Microcystin-LR sensitizes the Oncorhynchus mykiss intestinal epithelium and interacts with paralytic shellfish toxins to alter oxidative balance.

In the context of harmful algal blooms, fish can be exposed to the combined effects of more than one toxin. We studied the effects of consecutive exposure to Microcystin-LR (MCLR) in vivo and paralytic shellfish toxins (PST) ex vivo/in vitro (MCLR+PST) in the rainbow trout Oncorhynchus mykiss's middle intestine. We fed juvenile fish with MCLR incorporated in the feed every 12 h and euthanized them 48 h after the first feeding. Immediately, we removed the middle intestine to make ex vivo and in vitro preparations and exposed them to PST for one hour. We analyzed glutathione (GSH) and glutathione disulfide (GSSG) contents, glutathione S-transferase (GST), glutathione reductase (GR), catalase (CAT), and protein phosphatase 1 (PP1) activities in ex vivo intestinal strips; apical and basolateral ATP-biding cassette subfamily C (Abcc)-mediated transport in ex vivo everted and non- everted sacs; and reactive oxygen species (ROS) production in isolated enterocytes in vitro. MCLR+PST treatment decreased the GSH content, GSH/GSSG ratio, GST activity, and increased ROS production. GR activity remained unchanged, while CAT activity only increased in response to PST. MCLR inhibited PP1 activity and activated Abcc-mediated transport only at the basolateral side of the intestine. Our results show a combined effect of MCLR+PST on the oxidative balance in the O. mykiss middle intestine, which is not affected by the two toxins groups when applied individually. Basolateral Abcc transporters activation by MCLR treatment could lead to an increase in the absorption of toxicants (including MCLR) into the organism. Therefore, MCLR makes the O. mykiss middle intestine more sensitive to possibly co-occurring cyanotoxins like PST.

Effects of paralytic shellfish toxins on the middle intestine of Oncorhynchus mykiss: Glutathione metabolism, oxidative status, lysosomal function and ATP-binding cassette class C (ABCC) proteins activity.

We studied the absorption, cytotoxicity and oxidative stress markers of Paralytic Shellfish Toxins (PST) from three extracts from Alexandrium catenella and A. ostenfeldii, in middle Oncorhynchus mykiss intestine in vitro and ex vivo preparations. We measured glutathione (GSH) content, glutathione-S transferase (GST), glutathione reductase (GR) and catalase (CAT) enzymatic activity, and lipid peroxidation in isolated epithelium exposed to 0.13 and 1.3 µM PST. ROS production and lysosomal membrane stability (as neutral red retention time 50%, NRRT50) were analyzed in isolated enterocytes exposed to PST alone or plus 3 µM of the ABCC transport inhibitor MK571. In addition, the concentration-dependent effects of PST on NRRT50 were assayed in a concentration range from 0 to 1.3 µM PST. We studied the effects of three different PST extracts on the transport rate of the ABCC substrate DNP-SG by isolated epithelium. The extract with highest inhibition capacity was selected for studying polarized DNP-SG transport in everted and non-everted intestinal segments. We registered lower GSH content and GST activity, and higher GR activity, with no significant changes in CAT activity, lipid peroxidation or ROS level. PST exposure decreased NRRT50 in a concentration-depend manner (IC50 = 0.0045 µM), but PST effects were not augmented by addition of MK571. All the three PST extracts inhibited ABCC transport activity, but this inhibition was effective only when the toxins were applied to the apical side of the intestine and DNP-SG transport was measured at the basolateral side. Our results indicate that PST are absorbed by the enterocytes from the intestine lumen. Inside the enterocytes, these toxins decrease GSH content and inhibit the basolateral ABCC transporters affecting the normal functions of the cell. Furthermore, PST produce a strong cytotoxic effect to the enterocytes by damaging the lysosomal membrane, even at low, non-neurotoxic concentrations.

Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs).

