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TEGDMA Modulates Glutathione Transferase P1 Activity in Gingival Fibroblasts
1 Faculté de Chirurgie Dentaire, Laboratoire de Biologie et Physiopathologie Cranio-Faciale, 1 rue Maurice Arnoux, F-92120 Montrouge, France; Correspondence: * corresponding author, lenastan @ hotmail.com
Dental resinous materials can contain large amounts (from 30 to 50%) of triethylene-glycol-dimethacrylate (TEGDMA). This compound leaches into aqueous media and is toxic to dental pulp, as well as to gingival fibroblasts in vitro. To elucidate the mechanism of TEGDMA toxicity, we investigated the effects on glutathione (GSH) level and glutathione transferase P1 (GSTP1) activity in cultured human gingival fibroblasts. TEGDMA cytotoxic concentrations (from 0.5 to 2 mM) induced a depletion of GSH without formation of oxidized GSH (GSSG). In fibroblasts expressing the wild-type GSTP1, TEGDMA both inhibited and potentiated GSTP1 activity at high (IC50 = 1.1 mM) and low concentrations, respectively. In contrast, cells expressing the GSTP1 *A/*B variant showed a weak inhibition of GST activity only, associated with greater sensitivity to drug toxicity. Biochemical analysis of GSTP1 inhibition revealed that TEGDMA is a non-competitive antagonist with respect to GSH and substrate. Thus, TEGDMA interference with GSH and GSTP1 activity may contribute to dental-resin-induced adverse effects.
Key Words: TEGDMA • GST • glutathione • gingival fibroblasts
Triethylene glycol dimethacrylate (TEGDMA) is a common component of the bonding agents and resin composites used in dentistry for various purposes, including restorative dentistry. TEGDMA inclusion reduces viscosity of resin materials and enhances bond strengths to dentin. However, it is now recognized that TEGDMA monomers leach from resin composite after polymerization (Hume and Gerzina, 1996; Spahl et al., 1998), which may induce various deleterious effects, including inflammatory reactions, cytotoxicity, mutagenesis, and apoptosis (Schweikl and Schmalz, 1999; Geurtsen, 2000; Janke et al., 2003). TEGDMA is also one of the common sensitizers of contact dermatitis in dental personnel, and induces allergic responses (Ortengren, 2000). Previously, along with other groups, we have shown that the treatment of human gingival fibroblasts in vitro with TEGDMA, or with eluates from different dental restorative biomaterials containing TEGDMA, induces variable cytotoxic effects associated with a depletion of intracellular glutathione (Stanislawski et al., 1999, 2000, 2003; Engelmann et al., 2001). We further reported that TEGDMA-mediated toxic properties in human fibroblasts were associated with significant production of reactive oxygen species (ROS), which can be prevented by thiol-containing compounds such as N-acetyl cysteine and other anti-oxidant vitamins (Stanislawski et al., 2003). Although the mechanism of GSH depletion by TEGDMA remains unknown, various processes may potentially be involved, including oxidation of GSH by ROS, a direct TEGDMA interaction with GSH, or a glutathione transferase (GST)-mediated interaction. Mammalian GST is a multi-gene family of enzymes playing an important role in the maintenance of cellular redox status (Eaton and Bammler, 1999) and in the metabolism of a wide variety of electrophilic compounds with alkylating properties (Kensler, 1997). Four major (Alpha, Mu, Pi, and Theta) classes of soluble GST, 4 minors (Zeta, Sigma, Omega, and Kappa), and 1 microsomal GST have been described (Mannervik et al., 1992). Human fibroblasts most exclusively express the GSTP1 isoform, which is polymorphic due to an amino acid substitution Ile105Val within the active site of the enzyme (Zimniak et al., 1994). Although this substitution might affect both the enzyme activity and substrate affinity, the consequences of this polymorphism for drug cytotoxicity and modulation of GST activity by xenobiotics, such as TEGDMA, remain unknown. The interaction between TEGDMA and GSTP1 is still unknown and could be modulated by this polymorphism. On the basis of variability of TEGDMA toxicity on fibroblasts (Stanislawski et al., 2003), we hypothesized that TEGDMA acts differently, depending on GSTP1 isoforms. Thus, in this study, we determined the effects of TEGDMA on intracellular GSH, GSSG, and GST activity in human fibroblasts. The GST activity was further studied in fibroblasts corresponding to the three different GSTP1 variants at position 105 (GSTP1 *A/*A, GSTP1 *A/*B, and GSTP1 *B/*B) in relation to TEGDMA cytotoxic effects. Finally, the GSTP1-TEGDMA interaction was further characterized in a cell-free system.
