Effects on protein and mRNA expression levels of p53 induced by fluoride in human embryonic hepatocytes

Toxicology Letters 158 (2005) 158–163

Effects on protein and mRNA expression levels of p53 induced by fluoride in human embryonic hepatocytes A.G. Wang a,∗ , Q.L. Chu b , W.H. He a , T. Xia a , J.L. Liu a , M. Zhang a , A.K. Nussler c,1 , X.M. Chen a , K.D. Yang a,∗∗ a

c

Department of Environmental Health, School of Public Heath, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, PR China b Department of Preventive Medicine, Medical College of Hubei Vocational-Technology College, Xiaogan 432000, Hubei, PR China Department of General, Visceral and Transplantation Surgery, Virchow Clinic, Humboldt University of Berlin, Berlin 13353, Germany Received 8 January 2005; received in revised form 8 March 2005; accepted 14 March 2005 Available online 13 June 2005

Abstract We investigated the effects of protein and mRNA expression levels on p53 induced by fluoride in human embryo hepatocyte L-02 cells. The protein and mRNA levels of p53 in L-02 cells were measured after in vitro cultured L-02 was exposed to sodium fluoride at different doses (40, 80, and 160 ␮g/ml) for 24 h. The results showed that the cell survival rate of L-02 cells in the high dose fluoride group was significantly lower than that of the control group. The protein expression levels of p53 in the middle and high dose fluoride group were significantly higher than in the control group and elevated with increasing fluoride concentration. The mRNA expression levels of p53 in the fluoride groups were markedly higher than in the control group. The mRNA expression level of p53 in the high dose fluoride group was however lower compared to the middle dose fluoride group, but similar to the low dose fluoride group. These finding suggest that fluoride can decrease the L-02 cells survival rate and induce protein and mRNA expressions of p53; however, there is no consistency between the protein expression level of p53 and the mRNA expression level. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Fluoride; Human embryo hepatocytes; p53 Protein; p53 mRNA

1. Introduction ∗

Corresponding author. Tel.: +86 27 83692715; fax: +86 27 83692701. ∗∗ Corresponding author. E-mail address: [email protected] (A.G. Wang). 1 Present address: Fresenius Biotech GmbH, Division of Cell Therapy, Bad Homburg 61352, Germany.

Fluorosis, which seriously impairs human health, is prevalent in some parts of central and western China. Epidemiological evidence and experimental results have shown that fluorosis does not only cause

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adverse biochemical effects on structure and function of skeletal and teeth, but also of non-skeletal systems such as brain, liver, renal, and spinal cord. More recent studies have suggested that the toxicity of excessive fluoride is relative to apoptosis and can induce a variety of apoptosis cell types (Anuradha et al., 2001; Chen et al., 2002; Refsnes et al., 2003; Tokunaga et al., 2003; Wang et al., 2004). Although apoptosis induced by fluoride has become an important mechanism of fluoride toxicity (Zhang and Wu, 2003), the real apoptosis mechanism induced by fluoride is still unknown. Furthermore, there are conflicting reports with regard to excessive fluoride causing cancer. Some animal experimental results indicated that excessive fluoride could cause cancer (Toft, 1989), which has also been supported by epidemiological investigations (Persing, 1989). The p53 protein, a tumor suppressor, possesses a potent transactivation domain which regulates the transcription of a number of genes. The activities of some of these genes are associated to cell proliferation, cell differentiation, and DNA repair systems. The p53 protein acts as a “molecular police” and a critical molecular of these functions (Hall et al., 1996; Lane, 1992). When cells are subjected to DNA damage, the p53 protein binds with DNA, RNA polymerase and then regulates the expression of related genes as a nuclear transcriptional factor in order to regulate the cell cycle and promote cell apoptosis to maintain the integrity of the cellular genome. Some animal experiments have shown that p53 regulates cell apoptosis induced by fluorosis (Jiang, 1999; Hu et al., 2002). Moreover, wild type p53 acts as a tumor suppressor protein, whereas mutant p53 could increase function properties such as immortalization of primary tumor cells. Change or absence of wild type p53 was associated with comprehensive human tumors (Hollstein et al., 1991; Levine et al., 1991). The p53 gene mutation has been found in approximately 40%–50% of different human cancer cells (Vogelstein and Kinzler, 1992). Ramesh et al. (2001) reported that p53 mutation was found in patients with osteosarcoma with a high bone fluoride level (Ramesh et al., 2001). Due to the various effects of p53 in cell apoptosis and p53 mutation in tumor cells, we investigated the effects of different doses of sodium fluoride on protein and mRNA expression levels in human L-02 cells in vitro.

