1ST MEETING ON “NATURAL TOXINS” IN HONOR OF CESARE MONTECUCCO

September 6-7, 2018

AULA MAGNA, Vallisneri Building Via Ugo Bassi 58/B, Padova

Thousands of living organisms, ranging from bacteria to fungi, insects, vertebrates, and plants, produce toxins of different chemical nature. These toxins are collectively called natural toxins to distinguish them from the toxins and poisons synthetized by humans. The natural toxins act via different mechanisms; however, all of them result from their co-evolution with their target or the physiological function they affect. Therefore, the study of their structures and mechanisms of action has multiple values: a) to identify their presence in food or other biological products, b) to shed light on the pathogenesis of the possible diseases caused by these toxins, and, at the same time, c) to shed light on the physiological process that they alter; d) to identify antidotes/drugs/antibodies/vaccines and/or therapeutic protocols designed to prevent or neutralize the action of natural toxins.

SCIENTIFIC COMMITTEE

Chiara Dall’Asta, Carmela Dell’Aversano, Corrado L. Galli, Carlo A. Locatelli, Antonella Penna, Ornella Rossetto, Silvio Sosa, Emanuela Testai, Aurelia Tubaro

ORGANIZING COMMITTEE

Ornella Rossetto, Marco Pirazzini, Michela Rigoni (Università di Padova) Silvio Sosa, Marco Pelin, Laura Fusco (Università di Trieste)

PROGRAMME

September 6, 2018 AULA MAGNA, Vallisneri building, Via Ugo Bassi 58/B, Padova

14.00 Introduction: Corrado L. Galli – SITOX President

Aurelia Tubaro

MYCOTOXINS Chairpersons: Chiara Dall’Asta, Patrizia Hrelia 14.15-14.45 Opening Lecture: From MycoRed to MycoKey: key EU projects for the mycotoxin management in food and feed chains - Antonio MORETTI and Antonio F. LOGRIECO 14.45-15.00 Chiara Dall’Asta – Mycotoxins in food: new integrated approaches for the analysis and the mechanism of action 15.00-15.15 Luca Dellafiora – Computational toxicodynamics & in vitro toxicology: application and perspectives in the risk assessment on mycotoxins 15.15-15.30 General Discussion Coffee Break and Poster Session: 15.30-16.00

MARINE & FRESHWATER TOXINS Chairpersons: Emanuela Testai, Carmela Dell’Aversano 16. 00-16.20 Plenary Lecture: Tetrodotoxins in Europe – what is going on? – Andrew D. TURNER 16.20-16.35 Carmela Dell’Aversano – LC-HRMS studies on Ostreopsis-related toxins in algae, seafood and aerosols. What’s left? 16.35-16.50 Marco Pelin – Dermotoxicity of palytoxins, an emerging problem for human health 16.50-17.05 Emanuela Testai – Human health risk associated with cyanotoxin exposure 17.05-17.20 Anastasia Hiskia - Transformation products of cyanotoxins 17.20-17.35 Luciana Tartaglione - LC-High resolution MSn and LC-Tandem mass spectrometry for a data merging of a Greek cyanobacteria biomass from Lake Kastoria 17.35-18.00 General Discussion 18.00-19.30 SITOX Working Group on Natural Toxins meeting (Meeting Room, 1st floor)

September 7, 2018 AULA MAGNA, Vallisneri Building, Via Ugo Bassi 58/B, Padova

MISCELLANEOUS TOXINS

Chairpersons: Corrado L. Galli, Mark Poli 9.00-9.20 Opening Lecture: Structures and chemistry of the azaspiracids, and their consequences for azaspiracid toxicity - Christopher O. Miles 9.20-9.35 Aurelia Tubaro – Emerging marine toxins in temperate waters 9.35-9.50 Luc de Haro - Marine Toxicology: New challenges for Poison Control Centers 9.50-10.05 Antonella Penna – Molecular tools and approaches for the monitoring of harmful algal blooms 10.05-10.20 Mark Van Asten - Interpreting data from the use of the Phytoxigene QPCR assays in screening and monitoring for harmful algal blooms 10.20-10.35 Maura Manganelli – Are cyanotoxins in food an emerging risk? The results of an EFSA Project 10.35-10.50 Alessandra Ieri - Loxoscelism: presentation of a case series observed from the Medical Toxicology Unit and Poison Centre of Florence Careggi University Hospital 10.50-11.10 General Discussion Coffee Break and Poster Session: 11.10-11.30

PROTEIN TOXINS

Chairpersons: Cesare Montecucco, Carlo A. Locatelli 11.30-11.45 Ornella Rossetto – Many novel botulinum neurotoxins and botulinum-like toxins 11.45-12.00 Luca Bano – Non-neurotoxic clostridial toxins causing animal diseases 12.00-12.15 Davide Lonati – Molecular and toxicological study of Viper venoms neurotoxicity 12.15-12.30 Giulia Zanetti – Inhibitors of botulinum neurotoxins 12.30-12.45 Mauro Dalla Serra – Pore-forming toxins 12.45-13.10 General Discussion

13.10-13.30 Concluding Remarks (C. L. Galli and C. Montecucco) 13.30-15.30 Mark Van Asten – Demonstration of the Phytoxigene QPCR assay (Room at the 2nd floor, South-Sud side)

LIST OF CHAIRPERSONS AND SPEAKERS

Luca Bano - Istituto Zooprofilattico Sperimentale delle Venezie (Italy) Mauro Dalla Serra - Istituto di Biofisica, Consiglio Nazionale delle Ricerche (Italy) Chiara Dall’Asta – Università di Parma (Italy) Luc de Haro - Marseille Poison Centre (France) Carmela Dell’Aversano – Università Federico II di Napoli (Italy) Luca Dellafiora – Università di Parma (Italy) Corrado L. Galli – Università di Milano (Italy) Anastasia Hiskia - Institute of Nanoscience and Nanotechnology (Greece) Patrizia Hrelia – Università di Bologna (Italy) Alessandra Ieri - AOUC Hospital (Italy) Carlo A. Locatelli - Centro Antiveleni di Pavia (Italy) Antonio F. Logrieco - Consiglio Nazionale delle Ricerche (Italy) Davide Lonati – Centro Antiveleni di Pavia (Italy) Christopher O. Miles - National Research Council (Canada) Maura Manganelli – Istituto Superiore di Sanità (Italy) Cesare Montecucco – Università di Padova (Italy) Antonio Moretti – Consiglio Nazionale delle Ricerche (Italy) Marco Pelin - Università di Trieste (Italy) Mark Poli - U.S. Army Medical Research Institute of Infectious Diseases (USA) Antonella Penna – Università di Urbino Carlo Bo (Italy) Ornella Rossetto – Università di Padova (Italy) Luciana Tartaglione - Università Federico II di Napoli (Italy) Emanuela Testai – Istituto Superiore di Sanità (Italy) Aurelia Tubaro – Università di Trieste (Italy) Andrew D. Turner - Centre for Environment Fisheries and Aquaculture Science (UK) Mark Van Asten - University of New South Wales (Australia) Giulia Zanetti – Università di Padova (Italy)

LIST OF ABSTRACTS Non-neurotoxic clostridial toxins causing animal diseases

Luca Bano

Diagnostic and Microbiology Laboratory, Istituto Zooprofilattico Sperimentale delle Venezie

Clostridia are sporulating, anaerobic bacteria that are widely spread in the environment (soil, sediment, water, dust, spoils, litter, plant decomposition) including the digestive tract of humans and animals.

