Stonefish antivenom neutralises the inflammatory and cardiovascular effects induced by scorpionfish Scorpaena plumieri venom.

Venomous fish are often involved in human accidents and symptoms of envenomation include local (intense pain and swelling) and systemic effects (cardiovascular and neurological disorders). However the only commercially available antivenom is against the Indo-Pacific stonefish Synanceja trachynisStonefish Antivenom (SFAV). The aim of the present study was to evaluate the potential of SFAV in neutralising the in vivo effects of some toxic activities of scorpionfish Scorpaena plumieri venom (SpV), and the in vitro immuno cross-reactivity. The SpV (7.5-100 µg/animal) caused nociceptive and dose-dependent edematogenic responses in the mice footpad. In rats SpV (300 µg/kg, i.v.) produced immediate and transient increase in arterial blood pressure and decrease in heart rate. Prior incubation of SpV with SFAV (1 µg SpV/1 U SFAV) abolished the inflammatory response, and significantly attenuated the cardiovascular effects induced by SPV. Western blotting analysis on two-dimensional SDS-PAGE of S plumieri venom proteins using SFAV proved that the epitopes recognized by SFAV are shared with the ∼98 kDa proteins. This is the first report of venom similarities between Indo-Pacific and Atlantic venomous fish, suggesting that the SpV compound responsible for inflammatory and cardiovascular effects possesses similar biochemical and antigenic properties to those found in stonefish venom.

Cardiovascular effects of scorpionfish (Scorpaena plumieri) venom.

The aim of the present study was to investigate the cardiovascular activity of Scorpaena plumieri venom in both in vivo and in vitro models. In anesthetized rats, doses of the venom (14-216 microg protein/kg) induced a transient increase in the mean arterial pressure. However at higher dose (338 microg protein/kg) this effect was followed by a sudden hypotension and the animal evolved to death. The heart rate was temporarily increased and followed by bradycardia using doses > or =108 microg/kg. In isolated rat hearts the crude venom (5-80 microg protein) produced dose-dependent positive ventricular chronotropic, inotropic, lusitropic and coronary vasoconstriction responses. Partial purification of an active fraction (CF, cardiovascular fraction) which reproduced the cardiovascular effects induced by crude venom on isolated hearts was achieved by conventional gel filtration chromatography. Adrenergic blockades, prazosin and propranolol, significantly attenuated these responses. The coronary vasoconstriction response to CF was also attenuated by chemical endothelium denudation. In conclusion, the data showed that S. plumieri fish venom induces disorders in the cardiovascular system. It also suggests that alpha(1) and beta-adrenergic receptors, and the vascular endothelium, are involved at least partially, in these cardiac effects.

Antivenom action on renal effects induced by Thalassophryne nattereri venom

Thalassophryne nattereri (niquim) is a venomous fish responsible for numerous accidents involving fishermen in northern and northeastern Brazil. The aim of the present investigation was to evaluate the action of antivenom on renal effects caused by Thalassophryne nattereri venom. Isolated kidneys of Wistar rats were perfused with a previously dialyzed Krebs-Henseleit solution containing 6 g% bovine serum albumin. The antivenom action was studied through perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). The niquim venom (1 miug/mL), the antivenom alone (1 miug/mL) or the venom incubated with antivenom were added to the system 30 minutes after the beginning of each perfusion. Previous works have shown venom induced-alterations of renal function parameters. In the isolated rat Kidney, T. nattereri venom (1 miug/mL) increased the perfusion pressure and renal vascular resistance at 60, 90 and 120 minutes. UF and GFR also increased at 60, 90 and 120 minutes when compared with the control group; however, no effects were observed on the percent of sodium (% TNa more control equal 81.1 more or less 0.86; % TNa more 60 equal 78.04 more or less 1.18; % TNa more 90 equal -5.16 more or less 3.34; %TNa more 120 equal 79.49 more or less 0.87) and potassium (%TKcontrol equal 72.29 more or less 1.12; %TK more 60 equal 75.41 more or less 0.65; % TK more 90 equal 71.23 more or less 2.55; % TK more 120 equal 76.62 more or less 1.04) tubular transporto. The administration of the antivenom (1 miug/mL) incubated with venom (1 miug/mL) reduced the changes in PP, RVR, UF and GFR provoked by Thalassophryne nattereri venom. The group perfused with venom alone showed a moderate deposit of a proteinaceous material in the tubules and urinary space.(...)

Neutralizing antibodies obtained in a persistent immune response are effective against deleterious effects induced by the Thalassophryne nattereri fish venom.

Thalassophryne nattereri envenoming represents a great cost to North and Northeast Brazilian communities in terms of public healths, leisure and tourism. Victims rapidally develop symptoms as pain, local swelling, erythema followed by intense necrosis that persist for long days. The aim of this work was tested the immune competence of neutralizing antibodies in pre-immunized mice against principal toxic activities induced by venom. During the primary antibody response in mice, an elevation of IgG antibody levels was only observed on day 28. After boosting, high antibody levels were detected between days 49 and 70, with a 12-fold increase in IgG level over control values at day 49. We confirmed the in vitro neutralizing capacity of T. nattereri anti-venom against toxic effects and thereafter we show that neutralizing antibodies obtained in a persistent immune response are more effective, inclusive against edematous reaction. After boosting during the secondary response mice with high antibody levels do not present any alterations in venule or arteriole after topical application of venom on cremaster muscle. In addition, CK activity diminished in these mice with high neutralizing antibody levels corroborating the attenuation of the myonecrotic effect by venom. In addition, we determined the presence of high IgG antibodies levels in patients 6 months after injury by T. nattereri. In conclusion, the presence of neutralizing antibodies against to T. nattereri venom in the serum of pre-immunized mice could change the outcome of lesion at site of posterior envenoming. Antigen-specific antibodies of high affinity in consequence to specific immune response, dependent of T lymphocyte activation, could minimize the symptoms of intense and immediate inflammatory reaction caused by T. nattereri venom. These finding prompt us to the possibility of development of immune therapeutic strategies using specific anti-venom as an efficient intervention for protecting human victims.

Marine antivenoms.

There is an enormous diversity and complexity of venoms and poisons in marine animals. Fatalities have occurred from envenoming by sea snakes, jellyfish, venomous fish such as stonefish, cone snails, and blue-ringed octopus. Deaths have also followed ingestion of toxins in shellfish, puffer fish (Fugu), and ciguatoxin-containing fish. However antivenoms are generally only available for envenoming by certain sea snakes, the major Australian box jellyfish (Chironex fleckeri) and stonefish. There have been difficulties in characterizing the toxins of C. fleckeri venom, and there are conflicting animals studies on the efficacy of C. fleckeri antivenom. The vast majority of C. fleckeri stings are not life-threatening, with painful skin welts the major finding. However fatalities that do occur usually do so within 5 to 20 minutes of the sting. This unprecedented rapid onset of cardiotoxicity in clinical envenoming suggests that antivenom may need to be given very early (within minutes) and possibly in large doses if a life is to be saved. Forty years of anecdotal experience supports the beneficial effect of stonefish antivenom in relieving the excruciating pain after stonefish spine penetration. It remains uncertain whether stonefish antivenom is efficacious in stings from spines of other venomous fish, and the recommendation of giving the antivenom intramuscularly needs reassessment.

Stonefish (Synanceia spp.) antivenom neutralises the in vitro and in vivo cardiovascular activity of soldierfish (Gymnapistes marmoratus) venom.

The soldierfish (Gymnapistes marmoratus), which is related to the stonefish (Synanceia spp.), inhabits the western, southern and lower eastern coastlines of Australia. We have previously found that G. marmoratus venom possesses pharmacological activity similar to Synanceia trachynis venom (Hopkins, B.J., Hodgson, W.C., 1998. Cardiovascular studies on venom from the soldierfish (Gymnapistes marmoratus). Toxicon 36, 973-872; Church, J.E., Hodgson, W.C., 2000. Dose-dependent cardiovascular and neuromuscular effects of stonefish (Synanceja trachynis) venom. Toxicon 38, 391-407), namely an action at muscarinic receptors and adrenoceptors. The aim of this study was to determine the effectiveness of Synanceia antivenom in neutralising the in vitro and in vivo cardiovascular activity of G. marmoratus venom. Venom extract (0.4-12 microg protein/ml) caused dose- and endothelium-dependent relaxation in porcine U46619-precontracted coronary arteries. This relaxation was abolished by 10 min prior exposure of the tissue to Synanceia antivenom (3 units/ml). In rat paced (5 ms, 2 Hz, 7-12 V) left atria, G. marmoratus venom extract (40 microg protein/ml) produced a transient negative, followed by a sustained positive inotropic response. In spontaneously beating right atria, venom extract (40 microg protein/ml) produced similar changes in rate. Prior incubation of venom extract with Synanceia antivenom (1 unit/4 microg venom extract protein, 10 min) significantly attenuated both components of the inotropic response, and abolished the positive chronotropic response. In the anaesthetised rat, venom extract (400 microg protein/kg, i.v.) produced a transient depressor response, followed by a more sustained pressor response. Prior incubation of venom extract with Synanceia antivenom (1 unit/4 microg venom extract protein, 10 min) significantly attenuated both components of the in vivo response. As Synanceia antivenom neutralised the cardiovascular activity of G. marmoratus venom both in vitro and in vivo, we suggest that the venoms of the two species may share a similar component(s).

