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Cyclic imines (CIs) produced by microalgal species that accumulate in the food chains of marine organisms are novel biotoxins that do not belong to the classic group of marine biotoxins. In the past, CIs were found only in limited areas; however, in recent years, rapid changes in marine ecosystems have led to widespread CIs and increased exposure to toxic risks. In this study, we analyzed seven CI toxins, GYM-A, SPX (13-desmethyl spirolide C, 13, 19-dideMe spirolide C, 20-methyl spirolide G), and PnTX-E, F, and G, using LC/MRM-MS. Shellfish samples were purchased from a domestic Korean fish market (67 samples in 2021 and 216 samples in 2022). The entire body of the shellfish was ground and extracted with 50% methanol, followed by lipophilic-specific SPE. Only GYM-A, PnTX-G, and 13-desmethyl spirolide C were detected in all analyzed samples. The maximum concentrations of GYM-A is maximum 179 ppt (ng/kg) in Crassostrea nippona (March 2022), PnTX-G is maximum 7 ppt in Anadara broughtonii (April 2022), 13-desmethyl SPX C is maximum 58 ppt in Crassostrea nippona (April 2022). The southern coast exhibited the highest frequency of detection of these toxins, which was attributed to elevated sea-surface temperatures, aligned with conducive conditions for toxin-producing phytoplankton. According to the monitoring results, there were no significant CI toxins in the shellfish; however, it is important to monitor CI toxin accumulation in shellfish because of their high risk of toxicity. [ABSTRACT FROM AUTHOR]

An efficient strategy for rapid assembly of the complex substituted cyclohexene core that is present in several cyclic imine marine toxins is presented. Several of these toxins, including pinnatoxin A and recently discovered portimine A, have been the focus of much attention due to their fascinating biological activities. We demonstrate that the substituted cyclohexene-diene motif, which is a challenging feature to access synthetically, can be prepared through a stepwise Ireland–Claisen rearrangement/enyne metathesis procedure beginning from chiral esters. This approach enables a divergent strategy that can be implemented in syntheses of cyclic imines or derivatives thereof. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Active and passive sampling methods were employed over a four-month period, at a site off the South-West coast of Ireland, to characterise the occurrence of cyclic imines in the water column. The marine toxins 13-desmethyl-SPXC, 20-methyl SPXG toxins and pinnatoxin G were detected using active sampling from Diaion HP-20 resin. Seven water depths were sampled to determine stratification of the toxins in the water column using Solid Phase Adsorption and Toxin Tracking (SPATT). Both 13-desmethyl-SPXC and pinnatoxin G were detected using two different resin types; Diaion HP-20 and Amberlite XAD761. HP-20 proved more effective at accumulating the toxins, with a higher percentage of positive samples and a higher ratio of toxin adsorbed relative to XAD761. No temporal variation in toxin-quantities was detected, indicating that there was no change in density of causative algal species in the water column. Pinnatoxin G was detected more frequently from surface to 30 m depth, with a similar pattern observed for 13-desmethyl-SPXC occurrence using XAD761. No difference in the occurrence of 13-desmethyl-SPXC was observed between depths using HP-20 resin. This is the first reported incidence of pinnatoxin G in Irish waters and highlights cyclic imines as emerging toxins in European waters. [ABSTRACT FROM AUTHOR]

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 (n ACh Rs) at the neuromuscular junction. In the present study, binding affinities and modes of these pinnatoxin isomers at neuronal and muscle n ACh Rs 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 n ACh Rs, 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 n ACh Rs, with interactions potentially playing a role in pinnatoxin toxicity. [ABSTRACT FROM AUTHOR]

Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts. [ABSTRACT FROM AUTHOR]

