Various protective effects of N-acetylcysteine (NAC) against triethylene glycol dimethacrylate (TEGDMA)-induced cell damage have been demonstrated, but so far there is no evidence on NAC direct monomer detoxification mechanism. Here, we hypothesized that NAC might reduce TEGDMA cytotoxicity due to direct NAC adduct formation.
We measured the cytotoxic effects of TEGDMA in presence and in absence of NAC by MTT test. Then we analyzed the presence of TEGDMA–NAC adduct formation in extracellular and intracellular compartments by capillary electrophoresis–UV detection (CE–UV) and capillary electrophoresis–mass spectrometry (CE–MS) analytical techniques. Moreover, we quantified the effective intracellular and extracellular TEGDMA concentrations through HPLC in the presence and absence of 10 mmol/L NAC.
TEGDMA reduced 3T3 cell vitality in a dose- and time-dependent manner, while NAC decreased monomer cytotoxicity and extracellular monomer concentrations by a direct reaction with TEGDMA. The adducts between the two molecules were detected both in the presence and absence of cell. Moreover a signal ascribed to the methacrylic acid was present in the CE–UV electropherogram of cellular lysates obtained after incubation with TEGDMA.
Our results suggest that in vitro detoxification capability of NAC against TEGDMA-induced cell damage might occur also through the formation of NAC–TEGDMA adduct.
Methacrylic monomers such as 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) are largely present in resinous based materials (RBM) utilized in preventive and restorative dentistry, as well as in products used in the prosthodontic and orthodontic field. In a clinical situation, incomplete polymerization of these chemicals leads to the release of unreacted residual monomers which may cause adverse biological reactions such as pulp injury and allergic effects in adjacent oral tissues . TEGDMA is considered a major co-monomer eluted from even polymerized resin composites into an aqueous environment, and it has been shown to cause specific stress responses in various eukaryotic cells in vitro . According to the current paradigm, tightly regulated cellular signaling networks of vital importance are modulated by monomer-produced oxidative stress. This burden is a consequence of a TEGDMA-caused depletion of intracellular glutathione (GSH) which is in turn associated with an increase in the level of reactive oxygen species (ROS) . If the monomer-induced ROS production exceeds the capacities of the cells’ enzymatic and non-enzymatic antioxidant systems, it leads to the disruption of specific cell functions . The induction of DNA damage or cytotoxicity via apoptosis is a major stress response of cells targeted by TEGDMA but the disturbance of adequate responses of cells of the immune system, cell proliferation and differentiation or mineralization processes have been reported as well .
N-acetylcysteine (NAC) is an anti-oxidant agent taken up by cells and enzymatically deacetylated to l -cysteine. Thus, NAC is a precursor of GSH synthesis which increases the intracellular GSH reserves, and it can act as a direct oxidant scavenger as well . It has been demonstrated that cells are protected from TEGDMA-induced damage in the presence of non-enzymatic antioxidants such NAC . Interestingly, it was found that resin-induced oxidative stress is most likely causes inhibition of mineralization because NAC considerably reduced cytotoxicity of methacrylates to maintain osteoblastic viability and function . Noteworthy, it has been suggested that NAC exhibited its protective function against methacrylate-based adverse phenomena through various mechanisms including anti-oxidant activity, activation of NFkB, cell differentiation or by the formation of a Michael adduct . A similar reaction may occur between GSH and methacrylates leading to the GSH depletion. In this respect, the spontaneous formation of a complex between HEMA and GSH has been reported . We have recently shown that the ability of NAC to counteract HEMA-induced cell damage was at least in part due to the formation of HEMA–NAC adducts via Michael addition . The nucleophilic thiol group of NAC is easily added to the α,β-unsaturated carbonyl moiety of the HEMA monomer. TEGDMA as a bifunctional molecule contains the identical methacrylic moiety. Hence, we hypothesized that TEGDMA might covalently bind to NAC forming TEGDMA–NAC adducts which could contribute to mitigate the toxic effects of the monomer. We therefore analyzed the presence of TEGDMA–NAC adduct formation in extracellular and intracellular compartments by capillary electrophoresis–UV detection (CE–UV) and capillary electrophoresis–mass spectrometry (CE–MS) analytical techniques. Moreover, we measured the effective intracellular and extracellular TEGDMA concentrations through HPLC analysis as well as extracellular TEGDMA levels in the presence of NAC.