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2. Materials and methods 2.1. General chemicals We obtained L-02 cells from China Type culture collection at the Wuhan University and Fetal bovine serum, HEPES buffer, and DMEM cell culture powder were obtained from GIBCO BRL (Paisley, Scoltland). Trypsin, DAB, biascrylamide, RNAase, and HRP-conjugated sheep anti-mouse antibodies were purchased from Sigma (Diesenhofen, Germany), and acrylamide as well as protein marker from Life Technologies GmbH (Karlsruhe, Germany). RevertAidTM First Strand cDNA Synthesis Kit, dNTP Mix, Taq enzyme, and p53 mouse anti-human monoclonal antibodies were supplied by a Beijing corporation (Beijing, China). Sodium fluoride and all other reagents were obtained from Shanghai chemical reagent corporation (Shanghai, China) unless indicated otherwise. 2.2. L-02 cell culture and NaF treatment L-02 cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 15 mM Hepes, and antibiotic supplement (penicillin 100 U/ml and streptomycin 0.1 mg/ml) at 37 ◦ C in a humidified incubator with 95% air and 5% CO2 . Exponentially growing cells were divided into four groups and treated with PBS (the control group) and sodium fluoride at 40, 80, and 160 ␮g/ml (low, middle, and high dose of fluoride, respectively). After 24 h of incubation, the cells were harvested for Western blot and RT-PCR analysis. 2.3. MTT assay To assess the effect on cellular viability of different fluoride concentrations, the MTT assay was applied. Exponentially growing cells in culture 50-ml flasks were detached using 0.25% trypsin, and seeded into 96-well culture plates. After 20 h incubation with 5% CO2 at 37 ◦ C, sodium fluoride was added as described above. Three wells were filled with each sodium fluoride concentration and another three wells with non-seeding cells to act as a blank. Two hundred microliters of DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum was added to

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Cells of each group were trypsinized and pelleted by centrifugation at 800 rpm/min for 3 min. Cell pellet were washed with PBS, and then dissolved in 0.5 ml cell lysis solution containing 10 mmol/l Na2 EDTA, 0.1 mol/l NaCl, 10 ␮l PMSF, 10 mmol/l Tris, 10 ␮l Aprotin (1 ␮g/ml). The protein concentration was measured with the Bio-Red DC Protein Assay Kit. The total protein levels of each group were evenly adjusted, an equal amount of 2× SDS loading buffer was added, and then denatured at 98 ◦ C for 10 min. Once the lysates had cooled off at room temperature, they were stored at −20 ◦ C for further analysis. According to the method of Sambrook (Hsieh et al., 1997), 20 ␮l cell lysate (corresponding to 20 ␮g) of each group were loaded into wells with 10% SDS-polyacrylamide gel. After electrophoresis and thereafter eletrophoretic transfer to nitrocellulose membranes, blot were blocked with 5% non-fat milk for 1 h at room temperature. Membranes were probed with the p53 mouse anti-human monoclonal antibodies at 37 ◦ C for 2 h, washed four times with PBS, and incubated with secondary hrp-conjugated sheep anti-mouse antibodies at 37 ◦ C for 1 h. After the final wash, substrate DAB was added. The relative amounts of p53 were calculated from the scanning profiles, and analyzed by Gel-Pro Analyzer version 3.0 software.

