ciguatoxins and Neuroblastoma

Ciguatera fish poisoning (CFP) is a foodborne illness affecting > 50,000 people worldwide annually. It is caused by eating marine invertebrates and fish that have accumulated ciguatoxins (CTXs). Recently, the risk of CFP to human health, the local economy, and fishery resources have increased; therefore, detection methods are urgently needed. Functional assays for detecting ciguatoxins in fish include receptor binding (RBA) and neuroblastoma cell-based assay (N2a assay), which can detect all CTX congeners. In this study, we made these assays easier to use. For RBA, an assay was developed using a novel near-infrared fluorescent ligand, PREX710-BTX, to save valuable CTXs. In the N2a assay, a 1-day assay was developed with the same detection performance as the conventional 2-day assay. Additionally, in these assays, we used calibrated CTX standards from the Pacific determined by quantitative NMR for the first time to compare the relative potency of congeners, which differed significantly among previous studies. In the RBA, there was almost no difference in the binding affinity among congeners, showing that the differences in side chains, stereochemistry, and backbone structure of CTXs did not affect the binding affinity. However, this result did not correlate with the toxic equivalency factors (TEFs) based on acute toxicity in mice. In contrast, the N2a assay showed a good correlation with TEFs based on acute toxicity in mice, except for CTX3C. These findings, obtained with calibrated toxin standards, provide important insights into evaluating the total toxicity of CTXs using functional assays.

Topics: Animals; Ciguatera Poisoning; Ciguatoxins; Fishes; Humans; Mice; Neuroblastoma; Protein Binding

The growing concern about ciguatera fish poisoning (CF) due to the expansion of the microorganisms producing ciguatoxins (CTXs) increased the need to develop a reliable and fast method for ciguatoxin detection to guarantee food safety. Cytotoxicity assay on the N2a cells sensitized with ouabain (O) and veratridine (V) is routinely used in ciguatoxin detection; however, this method has not been standardized yet. This study demonstrated the low availability of sodium channels in the N2a cells, the great O/V damage to the cells and the cell detachment when the cell viability is evaluated by the classical cytotoxicity assay and confirmed the absence of toxic effects caused by CTXs alone when using the methods that do not require medium removal such as lactate dehydrogenase (LDH) and Alamar blue assays. Different cell lines were evaluated as alternatives, such as human neuroblastoma, which was not suitable for the CTX detection due to the greater sensitivity to O/V and low availability of sodium channels. However, the HEK293 Nav cell line expressing the α1.6 subunit of sodium channels was sensitive to the ciguatoxin without the sensitization with O/V due to its expression of sodium channels. In the case of sensitizing the cells with O/V, it was possible to detect the presence of the ciguatoxin by the classical cytotoxicity MTT method at concentrations as low as 0.0001 nM CTX3C, providing an alternative cell line for the detection of compounds that act on the sodium channels.

Topics: Animals; Ciguatera Poisoning; Ciguatoxins; HEK293 Cells; Humans; Mice; Neuroblastoma; Sodium Channels

Topics: Animals; Cell Line, Tumor; Ciguatoxins; Marine Toxins; Neuroblastoma; Ouabain; Oxocins; Veratridine

Topics: Animals; Biological Assay; Cell Line, Tumor; Cell Proliferation; Cell Survival; Ciguatoxins; Dose-Response Relationship, Drug; Fishes; Limit of Detection; Marine Toxins; Mice; Neuroblastoma; Neurons; Ouabain; Oxocins; Reproducibility of Results; Saxitoxin; Time Factors; Veratridine; Voltage-Gated Sodium Channel Agonists; Voltage-Gated Sodium Channels

