Cytotoxic effects of crotoxin from Crotalus durissus terrificus snake in canine mammary tumor cell lines

Background: Mammary gland tumors are the most prevalent neoplasm in intact female dogs, and they are good natural models to study comparative oncology. Most canine mammary malignancies, as in women, are commonly refractory to conventional therapies and demand continuous new therapeutic approaches. Crotalus durissus terrificus, also called rattlesnake, has more than 60 different proteins in its venom with multiple pharmaceutical uses, such as antitumor, antiviral, and antimicrobial action. Crotoxin, a potent β-neurotoxin formed by the junction of two subunits, a basic subunit (CB-PLA2) and an acidic subunit (crotapotin), has already been reported to have anticancer properties in different types of cancers. Methods: In this work, we describe the cytotoxic potential of crotoxin and its subunits compared to doxorubicin (drug of choice) in two canine mammary carcinoma cell lines. Results: Crotoxin, CB-PLA2, crotalic venom, and doxorubicin decreased cell viability and the ability to migrate in a dose-dependent manner, and crotapotin did not present an antitumoral effect. For all compounds, the predominant cell death mechanism was apoptosis. In addition, crotoxin did not show toxicity in normal canine mammary gland cells. Conclusion: Therefore, this work showed that crotoxin and CB-PLA2 had cytotoxic activity, migration inhibition, and pro-apoptotic potential in canine mammary gland carcinoma cell lines, making their possible use in cancer research.

Acidentes crotálicos no Brasil: atualidades e perspectivas / Crotalic accidents in Brazil: actualities and perspectives

Introdução: Os acidentes ofídicos são eventos negligenciados em países tropicais e em desenvolvimento, incluindo o Brasil. Serpentes do gênero Crotalus são aquelas que produzem os quadros de maior letalidade no país. Objetivos: Abordar os principais aspectos do acidente por Crotalus, com ênfase na biologia das serpentes, na condução clínica dos eventos mórbidos e nas propriedades terapêuticas da peçonha destes animais. Métodos: Revisão da literatura com estratégia de busca definida, a partir da utilização das bases PubMed, LILACS e SciELO. Resultados: A inoculação da peçonha crotálica produz sinais locais discretos, mas repercussões sistêmicas podem ocorrer, especialmente alterações neurológicas e insuficiência renal aguda. A avaliação laboratorial é importante para auxiliar na distinção de outros acidentes ofídicos e para estimar a gravidade do quadro. O soro anticrotálico precisa ser administrado o mais brevemente possível, a partir da estimativa da quantidade de peçonha inoculada. A maior parte dos agravos ocorre no período chuvoso, acometendo principalmente homens jovens que trabalham na zona rural. A adoção de medidas de proteção e a educação em saúde são estratégias pertinentes para a prevenção e a redução do número de casos. As peçonhas de Crotalus possuem ações antimicrobianas, antiagregantes plaquetárias e aplicabilidade em oftalmologia (estrabismo). Conclusão: O conhecimento dos diferentes aspectos dos acidentes crotálicos é essencial para a adequada abordagem diagnóstica e terapêutica de tais condições mórbidas. As propriedades farmacológicas de componentes da peçonha crotálica deverão ser melhor investigadas nos próximos anos, dadas as possibilidades de utilização para o tratamento de diferentes enfermidades humanas

Introduction: Ophidian accidents are neglected events in tropical and in developing countries, including Brazil. Serpents of the Crotalus genus (rattlesnakes) are those that produce the highest case-fatality in the country. Purpose: to address the main aspects of the accident caused by Crotalus, with emphasis on the snakes biology, the clinical approach to snake bites and the therapeutic properties of the venom of these animals. Methods: literature review with a defined search strategy, using the PubMed, LILACS and SciELO databases. Results: Inoculation of crotalic venom produces discrete local signs, but systemic repercussions can occur, especially neurological alterations and acute renal failure. Laboratory evaluation is important to help distinguish from other ophidian accidents and to estimate the severity of the condition. Anticrotalic serum must be administered as soon as possible, based on the estimated amount of inoculated venom. Most of the morbid events occur in the rainy season (higher temperature), mainly affecting young men who work in rural areas. The adoption of protective measures and health education, aimed at the population most commonly involved, are relevant strategies for preventing and reducing the number of cases. In addition, crotalic venoms have antimicrobial actions, antiplatelets functions and ophthalmological applicability (strabismus). Conclusion: Knowledge of the different aspects of crotalic injuries is essential for an adequate diagnostic and therapeutic approach to such morbid conditions. The pharmacological properties of crotalic venom components should be better investigated in the next few years, given the possibilities of their use for the treatment of different human diseases.

