Nickel (II) enhances benzo[a]pyrene diol epoxide-induced mutagenesis through inhibition of nucleotide excision repair in human cells: a possible mechanism for nickel (II)-induced carcinogenesis

Wenwei Hu1, Zhaohui Feng1 and Moon-shong Tang1,–4

1 Department of Environmental Medicine, 2 Department of Medicine and 3 Department of Pathology, New York University School of Medicine, Tuxedo, NY 10987, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nickel (II), a ubiquitous environmental and industrial contaminant, is a well-known human carcinogen, particularly in human lung cancer. Although by itself it is a weak mutagen, nickel (II) is able to significantly enhance the genotoxicity of other mutagens and carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and ultraviolet light. Certain human populations, especially cigarette smokers, are frequently exposed to both nickel (II) and PAHs. To understand the interplay of nickel (II) and PAHs in mutagenesis and human carcinogenesis, we used a shuttle vector mutagenicity assay to examine the effect of nickel (II) on (±) anti-7ß, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (BPDE)-induced mutagenesis in human cells. BPDE is an activated metabolite of benzo[a]pyrene (BP), a major carcinogen in cigarette smoke. The shuttle vector pSP189 modified with BPDE was transfected into human cells with and without nickel (II) exposure. We found that nickel (II) exposure significantly enhanced BPDE-induced mutation frequency, but did not change BPDE-induced mutational spectrum in the supF gene of pSP189 plasmids replicated in nucleotide excision repair (NER)-proficient human cells. However, the enhancing effect of nickel (II) on BPDE-induced mutation frequency was not observed in NER-deficient human XPA cells. We also found that nickel (II) exposure of human cells did not change the spontaneous mutation frequency of the supF gene in NER-proficient or NER-deficient human cells, indicating that nickel (II) did not affect the replication fidelity in human cells. Using a plasmid containing a luciferase reporter gene and a host cell reactivation assay, we have found that nickel (II) exposure greatly inhibited the repair of BPDE–DNA adducts in NER-proficient but not in NER-deficient cells. Together these results strongly suggest that nickel (II) can greatly enhance the mutagenicity and genotoxicity of PAHs by inhibiting the NER pathway in human cells, and this may constitute an important mechanism for nickel (II)-induced human carcinogenesis.

Abbreviations: BP, benzo[a]pyrene; BPDE, (±) anti-7ß, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene; CPD, cyclobutane pyrimidine dimer; hprt, hypoxanthine (guanine) phosphoribosyl-transferase; NER, nucleotide excision repair; PAH, polycyclic aromatic hydrocarbon; UV, ultraviolet


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nickel (II) is a ubiquitous environmental and industrial contaminant (1,2). It is widely used in industrial processes, such as electroplating and the manufacture of steel and batteries (1,2). Epidemiological studies have shown that occupational exposure to nickel (II) is strongly associated with a high incidence of human lung and nasal cancers (2,3). Nickel (II) can also cause cell transformation and induce tumors in animal models (47). Lung and kidney are the major target organs for both soluble and insoluble forms of nickel (II) in humans as well as rodents (3,5,8). Although nickel (II) is a well-known carcinogen in humans and animals, the underlying molecular mechanisms of nickel (II)-induced carcinogenesis are not yet well understood.

It has been shown that nickel (II) by itself causes little or no damage to naked DNA, and it is not mutagenic in bacterial test systems and only a weak mutagen in cultured mammalian cells (1,911). However, nickel (II) is able to significantly enhance the genotoxicity of some other DNA damaging agents. For example, it has been found that nickel chloride treatment can increase ultraviolet (UV) light-induced mutation frequencies of the hypoxanthine (guanine) phosphoribosyl-transferase (hprt) gene and sister-chromatid exchanges in V79 Chinese hamster cells, and can enhance morphological transformation induced by benzo[a]pyrene (BP), a major carcinogen in cigarette smoke, in hamster embryo cells (11,12). Intriguingly, however, nickel (II) treatment, under conditions that induce high levels of cytotoxicity, has been reported to reduce (±) anti-7ß, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (BPDE), an activated metabolite of BP-induced mutations in the hprt gene in human fibroblasts (13). It thus appears that in human cells, nickel (II) treatment induces pleiotropic effects that are influenced by the experimental conditions used.

Cigarette smoke contains a substantial amount of nickel; up to 600 ng of nickel have been found in the main stream of smoke generated per cigarette (14). The exact chemical form of nickel in cigarette mainstream smoke has not yet been established. Cigarette smoking is known to be the leading cause of human lung cancer; 90% of lung cancer-related deaths have been attributed to cigarette smoking (15,16). Polycyclic aromatic hydrocarbons (PAHs), the major carcinogens in cigarette smoke and widespread environmental pollutants present in all combustive products of organic materials have been suggested as being responsible for the initiation and development of human lung cancer (17). We have found recently that various diol epoxides of PAHs present in cigarette smoke, including BPDE, preferentially form DNA adducts at mutational hotspots in the human p53 and K-ras genes, the two most commonly mutated genes in smoking-related lung cancers (1821). Furthermore, we have also found that the DNA adducts formed at these mutational hotspots are poorly repaired (21,22). These findings provide us with direct molecular links between the cigarette carcinogen PAHs and lung carcinogenesis.

PAHs are strong mutagens in almost all biological systems. PAH–DNA adducts are able to block DNA replication and transcription and induce various mutations, notably G to T transversions (18,2326). Nickel (II), on the other hand, although by itself a weak mutagen, can enhance UV light-induced mutagenicity and carcinogenicity (1,11,27). These findings raise several interesting and important questions: what are the mechanisms underlying nickel (II)-induced carcinogenicity? Is nickel (II) a co-mutagen and/or co-carcinogen of PAHs? If so, then what are the mechanisms through which nickel (II) exerts its effects on PAH-induced mutagenesis and carcinogenesis? Since certain human populations, especially cigarette smokers, are frequently exposed to both nickel (II) and PAHs, answering these questions is crucial for understanding cigarette smoke-induced carcinogenesis.

