Tetrodotoxin (TTX) is starting to appear in molluscs from the European waters and is a hazard to seafood consumers. This toxin blocks sodium channels resulting in neuromuscular paralysis and even death. As a part of the risk assessment process leading to a safe seafood level for TTX, oral toxicity data are required. In this study, a 4-level Up and Down Procedure was designed in order to determine for the first time the oral lethal dose 50 (LD₅₀) and the No Observed Adverse Effect Level (NOAEL) in mice by using an accurate well-characterized TTX standard. Full article

Makaluvamines are pyrroloiminoquinones isolated from Zyzzya sponges. Until now, they have been described as topoisomerase II inhibitors with cytotoxic effects in diverse tumor cell lines. In the present work, seven makaluvamines were tested in several antioxidant assays in primary cortical neurons and neuroblastoma cells. Among the alkaloids studied, makaluvamine J was the most active in all the assays. This compound was able to reduce the mitochondrial damage elicited by the well-known stressor H₂O₂. The antioxidant properties of makaluvamine J are related to an improvement of the endogenous antioxidant defenses of glutathione and catalase. SHSY5Y assays proved that this compound acts as a Nrf2 activator leading to an improvement of antioxidant defenses. A low concentration of 10 nM is able to reduce the reactive oxygen species release and maintain a correct mitochondrial function. Based on these results, non-substituted nitrogen in the pyrrole plus the presence of a p-hydroxystyryl without a double bond seems to be the most active structure with a complete antioxidant effect in neuronal cells. Full article

Current regulation for marine toxins requires a monitoring method based on mass spectrometric analysis. This method is pre-targeted, hence after searching for pre-assigned masses, it identifies those compounds that were pre-defined with available calibrants. Therefore, the scope for detecting novel toxins which are not included in the monitoring protocol are very limited. In addition to this, there is a poor comprehension of the toxicity of some marine toxin groups. Also, the validity of the current approach is questioned by the lack of sufficient calibrants, and by the insufficient coverage by current legislation of the toxins reported to be present in shellfish. As an example, tetrodotoxin, palytoxin analogs, or cyclic imines are mentioned as indicators of gaps in the system that require a solid comprehension to assure consumers are protected. Full article

Paralytic shellfish toxins (PST) traditionally have been analyzed by liquid chromatography with either pre- or post-column derivatization and always with a silica-based stationary phase. This technique resulted in different methods that need more than one run to analyze the toxins. Furthermore, tetrodotoxin (TTX) was recently found in bivalves of northward locations in Europe due to climate change, so it is important to analyze it along with PST because their signs of toxicity are similar in the bioassay. The methods described here detail a new approach to eliminate different runs, by using a new porous graphitic carbon stationary phase. Firstly we describe the separation of 13 PST that belong to different groups, taking into account the side-chains of substituents, in one single run of less than 30 min with good reproducibility. The method was assayed in four shellfish matrices: mussel (Mytillus galloprovincialis), clam (Pecten maximus), scallop (Ruditapes decussatus) and oyster (Ostrea edulis). The results for all of the parameters studied are provided, and the detection limits for the majority of toxins were improved with regard to previous liquid chromatography methods: the lowest values were those for decarbamoyl-gonyautoxin 2 (dcGTX2) and gonyautoxin 2 (GTX2) in mussel (0.0001 mg saxitoxin (STX)·diHCl kg⁻¹ for each toxin), decarbamoyl-saxitoxin (dcSTX) in clam (0.0003 mg STX·diHCl kg⁻¹), N-sulfocarbamoyl-gonyautoxins 2 and 3 (C1 and C2) in scallop (0.0001 mg STX·diHCl kg⁻¹ for each toxin) and dcSTX (0.0003 mg STX·diHCl kg⁻¹ ) in oyster; gonyautoxin 2 (GTX2) showed the highest limit of detection in oyster (0.0366 mg STX·diHCl kg⁻¹). Secondly, we propose a modification of the method for the simultaneous analysis of PST and TTX, with some minor changes in the solvent gradient, although the detection limit for TTX does not allow its use nowadays for regulatory purposes. Full article

