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Yessotoxin (YTX), originally found in association with diarrhetic shellfish poisoning (DSP), caused neither intestinal fluid accumulation nor inhibition of protein phosphatase 2A. Orally, YTX was not lethal to mice at 1.0 mg/kg. The toxin showed weak cytotoxic and antifungal activities. Neither hemolytic nor ichthyotoxic effect was observed.
Two human cell lines have been used, HEp-2 and (de)differentiated Caco-2, derived from a larynx and a colon carcinoma, respectively, with the aim of evaluating and characterizing the cytotoxicity of okadaic acid (OA) and related toxins. Effects of OA and dinophysistoxin-1 (DTX-1) on cell viability (neutral red uptake) and on cell morphology/cytoskeleton structure have been observed in both cell lines, though at different time exposures and with different concentrations. The morphological alteration was detected earlier than the viability inhibition in HEp-2 cells with both toxins and in Caco-2 cells with DTX-1. HEp-2 cells have shown to be more sensitive than the intestinal cell line and thus possibly suitable for screening of contaminated samples, while Caco-2 cells could be used for further investigating the possible mechanisms involved in diarrhoeic shellfish poisoning (DSP) toxins.
Accumulation of paralytic shellfish poisoning (PSP) toxins by surfclams, Spisula solidissima, was studied over a period of two years at two inshore locations in southern Maine and at six stations on Georges Bank in the Gulf of Maine. Whole animals as well as individual tissues (siphon, mantle, digestive gland, foot, adductor muscle, gill) were analyzed for PSP-toxicity levels using the standard AOAC mouse bioassay. Analyses of gut contents were carried out on surfclams from both inshore and offshore locations to identify the type of particles ingested. Surfclams feed primarily on phytoplankton and detrital material characteristic of the overlying seawater and surface sediment. No evidence was found for any selection based on particle size or type. Elevated levels of PSP toxins were noted in surfclams from Georges Bank more than two years after initial toxification. Toxins were not evenly distributed among the various tissues of surfclams. Initially, maximum toxicity among surfclam tissues was found in digestive glands; however, subsequent analyses of samples collected later in the year indicated that toxicity in gill and mantle tissues had increased relative to initial values. No toxicity was detected in adductor muscles. Surfclams are characterized by a high variation in total toxin load among individual animals, with a tendency for decreasing variation as toxin levels increase. Archived data from the Main Department of Marine Resources revealed annual and seasonal patterns of toxin accumulation by surfclams, i.e., toxin accumulation is an annual event, with initial increases in toxicity usually occurring in early spring.
A colorimetric phosphatase-inhibition bioassay was developed for the quantitative measurement of okadaic acid (OA) the main diarrhetic toxin responsible for diarrhetic shellfish poisoning. The assay used an artificial substrate, paranitrophenylphosphate, and a semi-purified protein phosphatase PP2Ac containing extract prepared from rabbit muscle. Calibration dose-inhibition curves were constructed using standard OA and they permitted easy determination of the enzyme concentration Et in their linear portion. In the range of linearity, the slope increased when Et decreased, thus giving a detecting limit of 0.04 pmol in the reaction mixture (1 ml). The lowest assayable concentration of OA was 4 ng/ml in aqueous solutions and 40 ng/ml (i.e., 100 ng of OA per g of mussel tissue) in crude methanol mussels extracts. The intra and interassay coefficients of variation in the measurement of OA for the toxin spiked aqueous samples averaged, respectively, 7.7% and 3.7%, and interexperiments coefficients of variation for the toxin spiked mussel extracts averaged 4.6%. The presence of OA was ascertained by a method in which one assay was performed at two or three different levels of enzyme concentration. The rapidity, accuracy, reproducibility, specificity, and simplicity of the procedure provides a simple way to assay okadaic acid in buffered or complex solutions.
Mussels (Mytilus edulis) and clams (Ruditapes philippinarum) were contaminated experimentally using cultures of Alexandrium minutum, a toxic dinoflagellate isolated from French coastal waters. Experiments were carried out in continually flushed and open-circuit systems using Alexandrium densities of 100 to 700 cells/ml delivered to tanks containing the shellfish. All experiments indicated an inversion of the relative proportions of gonyautoxins (GTX2 and GTX3) in shellfish meat during decontamination, whereas saxitoxin (STX) only accumulated during mussel depuration. However, in mussels a density as low as 100 cells/ml led within 10 days to bioaccumulation of paralytic shellfish poisoning (PSP) toxins above the public health threshold. Similar results were observed in clams subjected to fivefold higher cell densities, indicating a less effective assimilation of the dinoflagellate than by mussel. Decontamination experiments on PSP toxin-contaminated mussels (360 micrograms STX eq./100 g or lower uptake) fed two nontoxic diets (1,000 and 10,000 cells/ml of Tetraselmis suesica) showed an appreciable reduction in the time needed to decrease toxin concentration below the accepted threshold for human consumption. We suggest that a simple relation can be established between initial toxicity, the concentration of nontoxic alga available, and the time required for depuration once decontamination kinetics becomes linear and corresponds to the inverse of contamination kinetics.
Brevetoxins are produced by the marine dinoflagellate Ptychodiscus brevis, an organism linked to red tide outbreaks, and the accompanying toxicity to marine animals and to neurotoxic shellfish poisoning in humans. Brevetoxins bind with high affinity to voltage-sensitive sodium channels and cause increased sodium ion conductance and nerve cell depolarization. The brevetoxin competitive binding assay with tritium-labeled brevetoxin 3 (3H-PbTx-3) and rat brain synaptosomes is a sensitive and specific assay for pure brevetoxins. Here we report that extracts of manatee, turtle, fish, and clam tissues contain components that interfere with the assay by cooperative, noncompetitive inhibition of 3H-PbTx-3 specific binding and increased nonspecific binding to synaptosomes. By determining the "apparent" toxin concentration ("[Toxin]") in the extract at several assay concentrations, a reasonable correction for the complex inhibition could be made using a semilog plot to extrapolate [Toxin] to zero extract concentration to obtain [Toxin]0. Spiking 4 extracts with 60 nM PbTx-3 caused [Toxin]0 to increase by 41 +/- 8 nM, indicating that the noncompetitive components did not prevent the assay of toxin but did reduce the accuracy of the result. Fourfold repetition of the assay of 4 samples gave standard deviations of 25 to 60% of the value of [Toxin]0, so the error can be fairly large, especially for samples with little toxin. Purification of an extract with a 1 g sample prep column of C-18 decreased the complex inhibition by about 3-fold but did not eliminate interference in the assay.
Sodium channels obtained from rat brain membrane preparations were coated onto microtiter plates and used to develop a direct solid-phase binding assay. The tritiated sodium channel blocker saxitoxin ([3H]-saxitoxin; STX) was used to detect toxins in paralytic shellfish poisoning (PSP) by measuring the competitive displacement of other toxins. With this assay the amount of STX and tetrodotoxin needed to displace 50% of bound [3H]STX was 1.7 and 1.76 ng/ml for buffer samples, respectively. In the direct solid-phase binding assays, the PSP toxins were effectively bound to the rat brain membranes. The IC50 of this assay for different PSP toxin solutions obtained from mussels contaminated in red tides ranged from 0.03 to 0.30 ng/ml. Therefore, this assay represents a potentially useful method for the detection of toxin-contaminated mussels.
Okadaic acid (OA), produced by marine phytoplankton, is the parent compound of a family of marine toxins responsible for diarrheic shellfish poisoning (DSP). A monoclonal antibody to OA (6/50) (Ab1) has been raised and in turn used for immunization of syngeneic animals. Mice inoculated with the 6/50 idiotype produced both anti-idiotypic antibodies (Ab2) and OA binding antibodies (Ab3). The selected anti-idiotypic antibody 1/59 bound to the immunizing 6/50 idiotype but not to F(ab')2 fragments of pooled normal mouse Ig. It inhibited the binding of OA to solid-phase attached F(ab')2 of 6/50 IgG as well as the binding of 6/50 IgG to a solid-phase bound OA. Like OA, 1/59 anti-idiotypic antibody inhibited protein phosphatase 1 and 2A catalytic subunits in a 32P-phosphorylase a phosphatase radioassay. Thus, 1/59 IgG is a novel internal image anti-idiotypic antibody (Ab2 beta) and can serve as a surrogate of OA in biological assays.
A BSTRACTBacteria have been implicated in the production of paralytic shellfish poison (PSP) toxins, which are normally associated with bloom-forming algal species, specifically toxic dinoflagellate algae. To clarify the role that these bacteria may play in the production of PSP toxins, it is desirable to identify and localize the bacteria associated with the dinoflagellates. 16S rRNA-targeted probes offer the possibility for both, and thus, probes have been made to putatively toxigenic bacteria isolated from the PSP-related dinoflagellate Alexandrium tamarense and tested for their specificity in dot blot and in situ hybridization experiments.
The purification and crystallization of T17, a toxin from Ptychodiscus brevis, is reported. The toxicity of this compound and a second toxin known as T34 are compared by i.v., i.p. and oral administration in mice. Both toxins produce symptoms characteristic of muscarnic stimulants; hypersalivation, rhinorrhea and excessive urination and defecation being the most commonly observed. T17, which is orally toxic, is believed to be the agent responsible for Neurotoxic Shellfish Poisoning.
A toxic strain of Aphanizomenon flos-aquae (NH-1), isolated from a toxic bloom in a pond in Durham, New Hampshire, has been mass cultured in the laboratory. The toxin was extracted by ultrasonic disruption of the cells and purified by; (a) filtration through a 10 kilodalton filter, and (b) chromatography on a strong cation exchange resin column using 0.01 M, then 0.1 M, pH 5, sodium acetate buffer followed by 0.75 M hydrochloric acid. Mouse assays and fluorescence generated by hydrogen peroxide oxidation were used to monitor the fractions. Only a nonfluorescent toxic peak followed immediately by a fluorescent less-toxic peak were detected, both eluting with the hydrochloric acid fractions. The toxins were identical in behavior to neosaxitoxin and saxitoxin, respectively, when compared with elution profiles of the paralytic shellfish poisons from Gonyaulax tamarensis var. excavata and by paper electrophoretic and thin-layer chromatographic comparisons. The toxin profile appears to be different from that of a previously isolated strain of A. flos-aquae from Kezar Lake.
Specimens of a xanthid crab Atergatis floridus were collected from two adjacent areas in Kabira Bay, Ishigaki Island, Okinawa and compared with respect to toxicity and toxin composition. 'Reef specimens', which were collected from the reefs of Kabira Bay, showed an average toxicity score of 380 MU/g as paralytic shellfish poison, and a toxin composition consisting mainly of saxitoxin and a neosaxitoxin-related substance. On the other hand, 'Kojima specimens', which were collected from a small island in the bay, showed an average toxicity of 38 MU/g as tetrodotoxin, and a toxin composition consisting mainly of tetrodotoxin and related substance(s).
In the presence of ouabain, veratridine enhances sodium influx in the mouse neuroblastoma cell line Neuro-2A (ATCC, CCL131), causing cellular swelling and subsequent death. Tetrodotoxin (puffer fish toxin) or saxitoxin (paralytic shellfish poison), both of which block the sodium channel of excitable membranes, antagonize this effect, enabling cell growth to continue. This phenomenon was used as the basis of a new assay for these toxins. It is also possible to estimate the quantity of TTX from the relationship between TTX concentration and percentage of living cells. This new method is simple, inexpensive, and sensitive, and may replace the conventional mouse bioassay.
This paper provides evidence for a protein component which appears to be involved in the seasonal resistance of small shore crabs, Hemigrapsus oregonesis and Hemigrapsus nudus to saxitoxin, a principle neurotoxin involved in paralytic shellfish poisoning (PSP). This unique protein complex was isolated and partially purified by ion exchange chromatography using DEAE-cellulose from visceral tissue extracts of resistant crabs. The complex was absent in control crabs that were sensitive to saxitoxin. In addition, the protein complex was induced in the crab after acute administration of low doses of saxitoxin. Results indicate that the protein complex is acidic in nature and has an apparent mol. wt of 145,000.
