Human envenoming by Lachesis muta muta venom, although infrequent, is rather severe, being characterized by pronounced local tissue damage and systemic dysfunctions. Studies on the pharmacological actions of L. m. muta venom are relatively scant and the direct actions of the crude venom and its purified phospholipase A2 (PLA2) have not been addressed using in vitro models. In this work, we investigated the cytotoxicity of L. m. muta venom and its purified PLA2 isoform LmTX-I in cultured Madin-Darby canine kidney (MDCK) and in a skeletal muscle (C2C12) cell lines. As revealed by neutral red dye uptake assay, the crude venom (10 or 100 μg/ml) induced a significant decrease in cell viability of MDCK cells. LmTX-I at the concentrations tested (70-270 μg/ml or 5-20 μM) displayed no cytotoxicity in both MDCK and C2C12 cell lines. Morphometric analysis of Feulgen nuclear reaction revealed a significant increase in chromatin condensation (pyknosis), apparent reduction in the number of mitotic nuclei and nuclear fragmentation of some MDCK cells after incubation with L. m. muta venom. Monolayer exposure to crude venom resulted in morphological changes as assessed by scanning electron microscopy. The staining with TRITC-labelled phalloidin showed a marked disarray of the actin stress fiber following L. m. muta venom exposure. In contrast, LmTX-I had no effect on nucleus and cell morphologies as well as on stress fiber organization. These results indicate that L. m. muta venom exerts toxic effects on cultured MDCK cells. The LmTX-I probably does not contribute per se to the direct venom cytotoxicity, these effects are mediated by metalloproteinases/disintegrins and other components of the venom. © 2006 Elsevier Ltd. All rights reserved.495678692Ahn, 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) Int. J. Biochem. Cell Biol., 29, pp. 911-919Alix, S.N., Woodbury, D.J., Phospholipase A2 action on planar lipid bilayers generates a small, transitory current that is voltage independent (1997) Biophys. J., 72, pp. 247-253Amaral, C.F.S., Da Silva, O.A., Godoy, P., Miranda, D., Renal cortical necrosis following Bothrops jararacussu snake bite (1985) Toxicon, 23, pp. 877-885Angulo, Y., Lomonte, B., Differential susceptibility of C2C12 myoblasts and myotubes to group II phospholipase A2 myotoxins from crotalid snake venoms (2005) Cell Biochem. Funct., 23, pp. 307-313Aragon-Ortiz, F., Brenes-Brenes, J.R., Gubensek, F., Characterization of a lectin-like protein isolated from Lachesis muta snake venom (1989) Rev. Biol. Trop., 37, pp. 79-83Arni, R.K., Ward, R.J., Phospholipase A2-a structural review (1996) Toxicon, 34, pp. 827-841Bieber, A.L., Ziolkowski, C., d' Avis, P.A., Rattlesnake toxins alter development of muscle cells in culture (1994) Ann. N.Y. Acad. Sci., 710, pp. 126-145Bjarnason, J.B., Fox, J.W., Hemorrhagic metalloproteinases from snake venoms (1994) Pharmacol. Therap., 62, pp. 325-372Brusés, J.L., Capaso, J., Katz, E., Pilar, G., Specific in vitro biological activity of snake venom myotoxins (1993) J. Neurochem., 60, pp. 1030-1042Bultrón, E., Thelestam, M., Gutiérrez, J.M., Effects on cultured mammalian cells of myotoxin III, a phospholipase A2 isolated from Bothrops asper (terciopelo) venom (1993) Biochim. Biophys. Acta, 1179, pp. 253-259Burdmann, E.A., Woronik, V., Prado, E.B.A., Abdulkader, R.C., Saldanha, L.B., Barreto, O.C.O., Marcondes, M., Snakebite-induced acute renal failure: an experimental model (1993) Am. J. Trop. Med. Hyg., 48, pp. 82-88Chan, A.B., Allen, C.N., Simmons, N.L., Parsons, M.E., Hirst, B.H., Resistance to acid of canine kidney (MDCK) and human colonic (T84) and ileo-caecal (HCT-8) adenocarcinoma epithelial cell monolayers in vitro (1989) Q. J. Exp. Physiol., 74, pp. 553-556Chioato, 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, pp. 