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PubMed2004

Acute toxicity of gymnodimine to mice.

The acute toxicity of the phycotoxin gymnodimine to female Swiss mice by intraperitoneal injection and by oral administration has been determined. Gymnodimine was highly toxic by injection, the LD50 being only 96 microg/kg. Animals either died within 10 min of injection or made a full recovery with no perceptible long-term effects. Gymnodimine was also toxic after oral administration by gavage (LD50 755 microg/kg), but was much less toxic when administered with food. No signs of toxicity were seen in mice voluntarily ingesting food containing gymnodimine at a level sufficient to give a dose of approximately 7500 microg/kg. Pre-treatment with physostigmine or neostigmine protected against injected gymnodimine, suggesting that the latter exerts its toxic effects via blockade of nicotinic receptors at the neuromuscular junction. The low toxicity of gymnodimine when ingested with food suggests that this compound is of low risk to humans, a conclusion that is consonant with anecdotal evidence for the absence of harmful effects in individuals consuming shellfish contaminated with gymnodimine.

Administration, Oral↗

PubMed2004

Studies of polyether toxins in the marine phytoplankton, Dinophysis acuta, in Ireland using multiple tandem mass spectrometry.

Diarretic shellfish poisoning (DSP) is a toxic syndrome associated with the consumption of bivalve molluscs. The DSP toxins are polyether compounds, which include okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs) and pectenotoxin seco acids (PTX2SAs). These toxins originate in marine dinoflagellates, including Dinophysis spp. Phytoplankton samples were collected from the southwest coast of Ireland and D. acuta was the predominant species. Monocultures of D. acuta cells were prepared by hand picking from microscope slides in order to confirm their toxin profiles. There was a remarkable consistency in the toxin profiles in all of the phytoplankton samples collected during the summer months, irrespective of location, depth or mesh size. Analysis using liquid chromatography-multiple tandem mass spectrometry (LC-MS/MS) revealed that DTX2 and OA were the predominant toxins at a consistent ratio. The average toxin composition was: DTX2 (53+/-5%), OA (26.5+/-2.3%) and total pectenotoxins (20.8+/-4.7%). Toxin profiles in D. acuta from Europe were distinctly different from those found in New Zealand, where PTX2 was the predominant toxin and DTX2 was absent.

Animals↗

PubMed2004

Yessotoxins in Norwegian blue mussels (Mytilus edulis): uptake from Protoceratium reticulatum, metabolism and depuration.

The Protoceratium reticulatum cell density at Flodevigen reached a maximum of 2200 cells/L on 16 May 2001. The levels of yessotoxins (YTXs) in blue mussels (Mytilus edulis) at the same site increased sharply by 14 May and peaked on 28 May, after which they steadily declined. No other algal species present showed a similar pattern of correspondence. Together with the recent finding that Norwegian strains of P. reticulatum produce YTXs, these results indicate that P. reticulatum causes yessotoxin (YTX) contamination of shellfish in Norway, and that only relatively low cell densities are necessary for this to occur. The mussels from Flodevigen were analyzed by LC-MS for YTX, 45-hydroxyYTX, carboxyYTX, and a new yessotoxin believed to be 45-hydroxycarboxyYTX, and by ELISA for YTXs. The seasonal variations in toxin content versus time measured by the two methods were qualitatively very similar, although the response in the ELISA was 3-9 times higher due to the antibodies detecting other YTXs that were not detected by the LC-MS method. Changes in the LC-MS profile for YTXs, and in the ratio of YTXs by LC-MS to YTXs by ELISA with time, were consistent with extensive metabolism of YTX in the mussels. Kinetic analysis of the LC-MS data showed an initial half-life of 20 days for YTX, and for YTX+45-hydroxyYTX, in the mussels. Similar analysis of the ELISA data gave a half-life of 24 days for YTXs. The depuration rate remained consistent over a 3-month period during which the temperature remained at 13-16 degrees C.

Animals↗

PubMed2005

Ultrasensitive detection of domoic acid in mouse blood by competitive ELISA using blood collection cards.

