Abstract
Introduction
The public interest steadily increases in the biological adverse effects caused by components released from resin-based dental restorations.
Objective
In this study, the cytotoxicity and the genotoxicity were investigated of following released components from dental resin restorations in human gingival fibroblasts (HGF): tetraethyleneglycol dimethacrylate (TEEGDMA), neopentylglycol dimethacrylate (Neopen), diphenyliodoniumchloride (DPIC), triphenyl-stibane (TPSB) and triphenylphosphane (TPP).
Methods
XTT based cell viability assay was used for cytotoxicity screening of substances. γ-H2AX assay was used for genotoxicity screening. In the γ-H2AX assay, HGFs were exposed to the substances for 6 h. Induced foci represent double DNA strand breaks (DSBs), which can induce ATM-dependent phosphorylation of the histone H 2 AX. Cell death effects (apoptosis and necrosis), induced by the substances were visually tested by the same investigator using the fluorescent microscope.
Results
All tested substances induced a dose-dependent loss of viability in HGFs. Following toxicity ranking among the substances at EC 50 -concentration were found in the XTT assay (mM, mean ± SEM; n = 5): DPIC > Neopen > TPSB > TPP > TEEGDMA.
DSB-foci per HGF-cell were obtained, when HGFs were exposed to the EC 50 -concentration of each substance in the following order (mean ± SEM; n = 3): DPIC > Neopen > TPSB > TPP > TEEGDMA.
Multi-foci cells (cells that contain more than 40 foci each) in 80 HGF-cells at EC 50 -concentration of each substance were found as follow (mean ± SEM; n = 3): DPIC > Neopen > TPP > TPSB > TEEGDMA.
Cell apoptosis contained in each substance at EC 50 -concentration in the following order (mean ± SEM; n = 3): DPIC > Neopen > TPSB > TPP >TEEGDMA. Cell necrosis contained in each substance at EC 50 -concentration in the following order (mean ± SEM; n = 3): DPIC > Neopen > TPSB > TPP > TEEGDMA.
Conclusion
Leached components from dental resin restorations can induce DNA DSBs and cell death effects in HGFs.
1
Introduction
The development and widespread use of new generations of resin-based dental restorative materials has allowed for the application of more conservative, esthetic, and long lasting restorative techniques . These adhesive techniques are extensively used in a wide variety of applications in dentistry, including restorative procedures, prosthodontics, orthodontics and preventive dentistry, making resin-based composites one of the most important groups of materials in dental practice. The main bulk of scientific and manufacturing effort during the past years has been focused on the improvement of the filler fraction of these materials, providing a great variety of new formulations in the micro- or nano-scale in an attempt to improve their mechanical and esthetic properties. On the other hand, little improvement has been offered with respect to the resinous matrix of these materials, which is based in the majority of commercially available products on methacrylate monomers . Most of resin matrices consist of a mixture of various methacrylate monomers, such as bis-phenol-A-glycidyl dimethacrylate (BisGMA) and urethane dimethacrylate (UDMA) in combination with co-monomers of lower viscosity such as, triethyleneglycol dimethacrylate (TEGDMA), tetraethyleneglycol dimethacrylate (TEEGDMA), ethyleneglycol dimethacrylate (EGDMA), neopentylglycol dimethacrylate (Neopen) or diethyleneglycol dimethacrylate (DEGDMA) , initiators as diphenyliodoniumchloride (DPIC) , comphorquinone (CQ), benzoyl peroxide (BPO), dimethylaminoethyl methacrylate (DMAEMA), dimethyl-para-toloidine (DMPT) and contaminants such as triphenyl-stibane (TPSB) and/or triphenylphosphane (TPP) . From the biological aspect, there are concerns about resin-monomers and additives biocompatibility and biochemical stability for both patients and dentists. In addition, there has been intensive research on the tumor initiation potential and long-lasting effects by hazardous xenobiotics in dental materials . The unpolymerized monomers/co-monomers and additives can be released from dental resin-based materials during insertion and even after polymerization by means of physical and chemical processes directly into the oral cavity , or via dentinal microchannels into the pulp where they can reach millimolar concentrations . In the pulp, they can damage the resident cells and/or can reach the blood-stream . Additionally, leached monomers/co-monomers and additives can be diluted by saliva and enter the intestine . After cellular absorption and metabolism of monomers/co-monomers, they can form radical intermediates, which can be metabolized to epoxy compounds as detected by human liver microsome assay . Epoxy intermediates have the potential to attack biomolecules (via radical formation), among where the DNA is the most critical target (e.g., the N7-position of guanine) . Monomers/co-monomers can induce mutagenic/carcinogenic effects in oral and other cells . In addition, monomers/co-monomers have the potential to increase the levels of reactive oxygen species (ROS) . ROS are reactive mediators of signaling cascades but, elevated levels of ROS can disrupt the cellular redox balance, resulting in oxidative DNA damage and apoptosis in mammalian cells . ROS can induce adverse toxic effects in the affected cells or in the organism and can induce cell mutagenicity and genotoxicity , as well as embryo toxicity and teratogenicity . In previous studies, it was shown that DPIC and/or its degradation products, exhibit cytotoxic and genotoxic activity in vivo and in vitro . Furthermore, leached components from dental resin composites can induce cytotoxic reactions in human gingival fibroblasts (HGFs) leading to reduced cell viability, plasma membrane damage, DNA fragmentation, and increased cell death .
