Imunomodulação por frações de veneno de Bothrops alternatus em células mononucleares de sangue periférico

Os adjuvantes são utilizados para melhorar a imunogenicidade de antígenos de uma vacina, de forma a auxiliar a retenção dos mesmos no organismo e/ou melhorar sua rota de entrega. Atualmente, os adjuvantes com uso predominante e com licença de uso são os sais de alumínio. Os venenos de serpentes são fontes ricas de proteínas e moléculas com potenciais tóxicos, mas também farmacológicos. A Bothrops alternatus, popularmente conhecida como Urutu-Cruzeiro, é uma serpente peçonhenta da família Viperidae que está distribuída em países da América do Sul e no Sul e Sudeste do território brasileiro. A ação inflamatória do veneno botrópico é causada por conjuntos de frações com especificidades diversas e substâncias heterogêneas. Essas frações induzem a liberação de substâncias complexas do processo inflamatório. O objetivo do presente estudo foi avaliar o efeito imunomodulador de frações de veneno de B. alternatus em culturas de células mononucleares do sangue periférico (PBMC) humano. Estudando as citocinas liberadas por essas células e isolando as frações que tiverem melhor função pró-inflamatória poderá ser possível avançar em estudos de possíveis novos adjuvantes. Foram realizados: Fracionamento do veneno por cromatografia de exclusão molecular; Dosagem protéica do veneno por método de BCA; Eletroforese em gel de poliacrilamida; Cultura de PBMC estimulada com as frações; Análise de viabilidade celular por ensaio de MTT; Dosagens de citocinas e quimiocinas por ensaio de CBA (Cytometric Bead Array). O fracionamento resultou em 10 frações, cujos perfis foram avaliados em gel de poliacrilamida 12,5%, corados com prata. Foram realizadas culturas de PBMC, que foram estimuladas com as frações de veneno em concentrações de 5 e 10 µg/mL. Com exceção da fração 3, as demais frações não apresentaram efeito citotóxico, avaliado pelo método de MTT. As frações 8, 9 e 10 induziram aumento da produção de TNF, IL-10, IL-6, IL-1β e IL-8 em relação aos controles negativos. Ao avançar com os estudos, talvez seja possível purificar tais frações em busca de uma molécula que possa ser utilizada como um possível novo adjuvante.

Platelet aggregation inhibitors from Bothrops alternatus snake venom.

Snake venoms are a complex mixture of proteins and peptides that can activate/inhibit platelet aggregation. Bothrops alternatus venom include three main families: metalloproteinases (SVMPs), serinoproteinases (SVSPs) and phospholipases A2 (PLA2s), among other minor components. In this work, we used inhibitor cocktails (containing Na2-EDTA, PMSF and/or pBPB) to investigate the effect of these three families and of baltergin (a PIII SVMP) on platelet aggregation by a turbidmetric method using a microplate reader. Cocktails 1 (active SVMPs) and 2 (active PLA2s) significantly reduced aggregation induced by ristocetin and collagen and by collagen and thrombin, respectively. Cocktail 3 (active SVSPs) showed a mild activation of aggregation, indicating the content of thrombin-like enzymes (TLEs) in this venom is low. Cocktail 4 (active minor components) displayed inhibitory effect with all agonists assayed (ristocetin, ADP, collagen and thrombin) but at higher IC50 values. Baltergin exhibited inhibitory effect when the catalytic domain was active for ristocetin-stimulated platelet aggregation and showed a non-enzymatic mechanism of inhibition when collagen was used as agonist. It was not able to disaggregate platelet thrombus. We conclude that B. alternatus venom is a source of natural inhibitors of platelet aggregation due to the action of SVMPs and PLA2s. Other minor components such as C-type lectins likely contribute to the antiplatelet effect. The interest in knowing the action of venom components on platelet function lies both in the understanding of the pathophysiology of snake bite envenomation and in their biotechnological application.

Photobiomodulation therapy on local effects induced by juvenile and adult venoms of Bothrops alternatus.