were incubated at 0 ◦ C for 5 min. After this, 0.2 ml chloroform was added, the tubes were shaken for 15 s, and put on ice for 2–3 min, then they were centrifuged at 12,000 × g for 15 min at 4 ◦ C. The colorless upper aqueous phase containing the RNA was transferred to a new EP tube without RNAase. An equal volume of isopropanol was added, and the RNA was precipitated by centrifugation. The RNA pellet was washed with 75% ethanol and recovered in water treated with diethylene pyrocarbonate (DEPC, 10–20 ␮l). RNA purity was tested by gel electrophoresis, showing an optical density ratio between 1.8 and 2.0. The RNA solution was conserved at −70 ◦ C for further analysis. Three micrograms of total RNA of each group were applied for RT-PCR. The primer pairs for human p53 were synthesized according to the gene sequence reported by Sambrook et al. (1993) (forward primer, 5 -CTG AGG TTG GCT CTG ACT GTA CCA CCA TCC-3 ; reverse primer, 5 -CTC ATT CAG CTC TCG GAA CAT CTC GAA GCG-3 ), giving an amplified product of 371 bp. ␤-Actin was used as an internal control in RT-PCR reaction with primers (forward, 5 -GGG TCA GAA GGA TTC CTA TG-3 ; reverse, 5 -GGT CTC AAA CAT GAT CTG GG-3 ), giving a PCR product of 237 bp. All of the steps for the reverse transcription and the subsequent amplification were performed in a single reaction tube according to the manufacturers protocol. The synthesis of first-strand cDNA was carried out at 70 ◦ C for 10 min. The PCR profile was 94 ◦ C for 45 s, 60 ◦ C for 45 s (␤-actin annealing at 52 ◦ C for 45 s), and 72 ◦ C for 2 min for 35 cycles, followed by 72 ◦ C for 7 min. After PCR, 2.5 ␮l of the reaction mix was analysed on 2% agarose gel with ethidium bromide (0.5 mg/ml). The level of p53 expression was measured by densitometric analysis and standardized by comparison to the ␤-actin control using a digital imaging and analysis system (Biocapt MV software).

2.5. RNA extraction and RT-PCR analysis

2.6. Statistical analysis

Cell samples were assayed for p53 mRNA expression via RT-PCR. Total RNA was extracted with Trizol reagent according to manufacturers instructions. The medium of each 50-ml flask was discarded, 1.0 ml Trizol was added and then placed in the EP tube without RNAase. To isolate the samples they

Statistical analysis results are represented as mean ± S.D. Statistical significance was assessed by ANOVA with subsequent Dunnett’s test using the SPSS (11.0) software on computer. A difference at P < 0.05 was considered statistically significant. In all experiments, assays were performed in triplicate.

all of them. After 24 h incubation, 20 ␮l of MTT (5 mg/ml) was added to all wells. After another 4 h, 150 ␮l of DMSO was added and shaken for 10 min. The optical density (OD) of each well was measured with a Benchmark at 490 nm. L-02 cell survival was calculated by the equation: LCS = (OD treated well − mean OD control wells/OD treated well) × 100%. 2.4. Western blot analysis

A.G. Wang et al. / Toxicology Letters 158 (2005) 158–163 Table 1 Effects of fluoride on human embryo hepatocytes Groups

Samples (n)

Cell viability (%)

Control (1× PBS) Low dose of fluoride (40 ␮g/ml) Middle dose of fluoride (80 ␮g/ml) High dose of fluoride (160 ␮g/ml)

3 3

100.00 ± 0.00 99.72 ± 1.93

3

95.28 ± 1.81

3

59.69 ± 1.18**

Exponentially growing L-02 cells were seeded into 96-well culture plates. Cells were treated with increasing concentration of fluoride (0, 40, 80, and 160 ␮g/ml) for 24 h, and cell viability was determined by MTT assay. Results are expressed as percentage of cell viability relative to the control value. Data are the mean ± S.D. of four independent experiments performed in triplicate. Compared to the control group significance difference: * P < 0.05, ** P<0.01.

