Ⅰ.INTRODUCTION
Cigarette smoke contains more than 6,000 hazardous chemicals and 40 or more carcinogens, including nitrosamine, and it is known as a major risk factor for oral and laryngeal cancers [1]. However, most carcinogens in cigarette smoke also exist in industrial worksites and in polluted air, indicating that the carcinogenic effect of cigarette is debatable. The relationship between smoking and carcinogenesis should be clarified by studying the carcinogens that exist only in tobacco smoke. NKK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) is the most abundant type of nitrosamine extracted from tobacco alkaloids and is a strong carcinogen that is found only in cigarette smoke [2]. Therefore, assessment of carcinogenic risk due to NKK is crucial for judging the carcinogenic potential of smoking.
Mercury chloride is the main component of dental amalgam and has been used for well over 100 years. A standard amalgam product contains approximately 50% of mercury [3]. There have been concerns for such high content of mercury in amalgam [4-6]. Mercury released from amalgam is transferred to the intestines along with food and is then distributed throughout the body via the bloodstream [7]. A recent report using human salivary gland tissue cells suggested the carcinogenic potency of mercury [8]. It has been reported that mercury dichloride induces DNA damage even at low concentrations that do not show a cytotoxic effect, suggesting carcinogenic relevance for mercury. The proportion of salivary gland tumors among head and neck cancers is increasing and the parotid gland is mostly affected in such cases [9-11]. The specific factors involved in the increasing incidence of salivary gland tumors are not yet known. It is critical to investigate the factors involved in increasing cases of salivary gland tumors for their prevention and treatment. Other risk factors involved in carcinogenesis such as smoking and drinking are well known, but since the incidence of salivary gland tumors is still increasing despite the absence of any direct influence of these factors, indicates the involvement of unknown factors or an interaction between known risk factors.
The present study focused on the interaction between carcinogenic risk factors that may be potent during longer periods of exposure. Most carcinogens have prolonged exposure times and subsequently bring about gradual carcinogenesis. A constant effect in the oral environment implies that the interaction between risk factors plays a major role in carcinogenesis of oral tumors. Thus, the present study intended to evaluate the carcinogenic potential of the interaction between NNK, the most potent and smoking-related among the carcinogenic nitrosamines in tobacco, and mercury, which has a chance of leakage in the dental amalgam.
Most oral squamous cell carcinomas are solid tumors and the primary targets of oral cancers are the epithelial cells in the oral mucosa [12]. Thus, studies using an epithelial cell model need to study the relation between NNK or mercury and oral cancers. The present study adopted a keratinocyte model, which is an epithelial cell type derived from the human skin tissue. Human skin tissue-derived epithelial cells are widely used to assess the biological effects of dental materials and are used as a substitute for oral cell models due to their similarities to the oral keratinocyte model and easier handling [13,14]. We have used an immortalized human epithelial cell model, which is capable of continuous cultivation while retaining normal cell properties, to assess the carcinogenic potential of continuous exposure to mercury in dental amalgam and nitrosamines in cigarette smoke [15,16].
The present study involved exposure of immortalized human epithelial cells to mercury and NNK in order to assess the potential interactions between NNK and mercury and their potential carcinogenicity by testing related indicators such as production of reactive oxygen species and apoptosis.
Ⅱ.MATERIAL and METHODS
Chemicals
HgCl2, NNK, and other chemicals were purchased from Sigma-Aldrich (St. Louis, USA). Cell culture media, including DMEM, were obtained from Gibco BRL (Gaithersburg, USA) and the ROS kit was purchased from Promega (Madison, USA).
Cell culture
Human epithelial cells (RHEK-1) immortalized by Ad12-SV40 at passage 164 were used for the study. Cells were cultured in Dulbecco’s modified Eagle medium media containing EGF, 10% FBS, hydrocortisone (5 μg/ml), antibiotics, etc. at 37°C in a 5% CO2 incubator. When the cells reached confluency, they were subcultured at a ratio of 1:3.
