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.

Involvement of phospholipase A2 in the neuromuscular blockade caused by coralsnake (Micrurus spp.) venoms in mouse phrenic nerve-diaphragm preparations in vitro.

In this work, we examined the neuromuscular blockade caused by venoms from four South-American coralsnakes (Micrurus altirostris - MA, M. corallinus - MC, M. spixii - MS, and M. dumerilii carinicauda - MDC) and the ability of varespladib (VPL), a phospholipase A2 (PLA2) inhibitor, to attenuate this blockade. PLA2 activity was determined using a colorimetric assay and a fixed amount of venom (10 µg). Neurotoxicity was assayed using a single concentration of venom (10 µg/ml) in mouse phrenic nerve-diaphragm (PND) preparations mounted for myographic recordings and then subjected to histological analysis. All venoms showed PLA2 activity, with MS and MA venoms having the highest (15.53 ± 1.9 A425 nm/min) and lowest (0.23 ± 0.14 A425 nm/min) activities, respectively. VPL (292 and 438 µM) inhibited the PLA2 activity of all venoms, although that of MA venom was least affected. All venoms caused neuromuscular blockade, with MS and MDC venoms causing the fastest and slowest 100% blockade [in 40 ± 3 min and 120 ± 6 min (n = 4), respectively]; MA and MC produced complete blockade within 90-100 min. Preincubation of venoms with 292 µM VPL attenuated the blockade to varying degrees: the greatest inhibition was seen with MDC venom and blockade by MS venom was unaffected by this inhibitor. These results indicate that PLA2 has a variable contribution to coralsnake venom-induced neuromuscular blockade in vitro, with the insensitivity of MS venom to VPL suggesting that blockade by this venom is mediated predominantly by post-synaptically-active α-neurotoxins.

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.

Effect of the phospholipase A2 inhibitor Varespladib, and its synergism with crotalic antivenom, on the neuromuscular blockade induced by Crotalus durissus terrificus venom (with and without crotamine) in mouse neuromuscular preparations.

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 A2 (PLA2) 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 PLA2 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 PLA2 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.

Comparison of biological activities of Tityus pachyurus venom from two Colombian regions.

BACKGROUND: In the present study, we have tested whether specimens of the medically relevant scorpion Tityus pachyurus, collected from two climatically and ecologically different regions, differ in the biological activities of the venom. METHODS: Scorpions were collected in Tolima and Huila, Colombia. Chemical profiles of the crude venom were obtained from 80 scorpions for each region, using SDS-PAGE and RP-HPLC. Assays for phospholipase A2, direct and indirect hemolytic, proteolytic, neuromuscular, antibacterial, and insecticidal activities were carried out. RESULTS: The electrophoretic profiles of venom from the two regions showed similar bands of 6-14 kDa, 36-45 kDa, 65 kDa and 97 kDa. However, bands between 36 kDa and 65 kDa were observed with more intensity in venoms from Tolima, and a 95 kDa band occurred only in venoms from Huila. The chromatographic profile of the venoms showed differences in the intensity of some peaks, which could be associated with changes in the abundance of some components between both populations. Phospholipase A2 and hemolytic activities were not observable, whereas both venoms showed proteolytic activity towards casein. Insecticidal activity of the venoms from both regions showed significant variation in potency, the bactericidal activity was variable and low for both venoms. Moreover, no differences were observed in the neuromuscular activity assay. CONCLUSION: Our results reveal some variation in the activity of the venom between both populations, which could be explained by the ecological adaptations like differences in feeding, altitude and/or diverse predator exposure. However more in-depth studies are necessary to determine the drivers behind the differences in venom composition and activities.

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.

Comparison of biological activities of Tityus pachyurus venom from two Colombian regions

Background: In the present study, we have tested whether specimens of the medically relevant scorpion Tityus pachyurus, collected from two climatically and ecologically different regions, differ in the biological activities of the venom. Methods: Scorpions were collected in Tolima and Huila, Colombia. Chemical profiles of the crude venom were obtained from 80 scorpions for each region, using SDS-PAGE and RP-HPLC. Assays for phospholipase A2, direct and indirect hemolytic, proteolytic, neuromuscular, antibacterial, and insecticidal activities were carried out. Results: The electrophoretic profiles of venom from the two regions showed similar bands of 6-14 kDa, 36-45 kDa, 65 kDa and 97 kDa. However, bands between 36 kDa and 65 kDa were observed with more intensity in venoms from Tolima, and a 95 kDa band occurred only in venoms from Huila. The chromatographic profile of the venoms showed differences in the intensity of some peaks, which could be associated with changes in the abundance of some components between both populations. Phospholipase A2 and hemolytic activities were not observable, whereas both venoms showed proteolytic activity towards casein. Insecticidal activity of the venoms from both regions showed significant variation in potency, the bactericidal activity was variable and low for both venoms. Moreover, no differences were observed in the neuromuscular activity assay. Conclusion: Our results reveal some variation in the activity of the venom between both populations, which could be explained by the ecological adaptations like differences in feeding, altitude and/or diverse predator exposure. However more in-depth studies are necessary to determine the drivers behind the differences in venom composition and activities.(AU)

