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Ichthyotoxicity of soft coral metabolites
246
Toxins '89 Symposium
of the toxins isolated from marine organisms may well be synthesized by symbiotic algae or bacteria. The molecular mechanism of action of the exotoxin from Pseudomonas pseudomallei, which is responsible for the disease melioidosis in Asia and in tropical Australia, was shown by Dr RAHMAH MOHAMED (Bangi, Malaysia) to involve ADP-ribosylation of elongation factor II, similar to that observed with diphtheria toxin. The mode of action of the fungal metabolite gliotoxin and its immunomodulating properties were described by Dr PAUL WARING (Canberra) . Gliotoxin shows selective toxicity towards immune cells, inducing apoptosis. It may prove clinically useful in treating tissue for transplantation, eliminating the need for systemic immunosuppression. Dr HEGARTY (CSIRO, Brisbane) described a compound (castanospermine) isolated from a local black bean tree which has potential as an anti-AIDS drug. The medicinal use of natural toxins was further extended by Dr GEOFFREY PIETERSZ (University of Melbourne) who described conjugates of toxins with tumor-specific monoclonal antibodies for use in cancer therapy. The papers presented on snake venoms highlighted the difficulty in identifying the culprit snake-the major species of concern in Australia being the Rough Scaled snake and the Taipan . Dr ALAN WHITAKER (Princess Alexandra Hospital, Brisbane) described hemostatically active components of Australian snakes, some of which may be useful therapeutically in preventing blood clotting . In addition many other interesting papers and posters were presented. The objectives of the symposium, to bring together scientists from a number of disciplines to discuss the current state and future directions of toxin research in Australia were realised in a very successful meeting which fostered interdisciplinary collaboration . We hope to hold future symposia at regular intervals, on this topic . DIANE WATTERs MARTIN LAVIN
ABSTRACTS
Abstracts are arranged alphabetically by first author and only include those actually presented at the meeting and only those related to toxinology. Ichthyotoxicity ojsoft coral metabolites . PORFIRIO M . Auxo, BRUCe F . BOWDEN and JOttx C . COLI. (Department of Chemistry and Biochemistry, James Cook University, Townsville, 4811, Queensland, Australia) . T~ Toxictrv of the Zoanthids Palythoa toxica and Palythoa tuberculosa, which contains palytoxin (MOORS and Sct~tteR, 1971, and probably the most toxic compound known from the marine environment) has led to investigations of toxicity in the Alcyonacea . This interest has resulted in numerous reports of cytotoxicity, references to antifouling properties against filamentoue algae (COLL et al ., 1987), barnacles (K~rx et al., 1986), and diatoms (BANDt7RRAOA aIId FENICAL, 1985 ; T~ttcLrrr et al., 1985) as well as reports of insecticidal (CeRnrt,t.nat., 1986) and antimicrobial (CARneu.uve et al., 1987) properties . Ichthyotoxicity of test samples against Gambusia arms is a convenient bioassay which has been used in numerous studies by toxinologists . Ichthyotoxicity tests measure the toxicity of compounds (dissolved or suspended in water) which are presumably absorbed by the gill membranes of the fish . Previous work (LeBeRRE et al ., 1986) has shown that approximately 50% of ( > 300) soft coral samples examined were toxic to Gambusia afftreis . A number of species of soft corals have now been examined in detail and ranked according to their ichthyotoxicity under the previously reported (LABARRE et ol., 1986) conditions . An extract of each coral was fractionated to yield a lipophilic, and a water-soluble fraction, and the relative toxicities of those fractions were determined . Studies on a Sarcophton species revealed that the concentration of the major diterpenoid metabolite (which was ichthyotoxic) was significantly higher in the actual coral polyp than in the surrounding ccenenchyme tissue; this result may be significant for the coral in terms of protection against predation by Chaetodons (Pisces) which selectively browse on soft coral polyps. Solutions of some pure terpenoid compounds isolated from several species of soft corals were tested for ichthyotoxicity at various
Toxins '89 Symposium
247
