YTX treatment induced apoptosis in B16F10 cells.

<p>B16F10 cells were either left untreated or were treated for indicated times with 10, 30, 50 and 100nM YTX. YTX solvent controls were performed and no effects were observed. Cells were then analyzed for Annexin-V and PI staining by flow cytometry and the percentage of live cells (Annexin V-FITC -/PI -), early apoptotic (Annexin V-FITC +/PI -) and late apoptotic or necrotic cells (Annexin V-FITC +/PI +) was determined. Mean ± SEM of three experiments. Significant differences between untreated and YTX-treated cells: (*) p≤0.05, (**) p≤0.01 and (***) p≤0.001.</p

YTX treatment induced apoptosis and necrosis in the RBL-2H3 cell line.

<p>RBL-2H3 cells were either left untreated or were treated for indicated times with 10, 30, 50 and 100nM YTX. YTX solvent controls were performed and no effects were observed. Cells were then analysed for Annexin V-FITC and PI staining by flow cytometry and the percentage of live cells (Annexin V-FITC -/ PI -), early apoptotic (Annexin V-FITC +/PI -) and late apoptotic or necrotic cells (Annexin V-FITC +/PI +) was determined. Data are the mean ± SEM of three experiments. Significant differences between untreated and YTX-treated cells: (*) p≤0.05, (**) p≤0.01 and (***) p≤0.001.</p

Effect of YTX on β-hexosaminidase release induced by IgE/Ag in RBL-2H3 and BMMCs.

<p>IgE-sensitized RBL-2H3 cells (A) or BMMCs (B) were incubated with 10 and 30nM YTX or vehicle (methanol) for 30 or 60min. Then YTX was removed (+) from the medium or not (-) and cells were stimulated for 45min with DNP-HSA antigen at the indicated concentrations. The percentage release of β-hexosaminidase was determined and compared to vehicle treated cells. No effect was observed after vehicle incubation. Data (percentage release) are the mean ± SEM of three experiments. Significant differences between DNP-HSA- (hatched) and DNP-HSA+YTX-treated cells: (*) p≤0.05.</p

Effect of YTX on RBL-2H3 and BMMCs cell viability.

<p>Cells were incubated with 10, 30, 50 and 100nM YTX for the indicated times and cell viability was assessed by the MTT assay. Corresponding controls with YTX solvent (methanol) were performed and cell viability was arbitrarily set to 100%. Of note, solvent did not significantly affect cell viability as compared to non-treated cells even at the highest concentration of vehicle. Data are the mean ± SEM of three experiments. Significant differences between untreated and YTX-treated cells: (*) p≤0.05, (**) p≤0.01 and (***) p≤0.001.</p

Effect of YTX on MEC1 and B16F10 cell line viability.

<p>The MEC1 and B16F10 cell lines were incubated with 10, 30, 50 and 100nM YTX for the indicated times and cell viability was assessed by the MTT assay at the indicated time points. Corresponding controls with YTX solvent were performed and cell viability was arbitrarily set to 100%. Of note, solvent did not significantly affect cell viability as compared to non-treated cells even at the highest concentration of vehicle. Data are the mean ± SEM of three experiments. Significant differences between untreated and YTX-treated cells: (*) p≤0.05, (**) p≤0.01 and (***) p≤0.001.</p

YTX treatment decreases tumour development in the B16F10 melanoma mouse model.

<p>(A) Mice were injected subcutaneous B16F10 melanoma cells and tumours were allowed to develop until they reached a volume of 50 mm³ achieved between days 5 and 10. Mice were then either left untreated or were treated with the indicated concentrations of YTX or vehicle, which was injected subcutaneous right next to the tumour. (B) Representative glucose uptake after FX Pro Kodak image analysis of untreated, vehicle and YTX-treated mice at the day of sacrifice. (C) Corresponding resected tumour weight of animals after sacrifice at day 12 post-1st treatment. (D) Corresponding weight of untreated, vehicle- and YTX-treated mice. (E-G) Results of the haematological analysis performed before mice sacrifice. Data are expressed as mean ± SEM. Significant differences between untreated and YTX: (*) p≤0.05 and (***) p≤0.001.</p

In Vitro Effects of Chronic Spirolide Treatment on Human Neuronal Stem Cell Differentiation and Cholinergic System Development

Spirolides (SPX) are marine toxins, produced by dinoflagellates that act as potent antagonists of nicotinic acetylcholine receptors. These compounds are not toxic for humans, and since there are no reports of human intoxications caused by this group of toxins they are not yet currently regulated in Europe. Currently 13-desmethyl spirolide C, 13,19-didesmethyl spirolide C, and 20-methyl spirolide G are commercially available as reference materials. Previous work in our laboratory has demonstrated that after 4 days of treatment of primary mice cortical neurons with 13-desmethyl spirolide C, the compound ameliorated the glutamate induced toxicity and increased acetylcholine levels and the expression of the acetylcholine synthesizing enzyme being useful both in vitro and in vivo to decrease the brain pathology associated with Alzheimer’s disease. In this work, we aimed to extend the study of the neuronal effects of spirolides in human neuronal cells. To this end, human neuronal progenitor cells CTX0E16 were employed to evaluate the in vitro effect of spirolides on neuronal development. The results presented here indicate that long-term exposure (30 days) of human neuronal stem cells to SPX compounds, at concentrations up to 50 nM, ameliorated the MPP⁺-induced neurotoxicity and increased the expression of neuritic and dendritic markers, the levels of the choline acetyltransferase enzyme and the protein levels of the α7 subunit of nicotinic acetylcholine receptors. These effects are presumably due to the previously described interaction of these compounds with nicotinic receptors containing both α7 and α4 subunits. All together, these data emphasize the idea that SPX could be attractive lead molecules against neurodegenerative disorders

