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Additive effects of TEGDMA and hydrogenperoxide on the cellular glutathione content of human gingival fibroblasts
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 921–926
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/dema
Additive effects of TEGDMA and hydrogenperoxide on the cellular glutathione content of human gingival fibroblasts Joachim Volk a , Gabriele Leyhausen a , Sami Dogan b , Werner Geurtsen b,∗ a b
Department of Conservative Dentistry and Periodontology, Medical University Hannover, D-30625 Hannover, Germany Department of Restorative Dentistry, Division of Operative Dentistry, University of Washington, Seattle, WA, USA
a r t i c l e
i n f o
Article history:
a b s t r a c t Objectives. Only few data are available about cytotoxic effects of leachable dental resin com-
Received 31 March 2006
pounds in combination with hydrogen peroxide (H2 O2 ) segregated from dental bleaching
Received in revised form
agents. Therefore, the purpose of this study was to evaluate the effects of various concentra-
9 August 2006
tions of triethylene-glycol dimethacrylate (TEGDMA) and H2 O2 on intracellular glutathione
Accepted 10 August 2006
levels (GSH) and viability of human gingival fibroblasts (HGF) that are primary target cells of cytotoxic actions of these substances. Methods. HGF were grown in 96-well plates for 24 h, treated with various concentrations
Keywords:
of TEGDMA (0.5–5.0 mM) for 24 h and subsequently for 90 min with 0.2 mM H2 O2 or culture
Gingival fibroblasts
medium (control). The relative intracellular GSH concentration was determined using a fluo-
TEGDMA
rescence assay with monobromobimane. Readings were normalized to cell numbers, which
H2 O2
were determined by a propidium iodide assay. Data were statistically analyzed by t-test and
Glutathione
ANOVA with Tukey’s post test. A significance level of p < 0.05 was used. Results. Exposure to TEGDMA reduced the viability of HGF at concentrations ≥1.0 mM. TEGDMA induced a decrease of the GSH pool in a concentration-dependent manner (p < 0.05). The depletion of GSH was correlated with a reduction of viability (p < 0.05) and the total cell number. Furthermore, a significant decrease of the intracellular GSH content was found when cells were exposed to TEGDMA in combination with H2 O2 , compared to experiments without H2 O2 . Significance. We conclude from our findings that TEGDMA and H2 O2 have additive adverse effects on GSH metabolism and cell viability. © 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Many patients, specifically adolescents and young adults, frequently use peroxide-based bleaching agents to lighten their teeth. The active ingredient within these products is primarily H2 O2 , which is either directly applied or liberated from carbamide peroxide (CP) through an intra-oral chemical reaction. The concentration of H2 O2 in bleaching materials varies significantly between 3.4 and 6.8% in home bleaching agents and
up to 35% in office bleaching agents [1,2]. Wattanapayungkul et al. [3] determined that during a 1-h-period a concentration of 20 mM H2 O2 was created in saliva from a 10% CP gel. McMillan et al. [4] found a maximum concentration of 2 mM H2 O2 , which was released from a 6.5% H2 O2 –gel during a period of 60 min. H2 O2 is slowly degraded in saliva. In fact, after 40 min, only 25% of the initially applied peroxide is disintegrated [5]. Likewise, the CP concentration only gradually declines at the tooth surface. After a period of 1 h, 70% of the initial concentration was
∗ Corresponding author at: Department of Restorative Dentistry, School of Dentistry, University of Washington, Box 357456, Seattle, WA 98195-7456, USA. Tel.: +1 206 543 5948; fax: +1 206 543 7783. E-mail address: [email protected] (W. Geurtsen). 0109-5641/$ – see front matter © 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2006.08.001
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still present [3], and after 2 h 50% of the initial concentration remained [6]. These data indicate that oral cells are exposed to high H2 O2 concentrations during bleaching therapies, which comprise a period of several weeks or even longer. H2 O2 readily penetrates cell membranes and generates various reactive oxygen species (ROS), such as the hydroxyl radical (• OH). This radical is highly reactive and is considered to be the most toxic ROS causing genetic instability [7]. The application of tooth-colored esthetic filling materials, specifically composite resins, increased dramatically during the past decade equivalent to the use of bleaching agents. The most important composite comonomer, triethylene-glycol dimethacrylate (TEGDMA), was frequently found in aqueous eluates from various products [8–10]. TEGDMA can easily penetrate cell membranes and subsequently react with intracellular molecules and structures [11]. TEGDMA reveals a high cytotoxicity [12,13], moderate genotoxic potency at low ‘subtoxic’ concentrations [14–16], and interferes with various metabolic pathways. Recently, it has been shown that non-toxic concentrations of TEGDMA cause a depletion of the important intracellular antioxidant glutathione (GSH, l␥-glutamyl-l-cysteinyl-glycine) within the very short period of 90 min to 4 h [17,18]. This reaction may subsequently cause severe cellular alterations in different cell types [13,17,19–21]. It was found, for instance, that the reduction of antioxidant GSH due to TEGDMA caused a significant increase of the endogenous H2 O2 concentration [11,16]. The significance of these data is that a persistent exposure of cells and tissues to low concentrations of water-leachable TEGDMA may have chronic negative effects, which may cause dysfunction or disease in time. In addition, GSH-depleted cells will be more susceptible to subsequent injuries from other toxic xenobiotics, for instance H2 O2 . Contrary to the rapid increase in clinical use of resinbased filling materials as well as bleaching agents, very little is known about the chemical–biological interactions of the highly reactive substances generated during a bleaching therapy with oral cells and tissues. Therefore, it was the objective of this study to investigate the hypothesis that TEGDMA and H2 O2 in combination will have additive cytotoxic effects on oral human cells. Primary human gingival fibroblasts were selected as important target cells of adverse reactions due to both substances. The relative intracellular GSH concentration was determined as parameter for the individual and combined cytotoxic activity of TEGDMA and GSH.
