Neurotoxic Shellfish Poisoning Research Articles

Acute Effects of Brevetoxin-3 Administered via Oral Gavage to Mice.

Brevetoxins (BTXs) constitute a family of lipid-soluble toxic cyclic polyethers mainly produced by Karenia brevis, which is the main vector for a foodborne syndrome known as neurotoxic shellfish poisoning (NSP) in humans. To prevent health risks associated with the consumption of contaminated shellfish in France, the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) recommended assessing the effects of BTXs via an acute oral toxicity study in rodents. Here, we investigated the effect of a single oral administration in both male and female mice with several doses of BTX-3 (100 to 1,500 µg kg-1 bw) during a 48 h observation period in order to provide toxicity data to be used as a starting point for establishing an acute oral reference dose (ARfD). We monitored biological parameters and observed symptomatology, revealing different effects of this toxin depending on the sex. Females were more sensitive than males to the impact of BTX-3 at the lowest doses on weight loss. For both males and females, BTX-3 induced a rapid, transient and dose-dependent decrease in body temperature, and a transient dose-dependent reduced muscle activity. Males were more sensitive to BTX-3 than females with more frequent observations of failures in the grip test, convulsive jaw movements, and tremors. BTX-3's impacts on symptomatology were rapid, appearing during the 2 h after administration, and were transient, disappearing 24 h after administration. The highest dose of BTX-3 administered in this study, 1,500 µg kg-1 bw, was more toxic to males, leading to the euthanasia of three out of five males only 4 h after administration. BTX-3 had no effect on water intake, and affected neither the plasma chemistry parameters nor the organs' weight. We identified potential points of departure that could be used to establish an ARfD (decrease in body weight, body temperature, and muscle activity).

Determination of neurotoxic shellfish poisoning toxins in shellfish by liquid chromatography-tandem mass spectrometry coupled with dispersive solid phase extraction

An innovative method based on dispersive solid phase extraction (d-SPE) in conjunction with LC-MS/MS had been developed for the simultaneous quantitative determination of three brevetoxins (BTXs), which can result in neurotoxic shellfish poisoning (NSP), in shellfish. The toxins were extracted with a 50 % acetonitrile (v/v) and cleaned by alumina-neutral sorbent. After chromatographic separation on a C18 column, the analytes were qualitatively and quantitatively detected using multiple reaction monitoring (MRM) in positive ionization mode. The created approach was validated by SANTE 11312/2021. The LOQs were 5 μg/kg for each toxin, below the advised regulatory limit of 800 μg BTX-2/kg. The mean recoveries of brevetoxins were in the range of 75.9 %–114.1 %, and the ranges of their intra- and inter-day precisions were 0.9–9.7 % and 0.6–7.2 %, respectively. The matrix effects for three BTXs in four shellfish matrices were in the range of 85.6 %–114.8 %. The method demonstrated great consistency and high sensitivity, and it can meet the requirements of daily monitoring.

Clam calamity: five concurrent cases of neurotoxic shellfish poisoning with varying presentations following ingestion of clams from Gulf of Mexico water contaminated with Karenia brevis confirmed by serum brevetoxin assays

Introduction Karinia brevis, a marine dinoflagellate, is the causative organism for “red-tide” on the east coast of Florida.This microbe produces brevetoxins, which bioaccumulate in filter feeding bivalve shellfish. In humans, inhalational exposure is common, while ingestion of contaminated shellfish is more rare. Ingested brevetoxin causes gastrointestinal and neurological symptoms collectively known as neurotoxic shellfish poisoning. Case cluster A group of tourists collected clams from a beach during a red tide event. The clams were soaked in brine, microwaved, and consumed for lunch. The index patient experienced seizure-like activity postprandially prompting the cohort to present for medical attention. Five people presented to the emergency department with neurotoxic shellfish poisoning-related symptoms. All patients received supportive care only. Symptoms resolved within 24 hours. Serum brevetoxin concentrations were reported for four patients. Discussion Ingestion of brevetoxin is rare but may become more common as the frequency and severity of “red-tide” events increase. In our cluster, each person consumed a different number of clams and presented with classic and some “non-classic” symptoms. A trend toward more severe symptoms with a larger number of clams ingested was observed. Conclusions This case cluster describes the clinical course of individuals after consumption of brevetoxin contaminated shellfish.