wncentrations. Several of these compounds exhibited ichthyotoxicity at various concentrations in the 5-10 ppm range. REFERENCES Bnxnuwe~+ce, M. M. and Frnncnc, W. (1985) J. Chem . Ecol. 12, 951-987. CARDELLINA, I. H. (1986) Pwe and Appl. Ckem . 58, 365-374. CtienE,uNe, J. H., HErmtucxsox, R. L. and M~rrxEUy K. P. (1987) Tetrahedron Lett . 28, 727. Coc.c., J. C., PxrcE, I. R., Kox~o, G. M. and BownEx, B. F. (1987) Mar. Biol. 96, 129-135. KEn7Ex, P. A., RH1NEfiART, K. L. and HoorEa, I. R. (1986) J. Org. Chem. 51, 4450~454 . LnB~exE, S., COLL, J. C. and S~rrweco, P. W. (1986) Biol. Bull. 171, 565-574. MooaE, R. E. and ScHEUEa, P. J. (1971) Science 172, 495-500. Ttitcsrr, N. M., BISHOP, S. S., McCoxrrEt.~, O. J. and YonEe, J. A. (1985) J. Chem. Ecol. 9, 817-830. Effect of Catostylus mosaicus venom on erythrocytes. Non Aztt.n (Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia). THE SCYPHOZOA family has been known to be the main cause of concern for the beach-goers and fishermen since they cause envenomation which may result in fatal, systemic or local syndromes. The jellyfish, Catostylus mosoicus, most commonly found around the coastal waters of the Malay Peninsula and the eastern Australian coastline seem to exhibit seasonal variation in their stinging ability, being more noxious during their breeding season (H~rsTEnn, 1978) Extract from the tentacle of Catostylus mosaicus was shown to lyre erythrocytes, with different species exhibiting different susceptibility . Erythrocytes from rabbit were most susceptible followed by those of rat and then human. The haemolytic activity increased in a sigmoid manner with increasing concentrations of the crude extract protein . The extract is devoid of hydrolytic enzymes normally present in venoms except for phospholipase A activity . The phospholipase A present was capable of hydrolysing phospholipids from erythrocyte membrane with the concomitant appearance of their lyso-derivative and fatty acid . The haemolytic component was not extractable with solvent since precipitation with acetone gave a precipitated fraction that was devoid of haemolytic activity while that of the supernatant fraction showed a 24 fold increase in the haemolytic activity . Fractionation of the ft+eeze~ried supernatant fraction gave two peaks of haemolytic activity with peak I having the major portion of haemolytic activity. The haemolytic activity of the crude extract was quite stable to heat since heating the extract at 100°C for one hr only reduced its activity to approximately 60%. However, upon purification, there was a loss in the stability of the haemolytic component. REFERENCE HaisiFwn, B. W. (1978) Phylum coelenterata . In : Poisonous and Venomous Marine Animals of the World, pp . 87-140. Princeton, NJ, U.S.A.: Darwin Press. Some biological andpathological effects of tiger .make venom. E. A. BENNETT (Department of Medical Laboratory Science, Qucensland University of Technology, Brisbane, Australia) . TtoEx snake venom contains a factor V-dependent prothrombin activator, two presyaaptic neurotoxins (mol . wt ce 14,000), a homologous non-neurotoxic protein and two postsynaptic neurotoxins (mol . wt ca 8,000) . The presynaptic toxins display myotoxlc and phospholipase A~ activities, the latter being invoked to explain the other actions (Hwatus and MecDornvEt.L, 1981). The two presynaptic toxins, here referred to as Ntx-a and Ntx-c, and the homologous proton of tiger snake venom, Ntx-b, were purified by gel filtration and ion exchange chromatography (BErrxerr and CoouumE, 1987). Ntz-a and Ntx-c are identical with notezin and Notechis II-5 (K~ecssox et al., 1972). Ntx-a and Ntx-c were treated with N-bromosuoc~nimi de (NBS) at pH 4 with almost total loss of neurotoxicity. The effects of the inactivated Ntx's on liver and muscle were studied and compared with the active toxins . NBS also effectively removed the cytotoxic and myolytic effects. The liver response is a non-specific one with differences between Ntx-a and Ntx-b detectable mainly at the ultramicroscopic level. The damage is not clinically significant and reversible . The damage is insufficient to produce large biochemical changes at least in the early stages of intoxication . A suggestion that these effects could provide an animal model for Reye's syndrome has been suggested (Btit-NEA et al., 1985; BNt-Nrw, personal communication). The suggestion is to be examined further . The muscle response is quite characteristic with early loss of myofibrillar integrity. Release of myoglobin can lead to kidney failure. With low dose of toxins, regeneration of tissue occurs (Hnxx13 et al., 1975). The effect of
