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Azaspiracids (AZAs) are lipid biotoxins produced by the marine dinoflagellates Azadinium and Amphidoma spp. that can accumulate in shellfish and cause food poisoning in humans. However, the mechanisms underlying the tolerance of shellfish to high levels of such toxins remain poorly understood. This study investigated the combined effects of detoxification metabolism and stress-related responses in scallops Chlamys farreri exposed to AZA. Scallops accumulated a maximum of 361.81 μg AZA1 eq/kg and 41.6 % AZA residue remained after 21 days of exposure. A range of AZA2 metabolites, including AZA19, AZA11, and AZA23, and trace levels of AZA2-GST, were detected. Total hemocyte counts significantly increased and ROS levels remained consistently high until gradually decreasing. Immune system activation mediated mitochondrial dysfunction and severe energy deficiency. DEGs increased over time, with key genes CYP2J6 and GPX6 contributing to AZA metabolism. These transcriptome and metabolic results identify the regulation of energy metabolism pathways, including inhibition of the TCA cycle and activation of carbohydrates, amino acids, and lipids. AZA also induced autophagy through the MAPK-AMPK signaling pathways, and primary inhibited PI3K/AKT to decrease mTOR pathway expression. Our results provide additional insights into the resistance of C. farreri to AZA, characterized by re-establishing redox homeostasis toward a more oxidative state., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

When humans consume seafood contaminated by lipophilic polyether phycotoxins, such as azaspiracids (AZAs), the toxins are mainly leached and absorbed in the small intestine, potentially causing intestinal damage. In this study, human intestinal epithelial Caco-2 cells were used to investigate the adverse effects of azaspiracid-2 (AZA-2) on human intestinal epithelial cells. Cell viability, apoptosis, oxidative damage and mitochondrial ultrastructure were investigated, and ribonucleic acid sequence (RNA-seq) analysis was applied to explore the potential mechanisms of AZA-2 toxicity to Caco-2 cells. Results showed that AZA-2 significantly reduced the proliferation of Caco-2 cells in a concentration-dependent response, and the 48 h EC 50 of AZA-2 was 12.65 nmol L -1 . AZA-2 can induce apoptosis in Caco-2 cells in a dose-dependent manner. Visible mitochondrial swelling, cristae disintegration, membrane rupture and autophagy were observed in Caco-2 cells exposed to AZA-2. Reactive oxygen species (ROS) and malondialdehyde (MDA) content were significantly increased in Caco-2 cells after 48 h of exposure to 1 and 10 nmol L -1 of AZA-2. Transcriptome analysis showed that KEGG pathways related to cellular oxidative damage and lipid metabolism were affected, mainly including mitophagy, oxidative phosphorylation, cholesterol metabolism, vitamin digestion and absorption, bile secretion and the peroxisome proliferator-activated receptor signaling pathway. The cytotoxic effects of AZA-2 on Caco-2 cells may be associated with ROS-mediated autophagy and apoptosis in mitochondrial cells. Results of this study improve understanding of the cytotoxicity and molecular mechanisms of AZA-2 on Caco-2 cells, which is significant for protecting human health.

The presence and impact of toxins have been detected in various regions worldwide ever since the discovery of azaspiracids (AZAs) in 1995. These toxins have had detrimental effects on marine resource utilization, marine environmental protection, and fishery production. Over the course of more than two decades of research and development, scientists from all over the world have conducted comprehensive studies on the in vivo metabolism, in vitro synthesis methods, pathogenic mechanisms, and toxicology of these toxins. This paper aims to provide a systematic introduction to the discovery, distribution, pathogenic mechanism, in vivo biosynthesis, and in vitro artificial synthesis of AZA toxins. Additionally, it will summarize various detection methods employed over the past 20 years, along with their advantages and disadvantages. This effort will contribute to the future development of rapid detection technologies and the invention of detection devices for AZAs in marine environmental samples.

Portimine, a recently identified cyclic imine produced by the dinoflagellate Vulcanodinium rugosum, has been described as a potent apoptotic agent in contrast to most of the cyclic imines that are well-known to be neurological toxins. As apoptosis can be a consequence of a high level of DNA lesions, we investigated the responses of portimine on several endpoints aimed at detecting DNA damage in the hepatic cell line HepaRG. Portimine induced phosphorylation of H2AX, which could possibly be consistent with the previously published induction of apoptosis with this toxin. In addition, detection of apoptosis through the activation of caspase-3, the induction of strand breaks detected by the comet assay as well as chromosome and genome mutations using the micronucleus assay were addressed. Surprisingly, portimine treatment resulted in increases in only γH2AX in differentiated HepaRG cells whereas no effects on the other endpoints were detected. These increases in γH2AX in the absence of genotoxic effects in the other tests could indicate that portimine could possibly induce a DNA replication stress and/or that the compound can be detoxified by the HepaRG cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Seafood represents a significant part of the human staple diet. In the recent years, the identification of emerging lipophilic marine toxins has increased, leading to the potential for consumers to be intoxicated by these toxins. In the present work, we investigate the presence of lipophilic marine toxins (both regulated and emerging) in commercial seafood products from non-European locations, including mussels Mytilus chilensis from Chile, clams Tawerea gayi and Metetrix lyrate from the Southeast Pacific and Vietnam, and food supplements based on mussels formulations of Perna canaliculus from New Zealand. All these products were purchased from European Union markets and they were analyzed by UPLC-MS/MS. Results showed the presence of the emerging pinnatoxin-G in mussels Mytilus chilensis at levels up to 5.2 µg/kg and azaspiracid-2 and pectenotoxin-2 in clams Tawera gayi up to 4.33 µg/kg and 10.88 µg/kg, respectively. This study confirms the presence of pinnatoxins in Chile, one of the major mussel producers worldwide. Chromatograms showed the presence of 13-desmethyl spirolide C in dietary supplements in the range of 33.2-97.9 µg/kg after an extraction with water and methanol from 0.39 g of the green lipped mussels powder. As far as we know, this constitutes the first time that an emerging cyclic imine toxin in dietary supplements is reported. Identifying new matrix, locations, and understanding emerging toxin distribution area are important for preventing the risks of spreading and contamination linked to these compounds.

Pinnatoxin G (PnTx-G) is a marine cyclic imine toxin produced by the dinoflagellate Vulcanodinium rugosum , frequently detected in edible shellfish from Ingril Lagoon (France). As other pinnatoxins, to date, no human poisonings ascribed to consumption of PnTx-G contaminated seafood have been reported, despite its potent antagonism at nicotinic acetylcholine receptors and its high and fast-acting toxicity after intraperitoneal or oral administration in mice. The hazard characterization of PnTx-G by oral exposure is limited to a single acute toxicity study recording lethality and clinical signs in non-fasted mice treated by gavage or through voluntary food ingestion, which showed differences in PnTx-G toxic potency. Thus, an acute toxicity study was carried out using 3 h-fasted CD-1 female mice, administered by gavage with PnTx-G (8-450 µg kg - 1 ). At the dose of 220 µg kg - 1 and above, the toxin induced a rapid onset of clinical signs (piloerection, prostration, hypothermia, abdominal breathing, paralysis of the hind limbs, and cyanosis), leading to the death of mice within 30 min. Except for moderate mucosal degeneration in the small intestine recorded at doses of 300 µg kg - 1 , the toxin did not induce significant morphological changes in the other main organs and tissues, or alterations in blood chemistry parameters. This acute oral toxicity study allowed to calculate an oral LD 50 for PnTx-G equal to 208 g kg - 1 (95% confidence limits: 155-281 µg kg - 1 ) and to estimate a provisional NOEL of 120 µg kg - 1 ., Competing Interests: The authors declare no conflict of interest.