Among more than 200 Clostridium species, 14 are considered as major pathogens in humans and/or animals: C. botulinum, C. chauvoei, C. colinum, C. difficile, C. haemolyticum, C. histolyticum, C. novyi, C. perfringens, C. piliforme, C. septicum, C. sordellii, C. spiroforme, and C. tetani.

Most of Clostridia are not pathogen by themselves, but through the production of toxins, which act locally and/or generally and are often coded by genes located on extra-chromosomal and/or mobile genetic elements.

Based on their mode of action, two main classes of bacterial protein toxins can be distinguished, firstly those which act on cell membrane, for example through a pore-forming activity or enzymatic modification of membrane components, and secondly those which have the property to enter cells and modify intracellular targets. Intracellularly active toxins contain at least three functional domains: receptor binding domain, translocation domain, and enzymatic domain.

The most important non-neurotoxins producing Clostridia involved in animal diseases are C. perfringens, C. sordellii, C. septicum and C. spiroforme. A pathologic role in the animal diseases has been well-established for some “recently-discovered” toxins such as the pore-forming NetB toxin produced by C. perfringens and responsible for the necrotic enteritis in chicken. Other toxins related to diseases in humans (eg. lethal toxin of C. sordelli, α-toxin of C. septicum, toxins produce by C. difficile) are still poorly studied as pathogens for animals.

Among the 18 different toxins produced by C. perfringens, β2 is drawing the attention of researchers because it appears involved in the pathogenesis of diseases that affect different animal species: enteroxaemia of ruminants and enteritis in piglets and hares.

For many years, Clostridia have been characterized based on the biochemical reactions and, more recently, of the detection of genetic markers of virulence. In the future, a proteomic approach will be needed to elucidate the mechanism of action and the antigenicity of the Clostridia toxins.

Biotoxins presence in Veneto Region: preliminary results of correlation between Dinophysis spp bloom and Okadaic acid in mussels

P. Bordin1, L.Bille 1, L. Contiero 1, A.Milandri2, L.Barco1, M.Pompei2 , M. Toson 1,P. Fumelli 3, E. Rossetti 4, P. Antonelli 1,M. Dalla Pozza 1, G. Arcangeli 1

1 Istituto Zooprofilattico Sperimentale delle Venezie – Legnaro (PD), Italy

2 Centro ricerche Marine – Cesenatico (FC), Italy

3Azienda ULSS n.5 Polesana – Adria (RO), Italy

4 Scardovari - Consorzio Cooperative Pescatori del Polesine –Scardovari (RO), Italy

Italy is the third European producer of marine molluscs and Veneto Region is one of the main shellfish production areas of the country. Lipophilic biotoxins accumulation in shellfish, in particularOkadaic acid (AO) and, to a lesser extent, Yessotoxins (YTX), has become more and more frequent in the recent years with consequent shellfish harvesting prohibition for long time. Furthermore, in 2017, during an Alexandriumspp bloom the presence of Paralytic Shellfish Poisoning (PSP) toxins, at levels below theEU Reg. limit [1], was detected for the first time in Truncate donax (D. trunculus) [2].

The aim of this work is to present the preliminary results of the implementation of an early warning system based on the monitoring of phytoplankton as an indicator of biotoxins situation for the management of shellfish production areas, as it is suggestedby the EU legislation on food hygiene [3]. According to the procedure, shellfish and sea water are systematically collected on a weekly basis in one fixed sampling point located in the Po River delta area, which is considered particularly at risk for biotoxins accumulation in mussels and tested for the presence of phytoplankton and marine toxins, respectively.Water samples arecollected by means a dividable hose,which allows to obtaininformation regarding the phytoplankton presence at three different depth. Phytoplankton harmful species are enumeratedaccording to BS EN 15204: 2006 "Water quality - Guidance standard on the enumeration of phytoplankton using inverted microscopy (Utermohl technique)". The quantification of lipophilic toxins in molluscs carried out using the LC-MS / MS method.

The results regarding the sampling activities performed during 2017 show that concentration of AO beyond the legal limit in mussels was detected after Dinophysis spp reached a concentration of 102 cell/L, which is considered as a“trigger level” in other countries [4]. Considering Dinophysis species composition, Dinophysis fortii seems to be main responsible of the high OA accumulation in mussels.

Preliminary results of the project highlight how the use of phytoplankton monitoring could be an important tool,which may contribute to save resources in terms of time and costs related to shellfish control, ensuring at the same time the consumer health. Therefore, it will be very important to extend the described sampling approach to all the Veneto Region mussel farms, in order to acquire the necessary knowledge in order to implement an Italian effective "early warning system” as already done by other European countries [5].

References

[1] Regulation (EC) No 854/2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Official Journal of the European Union, Series, L 226, 83e127, 25.06.04.

[2] S. Pigozzi, A.Ceredi, M. Pompei, P. Bordin, L. Bille, C. Dell’Aversano, L. Tartaglione, L. Sidari, B.Zanolin, A. Cacitti, L. Pelagatti,M. Ricci, A. Milandri “First detection of tetrodotoxin in shellfish from Northern Italy” Book of abstract of 6th International Symposium Marine and Freshwater Toxins Analysis October 22-25, 2017. Baiona, Spain, pag.60-61

[3] Regulation (EC) No 853/2004 laying down specific hygiene rules for food of animal origin. Official Journal of the European Communities, Series L, 139, 55e205. http://europa.eu.int/eurlex/pri/de/oj/dat/2004/l_139/l_13920040430de00550205.pdf, 30.4.04.

[4]Cefas, “Protocol for sampling and transport of water for the purpose of Official Control Monitoring of classified shellfish production areas under EU Regulation 854/2004”, Protocol version 7 – July 2015

[5]A. Silva, L. Pinto, S.M. Rodrigues, H. de Pablo, M. Santos, T. Moita, M. Mateus. A HAB warning system for shellfish harvesting in Portugal. Harmful Algae 53 (2016) 33–39

This project is financed by the Italian Ministry of Health RC 17/15

Pore-forming toxins

Mauro Dalla Serra

National Research Council of Italy - Institute of Biophysics, via Sommarive 18, 38123 Trento (Italy)

Pore-forming toxins (PFT) constitute the largest and best characterized class of protein toxins, accounting for more than 30% of all known bacterial protein toxins. They are potent virulence factors evolved during ancient competition among organisms for defense and/or attack purposes. Interestingly, similar structures and modes of action are also adopted by components of the immune system, like perforin and complement, and by antimicrobial peptides.

PFT are normally able to drill poorly selective nanometer-sized holes into the target cell membranes, causing cell death through osmotic imbalance.

Regarding the pore structure, two conformations have been described: a purely proteinic channel or a protein-lipid mixed arrangement. This second case is characterized by the co-presence of lipid and protein elements in the pore walls. Lipid heads could either intercalate between protein monomers or constitute the chord of arc shaped pores. In both cases the lipid lamellar structure is destroyed and lipids should bend, assuming a toroidal shape.

Here I will shortly discuss examples of both classes of functional pores formed by bacterial an animal toxins, investigated by some biophysical techniques, like electrophysiology, AFM, fluorescence. Experimental evidences supporting the ability to punch proteolipidic nanopores into lipid membranes will be presented.

Marine Toxicology: New challenges for Poison control Centers.

Luc de Haro

Marseille Poison Centre, Sainte Marguerite Hospital, 13009 Marseille, France.

