Toxicity of phospholipases A2 D49 (6-1 and 6-2) and K49 (Bj-VII) from Bothrops jararacussu venom

Abstract

Purified phospholipase A2 (PLA2) enzymes from Bothrops jararacussu snake venom were examined to evaluate NIH 3T3 and COS7 fibroblast cytotoxicity, as well as muscle myotoxic and inflammatory activities. Separation of fractions Bj-VII (from BthTX-I; a Lys49 PLA2 homolog) and 6-1 and 6-2 (from BthTX-II; an Asp49 PLA2) from B. jararacussu snake venom by SDS-PAGE in tricine buffer in the absence and presence of dithiothreitol revealed a homodimer with an estimated molecular mass of approximately 30 kDa (monomer mass approximately 15 kDa). This finding indicates that these toxins form dimeric complexes-a previously reported tendency among PLA2s. These toxins were assayed for viability with the MTT assay, which is used to examine the effects of phospholipases on the mitochondrial viability of cells. The toxins were also assayed for cytolysis of the fibroblast cell lines NIH 3T3 and COS7 by quantification of lactate dehydrogenase released into the medium. The results indicate that the PLA2s 6-1, 6-2 and the Bj-VII PLA2 homolog studied here induce moderate footpad edema and local myotoxicity. Moreover, exposure to these phospholipases led to a reduction in fibroblast viability; at the 1 muM dose of PLA2 tested, a reduction of 50% in cell viability was observed. The present findings indicate that the inflammatory activity observed in envenomation may be correlated with the cytotoxicity observed in fibroblasts.

Determination of Toxic Activities inBothropsspp. Snake Venoms Using Animal-Free Approaches: Correlation BetweenIn VitroVersusIn VivoAssays

“…The correlation between the levels of PLA 2 and edematogenic effects (r ¼ 0.94, p < .05) observed in this study is explained by the fact that PLA 2 s catalyze the hydrolysis of glycerophospholipids and promote the release of lysophospholipids and arachidonic acid, which are precursors of prostaglandins and leukotrienes, molecules involved in the inflammatory process characterized by increase by microvascular permeability formation, leukocyte recruitment into tissues and edema (Carvalho et al, 2013). Another example of edema formation through independent catalytic mechanisms by a PLA 2 isolated from B. atrox venom (Kanashiro et al, 2002) is the degranulation of mastocytes, histamine liberation and formation of edema (Bonfim et al, 2009). The highly significant correlation observed between PLA 2 activity and venom-induced edema, confirms our hypothesis that in vitro PLA 2 activity can be used instead in vivo edema activity tests.…”

Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A2 class

“…Recently, Bonfim et al showed that BthTX‐II presents a higher myotoxic effect than BthTX‐I (a Lys49 PLA 2 from Bothrops jararacussu snake venom) and attributed the additional potency to an indirect effect related to the catalytic activity exerted by this protein 63. On the other hand, our experiments have demonstrated that BthTX‐II is able to induce practically the same level of muscle damage in the absence of calcium as the Lys49‐PLA 2 BthTX‐I 89.…”

“…These toxins seem to be an exception among the classic Asp49‐PLA 2 s since they present reduced catalytic potency18 even though they conserve the residues of the catalytic network 59–62. BthTX‐II, a basic Asp49‐PLA 2 toxin from B. jararacussu , is also known for its edematogenic and hemolytic effects57, 58, 63 and for its ability to induce platelet aggregation and secretion through multiple signal transduction pathways 64. The residues between Thr112 and Pro121 from BthTX‐II primary sequence are assumed to be responsible for its myotoxic activity 30, 59…”

Purification and partial characterization of an anticoagulant PLA2 from the venom of Indian Daboia russelii that induces inflammation through upregulation of proinflammatory mediators

“…The degradation of membrane lipids owing to the enzymatic activity of PLA 2 enzymes leads to the release of intracellular contents, which cause oxidative stress as well as inflammatory response . PLA 2 enzymes like 6‐1 and 6‐2 (Asp49 PLA 2 ) and Bj‐VII (Lys49 PLA 2 ) isolated from the venom of Bothrops jararacussu are reported to exhibit mild edema in the footpad of tested mice . Interestingly, apart from the catalytically active, noncatalytic enzyme homologues (Lys49 residues) are also found to exhibit inflammatory reactions, suggesting that catalysis is not the sole criteria for exhibiting inflammation …”