Highlights
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Cyto- and genotoxicity of FIT monomer and Lucirin TPO photoinitiator were evaluated.
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FIT showed less cytotoxic and genotoxic effects than BisGMA.
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TPO showed 7 times higher cytotoxicity than CQ.
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Components of FIT-based mixtures exhibited an antagonistic cytotoxic effect.
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Components of BisGMA-based mixtures showed a synergistic cytotoxic effect.
Abstract
Objective
To compare cytotoxicity and genotoxicity of novel urethane-based monomer FIT-852 and monoacylphosphine oxide photoinitiator (Lucirin TPO) with conventional Bisphenol A-glycidyl-methacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) monomers and camphorquinone (CQ)/amine photoinitiator system, respectively. Moreover, we quantified and analyzed the combinatorial effects of individual substances in resin-based mixtures concerning the nature of the combinatorial effects.
Methods
Cytotoxic and genotoxic effects of BisGMA, FIT, TEGDMA, CQ, DMAEMA and TPO and their combined toxicity in four clinically relevant mixtures (FIT/TPO, FIT/CQ, BisGMA/TPO, BisGMA/CQ) were tested on human fetal lung fibroblasts MRC-5 using MTT and Comet assays. We assessed combination effects of monomers and photoinitiators on overall toxicity from the measured concentration-effect relationships. Combination index (CI) was calculated on the basis of the median-effect equation derived from the mass-action law principle.
Results
Individual substances showed decreasing cytotoxic effects in the following order: BisGMA>TPO>FIT>CQ>DMAEMA>TEGDMA. Experimental mixtures showed decreasing cytotoxic effects in the order BisGMA/TPO>BisGMA/CQ>FIT/CQ>FIT/TPO. FIT-based mixtures exhibited antagonistic cytotoxic effects between components while BisGMA-based mixtures demonstrated synergistic effects at ED 50. TPO amplified both antagonistic and synergistic cytotoxic effects in mixtures. Pure substances showed genotoxicity in the following order: TPO>BisGMA>FIT>CQ>TEGDMA. We did not detect the genotoxic potential of DMAEMA. The rank of genotoxic concentrations of the mixtures was: BisGMA/TPO>BisGMA/CQ>FIT/CQ>FIT/TPO.
Significance
Lower cytotoxicity and genotoxicity of FIT than BisGMA suggests its greater biocompatibility. Conversely, photoinitiator TPO was significantly more cytotoxic and genotoxic than both CQ and DMAEMA. CI values showed that components of FIT-based mixtures exhibit an antagonistic cytotoxic effect, while compontents of BisGMA-based mixtures show synergism.
1
Introduction
Residual monomers and other unbound substances from resin-based composites (RBCs) may reduce biocompatibility of RBCs via direct contact with oral tissues. Unbound components of composites diffuse through dentin into the pulp or elute into the oral cavity . These substances may also be systemically distributed throughout the organism, either being taken up by circulating blood or swallowed by saliva, consequently causing adverse systemic effects .
Several studies reported on the cytotoxicity, mutagenicity, estrogenicity, allergic reactions and systemic toxicity of RBC components . Other studies have shown adverse effects of monomers on cellular homeostasis even in sub-toxic concentrations . Investigations have also revealed that monomers and photoinitiators cause cellular redox imbalance increasing the levels of reactive oxygen species (ROS) and subsequent cell death via apoptosis as well as DNA damage .
Investigation on the biological effects of resin-based materials usually includes several most commonly used substances (monomers, oligomers, photoinitiators) acting separately , or eluates of commercial materials whose exact compositions remain undisclosed by the manufacturers . It is important to emphasize that two or more substances may exert significant cytotoxic and genotoxic effects alone, but in combination they may interact and produce toxic effects different from those expected from their sole actions . This necessitates the investigation of potentially combined biological effects of base monomers, co-monomers and photoinitiators. It is particularly important to discover synergistic toxic effects of mixed substances, producing higher biological effects than those coming from pure addition of their separate actions. Combined cytotoxic effects of different monomers and photoinitiators have not been studied extensively in the literature . Authors have mostly claimed synergistic effects by simple comparison of monomer mixtures. However, the synergism cannot be claimed by simple arithmetic addition of effects . The synergistic toxic effect of mixture of components occurs as a combination of effects’ magnitudes and their concentration dependences, the later obeying the mass-action low principle . These are both accounted in the so-called combination index (CI) which can be calculated when concentration-effect dependences of mixtures and their components are separately determined. Then, combinatorial toxic effect (synergy, addition, or antagonism) can be evaluated from the CI, not only for the concentrations of components in the investigated mixture, but for the range of component’s concentrations providing in this way general conclusions for the study. One should note that combinatorial effects may present different nature with different concentrations of components in the mixture; antagonism may occur with e.g. low-concentration of one component, but strong synergism when concentration of this component is increased.
