video time-lapse microscopy of phagocytosis and ... · was used to observe intracellular detail...

[CANCER RESEARCH 42, 2729-2735, July 1982]0008-5472/82/0042-OOOOS02.00

Video Time-Lapse Microscopy of Phagocytosis and Intracellular Fate ofCrystalline 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 carcinogeniccrystalline nickel sulfide (NiS) particles in Chinese hamsterovary cells were studied using time-lapse video recording withphase-contrast and bright-field optics. Crystalline NiS particles

were phagocytosed by Chinese hamster ovary cells in regionsof membrane ruffling. While these particles may remain boundto the cell surface for variable time intervals (min to hr), theirinternalization generally required only 7 to 10 min. Endocy-

tosed crystalline NiS particles exhibited saltatory motion, andlysosomes were observed to interact repeatedly with the particles in a manner similar to that observed during the digestionof macropinosomes. Particles were never observed to be exo-

cytosed from the cell, and with time, most of the internalizedparticles aggregated in the region around the nucleus. After24 to 48 hr, particle saltation decreased to a point where theparticle position became relatively fixed in the perinuclearregion, and in some instances, this was associated with aconspicuous vacuole formation around the particles. It is concluded that the uptake and distribution of crystalline NiS particles occur by normal endocytic and saltatory processes asoccur during the formation and breakdown of macropinosomes. The observed lysosomal interaction with phagocytosedcytoplasmic NiS may accelerate particulate nickel dissolutionallowing 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 inexperimental animals (17, 26, 27, 30) and of neoplastic transformation in primary cell cultures (9-12, 23). In contrast,

amorphous NiS exhibited no carcinogenic activity in experimental animals (27) and has been repeatedly shown to possessvery weak cell-transforming activity (9-12). Recently, Costa

and Mollenhauer (9,10) related the potent carcinogenic activityof the crystalline nickel sulfide compounds to their selectivephagocytosis by cell cultures, since amorphous NiS particlesof similar respirable size (< 5 jum) were not endocytosed.Further studies by Abbracchio et al. (1 -3) demonstrated thatcrystalline NiS particles had a negative surface potential ( â€”28mV), while amorphous NiS particles exhibited an overall posi-

' Primary funding for this research project was provided by the Office of

Research and Development. Environmental Protection Agency, under Grant R-808048. Additional funding was provided by Research Training Grant ES07090from the National Institute of Environmental Health Sciences and by ResearchContract DE-AS05-81 ER 60016 from the United States Department of Energy.The Environmental Protection Agency does not necessarily endorse any commercial products used in this study, and the conclusions represent the views ofthe authors and do not necessarily represent the opinions, policies, or recommendations of the Environmental Protection Agency. Additional support wasprovided by American Cancer Society Grant BC 334, and National Institute ofHealth Grant AM27078.

2 To whom requests for reprints should be addressed.

Received December 1, 1981 ; accepted March 30, 1982.

tive surface charge (+9 mV). Since the interation of the NiSparticles with the cell membrane involved the particle surface,it was proposed that the negative surface charge was directlyrelated to their endocytosis. The level of phagocytosis of crystalline NiS particles is not influenced by the components oftissue culture media or serum, since similar endocytosis ofcrystalline NiS occurs in a salts/glucose medium as in complete culture growth medium (1 -3,16). Recent studies by Heck

and Costa (16) lend further support to this hypothesis, sincechemical reduction of amorphous NiS particle surfaces withLiAlHi resulted in their avid endocytosis by cultured cells, andthese chemically reduced amorphous NiS particles caused anincidence of transformation equalling that of the crystalline NiSin keeping with the direct relationship between phagocytosisand transforming activity.