In literature there was little emphasis to the nature of biological damage caused by the selected substances on oral cells. The purpose of this study is to monitor the in vitro possible biological hazards caused by substances that may contained in most recent and widely used dental restorative material (composite resins) and eluted into oral cavity during and after polymerization. In this study, the polymerization initiator DPIC and contaminants TTP and TPSB were investigated for cytotoxicity, induction of DNA double strand breaks (DNA DSBs) and for possible cell death (via apoptosis/necrosis) compared to composite monomers TEEGDMA and Neopen, in human gingival fibroblasts (HGFs).
2
Materials and methods
2.1
Chemicals
TEEGMA, Neopen, DPIC, TPSB and TPP were obtained from Sigma–Aldrich (St. Louis, MO, USA). All chemicals and reagents were of the highest purity available. All substances are dissolved in dimethyl sulphoxide (DMSO, 99% purity; Merck, Darmstadt, Germany) and diluted with medium (final DMSO concentration: 1%). Control cells received either DMSO 1% in medium, or 1 mM hydrogen peroxide (H 2 O 2 ; VWR International, Darmstadt, Germany).
2.2
Cell culture and drug treatment
HGFs (Cat. No.: 1210412) were obtained from Provitro, Cell-Lining (Berlin, Germany). The HGFs (passage 9) were grown on 175 cm 2 cell culture flasks to approximately 75–85% confluence and maintained in an incubator with 5% CO 2 atmosphere at 100% humidity and 37 °C. Quantum 333 medium supplemented with l -glutamine and 1% antibiotic/antimycotic solution (10,000 U/ml penicillin, 25 mg/ml streptomycin sulphate, 25 mg/ml amphotericin B; PAA Laboratories, Cölbe, Germany) was used to culture HGFs. After reaching confluence, the cells were washed with Dulbecco’s phosphate buffered saline (PAA Laboratories) detached from the flasks by a brief treatment with trypsin/EDTA (PAA Laboratories).
2.3
XTT-based viability assay
Tetrazolium salt XTT (sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate) based cell viability assay was used, according to the method described in earlier studies , to determine the half-maximum effect concentrations (EC 50 ) for the tested compounds TEEGDMA, Neopen, DPIC, TPSB and TPP in HGFs and for the estimation of the substance concentrations for the use in the γ-H2AX-assay. HGFs at 20,000 cell/well were seeded into a 96-well microtiter plate in 0.1 ml medium, and then the cells were incubated for 24 h. Then the cells were treated with medium containing DPIC (0.1–30 mM), Neopen (0.1–10 mM), TPSB (0.1–30 mM), TPP (0.1–30 mM), or TEEGDMA (0.1–30 mM). Negative control cells received either medium alone, or medium + DMSO (final DMSO concentration: 1%). Positive control cells received 1% Triton X-100 (Sigma–Aldrich). After incubation for 20 h, the cell monolayer was washed and a mixture of XTT labeling reagent, in RPMI 1640, without phenol red and electron-coupling reagent (PMS [N-methyldibenzopyrazine methyl sulphate] in phosphate buffered saline) was added as recommended by the supplier (cell proliferation kit II; Roche Diagnostics Penzberg, Germany) 4 h before photometric analysis.
The formazan formation was quantified spectrophotometrically at 450 nm (reference wavelength 670 nm) using a micro-titer plate reader (Victor 3, Perkin Elmer Las, Jügesheim, Germany). All experiments were repeated five times.