Materials TEGDMA, human placental glutathione transferase Pi (GST-Pi), and other reagents were purchased from Sigma (St. Louis, MO, USA). For convenience, placental GST-Pi has been designated GSTP1.
Cell Culture and Cytotoxicity Assay
GST Genotyping, GSH, GSSG Levels, and GST Activity of Fibroblasts GST activity in fibroblast homogenates was measured in the presence of 1 mM CDNB (1-chloro-2,4-dinitrobenzene) and 1 mM GSH, as previously described (Habig et al., 1974). In some experiments, commercial GST was used at a final concentration of 0.22 unit/mL. We monitored the reaction spectrophotometrically by recording the increase in absorbance at 340 nm. For GSH and GST assays, reactions were allowed to proceed in initial rate conditions and were not influenced by TEGDMA. Protein content was determined with use of the Bio-Rad’s Protein Assays (Bio-Rad Laboratories Inc., Hercules, CA, USA). Data are expressed as the mean ± SD (standard deviation). Significant differences between groups of experiments were determined by one-way analysis of variance, followed by Student’s t test with a threshold of P < 0.05. All experiments were performed in triplicate and were repeated from 2 to 4 times.
TEGDMA-induced Depletion of GSH without Formation of GSSG in Fibroblasts and in a Cell-free System Treatment of fibroblasts with TEGDMA at a TD50 concentration, which provides 50% of cytotoxicity after 24 hrs—i.e., between 0.5 and 1.5 mM (Stanislawski et al., 2003)—induced a rapid time-dependent depletion of GSH (17% decrease within 15 min, 84% within 1 hr, P < 0.05 for both) and almost complete depletion within 3 hrs (Fig. 1A  ). The content of GSSG did not increase after six-hour treatment with TEGDMA. Direct incubation of TEGDMA with GSH (cell-free system) also resulted in GSH depletion without formation of GSSG. However, the GSH depletion rate in vitro was lower than in fibroblasts (25% decrease within 1 hr, P < 0.0001, Fig. 1B), suggesting that, in fibroblasts, an additional process (i.e., possibly GSTP1) catalyzed the GSH depletion. To explore this possibility, we added GSTP1 to the GSH+TEGDMA mixture in the cell-free system. In these conditions, an additional GSH depletion of 8 nmoles occurred within 1 hr (18 ± 1) (Fig. 1D). Unlike TEGDMA, reactive oxygen species induced oxidation of GSH to GSSG in vitro (Fig. 1C).
TEGDMA-induced Modulation of GST Activity in Fibroblasts and a Cell-free System GSTP1 is the GST predominantly expressed in human fibroblasts (Hansson et al., 1996). This enzyme has been shown to be polymorphic in human populations (Eaton et al., 1999). To explore GSTP1 polymorphism as a risk factor for fibroblast susceptibility to TEGDMA toxicity (Stanislawski et al., 2003), we genotyped human gingival fibroblasts for GSTP1 variants, and investigated the GST activity of the wild-type GSTP1 *A/*A and mutants GSTP1 *A/*B and GSTP1 *B/*B after TEGDMA treatment of fibroblasts. Treatment of fibroblasts expressing the GSTP1 *A/*A in the presence of high TEGDMA concentrations (from 0.5 to 2 mM) inhibited GST activity in a concentration-dependent manner (IC50 of 1.1 mM), whereas low concentrations (from 0.1 to 0.5 mM) increased GST activity only weakly (Fig. 2 ). The activity of GSTP1 *B/*B and *A/*B variants was not significantly increased by TEGDMA, whereas high concentrations (from 1 to 2 mM) induced a weak and stable inhibitory effect (approximately 20%), despite increasing concentrations of TEGDMA.
The activity of purified GSTP1 treated with 1 mM TEGDMA in the cell-free system also decreased in a time-dependent manner, starting at 75 min (32%, P < 0.01), with complete inhibition achieved after 3 hrs (Fig. 3A ). ‘Half-inhibition’ of GST activity occurred after 90 min of GST treatment, a duration which was chosen for all subsequent experiments. Low TEGDMA concentrations (from 0.1 to 0.4 mM) increased GST activity only weakly (from 18% to 20%, P < 0.05), whereas higher concentrations impaired GST activity, starting at 1 mM (24%, P<0.01), with almost complete inhibition obtained with 1.5 mM TEGDMA (Fig. 3B).
