Cell Biology and Toxicology. 2006; 22: 39–46. DOI: 10.1007/s10565-006-0018-z
C Springer 2006
Effects of hydrogen peroxide (H2 O2 ) on alkaline phosphatase activity and matrix mineralization of odontoblast and osteoblast cell lines D.H. Lee, B.-S. Lim, Y.-K. Lee and H.-C. Yang Department of Dental Biomaterials Science and Dental Research Institute, College of Dentistry, Seoul National University, Seoul, Korea Received 9 June 2005; accepted 10 October 2005
Keywords: hydrogen peroxide (H2 O2 ), MDPC-23, MC3T3-E1, alkaline phosphatase (ALP), extracellular mineralization Abstract Hydrogen peroxide (H2 O2 ), an oxidizing agent, has been widely used as a disinfectant. Recently, because of its reactive properties, H2 O2 has also been used as a tooth bleaching agent in dental care. This is a cause for concern because of adverse biological effects on the soft and hard tissues of the oral environment. To investigate the influence of H2 O2 on odontoblasts, the cells producing dentin in the pulp, we assessed cellular viability, generation of reactive oxygen species (ROS), alkaline phosphatase (ALP) activity, and nodule formation of an odontoblastic cell line (MDPC-23) after treatment with H2 O2 , and compared those with the effects on preosteoblastic MC3T3-E1 cells. Cytotoxic effects of H2 O2 began to appear at 0.3 mmol/L in both MDPC-23 and MC3T3-E1 cells. At that concentration, the accumulation of intracellular ROS was confirmed by a fluorescent probe, DCFH-DA. Although more ROS were detected in MDPC-23, the increasing pattern and rate are similar between the two cells. When the cells were treated with H2 O2 at concentrations below 0.3 mmol/L, MDPC-23 displayed a significant increase in ALP activity and mineralized bone matrix, while MC3T3-E1 cells showed adverse effects of H2 O2 . It is known that ROS are generally harmful by-products of aerobic life and represent the primary cause of aging and numerous diseases. These data, however, suggest that ROS can induce in vitro cell differentiation, and that they play a more complex role in cell physiology than simply causing oxidative damage. Abbreviations: ROS, reactive oxygen species; ALP, alkaline phosphatase; MTT, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide; DCFH-DA, 2 ,7 -dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescin Introduction Oxidative stress resulting in an increase of intracellular reactive oxygen species (ROS) has been implicated in various pathological processes, including aging, cancer, diabetes mellitus, atherosclerosis, and neurological degeneration
(Floyd, 1990; Ames and Shigenaga, 1992; Jenner 1994; Dandona et al., 1996). ROS such as hydrogen peroxide (H2 O2 ), hydroxyl radicals, and superoxide are known for their deleterious effects on various cellular components, including DNA, protein, and lipids. At cellular levels, ROS can cause the reduction of proliferation, apoptosis, cell cycle
40 arrest, and modulation of differentiation (Slater et al., 1995; Allen and Tresini, 2000; Shackelford et al., 2000). The effects of oxidative stress on cell differentiation vary according to the different types of cells. The osteoblastic differentiation of MC3TC-E1, a preosteoblastic cell line, was inhibited by H2 O2 . The stimulation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and nuclear factor-κB appeared to be involved (Bai et al., 2004). A recent study showed an ROS-stimulated receptor activator on the NF-κB ligand (RANKL) in osteoblasts, a critical osteoclastogenic factor, which suggests a new role of osteoblasts in oxidative stress-induced bone loss (Bai et al., 2005). In contrast to bone osteoblastic cells, H2 O2 enhanced the differentiation of osteoblastic calcifying vascular cells (CVC) (Mody et al., 2001). Stimulation of differentiation by oxidative stress was also seen in other cell types such as osteoclasts and hepatoma cells (Garrett et al., 1990; Ren et al., 1998), although the molecular mechanism underlining enhancement of differentiation is not yet fully understood. The use of H2 O2 as a tooth bleaching agent has increased, as aesthetic dental care attracts more public attention. H2 O2 (up to 35%) can be applied directly to the tooth surface, and carbamide peroxide can be also used to produce H2 O2 in water (Haywood, 