Jararacussu Snake Venom Research Articles

BjussuMP-II, a venom metalloproteinase, induces the release and cleavage of pro-inflammatory cytokines and disrupts human umbilical vein endothelial cells

Snake venom metalloproteases (SVMPs) are hydrolytic enzymes dependent on metal binding, primarily zinc (Zn2+), at their catalytic site. They are classified into three classes (P–I to P-III). BjussuMP-II, a P–I SVMP isolated from Bothrops jararacussu snake venom, has a molecular mass of 24 kDa. It exhibits inhibitory activity on platelet aggregation and hydrolyzes fibrinogen. TNF-α upregulates the expression of adhesion molecules on endothelial cell surfaces, promoting leukocyte adhesion and migration during inflammation. Literature indicates that SVMPs may cleave the TNF-α precursor, possibly due to significant homology between metalloproteases from mammalian extracellular matrix and SVMPs. This study aimed to investigate BjussuMP-II’s effects on human umbilical vein endothelial cells (HUVEC), focusing on viability, detachment, adhesion, release, and cleavage of TNF-α, IL-1β, IL-6, IL-8, and IL-10. HUVEC were incubated with BjussuMP-II (1.5–50 μg/mL) for 3–24 h. Viability was determined using LDH release, MTT metabolization, and 7AAD for membrane integrity. Adhesion and detachment were assessed by incubating cells with BjussuMP-II and staining with Giemsa. Cytokines were quantified in HUVEC supernatants using EIA. TNF-α cleavage was evaluated using supernatants from PMA-stimulated cells or recombinant TNF-α. Results demonstrated BjussuMP-II’s proteolytic activity on casein. It was not toxic to HUVEC at any concentration or duration studied but interfered with adhesion and promoted detachment. PMA induced TNF-α release by HUVEC, but this effect was not observed with BjussuMP-II, which cleaved TNF-α. Additionally, BjussuMP-II cleaved IL-1β, IL-6, and IL-10. These findings suggest that the zinc metalloprotease BjussuMP-II could be a valuable biotechnological tool for treating inflammatory disorders involving cytokine deregulation.

Evaluation of the Toxicological Effects from Bothrops Jararacussu Snake Venom on Zebrafish (Danio Rerio) Embryos and Larvae

Introduction: Approximately 90% of reported and identified cases of snakebites in Brazil are caused by species belonging to the Bothrops genus. These snakes have clinical rele-vance due to their venom composition, which contains substances capable of triggering local and systemic effects, leading to morbidities and/or mortality. Objective: The objective of this study was to evaluate the toxic and toxinological effects of Both-rops Jararacussu snake venom on zebrafish embryos and larvae. Methods: The stability of B. Jararacussu snake venom under the conditions used in the toxicity experiments in zebrafish embryos and larvae was evaluated on citrated human plasma. Zebrafish embryos and/or larvae mortality, morphological alterations, spontaneous tail movements and heartbeat caused by the venom were quantified within 96 hours. Toxicity parameters and activity of enzyme-related toxicity biomarkers were evaluated in zebrafish after 96 hours of semi-static exposure to the venom. Results: The results indicated that the venom causes toxicity in zebrafish embryos and larvae, in-ducing embryonic mortality, alteration in the number of spontaneous tail movements and activity of biomarker enzymes. The results suggested that the toxic effects caused by the venom in the ear-ly stages of zebrafish development are mediated, in part, by neurotoxic action, induction of oxida-tive and metabolic stress caused by low molecular weight components, and proteins present in this venom. Conclusion: Toxinological evaluations using the zebrafish as a model are scarce; however, this study presented promising results that encourage the development of future research in toxinology using this animal as a model organism.

A C-type lectin induces NLRP3 inflammasome activation via TLR4 interaction in human peripheral blood mononuclear cells.

