Analytical and functional profiles of paralytic shellfish toxins extracted from Semele proficua and Senilia senilis from Angola.

Paralytic shellfish poisoning is a foodborne illness that typically derive from the consumption of shellfish contaminated with saxitoxin-group of toxins produced by dinoflagellates of the genus Gymnodinium, Alexandrium and Pyrodinium. N-sulfocarbamoyl, carbamate and dicarbamoyl are the most abundant. In 2007 and 2008 some episodes of PSP occurred in Angola where there is not monitoring program for shellfish contamination with marine biotoxins. Therefore, ten samples extracted from Semele proficua from Luanda Bay and Senilia senilis from Mussulo Bay, were analyzed by HPLC finding saxitoxin, decarbamoylsaxitoxin and other three compounds that have an unusual profile different to the known hydrophilic PSP toxins were found in different amounts and combinations. These new compounds were not autofluorescent, and they presented much stronger response after peroxide oxidation than after periodate oxidation. The compounds appear as peaks eluted at 2.5 and 5.6 min after periodate oxidation and 8.2 min after peroxide oxidation. Electrophysiological studies revealed that none of the three unknown compounds had effect at cellular level by decreasing the maximum peak inward sodium currents by blocking voltage-gated sodium channels. Thus, not contributing to PSP intoxication. The presence in all samples of saxitoxin-group compounds poses a risk to human health and remarks the need to further explore the presence of new compounds that contaminate seafood, investigating their activity and developing monitoring programs.

Synergistic Effect of Brevetoxin BTX-3 and Ciguatoxin CTX3C in Human Voltage-Gated Nav1.6 Sodium Channels.

Emerging marine biotoxins such as ciguatoxins and brevetoxins have been widely and independently studied as food pollutants. Their maximum levels in food components were set without considering their possible synergistic effects as consequence of their coexistence in seafood and their action at the same cellular target. The absolute lack of data and regulations of the possible combined effects that both marine biotoxins may have raised the need to analyze their direct in vitro effects using electrophysiology techniques. The results presented in this study indicate that ciguatoxins and brevetoxins had a synergistic effect on human Nav1.6 voltage-gated sodium channels by hyperpolarizing their activation and inactivation states. The results presented here indicate that brevetoxin 3 (BTX-3) acts as partial agonist of human sodium channels, while ciguatoxin 3C (CTX3C) was a full agonist, explaining the differences in the effect of each toxin in the channel. Therefore, this work sets the cellular basis to further apply this type of studies to other food toxicants that may act synergistically and thus implement the corresponding regulatory limits considering their coexistence and the risks to human and animal health derived from it.

Acute toxicology report of the emerging marine biotoxin Brevetoxin 3 in mice: Food safety implications.

Brevetoxins (PbTxs) are emerging marine toxins that can lead to Neurotoxic Shellfish Poisoning in humans by the ingestion of contaminated seafood. Recent reports on brevetoxin detection in shellfish in regions where it has not been described before, arise the need of updated guidelines to ensure seafood consumers safety. Our aim was to provide toxicological data for brevetoxin 3 (PbTx3) by assessing oral toxicity in mice and comparing it with intraperitoneal administration. We followed an Up-and-Down procedure administering PbTx3 to mice and registering clinical signs, neuromuscular function, histopathology, and blood changes. Neuromuscular dysfunction like seizures and ataxia, as well as loss of limb strength were observed at 6 h. Performance and clinical signs largely improved at 24 h, time at which no blood biochemical or histological alterations were detected independently of the administration route. However, PbTx3 oral administration results in lower toxicity than intraperitoneal administration. Mortality was only observed at 4000 µg/kg bw PbTx3 administered via oral, but we still found toxicity clinical signs at low toxin doses. We could stablish an oral Lowest-Observable-Adverse-Effect-Level for PbTx3 of 100 µg/kg bw and an oral No-Observable-Adverse-Effect-Level of 10 µg/kg bw in mice. The data here reported should be considered in the evaluation of risks of PbTxs for human health.

Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection.

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.

Synergistic effect of environmental food pollutants: Pesticides and marine biotoxins.

