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The oceans harbor a great reservoir of molecules with unknown bioactivities, which could be useful for the treatment of illnesses that nowadays have no cure, such as neurodegenerative diseases. In this work, we evaluated the neuroprotective potential of the marine Fijian compounds tavarua deoxyriboside A and jasplakinolide against oxidative stress and neuroinflammation, crucial mechanisms in neurodegeneration. Both metabolites protected SH-SY5Y human neuroblastoma cells from H2O2 damage, improving mitochondrial function and activating the antioxidant systems of cells. These effects were mediated by their ability of inducing Nrf2 translocation. In BV2 microglial cells activated with lipopolysaccharide, Fijian metabolites also displayed promising results, decreasing the release of proinflammatory mediators (ROS, NO, cytokines) through the reduction of gp91 and NFkB–p65 expression. Finally, we performed a coculture among both cell lines, in which treatment with compounds protected SH-SY5Y cells from activated microglia, corroborating their neuroprotective effects. These results suggest that tavarua deoxyriboside A and jasplakinolide could be used as candidate molecules for further studies against neurodegeneration.

The chemical investigation of the sponge Stylissa aff. carteri collected around Futuna Islands in the Pacific Ocean led to the isolation of three new dimeric pyrrole 2-aminoimidazole alkaloids (PIAs). Futunamine (1) features an unprecedented pyrrolo[1,2-c]imidazole core, while two other new dimeric PIAs were identified as analogues of palau'amine. Together with other known PIAs isolated from this species, they were shown to exhibit anti-inflammatory and neuroprotective activities.

Gracilins are diterpenes derivative, isolated from the marine sponge Spongionella gracilis. Natural gracilins and synthetic derivatives have shown antioxidant, immunosuppressive, and neuroprotective capacities related to the affinity for cyclophilins. The aim of this work was to study anti-inflammatory and immunosuppressive pathways modulated by gracilin L and two synthetic analogues, compound 1 and 2, on a cellular model of inflammation. In this way, the murine BV2 microglia cell line was used. To carry out the experiments, microglia cells were pre-treated with compounds for 1 h and then stimulated with lipopolysaccharide for 24 h to determine reactive oxygen species production, mitochondrial membrane potential, the release of nitric oxide, interleukin-6 and tumor necrosis factor-α and the expression of Nuclear factor-erythroid 2-related factor 2, Nuclear Factor-κB, the inducible nitric oxide synthase, and the cyclophilin A. Finally, a co-culture of neuron SH-SY5Y and microglia BV2 cells was used to check the neuroprotective effect of these compounds. Cyclosporine A was used as a control of effect. The compounds were able to decrease inflammatory mediators, the expression of inflammatory target proteins as well as they activated anti-oxidative mechanism upon inflammatory conditions. For this reason, natural and synthetic gracilins could be interesting for developing anti-inflammatory drugs.

A new method based on a QuEChERS extraction followed by the ultra-high liquid chromatography tandem mass spectrometry (UPLC-MS/MS) detection has been developed for the analysis of mycotoxin in beer. The method allows the identification and quantification of 23 mycotoxins with different chemical characteristic including regulated, emerging and masked compounds. A sample treatment procedure involving a QuEChERS extraction and dispersive solid-phase clean-up steps was applied. This protocol involves a new approach based on a sample concentration before the extraction. The method was in-house validated in terms of limits of detection (LODs), limits of quantification (LOQs), linearity, repeatability and recoveries. For most compounds, recoveries ranged from 70% to 110% with LOQs (from 0.038 to 30.43 µg/L) lower than the maximum residue levels established in European regulations. In general, acceptable performance characteristics were obtained fulfilling the current legislation. Therefore, the proposed method is appropriate for routine analysis of beer.

Fusarium foetens, a pathogen of Begonia plants, has been recently described as a new fungal species. This Fusarium species causes a destructive vascular wilt disease which leads to the death of the plant. Moreover, Fusarium species are known to produce a huge variety of secondary metabolites such as mycotoxins and phytotoxins. Here, we studied the toxicogenic profile of one F. foetens strain, isolated from maize, employing two methods based on the use of ultra-performance liquid chromatography coupled to mass spectrometry-ion trap-time of flight detection. The mycotoxins beauvericin and fusaric acid were detected in a pure culture of F. foetens. In addition, four fusaric acid analogs (10,11-dihidroxyfusaric acid, hydroxyfusaric acid, dehydrofusaric acid, and a hydroxylated unsaturated fusaric acid analog) were tentatively identified on the basis of their accurate mass and fragmentation patterns. Therefore, these preliminary data indicate that F. foetens isolated from maize is able to produce Fusarium mycotoxins including beauvericin and fusaric acid.

This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the strategic project UID/04292/2020 granted to MARE—Marine and Environmental Sciences Centre, and UIDP/04046/2020 and UIDB/04046/2020 granted to BioISI—BioSystems and Integrative Sciences Institute, through POINT4PAC project (Oncologia de Precisão: Terapias e Tecnologias Inovadoras, SAICTPAC/0019/2015-LISBOA- 01-0145-FEDER-016405), through CROSS-ATLANTIC project (PTDC/BIA-OUT/29250/2017), co-financed by COMPETE (POCI-01-0145-FEDER-029250) and through Molecules for Health project (PTDC/BIA-BQM/28355/2017). This work was also funded by the Integrated Programme of SR&TD Smart Valorization of Endogenous Marine Biological Resources Under a Changing Climate (Centro-01-0145-FEDER-000018), co-funded by Centro 2020 Programme, Portugal 2020, European Union, through the European Regional Development Fund. Parkinsons Disease (PD) is the second most common neurodegenerative disease worldwide, and is characterized by a progressive degeneration of dopaminergic neurons. Without an effective treatment, it is crucial to find new therapeutic options to fight the neurodegenerative process, which may arise from marine resources. Accordingly, the goal of the present work was to evaluate the ability of the monoterpenoid lactone Loliolide, isolated from the green seaweed Codium tomentosum, to prevent neurological cell death mediated by the neurotoxin 6-hydroxydopamine (6-OHDA) on SH-SY5Y cells and their anti-inflammatory effects in RAW264.7 macrophages. Loliolide was obtained from the diethyl ether extract, purified through column chromatography and identified by NMR spectroscopy. The neuroprotective effects were evaluated by the MTT method. Cells’ exposure to 6-OHDA in the presence of Loliolide led to an increase of cells’ viability in 40%, and this effect was mediated by mitochondrial protection, reduction of oxidative stress condition and apoptosis, and inhibition of the NF-KB pathway. Additionally, Loliolide also suppressed nitric oxide production and inhibited the production of TNF- and IL-6 pro-inflammatory cytokines. The results suggest that Loliolide can inspire the development of new neuroprotective therapeutic agents and thus, more detailed studies should be considered to validate its pharmacological potential. info:eu-repo/semantics/publishedVersion

