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Ethnopharmacological Relevance: Senescent leaves of Heliotropium foertherianum Diane & Hilger (Boraginaceae) are traditionally used in the Pacific region to treat Ciguatera Fish Poisoning. This plant contains rosmarinic acid that is known for its multiple biological activities. In the present study, H. foertherianum aqueous extract, rosmarinic acid and its derivatives were evaluated for their capacity to reduce the effect of ciguatoxins., Materials and Methods: Aqueous extract of H. foertherianum leaves was prepared and studied for its effects against a Pacific ciguatoxin (P-CTX-1B) in the neuroblastoma cell assay and the receptor binding assay. Rosmarinic acid and six derivatives were also evaluated by means of these bioassays. For this purpose, we have developed an improved synthetic route for caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE)., Results: Both the aqueous extract of H. foertherianum leaves and rosmarinic acid showed inhibitory activities against a Pacific ciguatoxin in the above bioassays. Among all the molecules that were evaluated, rosmarinic acid was the most active compound., Conclusion: These results confirm further the potential of H. foertherianum in the treatment of Ciguatera Fish Poisoning., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Ciguatoxin (P-CTX-1B) from the dinoflagellate Gambierdiscus toxicus, belongs to the family of polyether neurotoxins responsible for the neurological poisoning disorder ciguatera. Although it is the most widespread marine-borne disease affecting humans, there is no current FDA-approved treatment available except for symptomatic therapies. In this paper, we report that P-CTX-1B promotes catecholamine secretion from bovine chromaffin cells, an effect that is insensitive to concomitant activation of capacitative Ca(2+) entry. Moreover, we confirm that brevenal, a polyether from the dinoflagellate Karenia brevis, blocks P-CTX-1B-induced catecholamine secretion. This effect is partially reversible. Our results therefore raise the prospect of finding functional antagonists for P-CTX-1B that could be useful for the treatment of ciguatera., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Ciguatera is a global food poisoning caused by the consumption of fish that have accumulated sodium channel activator toxins, ciguatoxins. At present, most diagnosed cases of ciguatera are treated with symptomatic and supportive remedies, and no specific therapy has been devised. Here we report that ciguatoxin CTX3C can be effectively neutralized in vitro and in vivo by simultaneous use of two anti-ciguatoxin monoclonal antibodies, providing the first rational approach toward directly preventing and treating ciguatera.

It is well established that the targeted receptor for ciguatoxin (CTX) in mammalian tissues is the sodium channel, affecting the influx of sodium into cells and altering the action potential and function of the cell. Since the syntheses of fragments of CTX has become available, our focus has been on the receptor functions of the west sphere AB and east sphere JKLM fragments using the neuroblastoma cell assay, guinea pig atrium assay, and the membrane immunobead assay (MIA). The data presented here suggest that the west sphere AB of the ciguatoxin molecule is the active portion and is responsible for the activation of the sodium channels., ((c) 2006 Wiley-Liss, Inc.)

The effects of 31 plant extracts, which most are traditionally used to treat ciguatera fish poisoning in the Pacific area, were studied on the cytotoxicity of mouse neuroblastoma cells produced by ouabain, veratridine and/or brevetoxin-3 or Pacific ciguatoxin-1. The cell viability was determined using a quantitative colorimetric method. A marked cytotoxicity of seven of the 31 plant extracts studied, was observed. Despite this, these plant extracts were suspected to contain active compound(s) against the cytotoxicity produced by brevetoxin (2 extracts), brevetoxin, ouabain and/or veratridine (3 extracts), or only against that of ouabain and/or veratridine (2 extracts). Among the 24 plant extracts that exhibited by themselves no cytotoxicity, 22 were active against the effect of brevetoxin or against that of both veratridine and brevetoxin. Similar results were obtained when the seven most active plant extracts were reassayed using ciguatoxin instead of brevetoxin. In conclusion, the present work reports the first activity assessment of some plant extracts, achieved in vitro on a quite large scale. The fact that 27 plant extracts were found to exert, in vitro, a protective effect against the action of ciguatoxin and/or brevetoxin, paves the way for finding new active compounds to treat ciguatera fish poisoning, provided these compounds also reverse the effects of sodium channel activators.

