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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.
The dinoflagellate Dinophysis acuta has been associated with various incidents of diarrhetic shellfish poisoning. A sample of Dinophysis acuta collected from New Zealand waters in 2002 was previously found to contain high levels of pectenotoxins, but only a very low level of the diarrhea-inducing okadaic acid (OA). After hydrolysis under basic conditions, however, the concentration of OA increased substantially, indicating the presence of conjugated forms of OA. Using various liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) techniques, a number of OA esters were detected in the original extract. The principal compound was identified as a C8 diol-ester of OA (OA-D8), which had been identified previously in another dinoflagellate, Prorocentrum lima. The retention time, as well as positive and negative ion MS, MS/MS and UV spectra of the D. acuta compound, matched exactly those of OA-D8 isolated from P. lima. In addition to OA-D8, several other novel OA esters were detected in the D. acuta but these have not yet been identified. This is the first report identifying the presence of OA esters in Dinophysis species.
The discovery of new molecules contributes to the development of basic scientific concepts, leads to valuable drug-oriented compounds, and suggests possible new pharmacological reagents. Newly discovered substances can even be responsible for the creation of new scientific fields. Due to the radically different habitats of marine organisms, several notable examples of secondary metabolites from marine organisms have been isolated. Two of the most remarkable properties of these compounds are their structural and physiological diversities. These bioactive compounds are candidates for drugs or biological probes for physiological studies. Palytoxin is a polyol compound that shows extreme acute toxicity. Halichondrins are remarkable antitumor macrolides from sponge. Pinnatoxins, potent shellfish poisons, cause food poisoning. This paper describes bioorganic studies on such newly discovered wonders of nature. Several bioactive marine alkaloids and important substances involved in dynamic ecological systems are also described.
Okadaic acid (OA), a major polyether toxin involved in diarrhetic shellfish poisoning (DSP), is a potent tumor promoter in rodent skin and glandular stomach and a specific inhibitor of the serine/threonine protein phosphatases PP1 and PP2A. A previous study, which used the cytokinesis-block micronucleus (CBMN) assay in CHO-K1 cells, showed that OA induced chromosome damage in the presence of a rat liver metabolic activation system (S9). To support OA biotransformation by S9, the same test system was performed, and DNA damage induced by OA was measured with and without metabolic activation as well as in the presence of heat-inactivated S9 fraction. The results showed that only in the presence of active S9 did OA significantly increased the frequency of micronucleated binucleated (MNBN) cells. After a 4-h treatment a 2- to 5-fold increase of MNBN cells was observed at 30 nM and at 50 nM of OA. However, without S9 or in the presence of heat-inactivated S9, OA did not induce any chromosome damage. We concluded that OA can be metabolically activated in vitro into metabolites that are more genotoxic. The CBMN assay coupled with fluorescence in situ hybridization (FISH) using a DNA probe for centromere detection was performed to discriminate between clastogenic (chromosome breakage) and aneugenic (chromosome loss) effects. FISH analysis showed that OA metabolites increased in a dose-dependent manner in centromere positive micronuclei (CEN+): 60% of CEN+ at 30 nM and 75% of CEN+ at 50 nM of OA. The uptake of OA into CHO-K1 cells and the biotransformation of the toxin are discussed.
A single run HPLC method utilizing ion exchange as the separation mode with a novel mobile phase system coupled to chemical postcolumn oxidation and fluorescence detection has been developed and demonstrated to be applicable to the quantitative analysis of paralytic shellfish poisons (PSPs) produced by Australian cyanobacteria (Anabaena circinalis) and other cyanobacteria. Both the cyanobacterial matrix and natural water constituents did not significantly affect the performance of this method. The daily precision of this method was adequate for it to be considered as a routine analytical tool for direct PSP analysis (prePSP concentration is not required) of cyanobacterial extracts and water bodies containing PSPs (C1, C2, GTX2, GTX3, NEO, STX) in the low parts per billion concentration range (10-70 ppb).
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.