Cell treatment
Human epithelial cells at 70% confluence were treated for 14 days with culture media containing mercury chloride (0.5, 1, 5, 10 μM) alone or simultaneously with NNK (100 μM). When cells reached 100% confluency, they were subcultured at a ratio of 1:3, and carcinogenic potential was evaluated at selected passage numbers. For analysis of ROS generation, apoptosis, and change in expression of inflammatory factors, cells were exposed to NNK or HgCl2 for 24 hours prior to the analysis
Analysis of mercury chloride and NNK induced cytotoxicity in human cells
The cytotoxic potential of mercury chloride was determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) assay, which measures the concentration of formazan formed by the reduction of MTT by the mitochondrial enzymes of living cells. Cells were grown in 96-well microplates with mercury chloride or NKK solution for 24 hours in a CO2 incubator and were then incubated with 10 μl MTT solution (5 mg/ml) for 5-6 hours. The resulting formazan was dissolved in 100 μl of 0.03 N HCl-isopropanol solution and absorbance was measured with a microplate reader at 540 nm. Cell survival rate was represented as the percentage of absorbance of wells containing mercury chloride or NNK divided by the absorbance of control untreated cells.
Measurement of cell density
Changes in cell contact inhibition were measured as the cell count per unit area when the cells reached confluency. Cell culture media were renewed after every 3 days at 5 x 103 cells/cm2 wells.
Soft agar colony formation
Noble agar (1.2 g) in 35 ml of distilled H2O (dH2O) was autoclaved for 30 minutes; 15 ml of dH2O, 24 ml of FBS, and 50 ml of 2X EMEM were added to this to prepare the 0.9% agar base. The agar base (5 ml) was dispensed in petri dishes and was kept overnight in a 37°C incubator. The top agar (0.36%) was prepared by autoclaving 0.9 of noble agar for 30 minutes and then adding 7.2 ml of dH2O, 7.2 ml of FBS, and 14.4 ml of 2X EMEM. A 2 ml volume of top agar containing 1 x 104 cells was plated on each of the prepared base agar dishes and the number of colonies greater than 0.3 mm in size was counted.
Measurement of cell aggregation
Base agar (5 ml) was plated on petri dishes as described in the soft agar assay. After overnight incubation at 37°C, 105 cells/dish were inoculated onto the plates in media containing 10% FBS, and the number and size of colonies greater than or equal to 1 mm were measured after 4 days of inoculation.
ROS assay
2’,7’-Dichlorofluorescin diacetate (DCFH-DA) is a lipophilic substance that can easily permeate into cells where it is converted to a fluorescent probe, DCF (2’,7’-dichlorofluorescin) by intracellular ROS. Thus, measurement of DCF is commonly used to measure intracellular ROS activity. Cells were exposed to mercury chloride in the presence or absence of NNK in 12-well culture plates for 24 hours, washed with Lock’s buffer, and treated with DCFH-DA for 15 minutes in a dark room. The cells were washed again, lysed with NaOH, and the cellular fluorescence was then measured with a luminescence spectrometer at the excitation wavelength of 488 nm and the emission wavelength of 525 nm [17].
Analysis of apoptosis
Apoptosis was quantified using the Cell Death Detection ELISA Plus kit (Roche, Germany) as described in the manufacturer’s manual. Cells were exposed to mercury chloride in the presence or absence of NNK for 24 hours. After removal of the media, cells were lysed with lysis buffer for 30 minutes and then transferred to 1.5 ml Eppendorf tubes to be centrifuged at 200g for 10 minutes. The supernatant containing oligonucleosomes was used to detect DNA fragmentation with a microplate reader at 405 nm.
RT-PCR
After extracting total RNA, poly-A positive transcripts were obtained by adding oligo d(T) primer to 1 μg of total RNA at 60°C for 5 minutes. After adding 30 μl of the Reverse Transcription mix (0.1 M DTT, RNA guard, RT-buffer, dNTP, dH2O, RT) to each sample, the samples were incubated at 37°C for 60 minutes and at 70°C for 10 minutes and then placed on ice. cDNA (2.5 μl) was added to 47.5 μl of PCR mix [PCR-buffer, dH20, Taq polymerase, 5’-primer, 3’-primer, dNTP-mix, (32p – labelled0).], and the PCRs with primerspecific annealing temperatures were carried out as described in Table 1. The samples were electrophoresed on 10% polyacrylamide gels, and the intensity of the bands was measured using ChemiDoc XRS (Bio-Rad).
Statistical analysis
The numerical data were compared using the Student’s t-test and a p-value of less than 0.05 was considered significant.
