Effects of three resin monomers on the cellular glutathione concentration of cultured human gingival fibroblasts

Dental Materials (2006) 22, 499–505

www.intl.elsevierhealth.com/journals/dema

Effects of three resin monomers on the cellular glutathione concentration of cultured human gingival fibroblasts J. Volka, J. Engelmanna, G. Leyhausena, W. Geurtsenb,* a

Department of Conservative Dentistry and Periodontology, Medical University Hannover, D-30625 Hannover, Germany b Department of Restorative Dentistry/Division of Operative Dentistry, School of Dentistry, University of Washington, P.O. Box 357456, Seattle, WA 98195 7456, USA Received 21 March 2005; received in revised form 9 June 2005; accepted 16 June 2005

KEYWORDS Gingival fibroblasts; TEGDMA; HEMA; UDMA; Glutathione

Summary Objectives: Oral and systemic cells are permanently exposed to various types of xenobiotics, such as dental restorative materials, which may subsequently cause adverse effects. Objective of the present investigation was to analyze the effects of three important resin monomers on the glutathione metabolism of human gingival fibroblasts after an incubation period of 4 h. Methods: Cells were exposed to various concentrations of 2-hydroxyethyl methacrylate (HEMA; 0.1–10 mM), triethylene-glycol dimethacrylate (TEGDMA; 0.05– 2.5 mM), and urethane dimethacrylate (UDMA; 0.005–0.25 mM). Subsequently, cellular glutathione (GSH) concentrations were determined after a treatment period of 4 h using the monobromobimane assay. Data were statistically evaluated using Tukey ANOVA with p!0.05. Results: GSH depletion was dependent on the type of the resin monomer: UDMAO TEGDMAOHEMA. The concentrations for a 50%-reduction of cellular GSH varied between 0.1 mM (0.05 mM) (UDMA), 0.33 mM (0.09 mM) (TEGDMA), and 1.6 mM (0.8 mM) (HEMA). Simultaneously, no decrease of cell numbers was found at any tested concentration. Significance: These data indicate that the investigated resins may cause cell damage due to depletion of intracellular GSH level even at low concentrations within a short period of time. The decrease of GSH is an early reaction, which is triggered prior to other cytotoxic alterations. Q 2005 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Introduction * Corresponding author. Tel.: C1 206 543 5948; fax: C1 206 543 7783. E-mail address: [email protected] (W. Geurtsen).

Residual monomers are released from many resinbased restorative materials in microgram to

0109-5641/$ - see front matter Q 2005 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2005.06.002

500 milligram amounts into the oral cavity after polymerisation [1–10]. Base monomers, such as urethane dimethacrylate (UDMA), and comonomers like triethyleneglycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA), are frequently found in aqueous eluates from polymerized dental resinous biomaterials. Previous studies revealed a variety of potential cytotoxic and metabolic effects due to leaching of these methacrylates from restorations, for instance tooth sensitivity [11], local immunological effects [12], chronic inflammatory reactions of human pulps [13–15], genotoxicity [16,17], and apoptosis [18–20]. Recently, it has been shown that HEMA and TEGDMA caused a depletion of the intracellular glutathione (GSH, L-g-glutamyl-L-cysteinylglycine) pool in different cell types [19,21–24]. GSH is a tripeptide present in virtually all animal cells [25]. In general, GSH is the most abundant intracellular thiol-type molecule with a low molecular weight, which is found in millimolar concentrations in mammalian cells. GSH is rapidly regenerated and thus an efficient protector against toxic influences from xenobiotics and endogenous detrimental metabolic substances, specifically reactive oxygen species (ROS). Therefore, GSH provides protection against oxidative damage [26]. On the other hand, an irreversible depletion of intracellular GSH may detrimentally impair the cells’ protective capacity against toxic influences by xenobiotics such as dental resin components, resulting in cell death [19]. Although few previous studies indicate that various monomeric substances may have a different effect on the initial and critical GSH depletion [19,21–24], no data about potential influences of the specific chemical characteristics of an individual resin component, in particular the number of reactive methacrylate groups of a monomer, on GSH depletion is available in the dental or medical literature. This information, however, would be very valuable for the understanding of the mechanisms resulting in a significant GSH exhaustion of exposed cells. Therefore, it was the objective of our study to analyze the effects of important monomers with one or two methacrylate groups on GSH metabolism and viability of primary human gingival fibroblasts, a cell-type, which is primarily exposed to toxic influences from released resin substances. The tested hypothesis was that compounds with two metharcylate groups cause a significantly more pronounced GSH depletion compared to substances with only one methacrylate group. HEMA was selected as representative of monomers with one

J. Volk et al. methacrylate group, whereas the other two investigated compounds TEGDMA and UDMA have two methacrylate groups.