2.
Materials and methods
2.1.
Materials
Dulbecco’s modified Eagle’s medium (DMEM), HEPES, penicillin, streptomycin, amphotericin, and fetal calf serum (FCS) were purchased from Biochrom (Berlin, Germany), NaHCO3 ¨ (Seelze, Germany), and trypsin/EDTA from Riedel de Haen from Sigma (Taufkirchen, Germany). triethylene-glycol dimethacrylate (TEGDMA) was a gift from VOCO (Cuxhaven, Germany). H2 O2 (30%) was purchased from Merck (Darmstadt, Germany), monobromobimane (MBBr) and Nonidet P-40 were purchased from Fluka (Seelze, Germany), propidiumiodide
(PI) and dimethylsulfoxide (DMSO) from Sigma (Taufkirchen, Germany), and Hank’s balanced salt solution (HBSS) from GIBCO BRL (Karlsruhe, Germany).
2.2.
Cell culture
Primary human gingival fibroblasts (HGF) were cultured from biopsies of healthy gingiva of permanent molars. Informed consent was obtained from the donors according to the guidelines of the Institutional Review Board. The biopsies were stored at 4 ◦ C for 24 h at most in HBSS supplemented with penicillin (100 U/mL), streptomycin (100 g/mL), and amphotericin (2.5 g/mL) prior to amplification. The tissue samples were placed into 25-cm2 tissue culture flasks and grown in DMEM culture medium with 4.5 g/L glucose, 10 mM HEPES, NaHCO3 (3.7 g/L), penicillin (100 U/mL), and streptomycin (100 g/mL), supplemented with 10% fetal calf serum (FCS) at 37 ◦ C and 10% CO2 . When outgrowth of cells was observed, the medium was replaced twice weekly until cells reached confluency. Cells were detached from the substrate by a brief treatment with trypsin/EDTA (0.25% trypsin, 0.02% EDTA) and cultured in 75-cm2 tissue flasks until confluent monolayers were re-obtained. Early passages were frozen in liquid nitrogen. All cultures were routinely tested for mycoplasma contamination by means of the mycoplasma detection kit Venor GeM (Minerva Biolabs, Berlin, Germany).
2.3.
Treatment of cells with TEGDMA and H2 O2
Stock solutions (200×) of TEGDMA were prepared in DMSO and freshly diluted in DMEM prior to each experiment. HGF from passages number 6 to 12 were seeded in 96-well plates (1 × 104 cells/well) and allowed to grow for 24 h. Then cells were washed with DMEM and exposed to TEGDMA for 24 h at concentrations between 0.5 and 5 mM. After washing, TEGDMA-treated cells were incubated for another 90 min with 0.2 mM H2 O2 and medium, but without TEGDMA. Control cultures were incubated during the initial 24 h with medium containing 0.5% DMSO. After washing control cells were either exposed to medium containing 0.2 mM H2 O2 or to medium containing 0.5% DMSO for additional 90 min.
2.4.
Glutathione assay
The relative intracellular GSH concentrations were determined using an assay with monobromobimane (MBBr) in microtiter plates as described previously [19]. Briefly, medium was removed from the wells after treatment of the cells. Monolayers were washed with HBSS. Then, MBBr in HBSS was added and after 35 min in the darkness, fluorescence intensity of the MBBr-GSH adduct was measured at the excitation wavelength (Ex) of 360 nm and the emission wavelength (Em) of 460 nm. Subsequently, PI was added for 20 min and the fluorescence (FPI ) of this stain was measured at Ex 530 nm/Em 645 nm. By adding the surfactant Nonidet P-40 for 20 min, all vital cells were lyzed and the fluorescence (Fmax ) was read again to determine the total cell number per well (PI stains only DNA of non-vital cells). Then MBBr-readings were normalized to cell numbers based on Fmax in the presence of the surfactant Non-
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Fig. 1 – Intracellular glutathione content of HGF exposed to: TEGDMA (), TEGDMA in combination with 0.2 mM H2 O2 (), and 0.2 mM H2 O2 ( ). Values in % of controls (normalized to cell numbers) are means ± standard deviations (n = 8). The indices represent the statistically significant differences compared to untreated cells (a), H2 O2 treated cells (b), and cells treated with TEGDMA (c) (p < 0.05).
Fig. 2 – Percentage of viable HGF exposed to TEGDMA (), TEGDMA in combination with 0.2 mM H2 O2 (), and 0.2 mM H2 O2 ( ). Values in % of controls are means ± standard deviations (n = 8). The indices represent the statistically significant differences compared to untreated cells (a), H2 O2 treated cells (b), and cells only treated with TEGDMA (c) (p < 0.05).
idet P-40. The PI-readings FPI and Fmax were used to estimate the vitality of cells as a function of membrane leakiness [19]. All readings were performed in a FLx 800 microplate fluorescence reader (BioTec, Neufahrn, Germany).
nificantly reduced the ED50 of TEGDMA with regard to GSH concentration and cell viability to a two- or even three-fold lower concentration (Table 1). The total cell number significantly decreased only at the highest TEGDMA concentration (5.0 mM) to 38.4% (±13.0%) (TEGDMA alone) or 37.0% (±10.4%)
2.5.
Statistical analysis
Assays were run at least eight times with six replicates each. Results were expressed as means + standard deviations. Statistical analysis was performed by t-test and ANOVA with Tukey’s post test. A significance level of p < 0.05 was used. Linear regression analysis (MS® Excel 2000) was applied to calculate the concentrations of TEGDMA alone and in combination with 0.2 mM H2 O2 that reduce the intracellular GSH concentration, cell viability, and total cell number to 50% of untreated control cells (=ED50 ).
