Biochemical and toxicological profiles of venoms from an adult female South American bushmaster (Lachesis muta rhombeata) and her offspring.

In this work, we compared the biochemical and toxicological profiles of venoms from an adult female specimen of Lachesis muta rhombeata (South American bushmaster) and her seven offspring born in captivity, based on SDS-PAGE, RP-HPLC, enzymatic, coagulant, and hemorrhagic assays. Although adult and juvenile venoms showed comparable SDS-PAGE profiles, juveniles lacked some chromatographic peaks compared with adult venom. Adult venom had higher proteolytic (caseinolytic) activity than juvenile venoms (p < 0.05), but there were no significant inter-venom variations in the esterase, PLA2, phosphodiesterase and L-amino acid oxidase (LAAO) activities, although the latter activity was highly variable among the venoms. Juveniles displayed higher coagulant activity on human plasma, with a minimum coagulant dose ∼42% lower than the adult venom (p < 0.05), but there were no age-related differences in thrombin-like activity. Adult venom was more fibrinogenolytic (based on the rate of fibrinogen chain degradation) and hemorrhagic than juvenile venoms (p < 0.05). The effective dose of Bothrops/Lachesis antivenom (produced by the Instituto Butantan) needed to neutralize the coagulant activity was ∼57% greater for juvenile venoms (p < 0.05), whereas antivenom did not attenuate the thrombin-like activity of juvenile and adult venoms. Antivenom significantly reduced the hemorrhagic activity of adult venom (400 µg/kg, i. d.), but not that of juvenile venoms. Overall, these data indicate a compositional and functional ontogenetic shift in L. m. rhombeata venom.

Cardiac Effects of Micrurus corallinus and Micrurus dumerilii carinicauda (Elapidae) Venoms and Neutralization by Brazilian Coralsnake Antivenom and Varespladib.

In this work, we examined the action of two South American coralsnake (Micrurus corallinus and Micrurus dumerilii carinicauda) venoms on rat heart function in the absence and presence of treatment with Brazilian coralsnake antivenom (CAV) and varespladib (VPL), a potent phospholipase A2 inhibitor. Anesthetized male Wistar rats were injected with saline (control) or a single dose of venom (1.5 mg/kg, i.m.) and monitored for alterations in echocardiographic parameters, serum CK-MB levels and cardiac histomorphology, the latter using a combination of fractal dimension and histopathological methods. Neither of the venoms caused cardiac functional alterations 2 h after venom injection; however, M. corallinus venom caused tachycardia 2 h after venom injection, with CAV (given i.p. at an antivenom:venom ratio of 1:1.5, v/w), VPL (0.5 mg/kg, i.p.) and CAV + VPL preventing this increase. Both venoms increased the cardiac lesional score and serum CK-MB levels compared to saline-treated rats, but only the combination of CAV + VPL prevented these alterations, although VPL alone was able to attenuate the increase in CK-MB caused by M. corallinus venom. Micrurus corallinus venom increased the heart fractal dimension measurement, but none of the treatments prevented this alteration. In conclusion, M. corallinus and M. d. carinicauda venoms caused no major cardiac functional alterations at the dose tested, although M. corallinus venom caused transient tachycardia. Both venoms caused some cardiac morphological damage, as indicated by histomorphological analyses and the increase in circulating CK-MB levels. These alterations were consistently attenuated by a combination of CAV and VPL.

Partial efficacy of a Brazilian coralsnake antivenom and varespladib in neutralizing distinct toxic effects induced by sublethal Micrurus dumerilii carinicauda envenoming in rats.

In this work, we reported the efficacy of a combination of Brazilian therapeutic coralsnake antivenom (CAV) and varespladib (phospholipase A2 inhibitor - VPL) in partially neutralizing selected toxic effects of Micrurus dumerilii carinicauda coralsnake venom in rats. Venom caused local myonecrosis and systemic neurotoxicity, nephrotoxicity, and hepatotoxicity within 2 h of injection. CAV and VPL administered separately failed to prevent most of these alterations. However, a combination of CAV plus VPL offered variable protection against venom-induced coagulation disturbances, leukocytosis, and renal-hepatic morphological alterations.

In vivo treatment with varespladib, a phospholipase A2 inhibitor, prevents the peripheral neurotoxicity and systemic disorders induced by Micrurus corallinus (coral snake) venom in rats.

