Using solid phase adsorption toxin tracking and extended local similarity analysis to monitor lipophilic shellfish toxins in a mussel culture ranch in the Yangtze River Estuary.

Harmful algal blooms (HABs) and their associated phycotoxins are increasing globally, posing great threats to local coastal ecosystems and human health. Nutrients have been carried by the freshwater Yangtze River and have entered the estuary, which was reported to be a biodiversity-rich but HAB-frequent region. Here, in situ solid phase adsorption toxin tracking (SPATT) was used to monitor lipophilic shellfish toxins (LSTs) in seawaters, and extended local similarity analysis (eLSA) was conducted to trace the temporal and special regions of those LSTs in a one-year trail in a mussel culture ranch in the Yangtze River Estuary. Nine analogs of LSTs, including okadaic acid (OA), dinophysistoxin-1 (DTX1), yessotoxin (YTX), homoyessotoxin (homoYTX), 45-OH-homoYTX, pectenotoxin-2 (PTX2), 7-epi-PTX2 seco acid (7-epi-PTX2sa), gymnodimine (GYM) and azaspiracids-3 (AZA3), were detected in seawater (SPATT) or rope farmed mussels. The concentrations of OA + DTX1 and homoYTX in mussels were positively correlated with those in SPATT samplers (Pearson test, p < 0.05), indicating that SPATT (with resin HP20) would be a good monitoring tool and potential indicator for OA + DTX1 and homoYTX in mussel Mytilus coruscus. The eLSA results indicated that late summer and early autumn were the most phycotoxin-contaminated seasons in the Yangtze River Estuary. OA + DTX1, homoYTX, PTX2 and GYM were most likely driven by the local growing HAB species in spring and summer, while Yangtze River diluted water may impact the accumulation of HAB species, causing potential phycotoxin contamination in the Yangtze River Estuary in autumn and winter. Together, the results showed that the mussel harvesting season, late summer and early autumn, would be the season with the greatest phycotoxin risk and would be the most contaminated by local growing toxic algae. Routine monitoring sites should be set up close to the local seawaters.

DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on nucleic acids.

Although ubiquitylation had traditionally been considered limited to proteins, the discovery of non-proteinaceous substrates (e.g. lipopolysaccharides and adenosine diphosphate ribose (ADPr)) challenged this perspective. Our recent study showed that DTX2 E3 ligase efficiently ubiquitylates ADPr. Here, we show that the ADPr ubiquitylation activity is also present in another DELTEX family member, DTX3L, analysed both as an isolated catalytic fragment and the full-length PARP9:DTX3L complex, suggesting that it is a general feature of the DELTEX family. Since structural predictions show that DTX3L possesses single-stranded nucleic acids binding ability and given the fact that nucleic acids have recently emerged as substrates for ADP-ribosylation, we asked whether DELTEX E3s might catalyse ubiquitylation of an ADPr moiety linked to nucleic acids. Indeed, we show that DTX3L and DTX2 are capable of ubiquitylating ADP-ribosylated DNA and RNA synthesized by PARPs, including PARP14. Furthermore, we demonstrate that the Ub-ADPr-nucleic acids conjugate can be reversed by two groups of hydrolases, which remove either the whole adduct (e.g. SARS-CoV-2 Mac1 or PARP14 macrodomain 1) or just the Ub (e.g. SARS-CoV-2 PLpro). Overall, this study reveals ADPr ubiquitylation as a general function of the DELTEX family E3s and presents the evidence of reversible ubiquitylation of ADP-ribosylated nucleic acids.

Identification of 24-O-ß-d-Glycosides and 7-Deoxy-Analogues of Okadaic Acid and Dinophysistoxin-1 and -2 in Extracts from Dinophysis Blooms, Dinophysis and Prorocentrum Cultures, and Shellfish in Europe, North America and Australasia.