Lectins are a large group of proteins found in many snake venoms. BjcuL is a C-type lectin from Bothrops jararacussu snake venom that does not present cytotoxicity action on human peripheral blood mononuclear cells (PBMCs) at concentrations of 5 and 10μg/mL. BjcuL demonstrates an immunomodulatory role in PBMCs with the production of pro- and anti-inflammatory cytokines (IL-2, IL-10, IFN-γ, IL-6, TNF-α, and IL-17) in addition to stimulate T cells to produce reactive oxygen species (ROS) that could play a role in the acute inflammatory reaction observed in the victims. Inflammasomes are an essential arm in cells of innate immunity to detect and sense a range of endogenous or exogenous, sterile, or infectious stimuli to elicit cellular responses and effector mechanisms. NLRP3 inflammasome is a significant target for this study, because the lectin is responsible for leukocyte activation stimulating the release of inflammatory mediators, which results in dynamic cellular responses to remove the detrimental process to the body in snakebites. Thus, this study aimed to investigate how isolated BjcuL fromB. jararacussuvenom affects NLRP3 inflammasome activation on PBMCs. For this, the cells were isolated by density gradient and incubated with BjcuL at different periods and concentrations for the evaluation of the activation of the NLRP3 inflammasome through gene and protein expressions of ASC, CASPASE-1, and NLRP3 by RT-qPCR, Western blot, and immunofluorescence, as well as the participation of Toll-like receptor 4 (TLR4) and ROS in the IL-1β production, a product resultant of the NLRP3 inflammasome activation. Herein, BjcuL interacts with TLR4 as demonstrated by in vitro and in silico studies and induces cytokines release via NF-κB signaling. By genic and protein expression assays, BjcuL activates NLRP3 inflammasome, and the pharmacological modulation with LPS-RS, an antagonist of TLR4; LPS-SM, an agonist of TLR4; MCC950, a specific NLRP3 inhibitor, and rotenone, an inhibitor of mitochondrial ROS, confirmed the participation of TLR4 and ROS in the NLRP3 inflammasome activation and IL-1β liberation. The effects of BjcuL on the regulation and activation of the NLRP3 inflammasome complex via TLR4 activation with ROS participation may be determinant for the development of the inflammatory local effects seen in snakebite victims. In addition, in silico together with in vitro studies provide information that may be useful in the rational design of TLR agonists as well as new adjuvants for immunomodulatory therapy.

Action of BjussuMP-II, a snake venom metalloproteinase isolated from Bothrops jararacussu venom, on human neutrophils

Snake venom metalloproteinases (SVMPs) are enzymatic proteins present in large amounts in snake venoms presenting proteolytic, hemorrhagic, and coagulant activities. BjussuMP-II, a class P–I SVMP, isolated from the Bothrops jararacussu snake venom does not have relevant hemorrhagic activity but presents fibrinolytic, fibrinogenolytic, antiplatelet, gelatinolytic, and collagenolytic action. This study aimed to verify the action of BjussuMP-II on human neutrophil functionality focusing on the lipid bodies formation and hydrogen peroxide production, the release of dsDNA through colorimetric and microscopic assays, and cytokines by immunoenzymatic assays. Results showed that BjussuMP-II at concentrations of 1.5 up to 50 μg/mL for 24 h is not toxic to human neutrophils using an MTT assay. Under non-cytotoxic concentrations, BjussuMP-II can induce an increase in the formation of lipid bodies, production of hydrogen peroxide and cytokines [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-8 (IL-8)] liberation and, the release of dsDNA to form NETs. Taken together, the data obtained show for the first time that BjussuMP-II has a pro-inflammatory action and activates human neutrophils that can contribute to local damage observed in snakebite victims.

Dynamics of action of a Lys-49 and an Asp-49 PLA2s on inflammasome NLRP3 activation in murine macrophages

Phospholipases A2 (PLA2s) are proteins found in snake venoms with hemolytic, anticoagulant, myotoxic, edematogenic, bactericidal and inflammatory actions. In Bothrops jararacussu snake venom were isolated a Lys49-PLA2 (BthTX-I) and an Asp49-PLA2 (BthTX-II) with myotoxic and inflammatory actions. Both PLA2s can activate the NLRP3 inflammasome, an intracytoplasmic platform that recognizes molecules released when tissue is damaged liberating IL-1β that contributes to the inflammatory response observed in envenoming. The dynamic of action of BthTX-I and BthTX-II in both thioglycollate (TG)-elicited macrophages and C2C12 myoblasts and the involvement of EP1 and EP2 receptors, and PGE2 in NLRP3 inflammasome activation were evaluated. Both toxins induced PGE2 liberation and inflammasome components (NLRP3, Caspase-1, ASC, IL-1β, and IL18), IL-6, P2X7, COX-1, COX-2, EP2 and EP4 gene expression in TG-elicited macrophages but not in C2C12 myoblasts. EP2 (PF04418948) and EP4 (GW627368X) inhibitors abolished this effect. Both PLA2s also induced NLRP3 inflammasome protein expression that was abolished with the inhibitors used. Immunofluorescence and IL-1β assays confirmed the NLRP3 activation in TG-elicited macrophages with the participation of both EP2 and EP4 receptors confirming their involvement in this effect. All in all, BthTX-I and BthTX-II activate macrophages and induce the NLRP3 inflammasome complex activation with the participation of the PGE2 via COX pathway and EP2 and EP4, both PGE2 receptors, contributing to the local inflammatory effects observed in envenoming.

Role of Toll-like receptors in local effects in a model of experimental envenoming induced by Bothrops jararacussu snake venom and by two phospholipases A2

Literature shows that phospholipases A2 isolated from snake venoms of the genus Bothrops are involved in the local inflammatory response. However, the mechanisms by which these enzymes trigger this process have not yet been clarified. Toll-Like receptors (TLRs) are transmembrane proteins that recognize pathogens associated molecular patterns (PAMPs), or even damage associated molecular patterns (DAMPs). After this recognition, an innate immune response is activated resulting in cytokines liberation contributing to inflammation. Thus, the purpose of this work was to study the participation of different TLRs during the local inflammatory process induced by B. jararacussu snake venom and by two isolated phospholipases A2, BthTX-I or BthTX-II, from this venom in a model of experimental envenoming. For this, sub-lethal doses of B. jararacussu venom (BjussuV), BthTX-I or BthTX-II were injected in the gastrocnemius muscle. Myotoxic activity was evaluated by histological analysis and by quantification of plasma levels of total-creatine kinase (CK). The pro-inflammatory cytokines TNF-α and IL-1β was measured in both muscle tissue homogenate and plasma. A quantification of the gene expression of TLRs 2, 4, 5 and 9 in muscle tissue homogenate was performed by the real-time polymerase chain reaction (RTq-PCR). According to the results, it can be observed that, when compared to the control, there was a significant increase of CK and TNF-α in the bloodstream of the animals injected with both BjussuV, BthTX-I and BthTX-II. In muscle tissue homogenate, it was observed a significant increase in both cytokines, TNF-α and IL-1β, levels compared to the control animals. The results point to an important increase in the gene expression of TLR2 and TLR4, suggesting that these TLRs can be important targets for the development of future therapies for local treatment for victims of snakebites.

Bothrops jararacussu snake venom lectin induces mast cell activation and vascular permeability enhance in an animal model

Bothrops species trigger an acute inflammatory response in victims, with activated leukocytes releasing several mediators that may contribute to local and systemic effects. The effects of BjcuL, a lectin isolated from B. jararacussu snake venom, on mast cells and vasopermeability were investigated in this study. BjcuL activates mast cells and increases vasopermeability through the involvement of histamine and platelet activating factor, which may play a role in the victims' acute inflammatory reaction.

Mutagenic and genotoxic activities of Phospholipase A2 Bothropstoxin-I from Bothrops jararacussu in Drosophila melanogaster and human cell lines

Phospholipase A2 Bothropstoxin-I (PLA2 BthTX-I) is a myotoxic Lys49-PLA2 from Bothrops jararacussu snake venom. In order to evaluate the DNA damage caused by BthTX-I, we used the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster and Comet assay in HUVEC and DU-145 cells. For SMART, different concentrations of BthTX-I (6.72 to 430 μg/mL) were used and no significant changes in the survival rate were observed. Significant frequency of mutant spots was observed for the ST cross at the highest concentration of BthTX-I due to recombinogenic activity. In the HB cross, BthTX-I increased the number of mutant spots at intermediate concentrations, being 53.75 μg/mL highly mutagenic and 107.5 μg/mL predominantly recombinogenic. The highest concentrations were neither mutagenic nor recombinogenic, which could indicate cytotoxicity in the wing cells of D. melanogaster. In vitro, all BthTX-I concentrations (1 to 50 μg/mL) induced decrease in HUVEC cell viability, as well as in DU-145 cells at concentrations of 10, 25, and 50 μg/mL. The comet assay showed that in HUVEC and DU-145 cells, all BthTX-I concentrations promoted increase of DNA damage. Further studies should be performed to elucidate the mechanism of action of PLA2 BthTX-I and its possible use in therapeutic strategies against cancer.

