Psammaplin A and Its Analogs Attenuate Oxidative Stress in Neuronal Cells through Peroxisome Proliferator-Activated Receptor γ Activation.

Psammaplins are sulfur containing bromotyrosine alkaloids that have shown antitumor activity through the inhibition of class I histone deacetylases (HDACs). The cytotoxic properties of psammaplin A (1), the parent compound, are related to peroxisome proliferator-activated receptor γ (PPARγ) activation, but the mechanism of action of its analogs psammaplin K (2) and bisaprasin (3) has not been elucidated. In this study, the protective effects against oxidative stress of compounds 1-3, isolated from the sponge Aplysinella rhax, were evaluated in SH-SY5Y cells. The compounds improved cell survival, recovered glutathione (GSH) content, and reduced reactive oxygen species (ROS) release at nanomolar concentrations. Psammaplins restored mitochondrial membrane potential by blocking mitochondrial permeability transition pore opening and reducing cyclophilin D expression. This effect was mediated by the capacity of 1-3 to activate PPARγ, enhancing gene expression of the antioxidant enzymes catalase, nuclear factor E2-related factor 2 (Nrf2), and glutathione peroxidase. Finally, HDAC3 activity was reduced by 1-3 under oxidative stress conditions. This work is the first description of the neuroprotective activity of 1 at low concentrations and the mechanism of action of 2 and 3. Moreover, it links for the first time the previously described effects of 1 in HDAC3 and PPARγ signaling, opening a new research field for the therapeutic potential of this compound family.

First description of adenosine production by Gnomoniopsis smithogilvyi, causal agent of chestnut brown rot.

Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) is the main causal agent of chestnut brown rot on sweet chestnut worldwide. The rotting of nuts leads to alterations in the organoleptic qualities and decreased fruit production, resulting in significant economic losses. In 2021, there was an important outbreak of chestnut rot in southern Galicia (Spanish northwest). The profile of secondary metabolites from G. smithogilvyi was studied, especially to determine its capability for producing mycotoxins, as happens with other rotting fungi, due to the possible consequences on the safety of chestnut consumption. Secondary metabolites produced by isolates of G. smithogilvyi growing in potato dextrose agar (PDA) medium were identified using liquid chromatography coupled with high-resolution mass spectrometry. Three metabolites with interesting pharmacological and phyto-toxicological properties were identified based on their exact mass and fragmentation patterns, namely adenosine, oxasetin, and phytosphingosine. The capacity of G. smithogilvyi to produce adenosine in PDA cultures was assessed, finding concentrations ranging from 176 to 834 µg/kg. Similarly, the production of mycotoxins was ruled out, indicating that the consumption of chestnuts with necrotic lesions does not pose a health risk to the consumer in terms of mycotoxins.

Synergistic Effect of Brevetoxin BTX-3 and Ciguatoxin CTX3C in Human Voltage-Gated Nav1.6 Sodium Channels.

Emerging marine biotoxins such as ciguatoxins and brevetoxins have been widely and independently studied as food pollutants. Their maximum levels in food components were set without considering their possible synergistic effects as consequence of their coexistence in seafood and their action at the same cellular target. The absolute lack of data and regulations of the possible combined effects that both marine biotoxins may have raised the need to analyze their direct in vitro effects using electrophysiology techniques. The results presented in this study indicate that ciguatoxins and brevetoxins had a synergistic effect on human Nav1.6 voltage-gated sodium channels by hyperpolarizing their activation and inactivation states. The results presented here indicate that brevetoxin 3 (BTX-3) acts as partial agonist of human sodium channels, while ciguatoxin 3C (CTX3C) was a full agonist, explaining the differences in the effect of each toxin in the channel. Therefore, this work sets the cellular basis to further apply this type of studies to other food toxicants that may act synergistically and thus implement the corresponding regulatory limits considering their coexistence and the risks to human and animal health derived from it.

Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection.

