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An individual non-motile (NM) cell was isolated from a surface sediment sample collected in Guangxi, China and subsequently established as a dinoflagellate strain in culture. The motile cells are 22.5–32.5 µm long and 20.0–30.0 µm wide, with a plate formula of Po, X, 4′, 3a, 7″, 6c, 5(?)s, 5″′, 2″″, fitting the description of Vulcanodinium rugosum. The Chinese strain shares 99.8%, 97.4% and 96.7% similarity (LSU sequence) with those from Australasia, France and Japan. Asexual division of V. rugosum takes place either in the thecate motile stage or within a NM division cell. Motile cells divided by binary fission inside the parent cell and transformed to NM division cells within 24 h. The NM cell underwent one to three consecutive divisions within the parent wall. The divisions were not always synchronous and neither was the release of motile cells from the NM cells. It generally took 4 to 6 days for the NM cells to complete one division. NM cells survived for 1 month at 4 °C in the dark, suggesting that they might play an important role in species dispersal. A novel pinnatoxin was detected at 20 pg cell−1 and no other known pinnatoxins (A–G) were detected. [ABSTRACT FROM PUBLISHER]

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.

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 Ca 2+ levels also dropped immediately upon pinnatoxin G treatment, which may relate to antagonism of nicotinic acetylcholine receptor-mediated Ca 2+ inflow. In conclusion, pinnatoxin G can decrease cancer cell viability, with both cytostatic and cytotoxic effects.

Portimine, a recently identified cyclic imine produced by the dinoflagellate Vulcanodinium rugosum, has been described as a potent apoptotic agent in contrast to most of the cyclic imines that are well-known to be neurological toxins. As apoptosis can be a consequence of a high level of DNA lesions, we investigated the responses of portimine on several endpoints aimed at detecting DNA damage in the hepatic cell line HepaRG. Portimine induced phosphorylation of H2AX, which could possibly be consistent with the previously published induction of apoptosis with this toxin. In addition, detection of apoptosis through the activation of caspase-3, the induction of strand breaks detected by the comet assay as well as chromosome and genome mutations using the micronucleus assay were addressed. Surprisingly, portimine treatment resulted in increases in only γH2AX in differentiated HepaRG cells whereas no effects on the other endpoints were detected. These increases in γH2AX in the absence of genotoxic effects in the other tests could indicate that portimine could possibly induce a DNA replication stress and/or that the compound can be detoxified by the HepaRG cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 (LD 50 ) for dhATX was 0.73 mg/kg, indicating a reduced toxicity compared to ATX (LD 50 of 0.23 mg/kg). However, by oral administration (both gavage and feeding), dhATX was more toxic than ATX (gavage LD 50 of 2.5 mg/kg for dhATX and 10.6 mg/kg for ATX; feeding LD 50 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 LD 50 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 (EC 50 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.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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 LC 50 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.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

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 α4β2 neuronal receptors, with an order of affinity of muscle type > α7 > α4β2. 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 α4β2 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 α4β2 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., (© 2015 International Society for Neurochemistry.)

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.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Palytoxin is a highly toxic non-proteinaceous marine natural product that can pass through the food chain and result in human illnesses. A recent review by the European Food Safety Authority concluded that palytoxin requires regulation in seafood and a limit of 30 μg kg⁻¹ for shellfish flesh was suggested. Current methods based on LC-MS detection of intact palytoxins do not have sufficient sensitivity to enforce this limit for palytoxin. To improve sensitivity for trace analysis, a novel screen approach has been developed that uses LC-MS/MS analysis of substructures generated by oxidative cleavage of vicinal diol groups present in the intact toxin. Oxidation of palytoxins, ovatoxins or ostreocins using periodic acid generates two nitrogen-containing aldehyde fragments; an amino aldehyde common to these toxins, and an amide aldehyde that may vary depending on toxin type. Conditions for micro-scale oxidation of palytoxin were optimised, which include a novel SPE cleanup and on-column oxidation step. Rapid analysis of cleavage fragments was established using LC-MS/MS. Linear calibrations were established for the amino aldehyde from a palytoxin reference standard, which is suitable for all known palytoxin-like compounds, and for the confirmatory amide aldehydes of palytoxin and ostreocin-D. Palytoxin recoveries (at 10 μg kg⁻¹) from shellfish and fish tissues were 114-119% (as amine aldehyde) and 90-115% (as amide aldehyde) with RSDs for both of ≤ 18% (all tissues, n = 12). The method LOD was determined to be approximately 1 ng mL⁻¹ and the LOQ 4 ng mL⁻¹, which corresponds to 10 μg kg⁻¹ in tissue (flesh of shellfish or fish). The method has potential for use in research and is sufficiently sensitive for regulatory testing, should it be required., (Copyright © 2012. Published by Elsevier Ltd.)

