The edematogenic effect of Micrurus lemniscatus venom is dependent on venom phospholipase A2 activity and modulated by non-neurogenic factors.

Coral snakes mainly cause neurotoxic symptoms in human envenomation, but experimental studies have already demonstrated several pharmacological activities in addition to these effects. This investigation was carried out with the aim of evaluating (1) non-neurogenic mechanisms involved in the inflammatory response induced by Micrurus lemniscatus venom (MLV) in rat hind paws, (2) participation of PLA2 in this response, and (3) neutralizing efficiency of commercial anti-elapid antivenom on edema. MLV promoted a rapid, significant increase in vascular permeability, influx of leukocytes, and disorganization of collagen bundles, as demonstrated by histological analysis. Several pretreatments were applied to establish the involvement of inflammatory mediators in MLV-induced edema (5 µg/paw). Treatment of animals with chlorpromazine reduced MLV-induced edema, indicating participation of TNF-α. However, the inefficiency of other pharmacological treatments suggests that eicosanoids, leukotrienes, and nitric oxide have no role in this type of edema formation. In contrast, PAF negatively modulates this venom-induced effect. MLV was recognized by anti-elapid serum, but this antivenom did not neutralize edema formation. Chemical modification of MLV with p-bromophenacyl bromide abrogated the phospholipase activity and markedly reduced edema, demonstrating PLA2 participation in MLV-induced edema. In conclusion, the non-neurogenic inflammatory profile of MLV is characterized by TNF-α-mediated edema, participation of PLA2 activity, and down-regulation by PAF. MLV induces an influx of leukocytes and destruction of collagen fibers at the site of its injection.

The edematogenic effect of Micrurus lemniscatus venom is dependent on venom Phospholipase A2 activity and modulated by non-neurogenic factors

Coral snakes mainly cause neurotoxic symptoms in human envenomation, but experimental studies have already demonstrated several pharmacological activities in addition to these effects. This investigation was carried out with the aim of evaluating (1) non-neurogenic mechanisms involved in the inflammatory response induced by Micrurus lemniscatus venom (MLV) in rat hind paws, (2) participation of PLA2 in this response, and (3) neutralizing efficiency of commercial anti-elapid antivenom on edema. MLV promoted a rapid, significant increase in vascular permeability, influx of leukocytes, and disorganization of collagen bundles, as demonstrated by histological analysis. Several pretreatments were applied to establish the involvement of inflammatory mediators in MLV-induced edema (5 µg/paw). Treatment of animals with chlorpromazine reduced MLV-induced edema, indicating participation of TNF-α. However, the inefficiency of other pharmacological treatments suggests that eicosanoids, leukotrienes, and nitric oxide have no role in this type of edema formation. In contrast, PAF negatively modulates this venom-induced effect. MLV was recognized by anti-elapid serum, but this antivenom did not neutralize edema formation. Chemical modification of MLV with p-bromophenacyl bromide abrogated the phospholipase activity and markedly reduced edema, demonstrating PLA2 participation in MLV-induced edema. In conclusion, the non-neurogenic inflammatory profile of MLV is characterized by TNF-α-mediated edema, participation of PLA2 activity, and down-regulation by PAF. MLV induces an influx of leukocytes and destruction of collagen fibers at the site of its injection.

Inflammatory mediators in the pronociceptive effects induced by Bothrops leucurus snake venom: The role of biogenic amines, nitric oxide, and eicosanoids.

