Neutralization of hyaluronidase and indirect hemolytic activities of Costa Rican snake venoms by a polyvalent antivenom

J. A. Gené, M. Gómez, J. M. Gutiérrez and L. Cerdas, Neutralization of hyaluronidase and indirect hemolytic activities of Costa Rican snake venoms by polyvalent antivenom. Toxicon23, 1015–1018, 1985. — The ability of a polyvalent antivenom produced in Costa Rica to neutralize hyaluronidase and indirect hemolytic activities of Costa Rican crotalid venoms was tested. Antivenom neutralized remarkably well hyaluronidase activity of all venoms tested, with ed₅₀ doses ranging from 2 to 45 μl antivenom/mg venom. Neutralization of indirect hemolytic activity was also achieved using the antivenom, with ed₅₀ values ranging from 332 to 716μl antivenom/mg venom.     

Bioactive proteins from stonefish venom

1. Of all the venomous fish known, the stonefish is one of the most commonly encountered by man. Studies on its venom started in the 1950s, but little work was performed after that until several groups revived interest in the venom in the 1980s after easier accessibility to the fish. 2. Stonefish venom is a mixture of proteins, containing several enzymes, including hyaluronidase of high specific activity. A purified stonefish hyaluronidase has been characterized. 3. Several of the effects of the crude venom have been isolated to a protein lethal factor that has cytolytic, neurotoxic and hypotensive activity. This protein is stonustoxin from Synanceja horrida, trachynilysin from Synanceja trachynis and verrucotoxin from Synanceja verrucosa. 4. The biochemical properties and activities of these protein lethal factors are reviewed.     

A new method for the detection of phospholipase A2 variants: identification of isozymes in the venoms of newborn and adult Bothrops asper (terciopelo) snakes

A new method for the identification of phospholipase A2 isozymes in snake venoms is described. The technique is based on the separation of the venom components by isoelectric focusing in agarose gels, transfer of the protein bands by diffusion onto nitrocellulose paper and detection of the phospholipolytic activity of the enzymes by a hemolytic assay either in agarose gels or by benzidine reaction on a solid matrix. Striking differences in the electrophoretic patterns of the phospholipase A2 isozymes between the Atlantic and Pacific venoms and between the newborn and adult venoms from Bothrops asper specimens were observed. The method allowed the detection of 9 different phospholipase A2 isozymes in the venom of adult Atlantic, 7 isozymes in the venom of adult Pacific, and 2-3 isozymes in the venoms of newborn specimens. Horse polyvalent antivenom varied in its capacity to neutralize the phospholipolytic activity of the different isozymes in the same venom and among different venoms.     

The hyaluronidase activities of some Southeast Asian snake venoms

The hyaluronidase activities of venoms of snakes indigenous to Southeast Asia were investigated. With the exception of the venom of the Malayan krait Bungarus candidus, the elapid venoms had either little or no hyaluronidase activities, whereas the viperid venoms possessed considerable activity. A component of Russell's viper venom with hyaluronidase activities had a mol. wt of approximately 14,000. Neither MP4, a monoclonal antibody raised against the purified Russell's viper venom hyaluronidase toxin, nor a monospecific polyclonal antivenom neutralized the hyaluronidase activities of this purified hyaluronidase component of crude Russell's viper venom. The Russell's viper venom hyaluronidase activities was labile on heating and storage. The significance of these observations to envenomation and antivenom production is discussed.     

Stonefish (Synanceia spp.) antivenom neutralises the in vitro and in vivo cardiovascular activity of soldierfish (Gymnapistes marmoratus) venom.