Azaspiracids (AZAs) are marine biotoxins including a variety of analogues. Recently, novel AZAs produced by the Mediterranean dinoflagellate Azadinium dexteroporum were discovered (AZA-54, AZA-55, 3-epi-AZA-7, AZA-56, AZA-57 and AZA-58) and their biological effects have not been investigated yet. This study aimed to identify the biological responses (biomarkers) induced in mussels Mytilus galloprovincialis after the bioaccumulation of AZAs from A. dexteroporum . Organisms were fed with A. dexteroporum for 21 days and subsequently subjected to a recovery period (normal diet) of 21 days. Exposed organisms accumulated AZA-54, 3-epi-AZA-7 and AZA-55, predominantly in the digestive gland. Mussels' haemocytes showed inhibition of phagocytosis activity, modulation of the composition of haemocytic subpopulation and damage to lysosomal membranes; the digestive tissue displayed thinned tubule walls, consumption of storage lipids and accumulation of lipofuscin. Slight genotoxic damage was also observed. No clear occurrence of oxidative stress and alteration of nervous activity was detected in AZA-accumulating mussels. Most of the altered parameters returned to control levels after the recovery phase. The toxic effects detected in M. galloprovincialis demonstrate a clear biological impact of the AZAs produced by A. dexteroporum , and could be used as early indicators of contamination associated with the ingestion of seafood.

Azaspiracids (AZAs) are microalgal toxins that can accumulate in shellfish and lead to human intoxications. To facilitate their study and subsequent biomonitoring, purification from microalgae rather than shellfish is preferable; however, challenges remain with respect to maximizing toxin yields. The impacts of temperature, growth media, and photoperiod on cell densities and toxin production in Azadinium spinosum were investigated. Final cell densities were similar at 10 and 18 °C, while toxin cell quotas were higher (~3.5-fold) at 10 °C. A comparison of culture media showed higher cell densities and AZA cell quotas (2.5-5-fold) in f10k compared to f/2 and L1 media. Photoperiod also showed differences, with lower cell densities in the 8:16 L:D treatment, while toxin cell quotas were similar for 12:12 and 8:16 L:D treatments but slightly lower for the 16:8 L:D treatment. AZA1, -2 and -33 were detected during the exponential phase, while some known and new AZAs were only detected once the stationary phase was reached. These compounds were additionally detected in field water samples during an AZA event.

Pinnatoxins (PnTXs) are emerging neurotoxins that were discovered about 30 years ago. They are solely produced by the marine dinoflagellate Vulcanodinium rugosum , and may be transferred into the food chain, as they have been found in various marine invertebrates, including bivalves. No human intoxication has been reported to date although acute toxicity was induced by PnTxs in rodents. LD 50 values have been estimated for the different PnTXs through the oral route. At sublethal doses, all symptoms are reversible, and no neurological sequelae are visible. These symptoms are consistent with impairment of central and peripheral cholinergic network functions. In fact, PnTXs are high-affinity competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Moreover, their lethal effects are consistent with the inhibition of muscle nAChRs, inducing respiratory distress and paralysis. Human intoxication by ingestion of PnTXs could result in various symptoms observed in episodes of poisoning with natural nAChR antagonists. This review updates the available data on PnTX toxicity with a focus on their mode of action on cholinergic networks and suggests the effects that could be extrapolated on human physiology.

Background: Azaspiracids (AZAs) are marine toxins that are produced by Azadinium and Amphidoma dinoflagellates that can contaminate edible shellfish inducing a foodborne poisoning in humans, which is characterized by gastrointestinal symptoms. Among these, AZA1, -2, and -3 are regulated in the European Union, being the most important in terms of occurrence and toxicity. In vivo studies in mice showed that, in addition to gastrointestinal effects, AZA1 induces liver alterations that are visible as a swollen organ, with the presence of hepatocellular fat droplets and vacuoles. Hence, an in vitro study was carried out to investigate the effects of AZA1, -2, and -3 on liver cells, using human non-tumor IHH hepatocytes., Results: The exposure of IHH cells to AZA1, -2, or -3 (5 × 10 -12 -1 × 10 -7 M) for 24 h did not affect the cell viability and proliferation (Sulforhodamine B assay and 3 H-Thymidine incorporation assay), but they induced a significant concentration-dependent increase of mitochondrial dehydrogenases activity (MTT reduction assay). This effect depends on the activity of mitochondrial electron transport chain complex I and II, being counteracted by rotenone and tenoyl trifluoroacetone, respectively. Furthermore, AZAs-increased mitochondrial dehydrogenase activity was almost totally suppressed in the K + -, Cl - -, and Na + -free media and sensitive to the specific inhibitors of K ATP and hERG potassium channels, Na + /K + , ATPase, and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels., Conclusions: These results suggest that AZA mitochondrial effects in hepatocytes derive from an imbalance of intracellular levels of K + and, in particular, Cl - ions, as demonstrated by the selective reduction of toxin effects by CFTR chloride channel inhibition.

Marine biotoxins are produced by aquatic microorganisms and accumulate in shellfish or finfish following the food web. These toxins usually reach human consumers by ingestion of contaminated seafood, although other exposure routes like inhalation or contact have also been reported and may cause serious illness. This review shows the current data regarding the symptoms of acute intoxication for several toxin classes, including paralytic toxins, amnesic toxins, ciguatoxins, brevetoxins, tetrodotoxins, diarrheic toxins, azaspiracids and palytoxins. The information available about chronic toxicity and relative potency of different analogs within a toxin class are also reported. The gaps of toxicological knowledge that should be studied to improve human health protection are discussed. In general, gathering of epidemiological data in humans, chronic toxicity studies and exploring relative potency by oral administration are critical to minimize human health risks related to these toxin classes in the near future.

Azaspiracids (AZAs) are marine biotoxins produced by the genera Azadinium and Amphidoma, pelagic marine dinoflagellates that may accumulate in shellfish resulting in human illness following consumption. The complexity of these toxins has been well documented, with more than 40 structural variants reported that are produced by dinoflagellates, result from metabolism in shellfish, or are extraction artifacts. Approximately 34 μg of a new AZA with MW 823 Da (AZA26 (3)) was isolated from blue mussels ( Mytilus edulis), and its structure determined by MS and NMR spectroscopy. AZA26, possibly a bioconversion product of AZA5, lacked the C-20-C-21 diol present in all AZAs reported thus far and had a 21,22-olefin and a keto group at C-23. Toxicological assessment of 3 using an in vitro model system based on Jurkat T lymphocyte cells showed the potency to be ∼30-fold lower than that of AZA1. The corresponding 21,22-dehydro-23-oxo-analogue of AZA10 (AZA28) and 21,22-dehydro analogues of AZA3, -4, -5, -6, -9, and -10 (AZA25, -48 (4), -60, -27, -49, and -61, respectively) were also identified by HRMS/MS, periodate cleavage reactivity, conversion from known analogues, and NMR (for 4 that was present in a partially purified sample of AZA7).

