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Crotoxin (CrTX) is a neurotoxin isolated from the venom of the South American rattlesnake. Previous studies demonstrated that CrTX was able to inhibit the activity of the growth factor receptor tyrosine kinase and that CrTX possesses potent antitumor activity when combined with Iressa, an epidermal growth factor receptor inhibitor. The aim of the present study was to determine the antitumor effect of CrTX and the combination of CrTX with Iressa in lung adenocarcinoma SPCA-1 cells. The results demonstrated that CrTX inhibited the cellular growth of SPCA-1 cells via G1 arrest and induction of apoptosis. In addition, the c-Jun N-terminal kinase pathway was important in CrTX-induced apoptosis in SPCA-1 cells. Notably, CrTX was able to significantly enhance the cytotoxic effects of Iressa in SPCA-1 cells. The in vivo antitumor assay demonstrated that treatment with either CrTX or Iressa significantly inhibited tumor growth, while the combination of CrTX and Iressa demonstrated the most significant antitumor activity, which was reflected by tumor weight and angiogenesis, presented as microvascular density. Therefore, the present study suggested that CrTX is a potential anti-lung cancer agent and sensitizer of Iressa. [ABSTRACT FROM AUTHOR]

Aim:Crotoxin (CrTX) is the primary toxin in South American rattlesnake (Crotalus durissus terrificus) venom, and exhibits antitumor and other pharmacological actions in vivo and in vitro. Here, we investigated the molecular mechanisms of the antitumor action of CrTX in human lung carcinoma cells in vitro.Methods:Human lung squamous carcinoma SK-MES-1 cells were tested. The cytotoxicity of CrTX was evaluated in both MTT and colony formation assays. Cell cycle was investigated with flow cytometry. Cell apoptosis was studied with Hoechst 33258 and Annexin V-FITC staining. The levels of relevant proteins were analyzed using Western blot assays.Results:CrTX (25, 50, 100 μmol/L) inhibited the growth and colony formation of SK-MES-1 cells in dose- and time-dependent manners. CrTX increased the proportion of S phase cells and dose-dependently induced cell apoptosis, accompanied by down-regulating the expression of proliferating cell nuclear antigen (PCNA), and increasing the level of cleaved caspase-3. Furthermore, CrTX dose-dependently increased the expression of autophagy-related proteins LC3-II and beclin 1, and decreased the level of p62 in the cells. Moreover, CrTX (50 μmol/L) significantly increased p38MAPK phosphorylation in the cells. Pretreatment of the cells with SB203580, a specific inhibitor of p38MAPK, blocked the inhibition of CrTX on cell proliferation, as well as CrTX-induced apoptosis and cleaved caspase-3 expression.Conclusion:The p38MAPK signaling pathway mediates CrTX-induced apoptosis and autophagy of human lung carcinoma SK-MES-1 cells in vitro. [ABSTRACT FROM AUTHOR]

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.)

Abstract: 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 &y& Elsevier]

Abstract: Crotoxin (CTX) is the main neurotoxic component of Crotalus durissus terrificus snake venom. It inhibits tumour growth and modulates the function of macrophages, which are essential cells in the tumour microenvironment. The present study investigated the effect of CTX on the secretory activity of monocultured macrophages and macrophages co-cultivated with LLC-WRC 256 cells. The effect of the macrophage secretory activities on tumour cell proliferation was also evaluated. Macrophages pre-treated with CTX (0.3 μg/mL) for 2 h were co-cultivated with LLC-WRC 256 cells, and the secretory activity of the macrophages was determined after 12, 24 and 48 h. The co-cultivation of CTX-treated macrophages with the tumour cells caused a 20% reduction in tumour cell proliferation. The production of both H2O2 and NO was increased by 41% and 29% after 24 or 48 h of co-cultivation, respectively, compared to the values for the co-cultures of macrophages of control. The level of secreted IL-1β increased by 3.7- and 3.2-fold after 12 h and 24 h of co-cultivation, respectively. Moreover, an increased level of LXA4 (25%) was observed after 24 h of co-cultivation, and a 2.3- and 2.1-fold increased level of 15-epi-LXA4 was observed after 24 h and 48 h, respectively. Boc-2, a selective antagonist of formyl peptide receptors, blocked both the stimulatory effect of CTX on the macrophage secretory activity and the inhibitory effect of these cells on tumour cell proliferation. Taken together, these results indicate that CTX enhanced the secretory activity of macrophages, which may contribute to the antitumour activity of these cells, and that activation of formyl peptide receptors appears to play a major role in this effect. [Copyright &y& Elsevier]

Abstract: Crotoxin, the main neurotoxic component of the venom of South American rattlesnake (Crotalus durissus terrificus), is reported to have potent antinociceptive activity. Several authors have shown mainly in behavioral pain models that crotoxin induces antinociceptive effects, supposed to be mediated by actions on the central nervous system. The antinociceptive effects of crotoxin (45 μg/kg ip) in rats were verified in this study by increased response latencies in a Hargreaves test and tail flick test. In addition, it was demonstrated that crotoxin does not lead to motor impairments during a rotarod test and open field test. The main objective, carried out by blood oxygen level dependent functional Magnetic Resonance Imaging (BOLD fMRI) in anesthetized rats, was to determine which specific brain structures are involved in these antinociceptive effects. Moreover, potential antihyperalgesic effects were investigated by inducing a local hyperalgesia on the left hind paw. Therefore, antinociceptive effects (right paw) and antihyperalgesic effects (left paw) of crotoxin were able to be differentiated. As a result, crotoxin exhibited dominant antihyperalgesic but also antinociceptive effects during pain stimulation. Reductions of BOLD signal already occurred in brain input structures but were most prominent in primary and secondary somatosensory cortices. In conclusion, BOLD fMRI in anesthetized rats proved to be a helpful tool in toxinology, particularly in unraveled mechanisms of modulating nociception in the central nervous system by (potential) analgesics like crotoxin. [Copyright &y& Elsevier]