Materials and methods Materials Dulbecco’s modified Eagle’s medium (DMEM), HEPES, penicillin, streptomycin and amphotericin, and fetal calf serum (FCS) were purchased from Biochrom (Berlin, Germany), NaHCO3 from Riedel de Hae ¨n (Seelze, Germany), and trypsin/EDTA from Sigma (Taufkirchen, Germany). Urethane dimethacrylate (UDMA), triethylene-glycol dimethacrylate (TEGDMA), and 2-hydroxyethyl methacrylate (HEMA) were gifts from VOCO (Cuxhaven, Germany) (Fig. 1). 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) was purchased from GIBCO BRL (Karlsruhe, Germany).

Cell cultures Primary human gingival fibroblasts (HGFs) 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 8C for 24 h at most in HBSS supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), and amphotericin (2.5 mg/ml) prior to amplification. The tissue samples were placed into 25-cm2 tissue culture flasks and grown in DMEM with 4.5 g/l glucose, 10 mM HEPES, NaHCO 3 (3.7 g/l), penicillin (100 U/ml), and streptomycin (100 mg/ml), supplemented with 10% fetal calf serum (FCS) at 37 8C 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).

Effects of monomers on GSH

Figure 1

501

Structural formula of the (co)monomers HEMA, TEGDMA, and UDMA.

Treatment of cells with monomers HGF from passages numbers 5–9 were seeded in 96well plates (1!104 cells/well) and allowed to grow for 44 h. Subsequently, HGFs were treated with various concentrations of HEMA (0.1–10 mM), TEGDMA (0.05–2.5 mM), or UDMA (0.005–0.5 mM) for 4 h. Stock solutions (200!) of the methacrylates were prepared in DMSO and were freshly diluted in DMEM prior to each experiment. The final concentration of DMSO did not exceed 0.5% (v/v). It was found in previous experiments that this DMSOconcentration is non-toxic for HGF (data not shown). Cells incubated with fresh medium containing 0.5% DMSO served as control.

Glutathione assays The relative intracellular GSH concentrations were determined using an assay with monobromobimane (MBBr) in microtiter plates as described previously [27]. 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, the fluorescence intensity of 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 measured again to determine the total cell number per well (PI stains DNA of non-vital cells only). MBBr-readings were then normalized to cell numbers based on Fmax in the presence of the surfactant Nonidet P-40. The PIreadings FPI and Fmax were used to estimate the vitality of cells as a function of membrane leakiness [27]. All readings were performed in a FLx 800 microplate fluorescence reader (BioTec, Neufahrn, Germany).

Statistical analysis Assays were run at least six times with each six replicates. Results were expressed as meansG standard deviations (SDs). Statistical analysis was performed by ANOVA with Tukey’s post test. P-values !0.05 were considered significant.

Results The effects of the three methacrylates (HEMA, TEGDMA, UDMA, Fig. 1) on the relative intracellular reduced glutathione content (GSH) of HGF are shown in Fig. 2(a)–(c). All monomers caused a dose-dependent depletion of intracellular GSH, and the ranking of the potency of the investigated resin substances to reduce the intracellular GSH pool to 50% (ED50) was UDMA (0.1G0.05 mM)OTEGDMA (0.33G0.09 mM)OHEMA (1.6G0.8 mM) (Table 1). HEMA induced a significant GSH depletion at

502

J. Volk et al. metabolism was caused by UDMA, which generated a pronounced decrease of cellular GSH at concentrations higher than 0.05 mM (67.5C15.9%), p! 0.001). None of the substances significantly reduced the total number of cells during the 4 h period of treatment (Table 2). However, UDMA significantly decreased the viability of the cells at concentrations of 0.25 mM and higher (p!0.05) (Fig. 3).

