Hemodynamic impairment induced by Crotoxin using in vivo and ex vivo approach in a rat model.

Crotalus durissus snakebite represent 10 % of snakebite cases in Brazil, which cardiovascular disorders are associated with severe cases. Considering crotoxin (CTX) as the major venom component, the present study aimed to evaluate the hemodynamic alterations induced by CTX using in vivo and ex vivo approaches in a rat model. In vivo cardiac function parameters were analyzed from anesthetized rats treated with CTX or saline only (Sham), along with serum creatine kinase MB (CK-MB) and lung myeloperoxidase. From the same animals, hearts were isolated and functional parameters evaluated in Langendorff method ex vivo. CTX binding to myoblast cell line in vitro were evaluated using confocal microscopy and flow cytometry. CTX was capable of reducing arterial and diastolic blood pressure, heart rate, along with left ventricle pressure development or decay during systole (LVdP/dtmax and LVdP/dtmin) in vivo, however no differences were found in the ex vivo approach, showing that intrinsic heart function was preserved. In vitro, CTX binding to myoblast cell line was mitigated by hexamethonium, a nicotinic acetylcholine receptor antagonist. The present study has shown that CTX induce hemodynamic failure in rats, which can help improve the clinical management of cardiovascular alterations during Crotalus durissus snakebite.

Immunomodulatory actions and epigenetic alterations induced by proteases from Bothrops snake venoms in human immune cells.

The aim of this study was to evaluate the immunomodulatory effects of two toxins from Bothrops snake venoms (the P-I metalloprotease Batroxase and the thrombin-like serine protease Moojase) on human peripheral blood mononuclear cells (PBMC), also investigating changes in the expression of genes related to epigenetic alterations and their immunotherapeutic potential. After 24 h of PBMC stimulation, Batroxase (2 µg/mL) and Moojase (4 µg/mL) increased some cytokine levels (including IL-6 and IL-10), but did not promote cell death processes (apoptosis/necrosis) or alterations in the global DNA methylation levels. Gene expression experiments (RT-qPCR) showed that most of the genes with altered transcript levels encode enzymes that act on histones, such as acetyltransferases (HAT1), deacetylases (HDACs), methyltransferases (DOT1L) or demethylases (KDM5B), indicating that these toxins may alter gene regulation through epigenetic changes mainly related to histones and to methyl-CpG binding proteins (MECP2). Subsequently, the immunotherapeutic potential of these toxins was evaluated using in vitro cytotoxicity assays with NK cells and K562 leukemic cells. Both toxins were able to potentiate the NK cell cytotoxic effects against K562 tumor cells, and the effect of Batroxase was dependent on the concomitant stimulus with IL-2, whereas Moojase increased the NK cytotoxicity independently of IL-2. Thus, Batroxase and Moojase presented interesting immunomodulatory effects that could be explored for the development of new strategies in anticancer immunotherapies.

Effects of crotoxin, a neurotoxin from Crotalus durissus terrificus snake venom, on human endothelial cells.

Vascular endothelium plays an important modulatory role due to the production of molecules that mediate vasomotricity, inflammation, and leukocyte adhesion and rolling. Here we addressed whether crotoxin (25-200 µg/mL) - the main component of Crotalus durissus terrificus snake venom - interferes with cell viability, apotosis/necrosis, and cell response to oxidative stress in human umbilical vein endothelial cells (HUVEC) in vitro. We also examined whether crotoxin alters the levels of interleukins, adhesion molecules, and endothelial vasoactive factors in HUVEC cells treated or not with lipopolysaccharide (LPS; 1 µg/mL; 24 h). Crotoxin was not cytotoxic towards HUVEC cells, and downregulated the LPS-induced production of adhesion molecules (VCAM-1, ICAM-1, and E-selectin), vasoactive factors (endothelin-1 and prostaglandin I2), and interleukins (IL-6, IL-8, and IL1ß), as well as protected cells against H2O2-induced oxidative stress. Hence, crotoxin played anti-inflammatory, antioxidant, immunomodulating, and vasoactive actions on HUVEC cells, in vitro. Considering that the initial stages of atherosclerosis is characterized by vasoconstriction, increased levels of adhesion molecules, inflammatory cytokines, and oxidative stress in the vascular endothelium; and crotoxin downmodulated all these events, our findings indicate that the actions of crotoxin here demonstrated suggest that it may have an anti-atherogenic action in vivo, which deserves to be tested in future studies.

