Extracellular requirements for the endocytosis of carcinogenic crystalline nickel sulfide particles by facultative phagocytes

Toxkmiog~ Letters, 12 (1982) 243-250 Elsevier Biomedical Press

243

EXTRACELLULAR REQUIREMENTS FOR THE ENDOCYTOSIS OF CARCINOGENIC CRYSTALLINE NICKEL SULFIDE PARTICLES BY FACULTATIVE PHAGOCYTES

J. DANIEL HECK* and MAX COSTA Division of Toxicology, Department of Pharmacology, The University of Texas, Medica! School at Houston, P.O. Box 20708, Houston, TX 77025 (U.S.A.) (Received February ist, 1982) (Accepted March 2nd, 1982)

SUMMARY Various culture medium components were examined for their effect upon the phagocytosis of carcinogenic crystalline and non-carcinogenic amorphous NiS by cultured fibroblastic cells using both a visual and radioactive assay for phagocytosis. Crystalline NiS was phagocytosed by cells in a simple salts/glucose maintenance medium to an extent similar to that observed in complex culture medium fortified with 10% fetal bovine serum (FBS), suggesting that serum proteins and other components in complex culture medium exert little influence upon the uptake of these heavy metal particles. Phagocytosis of crystalline NiS was shown to be highly dependent upon CaZ+ since omission of Ca2+ from the salts/glucose medium substantially reduced phagocytosis, while readdition of Caz+ stimulated uptake in a concentration-dependent manner. The uptake of the NiS particles was inhibited by trifluoperazine, a calmodulin antagonist, implicating intracellular Ca*+ in this phagocytosis process. Since the opposite surface charge of crystalline and amorphous NiS has been related to their different phagocytic uptake by cells whose primary function is not phagocytosis (facultative phagocytes), these results show that the culture medium components do not modify the surface charge of these particles in a way that significantly influences their uptake.

Particulate nickel compounds of low water solubility vary widely in their carcinogenic potency, in contrast to the soluble nickel compounds which as a group exhibit a more consistent but lower order of carcinogenicity [l-3]. Particles of *To whom reprint requests should be addressed. Abbreviations:

CHO, Chinese hamster ovary; FBS, fetal bovine serum.

0378-4274/82/~~/SO2.75

0 Elsevier Biomedical Press

244

essentially insoluble crystalline nickel sulfide compounds (NiS and NisSz) of respirable size (5 5 pm) induce transformation in cultured cells [4-71, are potent carcinogens in experimental animals f&10] and in the case of crystalline Ni3S.zand perhaps other nickel compounds may have been associated with respiratory cancers among occupationally exposed workers [2, 1l-131. In contrast, a number of other nickel compounds of similar size and similar low water solubility (Ni, Ni@z, NiO, amorphous NiS) exhibit lower potency in the transformation of cells in vitro [7] and in the induction of tumors in vivo (Ni, amorphous NiS) [2, 31. A growing body of experimental evidence 14, 5, 71 attests to the importance of the selective phagocytic uptake of the potently carcinogenic particulates by mammalian cells in contrast to the lack of such uptake of the non-carcinogenic compounds as the primary determinant of the carcinogenicity of these and perhaps other particulate metal compounds. For example, crystalline NiS particles which have a negative surface charge (zeta potential - 27 mV) are actively phagocytosed by a variety of cultured fibroblasts including primary cultures of Syrian hamster embryo cells which undergo morphological transformation following phagocytosis of these particles 14, 14-161. Positively charged amorphous NiS particles (zeta potential +9 mV) are not phagocytosed and do not induce transformation until their surface is changed to a more negative potential by reduction with LiAlH4 [14-161. The phenomenon of particle uptake by cells which do not have phagocytosis as their primary function (i.e. by ‘nonprofessional’ or ‘facultative’ phagocytes as opposed to macrophage) has not been extensively studied. In the light of the fact that these differences in phagocytosis between crystalline and amorphous NiS have been directly linked to their opposing surface charge [ 14-161, we have studied the influence of serum, complex growth medium and calcium on the uptake of these particles since extra cellular conditions may affect surface charge and thereby alter phagocytosis. Calcium ion concentration was the only condition found to exert a significant influence on the phagocytosis of crystalline NiS particles. MATERIALS AND METHODS

