Genotoxicity of Samples of Nickel Refinery Dust

Farrah Clemens*,{dagger} and Joseph R. Landolph*,{dagger},{ddagger},§,1

* Department of Molecular Microbiology and Immunology, {dagger} USC/Norris Comprehensive Cancer Center, and {ddagger} Department of Pathology, Keck School of Medicine; and § Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, California 90033

Received January 9, 2003; accepted February 11, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
At the International Nickel Company (INCO) nickel refinery in Clydach, Wales, U.K., which has operated since 1901, 365 respiratory cancers, including 85 nasal cancers and 280 lung cancers, have occurred in workers since the 1920s. From 1901 to 1923, incidences of these cancers were high. In 1923, the refining process was changed, eliminating a nickel arsenide, Ni5As2, called orcelite, from the refinery. Incidences of respiratory cancers decreased substantially from 1925 to 1930. Refinery dust samples were obtained in 1920 and in 1929; both of these samples contain primarily nickel oxide (NiO), but the 1920 sample also contains orcelite. The orcelite content of the 1920 sample is ~10%, while that of the 1929 sample is ~1%. We hypothesized that orcelite in the 1920 sample was partially responsible for inducing nasal and lung cancers in the refinery workers, and we tested this hypothesis. The 1920 and 1929 samples and orcelite were phagocytosed by cultured C3H/10T1/2 Cl 8 (10T1/2) mouse embryo cells to similar extents and were similarly cytotoxic to 10T1/2 cells. The 1920 sample and orcelite induced dose-dependent morphological transformation of 10T1/2 cells; the 1929 sample did not. The cell transforming ability of the 1920 sample, and therefore its probable carcinogenicity, correlates with induction of respiratory cancers in refinery workers exposed to orcelite-containing nickel refinery dust before 1923. Inability of the 1929 sample to induce morphological transformation correlates with decreased human respiratory tumor incidence at this plant after 1923. This data supports our hypothesis that orcelite in the 1920 refinery sample contributed to its carcinogenicity to nickel refinery workers.

Key Words: orcelite; nickel arsenide; morphological cell transformation; C3H/10T1/2 mouse embryo cells; green (high-temperature) nickel oxide.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nickel metal has important applications in alloys such as stainless steel, in nickel cadmium batteries, and in nickel plating. Nickel compounds are used in catalytic hydrogenation of fats and oils and in ceramic glazes and paints (IARC, 1976Go; NIOSH, 1977Go). In the past, in industries refining nickel ores, workers were exposed by inhalation to concentrations of approximately 1 mg/m3 of mixtures of specific soluble and insoluble nickel compounds (Morgan and Rouge, 1984Go). This correlates with increased incidences of nasal and respiratory cancer (IARC, 1990Go).

The International Nickel Company (INCO) refinery at Clydach, Wales, U.K., has operated since 1901. Before 1930, this refinery imported nickel ore as a matte from Canada and refined it. The matte consisted of nickel and copper sulfides, metallic nickel, and metallic copper, and cobalt sulfide, and the precious metals, silver, platinum, palladium, selenium, and tellurium (Draper et al., 1994aGo). The matte was crushed, fed into calciners, and oxidized at temperatures up to 780°C. The resultant material was then treated in vats of hot 12% sulfuric acid to remove the metallic copper and nickel. The remaining material was then filtered and dried, reduced with hydrogen gas, and reacted with carbon monoxide gas to form highly volatile, highly toxic nickel carbonyl. The nickel carbonyl was then passed over nucleating pellets of nickel at high temperatures to convert it into metallic nickel and carbon monoxide gas. These processes were repeated up to seven times to extract the maximal amounts of valuable metallic elements from the ore (Draper et al., 1994aGo).

Three hundred sixty-five cases of respiratory cancer have been reported at the Clydach nickel refinery since the 1920s, including 85 nasal cancers and 280 lung cancers (Draper et al., 1994bGo). The INCO refinery modified the nickel refining process after 1923, principally by eliminating arsenic contamination from the sulfuric acid, thereby also reducing or eliminating nickel arsenide from the recycling processes. Subsequent to reduction of nickel arsenide in the recycling process, the incidences of nasal and respiratory cancers greatly decreased from 1925 to 1930 (Draper et al., 1994aGo; Gurley et al., 1986Go; NIOSH, 1977Go; Sunderman and McCully, 1983Go).

Samples were obtained from this refinery in 1920 and 1929, and were archived. Both samples have a similar range of particle sizes, and mean equivalent particle diameters of 3.0 µm and 1.5 µm, respectively. Their main component is green (high-temperature, or HT) nickel oxide (NiO) (Draper et al., 1994bGo). A major difference between the two samples is their different contents of the nickel arsenide, orcelite. Sample 91 CLYD3, obtained in 1920, is a concentrate containing 37.4% nickel; its major components are bunsenite (NiO) or the isomorphous (Cu0.2•Ni0.8)O. Its minor components are a spinel structure of Fe2O3 or Fe3O4, a copper nickel oxide (NiO•CuO), and orcelite (Ni5•As2) (Table 1Go). Arsenic, which contaminated the sulfuric acid until 1923, became concentrated in the refinery processes and was found to constitute 10% of the 1920 sample (Draper et al., 1994bGo). The arsenic was in the form of a nickel arsenide, Ni5As2, called orcelite, which would make up a total of 25% of the 1920 sample (Draper et al., 1994bGo).


