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 nonenzymatic, 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. (C) 2010 Elsevier Ltd. All rights reserved.

Prevalence of Hepatozoon spp. (Apicomplexa, Hepatozoidae) among recently captured Brazilian snakes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystallization and preliminary X-ray crystallographic analysis of the heterodimeric crotoxin complex and the isolated subunits crotapotin and phospholipase A(2)

Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A(2) and a catalytically inactive acidic phospholipase A(2) analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. The crotoxin complex crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 angstrom, and diffracted to 1.75 angstrom resolution. The crystal of the phospholipase A(2) domain belongs to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22), with unit-cell parameters a = b = 38.7, c = 286.7 angstrom, and diffracted to 2.6 angstrom resolution. The crotapotin crystal diffracted to 2.3 angstrom resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.

Gabaergic-benzodiazepine system is involved in the crotoxin-induced anxiogenic effect

The behavioral effects of crotoxin (CTX), the major component of Crotalus durissus terrificus venom, were studied in rats submitted to the open field, holeboard, and social interaction tests. CTX (100, 250, and 500 mu g/kg, IP) was administered 2 h before the tests. In the open field, CTX reduced ambulation (250 mu g/kg) and rearing (250 and 500 mu g/kg) and increased grooming (100 and 250 mu g/kg) and freezing (250 mu g/kg). In the holeboard and social interaction, all the CTX doses evaluated decreased, respectively, head dip and head dipping, and social interaction time. The CTX-induced behavioral alterations could be attributed to its neuromuscular transmission blockade, but this possibility was ruled out because CTX (250 and 500 mu g/kg, IP, 2 h before the rotarod test) was unable to modify the rotarod performance of rats. The involvement of the benzodiazepine receptor in the CTX-induced behavioral alterations was investigated through the pretreatment (30 min before the tests, IP) of the animals with diazepam (1.2 mg/kg), or flumazenil (4 and 10 mg/kg). Both diazepam and flumazenil antagonized the CTX induced behavioral alterations in the open field, holeboard, and social interaction tests. This study demonstrated that: (1) CTX is an anxiogenic compound; and (2) the gabaergic-benzodiazepine system may play a role in the CTX-induced anxiogenic effect. (C) 1999 Elsevier B.V.

Crotoxin-induced behavioral effects in rats

Crotoxin is the major component of Crotalus durissus terrificus venom. In view of the presence of high-affinity specific binding sites for crotoxin in the brain, the objective of this work was to investigate whether crotoxin induces behavioral effects in the open-field and hole-board tests. Adult male Wistar rats (180-220 g) treated with crotoxin, 100, 250 and 500 mu g/kg, ip, administered 2 h before the test, presented statistically significant behavioral alterations (ANOVA for one-way classification complemented with Dunnet test, P<0.05). In the open-field test, 250 and 500 mu g/kg of crotoxin increased freezing (from 3.22 sec to 10.75 sec and 11.2 sec) and grooming (from 13.44 sec to 22.75 sec and 21.22 sec) and decreased ambulation (from 64.8 to 39.38 and 45.8). The dose of 500 mu g/kg also decreased rearing (from 24.9 to 17.5). In the hole-board test, 500 mu g/kg of crotoxin decreased head-dip count (from 6.33 to 4.00). All the crotoxin-induced behavioral effects were antagonized by an anxiolytic dose of diazepam (1.5 mg/kg, ip, 30 min before the tests). These results show that crotoxin reduced open-field activity and exploratory behavior as well. We suggest that these effects express an increased emotional state induced by this toxin.

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 (Co-60, 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 mu g/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 mu g/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 mu mol fatty acid released/min/mg protein, and that of the iCTX at 37 degrees C was 39 mu mol 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. 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Distribution of I-125-labeled crotamine in mice tissues

Crotamine is a strong basic polypeptide from Crotalus durissus terrificus (Cdt) venom composed of 42 amino acid residues tightly bound by three disulfide bonds. It causes skeletal muscle spasms leading to spastic paralysis of hind limbs in mice. The objective of this paper was to study the distribution of crotamine injected intraperitoneally (ip) in mice. Crotamine was purified from Cdt venom by gel filtration, followed by ion exchange chromatography, using a fast-performance liquid chromatography (FPLC) system. Purified crotamine was irradiated at 2 kGy in order to detoxify. Both native and irradiated proteins were labeled with 125, using chloramine T method, and separated by get filtration. Male Swiss mice were injected ip with 0.1 mL (2 x 10(6) cpm/mouse) of I-125 native or irradiated crotamine. At various time intervals, the animals were killed by ether inhalation and blood, spleen, liver, kidneys, brain, lungs, heart, and skeletal muscle were collected in order to determine the radioactivity content. The highest levels of radioactivity were found in the kidneys and the liver, and the lowest in the brain. (c) 2006 Elsevier Ltd. All rights reserved.

