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Crotalus neutralizing factor (CNF) is an endogenous glycoprotein from Crotalus durissus terrificus snake blood that inhibits secretory phospholipases A 2 (sPLA 2 ) from the Viperid but not from Elapid venoms (subgroups IA and IIA, respectively). In the present study, we demonstrated that CNF can inhibit group III-PLA 2 from bee venom by forming a stable enzyme-inhibitor complex. This finding opens up new possibilities for the potential use of CNF and/or CNF-based derivatives in the therapeutics of bee stings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In Colombia, South America, there is a subspecies of the South American rattlesnake Crotalus durissus , C. d. cumanensis , a snake of the Viperidae family, whose presence has been reduced due to the destruction of its habitat. It is an enigmatic snake from the group of pit vipers, venomous, with large articulated front fangs, special designs on its body, and a characteristic rattle on its tail. Unlike in Brazil, the occurrence of human envenomation by C. durisus in Colombia is very rare and contributes to less than 1% of envenomation caused by snakes. Its venom is a complex cocktail of proteins with different biological effects, which evolved with the purpose of paralyzing the prey, killing it, and starting its digestive process, as well as having defense functions. When its venom is injected into humans as the result of a bite, the victim presents with both local tissue damage and with systemic involvement, including a diverse degree of neurotoxic, myotoxic, nephrotoxic, and coagulopathic effects, among others. Its biological effects are being studied for use in human health, including the possible development of analgesic, muscle relaxant, anti-inflammatory, immunosuppressive, anti-infection, and antineoplastic drugs. Several groups of researchers in Brazil are very active in their contributions in this regard. In this work, a review is made of the most relevant biological and medical aspects related to the South American rattlesnake and of what may be of importance for a better understanding of the snake C. d. cumanensis , present in Colombia and Venezuela.

The most enigmatic group of rattlesnakes is the long-tailed rattlesnake group, consisting of three species: Crotalus ericsmithi, Crotalus lannomi and Crotalus stejnegeri. These species have been the least studied rattlesnakes in all aspects, and no study on the characterization of their venoms has been carried out to date. Our main objective was to investigate the proteomic composition, as well as some of the biochemical and toxic activities of these venoms, and their neutralization by commercial antivenom. The venom proteome of C. ericsmithi mainly contains metalloproteinases (SVMP; 49.3%), phospholipases A 2 (PLA 2 ; 26.2%), disintegrins (Dis; 12.6%), and snake venom serine proteases (SVSP; 6.8%), while C. lannomi venom mainly consists of SVMP (47.1%), PLA 2 (19.3%), Dis (18.9%), SVSP (6%) and l-amino acid oxidase (LAAO; 2.6%). For these venoms high lethality was recorded in mice, the most potent being that of C. lannomi (LD 50 of 0.99 μg/g body weight), followed by C. ericsmithi (1.30 μg/g) and finally C. stejnegeri (1.79 μg/g). The antivenoms Antivipmyn® from SILANES and Fabotherapic polyvalent antiviperin® from BIRMEX neutralized the lethal activity of the three venoms. Although this group of snakes is phylogenetically related to the C. viridis group, no neurotoxic components (crotoxin or crotoxin-like proteins) common in rattlesnakes were found in their venoms. This study expands current knowledge on the venoms of understudied snake species of the Mexican herpetofauna., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest associated with this manuscript., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Intraspecific variation in snake venoms has been widely documented worldwide. However, there are few studies on this subject in Mexico. Venom characterization studies provide important data used to predict clinical syndromes, to evaluate the efficacy of antivenoms and, in some cases, to improve immunogenic mixtures in the production of antivenoms. In the present work, we evaluated the intraspecific venom variation of Crotalus basiliscus, a rattlesnake of medical importance and whose venom is used in the immunization of horses to produce one of the Mexican antivenoms. Our results demonstrate that there is variation in biological and biochemical activities among adult venoms and that there is an ontogenetic change from juvenile to adult venoms. Juvenile venoms were more lethal and had higher percentages of crotamine and crotoxin, while adult venoms had higher percentages of snake venom metalloproteases (SVMPs). Additionally, we documented crotoxin-like PLA 2 variation in which specimens from Zacatecas, Sinaloa and Michoacán (except 1) lacked the neurotoxin, while the rest of the venoms had it. Finally, we evaluated the efficacy of three lots of Birmex antivenom and all three were able to neutralize the lethality of four representative venoms but were not able to neutralize crotamine. We also observed significant differences in the LD 50 values neutralized per vial among the different lots. Based on these results, we recommend including venoms containing crotamine in the production of antivenom for a better immunogenic mixture and to improve the homogeneity of lots., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