3. Results 3.1. Effect of fluoride on L-02 cell viability Table 1 demonstrates that the survival rate of L-02 treated cells by high dose of fluoride was significantly lower than in the control group (P < 0.01), although there was no statistical difference of cell viability between the low dose and middle dose compared with the control group (P > 0.05). 3.2. Effects of fluoride on p53 protein expression level in L-02 cells Fig. 1 shows the protein expression level of p53 in L-02 cells, which is exposed to different doses of sodium fluoride. Results from densitometric analyses

Fig. 1. Effects of different fluoride concentration on the protein expression level of p53 in human embryo hepatocytes. Exponentially growing L-02 cells were incubated with increasing concentration of fluoride (0, 40, 80, and 160 ␮g/ml) for 24 h. Cell extract of each concentration was prepared. 20 ␮l supernatant was loaded onto each lane for 10% SDS-polyacrylamide gel electrophoresis, followed by Western blot analysis with p53 mouse anti-human monoclonal antibody as described in Section 2. Results of one representative Western blot showed cells not exposed to fluoride (lanes 1 and 2); cells in lanes 3 and 4 were exposed to 40 mg/l fluoride; cells in lanes 5 and 6 cells were exposed to 80 mg/l fluoride; and cells in lanes 7 and 8 were exposed to 160 ␮g/ml fluoride.

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Table 2 Determination of protein expression levels on p53 treated with different doses of fluoride Groups

Samples (n)

Densitometric value

Control (1× PBS) Low dose of fluoride (40 ␮g/ml) Middle dose of fluoride (80 ␮g/ml) High dose of fluoride (160 ␮g/ml)

3 3

0.165 ± 0.024 0.242 ± 0.020

3

0.836 ± 0.325**

3

2.102 ± 0.298**

The results of Western blot were carried out as described in Fig. 1. The blots were scanned and the band intensity numbers were immediately read in a gel-pro Analyzer (3.0 version). The results were expressed as densitometric values. Data are the mean ± S.D. of triplicate experiments. In contrast to the control group, significance difference: * P < 0.05, ** P < 0.01.

of the intensity of the various bands are shown in Table 2. As shown in Fig. 1 and Table 2, the protein expression levels of p53 in the middle and high dose fluoride group were obviously higher than those in the control group (P < 0.01) and were elevated with increasing fluoride concentration. The protein expression level of p53, treated with a low dose of fluoride, was higher than that of the control group; however, the difference was not statistically significant (P > 0.05). 3.3. Effects of fluoride on p53 mRNA expression level in L-02 cells Results from densitometric analyses on the intensity of the various bands (Fig. 2) are shown in Table 3. As Table 3 Determination of mRNA expression levels on p53 exposed to different doses of fluoride Groups

Samples (n)

Ratio of densitometric value (p53/␤-actin)

Control (1× PBS) Low dose of fluoride (40 ␮g/ml) Medium dose of fluoride (80 ␮g/ml) High dose of fluoride (160 ␮g/ml)

5 5

1.204 ± 0.141 1.384 ± 0.127**

5

1.555 ± 0.107**

5

1.409 ± 0.092**

The results of RT-PCR are shown in Fig. 2 below. Bands were measured by densitometric analysis and standardized by comparison to the ␤-actin control using the Biocapt MV software analysis system. Data are the mean ± S.D. of triplicate experiments. In contrast to the control group, significance difference: * P<0.05, ** P<0.01.

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Fig. 2. Effects of different fluoride concentration on the mRNA expression level of p53 in human embryo hepatocytes by RT-PCR. The RNA extracted from L-02 cells was incubated with fluoride at increasing concentrations (40, 80, and 160 ␮g/ml) for 24 h, and 3.0 ␮g of each group was subjected to RT-PCR. An amplified band of 371 bp was obtained by using primers of p53. As a control, we also amplified 237 bp of ␤-actin gene. Results of one representative RT-PCR show: lane 1, marker; lane 2, fluoride free; lane 3, 40 ␮g/ml fluoride; lane 4, 80 ␮g/ml fluoride; lane 5, 160 ␮g/ml fluoride; lane 6, negative control.

shown in Fig. 2 and Table 3, the mRNA expression levels of p53 in L-02 cells, which were exposed to different doses of sodium fluoride, were markedly higher than those in the control group. In the low and the middle dose group, the mRNA expression levels of p53 were increased with a dose of fluoride. In contrast, the mRNA expression level of p53 in the high dose fluoride group was significantly higher than that in the control group (P < 0.01); compared however to the middle dose fluoride group, it seemed to decrease. The mRNA expression level of p53 in the high dose fluoride group was similar to the low dose fluoride group.

