Nickel-Induced Histone Hypoacetylation: The Role of Reactive Oxygen Species

Jiuhong Kang*,1, Yuntao Zhang*, Jie Chen{dagger}, Haifeng Chen{ddagger}, Changjun Lin*, Qin Wang* and Yingxian Ou{dagger}

* School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, China; {dagger} Department of Hematology, General Hospital of Lanzhou, Gansu 730000, China; and {ddagger} Institut de Topologie de Dynamique des Systemes, CNRS ESA 7986, Université Paris 7-Denis-Diderot, 1 Rue Guy de la Brosse, 75005 Paris, France

Received February 19, 2003; accepted April 24, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The carcinogenicity of specific insoluble nickel compounds is mainly due to their intracellular generation of Ni2+ ion and its suppression on gene transcription, while the inhibition of Ni2+ on histone acetylation plays an important role in the suppression or silencing of genes. Recent studies on Ni2+ and histone H4 acetylation suggest that Ni2+ inhibits the acetylation of histone H4 through binding with its N-terminal histidine-18. It is well known that bound Ni2+ readily produces reactive oxygen species (ROS) in vivo, a critical factor inversely related with the occurrence of resistance of mammalian cells to Ni2+. Thus, we tried to find the possible role of ROS in the induction of Ni2+ on histone acetylation in the present study. We found that a high concentration of Ni2+ (no less than 600 µM) caused a significant decrease of histone acetylation in human hepatoma cells. This inhibition was shown to result mainly from the influence of Ni2+ on the overall histone acetyltransferase (HAT) activity indicated by the histone acetylation assay with the presence of a specific histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). The in vitro HAT and HDAC assays further confirmed this result. At the same time, we found that the exposure of hepatoma cells to Ni2+ generated ROS. Coadministration of hydrogen peroxide with Ni2+ generated more ROS and more histone acetylation inhibition. Addition of the antioxidants 2-mercaptoethanol (2-ME) at 2 mM or N-acetyl-cysteine (NAC) at 1 mM, with Ni2+ together, completely suppressed ROS generation and significantly diminished the induced histone hypoacetylation. The data presented here prove that the ROS generation plays a role in the inhibition of histone acetylation, and, hence, the gene suppression and carcinogenesis caused by Ni2+ exposure, providing a new door for us to continuously understand the mechanism of ROS in the carcinogenicity of Ni2+ and the resistance of mammalian cells to Ni2+.

Key Words: Nickel; carcinogenesis; histone acetylation; histone acetyltransferase (HAT); histone deacetylase (HDAC); reactive oxygen species (ROS).


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In order of abundance in the earth’s crust, nickel ranks as the 24th element. Due to its abundance in all types of food (Nielson, 1987Go; Sunderman and Oskarsson, 1999), the daily dietary intake of nickel is estimated at more than triple the possible requirement of daily nutrition (Anke et al., 1995Go; Barceloux, 1999Go; Kirchgessner et al., 1982Go; Nielson, 1987Go; Schnegg et al., 1975). Additionally, because of the wide usage of metallic nickel and its compounds in modern industry, the high consumption of nickel-containing products inevitably leads to more diseases by nickel and its by-products at all stages of production, recycling and disposal, as suggested by the epidemiological studies, which indicate an increased risk of respiratory tract and nasal cancers in miners and workers in nickel refineries (Anderson, 1992Go; Easton et al., 1992Go; Roberts et al., 1992Go). It is well known that exposure to nickel compounds has adverse effects on human health and causes serious illness related to the carcinogenic activity of specific insoluble nickel compounds. Based upon the epidemiological studies, all nickel compounds except for metallic nickel are classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC) in 1990 (IARC, 1990Go). Thus, the molecular mechanisms of the carcinogenicity of specific insoluble nickel compounds have been focused on for a long time.