Knowledge Sharing to Reduce Toxin Exposure Risks from Harmful Algal Blooms: Global Networks and Political Barriers.

Harmful algal blooms (HABs) are a significant global environmental management challenge, especially with respect to microalgae that produce dangerous natural toxins. Examples of HAB toxin diseases with major global health impact include: ciguatera poisoning, paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP), diarrhetic shellfish poisoning (DSP), and neurotoxic (brevetoxin) shellfish poisoning (NSP). Such diseases affect communities globally and contribute to health inequalities within the United States and beyond. Sharing data and lessons learned about the factors determining bloom occurrence and associated exposure to contaminated seafood across locations can reduce public health risks. Knowledge sharing is particularly important as ongoing global environmental changes seem to alter the intensity, location, and timing of toxic HAB events, reducing the reliability of conventional guidance where toxin risks have been endemic and leading to emerging challenges in new settings. Political changes that disrupt membership in knowledge-sharing networks may impede efforts to share scientific expertise and best practices. In this commentary, we stress the importance of community and expert knowledge sharing for reducing HAB risks, both for vulnerable communities in the United States and globally. Considering the impacts of political changes, we note the indirect engagement sometimes required for continued participation in international coordination programs. As an example, we highlight how lessons learned from a Native-led toxin monitoring and testing program (the Southeast Alaska Tribal Ocean Research partnership) can inform programs in other settings. We also describe how international knowledge is mutually valuable for this program in Southeast Alaska.

Exposure-response relationship between K. brevis blooms and reporting of upper respiratory and neurotoxin-associated symptoms

In southwest Florida, Karenia brevis (K. brevis) blooms occur frequently, can be very intense and persist over several years. Individuals living in coastal communities around the Gulf of Mexico are particularly vulnerable to brevetoxins released by K. brevis in seawater and carried inland within marine aerosol. Exposure to K. brevis occurs during residential, recreational, and occupational activities and has been associated with upper respiratory tract (URT) symptoms in healthy and medically vulnerable individuals. Additionally, ingestion of brevetoxin-contaminated seafood causes neurotoxic shellfish poisoning (NSP), and severe headaches prompting emergency department visits which occur in excess during K. brevis blooms. The current study examined a dose-response relationship between K. brevis in coastal waters and URT and NSP-like symptoms and headaches among southwest Florida residents. Data on past medical history (PMH) and medical symptoms were collected from the participants (n = 258) in five southwest Florida counties between June 2019 to August 2021. A dose-response relationship was observed between K. brevis blooms and reporting of URT and NSP-like symptoms and headaches. Reporting of NSP-like symptoms was higher among participants with a PMH of migraines, chronic fatigue syndrome (CFS) and mild memory loss, while the association of headaches with K. brevis blooms was accentuated among individuals with a PMH of migraines. These results suggest further investigations into the threshold of aerosolized brevetoxin dose required to elicit URT, headaches and/or NSP-like symptoms. These symptoms ultimately cause significant public health safety concerns, primarily among vulnerable populations with preexisting neurological conditions.

Toxic Effects and Tumor Promotion Activity of Marine Phytoplankton Toxins: A Review.

Phytoplankton are photosynthetic microorganisms in aquatic environments that produce many bioactive substances. However, some of them are toxic to aquatic organisms via filter-feeding and are even poisonous to humans through the food chain. Human poisoning from these substances and their serious long-term consequences have resulted in several health threats, including cancer, skin disorders, and other diseases, which have been frequently documented. Seafood poisoning disorders triggered by phytoplankton toxins include paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), amnesic shellfish poisoning (ASP), diarrheic shellfish poisoning (DSP), ciguatera fish poisoning (CFP), and azaspiracid shellfish poisoning (AZP). Accordingly, identifying harmful shellfish poisoning and toxin-producing species and their detrimental effects is urgently required. Although the harmful effects of these toxins are well documented, their possible modes of action are insufficiently understood in terms of clinical symptoms. In this review, we summarize the current state of knowledge regarding phytoplankton toxins and their detrimental consequences, including tumor-promoting activity. The structure, source, and clinical symptoms caused by these toxins, as well as their molecular mechanisms of action on voltage-gated ion channels, are briefly discussed. Moreover, the possible stress-associated reactive oxygen species (ROS)-related modes of action are summarized. Finally, we describe the toxic effects of phytoplankton toxins and discuss future research in the field of stress-associated ROS-related toxicity. Moreover, these toxins can also be used in different pharmacological prospects and can be established as a potent pharmacophore in the near future.

