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.     

Anti-sera raised in rabbits against crotoxin and phospholipase A2 from Crotalus durissus cascavella venom neutralize the neurotoxicity of the venom and crotoxin.

Crotoxin, the principal neurotoxin in venom of the South American rattlesnakes Crotalus durissus terrificus and Crotalus durissus cascavella, contains a basic phospholipase A2 (PLA2) and an acidic protein, crotapotin. In this work, we examined the ability of rabbit anti-sera against crotoxin and its PLA2 subunit to neutralize the neurotoxicity of venom and crotoxin from C. d. cascavella in mouse phrenic nerve-diaphragm and chick biventer cervicis preparations. Immunoblotting showed that the anti-sera recognized C. d. cascavella crotoxin and PLA2. This was confirmed by ELISA, with both anti-sera having end-point dilutions of 3 x 10(-6). Anti-crotoxin serum neutralized the neuromuscular blockade in phrenic nerve-diaphragm muscle preparations at venom or crotoxin:anti-serum ratios of 1:2 and 1:3, respectively. Anti-PLA2 serum also neutralized this neuromuscular activity at a venom or crotoxin:anti-serum ratio of 1:1. In biventer cervicis preparations, the corresponding ratio for anti-crotoxin serum was 1:3 for venom and crotoxin, and 1:1 and 1:2 for anti-PLA2 serum. The neutralizing capacity of the sera in mouse preparations was comparable to that of commercial anti-serum raised against C. d. terrificus venom. These results show that anti-sera against crotoxin and PLA2 from C. d. cascavella venom neutralized the neuromuscular blockade induced by venom and crotoxin in both nerve-muscle preparations, with the anti-serum against crotoxin being slightly less potent than that against crotoxin.     

Cross-neutralization of the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms by antisera against crotoxin and phospholipase A2 from Crotalus durissus cascavella venom.

We have previously demonstrated that rabbit antisera raised against crotoxin from Crotalus durissus cascavella venom (cdc-crotoxin) and its PLA2 (cdc-PLA2) neutralized the neurotoxicity of this venom and its crotoxin. In this study, we examined the ability of these antisera to neutralize the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms and their major toxins, cdt-crotoxin and bothropstoxin-I (BthTX-I), respectively, in mouse isolated phrenic nerve-diaphragm preparations. Immunoblotting showed that antiserum to cdc-crotoxin recognized cdt-crotoxin and BthTX-I, while antiserum to cdc-PLA2 recognized cdt-PLA2 and BthTX-I. ELISA corroborated this cross-reactivity. Antiserum to cdc-crotoxin prevented the neuromuscular blockade caused by C. d. terrificus venom and its crotoxin at a venom/crotoxin:antiserum ratio of 1:3. Antiserum to cdc-PLA2 also neutralized the neuromuscular blockade caused by C. d. terrificus venom or its crotoxin at venom or toxin:antiserum ratios of 1:3 and 1:1, respectively. The neuromuscular blockade caused by B. jararacussu venom and BthTX-I was also neutralized by the antisera to cdc-crotoxin and cdc-PLA2 at a venom/toxin:antiserum ratio of 1:10 for both. Commercial equine antivenom raised against C. d. terrificus venom was effective in preventing the neuromuscular blockade typical of B. jararacussu venom (venom:antivenom ratio of 1:2), whereas for BthTX-I the ratio was 1:10. These results show that antiserum produced against PLA2, the major toxin in C. durissus cascavella venom, efficiently neutralized the neurotoxicity of C. d. terrificus and B. jararacussu venoms and their PLA2 toxins.     

Determination of Crotalus durissus cascavella venom components that induce renal toxicity in isolated rat kidneys.

