Synergism between basic Asp49 and Lys49 phospholipase A2 myotoxins of viperid snake venom in vitro and in vivo

artículo (arbitrado) -- Universidad de Costa Rica, Instituto de investigaciones Clodomiro Picado. 2014Two subtypes of phospholipases A2 (PLA2s) with the ability to induce myonecrosis, ‘Asp49’ and ‘Lys49’ myotoxins, often coexist in viperid snake venoms. Since the latter lack catalytic activity, two different mechanisms are involved in their myotoxicity. A synergism between Asp49 and Lys49 myotoxins from Bothrops asper was previously observed in vitro, enhancing Ca2+ entry and cell death when acting together upon C2C12 myotubes. These observations are extended for the first time in vivo, by demonstrating a clear enhancement of myonecrosis by the combined action of these two toxins in mice. In addition, novel aspects of their synergism were revealed using myotubes. Proportions of Asp49 myotoxin as low as 0.1% of the Lys49 myotoxin are sufficient to enhance cytotoxicity of the latter, but not the opposite. Sublytic amounts of Asp49 myotoxin also enhanced cytotoxicity of a synthetic peptide encompassing the toxic region of Lys49 myotoxin. Asp49 myotoxin rendered myotubes more susceptible to osmotic lysis, whereas Lys49 myotoxin did not. In contrast to myotoxic Asp49 PLA2, an acidic non-toxic PLA2 from the same venom did not markedly synergize with Lys49 myotoxin, revealing a functional difference between basic and acidic PLA2 enzymes. It is suggested that Asp49 myotoxins synergize with Lys49 myotoxins by virtue of their PLA2 activity. In addition to the membrane-destabilizing effect of this activity, Asp49 myotoxins may generate anionic patches of hydrolytic reaction products, facilitating electrostatic interactions with Lys49 myotoxins. These data provide new evidence for the evolutionary adaptive value of the two subtypes of PLA2 myotoxins acting synergistically in viperid venoms.Funding support by the Graduate Studies Program, Universidad de Costa Rica; International Centre for Genetic Engineering and Biotechnology, Italy (CRP/COS13-01); and Vicerrectoria de Investigacion, Universidad de Costa Rica (741-B4-100).UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Poor Regenerative Outcome after Skeletal Muscle Necrosis Induced by Bothrops asper Venom: Alterations in Microvasculature and Nerves

artículo (arbitrado) -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2011Background: Viperid snakebite envenoming is characterized by prominent local tissue damage, including muscle necrosis. A frequent outcome of such local pathology is deficient skeletal muscle regeneration, which causes muscle dysfunction, muscle loss and fibrosis, thus provoking permanent sequelae that greatly affect the quality of life of patients. The causes of such poor regenerative outcome of skeletal muscle after viperid snakebites are not fully understood. Methodology/Principal Findings: A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America. Gastrocnemius muscle was injected with either B. asper venom, a myotoxic phospholipase A2 (Mtx), a hemorrhagic metalloproteinase (SVMP), or saline solution. At various time intervals, during one month, tissue samples were collected and analyzed by histology, and by immunocytochemical and immunohistochemical techniques aimed at detecting muscle fibers, collagen, endothelial cells, myoblasts, myotubes, macrophages, TUNEL-positive nuclei, and axons. A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature. In contrast, poor regeneration, with fibrosis and atrophic fibers, occurred when muscle was injected with venom or SVMP, both of which provoke necrosis, microvascular damage leading to hemorrhage, and poor axonal regeneration. Conclusions/Significance: The deficient skeletal muscle regeneration after injection of B. asper venom is likely to depend on the widespread damage to the microvasculature, which affects the removal of necrotic debris by phagocytes, and the provision of nutrients and oxygen required for regeneration. In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model.This study was supported by NeTropica (grant 2-N-2008), by Vicerrectoría de Investigación, Universidad de Costa Rica (project 741-A7-604). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Envenomations by Bothrops and Crotalus Snakes Induce the Release of Mitochondrial Alarmins