In South America there are three snake genera with predominantly neurotoxic venoms: Crotalus, Micrurus and Hydrophis, which include nine species/subspecies, 97 species and a single marine species, respectively. Although accidents with neurotoxic venoms are less frequent than those with anticoagulant, cytotoxic or necrotic venoms (e.g. from Bothrops), they are of major public health importance. Venoms from genus Crotalus have been extensively studied, while data on the venoms from the other two genera are very limited, especially for Hydrophis. The venoms of North and South American Crotalus species show biochemical and physiopathological differences. The former species cause bothrops-like envenomation symptoms, while the latter mainly have neurotoxic and myotoxic effects, leading to respiratory paralysis and, occasionally, renal failure by myoglobinuria and death, often with no local lesions. Micrurus and Hydrophis also cause neurotoxic envenomations. Many studies have isolated, identified and characterized new enzymes and toxins, thus expanding the knowledge of snake venom composition. The present review summarizes the currently available information on neurotoxic venoms from South American snakes, with a focus on protein composition and toxicological properties. It also includes some comments concerning potential medical applications of elapid and crotalic toxins., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The proteomic, enzymatic, toxicological, and immunogenic profiles of the venom of C. d. pifanorum were studied. It was found that venom of C. d. pifanorum is composed of 63% phospholipases A 2 (PLA 2 s), 13% serine proteinases (SVSPs), 8% bradykinin-potentiating peptides (BPPs), 4% L-amino acid oxidases (LAAOs), 3% metalloproteinases (SVMPs), and other minor components. This composition allows the venom to exert lethal, PLA 2 , myotoxic, coagulant and defibrinogenating activities, but no azocaseinolytic or hemorrhagic activities. The addition of C. d. pifanorum venom to the group of venoms used as immunogens to produce the Central American antivenom PoliVal-ICP (i.e., venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys) resulted in 1) the expansion of the neutralization scope of the antivenom to cover the venom of C. d. pifanorum and other antigenically related venom (i.e., C. s. scutulatus venom), 2) improvement of the neutralizing potency towards the venom of C. simus, and 3) no significant reduction of the neutralization of venoms of B. asper and L. stenophrys. It was concluded that supplementation of the immunogens used to produce PoliVal-ICP with the venom of C. d. pifanorum is a viable alternative to expand the neutralization scope of the antivenom. BIOLOGICAL SIGNIFICANCE: The venom of Crotalus durissus pifanorum from Venezuela has a proteomic profile like those of other subspecies of the South American rattlesnake C. durissus, with predominance of phospholipases A 2 (especially crotoxin) and serine proteinases. This explains a toxicological profile characterized by neurotoxicity, myotoxicity, and coagulopathies, but being devoid of hemorrhagic activity. The antivenom used in Central America (PoliVal-ICP) includes the venom of C. simus, which has a different composition, in the immunizing strategy. Accordingly, this antivenom does not neutralize C. d. pifanorum venom. The addition of C. d. pifanorum venom to the immunizing mixture of PoliVal-ICP expands the neutralizing scope of this antivenom, to cover additional rattlesnake venoms, while not affecting the response to C. simus, Bothrops asper and Lachesis stenophrys venoms. This represents an improvement of the current PoliVal-ICP., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Snakebite in Mexico is commonly treated with an antivenom which uses Bothrops asper and Crotalus simus venoms as immunogens. Current taxonomic recommendations for the C. simus species complex suggest a novel endemic species from Mexico: Crotalus mictlantecuhtli. The aim of this report was to evaluate the immunogenic properties of C. mictlantecuhtli venom and its potential to generate polyclonal antibodies capable of neutralizing other pitviper venoms. We generated an experimental anti-Crotalus mictlantecuhtli serum, using the rabbit model, to test recognition and neutralizing capacity against the homologous venom as well as venoms from C. atrox, C.basiliscus, C. durissus terrificus, C. scutulatus salvini, C. tzabcan and Ophryacus sphenophrys. Pre-incubation neutralization experiments using our experimental serum showed positive results against venoms containing crotoxin, while venoms from two non-neurotoxic pit-vipers were not neutralized. Rescue experiments in mice showed that, when intravenously injected (i.v.), C. mictlantecuhtli venom is not neutralized by a maximum dose of Antivipmyn® and the experimental serum after 5 min of envenomation, albeit mice envenomated intraperitoneally (i.p.) and rescued i.v. with Antivipmyn® survived even at 50 min after envenomation. Our results highlight the importance of using the highly neurotoxic C. mictlantecuhtli venom to increase antivenom effectiveness against Mexican neurotoxic pitvipers., (Copyright © 2020. Published by Elsevier Ltd.)

