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.

Antivenomics as a tool to improve the neutralizing capacity of the crotalic antivenom: a study with crotamine

Background Snakebite treatment requires administration of an appropriate antivenom that should contain antibodies capable of neutralizing the venom. To achieve this goal, antivenom production must start from a suitable immunization protocol and proper venom mixtures. In Brazil, antivenom against South American rattlesnake (Crotalus durissus terrificus) bites is produced by public institutions based on the guidelines defined by the regulatory agency of the Brazilian Ministry of Health, ANVISA. However, each institution uses its own mixture of rattlesnake venom antigens. Previous works have shown that crotamine, a toxin found in Crolatus durissus venom, shows marked individual and populational variation. In addition, serum produced from crotamine-negative venoms fails to recognize this molecule. Methods In this work, we used an antivenomics approach to assess the cross-reactivity of crotalic antivenom manufactured by IVB towards crotamine-negative venom and a mixture of crotamine-negative/crotamine-positive venoms. Results We show that the venom mixture containing 20% crotamine and 57% crotoxin produced a strong immunogenic response in horses. Antivenom raised against this venom mixture reacted with most venom components including crotamine and crotoxin, in contrast to the antivenom raised against crotamine-negative venom. Conclusions These results indicate that venomic databases and antivenomics analysis provide a useful approach for choosing the better venom mixture for antibody production and for the subsequent screening of antivenom cross-reactivity with relevant snake venom components.(AU)

New insights into the structural characteristics of irradiated crotamine

Background:Since ionizing radiation has the potential to alter the molecular structure and affect the biologica properties of biomolecules, it has been successfully employed to attenuate animal toxins. The present study aimed to characterize the structural modifications on irradiated crotamine, a toxin from Crotalus durissus terrificus venom, using circular dichroism (CD), fluorescence, Fourier transformed infrared spectroscopy (FTIR), atomic force microscopy (AFM) and differential scanning calorimetry (DSC).Methods:A combination of size exclusion and ion-exchange chromatography was used to purify the peptide using crude venom. The pure toxin was then submitted to 2 kGy gamma irradiation doses from a cobalt-60 source. Native and irradiated crotamine were analyzed using a fluorescence spectrophotometer. Wavelength was fixed at 295 nm and fluorescence emission scans were collected from 300 to 400 nm. CD and FTIR techniques were used to identify the secondary structure of both samples. DSC analyses were performed at a starting temperature of 20 °C up to a final temperature of 90 °C. AFM provided a 3D profile of the surfaces of both crotamine forms adsorbed on mica.Results:Fluorescence spectroscopy showed that the quantum yield of the irradiated form decreased. CD spectra of native and irradiated crotamine solutions showed differences between the samples in wavelength, indicating that irradiation induced a transition of a small portion of the random coil regions towards an a-helical conformation. FTIR and CD showed that the native and irradiated crotamine spectra were different with regard to secondary structure. The thermodynamic analysis showed that irradiation caused changes in the calorimetric profile and CD showed that temperature-induced changes also occur in the secondary structure. Finally, AFM showed the possible formation of insoluble aggregates.Conclusions:Our results indicate that irradiation leads to progressive changes in the structure of the toxin, which could explain a decrease in myotoxic activity.(AU)

New insights into the structural characteristics of irradiated crotamine

Background: Since ionizing radiation has the potential to alter the molecular structure and affect the biologica properties of biomolecules, it has been successfully employed to attenuate animal toxins. The present study aimed to characterize the structural modifications on irradiated crotamine, a toxin from Crotalus durissus terrificus venom, using circular dichroism (CD), fluorescence, Fourier transformed infrared spectroscopy (FTIR), atomic force microscopy (AFM) and differential scanning calorimetry (DSC). Methods: A combination of size exclusion and ion-exchange chromatography was used to purify the peptide using crude venom. The pure toxin was then submitted to 2 kGy gamma irradiation doses from a cobalt-60 source. Native and irradiated crotamine were analyzed using a fluorescence spectrophotometer. Wavelength was fixed at 295 nm and fluorescence emission scans were collected from 300 to 400 nm. CD and FTIR techniques were used to identify the secondary structure of both samples. DSC analyses were performed at a starting temperature of 20 °C up to a final temperature of 90 °C. AFM provided a 3D profile of the surfaces of both crotamine forms adsorbed on mica. Results: Fluorescence spectroscopy showed that the quantum yield of the irradiated form decreased. CD spectra of native and irradiated crotamine solutions showed differences between the samples in wavelength, indicating that irradiation induced a transition of a small portion of the random coil regions towards an a-helical conformation. FTIR and CD showed that the native and irradiated crotamine spectra were different with regard to secondary structure. The thermodynamic analysis showed that irradiation caused changes in the calorimetric profile and CD showed that temperature-induced changes also occur in the secondary structure. Finally, AFM showed the possible formation of insoluble aggregates. Conclusions: Our results indicate that irradiation leads to progressive changes in the structure of the toxin, which could explain a decrease in myotoxic activity.

