Mode of Action of Ciguatera Toxins - ACS Symposium Series (ACS

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Mode

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A. M. LEGRAND and R. BAGNIS

Downloaded by UNIV OF MINNESOTA on September 4, 2016 | http://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch020

Institut Territorial de RecherchesMédicalesLouisMalardé,Papeete, Tahiti, French Polynesia Ciguatera fish poisoning involves one principal toxin, named ciguatoxin (1) and several secondary toxins including maitotoxin (2) and scaritoxin (3), which frequently coexist with ciguatoxin in marine organisms. A l l these toxins have a strong lethal potency on mice (4-8). Many pharmacological studies concern ciguatoxin, the chief pathogenic compound. Scarcer are the data about scaritoxin, a toxin specific of parrot fishes, while the mode of action of maitotoxin is actually the purpose of a deep research. This review summarizes the present knowledge. Ciguatoxin The first report on the pharmacological action of ciguatoxin was by Li (9), he observed in the anaesthetized rat a biphasic cardiovascular response with bradycardia and hypotension followed by tachycardia and hypertension. These biphasic effects resembled those of typical anticholinesterase poisons. In in vitro experiments with rabbit intestinal segments, L i observed that ciguatoxin extracts produced a marked inhibition of human and bovine erythrocyte cholinesterases. He concluded that the pharmacological action of ciguatoxin was due to cholinesterase inhibition. The intraperitoneal acute toxicity of several ciguatoxic extracts was found to be closely correlated with their in vitro anticholinesterase activity (10). A similar correlation was established between this in vitro anticholinesterase activity and pupillary miosis triggered by topical application in rabbits'eyes (11). Yet, later studies showed that the pharmacological action of ciguatoxin cannot result from cholinesterase inhibition. Indeed, Ogura (12) reported a non-competitive antagonism between neuromuscular action of toxic extracts and acetylcholine. The neuromuscular block induced by ciguatoxin in the rat sciatic nerve-gastrocnemius muscle preparation in vitro was antagonized by the anticholinesterase physostigmine (13). Rayner at al (14) have commented on the differences between the respiratory action of ciguatoxin and typical anticholinesterase effects. Ogura et al (15) have not observed with ciguatoxin extracts the typical electroencephalographic activation of anticholinesterase agent while they had obtained the 0097-6156/ 84/ 0262-0217506.00/ 0 © 1984 American Chemical Society

Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.


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effect with physostigmine. Further investigation of the in viXSiO anticholinesterase action of ciguatoxin has shown that the e f f e c t i s non-specific and rather corresponds with a widespread a n t i enzymatic a c t i v i t y (F6). Red blood c e l l cholinesterase assays from intravenously intoxicated rats (1_7) supported the conclusion that ciguatoxin i s not an in visoo cholinesterase i n h i b i t o r (18). Respiratory and cardiovascular e f f e c t s of ciguatoxin have been studied. Cheng dt at (19) reported that respiratory arrest induced by a l e t h a l dose of ciguatoxin i s caused by an i n h i b i t i o n of the central respiratory mechanism. L i ' s r e s u l t s (9) showing biphasic cardiovascular e f f e c t s , transient hypotension and bradycardia succeeded by hypertension and tachycardia, were c o n f i r med i n the rat by Rayner (18). This biphasic response remained unchanged following s p i n a l i z a t i o n , b i l a t e r a l vagotomy, adrenalectomy and nephrectomy. The hypotension and bradycardia were found to be antagonized by hexamethonium, atropine and hemicholinium, while the hypertension and tachycardia were suppressed by atropine i n doses greater than 10 mg/kg, 3-blocking agents, p r i o r reserpinization and tetrodotoxin. A s i m i l a r biphasic response was observed i n the rabbit by Laborit OX at (20). They noted that diethazine (a central a n t i c h o l i n e r g i c agent) hardly affected the responses, but calcium glutamate exerted a protective action. In the pentobarbital or a-chloralose anaesthetized cat, the same complex of e f f e c t s were observed with s l i g h t l y increased doses (21). The hypotension and bradycardia were suppressed by hexamethonium and atropine ; the hypertension and tachycardia were p a r t i a l l y suppressed by atropine, propranolol and clonidine, a central antihypertensive agent. Moreover, phentolamine was found to be able to antagonize the bradycardia and hypotensive phase as well as the tachycardia and hypertensive response. Further antagonistic action was obtained with prazosin and yohimbine, which are respectively a^-and ou-adrenergic blocking agents. Prazosin completly suppressed the action of the toxin while yohimbine only p a r t i a l l y antagonized the e f f e c t s . The r e s u l t s suggest that ciguatoxin has an a-adrenergic mimetic action, s p e c i a l l y at ai-adrenoceptors. Ciguatoxin e f f e c t s were investigated also on various isolated organs and tissues. Banner OX at (22, 22) tested semi-purified extracts of ciguatoxin on isolated toad s c i a t i c nerve-gastrocnemius muscle and guinea-pig phrenic nerve-diaphragm preparations. They concluded that while the action p o t e n t i a l of the nerve may be lost from long immersion i n a solution of ciguatoxin, the immediate e f f e c t was upon the nerve-muscle junction. Similar r e s u l t s were obtained more recently by M i l l e r (24). Ciguatoxin produced depolarization of the pedal ganglion c e l l s of the sea hare kpty&ija juLiana (25). I t increased the permeability of frog skin membrane to sodium ions (26) and depolarized muscle fibers of the frog (27, 28). This depolarization was antagonized by tetrodotoxin and C a ^ - r i c h medium. Ciguatoxin was also reported to decrease the amplitude of the action p o t e n t i a l of the nerve and block the neuromuscular conduction i n the s c i a t i c nerve-gastrocnemius preparation of the rat (29). F i n a l l y , the e f f e c t s of ciguatoxin on the resting membrane potential appear to r e s u l t from replacement of