Recent improvements of the organic matrix of RBCs include the development of new methacrylate monomers and photoinitiators. Polymerization shrinkage and potential risks associated with the toxicity of glycidyl dimethacrylate (BisGMA) and estrogenic effects of its precursor bisphenol-A , led to the development of bisphenol-A alternatives. Derivatives of urethane dimethacrylate (UDMA) and long-chain monomers with high molecular weight have already been used in several commercial low-shrinkage RBCs. Phosphine oxide photoinitiators are aimed at overcoming the yellowing effect of camphorquinone (CQ) and maintaining or improving polymerization efficiency without additional co-initiators .
Previous studies have reported comparable or better conversion, lower shrinkage and better color stability of commercial or experimental low-shrinkage RBCs compared to conventional ones. FIT-based experimental RBCs showed a higher degree of conversion and significantly lower shrinkage but lower Vickers hardness, flexural strength and modulus and lower color stability than BisGMA-based experimental RBCs. Lucirin TPO (TPO) photoinitiator exhibited better results over CQ/amine system in terms of polymerization efficiency and color stability .
Scarce literature data are available on toxicity of low-shrinkage monomers and novel photoinitiators. Van Landuyt et al. have reported stronger cytotoxic effect for TPO-containing than CQ/amine-containing adhesive. Eluates from two low-shrinkage RBCs, GC Kalore and Bisco Reflexion, have shown reduced cell viability . It is difficult to ascertain the toxic potential of certain components in multi-component commercial RBCs due to the often undisclosed composition or patent protection, especially in the case of novel, low-shrinkage monomers. Therefore, experimental mixtures seem to be a more appropriate model for biocompatibility testing.
In the present work, we aimed to compare cytotoxicity and genotoxicity of the novel, urethane-based, low-shrinkage monomer, FIT-852 (Code: FIT, Esstech Inc., Essington, PA, USA) and the alternative photoinitiator TPO to the most frequently used cross-linking monomer BisGMA, co-monomer triethylene glycol dimethacrylate (TEGDMA) and photoinitiator system CQ/amine. We used MTT and Comet assays to test cytotoxicity and genotoxicity, respectively, both being performed on fetal lung MRC-5 fibroblasts. Even though there are cell lines that could be considered as more convenient to evaluate dental materials, MRC-5 fibroblasts have already been used in several similar studies . This study focused on individual substances and clinically relevant mixtures. Moreover, we aimed to assess and quantify the combinatorial toxic effects of components present in these experimental mixtures, and to compare these combinatorial effects between different types of mixture compositions. The null hypotheses were: (1) there were no differences in cytotoxic and genotoxic effects of the tested pure substances and their mixtures (2) the mixture of monomers and photoinitiators does not influence their interaction and the total toxic capacity of the mixture.
2
Material and methods
2.1
Tested substances and mixtures
Table 1 contains the details of the tested monomers, photoinitiators and their clinically relevant mixtures. All ingredients were weighed on an analytical balance, accurate to 0.1 mg (ACCULAB ALC-110.4, Sartorius group, Goettingen, Germany). Monomers were homogenized in light-proof plastic tubes by slow rotation at 20 rpm for 24 h on a lab rotator (Stuart SB2, Bibby Scientific Ltd., Staffordshire, UK). Photoinitiators were added to monomer mixtures by dissolving in an ultrasonic bath for 1 min. All mixing was done in plastic tubes wrapped in aluminum foil to create dark conditions and prevent any chemical interaction. In the repeated experiments, we used freshly mixed substances and mixtures.
