Applied Cellular Radiobiology Department and
1 Veterinary Sciences Department, Armed Forces Radiobiology Research Institute, Bethesda, MD 20889-5603 and
2 Molecular Oncology Branch, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Abstract |
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Abbreviations: ALP, alkaline phosphatase; DU, depleted uranium; HMTA, heavy metaltungsten alloys; HOS, human osteosarcoma; MN, micronuclei; MN-CB, micronucleated cytokinesis-blocked; ROS, reactive oxygen species.
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Introduction |
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Several recent studies have investigated the potential health effects of militarily relevant heavy metals (15). These investigations have not only demonstrated the transforming ability (1) and mutagenicity (2) of DU, but also its neurotoxicity (5). In contrast, there is no information regarding the health effects of imbedded HMTAs. Studies have shown that occupational exposure to hard metal dust, a mixture of cobalt- and tungsten carbide-containing particles, is associated with development of different pulmonary diseases, including fibrosing alveolitis and lung cancer (6,7). The toxic properties of hard metal particles are not only attributed to an interaction between cobalt metal and carbide particles (8), but also to the production of hydroxyl radicals, which have been implicated in their genotoxic effects (9). The HMTAs used in military applications, however, are somewhat different from conventional hard metal. HMTA penetrators consist of a combination of tungsten, nickel and either cobalt or iron (tungsten >90%, nickel 16%, iron 16% or cobalt ~16%), in contrast to hard metal dust, which is a mixture of cobalt metal (510%) and tungsten carbide particles (>80%) (10). The differences in metal composition and percentages of hard metal particles and tungsten alloys used in military applications preclude the assumption that the biological effects of hard metal particles and tungsten alloy particles would be the same.
There are no studies that address the potential health effects of internalized tungsten or HMTAs in terms of genotoxicity, mutagenicity or carcinogenicity. The long-term health risks associated with internal chronic exposure to HMTA particles are not defined but are crucial to developing carcinogenesis risk standards for personnel who could be injured by HMTA shrapnel. Therefore, in view of carcinogenesis risk estimates and medical management questions relevant to possible future incidents of tungsten internalization, an examination of molecular and cellular effects, including the potential transforming ability of tungsten and tungsten alloys, are necessary to understanding the potential carcinogenic effects of this material. The use of cell culture models to investigate potential or known carcinogens can provide important insights into the cellular and molecular mechanisms of carcinogenesis.
The in vitro transformation assay has not previously been used to study the transforming ability of HMTAs. This assay has been widely used in conjunction with metal salts to assess the potential carcinogenicity of metal compounds (e.g. DU, nickel, chromium and lead) (1115). We have therefore chosen to use this assay to initiate an assessment of the potential carcinogenicity of HMTAs. The HOS TE85 cell line, an immortalized, non-tumorigenic, osteoblast-like cell line, has been successfully used to demonstrate the transformation of non-tumorigenic human cells to the tumorigenic phenotype by metals (1,13,14) and chemical carcinogens (161,17). Several metal powders were chosen for this study. A fine tungsten powder, pure crystalline nickel, iron and cobalt were used since they are the components of one of several possible tungsten alloys used in military applications. Crystalline NiS has previously been shown to transform cells in vitro (13) and was tested as a positive control. Tantalum oxide (Ta2O5) was also used for comparison since tantalum, which is widely used in prosthetic devices, is considered an inert metal with few reported toxic effects (5).
To understand the potential health effects of internalized HMTAs used in military applications, we compared the effects of various metals that compose these HMTAs on human cell survival, transformation and induction of DNA damage. Our data demonstrate, for the first time, that a mixture of the metals used in tungsten alloys can transform human cells to the tumorigenic phenotype, similar to results observed with some DU (1) and Ni compounds (1,1216). HMTA transformants form anchorage-independent colonies, show aberrant growth rates and produce tumors in athymic mice.
