Local and systemic effects of BtaMP-1, a new weakly hemorrhagic Snake Venom Metalloproteinase purified from Bothriopsis taeniata Snake Venom.

A new weak hemorrhagic metalloproteinase named BtaMP-1 was purified from Bothriopsis taeniata snake venom by molecular exclusion followed by anion exchange chromatographies. This protein showed a molecular mass of 25,968.16 Da and is composed of 218 amino acid residues. The multiple alignments of its partial amino acid sequence showed high structural identity with other P-I class SVMP. BtaMP-1 showed caseinolytic activity that was enhanced by Ca2+ ion, completely inhibited by chelating and reducing agents and can be classified as an α-fibrinogenolytic enzyme. Locally, BtaMP-1 induces hemorrhage and edema, but not myotoxicity. These findings were confirmed by histological analysis of mouse gastrocnemius muscle. "In vitro" studies suggest that BtaMP-1 induce cytotoxicity in myoblast C2C12 but not in the myotubes cell line. BtaMP-1 induced systemic alterations in mice with one MHD and two hours exposure; histological analysis of lungs showed hemorrhagic areas, congestion, and increase the thickness of alveolar septum. Also, this protein induced mild effects on kidney and disruption of coagulation by depletion of fibrinogen plasma levels. This work provides insights into the importance of BtaMP-1 biological effects in envenomation by Bothropsis taeniata snake venom and providing further evidence to understand the role of P-I class SVMP in ophidian envenomation.

Experimental poisoning of goats by Ipomoea carnea subsp. fistulosa in Argentina: a clinic and pathological correlation with special consideration on the central nervous system / Intoxicação experimental por Ipomoea carnea subsp. fistulosa em caprinos da Argentina: correlação clínico-patológica, com especial atenção ao sistema nervoso central

Ipomoea carnea subsp. fistulosa, aguapei or mandiyura, is responsible for lysosomal storage in goats. The shrub contains several alkaloids, mainly swansonine which inhibits lysosomal α-mannosidase and Golgi mannosidase II. Poisoning occurs by inhibition of these hydrolases. There is neuronal vacuolation, endocrine dysfunction, cardiovascular and gastrointestinal injury, and immune disorders. Clinical signs and pathology of the experimental poisoning of goats by Ipomoea carnea in Argentina are here described. Five goats received fresh leaves and stems of Ipomoea. At the beginning, the goats did not consume the plant, but later, it was preferred over any other forage. High dose induced rapid intoxication, whereas with low doses, the course of the toxicosis was more protracted. The goats were euthanized when they were recumbent. Cerebrum, cerebellum, medulla oblongata, pons and colliculi, were routinely processed for histology. In nine days, the following clinical signs developed: abnormal fascies, dilated nostrils and abnormal postures of the head, cephalic tremors and nystagmus, difficulty in standing. Subsequently, the goats had a tendency to fall, always to the left, with spastic convulsions. There was lack in coordination of voluntary movements due to Purkinje and deep nuclei neurons damage. The cochlear reflex originated hyperreflexia, abnormal posture, head movements and tremors. The withdrawal reflex produced flexor muscles hypersensitivity at the four legs, later depression and stupor. Abnormal responses to sounds were related to collicular lesions. Thalamic damage altered the withdrawal reflex, showing incomplete reaction. The observed cervical hair bristling was attributed to a thalamic regulated nociceptive response. Depression may be associated with agonists of lysergic acid contained in Ipomoea. These clinical signs were correlated with lesions in different parts of the CNS.

