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PubMed2006

Identification and characterization of a "biomarker of toxicity" from the proteome of the paralytic shellfish toxin-producing dinoflagellate Alexandrium tamarense (Dinophyceae).

The objective of this study was to identify and characterize a "biomarker of toxicity" from the proteome of Alexandrium tamarense, a paralytic shellfish toxin (PST)-producing dinoflagellate. A combination of 2-DE and MS approaches was employed to identify proteins of interest in the vegetative cells of several strains of A. tamarense with different toxin compositions and from different geographical locations. The electrophoretic analysis of the total water-soluble proteins from these toxic strains by 2-DE showed that several abundant proteins, namely AT-T1, AT-T2 and AT-T3, differing slightly in apparent Mr and pIs, were consistently present in all toxic strains of A. tamarense. Further analysis by MALDI-TOF MS and N-terminal amino acid sequencing revealed that they are isoforms of the same protein. Even more intriguing is that these proteins in A. tamarense have similar amino acid sequences and are closely related to a "biomarker of toxicity" previously reported in A. minutum. Unambiguous and highly species-specific identification was later achieved by comparing the PMFs of proteins in these two species. An initial attempt to characterize these proteins by generation of murine polyclonal antibodies against the AT-T1 protein was successful. Western blot analysis using the murine AT-T1-polycolonal antibodies identified all the toxic strains of A. tamarense and A. minutum, but not the nontoxic strain of A. tamarense. These results indicate that these protein characteristics for toxic strains are species-specific and that they are stable properties of the tested algae which are clearly distinguishable irrespective of geographical location and toxin composition. To our knowledge, this is the first study to demonstrate the use of polyclonal antibodies against marker proteins purified from 2-DE gels to distinguish different strains and species of the PST-producing dinoflagellate Alexandrium. It provides the basis for the production of monoclonal antibody probes against the "biomarkers of toxicity" for those dinoflagellates whose genome is incompletely characterized. Potentially, immunoassays could be developed to detect the presence of toxic algae in routine monitoring programs as well as to predict bloom development and movement.

Animals↗

PubMed1998

A high-throughput, microtiter plate assay for paralytic shellfish poisons using the saxitoxin-specific receptor, saxiphilin.

An isoform of the paralytic shellfish poison (PSP)-specific receptor saxiphilin, from the tropical centipede Ethmostigmus rubripes, was used as the basis for a radiometric, high-throughput, microtiter plate assay for this group of toxins. Characterization of the assay revealed that it was able to detect several representatives from the various structural PSP subgroups and yet was insensitive toward tetrodotoxin. To test the utility of the assay as a seafood toxin-monitoring tool, the assay was subjected to a variety of marine organism extracts, some of which were known to contain PSPs, and whole extract toxicity expressed as STX equivalents (STXeq) was measured by two methods: First, by comparison of values from a screening assay with a standard STX inhibition curve and, second, for highly active extracts, by calculation using the IC50 from a full inhibition curve of the extract. For extracts which could be quantified by both methods, there was almost 100% correlation between the derived values. STXeq derived by both methods from the bioassay highly correlated with absolute toxin quantities from HPLC analysis.

Amphibian Proteins↗

PubMed1981

1,3,5-Trichloro-2-(4-nitrophenoxy)benzene in fish, shellfish, and seawater in Tokyo Bay, 1977-1979.

Concentrations of 1,3,5-trichloro-2-(4-nitrophenoxy)benzene (CNP) were measured in goby-fish, sea bass, shellfish, and seawater samples collected in the coastal waters of Tokyo Bay. The concentrations of CNP found in goby-fish were in the range of 2.6 to 91,400 ppb in liver, 0.1 to 360 ppb in muscle, trace to 2,900 ppb in short-necked clam, and not detectable to 1.9 ppb in seawater. Although the concentrations in the samples varied with time and place of sampling, the maximum levels were found in the samples obtained in May or June of 1977, 1978, and 1979.

Animals↗

PubMed2005

Determination of paralytic shellfish poisoning toxins in cultured microalgae by high-performance liquid chromatography with fluorescence detection.