Biochemical and biological differences in the venom of Crotalus durissus cumanensis from three ecoregions of Colombia were evaluated. Rattlesnakes were collected from the geographic areas of Magdalena Medio (MM), Caribe (CA) and Orinoquía (OR). All three regionally distributed venoms contain proteases, PLA 2 s and the basic subunit of crotoxin. However, only crotamine was detected in the CA venom. The highest lethality, coagulant, phospholipase A 2 and hyaluronidase activities were found in the MM venom. Also, some differences, observed by western blot and immunoaffinity, were found in all three venoms when using commercial antivenoms. Furthermore, all three eco-regional venoms showed intraspecific variability, considering the differences in the abundance and intensity of their components, in addition to the activity and response to commercial antivenoms.

Abstract: The composition and toxicological profile of the venom of the rattlesnake Crotalus simus in Mexico was analyzed at the subspecies and individual levels. Venoms of the subspecies C. s. simus, C. s. culminatus and C. s. tzabcan greatly differ in the expression of the heterodimeric neurotoxin complex ‘crotoxin’, with highest concentrations in C. s. simus, followed by C. s. tzabcan, whereas the venom of C. s. culminatus is almost devoid of this neurotoxic PLA2. This explains the large variation in lethality (highest in C. s. simus, which also exerts higher myotoxicity). Coagulant activity on plasma and fibrinogen occurs with the venoms of C. s. simus and C. s. tzabcan, being absent in C. s. culminatus which, in turn, presents higher crotamine-like activity. Proteomic analysis closely correlates with toxicological profiles, since the venom of C. s. simus has high amounts of crotoxin and of serine proteinases, whereas the venom of C. s. culminatus presents higher amounts of metalloproteinases and crotamine. This complex pattern of intraspecies venom variation provides valuable information for the diagnosis and clinical management of envenoming by this species in Mexico, as well as for the preparation of venom pools for the production and quality control of antivenoms. Biological significance: This study describes the variation in venom composition and activities of the three subspecies of Crotalus simus from Mexico. Results demonstrate that there is a notorious difference in these venoms, particularly regarding the content of the potent neurotoxic phospholipase A2 complex ‘crotoxin’. In addition, other differences were observed regarding myotoxic and coagulant activities, and expression of the myotoxin ‘crotamine’. These findings have implications in, at least, three levels: (a) the adaptive role of variations in venom composition; (b) the possible differences in the clinical manifestations of envenomings by these subspecies in Mexico; and (c) the design of venom mixtures for the preparation of antivenoms effective in the neutralization of the venoms of the three subspecies. [Copyright &y& Elsevier]

Background: Understanding the processes that drive the evolution of snake venom is a topic of great research interest in molecular and evolutionary toxinology. Recent studies suggest that ontogenetic changes in venom composition are genetically controlled rather than environmentally induced. However, the molecular mechanisms underlying these changes remain elusive. Here we have explored the basis and level of regulation of the ontogenetic shift in the venom composition of the Central American rattlesnake, Crotalus s. simus using a combined proteomics and transcriptomics approach. Results: Proteomic analysis showed that the ontogenetic shift in the venom composition of C. s. simus is essentially characterized by a gradual reduction in the expression of serine proteinases and PLA2 molecules, particularly crotoxin, a β-neurotoxic heterodimeric PLA2, concominantly with an increment of PI and PIII metalloproteinases at age 9-18 months. Comparison of the transcriptional activity of the venom glands of neonate and adult C. s. simus specimens indicated that their transcriptomes exhibit indistinguisable toxin family profiles, suggesting that the elusive mechanism by which shared transcriptomes generate divergent venom phenotypes may operate post-transcriptionally. Specifically, miRNAs with frequency count of 1000 or greater exhibited an uneven distribution between the newborn and adult datasets. Of note, 590 copies of a miRNA targeting crotoxin B-subunit was exclusively found in the transcriptome of the adult snake, whereas 1185 copies of a miRNA complementary to a PIII-SVMP mRNA was uniquely present in the newborn dataset. These results support the view that age-dependent changes in the concentration of miRNA modulating the transition from a crotoxin-rich to a SVMP-rich venom from birth through adulhood can potentially explain what is observed in the proteomic analysis of the ontogenetic changes in the venom composition of C. s. simus. Conclusions: Existing snake venom toxins are the result of early recruitment events in the Toxicofera clade of reptiles by which ordinary genes were duplicated, and the new genes selectively expressed in the venom gland and amplified to multigene families with extensive neofunctionalization throughout the approximately 112-125 million years of ophidian evolution. Our findings support the view that understanding the phenotypic diversity of snake venoms requires a deep knowledge of the mechanisms regulating the transcriptional and translational activity of the venom gland. Our results suggest a functional role for miRNAs. The impact of specific miRNAs in the modulation of venom composition, and the integration of the mechanisms responsible for the generation of these miRNAs in the evolutionary landscape of the snake's venom gland, are further challenges for future research. [ABSTRACT FROM AUTHOR]

