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

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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 ∼30 kDa (monomer mass ∼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 μM 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.

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Fig. 1
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Abbreviations

CK:

Creatine kinase

LHD:

Lactate dehydrogenase

MTT:

3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

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Acknowledgments

The authors thank Mr. Paulo A. Baldasso for general technical help. This work was supported by CAPES and FAPESP.

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Authors and Affiliations

  1. Department of Biochemistry, Institute of Biology, State University of Campinas (Unicamp), P.O. Box 6109, 13083 970, Campinas, SP, Brazil

    V. L. Bonfim, D. D. de Carvalho, L. A. Ponce-Soto & S. Marangoni

  2. Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (Unicamp), P.O. Box 6109, 13083 970, Campinas, SP, Brazil

    L. A. Ponce-Soto

  3. Basic Institute of Bioscience (IBB), University of Taubaté (UNITAU), Taubaté, SP, Brazil

    B. H. Kassab

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  1. V. L. Bonfim
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  2. D. D. de Carvalho
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Correspondence to V. L. Bonfim.

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Bonfim, V.L., de Carvalho, D.D., Ponce-Soto, L.A. et al. Toxicity of phospholipases A2 D49 (6-1 and 6-2) and K49 (Bj-VII) from Bothrops jararacussu venom. Cell Biol Toxicol 25, 523–532 (2009). https://doi.org/10.1007/s10565-008-9106-6

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