A novel method for the determination of paralytic shellfish poisoning (PSP) toxins using high-performance liquid chromatography with fluorescence detection was developed. The fluorescent derivates of neosaxitoxin (neoSTX), saxitoxin (STX), gonyautoxins 1 and 4 (GTX1+4), and gonyautoxins 2 and 3 (GTX2+3) were separated on a muBondapak NH2 column (300 mm x 3.9 mm, 10 microm) using water and acetate buffer (pH 6.5) as the mobile phase (1.00 mL min(-1)) in gradient mode with fluorescence detection at 390 nm (excitation at 330 nm). The linear ranges of neoSTX, STX, GTX1+4 and GTX2+3 were 3.31-331, 0.952-95.2, 3.78-378 and 0.124-12.4 ng mL(-1), respectively. The detection limits of neoSTX, STX, GTX1+4 and GTX2+3 were 1.10, 0.32, 1.26 and 0.041 ng mL(-1), respectively. The method was successfully applied to the determination of PSP toxins in microalgae. The recoveries ranged from 88+/-2% to 107+/-4% and the relative standard deviations were 0.16% to 4.4%. The procedure is also environmentally friendly because no organic solvent is used in the mobile phase.

Amides↗

PubMed1993

Environmental analysis of polychlorinated terphenyls: distribution in shellfish from the Ebro Delta (Mediterranean).

Polychlorinated terphenyls (PCTs) have characteristics almost identical with those of polychlorinated biphenyls (PCBs) and have been used for analogous applications, but only sporadic reports of the occurrence of PCTs in the environment have been published. High-resolution gas chromatography with electron-capture detection (HRGC-ECD) and mass spectrometric detection in the selected ion monitoring mode was used to analyse samples for PCTs. The homologue distribution of Aroclor 5432, 5460, Leromoll 141 and the PCTs in samples of shellfish from the Ebro Delta (Catalonia, Spain) was established, taking into account the contribution of the [M-Cl2]+ fragments. Quantification was achieved by HRGC-ECD. Concentrations were between 790 and 3 ng/g (dry mass).

Animals↗

PubMed1994

Comparison of UV absorption and electrospray mass spectrometry for the high-performance liquid chromatographic determination of domoic acid in shellfish and biological samples.

Domoic acid, a neurotoxic amino acid produced by the marine diatom Nitchia pungens multiseries, was determined in samples of anchovies, razor clams, mussels, crab, rat serum, urine and feces by HPLC with UV absorption and electrospray (ESI) mass spectrometric (MS) detection. Shellfish samples were extracted with methanol-water followed by clean-up of the extracts with solid-phase extraction cartridges (strong anion or strong cation exchange). An aliquot of the fraction containing the domoic acid was analysed by HPLC. HPLC column size, mobile phase composition and flow-rate were selected so that essentially the same conditions could be used for both HPLC-UV and HPLC-ESI-MS with selected ion monitoring (SIM) determinations. These included the use of acetonitrile-water-formic acid as the mobile phase, at a flow-rate of 0.2 ml/min (split 13:1 for HPLC-ESI-MS-SIM, 10 microliters/min to the mass spectrometer). The results indicated that extracts found positive by the HPLC-UV method could be readily confirmed directly by HPLC-ESI-MS-SIM without additional sample treatment down to levels of 0.1 micrograms/g of domoic acid. This study demonstrates the use of HPLC-ESI-MS-SIM for the routine confirmation of domoic acid in a wide variety of samples.

Animals↗

PubMed1996

Liquid chromatographic determination of okadaic acid and dinophysistoxin-1 in shellfish after derivatization with 9-chloromethylanthracene.

The reagent 9-chloromethylanthracene was evaluated for derivatization of the diarrhetic shellfish poisons, okadaic acid and dinophysistoxin-1 (DTX-1), to form fluorescent products separable by liquid chromatography. The toxins were reacted with the reagent in acetonitrile in the presence of tetramethylammonium hydroxide for 1 h at 90 degrees C. The products were purified by using two silica solid-phase extraction cartridges before being determined by reversed-phase liquid chromatography with fluorescence detection. The results are comparable to those obtained using 9-anthryldiazomethane (ADAM) for okadaic acid and DTX-1 in mussel tissue. Detection limits were estimated to be about 70-100 ng/g hepatopancreas (equivalent to 12-20 ng/g whole tissue) for each toxin.

Animals↗

PubMed1987

Effect of shellfish consumption on cholesterol absorption in normolipidemic men.

