The increase in CK released from EDL muscles after addition of LOBE was considered to be indicative of direct myotoxic activity. CK activity was expressed as enzyme units released into the medium per gram of muscle (U/g). One enzyme unit was defined as the amount that catalyzes the transformation of 1 μmol of substrate per min at 25 °C. The genotoxic activity was detected in vivo using the model of envenomation described in

Subsection 2.3.1. The blood, liver, lungs, heart and kidneys were collected at 6, 12 and 48 h after LOBE injection (1 mg/kg, s.c.). The organs were gently homogenized in a cold PBS solution (2 mL) to obtain MAPK inhibitor a cell suspension. Total blood was used for the detection of DNA damage in lymphocytes. Genotoxicity was then evaluated using the comet assay. The alkaline comet assay was performed as described by Singh et al. (1988), with minor modifications (Azqueta et al., 2009 and Tice et al., 2000). Briefly, 20 μL of homogenized organs and blood were mixed with 0.75% low-melting point agarose and immediately spread onto a glass microscope slide that had been pre-coated with a layer

of 1% normal-melting point agarose. The slides were then incubated in an ice-cold lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM EDTA, 1% Triton X-100 and 10% DMSO, pH = 10.0; Gibco BRL, selleck kinase inhibitor Grand Island, Baricitinib NY) at 4 °C for at least 1 h to remove the cellular proteins and membranes, leaving the DNA as “nucleoids”. In the modified

version of comet assay, the slides were removed from the lysis solution and washed three times in enzyme buffer (40 mM HEPES, 100 mM KCl, 0.5 mM Na2-EDTA, and 0.2 mg/mL BSA, pH = 8.0), were drained and were incubated at 37 °C in this buffer with one of the following: 70 μL of Fpg (New England Biolabs, Beverly, MA, USA) at 100 mU per gel for 45 min (for the detection of oxidized purines) or 70 μL of Endo III (New England Biolabs, Beverly, MA, USA) at 100 mU per gel for 30 min (for the detection of oxidized pyrimidines). After lysis, the slides were placed in a horizontal electrophoresis unit that had been filled with fresh buffer (300 mM NaOH and 1 mM EDTA, pH > 13.0), which was left to cover the slides for 20 min at 4 °C to allow the DNA to unwind and reveal the expression of alkali-labile sites. Electrophoresis was conducted for 20 min at 25 V (78 V/cm) and 300 mA. All of the steps outlined above were performed under yellow light or in the dark to prevent additional DNA damage. The slides were then neutralized (0.4 M Tris, pH = 7.5), washed in double-distilled water and stained using a silver staining protocol, as described by Nadin et al. (2001). After the staining step, the gels were left to dry at room temperature overnight and were analyzed using an optical microscope.