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Background: Crotalus Neutralizing Factor (CNF) is a γ-type Phospholipase A2 (PLA2) inhibitor present in the blood of Crotalus durissus terrificus snake. Particularly, CNF inhibits the toxic action of Crotoxin (CTX), which is a major neurotoxin found in C. d. terrificus venom. CTX induces also myotoxic action and demonstrates high selectivity for skeletal muscle fibers. Consequently, CTX can diffuse beyond the site of infection, which can potentially evoke rhabdomyolysis. The present study has evaluated the effects of CTX on myoblasts and myotubes of muscle cells C2C12 in vitro and the effect of CNF on CTX-induced damage., Methods: Cytotoxicity assays were performed by measuring the mitochondrial enzyme dehydrogenase levels. Furthermore, creatine kinase and lactate dehydrogenase levels were used as indicators of muscle damage., Results: Crotoxin has been found to have cytotoxic effects on C2C12 myoblast cells, while CNF has not shown toxic effects on these cells. Furthermore, the findings have shown CNF (50 μg/mL) to abolish CTX toxicity in myoblasts. The myotubes, differentiated cells, showed no change in mitochondrial respiration when exposed to CNF or CTX, showing greater resistance to the toxic actions of crotoxin., Conclusion: The data have confirmed the potential of CNF as an anti-myotoxic agent to prevent CTX-damaged myoblasts and increase resistance to the toxic effects of crotoxin on differentiated cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Mammary gland tumors are the most prevalent neoplasm in intact female dogs, and they are good natural models to study comparative oncology. Most canine mammary malignancies, as in women, are commonly refractory to conventional therapies and demand continuous new therapeutic approaches. Crotalus durissus terrificus , also called rattlesnake, has more than 60 different proteins in its venom with multiple pharmaceutical uses, such as antitumor, antiviral, and antimicrobial action. Crotoxin, a potent β-neurotoxin formed by the junction of two subunits, a basic subunit (CB-PLA 2 ) and an acidic subunit (crotapotin), has already been reported to have anticancer properties in different types of cancers., Methods: In this work, we describe the cytotoxic potential of crotoxin and its subunits compared to doxorubicin (drug of choice) in two canine mammary carcinoma cell lines., Results: Crotoxin, CB-PLA 2 , crotalic venom, and doxorubicin decreased cell viability and the ability to migrate in a dose-dependent manner, and crotapotin did not present an antitumoral effect. For all compounds, the predominant cell death mechanism was apoptosis. In addition, crotoxin did not show toxicity in normal canine mammary gland cells., Conclusion: Therefore, this work showed that crotoxin and CB-PLA 2 had cytotoxic activity, migration inhibition, and pro-apoptotic potential in canine mammary gland carcinoma cell lines, making their possible use in cancer research., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Crotoxin (CTX) is a neurotoxin that is isolated from the venom of Crotalus durissus terrificus, which displays immunomodulatory, anti-inflammatory, and anti-tumoral effects. Previous research has demonstrated that CTX promotes the adherence of leukocytes to the endothelial cells in blood microcirculation and the high endothelial venules of lymph nodes, which reduces the number of blood cells and lymphocytes. Studies have also shown that these effects are mediated by lipoxygenase-derived mediators. However, the exact lipoxygenase-derived eicosanoid involved in the CTX effect on lymphocytes is yet to be characterized. As CTX stimulates lipoxin-derived mediators from macrophages and lymphocyte effector functions could be modulated by activating formyl peptide receptors, we aimed to investigate whether these receptors were involved in CTX-induced redistribution and functions of lymphocytes in rats. We used male Wistar rats treated with CTX to demonstrate that Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe), an antagonist of formyl peptide receptors, prevented CTX-induced decrease in the number of circulating lymphocytes and increased the expression of the lymphocyte adhesion molecule LFA1. CTX reduced the T and B lymphocyte functions, such as lymphocyte proliferation in response to the mitogen Concanavalin A and antibody production in response to BSA immunization, respectively, which was prevented by the administration of Boc2. Importantly, mesenteric lymph node lymphocytes from CTX-treated rats showed an increased release of 15-epi-LXA 4 . These results indicate that formyl peptide receptors mediate CTX-induced redistribution of lymphocytes and that 15-epi-LXA 4 is a key mediator of the immunosuppressive effects of CTX., 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 © 2022 Elsevier Ltd. All rights reserved.)

The venom of the South American rattlesnake Crotalus durissus terrificus causes an irreversible neuromuscular blockade in isolated preparations due to action of the presynaptically-acting heterodimeric phospholipase A 2 (PLA 2 ) crotoxin. Some populations of this subspecies contain, in addition to crotoxin, the toxin crotamine, which acts directly on muscle fibers. In this study we used C. d. terrificus venoms with (crot+) or without (crot-) crotamine to test whether Varespladib, a PLA 2 inhibitor, is able to abrogate the neuromuscular blockade induced by these venoms comparatively with crotalic antivenom. Mouse phrenic nerve-diaphragm preparations were exposed to venoms previously incubated with two different concentrations of Varepladib or antivenom, or with a mixture of these two agents, before addition to the bath. In another experimental setting, venoms were initially added to the system, followed by the addition of Varespladib or antivenom 10, 30, or 60 min after venom. At the highest concentrations tested, Varespladib and antivenom inhibited the action of the venom >80% and >70%, respectively. With lower concentrations the inhibition of neuromuscular blockade decreased, but when low doses of the two agents were incubated together with the venom, the inhibitory effect improved, underscoring a synergistic phenomenon. When added after venom, Varespladib was able to halt the progression of the neuromuscular blockade even when added at 60 min. Antivenom exhibited a lower ability to inhibit the toxic effect of the venoms in these conditions. In conclusion, the PLA 2 inhibitor Varespladib is highly effective at abrogating the neuromuscular blocking activity of crotamine-positive and crotamine-negative C. d. terrificus venoms and seems to act synergistically with antivenom., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The South American rattlesnake Crotalus durissus spp has a wide geographic distribution in Brazil. Although responsible for only a low proportion of ophidian accidents, it is considered one of the most medically important species of venomous snakes due to the high mortality rate (1.87%). Snake venom is a complex phenotype commonly subjected to individual intraspecific, ontogenetic and geographic variability. Compositional differences in pooled venom used in the immunization process may impact the efficacy of the antivenom. In order to assure standardized high-quality antivenom, the potency of each Brazilian crotalic antivenom batch is determined against the 'Brazilian Crotalic Reference Venom' (BCRV). BCRV is produced by Instituto Butantan using venom obtained from the first milking of recently wild-caught C. d. terrificus specimens brought to the Institute. The decrease in the number of snake donations experienced in recent years can become a threat to the production of future batches of BCRV. To evaluate the feasibility of using venom from long-term captive animals in the formulation of BCRV, we have compared the proteomic, biochemical and biological profiles of C. d. terrificus venom pooled from captive specimens (CVP- captive venom pool) and BCRV. Electrophoretic and venomics analyses revealed a very similar venom composition profile, but also certain differences in toxins abundance, with some low abundant protein families found only in BCRV. Enzymatic (L-amino acid oxidase, phospholipase A 2 and proteolytic) and biological (myotoxic and coagulant) activities showed higher values in CVP than in BCRV. CVP also possessed slightly higher lethal effect, although the Instituto Butantan crotalic antivenom showed equivalent potency neutralizing BCRV and CVP. Our results strongly suggest that venom from long-term captive C. d. terrificus might be a valid alternative to generate an immunization mixture of equivalent quality to the currently in use reference venom., (Copyright © 2020. Published by Elsevier Ltd.)

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.)