Crotoxin, a neurotoxin from Crotalus durissus terrificus snake venom, as a potential tool against thrombosis development.

Endothelium plays an important modulatory role due to the synthesis and secretion of molecules that act on hemostasis and fibrinolysis. As there are no literature data about the effect of crotoxin (CTX) - the main component of Crotalus durissus terrificus snake venom - on human endothelial cells, the present study examined how CTX (25-200 µg/mL) affects the endothelial production of the hemostatic factors antithrombin III, protein C, protein S, plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (t-PA), and vWF in human umbilical vein endothelial cells (HUVEC) in vitro. Production of hemostatic factors was assessed in HUVEC cells treated with CTX (25-200 µg/mL) in the presence or absence of lypopolysaccharide (LPS; 1 µg/mL; 24 h). We found that CTX alone did not exert pro- or anticoagulant effect on hemostasis, and pro- or antifibrinolytic effect on fibrinolysis. LPS alone had procoagulant (increased vWF and reduced protein C and S levels) and antifibrinolytic action (reduced t-PA and increased PAI-1 levels). However, CTX exerted anticoagulant and profibrinolytic action in the presence of LPS by lowering the levels of vWF and t-PA and elevating the levels of protein C and PAI-1. Therefore can be used as a potential tool against thrombosis development.

Kinetic investigations and stability studies of two Bothrops L-amino acid oxidases.

BACKGROUND: L-amino acid oxidases isolated from snake venoms (SV-LAAOs) are enzymes that have great therapeutic potential and are currently being investigated as tools for developing new strategies to treat various diseases, including cancer and bacterial infections. The main objective of this study was to make a brief evaluation of the enzymatic stability of two Bothrops LAAOs, one isolated from Bothrops jararacussu (BjussuLAAO-II) and the other from Bothrops moojeni (BmooLAAO-I) venoms. METHODS AND RESULTS: The enzymatic activity and stability of both LAAOs were evaluated by microplate colorimetric assays, for which BjussuLAAO-II and BmooLAAO-I were incubated with different L-amino acid substrates, in the presence of different ions, and at different pH ranges and temperatures. BjussuLAAO-II and BmooLAAO-I demonstrated higher affinity for hydrophobic amino acids, such as Phe and Leu. The two enzymes showed high enzymatic activity in a wide temperature range, from 25 to 75 °C, and presented optimum pH around 7.0. Additionally, Zn2+, Al3+, Cu2+ and Ni2+ ions negatively modulated the enzymatic activity of both LAAOs. As to stability, BjussuLAAO-II and BmooLAAO-I showed high enzymatic activity for 42 days stored at 4 °C in neutral pH solution. Moreover, the glycan portions of both LAAOs were analyzed by capillary electrophoresis, which revealed that BjussuLAAO-II presented two main glycan portions with relative masses of 7.78 and 8.13 CGU, while BmooLAAO-I showed three portions of 7.58, 7.94 and 8.37 CGU. CONCLUSIONS: Our results showed that, when stored properly, BjussuLAAO-II and BmooLAAO-I present enzymatic stability over a long time period, which is very important to allow the use of these enzymes in pharmacological studies of great impact in the medical field.

Cytotoxic and inflammatory potential of a phospholipase A2 from Bothrops jararaca snake venom.

