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Shellfish toxin research at the NRC Atlantic Research Laboratory.

Shellfish toxins are a global problem and the frequency of toxin outbreaks appears to be increasing. We in Canada are not immune from this threat and public health and the shellfish industry must be protected. The first documented report of shellfish poisoning in Canada appeared in 1793 and recounts the fatal poisoning of several crew members of Captain George Vancouver's expedition following consumption of contaminated mussels. Based on the symptoms described, it has been concluded that the causative agents were the paralytic shellfish poisons (PSPs), known even in Biblical times.

Although they have a less illustrious history, there are additional shellfish toxins of particular significance to Canada. The most recent, of course, is the Amnesic Shellfish Poison (ASP) domoic acid, a neuroexcitatory amino acid not previously associated with shellfish toxicity. There also exists a threat from a third group of toxins called the diarrhetic shellfish poisons (DSPs), which cause vomiting and diarrhea following ingestion. Although outbreaks of DSP poisoning have occurred in the cold-water areas of Japan, Europe and Scandinavia, there is no proof that an outbreak of DSP poisoning has ever occurred in Canada. However, DSP has been detected in shellfish from Maine and it clearly represents a possible threat to the shellfish industry in the Maritime provinces.

Since the domoic acid crisis, the National Research Council's Atlantic Research Laboratory (ARL) has devoted significant efforts to studying shellfish toxins of importance to Canada. These efforts are described in this article.

Paralytic Shellfish Poisons

One might assume that shellfish toxin research got its start at ARL with the identification of the neurotoxin domoic acid in tainted mussels from Prince Edward Island in late 1987. In fact, even before the PEI mussel crisis, work had begun on the PSPS, perhaps the most infamous group of marine toxins.

The PSPs comprise a group of about 12 water soluble, tetrahydropurine derivatives. These compounds are particularly awkward to handle since they are very hygroscopic and extremely sensitive to oxygen. Although known for centuries it was not until the seventies that the first structure, that of saxitoxin, was established. There were many reasons for the delay including the lack of good chromatography systems to separate these very basic, labile, water-soluble compounds, the difficulty in obtaining a suitable derivative for X-ray crystallography, and the presence of the unusual hydrated ketone function. However, as in so many events in science, once the principle concept is established, a rash of additional discoveries soon follow. Within a few years an entire suite of PSPs was discovered.

The two parent structures of the PSP family are saxitoxin and the N-hydroxylated compound neosaxitoxin. The remaining members of the group all possess either the saxitoxin or neosaxitoxin skeleton with varying degrees of sulphation. This is not uncommon in marine metabolites and it influences significantly the chemistry and toxicity of the various members.

Most of the PSPs have been found in shellfish, but they are not the de novo products of these marine invertebrates. Although suspected for a long time, it was not until the thirties that it was established conclusively that certain microalgae or phytoplankton are the real source of the toxins. The culprit microalgae are dinoflagellates belonging to the genera Alexandrium (formerly Gonyaulax), Pyrodinium, and Gymnodinium, which form massive blooms ('red tides') under certain climatic and oceanographic conditions. These microalgae are ingested by feeding shellfish and the PSPs are retained in the gut. Interestingly, the PSP profiles of the dinoflagellates and the shellfish are usually different, indicating catabolic modification of the toxins by the invertebrates. The PSP profile is also highly variable from strain to strain of dinoflagellate and the factors affecting this and indeed the mechanism which triggers PSP production is little understood. Recently, a Japanese group has proposed that the PSPs are not produced by the microalgae at all, but that they are the products of associated microbes. This theory has been questioned and it remains to be seen if it will be supported by the work of other investigators.

The PSPs would probably be just another group of exotic natural products if it were not for the fact that they are ingested and stored by shellfish, a marine delicacy which we enjoy eating. Until relatively recently when we learned the chemical nature of the toxins, there was no easy method of detecting them in seafood. Folklore and tribal customs played a vital part in warning of potential disaster as did feeding the suspect food to the family dog or cat. Nowadays, laboratory mice are injected intraperitoneally with shellfish extracts and, if PSPs are present in dangerous concentrations, the mice develop characteristic symptoms and quickly expire.

