A Review of Cyclic Imines in Shellfish: Worldwide Occurrence, Toxicity and Assessment of the Risk to Consumers.

Cyclic imines are a class of lipophilic shellfish toxins comprising gymnodimines, spirolides, pinnatoxins, portimines, pteriatoxins, prorocentrolides, spiro-prorocentrimine, symbiomines and kabirimine. They are structurally diverse, but all share an imine moiety as part of a bicyclic ring system. These compounds are produced by marine microalgal species and are characterized by the rapid death that they induce when injected into mice. Cyclic imines have been detected in a range of shellfish species collected from all over the world, which raises the question as to whether they present a food safety risk. The European Food Safety Authority (EFSA) considers them to be an emerging food safety issue, and in this review, the risk posed by these toxins to shellfish consumers is assessed by collating all available occurrence and toxicity data. Except for pinnatoxins, the risk posed to human health by the cyclic imines appears low, although this is based on only a limited dataset. For pinnatoxins, two different health-based guidance values have been proposed at which the concentration should not be exceeded in shellfish (268 and 23 µg PnTX/kg shellfish flesh), with the discrepancy caused by the application of different uncertainty factors. Pinnatoxins have been recorded globally in multiple shellfish species at concentrations of up to 54 times higher than the lower guidance figure. Despite this observation, pinnatoxins have not been associated with recorded human illness, so it appears that the lower guidance value may be conservative. However, there is insufficient data to generate a more robust guidance value, so additional occurrence data and toxicity information are needed.

Acute toxicity of decarbamoyl gonyautoxin 1&4 to mice by various routes of administration.

Saxitoxin and its derivatives, the paralytic shellfish toxins (PSTs), are well known to be toxic to humans, and maximum permitted levels in seafood have been established by regulatory authorities in many countries. Monitoring of PSTs is typically performed using chemical methods which quantify the concentration of the individual PST analogues, of which there are many. However, since the toxicities of analogues are different, they do not equally contribute to the overall toxicity of the sample. To account for these differences, toxicity equivalency factors (TEFs) need to be determined for each analogue and applied. Currently there are no established TEFs for decarbamoyl gonyautoxin 1&4 (dcGTX1&4), which occurs in some clam species such as Mactra chinensis contaminated with PSTs due to metabolism within the shellfish. In this study the median lethal dose of purified, equilibrated epimeric mixture of dcGTX1&4 has been determined by intraperitoneal injection (i.p.) (4.75 µmol/kg) and by feeding (34.9 µmol/kg). The most relevant route of exposure is orally with feeding being more representative of human consumption and more reliable than gavage. Based on the median lethal dose by feeding, a TEF of 0.1 is recommended for dcGTX1&4. Receptor binding activity and i.p. toxicity results showed dcGTX1&4 to be much less toxic than STX (140-170-fold). However, by feeding a much smaller difference in toxicity was observed with dcGTX1&4 being only 11-fold less toxic than STX. Analysis of the gut contents of mice dosed with dcGTX1&4 showed the presence of decarbamoyl gonyautoxin 2&3, decarbamoyl saxitoxin and decarbamoyl neosaxitoxin, all of which are of greater toxicity. This conversion of dcGTX1&4 within the digestive track to more toxic congeners may explain the high relative toxicity of dcGTX1&4 by feeding compared to that determined by i.p. and by sodium channel activity.

Cyclic Imine Pinnatoxin G is Cytotoxic to Cancer Cell Lines via Nicotinic Acetylcholine Receptor-Driven Classical Apoptosis.