Invasive Indo-Pacific lionfish (Pterois volitans) have rapidly expanded in the Western Atlantic over the past decade and have had a significant negative impact on reef fish biodiversity, habitat, and community structure, with lionfish out-competing native predators for resources. In an effort to reduce this population explosion, lionfish have been promoted for human consumption in the greater Caribbean region. This study examined whether the geographical expansion of the lionfish into a known ciguatera-endemic region can pose a human health threat for ciguatera fish poisoning (CFP). More than 180 lionfish were collected from waters surrounding the US Virgin Islands throughout 2010 and 2011. Ciguatoxin testing included an in vitro neuroblastoma cytotoxicity assay for composite toxicity assessment of sodium-channel toxins combined with confirmatory liquid chromatography tandem mass spectrometry. A 12% prevalence rate of ciguatoxic lionfish exceeding the FDA guidance level of 0.1 µg/kg C-CTX-1 equivalents was identified in fish from the U.S. Virgin Islands, highlighting a potential consumption risk in this region. This study presents the first evidence that the invasive lionfish, pose a direct human health risk for CFP and highlights the need for awareness and research on this food safety hazard in known endemic areas.

Topics: Animals; Atlantic Ocean; Biodiversity; Caribbean Region; Chromatography, High Pressure Liquid; Ciguatera Poisoning; Ciguatoxins; Ecosystem; Fishes; Food Safety; Humans; Indicators and Reagents; Marine Biology; Marine Toxins; Meat; Neuroblastoma; Predatory Behavior; Seafood; Sodium Channel Blockers; Tandem Mass Spectrometry; Toxicity Tests; United States Virgin Islands

Ciguatera fish poisoning (CFP), a disease caused by consuming fish that have accumulated ciguatoxins (CTXs) in their tissue, is regarded as the most prevalent form of intoxication in French Polynesia. Recently, the Australes, one of the least affected archipelago until the early 1980s, has shown a dramatic increase in its incidence rates in 2009 with unusual CFP cases. In the present work, potential health hazards associated with the proliferation of various marine phytoplankton species and the consumption of fish and marine invertebrates highly popular among local population were assessed in three Australes islands: Raivavae, Rurutu and Rapa. Extracts from the marine dinoflagellates Gambierdiscus, Ostreospis and mat-forming cyanobacteria as well as fish, giant clams and sea urchin samples were examined for the presence of CTXs and palytoxin (PLTX) by using the neuroblastoma cell-based assay (CBA-N2a). Cytotoxic responses observed with both standards (Pacific CTX-3C and PLTX) and targeted marine products indicate that CBA-N2a is a robust screening tool, with high sensitivity and good repeatability and reproducibility. In Rurutu and Raivavae islands, our main findings concern the presence of CTX-like compounds in giant clams and sea urchins, suggesting a second bio-accumulation route for CFP toxins in the ciguatera food chain. In Rapa, the potential CFP risk from Gambierdiscus bloom and fish was confirmed for the first time, with levels of CTXs found above the consumer advisory level of 0.01 ng Pacific CTX-1B g(-1) of flesh in three fish samples. However, despite the presence of trace level of PLTX in Ostreopsis natural assemblages of Rapa, no sign of PLTX accumulation is yet observed in tested fish samples. Because this multi-toxinic context is likely to emerge in most French Polynesian islands, CBA-N2a shows great potential for future applications in the algal- and toxin-based field monitoring programmes currently on hand locally.

Topics: Cell Line, Tumor; Ciguatera Poisoning; Ciguatoxins; Humans; Neuroblastoma; Polynesia; Reproducibility of Results; Seafood

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.

Topics: Administration, Oral; Animals; Area Under Curve; Biological Availability; Cell Line, Tumor; Cell Survival; Ciguatera Poisoning; Ciguatoxins; Half-Life; Injections, Intraperitoneal; Injections, Intravenous; Male; Mice; Neuroblastoma; Rats; Rats, Sprague-Dawley; Sodium Channels

