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[CANCER RESEARCH 45, 2320-2325, May 1985] Induction of Chromosomal Damage in Chinese Hamster Ovary Cells by Soluble and Particulate Nickel Compounds: Preferential Fragmentation of the Heterochromatic Long Arm of the X-Chromosome by Carcinogenic Crystalline NiS Particles1 Pramila Sen and Max Costa2 Department of Pharmacology, University of Texas Medical School at Houston, Houston, Texas 77025 ABSTRACT Treatment of intact Chinese hamster ovary cells with crystal line NiS and NiCI2 resulted in the induction of chromosomal aberrations which included gaps, breaks, and exchanges. The incidence of these aberrations increased in a time- and concen tration-dependent fashion. NiCI2 was more potent in inducing chromosomal aberrations in cells that were maintained with a salts/glucose medium during metal treatment than when cells were treated in culture growth medium. Chromosomal aberra tions induced by NiCI2 occurred randomly among the autosomal arms; however, the heterochromatic centromeric regions of the chromosomes were preferentially damaged. In addition to induc ing the same type of aberrations found with NiCI2, crystalline NiS particles also caused the selective fragmentation of the hetero chromatic long arms of the X-chromosomes. This fragmentation was attributed to the difference in the mechanism of delivery of nickel ions from phagocytized crystalline NiS particles which aggregate around the nuclear membrane and release large amounts of nickel ions from a dissolving phagocytized particle. Previous studies have demonstrated that treatment of intact cells with crystalline NiS particles produces a considerably higher level of nickel in the nucleus compared with similar exposure to water-soluble NiCI2. Since heterochromatin is known to form the inside lining of the interface nucleus, nickel ions, as they are solubilized from a phagocytized particle and enter the nucleus, are likely to encounter heterochromatin before they interact with euchromatin. In contrast, nickel ions derived from NiCI2 do not preferentially accumulate in the cell, and those ions that enter the cell are taken up by a nonphagocytic mechanism. It is proposed that when cells are treated with high levels of NiCI2 in an attempt to achieve the cellular levels of nickel produced by NiS phagocytosis, this overloading results in cytotoxic responses rather than the preferential fragmentation of heterochromatin observed with particles. Since liposome-mediated delivery of NiCI2 also results in fragmentation of the long arm of the Xchromosome, the selective breakage of heterochromatin by NiS particles may be due solely to the mechanism of Ni2+ delivery in cells. INTRODUCTION Nickel compounds represent well-established human carcino gens based upon epidemiológica! studies (1-3). Certain nickel 1This work was supported by Grant CA 24581 from the National Cancer Institute and by Contract DE AS05-81-ER 600 16 from the U.S. Department of Energy. 2 To whom requests for reprints should be addressed, at Department of Phar macology, University of Texas Medical School at Houston, P. 0. Box 20708, Houston, TX 77025. Received 11/6/84; revised 1/29/85; accepted 2/1/85. CANCER RESEARCH compounds such as crystalline nickel sulfide and subsulfide are extremely potent carcinogens in experimental animals (4-6), while others such as amorphous NiS exhibit less potency (5). In contrast to these particulate nickel compounds, water-soluble nickel compounds are not considered carcinogenic in experimen tal animals, even following multiple injections (7), and they are less potent in transforming cells compared with the crystalline NiS particles (8,9). Based upon studies in tissue culture, it has been proposed that the transforming and carcinogenic activity of crystalline Ni3S2, crystalline NiS, and amorphous NiS was proportional to their cellular uptake by potential cancer target cells that exhibited facultative phagocytic properties (not macrophages) (10, 11). The active phagocytosis of the potently carcinogenic crystalline Ni3S2 and NiS represents a highly efficient mechanism for deliv ering large quantities of nickel ions into the cells (12). Mounting experimental evidence attests to the importance of such a phag ocytic mechanism in cancer target cells as an initial step in the carcinogenicity of crystalline nickel sulfide particles in vivo (13, 14), although the cause-and-effect relationship of this first step has not been unequivocally proven (14). Following phagocytosis, the particles are solubilized in the cell, possibly as a result of the lysosomal acidification of the