To address these questions, we used a shuttle vector pSP189, containing a supF gene as a mutational target, to determine the effect of nickel (II) on BPDE–DNA adduct-induced mutagenesis in human cells. We found that nickel (II) exposure of human cells significantly enhanced BPDE–DNA adduct-induced mutation frequency of the supF gene, but did not change the mutational spectrum. Using a host cell reactivation assay with a plasmid containing a luciferase reporter gene, we further examined the possible mechanisms through which nickel (II) exerts its effects on the mutagenicity of BPDE– DNA adducts in human cells, and we found that nickel (II) greatly inhibited the nucleotide excision repair (NER) of BPDE–DNA adducts in human cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plasmids
The shuttle vector pSP189, which contains the supF gene as a mutational target, was kindly provided by Dr Michael Seidman (NIA, NIH, Baltimore, MD) (28). The pGL3-luciferase reporter vector containing a coding region for firefly luciferase (Catalog number E1741), and the pSV-ß-galactosidase control vector containing the bacterial lacZ gene, which codes for ß-galactosidase were purchased from Promega (Madison, WI).

Cells and cell cultures
SV-40-transformed NER-deficient human XPA fibroblasts (XP12BE) and NER-proficient human fibroblasts (GM00637) were obtained from NIGMS Human Genetic Cell Repository (Camden, NJ). These cells were grown in Minimum Essential Medium supplemented with 10% fetal bovine serum in a 5% CO2 humidified incubator.

Colony formation ability assay
Logarithmically growing NER-deficient human XPA fibroblasts (XP12BE) and NER-proficient human fibroblasts (GM00637) were incubated with various concentrations of nickel chloride, NiCl2 (Sigma, St Louis, MO), for 24 h at 37°C in an incubator. After incubation, cells were immediately trypsinized and 300 cells/dish were seeded to test for colony formation ability. After 9 days of incubation, colonies were fixed with methanol, stained with crystal violet and counted. Colony formation ability was calculated based on plating efficiency of NiCl2-treated cells versus plating efficiency of untreated control cells.

Modification of plasmids with BPDE
BPDE was purchased from Chemsyn Science Laboratories (Lenexa, KS). The plasmids pSP189 and pGL3-luciferase were modified with BPDE as described previously (29). Briefly, purified plasmids were modified with various concentrations of BPDE at 25°C for 2 h, and the reactions were stopped by repeated phenol and diethyl ether extractions to remove the unreacted BPDE. DNA was then ethanol precipitated and dissolved in TE [10 mM Tris, 1 mM EDTA (pH 7.5)].

SupF mutagenesis assay
Human NER-deficient XPA fibroblasts (XP12BE) and NER-proficient fibroblasts (GM00637) were grown to 70% confluence in 150 mm tissue-culture dishes. Various concentrations of NiCl2 was added to the culture medium, and the cells were then incubated at 37°C for 24 h. NiCl2 was then removed and the pSP189 plasmids with and without BPDE modification were transfected into cells using Lipofectamine (Invitrogen, Carslbad, CA), according to manufacturer's instructions. Briefly, cells were transfected with a mixture of plasmids (40 µg) and Lipofectamine for 6 h. After transfection, medium containing the transfection mixture was removed and cells were cultured in fresh medium in the presence of NiCl2 at the pre-treatment concentrations for another 60 h. The transfected plasmids were then rescued from the human cells by the alkaline lysis method described previously by Lee et al. (30). Briefly, cells were trypsinized, washed and resuspended in suspension buffer (50 mM Tris–HCl, pH 8.0, 10 mM EDTA, 100 µg/ml RNase A), mixed with lysis buffer (0.2 M NaOH, 1% SDS), incubated on ice for 15 min and neutralization buffer (3 M potassium acetate, pH 5.5) was added. After incubation at room temperature for 15 min, the mixture was centrifuged for 10 min at 16 000 g and the supernatant was extracted with phenol and chloroform and then precipitated with ethanol. The DNA was resuspended and treated with the DpnI restriction enzyme (New England Biolabs, Beverly, MA) to remove the unreplicated plasmids containing the bacterial adenine methylation pattern. The replicated plasmids were then electroporated into indicator MBM7070 bacteria, which carry a lacZ gene with an amber mutation. The transformed bacteria were plated on Luria–Bertani broth plates containing ampicillin (50 µg/ml), isopropyl ß-D-thiogalactoside (IPTG) (190 µg/ml) and 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-Gal) (0.8 mg/ml). After overnight incubation at 37°C, white and light blue mutant colonies were picked from the background of blue wild-type colonies and re-streaked; the plasmids were then extracted and purified using QIApre-spin Plasmid Kit (Qiagen, Valencia, CA). The sequences of the supF gene of mutant plasmids were determined with the primer 5'-GGC GAC ACG GAA ATG TTG AA-3' and CEQ Dye Terminator Cycle Sequencing Kit (Beckman Coulter, Fullerton, CA), using the CEQ 2000XL Automatic DNA Analyser (Beckman Coulter).