Exploring the ability of low-level laser irradiation to reduce myonecrosis and increase Myogenin transcription after Bothrops jararacussu envenomation.

Envenoming caused by snakebites is a very important neglected tropical disease worldwide. The myotoxic phospholipases present in the bothropic venom disrupt the sarcolemma and compromise the mechanisms of energy production, leading to myonecrosis. Photobiomodulation therapy (PBMT) has been used as an effective tool to treat diverse cases of injuries, such as snake venom-induced myonecrosis. Based on that, the aim of this study was to analyze the effects of PBMT through low-level laser irradiation (904nm) on the muscle regeneration after the myonecrosis induced by Bothrops jararacussu snake venom (Bjssu) injection, focusing on myogenic regulatory factors expression, such as Pax7, MyoD, and Myogenin (MyoG). Male Swiss mice (Mus musculus), 6-8-week-old, weighing 22 ± 3g were used. Single sub-lethal Bjssu dose or saline was injected into the right mice gastrocnemius muscle. At 3, 24, 48, and 72h after injections, mice were submitted to PBMT treatment. When finished the periods of 48 and 72h, mice were euthanized and the right gastrocnemius were collected for analyses. We observed extensive inflammatory infiltrate in all the groups submitted to Bjssu injections. PBMT was able to reduce the myonecrotic area at 48 and 72h after envenomation. There was a significant increase of MyoG mRNA expression at 72h after venom injection. The data suggest that beyond the protective effect promoted by PBMT against Bjssu-induced myonecrosis, the low-level laser irradiation was able to stimulate the satellite cells, thus enhancing the muscle repair by improving myogenic differentiation.

Muscle proteolysis via ubiquitin-proteasome system (UPS) is activated by BthTx-I Lys49 PLA2 but not by BthTx-II Asp49 PLA2 and Bothrops jararacussu venom

Bites by viperid snakes belonging to Bothrops genus produce fast and intense local edema, inflammation, bleeding and myonecrosis. In this study, we investigated the role of Myogenic Regulatory Factors (MRFs: MyoD; Myog), negatively regulated by GDF-8 (Myostatin), and ubiquitin-proteasome system pathway (UPS: MuRF-1; Fbx-32) in gastrocnemius muscle regeneration after Bothrops jararacussu snake venom (Bjussu) or its isolated phospholipase A2 myotoxins, BthTx-I (Lys-49 PLA2) and BthTx-II (Asp-49 PLA2) injection. Male Swiss mice received a single intra-gastrocnemius injection of crude Bjussu, at a dose/volume of 0.83 mg/kg/20 μl, and BthTx-I or BthTx-II, at a dose/volume of 2.5 mg/kg/20 μl. Control mice (Sham) received an injection of sterile saline solution (NaCl 0.9%; 20 μl). At 24, 48, 72 and 96 h post injection, right gastrocnemius was collected for protein expression analyses. Based on the temporal expressional dynamics of MyoD, Myog and GDF-8/Myostatin, it was possible to propose that the myogenesis pathway was impacted most badly by BthTx-II followed by BthTx-I and lastly by B. jararacussu venom, thus suggesting that catalytic activity has likely inhibitory role on the satellite cells-mediated reparative myogenesis pathway. Inversely, the catalytic activity seems to be not a determinant for the activation of proteins ubiquitination by MuRF-1 and Fbx-32/Atrogin-1 E3 proteasome ligases, given proteolysis pathway through UPS was activated neither after Bjussu, nor after BthTx-II, but just after the catalytically-inactive BthTx-I Lys-49 PLA2-homologue exposure. The findings of this study disclose interesting perspective for further mechanistic studies about pathways that take part in the atrophy and repair after permanent damage induced by bothropic snakebites.

Inflammation and coagulation crosstalk induced by BJcuL, a galactose-binding lectin isolated from Bothrops jararacussu snake venom

