Absorption and Effect of Azaspiracid-1 Over the Human Intestinal Barrier.

BACKGROUND: Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellates genera Azadinium and Amphidoma. These toxins cause azaspiracid poisoning (AZP), characterized by severe gastrointestinal illness in humans after the consumption of bivalve molluscs contaminated with AZAs. The main aim of the present study was to examine the consequences of human exposure to AZA1 by the study of absorption and effects of the toxin on Caco-2 cells, a reliable model of the human intestine. METHODS: The ability of AZA1 to cross the human intestinal epithelium has been evaluated by the Caco-2 transepithelial permeability assay. The toxin has been detected and quantified using a microsphere-based immunoassay. Cell alterations and ultrastructural effects has been observed with confocal and transmission electron microscopy Results: AZA1 was absorbed by Caco-2 cells in a dose-dependent way without affecting cell viability. However, modifications on occludin distribution detected by confocal microscopy imaging indicated a possible monolayer integrity disruption. Nevertheless, transmission electron microscopy imaging revealed ultrastructural damages at the nucleus and mitochondria with autophagosomes in the cytoplasm, however, tight junctions and microvilli remained unaffected. CONCLUSION: After the ingestion of molluscs with the AZA1, the toxin will be transported through the human intestinal barrier to blood causing damage on epithelial cells.

Subacute immunotoxicity of the marine phycotoxin yessotoxin in rats.

Yessotoxin (YTX) is a marine phycotoxin produced by dinoflagellates and accumulated in filter feeding shellfish. YTX content in shellfish is regulated by many food safety authorities to protect human health, although currently no human intoxication episodes have been unequivocally related to YTX presence in food. The immune system has been proposed as one of the target organs of YTX due to alterations of lymphoid tissues and cellular and humoral components. The aim of the present study was to explore subacute immunotoxicity of YTX in rats by evaluating the haematological response, inflammatory cytokine biomarkers and the presence of YTX-induced structural alterations in the spleen and thymus. The results showed that repeated administrations of YTX caused a decrease of lymphocyte percentage and an increase of neutrophil counts, a reduction in interleukine-6 (IL-6) plasmatic levels and histopathological splenic alterations in rats after four intraperitoneal injections of YTX at doses of 50 or 70 µg/kg that were administered every 4 days along a period of 15 days. Therefore, for the first time, subacute YTX-immunotoxicity is reported in rats, suggesting that repeated exposures to low amounts of YTX might also suppose a threat to human health, especially in immuno-compromised populations.

In vivo cardiomyocyte response to YTX- and AZA-1-induced damage: autophagy versus apoptosis.

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

Subacute Cardiotoxicity of Yessotoxin: In Vitro and in Vivo Studies.

Yessotoxin (YTX) is a marine phycotoxin produced by dinoflagellates and accumulated in filter feeding shellfish. Although no human intoxication episodes have been reported, YTX content in shellfish is regulated by many food safety authorities due to their worldwide distribution. YTXs have been related to ultrastructural heart damage in vivo, but the functional consequences in the long term have not been evaluated. In this study, we explored the accumulative cardiotoxic potential of YTX in vitro and in vivo. Preliminary in vitro evaluation of cardiotoxicity was based on the effect on hERG (human ether-a-go-go related gene) channel trafficking. In vivo experiments were performed in rats that received repeated administrations of YTX followed by recordings of electrocardiograms, arterial blood pressure, plasmatic cardiac biomarkers, and analysis of myocardium structure and ultrastructure. Our results showed that an exposure to 100 nM YTX for 12 or 24 h caused an increase of extracellular surface hERG channels. Furthermore, remarkable bradycardia and hypotension, structural heart alterations, and increased plasma levels of tissue inhibitor of metalloproteinases-1 were observed in rats after four intraperitoneal injections of YTX at doses of 50 or 70 µg/kg that were administered every 4 days along a period of 15 days. Therefore, and for the first time, YTX-induced subacute cardiotoxicity is supported by evidence of cardiovascular function alterations related to its repeated administration. Considering international criteria for marine toxin risk estimation and that the regulatory limit for YTX has been recently raised in many countries, YTX cardiotoxicity might pose a health risk to humans and especially to people with previous cardiovascular risk.

Heart Alterations after Domoic Acid Administration in Rats.

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

Subacute Cardiovascular Toxicity of the Marine Phycotoxin Azaspiracid-1 in Rats.

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

Acute cardiotoxicity evaluation of the marine biotoxins OA, DTX-1 and YTX.

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

Indole alkaloids from the Marquesan plant Rauvolfia nukuhivensis and their effects on ion channels.

In addition to the already reported nukuhivensiums 1 and 2, 11 indole alkaloids were isolated from the bark of the plant Rauvolfia nukuhivensis, growing in the Marquesas archipelago. The known sandwicine (3), isosandwicine (4), spegatrine (8), lochneram (9), flavopereirine (13) have been found in this plant together with the norsandwicine (5), isonorsandwicine (6), Nb-methylisosandwicine (7), 10-methoxypanarine (10), nortueiaoine (11), tueiaoine (12). The structure elucidation was performed on the basis of a deep exploration of the NMR and HRESIMS data as well as comparison with literature data for similar compounds. Norsandwicine, 10-methoxypanarine, tueiaoine, and more importantly nukuhivensiums, were shown to significantly induce a reduction of IKr amplitude (HERG current). Molecular modelling through docking was performed in order to illustrate this result.

In vitro chronic effects on hERG channel caused by the marine biotoxin azaspiracid-2.

Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellate Azadinium spinosum that accumulate in many shellfish species. Azaspiracid poisoning caused by AZA-contaminated seafood consumption is primarily manifested by diarrhea in humans. To protect human health, AZA-1, AZA-2 and AZA-3 content in seafood has been regulated by food safety authorities in many countries. Recently AZAs have been reported as a low/moderate hERG channel blockers. Furthermore AZA-2 has been related to arrhythmia appearance in rats, suggesting potential heart toxicity. In this study AZA-2 in vitro effects on hERG channel after chronic exposure are analyzed to further explore potential cardiotoxicity. The amount of hERG channel in the plasma membrane, hERG channel trafficking and hERG currents were evaluated up to 12 h of toxin exposure. In these conditions AZA-2 caused an increase of hERG levels in the plasma membrane, probably related to hERG retrograde trafficking impairment. Although this alteration did not translate into an increase of hERG channel-related current, more studies will be necessary to understand its mechanism and to know what consequences could have in vivo. These findings suggest that azaspiracids might have chronic cardiotoxicity related to hERG channel trafficking and they should not be overlooked when evaluating the threat to human health.

In vivo arrhythmogenicity of the marine biotoxin azaspiracid-2 in rats.

Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellate Azadinium spinosum that accumulate in several shellfish species. Azaspiracid poisoning episodes have been described in humans due to ingestion of AZA-contaminated seafood. Therefore, the contents of AZA-1, AZA-2 and AZA-3, the best-known analogs of the group, in shellfish destined to human consumption have been regulated by food safety authorities of many countries to protect human health. In vivo and in vitro toxicological studies have described effects of AZAs at different cellular levels and on several organs, however, AZA target remains unknown. Very recently, AZAs have been demonstrated to block the hERG cardiac potassium channel. In this study, we explored the potential cardiotoxicity of AZA-2 in vivo. The effects of AZA-2 on rat electrocardiogram (ECG) and cardiac biomarkers were evaluated for cardiotoxicity signs besides corroborating the hERG-blocking activity of AZA-2. Our results demonstrated that AZA-2 does not induce QT interval prolongation on rat ECGs in vivo, in spite of being an in vitro blocker of the hERG cardiac potassium channel. However, AZA-2 alters the heart electrical activity causing prolongation of PR intervals and the appearance of arrhythmias. More studies will be needed to clarify the mechanism by which AZA-2 causes these ECG alterations; however, the potential cardiotoxicity of AZAs demonstrated in this in vivo study should be taken into consideration when evaluating the possible threat that these toxins pose to human health, mainly for individuals with pre-existing cardiovascular disease when regulated toxin limits are exceeded.