Toxins '89 Symposium
of the toxins isolated from marine organisms may well be synthesized by symbiotic algae or bacteria. The molecular mechanism of action of the exotoxin from Pseudomonas pseudomallei, which is responsible for the disease melioidosis in Asia and in tropical Australia, was shown by Dr RAHMAH MOHAMED (Bangi, Malaysia) to involve ADP-ribosylation of elongation factor II, similar to that observed with diphtheria toxin. The mode of action of the fungal metabolite gliotoxin and its immunomodulating properties were described by Dr PAUL WARING (Canberra) . Gliotoxin shows selective toxicity towards immune cells, inducing apoptosis. It may prove clinically useful in treating tissue for transplantation, eliminating the need for systemic immunosuppression. Dr HEGARTY (CSIRO, Brisbane) described a compound (castanospermine) isolated from a local black bean tree which has potential as an anti-AIDS drug. The medicinal use of natural toxins was further extended by Dr GEOFFREY PIETERSZ (University of Melbourne) who described conjugates of toxins with tumor-specific monoclonal antibodies for use in cancer therapy. The papers presented on snake venoms highlighted the difficulty in identifying the culprit snake-the major species of concern in Australia being the Rough Scaled snake and the Taipan . Dr ALAN WHITAKER (Princess Alexandra Hospital, Brisbane) described hemostatically active components of Australian snakes, some of which may be useful therapeutically in preventing blood clotting . In addition many other interesting papers and posters were presented. The objectives of the symposium, to bring together scientists from a number of disciplines to discuss the current state and future directions of toxin research in Australia were realised in a very successful meeting which fostered interdisciplinary collaboration . We hope to hold future symposia at regular intervals, on this topic . DIANE WATTERs MARTIN LAVIN
ABSTRACTS
Abstracts are arranged alphabetically by first author and only include those actually presented at the meeting and only those related to toxinology. Ichthyotoxicity ojsoft coral metabolites . PORFIRIO M . Auxo, BRUCe F . BOWDEN and JOttx C . COLI. (Department of Chemistry and Biochemistry, James Cook University, Townsville, 4811, Queensland, Australia) . T~ Toxictrv of the Zoanthids Palythoa toxica and Palythoa tuberculosa, which contains palytoxin (MOORS and Sct~tteR, 1971, and probably the most toxic compound known from the marine environment) has led to investigations of toxicity in the Alcyonacea . This interest has resulted in numerous reports of cytotoxicity, references to antifouling properties against filamentoue algae (COLL et al ., 1987), barnacles (K~rx et al., 1986), and diatoms (BANDt7RRAOA aIId FENICAL, 1985 ; T~ttcLrrr et al., 1985) as well as reports of insecticidal (CeRnrt,t.nat., 1986) and antimicrobial (CARneu.uve et al., 1987) properties . Ichthyotoxicity of test samples against Gambusia arms is a convenient bioassay which has been used in numerous studies by toxinologists . Ichthyotoxicity tests measure the toxicity of compounds (dissolved or suspended in water) which are presumably absorbed by the gill membranes of the fish . Previous work (LeBeRRE et al ., 1986) has shown that approximately 50% of ( > 300) soft coral samples examined were toxic to Gambusia afftreis . A number of species of soft corals have now been examined in detail and ranked according to their ichthyotoxicity under the previously reported (LABARRE et ol., 1986) conditions . An extract of each coral was fractionated to yield a lipophilic, and a water-soluble fraction, and the relative toxicities of those fractions were determined . Studies on a Sarcophton species revealed that the concentration of the major diterpenoid metabolite (which was ichthyotoxic) was significantly higher in the actual coral polyp than in the surrounding ccenenchyme tissue; this result may be significant for the coral in terms of protection against predation by Chaetodons (Pisces) which selectively browse on soft coral polyps. Solutions of some pure terpenoid compounds isolated from several species of soft corals were tested for ichthyotoxicity at various
Toxins '89 Symposium
247