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TABLE 1 Comparison of the Components of the Refinery Dust Samples from the INCO Refinery, CLYD3 (1920), and CLYD23 (1929)
 
Sample 91 CLYD23, obtained in 1929, is a concentrate containing 26.6% nickel; its major components are bunsenite (NiO) and the isomorphous (Cu0.2•Ni0.8)O. Its minor components are tenorite (CuO), a copper nickel oxide (NiO•CuO), smaller amounts of other materials, and nickel arsenide (orcelite). Arsenic constitutes only 1% of the 1929 sample (Table 1Go).

We hypothesized that exposure of nickel refinery workers to orcelite, alone or in combination with the green (HT) NiO in the sample, caused nasal and respiratory cancer in these workers before 1923. To test this hypothesis, we studied the ability of these nickel samples to be phagocytosed into, and to induce cytotoxicity, chromosomal aberrations, and morphological cell transformation in, C3H/10T1/2 Cl 8 (10T1/2) cells, a mouse fibroblastic cell line derived from C3H mouse embryos (Reznikoff et al., 1973bGo). Carcinogenic insoluble nickel compounds (Miura et al., 1989Go), lead chromate (Patierno et al., 1988Go), aflatoxin B1 (Billings et al., 1979), polycyclic aromatic hydrocarbons (Landolph and Heidelberger, 1979Go; Reznikoff et al., 1973aGo), and aromatic amines (Landolph and Heidelberger, 1979Go) all induce morphological and neoplastic transformation in 10T1/2 cells.

Insoluble nickel subsulfide, crystalline nickel monosulfide, and green (HT) and black (low-temperature, or LT) nickel oxides are phagocytized into, and induce cytotoxicity, chromosomal aberrations, and morphological transformation in, SHE cells (Costa et al., 1982Go, 1994Go; Heck and Costa, 1983Go) and 10T1/2 cells (Landolph, 1989Go, 1990Go; 1999Go; Landolph et al., 2002Go; Miura et al., 1989Go) and induce cytotoxicity and anchorage independence in diploid human fibroblasts (Biedermann and Landolph, 1987Go). Insoluble nickel compounds generate oxygen radicals in mammalian cells, which likely contribute to induction of morphological transformation and the 130 changes in gene expression observed in nickel compound transformed 10T1/2 cell lines (Landolph, 1999Go; Landolph et al., 2002Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Samples studied.
Two dust samples taken from the Clydach refinery were obtained through the courtesy of Dr. John Duffus, University of Edinburgh, Scotland, U.K., and through the courtesy of Dr. Milton Parks and Dr. Sallie Williams of the nickel refinery at Clydach, Wales, U.K. The sample 91 CLYD3 was taken from the refinery in 1920, and the sample 92 CLYD23 was taken in 1929, and both samples were archived. Table 1Go shows the percentage of nickel and the other components in each sample. The 1920 sample contains a nickel arsenide compound, orcelite, in the stoichiometry of Ni5-xAs2. Pure nickel arsenide, Ni5As2, was obtained through the courtesy of Dr. William F. Sunderman, Jr., previously of the University of Connecticut (Sunderman and McCully, 1983Go), now of Middlebury University, Vermont.

The archived samples were stored by Dr. Morrell H. Draper at the University of Edinburgh, Consultant in Toxicology, 10 West Mayfield, Edinburgh, Scotland, U.K. Dr. Draper stored these samples in sterile, airtight containers free from contaminants. We obtained these samples from Dr. Draper, courtesy of Dr. Duffus, University of Edinburgh. We also stored these samples in sterile, airtight containers free from contaminants in the same manner as Dr. Draper. For each experiment, a fresh stock of the samples was weighed on the day of the experiment; the samples were suspended in acetone for sterilization and then used immediately. After weighing out the samples and suspending them in acetone, the treatment concentrations were vortexed for homogenization and then serially diluted from the stock. Immediately prior to adding the samples in acetone to the cell cultures, the samples were again vortexed to ensure homogeneity of the suspension.

Cells and cell culture.
C3H/10T1/2 Cl 8 (10T1/2) cells were grown in Basal Medium Eagles (BME) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Omega Scientific Company, Inc., Tarzana, CA). Lots of fetal calf serum (FCS) were prescreened to identify those that supported a plating efficiency of approximately 30% and a yield of morphological cell transformation greater than five foci/twenty dishes when cells were treated with 1 mg/ml of the carcinogen, 3-methylcholanthrene (MCA). These lots of FCS were purchased and used in these studies. 10T1/2 cells were used between passages five and ten, to minimize the yield of spontaneous morphological transformation. Cells were grown in 75 cm2 tissue culture flasks (VWR Company, San Francisco, CA) and were maintained in logarithmic phase of growth in incubators containing 5% CO2 and 95% air (v/v) and maintained at a temperature of 37°C in a humidified atmosphere (Landolph and Heidelberger, 1979Go; reviewed in Landolph, 1985Go; Miura et al., 1989Go; Patierno et al., 1988Go; Reznikoff et al., 1973aGo,bGo).

Assays to detect phagocytosis of insoluble nickel compounds.
Two thousand 10T1/2 cells were seeded into each 60 mm dish, and two dishes were seeded and used for each treatment condition. Twenty-four h after the cells were seeded, the samples were suspended in acetone and added to the cells. Twenty-five ml of the sample suspension was added to each dish containing 5 ml of BME medium plus 10% (v/v) fetal calf serum (the final concentration of acetone in the medium was 0.5% v/v). After treatment of cells with sample for 48 h, the medium was removed, and the cells were rinsed once with isotonic saline (0.9%), then fixed for 20 min with 100% ethanol, and stained with 1% crystal violet. The cells were then examined with a light microscope, and those cells with one or more vacuoles containing one or more particles of nickel sample in the cytoplasm were scored as phagocytosing cells. The data are reported as the percentage of cells containing at least one vacuole with a phagocytosed particle.