It has been hypothesized that the carcinogenic potency ofcrystalline NiS particles is derived from the release of ionicNi2+ inside the cell following their avid phagocytosis by facul

tative phagocytes (potential target cells for transformation capable of phagocytosis but not performing this function exclusively). Thus, particle dissolution is rate limiting in the deliveryof oncogenically active ionic nickel across the nuclear membrane and into the nucleus, since this membrane represents abarrier to the interaction of particulate crystalline NiS with DNA(9). Studies by Abbracchio ef al. (3) suggested that the dissolution of phagocytosed crystalline NiS particles was accelerated by several possible cytoplasmic events of which lysosomalinteraction was implicated as the most probable, since theacidic pH of the lysosomes could enhance the dissolution ofcrystalline NiS particles. The purpose of the present study was3-fold: (a) to further characterize the process by which crys

talline NiS particles are phagocytosed; (b) to observe cytoplasmic lysosome-particle interactions; and (c) to determine particlemovement and distribution inside the cell. For this study, wehave used the technique of video time-lapse microscopy toobtain a continuous record of the interaction of crystalline NiSwith the cell. Our results describe the phagocytosis processand lend support to the proposed lysosomal interaction withcytoplasmic crystalline NiS particles.

MATERIALS AND METHODS

Materials

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 Chemical 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

3The abbreviation used is: CHO, Chinese hamster ovary.

JULY 1982 2729

Research. on October 23, 2020. © 1982 American Association for Cancercancerres.aacrjournals.org Downloaded from

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R. M. Evans et al.

chamber were observed with a Leitz Diavert inverted microscope usinga x 63 oil lens (phase) N.A. 1.4 The platform of the Leitz microscopewas enclosed in a plexiglass box where the temperature was maintained at 37Â°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 tungstenhalogen lamp illuminator, a Ploemopak fluorescence vertical illuminator, 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). Thesignal from the camera was patched into a Panasonic time-lapsevideotape recorder (Model NV-8030) and a Panasonic video monitor(Model NV-5300). All observations reported here were with oil immersion x 63 objective (made with the Dvorak-Stotler chamber) andrecorded at regular speed or at an 18/1 time lapse on 0.5-inch Hitachivideo tape (R-176) (28).

Tape records of video images were photographed either from themonitor as single frames or during continuous playback. Polaroid blackand white photographs were taken using a Polaroid Model CU-5 landcamera (5-inch lens), equipped with a Polaroid Cathode Ray Tubehood (Model 88-49) (12-inch diagonal, at f32, 1/15 sec). Prints werealso made from 16-mm film converted from videotape.

Cell Culture. CHO cells grown in monolayer were maintained inMcCoy's Medium 5A supplemented with 10% fetal bovine serum in an

atmosphere of 5% CO2/95% air. Cells were removed from the mono-

layer by trypsinization, and approximately 1000 cells were introducedinto the Dvorak-Stotler chamber (sterile) with a disposable 5-ml Luer

tip plastic syringe. The chamber containing cells was placed in anincubator (37Â°)overnight to allow reattachment of the cells. Crystalline

NiS, acridine orange, or other treatment chemicals were injected intothe chamber on the following day as described in the legends to thefigures.

Crystalline NiS Treatment. Following attachment (overnight), 20 HQNiS (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 washedwith 5 ml of medium.

Acridine Orange Treatment. Cells were incubated in dark withacridine orange (1 tiQ/m\) for 30 min. The monolayer cells were thenwashed with 3 ml of medium free of acridine orange and allowed toremain in the dark for an additional 15 min prior to photoexcitation (UVrange) and viewing the resulting fluorescence.

Crystalline NiS Particle Identification. Crystalline NiS particlesusually 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 easilyidentified by their light opacity (Fig. 2d; Fig. 3d; Fig. 50). Once NiSparticles were unambiguously identified by bright field, phase contrastwas used to observe intracellular detail during time-lapse video record

ing.

