Intracellular glutathione: A main factor in TEGDMA-induced cytotoxicity?

Abstract

Objective

To evaluate whether the reduction/prevention of triethylene glycol dimethacrylate (TEGDMA)-induced decrease of intracellular glutathione (GSH) protects human periodontal ligament fibroblasts (HPLF) against cell death.

Methods

HPLF were preincubated for 30 min with exogenous GSH and then treated with TEGDMA (2.5 mM) with/without GSH (0.5–2.5–5 mM) for the following incubation exposure types: 6 h (GI); 6 h followed by 18 h recovery time in presence (GII) or absence (GIII) of exogenous GSH; 24 h without recovery time (GIV). TEGDMA-cytotoxicity and intracellular glutathione were assessed by Hoechst 33342 and monobromobimane (MBBr) assays. Data were statistically analyzed with Bonferroni ANOVA ( p < 0.05).

Results

Preincubation with exogenous GSH increased the intracellular GSH-concentration. TEGDMA was cytotoxic at all treatment times except at 6 h (GI) (94 ± 7% of control). In GII the treatment with TEGDMA alone (59 ± 7%) showed no different results to cultures exposed to TEGDMA and GSH. Exogenous GSH had no effect on the TEGDMA-induced cytotoxicity also in the GIII and GIV. Thus, a combined incubation with GSH did not prevent the cytotoxicity of TEGDMA, despite of a significant increase of intracellular GSH-concentration in the presence of exogenously supplied GSH.

Significance

The glutathione-decreasing effect of TEGDMA is not the major cause of TEGDMA-induced cytotoxicity, indicating more complex mechanisms, which are causative for TEGDMA-cytotoxicity in HPLF.

Introduction

Resinous materials are widely applied in modern dentistry, e.g., in preventive and restorative procedures, as well as prosthodontics and orthodontics. These materials contain various components, like different monomers and additives . Despite their improved physico-chemical properties, resinous materials still exhibit clinical disadvantages and may cause adverse reactions, such as pulp reactions and allergic effects . It has been documented that compounds of dental resins may induce severe (e.g., bisphenol A-glycidylmethacrylate (Bis-GMA); urethane dimethacrylate (UDMA); triethylene glycol dimethacrylate (TEGDMA)) or medium (e.g., 2-hydroxyethyl methacrylate (HEMA) and camphorquinone (CQ)) cytotoxicity due to the incomplete monomer–polymer conversion and the release of monomeric substances and additives into the oral environment after placement . Among the substances released, the (co)monomers have been identified as the main cause of cytotoxicity. Their effects have been evaluated in a variety of cell culture systems, including immortalized cell lines (3T3 fibroblasts, V79 chinese hamster lung fibroblasts, HaCaT keratinocytes, etc.), as well as human primary cells derived from skin, gingiva, pulp or periodontium .

Triethylene glycol dimethacrylate (TEGDMA) is one of the most important comonomers of resin matrices, which provides various beneficial physical properties . However, as mentioned before, TEGDMA is highly cytotoxic and moderately genotoxic. It is one of the main compounds leaching from polymerized resins . It can easily penetrate cell membranes and react with intracellular molecules and structures . Some authors have shown that TEGDMA may cause a depletion of the intracellular glutathione level (GSH) associated with an increased level of reactive oxygen species (ROS) . GSH is a tripeptide and the most abundant intracellular thiol-type molecule with a low molecular weight, found in mammalian cells. It is involved in cellular detoxification and maintenance of redox balance. After depletion, GSH-level rapidly regenerates . GSH is also an efficient protector against oxidative damages generated by various xenobiotics and endogenous detrimental metabolic substances, specifically ROS. When the GSH metabolism in cells is deregulated (e.g., by xenobiotics), the redox balance is disturbed, which is often associated with cell cycle delay and apoptosis . There are some indications that various antioxidants may prevent cell damage triggered by TEGDMA and other (co)monomers .