Inflammation and coagulopathies are important systemic events following snakebite. Snake venom galactoside-binding lectins (SVgalLs) are known modulators of the immune response with no direct effect on hemostasis. Considering the crosstalk between inflammation and coagulation, the present study investigated how BJcuL, a proinflammatory SVgalL isolated from Bothrops jararacussu venom, mediated the inflammation-induced procoagulant activity. We examined the proinflammatory cytokine production and procoagulant tissue factor (TF) activity in human whole blood and monocyte-rich cell suspension (MR-PBMC) treated with BJcuL. This lectin increased production of the cytokines TNF-α, IL-1β and IL-6, upregulated TF expression on the cell surface, and induced procoagulant activity. The proinflammatory behavior was mediated by the direct interaction between the lectin and toll-like receptor 4, via binding to β-galactoside-containing glycoconjugates on the cell surface, and activation of NFκ-B signaling. Interestingly, the BJcuL-induced inflammation was directly associated with the procoagulant activity of MR-PBMC cells. In whole blood culture, the lectin exhibited similar behavior, i.e. it induced cytokine production and MR-PBMC TF-mediated procoagulant activity. Therefore, the present study is the first report on the inflammation-induced procoagulant activity of SVgalLs, and it indicates that BJcuL is an important factor associated with coagulopathy in patients with snake envenomation.

Evaluation of self-assembled Bothrops jararacussu snake venom proteins cross-linked chitosan nanoparticles activity for use as a potential antibacterial

Evaluation of self-assembled Bothrops jararacussu snake venom proteins cross-linked chitosan nanoparticles activity for use as a potential antibacterial

Lectin isolated from Bothrops jararacussu venom induces IL-10 release by TCD4+ cells and TNF-α release by monocytes and natural killer cells.

BjcuL is a C-type lectin isolated from Bothrops jararacussu snake venom with specificity for binding β-d-galactose units. BjcuL is not toxic to human peripheral blood mononuclear cells (PBMCs), but it inhibits PBMC proliferation and stimulates these cells to produce superoxide anions and hydrogen peroxide primarily via lymphocyte stimulation; it does not stimulate the production of nitric oxide and PGE2 . The purpose of this study was to investigate the effect of BjcuL on PBMC activation with a focus on cytokine release modulating PBMC proliferation. The results showed for the first time that BjcuL coupled to FITC interacted with monocytes, B cells, natural killer (NK) cells, and with subpopulations of T cells. These cell-cell interactions can lead to cell activation and inflammatory cytokines release, such as IL-6 and TNF-α, as well as the anti-inflammatory cytokine IL-10. In addition, TNF-α release was attributed to NK cells and monocytes, whereas IL-10 was attributed to TCD4+ and Treg cells when stimulated by BjcuL. The temporal cytokines profile produced by cells when stimulated with this lectin allows us to assert that BjcuL has immunomodulatory activity in this context.

Inhibitory effects of Morus nigra L. (Moraceae) against local paw edema and mechanical hypernociception induced by Bothrops jararacussu snake venom in mice

BackgroundBothropic venoms cause intense local damage, pain, edema, and myonecrosis. Morus nigra L. (Moraceae) has several uses in folk medicine and can be a promising candidate for the treatment of several inflammatory disorders. Hypothesis/PurposeThe present study aims to evaluate the anti-inflammatory and antinociceptive effects of the ethanolic extract of Morus nigra L. (Mn-EtOH) on paw lesions induced by Bothrops jararacussu snake venom (BjcuV) in mice. MethodsUV–vis absorption of BjcuV was evaluated. A phytochemical study was performed, which led to the isolation and characterization of three compounds. These compounds were identified using spectrometric methods, namely LC–MS and NMR (1D and 2D), followed by the validation of their spectra with the data available in the literature. Further, the flavonoids i.e. rutin and quercetin (chemical markers of M. nigra), Mn-EtOH or Mn-EtOH-encapsulated electrospun fibers of Eudragit L100 (FB/Mn-EtOH), and Mn-EtOH-encapsulated microparticles of Eudragit L100 (MP/Mn-EtOH) were evaluated, in paw edema test induced by BjcuV. ResultsUV–vis spectra showed the presence of phospholipases A2 as component of BjcuV. The chemical examination resulted in the isolation of β-sitosterol, quercetin-3-O-glucopyranoside, and kaempferol-3-O-glucopyranoside. Mn-EtOH, FB/Mn-EtOH, MP/Mn-EtOH, rutin, and quercetin reduced the local edema induced by BjcuV. The Mn-EtOH also prevented edema provoked by serotonin and bradykinin. Moreover, it reduced paw edema and peritoneal leukocyte infiltration induced by carrageenan, and decreased the mechanical hypernociception of BjcuV. Mn-EtOH exerted anti-inflammatory and antinociceptive effects, possibly by the inhibition of leukocyte migration and the modulation of serotonin and bradykinin actions. This anti-inflammatory activity was maintained even upon incorporation of the M. nigra extract into the drug delivery systems (i.e., Mn-EtOH-encapsulated FBs and MPs of Eudragit L100). ConclusionThese results reinforce the therapeutic potential of M. nigra in the treatment of inflammatory conditions, in addition to, its role as a complementary treatment of snakebites.