3.
Results
The effects of TEGDMA (alone and in combination with H2 O2 ) on the relative intracellular glutathione content (GSH) of HGF, cell viability and total cell number are shown in Figs. 1–3. TEGDMA depleted the intracellular GSH pool (Fig. 1) dependent upon concentration and reduced the intracellular GSH pool at 1.9 mM (±0.6 mM) to 50% (ED50(GSH depletion) ; Table 1). TEGDMA reduced GSH significantly at a concentration of 0.5 mM (83.4 ± 9.8%, p < 0.05). Further, a significant decrease of GSH was found when cells were exposed both to TEGDMA (≥1.0 mM) and 0.2 mM H2 O2 , compared to assays without additional H2 O2 or to cultures treated only with 0.2 mM H2 O2 . The ED50 of the TEGDMA/H2 O2 treatment was more than twice as low (0.8 ± 0.3 mM) as the ED50 of TEGDMA alone (1.9 ± 0.6 mM) (Table 1). The depletion of GSH correlated with the reduction of viability and the total cell number (p < 0.05; Figs. 2 and 3). Exposure of cells to TEGDMA and H2 O2 resulted in a significant decrease of the ED50 regarding cell viability from 3.2 mM (±0.3 mM) to about 0.9 mM (±0.2 mM) compared to cells only treated with TEGDMA. H2 O2 at a concentration of 0.2 mM sig-
Fig. 3 – Percentage of total HGF exposed to TEGDMA (), TEGDMA in combination with 0.2 mM H2 O2 (), and 0.2 mM H2 O2 ( ). Values in % of controls are means ± standard deviations (n = 8). The indices represent the statistically significant differences compared to untreated cells (a), H2 O2 treated cells (b), and cells only treated with TEGDMA (c) (p < 0.05).
Table 1 – ED50 -values (mM) for MBBr and PI studies Assay GSH depletion (MBBr) Cell viability (PI) Total cell number (PI)
ED50 of TEGDMA [mM]
ED50 of TEGDMA + 0.2 mM H2 O2 [mM]
1.9 (0.6) 3.2 (0.3) 4.6 (0.8)
0.8 (0.3) 0.9 (0.2) 4.2 (0.8)
ED50 -concentrations (mM) of TEGDMA and TEGDMA in combination with 0.2 mM H2 O2 regarding the relative intracellular glutathione concentration in HGF (n = 8), decrease of cell viability (n = 6), and decrease of total cell number (n = 6). Cells were exposed to TEGDMA for 24 h followed by a subsequent treatment with 0.2 mM H2 O2 for 90 min. Values are means ± standard deviations (S.D.).
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(TEGDMA + H2 O2 ) compared to untreated cells. When cells were exposed solely to H2 O2 for 90 min from 0.001 mM up to a concentration of 0.2 mM H2 O2 , the number of vital cells was consistently higher than 80% of control cultures and total cell number was always higher than 90% of controls (data not shown).
4.
Discussion
Objective of this investigation was to analyze combined effects of a resin monomer and H2 O2 on cells’ GSH metabolism. Since various experiments clearly indicated that TEGDMA is the most important monomeric substance leaching from composite resins into aqueous environments in very high quantities it was selected for our study. The range of TEGDMA that was applied for our experiments reflects findings of previous studies. Specifically during the first days after application TEGDMA leaches from composites into aqueous media in amounts that exceed the highest concentration applied in our investigation [22,23]. This also applies to those levels of H2 O2 that we used for our experiments. Concentrations of up to 20 mM H2 O2 were created in vivo in saliva from a low-concentrated 10% CP gel that was applied in a bleaching tray [3]. An additional critical parameter that has not yet been considered in the dental literature is the increasing use of whitening oral health care or dietary products, such as tooth pastes or chewing gums. These materials are used by many patients for a long period of time or even indefinitely in order to secure the result of a previous bleaching therapy or to whiten their teeth additionally. The problem associated with these products is the uncontrolled and chronic release of highly active peroxides into the oral cavity. These aspects point out that many patients are potentially exposed to peroxides and GSH depleting resin monomers repeatedly and for a long period of time, specifically after placement of resin restorations. Recently the effects of structural characteristics of resin monomers (e.g. number of methacrylate groups) on cellular GSH depletion were analyzed. The findings of this study revealed that UDMA as well as HEMA may also significantly decrease cellular GSH levels [17]. UDMA, however, was found in aqueous extracts in smaller amounts only due its ‘hydrophobic nature’ [22]. HEMA on the other hand, is a main component in dentin adhesives rather than in composite resins. Therefore, HEMA will not be released in high quantities into the oral cavity like TEGDMA, the main comonomer in filling composites. Both investigated substances, TEGDMA and H2 O2 , readily penetrate cell membranes of oral tissues and alter the intracellular level of reduced glutathione (GSH) [16,18,19]. GSH is an efficient protector against toxic influences from xenobiotics and reactive oxygen species (ROS). The balance between reduced GSH and its oxidized form, GSSG, however, is an important mechanism whereby cells maintain redox homeostasis. An irreversible depletion of intracellular GSH will detrimentally impair the cells’ protection against subsequent toxic influences due to xenobiotics and exogenously as well as endogenously generated ROS. This may be either caused by higher concentrations of an individual substance, which depletes GSH [17,18] or by a combination of various materials
