The role of nickel and nickel-mediated reactive oxygen species in the mechanism of nickel carcinogenesis.

Increasing evidence demonstrates the reactive oxygen species (ROS) are implicated in metal carcinogenesis. Exposure of cultured Chinese hamster ovary (CHO) cells to several nickel compounds, i.e. NiS, Ni3S2, NiO (black and green), and NiCl2 has been shown to increase oxidation of 2',7-dichlorofluorescein to the fluorescent 2',7-dichlorofluorescein (DCF), suggesting that nickel compounds increased the concentration of oxidants in CHO cells. This fluorescence can be attenuated by addition of exogenous catalase to the extracellular media, indicating that H2O2 is one of the formed oxidants in this system. Fluorimetric measurements of chromogens following thiobarbituric acid reaction showed that nickel compounds also induce lipid peroxidation with a decreasing potency NiS, Ni3S2 > black NiO > green NiO > NiCl2. These results suggest that lipid hydroperoxides may also be produced through the action of nickel in intact cells. MgCl2, an antagonist of Ni-induced DNA strand breaks and cell transformation, has no effect on the formation of DCF fluorescence induced in CHO cells by nickel. The results suggest that nickel is an active inducer of ROS in intact mammalian cells and that the molecular mechanism of nickel carcinogenesis may involve multiple steps of nickel-mediated ROS.


Introduction
It is becoming apparent that reactive oxygen species (ROS) play an important role in the etiology of diverse human pathologies such as carcinogenesis (1,2), irradiation injury (3), and tumor promotion (4) as well as the normal process of aging (5). ROS, such as hydroxyl radical or metaloxo, and their subsequent reaction products, may be responsible for single-strand breaks in cellular DNA, as well as oxidation of DNA bases, chromosomal aberrations, and DNA-protein cross-links (2,6).
Increasing experimental evidence suggests that nickel-induced genotoxicity may also be mediated by oxygen radical intermediates (7-10). Ni compounds have been shown, for example, to produce reactive oxygen species through the interactions of nickel ions with protein ligands, such as the imidazole nitrogen of histidine (11 Incubation of calf thymus DNA with Ni3S2 or NiCl2 in the presence of H202 or ascorbate leads to the formation of oxidative base damage  and depurination of the DNA (12). When isolated human chromatin was treated with Ni(II) in the presence of H202, several oxidative DNA base modifications were demonstrated (13). Lipid peroxidation has also been reported to occur in lung tissue due to activation of alveolar macrophages after parenteral injection of NiCl2 in rats (14).
Although Ni can lead to oxidation of DNA bases in vitro, direct evidence for Niinduced oxidation in intact cells has only recently been reported (15). We found that Ni3S2 and NiCI2 caused increased oxidation of nonfluorescent 2',7'-dichlorofluorescin (DCFH) that resulted in formation of a measurable fluorescent product in intact Chinese hamster ovary(CHO) cells (15). Only very strong oxidants, such as H202 and organic hydroperoxides can oxidize DCFH, suggesting that Ni elevated the level of these oxidants in intact mammalian cells. In the present study, we compared the effect of exogenous catalase or MgCl2 on the capacity of several different Ni compounds to induce oxidation of DCFH. The capacity of these Ni compounds to induce lipid peroxidation was also studied.

Assay for Dichlorofluorescein
The principle of the assay is based upon the cellular uptake of nonpolar dichlorofluorescin diacetate (DCF-dAc, Kodak, Rochester, NY), which is then hydrolyzed to a polar product (DCFH) that is trapped within the cells (16). The optimal conditions for this assay with intact CHO cells were previously reported (15

Lipid Peroxidation
The levels of lipid peroxide in the nickeltreated CHO cells were measured by thiobarbituric acid (TBA) reactions, as previously described by Ohkawa et al. (17). CHO cells were seeded at 5 x 105 cells in 100 mm dishes in 10 ml of complete a-MEM medium. The next day, cells were treated with several Ni compounds for 24 hr, washed twice in ice-cold PBS and scraped with a rubber policeman. Cells were resuspended in PBS at 107 cells/ml and frozen at -70°C overnight.
After thawing 0.5 ml of the frozen cell suspension was mixed with 0.16 ml of 10% SDS (Bio Rad, Richmond, CA) and 3 ml of 0.4% TBA (Sigma) in 10% acetic acid (Fisher), pH 5. The mixture was adjusted to 4.0 ml with distilled water, and then heated to 90°C for 60 min. After cooling, 4 mL of butanol were added and the mixture was shaken vigorously. After centrifugation at 1500 rpm for 15 min, the fluorescence of TBA-related chromogens in the butanol phase was measured at an excitation wavelength of 515 nm (bandwidth 4.5 nm), and an emission wavelength of 553 nm (bandwidth 9.0 nm).