The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet a consensus protocol is still lacking. In this study, six key parameters of CBA-N2a were revisited: cell seeding densities, cell layer viability after 26 h growth, MTT incubation time, Ouabain and Veratridine treatment and solvent and matrix effects. A step-by-step protocol was defined identifying five viability controls for the validation of CBA-N2a results. Specific detection of two voltage gated sodium channel activators, pacific ciguatoxin (P-CTX3C) and brevetoxin (PbTx3) and two inhibitors, saxitoxin (STX) and decarbamoylsaxitoxin (dc-STX) was achieved, with EC50 values of 1.7 ± 0.35 pg/mL, 5.8 ± 0.9 ng/mL, 3 ± 0.5 ng/mL and 15.8 ± 3 ng/mL, respectively. When applied to the detection of ciguatoxin (CTX)-like toxicity in fish samples, limit of detection (LOD) and limit of quantification (LOQ) values were 0.031 ± 0.008 and 0.064 ± 0.016 ng P-CTX3C eq/g of flesh, respectively. Intra and inter-assays comparisons of viability controls, LOD, LOQ and toxicity in fish samples gave coefficients of variation (CVs) ranging from 3% to 29%. This improved test adaptable to either high throughput screening or composite toxicity estimation is a useful starting point for a standardization of the CBA-N2a in the field of marine toxin detection.

Effect of acute exposure of saxitoxin on development of zebrafish embryos (Danio rerio).

As a type of cyanobacterial toxins, saxitoxin (STX) is receiving great interest due to its increasing presence in waterbodies. However, the underlying mechanism of STX-induced adverse effect is poorly understood. Here, we examined the developmental toxicity and molecular mechanism induced by STX using zebrafish embryos as an animal model. The embryonic toxicity induced by STX was demonstrated by inhibition of embryo hatching, increase in mortality rate, abnormal heart rate, abnormalities in embryo morphology as well as defects in angiogenesis and common cardinal vein remodeling. STX induced embryonic DNA damage and cell apoptosis, which would be alleviated by antioxidant N-acetyl-L-cysteine. Additionally, STX significantly increased reactive oxygen species level, catalase activity and malondialdehyde content and decreased the activity of superoxide dismutase and glutathione content. STX also promoted the expression of vascular development-related genes DLL4 and VEGFC, and inhibited VEGFA expression. Furthermore, STX altered the transcriptional regulation of apoptosis-related genes (BAX, BCL-2, P53 and CASPASE 3). Taken together, STX induced adverse effect on development of zebrafish embryos, which might be associated with oxidative stress-induced apoptosis.

Improved Accuracy of Saxitoxin Measurement Using an Optimized Enzyme-Linked Immunosorbent Assay.

Paralytic shellfish poisoning (PSP) is precipitated by a family of toxins produced by harmful algae, which are consumed by filter-feeding and commercially popular shellfish. The toxins, including saxitoxin, neosaxitoxin, and gonyautoxins, accumulate in shellfish and cause intoxication when consumed by humans and animals. Symptoms can range from minor neurological dysfunction to respiratory distress and death. There are over 40 different chemical congeners of saxitoxin and its analogs, many of which are toxic and many of which have low toxicity or are non-toxic. This makes accurate toxicity assessment difficult and complicates decisions regarding whether or not shellfish are safe to consume. In this study, we describe a new antibody-based bioassay that is able to detect toxic congeners (saxitoxin, neosaxitoxin, and gonyautoxins) with little cross-reactivity with the low or non-toxic congeners (decarbamoylated or di-sulfated forms). The anti-saxitoxin antibody used in this assay detects saxitoxin and neosaxitoxin, the two most toxic congers equally well, but not the relatively highly toxic gonyautoxins. By incorporating an incubation step with L-cysteine, it is possible to convert a majority of the gonyautoxins present to saxitoxin and neosaxitoxin, which are readily detected. The assay is, therefore, capable of detecting the most toxic PSP congeners found in commercially relevant shellfish. The assay was validated against samples whose toxicity was determined using standard HPLC methods and yielded a strong linear agreement between the methods, with R2 values of 0.94-0.96. As ELISAs are rapid, inexpensive, and easy-to-use, this new commercially available PSP ELISA represents an advance in technology allowing better safety management of the seafood supply and the ability to screen large numbers of samples that can occur when monitoring is increased substantially in response to toxic bloom events.

Cytoprotection of lipoic acid against toxicity induced by saxitoxin in hippocampal cell line HT-22 through in silico modeling and in vitro assays.

