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Innovative Mode of Action Based in Vitro Assays for Detection of Marine Neurotoxins

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Innovative mode of action based in vitro assays for detection of marine neurotoxins Jonathan Nicolas Thesis committee Thesis advisor Prof. Dr I.M.C.M. Rietjens Professor of Toxicology Wageningen UR Thesis co-supervisors Dr P.J.M. Hendriksen Project leader, BU Toxicology and Bioassays RIKILT - Institute of Food Safety, Wageningen UR Dr T.F.H. Bovee Expertise group leader Bioassays & Biosensors, BU Toxicology and Bioassays RIKILT - Institute of Food Safety, Wageningen UR Other members Prof. Dr D. Parent-Massin, University of Western Brittany, Brest, France Dr P. Hess, French Research Institute for Exploitation of the Sea (IFREMER), Nantes, France Prof. Dr A.J. Murk, Wageningen UR Prof. Dr A. Piersma, National Institute for Public Health and the Environment (RIVM), Bilthoven This research was conducted under the auspices of the Graduate School VLAG (Avanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Innovative mode of action based in vitro assays for detection of marine neurotoxins Jonathan Nicolas Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. ir. A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday 07 October 2015 at 11 a.m. in the Aula. Jonathan Nicolas Innovative mode of action based in vitro assays for detection of marine neurotoxins, 214 pages, PhD thesis, Wageningen UR, Wageningen, NL (2015) With references, with summary in English ISBN: 978-94-6257-472-4 Contents Chapter 1 General introduction.......................................................................................7 Chapter 2 Marine biotoxins and associated outbreaks following seafood consumption: prevention and surveillance in the 21st century.........................................25 Chapter 3 Marine neurotoxins: state-of-the-art, bottlenecks and perspectives for mode of action based methods of detection in seafood........................................63 Chapter 4 In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing......95 Chapter 5 Exploration of new functional endpoints in neuro-2a cells for the detection of marine neurotoxins...................................................................................115 Chapter 6 Detection of marine neurotoxins in food safety testing using a multielectrode array...........................................................................................................133 Chapter 7 Broad and animal free in vitro detection of marine biotoxins in seafood products.....................................................................................................153 Chapter 8 General discussion, summary, list of abbreviations....................................183 Appendix Acknowledgements, curriculum vitae, list of publications and overview of completed training activities ...................................................................207 CHAPTER 1 General introduction Ingestion Inhalation Dermal absorption Chapter 1 1. General background 1 Marine biotoxins are naturally occurring chemicals produced by particular Table 1.1: Major groups of marine biotoxins and their main producers. phytoplankton species. Harmful algal blooms (HABs), i.e. rapid increases of the population of phytoplankton, occur at certain environmental conditions. HABs Biotoxin Syndrome Producer Reference Azaspiracid poi- can be harmful not only through the production of marine biotoxins, but HABs Azaspiracids Azadinium spinosum [58] soning (AZP) can also deplete oxygen from the water, block light to organisms living deeper in Karenia bicuneiformis, brevis, Neurologic shell- the water and can even clog fish gills. Because of the global warming, HABs are brevisulcata, concordia, cristata, Brevetoxins fish poisoning [2, 24] expected to take place more frequently and therefore represent a growing threat mikimotoi, papilionacea, selliformis (NSP) for public and environmental health. The majority of algae producing marine Chantonella cf. verruculosa biotoxins are the dinoflagellates, although only a minority of them produces Pseudo-nitzschia australis, calli- antha, cuspidata, delicatissima, toxins affecting other organisms. The main producers of marine biotoxins are Amnesic shellfish Domoic acid fraudulenta, galaxiae, multiseries, [59] poisoning (ASP) presented in Table 1.1. multistriata, pseudodelicatissima, Marine biotoxins are known to accumulate in seafood products such as punges, seriata, turgidula fish, crabs