BioMed Research International

Volume 2014 (2014), Article ID 895049, 5 pages

http://dx.doi.org/10.1155/2014/895049

Research Article

Protective Effect of Alpha-Tocopherol Isomer from Vitamin E against the H₂O₂ Induced Toxicity on Dental Pulp Cells

Fernanda da Silveira Vargas,¹ Diana Gabriela Soares,¹ Ana Paula Dias Ribeiro,² Josimeri Hebling,³ and Carlos Alberto De Souza Costa⁴

¹Oral Rehabilitation Program, Department of Dental Materials and Prosthodontics, University Estadual Paulista, UNESP, Araraquara School of Dentistry, Humaitá Street 1680, 14801-903 Araraquara, SP, Brazil

²Department of Operative Dentistry, Brasilia University, School of Dentistry, Darcy Ribeiro Center, 70910-900 Brasília, DF, Brazil

³Department of Orthodontics and Pediatric Dentistry, University Estadual Paulista, UNESP, Araraquara School of Dentistry, Humaitá Street 1680, 14801-903 Araraquara, SP, Brazil

⁴Department of Physiology and Pathology, University Estadual Paulista, UNESP, Araraquara School of Dentistry, Humaitá Street 1680, 14801-903 Araraquara, SP, Brazil

Received 30 April 2013; Accepted 28 October 2013; Published 21 January 2014

Academic Editor: Maha Zaki Rizk

Copyright © 2014 Fernanda da Silveira Vargas et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The aim of this study was to evaluate the protective effects of different concentrations of vitamin E alpha-tocopherol (α-T) isomer against the toxicity of hydrogen peroxide (H₂O₂) on dental pulp cells. The cells (MDPC-23) were seeded in 96-well plates for 72 hours, followed by treatment with 1, 3, 5, or 10 mM α-T for 60 minutes. They were then exposed or not to H₂O₂ for 30 minutes. In positive and negative control groups, the cells were exposed to culture medium with or without H₂O₂ (0.018%), respectively. Cell viability was evaluated by MTT assay (Kruskal-Wallis and Mann-Whitney tests; %). Significant reduction of cell viability (58.5%) was observed in positive control compared with the negative control. Cells pretreated with α-T at 1, 3, 5, and 10 mM concentrations and exposed to H₂O₂ had their viability decreased by 43%, 32%, 25%, and 27.5%, respectively. These values were significantly lower than those observed in the positive control, thereby showing a protective effect of α-T against the H₂O₂ toxicity. Overall, the vitamin E α-T isomer protected the immortalized MDPC-23 pulp cells against the toxic effects of H₂O₂. The most effective cell protection was provided by 5 and 10 mM concentrations of α-T.

1. Introduction

Hydrogen peroxide (H₂O₂) is a thermally instable chemical agent with high oxidative power, which dissociates into free radicals and other reactive oxygen species (ROS), such as hydroxyl radicals (OH⁻), singlet oxygen (O²⁻), and superoxide anion () [1]. This molecule has been widely used in dentistry to treat discolored teeth, because of its capability to oxidize the complex organic molecules of the dental structure that respond for the darker coloration of the teeth [2]. However, these highly oxidative molecules can diffuse through mineralized tooth structures, such as enamel and dentin, to reach the subjacent pulp tissue, a specialized connective tissue responsible for maintaining the tooth viability [3, 4]. The contact of the pulp cells with ROS results in oxidative stress generation, mainly because of the imbalance between the amount of ROS and endogenous antioxidants [1]. This oxidative stress damages the cell membrane and causes cell viability reduction, extracellular matrix degradation, inflammatory tissue reaction, and even pulpal necrosis [3–5].

The treatment of dental pulp cells with antioxidants has been proposed in order to prevent the oxidative damage from components leached by dental materials and bleaching gels, which are capable of diffusing across mineralized tissues of teeth [6, 7]. Vitamin E (VE) has a recognized anti-inflammatory and antioxidant activity in different cell lineages, such as fibroblast, osteoblasts, and neurons [8]. This kind of vitamin is composed of a blend of tocopherols and tocotrienols; however, the antioxidant action of VE is mediated by the alpha-tocopherol (α-T) isomer [9]. The α-T is capable of stabilizing cell membrane against reactive oxygen species (ROS) produced during normal cellular metabolic activities, preventing the chain propagation from the oxidative stress [10]. The protective activity of this molecule against the oxidative damage related to different conditions, as atherosclerosis, diabetes, Alzheimer, and Parkinson diseases, has been widely described [8]. In view of this, it was hypothesized that the VE antioxidant property may also protect pulp cells against the oxidative toxic effects caused by components leached by dental bleaching gels. Therefore, the aim of this study was to evaluate the protective effects of different concentrations of VE α-T isomer against the toxicity of H₂O₂ applied on the immortalized odontoblast-like MDPC-23 cell line.