Pinnatoxin G is a cyclic imine neurotoxin produced by dinoflagellates that has been reported in shellfish. Like other members of the pinnatoxin family, it has been shown to have its effects via antagonism of the nicotinic acetylcholine receptors, with preferential binding to the α7 subunit often upregulated in cancer. Because increased activity of α7 nicotinic acetylcholine receptors contributes to increased growth and resistance to apoptosis, the effect of pinnatoxin G on cancer cell viability was tested. In a panel of six cancer cell lines, all cell types lost viability, but HT29 colon cancer and LN18 and U373 glioma cell lines were more sensitive than MDA-MB-231 breast cancer cells, PC3 prostate cancer cells, and U87 glioma cells, correlating with expression levels of α7, α4, and α9 nicotinic acetylcholine receptors. Some loss of cell viability could be attributed to cell cycle arrest, but significant levels of classical apoptosis were found, characterized by caspase activity, phosphatidylserine exposure, mitochondrial membrane permeability, and fragmented DNA. Intracellular Ca2+ levels also dropped immediately upon pinnatoxin G treatment, which may relate to antagonism of nicotinic acetylcholine receptor-mediated Ca2+ inflow. In conclusion, pinnatoxin G can decrease cancer cell viability, with both cytostatic and cytotoxic effects.

Acute toxicity of dihydroanatoxin-a from Microcoleus autumnalis in comparison to anatoxin-a.

The cyanobacterium Microcoleus autumnalis grows as thick benthic mats in rivers and is becoming increasingly prevalent around the world. M. autumnalis can produce high concentrations of anatoxins and ingestion of benthic mats has led to multiple dog deaths over the past two decades. M. autumnalis produces a suite of different anatoxin congeners including anatoxin-a (ATX), dihydroanatoxin-a, (dhATX), homoanatoxin-a and dihydrohomoanatoxin-a. Benthic mat samples often contain high levels of dhATX, but there is little toxicology information on this congener. In the present study, natural versions of dhATX and ATX were purified from cyanobacteria to determine the acute toxicity by different routes of administration using mice. Nuclear magnetic resonance spectroscopy was used to confirm the putative structure of dhATX. By intraperitoneal (ip) injection, the median lethal dose (LD50) for dhATX was 0.73 mg/kg, indicating a reduced toxicity compared to ATX (LD50 of 0.23 mg/kg). However, by oral administration (both gavage and feeding), dhATX was more toxic than ATX (gavage LD50 of 2.5 mg/kg for dhATX and 10.6 mg/kg for ATX; feeding LD50 of 8 mg/kg for dhATX and 25 mg/kg for ATX). The relative nicotinic acetylcholine receptor-binding affinities of ATX and dhATX were determined using the Torpedo electroplaque assay which showed consistency with the relative toxicity determined by ip injection. This work highlights that toxicity studies based solely on ip injection may not yield LD50 values that are relevant to those derived via oral administration, and hence, do not provide a good estimate of the risk posed to human and animal health in situations where oral ingestion is the likely route of exposure. The high acute oral toxicity of dhATX, and its abundance in M. autumnalis proliferations, demonstrates that it is an important environmental contaminant that warrants further investigation.

Development of an LC-MS/MS method to simultaneously monitor maitotoxins and selected ciguatoxins in algal cultures and P-CTX-1B in fish.

Ciguatera fish poisoning is a serious human health issue that is highly localized to tropical and sub-tropical coastal areas, affecting many of the indigenous island communities intrinsically linked to reef systems for sustenance and trade. It is caused by the consumption of reef fish contaminated with ciguatoxins and is reported as the most common cause of non-bacterial food poisoning. The causative toxins bioaccumulate up the food web, from small herbivorous fish that graze on microalgae of the genus Gambierdiscus into the higher trophic level omnivorous and carnivorous fish predating on them. The number of Gambierdiscus species being described is increasing rapidly and the role of other toxins produced by this microalgal genus in ciguatera intoxications, such as maitotoxin, remains unclear. Ciguatoxins and maitotoxin are among the most potent marine toxins known and there are currently no methods of analysis that can simultaneously monitor these toxins with a high degree of specificity. To meet this need a rapid and selective ultra-performance liquid chromatography tandem mass spectrometry method has been developed to rapidly screen Gambierdiscus cultures and environmental sample device extracts for ciguatoxins and maitotoxins. A fast sample preparation method has also been developed to allow sensitive quantification of the potent ciguatoxin fish metabolite P-CTX-1B from fish extracts, and this method has been subjected to a small validation study. Novel aspects of this approach include the use of alkaline mobile phase for chromatographic separation and specific monitoring of the various toxins. This method has good potential to help evaluate ciguatera risk associated with Gambierdiscus and related microalgal species, and to help promote method development activities for this important and analytically challenging toxin class.