Abstract: Crotalus durissus terrificus, C. d. collilineatus, C. d. cascavella and C. d. marajoensis are responsible minor but severe snake bites in Brazil. The venoms of these snakes share the presence of crotoxin, a neurotoxin comprising of two associated components, crotapotin and phospholipase A2 (PLA2). Treatment of the victims with specific antiserum is the unique effective therapeutic measure. The ability of anti-Crotalus antisera produced by the routine using crude venom to immunize horses or purified crotoxin and PLA2 as individual immunogens was compared. Antisera obtained from horses immunized with C. durissus terrificus crude venom were able to recognize and neutralize not only the toxins presents in C. durissus terrificus, but also the ones present in the venoms from C. d. collilineatus, C. d. cascavella and C. d. marajoensis. Antisera from horses immunized with individual crotoxin or PLA2, although in lesser titers, were also able of recognizing the toxins in all four Crotalus species and neutralize the lethality of the C. d. terrificus venom. [Copyright &y& Elsevier]

Abstract: In this study, we evaluated the effect of the Crotalus durissus terrificus (Cdt) venom on the chronic paw edema induced by the injection of bacillus Calmette-Guérin (BCG) into the footpad of mice. The BCG injection evoked chronic edema, which was significantly diminished in animals treated subcutaneously (s.c.) with Cdt venom 1 h before or after the BCG injection. This inhibition persisted throughout the evaluation period (15 days). In mice injected with Cdt venom 6 or 11 days after injection of BCG, we observed a significant reduction in edema only in the period after the venom injection. While studying possible mechanisms involved in this inhibition, we observed that pre-treatment with dexamethasone, zileuton or Boc2 (a selective antagonist of formyl peptide receptors), but not with indomethacin, canceled out the inhibitory effect of Cdt venom on the edema induced by BCG. These results strongly suggest that this rattlesnake venom can stimulate the generation of mediators from the lipoxygenase pathway, which can down-regulate this chronic inflammatory edema. Using fractionated venom, the results indicated that crotoxin was the only component of Cdt venom responsible for this inhibitory effect. These results indicated that crotoxin, the main toxin of the C. durissus terrificus venom, has a significant inhibitory effect on BCG-induced chronic edema, possibly by generating anti-inflammatory mediators from the lipoxygenase pathway. [Copyright &y& Elsevier]

Aim:To investigate the antitumor actions of the Crotalus durissus neurotoxin (crotoxin) on human esophageal carcinoma (Eca-109) cells in vitro and transplanted esophageal Eca-109 tumors in nude mice.Methods:The growth-inhibitory effect was analyzed in Eca-109 cells using MTT assay. Cell morphology changes in nuclei were observed using Hoechst 33342 staining, while apoptosis and cell cycle distribution were examined by flow cytometry. RT-PCR was used to measure the Bcl-2, p15, and caspase-3 p17 gene expression levels. A tumor transplantation model was established by inoculation of Eca-109 cells were into female Balb/c nude mice. Crotoxin (25, 50, and 100 mg/kg) was subcutaneously injected into the transplanted tumors every 2 d for a total of 10 injections. Tumor size and weight were measured. Bcl-2, p15, and caspase-3 p17 protein expression in transplanted tumors was analyzed using Western blotting.Results:Crotoxin (25, 50, and 100 μg/mL) inhibited the growth of Eca-109 cells in a dose-dependent manner with inhibition rates of 22.9%, 35.8%, and 57.2%, respectively. Hoechst 33342 staining revealed apoptotic cells with pyknotic nuclear chromatin after crotoxin treatment. In Eca-109 cells, crotoxin induced apoptosis and G1 block, significantly upregulated the expression of p15 and caspase-3 p17 genes and downregulated the expression of Bcl-2 gene. Furthermore, crotoxin inhibited the growth of Eca-109 tumors in nude mice in a dose-dependent manner. Western blotting showed that crotoxin increased p15 and caspase-3 p17 protein levels and reduced Bcl-2 protein level in tumor specimens.Conclusion:Crotoxin inhibits the growth of Eca-109 cells in vitro via apoptosis induction and G1 block. Local administration of crotoxin inhibits the growth of subcutaneously transplanted Eca-109 cells in nude mice, possibly via increasing p15 and caspase-3 p17 protein expression and reducing Bcl-2 protein expression. [ABSTRACT FROM AUTHOR]

Crotoxin complex CA/CB and crotamine are the main toxins associated with Crotalus envenomation besides the enzymatic activities of phospholipases (PLA 2 ) and proteases. The neutralization at least of the crotoxin complex by neutralizing the subunit B could be a key in the production process of antivenoms against crotalids. Therefore, in this work, a Crotoxin B was recombinantly expressed to evaluate its capacity as an immunogen and its ability to produce neutralizing antibodies against crotalid venoms. A Crotoxin B transcript from Crotalus tzabcan was cloned into a pCR ® 2.1-TOPO vector (Invitrogen, Waltham, MA, USA) and subsequently expressed heterologously in bacteria. HisrCrotoxin B was extracted from inclusion bodies and refolded in vitro. The secondary structure of HisrCrotoxin B was comparable to the secondary structure of the native Crotoxin B, and it has PLA 2 activity as the native Crotoxin B. HisrCrotoxin B was used to immunize rabbits, and the obtained antibodies partially inhibited the activity of PLA 2 from C. tzabcan . The anti-HisrCrotoxin B antibodies neutralized the native Crotoxin B and the whole venoms from C. tzabcan, C. s. salvini , and C. mictlantecuhtli. Additionally, anti-HisrCrotoxin B antibodies recognized native Crotoxin B from different Crotalus species, and they could discriminate venom in species with high or low levels of or absence of Crotoxin B.