Progress on the investigation and monitoring of marine phycotoxins in China

Marine phycotoxins associated with paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), tetrodotoxin (TTX), palytoxin (PLTX) and neurotoxin β-N-methylamino-L-alanine (BMAA) have been investigated and routinely monitored along the coast of China. The mouse bioassay for monitoring of marine toxins has been progressively replaced by the enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which led to the discovery of many new hydrophilic and lipophilic marine toxins. PSP toxins have been detected in the whole of coastal waters of China, where they are the most serious marine toxins. PSP events in the Northern Yellow Sea, the Bohai Sea and the East China Sea are a cause of severe public health concern. Okadaic acid (OA) and dinophysistoxin-1 (DTX1), which are major toxin components associated with DSP, were mainly found in coastal waters of Zhejiang and Fujian provinces, and other lipophilic toxins, such as pectenotoxins, yessotoxins, azaspiracids, cyclic imines, and dinophysistoxin-2(DTX2) were detected in bivalves, seawater, sediment, as well as phytoplankton. CFP events mainly occurred in the South China Sea, while TTX events mainly occurred in Jiangsu, Zhejiang and Fujian provinces. Microalgae that produce PLTX and BMAA were found in the phytoplankton community along the coastal waters of China.

Chemodiversity of Brevetoxins and Other Potentially Toxic Metabolites Produced by Karenia spp. and Their Metabolic Products in Marine Organisms.

In recent decades, more than 130 potentially toxic metabolites originating from dinoflagellate species belonging to the genus Karenia or metabolized by marine organisms have been described. These metabolites include the well-known and large group of brevetoxins (BTXs), responsible for foodborne neurotoxic shellfish poisoning (NSP) and airborne respiratory symptoms in humans. Karenia spp. also produce brevenal, brevisamide and metabolites belonging to the hemi-brevetoxin, brevisin, tamulamide, gymnocin, gymnodimine, brevisulcenal and brevisulcatic acid groups. In this review, we summarize the available knowledge in the literature since 1977 on these various identified metabolites, whether they are produced directly by the producer organisms or biotransformed in marine organisms. Their structures and physicochemical properties are presented and discussed. Among future avenues of research, we highlight the need for more toxin occurrence data with analytical techniques, which can specifically determine the analogs present in samples. New metabolites have yet to be fully described, especially the groups of metabolites discovered in the last two decades (e.g tamulamides). Lastly, this work clarifies the different nomenclatures used in the literature and should help to harmonize practices in the future.

Guidance Level for Brevetoxins in French Shellfish

Brevetoxins (BTXs) are marine biotoxins responsible for neurotoxic shellfish poisoning (NSP) after ingestion of contaminated shellfish. NSP is characterized by neurological, gastrointestinal and/or cardiovascular symptoms. The main known producer of BTXs is the dinoflagellate Karenia brevis, but other microalgae are also suspected to synthesize BTX-like compounds. BTXs are currently not regulated in France and in Europe. In November 2018, they have been detected for the first time in France in mussels from a lagoon in the Corsica Island (Mediterranean Sea), as part of the network for monitoring the emergence of marine biotoxins in shellfish. To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish. Toxicological data were too limited to derive an acute oral reference dose (ARfD). Based on human case reports, we identified two lowest-observed-adverse-effect levels (LOAELs). A guidance level of 180 µg BTX-3 eq./kg shellfish meat is proposed, considering a protective default portion size of 400 g shellfish meat.