Noncholesterol marine sterols, abundant in molluscan shellfish, could inhibit cholesterol absorption. Eight normolipidemic males were fed for 3 weeks each three natural food diets in which a mixture of oysters and clams, crab, or chicken was served as the primary source of animal protein. The diets were equalized for caloric distribution, cholesterol, and n-3 fatty acids, leaving the noncholesterol marine sterols in the oyster/clam diet (444 mg/2,000 kcal) as the potential lipid-modifying variable. Cholesterol absorption was measured by plasma isotope ratio after doses of oral 14C- and intravenously infused 3H-labeled cholesterol. Cholesterol absorption was lower (42 +/- 4%) during the oyster/clam diet than during the chicken (54 +/- 3%, P less than 0.01) or crab (55 +/- 3%, P less than 0.01) diet periods. There was no difference between the chicken and crab diet periods. Total plasma cholesterol and triglycerides, very low density lipoprotein cholesterol and triglycerides, and low density and high density lipoprotein (HDL) cholesterol were not significantly different between any of the diets. The ratio of the HDL2-/HDL3-cholesterol was higher following the oyster/clam diet (0.46 +/- 0.09) than the chicken diet (0.32 +/- 0.06, P less than 0.05). Plasma and red cell membrane n-3 fatty acids were not significantly different among the three diets, but red cell membrane n-3 fatty acids increased as a function of time regardless of dietary sequence.

Adult↗

PubMed1988

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.

Action Potentials↗

PubMed1992

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.

Acylation↗

PubMed1994

Isolation and structure determination of a new marine neurotoxin from the New Zealand shellfish, Austrovenus stutchburyi.

A new marine neurotoxin was isolated from a water extract of the New Zealand shellfish, Austrovenus stutchburyi, by chromatography on columns of Sephadex G-15 and LH-20, followed by reverse-phase HPLC. It was identified as (4-methoxycarbonylbutyl)trimethyl-ammonium chloride, and this was confirmed by synthesis.

Animals↗

PubMed1994

Comparison of mouse bioassay, HPLC and enzyme immunoassay methods for determining diarrhetic shellfish poisoning toxins in mussels.

Mussel specimens (Mytilus galloprovincialis) collected from two different areas of the Adriatic Sea were analysed for diarrhoetic shellfish poisoning (DSP) toxin by three methods: mouse bioassay, the DSP Check enzyme immunoassay kit, and high-performance liquid chromatography (HPLC). The results obtained confirm that Yasumoto's mouse bioassay, capable of detecting all the components of the DSP group, is still necessary to determine the wholesomeness of the product. The ELISA method has not always given quantitatively reliable results. The HPLC method is advantageous in terms of sensitivity, accuracy, specificity and rapidity. However, its application is limited so far to the determination of okadaic acid in mussels.

Animals↗

PubMed1995

Detection of diarrhoetic shellfish toxins in mussels from Italy by ionspray liquid chromatography-mass spectrometry.

Direct detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and some of their related compounds in toxic mussels (Mytilus galloprovincialis) is reported using ionspray liquid chromatography-mass spectrometry (LC-ISP-MS). This was employed to analyse diarrhoetic shellfish poisoning (DSP) toxins in mussels collected from coastal areas of the northern and southern Adriatic Sea. DTX-1 was found in some samples from both the northern and southern Adriatic and this is the first report of the unambiguous identification of this toxin in Italian mussels. The low levels found indicate that this toxin did not play a significant role in toxicity in these samples. Okadaic acid was found in all the mussels examined, although its concentration was not always sufficient to account for DSP toxicity. Furthermore, two related compounds of OA were detected in all the samples and one related DTX-1 compound was observed in some samples from the northern Adriatic. All three compounds are still to be identified, but it is possible that these substances are involved in mussel DSP toxicity in the Adriatic Sea.

Animals↗

PubMed1994

Identification of Norwalk virus in artificially seeded shellfish and selected foods.

A rotavirus dsRNA purification protocol was adapted to extract Norwalk ssRNA from artificially contaminated shellfish, and a sensitive reverse transcription-polymerase chain reaction assay for Norwalk virus was devised to identify an estimated 20-200 genomic copies. The technique includes deproteinization with guanidinium isothiocyanate, adsorption of RNA to hydroxyapatite, and sequential precipitation with cetyltrimethylammonium bromide and ethanol. The protocol allows high recovery of viral RNA free of enzymatic inhibitors from oysters, clams, and a variety of food matrices. Norwalk virus sequences were copied and amplified by using primers selected from the polymerase gene. Digestion of the amplified products with restriction enzymes ensured the specificity of the test. This rapid and sensitive assay may significantly improve the prospect for the routine screening of the uncultivatable Norwalk virus in food stuffs.