Emerging marine biotoxins such as ciguatoxins and pyrethroid compounds, widely used in agriculture, are independently treated as environmental toxicants. Their maximum residue levels in food components are set without considering their possible synergistic effects as consequence of their interaction with the same cellular target. There is an absolute lack of data on the possible combined cellular effects that biological and chemical pollutants, may have. Nowadays, an increasing presence of ciguatoxins in European Coasts has been reported and these toxins can affect human health. Similarly, the increasing use of phytosanitary products for control of food plagues has raised exponentially during the last decades due to climate change. The lack of data and regulation evaluating the combined effect of environmental pollutants with the same molecular target led us to analyse their in vitro effects. In this work, the effects of ciguatoxins and pyrethroids in human sodium channels were investigated. The results presented in this study indicate that both types of compounds have a profound synergistic effect in voltage-dependent sodium channels. These food pollutants act by decreasing the maximum peak inward sodium currents and hyperpolarizing the sodium channels activation, effects that are boosted by the simultaneous presence of both compounds. A fact that highlights the need to re-evaluate their limits in feedstock as well as their potential in vivo toxicity considering that they act on the same cellular target. Moreover, this work sets the cellular basis to further apply this type of studies to other water and food pollutants that may act synergistically and thus implement the corresponding regulatory limits taking into account its presence in a healthy diet.

Intestinal secretory mechanisms in Okadaic acid induced diarrhoea.

Okadaic acid (OA) is an important marine lipophilic phycotoxin responsible for diarrhetic shellfish poisoning (DSP). This toxin inhibits protein phosphatases (PPs) like PP2A and PP1, though, this action does not explain OA-induced toxicity and symptoms. Intestinal epithelia comprise the defence barrier against external agents where transport of fluid and electrolytes from and to the lumen is a tightly regulated process. In some intoxications this balance becomes dysregulated appearing diarrhoea. Therefore, we evaluated diarrhoea in orally OA-treated mice as well as in mice pre-treated with several doses of cyproheptadine (CPH) and then treated with OA at different times. We assessed stools electrolytes and ultrastructural alteration of the intestine, particularly evaluating tight and adherens junctions. We detected increased chloride and sodium faecal concentrations in the OA-exposed group, suggesting a secretory diarrhoea. Pre-treatment with CPH maintains chloride concentration in values similar to control mice. Intestinal cytomorphological alterations were observed for OA mice, whereas CPH pre-treatment attenuated OA-induced damage in proximal colon and jejunum at 2 h. Conversely, tight junctions' distance was only affected by OA in jejunum at the moment diarrhoea occurred. In this study we found cellular mechanisms by which OA induced diarrhoea revealing the complex toxicity of this compound.

In vivo subchronic effects of ciguatoxin-related compounds, reevaluation of their toxicity.

Ciguatoxins are marine compounds that share a ladder-shaped polyether structure produced by dinoflagellates of the genus Gambierdiscus and Fukuyoa, and include maitotoxins (MTX1 and MTX3), ciguatoxins (CTX3C) and analogues (gambierone), components of one of the most frequent human foodborne illness diseases known as ciguatera fish poisoning. This disease was previously found primarily in tropical and subtropical areas but nowadays, the dinoflagellates producers of ciguatoxins had spread to European coasts. One decade ago, the European Food Safety Authority has raised the need to complete the toxicological available data for the ciguatoxin group of compounds. Thus, in this work, the in vivo effects of ciguatoxin-related compounds have been investigated using internationally adopted guidelines for the testing of chemicals. Intraperitoneal acute toxicity was tested for maitotoxin 1 at doses between 200 and 3200 ng/kg and the acute oral toxicity of Pacific Ciguatoxin CTX3C at 330 and 1050 ng/kg and maitotoxin 1 at 800 ng/kg were also evaluated showing not effects on mice survival after a 96 h observation period. Therefore, for the following experiments the oral subchronic doses were between 172 and 1760 ng/kg for gambierone, 10 and 102 ng/kg for Pacific Ciguatoxin CTX3C, 550 and 1760 ng/kg for maitotoxin 3 and 800, 2560 and 5000 ng/kg for maitotoxin 1. The results presented here raise the need to reevaluate the in vivo activity of these agents. Although the intraperitoneal lethal dose of maitotoxin 1 is assumed to be 50 ng/kg, without chemical purity identifications and description of the bioassay procedures, in this work, an intraperitoneal lethal dose of 1107 ng/kg was obtained. Therefore, the data presented here highlight the need to use a common procedure and certified reference material to clearly establish the levels of these environmental contaminants in food.

Current Trends and New Challenges in Marine Phycotoxins.