Bothrops snake envenomation is characterized by severe local manifestations such as pain, edema, inflammation, hemorrhage, and myonecrosis. Furthermore, it is described that venom from juvenile and adult snakes may have differences in their composition that can lead to differences in the evolution of the clinical manifestation of the victim. Photobiomodulation (PBM) has been shown to be an effective adjuvant therapy to serum therapy to reduce the local effects induced by bothropic snake venom. This study evaluated the effect of PBM on the local reaction, after Bothrops alternatus snake venom (BaV) injection, in its juvenile (BaJV) and adult (BaAV) stages. Balb/C mice were injected with the juvenile or adult venoms of BaV or saline solution (control group). PBM at a wavelength of 660 nm, 100 mW, 0.33 W/cm2, 40 s, and a 0.028 cm2 beam was applied transcutaneous to a single point with a radiant exposure of 4 J/cm2, 30 min after venom injection. Edema, inflammatory infiltrate, hyperalgesia, and myonecrosis were analyzed. Both venoms induced significant edema and myonecrosis in the gastrocnemius muscle. Hyperalgesia in the mice paw and a prominent leukocyte infiltrate into the peritoneum were also observed. PBM significantly reduced all evaluated parameters. In conclusion, PBM treatment was effective in reducing the local effects induced by B. alternatus venom at different stages of snake development and could be a useful tool as an adjuvant treatment for bothropic envenomation.

Effects of cilostazol, a Phosphodiesterase-3 inhibitor, on kidney function and redox imbalance in acute kidney injury caused by Bothrops alternatus venom.

The mechanisms of pathogenesis of acute kidney injury (AKI) in snakebites is multifactorial and involves hemodynamic disturbances, with release of free radical causing cytotoxic effects. The phosphodiesterase-3 (PDE3) inhibitor, Cilostazol, has been reported to provide protection against renal oxidative stress. OBJECTIVE: We evaluated the protective effects of cilostazol against Bothrops alternatus snake venom (BaV)-induced nephrotoxicity. METHODS: Wistar rat kidneys (n = 6, 260-300 g) were isolated and perfused with Krebs-Henseleit solution containing 6 g/100 mL of bovine serum albumin. After 30 min, the kidneys were perfused with BaV to a final concentration of 1 and 3 µg/mL, and subsequently evaluated for perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and percentage of electrolyte tubular sodium and chloride transport (%TNa+, %TCl-). Oxidative stress and renal histological analyses were performed. RESULTS: BaV caused a reduction in all the evaluated renal parameters (PP, RVR, GFR, UF, %TNa+, and %TCl-). Although only the effects on PP and UF were reversed with cilostazol treatment, the decrease in the malondialdehyde levels, without changes in glutathione levels, further reduced the venom-induced renal tissue changes. CONCLUSION: Our data suggest that PDE3 is involved in BaV-induced nephrotoxicity, as cilostazol administration significantly ameliorated these effects.

Traces of Bothrops snake venoms in necrotic muscle preclude myotube formation in vitro.

Deficient skeletal muscle regeneration, which often leads to permanent sequelae, is a common clinical finding in envenomations caused by snakes of the family Viperidae, such as those of Bothrops alternatus and B. diporus in South America. The causes of such poor muscle regenerative outcome are still incompletely understood. Using a murine experimental model of envenomation by the venoms of these two species, we assessed whether traces of venom components that remain in muscle tissue days after envenomation affect myoblasts and myotube formation in culture. The kinetics of drop in venom concentration in the tissue was assessed by ELISA and Western blot, and by the quantification of venom phospholipase A2 activity. A rapid drop of venom components was observed in muscle, although a band of 58-63 kDa remained even 168 h after venom injection, and venom phospholipase A2 activity was detected in muscle tissue days after envenomation. Muscle homogenates from envenomated animals were cytotoxic to myoblasts in culture and inhibited the formation of myotubes even in conditions where homogenates were devoid of cytotoxicity. These deleterious effects were abrogated when homogenates were incubated with antivenom. Our findings agree with previous observations with the venom of Bothrops asper and provide further evidence that one of the causes of the poor skeletal muscle regeneration after Bothrops sp venom-induced myonecrosis is the deleterious action on myogenic cells of traces of venom components remaining in the tissue.

