Brevisulcenal-F: a polycyclic ether toxin associated with massive fish-kills in New Zealand.

A novel marine toxin, brevisulcenal-F (KBT-F, from karenia brevisulcata toxin) was isolated from the dinoflagellate Karenia brevisulcata. A red tide of K. brevisulcata in Wellington Harbour, New Zealand, in 1998 was extremely toxic to fish and marine invertebrates and also caused respiratory distress in harbor bystanders. An extract of K. brevisulcata showed potent mouse lethality and cytotoxicity, and laboratory cultures of K. brevisulcata produced a range of novel lipid-soluble toxins. A lipid soluble toxin, KBT-F, was isolated from bulk cultures by using various column chromatographies. Chemical investigations showed that KBT-F has the molecular formula C(107)H(160)O(38) and a complex polycyclic ether nature. NMR and MS/MS analyses revealed the complete structure for KBT-F, which is characterized by a ladder-frame polyether scaffold, a 2-methylbut-2-enal terminus, and an unusual substituted dihydrofuran at the other terminus. The main section of the molecule has 17 contiguous 6- and 7-membered ether rings. The LD(50) (mouse i.p.) for KBT-F was 0.032 mg/kg.

Convenient large-scale purification of yessotoxin from Protoceratium reticulatum culture and isolation of a novel furanoyessotoxin.

Yessotoxins from a large-scale culture (226 L) of Protoceratium reticulatum strain CAWD129 were harvested by filtration followed by solid-phase extraction. The extract was purified by column chromatography over basic alumina and reverse-phase flash chromatography to afford pure yessotoxin (193 mg). Isolation of yessotoxin was greatly facilitated by selection of a strain which did not produce analogues that interfered with yessotoxin isolation. In addition to yessotoxin, numerous minor yessotoxins were detected by LC-MS in other fractions. From one of these, an early eluting minor analogue with the same molecular weight as yessotoxin and a similar mass spectrometric fragmentation pattern was isolated. This analogue was identified by NMR and mass spectrometry as a novel yessotoxin analogue containing a furan ring in the side chain. This finding reveals biosynthetic flexibility of the yessotoxin pathway in P. reticulatum and confirms earlier findings of production of many minor yessotoxin analogues by this alga. Production of these analogues appeared to be a constitutive trait of P. reticulatum CAWD129.

Identification of 45-hydroxy-46,47-dinoryessotoxin, 44-oxo-45,46,47-trinoryessotoxin, and 9-methyl-42,43,44,45,46,47,55-heptanor-38-en-41-oxoyessotoxin, and partial characterization of some minor yessotoxins, from Protoceratium reticulatum.

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.

Isolation and identification of a cis-C8-diol-ester of okadaic acid from Dinophysis acuta in New Zealand.

A cis-isomer of a C(8)-diol ester of okadaic acid (1) was isolated during large-scale purification of pectenotoxins (PTXs) from extracts of Dinophysis acuta collected from the west coast of South Island, New Zealand. The compound was identified by NMR spectroscopic and liquid chromatography-mass spectrometry (LC-MS) studies, and is the first reported cis-isomer of an okadaic acid C(8)-diol-ester identified in Dinophysis. The more abundant trans-C(8)-diol ester of okadaic acid (2) isolated from the same Dinophysis extract was rapidly hydrolyzed to okadaic acid in vitro by the supernatant from green-lipped mussel hepatopancreas.

Isolation of yessotoxin 32-O-[beta-L-arabinofuranosyl-(5'-->1'')-beta-L-arabinofuranoside] from Protoceratium reticulatum.

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.

Isolation and identification of pectenotoxins-13 and -14 from Dinophysis acuta in New Zealand.

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.

Production of 7-epi-pectenotoxin-2 seco acid and assessment of its acute toxicity to mice.