Metal compounds Amorphous and crystalline NiS were prepared in the previously described manner [7] by precipitation of amorphous NiS from a NiC12 solution using (NH&S. The amorphous precipitate was heated extensively in a vacuum to form crystalline NiS (low-temperature form, rhombohedral structure). The particles were ground with a Spex 5100 impact grinder (Spex Ind., Metuchen, NJ) and sized by membrane filtration to a mean particle diameter of 4 pm. Crystalline and amorphous 63NiS were prepared in a similar manner from 63NiC12(New England Nuclear, Boston MA) and were sized by passage through a 6 pm pore membrane filter (Nucleopore Corp., Pleasonton, CA). Crystalline Ni3$ was prepared by Dr. E. Rostiner of the University of Connecticut, Storrs, CT [4, 51. The crystalline structure of both

245

crystalline NiS and NisS2 was confirmed by powder X-ray diffraction. NiS lacked any distinct powder X-ray diffraction pattern.

Amorphous

Phagocytosis radioassay: effect of temperature Overnight cultures of approx. 1.10’ CHO cells were established in 25 cm2 culture flasks containing 5 ml of McCoy’s 5A medium supplemented with 10% FBS (Biolabs, Northbrook, IL). The flask caps were then sealed and half were retained in a 37 “C incubator while half were placed into a 4 “C refrigerator 15 min before metal particle treatments. Amorphous and crystalline 63NiS having specific activities of 4002 cpm/pg and 4289 cpm/pg, respectively, were applied to the flasks in O.l-ml ~01s. of sonicated 1 mg/ml suspensions in normal saline. Following a gentle agitation to disperse the particles in the culture medium, the capped flasks were incubated at 37’C or 4°C. At the specified timepoints, the medium was decanted and the cell monolayer was washed with three 5-ml vols. of ice-cold Puck’s Saline A, followed by one 5-ml vol. of saline A acidified with 0.1 N acetic acid, and finally with three more 5-ml vols. of saline A. The cells were then detached from the culture flask with 1 ml of 0.25% trypsin solution and triplicate 0.25 ml aliquots of the resulting cell suspension were applied to Nucleopore polyester filters (25 mm diameter, 10 pm pore) over a mild vacuum. The cells deposited on the filter were then washed with l-ml ~01s. in a three-step washing procedure similar to that described above. Preliminary trials demonstrated that this washing procedure was highly effective in removing ionic and particulate radioactivity from the 10 pm-pore filters following the application of these 63NiS particle preparations which had previously been sized by passage through a 6-pm filter. The filters retained the cells along with internalized 63NiS particles and were counted in 10 ml of 3a70B fluor (Research Products International, Elk Grove, IL) in a Packard 3255 liquid scintillation spectrometer. Phagocytosis radioassay: effect of medium components Overnight cultures of approx. 5. lo4 CHO cells were prepared in 60-mm culture dishes containing 5 ml of McCoy’s 5A medium supplemented with 10% FBS. 15 min prior to treatment with metal particles the medium was decanted, the monolayers were washed with 5 ml of saline A, and 5 ml of the appropriate medium was added to each plate. Our simple salts/glucose maintenance medium consisted of 50 mM HEPES, pH 7.2, 100 mM NaCl, 5 mM KCl, 5 mM glucose, and CaClz ranging from 0 to 2 mM concentration. Trifluoperazine (Smith, Kline and French), an antagonist of calmodulin, was added to some plates at a concentration of 10 PM to discern whether the effect of calcium on NiS phagocytosis was primarily intracellular or extracellular in nature. Phagocytic uptake of amorphous and crystalline 63NiS was allowed to proceed during a 30-min incubation at 37°C following treatment of the plates with 100 pl of a sonicated 500 pg/ml suspension of the metal particles in distilled water. Separation of surface-adsorbed and nonphago-