An enzyme capable of hydrolysing pectenotoxins (PTXs) and okadaic acid (OA) esters within the hepatopancreas of the Greenshell™ mussel Perna canaliculus was isolated and characterized. The enzyme was purified by sequential polyethylene glycol fractionation, anion exchange, hydrophobic interaction, gel filtration and hydroxyapatite chromatography. The enzyme was an acidic (pI ∼ 4.8), monomeric, 67 kDa, serine esterase with optimum activity at pH 8.0 and 25 °C. PTX2 and PTX1 were hydrolysed but the enzyme was inactive against PTX11, PTX6 and acid isomerised PTX2 and PTX11. PTX11 and PTX2b competitively inhibited PTX2 hydrolysis. The enzyme also hydrolysed short and medium chain length (C2-C10) 4-nitrophenyl-esters, okadaic acid C8-C10 diol esters and DTX1 7-O-palmitoyl ester (DTX3). MALDI-Tof MS/MS analysis showed that the enzyme had some homology with a juvenile hormone esterase from the Red Flour Beetle Tribolium castaneum, although BLAST searches of several data bases using de novo amino acid sequences failed to identify any homology with known proteins., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

Fast-acting cyclic imines belonging to the pinnatoxin and pteriatoxin group of toxins were originally identified in shellfish of the genera Pinna and Pteria in Japan, after food poisoning events in China linked to consumption of Pinna spp. Recently, a range of new and known pinnatoxin analogs has been identified in shellfish, sediment, and seawater samples from Australia and New Zealand. Although the structurally closely-related spirolide toxins are better known, and have a worldwide distribution including Norway and other parts of Europe, the presence of pinnatoxins has not been reported in European waters or shellfish. Here we report results from a survey of Norwegian blue mussels for the presence of pinnatoxins and spirolides, by LC-MS/MS analysis of extracts obtained as part of Norway's routine monitoring programme for regulated algal toxins during late autumn and early winter 2009. Spirolides and pinnatoxin G were widespread (pinnatoxin G (1), spirolide C (2), iso-spirolide C (3), 13-desmethylspirolide C (4), 13,19-didesmethylspirolide C (5), and 20-methylspirolide G (6) were detected in 69%, 13%, 60%, 22%, 33%, and 77%, respectively, of the shellfish samples) and, although levels were generally low, concentrations of up to 115 μg/kg of pinnatoxin G (1) and 226 μg/kg of 13-desmethylspirolide C (4) were detected. We also analyzed stored extracts from passive sampling disks deployed as part of a separate study in autumn 2007. All the stored extracts contained 20-methylspirolide G (which predominated at most locations), most contained pinnatoxin G (73%) and 13,19-didesmethylspirolide C (67%), but iso-spirolide C (36%) and 13-desmethylspirolide C (52%) were also detected in many of the samples. These results suggest that pinnatoxins may be much more widespread than previously suspected, and indicate that they or related compounds could be responsible for sporadic incidents of rapid-onset symptoms during mouse bioassays of shellfish in Europe and elsewhere. The toxicological significance of these levels of pinnatoxins and spirolides is at present unclear. However, although pinnatoxins appear to be less toxic than spirolides by intraperitoneal injection in the mouse bioassay, recently published preliminary toxicological data indicate that pinnatoxins may be as much as an order of magnitude more toxic than spirolides by oral ingestion via food., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Members of the cyclic imine group of toxins, gymnodimine and spirolides, have