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Materials and methods |
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Metal powders
The aim of the study was to examine the transforming capability of two HMTAs (tungsten/nickel/cobalt or tungsten/nickel/iron) currently used in military munitions. The weight percentage compositions of these alloys when used for military applications is approximately 9193% tungsten, 53% nickel and either 24% iron or 24% cobalt (10). Since the HMTAs used by the military are not commercially available, we used a mixture of these metals, in the same percentages used by the military, to model the particles of the alloys. In this study we therefore tested the effects of a pure mixture of these materials. The powders were obtained from Alfa Aesar (Ward Hill, MA). The following powders were used: (i) extrafine cobalt metal (Alfa Aesar 10455, 99.5% purity), median particles size (d50) 14 µm, called hereafter Co; (ii) extrafine nickel metal (Alfa Aesar 10256, 99% purity), d50 35 µm, called hereafter Ni; (iii) extrafine iron metal (Alfa Aesar 40337, 98% purity), d5013 µm, called hereafter Fe; (iv) extrafine tungsten metal (Alfa Aesar 10400, 99.9% purity), d50 13 µm, called hereafter W; (v) a pure mixture of W (92%), Ni (5%) and Co (3%) particles made in the laboratory without extensive milling, called hereafter rWNiCo; (vi) a pure mixture of W (92%), Ni (5%) and Fe (3%) particles made in the laboratory without extensive milling, called hereafter rWNiFe.
Prior to each experiment the insoluble metal particles were washed once in sterile H2O and again in acetone. They were then suspended in acetone, agitated with a magnetic stirring bar and dispensed into cell cultures. The suspensions were carefully mixed and dispersed before being added to the cells. Doseresponse experiments were conducted by altering the amounts of each metal powder, based upon its percentage of 100% of the total amount of powders. For example, 100 µg rWNiCo powder/ml consists of 92 µg W, 5 µg Ni and 3 µg Co, while 50 µg rWNiCo powder/ml consists of 46 µg W, 2.5 µg Ni and 1.5 µg Co. In this manner the total amount (weight) of metal powder mixture was varied while the ratios of the component metals were held constant.
Cellular survival assay
Cytotoxicity was assayed by measuring a reduction in plating efficiency. Exponentially growing cells were seeded at 104 cells/100 mm dish with three dishes per treatment group. Cultures were then treated 24 h later with 100 µl volumes of metal particles for 24 h. Cells were then rinsed with Dulbecco's phosphate-buffered saline. Cultures were detached with trypsin/EDTA and counted with a Coulter counter (Hialeah, FL). Appropriate numbers of cells (100 or 500) were then plated onto 60 mm diameter Petri dishes and cultures were returned to the incubator for 10 days to allow for colony formation. Cultures were then fixed with methanol and stained with 1% crystal violet. Plates with >15 colonies and colonies with >50 cells were counted as survivors.
Transformation and cell growth studies
For transformation assays 104 cells/100 mm dish were seeded and exposed to metal particles 24 h later, with three flasks per experiment. Prior to each experiment the insoluble metal particles were washed once in sterile H2O and again in acetone. They were then suspended in acetone, agitated with a magnetic stirring bar and dispensed into cell cultures. Immediately after the 24 h metal particle exposure cells were rinsed (three times with sterile serum-free medium), trypsinized, counted and seeded in 100 mm tissue culture dishes at a density calculated to yield ~95200 surviving cells per dish. The cultures were incubated for 5 weeks with weekly changes of nutrient medium. At the end of the incubation period cells were fixed, stained and examined for the appearance of transformed foci (18). Transformed foci were assayed using the criteria developed by Reznikoff et al. (18) and the modified scoring protocols were derived from Landolph (19) and the International Agency on Cancer Research/National Cancer Institute/Environmental Protection Agency (IARC/NCI/EPA) Working Group (20). The Working Group report indicated that: (i) because of the continuum of focus morphology, some foci can be intermediate (I/II or II/III) in character; (ii) these foci should be scored conservatively and assigned to the category of lower aggressive behavior. With HOS cells it was easy to distinguish between a type I and a type II focus, but somewhat difficult to differentiate between type II and type III foci. Therefore, in our experiments only type II and type II/type III foci were scored as transformants. In Table I the number of dishes with foci equals the number of foci of type II + type III. To determine the transformation frequency the Poisson formula is used, employing the first term of the Poisson distribution: Po = exp(mN), m = lnPo. Details of the transformation frequency calculations and the standard error have been described elsewhere (1,211,22) but are detailed below. Transformed foci are not contact inhibited and may be dislodged during refeeding, forming satellite colonies. To avoid this source of errors, the average number of transformed foci per dish (
) was computed from the proportion of dishes free of transformed colonies, f (i.e.
= lnf). To determine the transformation frequency, the following formula was used:
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This calculation has been widely used in radiation-induced neoplastic transformation studies to quantitatively compare transformation frequencies. The transformation data are presented from three independent experiments unless otherwise noted.
