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 ...

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

Crotoxin-induced behavioral effects in rats

Crotoxin is the major component of Crotalus durissus terrificus venom. In view of the presence of high-affinity specific binding sites for crotoxin in the brain, the objective of this work was to investigate whether crotoxin induces behavioral effects in the open-fleld and hole-board tests. Adult male Wistar rats (l80-220 g) treated with crotoxin, 1OO, 250 and 500 mug/kg, ip, administered 2 h before the test, presented statistically significant behavioral alterations (ANOVA for one-way classification complemented with Dunnet test, P<0.05). In the open-field test, 250 and 500 mug/kg of crotoxin increased freezing (from 3.22 sec to 10.75 sec) and grooming (from 13.44 sec to 22.75 sec and 21.22 sec) and decreased ambulation (from 64.8 to 39.38 and 45.8). The dose of 500 mug/kg also decreased rearing (from 24.9 to 17.5). In the hole-board test, 500 mug/kg of crotoxin decreased head-dip count (from 6.33 to 4.00). All the crotoxin- induced behavioral effects were antagonized by an anxiolytic dose of diazepam (1.5 mg/kg, ip, 30 min before the tests). These results show that crotoxin reduced open-field activity and exploratory behavior as well. We suggest that these effects express an increased emotional state induced by this toxin.

Neutralization of the effect of Crotalus durissus terrificus venom by gangliosides

We determined the ability of a mixture of gangliosides (16 percent) GDlb, 19 percent GT1b, 21 percent GM1, 40 percent GD1a) to neutralize the effect of Crotalus durissus terrificus (Cdt) venom in vitro and in vivo. Protection was indicated by the absence of muscular contractions, hind limb paralysis or death of BLB/c mice (16-18g) after receiving Cdt venom (1µgCdt venom containing 0.6 µg protein) at the doses indicated. A dose of Cdt venom above 0.9µg (ip) or 1 µg (im) induced muscular contraction and above 1.2 µg (ip) or 5.5 µg (im) the venom induced muscular contraction and hind limb paralysis. Cdt venom BOVE 2.5 µG (IP) OR 9 µg (im) induced all these symptoms and 95 to 100 percent death in experimental animals. The lethal dose 50 percent of the Cdt venom used was 8µ (im) and 1.5 µg (ip). In vitro studies, 4 mg gangliosides neutralized the effect of up to 1.5 µg Cdt venom. Quantities as low as 0.2 mg gangliosides were capable neutralizing 0.9 µg of Cdt venom in vitro. Intramuscular treatment with 1 mg gangliosides performed 60 min after the intramuscular injection of 5 µg Cdt venom protected 100 percent of the animals. In contrast, no protection was achieved with intraperitoneal treatment with gangliosides. The data show that gangliosides were effective in neutralizing the toxic effect induced by Crotalus durissus terrificus venom both in vitro and in vivo and that post-exposure intramuscular treatment with gangliosides could protect animals experimentally inoculated with the venom

Effects of crotoxin on the action potential kinetics of frog skeletal muscle

1. The effect of crotoxin on the action potential kinetics of frog (Rana catesbeiana; 60-80 g) skeletal muscle was studied using a modified Ringer solution containing 1.6 mM KCl. 2. Crotoxin affected the kinetics of the action potential in a dose-dependent manner (90 to 460 nM). At 230 nM, crotoxin prolonged the duration of the action potential (from 1.1 +/- 0.1 to 1.6 +/- 0.1 ms) and slowed the rates of depolarization (from 282.0 +/- 7.3 to 196.0 +/- 13.2 V/s) and repolarization (from -81.4 +/- 6.9 to -55.6 +/- 3.8 V/s), in a dose-dependent manner. Its phospholipase subunit (component B) was five times less effective. 3. No effect of crotoxin was observed in the presence of 2.5 mM KCl or when SrCl2 was substituted for CaCl2. Lowering the muscle temperature to 12 degrees C did not reduce the effect of crotoxin. 4. No effect on the passive membrane response to hyperpolarizing current pulses was observed, which implies no major effect on the membrane resistance and capacitance. 5. It is concluded that crotoxin reduces the Na+ current and slows down the repolarization mechanism. This effect is probably not dependent on the phospholipase A2 activity of crotoxin and is inhibited by the substitution of Sr2+ for Ca2+

