Crotoxin: novel activities for a classic beta-neurotoxin.

Crotoxin, the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, was the first snake venom protein to be purified and crystallized. Crotoxin is a heterodimeric beta-neurotoxin that consists of a weakly toxic basic phospholipase A(2) and a non-enzymatic, non-toxic acidic component (crotapotin). The classic biological activities normally attributed to crotoxin include neurotoxicity, myotoxicity, nephrotoxicity and cardiotoxicity. However, numerous studies in recent years have shown that crotoxin also has immunomodulatory, anti-inflammatory, anti-microbial, anti-tumor and analgesic actions. In this review, we describe the historical background to the discovery of crotoxin and its main toxic activities and then discuss recent structure-function studies and investigations that have led to the identification of novel pharmacological activities for the toxin.

Biological activities of a lectin from Bothrops jararacussu snake venom.

Snake venoms contain saccharide-binding lectins. In this work, we examined the biological activities of a lectin (BjcuL) purified from Bothrops jararacussu snake venom by chromatography on non-derivatized Sepharose 4B and Sephacryl S-200 HR. The protein, a homodimer with subunits of 14.5 kDa, gave a single immunoprecipitin line in immunoelectrophoresis and cross-reacted in ELISA with antivenoms raised against Bothrops spp. (lanceheads), Micrurus spp. (coral snakes), Crotalus durissus terrificus (South American rattlesnake), and arthropod (Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus) venoms. BjcuL agglutinated human formaldehyde-fixed erythrocytes at > or = 100 ng/ml and was inhibited by lactose and EDTA (> or = 2 mM) and high concentrations (> 100 mM) of glucose and sucrose, but not by N-acetylglucosamine. BjcuL had no direct hemolytic activity and was devoid of esterase, PLA2 and proteolytic activities. The lectin (up to 200 microg/ml) did not aggregate human platelet-rich plasma (PRP) or washed platelets (WP), nor did it alter the aggregation induced by ADP in PRP or by thrombin in WP. When injected into mouse hind paws, BjcuL (10-100 microg/paw) caused edema and increased vascular permeability, with a maximum effect after 1h that persisted for up to 6 h (edema) or gradually decreased after the peak interval (vascular permeability). No hemorrhage was observed in BjcuL-injected paws. In anesthetized rats, B. jararacussu venom (200 microg/kg, i.v.) produced sustained hypotension (maximum decrease of approximately 60%) whereas a similar dose of BjcuL decreased the blood pressure by approximately 15%, with a rapid return to the resting level.

The presynaptic activity of bothropstoxin-I, a myotoxin from Bothrops jararacussu snake venom.

Bothropstoxin-I from Bothrops jararacussu snake venom is a lysine-49 phospholipase A(2) with myotoxic and neurotoxic activities. In this study, we used mouse phrenic nerve-diaphragm preparations in the absence and presence of manganese (Mn(2+)), a presynaptic blocker, to investigate a possible presynaptic action of bothropstoxin-I. At concentrations of 0.9 mM and 1.8 mM, Mn(2+) produced 50% neuromuscular blockade in less than 4 min., which was spontaneously reversible at the lower concentration. Bothropstoxin-I (1.4 microM) irreversibly inhibited neuromuscular blockade by 50% in 31+/-4 min. (mean+/-S.E.M., n = 9). Pretreating preparations with 0.9 mM Mn(2+) prevented the blockade by bothropstoxin-I. When added after bothropstoxin-I, Mn(2+) produced its characteristic blockade and, after washing, the twitch tension returned to pre-Mn(2+) levels, indicating that bothropstoxin-I caused irreversible damage before the addition of Mn(2+). Electrophysiological measurements showed that a concentration of bothropstoxin-I (0.35 microM), which did not produce neuromuscular blockade, caused the appearance of giant miniature end-plate potentials with no change in the membrane resting potential but increased the quantal content. Preparations preincubated with Mn(2+) (0.9 mM, 30 min.) were protected against the depolarizing action of bothropstoxin-I (0.7 microM). These results show that, in addition to its well-known myotoxic effect, bothropstoxin-I also has a presynaptic action.

Pharmacological evidence for a presynaptic action of venoms from Bothrops insularis (jararaca ilhoa) and Bothrops neuwiedi (jararaca pintada).