been found to be potent antagonists of both muscle type and neuronal nicotinic acetylcholine receptors. These toxins exhibit fast acting toxicity in vivo, causing death within minutes by respiratory depression. This toxicity is shared by the novel cyclic imine pinnatoxins E and F, produced by marine dinoflagellates and recently isolated from New Zealand shellfish. However, there is currently very little data available regarding the mechanism of action for any of the pinnatoxins, and no data at all on the novel pinnatoxins E and F. The aim of the current study was to investigate potential antagonism of nicotinic acetylcholine receptors by pinnatoxins E and F using two in vitro tissue preparations. Compound muscle action potentials elicited by stimulation of the phrenic nerve were recorded from the hemidiaphragm in order to test effects on muscle type heteromeric nicotinic receptors, while effects on α7 homomeric neuronal nicotinic receptors were investigated by recording gamma oscillations in response to tetanic stimulation of the CA1 region of the hippocampus. Both a crude extract containing a mixture of pinnatoxins E and F, as well as pure pinnatoxin F, had no effect on gamma oscillation spectral density or spike count at any concentrations. Conversely, at these same concentrations, both crude and pure pinnatoxin caused an almost complete abolition of nerve-evoked hemidiaphragm action potential responses, without any effect on electrically-evoked (direct) responses. This neuromuscular block could not be reversed by neostigmine. These results show that pinnatoxins E and F block neuromuscular transmission and suggest that observed in vivo muscle paralysis by pinnatoxin is due to selective antagonism of muscle type nicotinic acetylcholine receptors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Immunoglobulin-containing food products and nutraceuticals such as bovine colostrum are of interest to consumers as they may provide health benefits. Commercial scale colostrum products are valued for their immunoglobulin G (IgG) content and therefore require accurate analysis. One of the most commonly used methods for determining total soluble IgG in colostrum products is based on affinity chromatography using a Protein G column and UV detection. This paper documents improvements to the accuracy of the Protein G analysis of IgG in colostrum products, especially those containing aggregated forms of IgG. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis confirmed that aggregated IgG measured by Protein G does not contain significant amounts of casein or other milk proteins. Size exclusion chromatography identified the content of soluble IgG as mainly monomeric IgG and aggregated material MW > 450 kDa with small amounts of dimer and trimer. The turbidity of the eluting IgG, mainly associated with aggregated IgG, had a significant effect on the quantitative results. Practical techniques were developed to correct affinity LC data for turbidity on an accurate, consistent, and efficient basis. The method was validated in two laboratories using a variety of colostrum powders. Precision for IgG was 2-3% (RSD(r)) and 3-12% (RSD(R)). Recovery was 100.2 ± 2.4% (mean ± RSD, n = 10). Greater amounts of aggregated IgG were solubilized by a higher solution:sample ratio and extended times of mixing or sonication, especially for freeze-dried material. It is concluded that the method without acid precipitation and with turbidity correction provides accurate, precise, and robust data for total soluble IgG and is suitable for product specification and quality control of colostrum products.

