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Samples of Mytilus galloprovincialis collected from shellfish aquaculture plans located in the Adriatic Sea were analysed for yessotoxin (YTX) by three methods, in vivo (Mouse Bioassay, MBA), in vitro (functional assay) and chemical test (Liquid Chromatography–Mass Spectrometry, LC–MS/MS). As YTX coexists with other phycotoxins in shellfish, namely the diarrhoetic shellfish poisoning, okadaic acid, dinophysistoxins, pectenotoxins, and azaspirazids, the MBA is not completely satisfactory because it is difficult to identify which toxin causes the death of the mice. So, the two other techniques were proposed to detect and quantify YTX and its analogues in order to avoid this problem. The global results showed no difference among the three methods and the correlation between the functional assay and LC–MS/MS was positive (Spearman r = 0.72). Both analytical methods demonstrated advantages; the functional assay is specific, very sensitive and correlates well with real toxicity, whereas LC–MS/MS is convenient because it allows the detection of YTX and some analogues which are currently included in the EU regulation. For this reason LC–MS/MS will become the official method starting 1st January 2015 ( Regulation 15/2011/EU ). Only four samples exceeded the current EU regulation limit of 1 mg of YTX equivalent kg −1 . However, all samples belonged to a monitoring program and they were not suitable for consumers.

Harmful algal blooms (HABs) occur nearly annually off the west coast of Florida and can impact both humans and wildlife, resulting in morbidity and increased mortality of marine animals including sea turtles. The key organism in Florida red tides is the dinoflagellate Karenia brevis that produces a suite of potent neurotoxins referred to as the brevetoxins (PbTx). Despite recent mortality events and rehabilitation efforts, still little is known about how the toxin directly impacts sea turtles, as they are not amenable to experimentation and what is known about toxin levels and distribution comes primarily from post-mortem data. In this study, we utilized the freshwater turtle Trachemys scripta and the diamondback terrapin, Malaclemys terrapin as model organisms to determine the distribution, clearance, and routes of excretion of the most common form of the toxin, brevetoxin-3, in turtles. Turtles were administered toxin via esophageal tube to mimic ingestion (33.48μg/kg PbTx-3, 3×/week for two weeks for a total of 7 doses) or by intratracheal instillation (10.53μg/kg, 3×/week for four weeks for a total of 12 doses) to mimic inhalation. Both oral and intratracheal administration of the toxin produced a suite of behavioral responses symptomatic of brevetoxicosis. The toxin distributed to all organ systems within 1h of administration but was rapidly cleared out over 24-48h, corresponding to a decline in clinical symptoms. Excretion appears to be primarily through conjugation to bile salts. Histopathological study revealed that the frequency of lesions varied within experimental groups with some turtles having no significant lesions at all, while similar lesions were found in a low number of control turtles suggesting another common factor(s) could be responsible. The overall goal of this research is better understand the impacts of brevetoxin on turtles in order to develop better treatment protocols for sea turtles exposed to HABs.

Massive algal proliferations known as Harmful Algal Blooms (HABs) represent one of the most important threats to coastal areas. Among them, the so-called Florida Red Tides (FRTs, caused by blooms of the dinoflagellate Karenia brevis and associated brevetoxins) are particularly detrimental in the southeastern U.S., causing high mortality rates and annual losses in excess of $40 million. The ability of marine organisms to cope with environmental stressors (including those produced during HABs) is influenced by genetic and epigenetic mechanisms, the latter resulting in phenotypic changes caused by heritable modifications in gene expression, without involving changes in the genetic (DNA) sequence. Yet, studies examining cause-effect relationships between environmental stressors, specific epigenetic mechanisms and subsequent responses are still lacking. The present work contributes to increase this knowledge by investigating the effects of Florida Red Tides on two types of mechanisms participating in the epigenetic memory of Eastern oysters: histone variants and DNA methylation. For that purpose, a HAB simulation was conducted in laboratory conditions, exposing oysters to increasing concentrations of K. brevis. The obtained results revealed, for the first time, the existence of H2A.X, H2A.Z and macroH2A genes in this organism, encoding histone variants potentially involved in the maintenance of genome integrity during responses to the genotoxic effect of brevetoxins. Additionally, an increase in H2A.X phosphorylation (γH2A.X, a marker of DNA damage) and a decrease in global DNA methylation were observed as the HAB simulation progressed. Overall, the present work provides a basis to better understand how epigenetic mechanisms participate in responses to environmental stress in marine invertebrates, opening new avenues to incorporate environmental epigenetics approaches into management and conservation programs.

Yessotoxin (YTX) is a marine phycotoxin produced by dinoflagellates and accumulated in filter feeding shellfish. YTX content in shellfish is regulated by many food safety authorities to protect human health, although currently no human intoxication episodes have been unequivocally related to YTX presence in food. The immune system has been proposed as one of the target organs of YTX due to alterations of lymphoid tissues and cellular and humoral components. The aim of the present study was to explore subacute immunotoxicity of YTX in rats by evaluating the haematological response, inflammatory cytokine biomarkers and the presence of YTX-induced structural alterations in the spleen and thymus. The results showed that repeated administrations of YTX caused a decrease of lymphocyte percentage and an increase of neutrophil counts, a reduction in interleukine-6 (IL-6) plasmatic levels and histopathological splenic alterations in rats after four intraperitoneal injections of YTX at doses of 50 or 70 μg/kg that were administered every 4 days along a period of 15 days. Therefore, for the first time, subacute YTX-immunotoxicity is reported in rats, suggesting that repeated exposures to low amounts of YTX might also suppose a threat to human health, especially in immuno-compromised populations.

Blooms of Karenia brevis (also called red tides) occur almost annually in the Gulf of Mexico. The health effects of the neurotoxins (i.e., brevetoxins) produced by this toxic dinoflagellate on marine turtles are poorly understood. Florida's Gulf Coast represents an important foraging and nesting area for a number of marine turtle species. Most studies investigating brevetoxin exposure in marine turtles thus far focus on dead and/or stranded individuals and rarely examine the effects in apparently "healthy" free-ranging individuals. From May-July 2014, one year after the last red tide bloom, we collected blood from nesting loggerhead sea turtles (Caretta caretta) on Casey Key, Florida USA. These organisms show both strong nesting and foraging site fidelity. The plasma was analyzed for brevetoxin concentrations in addition to a number of health and immune-related parameters in an effort to establish sublethal effects of this toxin. Lastly, from July-September 2014, we collected unhatched eggs and liver and yolk sacs from dead-in-nest hatchlings from nests laid by the sampled females and tested these samples for brevetoxin concentrations to determine maternal transfer and effects on reproductive success. Using a competitive enzyme-linked immunosorbent assay (ELISA), all plasma samples from nesting females tested positive for brevetoxin (reported as ng brevetoxin-3[PbTx-3] equivalents [eq]/mL) exposure (2.1-26.7ng PbTx-3eq/mL). Additionally, 100% of livers (1.4-13.3ng PbTx-3eq/mL) and yolk sacs (1.7-6.6ng PbTx-3eq/mL) from dead-in-nest hatchlings and 70% of eggs (