1991). The major side-effect of the bleaching procedure is tooth sensitivity caused by reversible histological damage to the pulp, providing indirect evidence for penetration of H2 O2 into pulp chambers (Dahl and Pallesen, 2003). Previous in vitro studies have also shown that H2 O2 or carbamide peroxide could penetrate into pulp chambers through enamel and dentin (Benetti et al., 2004; G¨okay et al., 2004; Sulieman et al., 2005). Thus, pulp cells and odontoblasts that reside in pulp chambers might be affected by the penetrating oxidizing reagent, leading to undesired side-effects. Odontoblasts, highly differentiated postmitotic cells, are able to produce dentin, composed of hydroxyapatite crystals and an organic matrix containing fibrous collagens, proteoglycans,
phosphoproteins, and phospholipids (Goldberg and Septier, 2002; Arana-Chavez and Massa, 2004). When odontoblasts and the dentin matrix are damaged or removed by caries or surgical dental procedures, there are two critical steps involved in re-forming dentin: differentiation of pulp cells to secondary odontoblasts and secretion of reparative dentin by the newly formed odontoblasts. The ability of odontoblasts to produce dentin can be assessed in vitro by alkaline phosphatase (ALP) activity and nodule formation, which is frequently used for the evaluation of osteoblastic differentiation. To explore the biological aspect of tooth bleaching, we evaluated the effects of H2 O2 on differentiation markers of an odontoblast-like cell line (MDPC-23) and compared them with the effects on MC3T3-E1 cells. The MDPC-23 immortalized cell line, originates from mouse fetal molar dental papillae and is found to express several odontoblast-specific proteins, such as dentin phosphoprotein and dentin sialoprotein (Hanks et al., 1998). We employed ALP and nodule formation ability as differentiation markers to investigate the effects of oxidative stress, which was assessed by a fluorescent probe after treatment with H2 O2 . Materials and methods Materials Cell culture medium and reagents were purchased from Gibco-BRL (Grand Island, NY, USA). All reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise noted. MC3T3-E1 cells were obtained from the RIKEN Cell Bank (Ibaraki, Japan). Cell cultures Cells of the odontoblast cell line MDPC-23 were maintained in Dulbecco’s modified Eagle’s medium (DMEM), containing 4.5 g/L glucose, 10% fetal bovine serum (FBS), and supplemented
41 with an antibiotic solution (100 U/ml penicillin-G and 100 μg/ml streptomycin) at 37◦ C in a humidified atmosphere (5% CO2 /95% air). The MC3T3E1 mouse preosteoblast cell line was grown in Eagle’s alpha minimum essential medium (α-MEM) containing 10% FBS, and supplemented as indicated above for DMEM.
was added and mixed thoroughly. The optical densities of formazan production were measured at 570 nm and 630 nm. The data represent at least three experiments, and are shown as the mean ± SD of quadruplicate wells.
Alkaline phosphatase activity assay
To determine the degree of mineralization in MDPC-23 and MC3T3-E1 cultures, they were stained with Alizarin red S. Cells were plated in 96-well plates in complete medium. At confluency, osteogenic differentiation was initiated using the complete medium supplemented with 10 mmol/L β-glycerophosphate and 50 μg/ml ascorbate. The medium was changed every 2 days up to 2 weeks. To demonstrate calcified nodules, cell cultures at day 14 were washed twice with PBS, fixed with 50% ethanol for 10 min, rehydrated with 1 ml of distilled water for 5 min, then stained with 200 μl 1% Alizarin red S (pH 4.0) for 3 min at room temperature. After staining, cultures were washed three times with distilled water, followed by 70% ethanol. To quantify matrix mineralization, the Alizarin red S-stained cultures were incubated with 100 mmol/L cetylpyridinium chloride for 1 h to solubilize and release calciumbound Alizarin red into the solution (Johnson et al., 2001). The absorbance of the released Aalizarin red S was measured at 570 nm using a microplate reader (Model 550, Bio-Rad). Data are expressed as units of Alizarin red S released per milligram of protein in each culture. The data represent at least three experiments, and are shown as the mean ± SD of quadruplicate wells.