4. Discussion More recent studies have found that p53 can mediate various gene expressions such as p21, c-myc, bcl-2, TGF-␤, IL-2, fas, and bax, and that it also plays an important role in cell proliferation and differentiation (Jiang, 1999). Furthermore, p53 acts as a key-regulating molecule during cell stress, which could cause different responses such as suppressing cell growth or apoptosis to cell emergency signals by transcription or non-transcription (Hall et al., 1996). When cells were subjected to DNA damage,

the p53 protein accumulated at the nuclear level showed G1/S and G2/M arrests, and discontinuation of cell proliferation. If this DNA damage was not repaired, p53 would activate genes inducing apoptosis transcription to cause cells entering into apoptosis. Our study shows that excessive fluoride not only decreases cell viability, but also induces cell damage and increases the expression of p53 on transcription and translation levels. A dose–effect relationship exists between the concentration of fluoride and the protein expression level. When L-02 cells were exposed to lower dose fluoride, the tendency of mutation of p53 was similar in both the mRNA and the protein expression levels. When cells were exposed to high dose fluoride (160 ␮g/ml), the mRNA expression level of p53 basically remained the same as in the low dose fluoride group (40 ␮g/ml); in the middle dose group (80 ␮g/ml), it however decreased, and compared to the control group it showed a significant difference. Normal p53 protein acts as a “molecular police” and monitors integration of cell genomes (Lane, 1992). Current studies have demonstrated that fluoride caused DNA damage (Wang et al., 2004; Hirano and Ando, 1997). When cells were exposed to low dose fluoride, could cause increasing transcription and translation levels of p53, which regulate cell cycle, repair damaged DNA, and maintain genome integrity (Fu et al., 2003). Furthermore, when mass damaged DNA binds with p53 protein, it influences the p53 protein conformation, covalence modification or binding with other proteins; thus, decreasing the degradation of p53 protein (Reed et al., 1995). This process indirectly results in the accumulation of p53 protein; it shows that p53 increases after DNA damage (Kastan et al., 1991). When stimulation of fluoride is further enhanced (up to 160 ␮g/ml fluoride), the accumulation of p53 protein in cells restricts the p53 mRNA expression (Fu et al., 2003). Cole et al. (1986) reported that L5178Y cells exposed to a physiological dose of sodium fluoride (1 mg/l), did not induce cells mutation. However, when cells were exposed to 100–150 mg/l for 16 h or 10–50 mg/l for 48 h, it induced the mutation of the TK site of L5178Y cells and showed a dose-depending effect. This suggests that a high concentration of fluoride could induce p53 gene mutation. Although mutation of p53 did not increase the mRNA expression level of p53, it could however result in a low p53 expression level (Milner and Medcalf, 1991; Haupt et al., 1997).

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In addition, we found that the half-life of mutant p53 protein was longer than wild p53 protein; thus, p53 degenerated slower (Vine et al., 1991). Our data indicates that excessive fluoride can induce p53 mRNA and protein expressions; however, the protein expression level of p53 was not consistent with the mRNA expression level of p53 in the high dose fluoride group. This finding indicates that excessive fluoride may cause p53 gene mutated; however, this still requires further investigations.

Acknowledgements The authors would like to thank Ms. S. Albrecht very much for the edition of the manuscript into proper English. The work was supported by grants from the National Nature Science Foundation of China (Nos. 30271155, and 30371250), and the China national key basic research and development program (No. 2002CB512908).

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