| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
RESEARCH REPORTS |
1 Faculté de Chirurgie Dentaire, Laboratoire de Biologie et Physiopathologie Cranio-Faciale, 1 rue Maurice Arnoux, F-92120 Montrouge, France;
2 Centre Universitaire des Saints-Pères, INSERM U 490, Toxicologie Moléculaire et Service de Biochimie, Hôpital Européen Georges Pompidou, Paris, France; and
3 Institut Cochin, CNRS UMR 8104, INSERM U 567, Paris, France;
* corresponding author, lenastan @ hotmail.com
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
KEY WORDS: TEGDMA • GST • glutathione • gingival fibroblasts
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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 & METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cell Culture and Cytotoxicity Assay
Gingival fibroblasts were obtained from premolars extracted during orthodontic treatment with informed consent of the patients, following French regulations as previously described (Stanislawski et al., 2000), and used between passage 2 and passage 6 for treatment with TEGDMA or vehicle (DMSO). The DMSO concentrations used varied from 0.004 to 0.17%. Cell viability was assessed by the MTT test and expressed as a percentage of that of control cells. TEGDMA contains 80 ppm hydroquinone, but we found that, at final concentrations (from 0.001 to 0.047), it showed no toxicity or effect on GSH in fibroblasts.
GST Genotyping, GSH, GSSG Levels, and GST Activity of Fibroblasts
DNA from human gingival fibroblasts was extracted by means of a commercial kit (Qiagen S.A., Courtaboeuf, France) and genotyped for GSTP1 as previously described (Stucker et al., 2002). GSH and GSSG levels in untreated and TEGDMA-treated cells were determined spectrophotometrically by the glutathione reductase recycling assay. GSSG was determined after GSH blocking with 2-vinylpyridine as described (Griffith, 1980). In control experiments, GSH depletion was induced in the presence of 0.05–0.35 mM H2O2 and 100 µM FeCl2.
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.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). 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).
|
). 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.
|
). ‘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).
|
TEGDMA-induced Cytotoxicity in Fibroblasts According to the GSTP1 Genotype
TEGDMA-mediated cytotoxicity of fibroblasts was not detectable for short periods of cell treatment, up to 6 hrs (data not shown). The concentration that caused 50% of toxicity (TD50) was therefore determined after 24 hrs of cell treatment with TEGDMA and showed no significant difference between the *A/*A group (1.09 ± 0.77 mM, n = 11) and the *A/*B group (1.05 ± 0.64 mM, n = 12) (Fig. 4). In contrast, the TD50 value of the *B/*B group (0.48 ± 0.2 mM, n = 5) was significantly lower (P < 0.05), with very little inter-individual variability (0.2 mM).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). 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 
,ß- unsaturated aldehydes (Iersel et al., 1997). However, the other 3 cysteine residues described in GSTP1 cannot be excluded (Tamai et al., 1990).
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.
|
ACKNOWLEDGMENTS |
|---|
Received February 10, 2004; Last revision August 31, 2004; Accepted September 17, 2004
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Berkhane K, Widersten M, Engström A, Kozarich JW, Mannervik B (1994). Detoxication of base propenals and other ,ß-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione S-transferases. Proc Natl Acad Sci USA 91:1480–1484.
Eaton DL, Bammler TK (1999). Concise review on the glutathione S-transferases and their significance to toxicology. Toxicol Sci 49:156–164.
Elovaara E, Kivistö H, Vainio H (1983). Effects of methyl methacrylate on non-protein thiols and drug metabolizing enzymes in rat liver and kidneys. Arch Toxicol 52:109–121.[ISI][Medline]
Engelmann J, Leyhausen G, Leibfritz D, Geurtsen W (2001). Metabolic effects of dental resin components in vitro detected by NMR spectroscopy. J Dent Res 80:869–875.
Fryer AA, Bianco A, Hepple M, Jones PW, Strang RC, Spiteri MA (2000). Polymorphism at the glutathione S-transferase GST P1 locus. A new marker of bronchial hyperresponsiveness and asthma. Am J Respir Crit Care Med 161:1437–1442.
Geukens S, Goossens A (2001). Occupational contact allergy to (meth)acrylates. Contact Derm 44:153–159.
Geurtsen W (2000). Biocompatibility of resin-modified filling materials. Crit Rev Oral Biol Med 11:333–355.[Abstract]
Griffith OW (1980). Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 106:207–212.[ISI][Medline]
Habig W, Pabst MJ, Jakoby WB (1974). Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J Biol Chem 249:7130–7139.