Bothrops leucurus is the major causative agent of venomous snakebites in Northeastern Brazil. Severe pain is the most frequent symptom in these envenomings, with an important inflammatory component. This work characterized the pronociceptive effects evoked by B. leucurus venom (BLV) in mice and the role of inflammatory mediators in these responses. The nociceptive behaviors were quantified by the modified formalin test. The mechanical hyperalgesia was assessed by the digital von Frey test. Pharmacological assays were performed with different antagonists and synthesis inhibitors to investigate the involvement of inflammatory mediators in both nociceptive events. BLV (1-15 µg/paw) injection in mice evoked intense and dose-dependent nociceptive behaviors that lasted for up to 1 h. BLV (10 µg/paw) also caused sustained mechanical hyperalgesia. Histamine and serotonin played a role in the nociception, but not in the BLV-induced mechanical hyperalgesia. Nitric oxide contributed to both responses, but only to the late stages of mechanical hyperalgesia. Eicosanoids were also present in both nociceptive responses. Prostanoid synthesis by COX-1 seemed to be more relevant for the nociception, whereas COX-2 had a more prominent role in the mechanical hyperalgesia. Leukotrienes were the most relevant mediators of BLV-induced mechanical hyperalgesia, hence inhibiting lipoxygenase pathway could be an efficient therapeutic strategy for pain management during envenoming. Our behavioral data demonstrates that BLV promotes nociceptive transmission mediated by biogenic amines, nitric oxide and eicosanoids, and nociceptor sensitization through nitric oxide and eicosanoids. Moreover, phospholipases A2 (PLA2), an important class of toxins present in bothropic venoms, appear to play an important role in the nociceptive and hypernociceptive response induced by BLV.

Cardiac effect induced by Crotalus durissus cascavella venom: Morphofunctional evidence and mechanism of action.

Envenoming, resulting from snake bites, is a global public health problem. The present study was undertaken to investigate the influence of Crotalus durissus cascavella (Cdcas) venom on cardiac activity and the mechanisms of action underlying its effect. To investigate the inotropic and chronotropic effects induced by Cdcas, studies were performed on the left and right atria. A series of tests were conducted to investigate whether the negative inotropic effect, induced by Cdcas, was related to cardiac damage. Cdcas venom (0.1-30 µg/mL) elicited a significant negative inotropic effect. The addition of Cdcas crude venom (7.5, 15 and 30 µg/mL) did not induce significant alterations in cell proliferation, nor in the enzymatic activity of total-CK and CKMB. Ultrastructural evaluation demonstrated that cardiac cells from isoproterenol and Cdcas groups revealed discreet swelling and displaced intermyofibrillar mitochondria with disorganization of the cristae. No change was observed in cardiac electrical activity in perfused isolated rat hearts with Cdcas. In addition, Cdcas reduced contractility in isolated cardiomyocytes from the rat left ventricle. The negative inotropic effect of Cdcas was reduced by l-NAME (100 µM), PTIO (100 µM), ODQ (10 µM) and KT5823 (1 µM), suggesting the participation of NO/cGMP/PKG pathway due to Cdcas. In non-anesthetized rats, Cdcas induced hypotension followed by bradycardia, the latter was also observed by ECG (anesthetized animals). Our results suggest that the negative inotropic effect induced by Cdcas venom is unrelated to cardiac toxicity, at least, at the concentrations tested; and occurs through of NO/cGMP/PKG pathway, likely leading to hypotension and bradycardia when administered in vivo.

New insights into the phylogeographic distribution of the 3FTx/PLA2 venom dichotomy across genus Micrurus in South America.