Role of crotoxin in coagulation: novel insights into anticoagulant mechanisms and impairment of inflammation-induced coagulation

Snake venom phospholipases A2 (svPLA2) are biologically active toxins, capable of triggering and modulating a wide range of biological functions. Among the svPLA2s, crotoxin (CTX) has been in the spotlight of bioprospecting research due to its role in modulating immune response and hemostasis. In the present study, novel anticoagulant mechanisms of CTX, and the modulation of inflammation-induced coagulation were investigated. Methods: CTX anticoagulant activity was evaluated using platelet poor plasma (PPP) and whole blood (WB), and also using isolated coagulation factors and complexes. The toxin modulation of procoagulant and pro-inflammatory effects was evaluated using the expression of tissue factor (TF) and cytokines in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMC) and in WB. Results: The results showed that CTX impaired clot formation in both PPP and WB, and was responsible for the inhibition of both intrinsic (TF/factor VIIa) and extrinsic (factor IXa/factor VIIIa) tenase complexes, but not for factor Xa and thrombin alone. In addition, the PLA2 mitigated the prothrombinase complex by modulating the coagulation phospholipid role in the complex. In regards to the inflammation-coagulation cross talk, the toxin was capable of reducing the production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and was followed by decreased levels of TF and procoagulant activity from LPS-treated PBMC either isolated or in WB. Conclusion: The results obtained in the present study recognize the toxin as a novel medicinal candidate to be applied in inflammatory diseases with coagulation disorders.(AU)

Immunotherapeutic potential of Crotoxin: anti-inflammatory and immunosuppressive properties

For the past 80 years, Crotoxin has become one of the most investigated isolated toxins from snake venoms, partially due to its major role as the main toxic component in the venom of the South American rattlesnake Crotalus durissus terrificus. However, in the past decades, progressive studies have led researchers to shift their focus on Crotoxin, opening novel perspectives and applications as a therapeutic approach. Although this toxin acts on a wide variety of biological events, the modulation of immune responses is considered as one of its most relevant behaviors. Therefore, the present review describes the scientific investigations on the capacity of Crotoxin to modulate anti-inflammatory and immunosuppressive responses, and its application as a medicinal immunopharmacological approach. In addition, this review will also discuss its mechanisms, involving cellular and molecular pathways, capable of improving pathological alterations related to immune-associated disorders.(AU)

Crotalus durissus terrificus crotapotin naturally displays preferred positions for amino acid substitutions

Background: Classically, Crotalus durissus terrificus (Cdt) venom can be described, according to chromatographic criteria, as a simple venom, composed of four major toxins, namely: gyroxin, crotamine, crotoxin and convulxin. Crotoxin is a non-covalent heterodimeric neurotoxin constituted of two subunits: an active phospholipase A2 and a chaperone protein, termed crotapotin. This molecule is composed of three peptide chains connected by seven disulfide bridges. Naturally occurring variants/isoforms of either crotoxin or crotapotin itself have already been reported. Methods: The crude Cdt venom was separated by using RP-HPLC and the toxins were identified by mass spectrometry (MS). Crotapotin was purified, reduced and alkylated in order to separate the peptide chains that were further analyzed by mass spectrometry and de novo peptide sequencing. Results: The RP-HPLC profile of the isolated crotapotin chains already indicated that the α chain would present isoforms, which was corroborated by the MS and tandem mass spectrometry analyses. Conclusion: It was possible to observe that the Cdt crotapotin displays a preferred amino acid substitution pattern present in the α chain, at positions 31 and 40. Moreover, substitutions could also be observed in ß and γ chains (one for each). The combinations of these four different peptides, with the already described chains, would produce ten different crotapotins, which is compatible to our previous observations for the Cdt venom.(AU)

Efecto citotóxico de fosfolipasas A2 del veneno de Crotalus durissus cumanensis de Colombia / Cytotoxic effect of A2 phospholipases of the venom of Crotalus durissus cumanensis from Colombia / Efeito citotóxico da fosfolipase A2 do veneno de Crotalus durissus cumanensis da Colômbia

Introducción. Los venenos de serpientes representan una fuente importante de proteínas y péptidos, los cuales exhiben diversas actividades biológicas, tales como antibacterianas, antiparasitarias, antivi-rales, antitumorales, antifúngicas y contra la agregación plaquetaria, entre otras.Las fosfolipasas A2 presentes en los venenos de serpientes son las proteínas más estudiadas en estos modelos. Se ha demostrado que las fosfolipasas A2, activas e inactivas, poseen actividad catalítica contra células tumorales. Objetivo. Aislar, purificar y caracterizar la fosfolipasa A2 del veneno de Crotalus durissus cumanensis para evaluar su actividad antitumoral in vitro. Materiales y métodos. El aislamiento, la purificación y la identificación de la crotoxina B se hizo mediante la cromatografía de exclusión molecular, la cromatografía líquida de alto rendimiento de fase inversa (Reversed Phase High-Performance Liquid Chromatography, RP-HPLC) y la espectrometría de masas. El efecto citotóxico sobre células tumorales (K562) y células normales (células mononucleares de sangre periférica) se determinó utilizando la técnica de MTT. Resultados. La separación y posterior identificación de la crotoxina B del veneno de C. d. cumanensis de Colombia, permitieron evidenciar que esta fosfolipasa A2 posee efecto citotóxico sobre las células mononucleares de sangre periférica con una dosis de 18,23 ± 0,57 µg/ml, mientras que, para las células K562, fue de 2,34 ± 0,199 µg/ml. Conclusiones. Los resultados sugieren la posibilidad de utilizar la crotoxina B aislada del veneno de C. d. cumanensis como un posible recurso terapéutico para su aplicación en humanos.