An occurrence of neurotoxic shellfish poisoning by consumption of gastropods contaminated with brevetoxins

Brevetoxins were confirmed in urine specimens from patients diagnosed with neurotoxic shellfish poisoning (NSP) after consumption of gastropods that were recreationally harvested from an area previously affected by a Karenia brevis bloom. Several species of gastropods (Triplofusus giganteus, Sinistrofulgur sinistrum, Cinctura hunteria, Strombus alatus, Fulguropsis spirata) and one clam (Macrocallista nimbosa) from the NSP implicated gastropod collection area (Jewfish Key, Sarasota Bay, Florida) were examined for brevetoxins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA). All gastropods and the clam were contaminated with brevetoxins. Composite B-type toxin concentrations in gastropods ranged from 1.1 to 198 μg BTX-3 equiv./g by ELISA, levels likely capable of causing NSP in consumers. Several brevetoxin metabolites previously characterized in molluscan shellfish were identified in these gastropods. Brevetoxin analog profiles by ELISA were similar in the gastropod species examined. This work documents the occurrence of NSP through consumption of a type of seafood not typically monitored in Florida to protect human health, demonstrating the need to better assess and communicate the risk of NSP to gastropod harvesters in Karenia brevis endemic areas.

Validation and Assessment of an Enzyme-Linked Immunosorbent Assay (Elisa) for Use in Monitoring and Managing Neurotoxic Shellfish Poisoning

Neurotoxic shellfish poisoning caused by the ingestion of shellfish contaminated with brevetoxins threatens human health and economies reliant on the Gulf of Mexico. In the United States, the only federally approved method to test shellfish for brevetoxins is a mouse bioassay. Although effective in protecting public health, this assay has considerable shortcomings, including low throughput, lack of sensitivity and specificity, and reliance on live animal testing. Here, a commercially available enzyme-linked immunosorbent assay (ELISA) (MARBIONC) was validated, and results of field samples tested using ELISA and the mouse bioassay were compared. In this validation of the ELISA method for eastern oysters Crassostrea virginica and clams (Mercenaria spp. and sunray venus Macrocallista nimbosa), ELISA demonstrated strong recoveries, specificity, and ruggedness. Quantitative results were obtained for more than 90% of the 526 field samples tested by ELISA compared with only 26% of the samples tested by mouse bioassay. When quantitative data were available for both assays, significant correlations across methods were found. Based on this work, in 2017, the Interstate Shellfish Sanitation Conference approved the limited use of the MARBIONC ELISA in neurotoxic shellfish poisoning management.

Overview of Australian and New Zealand harmful algal species occurrences and their societal impacts in the period 1985 to 2018, including a compilation of historic records

Similarities and differences between Australia and New Zealand in Harmful Algal species occurrences and Harmful Algal Events impacting on human society (HAEDAT) are reported and factors that explain their differences explored. Weekly monitoring of harmful phytoplankton and biotoxins commenced in Australia in 1986 and in New Zealand in 1993. Anecdotal historic HAB records in both countries are also catalogued. In Australia, unprecedented highly toxic Paralytic Shellfish Toxin (PST)-producing blooms of Alexandrium catenella have impacted the seafood industry along the 200 km east coast of Tasmania from 2012 to present. Toxic blooms in 1986-1993 by Gymnodinium catenatum in Tasmania were effectively mitigated by closing the affected area for shellfish farming, while a bloom by this same species in 2000 in New Zealand caused significant economic damage from restrictions on the movement of greenshell mussel spat. The biggest biotoxin event in New Zealand was an unexpected outbreak of Neurotoxic Shellfish Poisoning (NSP) in 1993 in Hauraki Gulf (putatively due to Karenia cf. mikimotoi) with 180 reported cases of human poisonings as well as reports of respiratory irritation north of Auckland. Strikingly, NSP never recurred in New Zealand since and no NSP events have ever been reported in Australia. In New Zealand, Paralytic Shellfish Poisoning (PSP) was the predominant seafood toxin syndrome, while in Australia Ciguatera Fish Poisoning (CFP) was the major reported seafood toxin syndrome, while no CFP has been recorded from consumption of New Zealand fish. In Australia, Diarrhetic Shellfish Poisoning (DSP) illnesses were recorded from two related outbreaks in 1997/98 following consumption of beach harvested clams (pipis) from a previously non-monitored area, whereas in New Zealand limited DSP illnesses are known. No human illnesses from Amnesic Shellfish Poisoning (ASP) have been reported in either Australia or New Zealand. Selected examples of HABs appearing and disappearing (NSP in New Zealand, Alexandrium catenella in Tasmania), species expanding their ranges (Noctiluca, Gambierdiscus), and reputed ballast water introductions (Gymnodinium catenatum) are discussed. Eutrophication has rarely been invoked as a cause except for confined estuaries and fish ponds and estuarine cyanobacterial blooms. No trend in the number of HAEDAT events from 1985 to 2018 was discernible.