A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole-genome mRNA expression of undifferentiated intestinal Caco-2 cells. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Azaspiracids (AZAs) are marine algal toxins that can be accumulated by edible shellfish to cause a foodborne gastrointestinal poisoning in humans. In the European Union, only AZA1, -2 and -3 are currently regulated and their concentration in shellfish is determined through their toxic equivalency factors (TEFs) derived from the intraperitoneal lethal potency in mice. Nevertheless, considering the potential human exposure by oral route, AZAs TEFs should be calculated by comparative oral toxicity data. Thus, the acute oral toxicity of AZA1, -2 and -3 was investigated in female CD-1 mice treated with different doses (AZA1: 135-1100μg/kg; AZA2 and AZA3: 300-1100μg/kg) and sacrificed after 24h or 14days. TEFs derived from the median lethal doses (LD 50 ) were 1.0, 0.7 and 0.5, respectively for AZA1, -2 and -3. In fact, after 24h from gavage administration, LD 50s were 443μg/kg (AZA1; 95% CL: 350-561μg/kg), 626μg/kg (AZA2; 95% CL: 430-911μg/kg) and 875μg/kg (AZA3; 95% CL: 757-1010μg/kg). Mice dead more than 5h after the treatment or those sacrificed after 24h (doses: ≥175μg AZA1/kg, ≥500μg AZA2/kg and ≥600μg AZA3/kg) showed enlarged pale liver, while increased serum markers of liver alteration were recorded even at the lowest doses. Blood chemistry revealed significantly increased serum levels of K + ions (≥500mg/kg), whereas light microscopy showed tissue changes in the gastrointestinal tract, liver and spleen. No lethality, macroscopic, tissue or haematological changes were recorded two weeks post exposure, indicating reversible toxic effects. LC-MS/MS analysis of the main organs showed a dose-dependency in gastrointestinal absorption of these toxins: at 24h, the highest levels were detected in the stomach and, in descending order, in the intestinal content, liver, small intestine, kidneys, lungs, large intestine, heart as well as detectable traces in the brain. After 14days, AZA1 and AZA2 were still detectable in almost all the organs and intestinal content., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The identification of a new suite of toxins, called azaspiracids (AZA), as the cause of human illnesses after the consumption of shellfish from the Irish west coast in 1995, resulted in interest in understanding the global distribution of these toxins and of species of the small dinoflagellate genus Azadinium, known to produce them. Clonal isolates of four species of Azadinium, A. poporum, A. cuneatum, A. obesum and A. dalianense were obtained from incubated sediment samples collected from Puget Sound, Washington State in 2016. These Azadinium species were identified using morphological characteristics confirmed by molecular phylogeny. Whereas AZA could not be detected in any strains of A. obesum, A. cuneatum and A. dalianense, all four strains of A. poporum produced a new azaspiracid toxin, based on LC-MS analysis, named AZA-59. The presence of AZA-59 was confirmed at low levels in situ using a solid phase resin deployed at several stations along the coastlines of Puget Sound. Using a combination of molecular methods for species detection and solid phase resin deployment to target shellfish monitoring of toxin at high-risk sites, the risk of azaspiracid shellfish poisoning can be minimized., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Yessotoxins (YTX) and azaspiracids (AZAs) are marine toxins produced by phytoplanktonic dinoflagellates that get accumulated in filter feeding shellfish and finally reach human consumers through the food web. Both toxin classes are worldwide distributed, and food safety authorities have regulated their content in shellfish in many countries. Recently, YTXs and AZAs have been described as compounds with subacute cardiotoxic potential in rats owed to alterations of the cardiovascular function and ultrastructural heart damage. These molecules are also well known in vitro inducers of cell death. The aim of this study was to explore the presence of cardiomyocyte death after repeated subacute exposure of rats to AZA-1 and YTX for 15 days. Because autophagy and apoptosis are often found in dying cardiomyocytes, several autophagic and apoptotic markers were determined by western blot in heart tissues of these rats. The results showed that hearts from YTX-treated rats presented increased levels of the autophagic markers microtubule-associated protein light chain 3-II (LC3-II) and beclin-1, nevertheless AZA-1-treated hearts evidenced increased levels of the apoptosis markers cleaved caspase-3 and -8, cleaved PARP and Fas ligand. Therefore, while YTX-induced damage to the heart triggers autophagic processes, apoptosis activation occurs in the case of AZA-1. For the first time, activation of cell death signals in cardiomyocytes is demonstrated for these toxins with in vivo experiments, which may be related to alterations of the cardiovascular function.

Azaspiracids (AZA) are a group of food poisoning phycotoxins that are known to accumulate in shellfish. They are produced by some species of the planktonic dinophycean taxon Amphidomataceae. Azaspiracids have been first discovered in Ireland but are now reported in shellfish from numerous global sites thus showing a wide distribution. In shellfish samples collected in 2009 near Huelva (Spain), AZA was also found along the Andalusian Atlantic coast for the first time. Analysis using LC-MS/MS revealed the presence of two different AZA analogues in different bivalve shellfish species (Chamelea gallina, Cerastoderma edule, Donax trunculus, and Solen vagina). In a number of samples, AZA levels exceeded the EU regulatory level of 160μg AZA-1 eq. kg -1 (reaching maximum levels of >500μg AZA-1 eq. kg -1 in Chamelea gallina and >250μg AZA-1 eq. kg -1 in Donax trunculus) causing closures of some local shellfish production areas. One dinophyte strain established from the local plankton during the AZA contamination period and determined as Amphidoma languida was in fact toxigenic, and its AZA profile disclosed it as the causative species: it contained AZA-2 as the main compound and the new compound AZA-43 initially detected in the shellfish. AZA-43 had the same mass as AZA-3, but produced different collision induced dissociation (CID) spectra. High resolution mass spectrometric measurements indicated that there is an unsaturation in the H, I ring system of AZA-43 distinguishing it from the classical AZA such as AZA-1, -2, and -3. Furthermore, the Spanish strain was different from the previously reported AZA profile of the species that consist of AZA-38 and AZ-39. In molecular phylogenetics, the Andalusian strain formed a monophyletic group together with other strains of Am. languida, but ITS sequences data revealed surprisingly high intragenomic variability. The first Andalusian case of AZA contamination of shellfish above the EU regulatory limit reported here clearly revealed the risk of azaspiracid poisoning (AZP) for this area and also for the Atlantic coast of Iberia and North Africa. The present study underlines the need for continuous monitoring of AZA and the organisms producing such toxins., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Azaspiracids (AZAs) are the most recently discovered group of biotoxins and are the cause of azaspiracid shellfish poisoning (AZP) in humans. To date over thirty analogues have been identified. However, toxicological studies of AZAs are limited due to the lack of availability of toxins and toxin standards. Most data available are on acute toxicity and there are no data available on genotoxicity of AZAs. This study presents an integrated approach investigating the genotoxic potential of AZA1-3 in cell culture systems using the Comet assay combined with assays to provide information on possible apoptotic processes, cytotoxicity and changes in cell number. Results demonstrate a time and dose dependent increase in DNA fragmentation in most cell lines, indicating a genotoxic effect of AZA1-3. However, a significant reduction in cell number and a clear shift from early to late apoptosis was observed for all analogues in Jurkat T cells and HepG-2 cells; CaCo-2 cells did not show a clear apoptotic profile. Late apoptotic/necrotic cells correlate well with the percentage of tail DNA for all analogues in all three cell lines. All data taken together indicate that AZA1-3 is not genotoxic per se and demonstrate apoptotic/necrotic processes to be involved to some extent in AZAs toxicity. The sensitivities of cell lines and the different potencies of AZA1-3 are in agreement with the literature available. The order of sensitivity for all three AZAs tested in the present study is, in increasing order, CaCo-2 cells < HepG-2 cells < Jurkat T cells. The order of potency of AZA1-3 varies among the cell lines., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Azaspiracids (AZAs) are marine toxins produced by Azadinium spinosum that get accumulated in filter feeding shellfish through the food-web. The first intoxication was described in The Netherlands in 1990, and since then several episodes have been reported worldwide. Azaspiracid-1, AZA-2, and AZA-3 presence in shellfish is regulated by food safety authorities of several countries to protect human health. Azaspiracids have been related to widespread organ damage, tumorogenic properties and acute heart rhythm alterations in vivo but the mechanism of action remains unknown. Azaspiracid toxicity kinetics in vivo and in vitro suggests accumulative effects. We studied subacute cardiotoxicity in vivo after repeated exposure to AZA-1 by evaluation of the ECG, arterial blood pressure, plasmatic heart damage biomarkers, and myocardium structure and ultrastructure. Our results showed that four administrations of AZA-1 along 15 days caused functional signs of heart failure and structural heart alterations in rats at doses ranging from 1 to 55 µg/kg. Azaspiracid-1 altered arterial blood pressure, tissue inhibitors of metalloproteinase-1 plasma levels, heart collagen deposition, and ultrastructure of the myocardium. Overall, these data indicate that repeated exposure to low amounts of AZA-1 causes cardiotoxicity, at doses that do not induce signs of other organic system toxicity. Remarkably, human exposure to AZAs considering current regulatory limits of these toxins may be dangerously close to clearly cardiotoxic doses in rats. These findings should be considered when human risk is estimated particularly in high cardiovascular risk subpopulations., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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.