Host cell reactivation assay
Human NER-deficient XPA fibroblasts (XP12BE) and NER-proficient fibroblasts (GM00637) were plated in triplicate in 60 mm dishes at a density of 3 x 105 cells/dish and exposed to various concentrations of NiCl2 at 37°C for 24 h. The cells were then transfected with 2 µg of pGL3-luciferase reporter plasmid modified with various concentrations of BPDE, using FuGENE 6 transfection reagent (Boehringer Mannheim, Indianapolis, IN). The untreated pSV-ß-galactosidase control vector (0.5 µg), a ß-galactosidase-expressing plasmid, was co-transfected into human cells as an internal control to normalize transfection efficiency. After transfection for 3 h, medium containing the transfection mixture was removed and cells were incubated in fresh medium in the presence of NiCl2 at the pre-treatment concentrations for another 24 h. Cells were then lysed with 600 µl of Reporter Lysis Buffer (Promega). Transient expression of luciferase was determined by mixing 50 µl of cell extract with 100 µl of Luciferase Assay Reagent (Promega) and measuring the light emission with a luminometer (Wallac 1420 Victor 2 multilable counter system, Gaithersburg, MD). Transient expression of ß-galactosidase was determined using the ß-galactosidase Enzyme Assay System (Promega). Values of luciferase expression were normalized to the ß-galactosidase control and averaged over the triplicates. Since the reporter gene will not be expressed unless BPDE–DNA adducts are repaired by cells, this assay can be used to detect the repair capacity of cells. The relative luciferase activity (i.e. reactivation of the damaged plasmids by the host cells) from BPDE-treated pGL3-luciferase reporter plasmids is expressed as a percentage of luciferase activity from untreated pGL3-luciferase reporter plasmids and is used to represent the repair capacity of cells.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been found that nickel (II) treatment can significantly enhance UV light-induced mutagenesis and BPDE-induced cell transformation, sister-chromatid exchange and ouabain-resistant mutations (11,12,27). On the other hand, it was reported that human cells treated first with nickel (II) and then with BPDE produced fewer hprt mutants than cells treated with BPDE alone (13). In contrast, human cells treated first with BPDE and then with nickel (II) produced much more hprt mutants than cells treated with BPDE alone (13). Intriguingly, the combination of BPDE and nickel (II) treatment, regardless of the sequence of treatment, caused similar increases in the level of cytotoxicity over either nickel (II) or BPDE treatment alone (13). Together these results suggest that nickel (II) may have two effects on cell physiology: (i) nickel (II) may affect the fidelity of DNA replication, particularly translesion synthesis and/or the efficiency of DNA repair and (ii) the cytotoxic effect of nickel (II) could be mostly independent of its genotoxic effect, and BPDE may enhance the cytotoxicity of nickel (II). This study attempted to determine the effect of nickel (II) treatment on BPDE–DNA adduct-induced mutagenesis and elucidate the mechanisms of how nickel (II) affects BPDE–DNA adduct-induced mutagenesis. We have chosen the shuttle vector pSP189–human cell transfection system for this study.

Nickel (II) induced cytotoxicity in NER-proficient and NER-deficient human fibroblasts
In order to choose a nickel (II) treatment condition that minimizes the cytotoxic effects of nickel (II) but still allows for detectable levels of effect on mutagenesis and DNA repair, we first determined the cytotoxicity of NiCl2 in NER-proficient human fibroblasts (GM00637) and NER-deficient XPA (XP12BE) fibroblasts. It has been established that the uptake of soluble nickel (II) by cells is a slow process and it takes 16–24 h to reach the plateau (9). We, therefore, treated the cells with various concentrations of NiCl2 for 24 h to achieve a maximal level of nickel (II) uptake by cells. The dose effect of NiCl2 cytotoxicity in NER-proficient and NER-deficient human fibroblasts is shown as colony formation ability in Table I. There are no statistically significant differences in NiCl2-induced cytotoxicity between NER-proficient and NER-deficient cells (P > 0.05). After 24 h exposure, the colony formation ability is only slightly decreased at the concentration of 300 µM NiCl2, and it drops to 76 ± 4% in NER-proficient and 69 ± 5% in NER-deficient human cells, respectively. Thereafter, it drops to 52 ± 6 and 39 ± 3% at 500 µM NiCl2 treatment, and to 23 ± 2 and 15 ± 4% at 750 µM NiCl2 treatment in NER-proficient and NER-deficient human cells, respectively. Based on the results presented in Table I, subtoxic dosage up to 300 µM NiCl2 was used in the following mutagenesis and repair studies.


View this table:
[in this window]
[in a new window]
 
Table I. Cytotoxicity of NiCl2 in human fibroblasts

 
Effect of nickel (II) on BPDE-induced mutation frequency in supF gene in NER-proficient and NER-deficient human fibroblasts
To determine the effect of nickel (II) on BPDE-induced mutagenesis in human cells, NER-proficient human fibroblasts (GM00637) were exposed to 50 and 300 µM of NiCl2 for 24 h, and then transfected with the shuttle vector pSP189 modified with 3 or 15 µM of BPDE. The transfected cells were further incubated in the presence of NiCl2 at the pre-treatment concentrations for another 60 h. The progeny plasmids were then rescued and transformed into indicator bacteria MBM7070 to identify plasmids carrying mutations in the supF gene. The mutation frequency of the supF gene was defined as the ratio of the number of white or light blue mutant colonies to the total number of bacterial colonies obtained on indicator plates containing IPTG and X-Gal. Results in Table II show that NiCl2 exposure does not change the mutation frequencies of the supF gene in BPDE-untreated pSP189 plasmids replicated in either NER-proficient or NER-deficient human fibroblasts. This result suggests that nickel (II) itself has a very weak mutagenic effect in human cells, which is consistent with previous reports (1,9, 3133). However, nickel (II) exposure shows enhancement of BPDE–DNA adduct-induced supF mutation frequency in pSP189 plasmids replicated in NER-proficient human fibroblasts. While 50 µM NiCl2 exposure shows no significant enhancing effects on BPDE–DNA adduct-induced supF mutation frequency, 300 µM NiCl2 exposure increases mutation frequency of supF gene from 10.4 ± 1.6 x 10-4 to 18.4 ± 2.1 x 10-4 (P < 0.05) for pSP189 plasmids treated with 3 µM BPDE, and from 37.6 ± 4.7 x 10-4 to 75.5 ± 3.5 x 10-4 (P < 0.001) for plasmids treated with 15 µM BPDE. These results demonstrate that NiCl2 exposure can significantly enhance the mutagenicity of BPDE in NER-proficient human fibroblasts.