Cell survival fraction was determined using the conventional clonogenic assay (1,16). Cytotoxicity and survival assays were conducted in parallel with each transformation assay as described (1,16). Several transformed foci were picked with cloning cylinders and expanded by mass culture to establish transformed clonal lines. The transformation doseresponse curve was determined as indicated above except that increasing concentrations of pure metal powders were used. The optimum dose level for transformation was selected following a preliminary toxicity test based upon colony-forming efficiency. The high dose selected resulted in >60% toxicity and the low dose selected resulted in minimal toxicity.
To determine saturation densities, cells were plated at 1x105 cells per 100 mm diameter plate in complete growth medium and monitored for growth as previously described (1,16). For the soft agar clonability assay cells were plated at a density of 2x103 cells per well in a 6-well plate sandwiched by 1 ml bottom agar (0.6%) and 1 ml top agar (0.3%). Cells were fed weekly by adding a new layer of top agar. Colonies >0.5 mm diameter were scored using a microscope after 2 weeks. The plating efficiency value for each clone represents the mean number of colonies scored from three wells. For both saturation density determination and plating efficiency in soft agar data are representative of three independent experiments. To determine DNA synthesis, incorporation of tritiated thymidine into DNA was measured as previously described (1).
Invasion through Matrigel
The ability of transformed cells to degrade and cross tissue barriers was assessed in an in vitro invasion assay that utilizes Matrigel, a reconstituted basement membrane (Collaborative Research, Waltham, MA). For qualitative evaluation of cell behavior 5x104 cells were plated onto 16 mm dishes (Costar, Cambridge, MA) which had been previously coated with 250 µl of Matrigel (10 mg/ml). The net-like formation characteristic of invasive cells occurred within 12 h; invasion into the Matrigel was evident after 4 days.
Alkaline phosphatase (ALP) activity
The percentage of cells exhibiting ALP activity on their cell surface was evaluated on cytospins obtained 4 days after cell seeding using a cytochemical method (Kit 86R; Sigma) (23). The percentage of cells was calculated on at least 350 cells. Cellular ALP activity was also analyzed 4 days after seeding. Approximately 106 cells were resuspended in homogenization buffer (1 mmol/l MgCl2, 1 mmol/l CaCl2, 20 mol/l ZnCl2, 0.1 mol/l NaCl, 0.1% Triton X-100, 0.05 mol/l TrisHCl, pH 7.4) and disrupted by gentle vortexing. The homogenate was used for the ALP assay, which was performed with p-nitrophenol phosphate as substrate as per the Kit 86R instructions. L/B/K ALP activity was normalized for the content of protein in the sample. Protein was measured using the Bradford method (1,2). One unit of L/B/K ALP activity is defined as the amount of enzyme capable of transforming 1 µmol substrate in 1 min at 25°C.
Tumorigenicity assay
Experiments were performed with 45 week old female athymic mice (Division of Cancer Treatment, NCI Animal Program, Frederick Cancer Research Facility, Frederick, MD). For this assay 5x106 cells in a 0.2 ml sterile suspension were injected s.c. in the right scapula. Animals were then observed for tumor growth at the sites of injection for 180 days. Tumor area was measured using a caliper measurement of two perpendicular diameters. When tumors were >100 mm2 the animal was killed.
Northern blot analysis and DNA probes
Cytoplasmic RNA was extracted from exponentially growing cells and separated by electrophoresis in 1% agaroseformaldehyde gels. RNA preparation and blotting onto nytran filters, hybridization with radiolabeled DNA probes and autoradiography were previously described (1,16). The ras probe, a SacI 2.9 kb fragment of the human ras gene, and a 1.8 kb fragment of the human ß-actin gene were obtained from Oncor (Gaithersburg, MD). 32P-labeled DNA probes were prepared using a random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN).
Micronuclei (MN) analysis
The induction of MN in control- and metal powder(s)-exposed cells was assessed using the conventional fluorescence plus Giemsa harlequin staining protocol (24). Following a 1 h exposure, the medium containing the metal powder was removed and cells were rinsed (three times with sterile serum-free medium) and incubated again at 37°C in complete medium. Mitomycin C was used as a positive control (24 h exposure). Cytochalasin B (6 µg/ml) was added after 24 h to block cytokinesis. At 48 h post-treatment cells were dropped onto slides using a cytospin (Shandon, St Louis, MO) for 5 min at 600 r.p.m. Slides were fixed with 5% Giemsa.