Anhydroexfoliamycin, a secondary metabolite from Streptomyces, has shown antioxidant properties in primary cortical neurons reducing neurodegenerative hallmarks diseases, both in vitro and in vivo models. Activated microglia, in the central nervous system, plays a crucial role in neuroinflammation and is associated with neurodegeneration. Therefore, the aim of the present study was to determine the anti-inflammatory and antioxidant potential of the anhydroexfoliamycin over microglia BV2 cells. Neuroinflammation was simulated by incubation of microglia cells in the presence of lipopolysaccharide to activate proinflammatory transduction pathways. Moreover, a coculture of neuron SH-SY5Y and microglia BV2 cells was used to evaluate the neuroprotective properties of the Streptomyces metabolite. When microglia cells were preincubated with anhydroexfoliamycin, proinflammatory pathways, such as the translocation of the nuclear factor κB, the phosphorylation of c-Jun N-terminal kinase, and the inducible nitric oxide synthase expression, were inhibited. In addition, intracellular reactive oxygen species generation and the liberation of nitric oxide, interleukin 6, and tumor necrosis factor α were also decreased. Besides, the Streptomyces-derived compound showed antioxidant properties promoting the translocation of the factor erythroid 2-related factor 2 and protecting the SH-SY5Y cells from the neurotoxic mediators released by activated microglia. The effects of this compound were at the same level as the immunosuppressive drug cyclosporine A. Therefore, these results indicate that anhydroexfoliamycin is a promising tool to control microglia-driven inflammation with therapeutic potential in neuroinflammation The research leading to these results has received funding from the following FEDER cofunded grants: Conselleria de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (Grant ED431C 2017/01); Ministerio de Ciencia e Innovación Grants ISCIII/PI16/01830 and ISCIII/PI19/01248; European Union Interreg AlertoxNet Grant EAPA-317-2016, Interreg Agritox Grant EAPA-998-2018, and Grant H2020 778069-EMERTOX. S.G. was supported by a fellowship from FIDIS, Spain SI

Since the potential anticancer activity of auranofin was discovered, gold compounds have attracted interest with a view to developing anticancer agents that follow cytotoxic mechanisms other than cisplatin. Two benzimidazole gold(I) derivatives containing triphenylphosphine (Au(pben)(PPh3)) (1) or triethylphosphine (Au(pben)(PEt3)) (2) were prepared and characterized by standard techniques. X-ray crystal structures for 1 and 2 were solved. The cytotoxicity of 1 and 2 was tested in human neuroblastoma SH-SY5Y cells. Cells were incubated with compounds for 24 h with concentrations ranging from 10 &micro, M to 1 nM, and the half-maximal inhibitory concentration (IC50) was determined. 1 and 2 showed an IC50 of 2.7 and 1.6 &micro, M, respectively. In order to better understand the type of cell death induced by compounds, neuroblastoma cells were stained with Annexin-FITC and propidium iodide. The fluorescence analysis revealed that compounds were inducing apoptosis, however, pre-treatment with the caspase inhibitor Z-VAD did not reduce cell death. Analysis of compound effects on caspase-3 activity and reactive oxygen species (ROS) production in SH-SY5Y cells revealed an antiproliferative ability mediated through oxidative stress and both caspase-dependent and caspase-independent mechanisms.

A simple method for the quantification of 40 mycotoxins in milk was developed. This method is based on a QuEChERS extraction followed by the ultra-high liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection, and allows the simultaneous analysis of regulated, emerging, and modified mycotoxins. A sample treatment procedure was optimized to include a concentration step for the analysis of some compounds such as aflatoxin M1. The method was in-house validated in terms of limits of detection (LODs), limits of quantification (LOQs), linearity, recoveries, and precision. LOQs lower than 10 ng/mL were obtained, and recoveries ranged from 61% to 120% with a precision, expressed as the relative standard deviation, lower than 15%. Therefore, acceptable performance characteristics were obtained fulfilling European regulations. The method was successfully applied for the quantification of mycotoxins in raw milk. It can be highlighted high occurrence of beauvericin and enniatins were found in low amounts.

In this work, four manganese(II) complexes derived from the ligands H2L1-H2L4, that incorporate dansyl or tosyl fluorescent dyes, have been investigated in term of their antioxidant properties. Two of the manganese(II) complexes have been newly prepared using the asymmetric half-salen ligand H2L2 and the thiosemicarbazone ligand H2L3. The four organic strands and the manganese complexes have been characterized by different analytical and spectroscopic techniques. The study of the antioxidant behaviour of these two new complexes and other two fluorophore-labelled analogues was tested in SH-SY5Y neuroblastoma cells. These four model complexes 1–4 were found to protect cells from oxidative damage in this human neuronal model, by reducing the release of reactive oxygen species. Complexes 1–4 significantly improved cell survival, with levels between 79.1 ± 0.8% and 130.9 ± 4.1%. Moreover, complexes 3 and 4 were able to restore the mitochondrial membrane potential at 1 μM, with 4 reaching levels higher than 85%, similar to the percentages obtained by the positive control agent cyclosporin A. The incorporation of the fluorescent label in the complexes allowed the study of their ability to enter the human neuroblastoma cells by confocal microscopy The research leading to these results has received funding from the following FEDER cofunded-grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01), 2018 GRC GI-1584 (ED431C 2018/13), MetalBIO Network (ED431D 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad IISCIII/PI19/001248. From Ministerio de Ciencia, Innovación y Universidades, MULTIMETDRUGS (RED2018-102471-T). From European Union, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX SI

So far, the Futuna Islands located in the Central Indo-Pacific Ocean have not been inventoried for their diversity in marine sponges and associated chemical diversity. As part of the Tara Pacific expedition, the first chemical investigation of the sponge Narrabeena nigra collected around the Futuna Islands yielded 18 brominated alkaloids: seven new bromotryptamine derivatives 1&ndash, 7 and one new bromotyramine derivative 8 together with 10 known metabolites of both families 9&ndash, 18. Their structures were deduced from extensive analyses of nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) data. In silico metabolite anticipation using the online tool MetWork revealed the presence of a key and minor biosynthetic intermediates. These 18 compounds showed almost no cytotoxic effect up to 10 &micro, M on human neuroblastoma SH-SY5Y and microglia BV2 cells, and some of them exhibited an interesting neuroprotective activity by reducing oxidative damage.

Mycotoxins are toxic compounds that can be present in feed, food and beverages. In this work, 25 magnetic nanostructured materials were developed to remove the main types of mycotoxins from liquid food matrices. The efficiency for binding mycotoxins from contaminated aqueous solutions was studied. Nanocomposites (diameters lower to 15 μm) composed of mixtures of activated carbon, bentonite and aluminium oxide were able to eliminate up to 87% of mycotoxins with an adsorption efficiency of 450 µg/g. On the other hand, spheres with sizes below 3 mm and composed by biopolymers and activated carbon or graphene oxide removed up to 70% of mycotoxins (adsorption of 598 ng/g). These particles were tested for beer detoxification, and spheres composed of alginate and activated carbon or pectin maintain the ability to eliminate toxins from this beverage. Hence, this technology could be a useful tool for the food industry.

