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 consumersThe research leading to USC results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01 and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016, and through Axencia Galega de Innovación, Spain, ITC-20133020 SINTOX. In addition from the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007–2013) under grant agreement 315285 CIGUATOOLS and 312184 PHARMASEA. Inés Rodriguez is supported by a fellowship from Subprograma de Formación de Personal Investigador (AGL2012-40185-CO2-01), Spain. In depth investigation of the toxic episodes leading to the results and publication of the present work was undertaken by the Greek National Reference Laboratory of Marine Biotoxins (NRLMB) to fulfill the requirements of EU Regulation 178/2002/EC (Articles 6 and 7) regarding risk analysis and communication and scientific information needed for risk assessment and EU Regulation 882/2004/EC (article 7) with regard to transparency and information to the public. Collaboration of all the staff of the NRLMB is greatly appreciated. Thanks are also expressed to all the Greek regional veterinary services for their contribution to the shellfish samplings and for provision of the seawater analyses results for the presence of potentially toxic microalgae. The use of cell counts’ data within the period 2006–2009 and 2012 regarding P. minimum presence in seawater, derived from the Greek “National Programme for Monitoring of Bivalve Molluscs’ Production Areas for the presence of Marine Biotoxins” and conducted by the Laboratory Unit of Toxic Marine Microalgae (LUTMM), Department of Biology, Aristotle University of Thessaloniki (scientific coordinator: G. Nikolaidis (until February 2010) and M. Arsenakis (March 2010–to date)), as well as the restrictions of LUTMM regarding the use of data produced within 2013–2015 due to contract terms and ISO 17025 requirements are acknowledgedS

Study of apoptosis in the rat lateral geniculate nucleus during development and following lesions of efferent and afferent connections of this area

