Mercedes R. Vieytes

Modulation of cytosolic calcium levels of human lymphocytes by yessotoxin, a novel marine phycotoxin!

Yessotoxin (YTX) is a polyether toxin of marine origin that has been classified among the diarrheic shellfish poisoning (DSP) toxins group due to its lipophilic nature. However, unlike other DSP toxins, YTX does not produce diarrhea and its mechanisms of action are unknown. We studied the effect of YTX on the cytosolic calcium levels of freshly isolated human lymphocytes by means of fluorescence imaging microscopy. We showed that YTX produced a calcium influx through nifedipine and SKF 96365 (1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenyl]-1H-imidazole hydrochloride)-sensitive channels. This Ca2+ entry was not affected by the DSP toxin okadaic acid, which inhibits protein phosphatases. In addition, YTX also produced an inhibition of capacitative calcium entry activated by thapsigargin or by preincubation in a Ca2+-free medium. This capacitative calcium entry was not sensitive to nifedipine. Furthermore, the inhibitory effect of YTX was dependent on the time of addition of the toxin. We suggest that YTX may interact with calcium channels in a way similar to that described for other polyether marine compounds such as brevetoxins and maitotoxin, although an involvement of other second messengers is also likely.

Azaspiracid-1, a potent, nonapoptotic new phycotoxin with several cell targets

This paper reports on potential cellular targets of azaspiracid-1 (AZ-1), a new phycotoxin that causes diarrhoeic and neurotoxic symptoms and whose mechanism of action is unknown. In excitable neuroblastoma cells, the systems studied were membrane potential, F-actin levels and mitochondrial membrane potential. AZ-1 does not modify mitochondrial activity but decreases F-actin concentration. These results indicate that the toxin does not have an apoptotic effect but uses actin for some of its effects. Therefore, cytoskeleton seems to be an important cellular target for AZ-1 effect. AZ-1 does not induce any modification in membrane potential, which does not support for neurotoxic effects. In human lymphocytes, cAMP, cytosolic calcium and cytosolic pH (pHi) levels were also studied. AZ-1 increases cytosolic calcium and cAMP levels and does not affect pHi (alkalinization). Cytosolic calcium increase seems to be dependent on both the release of calcium from intracellular Ca(2+) pools and the influx from extracellular media through Ni(2+)-blockable channels. AZ-1-induced Ca(2+) increase is negatively modulated by protein kinase C (PKC) activation, protein phosphatases 1 and 2A (PP1 and PP2A) inhibition and cAMP increasing agents. The effect of AZ-1 in cAMP is not extracellularly Ca(2+) dependent and insensitive to okadaic acid (OA).

In Vitro and in Vivo Evaluation of Paralytic Shellfish Poisoning Toxin Potency and the Influence of the pH of Extraction

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.

Maitotoxin-induced calcium entry in human lymphocytes: Modulation by yessotoxin, Ca2+ channel blockers and kinases

We have studied the effect of the ciguatera-related toxin maitotoxin (MTX) on the cytosolic free calcium concentration ([Ca(2+)]i) of human peripheral blood lymphocytes loaded with the fluorescent probe Fura2 and the regulation of MTX action by different drugs known to interfere in cellular Ca(2+) signalling mechanisms and by the marine phycotoxin yessotoxin (YTX). MTX produced a concentration-dependent elevation of [Ca(2+)]i in a Ca(2+)-containing medium. This effect was stimulated by pretreatment with YTX 1 microM and NiCl(2) 15 microM. The voltage-independent Ca(2+) channel antagonist 1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenyl]-1H-imidazole hydrochloride (SKF96365) blocked the MTX-induced [Ca(2+)]i elevation, while the L-type channel blocker nifedipine had no effect. Pretreatment with NiCl(2) or nifedipine did not modify YTX-induced potentiation of MTX effect, and SKF96365-induced inhibition was reduced in the presence of YTX, which suggest different pathways to act on [Ca(2+)]i. Preincubation with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H-89) or genistein (10 microM) also had no effect on the MTX-induced [Ca(2+)]i increment. In contrast, the PKC inhibitor bisindolilmaleimide I (GF109203X 1 microM) potentiated the MTX effect, whereas phosphatidylinositol (PI) 3-kinase inhibition with wortmannin (10 nM) reduced the MTX-elicited Ca(2+) entry. In summary, MTX produced Ca(2+) influx into human lymphocytes through a SKF96365-sensitive, nifedipine-insensitive pathway. The MTX-induced [Ca(2+)]i elevation was stimulated by the marine toxin YTX through a mechanism insensitive to SKF96365, nifedipine or NiCl(2). It was also stimulated by the divalent cation Ni(2+) and PKC inhibition and was partially inhibited by PI 3-kinase inhibition.

Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells

Palytoxin is one of the most complex and biggest molecules known to show extreme acute toxicity. The dinoflagellate Ostreopsis spp., the producer organism of palytoxin, has been shown to be distributed worldwide, thus making palytoxin an emerging toxin. Rat-derived hepatocytes (Clone 9) and BE (2)-M17 human neuroblastoma cells were used to test palytoxin or palytoxin-like compounds by measuring the cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity (as low as 0.2 ng/ml). This method was useful for toxin detection in Ostreopsis extracts and naturally contaminated mussel samples. A comparative study testing toxic mussel extracts by LC (liquid chromatography)-MS/MS (tandem MS), MBA (mouse bioassay), haemolysis neutralization assay and a cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds.

Modified mass action law-based model to correlate the solubility of solids and liquids in entrained supercritical carbon dioxide.

The solubility of solids and liquids in supercritical CO2 with added entrainers was modeled with a modified version of the equation of Chrastil to include the effect of entrainers. By considering the formation of the solute-entrainer-solvent complexes an equation is obtained which predicts an exponential increase of solubility with fluid density and/or entrainer concentration. The correlating model was tested by non-linear regression through a computerized iterative process for several systems where an entrainer was present. Four experimental parameters are easily regressed from experimental data, hence the corresponding properties of components such as chemical potentials or critical parameters are not needed. Instead of its simplicity, this thermodynamical model provided a good correlation of the solubility enhancement in the presence of entrainer effect.

Design and Synthesis of Skeletal Analogues of Gambierol: Attenuation of Amyloid-β and Tau Pathology with Voltage-Gated Potassium Channel and N-Methyl-d-aspartate Receptor Implications

Gambierol is a potent neurotoxin that belongs to the family of marine polycyclic ether natural products and primarily targets voltage-gated potassium channels (K(v) channels) in excitable membranes. Previous work in the chemistry of marine polycyclic ethers has suggested the critical importance of the full length of polycyclic ether skeleton for potent biological activity. Although we have previously investigated structure-activity relationships (SARs) of the peripheral functionalities of gambierol, it remained unclear whether the whole polycyclic ether skeleton is needed for its cellular activity. In this work, we designed and synthesized two truncated skeletal analogues of gambierol comprising the EFGH- and BCDEFGH-rings of the parent compound, both of which surprisingly showed similar potency to gambierol on voltage-gated potassium channels (K(v)) inhibition. Moreover, we examined the effect of these compounds in an in vitro model of Alzheimers disease (AD) obtained from triple transgenic (3xTg-AD) mice, which expresses amyloid beta (Aβ) accumulation and tau hyperphosphorylation. In vitro preincubation of the cells with the compounds resulted in significant inhibition of K(+) currents, a reduction in the extra- and intracellular levels of Aβ, and a decrease in the levels of hyperphosphorylated tau. In addition, pretreatment with these compounds reduced the steady-state level of the N-methyl-D-aspartate (NMDA) receptor subunit 2A without affecting the 2B subunit. The involvement of glutamate receptors was further suggested by the blockage of the effect of gambierol on tau hyperphosphorylation by glutamate receptor antagonists. The present study constitutes the first discovery of skeletally simplified, designed polycyclic ethers with potent cellular activity and demonstrates the utility of gambierol and its synthetic analogues as chemical probes for understanding the function of K(v) channels as well as the molecular mechanism of Aβ metabolism modulated by NMDA receptors.