Antifouling activity of portimine, select semisynthetic analogues, and other microalga-derived spirocyclic imines.

A range of natural products from marine invertebrates, bacteria and fungi have been assessed as leads for nature-inspired antifouling (AF) biocides, but little attention has been paid to microalgal-derived compounds. This study assessed the AF activity of the spirocyclic imine portimine (1), which is produced by the benthic mat-forming dinoflagellate Vulcanodinium rugosum. Portimine displayed potent AF activity in a panel of four macrofouling bioassays (EC50 0.06-62.5 ng ml-1), and this activity was distinct from that of the related compounds gymnodimine-A (2), 13-desmethyl spirolide C (3), and pinnatoxin-F (4). The proposed mechanism of action for portimine is induction of apoptosis, based on the observation that portimine inhibited macrofouling organisms at developmental stages known to involve apoptotic processes. Semisynthetic modification of select portions of the portimine molecule was subsequently undertaken. Observed changes in bioactivity of the resulting semisynthetic analogues of portimine were consistent with portimine's unprecedented 5-membered imine ring structure playing a central role in its AF activity.

Paralytic shellfish toxin producing Aphanizomenon gracile strains isolated from Lake Iznik, Turkey.

Aphanizomenon gracile is one of the most widespread Paralytic Shellfish Toxin (PST) producing cyanobacteria in freshwater bodies in the Northern Hemisphere. It has been shown to produce various PST congeners, including saxitoxin (STX), neosaxitoxin (NEO), decarbamoylsaxitoxin (dcSTX) and gonyautoxin 5 (GTX5) in Europe, North America and Asia. Three cyanobacteria strains were isolated in Lake Iznik in northwestern Turkey. Morphological characterization of these strains suggested all three strains conformed to classical taxonomic identification of A. gracile with some differences such as clumping of filaments, partially hyaline cells in some filaments and longer than usual vegetative cells. Sequences of 16S rRNA gene of these strains were placed within an A. gracile cluster including the majority of PST producing strains, confirming the identification of these strains as A. gracile. These new strains possessed saxitoxin biosynthesis genes sxtA, sxtG and their sequences clustered with those of other A. gracile. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis demonstrated the presence of NEO, STX, dcSTX and decarbamoylneosaxitoxin (dcNEO) in all strains. This is the first report of a PST producer in any water body in Turkey and first observation of dcNEO in an A. gracile culture.

Acute Toxicities of the Saxitoxin Congeners Gonyautoxin 5, Gonyautoxin 6, Decarbamoyl Gonyautoxin 2&3, Decarbamoyl Neosaxitoxin, C-1&2 and C-3&4 to Mice by Various Routes of Administration.

Paralytic shellfish poisoning results from consumption of seafood naturally contaminated by saxitoxin and its congeners, the paralytic shellfish toxins (PSTs). The levels of such toxins are regulated internationally, and maximum permitted concentrations in seafood have been established in many countries. A mouse bioassay is an approved method for estimating the levels of PSTs in seafood, but this is now being superseded in many countries by instrumental methods of analysis. Such analyses provide data on the levels of many PSTs in seafood, but for risk assessment, knowledge of the relative toxicities of the congeners is required. These are expressed as "Toxicity Equivalence Factors" (TEFs). At present, TEFs are largely based on relative specific activities following intraperitoneal injection in a mouse bioassay rather than on acute toxicity determinations. A more relevant parameter for comparison would be median lethal doses via oral administration, since this is the route through which humans are exposed to PSTs. In the present study, the median lethal doses of gonyautoxin 5, gonyautoxin 6, decarbamoyl neosaxitoxin and of equilibrium mixtures of decarbamoyl gonyautoxins 2&3, C1&2 and C3&4 by oral administration to mice have been determined and compared with toxicities via intraperitoneal injection. The results indicate that the TEFs of several of these substances require revision in order to more accurately reflect the risk these toxins present to human health.

The marine cytotoxin portimine is a potent and selective inducer of apoptosis.