Bordonein-L, a new L-amino acid oxidase from Crotalus durissus terrificus snake venom: isolation, preliminary characterization and enzyme stability

Background Crotalus durissus terrificus venom (CdtV) is one of the most studied snake venoms in Brazil. Despite presenting several well known proteins, its L-amino acid oxidase (LAAO) has not been studied previously. This study aimed to isolate, characterize and evaluate the enzyme stability of bordonein-L, an LAAO from CdtV.Methods The enzyme was isolated through cation exchange, gel filtration and affinity chromatography, followed by a reversed-phase fast protein liquid chromatography to confirm its purity. Subsequently, its N-terminal amino acid sequence was determined by Edman degradation. The enzyme activity and stability were evaluated by a microplate colorimetric assay and the molecular mass was estimated by SDS-PAGE using periodic acid-Schiff staining and determined by mass spectrometry.Results The first 39 N-terminal amino acid residues exhibited high identity with other snake venom L-amino acid oxidases. Bordonein-L is a homodimer glycoprotein of approximately 101 kDa evaluated by gel filtration. Its monomer presents around 53 kDa estimated by SDS-PAGE and 58,702 Da determined by MALDI-TOF mass spectrometry. The enzyme exhibited maximum activity at pH 7.0 and lost about 50 % of its activity after five days of storage at 4 °C. Bordonein-Ls activity was higher than the control when stored in 2.8 % mannitol or 8.5 % sucrose.Conclusions This research is pioneering in its isolation, characterization and enzyme stability evaluation of an LAAO from CdtV, denominated bordonein-L. These results are important because they increase the knowledge about stabilization of LAAOs, aiming to increase their shelf life. Since the maintenance of enzymatic activity after long periods of storage is essential to enable their biotechnological use as well as their functional studies.

In vitro activity of phospholipase A2 and of peptides from Crotalus durissus terrificus venom against amastigote and promastigote forms of Leishmania (L) infantum chagasi

Background American visceral leishmaniasis is caused by the intracellular parasiteLeishmania (L.) infantum chagasi, and transmitted by the sand fly Lutzomyia longipalpis. Since treatment is based on classical chemotherapeutics with significant side effects, the search for new drugs remains the greatest global challenge. Thus, this in vitro study aimed to evaluate the leishmanicidal effect ofCrotalus durissus terrificus venom fractions on promastigote and amastigote forms of Leishmania (L.) infantum chagasi. Methods Phospholipase A 2 (PLA 2 ) and a pool of peptide fraction ( 3 kDa) were purified from Crotalusvenom. Furthermore, promastigotes and peritoneal macrophages of mice infected by amastigotes were exposed to serial dilutions of the PLA 2 and peptides at intervals varying between 1.5625 g/mL and 200 g/mL. Both showed activity against promastigotes that varied according to the tested concentration and the time of incubation (24, 48 and 72 h). Results MTT assay for promastigotes showed IC 50 of 52.07 g/mL for PLA2 and 16.98 g/mL for the peptide fraction of the venom. The cytotoxicity assessment in peritoneal macrophages showed IC50 of 98 g/mL and 16.98 g/mL for PLA 2 and peptide by MTT assay, respectively. In peritoneal macrophages infected by Leishmania (L.) infantum chagasi amastigotes, the PLA 2 stimulated growth of parasites, and at higher doses reduced growth by 23 %. The peptide fraction prevented 43 % of the intracellular parasite growth at a dose of 16.98 g/mL, demonstrating the toxicity of this dose to macrophages. Both fractions stimulated H 2 O 2 production by macrophages but only PLA 2 was able to stimulate NO production. Conclusion We have demonstrated the in vitro leishmanicidal activity of the PLA2 and peptide fraction ofCrotalus venom. The results encourage further studies to describe the metabolic pathways involved in cell death, as well as the prospecting of molecules with antiparasitic activity present in the peptide fraction of Crotalus durissus terrificus venom.