Azaspiracid-1 is an algal toxin that accumulates in edible mussels, and ingestion may result in human illness as manifested by vomiting and diarrhoea. When injected into mice, it causes neurotoxicological symptoms and death. Although it is well known that azaspiracid-1 is toxic to most cells and cell lines, little is known about its biological target(s). A rat PC12 cell line, commonly used as a model for the peripheral nervous system, was used to study the neurotoxicological effects of azaspiracid-1. Azaspiracid-1 induced differentiation-related morphological changes followed by a latter cell death. The differentiated phenotype showed peripherin-labelled neurite-like processes simultaneously as a specific isoform of peripherin was down-regulated. The precise mechanism behind this down-regulation remains uncertain. However, this study provides new insights into the neurological effects of azaspiracid-1 and into the biological significance of specific isoforms of peripherin.

Two strains of Azadinium poporum, one from the Korean West coast and the other from the North Sea, were mass cultured for isolation of new azaspiracids. Approximately 0.9 mg of pure AZA-36 (1) and 1.3 mg of pure AZA-37 (2) were isolated from the Korean (870 L) and North Sea (120 L) strains, respectively. The structures were determined to be 3-hydroxy-8-methyl-39-demethyl-azaspiracid-1 (1) and 3-hydroxy-7,8-dihydro-39-demethyl-azaspiracid-1 (2) by ¹H- and (13)C-NMR. Using the Jurkat T lymphocyte cell toxicity assay, (1) and (2) were found to be 6- and 3-fold less toxic than AZA-1, respectively.

Spirolides are a large family of lipophilic marine toxins produced by dinoflagellates that have been detected in contaminated shellfish. Among them, 13,19-didesmethyl and 13-desmethyl spirolide C phycotoxins are widely distributed and their mode of action needs to be clearly defined. In order to further characterize the pharmacological profiles of these phycotoxins on various nicotinic acetylcholine receptor (nAChR) subtypes and to examine whether they act on muscarinic receptors (mAChRs), functional electrophysiological studies and competition binding experiments have been performed. While 13-desmethyl spirolide C interacted efficiently with sub-nanomolar affinities and low selectivity with muscular and neuronal nAChRs, 13,19-didesmethyl spirolide C was more selective of muscular and homopentameric α7 receptors and recognized only weakly neuronal heteropentameric receptors, especially the α4β2 subtype. Thus, the presence of an additional methyl group on the tetrahydropyran ring significantly modified the pharmacological profile of 13-desmethyl spirolide C by notably increasing its affinity on certain neuronal nAChRs. Structural explanations of this selectivity difference are proposed, based on molecular docking experiments modeling different spirolide-receptor complexes. In addition, the 2 spirolides interacted only with low micromolar affinities with the 5 mAChRs, highlighting that the toxicity of the spirolide C analogs is mainly due to their high inhibition potency on various peripheral and central nAChRs and not to their low ability to interact with mAChR subtypes., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Azaspiracids (AZAs) are marine biotoxins produced by dinoflagellates that can accumulate in shellfish, which if consumed can lead to poisoning events. AZA7-10, 7-10, were isolated from shellfish and their structures, previously proposed on the basis of only LC-MS/MS data, were confirmed by NMR spectroscopy. Purified AZA4-6, 4-6, and 7-10 were accurately quantitated by qNMR and used to assay cytotoxicity with Jurkat T lymphocyte cells for the first time. LC-MS(MS) molar response studies performed using isocratic and gradient elution in both selected ion monitoring and selected reaction monitoring modes showed that responses for the analogues ranged from 0.3 to 1.2 relative to AZA1, 1. All AZA analogues tested were cytotoxic to Jurkat T lymphocyte cells in a time- and concentration-dependent manner; however, there were distinct differences in their EC50 values, with the potencies for each analogue being: AZA6 > AZA8 > AZA1 > AZA4 ≈ AZA9 > AZA5 ≈ AZA10. This data contributes to the understanding of the structure-activity relationships of AZAs.

Azaspiracid (AZA) poisoning was unknown until 1995 when shellfish harvested in Ireland caused illness manifesting by vomiting and diarrhoea. Further in vivo/vitro studies showed neurotoxicity linked with AZA exposure. However, the biological target of the toxin which will help explain such potent neurological activity is still unknown. A region of Irish coastline was selected and shellfish were sampled and tested for AZA using mass spectrometry. An outbreak was identified in 2010 and samples collected before and after the contamination episode were compared for their metabolite profile using high resolution mass spectrometry. Twenty eight ions were identified at higher concentration in the contaminated samples. Stringent bioinformatic analysis revealed putative identifications for seven compounds including, glutarylcarnitine, a glutaric acid metabolite. Glutaric acid, the parent compound linked with human neurological manifestations was subjected to toxicological investigations but was found to have no specific effect on the sodium channel (as was the case with AZA). However in combination, glutaric acid (1 mM) and azaspiracid (50 nM) inhibited the activity of the sodium channel by over 50%. Glutaric acid was subsequently detected in all shellfish employed in the study. For the first time a viable mechanism for how AZA manifests itself as a toxin is presented.