A new multi-mycotoxin analysis method was developed to identify and quantify 22 mycotoxins in multiple feed matrices. This method is based on a QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction procedure followed by the ultra-high liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection. The QuEChERS extraction procedure was optimized for minimizing the matrix effect of maize. Obtained recoveries ranged from 67 % to 94 % and low LOQs (from 0.22 to 32.64 μg/kg) for regulated and emerging mycotoxins were obtained. Then, the method was expanded to seven raw materials and eight feedstuffs. In these matrices, the recovery for most mycotoxins was also high enough to fulfill the current legislation. The developed method was used for the analysis of 75 samples obtained from a nearby feed factory. Maize and maize-based products showed the highest occurrence of mycotoxins, although always below the legal limit. In addition, the ability of spheres of different composition and size to eliminate mycotoxins from raw materials and feedstuffs was tested. Up to 28 % of the mycotoxin content can be removed from matrices by using glass spheres of 2 mm of diameter. Therefore, a new process for the physical removal of mycotoxins was developed.

Marine and freshwater toxins contaminate water resources, shellfish and aquaculture products, causing a broad range of toxic effects in humans and animals. Different core-shell nanoparticles were tested as a new sorbent for removing marine and freshwater toxins from liquid media. Water solutions were contaminated with 20 μg/L of marine toxins and up to 50 μg/L of freshwater toxins and subsequently treated with 250 or 125 mg/L of nanoparticles. Under these conditions, carbon nanoparticles removed around 70% of saxitoxins, spirolides, and azaspiracids, and up to 38% of diarrheic shellfish poisoning toxins. In the case of freshwater toxins, the 85% of microcystin LR was eliminated; other cyclic peptide toxins were also removed in a high percentage. Marine toxins were adsorbed in the first 5 min of contact, while for freshwater toxins it was necessary 60 min to reach the maximum adsorption. Toxins were recovered by extraction from nanoparticles with different solvents. Gymnodinium catenatum, Prorocentrum lima, and Microcystis aeruginosa cultures were employed to test the ability of nanoparticles to adsorb toxins in a real environment, and the same efficacy to remove toxins was observed in these conditions. These results suggest the possibility of using the nanotechnology in the treatment of contaminated water or in chemical analysis applications.

Two new zoanthamine alkaloids, namely 3-acetoxynorzoanthamine (1) and 3-acetoxyzoanthamine (2), have been isolated from the zoantharian Zoanthus cf. pulchellus collected off the coast of the Santa Elena Peninsula, Ecuador, together with three known derivatives: zoanthamine, norzoanthamine, and 3-hydroxynorzoanthamine. The chemical structures of 1 and 2 were determined by interpretation of their 1D and 2D NMR data and comparison with literature data. This is the first report of zoanthamine-type alkaloids from Zoanthus cf. pulchellus collected in the Tropical Eastern Pacific. The neuroinflammatory activity of all the isolated compounds was evaluated in microglia BV-2 cells and high inhibitory effects were observed in reactive oxygen species (ROS) and nitric oxide (NO) generation The project is originally funded by the Secretaria de Educación Superior, Ciencia, Tecnología e Innovación (SENESCYT) in the framework of the PIC-14-CENAIM-001 Project Caracterización de la Biodiversidad Microbiológica y de Invertebrados de la Reserva Marina “El Pelado” a Escala Taxonómica, Metabolómica y Metagenómica para su Uso en Salud Humana y Animal. Part of this project (Grant-Aid Agreement No. PBA/MB/16/01) is carried out with the support of the Marine Institute and is funded under the Marine Research Programme by the Irish Government. P.O.G. and K.B.J. acknowledge NUI Galway for supporting part of their Ph.D. scholarship. The research leading to the results of the biological assays has received funding from the following FEDER cofunded-grants: Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01); CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC-2016-5507-2, ITC-20161072; European Union POCTEP 0161-Nanoeaters-1-E-1, Interreg AlertoxNet EAPA-317-2016, and H2020 778069-EMERTOX SI

Maitotoxins (MTX) are among the most potent marine toxins identified to date causing cell death trough massive calcium influx. However, the exact mechanism for the MTX-induced calcium entry and cytotoxicity is still unknown. In this work, the effect of MTX-1 on the cytosolic free calcium concentration and cellular viability of human neuronal stem cells was evaluated. MTX elicited a concentration-dependent decrease in cell viability which was already evident after 1 h of treatment with 0.25 nM MTX; however, at a concentration of 0.1 nM, the toxin did not cause cell death even after 14 days of exposure. Moreover, the toxin caused a concentration dependent rise in the cytosolic calcium concentration which was maximal at toxin concentrations of 1 nM and dependent on the presence of extracellular calcium on the bathing solution. Several pharmacological approaches were employed to evaluate the role of canonical transient potential receptor channels (TRPC) on the MTX effects. The results presented here lead to the identification of the TRPC4 channels as contributors to the MTX effects in human neuronal cells. Both, the calcium increase and the cytotoxicity of MTX were either fully (for the calcium increase) or partially (in the case of cytotoxicity) reverted by the blockade of canonical TRPC4 receptors with the selective antagonist ML204. Furthermore, the sodium proton exchanger blocker amiloride also partially inhibited the calcium rise and the cell death elicited by MTX while the combination of amiloride and ML204 fully prevented both the cytotoxicity and the calcium rise elicited by the toxin.

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 The research leading to USC results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01 and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016, and through Axencia Galega de Innovación, Spain, ITC-20133020 SINTOX. In addition from the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007–2013) under grant agreement 315285 CIGUATOOLS and 312184 PHARMASEA. Inés Rodriguez is supported by a fellowship from Subprograma de Formación de Personal Investigador (AGL2012-40185-CO2-01), Spain. In depth investigation of the toxic episodes leading to the results and publication of the present work was undertaken by the Greek National Reference Laboratory of Marine Biotoxins (NRLMB) to fulfill the requirements of EU Regulation 178/2002/EC (Articles 6 and 7) regarding risk analysis and communication and scientific information needed for risk assessment and EU Regulation 882/2004/EC (article 7) with regard to transparency and information to the public. Collaboration of all the staff of the NRLMB is greatly appreciated. Thanks are also expressed to all the Greek regional veterinary services for their contribution to the shellfish samplings and for provision of the seawater analyses results for the presence of potentially toxic microalgae. The use of cell counts’ data within the period 2006–2009 and 2012 regarding P. minimum presence in seawater, derived from the Greek “National Programme for Monitoring of Bivalve Molluscs’ Production Areas for the presence of Marine Biotoxins” and conducted by the Laboratory Unit of Toxic Marine Microalgae (LUTMM), Department of Biology, Aristotle University of Thessaloniki (scientific coordinator: G. Nikolaidis (until February 2010) and M. Arsenakis (March 2010–to date)), as well as the restrictions of LUTMM regarding the use of data produced within 2013–2015 due to contract terms and ISO 17025 requirements are acknowledged SI

Although voltage-gated sodium channels (Na v ) are the cellular target of paralytic shellfish poisoning (PSP) toxins and that patch clamp electrophysiology is the most effective way of studying direct interaction of molecules with these channels, nowadays, this technique is still reduced to more specific analysis due to the difficulties of transforming it in a reliable throughput system. Actual functional methods for PSP detection are based in binding assays using receptors but not functional Na v channels. Currently, the availability of automated patch clamp platforms and also of stably transfected cell lines with human Na v channels allow us to introduce this specific and selective method for fast screenings in marine toxin detection. Taking advantage of the accessibility to pure PSP standards, we calculated the toxicity equivalent factors (TEFs) for nine PSP analogs obtaining reliable TEFs in human targets to fulfill the deficiencies of the official analytic methods and to verify automated patch clamp technology as a fast and reliable screening method for marine toxins that interact with the sodium channel. The main observation of this work was the large variation of TEFs depending on the channel subtype selected, being remarkable the variation of potency in the 1.7 channel subtype and the suitability of Na v 1.6 and 1.2 channels for PSP screening.

