Toxicity, uptake, and mutagenicity of particulate and soluble nickel compounds.

Toxicity testing in AS52 cells (24-hr exposures) gave LC50 values of 2 to 130 micrograms Ni/ml for particulate nickel compounds and 45 to 60 micrograms Ni/ml for water-soluble salts (NiCl2, NiSO4, Ni(CH3COO)2). The Ni(OH)2, NiCO3, and sulfides (Ni3S2, Ni7S6, "amorphous NiS") exhibited similar toxicities (LC50's of 2 to 8 micrograms Ni/ml), while three nickel oxides were more variable and less toxic (LC50's of 18 to 130 micrograms Ni/ml). Most compounds displayed nuclear to cytoplasmic nickel ratios of approximately 1:1.5 to 1:5 (except approximately 1:20 for nickel salts). At the LC50's, a 75-fold range in exposure levels occurred compared to a 10-fold range in cytoplasmic and nuclear nickel concentrations, [Ni]. Cellular nickel distribution indicated three groupings: inert compounds (green NiO, lithium nickel oxide, relatively low nuclear and cytosolic [Ni]); water-soluble salts (very low nuclear [Ni]; high cytosolic [Ni]), and slightly soluble compounds (relatively high cytosolic and nuclear [Ni]). Nickel compounds are considered to be only weak or equivocal mutagens. In this study, a low but significant increase in mutation rate at the gpt locus was shown. Although the results would not be sufficient to deem nickel compounds mutagenic by traditional criteria, characterization by PCR analysis indicated that the spontaneous and nickel-induced mutants exhibited different and compound-specific mutational spectra (thus confirming nickel compound involvement). The results reported illustrate some of the methodologic problems involved in testing "weak" mutagens and indicate that alternative approaches may be necessary in classifying the mutagenicity of nickel and other compounds.


Introduction
Single injections of experimental animals with crystalline nickel compounds such as Ni3S2 and NiS have been found to result in a 90 to 100% incidence of tumors at the exposure site (1)(2)(3). By contrast, water soluble compounds (NiCl2, NiSO4) do not induce tumors, while amorphous NiS does so weakly. More recently, however, the repeated administration of nickel salts has produced a low incidence of malignancies in rats (4). In cell culture both crystalline and soluble compounds induce transformation, though soluble compounds must be present at much higher levels (2,(5)(6)(7). Amorphous NiS has not exhibited transforming activity. Cellular transformation and toxicity have been found to depend on This paper was presented at the Second International Meeting on Molecular Mechanisms of Metal Toxicity and Carcinogenicity held 10-17 January 1993 in Madonna di Campiglio, Italy.
Financial assistance was received from the Nickel Producers Environmental Research Association the ability of nickel to enter the cell by phagocytosis (8)(9)(10). Attempts to explain the mechanism of action and potency differences between various nickel compounds have lead to the "Nickel-Ion Hypothesis" (11,12). This hypothesis suggests that the nickel(II) ion is the active agent in nickel toxicity, mutagenesis and carcinogenesis, and that its intracellular concentration is a major determinant, irrespective of the nickel compound to which an organism is exposed.
Despite their tumorigenicity in man and animals, nickel compounds and many other metal compounds have not been found to be mutagenic in traditional in vitro test systems. In the established CHO/hprt mutation assay large deletions and rearrangements extending past the hprt locus may affect or delete neighboring genes necessary for survival, thus decreasing the ability to detect or quantify certain types of mutagens (13). An approach to circumvent this problem was to use AS52 cells, which are CHO cells modified by deleting most of the hprt gene and adding the corresponding bacterial xprt gene (gpt) to a somatic chromosome (14,15). Thus, in addition to base substitutions, frame shifts, small deletions, additions, and chromosome breakage and rearrangements normally measured in CHO cells (16), the new location of this gene is known to allow detection of mutations caused by X-irradia-tion (which causes large deletions). With this modification we hoped to be able to detect nickel-induced mutations, which are suspected to involve large DNA deletions or rearrangements.