The ouabain/veratridine-dependent neuroblastoma (neuro-2a) cell-based assay (CBA) was applied for the determination of the presence of ciguatoxin (CTX)-like compounds in ciguatera-suspected fish samples caught in the Canary Islands. In order to avoid matrix interferences the maximal concentration of wet weight fish tissue exposed to the neuro-2a cells was set at 20 mg tissue equivalent (TE) ml(-1) according to the sample preparation procedure applied. In the present study, the limit of quantification (LOQ) of CTX1B equivalents in fish extract was set at the limit of detection (LOD), being defined as the concentration of CTX1B equivalents inhibiting 20% cell viability (IC(20)). The LOQ was estimated as 0.0096 ng CTX1B eq.g TE(-1) with 23-31% variability between experiments. These values were deemed sufficient even though quantification given at the IC(50) (the concentration of CTX1B equivalents inhibiting 50% cell viability) is more accurate with a variability of 17-19% between experiments. Among the 13 fish samples tested, four fish samples were toxic to the neuro-2a cells with estimations of the content in CTX1B g(-1) of TE ranging from 0.058 (± 0.012) to 6.23 (± 0.713) ng CTX1B eq.g TE(-1). The high sensitivity and specificity of the assay for CTX1B confirmed its suitability as a screening tool of CTX-like compounds in fish extracts at levels that may cause ciguatera fish poisoning. Species identification of fish samples by DNA sequence analysis was conducted in order to confirm tentatively the identity of ciguatera risk species and it revealed some evidence of inadvertent misidentification. Results presented in this study are a contribution to the standardisation of the neuro-2a CBA and to the risk analysis for ciguatera in the Canary Islands.

Topics: Animals; Base Sequence; Cell Line, Tumor; Ciguatera Poisoning; Ciguatoxins; DNA Primers; Fishes; Humans; Inhibitory Concentration 50; Limit of Detection; Neuroblastoma; Spain

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.. 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).. 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.. These results confirm further the potential of H. foertherianum in the treatment of Ciguatera Fish Poisoning.

Topics: Animals; Cell Line, Tumor; Ciguatera Poisoning; Ciguatoxins; Cinnamates; Depsides; Heliotropium; Medicine, Traditional; Mice; Neuroblastoma; Pacific Islands; Phytotherapy; Plant Extracts; Plant Leaves; Rosmarinic Acid

The applicability of a new enzyme-linked immunoassay (ELISA) for detecting ciguatoxin (CTX) in fish tissue was evaluated by testing three fish species commonly implicated in ciguatera fish poisoning in Hawaii. A total of 164 individual almaco jack (Seriola rivoliana) and greater amberjack (S. dumerili) and a total of 175 individuals of the blue-spotted grouper (Cephalopholis argus) were caught at various locations in the Hawaiian Islands. Muscle tissue from each individual was assessed for the presence of CTX using two methods: a semi-quantitative ELISA that was recently developed for detecting picogram levels of CTX in fish extract and a neuroblastoma (NB) cell assay commonly used to screen for marine toxins in fish. Results of the tests were highly correlated, with the ELISA indicating the presence of CTX in 9.4% of all fish samples, and the NB assay indicating toxicity in 6.8% of the fish samples. We conclude that the ELISA produces reliable and accurate results that are consistent with those provided by the accepted NB assay and that the ELISA has potential for future applications in screening fish populations for CTX.

Topics: Animals; Biological Assay; Cell Survival; Ciguatera Poisoning; Ciguatoxins; Environmental Monitoring; Enzyme-Linked Immunosorbent Assay; Food Contamination; Mice; Muscle, Skeletal; Neuroblastoma; Perciformes; Reproducibility of Results; Seafood; Tumor Cells, Cultured

A sandwich enzyme-linked immunosorbent assay was developed to detect ciguatoxin (CTX) in fish tissue. The assay utilizes two antibodies, chicken immunoglobulin Y specific to the ABCD domain of CTX and a mouse monoclonal immunoglobulin G-horseradish peroxidase conjugate specific to the JKLM domain of CTX. The sensitivity, working range, cross reactivity, accuracy, precision, and reproducibility were examined.

Topics: Animals; Antibodies, Monoclonal; Biological Assay; Chickens; Ciguatera Poisoning; Ciguatoxins; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Immunoglobulins; Mice; Neuroblastoma; Poisons; Predictive Value of Tests; Reproducibility of Results; Seafood; Tumor Cells, Cultured

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.

