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Some biological and pathological effects of tiger snake venom
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
248
Toxins '89 Symposium
NBS illustrates a key role for tryptophan in the action of the neumtoxins . The cytolytic tissue responses are consistent with a role for the venom in prey digestion. REFERENCES BNt-Nra, L., NEUa~xN, M. G., Esctux, J. and ISHAY, J. S. (1985) Liver 5, 326. BENNrrIT, E. A. and Coop, I. F. (1987) Progress in Venom and Toxin Research : First Asia-Pacific Congress on Animal, Plant and Microbial Toxins, Singapore pp, 2619 . Hwxxts, J. B., Jofnvsox, M. A. and K~tet.ssoN, E. (1975) Clin. Exp . Pharm . Physiol. 2, 383. Htiuus, J. B. Snd MACDONELL, C. A. (1981) Toxicon 19, 419. Ktitt.ssox, E., E~>~e, D. and RYDEN, S. (1972) Toxicon 10, 405. Ciguatera toxin. Mtc~l. F. CAPRA (Department of Public Health and Nutrition, Queensland University of Technology, GPO Box 2434, Brisbane 4001, Australia) .
CIGUATOXIN (CI'X) is a powerful non-protein toxin (mow 0.45 itg/kg, mouse) believed to be produced by the epiphytic dinoflagellate, Gambierdiscus toxicus . There is as yet no full structural elucidation of the CTX although the mol. wt is known (1,111 Daltons) and molecular formulae of C33HnNO u and CxH,sO have been suggested. CTX is known to be a polycyclic ether compound with structural similarities to other marine natural products such as okadaic acid. Variations in human and animal symptoms after exposure to CTX have led a number of workers to suggest the occurrence of multiple ciguatera toxins . CTX enters the food chain via browsing and herbivorous fish to move through a hierarchy of carnivores becoming concentrated in higher carnivores such as barracuda, mackerel and coral trout. Consumption of these species by humans can lead to a severe form of food poisoning . Human ciguatera poisoning in Queensland has been estimated from epidemiological surveys in Maryborough-Hervey Bay and t sirns where the respective annual incidences were found to be 24/100,000 and 34/100,000 . Occasionally many people are poisoned by a few fish captured from a toxic area . In 1987, 63 people were poisoned in Sydney, after eating Spanish Mackerel that originated in Hervey Bay, Queensland . The type and duration of the Symptoms exhibited by the Sydney victims will be presented . The results of nerve conduction studies on some of the Sydney victims will also be presented. Significant changes in the supernormal period, refractory periods and conduction velocity were noted in human victims of CfX. CTX appears to exert its effects on animals by altering the movement of Na* across excitable membranes. CTX alters a number of nerve conduction parameters in both mammals and fish . Fish appear to have mechanisms which prevent CTX reaching the target sites on their nerves. Any elucidation of such mechanisms may hold the key to a more effective treatment of human ciguatera poisoning. Resiniferatoxin (RTXJ : a novel and extremely potent capsaicnoid from plants of the genus Euphorbia. D. J. oe VRIB4' and P. M. BLU~ERG (Molecular Mechanisms of Tumor Promotion Section National Cancer Institute, Bethesda, MD, U.S .A ., and 'Present Address: Toxicology Key Centre, R.M .LT., Melbourne, Victoria, Australia) .
Ptruvrs from the family Euphorbiaceae have provided the valuable biochemical tools, the phorbol esters, which through their action at protein kinase C have potent tumor promoting properties . These compounds are also irritants, and their isolation from natural sources has been guided by the activity of organic extracts of the plant in a mouse ear-reddening assay. A related diterpene, resiniferatoxin (RTX) was also identified in Euphorbia resinifera, E. Poissonü and E. unispina (EVerrs et al., 1976 ; HIItGEt~urnv et al., 1975). RTX was remarkable for its potency in the irritanty assay with a 50% effective dose of 1.6 x 10 - " moles per ear. RTX is irritant acutely while phorbol esters have a delayed onset. In addition, RTX shares none of the characteristic phorbol eater effects indicating it possesses a discrete mechanism of action . Capsaicin, the irritant component in fruit of various species of Capsicum shares with RTX a 4-hydroxy-3methoxyphenyl substituent. One well documented action of capsaicin is to produce a dramatic fall in body temperature . To further explore possible homology between these two classes of irritant compounds, their thermoregulatory actions were compared (DE Vten?s and BL1J~(BERG 1989). On s.c. injection into mice, both agents produced a decrease in body temperature of 8°C. The 50% effective dose for RTX was 3 ug/kg while that for capsaicin was 7 mg/kg. Tolerance to the hypothermic effects of both compounds readily developed and crosstolerance between the compounds was observed. The extreme potency of RTX at the capsaicin pharmacophore indicates it will provide a useful tool in understanding this site. REFERENCES ne Vx¢~s, D. J. SIId BLCTr~ERG, P. M. (1989) Lrfe Sci . 44, 711-715. Ev~ivs, F. J., and Sctn~r, R. J. (1976) Phytochemistry 15, 333--335 . Heaanvx~tix, M., AnoLrta, W. and HF,cICm, E. (1975) Tetrahedron Lett. 19, 1595-1598 .
