Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines

Yessotoxin (YTX) modulates cellular phosphodiesterases (PDEs). In this regard, opposite effects had been described in the tumor model K-562 cell line and fresh human lymphocytes in terms of cell viability, cyclic adenosine 3´,5´-cyclic monophosphate (cAMP) production and protein expression after YTX treatment. Studies in depth of the pathways activated by YTX in K-562 cell line, have demonstrated the activation of two different cell death types, apoptosis and autophagy after 24 and 48 hours of treatment, respectively. Furthermore, the key role of type 4A PDE (PDE4A) in both pathways activated by YTX was demonstrated. Therefore, taking into account the differences between cellular lines and fresh cells, a study of cell death pathways activated by YTX in a non-tumor cell line with mitotic activity, was performed. The cellular model used was the lymphoblastoid cell line that represents a non-tumor model with normal apoptotic and mitotic machinery. In this context, cell viability and cell proliferation, expression of proteins involved in cell death activated by YTX and mitochondrial mass, were studied after the incubation with the toxin. Opposite to the tumor model, no cell death activation was observed in lymphoblastoid cell line in the presence of YTX. In this sense, variations in apoptosis hallmarks were not detected in the lymphoblastoid cell line after YTX incubation, whereas this type I of programmed cell death was observed in K-526 cells. On the other hand, autophagy cell death was triggered in this cellular line, while other autophagic process is suggested in lymphoblastoid cells. These YTX effects are related to PDE4A in both cellular lines. In addition, while cell death is triggered in K-526 cells after YTX treatment, in lymphoblastoid cells the toxin stops cellular proliferation. These results point to YTX as a specific toxic compound of tumor cells, since in the non-tumor lymphoblastoid cell line, no cell death hallmarks are observed

Spongionella secondary metabolites protect mitochondrial function in cortical neurons against oxidative stress

Accepted: 8 January 2014 This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Acknowledgments The research leading to these results has received funding from the following FEDER cofunded-grants: From Ministerio de Ciencia y Tecnología, Spain: AGL2009-13581-CO2-01, AGL2012-40485-CO2-01. From Xunta de Galicia, Spain: 10PXIB261254 PR. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007–2013) under grant agreement Nos. 265896 BAMMBO, 265409 µAQUA, and 262649 BEADS, 315285 CIGUATOOLS and 312184 PHARMASEA. From the Atlantic Area Programme (Interreg IVB Trans-national): 2009-1/117 Pharmatlantic. MER thanks the Government of the Arab Republic of Egypt for a PhD Scholarship. MJ thanks the Scottish University Life Science Alliance which provided funding to set up the compound library.Peer reviewedPublisher PD

Pre-validación de un método de Cromatografía de Líquidos-Espectrometría de Masas para el análisis simultáneo de toxinas lipofílicas

[ESP] Las biotoxinas marinas de origen fitoplanctónico y de carácter lipofílico pueden acumularse en diferentes tipos de moluscos bivalvos, presentando un importante riesgo para la salud pública. La legislación de la UE, a través del Reglamento (CE) nº 2074/2005, establece los métodos de ensayos reconocidos para la detección de toxinas lipofílicas, señalando como método de referencia los ensayos biológicos e indicando posibles métodos alternativos al método de referencia, entre los que se encuentra la Cromatografía de líquidos acoplada a la Espectrometría de masas (LC-MS). Este Reglamento también indica la necesidad de sustituir lo antes posible los métodos biológicos por métodos de detección alternativos que hayan sido validados conforme a un protocolo acordado a nivel internacional. De acuerdo con esto, el Laboratorio Comunitario de Referencia de Biotoxinas Marinas (LCRBM) está actualmente coordinando, a nivel europeo, diversos estudios enfocados a la validación de un método de LC-MS para la determinación de toxinas lipofílicas. En el presente trabajo se describen los diferentes estudios realizados en la etapa de pre-validación del método, cuyo objetivo se centra en la optimización y estandarización de un protocolo para el análisis simultáneo de ácido ocadaico (AO) y dinofysistoxinas (DTXs), pectenotoxinas (PTXs), azaspirácidos (AZAs), yesotoxinas (YTXs) y espirólidos. A partir de los resultados obtenidos en esta etapa se elaboró un “Procedimiento Normalizado de Trabajo”, candidato a ser validado a través de un estudio colaborativo. Actualmente dicho procedimiento está en fase de evaluación y perfeccionamiento.Este trabajo ha sido financiado por DG SANCO (Comisión Europea, Bruselas), la Agencia Española de Seguridad Alimentaria y Nutrición (AESAN) y por el proyecto STREP FOOD-CT-2004-514055 (DETECTOX). Agradecer a INTECMAR (Instituto Tecnológico para o Control do Medio Mariño de Galicia) por facilitarnos muestras de moluscos naturalmente contaminados con toxinas lipofílicas

