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Ciguatera is a major circumtropical poisoning caused by the consumption of marine fish and invertebrates contaminated with ciguatoxins (CTXs): neurotoxins produced by endemic and benthic dinoflagellates which are biotransformed in the fish food-web. We provide a history of ciguatera research conducted over the past 70 years on ciguatoxins from the Pacific Ocean (P-CTXs) and Caribbean Sea (C-CTXs) and describe their main chemical, biochemical, and toxicological properties. Currently, there is no official method for the extraction and quantification of ciguatoxins, regardless their origin, mainly due to limited CTX-certified reference materials. In this review, the extraction and purification procedures of C-CTXs are investigated, considering specific objectives such as isolating reference materials, analysing fish toxin profiles, or ensuring food safety control. Certain in vitro assays may provide sufficient sensitivity to detect C-CTXs at sub-ppb levels in fish, but they do not allow for individual identification of CTXs. Recent advances in analysis using liquid chromatography coupled with low- or high-resolution mass spectrometry provide new opportunities to identify known C-CTXs, to gain structural insights into new analogues, and to quantify C-CTXs. Together, these methods reveal that ciguatera arises from a multiplicity of CTXs, although one major form (C-CTX-1) seems to dominate. However, questions arise regarding the abundance and instability of certain C-CTXs, which are further complicated by the wide array of CTX-producing dinoflagellates and fish vectors. Further research is needed to assess the toxic potential of the new C-CTX and their role in ciguatera fish poisoning. With the identification of C-CTXs in the coastal USA and Eastern Atlantic Ocean, the investigation of ciguatera fish poisoning is now a truly global effort.

Caribbean ciguatoxin-1 (C-CTX-1) induced, after about 1h exposure, muscle membrane depolarisation and repetitive post-synaptic action potentials (APs) in frog neuromuscular preparations. This depolarising effect was also observed in a Ca(2+)-free medium with a strong enhancement of spontaneous quantal transmitter release, compared with control conditions. The ciguatoxin-induced increase in release could be accelerated when Ca(2+) was present in the extracellular medium. C-CTX-1 also enhanced nerve-evoked quantal acetylcholine (ACh) release. At normal neuromuscular junctions loaded with the fluorescent dye FM1-43, C-CTX-1 induced swelling of nerve terminals, an effect that was reversed by hyperosmotic d-mannitol. In myelinated axons, C-CTX-1 increased nodal membrane excitability, inducing spontaneous and repetitive APs. Also, the toxin enlarged the repolarising phase of APs in control and tetraethylammonium-treated axons. Overall, our data suggest that C-CTX-1 affects nerve excitability and neurotransmitter release at nerve terminals. We conclude that C-CTX-1-induced up-regulation of Na(+) channels and the inhibition of K(+) channels, at low nanomolar concentrations, produce a variety of functional dysfunctions that are in part responsible for the human muscle skeletal symptoms observed in ciguatera. All these dysfunctions seem to result from the subtle balance between ionic currents, intracellular Na(+) and Ca(2+) concentrations, and engaged second messengers., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The mechanisms underlying the swelling of frog red blood cells (RBC), induced by Pacific (P-CTX-1) and Caribbean (C-CTX-1) ciguatoxins (CTXs), were investigated by measuring the length, width and surface of their elliptic shape. P-CTX-1 (0.5 to 5 nM) and C-CTX-1 (1 nM) induced RBC swelling within 60 min. The CTXs-induced RBC swelling was blocked by apamin (1 microM) and by Sr(2+) (1 mM). P-CTX-1-induced RBC swelling was prevented and inhibited by H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (27 microM), an inhibitor of soluble guanylate cyclase (sGC), and NOS blockade by NG methyl-l-arginine (l-NMA; 10 microM). Cytochalasin D (cytD, 10 microM) increased RBC surface and mimicked CTX effect but did not prevent the P-CTX-1-induced l-NMA-sensitive extra increase. Calculations revealed that P-CTX-1 and cytD increase RBC total surface envelop and volume. These data strongly suggest that the molecular mechanisms underlying CTXs-induced RBC swelling involve the NO pathway by an activation of the inducible NOS, leading to sGC activation which modulates intracellular cGMP and regulates L-type Ca(2+) channels. The resulting increase in intracellular Ca(2+) content, in turn, disrupts the actin cytoskeleton, which causes a water influx and triggers a Ca(2+)-activated K(+) current through SK2 isoform channels.