Ipomoea carnea subsp. fistulosa, aguapeí ou mandiyura, causa uma doença de depósito lisossomal em caprinos. A planta contém vários alcalóides, principalmente swansonine, que inibe uma α-mannosidase lisossomal e uma mannosidase II do Golgi. A intoxicação ocorre pela inibição dessas hidrolases. Há vacuolização neuronal, disfunção endócrina, lesões cardiovasculares e gastrointestinais e distúrbios imunológicos. No presente trabalho é descrita a intoxicação experimental por Ipomoea carnea subsp. fistulosa em caprinos da Argentina. Cinco cabras receberam folhas frescas e ramos de Ipomoea. No começo eles não consumiram a planta, mas depois ela foi preferida a qualquer outras forrageiras. Altas doses induziram uma intoxicação rápida, enquanto que com doses baixas demora mais tempo. As cabras foram sacrificadas quando ficavam em decúbito. Cérebro, cerebelo, medula oblonga, ponte e colículos foram processados rotineiramente para histologia. Aos nove dias, encontramos: fascie anormal, narinas dilatadas, posturas anormais da cabeça, tremores cefálicos e nistagmo, dificuldade para permanecer na estação. Posteriormente, presentearam a tendência de queda, sempre à esquerda, com convulsões espásticas. Os neurônios de Purkinje e os os núcleos profundos foram danificados. Como conseqüência, emergiu a falta de coordenação de movimentos voluntários. A hiper-reflexo coclear originou postura anormal, movimentos de cabeça e tremores. A retirada produz reflexos hipersensibilidade nos flexores dos quatro membros, depois depressão e letargia. As respostas anormais estão ligadas a lesões do colículo. Injurias no tálamo alteram o reflexo de retirada, mostrando uma reação incompleta. Os pelos eriçados da região cervical podem representar uma resposta nociceptiva também regulada pelo tálamo. A depressão pode estar associada com agonistas do ácido lisérgico presentes na Ipomoea. Esses sintomas estão relacionados com lesões em diferentes partes do SNC.

Neurotoxicological effects of a thrombin-like enzyme isolated from Crotalus durissus terrificus venom (preliminary study).

A thrombin-like enzyme, purified from the venom of Crotalus durissus terrificus by gel filtration and affinity chromatography, showed a single protein band in Sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) with a molecular weight of about 33kDa. Clear cellular morphological changes, deep ganglioside level modifications in some brain areas and behavioral alterations in pup rats injected with this protein were detected. Ganglioside composition, one of the chemical markers of brain maturation, was altered specially in the hypothalamus, hippocampus and prefrontal cortex. The most reliable behavioral effects were a delayed, maturation of the righting reflex, posture and motor response after treatment. These effects were consistent with the histological changes revealed in the cerebellum and prefrontal cortex of treated neonate rats, areas related to motor activities.

Equine laminitis: bites by Bothrops spp cause hoof lamellar pathology in the contralateral as well as in the bitten limb.

The envenoming caused by Bothrops snakebite includes local symptoms, such as pronounced edema, hemorrhage, intense pain, vesicles, blisters and myonecrosis. The principal systemic symptom consists in the alteration of blood clotting, due to fibrinogen consumption and platelet abnormalities. The horses involved in this study had this symptomatology and one of them exhibited symptoms consistent with laminitis in the bitten and in the contralateral limbs. Laminitis lesions were characterized by separation of the hoof lamellar basement membrane (BM) from basal cells of the epidermis. These results demonstrated that Bothrops snake venom can induce acute laminitis. We conclude that components of the venom, probably metalloproteinases, cause severe lesions in the hoof early in the envenoming process. Antivenom therapy must be initiated as soon as possible in order to prevent complications, not only to save the life of an envenomed horse, but also to avoid the dysfunctional sequels of laminitis.

[Renal injury in rats poisoned by venom of Bothrops neuwiedii diporus from Argentina]. / Daño renal en ratas inducido por veneno de Bothrops neuwiedii diporus de Argentina.