Discussion

Figure 2 Intracellular glutathione content in HGF treated with (a) HEMA, (b) TEGDMA, or (c) UDMA for 4 h in % of controls and normalized to cell numbers. Values are means (GSDs) (nZ6); *p!0.05, **p!0.005, ***p!0.001, statistically significant differences compared to controls.

a concentration of 0.5 mM (83.0C9.4%), p!0.005), TEGDMA caused an equivalent effect already at concentrations higher than 0.1 mM (75.3C11.0%), p!0.001). The most severe effect on cells’ GSH Table 1 ED50-concentrations (mM) of the (co) monomers as to the relative intracellular glutathione concentration in HGF treated for 4 h. (co)monomer

ED50 (mM)

HEMA TEGDMA UDMA

1.60 (0.80) 0.33 (0.09) 0.10 (0.05)

Values are means (SDs) (nZ6).

Our study revealed that micromolar concentrations of HEMA, TEGDMA, and UDMA deplete the intracellular GSH within a short period of time in a concentration-dependent manner (Fig. 2). The effect was up to 16-fold stronger for compounds with two methacrylate groups (TEGDMA, UDMA) compared to HEMA with one methacrylate group (Table 1). The concentrations causing a 50% reduction of the intracellular GSH content (ED50) ranged between 0.1 mM (UDMA), 0.33 mM (TEGDMA), and 1.6 mM (HEMA). Previous studies revealed that a depletion of GSH without subsequent recovery may lead to ‘oxidative stress’ and an inhibition and/or alteration of important cellular components, like lipid peroxidation in cell membranes, modification of transductional pathways or DNA damage [28–30]. Recent experiments in our laboratory demonstrated that individual resin monomers like TEGDMA triggered apoptosis in association with a marked GSH depletion [18,19]. Therefore, it may be hypothesized that low concentrations of various resin (co)monomers could accumulate intracellularly within a short period of time to an overall toxic level, which could then result in an irreversible exhaustion of the cells’ detoxifying GSH pool, thus finally causing apoptosis. Although it was found that TEGDMA can increase the concentration of reactive oxygen species (ROS) in human gingival fibroblasts the depletion of the cells’ GSH pool, which is also associated with TEGDMA does not seem to result primarily from an oxidative process. It should be rather caused by a direct interaction of TEGDMA and GSH, for instance due to a Michael-type reaction [23,27,31–33]. Recent studies revealed that initiating substances, like camphorquinone in combination with a co-initiating compound, generate high amounts of ROS in vitro and in vivo that can cause DNA strand breaks [34–36]. These findings, therefore, suggest that a combination of TEGDMA and initiating substances that are also released from dental resins in significant quantities

Effects of monomers on GSH

503

Table 2 Total cell number (% of control) after incubation with HEMA, TEGDMA, or UDMA for 4 h determined by PI staining of surfactant-treated cells (controlZ100%). HEMA (mM)

% of control

TEGDMA (mM)

% of control

UDMA (mM)

% of control

0.1 0.5 1.25 2.5 5.0 10.0

99 (10) 105 (11) 102 (11) 100 (8) 99 (12) 96 (16)

0.05 0.1 0.5 1.0 1.25 2.5

103 (12) 105 (11) 107 (10) 102 (7) 102 (10) 109 (14)

0.005 0.01 0.05 0.1 0.25 0.5*

107 104 113 113 112 124

(12) (11) (15) (16) (19) (17)

Values are means (GSDs) (nZ6,*nZ3).

after polymerization may exhibit a synergistic effect regarding the generation of ROS. It may be speculated that ROS caused by TEGDMA in association with another ROS-inducing resinous substance may directly affect the intracellular GSH concentration. Interestingly, it was recently reported that non-toxic concentrations of various resin substances including TEGDMA and camphorquinone altered the cellular GSH concentration, which might be indicative of oxidative stress [37]. In addition, it was observed that TEGDMA might act as non-competitive antagonist of glutathione transferase P1 that plays an important role in the cellular detoxification of xenobiotics [32]. Interestingly, P1 is almost exclusively expressed in human fibroblasts [38]. This enzyme reveals a hydrophobic substratebinding site in close vicinity to the glutathionebinding site that has a broad specificity for numerous hydrophobic compounds, such as TEGDMA [39]. Taken together, these data and our results indicate a rapid and very pronounced GSH decrease at low concentrations and thus support the hypothesis that TEGDMA can directly react with GSH, which may finally cause apoptosis [31]. Contrary to HEMA and TEGDMA, UDMA significantly reduced the viability of the cells at concentrations of R0.25 mM (p!0.05) (Fig. 3) associated with a marked depletion of the intracellular GSH pool to 33.7% (6.6%). Although HEMA and TEGDMA caused a more pronounced depletion of GSH at concentrations higher than 0.25 mM, no cytotoxic effects or reduced cell viability was observed due to incubation with these two compounds. A high cytotoxic potency of UDMA was also reported in previous studies [40]. These reports and our findings indicate that this base monomer may trigger various toxic mechanisms in addition to GSH depletion, such as interactions with cell membranes. The highly lipophilic monomer UDMA may penetrate cell membranes. Subsequently it may react with the membranes’ lipid layer resulting in a disorientation of the bilayer and membrane damage [41,42].

