Alvaro Antelo

Synthesis and Characterization of a New Gemini Surfactant Derived from 3α,12α-Dihydroxy-5β-cholan-24-amine (Steroid Residue) and Ethylenediamintetraacetic Acid (Spacer)

A new gemini steroid surfactant derived from 3alpha,12alpha-dihydroxy-5beta-cholan-24-amine (steroid residue) and ethylenediamintetraacetic acid (spacer) was synthesized and characterized in aqueous solution by surface tension, fluorescence intensity of pyrene, and light scattering (static and dynamic) measurements. These techniques evidence the existence of a threshold concentration (cac), below which a three layers film is formed at the air-water interface. Above the cac, two types of aggregates--micelles and vesicle-like aggregates--coexist in a metastable state. Filtration of a solution with a starting concentration of 2.6 mM (buffer 150 mM, pH 10) allows isolation of the micelles, which have an average aggregation number of 12, their density being 0.28 g cm(-3). Under conditions where only the vesicle-like aggregates are detected by dynamic light scattering, a value of 5.5 x 10(4) was obtained for their aggregation number at 30 microM, their density being 6.8 x 10(-4) g cm(-3). At high concentrations, the intensity ratio of the vibronic peaks of pyrene, I1/I3, (=0.68) is very close to published values for deoxycholate micelles, indicating that the probe is located in a region with a very low polarity and far from water. A hypothesis to explain the observed aggregation behavior (small aggregates are favored with increasing gemini concentration) is outlined.

Benefit of 13-desmethyl Spirolide C Treatment in Triple Transgenic Mouse Model of Alzheimer Disease: Beta-Amyloid and Neuronal Markers Improvement

Spirolides are marine toxins that are not currently in the routine monitoring assays. Nicotinic receptors seem to be the target of these compounds making them a promising pharmacological tool for related diseases as dementias as previously shown in vitro. In the present work, the bioavailability of 13-desMethyl spirolide C (13-desMeC) in the brain and in vivo effects were tested. Bioavailability was studied by ultra-performance liquid chromatography-mass spectrometry and its effect over Alzheimer hallmarks was studied by Proton magnetic resonance spectroscopy (H-MRS) and western blot. Only 2 minutes after its intraperitoneal injection it is found in brain and remains detectable even 24 hours post administration. Based on previous works that showed beneficial effects in an in vitro model of Alzheimers disease (AD), we studied the effect in the same mice, 3xTg-AD, in vivo. We found that 13-desMeC (11.9 ug/kg, i.p.) induced positive effects on AD markers with an increase in N-acetyl aspartate (NAA) levels. These results were supported by an increase in synaptophysin levels and also a decrease in the intracellular amyloid beta levels in the hippocampus of treated 3xTg- AD versus non treated mice remarking the positive effects of this molecule in a well known model of AD. These data indicate for the first time that 13-desMeC cross the blood brain barrier and shows in vivo beneficial effects against AD after administration of low intraperitoneal doses of this marine toxin. This toxin may inspire a novel medical treatment of age-related diseases.

Supramolecular structures generated by a p-tert-butylphenylamide derivative of deoxycholic acid. From planar sheets to tubular structures through helical ribbons.

The formation of supramolecular structures initiated by a p-tert-butylphenylamide derivative of deoxycholic acid (Na-t-butPhDC) is investigated. At 1.18 mM concentration of Na-t-butPhDC and 37 degrees C, initial flat ribbons are observed which self-transform into helical ribbons (with a mean pitch angle of 47 +/- 6 degrees) which finally originate molecular tubes with an external diameter of 241 +/- 28 nm. Most of the molecular tubes show helical markings with a pitch angle value of 45 +/- 4 degrees, in full agreement with predictions of simple models based on chiral elastic properties of the membrane. A lateral association mechanism is proposed to account for the growth of the external diameter (from 225 +/- 32 to 546 +/- 59 nm) of tubes with time at 3.99 mM.

Aggregation behavior of tetracarboxylic surfactants derived from cholic and deoxycholic acids and ethylenediaminetetraacetic acid.

The reaction of 3beta-aminoderivatives of cholic and deoxycholic acids (steroid residues) with dimethyl ester of ethylenediaminetetraacetic acid (bridge) leads to the formation of dimers carrying four carboxylic organic functions, two of them located on the side chain of each steroid residue and the other two on the bridge. As tetrasodium salts, these new compounds behave as surfactants and have been characterized by surface tension, fluorescence intensity of pyrene (as a probe), and static and dynamic light scattering measurements. Thermodynamic parameters for micellization were obtained from the dependence of the critical micelle concentration (cmc) with temperature. For both surfactants, the fraction of bound counterions is close to 0.5. The aggregation behavior is similar to one of their bile salt residues [i.e., sodium cholate (NaC) and sodium deoxycholate (NaDC)] and can be summarized as follows: (i) molecular areas at the interface for the new surfactants are fairly close to twice the value for a single molecule in a monolayer of natural bile salts; (ii) the environment where pyrene is solubilized is very apolar, as in natural bile salt aggregates; (iii) Gibbs free energies (per steroid residue) for micellization are not far from published values for NaC and NaDC, and the differences can be understood on the basis of less hydrophobicity of the new surfactants due to the charges in the bridge; and (iv) as for NaC and NaDC, aggregates have rather low aggregation numbers (which depend on the amount of added inert salt, NaCl). A structure based on the disklike model accepted for small bile salt aggregates is proposed.

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 Gambierdiscus toxicus. 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 ([Ca(2+)]c), intracellular pH (pHi), 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.

Acute Oral Toxicity of Tetrodotoxin in Mice: Determination of Lethal Dose 50 (LD50) and No Observed Adverse Effect Level (NOAEL)

Tetrodotoxin (TTX) is starting to appear in molluscs from the European waters and is a hazard to seafood consumers. This toxin blocks sodium channels resulting in neuromuscular paralysis and even death. As a part of the risk assessment process leading to a safe seafood level for TTX, oral toxicity data are required. In this study, a 4-level Up and Down Procedure was designed in order to determine for the first time the oral lethal dose 50 (LD50) and the No Observed Adverse Effect Level (NOAEL) in mice by using an accurate well-characterized TTX standard.

Liquid Chromatography with a Fluorimetric Detection Method for Analysis of Paralytic Shellfish Toxins and Tetrodotoxin Based on a Porous Graphitic Carbon Column

Paralytic shellfish toxins (PST) traditionally have been analyzed by liquid chromatography with either pre- or post-column derivatization and always with a silica-based stationary phase. This technique resulted in different methods that need more than one run to analyze the toxins. Furthermore, tetrodotoxin (TTX) was recently found in bivalves of northward locations in Europe due to climate change, so it is important to analyze it along with PST because their signs of toxicity are similar in the bioassay. The methods described here detail a new approach to eliminate different runs, by using a new porous graphitic carbon stationary phase. Firstly we describe the separation of 13 PST that belong to different groups, taking into account the side-chains of substituents, in one single run of less than 30 min with good reproducibility. The method was assayed in four shellfish matrices: mussel (Mytillus galloprovincialis), clam (Pecten maximus), scallop (Ruditapes decussatus) and oyster (Ostrea edulis). The results for all of the parameters studied are provided, and the detection limits for the majority of toxins were improved with regard to previous liquid chromatography methods: the lowest values were those for decarbamoyl-gonyautoxin 2 (dcGTX2) and gonyautoxin 2 (GTX2) in mussel (0.0001 mg saxitoxin (STX)·diHCl kg−1 for each toxin), decarbamoyl-saxitoxin (dcSTX) in clam (0.0003 mg STX·diHCl kg−1), N-sulfocarbamoyl-gonyautoxins 2 and 3 (C1 and C2) in scallop (0.0001 mg STX·diHCl kg−1 for each toxin) and dcSTX (0.0003 mg STX·diHCl kg−1 ) in oyster; gonyautoxin 2 (GTX2) showed the highest limit of detection in oyster (0.0366 mg STX·diHCl kg−1). Secondly, we propose a modification of the method for the simultaneous analysis of PST and TTX, with some minor changes in the solvent gradient, although the detection limit for TTX does not allow its use nowadays for regulatory purposes.

Evaluation of the impact of mild steaming and heat treatment on the concentration of okadaic acid, dinophysistoxin-2 and dinophysistoxin-3 in mussels

This study explores the effect of laboratory and industrial steaming on mussels with toxin concentrations above and below the legal limit. We used mild conditions for steaming, 100 °C for 5 min in industrial processing, and up to 20 min in small-scale laboratory steaming. Also, we studied the effect of heat on the toxin concentration of mussels obtained from two different locations and the effect of heat on the levels of dinophysistoxins 3 (DTX3) in both the mussel matrix and in pure form (7-O-palmitoyl okadaic ester and 7-O-palmytoleyl okadaic ester). The results show that the loss of water due to steaming was very small with a maximum of 9.5%, that the toxin content remained unchanged with no concentration effect or increase in toxicity, and that dinophysistoxins 3 was hydrolyzed or degraded to a certain extent under heat treatment. The use of liquid-certified matrix showed a 55% decrease of dinophysistoxins 3 after 10 min steaming, and a 50% reduction in total toxicity after treatment with an autoclave (121 °C for 20 min).

Rapid analysis of paralytic shellfish toxins and tetrodotoxins by liquid chromatography-tandem mass spectrometry using a porous graphitic carbon column

Although paralytic shellfish toxins (PSTs) have traditionally been analyzed by liquid chromatography with either pre- or post-column derivatization, and these methods have been validated successfully through inter-laboratory studies, mass spectrometry methods have also been described in literature for use in monitoring programs. However, methods using liquid chromatography coupled with mass spectrometry (LC-MS) need to be improved in terms of sensitivity, analyte recovery and retention time stability because of undesirable matrix effects. Furthermore, tetrodotoxin (TTX) has been found in northern European bivalves, so it is important to analyze TTX compounds alongside PSTs because characteristics of their toxicity are similar. This paper describes, for the first time, a chemical method that allows determination of PSTs, both hydrophilic and hydrophobic, alongside TTX and its analogue 4,9-anhydro tetrodotoxin (4,9-anhTTX) with LC-MS/MS using a Hypercarb® column. The method was validated for 13 hydrophilic PSTs and TTXs and was able to discriminate six hydrophobic PSTs in 20 min. The method was developed for four shellfish matrices: mussel (Mytillus galloprovincialis), clam (Ruditapes decussatus), scallop (Pecten maximus) and oyster (Ostrae edulis). Clean-up procedure used in this work allowed us to obtain good results for validation parameters for both PSTs and TTXs. No standards were available so strains of Gymnodinium catenatum (G. catenatum) were used instead.