Alkaline elution test
The DNA breakage potencies of the metal powders were examined using the rapid alkaline elution test based on the method of Kohn et al. (25) and using the Millipore Multiscreen Assay System as described by Anard and co-workers (26). A 96-well filtration plate with a hydrophilic polyvinylidene fluoride microporous membrane (pore size 0.65 µm) sealed to its bottom was used along with a vacuum manifold to remove solutions from the filters. Cells were labeled for 18 h with 1 µCi/ml [3H]TTP (ICN, Costa Mesa, CA) prior to analysis of DNA damage by alkaline elution. Approximately 7x104 labeled cells were dispersed into each well of the filtration plate and exposed to the different powders suspended in complete medium. After 1 h exposure 1 mM sodium formate was added and the cells were lysed in a solution containing 0.04 M Na4EDTA, 2 M NaCl, 0.2% N-lauroylsarcosine, 0.5 mg/ml proteinase K and 1 M sodium formate, pH 9.0. The DNA remaining on the filters was washed with a solution containing 0.02 mM Na4EDTA. Elution buffer (0.1 M tetrapropylammonium hydroxide, 0.02 M EDTA, pH 12.1) was added in a volume of 300 µl and the DNA was eluted by vacuum (~16 kPa) in a single fraction. The DNA breakage potency of the different powders was assessed by quantifying the radioactivity recovered with the eluted fraction.
Histopathology of tumors
For routine staining, tumor tissues were fixed in buffered 10% formalin, embedded in paraffin, sectioned and stained by routine hematoxylin and eosin methods (1,16).
Statistics
Statistics for the MN and alkaline elution assays were performed with the 2 test and the TukeyKramer multiple comparisons test, respectively.
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Results |
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Transformation of HOS cells by rWNiCo and rWNiFe: comparison with pure W, Ni, Co and Fe
To assess morphological cell transformation, the standard focus formation assay previously described by others for both C3H/10T1/2 and HOS cells was used (1,1119); we also previously used this assay to examine the effects of DU on HOS cells (1). Based on our toxicity and growth data we selected a non-toxic and non-cytostatic exposure of the two metal mixtures to examine their transforming potentials. The morphology of untreated and rWNiCo-treated HOS cells is shown in Figure 3. HOS cells exhibit a flat epithelial-like morphology and appear to grow in a monolayer (Figure 3A
). In contrast, exposure to rWNiCo caused a morphological change in HOS cells (Figure 3B
). Following treatment with rWNiCo and weekly changes of nutrient medium for 5 weeks, diffused type II foci appeared (Figure 3B
). The morphology of the foci is distinctly different from the surrounding cells (Figure 3b
). The foci exhibit a slight multi-layered pattern, which is somewhat different from the `piled up' appearance seen in transformed C3H10T1/2 cells (1,1618). Similar morphological changes were observed for cells exposed to rWNiFe (data not shown). The transforming potential of each pure metal was also tested. Table I
shows measured values for the transformation frequencies (normalized per surviving cell) for HOS cells treated with W (46 µg/ml), Ni (2.5 µg/ml), Co (1.5 µg/ml), Fe (1.5 µg/ml), rWNiCo (50200 µg powder/ml) and rWNiFe (50200 µg powder/ml). The data demonstrate that treatment with rWNiCo and rWNiFe resulted in a transformation frequency of 37.6 ± 3.90x104 and 40.1 ± 3.85x104, respectively. These increased transformation frequencies represent 8.90 ± 0.93- and 9.50 ± 0.91-fold increases in transformation frequency, respectively, compared with the frequency in untreated HOS cells. In comparison with rWNICo and rWNiFe, cellular exposure to the pure powders W, Co and Fe (1.5184 µg/ml) did not significantly increase the transformation frequency above untreated control levels (Table I
). Data are not shown for Co and Fe (3 and 6 µg/ml) and W (92 and 184 µg/ml). Incubation with pure nickel (2.5, 5, 10 µg/ml), however, did result in a small but statistically significant increase in transformation frequency (control, 4.21 ± 0.41x104; 2.5 µg/ml, 7.55 ± 0.75x104; 5 µg/ml, 9.11 ± 0.95 x104; 10 µg/ml, 10.55 ± 1.85x104). Data in Table I
are only shown for the lowest amount of pure nickel. In comparison, cellular exposure to a higher dose of the known transforming agent crystalline NiS (50 µg/ml) increased the transformation frequency to 32.5 ± 3.10x104. Not only has Ni exposure been epidemiologically linked to cancer but the ability of Ni to transform cells in vitro was previously demonstrated by this laboratory and others and is shown here for comparison (1,1214). In contrast, the biologically inert Ta2O5 did not induce an increase in HOS transformation frequency above untreated levels (Table I
). These data also demonstrated a metal dose-dependent increase in transformation frequency. Several rWNiCo- and rWNiFe-transformed foci were picked with cloning cylinders and expanded by mass culture to establish transformed clonal lines. A spontaneous focus arising from untreated HOS cells was also selected and expanded. These transformed clones were selected for growth analysis (invasiveness in Matrigel; saturation density; growth in agar), ALP activity and tumorigenicity.