Results and Discussion
Effects ofN10 and NIS on the Formation ofDCF in Intact CHO Cells Table 1 shows the effects of NiO (black and green) and NiS on the formation of DCF in intact CHO cells. Similar effects with Ni3S2 and NiCl2 were previously reported (15). The increased DCF fluorescence intensities induced by NiO   the physicochemical properties on the surface of the particles (18). Our studies demonstrate that Ni compounds caused an increase in cellular oxidants that may have the ability to convert Ni+2 to Ni+3, or damage DNA bases and induce DNA-protein cross-links, etc. (7)(8)(9). Although the oxidants induced by these Ni compounds have not as yet been identified, previous studies have shown that other hydroperoxides, in addition to H 202 were capable of oxidizing DCFH to DCF in vitro (15,19). Thus the DCF fluorescence assay allows detection of a whole spectrum of oxidants that are potentially formed in living cells following Ni treatment.

Efects of Catalase and MgC12 on Oxidation ofDCFH by NiCL2 or Ni S
The effects of catalase on the formation of fluorescent DCF induced by Ni3S2 are shown in Figure 1. Incubation with 10 jig/cm2 of Ni3S2 (6 hr) increased the level of oxidants in the intact CHO cells as previously reported (15). These doses were not very toxic as measured by plating efficiency (20). Catalase attenuated the fluorescence induced by Ni3S2 but had no effect on the control fluorescence level. These results show that H202 was one of the oxidants induced by nickel compounds. Although catalase cannot enter CHO cells, intracellular H202 can easily escape through the cell membrane and subsequently be decomposed by the catalase present in the extracellular medium.
Magnesium has been shown to be a potent inhibitor of nickel-induced cell transformation in vitro and reduces nickelinduced carcinogenesis in mice (21,22). Figure 2 shows that MgCl2 had no effect on the formation of oxidized DCF in intact CHO cells, even at a concentration of 20 mM for 5 hr. Magnesium only slightly inhibited nickel uptake by lung tissue (22,23).

Effets ofNickel Compounds on Lipid Peroxidation
Although strong oxidants such as hydroxyl radicals or metal oxo have been implicated as genotoxic, lipid peroxidation products such as malondialdehyde may also be mutagenic and carcinogenic (24). The effects of NiS, Ni3S2 and NiCl2 on the formation of TBA-related chromogens in intact CHO cells is shown in Figure 3. After 24-hr treatments, relatively water insoluble NiS and Ni3S2 induced lipid peroxidation whereas NiCl2 did not. Figure 4 shows that black and green NiO induced a dose-dependent formation of TBArelated chromogens. The capacity of nickel compounds to induce lipid peroxidation can be classified by decreasing potency: NiS, Ni3S2>black NiO>green NiO>NiCl2. This classification of the nickel compounds is also consistent with the results of cell transformation studies (6). The results indicate that lipid peroxides may play an important role in nickel carcinogenesis, although other studies in rat liver showed that lipid peroxidation was not causally related to genetic damage, as detected by DNA strand breakage (25).

Summary
In summary, the exposure of intact CHO cells to different Ni compounds resulted in increased formation of oxidants as detected by DCF formation. Both H202 and lipid hydroperoxides are induced by these Ni compounds. The increase of DCF fluorescence required only 3-hr exposures and the fluorescence induced by Ni3S2 could be attenuated by catalase. Lipid peroxidation was studied after 24-hr treatments with the Ni compounds. In contrast to insoluble nickel, highly water-soluble NiCI2 did not induce lipid peroxidation in CHO cells. Our results show that Ni is an active inducer of ROS in intact mammalian cells and that nickel carcinogenesis may involve multiple types of oxidative damage.