246

cytized particles from cells was accomplished proeedures described above.

by the washing and filtration

Phagocytosis assay by visual quantitation The effect of medium components such as serum and calcium upon phagocytic uptake of the crystalline and amorphous nickel compounds was assessed visually using the previously described procedure [7]. Culture dishes containing 5 - lo4 CHO cells were prepared and treated with the metal particles in a manner analogous to that employed for the phagocytosis radioassays. 15 min before application of the metal particle suspensions, the cell culture medium was poured off, the ceils were rinsed once with saline A, and the medium was replaced with either McCoy’s 5A medium, this me~um supplemented with 10% FBS, or with the simple salts/glucose maintenance medium containing calcium concentrations ranging from 0 to 2 mM, After a 4-h incubation period at 37 “C, the medium was decanted and the cells were fixed with 95% ethanol and stained with a crystal violet solution. Quantitation of phagocytosis was accomplished by counting the percentage of cells (500 cells in all areas of the plate) containing conspicuous vacuolated intracellular particles under light microscopic (400x) observation. CHO cells were used because internalized particles could easily be distinguished from particles adsorbed on the cell surface by the conspicuous vacuoles formed following endocytosis of the particles. Thioglycollate-stimulated peritoneal macrophage were also isolated from male mice by standard protocols and microscopic phagocytosis assays were performed in these cells following incubation with the nickel compounds for 2 h to 24 h as described above for the facultative phagocytes. RESULTS

The phagocytic uptake of crystalline and amorphous 63NiS over a period of 7 h at either 37 ‘C or 4 “C is depicted graphically in Fig. 1. Uptake of crystalline 63NiS proceeded throughout the 7-h period at 37%Zand was completely inhibited at 4”C, consistent with the hypothesis that the entry of N&derived nickel into the cell is primarily by an active, energy-requiring phagocytic process rather than a passive entry of Ni2+ following extracellular ~ssolution of the NiS particles. In contrast, amorphous NiS showed no signi~cant uptake during the entire 37 *C incubation period, au indication that these particles’ surfaces are lacking in a critical parameter essential to trigger the endocytotic mechanism in these fibroblastic cells. The results depicted in Table I demonstrate that both crystalline nickel sulfides tested were taken up by CHO cells to a significant extent in the complete McCoy’s 5A medium containing 10% FBS, in contrast to an extremely low uptake of similarly sized amorphous NiS particles. In the absence of serum, uptake of the crystalline nickel sulfides was in some cases reduced but remained significant, indicating that serum components such as proteins are not absolutely essential for

241

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the endocytosis of these particles. Phagocytic uptake of all of the metal particles tested was even more avid in the simple salts/glucose maintenance medium, while the large difference in uptake between those with crystalline structures and the amorphous compound was consistently observed under all conditions (Table I). An indication of the critical role of calcium in the uptake of the particles was seen in the results depicted in Table I which addition of the calmodulin antagonist trifluoperazine resulted in reduced uptake of all particle types in the salts/glucose medium. The effect of calcium on the phagocytosis of these particles was therefore investigated further in a study of particle uptake at calcium concentrations of 1 pM, 10 pM, 100 FM and 1 mM. Fig. 2 illustrates that the phagocytic uptake of crystalline NIS increased in response to increasing calcium concentration as measured by both the radioassay of 63NiS uptake and by visual quantitation of intracellular particles. Extensive cell detachment from the growth surface occurred in plates containing medium totally lacking in calcium and it was therefore not possible to quantitate particle phagocytosis by these cells. The uptake of these nickel compounds in mouse peritoneal macrophage was more difficult to quantitate than in the facultative phagocytes. However, at short time intervals (2 h) during which the uptake was linear crystalline NiS was phagocytosed

