Heat deactivation of the stonefish Synanceia horrida venom - implications for first-aid management.

OBJECTIVES: To investigate the effects of temperature and hot water immersion time on neutralising venom lethality of the Australian estuarine stonefish (Synanceia horrida). DESIGN: Depths of the spines were measured while venom was extracted from S. horrida individuals. The venom was then exposed to temperatures of 4°C, 37.0°C, 40.1°C, 42.3°C, 45.0°C, 47.7°C, 55.2°C, and 60.0°C for either five or 20 minutes incubation periods. Venom samples were added to cultured human cardiomyocytes and cell viability curves were produced using the ACEA's xCELLigence real-time cell monitoring system. MAIN OUTCOME MEASURES: Determination of venom lethality on cardiomyocytes at a range of temperatures. RESULTS: The average depth of the spine required to go into a victims' flesh before the venom gland compressed and expelled venom was 18 mm. Cardiomyocytes exposed to heat-treated venom for five minutes required higher temperatures to neutralise 99% of the venom, namely 44.6°C in comparison to 42.1°C with an incubation time of 20 minutes. CONCLUSION: This study supports the use of hot water immersion therapy in the treatment of S. horrida stings. It is suggested that due to the depth of the puncture wound longer incubation times should be sought to allow heat to penetrate the deeper portions of the dermis and effectively begin venom deactivation.

The anti-tumor effects of the recombinant toxin protein rLj-RGD3 from Lampetra japonica on pancreatic carcinoma Panc-1 cells in nude mice.

Recombinant Lampetra japonica RGD peptide (rLj-RGD3) is a soluble toxin protein with three RGD (Arg-Gly-Asp) motifs and a molecular weight of 13.5kDa. The aim of this study was to investigate the effects and mechanisms of rLj-RGD3 on tumor growth and survival in pancreatic carcinoma Panc-1 cell-bearing mice. A Panc-1 human pancreatic carcinoma-bearing nude mouse model was successfully generated, and the animals were treated with different doses of rLj-RGD3 for 3 weeks. The volume and weight of the subcutaneous tumors, the survival of the nude mice, histopathological changes, the intratumoral MVD, the number of apoptotic Panc-1 cells, and apoptosis-related proteins and gene expressions were determined. rLj-RGD3 significantly decreased the tumor volumes and weights, and the maximum tumor volume and weight IR values were 53.2% (p<0.001) and 55.9% (p<0.001), respectively. The life expectancy of Panc-1-bearing nude mice treated with rLj-RGD3 was increased by 56.3% (p<0.001). Meanwhile, rLj-RGD3 promoted the expression of Bax, caspase-3, and caspase-9 and inhibited Bcl-2 and VEGF expression. In addition, rLj-RGD3 did not change FAK, PI3K and Akt expression, but p-FAK, p-PI3K and p-Akt, levels were down-regulated. These results show that rLj-RGD3 induced potent anti-tumor activity in vivo and suppressed the growth of transplanted Panc-1 cells in a nude mouse model, implying that rLj-RGD3 may serve as a potent clinical therapeutic agent for human pancreatic carcinoma.

Low concentrations of the recombinant toxin protein rLj-RGD3 suppress TNF-α-induced human renal carcinoma cell invasion.

A hallmark of renal cell carcinoma (RCC) invasion is the degradation of the extracellular matrix (ECM) by the local production of gelatinase enzymes. Matrix metalloproteinase-9 (MMP-9)-induced cancer cell invasion is one of the pivotal steps in cancer metastasis. It has been reported that tumor necrosis factor-α (TNF-α), a regulator of MMP-9, can induce invasion in human renal carcinoma cells. Previous work in our laboratory has shown that rLj-RGD3, a recombinant RGD (Arg-Gly-Asp)-toxin protein from the buccal gland secretion of Lampetra japonica, possesses anti-tumor activity. In this study, we demonstrated that rLj-RGD3 suppressed TNF-α-induced MMP-9 secretion in 786-0 cells (human renal carcinoma cells). To investigate the regulatory effect of rLj-RGD3 on TNF-α-induced MMP-9 secretion, we pre-treated cells with rLj-RGD3. Interestingly, rLj-RGD3 had no significant effect on the constitutive secretion of MMPs. However, low concentrations of rLj-RGD3 decreased TNF-α-induced MMP-9 secretion. Functional studies revealed that rLj-RGD3 induced apoptosis and significantly inhibited the proliferation, migration, and invasion of 786-0 cells. Furthermore, the actin architecture in cells pre-treated with rLj-RGD3 was aggregated and disorganized. Our findings suggest that rLj-RGD3 may be used as a potential drug in renal cancer therapy.

Pardaxin, a fish antimicrobial peptide, exhibits antitumor activity toward murine fibrosarcoma in vitro and in vivo.

