 1981 ; accepted March 30, 1982.
Materials were obtained from the following sources: Dvorak-Stotler
chamber from Nicholson Precision Instruments, Inc., Bethesda, Md.;
crystalline NiS from Alfa Inorganics; acridine orange from Sigma Chem
ical Co., St. Louis, Mo.; McCoy's Medium 5A from Grand Island
Biological Co., Grand Island, N. Y.
Methods
Time-Lapse
Video Recording.
CHO3 cells in the Dvorak-Stotler
3 The abbreviation used is: CHO, Chinese hamster ovary.
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2729
R. M. Evans et al.
chamber were observed with a Leitz Diavert inverted microscope using
a x 63 oil lens (phase) N.A. 1.4 The platform of the Leitz microscope
was enclosed in a plexiglass box where the temperature was main
tained at 37°by a hair dryer (on warm setting) connected to a Yellow
Springs Instrument Co., Inc., Termistemp temperature
controller
(Model 71 A). The microscope was equipped with a 50-watt tungsten
halogen lamp illuminator, a Ploemopak fluorescence vertical illumina
tor, and a 100-watt mercury and 75-watt xenon epiilluminator (28).
An RCA (Model TC 1030) silicon-intensifier target television camera
was mounted directly by a Leitz adapter to the microscope (28). The
signal from the camera was patched into a Panasonic time-lapse
videotape recorder (Model NV-8030) and a Panasonic video monitor
(Model NV-5300). All observations reported here were with oil immer
sion x 63 objective (made with the Dvorak-Stotler chamber) and
recorded at regular speed or at an 18/1 time lapse on 0.5-inch Hitachi
video tape (R-176) (28).
Tape records of video images were photographed either from the
monitor as single frames or during continuous playback. Polaroid black
and white photographs were taken using a Polaroid Model CU-5 land
camera (5-inch lens), equipped with a Polaroid Cathode Ray Tube
hood (Model 88-49) (12-inch diagonal, at f32, 1/15 sec). Prints were
also made from 16-mm film converted from videotape.
Cell Culture. CHO cells grown in monolayer were maintained in
McCoy's Medium 5A supplemented with 10% fetal bovine serum in an
atmosphere
of 5% CO2/95%
layer by trypsinization,
into the Dvorak-Stotler
air. Cells were removed from the mono-
and approximately 1000 cells were introduced
chamber (sterile) with a disposable 5-ml Luer
tip plastic syringe. The chamber containing cells was placed in an
incubator (37°) overnight to allow reattachment of the cells. Crystalline
NiS, acridine orange, or other treatment chemicals were injected into
the chamber on the following day as described in the legends to the
figures.
Crystalline NiS Treatment. Following attachment (overnight), 20 HQ
NiS (mean particle size, 3 /im) per ml McCoy's Medium 5A were
charged
into the Dvorak-Stotler
chamber by sterile Luer tip syringe.
Cells were incubated with the NiS particles for 2 hr and then washed
with 5 ml of medium.
Acridine Orange Treatment. Cells were incubated in dark with
acridine orange (1 tiQ/m\) for 30 min. The monolayer cells were then
washed with 3 ml of medium free of acridine orange and allowed to
remain in the dark for an additional 15 min prior to photoexcitation (UV
range) and viewing the resulting fluorescence.
Crystalline NiS Particle Identification. Crystalline NiS particles
usually appear as bright spots when viewed by phase optics. However,
by removing the phase disc on the Abbe phase condenser, bright-field
conditions were created which allowed NiS particles to be easily
identified by their light opacity (Fig. 2d; Fig. 3d; Fig. 50). Once NiS
particles were unambiguously identified by bright field, phase contrast
was used to observe intracellular detail during time-lapse video record
arrow). Within 4 min (Fig. 1c), a phase-dense region begins to
form beneath the particle on the right. The particle on the left
has disappeared from focus as a result of its position on a
developing membrane ruffle (note the loss of the sharp demar
cation of the edge of the cell which could be observed previ
ously in Fig. 1, a and b). Internalization was observed frequently
at regions of active cellular ruffling. Fig. 1, d and e, demon
strates the progressive formation of a membrane envelope
around the particle (t = 5.7 to 6.0 min). Fig. 1, f and g, (t = 6.9
to 8.7 min) records the final passage of the particle to the
inside of the cell (total time = 8.7 min). The membrane ruffling
is greatly diminished in Fig. 1g as indicated by the return of the
clearly defined cellular edge with the remaining crystalline NiS
particle (short arrow) still bound to cell surface. Following
endocytosis, the particle moves from the site of uptake by a
process of saltatory motion, and the path of motion can be
followed in Fig. Mi. The internalized particle in Fig. ih (arrow)
was followed for an additional 10 min, at the end of which it
has traveled approximately 10 jum. The uptake of the crystalline
particle represents the first step of in vitro transformation
diagrammed in the model in Chart 1.