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for > 90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and < 900 Da were suspected to contribute to the overall toxicity.

Ciguatera is a common seafood poisoning in Western Atlantic and French West Indies. Ciguatera fish poisoning in the Caribbean is a public health problem. A toxicological study was carried out on 178 Caribbean fish specimens (26 species) captured off Guadeloupe and Saint Barthelemy between 1993 and 1999. The mouse bioassay and the chick feeding test were used to control fish edibility. Ciguatoxins presence was assumed when symptomatology was typical of ciguatera in mouse and chick. Fishes were classified in three groups: non toxic fish (edible), low toxic fish (not edible) and toxic fish (not edible). 75% of fishes were non toxic. Toxic fish specimens belonged to four families of high trophic level carnivores: Carangidae, Lutjanidae, Serranidae et Sphyraenidae. Percentages of toxic fishes to humans reached 55% for Caranx latus and 33% for Caranx bartholomaei and Caranx lugubris. Only a significant correlation between weight and toxicity was only found for C. latus and snappers. Small carnivorous groupers (Serranidae) were also toxic. Atoxic fish species were (a) pelagic fish (Coryphaena hippurus, Auxis thazard and Euthynnus pelamis), (b) invertebrates feeders (Malacanthus plumieri, Balistes vetula), (c) small high-risk fish or (d) fish of edible benthic fish families. Liver of four fishes (Mycteroperca venenosa, Caranx bartholomaei, Seriola rivoliana, Gymnothorax funebris) contained ciguatoxins at a significant level although their flesh was safe. This study confirms the usefulness of mouse and chick bioassays for sanitary control of fish.

A grey snapper (Lutjanus griseus), a grouper (Serranidae) and a black jack (Caranx lugubris) were implicated in three different ciguatera poisonings in Guadeloupe, French West Indies. A mouse bioassay indicated toxicity for each specimens: 0.5-1, > or = 1 and > 1 MUg g(-1), respectively. After purification by gel filtration chromatography, the samples were analysed by high-performance liquid chromatography coupled to mass spectrometry (LC-MS). The toxin profiles differ from one fish to another. C-CTX-1 was detected at 0.24, 0.90 and 13.8 ng g(-1) flesh in the snapper, grouper and jack, respectively. It contributed only to part of the whole toxicity determined by the mouse bioassay. Other toxins identified were C-CTX-2 (a C-CTX-1 epimer), three additional isomers of C-CTX-1 or-2, and five ciguatoxin congeners (C-CTX-1127, C-CTX-1143 and its isomer C-CTX-1143a, and C-CTX-1157 and its isomer C-CTX-1157b). Putative hydroxy-polyether-like compounds were also detected in the flesh of the grouper with [M+ + H]+ ions at m/z 851.51, 857.50, 875.51, 875.49 and 895.54 Da. Some of these compounds have the same mass range as some known dinoflagellate toxins. In conclusion, this study confirms the usefulness of LC-MS analysis to determine the ciguatoxins levels and the toxin profile in fish flesh hazardous to humans.

The effects of Caribbean ciguatoxin (C-CTX-1) isolated from horse-eye jack (Caranx latus) on the electrical and mechanical activities of frog auricle were studied. C-CTX-1 (1 pM-50 nM) dose-dependently shortened the duration of the plateau and the repolarizing phase of the action potential (AP). The AP shortening induced by C-CTX-1 (50 nM) was suppressed or prevented either by tetrodotoxin (TTX; 0.6 nM) or by atropine (0.1mM). C-CTX-1 (50 nM) prolonged the TTX (0.6 nM)-sensitive electrical response of the vagus nerve branches, which innervate the auricle. The C-CTX-1 (50 nM)-induced shortening of the plateau and of the repolarization phase were prevented or reversed by gallamine (20 microM) and pirenzepine (0.5 microM), respectively. C-CTX-1 (50 nM) decreased the amplitude of the peak contraction and shortened its duration. In the presence of gallamine (20 microM), C-CTX-1 decreased the amplitude of the peak contraction and shortened its duration in the presence of pirenzepine (0.5 microM). C-CTX-1 (50 nM) decreased the time constant of the relaxation phase of the peak contraction suggesting that it increased the Na(+)/Ca(2+) exchange activity. Acetylcholine (ACh; 1 pM) shortened APD, decreased the peak contraction and mimics the effects of C-CTX-1. In conclusion, the presented data show that C-CTX-1 released ACh from atrial cholinergic nerve terminals which activated M(1) and M(2) muscarinic receptors subtype (mAChR). Our findings suggest that M(1) and M(2) mAChR are present in frog atrial tissue and play a previously unrecognized role in the modulation of the AP duration and of the mechanical activity of cardiac tissue.

