Cytotoxic Activity Of A New Isoform L-Amino Acid Oxidase (Balt-LAAO-II) From Bothrops alternatus (Urutu) Snake Venom In Human Leukemic HL60 Cells

In this work we describe the isolation of a new isoform L-amino acid oxidase (LAAO) referred to as Balt-LAAO-II from Bothrops alternatus snake venom, which was highly purified using a combination of molecular exclusion (Sephadex G-75) and RP-HPLC chromatographics steps. When analyzed by SDS-PAGE, the purified Balt-LAAO-II presented a molecular weight of [~]66 kDa. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom L-amino acid oxidases. This enzyme induces in vitro cytotoxicity on cultured human leukemic HL60 cells. Cells were grown in RPMI medium and were incubated with isoform Balt-LAAO-II (1, 10 and 100 g/mL) for up to 72 h. All three concentrations of venom markedly decreased the cell viability from 6 h onwards based on the staining with propidium iodide, the reduction of 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and the uptake of neutral red. Flow cytometry showed that all isoform Balt-LAAO-II and whole venom concentrations induced apoptosis after 2-6 h of incubation. Morphological analysis of cells incubated with isoform Balt-LAAO-II and whole venom showed cell rounding and lysis that increased with the venom concentration and duration of incubation. These results show that isoform Balt-LAAO-II from venom Bothrops alternatus is cytotoxic to cultured HL60 cells and suggest that this damage may involve apoptotic and oxidative stress pathways.