Chemiluminescent Receptor Binding Assay for Ciguatoxins and Brevetoxins Using Acridinium Brevetoxin-B2

Ciguatera is the term for poisoning resulting from eating fish from tropical or subtropical regions. The causative toxins collectively named ciguatoxins (CTXs) widely differ in structures depending on their geographic origins, which range from the Pacific Ocean and the Indian Ocean to the Caribbean Sea. Neurotoxic shellfish poisoning (NSP) is caused by the ingestion of bivalve shellfish contaminated with brevetoxins (BTXs). Structurally, both CTXs and BTXs consist of fused ether rings aligned in a ladder shape. Pharmacologically, they bind at the same site (site-5) of voltage-gated sodium channels. However, the great structural diversity and the rare availability of reference toxins hinder LC-MS and ELISA methods, which operate on structure-based recognition. In this study, we prepared a chemiluminescent ligand, acridinium BTXB2 (ABTX), and tested its suitability for use in competitive binding assays to detect CTXs and BTXs. The affinity of ABTX to the rat brain synaptosome estimated by Ki (1.66 pM) was approximately two-fold higher than that of PbTx-3 (BTX3). In addition, the equilibrium dissociation constant (KD) was 0.84 nM, the maximum number of binding was 6.76 pmol toxin/mg protein, and the detection limit was 1.4 amol. The assays performed on samples spiked with CTX3C or BTXB4 (N-palmitoylBTXB2) at 0.2–1.0 ng CTX/g fish flesh, and 200–800 ng BTXB4/g shellfish showed a linear relationship between the theoretical and observed toxin amounts.

Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels.

Brevetoxins are produced by dinoflagellates such as Karenia brevis in warm-water red tides and cause neurotoxic shellfish poisoning. They bind to voltage-gated sodium channels at neurotoxin receptor 5, making the channels more active by shifting the voltage-dependence of activation to more negative potentials and by slowing the inactivation process. Previous work using photoaffinity labeling identified binding to the IS6 and IVS5 transmembrane segments of the channel α subunit. We used alanine-scanning mutagenesis to identify molecular determinants for brevetoxin binding in these regions as well as adjacent regions IVS5-SS1 and IVS6. Most of the mutant channels containing single alanine substitutions expressed functional protein in tsA-201 cells and bound to the radioligand [42-3H]-PbTx3. Binding affinity for the great majority of mutant channels was indistinguishable from wild type. However, transmembrane segments IS6, IVS5 and IVS6 each contained 2 to 4 amino acid positions where alanine substitution resulted in a 2–3-fold reduction in brevetoxin affinity, and additional mutations caused a similar increase in brevetoxin affinity. These findings are consistent with a model in which brevetoxin binds to a protein cleft comprising transmembrane segments IS6, IVS5 and IVS6 and makes multiple distributed interactions with these α helices. Determination of brevetoxin affinity for Nav1.2, Nav1.4 and Nav1.5 channels showed that Nav1.5 channels had a characteristic 5-fold reduction in affinity for brevetoxin relative to the other channel isoforms, suggesting the interaction with sodium channels is specific despite the distributed binding determinants.