Marine phycotoxins are a multiplicity of bioactive compounds which are produced by microalgae and bioaccumulate in the marine food web. Phycotoxins affect the ecosystem, pose a threat to human health, and have important economic effects on aquaculture and tourism worldwide. However, human health and food safety have been the primary concerns when considering the impacts of phycotoxins. Phycotoxins toxicity information, often used to set regulatory limits for these toxins in shellfish, lacks traceability of toxicity values highlighting the need for predefined toxicological criteria. Toxicity data together with adequate detection methods for monitoring procedures are crucial to protect human health. However, despite technological advances, there are still methodological uncertainties and high demand for universal phycotoxin detectors. This review focuses on these topics, including uncertainties of climate change, providing an overview of the current information as well as future perspectives.

Determination of the toxicity equivalency factors for ciguatoxins using human sodium channels.

Ciguatoxins (CTXs) which are produced by dinoflagellates of the genus Gambierdiscus and Fukuyoa and share a ladder-shaped polyether structure, are causative compounds of one of the most frequent foodborne illness disease known as ciguatera fish poisoning (CFP). CFP was initially found in tropical and subtropical areas but nowadays the dinoflagellates producers of ciguatoxins had spread to European coasts. Therefore, this raises the need of establishing toxicity equivalency factors for the different compounds that can contribute to ciguatera fish poisoning, since biological methods have been replaced by analytical techniques. Thus, in this work, the effects of six compounds causative of ciguatera, on their main target, the human voltage-gated sodium channels have been analyzed for the first time. The results presented here led to the conclusion that the order of potency was CTX1B, CTX3B, CTX4A, gambierol, gambierone and MTX3. Furthermore, the data indicate that the activation voltage of sodium channels is more sensitive to detect ciguatoxins than their effect on the peak sodium current amplitude.

Serotonin involvement in okadaic acid-induced diarrhoea in vivo.

The consumption of contaminated shellfish with okadaic acid (OA) group of toxins leads to diarrhoeic shellfish poisoning (DSP) characterized by a set of symptoms including nausea, vomiting and diarrhoea. These phycotoxins are Ser/Thr phosphatase inhibitors, which produce hyperphosphorylation in cellular proteins. However, this inhibition does not fully explain the symptomatology reported and other targets could be relevant to the toxicity. Previous studies have indicated a feasible involvement of the nervous system. We performed a set of in vivo approaches to elucidate whether neuropeptide Y (NPY), Peptide YY (PYY) or serotonin (5-HT) was implicated in the early OA-induced diarrhoea. Fasted Swiss female mice were administered NPY, PYY(3-36) or cyproheptadine intraperitoneal prior to oral OA treatment (250 µg/kg). A non-significant delay in diarrhoea onset was observed for NPY (107 µg/kg) and PYY(3-36) (1 mg/kg) pre-treatment. On the contrary, the serotonin antagonist cyproheptadine was able to block (10 mg/kg) or delay (0.1 and 1 mg/kg) diarrhoea onset suggesting a role of 5-HT. This is the first report of the possible involvement of serotonin in OA-induced poisoning.

Targeting Chloride Ion Channels: New Insights into the Mechanism of Action of the Marine Toxin Azaspiracid.

Azaspiracids (AZAs) are marine toxins produced by dinoflagellates belonging to the genera Azadinium and Amphidoma that caused human intoxications after consumption of contaminated fishery products, such as mussels. However, the exact mechanism for the AZA induced cytotoxic and neurotoxic effects is still unknown. In this study several pharmacological approaches were employed to evaluate the role of anion channels on the AZA effects that demonstrated that cellular anion dysregulation was involved in the toxic effects of these compounds. The results presented here demonstrated that volume regulated anion channels (VRACs) are affected by this group of toxins, and, because there is not any specific activator of VRACs besides the intracellular application of GTPγ-S molecule, this group of natural compounds could represent a powerful tool to analyze the role of these channels in cellular homeostasis. In addition to this, in this work, a detailed pharmacological approach was performed in order to elucidate the anion channels present in human HEK293 cells as well as their regulation by the marine toxins azaspiracids. Altogether, the data presented here demonstrated that the effect of azaspiracids in human cells was completely dependent on ATP-regulated anion channels, whose upregulation by these toxins could lead to regulatory volume decrease and underlie the reported toxicity of these compounds.

Partial Blockade of Human Voltage-Dependent Sodium Channels by the Marine Toxins Azaspiracids.