vacuole containing the endocytized particle (15). In fact, recent studies have shown a good correlation of cytotoxicity with the dissolution of phagocytized particulate nickel compounds at acidic pH (16). This intracellular solubilization is important for the nuclear uptake of ionic nickel, since particles of NiS cannot cross the nuclear membrane (8). Video intensification microscopy studies indicate that phagocytized crystalline nickel sulfide particles which are contained in highly acidified vacuoles aggregate around the nucleus (12). It has been shown that solubilized nickel is gener ated from these particles and that it enters the nucleus where it interacts with DNA. The nuclear levels of nickel were shown to be high in cells treated with crystalline NiS particles compared to cells treated with equivalent concentrations of NiCI2 (13, 14). Additionally, cells treated with crystalline NiS particles exhibited a greater proportion of nickel bound to DNA relative to that bound to protein in comparison with cells similarly treated with NiCI2 (13, 14). In addition to the binding of nickel to the DNA bases, lesions such as DNA-protein cross-links and single strand breaks are induced in the DNA by this metal ion (17, 18). The ability of Ni2+ to coordinate with protein and DNA in a highly stable ternary complex may indicate a preference for Ni2+ to remain in the nucleus (13,14,17,18). Since the formation of this Ni-DNA-protein complex is dependent upon the nuclear Ni2+ concentration and also upon the ability of cells to replicate their VOL. 45 MAY 1985 2320 SELECTIVE DAMAGE TO HETEROCHROMATIN DMA (13, 14), the nuclear formation of this complex is favored with NiS particles more than NiCI2 (see above). In the present study, we have examined and compared the effect of NiCI2 and crystalline NiS particles on the chromosomes of Chinese hamster ovary cells. Our results show that both compounds produce a number of chromosomal lesions, but in general, heterochromatic regions appear to be a prevalent site where chromosomes are damaged by ionic nickel. The phagocytized crystalline NiS particles appear to produce a more striking effect on heterochromatin compared with NiCI2, since the long arm of the X-chromosome is selectively fragmented by crystalline NiS treatment under conditions where there are no observable effects on other chromosomes. Water-soluble NiCI2 did not pro duce any similar fragmentation of the long arms of the X- BY NICKEL COMPOUNDS plating. Mitotic cells were plated into 100-mm plastic Retri dishes and were treated with either 100 or 500 MM NiCI2 for 2 hr in SGM, 1, 3, 5, and 11 h after the initial plating, for the analysis of aberrations induced by NiCI2 during early G,, late G,. early S, and late S phase, respectively. After each treatment, a 24-h recovery time was given. Mitotic cells were selected by Colcemid treatment (see above) and dislodged by gently pipetting the overlaying medium. The cells were collected by centrifugation at 1200 rpm, treated with a hypotonie solution (0.56% KCI) for 5 min at room temperature, and fixed in 3:1 methanol/glacial acetic acid fixative for 30 min with two changes of fixative. The cell suspension was finally dropped onto clean wet slides and air dried. Slides were stained with Giemsa and then mounted and subsequently scanned for qualitative and quantitative analysis of the chromosomal aberrations. Cell cycle position was monitored by mitotic index or [3H]thymidine labeling index. chromosome, but the centromeric regions of different chromo somes were often found to be involved in chromosomal aberra tions with either compound. It is proposed that the long arm of the X-chromosome is selectively damaged by crystalline NiS particles because the heterochromatin of this chromosome forms the bulk of an inside lining in the interface nucleus (15) and represents the first chromatin site nickel ions encounter as they enter the nucleus (12). The greater selectivity of NiS over NiCI2 to produce this fragmentation is thought to relate only to the concentration of nickel that can be delivered into the nucleus from localized particles surrounding the nuclear membrane. RESULTS Induction of Chromosomal Aberrations by NiCI2. Table 1 demonstrates the induction of chromosomal aberrations by NiCI2 in CHO cells maintained in a SGM or complete a-MEM medium. Treatment of cells for 2 h with NiCI2 followed by a 24-h recovery time resulted in a concentration-dependent induction of aberra tions. The effect occurred at a lower concentration when cells were treated with NiCI2 in a SGM compared with complete growth medium. This was due to the higher