The growing concern about ciguatera fish poisoning (CF) due to the expansion of the microorganisms producing ciguatoxins (CTXs) increased the need to develop a reliable and fast method for ciguatoxin detection to guarantee food safety. Cytotoxicity assay on the N2a cells sensitized with ouabain (O) and veratridine (V) is routinely used in ciguatoxin detection; however, this method has not been standardized yet. This study demonstrated the low availability of sodium channels in the N2a cells, the great O/V damage to the cells and the cell detachment when the cell viability is evaluated by the classical cytotoxicity assay and confirmed the absence of toxic effects caused by CTXs alone when using the methods that do not require medium removal such as lactate dehydrogenase (LDH) and Alamar blue assays. Different cell lines were evaluated as alternatives, such as human neuroblastoma, which was not suitable for the CTX detection due to the greater sensitivity to O/V and low availability of sodium channels. However, the HEK293 Nav cell line expressing the α1.6 subunit of sodium channels was sensitive to the ciguatoxin without the sensitization with O/V due to its expression of sodium channels. In the case of sensitizing the cells with O/V, it was possible to detect the presence of the ciguatoxin by the classical cytotoxicity MTT method at concentrations as low as 0.0001 nM CTX3C, providing an alternative cell line for the detection of compounds that act on the sodium channels.

Crosstalk between cyclophilins and T lymphocytes in coronary artery disease.

Cardiovascular diseases and atherosclerosis are currently some of the most widespread diseases of our time. Within cardiovascular disease, coronary artery disease and underlying atherosclerosis were recently linked with systemic and local inflammation. Cyclophilins participate in the initiation and progression of these inflammatory-related diseases. Cyclophilins are released into the extracellular space upon inflammatory stimuli and participate in the pathology of cardiovascular diseases. The cell surface receptor for extracellular cyclophilins, the CD147 receptor, also contributes to coronary artery disease pathogenesis. Nevertheless, the physiological relevance of cyclophilin's family and their receptor in cardiovascular diseases remains unclear. The present study aimed to better understand the role of cyclophilins in cardiovascular artery disease and their relationship with inflammation. Hence, cyclophilins and pro-inflammatory interleukins were measured in the serum of 30 subjects (divided into three groups according to coronary artery disease status: 10 patients with acute coronary syndrome, 10 patients with chronic coronary artery disease, and 10 control volunteers). In addition, cyclophilin levels and CD147 receptor expression were measured in T lymphocytes purified from these subjects. Cyclophilin A, B, and C, pro-inflammatory interleukins, and CD147 membrane expression were significantly elevated in patients with coronary artery disease.

In vivo subchronic effects of ciguatoxin-related compounds, reevaluation of their toxicity.

Ciguatoxins are marine compounds that share a ladder-shaped polyether structure produced by dinoflagellates of the genus Gambierdiscus and Fukuyoa, and include maitotoxins (MTX1 and MTX3), ciguatoxins (CTX3C) and analogues (gambierone), components of one of the most frequent human foodborne illness diseases known as ciguatera fish poisoning. This disease was previously found primarily in tropical and subtropical areas but nowadays, the dinoflagellates producers of ciguatoxins had spread to European coasts. One decade ago, the European Food Safety Authority has raised the need to complete the toxicological available data for the ciguatoxin group of compounds. Thus, in this work, the in vivo effects of ciguatoxin-related compounds have been investigated using internationally adopted guidelines for the testing of chemicals. Intraperitoneal acute toxicity was tested for maitotoxin 1 at doses between 200 and 3200 ng/kg and the acute oral toxicity of Pacific Ciguatoxin CTX3C at 330 and 1050 ng/kg and maitotoxin 1 at 800 ng/kg were also evaluated showing not effects on mice survival after a 96 h observation period. Therefore, for the following experiments the oral subchronic doses were between 172 and 1760 ng/kg for gambierone, 10 and 102 ng/kg for Pacific Ciguatoxin CTX3C, 550 and 1760 ng/kg for maitotoxin 3 and 800, 2560 and 5000 ng/kg for maitotoxin 1. The results presented here raise the need to reevaluate the in vivo activity of these agents. Although the intraperitoneal lethal dose of maitotoxin 1 is assumed to be 50 ng/kg, without chemical purity identifications and description of the bioassay procedures, in this work, an intraperitoneal lethal dose of 1107 ng/kg was obtained. Therefore, the data presented here highlight the need to use a common procedure and certified reference material to clearly establish the levels of these environmental contaminants in food.

Current Trends and New Challenges in Marine Phycotoxins.

