Inés Rodríguez

First Detection of Tetrodotoxin in Greek Shellfish by UPLC-MS/MS Potentially Linked to the Presence of the Dinoflagellate Prorocentrum minimum

During official shellfish control for the presence of marine biotoxins in Greece in year 2012, a series of unexplained positive mouse bioassays (MBA) for lipophilic toxins with nervous symptomatology prior to mice death was observed in mussels from Vistonikos Bay–Lagos, Rodopi. This atypical toxicity coincided with (a) absence or low levels of regulated and some non-regulated toxins in mussels and (b) the simultaneous presence of the potentially toxic microalgal species Prorocentrum minimum at levels up to 1.89 × 103 cells/L in the area’s seawater. Further analyses by different MBA protocols indicated that the unknown toxin was hydrophilic, whereas UPLC-MS/MS analyses revealed the presence of tetrodotoxins (TTXs) at levels up to 222.9 μg/kg. Reviewing of official control data from previous years (2006–2012) identified a number of sample cases with atypical positive to asymptomatic negative MBAs for lipophilic toxins in different Greek production areas, coinciding with periods of P. minimum blooms. UPLC-MS/MS analysis of retained sub-samples from these cases revealed that TTXs were already present in Greek shellfish since 2006, in concentrations ranging between 61.0 and 194.7 μg/kg. To our knowledge, this is the earliest reported detection of TTXs in European bivalve shellfish, while it is also the first work to indicate a possible link between presence of the toxic dinoflagellate P. minimum in seawater and that of TTXs in bivalves. Confirmed presence of TTX, a very heat-stable toxin, in filter-feeding mollusks of the Mediterranean Sea, even at lower levels to those inducing symptomatology to humans, indicates that this emerging risk should be seriously taken into account by the EU to protect the health of shellfish consumers.

First report of Ciguatoxins in two starfish species: Ophidiaster ophidianus and Marthasterias glacialis

Ciguatera fish poisoning (CFP) is a syndrome caused by the ingestion of fish contaminated with Ciguatoxins (CTXs). These phycotoxins are produced mainly by dinoflagellates that belong to the genus Gambierdiscus that are transformed in more toxic forms in predatory fish guts, and are more present in the Indo-Pacific and Caribbean areas. It is estimated that CFP causes per year more than 10,000 intoxications worldwide. With the rise of water temperature and anthropogenic intervention, it is important to study the prevalence of CFP in more temperate waters. Through inter- and subtidal sampling, 22 species of organisms were collected, in Madeira and Azores archipelagos and in the northwestern Moroccan coast, during September of 2012 and June and July of 2013. A total of 94 samples of 22 different species of bivalves, gastropods, echinoderms and crustaceans where analyzed by Ultra Performance Liquid Chromatography-Mass Spectometry-Ion Trap-Time of Flight (UPLC-MS-IT-TOF) and Ultra Performance Chromatography- Mass Spectrometry (UPLC-MS). Our main aim was to detect new vectors and ascertain if there were some geographical differences. We detected for the first time putative CTXs in echinoderms, in two starfish species—M. glacialis and O. ophidianus. We detected differences regarding uptake values by organisms and geographical location. Toxin amounts were significant, showing the importance and the need for continuity of these studies to gain more knowledge about the prevalence of these toxins, in order to better access human health risk. In addition, we suggest monitoring of these toxins should be extended to other vectors, starfish being a good alternative for protecting and accessing human health risk.

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.

The association of bacterial C9-based TTX-like compounds with Prorocentrum minimum opens new uncertainties about shellfish seafood safety

In 2012, Tetrodotoxin (TTX) was identified in mussels and linked to the presence of Prorocentrum minimum (P. minimum) in Greece. The connexion between TTX and P. minimum was further studied in this paper. First, the presence of TTX-producer bacteria, Vibrio and Pseudomonas spp, was confirmed in Greek mussels. In addition these samples showed high activity as inhibitors of sodium currents (INa). P. minimum was before associated with neurotoxic symptoms, however, the nature and structure of toxins produced by this dinoflagellate remains unknown. Three P. minimum strains, ccmp1529, ccmp2811 and ccmp2956, growing in different conditions of temperature, salinity and light were used to study the production of toxic compounds. Electrophysiological assays showed no effect of ccmp2811 strain on INa, while ccmp1529 and ccmp2956 strains were able to significantly reduce INa in the same way as TTX. In these samples two new compounds, m/z 265 and m/z 308, were identified and characterized by liquid chromatography tandem high-resolution mass spectrometry. Besides, two TTX-related bacteria, Roseobacter and Vibrio sp, were observed. These results show for the first time that P. minimum produce TTX-like compounds with a similar ion pattern and C9-base to TTX analogues and with the same effect on INa.