uptake of nickel into cells maintained in SGM. At least 100 metaphase cells were evaluated for each treatment time shown in each table. Control cells were routinely scored for each experiment; typical control aberrations are given in Table 1. Table 1 also shows aberrations in cells treated for 6 h with NiCI2; however, higher concentrations for this time interval or similar concentrations for longer treatment times resulted in no metaphases. As noted in the table, the majority of chromosomal aberrations observed consisted of gaps, breaks, and exchanges with rare occurrence of dicentrics and fragments. At 1 mwi NiCI2, 14.4% of all aberrations were found to be located in centromeric regions of the chromosomes. This is in contrast to a random expected value of 1 to 2% for MATERIALS AND METHODS CHO3 cells were grown as monolayer cultures in plastic Retri dishes with a-MEM (Grand Island Biological Co., Grand Island, NY). The medium was fortified with 10% fetal bovine serum (Armour Pharmaceutical Co., Kankakee, IL) and with penicillin (100 U/ml), fungizone (0.25 ¿jg/ml),and of streptomycin (100 nQ/m\). NiCI2 and crystalline NiS were purchased from Alfa Inorganics (Danvers, MA). Crystalline NiS used was the low temperature form (Millerite), which has a rhombohedral crystalline struc ture (8). Paniculate NiS was ground with an impact mill, and particles averaging 2 to 3 ^m in diameter were prepared by nucleopore filtration as described previously (8). Physiological concentrations of metal binding amino acids such as cysteine and histidine have been found to exert strong inhibitory effects on the toxicity and uptake of metal ions such as Ni2+ (19). They also Tabtel Concentrationdependenceand comparison of chromosomalaberrations induced by NiCli in Chinesehamster ovary cells Intact CHO cells were treated with NiCI2under the conditions shown. The metal was removed, and the cells were allowed to recover for 24 h prior to collection of mitotic cells, as described in "Materials and Methods." Previous studies have appear to account for the majority of inhibitory activity of whole serum toward uptake (19); therefore, in selected instances, cells were treated with NiCI2 while maintained in a simple SGM. Synchronized or log-phase shown that incubation of CHO cells in a SGM for at least 12 h does not affect plating efficiency and trypan blue exclusion (19, 27). cultures were placed in the SGM for relatively short time intervals (1 to 3 hr) only during the time of metal exposure. Previous studies have demonstrated that incubation of CHO cells in this SGM did not decrease cell viability or change other growth parameters (19). NiCI2 was dissolved in SGM just prior to use and was filter-sterilized by passage through a 0.45-Mtn millipore filter. Stock suspensions of crystalline NiS particles NiCI2con with aberrationsG"868141813281547111315612B971291722573 ot with centration damage multiple (%)9.1611.4"15.7"25.7"34.2*32.0"58.7"42.5"5.8C8.3C17.8C22.8C19.2e GIM)001101001005001000001005001000250500Treatment time (h)2222222224822266Cells damage000000150000000 were prepared by sonicating 2 to 3 ^m particles in ethanol, collecting and drying the sterilized particles, and resuspending them in a stock of sterile 0.9% NaCI solution. Chromosomes were prepared from Colcemidarrested (0.02 ^g/ml for 2 h) mitotic cells that had been treated with the nickel compounds. In order to study the cell cycle specificity of NiClr induced aberrations, cells were synchronized by the selective detach ment of mitotic cells. Cells were plated in flasks 12 h prior to mitotic selection. Four hr before selection of mitotic cells, Colcemid (0.02 ¿¿g/ml) was added, and the cells were washed two times with medium before * G. gaps; B, breaks; E, exchanges; D, dicentrics; F, fragments. 6 Treated in SGM. c Treated in «-MEMwith fetal bovine serum. 3The abbreviationsused are: CHO, Chinesehamster ovary; SGM, salts/glucose medium [50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid buffer, pH 7.2:100 mMNaCI:5 mw KCI:5 mw CaCI2];a-MEM, «-minimal essential medium. CANCER RESEARCH VOL. 45 MAY 1985 2321 SELECTIVE DAMAGE TO HETEROCHROMATIN centramene damages. Figure 1 illustrates the type of aberrations frequently seen with NiCI2. The broken arrow in Figure 1a dem onstrates an exchange induced by NiCI2, while the solid arrow illustrates a nickel-induced gap. The broken arrow in Figure 10 shows a centromeric exchange induced by NiCI2, while the other arrow illustrates a centromeric break. Cell Cycle-specific Induction of Chromosomal Aberrations by NiCI2. Table 2 shows the chromosomal aberrations induced by NiCI2 in CHO cells at