Cytotoxicity assays.
These assays were performed as we previously described them (reviewed in Landolph, 1985Go; Landolph and Heidelberger, 1979Go; Miura et al., 1989Go; Patierno et al., 1988Go; Verma et al., 2000). Briefly, 200 10T1/2 cells were seeded into each 60 mm dish in 5 ml of BME containing 10% FCS (v/v). Five dishes were seeded for each concentration of each sample to be studied. Twenty-four h after cells were seeded, the samples were suspended in acetone and added to the cells (the final concentration of acetone in the medium was 0.5%, v/v). Twenty-five ml of the sample suspension was then added to each dish containing 5 ml of BME with 10% FCS. Two negative controls, medium alone and medium plus 0.5% (v/v) acetone, were used in each experiment. After 48 h of treatment of cells with samples, the medium was removed and replaced with fresh medium containing 10% FCS. Cells were cultured for an additional five to seven days, until colonies became visible under the dissecting microscope. Eight to ten days after the cells were initially seeded, when living colonies were clearly visible by examination of the dishes under a dissecting microscope, the medium was removed, and the cells were rinsed once with isotonic saline (0.9%), fixed for 20 min with 100% methanol, and stained for 20 min with filtered 10% Giemsa stain. Colonies containing 20 or more cells in each dish were scored under the dissecting microscope.

The plating efficiency (PE) is the number of colonies (containing 20 or more cells) on the dishes eight days post seeding, divided by the total number of cells seeded (200 cells), multiplied by 100%. The average survival fraction (PE of treated cells/PE of acetone control cells) was calculated for each set of five dishes for each treatment and reported as mean ± standard deviation. Utilizing the relationship, S = exp(-kc), where S is the survival fraction, c is the concentration of the sample tested, and k is the slope of the dose-response curve, the average survival fractions for each treatment were plotted on a semilogarithmic scale (log concentrations versus dose) to determine the LC50 value (the concentration at which the survival fraction is reduced to 50%) for each sample in 10T1/2 cells.

Assays to detect chromosome aberrations.
The method used to detect chromosomal aberrations was that of Ishidate et al.(1977Go, 1981)Go. Fifty thousand 10T1/2 cells were seeded into each 60 mm dish, and two dishes were utilized to determine the induction chromosomal aberrations by each concentration of sample studied. Twenty-four h after the cells were seeded, the samples were suspended in acetone and added to the cells. Twenty-five ml of the sample suspension was added to each dish containing 5 ml of BME containing 10% FCS (the final concentration of acetone was 0.5% v/v in the medium). Cells were treated with nickel samples for 48 h; then cells were arrested in the metaphase stage by treating them with 0.02 mg/ml colcemid for 18 h prior to termination of nickel sample treatment. After treatment with nickel samples, cells were incubated with hypotonic solution (0.075 M KCl) for 20 min, then fixed in cold Carnoy’s fixative (3:1 v/v methanol: acetic acid). Fixed cells were dropped onto slides, air dried, and stained with 10% Giemsa stain. For each concentration of nickel sample studied, chromosomes from 100 cells treated with the nickel sample were examined by microscope and scored for various chromosomal aberrations.

Assays to detect morphological transformation.
These assays were conducted according to methods described by Reznikoff et al. (1973a)Go with modifications developed and used in our laboratory (reviewed in Landolph, 1985Go; Landolph and Heidelberger, 1979Go; Miura et al., 1989Go; Patierno et al., 1988Go). Two thousand cells were seeded into each 60 mm dish, and 20 dishes were used to determine the induction of morphological cell transformation by each concentration of each nickel sample studied. Twenty-four h after cells were seeded, the nickel samples were suspended in acetone and added to the cells (the final concentration of acetone was 0.5% v/v in the medium). Twenty-five ml of the sample suspension was added to each dish containing 5 ml of BME containing 10% FCS. MCA (1 mg/ml) was used as a positive control as a known inducer of morphological transformation (reviewed in Landolph, 1985Go; Landolph and Heidelberger, 1979Go; Miura et al., 1989Go; Patierno et al., 1988Go; Reznikoff et al., 1973aGo). Medium alone and medium plus acetone (0.5% v/v in the medium) were used as negative controls. After 48 h of treatment of cells with samples, the medium was removed and replaced with fresh medium not containing samples, and the medium was then replaced twice per week until cells became confluent, then once per week. The cells were cultured for a total of six weeks from the time they were seeded. Six weeks after the cells were initially seeded, the medium was removed, and the cells were rinsed once with isotonic saline (0.9%), then fixed for 20 min with 100% methanol, and stained for 20 min with filtered 10% Giemsa stain. Type II and type III foci were scored and tabulated as described by Reznikoff et al. (1973a)Go, with our more recent modifications for scoring heterogeneous foci and foci on the borderline between type I and type II and between type II and type III (Landolph and Heidelberger, 1979Go; Miura et al., 1989Go; Patierno et al., 1988Go; reviewed in Landolph, 1985Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phagocytosis of Nickel Refinery Samples and Orcelite
Since insoluble nickel compounds need to be phagocytosed by cells in order to manifest their cytotoxic and genotoxic properties (Costa et al., 1982Go; reviewed in Landolph, 1990Go; Miura et al., 1989Go), we first determined whether these insoluble nickel refinery samples were phagocytosed by 10T1/2 cells. To do this, we treated 10T1/2 cells with doses of the nickel samples ranging from 0.5 to 7.5 µg/ml. When 10T1/2 cells were treated with 0.5 to 7.5 µg/ml of nickel samples, they phagocytosed all three samples, 91 CLYD3 (1920), 91 CLYD23 (1929), and Ni5As2 (orcelite) (Figure 1Go). The phagocytosis of these three samples was reproducible in two separate experiments, so the results of both experiments were averaged and plotted in Figure 1Go. Over concentrations ranging from 0.5 to 2.5 µg/ml, there was a dose-dependent uptake of particles from the 1920 sample. At concentrations ranging from 2.5 to 7.5 µg/ml, there was no further increase in uptake of this sample, but a significant, dose-related decrease in phagocytic uptake. Uptake of the 1929 sample increased linearly with concentration, from 0.5 to 1.0 µg/ml, and then a more gradual increase in uptake with a lesser slope occurred between 2.5 and 7.5 µg/ml. Phagocytic uptake of the Ni5As2 sample occurred in a dose-dependent manner over the entire dose range from 0.5 to 7.5 µg/ml. The slope for uptake was greatest from 0 to 0.5 µg/ml, and then a lesser slope for uptake occurred from 0.5 to 7.5 µg/ml (Figure 1Go).