Neutralization of Thalassophryne nattereri (niquim) fish venom by an experimental antivenom.

T. nattereri (niquim) is a venomous fish involved in many human accidents in Brazil. The clinical picture includes mild local erythema, severe edema, intense pain and rapid progression to necrosis. The present therapy with anti-inflammatory and analgesic drugs is ineffective and, therefore, we decided to assess serum therapy as an alternative treatment using an experimental antivenom. The antivenom used was raised in rabbits showing an ELISA antibody titer of 1:8,192,000 and its ability to neutralize lethality, necrosis, nociception and edema was evaluated both by pre-incubating the venom with antivenom before injection into mice or by independent injections of venom and antivenom. Lethality was completely neutralized by pre-incubation (ED(50)=141.5 microl/mg) while necrosis and nociception were neutralized by pre-incubation or the independent injection of antivenom. Edema was only partially prevented even when large amounts of antivenom were used. These data suggest that antivenom may be a promising treatment for patients stung by T. nattereri and suggest the viability of producing a horse antivenom for use in clinical trials.

Dose-dependent cardiovascular and neuromuscular effects of stonefish (Synanceja trachynis) venom.

There has been recent debate regarding the labile nature of stonefish venoms and the pharmacology of their breakdown products. The present study examined the cardiovascular and neuromuscular effects of lyophilised venom, and conducted a preliminary investigation of freshly milked venom. Lyophilised venom (20 microg/ml) caused endothelium-dependent relaxation in rat aortae that was abolished by atropine (0.1 microM). In contrast, an endothelium-independent contractile response occurred in porcine coronary arteries. However, in the presence of atropine (10 nM), this became a relaxation response which was attenuated by the B2 antagonist FR-173657 (0.1 microM) or by a combination of idazoxan (1 microM) and propranolol (1 microM). In rat isolated atria, lyophilised venom (4 microg/ml) caused a biphasic inotropic response consisting of an initial decrease, and then increase, in force which were attenuated by atropine (0.5 microM) and propranolol (5 microM), respectively. The increase in force produced by venom was unaffected by reserpine pre-treatment suggesting a direct action at adrenoceptors. In the anaesthetised rat, lyophilised venom (1-300 microg/kg, i.v.), caused a dose-dependent depressor response, with a subsequent pressor response at higher concentrations (30-300 microg/kg, i.v.). In the presence of atropine (1 mg/kg, i.v.), the depressor response to venom was abolished, a transient pressor response unmasked and the secondary pressor response augmented. In the additional presence of prazosin (50 microg/kg, i.v.), the transient pressor response was abolished and the secondary pressor response attenuated. Lyophilised venom had no significant effect on nerve-evoked (10 microg/ml) or directly-evoked (100 microg/ml) twitches of the chick biventer cervicis muscle preparation. Milked venom (1 microl/ml) caused a biphasic response (i.e., an initial relaxation followed by contraction) in rat aortae, a contraction in porcine coronary arteries, complete cessation of rat isolated atrial activity and markedly inhibited both nerve-evoked and directly-evoked twitches of the chick biventer cervicis muscle preparation. In the anaesthetised rat, milked venom (15 microl/kg, i.v.) caused immediate cardiovascular collapse. It appears that the cardiovascular effects of stonefish venom are mediated by a dose-dependent action at muscarinic receptors and adrenoceptors.

Thalassophryne nattereri fish venom: biological and biochemical characterization and serum neutralization of its toxic activities.

Envenomation by Thalassophryne nattereri fishes are an important medical problem in northeast of Brazil, causing in human victims considerable pain and edema followed by necrosis. Venom obtained from fresh captured specimens of this fish was tested in vitro or in animal models for a better characterization of its toxic activities. Intradermal injection of the venom in the foot pad of mice induced local edema and hemorrhage followed a few hours later by necrosis. Subcutaneous injection of the venom induced systemic effects consisting in jerking motions, paralysis of hind limbs, erection of hair, rotational movements and violent convulsions followed by death. Dead animals showed hyperemia of the small intestine and lungs. The venom showed distinct edematous, necrotizing and hemolytic activities, a low level of hemorrhagic, myotoxic and proteolytic activities and no detectable phospholipase A2 activity. SDS-PAGE analysis of the crude venom showed at least 17 components with the major band located around Mw = 19,000. Almost all proteins stained by amido black were also revealed by Western blotting with antibodies to T. nattereri venom. Fractionation of the venom by either gel filtration or cation exchange chromatography resulted in a few distinct peaks but in both situations the biological activities were located in only one of the peaks which corresponded to basic proteins with approximately Mw = 47,000. Heating of the venom at 56 degrees C for 60 min completely destroyed its biological activities. All venom toxic activities except edema were completely neutralized after in vitro incubation with anti-T. nattereri serum.