Dinoflagellate species isolated from the green calcareous seaweed, Halimeda sp. J.V. Lamouroux, growing in Rarotongan lagoons, included Gambierdiscus australes Faust & Chinain, Coolia monotis Meunier, Amphidinium carterae Hulburth, Prorocentrum lima (Ehrenberg) Dodge, P. cf. maculosum Faust and species in the genus Ostreopsis Schmidt. Isolates were identified to species level by scanning electron microscopy and/or DNA sequence analysis. Culture extracts of G. australes isolate CAWD149 gave a response of 0.04 pg P-CTX-1 equiv. per cell by an N2A cytotoxicity assay (equivalent to ca 0.4 pg CTX-3C cell(-1)). However, ciguatoxins were not detected by LC-MS/MS. Partitioned fractions of the cell extracts potentially containing maitotoxin were found to be very toxic to mice after intraperitoneal (i.p.) injection. A. carterae was also of interest as extracts of mass cultures caused respiratory paralysis in mice at high doses, both by i.p. injection and by oral administration. The Rarotongan isolate fell into a different clade to New Zealand A. carterae isolates, based on DNA sequence analysis, and also had a different toxin profile. As A. carterae co-occurred with G. australes, it may contribute to human poisonings attributed to CTX and warrants further investigation. A crude extract of C. monotis was of low toxicity to mice by i.p. injection, and an extract of Ostreopsis sp. was negative in the palytoxin haemolysis neutralisation assay., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Investigations into a series of dog poisonings on beaches in Auckland, North Island, New Zealand, resulted in the identification of tetrodotoxin (TTX) in the grey side-gilled sea slug, Pleurobranchaea maculata. The levels of TTX in P. maculata, assayed by liquid chromatography-mass spectrometry (LC-MS) ranged from 91 to 850 mg kg(-1) with a median level of 365 mg kg(-1) (n = 12). In two of the dog poisoning cases, vomit and gastrointestinal contents were found to contain TTX. Adult P. maculata were maintained in aquaria for several weeks. Levels of TTX decreased only slightly with time. While in the aquaria, P. maculata spawned, with each individual producing 2-4 egg masses. The egg masses and 2-week old larvae also contained TTX. Tests for other marine toxins were negative and no other organisms from the area contained TTX. This is the first time TTX has been identified in New Zealand and the first detection of TTX in an opisthobranch., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Pinnatoxins and pteriatoxins are a group of cyclic imine toxins that have hitherto only been isolated from Japanese shellfish. As with other cyclic imine shellfish toxins, pinnatoxins cause rapid death in the mouse bioassay for lipophilic shellfish toxins, but there is no evidence directly linking these compounds to human illness. We have identified the known pinnatoxins A (1) and D (6), and the novel pinnatoxins E (7), F (8) and G (5), in a range of shellfish and environmental samples from Australia and New Zealand using LC-MS. After isolation from the digestive glands of Pacific oysters, the structures of the novel pinnatoxins were determined by mass spectrometry and NMR spectroscopy, and their LD(50) values were evaluated by ip administration to mice. Examination of the toxin structures, together with analysis of environmental samples, suggests that pinnatoxins F and G are produced separately in different dinoflagellates. Furthermore, it appears probable that pinnatoxin F (8) is the progenitor of pinnatoxins D (6) and E (7), and that pinnatoxin G (6) is the progenitor of both pinnatoxins A-C (1 and 2) and pteriatoxins A-C (3 and 4), via metabolic and hydrolytic transformations in shellfish.

Brevetoxins (BTXs) are a class of cyclic polyether toxins produced by the dinoflagellate Karenia brevis. These substances are subject to extensive conjugative metabolism in shellfish. BTX-B forms a conjugate with cysteine and is oxidized and reduced to yield BTX-B2, which is further modified by fatty acid addition via cysteine amide linkage to give biologically active brevetoxin metabolites. In this study, we evaluated the commonly used in vitro (ELISA, radioimmunoassay, receptor binding assay and N2A cytotoxicity assay) and in vivo mouse brevetoxin bioassays for the detection of the brevetoxin fatty acid conjugate N-palmitoylBTX-B2, and compared the results to those for dihydroBTX-B and BTX-B2. The receptor binding assay for N-palmitoylBTX-B2 showed comparable sensitivity to that for dihydroBTX-B, and an 11-fold higher sensitivity than for BTX-B2. Although the ELISA showed similarly high sensitivity to dihydroBTX-B and BTX-B2, with EC(50) values of ca. 0.26 ng/ml, it was 23 times less sensitive to N-palmitoylBTX-B2. On the other hand, the N2A cytotoxicity assay was highly sensitive to N-palmitoylBTX-B2, with an EC(50) of 0.15 ng/ml, but was 12- and 40-fold less sensitive to dihydroBTX-B and BTX-B2, respectively. The relative sensitivity of the N2A cytotoxicity assay for each of these metabolites paralleled that of the mouse bioassay (relative LD(50) values 1:20:30 for N-palmitoylBTX-B2:dihydroBTX-B:BTX-B2). We conclude that the most sensitive bioassay for dihydroBTX-B and BTX-B2 is the ELISA, whereas the N2A cytotoxicity assay is most sensitive for N-palmitoylBTX-B2., (Published by Elsevier Ltd.)