Envenomation by Crotalus durissus terrificus leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure (ARF). The most serious systemic change and primary cause of death is ARF. In this work, we used RP-HPLC to isolate crotoxin, convulxin and gyroxin from venom of the related subspecies Crotalus durissus cascavella and investigated the effects of these toxins on renal function in the isolated rat kidneys perfused with Krebs-Henseleit solution containing 6% of bovine serum albumin. The parameters studied included perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), percent of sodium tubular transport (%TNa(+)), percent of potassium tubular transport (%TK(+)) and percent of chloride tubular transport (%TCl(-)). Crotoxin (5 microg/ml) increased the PP, RVR, GFR, UF and decreased %TNa(+), %TK(+) and %TCl(-), with gyroxin (5 micro g/ml) the GFR remained stable during the 120 min of perfusion, whereas PP and RVR increased significantly and the %TNa(+), %TK(+) and %TCl(-) decreased significantly. Convulxin (5 micro g/ml) had no effect on renal function. Crotoxin caused alterations in all renal parameters. Gyroxin produced a minor effect compared to crotoxin. These results indicated that crotoxin is the main componenet responsible for acute nephrotoxicity caused by C. d. cascavella venom.     

Ability of rabbit antiserum against crotapotin to neutralize the neurotoxic, myotoxic and phospholipase A2 activities of crotoxin from Crotalus durissus cascavella snake venom.

The toxicity of crotoxin, the major toxin of Crotalus durissus terrificus (South American rattlesnake) venom, is mediated by its basic phospholipase A(2) (PLA(2)) subunit. This PLA(2) is non-covalently associated with crotapotin, an acidic, enzymatically inactive subunit of the crotoxin complex. In this work, rabbit antiserum raised against crotapotin purified from Crotalus durissus cascavella venom was tested for its ability to neutralize the neurotoxicity of this venom and its crotoxin in vitro. The ability of this antiserum to inhibit the enzymatic activity of the crotoxin complex and PLA(2) alone was also assessed, and its potency in preventing myotoxicity was compared with that of antisera raised against crotoxin and PLA(2). Antiserum to crotapotin partially neutralized the neuromuscular blockade caused by venom and crotoxin in electrically stimulated mouse phrenic nerve-hemidiaphragm preparations and prevented the venom-induced myotoxicity, but did not inhibit the enzymatic activity of crotoxin and purified PLA(2). In contrast, previous findings showed that antisera against crotoxin and PLA(2) from C. d. cascavella effectively neutralized the neuromuscular blockade and PLA(2) activity of this venom and its crotoxin. The partial neutralization of crotoxin-mediated neurotoxicity by antiserum to crotapotin probably reduced the binding of crotoxin to its receptor following interaction of the antiserum with the crotapotin moiety of the complex.     

Functional characterization of a basic D49 phospholipase A2 (LmTX-I) from the venom of the snake Lachesis muta muta (bushmaster).

The whole venom of Lachesis muta muta is preponderantly neurotoxic but moderately myotoxic on the chick biventer cervicis preparation (BCp). We have now examined these toxic activities of a basic phospholipase A(2), LmTX-I, isolated from the whole venom. LmTX-I caused a significant concentration-dependent neuromuscular blockade in the BCp. The time to produce 50% neuromuscular blockade was 14.7+/-0.75 min (30 microg/ml), 23.6+/-0.9 min (10 microg/ml), 34+/-1.7 min (2.5 microg/ml) and 39.2+/-3.6 min (1 microg/ml), (n=5/concentration; p<0.05). Complete blockade with all tested concentrations was not accompanied by inhibition of the response to ACh. At the highest concentration, LmTX-I (30 microg/ml) significantly reduced contractures elicited by exogenous KCl (20mM), increased the release of creatine kinase (1542.5+/-183.9 IU/L vs 442.7+/-39.8 IU/L for controls after 120 min, p<0.05), and induced the appearance of degenerating muscle fibers ( approximately 15%). Quantification of myonecrosis indicated 14.8+/-0.8 and 2.0+/-0.4%, with 30 and 10 microg/mlvenom concentration, respectively, against 1.07+/-0.4% for control preparations. The findings indicate that the basic PLA(2) present on venom from L. m. muta (LmTX-I) possesses a dominant neurotoxic action on isolated chick nerve-muscle preparations, whereas myotoxicity was mainly observed at the highest concentration used (30 microg/ml). These effects of LmTX-I closely reproduce the effects of the whole venom of L. m. muta in chick neuromuscular preparations.     

