Addressing the Analytical Challenges for the Detection of Ciguatoxins Using an Electrochemical Biosensor.

The importance of ciguatoxins (CTXs) in seafood safety and their emerging occurrence in locations far away from tropical areas highlight the need for simple and low-cost methods for the sensitive and rapid detection of these potent marine toxins to protect seafood consumers. Herein, an electrochemical immunosensor for the detection of CTXs is presented. A sandwich configuration is proposed, using magnetic beads (MBs) as immobilization supports for two capture antibodies, with their combination facilitating the detection of CTX1B, CTX3C, 54-deoxyCTX1B, and 51-hydroxyCTX3C. PolyHRP-streptavidin is used for the detection of the biotinylated detector antibody. Experimental conditions are first optimized using colorimetry, and these conditions are subsequently used for electrochemical detection on electrode arrays. Limits of detection at the pg/mL level are achieved for CTX1B and 51-hydroxyCTX3C. The applicability of the immunosensor to the analysis of fish samples is demonstrated, attaining detection of CTX1B at contents as low as 0.01 µg/kg and providing results in correlation with those obtained using mouse bioassay (MBA) and cell-based assay (CBA), and confirmed by liquid chromatography coupled to high-resolution mass spectrometry (LC-ESI-HRMS). This user-friendly bioanalytical tool for the rapid detection of CTXs can be used to mitigate ciguatera risk and contribute to the protection of consumer health.

Ciguatoxins and Maitotoxins in Extracts of Sixteen Gambierdiscus Isolates and One Fukuyoa Isolate from the South Pacific and Their Toxicity to Mice by Intraperitoneal and Oral Administration.

Ciguatoxins (CTXs), and possibly maitotoxins (MTXs), are responsible for Ciguatera Fish Poisoning, an important health problem for consumers of reef fish (such as inhabitants of islands in the South Pacific Ocean). The habitational range of the Gambierdiscus species is expanding, and new species are being discovered. In order to provide information on the potential health risk of the Gambierdiscus species, and one Fukuyoa species (found in the Cook Islands, the Kermadec Islands, mainland New Zealand, and New South Wales, Australia), 17 microalgae isolates were collected from these areas. Unialgal cultures were grown and extracts of the culture isolates were analysed for CTXs and MTXs by liquid chromatography tandem mass spectrometry (LC-MS/MS), and their toxicity to mice was determined by intraperitoneal and oral administration. An isolate of G. carpenteri contained neither CTXs nor MTXs, while 15 other isolates (including G. australes, G. cheloniae, G. pacificus, G.honu, and F. paulensis) contained only MTX-1 and/or MTX-3. An isolate of G. polynesiensis contained both CTXs and MTX-3. All the extracts were toxic to mice by intraperitoneal injection, but those containing only MTX-1 and/or -3 were much less toxic by oral administration. The extract of G. polynesiensis was highly toxic by both routes of administration.

Chronic ciguatoxin treatment induces synaptic scaling through voltage gated sodium channels in cortical neurons.

Ciguatoxins are sodium channels activators that cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents with long-term neurological alterations. In central neurons, chronic perturbations in activity induce homeostatic synaptic mechanisms that adjust the strength of excitatory synapses and modulate glutamate receptor expression in order to stabilize the overall activity. Immediate early genes, such as Arc and Egr1, are induced in response to activity changes and underlie the trafficking of glutamate receptors during neuronal homeostasis. To better understand the long lasting neurological consequences of ciguatera, it is important to establish the role that chronic changes in activity produced by ciguatoxins represent to central neurons. Here, the effect of a 30 min exposure of 10-13 days in vitro (DIV) cortical neurons to the synthetic ciguatoxin CTX 3C on Arc and Egr1 expression was evaluated using real-time polymerase chain reaction approaches. Since the toxin increased the mRNA levels of both Arc and Egr1, the effect of CTX 3C in NaV channels, membrane potential, firing activity, miniature excitatory postsynaptic currents (mEPSCs), and glutamate receptors expression in cortical neurons after a 24 h exposure was evaluated using electrophysiological and western blot approaches. The data presented here show that CTX 3C induced an upregulation of Arc and Egr1 that was prevented by previous coincubation of the neurons with the NaV channel blocker tetrodotoxin. In addition, chronic CTX 3C caused a concentration-dependent shift in the activation voltage of NaV channels to more negative potentials and produced membrane potential depolarization. Moreover, 24 h treatment of cortical neurons with 5 nM CTX 3C decreased neuronal firing and induced synaptic scaling mechanisms, as evidenced by a decrease in the amplitude of mEPSCs and downregulation in the protein level of glutamate receptors that was also prevented by tetrodotoxin. These findings identify an unanticipated role for ciguatoxin in the regulation of homeostatic plasticity in central neurons involving NaV channels and raise the possibility that some of the neurological symptoms of ciguatera might be explained by these compensatory mechanisms.