In vitro activity of phospholipase A 2 and of peptides from Crotalus durissus terrificus venom against amastigote and promastigote forms of Leishmania (L) infantum chagasi

Background American visceral leishmaniasis is caused by the intracellular parasiteLeishmania (L.) infantum chagasi, and transmitted by the sand fly Lutzomyia longipalpis. Since treatment is based on classical chemotherapeutics with significant side effects, the search for new drugs remains the greatest global challenge. Thus, this in vitro study aimed to evaluate the leishmanicidal effect ofCrotalus durissus terrificus venom fractions on promastigote and amastigote forms of Leishmania (L.) infantum chagasi. Methods Phospholipase A 2 (PLA 2 ) and a pool of peptide fraction (<3 kDa) were purified from Crotalusvenom. Furthermore, promastigotes and peritoneal macrophages of mice infected by amastigotes were exposed to serial dilutions of the PLA 2 and peptides at intervals varying between 1.5625 μg/mL and 200 μg/mL. Both showed activity against promastigotes that varied according to the tested concentration and the time of incubation (24, 48 and 72 h). Results MTT assay for promastigotes showed IC 50 of 52.07 μg/mL for PLA2 and 16.98 μg/mL for the peptide fraction of the venom. The cytotoxicity assessment in peritoneal macrophages showed IC50 of 98 μg/mL and 16.98 μg/mL for PLA 2 and peptide by MTT assay, respectively. In peritoneal macrophages infected by Leishmania (L.) infantum chagasi amastigotes, the PLA 2 stimulated growth of parasites, and at higher doses reduced growth by 23 %. The peptide fraction prevented 43 % of the intracellular parasite growth at a dose of 16.98 μg/mL, demonstrating the toxicity of this dose to macrophages. Both fractions stimulated H 2 O 2 production by macrophages but only PLA 2 was able to stimulate NO production. Conclusion We have demonstrated the in vitro leishmanicidal activity of the PLA2 and peptide fraction ofCrotalus venom. The results encourage further studies to describe the metabolic pathways involved in cell death, as well as the prospecting of molecules with antiparasitic activity present in the peptide fraction of Crotalus durissus terrificus venom.(AU)

Quadros clínico-patológicos do envenenamento ofídico por Crotalus durissus terrificus e Bothrops spp: em animais de produção / Clinical and pathological pictures of snakebite poisoning by Crotalus durissus terrificus and Bothrops spp: in livestock

Foi realizada uma revisão dos quadros clínico-patológicos causados pelos venenos de Crotalus durissus terrificus e Bothrops spp. em bovinos, búfalos, ovinos equinos e suínos. Foram compilados os dados obtidos pela experimentação em animais de produção encontrados na literatura e os obtidos através de experimentação realizada por nossa equipe. Também foram revisados os casos naturais de envenenamento ofídico comunicados. Em dois Quadros foram lançados os mais importantes dados dessas revisões, que revelou diversos aspectos interessantes: 1) em nossos experimentos, o veneno de Crotalus durissus terrificus, quando injetado por via subcutânea em cavalos, causou um edema acentuado no local da aplicação, ao contrário do que tem sido observado em todas as outras espécies animais, aspecto não relatado na literatura; 2) em nossos experimentos, o veneno de diversas espécies de Bothrops, quando injetado por via subcutânea em bovinos, ovinos e equinos, não causou edema como em geral é relatado na literatura, e sim hemorragias subcutâneas acentuadas no local da aplicação. Nos casos não fatais este sangue era reabsorvido em poucos dias sem deixar sequelas. Exceção foi a reação ao veneno de Bothrops jararacussu, que causou edema nos ovinos experimentais, e tumefação acentuada que resultou em fístula com eliminação de líquido seroso nos equinos experimentais. O objetivo do presente estudo visa contribuir para o aperfeiçoamento do diagnóstico de acidentes ofídicos em animais de produção.

A review was performed about the clinical and pathological pictures caused by the venoms of Crotalus durissus terrificus and Bothrops spp. in cattle, buffaloes, horses, sheep and swine. The data were compiled from experiments in livestock species found in the literature, from experimentation accomplished by our research group, and from communicated natural cases of snakebite poisoning. The most important data were placed on two Tables, the analysis of which revealed some interesting aspects: (1) in our experiments the venom of Crotalus durissus terrificus caused in horses severe edema at the site of subcutaneous injection, to the contrary as observed in all other experimental animal species, an aspect not recorded in the literature; (2) in our experiments the venom of Bothrops species in cattle, sheep and horses, injected subcutaneously, did not cause edema as generally reported in the literature, but caused severe subcutaneous hemorrhages at the injection site. In the non fatal cases the blood was reabsorbed in a few days without leaving sequelae; exception was the reaction to the venom of Bothrops jararacussu, which caused edema in the experimental sheep, and severe tumefaction resulting in fistulous elimination of serous liquid in the experimental horses. The aim of this study was to contribute for the diagnostic of snakebite accidents in livestock.