Marine phycotoxins are a multiplicity of bioactive compounds which are produced by microalgae and bioaccumulate in the marine food web. Phycotoxins affect the ecosystem, pose a threat to human health, and have important economic effects on aquaculture and tourism worldwide. However, human health and food safety have been the primary concerns when considering the impacts of phycotoxins. Phycotoxins toxicity information, often used to set regulatory limits for these toxins in shellfish, lacks traceability of toxicity values highlighting the need for predefined toxicological criteria. Toxicity data together with adequate detection methods for monitoring procedures are crucial to protect human health. However, despite technological advances, there are still methodological uncertainties and high demand for universal phycotoxin detectors. This review focuses on these topics, including uncertainties of climate change, providing an overview of the current information as well as future perspectives.

Bromoditerpenes from the Red Seaweed Sphaerococcus coronopifolius as Potential Cytotoxic Agents and Proteasome Inhibitors and Related Mechanisms of Action.

Seaweeds are a great source of compounds with cytotoxic properties with the potential to be used as anticancer agents. This study evaluated the cytotoxic and proteasome inhibitory activities of 12R-hydroxy-bromosphaerol, 12S-hydroxy-bromosphaerol, and bromosphaerol isolated from Sphaerococcus coronopifolius. The cytotoxicity was evaluated on malignant cell lines (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, and SK-MEL-28) using the MTT and LDH assays. The ability of compounds to stimulate the production of hydrogen peroxide (H2O2) and to induce mitochondrial dysfunction, the externalization of phosphatidylserine, Caspase-9 activity, and changes in nuclear morphology was also studied on MCF-7 cells. The ability to induce DNA damage was also studied on L929 fibroblasts. The proteasome inhibitory activity was estimated through molecular docking studies. The compounds exhibited IC50 values between 15.35 and 53.34 µM. 12R-hydroxy-bromosphaerol and 12S-hydroxy-bromosphaerol increased the H2O2 levels on MCF-7 cells, and bromosphaerol induced DNA damage on fibroblasts. All compounds promoted a depolarization of mitochondrial membrane potential, Caspase-9 activity, and nuclear condensation and fragmentation. The compounds have been shown to interact with the chymotrypsin-like catalytic site through molecular docking studies; however, only 12S-hydroxy-bromosphaerol evidenced interaction with ALA20 and SER169, key residues of the proteasome catalytic mechanism. Further studies should be outlined to deeply characterize and understand the potential of those bromoditerpenes for anticancer therapeutics.

Targeting Chloride Ion Channels: New Insights into the Mechanism of Action of the Marine Toxin Azaspiracid.

Azaspiracids (AZAs) are marine toxins produced by dinoflagellates belonging to the genera Azadinium and Amphidoma that caused human intoxications after consumption of contaminated fishery products, such as mussels. However, the exact mechanism for the AZA induced cytotoxic and neurotoxic effects is still unknown. In this study several pharmacological approaches were employed to evaluate the role of anion channels on the AZA effects that demonstrated that cellular anion dysregulation was involved in the toxic effects of these compounds. The results presented here demonstrated that volume regulated anion channels (VRACs) are affected by this group of toxins, and, because there is not any specific activator of VRACs besides the intracellular application of GTPγ-S molecule, this group of natural compounds could represent a powerful tool to analyze the role of these channels in cellular homeostasis. In addition to this, in this work, a detailed pharmacological approach was performed in order to elucidate the anion channels present in human HEK293 cells as well as their regulation by the marine toxins azaspiracids. Altogether, the data presented here demonstrated that the effect of azaspiracids in human cells was completely dependent on ATP-regulated anion channels, whose upregulation by these toxins could lead to regulatory volume decrease and underlie the reported toxicity of these compounds.

Serotonin involvement in okadaic acid-induced diarrhoea in vivo.

The consumption of contaminated shellfish with okadaic acid (OA) group of toxins leads to diarrhoeic shellfish poisoning (DSP) characterized by a set of symptoms including nausea, vomiting and diarrhoea. These phycotoxins are Ser/Thr phosphatase inhibitors, which produce hyperphosphorylation in cellular proteins. However, this inhibition does not fully explain the symptomatology reported and other targets could be relevant to the toxicity. Previous studies have indicated a feasible involvement of the nervous system. We performed a set of in vivo approaches to elucidate whether neuropeptide Y (NPY), Peptide YY (PYY) or serotonin (5-HT) was implicated in the early OA-induced diarrhoea. Fasted Swiss female mice were administered NPY, PYY(3-36) or cyproheptadine intraperitoneal prior to oral OA treatment (250 µg/kg). A non-significant delay in diarrhoea onset was observed for NPY (107 µg/kg) and PYY(3-36) (1 mg/kg) pre-treatment. On the contrary, the serotonin antagonist cyproheptadine was able to block (10 mg/kg) or delay (0.1 and 1 mg/kg) diarrhoea onset suggesting a role of 5-HT. This is the first report of the possible involvement of serotonin in OA-induced poisoning.