Recent studies in this field found that, as human and rodent carcinogens, nickel compounds display a strong potential to inactivate gene expression in spite of their weak mutagenicity. Thus, not gene mutation, but gene silencing is critical in nickel-induced carcinogenesis (Beyersmann, 2002Go; Cangul et al., 2002Go). In the process of gene expression regulation, chromatin remodeling is proved to be crucial because, in eukaryotes, binding of genomic DNA with histones in the nucleosome and folding of the chromatin pose a barrier to transcription, i.e., the structure of chromatin prevents the transcription machinery from interacting with promoter DNA sequences (Fry and Peterson, 2002Go). To overcome this, chromatin remodeling is triggered during gene transcription initiation mainly by two classes of enzymes: those that covalently modify nucleosomal histone proteins through acetylation and those that alter chromatin structure through the hydrolysis of adenosine triphosphate (ATP) (Fry and Peterson, 2001Go, 2002Go; Grunstein, 1997Go). Among them, histone acetylation and its related enzymes are continuously needed during gene transcription initiation and elongation. Generally, histone hyperacetylation leads to some genes activation or up-regulation, while histone hypoacetylation leads to inactivation or down-regulation of genes (Archer and Hodin, 1999Go; Klochendler-Yeivin and Yaniv, 2001Go).

Ni2+ has been found to inactivate the antiangionetic thrombospondin gene and a telomere marker gene by induction of histone H4 hypoacetylation and chromatin condensation (Broday et al., 1999Go; Salnikow et al., 1997Go). Further studies show that Ni2+ inhibits the acetylation of histone H4 through binding with its N-terminal histidine-18, which is located near the lysine residues for histone H4 acetylation (Broday et al., 2000Go; Zoroddu et al., 2000Go), i.e., histone H4 acetylation is inhibited by the histone H4-bound Ni2+. It is one well-known characteristic of bound-Ni2+ to produce reactive oxygen species (ROS) in vivo, and the generation of ROS by histone-bound Ni2+ has also been proved previously (Bal and Kasprzak, 2002Go). Since ROS are very reactive and react easily with surrounding protein, DNA, and other molecules in vivo, their generation likely plays important roles in Ni2+-induced gene expression alterations, carcinogenesis, and especially resistance of mammalian cells to Ni2+ (Denkhaus and Salnikow, 2002Go; Qu, 2001; Salnikow et al., 1994Go). Hence, we are interested in whether the generation of ROS in vivo, especially those generated by histone-bound Ni2+, can influence the acetylation modification of histones through reacting with histones or molecules around them, such as histone acetyltransferases (HAT) or histone deacetylases (HDAC) (Archer and Hodin, 1999Go; Klochendler-Yeivin and Yaniv, 2001Go), two classes of enzymes involve in the control of the state of histone acetylation. Our knowledge of the relation between ROS generation and Ni2+-induced histone hypoacetylation still remains unclear. To clarify this will provide a new approach to further understand the role of ROS generation in Ni2+-induced gene silencing, toxicity, and the resistance of mammalian cells to Ni2+.

Here, we suppose that Ni2+-induced generation of ROS may play a role in the carcinogenicity of specific nickel compounds by leading to the inhibition of histone acetylation. To address this hypothesis, effects of Ni2+ on histone acetylation alteration and ROS generation were studied in human hepatoma cells. Ni2+ treatment significantly inhibited histone acetylation through affecting the activity of HAT in cells, and the generation of ROS was induced by Ni2+ and partially involved in the induction of histone hypoacetylation. These results suggested a new approach, diminishing Ni2+-generated ROS, to prevent and cure Ni2+-related histone hypoacetylation, gene suppression, and diseases such as cancer.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and mycoplasma detection.
Human hepatoma cells (Hep3B) were maintained in RPMI-1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (equivalent to 100 units/ml and 100 mg/ml, respectively) at 37°C as monolayers in a humidified atmosphere containing 5% CO2. After culturing the cells (2 x106 cells/ml) for 24 h, the culture medium was aspirated and replaced with new medium containing Ni2+ and/or appropriate reagents where indicated. To ensure that the cells were free of mycoplasma, mycoplasmas in cultured cells were detected once at every two weeks by staining of DNA with 4',6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO) (Russel et al., 1975).