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FIG. 1. Phagocytic uptake of the 1920 and 1929 samples, and Ni5As2 by 10T1/2 cells: 2,000 cells were seeded into each 60 mm dish, using 2 dishes per concentration; 24 h after seeding, 25 ml of treatment concentrations were added to the dishes. The dishes were fixed and stained with Crystal Violet after 48 h. The dishes were then scored for cell-containing vacuoles with particles. Two experiments were done for each sample tested.

 
To compare uptake among these three samples, we compared the phagocytic uptake we determined for all three samples at 1.0 µg/ml and at 5.0 µg/ml, shown in Figure 1Go. In cells treated with the 1920 sample, at a concentration of 1.0 µg/ml, 5% of the cells contained vesicles with phagocytosed particles, and at 5.0 µg/ml, 8.0% of the cells contained phagocytosed particles. In cells treated with 1.0 µg/ml and 5.0 µg/ml concentrations of the 1929 sample, 7% and 7.5% of cells contained vesicles with phagocytosed particles, respectively. In cells treated with 1.0 µg/ml and 5.0 µg/ml of orcelite, 11%, and 19% of cells, respectively, contained phagocytosed particles. Hence, the relative abilities of these nickel-containing samples to be taken up by phagocytosis into 10T1/2 cells, when concentrations less than 5 mg/ml were considered, were orcelite > 1929 sample {approx} 1920 sample (Figure 1Go).

Cytotoxicity of Nickel-Containing Samples
The three nickel-containing samples — 91 CLYD3 (1920), 91 CLYD23 (1929), and Ni5As2 — all independently caused a dose-dependent cytotoxicity to 10T1/2 cells when the cells were treated with concentrations of these samples ranging from 0.1 to 7.5 µg/ml (Figure 2Go), which is over a similar concentration range that these samples were phagocytosed into 10T1/2 cells (Figure 1Go). The results of four separate cytotoxicity experiments were reproducible for each separate nickel-containing sample, so the results of the four experiments were averaged separately for each of the three samples and plotted (Figure 2Go). When 10T1/2 cells were treated with concentrations of the 1920 sample ranging from 0 to 7.5 µg/ml, the survival of the treated cells was reduced from 100% to 31% (Figure 2Go). The 1920 sample had a cytotoxicity similar to the other two samples in the concentration range from 0.1 to 2.0 µg/ml, but was found to be less cytotoxic than the other two samples studied at concentrations of 5.0 µg/ml and 7.5 µg/ml. The LC50 value for the survival of 10T1/2 cells treated with the 1920 sample was found to be 2.4 ± 0.3 µg/ml.



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FIG. 2. Plot of cell survival of 10T1/2 cells treated with the 1920 and 1929 samples, and Ni5As2: 200 cells were seeded into each 60mm dish, using 5 dishes per concentration; 24 h after seeding, 25ml of treatment concentrations were added to the dishes. After 48 h of treatment, the medium in the dishes was replaced. The dishes were fixed and stained with Giemsa stain 8–10 days post-seeding. The dishes were then scored for colonies containing 20 or more cells. The plating efficiencies were calculated and the survival fraction determined.

 
Treatment of 10T1/2 cells with concentrations of the 1929 sample ranging from 0 to 7.5 µg/ml reduced the survival fraction of 10T1/2 cells from 100% to 13%. The highest concentration of the 1929 sample used, 7.5 µg/ml, reduced the survival of 10T1/2 cells substantially, down to 15%, compared to the treatment of cells with the 1920 sample at the same dose, which only reduced the survival of the cells to 30%. The shape of this survival curve of 10T1/2 cells treated with the 1929 sample was exponential—a straight line in a semi-logarithmic plot of ln(s) versus concentration. The LC50 value for the 1929 sample, derived from the survival curve for the 1929 sample, was 1.7 ± 0.4 µg/ml.