We studied the variation in toxin profiles of purified extracts of 10 individual specimens and two pools of ciguateric Caranx latus. High-performance liquid chromatography/mass spectrometry (HPLC/MS) identified in all individual samples at least seven Caribbean ciguatoxins (C-CTXs) comprising C-CTX-1 and its epimer C-CTX-2 ([M+H](+) m/z 1141.58), and five new C-CTX congeners with pseudo-molecular ions at m/z 1141.58, 1143.60, 1157.57, 1159.58, and 1127.57. In some samples, additional C-CTX isomers were detected with [M+H](+) ions at m/z 1141.58 (two), 1143.60 (one) and 1157.57 (two). The two low-toxic pools contained only four to six ciguatoxins. The comparison in relative proportions of four different mass classes ([M+H](+) at m/z 1141, 1143, 1157 and 1127) showed that the group at m/z 1157 increased (2-20%) with flesh toxicity. More than 80% of group m/z 1141 comprised C-CTX-1, C-CTX-2 and their isomer C-CTX-1a whose level in this group correlated with fish toxicity. Contrary to low-toxic fishes, high-risk specimens had C-CTX-1 levels <50% and were subjected to large losses of activity on purification indicating that unstable ciguatoxins were present. A possible conversion of C-CTX-1 into C-CTX-1a was identified when flesh was cooked, without changes in toxicity. In conclusion, HPLC/MS characterised 12 C-CTXs accumulated by C. latus at variable levels.

We report the isolation and initial characterisation of Indian Ocean ciguatoxin (I-CTX) present in toxic lipid soluble extracts isolated from ciguateric fishes collected off the Republic of Mauritius in the Indian Ocean. Following i.p. injection of this extract, mice displayed symptoms that were similar, though not identical, to those produced by Pacific and Caribbean ciguatoxins (P-CTXs and C-CTXs). Using a radiolabelled brevetoxin (PbTx) binding assay and mouse bioassay guided fractionation, I-CTX was purified by Florisil, Sephadex LH-20 and TSK HW-40S chromatography with good recovery. Isolation to purity was not possible by preparative reversed phase high-performance liquid chromatography (HPLC) due to significant losses of toxicity. However, analytical reversed phase HPLC coupled to an electrospray mass spectrometry detector identified a [M + H](+) ion at m/z 1141.58 which co-eluted with activity that displaced [3H]-PbTx binding to rat brain. This mass corresponded to C-CTX-1, but the fragmentation pattern of I-CTX showed a different ratio of pseudo molecular and product ions. I-CTX was found to elute later than P-CTX-1 but was practically indistinguishable from C-CTX-1 on reversed phase HPLC, while the TSK HW-40S column chromatography differentiated I-CTX from the later eluting C-CTX-1. Taken together, these results indicate that I-CTX is a new ciguatoxin (CTX) responsible for ciguatera caused by reef fish in the Indian Ocean., (Copright 2002 Elsevier Science Ltd.)