Introduction. Snake venoms are an important source of proteins and peptides, which display various biological activities such as antibacterial, antiparasitic, antiviral, antitumor, antifungal and against platelet aggregation, among others.Phospholipases A2 present in snake venoms are the most studied proteins in these models. Active and inactive A2 phospholipases have been shown to possess catalytic activity against tumor cells. Objective. To isolate, purify and characterize the phospholipase A2 of the venom of Crotalus durissus cumanensis to evaluate its in vitro antitumor activity. Materials and methods. Isolation, purification and identification of crotoxin B was done with Size Exclusion Chromatography, Reversed Phase High-Performance Liquid Chromatography, RP-HPLC, and Mass Spectrometry. The cytotoxic effect on tumor cells (K562) and normal cells (peripheral blood mononuclear cells) was determined using the MTT technique. Results. The separation and subsequent identification of crotoxin B, found in the venom of C. d. cumanensis from Colombia, showed that this phospholipase A2 has a cytotoxic effect on peripheral blood mononuclear cells at a dose of 18.23 ± 0.57 µg / ml, whereas for K562 cells, it was 2.34 ± 0.199 µg/ml Conclusions. The results suggest the use of crotoxin B, isolated from the venom of C. d. cumanensis, as a possible therapeutic resource for human application.

Introdução. Os venenos da serpentes constituem uma importante fonte de proteínas e péptidos, os quais exibem várias actividades biológicas, tais como agentes antibacterianos, antiparasitárias, antivi-rais, antitumorais, antifúngicas e contra a agregação de plaquetas, entre outros. As fosfolipases A2 presentes no veneno da serpentes são as proteínas mais estudadas nestes modelos. Tem sido demostrado que as fosfolipases A2, activas e inactivas, possuem actividade catalítica contra células tumorais. Objetivo. Isolar, purificar e caracterizar a fosfolipase A2 do veneno da Crotalus durissus cumanensis para avaliar a sua actividade anti-umoral in vitro. Materiais e métodos. O isolamento, a purificação e identificação da crotoxina B foi realizada por cromatografia de exclusão molecular, cromatografia líquida de alta eficiência de fase reversa (Reversed Phase High-Performance Liquid Chromatography, RP-HPLC) e espectrometria de massa. O efeito cito-tóxico sobre células tumorais (K562) e células normais (células mononucleares do sangue periférico) foi determinada usando a técnica de MTT. Resultados. A Separação e subsequente identificação da crotoxina B do veneno da C. d. cumanensis da Colômbia, permitiu constatar que esta fosfolipase A2 tem um efeito citotóxico em células mono-nucleares de sangue periférico, com uma dose de 18,23 ± 0,57 µg/ ml, enquanto que para as células K562, foi 2,34 ± 0,199 ug/ml. Conclusões. Os resultados sugerem a possibilidade de utilizar crotoxina B isolada a partir do veneno da C. d. cumanensis como recurso para o potencial uso terapêutico em humanos.

Crotoxin in humans: analysis of the effects on extraocular and facial muscles / Crotoxina em humanos: estudo da ação em músculos extraoculares e faciais

PURPOSE: Crotoxin is the main neurotoxin of South American rattlesnake Crotalus durissus terrificus. The neurotoxic action is characterized by a presynaptic blockade. The purpose of this research is to assess the ability of crotoxin to induce temporary paralysis of extraocular and facial muscles in humans. METHODS: Doses of crotoxin used ranged from 2 to 5 units (U), each unit corresponding to one LD50. We first applied 2U of crotoxin in one of the extraocular muscles of 3 amaurotic individuals to be submitted to ocular evisceration. In the second stage, we applied crotoxin in 12 extraocular muscles of 9 patients with strabismic amblyopia. In the last stage, crotoxin was used in the treatment of blepharospasm in another 3 patients. RESULTS: No patient showed any systemic side effect or change in vision or any eye structure problem after the procedure. The only local side effects observed were slight conjunctival hyperemia, which recovered spontaneously. In 2 patients there was no change in ocular deviation after 2U crotoxin application. Limitation of the muscle action was observed in 8 of the 12 applications. The change in ocular deviation after application of 2U of crotoxin (9 injections) was in average 15.7 prism diopters (PD). When the dose was 4U (2 applications) the change was in average 37.5 PD and a single application of 5U produced a change of 16 PD in ocular deviation. This effect lasted from 1 to 3 months. Two of the 3 patients with blepharospasm had the hemifacial spasm improved with crotoxin, which returned after 2 months. CONCLUSIONS: This study provides data suggesting that crotoxin may be a useful new therapeutic option for the treatment of strabismus and blepharospasm. We expect that with further studies crotoxin could be an option for many other medical areas.