Movement of NiS Particles to Perinuclear Region. Fig. 2
shows the movement of internalized crystalline NiS particles
from a region of membrane ruffling to the perinuclear area. Fig.
2a shows a particle at 0 sec. Twelve min later, the particle has
traveled approximately 20/zm in distance and has encountered
a phase-dense lysosome (Fig. 2b). Fig. 2c documents the path
of particle from 0 time (*) to its position at 13.2 min. A brightfield micrograph at 24 min (Fig. 2d) shows the light-opaque
nickel particle in close proximity to the nucleus. The particle
also appears inside a large vacuole which may have formed as
a result of lysosomal fusion.
The movement of the particle, once inside the cells, is
characterized as saltatory movement. Saltatory motion is dis
tinguished from simple Brownian movement by sudden particle
movement, usually of many jum and at velocities in excess of
those that they may have had prior to saltation or discontinuous
jump (20).
aNiS
twACROSSLINK!»
IDNASTRAW
BREAKS
fWWTICN
^FIDELITY
ITREPLICATI».
tawmsm ABERRATIONS
ing.
aNiS_
TRANSFORMATION
RESULTS
CARCINOGENESIS
Uptake of Crystalline NiS Particles. Because of their
opaque nature, crystalline NiS particles are readily observable
during phagocytosis. Following 2-hr treatment of CHO cells
with NiS, cells were observed under bright-field optics to first
locate those cells bearing NiS particles attached to their sur
faces. Fig. 1 shows a sequence taken from videotape record
of the internalization of a crystalline NiS particle by a CHO cell.
Fig. 1a (f = 0) shows 2 surface-bound nickel particles which
were first located as 2 black spots by bright-field optics. With
phase contrast, both appear white in the figure. Fig. 1, a
through h, filmed at 18/1, illustrates the sequence of events
which occur during phagocytosis of one of these particles (long
2730
RUFFLING
OFNi** SOLUBJZED
AGGREGATION
LYSOSm
BINDING10nOVBOffF«m
SURFACE3-7HR
OFIKIEPNALIZO)
PARTICLES3-6 FRfflPARTICLE
AREA20-50
amine
IN1BKTION2-3
HRFtRINUOEAR WPASSAGE
J11NACTIVE
HINIPTAKEID-IS
HINSALTATORY
Chart 1. Proposed mechanism of crystalline NiS-induced cytotoxicity and
carcinogenesis. The crystalline NiS used in this study was the low-temperature
form (Millerite) which has a rhombohedral crystalline structure. This form of
crystalline NiS has been designated a or ßdepending upon which reference is
consulted (i.e., fora, see Ref. 14 and for ß,see Ref. 18). In our studies, we have
used the a designation; however, there is only one other form of crystalline NiS
that exists at room temperature (high-temperature form, hexagonal structure),
but a portion of this form eventually reverts to the more stable low-temperature
form within 3 months.
CANCER
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Video Microscopy
Lysosome-crystalline
NiS Interaction. Fig. 3 shows a se
quence from a video recording of lysosome-particle interaction.
Fig. 3, a to c, follows the movement of a lysosome from the cell
periphery toward a phagocytosed NiS particle and records its
binding to the particle-phagosome.
In later sequences (not
shown), the lysosomes were observed to detach from the
particle and then bind to the same particle. This process of
binding and detachment followed by reattachment was re
peated over and over. Fig. 3d definitively demonstrates the
presence of the large number of particles that this cell has
accumulated.
It is of interest to note the perinuclear aggregation of NiS
particles. The 2 largest particles in Fig. 3, near the nucleus,
moved only slightly during the entire sequence, which covered
17 min of real time.
Lysosome-Particle
Interaction
Studied by Acridine Or
ange. Lysosomes were identified by their phase-dense ap
pearance and their ability to accumulate acridine orange. Fig.
4 shows the interaction of 2 lysosomes with a NiS particle. Fig.
4, a and b, is of a video recording of a phase-contrast sequence
which shows the approach and interaction of the lysosomes
with the particle. The acridine orange-acquiring lysosomes are
easily distinguished by their fluorescence and can be seen to
be in close contact with the particle. It is difficult to distinguish
whether the fluorescence at the particle edge is the result of
fusion between lysosome and the membrane envelope contain
ing the particle or merely a reflection off the particle of the
lysosomal fluorescence. However, subsequent vacuolization of
some particles aggregated around the nucleus suggests that
fusion of lysosome with particles must occur.