Materials and Methods
Preparation and Characterization of Nkkd Compounds Nickel hydroxide was synthesized by slowly mixing 0.6 M NiSO4.7H20 and hot 1.4 M NaOH followed by centrifugation and filtration to collect the precipitate [modified from Kasprzak et al. (17)]. Heating at 80°C for 20 hr to dry and improve crystallinity gave a dark-green product. The sample was ground in a Spex 8000 Mixer/Mill (Spex Industries, Metucher, NJ) with tungsten carbide grinding vial and the light green powder was dry-sieved through a series of wire mesh sieves (#100, #200, #325, #400; respectively, 150, 75, 45, 38 pm openings). Particles not passing through the last sieve were reground and sieved, then wet sieved by suspending in water and filtering through 25 pm and 5 pim sieves. Particles passing through the final 5 pm sieve were collected by centrifugation. The Ni(OH)2 was dried at room temperature and then at 80°C for 5 hr.
NiCO3, which was to be essentially free of nickel hydroxide, was prepared by a procedure suggested by V. J. Zatka (per-  pim) air dried and ground were kept for characterization but not used in the in vivo tests. Amorphous NiS was prepared using a 15% NiCl2 solution and excess 22% (NH4)2S (10) in an acetate buffered system. The pH was measured periodically and glacial acetic acid added as required to keep the pH in the range of 4.0 to 6.0. The product was filtered and rinsed with water and acetone, dried at 1 10°C for 17 hr, then ground in the Spex grinder. Particles sieved and collected as described for the nickel hydroxide were sterilized in acetone and dried for 5 hr at 80°C. Black NiO, Li2Ni8010 (nickel oxide in which 2.39% Li stabilizes a Ni(III) content of 19.2%), crystalline a-Ni7S6, and crystalline nickel subsulphide (Ni3S2) were obtained from INCO Limited (Toronto, ON) and green NiO was purchased from Johnson Matthey Chemicals (Brampton, ON) (puratronic grade). These compounds were wet-sieved through a 25-pm sieve and 10-pm nylon Spectra/mesh disposable filter. Particles were collected by centrifugation, sterilized in acetone, centrifuged, and then dried for 5 hr at 800C.
High purity nickel chloride and nickel sulfate were purchased from BDH (Toronto, ON) (analar grade) and nickel acetate from Johnson Matthey (puratronic grade). Stock solutions were made in water, filter-sterilized through 0.22-pm filters, and stored in sterile glass bottles. NiCl2 and NiSO4 were prepared as 500 mM stock solutions, while Ni(CH3COO)2 was made as a 100 mM stock solution.
The nickel, sulfur, and minor constituent contents of the compounds provided by INCO (Table 1) by comparing lattice spacings and peak intensities to standard spectra available in the JCPDS data base (18). CG Culture AS52 cells derived by modification of the CHO-KI line were kindly provided by K. R. Tindall, National Institute of Environmental Health Sciences (Research Triangle Park, NC). These cells were grown in Ham's F12 medium (F12) with 5 to 10% fetal bovine serum (FBS). Dialyzed FBS was used in mutation testing procedures. Prior to mutagenicity studies, the medium was supplemented with 10 pg/ml mycophenolic acid, 25 pg/ml adenine, 50 pM thymidine, 250 pg/ml xanthine, and 3 pM aminopterin (MPA medium), to eliminate spontaneous mutants. Antibiotics were not added for routine culturing, though 100 U penicillin G, 100 pg streptomycin, and 0.25 pg amphotericin B per ml medium were employed in individual experiments. Cells were grown in a 37°C, 95 to 100% humidity, 5% CO2 (continuous flow) incubator, and routinely subcultured every 3 to 4 days. A doubling time of 14 Figure 2. Nickel toxicity curves in AS52 cells. AS52 cells were seeded in F12/10% FBS medium at 500 cells per 60mm diameter dish 2 days before treatment. The cells were exposed to the range of nickel compounds in serum-free medium at the concentrations indicated for 24 hr (or 5 hr exposure for nickel salts, as noted). The cells were rinsed to remove extracellular nickel, then incubated in fresh F12/10% FBS medium until colonies formed to 9 days). The   AS52 cells from a near confluent culture dish were released with trypsin, counted, diluted, and replated at 500 cells per 60 mm diameter dish in F 12 medium with 10% fetal bovine serum (F12/10). One to two days later, the medium was replaced with serum-free medium (F 12/0) and the test compound was added. After an exposure period of 24 hr, or 5 hr for nickel salts, the medium was removed and the cells rinsed to remove nickel not taken up by the cells. The F12/10 medium was renewed and then the cells were incubated for 6 to 8 days until colonies of sufficient size to be counted developed. Colonies were stained using a solution of 0.5% crystal violet in ethanol, thereby producing clearly visible, circular colonies of a deep violet color. These colonies were then counted using an automated colony counter and the number per dish relative to the number in untreated control culture dishes (relative % survival) was reported.