Neurotoxicity and reactive astrogliosis in the anterior cingulate cortex in acute ciguatera poisoning.

Ciguatoxins (CTXs) cause long-term disturbance of cerebral functions. The primary mechanism of neurotoxicity is related to their interaction with voltage-gated sodium channels. However, until now, the neurological targets for CTXs in the brain of intact animals have not been described. In our study, 1 day following oral exposure to 0.26 ng/g of Pacific ciguatoxin 1 (P-CTX-1), we performed in vivo electrophysiological recordings in the rat anterior cingulate cortex (ACC) and identified the increase in spontaneous firings and enhanced responses to visceral noxious stimulation. Local field recordings characterized the P-CTX-1-induced synaptic potentiation and blockage of the induction of electrical stimulation-induced long-term potentiation in the medial thalamus (MT)-ACC pathway. Furthermore, intracerebroventricular administration of P-CTX-1 at doses of 1.0, 5.0, and 10 nM produced a dose-dependent increase in ACC neuronal firings and MT-ACC synaptic transmission. Further studies showed upregulated Na(+) channel expression in astrocytes under pathological conditions. We hypothesized that the astrocytes might have been activated in the ciguatera poisoning in vivo. Increases in glial fibrillary acid protein expression were detected in reactive astrocytes in the rat ACC. The activation of astroglia was further indicated by activation of the gap junction protein connexin 43 and upregulation of excitatory amino acid transporter 2 expression suggesting that glutamate was normally rapidly cleared from the synaptic cleft during acute ciguatera poisoning. However, neurotoxicity and reactive astrogliosis were not detected in the ACC after 7 days of P-CTX-1 exposure. The present results are the first characterization of P-CTX-1-invoked brain cortex neuronal excitotoxicity in vivo and supported the theme that neuron and astroglia signals might play roles in acute ciguatera poisoning.

Bioavailability and intravenous toxicokinetic parameters for Pacific ciguatoxin P-CTX-1 in rats.

Ciguatoxins are sodium channel activator toxins responsible for ciguatera fish poisoning. In this study, we determined the toxicokinetic parameters of the Pacific ciguatoxin P-CTX-1 in rats after an intravenous (iv) dose of 0.13 ng P-CTX-1 per g of body weight. The ciguatoxin activity was assessed over time in blood using the sensitive functional Neuro2a assay. The data were analyzed with a two-compartmental model. After exposure, the ciguatoxin activity exhibited a rapid (alpha half-life of 6 min) and extensive distribution into tissues (apparent steady state volume of distribution of 7.8 L). Ciguatoxin elimination from blood was slower with a beta half-life estimated at 35.5 h. The toxicokinetic parameters determined from this study were compared to data previously obtained after oral and intraperitoneal exposure of rats to 0.26 ng P-CTX-1 per g of body weight. Maximal bioavailability was determined by the area under the concentration curve, and was used to calculate the absolute P-CTX-1 bioavailabilities for oral and intraperitoneal routes of exposures of 39% and 75%, respectively.

Toxicokinetics of the ciguatoxin P-CTX-1 in rats after intraperitoneal or oral administration.

Ciguatoxins are voltage-gated selective algal toxins responsible for ciguatera fish poisoning. In this study we evaluate the toxicokinetics of one of the most common ciguatoxins found in the Pacific, the P-CTX-1, in rat after an oral or intraperitoneal (ip) dose of 0.26 µg/kg body weight. We report levels of ciguatoxin activity assessed over time in blood, urine and feces, and at 4 days in liver, muscle and brain, using the functional in vitro N2A cytotoxicity assay. Following exposure, the ciguatoxin activity exhibited a rapid systemic absorption that was followed by a bi-exponential decline, and data best fit a two-compartment model analysis. Maximum blood concentrations were reached at 1.97 and 0.43 h after the oral and ip dose, respectively. Ciguatoxin elimination from blood was slow with terminal half lives (t(½)ß) estimated at 82 h for oral and 112 h for ip dosing. Ciguatoxin activity remained in liver, muscle and brain 96 h after ip and oral administration. While smaller amounts appeared in the urine, the main excretion route was feces, with peak rates reaching > 10 pg P-CTX-1 equivalents/h in both routes of administration. Assay guided fractionation showed the presence in the feces and liver of peaks of activity corresponding to the P-CTX-1 and to other less polar metabolites. In conclusion, biologically active ciguatoxins are detectable in blood, liver, muscle and brain, and continued to be excreted in urine and feces 4 days following exposure. Blood, as well as urine and feces may be useful matrices for low-invasive testing methods for ciguatera clinical cases.