DSP Toxin Distribution across Organs in Mice after Acute Oral Administration.

Okadaic acid (OA) and its main structural analogs dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) are marine lipophilic phycotoxins distributed worldwide that can be accumulated by edible shellfish and can cause diarrheic shellfish poisoning (DSP). In order to study their toxicokinetics, mice were treated with different doses of OA, DTX1, or DTX2 and signs of toxicity were recorded up to 24 h. Toxin distribution in the main organs from the gastrointestinal tract was assessed by liquid chromatography-mass spectrometry (LC/MS/MS) analysis. Our results indicate a dose-dependency in gastrointestinal absorption of these toxins. Twenty-four hours post-administration, the highest concentration of toxin was detected in the stomach and, in descending order, in the large intestine, small intestine, and liver. There was also a different toxicokinetic pathway between OA, DTX1, and DTX2. When the same toxin doses are compared, more OA than DTX1 is detected in the small intestine. OA and DTX1 showed similar concentrations in the stomach, liver, and large intestine tissues, but the amount of DTX2 is much lower in all these organs, providing information on DSP toxicokinetics for human safety assessment.

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 µM; 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 µM 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.

Partial Blockade of Human Voltage-Dependent Sodium Channels by the Marine Toxins Azaspiracids.

Azaspiracid toxins were first identified at the end of the last century in Irish mussels, and during the last two decades considerable cytotoxic and neurotoxic effects caused by these toxins have been described. Azaspiracids are synthesized by dinoflagellates and accumulate in several species of filter-feeding bivalve mollusks, thereby incorporating into the food chain and causing human intoxications. Among the cellular effects of azaspiracids, inhibition of spikes in neurons and hyperpolarization of the neuronal membrane potential have been reported; however, the underlying processes leading to these effects were never elucidated. In this regard, initial studies reported no activity of the toxin in neuronal voltage-gated sodium channels, and a recent work described no effect of azaspiracid-1 on the inactivation kinetics of voltage-gated sodium channels; however, the relationship between the known alterations of the cytoskeleton caused by these toxins and their effects on ion channels has never been evaluated. In this work, the cytotoxic effect of azaspiracids was evaluated in human cells as well as their activity on voltage-gated sodium channels and in cell morphology in order to unravel the cellular targets involved in the mechanism of action of this group of marine toxins. The data reported here demonstrate, for the first time, that both azaspiracid-1 and azaspiracid-2 caused a rapid concentration-dependent inhibition of the amplitude of voltage-gated sodium currents without affecting their inactivation kinetics, an effect that was increased after long-term treatment of the cells with the toxin. Simultaneously, long-term exposure of the cells to azaspiracids caused a profound alteration of the cell cytoskeleton and decreased the metabolic activity of human cells. Altogether, the data presented here indicate that the partial blockade of voltage-gated sodium channels by these toxins is not related with their effect on the actin cytoskeleton. However, since azaspiracids are common toxins in European waters, their effect on voltage-gated sodium channels, first reported here, should be considered to avoid synergistic toxicity with other marine toxins that are known potent blockers of sodium channels such as the saxitoxins and tetrodotoxins, but further studies are needed in order to elucidate how these compounds alter ion homeostasis.

Gracilin-Derivatives as Lead Compounds for Anti-inflammatory Effects.

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.

Futunamine, a Pyrrole-Imidazole Alkaloid from the Sponge Stylissa aff. carteri Collected off the Futuna Islands.

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.

Biological Activities of Cyclic and Acyclic B-Type Laxaphycins in SH-SY5Y Human Neuroblastoma Cells.

