Toxicity Of Phospholipases A2 D49 (6-1 And 6-2) And K49 (bj-vii) From Bothrops Jararacussu Venom

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. © 2008 Springer Science+Business Media B.V.256523532Ahn, M.Y., Lee, B.M., Kim, Y.S., Characterization and cytotoxicity of L-amino acid oxidase from the venom of king cobra (Ophiophagus hannah) (1997) International Journal of Biochemistry and Cell Biology, 29 (6), pp. 911-919. , DOI 10.1016/S1357-2725(97)00024-1, PII S1357272597000241Andriao-Escarso, S.H., Soares, A.M., Rodrigues, V.M., Angulo, Y., Diaz, C., Lomonte, B., Gutierrez, J.M., Giglio, J.R., Myotoxic phospholipases A2 in Bothrops snake venoms: Effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu (2000) Biochimie, 82 (8), pp. 755-763. , DOI 10.1016/S0300-9084(00)01150-0Angulo, Y., Lomonte, B., Differential susceptibility of C2C12 myoblasts and myotubes to group II phospholipase A2 myotoxins from crotalid snake venoms (2005) Cell Biochemistry and Function, 23 (5), pp. 307-313. , DOI 10.1002/cbf.1208Arni, R.K., Ward, R.J., Phospholipase A2 - A structural review (1996) Toxicon, 34 (8), pp. 827-841. , DOI 10.1016/0041-0101(96)00036-0Bonfim, V.L., Ponce-Soto, L.A., Novello, J.C., Marangoni, S., Cytotoxic action in myoblasts and myotubes (C2C12) and enzymatic characterization of a new phospholipase A2 isoform (Bj-V) from Bothrops jararacussu venom (2006) Protein and Peptide Letters, 13 (7), pp. 707-713. , http://docstore.ingenta.com/cgi-bin/ds_deliver/1/u/d/ISIS/30400784.1/ben / ppl/2006/00000013/00000007/art00010/22524866E18A17B711525244689785D28A12EB6053. pdf?link=http://www.ingentaconnect.com/error/delivery&format=pdf, DOI 10.2174/092986606777790520Bonfim, V.L., Toyama, M.H., Novello, J.C., Hyslop, S., Oliveira, C.R.B., Rodrigues-Simioni, L., Marangoni, S., Isolation and enzymatic characterization of a basic phospholipase A 2 from Bothrops jararacussu snake venom (2001) Journal of Protein Chemistry, 20 (3), pp. 239-245. , DOI 10.1023/A:1010956126585Bonventre, J.V., Sapirstein, A., Group IV cytosolic phospholipase A2 (PLA2) function: Insights from the knockout mouse (2002) Adv Exp Med Biol, 507, pp. 25-31. , 12664560Brazil VO. A defesa contra o ofidismo. São paulo: Pocai-Weiss 1911. 152De Castro, R.C., Landucci, E.C.T., Toyama, M.H., Giglio, J.R., Marangoni, S., De Nucci, G., Antunes, E., Leucocyte recruitment induced by type II phospholipases A2 into the rat pleural cavity (2000) Toxicon, 38 (12), pp. 1773-1785. , DOI 10.1016/S0041-0101(00)00107-0, PII S0041010100001070Chacur, M., Longo, I., Picolo, G., Gutierrez, J.M., Lomonte, B., Guerra, J.L., Teixeira, C.F.P., Cury, Y., Hyperalgesia induced by Asp49 and Lys49 phospholipases A2 from Bothrops asper snake venom: Pharmacological mediation and molecular determinants (2003) Toxicon, 41 (6), pp. 667-678. , DOI 10.1016/S0041-0101(03)00007-2Chaves, F., Barboza, M., Gutierrez, J.M., Pharmacological study of edema induced by venom of the Bothrops asper (Terciopelo) in mice (1995) Toxicon, 33, pp. 331-339. , 10.1016/0041-0101(94)00135-UChaves, F., Leon, G., Alvarado, V.H., Gutierrez, J.M., Pharmacological modulation of edema induced by Lys-49 and Asp-49 myotoxic phospholipases A2 isolated from the venom of the snake Bothrops asper (terciopelo) (1998) Toxicon, 36 (12), pp. 1861-1869. , DOI 10.1016/S0041-0101(98)00107-X, PII S004101019800107XChioato, L., De