The analgesic effect of crotoxin on neuropathic pain is mediated by central muscarinic receptors and 5-lipoxygenase-derived mediators

Crotoxin (CTX). a neurotoxin isolated from the venom of the South American rattlesnake Crotalus durissus terrificus. induces analgesia. In this study, we evaluated the antinociceptive effect of CTX in a model of neuropathic pain induced by rat sciatic nerve transection. Hyperalgesia was detected 2 h after nerve transection and persisted for 64 days. Immersion of proximal and distal nerve stumps in CTX solution (0.01 mM for 10 s), immediately after nerve transection, blocked hyperalgesia. The antinociceptive effect of CTX was long-lasting, since it was detected 2 h after treatment and persisted for 64 days. CTX also delayed, but did not block, neurectomy-induced neuroma formation. The effect of CTX was blocked by zileuton (100 mg/kg, p.o.) and atropine (10 mg/kg. i.p.), and reduced by yohimbine (2 mg/kg, i.p.) and methysergide (5 mg/kg, i.p.). on the other hand. indomethacin (4 mg/kg, i.v.). naloxone (1 mg/kg, i.p.). and N-methyl atropine (30 mg/kg, i.p.) did not interfere with the effect of CTX. These results indicate that CTX induces a long-lasting antinociceptive effect in neuropathic pain, which is mediated by activation of central muscarinic receptors and partially, by activation of alpha-adrenoceptors and 5-HT receptors. Eicosanoids derived from the lipoxygenase pathway modulate the action of crotoxin. (C) 2008 Elsevier B.V. All rights reserved.

Purificação e caracterização de proteínas de venenos de serpentes que interferem na cascata de coagulação sanguínea

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudos conformocionais de diversas proteínas em solução utilizando a técnica de espalhamento de raios-X à baixo ângulo-saxs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização morfológica, morfométrica e molecular de Hepatozoon spp. (Apicomplexa, Hepatozoidae) de serpentes brasileiras naturalmente infectadas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito da interação entre Bothropstoxina-I e Crotapotina na junção neuromuscular de camundongos “in vitro”: estágio em iniciação científica

A bothropstoxina-I (BthTX-I) é uma fosfolipase A2 (PLA2) Lys49 miotóxica isolada do veneno da Bothrops jararacussu. Embora seja desprovida de atividade neurotóxica in vivo, esta toxina bloqueia a transmissão neuromuscular in vitro. A relação entre as atividades miotóxica e paralisante da BthTX-I ainda não está esclarecida. A crotapotina corresponde à subunidade não-enzimática da crotoxina, principal fração tóxica do veneno da Crotalus durissus terrificus. Isoladamente a crotapotina é atóxica, porém atua como carreadora da PLA2 Asp49 da crotoxina, potencializando sua ação neurotóxica. Esta proteína também é capaz de se complexar com outras PLA2s (Asp49 ou Lys49) de venenos ofídicos, alterando suas toxicidades. Neste trabalho avaliamos a influência da crotapotina sobre o bloqueio neuromuscular e a atividade miotóxica da BthTX-I in vitro. Preparações do nervo frênico-músculo diafragma de camundongos machos foram montadas em cubas para o registro das contrações musculares evocadas direta e indiretamente. Cortes transversais do músculo foram submetidos à coloração por hematoxilina e eosina para a avaliação do padrão morfológico. A BthTX-I (1 μM) isoladamente, ou pré-incubada com crotapotina (2 M) à 35 ºC por 30 minutos, foram adicionadas às preparações. A análise dos dados foi realizada por testes não paramétricos (p<0.05). A BthTX-I induziu bloqueio irreversível e tempo-dependente das contrações musculares diretas e indiretas. O tempo para o bloqueio de 50% das contrações indiretas (18,98 ± 1,94 min, n=4) foi significativamente menor que o das diretas (45,97 ± 5,61 min, n=5). A pré-incubação com a crotapotina não alterou de forma significativa o bloqueio das contrações diretas ou indiretas induzidos pela BthTX-I. Isoladamente, a crotapotina não afetou as contrações... (Resumo completo, clicar acesso eletrônico abaixo)

Assessing the influence of mechanical ventilation on blood gases and blood pressure in rattlesnakes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

New insights into the structural characteristics of irradiated crotamine

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A new heterologous fibrin sealant as a scaffold to cartilage repair - experimental study and preliminary results

Autologous fibrin gel is commonly used as a scaffold for filling defects in articular cartilage. This biomaterial can also be used as a sealant to control small hemorrhages and is especially helpful in situations where tissue reparation capacity is limited. In particular, fibrin can act as a scaffold for various cell types because it can accommodate cell migration, differentiation, and proliferation. Despite knowledge of the advantages of this biomaterial and mastery of the techniques required for its application, the durability of several types of sealant at the site of injury remains questionable. Due to the importance of such data for evaluating the quality and efficiency of fibrin gel formulations on its use as a scaffold, this study sought to analyze the heterologous fibrin sealant developed from the venom of Crotalus durissus terrificus using studies in ovine experimental models. The fibrin gel developed from the venom of this snake was shown to act as a safe, stable, and durable scaffold for up to seven days, without causing adverse side effects. Fibrin gel produced from the venom of the Crotalus durissus terrificus snake possesses many clinical and surgical uses. It presents the potential to be used as a biomaterial to help repair skin lesions or control bleeding, and it may also be used as a scaffold when applied together with various cell types. The intralesional use of the fibrin gel from the venom of this snake may improve surgical and clinical treatments in addition to being inexpensive and adequately consistent, durable, and stable. The new heterologous fibrin sealant is a scaffold candidate to cartilage repair in this study.