Analysis ofNickel Content in Cells,

Nudei, and Cytsol
To get a measure of the biologically available nickel levels (as opposed to total nickel present), we set out to determine the dissolved nickel (nickel ion) levels in the cytosol and nuclei. The final procedure used to assess the cytosolic fraction and a nuclear fraction free of particulates is outlined in Figure 1. To assess possible contributions to the measured nickel levels from particulate dissolution during the cellular fractionation protocol outlined in Figure 1, simulation experiments were conducted for green NiO and Ni3S2. All samples were analyzed by electrothermal atomic absorption spectrometry (EAAS). A Perkin Elmer Atomic Absorption Spectrometer Model 703 (Rexdale, ON) furnished with a graphite furnace Model HGA-500 was used.

Mutagenicity Testing
Cells growing in MPA medium were replated in F12/10% FBS on day -2. On day 0 the medium was replaced with serum-free F12 and the nickel compound or ethylmethanesulfonate (EMS, positive control) was added. Test doses for particulate nickel compounds were chosen to give 20 to 80% survival based on previously determined toxicity curves. After an exposure period of 24 hr for particulates or 5-hr for nickel salts and EMS, the medium was removed and the cells rinsed with F12/5% FBS to remove nickel not taken up by the cells. For 5-hr exposures, fresh F12/10%   BAS52 cells grown in MPA medium to eliminate spontaneous mutations or loss of the gptgene were plated in F12/10% FBS 2 days ( 5.105 cells/100 mm dish) prior to treatment. Cells were exposed to the test compounds for 24 hr (or 5 hr for nickel salts, as indicated) in serum-free medium, then rinsed, trypsinized, and replated in F12/10% FBS. The cells were subcultured every 3 days as required during the expression period of 8 days. After this period, 6-thioguanine resistant mutants were selected by plating at 2x105 cells/100 mm dish in 10 ml F12/10% dialyzed FBS plus 10 pM 6-thioguanine. At this same time 500 cells/60 mm dish were plated in medium without thioguanine for determination of plating efficiency. After 10 to 15 days, dishes were rinsed with PBS and colonies stained and counted. The mutation frequency is expressed as the number of mutant colonies per million cells plated. Results are reported in this table for a single experiment based on 5 to 6 dishes per treatment condition with further replicates for negative controls as indicated. tTest doses were selected to correspond to formal survival rates of 80%, 65%, 50%, 35%, and 20%. See Table Al for amounts of the compound added. cTwo-tailed significance test. dSignificantly different from control rates by one-tailed ttest, at the 0.05 probability level or better. 'Significant at the 0.05 probability level or better.