Materials and methods
Chemicals and reagents
Thriethylene glycol dimethacrylate (TEGDMA), 3-(4,5-dimethyiazol-2-1)-2-5-diphenyl tetrazolium bromide (MTT), N-acetylcysteine (NAC), propidium iodide (PI), cell culture medium and reagents were purchased from Sigma Chemical Co. (Milan, Italy). Methanol (HPLC grade, Prolabo, France) and ultra-pure water (obtained by a P.Nix Power System apparatus, Human, Seoul, Korea) were used for HPLC analysis. Ammonia solution (28%, Carlo Erba, Milan, Italy), formic acid (98%, Mallinckrodt Baker B.V., Deventer, Holland) and sodium hydroxide pastilles (Merck, Darmstadt, Germany) were also utilized.
Effect of TEGDMA on cell vitality
Mouse 3T3 fibroblasts (Swiss albino mouse cell line, Istituto Zooprofilattico, Brescia, Italy) were grown in a 5% CO 2 atmosphere at 37 °C in DMEM (Dulbecco’s modified Eagle medium) with HEPES (10 mmol/L), glucose (1.0 g/L), NaHCO 3 (3.7 g/L), penicillin (100 units/mL), streptomycin (100 μg/mL) and 10% fetal calf serum.
Cytotoxic concentrations of TEGDMA were identified by the MTT assay. 3T3 fibroblasts were plated in a 96-well tissue culture dish at 8000 cells/well for 24 h. Next, the medium was removed and the cell monolayer was pre-incubated in the presence or absence of NAC (10 mmol/L) for 2 h: then, cell cultures were treated with TEGDMA (0, 0.5, 1.0, 1.5, 2.0 mmol/L) for 6 and 24 h in the presence or absence of NAC (10 mmol/L). After the exposure time, cells were washed twice with phosphate-buffered saline (PBS) and next incubated with 100 μL/well of a solution of MTT (0.5 mg/mL) in PBS at 37 °C for 1 h in a 5% CO 2 atmosphere. Finally, the MTT solution was replaced with 100 μL/well of DMSO and gently swirled for 10 min. The optical density in each well was immediately measured by a plate reader at 540 nm (Sunrise, Tecan, Switzerland). The results were expressed as the percentage of untreated cultures (=100%). Each experiment was performed at least five times in quadruplicate ( n = 5).
In addition, flow cytometry (FACScan, Becton–Dickinson, San Jose, CA, USA) was used to detect cell viability. Cells (1 × 10 5 ) were plated in culture dishes (35 mm) and incubated at 37 °C for 24 h. Cells were then exposed to TEGDMA (0–2 mmol/L) for 6 and 24 h in the presence or absence of 10 mmol/L NAC. After treatment, floating and adherent cells were collected, harvested by centrifugation and then washed once with phosphate-buffered saline. The vitality of cells in treated cell cultures and controls was assessed by the immediate stained with uptake of PI (1 μg/mL) into non-fixed cells after exposure, and subsequent flow-cytometric analyses based on an FSC-H/FL-3H profile. Viable cells (no staining) and death cells (PI staining) were detected and quantified as a percentage of the entire population. Data from at least three independent experiments, performed in duplicate, were analyzed using WinMDI Version 2.8 (The Scripps Research Institute, San Diego, CA, USA) ( n = 3).
Intracellular and extracellular concentrations of TEGDMA and metabolites
In order to determine the effect of NAC on intracellular and extracellular concentration of TEGDMA, 3T3 fibroblasts (1.0 × 10 6 ) were seeded in 25 cm 2 flasks, pre-incubated with NAC (10 mmol/L) for 2 h, and then treated with TEGDMA (0, 0.5, 1.0, and 2.0 mmol/L) for various time periods (10 min, 1 h, 2 h and 24 h) in the presence or absence of NAC (10 mmol/L). Cytosol from 3T3 fibroblasts was obtained after cell lysis by freezing (−80 °C) and thawing. Cellular lysates were centrifuged (20,000 × g , 15 min, 4 °C), and the supernatants were collected. The extracellular media were also centrifuged and filtered through a 0.45 μm syringe filter (Whatman, MaidstoneKent, UK).
Both samples were then analyzed using a JASCO HPLC system (2 PU-980 pumps, UV-970 UV/VIS detector and AS-1555 autosampler, JASCO Analytical Instruments, Mary’s Court Easton, MD, USA). The analyses were performed at a wavelength of 220 nm with a C-18 Supelco reversed phase column (150 mm × 4.7 mm, 5 μm) using an elution gradient of water (A) and methanol (B) starting from 35% to 75% of B (30 min), 0.7 mL/min flow, 50 μL injected volume. Each analysis was performed five times ( n = 5).
