Acidente crotálico seguido por primeiro episódio psicótico: um folclore clínico / Rattlesnake bite accident followed by first psychotic episode: a clinical folklore / Accidente crotálico seguido de primer episodio psicótico: un folclore clínico

Introdução: Acidentes ofídicos são doenças negligenciadas e constituem uma parcela importante da morbidade de pessoas em idade produtiva que vivem em zonas rurais. A maior parte dos seus efeitos a curto prazo é amplamente conhecida, especialmente aqueles de natureza clínica; no entanto, ainda se observa lacuna importante do conhecimento das consequências a longo prazo de tais agravos, notadamente as de ordem psíquica. Este artigo relata um caso de adoecimento mental subsequente a um acidente crotálico e gera reflexões de âmbito cultural e fisiopatológico a respeito das sequelas de tais eventos. Apresentação do caso: Trata-se de adolescente residente no interior baiano que foi vítima de mordedura por cascavel e teve necessidade de hospitalização em unidade de terapia intensiva. Observou-se que, mesmo após melhora clínica, iniciou com sintomas psicóticos prodrômicos e progrediu para piora mental grave, que culminou em internação psiquiátrica e diagnóstico de esquizofrenia no decorrer dos meses seguintes. Conclusões: Nota-se, neste caso, correlação direta entre esses dois eventos; mas, em razão da escassez de trabalhos científicos que abordem tais questões, depreende-se que é preciso investigar e estudar com maior profundidade possíveis associações entre acidentes crotálicos e psicoses.

Introduction: Snakebites are neglected diseases and constitute an important part of the morbidity of working-age people who live in rural areas. Most of their short-term effects are widely known, especially those of a clinical nature; however, there is still an important gap in the knowledge of the long-term consequences of such injuries, notably those of a psychotic nature. This article aims to report a case of mental illness subsequent to a rattlesnake bite accident and generate cultural and pathophysiological reflections regarding the consequences of such events. Case presentation: An adolescent residing in the interior of the state of Bahia was bitten by a rattlesnake and required hospitalization in an intensive care unit. It was observed that even after clinical improvement, the case started with prodromal psychotic symptoms and progressed to severe mental deterioration that culminated in psychiatric hospitalization and diagnosis of schizophrenia over the following months. Conclusions: In this case, there was a direct correlation between these two events, but because of the scarcity of scientific works that address such issues, it is necessary to investigate and study in greater depth possible associations between snakebite accidents and psychoses.

Introducción: Las mordeduras de serpientes son enfermedades desatendidas y constituyen una parte importante de la morbilidad de las personas en edad laboral que viven en zonas rurales. La mayoría de sus efectos a corto plazo son ampliamente conocidos, especialmente los de carácter clínico; sin embargo, todavía existe un importante vacío en el conocimiento de las consecuencias a largo plazo de este tipo de lesiones, en particular las de carácter psíquico. Este artículo tiene como objetivo informar un caso de enfermedad mental posterior a un accidente crotálico y generar reflexiones culturales y fisiopatológicas sobre las consecuencias de tales eventos. Presentación del caso: Se trata de un adolescente residente en el interior de Bahía que fue mordido por una serpiente cascabel y requirió hospitalización en unidad de cuidados intensivos. Se observó que, aún después de la mejoría clínica, comenzó con síntomas psicóticos prodrómicos y progresó a un deterioro mental severo que culminó con hospitalización psiquiátrica y diagnóstico de esquizofrenia en los meses siguientes. Conclusiones: En este caso, existe una correlación directa entre estos dos eventos pero, debido a la escasez de trabajos científicos que aborden tales cuestiones, parece necesario investigar y estudiar con mayor profundidad posibles asociaciones entre accidentes crotálicos y psicosis.

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.