This study explores the effect of laboratory and industrial steaming on mussels with toxin concentrations above and below the legal limit. We used mild conditions for steaming, 100 °C for 5 min in industrial processing, and up to 20 min in small-scale laboratory steaming. Also, we studied the effect of heat on the toxin concentration of mussels obtained from two different locations and the effect of heat on the levels of dinophysistoxins 3 (DTX3) in both the mussel matrix and in pure form (7-O-palmitoyl okadaic ester and 7-O-palmytoleyl okadaic ester). The results show that the loss of water due to steaming was very small with a maximum of 9.5%, that the toxin content remained unchanged with no concentration effect or increase in toxicity, and that dinophysistoxins 3 was hydrolyzed or degraded to a certain extent under heat treatment. The use of liquid-certified matrix showed a 55% decrease of dinophysistoxins 3 after 10 min steaming, and a 50% reduction in total toxicity after treatment with an autoclave (121 °C for 20 min). Full article

Domoic acid (DA) is one of the best known marine toxins, causative of important neurotoxic alterations. DA effects are documented both in wildlife and experimental assays, showing that this toxin causes severe injuries principally in the hippocampal area. In the present study we have addressed the long-term toxicological effects (30 days) of DA intraperitoneal administration in rats. Different histological techniques were employed in order to study DA toxicity in heart, an organ which has not been thoroughly studied after DA intoxication to date. The presence of DA was detected by immunohistochemical assays, and cellular alterations were observed both by optical and transmission electron microscopy. Although histological staining methods did not provide any observable tissue damage, transmission electron microscopy showed several injuries: a moderate lysis of myofibrils and loss of mitochondrial conformation. This is the first time the association between heart damage and the presence of the toxin has been observed. Full article

Yessotoxin (YTX) is a polyether compound produced by dinoflagellates and accumulated in filter feeding shellfish. No records about human intoxications induced by this compound have been published, however it is considered a toxin. Modifications in second messenger levels, protein levels, immune cells, cytoskeleton or activation of different cellular death types have been published as consequence of YTX exposure. This review summarizes the main intracellular pathways modulated by YTX and their pharmacological and therapeutic implications. Full article

Okadaic acid and its analogues are potent phosphatase inhibitors that cause Diarrheic Shellfish Poisoning (DSP) through the ingestion of contaminated shellfish by humans. This group of toxins is transmitted worldwide but the number of poisoning incidents has declined over the last 20 years due to legislation and monitoring programs that were implemented for bivalves. In the summer of 2012 and 2013, we collected a total of 101 samples of 22 different species that were made up of benthic and subtidal organisms such echinoderms, crustaceans, bivalves and gastropods from Madeira, São Miguel Island (Azores archipelago) and the northwestern coast of Morocco. The samples were analyzed by UPLC-MS/MS. Our main objective was to detect new vectors for these biotoxins. We can report nine new vectors for these toxins in the North Atlantic: Astropecten aranciacus, Arbacia lixula, Echinaster sepositus, Holothuria sanctori, Ophidiaster ophidianus, Onchidella celtica, Aplysia depilans, Patella spp., and Stramonita haemostoma. Differences in toxin contents among the species were found. Even though low concentrations were detected, the levels of toxins that were present, especially in edible species, indicate the importance of these types of studies. Routine monitoring should be extended to comprise a wider number of vectors other than for bivalves of okadaic acid and its analogues. Full article

