Paz Otero

First toxin profile of ciguateric fish in Madeira Arquipelago (Europe).

Ciguatera fish poisoning (CFP) is a human foodborne intoxication caused by ingestion of tropical fishes contaminated with the potent polyether toxins known as ciguatoxins (CTXs). These toxins are issued from Gambierdiscus species of dinoflagellates. Herbivorous fish accumulate these toxins in their musculature and viscera after ingesting dinoflagellates. Epidemiological studies showed that CFP has been present in areas between 35 degrees North and 35 degrees South latitude, mainly, Indo-pacific and Caribbean areas, but not in waters closed to European and African continent. In the present paper, a specimen of Seriola dumerili weighing 70 kg and a smaller Seriola fasciata specimen, captured in waters belonging to Selvagens Islands (Madeira Arquipelago), were analyzed. Fishes from this genus were implicated in previous suspected ciguatera poisoning outbreaks in the Portuguese Madeira Arquipelago in the North Atlantic Ocean. Analysis was performed by two approaches, a functional method using cerebellar granule cells and by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) method. The study was carried out in one portion of the tail muscle of Seriola fasciata and five parts of the body of Seriola dumerili (tail muscle, head, ventral muscle, mid muscle, and liver). The functional method consisted in the modification of the inward sodium current in cerebellar granule cells and the chemical method was a high resolution chromatography, which allowed elucidating the toxin profile in the samples. In addition, UPLC-MS technique was optimized and used for detecting and quantifying CTXs for the first time. After fish extraction and clean up, the chromatograms revealed the presence of CTX-1B at 1111.6 m/z, CTX-3C at 1023.5 m/z, a CTX analogue at 1040.6 m/z, and a CTX from the Caribbean or Indic waters at 1141.6 m/z. Therefore, the results obtained in the present paper for both methods confirm, for the first time, the presence of CTX in fish from Madeira Arquipelago.

Pharmacokinetic and toxicological data of spirolides after oral and intraperitoneal administration.

Spirolides are a kind of marine toxins included in the cyclic imine toxin group and produced by the dinoflagellate Alexandrium ostenfeldii. This study shows for the first time a complete and detailed description about the symptoms observed in mice when these toxins were intraperitoneal (i.p.) administered. It is also compared the i.p. toxicity of 13-desmethyl spirolide C (13-desMeC), 13,19-didesMeC (13,19-didesMeC) and 20-methyl spirolide G (20-Me-G) in experiments performed with highly purified toxins. The bioassay indicates that 13-desMeC and 13,19-didesMeC are extremely toxic compounds which have a LD(50) of 27.9μg/kg and 32.2μg/kg, respectively. However, when 20-MeG was i.p administrated with dose up 63.5μg/kg, no deaths were recorded. In order to evaluate the oral toxicity, spirolides were administered by gastric intubation into mice. Then, samples of blood, urine and faeces were collected and analyzed by liquid chromatography-mass spectrometry tandem (LC-MS/MS) technique. Spirolides appear in blood at 15min and in urine after 1h of being toxin administered. In summary, in this paper, it is provided new data about the toxicity, absorption, and excretion of spirolides in mouse. So far, little information is available on this item but necessary for spirolide regulation in the European Union (EU).

Effects of environmental regimens on the toxin profile of Alexandrium ostenfeldii

Environmental conditions are key factors in the development of marine toxic phytoplankton. Spirolides are marine toxins with a heptacyclic imine ring responsible for the toxicity in mice. Alexandrium ostenfeldii (A. ostenfeldii) is the main producer of these toxins, although this dinoflagellate often produces toxins belonging to the paralytic shellfish poisoning (PSP) group. The present study shows the first evidence that external environmental factors can influence the toxin profile produced by the dinoflagellate A. ostenfeldii. The species investigated is indigenous to the North Atlantic coast, and their cells grew under several environmental parameters. Toxin production was measured by means of liquid chromatography-mass spectrometry (LC-MS) and the chromatograms reflect the presence of two spirolides in all cultures; one in the region m/z 692.5, corresponding to 13-desmethyl spirolide C (13-desMeC) and the other in the region m/z 678.5, which corresponds to 13,19-didesmethyl spirolide C (13,19-didesMeC). The physical parameters studied were salinity, culture media, and photoperiod. The highest amount of toxin per cell was obtained when dinoflagellates grew in F/2 and Walne medium, 28 per thousand salinity, and 24 h of light. However, the highest proportion of 13,19-didesMeC with respect to 13-desMeC was achieved in L1 medium, 33 per thousand salinity, and 14:10 h light:dark. On the contrary, the highest proportion of 13-desMeC in cells was obtained when A. ostenfeldii was cultured in F/2 medium, 28 per thousand salinity, and the same photoperiod. Therefore, from these data the optimum conditions to culture A. ostenfeldii and to obtain the highest amount of spirolide per cell are shown. In addition, these environmental conditions can be considered a tool to predict and avoid A. ostenfeldii blooms.

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.

Detection of 13,19-didesmethyl C spirolide by fluorescence polarization using Torpedo electrocyte membranes.

Fluorescence polarization (FP) is a powerful tool for studying molecular interactions by monitoring changes in the apparent size of fluorescent molecules. In this paper, a previously described fluorescence polarization assay was used to detect 13,19-didesmethyl C spirolide. The assay is based on the competition of cyclic imine marine biotoxins with alpha-bungarotoxin for binding to nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata. The 13,19-didesmethyl C spirolide was detected in buffer and mussel matrix. The sensitivity of the assay for the 13,19-didesmethyl C spirolide and the 13-desmethyl C spirolide was similar. After an acetone/chloroform extraction of spiked mussel meat, the average recovery rate of 13,19-didesmethyl C spirolide was 77.7 +/- 1.9%. The quantification range for this toxin in mussel was 40-200 microg/kg of shellfish meat. This assay can be used to detect the spirolides 13,19-didesmethyl C spirolide and 13-desmethyl C spirolide, in shellfish as a screening assay.

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.

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.