Antileishmanial effects of γCdcPLI, a phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum, on Leishmania (Leishmania) amazonensis

BACKGROUND Leishmaniasis, a neglected disease caused by the parasite Leishmania, is treated with drugs associated with high toxicity and limited efficacy, in addition to constant reports of the emergence of resistant parasites. In this context, snake serums emerge as good candidates since they are natural sources with the potential to yield novel drugs. OBJECTIVES We aimed to show the antileishmanial effects of γCdcPLI, a phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum, against Leishmania (Leishmania) amazonensis. METHODS Promastigotes forms were exposed to γCdcPLI, and we assessed the parasite viability and cell cycle, as well as invasion and proliferation assays. FINDINGS Despite the low cytotoxicity effect on macrophages, our data indicate that γCdcPLI has a direct effect on parasites promoting an arrest in the G1 phase and reduction in the G2/M phase at the highest dose tested. Moreover, this PLA2 inhibitor reduced the parasite infectivity when promastigotes were pre-treated. Also, we demonstrated that the γCdcPLI treatment modulated the host cell environment impairing early and late steps of the parasitism. MAIN CONCLUSIONS γCdcPLI is an interesting tool for the discovery of new essential targets on the parasite, as well as an alternative compound to improve the effectiveness of the leishmaniasis treatment.

Early-Onset Respiratory Muscle Paralysis in Crotalic Envenomation: A Case Study

ABSTRACT Crotalic envenomation is responsible for approximately 8%-13% of ophidism cases in Brazil, yet it is associated with the highest mortality among snakes. We describe the case of a patient bitten by a rattlesnake who developed ventilatory muscle paralysis within hours after envenomation. While diaphragmatic paralysis is a rare late neurotoxic event following crotalic envenomation, in this case, paralysis occurred early but was rapidly reversed after antivenom administration. This report discusses potential contributing factors based on a comprehensive literature review.

Oxidative stress and changes in hematological and biochemical biomarkers in Crotalus durissus terrificus (South American rattlesnake) venom-induced acute kidney injury in rats / Estrés oxidativo y alteraciones en biomarcadores hematológicos y bioquímicos en lesión renal aguda inducida por el veneno de Crotalus durissus terrificus (cascabel de America del Sur) en ratones

Abstract Acute kidney injury (AKI) is the major cause of mortality following bites by the South American rattlesnake Crotalus durissus terrificus. We investigated the early onset of Crotalus durissus terrificus venom-induced AKI in rats within 2 h of venom injection and its attenuation by antivenom. Several biomarkers were used to monitor AKI in the absence or presence of antivenom. Male Wistar rats were divided into five groups (n=5 each): G1, rats injected with saline (control); G2, rats injected with venom (6 mg kg-1, intraperitoneally) and euthanized after 2 h to evaluate AKI; G3 and G4, rats injected with 0.9% sterile saline or antivenom 2 h after venom, respectively, and monitored until death or up to 24 h post-venom, and G5, rats injected with antivenom alone and monitored for 24 h. Blood, urine and renal tissue samples were collected immediately after death to assess oxidative stress, hematological and biochemical alterations, and renal histological damage. Venom caused AKI within 2 h (G2) that persisted for up to 8.2 ± 1.6 h (G3), as confirmed by increases in blood urea, creatinine, and renal proteinuria; these increases were attenuated by antivenom. There were no changes in blood protein concentrations in G2 and G3, whereas there were increases in blood reduced glutathione, glutathione peroxidase, and plasma TBARS (but not in catalase) that were attenuated to varying extents by antivenom. There were no marked changes in platelets or leukocytes, but an increase in erythrocytes after 8.2 h with venom alone was attenuated by antivenom. Renal glomerular and tubular damage was greatest after 2 h post-venom groups alone was attenuated by antivenom. Renal glomerular and tubular damage was greatest after 2 h post-venom and declined thereafter. Venom caused early-onset AKI, with variable effects on lipid peroxidation and oxidative stress. Antivenom attenuated the AKI, as shown by the decrease in blood urea and the normalization of proteinuria, without protecting against lipid peroxidation.