Baltetin: a new C-type lectin-like isolated from Bothrops alternatus snake venom which act as a platelet aggregation inhibiting.

C-type lectin-like proteins found in snake venom, known as snaclecs, have important effects on hemostasis through targeting membrane receptors, coagulation factors and other hemostatic proteins. Here, we present the isolation and functional characterization of a snaclec isolated from Bothrops alternatus venom, designated as Baltetin. We purified the protein in three chromatographic steps (anion-exchange, affinity and reversed-phase chromatography). Baltetin is a dimeric snaclec that is approximately 15 and 25 kDa under reducing and non-reducing conditions, respectively, as estimated by SDS-PAGE. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry and Edman degradation sequencing revealed that Baltetin is a heterodimer. The first 40 amino acid residues of the N-terminal region of Baltetin subunits share a high degree of sequence identity with other snaclecs. Baltetin had a specific, dose-dependent inhibitory effect on epinephrine-induced platelet aggregation in human platelet-rich plasma, inhibiting up to 69% of platelet aggregation. Analysis of the infrared spectra suggested that the interaction between Baltetin and platelets can be attributed to the formation of hydrogen bonds between the PO32- groups in the protein and PO2- groups in the platelet membrane. This interaction may lead to membrane lipid peroxidation, which prevents epinephrine from binding to its receptor. The present work suggests that Baltetin, a new C-type lectin-like protein isolated from B. alternatus venom, is the first snaclec to inhibit epinephrine-induced platelet aggregation. This could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders.

Variaciones de caracteres morfológicos dentro de la especie Bothrops alternatus / Morphological characters variations within the Bothrops alternatus species

Resumen El género Bothrops (yarará) es responsable de más del 95% de los accidentes por ofidios venenosos en Argentina. La mayoría de éstos son notificados en provincias del Nordeste y Noroeste. Bothrops alternatus, es la especie más frecuentementehallada, siendo el vipérido de gran talla más meridional del mundo y una de las especies de mayor importancia sanitaria en Argentina, pudiendo hallarse en relación a núcleos urbanos. Presentan en el dorso figuras arriñonadas características, que remedan atubos de teléfono o porotos unidos, completos o incompletos, hasta la forma fusionada. Presenta foseta loreal, órgano termore-ceptor ubicado entre el ojo y la fosa nasal. Su dentición es solenoglifa, dientes inoculadores huecos, con conducto cerrado, fijosa un hueso maxilar móvil. Si bien se describen variaciones entre animales, se considera una especie monotípica. Su identificaciónes importante dada la abundancia de especímenes y de hallazgos en regiones periurbanas.

Abstract The Bothrops genus is responsible for more than 95% of ophidian accidents in Argentina, most of these are reported in the Northeast and Northwest provinces. Bothrops alternatus, is the most frequent species of the genus Bothrops (yarará), themost southern large-scale viperid in the world and one of the most important species in Argentina, being able to be found in rela-tion to urban nucleus. Their characteristic features are kidney-like figures in the skin, that mimic phone tubes or beans, completeor incomplete, until the merged form. It presents a loreal pit, thermoreceptor organ located between the eye and the nostril. Itsdentition is solenoglyphous, hollow inoculating teeth, with closed duct, fixed to a mobile maxillary bone. Although variationsbetween animals are described, it is considered a monotypic species. The identification of this snake is important due their abun-dance in around urban regions and its frequency of finding.

Antiophidic activity of the secondary metabolite lupeol isolated from Zanthoxylum monogynum.