Does the rattle of Crotalus durissus terrificus reveal its dietary history?

Background Environmental devastation threatens the survival of many species, including venomous snakes such as the South American rattlesnake Crotalus durissus terrificus. This observation is based on the decrease of snakes collected and donated to Brazilian research institutes. Nevertheless, some individuals have managed to survive and procreate. The question is how these snakes are adapting in these new environmental conditions.Methods To answer it, the carbon-13 level of rattlesnakes and their feed (either laboratory or wild mice) was evaluated by isotope-ratio mass spectrometry. Thus, rattle segments from 16 adults and 15 offspring of captive snakes, and of three wild newborn C. d. terrificus were evaluated as well as 17 Mus musculus mice captured in traps, four live feeder mice and the ration offered to mice at animal houses.Results The isotopic exchange time of the captive adult snakes (n = 16) varied between 33 and 37 months and of captive-born animals (n = 15), until reaching a plateau of equilibrium, varied from 18 to 24 months. Regarding the captured Mus musculus (n = 17), 88.23% (n = 15) were from a C4 environment. Of the six rattle rings from offspring of captured C. d. terrificus, five were from a C4environment, whereas of the 170 rattle rings studied, 60% originated from a C3 environment and 40% from a C4. The same carbon-13 values were found in captive snakes.Conclusions Based on the present results, it can be inferred that most C. d. terrificus snakes (60%) fed animals from a C3environment; birds consist of an alimentary alternative for snakes, as well as rodents, small reptiles and amphibians; different venom compositions among snakes from the same region may be related to the food type; the primary rattle of offspring reflects the maternal diet during gestation; and, finally, the different rattle rings indicate the alimentary history of these animals.

Envenenamento crotálico em cães / Crotalic envenomation in dogs

No Brasil, foram notificados no ano de 2011 30.826 casos de acidentes ofídicos em seres humanos, sendo que destes, 71,9% foram serpentes do gênero Bothrops. Todavia, dados do Serviço de Toxicologia do Hospital João XXIII (Belo Horizonte, MG) reportam que a maior parte dos acidentes por serpentes são decorrentes da espécie Caudisona durissa (Crotalus durissus) (ANDRADE FILHO et al., 2013). Do mesmo modo, no Hospital Veterinário da Escola de Veterinária da Universidade Federal de Minas Gerais (UFMG), o percentual de atendimentos decorrentes de picadas com serpentes da espécie C. durissa no ano de 2012 até maio de 2013 foi de 90% (foi atendido um total de 10 animais envenenados por serpentes neste período e apenas um animal foi serpente Bothrops). Os diagnósticos caninos foram realizados com base na identificação da serpente, nas manifestações clínicas e achados laboratoriais.

Aspectos clínico-patológicos e laboratoriais do envenenamento crotálico experimental em equinos / Clinical and pathological aspects of experimental crotalic envenoming in horses

Descrevem-se os quadros clínico-patológicos e laboratoriais de equinos inoculados experimentalmente com a peçonha de Caudisona durissa terrificus (Crotalus durissus terrificus na antiga nomenclatura), com a finalidade de fornecer subsídios que favoreçam a compreensão desse tipo de acidente ofídico em equinos. O veneno liofilizado foi diluído em 1ml de solução salina a 0,9% e inoculado por via subcutânea em cinco equinos, nas doses de 0,12mg/kg (um animal), 0,066mg/kg (dois animais) e 0,03mg/kg (dois animais). O veneno causou a morte do equino que recebeu a dose de 0,12mg/kg e de um dos dois que receberam a dose de 0,066mg/kg, com evolução de 27h27min e 52h29min, respectivamente. O segundo animal que recebeu a dose de 0,066mg/kg também adoeceu, mas recuperou-se após 12 dias da inoculação. A dose de 0,03mg/kg determinou quadros não fatais do envenenamento, com período de evolução que variou entre 6 e 10 dias. O quadro clínico caracterizou-se por considerável aumento de volume no local de inoculação (escápula) que se estendeu por todo o membro, apatia e cabeça baixa, alterações locomotoras evidenciadas pelo arrastar das pinças no solo, decúbito e dificuldade para levantar, redução dos reflexos auricular, palatal, do lábio superior e de ameaça, e aumento das frequências cardíaca e respiratória. Os exames laboratoriais revelaram leucocitose por neutrofilia e linfocitose em apenas dois animais. Houve aumento das enzimas creatina quinase (CK), dehidrogenase láctica (DHL) e da ureia, e também redução nos níveis séricos de cálcio, fósforo e magnésio. O tempo de tromboplastina parcial ativada (TTPA) aumentou nos equinos que morreram. Os achados de necropsia foram edema do tecido subcutâneo em todo o membro em que foi aplicado o veneno, sufusões no epicárdio dos ventrículos cardíacos esquerdo e direito, e bexiga com áreas hemorrágicas em grande parte da mucosa. Ao exame histopatológico observaram-se fígado com moderada vacuolização difusa, afetando mais a zona intermediária do lóbulo hepático, leve dilatação dos sinusoides hepáticos em algumas áreas e rim com leve dilatação dos túbulos uriníferos, principalmente no córtex.

The clinic-pathological picture and laboratory findings in horses experimentally inoculated with the venom of Caudisona durissa terrificus (Crotalus durissus terrificus, according to the former nomenclature) are described. The purpose of this study was to contribute to the understanding of this type of snake accident in horses. The lyophilized venom was diluted into 1ml of a 0.9% saline solution and was inoculated subcutaneously into five horses, at the doses of 0.12mg/kg (one horse), 0.066mg/kg (two horses) and 0.03mg/kg (two horses). The venom caused death of the horse that had received 0.12mg/kg, and of one horse of the two that had received the dose of 0.066mg/kg. The clinical course varied from 27h27min to 52h29min. The second horse inoculated with 0.066mg/kg recovered within 12 days after inoculation. The dose of 0.03mg/kg had a course of 6 to 10 days, but did not cause fatal envenomation. The clinical picture in the horses was characterized by swelling of the inoculation site (shoulder) that spread to the whole leg, by apathy and lowered head, locomotory alterations shown by dragging of the hoves on the ground, decubitus and difficulty to get up, reduction of auricular, palatal, upperlip and threat reflexes, and increase of heart and breathing frequency. The laboratory examination revealed leukocytosis and lymphocytosis in two horses. There was increase of the creatine-kinase (CK), lactic dehydrogenase (DHL) and urea, and reduction in the seric levels of calcium, phosphorus and magnesium. The activated partial tromboplastina time (TTPA) increased in the horses that died. Postmortem findings were edema of the subcutaneous tissue of the whole leg into which the venom was inoculated, suffusions in the epicard of left and right heart ventricles, and bladder with hemorrhagic areas in its mucosa. Histopatologic examination revealed the liver parenchyma with diffuse moderate vacuolation affecting predominantly the intermediate area of the hepatic lobe, and slight dilation of the sinusoides in some areas, and slight dilation of the kidney tubules mainly in the cortex.

Neutralización heteróloga e interacción de amanitinas con venenos de Bothropsy Crotalus y de abeja / Heterologous neutralization and interaction of toxic amanitins with Bothrops and Crotalus venoms, and honey bee venom / Neutralização heteróloga e interação das amanitinas tóxicas com veneno de Bothrops e Crotalus e veneno de abelha

En el presente trabajo se examinó la interacción de las amanitinas de Amanita phalloides (Basidiomycetes) con los venenos de las serpientes Bothrops neuwiedi diporus ("yarará pequeña"), B. alternatus ("yarará grande"), Crotalus durissus terrificus ("serpiente de cascabel") y de la abeja mielera Apis mellifera. Se aplicaron las técnicas de Ouchterlony, inmunotransferencia, electroforesis rocket y electroforesis en gel de poliacrilamida a los anti-venenos y anti-toxinas obtenidos por inmunización en caballos y/o en conejos. Los anti­sueros de serpientes y las amanitinas reaccionaron en forma cruzada, así como el veneno de abeja y las amanitinas. Cuando los venenos de Bothrops neuwiedii diporus y Crotalus durissus terrificus se preincubaron con las amanitinas y se analizaron por electroforesis en gel de poliacrilamida-dodecilsulfato de sodio (dodecylsulfate-polyacrylamide gel electrophoresis: SDS-PAGE), algunas bandas de proteínas desaparecieron y otras se redujeron notablemente. Estos resultados revelan por primera vez la interacción y la degradación de las proteínas de los venenos de serpientes por las amanitinas. Por otra parte, la modificación del tiempo de coagulación de la sangre humana, debida a los venenos, se corrigió con los ciclopéptidos de Amanita. Estos resultados también se informan por primera vez en este trabajo. La presencia de polipéptidos tóxicos en los venenos de serpientes y abejas, así como en A. phalloides y la reactividad cruzada demostradas en este trabajo, sugieren la existencia de epítopos comunes a todos ellos. Teniendo en cuenta estas reacciones, el uso de anti-venenos heterólogos parece ser de utilidad en el tratamiento del envenenamiento.