Abstract: The antiplasmodial activity of phospholipases A2 (PLA2) isolated from different animals has been studied. We explored the in vitro anti Plasmodium falciparum effect of a fraction containing crotoxin, Crotoxin B and whole venom of the rattlesnake Crotalus durissus cumanensis. Fraction II (crotoxin complex) was obtained by size exclusion chromatography, whereas Crotoxin B was purified by RP-HPLC. The whole venom is active against the parasite at concentrations of 0.17±0.03μg/ml, fraction II at 0.76±0.17μg/ml and Crotoxin B at 0.6±0.04μg/ml. Differences were observed in the cytotoxic activity against peripheral mononuclear cells, with Crotoxin B exhibiting the highest cytotoxicity. The concentration of Crotoxin B required to exert cytotoxic activity was higher than that required to exert antiplasmodial activity. Lethality in mice confirmed the higher toxicity and neurotoxicity of whole venom and fraction II, whereas Crotoxin B was not lethal at the doses tested. These results suggest the potential of Crotoxin B as a lead compound for antimalarial activity. [Copyright &y& Elsevier]

Copyright of Arquivos Brasileiros de Oftalmologia is the property of Arquivos Brasileiros de Oftalmologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This review will focus on a description of the three-dimensional structures of two β-neurotoxins, the monomeric PLA 2 ammodytoxin from Vipera ammodytes ammodytes , and heterodimeric crotoxin from Crotalus durissus terrificus , and a detailed structural analysis of their multiple functional sites. We have recently determined at high resolution the crystal structures of two natural isoforms of ammodytoxin (AtxA and AtxC) ( Saul et al., 2010 ) which exhibit different toxicity profiles and different anticoagulant properties. Comparative structural analysis of these two PLA 2 isoforms, which differ only by two amino acid residues, allowed us to detect local conformational changes and delineate the role of critical residues in the anticoagulant and neurotoxic functions of these PLA 2 ( Saul et al., 2010 ). We have also determined, at 1.35 Å resolution, the crystal structure of heterodimeric crotoxin ( Faure et al., 2011 ). The three-dimensional structure of crotoxin revealed details of the binding interface between its acidic (CA) and basic (CB) subunits and allowed us to identify key residues involved in the stability and toxicity of this potent heterodimeric β-neurotoxin ( Faure et al., 2011 ). The precise spatial orientation of the three covalently linked polypeptide chains in the mature CA subunit complexed with CB helps us to understand the role played by critical residues of the CA subunit in the increased toxicity of the crotoxin complex. Since the CA subunit is a natural inhibitor of the catalytic and anticoagulant activities of CB, identification of the CA–CB binding interface describes residues involved in this inhibition. We propose future research directions based on knowledge of the recently reported 3D structures of crotoxin and ammodytoxin. [ABSTRACT FROM AUTHOR]

This work investigated the effect of gallium arsenide (GaAs) irradiation (power: 5 mW; intensity: 77.14 mW/cm, spot: 0.07 cm) on regenerating skeletal muscles damaged by crotoxin (CTX). Male C57Bl6 mice were divided into six groups ( n = 5 each): control, treated only with laser at doses of 1.5 J or 3 J, CTX-injured and, CTX-injured and treated with laser at doses of 1.5 J or 3 J. The injured groups received a CTX injection into the tibialis anterior (TA) muscle. After 3 days, TA muscles were submitted to GaAs irradiation at doses of 1.5 or 3 J (once a day, during 5 days) and were killed on the eighth day. Muscle histological sections were stained with hematoxylin and eosin (H&E) in order to determine the myofiber cross-sectional area (CSA), the previously injured muscle area (PIMA) and the area density of connective tissue. The gene expression of MyoD and myogenin was detected by real-time PCR. GaAs laser at a dose of 3 J, but not 1.5 J, significantly increased the CSA of regenerating myofibers and reduced the PIMA and the area density of intramuscular connective tissue of CTX-injured muscles. MyoD gene expression increased in the injured group treated with GaAs laser at a dose of 1.5 J. The CTX-injured, 3-J GaAs laser-treated, and the CTX-injured and treated with 3-J laser groups showed an increase in myogenin gene expression when compared to the control group. Our results suggest that GaAs laser treatment at a dose of 3 J improves skeletal muscle regeneration by accelerating the recovery of myofiber mass. [ABSTRACT FROM AUTHOR]

Copyright of Arquivos Brasileiros de Oftalmologia is the property of Arquivos Brasileiros de Oftalmologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract: Crotoxin, one of the major toxins of South American rattlesnake Crotalus durissus subspecies, is an heterodimeric complex composed of two distinct subunits: a basic phospholipase A2 (PLA2, CB) and an acidic nontoxic catalytically inactive protein, crotapotin (CA). It''s well known that CB has a high enzymatic activity; however the molecular aspects that determine this fact remain unknown. In this study, an in silico approach was used to predict the CA structure by homology modeling, and the crotoxin structure by means of molecular docking. CA structure was built using the software Modeller taking Crotalus atrox PLA2 (1PP2:R) as a template. Different criteria measured by Procheck, Verify 3D and ProSA were indicative of the reliability and the proper fold for the predicted structural model of CA. Then, a combination of this model and CB crystal structure was used to build the structure of crotoxin complex through rigid-body protein–protein docking. The crotoxin-3D model suggested that by means of hydrophobic and π–π stacking interactions, CA-Y24 and CA-F119 interact with CB-F24 and CB-F119, respectively. Those interactions could prevent the interfacial adsorption of the CB onto the lipid/water interface by blocking part of the interfacial binding surface of the PLA2. This fact could explain the differences regarding to enzymatic activity between the crotoxin complex and CB. In addition, the crotoxin-3D model showed solvent-exposed regions of CA that could bind the receptor expressed in target cells. [Copyright &y& Elsevier]