For many years this was the only method researchers had to identify or track PSPs. In the 50s, this bioassay was adopted by the Fisheries Inspection Branch of Fisheries and Oceans as a means of testing seafood for human consumption. The assay is reliable, well understood, and relatively simple to operate. The safe limit for PSPS, as set by Health and Welfare Canada, is 0.8 [mu]g/g of shellfish tissue. The mouse bioassay can reliably detect 0.4 [mu]g/g.

In recent years, there has been a demand in the 80s by both the regulatory agencies and the shellfish industry for a cheaper, more sensitive chemical assay that could be automated. In the eighties, James Sullivan of the US Food and Drug Administration (FDA) introduced an HPLC method which could separate PSPs using a resin-based reversed-phase column and a mobile phase containing ion-pairing reagents and buffers. The chromatography is remarkably effective, but the real trick is to detect the PSPs, since, in addition to their intractable nature, the toxins are UV-transparent and no stable, easily detected derivatives have been prepared that are suitable for chromatography. However, it is known that under alkaline conditions the PSPs are easily oxidized to fluorescent derivatives. By attaching a post-column reactor to the HPLC column, the separated PSPs are converted to their fluorescent derivatives and these are detected as they elute from the reactor. The system is complex and requires a great deal of familiarization and skill to make it work effectively. It is certainly not as simple to operate as the mouse bioassay, but it does satisfy the requirements of sensitivity (it is over 1000 times more sensitive than the bioassay), it has the potential for automation, and it provides important information about the nature and distribution of the toxins present.

The problem of detecting PSPs would end right here if it were not for the fact that the members of the PSP group oxidize at different rates. The presence of the N-hydroxyl group severely inhibits the oxidation rate and this unequal response means that it is impossible to quantitate any PSP mixture unless standards are available to calibrate the analytical system. Furthermore, since some PSPs are more toxic than others, accurate quantification becomes even more essential if the method is to have some practicality. This underscores the major problem of the HPLC-fluorescence approach since no PSP, other than saxitoxin, is available commercially. Once again, the reason for this state of affairs is attributable to the difficult chemistry of these compounds, the low concentration of toxins in dinoflagellates having the appropriate spectrum of toxins, and the difficulty in obtaining sufficient algal biomass for extraction of the toxins.

It was at this point that NRC entered the shellfish toxin arena. It was obvious that there was an urgent need for purified PSPS, not just for preparing standards, but also for investigating new chemical assay approaches and, through biotechnology, for developing other rapid and inexpensive tests. At ARL, the marine bioactives group headed by Jeffrey Wright, MCIC, had the expertise to isolate and characterize marine compounds and the Marine Analytical Chemistry Standards Programme (MACSP), led by David Jamieson, FCIC, was in the business of developing and supplying standards and reference materials to the scientific community. The idea to begin work on PSPs received strong encouragement from NRC's Committee on Marine Analytical Chemistry (CMAC). Furthermore, Peter Wangersky, MCIC, of the Oceanography Department at Dalhousie University had developed a turbidostat for continuous large-scale culture of marine microalgae. This seemed to be the ideal system to produce the dinoflagellates that would be required for the project. A two-year collaborative project was established with funding from the Federal Departments of Supply and Services (DSS), Fisheries and Oceans (DFO), National Health and Welfare (NHW), and the NRC. Wangersky would deliver a monthly supply of toxic dinoflagellate cells, and NRC chemists including Maurice Laycock, Ved Pathak and Wright were to extract, purify, and characterize the individual toxins.

Not surprisingly, there were difficulties. At first the yield of dinoflagellate cells was lower than expected, and the chemists quickly discovered the mysteries of PSP chemistry. Until this collaboration, only small University groups had studied PSPs, and then only at the [mu]g scale and usually in the search for new PSPs. The NRC scientists were trying to isolate, purify and characterize mg amounts of the toxins and the scale-up problems were significant. A vital factor in the NRC approach was the HPLC-fluorescence system which was used to monitor continuously the separation process. This was a significant improvement over previous approaches which generally used the mouse bioassay to track the toxins. Although the bioassay is a sensitive warning of toxicity, it provides no information about the composition or distribution of toxins in a mixture.

