Rabbit antivenom efficacy against myotoxic and neurotoxic activities of Bothrops jararacussu venom and Bothropstoxin-I

Bothrops jararacussu venom and its major toxin bothropstoxin-I (BthTX-I) possess myotoxic and neurotoxic properties. The efficacy of a rabbit antivenom raised against B. jararacussu venom in the neutralization of physiological, biochemical, and morphological changes induced by the venom and its major toxin BthTX-I was studied in mouse isolated phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations. The times required for 50 per cent neuromuscular blockade in PND and EDL preparations for venom were 70ñ11.5 (S.E.M., n=5) min and 58ñ8 (n=16) (50 µ/mL), and for BthTX-I 31ñ6 (n=3) min and 30ñ3 (n=5) min (20 µg/mL), respectively. After 120 min incubation, creatine kinase (CK) concentrations in solution containing the EDL preparations were 3464ñ346 U/L after exposure to venom (50 µg/mL, n=5) and 3422ñ135 U/L to BthTX-I (20µg/mL, n=4), respectively. Rabbit antivenom dose-dependently neutralized venom and toxin-induced neuromuscular blockade in both preparations and effectively prevented venom and toxin-induced CK release from EDL. Histological analysis showed that rabbit antivenom neutralized morphological damage caused by B.jararacussu venom and BthTX-I in EDL preparations. these results indicate that rabbit antivenom effectively neutralized the biological activities of B.jararacussu venom and BthTX-I.

Intraspecific variation in neurotoxic and myotoxic activities of Bothrops neuwiedii venoms

Snake venoms frequently vary in composition. In this work, we compared the neurotoxic and myotoxic activities of 16 lots of Bothrops neuwiedii venoms from different regions of Brazil, using chick biventer cervicis preparations. The neuromuscular blockade varied from 2 per cent to 100 per cent after 120 min incubation with venoms (50µg/ml). In all cases, this blockade was irreversible and concentration-dependent; at low concentrations (10-20 µg/ml), 15 of the 16 venom lots failed to abolish responses to acetylcholine (110µM), but blocked responses to KCI (13.4mM), and induced contracture. At 5-20µg/ml, the most active venom totally blocked twitch-tension without affecting responses to acetylcholine and KCI. Polyacrylamine gel electrophoresis for basic proteins showed that the most active samples contained a band that was absent in the less active venoms. These results indicate that there may be considerable intraspecific variation in the neurotoxic activity of B. ineuwiedii venoms, whereas myotoxic activity is less variable.

Neutralizing capacity of antisera raised in horses and rabbits against Crotalus durissus terrificus (South American rattlesnake) venom and its main toxin, crotoxin.

Crotalus durissus terrificus (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. We have investigated the ability of commercial equine antivenom and antivenoms raised in rabbits against C. d. terrificus venom and crotoxin to neutralize the physiological and morphological changes induced by this venom and crotoxin in electrically-stimulated phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations of mice. The time required to produce 50% neuromuscular blockade in the PND and EDL preparations was, respectively, 103+/-9 and 59+/-6 min for C. d. terrificus venom (10 microg/ml) and 75+/-9 and 110+/-7 min for crotoxin (10 microg/ml). The antivenoms dose-dependently inhibited this neuromuscular activity of the venom and crotoxin. At a venom:antivenom ratio of 1:3, the rabbit antivenoms were as effective as the commercial equine antivenom. The creatine kinase (CK) concentrations in the organ bath containing EDL muscle were 290 and 1020 U/l following a 120 min exposure to C. d. terrificus venom and crotoxin, respectively. All of the antivenoms neutralized the release of CK by crotoxin, but were ineffective against C. d. terrificus venom. Histological analysis of the two preparations showed that rabbit anticrotoxin antivenom protected against the myotoxic action of C. d. terrificus venom and crotoxin better than the other antivenoms. We conclude that antisera raised in rabbits are better than equine antiserum in neutralizing the neurotoxic and myotoxic activities of C. d. terrificus venom and crotoxin.

An unusual presynaptic action of Bothrops insularis snake venom mediated by phospholipase A2 fraction.

