* Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at the National Institute of Environmental Health Sciences;
Metals Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Maryland;
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
Received May 25, 2001; accepted August 3, 2001
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ABSTRACT |
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Key Words: nickel; arsenic; lipid peroxidation; free radical; DNA damage; apoptosis; GSH.
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INTRODUCTION |
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Nickel compounds are well-established human carcinogens (Costa and Klein, 1999; Salnikow et al., 1999
). However, the precise molecular mechanism of nickel carcinogenesis is undefined. It is known that many transition metals can damage DNA by facilitating production of free radicals generated by various mechanisms including Fenton chemistry (Sarkar, 1995
; Sunderman, 1989
). Nickel is a redox active metal and indirect damage due to generation of reactive oxygen species (ROS) is probably important in nickel toxicity and carcinogenesis (Kasprzak, 1991
). Nickel-mediated lipid peroxidation may be also involved in carcinogenesis. For example, it has been reported that products of nickel-induced lipid peroxidation can damage DNA in vitro (Ueda et al., 1985
). It has also been shown that hydroxyl radicals are involved in nickel-mediated lipid peroxidation, a finding that may have implications in the carcinogenicity of nickel compounds (Athar et al., 1987
). Furthermore, nickel impairs cellular defense mechanisms against peroxidation by depleting free-radical scavengers including glutathione (GSH), or by inhibiting catalase, superoxide dismutase, glutathione peroxidase, glutathione S-transferase, or other enzymes that protect against free-radical injury (Donskoy et al., 1986
; Sunderman, 1989
). Thus, nickel exposure disrupts cellular redox status as part of its adverse effects.
Apoptosis can be initiated by a variety of stimuli, including oxidants, ionizing radiation, chemotherapeutics, and toxic chemicals (Hockenbery et al., 1993). When apoptosis is the mode of cell death, initial stress-induced damage does not kill cells directly, but rather it triggers a signaling program that leads to "suicidal" cell death (Gabai et al., 1998
). ROS and oxidative damage have been implicated in the induction of apoptosis (Mates and Sanchez-Jimenez, 2000
). In this regard, oxidative damage to the deoxynucleotide pool and genomic DNA have also been associated with the initiation of the carcinogenic process (Mates and Sanchez-Jimenez, 2000
). For example, the oxidative product of dGTP, 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) has been reported to be highly mutagenic through misincorporation into DNA, causing AT-CG mutations (Bialkowski and Kasprzak, 1998
). In vivo promutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) levels are increased in animals exposed to redox-active carcinogenic metals such as nickel (Porter et al., 1996
), and nickel, in vitro, facilitates oxidation of guanine in 2'-deoxyguanosine in DNA, forming 8-oxo-dG (Kasprzak and Hernandez, 1989
).
Nickel toxicity can be modified by prior nickel exposure or by exposure to other metals. For instance, the development of tolerance to the dermal toxicity of nickel can be produced by oral nickel exposure in both humans (Panzani et al., 1995) and animals (Van Hoogstraten et al., 1993
). Similarly, cells exposed to nickel can develop and retain nickel resistance, which involves an altered oxidative stress response (Salnikow et al., 1994
). Nickel-resistant cells also show altered GSH metabolism (Salnikow et al., 1994
). As to other metals, the effects of manganese or magnesium in blocking nickel toxicity or carcinogenesis have been reported in several studies (Hong et al., 1997
; Judde et al., 1987
; Kasprzak et al., 1985
; Sunderman et al., 1974
, 1976
; Sunderman and McCully, 1983). Magnesium-induced tolerance to nickel is thought to occur by reducing the intracellular nickel concentrations and thereby reducing ROS (Hong et al., 1997
).
Thus, in the present work, we followed our initial observation of arsenic-induced acquired nickel cross-tolerance in CAsE cells (Romach et al., 2000) with additional study on the mechanism of nickel tolerance. This model provides the opportunity to study the mechanism of acquired nickel tolerance in the absence of prolonged pretreatment with elevated levels of nickel. The acquisition of tolerance to nickel could have implications in nickel carcinogenesis and metal-metal interactions in inorganic carcinogenesis.
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MATERIAL AND METHODS |
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Cell culture and treatment.
The TRL 1215 cell line was originally derived from the liver of 10-day-old Fischer F344 rats (Idoine et al., 1976). The cells are diploid and normally nontumorigenic. Cells were cultured as previously described (Zhao et al., 1997
). Control cells were cultured in William's E media containing 10% fetal bovine serum, while 0.5 µM arsenite was added to culture medium to induce transformation, which occurred at about 18 weeks of exposure (Zhao et al., 1997
). The cells chronically treated with arsenite (CAsE cells) show extensive evidence of malignant transformation, including the formation of invasive tumors with metastatic potential upon inoculation of these cells into nude mice (Zhao et al., 1997
). In all cases, CAsE cells were compared to passage-matched controls.