A method based on the combined use of ultraperformance liquid chromatography coupled to mass spectrometry–ion trap–time-of-flight (UPLC–MS–IT–TOF) detection was employed to identify the metabolite production of Aspergillus ochraceus, which is the major cause of food and feed contamination due to ochratoxin A. Under the proposed chromatographic conditions, seven metabolites belonging to the family of circumdatins were separated and identified. Their initial identification was performed through the exact molecular formula, as a function of their accurate mass. Collision-induced dissociation was applied to predict precursor and product ions, and the elemental composition of each compound was obtained. The elimination of nitrogenous groups followed by successive losses of carbonyl groups is the common fragmentation pathway of circumdatins. With the fragmentation data obtained, an UPLC–MS/MS method was created and optimized to detect circumdatins in corn samples.

Mycotoxins occur naturally as agricultural contaminants all over the world. The toxic effects of some of their metabolites are known and their presence regulated in food and feed. This paper describes two methods for the detection of toxins of type-A trichothecenes group, and their modified forms, using mass spectrometry. Ultra-performance liquid chromatography coupled to mass spectrometry-ion trap-time of flight (UPLC-MS-IT-TOF) was employed to characterize the fragmentation pathways of 10 type-A trichothecenes, and characteristic ions were tentatively identified in scan mode through their accurate masses. Unknown signals were detected in a F. sporotrichioides extract, which afterwards were identified as seven modified forms of neosolaniol (NEO) and T-2 toxin. Then, UPLC coupled to tandem mass spectrometry (MS/MS) was employed to develop a precursor ion scanning method that can be used as a screening tool to detect any modified type-A trichothecenes.

Monitoring the quality of freshwater is an important issue for public health. In the context of the European project μAqua, 150 samples were collected from several waters in France, Germany, Ireland, Italy, and Turkey for 2 yr. These samples were analyzed using 2 multitoxin detection methods previously developed: a microsphere-based method coupled to flow-cytometry, and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. The presence of microcystins, nodularin, domoic acid, cylindrospermopsin, and several analogues of anatoxin-a (ATX-a) was monitored. No traces of cylindrospermopsin or domoic acid were found in any of the environmental samples. Microcystin-LR and microcystin-RR were detected in 2 samples from Turkey and Germany. In the case of ATX-a derivatives, 75% of samples contained mainly H2 -ATX-a and small amounts of H2 -homoanatoxin-a, whereas ATX-a and homoanatoxin-a were found in only 1 sample. These results confirm the presence and wide distribution of dihydro derivatives of ATX-a toxins in European freshwaters. Environ Toxicol Chem 2017;36:645-654. © 2016 SETAC.

This chapter focuses on the analysis of the main natural toxins associated with food safety. Mycotoxins are produced primarily by fungi of the genera Fusarium, Aspergillus, Penicillium, Claviceps, and Alternaria. The main representatives are aflatoxins, ochratoxin A, patulin, trichothecenes, zearalenone, fumonisins, and ergot alkaloid. Marine toxins are produced primarily by dinoflagellates of several genera. The main toxins are classified on the basis of the main toxin content: okadaic acid and dinophysistoxins, azaspiracids, yessotoxins, pectenotoxins, domoic acid, and saxitoxin and gonyautoxins. Cyanotoxins are produced by blue-green microalgae classified according to their effect: hepatotoxins (microcystins (MCs), nodularins and cylindrospermopsins), neurotoxins (anatoxin-a and analogs, saxitoxins, and s-. N-methylamino-. l-alanine), and dermatotoxins (aplysiatoxins and lyngbyatoxins). Finally, tetrodotoxin is produced by bacteria and it is a concern in fish and shellfish. All these toxins are analyzed mostly by LC-MS, and the specific features of the analysis is discussed in the chapter.

In 2012, Tetrodotoxin (TTX) was identified in mussels and linked to the presence of Prorocentrum minimum (P. minimum) in Greece. The connexion between TTX and P. minimum was further studied in this paper. First, the presence of TTX-producer bacteria, Vibrio and Pseudomonas spp, was confirmed in Greek mussels. In addition these samples showed high activity as inhibitors of sodium currents (INa). P. minimum was before associated with neurotoxic symptoms, however, the nature and structure of toxins produced by this dinoflagellate remains unknown. Three P. minimum strains, ccmp1529, ccmp2811 and ccmp2956, growing in different conditions of temperature, salinity and light were used to study the production of toxic compounds. Electrophysiological assays showed no effect of ccmp2811 strain on INa, while ccmp1529 and ccmp2956 strains were able to significantly reduce INa in the same way as TTX. In these samples two new compounds, m/z 265 and m/z 308, were identified and characterized by liquid chromatography tandem high-resolution mass spectrometry. Besides, two TTX-related bacteria, Roseobacter and Vibrio sp, were observed. These results show for the first time that P. minimum produce TTX-like compounds with a similar ion pattern and C9-base to TTX analogues and with the same effect on INa Inés Rodríguez is supported by a fellowship from Subprograma de Formación de Personal Investigador MINECO (AGL2012-40185-CO2-01), Spain. The research leading to these results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA – Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEA. SI

Bromoalkaloids are secondary metabolites with a demonstrated high activity in several therapeutic areas. In this research, we probe the neuroprotective and antioxidant activities of hymenialdisine and hymenin. Both structures were tested in an oxidative stress cellular model, consisting of cortical neurons that are incubated with the oxidative stress inducer hydrogen peroxide and the tested compound. Several oxidation biomarkers were analyzed, and the results of the oxidative stress induced neurons in the presence and absence of bromoalkaloids were compared. Both compounds demonstrated significant neuroprotective ability under stress conditions at low nanomolar concentrations, with hymenialdisine highlighted for demonstrating a more complete protection. Also, the activity of hymenialdisine and hymenin was studied in the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, and, for the first time, these halogenated metabolites are described as Nrf2 inducers, reinforcing the antioxidant capacity observed and therefore opening a new path of investigation. These results, added to the previously described effect of this compound family in negatively modulating several kinases and proinflammatory cytokines, position hymenialdisine and hymenin as good candidates for the development of new drugs for neurodegenerative diseases.

