Jeffrey L.C. Wright

AN OUTBREAK OF DOMOIC ACID POISONING ATTRIBUTED TO THE PENNATE DIATOM PSEUDONITZSCHIA AUSTRALIS1

A bloom of the pennate diatom Pseudonitzschia australis Frenguelli (= Nitzschia pseudoseriata Hasle) occurring in Monterey Bay, California, in early September 1991 coincided with an episode of mortality in brown pelicans (Pelicanus occidentalis) and Brandts cormorants (Phalacrocorax penicillatus). High levels of domoic acid (DA), the amnesic shellfish poisoning toxin, were recorded in the plankton samples. Furthermore, high levels of DA, as well as numerous remnants of P. australis frustules, were found in the stomach contents of affected birds and in the visceral contents of local anchovies, a principal food source of seabirds. This is the first confirmed report of DA poisoning since the original 1987 episode in Atlantic Canada caused by Nitzschia pungens Grunow forma multiseries Hasle. It suggests another species of planktonic pennate diatom is capable of producing DA and that herbivorous finfish can act as vectors for this toxin.

CHARACTERIZATION OF BIOLOGICALLY INACTIVE SPIROLIDES E AND F : IDENTIFICATION OF THE SPIROLIDE PHARMACOPHORE

Abstract Two new spirolide derivatives, E and F, have been isolated in low yield from shellfish extracts. Absence of activity in the mouse bioassay of these derivatives, and of the secondary amine reduction product of spirolide B, identifies the spirolide pharmacophore as the cyclic imine moiety.

Detection of new 7-O-acyl derivatives of diarrhetic shellfish poisoning toxins by liquid chromatography-mass spectrometry☆

A novel method for the detection of acylated diarrhetic shellfish poisoning toxins is reported. Direct determination of these compounds is possible using high performance liquid chromatography coupled with ion-spray mass spectrometry. An extract, purified from the digestive glands of toxic mussels (Mytilus edulis) contaminated with okadaic acid, dinophysistoxin-1, and a recently reported analog, dinophysistoxin-2, was also shown to contain small amounts of dinophysistoxin-3, a mixture of 7-O-acyl ester derivatives of dinophysistoxin-1. In addition, acyl ester derivatives of okadaic acid and dinophysistoxin-2 were also detected by direct LC-MS analysis and confirmed by analysis of their hydrolysis products. This is the first report of the detection of other naturally occurring 7-O-acyl esters similar to dinophysistoxin-3.

Interaction of domoic acid and several derivatives with kainic acid and AMPA binding sites in rat brain.

We have determined the inhibitory potencies of domoic acid and a series of derivatives of domoic acid at kainic acid and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding sites in rat forebrain membranes. These derivatives of domoic acid differed in the configuration, stereochemistry, and degree of saturation of the side chain attached to C-4 of the prolyl ring. The binding data were analyzed in terms of one or two classes of sites as appropriate. Domoic acid and kainic acid displayed similar inhibition constants at [3H]kainic acid sites (IC50 = 5 and 7 nM, respectively). At both kainic acid and AMPA binding sites, all of the compounds tested were less potent than domoic acid itself. At high affinity [3H]kainic acid sites, the derivatives could be categorized into two groups; those with nanomolar affinity and those with micromolar affinity. All members of the former group possessed a side chain with the first double bond intact and in the Z (cis) configuration. The more distal atoms present in the extended side chain of domoic acid did not appear to contribute to the high affinity interaction with the kainic acid receptor. Although all the compounds tested were weaker inhibitors of [3H]AMPA binding compared to [3H]kainic acid binding, there was a high correlation between the rank order of potency of the seven domoic acid derivatives at [3H]kainic acid and at [3H]AMPA binding sites. The inhibition data for kainic acid at [3H]AMPA binding sites were described adequately in terms of a 1-site model, whereas the data for domoic acid required two classes of sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Responses of the sea urchin Strongylocentrotusdroebachiensis (O.F. Müller) to water-borne stimuli from potential predators and potential food algae

Abstract At temperatures of 10–12°C the sea urchin Strongylocentrotus droebachiensis (O.F. Muller),on 80% of the occasions tested, moved away from water that had passed over lobsters or crabs. At lower temperatures an increasing proportion of urchins remained inactive, but of those that were active, 80% still moved away from the scent of predators. At 10–12°C a similar proportion moved towards water that has passed over Laminaria, but at reduced temperatures a smaller proportion of the active urchins responded in this way. The attractant in Laminaria is an unstable, volatile hydrophobic compound with a low molecular weight.

Two new water-soluble dsp toxin derivatives from the dinoflagellate prorocentrum maculosum: possible storage and excretion products

Two novel water-soluble sulfated DSP toxin derivatives 4 and 5, are reported from the benthic dinoflagellate Prorocentrum maculosum. Their occurrence supports the idea that all DSP toxin-producing Prorocentrum species biosynthesize such compounds as a means of toxin storage, and perhaps as a means of eventually exporting them from the cell.