Neutralization of the pharmacological effects of bothropstoxin-I from Bothrops jararacussu (jararacu?u) venom by crotoxin antiserum and heparin.

Bothropstoxin-I (BthTX-I), the principal myotoxin of Bothrops jararacussu venom, is devoid of phospholipase A(2) (PLA(2)) activity but capable of blocking neuromuscular transmission in mouse nerve-muscle preparations. In this study, the ability of crotoxin antiserum and heparin in preventing the neurotoxic and myotoxic effects of BthTX-I was investigated. Phrenic nerve-diaphragm preparations (PND) stimulated indirectly with supramaximal stimuli (0.2 ms, 0.1 Hz) were incubated with BthTX-I (20 microg/ml) alone or with BthTX-I preincubated with antiserum or heparin for 30 min at 37 degrees C prior to testing. Control preparations were incubated with Tyrode solution, antiserum or heparin alone. BthTX-I (20 microg/ml) produced 50% neuromuscular blockade in the PND preparations in 31+/-4min, with complete blockade occurring in 120 min. The antiserum and heparin significantly prevented the neuromuscular blockade caused by BthTX-I (84 +/- 4% and 100% protection, respectively). Light microscopy examination of the muscles at the end of the 120 min incubation showed that BthTX-I damaged 48 +/- 6% of the fibers. Preincubating the toxin with antivenom significantly reduced the extent of this damage (only 15 +/- 4% of fibers affected, corresponding to 69% protection, P<0.01) whereas heparin offered no protection (34 +/- 7% of fibers affected, not significantly different from that seen with toxin alone). These results show that the antivenom was more effective in neutralizing the myotoxic effects of BthTX-I than was heparin.     

Neurotoxic and myotoxic actions from Lachesis muta muta (surucucu) whole venom on the mouse and chick nerve-muscle preparations.

Lachesis genus is one of the less studied among others from Viperidae's genera, mainly due to difficulties in obtaining the venom. Accidents by Lachesis snakes cause severe envenoming syndrome, eventually leading victims to shock. This work is part of a comprehensive study aimed at studying the venom and its effects. Herein the neurotoxicity and myotoxicity of L. muta muta venom were investigated on mouse phrenic nerve-diaphragm (PNDp) and chick biventer cervicis (BCp) preparations. For both preparations the time required to venom produces 50% neuromuscular blockade was indirectly concentration-dependent, being for PNDp: 117.6+/-6.5 min (20 microg/ml), 70.1+/-8.6 min (50 microg/ml) and 43.6+/-3.8 min (100 microg/ml), and for BCp: 28+/-1.8 min (50 microg/ml), 30.4+/-2.3 min (10 microg/ml), 50.4+/-4.3 min (5 microg/ml) and 75.2+/-0.7 min (2 microg/ml), (n=5/dose). In BCp, a venom dose of 50 microg/ml significantly reduced contractures elicited by exogenous acetylcholine (55 microM) and KCl (20 mM), as well as increased the release of creatine kinase (442.7+/-39.8 IU/l in controls vs 4322.6+/-395.2 IU/l, after 120 min of venom incubation (P<0.05). Quantification of myonecrosis in BCp indicated the doses 50 and 10 microg/ml as significantly myotoxic affecting 59.7+/-6.2%, and 20.8+/-1.2% of fibers, respectively, whereas 5 and 2 microg/ml that affected 13.5+/-0.8% and 5.4+/-0.6% of fibers, were considered weakly- and non-myotoxic, respectively. We concluded that there are neurotoxins present in the venom, the concentration of which governs its pre- (if low) or postsynaptic (if high) activity. Since myotoxicity in the avian preparation is negligible at lower venom doses, but not neurotoxicity, we suggest that this effect may contribute minimum to the venom neurotoxic effect. The BCp is more sensible than PNDp to Lachesis m. muta venom.     

Action of Micrurus dumerilii carinicauda coral snake venom on the mammalian neuromuscular junction.