Therefore, the objective of this study was to investigate the mechanism of TEGDMA-induced cytotoxicity in human periodontal ligament fibroblasts (HPLF) in the presence and absence of the antioxidant glutathione. The hypothesis set forth was that exogenous GSH may prevent or reduce TEGDMA-associated cytotoxicity in HPLF.

Materials and methods

Materials

Dulbecco’s modified Eagle’s medium (DMEM), HEPES, penicillin, streptomycin, and amphotericin were purchased from Biochrom (Berlin, Germany), fetal calf serum (FCS) from Lonza (Cologne, Germany), NAHCO 3 and Hoechst 33342™ (H 33342) from Riedel de Häen (Seelze, Germany), Trypsin/EDTA and dimethylsulfoxide (DMSO) from Sigma (Taufkirchen, Germany). Triethylene-glycol dimethacrylate (TEGDMA) was a gift from VOCO (Cuxhaven, Germany). Monobromobimane (MBBr), glutathione (GSH) and 2′,7′-dichlorofluorescine diacetate (DCFH-DA) were purchased from Fluka (Seelze, Germany), Hank’s balanced salt solution (HBSS) from GIBCO BRL (Karlsruhe, Germany), and methyl tetrazolium (MTT) from Sigma–Aldrich (Steinheim, Germany).

An FLx-800 spectrophotometer (Bio-Tek, Neufahrn, Germany) was used for all fluorometric measurements and a SpectraMax-250 reader (Molecular Devices, Ismaning/München, Germany) for the optical density measurements.

Tissue collection and cell culture

Primary human periodontal ligament fibroblasts (HPLF) were cultured from biopsies of healthy premolar and permanent molar teeth. Informed consent was obtained from all 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 μg/mL), and amphotericin (2.5 μg/mL) prior to amplification. The tissue samples were placed into 25-cm 2 tissue culture flasks and grown in DMEM culture medium with 4.5 g/L glucose, 10 mM HEPES, NaHCO 3 (3.7 g/L), penicillin (100 U/mL) and streptomycin (100 μg/mL), supplemented with 10% FCS at 37 °C and 10% CO 2 . When outgrowth of cells was observed, the medium was replaced twice weekly until cells reached confluence. Cells were detached from the substrate by a brief treatment with 0.25% trypsin in 0.02% EDTA and cultured in 75-cm 2 tissue flasks until confluent monolayers were 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).

Kinetics of GSH-uptake

To analyze a possible protective effect of GSH on TEGDMA-induced cytotoxicity, a pre-treatment with exogenous GSH was performed. Firstly, the optimal treatment period for maximum GSH uptake was determined. HPLF (from passages 4 to 10) were seeded in 96-well plates (1 × 10 4 cells/well) and allowed to grow for 24 h. Then cells were treated with GSH (0.5–5 mM) and incubated at 37 °C in a 10% CO 2 atmosphere for 30 min, 1 h, and 3 h. Immediately after the various times of treatment, the relative intracellular GSH concentrations were determined using an assay with monobromobimane (MBBr) (12.5 mg/5 mL DMSO). Cells were washed in 100 μL HBSS and incubated with 200 μL MBBr in the darkness for 35 min at room temperature prior to the fluorometric readings (excitation (Ex): 360 nm/emission (Em): 460 nm). All experiments were performed at least three times with six replicates each.

ED 50 determination of TEGDMA

Before application of GSH, TEGDMA was dissolved in ethanol and tested within a concentration range of 0.0625–2.5 mM for cytotoxicity. HPLF (10 4 cells/well) were placed into 96-well plates and cultured for 24 h. Then cells were incubated with different concentrations of TEGDMA for 24 h. After incubation, cell number was determined using the DNA-intercalating staining H 33342™. The fluorescence intensity was evaluated using a Bio-Tek FLx-800 fluorescence-luminescence reader (Ex: 360 nm/Em: 460 nm). Each assay, with 6 replicates each, was repeated three times to ensure reproducibility.