Previous studies have demonstrated the potential antiophidic activity of Zanthoxylum monogynum A.St.-Hil. a tree from the Rutaceae family native to South America. In this present contribution, we demonstrate the activity of the metabolite lupeol, a triterpenoid isolated from the stem bark of Z. monogynum against the harmful effects of the Bothrops alternatus venom. We investigated the antiophidic properties of lupeol, for this purpose, and use crude venom (Pb) incubated with lupeol in different concentrations, testing in vitro experiments and inoculated in mice for inhibitory evaluations in vivo. Besides, we tried to elucidate through the molecular dynamics the mechanism of action of lupeol with the bothropic thrombin-like toxin Jararacussin-I; the acidic phospholipase A2 toxin BthA-I from Bothrops jararacussu and the metalloproteinase toxin BmooMP-I from Bothrops moojeni. In our results, we demonstrated the potential inhibitory effect upon coagulant, phospholipasic and myotoxic activities of the bothropic venom, previously incubated with lupeol. We found that lupeol triterpenoid was able to partially inhibit local and systemic damage caused by snake venom toxins. Our in silico results demonstrate that lupeol is capable of interacting and altering the activity of the thrombin-like toxin Jararacussin-I, and capable of interacting with the BthA-I acidic PLA2, both toxins present in Bothrops snakes venom, thus demonstrating the pharmacological potential of this compound for the treatment of bothropic accidents.

Hemostatic evaluation of rabbits envenomed with Bothrops alternatus treated with anti-bothropic serum, desmopressin and tranexamin acid / Avaliação hemostática de coelhos envenenados com Bothrops alternatus e tratados com soro antibotrópico, desmopressina e ácido tranexâmico

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly composed of proteolytic enzymes. Although the antibothropic serum produced by the Brazilian Institutes is remarkably efficient, more studies are necessary, especially in veterinary medicine. The venom contain enzymes and non-enzymatic proteins that interfere with hemostasis leading to hemorrhage or even thrombosis. Possible treatment associations with known bothropic antivenom were the reason for the development of the present study. The aim of this study was to evaluate hemostasis alterations caused by Bothrops alternatus venom in rabbits followed by treatments with anti-bothropic serum, tranexamic acid and desmopressin. Twenty New Zealand rabbits were distributed into five groups (n=4) that were experimentally envenomed with 150mcg/kg of B. alternatus venom via intramuscular injection and treated as follow: Group 1 (G1) was the positive control and received venom and PBS/BSA; Group 2 (G2) was treated with tranexamic acid; Group 3 (G3) with desmopressin; Group 4 (G4) with tranexamic acid and anti-bothropic serum; and Group 5 (G5) with anti-bothropic serum and desmopressin. Blood samples were collected before venom administration, and one, four, eight and 12 hours after, for Partial activated partial thromboplastin time, Prothrombin Time, Thrombin Time and fibrinogen evaluation. Thrombin generation (TG) test was carried out with a pool of samples from final times (8 and 12h). At the end of 12h, all animals were euthanized and necropsy was conducted. Samples from muscle tissue, heart, lungs and kidney were analyzed. Classic coagulation tests showed no significant differences amongst groups and times. However, TG indicated that the venom causes a hypocoagulability state, which was not reversed by proposed treatments. Histology showed muscle inflammation, hemorrhage and necrosis, as well as hemorrhage in other tissues with no differences amongst groups. B. alternatus envenomation causes hypocoagulability detected by TG assay, but not through classical coagulation tests. The use of tranexamic acid and desmopressin for hemostasis stabilization after inoculation of the venom did not show advantage in coagulation restoration.(AU)