The effects of the diarrhetic shellfish poisoning toxins, okadaic acid and dinophysistoxin-1, on the growth of microalgae

The diarrhetic shellfish poisoning toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1) are potent phosphatase inhibitors produced by certain species of marine dinoflagellates. OA can cause hyperphosphorylation of a broad range of animal and higherpalnt proteins, but little is known regarding the effects of the DSP toxins on marine organisms or their biological function. A variety of microalgae, including a clone ofProrocentrum lima known to produce both OA and DTX-1, were incubated with solutions of OA and in one case DTX-1 or a combination of OA and DTX-1. OA inhibited the growth of all non-DSP-producing test species at micromolar concentrations, butP. lima was not affected even at much higher levels. This differential activity of OA suggests that the DSP toxins may play an allelopathic role and raises questions regarding the strategies adopted by DSP-producing dinoflagellates such asP. lima to avoid autotoxicity. The effects of DTX-1 on microalgal growth were found to be equivalentt to those of OA, and the effects of both toxins in combination were simply additive.

Dealing with seafood toxins: present approaches and future options

In most maritime countries, the seafood industry is an important source of revenue and accounts for a significant portion of the countrys food supply. However, certain seafood, in particular shellfish, may become contaminated with naturally occurring toxins. The existence of shellfish toxins has been known for centuries and early attempts to deal with this threat led to the creation of folklore rituals and religious customs among tribal and ethnic groups. Eventually it was found that these toxins are produced by microbial organisms, usually phytoplankton, that occur naturally in the ocean. In Canada, as well as other countries with cold or temperate coastal waters, three groups of toxins are of particular importance and these are the paralytic shellfish poisoning (PSP) toxins, the diarrhetic shellfish poisoning (DSP) toxins and amnesic shellfish poisoning (ASP) toxin. Since it is virtually impossible to prevent the occurrence of these toxins, the approach in modern times has been to monitor seafood for the presence or absence of toxins using a standardized mouse bioassay. However, not all toxins can be detected effectively by this method and there is a trend in several countries to dispense with the mouse bioassay. Furthermore, there are differences between countries as to the preferred method of sampling and analysis and there is not agreement between all countries concerning tolerance levels of the various toxins in seafood. These difficulties, the occurrence of new toxins, and the rapid expansion of the shellfish aquaculture industry have put considerable pressure upon the scientific community to develop new methods of coping with these toxins, including more sensitive chemical or biochemical assays that are rapid and inexpensive. The possibilities and options for coping with these ubiquitous toxins are presented and discussed.

Assignment of the relative stereochemistry of the spirolides, macrocyclic toxins isolated from shellfish and from the cultured dinoflagellate Alexandrium ostenfeldii

Abstract The relative stereochemistry of 13-desmethyl spirolide C, except for one chiral center, has been determined from NMR data by means of ConGen, a molecular modeling method which applies high-temperature molecular dynamics under distance constraints generated from NOESY and ROESY data. The method shows this spirolide to have the same relative stereochemistry as pinnatoxins A and D in the region of their common structure. Applicability of the ConGen method to molecules of this type is further justified by demonstrating that it yields the correct relative stereochemistry of the pinnatoxins when used with constraints generated from published data. The relative stereochemistries of spirolides B and D are also determined by comparisons of their NMR data with 13-desmethyl spirolide C and further application of ConGen.

Comparative toxicity of the diarrhetic shellfish poisons, okadaic acid, okadaic acid diol-ester and dinophysistoxin-4, to the diatom Thalassiosira weissflogii.

Quantitative structure-activity relationships were determined for the diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA), OA diol-ester and dinophysistoxin-4 (DTX-4), using a sensitive bioassay procedure with the diatom Thalassiosira weissflogii. OA diol-ester was found to be nearly as toxic as OA. This result contradicted the accepted idea that only the free acid toxins, such as DTX-1 and OA, are potent phosphatase inhibitors. Postassay analyses using liquid chromatography-mass spectrometry (LC-MS) of cultures incubated with OA diol-ester showed that the ester had partially decomposed to OA, which explained some but not all of the observed toxicity. The formation of OA during the bioassay raised the possibility that cells exposed to inactive DSP toxin esters could metabolically activate them. This was examined in an additional experiment which showed that the hydrolysis of both DTX-4 and OA diol-ester was spontaneous and apparently not mediated by the presence of T. weissflogii cells. However, cells of T. weissflogii challenged with OA diol-ester rapidly metabolized most of the toxin to a more water-soluble product. From interpretation of mass spectral data obtained using ion-spray LC-MS, the metabolite was identified as an oxygenated diol-ester of OA, implying that it was the product of a monooxygenase-detoxification pathway. It is postulated that OA diol-ester, as a lipid-soluble, uncharged molecule with a propensity to hydrolyse to OA, may facilitate the transfer of OA across cell walls and membranes.