Ⅲ.RESULTS
Analysis of cytotoxic potential of NNK and HgCl2 in human cells
The MTT assay was performed to evaluate the cytotoxicity of NNK and HgCl2 in human cells and to quantify the level of exposure. Thus, cytotoxicity as well as carcinogenic potential of the substances was assessed and optimal concentrations of exposure were simultaneously selected. For HgCl2, which showed significant cytotoxicity at 20 μM, exposure concentrations were selected as 0.5, 1, 5, and 10 μM (Fig 1-A). There was no significant decrease in cell viability below 200 μM NNK (Fig 1-B). Thus, 100 μM NNK was selected as the optimal dose, because this was the maximum dose that rendered intact cellular function without severe cytotoxicity.
Carcinogenicity and interaction between HgCl2 and NNK in human cells
Parameters of neoplastic cellular transformation, such as cell saturation density, soft-agar colony formation, and cell aggregation, were analyzed to examine the carcinogenic potential of mercury chloride and NNK. These parameters represent the characteristics of carcinogenic cells, including contact inhibition, anchorage independence, and cell adhesion, thereby predicting carcinogenesis. For analysis of saturation density and cell aggregation, with the exception of the maximum concentration (10 μM), none of the HgCl2 concentrations showed a significant increase in soft-agar colony formation (Table 2-A). The carcinogenic potential of mercury chloride was also observed in terms of cellular morphology and no foci were observed at all the concentrations tested. Foci were observed in all groups at passage 10, including the control group, indicating that the carcinogenic potential of mercury chloride could not be determined in this study (Table 3-A). The carcinogenic interaction between mercury chloride and NNK was also evaluated, and while the administration of NNK alone did not show any carcinogenic changes, exposure to NNK along with mercury chloride significantly increased cell aggregation and soft agar colony formation at 10 μm (Table 2-B). Cell morphology was studied to test the carcinogenic interaction between mercury chloride and NNK, and foci were observed to appear in passage 7 at 10 μm, which is the maximum concentration. Passage 9 also showed appearance of foci but this was not considered as increased carcinogenic potential, since, in this passage, foci appeared at all concentrations of NNK in the presence or absence of mercury chloride (Table 3-B).
Production of reactive oxygen species (ROS)
An imbalance of the oxidation-reduction system is regarded as the primary factor involved in mercury-induced carcinogenesis. Thus, ROS generation was measured to analyze the carcinogenic mechanism in this system. Exposure to 10 μM HgCl2 alone increased ROS production by 1.5 fold. Co-treatment with 100 μM NNK increased ROS production at 0.5, 1, 5, and 10 μM HgCl2 by 1.6, 1.5, 1.8, and 2.1 fold, respectively (Figure 2).
Influence of HgCl2 and NNK on apoptosis
The carcinogenic process in human epithelial cells includes suppressing normal cell apoptosis and providing a relatively advantageous environment for proliferation. Thus, ELISA using antibody against DNA-histone fragmentation was performed to detect the apoptotic effects of HgCl2 in the presence or absence of NNK. HgCl2 exposure alone increased DNA fragmentation by 1.5 fold at 10 μM but co-treatment with NNK revealed a significant increase in DNA fragmentation at 1, 5, and 10 μM HgCl2, suggesting that co-treatment increased apoptosis (Figure 3).
Changes in inflammatory factors and growthrelated factors
mRNA expression of interleukin-1, -6 and TNF-α was analyzed by RT-PCR. mRNA levels of IL-1 and IL-6 were found to increase. Co-treatment with NNK further increased the mRNA levels of IL-1 and IL-6 (Figure 4).