different stages of the cell cycle. There was very little aberration above background level exhibited dur ing the Gìphase (Table 2). During S phase and particularly late S phase, there was a striking increase in the frequency of chromosomal aberrations. These findings suggested that the DNA replication phase is more susceptible to the induction of chromosomal aberrations by NiCI2 and the aberration frequency observed in a log-phase population of cells may be due to the S phase population of that culture. It was also interesting to note that most of the aberrations were caused during late S phase, the cell cycle period when heterochromatic DNA replicates. Effect of Crystalline NiS Particles on the Induction of Chro mosomal Aberrations. Table 3 demonstrates that treatment of intact CHO cells with crystalline NiS results in a time- and concentration-dependent induction of chromosomal aberrations. The nature of the aberrations observed with crystalline NiS were very similar to those found with NiCI2 except that, with crystalline NiS particles, there was also a selective fragmentation of long arms of the X-chromosomes. Thus most of the NiS-induced fragmentation shown in Table 3 represents the heterochromatic long arms of the X-chromosomes. The induction of chromosomal aberrations by crystalline NiS particles was dependent upon time since, even at high concentrations, there were few aberrations seen at 6 h, but these progressively increased after 24 or 48 h. Cell cycle-specific BonEarly phase(1-3h)cLate d BY NICKEL COMPOUNDS Figure 2 illustrates the fragmentation of the long arms of the Xchromosome caused by crystalline NiS particles. Progressing from a to f in Figure 2, there was more striking fragmentation of the long arm of this chromosome compared to other chromo somes that are not heterochromatic. Even the short arm of the same X-chromosome did not exhibit fragmentation. At higher concentrations of NiS for longer exposure time intervals (I.e., 48 h), it was difficult to identify the chromosomes involved in the fragmentation because fragmentation was extensive. However, analysis of the intact chromosome morphology as well as the morphology of the partially fragmented chromosome indicated that the heterochromatic long arm of the X-chromosome was often involved in this fragmentation. As many as 19.5% of the total damaged cells had fragmentation after 48 h and, in most instances, these involved breakage of the long arm of the Xchromosome (Table 3). Fewer cells with fragmentations were found after 6- and 24-h treatments at all concentrations of crystalline NiS. The occurrence of selective fragmentation by NiS but not by NiCI2 treatment suggested that this might be due to a difference in the mechanism of delivery of nickel ions into the nucleus (13). To test this hypothesis, CHO cells were treated with nickel ionsaturated albumen which was encapsulated into the liposomes. This allowed the nickel to enter cells by a mechanism that may model the delivery of nickel particles into the cells. To prepare liposomes containing nickel-saturated protein, albumin was treated with NiCI2, and unbound nickel was removed by Sephadex G-10 chromatography.4 CHO cells in logarithmic growth phases were treated with various concentrations of this complex in a SGM and in complete a-MEM medium for time intervals ranging from 4 to 24 h. A minimum of 100 metaphase cells were analyzed for the presence or absence of chromosomal fragmen tation. Treatment of cells for 4 h with liposomes containing the Table 2 Ni-albumen complex (NiCI2 concentration of 400 UM) resulted in induction of chromosomal abbreviations by NiCI¡in CHO cells a maximum of 4.2% cells with fragmentation, which, in many Types of aberrations cases, could be attributed to fragmentation of the X-chromo NiClz (JIM)"100500100500100500100500damage (%)13.216.619.718.531.439.444.956.0G"21691516431519B131111630452427E2363351020D02326202F00001000 somes. Unencapsulated NiCI? alone, when added at similar or higher concentrations, did not produce any chromosomal frag mentation.4 Similarly, liposomes alone, liposomes with albumen, phase(3-5 G, h)Early phase(5-7 S h)Late phase(11-13h)Of S or albumen with NiCI2 alone, when added at similar or higher levels, did not cause any fragmentation of the long arm of the Xchromosome. Additional experiments were also conducted to examine whether the irritant effect of an internalized particle may produce fragmentation of the X-chromosome or other chromo- * Cells were treated in a SGM for 2 h. " G, gaps; B, breaks; E, exchanges; D, dicentrics; F, fragments. : Time interval following release from mitosis during which NiClz was added. 4 P. Sen et al., unpublished observations. Table 3 Induction of chromosomal aberrations by crystalline NiS in intact Chinese hamster ovary cells Crystalline NiS Types of aberrations Cells with mei na (h)666242424484848OBIIä (%)22.821.621.520.016.140.334.036.661.317.0G"1318141116221916388B2619242116313143497E36542681131D202313961 tion(%)0026377612 [ecumenitime Will]damage uunuciiif aiiuii(xQ/ml)510205102051020Untreatedi a G, gaps; B. breaks; E, exchanges; D, dicentrics. CANCER RESEARCH VOL. 45 MAY 1985 2322 Cells with SELECTIVE DAMAGE TO HETEROCHROMATIN somes. Cells were treated with activated charcoal particles at concentrations and for time intervals similar to those utilized for analysis of chromosomal fragmentation by crystalline NiS parti cles (see Table 3). Since activated charcoal particles were con siderably less dense than crystalline NiS particles, there was a greater particle exposure for a given mass of these particles compared with crystalline NiS particles. There were no fragmen tations of chromosomes induced by exposure of cells to acti vated charcoal, despite the active phagocytosis of these particles (data not shown). DISCUSSION The extent and nature of chromosomal damage induced by nickel compounds was dependent upon the method of delivery of nickel ions and upon the cell cycle position. NiCI2 was not readily taken up by cells in tissue culture media, due to the inhibitory effects of nickel binding amino acids such as cysteine and histidine present in tissue culture growth media (19). Thus, the potency of NiCI2 in inducing chromosomal aberrations was considerably increased when cultured cells were exposed to this metal ion in a minimal salts/glucose maintenance medium. Ex posure of cells to the minimal salts/glucose medium per se did not result in any measurable effects on a number of cellular functions (19). The mechanism of metal delivery also influenced the nature of the chromosomal lesion observed. Although soluble NiCI2 pro duced a high level of centromere-associated aberrations, it did not, under any conditions, cause fragmentation of the long arm of the X-chromosome as was observed following treatment with crystalline NiS particles. However, introduction of nickel-albumen complexes into cells by means of liposomes did produce heterochromatic fragmentation of the X-chromosome.4 In interphase cells, condensed heterochromatin forms the inner lining of the nuclear membrane (15). Thus, it is likely that the nickel ions liberated after the solubilization of NiS particles in perinuclear regions interact with heterochromatic DNA before interacting with euchromatic DNA. Since the long arm of the X-chromosome represents most of the heterochromatic DNA, it is likely to be the most extensively damaged by NiS particles. In contrast, ionic nickel from the NiCI2, which enters the cell less readily than the particles, is distributed throughout the cell, interacting with nu merous ligands in addition to DNA. Overloading of cells with NiCI2 cannot produce an incidence of transformation equivalent to the maximum possible transformation frequency found with crystalline NiS (8, 13,14). The incidence of transformation and the nuclear levels of Ni2+ are considerably greater with NiS particles compared to NiCI2 (8,13,14). Nickel ions are known to bind to the phosphate groups of DNA bases, but the ion also has affinity for purine bases (20). The presence of GC-rich, repetitive DNA in heterochromatin may cause more nickel ions to interact with DNA per unit area in such regions as compared to dispersed chromatin having unique or even moderately repe titious DNA sequences. Thus, the number of initial lesions per unit area in heterochromatin is expected to be many-fold greater than euchromatin, resulting in the higher number of aberrations in the heterochromatic regions. Another important factor affect ing the incidence of chromosomal aberrations was the fraction of cells in the late S phase of the cell cycle. According to the studies illustrated in Table 2, those cells in the late S phase of BY NICKEL COMPOUNDS the cell cycle represent the most susceptible cell cycle population for the induction of chromosomal lesion. Interestingly, NiCI2 and crystalline NiS have been shown to cause an S-phase cell cycle block (21), and this accumulation of cells in S phase may be one of the reasons why the chromosomal aberrations induced by crystalline NiS particles appeared to require longer exposure times (i.e., 24 h; Table 3). During S phase, cells are probably more susceptible to the DNA-damaging effects of nickel because DNA is unfolded and more accessible to nickel ion binding during its replication. A major lesion induced by nickel in the DNA is the DNA-protein cross-link (18). The formation of this lesion may involve the chemical reactivity of ionic nickel in forming a ternary complex with protein and DNA (22). The equilibrium binding constant of nickel for DNA indicates very weak affinity of the metal for DNA (22). The binding constant of nickel for protein indicates greater affinity of the metal for protein than for DNA. However, the formation of a ternary DNA-nickel-protein complex represents a way of maintaining nickel bound to DNA in a very stable complex (22). Our own studies have demonstrated that this ternary com plex will form in vitro only if nickel is added to DNA prior to the addition of nuclear protein (23). The formation of this ternary complex may cause preferential retention of nickel in the nucleus, making its entry into the nucleus irreversible. Additionally, the proteins cross-linked to DNA by nickel either in vitro or in the intact cells are the same proteins that are very tightly bound to the DNA (23). Therefore, nickel appears to cross-link proteins which have an inherently high binding affinity for DNA. Recent studies5 have demonstrated that the nickel-induced DNA-protein cross-link forms preferentially during the late S phase, and pro teins present in the heterochromatin fraction are extensively cross-linked to the DNA by nickel treatment of intact cells. The observed fragmentations of the long arm of the X-chromosome may result from incomplete replication of DNA in these crosslinked regions. Earlier studies have shown that the aberrations induced by carcinogenic and mutagenic agents are preferentially located in heterochromatic regions (24, 25). These studies have suggested that (a) concentration of repetitive DNA in these regions and (b) condensed state of heterochromatin to form chromocenters during interphase might be responsible for the increased aber rations in the heterochromatic regions of the chromosomes. Our results add support to these previous findings by demonstrating that the highly carcinogenic NiS particles produce extensive fragmentation of the heterochromatic long arm of the X-chro mosome. The effect appeared to be due to the mechanism of nickel delivery into cells. The significance of the heterochromatin fragmentation in relationship to the carcinogenesis of crystalline NiS is not known. It is, however, interesting to note the lack of mutagenicity exhibited by potently carcinogenic nickel com pounds in mammalian systems (26) which is consistent with a site of action in heterochromatin. Heterochromatic DNA has few actively transcribed genes and contains repetitive DNA. Little is known about the function of this type of DNA, and therefore it is difficult to ascertain the significance of the observed selective interaction of nickel with heterochromatin. 5 Patierno et al., unpublished observations. CANCER RESEARCH VOL. 45 MAY 1985 2323 SELECTIVE DAMAGE TO HETEROCHROMATIN ACKNOWLEDGMENTS The authors thank Faye Howard for secretarial assistance and S. R. Patierno for his criticism of this manuscript. The authors thank Dr. R. L. Juliano for his assistance in preparing the liposomes utilized in this study. The authors also thank Dr. S. Pathak and Dr. T. C. Hsu for helpful discussion. REFERENCES 1. Chovil, A., Sutherland, R. B., and Halliday, M. Respiratory cancer in a cohort of nickel sinter plant workers. Br. J. Ind. Med., 38: 327-333,1981. 2. Magnus, K., Andersen, A., and Hogetveit, A. C. Cancer of respiratory organs among workers at a nickel refinery in Norway. Int. J. Cancer, 3: 681-685, 1982. 3. Enterline, P. E. and Marsh, G. M. Mortality among workers in a nickel refinery and alloy manufacturing plant in West Virginia. J. Nati. Cancer Inst., 68; 925933. 1982. 4. Oilman, J. P. W. Metal carcinogenesis. II. A study of the carcinogenic activity of cobalt, copper, iron, and nickel compounds. Cancer Res., 22: 158-162, 1962. 5. Sunderman, F. W., Jr., and Maenza, R. M. Commun. Chem. Pathol. Pharmacol., 74:319-330, 1976. 6. Sunderman, F. W., Jr. Recent advances in metal carcinogenesis. 14: 93-122, 1984. 7. Kasprzak, K. S., Gabryel, P., and Jarczewska, K. Carcinogenicity of nickel (II) hydroxide and nickel sulfate in Wistar rats and its relation to the in vitro dissolution rates. Carcinogenesis (Lond.), 4: 275-279,1983. 8. Costa, M., Simmons-Hansen, J., Bedrossian, C. W. M.. Bonura, J., and Caprioli, R. M. Phagocytosis, cellular distribution, and carcinogenic activity of particulate nickel compounds in tissue culture. Cancer Res., 41: 2868-2876, 1981. 