BACKGROUND: Snake venom phospholipases A2 (PLA2s) have been reported to induce myotoxic, neurotoxic, hemolytic, edematogenic, cytotoxic and proinflammatory effects. This work aimed at the isolation and functional characterization of a PLA2 isolated from Bothrops jararaca venom, named BJ-PLA2-I. METHODS AND RESULTS: For its purification, three consecutive chromatographic steps were used (Sephacryl S-200, Source 15Q and Mono Q 5/50 GL). BJ-PLA2-I showed acidic characteristics, with pI~ 4.4 and molecular mass of 14.2 kDa. Sequencing resulted in 60 amino acid residues that showed high similarity to other Bothrops PLA2s, including 100% identity with BJ-PLA2, an Asp49 PLA2 previously isolated from B. jararaca venom. Being an Asp49 PLA2, BJ-PLA2-I showed high catalytic activity, and also inhibitory effects on the ADP-induced platelet aggregation. Its inflammatory characterization showed that BJ-PLA2-I was able to promote leukocyte migration in mice at different concentrations (5, 10 and 20 µg/mL) and also at different response periods (2, 4 and 24 h), mainly by stimulating neutrophil infiltration. Furthermore, increased levels of total proteins, IL-6, IL-1ß and PGE2 were observed in the inflammatory exudate induced by BJ-PLA2-I, while nitric oxide, TNF-α, IL-10 and LTB4 levels were not significantly altered. This toxin was also evaluated for its cytotoxic potential on normal (PBMC) and tumor cell lines (HL-60 and HepG2). Overall, BJ-PLA2-I (2.5-160 µg/mL) promoted low cytotoxicity, with cell viabilities mostly varying between 70 and 80% and significant values obtained for HL-60 and PBMC only at the highest concentrations of the toxin evaluated. CONCLUSIONS: BJ-PLA2-I was characterized as an acidic Asp49 PLA2 that induces acute local inflammation and low cytotoxicity. These results should contribute to elucidate the action mechanisms of snake venom PLA2s.

New Insights on Moojase, a Thrombin-Like Serine Protease from Bothrops moojeni Snake Venom.

Snake venom serine proteases (SVSPs) are enzymes that are capable of interfering in various parts of the blood coagulation cascade, which makes them interesting candidates for the development of new therapeutic drugs. Herein, we isolated and characterized Moojase, a potent coagulant enzyme from Bothrops moojeni snake venom. The toxin was isolated from the crude venom using a two-step chromatographic procedure. Moojase is a glycoprotein with N-linked glycans, molecular mass of 30.3 kDa and acidic character (pI 5.80⁻6.88). Sequencing of Moojase indicated that it is an isoform of Batroxobin. Moojase was able to clot platelet-poor plasma and fibrinogen solutions in a dose-dependent manner, indicating thrombin-like properties. Moojase also rapidly induced the proteolysis of the Aα chains of human fibrinogen, followed by the degradation of the Bß chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC analysis of its fibrinogenolysis confirmed the main generation of fibrinopeptide A. Moojase also induced the fibrinolysis of fibrin clots formed in vitro, and the aggregation of washed platelets, as well as significant amidolytic activity on substrates for thrombin, plasma kallikrein, factor Xia, and factor XIIa. Furthermore, thermofluor analyses and the esterase activity of Moojase demonstrated its very high stability at different pH buffers and temperatures. Thus, studies such as this for Moojase should increase knowledge on SVSPs, allowing their bioprospection as valuable prototypes in the development of new drugs, or as biotechnological tools.

BjSP, a novel serine protease from Bothrops jararaca snake venom that degrades fibrinogen without forming fibrin clots.

Snake venom serine proteases (SVSPs) are commonly described as capable of affecting hemostasis by interacting with several coagulation system components. In this study, we describe the isolation and characterization of BjSP from Bothrops jararaca snake venom, a serine protease with distinctive properties. This enzyme was isolated by three consecutive chromatographic steps and showed acidic character (pI 4.4), molecular mass of 28 kDa and N-carbohydrate content around 10%. Its partial amino acid sequence presented 100% identity to a serine protease cDNA clone previously identified from B. jararaca venom gland, but not yet isolated or characterized. BjSP was significantly inhibited by specific serine protease inhibitors and showed high stability at different pH values and temperatures. The enzyme displayed no effects on washed platelets, but was able to degrade fibrin clots in vitro and also the Aα and Bß chains of fibrinogen differently from thrombin, forming additional fibrinopeptides derived from the Bß chain, which should be related to its inability to coagulate fibrinogen solutions or platelet-poor plasma. In the mapping of catalytic subsites, the protease showed high hydrolytic specificity for tyrosine, especially in subsite S1. Additionally, its amidolytic activity on different chromogenic substrates suggests possible effects on other factors of the coagulation cascade. In conclusion, BjSP is a serine protease that acts nonspecifically on fibrinogen, generating different Bß fibrinopeptides and thus not forming fibrin clots. Its distinguished properties in comparison to most SVSPs stimulate further studies in an attempt to validate its potential as a defibrinogenating agent.