Micrurus is a monophyletic genus of venomous coral snakes of the family Elapidae. The ~80 recognized species within this genus are endemic to the Americas, and are distributed from southeastern United States to northern Argentina. Although relatively few bites are recorded due to their reclusive nature, semi-fossorial habits, and their occurrence in sparsely populated areas, coral snakes possess powerful venoms that target the cholinergic system and, if early treatment is missed, can cause neuromuscular paralysis, respiratory failure, and death by asphyxiation within hours of envenoming. The to-date proteomically characterized 18 micrurine venoms exhibit a puzzling phenotypic dichotomy, characterized by the toxin arsenal being dominated either by pre-synaptically acting PLA2s or post-synaptic 3FTxs, and a general, but imperfect, distributional pattern of these venom phenotypes along the North-South axis of the American continent. The lack of perfect phylogenetic clustering suggests that phylogeny may not be the sole factor driving the evolution of the divergent venom phenotypes across Micrurus venoms. To shed new light on the origin and expression pattern of the 3FTx/PLA2 venom dichotomy, we have conducted a comparative proteomics analysis of venoms from the Brazilian ribbon coral snake, Micrurus lemniscatus carvalhoi, sourced from different localities in the Brazilian states of São Paulo; the Caatinga coral snake, M. ibiboboca, from central Bahia state (Brazil); two Micrurus specimens of uncertain taxonomy collected in the Brazilian states of Alagoas and Rio de Janeiro; and the Western ribbon coral snake, M. l. helleri, from Leticia, the southernmost town of the Colombian Department of Amazonas. Venoms from São Paulo and Rio de Janeiro showed 3FTx-predominant phenotypes, while in venoms from Leticia, Alagoas and Bahia PLA2s represented the major toxin family. Comparative venom proteomics suggests that both Micrurus venom phenotypes exhibit a high degree of toxin evolvability. Mapping the 3FTx/PLA2 dichotomy across the Americas points to a phylogeographic pattern for venom phenotypes consistent with, but more complex than, the North-South distribution hypothesis anticipated in previous investigations. BIOLOGICAL SIGNIFICANCE: New World coral snakes (Micrurus: Elapidae) produce potent venoms that target pre- and post-synaptically cholinergic nerve terminals resulting in neuromuscular paralysis, and in severe envenomings, may lead to death from asphyxiation by respiratory arrest. Presynaptic ß-neurotoxins of group IA PLA2 protein subfamily and postsynaptic α-neurotoxins with 3FTx fold are the major components (>80%) of coral snake venoms. Micrurine venoms exhibit a puzzling phenotypic venom dichotomy, characterized by the dominant expression of either α- or ß-neurotoxins. The distribution of these alternative compositional profiles has been fragmentarily studied both across Micrurus phylogeny and along the North-South axis of the genus radiation in the American continent, from southern United States to Northern Argentina. The unpredictability of the neurotoxin profile across the distribution range of the coral snakes represents a difficulty for applying the most appropriate treatment upon a coral snakebite. A deep knowledge of the phylogeographic distribution and the evolution of dichotomic Micrurus venoms would be useful for tracing the evolutionary path to their present day phenotypes, rationalizing the patchy cross-reactivity of current Micrurus antivenoms, and improving the efficacy of antivenoms to neutralize coral snake envenomings.

Lemnitoxin, the major component of Micrurus lemniscatus coral snake venom, is a myotoxic and pro-inflammatory phospholipase A2.

The venom of Micrurus lemniscatus, a coral snake of wide geographical distribution in South America, was fractionated by reverse-phase HPLC and the fractions screened for phospholipase A2 (PLA2) activity. The major component of the venom, a PLA2, here referred to as 'Lemnitoxin', was isolated and characterized biochemically and toxicologically. It induces myotoxicity upon intramuscular or intravenous injection into mice. The amino acid residues Arg15, Ala100, Asn108, and a hydrophobic residue at position 109, which are characteristic of myotoxic class I phospholipases A2, are present in Lemnitoxin. This PLA2 is antigenically related to M. nigrocinctus nigroxin, Notechis scutatus notexin, Pseudechis australis mulgotoxin, and Pseudonaja textilis textilotoxin, as demonstrated with monoclonal and polyclonal antibodies. Lemnitoxin is highly selective in its targeting of cells, being cytotoxic for differentiated myotubes in vitro and muscle fibers in vivo, but not for undifferentiated myoblasts or endothelial cells. Lemnitoxin is not lethal after intravenous injection at doses up to 2µg/g in mice, evidencing its lack of significant neurotoxicity. Lemnitoxin displays anticoagulant effect on human plasma and proinflammatory activity also, as it induces paw edema and mast cell degranulation. Thus, the results of this work demonstrate that Lemnitoxin is a potent myotoxic and proinflammatory class I PLA2.

Coral snake bites (Micrurus spp.) in Brazil: a review of literature reports.