Toxic Action Reevaluation of Okadaic Acid, Dinophysistoxin-1 and Dinophysistoxin-2: Toxicity Equivalency Factors Based on the Oral Toxicity Study

Background/Aims: Okadaic acid (OA) and the structurally related compounds dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) are marine phycotoxins that cause diarrheic shellfish poisoning (DSP) in humans due to ingestion of contaminated shellfish. In order to guarantee consumer protection, the regulatory authorities have defined the maximum level of DSP toxins as 160 µg OA equivalent kg-1 shellfish meat. For risk assessment and overall toxicity determination, knowledge of the relative toxicities of each analogue is required. In absence of enough information from human intoxications, oral toxicity in mice is the most reliable data for establishing Toxicity Equivalence Factors (TEFs). Methods: Toxins were administered to mice by gavage, after that the symptomatology and mice mortality was registered over a period of 24 h. Organ damage data were collected at necropsy and transmission electron microscopy (TEM) was used for ultrastructural studies. Toxins in urine, feces and blood were analyzed by HPLC-MS/MS. The evaluation of in vitro potencies of OA, DTX1 and DTX2 was performed by the protein phosphatase 2A (PP2A) inhibition assay. Results: Mice that received DSP toxins by gavage showed diarrhea as the main symptom. Those toxins caused similar gastrointestinal alterations as well as intestine ultrastructural changes. However, DSP toxins did not modify tight junctions to trigger diarrhea. They had different toxicokinetics and toxic potency. The lethal dose 50 (LD50) was 487 µg kg-1 bw for DTX1, 760 µg kg-1 bw for OA and 2262 µg kg-1 bw for DTX2. Therefore, the oral TEF values are: OA = 1, DTX1 = 1.5 and DTX2 = 0.3. Conclusion: This is the first comparative study of DSP toxins performed with accurate well-characterized standards and based on acute toxicity data. Results confirmed that DTX1 is more toxic than OA by oral route while DTX2 is less toxic. Hence, the current TEFs based on intraperitoneal toxicity should be modified. Also, the generally accepted toxic mode of action of this group of toxins needs to be reevaluated

Spongionella Secondary Metabolites Regulate Store Operated Calcium Entry Modulating Mitochondrial Functioning in SH-SY5Y Neuroblastoma Cells

cknowledgements The research leading to these results has received funding from the following FEDER cofounded-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 OrdenaciónUniversitaria, GRC2013-016, and through AxenciaGalega de Innovación, Spain, ITC-20133020 SINTOX. 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. Jon Andoni Sánchez is supported by a fellowship from Plan Galego de Investigación e Crecemento, Xunta de Galicia, Spain.Peer reviewedPublisher PD

Actividades del Laboratorio Comunitario de Referencia de Biotoxinas Marinas

[ESP] El Laboratorio de Biotoxinas Marinas de la Agencia Española de Seguridad Alimentaria en Vigo es Laboratorio Comunitario de Referencia para el control de las biotoxinas marinas (LCRBM), lo que conlleva unas tareas de coordinación y liderazgo en el análisis de biotoxinas marinas en el marco de la Unión Europea. Por otro lado, el LCRBM es también el Laboratorio Nacional de Referencia (LNRBM) en España. Las funciones que como LCR y como LNR le asigna la legislación europea, incluyen labores de coordinación de actividades de los laboratorios europeos o nacionales, según el caso, en la aplicación de los métodos oficiales de análisis de biotoxinas marinas. Entre ellas, la organización de ensayos comparativos y su adecuado seguimiento de acuerdo con los protocolos internacionalmente reconocidos; así como la provisión de información, formación y asistencia científico-técnica a los laboratorios nacionales de control oficial, a los laboratorios de la red europea, a las autoridades competentes y a los Países Terceros. En el presente trabajo, se describen las diferentes actividades llevadas a cabo por el LCRBM, en especial, en cuanto a la organización de ensayos comparativos para la determinación de toxinas PSP y lipofílicas, dirigidos a la red europea y nacional, así como todas aquellas actividades encaminadas a la armonización de los métodos que se vienen aplicando y al desarrollo y validación de nuevos métodos analíticos alternativos al bioensayo en ratón, por tratarse de una urgente necesidad en este campo de la seguridad alimentaria.Este trabajo ha sido financiado por DG SANCO (Comisión Europea, Bruselas) y la Agencia Española de Seguridad Alimentaria y Nutrición (AESAN). El LCRBM quiere agradecer a INTECMAR (Instituto Tecnológico para o Control do Medio Mariño de Galicia) y a la asociación gallega de productores de mejillón SOCOMGAL por el suministro de material tóxico para estos estudios