248 TABLE I THE EFFECT OF MEDIUM COMPOSITION ON THE PHAGOCYTOSIS OF CRYSTALLINE NiS PARTICLES Compound (lo cLg/ml)

McCoy’s 5A Medium 10% FBS

No FBS

Crystalline NiS Amorphous NiS Crystalline NisS2

51.740708 0.45% 29.23%

58.77% 0% 27.14%

Salts/glucose medium (2 mM Cal+)

Salts/glucose medium (2 mM Ca*+) + trifluoperazine

55.53% 2.72% 35.04%

48.42% 0.43% 20.56%

(lo KM)

a Number of cells with nickel sulfide particles/total number examined. Cultured CHO cells were exposed to particles (5 4 am) of the nickel compounds shown in the table for 4 h. Similar findings were observed in several other experiments.

more than was amorphous NiS; at later time intervals the amorphous NiS particles entered a significant number of the macrophage, a result not observed with the facultative phagocytes despite prolonged exposure to high concentrations of amorphous NiS.

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249

DISCUSSION

Costa and Mollenhauer [4, 51 first proposed that the selective phagocytic uptake of particles of certain nickel compounds by potential target cells for in vitro transformation and in vivo carcinogenesis might account for their potency in inducing the expression of these aberrant cell growth patterns. This hypothesis was a logical extension of the observation that the potently transforming and carcinogenic crystalline nickel compounds NiS, NisS2, NisSez were avidly taken up by a variety of cultured mammalian cell types while other nickel compounds such as amorphous NiS which exhibit little or no carcinogenicity are not endocytosed to any appreciable extent [4, 51. Crystalline and amorphous NiS particles have proven to be extremely useful model compounds for the investigation of this phenomenon in that both compounds exhibit similar low water solubility, both can be prepared in similar sizes, and both have been tested for cell transformation and carcinogenesis: the crystalline form having been found to be characteristically potent in these assays and the amorphous form to be virtually inactive. Abbracchio et al. [15, 161 demonstrated a difference in surface charge between Nis particles having crystalline and amorphous structures with determinations of their zeta potentials of -27mV and +9 mV, respectively. Heck and Costa [14] observed that an alteration of the surface of the noncarcinogenic amorphous NiS particles by a vigorous reduction procedure rendered them more negative in charge and resulted in their increased phagocytic uptake by cultured cells and a concomitant potentiation of their in vitro transformation potency to a level similar to that characteristic of crystalline NiS. While the surface charge of any particle and any biological effects associated with that charge are a function of the components of the medium in which the cell and serum particle interact, the present report reveals that particle-adsorbed components such as proteins are not essential for the avid endocytosis of crystalline NiS particles by CHO cells. Such uptake occurs even in a simple minimal salts/glucose maintenance medium at levels comparable to those observed in a complex growth medium with or without serum supplementation, indicating that facultative phagocytes such as cultured CHO fibroblasts do not require opsonization of those particles as a prerequisite to uptake as is commonly the case with macrophage phagocytosis [17]. The fact that selective cellular endocytosis of carcinogenic nickel sulfide particles occurs with only the minimal requirements of physiological temperature and calcium concentrations implicates the operation of a highly specific and efficient endocytotic mechanism in the entry of such particles into the target cells of transformation and carcinogenesis. Macrophage, which are professional phagocytes, exhibit a similar selectivity in the uptake of amorphous and crystalline NiS during the early periods of exposure. However, unlike the facultative phagocytes, macrophage eventually take up substantial quantities of amorphous NIS particles.