The HPLC-fluorescence system at ARL was set up initially by Roger Guevremont, MCIC, and Michael Quigley, MCIC, who assembled a system that gave good separation and sensitivity to the saxitoxin standard. Although useful qualitative data were obtained, it was impossible to generate any reliable quantitative data without the necessary standards. Although there are fudge factors' in the literature, many of these values are suspect and can be used only as a first approximation. In late 1989, Stephen Ayer, MCIC, took over responsibility for the HPLC system and around this time ARL was invited to participate in an inter-laboratory comparison of the HPLC-fluorescence method organized by James Hungerford of the US FDA. This was a very useful exercise as it gave Ayer an opportunity to refine the HPLC system. The method is also being used by Greig Sim of the MACSP group to investigate the feasibility of producing a scallop-tissue reference material.

By the end of the two-year period of collaboration, the major biological problem of sufficient algal biomass had been solved. Also, Laycock had defined the chromatography methods, the recovery at each step had been improved considerably, and satisfactory conditions for storing and preserving the toxins had been established. Through another contract with Dalhousie, other strains of dinoflagellates with a different spectrum of PSPs are being cultivated. Soon, mg amounts of four different PSPs, namely, saxitoxin, neosaxitoxin, GTX-2 and C-2 will be on hand.

The availability of these PSPs is a significant step forward since they represent essentially all three chemical types of PSPs and they can be used to examine new analytical methods for their detection. While this project was underway, the laboratory acquired a Canadian-built Sciex API III IonSpray tandem mass spectrometer. This instrument can detect pg amounts of purified marine toxins when introduced by flow injection, but its real strength is the capability to analyze mixtures by attaching an LC interface to the spectrometer.

Unfortunately, the mobile phase buffer system used in the HPLC-fluorescence method cannot be adopted for LC-MS because the ion pairing reagents interfere with the mass spectrometry. This means an entirely new eluant system must be developed that is completely compatible with the mass spectrometry. Recently, Stephen Pleasance and Michael Quilliam, MCIC, have achieved some success in separating partially purified mixtures of PSPs by reversed-phase chromatography using water-acetonitrile mixtures and volatile buffers. Another very promising approach is to use capillary zone electrophoresis (CZE) coupled to the mass-spectrometer. Since PSPs carry a charge and can be separated by electrophoresis, CZE has the potential to produce extremely narrow bands of toxins, and so may be more practical and sensitive than the LC approach. Recently, CZE equipment and a CZE/MS interface have been acquired and Pleasance, Quilliam and Pierre Thibault have begun preliminary experiments on methods development.

The pure PSPs will also allow us to obtain more spectral data for these toxins. No optical data has been reported for any PSP other than saxitoxin, and although some NMR and MS data for PSPs have been recorded, more detailed information is required. The lack of IR data is somewhat surprising since it can be used to detect the presence or absence of sulphate groups, as well as the amide function and guanidine groups. In collaboration with ARL chemists, NMR is being studied by John Walter, FT-IR by Michael Falk, FCIC, and MS/MS by Pleasance, Quilliam, and Thibault together with Sherwood Hall and Jim Sphon of the US FDA.

Amnesic Shellfish Poisoning

If our entry into PSPs was carefully planned, then by contrast our introduction to ASP or domoic acid was hectic... The combined efforts of the staff at ARL in identifying this new toxin during the 1987 PEI mussel crisis are well documented. Since then, considerably more work has been done on this new shellfish toxin.

Domoic acid was isolated originally in Japan some 30 years ago from the red macroalga Chondria armata. It was chemically synthesized in the seventies and this led to a minor revision of the structure. Until the PEI mussel crisis, there had been little other interest in domoic acid or any of its derivatives. It was known to be a powerful insecticide, considerably more potent than DDT for example, and there were obscure reports of its properties as an anthelmintic. It was not known as a shellfish toxin, and indeed there was little indication of its profound effect on human health.