Filamentous cyanobacteria of the genus Phormidium have been rarely studied for their chemical diversity. For the first time, the cultivable Phormidium autumnale was shown to produce a prenylated cyclic peptide named autumnalamide (1). The structure of this peptide was fully determined after a deep exploration of the spectroscopic data, including NMR and HRMS. Interestingly, a prenyl moiety was located on the guanidine end of the arginine amino acid. The absolute configurations of most amino acids were assessed using enantioselective GC/MS analysis, with (13)C NMR modeling being used for the determination of d-arginine and d-proline. The effects of 1 on sodium and calcium fluxes were studied in SH-SY5Y and hNav 1.6 HEK cells. When the Ca(2+) influx was stimulated by thapsigargin, strong inhibition was observed in the presence of 1. As a consequence, this compound may act by disrupting the normal calcium uptake of this organelle, inducing the opening of the mitochondrial permeability transition pore, which results in the indirect blockade of store-operated channels.

HMC-1 are inflammatory cells that release vasoactive substances such as histamine. These cells have the c-kit receptor permanently activated in the membrane due to mutations in the proto-oncogene c-kit: Val-560 → Gly and Asp-816 → Val. Thus, there are two known cellular lines: HMC-1560 and HMC-1560,816. These mutations are involved in a disease called mastocitosys. In the present paper both lines were used to study the influence of cAMP/PKA/PDEs pathway on the histamine release and Ca2+ signaling since this pathway is often involved in these process. For this, the cells were preincubated with cAMP/PKA/PDEs modulators such as dibutyryl cAMP (dbcAMP), forskolin, H89, rolipram, IBMX, or imidazole and then stimulated with ionomycin. When cells were stimulated with agents that increase cAMP levels, the histamine release was not modified in HMC-1560 but decreased in HMC-1560,816 cells. The same happened when PKA was blocked. Furthermore, PDEs role on histamine release was independent of cAMP in HMC-1560 cells and possibly also in HMC-1560,816 cells. By contrast, the modulation of PKA and PDEs together changed the response in both cellular lines, therefore a relationship between them was suggested. All these modulatory effects on histamine release are Ca2+-independent. On the other hand, the effect of c-kit modulation on the cAMP/PKA/PDEs pathway was also checked. This receptor was blocked with STI571 (imatinib) and BMS-354825 (dasatinib), but only the last one caused a decrease in the cellular response to ionomycin. This article demonstrates for the first time than the cAMP/PKA/PDEs pathway is involved in the activation of HMC-1560 and HMC-1560,816 cells. J. Cell. Biochem. 115: 896–909, 2014. © 2013 Wiley Periodicals, Inc.

Bifurcaria bifurcata is a marine brown seaweed mainly found on the Atlantic coast. Herein, we report the antioxidant and neuroprotective activities of seven fractions (F1&ndash, F7) obtained by normal phase chromatography from the B. bifurcata dichloromethane extract, as well as of its two major isolated diterpenes. Total phenolic content of fractions was determined by the Folin&ndash, Ciocalteu method, while antioxidant activity was evaluated by the DPPH, ORAC, and FRAP assays. Neuroprotective effects were evaluated in a neurotoxic model induced by 6-hydroxydopamine (6-OHDA) in a human neuroblastoma cell line (SH-SY5Y), while the mechanisms associated to neuroprotection were investigated by the determination of mitochondrial membrane potential, H2O2 production, Caspase-3 activity, and by observation of DNA fragmentation. Fractions F4 and F5 exhibited the best neuroprotective and antioxidant activities, respectively. F4 fraction prevented changes in mitochondrial potential, and induced a reduction of H2O2 levels production and an increase in cell viability, suggesting that it may contain multi-target compounds acting on different pathways. Hence, this fraction was subjected to purification steps, affording the known diterpenes eleganolone and eleganonal. Both compounds exhibited antioxidant potential, being interesting candidates for further neuroprotective studies.

Marine toxins are a food safety concern worldwide. This review discusses current analytical methods for those toxins that are legally regulated in Europe, namely domoic acid, saxitoxin, okadaic acid, yessotoxin, pectenotoxin and azaspiracids, and all their analogues, and those that are not regularly monitored but are a threat to humans, such as tetrodotoxin, ciguatoxins and cyclic imines. Because of legislative changes that were implemented in 2011, most legally required methods are chromatographic. Saxitoxin and domoic acid are monitored by high-performance liquid chromatography with fluorescent and ultraviolet detection, respectively. All other toxins are monitored nowadays by means of chromatographic separation and mass spectrometry detection.

International audience; Previous works with autumnalamide reported that Store Operated Calcium (SOC) channels were blocked through mitochondrial modulation. In the present paper we studied the effect of autumnalamide on ionomycin Ca2+ fluxes. Thus, autumnalamide did not modify ionomycin-sensitive intracellular pools while the ionomycin-induced Ca2+ influx was blocked with similar potency whether the incubation was done before or after ionomycin-sensitive pools depletion. Nevertheless, autumnalamide was not able to inhibit ionomycin-induced Ca2+ influx once the membrane channels were activated. Moreover, the compound efficiently inhibited flufenamic acid (FFA) Ca2+ release induced in this organelle but no the next influx. Since in previous work the effect of autumnalamide was inhibited by cyclosporine A (CsA), structures that target this drug were studied. Therefore, the affinity of autumnalamide for cyclophilin D (Cyp D) was examined. The KD obtained for Cyp D- autumnalamide was 1.51 ± 1.399. Moreover, the KD for Cyp A- autumnalamide was calculated. The peptide had a similar order of Cyp A binding affinity than CsA (8.08 ± 1.23 and 6.85 ± 1.1 μM respectively). After testing autumnalamide-binding capacity for Cyp A, the activity of this compound on Cyp A pathway was tested. Thus, the effect on interleukin (IL)-2 release on activated T-lymphocytes was checked. Autumnalamide was able to reduce IL-2 levels near to T cells in resting conditions. Next, the effect over calcineurin and NFATc1 was also evaluated. While CsA inhibits both calcineurin and NFATc1, autumnalamide did not produce any effect. From these results we can conclude that, autumnalamide targeted mitochondrion and prevent T-cells from IL-2 production through the modulation of SOC Ca2+ channels.

