Age effects on the pharmacokinetics of tityustoxin from Tityus serrulatus scorpion venom in rats

The pharmacokinetics of scorpion venom and its toxins has been investigated in experimental models using adult animals, although, severe scorpion accidents are associated more frequently with children. We compared the effect of age on the pharmacokinetics of tityustoxin, one of the most active principles of Tityus serrulatus venom, in young male/female rats (21-22 days old, N = 5-8) and in adult male rats (150-160 days old, N = 5-8). Tityustoxin (6 µg) labeled with 99mTechnetium was administered subcutaneously to young and adult rats. The plasma concentration vs time data were subjected to non-compartmental pharmacokinetic analysis to obtain estimates of various pharmacokinetic parameters such as total body clearance (CL/F), distribution volume (Vd/F), area under the curve (AUC), and mean residence time. The data were analyzed with and without considering body weight. The data without correction for body weight showed a higher Cmax (62.30 ± 7.07 vs 12.71 ± 2.11 ng/ml, P < 0.05) and AUC (296.49 ± 21.09 vs 55.96 ± 5.41 ng h-1 ml-1, P < 0.05) and lower Tmax (0.64 ± 0.19 vs 2.44 ± 0.49 h, P < 0.05) in young rats. Furthermore, Vd/F (0.15 vs 0.42 l/kg) and CL/F (0.02 ± 0.001 vs 0.11 ± 0.01 l h-1 kg-1, P < 0.05) were lower in young rats. However, when the data were reanalyzed taking body weight into consideration, the Cmax (40.43 ± 3.25 vs 78.21 ± 11.23 ng kg-1 ml-1, P < 0.05) and AUC (182.27 ± 11.74 vs 344.62 ± 32.11 ng h-1 ml-1, P < 0.05) were lower in young rats. The clearance (0.03 ± 0.002 vs 0.02 ± 0.002 l h-1 kg-1, P < 0.05) and Vd/F (0.210 vs 0.067 l/kg) were higher in young rats. The raw data (not adjusted for body weight) strongly suggest that age plays a pivotal role in the disposition of tityustoxin. Furthermore, our results also indicate that the differences in the severity of symptoms observed in children and adults after scorpion envenomation can be explained in part by differences in the pharmacokinetics of the toxin.

Neutralization of the edema-forming, defibrinating and coagulant effects of Bothrops asper venom by extracts of plants used by healers in Colombia

We determined the neutralizing activity of 12 ethanolic extracts of plants against the edema-forming, defibrinating and coagulant effects of Bothrops asper venom in Swiss Webster mice. The material used consisted of the leaves and branches of Bixa orellana (Bixaceae), Ficus nymphaeifolia (Moraceae), Struthanthus orbicularis (Loranthaceae) and Gonzalagunia panamensis (Rubiaceae); the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); the whole plant of Pleopeltis percussa (Polypodiaceae) and Trichomanes elegans (Hymenophyllaceae); rhizomes of Renealmia alpinia (Zingiberaceae), Heliconia curtispatha (Heliconiaceae) and Dracontium croatii (Araceae), and the ripe fruit of Citrus limon (Rutaceae). After preincubation of varying amounts of each extract with either 1.0 µg venom for the edema-forming effect or 2.0 µg venom for the defibrinating effect, the mixture was injected subcutaneously (sc) into the right foot pad or intravenously into the tail, respectively, to groups of four mice (18-20 g). All extracts (6.2-200 µg/mouse) partially neutralized the edema-forming activity of venom in a dose-dependent manner (58-76% inhibition), with B. orellana, S. orbicularis, G. panamensis, B. rosademonte, and D. croatii showing the highest effect. Ten extracts (3.9-2000 µg/mouse) also showed 100% neutralizing ability against the defibrinating effect of venom, and nine prolonged the coagulation time induced by the venom. When the extracts were administered either before or after venom injection, the neutralization of the edema-forming effect was lower than 40% for all extracts, and none of them neutralized the defibrinating effect of venom. When they were administered in situ (sc at the same site 5 min after venom injection), the neutralization of edema increased for six extracts, reaching levels up to 64% for C. limon.

Thalidomide and pentoxifylline block the renal effects of supernatants of macrophages activated with Crotalus durissus cascavella venom

Because thalidomide and pentoxifylline inhibit the synthesis and release of tumor necrosis factor-alpha (TNF-alpha), we determined the effect of these drugs on the renal damage induced by supernatants of macrophages activated with Crotalus durissus cascavella venom in order to identify the role of TNF-alpha in the process. Rat peritoneal macrophages were collected with RPMI medium and stimulated in vitro with C.d. cascavella venom (10 µg/ml) in the absence and presence of thalidomide (15 µM) or pentoxifylline (500 µM) for 1 h and washed and kept in culture for 2 h. Supernatant (1 ml) was tested on an isolated perfused rat kidney (N = 6 for each group). The first 30 min of each experiment were used as control. The supernatant was added to the perfusion system. All experiments lasted 120 min. The toxic effect of the preparation of venom-stimulated macrophages on renal parameters was determined. At 120 min, thalidomide (Thalid) and pentoxifylline (Ptx) inhibited (P < 0.05) the increase in perfusion pressure caused by the venom (control = 114.0 ± 1.3; venom = 137.1 ± 1.5; Thalid = 121.0 ± 2.5; Ptx = 121.4 ± 4.0 mmHg), renal vascular resistance (control = 4.5 ± 0.2; venom = 7.3 ± 0.6; Thalid = 4.5 ± 0.9; Ptx = 4.8 ± 0.6 mmHg/ml g-1 min-1), urinary flow (control = 0.23 ± 0.001; venom = 0.44 ± 0.01; Thalid = 0.22 ± 0.007; Ptx = 0.21 ± 0.009 ml g-1 min-1), glomerular filtration rate (control = 0.72 ± 0.06; venom = 1.91 ± 0.11; Thalid = 0.75 ± 0.04; Ptx = 0.77 ± 0.05 ml g-1 min-1) and the decrease in percent tubular sodium transport (control = 77.0 ± 0.9; venom = 73.9 ± 0.66; Thalid = 76.6 ± 1.1; Ptx = 81.8 ± 2.0%), percent tubular chloride transport (control = 77.1 ± 1.2; venom = 71.4 ± 1.1; Thalid = 77.6 ± 1.7; Ptx = 76.8 ± 1.2%), and percent tubular potassium transport (control = 72.7 ± 1.1; venom = 63.0 ± 1.1; Thalid = 72.6 ± 1.0; Ptx = 74.8 ± 1.0%), 30 min before and during the stimulation of macrophages with C.d. cascavella venom. These data suggest the participation of TNF-alpha in the renal effects induced by supernatant of macrophages activated with C.d. cascavella venom.

Alternagin-C, a disintegrin-like protein from the venom of Bothrops alternatus, modulates alpha2ß1 integrin-mediated cell adhesion, migration and proliferation

The alpha2ß1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2ß1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC) and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of ~10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2ß1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2ß1 integrin.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR), we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49), from the venom of Crotalus durissus collilineatus (Cdc PLA2). The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.

Pulsed ultrasound therapy accelerates the recovery of skeletal muscle damage induced by Bothrops jararacussu venom

We studied the effect of pulsed ultrasound therapy (UST) and antibothropic polyvalent antivenom (PAV) on the regeneration of mouse extensor digitorum longus muscle following damage by Bothrops jararacussu venom. Animals (Swiss male and female mice weighing 25.0 ± 5.0 g; 5 animals per group) received a perimuscular injection of venom (1 mg/kg) and treatment with UST was started 1 h later (1 min/day, 3 MHz, 0.3 W/cm², pulsed mode). Three and 28 days after injection, muscles were dissected and processed for light microscopy. The venom caused complete degeneration of muscle fibers. UST alone and combined with PAV (1.0 mL/kg) partially protected these fibers, whereas muscles receiving no treatment showed disorganized fascicules and fibers with reduced diameter. Treatment with UST and PAV decreased the effects of the venom on creatine kinase content and motor activity (approximately 75 and 48%, respectively). Sonication of the venom solution immediately before application decreased the in vivo and ex vivo myotoxic activities (approximately 60 and 50%, respectively). The present data show that UST counteracts some effects of B. jararacussu venom, causing structural and functional improvement of the regenerated muscle after venom injury.