at lower concentrations, which was detected in the present study. An elevated level of reactive oxygen species is of specific importance as ROS are biologically highly active molecules that oxidize lipids, amino acids as well as carbohydrates and cause DNA damage. These reactions finally result in severe changes of cells’ functions leading to cell death or mutations [24–27]. It is confirmed by previous findings that in vitro exposure of fibroblasts to compounds leaching from dental materials like TEGDMA can cause apoptosis, necrosis or a combination of both [28]. But only few data are available about (toxic) effects of methacrylates leaching from resin-based filling materials in combination with H2 O2 that is segregated by dental bleaching agents. Therefore, we performed experiments with human primary gingival fibroblasts (HGF) to analyze their relative GSHcontent and viability due to a treatment with TEGDMA and a subsequent shorter treatment with H2 O2 only. This study design simulates in vivo conditions very well, because gingival and other oral mucosal cells may be exposed to lower TEGDMA concentrations for a longer period of time in association with an exposure to high concentrations of H2 O2 during bleaching therapies. HGF were selected because they are one of the primary target cells of potential injuries caused by the investigated substances in the oral cavity, where the released compounds are present at their highest concentrations. Based upon data from previous leaching studies and dependent on the product, the leached amounts of TEGDMA can vary between 0.9 and 3.0 mM during the initial 24 h after application of a resinous restorative material [10,29]. Within this range, a significant decrease of cellular viability and GSHcontent of the gingival fibroblasts were found in our experiments (Figs. 1 and 2). TEGDMA at the low concentration of 0.5 mM significantly decreased the GSH pool to approximately 84% of control, whereas, at a concentration of 1.9 mM the intracellular GSH level decreased to 50% of control values. No effect on cells’ viability was observed at a concentration of 0.5 mM. Bleaching therapies using hydrogenperoxide or its derivatives are used by many patients, specifically adolescents. These bleaching agents release considerable amounts of active hydrogenperoxide into saliva. For instance, concentrations of 20 mM H2 O2 due to the application of a 10% CP gel and of up to 2 mM H2 O2 after using a 6.5% H2 O2 –gel during a period of 60 min were found in saliva [3,4]. Importantly, Hannig et al. determined that only 25% of the initially applied peroxide is disintegrated within 40 min [5]. These findings reveal that H2 O2 is only slowly degraded in saliva. These data clearly indicate the need to analyze potential combined adverse reactions of hydrogenperoxide and important xenobiotics released from filling materials, specifically resin-based oral biomaterials. In order to investigate detrimental effects at low concentrations that can be present in the oral cavity during a regular bleaching treatment and the use of whitening tooth pastes or chewing gums for a long period of time the low amount of 0.2 mM H2 O2 was selected for our experiments. This concentration caused a moderate reduction of the number of vital cells, whereas, the decline of GSH level was much more pronounced (∼60% of control values) (Figs. 1 and 2). The subsequent simultaneous incubation of HGF with TEGDMA and H2 O2 significantly enhanced the toxic reaction at all tested
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 921–926
concentrations. Most important, the combination of 0.5 mM TEGDMA with 0.2 mM H2 O2 reduced the GSH pool to 50% of controls and the share of viable cells to 75% compared to untreated fibroblast cultures (Figs. 1 and 2). It may be concluded from these results that the GSH depletion was associated with a significant increase of the cell toxicity of TEGDMA. Scarce data about toxic effects of H2 O2 alone or in combination with various components leaching from restorative materials on oral cells have been published although high concentrations of these substances may be present in the oral cavity for an extended period of time. Most authors concentrated on the analysis of adverse reactions generated by individual resin substances, particularly TEGDMA. For example, Stanislawski et al. [13] reported a dramatic depletion of cellular GSH followed by an increase of ROS in human gingival and pulpal fibroblasts after incubation with TEGDMA. The elevated ROS level resulted in severe cytotoxitiy. Antioxidants prevented the TEGDMA-induced cytotoxicity while GSH depletion was only partially prevented [13,30]. This set of data indicates that the GSH-depletion is an important parameter associated with cytotoxicity. It is therefore hypothesized that the depletion of the cells’ GSH pool due to TEGDMA is caused by a direct interaction of TEGDMA and GSH, for instance due to a Michel-type reaction [13,18,19,31]. The combination of TEGDMA with 0.2 mM H2 O2 caused a clear additive toxic effect, even at the very low TEGDMA concentration of 0.5 mM. Reichl et al. [32] investigated the impact of TEGDMA and H2 O2 on the gluconeogenesis in rat kidney tubules. They found a marked synergistic inhibitory effect caused by the combination of both substances on the cellular energy metabolism.
5.
Conclusion
Taken together, our experiments provide evidence that TEGDMA in combination with hydrogenperoxide significantly interferes with the metabolism and viability of human oral cells already at very low concentrations that may be frequently present in the oral cavity. Our data also point out that this effect is at least partially due to a decrease of the intracellular GSH pool. Our hypothesis that a simultaneous exposure of oral cells to TEGDMA and H2 O2 causes an additive cytotoxic effect is clearly supported by our results. Based upon our findings as well as the pronounced ‘popularity’ of tooth-colored resin restorations and tooth whitening procedures on the one hand and the insufficient knowledge about adverse effects of hydrogenperoxide in combination with other important dental substances on the other hand it would be of outstanding importance to determine their potential biological–chemical interactions much more intensely. These data would be necessary for a meaningful risk assessment of bleaching therapies. Considering the pronounced interference of the investigated substances with primary oral human cells even at low concentrations it is recommended to avoid a simultaneous treatment of patients with new resin restorations and bleaching procedures. Patients should be also informed about potential biological risks associated with the use of whitening peroxidereleasing tooth pastes and chewing gums.
925
Acknowledgements Our thanks are due to Ms. A. Beckedorf for her excellent technical assistance. This study was supported by a grant of the ¨ Zahn-, Mund- und Kieferheilkunde Deutsche Gesellschaft fur (DGZMK).