No Brasil, as serpentes do gênero Bothrops são responsáveis por milhares de acidentes, e seus venenos são compostos principalmente de enzimas proteolíticas. Embora o soro antiofídico produzido pelos institutos brasileiros seja notavelmente eficiente, mais estudos são necessários, especialmente na medicina veterinária. O veneno contem enzimas e proteínas não-enzimáticas que interferem com a hemostasia levando a hemorragias ou trombose. A associação de outros tratamentos ao soro antibotrópico foi a razão para o desenvolvimento do presente estudo. O objetivo deste estudo foi avaliar as alterações da hemostasia causadas pelo veneno de Bothrops alternatus em coelhos, após tratamento com soro antibotrópico, ácido tranexâmico e desmopressina. Vinte coelhos da Nova Zelândia foram distribuídos em cinco grupos (n = 4) que foram submetidos a experimentos com 150mcg/kg de veneno de B. alternatus por injeção intramuscular. O Grupo 1 (G1) foi o controle positivo e recebeu veneno e PBS / BSA, enquanto o Grupo 2 (G2) foi tratado com ácido tranexâmico, o Grupo 3 (G3) com desmopressina, o Grupo 4 (G4) com ácido tranexâmico e soro antibotrópico, e o Grupo 5 (G5) com soro antibotrópico e desmopressina. As amostras de sangue foram coletadas antes da administração do veneno, e uma, quatro, oito e 12 horas após os tratamentos para realização de tempo de tromboplastina parcial ativada parcial (TTPa), tempo de protrombina (TP), tempo de trombina (TT) e mensuração de fibrinogênio. Para o ensaio de geração de trombina (TG) foi realizado com um pool de amostras nos tempos finais (8 e 12h). Ao final das 12h, todos os animais foram sacrificados e a necropsia foi realizada. Amostras de tecido muscular, coração, pulmões e rins foram analisadas. Os testes TTPa, TP, TT e fibrinogênio não mostraram diferenças significativas entre os grupos e os tempos. No entanto, o TG indicou que o veneno causa um estado de hipocoagulabilidade, que não foi revertido pelos tratamentos propostos. Na histologia, foram observadas inflamação muscular, hemorragia e necrose, além de hemorragia em outros tecidos, sem diferenças entre os grupos. O envenenamento por B. alternatus causa hipocoagulabilidade detectada mais precocemente pelo teste de geração de trombina. O uso de ácido tranexâmico e desmopressina para estabilização da hemostasia após a inoculação do veneno não mostrou vantagem na restauração da coagulação.(AU)

Bothrops alternatus Snake Venom Induces Cytokine Expression and Oxidative Stress on Renal Function.

BACKGROUND: Envenomation caused by Bothrops alternatus is common in Southern Brazil. Acute Kidney Injury occurs after Bothrops snakebite and more information is necessaryrequired to understand its mechanism. OBJECTIVE: The objective was to evaluate the effect of Bothrops alternatus venom (BaV) on renal cells and rat isolated kidney function. METHODS: Wistar rats (n = 6, weighing 260-320 g) were perfused with a Krebs-Henseleit solution containing 6 g 100 mL-1 of bovine serum albumin. After 30 minutes, the kidneys were perfused with BaV to a final concentration of 1 and 3 µgmL-1; and subsequently were evaluated for Perfusion Pressure (PP), Renal Vascular Resistance (RVR), Urinary Flow (UF), Glomerular Filtration Rate (GFR), and percentage of electrolyte tubular transport. Renal histological analysis, cytokine release, oxidative stress and cytotoxicity in renal proximal tubular cells were assessed. RESULTS: BaV reduced PP, RVR, GFR, UF, total and proximal sodium transport (%TNa+), and chloride (%TCl-) in the isolated kidney perfusion model. Histological analysis of perfused kidneys disclosed the presence of proteinaceous material in the glomeruli and renal tubules, vacuolar tubular epithelial cell degeneration, Bowman's capsule degeneration, swelling of glomerular epithelial cells, glomerular atrophy and degeneration, and the presence of intratubular protein. Cytokine release (TNF-α, IL-1ß, IL-10) and oxidative stress were increased in the kidneys. The viability of LLC-MK2 cells (IC50: 221.3 µg/mL) was decreased by BaV and necrosis was involved in cell death. CONCLUSION: These findings indicate that BaV modifies functional parameters in an isolated perfused kidney model and has cytotoxic effects on renal lineage cells.

First report on BaltCRP, a cysteine-rich secretory protein (CRISP) from Bothrops alternatus venom: Effects on potassium channels and inflammatory processes.

CRISPs represent a family of cysteine-rich secretory proteins with molecular mass between 20 and 30 kDa and a highly conserved specific pattern of 16 cysteine residues. In this work, we isolated and characterized a novel CRISP from Bothrops alternatus venom, named BaltCRP, also evaluating its effects on different isoforms of potassium channels (Kv1.1; Kv1.2; Kv1.3; Kv1.4; Kv1.5; Kv2.1; Kv10.1 and Shaker) and on inflammatory processes in vivo. This toxin has a molecular mass of 24.4 kDa and pI around 7.8. Electrophysiological experiments using voltage clamp techniques showed that BaltCRP can affect the currents of Kv1.1; Kv1.3; Kv2.1 and Shaker channels. In addition, BaltCRP induced inflammatory responses characterized by an increase of leukocytes in the peritoneal cavity of mice, also stimulating the production of mediators such IL-6, IL-1ß, IL-10, PGE2, PGD2, LTB4 and CysLTs. Altogether, these results demonstrated that BaltCRP can help understand the biological effects evoked by snake venom CRISPs, which could eventually lead to the development of new molecules with therapeutic potential.