RESULTS

Uptake of Crystalline NiS Particles. Because of theiropaque nature, crystalline NiS particles are readily observableduring phagocytosis. Following 2-hr treatment of CHO cellswith NiS, cells were observed under bright-field optics to firstlocate those cells bearing NiS particles attached to their surfaces. Fig. 1 shows a sequence taken from videotape recordof the internalization of a crystalline NiS particle by a CHO cell.Fig. 1a (f = 0) shows 2 surface-bound nickel particles whichwere first located as 2 black spots by bright-field optics. With

phase contrast, both appear white in the figure. Fig. 1, athrough h, filmed at 18/1, illustrates the sequence of eventswhich occur during phagocytosis of one of these particles (long

arrow). Within 4 min (Fig. 1c), a phase-dense region begins to

form beneath the particle on the right. The particle on the lefthas disappeared from focus as a result of its position on adeveloping membrane ruffle (note the loss of the sharp demarcation of the edge of the cell which could be observed previously in Fig. 1, a and b). Internalization was observed frequentlyat regions of active cellular ruffling. Fig. 1, d and e, demonstrates the progressive formation of a membrane envelopearound 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 theinside of the cell (total time = 8.7 min). The membrane rufflingis greatly diminished in Fig. 1g as indicated by the return of theclearly defined cellular edge with the remaining crystalline NiSparticle (short arrow) still bound to cell surface. Followingendocytosis, the particle moves from the site of uptake by aprocess of saltatory motion, and the path of motion can befollowed in Fig. Mi. The internalized particle in Fig. ih (arrow)was followed for an additional 10 min, at the end of which ithas traveled approximately 10 jum. The uptake of the crystallineparticle represents the first step of in vitro transformationdiagrammed in the model in Chart 1.

Movement of NiS Particles to Perinuclear Region. Fig. 2shows the movement of internalized crystalline NiS particlesfrom a region of membrane ruffling to the perinuclear area. Fig.2a shows a particle at 0 sec. Twelve min later, the particle hastraveled approximately 20/zm in distance and has encountereda phase-dense lysosome (Fig. 2b). Fig. 2c documents the pathof particle from 0 time (*) to its position at 13.2 min. A bright-

field micrograph at 24 min (Fig. 2d) shows the light-opaque

nickel particle in close proximity to the nucleus. The particlealso appears inside a large vacuole which may have formed asa result of lysosomal fusion.

The movement of the particle, once inside the cells, ischaracterized as saltatory movement. Saltatory motion is distinguished from simple Brownian movement by sudden particlemovement, usually of many jum and at velocities in excess ofthose that they may have had prior to saltation or discontinuousjump (20).

aNiS

aNiS_

RUFFLING

twACROSSLINK!Â»IDNASTRAWBREAKSfWWTICN^FIDELITYITREPLICATIÂ».tawmsm ABERRATIONS

TRANSFORMATION

CARCINOGENESIS

BINDING10

HINIPTAKEID-ISHINSALTATORY

nOVBOffFÂ«mamineAREA20-50

J11NACTIVE

LYSOSmIN1BKTION2-3

HRFtRINUOEAR

AGGREGATIONOFIKIEPNALIZO)PARTICLES3-6

WPASSAGE

OFNi** SOLUBJZED

FRfflPARTICLESURFACE3-7HR

Chart 1. Proposed mechanism of crystalline NiS-induced cytotoxicity andcarcinogenesis. The crystalline NiS used in this study was the low-temperatureform (Millerite) which has a rhombohedral crystalline structure. This form ofcrystalline NiS has been designated a or ÃŸdepending upon which reference isconsulted (i.e., fora, see Ref. 14 and for ÃŸ,see Ref. 18). In our studies, we haveused the a designation; however, there is only one other form of crystalline NiSthat exists at room temperature (high-temperature form, hexagonal structure),but a portion of this form eventually reverts to the more stable low-temperatureform within 3 months.

2730 CANCER RESEARCH VOL. 42



Video Microscopy of Selectively Phagocytosed NiS Particles

Lysosome-crystalline NiS Interaction. Fig. 3 shows a sequence from a video recording of lysosome-particle interaction.

Fig. 3, a to c, follows the movement of a lysosome from the cellperiphery toward a phagocytosed NiS particle and records itsbinding to the particle-phagosome. In later sequences (notshown), the lysosomes were observed to detach from theparticle and then bind to the same particle. This process ofbinding and detachment followed by reattachment was repeated over and over. Fig. 3d definitively demonstrates thepresence of the large number of particles that this cell hasaccumulated.