2. Materials and Methods

The H₂O₂ concentration capable of reducing the cell viability by approximately 50% (IC-50) was determined. For such purpose, solutions containing decreasing H₂O₂ concentrations were prepared (0.035%, 0.018%, 0.009%, and 0.045%) in serum-free DMEM (Dulbecco’s Modified Eagle’s Medium; Sigma Aldrich Corp., St. Louis, MO, USA). Then, odontoblast-like MDPC-23 cells were seeded in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco Co., Grand Island, NY, USA) and antibiotics (IU/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L glutamine; Gibco Co.), in 96-well plates (1 × 10⁴ cells/well) (Costar Corp., Cambridge, MA, USA) during 72 h at 37°C and 5% CO₂. After that, the DMEM was aspirated and 100 μL of the H₂O₂ solutions were applied on the cells during 30 minutes. Cell viability was evaluated by the cytochemical demonstration of the succinic dehydrogenase (SDH) enzyme using the methyl tetrazolium (MTT) assay (Gibco Co.) [3, 4]. The absorbance values of the groups (570 nm) were transformed into percentages of cell viability, considering the negative control group (DMEM) as having 100% of cell viability. The 0.018% H₂O₂ concentration resulted in 59% of cell viability reduction and was selected to evaluate the α-T protective effect against H₂O₂ aggression.

In order to evaluate the protective effect of α-T against H₂O₂ toxicity, four decreasing concentrations of this molecule (1, 3, 5, and 10 mM) were prepared by diluting a stock α-T solution (Sigma Chemical Co.) in DMEM with 5% dimethyl sulfoxide (DMSO). In this way, experimental groups were formed according to the treatment of the MDPC-23 cells with different α-T concentrations followed by exposition or not of the cells to a 0.018% H₂O₂ solution for 30 minutes. To evaluate α-T toxicity (α-T+ H₂O₂−), the α-T solutions were applied on cultured cells for 60 minutes; to evaluate α-T protective effect against H₂O₂ aggression, the solutions were applied for 60 minutes and then aspirated, followed by H₂O₂ application for 30 minutes (α-T+ H₂O₂+). In negative control group, DMEM containing 5% DMSO was applied (α-T− H₂O₂−) on the MDPC-23 cells. In positive control group, 0.018% H₂O₂ was applied on the cultured cells for 30 minutes. After treatments, the MTT assay was performed and percentages of cell viability for each experimental group were determined. Data were subjected Kruskal-Wallis complemented by the Mann-Whitney test. The significance level was set at 5% and the following null hypotheses were established: (1) H₂O₂ does not cause toxic effects to odontoblast-like cells; (2) α-T cannot eliminate or at least reduce the oxidative effects of H₂O₂. Three independent experiments were performed at different times to demonstrate the reproducibility of data, and, in each appointment, a total of six replicates () were used for each group.

3. Results

Table 1 shows the results for the H₂O₂ IC-50. The experimental groups used to assess the protective role of α-T against cell toxicity mediated by H₂O₂ are summarized in Table 2. Cell viability data obtained after cell treatment with α-T followed or not by exposure to H₂O₂ are shown in Table 3. Considering the negative control group (G1) as having 100% of cell viability, there was a 58.5% decrease in the positive control group (G2) that was lower than that observed in the experimental groups (). The cell viability reduction in groups G3, G4, G5, and G6, in which the MDPC-23 cells were treated with different concentrations of α-T, was 6%, 13%, 10%, and 14%, respectively. Despite being considered discrete, the cell viability reduction for G4, G5, and G6 was significant when compared with the negative control group (G1, ). In groups G7, G8, G9, and G10, in which the cultured cells were pretreated with α-T before being exposed to H₂O₂, the cell viability reduction was 43%, 32%, 25%, and 27.5%, respectively. The protective effect against H₂O₂ cytotoxicity observed in G7, G8, G9, and G10 was significantly higher when compared to the positive control group (G2) regardless of the α-T concentration (). G8, G9, and G10 presented the highest values of cell viability recovery, with no significant difference among them () (Table 3, rows). G9 and G10, which did not show significant difference when the cells were treated or not with H₂O₂ () (Table 3, columns), presented the best results for cell viability recovery.