Abstract: Dengue is the most important arbovirus in the world with an estimated of 50 million dengue infections occurring annually and approximately 2.5 billion people living in dengue endemic countries. Yellow fever is a viral hemorrhagic fever with high mortality that is transmitted by mosquitoes. Effective vaccines against yellow fever have been available for almost 70 years and are responsible for a significant reduction of occurrences of the disease worldwide; however, approximately 200,000 cases of yellow fever still occur annually, principally in Africa. Therefore, it is a public health priority to develop antiviral agents for treatment of these virus infections. Crotalus durissus terrificus snake, a South American rattlesnake, presents venom with several biologically actives molecules. In this study, we evaluated the antiviral activity of crude venom and isolated toxins from Crotalus durissus terrificus and found that phospholipases A2 showed a high inhibition of Yellow fever and dengue viruses in VERO E6 cells. [Copyright &y& Elsevier]

Abstract: The crystal structure of crotoxin, a potent presynaptic neurotoxin from Crotalus durissus terrificus, was solved at 1.35 Å resolution. It shows the architecture of the three disulfide-linked polypeptide chains (α, β, and γ) of the acidic subunit CA noncovalently complexed with the basic phospholipase A2 (PLA2) subunit CB. The unique structural scaffold of the association of the CA and CB subunits indicates that posttranslational cleavage of the pro-CA precursor is a prerequisite for the assembly of the CA–CB complex. These studies provide novel structural insights to explain the role of the CA subunit in the mechanism of action of crotoxin. The crystal structure of the highly toxic and stable CA2CBb complex crystallized here allows us to identify key amino acid residues responsible for significant differences in the pharmacological activities of the two classes of crotoxin complexes. In particular, we show that critical residues Trp31 and Trp70 of the CBb subunit establish intermolecular polar contacts with Asp99 and Asp89, respectively, of the β-chain of CA2 and contribute to the stability and toxicity of the CA2CBb complex. These interactions also lead to decreased PLA2 activity by partially blocking substrate access to the catalytic dyad and by masking several interfacial binding surface residues important for PLA2 interaction with phospholipids. Identification of the binding interface between the CA subunits and the CB subunits of crotoxin is important for the structure-based design of antineurotoxic inhibitors. Since crotoxin displays numerous physiological functions, including antitumoral properties, knowledge of its three-dimensional structure will be useful for the understanding of these diverse effects. [Copyright &y& Elsevier]

Abstract: Crotalus durissus terrificus venom and its main component, crotoxin (CTX), have the ability to down-modulate the immune system. Certain mechanisms mediated by cells and soluble factors of the immune system are responsible for the elimination of pathogenic molecules to ensure the specific protection against subsequent antigen contact. Accordingly, we evaluated the immunomodulatory effects of CTX on the immune response of mice that had been previously primed by immunisation with human serum albumin (HSA). CTX inoculation after HSA immunisation, along with complete Freund’s adjuvant (CFA) or Aluminium hydroxide (Alum) immunisation, was able to suppress anti-HSA IgG1 and IgG2a antibody production. We showed that the inhibitory effects of this toxin are not mediated by necrosis or apoptosis of any lymphoid cell population. Lower proliferation of T lymphocytes from mice immunised with HSA/CFA or HSA/Alum that received the toxin was observed in comparison to the mice that were only immunised. In conclusion, CTX is able to exert potent inhibitory effects on humoural and cellular responses induced by HSA immunisation, even when injected after an innate immune response has been initiated. [Copyright &y& Elsevier]

Abstract: In the present study, it was investigated which components are responsible for the anti-inflammatory properties of Crotalus durissus terrificus venom (CdtV). The effect of crotoxin, as well as of other CdtV components was evaluated on edema, cell migration and alterations in leukocyte–endothelium interactions induced by carrageenan. Crotoxin (40μgkg−1) was injected at different time periods before or after the injection of carrageenan (15mgkg−1) into the mouse hind paw, peritoneum or scrotum. Results showed that crotoxin, but not other CdtV components, significantly inhibited inflammatory edema and cell migration when administered before or after carrageenan injection in mice. This toxin also prevented the occurrence of alterations in leukocyte–endothelium interactions induced by carrageenan injection, such as the increase in adhered cells. In animals pretreated with Boc2 (a selective antagonist of formyl peptide receptors), crotoxin showed neither inhibitory effects on edema and cell migration, nor prevented alterations in leukocyte–endothelium interactions induced by carrageenan. These findings demonstrate that crotoxin is the component responsible for the long-lasting anti-inflammatory activity of crude C. durissus terrificus venom, and activation of formyl peptide receptors seems to play a major role in this effect. [Copyright &y& Elsevier]