FBS was added, the cells were incubated 19 to 24 hr, and were then rinsed once with phosphate-buffered saline (PBS). For 24-hr exposures, the cells were rinsed with PBS. Subcultures were made on day 1 and every 2 to 3 days thereafter to allow expression of the altered gene, with a larger number of cells replated from cultures in which a lower survival was expected, so that at the next replating approximately equal densities were reached. On day 9 the cells were replated at 2 x105 cells/100 mm dish (5 or 6 dishes per treatment condition) in 10 ml F12/10% dFBS (or 5% dFBS and 5% dialyzed calf serum) plus 10 pM 6-thioguanine (6-TG) to select for gpt mutants. At the same time, 500 cells/60 mm dish (3 dishes per treatment condition) were plated in the same medium without 6-TG for a plating-efficiency (PE) determination. After 8 days (day 17), the PE dishes were rinsed with PBS and stained with crystal violet. Following a selection period of 10 to 12 days the 6-TG-resistant colonies (loss of xanthine-guanine phosphoribosyl transferase (XPRT) activity at the gpt locus) were stained with crystal violet and counted.

Characterization ofMutants
Colonies were picked off plates and clonally expanded to approximately 106 cells. Approximately 0.5 to 1 pg of mutant DNA was amplified by the polymerase chain reaction (PCR) employing suitable primers encompassing the gpt gene. These primers constituted the starting points for the DNA synthesis (polymerization). The products were separated by electrophoresis on a 1% agarose gel, which was subsequently stained with ethidium bromide. Additional details are provided in Rossetto etal. (19).

Chaterization ofNickel Compounds
Results of the chemical and X-ray diffraction analysis of the nickel compounds are given in Table 1. The percent nickel was used to convert from pg compound/ml (as weighed out) to pg nickel/ml, the units reported in the tables and figures.
For Sample 1 (nickel hydroxide), the X-ray diffraction peaks were too broad to allow an accurate measurement of d values or the diffraction angles (20) Tables 3 and Al. Al-X 5sajyl Figure 4. Toxicity, mutagenicity, and uptake of NiSO4 in AS52 cells after 5 hr exposure. The data plotted in this summary figure were derived from those depicted in Figure 2J or compiled in Tables 3 and Al. amorphous NiS, according to the diffraction pattern contained a mixture of NiS2 and NiSO4.6H20. These are possible oxidation products in the formation of NiS, though they do not account for the analyzed nickel and sulfur contents. The high background in the diffraction pattern obtained suggested that a noncrystalline component was present, the most likely species being amorphous NiS (, 60 mole percent). For Sample 7, the complexity of the pattern prevented a definite identification, although a-Ni7S6 appeared to be the major component. Other nickel/sulfur compounds were probable, with NiS, anhydrous NiSO4, and Ni3S2 implicated. Sample 8 was definitively identified as Ni3S2 by both X-ray diffraction and chemical analysis.

Toxicity ofNiwkel Compounds
Dose-response curves showing the percent survival of the exposed cells (with respect to untreated control cells) versus the amount of nickel added to the culture medium are given in Figure 2 (2A to 2L) for the 11 compounds tested. The average percent survival and standard deviation depicted were calculated from the results of at least 3 replicate experiments. LC50 values (concentration which kills 50% of the exposed cells) were obtained from the respective graph for each compound tested and are given in Table 2 along with cellular nickel levels. To facilitate comparison of the various compounds, concentrations are expressed in units of pig Ni/ml.

Nickl Uptake
Appendix Table Al summarizes the nonparticulate ("dissolved") nickel contents of the cell cytosol (average of 4 experiments) and of the cell nuclei (average of 2 experiments). Exposure doses are the same as those used in the mutation experiments. For most of the compounds, a significant (p<0.05) relationship exists between administered dose and observed intracellular nickel concentrations.

Mutgenicity Testing
Representative mutation frequencies for 6thioguanine (6-TG) resistance induced by the various nickel compounds are summarized in Table 3. The mutation frequency represents the number of mutations observed per million cells plated. At the time of selection, the cells had recovered such that the PE of treated cultures were approximately the same (40 to 50%) as the negative (nonexposed) controls, regardless of the exposure dose of the nickel com-pound. The plating efficiencies of the EMS-treated cells (17 to 34% depending on dose) were significantly lower than the controls.