Pectenotoxins (PTXs) accumulate in shellfish feeding on dinoflagellates of the genus Dinophysis, so that humans can be exposed to these toxins through shellfish consumption. Some PTXs are toxic to experimental animals, whereas others are of much lower toxicity. Pectenotoxin-2, the most abundant PTX from most Dinophysis spp., is rapidly metabolized by most shellfish to a mixture of pectenotoxin-2 seco acid (2) and 7-epi-pectenotoxin-2 seco acid (1). A mixture of 1 and 2 was produced during purification of an extract from in vitro enzymatic hydrolysis of pectenotoxin-2. These were separated by preparative HPLC, and the structure of 1 was confirmed by one- and two-dimensional 1H and 13C NMR spectroscopy and LC-MS3 analyses. No toxic changes were recorded in mice injected intraperitoneally with 1 or 2 at a dose of 5000 microg/kg. PTX seco acids are therefore unlikely to be of consequence to human consumers at the concentrations found in contaminated shellfish.

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.

Polyhydroxylated amide analogs of yessotoxin from Protoceratium reticulatum.

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.

Isolation and identification of (44-R,S)-44,55-dihydroxyyessotoxin from Protoceratium reticulatum, and its occurrence in extracts of shellfish from New Zealand, Norway and Canada.

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.

Investigations into the cellular actions of the shellfish toxin gymnodimine and analogues.

The effects of the shellfish toxin gymnodimine and its analogues (gymnodimine acetate, gymnodimine methyl carbonate and gymnodamine) on cellular viability were tested using the Neuro2a neuroblastoma cell line. Concentrations of toxins up to 10µM had variable effects on reducing cell number as determined using the MTT assay and no effects on the expression of a number of signal transduction proteins (c-Jun, ATF-2, ATF-3) which are sensitive to cellular stress. However, pre-exposure of Neuro2a cells to 10µM concentrations of toxins for 24h greatly sensitized these cells to the apoptotic effects of another algal toxin, okadaic acid. These results suggest that gymnodimine and its analogues sensitize Neuro2a cells to cytotoxins and raise the possibility that algal blooms involving the production of both okadaic acid-type molecules and gymnodimine may generate greater cytotoxicity and pose a greater public health problem. Furthermore, our studies establish the Neuro2a cell line as a potentially high-throughput cellular system sensitive to the pharmacological effects of gymnodimine and analogues, and as a potential screen for algal-derived toxins.

Solid phase adsorption toxin tracking (SPATT): a new monitoring tool that simulates the biotoxin contamination of filter feeding bivalves.

A simple and sensitive in situ method for monitoring the occurrence of toxic algal blooms and shellfish contamination events has been developed. The technique involves the passive adsorption of biotoxins onto porous synthetic resin filled sachets (SPATT bags) and their subsequent extraction and analysis. The success of the method is founded on the observation that during algal blooms significant amounts of toxin, including the low polarity lipophilic compounds such as the pectenotoxins and the okadaic acid complex toxins, are dissolved in the seawater. The results of field trials during Dinophysis acuminata and Protoceratium reticulatum blooms are presented. These data prove the concept and demonstrate that the technique provides a means of forecasting shellfish contamination events and predicting the net accumulation of polyether toxins by mussels. As an early warning method it has many advantages over current monitoring techniques such as shellfish-flesh testing and phytoplankton monitoring. In contrast to the circumstantial evidence provided by genetic probe technologies and conventional phytoplankton monitoring methods, it directly targets the toxic compounds of interest. The extracts that are obtained for analysis lack many of the extraneous lipophilic materials in crude shellfish extracts so that many of the matrix problems associated with chemical and biological analysis of these extracts are eliminated. Analyses can confidently target parent compounds only, because analytical and toxicological uncertainties associated with the multiplicity of toxin analogues produced by in vivo biotransformation in shellfish tissues are reduced. Time integrated sampling provides a good simulation of biotoxin accumulation in filter feeders and the high sensitivity provides lengthy early warning and conservative estimates of contamination potential. The technique may reduce monitoring costs and provide improved spatial and temporal sampling opportunities. When coupled with appropriate analytical techniques (e.g. LC-MS/MS multi-toxin screens, ELISA assays, receptor binding assays), the technique has the potential to offer a universal early warning method for marine and freshwater micro-algae toxins.

