Carmen Alfonso

First Toxicity Report of Tetrodotoxin and 5,6,11-TrideoxyTTX in the Trumpet Shell Charonia lampas lampas in Europe

Tetrodotoxin (TTX) is one of the most potent toxins already isolated, which occurs in a wide variety of animals. In this work, the occurrence of TTX and analogues was examined using mass spectrometry, confocal microscopy, liquid chromatography-mass spectrometry (LC-MS), and mouse bioassay in a trumpet shell (Charonia lampas lampas) and in the fluids of a patient poisoned by consuming this shell. Retention time data in the LC-MS system within the enhanced mass spectrum (EMS) mode indicated the presence of TTX and the analogue 5,6,11-trideoxyTTX; the enhanced product ion (EPI) mode confirmed the existence of both toxins with the formation of characteristic daughter ions from the fragment pattern of each molecule. TTX and 5,6,11-trideoxyTTX were only detected in the digestive gland of the trumpet shell and also in the urine and serum of the patient. The concentration of 5,6,11-trideoxyTTX checked in the samples by LC-MS was 3 times higher than TTX. However, the results obtained by mouse bioassay showed that the analogue is much less toxic than TTX. In vitro toxicity was checked using cerebellar cells; in these experiments the trumpet shell sample showed high toxicity, but the level was lower than in vivo results probably due to some competition between analogues. This paper shows for first time the presence and toxicity of TTX and 5,6,11-trideoxyTTX in a trumpet shell collected in the European coasts. The LC-MS method is a useful tool to confirm the presence of TTX and the further identification of TTX analogues.

Comment on “Effect of Uncontrolled Factors in a Validated Liquid Chromatography–Tandem Mass Spectrometry Method Question Its Use as a Reference Method for Marine Toxins: Major Causes for Concern”

Chromatographic techniques coupled to mass spectrometry is the method of choice to replace the mouse bioassay (MBA) to detect marine toxins. This paper evaluates the influence of different parameters such as toxin solvents, mass spectrometric detection method, mobile-phase-solvent brands and equipment on okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) quantification. In addition, the study compares the results obtained when a toxin is quantified against its own calibration curve and with the calibration curve of the other analogues. The experiments were performed by liquid chromatography (LC) and ultraperformance liquid chromatography (UPLC) with tandem mass spectrometry detection (MS/MS). Three acetonitrile brands and two toxin solvents were employed, and three mass spectrometry detection methods were checked. One method that contains the transitions for azaspiracid-1 (AZA-1), azaspiracid-2 (AZA-2), azaspiracid-3(AZA-3), gimnodimine (GYM), 13-desmethyl spirolide C (SPX-1), pectenotoxin-2 (PTX-2), OA, DTX-1, DTX-2, yessotoxin (YTX), homoYTX, and 45-OH-YTX was compared in both instruments. This method operated in simultaneous positive and negative ionization mode. The other two mass methods operated only in negative ionization mode, one contains transitions to detect DTX-1, OA DTX-2, YTX, homoYTX, and 45-OH-YTX and the other only the transitions for the toxins under study OA, DTX-1, and DTX-2. With dependence on the equipment and mobile phase used, the amount of toxin quantified can be overestimated or underestimated, up to 44% for OA, 46% for DTX-1, and 48% for DTX-2. In addition, when a toxin was quantified using the calibration curve of the other analogues, the toxin amount obtained is different. The maximum variability was obtained when DTX-2 was quantified using either OA or a DTX-1 calibration curve. In this case, the overestimation was up to 88% using the OA calibration curve and up to 204% using the DTX-1 calibration curve. In summary, the correct quantification of DSP toxins by MS detection depends on multiple factors. Since these factors are not taken into account in a validated protocol, these results question the convenience of having MS/MS as a reference method for protecting consumers of marine toxins, moreover if toxicity of each group is considered independently and total toxicity is not summed anymore as it is in the MBA.

First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples

In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50-350 μg kg(-1) meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.