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid (1) and dinophysistoxin-2 (2) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24-O-ß-d-glucosides of 1 and 2 (4 and 5, respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1, 2, dinophysistoxin-1 (3), and a synthetic specimen of 7-deoxy-1 (7), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1-5, the 24-O-ß-d-glucoside of dinophysistoxin-1 (6), 7, 7-deoxy-2 (8), and 7-deoxy-3 (9) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum lima cultures was also examined by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and was found to contain 1, 3, 7, and 9. However, although 4-6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.

Determination of lipophilic marine biotoxins by liquid chromatography-tandem mass spectrometry in five shellfish species from Washington State, USA.

Low extraction efficiency (60-81%) of okadaic acid (OA) and dinophysistoxin 1 (DTX1) was obtained for 4 out of 5 shellfish species from Washington State (WA), USA, during application of a standard extraction method for determination of lipophilic marine biotoxins by LC-MS/MS as recommended by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). OA and total OA including esters, DTX1, DTX2, and total DTX including esters, azaspiracid 1, 2, and 3 (AZA1, AZA2, and AZA3), pectenotoxin 2 (PTX2), and yessotoxin (YTX) were the toxins examined. Matrix-matched standards prepared from the same control samples used for spike-and-recovery tests were employed to evaluate toxin extraction efficiency and sample clean-up procedures. We adjusted the EURLMB extraction method by either using an acidified methanol extraction or pre-cooking shellfish homogenates at 70 °C for 20 min before EURLMB extraction. Extraction efficiency was improved markedly for OA and DTX1 with both modified methods and for YTX with the pre-cooking step included. However, recoveries were lower for YTX using the acidified methanol extraction and for PTX2 in non-mussel samples with the pre-cooking step. A hexane wash was applied to clean water-diluted non-hydrolyzed samples and a hexane wash was combined with solid-phase extraction for cleaning hydrolyzed samples. Improved sample clean-up, combined with LC-MS/MS adjustments, enabled quantification of U.S. Food and Drug Administration-regulated toxins in five shellfish species from WA with acceptable accuracy using non-matrix matched calibration standards.

DSP Toxin Distribution across Organs in Mice after Acute Oral Administration.

Okadaic acid (OA) and its main structural analogs dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) are marine lipophilic phycotoxins distributed worldwide that can be accumulated by edible shellfish and can cause diarrheic shellfish poisoning (DSP). In order to study their toxicokinetics, mice were treated with different doses of OA, DTX1, or DTX2 and signs of toxicity were recorded up to 24 h. Toxin distribution in the main organs from the gastrointestinal tract was assessed by liquid chromatography-mass spectrometry (LC/MS/MS) analysis. Our results indicate a dose-dependency in gastrointestinal absorption of these toxins. Twenty-four hours post-administration, the highest concentration of toxin was detected in the stomach and, in descending order, in the large intestine, small intestine, and liver. There was also a different toxicokinetic pathway between OA, DTX1, and DTX2. When the same toxin doses are compared, more OA than DTX1 is detected in the small intestine. OA and DTX1 showed similar concentrations in the stomach, liver, and large intestine tissues, but the amount of DTX2 is much lower in all these organs, providing information on DSP toxicokinetics for human safety assessment.

Risk Assessment of Pectenotoxins in New Zealand Bivalve Molluscan Shellfish, 2009-2019.

Pectenotoxins (PTXs) are produced by Dinophysis spp., along with okadaic acid, dinophysistoxin 1, and dinophysistoxin 2. The okadaic acid group toxins cause diarrhetic shellfish poisoning (DSP), so are therefore regulated. New Zealand currently includes pectenotoxins within the DSP regulations. To determine the impact of this decision, shellfish biotoxin data collected between 2009 and 2019 were examined. They showed that 85 samples exceeded the DSP regulatory limit (0.45%) and that excluding pectenotoxins would have reduced this by 10% to 76 samples. The incidence (1.3%) and maximum concentrations of pectenotoxins (0.079 mg/kg) were also found to be low, well below the current European Food Safety Authority (EFSA) safe limit of 0.12 mg/kg. Inclusion within the DSP regulations is scientifically flawed, as pectenotoxins and okadaic acid have a different mechanism of action, meaning that their toxicities are not additive, which is the fundamental principle of grouping toxins. Furthermore, evaluation of the available toxicity data suggests that pectenotoxins have very low oral toxicity, with recent studies showing no oral toxicity in mice dosed with the PTX analogue PTX2 at 5000 µg/kg. No known human illnesses have been reported due to exposure to pectenotoxins in shellfish, a fact which combined with the toxicity data indicates that they pose negligible risk to humans. Regulatory policies should be commensurate with the level of risk, thus deregulation of PTXs ought to be considered, a stance already adopted by some countries.