Resumen La injuria o lesión renal aguda (LRA) es la mayor causa de mortalidad debido a las mordeduras por cascabeles Crotalus durissus terrificus. Se estudió la instalación precoz de LRA, en ratas, inducida por el veneno de Crotalus durissus terrificus después de 2 h de su inoculación y la atenuación por el antiveneno. Se utilizaron diversos biomarcadores para monitorear LRA en ausencia o presencia del antiveneno. Ratas Wistar machos fueron divididos en 5 grupos (n=5 por grupo): G1, ratas inoculadas con solución salina (control); G2, ratas inoculadas con veneno (6 mg kg-1 dosis, vía intraperitoneal), y sacrificadas después de 2 h para evaluar LRA; G3 y G4, ratas inoculadas con 0.9% de solución salina esterilizada o antiveneno luego de 2 h después de inoculado el veneno, respectivamente, y monitoreadas hasta su muerte o hasta 24 h después de inoculado el veneno; y G5, ratas inoculadas con antiveneno solo y monitoreadas durante 24 h. Las muestras de sangre, orina, y tejido renal fueron colectadas inmediatamente después de la muerte de los animales para evaluar estrés oxidativo, alteraciones hematológicas y bioquímicas, y daño histológico renal. El veneno causó LRA dentro de las 2 h (G2) persistiendo durante más de 8,2 ± 1,6 h (G3), estando esto confirmado por el incremento de urea sanguínea, creatinina, y proteinuria renal; estos aumentos disminuyeron con la aplicación del antiveneno. No se observaron alteraciones en las concentraciones de proteínas sanguíneas en G2 y G3, mientras que se encontraron incrementos en glutatión reducido sanguíneo, glutatión peroxidasa y TBARS plasmática (pero no en catalasa), que disminuyeron con la aplicación del antiveneno aunque en diferente grado. No ocurrieron alteraciones marcadas de plaquetas o leucocitos, mientras que el aumento de glóbulos rojos observado luego de 8,2 h de la inoculación con veneno, disminuyó con el antiveneno. El daño renal glomerular y tubular fue más importante luego de 2 h de la inoculación con veneno y posteriormente disminuyó. El veneno causó LRA precoz a las 2 h, con efectos variables sobre la peroxidación lipídica y el estrés oxidativo. El antiveneno redujo el daño renal, conforme lo demostrado por la disminución en la urea sanguínea y por la normalización de la proteinuria, aunque no se observó protección contra la peroxidación lipídica.

A new approach of immunotherapy against Crotalus snakes envenoming: ostrich (Struthio camelus) egg yolk antibodies (IgY-technology) / Un nuevo enfoque de inmunoterapia contra el envenenamiento de serpientes Crotalus: anticuerpos de yema de huevo de avestruz (Struthio camelus) (tecnología IgY)

Abstract Crotalid envenomation is a neglected collective health problem involving many countries in America, which need secure and inexpensive snake anti-venom treatments. Here, high antibody titers (IgY) were raised in the Ostrich (Struthio camelus) egg yolk by immunizing with the venom of Venezuelan venomous Crotalus snakes. Ostriches were immunized with a pool of venoms from common rattlesnake (Crotalus durissus cumanensis), Uracoan rattlesnake (Crotalus vegrandis), Guayana rattlesnake (Crotalus durissus ruruima) and black rattlesnake (Crotalus pifanorum). The anti-snake venom antibodies were prepared from egg yolk by the water dilution method, enriched by the addition of caprylic acid (CA) and precipitation with ammonium sulfate at 30% (W/V). The purity and molecular mass of the final product was satisfactory, yielding a single ∼ 175 kDa band in SDS-PAGE gels ran under non-reducing conditions. In the immunoblot analysis, specific binding of the antivenom was observed with most venom proteins. The LD50 was 16.5 g/mouse (825 μg/kg body weight). High titers of IgY against Crot/pool venom were shown by ELISA. The median effective dose (ED50) was 19.66 mg/2LD50. IgY antibodies neutralized efficiently the Crot/pool venom lethality. As far as we know, this is the first anti-snake venom produced in ostriches, which could make this technology an affordable alternative for low-income countries, since it is likely to produce manteniabout 2-4 g of IgY per ostrich egg. Hence, almost 400 g of IgY can be purified from only one ostrich during a year. In addition, there are enormous differences in the cost of investment in the maintenance of horses, from the points of view of infrastructure, feeding and veterinary care, in which the cost can reach USD 100 per animal per day, compared to a maintenance cost of USD 146 per month per producing bird. These results are encouraging and could easily be extrapolated to the manufacturing of other antivenoms and antitoxins as well, as they could be applied to the manufacturing of potential diagnostic tools.