The dorsal lateral geniculate nucleus (dLGN) receives its major afferent inputs from the retina and has organized reciprocal connections with the primary visual cortex. In this study we investigated cell death in the rat dLGN during development and following lesions of its connections. These lesions involved monocular enucleation and unilateral ablation of the visual cortex at different developmental stages. We identified dying cells using the TUNEL method for the in situ end labelling of DNA fragmentation. Electron microscopic analysis and immunohistochemistry for activated caspase-3 were used to confirm the apoptotic nature of cell death. TUNEL used in conjunction with immunofluorescence for neuronal nuclear protein (NeuN) or Glial Fibrillary Acidic Protein (GFAP) was applied to identify the phenotype of apoptotic cells. During development, dying cells in the dLGN were detected during the first postnatal week, demonstrating features typical of apoptosis. The overwhelming majority of apoptotic cells were neurons. Apoptotic neuronal death in the dLGN is temporally coordinated with major developmental events in this area, suggesting that apoptosis may play a fundamental role in the regulation of neuronal numbers in the dLGN and in the development and establishment of a functional neuronal network. Following monocular enucleation at birth and at later stages of development, cell death was significantly increased in the contralateral dLGN, which was of the apoptotic type. The overwhelming majority of apoptotic cells were neurons. The frequency of apoptotic cells was inversely related to the age of lesion, however, monocular enucleation did not induce any marked changes in the total neuronal population of the dLGN. Cortical lesions at birth and subsequent developmental stages, resulted in a massive increase of cell death in the ipsilateral dLGN, which was typical of apoptosis, and led to the almost complete elimination of the nucleus. The effects of cortical lesions were independent of the age of lesion. Neurons in the dLGN depend on the integrity of their connections for survival. Apoptotic cell death in the dLGN following lesions of its connections depends on the type and extent of the connectivity affected.Ο ραχιαίος έξω γονατώδης πυρήνας (dLGN) του θαλάμου στον επίμυ δέχεται προσαγωγές ίνες από τον αμφιβληστροειδή χιτώνα του ετεροπλάγιου, κυρίως, οφθαλμού και έχει οργανωμένες, αμοιβαίες συνδέσεις με τον ομοπλάγιο οπτικό φλοιό. Στην εργασία αυτή μελετήθηκε το φαινόμενο της απόπτωσης στον dLGN του επίμυος κατά την ανάπτυξη και μετά από βλάβες προσαγωγών και απαγωγών συστημάτων ινών της περιοχής αυτής. Ειδικότερα, οι βλάβες αυτές αφορούσαν στη χειρουργική αφαίρεση του βολβού του οφθαλμού ή του οπτικού φλοιού σε διάφορα στάδια της ανάπτυξης. Για την ταυτοποίηση και την καταμέτρηση των αποπτωτικών κυττάρων χρησιμοποιήθηκε η μέθοδος TUNEL, ενώ για την πιστοποίηση των μορφολογικών χαρακτηριστικών της απόπτωσης έγινε παρατήρηση υλικού με το ηλεκτρονικό μικροσκόπιο. Επιπλέον, χρησιμοποιήθηκε η μέθοδος της ανοσοϊστοχημείας για την ανίχνευση της ενεργοποιημένης κασπάσης-3, η οποία αποτελεί αξιόπιστο αποπτωτικό δείκτη. Για τον προσδιορισμό του φαινοτύπου των κυττάρων που αποπίπτουν εφαρμόστηκε η μέθοδος της διπλής σήμανσης που περιελάμβανε την ταυτόχρονη σήμανση τομών με τη μέθοδο TUNEL με φθορισμό για τη ανίχνευση των αποπτωτικών κυττάρων, σε συνδυασμό με τη μέθοδο του ανοσοφθορισμού για τη σήμανση νευρώνων ή αστροκυττάρων, με αντισώματα εναντίον της νευρωνο-ειδικής πρωτεΐνης NeuN ή της όξινης πρωτεΐνης των ινιδίων των αστροκυττάρων (GFAP). Τα αποτελέσματα έδειξαν ότι κατά την ανάπτυξη o κυτταρικός θάνατος στον dLGN του επίμυος είναι αποπτωτικού τύπου, παρατηρείται κατά τη διάρκεια της πρώτης μεταγεννητικής εβδομάδας, εκδηλώνεται κυρίως στους νευρώνες του πυρήνα και ότι πιθανώς να διαδραματίζει καθοριστικό ρόλο στη ρύθμιση του τελικού αριθμού των νευρώνων του dLGN και την εδραίωση των λειτουργικών του συνδέσεων. Η καταστροφή των προσαγωγών συνδέσεων του dLGN από τον αμφιβληστροειδή χιτώνα του οφθαλμού στον νεογέννητο και αναπτυσσόμενο επίμυ οδηγεί σε αύξηση της απόπτωσης των κυττάρων του ετεροπλάγιου dLGN, εκδηλώνεται κυρίως στους νευρώνες του πυρήνα, δεν οδηγεί σε μείωση του τελικού κυτταρικού πληθυσμού του dLGN και η μέγιστη τιμή της εξαρτάται από τον βαθμό ωρίμασης των κυττάρων του πυρήνα. Η καταστροφή των αμοιβαίων συνδέσεων του dLGN με τον οπτικό φλοιό στον νεογέννητο και στον αναπτυσσόμενο επίμυ προκαλεί τον αποπτωτικό θάνατο της πλειονότητας των κυττάρων του ομοπλάγιου dLGN, η οποία οδηγεί στην τελική υποπλασία του πυρήνα και είναι ανεξάρτητη από τον βαθμό ωρίμασης των κυττάρων του. Τα συμπεράσματα αυτά οδηγούν στην τελική διαπίστωση ότι μετά από βλάβες των συνδέσεων του dLGN στον νεογέννητο και στον αναπτυσσόμενο επίμυ, η επιβίωση ή η απόπτωση των κυττάρων του καθορίζεται από το είδος και την έκταση των συνδέσεων που καταστρέφονται

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