Skeletal muscle necrosis is a common manifestation of viperid snakebite envenomations. Venoms from snakes of the genus Bothrops, such as that of B. asper, induce muscle tissue damage at the site of venom injection, provoking severe local pathology which often results in permanent sequelae. In contrast, the venom of the South American rattlesnake Crotalus durissus terrificus, induces a clinical picture of systemic myotoxicity, i.e., rhabdomyolysis, together with neurotoxicity. It is known that molecules released from damaged muscle might act as ‘danger’ signals. These are known as ‘alarmins’, and contribute to the inflammatory reaction by activating the innate immune system. Here we show that the venoms of B. asper and C. d. terrificus release the mitochondrial markers mtDNA (from the matrix) and cytochrome c (Cyt c) from the intermembrane space, from ex vivo mouse tibialis anterior muscles. Cyt c was released to a similar extent by the two venoms whereas B. asper venom induced the release of higher amounts of mtDNA, thus reflecting hitherto some differences in their pathological action on muscle mitochondria. At variance, injection of these venoms in mice resulted in a different time-course of mtDNA release, with B. asper venom inducing an early onset increment in plasma levels and C. d. terrificus venom provoking a delayed release. We suggest that the release of mitochondrial ‘alarmins’ might contribute to the local and systemic inflammatory events characteristic of snakebite envenomations

Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis

Citation: Herrera C, Macêdo JKA, Feoli A, Escalante T, Rucavado A, Gutiérrez JM, et al. (2016) Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis. PLoS Negl Trop Dis 10(4): e0004599. doi:10.1371/journal. pntd.0004599The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A2, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM) and other extracellular matrix (ECM) proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs) or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms.Universidad de Costa Rica/[741-B4-660]/UCR/Costa RicaUniversidad de Costa Rica/[741-B6-125]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Antibothropic action of Casearia sylvestris Sw. (flacourtiaceae) extracts

Casearia sylvestris Sw., popularly known in Brazil as 'guacatonga', has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects22678479