Purification of five azaspiracids from mussel samples contaminated with DSP toxins and azaspiracids

Human intoxications during toxic episodes in shellfish are a very important concern for public health, as well as for economic interests of producer regions. Although initially each toxin appeared in a determined geographical zone, nowadays many of them are found in multiple places worldwide. In addition, more toxic compounds (new toxins or new analogs of known toxins) are being isolated and identified, which bring about new risks for public health. An example of this situation is the group of azaspiracids (AZAs). Initially these toxins were concentrated in Irish coasts but today appear in many different geographic locations; in the first toxic episode only three analogs were isolated, but now it is known that the group is comprised of at least eleven identified compounds. A substantial problem associated with all these new toxins is the extreme difficulty associated with the study of their toxic effects and mechanisms of action due to the very small quantities of purified toxin available. Therefore, the study of procedures to isolate them from contaminated shellfish or to synthesize them is of tremendous importance. In this paper we design a complete procedure to obtain AZAs analogs from mussels contaminated with DSP toxins and azaspiracids by means of three consecutive steps: an extraction procedure to remove toxins from shellfish, a solid phase extraction (SPE) to clean the samples and separate DSP toxins and AZAs, and a preparative HPLC to isolate each analog. In all the steps LC/MS is used to detect and quantify the toxins. Large amounts of AZA1, AZA2, AZA3, AZA4 and AZA5 were obtained by use of this procedure, which can be utilized in future studies relating to the toxins such as the production of certified materials and standards.

A comparative study of the effect of ciguatoxins on voltage-dependent Na+ and K+ channels in cerebellar neurons.

Ciguatera is a global disease caused by the consumption of certain warm-water fish (ciguateric fish) that have accumulated orally effective levels of sodium channel activator toxins (ciguatoxins) through the marine food chain. The effect of ciguatoxin standards and contaminated ciguatoxin samples was evaluated by electrophysiological recordings in cultured cerebellar neurons. The toxins affected both voltage-gated sodium (Nav) and potassium channels (Kv) although with different potencies. CTX 3C was the most active toxin blocking the peak inward sodium currents, followed by P-CTX 1B and 51-OH CTX 3C. In contrast, P-CTX 1B was more effective in blocking potassium currents. The analysis of six different samples of contaminated fish, in which a ciguatoxin analogue of mass 1040.6, not identical with the standard 51-OH CTX 3C, was the most prevalent compound, indicated an additive effect of the different ciguatoxins present in the samples. The results presented here constitute the first comparison of the potencies of three different purified ciguatoxins on sodium and potassium channels in the same neuronal preparation and indicate that electrophysiological recordings from cultured cerebellar neurons may provide a valuable tool to detect and quantify ciguatoxins in the very low nanomolar range.

Evaluation of Various pH and Temperature Conditions on the Stability of Azaspiracids and Their Importance in Preparative Isolation and Toxicological Studies

Azaspiracids (AZAs) are a group of shellfish toxins that were discovered in mussels from Irish waters in 1995. Because of the rare occurrence of poisoning incidents, the toxicity of the compounds is a continued matter of debate. Neither their mechanism of action nor their pharmacokinetic behavior has been elucidated, principally because of the lack of standards and reference tissues. Procedures to isolate AZAs from contaminated shellfish or to synthesize them have been developed; in particular, the procedures used for the preparative isolation of these toxins are currently being improved. The present paper describes the stability of AZAs in an array of pH and temperature conditions in methanolic solution, in shellfish tissue, and in aqueous mixtures of acids and shellfish tissues. Strong acids such as hydrochloric and formic acid led to rapid degradation of AZA1 at mM concentration, while the weaker acetic acid required harsher temperature conditions (70 degrees C) and greater concentrations to show similar effects. AZAs showed much greater stability in aqueous acidic mixtures with shellfish tissues, suggesting a significant protective effect of the matrix. A mechanism for the acid-catalyzed degradation is proposed, supported by mass spectral evidence from some of the degradation products. Strong bases (sodium hydroxide) also showed a detrimental effect on AZA1; however, weaker bases (ammonium hydroxide) did not lead to degradation over 24 h at room temperature. Finally, the toxic potential of acid degradation products of AZAs was found to be dramatically reduced compared to the parent compounds, as assessed through cytotoxicity.