Abstract: Crotoxin, the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, was the first snake venom protein to be purified and crystallized. Crotoxin is a heterodimeric β-neurotoxin that consists of a weakly toxic basic phospholipase A2 and a non-enzymatic, non-toxic acidic component (crotapotin). The classic biological activities normally attributed to crotoxin include neurotoxicity, myotoxicity, nephrotoxicity and cardiotoxicity. However, numerous studies in recent years have shown that crotoxin also has immunomodulatory, anti-inflammatory, anti-microbial, anti-tumor and analgesic actions. In this review, we describe the historical background to the discovery of crotoxin and its main toxic activities and then discuss recent structure–function studies and investigations that have led to the identification of novel pharmacological activities for the toxin. [Copyright &y& Elsevier]

Abstract: Crotoxin (CA.CB) is a β-neurotoxin from Crotalus durissus terrificus snake venom that is responsible for main envenomation effects upon biting by this snake. It is a heterodimer of an acidic protein (CA) devoid of any biological activity per se and a basic, enzymatically active, PLA2 counterpart (CB). Both lethal and enzymatic activities of crotoxin have been shown to be inhibited by CNF, a protein from the blood of C. d. terrificus snakes. CNF replaces CA in the CA.CB complex, forming a stable, non-toxic complex CNF.CB. The molecular sites involved in the tight interfacial protein–protein interactions in these PLA2-based complexes have not been clearly determined. To help address this question, we used the peptide arrays approach to map possible interfacial interaction sites in CA.CB and CNF.CB. Amino acid stretches putatively involved in these interactions were firstly identified in the primary structure of CB. Further analysis of the interfacial availability of these stretches in the presumed biologically active structure of CB, suggested two interaction main sites, located at the amino-terminus and β-wing regions. Peptide segments at the carboxyl-terminus of CB were also suggested to play a secondary role in the binding of both CA and CNF. [Copyright &y& Elsevier]

Abstract: A new PLA2 (Cdcum6) from crotoxin complex of Colombian Crotalus durissus cumanensis rattlesnake was purified using molecular exclusion chromatography and RP-HPLC. The molecular mass of Cdcum6 was determined by SDS-PAGE ∼14KDa and confirmed by MALDI-TOF (14321.98Da). The enzyme showed K m 6.0mM, V max 3.44nmol/min, optimum pH was 8.0 and temperature was between 30 and 45°C, and it had a strict requirement of Ca2+ for its activity. The N-terminal sequence of PLA2 was SLVQF EKMIK EVAGK NGVPWY. Comparison of amino acid sequence data with other PLA2 from South American Crotalus durissus rattlesnakes showed that Cdcum6 shares the highest sequence identity with Cdr13 an isoform PLA2 from Crotalus durissus ruruima, nevertheless, Cdcum6 showed high content of basic and hydrophobic amino acids. In mice, Cdcum6 presented higher LD50 than crotoxin complex from C. d. cumanensis. Additionally, Cdcum6 induced a conspicuous local myotoxic effect and moderate footpad edema; in vitro, it was antigoagulant in doses as low as 0.5μg/ml, and it was not cytotoxic on myoblast but Cdcum6 was able to lyse myotubes. [Copyright &y& Elsevier]

• In South America there are three snakes genera with abundant neurotoxic components venom. • Neurotoxic venoms have great medical importance because of their lethality. • There are only a few reports related to Micrurus or Hydrophis genera mainly. • High potential medical applications of elapid and crotalic toxins. • Summarizes the knowledge to date about South American snakes venom with neurotoxic components. 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. [ABSTRACT FROM AUTHOR]

Abstract: Crotoxin (CTX), a neurotoxin isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces analgesia. In this study, we evaluated the antinociceptive effect of CTX in a model of neuropathic pain induced by rat sciatic nerve transection. Hyperalgesia was detected 2 h after nerve transection and persisted for 64 days. Immersion of proximal and distal nerve stumps in CTX solution (0.01 mM for 10 s), immediately after nerve transection, blocked hyperalgesia. The antinociceptive effect of CTX was long-lasting, since it was detected 2 h after treatment and persisted for 64 days. CTX also delayed, but did not block, neurectomy-induced neuroma formation. The effect of CTX was blocked by zileuton (100 mg/kg, p.o.) and atropine (10 mg/kg, i.p.), and reduced by yohimbine (2 mg/kg, i.p.) and methysergide (5 mg/kg, i.p.). On the other hand, indomethacin (4 mg/kg, i.v.), naloxone (1 mg/kg, i.p.), and N-methyl atropine (30 mg/kg, i.p.) did not interfere with the effect of CTX. These results indicate that CTX induces a long-lasting antinociceptive effect in neuropathic pain, which is mediated by activation of central muscarinic receptors and partially, by activation of α-adrenoceptors and 5-HT receptors. Eicosanoids derived from the lipoxygenase pathway modulate the action of crotoxin. [Copyright &y& Elsevier]

Epithelial–mesenchymal transition (EMT) occurs in the early stages of embryonic development and plays a significant role in the migration and the differentiation of cells into various types of tissues of an organism. However, tumor cells, with altered form and function, use the EMT process to migrate and invade other tissues in the body. Several experimental (in vivo and in vitro) and clinical trial studies have shown the antitumor activity of crotoxin (CTX), a heterodimeric phospholipase A2 present in the Crotalus durissus terrificus venom. In this study, we show that CTX modulates the microenvironment of tumor cells. We have also evaluated the effect of CTX on the EMT process in the spheroid model. The invasion of type I collagen gels by heterospheroids (mix of MRC-5 and A549 cells constitutively prepared with 12.5 nM CTX), expression of EMT markers, and secretion of MMPs were analyzed. Western blotting analysis shows that CTX inhibits the expression of the mesenchymal markers, N-cadherin, α-SMA, and αv. This study provides evidence of CTX as a key modulator of the EMT process, and its antitumor action can be explored further for novel drug designing against metastatic cancer. [ABSTRACT FROM AUTHOR]