The measurements of ALP activity and protein content were performed as described previously (Kirsch et al., 1997). MDPC-23 odontoblasts and MC3T3-E1 osteoblasts, seeded in 96-well plates, were treated at 70% confluence with H2 O2 in media containing 0.5% FBS. Cells were lysed by sonication on 0.05% Triton X-100 in phosphatebuffered saline (PBS) for 60 s at room temperature. Total cellular ALP activity in the lysates was measured in 2-amino-2-methyl-1-propanol buffer, pH 10.3, with p-nitrophenyl phosphate as a substrate at 37◦ C. Reactions were terminated by the addition of 0.5 mol/L NaOH. Absorbance of the reaction was measured at 405 nm using a microplate reader (Model 550, Bio-Rad, Hercules, CA, USA). Total protein levels in the lysates were measured according to Bradford (1976) using the bovine serum albumin as a standard. ALP activity was expressed as nanomoles of p-nitrophenol liberated per microgram of total cellular protein. The data represent at least three experiments, and are shown as the mean ± SD of quadruplicate wells. Cell viability The cytotoxicity of H2 O2 was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates and treated in the same manner as the ALP activity assay. At the end of the incubation period, cells were washed with PBS, then 0.2 ml of medium and 20 μl of MTT solution (5 mg of MTT/ml PBS) were added to each well. After 4 h of incubation, 100 μl of acidisopropanol (0.04 mol/L HCl in isopropanol)
Alizarin red S staining
Oxidative stress measurements Production of ROS in cultured cells was quantified using the cell-permeant fluorescence probe 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA), as previously described (LeBel and Bondy, 1990). A stock solution of DCFH-DA (20 mmol/L) was stored at −20◦ C in DMSO. The probe was extemporaneously diluted in Hanks
42
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balanced salt solution (HBSS) with sodium bicarbonate and 5.5 mmol/L glucose. MDPC-23 and MC3T3-E1 cells were incubated in a 96-well microplate with 20 μmol/L DCFH-DA for 20 min at 37◦ C, washed three times with PBS, then treated in the presence or in the absence (control) of H2 O2 at 37◦ C. DCF dichlorofluorescin fluorescence was determined using a FLUOstar Optima microplate spectrofluorometer (BMG Labtechnologies, Offenburg, Germany) with excitation at 488 nm and emission at 530 nm. For H2 O2 , DCF fluorescence was measured over the course of 3 h. DCF fluorescence measurements were normalized to the cell number determined by the MTT assay. The number of cells per well of control and treated cells did not vary for up to 3 h of cell growth in the presence of H2 O2 .
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Statistical analysis of the data was performed by ttests or a one-way analysis of variance (ANOVA) followed by a multiple-comparison Tukey’s test, with the use of SigmaStat 2.0 software (SPSS, Chicago, IL, USA). Results ROS production in response to H2 O2 -induced oxidative stress in odontoblasts and osteoblasts Cellular responses elicited by H2 O2 depend upon the severity of the damage, which is further influenced by cell type, magnitude of the dose, and exposure (Martindale and Holbrook, 2002). In our experiments, MDPC-23 and MC3T3-E1 cells underwent severe cell death after high-dose H2 O2 (0.5 mmol/L) treatment for 1, 4, or 15 days as determined by the MTT assay. For low doses (0.1 or 0.2 mmol/L) of H2 O2 , the cell viability of MDPC23 and MC3T3-E1 was not significantly affected, as compared to the controls over the course of 14 days (Figure 1). Intracellular ROS production by 0.3 mmol/L H2 O2 was measured by DCF fluorescence in
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Figure 1. Cell viability of MC3T3-E1 (A) and MDPC-23 cells (B) treated with H2 O2 . Cells were treated in the absence (control) or presence of various concentrations of H2 O2 for 1, 4, or 15 days, and the viability was expressed as a percentage of the control cells (MTT assay). ∗ p < 0.05, ∗∗ p < 0.005; significant differences compared to controls. Data are the means ± SD of three independent experiments.
MDPC-23 and MC3T3-E1 over the course of 3 h (Figure 2). In response to H2 O2 , both cell types showed a similar pattern of fluorescence increase: a fast increase in the early phase before 30 min and a relatively slow increase after that. After 3 h of exposure to H2 O2 , DCF fluorescence increased 7.6 ± 0.2-fold and 18.8 ± 2.9-fold in MDPC-23 and in MC3T3-E1, respectively. Because we employed an indirect method to measure intracellular ROS with DCF, it is not possible to compare the absolute amounts of ROS between the different cell types. However, the rate of increase in fluorescence intensity after treatment suggests that MC3T3-E1 cells respond more sensitively to H2 O2 than do MDPC-23 cells.
43 A
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Figure 2. Increased accumulation of ROS in MC3T3-E1 (A) and MDPC-23 (B), stimulated by H2 O2 . MC3T3-E1 or MDPC-23 cells were cultured in 96-well plates and loaded with 20 μmol/L DCFH-DA for 20 min. The cells were then treated with 0.3 mmol/L H2 O2 . DCF fluorescence was monitored over the course of 3 h and normalized to cell number. Data are the means ± SD of three independent experiments.
ALP activity of odontoblasts and osteoblasts in response to oxidative stress To determine whether H2 O2 -induced oxidative stress has any considerable influence on the differentiation of odontoblasts and osteoblasts, we first tested the effects of H2 O2 on MDPC-23 and MC3T3-E1 cultures via ALP activity. Treatment of MC3T3-E1 cells with H2 O2 inhibited ALP activity dose-dependently with a maximal effect at 0.3 mmol/L. Exposure of MC3T3-E1 cells to H2 O2 (0.2 or 0.3 mmol/L) for 4 days significantly ( p < 0.01) decreased ALP activity compared to the control (Figure 3A). In contrast to the results
Figure 3. Effects of H2 O2 on ALP activity of MC3T3-E1 (A) and MDPC-23 (B). The cells were treated on alternate days for 4 days with the indicated concentrations of H2 O2 . ALP activity was measured from whole-cell lysates as described in Materials and Methods. ∗ p < 0.01, ∗∗ p < 0.005; significant differences compared to controls. Data are the means ± SD of three independent experiments.
with MC3T3-E1, 0.1 and 0.2 mmol/L of H2 O2 significantly induced the expression of ALP activity in MDPC-23 cultures (Figure 3B). These results suggest that low doses of H2 O2 suppress osteoblastic differentiation of MC3T3-E1 cells, whereas they induce differentiation of MDPC-23 odontoblasts. Mineralization in odontoblast and osteoblast cultures in response to oxidative stress An important criterion for the characterization of osteoblastic and odontoblastic cells is their ability to mineralize the collagenous matrix they secrete.
44 The extent of cellular differentiation of MC3T3E1 and MDPC-23 cells treated with H2 O2 was assessed by in vitro mineralization of the extracellular matrix in the presence of L-ascorbic acid and β-glycerophosphate. In both of the cell cultures tested, detectable amounts of mineralization of the extracellular matrix, judged by Alizarin red S staining, were produced (Figure 4). Consistent with the result of the ALP assay, MDPC-23 cultures treated with H2 O2 also showed increased numbers of nodules as indicated by the intense
Alizarin red S staining (Figure 4B). MDPC-23 cells treated in the presence of 0.2 and 0.3 mmol/L H2 O2 for 14 days exhibited a significant increase ( p < 0.005) in the mineralization of the extracellular matrix compared to the control (Figure 4B). In contrast, less Alizarin red S was detected in H2 O2 -treated MC3T3-E1 cultures (Figure 4A). MC3T3-E1 cells exhibited a slight decrease in Alizarin red S staining by treatment of H2 O2 at low doses (0.05 or 0.1 mmol/L), though this was not significantly different from the nontreated controls (Figure 4A).
Discussion
Figure 4. Extent of matrix mineralization in MC3T3-E1 (A) and MDPC-23 (B) cultures treated with H2 O2 . Each cell was cultured for 15 days with the complete medium (supplemented with 10 mmol/L β-glycerophosphate and 50 μg/ml ascorbate) in the absence or presence of H2 O2 . The cells were stained with Alizarin red S as described in Materials and Methods. Calcified nodules that appeared bright red were identified by light microscopy. To quantitate the Alizarin red S stain, each well was incubated with 100 mmol/L cetylpyridium chloride for 1 h. The Alizarin red stain released into solution was collected, and read as units of Alizarin red released (1 unit is equivalent to 1 unit optical density at 570 nm) per mg of protein. ∗ p < 0.05; significant difference compared to controls. Data are the means ± SD of three independent experiments.
Over the past decade, reduction–oxidation (redox) reactions that generate ROS, including H2 O2 , O− 2 and OH− , have been identified as important chemical mediators in the regulation of signal transduction processes involved in cell growth and differentiation. The purpose of this study was to evaluate the biological effects of H2 O2 , a major component of tooth-bleaching materials, on an odontoblastlike cell line (MDPC-23) and preosteoblastic cell line (MC3T3-E1) through the analysis of a differentiation marker. Our results suggest that the oxidative stress of H2 O2 has contrasting effects on the differentiation of odontoblasts and osteoblasts. H2 O2 enhanced the differentiation of MDPC-23, whereas it inhibited osteogenic differentiation in MC3T3-E1, as seen by its effects on an early differentiation marker, ALP activity. Furthermore, H2 O2 increased mineral formation, a late marker of differentiation, in MDPC-23 while inhibiting mineralization in MC3T3-E1 cells. The inhibition of osteogenic differentiation of MC3T3-E1 cells by H2 O2 in this study is consistent with the effects on primary rabbit bone marrow stromal cells (BMSC) and calvarial osteoblast cultures, where a large reduction of differentiation markers including ALP, type I collagen, and colonyforming unit–osteoprogenitor (CFU-O) formation were observed after exposure to H2 O2 (Bai et al., 2004).
45 In the case of MDPC-23 cells, H2 O2 treatment enhanced odontoblastic differentiation as shown in Figure 3 and 4. Because of the similar cellular functions employed in producing hard tissues, it is surprising that the odontoblastic cells responded to H2 O2 differently from the preosteoblastic cell lines. The effects of H2 O2 on differentiation of MDPC-23 cells could be due to gene regulation, cytodifferentiation or matrix mineralization. With only the results of this study, it is not clear what step is affected by H2 O2 . However, increase of ALPase activity suggests that a relatively earlier phase of differentiation is affected. The accelerated mineralization of matrix shown by nodule formation could be a consequence of other earlier factors affected, which might be confirmed by application of H2 O2 at different times throughout the differentiation process. Because of the toxicological effects deriving from its powerful oxidative reactivity, use of H2 O2 as a tooth bleaching agent should be evaluated for its safety in the human body, especially the oral cavity. Differentiation of pulp cells and the ability of odontoblasts to produce dentins are important steps in the recovery of a damaged tooth from bacterial or surgical injury. The fact that H2 O2 can penetrate into the pulp chamber led us to explore the adverse effects of tooth bleaching agents on pulp regeneration. However, our results suggest a favorable effect of H2 O2 on odontoblasts, enhancing their ability to produce dentin. Since the present study was performed with an immortalized cell line, it needs to be confirmed with primary cultures or in vivo animal experiments in the future.
Acknowledgments This study was supported by a research grant to H.-C. Yang from the Ministry of Health and Welfare, Republic of Korea (03-PJ1-PG3-205000045). The authors thank Dr. Seog Bae Oh at Seoul National University for generously providing of
the odontoblast cell line MDPC-23. The authors also thank Ms. Na Ryoung Kim for technical assistance during the course of our experiments.
References Allen RG, Tresini M. Oxidative stress and gene regulation. Free Rad Biol Med. 2000;28:463–99. Ames BN, Shigenaga MK. Oxidants are a major contributor to aging. Ann NY Acad Sci. 1992;663:85–96. Arana-Chavez VE, Massa LF. Odontoblast: the cells forming and maintaining dentin. Int J Biochem Cell Biol. 2004;36:1367– 73. Bai XC, Lu D, Bai J, et al. Oxidative stress inhibits osteoblastic differentiation on bone cells by ERK and NF-κB. Biochem Biophys Res Commun. 2004;314:197–207. Bai XC, Lu D, Liu AL, et al. Reactive oxygen species stimulates receptor activator of NF-κB ligand expression in osteoblast. J Biol Chem. 2005;280:17497–506. Benetti AR, Valera MC, Mancini MNG, et al. In vitro penetration of beaching agents into the pulp chamber. Int Endod J. 2004;37:120–4. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein– dye binding. Anal Biochem. 1976;72:248–54. Dahl JE, Pallesen U. Tooth bleaching—a crictical review of the biological aspects. Crit Rev Oral Biol Med. 2003;14:292–304. Dandona P, Thusu K, Cook S, et al. Oxidative damage to DNA in diabetes mellitus. Lancet. 1996;347:444–5. Floyd RA. Role of oxygen free radicals in carcinogenesis and brain ischemia. FASEB J. 1990;4:2587–97. Garrett IR, Boyce BF, Oreffo ROC, et al. Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro and in vivo. J Clin Invest. 1990;85:632–9. G¨okay O, M¨ujdeci A, Algın E. Peroxide penetration into the pulp from whitening strips. J Endod. 2004;30:887–9. Goldberg M, Septier D. Phospholipids in amelogenesis and dentinogenesis. Crit Rev Oral Biol Med. 2002;13:276–90. Hanks CT, Sun ZL, Fang DN, et al. Cloned 3T6 cell line from CD-1 mouse fetal molar dental papillae. Connect Tissue Res. 1998;37:233–49. Haywood VB. Nightguard vital bleaching, a history and product update. Part I. Esthet Dent Update. 1991;2:63–6. Jenner P. Oxidative damage in neurodegenerative disease. Lancet. 1994;344:796–8. Johnson K, Hashimoto S, Lotz M, et al. Up-regulated expression of the phosphodiesterase nucleotide pyrophosphatase family member PC-1 is a marker and pathogenic factor for knee meniscal cartilage matrix calcification. Arthritis Rheum.2001;44:1071–81. Kirsch T, Nah HD, Shapiro IM, Pacifici M. Regulated production of mineralization-competent matrix vesicles in hypertrophic chondrocytes. J Cell Biol.1997;137:1149–60. LeBel CP, Bondy SC. Sensitive and rapid quantification of oxygen reactive species formation in rat synaptosomes. Neurochem Int. 1990;17:435–41.
46 Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol. 2002;192:1– 15. Mody N, Parhami F, Sarafian TA, et al. Oxidative stress modulates osteoblastic differentiation of vascular and bone cells. Free Rad Biol Med. 2001;31:509–19. Ren J-G, Zheng R-L, Shi Y-M, et al. Apoptosis, redifferentiation and arresting proliferation simultaneously triggered by oxidative stress in human hepatoma cell. Cell Biol Int. 1998;22:41–9. Shackelford RE, Kaufmann WK, Paules RS. Oxidative stress and cell cycle checkpoint function. Free Rad Biol Med. 2000;28:1387– 404.
Slater AF, Stefan C, Nobel I, et al. Signalling mechanisms and oxidative stress in apoptosis. Toxicol Lett. 1995;82:149–53. Sulieman M, Addy M, Macdonald E, et al., The bleaching depth of a 35% hydrogen peroxide based in-office product: a study in vitro. J Dent. 2005;33:33–40.
Address for correspondence: Hyeong-Cheol Yang, Department of Dental Biomaterials Science, College of Dentistry, Seoul National University, 28 Yeonkun-dong, Chongro-ku, Seoul 110-749, Korea. E-mail:
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