Between 2010 and 2014, the Marseille poison center had been requested by various structures at the national and European levels to make a synthesis of the new dangers induced by the toxic marine organisms [1]. At that time, the French Metropolitan Coast was directly concerned by cumbersome toxic invaders: in Aquitaine with the Portuguese-man-of-war (Physalia sp.) swarming in summer [2]; in Brittany with the sea lettuce of the genus Ulva (mounds in putrefaction on the beaches produce hydrogen sulfide); in Provence with blooms of tropical dinoflagellates of the genus Ostreopsis producing "palytoxin-like" molecules able to contaminate the sea water but also the surrounding air [3]. If these health problems with heavy economic impacts persist a few years later, what more can we say in 2018? Undeniably, the initial concerns are confirmed: the direct (overexploitation of fishing sources, water pollution, etc.) or indirect (global warming) anthropogenic modifications of aquatic biotopes are at the origin of new marine toxicological problems to which poison control centers are faced. The list of these new medical challenges is long: the venomous lion fish of the Indian Ocean (genus Pterois) has invaded the West Indies and generates many envenomations [4]; the Red Sea Lessepsian pufferfish Lagocephalus sceleratus colonized the entire Mediterranean in record time (confirmed presence in Spain in 2017) and the consumption of its tetrodotoxin-rich flesh is potentially lethal; the consumption of sea violets of the genus Microcosmus is at the origin of unexplained cerebellar syndromes in Provence but also in Croatia; the presence of ciguatera is now confirmed at the gates of Europe (native cases reported for the first time in Madeira and the Canary Islands); soft corals of the genus Palythoa or Zoanthus have become undesirable guests in marine aquaria because these invasive invertebrates can induce severe respiratory and ocular symptoms in aquarists; Cyanobacterial blooms in fresh and/or brackish waters are multiplying with the production of neurotoxins or nephrotoxins with consequences in veterinary and human medicine... These subjects are fascinating but worrying because they are witnesses to ecological imbalances with tangible effects that we did not imagine a few years ago.

[1] Schmitt C, de Haro L. Clinical Marine Toxicology: A European perspective for clinical toxicologists and Poison Centres. Toxins 2013;5:1343-52

[2] Labadie M, Aldabe B, Ong N, et al. Portuguese man-of-war (Physalia physalis) envenomation on the Aquitaine coast of France. Clin Toxicol 2012;50:567-70

[3] Tichadou L, Glaizal M, Armengaud A, et al. Health impact of unicellular algae of the Ostreopsis genus blooms in the Mediterranean Sea: experience of the French Mediterranean coast surveillance network from 2006 to 2009. Clin Toxicol 2010;48:839-44

[4] Résière D, Cerland L, de Haro L, et al. Envenomation by the invasive Pterois volitans species in the French West Indies: a two-year prospective study in Martinique. Clin Toxicol 2016;54:1-6

Computational toxicodynamics & in vitro toxicology: application and perspectives in the risk assessment on mycotoxins

Luca Dellafiora

Department of Food and Drug, University of Parma, Parma, Italy

Recently, the risk assessment of mycotoxins of food origin moved from the one-molecule-one-target approach toward the group-based toxicology paradigm. Indeed, the old-fashion targeted testing of parental compounds is no longer considered supportive given the high number of diversely toxic metabolites arising from fungal (producers), plants (reservoirs) and animals (bystander targets) metabolism. In addition, the multi-target toxicity of certain mycotoxins highlighted the need to look beyond the typical biological targets considered in the past [1]. However, the urgency to carefully investigate such a scenario is not supported by the current metrological infrastructures as most of the mycotoxins analogues and metabolites are not commercially available. Therefore, nor their synthesis/purification neither the unbiased search of unexpected targets can be affordably accounted unless a hierarchical upstream analysis to prioritize experimental trials is applied. In this framework, hybrid in silico/in vitro approaches may represent affordable analytical tools to assess the toxicity of huge numbers of queries, as well as to explore their possible multi-target toxicity. The case studies of estrogenic activity of Alternaria mycotoxins and the targets fishing study of the fungal metabolite atromentin are presented as proofs of concept. In the first case, the effects of fungal and human metabolism on the estrogenicity of alternariol, the best characterized mycoestrogen from Alternaria species, were assessed. The in silico 3D receptor-modeling integrated data from alkaline phosphatase assay on Ishikawa cells and binding activity identifying methylation as a chemical modification enhancing estrogenicity [2]. In the second case, the poorly characterized fungal metabolite atromentin underwent a target fishing study to investigate potential biological (adverse) activity. A coupling of ligand- and structure-based virtual screening identified estrogen receptors and 17-β-hydroxysteroid dehydrogenase among the possible biological targets. The in vitro assessment confirmed for the first time its mild estrogenicity pinpointing the need to collect further data on occurrence, exposure and toxicity to characterize the possible risks for consumers [3].

Therefore, the analysis of mycotoxins toxicodynamic in silico coupled to in vitro studies proved to be effective to face the wealth of compounds to be tested and to explore the number of possible targets still undiscovered. Such approaches can significantly focus and reduce the number of in vivo trials, as well as refine toxicological investigations and data interpretation.

References:

[1] Dellafiora et al., 2018. Toxins. 10 (2), pii: E52

[2] Dellafiora et al., 2018. Food Chem. 248, 253-261

[3] Dellafiora et al., 2019. Food Chem. 270, 61-69

LC-HRMS studies on Ostreopsis-related toxins in algae, seafood and aerosols. What’s left?

Carmela Dell'Aversano,1,2 Luciana Tartaglione,1 Martino Forino,1 Antonella Penna,2,3 Silvia Casabianca,2,3 Samuela Capellacci,2,3 Cecilia Totti,4 Stefano Accoroni,4 Rossella Pistocchi,5 Franca Guerrini,5 Laura Pezzolesi,5 Giorgio Honsell,6 Marco Pelin, 7 Silvio Sosa,7 and Aurelia Tubaro7

1University of Napoli Federico II, Department of Pharmacy, Via D. Montesano 49, 80131 Napoli Italy

2 CoNISMa, Italian Interuniversity Consortium on Marine Sciences, Piazzale Flaminio 9, 00196, Rome, Italy

3Department of Biomolecular Sciences, University of Urbino, Viale Trieste 296, 61121 Pesaro, Italy

4Department of Life Science and Environment, Università Politecnica delle Marche, Ancona, Italy

5Department of Biological, Geological and Environmental Sciences, University of Bologna, Ravenna, Italy

6Department of Agricultural and Environmental Sciences, University of Udine, Udine, Italy

7Department of Life Science, University of Trieste, 34127 Trieste, Italy

Over the last decade massive blooms of the benthic dinoflagellate Ostreopsis cf. ovata, once confined to tropical and subtropical areas, have occurred in more temperate regions worldwide including the Mediterranean Sea. Concurrently, negative impacts on human health mainly due to inhalation of toxic aerosols and/or skin contact were observed together with death of benthic marine invertebrates.

When first toxic outbreaks related to O. cf. ovata occurred in 2005 along the Ligurian coasts (Italy), little was known on several aspects of the phenomenon. Although some Ostreopsis spp. were known to produce congeners of palytoxin (PLTX), O. cf. ovata was not known as a toxic species and its metabolic profile had never been investigated. Secondly, although PLTX itself was reported as one of the most potent non-protein marine toxins so far known and tentatively suggested as the causative agent of some fatal food poisonings in the tropics, it had never been suspected to exert toxicity through inhalation. Last but not least, the role of the environmental conditions on O. cf. ovata proliferation and toxin production had been poorly studied. Therefore, the need for increase knowledge on potential risks for humans and ecosystem stimulated research in the field.