Hansson J, Edgrein MR, Egyhazi S, Hao XY, Mannervik B, Ringborg U (1996). Increased cisplatin sensitivity of human fibroblasts from a subject with inherent glutathione deficiency. Acta Oncol 35:683–690.[ISI][Medline]
Harries LW, Stubbins MJ, Forman D, Howard GC, Wolf CR (1997). Identification of genetic polymorphisms at the glutathione S-transferase Pi locus and association with susceptibility to bladder, testicular and prostate cancer. Carcinogenesis (Lond) 18:641–644.
Hemmingsen A, Fryer AA, Strange RC, Demarzo N, Pozzato V, Jovine L, et al. (2001). Polymorphism at the glutathione S-transferase, GST P1 locus is associated with isocyanate-induced asthma: use of an ARMS-PCR assay for the simultaneous determination of I105-V105 and A114-V114 alleles. Chem Biol Inter 133:120–122.
Hume WR, Gerzina TM (1996). Bioavailability of components of resin-based materials which are applied to teeth. Crit Rev Oral Biol Med 7:172–179.
Iersel M, Ploemen JP, Lo Bello M, Federici G, van Bladeren PY (1997). Interactions of ,ß-unsaturated aldehydes and ketones with human glutathione S-transferase P1. Chem Biol Inter 108:67–78.[ISI][Medline]
Janke V, von Neuhoff N, Schlegelberger B, Leyhausen G, Geurtsen W (2003). TEGDMA causes apoptosis in primary human gingival fibroblasts. J Dent Res 82:814–818.
Kensler TW (1997). Chemoprevention by inducers of carcinogen detoxication enzymes. Env Health Persp 105(Suppl 4):964–970.[ISI][Medline]
Mannervik B, Awasthi YL, Board PG, Hayes JP, Dillio C, Ketterer B, et al. (1992). Nomenclature for human glutathione transferase (letter). Biochem J 282:305–306.
Oakley AJ, Lo Bello M, Nuccetelli M, Mazzeti A, Parker MW (1999). The ligandin (non-substrate) binding site of human Pi class glutathione transferase is located in the electrophile binding site (H-site). J Mol Biol 291:913–926.[ISI][Medline]
Ortengren U (2000). On composite resin materials. Degradation, erosion and possible adverse effects in dentists. Swed Dent J Suppl 141:1–61.
Schweikl H, Schmalz G (1999). Triethylene glycol dimethacrylate induces large deletions in the hprt gene of V79 cells. Mutat Res 438:71–78.[ISI][Medline]
Spahl W, Budzikiewicz H, Geurtsen W (1998). Determination of leachable components from four commercial dental composites by gas and liquid chromatography/mass spectrometry. J Dent 26:137–145.[ISI][Medline]
Stanislawski L, Daniau X, Lautie A, Goldberg M (1999). Factors responsible for pulp cell cytotoxicity induced by resin-modified glass ionomer cements. J Biomed Mater Res (Appl Biomater) 48:277–288.
Stanislawski L, Soheili-Majd E, Perianin A, Goldberg M (2000). Dental restorative biomaterials induce glutathione depletion in cultured human gingival fibroblasts: protective effect of N-acetyl cysteine. J Biomed Mater Res 51:469–474.[ISI][Medline]
Stanislawski L, Lefeuvre M, Bourd K, Soheili-Majd E, Goldberg M, Perianin A (2003). 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 66(A):476–482.
Stucker I, Hirvonen A, de Waziers I, Cabelguenne A, Mitrunen K, Cenee S, et al. (2002). Genetic polymorphisms of glutathione S-transferases as modulators of lung cancer susceptibility. Carcinogenesis 23:1475–1481.
Tamai K, Satoh K, Tsuchida S, Hatayama I, Maki T, Sato K (1990). Specific inactivation of glutathione S-transferases in class Pi by SH-modifiers. Biochem Bioph Res Commun 167:331–338.[ISI][Medline]
Zimniak P, Nanduri B, Pikula S, Bandorowicz-Pikula J, Singhal SS, Srivastava SK, et al. (1994). Naturally occuring human glutathione-S-transferase GST P1-1 isoforms with isoleucine and valine in position 104 differ in enzymatic properties. Eur J Biochem 204:893–900.
This article has been cited by other articles:
|
|
 |
|
  H. Schweikl, G. Spagnuolo, and G. Schmalz Genetic and cellular toxicology of dental resin monomers. J. Dent. Res., October 1, 2006; 85(10): 870 - 877. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| IADR Journals | Advances in Dental Research ® |
| Journal of Dental Research ® | Critical Reviews (1990-2004) |