In this study, we investigated the action of varespladib (VPL) alone or in combination with a coral snake antivenom (CAV) on the local and systemic effects induced by Micrurus corallinus venom in rats. Adult male Wistar rats were exposed to venom (1.5 mg/kg - i.m.) and immediately treated with CAV (antivenom:venom ratio 1:1.5 'v/w' - i.p.), VPL (0.5 mg/kg - i.p.), or both of these treatments. The animals were monitored for 120 min and then anesthetized to collect blood samples used for haematological and serum biochemical analysis; after euthanasia, skeletal muscle, renal and hepatic tissue samples were collected for histopathological analysis. M. corallinus venom caused local oedema without subcutaneous haemorrhage or apparent necrosis formation, although there was accentuated muscle morphological damage; none of the treatments prevented oedema formation but the combination of CAV and VPL reduced venom-induced myonecrosis. Venom caused neuromuscular paralysis and respiratory impairment in approximately 60 min following envenomation; CAV alone did not prevent the neurotoxic action, whereas VPL alone prevented neurotoxic symptoms developing as did the combination of CAV and VPL. Venom induced significant increase of serum CK and AST release, mostly due to local and systemic myotoxicity, which was partially prevented by the combination of CAV and VPL. The release of hepatotoxic serum biomarkers (LDH and ALP) induced by M. corallinus venom was not prevented by CAV and VPL when individually administered; their combination effectively prevented ALP release. The venom-induced nephrotoxicity (increase in serum creatinine concentration) was prevented by all the treatments. VPL alone or in combination with CAV significantly prevented the venom-induced lymphocytosis. In conclusion, VPL shows to be effective at preventing the neurotoxic, nephrotoxic, and inflammatory activities of M. corallinus venom. In addition, VPL acts synergistically with antivenom to prevent a number of systemic effects caused by M. corallinus venom.

Protective action of N-acetyl-L-cysteine associated with a polyvalent antivenom on the envenomation induced by Lachesis muta muta (South American bushmaster) in rats.

In this study, we examined the potential use of N-acetyl-L-cysteine (NAC) in association with a polyvalent antivenom and as stand-alone therapy to reduce the acute local and systemic effects induced by Lachesis muta muta venom in rats. Male Wistar rats (300-350 g) were exposed to L. m. muta venom (1.5 mg/kg - i.m.) and subsequently treated with anti-Bothrops/Lachesis serum (antivenom:venom ratio 1:3 'v/w' - i.p.) and NAC (150 mg/kg - i.p.) separately or in association; the animals were monitored for 120 min to assess changes in temperature, locomotor activity, local oedema formation and the prevalence of haemorrhaging. After this time, animals were anesthetized in order to collect blood samples through intracardiac puncture and then euthanized for collecting tissue samples; the hematological-biochemical and histopathological analyses were performed through conventional methods. L. m. muta venom produced pronounced local oedema, subcutaneous haemorrhage and myonecrosis, with both antivenom and NAC successfully reducing the extent of the myonecrotic lesion when individually administered; their association also prevented the occurrence of subcutaneous haemorrhage. Venom-induced creatine kinase (CK) release was significantly prevented by NAC alone or in combination with antivenom; NAC alone failed to reduce the release of hepatotoxic (alanine aminotransferase) and nephrotoxic (creatinine) serum biomarkers induced by L. m. muta venom. Venom induced significant increase of leucocytes which was also associated with an increase of neutrophils, eosinophils and monocytes; antivenom and NAC partially reduced these alterations, with NAC alone significantly preventing the increase of eosinophils whereas neither NAC or antivenom prevented the increase in monocytes. Venom did not induce changes in the erythrogram parameters. In the absence of a suitable antivenom, NAC has the potential to reduce a number of local and systemic effects caused by L. m. muta venom.

Action of Varespladib (LY-315920), a Phospholipase A2 Inhibitor, on the Enzymatic, Coagulant and Haemorrhagic Activities of Lachesis muta rhombeata (South-American Bushmaster) Venom.