Aggregation of Crystalline NiS Particles around the Nu
cleus. As was observed in Fig. 2, crystalline NiS particles,
following phagocytosis, move to the perinuclear region. With
time, aggregations of particles were observed around the nu
cleus. After 24 hr, their saltatory motion is greatly diminished,
and the particles remain relatively fixed in their positions. Phase
microscopy (Fig. 5a) suggested the presence of particles
around the nucleus, and bright-field microscopy (Fig. 5t>) con
firmed that the nuclear aggregated particles were in fact crys
talline NiS. The aggregation pattern shown in Fig. 5 is a typical
finding at times as long as 48 hr following endocytosis of
crystalline NiS particles. Particles were not observed to be
exocytosed from the cell at times to 48 hr.
DISCUSSION
The endocytosis of crystalline NiS particles and its intracellular fate are summarized in Chart 1. This scheme is based in
part on our video-recorded observation of uptake and lyso
somal interaction from 20 different CHO cells and from other
studies (1 -3, 8-11 ) reported by this laboratory. The first step
in NiS particle carcinogenesis is an endocytic process which
brings the particle into the cell enclosed in phagocytic vesicle.
The surface property of the NiS particle is an important deter
minant for phagocytosis. Abbracchio ef al. (1 -3) and Heck and
Costa (16) have demonstrated that crystalline NiS particles
used in this present study have a negative surface potential,
while amorphous NiS particles are characterized by a positive
surface potential. Video time-lapse studies of amorphous NiS
particle interaction with CHO cells show that these particles
reversibly bind to the cell surface and are not phagocytosed.
of Selectively Phagocytosed
NiS Particles
The binding and uptake of crystalline NiS particles appear to
occur at ruffling areas of the cell membrane. Since the cell
membrane has an overall net negative surface charge, it is
possible that, in these ruffling regions, the membrane charge
is altered, resulting in a favorable electrostatic interaction with
negatively charged crystalline NiS particles. Parallel uptake
studies performed in a simple salts/glucose
medium show
similar results and exclude the possibility that serum proteins,
amino acids, or other components modify the phagocytosis of
NiS particles (16). We have also observed that, once crystalline
NiS particles bind, they tend to remain attached to the cell
membrane unlike amorphous particles. These and many other
observations shown in Chart 1 and described in other parts of
this paper were easily made using time-lapse microscopy and
add considerable new information to our previous studies using
static light and electron microscopy (8, 9).
The phagocytosis of single NiS particles can be sequentially
followed in Fig. 1. As described by Silverstein et al. (24), the
cell surface membrane applies closely to the particle, so that
the initial phagosome usually corresponds in size and shape to
those of the particle (Fig. 1h). Fig. 1, c to e, records the
formation of phase-dense membrane folds around the particle.
Fig. U may represent the opening of the membrane invagina
tion just before final fusion of the membrane folds. The endo
cytic vesicle or phagosome of each particle taken up has its
own characteristic size and shape. Changes in vesicle size may
result from later fusion with lysosomes.
Following phagocytosis of crystalline NiS, there is a short lag
period during which there is limited lysosomal involvement
while the particle is actually moved from the ruffling region
(Fig. 2). This may loosely be defined as the second step in
nickel particle carcinogenesis in which the particle is moved
from the cell periphery to the perinuclear region, during which
time substantial lysosomal interaction occurs. In extended cel
lular processes (Fig. 2), particles are observed to saltate in a
longitudinal direction toward the nucleus. Similar observations
have been reported by Freed and Lebowitz (15) using timelapse cinemicrography of cultured Hela cells treated with car
bon particles. The latter studies presented evidence linking
long saltatory movements with microtubules (15). Linear arrays
of microtubules were proposed to be responsible for moving
particles to the cytocenter (i.e., in close proximity to the Golgi
apparatus and other structures of the smooth endoplasmic
reticulum). In our study, many particles were observed to
occasionally reverse direction of saltation, but the net move
ment of all particles was to the perinuclear region where, with
time, they aggregated around the nucleus.
The initial interaction of lysosomes with NiS particles was
similar to that observed by Willingham and Yamada (29) be
tween macropinosomes and lysosomes. This phenomenon,
termed piranhalysis, is characterized in mouse 3T3 fibroblasts
by the fragmentation of pinosomes mediated by repeated in
teraction with lysosomes. This piranhalytic interaction between
lysosomes and NiS-containing phagosome was observed in
this study. This period is schematically illustrated in Chart 1
which provides the average time interval of active lysosomal
attack.
This contact may result in the exposure of the particle to the
acidic content of lysosome. The chemical inference from these
findings and the studies of Abbracchio ef al. (1 -3) suggest that
this cytoplasmic interaction accelerates the intracellular dis-
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2731
R. M. Evans et al.
solution of participate crystalline NiS to ionic nickel.
Particle saltation became greatly reduced with time once the
particle reached the vicinity of the nucleus. After 24 hr, particle
movement became restricted, limited to a slight rocking action
of the particles in a relatively fixed position. No particles were
observed to be exocytosed from the cells, even after 48 hr. At
24 hr, distinct vacuoles could be detected around some parti
cles. Vacuolization may in part be due to the fusion of lysosomes with NiS-containing phagosome. The slight fluorescent
crescent at the edge of the particle in contact with the lysosome
(Fig. 5c) may represent lysosome content entering the phago
some in the region between the phagosome membrane and the
particle surface.