A Lineweaver-Burk plot of the data shows that the Km values for GSH was of the same order in the presence or absence of TEGDMA (0.66 ± 0.18 and 0.72 ± 0.28 mM, respectively). Km is the Michaelis constant, which corresponds to the substance concentration of substrate at which the rate of reaction is equal to one-half of the maximum rate. The Km values for control cells are close to those previously published (0.1 mM for GSH and from 0.8 to 3.0 mM for CDNB of GSTP1*A and GSTP1*B, respectively) (Zimniak et al., 1994). No change was noted in the Km values with respect to CDNB in the presence or absence of TEGDMA (0.58 ± 0.02 and 0.57 ± 0.03 mM, respectively). In contrast, the Vmax value (maximal activity of the enzyme) was decreased by approximately 63%. The TEGDMA IC50 values for GST inhibition in fibroblasts and in the cell-free system were similar (1.1 ± 0.32 and 1.12 ± 0.02 mM). (Ki values were 0.92 and 1.06 mM, respectively.)
TEGDMA-induced Cytotoxicity in Fibroblasts According to the GSTP1 Genotype
The present study provides new insights into the mechanism by which TEGDMA induces cytotoxicity of human gingival fibroblasts. Analysis of our data confirms that TEGDMA induced a depletion of GSH in fibroblasts (Engelmann et al., 2001; Stanislawski et al., 2003), a process that also occurred in a cell-free system (Fig. 1B  ). Although TEGDMA induced significant production of ROS in gingival fibroblasts (Stanislawski et al., 2003), the depletion of GSH induced by TEGDMA did not appear to result from an oxidative process, but might involve a direct interaction of TEGDMA with GSH. This is supported by our observation that TEGDMA, in contrast to ROS, failed to induce detectable formation of GSSG. It is also supported by the formation of an adduct GSH-TEGDMA, revealed by mass spectroscopy analysis (data not shown). In fibroblasts, GSH depletion was much greater and more rapid than in vitro and was accelerated in a cell-free system in the presence of GSTP1. This suggests that the GSH depletion may be catalyzed by GSTP1 in the cell. The GSTs possess a glutathione-binding site (G-site) displaying high specificity and a closely adjacent hydrophobic substrate-binding site (H-site) with a broad specificity toward hydrophobic compounds (Eaton and Bammler, 1999). GSTP1, the isoform predominantly expressed in human fibroblasts (Hansson et al., 1996), is polymorphic due to the substitution Ile105Val located close to the enzyme substrate-binding site (Zimniak et al., 1994). Under our experimental conditions, the activity of the homozygous wild-type GSTP1 was slightly higher than that of the homozygous mutant. Interestingly, the activity of this mutant was weakly inhibited in TEGDMA-treated fibroblasts (20%), in contrast to wild- type GSTP1, where it was strongly inhibited by approximately 80%. The difference in toxicity levels between gingival fibroblasts expressing GST P1*A and those expressing GST P1*B should be confirmed by in vitro experiments after wild-type and mutant cDNA transfection. Similarly, the use of purified enzymes could lead to analyses of the interactions between TEGDMA and GSTP1, and to kinetic studies of GSTP1 in the presence of GSH and TEGDMA. The relative "resistance" to TEGDMA indicates that the Ile105Val substitution could play a crucial role in the interaction between TEGDMA and GSTP1. The Ile105 residue appears to play a key role in modulation of GST activity by TEGDMA, since the mutation also abolished the potentiating effect, induced by low TEGDMA concentrations (0.1 mM), found here with the wild-type enzyme activity in fibroblasts. A modulator effect of TEGDMA, similar to that observed in fibroblasts with GSTP1 *A/*A, was also reproduced in a cell-free system, with a purified placental GSTP1 preparation containing approximately 51 and 6% of GSTP1 *A/*A and *B/*B, respectively (Harries et al., 1997).