Okadaic acid (OA), an algal toxin, is known to induce Diarrhetic Shellfish Poisoning and apoptosis in a variety of cell lines. One of the main targets of OA is the actin cytoskeleton which can be modulated by integrins and other structural proteins. In this paper we studied the role of these proteins and skeletal structures on OA-induced apoptosis in neuroblastoma cells. Results show that beta1 integrin and vinculin are down-regulated when cells were exposed to OA. We observed an interaction between talin and beta1 integrin that is impaired in OA treated cells.
Brevetoxins are natural neurotoxins that are produced by "red tide" algae. This class of compounds can cause neurotoxic shellfish poisoning and other health problems. Brevetoxin-2 is the most abundant among the nine brevetoxins that have been characterized, whereas brevetoxin-1 is the most toxic. In this study, brevetoxin-1 and brevetoxin-2 were incubated with rat liver hepatocytes and rat liver microsomes, respectively. After clean-up steps were taken to remove the proteins, samples were analyzed by liquid chromatography (LC) coupled with electrospray mass spectrometry (LC-MS). After incubation of brevetoxin-1, two metabolites were found: brevetoxin-1-M1 (molecular weight = 900 Da), and brevetoxin-1-M2 (molecular weight = 884 Da). The increase in molecular weight combined with evidence from tandem mass spectrometry showing an increased tendency for loss of water molecules, along with considerations of established precedents for chemical transformations led to the conclusion that brevetoxin-1-M1 was formed by converting one double bond in the E or F ring of brevetoxin-1 into a diol. The second metabolite (brevetoxin-1-M2) is proposed to be a hydrolysis product of brevetoxin-1 involving opening of the lactone ring with the addition of a water molecule. The incubation study of the other starting compound, brevetoxin-2, found two metabolites in the LC-ES-MS selected ion chromatogram. Brevetoxin-2-M1 (molecular weight = 912 Da) gave a large [M-H]- peak at m/z 911, and its product ion mass spectrum allowed the deduction that this metabolite was the hydrolysis product of brevetoxin-2 involving conversion of the lactone to a carboxylic acid and an alcohol. The second metabolite (brevetoxin-2-M2, molecular weight = 896 Da) was deduced to have the same structure as that of brevetoxin-3 based on identical chromatographic retention times and similar mass spectra as those obtained for a brevetoxin-3 standard.
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.
Dinophysis are species of dinoflagellates that cause diarrhetic shellfish poisoning. We have previously reported that they probably acquire plastids from cryptophytes in the environment, after which they bloom. Thus monitoring the intracellular plastid density in Dinophysis and the source cryptophytes occurring in the field should allow prediction of Dinophysis blooming. In this study the nucleotide sequences of the plastid-encoded small subunit ribosomal RNA gene and rbcL (encoding the large subunit of RuBisCO) from Dinophysis spp. were compared with those of cryptophytes, and genetic probes specific for the Dinophysis plastid were designed. Fluorescent in situ hybridization (FISH) showed that the probes bound specifically to Dinophysis plastids. Also, FISH on collected nanoplankton showed the presence of probe-hybridized eukaryotes, possibly cryptophytes with plastids identical to those of Dinophysis. These probes are useful not only as markers for plastid density and activity of Dinophysis, but also as tools for monitoring cryptophytes that may be sources of Dinophysis plastids.
A Real-time polymerase chain reaction (PCR) assay was designed and evaluated for rapid detection and quantification of the toxic dinoflagellates Alexandrium catenella and A. tamarense, which cause paralytic shellfish poisoning. Two sets of PCR primers and fluorogenic probes targeting these two species were derived from the sequence of 28S ribosomal DNA. PCR specificity was examined in closely related Alexandrium spp. and many other microalgae. A. catenella-specific primers and probe detected the PCR amplification only from A. catenella strains, and nonspecific signals were not detected from any microalgae. Also, A. tamarense-specific primers and probe also detected the targeted species, suggesting the strict species specificity of each PCR. This assay could detect one cell of each species, showing its high sensitivity. Moreover, using the developed standard curves, A. tamarense and A. catenella could be quantified in agreement with the quantification by optical microscopy. The performance characteristics of species specificity, sensitivity, and rapidity suggest that this method is applicable to the monitoring of the toxic A. tamarense and A. catenella.
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.