After the mussel toxin crisis, an initial priority was to develop a routine and robust chemical analytical method for detecting domoic acid. Fortunately, this toxin is much more amenable to detection than the PSPs and a rapid (5 min), sensitive method was developed quickly by Archie McCulloch, FCIC, Gavin McInnes, FCIC, Quilliam and Sim, based on reversed-phase HPLC and UV detection. The minimum detection of this method is 0.5 [mu]g/g of the toxin in shellfish tissue, well below the safe limit of 20 [mu]g/g set by NHW in 1988. It is important to point out that the latter level cannot be detected by the mouse bioassay hence the development of a more sensitive chemical assay was essential. In collaboration with Roger Pocklington, FCIC, from DFO, Joyce Milley and Quilliam have developed a trace analytical HPLC method using the FMOC derivative of domoic acid which is useful for detecting low levels of the toxin in seawater and phytoplankton. The minimum detection limit in this method is 15 pg/mL of seawater. The method is now being adapted to the analysis of shellfish tissue with a clean-up step to remove interfering endogenous compounds present in the tissue. Recently, the same clean-up has been employed in another method which uses CZE coupled with a UV detector.

Domoic acid is particularly well suited to FAB mass spectral analysis and this gave Pleasance, Quilliam and Thibault an opportunity to investigate other more sophisticated analytical methods using chromatography systems coupled with ionspray mass spectrometry. Indeed, reversed-phase LC-MS is a very sensitive method for domoic acid (0.1 [mu]g/g) and related compounds, and a CZE interface is now being developed which promises to be extremely sensitive.

Using domoic acid isolated from mussel tissue, the MACSP group have prepared an instrument calibration solution DACS-1(89 [mu]/mL in 90% aqueous acetonitrile). A shelf-stable mussel tissue reference material MUS-1(126 [mu]g/g), which is an homogenate of mussel tissue naturally contaminated with domoic acid, is also available. Both of these materials can be purchased from the MACSP at ARL.

Further analysis of mussel extracts or phytoplankton extracts has uncovered traces of other compounds with the same UV properties as domoic acid. These have been characterized by Falk, McInnes, Walter and Wright as the C6' diastereoisomer as well as the geometrical isomers of domoic acid, which are more conveniently obtained by photolysis of domoic acid. Other derivatives, including the dihydro- and tetrahydro- compounds have also been prepared and characterized, and are being used in a structure-function study.

Domoic acid is a glutamate agonist that displays marked neurotoxic properties in the mammalian central nervous system. It disrupts normal neurochemical transmission in the brain by binding to certain glutamate receptors, the so-called kainate receptors, of neuronal cells. This results in increased firing of the neurons and eventual rupture of the cell. A common feature of all such glutamate agonists which bind to these receptors is that a portion of their structure represents a conformationally restricted form of glutamic acid, and so differences in efficacy of binding to the receptor, and hence potency, can be attributed to differences in the chemistry of the side-chain.

In collaboration with Wright, two University of Toronto researchers, David Hampson and John McDonald, have undertaken a structure-function study using the naturally occurring isomers isolated from mussel tissue, as well as other compounds synthesized from domoic acid itself. Two aspects are being investigated. In the first, the binding efficiency of domoic acid derivatives to rat forebrain membrane is being measured, while in the second the electrophysiological response of neuron cells to challenges of these compounds is being measured. The timing of this study is very opportune since three independent groups have recently isolated the kainate receptor protein as well as the gene, thus the receptor itself will soon be available for study.

During the months following the mussel crisis, a group of ARL scientists including Steven Bates, Carolyn Bird, and Anthony de Freitas, MCIC, together with DFO colleagues, established the source of the toxin as the diatom Nitzschia pungens forma multiseries which formed a massive bloom off eastern PEI at the time the contaminated mussels were harvested. This is the first time a diatom has been implicated in shellfish toxicity. The global implications are significant since N. pungens is ubiquitous in coastal waters. Fortunately however, not all forms of N. pungens appear to produce domoic acid. For example, Bird and de Freitas have shown the nominate form (forma pungens) does not produce the toxin, nor does another common species, Nitzschia serriata.

A curious feature of the PEI bloom was that it persisted for two months or more instead of the two or three weeks that is normal for a phytoplankter. Grazing by herbivores (e.g. copepods or other crustaceans) is usually what determines the life span of a bloom, so it is possible that N. pungens or domoic acid has some antifeedant property that discourages herbivores. This idea is supported by the known insecticidal properties of domoic acid, and Anthony Windust and Wright have already demonstrated acute toxicity of domoic acid to local species of copepods. Other feeding experiments with the whole alga are currently underway.