The close proximity of the particles to the nucleus as shown
in Fig. 5 represents a third stage of the nickel particle carcinogenesis where the release of nickel ions would result in their
delivery across the nuclear membrane. Since examinations of
numerous electron micrographs of cultured cells treated with
crystalline NiS revealed that no particulate nickel entered the
nucleus (8, 9), yet other studies demonstrated the presence of
ionic nickel in the nucleus (12), dissolution of crystalline NiS
particles may be analogous to a carcinogenesis activation step,
and the involvement of lysosomes is key to this process. Ionic
nickel binding proteins such as metallothionien may accelerate
the dissolution of the particles by altering particle surface
equilibrium between dissolved and particulate nickel. The cytoplasmic dissolution of crystalline NiS particles would be
greatly accelerated by the acidic pH of the lysosomes and by
the rapid removal of ionic nickel on the particle surface through
its chelation to metal binding proteins.
Since ionic nickel appears to be the ultimate carcinogen in
mediating the genotoxic effects of crystalline NiS, it is note
worthy that ionic nickel can lead to DNA-protein cross-linking
(6), DNA strand breaks (6, 22), chromosomal aberrations (19),
decreased fidelity of transcription (25), and other genetic ef
fects (4) which would contribute to its induction of neoplastic
transformation (5, 7, 12, 13, 21). The reason for the potent
carcinogenic activity of the crystalline NiS particles may be
related to the way ionic nickel is delivered into the nucleus, or
alternatively, it may simply be a result of the high intracellular
nickel levels produced as a result of NiS particle phagocytosis
(12).
ACKNOWLEDGMENTS
We thank Linda Haygood for secretarial assistance.
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VOL. 42
Video Microscopy of Selectively Phagocytosed NiS Particles
Fig. 1. Phagocytosis of crystalline nickel sulfide particles. Particle phagocytosis was recorded on videotape at 18/1 time lapse from which Polaroid photographs
were taken. In a to h. there is a total time lapse of 12 min. In a (O min), 2 crystalline NiS particles (white appearance under phase contrast) become bound to the CHO
cell surface. One particle (tong arrow) will be phagocytosed; the other particle (short arrow) remains on the cell surface, x 1700.
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1982
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R. M. Evans et al.
*Osee
Fig. 2. Movement of internalized NiS particle to perinuclear region. Movement
of a single particle (black arrow) was followed for 24 min. a to c, phase-contrast
photographs in which the particle appears white; d. a bright-field view where the
particle appears dark. It can be seen in d that other particles have also entered
Fig. 3. Lysosomal interaction with internalized crystalline NiS particles. CHO
cells were treated with crystalline NiS (20 fig/ml) for 2 hr, washed, and incubated
for 16 hr in complete medium. Video time lapse was 18/1. Total real time elapsed
between a and d is 17 min. In a to d, the interaction of NiS particles (white arrow)
with a lysosome (black arrow) is followed, d, a bright-field view at 17 min. NiS
particles are dark in appearance (light opaque). The particle indicated by the
arrow is the same particle in a to c. P, particle; ¿.,
lysosome; N, nucleus, x 1700.
2734
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1982 American Association for Cancer Research.
VOL.
42
Video Microscopy
Fig. 4. Acridine orange staining of lysosome interacting with crystalline NiS
particles. CHO cells were first treated with 20 /¿gof crystalline NiS per ml for 2
hr, washed, and incubated for 3 additional hr before treating with acridine orange
(1 mg/ml) as described in "Materials and Methods.' a (O min) and b (40 sec),
the approach and binding of 2 lysosomes to a crystalline NiS particle video
recorded with phase-contrast optics; c (45 sec), the fluorescent lysosomes bound
to the particle, lys, lysosome; P. particle; N, nucleus, x 1700.
JULY
of Selectively Phagocytosed
NiS Particles
Fig. 5. Aggregation of crystalline NiS particles around nucleus. CHO cells
treated with crystalline NiS (20 /ig/ml) for 2 hr, washed, and incubated in fresh
medium for 48 hr. a, a phase-contrast photograph of NiS particles around the
nucleus; b, a bright-field photograph of the same cellular region. Bright-field
microscopy allows for easy identification of the particles, x 1700.
1982
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1982 American Association for Cancer Research.
2735
Video Time-Lapse Microscopy of Phagocytosis and Intracellular
Fate of Crystalline Nickel Sulfide Particles in Cultured
Mammalian Cells
R. Mark Evans, Peter J. A. Davies and Max Costa
Cancer Res 1982;42:2729-2735.
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