OBJETIVO: A crotoxina é a principal neurotoxina da cascavel sul-americana Crotalus durissus terrificus e sua ação neurotóxica caracteriza-se por um bloqueio pré-sináptico. O objetivo da pesquisa é avaliar a capacidade da crotoxina em induzir paralisia transitória de músculos extraoculares e faciais em seres humanos. MÉTODOS: As doses utilizadas de crotoxina foram de 2 a 5 unidades (U), sendo que cada unidade correspondia a uma DL-50. Na primeira etapa, aplicou-se 2U de crotoxina em músculos extraoculares de 3 indivíduos amauróticos, candidatos à evisceração. Na segunda etapa, realizaram-se 12 aplicações de crotoxina em músculos extraoculares de 9 indivíduos estrábicos e amblíopes. Na terceira e última etapa, utilizou-se a crotoxina para o tratamento do blefaroespasmo essencial em 3 indivíduos. RESULTADOS: Nenhum paciente demonstrou qualquer efeito sistêmico ou alteração da visão ou de qualquer estrutura ocular. O único efeito local adverso foi hiperemia conjuntival, que melhorou espontaneamente. Em 2 pacientes não houve alteração do desvio ocular após a aplicação de 2U de crotoxina. Observou-se em 8 das 12 aplicações, limitação do movimento ocular no campo de ação do músculo aplicado. A diminuição do desvio ocular com 2U crotoxina (9 aplicações) foi em média de 15,7 dioptrias prismáticas (DP); na dosagem de 4U (2 aplicações) foi em média de 37,5 DP e na única aplicação de 5U, obteve-se redução de 16 DP no desvio ocular. A alteração do alinhamento ocular manteve-se por 1 a 3 meses. Dois dos 3 pacientes portadores de blefaroespasmo apresentaram melhora dos espasmos hemifacias, os quais voltaram após 2 meses. CONCLUSÕES: Através dos resultados observados neste estudo, acreditamos que a crotoxina possa ser útil no tratamento do estrabismo e do blefaroespasmo. Novos estudos precisam ser realizados para confirmar a eficácia e a segurança da crotoxina como opção terapêutica para diversas áreas da medicina que atualmente utilizam a toxina botulínica.

Study of crotoxin on the induction of paralysis in extraocular muscle in animal model / Estudo da crotoxina na indução de paralisia da musculatura extraocular em modelo animal

PURPOSE: Crotoxin is the major toxin of the venom of the South American rattlesnake Crotalus durissus terrificus, capable of causing a blockade of the neurotransmitters at the neuromuscular junction. The objective of this study was to appraise the action and effectiveness of the crotoxin induced paralysis of the extraocular muscle and to compare its effects with the botulinum toxin type A (BT-A). METHODS: The crotoxin, with LD50 of 1.5 µg, was injected into the superior rectus muscle in ten New Zealand rabbits. The concentration variance was 0.015 up to 150 µg. Two rabbits received 2 units of botulinum toxin type A for comparative analysis. The evaluation of the paralysis was performed using serial electromyography. After the functional recovery of the muscles, which occurred after two months, six rabbits were sacrificed for anatomopathology study. RESULTS: The animals did not show any evidence of systemic toxicity. Transitory ptosis was observed in almost every animal and remained up to fourteen days. These toxins caused immediate blockade of the electrical potentials. The recovery was gradual in the average of one month with regeneration signs evident on the electromyography. The paralysis effect of the crotoxin on the muscle was proportional to its concentration. The changes with 1.5 µg crotoxin were similar to those produced by the botulinum toxin type A. The histopathology findings were localized to the site of the injection. No signs of muscle fiber's necrosis were seen in any sample. The alterations induced by crotoxin were also proportional to the concentration and similar to botulinum toxin type A in concentration of 1.5 µg. CONCLUSION: Crotoxin was able to induce transitory paralysis of the superior rectus muscle. This effect was characterized by reduction of action potentials and non-specific signs of fibrillation. Crotoxin, in concentration of 1.5 µg was able to induce similar effects as botulinum toxin type A.

OBJETIVO: A crotoxina é a principal toxina do veneno da cobra cascavel sul-americana Crotalus durissus terrificus e causa bloqueio da neurotransmissão na junção neuromuscular. O objetivo deste estudo foi avaliar a ação e aplicabilidade da crotoxina na indução de paralisia da musculatura extrínseca ocular, e comparar seus efeitos com os da toxina botulínica do tipo A (TB-A). MÉTODOS: A crotoxina, com DL50 de 1,5 µg, foi aplicada no músculo reto superior direito de dez coelhos da raça neozelandesa, em concentrações que variaram de 0,015 µg a 150 µg. Em dois coelhos, utilizou-se 2 unidades de toxina botulínica do tipo A para análise comparativa. A avaliação da paralisia foi realizada através de eletromiografia seriada. Após a recuperação, que ocorreu em dois meses, seis coelhos foram sacrificados para estudo anátomopatológico. RESULTADOS: Os animais não apresentaram sinais de intoxicação sistêmica. Ptose palpebral transitória foi observada em quase todos os animais e permaneceu por até 14 dias. As toxinas causaram um bloqueio imediato da captação dos potenciais elétricos. A recuperação foi gradativa no período aproximado de um mês, observando-se sinais evidentes de regeneração no registro eletromiográfico. Os efeitos da crotoxina na paralização do músculo injetado foram proporcionais à concentração. A crotoxina, na concentração de 1,5 µg, induziu alterações semelhantes às da toxina botulínica do tipo A. Os achados anátomo-patológicos foram localizados somente na região em que se aplicou as toxinas, não havendo necrose de fibras musculares em nenhuma amostra analisada. As alterações causadas pela crotoxina também foram proporcionais à concentração utilizada e similares a toxina botulínica do tipo A na concentração de 1,5 µg. CONCLUSÃO: A crotoxina foi capaz de induzir paralisia transitória do músculo reto superior. Este efeito foi caracterizado pela redução na amplitude dos potenciais de ação e sinais inespecíficos de fibrilação. Observou-se que a ação da crotoxina, em concentração de 1,5 µg, proporcionou efeito semelhante ao da toxina botulínica do tipo A.