It is of interest to note the perinuclear aggregation of NiSparticles. The 2 largest particles in Fig. 3, near the nucleus,moved only slightly during the entire sequence, which covered17 min of real time.

Lysosome-Particle Interaction Studied by Acridine Orange. 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 lysosomeswith the particle. The acridine orange-acquiring lysosomes are

easily distinguished by their fluorescence and can be seen tobe in close contact with the particle. It is difficult to distinguishwhether the fluorescence at the particle edge is the result offusion between lysosome and the membrane envelope containing the particle or merely a reflection off the particle of thelysosomal fluorescence. However, subsequent vacuolization ofsome particles aggregated around the nucleus suggests thatfusion of lysosome with particles must occur.

Aggregation of Crystalline NiS Particles around the Nucleus. As was observed in Fig. 2, crystalline NiS particles,following phagocytosis, move to the perinuclear region. Withtime, aggregations of particles were observed around the nucleus. After 24 hr, their saltatory motion is greatly diminished,and the particles remain relatively fixed in their positions. Phasemicroscopy (Fig. 5a) suggested the presence of particlesaround the nucleus, and bright-field microscopy (Fig. 5t>) con

firmed that the nuclear aggregated particles were in fact crystalline NiS. The aggregation pattern shown in Fig. 5 is a typicalfinding at times as long as 48 hr following endocytosis ofcrystalline NiS particles. Particles were not observed to beexocytosed from the cell at times to 48 hr.

DISCUSSION

The endocytosis of crystalline NiS particles and its intracel-

lular fate are summarized in Chart 1. This scheme is based inpart on our video-recorded observation of uptake and lyso

somal interaction from 20 different CHO cells and from otherstudies (1 -3, 8-11 ) reported by this laboratory. The first stepin NiS particle carcinogenesis is an endocytic process whichbrings the particle into the cell enclosed in phagocytic vesicle.The surface property of the NiS particle is an important determinant for phagocytosis. Abbracchio ef al. (1 -3) and Heck andCosta (16) have demonstrated that crystalline NiS particlesused in this present study have a negative surface potential,while amorphous NiS particles are characterized by a positivesurface potential. Video time-lapse studies of amorphous NiS

particle interaction with CHO cells show that these particlesreversibly bind to the cell surface and are not phagocytosed.

The binding and uptake of crystalline NiS particles appear tooccur at ruffling areas of the cell membrane. Since the cellmembrane has an overall net negative surface charge, it ispossible that, in these ruffling regions, the membrane chargeis altered, resulting in a favorable electrostatic interaction withnegatively charged crystalline NiS particles. Parallel uptakestudies performed in a simple salts/glucose medium showsimilar results and exclude the possibility that serum proteins,amino acids, or other components modify the phagocytosis ofNiS particles (16). We have also observed that, once crystallineNiS particles bind, they tend to remain attached to the cellmembrane unlike amorphous particles. These and many otherobservations shown in Chart 1 and described in other parts ofthis paper were easily made using time-lapse microscopy and

add considerable new information to our previous studies usingstatic light and electron microscopy (8, 9).

The phagocytosis of single NiS particles can be sequentiallyfollowed in Fig. 1. As described by Silverstein et al. (24), thecell surface membrane applies closely to the particle, so thatthe initial phagosome usually corresponds in size and shape tothose of the particle (Fig. 1h). Fig. 1, c to e, records theformation of phase-dense membrane folds around the particle.

Fig. U may represent the opening of the membrane invagination just before final fusion of the membrane folds. The endocytic vesicle or phagosome of each particle taken up has itsown characteristic size and shape. Changes in vesicle size mayresult from later fusion with lysosomes.