Pharmacological characterization of cnidarian extracts from the Caribbean Sea: evaluation of anti-snake venom and antitumor properties

BackgroundCnidarians produce toxins, which are composed of different polypeptides that induce pharmacological effects of biotechnological interest, such as antitumor, antiophidic and anti-clotting activities. This study aimed to evaluate toxicological activities and potential as antitumor and antiophidic agents contained in total extracts from five cnidarians: Millepora alcicornis, Stichodactyla helianthus, Plexaura homomalla, Bartholomea annulata and Condylactis gigantea (total and body wall).MethodsThe cnidarian extracts were evaluated by electrophoresis and for their phospholipase, proteolytic, hemorrhagic, coagulant, fibrinogenolytic, neuromuscular blocking, muscle-damaging, edema-inducing and cytotoxic activities.ResultsAll cnidarian extracts showed indirect hemolytic activity, but only S. helianthus induced direct hemolysis and neurotoxic effect. However, the hydrolysis of NBD-PC, a PLA2 substrate, was presented only by the C. gigantea (body wall) and S. helianthus. The extracts from P. homomalla and S. helianthus induced edema, while only C. gigantea and S. helianthus showed intensified myotoxic activity. The proteolytic activity upon casein and fibrinogen was presented mainly by B. annulata extract and all were unable to induce hemorrhage or fibrinogen coagulation. Cnidarian extracts were able to neutralize clotting induced by Bothrops jararacussu snake venom, except M. alcicornis. All cnidarian extracts were able to inhibit hemorrhagic activity induced by Bothrops moojeni venom. Only the C. gigantea (body wall) inhibited thrombin-induced coagulation. All cnidarian extracts showed antitumor effect against Jurkat cells, of which C. gigantea (body wall) and S. helianthus were the most active; however, only C. gigantea (body wall) and M. alcicornis were active against B16F10 cells.ConclusionThe cnidarian extracts analyzed showed relevant in vitro inhibitory potential over the activities induced by Bothrops venoms; these results may contribute to elucidate the possible mechanisms of interaction between cnidarian extracts and snake venoms.

The toxin BjussuLAAO-II induces oxidative stress and DNA damage, upregulates the inflammatory cytokine genes TNF and IL6, and downregulates the apoptotic-related genes BAX, BCL2 and RELA in human Caco-2 cells

Colorectal carcinoma is one of the most common cancers in adults. As chemotherapy, the first-choice treatment for colorectal carcinoma, is often infeasible due to acquired tumor resistance and several adverse effects, it is important to discover and explore new molecules with better therapeutic action. Snake venom toxins have shown promising results with high cytotoxicity against tumor cells, but their mechanisms of action remain unclear. Here we examined how BjussuLAAO-II, an L-amino acid oxidase isolated from Bothrops jararacussu snake venom, exerts cytotoxicity towards colorectal adenocarcinoma human cells (Caco-2) and human umbilical vein endothelial cell line (HUVEC). A 24-h treatment with BjussuLAAO-II at 0.25 − 5.00 μg/mL diminished cell viability by decreasing (i) mitochondrial activity, assessed by reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and resazurin; (ii) the activity of acid phosphatases; and (iii) lysosomal function, assessed by neutral red uptake. BjussuLAAO-II also increased intracellular levels of reactive oxygen species and DNA damage, as assessed by fluorescence and the comet assay, respectively. BjussuLAAO-II altered the expression of cell proliferation-related genes, as determined by RT-qPCR: it elevated the expression of the inflammatory cytokine genes TNF and IL6, and lowered the expression of the apoptotic-related genes BAX, BCL2, and RELA. Therefore, BjussuLAAO-II induces Caco-2 cells death by acting on multiple intracellular targets, providing important data for further studies to assess whether these effects are seen in both tumor and normal cells, with the aim of selecting this drug for possible therapeutic purposes in the future.