High accumulations of phytoplankton species that produce toxins are referred to as harmful algal blooms (HABs). HABs represent one of the most important sources of contamination in marine environments, as well as a serious threat to public health, fisheries, aquaculture-based industries, and tourism. Therefore, methods effectively controlling HABs with minimal impact on marine ecology are required. Marine dinoflagellates of the genera Dinophysis and Prorocentrum are representative producers of okadaic acid (OA) and dinophysistoxins responsible for the diarrhetic shellfish poisoning (DSP) which is a human intoxication caused by the consumption of shellfish that bioaccumulate those toxins. In this work we explore the use of natural clay for removing Prorocentrum lima. We evaluate the adsorption properties of clays in seawater containing the dinoflagellates. The experimental results confirmed the cell removal through the flocculation of algal and mineral particles leading to the formation of aggregates, which rapidly settle and further entrain cells during their descent. Moreover, the microscopy images of the samples enable one to observe the clays in aggregates of two or more cells where the mineral particles were bound to the outer membranes of the dinoflagellates. Therefore, this preliminary data offers promising results to use these clays for the mitigation of HABs. Full article

Ciguatera fish poisoning (CFP) is a syndrome caused by the ingestion of fish contaminated with Ciguatoxins (CTXs). These phycotoxins are produced mainly by dinoflagellates that belong to the genus Gambierdiscus that are transformed in more toxic forms in predatory fish guts, and are more present in the Indo-Pacific and Caribbean areas. It is estimated that CFP causes per year more than 10,000 intoxications worldwide. With the rise of water temperature and anthropogenic intervention, it is important to study the prevalence of CFP in more temperate waters. Through inter- and subtidal sampling, 22 species of organisms were collected, in Madeira and Azores archipelagos and in the northwestern Moroccan coast, during September of 2012 and June and July of 2013. A total of 94 samples of 22 different species of bivalves, gastropods, echinoderms and crustaceans where analyzed by Ultra Performance Liquid Chromatography-Mass Spectometry-Ion Trap-Time of Flight (UPLC-MS-IT-TOF) and Ultra Performance Chromatography- Mass Spectrometry (UPLC-MS). Our main aim was to detect new vectors and ascertain if there were some geographical differences. We detected for the first time putative CTXs in echinoderms, in two starfish species—M. glacialis and O. ophidianus. We detected differences regarding uptake values by organisms and geographical location. Toxin amounts were significant, showing the importance and the need for continuity of these studies to gain more knowledge about the prevalence of these toxins, in order to better access human health risk. In addition, we suggest monitoring of these toxins should be extended to other vectors, starfish being a good alternative for protecting and accessing human health risk. Full article

The Mediterranean marine sponge Crambe crambe is the source of two families of guanidine alkaloids known as crambescins and crambescidins. Some of the biological effects of crambescidins have been previously reported while crambescins have undergone little study. Taking this into account, we performed comparative transcriptome analysis to examine the effect of crambescin-C1 (CC1) on human tumor hepatocarcinoma cells HepG2 followed by validation experiments to confirm its predicted biological activities. We report herein that, while crambescin-A1 has a minor effect on these cells, CC1 protects them against oxidative injury by means of metallothionein induction even at low concentrations. Additionally, at high doses, CC1 arrests the HepG2 cell cycle in G0/G1 and thus inhibits tumor cell proliferation. The findings presented here provide the first detailed approach regarding the different effects of crambescins on tumor cells and provide a basis for future studies on other possible cellular mechanisms related to these bioactivities. Full article