Snakebites caused by Crotalus genus are the second most frequent in Brazil. Crotoxin is a beta-neurotoxin responsible for the main envenomation effects of Crotalus biting, while crotamine immobilizes the animal hind limbs, contributing to prey immobilization and to envenoming symptoms. As crotoxin and crotamine represent about 90% of Crotalus venom dry weight, these toxins are of great importance for antivenom therapy. In this sense, knowledge regarding the antigenicity/immunogenicity at the molecular level of these toxins can provide valuable information for the improvement of specific antivenoms. Therefore, the aims of this study are the identification of the B-cell epitopes from crotoxin and crotamine; and the characterization of the neutralizing potency of antibodies directed against the corresponding synthetic epitopes defined in the current study. Linear B-cell epitopes were identified using the Spot Synthesis technique probed with specific anti-C. d. terrificus venom horse IgG. One epitope of crotamine (F 12 PKEKICLPPSSDFGKMDCRW 32 ) and three of crotoxin (L 10 LVGVEGHLLQFNKMIKFETR 30 ; Y 43 CGWGGRGRPKDATDRCCFVH 63 and T 118 YKYGYMFYPDSRCRGPSETC 138 ) were identified. After synthesis in their soluble form, the peptides mixture correspondent to the mapped epitopes was entrapped in liposomes and used as immunogens for antibody production in rabbits. Anti-synthetic peptide antibodies were able to protect mice from the lethal activity of C. d. terrificus venom., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: The snakes in Venezuela vary in their different venom composition amid the species. In this sense, studies have been carried out elucidating mechanisms related to their immunostimulatory and/or immunosuppressive effects in vitro, measuring inhibition or stimulation on the mice spleen and lymph nodes lymphocytes under the rattlesnake ( Crotalus durissus cumanensis ) ( Cdc ) and mapanare ( Bothrops colombiensis ) crude venoms actions, and also its purified fraction crotoxin (CTX) ( Cdc ) and a semi-purified fraction (SPF) ( Bc ) activities. Material and methods: The stimulation of lymphocyte proliferation was carried out in the presence or absence of Concanavalin A (ConA) and lipopolysaccharides (LPS). Results: The lymphocyte response was measured by the Alamar Blue ® (Resazurin) assay, observing that the Crotalus crude venom increased basal proliferation in the spleen and lymph nodes, being also increased with ConA and LPS. CTX slightly decreased the proliferative response in the presence of mitogens. Both Bc venom and its SPF fraction had no significant effect on basal proliferation in the spleen and lymph nodes, but a decrease in the response with ConA was observed. These results suggest that CTX has an inhibitory action on lymphocyte proliferation, while Cdc crude venom has a stimulatory action on T and B cell populations. Bothrops colombiensis venom had no effect on these two types of cell populations. As it is known, lymphocytes are cells of enormous flexibility and can operate in diverse aspects, warranting that the correct immune response persists controlled. Conclusions: These results suggested that these different toxins can modulate lymphocyte functional activation toward an inhibitory or stimulatory state.