Venoms and Isolated Toxins from Snakes of Medical Impact in the Northeast Argentina: State of the Art. Potential Pharmacological Applications.

Among the ophidians that inhabit the Northeast of Argentina, the genus Bothrops such as B. alternatus and B. diporus species (also known as yararás) and Crotalus durisus terrificus (named cascabel), represent the most studied snake venom for more than thirty years. These two genera of venomous snakes account for the majority of poisonous snake envenomations and therefore, constitute a medical emergency in this region. This review presents a broad description of the compiled knowledge about venomous snakebite: its pathophysiological action, protein composition, isolated toxins, toxin synergism, toxin-antitoxin cross-reaction assays. Properties of some isolated toxins support a potential pharmacological application.

IgY-based antivenom against Bothrops alternatus: Production and neutralization efficacy.

Antivenom for the treatment of bothropic snakebite is a priority for public health institutions from Latin America. An alternative to the conventional antivenom production is based on the use of egg yolk antibodies - IgY-technology - by immunizing laying hens. In this study, we produced, characterized and assessed the efficacy of IgY-based antivenoms against B. alternatus venom. Immunochemical studies (reactivity, avidity and antigen recognition pattern) as well as antivenom efficacy assays were performed. After the 3rd immunization, levels of specific IgY reached a maximum that was maintained throughout the observation period, while avidity indexes of the extracts increased after the successive immunizations. Furthermore, IgY against B. alternatus recognized protein complexes of the venom with high (>40 kDa), medium (20-40 kDa) and low (<20 kDa) molecular weights. IgY antivenoms obtained after 8 immunizations neutralized 35.65 µg of B. alternatus venom per mg of antivenom, while specific activities values ranged from 0.28 to 0.42. In conclusion, we produced and characterized IgY antivenoms capable of neutralizing the lethal activity of B. alternatus venom at a preclinical level. Thus, IgY-technology may allow the production of effective and affordable antivenoms fulfilling the urgent needs of many countries where conventional manufacture is unable to provide enough availability of antivenoms.

Análise da variação sexual e ontogenética de venenos da espécie Bothrops alternatus Duméril, Bibron & Duméril, 1854. (Serpentes, Viperidae)

Snakes are complex mixtures whose main functions are the stoppage and the killing of prey as well as the initial promotion of digestion and their actions may vary according to the age, geographical distribution and individual character of each snake. The venom of Bothrops alternatus, as well as most of the botrópico venoms, has as main actions, proteolytic, coagulant and hemorrhagic activities. The objective of this study was to evaluate the possible variations of venoms of male and female snakes of the Bothrops alternatus species throughout the development, through biochemical characterization (protein dosage, SDS-PAGE electrophoresis, gelatinolytic activity, caseinolytic activity and coagulant activity) and biological characterization (hemorrhagic and myotoxic activity). As a result, protein content of male and female venoms did not show ontogenetic or sexual variation, with 90 to 100% protein content. The electrophoretic profiles of the venoms were similar, but a protein with an approximate molecular weight of 17.0 kDa varied between the sexes and as the snakes developed. By means of the SDS-PAGE zimography and caseinolytic activity, it was possible to observe that the proteolytic action of the female venoms was not very active, with action only in the third and fourth year, whereas the male venoms showed activity only in the first year of life. The B. alternatus venoms had the ability to coagulate citrated equine plasma in 60 seconds and venoms of both sexes were more coagulant in the first year of life, suffering a decline in action throughout development. In the hemorrhagic activity the venom samples presented sexual variation, and the ontogenetic variation was only observed in the venoms of males, where they were more active in the fourth year of age. The myotoxic activity of B. alternatus venoms showed that there is sexual variation, whereas ontogenetic variation was only identified in the venom of males, with oscillations of the specific activity values for the CK enzyme. By means of the joint analysis of the data generated, we conclude that there is sexual and ontogenetic variation in venoms of the species B. alternatus and that several factors can influence in the triggering of their actions and activities, since environmental and genetic factors are the key to the maintenance species in the habitat in which they live.