A phospholipase A2-containing fraction was isolated from the venom of Bothrops insularis by a combination of gel filtration on Sephadex G-150 and ion exchange chromatography on DEAE-Sephadex. Peak IV of the latter chromatography containing all of the phospholipase A2 (PLA2) activity, was assayed on isolated neuromuscular preparations. In the mouse phrenic nerve-diaphragm incubated in Tyrode at 37 degrees C, the PLA2 fraction produced an initial increase in the twitch tension and in the frequency of the mepps, followed by a dose-dependent, irreversible blockade. The replacement of 1.8 mM Ca2+ by 4 mM Sr2 inhibited the neuromuscular blocking effect of the fraction. In the chick hiventer cervicis preparation incubated with Krebs solution at 37 degrees C, the PLA2 fraction induced blockade but did not affect the response to acetylcholine and K+, excluding the involvement of post-synaptic and direct muscular effects. A low temperature (18-22 degrees C) incubation prevented the neuromuscular effect from developing. These results suggest that the PLA2-containing fraction acts predominantly at presynaptic sites at the neuromuscular junction. This fraction also accounts for most of the pharmacological effects of the crude venom.

Characterization of a myotoxin from the Duvernoy's gland secretion of the xenodontine colubrid Philodryas olfersii (green snake): effects on striated muscle and the neuromuscular junction.

A myotoxin has been isolated from the Duvernoy's gland (DG) secretion of the xenodontine colubrid Philodrvas olfersii (green snake) by gel filtration on Sephadex G-100 SF. Under non-reducing and reducing conditions in SDS-PAGE, the myotoxin migrates as a single band with a mol. wt. of 20000. The toxin has 182 amino acid residues (approximately 20% acidic), a pI of 4.8 and a blocked N-terminal. In the chick biventer cervicis preparation, P. olfersii myotoxin partially blocks potassium-evoked contractures without affecting either the twitch-tension resulting from indirect stimulation or the contractures evoked by acetylcholine. Both the DG secretion and the myotoxin increase the serum creatine kinase (CK) levels of mice and stimulate the release of CK from the biventer cervicis preparation in a dose- and time-dependent manner. The varying degrees of muscle cell lysis and extensive widening of the intercellular spaces caused by the DG secretion are reproduced by the myotoxin, with the exception that in the latter the partial or total loss of transverse muscle striations is restricted to the muscle periphery. This myotoxin is the first such protein to be characterized from a DG secretion.

Novel effects of guanidine on the neuromuscular junction.

1. The effects of guanidine on the isolated mouse phrenic nerve diaphragm (MPND) and chick biventer cervicis (CBC) neuromuscular preparations were determined by myographic and electrophysiological methods. 2. Guanidine at concentrations of 5-10 mM induced an initial facilitation followed by neuromuscular blockade in both preparations. In the isolated MPND such blockade was associated with the abolition of miniature end-plate potentials (MEPPs), but in the CBC the acetylcholine-induced contracture remained unimpaired. After guanidine removal, a heretofore undescribed pronounced facilitation of neuromuscular transmission associated with an increase in MEPP frequency was observed. Simultaneously, the muscular contractions exhibited delayed relaxation and aftercontractions. 3. The K+ channel opener, cromakalim (100-200 microM) inhibited both the well-described initial and the novel postremoval facilitatory effects of guanidine in a concentration-dependent manner. These findings are consistent with the proposal that guanidine blocks K+ channels in motor nerve endings. 4. The guanidine-induced NMB was reverted by increasing the Ca2+ concentration (1.8-5 mM) in the nutritive solution. 5. Tetrodotoxin (TTX, 1.56 microM) did not influence the increase in MEPPS frequency induced by guanidine (10 mM) but did reduce the rise in MEPPS frequency observed after guanidine removal. 6. The present findings indicate that the effects of guanidine on the neuromuscular junction are more complex than currently described because they include a neuromuscular blockade and a post-removal facilitation previously unreported in the literature.

The effects of Duvernoy's gland secretion from the xenodontine colubrid Philodryas olfersii on striated muscle and the neuromuscular junction: partial characterization of a neuromuscular fraction.