869-883Clarke, P.G., Developmental cell death: morphological diversity and multiple mechanisms (1990) Anat. Embryol., 181, pp. 195-213Collares-Buzato, C.B., McEwan, G.T.A., Jepson, M.A., Simmons, N.L., Hirst, B.H., Paracellular barrier and junctional protein distribution depend on basolateral extracellular Ca2+ in cultured epithelia (1994) Biochim. Biophys. Acta, 1222, pp. 147-158Collares-Buzato, C.B., Jepson, M.A., McEwan, G.T.A., Simmons, N.L., Hirst, B.H., Co-culture of two MDCK strains with distinct junctional protein expression: A model for intercellular junction rearrangement and cell sorting (1998) Cell Tissue Res, 291, pp. 267-276Collares-Buzato, C.B., Le Sueur, L.P., Cruz-Höfling, M.A., Impairment of the cell-to-matrix adhesion and cytotoxicity induced by Bothrops moojeni snake venom in cultured renal tubular epithelia (2002) Toxicol. Appl. Pharmacol., 181, pp. 124-132Damico, D.C.S., Bueno, L.G.F., Rodrigues-Simioni, L., Marangoni, S., Cruz-Höfling, M.A., Novello, J.C., Neurotoxic and myotoxic actions from Lachesis muta muta (surucucu) whole venom on the mouse and chick nerve-muscle preparations (2005) Toxicon, 46, pp. 222-229Damico, D.C.S., Lilla, S., de Nucci, G., Ponce-Soto, L.A., Winck, F.V., Novello, J.C., Marangoni, S., Biochemical and enzymatic characterization of two basic Asp49 phospholipase A2 isoforms from Lachesis muta muta (Surucucu) venom (2005) Biochim. Biophys. Acta, 1726, pp. 75-86Damico, D.C.S., Bueno, L.G.F., Rodrigues-Simioni, L., Marangoni, S., Cruz-Höfling, M.A., Novello, J.C., Functional characterization of a basic D49 phospholipase A2 (LmTX-I) from the venom of the snake Lachesis muta muta (bushmaster) (2006) Toxicon, 47, pp. 759-765De Moura, R.S., Aguiar, A.S., Melgarejo, A.R., De Carvalho, C.R.M., Pharmacological aspects of mouse hind-paw oedema induced by Lachesis muta rhombeata venom (1998) Toxicon, 36, pp. 771-780Diniz, M.R., Oliveira, E.B., Purification and properties of a Kininogenin from the venom of Lachesis muta (bushmaster) (1992) Toxicon, 30, pp. 247-258Dixon, 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, pp. 1230-1237Ebisui, C., Tsujinaka, T., Morimoto, T., Fujita, J., Ogawa, A., Ishidoh, K., Kominami, E., Monden, M., Changes of proteasomes and cathepsins activities and their expression during differentiation of C2C12 myoblasts (1995) J. Biochem. (Tokyo), 117, pp. 1088-1094Fuly, A.L., Machado, O.L., Alves, E.W., Carlini, C.R., Mechanism of inhibitory action on platelet activation of a phospholipase A2 isolated from Lachesis muta (Bushmaster) snake venom (1997) Thromb. Haemost., 78, pp. 1372-1380Fuly, A.L., Calil-Elias, S., Zingali, R.B., Guimarães, J.A., Melo, P.A., Myotoxic activity of an acidic phospholipase A2 isolated from Lachesis muta (Bushmaster) snake venom (2000) Toxicon, 38, pp. 961-972Fuly, A.L., de Miranda, A.L., Zingali, R.B., Guimarães, J.A., Purification and characterization of a phospholipase A2 isoenzyme isolated from Lachesis muta snake venom (2002) Biochem. Pharmacol., 63, pp. 1589-1597Gallagher, P.G., Bao, Y., Serrano, S.M.T., Kamiguti, A.S., Theakston, R.D.G., Fox, J.W., Use of microarrays for investigating the subcytotoxic effects of snake venoms: insights into venom-induced apoptosis in human umbilical vein endothelial cells (2003) Toxicon, 41, pp. 429-440Giovanni-De-Simone, S., Aguiar, A.S., Gimenez, A.R., Novellino, K., de Moura, R.S., Purification, properties, and N-terminal amino acid sequence of a kallikrein-like enzyme from the venom of Lachesis muta rhombeata (Bushmaster) (1997) J. Protein Chem., 16, pp. 809-818Gowda, T.V., Middlebrook, J.L., Effects of myonecrotic snake venom phospholipase A2 toxins on cultured muscle cells (1993) Toxicon, 31, pp. 267-278Gutiérrez, J.M., Lomonte, B., Phospholipase A2 myotoxins from Bothrops