Efeito do veneno de Crotalus durissus terrificus no edema crônico induzido na pata de camundongos pela injeção de bacilo Calmette-Guérin (BCG) / Effect of the Crotalus durissus terrificus in edema chronic induced in mice paw by the injection of bacilo Calmette-Guérin (BCG)

Diferentemente do observado em outros envenenamentos por serpentes da família Viperidae, nos causados pela Cdt não se observam sinais inflamatórios significativos no local da picada. Estudos prévios mostram que o VCdt inibe a resposta inflamatória aguda e algumas atividades biológicas de macrófagos, principal célula do processo inflamatório crônico. Nesse trabalho verificou-se o efeito do VCdt sobre o edema de pata crônico induzido pela injeção intraplantar de BCG em camundongos. Na concentração utilizada, o BCG evoca um edema crônico que é significativamente inibido pelo pré-tratamento dos animais com o VCdt (s.c.). Essa inibição persistiu por todo o período estudado (15 dias). O grupo que recebeu o veneno 1 h após a injeção de BCG também apresentou perfil de edema inibido em relação ao grupo controle, de forma semelhante ao observado no grupo pré-tratado com o veneno. Os grupos tratados com o VCdt 6 ou 11 dias após a injeção do BCG apresentavam edema de magnitude semelhante até o dia da injeção do veneno, sendo observada uma inibição significativa desse edema no dias subseqüentes à injeção do veneno. Uma vez constatado esse efeito inibitório do VCdt sobre o edema inflamatório crônico induzido pelo BCG, estudou-se qual a fração do veneno seria responsável por esse efeito. Os resultados indicam que a crotoxina, e não outros componentes deste veneno seja a responsável por essa inibição. Estudando-se possíveis mecanismos envolvidos nessa inibição, a reversão da inibição induzida pelo VCdt observada em grupos pré-tratados com dexametazona e zileuton, sugerem fortemente a participação de mediadores originados na via das lipoxigenases, no efeito inibitório do VCdt sobre esse modelo de inflamação crônica. Em conclusão, os resultados indicam que a crotoxina do VCdt possua uma significativa ação inibitória sobre o edema crônico induzido pelo BCG, possivelmente pela geração de mediadores antiinflamatórios da via das lipoxigenases.

Avaliação do efeito da crotoxina em animais portadores de encefalomielite autoimune experimental, um modelo de animais de esclerose múltipla / Evaluation of crotoxin effect in the experimental autoimune encephalomyelitis, an animal model of multiple sclerosis

A esclerose múltipla é uma doença inflamatória crônica, de origem autoimune, que acarreta diversas alterações motoras, cognitivas e sensitivas. Dentre as alterações sensitivas, a dor é um dos graves problemas que afetam pessoas portadoras desta doença, interferindo com diversos aspectos da vida do paciente. É importante ressaltar que a esclerose múltipla não tem cura, sendo que a terapêutica se concentra nas ações que retardam a progressão da doença e promovem o alívio dos sintomas, melhorando a qualidade da vida do paciente...

Multiple sclerosis is a Central Nervous System Inflamatory demyelinating disease that has as primary symptomps losses of sensory, cognitive and motor functions. Among the sensory alternations, pain is one of the major concern, afecting various aspects of the patients lives...

Renal and vascular effects of Crotalus durissus cumanensis venom and its crotoxin fraction

In this study, we evaluated the actions of Crotalus durissus cumanensis venom (CDCmV), and its crotoxin (Crtx) fraction, on renal and vascular functions in Wistar rats. In isolated perfused kidneys, CDCmV (10 µg/mL) significantly increased the perfusion pressure (PP) from 110.7 ± 2.4 to 125.3 ± 2.8 mmHg after 30 minutes. This effect was accompanied by an increased renal vascular resistance (RVR) from 5.4 ± 0.1 to 6.2 ± 0.2 mmHg/mL.g-1.min-1. We observed decreases in urinary flow (UF) from 0.13 ± 0.01 to 0.05 ± 001 mL.g-1.min-1 and glomerular filtration rate (GFR) from 0.66 ± 0.06 to 0.18 ± 0.02 mL.g-1.min-1. Crtx did not change PP or RVR, but diminished GFR (from 0.65 ± 0.05 to 0.26 ± 003 mL.g-1.min-1) and UF (from 0.11 ± 0.008 to 0.09 ± 0.008 mL.g-1.min-1). Both CDCmV and Crtx reduced the percentage of tubular transport of sodium, chloride and potassium. The cytotoxicity of these substances against MDCK cells was tested by the MTT method: only CDCmV caused a decrease in the cell viability with an IC50 of 5.4 µg/mL. In endothelium-intact isolated aortic rings, CDCmV (0.1 to 30 µg/mL) increased the sustained phenylephrine-induced contraction to a value of 130.0 ± 6.6 percent of its corresponding control, but showed a relaxant effect in endothelium-denuded preparations. Similar results were observed in aortic rings contracted with potassium (40 mM). Crtx was ineffective in aortic ring assays. Thus, it is reasonable to suggest that the renal effects induced by the CDCmV may be due to its influence on the endothelium's ability to release factors that can alter the contractile behavior of vascular smooth muscle. In conclusion, CDCmV is toxic to kidney cells. It changes parameters of the renal function including the glomerular filtration rate, renal vascular resistance and tubular transport. The actions induced by CDCmV also involve endothelium-dependent vasoactive properties. Their effects may be only partially attributed to Crtx.