Exposure of cells to Ni2+ and other agents.
NiCl2 (Approx. 98%), H2O2, 2-mercaptoethanol (2-ME) and N-acetyl-cysteine (NAC) were obtained from Sigma Chemical Co. (St. Louis, MO). NiCl2 and NAC stock solution (1 M) in distilled water were filtered through a sterile, pathogen-free nylon filter (pore size: 0.22 µm; MSI Inc., MA), the pH of stock solutions was carefully adjusted to 7.2 using 0.5 N HCl and 1 N NaOH before filtration. A freshly prepared stock solution was mixed with RPMI-1640 at indicated concentrations before use.

Histone purification.
Preparation of histones from Hep3B cells was done according to Cousens et al. (1979)Go with the following modifications: the washed cells were suspended in lysis buffer (Cousens et al., 1979Go) containing trichostatin A (TSA) (100 ng/ml) and PMSF (1 mM). After pipetting up and down for 20 times, the nuclei were washed three times in the lysis buffer and once in 10 mM Tris and 13 mM EDTA (pH 7.4). The histones were extracted from the pellet in 0.4 N H2SO4. After centrifugation, the histones in the supernatant were collected by cold-acetone precipitation, air-dried, then suspended in 4 M urea and stored at -20°C before use.

Western blotting analysis of histone H4 acetylation.
Equal amounts of purified histones (30 mg/lane) were subjected to SDS–PAGE on 15% polyacrylamide gels and were electrophoretically transferred to a nitrocellulose membrane. Nitrocellulose blots were blocked with 5% milk in TTBS (Tris-buffered saline plus 0.05% Tween 20, pH 7.5) and incubated overnight at 4°C with a specific antibody for histone H4-acetyl-lysines in TTBS containing 5% milk. After incubation with horseradish peroxidase-conjugated secondary antibody, immunoreactivity was visualized by means of enhanced chemiluminescence (ECL, Amersham, UK).

Histone acetylation assay.
Cells were plated at a density of 2 x 106 cells/ml and exposed to 10 mCi/ml 3H-acetate (5.0 Ci/mmol, Amersham, UK). After incubation for 10 min at 37°C, the cells were stimulated with Ni2+ or/and other agents for the indicated times in the absence or presence of TSA (Sigma, St. Louis, MO, USA). Then histones were purified and 3H-labelled histones were determined by liquid scintillation counting.

HDAC assay.
An HDAC assay was performed as previously reported (Ito et al., 2000Go). Radiolabeled histones were prepared from Hep3B cells following incubation with TSA (100 ng/ml, 6 h) in the presence of 0.1 µCi/ml 3H-acetate. The histones were purified, dried, and resuspended in 50 mM Tris-HCl buffer (pH 8.0). Crude HDAC preparations were extracted from total cellular homogenates and then incubated with 0–1000 µM Ni2+ and the 3H-labelled histones in 50 mM Tris-HCl buffer (pH 8.0) for 30 min at 37°C before the reaction was terminated by the addition of 1 N HCl-0.4 N acetic acid. The released 3H-labelled acetic acid was extracted from the reaction mixture and then determined by liquid scintillation counting.

HAT assay.
Crude HAT preparations were extracted from total cellular homogenates of Hep3B cells, and the in vitro acetylation assay was performed as described previously (Chicoine et al., 1987Go; Ito et al., 2000Go). A typical HAT assay was performed using a 50-µl reaction mixture containing: histone protein (20 mg of core histones extracted from Hep3B cells), 20 ml of crude HAT extract (from 1 x 107 cells), 0–1000 µM Ni2+, 100 ng/ml TSA, 10 mM HEPES (pH 7.8), 4% glycerol and 0.1 µCi 3H-acetyl-CoA (5.6 Ci/mmol, Amersham, UK). Reactions were initiated by the addition of 3H-acetyl-CoA to the mixture, followed by incubation for 1 h at 30°C. After incubation, the reaction mixture was spotted onto Whatman p81 phosphocellulose paper (Whatman), washed extensively with 0.2 M sodium carbonate buffer (pH 9.2), and then briefly washed with acetone. The dried filters were counted by liquid scintillation.