Portimine is a recently discovered member of a class of marine micro-algal toxins called cyclic imines. In dramatic contrast to related compounds in this toxin class, portimine has very low acute toxicity to mice but is highly cytotoxic to cultured cells. In this study we show that portimine kills human Jurkat T-lymphoma cells and mouse embryonic fibroblasts (MEFs), with LC50 values of 6 and 2.5 nM respectively. Treated cells displayed rapid caspase activation and phosphatidylserine exposure, indicative of apoptotic cell death. Jurkat cells overexpressing the anti-apoptotic protein Bcl-2 or Bax/Bak knockout MEFs were completely protected from portimine. This protection was apparent even at high concentrations of portimine, with no evidence of necrotic cell death, indicating that portimine is a selective chemical inducer of apoptosis. Treatment of the Bcl-2-overexpressing cells with both portimine and the Bcl-2 inhibitor ABT-737 proved a powerful combination, causing >90 % death. We conclude that portimine is one of the most potent naturally derived inducers of apoptosis to be discovered, and it displays strong selectivity for the induction of apoptotic pathways.

Algal toxins and producers in the marine waters of Qatar, Arabian Gulf.

Harmful Algal Bloom species are ubiquitous and their blooms occur in the Arabian Gulf. In this study, two cruises were performed in 2012 and 2013 to collect phytoplankton samples from 4 sites in the Arabian Gulf. Toxin analyses of phytoplankton samples for 32 algal toxins from 5 different toxin groups were conducted on the samples using both enzyme linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results demonstrated, for the first time, the presence of paralytic shellfish toxins (PSTs), diarrhetic shellfish toxin (DST), amnesic shellfish toxin (AST), cyclic imines (CIs) and polyether-lactone toxins in freeze-dried phytoplankton samples. Four Vulcanodinium rugosum cultures were established from field samples and these proved to contain between 603 and 981 ng pinnatoxin (PnTx) H per mg dry weight in addition to being positive for portimine. These strains from Qatar clustered with strains from Japan and Florida based on large subunit rRNA and rRNA internal transcribed spacer gene sequences.

Metamorphosis of the invasive ascidian Ciona savignyi: environmental variables and chemical exposure.

In this study, the effects of environmental variables on larval metamorphosis of the solitary ascidian Ciona savignyi were investigated in a laboratory setting. The progression of metamorphic changes were tracked under various temperature, photoperiod, substrate, larval density, and vessel size regimes. Metamorphosis was maximised at 18 °C, 12:12 h subdued light:dark, smooth polystyrene substrate, and 10 larvae mL(-1) in a twelve-well tissue culture plate. Eliminating the air-water interface by filling culture vessels to capacity further increased the proportion of metamorphosed larvae; 87 ± 5% of larvae completed metamorphosis within 5 days compared to 45 ± 5% in control wells. The effects of the reference antifouling compounds polygodial, portimine, oroidin, chlorothalonil, and tolylfluanid on C. savignyi were subsequently determined, highlighting (1) the sensitivity of C. savignyi metamorphosis to chemical exposure and (2) the potential to use C. savignyi larvae to screen for bioactivity in an optimised laboratory setting. The compounds were bioactive in the low ng mL(-1) to high µg mL(-1) range. Polygodial was chosen for additional investigations, where it was shown that mean reductions in the proportions of larvae reaching stage E were highly repeatable both within (repeatability = 14 ± 9%) and between (intermediate precision = 17 ± 3%) independent experiments. An environmental extract had no effect on the larvae but exposing larvae to both the extract and polygodial reduced potency relative to polygodial alone. This change in potency stresses the need for caution when working with complex samples, as is routinely implemented when isolating natural compounds from their biological source. Overall, the outcomes of this study highlight the sensitivity of C. savignyi metamorphosis to environmental variations and chemical exposure.

The use of a mucus trap by Dinophysis acuta for the capture of Mesodinium rubrum prey under culture conditions.