Comparison of biological activities of Tityus pachyurus venom from two Colombian regions

Abstract Background: In the present study, we have tested whether specimens of the medically relevant scorpion Tityus pachyurus, collected from two climatically and ecologically different regions, differ in the biological activities of the venom. Methods: Scorpions were collected in Tolima and Huila, Colombia. Chemical profiles of the crude venom were obtained from 80 scorpions for each region, using SDS-PAGE and RP-HPLC. Assays for phospholipase A2, direct and indirect hemolytic, proteolytic, neuromuscular, antibacterial, and insecticidal activities were carried out. Results: The electrophoretic profiles of venom from the two regions showed similar bands of 6-14 kDa, 36-45 kDa, 65 kDa and 97 kDa. However, bands between 36 kDa and 65 kDa were observed with more intensity in venoms from Tolima, and a 95 kDa band occurred only in venoms from Huila. The chromatographic profile of the venoms showed differences in the intensity of some peaks, which could be associated with changes in the abundance of some components between both populations. Phospholipase A2 and hemolytic activities were not observable, whereas both venoms showed proteolytic activity towards casein. Insecticidal activity of the venoms from both regions showed significant variation in potency, the bactericidal activity was variable and low for both venoms. Moreover, no differences were observed in the neuromuscular activity assay. Conclusion: Our results reveal some variation in the activity of the venom between both populations, which could be explained by the ecological adaptations like differences in feeding, altitude and/or diverse predator exposure. However more in-depth studies are necessary to determine the drivers behind the differences in venom composition and activities.

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.

Toxinological characterization of venom from Leptodeira annulata (Banded cat-eyed snake; Dipsadidae, Imantodini).

We investigated the histology of Duvernoy's venom gland and the biochemical and biological activities of Leptodeira annulata snake venom. The venom gland had a lobular organization, with secretory tubules formed by serous epithelial cells surrounding each lobular duct. The latter drained into a common lobular duct and subsequently into a central cistern. In contrast, the supralabial gland was mucous in nature. SDS-PAGE revealed a profile of venom components that differed from pitviper (Bothrops spp.) venoms. RP-HPLC also revealed greater complexity of this venom compared to Bothrops venoms. The venom had no esterase, l-amino acid oxidase or thrombin-like activity, but was proteolytic towards elastin-Congo red, fibrin, fibrinogen, gelatin and hide powder azure. The venom showed strong α-fibrinogenase and fibrinolytic activities and reduced the rate and extent of plasma recalcification. The proteolytic activity was inhibited by EDTA and 1,10-phenanthroline (metalloproteinase inhibitors) but not by AEBSF and PMSF (serine proteinase inhibitors). The venom had phospholipase A2 (PLA2) activity that was inhibited by varespladib. The venom cross-reacted with antivenoms to lancehead (Bothrops spp.), coralsnake (Micrurus spp.) and rattlesnake (Crotalus durissus terrificus) venoms. The venom did not aggregate rat platelets or inhibit collagen-induced aggregation, but partially inhibited thrombin-induced aggregation. The venom was hemorrhagic (inhibited by EDTA) and increased the vascular permeability (inhibited by varespladib) in rat dorsal skin. In gastrocnemius muscle, the venom caused myonecrosis and increased serum creatine kinase concentrations. In conclusion, L. annulata venom has various enzymatic and biological activities, with the local effects being mediated primarily by metalloproteinases and PLA2.

Biochemical characterization of venom from Pseudoboa neuwiedii (Neuwied's false boa; Xenodontinae; Pseudoboini).

In this work, we examined the proteolytic and phospholipase A2 (PLA2) activities of venom from the opisthoglyphous colubrid Pseudoboa neuwiedii. Proteolytic activity (3 and 10 µg of venom) was comparable to that of Bothrops neuwiedii venom but less than Bothrops atrox. This activity was inhibited by EDTA and 1,10-phenanthroline but only slightly affected (≤30% inhibition) by PMSF and AEBSF, indicating it was mediated by snake venom metalloproteinases (SVMPs). The pH and temperature optima for proteolytic activity were 8.0 and 37 °C, respectively. The venom had no esterase activity, whereas PLA2 activity was similar to B. atrox, greater than B. neuwiedii but less than B. jararacussu. SDS-PAGE revealed venom proteins >100 kDa, 45-70 kDa, 21-24 kDa and ~15 kDa, and mass spectrometry of protein bands revealed SVMPs, cysteine-rich secretory proteins (CRISPs) and PLA2, but no serine proteinases. In gelatin zymography, the most active bands occurred at 65-68 kDa (seen with 0.05-0.25 µg of venom). Caseinolytic activity occurred at 50-66 kDa and was generally weaker than gelatinolytic activity. RP-HPLC of venom yielded 15 peaks, five of which showed gelatinolytic activity; peak 7 was the most active and apparently contained a P-III class SVMP. The venom showed α-fibrinogenase activity, without affecting the ß and γ chains; this activity was inhibited by EDTA and 1,10-phenanthroline. The venom did not clot rat citrated plasma but reduced the rate and extent of coagulation after plasma recalcification. In conclusion, P. neuwiedii venom is highly proteolytic and could potentially affect coagulation in vivo by degrading fibrinogen via SVMPs.

