2.6. Genotoxicity Assessment

Primary and oxidative DNA damage were assessed by the standard alkaline and formamidopyrimidine-DNA glycosylase (FPG)-modified comet assay versions, respectively. Cells were collected using a cell scrapper after 24 h of submerged or ALI exposure. ALI samples were suspended in cryoprotective medium (cell culture medium supplemented with 10% DMSO) and frozen at −80 °C until analysis. Cells from submerged exposures were washed 2× with PBS pH 7.4, scrapped and suspended in PBS. For submerged conditions, cells exposed to 500 µM MMS and 2.5 mM of KBrO₃ for 30 min were included as PC of the primary and oxidative DNA damage, respectively, whereas for ALI cells exposed to 1 mM H₂O₂ for 30 min were used as PC. Cells were counted in a Neubauer’s chamber and 6.0 × 10³ cells were transferred to a microcentrifuge tube and centrifuged at 700× g for 5 min. Supernatant was removed and cells were resuspended in 100 μL of 1% LMP agarose. Five microliters were placed onto microscope slides precoated with 1% NMP, using a high-throughput system of 12-minigel comet assay unit (Severn Biotech Ltd.^®, Kidderminster, UK). Three slides were prepared, one for the standard alkaline comet assay and two for the enzyme-modified version (with or without FPG-enzyme), and duplicates of each sample were added to each slide. The alkaline comet assay procedure was performed as previously described (Bessa et al., 2019). After agarose solidification at 4 °C for 5 min, slides were immersed in ice-cold lysis solution (2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris-base, 10 M NaOH, pH 10, 1% Triton-X 100) during 1 h at 4 °C, protected from light. After lysis, FPG-modified comet assay slides were washed three times for 5 min with buffer F (0.1 M KCl, 0.5 mM Na₂EDTA, 40 mM HEPES, 0.2 mg/mL BSA, pH 8) prior incubation for 30 min at 37 °C with 2.7 U/mL of FPG enzyme or with buffer F alone. After incubation, FPG and buffer F slides were washed with PBS pH 7.4. The alkaline comet assay slides were washed 3 times with PBS pH 7.4 for 5 min. For DNA unwinding, all slides were immersed in electrophoresis solution (1 mM Na₂EDTA, 0.3 M NaOH, pH 13) for 40 min at 4 °C, followed by electrophoresis in the same solution for 30 min at a constant 25 V (0.9 V/cm) and 400 mA. At the end of electrophoresis, slides were neutralised and fixed as described elsewhere [42]. For the comet scoring, slides were initially hydrated in Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM Na₂EDTA, pH 7.5–8) and then stained with 1:10,000 dilution of SYBR^® Gold in TE buffer for 40 min at room temperature. Comets were visualised in a Motic BA410 ELITE series microscope equipped with a complete EPI-fluorescence kit and scored using the Comet Assay IV image analysis software (Perceptive Instruments, Staffordshire, UK). At least 100 cells/experimental group (50 in each replicate gel) were scored and the mean of the percentage of DNA in the comet tail (% tail intensity) was used as a DNA damage descriptor.