During official shellfish control for the presence of marine biotoxins in Greece in year 2012, a series of unexplained positive mouse bioassays (MBA) for lipophilic toxins with nervous symptomatology prior to mice death was observed in mussels from Vistonikos Bay–Lagos, Rodopi. This atypical toxicity coincided with (a) absence or low levels of regulated and some non-regulated toxins in mussels and (b) the simultaneous presence of the potentially toxic microalgal species Prorocentrum minimum at levels up to 1.89 × 10³ cells/L in the area’s seawater. Further analyses by different MBA protocols indicated that the unknown toxin was hydrophilic, whereas UPLC-MS/MS analyses revealed the presence of tetrodotoxins (TTXs) at levels up to 222.9 μg/kg. Reviewing of official control data from previous years (2006–2012) identified a number of sample cases with atypical positive to asymptomatic negative MBAs for lipophilic toxins in different Greek production areas, coinciding with periods of P. minimum blooms. UPLC-MS/MS analysis of retained sub-samples from these cases revealed that TTXs were already present in Greek shellfish since 2006, in concentrations ranging between 61.0 and 194.7 μg/kg. To our knowledge, this is the earliest reported detection of TTXs in European bivalve shellfish, while it is also the first work to indicate a possible link between presence of the toxic dinoflagellate P. minimum in seawater and that of TTXs in bivalves. Confirmed presence of TTX, a very heat-stable toxin, in filter-feeding mollusks of the Mediterranean Sea, even at lower levels to those inducing symptomatology to humans, indicates that this emerging risk should be seriously taken into account by the EU to protect the health of shellfish consumers. Full article

The separation of PSP toxins using liquid chromatography with a post-column oxidation fluorescence detection method was performed with different matrices. The separation of PSP toxins depends on several factors, and it is crucial to take into account the presence of interfering matrix peaks to produce a good separation. The matrix peaks are not always the same, which is a significant issue when it comes to producing good, reliable results regarding resolution and toxicity information. Different real shellfish matrices (mussel, scallop, clam and oyster) were studied, and it was seen that the interference is not the same for each individual matrix. It also depends on the species, sampling location and the date of collection. It was proposed that separation should be accomplished taking into account the type of matrix, as well as the concentration of heptane sulfonate in both solvents, since the mobile phase varies regarding the matrix. Scallop and oyster matrices needed a decrease in the concentration of heptane sulfonate to separate GTX4 from matrix peaks, as well as dcGTX3 for oysters, with a concentration of 6.5 mM for solvent A and 6.25 mM for solvent B. For mussel and clam matrices, interfering peaks are not as large as they are in the other group, and the heptane sulfonate concentration was 8.25 mM for both solvents. Also, for scallops and oysters, matrix interferences depend not only on the sampling site but also on the date of collection as well as the species; for mussels and clams, differences are noted only when the sampling site varies. Full article

Phycotoxins are marine toxins produced by phytoplankton that can get accumulated in filter feeding shellfish. Human intoxication episodes occur due to contaminated seafood consumption. Okadaic acid (OA) and dynophysistoxins (DTXs) are phycotoxins responsible for a severe gastrointestinal syndrome called diarrheic shellfish poisoning (DSP). Yessotoxins (YTXs) are marine toxins initially included in the DSP class but currently classified as a separated group. Food safety authorities from several countries have regulated the content of DSPs and YTXs in shellfish to protect human health. In mice, OA and YTX have been associated with ultrastructural heart damage in vivo. Therefore, this study explored the potential of OA, DTX-1 and YTX to cause acute heart toxicity. Cardiotoxicity was evaluated in vitro by measuring hERG (human èter-a-go-go gene) channel activity and in vivo using electrocardiogram (ECG) recordings and cardiac damage biomarkers. The results demonstrated that these toxins do not exert acute effects on hERG channel activity. Additionally, in vivo experiments showed that these compounds do not alter cardiac biomarkers and ECG in rats acutely. Despite the ultrastructural damage to the heart reported for these toxins, no acute alterations of heart function have been detected in vivo, suggesting a functional compensation in the short term. Full article