| Substances | Molecular weight (g/mol) | Tested concentrations | ||
|---|---|---|---|---|
| Cytotoxicity | Genotoxicity (μg mL −1 ) | |||
| (mmol L −1 ) | (μg mL −1 ) | |||
| BisGMA | 512.59 | 0.001, 0.005, 0.05, 0.1, 1, 2.5, 5 | 0.5, 2.6, 25.4, 51.3, 512.6, 1281.5 | 0.02, 0.1, 0.5, 2.5 × 10 −3 |
| FIT | 1200 a | 0.001, 0.005, 0.05, 0.1, 0.5 | 1.2, 6.0, 60.0, 120.0, 600.0 | 0.04, 0.2, 1, 5 |
| TEGDMA | 286.32 | 0.05, 0.1, 0.5, 1, 2.5 | 14.3, 28.6, 143.2, 286.3, 715.8 | 2, 10, 50, 250 |
| CQ | 166.217 | 0.05, 0.1, 0.5, 1, 2.5, 5 | 8.3, 16.6, 83.1, 166.2, 415.5, 831.0 | 0.6, 3, 15, 75 |
| DMAEMA | 157.21 | 0.1, 0.5, 1, 2.5, 5 | 15.7, 78.6, 157.2, 393.0, 786.0 | 0.6, 3, 15, 75 |
| TPO | 348.37 | 0.005, 0.05, 0.1, 0.5, 1, 2.5 | 1.7, 17.4, 34.8, 174.0, 348.0, 870.0 | 0.008, 0.04, 0.2, 1 × 10 −3 |
| Mixtures | Composition | Concentrations of the tested mixtures (μg mL −1 ) | ||
|---|---|---|---|---|
| Monomers (99 wt%) | Photoinitiators (1 wt%) | |||
| FIT/CQ | FIT 70 wt% TEGDMA 30 wt% |
CQ 0.2 wt% DMAEMA 0.8 wt% |
5, 10, 25, 50, 75, 100, 125,150, 200 | 0.5, 1, 5, 20, |
| FIT/TPO | FIT 70 wt% TEGDMA 30 wt% |
TPO 1 wt% | 5, 10, 25, 50, 75, 100, 150, 200, 225 | 0.1, 1, 5, 20 |
| BisGMA/CQ | BisGMA 70 wt% TEGDMA 30 wt% |
CQ 0.2 wt% DMAEMA 0.8 wt% |
0.5, 1, 5, 10, 25, 50, 75 | 0.05, 0.1, 5, 1 |
| BisGMA/TPO | BisGMA 70 wt% TEGDMA 30 wt% |
TPO 1 wt% | 0.1, 0.5, 1, 5, 10, 25, | 0.01, 0.05, 1, 0.1, 0.5 |
a According to information received from manufacturers molecular weight FIT is 1100-1200.
Monomers, photoinitiators and mixtures were dissolved in ethanol (absolute, ≥99.8%, Sigma-Aldrich) and then serial dilutions were prepared as shown in Table 1 . Pilot experiments showed that 1% of ethanol in cultured media caused no cytotoxicity and was used as control.
2.2
Eukaryotic cell cultures, media and growth conditions
We used human cell line of fetal lung fibroblasts MRC-5 (ECACC No. 84101801), passages 18–25, provided from the Oncology Institute of Vojvodina, Serbia. The cells were grown in Dulbecco’s Modified Eagle’s Minimal Essential Medium ( DMEM) with 4.5% glucose and 2 mmol × L −1 l -glutamine, supplemented with heat inactivated 10% fetal bovine serum, penicillin G (100 U mL −1 ) and streptomycin (100 μg × mL −1 ), at 37 °C with 5% CO 2 and 100% humidity. Cell line grew attached to the surface and a single cell suspension was obtained using 0.1% trypsin with 0.01% EDTA. We purchased all media, enzymes and supplements for cell cultures from PAA Laboratories GmbH, Austria.
2.3
Cytotoxicity testing
We tested cytotoxicity of monomers, photoinitiators and mixtures using the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); (Sigma-Aldrich Chemie Gmbh, Munich, Germany, CAS No 298-93-1) reduction assay . MRC-5 cells were seeded onto 96-well plates at a density of 5 × 10 4 cells/well and incubated 24 h to attach. The medium was then replaced with fresh complete medium containing different concentrations of the tested materials ( Table 1 ).
After incubation for 24 h the cell’s monolayers were inspected with inverted microscope. After that the medium containing test substances were removed and MTT was added in each well, adjusting its final concentration to 0.5 mg/mL, and the plates were incubated for additional 3 h. After that the medium was removed, and the formazan crystals were dissolved in dimethyl sulfoxide (DMSO). The optical density (OD) was measured at 570 nm using a microplate reading spectrophotometer (Thermo Scientific Multiscan FC, Finland). Cell viability was determined by comparing the OD of the wells containing treated cells to that of the vehicle (1% ethanol) treated cells. Eight individual wells were measured per treatment point because previous studies showed that 8 replicas per treatment point when using multichannel (8-channel) pipettes minimized the differences in cell numbers per well . At least two independent experiments were conducted. Student’s t-test was used for statistical analysis of the obtained data.