Manila clam stock from Arcachon Bay, France, is declining, as is commercial harvest. To understand the role of environmental biotic interactions in this decrease, effects of a toxic dinoflagellate, Alexandrium ostenfeldii, which blooms regularly in Arcachon bay, and the interaction with perkinsosis on clam physiology were investigated. Manila clams from Arcachon Bay, with variable natural levels of perkinsosis, were exposed for seven days to a mix of the nutritious microalga T-Iso and the toxic dinoflagellate A. ostenfeldii, a producer of spirolides, followed by seven days of depuration fed only T-Iso. Following sacrifice and quantification of protozoan parasite Perkinsus olseni burden, clams were divided into two groups according to intensity of the infection ("Light-Moderate" and "Moderate-Heavy"). Hemocyte and plasma responses, digestive enzyme activities, antioxidant enzyme activities in gills, and histopathological responses were analyzed. Reactive oxygen species (ROS) production in hemocytes and catalase (CAT) activity in gills increased with P. olseni intensity of infection in control clams fed T-Iso, but did not vary among A. ostenfeldii-exposed clams. Exposure to A. ostenfeldii caused tissue alterations associated with an inflammatory response and modifications in hemocyte morphology. In the gills, superoxide dismutase (SOD) activity decreased, and an increase in brown cell occurrence was seen, suggesting oxidative stress. Observations of hemocytes and brown cells in tissues during exposure and depuration suggest involvement of both cell types in detoxication processes. Results suggest that exposure to A. ostenfeldii disrupted the pro-/anti-oxidant response of clams to heavy P. olseni intensity. In addition, depressed mitochondrial membrane potential (MMP) in hemocytes of clams exposed to A. ostenfeldii suggests that mitochondrial functions are regulated to maintain homeostasis of digestive enzyme activity and condition index., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Since azaspiracid-1 (AZA1) was identified in 1998, the number of AZA analogues has increased to over 30. The development of an LC-MS method using a neutral mobile phase led to the discovery of isomers of AZA1, AZA2, and AZA3, present at ~2-16% of the parent analogues in phytoplankton and shellfish samples. Under acidic mobile phase conditions, isomers and their parents are not separated. Stability studies showed that these isomers were spontaneous epimerization products whose formation is accelerated with the application of heat. The AZA1 isomer was isolated from contaminated shellfish and identified as 37-epi-AZA1 by nuclear magnetic resonance (NMR) spectroscopy and chemical analyses. Similar analysis indicated that the isomers of AZA2 and AZA3 corresponded to 37-epi-AZA2 and 37-epi-AZA3, respectively. The 37-epimers were found to exist in equilibrium with the parent compounds in solution. 37-epi-AZA1 was quantitated by NMR, and relative molar response studies were performed to determine the potential differences in LC-MS response of AZA1 and 37-epi-AZA1. Toxicological effects were determined using Jurkat T lymphocyte cells as an in vitro cell model. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that 37-epi-AZA1 elicited a lethal response that was both concentration- and time-dependent, with EC50 values in the subnanomolar range. On the basis of EC50 comparisons, 37-epi-AZA1 was 5.1-fold more potent than AZA1. This data suggests that the presence of these epimers in seafood products should be considered in the analysis of AZAs for regulatory purposes.

Following a review of official control data on shellfish in France, Ingril Lagoon had been identified as a site where positive mouse bioassays for lipophilic toxins had been repeatedly observed. These unexplained mouse bioassays, also called atypical toxicity, coincided with an absence of regulated toxins and rapid death times in mice observed in the assay. The present study describes pinnatoxin G as the main compound responsible for the toxicity observed using the mouse bioassay for lipophilic toxins. Using a well-characterised standard for pinnatoxin G, LC-MS/MS analysis of mussel samples collected from 2009 to 2012 revealed regular occurrences of pinnatoxin G at levels sufficient to account for the toxicity in the mouse bioassays. Baseline levels of pinnatoxin G from May to October usually exceeded 40 μg kg(-1) in whole flesh, with a maximum in September 2010 of around 1200 μg kg(-1). These concentrations were much greater than those at the other 10 sites selected for vigilance testing, where concentrations did not exceed 10 μg kg(-1) in a 3-month survey from April to July 2010, and where rapid mouse deaths were not typically observed. Mussels were always more contaminated than clams, confirming that mussel is a good sentinel species for pinnatoxins. Profiles in mussels and clams were similar, with the concentration of pinnatoxin A less than 2% that of pinnatoxin G, and pteriatoxins were only present in non-quantifiable traces. Esters of pinnatoxin G could not be detected by analysis of extracts before and after alkaline hydrolysis. Analysis with a receptor-binding assay showed that natural pinnatoxin G was similarly active on the nicotinic acetylcholine receptor as chemically synthesized pinnatoxin G. Culture of Vulcanodinium rugosum, previously isolated from Ingril lagoon, confirmed that this alga is a pinnatoxin G producer (4.7 pg cell(-1)). Absence of this organism from the water column during prolonged periods of shellfish contamination and the dominance of non-motile life stages of V. rugosum both suggest that further studies will be required to fully describe the ecology of this organism and the accumulation of pinnatoxins in shellfish., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Gymnodimine-A and 13-desmethyl spirolide C are marine toxins belonging to the cyclic imine group produced by Karenia selliformis and Alexandrium ostenfeldii/peruvianum dinoflagellates, respectively. The aim of this work was to analyze the pharmacological properties of both toxins (at sub-lethal doses) on neuromuscular excitability, when injected locally to isoflurane-anesthetized mice, using a multimodal minimally-invasive in vivo electrophysiological approach. The main effect of both toxins was a marked reversible time- and dose-dependent decrease of the compound muscle action potential recorded from the tail muscle in response to caudal motor nerve stimulation. The dose-response curves of gymnodimine-A and 13-desmethyl spirolide C effect on the maximal amplitude of compound muscle action potential revealed 50% inhibitory doses of 51 ng/mouse (i.e. 1.6 μg/kg or 3.3 nmol/kg mouse) and 0.18 ng/mouse (i.e. 6 ng/kg or 0.01 nmol/kg mouse), respectively. The blocking effect occurred without significant modification of motor nerve excitability parameters. It is concluded that the inhibition of the mouse compound muscle action potential, induced by gymnodimine-A and 13-desmethyl spirolide C, results from an action of these toxins at the level of the skeletal neuromuscular junction, since both cyclic imine toxins are known to interact and block muscle-type nicotinic acetylcholine receptors. In the present in vivo study, 13-desmethyl spirolide C was about 300 fold more active than gymnodimine-A on equimolar basis. The present in vivo approach, associated to recent progress in chemical synthesis of cyclic imine toxins, paves the way for more detailed structure-activity studies to obtain new and more potent synthetic analogs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Azaspiracid-1 (AZA-1) inhibits endocytosis, but the consequences of this alteration on cellular processes are unknown. We hypothesized that the inhibition of endocytosis is a key step of the mode of action of AZA-1, leading to perturbation of cellular processes dependent on proper functioning of endocytic machinery. We tested this working hypothesis by probing whether AZA-1 can alter the maturation of cathepsin D in MCF-7 epithelial cells, as a model system. We found that cell treatment with AZA-1 inhibited the conversion of 52 kDa procathepsin D into the mature 30 kDa protein. The effects induced by AZA-1 were similar to those elicited by chlorpromazine and other agents preventing proper maturation of lysosomal enzymes, indicating that the inhibition of endocytic transfer of proforms to late endosomes/lysosomess is responsible for the effect induced by the toxin. By immunofluorescence microscopy, we found no colocalization of cathepsin D and the early endosomal marker EEA-1 in control cells, where most of cathepsin D resides in late endosomes/lysosomes. Co-localization of cathepsin D and EEA-1 immunoreactivity, in turn, was found in cells exposed to AZA-1, indicating that the toxin blocks protein maturation at the early steps of endocytosis, causing accumulation of procathepsin D in early endosomes. The molecular alteration induced by AZA-1 involved both secreted and intracellular pools of procathepsin D, showing that the toxin effect does not result from a general impairment of vesicular trafficking but is the outcome of a perturbed centripetal process. Furthermore, AZA-1 was found to inhibit procathepsin D maturation also in normal fibroblasts, showing that this molecular response is induced by this toxin in different cell types. The data we obtained corroborated our hypothesis and provide a unified molecular frame for the mode of action of AZAs in animal models, involving a primary alteration of endocytic processes.

Pinnatoxins belong to the cyclic imine (CI) group of marine toxins with a unique toxicological profile. The need for structural integrity of the aliphatic 7-membered cyclic imine for the potent bioactivity of pinnatoxins has been experimentally demonstrated. In this study, we probe interconversion of the natural cyclic imine and its open form, pinnatoxin A amino ketone (PnTX AK), under physiologically relevant aqueous conditions. Our studies demonstrate the high stability of PnTX A. The unusual stability of the imine ring in PnTX A has implications for its oral toxicity and detoxification. These studies, as well the access to PnTX amino ketone, were enabled by the total synthesis of (+)-pinnatoxin A completed previously in our laboratory.