Azaspiracid toxins were first identified at the end of the last century in Irish mussels, and during the last two decades considerable cytotoxic and neurotoxic effects caused by these toxins have been described. Azaspiracids are synthesized by dinoflagellates and accumulate in several species of filter-feeding bivalve mollusks, thereby incorporating into the food chain and causing human intoxications. Among the cellular effects of azaspiracids, inhibition of spikes in neurons and hyperpolarization of the neuronal membrane potential have been reported; however, the underlying processes leading to these effects were never elucidated. In this regard, initial studies reported no activity of the toxin in neuronal voltage-gated sodium channels, and a recent work described no effect of azaspiracid-1 on the inactivation kinetics of voltage-gated sodium channels; however, the relationship between the known alterations of the cytoskeleton caused by these toxins and their effects on ion channels has never been evaluated. In this work, the cytotoxic effect of azaspiracids was evaluated in human cells as well as their activity on voltage-gated sodium channels and in cell morphology in order to unravel the cellular targets involved in the mechanism of action of this group of marine toxins. The data reported here demonstrate, for the first time, that both azaspiracid-1 and azaspiracid-2 caused a rapid concentration-dependent inhibition of the amplitude of voltage-gated sodium currents without affecting their inactivation kinetics, an effect that was increased after long-term treatment of the cells with the toxin. Simultaneously, long-term exposure of the cells to azaspiracids caused a profound alteration of the cell cytoskeleton and decreased the metabolic activity of human cells. Altogether, the data presented here indicate that the partial blockade of voltage-gated sodium channels by these toxins is not related with their effect on the actin cytoskeleton. However, since azaspiracids are common toxins in European waters, their effect on voltage-gated sodium channels, first reported here, should be considered to avoid synergistic toxicity with other marine toxins that are known potent blockers of sodium channels such as the saxitoxins and tetrodotoxins, but further studies are needed in order to elucidate how these compounds alter ion homeostasis.

Detection of Cyclic Imine Toxins in Dietary Supplements of Green Lipped Mussels (Perna canaliculus) and in Shellfish Mytilus chilensis.

Seafood represents a significant part of the human staple diet. In the recent years, the identification of emerging lipophilic marine toxins has increased, leading to the potential for consumers to be intoxicated by these toxins. In the present work, we investigate the presence of lipophilic marine toxins (both regulated and emerging) in commercial seafood products from non-European locations, including mussels Mytilus chilensis from Chile, clams Tawerea gayi and Metetrix lyrate from the Southeast Pacific and Vietnam, and food supplements based on mussels formulations of Perna canaliculus from New Zealand. All these products were purchased from European Union markets and they were analyzed by UPLC-MS/MS. Results showed the presence of the emerging pinnatoxin-G in mussels Mytilus chilensis at levels up to 5.2 µg/kg and azaspiracid-2 and pectenotoxin-2 in clams Tawera gayi up to 4.33 µg/kg and 10.88 µg/kg, respectively. This study confirms the presence of pinnatoxins in Chile, one of the major mussel producers worldwide. Chromatograms showed the presence of 13-desmethyl spirolide C in dietary supplements in the range of 33.2-97.9 µg/kg after an extraction with water and methanol from 0.39 g of the green lipped mussels powder. As far as we know, this constitutes the first time that an emerging cyclic imine toxin in dietary supplements is reported. Identifying new matrix, locations, and understanding emerging toxin distribution area are important for preventing the risks of spreading and contamination linked to these compounds.

In Vivo Evaluation of the Chronic Oral Toxicity of the Marine Toxin Palytoxin.

Palytoxin (PLTX) is one of the most poisonous substances known to date and considered as an emergent toxin in Europe. Palytoxin binds to the Na+-K+ ATPase, converting the enzyme in a permeant cation channel. This toxin is known for causing human fatal intoxications associated with the consumption of contaminated fish and crustaceans such as crabs, groupers, mackerel, and parrotfish. Human intoxications by PLTX after consumption of contaminated fishery products are a serious health issue and can be fatal. Different reports have previously explored the acute oral toxicity of PLTX in mice. Although the presence of palytoxin in marine products is currently not regulated in Europe, the European Food Safety Authority expressed its opinion on PLTX and demanded assessment for chronic toxicity studies of this potent marine toxin. In this study, the chronic toxicity of palytoxin was evaluated after oral administration to mice by gavage during a 28-day period. After chronic exposure of mice to the toxin, a lethal dose 50 (LD50) of 0.44 µg/kg of PLTX and a No-Observed-Adverse-Effect Level (NOAEL) of 0.03 µg/kg for repeated daily oral administration of PLTX were determined. These results indicate a much higher chronic toxicity of PLTX and a lower NOAEL than that previously described in shorter treatment periods, pointing out the need to further reevaluate the levels of this compound in marine products.