wncentrations. Several of these compounds exhibited ichthyotoxicity at various concentrations in the 5-10 ppm range. REFERENCES Bnxnuwe~+ce, M. M. and Frnncnc, W. (1985) J. Chem . Ecol. 12, 951-987. CARDELLINA, I. H. (1986) Pwe and Appl. Ckem . 58, 365-374. CtienE,uNe, J. H., HErmtucxsox, R. L. and M~rrxEUy K. P. (1987) Tetrahedron Lett . 28, 727. Coc.c., J. C., PxrcE, I. R., Kox~o, G. M. and BownEx, B. F. (1987) Mar. Biol. 96, 129-135. KEn7Ex, P. A., RH1NEfiART, K. L. and HoorEa, I. R. (1986) J. Org. Chem. 51, 4450~454 . LnB~exE, S., COLL, J. C. and S~rrweco, P. W. (1986) Biol. Bull. 171, 565-574. MooaE, R. E. and ScHEUEa, P. J. (1971) Science 172, 495-500. Ttitcsrr, N. M., BISHOP, S. S., McCoxrrEt.~, O. J. and YonEe, J. A. (1985) J. Chem. Ecol. 9, 817-830. Effect of Catostylus mosaicus venom on erythrocytes. Non Aztt.n (Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia). THE SCYPHOZOA family has been known to be the main cause of concern for the beach-goers and fishermen since they cause envenomation which may result in fatal, systemic or local syndromes. The jellyfish, Catostylus mosoicus, most commonly found around the coastal waters of the Malay Peninsula and the eastern Australian coastline seem to exhibit seasonal variation in their stinging ability, being more noxious during their breeding season (H~rsTEnn, 1978) Extract from the tentacle of Catostylus mosaicus was shown to lyre erythrocytes, with different species exhibiting different susceptibility . Erythrocytes from rabbit were most susceptible followed by those of rat and then human. The haemolytic activity increased in a sigmoid manner with increasing concentrations of the crude extract protein . The extract is devoid of hydrolytic enzymes normally present in venoms except for phospholipase A activity . The phospholipase A present was capable of hydrolysing phospholipids from erythrocyte membrane with the concomitant appearance of their lyso-derivative and fatty acid . The haemolytic component was not extractable with solvent since precipitation with acetone gave a precipitated fraction that was devoid of haemolytic activity while that of the supernatant fraction showed a 24 fold increase in the haemolytic activity . Fractionation of the ft+eeze~ried supernatant fraction gave two peaks of haemolytic activity with peak I having the major portion of haemolytic activity. The haemolytic activity of the crude extract was quite stable to heat since heating the extract at 100°C for one hr only reduced its activity to approximately 60%. However, upon purification, there was a loss in the stability of the haemolytic component. REFERENCE HaisiFwn, B. W. (1978) Phylum coelenterata . In : Poisonous and Venomous Marine Animals of the World, pp . 87-140. Princeton, NJ, U.S.A.: Darwin Press. Some biological andpathological effects of tiger .make venom. E. A. BENNETT (Department of Medical Laboratory Science, Qucensland University of Technology, Brisbane, Australia) . TtoEx snake venom contains a factor V-dependent prothrombin activator, two presyaaptic neurotoxins (mol . wt ce 14,000), a homologous non-neurotoxic protein and two postsynaptic neurotoxins (mol . wt ca 8,000) . The presynaptic toxins display myotoxlc and phospholipase A~ activities, the latter being invoked to explain the other actions (Hwatus and MecDornvEt.L, 1981). The two presynaptic toxins, here referred to as Ntx-a and Ntx-c, and the homologous proton of tiger snake venom, Ntx-b, were purified by gel filtration and ion exchange chromatography (BErrxerr and CoouumE, 1987). Ntz-a and Ntx-c are identical with notezin and Notechis II-5 (K~ecssox et al., 1972). Ntx-a and Ntx-c were treated with N-bromosuoc~nimi de (NBS) at pH 4 with almost total loss of neurotoxicity. The effects of the inactivated Ntx's on liver and muscle were studied and compared with the active toxins . NBS also effectively removed the cytotoxic and myolytic effects. The liver response is a non-specific one with differences between Ntx-a and Ntx-b detectable mainly at the ultramicroscopic level. The damage is not clinically significant and reversible . The damage is insufficient to produce large biochemical changes at least in the early stages of intoxication . A suggestion that these effects could provide an animal model for Reye's syndrome has been suggested (Btit-NEA et al., 1985; BNt-Nrw, personal communication). The suggestion is to be examined further . The muscle response is quite characteristic with early loss of myofibrillar integrity. Release of myoglobin can lead to kidney failure. With low dose of toxins, regeneration of tissue occurs (Hnxx13 et al., 1975). The effect of