Abstract: This work investigates the influence of heat shock proteins (HSPs) on necrosis and subsequent skeletal muscle regeneration induced by crotoxin (CTX), the major component of Crotalus durissus terrificus venom. Mice were treated with radicicol, a HSP inductor, followed by an intramuscular injection of CTX into the gastrocnemius muscle. Treated groups were sacrificed 1, 10 and 21 days after CTX injection. Muscle histological sections were stained with toluidine blue and assayed for acid phosphatase or immunostained with either neuronal cell adhesion molecule (NCAM) or neonatal myosin heavy chain (MHCn). Muscle samples were also submitted to Western blotting analysis. The results show that CTX alone and CTX combined with radicicol induced a similar degree of myofiber necrosis. CTX-injured muscles treated with radicicol had increased cross-sectional areas at 10 and 21 days post-lesion compared with untreated CTX-injured muscles. Additionally, radicicol significantly increased the number of NCAM-positive satellite cells in the gastrocnemius at one day post-CTX injury. CTX-injured muscles treated with radicicol contained more MHCn-positive regenerating myofibers compared with untreated CTX-injured muscles. These results suggest that HSPs contribute to the regeneration of myofibers damaged by CTX. Additionally, further studies should investigate the potential therapeutic effects of radicicol in skeletal muscles affected by Crotalus venom. [Copyright &y& Elsevier]

Abstract: Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom and modulates immune and inflammatory responses, interfering with the activity of leukocytes. In the present work, the effects of crotoxin on the number of blood and lymphatic leukocytes and on lymph nodes and spleen lymphocytes population were investigated. The toxin s.c. administered to male Wistar rats, decreases the number of lymphocytes in blood and lymph circulation and increases the content of B and T-lymphocytes in lymph nodes. These effects were detected 1–2h after treatment. The crotoxin molecule is composed of two subunits, an acidic non-toxic polypeptide, named crotapotin and a toxic basic phospholipase A2 (PLA2). PLA2, but not crotapotin, decreased the number of circulating blood and lymph lymphocytes. Crotoxin promotes leukocyte adherence to endothelial cells of blood microcirculation and to lymph node high endothelial venules, which might contribute to the drop in the number of circulating lymphocytes. Crotoxin increases expression of the adhesion molecule LFA-1 in lymphocytes. The changes in the expression of the adhesion molecule might contribute, at least in part, for the increased leukocyte adhesion to endothelium. Zileuton, a 5-lipoxygenase inhibitor, blocked the decrease in the number of circulating leukocytes induced by crotoxin and also abolished the changes observed in leukocyte–endothelial interactions, suggesting the involvement of lipoxygenase-derived mediators in the effects of the toxin. [Copyright &y& Elsevier]

Abstract: The toxicity of crotoxin, the major toxin of Crotalus durissus terrificus (South American rattlesnake) venom, is mediated by its basic phospholipase A2 (PLA2) subunit. This PLA2 is non-covalently associated with crotapotin, an acidic, enzymatically inactive subunit of the crotoxin complex. In this work, rabbit antiserum raised against crotapotin purified from Crotalus durissus cascavella venom was tested for its ability to neutralize the neurotoxicity of this venom and its crotoxin in vitro. The ability of this antiserum to inhibit the enzymatic activity of the crotoxin complex and PLA2 alone was also assessed, and its potency in preventing myotoxicity was compared with that of antisera raised against crotoxin and PLA2. Antiserum to crotapotin partially neutralized the neuromuscular blockade caused by venom and crotoxin in electrically stimulated mouse phrenic nerve-hemidiaphragm preparations and prevented the venom-induced myotoxicity, but did not inhibit the enzymatic activity of crotoxin and purified PLA2. In contrast, previous findings showed that antisera against crotoxin and PLA2 from C. d. cascavella effectively neutralized the neuromuscular blockade and PLA2 activity of this venom and its crotoxin. The partial neutralization of crotoxin-mediated neurotoxicity by antiserum to crotapotin probably reduced the binding of crotoxin to its receptor following interaction of the antiserum with the crotapotin moiety of the complex. [Copyright &y& Elsevier]

Abstract: Crotoxin (Cro), the principal neurotoxic component of Crotalus durissus terrificus, has been previously reported to have a behavioral analgesic effect in rats and mice. The present study investigated electrophysiologically the effect of Cro on pain-evoked unit discharge of neurons in thalamic parafascicular nucleus (Pf) and underlying mechanisms of its effect. The electrical discharge of Pf neurons was recorded with the microelectrode technique in rats. Intracerebroventricular (icv) injection of Cro at 0.25, 0.45 and 0.65μg/kg resulted in a dose-dependent inhibitory effect on the pain-evoked discharge of Pf neurons. The discharge frequency and the discharge duration significantly (P<0.05) decreased after Cro administration. This inhibitory effect was significantly (P<0.05) attenuated after pretreatment with para-chlorophenylalanine (pCPA), or electrolytic lesion of dorsal raphe (DR) nucleus. In contrast, icv injection of atropine (muscarinic receptor antagonist, 5μg) or naloxone (opioid receptor antagonist, 4μg) had no effect on Cro-induced inhibition of discharge of Pf neurons. The results suggested that Cro has an analgesic effect, which is mediated, at least partially, by the central serotonergic system. [Copyright &y& Elsevier]