The basis for the neuroprotectant effect of D-mannitol in reducing the sensory neurological disturbances seen in ciguatera poisoning, is unclear. Pacific ciguatoxin-1 (P-CTX-1), at a concentration 10 nM, caused a statistically significant swelling of rat sensory dorsal root ganglia (DRG) neurons that was reversed by hyperosmolar 50 mM D-mannitol. However, using electron paramagnetic resonance (EPR) spectroscopy, it was found that P-CTX-1 failed to generate hydroxyl free radicals at concentrations of toxin that caused profound effects on neuronal excitability. Whole-cell patch-clamp recordings from DRG neurons revealed that both hyper- and iso-osmolar 50 mM D-mannitol prevented the membrane depolarisation and repetitive firing of action potentials induced by P-CTX-1. In addition, both hyper- and iso-osmolar 50 mM D-mannitol prevented the hyperpolarising shift in steady-state inactivation and the rise in leakage current through tetrodotoxin (TTX)-sensitive Na(v) channels, as well as the increased rate of recovery from inactivation of TTX-resistant Na(v) channels induced by P-CTX-1. D-Mannitol also reduced, but did not prevent, the inhibition of peak TTX-sensitive and TTX-resistant I(Na) amplitude by P-CTX-1. Additional experiments using hyper- and iso-osmolar D-sorbitol, hyperosmolar sucrose and the free radical scavenging agents Trolox and L-ascorbic acid showed that these agents, unlike D-mannitol, failed to prevent the effects of P-CTX-1 on spike electrogenesis and Na(v) channel gating. These selective actions of D-mannitol indicate that it does not act purely as an osmotic agent to reduce swelling of nerves, but involves a more complex action dependent on the Na(v) channel subtype, possibly to alter or reduce toxin association.

The present study investigated the actions of the polyether marine toxin Pacific ciguatoxin-1 (P-CTX-1) on neuronal excitability in rat dorsal root ganglion (DRG) neurons using patch-clamp recording techniques. Under current-clamp conditions, bath application of 2-20 nM P-CTX-1 caused a rapid, concentration-dependent depolarization of the resting membrane potential in neurons expressing tetrodotoxin (TTX)-sensitive voltage-gated sodium (Nav) channels. This action was completely suppressed by the addition of 200 nM TTX to the external solution, indicating that this effect was mediated through TTX-sensitive Nav channels. In addition, P-CTX-1 also prolonged action potential and afterhyperpolarization (AHP) duration. In a subpopulation of neurons, P-CTX-1 also produced tonic action potential firing, an effect that was not accompanied by significant oscillation of the resting membrane potential. Conversely, in neurons expressing TTX-resistant Nav currents, P-CTX-1 failed to alter any parameter of neuronal excitability examined in this study. Under voltage-clamp conditions in rat DRG neurons, P-CTX-1 inhibited both delayed-rectifier and 'A-type' potassium currents in a dose-dependent manner, actions that occurred in the absence of alterations to the voltage dependence of activation. These actions appear to underlie the prolongation of the action potential and AHP, and contribute to repetitive firing. These data indicate that a block of potassium channels contributes to the increase in neuronal excitability, associated with a modulation of Nav channel gating, observed clinically in response to ciguatera poisoning.