A capture mechanism observed in a culture of the dinoflagellate Dinophysis acuta when preying on the ciliate Mesodinium rubrum (also sometimes referred to as Myrionecta rubra) is described. The dinoflagellate released cohesive clumps of mucilage into the culture media. When M. rubrum cells came into contact with this mucilage, they were immediately immobilized, but remained alive for a short period of time. Observations of D. acuta cells 'visiting and probing' trapped M. rubrum cells were made and at a critical point D. acuta cells removed individual M. rubrum cells from the mucus to swim away with them. The removal of M. rubrum from the mucus coincided with the cells losing all their cilia and becoming swollen, presumably signifying the death of the cell. These changes may enable the D. acuta peduncle to penetrate the ciliate cell cortex. It is hypothesized that toxins produced by D. acuta play a role in the immobilization process within the mucilage trap.

Pinnatoxins E, F and G target multiple nicotinic receptor subtypes.

Pinnatoxins are members of the cyclic imine group of marine phycotoxins that are highly toxic in in vivo rodent bioassays, causing rapid death due to respiratory depression. Recent studies have shown that pinnatoxins E, F and G, found in New Zealand and Australian shellfish, act as antagonists at muscle-type nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction. In the present study, binding affinities and modes of these pinnatoxin isomers at neuronal and muscle nAChRs were assessed using radioligand binding, electrophysiological and molecular modelling techniques. Radioligand-binding studies revealed that all three pinnatoxins bound with high affinity to muscle-type nAChRs, as well as to the α7 and α4ß2 neuronal receptors, with an order of affinity of muscle type > α7 > α4ß2. The rank order of potency at all receptors was pinnatoxin F > G > E. Pinnatoxins F and G also antagonized ACh-evoked responses in α7 and α4ß2 neuronal receptors expressed in Xenopus oocytes. Molecular modelling revealed that pinnatoxins E, F and G make multiple hydrogen bond interactions with the binding site of muscle-type and α7 receptors, with few interactions at the α4ß2 binding site, reflecting the binding affinity and functional data. This study shows for the first time that pinnatoxins E, F and G bind to, and functionally antagonize neuronal nAChRs, with interactions potentially playing a role in pinnatoxin toxicity.

Single-Laboratory Validation of a Multitoxin Ultra-Performance LC-Hydrophilic Interaction LC-MS/MS Method for Quantitation of Paralytic Shellfish Toxins in Bivalve Shellfish.

A single-laboratory validation study was conducted for the hydrophilic interaction-LC-MS/MS analysis of paralytic shellfish toxins (PSTs) in bivalve shellfish. The method was developed as an alternative to the precolumn oxidation AOAC 2005.06 and postcolumn oxidation AOAC 2011.02 LC with fluorescence detection methods, receptor binding assay AOAC 2011.27, as well as the mouse bioassay AOAC 959.08. PSTs assessed were saxitoxin, neosaxitoxin, deoxydecarbamoylsaxitoxin, decarbamoylsaxitoxin, decarbamoylneosaxitoxin, gonyautoxins 1-6, decarbamoylgonyautoxins 2-3, and N-sulfocarbamoyl gonyautoxins 2&3. The method also included the determination of decarbamoylgonyautoxins 1&4, N-sulfocarbamoyl gonyautoxins 1&4, and M toxins. Twelve commercially produced bivalve species from both New Zealand and the United Kingdom were assessed, including mussels, oysters, scallops, and clams. Validation studies demonstrated acceptable method performance characteristics for specificity, linearity, recovery, repeatability, and within-laboratory reproducibility. LOD and LOQ were significantly improved in comparison to current fluorescence-based detection methods, and the method was shown to be rugged. The method performed well in comparison to AOAC 2005.06, with evidence obtained from both comparative analysis of 1141 PST-contaminated samples and successful participation in proficiency testing schemes. The method is suitable for use in regulatory testing and will be submitted for an AOAC collaborative study.

Development of a sensitive and selective liquid chromatography-mass spectrometry method for high throughput analysis of paralytic shellfish toxins using graphitised carbon solid phase extraction.