The mutation frequency of nonexposed cells observed in separate experiments appears to adhere to a normal statistical distribution. In 4 mutation experiments, 12 independent assessments (69 dishes total) yielded a spontaneous mutation rate of 26 ± 2.5 (mean ± sd; range 0-55). The statistical significance of the increase in mutation frequency after nickel exposure was assessed by comparison to the negative control data (no nickel added). Since the mutation frequency reported for the test compounds is based on the average of 5 to 6 replicate dishes in the same experiment, the standard deviation and the 95% confidence interval for each exposure condition were calculated; and are denoted as upper and lower limits in Table 3. A comparison of the mean mutation frequency with that of nonexposed (negative) control cultures using the one-tailed Student's t-test was employed to determine the significance of the nickel-induced mutation rates. For each compound, the results were also tested for the significance of the correlation between the administered nickel dose and the induced mutation rate. For this comparison, the correlation coefficient (r) was calculated and the significance determined using standard two-tailed statistical tables.
In Figures 3 and 4, the mutagenicity and nickel uptake data are plotted in conjunction with the cytoxicity results for Ni3S2 and NiSO4. As is evident from the data in Table 3, the increase in mutation frequency with intracellular nickel uptake is significant (p<0.05) only for Ni(OH)2, "amorphous NiS", Ni3S2, and NiSO4.

Mutant Ch izton
Sequencing and restriction analysis of the two PCR products observed revealed that the smaller (lower relative molecular mass) band corresponds to the functional gpt locus, while the larger band is functionally inactive [designated as a pseudogene, (19)]. Northern blot analysis of mRNA and reverse transcriptase PCR (RT-PCR) analysis of both mRNA and total RNA confirms this (data not shown). Analysis of the amplification products on an agarose gel (1%) revealed 3 distinct alterations at the gpdpseudogene loci: both PCR DNA bands remain intact; the smaller DNA band (corresponding to the functional gpt gene) is absent; or both bands are absent. Repetition of the PCR work has confirmed Environmental Health Perspectives the qualitative characterization of the observed mutants reported in Figure 5. In addition, re-evaluation of the mutants with a second primer set encompassing the gpt gene and larger stretches of DNA both upstream and downstream of the gene has also corroborated the depicted mutation profiles. Details are given in Rossetto et al. (19).

Discussion and Conclusions
Major Finding X-ray diffraction characterization of "amorphous NiS" synthesized from NiCl2 and (NH4)2S in the presence of air illustrates the difficulty of preparing a pure product without impurities such as NiS2 and NiSO4, at least under the conditions employed. The synthesis procedure used differed from that ofAbbracchio et al. (10) only in our use of an acetate buffered medium. Both the cytotoxicity (LD50 = 4.1 pg Ni/ml) and mutagenicity of "amorphous NiS" suggest that crystalline NiS2 is a sulfide of high cytotoxic and genotoxic potency. Carcinogenicity studies of NiS2 in rats support this conclusion (3). In view of the X-ray diffraction evidence for the presence of NiS2, the alternate explanation that the unexpected activity of amorphous nickel sulfide was due to negative surface charges, which is known to make it biologically active (22), is less likely.
Contributions to the measured nickel levels from particulate dissolution during the cellular fractionation process is judged to be small. Simulation experiments incorporating the protocol oudined in Figure 1 (i.e., same buffers, incubation periods, and temperatures) indicated that little of the particulates dissolved (0.01% for green NiO and 0.9% for Ni3S2), in agreement with the known serum dissolution halftimes (T50) of >11 years and 28 days, respectively ( Table 2). In contrast to the other particulate nickel compounds studied, there was no visible evidence of residual particulate material during the manipulations summarized in Figure 1 for Ni(OH)2 (T50<1 day, Table 2) and NiCO3, both of which are reported to be slightly soluble in water, rather than insoluble (23).