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calcium by ciguatoxin at receptor sites which regulate steady-state sodium permeability (28). Consequently, low concentrations of c i guatoxin increase the e x c i t a b i l i t y of excitable membranes while a conduction block could be presumed to result,at higher maintained concentrations, from gradually increasing internal sodium concentration. In mammalian isolated a t r i a , ciguatoxin induced a biphasic response. Oshika (30) observed i n rat and rabbit a t r i a a transitory negative inotropic phase, antagonized by atropine and hemicholinium, followed by a positive inotropic phase antagonized by MJ-1999, guanethidine and pre-treatment with reserpine. In guinea-pig a t r i a , only p o s i t i v e chronotropic and inotropic effects were reported (31). The l a t t e r were p a r t i a l l y antagonized by propranolol and phentolamine. In the rat myocardium, our studies showed at very low concentrations a p o s i t i v e chronotropic e f f e c t in right isolated a t r i a and a negative inotropic e f f e c t i n paced l e f t isolated a t r i a . Higher concentrations produced a decrease of the spontaneous rate of the right a t r i a while i n the l e f t a t r i a a biphasic response was observed, a transitory negative inotropic e f f e c t followed by a p o s i t i v e one. Negative inotropic and chronotropic effects were antagonized by atropine. Positive chronotropic response was suppressed by p r i o r r e s e r p i n i z a t i o n and propranolol while p o s i t i v e inotropic one was only p a r t i a l l y modified by r e serpine pretreatment, propranolol and phentolamine (32). These r e s u l t s , as those of Oshika (30) indicate both cholinergic and adrenergic action of ciguatoxin i n the isolated rat a t r i a , suggesting a marked release of acetylcholine and a small release of catecholamines at the nerve endings. Yet, other mechanisms i n addition to catecholamines release seems to be involved i n the positive inotropic response. Preliminary experiments of us showed that the more p u r i f i e d the ciguatoxic extract was, the less important the p o s i t i v e inotropic e f f e c t (32). As Rayner and Szekerczes reported an i n h i b i t i o n of the Na -K ATP-ase of human erythrocyte ghosts by crude extracts and none by p u r i f i e d extracts (33), thus, the possible presence of a cardiotonic contaminant i n p a r t i a l l y p u r i f i e d extracts has to be taken i n consideration. Ciguatoxin action was studied on smooth muscle. Miyahara and Shibata (34) investigated the e f f e c t s of the toxin on the i n h i b i tory mechanism of the guinea-pig tdZVUR COJLCixm. They demonstrated that ciguatoxin exerts a prominent calcium-sensitive action on nerve terminals and transmitter release even before a f f e c t i n g the r e a c t i v i t y of the neuro-effector organ. In the guinea-pig VCtt> da^QJiOM, ciguatoxin caused a potent excitatory e f f e c t antagonized by reserpine, guanethidine, phentolamine Ca-free medium and tetrodotoxin (35-37). This action was considered to be due p a r t i a l l y to a norepinephrine release and mainly to a supersensitivity of the post-synaptic membrane. Moreover, the authors reported an i n h i b i t o r y e f f e c t on the c o n t r a c t i l e response to transmural stimulation. They suggest that this e f f e c t might result from a strong nerve membrane depolarizing action of c i guatoxin which i n h i b i t s the stimulation-induced action p o t e n t i a l (35). In guinea-pig AjLaum, ciguatoxin extracts strongly decreased the e f f e c t of exogenous acetylcholine and histamine, while i n spontaneously active rat jdniinum preparation they inhibited