Acute renal failure is one of the systemic complications that can be found in bothropic accidents. In this study the effects on male Wistar rats induced by the venom of Bothrops neuwiedii diporus were evaluated. The histopathology revealed acute tubular necrosis, lesions firstly were observed 3 hours post inoculation of 700 microg of venom. Cortical kidney congestion and granulohialin degeneration of tubular epithelial cells were observed, these lesions achieved a maximum at 24 hours after inoculation. Tubular epithelial hidropic degeneration and dilatation of tubular lumen with hyalin casts were present inclusive up to 4 weeks after inoculation. Biochemical parameter values associated with kidney renal failure were increased 6 hours after venom inoculation (urea: 1.10+/-0.22 g/dl; creatinine: 19.60+/-1.51 mg/dl), but at the end of the first week they decreased till normal values. The urinary density was lower than normal value: 1.005+/-0.001 (p<0.001) and at the end of the first month they oscillated between 1.005 and 1.060 (p<0.001). Renal injury induced by B. neuwiedii diporus could be better appreciated by histopathology than by the routine laboratory assays.

Dano renal en ratas inducido por veneno de Bothrops neuwiedii diporus de Argentina / Renal injury in rats poisoned by venom of Bothrops neuwiedii diporus from Argentina

La insuficiencia renal aguda de las complicaciones sistémicas más frecuentes después de un accidente ofídico. En este estudio se evalúan los efectos que el veneno de Bothrops neuwiedii diporus produce en el riñón de ratas machos de la cepa Wistar. La histopatología permitió comprobar el desarrollo de necrosis tubular aguda; las lesiones iniciales se observaron a las 3 horas de la inoculación de una dosis de 700 microg del veneno, observándose en corteza renal congestión y degeneración granulohialina de las células epiteliales tubulares, acompañadas de dilatación y cilindros hialinos en la luz tubular. A las 24 horas se presentó necrosis tubular aguda en una superficie extensa de la corteza sin daño de la menbrana basal tubular. Las lesiones de degeneración turbia de células epiteliales tubulares, dilatación de la luz tubular y cilindros hialinos se mantuvieron presentes hasta las 4 semanas post-inoculación. Si bien los parámetros de la bioquímica clínica asociados con insuficiencia ranal aguda aumentaron a las 6 horas de la administración de veneno (urea: 1.10+/-0.22 g/dl; creatinina: 19.60+/-1.51 mg/dl), a la semana descendieron a valores normales. Las densidades urinarias, en cambio, a la semana se matuvieron más bajas que lo normal, 1.005+/-0.001 (p<0.001) y al mes oscilaron entre 1.005 y 1.060 (p<0.001). El daño renal producido por el veneno de B. neuwiedii diporus pudo ser valorado mejor histopatológicamente quepoe las técnicas convencionales de laboratorio.

Edematogenic and myotoxic activities of the Duvernoy's gland secretion of Philodryas olfersii from the north-east region of Argentina.

Philodryas olfersii is found in South America, from Amazonas to Patagonia. It is important to characterize the venom of P. olfersii, who inhabits the North-East region of Argentina, since snake venoms are known to exhibit considerable variability in composition and biological activities. In this work, mice weighing 18-20 g (n = 4 for each experimental group) were used. For the edematogenic activity mice were injected s.c. in the right foot pad with 50 microl of solutions containing different amounts of venom, whereas the left foot pad was injected with 50 microl of PBS. Two hours after injection mice were killed by cervical dislocation and both feet were cut off and weighed individually. For the myotoxic activity mice were injected i.m. with 100 microl of solutions containing 40 microg of venom. Blood samples were extracted after 1, 3, 6, 8, 10, 12, 14, 16 and 24 h of venom injection to determinate serum CPK activity and mice were sacrificed at the same time intervals to obtain the inoculated gastrocnemius muscle. They were fixed with Bouin solution and stained with Hematoxylin-Eosin. Results showed that P. olfersii venom exhibits a high edematogenic activity (MED = 0.31 microg) and a moderate myotoxic activity. Myonecrosis reached its highest level after 12 h of venom injection as shown by plasmatic CPK levels (5,401 +/- 330 IU/l) and microscopic assay. It demonstrates the potential toxicity of the venom of P. olfersii, who inhabits the North-East region of Argentina. It also reinforces the original warning concerning the potential danger of bites by colubrids.