Azadinium spinosum, a small toxic dinoflagellate, was recently isolated and identified as a primary producer of azaspiracid toxins (AZAs). Previous experiments related to AZA accumulation in blue mussels upon direct feeding with A. spinosum revealed increased mussel mortality and had negative effects on the thickness of the digestive gland tubules. Therefore we conducted follow up experiments in order to study effects of A. spinosum on mussel feeding behaviour. Individual assessment of mussel feeding time activity (FTA), clearance rate (CR), filtration rate (TFR), absorption rate (AR), faeces and pseudofaeces production were carried out on mussel fed either toxic (A. spinosum) or non-toxic (Isochrisis aff. galbana (T-Iso)) diets. Furthermore, AZA accumulation and biotransformation in mussels were followed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A. spinosum had a significant effect on mussel feeding behaviour compared to T-Iso: CR was lower by a factor of 6, FTA by a factor of 5, TFR by a factor of 3 and AR even decreased to negative values for the last day of exposure. Even so, a rapid AZA accumulation was observed during the first hours of the trial; less than 6h of feeding were required to reach AZA concentration in mussel above regulatory level. In consistence with physiological observations, AZA concentration of about 200 μg kg(-1) did not increase further until the end of the study. AZA bioconversion was also found to be a fast process: after 3h of exposure AZA17, -19 and AZA7-10 were already found, with a proportion of AZA17 equal to AZA2. These results show a negative effect of A. spinosum on blue mussel feeding activity and indicate a possible regulation of AZA uptake by decreasing filtration and increasing pseudofaeces production., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Toxins from the okadaic acid (OA) and azaspiracid (AZA) group cause considerable negative health effects in consumers when present in shellfish above certain levels. The main symptoms, dominated by diarrhoea, are caused by damage to the gastrointestinal (GI) tract. Even though OA and AZAs exert toxicity via different mechanisms, it is important to find out whether they may enhance the health effects if present together since they act on the same organs and are regulated individually. In this study, the main issue was the possibility of enhanced lethality in mice upon combined oral exposure to OA and AZA1. In addition, pathological effects in several organs and effects on absorption from the GI tract were studied. Although the number of mice was small due to low availability of AZA1, the results indicate no additive or synergistic effect on lethality when AZA1 and OA were given together. Similar lack of increased toxicity was observed concerning pathological effects that were restricted to the GI-tract. OA and AZA1 were absorbed from the GI-tract to a very low degree, and when given together, uptake was reduced. Taken together, these results indicate that the present practice of regulating toxins from the OA and AZA group individually does not present an unwanted increased risk for consumers of shellfish., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Azaspiracids (AZA) are polyether marine dinoflagellate toxins that accumulate in shellfish and represent an emerging human health risk. Although human exposure is primarily manifested by severe and protracted diarrhea, this toxin class has been shown to be highly cytotoxic, a teratogen to developing fish, and a possible carcinogen in mice. Until now, AZA's molecular target has not yet been determined. Using three independent methods (voltage clamp, channel binding assay, and thallium flux assay), we have for the first time demonstrated that AZA1, AZA2, and AZA3 each bind to and block the hERG (human ether-à-go-go related gene) potassium channel heterologously expressed in HEK-293 mammalian cells. Inhibition of K(+) current for each AZA analogue was concentration-dependent (IC(50) value range: 0.64-0.84 μM). The mechanism of hERG channel inhibition by AZA1 was investigated further in Xenopus oocytes where it was shown to be an open-state-dependent blocker and, using mutant channels, to interact with F656 but not with Y652 within the S6 transmembrane domain that forms the channel's central pore. AZA1, AZA2, and AZA3 were each shown to inhibit [(3)H]dofetilide binding to the hERG channel and thallium ion flux through the channel (IC(50) value range: 2.1-6.6 μM). AZA1 did not block the K(+) current of the closely related EAG1 channel. Collectively, these data suggest that the AZAs physically block the K(+) conductance pathway of hERG1 channels by occluding the cytoplasmic mouth of the open pore. Although the concentrations necessary to block hERG channels are relatively high, AZA-induced blockage may prove to contribute to the toxicological properties of the AZAs.

Although there has been much progress with regard to marine toxins from dinoflagellates, much remains to be done. Because these compounds are a seafood consumer risk, the demands cover from legislative to scientific aspects. Legislation is required for all new toxins that appear in the coasts. On the other hand, it is important to understand the toxicity of the different analogues, in terms of both the relative toxicity to reference compounds and the mechanism of toxicity itself, both acute and long-term. For this, a uniform approach to do toxic studies is necessary, especially acute toxicity. The need for pure standards in sufficient supply and the understanding of the mode of action of some of the compounds (such as yessotoxin or azaspiracids) will help the development of another important field, the use of marine toxins as drug leads, and the chemistry around them.

Azadinium spinosum (Elbrächter and Tillmann), a small marine dinoflagellate, has been recently described as a de novo producer of azaspiracid-1 and -2 (AZA1 and -2) diarrhoeic toxins. A culture of A. spinosum was established in our laboratory and optimised for pilot-scale production of this organism, to evaluate and understand AZA1 and -2 accumulation and biotransformation in blue mussels (Mytilus edulis) fed with A. spinosum. Adult mussels were continuously exposed to A. spinosum over 1 week in 160 L cylindrical conical tanks. Three different diets were tested for contamination: 5000, 10 000 cells mL(-1) of A. spinosum and a mixture of 5000 cells mL(-1) of A. spinosum with 5000 cells mL(-1) of Isochrysis aff. galbana (T-Iso, CCAP 927/14). During the subsequent period of detoxification (2 weeks), contaminated mussels were continuously fed with 5000 cells mL(-1) of T-Iso. Kinetics of accumulation, detoxification and biotransformation were evaluated, as well as the toxin distribution and the effect of A. spinosum on mussel digestive gland tubules. M. edulis fed on A. spinosum in the three tested conditions; this finding confirmed our recent experiments feeding A. spinosum to mussels. The original algal toxins AZA1 and -2, as well as mussel metabolites AZA3 to 12, -17, -19, -21 and -23 were found during these trials. After as little as 6 h, azaspiracid contents in mussels reached the EU regulatory limit, and metabolites were observed in all conditions at approximately 25% of the total AZA content. This fraction exceeded 50% after 24 h, and continued to increase until the end of the study. AZA17 and -19 were found to be the main metabolites, with AZA17 concentrations estimated in the same order of magnitude as that of the main algal toxin, AZA1., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Azaspiracids (AZAs) are polyether marine dinoflagellate toxins that accumulate in shellfish and represent an emerging human health risk. Although there have been no deaths associated with the AZA toxins, humans exposed to AZAs experience severe gastrointestinal symptoms. This toxin class has been shown to be highly cytotoxic, a teratogen to developing fish, and a possible carcinogen in mice. Just recently, the AZAs have been shown to be potassium channel inhibitors. This report employed multiple human cell lines [Jurkat T lymphocytes, Caco-2 intestinal cells, and BE(2)-M17 neuroblastoma cells] in characterizing cytotoxicity and pathways of apoptosis. Cytotoxicity experiments were consistent with published literature that has shown that AZA1 is cytotoxic in both a concentration- and time-dependent manner to each cell type tested, with mean EC(50) values ranging between 1.1 and 7.4 nM. Despite the absence of morphological indices indicating apoptosis, caspase-3/7 activity was higher in all cell types treated with AZA1. Furthermore, in T lymphocytes, the most sensitive cell type, the activities of initiator caspase-2 and caspase-10 and concentrations of intracellular cytochrome c were elevated. DNA fragmentation was also observed for T lymphocytes exposed to AZA1-AZA3. Collectively, our data confirm that AZA1 was highly cytotoxic to multiple cell types and that cells exposed to AZA1 underwent atypical apoptosis, possibly in conjunction with necrotic cytotoxicity.