Measurement of intracellular ROS generation.
The level of intracellular ROS was measured by the alteration of fluorescence resulting from oxidation of 29,79-dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, Eugene, OR) (LeBel, 1992). DCFH-DA was dissolved in DMSO to a final concentration of 20 mM before use. For the measurement of ROS, cells were incubated with 10 µM DCFH-DA at 37°C for 30 min, then the excess DCFH-DA was washed with RPMI-1640 media prior to the treatment with Ni2+ and/or other reagents for a time period indicated in the figure legends. The intensity of fluorescence was recorded using a flow cytometry (Becton Dickenson), with an excitation filter of 485 nm and an emission filter 535 nm. The ROS level was calculated as a ratio: ROS = mean intensity of exposed cells: mean intensity of unexposed cells.

Miscellaneous.
Protein concentrations were determined with the BCA protein assay (Pierce, USA) using bovine serum albumin as a standard. Statistical analysis was performed by analysis of variance (ANOVA post-hoc Bonferroni), and p values less than 0.05 or 0.01 were denoted as * or **, respectively.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of Ni2+ on Histone Acetylation in Hep3B Cells
Histone acetylation occurs in the lysine residues within the basic N-termini of core histones H2A, H2B, H3 and H4 (Klochendler-Yeivin and Yaniv, 2001Go; Salnikow et al., 1997Go), and the level of histone H4 acetylation has been reported to reflect the overall level of histone acetylation (Graham et al., 1991Go; Klochendler-Yeivin and Yaniv, 2001Go; Zoroddu et al., 2000Go). Thus, we first tested the effect of Ni2+ on histone H4 acetylation by Western blotting. As shown in Figure 1Go, Ni2+ exposure resulted in a dose- and time-dependent decrease in histone H4 acetylation. Then we quantitated the influence of Ni2+ on global histone acetylation by using the histone acetylation assay in the presence of 3H-acetate. Like those of Western blotting, both experiments, in Figures 2A and 2BGo, again indicated a dose- and time-dependent inhibitive effect of Ni2+ on histone acetylation. In both Western blotting and the histone acetylation assay, the reduction in acetylation of histone H4 and global histones showed a similar trend; thus we studied the links between Ni2+-generated ROS and histone hypoacetylation through quantitating the decrease of global histone acetylation. Simply, the reduction of acetylation was observed at concentrations no less than 600 µM, and a marked reduction was observed at a concentration of 1000 µM (the reduction rate is about 30% in Fig. 2AGo). The inhibition of histone acetylation was first apparent 2 h after exposure to 1000 mM Ni2+, and the hypoacetylation was sustained for 24 h.



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FIG. 1. Ni2+ inhibits histone H4 acetylation in Hep3B cells. (A) Hep3B cells were incubated with different concentrations of Ni2+ for 8 h; lanes 1–6: 0, 200, 400, 600, 800, 1000 µM of Ni2+, respectively. (B) Cells treated with 1000 µM Ni2+ for the indicated times; lanes 1–7: 0, 2, 4, 8, 12, 18, 24 h, respectively. Histones were isolated and separated on 15% SDS–PAGE gels (30 µg per lane) and then subjected to Western blotting with an antibody for acetylated Histone H4; see the upper panels in (A) and (B). 30 µg of histones used in the experiments were stained with Coomassie Blue—the lower panels in (A) and (B)—to confirm the equal amount of histones were added in each lane.

 


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FIG. 2. Ni2+ induced global histone hypoacetylation in Hep3B cells. Effect of Ni2+ on histone acetylation in the presence of 3H-acetate was quantified by liquid scintillation. Cells (2 x 106) were exposed to the indicated concentrations of Ni2+ for 8 h in the absence (A) or presence (C) of 0.3 µM TSA; or exposed to 1000 µM of Ni2+ for the indicated times in the absence (B) or presence (D) of TSA. Means ± SD of three parallel experiments were indicated. n =3x3 cultures per conditions, *p < 0.05, **p < 0.01 versus the control group.