The venoms of coral snakes (mainly Micrurus species) have pre- and/or postsynaptic actions, but only a few of these have been studied in detail. We have investigated the effects of Micrurus dumerilii carinicauda coral snake venom on neurotransmission in rat isolated phrenic nerve-diaphragm muscle and chick biventer cervicis preparations stimulated directly or indirectly. M. d. carinicauda venom (5 or 10 microg/ml) produced neuromuscular blockade in rat (85-90% in 291.8+/-7.3 min and 108.3+/-13.8, respectively; n=5) and avian (95.0+/-2.0 min; 5 microg/ml, n=5) preparations. Neostigmine (5.8 microM) and 3,4-diaminopyridine (230 microM) partially reversed the venom-induced neuromuscular blockade in rat nerve-muscle preparations. In neither preparation did the venom depress the twitch response elicited by direct muscle stimulation. The contractures induced by acetylcholine in chick preparations were inhibited by the venom (95-100%; n=4; p<0.05). In rat preparations, the venom produced a progressive decrease in the amplitude of miniature end-plate potentials (m.e.p.ps control frequency=69.3+/-5.0/min and control amplitude=0.4+/-0.2 mV) until these were abolished. Neostigmine (5.8 microM) and 3,4-diaminopyridine (230 microM) partially antagonized this blockade of m.e.p.ps. The resting membrane potential was not altered with the venom (10 microg/ml). M. d. carinicauda venom produced dose-dependent morphological changes in indirectly stimulated mammal preparations. Twenty-five per cent of muscle fibers were affected by a venom concentration of 5 microg/ml, whilst 60.7% were damaged by 10 microg of venom/ml. In biventer cervicis preparations, the morphological changes were slower in onset and were generally characterized by undulating fibers and, to a lesser extent, by zones of disintegrating myofibrils. A venom concentration of 5 microg/ml damaged 52.2% of the fibers. These findings indicate that M. d. carinicauda venom has neurotoxic and myotoxic effects and that the neuromuscular blockade involves mainly a postsynaptic action.     

Serum kinetics of crotoxin from Crotalus durissus terrificus venom in mice: evidence for a rapid clearance.

We report on an ELISA for the detection of crotoxin with a detection limit of 1-3 pg/ml of sample. Cross-reactivity with other animal venoms occurred only at concentrations above 1 microgram/ml. Serum kinetics of crotoxin were investigated in BALB/c mice after a single 10 micrograms s.c. dose of venom obtained from Crotalus durissus terrificus. Crotoxin levels were 254 +/- 141 ng/ml serum (X +/- S.E.) 15 min after venom injection, 3.9 +/- 0.5 ng/ml serum at 30 min and undetectable thereafter. The rapid clearance of crotoxin from the serum suggests that the test may be unsuitable for the clinical management of envenomation victims.     

Influence of temperature upon effects of crotoxin and gamma-irradiated crotoxin at rat neuromuscular transmission

The influence of temperature upon the effects of crotoxin (CTX), from Crotalus durissus terrificus venom, and gamma-irradiated (60Co, 2000 Gy) crotoxin (iCTX) was studied in rat neuromuscular transmission 'in vitro'. Indirect twitches were evoked in the phrenic-diaphragm preparation by supramaximal strength pulses with a duration of 0.5 ms and frequency of 0.5 Hz. The phospholipase A(2) (PLA(2)) enzymatic activity of CTX and iCTX was assayed against phosphadityl choline in Triton X-100. At 27 degrees C, CTX (14 microg/ml) did not affect the amplitude of indirectly evoked twitches. However, at 37 degrees C, CTX induced a time-dependent blockade of the neuromuscular transmission that started at 90 min and was completed within 240 min. iCTX (14 microg/ml) was inneffective on the neuromuscular transmission either at 27 or 37 degrees C. The PLA(2) enzymatic activity of CTX at 37 degrees C was 84 and that at 27 degrees C was 27 micromol fatty acid released/min/mg protein, and that of the iCTX at 37 degrees C was 39 micromol fatty acid released/min/mg protein. Thus, it was concluded that the mechanism of detoxification of CTX by gamma radiation at the neuromuscular level relies on the loss of its PLA(2) enzymatic activity.     

A comparative study of biological activities of crotoxin and CB fraction of venoms from Crotalus durissus terrificus, Crotalus durissus cascavella and Crotalus durissus collilineatus.

