HEMA (CAS 868-77-9), Bis-GMA (CAS 1565-94-2), gradisol and RNase A, low melting point (LMP) and normal melting point (NMP) agarose, phosphate buffered saline (PBS), DAPI (4′,6-diamidino-2-phenylindole), dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), MTT, lectin, penicillin, streptomycin, sodium ascorbate, Bradford reagent were from Sigma Chemicals (St. Loius, MO, USA). hOGG1 was purchased from New England Biolabs (Herts, UK). Melatonin (5-methoxy-N-acetyltryptamine) was provided by R.J. Reiter of University of Texas Health Science Center. Quantum 333 medium, Dulbecco’s phosphate buffered saline (DPBS), trypsin and EDTA were from PAA Laboratories GmbH (Cölbe, Germany). Methanol-free formaldehyde solution was from Thermo Fisher Scientific, Worcester, MA, USA. Mouse monoclonal anti-γ-H2AX primary antibody, 1:100 dilution, anti-phospho-histone H2A.X (Ser139) clone JBW301, was obtained from Upstate (Charlotesville, VA, USA). Alexa Fluor 488 secondary antibody, 1:100 dilution, conjugated goat anti-mouse IgG was from Molecular Probes (Eugene, OR, USA). Cell viability kit was purchased in BD Biosciences (San Jose, CA, USA). All other chemicals were of the highest commercial grade available.
Cells and treatment
HGFs cell line was purchased from Provitro (Berlin, Germany). The cells were grown in Quantum 333 medium containing l-glutamine and supplemented with 1% antibiotic–antimycotic solution (10,000 U/ml penicillin, 10 mg/ml streptomycin sulphate, 25 μg/ml amphotericin B) in 75 cm2 cell culture flasks to approximately 75–80% confluence and maintained in an incubator with 5% CO2 atmosphere at 100% humidity at 37°C. After reaching confluence, the cells were washed with DPBS, detached from the flasks by a brief treatment with 0.05% trypsin-0.02% EDTA.
The model adhesive consisted of HEMA and Bis-GMA at 45/55% w/w with 8% water based on the total final weight of the mixture . To obtain a well-mixed resin we applied extensive shaking and sonication. The mixture was diluted with the cells medium to the concentrations desired in the experiments on DNA damage. HGFs were exposed to HEMA/Bis-GMA mixture at appropriate concentrations for 6 h at 37°C. We incubate HGFs with HEMA/Bis-GMA mixture, with reference to Bis/GMA at 0.01, 0.25, 0.05, 0.1 and 0.2 mM in DNA damage experiments and at 0.1 and 1.0 mM in apoptosis and cell cycle experiments. In the experiment with antioxidants, the exposure to HEMA/Bis-GMA was preceded by an 1 h incubation with sodium ascorbate or melatonin at 37°C. After the incubation, the suspension of the cells was centrifuged to remove free antioxidants. Each DNA damage experiment included a positive control, which was hydrogen peroxide at 20 μM for 15 min on ice . In the H2AX histone phosphorylation experiment the concentration of hydrogen peroxide was 1 mM.
Assessment of oxidative DNA damage
The human hOGG1, the primary enzyme for the repair of 8-oxoGua, was used to assess the extent of oxidative modification to the DNA bases [22, 23]. The enzyme nicks the DNA strand at the 8-oxoGua sites, producing single-strand breaks (SSBs) which can be easily detected by the alkaline comet assay.
The comet assay was performed under alkaline conditions essentially according to the procedure of Singh et al.  with modifications  as described previously . A freshly prepared suspension of cells in 0.75% LMP agarose dissolved in PBS was spread onto microscope slides precoated with 0.5% NMP agarose. The cells were then lysed for 1 h at 4°C in a buffer consisting of 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, 10 mM Tris, pH 10. After lysis, the slides were placed in an electrophoresis unit, the DNA was allowed to unwind for 20 min in the electrophoretic solution consisting of 300 mM NaOH, 1 mM EDTA, pH >13. Electrophoresis was conducted at 4°C (the temperature of the running buffer did not exceed 12°C) for 20 min at an electric field strength of 0.73 V/cm (290 mA).
The slides were then washed in water, drained and stained with 2 μg/ml DAPI and covered with cover slips. To prevent additional DNA damage, all the steps described above were conducted under dimmed light or in the dark. The slides were observed at ×200 magnification using an Eclipse fluorescence microscope (Nikon, Tokyo, Japan) attached to a COHU 4910 video camera (Cohu, Inc., San Diego, CA, USA) equipped with a UV-1 filter block consisting of an excitation filter (359 nm) and barrier filter (461 nm) and connected to a personal-computer-based image analysis system, Lucia-Comet v. 4.51 (Laboratory Imaging, Praha, Czech Republic). A hundred images was randomly selected from each sample and the comet tail DNA (% tail DNA) was measured. Each experiment was repeated three times. % tail DNA is positively correlated with the level of DNA breakage or/and alkali labile sites and is negatively correlated with the level of DNA crosslinks in the alkaline version of the comet assay . In the pH 12.1 and neutral version, it is positively correlated with strand breaks and DSBs, respectively. The mean value of the % tail DNA in a particular sample was taken as an index of the DNA damage in this sample.
After incubation with HEMA/Bis-GMA and cell lysis the slides from the comet assay were washed three times in the enzyme buffer containing 40 mM HEPES–KOH, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin, pH 8.0 for 5 min each time and drained. The agarose on slides was covered with 30 μl of the enzyme buffer either with or without hOGG1 at 1 μg/ml, sealed with a cover glass and incubated for 10 min at 37°C . The slides were processed as described in “Cells and treatment” section. To check the ability of the enzyme to recognize the DNA oxidative damage, we exposed HGF to 20 μM hydrogen peroxide for 10 min on ice (positive control). We compared the values obtained for the hOGG1 enzyme with the control containing only enzyme buffer.