Studies on Venom of the fish Scatophagus Argus from Cochin Estuary-A Biochemical Approach

Studies reveal the presence of enzymes and different proteins in the venom of S.argus. The present study detected the presence of phosphodiesterase in S. argus venom. S. argus venom has displayed the presence of micromolar concentration of acetylcholine. Phospholipase activity in S. argus venom shows values below the detection threshold indicating that the venom does not possess this enzyme. The proteolylic activity of S. argus venom on casein and gelatin were assayed due to the probable involvement of proteases in causing the instability of biological activities of the fish venom. Caseinase and gelatinase enzymes were detected in S. argus venom. Though exact relationships of these enzymes and proteins in envenomation are not traced, the involvement of enzymes in envenomation cannot be ruled out. Further studies are required to find the mechanism of action of these enzymes and proteins present in S. argus venom. The present study opens new dimensions for isolation of the lethal compound present in S. argus venom. The preliminary study carried out here shows the presence of a lethal factor between 6.5 KDa - 68 KDa. Studies conclude that fish venom possesses many bioactive substances, especially peptides, proteases and enzymes that bind with high affinity to physiological targets and can be trapped for therapeutic purposes in the near future. Even though this study reveals the conundrums of S. argus venom, it opens new vistas of research on the venom components and the application and design of the venom as a drug.

Structure of the snake-venom toxin convulxin

Snake venoms contain a number of proteins that interact with components of the haemostatic system that promote or inhibit events leading to blood- clot formation. The snake- venom protein convulxin ( Cvx) binds glycoprotein ( GP) VI, the platelet receptor for collagen, and triggers signal transduction. Here, the 2.7 Angstrom resolution crystal structure of Cvx is presented. In common with other members of this snake-venom protein family, Cvx is an alphabeta- heterodimer and conforms to the C- type lectin- fold topology. Comparison with other family members allows a set of Cvx residues that form a concave surface to be putatively implicated in GPVI binding. Unlike other family members, with the exception of flavocetin- A ( FL- A), Cvx forms an (alphabeta)(4) tetramer. This oligomeric structure is consistent with Cvx clustering GPVI molecules on the surface of platelets and as a result promoting signal transduction activity. The Cvx structure and the location of the putative binding sites suggest a model for this multimeric signalling assembly.

Purification and functional characterisation of rhiminopeptidase A, a novel aminopeptidase from the venom of Bitis gabonica rhinoceros

This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites.

Purification and functional characterisation of Rhinocerase, a novel serine protease from the venom of Bitis gabonica rhinoceros

Background: Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders. Methodology/Principal Findings: In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36kDa, which reduces to 31kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading α and β chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate. Conclusions/Significance: A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.

Evolutionary analysis of novel serine proteases in the venom gland transcriptome of Bitis gabonica rhinoceros

Background: Serine proteases are major components of viper venom and target various stages of the blood coagulation system in victims and prey. A better understanding of the diversity of serine proteases and other enzymes present in snake venom will help to understand how the complexity of snake venom has evolved and will aid the development of novel therapeutics for treating snake bites. Methodology and Principal Findings: Four serine protease-encoding genes from the venom gland transcriptome of Bitis gabonica rhinoceros were amplified and sequenced. Mass spectrometry suggests the four enzymes corresponding to these genes are present in the venom of B. g. rhinoceros. Two of the enzymes, rhinocerases 2 and 3 have substitutions to two of the serine protease catalytic triad residues and are thus unlikely to be catalytically active, though they may have evolved other toxic functions. The other two enzymes, rhinocerases 4 and 5, have classical serine protease catalytic triad residues and thus are likely to be catalytically active, however they have glycine rather than the more typical aspartic acid at the base of the primary specificity pocket (position 189). Based on a detailed analysis of these sequences we suggest that alternative splicing together with individual amino acid mutations may have been involved in their evolution. Changes within amino acid segments which were previously proposed to undergo accelerated change in venom serine proteases have also been observed. Conclusions and Significance: Our study provides further insight into the diversity of serine protease isoforms present within snake venom and discusses their possible functions and how they may have evolved. These multiple serine protease isoforms with different substrate specificities may enhance the envenomation effects and help the snake to adapt to new habitats and diets. Our findings have potential for helping the future development of improved therapeutics for snake bites.