Improved Isolation Procedures for Okadaic Acid Group Toxins from Shellfish (Mytilus edulis) and Microalgae (Prorocentrum lima).

Okadaic acid (OA) group toxins may accumulate in shellfish and can result in diarrhetic shellfish poisoning when consumed by humans, and are therefore regulated. Purified toxins are required for the production of certified reference materials used to accurately quantitate toxin levels in shellfish and water samples, and for other research purposes. An improved procedure was developed for the isolation of dinophysistoxin-2 (DTX2) from shellfish (M. edulis), reducing the number of purification steps from eight to five, thereby increasing recoveries to ~68%, compared to ~40% in a previously reported method, and a purity of >95%. Cell densities and toxin production were monitored in cultures of Prorocentrum lima, that produced OA, DTX1, and their esters, over ~1.5 years with maximum cell densities of ~70,000 cells mL-1 observed. Toxin accumulation progressively increased over the study period, to ~0.7 and 2.1 mg L-1 of OA and DTX1 (including their esters), respectively, providing information on appropriate harvesting times. A procedure for the purification of OA and DTX1 from the harvested biomass was developed employing four purification steps, with recoveries of ~76% and purities of >95% being achieved. Purities were confirmed by LC-HRMS, LC-UV, and NMR spectroscopy. Additional stability observations led to a better understanding of the chemistry of these toxins.

Differences in Toxic Response Induced by Three Variants of the Diarrheic Shellfish Poisoning Phycotoxins in Human Intestinal Epithelial Caco-2 Cells.

Diarrheic shellfish poisoning (DSP) is caused by the consumption of shellfish contaminated with a group of phycotoxins that includes okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2). These toxins are inhibitors of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), but show distinct levels of toxicity. Aside from a difference in protein phosphatases (PP) inhibition potency that would explain these differences in toxicity, others mechanisms of action are thought to be involved. Therefore, we investigated and compared which mechanisms are involved in the toxicity of these three analogues. As the intestine is one of the target organs, we studied the transcriptomic profiles of human intestinal epithelial Caco-2 cells exposed to OA, DTX-1, and DTX-2. The pathways specifically affected by each toxin treatment were further confirmed through the expression of key genes and markers of toxicity. Our results did not identify any distinct biological mechanism for OA and DTX-2. However, only DTX-1 induced up-regulation of the MAPK transduction signalling pathway, and down-regulation of gene products involved in the regulation of DNA repair. As a consequence, based on transcriptomic results, we demonstrated that the higher toxicity of DTX-1 compared to OA and DTX-2 was consistent with certain specific pathways involved in intestinal cell response.

Response of fatty acids and lipid metabolism enzymes during accumulation, depuration and esterification of diarrhetic shellfish toxins in mussels (Mytilus galloprovincialis).

Bivalve mollusks accumulate diarrhetic shellfish toxins (DSTs) from toxigenic microalgae, thus posing a threat to human health by acting as a vector of toxins to consumers. In bivalves, free forms of DSTs can be esterified with fatty acids at the C-7 site to form acyl esters (DTX3), presumably a detoxification mechanism for bivalves. However, the effects of esterification of DSTs on fatty acid metabolism in mollusks remain poorly understood. In this study, mussels (Mytilus galloprovincialis) were fed the DST-producing dinoflagellate Prorocentrum lima for 10 days followed by an additional 10-days depuration in filtered seawater to track the variation in quantity and composition of DST acyl esters and fatty acids. A variety of esters of okadaic acid (OA) and dinophysistoxin-1 (DTX1) were mainly formed in the digestive gland (DG), although trace amounts of esters also appeared in muscle tissue. A large relative amount of OA (60%-84%) and DTX1 (80%-92%) was esterified to DTX3 in the visceral mass (referred to as digestive gland, DG), and the major ester acyl chains were C16:0, C16:1, C18:0, C18:1, C20:1 and C20:2. The DG and muscle tissues showed pronounced differences in fatty acid content and composition during both feeding and depuration periods. In the DG, fatty acid content gradually decreased in parallel with increasing accumulation and esterification of DSTs. The decline in fatty acids was accelerated during depuration without food. This reduction in the content of important polyunsaturated fatty acids, especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), would lead to a reduction in the nutritional value of mussels. Enzymes involved in lipid metabolism, including acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), lipoprotein lipase (LPL) and hepatic lipase (HL), were actively involved in the metabolism of fatty acids in the DG, whereas their activities were weak in muscle tissue during the feeding period. This study helps to improve the understanding of interactions between the esterification of DSTs and fatty acid dynamics in bivalve mollusks.

Dihydrodinophysistoxin-1 Produced by Dinophysis norvegica in the Gulf of Maine, USA and Its Accumulation in Shellfish.

Dihydrodinophysistoxin-1 (dihydro-DTX1, (M-H)-m/z 819.5), described previously from a marine sponge but never identified as to its biological source or described in shellfish, was detected in multiple species of commercial shellfish collected from the central coast of the Gulf of Maine, USA in 2016 and in 2018 during blooms of the dinoflagellate Dinophysis norvegica. Toxin screening by protein phosphatase inhibition (PPIA) first detected the presence of diarrhetic shellfish poisoning-like bioactivity; however, confirmatory analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) failed to detect okadaic acid (OA, (M-H)-m/z 803.5), dinophysistoxin-1 (DTX1, (M-H)-m/z 817.5), or dinophysistoxin-2 (DTX2, (M-H)-m/z 803.5) in samples collected during the bloom. Bioactivity-guided fractionation followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) tentatively identified dihydro-DTX1 in the PPIA active fraction. LC-MS/MS measurements showed an absence of OA, DTX1, and DTX2, but confirmed the presence of dihydro-DTX1 in shellfish during blooms of D. norvegica in both years, with results correlating well with PPIA testing. Two laboratory cultures of D. norvegica isolated from the 2018 bloom were found to produce dihydro-DTX1 as the sole DSP toxin, confirming the source of this compound in shellfish. Estimated concentrations of dihydro-DTX1 were >0.16 ppm in multiple shellfish species (max. 1.1 ppm) during the blooms in 2016 and 2018. Assuming an equivalent potency and molar response to DTX1, the authority initiated precautionary shellfish harvesting closures in both years. To date, no illnesses have been associated with the presence of dihydro-DTX1 in shellfish in the Gulf of Maine region and studies are underway to determine the potency of this new toxin relative to the currently regulated DSP toxins in order to develop appropriate management guidance.

Toxicity and differential oxidative stress effects on zebrafish larvae following exposure to toxins from the okadaic acid group.

Okadaic acid-group (OA-group) is a set of lipophilic toxins produced only in seawater by species of the Dinophysis and Prorocentrum genera, and characterized globally by being associated with harmful algal blooms (HABs). The diarrhetic shellfish poisoning toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1) are the most prevalent toxic analogues making up the OA-group, which jeopardize environmental safety and human health through consumption of hydrobiological organisms contaminated with these toxins that produce diarrhetic shellfish poisoning (DSP) syndrome in humans. Consequently, a regulatory limit of 160 µg of OA-group/kg was established for marine resources (bivalves). The aim of this study was to investigate effects varying concentrations of 1-15 µg/ml OA or DTX-1 on toxicity, development, and oxidative damage in zebrafish larvae (Danio rerio). After determining the lethal concentration 50 (LC50) in zebrafish larvae of 10 and 7 µg/ml (24 h) and effective concentration 50 (EC50) of 8 and 6 µg/ml (24 h), different concentrations (5, 6.5, or 8 µg/ml of OA and 4, 4.5, or 6 µg/ml of DTX-1) were used to examine the effects of these toxins on oxidative damage to larvae at different time points between 24 and 120 hpf. Macroscopic evaluation during the exposure period showed alterations in zebrafish including pericardial edema, cyclopia, shortening in the anteroposterior axis, and developmental delay. The activity levels of biochemical biomarkers superoxide dismutase (SOD) and catalase (CAT) demonstrated a concentration-dependent decrease while glutathione peroxidase (GPx) and glutathione reductase (GR) were markedly elevated. In addition, increased levels of oxidative damage (malondialdehyde and carbonyl content) were detected following toxin exposure. Data demonstrate that high concentrations of OA and DTX-1produced pathological damage in the early stages of development <48 h post-fertilization (hpf) associated with oxidative damage.

In vitro biotransformation of OA-group and PTX-group toxins in visceral and non-visceral tissues of Mytilus chilensis and Ameghinomya antiqua.

Lipophilic marine toxins (LMTs) are made up of multiple groups of toxic analogues, which are characterised by different levels of cellular and toxic action. The most prevalent groups in the southern Pacific zone are: a) okadaic acid group (OA-group) which consists of okadaic acid (OA) and dinophysistoxin-1 (DTX-1); and, b) pectenotoxin-2 (PTX2) group which consists of pectenotoxin-2 (PTX-2). The main objective of our study was to examine in vitro biotransformation of OA-group and PTX-group in the tissues of two endemic species of bivalves from southern Chile; blue mussels (Mytilus chilensis) and clams (Ameghinomya antiqua). The biotransformation processes of both groups were only detected in the digestive glands of both species using LC-MS/MS. The most frequently detected analogues were acyl derivatives (≈2.0 ± 0.1 µg ml-1) for OA-group and PTX-2SA (≈1.4 ± 0.1 µg ml-1) for PTX-group, with a higher percentage of biotransformation for OA-group (p < .001). In addition, simultaneous incubations of the different analogues (OA/PTX-2; DTX-1/PTX-2 and OA/DTX-1/PTX-2) did not show any interaction between the biotransformation processes. These results show that the toxicological variability of endemic species leads to biotransformation of the profile of toxins, so that these new analogues may affect people's health.

An okadaic acid fragment analogue prevents nicotine-induced resistance to cisplatin by recovering PP2A activity in non-small cell lung cancer cells.

We herein report the design, synthesis, and functional impact of an okadaic acid (OA) small analogue, ITH12680, which restores the activity of phosphoprotein phosphatase 2A (PP2A), whose deficient activity has been implicated in nicotine-mediated tumor progression and chemoresistance in non-small cell lung cancer (NSCLC). For its design, we paid attention to the structure of the PP2A-OA complex, where the C16-C38 OA fragment confers PP2A affinity and selectivity, but it is not involved in the inhibitory effect. Confirming this hypothesis, PP2A activity was not inhibited by ITH12680. By contrast, the compound partially restored OA-exerted PP2A inhibition in vitro. Moreover, flow cytometry and immunoblotting experiments revealed that ITH12680 reversed nicotine-induced cisplatin resistance in NSCLC cells, as it prevented nicotine-induced reduction of Bax expression and inhibited nicotine-mediated activation of cell survival and proliferation kinases, Akt and ERK1/2. Our findings suggest that the rescue of nicotine-inhibited PP2A activity could diminish the resistance to cisplatin treatment observed in NSCLC patients who continue smoking.

Toxins of Okadaic Acid-Group Increase Malignant Properties in Cells of Colon Cancer.

Diarrhetic shellfish poisoning (DSP) is a syndrome caused by the intake of shellfish contaminated with a group of lipophilic and thermostable toxins, which consists of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2). These toxins are potent protein Ser/Thr phosphatase inhibitors, mainly type 1 protein phosphatase (PP1) and type 2A protein phosphatase (PP2A). Different effects have been reported at the cellular, molecular and genetic levels. In this study, changes in cell survival and cell mobility induced by OA, DTX-1 and DTX-2 were determined in epithelial cell lines of the colon and colon cancer. The cell viability results showed that tumoral cell lines were more resistant to toxins than the nontumoral cell line. The results of the functional assays for testing cell migration, evaluation of cell death and the expression of proteins associated with cell adhesion showed a dual effect of toxins since in the nontumoral cell line, a greater induction of cell death, presumably by anoikis, was detected. In the tumoral cell lines, there was an induction of a more aggressive phenotype characterized by increased resistance to toxins, increased migration and increased FAK activation. In tumoral cell lines of colon cancer, OA, DTX-1/DTX-2 induce a more aggressive phenotype.

A Long-Term Time Series of Dinophysis acuminata Blooms and Associated Shellfish Toxin Contamination in Port Underwood, Marlborough Sounds, New Zealand.

Blooms of the dinoflagellate Dinophysis acuminata occur every year in an important mussel cultivation area in Port Underwood, Marlborough Sounds, New Zealand. Annual maximum cell numbers range from 1500⁻75,000 cells L-1 and over 25 years of weekly monitoring the D. acuminata bloom has never failed to exhibit peaks in abundance at some time between spring and autumn. During winter (June⁻August) the dinoflagellate is often undetectable, or at low levels (≤100 cells L-1), and the risk of diarrhetic shellfish poisoning (DSP)-toxin contamination over this period is negligible. Bloom occurrence may be coupled to the abundance of D. acuminata prey (Mesodinium sp.) but the mechanism by which it maintains its long-term residence in this hydrologically dynamic environment is unknown. The toxin profile of D. acuminata is dominated by pectenotoxin-2 (PTX-2) and dinophysistoxin-1 (DTX-1), but the cellular toxin content is low. It is rare that free DTX-1 is detected in mussels as this is invariably exclusively present as fatty acid-esters. In only five out of >2500 mussel samples over 16 years have the levels of total DTX-1 marginally exceeded the regulated level of 0.16 mg kg-1. It is also rare that free PTX-2 is detected in mussels, as it is generally only present in its hydrolysed non-toxic PTX-2 seco acid form. The D. acuminata alert level of 1000 cells L-1 is often exceeded without DTX-1 residues increasing appreciably, and this level is considered too conservative.

Toxin Profiles of Okadaic Acid Analogues and Other Lipophilic Toxins in Dinophysis from Japanese Coastal Waters.

The identification and quantification of okadaic acid (OA)/dinophysistoxin (DTX) analogues and pectenotoxins (PTXs) in Dinophysis samples collected from coastal locations around Japan were evaluated by liquid chromatography mass spectrometry. The species identified and analyzed included Dinophysis fortii, D. acuminata, D. mitra (Phalacroma mitra), D. norvegica, D. infundibulus, D. tripos, D. caudata, D. rotundata (Phalacroma rotundatum), and D. rudgei. The dominant toxin found in D. acuminata was PTX2 although some samples contained DTX1 as a minor toxin. D. acuminata specimens isolated from the southwestern regions (Takada and Hiroshima) showed characteristic toxin profiles, with only OA detected in samples collected from Takada. In contrast, both OA and DTX1, in addition to a larger proportion of PTX2, were detected in D. acuminata from Hiroshima. D. fortii showed a toxin profile dominated by PTX2 although this species had higher levels of DTX1 than D. acuminata. OA was detected as a minor toxin in some D. fortii samples collected from Yakumo, Noheji, and Hakata. PTX2 was also the dominant toxin found among other Dinophysis species analyzed, such as D. norvegica, D. tripos, and D. caudata, although some pooled picked cells of these species contained trace levels of OA or DTX1. The results obtained in this study re-confirm that cellular toxin content and profiles are different even among strains of the same species.

First identification of a C9-diol-ester of okadaic acid in Dinophysis acuta from Galician Rías Baixas (NW Spain).

Dinoflagellates of the genus Dinophysis produce okadaic acid (OA) and analogues. Cultures of a strain of Dinophysis acuta isolated from the Galician Rías contained high levels of pectenotoxins but little OA. Isomeric forms of the latter were suspected as the concentration of OA increased after alkaline hydrolysis. High resolution mass spectra of candidate compounds suggest the presence of an OA-C9-diol ester, reported for the first time in Dinophysis acuta.

Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis.

Ricin, Shiga toxin, exotoxin A, and diphtheria toxin are AB-type protein toxins that act within the host cytosol and kill the host cell through pathways involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. Intoxication is therefore viewed as an irreversible process. Using flow cytometry and a fluorescent reporter system to monitor protein synthesis, we show a single molecule of cytosolic toxin is not sufficient for complete inhibition of protein synthesis or cell death. Furthermore, cells can recover from intoxication: cells with a partial loss of protein synthesis will, upon removal of the toxin, increase the level of protein production and survive the toxin challenge. Thus, in contrast to the prevailing model, ongoing toxin delivery to the cytosol appears to be required for the death of cells exposed to sub-optimal toxin concentrations.

Characterization of the dinophysistoxin-2 acute oral toxicity in mice to define the Toxicity Equivalency Factor.

Ingestion of shellfish with dinophysistoxin-2 (DTX2) can lead to diarrheic shellfish poisoning (DSP). The official control method of DSP toxins in seafood is the liquid chromatography-mass spectrometry analysis (LC-MS). However in order to calculate the total toxicity of shellfish, the concentration of each compound must be multiplied by individual Toxicity Equivalency Factor (TEF). Considering that TEFs caused some controversy and the scarce information about DTX2 toxicity, the aim of this study was to characterize the oral toxicity of DTX2 in mice. A 4-Level Up and Down Procedure allowed the characterization of DTX2 effects and the estimation of DTX2 oral TEF based on determination of the lethal dose 50 (LD50). DTX2 passed the gastrointestinal barrier and was detected in urine and feces. Acute toxicity symptoms include diarrhea and motionless, however anatomopathology study and ultrastructural images restricted the toxin effects to the gastrointestinal tract. Nevertheless enterocytes microvilli and tight junctions were not altered, disconnecting DTX2 diarrheic effects from paracellular epithelial permeability. This is the first report of DTX2 oral LD50 (2262 µg/kg BW) indicating that its TEF is about 0.4. This result suggests reevaluation of the present TEFs for the DSP toxins to better determine the actual risk to seafood consumers.

Assimilation, Accumulation, and Metabolism of Dinophysistoxins (DTXs) and Pectenotoxins (PTXs) in the Several Tissues of Japanese Scallop Patinopecten yessoensis.

Japanese scallops, Patinopecten yessoensis, were fed with the toxic dinoflagellate Dinophysis fortii to elucidate the relative magnitude of assimilation, accumulation, and metabolism of diarrhetic shellfish toxins (DSTs) and pectenotoxins (PTXs). Three individual scallops were separately exposed to cultured D. fortii for four days. The average cell number of D. fortii assimilated by each individual scallop was 7.7 × 105. Dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2) and their metabolites were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS) and the toxin content in individual tissues (digestive gland, adductor muscle, gill, gonad, mantle, and the others), feces and the seawater medium were quantified. Toxins were almost exclusively accumulated in the digestive gland with only low levels being detected in the gills, mantles, gonads, and adductor muscles. DTX1 and PTX2 were the dominant toxins in the D. fortii cells fed to the scallops, whereas the dominant toxins detected in the digestive gland of scallops were PTX6 and esterified acyl-O-DTX1 (DTX3). In other tissues PTX2 was the dominant toxin observed. The ratio of accumulated to assimilated toxins was 21%-39% and 7%-23% for PTXs and DTXs respectively. Approximately 54%-75% of PTX2 and 52%-70% of DTX1 assimilated by the scallops was directly excreted into the seawater mainly without metabolic transformation.