Partial purification of Lophozozymus pictor toxin

An aqueous extract of a coral reef crab Lophozozymus pictor was subjected to a 6-stage purification and chemical testing procedure. The semi-purified toxin had a molecular weight between 1000 and 5000 and gave reactions that suggested it contained free amino and phenolic groups. The ₅₀ for mice was 377 μg/kg. The dose-death time relationships for the crab toxin, saxitoxin and tetrodotoxin were determined. The relationship for the crab toxin differed markedly from those for the other two toxins, the crab toxin being slower in its action.     

Resistance of nerves from certain toxic crabs to paralytic shellfish poison and tetrodotoxin

The inhibitory effect of paralytic shellfish poison and tetrodotoxin on nerves from toxic and nontoxic crabs was examined. The toxins at concentrations of 10(-3) - 10(-4) M partially or completely inhibited the action potential of nerves isolated from the legs of toxic crab species (Zosimus aeneus, Atergatis floridus and Platypodia granulosa), but had no effect at 10(-6) M, the concentration at which the action potential of nerves from a nontoxic crab (Plagusia dentipes) was inhibited completely. A xanthid crab Daira perlata was intermediate in respect to the resistance to toxins. These results agree with the previous results obtained by i.p. administration of both toxins into those crabs.     

Differences between the effects of saxitoxin (paralytic shellfish poison) and tetrodotoxin on the frog neuromuscular junction

1. End-plate potentials (e.p.p.) have been recorded from the neuromuscular junctions of frog sartorius and extensor longus dig. IV muscles, using intracellular micropipettes. Either curare or MgCl₂ were present in the Ringer solution, to keep the e.p.p. amplitude below the threshold for a muscle action potential and contraction.     

Serological cross-reactions between crab saxitoxin-induced protein and paralytic shellfish poison-contaminated shellfish

A polyclonal antiserum generated against crab saxitoxin-induced protein was tested against paralytic shellfish poison (PSP)-contaminated crabs and shellfish. Antibody-reactive proteins in PSP-contaminated bivalve mollusc extracts were localized using SDS-PAGE and immunoblotting. PSP-contaminated clams and oysters possessed a higher degree of immunoreactivity to the saxitoxin-induced protein found in PSP-resistant crabs than their respective non-contaminated controls.     

Accumulation of paralytic shellfish poisoning toxins in the edible shore crab Telmessus acutidens.

Several shellfishes including the crab Telmessus acutidens and its prey bivalve Mytilus galloprovincialis were collected at Onahama in Japan to investigate the accumulation of paralytic shellfish poisoning (PSP) toxins during the blooming season of toxic dinoflagellates. The toxicity of the viscera of T. acutidens collected in 1999 was 30.0 and 80.0 MU/g, and that of M. galloprovincialis was 9.6 MU/g by mouse bioassay. PSP toxins in the crab viscera were identified by HPLC-FLD and ESI-MS, so this is the first observation of PSP toxins in T. acutidens. Carbamate toxins (GTX1-4, and STX) were the major component in the crab as well as in the mussels, and accounted for over 60% on a molar basis. However, the ratio of the N1-OH toxin to N1-H toxin of the crabs were largely different from that of the mussels, and a reductive conversion of the toxins in T. acutidens is concluded as the probable cause. In 2000, PSP toxins were also detected in both crabs and mussels, though the contents were very low compared with the levels observed in 1999. The difference in the toxin abundance suggests that the toxin content in the crab was affected by those of the prey.     

Occurrence of paralytic shellfish poison (PSP) in the starfish Asterina pectinifera collected from the Kure Bay, Hiroshima Prefecture, Japan.

Assays were made for paralytic toxicity of marine invertebrates inhabiting at the coasts of Hiroshima Bay, where the infestation of bivalves such as cultured oysters with paralytic shellfish poison (PSP) has been occurred. The starfish Asterina pectinifera collected at the estuary of Nikoh River, Hiroshima Bay, was found to contain moderate levels of paralytic toxicity. Its highest toxicities as PSP found on July 30, 1999 were 12.5 MU/g for whole body, 11.0 MU/g for integument tissues and 3.9 MU/g for viscera, respectively. The toxicity of integument was changed from 3.6 to 11.0 MU/g in 1 year. Its paralytic toxin principles were identified as PSP toxins, composing mainly from saxitoxin (STX) group toxins such as carbamoyl-N-hydroxy neosaxitoxin (hyneoSTX), and STX, by HPLC and LC-MS, accounting for over 90 mol%. The PSP toxins contained in the starfish A. pectinifera considered to be transferred from bivalves or detritus living in the same area, which were contaminated with PSP. However, the involved pathway may be different from that of Asterias amurensis which was infested directly through food chain from its food bivalves, for its toxin pattern.     

Cross-reacting antigens in the butter clam (Saxidoma giganteus) and their relationship to total paralytic shellfish poison toxicity.

A specific protein with an apparent mol. wt of 23,000 was identified in foot homogenate derived from paralytic shellfish poisoning (PSP) contaminated butter clams and was found to cross-react with crab-saxitoxin-induced protein (SIP) antiserum. Antiserum, once cross-absorbed against non-toxic shellfish material, was incubated with tissue homogenate derived from 52 butter clams with varying total PSP toxicities in a prototype ELISA. A significant (r = 0.83; P less than 0.001) correlation existed between soluble clam antigen content in foot homogenate and total PSP toxicity; the latter measured by the mouse lethality bioassay. From the ELISA results, a soluble antigen threshold of 0.1% total protein was successfully used to distinguish between PSP toxic and non-toxic butter clams. It is proposed that this type of screening assay could be used in conjunction with the standard mouse bioassay to increase PSP monitoring and potentially reduce unnecessary animal testing.     

A rapid and sensitive assay for paralytic shellfish poison (PSP) toxins using mouse brain synaptoneurosomes.

A membrane potential assay using mouse brain synaptoneurosomes was evaluated for the determination of paralytic shellfish poison (PSP) toxin content of mussels and other bivalve species important to the shellfish industry. The assay relies on the ability of PSP toxins to block veratridine-induced depolarization of synaptoneurosomes. Changes in the membrane potential of synaptoneurosomes were monitored using the voltage-sensitive fluorescent probe rhodamine 6G. Standard saxitoxin was found to be a potent inhibitor of the membrane depolarizing effects of the sodium channel activator veratridine (I(50) ca. 4 nM). Likewise, shellfish extracts containing PSP toxins inhibited veratridine-induced depolarization. Neither saxitoxin or shellfish extracts had any discernible effect on the resting membrane potential of synaptoneurosomes. When synaptoneurosomal results for extracts of mussels (n=120) and other shellfish (n=29) were correlated with official mouse toxicity assay data there was very good agreement (r(2)=0.84 and 0.86, respectively), indicating that the in vitro assay has utility for a variety of commercially relevant shellfish species. Our investigation suggests that the mouse synaptoneurosome assay is of similar sensitivity to the official CD1 mouse toxicity assay. The synaptoneurosome fraction can be prepared quickly (approx. 40 min) and an individual assay takes less than 7 min. Since 20 such assays can be performed using material from a single CD1 mouse brain, there is considerable opportunity for reducing the number of animals required in conventional PSP monitoring while retaining the same animal system.     

Dinoflagellate Gymnodinium catenatum as the source of paralytic shellfish toxins in Tasmanian shellfish

Paralytic shellfish toxins in both cultured cells and natural phytoplankton blooms of the dinoflagellate Gymnodinium catenatum from inshore Tasmanian waters (Australia) were analyzed by high performance liquid chromatography, thin layer chromatography and electrophoresis techniques. The dinoflagellate toxins were dominated by low potency sulfocarbamoyl saxitoxin derivatives (98-99 mole% in total), including gonyautoxin VIII (C2) and its epimer (C1) and sulfocarbamoyl gonyautoxins I and IV (C3 and C4). Mussels and oysters contaminated by the dinoflagellate showed similar toxins, but contained larger proportions of C3 (40-57 mole%) and more potent carbamate toxins (7-23 mole% total).     

Transport of the organic cations gonyautoxin 2/3 epimers, a paralytic shellfish poison toxin, through the human and rat intestinal epitheliums.

The aim of this work is to study the mechanisms involved in gonyautoxins (GTXs) intestinal absorption. For this purpose, we studied the transport of GTX 2/3 epimers by intestinal epithelial cell lines (IEC-6 and Caco-2) cultured on polycarbonate filters. Specific transport was calculated by subtracting from the flux of GTX 2/3 measured at 37 degrees C that occurring at 4 degrees C, this being an indication of transcellular transport. The transcellular apical-to-basolateral (A-B) flux in Caco-2 cell monolayers, was greater than that in the opposite direction, suggesting the involvement of an active transport system favoring the absorption of the toxin. However, in IEC-6 cells the transcellular basolateral-to-apical (B-A) specific transport of the toxin was greater than that in the opposite direction. The A-B and B-A fluxes were, respectively, 127 +/- 26 and 205 +/- 23 nmol/min, suggesting the presence of a prevalent secretive process of the toxin in IEC-6 cells. The A-B transport of GTX 2/3 epimers in Caco-2 cells, but not in IEC-6 cells, was partially Na(+)-dependent and significantly inhibited by adenosine. TEA and verapamil in both Caco-2 and IEC-6 cells failed to affect the A-B and B-A transport of GTX 2/3 epimers. Cyanine in IEC-6 cells, but not in Caco-2 cells, increased the A-B flux of the toxin, suggesting the involvement of the organic cation transporter in the absorption of GTX 2/3 epimers. The mitochondrial energetic uncoupler 2,4-dinitrophenol significantly inhibited the A-B and the B-A transport in both cell lines. In conclusion, IEC-6 cells secrete actively the toxins, whereas Caco-2 cells were found to absorb the toxins in a process that was inhibited in the presence of adenosine and the absorption was dependent of Na(+).     