An Italian multidisciplinary network was created, including scientists from the Academia as well as operators of the regional environmental protection agencies and food safety laboratories. This joint effort led to clarify many of the aspects related to the Ostreopsis phenomenon that still represent one of the major threats to humans in the Mediterranean area. This presentation is meant to summarize the results of our studies on O. cf. ovata, highlighting inter- and intra-specific variability of the toxin profiles, structural variability of the detected toxins and, in some cases, linking such differences to the risk that PLTX congeners pose to humans following inhalatory, dermal and oral exposure. Phylogenetic relationships among many isolates of Mediterranean O. cf. ovata were investigated as well as innovative molecular qPCR based assays was developed for monitoring activities. The methodological approach, besides addressing many of the palytoxin-related issues, may serve as template for facing in due time any emerging toxin-related threat to humans.

Characterisation of the Cyanobacterial Bloom in the Lake Varese

Chiara Facca1 Diana Conduto António 2, Patrizia Pretto3, Armin Lahm4, Robert Loo21, Isabella Sanseverino2, Simona Tavazzi2, Helle Skejo2, Andrea Beghi5, Franca Pandolfi5, Pietro Genoni5 and Teresa Lettieri2*

1University Ca' Foscari Venezia, Department of Environmental Science, Informatics and Statistics, via Torino 155, 30172 Mestre (VE)-Italy

2European Commission, Joint Research Centre (JRC), Directorate D – Sustainable Resources, Water and Marine Resources,Via E. Fermi, 2749, I-21027 Ispra (VA) – Italy

3Biosearch Ambiente srl, Via Tetti Gai, 59, 10091, Alpignano (TO), Italy

4 Bioinformatics project support, P.za S.M. Liberatrice 18 ,00153 Roma,, Italy

5ARPA, Agenzia Regionale per la Protezione dell’Ambiente della Lombardia, Regione Lombardia, Varese, Lombardia,

Cyanobacteria are photosynthetic bacteria which are mostly found in freshwater systems. Increasing antrophogenic activities leading to eutrophication phenomena on aquatic ecosystems favoured a higher frequency and intensity of blooms. Such event can seriously compromise the quality of drinking and recreational water. The present study describes the phytoplankton occurrences in Lake Varese before, during and after the cyanobacteria bloom in 2017 by means of weekly samplings at different water column depths. Both metagenomics and microscopy analyses were carried out to describe the blooms. Phytoplankton cell abundances varied between 1.83 and 165.12 x 106 cells/L, being the lowest in July and the highest in October at the deepest layers, where Cyanophyceae represented 90% of the total phytoplankton community. At the surface the temporal trend was the opposite and the community appeared more diverse being composed as follow: 41% Cyanophyceae, 22% diatoms, 17% Chlorophyceae, 12%Cryptophyceae and 7% Dinophyceae.

Transformation products of cyanotoxins Anastasia Hiskia, Triantafyllos Kaloudis, Theodoros M. Triantis Institute of Nanoscience and Nanotechnology, NCSR Demokritos, Athens, Greece Freshwater cyanobacterial metabolites comprise a wide range of molecules that include some potent toxins, the cyanotoxins. Following their release into water, cyanotoxins can interact with naturally existing matter and organisms through complex chemical, biological and biochemical pathways. During water treatment, cyanotoxins can be degraded by conventional disinfectants and chemicals or by reactive oxygen species (ROS) when advanced oxidation processes (AOPs) are applied. Understanding the fate of cyanotoxins in the environment and during water treatment and identifying the related transformation products is imperative in order to predict their transport, persistence, reactions and toxicity under various conditions. An overview of the available data on transformation products of various classes of cyanotoxins (microcystins, cylindrospermopsin, anatoxin-a) in the environment and during water treatment processes (e.g. chlorination, ozonation, AOPs) is presented. The presentation focuses on ROS-driven transformation processes that are common to various emerging AOPs for drinking water treatment (e.g. UV/ozone, UV/peroxide, UV/Cl2, persulfate, UV/TiO2). As many aspects regarding the fate of cyanotoxins in the environment and during water treatment are not fully understood, research gaps and further research needs are discussed in order to elucidate their complex transformation pathways and the impacts of transformation products on the environment and on human health.

Loxoscelism: presentation of a case series observed from the Medical Toxicology Unit and Poison Centre of Florence Careggi University Hospital Ieri Alessandraa, Gambassi Francescoa, Zotto Alessandrob, Mannaioni Guidoa,b, Masini Emanuelaa,b

a) AOUC Hospital, Medical Toxicology Unit, Florence, Italy.

b) Department of NEUROFARBA, Pharmacology Section, University of Florence, Florence, Italy.

Loxosceles rufescens is one of the spiders of the Mediterranean area potentially dangerous for human health. It is frequently observable in houses, particularly at night and during warmer months. The venom contains toxins that cause necrosis and haemolysis responsible of dermo-necrotics wounds, sometimes with severe clinical presentation. The visceral-cutaneous loxoscelism is instead rare in Italy and presents itself with systemic symptoms such asthenia, fever, malaise, measles-like rash and, in severe cases, with haemolysis, jaundice, rhabdomyolysis and disseminated intravascular coagulation (DIC). We studied 132 cases of supposed Loxosceles rufescens bite that came to our attention between January 2013 and June 2018 (Poison Centre telephone consultations and clinical visits). The case series was stratified by age, gender, spider identification and clinical presentation. The wound was evaluated following the typical clinical evolution: erythema, vesicles, ischemia and necrosis. The presence of systemic symptoms like the measles-like generalized rash, fever, lymphangitis, lymphadenopathy, joints and muscular pain was carefully monitored until full recovery. Diagnosis was mainly presumptive because the bite is initially painless and the identification of the spider, captured or killed, from patients or medical caregivers, is rare. It is based on epidemiological criteria, such the presence of the spider in the geographical area, anamnesis, late onset of pain, clinical evolution of the wound. The differential diagnosis from other conditions is difficult and may overrate the cases of necrotic arachnoidism. Treatment consists in antibiotics, corticosteroids, antihistamines, low-molecular-weight heparin (LMWH), and local care of necrotic wounds [1]. [1] Boissiere F., Masson R., Fluieraru S., Vitse J., Dessena L., Lefevre M., Bekara F., Herlin C. Le loxoscelisme cutané, à propos d’une observation exceptionnelle de 9 cas consécutifs. Ann. Chir. Plast. Esthet., 61, 811-819,2016.

Microcystin-LR toxicity in primary rat cortical cultures Maja Justin, Klara Bulc Rozman, Dušan Šuput Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška cesta 4, Ljubljana, Slovenia. Industrialisation, intensive farming and climate changes have increased the occurrence of cyanobacterial blooms and consequently the occurrence of their toxins (e.g. microcystins) in our environment. Acute exposure to microcystins (MC) leads to massive uptake of MC by cells expressing organic anion transporting polypeptides (Oatp), especially the liver. This results in cytoskeleton collapse and intrahepatic haemorrhage. Although MCs are considered primarily as hepatotoxins, several intoxicated individuals in Caruaru incident exhibited a variety of neurological symptoms. Our previous research showed MC also induce DNA damage in the brain. In order to clarify the apparent neurotoxicity of MC we investigated the effects of MC-LR on primary cultures of cortical neurons and primary mixed cultures of cortical neurons and astrocytes. MC-LR induced toxicity was time- and dose-dependent. The effect of MC-LR was prominent in mixed cultures where cell viability and cell number declined the most. Affected cells were rounded and lost their protrusions. Annexin-V labelling confirmed the apoptotic death of those cells. Labelling of neuron specific beta III tubulin and astrocyte specific glial fibrillary acidic protein (GFAP) showed little changes in neuronal morphology while astrocytes shrunk and their protrusions shortened. The results are in accordance with higher expression of Slco1b2 in mixed cultures, since Slco1b2 encodes the rodent Oatp1b2 that has been proven to transport MC-LR across cell membrane. This study shows that astrocytes in culture are more susceptible to MC-LR induced toxicity than neurons. The loss of astrocytic homeostatic function could therefore be expected in the brain of intoxicated animals or individuals.

Molecular and toxicological study of Viper venoms neurotoxicity

Davide Lonati, Giulia Zanetti*, Elisa Duregotti*, Carlo Alessandro Locatelli, Ornella Rossetto*, Marco Pirazzini*

Istituti Clinici Scientifici Maugeri, IRCCS Maugeri Hospital and University of Pavia, Poison Control Centre and National Toxicology Information Centre - Toxicology Unit, Pavia, 27100, Italy

*University of Padova, Department of Biomedical Sciences, Padova, 35131, Italy

Four different venomous snakes are potentially dangerous to humans and may require immediate attention in Italy: Vipera aspis, Vipera berus, Vipera ammodytes and the small snake Vipera ursinii. The typical syndrome of envenomation is characterized by local symptoms (pain, edema, swelling and local necrosis in some case) and by systemic effects such as gastrointestinal manifestations, chest pain associated in some cases with unspecific ECG changes, hypotension, and coagulopathy. Single cases presenting neurotoxicity have been reported in Italy and they are related to V. aspis aspis and V. aspis francisciredi. Clinical study conducted by Pavia Poison Control Centre and National Toxicology Information Centre described a largest case series presenting with neurologic manifestations due to Italian viper envenomation. The main objectives of the clinical study were to characterize neurotoxic effects of the venom (including time onset/resolution and association with local and systemic symptoms) as well as to evaluate the clinical response to antivenom treatment.

Neurotoxic effects developed from 2 to 24 hours after the viper bite. Notably, more than half of cases (58.3%) manifested neurologic manifestations at least 11 hours after the bite (in 6 of them 20 hours after the bite). Our data demonstrate late appearance of neurologic symptoms that may be associated with only mild local swelling (41.6% of the cases), which represents the unique systemic manifestation (37.5% of the cases). These findings suggest that patients may need to be observed at least 24 hours for delayed neurologic effects. Antidote was intravenously administered in 19 (79.2%) patients. Neurotoxic effects have been reversible in all cases and may be the unique systemic manifestation of envenomation. Considering this last important clinical aspect, antidotic treatment of patients considered as GSS 2 only for neurotoxic effects (with mild local effects) may not be necessary. Another clinical observation is that patients bitten by Vipera berus manifested the classical envenomation hallmark without neurological manifestations.

Following this observation, teamwork with the group of researchers in Padova allowed to demonstrate the different composition of the viper poison in the different species present in Italy.

Even though it occurs rarely, neurotoxicity has historically been associated with V. ammodytes as its venom contains “snake presynaptic PLA2 (phospholipase A2) neurotoxins” (SPANs), like vipoxin, vaspin and ammodytoxin. More recently, genetic and proteomic analyses have shown that also V. aspis venom contains PLA2 components which may be responsible for the neurological symptoms developed by bitten patients. Interestingly, although PLA2s have been detected also in V. berus venoms there is general consensus in considering this adder as not neurotoxic. This suggests that the PLA2s within V. ammodytes, V. aspis and V. berus venoms are functionally and possibly antigenically different and therefore may be variably susceptible to neutralization by available antisera. Researchers of the Department of Biomedical Science, University of Padova, characterized the toxicological profile of Vipera aspis and Vipera berus venoms in vivo in mice and tested the effectiveness of two antivenoms, commonly used as antidotes, in counteracting the specific activities of the two venoms. Neurotoxic effects were registered only in Vipera aspis venom: the toxic effect is due to the degeneration of peripheral nerve terminals at the NMJ and this phenomenon is not neutralized by the two tested antisera. These results indicate that the effectiveness of different antisera is strongly influenced by the variable composition of the venoms and reinforce the arguments supporting the use of polyvalent antivenoms (better if produced with the venom of local vipera species).

Rererence:

1. Lonati D, Giampreti A, Rossetto O, Petrolini VM, Vecchio S, Buscaglia E, Mazzoleni M, Chiara F, Aloise M, Gentilli A, Montecucco C, Coccini T, Locatelli CA. Neurotoxicity of European viperids in Italy: Pavia Poison Control Centre case series 2001-2011. Clin Toxicol (Phila). 2014; 52:269-276.

2. Lamb T, de Haro L, Lonati D, Brvar M, Eddleston M. Antivenom for European Vipera species envenoming. Clin Toxicol (Phila). 2017; 55:557-568.

3. Zanetti G, Duregotti E, Locatelli CA, Giampreti A, Lonati D, Rossetto O, Pirazzini M. Variability in venom composition of European viper subspecies limits the cross-effectiveness of antivenoms. Sci Rep. 2018; 8:9818.

Structures and chemistry of the azaspiracids, and their consequences for azaspiracid toxicity

Christopher O. Miles

National Research Council Canada, 1411Oxford Street, Halifax, B3H 3Z1 Nova Scotia, Canada

Since azaspiracids were first identified in 1998, around 60 azaspiracid analogues have been reported from around the world, including parts of Europe, Asia, North and South America, including the Atlantic and Pacific Oceans and the Mediterranean Sea. Some of these azaspiracids are produced by marine microalgae, while others are metabolites produced by filter-feeding shellfish, and still others are formed abiotically and some may even be considered artefactual. Azaspiracids also accumulate in crabs, suggesting transfer up the food chain, with additional metabolism likely to occur. Azaspiracids cause severe gastrointestinal symptoms in humans, but although azaspiracids display neurotoxicity and other effects in vitro, a molecular target for the effects on the human digestive tract has not yet been firmly identified. The structure of the azaspiracid skeleton was first proposed in 1998, and was revised in 2004, but only in 2018 was the correct chemical structure established through a combination of NMR and LC-MS studies of natural and synthetic azaspiracid-3. Correct chemical structures are a prerequisite for establishing valid structure–activity relationships (SARs), and this SAR information can then be used by regulators to design a regulatory framework that protects consumers from azaspiracid poisoning while minimizing the disruption to seafood producers. The same SAR information can also be used by chemists to design analytical methods capable of detecting all azaspiracid analogues that are capable of causing human poisoning regardless of whether the toxin is a known azaspiracid or a novel analogue or metabolite. This is important, as novel azaspiracids have been found in both Italy and the USA without any of the currently regulated azaspiracids (azaspiracid-1, -2, and -3) being present.

This presentation will give an overview of the origins, chemistry, structure–activity and analytical methods for the azaspiracid group of toxins, and possible strategies for identifying the molecular targets of the azaspiracids.