Outbreaks, toxicology, and analytical methods of marine toxins in seafood

The consumption of seafood with marine toxins can cause intoxications of varied severity and often-fatal symptoms which may appear in few minutes. Marine toxins are resistant to cooking and freezing, posing a serious hazard to public health. They are usually classified as paralytic, neurotoxic, amnesic, or diarrheic shellfish poisoning. Azaspiracids are a separate group whose poisoning symptoms resemble the diarrheic group. Moreover, some fish species may be contaminated with tetrodotoxin and ciguatoxins. Seafood and seawater control is practiced to protect the consumers’ health and to avoid damages to the fishing economy, due to closures of cultivated areas. Some of the main outbreaks and toxicological properties of marine toxins, as well as the main analytical control tools available were reviewed.

Phycotoxins in Marine Shellfish: Origin, Occurrence and Effects on Humans.

Massive phytoplankton proliferation, and the consequent release of toxic metabolites, can be responsible for seafood poisoning outbreaks: filter-feeding mollusks, such as shellfish, mussels, oysters or clams, can accumulate these toxins throughout the food chain and present a threat for consumers’ health. Particular environmental and climatic conditions favor this natural phenomenon, called harmful algal blooms (HABs); the phytoplankton species mostly involved in these toxic events are dinoflagellates or diatoms belonging to the genera Alexandrium, Gymnodinium, Dinophysis, and Pseudo-nitzschia. Substantial economic losses ensue after HABs occurrence: the sectors mainly affected include commercial fisheries, tourism, recreational activities, and public health monitoring and management. A wide range of symptoms, from digestive to nervous, are associated to human intoxication by biotoxins, characterizing different and specific syndromes, called paralytic shellfish poisoning, amnesic shellfish poisoning, diarrhetic shellfish poisoning, and neurotoxic shellfish poisoning. This review provides a complete and updated survey of phycotoxins usually found in marine invertebrate organisms and their relevant properties, gathering information about the origin, the species where they were found, as well as their mechanism of action and main effects on humans.

Risk assessment indexes for shellfish poisoning outbreak caused by red tide

To establish the indexes and weights of risk assessment of shellfish poisoning outbreak caused by red tide. The risk assessment indexes were developed with the methods of literature review, brainstorm and expert consultation, and the weights of indexes were calculated by the method of analytic hierarchy process. The established indexes contained the risk possibility, impacts of public health, population vulnerability and resilience. The relative risk indexes(integrated risk indexes) of different shellfish poisoning were computed by combining hierarchy process and TOPSIS methods. Moreover, the weights of indexes were further used to generate absolute risk values by multiplying indexes. Four primary indexes and 17 secondary indexes were identified for risk assessment of shellfish poisoning outbreak. Of 17 secondary indexes, the knowing rate of shellfish poisoning, medical accessibility, the number of people being affected, laboratory testing capacity and the habits of eating seafood of local residents had relatively large weights (0.0876, 0.0840, 0.0716, 0.0703 and 0.0644, respectively), which accounted for nearly 38% of the total weight. All consistency ratio (CR) were less than 0.1. The index system was applied in Cangnan county of Zhejiang province. The results showed the relative risk indexes of paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), neurotoxic shellfish poisoning (NSP) and amnesic shellfish poisoning (ASP) were 0.4526, 0.7116, 0.1657 and 0.2884, and the absolute risk values were 0.2542, 0.2668, 0.1907 and 0.2184, respectively. The risk orders of the 4 kinds of shellfish poisoning sorted by relative risk indexes and absolute risk values were consistent. The indexes and weights of risk assessment of shellfish poisoning outbreak caused by red tide are established, which can provide scientific advice for prevention and control of shellfish poisoning outbreak.