13-Desmethyl spirolide-C is neuroprotective and reduces intracellular Aβ and hyperphosphorylated tau in vitro.

Spirolides are marine compounds of the cyclic imine group. Although the mechanism of action is not fully elucidated yet, cholinergic (muscarinic and nicotinic) receptors have been proposed as the main targets of these toxins. In this study we examined the effect of 13-desmethyl spirolide-C (SPX) on amyloid-beta (Aβ) accumulation and tau hyperphosphorylation in a neuronal model from triple transgenic mice (3xTg) for Alzheimer disease (AD). In vitro treatment of 3xTg cortical neurons with SPX reduced intracellular Aβ accumulation and the levels of phosphorylated tau. SPX treatment did not affect the steady-state levels of neither the M1 and M2 muscarinic nor the α7 nicotinic acetylcholine receptors (AChRs), while it decreased the amplitude of acetylcholine-evoked responses and increased ACh (acetylcholine) levels in 3xTg neurons. Additionally, SPX treatment decreased the levels of two protein kinases involved in tau phosphorylation, glycogen synthase kinase 3β (GSK-3β) and extracellular-regulated kinase (ERK). Also SPX abolished the glutamate-induced neurotoxicity in both control and 3xTg neurons. The results presented here constitute the first report indicating that exposure of 3xTg neurons to nontoxic concentrations of SPX produces a simultaneous reduction in the main pathological characteristics of AD. In spite of the few reports analyzing the mode of action of the toxin we suggest that SPX could ameliorate AD pathology increasing the intracellular ACh levels and simultaneously diminishing the levels of kinases involved in tau phosphorylation.

“Fluorescent glycogen” formation with sensibility for in vivo and in vitro detection

There are presently many methods of detecting complex carbohydrates, and particularly glycogen. However most of them require radioisotopes or destruction of the tissue and hydrolysis of glycogen to glucose. Here we present a new method based on the incorporation of 2-NBDG (2-{N-[7-nitrobenz-2-oxa-1, 3-diazol 4-yl] amino}-2-deoxyglucose), a d-glucose fluorescent derivative, into glycogen. Two kinds of approaches were carried out by using Clone 9 rat hepatocytes as a cellular model; (1) Incubation of cell lysates with 2-NBDG, carbohydrate precipitation in filters and measurement of fluorescence in a microplate reader (2) Incubation of living hepatocytes with 2-NBDG and recording of fluorescence images by confocal microscopy. 2-NBDG labeled glycogen in both approaches. We confirmed this fact by comparison to the labeling obtained with a specific monoclonal anti-glycogen antibody. Also drugs that trigger glycogen synthesis or degradation induced an increase or decrease of fluorescence, respectively. This is a simple but efficient method of detecting glycogen with 2-NBDG. It could be used to record changes in glycogen stores in living cells and cell-free systems and opens the prospect of understanding the role of this important energy reserve under various physiological and pathophysiological conditions.

Yessotoxin induces ER-stress followed by autophagic cell death in glioma cells mediated by mTOR and BNIP3.

Yessotoxin at nanomolar concentrations can induce programmed cell death in different model systems. Paraptosis-like cell death induced by YTX in BC3H1 cells, which are insensitive to several caspase inhibitors, has also been reported. This makes yessotoxin of interest in the search of molecules that target cancer cells vulnerabilities when resistance to apoptosis is observed. To better understand the effect of this molecule at the molecular level on tumor cells, we conducted a transcriptomic analysis using 3 human glioma cell lines with different sensitivities to yessotoxin. We show that the toxin induces a deregulation of the lipid metabolism in glioma cells as a consequence of induction of endoplasmic reticulum stress. The endoplasmic reticulum stress in turn arrests the cell cycle and inhibits the protein synthesis. In the three cell lines used we show that YTX induces autophagy, which is involved in cell death. The sensibility of the cell lines used towards autophagic cell death was related to their doubling time, being more resistant the cell line with the lowest proliferation rate. The involvement of mTOR and BNIP3 in the autophagy induction was also determined.