From MycoRed to MycoKey: key EU projects for the mycotoxin management in food and feed chains

Antonio Moretti and Antonio Francesco Logrieco

Institute of Sciences of Food Production (ISPA), Research National Council (CNR), Via Amendola 122/O, 70126, Bari, Italy

Among the emerging issues in food safety, the increase of plant diseases associated to the occurrence of mycotoxigenic fungal species is of major importance. As a result of their secondary metabolism, these fungi can produce mycotoxins, which are low-molecular-weight toxic compounds, provided of a broad range of biological activities. The consumption of mycotoxin-contaminated food can have multiple consequences on both human and animal health worldwide. Mycotoxins occur naturally and are the most prevalent source of food related health risks in field crops of high agro-food interest. Many of these crops, such as cereals, can suffers of a devastating worldwide diseases, often caused by a complex of fungal toxigenic species producing a wide range of mycotoxins, that can be accumulated in the final products. Management of good agricultural practices in the pre-harvest is a key issue for minimizing the risk of mycotoxin accumulation in crops before the harvest. Such practices can involve crop rotation, tillage, proper fertilization and distribution of fungicides or biological control agents, variety selection, timely planting and harvest. On the other hand, the reduction of mycotoxins along the agro-food chains is also highly depending from a correct post-harvest management such as separation of the infected crop products from the healthy material. Along the 7th Framework Large collaborative project of European Union “MycoRed” “Novel integrated strategies for worldwide mycotoxin reduction in food and feed chains”, and the Horizon 2020 European Union project “Integrated and Innovative Key actions for mycotoxin management in the food and feed chain” “Mycokey”, both coordinated by ISPA, an integrated management of pre-and post harvest practices for reducing the risk of contamination in crops have been and are in development. Moreover, further studies to evaluate the role played by the so called “masked” mycotoxins, in the final toxicity of food products are also deeply carried out. An update of the main scientific activities performed in both Projects will be provided, to define the main scenarios characterizing the work in progress on the mycotoxins in Europe and at worldwide level.

Monitoring of hydrophilic and lipophilic marine biotoxins in the Gulf of La Spezia from 2015 to 2018. R. Mua, V. Ciccotelli, L. A. Masiello, V. Savio, B. Vivaldi, C. Ercolini Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta (Genova, Italy) The Genoa chemical laboratory monitors ecotoxicology quality of bivalve molluscs coming from Gulf of La Spezia according to the plan “Health Monitoring and Surveillance on the production, purification and marketing of live bivalve molluscs”. The plan includes eight monitoring stations with monthly sample rate in winter and fortnightly in summer. The check is performed according to EC Regulation 853/2004 that establishes the biotoxin maximum concentration levels in shellfish. Paralytic shellfish poisoning toxins are determined by the mouse assay biological method. Determination of domoic acid (DA) are performed with HPLC-DAD, this method was validated according to “AESAN EU-RL-MB Domoic acid, version 1”. The linearity was achieved in the range of 0.4 mg/L to 8.0 mg/L corresponding in matrix from 2 to 40 mg/Kg with correlation coefficients (R2) > 0.990. The LOQ was 5 mg/Kg, repeatability and reproducibility (expressed as RSD%) were respectively < 7.7% and < 12.7 with uncertainty ranged from 23% at LOQ level to 17% at maximum level. Lipophilic marine biotoxins as Okadaic Acid and its derivatives (OAs), Yessotoxins (YTXs), Azaspiracid (AZAs) are quantified with LC-MS/MS. The method was tested on a secondary validation according to “EU-RL-MB: Sop for lipophilic marine biotoxins, version 5”. The LOQs were 20 μg/kg for OAs and AZAs and 50 μg/kg for YTXs with repeatability < 8.5% for all species. The monitoring results show that most of the detected toxins in the gulf belong to the OAs and YTXs family and their progress do not follow a seasonal trend. All samples analysed are compliant except two samples taken in new sowing areas (these mussel sowing came from Sardinia) that showing AO levels slightly above the allowed limit, respectively 165.8 µg/Kg and 162.6 µg/Kg. Despite these non-conformities the monitoring highlights that there is no risk related to La Spezia Gulf bivalve mussel consumption.

Dermotoxicity of palytoxins, an emerging problem for human health

Marco Pelin

Department of Life Sciences, Unversity of Trieste, Trieste, Italy

Palytoxin (PLTX) and its analogues are non-proteinaceous marine toxins identified in Palythoa corals, Ostreopsis dinoflagellates and Trichodesmium cyanobacteria. Cutaneous contact to seawater and/or marine organisms contaminated by PLTXs has been associated with skin irritation and inflammatory reactions, suggesting the skin as one of the targets of these toxins. However, dermotoxicity of PLTX is still a rather underestimated effect, even though it seems to be an increasing sanitary problem.

To elucidate the effects of PLTXs at the cutaneous level, an in depth in vitro study has carried on the HaCaT cell line, spontaneously immortalized non-tumor human skin keratinocytes. Using a panel of different analysis, the mechanism of PLTX cutaneous toxicity from the toxin binding to HaCaT cells up to cell death has been elucidated. In addition, the effects of PLTX on skin inflammation has been studied.

On the whole, this contribution will summarize the results obtained over the last 10 years, that allowed the elucidation of PLTX effects at the cutaneous level.

Molecular tools and approaches for the monitoring of harmful algal blooms PENNA, Antonella1; CASABIANCA, Silvia1; CAPELLACCI, Samuela1; VALBI, Eleonora1; DELL’AVERSANO Carmen2; TARTAGLIONE, Luciana2; GIACOBBE, Maria Grazia3; FRAGA, Santiago4, RIOBO’, Pilar4, VILA, Magda5; RICCI, Fabio1; SCARDI, Michele6 (1) Dept. of Biomolecular Sciences, University of Urbino, Italy, (2) Dept. of Pharmacy, University of Napoli Federico II, Italy, (3) IAMC CNR, Messina, Italy, (4) CSIC IEO, Vigo, Spain, (5) ICM CSIC, Barcelona, Spain, (6) Dept. of Biology, University of Tor Vergata, Rome The toxic blooms cause serious impacts to human health, marine environment and economic maritime activities at many coastal sites worldwide. Therefore, there is a urgent need for new methods not only for rapid and accurate detection and count of HAB species, but also for species-specific identification and reliable quantification of cell densities, the ultimate goal being the development of early warning and forecasting systems for HABs. Phylogenetic relationships and genetic population studies proved the identity of new species and allowed to gain new insights into phytoplankton assemblage structure in the Mediterranean Sea. Genus- and species-specific primers and probes designed on rDNA ribosomal and saxitoxin genes allowed to develop and apply new identification and counting qPCR and microarray based assays, which proved to be more rapid, sensitive and specific when applied in various substrates, such as the water column, hard and soft bottoms and aerosol. In the recent aquaculture system investigated for the PSP toxin producing species, the sxtA1 gene qPCR assay can support the analytical methods for STX determination in seawater and shellfish especially at early warning stage of toxic blooms. Further, predictive models can play an important role in managing and forecasting HABs. Models based on Machine Learning techniques and principally those based on Random Forests are very promising both at regional and at wider scale.