Following phagocytosis of crystalline NiS, there is a short lagperiod during which there is limited lysosomal involvementwhile the particle is actually moved from the ruffling region(Fig. 2). This may loosely be defined as the second step innickel particle carcinogenesis in which the particle is movedfrom the cell periphery to the perinuclear region, during whichtime substantial lysosomal interaction occurs. In extended cellular processes (Fig. 2), particles are observed to saltate in alongitudinal direction toward the nucleus. Similar observationshave been reported by Freed and Lebowitz (15) using time-

lapse cinemicrography of cultured Hela cells treated with carbon particles. The latter studies presented evidence linkinglong saltatory movements with microtubules (15). Linear arraysof microtubules were proposed to be responsible for movingparticles to the cytocenter (i.e., in close proximity to the Golgiapparatus and other structures of the smooth endoplasmicreticulum). In our study, many particles were observed tooccasionally reverse direction of saltation, but the net movement of all particles was to the perinuclear region where, withtime, they aggregated around the nucleus.

The initial interaction of lysosomes with NiS particles wassimilar to that observed by Willingham and Yamada (29) between macropinosomes and lysosomes. This phenomenon,termed piranhalysis, is characterized in mouse 3T3 fibroblastsby the fragmentation of pinosomes mediated by repeated interaction with lysosomes. This piranhalytic interaction betweenlysosomes and NiS-containing phagosome was observed in

this study. This period is schematically illustrated in Chart 1which provides the average time interval of active lysosomalattack.

This contact may result in the exposure of the particle to theacidic content of lysosome. The chemical inference from thesefindings and the studies of Abbracchio ef al. (1 -3) suggest thatthis cytoplasmic interaction accelerates the intracellular dis-

JULY 1982 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, particlemovement became restricted, limited to a slight rocking actionof the particles in a relatively fixed position. No particles wereobserved to be exocytosed from the cells, even after 48 hr. At24 hr, distinct vacuoles could be detected around some particles. Vacuolization may in part be due to the fusion of lyso-somes 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 phagosome in the region between the phagosome membrane and theparticle surface.

The close proximity of the particles to the nucleus as shownin Fig. 5 represents a third stage of the nickel particle carci-nogenesis where the release of nickel ions would result in theirdelivery across the nuclear membrane. Since examinations ofnumerous electron micrographs of cultured cells treated withcrystalline NiS revealed that no particulate nickel entered thenucleus (8, 9), yet other studies demonstrated the presence ofionic nickel in the nucleus (12), dissolution of crystalline NiSparticles may be analogous to a carcinogenesis activation step,and the involvement of lysosomes is key to this process. Ionicnickel binding proteins such as metallothionien may acceleratethe dissolution of the particles by altering particle surfaceequilibrium between dissolved and particulate nickel. The cy-toplasmic dissolution of crystalline NiS particles would begreatly accelerated by the acidic pH of the lysosomes and bythe rapid removal of ionic nickel on the particle surface throughits chelation to metal binding proteins.

Since ionic nickel appears to be the ultimate carcinogen inmediating the genotoxic effects of crystalline NiS, it is noteworthy 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 effects (4) which would contribute to its induction of neoplastictransformation (5, 7, 12, 13, 21). The reason for the potentcarcinogenic activity of the crystalline NiS particles may berelated to the way ionic nickel is delivered into the nucleus, oralternatively, it may simply be a result of the high intracellularnickel levels produced as a result of NiS particle phagocytosis(12).

ACKNOWLEDGMENTS

We thank Linda Haygood for secretarial assistance.

REFERENCES

1. Abbracchio, M. P., Bonura, J., Caprioli, R. M., and Costa, M. Bindingpatterns of carcinogenic and non-carcinogenic nickel particles to variouscellulose papers. Toxicologist. 1: 562. 1981.

2. Abbracchio, M. P., Heck. J. D., Caprioli, R. M., and Costa, M. Differences insurface properties of amorphous and crystalline metal Sulfides may explaintheir toxicological potency. Chemosphere, 10(8): 897-908, 1981.