Metabolic integrity assay.
The Promega Cell Titer 96 Non-Radioactive Cell Proliferation Assay kit was used to determine acute cytotoxicity of nickel or hydrogen peroxide in cells as defined by metabolic integrity. This assay measures the amount of formazan produced by metabolic conversion of Owen's reagent [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, MTS] by dehydrogenase enzymes found in the mitochondria of metabolically active cells. The quantity of formazan product, as measured by absorbance at 490 nm, is directly proportional to the number of living cells. A minimum of 4 replicates of 10,000 cells per well were plated in 96-well plates and allowed to adhere to the plate for 24 h, at which time the media was removed and replaced with fresh media containing the nickel or hydrogen peroxide. Cells were then incubated for an additional 24 h and cell viability was determined (Romach et al., 2000). In another series of experiments, cells were pretreated with BSO (100 µM), an inhibitor of GSH synthesis, for 6 h prior to nickel exposure, and then cell viability was determined. The LC50 values were determined from analysis of the linear portion of the metabolic integrity curves and compared between CAsE and control cells.
Lipid peroxidation.
Lipid peroxidation was measured by formation of thiobarbituric acid (TBA) reactive substances (TBARS) after the method of Jacobi et al. (1999). After treatment with NiCl2 for 24 h, cells were washed with PBS, harvested and lysed. Malondialdehyde (MDA), obtained by acid hydrolysis of 1,1,3,3-tetra-ethoxy-propane (TEP), was used as the standard for the quantification of TBARS. TBA was added to each tube and vortexed. The reaction mixture was incubated at 90°C for 20 min and the reaction was stopped by placing samples on ice. TBARS were subsequently extracted with 1.5 ml n-butanol and the phases were separated at 300 x g centrifugation for 5 min. Fluorescence was measured in the n-butanol phase by an Aminco-Bowman spectrophotofluorometer using an excitation wavelength of 515 nm and emission wavelength of 550 nm. Data were expressed as pmol TBARS per mg protein.
Determination of 8-oxo-dG level in nuclear DNA.
The level of 8-oxo-dG in nuclear DNA was determined using the enzymatic hydrolysis procedure according to Adachi et al. (1995). After treatment with nickel for 24 h, cells were washed with PBS and harvested by trypsinization. Separate samples of 8-oxo-dG and dG were used as standards. DNA hydrolysates were analyzed by an HPLC system consisting of a Waters 2690 Separation Module, a Waters 490E Programmable Multiwavelength Detector (Waters Co, Milford, MA, USA), an ESA (Chelmsford, MA) Coulochem II 5200A electrochemical detector (guard cell: 700 mV, standard analytical cell model 5010: working electrode E1 at 300 mV), and a Supelcosil LC-18-S column (250 x 4.6 mm, 5 µm grain; Supelco, Switzerland) equipped with a 2-cm guard column. Twenty-µl aliquots of the DNA hydrolysates were chromatographed isocratically at a flow rate of 1 ml/min, using 100 mM sodium acetate-orthophosphoric acid, pH 5.2/methanol (92:8) as the eluent. The system was controlled and chromatograms were acquired and integrated by a Millennium Chromatography Manager.
Electron spin resonance (ESR) measurements.
The generation of free radicals from the control or nickel-treated cells was investigated by ESR utilizing DMPO as a spin trap agent. Cells and the various reactants were mixed in a final volume of 300 µl. To help define the nature of the radical generated by nickel, SOD and catalase were diluted with PBS in the presence of DFO (100 µM). ESR recording was performed at room temperature using a quartz flat cell and a Bruker EMX spectrometer with a Super High Q cavity. The instrumental conditions are indicated in the figure legends. Spectra were recorded via computer interfaced to the spectrometer. Hyperfine coupling constants were determined with a spectral simulation program that is available to the public from the NIEHS/NIH on the internet (http://epr.niehs.nih.gov).
Determination of apoptosis by flow cytometry.
Detection of phosphatidylserine on the outer leaflet of apoptotic cells was performed using Annexin V and propidium iodide according to the manufacturer's recommendations. For each sample, 10,000 cells were examined by flow cytometry using a Becton Dickinson FACSort. The percent of apoptotic cells was determined by statistical analysis of the various dot plots using CellQuest software (Vermes et al., 1995).
Measurement of intracellular GSH.
After treatment with nickel for 24 h, cells were washed with PBS, harvested by trypsinization, lysed, and cellular debris were precipitated with perchloric acid. The level of intracellular GSH was determined in triplicate cell pellets using the fluorometric method of Hissin and Hiff (1976). Values were normalized to cell number.