Estudios de toxicidad del senecio grisebachii en ratones e identificación de componentes volatiles potencialmente toxicos / Toxicity studies of senecio grisebachii in mice and potentially toxic volatile components identification

El Senecio grisebachii es una planta de amplia distribución en el nordeste argentino que causa intoxicaciones en animales. En nuestro trabajo se alimentaron ratones con flores y hojas secas (20 por ciento de la ración) El vegetal causó intoxicación que se manisfestó por cambios en el comportamiento, perdida de peso y la histopatología revelò megalocitos y degeneración hidrópìca en zona centro lobulillar y periportal con mayor tiempo de consumo del vegetal. De distintas partes del vegetal se aislaron compuestos volátiles aún no detectados en esta planta, analizados por cromatografía gaseosa e identificados por espectrometría de masas. Los componentes identificados fueron: indol, b-mirceno, o-cimeno, ylangeno, cariofileno y a-cariofileno, que podrían sumarse a los tóxicos ya conocidos, los alcaloides pirrolizidinicos.

Efecto proteolítico de venenos de serpientes del género bothrops (yarará) de la Argentina y su neutralización por un antiveneno bivalente / Proteolityc effect of bothrops snake venom (yarará) from Argentina and neutralization by bivalent antivenom

Se estudió la actividad proteolítica de los venenos del género Bothrops de diferentes especies que habitan el litoral argentino y su neutralización por un suero hiperinmune bivalente producido en la Argentina. Se midió la actividad proteolíticas sobre caseína y se determinó la dosis proteolítica mínima (DPM), y la dosis efectiva 50 (DE) del suero bivalente frente a cada veneno. Las actividades proteolíticas de los venenos B, alternatus, B. neuwiedii, B. jararaca, B. jararacussu y B. moojeni fueron inferiores a las que presentan otras especies de Bothrops que habitan la región de America Central. Los resultados de la DE mostraron que el suero fue efectivo para neutralizar tal actividad en todos ellos, a pesar de que el suero utilizado en estas experiencias era específico para B. alternatus y B. neuwiedii.

Lesines locales y sistemicas inducidas por veneno de Bothrops alternatus (víbora de la cruz) de Argentina / Local and sistemic lesions induced by Bothrops alternatus venom (víbora de la cruz) of Argentine

Se inocularon ratas de 220+= 20 g de peso, en grupos de 5 animales, con 800 ug de veneno de Bothrops alternatus de Argentina desecado y homogeneizado, diluído en 0,1 ml de solución salina, pore via intramuscular. Para la obtención de sangre y su posterior sacrificio, se anestesiaron a las 3, 9 y 24 horas, tomándose muestras del músculo inoculado, hígado y riñón. Se realizaron determinaciones enzimáticas y los tejidos se procesaron para histopatología. A las 3 horas, se observaron necrosis de fibras musculares confirmadas por métodos histoquímicos, hemorragia e infiltrado inflamatorio, los que se intensificaron a las 9 y 24 horas. Paralelamente se observó un incremento plasmático de lñas actividades enzimáticas de creatin fosfoquinasa (CPK), aspartato aminotransferasa (AST) y la alanina aminotransferasa (ALT); siendo máximo el aumento de CPK entre las 3 y 9 horas. La respuesta inflamatoria estudiada en ratón, mostró una reacción rápida cuya recuperación fue lenta. La necrosis de fibras musculares fue acompañada por peroxidación de lípidos y precipitación de calcio en las células. Las lesiones de te4jido hepático no fueron relevantes y en riñón se detectaron alteraciones en zona yuxtamedular y en el intersticio cortical

Intoxicación por veneno de crotalus durissus terrificus (cascabel) en ratas / Crotalus durissus terrificus (cascabel) nenom poisoning in rats