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a c t i v i t y (24). In h e l i c a l s t r i p s of rabbit thoracic aorta, we observed that ciguatoxin s l i g h t l y potentiated the contraction induced by norepinephrine. On the other hand, a ciguatoxin-induced contraction was obtained. This contraction was completly i n h i b i t e d by phentolamine and prazosin, p a r t i a l l y by yohimbine. The results confirm that ciguatoxin has a marked mimetic a c t i v i t y at the ai-adrenoceptors. In conclusion, the complex description of the e f f e c t s observed on various preparations and t h e i r succession following slow dosage increase indicate that ciguatoxin acts at many target s i t e s of s l i g h t l y d i f f e r i n g s e n s i t i v i t y . The reported action of ciguatoxin on the resting membrane potential suggests that the various e f f e c t s observed may be the r e s u l t of a d i r e c t depolarizing action on excitable membranes. Scaritoxin About s c a r i t o x i n , the following results were reported. This toxin was found to depress the oxidative metabolic process i n the r a t brain (20) and to have a depolarizing action on excitable membranes (38). In the guinea-pig a t r i a , s c a r i t o x i n caused a marked potentiation of the acetylcholine negative inotropic and chronotropic e f f e c t s (39). In rat a t r i a , we observed biphasic inotropic and chronotropic e f f e c t s s i m i l a r to those of ciguatoxin. Negative inotropic and chronotropic e f f e c t s were antagonized by atropine. The p o s i t i v e chronotropic response was suppressed by reserpine pretreatment or by propranolol, while the p o s i t i v e inotropic e f f e c t was only p a r t i a l l y modified by reserpinization, propranolol and phentolamine (32). In the pentobarbital anaesthetized cat, s c a r i toxin exerted respiratory and cardiovascular e f f e c t s s i m i l a r to those of ciguatoxin (21). Although data about s c a r i t o x i n are l i m i ted, i t seems that this toxin has a pharmacological mode of action very close to that of ciguatoxin. I t may be speculated that c i guatoxin and s c a r i t o x i n are related compounds, s c a r i t o x i n r e s u l ting from ciguatoxin metabolic transformation i n some f i s h species. Maitotoxin Respiratory and cardiovascular e f f e c t s of maitotoxin have been studied i n pentobarbital anaesthetized cats (21). Sublethal doses of maitotoxin induced an important hyperventilation phase, hypertension and a transitory tachycardia followed by s l i g h t bradycardia. Higher dosage caused respiratory depression, cardiac arrhythmias and tachycardia leading to cardiac f a i l u r e . A r t i f i c i a l r e s p i r a t i o n did not modify the cardiac responses. Maitotoxin e f f e c t s were investigated also by hi VAJ&LO experiments. Miyahara OX at (31) reported a biphasic response i n the guinea-pig a t r i a , an i n i t i a l p o s i t i v e response followed by a progressive decrease of both the rate and the force of contractions. In the r a t , we observed a decrease of the spontaneous frequency of right a t r i a l preparations and biphasic inotropic e f f e c t s i n l e f t a t r i a l preparations. Negative e f f e c t s were not antagonized by atropine. The p o s i t i v e inotropic e f f e c t was modified very l i t t l e by p r i o r reserpinization or p r i o r exposure to propranolol and phentolamine but was sensitive to Mn ions. On the other hand, 2+