Animals↗

PubMed1994

Diarrhetic shellfish poison in mussels: comparison of methods of detection and determination of the effectiveness of depuration.

The lack of specificity of the Yasumoto mouse bioassay for Diarrhetic Shellfish Poisoning (DSP) toxin and the possibility of obtaining false positive results, prompted a search for a suitable method to confirm doubtful results. The mouse bioassay, an enzyme-linked immunoassay (ELISA) and a tissue culture bioassay were compared; in addition the efficiency of depuration of toxic mussels in ozonized water was evaluated. DSP assay using tissue cultures was found suitable for confirming results obtained using the mouse bioassay, particularly where interpretation was difficult. The ELISA kit gave satisfactory results, but it responded only to okadaic acid and dinophysistoxin 1. Depuration reduced toxicity in some cases after 3 days of treatment.

Animals↗

PubMed1989

Kynurenic acid protects against gastroduodenal ulceration in mice injected with extracts from poisonous Atlantic shellfish.

1. Mice were treated with an extract prepared from poisonous Atlantic mussels. 2. Gastric and duodenal ulcers, duodenal hyperemia and peritoneal ascites resulted from administration of the shellfish extract, with an LD84 of 1.0 ml. 3. Kynurenic acid, an antagonist at excitatory amino acid receptors, protected significantly against gastroduodenal ulcers, ascites and hyperemia when given at 60 or 75 min post-extract. 4. It is likely that the gastrointestinal damage evoked by this extract is due to its domoic acid content and that kynurenic acid may prove useful against domoic acid-induced gastropathy.

Animals↗

PubMed2006

Distribution of organochlorine pesticides and heavy metal residues in fish and shellfish from Calicut region, Kerala, India.

Fish collected from five different locations from the Calicut region, India were analysed for the levels of organochlorine pesticides (OCPs) and heavy metal (HM) residues in order to elucidate the status of these chemical contaminants in fish and shellfish meant for human consumption. The highest concentrations of OCPs detected in the edible portion of fish were 10.47, 70.57 and 28.35 ng g-1 wet weight, in marine, brackish water and freshwater, respectively. BHC and heptachlor epoxide formed the major share of OCPs in the marine fish while BHCs contributed to the major share in the freshwater and brackish water fish. The DDT (sum of DDT and its metabolites) ranged from 0.05 to 8 ng g-1 in the samples irrespective of the habitat. Among the HMs, Cu, Zn, Mn and Cr were present in the samples irrespective of the place of collection. About 22% of the samples from the freshwater area contained marginally higher lead content above the legal limit of 0.2-0.4 microg g-1. The Cr content ranged from 0.1 to 1.1 microg g-1 in the fish with marginally higher content in the fish from freshwater and marine regions (0.55-1.1 microg g-1). Mercury was detected in the samples (55%) from marine habitat only. The concentrations of OCPs and HMs in the samples, in general, were below the maximum residual level recommended by various organizations.

Animals↗

PubMed2005

Hydrophilic interaction liquid chromatography--mass spectrometry for the analysis of paralytic shellfish poisoning (PSP) toxins.

Hydrophilic interaction liquid chromatography (HILIC) was examined for the separation of paralytic shellfish poisoning (PSP) toxins using the stationary phase TSK-gel Amide-80. The parameters tested included type of organic modifier and percentage in the mobile phase, buffer concentration, pH, flow rate and column temperature. Using mass spectrometric (MS) detection, the HILIC column allowed the determination of all the major PSP toxins in one 30 min analysis with a high degree of selectivity and sensitivity. The high percentage of organic modifier in the mobile phase and the omission of ion pairing reagents, both favored in HILIC, provided limits of detection (LOD) in the range 50-100 nM in selected ion monitoring (SIM) mode on a single quadrupole LC-MS system. LOD in selected reaction monitoring (SRM) mode on a sensitive triple quadrupole system were as low as 5-30 nM. Excellent linearity of response was observed.

Animals↗