The most abundant protein families in viper venoms are Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs) and Phospholipases (PLA 2 s). These are primarily responsible for the pathophysiology caused by the bite of pit-vipers; however, there are few studies that analyze the pharmacokinetics (PK) of whole venom (WV) and its protein families. We studied the pathophysiology, PK profile and differential absorption of representative toxins from venom of Neotropical Rattlesnake ( Crotalus simus ) in a large animal model (ovine). Toxins studied included crotoxin (the main lethal component), which causes moderate to severe neurotoxicity; SVSPs, which deplete fibrinogen; and SVMPs, which cause local tissue damage and local and systemic hemorrhage. We found that Whole Venom (WV) was highly bioavailable (86%) 60 h following intramuscular (IM) injection, and extrapolation suggests that bioavailability may be as high as 92%. PK profiles of individual toxins were consistent with their physicochemical properties and expected clinical effects. Lymph cannulated animals absorbed 1.9% of WV through lymph during the first 12 h. Crotoxin was minimally detectable in serum after intravenous (IV) injection; however, following IM injection it was detected in lymph but not in blood. This suggests that crotoxin is quickly released from the blood toward its tissue targets.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED 50 and LD 50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a β-neurotoxin phospholipase A 2 -like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED 50 ) was 0.94 (p value < 0.05). In conclusion, slowness in spontaneous thrombin/fibrin generation even after recalcification elicit time lapse sufficient for elaboration of one dose-response curve to pro- or anti-coagulant agonists in CPS. We propose this methodology as an alternative and sensitive assay for assessing antivenom neutralizing ability in plasma of immunized horses as well as for in-process quality control., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Neutrophils have a critical role in the innate immune response; these cells represent the primary line of defense against invading pathogens or tissue injury. Crotoxin (CTX), the major toxin of the South American rattlesnake (Crotalus durissus terrificus) venom, presents longstanding anti-inflammatory properties, inhibiting neutrophil migration and phagocytosis by peritoneal neutrophils for 14 days. Herein, to elucidate these sustained inhibitory effects induced by CTX, we performed in vitro and in vivo studies evaluating the functionality of bone marrow neutrophils and possible molecular mechanisms associated with these effects. CTX inhibited the processes of chemotaxis, adhesion to fibronectin, and phagocytosis of opsonized particles; however, it did not affect ROS production or degranulation in bone marrow neutrophils. To understand the molecular mechanisms that orchestrate this effect, we investigated the expression of CR3 on the neutrophil surface and the total expression and activity of signaling proteins from the Syk-GTPase pathway, which is involved in actin polymerization. CTX down-regulated both subunits of CR3, as well as, the activity of Syk, Vav1, Cdc42, Rac1 and RhoA, and the expression of the subunit 1B from Arp2/3. Together, our findings demonstrated that CTX inhibits the functionally of bone marrow neutrophils and that this effect may be associated with an impairment of the Syk-GTPase pathway. This study demonstrates, for the first time, that the sustained down-modulatory effect of CTX on circulating and peritoneal neutrophils is associated with functional modifications of neutrophils still in the bone marrow, and it also contributes to a better understanding of the anti-inflammatory effect of CTX., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Deletion of Phe508 in the nucleotide binding domain (∆F508-NBD1) of the cystic fibrosis transmembrane regulator (CFTR; a cyclic AMP-regulated chloride channel) is the most frequent mutation associated with cystic fibrosis. This mutation affects the maturation and gating of CFTR protein. The search for new high-affinity ligands of CFTR acting as dual modulators (correctors/activators) presents a major challenge in the pharmacology of cystic fibrosis. Snake venoms are a rich source of natural multifunctional proteins, potential binders of ion channels. In this study, we identified the CB subunit of crotoxin from Crotalus durissus terrificus as a new ligand and allosteric modulator of CFTR. We showed that CB interacts with NBD1 of both wild type and ∆F508CFTR and increases their chloride channel currents. The potentiating effect of CB on CFTR activity was demonstrated using electrophysiological techniques in Xenopus laevis oocytes, in CFTR-HeLa cells, and ex vivo in mouse colon tissue. The correcting effect of CB was shown by functional rescue of CFTR activity after 24-h ΔF508CFTR treatments with CB. Moreover, the presence of fully glycosylated CFTR was observed. Molecular docking allowed us to propose a model of the complex involving of the ABCβ and F1-like ATP-binding subdomains of ΔF508-NBD1. Hydrogen-deuterium exchange analysis confirmed stabilization in these regions, also showing allosteric stabilization in two other distal regions. Surface plasmon resonance competition studies showed that CB disrupts the ∆F508CFTR-cytokeratin 8 complex, allowing for the escape of ∆F508CFTR from degradation. Therefore CB, as a dual modulator of ΔF508CFTR, constitutes a template for the development of new anti-CF agents., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Crotoxin (Crtx), the main toxin in the venom of Crotalus durissus terrificus snake, is a heterodimer with a basic subunit, CB, and an acidic subunit, CA. CB is a phospholipase A2 that depends on CA to specifically bind to the cell membrane. This toxin acts in the central nervous system (CNS) causing chronic seizure effects and other cytotoxic effects. Here, we report its action on glutamate release in rat cerebral cortex synaptosomes. Aiming at a better understanding of the mechanism of action of Crtx, calcium channel blockers were used and internalization studies were performed in cerebellar granule neurons. Our results show that Crtx induces calcium-dependent glutamate release via N and P/Q calcium channels. In addition, the CB subunit of Crtx is shown to be internalized. This internalization does not depend on the presence of CA subunit neither on the PLA2 activity of CB. A correlation between CB internalization and glutamate release remains to be established., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In accidents involving Crotalus snakes, the crotoxin complex (CTX) plays lethal action due to its neurotoxic activity. On the other hand, CTX have potential biotechnological application due to its anti-tumoral, anti-inflammatory, antimicrobial, analgesic and immunomodulatory properties. CTX is a heterodimer composed of Crotoxin A (CA or crotapotin), the acidic nontoxic and non-enzymatic component and; Crotoxin B (CB), a basic, toxic and catalytic PLA 2. Currently, there are two classes of CTX isoforms, whose differences in their biological activities have been attributed to features presented in CB isoforms. Here, we present the crystal structure of CB isolated from the Crotalus durissus collilineatus venom. It amino acid sequence was assigned using the SEQUENCE SLIDER software, which revealed that the crystal structure is a heterodimer composed of two new CB isoforms (col CB-A and col CB-B). Bioinformatic and biophysical analyses showed that the toxin forms a tetrameric assembly in solution similar to CB from Crotalus durissus terrificus venom, despite some differences observed at the dimeric interface. By the previously proposed classification, the col CB-B presents features of the class I isoforms while col CB-A cannot be classified into classes I and II based on its amino acid sequence. Due to similar features observed for other CB isoforms found in the NCBI database and the results obtained for col CB-A, we suggest that there are more than two classes of CTX and CB isoforms in crotalic venoms. • The crystal structure of CB from C. d. collilineatus venom was obtained. • Crystal structure is a heterodimer composed of two new CB isoforms. • Bioinformatic and biophysical analyses showed that the toxin is tetrameric. • col CB-A isoform cannot be classified into classes I and II based on its sequence. • We hypothesized that there are more than two classes of CB isoforms. [ABSTRACT FROM AUTHOR]