In the present work, the interaction of the amanitins of Amanita phalloides (Basidiomycetes) with the venoms of Bothrops neuwiedi diporus ("small yarará snake"), B. alternatus ("big yarará"), Crotalus durissus terrificus ("rattlesnake"), and honey bee Apis mellifera was examined. Ouchterlony, immunotransfer, rocket-electrophoresis, and polyacrylamide gel electrophoresis techniques were applied to anti-venoms and anti-toxins obtained by immunization in horses and/or in rabbits. Snake antisera and amanitins cross-reacted as well as bee venom and amanitins. When venoms of Bothrops neuwiedii diporus and Crotalus durissus terrificus were preincubated with amanitins and analysed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), some protein bands disappeared and others were significantly reduced. These results reveal for the first time the interaction and degradation of proteins in snake venoms by amanitins. Moreover, the modification of the human blood clotting time due to snake venoms was corrected by the Amanita cyclopeptides. These results are also reported for the first time in this work. The occurrence of toxic polypeptides in the snake and bee venoms as well as in A. phalloides, and the cross-reactivity demostrated herein, suggest the occurrence of epitopes common to all of them. Taking into account these reactions,the use of heterologous anti-venoms seems to be of value in envenomation treatment.

No presente trabalho foi examinada a interação das amanitinas de Amanita phalloides (Basidiomy­cetes) com os venenos das serpentes Bothrops neuwiedi diporus ("jararaca-cruzeira"), B. alternatus ("urutu"), Crotalus durissus terrificus ("serpente cascavel") e da abelha-europeia Apis mellifera. Foram aplicadas as técnicas de Ouchterlony, imunotransferência, eletroforese rocket e eletroforese em gel de poliacrilamida aos anti-venenos e anti-toxinas obtidos por imunização em cavalos e/ou em coelhos. Os anti-soros de serpentes e as amanitinas reagiram em forma cruzada, bem como o veneno de abelha e as amanitinas. Quando os venenos de Bothrops neuwiedii diporus e Crotalus durissus terrificus foram incubados previamente com as amanitinas e foram analisados por eletroforese em gel de poliacrilamida-dodecilsulfato de sódio (dodecylsulfate-polyacrylamide gel electrophoresis: SDS-PAGE), algumas faixas de proteínas desapareceram e outras se reduziram notavelmente. Estes resultados revelam por primeira vez a interação e a degradação das proteínas dos venenos de serpentes pelas amanitinas. Por outra parte, a modificação do tempo de coagulação do sangue humano, devido aos venenos, se corrigiu com os ciclopeptídeos de Amanita. Estes resultados também se informam por primeira vez neste trabalho. A presença de polipeptídeos tóxicos nos venenos de serpentes e abelhas, bem como em A. phalloides e a reatividade cruzada demonstradas neste trabalho, sugerem a existência de epítopos comuns a todos eles. Levando em consideração estas reações, o uso de anti-venenos heterólogos parece ser de utilidade no tratamento do envenenamento.

Mutagenicity induced by the hydroalcoholic extract of the medicinal plant Plathymenia reticulata Benth

Plathymenia reticulata Benth has an anti-inflammatory effect and is capable of neutralizing the neuromuscular blockade induced by Bothrops jararacussu or Crotalus durissus terrificus venoms, probably by precipitating venom proteins (an effect caused by plant tannins). The present study aimed to evaluate the mutagenic activity of P. reticulata by using the Salmonella mutagenicity assay (Ames test) and the micronucleus test in CHO-K1 cells. P. reticulata extract concentrations of 2.84, 5.68, 11.37, and 19.90 mg/plate were assayed by the Ames test using TA97a, TA98, TA100 and TA102 bacterial strains, with (+S9) and without (-S9) metabolic activation. Concentrations of 5, 1.6 and 0.5 ìg/mL of P. reticulata extract were used for the micronucleus test. P. reticulata extract was mutagenic to TA98 (-S9) and showed signs of mutagenic activity in TA97a and TA102 (both -S9) strains. Micronucleus test CBPI values showed that the endogenous metabolic system increased the number of viable cells when compared to the non-activated samples and the micronucleus frequency increased when the cells were treated in the absence of S9. We concluded that P. reticulata extract may present direct mutagenic properties.

Avaliação da interação de compostos ativos hidratantes com modelo de biomembrana de Crotalus durissus por meio de calorimetria exploratória diferencial e espectroscopia RAMAN / Study of the interaction of moisturizers with a Crotalus durissus biomembrane by differential scanning calorimetry and RAMAN spectroscopy

O objetivo foi verificar as alterações no estrato córneo em modelos alternativos de membrana após a aplicação de ativos hidratantes envolvendo métodos biofísicos. O modelo de biomembrana utilizado foi a muda de pele de Crotalus durissus e os ativos hidratantes foram: uréia, silício orgânico, extrato vegetal de Imperata cylindrica, reação de xilitol e glicose e componentes de NMF. Os resultados da avaliação das alterações do modelo por meio de Espectroscopia Raman com Transformada de Fourier sugerem que os ativos hidratantes confirmam segurança necessária, pois não alteraram de forma acentuada a estrutura do estrato córneo. Utilizando-se Calorimetria Exploratória Diferencial pode-se indicar que a solução de silício orgânico e o gel hidrofílico com uréia apresentaram melhor poder hidratante.

The objective of this research was to use biophysical techniques to investigate the alterations induced in a biomembrane model of the stratum corneum by the application of moisturizers. The biomembrane was obtained from the skin shed by the rattlesnake Crotalus durissus and the active moisturizing compounds were: urea, dimethylsilanol hyaluronate, Imperata cylindrical plant extract, carbohydrates and natural moisturizing factors (NMF components). Results from FT-Raman spectroscopy suggested that the moisturizers were safe, since they did not promote modifications in the structure of the stratum corneum. Differential scanning calorimetry results indicated that the solution containing the organic silicon compound and the gel with urea showed the best hydrating effects on the stratum corneum.

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.

Aspectos clínico-patológicos e laboratoriais do envenenamento crotálico experimental em bovinos / Clinical and pathological aspects of experimental Crotalus poisoning in cattle

Reproduziu-se experimentalmente o envenenamento crotálico, através da inoculação, por via subcutânea, do veneno de Crotalus durissus terrificus (cascavel sul-americana) em dez bovinos mestiços. Dois animais foram utilizados como controle. O bovino que recebeu dose de 0,03mg/kg de peso corporal, morreu 7h40min após a inoculação. A dose de 0,015mg/kg causou a morte em quatro de sete bovinos inoculados, enquanto os dois animais que receberam 0,0075mg/kg adoeceram discretamente e se recuperaram. Os sintomas tiveram início entre 1h30min e 13h45min após a inoculação. A evolução oscilou entre 5h25min e 45h para os animais que morreram e entre 33h15min e 17 dias entre os animais que se recuperaram. Os principais sinais nervosos observados foram diminuição da resposta aos estímulos externos, reflexos hipotônicos, arrastar dos cascos no solo, aparente apatia, paralisia do globo ocular e da língua, decúbito esternal e lateral. Verificaram-se também adipsia e, por vezes, petéquias nas mucosas vaginal e conjuntival. Houve discreto a moderado aumento do tempo de sangramento e moderado aumento do tempo de tromboplastina parcial ativada. Houve moderada leucocitose com neutrofilia, linfopenia relativa, eosinopenia, monocitose e discreto aumento do número de bastões. Foi evidenciado significativo aumento dos níveis séricos de creatinaquinase, contudo, não foram observadas alterações significativas através da urinálise. À necropsia constataram-se edema quase imperceptível no local da inoculação, discretas petéquias e sufusões no epicárdio, omento, vesícula biliar e mucosa da bexiga em alguns dos animais envenenados experimentalmente. Os exames histopatológicos revelaram necrose (hialinização) de grupos de miócitos ou em miócitos isolados em dez diferentes músculos esqueléticos examinados, próximos ou distantes do local de inoculação em todos os animais necropsiados. Concluí-se que o envenenamento por Crotalus Sul-americanas em bovinos não cursa com mioglobinúria...