Routine regulatory monitoring of paralytic shellfish toxins (PST) commonly employs oxidative derivitisation and complex liquid chromatography fluorescence detection methods (LC-FL). The pre-column oxidation LC-FL method is currently implemented in New Zealand and the United Kingdom. When using this method positive samples are fractionated and two different oxidations are required to confirm the identity and quantity of each PST analogue present. There is a need for alternative methods that are simpler, provide faster turnaround times and have improved detection limits. Hydrophilic interaction liquid chromatography (HILIC) HPLC-MS/MS analysis of PST has been used for research purposes, but high detection limits and substantial sample matrix issues have prevented it from becoming a viable alternative for routine monitoring purposes. We have developed a HILIC UPLC-MS/MS method for paralytic shellfish toxins with an optimised desalting clean-up procedure on inexpensive carbon solid phase extraction cartridges for reduction of matrix interferences. This represents a major technical breakthrough and allows sensitive, selective and rapid analysis of paralytic shellfish toxins from a variety of sample types, including many commercially produced bivalve molluscan shellfish species. Additionally, this analytical approach avoids the need for complex calculations to determine sample toxicity, as unlike other methods each PST analogue is able to be quantified as a single resolved peak. This article presents the method development and optimisation information. A thorough single laboratory validation study has subsequently been performed and this data will be presented elsewhere.

Paralytic shellfish toxins, including deoxydecarbamoyl-STX, in wild-caught Tasmanian abalone (Haliotis rubra).

For the first time wild-caught Tasmanian abalone, Haliotis rubra, have been reported to contain paralytic shellfish toxins (PSTs). This observation followed blooms of the toxic dinoflagellate Gymnodinium catenatum. No illnesses were reported, but harvesting restrictions were enforced in commercial areas. Abalone were assayed using HPLC-FLD methodology based on AOAC official method 2005.06. An uncommon congener, deoxydecarbamoyl-STX (doSTX), was observed in addition to regulated PSTs as unassigned chromatographic peaks. A quantitative reference material was prepared from contaminated Tasmanian abalone viscera and ampouled at 54.2 µmol/L. The LD50 of doSTX via intraperitoneal injection was 1069 nmol/kg (95% confidence limits 983-1100 nmol/kg), indicating it is nearly 40 times less toxic than STX. A toxicity equivalence factor of 0.042 was generated using the mouse bioassay. Levels of PSTs varied among individuals from the same site, although the toxin profile remained relatively consistent. In the foot tissue, STX, decarbamoyl-STX and doSTX were identified. On a molar basis doSTX was the dominant congener in both foot and viscera samples. The viscera toxin profile was more complex, with other less toxic PST congeners observed and was similar to mussels from the same site. This finding implicates localised dinoflagellate blooms as the PST source in Tasmanian abalone.

In vitro labelling of muscle type nicotinic receptors using a fluorophore-conjugated pinnatoxin F derivative.

Fluorescent molecules are regularly utilised to study ligand-receptor interactions. Many ligands for nicotinic receptors have been conjugated with fluorophores to study receptor kinetics, recycling and ligand binding characteristics. These include small agonist molecules, as well as large peptidic antagonists. However, no small molecule antagonists have been investigated using this method. Pinnatoxin F is a newly discovered non-peptidic muscle type nicotinic receptor antagonist produced by the marine dinoflagellate species Vulcanodinium rugosum. This molecule has the potential for conjugation to a fluorophore, allowing subsequent visualisation of interactions with nicotinic receptors. Pinnatoxin F was modified by addition of diaminopolyether spacers, to which a fluorophore (VivoTag(®) 645) was conjugated. The fluorescent pinnatoxin was then applied to muscle sections from thy1-YFP-H transgenic mice, which express YFP in motor nerves, to allow direct visualization of fluorescent binding at the neuromuscular junction. The addition of both the diaminopolyether spacer and the VivoTag(®) 645 reduced the potency of pinnatoxin F, as evidenced by a reduction in in vitro neuromuscular blocking activity and in vivo toxicity. Despite this reduced potency, the fluorescent molecule selectively labelled endplate regions in thy1-YFP mouse muscle sections and this labelling was inhibited by pre-exposure of muscle sections to native pinnatoxin F or the nicotinic antagonist α-bungarotoxin. This study proves nicotinic receptor binding activity of pinnatoxin F and is the first example of a fluorophore-conjugated small-molecule antagonist for nicotinic receptors. These results indicate the potential for other small-molecule nicotinic receptor antagonists to be fluorescently labelled and used as probes for specific nicotinic receptor subtypes.