Palytoxin detection and quantification using the fluorescence polarization technique.

Palytoxin (PLT) is a highly toxic nonpeptidic marine natural product, with a complex chemical structure. Its mechanism of action targets Na,K-ATPase. Fluorescence polarization (FP) is a spectroscopic technique that can be used to determine molecular interactions. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, FP was used to develop a detection method based on the interaction between the Na,K-ATPase and the PLT. The Na,K-ATPase was labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of protein-dye conjugate was measured when the amount of PLT in the medium was modified. The assay protocol was first developed using ouabain as a binding molecule. The final result was a straight line that correlates FP units and PLT concentration. Within this line the PLT equivalents in a natural sample can be quantified. A selective cleaning procedure to mussel samples and dinoflagellates cultures was also developed to avoid the matrix effect. The LOQ (limit of quantification) of the method is 10nM and the LOD (limit of detection) is 2 nM. This new PLT detection method is easier, faster, and more reliable than the other methods described to date.

Azaspiracid substituent at C1 is relevant to in vitro toxicity

The azaspiracids are a group of marine toxins recently described that currently includes 20 analogues. Not much is known about their mechanism of action, although effects on some cellular functions have been found in vitro. We used the reported effects on cell viability, actin cytoskeleton, and caspase activation to study the structure-activity relationship of AZA-1 and AZA-2 and the role of the carboxylic acid moiety in toxicity. AZA-1, AZA-2, and the synthetic AZA-2-methyl ester (AZA-2-ME), where the C1 carboxylic acid moiety of AZA-2 was esterified to the corresponding methyl ester moiety, induced a reduction of cell viability in neuroblastoma and hepatocyte cell lines with similar potency and kinetics. Interestingly, the mast cell line HMC-1 was resistant to AZA-induced cytotoxicity. Actin cytoskeleton alterations and caspase activation appeared after treatment with AZA-1, AZA-2, AZA-2-ME, and biotin-AZA-2 (AZA-2 labeled with biotin at C1) in neuroblastoma cells with similar qualitative, quantitative, and kinetics characteristics. Irreversibility of AZA effects on the actin cytoskeleton and cell morphology after short incubations with the toxin were common to AZA-1, AZA-2, and AZA-2-ME; however, 10-fold higher concentrations of biotin-AZA-2 were needed for irreversible effects. AZA-2-ME was rapidly metabolized in the cell to AZA-2, while transformation of biotin-AZA-2 into AZA-2 was less efficient, which explains the different potency in short exposure times. The moiety present at C1 is related to AZA toxicity in vitro. However, the presence of a methyl moiety at C8 is irrelevant to AZA toxicity since AZA-1 and AZA-2 were equipotent regardless of the readout effect.

Gambierone, a Ladder-Shaped Polyether from the Dinoflagellate Gambierdiscus belizeanus.

A new natural product named gambierone (1) was isolated from the cultured dinoflagellate Gambierdiscus belizeanus. The structure of this compound features an unprecedented polyether skeleton and an unusual right-hand side chain. Its relative configuration was fully determined by interpretation of ROESY experiment and comparison between experimental and theoretical NMR data. Although the succession of cycles has no chemical similarity with ciguatoxins, 1 has a molecular formula and biological activity similar to those of CTX-3C, although much lower in intensity.

Surface Plasmon Resonance Biosensor Method for Palytoxin Detection Based on Na + ,K + -ATPase Affinity

Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na+,K+-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na+,K+-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (kobs). From the representation of kobs versus PLTX concentration, the kinetic equilibrium dissociation constant (KD) for the PLTX-Na+,K+-ATPase association can be calculated. The value of this constant is KD = 6.38 × 10−7 ± 6.67 × 10−8 M PLTX. In this way the PLTX-Na+,K+-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods.