Involvement of oxidative stress in mutagenicity and apoptosis caused by dental resin monomers in cell cultures
Introduction
Incomplete polymerization of dental resin composites and resin-based bonding agents under clinical conditions result in unreacted resin monomers that may be released from the resin matrix into the aqueous environment of oral cavity [1]. Triethyleneglycol dimethacrylate (TEGDMA), a major monomer of resin matrix, especially in dentin bonding agents (30–50%), was found to be a main leachable compound from the less polymerized resin, [2] which caused public concern for any adverse biological effects of the material.
Cytotoxicity of the resin monomers has been evaluated with permanent cell lines or a primary culture of fibroblasts from various origins such as pulp, gingival, and periodontal ligament [3], [4], [5], [6]. In general, the cytotoxicity of important resin monomers might be ranked in decreasing order as follows: bisphenol A-diglycidyl dimethacrylate (Bis-GMA), urethane dimethacrylate (UDMA), TEGDMA, 2-hydroxyehtyl methacrylate (HEMA), and methyl methacrylate (MMA). A previous study of the structure–toxicity relationship suggested the lipophilicity of substituents affected cytotoxicity of the acrylates and methacrylates [7]. Among those resin monomers, the underlying mechanism of the cytotoxic effect of TEGDMA has been relatively well studied because of its high potential for leaching from the resin matrix. It was reported that TEGDMA affected glutathione transferase P1 activity of human gingival fibroblasts [8] and induced extensive reduction of intracellular glutathione (GSH), a major intracellular reducing agent, at cytotoxic concentrations [9], [10]. Reactive oxygen species (ROS) were also found to generate after early depletion of GSH, which was prevented by antioxidants [11]. Therefore, it seems clear that oxidative stress is involved in the cytotoxicity of TEGDMA. HEMA, a less cytotoxic resin monomer, was also found to deplete GSH with simultaneous production of ROS [12], [13], although the intracellular target for cytotoxicity still remains to be elusive.
There have been several evidences showing genotoxicity of several resin monomers. An Ames test with Salmonella typhimurium revealed that glycidyl methacrylate (GMA) could cause mutagenicity through base-pair substitution and frame shift of the nucleotide [14]. An increase in micronuclei was also observed in V79 Chinese hamster lung fibroblasts after treatment with bisphenol A, GMA, TEGDMA, and HEMA at subtoxic concentrations [15]. Furthermore, TEGDMA was reported to induce large nucleotide sequence deletions in the hypoxanthine-guanidine phosphoribosyltransferase (HPRT) gene of the V79 cells [16]. In a comet assay with human lymphocytes, DNA migration was enhanced by various resin monomers including TEGDMA and HEMA [17]. In spite of those results, the mechanism of their genotoxicity has not been completely elucidated. Direct interaction between nucleotides and resin monomers, production of DNA damaging intermediates, or inhibition of DNA repair systems might be responsible for the mutagenicity of resin monomers.
Although oxidative stress cannot always be regarded as a mutagenic condition, the chemicals generating intracellular oxidative stress or ROS have been known as potential mutagens or carcinogens [18]. The protective effects of antioxidant enzymes and reagents against genotoxicity also indicate the involvement of ROS in oxidative DNA damages, resulting in mutagenicity [19]. The findings that showed oxidative stress was caused by TEGDMA and HEMA led the authors to consider a potential role of oxidative stress in the mutagenicity of the resin monomers. To test this possibility, the effects of NAC on the formation of micronuclei by TEGDMA and HEMA in V79-4 cells were evaluated. The reasons for selecting V79-4 cells in the mutagenicity tests of this study are stable karyotype, short cell cycle, high plate efficiency, and easiness to get results comparable to numerous previous data obtained with V79 cells. GMA as a test monomer was also chosen because the cytotoxicity of the monomer was relieved by addition of NAC in our preliminary study.
Recently, the induction of apoptosis by TEGDMA and BisGMA was demonstrated in human gingival fibroblasts [20], [21]. Both necrosis and apoptosis appeared in TEGDMA-treated primary human pulp cells [22]. In an attempt to reveal the mechanism underlining the apoptic cell death, it was found that apoptosis was enhanced by a suggested inhibitory effect of TEGDMA on phosphatidylinositol 3-kinase cells in primary human pulp cells [22]. Although ROS are able to initiate apoptosis, the activity of the ROS-related signaling molecule, MAP kinase ERK1/2, was not altered by TEGDMA [22]. Therefore, it is uncertain that glutathione depletion and oxidative stress are responsible for TEGDMA-induced apoptosis. The authors tested the possibility that apoptosis, as well as mutagenicity, is mediated by the oxidative stress induced by the resin monomers.
Section snippets
Chemicals
GMA, TEGDMA, and HEMA were purchased from Aldrich (Aldrich Chemical Company, Deisenhofen, Germany). An annexin V-FITC apoptosis detection kit was purchased from BD Biosciences (San Diego, CA, USA). Cell culture medium and reagents were from GIBCO-BRL (Grand Island, NY, USA). All reagents were purchased from Sigma (Sigma Chemical Company, St. Louis, MO, USA) unless otherwise noted.
Cell cultures
V79-4 Chinese hamster lung fibroblasts (American Type Culture Collection, Rockville, MD, USA) and rat clonal dental
Resin monomer-induced cytotoxicities and protective effects of NAC
V79-4 fibroblasts and RPC-C2A pulp cells were exposed to increasing concentrations of resin monomers, and the cytotoxic activities were analyzed by an MTT. Cytotoxic effects of three resin monomers on V79-4 and RPC-C2A cells are shown in Fig. 1, Fig. 2. All monomers exhibited a dose-dependent effect, and the ranking of those cytotoxicities indicated by TC50 (mM) was GMA (V79-4, 0.49; RPC-C2A, 0.31) > TEGDMA (V79-4, 2.0; RPC-C2A, 3.92) > HEMA (V79-4, 5.8; RPC-C2A, 13.46). Considerable differences in
Discussion
Apoptosis, as well as mutagenicity, of dental resin monomers may be mediated by oxidative stress. The study showed that resin monomers (GMA, TEDGMA, and HEMA)-induced cytotoxicity is associated with oxidative stress, in agreement with previous studies [8], [11], [25]. Information on toxic effects caused in cells isolated from target tissues of dental restorative materials might be more specific. Many attempts have been made in the past to compare the responses of a continuous cell line (L929,
Acknowledgements
This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (03-PJ1-PG1-CH09-0001).
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D.H. Lee and B.-S. Lim equally contributed to this work.