Phlogiellus bundokalbo spider venom: cytotoxic fractions against human lung adenocarcinoma (A549) cells

Spider venom is a potential source of pharmacologically important compounds. Previous studies on spider venoms reported the presence of bioactive molecules that possess cell-modulating activities. Despite these claims, sparse scientific evidence is available on the cytotoxic mechanisms in relation to the components of the spider venom. In this study, we aimed to determine the cytotoxic fractions of the spider venom extracted from Phlogiellus bundokalbo and to ascertain the possible mechanism of toxicity towards human lung adenocarcinoma (A549) cells. Methods: Spider venom was extracted by electrostimulation. Components of the extracted venom were separated by reversed-phase high performance liquid chromatography (RP-HPLC) using a linear gradient of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 95% acetonitrile (ACN). Cytotoxic activity was evaluated by the MTT assay. Apoptotic or necrotic cell death was assessed by microscopic evaluation in the presence of Hoechst 33342 and Annexin V, Alexa FluorTM 488 conjugate fluorescent stains, and caspase activation assay. Phospholipase A2 (PLA2) activity of the cytotoxic fractions were also measured. Results: We observed and isolated six fractions from the venom of P. bundokalbo collected from Aurora, Zamboanga del Sur. Four of these fractions displayed cytotoxic activities. Fractions AT5-1, AT5-3, and AT5-4 were found to be apoptotic while AT5-6, the least polar among the cytotoxic components, was observed to induce necrosis. PLA2 activity also showed cytotoxicity in all fractions but presented no relationship between specific activity of PLA2 and cytotoxicity. Conclusion: The venom of P. bundokalbo spider, an endemic tarantula species in the Philippines, contains components that were able to induce either apoptosis or necrosis in A549 cells.(AU)

Pharmacological characterization of venoms from three theraphosid spiders: Poecilotheria regalis, Ceratogyrus darlingi and Brachypelma epicureanum

Background Tarantulas (Theraphosidae) represent an important source of novel biologically active compounds that target a variety of ion channels and cell receptors in both insects and mammals. In this study, we evaluate and compare the pharmacological activity of venoms from three taxonomically different theraphosid spiders bred in captivity: Poecilotheria regalis, an aggressive arboreal tarantula from southeastern India; Ceratogyrus darlingi, an aggressive tarantula from southern Africa; and Brachypelma epicureanum, a docile tarantula from the Yucatan dry forest of Mexico. Prior to this study, no research had been conducted with regard to the composition and pharmacological activity of these venoms. Methods The pharmacological characterization of the venoms was described for the first time by the assessment of their toxicity in crickets (LD50) along with their nociceptive (by using the formalin test), hyaluronidase, phospholipase A2, edematogenic and caseinolytic activity. Results P. regalis and B. epicureanum venoms induced a similar lethal effect on crickets (LD50 = 5.23 ± 3.1 and 14.4 ± 5.0 μg protein/g 48 h post-injection, respectively), whereas C. darlingi venom (119.4 ± 29.5 μg protein/g 48 h post-injection) was significantly less lethal than the other two venoms. All three venoms induced similar edematogenic activity on rats but did not induce nociceptive behavior. The assessment of enzymatic activity indicated that P. regalis venom induces significantly higher hyaluronidase activity (27.6 ± 0.9 TRU/mg) than both C. darlingi (99.7 ± 1.9 TRU/mg) and B. epicureanum (99.6 ± 1.6 TRU/mg); these latter venoms did not display phospholipase A2or caseinolytic activity. Conclusions This study demonstrates that these theraphosid spiders of different habitats produce venoms with different activities. P. regalis venom displays a high level of hyaluronidase activity, which may be associated with its potentially medically significant bite.

Biochemical and pharmacological study of venom of the wolf spider Lycosa singoriensis

The wolf spider Lycosa singoriensis is a large and venomous spider distributed throughout northwestern China. Like other spider venoms, the wolf spider venom is a chemical cocktail. Its protein content is 0.659 mg protein/mg crude venom as determined by the Lowry method. MALDI-TOF analysis revealed that the venom peptides are highly diverse and may be divided into three groups characterized by three independent molecular ranges: 2,000 to 2,500 Da, 4,800 to 5,500 Da and 7,000 to 8,000 Da, respectively. This molecular distribution differs substantially from those of most spider venoms studied so far. This wolf spider venom has low neurotoxic action on mice, but it can induce hemolysis of human erythrocytes. Furthermore, the venom shows antimicrobial activity against prokaryotic and eukaryotic cells.(AU)

Use of neurotoxins to study Ca2+ channel functions

The article contains a brief review on the properties and classification of voltage-dependent Ca2+ channels and on the organic blockers of the different channel types. The effects of peptide toxins from the venoms of Conus sp and of the spider Agelenopsis aperta on high voltage-activated Ca2+ channels are discussed. In addition, we present preliminary data on a novel peptide toxin purified from the venom of the spider Phoneutria nigriventer, which is a powerful blocker of L-and N-type Ca2+ channels.