Antibothropic Action Of Casearia Sylvestris Sw. (flacourtiaceae) Extracts

Casearia sylvestris Sw., popularly known in Brazil as 'guaçatonga', has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C. sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects. Copyright © 2008 John Wiley & Sons, Ltd.226784790Bjarnason, J.B., Fox, J.W., Hemorrhagic metalloproteinases from snake venoms (1994) Pharmacol Ther, 62, pp. 325-372Borges, M.H., Soares, A.M., Rodrigues, V.M., Effects of aqueous extract of Casearia sylvestris (Flacourtiaceae) on actions of snake and bee venoms and on activity of phospholipases A 2 (2000) Comp Biochem Physiol B Biochem Mol Biol, 127, pp. 21-30Borges, M.H., Soares, A.M., Rodrigues, V.M., Neutralization of proteases from Bothrops snake venoms by aqueous extract from Casearia sylvestris (Flacourtiaceae) (2001) Toxicon, 39, pp. 1863-1869Bülbring, E., Observation on the isolated phrenic nerve diaphragm preparation of the rat (1946) Br J Pharmacol, 1, pp. 38-61De Oliveira, M., Cavalcante, W.L., Arruda, E.A., Melo, P.A., Dal-Pai Silva, M., Gallaci, M., Antagonism of myotoxic and paralyzing activities of bothropstoxin-l by suramin (2003) Toxicon, 42, pp. 373-379Girish, K.S., Kemparaju, K., Inhibition of Naja naja venom hyaluronidase by plant-derived bioactive components and polysaccharides (2005) Biochemistry (Moscow), 70, pp. 948-952Gutierrez, J.M., Romero, M., Díaz, C., Borkow, G., Ovadia, M., Isolation and characterization of a metalloproteinase with weak hemorrhagic activity from the venom of the snake Bothrops asper (terciopelo) (1995) Toxicon, 33, pp. 19-29Harbone, J.B., (1998) Phytochemical Methods: A Guide to Modern Techniques of Plants Analysis, , 3rd edn, Chapman & Hall: LondonHavsteen, B., Flavonoids, a class of natural products of high pharmacological potency (1983) Biochem Pharmacol, 32, pp. 1141-1148Heluany, N.F., Homsi-Brandeburgo, M., Giglio, J.R., Prado-Franceschi, J., Rodrigues-Simioni, L., Effects induced by bothrop-stoxin, a component from Bothrops jararacussu snake venom, on mouse and chick muscle preparations (1992) Toxicon, 30, pp. 1203-1210Iwanaga, S., Suzuki, T., Enzymes in snake venoms (1979) Snake Venoms. Handbook of Experimental Pharmacology, pp. 61-158. , Lee CY ed, Springer: New YorkJin, G.Z., Yamagata, Y., Tomita, K., Structure of rutin pentamethanol (1990) Chem Pharm Bull, 38, pp. 297-300Khan, A.A., Ashfaq, A., Ali, M.N., (1979) Pharmacognostic Studies of Selected Indigenous Plants of Pakistan, , Pakistan Forest Institute. Spinezer Printers: PeshawarMahmood A, Ahmad M, Jabeen A, Zafar M, Nadeem S. 2005. Pharmacognostic studies of some indigenous medicinal plants of Pakistan. Available in http://www.siu.edu/∼ebl/leaflets/abid.htm. Access in 06/03/2005Maistro, E.L., Carvalho, J.C., Mantovani, M.S., Evaluation of the genotoxic potential of the Casearia sylvestris extract on HTC and V79 cells by the comet assay (2004) Toxicol In Vitro, 18, pp. 337-342Markland, F.S., Snake venoms and the hemostatic system (1998) Toxicon, 36, pp. 1749-1800Matrisian, L.M., The matrix-degrading metalloproteinases (1992) BioEssays, 14, pp. 455-463Milani Junior, R., Jorge, M.T., de Campos, F.P., Snake bites by the jararacuçu (Bothrops jararacussu): Clinicopatho-logical studies of 29 proven cases in Sao Paulo State, Brazil (1997) Q J Med, 90, pp. 323-334(2001) Manual de Diagnóstico e Tratamento de Acidentes por Animais Pegonhentos, , Ministério da Saúde do Brasil, 2nd edn. Fundação Nacional da Saúde: BrasíliaMors, W.B., Nascimento, M.C., Pereira, B.M., Pereira, N.A., Plant natural products active against snake bite - the molecular approach (2000) Phytochemistry, 55, pp. 627-642Oshima-Franco, Y., Alves, C.M.V., Andréo Filho, N., Neutralization of the neuromuscular activity of bothropstoxin-l, a myotoxin from Bothrops jararacussu snake venom, by a hydroalcoholic extract of Casearia sylvestris Sw. (guaçatonga) (2005) J Venom Anim Toxins Trop Dis, 11, pp. 465-478Oshima-Franco, Y., Hyslop, S., Cintra, A.C.O., Giglio, J.R., Cruz-Höfling, M.A., Rodrigues-Simioni, L., Neutralizing capacity of commercial bothropic antivenom against Bothrops jararacussu venom and bothropstoxin-l (2000) Muscle Nerve, 23, pp. 1832-1839Oshima-Franco, Y., Leite, G.B., Dal Belo, C.A., The presynaptic activity of bothropstoxin-l, a myotoxin from Bothrops jararacussu snake venom (2004) Basic Clin Pharmacol Toxicol, 95, pp. 175-182Oshima-Franco, Y., Leite, G.B., Silva, G.H., Neutralization of the pharmacological effects of bothropstoxin-l from Bothrops jararacussu (jararacuçu) venom by crotoxin antiserum and heparin (2001) Toxicon, 39, pp. 1477-1485Oshima-Franco, Y., Leite, G.B., Valério, A.A., Rabbit antivenom efficacy against myotoxic and neurotoxic activities of Bothrops jararacussu venom and bothropstoxin-l (2002) J Venom Anim Toxins, 8, pp. 226-243Pessini, A.C., Takao, T.T., Cavalheiro, E.C., A hyaluronidase from Tityus serrulatus scorpion venom: Isolation, characterization and inhibition by flavonoids (2001) Toxicon, 39, pp. 1495-1504Randazzo-Moura, P., Leite, G.B., Silva, G.H., Study of myotoxicity of bothropstoxin-l (BthTX-l) using manganese (Mn 2+) in mouse phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations (2006) Braz J Morphol Sci, 23, pp. 171-184Rates, S.M.K., Plants as source of drugs (2001) Toxicon, 39, pp. 603-613Rodrigues-Simioni, L., Borgese, N., Ceccarelli, B., The effects of Bothrops jararacussu venom and its components on frog nerve-muscle preparation (1983) Neuroscience, 10, pp. 475-489Rodrigues-Simioni, L., Prado-Franceschi, J., Cintra, A.C.O., Giglio, J.R., Jiang, M.S., Fletcher, J.E., No role for enzymatic activity or dantrolene-sensitive Ca 2+ stores in the muscular effects of bothropstoxin, a Lys49 phospholipase A 2 myotoxin (1995) Toxicon, 33, pp. 1479-1489Rosenfeld, G., Symptomatology, pathology and treatment of snakebites in South America (1971) Venomous Animals and their Venoms, pp. 345-384. , Bucherl W, Buckley E eds, Academic Press: New YorkRucavado, A., Escalante, T., Gutiérrez, J.M., Effect of the metalloproteinase inhibitor batimastat in the systemic toxicity induced by Bothrops asper snake venom: Understanding the role of metalloproteinases in envenomation (2004) Toxicon, 43, pp. 417-424Selistre, H.S., Queiroz, L.S., Cunha, O.A., De Souza, G.E., Giglio, J.R., Isolation and characterization of hemorrhagic, myonecrotic and edema-inducing toxins from Bothrops insularis (jararaca ilhoa) snake venom (1990) Toxicon, 28, pp. 261-273Simōes CMO, Schenkel EP, Gosmann G, Mello JCP, Mentz LA, Petrovick PR. 2004. Farmacognosia: da Planta ao Medicamento, 5th edn. UFRGS/UFSC: Porto Alegre/FlorianópolisSoares, A.M., Oshima-Franco, Y., Vieira, C.A., Mn 2+ ions reduce the enzymatic and pharmacological activities of bothropstoxin-l, a myotoxic Lys49 phospholipase A 2 homologue from Bothrops jararacussu snake venom (2002) Int J Biochem Cell Biol, 34, pp. 668-677Yingprasertchai, S., Bunyasrisawt, S., Ratanabanangkoon, K., Hyaluronidase inhibitors (sodium cromoglycate and sodium auro-thiomalate) reduce the local tissue damage and prolong the survival time of mice injected with Naja kaouthia and Calloselasma rhodostoma venoms (2003) Toxicon, 42, pp. 635-64

