Isolation and functional characterization of proinflammatory acidic phospholipase A(2) from Bothrops leucurus snake venom

In the present study, an acidic PLA(2), designated BI-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000 Da and pl was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9 U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-alpha, IL-1 beta and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA2 induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism. (C) 2011 Elsevier Inc. All rights reserved.

Antimicrobial activity of an L-amino acid oxidase isolated from Bothrops leucurus snake venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Bothrops leucurus venom induces nephrotoxicity in the isolated perfused kidney and cultured renal tubular epithelia

Bites from snake (Bothrops genus) cause local tissue damage and systemic complications, which include alterations such as hemostatic system and acute renal failure (ARF). Recent studies suggest that ARF pathogenesis in snakebite envenomation is multifactorial and involves hemodynamic disturbances, immunologic reactions and direct nephrotoxicity. The aim of the work was to investigate the effects of the Bothrops leucurus venom (BlV) in the renal perfusion system and in cultured renal tubular cells of the type MDCK (Madin-Darby Canine kidney). BlV (10 μg/mL) reduced the perfusion pressure at 90 and 120 min. The renal vascular resistance (RVR) decreased at 120 min of perfusion. The effect on urinary flow (UF) and glomerular filtration rate (GFR) started 30 min after BlV infusion, was transient and returned to normal at 120 min of perfusion. It was also observed a decrease on percentual tubular transport of sodium (%TNa+) at 120 min and of chloride (%TCl-) at 60 and 90 min. The treatment with BlV caused decrease in cell viability to the lowest concentration tested with an IC50 of 1.25 μg/mL. Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by necrosis. However, a cell death process may involve apoptosis in lower concentrations. BlV treatment (1.25 μg/mL) led to significant depolarization of the mitochondrial membrane potential and, indeed, we found an increase in the expression of cell death genes in the lower concentrations tested. The venom also evoked an increase in the cytosolic Ca2+ in a concentration dependent manner, indicating that Ca2+ may participate in the venom of B. leucurus effect. The characterization of the effects in the isolated kidney and renal tubular cells gives strong evidences that the acute renal failure induced by this venom is a result of the direct nephrotoxicity which may involve the cell death mechanism. © 2012.

Purificação e caracterização parcial de proteínas presentes no veneno de Bothrops leucurus

Pós-graduação em Microbiologia - IBILCE

Is rainfall seasonality important for reproductive strategies in viviparous Neotropical pit vipers? A case study with Bothrops leucurus from the Brazilian Atlantic Forest

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

L-Aminoacid oxidase from bothrops leucurus venom Induces nephrotoxicity via apoptosis and necrosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Accidents caused by Bothrops and Bothropoides in the State of Paraiba: epidemiological and clinical aspects

INTRODUCTION: Bothrops and Bothropoides snakes cause 70% of the ophidic accidents in Brazil. The species that cause ophidic accidents in State of Paraíba are Bothropoides erythromelas, Bothrops leucurus and Bothropoides neuwiedi. METHODS: This is a prospective and transverse study, following a quantitative approach of accidents involving Bothrops and Bothropoides admitted to the Toxicological Assistance and Information Centers of Campina Grande and João Pessoa (Ceatox-CG and Ceatox-JP), aimed at identifying the epidemiological and clinical profile of such accidents. All of the patients admitted had medical diagnoses and were monitored at Ceatox-CG or Ceatox-JP. RESULTS: The genera Bothrops and Bothropoides caused 91.7% of the ophidic accidents reported. Snake bites were frequent in men (75.1%), rural workers (65.1%), literate individuals (69%) between 11 and 20 years-old (21.7%), and toes the most common area attacked (52.7%). Most (86.6%) patients were admitted within 6 hours after the accident/bite, with a predominance of mild cases (64.6%). The annual occurrence in Paraíba was 5.5 accidents/100,000 inhabitants and lethality was 0.2%. CONCLUSIONS: Positive changes in the profiles of these accidents were verified, such as the non-application of inadequate solutions, including the use of tourniquet, coffee grounds, garlic, suction and/or cutting the bitten area. Moreover, the Itinerant Laboratory project, linked to Paraíba State University in partnership with Ceatox-CG, has contributed positively, providing several cities of the state with information regarding the prevention of accidents involving venomous animals. The local press has also contributed, reporting the educational work developed by the centers.

Biologia reprodutiva de três espécies de serpentes da Família Viperiade da região neotropical

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

BthMP: a new weakly hemorrhagic metalloproteinase from Bothrops moojeni snake venom

In this work, a new weakly hemorrhagic metalloproteinase (BthMP) was purified from Bothrops moojeni snake venom. This enzyme was homogeneous by native and SDS-PAGE. It showed a polypeptide chain of 23.5 kDa, pI=7.1, and N-terminal blocked. BthMP is comprised of high proteolytic activity on casein, fibrin and bovine fibrinogen, with no coagulating, esterase or phospholipase A(2) activities; it was inhibited by EDTA, EGTA and 1,10-phenanthroline and maintained its activity on pH from 7.0 to 9.0 and temperature from 5-40 degrees C. Assays with metal ions showed that Ca(2+) is an activator, whereas Zn(2+) and Hg(2+) inhibited about 50 and 80% of its activity, respectively. The edema evidenced the important role of the toxin in the inflammatory activity of the venom. BthMP also caused unclotting, and provoked histological alterations in the gastrocnemius muscle of mice inducing hemorrhage, necrosis and leukocytic infiltrate. The molecular mass and the inhibition assays suggest that the metal loproteinase BthMP belongs to class P-I of SVMPs. (c) 2008 Elsevier Ltd. All rights reserved.