Modificaciones en los marcadores enzimáticos hepáticos promovidos por veneno de ejemplares jóvenes de crotalus durissus cumanesis / Modifications in the enzymatic liver markers promoted by the venom of joung cotralus durissus cumanesis

Se determinaron los cambios séricos de ALT, AST, FA, promovidos por veneno de ejemplares jóvenes Crotalus durissus cumanensis. Ratones Balb/C inoculados con 0,75 mg proteínas de veneno/kg vía intraperitoneal y controles con ssf. Se obtuvieron muestras a diferentes intervalos post-inyección (1, 3, 6, 12 y 24 h) determinándose los niveles séricos de: ALT, AST, y FA, posterior al sacrificio se tomaron muestras de hígado para la valoración histopatológica. Los valores de ATL, AST y FA se incrementaron significativamente, siendo más temprano el aumento de AST (1h) y más tardío el de la FA (24). El hallazgo histopatológico del tejido hepático revelo tumefacción celular severa, células de Kuffer activadas y necrosis coagulativa focal. El incremento de los marcadores enzimáticos ALT, AST, y FA, paralelo a los daños histológicos encontrados en este estudio, sugieren daño hepato-biliar, adicionalmente, los niveles séricos de AST podrían indicar daño al tejido muscular esquelético y/o cardíaco

To determinate the effect of the venom of young Crotalus durissus cumanensis on levels of ALT, AST and AP, Balb/C mice were inoculated intraperitoneal with 0,75 mg/Kg of purified venom. Serum samples were obtained at different post-injection intervals (1, 3, 6, 12, 24h) and serum levels of ALT, AST and AP were determined. A significative difference (P ‹0,01) was observed in serum levels of ALT at 6h and at 24h post-venom injection, with a maximum of 587,64 U/L at 24 h, when compared with controls. In addition, AST shows a significative elevation between 1h and 12h post venom injection, with a maximum level of 2327 U/L at 3 h, while AP only showed significant differences at 24 h post-venom injection. These results suggest that the venom of young Crotalus durissus cumanensis induced liver-biliar injury, with a possible effect on skeletal muscle and/ or cardiac tissue

Crotalus durissus terrificus venom as a source of antitumoral agents

The basic knowledge on neoplasms is increasing quickly; however, few advances have been achieved in clinical therapy against tumors. For this reason, the development of alternative drugs is relevant in the attempt to improve prognosis and to increase patients' survival. Snake venoms are natural sources of bioactive substances with therapeutic potential. The objective of this work was to identify and characterize the antitumoral effect of Crotalus durissus terrificus venom (CV) and its polypeptide, crotoxin, on benign and malignant tumors, respectively, pituitary adenoma and glioblastoma. The results demonstrated that CV possess a powerful antitumoral effect on benign (pituitary adenoma) and malignant (glioblastoma multiforme) tumors with IC50 values of 0.96 ± 0.11 µg/mL and 2.15 ± 0.2 µg/mL, respectively. This antitumoral effect is cell-cycle-specific and dependent on extracellular calcium, an important factor for crotoxin phospholipase A2 activity. The CV antitumoral effect can be ascribed, at least partially, to the polypeptide crotoxin that also induced brain tumor cell death. In spite of the known CV nephrotoxicity and neurotoxicity, acute treatment with its antitumoral dose established in vitro was not found to be toxic to the analyzed animals. These results indicate the biotechnological potential of CV as a source of pharmaceutical templates for cancer therapy.

Estudo comparativo da ação da toxina botulínica tipo A e da crotoxina sobre as células satélites da musculatura extrínseca ocular em modelo animal / A comparative study of the effects of type A botulinum toxin and crotoxin on satellite cells of extraocular muscles in rabbits