Table 1: Results of the viability of the MDPC-23 cells exposed to different hydrogen peroxide (H₂O₂) concentrations for determination of the IC-50.

H₂O2 concentration
Cell viability (%)

0
100

0.035%
5

0.018%
41

0.009%
77

0.0045%
72

Table 2: Control and experimental groups () formed according to the treatment of the MDPC-23 cells with different alpha-tocopherol (-T) concentrations followed by exposure or not to hydrogen peroxide (H₂O₂).

Groups
Treatment

G1
(-T− H₂O₂−)

G2
(-T−  H₂O₂+)

G3
(1 mM+ H₂O₂−)

G4
(3 mM+ H₂O₂−)

G5
(5 mM+ H₂O₂−)

G6
(10 mM+ H₂O₂−)

G7
(1 mM+ H₂O₂+)

G8
(3 mM+ H₂O₂+)

G9
(5 mM+ H₂O₂+)

G10
(10 mM+ H₂O₂+)

Table 3: Percentage of viability of MDPC-23 cells treated with different alpha-tocopherol concentrations followed by exposure or not to hydrogen peroxide.

H₂O₂
Alpha-tocopherol concentrations

0
1 mM
3 mM
5 mM
10 mM

0%
100.5 (97–104)a,A,G1
94 (87–100)ab,A,G3
87 (86–91)bc,A,G4
90 (79–92)bc,AB,G5
86 (77–88)c,AB,G6

0.018%
41.5 (36–43.5)a,B,G2
57 (52–60)b,B,G7
68 (64–73)bc,B,G8
75 (67–84)c,A,G9
72.5 (69–78)c,A,G10

Lowercase letters permit comparisons within rows while uppercase letters permit comparisons within columns. Groups identified with the same letters do not differ significantly (Mann-Whitney test, ).

Based on the fact that H₂O₂ caused toxic effects to the cultured odontoblast-like cells and that α-T reduced the oxidative effects of this unstable chemical agent to the immortalized pulp cell line, both null hypotheses presented in this study were rejected.

4. Discussion

In spite of being very popular in dental offices, vital tooth bleaching has been associated with postoperative sensitivity and pulpal damage [3–5]. In view of this, different therapies have been suggested to minimize these adverse effects, including pretreatment with antioxidant agents to reduce the oxidative stress generated by bleaching gel components to the pulp cells [7]. In the present study, the biological activity of VE α-T isomer against the toxic effects of H₂O₂ to MDPC-23 cells was evaluated. This specific kind of pulp cell, which presents odontoblast phenotype, was used in this study because in mammalian teeth odontoblasts are organized in a monolayer to underlie the dentinal tissue. Therefore, odontoblasts are the first pulp cells to be reached by components of dental products capable of diffusing through enamel and dentin [11]. In addition, for over a decade, this immortalized pulp cell line has widely been used to evaluate the cytotoxicity of different dental products and their isolated chemical components [3, 4, 7].

It was shown that all concentrations of α-T assessed in the present study presented cell-protective effect. The MDPC-23 cells pretreated with α-T for 60 minutes and exposed to the H₂O₂ showed higher viability compared with the group exposed only to H₂O₂ (G2, positive control). This protective effect of α-T against oxidizing agents was reported in previous investigations [9, 10]. A recent study demonstrated that the combination of vitamins E and C protected brain cells against the toxic effects induced by diazinon, a widely used pesticide in agriculture that causes brain oxidative stress [12]. Another in vivo study found that VE plus selenium acted as a potent antioxidant agent, reducing the oxidative stress in pregnant rats and preventing the development of gestational diabetes mellitus [13]. It is known that VE is composed of a mixture of tocopherols and tocotrienols [8], which can be distinguished from each other by the lateral chain unsaturation. It has been described that α-T is the compound responsible for great part of the VE antioxidant action [14]. According to previous studies, α-T is the predominant component of biomembranes, being effective in electron donation due to the orthoposition of its methyl group, compared with the other VE isomers [15]. Therefore, α-T can prevent oxidative stress propagation and stabilize the cell membrane, thus preventing the disruption of the amphipathic balance of this cell structure [14]. Antioxidants such as α-T can stop free radicals by donating one of their electrons to the free radical. However, α-T does not become a new free radical because it remains stable before and after donating the electron, which characterizes its antioxidant action [14]. It has also been shown that VE can prevent diseases such as atherosclerosis as well as cardiovascular and inflammatory disorders [16]. Some researchers have reported that VE is directly involved in the maintenance of the balance of oxidative reactions generated during the inflammation [17–20]. The authors showed that this kind of vitamin can block nitric oxide synthase (iNOS), COX-2 expression, and the NF-B signaling pathway in cultured monocytes stimulated by E. coli LPS. Additionally, VE was capable of inhibiting the synthesis of PGE2 and inflammatory cytokines, such as TNF-a, IL-4, and IL-8. Therefore, one can consider that VE has a broad therapeutic potential. The present investigation revealed that cells exposed only to H₂O₂ (G2) presented a 58.5% reduction in cell viability. The toxic effect of H₂O₂ was also reported in previous studies in which the authors evaluated the trans-enamel and trans-dentinal cytotoxicity of high concentrations of H₂O₂ on odontoblast-like cells [3, 4]. On the other hand, the treatment of MDPC-23 cells with different concentrations of α-T prior to their exposition to H₂O₂ increased the cell viability by 16–33.5%.