Harmful Algal Blooms (HAB) are complex to manage due to their intermittent nature and their severe impact on the economy and human health. The conditions which promote HAB have not yet been fully explained, though climate change and anthropogenic intervention are pointed as significant factors. The rise of water temperature, the opening of new sea canals and the introduction of ship ballast waters all contribute to the dispersion and establishment of toxin-producing invasive species that promote the settling of emergent toxins in the food-chain. Tetrodotoxin, ciguatoxin, palytoxin and cyclic imines are commonly reported in warm waters but have also caused poisoning incidents in temperate zones. There is evidence that monitoring for these toxins exclusively in bivalves is simplistic and underestimates the risk to public health, since new vectors have been reported for these toxins and as well for regulated toxins such as PSTs and DSTs. In order to avoid public health impacts, there is a need for adequate monitoring programs, a need for establishing appropriate legislation, and a need for optimizing effective methods of analysis. In this review, we will compile evidence concerning emergent marine toxins and provide data that may indicate the need to restructure the current monitoring programs of HAB. Full article

The marine habitat provides a large number of structurally-diverse bioactive compounds for drug development. Marine sponges have been studied over many years and are found to be a rich source of these bioactive chemicals. This study is focused on the evaluation of the activity of six diterpene derivatives isolated from Spongionella sp. on mitochondrial function using an oxidative in vitro stress model. The test compounds include the Gracilins (A, H, K, J and L) and tetrahydroaplysulphurin-1. Compounds were co-incubated with hydrogen peroxide for 12 hours to determine their protective capacities and their effect on markers of apoptosis and Nrf2/ARE pathways was evaluated. Results conclude that Gracilins preserve neurons against oxidative damage, and that in particular, tetrahydroaplysulphurin-1 shows a complete neuroprotective activity. Oxidative stress is linked to mitochondrial dysfunction and consequently to neurodegenerative disorders like Parkinson and Alzheimer diseases, Friedreich ataxia or Amyotrophic lateral sclerosis. This neuroprotection against oxidation conditions suggest that these metabolites could be interesting lead candidates in drug development for neurodegenerative diseases. Full article

Anatoxin-a (ATX) is a potent neurotoxin produced by several species of Anabaena spp. Cyanobacteria blooms around the world have been increasing in recent years; therefore, it is urgent to develop sensitive techniques that unequivocally confirm the presence of these toxins in fresh water and cyanobacterial samples. In addition, the identification of different ATX analogues is essential to later determine its toxicity. In this paper we designed a fluorescent polarization (FP) method to detect ATXs in water samples. A nicotinic acetylcholine receptor (nAChR) labeled with a fluorescein derivative was used to develop this assay. Data showed a direct relationship between the amount of toxin in a sample and the changes in the polarization degree of the emitted light by the labeled nAChR, indicating an interaction between the two molecules. This method was used to measure the amount of ATX in three Anabaena spp. cultures. Results indicate that it is a good method to show ATXs presence in algal samples. In order to check the toxin profile of Anabaena cultures a LC-MS/MS method was also developed. Within this new method, ATX-a, retention time (RT) 5 min, and three other molecules with a mass m/z 180.1 eluting at 4.14 min, 5.90 min and 7.14 min with MS/MS spectra characteristic of ATX toxin group not previously identified were detected in the Anabaena spp. cultures. These ATX analogues may have an important role in the toxicity of the sample. Full article

Okadaic acid (OA) and its analogues, dinophysistoxin 1 (DTX1) and dinophysistoxin 2 (DTX2), are lipophilic and heat-stable marine toxins produced by dinoflagellates, which can accumulate in filter-feeding bivalves. These toxins cause diarrheic shellfish poisoning (DSP) in humans shortly after the ingestion of contaminated seafood. Studies carried out in mice indicated that DSP poisonous are toxic towards experimental animals with a lethal oral dose 2–10 times higher than the intraperitoneal (i.p.) lethal dose. The focus of this work was to study the absorption of OA, DTX1 and DTX2 through the human gut barrier using differentiated Caco-2 cells. Furthermore, we compared cytotoxicity parameters. Our data revealed that cellular viability was not compromised by toxin concentrations up to 1 μM for 72 h. Okadaic acid and DTX2 induced no significant damage; nevertheless, DTX1 was able to disrupt the integrity of Caco-2 monolayers at concentrations above 50 nM. In addition, confocal microscopy imaging confirmed that the tight-junction protein, occludin, was affected by DTX1. Permeability assays revealed that only DTX1 was able to significantly cross the intestinal epithelium at concentrations above 100 nM. These data suggest a higher oral toxicity of DTX1 compared to OA and DTX2. Full article

Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na⁺,K⁺-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na⁺,K⁺-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na⁺,K⁺-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (k_obs). From the representation of k_obs versus PLTX concentration, the kinetic equilibrium dissociation constant (K_D) for the PLTX-Na⁺,K⁺-ATPase association can be calculated. The value of this constant is K_D = 6.38 × 10⁻⁷ ± 6.67 × 10⁻⁸ M PLTX. In this way the PLTX-Na⁺,K⁺-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods. Full article

In vivo, after administration by gavage to mice and rats, okadaic acid has been reported to produce lesions in liver, small intestine and forestomach. Because several reports differ in the damage detected in different organs, and on okadaic acid distribution after consumption, we determined the toxicity of this compound after oral administration to mice. After 24 hours, histopathological examination showed necrotic foci and lipid vacuoles in the livers of intoxicated animals. By immunohistochemical analysis, we detected this toxin in the liver and kidneys of intoxicated animals. Okadaic acid induces oxidative stress and can be activated in vitro into reactive compounds by the post-mitochondrial S9 fraction, so we studied the okadaic effect on the gene expression of antioxidant and phase II detoxifying enzymes in liver. We observed a downregulation in the expression of these enzymes and a reduction of protein expression of catalase and superoxide dismutase 1 in intoxicated animals. Full article

In this paper, we show the effect of crambescidin-816, -800, and -830 on Saccharomyces cerevisiae viability. We determined that, of the three molecules tested, crambescidin-816 was the most potent. Based on this result, we continued by determining the effect of crambescidin-816 on the cell cycle of this yeast. The compound induced cell cycle arrest in G2/M followed by an increase in cell DNA content and size. When the type of cell death was analyzed, we observed that crambescidin-816 induced apoptosis. The antifungal effect indicates that crambescidins, and mostly crambescidin-816, could serve as a lead compound to fight fungal infections. Full article

The prevalence of poisoning events due to harmful algal blooms (HABs) has declined during the last two decades through monitoring programs and legislation, implemented mainly for bivalves. However, new toxin vectors and emergent toxins pose a challenge to public health. Several locations on the Portuguese coast were surveyed between 2009 and 2010 for three distinct biotoxin groups [saxitoxin (PST), spirolide (SPX) and okadaic acid (OA)], in 14 benthic species of mollusks and echinoderms. Our main goals were to detect new vectors and unravel the seasonal and geographical patterns of these toxins. PSTs were analyzed by the Lawrence method, SPXs by LC-MS/MS, and OA by LC-MS/MS and UPLC-MS/MS. We report 16 new vectors for these toxins in the North Atlantic. There were differences in toxin contents among species, but no significant geographical or seasonal patterns were found. Our results suggest that legislation should be adjusted to extend the monitoring of marine toxins to a wider range of species besides edible bivalves. Full article

Marine toxins are currently monitored by means of a bioassay that requires the use of many mice, which poses a technical and ethical problem in many countries. With the exception of domoic acid, there is a legal requirement for the presence of other toxins (yessotoxin, saxitoxin and analogs, okadaic acid and analogs, pectenotoxins and azaspiracids) in seafood to be controlled by bioassay, but other toxins, such as palytoxin, cyclic imines, ciguatera and tetrodotoxin are potentially present in European food and there are no legal requirements or technical approaches available to identify their presence. The need for alternative methods to the bioassay is clearly important, and biosensors have become in recent years a feasible alternative to animal sacrifice. This review will discuss the advantages and disadvantages of using biosensors as alternatives to animal assays for marine toxins, with particular focus on surface plasmon resonance (SPR) technology. Full article