Resumen El envenenamiento por crotálidos es un problema de salud colectiva desatendido, que involucra a muchos países del continente americano, los cuales necesitan tratamientos seguros y económicos. En este trabajo, se obtuvieron títulos altos de anticuerpos (IgY) producidos en yema de huevo de avestruz (Struthio camelus) mediante la inmunización con el veneno de serpientes venezolanas del genero Crotalus. Se inmunizaron avestruces con una colección de veneno de serpientes de cascabel común (Crotalus durissus cumanensis), cascabel de Uracoa (Crotalus vegrandis), cascabel de Guayana (Crotalus durissus ruruima) y cascabel negra (Crotalus pifanorum). Los anticuerpos anti-veneno de serpiente se prepararon a partir de yema de huevo por el método de dilución en agua, enriquecidos mediante la adición de ácido caprílico (CA), seguido de una precipitación con sulfato de amonio al 30% (P/V). La pureza y masa molecular de los anticuerpos (IgY) se definieron mediante ensayos de SDS-PAGE nativos y las masas moleculares se establecieron electroforéticamente, obteniéndose una única banda de IgY de ∼ 175 kDa. El análisis de inmunotransferencia mostró la unión específica del antiveneno con la mayoría de las proteínas del veneno. La DL50 fue de 16,5 μg/ratón (825 μg / kg de peso corporal); Se mostraron títulos altos de IgY contra el veneno de Crot / pool mediante ELISA. La dosis mediana efectiva (DE50) fue de 19,66 mg/2 LD50. Los anticuerpos IgY neutralizaron eficazmente la letalidad del veneno de Crot / pool. Hasta donde sabemos, se trata del primer antídoto de serpiente producido en avestruces, lo que podría abaratar la producción de este tratamiento en países del tercer mundo. Ya que es probable que se obtengan alrededor de 2-4 g de IgY por huevo de avestruz. Por lo tanto, se podrían purificar casi 400 g de IgY de un solo avestruz durante un año. Asimismo, debido a las enormes diferencias en el costo de inversión en el mantenimiento de los caballos desde el punto de vista de infraestructura, alimentación y atención veterinaria, en los que el costo puede llegar a los 100 USD por día, frente a los 146 USD por mes de mantenimiento de la producción de aves. Estos resultados abren un campo terapéutico, para la fabricación de otros antivenenos contra un amplio espectro de toxinas y también como probables herramientas de diagnóstico.

Antineoplastic properties and pharmacological applications of Crotalus durissus terrificus snake venom

ABSTRACT Snake toxins are widely studied owing to their importance in snakebite accidents, a serious public health issue in tropical countries, and their broad therapeutic potential. Isolated fractions from venom produced by snakes of the genus Crotalus sp. present a wide variety of pharmacological uses such as antifungal, antiviral, antibacterial, and antitumor properties, among other therapeutic potentialities. Given the direct effect of this venom on tumor cells, isolation of its compounds is important for the characterization of its anticarcinogenic actions. Crotalus durissus terrificus venom and its toxins have been widely evaluated as potential candidates for the development of new antineoplastic therapies that are efficient against different tumor lines and cellular targets. This review highlights the venom toxins of this species, with a focus on their antineoplastic properties.