The survival curve of 10T1/2 cells treated with 0.5 to 7.5 µg/ml of orcelite was similar to that of cells treated with the 1929 sample and was also crudely exponential. The survival of 10T1/2 cells was reduced from 100% to 11% as the cells were treated with concentrations of orcelite ranging from 0 to 7.5 µg/ml (Figure 2Go). The LC50 value derived from the orcelite survival curve, however, is closer in value to that of the 1920 sample, in that it was determined to be 2.4 ± 0.2 µg/ml. Treatment of 10T1/2 cells with orcelite and the 1929 sample resulted in roughly semilogarithmic survival curves. The slopes of these survival curves, calculated as if the curves were all exponential, were -6.7 for the 1920 sample, -10.0 for the 1929 sample, and -11.0 for the orcelite sample. Since the cytotoxicity curves in cells treated with the 1920 sample were curvilinear, we utilized both the overall shape of the curves and the LC50 values in evaluating the relative cytotoxic potencies among these samples. Hence, the relative cytotoxic potential of these samples, based on the overall shape of the survival curves, was 1929 sample (slope = -10.0, LC50 = 1.7 µg/ml) > Ni5As2 (slope = -11.0, LC50 = 2.4 µg/ml) > 1920 sample (slope = -6.7, LC50 = 2.4 µg/ml).

Induction of Chromosomal Aberrations in 10T1/2 Cells by Nickel-Containing Samples
In 10T1/2 cells treated with the negative control conditions (medium only or with 0.5% acetone), the percentage with chromosomal aberrations was 2% and 3%, respectively. The positive control, mitomycin C (MMC) at a concentration of 1 µg/ml, caused a strong yield of chromosomal aberrations, including breaks, fragments, dicentrics, translocations, and satellite associations (Table 2Go). In cells treated with 1 µg/ml of MMC, 20% of the cells had chromosomal aberrations, which was an eightfold increase in the percentage of cells bearing chromosomal aberrations over that in the average of the two control groups (2.5%).


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TABLE 2 Chromosome Aberrations Assay
 
Treatment of 10T1/2 cells separately with all three samples, 91 CLYD3 (1920), 91 CLYD23 (1929), and Ni5As2, over the concentration range from 0.5 to 7.5 µg/ml, increased the frequency of chromosomal aberrations in these cells over the frequency of chromosomal aberrations in control (medium or acetone-treated) cells (Table 2Go). Breaks, fragments, dicentrics, translocations, satellite associations, and rings were all induced in 10T1/2 cells treated with these nickel-containing samples. Breaks, fragments, and satellite associations were the most common types of chromosomal aberrations induced, and dicentrics and translocations were induced, but less frequently (Table 2Go). In 10T1/2 cells treated with 0.5 µg/ml, 1.0 µg/ml, 2.5 µg/ml, 5.0 µg/ml, and 7.5 µg/ml of the 1920 sample, the percentage of cells bearing chromosomal aberrations was 4%, 1%, 6%, 8%, and 7%, respectively (Table 2Go). The greatest amount of chromosomal aberrations occurred in cells treated with concentrations of 2.5 µg/ml, 5.0 µg/ml, and 7.5 µg/ml, in which 6%, 8% and 7% of the cells contained aberrations, which represents increases of 2.4-fold, 2.9-fold, and 3.2-fold, respectively (Table 2Go). There was no dose-dependence to the induction of chromosomal aberrations in cells treated with the 1920 sample.

Chromosomal aberrations were also induced in 10T1/2 cells treated with the 1929 sample. The percentage of cells with chromosomal aberrations increased from 2% and 3% in control (medium only or acetone only treated) cells to 3%, 4%, 4%, 4%, 4%, and 6% in cells treated with 0.5 µg/ml, 1.0 µg/ml, 2.5 µg/ml, 5.0 µg/ml, and 7.5 µg/ml of the 1929 sample, respectively (Table 2Go). In cells treated with 0.5 to 5.0 µg/ml, this represented a 1.6-fold increase, and in cells treated with the highest concentration, 7.5 µg/ml, a 2.4-fold increase in the percentage of cells with chromosomal aberrations. While the percentage of cells with chromosomal aberrations was higher in cells treated with the 1929 sample, the induction of chromosomal aberrations was not dose-dependent (Table 2Go).

No increase in the percentage of cells with chromosomal aberrations was detected in 10T1/2 cells treated with 0.5 µg/ml of orcelite (Table 2Go). In cells treated with Ni5As2 at concentrations of 1.0 µg/ml, 2.5 µg/ml, 5.0 µg/ml, and 7.5 µg/ml, small increases in chromosomal aberrations were observed, 4%, 7%, 6%, and 8%, respectively, similar to the small increases in chromosomal aberrations detected in 10T1/2 cells treated with the 1920 or 1929 samples (Table 2Go). These increases represented 1.6-fold, 2.8-fold, 2.4-fold, and 3.2-fold increases in the percentage of cells with chromosomal aberrations, and again, these increases were not strictly dose dependent.

Induction of Morphological Cell Transformation by Nickel-Containing Refinery Samples and Orcelite
Interestingly, the 1920 sample induced strong morphological transformation in 10T1/2 cells in the form of both type II and type III foci (Table 3Go, Figure 3Go). The yield of total type II plus type III foci increased in a dose-dependent manner in cells treated with 0.1 to 1.0 µg/ml of the 1920 sample, and this portion of the curve had a slope (calculated from the linear portion of the curve) of 4.5. This curve plateaued, when cells were treated with concentrations from 1.0 to 5.0 µg/ml, and increased again slightly when cells treated with 7.5 µg/ml of this sample (Figure 3Go). The second slope of this curve, from 5.0 to 7.5 µg/ml, was 1.60. An overall slope for this curve, assuming linearity, would be 0.75.


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TABLE 3 Number of Type II and III Foci Induced by 1920 and 1929 Samples, and by Ni5As2
 


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FIG. 3. Morphological transformation of 10T1/2 cells by the 1920 and 1929 samples, and Ni5As2: 2,000 cells were seeded into each 60mm dish, using 20 dishes per concentration. 24 h after seeding, 25ml of treatment concentrations were added to the dishes. After 48 h of treatment, the medium in the dishes was replaced, thereafter the medium was replaced every week. The dishes were fixed and stained with Giemsa stain 6–7 weeks post-seeding. The dishes were then scored for Type II and Type III foci. Data are plotted from values in Table 3Go and are the average value from two separate experiments.

