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Accumulation and transformation of DSP toxins in mussels Mytilus galloprovincialis during a toxic episode caused by Dinophysis acuminata.

The time course of several outbreaks of the diarrhetic shellfish poisoning (DSP) producer Dinophysis acuminata and the consequent kinetic of accumulation and loss of toxins in mussels Mytilus galloprovincialis feeding on them was studied. Samples of mussels and seawater were frequently (2-3 times a week) collected from a raft in the Ri;a de Vigo. DSP toxins content of mussels and water was analyzed by HPLC-FD and phytoplankton was quantified in an inverted light microscope. Only okadaic acid (OA) and some of its conjugated forms (OA CF), estimated by enzymatic hydrolysis, were found in the plankton samples obtained, comprised mainly of D. acuminata cells. The main accumulated form in mussels was OA reaching a maximum of 10.1 microg OA g(-1) in the digestive gland (d.g.) in 16 days, falling below the quarantine level (ca. 2 microg OA g(-1) d.g.) by 45 days. The low polarity conjugated forms (LPCF), estimated by hexane extraction, accounted for 6.2% of the total toxin burden of the mussels. To quantify the rates of the processes involved in the accumulation, transformation and loss of the toxins, two dynamic models, a one-compartment and a two-compartment, including OA and its conjugated forms as variables were designed and implemented. The one-compartment model provided a good fit to the OA and LPCF actual data (r(2)=0.92 and r(2)=0.94, respectively). The two-compartment model did not fit the data markedly better than its one-compartment counterpart (r(2)=0.93 and r(2)=0.95, for OA and LPCF, respectively). High hydrolysis rates were estimated for most of the OA CF, which means that these forms came largely from the ingested plankton. The low estimated acylation rates support the previous point and suggest that the formation of LPCF by direct acylation of the OA is of little importance in M. galloprovincialis. Only in cases where the intoxication period is very long, can the formed acyl-derivatives be important, because they seem to accumulate for a long time in the mussels, as suggested by the low hydrolysis and depuration rates estimated from model fitting.

Animals↗

Mussel blood cells, resistant to the cytotoxic effects of okadaic acid, do not express cell membrane p-glycoprotein activity (multixenobiotic resistance).

Okadaic acid (OA) is a dinoflagellate toxin, accumulating in shellfish and causing diarrhetic shellfish poisoning (DSP) in humans. OA is a highly cytotoxic agent in most cell lines because of its inhibiting properties of protein phosphatases. So far, the cytotoxicity of OA in mussels, the main vectors of DSP, has not been investigated. In this paper, the viability of mussel (Mytilus edulis) blood cells incubated in 10 nM-1 microM OA was studied. After 72 h of exposure, viability was reduced to 54% in 1 microM OA compared with 88% in control cells. This yielded a LC50 of >1 microM for OA, which is 30-1000-times higher compared with other cell types. It was hypothesised that P-glycoprotein (p-gp) activity (multixenobiotic resistance, MXR) contributed to the resistance to OA. Vincristine and rhodamine B was used as p-gp substrates and verapamil or staurosporine (ST) as inhibitors of p-gp transport. However, no indications of cell membrane p-gp activity were detected. Instead, experimental observations led to the conclusion that a MXR transport system was present within lysosomal membranes. Various concentrations of OA did not affect the dynamics of vincristine in blood cells. As a positive control for the assay, p-gp activity was measured in mussel gill tissue. The efflux of rhodamine B was reduced by verapamil, which is, considered evidence for cell membrane p-gp activity, thus the accuracy of the method was confirmed. Rhodamine B efflux was also reduced by OA in gill tissue, which suggested that OA is either a competitive substrate or inhibitor of p-gp activity. When the volume of the lysosomal compartment was measured in blood cells pre-exposed to OA, a significant increase was detected compared with control cells. It was proposed that uptake and storage of OA within the lysosomal system might protect mussel blood cells from the cytotoxic effects of this compound.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Recent advances in the study of epigenetic effects induced by the phycotoxin okadaic acid.

Okadaic acid (OA) is a phycotoxin produced by dinoflagellates. It accumulates in the digestive tracts of shellfish causing diarrhetic shellfish poisoning (DSP) in consumers. OA is a tumour promoter, and an inhibitor of both protein phosphatases and protein synthesis. OA induces DNA adducts, suggesting it may be carcinogenic. Since the Ames test without S(9) was negative, but a mutagenesis test was positive in mammalian cells, the question as to whether its molecular mechanism is genotoxic or epigenetic became unavoidable. Therefore, experiments were performed to search for epigenetic effects, since evidence for DNA-adduct formation using the gamma-(32)P-ATP post-labelling method was not obtained. We found that OA is a potent inducer of lipid peroxidation in human intestinal cells (Caco-2) at low concentrations (0.75-7.5 ng/ml versus IC50 of 15 ng/ml) with increased rates of 8-OH-dG and m(5)dC formation causing CG to AT transversion mutations and gene deregulation, respectively. The transcription and translation of connexin 43-specific mRNA were inhibited, and 3H-uridine incorporation in RNA was concomitantly increased. Consequently gap junction intracellular communication (GJIC) was inhibited, making possible cellular anarchic proliferation. Higher OA concentrations also disorganized the cellular cytoskeleton, since both actin and tubulin formations were impaired. Our results suggest that OA may induce tumours via an epigenetic mechanism.

Caco-2 Cells↗

Neurological disorders and travel.

Travel is associated with a number of neurological disorders that can be divided into two categories: (1) Neurological infections including encephalitides, neurotuberculosis, neurobrucellosis, cysticercosis and trichinosis. Some of these disorders can be prevented by vaccinations, such as Japanese B encephalitis and rabies, some by the use of insect repellents and some by avoiding raw milk products and undercooked meat. (2) Non-infective neurological disorders, such as acute mountain sickness and high altitude cerebral oedema, problems occurring during air travel such as syncope, seizures, strokes, nerve compression, barotrauma and vertigo, motion sickness and foodborne neurotoxic disorders such as ciguatera, shellfish poisoning and intoxication by cassava. This group of diseases and disorders could be prevented if the traveller knows about them, applies simple physiological rules, takes some specific medications and knows how to avoid intoxications in certain geographical areas. Meningococcal meningitis, malaria and jet lag syndrome are extensively discussed in other articles of this issue. The discussion in this paper will be limited to the other disorders.

Aerospace Medicine↗

Exciton coupled circular dichroic studies of self-assembled brevetoxin-porphyrin conjugates in lipid bilayers and polar solvents.

BACKGROUND: Brevetoxins, involved in the 'red tide' as well as shellfish poisoning, are known to bind to cell membranes and membrane proteins. Brevetoxin B (BTX-B) interacts specifically with neuronal sodium channels. We recently found that BTX also induces selective ion movements across lipid bilayers through transmembrane BTX self-assemblies. RESULTS: We examined the self-assembly of several BTX derivatives in the presence and absence of cations and lipid bilayers using the powerful porphyrin chromophores as circular dichroism labels. BTX derivatives self-assemble into tubes, which can bind to metals both when soluble and when inserted into the bilayer to form transmembrane pores. Depending on the tendency of the BTX derivative to self-aggregate (the critical 'micelle' concentration, cmc), it may aggregate in solution before membrane insertion, or may insert itself into the membrane as a monomer before assembling the pore. CONCLUSIONS: The active BTX-B complex in lipid bilayers is a cyclic, transmembrane self-assembly consisting of antiparallel aligned BTX molecules that can mediate selective ion movement through membranes. The differences in pore formation mechanisms between BTX derivatives may be reflected in differences in pore formation by natural BTX variants, perhaps explaining their varying levels of toxicity.

Circular Dichroism↗

Domoic acid: a fascinating marine toxin.

There are indications that toxic algal blooms are increasing because of pollution of coastal waters and worldwide shipping. This mini-review deals with the marine biotoxin domoic acid, also known as amnesic shellfish poison, and its main producing pennate diatom genus Pseudo-nitzschia (Bacillariophyceae). Besides contamination of seafood, these organisms have also been involved in human and marine wildlife mortality. The article aims to give an overview of all biological and environmental factors that should be considered when trying to evaluate a possible increase in toxic blooms of Pseudo-nitzschia spp. Pseudo-nitzschia blooms characteristically occur in a low light regime, at a time when the temperature is falling and at a wide range of salinities. Laboratory studies have shown that the production of domoic acid, a water-soluble amino acid, is related to silicon, phosphorus, nitrogen and trace metal (mainly iron) availability. Domoic acid has no known function in defence or primary metabolism; a role in excretion of excess photosynthetic energy or as a binding ligand for trace metals is suggested. The variability in domoic acid production by different Pseudo-nitzschia spp., or the presence of toxic and non-toxic strains of the same species, cannot be explained. The conclusion is drawn that an increase in toxic blooms of Pseudo-nitzschia spp. might be possible, especially because of the expected increase in nutrient availability from pollution and desert dust. Global warming may have an influence as well by lengthening the growth period for Pseudo-nitzschia, enlarging their global distribution and increasing the dust load through desertification.

Journal Article↗

Development of an ultrasensitive immunoassay for rapid measurement of okadaic acid and its isomers.

This report highlights the characteristics of an okadaic acid immunoassay with limits of detection in the subfemtomole range. Two different immunoassay formats were investigated and their characteristics compared in relation to linear ranges, limits of detection, and cross-reactivity with other seafood toxins present in water and/or mussel samples. The developed ELISA system can be manipulated to quantitatively measure total diarrhetic shellfish poisoning (DSP) content or for okadaic acid and dinophysistoxin-1 individual concentrations by variation of the format of the immunoassay. Real mussel samples were validated in percentage recovery test. Calibration curves were established, and aliquots of real samples were tested. Very good recoveries were attained, highlighting the validity of the ELISA system to accurately determine the DSP concentration in mussel samples.

Animals↗

Structure and stereochemistry of a new cytotoxic polychlorinated sulfolipid from Adriatic shellfish.

A detailed analysis of the causative toxins contained in the hepatopancreas of toxic mussels from the northern Adriatic sea has been carried out. Along with some DSP (diarrhetic shellfish poisoning) type toxins, such as okadaic acid, yessotoxin, and their derivatives, which are involved in a number of human intoxications throughout the world, we have now isolated a new cytotoxin, a polychlorinated sulfolipid 1, whose gross structure has been elucidated by spectral analysis, including various 2D NMR techniques. The relative stereochemistry of 1 was elucidated by successful application of the J-based configuration analysis developed for acyclic compounds using carbon-proton spin-coupling constants ((2,3)J(C,H)) and proton-proton spin-coupling constants ((3)J(H,H)); its absolute stereochemistry was established by the Mosher method. Compound 1 possesses in vitro cytotoxicity against WEHI 164 and RAW 264.7 cells.

Animals↗

Utilization of Folin-Ciocalteu phenol reagent for the detection of certain nitrogen compounds.

To determine in more detail the reaction of Folin-Ciocalteu phenol reagent with nitrogen compounds, a number of hydroxylamine-related compounds and a large number of guanidine-containing compounds were tested. In general, guanidine compounds did not react strongly unless they were hydroxyamino or hydrazino derivatives. The non-hydroxyamino paralytic shellfish poison saxitoxin, however, reacted to a significant extent. This may be due to the presence of a five-membered ring structure and its analogy to 2-aminopurines, which react strongly. A number of simpler amines were also tested. Tertiary aliphatic amines, but not primary, secondary, or quaternary amines, reacted strongly with the reagent. Primary, secondary, and tertiary aromatic amines all reacted strongly with the reagent. Reactivity was extended to pyrroles and indole derivatives but not to imidazole and benzimidazole derivatives. Defining the reactivity of Folin-Ciocalteau phenol reagent with nitrogen compounds extends the usefulness of the reagent for the detection and determination of certain nitrogen compounds in basic extracts by colorimetric means and by thin-layer chromatography.

Amides↗

Determination of domoic acid in phytoplankton by high-performance liquid chromatography of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivative.

Domoic acid is the toxin that can cause amnesic shellfish poisoning. In this work, a new precolumn derivatization reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, was used to react with domoic acid to form a stable unsymmetrical urea derivative, which was readily analyzed by reversed-phase HPLC with fluorescence detection. The one-step derivatizing method is rapid and straightforward. The method was applied to detect domoic acid in phytoplankton using a 3.9 x 150 mm Nova-Pak, 4 microm, C(18) column.

Aminoquinolines↗

Studies of the biosynthesis of DTX-5a and DTX-5b by the dinoflagellate Prorocentrum maculosum: regiospecificity of the putative Baeyer-Villigerase and insertion of a single amino acid in a polyketide chain.

The biosynthetic origins of the diarrhetic shellfish poisoning toxins DTX-5a and DTX-5b have been elucidated by supplementing cultures of the producing organism Prorocentrum maculosum with stable isotope labeled precursors and determining the incorporation patterns by 13C NMR spectroscopy. The amino acid residue in the sulfated side chain is found to originate from glycine, and oxygen insertion in the chain is shown to occur after polyketide formation.

Amino Acids↗

Identification and Characterization of Pectenotoxin (PTX) 4 and PTX7 as Spiroketal Stereoisomers of Two Previously Reported Pectenotoxins.

Pectenotoxins (PTXs) isolated from the scallop Patinopecten yessoensis were shown to be involved in an episode of diarrhetic shellfish poisoning (DSP). A total of eight analogues (PTX1-PTX7 and PTX10) have been isolated to date, and the structures of four of these analogues (PTX1, PTX2, PTX3, and PTX6) have already been elucidated. Here, we report the characterization of PTX4 and PTX7 as 7-epi-PTX1 and 7-epi-PTX6, respectively, on the basis of NMR data and an acid-catalyzed chemical interconversion. The structures of two new artifacts, PTX8 and PTX9, produced following this treatment are also reported.

Journal Article↗

Isolation and structure of prorocentrolide B, a fast-acting toxin from Prorocentrum maculosum.

A new toxin, prorocentrolide B (1), has been isolated following bioassay-guided fractionation of a BuOH extract of the tropical dinoflagellate, Prorocentrum maculosum Faust. This compound produces a rapid toxic response in the mouse bioassay, a type of activity not accounted for by other diarrhetic shellfish poisoning toxins produced by P. maculosum. The structure 1 was established by NMR and MS and is similar to prorocentrolide (2), a toxin from a strain of Prorocentrum lima. NMR data and the modeling program ConGen have been used to establish the relative stereochemistry of some individual ether ring systems and the hexahydroisoquinoline ring.

Animals↗

Three novel hydroxybenzoate saxitoxin analogues isolated from the dinoflagellate Gymnodinium catenatum.

In a recent survey of paralytic shellfish poisoning (PSP) toxins in Gymnodinium catenatum Graham extracts, using LC with postcolumn oxidation and fluorescence detection, three novel saxitoxin analogues were revealed in isolates from several locations, including Australian waters. We have named them as G. catenatum toxins, GC1 (1), GC2 (2), and GC3 (3). The compounds were isolated from a culture of the Australian strain by LC-MS-guided fractionation employing a C18-silica column and hydrophilic interaction chromatography. The unusual structures of these novel compounds were characterized by low- and high-resolution MS, MS/MS, and NMR spectroscopy. GC3 (3) was found to be the 4-hydroxybenzoate ester derivative of decarbamoylsaxitoxin, while GC1 (1) and GC2 (2) are the epimeric 11-hydroxysulfate derivatives of GC3 (3).

Animals↗

An endogenous annual clock in the toxic marine dinoflagellate Gonyaulax tamarensis.

Blooms of the toxic dinoflagellate Gonyaulax tamarensis (synonyms Protogonyaulax tamarensis and Alexandrium tamarense) cause outbreaks of paralytic shellfish poisoning (PSP) in coastal waters throughout the world. In the Gulf of Maine, episodes occur between April and November, a seasonality due in part to life-cycle alternations between motile, vegetative cells and resting cysts which overwinter in bottom sediments. Newly formed cysts have a mandatory 2-6 month dormancy period during which germination is not possible, but once mature, the resting state will continue if temperatures are unfavourable or oxygen is unavailable. We now report another factor controlling germination of cysts of G. tamarensis from deep coastal waters--an endogenous annual clock that can override an otherwise favourable environment for germination. Similar annual variability in germination has not been observed for cysts of this species from shallow estuaries. These results represent the first conclusive demonstration of an endogenous circannual rhythm in a marine plant. They are evolutionarily and ecologically significant because an endogenous annual clock can lead to the release of motile cells into deep and relatively invariant bottom waters at those times when temperature and light at the surface are suitable for growth. In shallow waters where seasonal variability is large and extends to bottom sediments, a strategy similar to that of the seeds of terrestrial plants would be more appropriate, namely a direct coupling between germination and the external environment.

Biological Clocks↗

Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism.

Okadaic acid is a polyether derivative of 38-carbon fatty acid, and is implicated as the causative agent of diarrhetic shellfish poisoning. It is a potent tumour promoter that is not an activator of protein kinase C, but is a powerful inhibitor of protein phosphatases-1 and -2A (PP1 and PP2A) in vitro. We report here that okadaic acid rapidly stimulates protein phosphorylation in intact cells, and behaves like a specific protein phosphatase inhibitor in a variety of metabolic processes. Our results indicate that PP1 and PP2A are the dominant protein phosphatases acting on a wide range of phosphoproteins in vivo. We also find that okadaic acid mimics the effect of insulin on glucose transport in adipocytes, which suggests that this process is stimulated by a serine/threonine phosphorylation event.

Acetyl-CoA Carboxylase↗

Phylogeography of the invasive cyanobacterium Cylindrospermopsis raciborskii.

Cylindrospermopsis raciborskii is a planktonic freshwater cyanobacterium that has become increasingly prevalent in tropical and temperate water bodies world-wide. This species is of concern from a water-quality perspective because of its known ability to produce toxins that can affect the health of humans and other animals. This study investigates genetic variation between strains of C. raciborskii isolated from freshwater rivers and reservoirs in Australia, Brazil, Germany, Hungary, Portugal and the USA. Strains were first characterized by analysis of their 16S rRNA gene nucleotide sequences and were found to have a sequence divergence of 99.1%. A phylogenetic tree, constructed using the 16S rRNA gene sequences showed that strains grouped into Australian, European and North/South American phylotypes. To investigate further the observed separation of strains into geographically distinct groups, we applied a cyanobacterium-specific short tandem repeat sequence technique, HIP1. An electrophoretic comparison of the HIP1 polymerase chain reaction products showed clear distinctions between the C. raciborskii strains. A phylogenetic tree, based on the repeat element banding patterns, also revealed three distinct groups of C. raciborskii strains. The first group consisted of strains from the USA and Brazil; the second comprised European strains from Germany, Hungary and Portugal; and the third were strains from Australia. In general, between-country variation was greater than within-country variation, indicating that this fingerprinting technique can successfully distinguish C. raciborskii strains taken from different global locations. The relationship between toxicity and the observed HIP1 polymerase chain reaction fingerprint profiles was less clear, although it is interesting to note that of the strains analysed in this study, only Australian strains are known to produce cylindrospermopsin and only Brazilian strains have been reported to produce paralytic shellfish poisoning toxins.

Alkaloids↗

Crystal structure of the tumor-promoter okadaic acid bound to protein phosphatase-1.

Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processes such as glycogen metabolism, cell cycle regulation, smooth muscle contraction, and protein synthesis. Microorganisms produce a variety of inhibitors of PP1, which include the microcystin class of inhibitors and okadaic acid, the latter being the major cause of diarrhetic shellfish poisoning and a powerful tumor promoter. We have determined the crystal structure of the molecular complex of okadaic acid bound to PP1 to a resolution of 1.9 A. This structure reveals that the acid binds in a hydrophobic groove adjacent to the active site of the protein and interacts with basic residues within the active site. Okadaic acid exhibits a cyclic structure, which is maintained via an intramolecular hydrogen bond. This is reminiscent of other macrocyclic protein phosphatase inhibitors. The inhibitor-bound enzyme shows very little conformational change when compared with two other PP1 structures, except in the inhibitor-sensitive beta12-beta13 loop region. The selectivity of okadaic acid for protein phosphatases-1 and -2A but not PP-2B (calcineurin) may be reassessed in light of this study.

Carcinogens↗