Ciguatera is a significant food-borne disease caused by potent polyether toxins (ciguatoxins) which accumulate in the flesh of ciguateric reef fish at risk levels > 0.1 ppb for Pacific ciguatoxins. Research on ciguatera has been severely hindered by the lack of analytical methods that detect and characterize low levels of ciguatoxin in crude extracts of fish. Here we report a new procedure for ciguatoxin analysis based on gradient reversed-phase HPLC/tandem mass spectrometry (HPLC/MS/MS). The method gave a linear response to pure Pacific and Caribbean ciguatoxins (P-CTX-1 and C-CTX-1) and the structurally related brevetoxin (PbTx-2) spiked into crude extracts of fish. Levels equivalent to 40 ppt P-CTX-1, 100 ppt C-CTX-1, and 200 ppt PbTx-2 in fish flesh could be detected by HPLC/MS/MS. Using P-CTX-1 as an internal standard, the analysis of extracts of 30 ciguateric fish from the Caribbean Sea (8 toxic, 12 borderline, and 10 nontoxic by mouse bioassay) confirmed the reliability of the method and allowed an estimated risk level of > 0.25 ppb C-CTX-1 to be established. HPLC/MS/MS provides a sensitive analytical approach, not previously available, for the determination of Pacific and Caribbean ciguatoxins at sub-ppb levels in fish flesh.

The toxins involved in ciguatera (fish poisoning) in the Caribbean Sea were isolated from Caranx latus, a pelagic fish often implicated in ciguatera in the Caribbean region, and purified by mouse bioassay directed fractionation. Five toxins were separated by reverse-phase high-performance liquid chromatography (HPLC). In order of increasing hydrophobicity, these toxins included a sleep-inducing fraction (< 1% of total toxicity), a major Caribbean ciguatoxin (C-CTX-1, 65% of toxicity), a minor Caribbean ciguatoxin (C-CTX-2, 13% of toxicity), a minor toxin (approximately 1% of toxicity) and a hydrophobic, fast-acting toxin (approximately 19% of toxicity). The i.p. injection into mice of each toxin induced signs typical of site-5 sodium channel activator toxins such as the Pacific ciguatoxins and brevetoxins. C-CTX-1 and C-CTX-2 were purified to homogeneity (LD50 = 3.6 and approximately 1 microgram/kg, respectively) and subjected to ion spray mass spectrometry. Both lost up to five H2O molecules and each had a [M+H]+ ion, m/z 1141.7, suggesting that C-CTX-1 and -2 are diastereomers that differ from the Pacific family of ciguatoxins. Turbo-assisted HPLC-mass spectrometry identified C-CTX-1, C-CTX-2 and three C-CTX-1-related compounds in an enriched fraction but no Pacific ciguatoxins were detected. The presence of different families of ciguatoxins in ciguateric fish from the Caribbean Sea and Pacific Ocean probably underlies the clinical differences in the ciguatera syndrome reported in these two regions. A Caribbean strain of the benthic dinoflagellate, Gambierdiscus toxicus, is suspected as source of these ciguatoxins. The extent to which these toxins are biotransformed as they pass through the marine food chain remains to be determined.

Ciguatera poisoning was studied in the chicken where it gave rise to internal hypersalivation, acute motor ataxia, low rectal temperature and arrested growth. LD50 figures indicated that the chicken is two to five time at least more sensitive to ciguatoxin than the mouse. So the chick is very convenient for laboratory studies in ciguatera research.

A total of 259 specimens of 32 potentially ciguatoxic fish species from St-Barthelemy, Leeward Islands in the Caribbean sea were checked for ciguatoxin. It was found that flesh and viscera from 229 specimens belonging to 27 species (essentially bottom-dwelling species) were ciguatoxic. No correlation between toxicity and species size or diet was demonstrated since five of the six highly ciguatoxic species Seriola dumerili, Caranx latus, Caranx bartholomaei, Caranx ruber and Scomberomorus cavalla are large roving predators feeding mainly on fishes while the last species (Alphestes afer) is a benthic resident of very small size (less than 300 g) which feeds mainly on invertebrates. Small (less than 1 kg) benthic carnivorous fishes (invertebrate feeders), such as Bodianus rufus, Priacanthus arenatus, Mulloidichtys martinicus, Malacanthus plumieri and Halichoeres radiatus, are significantly ciguatoxic and play an important role in the transmission of ciguatoxin in the food chain. Other ciguatoxic species included large carnivorous fishes and the small surgeon Acanthurus bahianus. Furthermore, additional results suggest that most of edible benthic species from Saint-Barthelemy harbour ciguatoxin as well, though at an extremely low level. The ciguatera food chain in the Caribbean sea is therefore comparable with that described in the Pacific Ocean, except concerning small invertebrate feeders since their role in this chain is demonstrated for the first time.