Simultaneous determination of neurotoxic shellfish toxins (brevetoxins) in commercial shellfish by liquid chromatography tandem mass spectrometry

An analytical method using liquid chromatography tandem mass spectrometry has been developed for the determination of neurotoxic shellfish poisoning (NSP) toxins (brevetoxins) in shellfish. The tested parameters included extraction solvent, clean-up method, mobile phase and mass spectrometric detection parameters including multiple reaction monitoring (MRM) conditions. The toxins were extracted with 80% methanol in water and cleaned up using Strata-X SPE cartridges. Chromatographic separation of brevetoxins (PbTx-1, PbTx-2 and PbTx-3) was carried out on a C18 column (100 mm × 2.1 mm, 2.6 μm) with gradient elution. The developed method was validated according to the requirements of SANTE 11945/2015. The average recoveries of the brevetoxins ranged from 75.9% to 105.8%, the intra-day precisions were in the range of 0.4–8.6%, the inter-day precisions were in the range of 0.7–6.3% and the limits of quantification (LOQs) were 25 μg/kg for each toxin. The validated method was successfully applied to real shellfish samples, including mussel, oyster and blood cockle. Overall, the method has shown excellent sensitivity and reproducibility, and it could be used as a reference method for the monitoring of individual brevetoxins in shellfish samples.

Role of Biomarkers in Monitoring Brevetoxins in Karenia brevis Exposed Shellfish.

Monitoring and management programs for marine toxins in seafood depend on efficient detection tools for their success in protecting public health. Here we review current methods of detection for neurotoxic shellfish poisoning (NSP) toxins, and current knowledge in brevetoxin metabolism in shellfish. In addition, we discuss a novel approach to developing monitoring tools for NSP toxins in molluscan shellfish. NSP is a seafood-borne disease caused by the consumption of brevetoxin-contaminated shellfish. Brevetoxins are a suite of cyclic polyether compounds found in blooms of the marine dinoflagellate Karenia brevis (K. brevis) and are potent neurotoxins. Preventive controls for NSP in the U.S. currently rely upon environmental monitoring of K. brevis blooms and assessment of their shellfish toxicity by mouse bioassay. The mouse bioassay for NSP approved by National Shellfish Sanitation Program was developed in the 1960s when very little information on the structural and toxicological properties of brevetoxins in algae and shellfish was available. Alternative methods to mouse bioassay based on current scientific knowledge in the area are needed for monitoring NSP toxins. It is now established that brevetoxins are metabolized extensively in shellfish. Algal brevetoxins undergo oxidation and reduction, as well as conjugation with fatty acids and amino acids in shellfish. Recently, three metabolites have been identified as biomarkers of brevetoxin exposure and toxicity in Eastern oyster (Crassostrea virginica) and hard clam (Mercenaria sp.). The role of these biomarkers in monitoring NSP toxins in K. brevis exposed molluscan shellfish is reviewed. Comparisons of biomarker levels by liquid chromatography-mass spectrometry (LC-MS) with composite toxin as measured by enzyme linked immunosorbent assay (ELISA), and shellfish toxicity by mouse bioassay, support the application of these biomarkers as a dynamic and powerful approach for monitoring brevetoxins in shellfish and prevention of NSP.

Ladder-Shaped Ion Channel Ligands: Current State of Knowledge.

Ciguatoxins (CTX) and brevetoxins (BTX) are polycyclic ethereal compounds biosynthesized by the worldwide distributed planktonic and epibenthic dinoflagellates of Gambierdiscus and Karenia genera, correspondingly. Ciguatera, evoked by CTXs, is a type of ichthyosarcotoxism, which involves a variety of gastrointestinal and neurological symptoms, while BTXs cause so-called neurotoxic shellfish poisoning. Both types of toxins are reviewed together because of similar mechanisms of their action. These are the only molecules known to activate voltage-sensitive Na+-channels in mammals through a specific interaction with site 5 of its α-subunit and may compete for it, which results in an increase in neuronal excitability, neurotransmitter release and impairment of synaptic vesicle recycling. Most marine ciguatoxins potentiate Nav channels, but a considerable number of them, such as gambierol and maitotoxin, have been shown to affect another ion channel. Although the extrinsic function of these toxins is probably associated with the function of a feeding deterrent, it was suggested that their intrinsic function is coupled with the regulation of photosynthesis via light-harvesting complex II and thioredoxin. Antagonistic effects of BTXs and brevenal may provide evidence of their participation as positive and negative regulators of this mechanism.