Monitoring of eastern blue mussels (Mytilus edulis), contaminated with domoic acid, involved mouse bioassays and quantitative analysis using HPLC. Mice undergo a typical scratching syndrome at sublethal as well as lethal doses of domoic acid. The onset of scratching behaviour and time of death in mice were inversely related to the dosage of domoic acid. An LD50 (i.p.) of 3.6 mg domoic acid/kg mouse was calculated. Toxic mussels held in tanks and flushed with uncontaminated sea water showed a decline in domoic acid concentration in mussel tissue with time. In addition, domoic acid concentrations in mussels from two infected rivers declined to negligible levels in 40-50 days under normal environmental conditions. The bulk of domoic acid and toxicity was located in the hepatopancreas which also contained large amounts of chlorophyll-A, an algae biomass indicator, relative to control mussels. These results support the conclusion that domoic acid was the primary causative factor in the shellfish poisonings from Prince Edward Island mussels in late 1987.
Okadaic acid and dinophysistoxin-1, the principal toxic components in diarrhetic shellfish poisoning, may be detected by high-performance liquid chromatography and fluorometric measurement as 9-anthrylmethyl esters. However, "greasy" samples may occur and the fluorescent reagent 9-anthryldiazomethane may decompose during storage, resulting in impurities that may seriously interfere with quantitative determination. Ultrasonic treatment of the samples during derivatization with 9-anthryldiazomethane was found to improve reproducibility. This may result from increased access to reactive sites on toxins by 9-anthryldiazomethane due to disruption of micelles formed by toxins and other partly hydrophobic compounds. A procedure for cleaning the derivatized samples, using a 0.1 g silica cartridge column and different eluent compositions from that reported by LEE et al. (1987), was found to facilitate chromatogram interpretation. Deoxycholic acid, a commercial available bile acid, was found to be an acceptable internal standard. The 9-anthrylmethyl esters of okadaic acid, dinophysistoxin-1 and deoxycholic acid, were stable at 4 degrees C for at least seven days when stored dry or in methanol.
The shore crab (Hemigrapsus sanguineus) is highly resistant to tetrodotoxin (TTX) although it contains no detectable amount of TTX (less than 5 MU/g, where 1 MU is defined as the amount of TTX killing a 20 g mouse in 30 min). Its body fluid was examined for neutralizing effects against the lethal activity of TTX. When the mixture of the body fluid and TTX was injected i.p. into mice, the lethal activity of TTX was significantly reduced; 1 ml of the body fluid was evaluated to neutralize 3.6-4.0 MU of TTX. Higher neutralizing activity (7.2-12.5 MU/ml of the body fluid) was exhibited by i.v. administration of the body fluid into mice before or after i.p. challenge of TTX. The lethal effect of paralytic shellfish poisons was not counteracted by the body fluid. Analysis by gel filtration on Sepharose 6B revealed that the body fluid contained TTX-binding high mol. wt substances (> 2,000,000) responsible for the neutralizing activity of the body fluid against TTX, which accounts for the high resistibility of the crab to TTX. When the crude toxin extracted from the liver of puffer (Takifugu niphobles) was mixed with the body fluid and chromatographed on Sepharose 6B, almost pure TTX was obtained from the fractions containing the TTX-binding high mol. wt substances, suggesting that the TTX-binding high mol. wt substances could be useful in purification of TTX from biological samples.
Okadaic acid (OA), the main toxin responsible for diarrhoeic shellfish poisoning (DSP) has high cytotoxicity for KB cell cultures (apparent after 3 hr of contact), facilitating rapid detection in contaminated mussels. We developed a method to determine the minimal active concentration (MAC) based on direct microscopic study of toxin-induced changes in cell morphology. A high correlation was found between the MAC of tested extracts and corresponding OA concentrations in mussel hepatopancreas as measured by high performance liquid chromatography. This technique is rapid and reproducible and does not require the use of living animals.