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

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

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

A novel activity on thymocytes cells exerted by the rattlesnake (Crotalus durissus cumanensis) venom / Nueva actividad de los timocitos estimulada por el veneno de la serpiente de cascabel (Crotalus durissus cumanensis)

Abstract. Introduction: The thymus is active mainly during the neonatal and pre-adolescent periods. Objective: To test naïve thymocytes proliferation and monocytes stimulation. Materials and methods: We collected fresh thymus tissue from neonate mice after surgery. Suspension cells were coated onto Ficoll-Hypaque support. The obtained cells (thymocytes) were cultured measuring the proliferation of naïve T cells stimulated by Crotalus durissus cumanensis (Cdc) venom at sub-lethal doses (20 ng). Then, we supplemented the wells with AlamarBlue™ and incubated them for 5 h to test their proliferation. Mononuclear cells from mice peripheral blood were collected and layered onto the support of the Ficoll-Hypaque solution. We added the thymocytes actively dividing (25 x 105 cells) from cultures stimulated with Cdc venom at 20 ng/well to cultured monocytes freshly obtained from the Ficoll-Hypaque separation. Both cell populations were incubated for 36 h until monocytes matured to macrophages. Results: The naïve thymocytes rapidly proliferated after stimulation with the Cdc venom (NTCdc) and these successively induced the maturation and function of monocytes progenitor cells to mature macrophages, which ingested Chinese ink. Conclusions: The naïve thymocytes proliferated by stimulation with the Cdc venom and subsequently the NT/Cdc induced the rapid maturation and function of monocytes progenitor cells becoming mature macrophages with their phenotypic characteristics.

Resumen. Introducción. El timo es activo principalmente durante los períodos neonatal y preadolescente. Objetivo. Probar la proliferación de los timocitos tempranos y la estimulación de monocitos que producen. Materiales y métodos. Se recogió tejido de timo fresco después de la cirugía de ratones recién nacidos. La suspensión de células se colocó sobre un soporte de Ficoll-Hypaque. Las células obtenidas (timocitos) se cultivaron y se midió la proliferación de células T vírgenes estimuladas por el veneno de Crotalus durissus cumanensis (Cdc) en dosis subletales (20 ng). A continuación, se agregó AlamarBlue™ a los pocillos y se incubaron durante 5 horas para evaluar la proliferación. Se recogieron células mononucleares de sangre periférica de ratones y se colocaron sobre un soporte de solución de Ficoll-Hypaque. Los timocitos que se dividieron activamente (25 x 105 células) a partir de los cultivos estimulados con veneno de Cdc (20 ng/pocillo) y se agregaron a los cultivos de monocitos recién obtenidos de la separación en la solución de Ficoll-Hypaque. Ambas poblaciones celulares se incubaron durante 36 horas hasta que los monocitos maduraron a macrófagos. Resultados Los timocitos tempranos experimentaron una rápida proliferación estimulada por el veneno de Cdc (NTCdc) y, posteriormente, indujeron la maduración y la función de las células progenitoras de monocitos, los cuales maduraron a macrófagos, que se tiñeron con tinta china. Conclusiones. Los timocitos tempranos proliferaron con la estimulación del veneno de Cdc y, posteriormente, el NT/Cdc indujo la maduración rápida y la función de las células progenitoras de monocitos, transformándose en macrófagos con sus características fenotípicas.

Analysis of the protein profile of the venoms of snakes Bothrops asper, Bothrocophias myersi and Crotalus durissus from the Colombian Andean Region obtained by RP-HPLC / Análisis del perfil proteico de los venenos de Bothrops asper, Bothrocophias myersi y Crotalus durissus serpientes de la Región Andina en Colombia obtenidos por RP-HPLC

ABSTRACT Snake venoms comprise a highly complex mixture of proteins, and there is also a high interspecific and intraspecific variability in their composition, even in the same region. Our aim was to compare the composition of the venoms of Bothrocophias myersi, Crotalus durissus, and Bothrops asper, snakes from the Colombian Andean region by Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC). The venoms were given to the research group under an agreement with Fundación Zoológica de Cali. The venoms pool was obtained by manual extraction, lyophilized and frozen. The venom protein was quantified by direct measurement with Nanodrop® 280 nm. The protein composition was established by RP-HPLC, using a Lichosper 100 RP, C18 column (250X4 mm) with a pore size of 5-m, as well as by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The highest quantity of protein was found in the venom of B. myersi (108.6 mg/ mL) followed by C. durissus (78.1 mg/mL) and B. asper (74.1 mg/mL). All venoms showed bands of 15 and 50 KDa by using SDS-PAGE. B. myersi venom chromatogram exhibited 16 peaks by RP-HPLC. We conclude that the composition of the three venoms is quite similar, being phospholipase A2 the common protein therein, and together with metalloproteinases they were the most abundant protein families in the venom of B. myersi. SDS-PAGE and RP-HPLC techniques allow a first approach to the profile of the venoms, which in turn could clarify the clinical syndrome produced.