These similarities indicate that TEGDMA may likely modulate the activity of GSTP1 directly in vivo. In the in vitro model for GST inhibition, we found no changes in the Km values in the presence or absence of TEGDMA, whereas the Vmax value was decreased, which indicates that TEGDMA acts as a non-competitive GST antagonist with respect to GSH and CDNB. This suggests that TEGDMA may interact in the vicinity of the substrate-binding site or with a distinct binding site. Thus, in addition to their role in catalyzing the conjugation of electrophilic substrates to GSH, the GSTs are able to bind a wide range of endogenous and exogenous ligands non-catalytically. In this context, a second non-substrate (ligand)-binding site located in, or adjacent to, the H-site was recently described for GSTP1 (Oakley et al., 1999). The binding of various ligand molecules to this binding site induced a non-competitive inhibition toward the substrate CDNB. Alternatively, TEGDMA interaction with GST may occur through direct binding with SH residues. This is supported by our cell-free system study, indicating GSH depletion without GSSG formation. One potential candidate for this interaction is the cysteine residue located in the substrate (H) binding site (Cys-47), as suggested by previous works on the inhibition of GST P1 by The biological relevance of the modulation of GST activity by TEGDMA remains unknown. Methacrylate metabolism occurs mainly through ester hydrolysis, and GST-mediated detoxification occurs only when the preference pathway is blocked, or when the cellular concentration of methacrylate is high (Elovaara et al., 1983). In this study, we showed that fibroblasts with GSTP1 *A/*A variant are less sensitive to TEGDMA toxicity than are cells with GSTP1 *B/*B. In contrast, GSTP1 *A/*A activity was markedly modulated, unlike the GSTP1 *B/*B variant, which showed only weak inhibition. These observations suggest that GSTP1 *A/*A may have a protective effect. Although the molecular mechanism of this phenomenon remains to be elucidated, it is tempting to postulate that GSTP1 *B/*B may be less effective than GSTP1 *A/*A in detoxifying TEGDMA. Besides detoxifying TEGDMA, GSTP1 may protect the cells by other mechanisms. Indeed, GSTP1 plays an important role in detoxification of base propenals, which is the natural degradation product of DNA (Berkhane et al., 1994). TEGDMA has been reported to induce large DNA sequence deletions, gene mutations at the hprt locus, DNA fragmentation (Schweikl and Schmalz, 1999), and apoptosis, although the respective contributions of TEGDMA-induced modulation of GSTP1, GSH depletion, and ROS production in DNA alterations remain to be elucidated. Recent reports have showed that the GSTP1 monomer is an inhibitor of c-Jun N-terminal kinase, a member of the mitogen-activated protein (MAP) which plays an important role in transcriptional activities (Adler et al., 1999). This inhibition is lost during oxidative stress, because of covalent dimerization of GSTP1 monomers. The resulting effect on c-Jun will affect proliferation and expression of cell-cycle regulators and apoptosis. Besides toxicity, TEGDMA produces various allergic reactions, such as dermatitis and respiratory hypersensivity, which are important problems in patients, particularly in dental personnel (Geukens and Goossens, 2001). On the other hand, polymorphism of the GSTP1 has been associated with respiratory allergies and the variant GSTP1 *B/*B with a protective role (Fryer et al., 2000; Hemmingsen et al., 2001). Whether the TEGDMA-induced allergic response is also related to GSTP1 polymorphism may be of clinical interest. In conclusion, we show here that TEGDMA induces a depletion of GSH and modulates the GSTP1 activity in both fibroblasts and a cell-free system. This effect on enzyme activity is significantly more marked in the wild-type enzyme compared with the mutant one. Biochemical analysis of TEGDMA interaction with GSTP1 identifies TEGDMA as a non-competitive antagonist of GSTP1. Analysis of our data strongly suggests that GSTP1 polymorphism could be involved in inter-individual susceptibility to TEGDMA cytotoxicity.
This investigation was supported by the Ministry of Education. We thank Mr. O. Laprevote and Mr. V. Guérineau (Laboratoire de spectrométrie de masse, Institut de chimie des substances naturelles, CNRS, Gif sur Yvette, France) for mass spectroscopy analysis. We also thank Dr. Nivet (Hôpital de Saint Cloud) for providing samples of gingival tissue. Received for publication February 10, 2004. Revision received August 31, 2004. Accepted for publication September 17, 2004.
Journal of Dental Research, Vol. 83, No. 12,
914-919 (2004) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
,β- unsaturated aldehydes (