Oral Chronic Toxicity of the Safe Tetrodotoxin Dose Proposed by the European Food Safety Authority and Its Additive Effect with Saxitoxin.

Tetrodotoxin (TTX) is a potent natural toxin causative of human food intoxications that shares its mechanism of action with the paralytic shellfish toxin saxitoxin (STX). Both toxins act as potent blockers of voltage-gated sodium channels. Although human intoxications by TTX were initially described in Japan, nowadays increasing concern about the regulation of this toxin in Europe has emerged due to its detection in fish and mollusks captured in European waters. Currently, TTX is only regularly monitored in Dutch fishery products. However, the European Food Safety Authority (EFSA) has established a safety level of 44 µg/kg TTX as the amount of toxin that did not cause adverse effects in humans. This level was extrapolated considering initial data on its acute oral toxicity and EFSA remarked the need for chronic toxicity studies to further reduce the uncertainty of future toxin regulations. Thus, in this work, we evaluated the oral chronic toxicity of TTX using the safety levels initially recommended by EFSA in order to exclude potential human health risks associated with the worldwide expanding presence of TTX. Using internationally recommended guidelines for the assessment of oral chronic toxicity, the data provided here support the proposed safety level for TTX as low enough to prevent human adverse effects of TTX even after chronic daily exposure to the toxin. However, the combination of TTX with STX at doses above the maximal exposure level of 5.3 µg/kg body weight derived by EFSA increased the lethality of TTX, thus confirming that both TTX and paralytic shellfish toxins should be taken into account to assess human health risks.

Reevaluation of the acute toxicity of palytoxin in mice: Determination of lethal dose 50 (LD50) and No-observed-adverse-effect level (NOAEL).

Palytoxin is an emergent toxin in Europe and one of the most toxic substances know to date. The toxin disrupts the physiological functioning of the Na+/K+-ATPase converting the enzyme in a permeant cation channel. Human intoxications by PLTX after consumption of contaminated fishery products are a serious health issue and can be fatal. Several reports have previously investigated the oral and intraperitoneal toxicity of PLTX in mice. However, in all cases short observation periods (24 and 48 h) after toxin administration were evaluated. In this work, single oral or intraperitoneal doses of PLTX were administered to healthy mice and surviving animals were followed up for 96 h. The data obtained here allowed us to calculate the oral and intraperitoneal lethal doses 50 (LD50) which were in the range of the values previously described. Surprisingly, the oral NOAEL for PLTX was more than 10 times lower than that previously described, a fact that indicates the need for the reevaluation of the levels of the toxin in edible fishery products.

Chronic In Vivo Effects of Repeated Exposure to Low Oral Doses of Tetrodotoxin: Preliminary Evidence of Nephrotoxicity and Cardiotoxicity.

Tetrodotoxin (TTX) is one of the most potent naturally occurring neurotoxins. InitiallyTTX was associated with human food intoxications in Japan, but nowadays, concerns about thehuman health risks posed by TTX have increased in Europe after the identification of the toxin infish, marine gastropods, and bivalves captured in European waters. Even when TTX monitoring isnot currently performed in Europe, an acute oral no observable effect level (NOAEL) of 75 µg/kghas been recently established but, to date, no studies evaluating the chronic oral toxicity of TTXhave been released, even when EFSA has highlighted the need for them. Thus, in this work, thechronic effects of low oral TTX doses (below the acute lethal dose 50) were evaluated followinginternationally adopted guidelines. The results presented here demonstrate that low oral doses ofTTX have deleterious effects on renal and cardiac tissues. Moreover, alterations in bloodbiochemistry parameters, urine production, and urinalysis data were already detected at the oraldose of 75 µg/kg after the 28 days exposure. Thus, the data presented here constitute an initialapproach for the chronic evaluation of the in vivo toxicity of tetrodotoxin after its ingestion throughcontaminated fishery products.

Structure Elucidation and Biological Evaluation of Maitotoxin-3, a Homologue of Gambierone, from Gambierdiscus belizeanus.

Gambierdiscus species are the producers of the marine toxins ciguatoxins and maitotoxins which cause worldwide human intoxications recognized as Ciguatera Fish Poisoning. A deep chemical investigation of a cultured strain of G. belizeanus, collected in the Caribbean Sea, led to the identification of a structural homologue of the recently described gambierone isolated from the same strain. The structure was elucidated mainly by comparison of NMR and MS data with those of gambierone and ascertained by 2D NMR data analyses. Gratifyingly, a close inspection of the MS data of the new 44-methylgambierone suggests that this toxin would actually correspond to the structure of maitotoxin-3 (MTX3, m/z 1039.4957 for the protonated adduct) detected in 1994 in a Pacific strain of Gambierdiscus and recently shown in routine monitoring programs. Therefore, this work provides for the first time the chemical identification of the MTX3 molecule by NMR. Furthermore, biological data confirmed the similar activities of both gambierone and 44-methylgambierone. Both gambierone and MTX3 induced a small increase in the cytosolic calcium concentration but only MTX3 caused cell cytotoxicity at micromolar concentrations. Moreover, chronic exposure of human cortical neurons to either gambierone or MTX3 altered the expression of ionotropic glutamate receptors, an effect already described before for the synthetic ciguatoxin CTX3C. However, even when gambierone and MTX3 affected glutamate receptor expression in a similar manner their effect on receptor expression differed from that of CTX3C, since both toxins decreased AMPA receptor levels while increasing N-methyl-d-aspartate (NMDA) receptor protein. Thus, further studies should be pursued to clarify the similarities and differences in the biological activity between the known ciguatoxins and the new identified molecule as well as its contribution to the neurological symptoms of ciguatera.

Synergistic Effect of Transient Receptor Potential Antagonist and Amiloride against Maitotoxin Induced Calcium Increase and Cytotoxicity in Human Neuronal Stem Cells.

Maitotoxins (MTX) are among the most potent marine toxins identified to date causing cell death trough massive calcium influx. However, the exact mechanism for the MTX-induced calcium entry and cytotoxicity is still unknown. In this work, the effect of MTX-1 on the cytosolic free calcium concentration and cellular viability of human neuronal stem cells was evaluated. MTX elicited a concentration-dependent decrease in cell viability which was already evident after 1 h of treatment with 0.25 nM MTX; however, at a concentration of 0.1 nM, the toxin did not cause cell death even after 14 days of exposure. Moreover, the toxin caused a concentration dependent rise in the cytosolic calcium concentration which was maximal at toxin concentrations of 1 nM and dependent on the presence of extracellular calcium on the bathing solution. Several pharmacological approaches were employed to evaluate the role of canonical transient potential receptor channels (TRPC) on the MTX effects. The results presented here lead to the identification of the TRPC4 channels as contributors to the MTX effects in human neuronal cells. Both, the calcium increase and the cytotoxicity of MTX were either fully (for the calcium increase) or partially (in the case of cytotoxicity) reverted by the blockade of canonical TRPC4 receptors with the selective antagonist ML204. Furthermore, the sodium proton exchanger blocker amiloride also partially inhibited the calcium rise and the cell death elicited by MTX while the combination of amiloride and ML204 fully prevented both the cytotoxicity and the calcium rise elicited by the toxin.

Transcriptomic Analysis of Ciguatoxin-Induced Changes in Gene Expression in Primary Cultures of Mice Cortical Neurons.

Ciguatoxins are polyether marine toxins that act as sodium channel activators. These toxins cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents several symptoms in humans including long-term neurological alterations. Earlier work has shown that both acute and chronic exposure of primary cortical neurons to synthetic ciguatoxin CTX3C have profound impacts on neuronal function. Thus, the present work aimed to identify relevant neuronal genes and metabolic pathways that could be altered by ciguatoxin exposure. To study the effect of ciguatoxins in primary neurons in culture, we performed a transcriptomic analysis using whole mouse genome microarrays, for primary cortical neurons exposed during 6, 24, or 72 h in culture to CTX3C. Here, we have shown that the effects of the toxin on gene expression differ with the exposure time. The results presented here have identified several relevant genes and pathways related to the effect of ciguatoxins on neurons and may assist in future research or even treatment of ciguatera. Moreover, we demonstrated that the effects of the toxin on gene expression were exclusively consequential of its action as a voltage-gated sodium channel activator, since all the effects of CTX3C were avoided by preincubation of the neurons with the sodium channel blocker tetrodotoxin.