 
Surprisingly, the 1929 sample did not induce any focus formation at all in 10T1/2 cells. This was consistent with the epidemiological data, which indicated a substantial reduction in human nasal and respiratory cancer after 1929 in the nickel refinery workers at Clydach.

No foci were induced when 10T1/2 cells were treated with 0.5 µg/ml or with 1.0 µg/ml of orcelite (Table 3Go, Figure 3Go). Orcelite, which was used as a positive control, also induced a high yield of morphological transformation in 10T1/2 cells (Figure 3Go). The induction of foci did occur in a dose-dependent manner in cells treated with orcelite over the concentration range from 2.5 to 7.5 µg/ml (Figure 3Go). The overall slope of this curve, assuming linearity of the dose-response curve, was 3.41. The yield of foci in cells treated with the highest concentration of 7.5 µg/ml of orcelite was 26 foci/20 dishes, which included the detection of 13 type III foci (Figure 3Go, Table 3Go). Therefore, the potency of these samples in inducing morphological cell transformation was orcelite (slope = 3.40) > 1920 sample (slope = 0.75) >>> 1929 sample (slope = 0, did not induce cell transformation), although the 1920 sample was more strongly transforming at the lower concentrations, from 0.5 to 2.0 µg/ml (Figure 3Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of the phagocytosis assays show that the 1920 sample was taken up by phagocytosis into 10T1/2 cells. Phagocytosis of this sample was linear at doses ranging from 0.5 to 2.5 µg/ml, but at 2.5 µg/ml and higher concentrations there was no increase in uptake. There were few cells containing phagocytosed particles at the highest dose tested, 7.5 µg/ml. This indicates that, while 30% of the cells survive after treatment with 7.5 µg/ml, most of these do not phagocytose the 1920 sample. The results from the four cytotoxicity experiments show that the 1920 sample caused a dose-dependent cytotoxicity with an LC50 value of 2.4 µg/ml and a slope of -6.6. Results from the chromosomal aberrations experiments indicate that the 1920 sample also induced chromosomal aberrations, mostly breaks, fragments, and satellite associations, along with a few of the less common aberrations, dicentrics, and translocations. The frequency of these chromosomal aberrations increased as 10T1/2 cells were treated with increasing concentrations of the 1920 sample, but the increases were not dose-dependent, nor were there large differences in the yield of chromosomal aberrations in cells treated with the different treatments. Based on the history of cancer induction in the nickel refinery workers, we hypothesized that the 1920 sample would be able to induce morphological cell transformation, while the 1929 sample would not. On the other hand, previous studies in our laboratory have shown that the major component of both samples, green (HT) NiO, is also an efficient inducer of morphological transformation in 10T1/2 cells (Landolph, 1985Go; Miura et al., 1989Go). Interestingly, our results show that the 1920 sample is capable of inducing strong (type II and type III foci) and dose-dependent morphological transformation in 10T1/2 cells, and the results were reproducible in separate experiments.

10T1/2 cells also phagocytosed particles of the 1929 sample in a dose-dependent manner. Phagocytic uptake of these particles increased, with the largest slope between 0 and 1.0 µg/ml. At concentrations from 1.0 to 7.5 µg/ml, phagocytic uptake of particles still increased, but the slope of this increase was smaller than that from 0 to 1.0 µg/ml. Over concentrations at which the cells phagocytosed the 1929 sample, this sample also caused a dose-dependent cytotoxicity to 10T1/2 cells, with a slope of -10.0 and an LC50 value of 1.7 µg/ml. The contents of both the 1920 and the 1929 refinery samples are clearly cytotoxic to 10T1/2 cells, consistent with the fact that green (HT) NiO is the main component in both the 1920 and 1929 samples. The cytotoxicity of the 1929 sample was similar to that of the 1920 sample in the dose range from 0.1 to 2.0 µg/ml. At concentrations of 5.0 µg/ml and 7.5 µg/ml, the 1929 sample was actually more cytotoxic than the 1920 sample. The 1929 sample also induced a small amount of chromosomal aberrations, including fragments, translocations, satellite associations, and a few dicentric chromosomes. The frequency of these chromosomal aberrations increased in 10T1/2 cells treated separately with all three nickel-containing samples, but the increases in chromosomal aberrations were not dose-dependent. However, there was not a significant difference between the ability of the 1920 or 1929 samples to induce chromosomal aberrations in 10T1/2 cells. Consistent with the epidemiological data, the 1929 sample did not induce any morphological transformation at all in 10T1/2 cells. This would confirm our hypothesis that the orcelite component of the 1920 sample is one cause of the cancers observed in the refinery workers prior to 1923.

Orcelite was also phagocytosed into 10T1/2 cells in a dose-dependent manner. The percentage of 10T1/2 cells phagocytosing orcelite particles increased from 0% at 0 µg/ml to 23% at the highest dose of 7.5 µg/ml. Orcelite caused a dose-dependent cytotoxicity to 10T1/2 cells that was similar to the cytotoxicity caused by the 1929 sample. The LC50 derived from the survival curve of 10T1/2 cells treated with orcelite, 2.4 µg/ml, was identical to that of the 1920 sample, but the shapes of the survival curves were different. Orcelite was more cytotoxic to 10T1/2 cells, with a slope of -11.0, compared to the survival curve for the 1920 sample, which was overall less cytotoxic and had a slope of -6.60. Results from the chromosomal aberrations experiments indicate that orcelite induced chromosomal aberrations. There was an increase in the induction of chromosomal aberrations, but not in a dose-dependent manner, from 0 to 7.5 µg/ml. Orcelite also induced strong (type II and type III) and dose-dependent morphological transformation of 10T1/2 cells, suggesting that the orcelite in the 1920 sample is partially responsible for the induction of nasal cancer and lung cancer in the workers in the Clydach refinery who were exposed to the refinery dust prior to 1923.