Statistical analysis.
Student's t-test or ANOVA with subsequent Dunnett's or Duncan's test were used as appropriate. All values are expressed as mean ± SEM of 3 or more replications. Differences were considered significant at p < 0.05.
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RESULTS |
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DISCUSSION |
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Apoptosis and cancer can often be viewed as opposing phenomena in cell population dynamics (Mates and Sanchez-Jimenez, 2000). In fact, evidence indicates that tumor formation can be associated with inhibition of apoptosis (Mates and Sanchez-Jimenez, 2000
) while cancer chemotherapeutics often act by inducing apoptosis in cancer cells. Diminished apoptosis is characteristic for several cancers and autoimmune disorders (Buzard and Kasprzak, 2000
). ROS have been widely reported to play a key role in cancer (Costa and Klein, 1999
) and production of ROS and oxidative damage are implicated in the induction of apoptosis. Thus, ROS production, oxidative injury, and apoptosis are strongly related (Mates and Sanchez-Jimenez, 2000
). The present study demonstrated that nickel-induced apoptosis was markedly reduced in nickel-resistant CAsE cells. Whether or not, and how the apoptotic block after nickel exposure might affect nickel carcinogenesis are unknown, but the perturbation of apoptosis is thought to contribute to the carcinogenic process (Mates and Sanchez-Jimenez, 2000
) either through enhanced initiation or progression. Thus, the possibility exists that prior or concurrent exposure to arsenic may actually enhance the carcinogenic effects of nickel. Because arsenic exposure clearly perturbed the genotoxic effects of nickel, the reduced genotoxicity in itself would reduce the stimulus for apoptosis, which in turn would inhibit nickel carcinogenesis. The results of the present study clearly show that the interplay between multiple exposures to inorganic carcinogens may be critical to eventual carcinogenic outcome, but this interplay can be quite complex. Perhaps a series of toxicity and carcinogenicity studies in vivo using various combined administration regiments of arsenic and nickel would shed further light on this issue.
GSH is involved in metal detoxification and is critical to the maintenance of the cellular redox status (Costa and Klein, 1999; Mates and Sanchez-Jimenez, 2000
). Depletion of GSH is a marker of oxidative stress (Spear and Aust, 1995
). With regard to nickel, hepatic GSH content is dramatically reduced after nickel injection in vivo (Rodriguez et al., 1991
) and is similarly depleted in nickel exposed cells in vitro (Li et al., 1993
). Interestingly, it has been reported that cells made nickel-resistant by nickel exposure had 1.8-fold higher basal levels of GSH than did the corresponding wild-type cells (Salnikow et al., 1994
). In agreement with the results of Salnikow et al. (1994), the present study demonstrated that intracellular reduced GSH levels were significantly increased about 1.6-fold in the nickel resistant CAsE cells. In addition, depletion of GSH negated nickel resistance in CAsE cells. Prior work has also shown that superoxide dismutase (SOD), an important component of the cellular antioxidant defense systems, is markedly up-regulated in CAsE cells (Chen et al., 2001
). Furthermore, resistance to hydrogen peroxide was quite pronounced in CAsE cells in the present study. Taken together, these data suggested that these nickel-resistant cells likely have an enhanced defense against oxidative stress, which is generalized and not simply specific to nickel. This supposition is supported by the present results indicating reduced nickel-induced radical formation, lipid peroxidation, and oxidative DNA damage as well as resistance to hydrogen peroxide in CAsE cells. Cells made resistant to nickel by nickel-exposure likewise show generalized resistance to oxidative stress (Salnikow et al., 1994
). Again, it is of interest in the present case where nickel tolerance developed in the absence of elevated nickel exposure. Data are emerging indicating that arsenic itself can induce oxidative stress in cells (Chen et al., 2001
) and in animals (Liu et al., 2001
), and this may be an important factor in arsenic toxicity. CAsE cells are quite tolerant to arsenic (Romach et al., 2000
), and it may be that a generalized tolerance to oxidative stress has developed in these cells that translates into tolerance to oxidative stress induced by nickel.
In summary, chronic arsenic exposure induced a marked cross-tolerance to nickel-induced cytotoxicity, genotoxicity, and apoptosis. This appears to be due to a generalized resistance to oxidative stress involving, at least in part, increased GSH levels and, perhaps, antioxidant enzymes. The observed acquired resistance to nickel may constitute a novel and important model to study the mechanisms of nickel carcinogenesis and metal-metal interactions in inorganic carcinogenesis.
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ACKNOWLEDGMENTS |
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NOTES |
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