Laxaphycins are a family of non-ribosomal lipopeptides that have been isolated from several cyanobacteria. Some of these compounds have presented cytotoxic activities, but their mechanism of action is poorly understood. In this work, the already described laxaphycins B and B3, and acyclolaxaphycins B and B3 were isolated from the marine cyanobacteria Anabaena torulosa. Moreover, two new acyclic compounds, [des-(Ala4-Hle5)] acyclolaxaphycins B and B3, were purified from the herviborous gastropod Stylocheilus striatus, with this being the first description of biotransformed laxaphycins. The structure of these new compounds was elucidated, together with the absolute configuration of acyclolaxaphycins B and B3. The bioactivities of the six peptides were determined in SH-SY5Y human neuroblastoma cells. Laxaphycins B and B3 were cytotoxic (IC50: 1.8 and 0.8 µM, respectively) through the induction of apoptosis. In comparison, acyclic laxaphycins did not show cytotoxicity but affected mitochondrial functioning, so their effect on autophagy-related protein expression was analyzed, finding that acyclic peptides affected this process by increasing AMPK phosphorylation and inhibiting mTOR. This work confirms the pro-apoptotic properties of cyclic laxaphycins B and is the first report indicating the effects on autophagy of their acyclic analogs. Moreover, gastropod-derived compounds presented ring opening and amino-acids deletion, a biotransformation that had not been previously described.

Caniferolide A, a Macrolide from Streptomyces caniferus, Attenuates Neuroinflammation, Oxidative Stress, Amyloid-Beta, and Tau Pathology in Vitro.

The macrolide caniferolide A was isolated from extracts of a culture of the marine-derived actinomycete Streptomyces caniferus, and its ability to ameliorate Alzheimer's disease (AD) hallmarks was determined. The compound reduced neuroinflammatory markers in BV2 microglial cells activated with lipopolysaccharide (LPS), being able to block NFκB-p65 translocation to the nucleus and to activate the Nrf2 pathway. It also produced a decrease in pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), reactive oxygen species (ROS) and nitric oxide release and inhibited iNOS, JNK, and p38 activities. Moreover, the compound blocked BACE1 activity and attenuated Aß-activation of microglia by drastically diminishing ROS levels. The phosphorylated state of the tau protein was evaluated in SH-SY5Y tau441 cells. Caniferolide A reduced Thr212 and Ser214 phosphorylation by targeting p38 and JNK MAPK kinases. On the other side, the antioxidant properties of the macrolide were determined in an oxidative stress model with SH-SY5Y cells treated with H2O2. The compound diminished ROS levels and increased cell viability and GSH content by activating the nuclear factor Nrf2. Finally, the neuroprotective ability of the compound was confirmed in two trans-well coculture systems with activated BV2 cells (both with LPS and Aß) and wild type and transfected SH-SY5Y cells. The addition of caniferolide A to microglial cells produced a significant increase in the survival of neuroblastoma in both cases. These results indicate that the compound is able to target many pathological markers of AD, suggesting that caniferolide A could be an interesting drug lead for a polypharmacological approach to the illness.

Structure and biological evaluation of new cyclic and acyclic laxaphycin-A type peptides.

Five new laxaphycins were isolated and fully characterised from the bloom forming cyanobacteria Anabaena torulosa sampled from Moorea, French Polynesia: three acyclic laxaphycin A-type peptides, acyclolaxaphycin A (1), [des-Gly11]acyclolaxaphycin A (2) and [des-(Leu10-Gly11)]acyclolaxaphycin A (3), as well as two cyclic ones, [l-Val8]laxaphycin A (4) and [d-Val9]laxaphycin A (5). The absolute configuration of the amino acids, established using advanced Marfey's analysis for compounds 2-5, highlights a conserved stereochemistry at the Cα carbons of the peptide ring that is characteristic of this family. To the best of our knowledge, this is the first report of acyclic analogues within the laxaphycin A-type peptides. Whether these linear laxaphycins with the aliphatic ß-amino acid on the N-terminal are biosynthetic precursors or compounds obtained after enzymatic hydrolysis of the macrocycle is discussed. Biological evaluation of the new compounds together with the already known laxaphycin A shows that [l-Val8]laxaphycin A, [d-Val9]laxaphycin A and [des-Gly11]acyclolaxaphycin induce cellular toxicity whereas laxaphycin A and des-[(Leu10-Gly11)]acyclolaxaphycin A do not affect the cellular viability. An analysis of cellular death shows that the active peptides do not induce apoptosis or necrosis but instead, involve the autophagy pathway.

Bromotryptamine and Bromotyramine Derivatives from the Tropical Southwestern Pacific Sponge Narrabeena nigra.

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 1-7 and one new bromotyramine derivative 8 together with 10 known metabolites of both families 9-18. 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 µM on human neuroblastoma SH-SY5Y and microglia BV2 cells, and some of them exhibited an interesting neuroprotective activity by reducing oxidative damage.

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.