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maitotoxin was found to strongly i n h i b i t the Na -K ATP-ase from microsomes of cat and human kidneys (40). Takahashi oX at (41,42) tested maitotoxin i n a rat pheochromocytoma c e l l l i n e , and observed a profound increase i n C a i n f l u x and a C a dependent release of (3H)-norepinephrine and-dopamine. These effects were not modified by tetrodotoxin or Na-free medium but were inhibited by Mn , verapamil, nicardipine and tetracaine, suggesting that maitotoxin activated the voltage-dependent calcium channel. Similar action was observed i n the guinea-pig isolated XJLoJim, taenia caa&l and V06 dz^QAQM and i n the rabbit thoracic aorta (43-45). The authors reported that maitotoxin had a l i t t l e e f f e c t on the Na -K -ATPase from porcine cerebral cortex (45). Recently, we studied maitotoxin e f f e c t s on the action potentiaT~of isolated perfused rat hearts to determine the existence of a d i r e c t action on the myocardium. An increase i n amplitude and duration of the ventricular action potential plateau was observed at low dosage. Higher doses caused a decrease of the spike amplitude and of the maximum rate of r i s e of the action potential while the lengthening of the plateau became more marked. These same effects appeared also i n hearts from reserpinized animals. They did not develop during perfusion with Mn ions, verapamil and low-calcium solution. Addition of Mn or verapamil, low-calcium solution or Na-rich medium reversed to a large extent the effects of maitotoxin. These results can be taken as arguments i n favor of an action involving calcium movements and/or calcium conductance. Although the mode of action of maitotoxin i s not yet completly elucidated, this compound appears to have a s p e c i f i c c e l l u l a r action. I t could become an useful tool for biochemical and pharmacological studies. 2 +

2 +

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The above-reviewed pharmacological results show some s l i g h t d i s crepancies s p e c i a l l y with ciguatoxin. These discrepancies may be explained partly by the v a r i a b i l i t y of the sample purity and p a r t l y by the presence i n the extracts of secondary toxins. The complex description of the effects observed experimentally can explain the polymorphism of the c l i n i c a l features. These pharmacological data can help the physician to improvise an appropriate treatment in ciguatera f i s h poisoning. Acknowledgments The l o c a l data have been obtained with the f i n a n c i a l a i d of both the French Ministry of Industry and Research and the T e r r i t o r i a l Government of French Polynesia.

Literature cited 1. Scheuer, P.J. ; Takahashi, W. ; Tsutsumi, J. ; Yoshida, T. Science (Wash. DC), 1967, 21, 1267-8. 2. Yasumoto, T. ; Bagnis, R. ; Vernoux, J.P. Bull. Jap. Soc. Sci. Fish., 1976, 42, 359-65. 3. Chungue, E. ; Bagnis, R. ; Fusetani, N. ; Hashimoto, Y. Toxicon, 1976, 15, 89-95. 4. Banner, A.H. ; Scheuer, P.J. ; Sasaki, S. ; Helfrich, P. Alender, C.B. Ann. N.Y. Acad. Sci. 1960, 90, 770-87. 5. Yasumoto, T. ; Scheuer, P.J. Toxicon, 1969, 7, 273-6.