Many novel botulinum neurotoxins and botulinum-like toxins

Rossetto O.1, Pirazzini M.1, Zanetti G.1 and Montecucco C.1,2

1. Department of Biomedical Science University of Padova, Italy

2. CNR Institute of Neuroscience, University of Padova, Italy

Botulinum neurotoxins (BoNTs) are the etiologic agents of botulism, a reversible, yet potentially lethal, neuroparalytic syndrome specifically affecting vertebrates. They are produced by anaerobic bacteria of the genus Clostridium and are the most poisonous toxins known [1, 2]. Such a potency is due to the specific capability of BoNTs to block peripheral neurotransmission by inactivating the molecular machinery responsible for neurotransmitter release at peripheral nerve terminals. Extreme potency and exquisite neurospecificity make BoNTs potential bioweapons on one side and remarkable therapeutics on the other side. Traditionally, seven different BoNTs were known, classified according to their immunological properties. Thanks to the widespread diffusion of fast and cheap next generation sequencing techniques, it is now clear that neurotoxigenic clostridia have considerable genetic heterogeneity and most importantly, toxin sequence variability. Accordingly, many botulinum neurotoxin variants have been identified and many others are likely being discovered. More interestingly, BoNT-like genes and toxins have been found in non-clostridial species. These novelties are now reviving the interest for BoNT basic and applied research and this booming field will be discussed in relation to the recent advances.

References

[1] Rossetto O., Pirazzini M., Montecucco C. (2014). Botulinum Neurotoxins: recent genetic, structural and mechanistic insights. Nature Reviews Microbiology.12(8):535-549.

[2] Pirazzini M, Rossetto O, Eleopra R, Montecucco C. Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology. Pharmacol Rev. 2017, 69(2):200-235.

LC-High resolution MSn and LC-Tandem mass spectrometry for a data merging of a Greek cyanobacteria biomass from Lake Kastoria Luciana Tartaglione1, Carmela Dell’Aversano1, Sevasti-Kiriaki Zervou2, Hanna Mazur-Marzec3, Anastasia Hiskia2, Triantafyllos Kaloudis4 1University of Naples Federico II, Via D. Montesano 49, 80131, Napoli, Italy 2Institute of Nanoscience and Nanotechnology, NCSR Demokritos, 15310 Ag. Paraskevi Athens, Greece 3Institute of Oceanography, University of Gdansk, Al. Marszałka J. Piłsudskiego 46, 81378, Gdynia, Poland 4EYDAP SA, Water Quality Control Department, Athens, Greece Microcystins (MCs) are a large group of structurally related compounds produced by cyanobacteria belonging to different genera, including the planktonic Microcystis, Planktothrix, Anabaena species, and the benthic Oscillatoria. There are more than 240 cyclic peptides and they have often been implicated in accidental human and animal poisonings along lake and estuarine shores. Nodularins are other cyanobacterial toxins produced by the species Nodularia spumigena sharing structural features with MCs, which occurs in brackish waters, essentially in the Baltic Sea, Australia, and New Zealand. All these compounds are potent inhibitors of serine/threonine protein phosphatases 1 and 2A, with hepatocytes being their final molecular target. A number of cyanopeptolins and micropeptines are known from literature to be produced by cyanobacteria, some of them toxic too. In the present study we analyzed a biomass sample collected in September 2014 from Lake of Kastoria (Greece) during a cyanobacterial bloom by using two different approaches: a targeted analysis by LC-tandem mass spectrometry at unit resolution and an untargeted approach by LC-High resolution LTQ Orbitrap MSn. A good correlation between the two approaches emerged but from untargeted analyses the presence of some MC variants that were not detected by LC-MS/MS emerged together with the presence of potentially new cyanopetolins.

Human health risks associated to cyanotoxins exposure.

Emanuela Testai, F.M. Buratti, E. Funari, M. Manganelli, S. Scardala, M. Stefanelli, S. Vichi

Istituto Superiore di Sanità- Environment and Health Department- Rome – Italy

Since 3.5 billion years ago, when cyanobacteria were already present on the earth, they have colonized almost all terrestrial and aquatic ecosystems, mainly but not exclusively freshwaters, where they can grow up to very high densities, forming blooms and scums. Cyanobacteria produce a high number of bioactive molecules, among which cyanotoxins, such as microcystins (the group for which more data are available), nodularins, cylindrospermopsin and some neurotoxins (with anatoxins and saxitoxins being the most studied). Increasing occurrence of blooms, in terms of extension and frequency, associated with excess of nutrients due to anthropogenic activities and climate changes, has given rise to some concern for human health and animal life exposed to cyanotoxins. Numerous cases of lethal poisonings have been associated with cyanotoxins ingestion in wild animal and livestock. In humans few episodes of lethal or severe human poisonings have been recorded after acute or short-term exposure; the most serious known episode associated with human exposure to cyanotoxins occurred in Brazil, where 56 out of 130 hemodialyzed patients died after treatment with water accidentally contaminated with microcystins. However, the repeated/chronic exposure to low cyanotoxin levels I considered to be, at least in medium-moderate income countries, the critical issue. In those areas where people are more dependent from stored (in some cases desalinated) drinking water in reservoir, such as arid and developing countries, this problem seems to be of higher concern. Humans may be indeed orally exposed to cyanotoxins by drinking contaminated water, through consumption of cyanotoxin-containing freshwater fish, crops, vegetables and food supplements, or by ingesting water during recreational activities. During recreational, sport and professional activities (i.e., fishing) in contaminated waters dermal and inhalation exposure may also occur.

The cyanotoxins known and studied so far have a large spectrum of toxic effects from acute gastroenteritis and flu-like symptoms, to hepato- and nephro-toxicity, neurotoxic effects and tumor promotion, depending on the toxin involved and the exposure scenarios. Cyanotoxins have been hypothetically associated with the development of neurodegenerative diseases but their role

is still very controversial. Despite the relevance of these effects, data on the kinetic behavior, toxicological profile and exposure levels are still scant and often limited to few variants within each cyanotoxin group. Nevertheless it is possible to use the available data to derive some reference values, although with a high degree of uncertainty, and the WHO organization has derived health based values and considerations on the risks for human health can be drawn to protect the potentially exposed populations.

Tetrodotoxins in Europe – what is going on?

Andrew D. Turner1, Monika Dhanji-Rapkova1, Andy Powell1, Karl Dean1, Guiseppe Polito2, Carmen Dell’Aversano2, Luciana Tartaglione2, Mariagrazia Giacobbe3, Antonella Penna4, Myriam Algoet1, Craig Baker-Austin1

1Cefas, Barrack Road, Weymouth, Dorset, United Kingdom

2Department of Pharmacy, School of Medicine and Surgery, University of Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy

3Centro Nazionale delle ricerche, Messina, Italy

4University of Urbino, Italy

Tetrodotoxin (TTX) poisoning is the most commonly-occurring lethal marine poisoning in the world, with the toxin being found in the organs of fish from the Tetraodontidae family, including most commonly the Puffer fish. Whilst the exact source of the toxins are unknown, they are recognised by many as being produced by a range of bacterial species. These subsequently appear to accumulate through the food chain and enter the fish as well as into gastropods, crustaceans, amphibians and octopus. Tetrodotoxins (TTXs) are generally associated with marine species harvested from warm tropical/sub-tropical regions of the world and until 2013 there were no reports of tetrodotoxins found anywhere in European bivalve molluscs.

During 2013-2014, TTXs were detected in shellfish from two shallow marine sites in Southern England, resulting in the first ever detection and confirmation of TTX in bivalves from anywhere in European waters. The methods utilised involved LC-MS/MS quantitation and were also used to confirm the presence of TTX in cultures of Vibrio species isolated from molluscs from the same region, providing strong evidence for bacterial production of these toxins. This work was followed up with a screen of shellfish around the UK, particularly focussing on southern England, with results showing a prevalence of toxins in shallow, warmer water settings.

Since this time, more countries have begun to assess shellfish for the presence of these potentially fatal toxins. TTXs have been reported in Greece, the Netherlands and more recently Spain. In addition, investigations into the presence of paralytic shellfish poisoning toxins and TTXs in Sicily have revealed for the first time TTXs in Italian shellfish.