3. Abbracchio, M. P.. Simmons-Hansen, J., and Costa. M. Cytoplasmic dissolution of phagocytized crystalline nickel sulfide particles: a prerequisite fornuclear uptake of nickel. J. Toxicol. Environ. Health, in press, 1982.

4. Amascher, D. E., and Paillet, S. C. Induction of trifluorothymidine-resistant

mutants by metal ions in L5178Y/TKV~ cells. MutÃ¢t. Res., 78. 279-288,

1980.5. Casto, B., Meyers, J. C., and Dipaolo, J. A. Enhancement of viral transfor

mation for evaluation of the carcinogenic or mutagenic potential of inorganicmetal salts. Cancer Res., 39. 193-198. 1978.

6. Ciccarelli, R. B.. Hampton, J. H.. and Jennette, K. W. Nickel carbonateinduces DNA-protein crosslinks and DNA strand breaks in rat kidney. CancerLett., 12: 349-354, 1981.

7. Costa, M. Metal Carcinogenesis Testing: Principles and in Vitro Methods.Clifton, N. J.: Humana Press, 1980.

8. Costa, M., Jones, M. K., and Lindberg. O. Metal carcinogenesis in tissueculture systems. In: A. E. Marteli (ed.), Inorganic Chemistry in Biology andMedicine, American Chemical Society Symposium Series 140. pp. 45-47.Washington, D.C., 1980.

9. Costa, M., and Mollenhauer, H. H. Phagocytosis of nickel subsulfide particlesduring the early stages of neoplastic transformation in tissue culture. CancerRes., 40: 2688-2694, 1980.

10. Costa, M., and Mollenhauer, H. H. Carcinogenic activity of particulate nickelcompounds is proportional to their cellular uptake. Science (Wash. D. C.),209. 515-517, 1980.

11. Costa, M., Nye, J. S., Sunderman, F. W., Jr., Allpass, P. R., and Gondos, B.Induction of sarcomas in nude mice by implantation of Syrian hamster fetalcells exposed in vitro to nickel subsulfide. Cancer Res., 39. 3591-3597,1979.

12. Costa, M., Simmons-Hansen, J., Bedrossian, C. W. M., Bonura, J., and

Caprioli, R. M. Phagocytosis, cellular distribution, and carcinogenic activityof particulate nickel compounds in tissue culture. Cancer Res.. 41: 2868-2876, 1981.

13. DiPaolo, J. A., and Casto, B. C. Quantitative studies of in vitro morphologicaltransformation of Syrian hamster cells by inorganic metal salts. Cancer Res.,39: 1008-1013, 1979.

14. Eckerline, P., and Kandier, H. Structure Data in Elements and IntermetallicPhase S, Vol. 6, p. 785. New York: Springer Verlag. 1971.

15. Freed, J. J., and Lebowitz, M. M. The association of a class of saltatorymovements with microtubules in cultured cells. J. Cell Biol., 45: 334-354,1970.

16. Heck, J. D., and Costa, M. Surface reduction of amorphous NiS particlespotentiates their phagocytosis and subsequent induction of morphologicaltransformation in Syrian hamster embryo cells. Cancer Lett., 15: 19-26,1982.

17. Jasmin, G., and Solymoss, B. The topical effects of nickel subsulfide onrenal parenchyma. In: G. N. Schrauzer (ed.). Inorganic and NutritionalAspects of Cancer, pp. 69-83. New York: Plenum Publishing Corp., 1978.

18. Kullerud, G., and Yund. R. A. The Ni-S system and related minerals. J.Petrol. 3. 126-275, 1962.

19. Nishimura, M., and Umeda, M. Induction of chromosomal aberrations incultured mammalian cells by nickel compounds. MutÃ¢t.Res., 68: 337-349,1979.

20. Rebhun, L. I. Polarized intracellular particle transport: saltatory movementsand cytoplasmic streaming. Int. Rev. Cytol., 32: 93-137. 1972.

21. Rivedal, E.. and Sanner, T. Sinergistic effect on morphological transformation of hamster embryo cells by nickel sulphate and benz(a)pyrene. CancerLett., 8. 203-208, 1980.