Saxitoxins (STXs) are potent neurotoxins that block voltage-gated channels in neurons and induce cytotoxicity. These toxins not only can generate reactive oxygen species but also can alter antioxidant levels, promoting oxidative stress. Under this pro-oxidant situation, the use of the antioxidant lipoic acid (LA) can represent a chemoprotective alternative to minimize the deleterious effects induced by neurotoxins as STXs. P-glycoprotein (P-gp) is a well-known ATP-binding cassette (ABC) transporter that plays a crucial role in the extrusion of toxic substances, decreasing their accumulation and potential intracellular effects in virtue of its broad substrate specificity, its expression in many excretory tissues and its large efflux capacity. The interaction of STXs with LA was evaluated by ab initio simulation, molecular docking and bioassays using the cell line HT-22. The interaction of STXs with LA occurs by physisorption. Molecular docking indicated that STXs can be a substrate of P-gp and, estimating the Free Energy of Binding (FEB), LA has lower amino acids residues binding sites, similar to verapamil, while STX and STX+LA_1 have similar amino acids residues and binding sites with similar FEB between this ligands.Cells were exposed to STXs and LA for 30min and 24h. LA treatment minimizes STX cytotoxicity, evaluated by trypan blue and MTT assay and both STX and STX-LA treatments were efficient to induce P-gp activity measured by rhodamine 123 dye extrusion. LA and STX+LA treatments induced low reactive oxygen species levels and low oxygen consumption. Based on our results, it can be concluded that LA was able to induce cytoprotection, including induction of cellular glutathione levels, and that STX+LA interaction reduced toxicity effects induced by STX. Overall, the in vitro results corroborated the semi-empirical evidences found using density functional theory ab initio simulation and molecular docking.

Uncovering the proteome response of murine neuroblastoma cells against low-dose exposure to saxitoxin.

The potent neurotoxin saxitoxin produced by both marine and freshwater phytoplankton causes paralytic shellfish poisoning syndrome. The toxicity and mode of action of the acute exposure of high-dose saxitoxin have been intensively studied for decades; however, the potential risk of exposure of low-dose saxitoxin remained to be uncovered. Here we present a proteomics study of murine neuroblastoma N2A cell with low-dose saxitoxin exposure (1 nM and 10 nM, 24-h intoxication). Differential proteins were profiled by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). A total of 9 proteins, including 14-3-3 beta (1433B), alpha enolase (ENO1) and cofilin 2 (CFL2), were altered by the low-dose saxitoxin exposure. We further validated the expressions of 1433B, ENO1 and CFL2 by Western blot analysis and the enzyme-linked immunosorbent assay. These 9 proteins involve cell apoptotic pathways, cell skeleton maintenance, membrane potentials and mitochondrial functions. Modulation of these 9 proteins by low-dose saxitoxin exposure could correlate to the reports on genotoxicity and neurotoxicity induced by saxitoxin. This study also suggested other potential risks of saxitoxin.

Comparative studies of saxitoxin (STX) -induced cytotoxicity in Neuro-2a and RTG-2 cell lines: An explanation with respect to changes in ROS.

Saxitoxin (STX), a paralytic shellfish toxin (PST) produced from toxic bloom-forming dinoflagellates, was selected to comparatively investigate the induction of cytotoxicity and apoptosis and a possible mechanism based on changes in the antioxidant defence system of two cellular strains: the mouse neuroblastoma cell line Neuro-2a and the rainbow trout fish cell line RTG-2. Increasing concentrations of STX (0-256 nM) presented little cytotoxic or apoptotic effects on the two cell lines. Measurements of cellular viability, lethal ratio and LDH leakage showed slight changes in Neuro-2a and RTG-2 cells (p > 0.05), and similar results were observed for cellular morphology and apoptotic rates. The contents of the main reactive oxygen species (ROS) components, superoxide anion (O2-) and hydrogen peroxide (H2O2), were markedly increased in Neuro-2a cell with STX exposure at middle (15 nM) and high (150 nM) concentrations (p < 0.05), and the simultaneous increase of the ratio of reduced/oxidized glutathione (GSH/GSSG) (p < 0.05) inferred the occurrence of oxidative stress. However, little difference was observed in all treated groups of RTG-2 cells. The activities of three antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), were significantly enhanced in Neuro-2a cells in the middle and high concentration groups (p < 0.05), while glutathione peroxidase (GPX) obviously decreased (p < 0.05) in all treated groups. Little change was found in RTG-2 cells with the same exposures. These results provided evidence that STX exposure altered the redox status of Neuro-2a cells and resulted in oxidative stress, but the same exposure exerted little effect on RTG-2 cells. Therefore, Neuro-2a cells are more sensitive than reproductive cells to STX exposure, and the antioxidant systems appears to be partly responsible for this differentiation response.