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) This work could not have been done without the kind collaboration of Pescados Marcelino. The research leading to these results has received funding from the following FEDER cofunded grants from CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, Xunta de Galicia Axencia Galega de Innovación, ITC-20133020 SINTOX, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016; from CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD; from the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007–2013) under grant agreement 312184 PHARMASEA SI

The presence of tetrodotoxin (TTX) and its analogues, 4-epiTTX, 4,9-anhydroTTX, 5-deoxyTTX, 11-deoxyTTX, 5,6,11-trideoxyTTX, 11-norTTX-6(S)-ol, and 11-norTTX-6(R)-ol was investigated for the first time in five different tissues (liver, gonads, gastrointestinal tract, muscle, and skin) of six specimens of the marine puffer fish Lagocephalus sceleratus from European waters (Aegean Sea, Greece) by using liquid chromatography coupled to electrospray ionisation mass spectrometry operating in the conventional mode in addition to low-energy collision dissociation tandem mass spectrometry (CID-MS/MS). Two isomers of 5,6,11-trideoxyTTX were detected in all specimens as the major TTX analogues, followed by 11-deoxyTTX, 11-norTTX-6(S)-ol, and TTX. However, minor amounts of 4-epiTTX, 4,9-anhydroTTX, 5-deoxyTTX, and 11-norTTX-6(R)-ol were also found in most of the tested tissues. In all puffer fish specimens, gonads, gastrointestinal tract, and liver contained the highest toxin levels, whereas muscle and skin contained lower amounts. Toxin distribution within the tissues of the six L. sceleratus specimens was different depending on fish size, area, and season where fish were caught. The LC-ESI-CID-MS/MS analysis employed is proposed as a suitable technique for determination of TTX and its analogues with a low detection limit (0.08 μg/g).

Protein phosphatase 2A is the major enzyme that dephosphorylates the serine/threonine residues of proteins in the cytoplasm of animal cells. This phosphatase is most strongly inhibited by okadaic acid. Besides okadaic acid, several other toxins and antibiotics have been shown to inhibit protein phosphatase 2A, including microsystin-LR, calyculin-A, tautomycib, nodularin, cantharidine, and fostriecin. This makes protein phosphatase 2A a valuable tool for detecting and assaying these toxins. High-scale production of active protein phosphatase 2A requires processing kilograms of animal tissue and involves several chromatographic steps. To avoid this, in this work we report the recombinant expression and characterization of the active catalytic subunit α of the protein phosphatase 2A in Trichoplusia ni insect larvae. Larvae were infected with baculovirus carrying the coding sequence for the catalytic subunit α of protein phosphatase 2A under the control of the polyhedrin promoter and containing a poly-His tag in the carboxyl end. The catalytic subunit was identified in the infected larvae extracts, and it was calculated to be present at 250 μg per gram of infected larvae, by western blot. Affinity chromatography was used for protein purification. Protein purity was determined by western blot. The activity of the enzyme, determined by the p-nitrophenyl phosphate method, was 94 μmol/min/mg of purified protein. The catalytic subunit was further characterized by inhibition with okadaic acid and dinophysis toxin 2. The results presented in this work show that this method allows the production of large quantities of the active enzyme cost-effectively. Also, the enzyme activity was stable up to 2 months at −20 °C.

International audience; In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50-350 μg kg(-1) meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.

The human mast cell lines HMC-1(560) and HMC-1(560,816) were used to study histamine release, Ca(2+) signaling and protein kinase C (PKC) localization and expression, with phorbol 12-myristate 13-acetate (PMA). Both sublines carry activating mutations in the proto-oncogene of c-kit that cause autophosphorylation and permanent c-kit tyrosine kinase activation. Both have the Gly-560 → Val mutation but only the second carries the Asp-816 → Val mutation. In this study, it was observed that the stimulation of PKC has different effects in HMC-1(560) and HMC-1(560,816) and this would be related to the difference in activating mutations in both mast cell lines. PKC activation increases ionomycin-induced histamine release in HMC-1(560) . This article demonstrates an opposite histamine response in HMC-1(560,816) cells, even though classical PKCs are the family of isozymes responsible for this effect in both cellular lines. Furthermore, it can be observed that upon cell stimulation with PMA, primarily cytosolic PKC translocates to the nucleous in HMC-1(560,816) cells, but not in HMC-1(560) cell line.

Chromatographic techniques coupled to mass spectrometry is the method of choice to replace the mouse bioassay (MBA) to detect marine toxins. This paper evaluates the influence of different parameters such as toxin solvents, mass spectrometric detection method, mobile-phase-solvent brands and equipment on okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) quantification. In addition, the study compares the results obtained when a toxin is quantified against its own calibration curve and with the calibration curve of the other analogues. The experiments were performed by liquid chromatography (LC) and ultraperformance liquid chromatography (UPLC) with tandem mass spectrometry detection (MS/MS). Three acetonitrile brands and two toxin solvents were employed, and three mass spectrometry detection methods were checked. One method that contains the transitions for azaspiracid-1 (AZA-1), azaspiracid-2 (AZA-2), azaspiracid-3(AZA-3), gimnodimine (GYM), 13-desmethyl spirolide C (SPX-1), pectenotoxin-2 (PTX-2), OA, DTX-1, DTX-2, yessotoxin (YTX), homoYTX, and 45-OH-YTX was compared in both instruments. This method operated in simultaneous positive and negative ionization mode. The other two mass methods operated only in negative ionization mode, one contains transitions to detect DTX-1, OA DTX-2, YTX, homoYTX, and 45-OH-YTX and the other only the transitions for the toxins under study OA, DTX-1, and DTX-2. With dependence on the equipment and mobile phase used, the amount of toxin quantified can be overestimated or underestimated, up to 44% for OA, 46% for DTX-1, and 48% for DTX-2. In addition, when a toxin was quantified using the calibration curve of the other analogues, the toxin amount obtained is different. The maximum variability was obtained when DTX-2 was quantified using either OA or a DTX-1 calibration curve. In this case, the overestimation was up to 88% using the OA calibration curve and up to 204% using the DTX-1 calibration curve. In summary, the correct quantification of DSP toxins by MS detection depends on multiple factors. Since these factors are not taken into account in a validated protocol, these results question the convenience of having MS/MS as a reference method for protecting consumers of marine toxins, moreover if toxicity of each group is considered independently and total toxicity is not summed anymore as it is in the MBA.

Palytoxin is a large and complex polyhydroxylated molecule with potent neurotoxic activity. Dinoflagellates from the Ostreopsis genera were demonstrated to be producers of this compound and analogues. Even though initially palytoxin appearance was restricted to tropical areas, the recent occurrence of Ostreopsis outbreaks in Mediterranean Sea point to a worldwide dissemination probably related to climatic change. Those dinoflagellates can bioaccumulate in shellfish, especially in filter-feeding mollusks and have been involved in damaging effects in seafood or human toxic outbreaks. The present study describes palytoxins effect on metabolic activity of mantle and hepatopancreas cells from the mussel Mytilus galloprovincialis Lmk. Our results indicate that palytoxin is highly cytotoxic to mussel cells; unlike it happens with other toxins more common in European coasts such as okadaic acid and azaspiracid. These findings have a special significance for the marine environment and aquiculture since they are evidence for the ability of palytoxin to affect the integrity of bivalve mollusks that are not adapted to the presence of this toxin.