Distribution of paralytic toxins in California shellfish

Samples of Saxidomus nuttali and Mytilus californianus collected during the 1981 dinoflagellate bloom at Bodega Bay, California, were analyzed for the presence of paralytic toxins. Neck tissue of S. nuttali contained saxitoxin (STX) and neoSTX (95% of the total toxicity), whereas the bodies contained neoSTX and a mixture of the gonyautoxins. In a sample of M. californianus the presence of neoSTX and the gonyautoxins was demonstrated, whereas a second sample, collected at a different site, contained almost exclusively neoSTX.     

Toxic effects, pharmacokinetics and clearance of saxitoxin, a component of paralytic shellfish poison (PSP), in cats.

Saxitoxin (STX) was the first known and most studied toxic component of paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na+ channel. Although the toxin's mechanism of action is well known at the molecular level, there are still many unresolved questions about its pharmacokinetics and the PSP intoxication syndrome in mammals. Some of these questions are addressed in the present paper, which describes an experimental design which allowed us to follow the dynamics of STX poisoning in vivo. Adult cats were anaesthetized and permanently coupled to artificial ventilation, they were then intravenously injected with Low (2.7 microg of STX/kg) and high doses (10 microg of STX/kg) of toxin. Cardiovascular parameters such as blood pressure and electrocardiograms were recorded, urine and blood samples were collected during the four hours of experimental time. In order to quantify mass amount of STX, we used the post-column derivatization HPLC method. Urine and blood samples were cleansed using a C-18 Sep-Pack cartridge and ultrafree microcentrifuge filters. At the end of each experiment, the animals were killed and tissue samples from brain, liver, spleen and medulla oblongata were extracted to measure the amount of STX. As compared to control period, Low doses of STX made no difference in hemodynamics parameters. In contrast, high doses drastically reduced blood pressure, produced myocardial failure and finally cardiac arrest. Administration of 2.5 microg/kg x min of dobutamine restored hemodynamics parameters and allowed the animal to overcome the shock. With high doses, the calculated STX renal clearance in cats is 0.81 ml/min x kg(-1). This valued corresponds to 20.25% of the reported inulin renal clearance. Nevertheless with Low doses the STX renal clearance is 3.99 ml/min x kg(-1). This data suggest that in cats with normal cardiovascular parameters and diuresis, the STX excretion mainly involves glomerular filtration. During experimental time, no PSP toxins other than STX was detected in the body fluids and tissue samples analyzed, indicating that the mammals can not metabolize this molecule. STX was found in intensely irrigated organs such as the liver and spleen but also in the central nervous system (brain and medulla oblongata), showing that STX was capable of crossing the blood brain barrier.     

Occurrence of paralytic shellfish toxins in Cambodian Mekong pufferfish Tetraodon turgidus: selective toxin accumulation in the skin.

The toxicity of two species of wild Cambodian freshwater pufferfish of the genus Tetraodon, T. turgidus and Tetraodon sp., was investigated. Tetraodon sp. was non-toxic. The toxicity of T. turgidus was localized mainly in the skin and ovary. Paralytic shellfish toxins (PSTs), comprising saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX), account for approximately 85% of the total toxicity. Artificially reared specimens of the same species were non-toxic. When PST (dcSTX, 50 MU/individual) was administered intramuscularly into cultured specimens, toxins were transferred via the blood from the muscle into other body tissues, especially the skin. The majority (92.8%) of the toxin remaining in the body accumulated in the skin within 48h. When the same dosage of tetrodotoxin (TTX) was similarly administered, all specimens died within 3-4h, suggesting that this species is not resistant to TTX. Toxin analysis in the dead specimens revealed that more than half of the administered TTX remained in the muscle and a small amount was transferred into the skin. The presence of both toxic and non-toxic wild specimens in the same species indicates that PSTs of T. turgidus are derived from an exogenous origin, and are selectively transferred via the blood into the skin, where the toxins accumulate.     

The occurrence of paralytic shellfish toxins in two species of xanthid crab from Suva barrier reef, Fiji Islands

Five species of crabs commonly occurring on Suva barrier reef, Fiji Islands, were tested for the presence of paralytic shellfish toxins. All 35 specimens of Atergatis floridus and all 18 specimens of Zosimus aeneus tested were lethal to mice, whilst none of 12 specimens of Carpilius maculatus, 8 of C. convexus and 10 of Eriphia sebana tested were lethal. A. floridus contained saxitoxin (55--60%), neosaxitoxin (35--40%), gonyautoxin-II (less than 5%) and a new toxin previously found in a toxic dinoflagellate, Pyrodinium bahamense var. compressa, and tentatively coded PBT (1%). Z. aeneus contained the same components, with additional trace amounts of gonyautoxin-I and III, but neosaxitoxin was the major component in this species. Comparison with the results of testing Okinawan specimens of Z. aeneus, A. floridus and Platipodia granulosa suggests that the toxin profile is specific to species.     

Transfer and metabolism of paralytic shellfish poisoning from scallop (Chlamys nobilis) to spiny lobster (Panulirus stimpsoni)

The transfer and transformation of paralytic shellfish poisoning (PSP) from scallop Chlamys nobilis to spiny lobster Panulirus stimpsoni were investigated in the present study. The results demonstrate that transfer and transformation of PSP toxins occurred when Panulirus stimpsoni were fed with toxic viscera of Chlamys nobilis, but depurated with non-toxic squids. Additionally, only the lobster hepatopancreas were found to contain PSP, and the toxin profiles were the same with those in the viscera of the scallop, including carbamate toxins (GTX₁₋₃), N-sulfocarbamoyl toxins (C₁₊₂ and B₁) and decarbamoyl toxins (dcGTX₂₊₃). Unlike the lobster, the scallop contained more than β toxins. After being fed with toxic Chlamys nobili for 6 d, Panulirus stimpsoni selectively accumulated N-sulfocarbamoyl toxins with low toxicity. However, when they were depurated with non-toxic squid, N-sulfocarbamoyl toxins tended to transform into carbamate toxins with higher toxicity. The concentration of dcGTX₂₊₃ in Panulirus stimpsoni decreased significantly and wasn’t detectable after depuration for 6 d, which was likely due to their initial low accumulation of toxins. These results reveal that PSP could be transferred and transformed in Crustaceans along the given food chain under the conditions of laboratory, but there are many questions remained to be solved, and the further studies should be carried out.     

Study of paralytic shellfish poisoning toxin profile in shellfish from the Mediterranean shore of Morocco

Since 1992, a monitoring program for bivalve molluscs contaminated by algal toxins was established at different stations along the Mediterranean Moroccan shores. The monitored stations were tested every 2 weeks. The presence of toxicity was determined using the mouse bioassay method. Toxin profile was carried out by HPLC/FD in selected contaminated tissues. According to the outcomes of this surveillance from 1994 to 1999, reliable information on toxicity of shellfish was obtained. They indicate that PSP is a recurrent toxicity in molluscs along the Mediterranean shore of Morocco. It has been noted a difference of PSP accumulation among individual shellfish. The cockle (Achanthocardia tuberculatum) presents toxicity throughout the year, while other specimens from the same area such as clam (Callista chione), warty venus (Venus gallina) and marine beans (Donax trunculus) accumulate it seasonally from January to April, after which they depurate the toxin. Moreover, the study of toxin profiles among individual shellfish was undertaken. It was found that shellfish presented a complex profile pointing to contamination by Gymnodinium catenatum.     

Determination of paralytic shellfish toxins in Portuguese shellfish by automated pre-column oxidation.

Automated pre-column oxidation (the method of Lawrence) was implemented on a routine basis since the end of 1996 to study paralytic shellfish poisoning (PSP) toxins in Portuguese shellfish. Liquid chromatography confirmed the presence of PSP toxins when the known toxic algae were present: Gymnodinium catenatum and/or Alexandrium cf. lusitanicum. On the other side, it has eliminated PSP toxins as a possible recurrent contaminant in oysters from Sado estuary. These oysters were already known to contain high levels of some metals (mainly zinc, copper and cadmium) due to their location in a contaminated area and their particular physiology prone to accumulate metals. The presence of PSP toxins in Scrobicularia plana from Mondego estuary and Tellina crassa from the northern coast, during the absence of the above toxic microalgae in the water column, was confirmed. Unlike other shellfish, these two genera have the feeding habit of aspirating more sediment than organisms in suspension, and probably ingest from the sediment resting cysts of PSP producing microalgae. This is another route of contamination that may help to explain why after a bloom certain shellfish species maintain toxicity for long periods. The method revealed to have a fast implementation on a daily basis, short analysis time (around 20 min between samples), high sensitivity and robustness, and therefore, it is one of the best HPLC methods for screening a large number of shellfish samples for monitoring purposes.