2.4
γ-H2AX immunofluorescence
DNA-DSBs formation was tested in HGFs unexposed and exposed to TEEGDMA, Neopen, DPIC, TPSB, and TPP by the method of γ-H2AX focus assay. For this microscopic assay, 12 mm round cover slips (Carl Roth, Karlsruhe, Germany) were cleaned in 1 N HCl and distributed into a 24-well plate. In each well medium, HGFs were seeded at 7 × 10 4 cell/ml and followed by overnight incubation at 37 °C. HGFs were exposed to medium containing substances in the following concentrations (mM, corresponding to EC 50 , 1/3 EC 50 and 1/10 EC 50 values, received from the XTT assay): TEEGDMA (4.1, 1.4, 0.41), Neopen (1.1, 0.367, 0.11), DPIC (0.9, 0.3, 0.09), TPSB (1.8, 0.6, 1.8) and TPP (2.4, 0.8, 0.24) for 6 h. Negative controls received either medium alone, or medium + DMSO (final DMSO concentration: 1%). Positive controls received 1 mM H 2 O 2 + medium, or 1 mM H 2 O 2 + medium + DMSO (final DMSO concentration: 1%) for 10 min. For immunofluorescent staining, cells were first washed 2 × 5 min with PBS, fixed by adding 0.5 ml ice-cold 4% paraformaldehyde in PBS for 5 min at 4 °C, then washed with cold PBS (4 °C) for 4 × 2 min, and permeabilized for 15 min with 0.5 ml of triton-citrate buffer (0.1% sodium citrate, 0.1% Triton X-100) at 4 °C. After washing 4 × 2 min with PBS, cells were blocked for 20 min with 0.2 ml of serum-free blocking buffer (Dako, Hamburg, Germany) per well at 25 °C. Thereafter, cells were incubated with mouse monoclonal anti γ-H2AX (Millipore, Billerica, MA, USA) at 1:1300 dilution in antibody diluent (0.3 ml per well) (Dako) at 4 °C overnight. After 4 × 5 min washes with PBS at 4 °C, cells were incubated with FluoroLink Cy3-labeled goat anti-mouse secondary antibody (GE Healthcare, Munich, Germany) at a dilution of 1:1300 in antibody diluent (0.3 ml per well) for 1 h at 25 °C in the dark. HGFs were then washed 2 × 5 min in PBS, and rinsed 5 min with deionised water at 25 °C. Finally, the cover slips were each placed on 0.2 ml of a mixture of 2 ml Prolong antifade and DAPI (Invitrogen) (76 × 26 mm; Carl Roth, Invitrogen, Karlsruhe, Germany) on a glass slide.
2.5
Apoptosis and necrosis
Apoptosis and necrosis found at EC 50 concentrations of TEEGDMA, Neopen, DPIC, TPSB, and TPP were visually tested in HGFs, exposed to the substances, using the method of γ-H2AX focus assay by the same investigator by eye down the fluorescent microscope. The morphological changes (monitored under microscope) associated with necrosis, which represents cell membrane rupture and lysis of the organized cell structure. Apoptosis represents fragmentation of the cytoplasm and nucleus in the target cell while the normal organelle structure is maintained ( Figs. 6 and 7 ).
2.6
Image acquisition
HGFs were investigated using a Zeiss Axioplan 2 imaging fluorescence microscope (Zeiss, Göttingen, Germany) equipped with a motorized filter wheel and appropriate filters for excitation of red, green and blue fluorescence. Images were obtained using a 63× and a 100× Plan-Neofluar oil immersion objective (Zeiss) and the ISIS fluorescence imaging system (MetaSystems, Altlussheim, Germany).
2.7
Data analysis
2.7.1
XTT test
The values performed from the XTT-based viability assay were calculated as percentage of the 100% control values, using Graph Pad Prism 4 (Graph Pad Software Inc., San Diego, USA), where they were plotted on a concentration log-scale and range of the maximum slope were comprised. Half-maximum-effect substance concentration at the maximum slope was revealed as EC 50 . The EC 50 values were obtained as half-maximum-effect concentrations from the fitted curves. Data are presented as means ± standard error of the mean (SEM). Each experiment was repeated five times. The statistical significance ( p < 0.05) of the differences between the experimental groups was checked using the Student’s t -test, corrected according to Bonferroni–Holm .
2.8
γ-H2AX test
For quantitative analysis of the γ-H2AX test, DSBs (foci) were counted by the same investigator by eye down using the fluorescence microscope (100× objective). Disrupted cells were excluded from the analysis. Cell counting was performed until at least 80 cells were reached, as described in previous study . Each experiment was repeated 3 times. The mean number of cells was scored and the standard error of the mean was calculated. Values were compared using the Student’s t -test ( p < 0.05), corrected according to Bonferroni–Holm. . If one cell contains 40 or more foci, it will be counted as multi-foci cell .