snake venoms (1997) Venom Phospholipase A2 Enzymes: Structure, Function, and Mechanism, pp. 321-352. , Kini R.M. (Ed), Wiley, EnglandGutiérrez, J.M., Ownby, C.L., Skeletal muscle degeneration induced by venom phospholipases A2: insights into the mechanisms of local and systemic myotoxicity (2003) Toxicon, 42, pp. 915-931Gutiérrez, J.M., Rucavado, A., Snake venom metalloproteinases: their role in the pathogenesis of local tissue damage (2000) Biochimie, 82, pp. 841-850Harris, J.B., Myotoxic phospholipases A2 and the regeneration of skeletal muscles (2003) Toxicon, 42, pp. 933-945Incerpi, S., de Vito, P., Luly, P., Rufini, S., Effect of ammodytin L from Vipera ammodytes on L-6 cells from rat skeletal muscle (1995) Biochim. Biophys. Acta, 1268, pp. 137-142Jorge, M.T., Sano-Martins, I.S., Tomy, S.C., Castro, S.C.B., Ferrari, R.A., Ribeiro, L.A., Warrell, D.A., Snakebite by the bushmaster (Lachesis muta) in Brazil: case report and review of the literature (1997) Toxicon, 35, pp. 545-554Kamiguti, A.S., Zuzel, M., Theakston, R.D., Snake venom metalloproteinases and disintegrins: interactions with cells (1998) Braz. J. Med. Biol. Res., 31, pp. 853-862Kini, R.M., Phospholipase A2-a complex multifunctional protein puzzle (1997) Enzymes: Structure, Function and Mechanism, pp. 1-28. , Kini R.M. (Ed), Wiley, Chichester, EnglandKotyk, A., Janacek, K., Membrane transport an interdisciplinary approach (1977) Biomembranes, 9, pp. 3-348Krizaj, I., Bieber, A.L., Ritonja, A., Gubensek, F., The primary structure of ammodytin L, a myotoxic phospholipase A2 homologue from Vipera ammodytes venom (1991) Eur. J. Biochem., 202, pp. 1165-1168Kudo, I., Murakami, M., Phospholipase A2 enzymes (2002) Prostag. Lipid Mediat., 68-69, pp. 3-58Leite, R.S., Franco, W., Ownby, C.L., Selistre-de-Araújo, H.S., Effects of ACL myotoxin, a Lys49 phospholipase A (2) from Agkistrodon contortrix laticinctus snake venom, on water transport in the isolated toad urinary bladder (2004) Toxicon, 43, pp. 77-83Li, Z.Y., Yu, T.F., Lian, E.C., Purification and characterization of l-amino acid oxidase from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation (1994) Toxicon, 32, pp. 1349-1358Lo, C.-M., Keese, C.R., Giaever, I., Cell-substrate contact: another factor may influence transepithelial electrical resistance of cell layers cultured on permeable filters (1999) Exp. Cell Res., 250, pp. 576-580Lomonte, B., Tarkowski, A., Hanson, L.A., Broad cytolytic specificity of myotoxin II, a lysine-49 phospholipase A2 of Bothrops asper snake venom (1994) Toxicon, 32, pp. 1359-1369Lomonte, B., Tarkowski, A., Bagge, U., Hanson, L.A., Neutralization of the cytolytic and myotoxic activities of phospholipases A2 from Bothrops asper snake venom by glycosaminoglycans of the heparin/heparan sulfate family (1994) Biochem. Pharmacol., 47, pp. 1509-1518Lomonte, 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, pp. 145-158Lomonte, B., Ângulo, Y., Calderón, L., An overview of lysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action (2003) Toxicon, 42, pp. 885-901Magalhaes, A., Ferreira, R.N., Richardson, M., Gontijo, S., Yarleque, A., Magalhaes, H.P.B., Bloch, C., Sanchez, E.F., Coagulant thrombin-like enzymes from the venoms of Brazilian and Peruvian bushmaster (Lachesis muta muta) snakes (2003) Comp. Biochem. Physiol. B: Biochem. Mol. Biol., 136, pp. 255-266Masuda, S., Araki, S., Yamamoto, T., Kaji, K., Hayashi, H., Purification of a vascular apoptosis-inducing factor from hemorrhagic snake venom (1997) Biochem. Biophys. Res. Commun., 235, pp. 59-63Mebs, D., Ownby, C.L., Myotoxic components of snake venoms: their biochemical and biological activities (1990) Pharmacol. Ther., 48, pp. 223-236Núnez, 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, pp. 