Domoic acid (DA), an analog of the excitatory amino acid glutamate, is produced by the diatom genus Pseudo-nitzschia and acts as a neurotoxin in humans. During diatom blooms, DA can contaminate shellfish, as well as other filter feeding organisms, and can be transferred by ingestion to higher trophic levels, including marine mammals and humans. The prevalence of this algal toxin and its effects on protected species makes measurement of domoic acid in living animals a necessary biomonitoring tool for the near future. Blood collection cards have already been used for the sampling, extraction and detection of brevetoxin in blood from exposed laboratory animals and, more recently, marine mammals. However, a difficulty unique to measuring DA in blood is the rapid rate (>95% in 2h) at which it is cleared from blood. To meet this challenge, a direct competitive ELISA (cELISA), a method of detection with extremely high sensitivity and specificity, was used to analyze the blood of DA-exposed mice after extraction from the blood collection cards. More than 99% of DA was cleared from blood within 4h post dosage; however, domoic acid was still quantifiable (>0.7ngml(-1)) at 4h from blood spot extracts and still detectable at 24h when compared to control blood spots. By using this highly sensitive assay in conjunction with the use of blood spot cards for easy blood sample extraction, this method could be a very effective means of biomonitoring domoic acid in marine mammals in the field, as well as human populations.

Animals↗

PubMed2005

Use of two detection methods to discriminate ciguatoxins from brevetoxins: application to great barracuda from Florida Keys.

In Florida (USA), numerous cases of human ciguatera fish poisoning, as well as neurotoxic shellfish poisoning following consumption of local seafood products, have been reported. By using in parallel, the sodium channel receptor binding assay (RBA), and the ouabain/veratridine-dependent cytotoxicity assay (N2A assay), we established criteria to identify, detect, and quantify ciguatoxins in fish extracts, with a brevetoxin as internal standard. Results showed that the Caribbean ciguatoxin C-CTX-1 exhibited an 8-fold higher potency in the RBA than brevetoxins and, a 440 and 2300-fold higher potency in the N2A assay than PbTx-1 and PbTx-3, respectively. Moreover, a sensitivity comparison between assays revealed that the N2A assay was more sensitive (12-fold) for ciguatoxin analysis, whereas the RBA was more sensitive (3-24-fold) for brevetoxins analysis. Based on the relative potency between toxins and the opposite sensitivity of both assays we have used the RBA and the N2A assay to screen great barracuda (Sphyraena barracuda) collected from the Florida Keys for ciguatoxins and brevetoxins. Fish extract analysis showed a sodium channel-dependent activity consistent with the presence of ciguatoxins, and not brevetoxins. Among 40 barracudas analyzed, 60% contained ciguatoxin levels in their liver measurable by the N2A assay with the most toxic fish containing 2.1ppb C-CTX-1 equivalents.

Animals↗

PubMed2005

Comparative study on differential accumulation of PSP toxins between cockle (Acanthocardia tuberculatum) and sweet clam (Callista chione).

At the western Mediterranean coast of Morocco, the cockle (Acanthocardia tuberculatum) contained persistent high levels of paralytic shellfish toxins for several years, while other bivalve molluscs such as sweet clam (Callista chione) from the same vicinity were contaminated seasonally to a much lesser extent. In order to understand the causes of this prolonged contamination, a comparative study on PSP decontamination between sweet clam and cockle was conducted from November 2001 until June 2002. PSP toxicity was analysed by automated pre-column oxidation (Prechromatographic oxidation and LC-FD) in several organs of both species, namely digestive gland, foot, gill, mantle, muscle and siphon for sweet clams. The results showed that cockle sequester PSP toxins preferably in non-visceral organs (Foot, gill and mantle) contrary to sweet clam that sequester them in visceral tissues (digestive gland). The toxin profile of cockle organs indicated dominance of dcSTX, whereas sweet clam tissues contained especially C-toxins. Substantial differences in toxin profile between cockle and sweet clam, from the same area as well as from the composition of PSP toxin producer, Gymnodinium catenatum, confirm the bioconversion of PSP toxins in cockle.

Animals↗

PubMed2006

Differential dynamics of dinophysistoxins and pectenotoxins, part II: offshore bivalve species.