9. DiPaolo, J. A. and Casto, B. C. Quantitative studies of in vitro morphological transformation of Syrian hamster cells by inorganic metal salts. Cancer Res., 39: 1008-1013,1979. 10. Costa, M. and Mollenhauer, H. H. Carcinogenic activity of particulate nickel compounds is proportional to their cellular uptake. Science (Wash. DC), 209: 515-517,1980. 11. Costa, M. and Mollenhauer, H. H. Phagocytosis of nickel subsulfide particles during the early stage of neoplastic transformation in tissue culture. Cancer Res., 40: 2688-2694,1980. BY NICKEL COMPOUNDS 12. Evans, R. M., Davies, P. J. A., and Costa, M. Video time-lapse microscopy of phagocytosis and ¡ntracellular fate of crystalline nickel sulfide particles in cultured mammalian cells. Cancer Res., 42: 2729-2735,1982. 13. Costa, M. Sequential events in the induction of transformation in cell culture by specific nickel compounds. Biol. Trace Element Res., 5: 285-295,1983. 14. Costa, M., and Heck, J. D. Perspectives on the mechanism of nickel carcino genesis. Adv. Inorg. Biochem., in press, 1985. 15. Hsu, T. C. Chromosome structure a possible function of constitutive hetercchromatin: the bodyguard hypothesis. Genetics, 79:137-150,1975. 16. Hansen, K. Solubility of metallic nickel and nickel oxides in a biological fluid at different pH levels. IUPAC Nickel Toxicology Conference, Paris, France, Ab stract 12, p. 12,1984. 17. Ciccarelli, R. B., Hampton, T. H., and Jennette, K. W. Nickel carbonate induces DNA-protein cross-links and DNA strand breaks in rat kidney. Cancer Lett., 72:349-354,1981. 18. Ciccarelli, R. B. and Wetterhahn, K. E. Nickel distribution and DNA lesions induced in rat tissues by the carcinogen nickel carbonate. Cancer Res., 42: 3544-3549,1982. 19. Abbracchio, M. P., Evans, R. M., Heck, J. D., Cantoni, O., and Costa, M. The regulation of ionic nickel uptake and cytotoxicity by specific amino acids and serum components. Biol. Trace Element Res., 4: 289-301, 1982. 20. Eichhorn, G. L. The effect of metal ions on the structure and function of nucleic acids. Adv. Inorg. Biochem., 3:1-46, 1981. 21. Costa, M., Cantoni, 0., deMars, M., and Swartzendruber, D. E. Toxic metals produce an S-phase-specific cell cycle block. Res. Commun. Chem. Pathol. Pharmacol., 38: 405-419, 1982. 22. Lee, J. E., Ciccarelli, R. B., and Jennette, K. W. Solubilization of the carcinogen nickel subfulsife and its interaction with deoxyribonucleic acid and protein. Biochemistry, 21: 771-778, 1982. 23. Kraker, A. J. and Costa, M. Cross-linking of Chinese hamster ovary cell nuclear proteins to DNA by Ni2* in vitro. Fed. Proc., 43: 2032,1984. 24. Natarajan, A. T. and Ahnstrom, G. Heterochromatin and chromosome aberra tions. Chromosoma (Beri.), 28: 48-61,1969. 25. Natarajan, A. T. and Schmid, W. Differential response of constitutive and facultative heterochromatin in the manifestation of mitomycin induced chro mosome aberrations in Chinese hamster cells in vitro. Chromosoma (Beri.), 33:48-62,1971. 26. Heck, J. D. and Costa, M. A review of the in vitro assessment of the toxicity of metal compounds. II. Mutagenesis. Biol. Trace Element Res., 4: 319-330, 1983. 27. Costa, M. The regulation of omithine decarboxylase activity in intact normal and transformed cells maintained with a minimal salts/glucose medium. Life Sci., 25:2113-2124,1979. ** I / \ b Fig. 1. Photograph illustrating NiCI2-induced chromosomal damage in CHO cells. Cells in both a and b were treated for 2 h with 500 MM NiCI2 in a salts/glucose medium. The cells were transferred from complete growth medium to the salts/glucose medium prior to the addition of NiCI2 (see "Materials and Methods"). Following treatment, the metal compound was washed from the cellular monolayer, and the cultures were placed in complete culture medium for 24 h. At the end of this time interval, mitotic cells were collected, and chromosomes were prepared as described in "Materials and Methods." The broken arrow in a illustrates a chromatid exchange, while the solid arrow shows a chromatid gap. x 1600. The broken arrow in b shows a centromere exchange, while the solid arrow depicts a centramene break, x 1600. CANCER RESEARCH VOL. 45 MAY 1985 2324 V^i X Fig. 2. Selective fragmentation of the long arm of the X-chromosome of CHO cells by crystalline NiS particles. CHO cells were treated with crystalline NiS particles (20 ¿ig/ml)for 48 h in complete culture medium. Following this treatment, the cultures treated with the particles were washed, and mitotic cells were collected by a 2-h incubation with Colcemid (see "Materials and Methods"). The arrows in a to f show the fragmentation of the long arm of the X-chromosome. x 1600.