As illustrated by the toxicity curves of Figure 2A to 2L and the LC50 data in Table 2, there are significant differences in the toxicity of the various nickel compounds tested. For particulate compounds, the range of LC50 values is from 2 pg Ni/ml for Ni(OH)2 to 130 pg Ni/ml for the green NiO. For the water-soluble salts, the LC50 increased from 45 to 60 pg Ni/ml for 24-hr exposures to 120 to 125 pg Ni/ml for 5-hr exposure. The (Ni(OH)2, carbonate, and sulfides show similar toxicities, while the nickel oxides are shown to be less toxic, with the potency depending on the compound tested (green, black, or containing Ni(III)/Li). The cytosolic and nuclear nickel contents of the compounds at a given cytotoxicity level show considerably less variation than the administered dose. Thus at LC50, the cytosolic nickel levels observed were in the range of 35 to 250 ng/106 cells (NiCO3 exempted), while levels of 6 to 80 ng/106 cells were observed in the nuclear fraction (Tables 2,A1). The particulate compounds with the lowest toxicity (green NiO and NiO with Ni(III)/Li), and therefore necessitating the highest extracellular nickel concentrations to give measurable cell toxicity, show the lowest intracellular nickel levels. Because considerable cytotoxicity occurred for these 2 compounds, an influence other than dissolved nickel in cells appears to contribute to the loss of cell viability.
The compounds exhibiting the strongest mutagenic responses-Ni(OH)2 "amorphous NiS," and Ni3S2, have the highest nuclear nickel levels and the lowest administered doses. Although nickel compounds appear to be only weak mutagens, the present results do indicate a significant increase (p<0.05) in mutation rate for the nickel compounds, although not consistently at all doses tested. The Ni(OH)2, Ni3S2, and "amorphous NiS," which not only exhibited evidence for a significant dose-response (p<0.01 or p<0.05) but also increased mutation frequencies relative to control cultures (p<0.025), might be termed mutagenic by generally accepted criteria requiring both observations to occur. The water soluble nickel salts at the majority of the administered doses produced significant increases (p<0.05) in mutation rate and raised cytosolic nickel levels, but had relatively low nickel concentrations in the nuclear compartment.
[Relatively low nuclear concentrations of nickel have also been observed by T. Malinski (Oakland University, Rochester, MI) when cells were exposed to nickel salts (personal communication)]. A number of interpretations of these observations are possible.
From the PCR characterization, three qualitative classes of mutations were observed ( Figure 5). Sequence analysis of a number of EMS mutants has shown that point mutations are members of this first family (both PCR gpt bands present). In addition, small deletions of several base pairs or frameshift mutations theoretically would also be scored in this first class. Deletions of all or part of the gpt locus are assigned to the second empirical type (small band absent), while multilocus deletions or damage are believed to correspond to the third category of mutants (both bands absent). Although it can be postulated that small mutations (i.e., point, frameshift, or deletion) in critical regions of primer annealing may result in similar profiles, the corroboration of mutation profiles utilizing a second distinct set of PCR primers argues against this conclusion. (Note that in this discussion gene deletion denotes an operational definition, i.e., the inability of a DNA segment to be amplified by PCR, and does not necessarily imply the total physical absence of a gene.) The profile of damage inflicted was found to depend on the nature of the nickel compound. Ni(OH)2 and NiSO4 induced substantial increases in deletions in all or part of the gpt gene compared to EMS and spontaneous mutants. Thus, although the mutagenicity testing showed the nickel compounds to be weakly mutagenic, they were able to induce mutations with molecular lesion profiles distinct from spontaneous changes. Perhaps, as in wild-type CHO  Designation of mutant types was based on an examination of PCR amplification products on standard 1% agarose gels. Three types of gel patterns were observed as indicated in the key shown above: (a), both PCR bands present (point mutation, small deletion, or frameshift) or (b), the smaller band not observed (active gene absent) or (c), both PCR bands not observed (active gene and pseudogene absent). Control mutants denote those arising spontaneously in the absence of any test chemical.