Oliveira, A.H.R., Ruller, J.M., Ward, R.J., Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2 (2002) Biochem J, 366, pp. 971-976. , 12079495Chioato, L., Ward, R.J., Mapping structural determinants of biological activities in snake venom phospholipases A2 by sequence analysis and site directed mutagenesis (2003) Toxicon, 42 (8), pp. 869-883. , DOI 10.1016/j.toxicon.2003.11.027Cintra, A.C.O., Marangoni, S., Oliveira, B., Giglio, J.R., Bothropstoxin-I: Amino acid sequence and function (1993) Journal of Protein Chemistry, 12 (1), pp. 57-64. , DOI 10.1007/BF01024915Daniele, J.J., Bianco, I.D., Delgado, C., Carrillo, D.B., Fidelio, G.D., A new phospholipase A2 isoform isolated from Bothrops neuwiedii (Yarara chica) venom with novel kinetic and chromatographic properties (1997) Toxicon, 35 (8), pp. 1205-1215. , DOI 10.1016/S0041-0101(97)00023-8, PII S0041010197000238De Carvalho, D.D., Costa, F.T.M., Duran, N., Haun, M., Cytotoxic activity of violacein in human colon cancer cells (2006) Toxicology in Vitro, 20 (8), pp. 1514-1521. , DOI 10.1016/j.tiv.2006.06.007, PII S0887233306001202De Faria, L., Antunes, E., Bon, C., Lobo De Araujo, A., Pharmacological characterization of the rat paw edema induced by Bothrops lanceolatus (Fer de lance) venom (2001) Toxicon, 39 (6), pp. 825-830. , DOI 10.1016/S0041-0101(00)00213-0, PII S0041010100002130Díaz, C., Gutiérrez, J.M., Lomonte, B., Isolation and characterization of basic myotoxic phospholipases A 2 from Bothrops godmani (Godman's pit viper) snake venom (1992) Arch Biochem Biophys, 298 (1), pp. 135-142. , 10.1016/0003-9861(92)90104-5 1524423Diaz-Oreiro, C., Gutierrez, J.M., Chemical modification of histidine and lysine residues of myotoxic phospholipases A2 isolated from Bothrops asper and Bothrops godmani snake venoms: Effects on enzymatic and pharmacological properties (1997) Toxicon, 35 (2), pp. 241-252. , DOI 10.1016/S0041-0101(96)00128-6, PII S0041010196001286Dijkstra, B.W., Drenth, J., Kalk, K.H., Active site and catalytic mechanism of phospholipase A2 (1981) Nature, 289 (5798), pp. 604-606. , DOI 10.1038/289604a0Dijkstra, B.W., Renetseder, R., Kalk, K.H., Wim, G.J., Holand, D.J., Structure of porcine pancreatic phospholipase A2 at 26 a resolution and comparison with bovine phospholipase A2 (1983) J Mol Biol, 168, pp. 163-179. , 10.1016/S0022-2836(83)80328-3 6876174Dixon, R.W., Harris, J.B., Myotoxic activity of the toxic phospholipase, notexin, from the venom of the Australian tiger snake (1996) J Neuropathol Exp Neurol, 55 (12), pp. 1230-1237. , 10.1097/00005072-199612000-00006 8957446Gutiérrez, J.M., Lomonte, B., Venom phospholipase A2 enzymes: Structure, function and mechanism (1997) Chichester, , WileyGutierrez, J.M., Ownby, C.L., Skeletal muscle degeneration induced by venom phospholipases A 2: Insights into the mechanisms of local and systemic myotoxicity (2003) Toxicon, 42 (8), pp. 915-931. , DOI 10.1016/j.toxicon.2003.11.005Gutiérrez, J.M., Ponce-Soto, L.A., Marangoni, S., Lomonte, B., Systemic and local myotoxicity induced by snake venom group II phospholipases A2: Comparison between crotoxin, crotoxin B and a Lys49 PLA2 homologue (2008) Toxicon, 51 (1), pp. 80-92. , 10.1016/j.toxicon.2007.08.007 17915277Gutierrez, J.M., Ownby, C.L., Skeletal muscle degeneration induced by venom phospholipases A 2: Insights into the mechanisms of local and systemic myotoxicity (2003) Toxicon, 42 (8), pp. 915-931. , DOI 