1. Cigautoxins (CTX) were extracted from flesh and viscera of seven large roving predatory fishes: Caranx bartholomaei, Caranx latus, Seriola dumerili, Alectis crinitus, Scomberomorus cavalla, Sphyraena barracuda and Gymnothorax funebris. 2. Generally each extract consisted of close-related CTX which were separated according to their polarity by Florisil column chromatography into a fast-acting CTX containing group and a slow-acting CTX containing group. 3. The shortest survival time of mice (ts) was low for the former group (less than 10 min) and high for the latter (greater than or equal to 29 min). 4. The level of purity had no influence on the range of ts values. The presence of these two CTX groups in different extracts did not from experimental conditions. 5. Attempts to convert fast-acting CTX to slow-acting CTX and vice-versa were negative. G. funebris and S. barracuda had an especially high content of unstable fast-acting CTX. 6. Purification of the slow-acting CTX was achieved by fast elution chromatography and Sephadex LH20 gel filtration. 7. The ts values of these CTX were identical for five species (40-44 min) but not for S. barracuda (29-32 min). 8. Thus ciguatoxic extracts from Caribbean fish were composed of several close-related CTX.

Ciguatoxin-like substances were extracted from the viscera or the flesh of eight Caribbean fish species, including small invertebrate feeders and large carnivores. The had similar properties, i.e. pharmacological action, solubility, chromatographic behaviour on silicic acid or Sephadex LH 20 column, stability in a weak acid solution and instability in alkaline medium. However, Florisil column and thin-layer chromatography showed different ciguatoxins whose number depended on tissue or species but not on fish trophic level. Less polar ciguatoxins appeared in salted and dried flesh. Thus, fish ciguatoxins are believed to be closely related substances, possibly changing in structure according to particular experimental conditions.

Chick poisoning induced by oral administration of toxic fish tissues or extracts gave rise to internal hypersalivation, decrease in weight and acute motor ataxia. Detoxification was low and repeated administration therefore led to toxin accumulation. Response of the chicken to liver feeding was roughly quantitative; so liver, which is the most potential toxic tissue, may be used for a preventive screening test in ciguatera-endemic areas.

This report deals with some of the biological and chemical properties of a liposoluble poison extracted from Caranx bartholomaei (yellow jack), a toxic fish from the French West Indies (St Barth's island). Within a single fish, poison concentration is higher in the viscera; toxicity level is uniform throughout the flesh. In the few specimens tested, liver toxicity variations parallel those of flesh. The poison is heat stable so that cooking does not impair the toxicity. It is soluble in acetone, diethyl ether, chloroform, benzene, methanol and ethanol, but insoluble in n. hexane. Crude toxin injection or ingestion induces ciguateric disease in cats, mice or newly born chicken. Crude toxin stability is good after 30 minutes at 90 degrees C in a 0,5 N solution of a weak acid but not of a weak base. In the same conditions, loss of activity is nearly complete with a strong acid or a strong base. Moreover, rapid alkali treatment at room temperature destroys more than 50 per cent of the toxicity. The toxin is eluted by chloroform-methanol (9:1) from a silicic acid column and by acetone-methanol (9,5:0,5) from a Florisil column. DEAE cellulose column chromatography clearly separates the toxin into two lethal components; but thin layer chromatography of crude or fractionated toxin indicates only one toxic band in three different solvent systems. As a conclusion, in spite of a few differences related to instability in alkalin medium and elution from DEAE cellulose, this poison is quite similar to those carried by ciguatoxic fishes from the Pacific area. The differences noted above could be accounted for either by a specific metabolism of the species studied or by differences in the causative agent due to geographical location, or both.

Ciguatera toxins were extracted from the tissues of 36 poisonous fishes including 9 dangerous species collected in the Caribbean. Toxicity assays were carried out in mice and the distinctive symptoms of ciguatera poisoning were observed. In a single fish, ciguatoxin was found in the blood, flesh, gonads, gills, heart, skin and bones. The concentration was highest in the viscera and in particular in the liver, kidney and spleen. The ratios of the toxin concentrations of the liver or viscera to that of the flesh were high and varied with the species suggesting that the toxin is stored in different ways in different fish. Subcellular fractionation of liver cells revealed that most of the ciguatoxin was attached to cytoplasmic proteins and that some toxin was probably bound to the membranes.