Abstract: This study explored the toxic effects of crotoxin isolated from Crotalus durissus cumanensis venom on the ultrastructure of mice cardiac autonomic nervous system. Mice were intravenously injected with saline (control group) and crotoxin diluted in saline venom (study group) at a dose of 0.107mg/kg mouse body weight. Samples from the inter-ventricular septum were prepared for electron microscopy after 6h (G1), 12h (G2), 24h (G3) and 48h (G4). The G1 group showed some cardiomyocyte with pleomorphic mitochondria. Capillary swollen walls, nerve cholinergic endings with depleted acetylcholine vesicles in their interior and other depletions were observed. A space completely lacking in contractile elements was noticed. The G2 group demonstrated a myelinic figure, a subsarcolemic region with few myofibrils and nervous cholinergic terminal with scarce vacuoles in their interior. The G3 group demonstrated a structure with a depleted axonic terminal, mitochondrias varying in size and enhanced electron density. In addition, muscular fibers with myofibrillar structure disorganization, a depleted nervous structure surrounded by a Schwann cell along with an abundance of natriuretic peptides, were seen. An amyelinic terminal with depleted Schwann cell and with scarce vesicles was also observed. Finally, axonic lysis with autophagic vacuoles in their interior and condensed mitochondria was observed in the G4 group. This work describes the first report of ultrastructural damage caused by crotoxin on mice cardiac autonomic nervous system. [Copyright &y& Elsevier]

Abstract: Antigens of Bothrops jararacussu snake venom cross-reacting with specific antibodies against crotoxin, an Asp49 PLA2-containing heterodimeric complex from Crotalus durissus terrificus snake venom, were purified by two steps of immunoaffinity chromatography. The resulting fraction (Bj-F) was shown to be non-toxic (to mice and rabbits) and immunogenic to rabbits. Antibodies raised against Bj-F were able to protect mice against the lethal effect of both B. jararacussu and Crotalus durissus terrificus snake venoms. Then, the procedure developed showed to be useful for the rapid preparation of an antigen able to elicit neutralizing antibodies against the lethal activities of both venoms. Further fractionation of Bj-F revealed the concomitant presence of two major components: BJcuL, a lectin present in B. jararacussu venom, and BthTX-I, a Lys49 PLA2 homolog, besides other molecules in minor amounts. Our data are discussed and raise the point that the presence of unrelated molecules may be taken into account when immuno-based methods are considered for purification purposes. [Copyright &y& Elsevier]

Aim: To investigate the role of crotoxin (CrTX)-induced autophagy in the death of MCF-7 cells, a caspase-3-deficient, human breast cancer cell line. Methods: Cultured MCF-7 cells were treated with various doses of CrTX, a phospholipase A2 (PLA2) isolated from the venom of the South American rattlesnake, Crotalus durissus terrificus. The cytotoxicity of CrTX in the presence and absence of caspase inhibitors was measured with methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) leakage assays. The activation of autophagy was determined with transmission electron microscope and monodansylcadaverin (MDC) labeling. The upregulation of lysosomal enzymes, the release of cyto-chrome c (cyto-c), and the nuclear translocation of the apoptosis inducing factor (AIF) were examined by immunoblotting and immunofluorescence. Results: CrTX inhibited the viability of MCF-7 cells in a dose- and time-dependent manner. CrTX-activated autophagy was revealed by punctuate MDC labeling, and an increase in the formation of autophagosomes as well as apoptosis, as evidenced by nuclear condensation and fragmentation. The activation of cathepsin B, D, and L, in addition to the release of cytochrome c and the relocation of AIF into nuclei, were observed after CrTX treatment. Autophagy inhibitors 3-methyladenine (3-MA), NH4Cl, and the pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-Vad-fmk), attenuated CrTX-induced cell death. Conclusion: An autophagic mechanism contributes to the apoptosis of MCF-7 cells induced by CrTX. [ABSTRACT FROM AUTHOR]

Abstract: The venom of Crotalus durissus terrificus is reported to have analgesic activity and the administration of Crotoxin (Cro) to cancer patients is reported to reduce the consumption of analgesics. This study investigated the analgesia induced by Cro and the effects of atropine and naloxone on the antinociceptive activity of Cro in mice and rats. The results showed that Cro at 66.5, 44.3 and 29.5μg/kg (ip) exhibited a dose-dependent analgesic action in mice using the hotplate and acetic acid writhing tests. Cro at 44.3μg/kg (ip) had significant analgesic action in the rat tail-flick test. In the mouse acetic acid-writhing test, intracerebral ventricular administration of Cro 0.3μg/kg produced marked analgesic effects. Microinjection of Cro (0.15μg/kg) into the periaqueductal gray area also elicited a robust analgesic action in rat hotplate test. Atropine at 0.5mg/kg (im) or 10mg/kg (ip) or naloxone at 3mg/kg (ip) failed to block the analgesic effects of Cro. These results suggest that Cro has analgesic effects mediated by an action on the central nervous system. The muscarinic and opioid receptors are not involved in the antinociceptive effects of Cro. [Copyright &y& Elsevier]