Seafood in many parts of the world may become contaminated with high levels of domoic acid and domoic acid isomers, and such seafood has been shown to cause toxic effects in humans and in marine animals. Domoic acid itself has been held responsible for the observed effects, although the possible contribution of the isomers to toxicity has not been investigated. In the present study, the acute intraperitoneal toxicity of isodomoic acid C in mice was found to be lower than that of domoic acid. Furthermore, the severities of the behavioural changes induced by isodomoic acids A, B and C were all much lower than that of domoic acid itself, suggesting that these substances pose relatively little risk to human or animal health.

Lipophilic phycotoxins in size-fractionated plankton net tows (20 mum mesh-size) were measured on-board during a month-long oceanographic cruise in North Sea coastal waters. Tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS) was employed for the detection and quantification of a broad spectrum of known and putative phycotoxins. For pectenotoxins (PTXs) the following ion masses ([M + NH(4)](+)) were monitored: m/z 876 for PTX-2, m/z 892 for PTX-11 and PTX-13, and m/z 874 for PTX-12 and PTX-14. The PTX levels in net plankton were highest along the Danish north coast, but levels over 50 ng per net tow were also detected on the southern Scottish East coast and in the northern Skagerrak. Abundance of PTXs was highly correlated with the occurrence of the marine dinoflagellate Dinophysis spp. Whereas in the eastern North Sea PTX-2 was the most abundant PTX, in the western North Sea PTX-1 was the major component, but it was also present in lower proportions in the Norwegian and Danish waters than in the western North Sea. Isobaric PTX-11 was absent or only detected at trace levels throughout the entire cruise, and PTX-13 and PTX-14 were not detected at all. The identity of PTX-1 was confirmed by comparison of retention time and mass spectrum of the North Sea phytoplankton sample to PTX-1 previously isolated from shellfish. Statistical analysis showed the best correlation between the occurrence of PTX-1 and Dinophysis acuminata cell concentration. Nevertheless, we could not rule out the possibility of metabolic transformations of PTXs by organisms that have grazed upon Dinophysis. Such biotransformations could conceivably occur in heterotrophic dinoflagellates or ciliates, or even via oxidation in copepod fecal pellets. In any case, this study confirmed the presence of PTX-1 in the plankton and is the first definitive report of this toxin in the North Sea.

The microcystin (MC) content and cyanobacterial community structure of Antarctic microbial mat samples collected from 40 ponds, lakes, and hydroterrestrial environments were investigated. Samples were collected from Bratina Island and four of the Dry Valleys, Wright, Victoria, Miers, and Marshall. Enzyme-linked immunosorbent assays (ELISAs), liquid chromatography-mass spectrometry (LC-MS), and protein phosphatase 2A (PP-2A) inhibition assays resulted in the identification of low levels (1 to 16 mg/kg [dry weight]) of MCs in all samples. A plot of indicative potencies of MCs (PP-2A inhibition assay/ELISA ratio) versus total MCs (ELISA) showed a general decrease in potency, as total MC levels increased, and a clustering of values from discrete geographic locations. LC-tandem MS analysis on selected samples identified eight novel MC congeners. The low-energy collisional activation spectra were consistent with variants of [D-Asp(3)] MC-RR and [D-Asp(3)] MC-LR containing glycine [Gly(1)] rather than alanine and combinations of homoarginine [hAr(2)] or acetyldemethyl 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (acetyldemethyl ADDA) [ADMAdda(5)] substitutions. Nostoc sp. was identified as a MC producer using PCR amplification of a region of the 16S rRNA gene and the aminotransferase domain of the mcyE gene. Automated ribosomal intergenic spacer analysis (ARISA) was undertaken to enable a comparison of cyanobacterial mat community structure from distant geographical locations. Two-dimensional multidimensional scaling ordination analysis of the ARISA data showed that in general, samples from the same geographic location tended to cluster together. ARISA also enabled the putative identification of the MC-producing Nostoc sp. from multiple samples.