Harmful algal blooms are increasing in frequency and extent worldwide and occur nearly annually off the west coast of Florida where they affect both humans and wildlife. The dinoflagellate Karenia brevis is a key organism in Florida red tides that produces a suite of potent neurotoxins collectively referred to as the brevetoxins (PbTx). Brevetoxins bind to and open voltage gated sodium channels (VGSC), increasing cell permeability in excitable cells and depolarizing nerve and muscle tissue. Exposed animals may thus show muscular and neurological symptoms including head bobbing, muscle twitching, paralysis, and coma; large HABs can result in significant morbidity and mortality of marine life, including fish, birds, marine mammals, and sea turtles. Brevetoxicosis however is difficult to treat in endangered sea turtles as the physiological impacts have not been investigated and the magnitude and duration of brevetoxin exposure are generally unknown. In this study we used the freshwater turtle Trachemys scripta as a model organism to investigate the effects of the specific brevetoxin PbTx-3 in the turtle brain. Primary turtle neuronal cell cultures were exposed to a range of PbTx-3 concentrations to determine excitotoxicity. Agonists and antagonists of voltage-gated sodium channels and downstream targets were utilized to confirm the toxin's mode of action. We found that turtle neurons are highly resistant to PbTx-3; while cell viability decreased in a dose dependent manner across PbTx-3 concentrations of 100-2000nM, the EC50 was significantly higher than has been reported in mammalian neurons. PbTx-3 exposure resulted in significant Ca2+ influx, which could be fully abrogated by the VGSC antagonist tetrodotoxin, NMDA receptor blocker MK-801, and tetanus toxin, indicating that the mode of action in turtle neurons is the same as in mammalian cells. As both turtle and mammalian VGSCs have a high affinity for PbTx-3, we suggest that the high resistance of the turtle neuron to PbTx-3 may be related to its ability to withstand anoxic depolarization. The ultimate goal of this work is to design treatment protocols for sea turtles exposed to red tides worldwide.

ResumenEl aumento de la resistencia bacteriana a los antibióticos ha causado preocupación a nivel mundial, por lo que se ha promovido la búsqueda de nuevos compuestos. Debido a su abundancia y diversidad, el fitoplancton marino constituye una importante fuente potencial de tales compuestos. La investigación sobre dinoflagelados ha llevado al descubrimiento de inhibidores de crecimiento bacteriano. El dinoflagelado marino Lingulodinium polyedrum causa proliferaciones algales en diferentes regiones del mundo, incluyendo México, y también se sabe que regula el crecimiento de otras especies en las aguas costeras. En este trabajo, se investiga la taxonomía de este dinoflagelado y se caracteriza la capacidad de sus extractos para inhibir el crecimiento de dos bacterias de importancia médica (Vibrio vulnificus y Staphylococcus aureus) en placas de cultivo de agar. La caracterización taxonómica se realizó por PCR y amplificación del gen de ITS, y se confirmó que la especie aislada en la costa del Pacífico de México fue L. polyedrum. Para demostrar el efecto inhibidor de los extractos de L. polyedrum, los cultivos se cosecharon por centrifugación. Los pellets de tres abundancias celulares se extrajeron con agua, metanol, hexano y cloroformo. Los experimentos en V. vulnificus mostraron una inhibición alta del crecimiento para los cuatro extractos, variando entre 77 y 98 %. Sorprendentemente, la inhibición del crecimiento fue menor cuando los extractos se originaron a partir de una mayor abundancia de células L. polyedrum, varía de 0 a 34 %. Para S. aureus, la inhibición del crecimiento también fue alta, pero no estadísticamente diferente para todos los extractos y abundancias de células, con un rango de 62 hasta 99 %. Esto resultados son prometedores para futuras aplicaciones farmacológicas. La cepa mexicana de L. polyedrum no produjo yesotoxinas detectables. AbstractThe increased bacterial resistance to antibiotics has caused global concern, prompting the search for new compounds. Because of their abundance and diversity, marine phytoplankton are an important potential source of such compounds. Research on dinoflagellates has led to the discovery of inhibitors of bacterial growth. The marine dinoflagellate Lingulodinium polyedrum blooms in different regions of the world, including Mexico, and is also known to regulate the growth of other species in coastal waters. Here, we investigated the taxonomy of this dinoflagellate and characterized the ability of its extracts to inhibit the growth of two bacteria of medical importance (Vibrio vulnificus and Staphylococcus aureus). Taxonomic characterization was performed by PCR and gene amplification of ITS, and confirmed that the species isolated off the Pacific coast of Mexico was L. polyedrum. To prove the inhibitory effect of L. polyedrum extracts, cultures were harvested by centrifugation. Pellets from three cellular abundances were extracted with water, methanol, hexane and chloroform. The experiments on V. vulnificus showed a high growth inhibition for the four extracts, ranging from 77 to 98 %. Surprisingly, the growth inhibition was lower when the extracts originated from a higher L. polyedrum cell abundance, ranging from 0 to 34 %. For S. aureus, the growth inhibition was also high, but not statistically different for all extracts and cell abundances, ranging from 62 to 99 %. This study obtained promising results for future pharmacological applications. Our Mexican strain of L. polyedrum did not produce any detectable yessotoxins. Rev. Biol. Trop. 64 (2): 805-816. Epub 2016 June 01.

© 2016 Phycological Society of America Gambierdiscus is a genus of benthic dinoflagellates found worldwide. Some species produce neurotoxins (maitotoxins and ciguatoxins) that bioaccumulate and cause ciguatera fish poisoning (CFP), a potentially fatal food-borne illness that is common worldwide in tropical regions. The investigation of toxigenic species of Gambierdiscus in CFP endemic regions in Australia is necessary as a first step to determine which species of Gambierdiscus are related to CFP cases occurring in this region. In this study, we characterized five strains of Gambierdiscus collected from Heron Island, Australia, a region in which ciguatera is endemic. Clonal cultures were assessed using (i) light microscopy; (ii) scanning electron microscopy; (iii) DNA sequencing based on the nuclear encoded ribosomal 18S and D8-D10 28S regions; (iv) toxicity via mouse bioassay; and (v) toxin profile as determined by Liquid Chromatography-Mass Spectrometry. Both the morphological and phylogenetic data indicated that these strains represent a new species of Gambierdiscus, G. lapillus sp. nov. (plate formula Po, 3′, 0a, 7″, 6c, 7-8s, 5‴, 0p, 2″″ and distinctive by size and hatchet-shaped 2′ plate). Culture extracts were found to be toxic using the mouse bioassay. Using chemical analysis, it was determined that they did not contain maitotoxin (MTX1) or known algal-derived ciguatoxin analogs (CTX3B, 3C, CTX4A, 4B), but that they contained putative MTX3, and likely other unknown compounds.

International audience; The aim of this study was to investigate (i) the cytotoxic effects of lipophilic phycotoxins, including okadaic acid (OA) and dinophysistoxin-1 and -2 (DTX-1 and DTX-2), pectenotoxin-2 (PTX-2), yessotoxin (YTX), spirolide (SPX), and azaspiracids-1, -2 and -3 (AZA-1, AZA-2 and AZA-3), in human HepaRG cells using a multiparametric high content analysis approach, (ii) the ability of nine lipophilic phycotoxins to act as PXR agonists in a HepG2-PXR cell line, (iii) their potential to induce CYP450 activity, and (iv) the role of CYP3A4 in cytotoxicity induced by lipophilic phycotoxins. Our results indicate that while OA, DTX-1 and DTX-2 activated PXR-dependent transcriptional activity in HepG2 cells, no increase of CYP450 (1A2, 3A4, 2C9, 2C19) activities were observed in HepaRG cell following a 72h treatment with these toxins. Multiparametric analysis showed that OA, DTX-1, DTX-2, and PTX-2 were highly cytotoxic in HepaRG cells; inducing cell loss, activation of caspase-3 and gamma-H2AX formation. However, no toxicity was observed for YTX, SPX, and AZAs. Moreover, we found that inhibition of CYP3A4 activity by ketoconazole enhances the toxic effects of OA, DTX-1, DTX-2, and PTX-2 in HepaRG cells. Taken together, these results suggest that CYP3A4-mediated metabolism of some lipophilic phycotoxins decreases their in vitro toxicity.

International audience; Phycotoxins are monitored in seafood because they can cause food poisonings in humans. Phycotoxins do not only occur singly but also as mixtures in shellfish. The aim of this study was to evaluate the in vitro toxic interactions of binary combinations of three lipophilic phycotoxins commonly found in Europe (okadaic acid (OA), yessotoxin (YTX) and azaspiracid-1 (AZA-1)) using the neutral red uptake assay on two human intestinal cell models, Caco-2 and the human intestinal epithelial crypt-like cells (HIEC). Based on the cytotoxicity of individual toxins, we studied the interactions between toxins in binary mixtures using the combination index-isobologram equation, a method widely used in pharmacology to study drug interactions. This method quantitatively classifies interactions between toxins in mixtures as synergistic, additive or antagonistic. AZA-1/OA, and YTX/OA mixtures showed increasing antagonism with increasing toxin concentrations. In contrast, the AZA-1/YTX mixture showed increasing synergism with increasing concentrations, especially for mixtures with high YTX concentrations. These results highlight the hazard potency of AZA-1/YTX mixtures with regard to seafood intoxication.

Octacyclic polyethers that correspond to the CDEFGHIJ-ring system of yessotoxin as well as G- and/or I-ring-modified analogues were synthesized in a divergent manner, starting from a common intermediate, using an [X + 2 + Y]-type convergent method. Reaction of a triflate with the oxiranyl anion generated from an epoxy sulfone, followed by ring expansion, allowed for the incorporation of medium-sized ring ethers into the key intermediate. Subsequent acetal formation and reductive etherification afforded various octacycles containing seven- and eight-membered ether rings.

The occurrence and intensity of harmful algal blooms (HABs) have been increasing globally during the past few decades. The impact of these events on seawater desalination facilities has become an important topic in recent years due to enhanced societal interest and reliance on this technology for augmenting world water supplies. A variety of harmful bloom-forming species of microalgae occur in southern California, as well as many other locations throughout the world, and several of these species are known to produce potent neurotoxins. These algal toxins can cause a myriad of human health issues, including death, when ingested via contaminated seafood. This study was designed to investigate the impact that algal toxin presence may have on both the intake and reverse osmosis (RO) desalination process; most importantly, whether or not the naturally occurring algal toxins can pass through the RO membrane and into the desalination product. Bench-scale RO experiments were conducted to explore the potential of extracellular algal toxins contaminating the RO product. Concentrations exceeding maximal values previously reported during natural blooms were used in the laboratory experiments, with treatments comprised of 50 μg/L of domoic acid (DA), 2 μg/L of saxitoxin (STX) and 20 μg/L of brevetoxin (PbTx). None of the algal toxins used in the bench-scale experiments were detectable in the desalinated product water. Monitoring for intracellular and extracellular concentrations of DA, STX, PbTx and okadaic acid (OA) within the intake and desalinated water from a pilot RO desalination plant in El Segundo, CA, was conducted from 2005 to 2009. During the five-year monitoring period, DA and STX were detected sporadically in the intake waters but never in the desalinated water. PbTx and OA were not detected in either the intake or desalinated water. The results of this study demonstrate the potential for HAB toxins to be inducted into coastal RO intake facilities, and the ability of typical RO operations to effectively remove these toxins.

Yessotoxin (YTX) and okadaic acid (OA), algal toxins accumulated in edible shellfish, were previously shown to induce a specific and reversible T Cell Receptor (TCR) down-regulation in T lymphocyte EL-4 cells, in a time and concentration-dependent manner, via protein kinase C (PKC) and serine/threonine protein phosphatase 2A (PP2A) activities. In this study we have evaluated the development of other signs of toxicity induced by low concentrations of YTX or OA for 3 days of treatment. Concentrations of YTX as low as 1 nM decreased a 35% the concentration of viable cells after 48 h exposure to the toxin, while concentrations as little as 5 nM YTX or OA were sufficient to induce membrane blebbing. The concentration of YTX that produced after 24 h of incubation a 50% reduction in maximum cell viability (EC50₂₄) was approximately 46 nM, whereas with OA over 75% of the cells were still viable after exposure to 100 nM OA. According to our results, the cytoskeleton of EL-4 cells seems to be a cell component particularly sensitive to YTX and OA with disruption of F-actin cytoskeleton in these cells treated with concentrations of YTX or OA as low as 5 nM at 48 h incubation. Toxicity by YTX or OA involved typical hallmarks of apoptosis and an increase of reactive oxygen species (ROS) production. The cytotoxic effects of YTX and OA reported here, and the previously demonstrated potential of these toxins to regulate the activity of EL-4 cells through the regulation of TCR expression, rise reasonable concern about possible risks for human health associated to the chronic exposure to low amounts of YTX or OA itself or enhanced by the presence of other shellfish toxins specially by a population potentially at risk such as immunocompromised patients.

Yessotoxin (YTX) is a marine polyether toxin previously described as a phosphodiesterase (PDE) activator in fresh human lymphocytes. This toxin induces a decrease of adenosine 3′,5′-cyclic monophosphate (cAMP) levels in fresh human lymphocytes in a medium with calcium (Ca2+), whereas the contrary effect has been observed in a Ca2+-free medium. In the present article, the effect of YTX in K-562 lymphocytes cell line has been analysed. Surprisingly, results obtained in K-562 cell line are completely opposite than in fresh human lymphocytes, since in K-562 cells YTX induces an increase of cAMP levels. YTX cytotoxicity was also studied in both K-562 cell line and fresh human lymphocytes. Results demonstrate that YTX does not modify fresh human lymphocytes viability, whereas in K-562 cells, YTX has a highly cytotoxic effect. It has been described in a previous study that YTX induces a small cytosolic Ca2+ increase in fresh human lymphocytes but no effect was observed on Ca2+ pools depletion in these cells. However, our results show that, in K-562 cells, YTX has no effect on cytosolic Ca2+ levels in a medium with Ca2+ and induces an increase on Ca2+ pools depletion followed by a Ca2+ influx. As far as Ca2+ modulation is concerned these results demonstrate that YTX has a clear opposite effect in tumoural and fresh human lymphocytes. In addition, intracellular Ca2+ reservoirs affected by YTX are different than thapsigargin-sensible pools. Furthermore, YTX-dependent Ca2+ pools depletion was abolished by cAMP analogue (dibutyryl cAMP), phosphodiesterase-4 (PDE4) inhibitor (rolipram), protein kinase A inhibitor (H89) and oxidative phosphorylation uncoupler carbonyl cyanide p-(trifluoromethoxy) (FCCP) treatments. This evidences the crosstalks between Ca2+, YTX and cAMP pathways. Also, results obtain demonstrate that YTX-dependent Ca2+ influx was only abolished by FCCP pre-treatment, which indicates a link between YTX and mitochondria in K-562 cell line. Cytosolic expression of A-kinase anchor proteins (AKAPs), the proteins which integrates phosphodiesterases (PDEs) and PKA to the mitochondria, was determined in both cell models. On the one hand, in human fresh lymphocytes, YTX increases AKAP149 cytosolic expression. This fact is accompanied with a decrease in cAMP levels, and therefore PDEs activation, which finally leads to cell survival. On the other hand, in tumoural lymphocytes, YTX has an opposite effect since decreases AKAP149 cytosolic expression and increase cAMP levels which leads to cell death. This is the first time that YTX and mitochondrial AKAPs proteins relationship is characterised. J. Cell. Biochem. 113: 3752–3761, 2012. © 2012 Wiley Periodicals, Inc.

A facile and feasible magneto-controlled immunosensing platform was designed for sensitive electrochemical immunoassay of brevetoxin B (BTX-2) in seafood by using guanine-assembled graphene nanoribbons (GGNRs) as molecular tags on a home-made magnetic carbon paste electrode. Initially, monoclonal mouse anti-BTX-2 antibodies were covalently immobilized on the surface of magnetic beads, which were used as the immunosensing probes for the capture of BTX-2. The recognition elements were prepared by chemical modification of bovine serum albumin-BTX-2 conjugates (BTX-2-BSA) with the GGNRs. Based on a competitive-type immunoassay format, the formed magnetic immunocomplex was integrated on the electrode with an external magnet, followed by determination in pH 6.5 phosphate-buffered solution containing 2 μM Ru(bpy) 3 Cl 2 . Under optimal conditions, the electrochemical signals decreased with the increasing BTX-2 concentrations in the sample. The dynamic concentration range spanned from 1.0 pg mL −1 to 10 ng mL −1 with a detection limit of 1.0 pg mL −1 BTX-2. Inter- and intra-batch assay precisions were substantially improved by resorting to the GGNR manifold. The method featured unbiased identification of negative (blank) and positive samples. No significant differences at the 95% confidence level were encountered in the analysis of 12 spiked samples including S. constricta, M. senhousia and T. granosa between the electrochemical immunoassay and commercially available enzyme-linked immunosorbent assay (ELISA) for determination of BTX-2.

More than 200 people in China suffered illness with symptoms of diarrhetic shellfish poisoning (DSP) following consumption of mussels (Mytilus galloprovincialis). The event occurred in the cities of Ningbo and Ningde near the East China Sea in May, 2011. LC-MS/MS analysis showed that high concentrations of okadaic acid, dinophysistoxin-1, and their acyl esters were responsible for the incidents. The total concentration was more than 40 times the EU regulatory limit of 160 μg OA eq./kg. Pectentoxin-2 and its seco-acids were also present in the mussels. Additionally, yessotoxins were found to be responsible for positive mouse bioassay results on scallop (Patinopecten yessoensis) and oyster (Crassostrea talienwhanensis) samples collected from the North Yellow Sea in June, 2010. This work shows that high levels of lipophilic toxins can accumulate in shellfish from the Chinese coast and it emphasises that adequate chemical analytical methodologies are needed for monitoring purposes. Further research is required to broaden the knowledge on the occurrence of lipophilic toxins in Chinese shellfish.

This work proposes to use the marine algal toxin yessotoxin (YTX) to establish reference model experiments to explore medically valuable effects from induction of multiple cell death pathways. YTX is one of few toxins reported to make such induction. It is a small molecule compound which at low concentrations can induce apoptosis in primary cultures, many types of cells and cell lines. It can also induce a non-apoptotic form of programmed cell death in BC3H1 myoblast cell lines. The present contribution reviews arguments that this type of induction may have principal interest outside this particular example. One principal effect of medical interest may be that cancer cells will not so easily adapt to the synergistic effects from induction of more than one death pathway as compared to induction of only apoptosis.

A single-laboratory validation is reported for an LC/MS/MS quantification of six brevetoxins in four matrixes (GreenshellTM mussel, eastern oyster, hard clam, and Pacific oyster). Recovery and precision data were collected from seven analytical batches using shellfish flesh at 0.05 mg/kg. Method recoveries and within-laboratory reproducibility ranged from 73 to 112%, with an RSD between 14 and 18% for brevetoxin-3, brevetoxin B5, brevetoxin B2, and S-desoxy brevetoxin B2. The recovery and within-laboratory reproducibility for brevetoxin-2 was 61%, with an RSD of 27%. Brevetoxin B1 gave an RSD of 12%, but no reference material was available and this toxin was only recorded in a hard clam sample naturally contaminated with brevetoxins. One naturally contaminated sample of each shellfish matrix, with brevetoxin levels ranging from 0.012 to 9.9 mg/kg, was tested in multiple batches, and the RSDs were similar to those for fortified samples at 0.05 mg/kg. Comparisons with limited data for the neurotoxic shellfish poisoning mouse bioassay for four naturally contaminated shellfish samples showed that the regulatory action limit of 0.8 mg/kg is conservative with respect to the bioassay regulatory limit of 20 mouse units/100 g.

This study shows that BC3H1 myoblast cell lines exposed to 100 nM yessotoxin (YTX) undergo a form of programmed cell death distinct from apoptosis and with features resembling paraptosis. Morphologically, cells treated with YTX reveal extensive cytoplasmic vacuolation, mitochondrial and endoplasmic reticulum swelling, uncondensed chromatin and cytoskeletal alterations. DNA electrophoresis evidences lack of DNA fragmentation and Western blotting analysis demonstrates activation of the mitogen-activated protein kinase JNK/SAPK1. Further characterisation of this form of programmed cell death may have interest within medicine and cancer therapy.

Although brevetoxins (PbTxs) produced by the marine dinoflagellate Karenia brevis are known to be absorbed across gill membranes and exert their acute toxic effects through an ion-channel mediated pathway in neural tissue, the exact biochemical mechanism concerning PbTxs neurotoxicity in neural tissue and gas-exchange organs has not been well elucidated. In this study, we calculated the LC(50) value of PbTx-1 using the medaka fish model, and presented the molecular responses of sub-acute exposure to PbTx-1 with proteomic method. By adopting two-dimensional electrophoresis, the abundances of 14 and 24 proteins were found to be remarkably altered in the gills and brains, respectively, in response to toxin exposure. Thirteen gill and twenty brain proteins were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis. These proteins could be categorized into diverse functional classes such as cell structure, macromolecule metabolism, signal transduction and neurotransmitter release. These findings can help to elucidate the possible pathways by which aquatic toxins affect marine organisms within target organs.

During a Florida red tide, brevetoxins (PbTxs) produced by Karenia brevis become aerosolized and can cause both immediate and prolonged airway symptoms in humans, especially in those with preexisting airway disease (e.g., asthma). Although environmental monitoring indicates that toxins remain airborne for up to 4 consecutive days, there is little information on airway responses after multiple-day exposures.To delineate putative mechanisms leading to pulmonary dysfunction after PbTx exposure, we studied airway responses before and after multiple exposures to aerosol PbTx-3, the most potent PbTx produced, in nonallergic (healthy) and in allergic sheep, which serve as a surrogate for patients with compromised airways.Both groups were exposed to 20 breaths of increasing concentrations of PbTx-3 (30-300 pg/mL) for 4 consecutive days. Airway responsiveness to carbachol (1 and 8 days after) and airway inflammation as assessed by bronchoalveolar lavage (0 and 7 days after) were measured.Both groups developed airway hyperresponsiveness (AHR) 1 day after challenge; the severity was concentration dependent and more severe in the allergic group. AHR remained after 8 days, but the difference in the severity between the groups was lost. Both groups developed an inflammatory response after exposure to 300 pg/mL PbTx-3. Immediately after exposure, lung neutrophilia was prominent. This neutrophilia persisted for 7 days in addition to increases in total cells and macrophages.Repeated exposures to PbTx-3 result in prolonged AHR and lung inflammation. These pathophysiologic responses could be underlying contributors to the prolonged respiratory symptoms in humans after red tides.

This paper describes the problem of public health monitoring for waterborne disease outbreaks using disparate evidence from health surveillance data streams and environmental sensors. We present a combined monitoring approach along with examples from a recent project at the Johns Hopkins University Applied Physics Laboratory in collaboration with the U.S. Environmental Protection Agency. The project objective was to build a module for the Electronic Surveillance System for the Early Notification of Community-based Epidemics (ESSENCE) to include water quality data with health indicator data for the early detection of waterborne disease outbreaks. The basic question in the fused surveillance application is 'What is the likelihood of the public health threat of interest given recent information from available sources of evidence?' For a scientific perspective, we formulate this question in terms of the estimation of positive predictive value customary in classical epidemiology, and we present a solution framework using Bayesian Networks (BN). An overview of the BN approach presents advantages, disadvantages, and required adaptations needed for a fused surveillance capability that is scalable and robust relative to the practical data environment. In the BN project, we built a top-level health/water-quality fusion BN informed by separate waterborne-disease-related networks for the detection of water contamination and human health effects. Elements of the art of developing networks appropriate to this environment are discussed with examples. Results of applying these networks to a simulated contamination scenario are presented.

A sensitive electrochemical immunosensor for the fast screening of brevetoxin B (BTX-2) in food samples was developed by means of immobilizing BTX-2–bovine serum albumin conjugate (BTX-2–BSA) on the gold nanoparticles-decorated amine-terminated poly(amidoamine) dendrimers (AuNP–PAADs). The presence of gold nanoparticles greatly improved the conductivity of the PAADs, and three-dimensional PAADs increased the surface coverage of the biomolecules on the electrode. Under optimal conditions, three types of immunosensor, i.e. with AuNPs, PAADs, or AuNP–PAADs, were used for the determination of BTX-2 in a competitive-type immunoassay format using horseradish peroxidase-labeled anti -BTX antibodies (HRP– anti -BTX-2) as trace in the H 2 O 2 – o -phenylenediamine ( o -PD) system. A low detection limit (LOD) of 0.01 ng/mL and a wide dynamic working linear range of 0.03–8 ng/mL BTX-2 using AuNP–PAADs as matrices were obtained in comparison with those of only using AuNP or PAADs. Intra-batch assay precision was substantially improved by resorting to the AuNP–PAADs manifold. The proposed method features unbiased identification of negative (blank) and positive samples. No significant differences were encountered in the analysis of the spiking real samples between the electrochemical immunosensor and liquid chromatography for the determination of BTX-2. Importantly, this method provided a biocompatible immobilization and a promising immunosensing platform for analytes with small molecules in the analysis and detection of food safety.

A synthesis of the WXYZA' domain (7) of the marine neurotoxin maitotoxin (1) is reported. The convergent synthetic strategy involves construction of key building blocks 11 and 12, their coupling, and the elaboration of the resulting ester (10) to the target molecule through a ring-closing metathesis and a hydroxy dithioketal cyclization as the key steps. For the construction of fragment 11, the Noyori reduction/Achmatowicz rearrangement and hydroxy epoxide opening technologies were applied (starting from furfuryl alcohol (13)), whereas for the synthesis of fragment 12, a carbohydrate-based approach was adopted (starting from 2-deoxy-D-ribose (14)). The synthesized WXYZA' domain (7) of maitotoxin (1) exhibited the expected (13)C NMR chemical shifts, supporting the originally assigned structure of the corresponding region of the natural product.

Brevetoxins are neurotoxins produced by marine dinoflagellates, primarily Karenia brevis, and can cause intoxication and even mortality of marine species, affect human health through the consumption of brevetoxin-contaminated shellfish, and effect respiratory irritation through aerosol exposure at coastal areas. Brevetoxin-A and brevetoxin-B, the major brevetoxins produced in algae, are metabolized to a series of amino acid and peptide-related derivatives in shellfish through the reactions of the amino acid residue cysteine with an α,β-unsaturated aldehyde group. In this paper, covalent interactions between brevetoxin and proteins were investigated using brevetoxin-B and human serum albumin (HSA) as a model. It is demonstrated that both noncovalent and covalent interactions can occur between brevetoxin-B and HSA with in vitro experiments. Covalent adducts of brevetoxin-B and HSA were generated under physiological conditions and reduced with sodium borohydride based on the reaction conditions of single amino acid residues with brevetoxin-B. LC/MS analysis of toxin-treated HSA recognized the formation of the intact protein adducts with primarily one and two toxin molecules attached to one HSA molecule. HSA treated with/without brevetoxin-B was digested with trypsin, trypsin following chymotrypsin, and Pronase, respectively, for LC/MS analysis of adduction sites. Brevetoxin-B was found to react primarily with Cys(34) and His(3) and with His and Lys at other sites of HSA with variable reactivity and with Lys in general the least reactive.

The increasing frequency of devastating blooms of the harmful dinoflagellate Karenia brevis has motivated investigations into understanding bloom dynamics and the potential for mitigation. Previous findings indicate that waterborne concentrations of the most abundant brevetoxin (brevetoxin B or PbTx-2) associated with these blooms decrease in the presence of other phytoplankton species. The current study explores the mechanism of brevetoxin removal from seawater upon exposure to phytoplankton competitors. Live phytoplankton removed waterborne brevetoxins more rapidly than lysates, but phytoplankton did not need to be in a state of active metabolism. Biomolecules, probably proteins, exuded from phytoplankton appeared to be responsible for the loss of brevetoxins, either by irreversible complexation or by degradation. Selective removal of PbTx-2 and -1, but not PbTx-3, -9 or BTX-B5, by cultured phytoplankton revealed that brevetoxin removal is dependent upon the presence of an α,β-unsaturated aldehyde functionality. The mechanism of biotransformation appears to be common among phytoplankton, since members of various taxonomic groups including diatoms, dinoflagellates, and a cryptophyte each caused 75-90% decrease in PbTx-2 concentration, as did a generic protein (bovine serum albumin) added to seawater at high concentration. These findings support the concept of potentially using competitor phytoplankton species or compounds derived from phytoplankton as biocontrol agents for waterborne toxins associated with red tide.

An expedient enantioselective synthesis of the Δ(4)-oxocene cores present in (+)-laurencin and (+)-prelaureatin was accomplished in eight steps via a novel one-pot regio- and stereoselective ring cyclization-fragmentation-expansion cascade from the tetrahydrofuran precursors which were prepared by stereocontrolled cyclization from vinylsilanes. This process is highlighted by an intramolecular oxo-carbenoid insertion and a β-silyl fragmentation sequence.

Brevetoxins, the algal toxins produced by Karenia brevis during red tide blooms, adversely impact health following ingestion or inhalation. Inhalation of brevetoxins results in a variety of acute symptoms including coughing, wheezing, and shortness of breath. Analysis of manatee lungs following death by purported brevetoxicosis has identified brevetoxin accumulation within macrophages, with pathological manifestions of lung congestion, inflammation, and edema. The goals of this work were to specifically examine effects of brevetoxin-2 on alveolar macrophages, a key cell in responding to toxins in the lung, as well as to determine if brevetoxin-2 results in an inflammatory response and/or direct cytotoxicity. Exposure of an alveolar macrophage cell line (MH-S) to an environmentally and physiologically relevant dose of brevetoxin-2 (0.5 μg/ml) did not significantly alter cellular growth over a 24 h time period. However, exposure of MH-S cells to brevetoxin-2 for 6 h increased phagocytosis of latex beads, increased secretion of interleukin (IL)-2, IL-4, and tumor necrosis factor-α, and decreased secretion of IL-5. Very few changes were seen in gene expression following 3 or 6 h exposure to brevetoxin-2. These results show that brevetoxin-2 induced an immune response indicative of inflammation in an alveolar macrophage cell line, indicating that inhalation of brevetoxin-2 may lead to lung inflammation through an alveolar macrophage-initiated pathway.

Brevetoxins are persistent, bioaccumulative, lipophilic polyether neurotoxins synthesized by Karenia brevis, a harmful algal bloom (HAB) dinoflagellate. Although some marine organisms accumulate potentially harmful levels of brevetoxins, little is known about neurotoxic effects in wild populations. Here, tissue (i.e., liver, kidney, muscle, intestine, gill, brain) brevetoxin levels (as ng PbTx-3 eq/g) and four neurochemical biomarkers (monoamine oxidase, MAO; cholinesterase, ChE; muscarinic cholinergic receptor, mAChR; N-methyl-d-aspartic acid receptor, NMDAR) were compared between eleven lemon sharks collected during a K. brevis bloom and eighteen lemon sharks not exposed to a bloom (controls) in a case-control manner. Brevetoxin levels in tissues were significantly higher in HAB-exposed sharks when compared to controls, and tissue levels (e.g., 277-3112 ng/g in livers, 429-2833 ng/g in gills) in HAB-exposed sharks were comparable to levels detected in a shark (e.g., 1223 ng/g in liver, 930 ng/g in gill) that died presumably of toxin exposure. Further, there were significant correlations between brain brevetoxin levels and ChE activity (r=-0.41; p

Herein, we describe advancements in monitoring of brevetoxins in molluscan shellfish, with respect to exposure management and control of neurotoxic shellfish poisoning (NSP). Current knowledge of the fate of brevetoxins in molluscan shellfish, and the toxic potency of brevetoxin metabolites, is presented. We review rapid assays for measuring composite brevetoxins, and methodology for measuring constituent brevetoxins, in contaminated shellfish. The applicability of in vitro methods for estimating brevetoxin burden and composite toxicity in shellfish is assessed. Specific and measurable biomarkers of brevetoxin exposure and toxicity in shellfish, and of human intoxication, are described. Their utility in regulatory monitoring of toxic shellfish and in clinical diagnosis of NSP is evaluated.

27 páginas, 4 tablas, 4 figuras, Diarrhetic Shellfish Poisoning (DSP) is a specific type of food poisoning, characterized by severe gastrointestinal illness due to the ingestion of filter feeding bivalves contaminated with a specific suite of toxins. It is known that the problem is worldwide and three chemically different groups of toxins have been historically associated with DSP syndrome: okadaic acid (OA) and dinophysistoxins (DTXs), pectenotoxins (PTXs) and yessotoxins (YTXs). PTXs and YTXs have been considered as DSP toxins because they can be detected with the bioassays used for the toxins of the okadaic acid group, but diarrhegenic effects have only been proven for OA and DTXs. Whereas, some PTXs causes liver necrosis and YTXs damages cardiac muscle after intraperitoneal injection into mice. On the other hand, azaspiracids (AZAs) have never been included in the DSP group, but they cause diarrhoea in humans. This review summarizes the origin, characterization, structure, activity, mechanism of action, clinical symptoms, method for analysis, potential risk, regulation and perspectives of DSP and associated toxins produced by marine dinoflagellates., This work was funded b yGrants AGL2005-07924-C04- 01/02 and CTQ2008-0754-C04-01/04 from the MEC of Spain.

Adriatoxin-B (ATX-B) is the first C13 terminal truncated YTX analogous metabolite that was isolated from large-scale cultures of the dinoflagellate Protoceratium reticulatum. The structure and conformational behavior of ATX-B was elucidated by a combination of NMR spectroscopy experiments and a conformational analysis. The isolation of this metabolite, which possesses a clear structural relationship with adriatoxin, supports the idea that the real source of these cut down substances is in the microalgae and not in the mollusks, via metabolic degradation like it has been assumed until now.

Brevetoxins (PbTx) are sodium channel neurotoxins produced by the marine dinoflagellate Karenia brevis during red tide blooms. Inhalation of PbTx in normal individuals and individuals with pre-existing airways disease results in adverse airway symptoms including bronchoconstriction. In animal models of allergic inflammation, inhalation of PbTx results in a histamine H₁-mediated bronchoconstriction suggestive of mast cell activation. How mast cells would respond directly to PbTx is unknown. We thus explored the activation of mouse bone marrow-derived mast cells (BMMCs) following exposure to purified PbTx-2. Following in vitro exposure to PbTx-2, we examined cellular viability, mast cell degranulation (β-hexosaminidase release), intracellular Ca²+ and Na+ flux, and the production of inflammatory mediators (IL-6). PbTx-2 induced significant cellular toxicity within 24 h as measured by LDH release and Annexin-V staining. However, within 1 h of exposure, PbTx-2 induced BMMC degranulation and an increase in IL-6 mRNA expression independent of the high-affinity IgE receptor (FcεRI) stimulation. Activation of BMMCs by PbTx-2 was associated with altered intracellular Ca²+ and Na+ levels. Brevenal, a naturally produced compound that antagonizes the activity of PbTx, prevented changes in intracellular Na+ levels but did not alter activation of BMMCs by PbTx-2. These findings demonstrate that PbTx-2 activates mast cells independent of FcεRI providing insight into critical events in the pathogenesis and a potential therapeutic target in brevetoxin-induced airway symptoms.

The red-tide dinoflagellate Protoceratium reticulatum is shown to be protected against turbulence-associated damage by the use of the additives Pluronic F68 (PF68) and carboxymethyl cellulose (CMC) in the culture medium. Relative to agitated controls, these additives had a dose-dependent protective effect at concentrations of up to 0.4 and 0.5 g L(-1) for CMC and F68, respectively. In static cultures, these additives inhibited growth directly or indirectly at a concentration of0.5 g L(-1). Compared to CMC, PF68 was a better protectant overall. Cell-specific production of yessotoxins was enhanced under elevated shear stress regimens so long as the turbulence intensity was insufficient to damage the cells outright. Shear-induced production of reactive oxygen species and direct effects of turbulence on the cell cycle contributed to the observed shear effects.

Brevetoxins are potent neurotoxins that exert their toxicity through activation of voltage-gated sodium channels. Exposure to brevetoxins cause severe respiratory inflammation in marine mammals and humans. Brevetoxin activation of voltage-gated sodium channels on immune cells can lead to several biological responses including cell proliferation, gene transcription, cytokine production and even apoptosis. Jurkat E6-1 T cells were treated with brevetoxin 2 for 4 hours at a dose previously shown to induce apoptosis and DNA damage. Changes in gene expression were then assessed via PCR arrays. Gene expression analysis revealed significant change in expression of 17 genes related to apoptosis, 21 genes related to DNA damage signaling, and 19 genes encoding common cytokines. The gene expression data supports the idea that brevetoxins trigger complex reactions involving both inflammation and cell death.

Blooms of the toxic dinoflagellate, Karenia brevis, occur almost annually off the Florida coast. These blooms, commonly called "red tides", produce a group of neurotoxins collectively termed brevetoxins. Many species of sealife, including sea turtles, are severely impacted by brevetoxin exposure. Effects of brevetoxins on immune cells were investigated in rescued loggerhead sea turtles, Caretta caretta, as well as through in vitro experiments using peripheral blood leukocytes (PBL) collected from captive sea turtles. In rescued animals, plasma brevetoxin concentrations were measured using a competitive ELISA. Plasma lysozyme activity was measured using a turbidity assay. Lysozyme activity correlated positively with plasma brevetoxin concentrations. Differential expression of genes affected by brevetoxin exposure was determined using two separate suppression subtractive hybridization experiments. In one experiment, genes from PBL collected from sea turtles rescued from red tide toxin exposure were compared to genes from PBL collected from healthy captive loggerhead sea turtles. In the second experiment, PBL from healthy captive loggerhead sea turtles were exposed to brevetoxin (500 ng PbTx-2/ml) in vitro for 18 h and compared to unexposed PBL. Results from the subtraction hybridization experiment conducted with red tide rescued sea turtle PBL indicated that genes involved in oxidative stress or xenobiotic metabolism were up-regulated. Using quantitative real-time PCR, a greater than 2-fold increase in superoxide dismutase and thioredoxin and greater than 10-fold increase in expression of thiopurine S-methyltransferase were observed. Results from the in vitro subtraction hybridization experiment indicated that genes coding for cytochrome c oxidases were the major up-regulated genes. Using quantitative real-time PCR, a greater than 8-fold increase in expression of beta-tubulin and greater than 3-fold increase in expression of ubiquinol were observed. Brevetoxin exposure may have significant implications for immune function in loggerhead sea turtles.

Red tide aerosols containing aerosolized brevetoxins are produced during the red tide bloom and transported by wind to coastal areas of Florida. This study reports the characterization of Florida red tide aerosols in human volunteer studies, in which an asthma cohort spent 1h on Siesta Beach (Sarasota, Florida) during aerosolized red tide events and non-exposure periods. Aerosol concentrations, brevetoxin levels, and particle size distribution were measured. Hourly filter samples were taken and analyzed for brevetoxin and NaCl concentrations. In addition, the aerosol mass concentration was monitored in real time. The results indicated that during a non-exposure period in October 2004, no brevetoxin was detected in the water, resulting in non-detectable levels of brevetoxin in the aerosol. In March 2005, the time-averaged concentrations of brevetoxins in water samples were moderate, in the range of 5-10 microg/L, and the corresponding brevetoxin level of Florida red tide aerosol ranged between 21 and 39 ng/m(3). The temporal profiles of red tide aerosol concentration in terms of mass, NaCl, and brevetoxin were in good agreement, indicating that NaCl and brevetoxins are components of the red tide aerosol. By continuously monitoring the marine aerosol and wind direction at Siesta Beach, we observed that the marine aerosol concentration varied as the wind direction changed. The temporal profile of the Florida red tide aerosol during a sampling period could be explained generally with the variation of wind direction.

Maitotoxin (1) continues to fascinate scientists not only because of its size and potent neurotoxicity but also due to its molecular architecture. To provide further support for its structure and facilitate fragment-based biological studies, we developed an efficient chemical synthesis of the ABCDEFG segment 3 of maitotoxin. (13)C NMR chemical shift comparisons of synthetic 3 with the corresponding values for the same carbons of maitotoxin revealed a close match, providing compelling evidence for the correctness of the originally assigned structure to this polycyclic system of the natural product. The synthetic strategy for the synthesis of 3 relied heavily on our previously developed furan-based technology involving sequential Noyori asymmetric reduction and Achmatowicz rearrangement for the construction of the required tetrahydropyran building blocks, and employed a B-alkyl Suzuki coupling and a Horner-Wadsworth-Emmons olefination to accomplish their assembly and elaboration to the final target molecule.

Florida red tides occur annually in the Gulf of Mexico from blooms of the marine dinoflagellate, Karenia brevis, which produces highly potent natural polyether toxins, brevetoxins. Several epidemiologic studies have demonstrated that human exposure to red tide aerosol could result in increased respiratory symptoms. Environmental monitoring of aerosolized brevetoxins was performed using a high-volume sampler taken hourly at fixed locations on Siesta Beach, Florida. Personal exposure was monitored using personal air samplers and taking nasal swab samples from the subjects who were instructed to spend 1 hr on Sarasota Beach during two sampling periods of an active Florida red tide event in March 2005, and in May 2008 when there was no red tide. Results showed that the aerosolized brevetoxins from the personal sampler were in modest agreement with the environmental concentration taken from a high-volume sampler. Analysis of nasal swab samples for brevetoxins demonstrated 68% positive samples in the March 2005 sampling period when air concentrations of brevetoxins were between 50 to 120 ng/m(3) measured with the high-volume sampler. No swab samples showed detectable levels of brevetoxins in the May 2008 study, when all personal samples were below the limit of detection. However, there were no statistical correlations between the amounts of brevetoxins detected in the swab samples with either the environmental or personal concentration. Results showed that the personal sample might provide an estimate of individual exposure level. Nasal swab samples showed that brevetoxins indeed were inhaled and deposited in the nasal passage during the March 2005 red tide event.

In this review, we focus on processes, organs and systems targeted by the marine toxins yessotoxin (YTX), okadaic acid (OA) and palytoxin (PTX). The effects of YTX and their basis are analyzed from data collected in the mollusc Mytilus galloprovincialis, the annelid Enchytraeus crypticus, Swiss CD1 mice and invertebrate and vertebrate cell cultures. OA and PTX, two toxins with a better established mode of action, are analyzed with regard to their effects on development. The amphibian Xenopus laevis is used as a model, and the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) as the experimental protocol.

Brevetoxin-1 (BTX-1), a marine toxin mostly produced by the dinoflagellatae Karenia brevis, has caused the death of marine organisms and has had numerous toxicological effects on human health. Hence, it is very necessary to develop a rapid, economical, and reliable immunoassay method for BTX-1 detection. In this study, two kinds of complete antigen were synthesized using the succinic anhydride and isobutyl chloroformate two-step methods. Conjugate BTX-1-OVA was used as an antigen for mice immunization, and BTX-1-BSA for measuring the titer of the produced antibodies. A hybridoma cell line 6C6 stably secreting monoclonal antibody (mAb) against BTX-1 was obtained by fusing SP2/0 myeloma cells with the spleen cells from the immunized mouse. The hybridoma 6C6 was injected into the abdomen of BALB/c mice to obtain ascites, and the anti-BTX-1 mAb was harvested from ascites by precipitation with caprylic acid/ammonium sulfate (CA-AS). The anti-BTX-1 mAb was identified as an IgG1 subtype, and the cross-reactivity results showed that anti-BTX-1 mAb was highly specific to BTX-1 with the affinity of 1.06 × 108 L/mol. The indirect competitive ELISA results indicated that the linear range for BTX-1 detection was 14–263 ng/mL with IC50 of 60 ng/mL, and a detection limit of 14 ng/mL. The average recovery rate from the spiked samples was 88 ± 2% in intra-assay and 89 ± 2% in inter-assay. The limit of detection (LOD) using the colloidal gold strip was 200 ng/mL with high specificity. Therefore, the anti-BTX-1 mAb can be used to detect BTX-1 in shellfish and other related samples.

This work describes the development and applicability of two functional assays for the detection of yessotoxin (YTX), a polycyclic ether marine toxin produced by dinoflagellates. The assays are based on the interaction between this toxin and the phosphodiesterase (PDE) enzyme and the subsequent measurement of the enzyme activity by colorimetric and electrochemical methods. Firstly, several enzyme substrates were tested in order to select those able to be detected by colorimetry or electrochemistry after enzymatic hydrolysis. The substrates that provided the highest absorbance values and density currents were p-nitrophenyl phenylphosphonate and alpha-naphthyl phosphate, respectively. After optimisation of the experimental parameters, limits of detection of 0.8 and 0.6 microM were attained by colorimetry and electrochemistry, respectively. An inhibitory effect of YTX on the PDE activity was observed. The assays have been applied to the analysis of YTX production by Protoceratium reticulatum cultures, and results were compared with liquid chromatography-tandem mass spectrometry analysis.

Maitotoxin (MTX) activates Ca2+-permeable nonselective cation channels and causes a dramatic increase in cytosolic free Ca2+concentration ([Ca2+]i) in every cell examined to date, but the molecular identity of the channels involved remains unknown. A clue came from studies of a structurally related marine toxin called palytoxin (PTX). PTX binds to the plasmalemmal Na+-K+-ATPase (NKA) and converts the Na+pump into a nonselective cation channel. Given the high permeability of the MTX channel for Ca2+, we considered the possibility that MTX may bind to the plasmalemmal Ca2+-ATPase (PMCA) pump, and like PTX, convert the pump into a channel. To test this hypothesis, the PMCA was overexpressed in Spodoptera frugiperda (Sf9) insect cells and in human embryonic kidneys (HEK) 293 cells. In both cell types, enhanced expression of the PMCA was associated with a significant increase in MTX-induced whole cell membrane currents. The effect of MTX on whole cell currents in both wild-type and PMCA overexpressing HEK cells was sensitive to pump ligands including Ca2+and ATP. MTX-induced currents were significantly reduced by knockdown of PMCA1 in HEK cells using small interfering RNA or in mouse embryonic fibroblasts from genetically modified mice with the PMCA1(+/−) PMCA4(−/−) genotype. Finally, PMCA catalytic activity (i.e., Ca2+-ATPase) in isolated membranes, or in purified PMCA preparations, was inhibited by MTX. Together, these results suggest that MTX binds to and converts the PMCA pump into a Ca2+-permeable nonselective cation channel.

Brevetoxins are polyether toxins produced by the dinoflagellate Karenia brevis that are released into the air and are known to cause respiratory hemorrhage in manatees and irritation in humans. Brevetoxin has been previously reported to cause DNA breakage and chromosomal aberrations in in vitro cell assays. The toxin is subject to epoxidation reaction and the formation of nucleic acid adducts in cultured lung fibroblasts and in lung tissue after intratracheal administration to rats. We have exposed rats intratracheally to brevetoxin B (45 microg/kg) and analyzed liver cells for DNA fragmentation using a comet assay. Brevetoxin B (PbTx2) treated rats showed a two to three-fold increase in the amount of DNA in the comet tails, indicating that brevetoxin has in vivo clastogenic activity. We next tested brevetoxin B for mutagenic activity using the Ames 98/100 mutagenesis assay. Brevetoxin B at concentrations from 0.064 to 200 microg/mL failed to cause histidine revertants. Oxidative metabolism of brevetoxin B resulting from Aroclor 1259-induced rat liver microsomes also failed to cause histidine revertants. Finally, direct application of the brevetoxin B epoxide (PbTx6) in the Ames 98/100 assay at concentrations from 0.064 to 200 microg/mL failed to induce histidine revertants. These studies indicate that brevetoxin B retains clastogenic activity after intratracheal administration to the rat. Although brevetoxin B has been shown to form nucleic acid adducts in the lung, neither brevetoxin B nor its epoxide metabolite has mutagenic potential as assessed by the Ames 98/100 test.

Brevetoxins are a group of natural neurotoxins characterized by polyether ring systems that are found in the blooms of red tide algae. In a conventional water/organic solvent system, without any other additives, deprotonated molecules of brevetoxins do not appear in high abundance in negative mode electrospray mass spectrometry (ES-MS) due to lack of acidic functional groups; thus, they are not well-suited for collision-induced decomposition (CID) experiments in negative mode electrospray. In this study, several anions were tested for their abilities to form anionic adducts by mixing ammonium salts of these anions with brevetoxin-2 and brevetoxin-3. Under CID, [M + Cl](-), [M + Br](-), [M + OAc](-), [M + HCOO](-), and [M + NO(3)](-) adducts all produced only the respective anions in CID experiments and thus provided no structural information. In contrast, upon CID, both [M + F](-) and [M + HCO(3)](-) precursor adducts gave structurally informative fragment peaks that exhibited similarities to those of [M - H](-) ions, which indicated that the first step in gas-phase decomposition of these anionic adducts was loss of neutral HF or H(2)CO(3) molecules, respectively, leaving deprotonated brevetoxin ([M - H](-)) to undergo consecutive fragmentations. In comparison to bicarbonate, fluoride formed adducts in higher abundance and provided more fragment peaks, thus more structural information, in MS/MS experiments. It is therefore the anion of choice to study brevetoxins in negative mode electrospray mass spectrometry using the anion attachment approach. The detailed fragmentation mechanisms are discussed, and diagnostic product ions are proposed for the brevetoxin-2 side chain (m/z 95, 133, and 151), the brevetoxin-3 side chain (m/z 97, 135, and 153), and the type-B brevetoxin backbone (m/z 725, 739, 741, and 797). To test the developed methodology, a sample of brevetoxin-2 subjected to in vitro microsomal incubation was analyzed, and a reduction product of the substrate was confirmed to have the structure of brevetoxin-3.

Apoptosis and oncotic necrosis in neuronal and glial cells have been documented in many neurological diseases. Distinguishing between these two major types of cell death in different neurological diseases is needed in order to better reveal the injury mechanisms so as to open up opportunities for therapy development. Accumulating evidence suggests apoptosis and oncosis epitomize the extreme ends of a broad spectrum of morphological and biochemical events. Biochemical markers that can distinguish between the calpain and caspase dominated types of cell death would help in this process. In this study, three chemical agents, maitotoxin (MTX), staurosporine (STS) and thylenediaminetetraacetic acid (EDTA), were used to induce different types of cell death in PC12 neuronal-like cells. MTX-induced necrosis, as determined by the increased levels of calpain-specific cleaved fragments of spectrin by antibodies specific to the calpain-cleaved 150 kDa alphaII-spectrin breakdown product (SBDP150) and 145 kDa alphaII-spectrin breakdown product (SBDP145). In this paradigm, there were no detectable SBDP150i and SBDP120 fragments as determined by antibodies specific to the caspase-cleaved specific fragments similar to those seen in the EDTA-mediated apoptotic PC-12 cells. In contrast to the calpain specific MTX necrosis treatment and the caspase EDTA apoptotic treatment is the STS treatment which induced both proteases as shown by the increase in all the SBDP fragments. Furthermore, compared to SBDP150, SBDP145 appears to be a more specific and sensitive biomarker for calpain activation. Taken together, our results suggested calpains and caspases which dominate the two major types of cell death could be independently discriminated by specifically examining the multiple alphaII-spectrin cleavage breakdown products.