Single and combined effects of microcystin- and saxitoxin-producing cyanobacteria on the fitness and antioxidant defenses of cladocerans.

Cyanobacteria produce different toxic compounds that affect animal life, among them hepatotoxins and neurotoxins. Because cyanobacteria are able to produce a variety of toxic compounds at the same time, organisms may be, generally, subjected to their combined action. In the present study, we demonstrate the single and combined effects on cladocerans of cyanobacteria that produce microcystins (hepatotoxins) and saxitoxins (neurotoxins). Animals were exposed (either singly or combined) to 2 strains of cyanobacteria isolated from the same environment (Funil Reservoir, Rio de Janeiro, Brazil). The effects on clearance rate, mobility, survivorship, fecundity, population increase rate (r), and the antioxidant enzymes glutathione-S-transferase (GST) and catalase (CAT) were measured. Cladoceran species showed a variety of responses to cyanobacterial exposures, going from no effect to impairment of swimming movement, lower survivorship, fecundity, and general fitness (r). Animals ingested cyanobacteria in all treatments, although at lower rates than good food (green algae). Antioxidant defense responses were in accordance with fitness responses, suggesting that oxidative stress may be related to such effects. The present study emphasizes the need for testing combined actions of different classes of toxins, because this is often, and most likely, the scenario in a more eutrophic world with global climatic changes. Environ Toxicol Chem 2017;36:2689-2697. © 2017 SETAC.

Extended Low-Dose Exposure to Saxitoxin Inhibits Neurite Outgrowth in Model Neuronal Cells.

The potent neurotoxin saxitoxin (STX) belongs to a group of structurally related analogues produced by both marine and freshwater phytoplankton. The toxins act by blocking voltage-gated sodium channels stopping the inflow of sodium ions and the generation of action potentials. Exposure from marine sources occurs as a result of consuming shellfish which have concentrated the toxins, and freshwater exposure can occur from drinking water although there have been no acute poisonings from the latter source to date. Previously, the majority of research into this group of toxins, collectively known as the paralytic shellfish toxins, has focused on acute exposure resulting in paralytic shellfish poisoning. While acute exposure guidelines exist for both sources, there are no chronic exposure guidelines and there has been minimal research into this pattern of exposure despite the known role of electrical activity in neurogenesis. We aimed to investigate this pattern of exposure and its potential effects on neurodevelopment using model neuronal cells. PC12 and SH-SY5Y cells were exposed to STX (0.25-3 µg/l) for 7 days, after which time they were stained with TRITC-Phalloidin, to observe adverse morphological effects. Cells exposed to STX had a significant decrease (18-85%) in long axonlike projections, instead exhibiting a significant increase in shorter projections classified as filopodia (p < 0.05). The results suggest that extended low-dose exposure to STX can inhibit proper neurite outgrowth at concentrations well below guideline levels for both sources of exposure making it a potential public health concern.

Uncovering the Complex Transcriptome Response of Mytilus chilensis against Saxitoxin: Implications of Harmful Algal Blooms on Mussel Populations.

Saxitoxin (STX), a principal phycotoxin contributing to paralytic shellfish poisoning, is largely produced by marine microalgae of the genus Alexandrium. This toxin affects a wide range of species, inducing massive deaths in fish and other marine species. However, marine bivalves can resist and accumulate paralytic shellfish poisons. Despite numerous studies on the impact of STX in marine bivalves, knowledge regarding STX recognition at molecular level by benthic species remains scarce. Therefore, the aim of this study was to identify novel genes that interact with STX in the Chilean mussel Mytilus chilensis. For this, RNA-seq and RT-qPCR approaches were used to evaluate the transcriptomic response of M. chilensis to a purified STX as well as in vivo Alexandrium catenella exposure. Approximately 800 million reads were assembled, generating 138,883 contigs that were blasted against the UniProt Mollusca database. Pattern Recognition Receptors (PRRs) involved in mussel immunity, such as Toll-like receptors, tumor necrosis factor receptors, and scavenger-like receptors were found to be strongly upregulated at 8 and 16 h post-STX injection. These results suggest an involvement of PRRs in the response to STX, as well as identifying potential, novel STX-interacting receptors in this Chilean mussel. This study is the first transcriptomic overview of the STX-response in the edible species M. chilensis. However, the most significant contribution of this work is the identification of immune receptors and pathways potentially involved in the recognition and defense against STX's toxicity and its impact of harmful algae blooms on wild and cultivated mussel populations.