In Brazil, the Crotalus durissus terrificus subspecie is the most studied, particularly concerning its crotoxin. Crotoxin is the major toxic component of the South American rattlesnake Crotalus durissus venom. It is composed of two different subunits, CA called crotapotin and CB weakly toxic phospholipase A2 with high enzymatic activity. In this paper, we decided to make a study of the main toxic characteristics of crotoxin (CTX) and CB fraction from the other subspecies, Crotalus durissus cascavella and of Crotalus durissus collilineatus, in comparison with those of C. d. terrificus. Ours results have shown that the venoms presented similar chromatographic profiles and the purified fractions were free of contaminants. Regarding the toxic activities, the DL50 of the crotoxins showed no significant differences between the subspecies. The smaller toxicity of CB indicated that the toxicity of the crotoxin complex depends on the interaction between CA and CB. CTX and fraction CB of the three species of Crotalus showed negligible proteolytic activity. C. d. terrificus CTX presented higher PLA2 activity when compared with the others two subspecies. The oedema induced by CB developed later than the CTX and reached its peak 3 h after the injection. The myotoxic activity was determined by assaying serum CK levels. Mice injected with CTX of C. d. terrificus presented greater myotoxic activity compared to the others. The myotoxic activity of CB from the three subspecies was lower than the activity of the crotoxin, reinforcing the idea that the fraction CA increases the toxicity of CB.     

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.     

Renal effects of supernatant from macrophages activated by Crotalus durissus cascavella venom: the role of phospholipase A2 and cyclooxygenase

In Brazil, the genus Crotalus is responsible for approximately 1500 cases of snakebite annually. The most common complication in the lethal cases is acute renal failure, although the mechanisms of the damaging effects are not totally understood. In this work, we have examined the renal effects caused by a supernatant of macrophages stimulated by Crotalus durissus cascavella venom as well the potential role of phospholipase A2 and cyclo-oxygenase. Rat peritoneal macrophages were collected and placed in a RPMI medium and stimulated by crude Crotalus durissus cascavella venom (1, 3 or 10 microg/ml) for 1 hr. They were then washed and kept in a culture for 2 hr. The supernatant (1 ml) was tested in an isolated perfused rat kidney. The first 30 min. of each experiment were used as an internal control, and the supernatant was added to the system after this period. All experiments lasted 120 min. A study of toxic effect on perfusion pressure, glomerular filtration rate, urinary flow percent of sodium tubular transport and percent of proximal tubular sodium transport was made. The lowest concentration of venom (1 microg/ml) was not statistically different from the control values. The most intense effects were seen at 10 microg/ml for all renal parameters. The infusion of the supernatant of macrophages stimulated with crude venom (3 or 10 microg/ml) increased the perfusion pressure, glomerular filtration rate and urinary flow, decreased the percent of sodium tubular transport and percent of proximal tubular sodium transport. Dexamethasone (10 microM) and quinacrine (10 microM) provided protection against the effect of the venom on glomerular filtration rate, urinary flow, percent of sodium tubular transport, percent of proximal tubular sodium transport and perfusion pressure. Indomethacin (10 microM) and nordiidroguaretic acid (1 microM) reversed almost all functional changes, except those of the perfusion pressure. These results suggest that macrophages stimulated with Crotalus durissus cascavella venom release mediators capable of promoting nephrotoxicity in vitro. Moreover, phospholipase A2 and cyclooxygenase products are involved in these biologic effects.     

Biochemical and pharmacological similarities between the venoms of newborn Crotalus durissus durissus and adult Crotalus durissus terrificus rattlesnakes.

A comparative study was performed with the venoms of newborn Crotalus durissus durissus, adult Crotalus durissus terrificus and adult Crotalus durissus durissus snakes. Venom of newborn specimens of C.d. durissus is very similar to that of adult specimens of C.d. terrificus, since they have strong lethal and myotoxic activities, and weak proteolytic, hemorrhagic and edema-forming effects, in contrast to venom of adult specimens of C.d. durissus. In addition, the two former venoms have high amounts of the neurotoxic complex crotoxin, whereas venom from adult C.d. durissus has a low concentration of crotoxin. Electrophoretic analysis corroborates the strong similarities between the former two venoms. It is concluded that venom of newborn C.d. durissus contains high concentrations of crotoxin and low amounts of hemorrhagic and proteolytic components, and that a drastic ontogenetic change takes place in the venom composition of this subspecies.     