The effect of Philodryas olfersii Duvernoy's secretion was studied in vivo in mice and chicks as well as in the mouse phrenic nerve-diaphragm and the chick biventer cervicis preparations. The whole secretion (20-40 micrograms/ml) increased the creatine kinase (CK) levels in mice but had no effect on the mouse phrenic nerve-diaphragm preparation. In the chick, the secretion caused head drop and paresia as well as irreversible blockade of the twitch-tension evoked by indirect stimulation in the chick biventer cervicis preparation (50% paralysis in 34.5 +/- 2.7 min, n = 4). The secretion also caused muscle contracture (30% of the maximal twitch-tension generated) after a latency of nearly 9 min. Following fractionation on a Superose 12 FPLC column, the neuromuscular activity was recovered in the high mol. wt fraction (Peak I). At a concentration of 10 micrograms/ml in the chick biventer cervicis preparation, Peak I caused 50% paralysis within 18.5 +/- 3.0 min (n = 4), and evoked a strong contracture (70% of the maximal twitch-tension generated). The contractile responses of the chick preparation to ACh and KCL were partially blocked (90%) by the whole secretion and totally blocked by Peak I. CK release was increased by the whole secretion but not by Peak I. The whole secretion also produced various degrees of muscle cell lysis and extensive widening of the intercellular spaces. The latter showed a loosely arranged membranous network. In general, Peak I caused only minor morphological alterations compared with the whole secretion, although these were still significantly different from those observed in the control preparations. The changes principally involved hypercontraction of the muscle fibers. Based on the above results, we conclude that Peak I contains the factor(s) responsible for the in vitro effects on neuromuscular transmission, whereas the direct myotoxic effect is apparently caused by at least one other component of the Duvernoy's secretion.

No role for enzymatic activity or dantrolene-sensitive Ca2+ stores in the muscular effects of bothropstoxin, a Lys49 phospholipase A2 myotoxin.

The role of low levels of phospholipase A2 (PLA2) activity and intracellular Ca2+ stores in the pharmacological action of bothropstoxin (BthTX), a myotoxic Lys49 PLA2 homologue isolated from the venom of Bothrops jararacussu, was investigated. We examined the muscular effects of BthTX in the mouse diaphragm and its PLA2 activity in radiolabeled human and rat primary cultures of skeletal muscle. Although it is a Lys49 PLA2 homologue, BthTX had a low, but easily detectable, level of enzymatic activity relative to two Asp49 PLA2 enzymes from Naja naja kaouthia and Naja naja atra venoms, and this activity was reduced by about 85% in the presence of Sr2+ (4.0 mM). However, the replacement of 1.8 mM Ca2+ by 4 mM Sr2+ did not alter the BthTX-induced contracture and blockade of the muscle twitch tension. In addition, Sr2+ decreased by 50% the time required to cause 50% paralysis, and evoked approximately a four-fold increase in the number of spontaneous spikes. In isolated sarcoplasmic reticulum preparations, BthTX opened the intracellular Ca2+ release channel (ryanodine receptor) and lowered the threshold of Ca(2+)-induced Ca2+ release by a second, as yet unidentified, mechanism. However, in intact muscle, dantrolene, an antagonist of some forms of intracellular Ca2+ release, had no effect on the actions of BthTX. These findings do not support any role for the low levels of PLA2 activity, or dantrolene-sensitive intracellular Ca2+ stores, in the action of BthTX. The mechanism whereby Sr2+ stimulates the pharmacological activity of BthTX remains to be clarified.

Lipoxygenase-derived mediators may be involved in in vivo neutrophil migration induced by Bothrops erythromelas and Bothrops alternatus venoms.

Bothrops erythromelas (BEV) and B. alternatus (BAV) venoms induced a dose-dependent neutrophil migration when injected into rat peritoneal cavities (20-160 micrograms/cavity). These venoms (80 micrograms/rat) also induced neutrophil migration in the air pouch model of inflammation. This migratory response seemed to be related to the phospholipase A2 (PLA2) activity of the venoms. BAV had approximately two times more PLA2 activity than BEV, and the neutrophil migration induced by the former venom was two to three-fold greater than that observed with the latter. Heated (90 degrees C for 5 min) BEV lost about 50% of its PLA2 activity and this was accompanied by a corresponding loss in the ability to induce neutrophil chemotaxis. Dexamethasone (0.5 mg/kg, s.c.), an indirect inhibitor of PLA2 activity, also abolished the neutrophil migration induced by both venoms. Since NDGA (100 mg/kg, s.c.) and dexamethasone, but not indomethacin (2 mg/kg, s.c.), strongly reduced the neutrophil migration induced by both bothropic venoms, it is suggested that arachidonate-derived lipoxygenase metabolites such as leukotriene B4 act as the chemotactic mediators. Macrophages could be the main cellular source of such metabolites since they are the predominant resident cells in the rat air pouch, and the migratory response of BEV and BAV into peritoneal cavities was potentiated in rats pretreated with thioglycollate. The neutrophil migration induced by BEV and BAV was not due to endotoxin contamination since heated BEV showed no effect and polymyxin B-treated BAV still remained active.