Treatment of cells with TEGDMA and GSH

Stock solutions of TEGDMA were prepared in 0.25% ethanol, stock solutions of GSH in distilled water. Both preparations were freshly diluted in DMEM prior to each treatment. HPLF between passages 4–10 were seeded in 96-well plates (1 × 10 4 cells/well) and allowed to grow for 24 h. Then cells were exposed to TEGDMA (2.5 mM (ED 50 )) in the absence or presence of various concentrations of GSH (0.5, 2.5, and 5 mM) and divided in groups according to the time of treatment.

  • Group I : Initially, cells were exposed to exogenous GSH at different concentrations for 30 min, and subsequently treated with 2.5 mM TEGDMA alone or in combination with the different concentrations of GSH for another 6 h.

  • Group II : Cells were exposed to exogenous GSH at different concentrations for 30 min, and then treated for 6 h with TEGDMA alone or in combination with the different concentrations of GSH. Finally, the cells were incubated with medium and GSH (0.5, 2.5, and 5 mM) for additional 18 h (recovery time).

  • Group III : Cells were exposed to GSH at different concentrations for 30 min, and then treated for 6 h with TEGDMA alone or in combination with different concentrations of GSH. Finally, the cells were incubated with fresh medium (without GSH) for additional 18 h (recovery time).

  • Group IV : The cells were exposed for 30 min to GSH at different concentrations, and then treated for 24 h with TEGDMA (2.5 mM) alone or in combination with different concentrations of GSH.

All treatment solutions contained 0.25% ethanol. Untreated cells incubated in growth medium with 0.25% ethanol (c1) and in growth medium without ethanol (c2 = 100% cell growth) served as solvent control and growth control.

After each treatment time, medium was removed and cells were stained with the fluorescent dyes.

Cytotoxicity assays

Determination of total cell numbers using H33342

After the treatment, the cytotoxicity of the substances was measured by the chromatin staining and membrane-permeable dye H 33342™. Medium containing the tested solutions was removed and 100 μL/well of H 33342™ (1 mg/mL) in PBS was added. After 30 min of incubation with the dye (37 °C in 10% CO 2 ), cells were washed with 0.2 mL and 0.1 mL PBS and the fluorescence intensity was evaluated using a Bio-Tek FLx-800 fluorescence reader (Ex: 360 nm/Em: 460 nm). The fluorescence of treated cells and of c1 (culture medium with 0.25% ethanol) was compared to c2 (100% growth control). Each assay with six replicates each was repeated three times to ensure reproducibility.

GSH determination

Parallel to the cytotoxicity tests, the influence of TEGDMA on the relative intracellular GSH concentrations was determined using an assay with monobromobimane (MBBr) (12.5 mg/5 mL DMSO). Medium was removed from the wells after the treatments (GI–GIV) and monolayers were washed with HBSS. Then, MBBr (200 μL) in HBSS was added. After 35 min in the darkness, the fluorescence intensity of the MBBr–GSH adduct was measured using the Bio-Tek FLx-800 fluorescence reader (Ex: 360 nm/Em: 460 nm).

Reactive oxygen species (ROS)

The production of reactive oxygen species (ROS) in cultured cells was quantified using the cell-permeable fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA). A stock solution of DCFH-DA (8 mM) was stored at −20 °C in DMSO.

In order to measure the ROS formation induced by TEGDMA, HPLF (1 × 10 4 cells/well) were stained with 200 μL of DCFH-DA (5 μL/mL) in HBSS for 20 min at 37 °C in a 10% CO 2 atmosphere. After incubation, cells were washed with HBSS and incubated with TEGDMA (2.5 mM) in the presence or absence of different concentrations of GSH (0.5–2.5–5 mM) for 4 h. DCF fluorescence was read directly after addition of the substances and then every 15 min (up to 4 h) at the excitation wavelength (Ex) of 485 nm and the emission wavelength (Em) of 528 nm. Maximum rate of fluorescence increase ( V max ) of each well was normalized to cell numbers, which were determined by H 33342™, as already described.

Statistical analysis

Assays were run at least three times with six replicates each. Results were expressed as means ± standard deviations (SD). Statistical analysis was performed by ANOVA with the Bonferroni’s post test, or Student t test. A significance level of p < 0.05 was applied.