LTB4 and PGE2 modulate the release of MIP-1α and IL-1ß by cells stimulated with Bothrops snake venoms.

Envenomation by Bothrops snakes promotes the release of inflammatory mediators, whose effects during this context are not well understood. These mediators include chemokines, cytokines and eicosanoids. Indeed, Bothrops snake envenomation results in local and systemic perturbations, including leukocyte recruitment, edema, pain and extensive tissue damage. Recently, our group demonstrated that leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) regulate macrophage production of interleukin-1ß (IL-1ß) induced by scorpion venom. Whether these molecular mechanisms also affect host cell responses to snake venoms, such as those from B. jararaca or B. jararacussu, is unknown. In this study, we demonstrate that B. jararaca or B. jararacussu venoms induce macrophage inflammatory protein 1-alpha (MIP-1α) and IL-1ß production by THP-1 (cell line of human peripheral blood monocytes) and AMJ2-C11 (cell line of mouse alveolar macrophage). We also show that venoms from both Bothrops species induce NF-κB activation in THP-1 cells. Furthermore, we observed that treatment of THP-1 and AMJ2-C11 cell lines with exogenous PGE2 reduces MIP-1α production, while increasing IL-1ß production in cells stimulated by B. jararaca or B. jararacussu venoms. Interestingly, exogenous LTB4 had the opposite effect by reducing IL-1ß and increasing MIP-1α release. Our results suggest that, differential eicosanoid metabolism in myeloid cells is tightly associated with the production of cytokines and chemokines after stimulation with B. jararaca or B. jararacussu venoms.

CR-LAAO causes genotoxic damage in HepG2 tumor cells by oxidative stress.

Snake venom L-amino acid oxidases (SV-LAAOs) are enzymes of great interest in research due to their many biological effects with therapeutic potential. CR-LAAO, an L-amino acid oxidase from Calloselasma rhodostoma snake venom, is a well described SV-LAAO with immunomodulatory, antiparasitic, microbicidal, and antitumor effects. In this study, we evaluated the genotoxic potential of this enzyme in human peripheral blood mononuclear cells (PBMC) and HepG2 tumor cells, as well as its interaction with these cells, its impact on the expression of DNA repair and antioxidant pathway genes, and reactive oxygen species (ROS)-induced intracellular production. Flow cytometry analysis of FITC-labelled CR-LAAO showed higher specificity of interaction with HepG2 cells than PBMC. Moreover, CR-LAAO significantly increased intracellular levels of ROS only in HepG2 tumor cells, as assessed by fluorescence. CR-LAAO also induced genotoxicity in HepG2 cells and PBMC after 4 h of stimulus, with DNA damages persisting in HepG2 cells after 24 h. To investigate the molecular basis underlying the genotoxicity attributed to CR-LAAO, we analyzed the expression profile (mRNA levels) of 44 genes involved in DNA repair and antioxidant pathways in HepG2 cells by RT2 Profiler polymerase chain reaction array. CR-LAAO altered the tumor cell expression of DNA repair genes, with two downregulated (XRCC4 and TOPBP1) and three upregulated (ERCC6, RAD52 and CDKN1) genes. In addition, two genes of the antioxidant pathway were upregulated (GPX3 and MPO), probably in an attempt to protect tumor cells from oxidative damage. In conclusion, our data suggest that CR-LAAO possesses higher binding affinity to HepG2 tumor cells than to PBMC, its genotoxic mechanism is possibly caused by the oxidative stress related to the production of H2O2, and is also capable of modulating genes related to the DNA repair system and antioxidant pathways.

A synthetic snake-venom-based tripeptide (Glu-Val-Trp) protects PC12 cells from MPP+ toxicity by activating the NGF-signaling pathway.

Venom small peptides that target neurotrophin receptors might be beneficial in neurodegeneration, including Parkinsons disease (PD). Their small size, ease of synthesis, structural stability and target selectivity make them important tools to overcome the limitations of endogenous neurotrophins as therapeutic agents. Additionally, they might be optimized to improve resistance to enzymatic degradation, bioavailability, potency and, mainly, lipophilicity, important to cross the blood brain barrier (BBB). Here, we evaluated the neuroprotective effects and mechanisms of the synthetic snake-venom-based peptide p-BTX-I (Glu-Val-Trp) in PC12 cells treated with MPP+ (1-methyl-4-phenylpyridinium), a dopaminergic neurotoxin that induces Parkinsonism in vivo. The peptide p-BTX-I induced neuritogenesis, which was reduced by (i) k252a, antagonist of the NGF-selective receptor, trkA (tropomyosin receptor kinase A); (ii) LY294002, inhibitor of the PI3 K/AKT pathway and (iii) U0126, inhibitor of the MAPK-ERK pathway. Besides that, p-BTX-I also increased the expression of GAP-43 and synapsin, which are molecular markers of axonal growth and synaptic communication. In addition, the peptide increased the viability and differentiation of cells exposed to MPP+, known to inhibit neuritogenesis. Altogether, our findings suggest that the synthetic peptide p-BTX-I protects PC12 cells from MPP+ toxicity by a mechanism that mimics the neurotrophic action of NGF. Therefore, the molecular structure of p-BTX-I might be relevant in the development of drugs aimed at restoring the axonal connectivity in neurodegenerative processes.

An overview of the immune modulating effects of enzymatic toxins from snake venoms.

Snake venoms are complex mixtures of organic and inorganic compounds, including proteins belonging to the protease (serine and metalloproteinases), oxidase (L-amino acid oxidases), and phospholipase (especially phospholipases A2) enzyme classes. These toxins account for the serious deleterious effects of snake envenomations, such as tissue necrosis, neurotoxicity, and hemorrhage. In addition to their toxic effects, snake venom toxins have served as important tools for investigating the mechanisms underlying envenomation and discovering new pharmacologically active compounds with immunotherapeutic potential. In this sense, the present review discusses the new findings and therapeutic perspectives in the immune modulating potential of enzymatic toxins from snake venoms belonging to the classes metalloproteinase, serine protease, L-amino acid oxidase, and phospholipase A2.

Kinetic investigations and stability studies of two Bothrops L-amino acid oxidases

L-amino acid oxidases isolated from snake venoms (SV-LAAOs) are enzymes that have great therapeutic potential and are currently being investigated as tools for developing new strategies to treat various diseases, including cancer and bacterial infections. The main objective of this study was to make a brief evaluation of the enzymatic stability of two Bothrops LAAOs, one isolated from Bothrops jararacussu (BjussuLAAO-II) and the other from Bothrops moojeni (BmooLAAO-I) venoms. Methods and results: The enzymatic activity and stability of both LAAOs were evaluated by microplate colorimetric assays, for which BjussuLAAO-II and BmooLAAO-I were incubated with different L-amino acid substrates, in the presence of different ions, and at different pH ranges and temperatures. BjussuLAAO-II and BmooLAAO-I demonstrated higher affinity for hydrophobic amino acids, such as Phe and Leu. The two enzymes showed high enzymatic activity in a wide temperature range, from 25 to 75 °C, and presented optimum pH around 7.0. Additionally, Zn2+, Al3+, Cu2+ and Ni2+ ions negatively modulated the enzymatic activity of both LAAOs. As to stability, BjussuLAAO-II and BmooLAAO-I showed high enzymatic activity for 42 days stored at 4°C in neutral pH solution. Moreover, the glycan portions of both LAAOs were analyzed by capillary electrophoresis, which revealed that BjussuLAAO-II presented two main glycan portions with relative masses of 7.78 and 8.13 CGU, while BmooLAAO-I showed three portions of 7.58, 7.94 and 8.37 CGU. Conclusions: Our results showed that, when stored properly, BjussuLAAO-II and BmooLAAO-I present enzymatic stability over a long time period, which is very important to allow the use of these enzymes in pharmacological studies of great impact in the medical field.(AU)

Cytotoxic and inflammatory potential of a phospholipase A2 from Bothrops jararaca snake venom

Snake venom phospholipases A2 (PLA2s) have been reported to induce myotoxic, neurotoxic, hemolytic, edematogenic, cytotoxic and proinflammatory effects. This work aimed at the isolation and functional characterization of a PLA2 isolated from Bothrops jararaca venom, named BJ-PLA2-I. Methods and Results: For its purification, three consecutive chromatographic steps were used (Sephacryl S-200, Source 15Q and Mono Q 5/50 GL). BJ-PLA2-I showed acidic characteristics, with pI~4.4 and molecular mass of 14. 2 kDa. Sequencing resulted in 60 amino acid residues that showed high similarity to other Bothrops PLA2s, including 100% identity with BJ-PLA2, an Asp49 PLA2 previously isolated from B. jararaca venom. Being an Asp49 PLA2, BJ-PLA2-I showed high catalytic activity, and also inhibitory effects on the ADP-induced platelet aggregation. Its inflammatory characterization showed that BJ-PLA2-I was able to promote leukocyte migration in mice at different concentrations (5, 10 and 20 µg/mL) and also at different response periods (2, 4 and 24 h), mainly by stimulating neutrophil infiltration. Furthermore, increased levels of total proteins, IL-6, IL-1 ß and PGE2 were observed in the inflammatory exudate induced by BJ-PLA2-I, while nitric oxide, TNF-α, IL-10 and LTB4 levels were not significantly altered. This toxin was also evaluated for its cytotoxic potential on normal (PBMC) and tumor cell lines (HL-60 and HepG2). Overall, BJ-PLA2-I (2.5-160 µg/mL) promoted low cytotoxicity, with cell viabilities mostly varying between 70 and 80% and significant values obtained for HL-60 and PBMC only at the highest concentrations of the toxin evaluated. Conclusions: BJ-PLA2-I was characterized as an acidic Asp49 PLA2 that induces acute local inflammation and low cytotoxicity. These results should contribute to elucidate the action mechanisms of snake venom PLA2s.(AU)

A new l-amino acid oxidase from Bothrops jararacussu snake venom: Isolation, partial characterization, and assessment of pro-apoptotic and antiprotozoal activities.

A new l-amino acid oxidase (LAAO) from Bothrops jararacussu venom (BjussuLAAO-II) was isolated by using a three-step chromatographic procedure based on molecular exclusion, hydrophobicity, and affinity. BjussuLAAO-II is an acidic enzyme with pI=3.9 and molecular mass=60.36kDa that represents 0.3% of the venom proteins and exhibits high enzymatic activity (4884.53U/mg/mim). We determined part of the primary sequence of BjussuLAAO-II by identifying 96 amino acids, from which 34 compose the N-terminal of the enzyme (ADDRNPLEECFRETDYEEFLEIARNGLSDTDNPK). Multiple alignment of the partial BjussuLAAO-II sequence with LAAOs deposited in the NCBI database revealed high similarity (95-97%) with other LAAOs isolated from Bothrops snake venoms. BjussuLAAO-II exerted a strong antiprotozoal effect against Leishmania amazonensis (IC50=4.56µg/mL) and Trypanosoma cruzi (IC50=4.85µg/mL). This toxin also induced cytotoxicity (IC50=1.80µg/mL) and apoptosis in MCF7 cells (a human breast adenocarcinoma cell line) by activating the intrinsic and extrinsic apoptosis pathways, but were not cytotoxic towards MCF10A cells (a non-tumorigenic human breast epithelial cell line). The results reported herein add important knowledge to the field of Toxinology, especially for the development of new therapeutic agents.

Disseminated intravascular coagulation caused by moojenactivase, a procoagulant snake venom metalloprotease.

Snake venom toxins that activate coagulation factors are key players in the process of venom-induced coagulopathy, and account for severe clinical manifestations. The present study applies a variety of biochemical, hematological, and histopathological approaches to broadly investigate the intravascular and systemic effects of moojenactivase (MooA), the first described PIIId subclass metalloprotease isolated from Bothrops sp. venom that activates coagulation factors. MooA induced consumption coagulopathy with high toxic potency, characterized by prolongation of prothrombin and activated partial thromboplastin time, consumption of fibrinogen and the plasma coagulation factors X and II, and thrombocytopenia. MooA promoted leukocytosis and expression of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, accompanied by tissue factor-dependent procoagulant activity in peripheral blood mononuclear cells. This metalloprotease also caused intravascular hemolysis, elevated plasma levels of creatine kinase-MB, aspartate transaminase, and urea/creatinine, and induced morphopathological alterations in erythrocytes, heart, kidney, and lungs associated with thrombosis and hemorrhage. Diagnosis of MooA-induced disseminated intravascular coagulation represents an important approach to better understand the pathophysiology of Bothrops envenomation and develop novel therapeutic strategies targeting hemostatic disturbances.

Immune cells and mediators involved in the inflammatory responses induced by a P-I metalloprotease and a phospholipase A2 from Bothrops atrox venom.

Bothrops envenomations can promote severe inflammatory responses by inducing edema, pain, leukocyte recruitment and release of chemical mediators by local cells. In the present study, two toxins from Bothrops atrox venom (the P-I metalloprotease Batroxase and the acidic phospholipase A2 BatroxPLA2) were evaluated in relation to their inflammatory effects induced in vivo and in vitro, mainly focusing on the participation of different immune cells and inflammatory mediators. Both toxins mainly promoted acute inflammatory responses with significant recruitment of neutrophils in the early hours (1-4h) after administration into the peritoneal cavity of C57BL/6 mice, and increased infiltration of mononuclear cells especially after 24h. Among the mediators induced by both toxins are IL-6, IL-10 and PGE2, with Batroxase also inducing the release of L-1ß, and BatroxPLA2 of LTB4 and CysLTs. These responses pointed to possible involvement of immune cells such as macrophages and mast cells, which were then evaluated in vitro. Mice peritoneal macrophages stimulated with Batroxase produced significant levels of IL-6, IL-1ß, PGE2 and LTB4, whereas stimulus with BatroxPLA2 induced increases of IL-6, PGE2 and LTB4. Furthermore, both toxins were able to stimulate degranulation of RBL-2H3 mast cells, but with distinct concentration-dependent effects. Altogether, these results indicated that Batroxase and BatroxPLA2 promoted local and acute inflammatory responses related to macrophages and mast cells and to the production of several mediators. Our findings should contribute for better understanding the different mechanisms of toxicity induced by P-I metalloproteases and phospholipases A2 after snakebite envenomations.

CR-LAAO, an L-amino acid oxidase from Calloselasma rhodostoma venom, as a potential tool for developing novel immunotherapeutic strategies against cancer.

L-amino acid oxidases from snake venoms have been described to possess various biological functions. In this study, we investigated the inflammatory responses induced in vivo and in vitro by CR-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodostoma venom, and its antitumor potential. CR-LAAO induced acute inflammatory responses in vivo, with recruitment of neutrophils and release of IL-6, IL-1ß, LTB4 and PGE2. In vitro, IL-6 and IL-1ß production by peritoneal macrophages stimulated with CR-LAAO was dependent of the activation of the Toll-like receptors TLR2 and TLR4. In addition, CR-LAAO promoted apoptosis of HL-60 and HepG2 tumor cells mediated by the release of hydrogen peroxide and activation of immune cells, resulting in oxidative stress and production of IL-6 and IL-1ß that triggered a series of events, such as activation of caspase 8, 9 and 3, and the expression of the pro-apoptotic gene BAX. We also observed that CR-LAAO modulated the cell cycle of these tumor cells, promoting delay in the G0/G1 and S phases. Taken together, our results suggest that CR-LAAO could serve as a potential tool for the development of novel immunotherapeutic strategies against cancer, since this toxin promoted apoptosis of tumor cells and also activated immune cells against them.

Crystal structure and molecular dynamics studies of L-amino acid oxidase from Bothrops atrox.

L-amino acid oxidases (LAAOs) are dimeric flavoproteins that catalyze the deamination of L-amino acid to α-keto acid, producing ammonia and hydrogen peroxide. In this study, we report the crystal structure and molecular dynamics simulations of LAAO from the venom of Bothrops atrox (BatroxLAAO). BatroxLAAO presents several biological and pharmacological properties with promising biomedical applications. BatroxLAAO structure contains the highly conserved structural pattern of LAAOs comprising a FAD-binding domain, substrate-binding domain and helical domain, and a dimeric arrangement that can be stabilized by zinc. Also, molecular dynamics results show an asymmetric behavior, and a direct communication between FAD- and substrate-binding domains of counterpart subunits. These findings shed light on the structural role of dimerization to catalytic mechanism of SV-LAAOs.

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.