RESUMEN Los venenos de las serpientes comprenden una mezcla compleja de proteínas, y existe una alta variabilidad interespecífica e intra-específica en su composición, incluso en la misma región. Nuestro objetivo fue comparar la composición de los venenos de Bothrocophias myersi, Crotalus durissus y Bothrops asper de la región andina de Colombia, mediante cromatografía líquida de alta eficiencia en fase reversa (RP-HPLC). Los venenos fueron entregados al grupo de investigación mediante un convenio con la Fundación Zoológica de Cali. El pool de venenos fue obtenido por extracción manual, liofilizado y congelado. La proteína de los venenos fue cuantificada por Absorbancia 280nm por medición directa con Nanodrop®. La composición proteica se estableció por RP-HPLC, utilizando una columna Lichosper 100 RP, C18 (250X4 mm) con un tamaño de poro de 5-jm, así como por electroforesis en gel dodecil sulfato de sodio-poliacrilamida (SDS-PAGE). La mayor cantidad de proteínas se encontró en el veneno de B. myersi (108.6 mg/mL), seguido de C. durissus (78.1 mg/mL) y B. asper (74.1 mg/mL). Todos los venenos mostraron bandas de 15 y 50 KDa por SDS-PAGE. El cromatograma de B. myersi exhibió 16 picos por RP-HPLC. Concluimos que la composición de los tres venenos es bastante similar, siendo la fosfolipasa A2 la proteína común en estos y junto con las metaloproteinasas fueron las familias de proteínas más abundantes en el veneno de B. myersi. Las técnicas de SDS-PAGE y el RP-HPLC permiten un primer acercamiento al perfil de los venenos, lo que a su vez podría contribuir a esclarecer el síndrome clínico producido.

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.

Qualitative and quantitative ultrastructural analysis of the mitochondria from adrenal gland cortex under the action of viperidae family snake venoms / Análisis ultrastructural cualitativo y cuantitativo de la mitocondria de la glándula adrenal bajo la acción de los venenos de serpientes de la familia viperidae

SUMMARY: The Viperidae venoms are composed of a mixture of constituents with enzymatic and non-enzymatic actions, which act on ultrastructural components of cells and tissues. Here, the number of mitochondria, mitochondrial area and the number of mitochondrial cristae from adrenal glands cortex treated with snake venoms were tested after 3, 6 and 24 hours of venom injections. The mitochondria quantitative changes showed a statistically significant decrease, in the number of mitochondria past 3, 6 and 24 h. There was an increase in the mitochondrial area after 6 h, where Crotalus vegrandis venom did not present significant differences with Crotalus pifanorum or Bothrops venezuelensis venoms. After 24 h, there was an escalation of mitochondrial area in all tested venoms. The number of mitochondrial cristae after 3 h did not present important differences with the control treatment. After 6 h, the number of mitochondrial cristae initiated to decrease under the activities of the 3 venoms action, until 24 h of observation. In the qualitative observations it was possible to witness an intense damage of the mitochondria, with loss and swelling of membranes, disappearance of cristae and the appearance of myelin figures, which started at 3 h after the Crotalus and Bothrops venoms injections. These damages probably were due to cytotoxic effects of phospholipases, metalloproteases and/or other proteolytic activities present in Viperidae snake venoms, being more evident in Crotalus venoms. As far as we know, these results define a novel finding that suggest that Viperidae snake venoms are extremely toxic to mammalian mitochondria.