ACKNOWLEDGEMENTS

We thank Ms. Linda Haygood for secretarial assistance. This work was supported by Grant No. R 808048 from the US. Env. Protection Agency, by research training grant No. ES 07090 from the National Institute of Environmental Health Sciences, and by contract No. DE-A50581ER 60016 from the U.S. Dept. of Energy. The EPA does not necessarily endorse any commercial products used in this study and the conclusions represent the views of the authors and do not represent the opinions, policies or recommendations of the EPA. REFERENCES 1 M. Costa, Metal Carcinogenesis Testing: Principles and In Vitro Methods, Humana Press, Clifton, NJ, 1980. 2 F.W. Sunderman Jr., Carcinogenicity and anticarcinogenicity of metal compounds, in P. Emmelot and E. Kriek (Eds.), Environmental Carcinogenesis, Elsevier/North-Holland Biomedical Press, Amsterdam, 1979. 3 F.W. Sunderman Jr. and R.M. Maenza, Comparisons of carcinogenicities of nickel compounds in rats, Res. Commun. Chem. Pathol. Pharmacol. 14 (1976) 319-330. 4 M. Costa and H.H. Mollenhauer, Carcinogenic activity of particulate nickel compounds is proportional to their cellular uptake, Science, 209 (1980) 515-517. 5 M. Costa and H.H. Mollenhauer, Phagocytosis of nickel subsulfide particles during the early stages of neoplastic transformation in tissue culture, Cancer Res., 40 (1980) 2688-2694. 6 M. Costa, J.S. Nye, F.W. Sunderman Jr., P.R. Allpass and B. Gondos, Induction of sarcomas in nude mice by impl~tation of Syrian hamster fetal cells exposed in vitro to nickel subsulfide, Cancer Res., 39 (1979) 3591-3597. 7 M. Costa, M.P. Abbracchio and J. Simmons-Hansen, Factors influencing the phagocytosis, neoplastic transformation and cytotoxicity of particulate nickel compounds in tissue culture systems, Toxicol. Appl. Pharmacol., 60 (1981) 313-323. 8 J.P.W. Gilman, Metal carcinogenesis, II. A study of the carcinogenic activity of cobalt, copper, iron and nickel compounds, Cancer Res., 22 (1962) 158-162. 9 G. Jasmin, and J.L. Riopelle, Renal carcinomas and erythrocytosis in rats following intrarenal injection of nickel subsulfide, Lab. Invest., 35, (1976) 71-78. 10 J.P.W. Gilman, Muscle tumorigenesis, in Proc. Sixth Canadian Cancer Conference, Honey Harbor Ontario, Pergamon, New York, 1965, pp. 209-223. 11 R. Lessard, D. Reed, B. Maheux and J. Lainbert, Lung cancer in New Caiedonia: A nickel smelting island, J. Occup. Med., 20 (1978) 815-517. 12 R. Doll, J.D. Mathews and L.G. Morgan, Cancers of the lung and nasal sinuses in nickel workers: A reassessment of the period of risk, Br. J. Indust. Med., 34 (1977) 102-105. 13 L. Kreyberg, Lung cancer in a nickel refinery, Br. J. Indust. Med., 35 (1978) 109-116. 14 J.D. Heck and M. Costa, Surface reduction of amorphous NiS particles potentiates their phagocytosis and subsequent induction of morphological transformation in Syrian hamster embryo cells, Cancer Lett., 15 (1982) 19-26. 15 M.P. Abbracchio, J.D. Heck and M. Costa, The phagocytosis and transforming activity of crystalline metal sulfide particles is related to their negative surface charge, Carcinogenesis, 3 (1982) 175-180. 16 M.P. Abbracchio, J.D. Heck, R.M. Caprioli and M. Costa, Differences in surface properties of amorphous and crystalline metal sulfides may explain their toxicological potency, Chemosphere, 10 (1981) 847-858, 17 F. Ulrich, Phagocytosis of E. coli by enzyme treated alveolar macrophag~, Am. J. Physiol., 220 (1971) 958-966.