DNA DSBs assay
The neutral comet assay was used to screen for DSBs in HGFs . In this version of the assay electrophoresis was run in a buffer consisting of 100 mM Tris and 300 mM sodium acetate at pH adjusted to 9.0 by glacial acetic acid. Electrophoresis was conducted for 60 min, after a 20 min equilibrium period, at electric field strength of 0.41 V/cm (50 mA) at 4°C. The slides were then proceeded as described in “Assessment of oxidative DNA damage” section. In this version, the mean value of the % tail DNA in a particular sample was taken as an index of the DNA DSBs in this sample.
The ability of the methacrylate monomers of the HEMA/Bis-GMA model adhesive to induce DSBs was confirmed and further analyzed by the immunofluorescence assay for the phosphorylation of the H2AX histone . HGFs were grown to approximately 75–80% confluence in 6-well plates. The medium was changed 24 h before incubation with the mixture with at 100 μM Bis-GMA. After the incubation, the cells were trypsinized with 500 μl trypsin–EDTA, washed with 1 ml medium and collected in 1.5 ml tubes. For immunofluorescent staining, cells (1–2 × 106) were washed in DPBS by centrifugation (300×g for 5 min at room temperature), fixed by 1 ml ice-cold 1% methanol-free formaldehyde in DPBS and incubated on ice for 15 min. Cells were centrifuged (300×g, 5 min, room temperature) and permeabilized with 80% ethanol in distilled water and kept at −20°C for 2 h until further staining. Cells were then washed three times with 1% BSA/0.2% Triton X-100/PBS (BTP) solution and stained with mouse monoclonal anti γ-H2AX primary antibody and incubated overnight at 4°C. Then, HGFs were washed three times with BTP solution and incubated with Alexa Fluor 488 secondary antibody for 1 h at room temperature in the dark. After the incubation cells were washed in BTP and counterstained with propidium iodide (PI, 5 μg/ml in DPBS in the presence of 100 μg/ml of RNase A) and incubated for 30 min at room temperature in the dark. Cells stained with Alexa Fluor 488 and PI were analyzed with LSRII flow cytometer (Becton–Dickinson Biologicals, San Jose, CA, USA) by measuring the intensity of green (530 ± 20 nm) and red (>600 nm) fluorescence of the cells. DNA content (red fluorescence of DNA-bound PI) was plotted on the x-axis and the level of γ-H2AX immunofluorescence (green fluorescence—Alexa Fluor 488) was plotted on the y-axis. Logarithmic Alexa Fluor 488 fluorescence was plotted versus linear PI fluorescence using FlowJo analysis software (TreeStar, Ashland, OR, USA). Untreated controls were used to set the threshold gating to determine the percentage of γ-H2AX positive cells. Intensity of cellular γ-H2AX immunofluorescence measured by flow cytometry is positively correlated with the level of DSBs and was used to quantify their extent [30, 31].
To examine DNA repair, cells after a 10 min pre-treatment with hydrogen peroxide at 100 μM on ice were washed and resuspended in a fresh medium containing HEMA/Bis-GMA at 10 μM Bis-GMA preheated to 37°C. Aliquots of the suspension were taken immediately and 30, 60, 90, and 120 min later. Placing the samples in an ice bath stopped the repair activity of cells. The kinetics of DNA repair was quantified by determination the extent of residual DNA damage at each time-point with using the comet assay.
The BD Annexin V-FITC Apoptosis Detection Kit I was used to measure apoptosis. The kit contains Annexin V conjugated to the flurochrome FITC that has affinity for phosphatidylserine, which is transferred through cell membrane in the earlier stages of apoptosis. Propidium iodine was used to distinguish early apoptotic cells from cells undergoing late apoptosis or necrosis. Cells that are viable are Annexin V-FITC and PI negative, cells that are in early apoptosis are Annexin-FITC positive and PI negative, cells that are in late apoptosis are both Annexin-FITC and PI positive, cells already dead are only PI positive. After 6 h of incubation with HEMA/Bis-GMA, cells were washed in cold medium and resuspended in 1× binding buffer at 106 cells/ml. 5 μl of Annexin V-FITC and 5 μl of PI were added to an aliquot of 100 μl (105 cells) of cells suspension, gently mixed by pipetting and incubated for 30 min at room temperature in the dark. Next, 400 μl of 1× binding buffer was added to each tube and samples were analyzed by flow cytometry. Each experiment had a negative, positive and unstained control sample. About 10,000 events were counted per sample. The apoptosis ratio was calculated as a percent of apoptotic cells in a sample.
The CycleTEST PLUS DNA Reagent Kit was used to determine the DNA index (DI) and cell-cycle phase distributions. Nuclei were isolated, stained with propidium iodine and afterward analyzed on the LSRII flow cytometer according to the manufacturer instruction. The DI was calculated by dividing the mean of the relative content of the exposed G0/G1 population by the mean of the control G0/G1 population. Results were analyzed by FlowJo software, v. 7.2.4.
The values in this study were expressed as mean ± SEM from three experiments, i.e. the data from three experiments were pooled and the statistical parameters were calculated. The Mann–Whitney test was used to determine differences between samples with distributions departing from normality. The differences between samples with the normal distribution were evaluated by applying the Student’s t test. Data analysis was performed using SigmaStat software (v. 3.0.0, SPSS, Chicago, USA).