Background and purpose: Okadaic acid (OA) and microcystins (MCs) are structurally different toxins with the same mechanism of action, inhibition of serine/threonine protein phosphatases (PPs). Methyl okadaate (MeOk), a methyl ester derivative of OA, was considered almost inactive due to its weak inhibition of PP1 and PP2A. Here, we have investigated the activity and potency of MeOk in hepatic cells in comparison with that of OA and MCs. Experimental approach: We tested the effects of MeOK, OA and microcystin-leucine and arginine (MC-LR) on the metabolic rate, the actin cytoskeleton and glucose uptake in a rat hepatocyte cell line (Clone 9) and in primary cultured rat hepatocytes. PP2A was assayed to compare OA and MeOk activity. Key results: MeOk disrupted the actin cytoskeleton and depressed the metabolic rate of both types of rat hepatocytes, being six-fold less potent than OA in Clone 9 cells but nearly six-fold more potent in primary cultured hepatocytes. However, unlike OA, MeOk did not change glucose uptake in these cells, suggesting a weak inhibition of PP2A, as confirmed in direct assays of PP2A activity. Conclusions and implications: Although MeOk was originally described as a weakly bioactive molecule, it clearly depressed the metabolic rate and disrupted the cytoskeleton in primary and immortalized rat hepatocytes. Furthermore, MeOk affected primary hepatocytes at much lower concentrations than those affecting immortalized cells. These effects were unrelated to PP2A inhibition. Our results suggest the risk to public health from MeOk in foodstuffs should be re-evaluated.

Tetrodotoxin (TTX) is one of the most potent toxins already isolated, which occurs in a wide variety of animals. In this work, the occurrence of TTX and analogues was examined using mass spectrometry, confocal microscopy, liquid chromatography-mass spectrometry (LC-MS), and mouse bioassay in a trumpet shell (Charonia lampas lampas) and in the fluids of a patient poisoned by consuming this shell. Retention time data in the LC-MS system within the enhanced mass spectrum (EMS) mode indicated the presence of TTX and the analogue 5,6,11-trideoxyTTX; the enhanced product ion (EPI) mode confirmed the existence of both toxins with the formation of characteristic daughter ions from the fragment pattern of each molecule. TTX and 5,6,11-trideoxyTTX were only detected in the digestive gland of the trumpet shell and also in the urine and serum of the patient. The concentration of 5,6,11-trideoxyTTX checked in the samples by LC-MS was 3 times higher than TTX. However, the results obtained by mouse bioassay showed that the analogue is much less toxic than TTX. In vitro toxicity was checked using cerebellar cells; in these experiments the trumpet shell sample showed high toxicity, but the level was lower than in vivo results probably due to some competition between analogues. This paper shows for first time the presence and toxicity of TTX and 5,6,11-trideoxyTTX in a trumpet shell collected in the European coasts. The LC-MS method is a useful tool to confirm the presence of TTX and the further identification of TTX analogues.

Paralytic shellfish poisoning (PSP) is one of the most severe forms of food poisoning. The toxins responsible for this poisoning are natural compounds, which cause the arrest of action potential propagation by binding to voltage-gated Na+ channels. Several standards for PSP toxins are nowadays commercially available; however, there is not accessible data on the biological activity of the toxins present on this standards and their in vivo toxicity. We have developed an in vitro quantification method for PSP toxins using cultured neurons and compared the potency of the commercial PSP toxin standards in this system with their relative toxicity by mouse bioassay. The in vitro potencies of the PSP toxin standards were saxitoxin (STX)decarbamoylsaxitoxin (dcSTX) = neosaxitoxin (NeoSTX)gonyautoxins 1, 4 (GTX1,4)decarbamoylneosaxitoxin (dcNeoSTX)gonyautoxins 2, 3 (GTX2,3)decarbamoylgonyautoxins 2, 3 (dcGTX2,3)gonyautoxin 5 (GTX5). The data in vitro correlated well with the toxicity values obtained by mouse bioassay. Using this in vitro model we also provide the first data evaluating the potencies of PSP toxins after extraction in acidic pHs, indicating that the toxicity of the sample increases in acidic conditions. This observation correlated well with the chemical transformations undergone by contaminated samples treated in several acidic conditions as corroborated by high-performance liquid chromatography (HPLC) detection of the toxins. Therefore, a variation of 2 units in the pH during PSP extraction may lead to large discrepancies regarding sample lethality during official PSP control in different countries. The results presented here constitute the first comprehensive and revised data on the potency of PSP toxins in vitro and their in vivo toxicity.

The phycotoxin palytoxin (PTX) binds to Na,K-ATPase, inhibiting its activity and converting the pump into a channel. These mechanisms are poorly understood. We examined the effect of PTX on membrane potential ( E m ), intracellular calcium concentration ([Ca 2+ ] i ) and intracellular pH (pH i ) in primary cultures of cerebellar granule cells (CGC) and compared PTX and ouabain actions in the same cellular parameters. In this system, PTX caused depolarization, intracellular calcium increase and acidification. This is similar to the effect of ouabain. Preincubation of the cells with ouabain, before addition of PTX, altered E m , [Ca 2+ ] i , and pH i in a fashion similar to that of ouabain alone. This suggest a direct interaction of PTX with the Na,K-ATPase. Therefore, we used a resonant mirror biosensor to evaluate the binding of PTX and ouabain to immobilized Na,K-ATPase. Ouabain binding to immobilized Na,K-ATPase was concentration-dependent. No binding of PTX to Na,K-ATPase was observed with up to 10 μM, or with PTX addition in the presence of ATP. The fact that ouabain binds to the pump in an immobilized conformation whereas not binding of PTX was observed indicates that PTX and ouabain do not share the same binding site, and PTX binding may require the tridimensional pump structure.

Yessotoxin (YTX) and its analogues are a newly recognized group of toxins with increased presence in shellfish in recent years. They can be quantified by various functional assays due to their interaction with phosphodiesterases (PDEs). One of these assays detects the binding between the YTX and the fluorescently labeled PDE I using fluorescence polarization, a spectroscopic technique based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. The aim of this study was to develop a YTX extraction procedure from mussels that does not interfere with this detection method. YTX concentrations were measured in spiked mussel extracts obtained through use of different extraction methods and cleaning procedures. The percentages of toxin recovery in various steps of the processes were calculated using these concentrations. Six extraction methods and two cleaning steps were used and no matrix effects and high toxin recoveries were obtained in two cases. One case used acetone as extraction solvent followed by three dichloromethane partitions and the other case used methanol. The cleaning procedure includes a silica cartridge and a 10,000 NMWL filter. Finally these two extraction-cleaning-detection methods were applied to a naturally contaminated mussel sample and results showed that not only YTX but also homoYTX and hydroxyYTX can be quantified with a 85-90% recovery.