Topics: Animals; Cell Line, Tumor; Ciguatoxins; Epitopes; Guinea Pigs; Heart Atria; Male; Mice; Myocardial Contraction; Neuroblastoma; Neurons; Seafood; Sodium Channels; Structure-Activity Relationship; Swine

Gambierol is a polycyclic ether toxin with the same biogenetic origin as ciguatoxins. Gambierol has been associated with neurological symptoms in humans even though its mechanism of action has not been fully characterized.. We studied the effect of gambierol in human neuroblastoma cells by using bis-oxonol to measure membrane potential and FURA-2 to monitor intracellular calcium.. We found that this toxin: i) produced a membrane depolarization, ii) potentiated the effect of veratridine on membrane potential iii) decreased ciguatoxin-induced depolarization and iv) increased cytosolic calcium in neuroblastoma cells.. These results indicate that gambierol modulate ion fluxes by acting as a partial agonist of sodium channels.

Topics: Calcium; Cell Polarity; Ciguatoxins; Ethers, Cyclic; Humans; Membrane Potentials; Neuroblastoma; Neurons; Polycyclic Compounds; Saxitoxin; Sodium Channels; Sodium-Calcium Exchanger; Tumor Cells, Cultured; Veratridine

Ciguatera is a human food poisoning caused by consumption of tropical and subtropical fish that have, through their diet, accumulated ciguatoxins in their tissues. This study used laboratory mice to investigate the potential to apply blood collection cards to biomonitor ciguatoxin exposure. Quantitation by the neuroblastoma cytotoxicity assay of Caribbean ciguatoxin (C-CTX-1) spiked into mice blood was made with good precision and recovery. The blood collected from mice exposed to a sublethal dose of Caribbean ciguatoxic extract (0.59 ng/g C-CTX-1 equivalents) was analyzed and found to contain detectable toxin levels at least 12 h post-exposure. Calculated concentration varied from 0.25 ng/ml at 30 min post-exposure to 0.12 ng/ml at 12 h. A dose response mice exposure revealed a linear dose-dependent increase of ciguatoxin activity in mice blood, with more polar ciguatoxin congeners contributing to 89% of the total toxicity. Finally, the toxin measurement in mice blood exposed to toxic extracts from the Indian Ocean or from the Pacific Ocean showed that the blood collection card method could be extended to each of the three known ciguatoxin families (C-CTX, I-CTX and P-CTX). The low matrix effect of extracted dried-blood samples (used at 1:10 or 1:20 dilution) and the high sensitivity of the neuroblastoma assay (limit of detection 0.006 ng/ml C-CTX-1), determined that the blood collection card method is suitable to monitor ciguatoxin at sublethal doses in mice and opens the potential to be a useful procedure for fish screening, environmental risk assessment or clinical diagnosis of ciguatera fish poisoning in humans or marine mammals.

Topics: Animals; Ciguatera Poisoning; Ciguatoxins; Dose-Response Relationship, Drug; Environmental Exposure; Gas Chromatography-Mass Spectrometry; Mice; Models, Animal; Neuroblastoma; Pacific States; Risk Assessment; Time Factors

Clinical reports and descriptions of chronic fatigue syndrome (CFS) and chronic ciguatera fish poisoning (CCFP) show great similarities in clinical symptomology. These similarities in the literature suggested the exploration of lipids in sera of CFS, CCFP, and other diseases with the membrane immunobead assay (MIA), which is typically used for screening ciguateric ocean fish. Sera from patients with other diseases, including hepatitis B, cancer, and diabetes, were included to assess the degree of specificity involved. Sera were treated with acetone in a ratio of 1 part serum to 4 parts acetone. The suspension was centrifuged, and the acetone layer was evaporated. The residue was weighed and redissolved in 1.0 mL methanol and tested by the MIA, undiluted and titered to 1:160. The undiluted acetone fraction of the 37 normal showed +/- activity to +activity with 16 no titer, 15 with 1:5 titer and two with 1:10 titer, and four with > or =1:40 titers. One hundred fifteen CFS sera showed 1 with 1+ and 114 with 2+ activity in the undiluted samples, 1 with 1:10 titer, 3 with 1:20 titer, 31 with 1:40 titer, 50 with 1:80 titer, and 30 with 160 titer. Thus 95.6% of the samples had > or =1:40 titer. Eight hepatitis B sera samples had > or =1:40 titers. Four CCFP samples had > or =1:40 titers. Three of 16 cancer samples had 1:40 titer. These data are summarized in Fig. 1. As shown in Table 1, a significant increase (P<0.001) in the chronic phase lipids (CPLs) was shown relative to the normal group. A preliminary chemical study in C18 octadecylsilyl columns showed all fractions (100% chloroform, 9:1 chloroform : methanol, 1:1 chloroform : methanol, and 100% methanol) to contain lipids reactive to MAb-CTX with different intensities. Prostaglandins were shown in 100% methanol fraction. Competitive MIA with crude fish ciguatoxin and CFS with synthetic JKLM ciguatoxin epitope suggested similarities in structure with ciguatoxin. This was compatible with the neuroblastoma assay demonstrated in the C(18) column fractions 9:1 and 1:1, chloroform : methanol solvents.