The mechanisms by which orcelite induces morphological transformation are not known at this time and require further investigation. Orcelite is a unique carcinogen, containing both Ni(+2) cations and As(-5) anions together on an insoluble particle. We hypothesize that both nickel cations and arsenide anions play a role in the mechanisms by which orcelite induces morphological transformation. There could be a synergism between the transforming activity of nickel cations and arsenide anions in the induction of morphological cell transformation. Studies adding nickel compounds, and arsenide compounds with other cations, to cell cultures and measuring the resultant yield of cell transformation should answer this question. We speculate that phagocytosis of orcelite by 10T1/2 cells and consequent dissolution of orcelite generates intracellular nickel ions. The binding of nickel ions to cellular protein, followed by reaction with endogenous hydrogen peroxide formed by cellular metabolism, could lead to oxygen radical generation and chromosome breakage, which could cause mutation in and activaton of proto-oncogenes into activated oncogenes (reviewed in Landolph, 1989Go, 1990Go, 1999Go; Landolph et al., 2002Go). Intracellular generation of nickel ions also probably leads to conformation changes in chromatin (Costa et al., 1994Go), which can lead to methylation of tumor suppressor genes (Costa et al., 1994Go). Ni(+2) ion-induced chromosome breakage could also lead to chromosome breakage, leading to deletion of chromosomes or their fragments bearing tumor suppressor genes (Landolph, 1989Go, 1990Go, 1999Go; Landolph et al., 2002Go). We believe that As(-5) anions also contribute to the cell transforming ability of orcelite, by as-yet-unknown mechanisms. Ni(+2) ions and arsenide anions could be comutagens or cocarcinogens, or could act synergistically to induce cell transformation and carcinogenesis. This could provide a strong carcinogenic potential to the orcelite particles. Further research is needed to answer these questions and, in particular, to determine the importance of the arsenic moiety in the orcelite in the induction of morphological cell transformation by orcelite.

Since both refinery samples are taken up into 10T1/2 cells by phagocytosis, induce cytotoxicity in the cells, and also induce similar levels of chromosomal aberrations in the cells, but exposure to only the 1920 sample correlates with an increased risk of nasal and lung cancers in the refinery workers, the data from the transformation assays are clearly more important to the issue of human cancer induction. The induction of morphological transformation in our standard transformation assay in 10T1/2 cells by the 1920 sample, but not by the 1929 sample, provided the most concrete support to our hypothesis that the 1920 sample contained carcinogenic potential. Therefore, the 1920 sample is likely to be carcinogenic, and the 1929 sample is likely not to be carcinogenic, based on the results of our studies demonstrating induction of morphological transformation in 10T1/2 cells by the 1920 sample but not by the 1929 sample. There was no correlation between induction of chromosomal aberrations and transforming abilities of these compounds. Chromosome aberrations might therefore be necessary, but not sufficient on their own, to induce morphological transformation in 10T1/2 cells.

The induction of morphological transformation in 10T1/2 cells by the 1920 sample and by orcelite was dose dependent. The shape of the dose-response curve for induction of morphological transformation by the 1920 sample, however, was very complex. We believe that the green (HT) NiO present in the 1920 sample may cause much of the initial increase in foci in cells treated with the 1920 sample over the concentration range from 0.1 to 1.0 µg/ml, while the Ni5As2 contributes an increasing amount to the induction of morphological transformation over the concentration range from 1.0 to 7.5 µg/ml. However, these nickel refinery samples are very complex. The 1920 sample consists of green (HT) NiO and a copper-nickel oxide as major components, and orcelite, iron oxides, and a second nickel oxide-copper oxide as minor components. There could be complex interactions among the green (HT) NiO, the copper-nickel oxide, the iron oxides, the nickel oxide-copper oxide, and the orcelite in the induction of cell transformation, both in terms of their physical interactions in the sample itself and in terms of the genetic damage they could cause to 10T1/2 cells. It is possible that the copper-nickel oxide, the iron oxides, and the nickel oxide–copper oxide components of this sample also contribute to the induction of morphological transformation. Experiments mixing various components of these samples together to attempt to reproduce the complex dose-response curve of the 1920 sample are in progress in our laboratory.

The dose-dependent increase of morphological transformation in 10T1/2 cells treated with the 1920 sample does correlate with the epidemiological data, indicating that there was an increased incidence of nasal tumors and lung tumors in refinery workers exposed to refinery dust prior to 1923. Apparently, the ability of the 1920 sample to induce morphological transformation in 10T1/2 cells is stable over many years. The inability of the 1929 sample to induce foci in 10T1/2 cells correlates with the substantial decrease in the incidence of cancers in the refinery workers after 1929. We do not know why the NiO in the 1929 sample did not induce either morphological transformation of 10T1/2 cells in vitro or tumors in the workers at the Clydach refinery after 1923. It is possible that the NiO within the 1929 sample alone is not present in a sufficiently high amount to induce cell transformation, and/or that the complex matrix of the 1929 sample suppresses the cell transforming effect of the green (HT) NiO in the 1929 sample. Further studies to address this question are in progress.