10.1016/j.toxicon.2003.11.005Hirabayashi, T., Murayama, T., Shimizu, T., Regulatory mechanism and physiological role of cytosolic phospholipase A2 (2004) Biological and Pharmaceutical Bulletin, 27 (8), pp. 1168-1173. , http://www.jstage.jst.go.jp/article/bpb/27/8/1168/_pdf, DOI 10.1248/bpb.27.1168Jung, Y.K., Miura, M., Yuan, J., Suppression of interleukin-1β-converting enzyme-mediated cell death by insulin-like growth factor J (1996) Biol Chem, 271, pp. 5112-5117. , 10.1074/jbc.271.9.5112Kanashiro, M.M., De Escocard, R.C.M., Petretski, J.H., Prates, M.V., Alves, E.W., Machado, O.L.T., Da Silva, W.D., Kipnis, T.L., Biochemical and biological properties of phospholipases A2 from Bothrops atrox snake venom (2002) Biochemical Pharmacology, 64 (7), pp. 1179-1186. , DOI 10.1016/S0006-2952(02)01288-1, PII S0006295202012881Kato, T., Ishiwata, M., Nagai, T., Mitochondrial calcium response in human transformed lymphoblastoid cells (2002) Life Sciences, 71 (5), pp. 581-590. , DOI 10.1016/S0024-3205(02)01725-3, PII S0024320502017253Kini, R.M., Evans, H.J., A common cytolytic region in myotoxins, hemolysins, cardiotoxins and antibacterial peptides (1989) Int J Pept Protein Res, 34, pp. 277-286. , 2599766Kini, R.M., Excitement ahead: Structure, function and mechanism of snake venom phospholipase A2 enzymes (2003) Toxicon, 42 (8), pp. 827-840. , DOI 10.1016/j.toxicon.2003.11.002Kudo, I., Murakami, M., Phospholipase A2 enzymes (2002) Prostaglandins and Other Lipid Mediators, 68-69, pp. 3-58. , DOI 10.1016/S0090-6980(02)00020-5, PII S0090698002000205Lambeau, G., Lazdunski, M., Receptors for a growing family of secreted phospholipases A2 (1999) Trends in Pharmacological Sciences, 20 (4), pp. 162-170. , DOI 10.1016/S0165-6147(99)01300-0, PII S0165614799013000Landucci, E.C.T., Castro, R.C., Toyama, M.H., Marangoni, S., Oliveira, B., Antunes, E., De Nucci, G., Inflammatory effects induced by piratoxin I, a PLA2 homologue isolated from Bothrops pirajai snake venom (1996) Programas e Resumos da IV Simpósio da Sociedade Brasileira de Toxinologí, p. 160Lomonte, B., Angulo, Y., Calderon, L., An overview of lysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action (2003) Toxicon, 42 (8), pp. 885-901. , DOI 10.1016/j.toxicon.2003.11.008Lomonte, B., Angulo, Y., Rufini, S., Cho, W., Giglio, J.R., Ohno, M., Daniele, J.J., Gutierrez, J.M., Comparative study of the cytolytic activity of myotoxic phospholipases A2 on mouse endothelial (tEnd) and skeletal muscle (C2C12) cells in vitro (1999) Toxicon, 37 (1), pp. 145-158. , DOI 10.1016/S0041-0101(98)00171-8, PII S0041010198001718Lomonte, B., Moreno, E., Tarkowski, A., Hanson, L.A., MacCarana, M., Neutralizing interaction between heparins and myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper snake venom. Identification of a heparin-binding and cytolytic toxin region by the use of synthetic peptides and molecular modeling (1994) J Biol Chem, 252 (47), pp. 29867-29873Mora, R., Valverde, B., Diaz, C., Lomonte, B., Gutierrez, J.M., A Lys49 phospholipase A2 homologue from Bothrops asper snake venom induces proliferation, apoptosis and necrosis in a lymphoblastoid cell line (2005) Toxicon, 45 (5), pp. 651-660. , DOI 10.1016/j.toxicon.2005.01.008Nuez, C.E., Angulo, Y., Lomonte, B., Identification of the myotoxic site of the Lys49 phospholipase A 2 from Agkistrodon piscivorus piscivorus snake venom: Synthetic C-terminal peptides