RESUMEN: Los venenos de Viperidae tienen acciones enzimáticas y no enzimáticas, que actúan sobre la estructura celular. Aquí se probaron, a las 3, 6 y 24 horas de la inyección del veneno, el número de mitocondrias, el área mitocondrial y el número de crestas mitocondriales de la corteza de las glándulas adrenales. Los cambios cuantitativos de las mitocondrias mostraron una disminución en el número de mitocondrias a las 3, 6 y 24 h. Hubo un aumento en el área mitocondrial a las 6 h, donde el veneno de la serpiente Crotalus vegrandis no presentó diferencias significativas con los venenos de Crotalus pifanorum o Bothrops venezuelensis. Después de 24 h, hubo un aumento del área mitocondrial en todos los venenos. El número de crestas mitocondriales a las 3 h no presentó alteraciones o diferencias importantes con el tratamiento de control. Después de 6 h, el número de crestas mitocondriales comenzó a disminuir bajo la acción de los 3 venenos, hasta las 24 h de observación. En las observaciones cualitativas se observó un daño intenso de las mitocondrias, con pérdida y edema de las membranas, desaparición de las cristae y aparición de figuras mielínicas, que comenzó a las 3 h después de las inyecciones de veneno de Crotalus y Bothrops. Estos daños se debieron factiblemente a los efectos citotóxicos de componentes proteolíticos de los venenos. Creemos que estos resultados definen un nuevo y original hallazgo, que sugiere que los venenos de serpiente Viperidae son extremadamente tóxicos para las mitocondrias de mamíferos.

Geographic variation of individual venom profile of Crotalus durissus snakes

South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.(AU)

Local and systemic effects caused by Crotalus durissus terrificus, Crotalus durissus collilineatus, and Crotalus durissus cascavella snake venoms in swiss mice

Abstract INTRODUCTION: Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation. METHODS: Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated. RESULTS: The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP. CONCLUSIONS: Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms.

Antivenomic approach of different Crotalus durissus collilineatus venoms

Our group has previously performed a proteomic study verifying that individual variations can occur among Crotalus durissus collilineatus venoms. These variations may lead to differences in venom toxicity and may result in lack of neutralization of some components by antivenom. In this way, this study aimed to evaluate the Brazilian anticrotalic serum capacity in recognizing twenty-two Crotalus durissus collilineatus venoms, as well as their fractions. Methods: The indirect enzyme-linked immunosorbent assay (ELISA) was chosen to evaluate the efficacy of heterologous anticrotalic serum produced by Instituto Butantan (Brazil) in recognizing the twenty-two Crotalus durissus collilineatus venoms and the pool of them. Moreover, the venom pool was fractionated using reversed-phase fast protein liquid chromatography (RP-FPLC) and the obtained fractions were analyzed concerning antivenom recognition. Results: Evaluation of venom variability by ELISA showed that all venom samples were recognized by the Brazilian anticrotalic antivenom. However, some particular venom fractions were poorly recognized. Conclusion: This study demonstrated that the Brazilian anticrotalic serum recognizes all the different twenty-two venoms of C. d. collilineatus and their fractions, although in a quantitatively different way, which may impact the effectiveness of the antivenom therapy. These results confirm the need to use a pool of venoms with the greatest possible variability in the preparation of antivenoms, in order to improve their effectiveness.(AU)

Immunotherapeutic potential of Crotoxin: anti-inflammatory and immunosuppressive properties

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

Cell migration inhibition activity of a non-RGD disintegrin from Crotalus durissus collilineatus venom