Azaspiracids (AZA) are polyether marine toxins of dinoflagellate origin that accumulate in shellfish and represent an emerging human health risk. Although monitored and regulated in many European and Asian countries, there are no monitoring programs or regulatory requirements in the United States for this toxin group. This did not prove to be a problem until June 2009 when AZAs were identified in US seafood for the first time resulting in human intoxications and further expanding their global distribution. Efforts are now underway in several laboratories to better define the effects and mechanism(s) of action for the AZAs. Our investigations have employed Jurkat T lymphocyte cells as an in vitro model to characterize the toxicological effects of AZA1, AZA2, and AZA3. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that AZA1, AZA2, and AZA3 each elicited a lethal response that was both concentration- and time-dependent, with EC(50) values in the sub- to low nanomolar range. On the basis of EC(50) comparisons, the order of potency was as follows: AZA2 > AZA3 > AZA1, with toxic equivalence factors (TEFs) relative to AZA1 of 8.3-fold and 4.5-fold greater for AZA2 and AZA3, respectively. Image analysis of exposed cells using Nomarski differential interference contrast (DIC) imaging and fluorescent imaging of cellular actin indicated that the morphological effects of AZA1 on this cell type are unique relative to the effects of AZA2 and AZA3. Collectively, our data support the growing body of evidence suggesting that natural analogues of AZA are highly potent and that they may have multiple molecular targets., (© 2012 American Chemical Society)

Spirolides are marine phycotoxins produced by the dinoflagellates Alexandrium ostenfeldii and A. peruvianum. Here we report that 13-desmethyl spirolide C shows little cytotoxicity when incubated with various cultured mammalian cell lines. When administered to mice by intraperitoneal (ip) injection, however, this substance was highly toxic, with an LD(50) value of 6.9 µg/kg body weight (BW), showing that such in vitro cytotoxicity tests are not appropriate for predicting the in vivo toxicity of this toxin. Four other spirolides, A, B, C, and 20-methyl spirolide G, were also toxic to mice by ip injection, with LD(50) values of 37, 99, 8.0 and 8.0 µg/kg BW respectively. However, the acute toxicities of these compounds were lower by at least an order of magnitude when administration by gavage and their toxic effects were further diminished when administered with food. These results have implications for future studies of the toxicology of these marine toxins and the risk assessment of human exposure.

Fast-acting cyclic imines belonging to the pinnatoxin and pteriatoxin group of toxins were originally identified in shellfish of the genera Pinna and Pteria in Japan, after food poisoning events in China linked to consumption of Pinna spp. Recently, a range of new and known pinnatoxin analogs has been identified in shellfish, sediment, and seawater samples from Australia and New Zealand. Although the structurally closely-related spirolide toxins are better known, and have a worldwide distribution including Norway and other parts of Europe, the presence of pinnatoxins has not been reported in European waters or shellfish. Here we report results from a survey of Norwegian blue mussels for the presence of pinnatoxins and spirolides, by LC-MS/MS analysis of extracts obtained as part of Norway's routine monitoring programme for regulated algal toxins during late autumn and early winter 2009. Spirolides and pinnatoxin G were widespread (pinnatoxin G (1), spirolide C (2), iso-spirolide C (3), 13-desmethylspirolide C (4), 13,19-didesmethylspirolide C (5), and 20-methylspirolide G (6) were detected in 69%, 13%, 60%, 22%, 33%, and 77%, respectively, of the shellfish samples) and, although levels were generally low, concentrations of up to 115 μg/kg of pinnatoxin G (1) and 226 μg/kg of 13-desmethylspirolide C (4) were detected. We also analyzed stored extracts from passive sampling disks deployed as part of a separate study in autumn 2007. All the stored extracts contained 20-methylspirolide G (which predominated at most locations), most contained pinnatoxin G (73%) and 13,19-didesmethylspirolide C (67%), but iso-spirolide C (36%) and 13-desmethylspirolide C (52%) were also detected in many of the samples. These results suggest that pinnatoxins may be much more widespread than previously suspected, and indicate that they or related compounds could be responsible for sporadic incidents of rapid-onset symptoms during mouse bioassays of shellfish in Europe and elsewhere. The toxicological significance of these levels of pinnatoxins and spirolides is at present unclear. However, although pinnatoxins appear to be less toxic than spirolides by intraperitoneal injection in the mouse bioassay, recently published preliminary toxicological data indicate that pinnatoxins may be as much as an order of magnitude more toxic than spirolides by oral ingestion via food., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In recent years, there has been an increase in the production of shellfish and in global demand for seafood as nutritious and healthy food. Unfortunately, a significant number of incidences of shellfish poisoning occur worldwide, and microalgae that produce phycotoxins are responsible for most of these. Phycotoxins include several groups of small to medium sized natural products with molecular masses ranging from 300 to over 3000 Da. Cyclic imines (CIs) are a recently discovered group of marine biotoxins characterized by their fast acting toxicity, inducing a characteristic rapid death in the intraperitoneal mouse bioassay. These toxins are macrocyclic compounds with imine (carbon-nitrogen double bond) and spiro-linked ether moieties. They are grouped together due to the imino group functioning as their common pharmacore and due to the similarities in their intraperitoneal toxicity in mice. Spirolides (SPXs) are the largest group of CIs cyclic imines that together with gymnodimines (GYMs) are best characterized. Although the amount of cyclic imines in shellfish is not regulated and these substances have not been categorically linked to human intoxication, they trigger high intraperitoneal toxicity in rodents. In this review, the corresponding chemical structures of each member of the CIs and their derivatives are reviewed as well as all the data accumulated on their mechanism of action at cellular level.

Currently, there are no fast in vitro broad spectrum screening bioassays for the detection of marine toxins. The aim of this study was to develop such an assay. In gene expression profiling experiments 17 marker genes were provisionally selected that were differentially regulated in human intestinal Caco-2 cells upon exposure to the lipophilic shellfish poisons azaspiracid-1 (AZA1) or dinophysis toxin-1 (DTX1). These 17 genes together with two control genes were the basis for the design of a tailored microarray platform for the detection of these marine toxins and potentially others. Five out of the 17 selected marker genes on this dedicated DNA microarray gave clear signals, whereby the resulting fingerprints could be used to detect these toxins. CEACAM1, DDIT4, and TUBB3 were up-regulated by both AZA1 and DTX1, TRIB3 was up-regulated by AZA1 only, and OSR2 by DTX1 only. Analysis by singleplex qRT-PCR revealed the up- and down-regulation of the selected RGS16 and NPPB marker genes by DTX1, that were not envisioned by the new developed dedicated array. The qRT-PCR targeting the DDIT4, RSG16 and NPPB genes thus already resulted in a specific pattern for AZA1 and DTX1 indicating that for this specific case qRT-PCR might a be more suitable approach than a dedicated array.

