Bothrops Jararaca Research Articles

Potential utilization of a lambda carrageenan polysaccharide, derived from a cultivated, clonal strain of the red seaweed Chondrus crispus (Irish moss) against toxic actions of venom of Bothrops jararaca and B. jararacussu snakes

Snakebites are a serious occupational problem affecting rural populations of tropical and sub-tropical, developing countries. Envenomation caused by the snakes Bothrops jararaca and B. jararacussu is characterized by local pain, edema, hemorrhage, tissue necrosis, and death. Despite the fact that antivenom may prevent the death of bite victims, treatment does not prevent tissue necrosis, often leading to amputation or deformity of the victim’s affected limb.Therefore, more efficient therapies need to be investigated. In this work, we tested the ability of a carrageenan galactan polysaccharide, isolated from a clonal strain of the red alga, Chondrus crispus (commonly known as Irish moss) to inhibit toxic, in vitro (coagulation, hemolytic and proteolytic) or in vivo (hemorrhagic, edematogenic, myotoxic and lethal) activities of B. jararaca or B. jararacussu venom. When the polysaccharide was mixed together with the venoms, inhibition of their toxic activities was achieved, but with different potencies. Moreover, inhibition of hemorrhage, edema, lethality, or myotoxicity was observed, even if the polysaccharide was injected before or after the injection of venoms, regardless of the route of administration (i.e., intravenous, subcutaneous, and intraperitoneal). A gel formulation containing the polysaccharide of C. crispus also protected mice from hemorrhage after administration of the venoms. Thus, the lambda carrageenan polysaccharide, as produced by a strain of C. crispus, may aid antivenom to block the toxic activities of B. jararaca and B. jararacussu venom, as well as to aid in the development of a more efficient therapy for envenomation by these venomous snakes.

A New Species of Snail-Eating Snake, Dipsas Cope, 1860 (Serpentes: Colubridae: Dipsadinae), from the Atlantic Forest of Brazil

We describe a new species of snail-eating snake from two localities within the Atlantic Forest of the states of Bahia and Minas Gerais, Brazil. The new species differs remarkably from all congeners by its dorsal pattern consisting of sharply bordered triangles resembling the pattern of sympatric pitvipers, more precisely Bothrops jararaca and B. pirajai. Parameters of external morphology of the new species resemble those of the Dipsas incerta species group. Its scalation, body/head shape, and color pattern are most similar to D. alternans, a species known to occur 360 km farther south, across the Rio Doce river. The new species differs from D. alternans by exhibiting triangular dorsal blotches and a higher number of pileus blotches, by the distribution of ventral spots and morphology of the supratemporal and premaxillae, as well as in hemipenial morphology. We also provide comparisons of the new species with all sympatric and/or parapatric congeners. The finding of a new snake species in the Atlantic rainforest of southern Bahia and adjacent Minas Gerais is particularly surprising as this region is easily accessible and represents a well-sampled area regarding herpetological surveys in the last decades.

Cleavage of proteoglycans, plasma proteins and the platelet-derived growth factor receptor in the hemorrhagic process induced by snake venom metalloproteinases

Envenoming by viperid snakes results in a complex pattern of tissue damage, including hemorrhage, which in severe cases may lead to permanent sequelae. Snake venom metalloproteinases (SVMPs) are main players in this pathogenesis, acting synergistically upon different mammalian proteomes. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca, induces severe local hemorrhage at pmol doses in a murine model. Our hypothesis is that in a complex scenario of tissue damage, HF3 triggers proteolytic cascades by acting on a partially known substrate repertoire. Here, we focused on the hypothesis that different proteoglycans, plasma proteins, and the platelet derived growth factor receptor (PDGFR) could be involved in the HF3-induced hemorrhagic process. In surface plasmon resonance assays, various proteoglycans were demonstrated to interact with HF3, and their incubation with HF3 showed degradation or limited proteolysis. Likewise, Western blot analysis showed in vivo degradation of biglycan, decorin, glypican, lumican and syndecan in the HF3-induced hemorrhagic process. Moreover, antithrombin III, complement components C3 and C4, factor II and plasminogen were cleaved in vitro by HF3. Notably, HF3 cleaved PDGFR (alpha and beta) and PDGF in vitro, while both receptor forms were detected as cleaved in vivo in the hemorrhagic process induced by HF3. These findings outline the multifactorial character of SVMP-induced tissue damage, including the transient activation of tissue proteinases, and underscore for the first time that endothelial glycocalyx proteoglycans and PDGFR are targets of SVMPs in the disruption of microvasculature integrity and generation of hemorrhage.