Crotalus poisoning was experimentally reproduced by subcutaneous inoculation of Crotalus durissus terrificus (South American rattlesnake) venom into 10 clinically healthy mixed bred 12 to 36-month-old cattle, weighing 125 to 449 kg. Two animals were used as controls. The animal that received a dose of 0.03mg/kg body weight died 7h40min after inoculation. A 0.015mg/kg dose provoked death in 4 out of 7 young oxen. Two animals given 0.0075mg/kg became slightly sick and recovered. Onset of symptoms occurred from 1h30min to 13h45min after inoculation. The clinical course varied from 5h25min to 45h for animals that died, and from 33h15min to 17 days for animals that recovered. The main nervous signs observed were diminished response to external stimuli, hypotonic reflexes, dragging of the hooves, apathy, difficulties in moving around obstacles, ocular globe paralysis, lateral and sternal decubitus, and tongue paralysis. Adipsia and sometimes petechiae in the conjunctival and vaginal mucosa were observed. A slight to moderate increase in bleeding time was noted in 6 animals, and a moderate increase in partial thromboplastin time was found in 7 others. Moderate leukocytosis with neutrophilia, relative lymphopenia, eosinopenia, and monocytosis was found. There was a significant increase in creatine kinase serum levels of a ten-fold order. No significant alterations were revealed by urinalysis. Necropsy revealed minimal edema at the inoculation site, few petechiae and equimoses in the epicardium, omentum, biliary vesicle and bladder mucosa of some animals. Histopathological examination revealed necrosis (hyalinization) of groups or isolated myocytes in different muscles examined, both near and far from the inoculation site, in all animals. The diagnosis of Crotalus poisoning and its differentiation from diseases causing paralysis and muscular necrosis in cattle in Brazil are discussed.

Young ovine death during hyperimmunization: crotalic envenomation or copper toxicosis?

The unfavorable evolution of a young ovine during hyperimmunization process with Crotalus durissus terrificus venom was investigated in order to differentiate its origin between ophidic envenomation and copper toxicosis. Clinical, laboratory, necroscopic and histological exams as well as evaluation and measurement of heavy metals (copper) in the kidneys and in the liver were carried out. Blood counts revealed anemia and serological tests showed high levels of blood urea nitrogen, creatinine, aspartate aminotransferase, creatine phosphokinase, total bilirubin and indirect bilirubin; which indicates liver, kidney and skeletal muscle damages. At necropsy, the animal presented hepatopathy and nephropathy. Histological examination revealed renal and hepatic features that may imply copper intoxication. Copper levels were 237.8 µg/g in the liver and 51.2 µg/g in the kidneys. Although the amount of metal found in both organs was below the level that can cause death, according to the literature, anatomopathological signs were suggestive of copper intoxication. Therefore, the hypothesis of metal toxicosis during the hyperimmunization process became more consistent than the crotalic envenomation one.(AU)

Frequency of rhabdiasid nematodes in wild Crotalus durissus terrificus (serpentes, viperidae) from Botucatu region, São Paulo state, Brazil

The aim of the present study was to evaluate the frequency of rhabdiasid nematodes in recently captured Crotalus durissus terrificus snakes from São Paulo State, Brazil. Fifty snakes (34 males and 16 females) were studied and each one was evaluated for the presence of that nematode at the moment of receipt at the Institution and after 90 days of quarantine inside individual cages. Tracheopulmonary washeswere examined. Snakes that died during quarantine underwent necropsy and lung examination. Analysis of the results obtained at the two evaluation times (0 and 90 days), in addition to the data obtained during necropsies, showed that 44 percent (18 males and 4 females) of the C. d. terrificus snakes were naturally infected by rhabdiasid nematodes. These data demonstrate the parasitism level in natural conditions and are important for the sanitary handling of these reptiles in captivity.

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 ...

Clinical and hematological alterations in dogs during experimental envenomation with Crotalus durissus terrificus venom and treated with antiophidic serum

The present work aimed to evaluate the clinical and hematological aspects during experimental envenomation by Crotalus durissus terrificus in dogs treated with different antiophidic serum doses. Sixteen dogs were divided into two groups of eight animals each. Group I received 1mg/kg venom subcutaneously and 30mg antiophidic serum intravenously; Group II received 1mg/kg venom subcutaneously and 60mg antiophidic serum intravenously. In the clinical evaluation, we observed ataxia, moderate sedation, dilated pupils, sialorrhea, flaccid paralysis of mandibular muscles, and discreet edema at the site of venom inoculation. Evaluating red and white blood cells, we observed a decrease of hemoglobins, globular volume and erythrocytes, and an increase of plasmatic proteins, leukocytes, neutrophils, monocytes and lymphocytes. Clotting time increased and there was blood incoagulability with return to normal clotting time six hours after antiophidic serum administration. Animals treated with six antiophidic serum flasks had a faster recovery than the animals that received three serum flasks