Antibothropic action of Casearia sylvestris Sw. (flacourtiaceae) extracts

Casearia sylvestris Sw., popularly known in Brazil as `guacatonga`, has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects. Copyright (C) 2008 John Wiley & Sons, Ltd

Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A(2) class

Phospholipases A(2) (PLA(2)s) are enzymes responsible for membrane disruption through Ca2+-dependent hydrolysis of phospholipids. Lys49-PLA(2)s are well-characterized homologue PLA(2)s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA(2)s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49-PLA(2) (BthTX-II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49-PLA(2) conserves all the residues responsible for Ca2+ coordination and of the catalytic network, features thought to be fundamental for PLA(2) enzymatic activity. Previous crystallographic studies of apo BthTX-II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX-II is not catalytic based on an in vitro cell viability assay and time-lapse experiments on C2C12 myotube cell cultures, X-ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX-II is devoid of catalytic activity, as already observed for Lys49-PLA(2)s. Crystallographic studies of the complex BthTX-II/Ca2+ show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca2+ are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX-II is more phylogenetically related to Lys49-PLA(2)s than to other Asp49-PLA(2)s, thus allowing Crotalinae subfamily PLA(2)s to be classified into two main branches: a catalytic and a myotoxic one. Proteins 2011; 79: 61-78. (C) 2010 Wiley-Liss, Inc.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq