Intraspecific Differences in the Venom of Crotalus durissus cumanensis from Colombia.

Biochemical and biological differences in the venom of Crotalus durissus cumanensis from three ecoregions of Colombia were evaluated. Rattlesnakes were collected from the geographic areas of Magdalena Medio (MM), Caribe (CA) and Orinoquía (OR). All three regionally distributed venoms contain proteases, PLA2s and the basic subunit of crotoxin. However, only crotamine was detected in the CA venom. The highest lethality, coagulant, phospholipase A2 and hyaluronidase activities were found in the MM venom. Also, some differences, observed by western blot and immunoaffinity, were found in all three venoms when using commercial antivenoms. Furthermore, all three eco-regional venoms showed intraspecific variability, considering the differences in the abundance and intensity of their components, in addition to the activity and response to commercial antivenoms.

Immunorecognition and Neutralization of Crotalus durissus cumanensis Venom by a Commercial Antivenom Produced in Colombia.

In Colombia, on average 2.9% of the nearly 5600 snakebite events that occur annually involve the rattlesnake Crotalus durissus cumanensis. The envenomation by this snake is mainly characterized by neurotoxicity and the main toxin is crotoxin (~64.7% of the total venom). The Instituto Nacional de Salud (INS) produces a polyvalent antivenom aimed at the treatment of bothropic, crotalid, and lachesic envenomations; nonetheless, its immune reactivity profile and neutralizing capacity over biological activities of the C. d. cumanensis venom has been poorly evaluated. In this sense, the study aims: (1) to describe an in-depth exploration of its immunoreactivity through second-generation antivenomics and HPLC fraction-specific ELISA immunoprofiles; and (2) to evaluate the neutralization pattern of the rattlesnake venom in vitro and in vivo biological activities. The results obtained showed a variable recognition of crotoxin subunits, in addition to a molecular mass-dependent immunoreactivity pattern in which the disintegrins were not recognized, and snake venom metalloproteinases and L-amino acid oxidases were the most recognized. Additionally, a high neutralization of proteolytic and coagulant activities was observed, but not over the PLA2 activity. Further, the median effective dose against C. d. cumanensis venom lethality was 962 µL of antivenom per mg of venom. In conclusion, (1) the antivenom recognition over the crotoxin and the disintegrins of the C. d. cumanensis should be improved, thus aiming upcoming efforts for the exploration of new techniques and approaches in antivenom production in Colombia, and (2) the neutralization activity of the antivenom seems to follow the molecular mass-dependent recognition pattern, although other explanations should be explored.

Efectos bioquímicos e histopatológicos en ratas inducidos por el veneno de Crotalus durissus cumanensis del municipio Falcón, estado Falcón, Venezuela / Biochemical and Histopathological Effects in rats Induced by the Poison of Crotalus durissus cumanensis from the Falcón Municipality, Falcón State, Venezuela

Resumen El accidente ofídico representa un importante problema de salud pública en las zonas tropicales y subtropicales del mundo. La cascabel común (Crotalus durissus cumanensis) es la serpiente del género Crotalus más abundante en Venezuela. El objetivo fue determinar los cambios séricos en la alanino aminotransferasa (ALT), aspartato aminotransferasa (AST), lactato deshidrogenasa (LDH) y creatina fosfocinasa (CK) en ratas, inducidos por el veneno de Crotalus durissus cumanensis, así como el establecimiento de los efectos histopatológicos. Se prepararon cinco grupos experimentales con tres ratas cada uno, reservando un grupo control placebo (G1), el cual fue tratado por ruta IP con 100 µL de solución salina fisiológica estéril (SSFe). Al resto de los animales se les administraron 50 µg de veneno nativo por la misma vía. Estos fueron sacrificados a las 1 (G2), 3 (G3), 6 (G4) y 9 (G5) horas posinoculación, con tomas de muestras séricas para determinaciones de las referidas enzimas y de diferentes órganos para estudios histopatológicos (corazón, músculo esquelético, hígado y riñón). Se observó un incremento en la ALT (p < 0,005) a partir de la primera hora posinyección, con un pico a la novena hora de 147 ± 8 UI/L; para la AST (p < 0,005) a partir de la primera hora posinyección con concentración pico a la sexta hora (293 ± 8 UI/L). De la LDH (p < 0,005) se registró un pico máximo de 2700 ± 8 UI/L a la novena hora posinyección. La CK (p < 0,005) mostró un pico máximo de concentración a la sexta hora (1489,66 ± 8,5 UI/L). Los resultados histopatológicos en tejido cardíaco mostraron hiperemia, congestión y necrosis de Zenker; en músculo esquelético, hiperemia, infiltrado inflamatorio, necrosis de Zenker; en hígado, extravasación de glóbulos rojos, congestión de sinusoides, telangiectasia e infiltrado inflamatorio en vena centrolobulillar. El riñón mostró hemorragia en túbulos contorneados, infiltrado inflamatorio, colapso de los túbulos hasta la pérdida de la arquitectura del órgano. Los efectos observados fueron tiempo-dependientes. Los resultados pueden indicar un posible efecto miotóxico y hepatotóxico.

Abstract The ophidian accident represents a major public health problem in the tropical and subtropical areas of the world. The common rattlesnake (Crotalus durissus cumanensis) is the most abundant snake of the genus Crotalus in Venezuela. The objective was to determine the serum changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine phosphokinase (CK) in rats, induced by the poison of Crotalus durissus cumanensis, as well as the establishment of histopathological effects. Five experimental groups were prepared with 3 rats each, reserving a placebo control group (G1), which was treated by IP route with 100 µL of sterile Physiological Saline Solution (SSFe), the rest of the animals were administered 50 µg of venom native by the same route, which were sacrificed at 1 (G2), 3 (G3), 6 (G4) and 9 (G5) hours post-inoculation, with serum samples taken for determinations of said enzymes, as well as different orgns for histopathological studies (heart, skeletal muscle, liver and kidney). An increase in ALT (p <0.005) was observed from the first hour post injection, with a peak at the ninth hour of 147 ± 8 IU / L; for AST (p <0.005) from the first hour post-injection with peak concentration at the sixth hour (293 ± 8 IU / L); with respect to LDH (p <0.005), a maximum peak of 2700 ± 8 IU / L was recorded at the ninth hour post-injection. CK (p <0.005) registered a maximum concentration peak at the sixth hour (1489.66 ± 8.5 IU / L). Histopathological results in cardiac tissue showed Zenker hyperemia, congestion, and necrosis; in skeletal muscle hyperemia, inflammatory infiltrate, Zenker's necrosis; in liver extravasation of red blood cells, sinus congestion, telangiectasia and inflammatory infiltrate in the centrilobular vein; the kidney showed hemorrhage in contoured tubules, inflammatory infiltrate, collapse of the tubules until loss of the organ architecture. The observed effects were time-dependent. The results could indicate a possible myotoxic and hepatotoxic effect.

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.

Inhibitory effects of Swietenia macrophylla on myotoxic phospholipases A2

Activity-guided fractionation of an ethanol-soluble extract of the leaves of Swietenia macrophylla King, Meliaceae, led to several fractions. As a result, sample Sm13-16, 23 had the most promising activity against phospholipases A2 (PLA2), Asp49 and Lys49 types. This fraction inhibited PLA2 activity of the Asp49 PLA2, when aggregated substrate was used. On the other hand, this activity was weakly neutralized when monodispersed substrate was used. In addition, Sm13-16, 23 inhibited, in a dose dependent manner, the cytotoxicity, myotoxicity and edema induced by PLA2s, as well as the anticoagulant activity of Asp49 PLA2. Overall, this fraction exhibited a better inhibition of the toxic activities induced by the Lys49 PLA2 than those caused by the Asp49 PLA2. The spectral data of Sm13-16, 23 suggested the presence of aromatic compounds (UV λ max (nm) 655, 266, and 219; IR λ max KBr (cm-1): ~ 3600-3000 (OH), 2923.07 and 1438.90 (C-H), 1656.69 (C = O), 1618.63 and 1607.67 (C-O), 1285.47772.60). We suggest that phenolic compounds could interact and inhibit the toxins by several mechanisms. Further analysis of the compounds present in the active fraction could be a relevant contribution in the treatment of accidents caused by snake envenomation.

Estudio clínico-epidemiológico de alteraciones hemorrágicas y necróticas causadas por el veneno de la serpiente de cascabel (crotalus durissus cumanensis) en pacientes venezolanos / Clinical and epidemiological study of hemorrhagic and necrotic alterations caused by the rattlesnake (crotalus durissus cumanensis) venom in venezuelan patients

Se describieron los efectos hemorrágicos, necróticos y edematosos de 135 pacientes provenientes de los estados Miranda, Aragua, Vargas y Distrito Capital, Venezuela, ocasionados por la mordedura de la serpiente cascabel común venezolana (Crotalus durissus cumanensis), durante los años 1998-2008. Los trastornos hemorrágicos, que tradicionalmente eran casi imperceptibles en los Crotalus venezolanos, hemos encontrado que hay evidencias francas de manifestaciones clínicas como: afibrinogenemia, alargamiento del tiempo de coagulación manual (TCM), tiempo parcial de tromboplastina (TTP) y tiempo de protrombina (TP), lo cual indica la presencia de estas fracciones hemorrágicas en el veneno de cascabeles nacionales. Se apreciaron diferencias entre ambos sexos, siendo predominante en el sexo masculino (82%). Sin embargo ha habido un aumento de incidencia significativa en el sexo femenino (17%). Por grupo etario, se observó predominancia entre 11 a 30 años de edad, en ambos sexos. El sitio de mordedura mayormente afectado fue el miembro superior (58,5%), con un porcentaje no menos significativo de miembros inferiores (40,7%). Estos hallazgos, permiten sugerir que el veneno de algunas serpientes cascabeles comunes en Venezuela, poseen un efecto sistémico sobre el músculo esquelético, y también efectos sobre capilares que generan edema, fenómenos hemorrágicos y necrosis, que habían pasado desapercibidos.

The bleeding, necrotic and edematous Snake bite effects from 135 patients of Miranda, Aragua, Vargas States and Capital District (Venezuela), caused by the Venezuelan common rattlesnake (Crotalus durissus cumanensis) from 1998 to 2008 were described. In bleeding disorders, which traditionally were almost imperceptible in Venezuelan Crotalus, we have found reliable evidence of clinical manifestations such as: afibrinogenemia, lengthening of the manual time of coagulation (MTC), and Partial Time of Thromboplastin (PTT) and Prothrombin time (PT), which indicates the presence of hemorrhagic fractions in the Venezuelan rattlesnake’s venoms. There were differences between the sexes, still predominant in male (82%). However, there has been an increase of significant impact on female (17%). By age, there was prevalence between 11 and 30 years old, both male and female. The mostly affected bite si te was upper limb (58,5%), with a no less significant percentage of lower limbs (40,7%). These findings, allowed us to suggest that some rattlesnake venoms have a systemic effect on skeletal muscle, and also effects on capillaries that generate swelling, hemorrhagic phenomena and necrosis.

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.

Biochemical comparison of venoms from young Colombian Crotalus durissus cumanensis and their parents

Crotalus durissus cumanensis, a rattlesnake endemic to Colombia and Venezuela, is considered one of the most lethal snake species in Latin America. The aim of the present study was to compare the protein content and biological activity of the venom obtained from eight specimens of C. durissus cumanensis, namely two adults from different localities of Colombia and six offspring born in captivity. Protein profiles of crude venoms were analyzed by SDS-PAGE and RP-HPLC, and biological activities were evaluated for lethality, edema, defibrination, hemolytic and coagulant activities to assess individual venoms of adults and a pool of young snake venoms. Transient edema appeared rapidly after venom inoculation, whereas hemorrhagic effect was not observed. Differences in protein profiles, lethality, hemolytic, coagulant and defibrinating activities between both adult snake venoms were observed; those from the mother snake exhibited higher activities. Venoms from young snakes were similar to the one obtained from the mother, but the coagulant effect was stronger in offspring venoms. Notably, biological effects of the father snake venom were not comparable to those previously described for C. durissus cumanensis from Venezuela and C. durissus terrificus from Brazil, confirming the high variability of the venom from Crotalus species.(AU)