Whereas the presynaptic action of Crotalus durissus terrificus venom is well-established, Bothrops venoms have historically been considered to have only postsynaptic and muscular effects. However, some studies have also suggested a presynaptic action for these venoms. In this work, we used chick biventer cervicis preparations to compare the presynaptic actions of two Bothrops venoms (B. insularis and B. neuwiedi) with that of C. d. terrificus venom. At 10 microg/ml, all venoms produced irreversible blockade of the twitch tension responses, with no reduction in acetylcholine (ACh)-induced contractures and only a slight decrease in potassium induced-contractures. The times (in min) required to produce 50% neuromuscular blockade (C. d. terrificus: 16.3+/-0.7, n = 8; B. insularis: 30.0+/-1.9, n = 5; B. neuwiedi: 42.0+/-2.0, n = 8; mean +/- SEM) were significantly different among the venoms (p < 0.01). Lowering the temperature at which the experiments were done (from 37 to 24 degrees C) prevented neuromuscular blockade by the three venoms, indicating that enzyme activity may be involved in this response. At concentrations capable of causing complete neuromuscular blockade, creatine kinase release remained close to levels seen in control preparations incubated with Krebs solution alone (500-1200 IU/l). Commercial crotalic antivenom, but not bothropic antivenom, protected against the neuromuscular blockade caused by B. insularis and B. neuwiedi venoms. These observations indicate that bothropic venoms may contain components which act presynaptically in a manner similar to C. d. terrificus venom, and that at low venom concentrations a direct action on skeletal muscle does not contribute to this presynaptic neurotoxicity.

Effect of Bothrops leucurus venom in chick biventer cervicis preparations.

Bothrops leucurus is a poorly studied pitviper found in northeastern Brazil. We examined the action of B. leucurus venom (5-100 microg/ml) on contractile responses in chick biventer cervicis preparations. Muscle damage was assessed by quantifying the release of creatine kinase (CK) and by histological analysis. B. leucurus venom dose-dependently inhibited the contractile responses of indirectly stimulated preparations, the maximum inhibition with 100 microg of venom/ml being 74.0+/-6.6% (mean+/-SEM) after 120 min. The venom also reduced contractures to exogenous acetylcholine (55 and 110 microM) and K(+) (13.4mM) (85-100% reduction with 100 microg of venom/ml) and increased the release of CK (348+/-139 U/ml in controls vs 1260+/-263 U/ml with 20 microg of venom/ml after 120 min, p<0.05). The accompanying morphological changes included multivacuolated, swollen, amorphous fibers and agglutinated myofibrils. These results indicate that B. leucurus venom can adversely affect neuromuscular transmission and produce muscle damage in avian preparations.

Biophysical, histopathological and pharmacological characterization of crotamine isoforms F22 and F32.

Two major crotamine isoforms (F22 and F32) were obtained after three chromatographic steps and were assayed in mouse phrenic nerve-diaphragm preparations. F32 and F22 (0.5 microg/ml, n=4) produced a facilitatory effect, which increased isometric twitch-tension by 300 and 230%, respectively, after a 120 min incubation. At a concentration of 0.1 microg/ml, both isoforms increased the twitch-tension by about 160%. However, when the isoforms were co-incubated (final concentration, 0.5 microg/ml) for 30 min prior to testing, they did not cause the facilitation seen with > or =0.1 microg/ml of each isoform alone. Histologically, F32 and F22 at 0.5 and 1 microg/ml were quantitatively alike in inducing tissue myonecrosis. However, a mixture of the two isoforms (final concentration, 0.5 microg/ml) significantly attenuated the damage seen with either toxin alone. Mass spectrometry analysis showed that the isoforms had the same molecular mass (4.8 kDa) and that they existed as monomers with a highly stable structure. These results indicate that F22 and F32 acted on muscle cells of the mouse phrenic-nerve diaphragm preparation through similar mechanisms. Since the isoforms did not produce the expected summation in the increase in muscle twitch-tension, it is possible that they may have different affinities for the sodium channel subunits.

Neuromuscular action of Bothrops lanceolatus (Fer de lance) venom and a caseinolytic fraction.

A protein capable of inducing neuromuscular blockade in avian preparations and of depolarizing mouse diaphragm muscle was isolated from Bothrops lanceolatus venom using gel filtration and ion-exchange chromatography. The purified protein was a single chain polypeptide with an estimated molecular mass of 27.5 kDa by SDS-PAGE and had caseinolytic activity (13.3 units/mg), but no phospholipase A(2). B.lanceolatus venom (50 micro g/ml) and the caseinolytic protein (20 micro g/ml) produced contracture and progressive irreversible blockade (50% in 25+/-5 min (SEM) and 45+/-15 min, respectively), in indirectly stimulated chick biventer cervicis preparations. The contractile responses to acetylcholine (ACh; 37 and 74 micro M, n=6) were inhibited by venom and the caseinolytic protein, whereas those to potassium (13.4mM, n=6) were not. Membrane resting potential measurements in mouse hemidiaphragm preparations showed that B.lanceolatus venom and the purified protein caused depolarization which was prevented by D-tubocurarine (14.6mM). The venom produced a slight increase in the amplitude and frequency of miniature end-plate potentials, but this effect was not seen with the purified fraction. These results suggest that the purified protein acts exclusively post-synaptically.

Lethal factor to mice produced by Escherichia coli isolated from chickens with swollen head syndrome.

A lethal toxin similar to Bacillus cererus lethal toxin was detected in the culture supernatants of Escherichia coli isolated from chickens with swollen head syndrome. The lethal activity was heat-labile, protease-sensitive and killed mice within 10 min. The light microscopy of the histopathological studies revealed that the principal organ affected by this toxin was the lung but the liver and kidneys also showed lesions. The relevance of this lethal activity from E. coli remains to be determined.