Ocorrência de acidentes provocados por peixes peçonhentos / The occurrence of accidents caused by poisonous fish species

O acidente provocado por peixes peçonhentos ou acantóxicos denomina-se ictismo. Peixe peçonhento ou acantóxico é aquele que possui glândulas epidérmicas de veneno associadas com esporões em nanadeiras e cauda. Há poucos estudos sobre os casos de ictismo no Brasil, embora existam aproximadamente 130 espécies de peixes que provocam acidentes entre os pescadores, mergulhadores e banhitas...

Antivenom use in Australia. Premedication, adverse reactions and the use of venom detection kits.

OBJECTIVES: To analyse reports of antivenom use and sequelae in Australia from July 1 1989 to June 30 1990. The value of snake venom detection kits (VDKs) was also analysed. METHODS: Information was obtained from antivenom usage reports returned to the Commonwealth Serum Laboratories and from personal letters sent to those reporting doctors. Information on VDKs was obtained from antivenom usage reports or from questionnaires packaged with VDKs. RESULTS: Reported antivenoms used were: red-back spider, 258 cases; funnel-web spider, 3 cases; stonefish, 26 cases; box jellyfish, 6 cases; snake, 91 cases. Immediate reactions followed administration of red-back spider antivenom in only two patients and snake antivenoms in four patients. Delayed reactions (serum sickness) followed use of red-back spider antivenom in three patients, stonefish antivenom in two, and snake antivenoms in three. No reaction was life-threatening. Premedication was used in the majority of red-back spider bites and in 66 of 86 snake bites. Oral corticosteroids were given prophylactically after some uses of snake antivenom to prevent serum sickness. VDKs were used in 181 cases of snake bite and were reported as being useful in selecting appropriate snake antivenom in 31% of cases of antivenom use. (Only 10 of these 181 were also reported on the antivenom usage report.) Appropriate first aid was given in 61% of cases. There were 50% fewer snake bites reported than 10 years ago. CONCLUSIONS: Antivenoms in Australia are well tolerated with few immediate or delayed reactions. The use of premedication and prophylactic oral corticosteroids for four to five days after antivenom administration may be responsible for this low reaction rate. VDK results help select the appropriate antivenom; however, in some cases positive results were obtained from urine samples from patients with no symptoms of envenomation.

Pardaxin-stimulated calcium uptake in PC12 cells is blocked by cadmium and is not mediated by L-type calcium channels.

Pardaxin is an excitatory neurotoxin which triggers neurotransmitter release as a result of voltage-dependent pore formation within the neuronal membrane. We have used several pharmacological manipulations of calcium influx to characterize pardaxin pore activity in PC12 cells in culture. Pardaxin stimulates the uptake of radioactive calcium into PC12 cells in a dose dependent fashion (ED50 of 0.4 microM). This stimulation is partially inhibited by nifedipine, a blocker of L-type calcium channels. Effective blockade of pardaxin stimulation was produced by the inorganic calcium channel blockers cadmium (IC50 of 10 microM) and nickel (2 mM). Homologous down regulation of L-calcium channels by the agonist Bay K-8644, inhibited the subsequent stimulation of calcium uptake by this drug, but not by pardaxin. A fluorometric analysis of pardaxin pore formation in unilamellar large liposomes indicates pardaxin pores are blocked by cadmium (10-200 microM). These data distinguish between pardaxin pores and L-type calcium channels in PC12 cells. We suggest pardaxin as a pharmacological ionophore tool to modulate neuronal calcium homeostasis and neurotransmitter release.

Vasoconstrictor components in the Arabian Gulf catfish (Arius thalassinus, Ruppell) proteinaceous skin secretion.

The Arabian Gulf catfish (Arius thalassinus, Ruppell) produces toxic substances from its skin and from venom glands located near the base of the pectoral fins. Investigation of the pharmacological properties of the skin toxin have previously shown cholinergic vasoconstrictor activity in umbilical arteries. Cholinergic vasoconstriction was confirmed in sheep renal arteries. This activity was partially blocked by atropine, while most of the residual contraction was eliminated by simultaneous addition of indomethacin. Skin toxin treatment of arterial specimens caused a release of prostaglandin (PGE2, TXB2 and 6-keto-PGF1 alpha) into the organ bath. Prostaglandin release was blocked by pretreatment with indomethacin. Heat denaturation of skin toxin caused a loss of only the indomethacin-sensitive muscle contraction activity; most of the residual activity was blocked by atropine.