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PubMed2004

Microcystin-LR and okadaic acid-induced cellular effects: a dualistic response.

Microcystins, potent heptapeptide hepatotoxins produced by certain bloom-forming cyanobacteria, are strong protein phosphatase inhibitors. They covalently bind the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A), thereby influencing regulation of cellular protein phosphorylation. The paralytic shellfish poison, okadaic acid, is also a potent inhibitor of these PPs. Inhibition of PP1 and PP2A has a dualistic effect on cells exposed to okadaic acid or microcystin-LR, with both apoptosis and increased cellular proliferation being reported. This review summarises the existing data on the molecular effects of microcystin-LR inhibition of PP1 and PP2A both in vivo and in vitro, and where possible, compares this to the action of okadaic acid.

Animals↗
PubMed2002

Direct detection of yessotoxin and its analogues by liquid chromatography coupled with electrospray ion trap mass spectrometry.

A liquid chromatography mass spectrometry (LC-MS) method is proposed for the sensitive, specific and direct detection of yessotoxin and its analogues, marine biotoxins which are associated with diarrhetic shellfish poisoning (DSP) and which have been found in the North Adriatic sea since 1995. The LC-MS method provided a detection limit of 70 pg for yessotoxin in full scan mode and was applied to determine the toxic profiles of a number of extracts or partially purified fractions of toxic mussels collected along the Emilia Romagna coasts (Italy) in the period 1995-1999. Detection of a desulfo-yessotoxin derivative from Mytilus galloprovincialis collected in 1998 is also reported.

Animals↗
PubMed1998

Solid-phase extraction and high-performance liquid chromatography procedures for the analysis of paralytic shellfish toxins.

Paralytic shellfish poisoning (PSP) toxins are produced by certain dinoflagellate species such as Gymnodinium catenatum and Alexandrium tamarensis, during certain periods of the year influenced by several environmental factors, affecting the aquaculture industry and mainly bivalve molluscs. HPLC with fluorescence detection is a powerful analytical technique for the analysis of such toxins; several HPLC alternatives have been developed in order to improve the liquid chromatographic analysis, but due to the complexity of the sample matrix, important work has been focused recently on the clean-up of samples prior to HPLC analysis. Solid-phase extraction procedures offer advantages for this clean-up. In this work we focus on the study of three different clean-up methods prior to HPLC with fluorescence detection analysis of PSP toxin present in contaminated mussel samples; by spiking uncontaminated mussel samples with two different PSP toxin standards and by calculating the recovery values for these experiments. These recoveries must be taken into account in order to quantify the exact amount of PSP toxins present in the contaminated samples.

Animals↗
PubMedDate not supplied

Expression and characterization of Flag-epitope- and hexahistidine-tagged derivatives of saxiphilin for use in detection and assay of saxitoxin.

Saxiphilin is a plasma protein from the bullfrog (Rana catesbiana) that binds saxitoxin (STX), a causative agent of paralytic shellfish poisoning. Saxiphilin is homologous to transferrin and consists of two internally homologous domains called the N-lobe and the C-lobe. STX binds to a single site in the C-lobe of saxiphilin. In this study, cloned genes coding for recombinant saxiphilin and C-lobe saxiphilin were modified to contain two tandemly located affinity tags, Flag epitope (DYKDDDDK) and His(6) (HHHHHH), at the protein C-terminus and were expressed in cultured insect cells using baculovirus vectors. Both tagged proteins are readily detected on immunoblots by anti-Flag monoclonal antibody. Flag-His(6)-tagged saxiphilin was purified to homogeneity using Ni(2+)-chelate affinity chromatography and Heparin Sepharose chromatography. Equilibrium analysis of [3H]STX binding to tagged saxiphilin and tagged C-lobe saxiphilin gave K(D) values of 0.75 and 2.7 nM, respectively. Flag-His(6)-tagged saxiphilin was also utilized in a microtiter well solid-phase assay with Reacti-bind metal chelate plates to measure [3H]STX binding and binding competition by unlabeled STX. Such Flag-His(6)-tagged derivatives of saxiphilin have many possible applications in the assay of STX and related toxinological research.

Amphibian Proteins↗
PubMed2001

Possible source of tetrodotoxin in the starfish Astropecten scoparius.

The seasonal variations of toxicity and stomach contents in toxic starfish Astropecten scoparius were detected. The average highest specimen toxicity, expressed as tetrodotoxin (TTX), was 16,821 mouse units (MU). The toxin was composed of TTX only, except April's sample containing mainly TTX along with minor paralytic shellfish poisons. The composition in the stomach of less and more toxic starfish was mainly Veremolpa scabra and Umborium suturale, respectively. The toxicities of U. suturale and V. scabra, found in the starfish stomach were 65 and 33 MU, respectively. For those collected from the coastal waters, U. suturale was toxic with average toxicity value of 77 MU/g, but V. scabra was nontoxic. The toxin in the specimens of U. suturale collected from either the digestive gland of starfish or the coastal waters, was TTX and anhydroTTX only. It indicates that the starfish A. scoparius might mainly accumulate high amount of TTX from U. suturale. Furthermore, both small gastropods U. suturale and Natica psuestes are first reported to contain TTX.

Animals↗
PubMed2001

Delayed haemolytic activity by the freshwater puffer Tetraodon sp. toxin.

In order to elucidate the toxin composition of the freshwater puffer in Bangladesh, about 230 specimens of Tetraodon sp. were collected from 1997 to 1999 and extracted. After partitioning the toxins between an aqueous layer and a 1-butanol layer, the toxin in the aqueous layer was characterized as paralytic shellfish poison (PSP) (data not shown), while the toxin in the 1-butanol layer was identified as palytoxin (PTX) or PTX-like substance based on the delayed haemolytic activity which was inhibited by an anti-PTX antibody and ouabain (g-strophanthin). This is the first report on the occurrence of PTX or PTX-like substance(s) in puffer fish.

Acrylamides↗
PubMed2001

The validity of two HPLC methods and a colorimetric PP2A assay related to the mouse bioassay in quantification of diarrhetic toxins in blue mussels (Mytilus edulis).

Validity of two HPLC methods and a PP2A assay in relation to the mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins was evaluated. The mouse bioassay for DSP toxins was performed on a total of 177 mussel samples from the Sognefjord, Norway, using diethyl ether in the final step of extraction. For fluorimetric HPLC analyses, either 4-bromomethyl-7-methoxycoumarin (BrMMC) or 9-anthryl diazomethane (ADAM) were used for analysis of 48 and 118 of the samples, respectively. The colorimetric PP2A inhibition assay was performed on all 177 samples that were analysed with the mouse bioassay. When comparing the HPLC-BrMMC, the HPLC-ADAM and the PP2A assays with the mouse bioassay, cut off values of < or =4, 5 and 6 microg okadaic acid (OA) equivalents (eq.)/5 g digestive gland (DG) was used. With reference to the results from the mouse bioassay, the total number of failure and correct classification by HPLC-ADAM and the PP2A method was compared for the three cut off values. No significant differences between the methods were detected. However, all differences were found in favour of HPLC-ADAM. All three methods could replace the mouse bioassay in detecting levels of diarrhetic toxins approved internationally for safe consumption of mussels. However, HPLC-ADAM seems to be the method of choice.

Animals↗
PubMed2002

Evaluation of marine biotoxin's accumulation by Acanthocardia tuberculatum from Algarve, Portugal.

Acanthocardia tuberculatum is a bivalve mollusc that presents recurrent problems of paralytic shellfish poisoning (PSP) contamination in the Mediterranean coasts of Spain and Morocco. Although not commercially exploited from the Portuguese south coast, it represents an alternative for reducing the harvest pressure on species presently exploited. Evaluation of accumulation of marine biotoxins was carried out by HPLC in this species, harvested during a campaign carried out in April 2001 aimed at evaluating bivalve's resources that covered the entire Portuguese south coast.PSP toxins were studied by automated pre-column oxidation. Toxins were found in specimens from all stations, but always under the regulatory limit of 80 microg STX eq./100g. Saxitoxin and decarbamoyl-saxitoxin were the only PSP toxins unambiguously identified. In commercially exploited species, saxitoxin and analogues were not detected. The amnesic toxin domoic acid was found, but in levels similar to those found in species commercially exploited, and always under the regulatory limit of 20 microg/g. A fast elimination, and not a prolonged retention, of domoic acid seems to occur in A. tuberculatum in view of the levels close to or higher than 20 microg/g found in commercial bivalves harvested the two preceding months. The diarrhoeic toxin okadaic acid was found at trace levels much under the allowable level, similar to what was happening with other species harvested during the same period.

Animals↗
PubMed2002

A tetrodotoxin-binding protein in the hemolymph of shore crab Hemigrapsus sanguineus: purification and properties.

The shore crab Hemigrapsus sanguineus hemolymph contains soluble proteins that bind tetrodotoxin (TTX) and are responsible for high resistance of the crab to TTX. The TTX-binding protein was purified from the hemolymph by ultrafiltration, lectin affinity chromatography and gel filtration HPLC. The purified protein gave only one band in native-polyacrylamide gel electrophoresis (PAGE), confirming its homogeneity. Its molecular weight was estimated to be about 400k by gel filtration HPLC, while it was estimated to be about 82k under non-reducing conditions and about 72 and 82k under reducing conditions by SDS-PAGE, indicating that the TTX-binding protein was composed of at least two distinct subunits. The TTX-binding protein was an acidic glycoprotein with pI 3.5, abundant in Asp and Glu but absent in Trp, and contained 6% reducing sugar and 12% amino sugar. The protein selectively bound to TTX, with a neutralizing ability of 6.7 mouse unit TTX/mg protein, but not to paralytic shellfish poisoning toxins. However, its neutralizing activity was almost lost by treatments with enzymes (protease XIV, thermolysin, trypsin, amyloglucosidase and alpha-amylase) and denaturing agents (1% SDS, 1% dithiothreitol, 8 M urea and 6 M guanidine hydrochloride), suggesting the involvement of both proteinaceous and sugar moieties in the binding to TTX and the importance of the steric conformation of the TTX-binding protein.

Amino Acids↗
PubMed2002

Emerita analoga (Stimpson)--possible new indicator species for the phycotoxin domoic acid in California coastal waters.

Blooms of domoic acid (DA) synthesizing diatoms (Pseudo-nitzschia spp.) have been associated with the death and injury of hundreds of marine shorebirds and mammals, exposed humans to potentially serious health risks, and threatened to significantly impact coastal fisheries and commerce dependent on marine resources. While indicator organisms are widely utilized to monitor for marine biotoxins like paralytic shellfish poisoning toxins, a reliable intertidal indicator species to monitor DA remains to be identified. Here we evaluate and confirm the utility of the common sand crab (Emerita analoga) as an indicator for DA in comparison with sea mussels (Mytilus californianus). Mussels and sand crabs, collected from natural populations in Santa Cruz, California (April 1999-February 2000), were tested for DA using the HPLC-UV method. Toxin loads in sand crabs ranged from below detectable limits to 13.4 micro g DA g(-1) and coincided with the abundance of DA producing Pseudo-nitzschia species nearshore. Toxin levels in mussels collected during the study period were below HPLC-UV detectable limits. The rise and fall of DA in sand crabs in synchrony with Pseudo-nitzschia abundance, combined with this common intertidal species' accessibility and ease of DA extraction, clearly indicate the utility of sand crabs as a reliable, cost-effective monitoring tool for DA in the nearshore coastal environment.

Animals↗
PubMed2003

GTX(4) imposters: characterization of fluorescent compounds synthesized by Pseudomonas stutzeri SF/PS and Pseudomonas/Alteromonas PTB-1, symbionts of saxitoxin-producing Alexandrium spp.

Saxitoxins, the etiological agent of paralytic shellfish poisoning, are synthesized by dinoflagellates and cyanobacteria. Several reports indicate that bacteria are capable of saxitoxin synthesis. Two bacterial strains were isolated from saxitoxin-producing dinoflagellates, Alexandrium tamarense and A. lusitanicum (=Alexandrium minutum), and grown under a variety of culture conditions including those previously reported to induce saxitoxin synthesis in bacteria. Five fluorescent compounds were accumulated by the bacteria that had HPLC-FLD retention times similar to a reference standard of GTX(4), one of the saxitoxin congeners. However, we were unable to detect GTX(1), the epimeric partner of GTX(4), in the bacterial samples. The GTX(4) standard was hydrolyzed by NaOH/heat treatment but four of the bacterial compounds were stable. Unlike GTX(4), none of the five bacterial compounds were detectable by HPLC-FLD following electrochemical oxidation. The fluorescence emission spectrum of each of the five bacterial compounds was unique and readily discernable from the spectrum of GTX(4). None of the samples containing the putative GTX(4) toxin yielded positive results when analyzed by a 3H-saxitoxin receptor-binding assay for saxitoxin-like activity. We cannot rule out the possibility that these bacteria produce saxitoxins, however, our data clearly demonstrate that they accumulate at least five different fluorescent compounds that could be easily mistaken for GTX(4). We conclude that these five fluorescent compounds are GTX(4) imposters and that fluorescence scanning and chemical/heat stability should, at a minimum, be incorporated into HPLC-FLD protocols for identification of saxitoxins.

Animals↗
PubMed2003

Cylindrospermopsin occurrence in two German lakes and preliminary assessment of toxicity and toxin production of Cylindrospermopsis raciborskii (Cyanobacteria) isolates.

Cylindrospermopsis raciborskii, a freshwater cyanobacterium of tropical origin, is not only increasingly found in (sub) tropical water bodies, but also in temperate regions. Since this species may produce potent toxins such as cylindrospermopsin (CYN) and paralytic shellfish poisons, its massive occurrence in water bodies used as drinking water sources or for recreation is of major concern. The proliferation of C. raciborskii in German water bodies has been documented for the past decade. We investigated the occurrence of CYN in field populations and isolates of C. raciborskii from two lakes, and assessed the toxicity of culture isolates using the mouse bioassay, primary rat hepatocytes and human derived cell lines. We show for the first time the occurrence of CYN in German water bodies. None of seven isolates of C. raciborskii contained CYN, however, all isolates were toxic to primary rat hepatocytes, human hepatoblastoma (HEP-G2) and human colon adenocarcinoma (CACO-2) cells. Methanolic extracts were more toxic than aqueous extracts. Three isolates tested in the mouse bioassay were toxic at a concentration of 800 mg kg(-1) showing liver and spleen damage and inflammation of the intestine. These results give strong evidence that the German isolates of C. raciborskii contain currently not identified or unknown toxins.

Alkaloids↗
PubMed1997

Determination of okadaic acid by micellar electrokinetic chromatography with ultraviolet detection.

Micellar electrokinetic chromatography (MEKC) with ultraviolet (UV) detection was applied for the determination of non-derivatized phycotoxins associated with diarrhoetic shellfish poisoning. A detection limit for 40 pg of okadaic acid (OA) was achieved. The UV intensities of this toxin measured at 200 nm showed good linearity in the range 40-640 pg. OA was detected in mussels spiked with 10 ng/g whole tissue. The presence of OA and dinophysistoxin-2 was observed in the crude extract of the dinoflagellate Prorocentrum lima.

Animals↗
PubMed1997

A rapid tissue culture assay for the detection of okadaic acid and related compounds in mussels.

Okadaic acid, the main toxin of the diarrhoetic shellfish poisoning (DSP) group, shows high cytotoxicity to Buffalo green monkey (BGM) kidney cell cultures after different times according to concentration. A rapid method of detecting the presence of okadaic acid and related compounds in contaminated mussels by means of a cytotoxicity test is presented. The method is based on direct microscopic observation of toxin-induced morphological changes in BGM cell cultures. A high correlation (r = 0.950; P < 0.001) was found between the official mouse bioassay and the cytotoxicity test conducted on naturally contaminated samples of Mytilus galloprovincialis.

Animals↗
PubMed1998

Okadaic acid production from the marine dinoflagellate Prorocentrum belizeanum Faust isolated from the Belizean coral reef ecosystem.

A species of epiphytic Prorocentrum (Dinophyta, Prorocentrales, collected from the macroalga Dictyota dichotoma, was brought into culture. Based on morphological characteristics, this isolate was identified as Prorocentrum belizeanum. Analysis of the culture extract using high-performance liquid chromatography and liquid chromatography-mass spectrometry indicated the production of okadaic acid, a toxic polyether linked with the human diseases, diarrhetic shellfish poisoning and ciguatera fish poisoning.

Animals↗
PubMed1998

Cross-reactivity of an anti-okadaic acid antibody to dinophysistoxin-4 (DTX-4), dinophysistoxin-5 (DTX-5), and an okadaic acid diol ester.

The cross-reactivity of the 6/50 monoclonal anti-okadaic acid antibody (mAb) to the recently discovered diarrhetic shellfish poisoning (DSP) metabolites dinophysistoxin-4 (DTX-4), dinophysistoxin-5 (DTX-5), and an okadaic acid (OA) diol ester was determined using a competitive indirect enzyme-linked immunosorbent assay (ELISA). The reactivity of the antibody to these molecules was compared to that with OA; the 6/50 mAb recognized all of these DSP compounds with equal sensitivity within the working range of the antibody (10-100 nM for OA). This confirms the ability of the antibody to detect all DSP compounds when used in analyses including ELISA and immunocytochemistry.

Animals↗
PubMed1998

Use of a channel biosensor for the assay of paralytic shellfish toxins.

Gonyautoxin (GTX), saxitoxin (STX) and tetrodotoxin (TTX), also known as paralytic shellfish poisons (PSP), block Na+ channels, including those in the frog bladder membrane. A tissue biosensor has been developed, consisting of a Na+ electrode covered with a frog bladder membrane integrated within a flow cell. The direction of Na+ transfer, investigated in the absence of Na+ channel blockers, established that active transport of Na+ occurs across the frogs bladder membrane from the internal to the external face. Transfer was shown to be TTX sensitive. The tissue sensor response to each of the different PSP was recorded and the results compared with toxicities determined by the standard mouse bio-assay. Using high concentrations of TTX from the puffer fish Takifugu niphobles, a linear correlation was found between the results from the two assay systems. However, the tissue biosensor system was also able to detect very low concentrations of TTX in samples from two species of puffer fish (Takifugu niphobles and Takifugu pardalis) at concentrations below the detection limit of the mouse bio-assay.

Animals↗