TEGDMA was identified by comparison of its retention time with that of the pure monomer in the same HPLC conditions. To evaluate the concentration of the monomer each analysis was preceded by the construction of a calibration line obtained with solutions of TEGDMA in acetonitrile (CH 3 CN) at concentrations starting from 0.03 mmol/L to 5.0 mmol/L. To verify if the detectability and the signal intensity of the monomer are the same in the analysis of a complex mixture like DMEM or cellular lysates, seven DMEM samples and seven cellular lysates were spiked with TEGDMA at concentrations starting from 0.03 mmol/L to 5.0 mmol/L.
In order to detect cellular metabolites, sub-confluenting 3T3 fibroblasts were treated with 1.0 mmol/L TEGDMA for different time periods (10 min, 1 h, 2 h, and 24 h), cytosol was collected as described, and analyzed by CE–UV technique.
Analysis of NAC–TEGDMA Adducts
A solution of NAC in H 2 O (4 mmol/L, 3 mL) was mixed with an ammonium acetate 0.1 mmol/L solution to reach pH 7.0. Subsequently the neutralized NAC solution was incubated at 37 °C with commercially pure TEGDMA corresponding to a final 0.5 mmol/L concentration in the reaction mixture. This reaction mixture was immediately and after 24 h analyzed by HPLC (as described above), CE–UV and CE–MS, and solutions containing NAC or TEGDMA separately were included as well. Each analysis was performed five times ( n = 5).
All analyses were performed with a Capillary Electrophoresis automated system (Agilent Technologies, Waldbronn, Germany) equipped with a diode array UV detector and external nitrogen supply and coupled to an Esquire 3000 plus mass spectrometer (BrukerDaltonics, Bremen, Germany) via a coaxial sheath liquid electrospray-ionization (ESI) interface (Agilent Technologies, Waldbronn, Germany). Sheath liquid was delivered by an external syringe pump (Cole Palmer, Vernon Hills, IL, USA) at constant flow rate. All experiments were performed in 50 μm internal diameter and 375 μm outer diameter fused silica uncoated capillary (Composite Metal Services, Hallow, Worcs., UK) of different length as specified below. New capillaries were conditioned as follows: (1) water (6 bar, 2 min), (2) 0.1 mol/L sodium hydroxide (4 bar, 20 min), (3) water (6 bar, 5 min). Between runs the capillary was rinsed with CH 3 CN 70% (1 min, 6 bar), H 2 O (0.7 min, 6 bar), 0.1 mol/L sodium hydroxide (0.7 min, 6 bar), H 2 O (2 min, 6 bar), and background electrolyte (BGE) (1.5 min, 6 bar). The temperature of the CE cartridge was set at 25 °C.
Capillary electrophoresis–UV detection (CE–UV)
CE–UV analyses were performed using a 63 cm total length capillary (55 cm effective length). The BGE was 25 mmol/L ammonium acetate titrated to pH 8.1 with ammonium hydroxide. Samples were injected at the anodic end by pressure application at 50 mbar × 10 s, followed by BGE injection at 50 mbar × 15 s. Running voltage was 30 kV.
Capillary electrophoresis–mass spectrometry (CE–MS)
CE–MS experiments were performed with a 89 cm total effective length capillary with on-column UV detection at 21.5 cm from the inlet side. BGE was 12.5 mmol/L acetic acid titrated to pH 8.1 with ammonium hydroxide. The sheath liquid (25 mmol/L ammonium acetate, pH 8.1/CH 3 OH, 30:70, v/v) was delivered at 200 μL/h. Nebulizer and dry gas, both nitrogen, were set at 6 psi and 4.0 L/min, respectively. Dry gas temperature was 250 °C. MS scan range was 60–500 m / z , using a maximum accumulation time of 200 ms and a set target value of 25,000 with the activation of the ion charge control (ICC) function. Mass spectrometry capillary voltage was 4500 V in positive ESI ionization mode. The protrusion of the CE capillary from the ESI needle (capillary exit) was regulated by using the tally marks on the micro-regulation screw on the ESI needle interface and optimized at the value of +2. All remaining conditions were the same as in CE–UV.
Flow injection analysis (FIA) was performed using the same system and capillary used for CE–MS with the procedure described in previous papers . The CE apparatus was used to introduce the sample in the MS source by flowing throw the capillary by pressure application (1 bar, instrument flush function) at the CE inlet capillary end. In FIA analysis the sheath liquid flow rate was raised to 240 μL/h. Mass spectrometry capillary voltage was 4500 V in positive and 4300 V in negative ESI ionization mode, respectively. All remaining conditions were the same as in CE–MS.
Data from all experiments were summarized as means ± standard deviation (SD) and differences between means were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. The level of significance was set at p < 0.05.