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Biological characterization of the transformed phenotype
Alteration in growth control is critical to neoplastic transformation and therefore the rWNiCo- and rWNiFe-transformed clones were further characterized by quantitative differences in growth properties associated with the neoplastic phenotype, e.g. saturation density and soft agar colony-forming efficiency. Additionally, ALP activity and tumorigenicity were assessed in untreated and transformed HOS cells.
The saturation densities of rWNiCo- and rWNIFe-transformed cells were approximately three times higher than that of the parental HOS cells (Table II). Saturation density data obtained for cells treated with W, Ni, Co and Fe powder were similar to that for parental HOS cells. An assessment of anchorage-independent growth ability was also done. A comparison of the transformants ability to grow in soft agar revealed that the rWNiCo and rWNiFe transformants generated colonies within 1 week with colony-forming efficiencies of 34 and 47%, respectively. Parental HOS cells and cells treated with the pure metals did not form a significant number of colonies in soft agar (Table II
). We have previously shown that DU, N-methyl-N'-nitro-N-nitrosoguanidine and EJ-ras transformants formed soft agar colonies whose size and frequency were comparable with those observed with the rWNiCo and rWNiFe transformants (1,15).
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Inoculation of athymic nude mice with rWNiCo and rWNiFe transformants resulted in the development of animal tumors at the site of injection within 4 weeks (Table III). In contrast, parental HOS cells and cells treated with W, Ni, Co or Fe (46, 2.5, 1.5 and 1.5 µg powder/ml, respectively) injected into nude mice did not result in tumor formation during a period of 6 months after cell inoculation. Tumor tissue was excised and used for histochemical and immunohistochemical analyses. Some tumor tissue was also used to establish cell lines for further study.
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Discussion |
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Our studies demonstrate for the first time that the malignant transformation of immortalized human cells can be achieved by exposure to a mixture of W, Ni and Co or a mixture of W, Ni and Fe. Transformants with both metal mixtures showed morphological changes, anchorage-independent growth in soft agar, induced tumors when transplanted into nude mice and exhibited alterations in ras oncogene expression. Cellular exposure to these mixtures also induced DNA breakage and chromosomal aberrations, i.e. MN induction, indicating that these mixtures are genotoxic. This is the first report to demonstrate not only that a tungsten alloy mixture can transform human cells, but also that, based upon equivalent concentrations and metal toxicities, the magnitude of this transformation is ~1.3-fold greater than that observed for NiS, a well-known transforming agent and carcinogen (13). Furthermore, both rWNiCo and rWNiFe caused a dose-dependent increase in the transformation frequency of HOS cells. Since there are little data regarding the potential tumorigenic and genotoxic effects of internalized tungsten and tungsten alloys, these results are important to the understanding of the mechanism of the potential late health effects, i.e. carcinogenicity, of tungsten alloys used in military applications.
These studies also confirm previous results demonstrating that HOS cells can be used to assess quantitative transformation in addition to morphological and neoplastic transformation (1,13,14,16). Morphological transformation studies have primarily involved rodent cell lines such as C3H and 10T1/2 (11,15). As with our previous DU studies (1), our current results again demonstrate that human cell models are available not only for morphological but also for quantitative transformation studies. The current studies with tungsten alloy mixtures demonstrate another step in understanding the transformation process of HOS cells.