22. Robison, S. H., and Costa, M. The induction of DNA strand breakage bynickel compounds in cultured. Chinese hamster ovary cells. Cancer Lett..15.: 35-40. 1982.

23. Saxholm. H. J. K., Reith, A., and Brogger, A. Oncogenic transformation andcell lysis in C3H/10T '/4 cells and increased sister chromatid exchange inhuman lymphocytes by nickel subsulfide. Cancer Res.. 41: 4136-4139,1981.

24. Silverstein, S. C., Steinman, R. M., and Cohn, Z. A. Endocytosis. Annu. Rev.Biochem., 46. 669-722, 1977.

25. Sirover, M. A., and Loeb, L. A. Infidelity of DNA synthesis in vitro: screeningfor potential metal mutagens or carcinogens. Science (Wash. D. C.), 145:1434-1436, 1976.

26. Sunderman. F. W., Jr. Carcinogenicity and anticarcinogenicity of metalcompounds. In: P. Emmelot and E. Kreek (ed.), Environmental Carcinogenesis, pp. 165-192. New York: Elsevier North-Holland, 1979.

27. Sunderman, F. W., Jr., and Maenza, R. M. Comparisons of carcinogenicitiesof nickel compounds in rats. Res. Commun. Chem. Pathol. Pharmacol., 14:319-330, 1976.

28. Willingham, M. C., and Pastan, I. The visualization of fluorescent proteins inliving cells by video intensification microscopy. Cell, J3. 501-507, 1978.

29. Willingham, M. C., and Yamada, S. S. A mechanism for destruction ofpinosomes in cultured fibroblasts. Piranhalysis. J. Cell Biol., 78: 480-487,1978.

30. Yarita, T., and Nettesheim, P. Carcinogenicity of nickel subsulfide for respiratory tract mucosa. Cancer Res., 38. 3140-3145. 1978.





Fig. 1. Phagocytosis of crystalline nickel sulfide particles. Particle phagocytosis was recorded on videotape at 18/1 time lapse from which Polaroid photographswere 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 CHOcell surface. One particle (tong arrow) will be phagocytosed; the other particle (short arrow) remains on the cell surface, x 1700.

JULY 1982 2733



R. M. Evans et al.

*Osee

Fig. 2. Movement of internalized NiS particle to perinuclear region. Movementof a single particle (black arrow) was followed for 24 min. a to c, phase-contrastphotographs in which the particle appears white; d. a bright-field view where theparticle appears dark. It can be seen in d that other particles have also entered

Fig. 3. Lysosomal interaction with internalized crystalline NiS particles. CHOcells were treated with crystalline NiS (20 fig/ml) for 2 hr, washed, and incubatedfor 16 hr in complete medium. Video time lapse was 18/1. Total real time elapsedbetween 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. NiSparticles are dark in appearance (light opaque). The particle indicated by thearrow is the same particle in a to c. P, particle; Â¿.,lysosome; N, nucleus, x 1700.





Fig. 4. Acridine orange staining of lysosome interacting with crystalline NiSparticles. CHO cells were first treated with 20 /Â¿gof crystalline NiS per ml for 2hr, 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 videorecorded with phase-contrast optics; c (45 sec), the fluorescent lysosomes boundto the particle, lys, lysosome; P. particle; N, nucleus, x 1700.

Fig. 5. Aggregation of crystalline NiS particles around nucleus. CHO cellstreated with crystalline NiS (20 /ig/ml) for 2 hr, washed, and incubated in freshmedium for 48 hr. a, a phase-contrast photograph of NiS particles around thenucleus; b, a bright-field photograph of the same cellular region. Bright-fieldmicroscopy allows for easy identification of the particles, x 1700.

JULY 1982 2735



1982;42:2729-2735. Cancer Res R. Mark Evans, Peter J. A. Davies and Max Costa Mammalian CellsFate of Crystalline Nickel Sulfide Particles in Cultured Video Time-Lapse Microscopy of Phagocytosis and Intracellular

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