A survey on some biochemical and pharmacological activities of venom from two Colombian colubrid snakes, Erythrolamprus bizona (Double-banded coral snake mimic) and Pseudoboa neuwiedii (Neuwied's false boa).

Colombian colubrid snake venoms have been poorly studied. They represent a great resource of biological, ecological, toxinological and pharmacological research. We assessed some enzymatic properties and neuromuscular effects of Erythrolamprus bizona and Pseudoboa neuwiedii venoms from Colombia. Proteolytic, amidolytic and phospholipase A2 (PLA2) activities were analyzed using colorimetric assays and the neuromuscular activity was analyzed in chick biventer cervicis (BC) preparations. The venom of both species showed very low PLA2 and amidolytic activities; however, both exhibited high proteolytic activity, which in E. bizona venom surpassed that of P. neuwiedii venom. E. bizona and P. neuwiedii venoms provoked partial neuromuscular blockade, which was more prominent in P. neuwiedii venom. E. bizona venom (30 µg/ml) induced a significant potentiation of the contracture response to exogenous ACh (110 µM), which was not accompanied by twitch height alteration, whereas the highest venom concentration (100 µg/ml) inhibited contracture responses to both ACh and KCl (40 mM). In contrast, P. neuwiedii venom (30 and 100 µg/ml) caused significant reduction in the contracture responses to exogenous ACh and KCl. The morphological analyses showed high myotoxic effects in the muscle fibers of BC incubated with either venoms; however, they are more prominent in the P. neuwiedii venom. Our results suggest that the myotoxicity of the venom of the two Colombian species can be ascribed to their high proteolytic activity. An interesting data was the potentiation of the ACh-induced contracture, but not the twitch height, caused by E. bizona venom, at a concentration that is harmless to muscle fibers integrity. This phenomenon remains to be further elucidated, and suggest that a possible involvement of post-synaptic receptors cannot be discarded. This work is a contribution to expand the knowledge on colubrid venoms; it allows envisaging that the two venoms offer the potential to go further in the identification of their components and biological targets.

Biological activities of Leptodeira annulata (banded cat-eyed snake) venom on vertebrate neuromuscular preparations.

The physiological properties of colubrid snake venoms are largely unknown and less frequently investigated. In this study, we assessed the enzymatic properties and biological activities of Leptodeira annulata (banded cat-eyed snake) venom, an opistoglyphous snake from Colombia. The proteolytic, phospholipase A2 and amidolytic activities are assessed using colorimetric assays and the biological activities were analyzed in avian and mammalian neuromuscular preparations. L. annulata venom caused neuromuscular blockade in chick biventer cervicis (BC) preparations (40± 15% and 50± 3% of twitch reduction for 30 and 100 µg/ml, respectively; p < 0.05) following 120 incubation; 10 µg/ml of venom did not induce blockade. There was a mild reduction in contracture response to exogenous acetylcholine (110 µM) in BC preparations exposed to 10 and 30 µg of venom/ml (∼4% and ∼32% of reduction, respectively, p > 0.05, n = 4) compared to basal values whereas the highest concentration (100 µg/ml) abolished it after 120 min. The venom caused a significant reduction in contracture response elicited by KCl (∼58 and ∼90 of reduction for 30 and 100 µg/ml, respectively, p < 0.05, n = 4). In mouse phrenic nerve-diaphragm (PND) preparations, L. annulata venom induced a progressive muscle membrane depolarization [from -85.9 ± 1.6 mV (t0) to -72.2 ± 2.9 mV (t120), p < 0.05, n = 4); the postsynaptic receptors remained functional as shown by carbachol-induced depolarization. The morphological analyses showed a concentration-dependent number of pathological states in muscle fibers from both BC and PND preparations pre-exposed to venom. The venom showed high proteolytic activity and low phospholipase A2 activity; there was no evidence for serine protease activity. These results indicate that the neuromuscular effect induced by L. annulata venom resulted from damaged muscle fibers that lead to the blockade of twitches response. The findings suggest that the myotoxicity might be related to the presence of metalloproteases in this venom.