HEMA with only one methacrylate group caused a significantly lower effect on the GSH metabolism. These data coincides with previous unspecific cytotoxicity studies, which consistently indicated higher ED50-concentrations and thus less severe biological–chemical interactions of this substance compared to TEGDMA or UDMA [7]. Only little information about specific cytotoxic mechanisms of HEMA is available in the dental literature. Chang et al. [43] showed that HEMA produced growth inhibition of human pulp fibroblasts and human gingival S–G cells in a dose-dependent manner, which may be partially explained by induction of cell cycle perturbation. In accordance with our results they found a GSH depletion, increased ROS production, and apoptosis in cells exposed to HEMA. Reichl et al. [44,45] hypothesized that HEMA and TEGDMA are intracellularly metabolized yielding oxidative radical metabolites. Walther et al. [46] confirmed this effect. They found that various antioxidative substances diminished acute toxic effects of both methacrylates. But this effect was much less detrimental in cells treated with HEMA compared to TEGDMA-incubated cells. These data also show a significant contribution of the number of methacrylate groups to the toxic potency of a resin component.

Figure 3 Percentage of vital HGF after 4 h treatment with HEMA, TEGDMA, or UDMA in % of controls. Values are means (SD) (nZ6); *p!0.05, statistically significant differences compared to controls.

504 Taken together, our findings demonstrate that all investigated resin monomers influence the metabolism of primary human gingival fibroblasts. The decrease of the intracellular GSH pool of HGF due to the tested compounds is an early and rapid reaction preceding other cytotoxic alterations, such as cell growth inhibition. In addition, our results clearly indicate that this process is associated with chemical characteristics, specifically the number of methacrylate groups.

Conclusions Resin-modified materials release high quantities of various methacrylates or initiating substances into aqueous environments shortly after polymerization. Subsequently, lower amounts of these ingredients are segregated over an extended period of time [7,47]. These compounds may cause additive or, in case of different mechanistic reactions, synergistic toxic effects on the cellular GSH metabolism. Thus, combinations of various individual resin compounds could generate longterm toxic reactions even at low or non-toxic concentrations, which may be causative for the observed chronic inflammatory reactions of human pulps exposed to resin materials [13–15,48] or other oral and systemic tissues.

Acknowledgements Our thanks are due to Ms A. Beckedorf for her excellent technical assistance. This study was supported by a grant of the DFG (Le 851/2-2).

References [1] Gerzina TM, Hume WR. Effect of hydrostatic pressure on the diffusion of monomers through dentin in vitro. J Dent Res 1995;74:369–73. [2] Gerzina TM, Hume WR. Diffusion of monomers from bonding resin–resin composite combinations through dentine in vitro. J Dent 1996;24:125–8. [3] Hume WR, Gerzia TM. Bioavailability of components of resin-based materials which are applied to teeth. Crit Rev Oral Biol Med 1996;7:172–9. [4] Geurtsen W. Substances released from dental resin composites and glass ionomer cements. Eur J Oral Sci 1998;106:687–95. [5] Lee SY, Huang HM, Lin CY, Shih YH. Leached components from dental composites in oral simulating fluids and the resultant composite strengths. J Oral Rehabil 1998;25: 575–88.