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Chungue, E. Ph.D. Thesis, University of Montpellier, France, 1977. Kimura, L.H. ; Hokama, Y. ; Abad, M.A. ; Oyama, M. ; Miyahara, J.T. Toxicon 1982, 20, 907-12. Hoffman, P.A. ; Grande , H.R. ; Mc Millan, J.P. Toxicon 1983, 21, 363-9. Li, K.M. Science 1965, 147, 1580-1. Banner, A.H. ; In "Animal Toxins" ; Russel, F.E. and Saunders, p.R., Ed. ; Pergamon Press : London, 1967 ; pp.157-65. Kosaki, T. ; Stephens, J. Fedn. Proc. Fedn. Am. Socs Exp. Biol. 1967, 26, 322. Ogura, Y. Report to 2nd Annual Conference on Marine Toxins, University of Hawaii, 1967. Kosaki, T.I. ; Anderson, H.H. Toxicon 1968, 6, 55-8. Rayner, M.D. ; Kosaki, T.I. ; Fellmeth, E.L. Science (Washington. D.C.) 1968, 160, 70-1. Ogura, Y. ; Nara, J. ; Yoshida, T. Toxicon 1968, 6, 131-40. Baslow, M.H. ; Rayner, M.D. Proc. 4th Int. Congr. Pharmac., 1969, p. 180. Rayner, M.D. ; Baslow, M.H. ; Kosaki, T.I. J. Fish. Res. Bd. Can. 1969, 26, 2 208. Rayner, M.D. In "Drugs from the Sea Conference Proceedings", Marine Technology Society : Washington, D.C., 1969, pp.345-50. Cheng, K.K. ; Li, K.M. ; Quintillis, Y.H. J. Path. 1969, 97, 89-92. Laborit, H. ; Baron, C. ; Ferran, C. ; Laborit, G. Agressologie, 1979, 20, 81-96. Legrand, A.M. ; Galonnier, M. ; Bagnis, R. Toxicon, 1982, 20, 311-5. Banner, A.H. ; Helfrich, P. ; Scheuer, P.J. ; Yoshida, T. Proc. 16th Gulf. Caribbean Fish. Inst., 1963, pp. 84-98. Banner, A.H. ; Shaw, S.W. ; Alender, C.B. ; Helfrich, P. South Pacific Commission, 1963, Technic Report 141, 17 p. Miller, D.M. ; Dickey, R.W. ; Tindall, D.R. Fedn. Proc. Fedn. Am. Socs. Exp. Biol. 1982, 41, 1561. Boyarsky, L.L. ; Rayner, M.D. Proc. Soc. Exp. Biol. Med. 1970, 134, 332-5. Setliff, J.A. ; Rayner, M.D. ; Hong, S.K. Toxic. Appl. Pharmac. 1971, 18, 676-84. Rayner, M.D. ; Kosaki, T.I. Fedn. Proc. Fedn. Am. Socs. Exp. Biol., 1970, 29, 548. Rayner, M.D. Fedn. Proc. Fedn. Am. Socs. Exp. Biol. 1972, 31, 1139-1145. Faucomprez, C. ; Ferezou, J.P. ; Bagnis, R. ; Chanfour, B. ; Niaussat, P.M. ; Drouet, J. Bull. Soc. Path. Exot. 1975, 68, 106-15. Ohshika, H. Toxicon 1971, 9, 337-43. Miyahara, J.T. ; Akau, C.K. ; Yasumoto, T. Res. Comm. Chem. Path. Pharmac., 1979, 25, 177-80. Legrand, A.M. ; Bagnis, R. Toxicon 1984, in press. Rayner, M.D. ; Szekerczes, J. Toxic. Appl. Pharmac. 1973, 24, 489-96. Miyahara, J.T. ; Shibata, S. Fedn. Proc. Fedn. Am. Socs. Exp. Biol. 1976, 35, 842.



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35. Ohizumi, Y. ; Shibata, S. ; Tachibana, K. J . Pharmac. Exp. Ther. 1981, 217, 475-80. 36. Ohizumi, Y. ; Ishida, Y. ; Shibata, S. Proc. 4th Int. Symp. Vascular Neuroeffector Mechanisms,1983, pp. 301-4. 37. Ohizumi, Y. ; Ishida, Y. ; Shibata, S. J . Pharmac. Exp. Ther., 1982, 221, 748-52. 38. Rayner, M.D., Personal Communication presented to the South P a c i f i c Commission, 1977. 39. Rentler, J.F. Thesis of Veterinary Surgeon, University of Lyon, France, 1980. 40. Bergmann, J.S. ; Nechay, B.R. Fedn. Proc. Fedn. Am. Socs. Exp. B i o l . 1982, 41, 1562. 41. Takahashi, M. ; Ohizumi, Y. ; Yasumoto, T. J . B i o l . Chem., 1982, 257, 7287-9. 42. Takahashi, M. ; Tatsumi, M. ; Ohizumi, Y. ; Yasumoto , T. J. B i o l . Chem., 1983, 258, 10944-9. 43. Ohizumi, Y. ; Yasumoto, T. Br. J . Pharmac., 1983, 79, 3-5. 44. Ohizumi, Y. ; Kajiwara, A. ; Yasumoto, T. J . Pharmac. Exp. Ther., 1983, 227, 199-204. 45. Ohizumi, Y. ; Yasumoto, T. J . Physiol. (London), 1983, 337, 711-21 . RECEIVED

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Mode of Action of Ciguatera Toxins - ACS Symposium Series (ACS

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