01%), which was used as a non-specific, non-biological positive control. Taking the muscle injury induced by Triton X-100 to be 100%, the myotoxic damage of the B. jararaca and L. obliqua venoms reached 58.8% and 39.6%, respectively, in our experimental conditions.

Because the maintenance of genomic stability is essential for cellular function, we measured the genotoxic effects induced by L. obliqua experimental envenomation in vivo ( Fig. 6). In the first set of experiments, DNA damage in the different organs and lymphocytes of rats 12 h after LOBE injection (1 mg/kg, s.c.) was assessed using the alkaline comet assay. For all samples, cell viability was evaluated using PLX4720 the trypan blue exclusion method

and was found to be greater than 90% in every experiment. The internal controls for the comet assay, using human blood cells, showed low damage in the negative control (DI = 0–10) and high damage in the positive control (DI = 180–300), thus validating the test conditions. As expected, exposure of the lymphocytes, heart, lungs, liver and kidney cells that had been isolated from normal animals to methyl methanesulfonate (MMS), which was used as positive control, resulted in a significant increase in DNA damage (not shown). As shown in CHIR-99021 manufacturer Fig. 6A, envenomed rats displayed high levels of DNA damage in the cells of all organs evaluated, as well as in the lymphocytes. The damage levels in the cells of the control Dichloromethane dehalogenase animals (those that had been injected with PBS) did not change significantly. The damage index in lymphocytes and kidneys reached levels that were 6.4 and 5.4 times higher than the levels in their respective controls. In another set of experiments, the kidneys were chosen to determine the temporal pattern of DNA damage at distinct time points after LOBE injection. In such cases, kidneys were selected because they had the highest damage index among the organs examined and also due to the high incidence of renal injury observed in human patients (Gamborgi et al., 2006). At 6 h,

kidney DNA damage had increased, reaching a maximal level at 12 h. After 48 h, the damage index decreased but was still significantly different from the controls (Fig. 6B). In order to verify the oxidative nature of the DNA damage detected in the kidney cells of LOBE-injected rats, we carried out a modified comet assay. While the alkaline test normally detects primarily repairable DNA single- and double-strand breaks and alkali-labile sites, the modified version is more specific to oxidative damage than the standard method. The modified version includes an incubation step with lesion-specific endonucleases that recognize resultant abasic sites and convert them into single-strand breaks. In the present study, we used Fpg, which is specific for oxidized purines, and Endo III, which targets oxidized pyrimidines.