CONTEXT: In the Americas, the main representatives of the family Elapidae are coral snakes of the genus Micrurus, of which 33 species are in Brazil. They are the smallest cause of venomous snakebite in Brazil. We analyzed literature reports of coral snake bites in Brazil from 1867 to 2014, and provide a brief review of case series and reports of coral snake bites in the Americas in general. METHODS: Only reports with clinical descriptions of envenomation were included. The variables recorded included identification of the offending snake, patient's age, sex, bite site, clinical manifestations, treatment, including antivenom and anticholinesterase drugs, and general evolution of the cases. 30 published reports describing bites caused by Micrurus spp. in Brazil were identified and involved 194 distinct cases. Since no information on the clinical manifestations was available in 44 cases, the analysis was restricted to 25 reports (150 cases). RESULTS: Most patients were from southern (61.3%; primarily Santa Catarina state, 60%) and southeastern (20%) Brazil and were male (70.7%), with a median age of 27 years (interquartile interval = 18 to 40 years). The offending snakes were described in 59 cases (M. corallinus 36, M. frontalis 12, M. lemniscatus 5, M. hemprichi 2, M. filiformis 1, M. ibiboboca 1, M. spixii 1 and M. surinamensis 1); in 22 cases only the genus (Micrurus spp.) was reported. Of the 143 cases in which the bite site was recorded, most involved the hands (46.2%) and feet (26.6%). The main clinical features were local numbness/paresthesia (52.7%), local pain (48%), palpebral ptosis (33.3%), dizziness (26.7%), blurred vision (20.7%), weakness (20%), slight local edema (16%), erythema (16%), dysphagia (14.7%), dyspnea (11.3%), inability to walk (10.7%), myalgia (9.3%), salivation (8%) and respiratory failure (4.3%). Fang marks were described in 47.3% of cases and 14% of bites were classified as asymptomatic. A slight increase in total blood creatine kinase was reported in 3 children, suggesting mild myotoxicity. Therapeutic procedures included coral snake antivenom (77.3%), anticholinesterase drugs (6%), and mechanical ventilation (3.3%). Two patients reported in 1933 developed paralysis/respiratory failure and died 6 h and 17 h post-bite. Four more deaths probably caused by coral snakes were reported (2 in 1867, 1 in 1959, 1 in 1962), but no clinical information was available. DISCUSSION: Neuromuscular blockade was the hallmark of systemic envenomation by Micrurus spp., with signs of myasthenia such as weakness and ptosis that may evolve to paralysis and respiratory failure. Local features, mainly numbness/paresthesia and pain, were frequently reported, with the pain being intense in some cases. Although myotoxicity has been detected in experimental studies with Micrurus spp. venoms, few human reports described laboratory findings compatible with myotoxicity. CONCLUSION: Most coral snake bites reported in Brazil were caused by M. corallinus and M. frontalis, with several patients showing signs of acute myasthenia. Serious complications such as paralysis with respiratory failure were observed but comparatively rare. The deaths occurred where respiratory support (mechanical ventilation) was unavailable when needed.

Avaliação da DL50 e edema pulmonar induzido pelo veneno de Tityus serrulatus (Scorpiones; Buthidae) procedente da Bahia, Brasil / x

O escorpião Tityus serrulatus é conhecido como a espécie mais importante, do ponto de vista médico, pois é o que causa os acidentes mais graves registrados para o território brasileiro. Este trabalho trata da caracterização do veneno do escorpião T. serrulatus, através da obtenção da DL50 e determinação da capacidade em induzir edema pulmonar, em ratos. O veneno foi obtido através de estímulo elétrico. A toxicidade foi determinada através da avaliação da DL50, pelo método de Finney (1971). Os valores demonstraram a baixa toxicidade do veneno (96,16 mg/camundongo), que corresponde a 3 - 7 vezes menos tóxico que venenos de espécimes de outras regiões do Brasil. O veneno testado também não induziu edema pulmonar, avaliado através da diferença entre o peso do pulmão de animais experimentais e controle. Estes resultados demonstram uma variação do veneno de T. serrulatus e poderia explicar a ausência de óbitos e do registro de edema pulmonar nos pacientes picados nestas regiões do Estado da Bahia, Brasil.

Tityus serrulatus is the most important scorpion species, which cause most accidents and induces the most serious forms of poisoning in Brazil. In the present study we investigate the toxicity and pulmonary-edema induced ability of specimens from the metropolitan region of Salvador (RMS) and south-central Bahia (SCB), state of Bahia, Brazil. Male Swiss mice (18-22g) had been used to evaluate the toxicity by the Finney method (1971). The LDs50 tested in mice weighing 18-20 g was 96,16 mg/mice. This value represents 3 to 7 times lesser than the toxicity of T. serrulatus from other regions of Brazil. Also, the venom does not induce pulmonary edema, as assessed by the weight difference between the tested and control lung. Our results demonstrate marked variation in the lethal and the pulmonary-induced edema of Tityus serrulatus venom from RMS and SCB. These results could explain the absence of death and pulmonary complications of scorpion envenomation in some regions of Bahia, Brazil.