Effect of Bothrops insularis venom on the mouse and chick nerve-muscle preparation.

The effects of Bothrops insularis venom were examined in vivo in mice and chicks and in vitro using the mouse phrenic nerve diaphragm and chick biventer cervicis muscle preparations. Incubation of the indirectly or directly stimulated mouse preparation with B. insularis venom (20-80 micrograms/ml) produced an initial increase in twitch tension followed by irreversible blockade. With direct stimulation in the presence of D-tubocurarine, no increase in twitch tension was observed prior to the onset of blockade. A venom-induced effect on presynaptic activity was suggested by the marked increase in the frequency of the mepps recorded in vitro 5-15 min after venom addition. A direct muscular effect was shown by the dose- and time-dependent reduction in the resting membrane potential of the diaphragm. Chick preparations were more sensitive than those of the mouse. In the isolated chick biventer cervicis muscle preparation, B. insularis venom induced a contracture and a dose-dependent block of responses to indirect stimulation. At low venom concentrations (1-5 micrograms/ml), no significant release of creatine kinase (CK) was observed from this preparation. However, a dose-dependent release of CK was detected at higher doses (10-80 micrograms/ml). For morphological studies, B. insularis venom was injected into the chick left pectoralis muscle. At low doses (0.4 microgram), only an inflammatory reaction was present, while at high doses (20-80 micrograms) increasing numbers of necrotic fibers were observed as well as occlusive thrombosis and hemorrhage. The muscular effect, also observed on the incubated muscle, points to a direct myolytic action of the whole venom.

Effects induced by bothropstoxin, a component from Bothrops jararacussu snake venom, on mouse and chick muscle preparations.

Bothropstoxin, a 13,700 mol. wt myotoxic phospholipase homologue isolated from the venom of Bothrops jararacussu and devoid of PLA2, proteolytic or hemolytic activities, inhibited muscle twitch tension, evoked either directly or indirectly through stimulation of the motor nerve in the mouse phrenic-diaphragm preparations. The compound action potential of the muscle was also abolished with a similar time course. In addition, the toxin (0.7 mM) evoked membrane depolarization which was inhibited in the presence of 10 mM Ca2+. In chick biventer cervicis muscle, the toxin (2 mM) induced a contracture that reached its maximum amplitude in 44.8 +/- 15.6 min (n = 6) and was not blocked by either d-tubocurarine or tetrodotoxin. The time to maximum amplitude was reduced to 5.5 +/- 1.0 min (n = 4) in nominally Ca(2+)-free Krebs solution and was completely abolished in Ca(2+)-free Krebs solution containing 1 mM EGTA.

Effect of metronidazole on platelet aggregation.

The effects of metronidazole on platelet aggregation induced by several agonists were studied on guinea pig and human platelet-rich plasma. Metronidazole (0.25-2.5 mg/ml) showed a dose-dependent inhibitory effect on in vitro aggregation of platelets from either source induced by adenosine-5-diphosphate (ADP), platelet aggregating factor (PAF), epinephrine, collagen or convulxin (extracted from Crotalus durissus terrificus venom). Total or partial inhibition was also detected in metronidazole pre-treated guinea pig platelet-rich plasma when challenged with PAF or ADP 45 and 135 min after oral treatment, respectively. This inhibition was both dose and time dependent.

Cross-neutralization on the histamine-releasing activity of snake venoms.

This paper investigates the neutralizing effect of crotalic and bothropic antivenoms on the histamine-releasing activity of three different Brazilian snake venoms (Crotalus durissus terrificus, Bothrops jararacussu and Bothrops alternatus). This relative histamine-releasing activity was determined on peritoneal mixed cells of rats. C. d. terrificus venom was the most efficient histamine-releasing agent (ED50 = 1.25 micrograms/ml) followed by B. jararacussu (ED50 = 1.97 ug/ml) and B. alternatus (ED50 = 15.1 ug/ml) venoms. The histamine-releasing effects eventually produced by antivenom sera (antivenoms) were avoided by the use of small volumes of each one (10-20 ul). Both the monovalent crotalic and the polyvalent bothropic antivenoms were able to neutralize the histamine-releasing activity of the three venoms without specificity. This cross-reaction suggests that the main histamine-releasing factor (s) present in these venoms has similar antigenic constitution.