Clinical and Epidemiological Aspects of Accidents by Venomous Animals in Mâncio Lima, a Western Amazonian City

Aims: To characterize the frequency and clinical characteristics of venomous animals’ accidents in Mâncio Lima, Acre.
 Study Design: A cross-sectional study.
 Place and Duration of Study: The study took place in Mâncio Lima, Acre, Brazil, between 2013 and 2015.
 Methodology: We included a cohort of 350 households (estimated to be 1,500 people of all ages) in the urban area of Mâncio Lima. The following questionnaires were applied: I. Occurrence of accidents by venomous animals and clinical characterization of accidents; II. The detailed description of households.
 Results: There were 111 (8%) accidents with snakes, 138 (9.9%) accidents with scorpions, 108 (7.8%) accidents with spiders and 99 (7.1%) accidents with stingrays. Bothrops jararaca was the most cited snake, being edema (local and systemic) and muscular pain the main symptoms. In relation to scorpionism and arachnidism, the hands were the body site of the greatest number of injuries, with local pain/tingling and pain/blistering being the main symptoms, respectively. Accidents by stingrays occurred mostly in the shallow part of the river; feet and legs were the main body sites affected and local pain/bleeding were the main symptoms.
 Conclusion: In Mâncio Lima, there was a severe frequency of envenomation in the population, specifically in low-income brown/black male rural workers with low educational level. A counter action is required with public health measures that protects the inhabitants of the region, offering greater hospital care and wide application of serum for everyone who needs it. It is also important to educate rural workers on venomous animals and preventive measures to avoid accidents.

Anti-inflammatory effect of Bougainvillea × buttiana (Var. Orange) extract in experimental Bothrops jararaca envenomation

Background: Bougainvillea x buttiana is a plant used in folk Mexican medicine to treat different inflammatory diseases. Objective: In this study, the anti-inflammatory effect of B. x buttiana orange extract (BxbO) was evaluated on edema formation, cytokine production, and lethality in mice in response to venom of Bothrops jararaca (VBj) snake. Materials and Methods: The BxbO extract was tested in vitro to determine its effect on phospholipase A2(PLA2) and in vivo for the formation of edema, the paw edema model was used, as well as the toxicity of the extract and the production of cytokines. Lethality induced by VBj, the survival percentage, was calculated. Results: BxbO extract significantly inhibited in vitro PLA2and in vivo, blocked the edema formation and cytokine production, and prevented lethality induced by VBj. The constituents of BxbO may bind to components of VBj, such as PLA2, thereby blocking the proteolytic action of the venom. In animals treated with BxbO extract injected 1 h after the venom injection, no difference was observed in the cytokine secretion. In contrast, for all mice treated with BxbO extract for 1 h before VBj administration or together with VBj, the pro-inflammatory cytokine secretion in the serum was attenuated and an exacerbated production anti-inflammatory cytokine. In the presence of the BxbO extract injected 1 h before the VBj injection or together with the VBj injection, mortality was significantly lower. Conclusion: Altogether, our results show that BxbO extract can inhibit the local and systemic activities of VBj. However, new studies are still required to identify the interaction mechanisms between bioactive compounds and cellular components.

Extracellular vesicles from Bothrops jararaca venom: Composition and initial assessment of biological functions

Extracellular vesicles from Bothrops jararaca venom: Composition and initial assessment of biological functions

Hemorrhagic metalloproteinase HF3 from Bothrops jararaca venom: Obtention of recombinant domains dc using a cell-free expression system, and interaction with endothelial cells

Hemorrhagic metalloproteinase HF3 from Bothrops jararaca venom: Obtention of recombinant domains dc using a cell-free expression system, and interaction with endothelial cells

Characterization of microvesicles released from human whole blood after incubation with Bothrops jararaca venom: An in vitro preliminary study

Characterization of microvesicles released from human whole blood after incubation with Bothrops jararaca venom: An in vitro preliminary study