2.4
Genotoxicity testing
2.4.1
Genotoxicity assay
We evaluated genotoxicity of monomers, photoinitiators and mixtures using Comet assay. The preliminary monitoring of genotoxicity was performed after exposure of MRC-5 cells to each material for 24 h, at 37 °C, 5% CO 2 and 100% humidity. A potent mutagen 4-nitroquinoline-1-oxide (4-NQO, Cas No. N-8141, Sigma Aldrich) was used as a positive control. Genotoxicity was tested by exposure of the cells for 24 h to the non-cytotoxic concentrations of individual substances and their clinically relevant mixtures, as shown in Table 1 . Different concentrations were used for cytotoxicity and genotoxicity testing as the latter depend on the results of the former testing (screening of genotoxicity is performed with non-cytotoxic concentrations, i.e. the maximal toxicity shoud be ≤20%). Three independent experiments were performed.
2.4.2
Comet assay
We performed the alkaline Comet assay as described by Singh et al. and Collins . The microscopic slides were pre-coated with 1% normal melting point (NMP) agarose (Eurobio, France) and air-dried for 24 h at room temperature. Single cell suspension was obtained by 0.1% trypsin solution. In order to confirm the cell viability used in Comet assay, we previously checked it with trypan-blue exclusion assay. Cell suspension (30 μL) was mixed with 70 μL of 1% low melting point (LMP) agarose (Bio-Rad Laboratories, USA) and added to the slides that had previously been coated with one layer of 1% NMP agarose. The slides were covered with glass cover slips, placed at 4 °C for 5 min and after that the cover slips were gently removed, and the slides were submerged into ice-cold lysing solution (2.5 mol L −1 NaCl, 0.1 mol L −1 EDTA, 0.01 mol L −1 Tris, 1% Triton X-100, pH 10) and placed at 4 °C for at least 1 h.
After lysis, the slides were placed in a horizontal gel electrophoresis chamber, loaded with freshly made ice-cold electrophoresis solution (300 mmol L −1 NaOH, 1 mmol L −1 EDTA, pH 13). The slides were kept in this solution for 20 min at 4 °C to allow DNA unwinding and expression of alkali-labile sites. Following that, the samples were electrophoresed for 20 min at 25 V and 300 mA at 4 °C.
After electrophoresis, the slides were neutralized with 0.4 mol L −1 Tris buffer (pH 7.5) for 15 min at 4 °C. Each slide was stained with ethidium bromide (5 μg mL −1 ) and visualized using fluorescence microscope (Leica, DMLS, Austria) with an excitation filter of 510–560 nm, barrier filter of 590 nm, at 400 × magnification. We used image analysis software (Comet Assay IV, Version 4.2, 2007, Perceptive Instruments, UK) for analysis of comets. Although the results of Comet assay could be monitored by using different endpoints, the most useful parameter is tail intensity (TI, % of DNA in comet tail), since it bears a linear relationship to DNA damage frequency, is relatively unaffected by threshold settings, and allows discrimination of damage over the widest possible range . Accordingly, TIs were scored as a reflection of DNA damage. Fifty nuclei per experimental point in each of the three independent experiments were analyzed; the tail intensities (TI, % of DNA in comet tail) were scored as a reflection of DNA damage. The one-way analysis of variance (non-parametric ANOVA, Mann–Whitney U test) was employed for statistical analysis. The difference was considered significant when p < 0.05.
2.5
Calculation of combinatiorial toxic effects
We assessed the combinatorial effects of monomers and photoinitiators on overall cytotoxicity and genotoxicity from the measured concentration-effect relationships using CompuSyn software (v.1.0, ComboSyn Inc, Paramus, USA). Combination index (CI) allows quantitative determination of component interactions, where CI < 1, = 1, and >1 indicate synergism, additive effect, and antagonism, respectively. CI was calculated on the basis of the median-effect equation derived from the mass-action law principle :
f a f u = ( D D m ) m ,
where D is the concentration of a component, f a is the fraction of cells affected by D, and f u is the fraction of unaffected cells ( f u = 1 − f a). Dm (ED 50 ) is the median-effect concentration that inhibits the system under study by 50%, and m is the coefficient signifying the shape of the concentration-effect relationship, where m = 1, >1, and <1 indicate hyperbolic, sigmoidal, and flat sigmoidal concentration-effect curves, respectively .
Alternatively, Eq. (1) can be written in the following form:
log f a f u = m ⋅ log D − m ⋅ log D m ,
as a basis of the median-effect plot where m is the slope and Dm is the antilog of the x -intercept.
CI for n-component combination at x % inhibition is :
( C I ) x = ∑ j = 1 n ( D x ) 1 − n D j ∑ 1 n D j D m j ( ( f a x ) j 1 − ( f a x ) j ) m j ,
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