Monitoring of freshwater toxins in European environmental waters by using novel multi-detection methods

Monitoring the quality of freshwater is an important issue for public health. In the context of the European project μAqua, 150 samples were collected from several waters in France, Germany, Ireland, Italy, and Turkey for 2 yr. These samples were analyzed using 2 multitoxin detection methods previously developed: a microsphere-based method coupled to flow-cytometry, and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. The presence of microcystins, nodularin, domoic acid, cylindrospermopsin, and several analogues of anatoxin-a (ATX-a) was monitored. No traces of cylindrospermopsin or domoic acid were found in any of the environmental samples. Microcystin-LR and microcystin-RR were detected in 2 samples from Turkey and Germany. In the case of ATX-a derivatives, 75% of samples contained mainly H2 -ATX-a and small amounts of H2 -homoanatoxin-a, whereas ATX-a and homoanatoxin-a were found in only 1 sample. These results confirm the presence and wide distribution of dihydro derivatives of ATX-a toxins in European freshwaters. Environ Toxicol Chem 2017;36:645-654.

How Safe Is Safe for Marine Toxins Monitoring

Current regulation for marine toxins requires a monitoring method based on mass spectrometric analysis. This method is pre-targeted, hence after searching for pre-assigned masses, it identifies those compounds that were pre-defined with available calibrants. Therefore, the scope for detecting novel toxins which are not included in the monitoring protocol are very limited. In addition to this, there is a poor comprehension of the toxicity of some marine toxin groups. Also, the validity of the current approach is questioned by the lack of sufficient calibrants, and by the insufficient coverage by current legislation of the toxins reported to be present in shellfish. As an example, tetrodotoxin, palytoxin analogs, or cyclic imines are mentioned as indicators of gaps in the system that require a solid comprehension to assure consumers are protected.

Tetracyclic Truncated Analogue of the Marine Toxin Gambierol Modifies NMDA, Tau, and Amyloid β Expression in Mice Brains: Implications in AD Pathology

Gambierol and its two, tetra- and heptacyclic, analogues have been previously proved as promising molecules for the modulation of Alzheimers disease (AD) hallmarks in primary cortical neurons of 3xTg-AD fetuses. In this work, the effect of the tetracyclic analogue of gambierol was tested in vivo in 3xTg-AD mice (10 months old) after 1 month of weekly treatment with 50 μg/kg. Adverse effects were not reported throughout the whole treatment period and no pathological signs were observed for the analyzed organs. The compound was found in brain samples after intraperitoneal injection. The tetracyclic analogue of gambierol elicited a decrease of amyloid β1-42 levels and a dose-dependent inhibition of β-secretase enzyme-1 activity. Moreover, this compound also reduced the phosphorylation of tau at the 181 and 159/163 residues with an increase of the inactive isoform of the glycogen synthase kinase-3β. In accordance with our in vitro neuronal model, this compound produced a reduction in the N2A subunit of the N-methyl-d-aspartate (NMDA) receptor. The combined effect of this compound on amyloid β1-42 and tau phosphorylation represents a multitarget therapeutic approach for AD which might be more effective for this multifactorial and complex neurodegenerative disease than the current treatments.

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).

Characterization of the dinophysistoxin-2 acute oral toxicity in mice to define the Toxicity Equivalency Factor

Ingestion of shellfish with dinophysistoxin-2 (DTX2) can lead to diarrheic shellfish poisoning (DSP). The official control method of DSP toxins in seafood is the liquid chromatography-mass spectrometry analysis (LC-MS). However in order to calculate the total toxicity of shellfish, the concentration of each compound must be multiplied by individual Toxicity Equivalency Factor (TEF). Considering that TEFs caused some controversy and the scarce information about DTX2 toxicity, the aim of this study was to characterize the oral toxicity of DTX2 in mice. A 4-Level Up and Down Procedure allowed the characterization of DTX2 effects and the estimation of DTX2 oral TEF based on determination of the lethal dose 50 (LD50). DTX2 passed the gastrointestinal barrier and was detected in urine and feces. Acute toxicity symptoms include diarrhea and motionless, however anatomopathology study and ultrastructural images restricted the toxin effects to the gastrointestinal tract. Nevertheless enterocytes microvilli and tight junctions were not altered, disconnecting DTX2 diarrheic effects from paracellular epithelial permeability. This is the first report of DTX2 oral LD50 (2262xa0μg/kgxa0BW) indicating that its TEF is about 0.4. This result suggests reevaluation of the present TEFs for the DSP toxins to better determine the actual risk to seafood consumers.

Analysis of natural toxins by liquid chromatography

Abstract This chapter focuses on the analysis of the main natural toxins associated with food safety. Mycotoxins are produced primarily by fungi of the genera Fusarium, Aspergillus, Penicillium, Claviceps, and Alternaria. The main representatives are aflatoxins, ochratoxin A, patulin, trichothecenes, zearalenone, fumonisins, and ergot alkaloid. Marine toxins are produced primarily by dinoflagellates of several genera. The main toxins are classified on the basis of the main toxin content: okadaic acid and dinophysistoxins, azaspiracids, yessotoxins, pectenotoxins, domoic acid, and saxitoxin and gonyautoxins. Cyanotoxins are produced by blue-green microalgae classified according to their effect: hepatotoxins (microcystins (MCs), nodularins and cylindrospermopsins), neurotoxins (anatoxin-a and analogs, saxitoxins, and s-N-methylamino- l -alanine), and dermatotoxins (aplysiatoxins and lyngbyatoxins). Finally, tetrodotoxin is produced by bacteria and it is a concern in fish and shellfish. All these toxins are analyzed mostly by LC-MS, and the specific features of the analysis is discussed in the chapter.