Renal effects induced by HF3, a metalloproteinase isolated from Bothrops jararaca venom, in a murine model

Renal effects induced by HF3, a metalloproteinase isolated from Bothrops jararaca venom, in a murine model

Ability of tannic acid administered intraperitoneally against the lethality of Bothrops jararaca venom

Ability of tannic acid administered intraperitoneally against the lethality of Bothrops jararaca venom

The venom variability of the Bothrops jararaca complex and its correlation with the speciation processes in continental islands

The venom variability of the Bothrops jararaca complex and its correlation with the speciation processes in continental islands

Cytotoxic mechanism of Bothrops jararaca venom mediated by mitochondrial depolarization

Snake venoms are natural sources of proteins and peptides with several biological activities. Therefore, this study aimed to evaluate the <em>in vitro</em> toxicity of crude <em>Bothrops jararaca</em> (<em>B. jararaca</em>) venom on different human tumor cells and investigate its action mechanism.

Potential use of extract of the plant Schwartiza brasiliensis (choisy) bedell ex gir.-Cañas against the toxic effects of the venom of Bothrops jararaca or B. jararacussu

Envenomation by snakes is a worldwide health public issue, and antivenoms are less efficient in neutralizing local toxic effects. Thus, more efficient therapies to treat patients deserve attention, and plants have been extensively tested. So, the aim of this work was to evaluate the effect of the aqueous fraction of the plant Schwartzia brasiliensis to inhibit some toxic activities of Bothrops jararaca or B. jararacussu venom. S. brasiliensis inhibited coagulant, hemolytic, proteolytic, hemorrhagic, edematogenic, and lethal activities of both venoms, regardless if plant was mixed together with venoms or injected after them as well as the route of administration (intravenous, oral or subcutaneous) of the plant. The S. brasiliensis extract showed no toxicity to mice or red blood cells. Thus, S. brasiliensis may be useful as an alternative treatment for snakebite envenomation and aid antivenom therapy to neutralize relevant toxic activities in patients bitten by Bothrops species.

Effect of Snake Venom Metalloprotease Desintegrin in Hepatocarcinoma Tumor Cells

Hepatocellular carcinoma is the third leading cause of cancer-related death in the world. This cancer is associated with cirrhosis following the hepatitis B or C virus infection, alcohol addiction, metabolic liver disease and exposure to dietary toxins such as aflatoxins and aristolochic acid. Studies demonstrate the integration of the HBV genome into liver cell DNA, including cases of patients with HBV-negative serology. Despite advances in prevention techniques, screening, and technology in cancer diagnosis and treatments, the incidence and mortality remain worrisome. Therefore, this research is significant due to the contribution of the development of new biological agents that can be used as monotherapy or adjuvant chemotherapy. Jararhagin, a snake toxin isolated from Bothrops jararaca venom, has been the subject of many studies seeking alternatives for the treatment of cancer. This protein contains the cysteine-rich disintegrin-like metalloproteinase domains and desirable functions to combat tumor cells, such as promoting acute inflammation, damaging the vascular endothelium through the zinc-dependent catalytic domain (responsible for hemorrhagic function) and enzymatically degrading the constituents of the endothelial basement membrane. Due to the antitumor effects of jararhagin presented in previous research, this study aimed to describe the possible antitumor effects of this snake metalloprotease in the murine liver tumor, intending to propose a new therapeutic option in the human liver tumor.

Engineered protein containing crotoxin epitopes induces neutralizing antibodies in immunized rabbits

Crotoxin (Ctx) is the main lethal component of Crotalus durissus terrificus venom. It is a neurotoxin, composed of two subunits associated by noncovalent interactions, the non-toxic acid subunit (CA), named Crotapotin, and the basic subunit (CB), with phospholipase A2 (PLA2) activity. Employing the SPOT synthesis technique, we determined two epitopes located in the C-terminal of each Ctx subunit. In addition, 3 other epitopes were mapped in different regions of Ctx using subcutaneous spot implants surgically inserted in mice. All epitopes mapped here were expressed together as recombinant multi-epitopic protein (rMEPCtx), which was used to immunize New Zealand rabbits. Anti-rMEPCtx rabbit serum cross-reacted with Ctx and crude venoms from C. d. terrificus, Crotalus durissus ruruima, Peruvian C. durissus and Bothrops jararaca (with lower intensity). Furthermore, anti-rMEPCtx serum was able to neutralize Ctx lethal activity. As the recombinant multiepitopic protein is not toxic, it can be administered in larger doses without causing adverse effects on the immunized animals health. Therefore, our work evidences the identification of neutralizing epitopes of Ctx and support the use of recombinant multiepitopic proteins as an innovation to immunotherapeutics production.