 
Effect of Ni2+ on HAT and HDAC Activity
HAT and HDAC are two metabolic enzymes responsible for the status of histone acetylation. To determine which enzyme is involved in the histone hypoacetylation resulting from the Ni2+ treatment, histone acetylation levels in cells were first examined in the presence of an HDAC inhibitor, TSA. Compared with the data without the presence of TSA (Figs. 2A,BGo), Figures 2C and 2DGo indicated that Ni2+ similarly inhibited histone acetylation in the absence or presence of 0.3 µM TSA, demonstrating that deacetylation by HDAC is not involved in the Ni2+-mediated histone hypoacetylation. When in vitro HAT and HDAC assays were carried out with Ni2+, HDAC activity remained unaltered, but HAT activity was inhibited in a dose-dependent manner (Figs. 3A,BGo), providing an evidence for the direct inhibition of HAT activity in vitro by Ni2+. Collectively, these results suggest that Ni2+ induced histone hypoacetylation in vivo by the direct or indirect inhibition of HAT activity.



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FIG. 3. The effect of Ni2+ on HAT and HDAC activities. Crude HAT and HDAC preparations were extracted from total cellular homogenates of Hep3B. Activities of HAT and HDAC in the presence of various concentrations of Ni2+ as indicated were determined as described in Materials and Methods. Experiments were triplicate and similar results were gotten. The means of three parallel experiments of Ni2+ on HAT (A) or HDAC (B) are plotted. *p < 0.05, **p < 0.01 versus the control group.

 
ROS Generation in Hep3B Cells Exposed to Ni2+
It has been previously shown that Ni2+ produced ROS in biological systems (Bal and Kasprzak, 2002Go; Denkhaus and Salnikow, 2002Go). To evaluate the induction of ROS in Ni2+-treated cells, cells preloaded with DCFH-DA were exposed to Ni2+ for the indicated times. DCFH-DA is commonly used to detect the generation of reactive oxygen intermediates in cells (LeBel et al., 1992Go). Figures 4A and 4BGo showed a dose- and time-dependent increase in ROS generation when Hep3B cells were incubated with Ni2+. Based on the Figure 4AGo, the exposure of cells to Ni2+ at a concentration of no less than 600 µM led to a very significant induction of ROS generation, and an about 2.6-fold increase of ROS compared with basal levels was found in cells exposed for 8 h to 1000 µM Ni2+.



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FIG. 4. The effect of Ni2+ on ROS generation. Relative ROS generation in Hep3B cells exposed to different concentrations of Ni2+ for 8 h (A), or exposed to 1000 µM of Ni2+ for the indicated times (B). All data represent mean values of six independent measurements ± SD. *p < 0.05, **p < 0.01 versus the control group in (A).

 
ROS Generation Played a Role in the Induction of Histone Hypoacetylation
To evaluate whether ROS participate in the induction of histone hypoacetylation, we used H2O2 or the antioxidants 2-ME and NAC to enhance or diminish the Ni2+-induced ROS generation. Neither the addition of H2O2, with a concentration of 20 or 50 µM alone, nor the addition of 2-ME (1 or 2 mM) or NAC (0.5 mM or 1 mM) induced a significant ROS generation or histone acetylation alteration in Hep3B cells compared to the relative control group (data not shown). On the contrary, H2O2, 2-ME, or NAC was efficient at different concentrations in enhancing or diminishing the ROS generated by Ni2+ (Fig. 5AGo). H2O2 at 50 µM, 2-ME at 2 mM, or NAC at 1 mM also efficiently enhanced or suppressed the inhibition of histone acetylation triggered by Ni2+ (Fig. 5BGo). When effects of H2O2, 2-ME, or NAC on HAT activity were studied, the above result was confirmed by the finding that the inhibition of Ni2+ on HAT activity was effectively enhanced or diminished by H2O2 (50 µM), 2-ME (2 mM), or NAC (1 mM) (Fig. 5CGo). Again, the addition of H2O2, 2-ME or NAC alone did not cause any significant alteration on the activity of HAT in our system (data not shown). These data herein indicate an important role of ROS in the induction of histone hypoacetylation caused by Ni2+.