View this table:
[in this window]
[in a new window]
 
Table II. BPDE-induced mutation frequencies of supF gene in pSP189 plasmids replicated in NER-proficient human fibroblasts and NER-deficient XPA fibroblasts with or without NiCl2 exposure

 
Two possible mechanisms can account for the enhancement of BPDE-induced mutagenesis by nickel (II) in human cells: intracellular nickel (II) may affect the repair efficiency of BPDE–DNA adducts, and/or may affect the fidelity of DNA replication. As nickel (II) exposure does not change the mutation frequency of untreated pSP189 plasmids replicated in either NER-proficient or NER-deficient human cells, it is unlikely that the enhancement of BPDE-induced mutagenesis in nickel (II)-treated cells is due to the decrease in DNA replication fidelity in nickel (II)-treated cells.

NER is the major pathway for repairing bulky BPDE–DNA adducts in human cells, the enhancement of BPDE-induced mutagenesis by nickel (II) in human cells could be caused by the inhibition of NER by nickel (II). If this is the case, then nickel (II) treatment of NER-deficient cells should not affect BPDE-induced mutagenesis in NER-deficient cells. Results in Table II and Figure 1 show that the mutation frequency of the supF gene in BPDE-treated plasmids replicated in NER-deficient XPA fibroblasts without NiCl2 exposure is much higher than the mutation frequency observed in BPDE-treated plasmids replicated in NER-proficient human fibroblasts without NiCl2 exposure; these results are consistent with the current understanding that NER is the major pathway for repairing BPDE–DNA adducts (34,35). The results, however, also clearly show that BPDE–DNA adducts induce the same level of supF mutation frequency in XPA fibroblasts with and without NiCl2 treatment. These findings that NiCl2 exposure can enhance BPDE–DNA adduct-induced mutagenesis in NER-proficient human fibroblasts but not in NER-deficient XPA fibroblasts strongly suggest that the enhancement of BPDE–DNA adduct-induced mutagenesis by nickel (II) is the result of inhibition of the NER of BPDE–DNA adducts by nickel (II) in human cells.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1. The effect of NiCl2 exposure on the BPDE–DNA adduct-induced supF mutation frequency in NER-proficient (GM00637) (NER+) and NER-deficient XPA (XP12BE) (NER–) human fibroblasts. The pSP189 plasmids modified with different concentrations of BPDE (0, 3 and 15 µM) were transfected into human cells with (solid lines, +Ni) or without (broken lines, –Ni) exposure to NiCl2 (300 µM) for 24 h. The BPDE–DNA adduct-induced supF mutation frequency in the plasmids under these different conditions were assayed as described in Materials and methods. The data represent three independent experiments.

 
Effect of nickel (II) on the repair of BPDE–DNA adducts in human cells
To further test the possibility that the enhancement of BPDE–DNA adduct-induced mutagenesis by nickel (II) is caused by the inhibition of NER, a host cell reactivation assay was used to investigate the repair of BPDE–DNA adducts in both NiCl2-treated and -untreated human cells. Plasmids containing a luciferase reporter gene (pGL3-luciferase) were modified with different concentrations of BPDE and transfected into NER-proficient and NER-deficient human cells with or without 300 µM NiCl2 exposure, and the luciferase activity, which is proportional to the extent of expression of the reporter gene, was checked 24 h after transfection. As the reporter gene will not express unless BPDE–DNA adducts are repaired by cells, the luciferase activity represents the extent of BPDE–DNA adduct repair, which in turn reflects the cellular repair capacity of BPDE–DNA adduct (36,37). The relative luciferase activity detected in NER-proficient and NER-deficient human cells with or without NiCl2 treatment is presented in Figure 2. The results show that when BPDE-modified plasmids were transfected into cells without NiCl2 treatment, much higher luciferase activities were detected in NER-proficient cells than in NER-deficient cells, which indicates that the NER-proficient human cells can efficiently repair the BPDE–DNA adducts from luciferase reporter plasmids while NER-deficient XPA cells do not, and that NER is a major pathway for the repair of BPDE–DNA adducts. However, when these BPDE-modified plasmids were transfected into NiCl2-treated NER-proficient cells, much lower luciferase activities were detected as compared with NER-proficient cells without NiCl2 treatment. In contrast, the same relative luciferase activities were detected when these BPDE-modified plasmids were transfected into NER-deficient cells with and without NiCl2 treatment. These results are consistent with the above conclusion that NiCl2 treatment inhibits DNA repair capacity and that NER is the target for nickel (II)-mediated repair inhibition.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. The effect of NiCl2 exposure on host cell reactivation of luciferase reporter gene modified with BPDE in NER-proficient (GM00637) (NER+) and NER-deficient XPA (XP12BE) (NER–) human fibroblasts. BPDE-modified luciferase reporter plasmids (pGL3-luciferase) were transfected into NER-proficient and NER-deficient human fibroblasts with (solid lines, +Ni) or without (broken lines, –Ni) 300 µM NiCl2 exposure for 24 h. After transfection for 3 h, cells were incubated in fresh medium for 24 h to allow DNA repair. Cells were then harvested and lysed, and the luciferase activity in the cells was measured. The relative luciferase activity, which represents the relative extent of BPDE–DNA adduct repair at each condition, was determined as the percentage of luciferase activity expressed from BPDE-treated plasmids to that from untreated plasmids. The data represent three independent experiments.