1587-1594Ogawa, T., Kitajima, M., Nakashima, K., Sasaki, Y., Ohno, M., Molecular evolution of group II phospholipases A2 (1995) J. Mol. Evol., 41, pp. 867-877Ohsaka, A., Hemorrhagic, necrotizing and edema-forming effects of snake venoms (1979) Handbook of Experimental Pharmacology, 52, pp. 480-546. , Lee C.Y. (Ed), Springer, BerlinOtero, R., Tobón, G.S., Gómez, L.F., Osorio, R., Valderrama, R., Hoyos, D., Urreta, J.E., Arboleda, J.J., Accidente ofídico in Antioquia y Chocó. Aspectos clínicos y epidemiológicos (marzo de 1989-febrero de 1990) (1992) Acta Med. Colombiana, 17, pp. 229-249Peixoto, E.B., Collares-Buzato, C.B., Protamine-induced epithelial barrier disruption involves rearrangement of cytoskeleton and decreased tight junction-associated protein expression in cultured MDCK strains (2005) Cell Struct. Funct., 29, pp. 165-178Powell, D.W., Barrier function of epithelia (1981) Am. J. Physiol. (Gastrointest. Liver Physiol.), 4, pp. 275-288Rucavado, A., Flores-Sánchez, E., Franceschi, A., Magalhães, A., Gutiérrez, J.M., Characterization of the local tissue damage induced by LHF-II, a metalloproteinase with weak hemorrhagic activity isolated from Lachesis muta muta snake venom (1999) Toxicon, 37, pp. 1297-1312Sanchez, E.O., Magalhães, A., Purification and partial characterization of an l-amino acid oxidase from bushmaster snake (Surucucu Pico de Jaca) Lachesis muta muta venom (1991) Braz. J. Med. Biol. Res., 24, pp. 249-260Sanchez, E.F., Magalhães, A., Diniz, C.R., Purification of a hemorrhagic factor (LHF-I) from the venom of the bushmaster snake (Lachesis muta muta) (1987) Toxicon, 25, pp. 611-619Sanchez, E.F., Cordeiro, M.N., Oliveira, E.B., Juliano, L., Prado, E.S., Diniz, C.R., Proteolytic specificity of two hemorrhagic factors, LHF-I and LHF-II, isolated from the venom of the bushmaster snake (Lachesis muta muta) (1995) Toxicon, 33, pp. 1061-1070Sanchez, E.F., Costa, M.I.E., Chavez-Olortegui, C., Assakura, M., Mandelbaum, F.R., Diniz, C.R., Characterization of a hemorrhagic factor, LHF-I, isolated from the bushmaster snake (Lachesis muta muta) venom (1995) Toxicon, 33, pp. 1653-1667Sanchez, E.F., Santos, C.I., Magalhaes, A., Diniz, C.R., Figueiredo, S., Gilroy, J., Richardson, M., Isolation of a proteinase with plasminogen-activating activity from Lachesis muta muta (Bushmaster) snake venom (2000) Arch. Biochem. Biophys., 378, pp. 131-141Sawai, T., Lampman, R., Hua, Y., Segura, B., Drongowski, R.A., Coran, A.G., Harmon, C.M., Lysophosphatidylcholine alters enterocyte monolayer permeability via a protein kinase C/Ca2+ mechanism (2002) Pediatr. Surg. Int., 18, pp. 591-594Schwerdt, G., Freudinger, R., Schuster, C., Silbernagl, S., Gekle, M., Inhibition of mitochondria and extracellular acidification enhance ochratoxin A-induced apoptosis in renal collecting duct-derived MDCK-C7 Cells (2004) Cell Physiol. Biochem., 14, pp. 47-56Suzuki, 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. 1260-1264Torii, S., Naito, M., Tsuruo, T., Apoxin I, a novel apoptosis inducing factor with l-amino acid oxidase activity purified from Western diamondback rattlesnake venom (1997) J. Biol. Chem., 272, pp. 9539-9542Torres, J.R., Torres, A.M., Arroyo-Parejo, M.A., Coagulation disorders in bushmaster envenomation (Letter) (1995) Lancet, 346, pp. 449-450Toyama, M.H., Toyama, D.O., Passero, L.F.D., Laurenti, M.D., Corbett, C.E., Tomokane, T.Y., Fonseca, F.V., Fonteles, H.S.A., Isolation of a new l-amino acid oxidase from Crotalus durissus cascavella venom (2006) Toxicon, 47, pp. 47-57Watlington, C.O., Smith, T.C., Huf, E.G., Direct electrical currents in metabolizing epithelial membranes (1970) Exp. Physiol. Biochem., 3, pp. 49-159Wiese, A.G., Pacifici, R.E., Davies, K.J., Transient adaptation of oxidative stress in mammalian cells (1995) Arch. Biochem. Biophys., 318, pp. 231-240