The transport of microalgae in the ballast tanks of shipping vessels is of global concern because many algal species can survive in ballast tanks as nonmotile or cyst stages increasing the likelihood of introductions into foreign ports. In 2004 a peridinoid dinoflagellate was collected and isolated from ballast residuals of the merchant vessel Southern Fighter in Port Tampa Bay, Florida, USA. Light and electron microscopy of the motile and nonmotile peridinoid cells indicated the dinoflagellate was Vulcanodinium rugosum . Bayesian and maximum-parsimony (MP) phylogenetic analyses of V. rugosum strain CCFWC516 showed that sequenced regions of its LSU and ITS matched those of V. rugosum strains from Japan but were divergent from those strains from New Zealand and France. LC–MS analyses indicated that strain CCFWC516 did not produce the neurotoxin pinnatoxin, an ability that has been reported for other strains of this species. Analyses did show, however, that strain CCFWC516 did produce portimine, a cyclic imine produced by all other strains of this species. The M/V Southern Fighter visited numerous ports along the coast of Japan and the North Sea before sailing to Florida. The phylogenetic match to Japanese strains, as well as the ship's ballast exchange history, suggests that strain CCFWC516 originated from Japan. In light of the increase in global shipping traffic, increases in vessel size and capacity, and the increased connectivity between the Pacific Ocean and the Caribbean and Gulf of Mexico that will result from the widening of the Panama Canal, the introduction of nonnative, harmful algal species is an area of heightened concern and calls for increased vigilance. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

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]

Abstract: Recent studies showing the presence of pinnatoxins in a range of shellfish from New Zealand and Australia () prompted an investigation into historical mouse bioassay positives from Rangaunu Harbour, Northland, New Zealand. Eleven shellfish samples collected from the harbour between 1993 and 2008 were found to contain pinnatoxins as shown by liquid chromatography–mass spectrometric (LC–MS) analysis. Reanalysis of data from a quantitative mouse bioassay using an acetone extraction gave excellent correlation to pinnatoxin levels for seven of the nine samples tested. The cause of positive mouse bioassay results when using diethyl ether was not confirmed. A telephone survey of 22 Rangaunu Harbour residents confirmed that they had regularly consumed shellfish and other seafood collected from the harbour. Consumption was on average 2.6 times/week over 29 years. There were no reports of any associated illnesses. [Copyright &y& Elsevier]

Abstract: 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 &y& Elsevier]

Abstract: 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 &y& Elsevier]

The article presents a study which investigates the production of pinnatoxins E, F, and G through scrippsielloid dinoflagellates. It states that the cysts of dinoflagellates were isolated in December 2009 from surface from the sediments linked with eel grass gathered at Franklin Harbour in South Australia and were injected to mice. Results show that the Australian isolates are the first to report of a micro-alga producing pinnatoxin G and the extracts of all isolates were highly toxic to mice.

Abstract: A peridinoid dinoflagellate was newly identified as the producer of pinnatoxins E (0–3.7pgcell−1) and F (0.3–20.1pgcell−1), as determined by LC–MS analysis of extracts of eight strains of the organism. The cyst-forming, thecate dinoflagellate was isolated from surface sediments associated with eel grass beds and mangroves in Rangaunu Harbour, Northland, New Zealand. Extracts of mass cultures of the dinoflagellate were tested for toxicity in mice by intraperitoneal injection, gavage and voluntary consumption. The LD50 values were 1.33, 2.33 and 5.95mg/kg respectively. [Copyright &y& Elsevier]

Pinnatoxin H was isolated from a culture of the dinoflagellate Vulcanodinium rugosum isolated from the South China Sea. The structure of pinnatoxin H was elucidated by LC–MS/MS and NMR spectroscopy. It was found to have the same macrocyclic structure and substituents as pinnatoxins D, E and F, but the side chain on the cyclohexenyl ring was an ethenyl group, as found in pinnatoxin G and portimine. The observation that this strain of V. rugosum produced only pinnatoxin H and portimine is consistent with previous findings that the profile of pinnatoxins can vary significantly among strains. The acute toxicity of pinnatoxin H to mice was 67 μg/kg (intraperitoneal) and 163 μg/kg (gavage). [ABSTRACT FROM AUTHOR]

Abstract: Portimine, a new polycyclic ether toxin containing a cyclic imine moiety, was isolated from the marine benthic dinoflagellate Vulcanodinium rugosum collected from Northland, New Zealand. The structure of portimine, including the relative configurations, was elucidated by spectroscopic analyses. The cyclic imine moiety consists of an unprecedented five-membered ring with a spiro-link to a cyclohexene ring. This is the only structural similarity to the pinnatoxin group of polycyclic ethers also produced by V. rugosum, which all contain a six-membered cyclic imine ring. The LD50 of portimine to mice by intraperitoneal injection was 1570μg/kg, indicating a much lower toxicity than many other cyclic imine shellfish toxins. In contrast, portimine was highly toxic to mammalian cells in vitro with an LC50 to P388 cells of 2.7nM, and activation of caspases indicating apoptotic activity. [Copyright &y& Elsevier]