Despite the important protective effect, α-T alone caused a slight cell viability reduction in those groups in which the cells were not exposed to H₂O₂ (G3 to G6). It was shown that 1 mM α-T concentration was statistically similar to the control (G1). On the other hand, 3, 5, and 10 mM α-T concentrations were significantly different from G1. These data suggest that an increase of the α-T concentration available to the cells might cause a prooxidant action of this VE isomer, resulting in reduction in the viability of the treated cells. Some studies have demonstrated the prooxidant action of α-T at high concentrations or in the presence of heavy metals or peroxides [21–23]. These findings could explain the results observed in those groups in which the MDPC-23 cells were exposed only to α-T (G3 to G6). However, while a slight prooxidant action of α-T was observed (6–14% cell viability reduction), this molecule was capable of minimizing the oxidant effect caused by H₂O₂ on cultured MDPC-23 cells (G7 to G10). The most relevant protective effects were obtained with 5 mM (G9) and 10 mM (G10) α-T concentrations, in which 33.5 and 31% of cell viability recovery were observed, respectively. Since no significant difference was found between G9 and G10, it may be suggested that the best α-T concentration for pretreatment of odontoblast-like cells would be 5 mM. This is not only because of the protective effect of this molecule against the H₂O₂ cell damage but also due to its slight toxicity (G5–10% cell viability reduction).

Overall, this in vitro study demonstrated the potential of α-T as an antioxidant agent because this VE isomer was capable of protecting pulp cells against the harmful effects of H₂O₂, which is the main active component of tooth bleaching gels. Although the present laboratory-based results cannot be directly extrapolated to clinical situation, the original data obtained under the tested experimental conditions are promising.

5. Conclusion

It can be concluded that previous exposition of odontoblast-like MDPC-23 pulp cells to VE α-T isomer protects this cell line against the toxic effects generated by hydrogen peroxide in vitro. These data can drive further in vivo studies with the purpose of establishing specific therapies capable of preventing or at least minimizing the pulpal damage caused by tooth bleaching techniques widely used in dentistry. This may avoid the postbleaching tooth sensitivity, making this esthetic clinical procedure safer and more comfortable to the patients.

Conflict of Interests

The authors have no conflict of interests.

Acknowledgments

The authors acknowledge the partial support by the Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (Grant no. 301291/2010-1), Fundação Amparo à Pesquisa do Estado de São Paulo-FAPESP (Grant nos. 2011/15366-5 and 2011/12938-8) and FUNDUNESP (Grant no. 0024/021/13-PROPe-CDC).

References

V. Cecarini, J. Gee, E. Fioretti et al., “Protein oxidation and cellular homeostasis: emphasis on metabolism,” Biochimica et Biophysica Acta, vol. 1773, no. 2, pp. 93–104, 2007.

M. A. M. Sulieman, “An overview of tooth-bleaching techniques: chemistry, safety and efficacy,” Periodontology 2000, vol. 48, no. 1, pp. 148–169, 2008.

F. Z. Trindade, A. P. D. Ribeiro, N. T. Sacono et al., “Trans-enamel and trans-dentinal cytotoxic effects of a 35% H₂O₂ bleaching gel on cultured odontoblast cell lines after consecutive applications,” International Endodontic Journal, vol. 42, no. 6, pp. 516–524, 2009.

D. G. Soares, A. P. Ribeiro, F. da Silveira Vargas, J. Hebling, and C. A. de Souza Costa, “Efficacy and cytotoxicity of a bleaching gel after short application times on dental enamel,” Clinical Oral Investigations, vol. 17, no. 8, pp. 1901–1909, 2013.

C. A. de Souza Costa, H. Riehl, J. F. Kina, N. T. Sacono, and J. Hebling, “Human pulp responses to in-office tooth bleaching,” Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology, vol. 109, no. 4, pp. e59–e64, 2010.

E. S. Majd, M. Goldberg, and L. Stanislawski, “In vitro effects of ascorbate and Trolox on the biocompatibility of dental restorative materials,” Biomaterials, vol. 24, no. 1, pp. 3–9, 2003.

A. F. Lima, F. C. R. Lessa, M. N. Gasparoto Mancini, J. Hebling, C. A. de Souza Costa, and G. M. Marchi, “Transdentinal protective role of sodium ascorbate against the cytopathic effects of H₂O₂ released from bleaching agents,” Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology, vol. 109, no. 4, pp. e70–e76, 2010.

B. B. Aggarwal, C. Sundaram, S. Prasad, and R. Kannappan, “Tocotrienols, the vitamin E of the 21st century: its potential against cancer and other chronic diseases,” Biochemical Pharmacology, vol. 80, no. 11, pp. 1613–1631, 2010.

N. Lu, W. Chen, and Y. Peng, “Effects of glutathione, Trolox and desferrioxamine on hemoglobin-induced protein oxidative damage: anti-oxidant or pro-oxidant?” European Journal of Pharmacology, vol. 659, no. 2-3, pp. 95–101, 2011.

J. Upadhyay and K. Misra, “Towards the interaction mechanism of tocopherols and tocotrienols (vitamin E) with selected metabolizing enzymes,” Bioinformation, vol. 3, no. 8, pp. 326–331, 2009.

M. Goldberg and A. J. Smith, “Cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering,” Critical Review of Oral Biologics and Medicine, vol. 15, no. 1, pp. 13–12, 2004.

N. Yilmaz, M. Yilmaz, and I. Altuntas, “Diazinon-induced brain toxicity and protection by vitamins e plus C,” Toxicology and Industrial Health, vol. 28, no. 1, pp. 51–57, 2012.

M. Guney, E. Erdemoglu, and T. Mungan, “Selenium-vitamin E combination and melatonin modulates diabetes-induced blood oxidative damage and fetal outcomes in pregnant rats,” Biological Trace Element Research, vol. 143, no. 2, pp. 1091–1102, 2011.

X. Wang and P. J. Quinn, “Vitamin E and its function in membranes,” Progress in Lipid Research, vol. 38, no. 4, pp. 309–336, 1999.

R. S. Parker, “Dietary and biochemical aspects of vitamin E,” Advances in Food and Nutrition Research, vol. 33, pp. 157–232, 1989.

E. Niki, “Do free radicals play causal role in atherosclerosis? Low density lipoprotein oxidation and vitamin E revisited,” Journal of Clinical Biochemistry and Nutrition, vol. 48, no. 1, pp. 3–7, 2011.

E. S. Wintergerst, S. Maggini, and D. H. Hornig, “Contribution of selected vitamins and trace elements to immune function,” Annals of Nutrition and Metabolism, vol. 51, no. 4, pp. 301–323, 2007.

E. Reiter, Q. Jiang, and S. Christen, “Anti-inflammatory properties of α- and γ-tocopherol,” Molecular Aspects of Medicine, vol. 28, no. 5-6, pp. 668–691, 2007.

Q. Jiang, X. Yin, M. A. Lil, M. L. Danielson, H. Freiser, and J. Huang, “Long-chain carboxychromanols, metabolites of vitamin E, are potent inhibitors of cyclooxygenases,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 51, pp. 20464–20469, 2008.

D. Wu and S. N. Meydani, “Age-associated changes in immune and inflammatory responses: impact of vitamin E intervention,” Journal of Leukocyte Biology, vol. 84, no. 4, pp. 900–914, 2008.

V. W. Bowry, K. U. Ingold, and R. Stocker, “Vitamin E in human low-density lipoprotein. When and how this antioxidant becomes a pro-oxidant,” Biochemical Journal, vol. 288, no. 2, pp. 341–344, 1992.

M. Tokuda and M. Takeuchi, “Effects of excess doses of α-tocopherol on the lipids and function of rainbow trout liver,” Journal of Nutritional Science and Vitaminology, vol. 41, no. 1, pp. 25–32, 1995.

P. Pearson, S. A. Lewis, J. Britton, I. S. Young, and A. Fogarty, “The pro-oxidant activity of high-dose vitamin E supplements in vivo,” BioDrugs, vol. 20, no. 5, pp. 271–273, 2006.