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).