Convulxin does not induce convulsions when injected into the rat dorsal hippocampus.

The search for new and selective neurotoxins has attracted several efforts in the recent years. Convulxin, a neurotoxin isolated from the Crotalus durissus terrificus venom, was reported to elicit convulsions upon intravenous injection in mice and cats. Therefore we have studied the behavioral, electroencephalographic and neuropathological effects of convulxin when microinjected into the dorsal hippocampus of rats. To test the hypothesis that substances released by convulxin from platelets and not convulxin itself is the convulsive agent, we injected a mixture of convulxin and platelet rich plasma. Results demonstrated that neither convulxin nor convulxin/platelet rich plasma exhibited neurotoxic effects upon intrahippocampal injection.

Muscle necrosis and regeneration after envenomation by Bothrops jararacussu snake venom.

The lesions caused by sublethal doses of Bothrops jararacussu venom injected into tibialis anterior (tib. ant.) muscles of mice were studied with paraffin sections. Doses of 5 and 20 micrograms produced a large area of necrosis in tib. ant., but hardly affected neighbouring muscles. Phagocytosis of necrotic remnants was followed by marked regeneration of the muscle fibres. Within two weeks of the 5 micrograms dose there was recovery to near normal appearance and slight fibrosis. With 20 micrograms, a circumscribed scar and stronger interstitial fibrosis developed in the tib. ant. Most regenerated muscle fibres were small, but varied in diameter, retained central nuclei for three months (the longest survival) and were surrounded by collagen. Doses of 80 and 200 micrograms produced widespread coagulative necrosis of tib. ant., though neighbouring leg muscles were relatively spared. Myonecrosis was evident microscopically at 10 min, and over the next week the necrotic muscle remained acellular and devoid of inflammatory reaction except at the very edge. Blood vessels within and outside tib. ant. often became hyalinized and thrombosed. Phagocytosis of debris proceeded from the periphery, and after two weeks the muscle was replaced by fibro-adipose tissue. There was little if any muscle fibre regeneration. Abscesses developed in the vicinity of the injection site in several mice receiving high venom doses, but never after low doses or saline. Muscle necrosis after B. jararacussu venom seems due primarily to direct action of the venom, though vascular thrombosis and ischaemia may contribute. The venom can cause fibrosis of muscle and hinder or prevent muscle fibre regeneration.

Activation of guinea-pig platelets induced by convulxin, a substance extracted from the venom of Crotalus durissus cascavella.

Convulxin (Cx), a component of the venom of the snake Crotalus durissus cascavella, induced the concentration-dependent aggregation of guinea-pig platelets when used at and above 50 +/- 5 ng/ml, accompanied by the release of ATP and by the formation of thromboxanes (Tx). Platelet activation by Cx was not due to potential contaminants found in the crude snake venom, such as phospholipase A2 and clotting enzymes. Aspirin (50-100 microM) failed to interfere with the platelet effects of Cx, demonstrating independence from cyclo-oxygenase. In contrast, indomethacin (50 microM) displayed a distinct inhibitory activity on the effects of Cx, as compared to aspirin, and thus exerts cyclo-oxygenase-independent effects on platelet activation. The ADP scavenger creatine phosphate/creatine phosphokinase (CP/CPK) inhibited aggregation by Cx used at concentrations below 6-8 times the threshold, but failed to interfere with higher amounts. Platelet aggregation by Cx was inhibited and reversed once established by EDTA (5mM) and by prostacyclin (0.1-1 microM). Cx-induced activation of platelets is thus Ca2+-dependent and liable to control by the adenylate cyclase-cyclic AMP system. Convulxin induced hypotension, bronchoconstriction and thrombocytopenia when injected i.v. to the anesthetised guinea pig at 0.3-3 microgram/kg. Aspirin and indomethacin (20 and 5 mg/kg respectively) mepyramine and methysergide (02. mg/kg) failed to interfere with these effects, but the combination of either aspirin or indomethacin with methysergide and mepyramine, suppressed the bronchial effects of Cx, leaving the hypotensive and thrombopenic effects unchanged. This synergism remains unexplained. Bronchoconstriction was platelet-dependent, being suppressed by platelet depletion with antiplatelet serum or by i.v. prostacyclin (1-10 microgram/kg).