Topics: Acute Disease; Animals; Antibodies, Monoclonal; Cell Line, Tumor; Cell Survival; Chronic Disease; Ciguatera Poisoning; Ciguatoxins; Epitopes; Fatigue Syndrome, Chronic; Female; Hepatitis B; Humans; Lipids; Male; Mice; Neoplasms; Neuroblastoma

Ciguatoxins (CTXs) and brevetoxins (PbTxs) modify the activation and inactivation processes of voltage-sensitive sodium channels (VSSC). In this study, the specific binding to rat brain synaptosomes of two commercial PbTxs, five purified CTXs and their derivatives was evaluated in competition with various concentrations of radiolabelled brevetoxin ([3H]PbTx-3). The results indicate that all CTXs bind specifically and with high affinity to sodium channels. Statistical analysis of the calculated inhibition constants identified two classes of toxins: the PbTxs and the less polar CTXs, and a group of CTXs of very high affinity. Relatively small chemical differences between the CTXs gave rise to significant differences in their affinity to the rat brain sodium channels. Cytotoxic effects associated with sodium channel activation were evaluated for the two classes of toxins on murine neuroblastoma cells, and their acute toxicity was determined in mice. CTXs have shown high affinities to VSSC of rat brain membranes and strong cytotoxic effects on neuroblastoma cells which correlate with their very low LD50 in mice. For PbTxs, it is different. Although binding with high affinity to VSSC and giving rise to significant cytotoxic effects, they are known to be poorly toxic intraperitoneally to mice. Furthermore, within the CTXs family, even though the most toxic compound (CTX-1B) has the highest affinity and the less toxic one (CTX-4B) the lowest affinity, a detailed analysis of the data pointed out a complex situation: (i) high affinity and toxicity seem to be related to the hydroxylation of the molecule on the A-ring rather than to the backbone type, (ii) acute toxicity in mice does not follow exactly the sodium-dependent cytotoxicity on neuroblastoma cells. These data suggest that the high toxicity of CTXs is related to sodium-dependent disturbances of the excitable membranes but might also involve other cellular mechanisms.

Topics: Animals; Brain Neoplasms; Ciguatoxins; Female; In Vitro Techniques; Ion Channel Gating; Lethal Dose 50; Male; Marine Toxins; Mice; Neuroblastoma; Neurotoxins; Oxocins; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Structure-Activity Relationship; Synaptosomes

We have modified the cell-based directed cytotoxicity assay for sodium channel and calcium channel active phycotoxins using a c-fos-luciferase reporter gene construct. In this report we describe the conceptual basis to the development of reporter gene assays for algal-derived toxins and summarize both published and unpublished data using this method. N2A mouse neuroblastoma cells, which express voltage-dependent sodium channels, were stably transfected with the reporter gene c-fos-luc, which contains the firefly luciferase gene under the transcriptional regulation of the human c-fos response element. The characteristics of the N2A reporter gene assay were determined by dose response with brevetoxin and ciguatoxin. Brevetoxin-1 and ciguatoxin-1 induced c-fos-luc with an EC50 of 4.6 and 3.0 ng ml(-1), respectively. Saxitoxin caused a concentration-dependent inhibition of brevetoxin-1 induction of c-fos-luc with an EC50 of 3.5 ng ml(-1). GH4C1 rat pituitary cells, which lack voltage-dependent sodium channels but express voltage-dependent calcium channels, were also stably transfected with the c-fos-luc. GH4C1 cells expressing c-fos-luciferase were responsive to maitotoxin (1 ng ml(-1)) and a putative toxin produced by Pfiesteria piscicida. Although reporter gene assays are not designed to replace existing detection methods used to measure toxin activity in seafood, they do provide a valuable means to screen algal cultures for toxin activity, to conduct assay-guided fractionation and to characterize pharmacologic properties of algal toxins.

Topics: Animals; Biological Assay; Ciguatoxins; Dose-Response Relationship, Drug; Genes, fos; Genes, Reporter; Luciferases; Marine Toxins; Mice; Neuroblastoma; Neurotoxins; Oxocins; Pfiesteria piscicida; Pituitary Gland; Proto-Oncogene Proteins c-fos; Rats; RNA, Messenger; Saxitoxin; Transfection; Tumor Cells, Cultured

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.

Topics: Animals; Bradykinin; Calcium; Ciguatoxins; Eels; Fluorometry; Fura-2; Glioma; Hybrid Cells; Mice; Neuroblastoma; Rats; Sodium; Tetrodotoxin; Tumor Cells, Cultured

In the present study we have developed an assay for the detection of sodium channel-specific marine toxins based upon mitochondrial dehydrogenase activity in the presence of veratridine and ouabain. This cell bioassay allows detection of either sodium channel enhancers, such as the brevetoxins and the ciguatoxins, or sodium channel blocking agents, such as the saxitoxins. The assay responds in a dose dependent manner and differentiates the toxic activity as either sodium channel blocking or enhancing. In addition, the assay is highly sensitive, with present detection limits of 2 ng/ml for either saxitoxins or brevetoxins (PbTx-1 and PbTx-3). Assay response to a ciguatoxic extract and to brevetoxins is rapid, allowing dose dependent detection within 4 to 6 h. The method is simple, utilizes readily available reagents, uses substantially less sample than required for mouse bioassay, and is well within the scope of even modest tissue culture facilities. This cell-based protocol has the potential to serve as an alternate and complementary method to the standard mouse bioassay.

Topics: Animals; Biological Assay; Cell Line; Cell Survival; Ciguatoxins; Marine Toxins; Mice; Neuroblastoma; Neurotoxins; Oxocins; Saxitoxin; Sodium Channels; Tetrazolium Salts; Tumor Cells, Cultured

Topics: Animals; Calcium; Ciguatoxins; Glioma; Hybrid Cells; Mice; Neuroblastoma; Neurotransmitter Agents; Rats; Synaptic Vesicles; Tetrodotoxin; Tumor Cells, Cultured

Purified ciguatoxin at 0.1 to 10 ng/ml inhibits the net accumulation of neurotransmitters (gamma-aminobutyric acid and dopamine) by brain synaptosomes. This action is due to a stimulation of neurotransmitter release. The half-maximum effect of the toxin is observed at 0.62 ng/ml. The effect of ciguatoxin is completely inhibited by tetrodotoxin (K0.5 = 4 nM). Electrophysiological studies on neuroblastoma cells indicate that ciguatoxin induces a membrane depolarization which is prevented by tetrodotoxin and which is due to an action that increases Na+ permeability. Under appropriate conditions ciguatoxin creates spontaneous oscillations in the membrane polarization level and repeated action potentials. Ciguatoxin stimulates 22Na+ entry through the voltage-dependent Na+ channels of neuroblastoma cells and rat skeletal myoblasts when it is used in synergy with veratridine, batrachotoxin, pyrethroids, sea anemone, or scorpion toxins. The half-maximum effect of ciguatoxin on 22Na+ flux in the presence of veratridine occurs at a concentration of 0.5 ng/ml. Stimulation of 22Na+ flux by ciguatoxin is abolished by tetrodotoxin. These results taken together indicate that ciguatoxin belongs to a new class of toxins acting on Na+ channels.

Topics: Animals; Brain; Cell Line; Ciguatoxins; Dopamine; Fishes; gamma-Aminobutyric Acid; Ion Channels; Kinetics; Marine Toxins; Mice; Muscles; Neuroblastoma; Rats; Rats, Inbred Strains; Sodium; Sodium-Potassium-Exchanging ATPase; Synaptosomes