Volume 102, Supplement 3, September 1994 75 circumvents the observation of strongly positive mutagenic responses in AS52 cells.

Interpretations
An examination of the nickel uptake and cytotoxicity data suggests that, as a first approximation, the "Nickel-Ion Hypothesis" holds. While administered LC50 doses differed by a factor of 75 for the 11 nickel compounds studied, the corresponding intracellular (cytosolic and nuclear), nonparticulate nickel concentrations varied only by about a factor of 10. (The cytosolic nickel levels for NiCO3 are excluded in this analysis; below.) The observed cytosolic nickel levels did not correlate with the LC50 values, while the nuclear concentrations did (p<0.01) (Tables 2,A1). By comparing the administered doses with the measured nickel levels in the cytosol and nucleus, one can see that the Ni(OH)2, NiCO3, "amorphous NiS" and Ni3S2 were the most efficient in delivering nickel to cells (measured as nonparticulate or "dissolved" form) and were also the most toxic. This observation is consistent with the Nickel-Ion Hypothesis. The fact that all the nickel compounds produce mutagenic responses (p<0.05) supports the notion of the Nickel-Ion Hypothesis that all nickel compounds are potentially genotoxic if they release nickel(II) ions.
It is reasonable to suggest that the measured, nonparticulate (dissolved) cytosolic nickel represents different compartments for the uptake from salt solutions than for the incorporation of particulate nickel compounds. It is known that phagocytized particulates of Ni3S2 are often present in cytoplasmic vacuoles (5,24). It is hypothesized that within vacuoles particulates undergo dissolution and that nickel(II) ions can be released from them to the cytosol (12,25). Presumably, the lysing step employed in releasing cytosolic nickel in the present study (see Figure 1) makes available for measurement nickel(II) ions present in both vacuoles and the cytosol proper. On the other hand, nickel(II) ions taken up from salt solutions [perhaps via calcium channels, (26)] may be expected to occur only in the cytosol proper, likely bound to proteins and low-molecular-mass ligands. In this case, only this pool is suspected of being released in the cell-membrane lysing step.
It is obvious from the present study that factors other than intracellular nickel concentration contribute to the cytotoxicity. Nickel compounds are known to exhibit differential surface activities which, for example, result in quite different abilities to induce haemolysis in human erythrocytes (27). Consequently, physicochemical surface or cell interactions probably contribute to cell injury. Such an effect may be especially important when relatively large doses are required, such as for the green and Ni(III)/Li nickel oxides.
Nickel-induced mutagenicity also seems to have a number of determinants. This can be seen by comparing the mutagenicity and the cytosolic/nuclear nickel levels for the particulate compounds with the water-soluble nickel salts (Tables 3,Al). Consequently, low nickel levels relative to those found in the cytosol fraction are observed in the nuclei of cells exposed to the chloride, sulfate, or acetate salts (and perhaps also to the carbonate, which appears to be the most soluble of the particulate compounds). In contrast, the cytosolic concentrations are substantially elevated for these compounds. A number of implications and interpretations flow from this observation.