Brevetoxins are neurotoxins associated with blooms of marine algae such as Karenia brevis and can accumulate in the marine food chain, causing intoxication of marine animals and people consuming seafood. Brevetoxin-B2 ( 5) is a toxic metabolite produced in shellfish exposed to algae that contain brevetoxin-B ( 1). S-Desoxybrevetoxin-B2 ( 4) has been proposed as a cometabolite produced during this transformation, and while LC-MS analyses suggest its presence in shellfish, it has not yet been isolated and characterized. Studies on these materials are severely constrained by the difficulty of obtaining and purifying them from natural sources. We have developed a convenient one-pot conversion of commercially available brevetoxin-B ( 1) into S-desoxybrevetoxin-B2 ( 4), and a simple method for converting 4 into brevetoxin-B2 ( 5). Full NMR and mass-spectral characterization of 4 and 5 confirmed their structures and showed that the ratio of diastereoisomers in the synthetic 4 and 5 was similar to that observed in naturally contaminated shellfish. The LD 50 values for 4, 5, and dihydrobrevetoxin-B ( 6) by ip injection in mice were 211, 400, and 250 microg/kg, respectively. The methodology for synthesis of brevetoxin metabolites should greatly facilitate toxicological, biochemical and immunochemical studies of these substances, as well as the production of analytical standards.

Several natural isomers of the seizurogenic neurotoxin domoic acid (DA) have been found to occur at up to mg/kg levels in shellfish. The aim of the current study was to assess the neurotoxic potency of isodomoic acids A and C (Iso-A and Iso-C), recently isolated from commercial shellfish. Hippocampal slices were obtained from young adult rats and maintained in a tissue recording chamber. Synaptically evoked population spikes were recorded in region CA1 before and after exposure to DA or its isomers. Both Iso-A and Iso-C produced transient neuronal hyperexcitability followed by a dose-dependent suppression of population spikes, but were, respectively, 4- and 20-fold less potent than DA (spike area: EC50 DA=237 nM; Iso-A=939 nM; Iso-C=4.6 microM). In the hippocampus, DA preconditioning induces tolerance to subsequent DA toxicity. However, in the present study neither Iso-A nor Iso-C were effective as preconditioning agents. Competitive binding studies using homomeric GluR6 kainate (kainic acid, KA) receptors showed the affinity of Iso-A to be 40-fold lower than DA (Ki DA=3.35 nM; Iso-A=130 nM). Together with earlier work showing Iso-C affinity at GluR6 receptors to be 240-fold lower than DA, our results suggest that neuroexcitatory effects of Iso-A in CA1 may involve both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and KA receptors, while Iso-C likely involves the activation of AMPA receptors alone.

In November 2005, at least five dogs died rapidly after contact with water from the Hutt River (lower North Island, New Zealand). Necropsy performed 24h later on one of the dogs (a 20-month-old Labrador) revealed few findings of interest, except for copious amounts of froth in the respiratory tract down to the bifurcation of the trachea and large quantities of algal material in the dog's stomach. Low and relatively stable flows in the Hutt River during spring had resulted in the proliferation of benthic cyanobacteria that formed large black/brown mats along the river edge. Samples from the Labrador's stomach contents and cyanobacterial mats were analysed microscopically and screened using chemical and biochemical assays for cyanotoxins: anatoxin-a, homoanatoxin-a, cylindrospermopsins, saxitoxins and microcystins. Liquid chromatography-mass spectrometry (LC-MS) confirmed the presence of the neurotoxic cyanotoxins anatoxin-a and homoanatoxin-a and their degradation products, dihydro-anatoxin-a and dihydro-homoanatoxin-a. This is the first report of homoanatoxin-a and associated degradation product in New Zealand. Based on morphology, the causative species was identified as Phormidium sp. Subsequent phylogenetic analysis of 16S rRNA gene sequences demonstrated that the causative organism was most similar to Phormidium autumnale. Further investigations led to the detection of homoanatoxin-a and anatoxin-a in cyanobacterial mats from four other rivers in the Wellington region (lower North Island, New Zealand). Access restrictions were placed on over 60% of river catchments in the western Wellington region, severely affecting recreational users.