Informe especial: estudios tendientes a determinar las posibles propiedades antitumorales del veneno de cobra y del complejo crotoxina A y B / Special report: studies to determine the possible antitumoral properities of Cobra venom and of crotoxin complex A and B

La presente investigación fue encomendada por el CONICET (Consejo Nacional de Investigaciones Cientificas y Técnicas) para determinar las posibles propriedades antineoplásicas del veneno de Cobra (Naja Naja Siamensis) VC) y del Complejo Crotoxina a y B (CCAB), purificado a partir del veneno de Crotalus durissus terificus (cascavel sudamericana). La coordinación de las investigaciones estuvo a cargo de los Dres. Baldi y Mordoh, y la ejecución de la parte experimental fue llevada a cabo por los restantes autores. Se utilizaron diversos sistemas experimentales: 1) lúneas celulares in vitro: de origen murino, normales (3T3 A31), o transformadas (M-A31, Ki-A31 y BP-A31) o de origen humano (adenocarcinoma mamario: MCF-7 y T47D). el efecto de las drogas fue determinado a 1, 10 y 100 ng/ml de VC y CCAB, solas o en combinación. En ninguna de las líneas celulares mencionadas se observaron cambios significativos en la velocidad de crecimiento o en la morfología celular; 2) desarrollo in vivo del Sarcoma 180 (S180) en ratones Balb/c: el VC a dosis de 21 ó 26 ng/g (inyecciones i.p. a los días 10, 11, 20, 21, 30 y 31 post-inoculo tumoral) no afectaron el crecimiento ni la sobrevida de los animales. Dosis de CCAB de 6 ng/g o 9 ng/g ]con el mismo esquema de inoculación anterior) no afectaron el crecimiento de s180 hasta los 25 días de desarrollo tumoral. Entre los días 25 y 30 la dosis de 9 ng/g determinó una evolución tumoral más rápida. La combinación de 13 ng/g VC y 4,5 ng/g CCAB con iguales tiempos de inyección no afectaron el desarrollo tumoral. La histología de los tumores de los animales tratados y controles no evidenció cambios significativos; 3) desarrollo in vivo del carcinoma de Ehrlich en ratones Swiss: se probó el efecto de CCAB (9 ng/g inyectados i.p. a los días 8, 9, 16, y 17) sin obtenerse modificación del desarrollo tumoral;...

Mecanismo de acción de miotoxinas aisladas de venenos de serpientes / Mechanism of action of myotoxins isolated from snake venoms

Desde los puntos de vista bioquímico y farmacológico, las miotoxinas aisladas de venenos de serpientes se ubican en cuatro grupos: (1) Fosfolipasas A miotóxicas, (2) miotoxinas básicas de bajo peso molecular, (3) cardiotoxinas de venenos elapídeos y (4) miotoxinas hemorrágicas. Las fosfolipasas miotóxicas notexinam taipoxina, crotoxina y miotoxina de Bothrops asper afectan inicialmente la integridad de la membrana plasmática, induciéndose un influjo de calcio que culmina con la muerte celular. Las miotoxinas básicas de bajo peso molecular crotamina y miotoxina a actúan específicamente en los canales de sodio del sarcolema, induciendo un influjo de sodio que trae como consecuencia despolarización y contracción muscular y vacuolización del retículo sarcoplásmico. Las cardiotoxinas son polipéptidos básicos capaces de desorganizar la estructura de las membranas, siendo su acción miotóxica una consecuencia de la alteración drástica que las mismas inducen en el sarcolema del músculo esquelético. Finalmente, dos componentes hemorrágicos (toxina hemorrágica b y viriditoxina) poseen actividad miotóxica, habiendose sugerido que este efecto es una consecuencia de la isquemia tisular resultante de la acción hemorrágica de estos componentes