OBJETIVOS: Avaliar o efeito da toxina botulínica do tipo A e da crotoxina na ativação de células satélites das fibras musculares de retos superiores de coelhos. MÉTODOS: Os músculos retos superiores do olho direito de 29 coelhos machos albinos neozelandeses foram inoculados com toxina botulínica do tipo A ou com crotoxina, em diferentes doses. Os músculos retos superiores contralaterais de cada animal foram inoculados com solução salina em volume igual ao das toxinas. Os animais foram sacrificados 12, 18 ou 25 dias após as aplicações. Os olhos foram enucleados e cada músculo foi preparado para análise imunoistoquímica, com marcadores de células satélites. Foi realizada contagem dos núcleos corados pelos marcadores a cada cem miofibras. RESULTADOS: A aplicação de toxina botulínica e de crotoxina provocou um aumento no número de células satélites ativadas e em proliferação nos músculos retos superiores. Uma maior ativação foi observada após a aplicação de crotoxina, embora, estatisticamente, a diferença do efeito de ativação entre os grupos botoxina e crotoxina não tenha sido significativa. Nos grupos botoxina e crotoxina, não houve correlação estatisticamente significativa entre a dose e o volume aplicados e o aumento na ativação das células. O tempo de vida após a aplicação contribuiu para o aumento das células ativadas nos grupos. CONCLUSÃO: A observação de maior desorganização na estrutura muscular e de sinais de regeneração mais evidentes no grupo crotoxina parece estar correlacionada ao aumento de células satélites ativadas.

PURPOSE: To evaluate the effect of botulinum toxin A and crotoxin on satellite cell activation in the muscle fibers of superior rectus muscles of rabbits. METHODS: The superior rectus muscles in the right eyes of 29 male, albino, New Zealand rabbits were inoculated with different doses of botulinum toxin A or crotoxin. The contra-lateral superior rectus muscles in each rabbit were inoculated with the same volume of saline solution only. The animals were sacrificed either 12, 18 or 25 days after the inoculation. The eyes were enucleated and subsequently, each muscle was prepared for immunohistochemical analysis, using satellite cell markers. The positive nuclei, revealed by the markers in each 100 myofibers, were counted. RESULTS: The application of the botulinum toxin A and crotoxina triggered a more significant increase satellite cell activation and proliferation in right superior rectus muscles in rabbits when compared with a saline solution inoculation in the contralateral muscles. Greater cell activation was observed after crotoxin application, although, statistically, the difference in the effects of this activation between the botoxin and crotoxin groups was not significant. There was no statistically significant correlation between the dose and the volume applied and resulting cell activation in the botoxin and crotoxin groups. Post-application survival time contributed to the increase in activated satellite cells in all groups. CONCLUSION: The observed increase in disorganization in the muscle structure, together with more obvious signs of regeneration in the crotoxina group, suggests a correlation with the increase in satellite cell activation.

Rabbit IgG antibodies against Phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom

Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 µg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 µg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms

El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 µg de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 µg.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes

Estudo comparativo: bioquimico e imunofarmacologico do veneno total, da crotoxina e suas isoformas de crotapotina e PLA2 de crotalus durissus terrificus. C. d. ruruima C.d. cascavella e C. d. collilineatus / Comparative study: biochemical and Immunopharmacology of the whole venom, crotoxin and of its isoforms crotapotina and PLA2 from Crotalus durissus terrificus. C. d. ruruima C.D. cascavella and C. d. collilineatus

Este trabalho está dividido em três momentos, o primeiro trata-se de um estudo comparativo do efeito dos venenos brutos de Crotalus durissus terrificus; C. d. ruruima; C. d. cascavella e C. d. collilineatus, bem como da crotoxina e da crotamina sobre a junção neuromuscular. O veneno C. d. ruruima foi purificado pela primeira vez através de HPLC em uma coluna Protein Pack SW 300, onde foi obtida a crotoxina e a crotamina (os demais venenos já foram purificados e os dados publicados). O estudo neurotóxico foi realizado em duas preparações nervo-frênico diafragma de camundongo e biventer cervicis de pintainho. Dos experimentos realizados em mamíferos e aves obteve-se o efeito neurotóxico esperado nas concentrações de 10 mg/ml. Os venenos brutos e as crotoxinas das serpentes C. d. terrificus e C. d. ruruima em preparações de mamífero produziram aumento da amplitude seguido de bloqueio total da resposta contrátil, diferentemente dos venenos e das crotoxinas das serpentes C. d. cascaavella e C. d. collilineatus, que causaram apenas bloqueio total neuromuscular. Em preparações de aves os quatro venenos e crotoxinas estudados induziram bloqueio total, porém sem causar facilitação. As crotaminas-positivas, estudadas em nervo...

This work is divided at three moments, the first one if terrificus deals with a comparative study of the effect of the crude venoms of Crotalus durissus; C. d. ruruima; C. d. cascavella and C. d. collilineatus, as well as of the crotoxin and the crotamine on the junction neuromuscular. Venoms C. d. ruruima was purified for the first time through HPLC in a column Protein Pack sw 300, where it was gotten the crotoxin and the crotamine (the too much venoms already had been purified and the published data). The neurotoxic study nerve-frênico was carried through in two preparations diaphragm of mouse and to chick biventer cervicis. The experiments carried through in mammals and birds the waited neurotoxic effect in the was gotten concentrations of 10 mg/ml. The crude venoms and the crotoxin of rastnaks C. d. terrificus and C. d. ruruima in preparations of mammal had produced increase of the followed amplitude of complete blockade of the contractil reply, differently of the venoms and the crotoxin of rastnakes C. d. cascavella and C. d. collilineatus, that they had caused only complete blockade to neuromuscular. In preparations of birds the four studied venoms and crotoxin had induced complete blockade, however without causing facilitation. The crotamine-positive, studied in nerve-frenico diaphragm of mouse of snakes C. d. terrificus and C. d. ruruima, in the concentration of 10 mg/ml, had caused facilitatori effect returning to the 120 after amplitude have controlled min of incubation. When crotoxin ...