Neuromuscular blocking activity of pinnatoxins E, F and G.

Pinnatoxins are produced by dinoflagellates and belong to the cyclic imine family of toxins. They are fast-acting and highly toxic when administered in vivo in rodent bioassays, causing death by respiratory depression within minutes. Studies have revealed that some cyclic imine toxins cause their toxicity by antagonizing both muscle type and heteromeric and homomeric neuronal nicotinic acetylcholine receptors (nAChRs). Pinnatoxins E, F and G all display potent toxicity in in vivo bioassays, with symptoms of toxicity similar to other cyclic imine toxins. However, very little work has been done on the mechanism of action of these pinnatoxin isomers. Thus the aim of the current study was to investigate the rank order of potency and mechanism of action of pinnatoxins E, F and G. The effects of pinnatoxin E, F and G on in vitro rat hemidiaphragm preparations were investigated using twitch tension and electrophysiological techniques to determine the effects of these toxins on cholinergic transmission at the neuromuscular junction. Pinnatoxins E, F and G all produced concentration-dependent reductions in the nerve evoked twitch response of the rat hemidiaphragm, with IC50 values ranging from 11 to 53 nM and a rank order of potency of F > G > E. Only complete washout of pinnatoxin E was evident, with pinnatoxins F and G displaying slow and incomplete washout profiles. Pinnatoxins F and G also reduced the amplitudes of spontaneous miniature endplate potentials and evoked endplate potentials at the neuromuscular junction, without affecting miniature endplate potential frequency or the resting membrane potential of the muscle fibres. These results show that pinnatoxins E, F and G are all potent neuromuscular blocking agents and cause toxicity by acting as antagonists at muscle type nicotinic acetylcholine receptors.

Determination of brevetoxins in shellfish by LC/MS/MS: single-laboratory validation.

A single-laboratory validation is reported for an LC/MS/MS quantification of six brevetoxins in four matrixes (Greenshell mussel, eastern oyster, hard clam, and Pacific oyster). Recovery and precision data were collected from seven analytical batches using shellfish flesh at 0.05 mg/kg. Method recoveries and within-laboratory reproducibility ranged from 73 to 112%, with an RSD between 14 and 18% for brevetoxin-3, brevetoxin B5, brevetoxin B2, and S-desoxy brevetoxin B2. The recovery and within-laboratory reproducibility for brevetoxin-2 was 61%, with an RSD of 27%. Brevetoxin B1 gave an RSD of 12%, but no reference material was available and this toxin was only recorded in a hard clam sample naturally contaminated with brevetoxins. One naturally contaminated sample of each shellfish matrix, with brevetoxin levels ranging from 0.012 to 9.9 mg/kg, was tested in multiple batches, and the RSDs were similar to those for fortified samples at 0.05 mg/kg. Comparisons with limited data for the neurotoxic shellfish poisoning mouse bioassay for four naturally contaminated shellfish samples showed that the regulatory action limit of 0.8 mg/kg is conservative with respect to the bioassay regulatory limit of 20 mouse units/100 g.

Acute toxicity of pinnatoxins E, F and G to mice.

The acute toxicities to mice of pinnatoxins E, F and G, members of the cyclic imine group of phycotoxins, by intraperitoneal injection and/or oral administration, have been determined. These substances were all very toxic by intraperitoneal injection, with LD(50) values between 12.7 and 57 µg/kg. Pinnatoxin E was much less toxic by oral administration than by intraperitoneal injection, but this was not the case for pinnatoxin F. The median lethal doses of the latter substance by gavage and by voluntary intake were only 2 and 4 times higher than that by injection. The high oral toxicity of pinnatoxin F raises concerns as to the possibility of adverse effects of this substance in shellfish consumers, although it should be noted that no toxic effects in humans have been recorded with pinnatoxins or with any other compound of the cyclic imine group.