2.8. Comet Assay

The comet assay was performed following the procedure previously described by Collins and Azqueta [42], with minor modifications.

Each experiment consisted in a negative control (untreated cells), a positive control (1.25 mM KBrO₃), and 5 concentrations of silver-kaolin formulation or 5 concentrations of the corresponding silver-kaolin release (see Section 2.3). The following concentrations of silver-kaolin formulations were assayed: 0.02, 0.06, 0.17, 0.5, and 1.5 mg/mL for the 3 h treatment and 0.01, 0.02, 0.05, 0.17 and 0.5 mg/mL for the 24 h one. In the short treatment, a total of 6 × 10⁵ cells were exposed to each testing condition for 3 h in a volume of 1 mL in 12-well plates. In the long treatment, a total of 3 × 10⁵ cells were exposed to each testing condition for 24 h in a volume of 1 mL in 12-well plates. When cells were exposed to KBrO₃, treatment always lasted 3 h.

After treatment, cells were centrifuged (5 min, 293× g, 4 °C), diluted in culture medium to 1 × 10⁶ cells/mL, and mixed with 1% low melting point (LMP) agarose (dissolved in PBS), achieving 0.8% LMP agarose. Two drops of 70 µL of the cell suspension per slide were placed on 1% standard agarose pre-coated and a 20 × 20 mm coverslip was placed on top of each drop. Three identical slides were prepared for each testing condition. Slides were kept immersed for 1 h in lysis solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 1% Triton X-100, adjusted to pH 10 with NaOH) at 4 °C. Then, two slides per testing condition were washed with buffer F (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/mL BSA, pH 8) three times (5 min each). Afterwards, 45 µL of buffer F or Fpg enzyme (previously titrated [43]) was added to each gel of their corresponding set of slides, and 22 × 22 mm coverslips were put on top of each gel. Fpg and buffer F slides were incubated in a humidified atmosphere, at 37 °C for 1 h. After that, the coverslips were removed and the slides (including the set of slides kept in lysis solution) were immersed in electrophoresis solution (1 mM EDTA, 0.3 M NaOH, pH > 13) for 40 min at 4 °C. Then, slides were subjected to electrophoresis (1 V/cm) for 20 min, at 4 °C and neutralized by washing them with PBS followed by distilled water (10 min, 4 °C each wash). Finally, each gel was stained with 30 µL of 1 µg/mL DAPI solution and comets were analyzed by a fluorescent microscope (Nikon Eclipse 50 i, Tokyo, Japan) using the image analysis system Comet Assay IV (Perceptive instruments, Bury Saint Edmunds, UK). A total of 100 randomly selected cells were analyzed per slide, 50 cells of each duplicate gel. The DNA damage indicator used was tail DNA intensity (% DNA in tail). The % DNA in tail of the 50 comets analyzed per gel was calculated and then, the mean of both medians of each slide was obtained. The slides which remained immersed in lysis solution were used to assess the strand breaks (SBs) and alkali labile sites (ALS). The difference between the median % DNA in tail of the Fpg-treated slides and the one of the buffer F-treated ones was used to calculate the net Fpg-sensitive sites.

Statistical analysis was carried out with the Stata 12.0 software. Three independent experiments were carried out and the mean and SD of each testing condition were obtained. The % DNA in tail of each testing condition was statistically compared with the negative control through the Kruskal–Wallis test. Statistical significance was set at p < 0.05. Minimum Information for Reporting Comet Assay (MIRCA) recommendations were followed in this manuscript [44].