from Lys49, but not from Asp49 myotoxins, exert membrane-damaging activities (2001) Toxicon, 39 (10), pp. 1587-1594. , DOI 10.1016/S0041-0101(01)00141-6, PII S0041010101001416Ponce-Soto, L.A., Baldasso, P.A., Romero-Vargas, F.F., Winck, F.V., Novello, J.C., Marangoni, S., Biochemical, pharmacological and structural characterization of two PLA2 isoforms Cdr-12 and Cdr-13 from Crotalus durissus ruruima snake venom (2007) Biochim Biophys Acta, 251-726 (1), pp. 75-86Ponce-Soto, L.A., Bonfim, V.L., Rodrigues-Simioni, L., Novello, J.C., Marangoni, S., Determination of primary structure of two isoforms 6-1 and 6-2 PLA 2 D49 from Bothrops jararacussu snake venom and neurotoxic characterization using in vitro neuromuscular preparation (2006) Protein Journal, 25 (2), pp. 147-155. , DOI 10.1007/s10930-006-0006-4Ponraj, D., Gopalakrishnakone, P., Establishment of an animal model for myoglobinuria by use of a myotoxin from Pseudechis australis (king brown snake) venom in mice (1996) Lab Anim Sci, 46 (4), pp. 393-398. , 8872989Rosenberg, P., Lethal potency of snake venom phospholipase A2 enzymes. Pitfalls to avoid in the study of correlations between enzymatic activity and pharmacological properties of phospholipase A2 enzymes (1997) Venom Phospholipase A2 Enzymes: Structure, Function and Mechanism, pp. 129-154. , R.M. Kini (eds). Wiley New YorkRuffini, S., Cesaroni, M., Balestro, N., Luly, P., Proliferative effect of ammodytin L from the venom of Vipera ammodytes on 208F rat fibroblasts in culture (1996) Biochem J, 320, pp. 467-472Salvini, T.F., Amaral, A.C., Miyabara, E.H., Turri, J.A.O., Danella, P.M., Selistre De Araujo, H.S., Systemic skeletal muscle necrosis induced by crotoxin (2001) Toxicon, 39 (8), pp. 1141-1149. , DOI 10.1016/S0041-0101(00)00245-2, PII S0041010100002452Serhan, C.N., Eicosanoids in leukocyte function (1994) Curr Opin Hematol, 1, pp. 69-77. , 9371262Six, D.A., Dennis, E.A., The expanding superfamily of phospholipase A(2) enzymes: Classification and characterization (2000) Biochim Biophys Acta, 1488, pp. 1-19. , 11080672Soares, A.M., Rodrigues, V.M., Homsi-Brandeburgo, M.I., Toyama, M.H., Lombardi, F.R., Arni, R.K., Giglio, J.R., A rapid procedure for the isolation of the LYS-49 myotoxin II from bothrops moojeni (caissaca) venom: Biochemical characterization, crystallization, myotoxic and edematogenic activity (1998) Toxicon, 36 (3), pp. 503-514. , DOI 10.1016/S0041-0101(97)00133-5, PII S0041010197001335Suzuki, K., Nakamura, M., Hatanaka, Y., Kayanoki, Y., Tatsumi, H., Taniguchi, N., Induction of apoptotic cell death in human endothelial cells treated with snake venom: Implication of intracellular reactive oxygen species and protective effects of glutathione and superoxide dismutases (1997) J Biochem, 122, pp. 1260-1264. , 9498574Todaro, G.J., Green, H., Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines (1963) J Cell Biol, 17, pp. 299-313. , 10.1083/jcb.17.2.299 13985244Vane, J., Control of the circulation by endothelial mediators. Inaugural G.B. west memorial lecture (1993) Int Arch Allergy Immunol, 101, pp. 333-345. , 10.1159/000236474 8353456Yamakawa, M., Nozaky, M., Hokama, Z., Fractionation of sakishimahabu (Trimeresurus elegans) venom and lethal, hemorrhagic and edema-forming activities of the fractions (1976) Toxins: Animal, Plant and Microbial, pp. 97-109. , A. Ohsaka K. Hayashi Y. Sawai (eds). Plenum New Yor