Neurotoxic and myotoxic actions of Naja naja kaouthia venom on skeletal muscle in vitro.

The neuromuscular and skeletal muscle actions of Naja naja kaouthia snake venom were studied in mammalian (rat left hemidiaphragm) and avian (chick biventer cervicis) nerve-muscle preparations. The venom (5 and 10 micro g/ml) produced neuromuscular blockade (85% in 36.8+/-2.0min, mean+/-SEM, n=5, and 18+/-0.6min, n=3, p<0.01, respectively) in the rat preparation. That the phospholipase A(2) (PLA(2)) activity of the venom is involved in this effect was evaluated by inhibiting this enzyme with p-bromophenacyl bromide. This resulted in significantly (p<0.01) increasing the time required for 85% blockade with 5 and 10 micro g/ml to 54+/-4.6min (n=3) and 29+/-0.6min (n=3), respectively. In chick preparations, the venom (5 micro g/ml) produced neuromuscular blockade in 14.0+/-1.8min (n=5). The contractures to exogenous acetylcholine were completely inhibited by the venom, whereas those to 134 micro M KCl were partially blocked in chick preparations (n=4, n=3, respectively). The venom (5 micro g/ml) produced a progressive decrease in the amplitude of miniature end-plate potentials (m.e.p.ps) in the rat hemidiaphragm, but did not alter the resting membrane potential at 5 micro g/ml. Neostigmine (5.8 micro M) immediate and partially reversed the 85% blockade produced by venom (61%, n=3) in rat preparations, as did 4-aminopyridine (53 micro M) ( approximately 59%, n=3). The 4-aminopyridine and neostigmine also restored the m.e.p.ps to pre-venom (control) values. In rat preparations, the venom damaged 47%+/-11% and 62.7+/-3.6% of the muscle fibers at concentrations of 5 and 10 micro g/ml, respectively. For venom in which PLA(2) activity was inhibited, the corresponding values were 38+/-11.8% (5 micro g/ml) and 67+/-9.6% (10 micro g/ml). These findings suggest a post-synaptic neurotoxic action for N. n. kaouthia venom, and that inhibiting phospholipase activity of the venom reduces significantly the neuromuscular block but not the direct myotoxicity.     

Structural and biological characterization of a crotapotin isoform isolated from Crotalus durissus cascavella venom.

Envenoming by Crotalus durissus subspecies leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure. The most serious systemic alteration and primary cause of death after snakebite is acute renal failure. In this work, we isolated crotapotin, an acid component (Crtp) of crotoxin from Crotalus durissus cascavella venom and we investigated its bactericidal and pro-inflammatory activities as well as its renal effects in rat isolated perfused kidneys. Crtp was bactericidal to the Gram-negative species Xanthomonas axonopodis pv. passiflorae, but was less effective against the Gram-positive Claribacteri ssp, probably because of differences in the cell wall composition. Crtp showed a high amino acid sequence homology with other Crtps described in the literature (around of 90%) and its A and B chains had high conserved regions corresponding to the calcium-binding loop, catalytic site and helix 3 of PLA2. The Crtp showed moderate pro-inflammatory activity and increased significantly the inflammation evoked by PLA2 when co-injected or co-incubated with PLA2. The renal parameters evaluated included the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR) and percent of sodium tubular transport (%TNa+). Crotapotin (5 microg/ml) significantly increased the PP and RVR, whereas the GFR, UF and %TNa+ were unaffected. These results suggest that crotoxin is the main venom component responsible for nephrotoxicity and crotapotin contributes little to this phenomenom. The biological and bactericidal actions of Crtp also suggest that this protein may have functions other than simply acting as a chaperone for PLA2.     

A pharmacological examination of venoms from three species of death adder (Acanthophis antarcticus, Acanthophis praelongus and Acanthophis pyrrhus).