Cyanobacterial blooms in New Zealand surface water resources have been surveyed and, in response to strict new standards for drinking water, more intensive monitoring for cyanotoxins has been initiated. Aphanizomenon issatschenkoi was recently identified in a New Zealand lake and was found to produce the potent neurotoxin anatoxin-a (ATX). A strain of Aph. issatschenkoi (CAWBG02) was cultured for ATX production and a novel derivative of ATX was found to account for a high proportion of the toxin content in the Aph. issatschenkoi cells. Spectroscopic data (LC-UV, liquid chromatography with ultraviolet absorption detection; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LC-HRMS, liquid chromatography with high resolution mass spectrometry) identified this derivative as 11-carboxyl anatoxin-a. Although precursors with a carboxyl group on C11 have been postulated in the biosynthetic pathway for ATX from amino acids and acetate, this is the first identification of a specific intermediate. The production of ATX and the intermediate by Aph. issatschenkoi was studied under different growth conditions. Concentrations of ATX and the intermediate increased in the aerated culture to 170 microg/L and 330 microg/L, respectively, at 21 days (18 x 10(9) cells/L). Cell concentrations did not markedly increase during subsequent growth to 37 days. ATX concentrations decreased, and 11-carboxyl ATX concentrations continued to increase during this period. Toxin production by Aph. issatschenkoi cells was maximal at 6 days of growth (0.08-0.09 pg/cell each; 2.3 x 10(8) cells/L). Other ATX analogues and metabolites were not detected in the cultures. Freeze-thawing of cultures resulted in complete conversion of the intermediate to ATX with a half-life of 5 min, and this conversion was inhibited by acidification, heating of the culture to 100 degrees C, or addition of methanol. The implications of the findings for mechanisms of biosynthesis of anatoxins by cyanobacteria and for monitoring of water bodies for cyanotoxins are discussed.

Two novel pectenotoxins (PTXs), PTX-13 and -14, were isolated from extracts of Dinophysis acuta collected from the west coast of South Island, New Zealand. The compounds were identified as oxidized analogues of PTX-2 by NMR spectroscopic and LC-MS studies. PTX-13 (32R-hydroxyPTX-2) corresponds to the unidentified analogue PTX-11x reported by [Suzuki et al., 2003. Liquid chromatography-mass spectrometry of spiroketal stereoisomers of pectenotoxins and the analysis of novel pectenotoxin isomers in the toxic dinoflagellate Dinophysis acuta from New Zealand. J. Chromatogr. A 992, 141-150]. PTX-13 underwent slow deuteration at the 13beta-position during NMR analysis. PTX-14 corresponds to the 32,36-dehydration product of PTX-13, and may be an artifact.

Yessotoxin 32-O-[beta-L-arabinofuranosyl-(5'-->1'')-beta-L-arabinofuranoside] (3) was isolated from extracts of Protoceratium reticulatum during a large scale isolation of yessotoxin (1). The structure was characterized by mass spectrometry and NMR spectroscopy. Di-glycoside-3, along with the corresponding mono-glycoside (2) were detected in cultures of P. reticulatum originating from Europe and New Zealand, suggesting that production of arabinosides of 1 is a normal feature of this alga. Formation of multiply charged anions and fragmentation of 3 occurred much more readily than for 1 and 2 under the LC-MS conditions used in this study.

Preparative HPLC purification of a side-fraction obtained during purification of 44,55-dihydroxyyessotoxin (6) afforded fractions containing previously unidentified yessotoxin analogues. Careful analysis of these fractions by HPLC-UV, LC-MS3, and NMR spectroscopy, revealed the identities of some of these analogues as 45-hydroxy-46,47-dinoryessotoxin (1), 44-oxo-45,46,47-trinoryessotoxin (2) and 9-methyl-42,43,44,45,46,47,55-heptanor-38-en-41-oxoyessotoxin (5). Numerous other analogues were present but could only be characterized by HPLC-UV and LC-MS3 due to their low abundance. The HPLC-UV and LC-MS3 data confirm the presence of large numbers of yessotoxin analogues, some of which may be oxidative degradation products, in extracts of Protoceratium reticulatum. Compound-1 is the first 46,47-dinoryessotoxin to be identified.