Matrix effects on a cell-based assay used for the detection of paralytic shellfish toxins in bivalve shellfish samples.

Detecting marine biotoxins such as paralytic shellfish toxins (PSTs) is essential to ensuring the safety of seafood. The mouse bioassay is the internationally accepted method for monitoring PSTs, but technical and ethical issues have led to a search for new detection methods. The mouse neuroblastoma cell-based assay (Neuro-2a CBA) using ouabain and veratridine (O/V) has proven useful for the detection of PSTs. However, CBAs are sensitive to shellfish-associated matrix interferences. As the extraction method highly influences matrix interferences, this study compared three extraction protocols: Association of Official Analytical Chemists (AOAC) 2005.06, AOAC 2011.02 and an alternative liquid-liquid method. These methods were used to assess the matrix effect of extracts from four commercially important bivalve species (Chilean mussel, Magellan mussel, clam and Pacific oyster) in Neuro-2a CBA. Extracts from all three protocols caused a toxic effect in Neuro-2a cells (without O/V) when tested at a concentration of 25 mg of tissue-equivalent (TE) ml(-1). The greatest toxicity was obtained through the AOAC 2011.02 protocol, especially for the Chilean mussel and Pacific oyster extracts. Similar toxicity levels (less than 15%) were observed in all extracts at 3.1 mg TE ml(-1). When assessed in Neuro-2a CBA, AOAC 2005.06 extracts presented the lowest matrix interferences, while the highest interferences were observed for AOAC 2011.02 in Magellan mussel and clam extracts. Finally, the AOAC 2005.06 and alternative protocols were compared using Chilean mussel samples fortified with 40 and 80 µg STX per 100 g meat. The AOAC 2005.06 method demonstrated better results. In conclusion, the AOAC 2005.06 extracts exhibited the fewest interferences in the Neuro-2a CBA. Therefore, this extraction method should be considered for the implementation of Neuro-2a CBA as a high-throughput screening methodology for PST detection.

Exploration of new functional endpoints in neuro-2a cells for the detection of the marine biotoxins saxitoxin, palytoxin and tetrodotoxin.

Marine neurotoxins accumulate in seafood and therewith represent a threat for consumers. At the European level, the use of in vivo bioassays is banned from 2015 onwards, except for the control of production areas. Cytotoxicity in the neuro-2a assay has been shown a promising in vitro alternative. However, given that cytotoxicity may be sensitive to confounding factors the current study investigates the suitability of functional endpoints as alternatives to cytotoxicity for the detection of marine neurotoxins. Microarray analyses were performed following exposure of neuro-2a cells to three marine neurotoxins (palytoxin (PlTx), saxitoxin (STX) and tetrodotoxin (TTX)) to identify genes up- or down-regulated that can be used as biomarkers for screening purposes. In addition to microarrays, the voltage dependent fluorescent probe bisoxonol was used to assess changes in cellular membrane potential. Biomarkers based on mRNA expression were detected for PlTx but not for STX and TTX. STX and TTX decreased the fluorescence of bisoxonol while PlTx showed no effect. When using cytotoxicity as the read out the neuro-2a assay detects these three neurotoxins at similar concentrations. Therefore it is concluded that the newly investigated endpoints in the neuro-2a assay are not preferred over cytotoxicity in a suitable broad and sensitive bioassay for the detection of marine neurotoxins in real practice.

Evaluation of Cytotoxicity and Cell Death Induced In Vitro by Saxitoxin in Mammalian Cells.

Since the cyanotoxin saxitoxin (STX) is a neurotoxin and induces ecological changes in aquatic environments, a potential risk to public and environmental health exists. However, data on STX-mediated cytotoxic and genotoxic effects are still scare. In order to gain a better understanding of the effects of this toxin, the cytotoxic and genotoxic potential of STX was examined in two mammalian cell lines. Neuro 2A (N2A), a neuroblastoma mouse cell line, and Vero cell line, derived from Vero green monkey kidney cells, were exposed to several concentrations of STX ranging from 0.5 to 64 nM to determine cell viability, induction of apoptosis (DNA fragmentation assay), and formation of micronuclei (MN) (cytokinesis-block micronucleus assay; CBMN) following 24 h of incubation. The half maximal effective concentration (EC50) values for STX calculated in cell viability tests were 1.01 nM for N2A and 0.82 nM for Vero cells. With increasing STX concentration there was evidence of DNA fragmentation indicating apoptosis induction in Vero cells with a 50% increase in DNA fragmentation compared to control at the highest STX concentration tested (3 nM). The results demonstrated no significant changes in the frequency of micronucleated binucleated cells in N2A and Vero cells exposed to STX, indicating the absence of genotoxicity under these test conditions. There was no apparent cellular necrosis as evidenced by a lack of formation of multinucleated cells. In conclusion, data reported herein demonstrate that STX produced death of both cell types tested through an apoptotic process.

A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation.

Saxitoxin (STX), a member of the family of paralytic shellfish poisoning toxins, poses toxicological and ecotoxicological risks. To develop an analytical recognition element for STX, a DNA aptamer (APT(STX1)) was previously discovered via an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by Handy et al. Our study focused on generating an improved aptamer based on APT(STX1) through rational site-directed mutation and truncation. In this study, we generated the aptamer, M-30f, with a 30-fold higher affinity for STX compared with APT(STX1). The Kd value for M-30f was 133 nM, which was calculated by Bio-Layer Interferometry. After optimization, we detected and compared the interaction of STX with aptamers (APT(STX1) or M-30f) through several techniques (ELISA, cell bioassay, and mouse bioassay). Both aptamers' STX-binding ability was demonstrated in all three methods. Moreover, M-30f performs better than its parent sequence with higher suppressive activity against STX. As a molecular recognition element, M-30f has good prospects for practical application.

Morbidity and mortality in stranded Cook Inlet beluga whales Delphinapterus leucas.

The endangered Cook Inlet (Alaska, USA) stock of beluga whales Delphinapterus leucas declined 47% between 1994 and 1998, from an estimated 653 whales to 347 whales, with a continued decline to approximately 312 in 2012. Between 1998 and 2013, 164 known dead strandings were reported by the National Marine Fisheries Service. Only 38 of these animals, or 23% of the known stranded carcasses, were necropsied. Carcasses were found between April and October. The majority of animals necropsied were adults (n=25), followed by juveniles (n=6), calves (n=3), and aborted fetuses (n=4). Eight of the 11 mature females were pregnant, post-partum, or lactating. Many (82%) of these belugas were in moderate to advanced autolysis, which hampered determination of a cause of death (COD). Each animal had a single primary COD assigned within a broad set of categories. The CODs were unknown (29%), trauma (18%), perinatal mortality (13%), mass stranding (13%), single stranding (11%), malnutrition (8%), or disease (8%). Other disease processes were coded as contributory or incidental to COD. Multiple animals had mild to moderate verminous pneumonia due to Stenurus arctomarinus, renal granulomas due to Crassicauda giliakiana, and ulcerative gastritis due to Anisakis sp. Each stranding affords a unique opportunity to obtain natural history data and evidence of human interactions, and, by long-term monitoring, to characterize pathologies of importance to individual and population health.

A tyrosine-containing analog of mu-conotoxin GIIIA as ligand in the receptor binding assay for paralytic shellfish poisons.

Development of novel analytical tools to detect marine biotoxins has been warranted in view of the apparent global pervasiveness of algal-derived shellfish poisoning, and the limitations of existing methods. Here, we describe the initial phase in the development and evaluation of a tyrosine-containing analog of µ-conotoxin (µ-CTX) GIIIA as an alternative to saxitoxin (STX) in a receptor binding assay (RBA) for paralytic shellfish poisons. The peptide analog was synthesized and characterized for structure and bioactivity. The major product of oxidation elicited paralytic symptoms in mice at a minimum dose of 1.31 mg kg(-1) (i.p.). Mass spectrometry analysis of the bioactive peptide gave a molecular mass of 2637.52 Da that was close to the predicted value. Iodination via chloramine-T produced non-, mono- and di-iodinated peptides (respectively, NIP, MIP and DIP). Competition assays against (3)H-STX revealed higher Ki and EC50 (P < 0.0001, ANOVA) indicating reduced affinity for the receptor, and limited displacement of receptor-bound STX. However, subsequent use of MIP may extend the application of RBA to detect small changes in toxin levels owing to its likely enhanced displacement by STX. This may be useful in analyzing samples with toxicities near the regulatory limit, or in establishing baseline values in high risk environments.

In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing.

The present study investigated if and to what extent murine stem cell-derived beating cardiomyocytes within embryoid bodies can be used as a broad screening in vitro assay for neurotoxicity testing, replacing for example in vivo tests for marine neurotoxins. Effect of nine model compounds, acting on either the Na(+), K(+), or Ca(2+) channels or the Na(+)/K(+) ATP-ase pump, on the beating was assessed. Diphenhydramine, veratridine, isradipine, verapamil and ouabain induced specific beating arrests that were reversible and none of the concentrations tested induced cytotoxicity. Three K(+) channel blockers, amiodarone, clofilium and sematilide, and the Na(+)/K(+) ATPase pump inhibitor digoxin had no specific effect on the beating. In addition, two marine neurotoxins i.e. saxitoxin and tetrodotoxin elicited specific beating arrests in cardiomyocytes. Comparison of the results obtained with cardiomyocytes to those obtained with the neuroblastoma neuro-2a assay revealed that the cardiomyocytes were generally somewhat more sensitive for the model compounds affecting Na(+) and Ca(2+) channels, but less sensitive for the compounds affecting K(+) channels. The stem cell-derived cardiomyocytes were not as sensitive as the neuroblastoma neuro-2a assay for saxitoxin and tetrodotoxin. It is concluded that the murine stem cell-derived beating cardiomyocytes provide a sensitive model for detection of specific neurotoxins and that the neuroblastoma neuro-2a assay may be a more promising cell-based assay for the screening of marine biotoxins.

Hapalindoles from the cyanobacterium fischerella: potential sodium channel modulators.

Hapalindoles make up a large group of bioactive metabolites of the cyanobacterial order Stigonematales. 12-epi-Hapalindole E isonitrile, 12-epi-hapalindole C isonitrile, 12-epi-hapalindole J isonitrile, and hapalindole L from Fischerella are acutely toxic for insect larvae; however, the biochemical targets responsible for the biological activities of hapalindoles are not understood. We describe here the electron impact mass spectra of these four hapalindole congeners; their structures were confirmed by nuclear magnetic resonance spectroscopy. In combination with the presented mass spectra of (15)N-labeled species and their retention times on a gas chromatography capillary column, a rapid and reliable determination should be possible in future research. The bioactivity of these hapalindoles was tested on mammalian cells focusing on their effects in the BE(2)-M17 excitable human neuroblastoma cell line. The fluorescent dye Alamar Blue was applied to monitor cytotoxicity, fura-2 to evaluate changes in the cytosolic calcium concentrations, and bis-oxonol to detect effects on membrane potential. Data showed that the hapalindoles did not affect cell viability of the neuroblastoma cells, even when they were incubated for 72 h. Neither depolarization nor initiation of calcium influx was observed in the cells upon hapalindole treatment. However, the data provide evidence that hapalindoles are sodium channel-modulating neurotoxins. They inhibited veratridine-induced depolarization in a manner similar to that of neosaxitoxin. Our data suggest hapalindoles should be added to the growing number of neurotoxic secondary metabolites, such as saxitoxins and anatoxins, already known in freshwater cyanobacteria. As stable congeners, hapalindoles may be a risk in freshwater ecosystems or agricultural water usage and should therefore be considered in water quality assessment.

Apoptosis of hemocytes from lions-paw scallop Nodipecten subnodosus induced with paralyzing shellfish poison from Gymnodinium catenatum.

The toxic dinoflagellate Gymnodinium catenatum produces paralyzing shellfish poisons (PSPs) that are consumed and accumulated by bivalves. Previously, we recorded a decrease in hemocytes 24h after injection of PSPs (gonyautoxin 2/3 epimers, GTX2/3) in the adductor muscle in the lions-paw scallop Nodipecten subnodosus. In this work, qualitative and quantitative analyses, in in vivo and in vitro experiments, revealed that the lower count of hemocytes results from cells undergoing typical apoptosis when exposed to GTX 2/3 epimers. This includes visible morphological alterations of the cytoplasmic membrane, damage to the nuclear membrane, condensation of chromatin, DNA fragmentation, and release of DNA fragments into the cytoplasm. Induction of apoptosis was accompanied by phosphatidylserine exposure to the outer cell membrane and activation of cysteine-aspartic proteases, caspase 3 and caspase 8. Addition of an inhibitor of caspase to the medium suppressed activation in hemocytes exposed to the toxins, suggesting that cell death was induced by a caspase-dependent apoptotic pathway. The results are important for future investigation of the scallop's immune system and should provide new insights into apoptotic processes in immune cells of scallops exposed to PSPs.