The cells were submitted to 4 °C for 10 min, and after that, the coverslips were removed and the slides immersed in lyses solution (containing 0.25 M NaCl, 100 mM EDTA, 10 mM Trizma base, pH 10 adjusted with 10 M NaOH, 5% DMSO and 1% Triton X-100), remaining there for 2 h, being this procedure responsible for the achievement of the nucleoid. Doxorubicin (Bergamo Ltda) (2 μg/mL) was used as a positive control. All the procedures described
above and the electrophoresis were carried out in Stem Cell Compound Library the dark. Before the electrophoretic run, the slides were kept in electrophoresis solution (300 mM sodium hydroxide and 1 mM EDTA, pH 13) for 20 min at 4 °C. The electrophoretic run was programmed at 25 V and 300 mA, and the run time was fixed as 25 min. After the run, the slides were immersed in neutralization solution
(0.4 M Tris–HCl, pH 7.4) for 10 min, dried at room temperature and fixed with 100% ethanol for 3 min. The coloration was performed with ethidium bromide solution at 20 μg/mL. To that end, 100 μL of this solution was placed over each slide, protected from light, covered with a coverslip and immediately analyzed by fluorescence microscopy at 400X. Comet standards were analyzed by visual scores according to Collins et al. (1993), with minimal modifications as previously described (Marcussi et al., 2011). The cells analyzed were classified by DNA injury extent in 5 classes: class 0, without damage (damage <5%); class 1, low level of damage (5–20%); class 2, medium
level of damage (20–40%); class 3, high level of damage (40–95%) and class 4, ZVADFMK totally damaged (damage> 95%). In order to perform comparative analysis, data were calculated with arbitrary units as described by Collins (2004). Data are presented as means with standard deviations (mean ± S.D.). A p value of less than 0.05 was deemed to be statistically significant (Kruskal–Wallis). Initially, cell viability tests were performed using a concentration response curve, before carrying out the micronucleus and comet the tests, in order to determine the quantities of venoms or toxins which allowed the evaluation of the DNA damage without affecting the cell cycles or inducing cell death. The effective doses chosen were 5, 15 and 30 μg/mL. As positive control the mutagenic and antineoplastic drug Cisplatin was used (6 μg/mL). The micronucleus test indicated that BthTX-I and BthTX-II from B. jararacussu and BatxLAAO from B. atrox were potentially genotoxic as there were more than 2 MN/1000 BN cells for a mean of 6 experiments with 30 μg/mL (7.6, 8.7 and 6.6 respectively) ( Table 1), suggesting a potential genotoxic effect. Concerning the crude venoms, only B. jararacussu and B. atrox showed to be potentially genotoxic, yielding (at 30 μg/mL) an average of 6 and 7.3 MN/1000 BN cells in 6 experiments performed with lymphocytes isolated from the blood of the 6 volunteers ( Table 2). These results confirm the significance of myotoxins and LAAOs in the composition of B. jararacussu and B. atrox venom.