Os venenos ofídicos são misturas complexas e tem como funções principais a paralisação e o abate da presa, bem como a promoção inicial da digestão e suas ações podem variar de acordo com a idade, a distribuição geográfica e o caráter individual de cada serpente. O veneno de Bothrops alternatus, assim como a maioria dos venenos botrópicos, tem como principais ações, as atividades proteolítica, coagulante e hemorrágica. O objetivo desse estudo foi avaliar as possíveis variações dos venenos de serpentes machos e fêmeas da espécie Bothrops alternatus ao longo do desenvolvimento, através de caracterização bioquímica (dosagem de proteínas, eletroforese em SDS-PAGE, atividade gelatinolítica, atividade caseinolítica e atividade coagulante) e caracterização biológica (atividade hemorrágica e miotóxica). Como resulados, teor proteico dos venenos de machos e fêmeas não apresentou variação ontogenética ou sexual, com teor entre 90 e 100% de proteína. Os perfis eletroforéticos dos venenos foram semelhantes, porém uma proteína com peso molecular aproximado de 17.0 kDa variou entre os sexos e conforme o desenvolvimento das serpentes. Por meio das técnicas de zimografia em SDS-PAGE e atividade caseinolítica foi possível observar que a ação proteolítica dos venenos de fêmeas apresentou-se pouco ativo, com ação somente no terceiro e quarto ano, enquanto que os venenos de machos mostraram atividade somente no primeiro ano de vida. Os venenos de B. alternatus tiveram a capacidade de coagular o plasma equino citratado em 60 segundos e os venenos de ambos os sexos foram mais coagulantes no primeiro ano de vida, sofrendo um declínio na ação ao longo do desenvolvimento. Na atividade hemorrágica as amostras de venenos apresentaram variação sexual, sendo a variação ontogenética só observada nos venenos de machos, onde foram mais ativos no quarto ano de idade. A atividade miotóxica dos venenos de B. alternatus mostrou que existe variação sexual, enquanto que a variação ontogenética só foi identificada nos venenos de machos, apresentando oscilações dos valores da atividade específica para a enzima CK. Por meio da análise conjunta dos dados gerados, concluímos que existe variação sexual e ontogenética em venenos da espécie B. alternatus e que diversos fatores podem influenciar no desencadear de suas ações e atividades, visto que, fatores ambientais e genéticos são a chave para a manutenção das espécies no habitat em que vivem.

BaltPLA2: A New Phospholipase A2 from Bothrops Alternatus Snake Venom with Antiplatelet Aggregation Activity.

BACKGROUND: In last decades, snake venoms have aroused great interest of the medicine due to the pathophysiological effects caused by their toxins. These include the phospholipases A2, low molecular weight proteins capable of causing haemorrhagic, myotoxic, inflammatory and neurotoxic effects after an ophidian accident. The present work describes the isolation and biochemical characterization of a new PLA2 isolated from the B. alternatus snake venom, which was named BaltPLA2. METHOD: The rapid and efficient purification of this toxin was performed using only two chromatography steps (anion exchange and hydrophobic chromatography). RESULTS: BaltPLA2 is an acidic protein (pI 4.4) with an apparent molecular mass of 17000 (SDSPAGE) and 14074.74 Da (MALDI TOF/TOF). Analysis of fragments ion by MS / MS showed the following internal amino acid sequence SGVIICGEGTPCEK, which did not exhibit homology with other PLA2 from the same venom. BaltPLA2 is a catalytically active, which displayed an anticoagulant action, inhibition of platelet aggregation induced by epinephrine (~ 80%) and ADP (24%). BaltPLA2 also was able to induce myonecrosis and the release of cytokines (IL-10, IL-12 and TNF- α) in macrophages culture. CONCLUSION: The results presented in this work greatly contribute to a better understanding of the mechanism of enzymatic and pharmacological actions of PLA2s from snake venoms and they may contribute to its application in medical research.