In-depth transcriptome reveals the potential biotechnological application of Bothrops jararaca venom gland.

Background:Lack of complete genomic data of Bothrops jararaca impedes molecular biology research focusing on biotechnological applications of venom gland components. Identification of full-length coding regions of genes is crucial for the correct molecular cloning design. Methods:RNA was extracted from the venom gland of one adult female specimen of Bothrops jararaca. Deep sequencing of the mRNA library was performed using Illumina NextSeq 500 platform. De novo assembly of B. jararaca transcriptome was done using Trinity. Annotation was performed using Blast2GO. All predicted proteins after clustering step were blasted against non-redundant protein database of NCBI using BLASTP. Metabolic pathways present in the transcriptome were annotated using the KAAS-KEGG Automatic Annotation Server. Toxins were identified in the B. jararaca predicted proteome using BLASTP against all protein sequences obtained from Animal Toxin Annotation Project from Uniprot KB/Swiss-Pro database. Figures and data visualization were performed using ggplot2 package in R language environment. Results:We described the in-depth transcriptome analysis of B. jararaca venom gland, in which 76,765 de novo assembled isoforms, 96,044 transcribed genes and 41,196 unique proteins were identified. The most abundant transcript was the zinc metalloproteinase-disintegrin-like jararhagin. Moreover, we identified 78 distinct functional classes of proteins, including toxins, inhibitors and tumor suppressors. Other venom proteins identified were the hemolytic lethal factors stonustoxin and verrucotoxin. Conclusion:It is believed that the application of deep sequencing to the analysis of snake venom transcriptomes may represent invaluable insight on their biotechnological potential focusing on candidate molecules.

Toxicological effects of bioactive peptide fractions obtained from Bothrops jararaca snake venom on the structure and function of mouse seminiferous epithelium.

Background:Pathogenesis of Bothrops envenomations is complex and despite numerous studies on the effects of this snake venom on various biological systems, relatively little is known about such effects on the male reproductive system. In the present study, the toxicological outcomes of the low molecular weight fraction (LMWF) of B. jararaca snake venom - containing a range of bioactive peptides - were investigated on the dynamics and structure of the seminiferous epithelium and 15P-1 Sertoli cells viability.Methods:LMWF (5 µg/dose per testis) venom was administered in male Swiss mice by intratesticular (i.t.) injection. Seven days after this procedure, the testes were collected for morphological and morphometric evaluation, distribution of claudin-1 in the seminiferous epithelium by immunohistochemical analyses of testes, and the nitric oxide (NO) levels were evaluated in the total extract of the testis protein. In addition, the toxicological effects of LMWF and crude venom (CV) were analyzed on the 15P-1 Sertoli cell culture.Results:LMWF induced changes in the structure and function of the seminiferous epithelium without altering claudin-1 distribution. LMWF effects were characterized especially by lost cells in the adluminal compartment of epithelium (spermatocytes in pachytene, preleptotene spermatocytes, zygotene spermatocytes, and round spermatid) and different stages of the seminiferous epithelium cycle. LMWF also increased the NO levels in the total extract of the testis protein and was not cytotoxic in concentrations and time tested in the present study. However, CV showed cytotoxicity at 10 μg/mL from 6 to 48 h of treatment.Conclusions: The major finding of the present study was that the LMWF inhibited spermatozoa production; principally in the spermiogenesis stage without altering claudin-1 distribution in the basal compartment. Moreover, NO increased by LMWF induce open of complexes junctions and release the germ cells of the adluminal compartment to the seminiferous tubule.