Repeat exposure to ciguatoxin leads to enhanced and sustained thermoregulatory, pain threshold and motor activity responses in mice: relationship to blood ciguatoxin concentrations.

Ciguatera is a common illness in tropical and subtropical regions that manifests in complex and long-lived symptoms which are more severe in subsequent exposures. This study measures central and peripheral neurologic signs, in parallel with blood toxin levels, in mice exposed once or twice (at 3 days interval) to a sublethal dose of ciguatoxin P-CTX-1 (0.26ng/g via i.p.). Mice were implanted with radiotransmitters to monitor motor activity and core temperature. A single exposure to ciguatoxin elicited an immediate and transient decrease in motor activity and temperature, and subsequent long-lasting thermoregulatory dysfunction resulting in stabilized body temperature around 36.0 degrees C with no observable circadian rhythm. The hypothermic response and the reduced activity were enhanced with a second exposure with 30% of the mice dying within 7h. Measurement of the peripheral nervous system by the tail flick assay revealed increased latency with a single ciguatoxin exposure, and a greater effect following the second exposure. Toxin was measurable in blood up to 3 days following the first exposure; at the 1h time point the concentrations were significantly elevated after a second exposure. These findings indicate an early response to ciguatoxin manifest in a central response to lower body temperature and reduce motor activity and a more persistent effect on the peripheral system leading to spinal heat antinociception and delayed fever-like response. The greater neurological response to a second ciguatoxin exposure was associated with elevated concentrations of ciguatoxin in the blood solely over the first hour of exposure. In conclusion, a single exposure to toxin exerts a significant neurological response which may be enhanced with subsequent exposure.

Uptake, tissue distribution, and excretion of brevetoxin-3 administered to mice by intratracheal instillation.

Brevetoxins are a family of potent lipid-soluble neurotoxins produced by the dinoflagellate Karenia brevis, the organism responsible for Florida red tide. Brevetoxins aerosolized by surf and wind produce irritation of the eyes, nose, and throat in people on or near red tide-affected beaches. The effects of chronic exposures to brevetoxins on healthy and health-compromised individuals are not known. The purpose of this study was to investigate the pulmonary uptake, tissue distribution, and excretion of polyether brevetoxin-3 in mice, a rodent model for investigating the potential systemic adverse health effects associated with repeated brevetoxin inhalation. Male CBA/CaJ mice were administered [3H]brevetoxin-3 by intratracheal instillation. Groups of 3 mice were sacrificed immediately after instillation and at 0.5, 3, 6, 12, 24, 48, and 96 h postinstillation. Four additional mice were placed into metabolism cages for excreta collection up to 168 h postinstillation. Brevetoxin-3 distributed rapidly to all tissues, with the highest initial doses in the liver and gastrointestinal tract. Elimination half-times ranged from approximately 28 h for fat, heart, intestines, kidneys, liver, and muscle to approximately 90 h for brain and testes. The total dose to tissue ranged from 39 ng brevetoxin equivalents-h/g for testes to 406 ng brevetoxin equivalents-h/g for liver. Approximately 90% of excretion had occurred within 96 h, with 11 and 64% of the initial brevetoxin dose excreted in urine and feces, respectively. These results are consistent with earlier reports of rapid absorption and widespread tissue distribution of brevetoxins in rats.

Type B brevetoxins show tissue selectivity for voltage-gated sodium channels: comparison of brain, skeletal muscle and cardiac sodium channels.