Effects of Bothrops alternatus venom in zebrafish: a histopathological study.

Zebrafish is an excellent model organism for studying tissue alterations caused by Bothrops alternatus venom (BAV) and for screening new anti-venom drugs. To study tissue alterations following exposure to BAV and the roles that glucocorticoids play in these tissue reactions, zebrafish were randomly divided into five groups: the free injection control group (FIC), the phosphate-buffered saline injection control group (PIC), the venom injected group (VI), the group treated with dexamethasone 1 h before venom injection (D1hBVI) and the group treated with dexamethasone 1 h after venom injection (D1hAVI). The concentration of BAV injected was 0.13 mg/mL and each fish received an injection of 20 µL. Body weight measurements and histopathological characteristics of the gills, kidneys, liver, and intestine were determined. Histopathological analyses showed necrosis, inflammation and weight gain in animals that received BAV. The histological alteration indices of the gills, liver, kidneys, and intestines were statistically higher in the animal groups treated with BAV. These alteration indices were lower in the D1hBVI and D1hAVI groups compared to the group treated with BAV alone. The D1hBVI group is presented with minor alterations. A significant difference in the histological alterations index was observed in the intestinal tissue of the FIC group compared to the PIC group. Cumulatively, zebrafish may serve as a useful biomarker for alterations induced by BAV. Interestingly, dexamethasone reduced the damage caused by BAV in the organs studied, which suggests that zebrafish might be useful for screening new drugs that can mitigate tissue damage caused by snakebites.

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom.

BACKGROUND: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. METHODS: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. RESULTS: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO32- groups, present in BaltDC, form hydrogen bonds with the PO2- groups present in the non-lipid portion of the membrane platelets. CONCLUSIONS: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.

Apoptosis induced by a snake venom metalloproteinase from Bothrops alternatus venom in C2C12 muscle cells.

In this study, the apoptosis inducing effects of baltergin as well as its influence on cell adhesion and migration on muscles cells in vitro were studied. Morphological analysis made by scanning electron and phase contrast microscopy demonstrated typical futures of programmed cell death, apoptosis. This mechanism was confirmed by fluorescence staining, molecular analysis of endonuclease activity and increased mRNA expression level of two representative genes (p53 and bax). On the other hand, baltergin exert an inhibition effect on myoblast cell adhesion and migration in vitro probably through a mechanism that involves the interaction of this enzyme with cell integrins. In conclusion, our results suggest that the absence of appropriate extracellular matrix contacts triggers anoikis. Therefore, this is the first report that demonstrated the mechanism of programmed cell death triggered by baltergin, a PIII metalloprotease isolated from Bothrops alternatus venom, in a myoblast cell line.

Isolation and characterization of a novel metalloprotease inhibitor from Bothrops alternatus snake serum.

Resistance of snakes and some other animals to snake envenomation has been attributed to soluble factors present in their tissues. Here we report the isolation of a novel metalloprotease inhibitor from Bothrops alternatus snake serum (named BaltMPI) with high purity, using a four-step chromatographic method. BaltMPI has molecular weights of 60.5 and 42.4kDa, as determined by SDS-PAGE and mass spectrometry, respectively, and pI=5.27. The first 60 amino acids from the N-terminal region of BaltMPI, determined by Edman's degradation, showed high homology (97%) with the snake venom metalloprotease inhibitor (SVMPI) BJ46a and other SVMPIs (78-82%). The chromatographic fractions and purified BaltMPI exhibited anti-hemorrhagic activity against Batroxase and BjussuMP-I. BaltMPI was stable over wide ranges of pH (1, 5, 8, and 9) and temperature (-80, -20, 4, 60, and 100°C), and suppressed the fibrinogenolytic, fibrinolytic, and azocaseinolytic activities of Batroxase. BaltMPI specifically inhibited the activity of metalloproteases, without affecting the activity of serine proteases. Together, our results suggest that BaltMPI and other SVMPIs are promising molecules for the treatment of snake envenomation, in particular that caused by Bothrops sp.