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available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/dema
Additive effects of TEGDMA and hydrogenperoxide on the cellular glutathione content of human gingival fibroblasts Joachim Volk a , Gabriele Leyhausen a , Sami Dogan b , Werner Geurtsen b,∗ a b
Department of Conservative Dentistry and Periodontology, Medical University Hannover, D-30625 Hannover, Germany Department of Restorative Dentistry, Division of Operative Dentistry, University of Washington, Seattle, WA, USA
a r t i c l e
i n f o
Article history:
a b s t r a c t Objectives. Only few data are available about cytotoxic effects of leachable dental resin com-
Received 31 March 2006
pounds in combination with hydrogen peroxide (H2 O2 ) segregated from dental bleaching
Received in revised form
agents. Therefore, the purpose of this study was to evaluate the effects of various concentra-
9 August 2006
tions of triethylene-glycol dimethacrylate (TEGDMA) and H2 O2 on intracellular glutathione
Accepted 10 August 2006
levels (GSH) and viability of human gingival fibroblasts (HGF) that are primary target cells of cytotoxic actions of these substances. Methods. HGF were grown in 96-well plates for 24 h, treated with various concentrations
Keywords:
of TEGDMA (0.5–5.0 mM) for 24 h and subsequently for 90 min with 0.2 mM H2 O2 or culture
Gingival fibroblasts
medium (control). The relative intracellular GSH concentration was determined using a fluo-
TEGDMA
rescence assay with monobromobimane. Readings were normalized to cell numbers, which
H2 O2
were determined by a propidium iodide assay. Data were statistically analyzed by t-test and
Glutathione
ANOVA with Tukey’s post test. A significance level of p < 0.05 was used. Results. Exposure to TEGDMA reduced the viability of HGF at concentrations ≥1.0 mM. TEGDMA induced a decrease of the GSH pool in a concentration-dependent manner (p < 0.05). The depletion of GSH was correlated with a reduction of viability (p < 0.05) and the total cell number. Furthermore, a significant decrease of the intracellular GSH content was found when cells were exposed to TEGDMA in combination with H2 O2 , compared to experiments without H2 O2 . Significance. We conclude from our findings that TEGDMA and H2 O2 have additive adverse effects on GSH metabolism and cell viability. © 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Many patients, specifically adolescents and young adults, frequently use peroxide-based bleaching agents to lighten their teeth. The active ingredient within these products is primarily H2 O2 , which is either directly applied or liberated from carbamide peroxide (CP) through an intra-oral chemical reaction. The concentration of H2 O2 in bleaching materials varies significantly between 3.4 and 6.8% in home bleaching agents and
up to 35% in office bleaching agents [1,2]. Wattanapayungkul et al. [3] determined that during a 1-h-period a concentration of 20 mM H2 O2 was created in saliva from a 10% CP gel. McMillan et al. [4] found a maximum concentration of 2 mM H2 O2 , which was released from a 6.5% H2 O2 –gel during a period of 60 min. H2 O2 is slowly degraded in saliva. In fact, after 40 min, only 25% of the initially applied peroxide is disintegrated [5]. Likewise, the CP concentration only gradually declines at the tooth surface. After a period of 1 h, 70% of the initial concentration was
∗ Corresponding author at: Department of Restorative Dentistry, School of Dentistry, University of Washington, Box 357456, Seattle, WA 98195-7456, USA. Tel.: +1 206 543 5948; fax: +1 206 543 7783. E-mail address: [email protected] (W. Geurtsen). 0109-5641/$ – see front matter © 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2006.08.001
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still present [3], and after 2 h 50% of the initial concentration remained [6]. These data indicate that oral cells are exposed to high H2 O2 concentrations during bleaching therapies, which comprise a period of several weeks or even longer. H2 O2 readily penetrates cell membranes and generates various reactive oxygen species (ROS), such as the hydroxyl radical (• OH). This radical is highly reactive and is considered to be the most toxic ROS causing genetic instability [7]. The application of tooth-colored esthetic filling materials, specifically composite resins, increased dramatically during the past decade equivalent to the use of bleaching agents. The most important composite comonomer, triethylene-glycol dimethacrylate (TEGDMA), was frequently found in aqueous eluates from various products [8–10]. TEGDMA can easily penetrate cell membranes and subsequently react with intracellular molecules and structures [11]. TEGDMA reveals a high cytotoxicity [12,13], moderate genotoxic potency at low ‘subtoxic’ concentrations [14–16], and interferes with various metabolic pathways. Recently, it has been shown that non-toxic concentrations of TEGDMA cause a depletion of the important intracellular antioxidant glutathione (GSH, l␥-glutamyl-l-cysteinyl-glycine) within the very short period of 90 min to 4 h [17,18]. This reaction may subsequently cause severe cellular alterations in different cell types [13,17,19–21]. It was found, for instance, that the reduction of antioxidant GSH due to TEGDMA caused a significant increase of the endogenous H2 O2 concentration [11,16]. The significance of these data is that a persistent exposure of cells and tissues to low concentrations of water-leachable TEGDMA may have chronic negative effects, which may cause dysfunction or disease in time. In addition, GSH-depleted cells will be more susceptible to subsequent injuries from other toxic xenobiotics, for instance H2 O2 . Contrary to the rapid increase in clinical use of resinbased filling materials as well as bleaching agents, very little is known about the chemical–biological interactions of the highly reactive substances generated during a bleaching therapy with oral cells and tissues. Therefore, it was the objective of this study to investigate the hypothesis that TEGDMA and H2 O2 in combination will have additive cytotoxic effects on oral human cells. Primary human gingival fibroblasts were selected as important target cells of adverse reactions due to both substances. The relative intracellular GSH concentration was determined as parameter for the individual and combined cytotoxic activity of TEGDMA and GSH.