Materials and methods

Materials

Dulbecco’s modified Eagle’s medium (DMEM), HEPES, penicillin, streptomycin, and amphotericin were purchased from Biochrom (Berlin, Germany), fetal calf serum (FCS) from Lonza (Cologne, Germany), NAHCO 3 and Hoechst 33342™ (H 33342) from Riedel de Häen (Seelze, Germany), Trypsin/EDTA and dimethylsulfoxide (DMSO) from Sigma (Taufkirchen, Germany). Triethylene-glycol dimethacrylate (TEGDMA) was a gift from VOCO (Cuxhaven, Germany). Monobromobimane (MBBr), glutathione (GSH) and 2′,7′-dichlorofluorescine diacetate (DCFH-DA) were purchased from Fluka (Seelze, Germany), Hank’s balanced salt solution (HBSS) from GIBCO BRL (Karlsruhe, Germany), and methyl tetrazolium (MTT) from Sigma–Aldrich (Steinheim, Germany).

An FLx-800 spectrophotometer (Bio-Tek, Neufahrn, Germany) was used for all fluorometric measurements and a SpectraMax-250 reader (Molecular Devices, Ismaning/München, Germany) for the optical density measurements.

Tissue collection and cell culture

Primary human periodontal ligament fibroblasts (HPLF) were cultured from biopsies of healthy premolar and permanent molar teeth. Informed consent was obtained from all 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 μg/mL), and amphotericin (2.5 μg/mL) prior to amplification. The tissue samples were placed into 25-cm 2 tissue culture flasks and grown in DMEM culture medium with 4.5 g/L glucose, 10 mM HEPES, NaHCO 3 (3.7 g/L), penicillin (100 U/mL) and streptomycin (100 μg/mL), supplemented with 10% FCS at 37 °C and 10% CO 2 . When outgrowth of cells was observed, the medium was replaced twice weekly until cells reached confluence. Cells were detached from the substrate by a brief treatment with 0.25% trypsin in 0.02% EDTA and cultured in 75-cm 2 tissue flasks until confluent monolayers were 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).

Kinetics of GSH-uptake

To analyze a possible protective effect of GSH on TEGDMA-induced cytotoxicity, a pre-treatment with exogenous GSH was performed. Firstly, the optimal treatment period for maximum GSH uptake was determined. HPLF (from passages 4 to 10) were seeded in 96-well plates (1 × 10 4 cells/well) and allowed to grow for 24 h. Then cells were treated with GSH (0.5–5 mM) and incubated at 37 °C in a 10% CO 2 atmosphere for 30 min, 1 h, and 3 h. Immediately after the various times of treatment, the relative intracellular GSH concentrations were determined using an assay with monobromobimane (MBBr) (12.5 mg/5 mL DMSO). Cells were washed in 100 μL HBSS and incubated with 200 μL MBBr in the darkness for 35 min at room temperature prior to the fluorometric readings (excitation (Ex): 360 nm/emission (Em): 460 nm). All experiments were performed at least three times with six replicates each.

ED 50 determination of TEGDMA

Before application of GSH, TEGDMA was dissolved in ethanol and tested within a concentration range of 0.0625–2.5 mM for cytotoxicity. HPLF (10 4 cells/well) were placed into 96-well plates and cultured for 24 h. Then cells were incubated with different concentrations of TEGDMA for 24 h. After incubation, cell number was determined using the DNA-intercalating staining H 33342™. The fluorescence intensity was evaluated using a Bio-Tek FLx-800 fluorescence-luminescence reader (Ex: 360 nm/Em: 460 nm). Each assay, with 6 replicates each, was repeated three times to ensure reproducibility.

Treatment of cells with TEGDMA and GSH

Stock solutions of TEGDMA were prepared in 0.25% ethanol, stock solutions of GSH in distilled water. Both preparations were freshly diluted in DMEM prior to each treatment. HPLF between passages 4–10 were seeded in 96-well plates (1 × 10 4 cells/well) and allowed to grow for 24 h. Then cells were exposed to TEGDMA (2.5 mM (ED 50 )) in the absence or presence of various concentrations of GSH (0.5, 2.5, and 5 mM) and divided in groups according to the time of treatment.