Inhibition of L-glutamate and GABA synaptosome uptake by crotoxin, the major neurotoxin from Crotalus durissus terrificus venom

This paper describes a brief study on the crotoxin mechanism of action, regarding the transport of GABA and L-glutamate in rats cortico-cerebral synaptosomes and in heterologous systems, such as COS-7 cells expressing gabaergic transporters, and C6 glioma cells and Xenopus oocytes expressing glutamatergic transporters. Crotoxin concentrations over 1 µM caused an inhibitory effect of ³H-L-glutamate and ³H-GABA, and reversibly inhibited L-glutamate uptake by C6 glioma cells. When COS-7 cells were assayed, no inhibition of the ³H-GABA transport could be evidenced. Crotoxin kept its inhibitory effect on neurotransmitters uptake even when Ca2+ ions were removed from the medium, therefore, independently of its PLA2 activity. In addition, high concentrations (2 mM) of BPB did not avoid the action of crotoxin on the neurotransmitters uptake. Crotoxin also inhibited ³H-L-glutamate, independently on Na+ channel blockade by TTX. In addition, an evaluation of the lactic dehydrogenase activity indicated that uptake inhibition does not involve a hydrolytic action of crotoxin upon the membrane. We may also suggest that crotoxin acts, at least partially, altering the electrogenic equilibrium, as evidenced by confocal microscopy, when a fluorescent probe was used to verify cell permeability on C6 glioma cells in presence of crotoxin.

Estrategias de la creencia en situaciones críticas: el cáncer y la crotoxina en Buenos Aires a mediados de los años ochenta / Strategies of the belief in critical situations: cancer and crotoxin in Buenos Aires in the 80's

Explora los avatares que acompañaron en Buenos Aires a mediados de la década de 1980 la movilización de enfermos de cáncer interesados en acceder a un tratamiento descalificado por el establishment médico y científico.

Actions of Crotalus durissus terrificus venom and crotoxin on the isolated rat kidney

Many studies have reported the occurrence of lethal acute renal failure after snakebites. The aim of the present investigation was to determine alterations in renal function produced by Crotalus durissus terrificus venom and crotoxin as well as the histological alterations induced by these venoms. Isolated kidneys from Wistar rats weighing 240 to 280 g were perfused with Krebs-Henseleit solution containing 6 g percent of previously dialyzed bovine serum albumin. The effects of Crotalus durissus terrificus venom and crotoxin were studied on glomerular filtration rate (GFR), urinary flow (UF), perfusion pressure (PP) and percentage sodium tubular transport ( percentTNa+). The infusion of Crotalus durissus terrificus venom (10 æg/ml) and crotoxin (10 æg/ml) increased GFR (control80 = 0.78 + or - 0.07, venom80 = 1.1 + or - 0.07, crotoxin80 = 2.0 + or - 0.05 ml g-1 min-1, P<0.05) and UF (control80 = 0.20 + or - 0.02, venom80 = 0.32 + or - 0.03, crotoxin80 = 0.70 + or - 0.05 ml g-1 min-1, P<0.05), and decreased percentTNa+ (control100 = 75.0 + or - 2.3, venom100 = 62.9 + or - 1.0, crotoxin80 = 69.0 + or - 1.0 ml g-1 min-1, P<0.05). The infusion of crude venom tended to reduce PP, although the effect was not significant, whereas with crotoxin PP remained stable during the 100 min of perfusion. The kidneys perfused with crude venom and crotoxin showed abundant protein material in the urinary space and tubules. We conclude that Crotalus durissus terrificus venom and crotoxin, its major component, cause acute nephrotoxicity in the isolated rat kidney. The current experiments demonstrate a direct effect of venom and crotoxin on the perfused isolated kidney

Crotoxin, the major toxin from the rattlesnake Crotalus durissus terrificus, inhibits 3H-choline uptake in guinea pig ileum

We examined the effect of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus, on the uptake of 3H-choline in minces of smooth muscle myenteric plexus from guinea pig ileum. In the concentration range used (0.03-1 ÁM) and up to 10 min of treatment, crotoxin decreased 3H-choline uptake by 50-75 percent compared to control. This inhibition was time dependent and did not seem to be associated with the disruption of the neuronal membrane, because at least for the first 20 min of tissue exposure to the toxin (up to 1 ÁM) the levels of lactate dehydrogenase (LDH) released into the supernatant were similar to those of controls. Higher concentrations of crotoxin or more extensive incubation times with this toxin resulted in elevation of LDH activity detected in the assay supernatant. The inhibitory effect of crotoxin on 3H-choline uptake seems to be associated with its phospholipase activity since the equimolar substitution of Sr2+ for Ca2+ in the incubation medium or the modification of the toxin with p-bromophenacyl bromide substantially decreased this effect. Our results show that crotoxin inhibits 3H-choline uptake with high affinity (EC25 = 10 +/- 5 nM). We suggest that this inhibition could explain, at least in part, the blocking effect of crotoxin on neurotransmission