2.
Materials and methods
2.1.
Materials
Dulbecco’s modified Eagle’s medium (DMEM), HEPES, penicillin, streptomycin, amphotericin, and fetal calf serum (FCS) were purchased from Biochrom (Berlin, Germany), NaHCO3 ¨ (Seelze, Germany), and trypsin/EDTA from Riedel de Haen from Sigma (Taufkirchen, Germany). triethylene-glycol dimethacrylate (TEGDMA) was a gift from VOCO (Cuxhaven, Germany). H2 O2 (30%) was purchased from Merck (Darmstadt, Germany), monobromobimane (MBBr) and Nonidet P-40 were purchased from Fluka (Seelze, Germany), propidiumiodide
(PI) and dimethylsulfoxide (DMSO) from Sigma (Taufkirchen, Germany), and Hank’s balanced salt solution (HBSS) from GIBCO BRL (Karlsruhe, Germany).
2.2.
Cell culture
Primary human gingival fibroblasts (HGF) were cultured from biopsies of healthy gingiva of permanent molars. Informed consent was obtained from the donors according to the guidelines of the Institutional Review Board. The biopsies were stored at 4 ◦ C for 24 h at most in HBSS supplemented with penicillin (100 U/mL), streptomycin (100 g/mL), and amphotericin (2.5 g/mL) prior to amplification. The tissue samples were placed into 25-cm2 tissue culture flasks and grown in DMEM culture medium with 4.5 g/L glucose, 10 mM HEPES, NaHCO3 (3.7 g/L), penicillin (100 U/mL), and streptomycin (100 g/mL), supplemented with 10% fetal calf serum (FCS) at 37 ◦ C and 10% CO2 . When outgrowth of cells was observed, the medium was replaced twice weekly until cells reached confluency. Cells were detached from the substrate by a brief treatment with trypsin/EDTA (0.25% trypsin, 0.02% EDTA) and cultured in 75-cm2 tissue flasks until confluent monolayers were re-obtained. Early passages were frozen in liquid nitrogen. All cultures were routinely tested for mycoplasma contamination by means of the mycoplasma detection kit Venor GeM (Minerva Biolabs, Berlin, Germany).
2.3.
Treatment of cells with TEGDMA and H2 O2
Stock solutions (200×) of TEGDMA were prepared in DMSO and freshly diluted in DMEM prior to each experiment. HGF from passages number 6 to 12 were seeded in 96-well plates (1 × 104 cells/well) and allowed to grow for 24 h. Then cells were washed with DMEM and exposed to TEGDMA for 24 h at concentrations between 0.5 and 5 mM. After washing, TEGDMA-treated cells were incubated for another 90 min with 0.2 mM H2 O2 and medium, but without TEGDMA. Control cultures were incubated during the initial 24 h with medium containing 0.5% DMSO. After washing control cells were either exposed to medium containing 0.2 mM H2 O2 or to medium containing 0.5% DMSO for additional 90 min.
2.4.
Glutathione assay
The relative intracellular GSH concentrations were determined using an assay with monobromobimane (MBBr) in microtiter plates as described previously [19]. Briefly, medium was removed from the wells after treatment of the cells. Monolayers were washed with HBSS. Then, MBBr in HBSS was added and after 35 min in the darkness, fluorescence intensity of the MBBr-GSH adduct was measured at the excitation wavelength (Ex) of 360 nm and the emission wavelength (Em) of 460 nm. Subsequently, PI was added for 20 min and the fluorescence (FPI ) of this stain was measured at Ex 530 nm/Em 645 nm. By adding the surfactant Nonidet P-40 for 20 min, all vital cells were lyzed and the fluorescence (Fmax ) was read again to determine the total cell number per well (PI stains only DNA of non-vital cells). Then MBBr-readings were normalized to cell numbers based on Fmax in the presence of the surfactant Non-
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Fig. 1 – Intracellular glutathione content of HGF exposed to: TEGDMA (), TEGDMA in combination with 0.2 mM H2 O2 (), and 0.2 mM H2 O2 ( ). Values in % of controls (normalized to cell numbers) are means ± standard deviations (n = 8). The indices represent the statistically significant differences compared to untreated cells (a), H2 O2 treated cells (b), and cells treated with TEGDMA (c) (p < 0.05).
Fig. 2 – Percentage of viable HGF exposed to TEGDMA (), TEGDMA in combination with 0.2 mM H2 O2 (), and 0.2 mM H2 O2 ( ). Values in % of controls are means ± standard deviations (n = 8). The indices represent the statistically significant differences compared to untreated cells (a), H2 O2 treated cells (b), and cells only treated with TEGDMA (c) (p < 0.05).
idet P-40. The PI-readings FPI and Fmax were used to estimate the vitality of cells as a function of membrane leakiness [19]. All readings were performed in a FLx 800 microplate fluorescence reader (BioTec, Neufahrn, Germany).
nificantly reduced the ED50 of TEGDMA with regard to GSH concentration and cell viability to a two- or even three-fold lower concentration (Table 1). The total cell number significantly decreased only at the highest TEGDMA concentration (5.0 mM) to 38.4% (±13.0%) (TEGDMA alone) or 37.0% (±10.4%)
2.5.
Statistical analysis
Assays were run at least eight times with six replicates each. Results were expressed as means + standard deviations. Statistical analysis was performed by t-test and ANOVA with Tukey’s post test. A significance level of p < 0.05 was used. Linear regression analysis (MS® Excel 2000) was applied to calculate the concentrations of TEGDMA alone and in combination with 0.2 mM H2 O2 that reduce the intracellular GSH concentration, cell viability, and total cell number to 50% of untreated control cells (=ED50 ).
3.
Results
The effects of TEGDMA (alone and in combination with H2 O2 ) on the relative intracellular glutathione content (GSH) of HGF, cell viability and total cell number are shown in Figs. 1–3. TEGDMA depleted the intracellular GSH pool (Fig. 1) dependent upon concentration and reduced the intracellular GSH pool at 1.9 mM (±0.6 mM) to 50% (ED50(GSH depletion) ; Table 1). TEGDMA reduced GSH significantly at a concentration of 0.5 mM (83.4 ± 9.8%, p < 0.05). Further, a significant decrease of GSH was found when cells were exposed both to TEGDMA (≥1.0 mM) and 0.2 mM H2 O2 , compared to assays without additional H2 O2 or to cultures treated only with 0.2 mM H2 O2 . The ED50 of the TEGDMA/H2 O2 treatment was more than twice as low (0.8 ± 0.3 mM) as the ED50 of TEGDMA alone (1.9 ± 0.6 mM) (Table 1). The depletion of GSH correlated with the reduction of viability and the total cell number (p < 0.05; Figs. 2 and 3). Exposure of cells to TEGDMA and H2 O2 resulted in a significant decrease of the ED50 regarding cell viability from 3.2 mM (±0.3 mM) to about 0.9 mM (±0.2 mM) compared to cells only treated with TEGDMA. H2 O2 at a concentration of 0.2 mM sig-
Fig. 3 – Percentage of total HGF exposed to TEGDMA (), TEGDMA in combination with 0.2 mM H2 O2 (), and 0.2 mM H2 O2 ( ). Values in % of controls are means ± standard deviations (n = 8). The indices represent the statistically significant differences compared to untreated cells (a), H2 O2 treated cells (b), and cells only treated with TEGDMA (c) (p < 0.05).
Table 1 – ED50 -values (mM) for MBBr and PI studies Assay GSH depletion (MBBr) Cell viability (PI) Total cell number (PI)
ED50 of TEGDMA [mM]
ED50 of TEGDMA + 0.2 mM H2 O2 [mM]
1.9 (0.6) 3.2 (0.3) 4.6 (0.8)
0.8 (0.3) 0.9 (0.2) 4.2 (0.8)
ED50 -concentrations (mM) of TEGDMA and TEGDMA in combination with 0.2 mM H2 O2 regarding the relative intracellular glutathione concentration in HGF (n = 8), decrease of cell viability (n = 6), and decrease of total cell number (n = 6). Cells were exposed to TEGDMA for 24 h followed by a subsequent treatment with 0.2 mM H2 O2 for 90 min. Values are means ± standard deviations (S.D.).
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(TEGDMA + H2 O2 ) compared to untreated cells. When cells were exposed solely to H2 O2 for 90 min from 0.001 mM up to a concentration of 0.2 mM H2 O2 , the number of vital cells was consistently higher than 80% of control cultures and total cell number was always higher than 90% of controls (data not shown).
4.
Discussion
Objective of this investigation was to analyze combined effects of a resin monomer and H2 O2 on cells’ GSH metabolism. Since various experiments clearly indicated that TEGDMA is the most important monomeric substance leaching from composite resins into aqueous environments in very high quantities it was selected for our study. The range of TEGDMA that was applied for our experiments reflects findings of previous studies. Specifically during the first days after application TEGDMA leaches from composites into aqueous media in amounts that exceed the highest concentration applied in our investigation [22,23]. This also applies to those levels of H2 O2 that we used for our experiments. Concentrations of up to 20 mM H2 O2 were created in vivo in saliva from a low-concentrated 10% CP gel that was applied in a bleaching tray [3]. An additional critical parameter that has not yet been considered in the dental literature is the increasing use of whitening oral health care or dietary products, such as tooth pastes or chewing gums. These materials are used by many patients for a long period of time or even indefinitely in order to secure the result of a previous bleaching therapy or to whiten their teeth additionally. The problem associated with these products is the uncontrolled and chronic release of highly active peroxides into the oral cavity. These aspects point out that many patients are potentially exposed to peroxides and GSH depleting resin monomers repeatedly and for a long period of time, specifically after placement of resin restorations. Recently the effects of structural characteristics of resin monomers (e.g. number of methacrylate groups) on cellular GSH depletion were analyzed. The findings of this study revealed that UDMA as well as HEMA may also significantly decrease cellular GSH levels [17]. UDMA, however, was found in aqueous extracts in smaller amounts only due its ‘hydrophobic nature’ [22]. HEMA on the other hand, is a main component in dentin adhesives rather than in composite resins. Therefore, HEMA will not be released in high quantities into the oral cavity like TEGDMA, the main comonomer in filling composites. Both investigated substances, TEGDMA and H2 O2 , readily penetrate cell membranes of oral tissues and alter the intracellular level of reduced glutathione (GSH) [16,18,19]. GSH is an efficient protector against toxic influences from xenobiotics and reactive oxygen species (ROS). The balance between reduced GSH and its oxidized form, GSSG, however, is an important mechanism whereby cells maintain redox homeostasis. An irreversible depletion of intracellular GSH will detrimentally impair the cells’ protection against subsequent toxic influences due to xenobiotics and exogenously as well as endogenously generated ROS. This may be either caused by higher concentrations of an individual substance, which depletes GSH [17,18] or by a combination of various materials