The effects of hyperosmolar D-mannitol were studied on single frog myelinated nerve fibres previously poisoned with Caribbean ciguatoxin-1 (C-CTX-1), a new toxin isolated from the pelagic fish Caranx latus inhabiting the Caribbean region. In current-clamped myelinated axons, C-CTX-1 (50-120 nM) caused spontaneous and repetitive action potential discharges after a short delay. In addition, the toxin produced a marked swelling of nodes of Ranvier of myelinated axons that reached a steady state within about 90 min, as revealed by using confocal laser scanning microscopy. The increased excitability and the nodal swelling caused by C-CTX-1 were prevented or reversed by an external hyperosmotic solution containing 100 mM D-mannitol. Moreover, the C-CTX-1-induced nodal swelling was completely prevented by the blockade of voltage-sensitive sodium channels by tetrodotoxin (TTX). It is suggested that C-CTX-1, by increasing nerve membrane excitability, enhances Na(+) entry into nodes of Ranvier through TTX-sensitive sodium channels, which directly or indirectly disturb the osmotic equilibrium between intra- and extra-axonal media resulting in an influx of water that was responsible for the long-lasting nodal swelling. The fact, that hyperosmolar D-mannitol either reversed or prevented the neurocellular actions of C-CTX-1, is of particular interest since it provides the rational basis for its use to treat the neurological symptoms of ciguatera fish poisoning in the Caribbean area.

Measurement of intracellular Ca2+ concentration ([Ca2+]i) in cultured mouse NG108-15 neuroblastoma x glioma hybrid cells, using the fluorescent probe fura-2, revealed that 5-25 nM ciguatoxin (CTX) increased [Ca2+]i either in cells bathed in standard medium or after removal of external Ca2+ by a Ca(2+)-free medium supplemented with EGTA. Tetrodotoxin prevented the CTX increased [Ca2+]i suggesting that CTX-induced mobilization of intracellular Ca2+ depends on Na+ influx through voltage-gated Na channels. CTX-induced Ca2+ mobilization prevented subsequent action of bradykinin (1 microM) suggesting that CTX stimulates the inositol 1,4,5-trisphosphate-releasable Ca2+ store.

Mannitol (1 g/kg i.v.) is currently the treatment of choice for acute ciguatera, but confirmation of this treatment's apparent efficacy awaits further experimental or controlled clinical evidence. In mice, mannitol (1 g/kg i.v.) administered before or after i.p. ciguatoxin did not influence the signs of intoxication or the time to death. The effects of oral ciguatoxin differed from those following i.p. ciguatoxin, but again i.v. mannitol provided no detectable benefit. Development of hypothermia was rapid in mice receiving i.p. or oral ciguatoxin and was unaffected by i.v. mannitol. A sublethal i.p. dose of ciguatoxin initially retarded (day 0-4) but then accelerated (day 4-12) the growth of mice. Mannitol (i.v.) had no influence on these effects of ciguatoxin on the growth of mice. Ciguatoxin inhibited responses of isolated diaphragms to nerve stimulation (ED50 = 9 x 10(-11) M), while directly stimulated diaphragms were inhibited by five-fold higher concentrations. Mannitol (50 mM) added to the organ bath did not influence the ciguatoxin-induced inhibition of diaphragm responses to nerve stimulation in vitro. Responses of isolated diaphragm to nerve stimulation were normal in preparations removed from ciguatoxin-treated mice displaying pronounced dyspnoea (gasping). However, responses to nerve stimulation were reduced in preparations removed from mice immediately following death from ciguatoxin. Mannitol (i.v.) partially protected the phrenic nerve-diaphragm from this effect of ciguatoxin in vivo. We conclude that the lethal effects of ciguatoxin in mice probably stem from a central action, and suggest that species differences may account for the absence of any marked beneficial effect of i.v. mannitol in the mouse model for ciguatera in humans.

