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 study, 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 chromatographic steps. SDS-PAGE analysis showed that purified Balt-LAAO-II had a molecular weight of similar to 66 kDa. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to those of other snake venom l-Amino acid oxidases. This enzyme induces in vitro cytotoxicity in cultured human leukemic HL60 cells. Cells were grown in RPMI medium and incubated with the isoform Balt-LAAO-II (1, 10, and 100 mu g/mL) for up to 72 h. All three concentrations of venom markedly decreased cell viability from 6 h onwards based on 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 isoforms of Balt-LAAO-II and whole venom concentrations induced apoptosis after 2-6 h of incubation. Morphological analysis of cells incubated with the isoform Balt-LAAO-II and whole venom showed cell rounding and lysis, which increased with venom concentration and duration of incubation. These results show that the isoform Balt-LAAO-II from venom Bothrops alternatus is cytotoxic to cultured HL60 cells, suggesting that this damage may involve the apoptotic and oxidative stress pathways.