2.9
Statistical analysis of apoptotic and necrotic cells
Apoptotic and necrotic HGF cells were counted using the fluorescence microscope (100× objective). Disrupted cells were excluded from the analysis. Cell counting was performed within 80 cells. Each experiment was repeated 3 times. The mean number of apoptotic and necrotic cells was scored and the standard error of the mean was calculated. The statistical significance ( p < 0.05) of the differences between the experimental groups was compared using the Student’s t -test, corrected according to Bonferroni–Holm .
2
Materials and methods
2.1
Chemicals
TEEGMA, Neopen, DPIC, TPSB and TPP were obtained from Sigma–Aldrich (St. Louis, MO, USA). All chemicals and reagents were of the highest purity available. All substances are dissolved in dimethyl sulphoxide (DMSO, 99% purity; Merck, Darmstadt, Germany) and diluted with medium (final DMSO concentration: 1%). Control cells received either DMSO 1% in medium, or 1 mM hydrogen peroxide (H 2 O 2 ; VWR International, Darmstadt, Germany).
2.2
Cell culture and drug treatment
HGFs (Cat. No.: 1210412) were obtained from Provitro, Cell-Lining (Berlin, Germany). The HGFs (passage 9) were grown on 175 cm 2 cell culture flasks to approximately 75–85% confluence and maintained in an incubator with 5% CO 2 atmosphere at 100% humidity and 37 °C. Quantum 333 medium supplemented with l -glutamine and 1% antibiotic/antimycotic solution (10,000 U/ml penicillin, 25 mg/ml streptomycin sulphate, 25 mg/ml amphotericin B; PAA Laboratories, Cölbe, Germany) was used to culture HGFs. After reaching confluence, the cells were washed with Dulbecco’s phosphate buffered saline (PAA Laboratories) detached from the flasks by a brief treatment with trypsin/EDTA (PAA Laboratories).
2.3
XTT-based viability assay
Tetrazolium salt XTT (sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate) based cell viability assay was used, according to the method described in earlier studies , to determine the half-maximum effect concentrations (EC 50 ) for the tested compounds TEEGDMA, Neopen, DPIC, TPSB and TPP in HGFs and for the estimation of the substance concentrations for the use in the γ-H2AX-assay. HGFs at 20,000 cell/well were seeded into a 96-well microtiter plate in 0.1 ml medium, and then the cells were incubated for 24 h. Then the cells were treated with medium containing DPIC (0.1–30 mM), Neopen (0.1–10 mM), TPSB (0.1–30 mM), TPP (0.1–30 mM), or TEEGDMA (0.1–30 mM). Negative control cells received either medium alone, or medium + DMSO (final DMSO concentration: 1%). Positive control cells received 1% Triton X-100 (Sigma–Aldrich). After incubation for 20 h, the cell monolayer was washed and a mixture of XTT labeling reagent, in RPMI 1640, without phenol red and electron-coupling reagent (PMS [N-methyldibenzopyrazine methyl sulphate] in phosphate buffered saline) was added as recommended by the supplier (cell proliferation kit II; Roche Diagnostics Penzberg, Germany) 4 h before photometric analysis.
The formazan formation was quantified spectrophotometrically at 450 nm (reference wavelength 670 nm) using a micro-titer plate reader (Victor 3, Perkin Elmer Las, Jügesheim, Germany). All experiments were repeated five times.