For many years, the presence of yessotoxins (YTXs) in shellfish has contributed to the outcome of the traditional mouse bioassay and has on many occasions caused closure of shellfisheries. Since YTXs do not appear to cause diarrhoea in man and exert low oral toxicity in animal experiments, it has been suggested that they should be removed from regulation. Before doing so, it is important to determine whether the oral toxicity of YTXs is enhanced when present together with shellfish toxins known to cause damage to the gastrointestinal tract. Consequently, mice were given high doses of YTX, at 1 or 5 mg/kg body weight, either alone or together with azaspiracid-1 (AZA1) at 200 μg/kg. The latter has been shown to induce damage to the small intestine at this level. The combined exposure caused no clinical effects, and no pathological changes were observed in internal organs. These results correspond well with the very low levels of YTX detected in internal organs by means of LC-MS/MS and ELISA after dosing. Indeed, the very low absorption of YTX when given alone remained largely unchanged when YTX was administered in combination with AZA1. Thus, the oral toxicity of YTX is not enhanced in the presence of sub-lethal levels of AZA1., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Azaspiracid Poisoning (AZP) is a human toxic syndrome which is associated with the consumption of bivalve shellfish. Unlike other shellfish, mussels contain a large array of azaspiracid analogs, many of which are suspected bioconversion products. These studies were conducted to elucidate the metabolic pathways of azaspiracid (AZA1) in the blue mussel (Mytilus edulis) and revealed that the main biotransformation product was the more toxic demethyl analog, AZA3. To elucidate the mechanism of this C-demethylation, an unprecedented xenobiotic bioconversion step in shellfish, AZA1 was fed to mussels that contained no detectable azaspiracids. Triple quadrupole mass spectrometry (MS) and high resolution Orbitrap MS were used to determine the uptake of AZA1 and the toxin profiles in three tissue compartments of mussels. The second most abundant bioconversion product was identified as AZA17, a carboxyl analog of AZA3, which is a key intermediate in the formation of AZA3. Also, two pairs of isomeric hydroxyl analogs, AZA4/AZA5 and AZA7/AZA8, have been confirmed as bioconversion products for the first time. Ultra high resolution (100 k) MS studies showed that the most probable structural assignment for AZA17 is 22-carboxy-AZA3 and a mechanism for its facile decarboxylation to form AZA3 has been proposed.

Alexandrium ostenfeldii is a widespread toxic dinoflagellate that has recently bloomed across the Adriatic Sea, seriously threatening both shellfish consumers and aquacultures. In 2007 we reported on preliminary studies carried out on field samples and cultures of A. ostenfeldii. At the time, along with three major spirolides - among which 27-hydroxy-13,19-didesmethyl spirolide C (3) proved to be a novel compound - a number of new minor spirolides were detected. Unfortunately, for all of them only Mass Spectrometry-based structural hypotheses could be ventured due to their very small amount. In the present paper we report on isolation and High Resolution Mass Spectrometry- and NMR-based structural elucidation of two of those minor spirolides detected in our previous study., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The main aim of this study was to examine absorption and pathological effects of a single sub-lethal dose of the marine biotoxin azaspiracid-1 (AZA1) in mice after oral intubation. When the mice received AZA1 at doses of 100, 200 or 300 μg/kg body weight (b.w.), the toxin was absorbed dose-dependently. Highest concentrations after 24 h were detected in kidneys, spleen and lungs, followed by liver and heart. Only trace amounts were seen in the brain. After seven days, the toxin level had dropped significantly in all organs except for the kidneys. The amount of toxin absorbed was highest in the liver, followed by kidneys, lungs, spleen and heart and the total amount of toxin in the internal organs analysed after 24 h was estimated to be only about 2% of the total amount given for all three dose groups. Pathological changes were only detected in the upper part of the small intestine (duodenum), consisting of mild cellular detachment in the tips of the villi, expansion of the crypts and necrotic changes in lamina propria. In a previous study very long persistence of damage to the gastrointestinal tract by repeated exposures to AZA toxins was reported. In our study, full recovery from the pathological changes was observed seven days after a single exposure to AZA1 at the doses applied., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The effect of azaspiracid-1 (AZA-1) on the plasma membrane proteins E-cadherin, Na(+)/K(+)-ATPase, and prolactin receptor (R(prl)) has been investigated in MCF-7 cells. Cell treatment for 24 h with 1nM AZA-1 induced the accumulation of a proteolytic fragment of E-cadherin and significant increases in the levels of Na(+)/K(+)-ATPase and R(prl) at the level of membranous structures. The effect induced by AZA-1 was mimicked by latrunculin A, suggesting that the toxin might act by blocking the endocytosis of plasma membrane proteins. The exposure of intact cells to a biotinylation reagent that does not permeate the plasma membrane provided data showing that AZA-1 treatment of MCF-7 cells doubled the levels of total protein located on the cell surface. The exposure of intact cells to exogenous proteases (trypsin and proteinase K) showed that AZA-1 treatment of MCF-7 cells modifies the availability of the three membrane protein markers to proteolytic attacks, providing evidence that significant portions of the protein pools are located in structures that are not exposed to the cell surface after the treatment with AZA-1. Distinct subcellular locations of the membrane protein markers in MCF-7 cells exposed to AZA-1 were confirmed by immunofluorescence microscopy. Direct evidence that AZA-1 inhibits endocytosis was obtained by showing that AZA-1 blocked the intracellular transfer of E-cadherin-bound antibody in MCF-7 cells. The effects of AZA-1 on the E-cadherin system were confirmed in Caco-2 and Madin Darby canine kidney epithelial cell lines. We conclude that AZA-1 inhibits endocytosis of plasma membrane proteins in epithelial cells.

Five major human toxic syndromes caused by the consumption of shellfish contaminated by algal toxins are presented. The increased risks to humans of shellfish toxicity from the prevalence of harmful algal blooms (HABs) may be a consequence of large-scale ecological changes from anthropogenic activities, especially increased eutrophication, marine transport and aquaculture, and global climate change. Improvements in toxin detection methods and increased toxin surveillance programmes are positive developments in limiting human exposure to shellfish toxins.

Azaspiracids (AZAs) are a novel group of marine phycotoxins that have been associated with severe human intoxication. We found that AZA-1 exposure increased lactate dehydrogense (LDH) efflux in murine neocortical neurons. AZA-1 also produced nuclear condensation and stimulated caspase-3 activity with an half maximal effective concentration (EC(50)) value of 25.8 nM. These data indicate that AZA-1 triggers neuronal death in neocortical neurons by both necrotic and apoptotic mechanisms. An evaluation of the structure-activity relationships of AZA analogs on LDH efflux and caspase-3 activation demonstrated that the full structure of AZAs was required to produce necrotic or apoptotic cell death. The similar potencies of AZA-1 to stimulate LDH efflux and caspase-3 activation and the parallel structure-activity relationships of azaspiracid analogs in the two assays are consistent with a common molecular target for both responses. To explore the molecular mechanism for AZA-1-induced neurotoxicity, we assessed the influence of AZA-1 on Ca(2+) homeostasis. AZA-1 suppressed spontaneous Ca(2+) oscillations (EC(50) = 445 nM) in neocortical neurons. A distinct structure-activity profile was found for inhibition of Ca(2+) oscillations where both the full structure as well as analogs containing only the FGHI domain attached to a phenyl glycine methyl ester moiety were potent inhibitors. The molecular targets for inhibition of spontaneous Ca(2+) oscillations and neurotoxicity may therefore differ. The caspase protease inhibitor Z-VAD-FMK produced a complete elimination of AZA-1-induced LDH efflux and nuclear condensation in neocortical neurons. Although the molecular target for AZA-induced neurotoxicity remains to be established, these results demonstrate that the observed neurotoxicity is dependent on a caspase signaling pathway.