The soldierfish (Gymnapistes marmoratus), which is related to the stonefish (Synanceia spp.), inhabits the western, southern and lower eastern coastlines of Australia. We have previously found that G. marmoratus venom possesses pharmacological activity similar to Synanceia trachynis venom (Hopkins, B.J., Hodgson, W.C., 1998. Cardiovascular studies on venom from the soldierfish (Gymnapistes marmoratus). Toxicon 36, 973-872; Church, J.E., Hodgson, W.C., 2000. Dose-dependent cardiovascular and neuromuscular effects of stonefish (Synanceja trachynis) venom. Toxicon 38, 391-407), namely an action at muscarinic receptors and adrenoceptors. The aim of this study was to determine the effectiveness of Synanceia antivenom in neutralising the in vitro and in vivo cardiovascular activity of G. marmoratus venom. Venom extract (0.4-12 microg protein/ml) caused dose- and endothelium-dependent relaxation in porcine U46619-precontracted coronary arteries. This relaxation was abolished by 10 min prior exposure of the tissue to Synanceia antivenom (3 units/ml). In rat paced (5 ms, 2 Hz, 7-12 V) left atria, G. marmoratus venom extract (40 microg protein/ml) produced a transient negative, followed by a sustained positive inotropic response. In spontaneously beating right atria, venom extract (40 microg protein/ml) produced similar changes in rate. Prior incubation of venom extract with Synanceia antivenom (1 unit/4 microg venom extract protein, 10 min) significantly attenuated both components of the inotropic response, and abolished the positive chronotropic response. In the anaesthetised rat, venom extract (400 microg protein/kg, i.v.) produced a transient depressor response, followed by a more sustained pressor response. Prior incubation of venom extract with Synanceia antivenom (1 unit/4 microg venom extract protein, 10 min) significantly attenuated both components of the in vivo response. As Synanceia antivenom neutralised the cardiovascular activity of G. marmoratus venom both in vitro and in vivo, we suggest that the venoms of the two species may share a similar component(s).     

Marine antivenoms

There is an enormous diversity and complexity of venoms and poisons in marine animals. Fatalities have occurred from envenoming by sea snakes, jellyfish, venomous fish such as stonefish, cone snails, and blue-ringed octopus. Deaths have also followed ingestion of toxins in shellfish, puffer fish (Fugu), and ciguatoxin-containing fish. However antivenoms are generally only available for envenoming by certain sea snakes, the major Australian box jellyfish (Chironex fleckeri) and stonefish. There have been difficulties in characterizing the toxins of C. fleckeri venom, and there are conflicting animals studies on the efficacy of C. fleckeri antivenom. The vast majority of C. fleckeri stings are not life-threatening, with painful skin welts the major finding. However fatalities that do occur usually do so within 5 to 20 minutes of the sting. This unprecedented rapid onset of cardiotoxicity in clinical envenoming suggests that antivenom may need to be given very early (within minutes) and possibly in large doses if a life is to be saved. Forty years of anecdotal experience supports the beneficial effect of stonefish antivenom in relieving the excruciating pain after stonefish spine penetration. It remains uncertain whether stonefish antivenom is efficacious in stings from spines of other venomous fish, and the recommendation of giving the antivenom intramuscularly needs reassessment.     

A potent vasoactive cytolysin isolated from Scorpaena plumieri scorpionfish venom

A new vasoactive cytolytic toxin, referred to as Sp-CTx, has been purified from the venom of the scorpionfish Scorpaena plumieri by a combination of gel filtration and anion exchange chromatographies. An estimation of Sp-CTx native molecular mass, performed by size exclusion chromatography, demonstrated that it is a 121 kDa protein. Further physicochemical studies revealed its glycoproteic nature and dimeric constitution, comprising subunits of approximately 65 kDa (MALDI-TOF-MS). Such protein has proved to possess a potent hemolytic activity on washed rabbit erythrocytes (EC₅₀ 0.46 nM), whose effect was strongly reduced after treatment with antivenom raised against stonefish venom - Synanceja trachynis (SFAV). This cross-reactivity has been confirmed by western blotting. Like S. plumieri whole venom (100 μg/mL), Sp-CTx (1-50 nM) caused a biphasic response on phenylephrine pre-contracted rat aortic rings, characterized by an endothelium- and dose-dependent relaxation phase followed by a contractile phase. The vasorelaxant activity has been abolished by l-NAME, demonstrating the involvement of nitric oxide on the response. We report here the first isolation of a cytolytic/vasoactive protein from scorpionfish venom and the data provided suggest structural and functional similarities between Sp-CTx and previously published stonefish hemolytic toxins.     

Partial purification of Chironex fleckeri (sea wasp) venom by immunochromatography with antivenom

Chironex fleckeri crude venom was partially purified using immobilized commercially available ovoid antivenom. The antibody preparation reacted with lethal, hemolytic, dermonecrotic and mouse writhing (pain) factors in the crude venom. The lethal activity was purified five fold, while the specific eluate contained lower quantities of hemolytic, dermonecrotic and mouse writhing activities than did the crude venom.     