Diarrhetic Shellfish Poisons

Following the domoic acid crisis, it seemed important that methods should be developed to detect and analyze other known shellfish toxins that might be of relevance to Canadian public health and the Canadian shellfish industry. Immediate candidates for such a study were the DSPs, a recently identified group of lipidsoluble shellfish toxins that are notably different in chemistry from the PSPs and ASP.

The parent compound of the group is okadaic acid, a complex cyclic polyether isolated originally from the sponge Halichondria okadaii. Other related molecules have been isolated from sponge sources, including DTX-1 and the episulphide derivative ancanthifolicin. Yasumoto's group has isolated these metabolites from contaminated shellfish and established that they are the causative agents in shellfish poisoning outbreaks which have plagued Japan for years. Such incidents of DSP outbreaks have also occurred in Europe, Scandinavia and South America. The DSPS, as their name might suggest, cause diarrhea and vomiting following ingestion but no deaths have been attributed to them.

Clearly the ability to recognize and detect low levels of DSPs is of paramount importance. Under a two-year, $1-million collaborative research agreement, NRC and Fenwick Laboratories of Halifax will develop ways to detect and identify DSPs and related compounds. As for many toxins, the presence of DSPs is usually established using a mouse or rat bioassay. However, the test in this case is complicated and subjective and quite unsuitable for routine screening. For many reasons, (low toxin concentration in cells or tissue, lack of significant chromophore, structural complexity and labile nature) the analysis of DSPs by instrumental methods is difficult. A published HPLC method uses fluorescence detection after derivatisation with 9-anthryldiazomethane (ADAM) but there are drawbacks to this method due to the highly reactive nature of the reagent. Nevertheless, the method has been successfully implemented at ARL by Fenwick Laboratories scientist Julie Marr and Quilliam, and is used routinely to monitor okadaic acid in phytoplankton cultures. In more complex matrices such as shellfish extracts, a special clean-up step before derivatisation will be required to remove interferences. The analytical research effort of Marr, Pleasance and Quilliam is focused on three aspects: improvement of the ADAM method and investigation of other derivatisation methods, development of a clean-up method suitable for trace levels of DSP, and development of a confirmatory method using LC-MS.

Once again, it was necessary to obtain sufficient amounts of the toxin for further study and methods development. In this case two sources were used, Halichondria melanodociae or 'Black Sponge', and a benthic dinoflagellate Prorocentrum concavum which is being cultivated at ARL by de Freitas.

The published chemical isolation scheme has been streamlined considerably by Fenwick scientist Tingmo Hu and Wright so that high quality okadaic acid is now readily obtained from either sponge or phytoplankton. Interestingly, during the isolation process, LC-MS analyses of okadaic acid fractions have uncovered traces of unknown but related compounds. Structural characterization of these compounds is underway.

Although they have been isolated from sponge tissue, it is unlikely that DSPs are true sponge metabolites. Instead dinoflagellates are the actual source of DSPs in nature, but these phytoplankton are often found in the canals of filter-feeding sponges. Several dinoflagellates belonging to the genus types Prorocentrum and Dinophysis have been implicated in DSP poisoning incidents and these phytoplankton have been found in waters off the eastern Canadian and north eastern US coastline. Clearly the work on DSP is important in the Canadian context

Future Directions

The long-term objective of the NRC research on shellfish toxins is to support the regulatory agencies and the shellfish industry by developing rapid, reliable, and economical tests for toxicity. Much of the work described here is the groundwork for achieving this objective. The specific goal of an economical 'stick-test' has not yet been reached, nor will it be without the purified toxins and the chemical analytical methods to verify such tests. It is the aim of NRC scientists, working in collaboration with industry, to reach this goal as quickly as possible.
COPYRIGHT 1990 Chemical Institute of Canada
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Title Annotation:Canada's National Research Council
Author:Wright, Jeffrey L.C.
Publication:Canadian Chemical News
Date:May 1, 1990
Previous Article:Marine biotechnology: a new focus at the National Research Council's Atlantic Research Laboratory in Halifax.
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