The absolute refractory period, relative refractory period, and the duration and magnitude of the supernormal period were measured after incubation of fish nerves with ciguatoxin and other channel modifying compounds, tetrodotoxin, veratridine, verapamil, and lignocaine. In vitro electrophysiological studies were carried out on the lateral line nerve of the whiting, Sillago ciliata Cuvier. Electrophysiological changes in fish nerves after exposure to ciguatoxin (0.3 MU.ml-1) and veratridine (1 x 10(-5) M) are similar to changes that occur in mammalian nerves and include an increase in the absolute refractory period, the relative refractory period, and the magnitude and duration of supernormality. The effects of ciguatoxin (0.3 MU.ml-1) in fish nerves were antagonised by tetrodotoxin (5 x 10(-10) M), verapamil (5 x 10(-7) M), and lignocaine (1 x 10(-5) g/ml-1). The nerves of Sillago ciliata used in this study responded to ciguatoxin and its antagonists in a similar manner to mammalian nerves, suggesting that these teleost nerves have no specific electrophysiological mechanism to cope with this toxin.

The mode of action of ciguatoxin (CTX) on the isolated atrial and papillary muscle of the guinea-pig heart was investigated using conventional methods for the measurement of mechanical and electrophysiological parameters. CTX induced positive inotropic and positive klinotropic responses in atrial and papillary muscles. Each response consisted of two phases. The initial positive inotropic response developed rapidly and resulted from the previously reported indirect action of CTX. The second phase of positive inotropy developed more slowly and was well maintained at doses of CTX up to 0.15 mouse units/ml in atria and up to 0.8 M.U./ml in papillary muscles. This phase was found to result from a direct action of CTX on the myocardium which was not reversed by washing. Tetrodotoxin (TTX) reversed the positive inotropic effects stemming from the direct action of CTX. The (-) and (+) enantiomers of propranolol were equally effective in inhibiting the direct effect of CTX. These antagonists did not displace CTX from the myocardium. CTX induced a TTX-sensitive depolarization of stimulated or quiescent atrial cells. All the effects of CTX on the atrial action potential were reversed by TTX. It was therefore concluded that CTX opens voltage dependent Na+ channels. CTX bound equally to resting and K+-depolarized Na+ channels but there were indications that electrical stimulation enhanced the rate of CTX binding. CTX overrides the positive staircase effect of increasing stimulation frequency. Na+ channels found in the atria which were particularly sensitive to TTX did not play a prominent role in mediating the CTX effect. CTX appeared to have little effect on the normal Na+ channel inactivation process. CTX did not restore contractions in the K+-depolarized cardiac muscles examined. The sensitivity of CTX action to TTX distinguished it from cardiac glycoside activity. Established mechanisms of Na+/Ca2+ exchange and Ca2+-induced release of Ca2+ can explain the link between CTX-induced increase of intracellular [Na+] and the positive inotropic response.

1. Ciguatoxin (CTX) caused a dose-dependent increase in the contractile force of the guinea-pig isolated left atria at concentrations ranging from 0.1 to 10 ng ml-1 with the ED50 value of 0.5 ng ml-1. 2. In the atria, tetrodotoxin (5 x 10(-7) M) inhibited markedly the inotropic action of CTX. The inotropic effect of CTX at low concentrations was abolished by practolol (10(-5) M) and reserpine (2 mg kg-1 daily, for 3 days), whereas that of CTX at high concentrations was partially inhibited by both drugs. 3. In single atrial cells, CTX (3 ng ml-1) produced a marked increase in the amplitude of longitudinal contractions. 4. CTX (3 ng ml-1) caused marked prolongation in the falling phase of action potentials of atrial strips without affecting the maximum rate of rise of action potentials and membrane resting potentials. The effect of CTX on action potentials was abolished by tetrodotoxin (10(-6) M). 5. The whole-cell patch-clamp experiments on myocytes revealed that CTX (20 ng ml-1) shifted the current-voltage curve of Na inward currents by 40 mV in the negative direction. CTX caused a small sustained Na inward current even at resting membrane potentials. 6. These results suggest that the inotropic action of lower concentrations of CTX is primarily due to an indirect action via noradrenaline release, whereas that of higher concentrations is caused not only by an indirect action but also by a direct action on voltage-dependent Na channels of cardiac muscle. It is also suggested that CTX activates cardiac muscle Na channels by modifying the voltage-dependence of channel activation to increase Na inward currents, thus producing cardiotonic actions.