Se inocularon ratas de 180 a 220 g de peso con 120 ug/100 g de neneno desecado de Crotalus durissus terrrificus de Argentina, en 0,1 ml de soloución salina, por via i.m en grupos de 5 animales. Para la obtención de sangre y posterior sacrificio fueron anestesiados a las 3, 9 y 24 horas, tomándose muestras del músculo inoculado, del contralateral, de hígado y riñón. Se realizaron determinaciones enzimáticas y los tejidos se procesaron para histopatología. Se observó daño del tejido muscular esquelético a partir de las 3 horas, con necrosis miolítica y coagulativa. Paralelamente se pbservó un aumento plasmático de CK, AST yALT, siendo el aumento de CK máximo a las 9 horas posteriores a la inoculación. El tejido hepático se vió afectado, con congestión y dilastación de los vasos sanguíneos grandes. Los sinusoides periféricos a tales vasos presentaron hemosiderina a las 24 horas; las pequeñas vénulas contenian trombos. Los incrementos de AST y alt fueron máximos a las 24 horas. En tejido renal se observó congentión cortical a partir de 3 horas. A las 24 horas la congestión se extendió a las zonas yuxtamedulares con escasa cantidad de cilindros hialinos, Todos los animales manifestaron alteraciones neurológicasd con ptosis palpebral y midriasis bilateral, dificultad respiratoria, insensibilidad y parálisis progresiva. Se demostró que Crotalus durissus terrrificus de Argentina no solo posee actividad neurotóxica, sino también miolítica, hepatotóxica y nefrotóxica

Hemorragia inducida por venenos de serpientes de Argentina / Hemorrhage induced by snake venoms in Argentina

Se inocularon ratones de 18 a 20 g de peso por vía intradérmica con 0.1 ml de diluciones seriadas de veneno en solución salina amortiguada a pH 7.2. Se conformaron grupos de 4 animales los que se sacrificaron a las 2 horas de la inoculación. Se quitó la piel y se midió el área hemorrágica. Se utilizaron los venenos de Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu y Bothrop neuwiedii. Todos ellos presentaron actividad hemorrágica. Crotalus durissus terrifcus no manifestó actividad hemorrágica.

Actividades hemorrágicas y coagulante del veneno de Bothrops alternatus de Argentina / Haemorrhagic and clotting activities of Argentina B. alternatus venon

Se trabajó con veneno crudo de Bothrops alternatus para determinar sus actividades hemorrágica y coagulante. Para hallar la actividad hemorrágica se inocularon ratones, en forma intradérmica, con diluciones seriadas de veneno, en un volumen de 0,1 ml de solución salina amortiguada con fosfato, pH 7,2. Luego de 2 horas, se sacrificaron con éter y se midió el área hemorrágica. La Dosis Hemorrágica Mínima (DHM) es la cantidad de veneno que produce un área hemorrágica mínima de 10 mm de diámetro, para el veneno de B. alternatus fue de 3,6 µg. El estudio de la Dosis Coagulante Mínima (DCM) se realizó confrontando diluciones seriadas de veneno con plasma humano. La DMC, capaz de coagular el plasma humano en 60 seg, fue de 14,5 µg/ml. Al igual que otras especies del género Bothrops de Latinoamérica, el veneno de B. alternatus de Argentina posee marcada actividad hemorrágica y coagulante

Efectos del veneno de Bothrops alternatus de Argentina sobre músculo y distintos organos de ratones / Effect of Bothrops alternatus poison of Argentina on muscle and different organs in mices

Se inucularon ratones de 18 1 20 g de peso, en el músculo gastrocnemius con 50 mug de veneno de Bothrops alternatus, en 0,1 ml de solución fisiológica. Se conformaron grupos de 5 animales los que fueron sacrificados a las 3,6 y 12 hs posteriores a la inoculación. Se extrajo el músculo inoculado y muestras de diferentes órganos, los que fueron fijados en Bouin y procesados para histopatología. El veneno provocó una reacción inflamatoria local intensa con gran edema, congestión y hemorragia. La observación microscópica del tejido muscular, mostró minecrosis de tipo coagulativa y miolítica. A las 3 y 6 hs de exposición, en tejido hepático se observó tumefacción celular y degeneración hidrópica en reas de venas centrales. En el tejido renal se presentó congestión cortical, tumefacción hidrópica de túbulos contorneados proximales y distales. En cambio no se hallaron anormalidades en corazón, pulmón y cerebro.