2.5.1. Comet assay to assess DNA strand breaks
The comet assay was performed under alkaline conditions as described by Collins et al. (2023). Blood was collected using a tip and a micropipette and placed in heparinized and refrigerated microtubes, while liver samples were dissected and immersed in refrigerated phosphate buffer (PBS). Liver samples of about 3x3mm were individually homogenized with the aid of a syringe, through the back and forth movement, in order to obtain a cell suspension.
Blood cells (5 μL whole blood aliquots) and dissociated liver cell suspensions (10 μL aliquots) were embedded in low melting point agarose (0.75%, w/v, 115 μL or 110 μL, respectively). The mixture was added to a microscope slide pre-coated with 1.5% normal melting point agarose, covered with a coverslip and then placed in the refrigerator for approximately 5 minutes at 4°C for solidification. Soon after, the coverslips were carefully removed and the slides immersed in lysis buffer (2.5M NaCl, 100mM EDTA and 10mM Tris, pH 10.0-10.5, with the addition of 1% Triton X - 100 and 10% DMSO) at 4ºC for a minimum of 12 hours and a maximum of 48 hours.
The slides were incubated in alkaline solution (300mM NaOH and 1mM EDTA, pH>13) for 20 minutes for DNA unfolding, followed by electrophoresis at ~ 1V/cm for approximately 20 minutes. All these steps were performed under weak yellow indirect light. Afterwards, the slides were neutralized with 0.4M Tris (pH 7.5) and, at the end, the DNA was stained by Syber Gold (1:10.000) (Invitrogen, USA) for further analysis.
Evaluation of 100 cells per individual and per tissue (50 cells in each duplicated slide) was performed. Resulting comet shapes were visually evaluated, being classified into five classes, according to the shape and size of the comet tail, with the classification for absence of tail considered 0, up to 4 when almost all DNA has moved to the tail, leaving a tiny comet head (Collins et al., 1997). In this way, we have a Damage Index for each animal ranging from zero (100 X 0 = 0; 100 cells observed completely without damage) to 400 (100 X 4 = 400; 100 cells observed with maximum damage).
International guidelines and recommendations for the comet assay consider the visual scoring of 100 comets to be a well-validated assessment method. It has a high correlation with computer image analysis (Collins et al., 1997). Negative and positive controls in in whole peripheral blood cells from healthy individuals were used for each assay to ensure the reliability of the procedure. Damage index of negative control = 13.00 ± 4.6 and Damage index of positive control: 105.30 ± 22.7). All slides were coded for blind analysis. The aspects of the comet assay procedures were reported following the guidelines of MIRCA (Minimum Information for Reporting on the Comet Assay) (Moller et al., 2020).