Photobiomodulation of local alterations induced by BthTX-I, a phospholipase A2 myotoxin from Bothrops jararacussu snake venom: In vivo and in vitro evaluation

This report describes the effect of photobiomodulation (PBM) on edema formation, leukocyte influx, prostaglandin E2 (PGE2) biosynthesis and cytotoxicity caused by bothropstoxin-I (BthTX-I), a phospholipase A2 (PLA2) homologue isolated from Bothrops jararacussu snake venom. Swiss mice or C2C12 cells were irradiated with low-level laser (LLL) at 685nm wavelength, an energy density of 4.6J/cm2 and an irradiation time of 13s. To evaluate the effect on edema formation and leukocyte influx, LLL was applied to the site of inoculation 30min and 3h post-injection. C2C12 cells were exposed to BthTX-I and immediately irradiated. PBM significantly reduced paw edema formation, peritoneal leukocyte influx and PGE2 synthesis, but increased the viability of C2C12 muscle cells after BthTX-I incubation. These findings demonstrate that PBM attenuated the inflammatory events induced by BthTX-I. The attenuation of PGE2 synthesis could be an important factor in the reduced inflammatory response caused by laser irradiation. The ability of LLL irradiation to protect muscle cells against the deleterious effects of BthTX-I may indicate preservation of the plasma membrane.

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.

Structural and binding studies of a C-type galactose-binding lectin from Bothrops jararacussu snake venom

BJcuL is a snake venom galactoside-binding lectin (SVgalL) isolated from Bothrops jararacussu and is involved in a wide variety of biological activities including triggering of pro-inflammatory response, disruption of microbial biofilm structure and induction of apoptosis. In the present work, we determined the crystallographic structure of BJcuL, the first holo structure of a SVgalL, and introduced the fluorescence-based thermal stability assay (Thermofluor) as a tool for screening and characterization of the binding mechanism of SVgalL ligands. BJcuL structure revealed the existence of a porous and flexible decameric arrangement composed of disulfide-linked dimers related by a five-fold symmetry. Each monomer contains the canonical carbohydrate recognition domain, a calcium ion required for BJcuL lectinic activity and a sodium ion required for protein stabilization. BJcuL thermostability was found to be induced by calcium ion and galactoside sugars which exhibit hyperbolic saturation profiles dependent on ligand concentration. Serendipitously, the gentamicin group of aminoglycoside antibiotics (gAGAs) was also identified as BJcuL ligands. On contrast, gAGAs exhibited a sigmoidal saturation profile compatible with a cooperative mechanism of binding. Thermofluor, hemagglutination inhibition assay and molecular docking strategies were used to identify a distinct binding site in BJcuL localized at the dimeric interface near the fully conserved intermolecular Cys86-Cys86 disulfide bond. The hybrid approach used in the present work provided novel insights into structural behavior and functional diversification of SVgaLs.

Liposomes containing an ASP49-phospholipase A2 from Bothrops jararacussu snake venom as experimental therapy against cutaneous leishmaniasis

The aim of this study was to evaluate in vitro anti-Leishmania amazonensis activity of a Phospholipase A2 (Asp49-PLA2) isolated from the venom of Bothrops jararacussu and its encapsulated form. Asp49-PLA2 (2mg/mL) was added to a lipid mixture, solubilized in chloroform and dried under nitrogen flow. The lipid vesicles were formed homogeneously using the extrusion method, physicochemically characterized by their diameters, zeta potentials, encapsulation rate and also submitted to a molecular docking in silico analysis. The activity of Asp49-liposomes was evaluated in vitro against promastigote forms of L. amazonensis and J774 macrophages. Parasite and macrophage viabilities using MTT method were assessed after 48h incubation. L. amazonensis-infected macrophages were also incubated with encapsulated Asp49 and in solution form. The amastigote forms were counted inside the infected macrophages and the culture supernatants were collected for nitrites and TNF-α quantifications. Asp49-PLA2 in solution form displayed an anti-Leishmania concentration-dependent effect. Asp49-liposomes were able to reduce 78% of promastigote forms and preserved 82% of J774 macrophages' viability. After 48h of incubation with nanoencapsulated Asp49-PLA2 there was a significant reduction in the number of amastigotes (55%; p<0.05) compared to the control group. When the macrophages were infected and incubated with Asp49-liposomes a significant increase in the production of nitrites and TNF-α was observed when compared to infected cells alone. The results indicated that the liposomal system achieved in this study is a promising tool to enhance microbicidal activity of the infected macrophages, conferring a biotechnological therapeutic approach against experimental leishmaniasis.