The growing problem of antibiotic resistance among bacteria requires searching for new therapeutic agents with bacteriostatic and/or bactericidal properties. Crotoxin is a β-neurotoxin from the venom of the Crotalus durissus terrificus. It is composed of two subunits: CA (non-active) and CB (with phospholipase A2 activity). It has already been shown that the isolated CB, but not the CA, subunit of crotoxin exhibits an antibacterial activity towards a variety of Gram-positive and Gram-negative bacterial species. However, no studies on the whole crotoxin complex have been carried out so far. We tested the antibacterial properties of crotoxin, as well as its isolated CB subunit, towards Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6535, Micrococcus luteus ATCC 10240, Escherichia coli ATCC 25922, Escherichia coli ATCC 8739, and Pseudomonas aeruginosa ATCC 10145. Both toxins exhibited antibacterial properties only against Micrococcus luteus ATCC 10240. Crotoxin showed only bacteriostatic activity with a MIC of 46 µM, while the CB subunit acted as both a bacteriostatic and bactericidal agent with a MIC = MBC = 0.21 μM. The bacteriostatic effect of the toxins was independent of the enzymatic activity of the CB subunit. Bactericidal properties, however, require phospholipase A2 activity. Both toxins reduced bacteria viability at the MIC by 72% and 85% for crotoxin- and CB-treated bacteria, respectively. The membrane permeability increased approximately three times within the first hour of incubation with toxins; afterwards, either no significant changes or a decrease of membrane permeability, compared to the control cells, were observed. We isolated a single, approximately 30 kDa bacterial wall protein which belongs to the NlpC/P60 family that interacts with crotoxin leading to the inhibition of bacterial growth. Neither crotoxin nor the CB subunit showed any cytotoxic properties to human fibroblasts at the MIC during the three-day incubation. [ABSTRACT FROM AUTHOR]

Here we report, for the first time, a natural hybrid between Crotalus atrox and C. mictlantecuhtli based on intermediate characteristics of the external morphology and venom. Morphologically, the individual had characteristics of both parent species. The hybrid's venom exhibited an intermediate composition including the presence of crotoxin which has never been documented in C. atrox but is well documented in C. mictlantecuhtli. The hybrid's venom was highly toxic and showed an intermediate proteolytic activity between the parental species. The two Mexican antivenoms were able to neutralize the hybrid's venom's lethality. [Display omitted] • Hybridization between Crotalus atrox and C. mictlantecuhtli is documented for the first time. • The external morphology of the hybrid exhibited characteristics of both parental species. • The venom of the hybrid presented intermediate venom composition between C. atrox and C. mictlantecuhtli. • The hybrid's venom has high lethal activity due to the presence of a crotoxin-like molecule. [ABSTRACT FROM AUTHOR]

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Introduction: Ophidian accidents are neglected events in tropical and in developing countries, including Brazil. Serpents of the Crotalus genus (rattlesnakes) are those that produce the highest case-fatality in the country. Purpose: to address the main aspects of the accident caused by Crotalus, with emphasis on the snakes biology, the clinical approach to snake bites and the therapeutic properties of the venom of these animals. Methods: literature review with a defined search strategy, using the PubMed, LILACS and SciELO databases. Results: Inoculation of crotalic venom produces discrete local signs, but systemic repercussions can occur, especially neurological alterations and acute renal failure. Laboratory evaluation is important to help distinguish from other ophidian accidents and to estimate the severity of the condition. Anticrotalic serum must be administered as soon as possible, based on the estimated amount of inoculated venom. Most of the morbid events occur in the rainy season (higher temperature), mainly affecting young men who work in rural areas. The adoption of protective measures and health education, aimed at the population most commonly involved, are relevant strategies for preventing and reducing the number of cases. In addition, crotalic venoms have antimicrobial actions, antiplatelets functions and ophthalmological applicability (strabismus). Conclusion: Knowledge of the different aspects of crotalic injuries is essential for an adequate diagnostic and therapeutic approach to such morbid conditions. The pharmacological properties of crotalic venom components should be better investigated in the next few years, given the possibilities of their use for the treatment of different human diseases.

Varespladib (LY315920) is a synthetic phospholipase A 2 (PLA 2) inhibitor that has been demonstrating antiophidic potential against snake venoms that present PLA 2 neurotoxins. In this study, we evaluate the capacity of Varespladib to inhibit the neuromuscular effects of crotoxin (CTX), the main toxic component of Crotalus durissus terrificus snake venom, and its PLA 2 subunit (CB). We performed a myographic study to compare the neuromuscular effects of CTX or CB and the mixture of these substances plus Varespladib in mice phrenic nerve-diaphragm muscle preparations. CTX (5 μg/mL), CB (20 μg/mL), or toxin-inhibitor mixtures pre-incubated with different concentration ratios of Varespladib (1:0.25; 1:0.5; 1:1; w/w) were added to the preparations and maintained throughout the experimentation period. Myotoxicity was assessed by light microscopic analysis of diaphragm muscle after myographic study. CTX and CB blocked the nerve-evoked twitches, and only CTX induced histological alterations in diaphragm muscle. Pre-incubation with Varespladib abolished the muscle-paralyzing activity of CTX and CB, and also the muscle-damaging activity of CTX. These findings emphasize the clinical potential of Varespladib in mitigating the toxic effects of C. d. terrificus snakebites and as a research tool to advance the knowledge of the mechanism of action of snake toxins. • Crotoxin and CB induce a time-dependent blockade of ex vivo neuromuscular preparations. • Crotoxin promotes myotoxicity in mice diaphragm muscle. • Varespladib prevents the neuromuscular blockade induced by Crotoxin and CB. • Varespladib neutralizes the myotoxicity of CTX. • Varespladib is an important tool for the study of snake PLA 2 toxins. [ABSTRACT FROM AUTHOR]

Crotalus Neutralizing Factor (CNF) was the first phospholipase A 2 inhibitor isolated from the plasma of the South American rattlesnake (Crotalus duri ssus terrificus). Previous biochemical and biophysical studies demonstrate an interaction of CNF with Crotoxin (CTX), the main toxic component in the venom of these snakes. CTX promotes the blockade of neuromuscular transmission by a sum of neurotoxic and myotoxic activities. However, the ability of CNF to inhibit these activities has not been shown until the present study. We performed a myographic study to compare the neuromuscular effects of CTX and the mixture CTX plus CNF in mice phrenic nerve-diaphragm muscle preparations. CTX (5 μg/mL) alone, or pre-incubated with CNF (5, 20 or 50 μg/mL) for 15 min was added to the preparations and maintained throughout the experimentation period. Myotoxicity was assessed by light microscopic analysis of diaphragm muscle after myographic study. CTX (5 μg/mL) blocked both indirectly and directly evoked twitches in neuromuscular preparations. In addition, CTX induced histological alterations in diaphragm muscle. Pre-incubation with CNF (50 μg/mL) abolished both the muscle-paralyzing and muscle-damaging activities of CTX. Therefore, the present study confirms, through functional studies, the antiophidic potential of CNF. Image 1 • Crotalus Neutralizing Factor inhibits the ex vivo toxic activities of Crotoxin in neuromuscular preparations of mice. • Neurotoxicity of Crotoxin is neutralized by preincubation with Crotalus Neutralizing Factor. • Myotoxicity of Crotoxin is neutralized by preincubation with Crotalus Neutralizing Factor. • Crotalus Neutralizing Factor is a valuable pharmacological tool for studies of Crotoxin action. [ABSTRACT FROM AUTHOR]

Phospholipases A 2 (PLA 2 s) are main components of snake venoms. Several snake species possess endogenous PLA 2 inhibitors in their circulating blood, which are generally known as sbPLIs (an acronym for s nake b lood p hospho l ipase A 2 i nhibitors). The sbPLIs are categorized in three classes (alpha, beta or gamma) depending on the existence of distinguishing protein domains in their structure. The Crotalus durrissus terrificus venom has a highly neurotoxic PLA 2 - crotoxin (CTX) - in its composition and the self-protection of the snake is mainly ensured by a sbγPLI named CNF (standing for C rotalus n eutralizing f actor). In an attempt to find smaller molecules able to inhibit the catalytic activity of CTX, in the present study we used linear peptide arrays to identify CNF segments possibly involved in the interaction with the toxin. Five reacting segments were identified as possible interacting regions. The target peptides were synthesized and located in the in silico CNF structure. Although all of them are exposed to the solvent, high concentrations were needed to inhibit the PLA 2 activity of the whole venom or CTX. Limitations of the methodology employed and particular characteristics of CTX inhibition by CNF are discussed. [Display omitted] • Five reacting peptides were identified in CNF-CTX interaction by linear peptide arrays. • All peptides are exposed to the solvent in CNF structure. • Two of them inhibited the PLA 2 activity of CTX in the mM range. [ABSTRACT FROM AUTHOR]

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 IL1β), as well as protected cells against H 2 O 2 –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. [ABSTRACT FROM AUTHOR]

Cancer is the second leading cause of death worldwide, and despite the effort of standard treatments, the search for new tools against this disease is necessary. Importantly, it is known that the tumor microenvironment plays a crucial role in tumor initiation, progression, and response to therapies. Therefore, studies of potential drugs that act on these components are as critical as studies regarding antiproliferative substances. Through the years, studies of several natural products, including animal toxins, have been conducted to guide the development of medical compounds. In this review, we present the remarkable antitumor activities of crotoxin, a toxin from the rattlesnake Crotalus durissus terrificus , highlighting its effects on cancer cells and in the modulation of relevant elements in the tumor microenvironment as well as the clinical trials conducted with this compound. In summary, crotoxin acts through several mechanisms of action, such as activation of apoptosis, induction of cell cycle arrest, inhibition of metastasis, and decrease of tumor growth, in different tumor types. Crotoxin also modulates tumor-associated fibroblasts, endothelial cells, and immune cells, which contribute to its antitumoral effects. In addition, preliminary clinical studies confirm the promising results of crotoxin and support its potential future use as an anticancer drug. [ABSTRACT FROM AUTHOR]