Different dinophysistoxin's profiles have been found repeatedly amongst some offshore bivalve molluscs. Species such as the clam, Spisula solida, esterify dinophysistoxins to a great extent, and contain always more okadaic acid (OA) than dinophysistoxin-2 (DTX2). In contrast, the clam Donax trunculus has a much higher percentage of non-esterified toxins, and often contains more DTX2 than OA. A detoxification experiment with D. trunculus and S. solida showed that the higher percentage of DTX2 in Donax was due to this toxin being eliminated more slowly than OA. The ester analogues of OA and DTX2 were eliminated faster than free OA and free DTX2 in D. trunculus. As D. trunculus esterifies OA to a greater extent than DTX2, the greater proportion of the free form of DTX2 explains why there is a gradual increase in total DTX2 over time. This slow elimination of free toxins contributes to wild D. trunculus specimens being, on average, six times more toxic than S. solida specimens on the Portuguese south coast. The commercial shellfish species more often monitored along the coast of Portugal between 2003 and 2004 (eight species in total) were examined for DTX2 content. The maximal percentage of total DTX2 in the total DSP toxins (OA + DTX2) was 40% in all species (clams, razors, cockles, oysters) except D. trunculus and Mytilus galloprovinciallis, in which it reached 70-90%. This phenomenon is seen only when contamination is due to the microalga Dinophysis acuta. The other important DSP-producer, Dinophysis acuminata, causes contamination only with OA. Current data suggest that D. acuta along the Portuguese coast always produces OA and DTX2 in a fixed ratio of 60:40. When this alga is dominant, contamination of bivalves occurs in a OA/DTX2 ratio of 60:40. In bivalves in which these toxins are esterified to a great extent, this profile is maintained due to the parallel elimination of OA and DTX2 esters at similar rates. However, in species with lower esterification, the relative proportion of OA and DTX2 rapidly decreases due to the selective retention of free DTX2. Elimination of pectenotoxin-2 seco acids (PTX2sa), the main pectenotoxin form found in both offshore species studied, followed an exponential decay. The half-lives of PTX2sas found in D. trunculus and S. solida were similar to that found in a previous study with the cockle, Cerastoderma edule, but longer than found in the blue mussel M. galloprovinciallis.

Animals↗

PubMed2006

Tissue distribution and effects of heat treatments on the content of domoic acid in blue mussels, Mytilus edulis.

The effect of heat treatment on domoic acid (DA) content in soft tissues of mussels Mytilus edulis was investigated using high performance liquid chromatography. DA concentrations in whole flesh, hepatopancreas and tissue remainder were measured in fresh, steamed and autoclaved mussel flesh. Relative decreases in DA and tissue fluid following heat treatments of whole flesh were similar resulting in approximately equal concentrations of DA pre- and post-treatment. DA concentration decreased in the hepatopancreas and increased in tissue remainder suggesting some organ disruption of mussels during heat treatment. These findings suggest that heat treatments using either conventional steaming or autoclaving at 121 degrees C are not appropriate techniques to reduce DA in mussels during commercial processing. We also conclude that sample pre-treatment has a minimal effect on the result of a DA analysis on whole mussel tissues. The stability of DA at different temperatures within a shellfish matrix was separately tested. Reductions in DA concentration (ca. 3-7%) compensate for some of the discrepancies between what was found in the cooking fluids in the initial study and what was expected based on the whole flesh concentration of the uncooked material.

Animals↗

PubMed2006

Toxin profile of Alexandrium ostenfeldii (Dinophyceae) from the Northern Adriatic Sea revealed by liquid chromatography-mass spectrometry.

This paper reports on the first occurrence of fairly high numbers of Alexandrium ostenfeldii along the Emilia Romagna coasts (Italy). Detailed liquid chromatography-mass spectrometry (LC-MS) analyses of the toxin profile were performed on a strain of the organism collected in November 2003, isolated during the event and grown in culture. Selected ion monitoring (SIM) and multiple reaction monitoring (MRM) experiments were carried out for detection of spirolides and paralytic shellfish poisoning (PSP) toxins. They revealed that the Adriatic A. ostenfeldii produces mainly spirolide 13-desmethyl C at levels of 3.7 pg/cell but not PSP toxins. Interestingly, low levels of some spirolide isomers that have not been reported so far in other strains of the dinoflagellate were also detected. This represents the first report of spirolide-type toxins in the Adriatic Sea.

Animals↗

PubMed2006

DTX5c, a new OA sulphate ester derivative from cultures of Prorocentrum belizeanum.

Prorocentrum belizeanum is a dinoflagellate known for its okadaic acid (OA) and dinophysitoxins (DTXs) production, both OA and DTX are polyether toxins of the Diarrhetic Shellfish Poisoning (DSP) group. We have recently published the isolation of a new diol-ester of okadaic acid from cultures of P. belizeanum. On this occasion we present a new sulphated water-soluble derivative, DTX-5c, isolated from this microalga, whose structure was established on the basis of its spectroscopical data.

Animals↗

PubMed2006

Detection of harmful cyanobacteria and their toxins by both PCR amplification and LC-MS during a bloom event.

We briefly report here the occurrence of toxic blooms in the eutrophic reservoir Billings, São Paulo city, Brazil. Water samples were collected in May 2004, during a cyanobacterial bloom. The presence of toxic species was confirmed by using PCR amplifications of a fragment region of genes encoding microcystin synthetase-mcyB. The determination of toxins was performed by liquid chromatography coupled with mass spectrometry (LC-MS). LC-MS analyses of the toxins from the bloom revealed variants of microcystins (MC), such as MC-LR, MC-RR and MC-YR. HPLC-FLD was used to determine the paralytic shellfish poisoning (PSP) saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 (GTX2) and 3 (GTX3). GTX2, GTX3 and NEO were detected for the first time in a natural sample from Billings reservoir. These results are a contribution to the knowledge of the biogeography of toxic cyanobacteria and their toxins, specifically in São Paulo.

Bacterial Toxins↗

PubMed2006

Accumulation and depuration profiles of PSP toxins in the short-necked clam Tapes japonica fed with the toxic dinoflagellate Alexandrium catenella.

A toxic dinoflagellate responsible for paralytic shellfish poisoning (PSP), Alexandrium catenella (Ac) was fed to the short-necked clam Tapes japonica, and the accumulation and depuration profiles of PSP toxins were investigated by means of high-performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD). The short-necked clams ingested more than 99% of the Ac cells (4 x 10(7)cells) supplied once at the beginning of experiment, and accumulated a maximal amount of toxin (185 nmol/10 clams) after 12h. The rate of toxin accumulation at that time was 23%, which rapidly decreased thereafter. Composition of the PSP toxin accumulated in the clams obviously different from that of Ac even 0.5h after the cell supply, the proportion of C1+2 being much higher than in Ac, although the reason remains to be elucidated. In contrast, a higher ratio of gonyautoxin (GTX)1+4 than in Ac was detected in the toxin profiles of clam excrements. The variation in toxin composition derived presumably from the transformation of toxin analogues in clams was observed from 0.5h, such as reversal of the ratio of C1 to C2, and appearance of carbamate (saxitoxin (STX), neoSTX and GTX2, 3) and decarbamoyl (dc) derivatives (dcSTX and dcGTX2, 3), which were undetectable in Ac cells. The total amount of toxin distributed over Ac cells, clams and their excrements gradually declined, and only 1% of supplied toxin was detected at the end of experiment.

Animals↗

PubMed2006

The marine biologist--Bob Endean.

Bob Endean was a dedicated marine biologist with an extensive knowledge of coral reef communities in the Great Barrier Reef and fauna in subtropical Queensland waters. He commenced a study of venomous and poisonous marine animals dangerous to man at a time when the field was new, employing a variety of techniques to investigate the venom apparatus, mode of delivery of venom or toxin, mode of toxic action on excitable tissues, and biochemistry of venom or toxin. Determination of the pharmacological properties of crude venom from Conus marine snails advanced characterization of conotoxins by later workers. A study of four types of nematocysts from the box-jellyfish Chironex fleckeri provided information as to their structure, function, and mechanism of discharge; myotoxins T1 and T2 were isolated from microbasic mastigophores. Endean studied poisonous stonefish (Synanceia trachynis) and, with Ann Cameron, scorpionfish (Notesthes robusta); investigations of ciguatera and of paralytic shellfish poisoning were initiated. He organized the collection of Australian frogs which led to the isolation of caerulein by Erspamer in Italy. Endean highlighted the ecological danger of the population explosion of the crown-of-thorns starfish (Acanthaster planci) and provided the impetus for the creation of the Great Barrier Reef Marine Park.

Animals↗

PubMed2006

Comparative effects of the toxic dinoflagellate Karenia brevis on clearance rates in juveniles of four bivalve molluscs from Florida, USA.

The effects of Karenia brevis (Gymnodiniales, Gymnodiniaceae) on the feeding activity of juveniles of four species of bivalve mollusc were examined in the laboratory to assess the potential impacts on these important shellfish populations from Florida. Clearance rates were determined under short-term (one hour) static and long-term (two days) flow-through conditions using both whole and lysed cultures of K. brevis. Under short-term conditions, the bay scallop, Argopecten irradians, was the most sensitive species, exhibiting a 79% reduction in clearance rate at 1000 cells ml(-1) of whole K. brevis culture compared to the control (no K. brevis). The eastern oyster, Crassostrea virginica, was the least responsive, showing a 38% reduction in clearance rate between the same treatments. The green mussel, Perna viridis, and the northern quahog, Mercenaria mercenaria, displayed intermediate responses. Similar results were also observed during long-term exposures to a continuous supply of K. brevis. Bay scallops showed a significant decline in clearance rate at 100 cells ml(-1) after 24h exposure; clearance rate of oysters was not affected by K. brevis at this concentration. No mortality was observed for any species during these brief exposures. The prospect for recovery of bay scallop populations in Florida estuaries where they were once abundant may be hampered by recurring blooms of K. brevis. Reduced clearance rates in M. mercenaria at high K. brevis densities could translate into poor growth of cultured Florida hard clams. On the other hand, P. viridis, which also showed reduced clearance rates at high K. brevis concentrations, might be negatively impacted by K. brevis blooms, thereby affecting their ability to spread into estuaries hampered by recurring toxic algal blooms.

Analysis of Variance↗

PubMed2005

Thermal tolerance of the invasive oyster Crassostrea gigas: feasibility of heat treatment as an antifouling option.

Pacific oysters, Crassostrea gigas are traditionally considered shellfish of great fishery and aquaculture value. For these reasons they are introduced worldwide. Recently there has been increasing reports about the prevalence of C. gigas as biofouling organism in cooling water systems. In the absence of relevant data on the susceptibility of oysters to commonly employed antifouling techniques such as heat treatment, it was presumed that oysters would be controlled by treatment programmes directed against other major fouling organisms. The present study was carried out to test the above hypothesis, and results showed that C. gigas has an upper temperature tolerance that is much higher than other major marine fouling animals including blue mussel Mytilus edulis. Apparently, temperature regimes presently used in heat treatment of cooling water systems fouled by mussels need to be increased, if C. gigas are to be controlled effectively. Our results also indicate that previous exposure of C. gigas to sublethal high temperatures could make them more resistant to subsequent thermal treatment, an aspect that should be taken into account when heat treatment is used as a fouling control option against oyster fouling.

Animals↗

PubMed2004

An intervention analysis for the reduction of exposure to methylmercury from the consumption of seafood by women of child-bearing age.

A previously developed exposure model was used [Risk Anal. 22 (2002) 689] to assess the effectiveness of various advisory scenarios on minimizing mercury (Hg) blood levels via the consumption of commercial seafood, both finfish and shellfish. This exposure model was developed to predict levels of Hg in blood in women of child-bearing age in the US based on the frequency of seafood consumption, the amount of seafood consumed per serving, and the types of seafood consumed. Steady-state relationships that employed descriptive statistics to account for toxicokinetic variation were used to predict levels of Hg in blood. The model incorporates an uncertainty dimension that is intended to represent the range of plausible interpretations of the data. The predictability of the model was confirmed via the use of National Health and Nutrition Examination Survey (NHANES) blood Hg data. In the present analysis, the model was used to predict the impact of limitations in the amount or types of seafood consumed on blood Hg levels. Specifically, simulations for various advisory scenarios were developed on the basis of limitations on total consumption of seafood, elimination of the consumption of certain species altogether, and/or a combination of both. In the baseline model, the median (uncertainty) estimates for the 50th, 95th, and 99th per capita population percentiles were 1.25, 8.2, and 16.1 ppb blood Hg, respectively. After restriction of seafood consumption to no more than 12 oz/week, the median (uncertainty) estimates for the 50th, 95th, and 99th per capita population percentiles were 1.22, 6.8, and 10.6 ppb blood Hg, respectively. Elimination of MeHg species, with average concentrations above 0.6 ppm, resulted in very modest decrements in Hg blood levels, in comparison to either the baseline or the reduced consumption scenarios. These results suggest that strategies to reduce MeHg exposure by reducing the amount of fish consumed (e.g., 12 oz/week) are more effective at eliminating the high end of the exposure distribution than are strategies intended to change the types of fish consumed.

Adult↗

PubMed2003

New insights into the role of the adenosine triphosphate-binding cassette transporters in high-density lipoprotein metabolism and reverse cholesterol transport.

Four adenosine triphosphate-binding cassette (ABC) transporters-ABCA1, ABCG1, ABCG5, and ABCG8-have been identified and shown to modulate cholesterol and lipoprotein metabolism. Recent analyses of ABCA1 indicate that upregulation of ABCA1 in the liver and macrophages of transgenic mice is associated with increased plasma high-density lipoprotein (HDL) cholesterol levels, increased net flux of cholesterol to the liver, and reduced diet-induced atherosclerosis. In ABCA1 transgenic mice, the enhanced expression of hepatic ABCA1 transporters is associated with increased plasma HDL cholesterol levels, suggesting that the liver plays an important role in the levels of plasma HDL cholesterol. Overexpression of ABCG1 in the liver of mice using recombinant ABCG1 vectors results in decreased plasma HDL levels and indicates that ABCG1 can modulate plasma lipoprotein levels in vivo. The potential importance of ABCG1 in reverse cholesterol transport has not been definitively established. Studies in patients with sitosterolemia have identified 2 major new transporters, ABCG5 and ABCG8, that play a pivotal role in the regulation of intestinal cholesterol, plant, and shellfish absorption. Modulation of the expression of ABCG5 and ABCG8 represents an important new mechanism in the regulation of cholesterol absorption in the intestine. The ABC transporters currently represent excellent targets for the development of new drugs for the treatment of patients with increased risk of premature cardiovascular disease.

ATP-Binding Cassette Transporters↗

PubMed2001

Modulation of cytosolic calcium levels of human lymphocytes by yessotoxin, a novel marine phycotoxin.

Yessotoxin (YTX) is a polyether toxin of marine origin that has been classified among the diarrheic shellfish poisoning (DSP) toxins group due to its lipophilic nature. However, unlike other DSP toxins, YTX does not produce diarrhea and its mechanisms of action are unknown. We studied the effect of YTX on the cytosolic calcium levels of freshly isolated human lymphocytes by means of fluorescence imaging microscopy. We showed that YTX produced a calcium influx through nifedipine and SKF 96365 (1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenyl]-1H-imidazole hydrochloride)-sensitive channels. This Ca2+ entry was not affected by the DSP toxin okadaic acid, which inhibits protein phosphatases. In addition, YTX also produced an inhibition of capacitative calcium entry activated by thapsigargin or by preincubation in a Ca2+-free medium. This capacitative calcium entry was not sensitive to nifedipine. Furthermore, the inhibitory effect of YTX was dependent on the time of addition of the toxin. We suggest that YTX may interact with calcium channels in a way similar to that described for other polyether marine compounds such as brevetoxins and maitotoxin, although an involvement of other second messengers is also likely.

Biological Transport↗