Effect of Phoneutria nigriventer spider venom on gastric emptying in rats

The effect of Phoneutria nigriventer spider venom (PNV) on the gastric emptying of liquids was studied in 240 young adult Wistar rats (2-3 months of age) divided into subgroups of 8 animals each. The study was performed in 3 stages. Initially, PNV was injected into rats at doses of 0.19, 0.38 or 0.76 mg/kg and the effect on gastric emptying was assessed 30 min later. In the second stage, a time-course study was performed by injecting 0.76 mg PNV/kg and measuring the effect on gastric emptying 15, 60 and 120 min post-venom. In the last stage, in order to investigate the possible mechanisms of PNV influence on gastric emptying, one group of rats underwent subdiaphragmatic vagotomy and then received 0.76 mg PNV/kg while three other groups were pretreated iv with either prazosin (0.4 mg/kg), domperidone (1.0 mg/kg) or propranolol (0.6 mg/kg) and then given 0.38 or 0.76 mg PNV/kg. In this last stage, gastric retention was measured 30 min post-venom. Each animal received a saline test meal solution containing phenol red as a marker (60 microg/ml). Ten min after administering the test meal by gavage, gastric retention was determined by measuring the residual test meal marker concentration and the animals were sacrificed. PNV (0.76 mg/kg) provoked a significant delay in gastric emptying of liquids 15, 30 and 60 min after its administration. Propranolol partially interfered with gastric emptying in rats that had received 0.38 and 0.76 mg PNV/kg. Vagotomy and pretreatment of the rats with prazosin and domperidone had no effect. We conclude that the delay in the liquid gastric emptying observed in severely envenomed rats was probably due, at least in part, to a venom-stimulated release of catecholamines which inhibited gastric motility by activating smooth muscle beta-adrenergic receptors.

Effects of Phoneutria nigriventer venom on rabbit vascular smooth muscle

1. The effects of Phneutria nigriventer venom (PNV) on rabbit vascular smooth muscle have been investigated. De-endothelialized vascular strips were superfused in a cascade system with oxygenated (95 per cent O2 + 5 per cent CO2) Krebs solution at 37 degrees C. 2. Phoneutria nigriventer venom (0.3-30 micrograms) produced dose-dependent and short-lived contractions of both venous (cava, mesenteric and jugular veins) and arterial (pulmonary and mesenteric arteries) tissues. 3. Methysergide (5.0 microM) did not significantly affect PNV-induced contractions in venous tissues (cava and mesenteric veins) or pulmonary artery, indicating that serotonin is not involved in the contraction. This was confirmed when PNV was dialyzed (24-48 h) since the contracting activity was still observed on the above tissues. In addition, the spasmogenic activity induced by dialyzed PNV was greatly reduced by incubating the venom with trypsin. 4. Neither tetrodotoxin (3.0 microM) nor phenoxybenzamine (0.05 microM) significantly affected PNV-induced contractions, suggesting that voltage-dependent sodium channel activation or endogenous catecholamine release from autonomic nerve endings on the vascular walls do not play a role in the response to PNV. 5. Our results demonstrate that PNV contains non-dialyzable components, probably peptides, that are responsible for the contractile activity on rabbit veins and pulmonary artery strips

Proyecto cooperativo interuniversitario UPCH-UNMSM. Loxoscelismo experimental: aspectos farmacológicos y anatomopatológicos / Interuniversitary cooperative project UPCH-UNMSM. Experimental loxoscelism: pharmacological and anatomopathological aspects

Se obtuvo veneno de arañas Loxosceles sp. procedente de dos localidades del Departamento de Lima (Perú): Urb. Santa María de Chosica y Santa Rosa de Quives (Km. 60). La técnica de estimulación eléctrica para obtención de veneno fue modificado con la finalidad de descartar la contaminación con regurgitado gástrico, obteniéndose 0.33 a 0.76 ul/araña adulta, el que fue utilizado en los ensayos de toxicidad aguda en conejos, y en los estudios bioquímicos. Un segundo método alternativo para la obtención de extractos tóxicos utilizado fue la microdiseccción y homogenización glandular total. La dosis letal media, fue estimado a diferentes intervalos de tiempo, utilizando un programa computarizado del método de transformación en probits. La LD50 intraperitoneal y endovenosa obtenida en ratones, fue de 0.299 y 0.165 glándulas/ratón, respectivamente. Un método de envenenamiento por picadura directa fue desarrollado para el estudio de toxicidad aguda en conejos, y los resultados fueron comparados con los producidos por inyección del veneno obtenido mediante estimulación eléctrica. Los efectos cutáneos del Loxoscelismo en conejos fueron tipificados y biopsias de lesiones en diferentes estadíos evolutivos fueron analizados mediante estudio histológico. El cuadro sistémico en conejos estuvo caracterizado por hemoproteinuria, ausencia de hematuria, lesiones cutáneas variables y muerte. Se determinó la potencia neutralizante in vitro del suero antiloxoscélico comercial (INS, Perú), demostrándose que 0.3ml. del suero, inactivan los efectos letales de una glándula de veneno loxoscélico. Se descarta la probable acción protectora del suero de rata sobre el efecto letal del veneno loxoscélico en el ratón.