The Crotalus durissus terrificus rattlesnake venom, its main toxin, crotoxin (CTX), and its crotapotin (CA) and phospholipase A2 (CB) subunits modulate the immune system. Formyl peptide receptors (FPRs) and lipoxin A4 (LXA4) are involved in CTX's effect on macrophages and neutrophils. Dendritic cells (DCs) are plasticity cells involved in the induction of adaptive immunity and tolerance maintenance. Therefore, we evaluated the effect of CTX, CA or CB on the maturation of DCs derived from murine bone marrow (BM). According to data, CTX and CB-but not CA-induced an increase of MHC-II, but not costimulatory molecules on DCs. Furthermore, CTX and CB inhibited the expression of costimulatory and MHC-II molecules, secretion of proinflammatory cytokines and NF-κBp65 and p38/ERK1/2-MAPK signaling pathways by LPS-incubated DCs. Differently, CTX and CB induced IL-10, PGE2 and LXA4 secretion in LPS-incubated DCs. Lower proliferation and IL-2 secretion were verified in coculture of CD3+ cells and DCs incubated with LPS plus CTX or CB compared with LPS-incubated DCs. The effect of CTX and CB on DCs was abolished in cultures incubated with a FPRs antagonist. Hence, CTX and CB exert a modulation on functional activity of DCs; we also checked the involvement the FPR family on cell activities. [ABSTRACT FROM AUTHOR]

Crotoxin is a heterodimeric ß-neurotoxin isolated from the venom of Crotalus durissus terrificus, consisting of two, non-covalently bound subunits, crotapotin (CA) and crotoxin B (CB). Both subunits present four different isoforms, consequently there are up to 16 different crotoxin complexes, each with different activity levels. The biological activities usually associated with crotoxin include neurotoxicity, myotoxicity and cardiotoxicity, however several other important biochemical and pharmacological effects of crotoxin have been observed, such as the antibacterial, antiviral, antiinflammatory, antitumoral and analgesic activity. In this study we present the production of recombinant crotoxin B (isoform C). Expression of the protein was carried out using E. coli RosettaTM (DE3)pLysS strain, and the obtained protein was separated and purified using Ni2+-affinity chromatography. To the best of our knowledge the developed method represents the first reported method for obtaining the crotoxin B (isoform C) through heterologous expression using an efficient and cost-effective procedure. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is one of the main causes of liver disease and transplantation worldwide. Current therapy is expensive, presents additional side effects and viral resistance has been described. Therefore, studies for developing more efficient antivirals against HCV are needed. Compounds isolated from animal venoms have shown antiviral activity against some viruses such as Dengue virus, Yellow fever virus and Measles virus. In this study, we evaluated the effect of the complex crotoxin (CX) and its subunits crotapotin (CP) and phospholipase A2 (PLA2-CB) isolated from the venom of Crotalus durissus terrificus on HCV life cycle. Huh 7.5 cells were infected with HCVcc JFH-1 strain in the presence or absence of these toxins and virus was titrated by focus formation units assay or by qPCR. Toxins were added to the cells at different time points depending on the stage of virus life cycle to be evaluated. The results showed that treatment with PLA2-CB inhibited HCV entry and replication but no effect on HCV release was observed. CX reduced virus entry and release but not replication. By treating cells with CP, an antiviral effect was observed on HCV release, the only stage inhibited by this compound. Our data demonstrated the multiple antiviral effects of toxins from animal venoms on HCV life cycle. [ABSTRACT FROM AUTHOR]

Background and purpose Crotoxin (CTX), a heterodimeric phospholipase A 2 (PLA 2 ) neurotoxin from Crotalus durissus terrificus snake venom, promotes irreversible blockade of neuromuscular transmission. Indirect electrophysiological evidence suggests that CTX exerts a primary inhibitory action on transmitter exocytosis, yet contribution of a postsynaptic action of the toxin resulting from nicotinic receptor desensitization cannot be excluded. Here, we examined the blocking effect of CTX on nerve-evoked transmitter release measured directly using radioisotope neurochemistry and video microscopy with the FM4-64 fluorescent dye. Experimental approach Experiments were conducted using mice phrenic-diaphragm preparations. Real-time fluorescence video microscopy and liquid scintillation spectrometry techniques were used to detect transmitter exocytosis and nerve-evoked [ 3 H]-acetylcholine ([ 3 H]ACh) release, respectively. Nerve-evoked myographic recordings were also carried out for comparison purposes. Key results Both CTX (5 μg/mL) and its basic PLA 2 subunit (CB, 20 μg/mL) had biphasic effects on nerve-evoked transmitter exocytosis characterized by a transient initial facilitation followed by a sustained decay. CTX and CB reduced nerve-evoked [ 3 H]ACh release by 60% and 69%, respectively, but only the heterodimer, CTX, decreased the amplitude of nerve-evoked muscle twitches. Conclusion and implications Data show that CTX exerts a presynaptic inhibitory action on ACh release that is highly dependent on its intrinsic PLA 2 activity. Given the high safety margin of the neuromuscular transmission, one may argue that the presynaptic block caused by the toxin is not enough to produce muscle paralysis unless a concurrent postsynaptic inhibitory action is also exerted by the CTX heterodimer. [ABSTRACT FROM AUTHOR]