Tavarua Deoxyriboside A and Jasplakinolide as Potential Neuroprotective Agents: Effects on Cellular Models of Oxidative Stress and Neuroinflammation

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

Gracilin A Derivatives Target Early Events in Alzheimer’s Disease: inVitro Effects on Neuroinflammation and Oxidative Stress

The search for compounds capable of targeting early pathological changes of Alzheimer̀s disease (AD), such as oxidative stress and neuroinflammation, is an important challenge. Gracilin A derivatives were recently synthesized, using a pharmacophore-directed retrosynthesis (PDR) strategy, and found to possess potent neuroprotective effects. In this work, the previously described derivatives 1–7 which demonstrated mitochondrial-mediated, antioxidant effects were chosen for further study. The ability of compounds to modulate the expression of antioxidant genes (CAT, GPx, SODs, and Nrf2) was determined in SH-SY5Y cells, and the simplified derivatives 2 and 3 were found to be the most effective. The anti-neuroinflammatory properties of all derivatives were assessed in BV2 microglial cells activated with lipopolysaccharide (LPS). Several derivatives decreased the release of cytokines (Il-1β, IL-6, GM-CSF, and TNF-α) and other damaging molecules (ROS, NO) and also regulated the translocation of Nrf2 and NFκB, and reduced p38 activation. These protective effects were confirmed in a trans-well coculture with BV2 and SH-SY5Y cells and several derivatives increased SH-SY5Y survival. This present work demonstrates the neuroprotective properties of gracilin A derivatives, making them promising candidate drugs for AD. Particularly, derivatives 2 and 3 showed the greatest potential as lead compounds for further developmentThe research leading to these results has received funding fromthe following FEDER cofunded-grants. From Consellería de Cultura, Educación e Ordenación Universitaria Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From 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. From European Union POCTEP 0161-Nanoeaters−1-E-1, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018 and H2020 778069-EMERTOX. Support from NIH (R37GM052964 to D.R.) and the Robert A. Welch Foundation (AA-1280 to D.R.) is also gratefully acknowledged.This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Chemical neuroscience, copyright © 2019 American Chemical Society, after peer review and technical editing by the publisher.S

Cytotoxic mechanism of sphaerodactylomelol, an uncommon bromoditerpene isolated from sphaerococcus coronopifolius

Marine natural products have exhibited uncommon chemical structures with relevant antitumor properties highlighting their potential to inspire the development of new anticancer agents. The goal of this work was to study the antitumor activities of the brominated diterpene sphaerodactylomelol, a rare example of the dactylomelane family. Cytotoxicity (10–100 M; 24 h) was evaluated on tumor cells (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, SK-ML-28) and the effects estimated by MTT assay. Hydrogen peroxide (H2O2) levels and apoptosis biomarkers (membrane translocation of phosphatidylserine, depolarization of mitochondrial membrane potential, Caspase-9 activity, and DNA condensation and/or fragmentation) were studied in the breast adenocarcinoma cellular model (MCF-7) and its genotoxicity on mouse fibroblasts (L929). Sphaerodactylomelol displayed an IC50 range between 33.04 and 89.41 M without selective activity for a specific tumor tissue. The cells’ viability decrease was accompanied by an increase on H2O2 production, a depolarization of mitochondrial membrane potential and an increase of Caspase-9 activity and DNA fragmentation. However, the DNA damage studies in L929 non-malignant cell line suggested that this compound is not genotoxic for normal fibroblasts. Overall, the results suggest that the cytotoxicity of sphaerodactylomelol seems to be mediated by an increase of H2O2 levels and downstream apoptosis.info:eu-repo/semantics/publishedVersio