A complex biogeographic history of diversification in Neotropical lancehead pitvipers (Serpentes, Viperidae)

AbstractBased on the literature, we had predicted that the diversification within the Neotropical snake genusBothropsoccurred along a latitudinal gradient from north to south, with diversification into unoccupied niches through ecological opportunity, not correlated with geoclimatic events. Using a dated phylogeny and estimating likelihoods of ancestral states at cladogenesis events, we reconstructed ancestral areas and assessed major events of the diversification ofBothropsclades, and we also discuss systematic implications for this group. Based on the phylogeny we produced,B. lojanuswas not considered as part of the genusBothropssince the results recovered this species nested within theBothrocophiasclade. We infer that the diversification of the MioceneBothrops pictusandBothrops alternatusclades may be related to the uplift of the western slopes of the Andes and the Argentinian Patagonian Andes, respectively. The PlioceneBothrops taeniatusandBothrops osborneiclades may be related to the uplift of the eastern and northern Andes, respectively. The Plio‐PleistoceneBothrops neuwiediclade may be related to the habitat transitions from a warmer and forested environment to a cooler and open landscape; theBothrops jararaca(i.e. island endemic species) andBothrops lanceolatusclades to over‐water dispersal with island speciation; andBothrops atroxclade to the appearance of the Panamanian land bridge. We found that a multitemporal and multidirectional history of diversification may be correlated with geoclimatic and dispersalist events. We argue that the vacant niche hypothesis by itself does not explainBothropsdiversification.

Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain

Snake venom metalloproteinases (SVMPs) are key toxins involved in local inflammatory reactions after snakebites. This study aimed to investigate the effect of SVMP domains on the alterations in leukocyte-endothelium interactions in the microcirculation of mouse cremaster muscle. We studied three toxins: BnP1, a PI-toxin isolated from Bothrops neuwiedi venom, which only bears a catalytic domain; Jararhagin (Jar), a PIII-toxin isolated from Bothrops jararaca venom with a catalytic domain, as well as ECD-disintegrin and cysteine-rich domains; and Jar-C, which is produced from the autolysis of Jar and devoid of a catalytic domain. All these toxins induced an increase in the adhesion and migration of leukocytes. By inhibiting the catalytic activity of Jar and BnP1 with 1.10-phenanthroline (oPhe), leukocytes were no longer recruited. Circular dichroism analysis showed structural changes in oPhe-treated Jar, but these changes were not enough to prevent the binding of Jar to collagen, which occurred through the ECD-disintegrin domain. The results showed that the catalytic domain of SVMPs is the principal domain responsible for the induction of leukocyte recruitment and suggest that the other domains could also present inflammatory potential only when devoid of the catalytic domain, as with Jar-C.

Utilization of the Plant Clusia Fluminensis Planch & Triana Against Some Toxic Activities of the Venom of Bothrops jararaca and B. jararacussu Snake Venom Toxic Activities.

In Brazil, the Bothrops genus accounts for 87% of registered snakebites, which are characterized by hemorrhage, tissue necrosis, hemostatic disturbances, and death. The treatment recommended by governments is the administration of specific antivenoms. Although antivenom efficiently prevents venom-induced lethality, it has limited efficacy in terms of preventing local tissue damage. Thus, researchers are seeking alternative therapies able to inhibit the main toxic effects of venoms, without compromising safety. The study aimed to test the ability of aqueous extracts of leaves, stems, and fruits of the plant Clusia fluminensis to neutralize some toxic effects induced by the venoms of Bothrops jararaca and Bothrops jararacussu. The plant extracts were incubated with venoms for 30 min. at 25 °C, and then in vitro (coagulant and proteolytic) and in vivo (hemorrhagic, myotoxic, and edematogenic) activities were evaluated. In addition, the extracts were administered to animals (by oral, intravenous or subcutaneous routes) before or after the injection of venom samples, and then hemorrhage and edema assays were performed. In addition, a gel solution of the fruit extract was produced and tested in terms of reducing hemorrhage effects. A chemical prospection was performed to identify the main classes of compounds present in the extracts. All the extracts inhibited the activities of the two venoms, regardless of the experimental protocol or route of administration of the extracts. Moreover, the gel of the fruit extract inhibited the venom-induced-hemorrhage. The extracts comprised of tannins, flavonoids, saponins, steroids, and terpenoids. Antivenom properties of C. fluminensis extracts deserve further investigation in order to gain detailed knowledge regarding the neutralization profile of these extracts.