This presentation will attempt to summarise the recent findings concerning the presence and potential risk of TTX occurrence to the European shellfish industry. It will present results from a number of different studies conducted at Cefas and in collaboration with the University of Napoli over the past three years. The results from these studies will be presented together with an assessment of factors which seem to increase the risk of TTX uptake in certain specific areas of the country. More recent work will also be discussed, including the extension of the screening activities in England to more recent years, as well as a series of investigations focussed on the unexpected presence of toxins in benthic marine organisms, including one notable example of a newly identified invasive species from southern Europe and Japan. These results indicate that the pathways to TTX accumulation in shellfish may be much more complex than originally assumed.

Interpreting data from the use of the Phytoxigene QPCR assays in screening and monitoring for harmful algal blooms Mark Van Asten1,2 1 Diagnostic Technology, Suite 45, 7 Narabang Way, Belrose, Sydney 2085, Australia. 2 University of New South Wales, School of Biotechnology and Biomolecular Sciences, Sydney, Australia. Since May 2016 the Ohio Environmental Protection Agency has utilized the Phytoxigene CyanoDTec qPCR assay as part of their integrated HAB monitoring strategy for public water systems. The CyanoDTec multi-plex qPCR assay CyanoDTec was utilised to screen for the presence and level of toxin genes responsible for the production of microcystin,/nodularin (mcyE), cylindrospermopsin (CryA) and saxitoxin (SxtA). The assay also identified and quantified total cyanobacteria using a 16s rRNA gene. Water samples have been collected for all 119 public water systems (PWSs) since the implementation of the assay and analyzed along with a microcystin ELISA test. ELISA analysis for saxitoxins and cylindrospermopsin was also conducted if SxtA or CyrA genes were detected. McyE was detected in source waters for 57 PWSs and six inland lakes and microcystins were detected at 45 PWSs and five inland lakes. 2% of PWS samples and 15% of inland lake samples had microcystins detections without corresponding mcyE detections. At several sites, detection of mcyE preceded microcystins detections by 1-4 weeks. SxtA was detected at 33 PWSs and 14 inland lakes, and saxitoxins were detected at 15 PWSs and 10 inland lakes. At one PWS, mcyE, SxtA, and CyrA were all detected, demonstrating multi-plex assay functionality. Results from the assay have demonstrated a utility of the assay and showed it out-performed cyanobacteria cell counts as a predictor for inland lake cyanotoxin production.

Are cyanotoxins in food an emerging risk? The results of an EFSA Project Susanna Vichi, F.M. Buratti, E. Funari, M. Manganelli, S. Scardala, M. Stefanelli, E. Testai Istituto Superiore di Sanità- Environment and Health Department- Rome –Italy Cyanobacterial blooms extension and frequency are increasing, following anthropogenic activities and climate changes. The spreading of cyanobacteria in coastal and estuarine waters and the identification of marine species represent an additional reason of concern since cyanotoxins may accumulate through ingestion of cyanobacterial cells or contaminated zooplankton in coastal edible aquatic vertebrates and invertebrates such as fish, mussels, other crustaceans that are not routinely checked for their presence to provide a ‘safe’ product to consumers. In order to understand the possible risk for the potentially exposed population a project was launched by EFSA to conduct an extensive literature search to collect data on the occurrence of different cyanotoxins in food matrices, the analytical methods for their detection and their toxicological profile. The analysis of collected papers indicated that most of them are focused on a single microcystin (MC) variant (MC-LR) out of the more than 100 MC known. Many studies on occurrence are affected by limited quality, due to analytical drawbacks in the detection methods, and were not considered in the exposure assessment. The availability of reliable analytical method is one of the major issues considering the topic of cyanotoxins contaminations of complex matrix, as blue-green algae supplements (BGAS), plants and aquatic organisms (fish, molluscs, crustaceans) are. In addition some toxins such as MCs and β-N-methylamino-L-alanine (BMAA) are known to exist as free toxins or bound to proteins; in the absence of an indication about the detection of free, bound or total toxin form, results cannot be compared. The data collected show the broad prevalence of MC in aquatic edible species from European and non-European countries confirming that accumulation of MCs is a global concern but, due to spotted results in different species, increasing variability, no firm conclusions could be drawn. Toxicity studies useful for the derivation of health based reference values are few, being many of them carried out using i.p. injection, which is poorly representative of actual human exposure. In addition, those toxicological studies carried out with poorly characterised cyanobacterial extracts or focused on single parameters, using a single dose, devoted to elucidation of mechanism of action, reporting qualitative description of effects were not used for data extraction. Regarding the relevant exposure scenarios, built up using the consumption of fish/shellfish and food supplement (BGAS) as reported in the EFSA FoodEx, the possibility of risky exposure is evidenced for fish and shell-fish consumers and for blue-green algae supplements as well in relation to MC contamination, although being the available data on exposure very limited, no definite conclusion on the health risks for the exposed population could be drawn. The project results indicate that it is highly recommended to collect more data on both exposure and toxicological profile of toxins.

Inhibitors of botulinum neurotoxins

Giulia Zanetti1, Marco Pirazzini1, Domenico Azarnia Tehran1, Thomas Binz2, Eric Johnson3, Ornella Rossetto1, and Cesare Montecucco1,4

1Department of Biomedical Sciences and 4National Research Council Institute of Neuroscience, University of Padova, Via Ugo Bassi 58/B, 35121 Padova, Italy

2Institut für Biochemie, Medizinische Hochschule Hannover, 30623 Hannover, Germany

3Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, WI, USA

Botulinum Neurotoxins (BoNTs) are a growing family of protein neurotoxins causing the neuroparalytic syndrome of botulism. BoNTs are classified as serotypes and indicated with an alphabetical letter from A to G (BoNT/A to /G) due to their different antigenicity. Moreover, each serotype includes many subtypes (BoNT/A1, /A2, etc.) existing in hundreds of different variants which are differently cross-reactive with existing antisera raised against the parental toxin. This calls for the research of novel antidotes, effective regardless of BoNT’s immunological properties. This can be achieved by interfering with conserved steps of their mechanism of action.

BoNTs are di-chain toxins where a heavy chain mediating the neuronal targeting is disulphide linked to a metalloprotease light chain responsible for the intraneuronal cleavage of SNARE proteins, the enzymatic activity causing the neuroparalysis. Reduction of the interchain disulphide bond is conditio sine qua non to enable the intraneuronal metalloprotease activity of all BoNTs and thus offers a good target to develop inhibitors. We found that the thioredoxin - thioredoxin reductase system (Trx-TrxR) mediates this step and that it is highly expressed at the NMJ, the major site of action of BoNTs. Moreover, Trx-TrxR is associated to the cytosolic side of synaptic vesicles, the organelles exploited by BoNTs as Trojan horses to penetrate into neurons, where the disulphide reduction is expected to occur. Using a pharmacological approach we found that specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner. The effective concentration range of inhibitors is similar for the different BoNTs, indicating that these neurotoxins are comparably dependent on disulphide reduction and that the Trx-TrxR system is part of the basic uptake mechanism of all BoNTs. Most importantly, Trx-TrxR inhibitors are very effective in blocking the neuroparalytic activity of BoNTs, regardless of the serotype, upon a local and a systemic (mimicking the clinical botulism) intoxication. This suggest that these compounds are lead candidates for the development of preventive and therapeutic drugs for human botulism.