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FIG. 5. The effect of H2O2, 2-ME or NAC on Ni2+-induced ROS generation and histone hypoacetylation. (A), Relative generation of ROS in Hep3B cells exposed to 1000 µM Ni2+ alone or in combination with H2O2, 2-ME, or NAC at the indicated concentrations for 8 h. ROS generation was quantified and analyzed as described in Figure 4Go. (B), Histone acetylation in cells exposed to 1000 µM Ni2+ alone or in combination with H2O2, 2-ME, or NAC for 8 h. Histone acetylation measurement and analysis were performed as described in Fig. 2Go. (C) HAT activity in vitro treated with 1000 µM Ni2+ alone or in combination with H2O2, 2-ME, or NAC at the indicated concentrations. HAT activity assay was performed as described in Fig. 3BGo. *p < 0.05, **p < 0.01 versus the 1000 µM Ni2+ group in (A)–(C).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies suggest that through binding with histones, Ni2+ can promote ROS generation and induce histone hypoacetylation (Bal and Kasprzak, 2002Go; Broday et al., 2000Go; Zoroddu et al., 2000Go). Considering that ROS are active and easily react with surrounding molecules, there probably exists some linkage between Ni2+-induced ROS generation and histone hypoacetylation, but to our knowledge, no information is currently available about that.

The carcinogenicity of specific insoluble nickel compounds, such as crystalline nickel sulfide (NiS) and subsulfide (Ni3S2), is believed due to the ability of cells to easily phagocytize and dissolve these compounds (Denkhaus et al., 2002; Kargacin et al., 1993Go). Accurately, it is the accumulation of the Ni2+ ion inside the cell that appears to determine the carcinogenic potencies of different nickel compounds. Although soluble nickel salts enter cells poorly, NiCl2 has been proved to be able to partially enter cells and concentration-dependently increase the Ni2+ ion accumulation inside cells, and hence NiCl2 is often used to study the carcinogenic mechanism of nickel compounds (Broday et al., 2000Go; Kargacin et al., 1993Go). In this study NiCl2 was chosen as a suitable agent for studying the role of ROS generation in Ni2+-induced histone hypoacetylation.

Exposure of hepatoma cells to Ni2+ resulted in a decreased histone acetylation in cells, as detected by Western blotting and in vivo histone acetylation assays. Two possible mechanisms can be proposed to explain the histone hypoacetylation resulting from Ni2+-exposure. One model postulates that Ni2+ inhibits the acetylation of histones by the direct or indirect down-regulation of overall HAT activity. The other model, on the contrary, involves the promoting of Ni2+ on the deacetylation of acetylated histones through the up-regulation of HDAC activity. The present data show that HDAC activity is not required for the inhibition of Ni2+ on histone acetylation, because TSA, an inhibitor of HDAC (Koyama et al., 2000Go), had no effect on the inhibition of histone acetylation triggered by Ni2+. Therefore we conclude that the decreased levels of histone acetylation must reflect the direct or indirect repression of HAT activity by Ni2+. This conclusion is confirmed by in vitro HAT and HDAC assays using an hepatoma cell extract, where the activity of HAT, but not that of HDAC, was inhibited by Ni2+ in a dose-dependent manner. Although additional work is needed to clarify how Ni2+ affects HAT activity, we proved that Ni2+ induced histone hypoacetylation through inhibiting HAT activity in hepatoma cells.