The simplest interpretation is that intracellular compartmentalization of nickel results in different mechanisms of genotoxicity. It is tempting to assign the mutagenicity of nickel salts to high levels of nickel in the cytoplasm, which, through indirect effects damage the genome. One such indirect mechanism might be related to the demonstrated ability of certain nickel(II) complexes of natural ligands to participate in active oxygen biochemistry (12,(28)(29)(30)(31). Such processes generate radical species that are known to damage DNA. By contrast, high concentrations of nickel(II) ions in the nucleus are capable, on binding to DNA and nuclear proteins, of damaging DNA [e.g., depurination, (26)], or inducing changes in chromosomal conformation that may result in permanent changes in gene expression (12,29,32,33). These interpretations receive strong support from the work of Sen and Costa (25), who demonstrated that the pathway of delivery of nickel to CHO cells determined the type of interaction with chromosomes. Thus nickel(II) salts, like crystalline NiS, induced chromosomal aberrations (gaps, breaks, and exchanges), but only the particulate compounds produced X-chromosome fragmentation. However, when nickel(II) was delivered to the cell in liposomes (as NiCl2 or the nickel(II) complex of bovine serum albumin), X-chromosome fragmentation was also observed. Our work corroborates these observations by Sen and Costa (25). The measured nonparticulate nickel concentrations support the notion that differ-ential intracellular compartmentallzatlon of nickel determines such differences in genotoxic responses.
The response to NiCO3 warrants additional comment. Crystallinity has generally been accepted as a prerequisite for the uptake of nickel compounds by phagocytosis. Although poorly crystalline by X-ray diffraction analysis, the NiCO3 used in our cytotoxicity and mutagenicity studies was considerably more efficient than the watersoluble salts in delivering nickel to the cells. Of course, microcrystallinity cannot be ruled out. Like the salts, the nuclear fraction of nickel in this case was also found to be small compared to the cytosolic levels observed, which is consistent with its solubility. This suggests that the NiCO3 induced mutagenicity, at least in part, in a fashion similar to the salts. Perhaps on uptake, the degree of vacuolation is less, or the extent of dissolution in vacuoles greater, than for the other solids. Nonvacuolized Ni3S2 has been reported in CHO cells (5,24). Conclusions In conclusion, our work has demonstrated that nonparticulate, intracellular nickel(II) concentrations, irrespective of the parent compound, constitute an important determinant in the cytotoxicity and mutagenicity responses by AS52 CHO cells to nickel compounds. This finding is consistent with the Nickel-Ion Hypothesis. However, this hypothesis must be modified to allow for differential compartmentalization of nonparticulate dissolved nickel within the cell that is dependent on the nickel-uptake pathway, as well as mechanical cell surface effects in case of relatively nontoxic compounds. The uptake of particulates by phagocytosis appears to elevate the measured, nonparticulate nickel levels in both the cytosol and the nucleus, while nickel from salts accumulates preferentially in the cytosol. These results suggest that cytosolic and nudear nickel accumulations (in nonparticulate form) appear to produce mutations by different mechanisms. The mutant profile analysis suggests that induction of mutations at levels only slighdy above background may indeed be indicative of the mutagenic potential of nickel compounds. It appears that there are methodologic problems involved in testing "weak" mutagens and that alternative approaches are necessary in classifying the mutagenicity of nickel and other compounds. The PCR analysis reported illustrates the potential of employing molecular biology techniques in a complementary manner.
Environmental Health Perspectives   'AS52 cells were seeded in F12/10% FBS medium at 0.5-1.0*106 cells per 100-mm diameter dish 1 to 2 days before treatment. The cells were exposed to the range of nickel compounds in serum-free medium at the concentrations indicated for 24 hr (or 5-hr exposure for nickel salts as noted). The cells were rinsed to remove extracellular nickel and then trypsinized, pelleted, rinsed, and counted. The cells were lysed in hypotonic buffer + Nonidet P-40, and centrifuged to pellet the nuclei and particulates. The supernatant (referred to as the cytosol) was kept for analysis. The pellet was digested with DNAse followed by Proteinase K/SDS and the particulates removed by centrifugation. The supernatant consisting of the nuclear nickel was analyzed for nickel content. Averages and standard deviations (std) were obtained based on four replicate experiments for the cytosolic nickel and two experiments for the nuclear nickel content.. bTest doses were selected to correspond to formal survival rates of 80%, 65%, 50%, 35%, and 20%. cSignificant at the .01 probability level dSignificant at the .05 probability level.