Brevetoxins and ciguatoxins are two classes of phycotoxins which exert their toxic effect by binding to site-5 of voltage-gated sodium channels. Sodium channels, a family of at least 10 structurally different proteins, are responsible for the rising phase of the action potential in membranes of neuronal, cardiac and muscular excitable cells. This work is a comparative study of the binding properties and the cytotoxic effects of ciguatoxins and brevetoxins on human embryonic cells (HEK) stably expressing either the skeletal muscle (Na(v)1.4), or the cardiac (Na(v)1.5) sodium channel alpha-subunit isoforms. We report that type A (PbTx-1) and type B (PbTx-3 and PbTx-2) brevetoxins as well as ciguatoxins target both cardiac and muscle channels; type B brevetoxins show isoform selectivity, presenting a lower affinity for the heart than the skeletal muscle channel. The lower selectivity of type B brevetoxins for heart sodium channels may result from a more rigid backbone structure than is found in type A brevetoxins and ciguatoxins.

Pathological effects on mice by gambierol, possibly one of the ciguatera toxins.

Gambierol was isolated from Gambierdiscus toxicus, which causes ciguatera fish poisoning. The acute toxicological effects induced in mice by synthesized gambierol were studied. The lethal doses were about 80 microg/kg by i.p. and i.v., and 150 microg/kg by p.o. The main injury by this toxin was observed in the lung, and secondary in the heart, resulting in systemic congestion. Another toxic effect was seen in the stomach, inducing hypersecretion and ulceration. With survival from the severe stage during the initial 3 h, recovery was favorable, especially after 4 days. Additional effects were not evident during 1-week post-administration observation.

Morphological observations of diarrhea in mice caused by experimental ciguatoxicosis.

Diarrhea and morphological influences on digestive tracts caused by ciguatoxin (CTX) were observed in mice microscopically. The lethal doses and clinical symptoms caused by i.p. administration were almost the same as those by p.o., the prominent difference being diarrhea that was caused only by i.p. route. The diarrhea was caused by a dose from 1/7 MU to 1 MU of CTX (MU: mouse unit, to kill a mouse of 15 g in 24 hr, corresponding to 7 ng of pure CTX), but not at lower or higher doses. In this study, we used an i.p. dose of 4/5 MU (10.4 ng/28 g). Diarrhea started within 10 min after administration and lasted until 30 min. The changes were observed in the large intestine; namely, CTX accelerated mucus secretion and peristalsis in the colon and stimulated defecation at the rectum, resulting in prominent diarrhea. In the colon, a large quantity of mucus was secreted from even immature goblet cells, and epithelial cell damages were observed in the upper portion of the large intestine but not in the latter half. The morphological changes caused by CTX in the upper portion of the large intestine were similar to those seen with cholera toxin.

Dinoflagelados (pyrrhophyta) del archipiélago los Roques (Venezuela): familias prorocentraceae y ostreopsidaceae / Dinoflagellates (pyrrhophyta) from the los Roques archipielago (Venezuela): families prorocentraceae and ostreopsidaceae

Se examinaron muestras provenientes del Parque Nacional Archipiélago Los Roques, en el Mar Caribe Sur-Oriental (Venezuela), mediante microscopía óptica. Se describen quince (15) especies de dinoflagelados (Pyrrhophyta) pertenecientes a las familias Procentraceae y Ostreopsidaceae. Los símbolos (*) y (**) señalan nuevos registros para Venezuela y el Mar Caribe respectivamente. Se encontraron diez especies para Prorocetraceae: Mesoporos perforatus (Gran) Lillick, Prorocentrum compressum (Bailey) Abé ex Dodge, *Prorocentrum concavum Fukuyo, *Prorocentrum emarginatum Fukuyo, *Prorocentrum gracile Schütt, Prorocentrum lima (Ehrenberg) Dodge, Prorocentrum micans Ehrenberg, *Prorocentrum rhathymun loeblich III, Sherley & Schmidt, *Prorocentrum scutellum Schröder y Prorocentrum triestinum Schiller. En Ostreopsidaceae se encontraron cinco especies: Coolia monotis Meunier, *Gambierdiscus toxicus Adachi&Fukuyo, ** Ostreopsis lenticularis Schmidt, *Ostreopsis ovata Fukuyo y **Ostreopsis siamensis Schmidt. Finalmente se presenta una clave para las especies Prorocentrum y Ostreopsis encontradas en este estudio.

Ciguatera and mannitol: in vivo and in vitro assessment in mice.

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.

[Biological assay of ciguatoxin in the chicken: analysis of the symptoms induced and toxicity of extracts of ciguatoxic chickens of the island of Saint Barthelemy]. / Controle biologique de la ciguatoxine chez le poussin: analyse des symptômes induits et de la toxicité d'extraits de poissons ciguatoxiques de l'île de Saint-Barthélémy.

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.