 
We also determined the effect of concentration of nickel (II) on BPDE–DNA adduct repair in NER-proficient cells. Luciferase activities were determined in cells first treated with different concentrations of NiCl2 and then transfected with plasmids modified with 15 µM BPDE. Results in Figure 3 show that nickel (II) exerts a dose-dependent repair inhibition effect. Compared with cells without NiCl2 exposure, at the concentration of 100 µM, NiCl2 decreases relative repair capacity to 79 ± 7%; and at 300 µM, NiCl2 decreases relative repair capacity to 14 ± 4%. However, the luciferase activity does not change significantly in cells treated with <50 µM NiCl2. These results are consistent with the observations, shown in Table II, that treatment of host cells with 50 µM does not enhance BPDE–DNA adduct-induced mutation frequency, and only when host cells are treated with NiCl2 at higher concentrations which are inhibitory to NER, BPDE– DNA adduct-induced mutation frequency is enhanced.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3. Effect of exposure to different concentrations of NiCl2 on the capacity of host cell reactivation in NER-proficient human fibroblasts. Plasmids containing a luciferase reporter (pGL3-luciferase) were modified with 15 µM BPDE and transfected into NER-proficient (GM00637) human fibroblasts exposed to various concentrations of NiCl2 for 24 h. After transfection for 3 h, the cells were incubated in fresh medium for another 24 h. Cells were then harvested and lysed, and the luciferase activity in the cells was measured. The relative luciferase activity expressed from BPDE-treated plasmids was determined as the percentage of luciferase activity expressed from untreated plasmids. The relative repair capacity was calculated as the percentage of the relative luciferase activity of the plasmids transfected in cells with NiCl2 exposure to that in cells without NiCl2 exposure. The data represent three independent experiments.

 
Effect of nickel (II) exposure on the mutational spectrum of the supF gene in BPDE-treated pSP189 plasmids replicated in NER-proficient and NER-deficient human fibroblasts
Previously, we have found that in the human p53 gene and K-ras genes, not only does BPDE preferentially form DNA adducts at positions corresponding to the major mutational hotspots in smoking-related lung cancers, but also BPDE– DNA adducts formed at these positions are poorly repaired (1922). Since NER is the major pathway for the repair of BPDE–DNA adducts, it is possible that the inhibition of the NER by nickel (II) may have sequence preference. If this is the case, NiCl2 exposure may change the mutational spectrum induced by BPDE–DNA adducts. To test this possibility the BPDE–DNA adduct-induced mutational spectrum of the supF gene in human cells with or without NiCl2 treatment was analyzed. Plasmids containing mutant supF were purified from 74 and 68 independent mutant pSP189 plasmids recovered after passage of the BPDE-modified pSP189 plasmids through NER-proficient human fibroblast cells with or without 300 µM NiCl2 exposure, and the supF gene in these plasmids was sequenced. The types of mutations and the mutational spectrum induced by BPDE–DNA adducts in the supF gene are presented in Table III and Figure 4. Several features are worth noting: (i) BPDE–DNA adducts induce the same kinds of mutations in cells with or without NiCl2 exposure, and there is no significant difference in the types of mutations between cells with and without NiCl2 exposure, (ii) G:C to T:A transversions are the predominant types of mutations induced by BPDE–DNA adducts in cells with or without NiCl2 exposure (61 and 63%, respectively) and (iii) there is no significant difference in the distribution of mutations induced by BPDE–DNA adducts between cells with or without NiCl2 exposure. These results demonstrate that repair inhibition by nickel (II) does not have a sequence preference and suggest that nickel (II) inhibits NER in general.


View this table:
[in this window]
[in a new window]
 
Table III. Types of mutations in the supF gene in BPDE-treated pSP189 plasmids replicated in NER-proficient human fibroblasts (GM00637) with or without NiCl2 exposure

 


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4. BPDE–DNA adduct-induced mutational spectrum in the supF gene in pSP189 plasmids replicated in NER-proficient (GM00637) human fibroblasts with (lower panel) or without (upper panel) 300 µM NiCl2 exposure. Methods for NiCl2 exposure, transfection and supF gene mutation detection are described in Materials and methods. The mutation frequency in the supF gene of pSP189 plasmid replicated in NER-proficient (GM00637) human fibroblasts with NiCl2 exposure is 75.5 ± 3.5 x 10-4, and the mutation frequency is 38.9 ± 4.2 x 10-4 in human fibroblasts without NiCl2 exposure. The mutant plasmids were purified (74 mutant plasmids from cells with NiCl2 exposure and 68 mutants plasmids from cells without NiCl2 exposure) and the supF gene in these plasmids was sequenced. X, single base deletions; +, single base insertions; #, *, _, multiple mutations (two mutations occurring in the same plasmid).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Both nickel (II) and PAHs are environmental contaminants that are also abundant in cigarette smoke (1,2,37). It is well established that PAHs are mutagenic and tumorigenic, and the interactions of metabolically activated PAHs with DNA may be the trigger for mutagenesis and carcinogenesis (17,18). Although nickel (II)-containing compounds are weak mutagens, it has been long recognized that nickel (II)-containing compounds are carcinogens for both human and animal (1,911). However, the mechanisms of nickel (II) carcinogenicity remain unclear. It has been shown that nickel (II) can induce DNA–protein crosslinks and oxidative DNA damage, change DNA methylation patterns and interfere with the repair of oxidative DNA damage and UV light-induced cyclobutane pyrimidine dimers (CPDs) (3844); these effects have been suggested to correlate with the carcinogenicity of nickel (II)-containing compounds.