2.4
γ-H2AX immunofluorescence
DNA-DSBs formation was tested in HGFs unexposed and exposed to TEEGDMA, Neopen, DPIC, TPSB, and TPP by the method of γ-H2AX focus assay. For this microscopic assay, 12 mm round cover slips (Carl Roth, Karlsruhe, Germany) were cleaned in 1 N HCl and distributed into a 24-well plate. In each well medium, HGFs were seeded at 7 × 10 4 cell/ml and followed by overnight incubation at 37 °C. HGFs were exposed to medium containing substances in the following concentrations (mM, corresponding to EC 50 , 1/3 EC 50 and 1/10 EC 50 values, received from the XTT assay): TEEGDMA (4.1, 1.4, 0.41), Neopen (1.1, 0.367, 0.11), DPIC (0.9, 0.3, 0.09), TPSB (1.8, 0.6, 1.8) and TPP (2.4, 0.8, 0.24) for 6 h. Negative controls received either medium alone, or medium + DMSO (final DMSO concentration: 1%). Positive controls received 1 mM H 2 O 2 + medium, or 1 mM H 2 O 2 + medium + DMSO (final DMSO concentration: 1%) for 10 min. For immunofluorescent staining, cells were first washed 2 × 5 min with PBS, fixed by adding 0.5 ml ice-cold 4% paraformaldehyde in PBS for 5 min at 4 °C, then washed with cold PBS (4 °C) for 4 × 2 min, and permeabilized for 15 min with 0.5 ml of triton-citrate buffer (0.1% sodium citrate, 0.1% Triton X-100) at 4 °C. After washing 4 × 2 min with PBS, cells were blocked for 20 min with 0.2 ml of serum-free blocking buffer (Dako, Hamburg, Germany) per well at 25 °C. Thereafter, cells were incubated with mouse monoclonal anti γ-H2AX (Millipore, Billerica, MA, USA) at 1:1300 dilution in antibody diluent (0.3 ml per well) (Dako) at 4 °C overnight. After 4 × 5 min washes with PBS at 4 °C, cells were incubated with FluoroLink Cy3-labeled goat anti-mouse secondary antibody (GE Healthcare, Munich, Germany) at a dilution of 1:1300 in antibody diluent (0.3 ml per well) for 1 h at 25 °C in the dark. HGFs were then washed 2 × 5 min in PBS, and rinsed 5 min with deionised water at 25 °C. Finally, the cover slips were each placed on 0.2 ml of a mixture of 2 ml Prolong antifade and DAPI (Invitrogen) (76 × 26 mm; Carl Roth, Invitrogen, Karlsruhe, Germany) on a glass slide.
2.5
Apoptosis and necrosis
Apoptosis and necrosis found at EC 50 concentrations of TEEGDMA, Neopen, DPIC, TPSB, and TPP were visually tested in HGFs, exposed to the substances, using the method of γ-H2AX focus assay by the same investigator by eye down the fluorescent microscope. The morphological changes (monitored under microscope) associated with necrosis, which represents cell membrane rupture and lysis of the organized cell structure. Apoptosis represents fragmentation of the cytoplasm and nucleus in the target cell while the normal organelle structure is maintained ( Figs. 6 and 7 ).
2.6
Image acquisition
HGFs were investigated using a Zeiss Axioplan 2 imaging fluorescence microscope (Zeiss, Göttingen, Germany) equipped with a motorized filter wheel and appropriate filters for excitation of red, green and blue fluorescence. Images were obtained using a 63× and a 100× Plan-Neofluar oil immersion objective (Zeiss) and the ISIS fluorescence imaging system (MetaSystems, Altlussheim, Germany).
2.7
Data analysis
2.7.1
XTT test
The values performed from the XTT-based viability assay were calculated as percentage of the 100% control values, using Graph Pad Prism 4 (Graph Pad Software Inc., San Diego, USA), where they were plotted on a concentration log-scale and range of the maximum slope were comprised. Half-maximum-effect substance concentration at the maximum slope was revealed as EC 50 . The EC 50 values were obtained as half-maximum-effect concentrations from the fitted curves. Data are presented as means ± standard error of the mean (SEM). Each experiment was repeated five times. The statistical significance ( p < 0.05) of the differences between the experimental groups was checked using the Student’s t -test, corrected according to Bonferroni–Holm .
2.8
γ-H2AX test
For quantitative analysis of the γ-H2AX test, DSBs (foci) were counted by the same investigator by eye down using the fluorescence microscope (100× objective). Disrupted cells were excluded from the analysis. Cell counting was performed until at least 80 cells were reached, as described in previous study . Each experiment was repeated 3 times. The mean number of cells was scored and the standard error of the mean was calculated. Values were compared using the Student’s t -test ( p < 0.05), corrected according to Bonferroni–Holm. . If one cell contains 40 or more foci, it will be counted as multi-foci cell .
2.9
Statistical analysis of apoptotic and necrotic cells
Apoptotic and necrotic HGF cells were counted using the fluorescence microscope (100× objective). Disrupted cells were excluded from the analysis. Cell counting was performed within 80 cells. Each experiment was repeated 3 times. The mean number of apoptotic and necrotic cells was scored and the standard error of the mean was calculated. The statistical significance ( p < 0.05) of the differences between the experimental groups was compared using the Student’s t -test, corrected according to Bonferroni–Holm .
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