The precise mechanism(s) by which rWNiCo and rWNiFe induce transformation in HOS cells is not fully known, although the data suggest several possibilities. Alterations in specific oncogenes (e.g. ras) and/or inactivation of tumor suppressor genes (e.g. p53) involved in the conversion of these cells to the malignant phenotype have been considered. HOS cells contain a mutation at codon 156 of the p53 gene, resulting in a mutated form of p53 protein that is believed to be partially responsible for their immortalization (28). Since neoplastic conversion is postulated to result from a multi-step process involving cell immortalization and gene alterations (27,2931), transformation of immortalized HOS cells by rWNiCo and rWNiFe to the malignant phenotype may involve other cellular oncogenes in this process. Our data demonstrate that the ras oncogene was activated in the transformation process induced by both rWNiCo and rWNiFe exposure. The ras oncogenes have been implicated in both chemical- and radiation-induced animal tumors (1,2932) and in spontaneous human tumors (33). Studies also show that the c-myc protooncogene plays an important role in the nickel- and lead-induced transformation of mammalian cells, possibly by stabilizing protooncogene RNAs (34,35). Additionally, effects on tumor suppressor proteins have also been observed in metal-transformed clones. DU- (1) and Ni-induced (1,12) transformations of HOS cells were both shown to affect the encoded protein of the Rb tumor suppressor gene by altering phosphorylation of Rb protein in the transformed clones. Similarly, long-term exposure of human epithelial cells to nickel resulted in p53 gene point mutations (35). Considering these findings, we speculate that the transformation of human cells by tungsten alloy mixtures may result from a conversion process that definitely involves activation of tumor-promoting gene(s) and that may involve alterations in tumor suppressor proteins. This hypothesis awaits further testing.
Histological and histochemical analyses of tumors formed by both the rWNiCo- and rWNiFe-transformed cells indicated a gland-like pattern with random calcium deposition rather than an osteogenic sarcoma pattern of growth. Specifically, this histological tumor examination revealed an infiltrative neoplasm consisting of cuboidal cells forming acini, papillary fronds and tubules with no neoplastic spindled cells present, which is consistent with a diagnosis of adenocarcinoma. The finding of a malignant epithelial neoplasm rather than a sarcoma, a malignant mesenchymal neoplasm, was not expected. However, transformation of a spindled cell line, like HOS, with subsequent transplantation into nude mice may result in dedifferentiation at some point, resulting in growth of an epithelial neoplasm. A previous study of NiS-induced transformation of HOS cells reported the same unexpected histomorphology of neoplasms in recipient nude mice; that study also included supportive immunohistochemistry with positive cytokeratin and positive carcinoembryonic antigen staining, both epithelial markers (14). Furthermore, in our study untreated HOS cells formed ALP-positive foci which calcified on extended culture, while the HMTA-transformed cells lacked the ability to form multilayered cellular structures and ALP-positive foci. Changes in ALP activity have been shown to be associated with expression of the malignant phenotype in human osteosarcoma cells (23). Additionally, both rWNiCo- and rWNiFe-transformed cells deposited calcium randomly in areas showing clumped growth of cells. Taken together, these observations suggest that HMTA-transformed cells had dedifferentiated and lost some osteoblastic characteristics. The similarity of our results to previous studies of HOS transformation and tumorigenesis strongly suggests that HOS tumorigenic cells dedifferentiate in vivo (1,14).
As indicated previously, the tungsten alloy mixtures and the pure metals (except Ni) have not been tested for transforming potential. However, studies addressing hard metal lung disease examined the cytotoxic and cytogenetic effects of Co, WC and a WC + Co mixture (36,37). The results demonstrated that WC + Co had a greater cellular toxicity than pure Co metal particles (8,9) and that cellular uptake of Co was enhanced when it was present in the form of WC + Co. The increased toxicity did not result from the increased bioavailability of Co, however, and it appears that WC + Co behaved as a specific toxic entity (9,3537). Similarly, our data also show that the tungsten alloy metal mixtures W/Ni/Co and W/Ni/Fe exhibit a greater toxicity than that of any of the pure metals. We have not yet determined whether there is any enhanced bioavailabilty of any of the pure metals in the mixture or whether this potentially increased bioavailability would affect the toxicity or transforming ability of the tungsten alloy mixtures. Ongoing studies on HMTA uptake and bioavailability may help to answer these questions.