J. Volk et al. [6] Pelka M, Distler W, Petschelt A. Elution parameters and HPLC-detection of single components from resin composite. Clin Oral Investig 1999;3:194–200. [7] Geurtsen W. Biocompatibility of resin-modified filling materials. Crit Rev Oral Biol Med 2000;11:333–55. [8] Munksgaard EC, Peutzfeldt A, Asmussen E. Elution of TEGDMA and BisGMA from a resin and a resin composite cured with halogen or plasma light. Eur J Oral Sci 2000;108: 341–5. [9] Michelsen VB, Lygre H, Skalevik R, Tveit AB, Solheim E. Identification of organic eluates from four polymer-based dental filling materials. Eur J Oral Sci 2003;111:263–71. [10] Issa Y, Watts DC, Brunton PA, Waters CM, Duxbury AJ. Resin composite monomers alter MTT and LDH activity of human gingival fibroblasts in vitro. Dent Mater 2004;20:12–20. [11] Unemori M, Matsuya Y, Akashi A, Goto Y, Akamine A. Composite resin restoration and postoperative sensitivity: clinical follow-up in an undergraduate program. J Dent 2001;29:7–13. [12] Jontell M, Hanks CT, Bratel J, Bergenholtz G. Effects of unpolymerized resin components on the function of accessory cells derived from the rat incisor pulp. J Dent Res 1995;74:1162–7. [13] Costa CA, Teixeira HM, do Nascimento AB, Hebling J. Biocompatibility of an adhesive system and 2-hydroxyethylmethacrylate. ASDC J Dent Child 1999;66:337–42 see also p. 294. [14] Costa CA, Hebling J, Hanks CT. Current status of pulp capping with dentin adhesive systems: a review. Dent Mater 2000;16:188–97. [15] Costa CA, Giro EM, do Nascimento AB, Teixeira HM, Hebling J. Short-term evaluation of the pulpo-dentin complex response to a resin-modified glass-ionomer cement and a bonding agent applied in deep cavities. Dent Mater 2003;19:739–46. [16] Schweikl H, Schmalz G. Triethylene glycol dimethacrylate induces large deletions in the hprt gene of V79 cells. Mutat Res 1999;438:71–8. [17] Kleinsasser NH, Wallner BC, Harreus UA, Kleinjung T, Folwaczny M, Hickel R, et al. Genotoxicity and cytotoxicity of dental materials in human lymphocytes as assessed by the single cell microgel electrophoresis (comet) assay. J Dent 2004;32:229–34. [18] Janke V, von Neuhoff N, Schlegelberger B, Leyhausen G, Geurtsen W. TEGDMA causes apoptosis in primary human gingival fibroblasts. J Dent Res 2003;82:814–8. [19] Engelmann J, Janke V, Volk J, Leyhausen G, von Neuhoff N, Schlegelberger B, et al. Effects of BisGMA on glutathione metabolism and apoptosis in human gingival fibroblasts in vitro. Biomaterials 2004;25:4573–80. [20] Paranjpe A, Bordador LC, Wang MY, Hume WR, Jewett A. Resin monomer 2-Hydroxyethyl methacrylate (HEMA) is a potent inducer of apoptotic cell death in human and mouse cells. J Dent Res 2005;84:172–7. [21] Engelmann J, Leyhausen G, Leibfritz D, Geurtsen W. Metabolic effects of dental resin components in vitro detected by NMR spectroscopy. J Dent Res 2001;80:869–75. [22] Stanislawski L, Soheili-Majd E, Perianin A, Goldberg M. Dental restorative biomaterials induce glutathione depletion in cultured human gingival fibroblast: protective effect of N-acetyl cysteine. J Biomed Mater Res 2000;51: 469–74. [23] Stanislawski L, Lefeuvre M, Bourd K, Soheili-Majd E, Goldberg M, Pe ´rianin A. TEGDMA-induced toxicity in human fibroblasts is associated with early and drastic glutathione depletion with subsequent production of oxygen reactive species. J Biomed Mater Res 2003;66A:476–82.