Two related thrombin-like enzymes present in Bothrops atrox venom

This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical Km on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80% homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii.

An Isoflavone From Dipteryx Alata Vogel Is Active Against The In Vitro Neuromuscular Paralysis Of Bothrops Jararacussu Snake Venom And Bothropstoxin I, And Prevents Venom-induced Myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 μg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 μg/mL) caused irreversible paralysis. Preincubation of TM (200 μg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

Neutralisation Of The Pharmacological Activities Of Bothrops Alternatus Venom By Anti-pla2 Iggs.

Basic phospholipases A2 (PLA2) are toxic and induce a wide spectrum of pharmacological effects, although the acidic enzyme types are not lethal or cause low lethality. Therefore, it is challenging to elucidate the mechanism of action of acidic phospholipases. This study used the acidic non-toxic Ba SpII RP4 PLA2 from Bothrops alternatus as an antigen to develop anti-PLA2 IgG antibodies in rabbits and used in vivo assays to examine the changes in crude venom when pre-incubated with these antibodies. Using Ouchterlony and western blot analyses on B. alternatus venom, we examined the specificity and sensitivity of phospholipase A2 recognition by the specific antibodies (anti-PLA2 IgG). Neutralisation assays using a non-toxic PLA2 antigen revealed unexpected results. The (indirect) haemolytic activity of whole venom was completely inhibited, and all catalytically active phospholipases A2 were blocked. Myotoxicity and lethality were reduced when the crude venom was pre-incubated with anti-PLA2 immunoglobulins. CK levels in the skeletal muscle were significantly reduced at 6 h, and the muscular damage was more significant at this time-point compared to 3 and 12 h. When four times the LD50 was used (224 μg), half the animals treated with the venom-anti PLA2 IgG mixture survived after 48 h. All assays performed with the specific antibodies revealed that Ba SpII RP4 PLA2 had a synergistic effect on whole-venom toxicity. IgG antibodies against the venom of the Argentinean species B. alternatus represent a valuable tool for elucidation of the roles of acidic PLA2 that appear to have purely digestive roles and for further studies on immunotherapy and snake envenoming in affected areas in Argentina and Brazil.

Rat Atrial Responses To Bothrops Jararacussu (jararacuçu) Snake Venom.

Envenoming by the pitviper Bothrops jararacussu produces cardiovascular alterations, including coagulopathy, systemic hemorrhage, hypotension, circulatory shock and renal failure. In this work, we examined the activity of this venom in rat isolated right atria. Incubation with venom (0.025, 0.05, 0.1 and 0.2mg/ml) caused concentration-dependent muscle contracture that was not reversed by washing. Muscle damage was seen histologically and confirmed by quantification of creatine kinase-MB (CK-MB) release. Heating and preincubation of venom with p-bromophenacyl bromide (a phospholipase A2 inhibitor) abolished the venom-induced contracture and muscle damage. In contrast, indomethacin, a non-selective inhibitor of cyclooxygenase, and verapamil, a voltage-gated Ca(2+) channel blocker, did not affect the responses to venom. Preincubation of venom with Bothrops or Bothrops/Crotalus antivenom or the addition of antivenom soon after venom attenuated the venom-induced changes in atrial function and tissue damage. These results indicate that B. jararacussu venom adversely affected rat atrial contractile activity and muscle organization through the action of venom PLA2; these venom-induced alterations were attenuated by antivenom.

Neuromuscular Activity Of Bothrops Fonsecai Snake Venom In Vertebrate Preparations.

The neuromuscular activity of venom from Bothrops fonsecai, a lancehead endemic to southeastern Brazil, was investigated. Chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND) preparations were used for myographic recordings and mouse diaphragm muscle was used for membrane resting potential (RP) and miniature end-plate potential (MEPP) recordings. Creatine kinase release and muscle damage were also assessed. In CBC, venom (40, 80 and 160μg/ml) produced concentration- and time-dependent neuromuscular blockade (50% blockade in 85±9 min and 73±8 min with 80 and 160μg/ml, respectively) and attenuated the contractures to 110μM ACh (78-100% inhibition) and 40mM KCl (45-90% inhibition). The venom-induced decrease in twitch-tension in curarized, directly-stimulated preparations was similar to that in indirectly stimulated preparations. Venom (100 and 200μg/ml) also caused blockade in PND preparations (50% blockade in 94±13 min and 49±8 min with 100 and 200μg/ml, respectively) but did not alter the RP or MEPP amplitude. In CBC, venom caused creatine kinase release and myonecrosis. The venom-induced decrease in twitch-tension and in the contractures to ACh and K(+) were abolished by preincubating venom with commercial antivenom. These findings indicate that Bothrops fonsecai venom interferes with neuromuscular transmission essentially through postsynaptic muscle damage that affects responses to ACh and KCl. These actions are effectively prevented by commercial antivenom.

Disarray Of Glomerular And Tubular Cell Adhesion Molecules In The Course Of Experimental Bothrops Moojeni Envenomation.

In this study, we show that administration of Bothrops moojeni venom in rats induces a general disturbance in the distribution and content of the tight junctional protein ZO-1, the cell-matrix receptor beta 1 integrin, the cytoskeletal proteins, vinculin and F-actin, and of the extracellular matrix component laminin in renal corpuscles and cortical nephron tubules. These findings suggest that cell-cell and cell-matrix adhesion proteins may be molecular targets in the B. moojeni-induced kidney injury.