The antitumor activity of pardaxin, a fish antimicrobial peptide, has not been previously examined in in vitro and in vivo systems for treating murine fibrosarcoma. In this study, the antitumor activity of synthetic pardaxin was tested using murine MN-11 tumor cells as the study model. We show that pardaxin inhibits the proliferation of MN-11 cells and reduces colony formation in a soft agar assay. Transmission electron microscopy (TEM) showed that pardaxin altered the membrane structure similar to what a lytic peptide does, and also produced apoptotic features, such as hollow mitochondria, nuclear condensation, and disrupted cell membranes. A qRT-PCR and ELISA showed that pardaxin induced apoptosis, activated caspase-7 and interleukin (IL)-7r, and downregulated caspase-9, ATF 3, SOCS3, STAT3, cathelicidin, p65, and interferon (IFN)-γ suggesting that pardaxin induces apoptosis through the death receptor/nuclear factor (NF)-κB signaling pathway after 14 days of treatment in tumor-bearing mice. An antitumor effect was observed when pardaxin (25 mg/kg; 0.5 mg/day) was used to treat mice for 14 days, which caused significant inhibition of MN-11 cell growth in mice. Overall, these results indicate that pardaxin has the potential to be a novel therapeutic agent to treat fibrosarcomas.

Structural and biological characterization of Nattectin, a new C-type lectin from the venomous fish Thalassophryne nattereri.

Lectins are glycan-binding receptors that recognize glycan epitopes on foreign pathogens and in the host systems. They can be involved in functions that include innate immunity, development, immune regulation and homeostasis. Several lectins have been purified and characterized from fish species. In this work, using cation-exchange chromatography, a galactose-specific lectin belonging to the family of C-type lectins was isolated from the venom of the Brazilian venomous fish Thalassophryne nattereri. Nattectin is a basic, non-glycosilated, 15 kDa monomeric protein. It exhibits hemagglutination activity that is independent of Ca(2+). We also demonstrated a lectin activity for Nattectin in the innate immune system, especially in neutrophil mobilization in mice, indicating that marine organisms are source of immunomodulator agents.

Verrucotoxin, a stonefish venom, modulates calcium channel activity in guinea-pig ventricular myocytes.

BACKGROUND AND PURPOSE: Stonefish (Synanceia genus) are commonly found in shallow waters of the Pacific and Indian Oceans. The venom of stonefish is stored in the dorsal fine spines and contains a proteinaceous toxin, verrucotoxin (VTX). The stings produced by the spines induce intense pain, respiratory weakness, damage to the cardiovascular system, convulsions and paralysis, sometimes leading to death. Although there are many studies on VTX, the mechanism(s) underlying the VTX-mediated cardiotoxicity is not yet fully understood. The aim of this study was to investigate the modulation of ion channels in cardiac tissue by VTX. EXPERIMENTAL APPROACH: The effects of VTX on changes in the voltage or current in guinea-pig ventricular myocytes were investigated using a patch clamp method. KEY RESULTS: VTX (10 microg ml(-1)) prolonged the action potential duration by 2.5-fold. VTX increased L-type Ca(2+) currents (I (Ca(L))) in a concentration-dependent manner with a EC(50) value of 7 microg ml(-1) and a maximum increase of 3.1-fold. The non-selective beta-adrenoceptor antagonist, propranolol (1 microM) and the selective beta(1)-adrenoceptor antagonist, CGP20712A (10 microM) each abolished the effect of VTX (100 microg ml(-1)) on I (Ca(L)). Furthermore, the protein kinase A (PKA) antagonists H-89 (10 microM) and Rp-8-Br-cAMPS (30 microM) inhibited the effect of VTX on I (Ca(L)). CONCLUSIONS AND IMPLICATIONS: VTX modulates Ca(2+) channel activity through the beta-adrenoceptor-cAMP-PKA pathway.

Skeletal muscle necrosis and regeneration after injection of Thalassophryne nattereri (niquim) fish venom in mice.

Stings by Thalassophryne nattereri are responsible for envenomation of fishermen in north-eastern Brazil. Its venom induces prominent local tissue damage, characterized by pain, oedema and necrosis. The pathogenesis of acute muscle damage induced by T. nattereri venom was studied in mice. Intramuscular injection induced myonecrosis within the first hours. Some muscle cells presented a hypercontracted morphology, but most necrotic fibres were not hypercontracted, being instead characterized by a disorganization of myofibrils, with Z line loss, mitochondrial swelling and sarcolemmal disruption. In addition, thrombosis was observed histologically in venules and veins, together with vascular congestion and stasis, evidenced by intravital microscopy. Venom induced a rapid increment in serum creatine kinase (CK) levels, concomitant with a reduction in gastrocnemius muscle CK activity, whereas no increments in muscle lactic acid were detected. A rapid cytolytic effect was induced by the venom on C2C12 murine myoblasts in culture. The inflammatory reaction in affected muscle was characterized by oedema and scarce cellular infiltrate of polymorphonuclear leucocytes and macrophages, with a consequent delay in the removal of necrotic material. Skeletal muscle regeneration was partially impaired, as evidenced by the presence of regenerating fibres of variable size and by the increase of fibrotic tissue in endomysium and perimysium. It is suggested that T. nattereri venom affects muscle fibres by a direct cytotoxic effect, and that the vascular alterations described preclude a successful regenerative process.

Evidence for adrenergic and tachykinin activity in venom of the stonefish (Synanceja trachynis).

The aim of the present study was to investigate previously suggested adrenergic and tachykinin activity, as well as the cardiovascular effects, of venom from the stonefish (Synanceja trachynis). Stonefish venom (60-120 micrograms/kg, i.v.) produced dose-dependent bronchoconstriction in anaesthetised guinea-pigs. This response (100 micrograms/kg, i.v.) was significantly reduced by the neurokinin 1 (NK1) receptor antagonist CP-99,994 (1 mg/kg, i.v.). Contractile responses to venom (4 micrograms/ml) of guinea-pig isolated ileum (GPI) were significantly inhibited by a combination of the sodium channel blocking drug tetrodotoxin (1 microM) and the ganglion blocking drug mecamylamine (10 microM). However, subsequent administration of CP-99,994 (0.1 microM) did not produce further inhibition. Endogenous tachykinin depletion with capsaicin (1 microM) also significantly attenuated responses to venom (4 micrograms/ml) in GPI. Venom (4 micrograms/ml) produced increases in rate and force of contraction of rat spontaneously beating isolated atria which were significantly inhibited by the beta-adrenoceptor antagonist propranolol (5 microM) but not by noradrenergic transmitter depletion with reserpine (4.5 mg/kg, i.p.). In the presence of the alpha 1-adrenoceptor antagonist prazosin (0.3 microM), venom (6 micrograms/ml) significantly inhibited electrically evoked twitches of prostatic segments of rat vas deferens. The inhibitory effect of venom was significantly reduced by the alpha 2-adrenoceptor antagonist idazoxan (1 microM) but not by propranolol (5 microM) or the neurokinin 2 (NK2) receptor antagonist SR-48,968 (0.1 microM). Venom (60-120 micrograms/kg, i.v.) produced dose-dependent increases in mean arterial blood pressure in anaesthetised rats. This pressor response (60 micrograms/kg, i.v.) was significantly reduced by prazosin (10-50 micrograms/kg, i.v.) and the leukotriene receptor antagonist SB205312 (1 mg/kg, i.v.), significantly increased by propranolol (2 mg/kg, i.v.), but not significantly affected by the cyclo-oxygenase inhibitor indomethacin (10 mg/kg, i.v.) or the thromboxane A2/prostaglandin H2 (TP) receptor antagonist GR32191B (1 mg/kg, i.v.). Pressor responses to venom (100 micrograms/kg, i.v.) were also observed in anaesthetised rabbits. These results suggest that stonefish venom contains a component capable of stimulating the release of endogenous tachykinins with subsequent activity at NK1 receptors. The venom also appears to act via stimulation of sodium channels on sensory nerves. The venom also has activity at alpha 2-adrenoceptors and a direct action at beta-adrenoceptors. The effect of venom on blood pressure of anaesthetised rats appears to include a pressor component that is mediated, in part,by alpha-adrenoceptors and leukotriene receptors, and a depressor component that is mediated by beta-adrenoceptors. However, the pressor response does not involve action at TP receptors, or require the production of cyclo-oxygenase metabolites.

Does crude venom of the stonefish (Synanceia verrucosa) activate beta-adrenoceptors in the frog heart muscle?

Venom isolated from the stonefish Synanceia verrucosa was assayed in concentrations of 0.07 and 5.7 micrograms/ml on frog atrial fibres and myocytes. Venom, less than 2.9 micrograms/ml, dose-dependently increased the amplitude and the duration of the stimulated peak tension, lengthened the time constant of the relaxation phase and shortened the duration of the action potential (AP). The concentration of venom 5.7 micrograms/ml decreased the amplitude of the peak tension, induced a contracture, reduced the amplitude of the plateau and shortened its duration as well as the repolarizing phase of the AP. The positive inotropic effect induced by the venom (2.9 micrograms/ml) on the contraction was inhibited dose-dependently by propranolol but was unchanged by the alpha-adrenergic antagonists urapidil and yohimbine, the adenyl cyclase activity remaining sensitive to forskolin. Venom, adrenalin and propranolol competed for a common site. Venom (2.9 micrograms/ml) increased both the Ca and the delayed outward K currents of enzymatically isolated atrial myocytes. The data suggest that the venom activates adrenoceptors, essentially beta-adrenoceptors.