Cigarette smokers and certain human populations are constantly exposed to both PAHs and nickel (II). It is thus important to understand the biological effects that result from exposure to both agents, such as cytotoxicity, mutagenicity, cell transformation and carcinogenicity. While it has been established that cells exposed to these two agents suffer higher than additive cytotoxicity regardless of the sequence of treatment with these two agents, there are conflicting reports regarding the effect of nickel (II) on PAH-induced mutagenesis (12,13). A co-mutagenic effect of NiSO4 with BP has been detected in a somatic mutant resistant to ouabain in primary cultures of Syrian hamster embryos (12). On the other hand, a recent report measuring the mutation frequency at the hprt gene found that Ni3S2 pre-treatment was protective towards BPDE-induced mutagenicity in human fibroblasts (13). The same report, however, also showed that when cells were treated with both nickel (II) and BPDE simultaneously the mutation frequency was synergistically increased (13). However, the conditions used in the latter study led to very low colony formation ability of human cells after cells were combining treated with Ni3S2 and BPDE, and seem to be inadequate for measuring mutation frequency at the hprt locus. In this study, we readdressed this question and determined the effects of nickel (II) on mutagenicity of PAHs in human cells, using a shuttle-vector mutagenicity assay. Our results demonstrate that nickel (II) exposure can enhance the mutagenicity of BPDE–DNA adducts in human cells, and this effect is mainly the result of inhibition of NER by nickel (II). Most recently, using high performance liquid chromatography/fluorescence assay, Hartwig and his colleagues have also shown that nickel (II) can inhibit the removal of BPDE–DNA adducts in human cells (45).

Our conclusion that nickel (II) does not affect the fidelity of DNA replication in human cells is based on the following three findings: (i) nickel (II) exposure does not enhance the mutation frequency in supF of control pSP189 plasmids, (ii) nickel (II) exposure does not change the mutational spectrum of supF in BPDE-treated pSP189 plasmids in NER-proficient cells and (iii) nickel (II) exposure does not change BPDE-induced mutation frequency or mutational spectrum in NER-deficient XPA cells. Using a host-cell reactivation assay, we have found that nickel (II) exposure can greatly inhibit the NER capacity for BPDE–DNA damage in human cells. Together these results lead us to conclude that the enhancement of nickel (II) on BPDE–DNA adduct-induced mutagenesis is caused by its inhibition of the repair of BPDE–DNA adducts rather than through an effect on the fidelity of DNA replication.

The underlying mechanisms of the inhibition of repair for BPDE–DNA adducts by nickel (II) are not yet understood. It has been shown that nickel (II) can interfere with the initial incision and ligation steps of NER pathway in the repair of UV light-induced CPD, and the ligation of DNA strand breaks in the repair of radiation-induced DNA double-strand breaks (41,42). Nickel (II) can also inhibit repair of oxidative DNA damage, which presumably is repaired by base excision repair pathway (44). The inhibitory effect of nickel (II) on such broad repair mechanisms at different steps of repair raises the possibility that nickel (II) exerts its inhibitory effect not through a specific protein but instead through co-factors that are necessary for different repair steps. Magnesium and zinc ions are two probable candidates. Magnesium ion mediates DNA- protein interactions in the damage recognition and incision step and is also a co-factor for DNA polymerases and DNA ligases in the polymerization and/or ligation steps in DNA repair (46). Several repair proteins such as XPA and NEH1 contain zinc fingers, and zinc is thus essential for maintaining the structural integrity of these proteins (4649). It is possible that nickel (II) competes with and displaces essential metal ions involved in DNA repair, such as magnesium and zinc (9,38,46). It has been found that the inhibitory effect of nickel (II) on CPD repair can be partially reversed by the addition of magnesium (9,46). Nickel (II) has also been found to be able to displace zinc ion from zinc finger structures (50). As XPA, a member of the protein complex of the NER pathway, that participates in the assembly of the damage recognition/incision complex, contains a zinc finger structure, the competition of zinc ion by nickel (II) might be relevant to the inhibition of DNA repair by nickel (II) (47). Since nickel (II) is able to directly interact with amino acids and protein, it is also possible that nickel (II) directly interacts with repair proteins and proteins involved in regulating NER, and impairs their function in NER.

In summary, we have demonstrated that nickel (II) can greatly enhance BPDE–DNA adduct-induced mutagenicity through inhibition of the NER pathway in human cells, and our results suggest that this inhibition by nickel (II) of DNA repair may play an important role in nickel (II)-induced human carcinogenesis, especially human lung carcinogenesis.