The mechanism by which rWNiCo and rWNiFe induce cell transformation in vitro appears to involve, at least partially, direct damage to the genetic material, manifested as increased DNA breakage and chromosomal aberrations (i.e. MN). Our data clearly show that direct DNA damage and induction of chromosomal aberrations result from cellular exposure to the tungsten alloy mixtures. In contrast, only the highest doses of pure W, Ni and Co induced any significant increase in DNA breakage above background. Ni and Co were previously shown to be genotoxic at higher, toxic concentrations (38), so our data with Ni and Co at low, non-toxic doses are not too surprising. The HMTA mixtures demonstrate a synergistic increase in DNA breakage when the pure metals are mixed together to compose the tungsten alloy mixture. In contrast, MN induction by the HMTA mixtures, while significantly greater than the level of MN induction by each pure metal, did not exhibit a synergistic relationship. The MN test may be somewhat less sensitive in assessing the synergistic DNA-damaging potential of the mixture demonstrated by the DNA breakage assay. The MN assay does, however, detect chromosomal aberrations and not just repairable DNA breakage. Therefore, the combination of the DNA single-strand break and MN assays provides a better understanding of the mechanisms responsible for the genotoxic nature of the HMTA mixtures.
Other mechanisms may be involved by which rWNiCo and rWNiFe induce cell transformation in vitro. Studies have demonstrated that non-genotoxic, carcinogenic metals like lead may induce transformation via an indirect mechanism such as changes in DNA conformation or enzymatic disturbances (39). Other studies have also demonstrated that inhibition of DNA replication, leading to an elevation in sister chromatid exchanges, may be involved in metal-induced transformation (40,41). All of these mechanisms could potentially be involved in the HMTA transformation process, since transformation induced by metals like Ni appears to involve multiple mechanisms essential to the neoplastic process. For example, the involvement of epigenetic mechanisms of action has also been postulated for Ni. Chromatin condensation, de novo methylation and the resultant aberrant genetic activity may be partially responsible for the carcinogenic action of Ni (34,42). Similarly, recent unpublished data from our laboratory demonstrate that cellular exposure to rWNiCo resulted in hypermethylation of DNA (unpublished data). Although we do not have any direct evidence that neoplastic transformation by the HMTA mixtures involves epigenetic mechanisms, we cannot rule out their potential involvement in the transformation process.
Another possible mechanism in HMTA mixture transformation may involve reactive oxygen species (ROS). The formation of oxygen radicals in metal transformation is well documented, however, for some metals the contribution of these free radicals to the carcinogenic process is somewhat unclear (38). For Ni it is clear that ROS are involved. On a cellular level, Ni has been shown to increase protein oxidation and induce formation of cell oxidants (42) and the binding of Ni to peptides increases its accessibility to critical DNA sites. Animal studies have confirmed the role of Ni and Ni-mediated ROS in the mechanism of Ni carcinogenesis (43). Similarly, Fe potentiates oxygen toxicity via the Fenton reaction, producing hydroxyl radicals and inducing oxidative stress (44). Animal carcinogenesis studies provide evidence that high doses of Fe are also carcinogenic (45). In contrast, Co, which also generates substantial amounts of oxygen radicals, is not mutagenic or carcinogenic (46). When Co and WC particles are mixed together, however, hydroxyl radicals are generated that contribute to the mixture's induction of DNA damage (9,37). Epidemiological studies demonstrate that occupational exposure to hard metals may cause different types of lung diseases (6,7), including cancer (47). Based on these findings with other metals, it is interesting to speculate that another mechanism by which the HMTA mixtures induce transformation, chromosomal aberrations and DNA breakage may involve oxidative damage. This hypothesis awaits further testing.
In summary, we used a model system of in vitro human osteoblast cells exposed for 24 h to rWNiCo (50 µg powder/ml) and rWNiFe (50 µg powder/ml) to assess the relative transforming potential of HMTAs in an effort to better understand the potential health risks from long-term exposure to internalized HMTAs. Cellular exposure to HMTAs induces neoplastic transformation. These HMTA transformants are characterized by anchorage-independent growth, tumor formation in nude mice and expression of high levels of the K-ras oncogene. The mechanism by which rWNiCo and rWNiFe induce cell transformation in vitro appears to involve, at least partially, direct damage to the genetic material manifested as increased DNA breakage or chromosomal aberrations (i.e. MN). These HMTAs appear to have transforming ability somewhat greater than that of many other trace heavy metals which also induce neoplastic cell transformation in vitro as well as cause tumor formation in animals (42). While additional animal studies are needed to address the effect of protracted exposure and tumor induction in vivo, the implication from our model system study is that the risk of neoplastic induction from internalized HMTA exposure may be similar to other biologically reactive and carcinogenic heavy metal compounds (e.g. Ni).
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Notes |
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Acknowledgments |
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References |
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