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
248
Toxins '89 Symposium
NBS illustrates a key role for tryptophan in the action of the neumtoxins . The cytolytic tissue responses are consistent with a role for the venom in prey digestion. REFERENCES BNt-Nra, L., NEUa~xN, M. G., Esctux, J. and ISHAY, J. S. (1985) Liver 5, 326. BENNrrIT, E. A. and Coop, I. F. (1987) Progress in Venom and Toxin Research : First Asia-Pacific Congress on Animal, Plant and Microbial Toxins, Singapore pp, 2619 . Hwxxts, J. B., Jofnvsox, M. A. and K~tet.ssoN, E. (1975) Clin. Exp . Pharm . Physiol. 2, 383. Htiuus, J. B. Snd MACDONELL, C. A. (1981) Toxicon 19, 419. Ktitt.ssox, E., E~>~e, D. and RYDEN, S. (1972) Toxicon 10, 405. Ciguatera toxin. Mtc~l. F. CAPRA (Department of Public Health and Nutrition, Queensland University of Technology, GPO Box 2434, Brisbane 4001, Australia) .
CIGUATOXIN (CI'X) is a powerful non-protein toxin (mow 0.45 itg/kg, mouse) believed to be produced by the epiphytic dinoflagellate, Gambierdiscus toxicus . There is as yet no full structural elucidation of the CTX although the mol. wt is known (1,111 Daltons) and molecular formulae of C33HnNO u and CxH,sO have been suggested. CTX is known to be a polycyclic ether compound with structural similarities to other marine natural products such as okadaic acid. Variations in human and animal symptoms after exposure to CTX have led a number of workers to suggest the occurrence of multiple ciguatera toxins . CTX enters the food chain via browsing and herbivorous fish to move through a hierarchy of carnivores becoming concentrated in higher carnivores such as barracuda, mackerel and coral trout. Consumption of these species by humans can lead to a severe form of food poisoning . Human ciguatera poisoning in Queensland has been estimated from epidemiological surveys in Maryborough-Hervey Bay and t sirns where the respective annual incidences were found to be 24/100,000 and 34/100,000 . Occasionally many people are poisoned by a few fish captured from a toxic area . In 1987, 63 people were poisoned in Sydney, after eating Spanish Mackerel that originated in Hervey Bay, Queensland . The type and duration of the Symptoms exhibited by the Sydney victims will be presented . The results of nerve conduction studies on some of the Sydney victims will also be presented. Significant changes in the supernormal period, refractory periods and conduction velocity were noted in human victims of CfX. CTX appears to exert its effects on animals by altering the movement of Na* across excitable membranes. CTX alters a number of nerve conduction parameters in both mammals and fish . Fish appear to have mechanisms which prevent CTX reaching the target sites on their nerves. Any elucidation of such mechanisms may hold the key to a more effective treatment of human ciguatera poisoning. Resiniferatoxin (RTXJ : a novel and extremely potent capsaicnoid from plants of the genus Euphorbia. D. J. oe VRIB4' and P. M. BLU~ERG (Molecular Mechanisms of Tumor Promotion Section National Cancer Institute, Bethesda, MD, U.S .A ., and 'Present Address: Toxicology Key Centre, R.M .LT., Melbourne, Victoria, Australia) .
Ptruvrs from the family Euphorbiaceae have provided the valuable biochemical tools, the phorbol esters, which through their action at protein kinase C have potent tumor promoting properties . These compounds are also irritants, and their isolation from natural sources has been guided by the activity of organic extracts of the plant in a mouse ear-reddening assay. A related diterpene, resiniferatoxin (RTX) was also identified in Euphorbia resinifera, E. Poissonü and E. unispina (EVerrs et al., 1976 ; HIItGEt~urnv et al., 1975). RTX was remarkable for its potency in the irritanty assay with a 50% effective dose of 1.6 x 10 - " moles per ear. RTX is irritant acutely while phorbol esters have a delayed onset. In addition, RTX shares none of the characteristic phorbol eater effects indicating it possesses a discrete mechanism of action . Capsaicin, the irritant component in fruit of various species of Capsicum shares with RTX a 4-hydroxy-3methoxyphenyl substituent. One well documented action of capsaicin is to produce a dramatic fall in body temperature . To further explore possible homology between these two classes of irritant compounds, their thermoregulatory actions were compared (DE Vten?s and BL1J~(BERG 1989). On s.c. injection into mice, both agents produced a decrease in body temperature of 8°C. The 50% effective dose for RTX was 3 ug/kg while that for capsaicin was 7 mg/kg. Tolerance to the hypothermic effects of both compounds readily developed and crosstolerance between the compounds was observed. The extreme potency of RTX at the capsaicin pharmacophore indicates it will provide a useful tool in understanding this site. REFERENCES ne Vx¢~s, D. J. SIId BLCTr~ERG, P. M. (1989) Lrfe Sci . 44, 711-715. Ev~ivs, F. J., and Sctn~r, R. J. (1976) Phytochemistry 15, 333--335 . Heaanvx~tix, M., AnoLrta, W. and HF,cICm, E. (1975) Tetrahedron Lett. 19, 1595-1598 .