    Notes
 
4 To whom correspondence should be addressed Email: tang{at}env.med.nyu.edu Back


    Acknowledgments
 
We wish to thank Dr Chuanshu Huang for technical assistance of measuring the expression of luciferase. We wish to thank Drs Yen-Yee Tang and Opinder S.Bhanot for their critical review of this manuscript. This work was supported by Public Health Service grants ES03124, ES10344, ES00260 and CA99007.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Hartwig,A. (1995) Current aspects in metal genotoxicity. Biometals, 8, 3–11.[ISI][Medline]
  2. Grandjean,P., Andersen,O. and Nielsen,G.D. (1988) Carcinogenicity of occupational nickel exposures: an evaluation of the epidemiological evidence. Am. J. Ind. Med., 13, 193–209.[ISI][Medline]
  3. Sunderman,F.W.,Jr. (2001) Nasal toxicity, carcinogenicity and olfactory uptake of metals. Ann. Clin. Lab. Sci., 31, 3–24.[Abstract/Free Full Text]
  4. Kasprzak,K.S., Gabryel,P. and Jarczewska,K. (1983) Carcinogenicity of nickel (II) hydroxides and nickel (II) sulfate in Wistar rats and its relation to the in vitro dissolution rates. Carcinogenesis, 4, 275–279.[ISI][Medline]
  5. Clary,J.J. (1975) Nickel chloride induced metabolic changes in the rat and guinea pig. Toxicol. Appl. Pharmacol., 31, 55–65.[ISI][Medline]
  6. Luckey,T.D. and Venugopal,B. (1977) pT, a new classification system for toxic compounds. J. Toxicol. Environ. Health, 2, 633–638.[ISI][Medline]
  7. IARC (1976) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man: Cadmium, Nickel, Some Epoxides, Miscellaneous Industrial Chemicals and General Considerations on Volatile Anaesthetics. IARC Scientific Publications, IARC, Lyon, vol. 11.
  8. Parker,K. and Sunderman,F.W.,Jr (1974) Distribution of 63Ni in rabbit tissues following intravenous injections of 63NiCl2. Res. Commun. Chem. Pathol. Pharmacol., 7, 755–762.[ISI][Medline]
  9. Hartwig,A., Mullenders,L.H., Schlepegrell,R., Kasten,U. and Beyersmann,D. (1994) Nickel (II) interferes with the incision step in nucleotide excision repair in mammalian cells. Cancer Res., 54, 4045–4051.[Abstract]
  10. IARC (1990) Monographs on the Evaluation of the Carcinogenic Risks to Humans, Chromium, Nickel and Welding. IARC Scientific Publications, IARC, Lyon, vol. 49.
  11. Hartwig,A. and Beyersmann,D. (1989) Enhancement of UV-induced mutagenesis and sister-chromatid exchanges by nickel ions in V79 cells: evidence for inhibition of DNA repair. Mutat. Res., 217, 65–73.[ISI][Medline]
  12. Rivedal,E. and Sanner,T. (1980) Potentiating effect of cigarette smoke extract on morphological transformation of hamster embryo cells by benzo[alpha]pyrene. Cancer Lett., 10, 193–198.[ISI][Medline]
  13. Hamdan,S., Morse,B. and Reinhold,D. (1999) Nickel subsulfide is similar to potassium dichromate in protecting normal human fibroblasts from the mutagenic effects of benzo[a]pyrene diolepoxide. Environ. Mol. Mutagen., 33, 211–218.[CrossRef][ISI][Medline]
  14. Hoffmann,D. and Hecht,S.S. (1990) Advances in tobacco carcinogenesis. In: Cooper,C.S. and Grover,P.L. (eds) Handbook of Experimental Pharmacology. Springer-Verlag, Berlin, pp. 70–74.
  15. Devesa,S.S., Grauman,D.J., Blot,W.J. and Fraumeni,J.F.,Jr (1999) Cancer surveillance series: changing geographic patterns of lung cancer mortality in the United States, 1950 through 1994. J. Natl Cancer Inst., 91, 1040–1050.[Abstract/Free Full Text]
  16. Miller,B.A., Ries,L.A.G., Hankey,B.F., Kosary,C.L., Harras,A., Devesa,S.S. and Edwards,B.K. (eds) (1993) SEER Cancer Statistics Review, 1973–1990. National Cancer institute. NIH Publications No. 93–2789. Department of Health and Human Services, Bethesda, MD.
  17. Hecht,S.S., Carmella,S.G., Murphy,S.E., Foiles,P.G. and Chung,F.L. (1993) Carcinogen biomarkers related to smoking and upper aerodigestive tract cancer. J. Cell. Biochem. Suppl., 17F, 27–35.[Medline]
  18. Hainaut,P. and Pfeifer,G.P. (2001) TP53 mutational spectrum in lung cancers and mutagenic signature of components of tobacco smoke: lessons from the IARC TP53 mutation database. Carcinogenesis, 22, 367–374.[Abstract/Free Full Text]
  19. Denissenko,M.F., Pao,A., Tang,M.-s. and Pfeifer,G.P. (1996) Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Science, 274, 430–432.[Abstract/Free Full Text]
  20. Smith,L.E., Denissenko,M.F., Bennett,W.P., Li,H., Amin,S., Tang,M.-s. and Pfeifer,G.P. (2000) Targeting of lung cancer mutational hotspots by polycyclic aromatic hydrocarbons. J. Natl Cancer. Inst., 92, 803–811.[Abstract/Free Full Text]
  21. Feng,Z., Hu,W., Chen,J.X., Pao,A., Li,H., Rom,W., Hung,M.C. and Tang,M.-s. (2002) Preferential DNA damage and poor repair determine ras gene mutational hotspot in human cancer. J. Natl Cancer Inst., 94, 1527–1536.[Abstract/Free Full Text]
  22. Denissenko,M.F., Pao,A., Pfeifer,G.P. and Tang,M.-s. (1998) Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers. Oncogene, 16, 1241–1247.[CrossRef][ISI][Medline]
  23. Hruszkewycz,A.M., Canella,K.A., Peltonen,K., Kotrappa,L. and Dipple,A. (1992) DNA polymerase action on benzo[a]pyrene-DNA adducts. Carcinogenesis, 13, 2347–2352.[Abstract]
  24. Choi,D.J., Roth,R.B., Liu,T., Geacintov,N.E. and Scicchitano,D. (1996) Incorrect base insertion and prematurely terminated transcripts during T7 RNA polymerase transcription elongation past benzo(a)pyrene diol epoxide-modified DNA. J. Mol. Biol., 264, 213–219.[CrossRef][ISI][Medline]