Stonefish (Synanceia trachynis) Antivenom: In Vitro Efficacy and Clinical Use

Certain species of fish have long been recognised as venomous. Although venomous fish do not represent a substantial source of human mortality, they are responsible for a number of envenomations each year that are serious enough to warrant clinical treatment. To the author's knowledge, the only fish antivenom still commercially available is the stonefish antivenom produced by CSL Ltd. in Australia. This antivenom consists of the purified F(ab)₂ fragment of equine IgG antibodies raised against the venom of Synanceia trachynis. The antivenom is cheap, and effective in neutralising all known clinical effects of serious S. trachynis envenomation. In addition, there is experimental evidence that stonefish antivenom neutralises the pharmacological effects of other fish venoms, particularly those of the lionfish (Pterois volitans) and the soldierfish (Gymnapistes marmoratus), as well as displaying cross‐reactivity with them in Western immuno‐blotting. The potential therefore exists for the use of stonefish antivenom in the treatment of severe envenomations by species of fish other than Synanceia spp.     

An electrophoretic study on phospholipase A2 isoenzymes in the venoms of Central American crotaline snakes.

The number and isoelectric points of phospholipase A2 isoenzymes were studied in the venoms of 12 Central American crotaline snakes of the genera Bothrops, Crotalus, Lachesis and Agkistrodon. The study was carried out by using a methodology based on electrophoretic separation of venoms, transfer to nitrocellulose and detection of activity of the bands by an indirect hemolytic assay in agarose-erythrocyte-egg yolk gels. All venoms tested had indirect hemolytic activity, although they varied in the number and isoelectric point of their phospholipases A2. Most venoms had predominantly acidic isoenzymes, with the exception of A. bilineatus which had mainly basic isoenzymes and B. schlegelii which had both acidic and basic isoenzymes. Analysis of interindividual variability in B. asper venom demonstrated that two phospholipase A2 isoenzymes are present in some venoms but absent in others. Polyvalent antivenom was effective in neutralizing phospholipase A2 activity of the 12 venoms tested, when venoms and antivenom were incubated in the fluid phase. This work demonstrates a conspicuous interspecific variability in the number and isoelectric points of phospholipases A2 present in Central American crotaline snake venoms.     

An in vitro comparative study upon the toxic properties of the venoms from Hemiscorpius lepturus, Androctonus crassicauda and Mesobuthus eupeus scorpions

The aim of the present study was to compare the toxic effects of the venoms from Hemiscorpius lepturus (H. lepturus), Androctonus crassicauda (A. crassicauda) and Mesobuthus eupeus (M. eupeus). For this purpose, three in vitro models were employed to compare the toxic effects of various concentrations of the venoms from these three scorpions, namely: hemolytic potential using human RBCs, phospholipase activity using Saubouraud's dextrose agar (SDA) supplemented with 2% egg yolk and lactate dehydrogenase (LDH) enzyme releasing effect using K562 leukemia cell line. In addition, the neutralizing effectiveness of the antivenom against these toxic properties was assessed. The results showed that, unlike the venoms from A. crassicauda and M. eupeus, the venom from H. lepturus produced dose-dependent lysis of human RBCs and showed phospholipase activity. However, all the tested venoms showed variable degrees of LDH releasing properties. The venom from H. lepturus had highest and the venom from M. eupeus had the lowest LDH releasing effect. The antivenom effectively inhibited all the tested toxicities. In conclusion, these results suggest that the venoms from the studied scorpions have variable toxic properties, which may explain the underlying reason for the differences in their clinical manifestations. In addition, the antivenom was effective in neutralizing all the tested toxic effects.     

Neutralization, by a monospecific Bothrops lanceolatus antivenom, of toxic activities induced by homologous and heterologous Bothírops snake venoms.

A monospecific Bothrops lanceolatus antivenom, currently used in Martinique, was tested for its efficacy in the neutralization of several toxic and enzymatic activities of the venoms of B. lanceolatus, B. atrox and B. asper. When tested by the i.p. route in mice, B. lanceolatus venom had an LD50 of 12.8 microg/g. In addition, it induced local tissue damage (hemorrhage, edema and myotoxicity) and showed indirect hemolytic activity, but was devoid of coagulant effect on human plasma in vitro and of defibrinating activity in mice. Antivenom was fully effective in the neutralization of lethal, hemorrhagic, edema-forming, myotoxic and indirect hemolytic effects of B. lanceolatus venom in assays involving preincubation of venom and antivenom. When tested against the venoms of B. asper and B. atrox, the antivenom completely neutralized the lethal, hemorrhagic, myotoxic and indirect hemolytic effects, and was partially effective in neutralizing edema-forming activity. In contrast, the antivenom was ineffective in the neutralization of in vitro coagulant and in vivo defibrinating effects induced by these two venoms.     