Varespladib (VPL) was primarily developed to treat inflammatory disturbances associated with high levels of serum phospholipase A2 (PLA2). VPL has also demonstrated to be a potential antivenom support agent to prevent PLA2-dependent effects produced by snake venoms. In this study, we examined the action of VPL on the coagulant, haemorrhagic and enzymatic activities of Lachesis muta rhombeata (South-American bushmaster) venom. Conventional colorimetric enzymatic assays were performed for PLA2, caseinolytic and esterasic activities; in vitro coagulant activities for prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in rat citrated plasma through a quick timer coagulometer, whereas the dimensions of haemorrhagic haloes obtained after i.d. injections of venom in Wistar rats were determined using ImageJ software. Venom (1 mg/ml) exhibited accentuated enzymatic activities for proteases and PLA2 in vitro, with VPL abolishing the PLA2 activity from 0.01 mM; VPL did not affect caseinolytic and esterasic activities at any tested concentrations (0.001-1 mM). In rat citrated plasma in vitro, VPL (1 mM) alone efficiently prevented the venom (1 mg/ml)-induced procoagulant disorder associated to extrinsic (PT) pathway, whereas its association with a commercial antivenom successfully prevented changes in both intrinsic (aPTT) and extrinsic (PT) pathways; commercial antivenom by itself failed to avoid the procoagulant disorders by this venom. Venom (0.5 mg/kg)-induced hemorrhagic activity was slightly reduced by VPL (1 mM) alone or combined with antivenom (antivenom:venom ratio 1:3 'v/w') in rats, with antivenom alone producing no protective action on this parameter. In conclusion, VPL does not inhibit other major enzymatic groups of L. m. rhombeata venom, with its high PLA2 antagonize activity efficaciously preventing the venom-induced coagulation disturbances.

A morphological and molecular study of Hydrodynastes gigas (Serpentes, Dipsadidae), a widespread species from South America.

BACKGROUND: Studies with integrative approaches (based on different lines of evidence) are fundamental for understanding the diversity of organisms. Different data sources can improve the understanding of the taxonomy and evolution of snakes. We used this integrative approach to verify the taxonomic status of Hydrodynastes gigas (Duméril, Bibron & Duméril, 1854), given its wide distribution throughout South America, including the validity of the recently described Hydrodynastes melanogigas Franco, Fernandes & Bentim, 2007. METHODS: We performed a phylogenetic analysis of Bayesian Inference with mtDNA 16S and Cytb, and nuDNA Cmos and NT3 concatenated (1,902 bp). In addition, we performed traditional morphometric analyses, meristic, hemipenis morphology and coloration pattern of H. gigas and H. melanogigas. RESULTS: According to molecular and morphological characters, H. gigas is widely distributed throughout South America. We found no evidence to support that H. gigas and H. melanogigas species are distinct lineages, therefore, H. melanogigas is a junior synonym of H. gigas. Thus, the melanic pattern of H. melanogigas is the result of a polymorphism of H. gigas. Melanic populations of H. gigas can be found in the Tocantins-Araguaia basin.

Cardiovascular activity of Micrurus lemniscatus lemniscatus (South American coralsnake) venom.

Envenomation by coralsnakes (Micrurus spp.) is characterized by blockade of peripheral neurotransmission mediated by the presence of α- and ß-neurotoxins. However, little is known about their cardiovascular activity. Micrurus lemniscatus lemniscatus is a coralsnake found in the Amazon basin and occasionally causes envenomation in humans. In this study, we examined the hemodynamic, vascular and atrial responses to M. l. lemniscatus venom. Anesthetized rats were used for hemodynamic and electrocardiogram (ECG) recordings; in vitro experiments were carried out in rat isolated thoracic aorta and atria preparations. In vivo, venom (0.1 and 0.3 mg/kg) caused immediate and persistent hypotension that was maximal within the first minute with both doses being lethal after ~40 and ~20 min, respectively. ECG, heart and respiratory rates were not altered during the transient hypotension phase induced by venom but all altered prior to death. There was no evidence of myonecrosis in cardiac muscle tissue, pulmonary hemorrhage nor thrombosis in anesthetized rats exposed to venom. In vitro, venom (10 µg/ml) did not contract aortic strips nor affected the maximal responses to pre-contraction with phenylephrine (PE, 0.0001-30 µM) in strips with and without endothelium. However, venom (10 µg/ml) relaxed aortic strips with endothelium pre-contracted with PE. In aortic strips pre-contracted with PE, venom prevented acetylcholine (0.0001-30 µM)-induced relaxation in strips with endothelium without affecting relaxation induced by sodium nitroprusside (0.1-100 nM) in strips without endothelium. Venom (30 µg/ml) produced a transient increase of atrial contractile force without affecting atrial rate. Taken together these findings indicate a predominantly vascular action of the venom, most likely involving toxins interacting with muscarinic receptors.

Taxonomy of the Micrurus spixii species complex (Serpentes, Elapidae).