  • Group I : Initially, cells were exposed to exogenous GSH at different concentrations for 30 min, and subsequently treated with 2.5 mM TEGDMA alone or in combination with the different concentrations of GSH for another 6 h.

  • Group II : Cells were exposed to exogenous GSH at different concentrations for 30 min, and then treated for 6 h with TEGDMA alone or in combination with the different concentrations of GSH. Finally, the cells were incubated with medium and GSH (0.5, 2.5, and 5 mM) for additional 18 h (recovery time).

  • Group III : Cells were exposed to GSH at different concentrations for 30 min, and then treated for 6 h with TEGDMA alone or in combination with different concentrations of GSH. Finally, the cells were incubated with fresh medium (without GSH) for additional 18 h (recovery time).

  • Group IV : The cells were exposed for 30 min to GSH at different concentrations, and then treated for 24 h with TEGDMA (2.5 mM) alone or in combination with different concentrations of GSH.

All treatment solutions contained 0.25% ethanol. Untreated cells incubated in growth medium with 0.25% ethanol (c1) and in growth medium without ethanol (c2 = 100% cell growth) served as solvent control and growth control.

After each treatment time, medium was removed and cells were stained with the fluorescent dyes.

Cytotoxicity assays

Determination of total cell numbers using H33342

After the treatment, the cytotoxicity of the substances was measured by the chromatin staining and membrane-permeable dye H 33342™. Medium containing the tested solutions was removed and 100 μL/well of H 33342™ (1 mg/mL) in PBS was added. After 30 min of incubation with the dye (37 °C in 10% CO 2 ), cells were washed with 0.2 mL and 0.1 mL PBS and the fluorescence intensity was evaluated using a Bio-Tek FLx-800 fluorescence reader (Ex: 360 nm/Em: 460 nm). The fluorescence of treated cells and of c1 (culture medium with 0.25% ethanol) was compared to c2 (100% growth control). Each assay with six replicates each was repeated three times to ensure reproducibility.

GSH determination

Parallel to the cytotoxicity tests, the influence of TEGDMA on the relative intracellular GSH concentrations was determined using an assay with monobromobimane (MBBr) (12.5 mg/5 mL DMSO). Medium was removed from the wells after the treatments (GI–GIV) and monolayers were washed with HBSS. Then, MBBr (200 μL) in HBSS was added. After 35 min in the darkness, the fluorescence intensity of the MBBr–GSH adduct was measured using the Bio-Tek FLx-800 fluorescence reader (Ex: 360 nm/Em: 460 nm).

Reactive oxygen species (ROS)

The production of reactive oxygen species (ROS) in cultured cells was quantified using the cell-permeable fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA). A stock solution of DCFH-DA (8 mM) was stored at −20 °C in DMSO.

In order to measure the ROS formation induced by TEGDMA, HPLF (1 × 10 4 cells/well) were stained with 200 μL of DCFH-DA (5 μL/mL) in HBSS for 20 min at 37 °C in a 10% CO 2 atmosphere. After incubation, cells were washed with HBSS and incubated with TEGDMA (2.5 mM) in the presence or absence of different concentrations of GSH (0.5–2.5–5 mM) for 4 h. DCF fluorescence was read directly after addition of the substances and then every 15 min (up to 4 h) at the excitation wavelength (Ex) of 485 nm and the emission wavelength (Em) of 528 nm. Maximum rate of fluorescence increase ( V max ) of each well was normalized to cell numbers, which were determined by H 33342™, as already described.

Statistical analysis

Assays were run at least three times with six replicates each. Results were expressed as means ± standard deviations (SD). Statistical analysis was performed by ANOVA with the Bonferroni’s post test, or Student t test. A significance level of p < 0.05 was applied.

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Nov 28, 2017 | Posted by in Dental Materials | Comments Off on Intracellular glutathione: A main factor in TEGDMA-induced cytotoxicity?
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