In 1987 a large-scale incident of human poisoning in Canada was traced to commercial mussels contaminated with domoic acid (DOM). Since then, routine screening of shellfish domoic acid content has been carried out using a variety of assays, with liquid chromatography using ultraviolet absorbance detection (LC-UV) or mass spectrometric detection (LC-MS) being the currently accepted standard methodologies. Recently, a highly specific competitive enzyme-linked immunosorbent assay (cELISA) has been developed for the detection and analysis of DOM in commercial shellfish, but its accuracy relative to LC methods has not been independently verified in mammalian tissues. In this study we demonstrate that measurement of rat serum DOM concentration by cELISA gives a good correlation (r2 = 0.993) across a broad range of concentrations when compared to LC-MS analysis, with only a small (15%) overestimation of sample DOM content. In addition, we have developed an extraction method for analysis of DOM in rat brain by cELISA which yields complete recovery across a range of sample dilutions.

44,55-Dihydroxyyessotoxin (1) was isolated from extracts of Protoceratium reticulatum and identified by analysis of its one- and two-dimensional NMR and mass spectra. In addition, LC-MS methods revealed the presence of compounds tentatively identified as (44-R,S)-44,55-dihydroxy-41a-homoyessotoxin (2) and (44-R,S)-44,55-dihydroxy-9-methyl-41a-homoyessotoxin (3). LC-MS analyses indicate that 1 is a constituent of P. reticulatum in New Zealand and Norway, and it was present in three species of mussels from New Zealand, Norway, and Canada.

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).

An unusual isomer of domoic acid (1), isodomoic acid C (2), has been found in New Zealand shellfish contaminated by amnesic shellfish poisoning (ASP) toxins and was shown to be produced by a local strain of the pennate diatom Pseudo-nitzschia australis. A bulk culture of this strain was used to isolate 2. The structure was determined from spectroscopic data and was shown to correspond to that of 2 from a Japanese red seaweed, the only other reported occurrence of this compound. The affinity of 2 for GluR6 glutamate receptors was 240-fold lower than for 1, indicating low neurotoxic potential.

Two analogs of yessotoxin were isolated from extracts of a culture of Protoceratium reticulatum. The structures of the analogs were identified as trihydroxylated amides of 41a-homoyessotoxin (1) and 9-methyl-41a-homoyessotoxin (2) by one- and two-dimensional 1H and 13C NMR spectroscopy and LC-MS3 analyses. Structures were further confirmed by micro-scale chemical conversions combined with LC-MS3 analyses. No toxic effects were recorded in mice injected intraperitoneally with 2 at a dose of 5000 microg/kg.

A liquid chromatography/mass spectrometry (LC/MS) method for amnesic shellfish poisoning toxins in shellfish was developed and validated. Tissue homogenate (4 g) was extracted with 16 mL methanol-water (1 + 1, v/v). Dilution into acetonitrile-water (1 + 9, v/v) was followed by C18 solid-phase extraction cleanup. Domoic acid (DA) and epi-domoic acid were determined by LC/MS/MS with electrospray ionization and multiple reaction monitoring. External calibration was performed with dilutions of a certified reference standard. Advantages of this method include speed, lower detection limits, and a very high degree of specificity. The LC/MS response was highly linear, and there were no significant interferences to the determination of DA. Formal method validation was performed on 4 shellfish species. Fortification studies gave recoveries (mean +/- SD; n = 24) of 93 +/- 14% at 1 mg/kg, and 93.3 +/- 7.6% at 20 mg/kg over all the species. Analysis of a mussel certified reference material showed the bias as < 5%. The limits of detection and quantitation were 0.15 and 0.5 mg/kg, respectively. Routine application of the method over 4 months gave a recovery for the QC sample (1 mg/kg fortified blank mussel homogenate) run with each batch of 88.9 +/- 5.5% (mean +/- SD; n = 37). The total uncertainty of measurement results were estimated as 0.12 (12%) at 0.25-5 mg/kg and 0.079 (7.9%) at 5-50 mg/kg. The major contribution to the uncertainty was the repeatability of the LC/MS determination, probably arising from subtle matrix effects.