Biodistribution studies of bee venom and spider toxin using radiotracers

The use of radiotracers allows the understanding of the bioavailability process, biodistribution, and kinetics of any molecule labelled with an isotope, which does not alter the molecule's biological properties. In this work, technetium-99m and iodine-125 were chosen as radiotracers for biodistribution studies in mice using bee (Apis mellifera) venom and a toxin (PnTX2-6) from the Brazilian "armed" spider (Phoneutria nigriventer) venom. Incorporated radioactivity was measured in the blood, brain, heart, lung, liver, kidney, adrenal gland, spleen, stomach, testicle, intestine, muscle, and thyroid gland. Results provided the blood kinetic parameter, and different organs distribution rates.(AU)

Effects of 60Co gamma radiation on crotamine

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3 percent, but did not eliminate it

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications

Effect of gamma irradiation on the behavioral properties of crotoxin

Crotoxin has been detoxified with gamma radiation in order to improve crotalic antiserum production. Nevertheless, present knowledge of the biological characteristics of irradiated crotoxin is insufficient to propose it as an immunizing agent. Crotoxin is known to increase the emotional state of rats and to decrease their exploratory behavior (Moreira EG, Nascimento N, Rosa GJM, Rogero JR and Vassilieff VS (1996) Brazilian Journal of Medical and Biological Research, 29: 629-632). Therefore, we decided 1) to evaluate the effects of crotoxin in the social interaction test, which has been widely used for the evaluation of anxiogenic drugs, and 2) to determine if irradiated crotoxin induces behavioral alterations similar to those of crotoxin in the social interaction, open-field and hole-board tests. Male Wistar rats (l8O-220 g) were used. Crotoxin (100, 250, and 500 mug/kg) was injected intraperitoneally 2 h before the social interaction test. Similarly, irradiated crotoxin (2000 Gy gamma radiation from a 60Co source) was administered at the doses of 100, 250, and 500 mug/kg for the hole-board test, and at the doses of 1000 and 2500 mug/kg for the open-field and social interaction tests. ANOVA complemented with the Dunnett test was used for statistical analysis (P<0.05). Crotoxin decreased the social interaction time(s) at the doses of 1OO, 250 and 500 mug/kg (means + SEM) from 51.6 ñ 4.4 to 32.6 ñ 3.7,28.0 ñ 3.6 and 31.6 ñ 4.4, respectively. Irradiated crotoxin did not induce behavioral alterations. These results indicate that 1) crotoxin may be an anxiogenic compound, and 2) in contrast to crotoxin, irradiated crotoxin was unable to induce behavioral alterations, which makes it a promising compound for the production of crotalic antiserum.

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-fleld and hole-board tests. Adult male Wistar rats (l80-220 g) treated with crotoxin, 1OO, 250 and 500 mug/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 mug/kg of crotoxin increased freezing (from 3.22 sec to 10.75 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 mug/kg also decreased rearing (from 24.9 to 17.5). In the hole-board test, 500 mug/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.

Pathophysiological observations on calves concurrently infected with Cooperia punctata and Haemonchus placei

Bezerros da raça Holandesa preta e branca receberam diariamente, via oral, 18.000 larvas infectantes de C. punctata e 2.000 H. placei por um período de 14 dias. Vinte e três dias após o início da infecçäo, os bezerros foram injetados com 12S l-albumina para realizaçäo do estudo do metabolismo da albumina. O número médio de vermes recuperados na necrópsia foi de 42.275 mais ou menos 20.598 para C. punctata e 6.075 mais ou menos 1.407 para H. placei. Os bezerros infectados perderam peso durante o período experimental, e apresentaram diminuiçäo significativa na meia vida biológica da 12S l-albumina (P<0,05). A taxa de catabolismo e do "clearance" fecal da albumina e a distribuiçäo da albumina do "pool" extra para o intravascular näo foram estatisticamente significativos. Os animais infectados apresentaram hipoalbuminemia e um ligeiro aumento no volume plasmático. Apesar destas alteraçöes, os sinais clínicos foram amenos

Immunochemical detection of purified crotoxin from crotalus durissus terrificus venom in the motor end plate of striaqted muscle in CBA/J mice

Electrophysiological studies of crotoxin, a potent neurotoxic fraction from Crotalus durissus terrificus venom, have shown a pre- and post-synaptic action. In order to determine the specific site of this activity, we performed an immunohistochemicaql analysis on striated muscle from CBA/J mice, injected with crotoxin (5LD50), iv, using a single-step immunoperoxidase assay with peroxidase-conjugated antibodies to whole venom. Control muscle and muscle obtained from treated animals 15 min after injection showed no staining. However, 30 min after injection, the neuromuscular motor end plate of mice who received crotoxin was clearly stained, including thin terminations, without any morphological alteration. Sixty min after administration, the motor end plate was no longer intact, but only roughly formed stained areas without clearly identifiable structures were present. These data show specific crotoxin binding to neural end plates in striated muscle, with a subsequent toxin-induced degeneration of this structure

Detoxification of the crotoxin complex by gamma radiation

Crotoxin, the major neurotoxin of the South American rattlesnake venom (2 mg protein/ml in 0.85% NaCl) was irradiated with a Co-60 gamma source at a dose rate of 1100 Gy/h and at doses of 250, 500, 1000, 1500 and 2000 Gy. Irradiated crotoxin was analyzed for free SH-groups, protein concentration, electrophoretic profile (SDS-PAGE), 50% lethal dose (LD50) in mice, and antigenicity against crotalic antiserum by diffusion immunoassay. Irradiation led to the formation of protein aggregates and solubility was reduced at doses of 1000 Gy or higher. The LD50 increased about 2-fold for 1000 Gy and 3-5-fold for 1500 Gy. However, the antigenic response was not changed as judged by the capacity of irradiated protein receiving up to 1000 Gy to react with anti-Crotalus durissus terrificus venom horse serum. The dose of 1000 Gy cleaved 0.95 disulfide bridges/mol and 1500 Gy cleaved 1.42 bridges/mol, indicating the importance of disulfide bond integrity for toxicity