Historically, the Micrurus spixii species complex comprises four subspecies (M. s. spixii, M. s. martiusi, M. s. obscurus, and M. s. princeps), however, recently, some authors have considered only two full species (M. spixii and M. obscurus). In this paper, we report data on meristic, morphometric, color pattern in life and after preservation, cranial osteology, and hemipenis morphology to Micrurus spixii species complex. We examined 358 specimens throughout the geographical distribution, including the type-series of all taxa. Differences in color pattern and morphology of hemipenis and skull support the existence of only two diagnosable taxa, M. spixii and M. obscurus, both considered here as a full species. We corroborate the synonymy of Micrurus s. martiusi and M. spixii based on wide overlap of meristic and morphometric characters, as well as the similarities of color pattern, hemipenis and skull characters. We can distinguished M. spixii from M. obscurus (in parenthesis) by the combination of the following characters: black cephalic cap (vs. cephalic cap absent, with red parietal region), hemipenial body with spines dispersed on the asulcate surface (vs. spines arranged in rows on the asulcate surface), capitate condition of hemipenis (vs. organ partially-capitate), narrow parietal bone with posterior angular borders (vs. enlarged parietal bone with elliptical posterior border), and relatively long venom inoculating fangs (vs. relatively short venom inoculating fangs).

Neurotoxicity of Micrurus lemniscatus lemniscatus (South American coralsnake) venom in vertebrate neuromuscular preparations in vitro and neutralization by antivenom.

We investigated the effect of South American coralsnake (Micrurus lemniscatus lemniscatus) venom on neurotransmission in vertebrate nerve-muscle preparations in vitro. The venom (0.1-30 µg/ml) showed calcium-dependent PLA2 activity and caused irreversible neuromuscular blockade in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, contractures to exogenous acetylcholine and carbachol (CCh), but not KCl, were abolished by venom concentrations ≥ 0.3 µg/ml; in PND preparations, the amplitude of the tetanic response was progressively attenuated, but with little tetanic fade. In low Ca2+ physiological solution, venom (10 µg/ml) caused neuromuscular blockade in PND preparations within ~ 10 min that was reversible by washing; the addition of Ca2+ immediately after the blockade temporarily restored the twitch responses, but did not prevent the progression to irreversible blockade. Venom (10 µg/ml) did not depolarize diaphragm muscle, prevent depolarization by CCh, or cause muscle contracture or histological damage. Venom (3 µg/ml) had a biphasic effect on the frequency of miniature end-plate potentials, but did not affect their amplitude; there was a progressive decrease in the amplitude of evoked end-plate potentials. The amplitude of compound action potentials in mouse sciatic nerve was unaffected by venom (10 µg/ml). Pre-incubation of venom with coralsnake antivenom (Instituto Butantan) at the recommended antivenom:venom ratio did not neutralize the neuromuscular blockade in PND preparations, but total neutralization was achieved with a tenfold greater volume of antivenom. The addition of antivenom after 50% and 80% blockade restored the twitch responses. These results show that M. lemniscatus lemniscatus venom causes potent, irreversible neuromuscular blockade, without myonecrosis. This blockade is apparently mediated by pre- and postsynaptic neurotoxins and can be reversed by coralsnake antivenom.

Antigenotoxicity protection of Carapa guianensis oil against mitomycin C and cyclophosphamide in mouse bone marrow.

The aim of this study was to evaluate the possible protective of C. guianensis oil against MMC and CP, which are direct- and indirect-acting chemical mutagens, using the micronucleus test. Three experiments were performed. First the C. guianensis oil was co-administered to mice at doses of 250, 500 and 1000 mg/kg bw with 4 mg/kg bw MMC or 50 mg/kg bw CP. Second, the mutagenic drug (CP) was administered ip 50 mg/kg bw and after 6 and 12 hours 250 and 500 mg/kg bw of C. guianensis oil were administered. In the last, C. guianensis oil was administrated (250 and 500 mg/kg bw) during five days and after it was administered ip 50 mg/kg bw CP. The results obtained showed that the C. guianensis oil is not cytotoxic neither genotoxic to mouse bone marrow. Regarding the antimutagenic effect, all doses of C. guianensis oil were significantly (p < 0.05) effective in reducing the frequency of micronucleated polychromatic erythrocytes, when compared with MMC or CP alone. Based on these results, our results suggest that the C. guianensis oil shows medicinal potential as an antimutagenic agent, modulating the mutagenicity caused by both direct- and indirect-acting chemical mutagens, in a mammalian model.

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.