Biochemical comparison of venoms from young Colombian Crotalus durissus cumanensis and their parents

Crotalus durissus cumanensis, a rattlesnake endemic to Colombia and Venezuela, is considered one of the most lethal snake species in Latin America. The aim of the present study was to compare the protein content and biological activity of the venom obtained from eight specimens of C. durissus cumanensis, namely two adults from different localities of Colombia and six offspring born in captivity. Protein profiles of crude venoms were analyzed by SDS-PAGE and RP-HPLC, and biological activities were evaluated for lethality, edema, defibrination, hemolytic and coagulant activities to assess individual venoms of adults and a pool of young snake venoms. Transient edema appeared rapidly after venom inoculation, whereas hemorrhagic effect was not observed. Differences in protein profiles, lethality, hemolytic, coagulant and defibrinating activities between both adult snake venoms were observed; those from the mother snake exhibited higher activities. Venoms from young snakes were similar to the one obtained from the mother, but the coagulant effect was stronger in offspring venoms. Notably, biological effects of the father snake venom were not comparable to those previously described for C. durissus cumanensis from Venezuela and C. durissus terrificus from Brazil, confirming the high variability of the venom from Crotalus species.

Caracterización Toxinológica del Veneno Total de la Serpiente de Cascabel Crotalus durissus cumanensis (VIPERIDAE), presente en la localidad de Porshoure, Guajira Venezolana / Toxinological Characterization of the Whole Venom of the Rattlesnake Crotalus durissus cumanensis (Viperidae) from Porshoure, Venezuelan Guajira

En el presente trabajo se caracterizó toxinológicamente el veneno total de la serpiente de cascabel Crotalus durissus cumanensis presente en la localidad de Porshoure, Guajira venezolana, determinando: dosis letal cincuenta (DL50), actividad hemorrágica, actividad edematizante, actividad hemolítica indirecta, actividad coagulante sobre plasma humano (in vitro) y actividad desfibrinante (in vivo), según las metodologías establecidas por la Organización Mundial de la Salud. Los resultados obtenidos mostraron que el veneno de esta subespecie es altamente tóxico, de naturaleza neurotóxica y coagulante. A nivel local produjo poca hemorragia y edema al compararlo con el veneno de otras serpientes de la misma subespecie, entre ellas Crotalus durissus cumanensis de la Villa del Rosario, estado Zulia; Crotalus durissus terrificus y Crotalus durissus durissus de Costa Rica y Brazil; Crotalus durissus terrificus de Colombia; Crotalus durissus ruruima, Crotalus durissus cascavella, Crotalus durissus collilineatus, entre otras. También resultó ser poco hemolítico y con características desfibrinantes (in vivo); efecto que hasta ahora no había sido reportado para serpientes pertenecientes al género Crotalus, lo que sin duda alguna constituye un valioso aporte para el diagnóstico y tratamiento del accidente crotálico en Venezuela.

The whole venom of the rattlesnake Crotalus durissus cumanensis (Viperidae) from Porshoure, Venezuelan Guajira, was characterized toxinologically in this research, determining: lethal dose 50 (LD50), bleeding, edematic and hemolytic activity, coagulant activity on human plasma (in vitro) and fibrinolytic activity, according to the methodologies outlined by the World Health Organization. The results demonstrated that the venom of the studied species is highly toxic, neurotoxic and coagulant. Local effects such as the hemorrhage and edema were not highly marked in comparison with other snakes’ venoms of the same subspecies as Crotalus durissus cumanensis from La Villa del Rosario, Zulia state; Crotalus durissus terrificus from Costa Rica and Crotalus durissus durissus from Brazil; Crotalus durissus terrificus from Colombia and Crotalus durissus ruruima, Crotalus durissus cascavella and Crotalus durissus collilineatus. The venom was slightly hemolytic and with fibrinolytic characteristic (in vivo). This effect has not yet been described within the genus Crotalus, what is a significant addition to the information of diagnosis and treatment of the rattlesnake bites in Venezuela.