The azaspiracids (AZAs) are a group of marine toxins implicated in several intoxications whose mechanism of action is unknown. These phycotoxins include the five compounds shown in : AZA-1 (1), AZA-2 (2), AZA-3 (3), AZA-4 (4), and AZA-5 (5). The aim of this work was to study the effects of the five naturally occurring azaspiracids (AZA-1 to -5, Fig. 1) and four synthetic analogues (6-9, Fig. 2) on intracellular pH, and the influence of Ca2+ upon this effect. The AZAs (1-5) were found to modulate cytosolic Ca2+ levels in human lymphocytes, while some of them, but not all, had effects on the intracellular pH. AZA-1 (1) and AZA-2 (2) did not modify intracellular pH in a Ca2+-containing or a Ca2+-free medium. AZA-3 (3) increased intracellular pH by 0.16 units in the presence of extracellular Ca2+, an effect that was blocked when a 1 mM solution of Ni2+ was added. In a Ca2+-free medium, the increase in pH induced by AZA-3 (3) was reduced to 0.08 pH units. AZA-4 (4) inhibited the basal pH increase even in the presence of a 1 mM solution of Ni2+. In a Ca2+-free medium, the inhibition caused by AZA-4 (4) was small, but when Ca2+ was added back to the medium, the pH basal increase was again significantly inhibited. The alkalinization was also inhibited when AZA-4 (4) was added simultaneously, 10 min before or 10 min after thapsigargin (Tg), and also when the Ca2+-influx induced by Tg was inhibited by Ni2+. AZA-5 (5), on the other hand, did not modulate the intracellular pH profile in either a Ca2+-containing or a Ca2+-free medium. Finally, we investigated four synthetic analogues (6-9, Fig. 2) whose structures were based on the four originally proposed structures of azaspiracid-1, with an opened E-ring. Compound 6 induced a small cytosolic Ca2+ increase, but did not modify intracellular pH in saline solution. In a Ca2+-free medium, compound 6 blocked the pH fall when Ca2+ was added back to the medium. Compound 7 also did not modify intracellular pH in saline solutions, however it significantly blocked basal pH increases in a Ca2+-free medium. Compound 8 did not alter intracellular pH, however compound 9 induced a small acidification when Ca2+ was present in the extracellular medium. These results point to a structure-activity relationship in AZAs pH effect that affects the modulation and the coupling of intracellular pH and Ca2+.

Yessotoxins (YTXs) are disulfated polyether toxins that were first isolated from scallops in Japan. It has been proposed that these toxins activate cellular phosphodiesterases (PDEs). The interaction between YTX and PDEs was confirmed by resonant biosensor and fluorescence polarization studies. The aim of this work is to study the specificity of different PDEs for YTX binding. Association measurements are done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers from the sensor surface where the association PDEs-YTX takes place. We use aminosilane cuvettes, where exonuclease Phosphodiesterase I from Crotalus atrox (PDE I), exonuclease Phosphodiesterase II from bovine spleen (PDE II), or phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) are immobilized. Over immobilized exonuclease PDE I and exonuclease PDE II are added different amounts of YTX, and typical association curve profiles are observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration, the association rate constant (k(ass)) and the dissociation rate constant (k(diss)) are obtained. From these values, the kinetic equilibrium dissociation constant (K(D)) of the YTX-PDE association can be calculated, indicating the affinity between them. The specificity of cyclic nucleotide PDE families is studied using different inhibitors that are added over immobilized cyclic nucleotide PDEs. In these conditions, changes in the association PDEs-YTX curves are detected. The results show YTX affinity by cyclic nucleotide PDE 1, PDE 3, PDE 4, and exonuclease PDE I.

The human mast cell line (HMC-1) has been used to study the relationship between intracellular pH and cytosolic calcium (Ca 2+ ) in mast cells. Thapsigargin (TG) caused store-operated Ca 2+ entry, that is enhanced by the PKC activator PMA. NH 4 Cl-induced alkalinization showed an inhibitory effect on TG-sensitive stores depletion (not on TG-insensitive stores), and also on final cytosolic Ca 2+ levels reached in response to both TG and the ionophore ionomycin. Loperamide, a positive modulator of store-operated channels, induced a slight Ca 2+ entry by itself, and also increased TG-induced Ca 2+ entry. This enhancement was not enough to reverse the inhibitory effect of NH 4 Cl-induced alkalinization. When comparing the effect of NH 4 Cl-induced alkalinization on Ca 2+ levels, with those observed using Ca 2+ channel blockers (namely Ni 2+ and SKF-96365), cytosolic profiles for this ion are different, either in modified saline solution or in HCO 3 - -free medium. Thus, it seems unlikely that the inhibitory effect of NH 4 Cl-induced alkalinization on Ca 2+ is taking place by blockage of Ca 2+ entry. Furthermore, inhibition of the plasma membrane Ca 2+ -ATPase (an important mechanism for Ca 2+ efflux) with sodium orthovanadate (SO) matches with the inhibition of the negative effect on Ca 2+ levels elicited by NH 4 Cl. Data indicate that NH 4 Cl-induced alkalinization might be activating Ca 2+ efflux from the cell, by stimulation of the plasma membrane Ca 2+ -ATPase, and also confirm our previous finding that Ca 2+ is a secondary signal to activate HMC-1 cells. © 2006 Wiley-Liss, Inc.

Yessotoxin (YTX) is a polycyclic ether toxin produced by phytoplanktonic microalgae from the group of dinoflagellates. It has been shown that YTX increases the 3′,5′-cyclic nucleotide phosphodiesterases (PDEs) activity and that there is a binding between these proteins and the toxin. Fluorescence polarization (FP) is a spectroscopic technique that can be used to study the interactions between molecules. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, the FP is applied to the study of the interaction between YTX and phosphodiesterases I and II (PDE I and II). The phosphodiesterases are labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of the protein–dye conjugate is measured when the YTX concentration in the medium increases. The results show that in both cases the fluorescence polarization of the conjugates decreases when they bind to YTX. For the PDE I, it is possible to draw a Gaussian curve or a straight line that relates the two variables (FP and YTX concentration). The concentration of this toxin in a spiked mussel extract (which contains the conjugate) can be quantified measuring its FP and using the equations of those lines. Different extraction methods are tried in this study, and those that can be used to obtain an appropriate mussel extract to be quantified with this technique are determined.

Yessotoxin (YTX) is a disulfated polyether toxin produced by phytoplanktonic microalgae from the dinoflagellates group. YTX has structural similarities to maitotoxin or brevetoxins, and the mechanism of action for YTX is specific and related to calcium modulation. Some studies show that YTX decreases adenosine 3',5'-cyclic monophosphate (cAMP) levels in human lymphocytes. This effect is calcium-dependent, and phosphodiesterases (PDEs) seem to be involved. This point was recently confirmed by studying kinetic constants of YTX-PDEs binding. The aim of this work is to compare the ability of YTX and analogues to bind immobilized PDEs by using a resonant mirror biosensor. These instruments measure biomolecular interactions in real time, without labeling, and allow detailed investigation of the reaction kinetics by analysis of the resultant signals. YTX and the derivatives, 45-hydroxyyessotoxin, carboxyyessotoxin, and yessotoxin-45-(S)-a-methoxy-a-trifluormethylphenylacetate, were added over immobilized PDEs. The kinetic constants obtained from these bindings indicate a relationship between toxin structure and affinity. Since the presence of the functional groups in the side chain decreased the toxic YTX effect and the PDEs-YTX association, our results show that the side chain plays an important role in the YTX affinity for PDEs.