Like in other systems (Bal and Kasprzak, 2002Go; Denkhaus and Salnikow, 2002Go; Salnikow et al., 2000Go), Ni2+ treatment also led to an increased ROS generation in cells, and the addition of H2O2 in Ni2+-treated cells significantly enhanced the ROS generation, whereas addition of 2-ME or NAC diminished that. A direct linkage between Ni2+-generated ROS and histone hypoacetylation was proved by the results that Ni2+-triggered histone hypoacetylation was efficiently enhanced or suppressed by the enhancement or diminution of ROS generation. This linkage is also confirmed by the HAT assay, where the enhancement or diminution of Ni2+-generated ROS again displayed an efficient enhancing or suppressing effect on the HAT inhibition triggered by Ni2+. The mechanism underlying the inhibition of HAT activity by ROS is unclear; one hypothesis is that the activity of HAT may be weakened by ROS through oxidative modification of critical cysteine or histidine residues in HAT proteins. This hypothesis seems extremely possible in Hep3B cells, since some of HAT proteins such as p300/CBP contain critical cysteine- and histidine-rich domains and have been found expressing in Hep3B cells (Arany et al., 1996Go; Newton et al., 2000Go). Moreover, this hypothesis is supported by our unpublished data, where the addition of H2O2 inhibited the HAT activity of exogenously introduced p300 in Hep3B cells. Our study also suggests that the generation of ROS is not the whole cause of histone hypoacetylation triggered by Ni2+, since H2O2 at 20 µM, 2-ME at 1 mM, or NAC at 0.5 mM did not enhance or suppress the histone hypoacetylation induced by Ni2+, although they significantly affected the generation of ROS. It suggests that Ni2+ inhibits histone acetylation through a multi-pathway, at least through inhibiting HAT activity by ROS generation (proved by us) and through affecting the access of HAT proteins to histones by the binding of Ni2+ with histones (proved by Broday et al., 2000Go). Although there exist other mechanisms, these data herein clearly prove that Ni2+-generated ROS plays a role in the inhibition of Ni2+ on histone acetylation.

The toxicity of Ni2+, especially the carcinogenicity of specific insoluble nickel compounds, is believed mainly resulting from the suppression of gene transcription and expression (Beyersmann, 2002Go; Cangul et al., 2002Go), while the resistance of mammalian cells to Ni2+ is closely allied to their defense capability against the generation of ROS triggered by Ni2+ (Qu et al., 2001Go; Salnikow et al., 1994Go), i.e., mammalian cells diminish the effects of Ni2+ through diminishing ROS generation. Thus, there must exist a linkage between Ni2+-induced gene suppression and ROS generation. Insofar as we are aware, this study constitutes the first report that Ni2+-induced ROS generation is involved in the inhibition of histone acetylation. The linkage between ROS generation and histone acetylation inhibition opens a new window for us to understand the role of ROS in the toxicity, especially the carcinogenicity of Ni2+ and the resistance of mammalian cells to Ni2+. Presumably, Ni2+ induces carcinogenesis through ROS generation, which inhibits histone acetylating and, hence, suppresses genes expression (Fig. 6Go). And whether a mammalian cell is sensitive or resistant to Ni2+ is probably determined by its defense ability against Ni2+-induced gene suppression, while ROS generation plays an important role in this suppression through inhibiting histone acetylation.



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FIG. 6. Schematic diagram of the possible role of ROS generation on the Ni2+-induced carcinogenesis.

 
Although as we have mentioned above, only high concentrations of NiCl2 possessed the activities of generating ROS and inhibiting histone acetylation, i.e., at 600 or 1000 µM, our study can indeed be very relevant to the mechanisms of carcinogenesis induced by specific insoluble nickel compounds. This is because soluble nickel salts enters cells poorly. Kargacin et al. (1993)Go found that the cytotoxicity of 600 µM of soluble nickel chloride was only equivalent to approximately 20 µM of nickel subsulfide.

In summary, we conclude that ROS production is increased during exposure of cells to Ni2+, and this production of ROS is involved in the inhibition of histone acetylation. Ni2+ induces histone hypoacetylation through a multi-pathway, at least through affecting the HAT activity by ROS generation and influencing the access of HAT with histones. These data suggest a possibly useful method to prevent nickel-induced histone hypoacetylation, gene silencing, and related diseases (cancer, etc.) through improving the antioxidative potential of people, especially those working in a high nickel-exposed environment.


    ACKNOWLEDGMENTS
 
This project was supported by SRF for ROCS, SEM, and the Doctoral Program of Lanzhou University, China.


    NOTES
 
1 To whom correspondence should be addressed. Fax: 86-931-891-2561. E-mail: kangjiuhong{at}lzu.edu.cn. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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