This study investigated the antineoplastic effects of crotoxin isolated from snake venom of the South American Crotalus durissus terrificus in oral cancer cell lines and in an animal model of chemically induced oral cancer. We analyzed cell viability and death, clonogenic formation, DNA fragmentation, migration assay, and gene expression of MMP2, MMP9, COL1A1, and CASP3. In the animal model, after induction of oral cancer by 4-nitroquinoline-1-oxide carcinogen, mice were treated with crotoxin to investigate its effects on tumor development in tongue and oral mucosa. Crotoxin inhibited cell proliferation, viability, colony formation, and migration, favoring cell death. Furthermore, crotoxin increased caspase-3 expression, decreased Ki-67 protein and mRNA expression of MMP2, MMP9, and COL1A1. Mice treated with crotoxin at 10 μg/kg did not alter biochemical parameters total cholesterol, very-low-density lipoprotein, high-density lipoprotein, liver transaminases, glycemia, creatinine, and urea. Crotoxin treatment significantly reduced the frequency of oral squamous cell carcinoma lesions by 50%. Thus, this study highlights crotoxin as a promising chemotherapeutic substance, considering its effects on controlling the neoplastic cell population, reducing cell migration, and inhibiting tumor development. Clinical studies are necessary to understand better the impact of crotoxin as a potential adjuvant therapeutic agent for oral cancer patients. [Display omitted] • Crotoxin inhibits proliferation in oral cancer cells and promotes cell death. • Crotoxin decreases migration in oral cancer cells. • Crotoxin does not impair biochemical parameters or body weight in mice. • Crotoxin reduces lesions of oral squamous cell carcinoma in mice. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases (IBD) is the result of dysregulation of mucosal innate and adaptive immune responses. Factors such as genetic, microbial and environmental are involved in the development of these disorders. Accordingly, animal models that mimic human diseases are tools for the understanding the immunological processes of the IBD as well as to evaluate new therapeutic strategies. Crotoxin (CTX) is the main component of Crotalus durissus terrificus snake venom and has an immunomodulatory effect. Thus, we aimed to evaluate the modulatory effect of CTX in a murine model of colitis induced by 2,4,6- trinitrobenzene sulfonic acid (TNBS). The CTX was administered intraperitoneally 18 hours after the TNBS intrarectal instillation in BALB/c mice. The CTX administration resulted in decreased weight loss, disease activity index (DAI), macroscopic tissue damage, histopathological score and myeloperoxidase (MPO) activity analyzed after 4 days of acute TNBS colitis. Furthermore, the levels of TNF-α, IL-1β and IL-6 were lower in colon tissue homogenates of TNBS-mice that received the CTX when compared with untreated TNBS mice. The analysis of distinct cell populations obtained from the intestinal lamina propria showed that CTX reduced the number of group 3 innate lymphoid cells (ILC3) and Th17 population; CTX decreased IL-17 secretion but did not alter the frequency of CD4+Tbet+ T cells induced by TNBS instillation in mice. In contrast, increased CD4+FoxP3+ cell population as well as secretion of TGF-β, prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) was observed in TNBS-colitis mice treated with CTX compared with untreated TNBS-colitis mice. In conclusion, the CTX is able to modulate the intestinal acute inflammatory response induced by TNBS, resulting in the improvement of clinical status of the mice. This effect of CTX is complex and involves the suppression of the pro-inflammatory environment elicited by intrarectal instillation of TNBS due to the induction of a local anti-inflammatory profile in mice. [ABSTRACT FROM AUTHOR]

A myographic study was performed to compare the neuromuscular effects of venoms and crotoxin-like proteins from Crotalus durissus ruruima and Crotalus durissus cumanensis in mice phrenic-diaphragm preparation. It was concluded that both venoms present neurotoxic activity as a consequence of their crotoxin content. Furthermore, crotoxin from C.d. cumanensis is more potent than that from C.d. ruruima venom. At the concentration range in which both venoms express neurotoxic activity, only C.d. cumanensis venom also manifest a direct myotoxic effect that probably involves the synergic participation of other components than crotoxin. [ABSTRACT FROM AUTHOR]