Ⅳ.DISCUSSION
Dental amalgam has been identified as the single largest source of continuous mercury exposure, and the mercury released from dental amalgam can irritate the oral mucosa, promote epithelial cell proliferation, or induce a hypersensitive reaction. The harmfulness of mercury release from amalgam has been debated for decades [18]. The carcinogen NNK is directly related to several tumors induced by smoking, and smokers inevitably exposed to NNK continually. This study aimed to evaluate the carcinogenic potential of the mercury component in dental amalgam by using a human epithelial cell model and to study the carcinogenicity related to smoking, thereby providing scientific data for judging the risk of simultaneous exposure to amalgam and cigarette smoke. All HgCl2 concentrations tested, except the maximum concentration 10 μM, led to a significant increase in soft-agar colony formation whereas saturation density and cell aggregation were not affected. Co-treatment with NNK, a well-known carcinogen in cigarette smoke, significantly increased soft agar colony formation and cell aggregation. Administration of 10 μM mercury chloride alone only showed an increase in soft agar colony formation, whereas co-treatment with NNK brought about significant increases in both parameters, suggesting a carcinogenic interaction between NNK and mercury chloride. However, their individual carcinogenic potential seems to be weak as carcinogenic changes were observed only at the maximum concentration and foci, which are indicators of tumor induction strength, only appeared at the maximum concentration in passage 7. A further study should diversify the concentrations and the exposure time to determine the actual concentration during exposure and the extent to which the interaction affects carcinogenesis in an in vivo system.
We confirmed that a significant amount of ROS is generated at 10 μM of mercury chloride. Excessive production of ROS is known as an essential mediator of mercury chloride toxicity [19]. De Flora et al suggested that mercury did not induce point mutations in bacteria but promoted c-mitosis in eukaryotes by attaching to SH-groups to act as a mitotic spindle inhibitor [20]. Schurz et al reported that the mechanism of mercury action was through increased ROS and that mercury itself did not damage DNA [21]. The present study presumed that increased tumorigenicity by mercury chloride was related to ROS production. ROS generation was found to increase upon co-treatment with NNK and mercury chloride and an increase in the carcinogenic potential of human cells was also seen. The fact that increased ROS generation corresponds to increased carcinogenicity implies that ROS plays a major role in the carcinogenic process in human cells. Thus continuous exposure to mercury as in dental amalgam might interact with NNK in cigarette smoke to promote ROS production in the oral cavity, thus providing a vulnerable environment for oral cells to undergo carcinogenesis.
ROS plays an important role in carcinogenesis by directly participating in the DNA damage process, such as by producing DNA adducts [22]. ROS is especially known to alter the signal transduction system to provoke various changes in cell regulation. ROS is also involved in apoptosis by activating signal transduction systems such as ERK-1, -2, Jun NH2-terminal kinase, MAPK, etc [23].
The apoptotic process balances with cell proliferation to ensure normal tissue functioning. Carcinogenic cells have a defective apoptotic machinery and sufficient cell death does not occur. Therefore, defective cells are more susceptible to proliferation than normal cells. Alterations in the apoptotic process thus represent a major causative factor in cancer development and progression. In this study, 10 μM of mercury chloride promoted apoptosis in human cells, suggesting that at higher concentrations, mercury chloride seems to alter apoptosis as well as cellular carcinogenic parameters. This apoptotic process of mercury chloride was increased even more in the presence of NNK. The synergistic effect on apoptosis by NNK showed a similar pattern to the phenomenon where NNK promotes carcinogenesis upon co-treatment with mercury chloride. Thus, the apoptotic synergism of these substances is likely to be related to carcinogenesis. An increase in apoptosis is thought to indicate an interrelation with carcinogenesis. ROS generation is closely related to apoptosis and thus, increased apoptosis by mercury chloride seems to be associated with ROS [24]. ROS generation can influence the late cell cycle including apoptosis, suggesting that ROS production by mercury chloride may alter the signal transduction system and this altered cell regulation is likely to provoke apoptosis. We need to confirm whether this is applicable to human epithelial cells by using a more specific ROS inhibitor in the future.
The mRNA expression of interleukin-1, -6 and TNF-α was analyzed by RT-PCR. The present study showed a slight increase in the mRNA levels of IL-1 and IL-6 upon treatment with mercury chloride alone and an additional increase in these mRNA levels following co-treatment with NNK. These results suggest that mercury chloride may participate in modulating the expression of inflammatory cytokines in an additive fashion with NNK. Further quantitative studies on the expression and amount of inflammatory cytokine production need to be conducted.
The present study suggests an influence of the mercury in dental amalgam on human epithelial cells and its synergistic effect with smoking. Moreover, it would provide scientific evidence for the risk of amalgam use as well as data for smoking management to prevent oral cancer. Since this study adopted a human epithelial cell model that is closely associated with oral cancer, it could provide useful information regarding the relation of dental amalgam and smoking with oral cancer.