The common (A. antarcticus), northern (A. praelongus) and desert (A. pyrrhus) death adders are species belonging to the Acanthophis genus. The present study compared some pharmacological aspects of the venoms of these species and examined the in vitro efficacy of death adder antivenom. Neurotoxicity was determined by the time to produce 90% inhibition (t(90)) of indirect (0.1 Hz, 0.2 ms, supramaximal voltage) twitches in the chick biventer cervicis nerve-muscle (3-10 microg/ml) and mouse phrenic nerve-diaphragm (10 microg/ml) preparations. A. praelongus venom was significantly less neurotoxic than A. antarcticus venom but was not significantly different from A. pyrrhus venom. In the biventer muscle, all three venoms (3-10 microg/ml) abolished responses to exogenous ACh (1 mM) and carbachol (20 microM), but not KCl (40 mM), indicating activity at post-synaptic nicotinic receptors. All venoms (30 microg/ml) failed to produce significant inhibition of direct twitches (0.1 Hz, 2.0 ms, supramaximal voltage) in the chick biventer cervicis nerve-muscle preparation. However, A. praelongus (30 microg/ml) venom initiated a significant direct contracture of muscle, indicative of some myotoxic activity. The prior (10 min) administration of death adder antivenom (1 unit/ml), which is raised against A. antarcticus venom, markedly attenuated the twitch blockade produced by all venoms (10 microg/ml). Administration of antivenom (1.5 units/ml) at t(90) markedly reversed, over a period of 4 h, the inhibition of twitches produced by A. praelongus (3 microg/ml, 72+/-6% recovery) and A. pyrrhus (3 microg/ml, 51+/-9% recovery) but was less effective against A. antarcticus venom (3 microg/ml, 22+/-7% recovery). These results suggest that all three venoms contain postsynaptic neurotoxins. Death adder antivenom displayed differing efficacy against the in vitro neurotoxicity of the three venoms.     

Role of nitric oxide in myotoxic activity induced by crotoxin in vivo.

This study was aimed to determine the role of nitric oxide on the skeletal myotoxic activity induced by crotoxin, the major component of the venom of Crotalus durissus terrificus. Rats were treated with N(G)-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of nitric oxide synthase or vehicle for 4 days, and on the 5th day received an intramuscular injection of crotoxin into the tibialis anterior muscle. Rats were also treated with aminoguanidine bicarbonate salt or 7-nitroindazole, inhibitors of the inducible and neuronal isoforms of nitric oxide synthase, respectively, for 4 days and on the 5th day injected with crotoxin. All treated groups were sacrificed 24 h after injection of crotoxin. Tibialis anterior and soleus muscles were removed, frozen and stored in liquid nitrogen. Histological sections were stained with toluidine blue and assayed for acid phosphatase. The results show that L-NAME significantly minimizes myonecrosis induced by crotoxin and both aminoguanidine and 7-nitroindazole partially prevented myonecrosis induced by crotoxin. Based on the present results we conclude that nitric oxide is a very important intracellular signaling molecule that mediates crotoxin myotoxic activity.     

Effect of heating on the toxic, immunogenic and immunosuppressive activities of Crotalus durissus terrificus venom.

The venom of South American rattlesnake Crotalus durissus terrificus is very toxic but poorly immunogenic and it has an immunosuppressive ability. The heating of venom at 56, 70 or 100 degrees C for 30 min caused a diminution in the lethal, phospholipase A(2) and myotoxic activities. SDS-PAGE analysis of the heated venom showed that the proteins of higher molecular weights were the most affected by heating whereas proteins with lower molecular weights (20,000-14, 000) were the most resistant to heating. The immunosuppressive effect was studied in mice immunized with human serum albumin (HSA) 1 h after receiving either heated venom or heated crotoxin. The heating of venom at 70 or 100 degrees C reduced its immunosuppressive effect whereas crotoxin had its suppressive effect reduced only when heated at 100 degrees C. The heating of venom at 56, 70 or 100 degrees C did not change its immunogenicity.These results suggest that heat treatment may be a useful technique to help in the production of antiserum to crotalid venom.