Induction of morphological transformation in fibroblastic cells is one of the early steps in the overall mechanism by which the control mechanisms of normal cells are degraded, and they are converted into tumor cells. In fibroblastic cell systems, chemical carcinogens and ionizing radiation usually induce morphological transformation first. A further step that occurs in transformed cell lines derived from foci of morphologically transformed cells induced by chemical carcinogens is anchorage independence, or ability of the cells to grow in soft agar or agarose (reviewed in Landolph, 1985Go). If primary cell cultures are used, such as Syrian hamster embryo (SHE) cells, then the cells will senesce, but some will escape from senescence. In spontaneously immortalized cells such as C3H/10T1/2 cells, focus formation is followed by anchorage independence. The development of tumorigenicity in fibroblastic cell systems, such as C3H/10T1/2 cells, SHE cells, and Balb/c 3T3 cells, is the last step in overall neoplastic cell transformation. (reviewed in Landolph, 1985Go, 1989Go, 1990Go, 1999Go; Landolph et al., 2002Go).

We used C3H/10T1/2 Cl 8 (10T1/2) mouse embryo cells for these studies for a number of reasons. Firstly, 10T1/2 cells are spontaneously immortalized but not otherwise transformed, allowing the cells to be frozen and thawed to use as necessary, which is convenient (Reznikoff et al., 1973bGo). Second, the cloning and preservation of these cells in large amounts allows the investigator to utilize cell stocks that are uniform, making the experiments for chemically induced morphological transformation reproducible (Landolph and Heidelberger, 1979Go; Reznikoff et al., 1973aGo,bGo). Third, these cells have a very low frequency of spontaneous transformation and a reproducible and dose-dependent frequency of morphological transformation when treated with chemical carcinogens or ionizing radiations (reviewed in Landolph, 1985Go). Transformed foci are very easy to score in this cell system, since they are very definitive and stain a dark blue or purplish color against a weakly staining, contact-inhibited monolayer (Reznikoff et al., 1973aGo,bGo; reviewed in Landolph, 1985Go, 1989Go, 1990Go, 1999Go; Landolph and Heidelberger, 1979Go; Landolph et al., 2002Go). When chemically induced foci of transformed cells are cloned in the living state and expanded into transformed cell lines, many of these transformed cell lines are able to form tumors when injected into immunosuppressed or nude mice (reviewed in Landolph, 1985Go; Miura et al., 1989Go; Patierno et al., 1988Go; Reznikoff et al., 1973aGo,bGo). In addition, our laboratory has shown that various insoluble nickel compounds, such as green nickel oxide, black nickel oxide, crystalline nickel monosulfide, and nickel subsulfide, all induce a dose-dependent and reproducible yield of type II and type III foci of transformed cells (reviewed in Landolph, 1989Go, 1990Go, 1999Go; Landolph et al., 2002Go; Miura et al., 1989Go; Verma et al., in pressGo). For all these reasons, we chose to use C3H/0T1/2 Cl 8 mouse embryo fibroblasts for these studies.

We note that both the 1920 sample and orcelite induced not only type II foci but also type III foci. Both type II and type II foci give rise to transformed cell lines, a fraction of which can form tumors when injected into Balb/c nude mice (reviewed in Landolph, 1985Go; Miura et al., 1989Go). Type III foci are the most aberrant of transformed foci in terms of growth patterns and ability to grow in soft agar, and they give rise to transformed cell lines that frequently form tumors when injected into Balb/c nude mice (reviewed in Landolph, 1985Go). Future work will report a more detailed characterization of the biological properties of the transformed cell lines induced by the 1920 sample and by orcelite, including a determination of their tumorigenicity.

These studies indicate that the assay for morphological transformation in 10T1/2 cells can be used to predict the carcinogenicity of pure insoluble nickel compounds and the carcinogenicity of complex nickel-containing samples such as those studied here.


    ACKNOWLEDGMENTS
 
This work was supported by grant ES03341 from the National Institute of Environmental Health Sciences to J.R L., by Training grant 5T32 CA09320 from the National Cancer Institute of the U.S.N.I.H. (P.I., J.R.L.), and by Training Grant 5 T32 AI078078 to the Department of Molecular Microbiology and Immunology of the Keck School of Medicine, U.S.C., from the National Institute of Allergy and Infectious Disease from the U.S.N.I.H., by core grant 5 P30 CA143089 from the National Cancer Institute to the USC/Norris Comprehensive Cancer Center, and by internal funding from the Department of Molecular Microbiology and Immunology and from the USC/Norris Comprehensive Cancer Center to J.R.L. F.C. was supported by a predoctoral fellowship from training grant T32 CA09569 from National Cancer Institute and by a fellowship from training grant 5 T32 AI078078 from the National Institute of Allergy and Infectious Diseases to the Department of Molecular Microbiology and Immunology of the Keck School of Medicine of the University of Southern California. The authors would like to acknowledge the generosity of Dr. John Duffus and Dr. Morrell H. Draper of the University of Edinburgh, Scotland, and the generosity of Dr. Sallie Williams and Dr. Milton Parks of the Clydach Nickel Refinery in Clydach, Wales, U.K., for providing the l920 and l929 samples used in this study and for their helpful and insightful discussions.


    NOTES
 
This work formed part of the requirements for the Ph.D. thesis for F.C. in the Department of Molecular Microbiology and Immunology at the Keck School of Medicine at the University of Southern California.

1 To whom all correspondence and requests for reprints should be addressed at Cancer Research Laboratory, Room #218, 1303 North Mission Road, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033. E-mail: landolph{at}hsc.usc.edu. Back


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 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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