  25. Yang,Y.L., Maher,V.M. and McCormick,J. (1987) Kinds of mutations formed when a shuttle vector containing adducts of (+/–)-7ß, 8-dihydroxy-9, 10-epoxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene replicates in human cells. Proc. Natl Acad. Sci. USA, 84, 3787–3791.[Abstract]
  26. Greenbaltt,M.S., Bennett,W.P., Hollstein,M. and Harris,C.C. (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res., 54, 4855–4878.[ISI][Medline]
  27. Hartwig,A. and Beyersmann,D. (1989) Comutagenicity and inhibition of DNA repair by metal ions in mammalian cells. Biol. Trace Elem. Res., 21, 359–365.[ISI][Medline]
  28. Canella,K.A. and Seidman,M.M. (2000) Mutation spectra in supF: approaches to elucidating sequence context effects. Mutat. Res., 450, 61–73.[ISI][Medline]
  29. Feng,Z., Hu,W., Rom,W.N., Beland,F.A. and Tang,M.-s. (2002) N-Hydroxy-4-aminobiphenyl-DNA binding in human p53 gene: sequence preference and the effect of C5 cytosine methylation. Biochemistry, 41, 6414–6421.[CrossRef][ISI][Medline]
  30. Lee,D.H., O'Connor,T.R. and Pfeifer,G.P. (2002) Oxidative DNA damage induced by copper and hydrogen peroxide promotes CG->TT tandem mutations at methylated CpG dinucleotides in nucleotide excision repair-deficient cells. Nucleic Acids Res., 30, 3566–3573.[Abstract/Free Full Text]
  31. Christie,N. and Katsifis,S.P. (1990) Nickel carcinogenesis. In Foulkes,E.C. (ed.), Biological Effects of Heavy Metals. CRC Press, Inc., Boca Raton, FL, vol. II, pp. 95–128.
  32. Sen,P. and Costa,M. (1985) Induction of chromosomal damage in Chinese hamster ovary cells by soluble and particulate nickel compounds: preferential fragmentation of the heterochromatic long arm of the X-chromosome by carcinogenic crystalline NiS particles. Cancer Res., 45, 2320–2325.[Abstract]
  33. Lee,Y.W., Klein,C.B., Kargacin,B., Salnikow,K., Kitahara,J., Dowjat,K., Zhitkovich,A., Christie,N.T. and Costa,M. (1995) Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens. Mol. Cell. Biol., 15, 2547–2557.[Abstract]
  34. Friedberg,E.C., Walker,G.C. and Siede,W. (1995) DNA Repair and Mutagenesis. ASM Press, Washington, DC, pp. 283–310.
  35. Tang,M.-s., Pierce,J.R., Doisy,R.P., Nazimiec,M.E. and MacLeod,M.C. (1992) Differences and similarities in the repair of two benzo[a]pyrene diol epoxide isomers induced DNA adducts by uvrA, uvrB and uvrC gene products. Biochemistry, 31, 8429–8436.[ISI][Medline]
  36. Jia,L., Wang,X.W. and Harris,C.C. (1999) Hepatitis B virus X protein inhibits nucleotide excision repair. Int. J. Cancer, 80, 875–879.[CrossRef][ISI][Medline]
  37. Wani,M.A., Wani,G., Yao,J., Zhu,Q. and Wani,A.A. (2002) Human cells deficient in p53 regulated p21 (waf1/cip1) expression exhibit normal nucleotide excision repair of UV-induced DNA damage. Carcinogenesis, 23, 403–410.[Abstract/Free Full Text]
  38. Harvey,R.G. (1991) Polycyclic aromatic hydrocarbons. Chemistry and Carcinogenicity. Cambridge University Press, Cambridge, UK, p. 396.
  39. Chakrabarti,S.K., Bai,C. and Subramanian,K.S. (2001) DNA-protein crosslinks induced by nickel compounds in isolated rat lymphocytes: role of reactive oxygen species and specific amino acids. Toxicol. Appl. Pharmacol., 170, 153–165.[CrossRef][ISI][Medline]
  40. Kawanishi,S., Inoue,S., Oikawa,S., Yamashita,N., Toyokuni,S., Kawanishi,M. and Nishino,K. (2001) Oxidative DNA damage in cultured cells and rat lungs by carcinogenic nickel compounds. Free Radic. Biol. Med., 31, 108–116.[CrossRef][ISI][Medline]
  41. Klein,C.B., Conway,K., Wang,X.W., Bhamra,R.K., Lin,X.H., Cohen,M.D., Annab,L., Barrett,J.C. and Costa,M. (1991) Senescence of nickel-transformed cells by an X chromosome: possible epigenetic control. Science, 251, 796–799.[ISI][Medline]
  42. Hartmann,M. and Hartwig,A. (1998) Disturbance of DNA damage recognition after UV-irradiation by nickel (II) and cadmium (II) in mammalian cells. Carcinogenesis, 19, 617–621.[Abstract]
  43. Takahashi,S., Takeda,E., Kubota,Y. and Okayasu,R. (2000) Inhibition of repair of radiation-induced DNA double-strand breaks by nickel and arsenite. Radiat. Res., 154, 686–691.[ISI][Medline]
  44. Dally,H. and Hartwig,A. (1997) Induction and repair inhibition of oxidative DNA damage by nickel (II) and cadmium (II) in mammalian cells. Carcinogenesis, 18, 1021–1026.[Abstract]
  45. Schwerdtle,T., Seidel,A. and Hartwig,A. (2002) Effect of soluble and particulate nickel compounds on the formation and repair of stable benzo[a]pyrene DNA adducts in human lung cells. Carcinogenesis, 23, 47–53.[Abstract/Free Full Text]
  46. Hartwig,A. (2001) Role of magnesium in genomic stability. Mutat. Res., 475, 113–121.[ISI][Medline]
  47. Bal,W., Schwerdtle,T. and Hartwig,A. (2003) Mechanism of nickel assault on the zinc finger of DNA repair protein XPA. Chem. Res. Toxicol., 16, 242–248.[CrossRef][ISI][Medline]
  48. Asmuss,M., Mullenders,L.H., Eker,A. and Hartwig,A. (2000) Differential effects of toxic metal compounds on the activities of Fpg and XPA, two zinc finger proteins involved in DNA repair. Carcinogenesis, 21, 2097–2104.[Abstract/Free Full Text]
  49. Hazra,T. K., Izumi,T., Boldogh,I., Imhoff,B., Kow,Y.W., Jaruga,P., Dizdaroglu,M. and Mitra,S. (2002) Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA. Proc. Natl Acad. Sci. USA, 99, 3523–3528.[Abstract/Free Full Text]
  50. Hartwig,A., Schlepegrell,R., Dally,H. and Hartmann,M. (1996) Interaction of carcinogenic metal compounds with deoxyribonucleic acid repair processes. Ann. Clin. Lab. Sci., 26, 31–38.[Abstract]
Received August 26, 2003; revised October 23, 2003; accepted October 28, 2003.