Acute toxicity of gymnodimine to mice.

The acute toxicity of the phycotoxin gymnodimine to female Swiss mice by intraperitoneal injection and by oral administration has been determined. Gymnodimine was highly toxic by injection, the LD50 being only 96 microg/kg. Animals either died within 10 min of injection or made a full recovery with no perceptible long-term effects. Gymnodimine was also toxic after oral administration by gavage (LD50 755 microg/kg), but was much less toxic when administered with food. No signs of toxicity were seen in mice voluntarily ingesting food containing gymnodimine at a level sufficient to give a dose of approximately 7500 microg/kg. Pre-treatment with physostigmine or neostigmine protected against injected gymnodimine, suggesting that the latter exerts its toxic effects via blockade of nicotinic receptors at the neuromuscular junction. The low toxicity of gymnodimine when ingested with food suggests that this compound is of low risk to humans, a conclusion that is consonant with anecdotal evidence for the absence of harmful effects in individuals consuming shellfish contaminated with gymnodimine.

Complex toxin profiles in phytoplankton and Greenshell mussels (Perna canaliculus), revealed by LC-MS/MS analysis.

Toxin profiles were determined in phytoplankton cell concentrates and Greenshell mussels (Perna canaliculus) exposed to a dinoflagellate bloom dominated by Dinophysis acuta and Protoceratium reticulatum. This was achieved by using a method for the simultaneous identification and quantification of a variety of micro-algal toxins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionisation (+/-) and monitoring of daughter ions in multiple reaction modes. Plankton concentrates and shellfish contained high levels of yessotoxins (YTXs) and pectenotoxins (PTXs) and low levels of okadaic acid (OA). A high proportion (>87%) of the OA in both plankton and shellfish was released by alkaline hydrolysis. An isomer of pectenotoxin 1 (PTX1i) was nearly as abundant as pectenotoxin 2 (PTX2) in the plankton and shellfish, and the latter contained high levels of their respective seco acids. DTX1, DTX2, and PTX6 were not detected. MS-MS experiments revealed that the shellfish contained several other oxygenated metabolites of YTX in addition to 45-hydroxy yessotoxin (45OH-YTX). Gymnodimine (GYM) was present in the shellfish but not plankton and it was probably the residue from a previous GYM contamination event. Unlike the other toxins, GYM was concentrated in tissues outside the digestive gland and levels did not decrease over 5 months. The depuration rates of YTX and PTXs from mussels were modelled.

Isolation of a 1,3-enone isomer of heptanor-41-oxoyessotoxin from Protoceratium reticulatum cultures.

The 1,3-enone isomer (1) of heptanor-41-oxoyessotoxin (2) was isolated from extracts of Protoceratium reticulatum during large-scale production of yessotoxin (4). We found that 2 readily isomerizes to 1 in the presence of dilute ammonia and present evidence for the existence of 40-epi-2 (3) that also isomerizes to 1. 1-3 were detected by LC-MS methods both in extracts of P. reticulatum cultures and in mussels contaminated with yessotoxins. The isomerization of 2 and 3 into 1 occurs so readily that purification on basic alumina needs to be conducted carefully. No toxic effects were recorded in mice injected intraperitoneally with 1 at a dose of 5,000 microg/kg.

Isolation of pectenotoxin-2 from Dinophysis acuta and its conversion to pectenotoxin-2 seco acid, and preliminary assessment of their acute toxicities.