Protein Synthesis Inhibition and Oxidative Stress Induced by Cylindrospermopsin Elicit Apoptosis in Primary Rat Hepatocytes

The increasing presence of cyanotoxin producers in several regions of the world is hazardous for humans and animals. Cylindrospermopsin (CYN) is nowadays recognized as a widely distributed freshwater cyanobacterial toxin. This toxin has been shown to induce protein synthesis inhibition as well as inhibition of glutathione synthesis. Given that the liver seems to be the main target of cylindrospermopsin, in this work we used cultures of primary rat hepatocytes to study the type of cell death induced by CYN nanomolar concentrations. The involvement of reactive oxygen species in toxin induced cell death, the relationship between protein synthesis inhibition and toxicity, and the cell endogenous antioxidant response regulation were studied. We show that cylindrospermopsin induces apoptosis in primary rat hepatocytes. At the concentrations used in this work, protein synthesis inhibition and oxidative stress were involved in the cytotoxic effect elicited by the toxin. Finally, activation of the cell antioxidant response was observed at the transcriptional and translational levels

First Identification of Palytoxin-Like Molecules in the Atlantic Coral Species <i>Palythoa canariensis</i>

Palytoxin (PLTX) is a complex marine toxin produced by Zoanthids (<i>Palyhtoa</i>), dinoflagellates (<i>Ostreopsis</i>), and cyanobacteria (<i>Trichodesmium</i>). Contact with PLTX-like compounds present in aerosols or marine organisms has been associated with adverse effects on humans. The worldwide distribution of producer species and seafood contaminated with PLTX-like molecules illustrates the global threat to human health. The identification of species capable of palytoxin production is critical for human safety. We studied the presence of PLTX analogues in <i>Palythoa canariensis</i>, a coral species collected in the Atlantic Ocean never described as a PLTX-producer before. Two methodologies were used for the detection of these toxins: a microsphere-based immunoassay that offered an estimation of the content of PLTX-like molecules in a <i>Palythoa canariensis</i> extract and an ultrahigh-pressure liquid chromatography coupled to an ion trap with a time-of-flight mass spectrometer (UPLC-IT-TOF-MS) that allowed the characterization of the toxin profile. The results demonstrated the presence of PLTX, hydroxy-PLTX and, at least, two additional compounds with PLTX-like profile in the <i>Palythoa canariensis</i> sample. The PLTX content was estimated in 0.27 mg/g of lyophilized coral using UPLC-IT-TOF-MS. Therefore, this work demonstrates that <i>Palythoa canariensis</i> produces a mixture of PLTX-like molecules. This is of special relevance to safeguard human health considering <i>Palythoa</i> species are commonly used for decoration by aquarium hobbyists

Differential Effects of Ciguatoxin and Maitotoxin in Primary Cultures of Cortical Neurons

Ciguatoxins (CTXs) and maitotoxins (MTXs) are polyether ladder shaped toxins derived from the dinoflagellate <i>Gambierdiscus toxicus</i>. Despite the fact that MTXs are 3 times larger than CTXs, part of the structure of MTXs resembles that of CTXs. To date, the synthetic ciguatoxin, CTX 3C has been reported to activate voltage-gated sodium channels, whereas the main effect of MTX is inducing calcium influx into the cell leading to cell death. However, there is a lack of information regarding the effects of these toxins in a common cellular model. Here, in order to have an overview of the main effects of these toxins in mice cortical neurons, we examined the effects of MTX and the synthetic ciguatoxin CTX 3C on the main voltage dependent ion channels in neurons, sodium, potassium, and calcium channels as well as on membrane potential, cytosolic calcium concentration ([Ca²⁺]_c), intracellular pH (pH_i), and neuronal viability. Regarding voltage-gated ion channels, neither CTX 3C nor MTX affected voltage-gated calcium or potassium channels, but while CTX 3C had a large effect on voltage-gated sodium channels (VGSC) by shifting the activation and inactivation curves to more hyperpolarized potentials and decreasing peak sodium channel amplitude, MTX, at 5 nM, had no effect on VGSC activation and inactivation but decreased peak sodium current amplitude. Other major differences between both toxins were the massive calcium influx and intracellular acidification produced by MTX but not by CTX 3C. Indeed, the novel finding that MTX produces acidosis supports a pathway recently described in which MTX produces calcium influx via the sodium–hydrogen exchanger (NHX). For the first time, we found that VGSC blockers partially blocked the MTX-induced calcium influx, intracellular acidification, and protected against the short-term MTX-induced cytotoxicity. The results presented here provide the first report that shows the comparative effects of two prototypical ciguatera toxins, CTX 3C and MTX, in a neuronal model. We hypothesize that the analogies and differences in the bioactivity of these two toxins, produced by the same microorganism, may be strongly linked to their chemical structure