Effects of monomers on GSH [24] Soheili-Majd E, Goldberg M, Stanislawski L. In vitro effects of ascorbate and Trolox on the biocompatibility of dental restorative materials. Biomaterials 2003;24:3–9. [25] Meister A, Anderson ME. Glutathione. Annu Rev Biochem 1983;52:711–60. [26] Dickinson DA, Forman HJ. Cellular glutathione and thiols metabolism. Biochem Pharmacol 2002;64:1019–26. [27] Engelmann J, Leyhausen G, Leibfritz D, Geurtsen W. Effect of TEGDMA on the intracellular glutathione concentration of human gingival fibroblasts. J Biomed Mater Res (Appl Biomater) 2002;63:746–51. [28] Cejas P, Casado E, Belda-Iniesta C, De Castro J, Espinosa E, Redondo A, et al. Implications of oxidative stress and cell membrane lipid peroxidation in human cancer. Cancer Causes Control 2004;15:707–19. [29] Miyamoto Y, Koh YH, Park YS, Fujiwara N, Sakiyama H, Misonou Y, et al. Oxidative stress caused by inactivation of glutathione peroxidase and adaptive responses. Biol Chem 2003;384:567–74. [30] Lander HM, Tauras JM, Ogiste JS, Hori O, Moss RA, Schmidt AM. Activation of the receptor for advanced glycation end products triggers a p21(ras)-dependent mitogen-activated protein kinase pathway regulated by oxidant stress. J Biol Chem 1997;272:17810–4. [31] Geurtsen W, Leyhausen G. Chemical–biological interactions of the resin monomer triethyleneglycol-dimethacrylate (TEGDMA). J Dent Res 2001;80:2046–50. [32] Lefeuvre M, Bourd K, Loriot MA, Goldberg M, Beaune P, Pe ´rianin A, et al. TEGDMA modulates glutathione transferase P1 activity in gingival fibroblasts. J Dent Res 2004;83:914–9. [33] Engelmann J, Volk J, Leyhausen G, Geurtsen W. ROSformation and glutathione level in human oral fibroblasts exposed to TEGDMA and camphorquinone, in press. [34] Pagoria D, Lee A, Geurtsen W. The effect of camphorquinone (CQ) and CQ-related photosensitizers on the generation of reactive oxygen species and the production of oxidative DNA damage. Biomaterials 2005;26:4091–9. [35] Winter K, Pagoria D, Geurtsen W. The effect of antioxidants on oxidative DNA damage induced by visible-light-irradiated camphorquinone/N,N-dimethyl-p-toluidine. Biomaterials 2005;26:5321–9. [36] Pagoria D, Geurtsen W. The effect of N-acetyl-L-cysteine and ascorbic acid on visible-light-irradiated camphorquinone/N,N-dimethyl-p-toluidine-induced oxidative stress in two immortalized cell lines. Biomaterials, in press.

505 [37] Noda M, Wataha JC, Lewis JB, Kaga M, Lockwood PE, Messer RL, et al. Dental adhesive compounds alter glutathione levels but not glutathione redox balance in human THP-1 monocytic cells. J Biomed Mater Res B Appl Biomater 2005;73:308–14. [38] Hansson J, Edgren MR, Egyhazi S, Hao XY, Mannervik B, Ringborg U. Increased cisplatin sensitivity of human fibroblasts from a subject with inherent glutathione deficiency. Acta Oncol 1996;35:683–90. [39] Eaton DL, Bammler TK. Concise review of the glutathione S-transferases and their significance to toxicology. Toxicol Sci 1999;49:156–64. [40] Geurtsen W, Lehmann F, Spahl W, Leyhausen G. Cytotoxicity of 35 dental resin composite monomers/additives in permanent 3T3 and three human primary fibroblast cultures. J Biomed Mater Res 1998;41:474–80. [41] Fujisawa S, Kadoma Y, Komoda Y. Changes in 1H-NMR chemical shifts of Bis-GMA and its related methacrylates induced by their interaction with phosphatidylcholine/cholesterol liposomes. Dent Mater J 1991;10:121–7. [42] Ahlers J, Cascorbi I, Foret M, Gies A, Kohler M, Pauli W, et al. Interaction with functional membrane proteins—a common mechanism of toxicity for lipophilic environmental chemicals? Comp Biochem Physiol C 1991;100:111–3. [43] Chang HH, Guo MK, Kasten FH, Chang MC, Huang GF, Wang YL, et al. Stimulation of glutathione depletion, ROS production and cell cycle arrest of dental pulp cells and gingival epithelial cells by HEMA. Biomaterials 2005;26: 745–53. [44] Reichl FX, Durner J, Hickel R, Kunzelmann KH, Jewett A, Wang MY, et al. Distribution and excretion of TEGDMA in guinea pigs and mice. J Dent Res 2001;80:1412–5. [45] Reichl FX, Durner J, Kehe K, Manhart J, Folwaczny M, Kleinsasser N, et al. Toxicokinetic of HEMA in guinea pigs. J Dent 2002;30:353–8. [46] Walther UI, Siagian II W, alther SC, Reichl FX, Hickel R. Antioxidative vitamins decrease cytotoxicity of HEMA and TEGDMA in cultured cell lines. Arch Oral Biol 2004;49: 125–31. [47] Mazzaoui SA, Burrow MF, Tyas MJ, Rooney FR, Capon RJ. Long-term quantification of the release of monomers from dental resin composites and a resin-modified glass ionomer cement. Biomed Mater Res 2002;63:299–305. [48] Hebling J, Giro EM, Costa CA. Biocompatibility of an adhesive system applied to exposed human dental pulp. J Endod 1999;25:676–82.