In recent decades, snake venom disintegrins have received special attention due to their potential use in anticancer therapy. Disintegrins are small and cysteine-rich proteins present in snake venoms and can interact with specific integrins to inhibit their activities in cell-cell and cell-ECM interactions. These molecules, known to inhibit platelet aggregation, are also capable of interacting with certain cancer-related integrins, and may interfere in important processes involved in carcinogenesis. Therefore, disintegrin from Crotalus durissus collilineatus venom was isolated, structurally characterized and evaluated for its toxicity and ability to interfere with cell proliferation and migration in MDA-MB-231, a human breast cancer cell line. Methods: Based on previous studies, disintegrin was isolated by FPLC, through two chromatographic steps, both on reversed phase C-18 columns. The isolated disintegrin was structurally characterized by Tris-TricineSDS-PAGE, mass spectrometry and N-terminal sequencing. For the functional assays, MTT and wound-healing assays were performed in order to investigate cytotoxicity and effect on cell migration in vitro, respectively. Results: Disintegrin presented a molecular mass of 7287.4 Da and its amino acid sequence shared similarity with the disintegrin domain of P-II metalloproteases. Using functional assays, the disintegrin showed low cytotoxicity (15% and 17%, at 3 and 6 µg/mL, respectively) after 24 h of incubation and in the wound-healing assay, the disintegrin (3 µg/mL) was able to significantly inhibit cell migration (24%, p < 0.05), compared to negative control. Conclusion: Thus, our results demonstrate that non-RGD disintegrin from C. d. collilineatus induces low cytotoxicity and inhibits migration of human breast cancer cells. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved in an important integrin family that highlights molecular aspects of tumorigenesis. Also, non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anticancer drugs.(AU)

Preclinical efficacy against toxic activities of medically relevant Bothrops sp. (Serpentes: Viperidae) snake venoms by a polyspecific antivenom produced in Mexico / Eficacia de un antiveneno poliespecífico producido en México para neutralizar, a nivel preclínico, las actividades tóxicas de venenos de Bothrops sp (Serpentes: Viperidae) de importancia médica

Abstract:The assessment of the preclinical neutralizing ability of antivenoms in Latin America is necessary to determine their scope of efficacy. This study was aimed at analyzing the neutralizing efficacy of a polyspecific bothropic-crotalic antivenom manufactured by BIRMEX in Mexico against lethal, hemorrhagic, defibrinogenating and in vitro coagulant activities of the venoms of Bothrops jararaca (Brazil), B. atrox (Perú and Colombia), B. diporus (Argentina), B. mattogrossensis (Bolivia), and B. asper (Costa Rica). Standard laboratory tests to determine these activities were used. In agreement with previous studies with bothropic antivenoms in Latin America, a pattern of cross-neutralization of heterologous venoms was observed. However, the antivenom had low neutralizing potency against defibrinogenating effect of the venoms of B. atrox (Colombia) and B. asper (Costa Rica), and failed to neutralize the in vitro coagulant activity of the venom of B. asper (Costa Rica) at the highest antivenom/venom ratio tested. It is concluded that, with the exception of coagulant and defibrinogenating activities of B. asper (Costa Rica) venom, this antivenom neutralizes toxic effects of various Bothrops sp venoms. Future studies are necessary to assess the efficacy of this antivenom against other viperid venoms. Rev. Biol. Trop. 65 (1): 345-350. Epub 2017 March 01.

ResumenEs necesario estudiar a nivel preclínico la capacidad neutralizante de los antivenenos producidos en América Latina, para conocer su espectro de cobertura. En este estudio se analizó la eficacia preclínica de un antiveneno poliespecífico botrópico-crotálico producido por BIRMEX, en México, para neutralizar los efectos letal, hemorrágico, desfibrinogenante y coagulante in vitro de los venenos de Bothrops jararaca (Brasil), B. atrox (Perú y Colombia), B. diporus (Argentina), B. mattogrossensis (Bolivia) y B. asper (Costa Rica). Se emplearon metodologías de laboratorio estándar en los análisis. En consonancia con estudios anteriores con diversos antivenenos botrópicos en América Latina, se observó un amplio patrón de neutralización de estos venenos heterólogos en la mayoría de los efectos estudiados. Sin embargo, el antiveneno mostró una baja capacidad neutralizante contra el efecto desfibrinogenante de los venenos de B. atrox (Colombia) y B. asper (Costa Rica) y no neutralizó la actividad coagulante in vitro del veneno de B. asper (Costa Rica) a la máxima razón antiveneno/ veneno empleada.

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)

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

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