Antibody neutralization of a myotoxin from the venom of Bothrops asper (terciopelo)

Neutralization of biological activities of B. asper myotoxin by a monospecific anti-myotoxin rabbit serum and polyvalent antivenom was studied. Both antisera neutralized myotoxic, phospholipase A and lethal activities of myotoxin, whereas edema-forming activity of myotoxin was not neutralized by these antisera. Anti-myotoxin rabbit serum neutralized most of the myotoxic activity of B. asper venom, but did not neutralize phospholipase A activity of this venom. Neutralization curves showed that myotoxicity induced by myotoxin persisted at antivenom/toxin or antiserum/toxin ratios at which phospholipase A activity was completely neutralized. This suggests that myotoxicity does not depend upon enzymatic activity of the toxin. Antiserum to myotoxin neutralized approximately 70% of myonecrosis induced by crude B. asper venom. This demonstrates that myotoxin is the main factor responsible for the development of myonecrosis in envenomations by B. asper.     

Action of a thionin isolated from nuts of Pyrularia pubera on human erythrocytes

Pyrularia thionin is a strongly basic peptide of 47 amino acids isolated from Pyrularia pubera. This peptide, a member of the thionin family, is hemolytic, cytotoxic and neurotoxic. The characteristics of the hemolytic activity on human erythrocytes are as follows: (1) the peptide does not itself have any phospholipase activity in a micellar assay system with egg yolk phosphatidylcholine, as evidenced by a lack of pH change or uptake of oxygen in the presence of lipoxidase; (2) erythrocyte membranes treated with thionin, however, show a low level of oxygen uptake in the presence of lipoxidase as a consequence of fatty acid release, and this activity is synergistic with that of bee venom phospholipase A2; (3) hemolysis caused by thionin is synergistic with added bee venom phospholipase A2; (4) kinetic analysis of the hemolytic assay reveals that the reaction follows Michaelis-Menten kinetics, being saturable with thionin with a Km of 1.6 microM; (5) binding studies with 125I-thionin show by Scatchard analysis a Kd value of 2.1 microM; (6) although iodinated thionin is inactive in the hemolysis assay, it acts as a competitive inhibitor to native thionin in the hemolytic assay; the inhibitor constant, Ki, for this reaction is 7.0 microM; and (7) Ca2+ above 1 mM inhibits the reaction. All the data are consistent with thionin binding to a receptor, most likely a protein, on the erythrocyte membrane, leading to the release of free fatty acids, most likely by activation of phospholipase A2. The release of fatty acids is itself not sufficient to explain the hemolytic reaction.     

Capacity of Thai green pit viper antivenom to neutralize the venoms of Thai Trimeresurus snakes and comparison of biological activities of these venoms

The capacity of Thai green pit viper antivenom raised to Trimeresurus albolabris to neutralize the venoms from six species of Trimeresurus sp. in Thailand has been examined. They were Trimeresurus albolabris, T. macrops, T. popeiorum, T. hageni, T. purpureomaculatus, and T. kanburiensis. The antivenom neutralized lethal and hemorrhagic activities of all these venoms. The capacity of antivenom to neutralize lethal toxicity of the venom was expressed as the amounts (mg) of snake venom neutralized by 1 ml of the antivenom. The largest capacity was found with the homologous venom. Results of immunodiffusion, immunoblotting, and antigen-antibody complex formation experiments supported the results of neutralization experiments. Several biological activities of the Trimeresurus venoms were also examined and compared. They were lethal, hemorrhagic, proteolytic, phospholipase A, arginine ester hydrolyse, and thrombin activities. There was no correlation between the ratios of lethal toxicity and hemorrhagic activity, lethal toxicity and phospholipase A activity, as well as hemorrhagic activity and proteolytic activity.     

The purification of acid phosphatase from honey bee venom (Apis mellifica)

Acid phosphatase from bee venom was purified by a combination of saturated ammonium sulphate precipitation, gel filtration and ion exchange chromatography. The final product which is a glycoprotein contained less than 0.1% phospholipase A2 or hyaluronidase activity and existed in two molecular weight (96,000 and 45,000) forms. Acid phosphatase is a potent allergen, in bee venom allergic patients, which is capable of releasing histamine from sensitized human basophils and of inducing a wheal and flare reactions in sensitized human skin.     

Detection of a cytolytic toxin in the venom of the stonefish (Synanceia trachynis)

The venom of the stonefish, Synanceia trachynis, contains a cytolytic toxin which is antigenic and ammonium sulfate-precipitable, and has a pI of ca 5.7 and an Mr of ca 158,000. The toxin is a potent but narrow-spectrum cytolysin which is lytic in vitro for rabbit, dog, rat, and guinea pig erythrocytes, in that order, but is largely or completely inactive against sheep, cow, human, monkey, mouse, goat, horse, burro and cat erythrocytes. Fractionation of the venom by molecular sieve fast protein liquid chromatography and isoelectric focusing did not separate the haemolytic activity from the venom's lethal and vascular permeability-increasing activities. Also, the three activities were destroyed by heat, proteases, Congo red, potassium permanganate and stonefish antivenoms. The results suggest that the haemolytic, lethal and vascular permeability-increasing activities of stonefish venom are properties of the same molecule, a previously unrecognized, membrane-damaging protein toxin.     

An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica

The ability of several batches of polyvalent antivenom to neutralize indirect hemolytic activity of Bothrops asper venom was studied using a sensitive plate test. All samples of antivenom tested effectively neutralized this activity. A highly significant correlation was observed between neutralization of indirect hemolysis and neutralization of lethal activity. This simple and sensitive in vitro test could be used to monitor antibody levels in horses immunized to produce polyvalent antivenom.     

Toxic activities of Brazilian centipede venoms

Centipedes have a venom gland connected to a pair of forceps, which are used to arrest preys. Human victims bitten by centipedes usually manifest burning pain, paresthesia and edema, which may develop into superficial necrosis. The aim of this work was to characterize and compare toxic activities found in venoms of three species of Brazilian centipedes-Otostigmus pradoi, Cryptops iheringi and Scolopendra viridicornis. By SDS-PAGE (4-20%), important differences were noticed among venoms (between 7 and 205kDa). Few bands showed feeble caseinolytic, fibrinogenolytic and gelatinolytic activities by zymography, but strong hyaluronidase activity was observed in S. viridicornis and O. pradoi venoms. In addition, such activities could be inhibited by o-phenanthroline, indicating that these enzymes are metalloproteinases. All venoms induced nociception, edema and myotoxicity in mice, but only S. viridicornis induced mild hemorrhagic activity. No coagulant activity was detected in centipede venoms. Low phospholipase A(2) activity was observed exclusively in S. viridicornis and O. pradoi venoms, but these venoms had intense direct hemolytic activity on human erythrocytes. Cross-reactivity among venoms was observed using species-specific sera raised in rabbits. Differences were noticed among centipede venoms, but S. viridicornis is indeed the most toxic venom and thereby it could induce a more severe envenomation.     

Efficacy of antivenom against the procoagulant effect of Australian brown snake (Pseudonaja sp.) venom: in vivo and in vitro studies.

Snake venom induced consumption coagulopathy (VICC) is a common complication of snake bite due to prothrombin activators or thrombin-like enzymes in the venom. This study aimed to determine the efficacy and dose of antivenom for treating VICC in patients envenomed by brown snakes (Pseudonaja spp.), including in vitro coagulation studies. In serial blood samples from patients with brown snake envenoming, venom and antivenom concentrations were measured using enzyme immunoassays. In vitro mixtures of brown snake venom and antivenom were used to investigate antivenom binding, neutralisation of prothrombin activity, prevention of venom-mediated clotting and effect on thrombin generation parameters using a thrombinoscope. In 27 envenomed patients the median venom concentration was 20 ng/mL (Interquartile range[IQR]:12-44 ng/mL) prior to antivenom and was not detected after antivenom administration, including 9 patients given one vial. In vitro, 200 microg/mL of antivenom bound all free venom at venom concentrations seen in patients. In vitro prothrombinase activity of the venom (using a chromogenic substrate) was not neutralised by antivenom. However, for venom concentrations seen in humans, 100 microg/mL of antivenom prevented venom clotting activity in human plasma and 479 microg/mL neutralised procoagulant venom activity measured by triggering thrombin generation. One vial of antivenom appears to be sufficient to bind and neutralise all venom in patients with severe brown snake envenoming.