We have developed a simple and effective method for isolating pectenotoxin-2 (PTX-2) from Dinophysis cells collected from a natural bloom. A two-step extraction procedure followed by two column chromatography steps produced PTX-2 in high purity suitable for use as an analytical standard and for toxicological studies. Incubation of purified PTX-2 with the supernatant from ultracentrifuged blue (Mytilus edulis) or Greenshell (Perna canaliculus) mussel hepatopancreas homogenate caused rapid conversion to pectenotoxin-2 seco acid (PTX-2 SA). Purification of PTX-2 SA was achieved by solvent extraction followed by column chromatography. PTX-2 and PTX-2 SA were fully characterized by LC-MS and NMR, and full (1)H and (13)C NMR assignments were obtained. Okadaic acid C(8)-diol ester was isolated during the purification of PTX-2, and its identity confirmed by NMR and LC-MS analyses. Pectenotoxin-2 seco acid methyl ester, identified by LC-MS, was also produced during the hydrolytic procedure due to the presence of methanol. PTX-2 was acutely toxic to mice by i.p. injection (LD(50)=219 microg/kg) but no effects were seen with PTX-2 SA at 5000 microg/kg. Neither PTX-2 nor PTX-2 SA was overtly toxic to mice by the oral route at doses up to 5000 microg/kg. No diarrhea was observed in mice dosed with either compound, suggesting that pectenotoxins do not belong in the diarrhetic shellfish poison group.

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.

The acid-catalyzed inter-conversion of spiroketal isomers of pectenotoxins PTX1, PTX6 and PTX2 were studied by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS-MS). Using a C8-silica reversed-phase column and a mobile phase of aqueous acetonitrile containing 2 mM ammonium formate and 50 mM formic acid, the known spiroketal stereoisomers of PTX1 eluted in order of PTX1, PTX4 and PTX8, while those of PTX6 eluted in the order PTX6, PTX7 and PTX9. Acid treatment of PTX2 yielded two novel spiroketal stereoisomers, which have been named PTX2b and PTX2c. LC-MS-MS spectra obtained for the [M+NH4]- ions of PTX2, PTX2b and PTX2c were essentially identical. As an application of the LC-MS-MS methodology, a sample of the toxic dinoflagellate Dinophysis acuta collected from the coast of New Zealand was analyzed for pectenotoxins. PTX2 and a new pectenotoxin, which has been named PTX11, were detected as the most predominant compounds. Novel PTX2 and PTX11 isomers were also found in the D. acuta although the levels of these compounds were low.

Brevisulcatic acids, marine ladder-frame polyethers from the red tide dinoflagellate Karenia brevisulcata in New Zealand.

The isolation and structural determination of new marine ladder-frame polyethers, brevisulcatic acids-1 (1) and -4 (2) are reported. Brevisulcatic acids were isolated from the dinoflagellate Karenia brevisulcata, which was identified as the causative species of a major red tide event in New Zealand in 1998. The ether ring composition and a ß-hydroxy, γ-methylene valeric acid side chain of 1 and 2 are common, but 2 has a γ-lactone as the 5-membered A-ring while 1 is the seco acid analogue. Compound 2 has structural and bioactivity similarities to brevetoxin A.

Identification of pectenotoxin-11 as 34S-hydroxypectenotoxin-2, a new pectenotoxin analogue in the toxic dinoflagellate Dinophysis acuta from New Zealand.

A new pectenotoxin, which has been named pectenotoxin-11 (PTX11), was isolated from the dinoflagellate Dinophysis acuta collected from the west coast of New Zealand. The structure of PTX11 was determined as 34S-hydroxypectenotoxin-2 by tandem mass spectrometry and UV and NMR spectroscopy. PTX11 appears to be only the third pectenotoxin identified as a natural biosynthetic product from algae after pectenotoxin-2 and pectenotoxin-12. The LD50 of PTX11 determined by mouse intraperitoneal injection was 244 microg/kg. The LD(min) of PTX11 in these experiments was 250 microg/kg. No signs of toxicity were recorded in mice following an oral dose of PTX11 at 5000 microg/kg. No diarrhea was observed in any of the animals administered with the test substance by either route of administration. Unlike pectenotoxin-2 (PTX2), PTX11 was not readily hydrolyzed to its corresponding seco acid by enzymes from homogenized green-lipped mussel (Perna canaliculus) hepatopancreas.