at lower concentrations, which was detected in the present study. An elevated level of reactive oxygen species is of specific importance as ROS are biologically highly active molecules that oxidize lipids, amino acids as well as carbohydrates and cause DNA damage. These reactions finally result in severe changes of cells’ functions leading to cell death or mutations [24–27]. It is confirmed by previous findings that in vitro exposure of fibroblasts to compounds leaching from dental materials like TEGDMA can cause apoptosis, necrosis or a combination of both [28]. But only few data are available about (toxic) effects of methacrylates leaching from resin-based filling materials in combination with H2 O2 that is segregated by dental bleaching agents. Therefore, we performed experiments with human primary gingival fibroblasts (HGF) to analyze their relative GSHcontent and viability due to a treatment with TEGDMA and a subsequent shorter treatment with H2 O2 only. This study design simulates in vivo conditions very well, because gingival and other oral mucosal cells may be exposed to lower TEGDMA concentrations for a longer period of time in association with an exposure to high concentrations of H2 O2 during bleaching therapies. HGF were selected because they are one of the primary target cells of potential injuries caused by the investigated substances in the oral cavity, where the released compounds are present at their highest concentrations. Based upon data from previous leaching studies and dependent on the product, the leached amounts of TEGDMA can vary between 0.9 and 3.0 mM during the initial 24 h after application of a resinous restorative material [10,29]. Within this range, a significant decrease of cellular viability and GSHcontent of the gingival fibroblasts were found in our experiments (Figs. 1 and 2). TEGDMA at the low concentration of 0.5 mM significantly decreased the GSH pool to approximately 84% of control, whereas, at a concentration of 1.9 mM the intracellular GSH level decreased to 50% of control values. No effect on cells’ viability was observed at a concentration of 0.5 mM. Bleaching therapies using hydrogenperoxide or its derivatives are used by many patients, specifically adolescents. These bleaching agents release considerable amounts of active hydrogenperoxide into saliva. For instance, concentrations of 20 mM H2 O2 due to the application of a 10% CP gel and of up to 2 mM H2 O2 after using a 6.5% H2 O2 –gel during a period of 60 min were found in saliva [3,4]. Importantly, Hannig et al. determined that only 25% of the initially applied peroxide is disintegrated within 40 min [5]. These findings reveal that H2 O2 is only slowly degraded in saliva. These data clearly indicate the need to analyze potential combined adverse reactions of hydrogenperoxide and important xenobiotics released from filling materials, specifically resin-based oral biomaterials. In order to investigate detrimental effects at low concentrations that can be present in the oral cavity during a regular bleaching treatment and the use of whitening tooth pastes or chewing gums for a long period of time the low amount of 0.2 mM H2 O2 was selected for our experiments. This concentration caused a moderate reduction of the number of vital cells, whereas, the decline of GSH level was much more pronounced (∼60% of control values) (Figs. 1 and 2). The subsequent simultaneous incubation of HGF with TEGDMA and H2 O2 significantly enhanced the toxic reaction at all tested
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 921–926
concentrations. Most important, the combination of 0.5 mM TEGDMA with 0.2 mM H2 O2 reduced the GSH pool to 50% of controls and the share of viable cells to 75% compared to untreated fibroblast cultures (Figs. 1 and 2). It may be concluded from these results that the GSH depletion was associated with a significant increase of the cell toxicity of TEGDMA. Scarce data about toxic effects of H2 O2 alone or in combination with various components leaching from restorative materials on oral cells have been published although high concentrations of these substances may be present in the oral cavity for an extended period of time. Most authors concentrated on the analysis of adverse reactions generated by individual resin substances, particularly TEGDMA. For example, Stanislawski et al. [13] reported a dramatic depletion of cellular GSH followed by an increase of ROS in human gingival and pulpal fibroblasts after incubation with TEGDMA. The elevated ROS level resulted in severe cytotoxitiy. Antioxidants prevented the TEGDMA-induced cytotoxicity while GSH depletion was only partially prevented [13,30]. This set of data indicates that the GSH-depletion is an important parameter associated with cytotoxicity. It is therefore hypothesized that the depletion of the cells’ GSH pool due to TEGDMA is caused by a direct interaction of TEGDMA and GSH, for instance due to a Michel-type reaction [13,18,19,31]. The combination of TEGDMA with 0.2 mM H2 O2 caused a clear additive toxic effect, even at the very low TEGDMA concentration of 0.5 mM. Reichl et al. [32] investigated the impact of TEGDMA and H2 O2 on the gluconeogenesis in rat kidney tubules. They found a marked synergistic inhibitory effect caused by the combination of both substances on the cellular energy metabolism.
5.
Conclusion
Taken together, our experiments provide evidence that TEGDMA in combination with hydrogenperoxide significantly interferes with the metabolism and viability of human oral cells already at very low concentrations that may be frequently present in the oral cavity. Our data also point out that this effect is at least partially due to a decrease of the intracellular GSH pool. Our hypothesis that a simultaneous exposure of oral cells to TEGDMA and H2 O2 causes an additive cytotoxic effect is clearly supported by our results. Based upon our findings as well as the pronounced ‘popularity’ of tooth-colored resin restorations and tooth whitening procedures on the one hand and the insufficient knowledge about adverse effects of hydrogenperoxide in combination with other important dental substances on the other hand it would be of outstanding importance to determine their potential biological–chemical interactions much more intensely. These data would be necessary for a meaningful risk assessment of bleaching therapies. Considering the pronounced interference of the investigated substances with primary oral human cells even at low concentrations it is recommended to avoid a simultaneous treatment of patients with new resin restorations and bleaching procedures. Patients should be also informed about potential biological risks associated with the use of whitening peroxidereleasing tooth pastes and chewing gums.
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Acknowledgements Our thanks are due to Ms. A. Beckedorf for her excellent technical assistance. This study was supported by a grant of the ¨ Zahn-, Mund- und Kieferheilkunde Deutsche Gesellschaft fur (DGZMK).
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