and filter feeding bivalves. According to the Food and Agriculture Karenia selliforme Gymnodimines - [60, 61] Organization, the consumption of seafood in Europe records a constant increase Gymnodinium mikimotoi Diarrhetic shell- from one year to another (see chapter 2). Marine biotoxins accumulate in the Phalacroma rotundatum Okadaic acids fish poisoning [62, 63] digestive gland of shellfish and do not harm the shellfish itself. However, marine Prorocentrum arenarium, lima (DSP) biotoxins represent a threat for consumers and monitoring seafood for their Alexandrium spp. Paralytic shellfish presence is important (Fig. 1.1). Saxitoxins Gymnodinium catenatum [64-66] poisoning (PSP) Pyrodinium bahamense Alexandrium ostenfeldii, peruvia- Spirolides - [67, 68] num Diarrhetic shell- Protoceratium reticulatum Yessotoxins fish poisoning Lingulodinium polyedrum [24, 69] (DSP) Gonyaulax polyhedra 8 Ingestion Inhalation Dermal absorption General introduction Table 1.1: Major groups of marine biotoxins and their main producers. 1 Biotoxin Syndrome Producer Reference Azaspiracid poi- Azaspiracids Azadinium spinosum [58] soning (AZP) Karenia bicuneiformis, brevis, Neurologic shell- brevisulcata, concordia, cristata, Brevetoxins fish poisoning [2, 24] mikimotoi, papilionacea, selliformis (NSP) Chantonella cf. verruculosa Pseudo-nitzschia australis, calli- antha, cuspidata, delicatissima, Amnesic shellfish Domoic acid fraudulenta, galaxiae, multiseries, [59] poisoning (ASP) multistriata, pseudodelicatissima, punges, seriata, turgidula Karenia selliforme Gymnodimines - [60, 61] Gymnodinium mikimotoi Diarrhetic shell- Phalacroma rotundatum Okadaic acids fish poisoning [62, 63] Prorocentrum arenarium, lima (DSP) Alexandrium spp. Paralytic shellfish Saxitoxins Gymnodinium catenatum [64-66] poisoning (PSP) Pyrodinium bahamense Alexandrium ostenfeldii, peruvia- Spirolides - [67, 68] num Diarrhetic shell- Protoceratium reticulatum Yessotoxins fish poisoning Lingulodinium polyedrum [24, 69] (DSP) Gonyaulax polyhedra 9 Chapter 1 1 Figure 1.1. Different routes of human exposure to marine biotoxins. Humans are exposed to marine biotoxins mainly through consumption of contaminated seafood. The main syndromes that can occur following consumption of seafood contaminated with marine biotoxins are Amnesic Shellfish Poisoning (ASP), Azaspiracid Shellfish Poisoning (AZP), Ciguatera Fish Poisoning (CFP), Diarrhetic Shellfish Poisoning (DSP), Neurologic Shellfish Poisoning (NSP) and Paralytic Shellfish Poisoning (PSP). The symptoms associated with the consumption of seafood contaminated with marine biotoxins vary from tingling or numbness around the lips to gastrointestinal disturbances, paralysis and in severe cases death [1, 2]. Besides the impact on health that may arise from the consumption of contaminated shellfish, it is estimated that there is a loss to the tourism and shellfish industry of about 900 million euros per year due to HABs [3]. It is therefore important to develop effective strategy plans to limit their impact. To do so, monitoring programs have been put in place and regulatory limits have been established in order to protect seafood consumers (see chapter 2). 10 General introduction 2. Legislation Different regulations and surveillance approaches are being applied in countries, 1 mainly depending on the type of toxins present in their coastal waters. However, due to globalization, i.e. increase in imports and exports, countries have to adapt to the legislations established in other and new markets. For example, while CFP was mostly reported in the Pacific Islands in the twentieth century, recent intoxications were reported in France and Germany, due to import of seafood products (see chapter 3). Regulation 853/2004 is another example, where production areas in Europe should be closed as long as necessary when toxic phytoplankton is detected to ensure consumer safety, and seafood can be transferred to a toxic phytoplankton-free area to allow detoxification prior to market release. International trading therefore requires the establishment of specialized structures, e.g. national reference laboratories for the detection of a wide range of marine biotoxins that may end up on consumers plates. Requirements before commercializing seafood are briefly summarized in Figure 1.2. 11 Product rejected Screening of seafood for presence of marine biotoxins Detoxification possible by moving batches to toxin-free area Destruction of batches Monitoring programs to be put in place Release on the market Negative for regulated marine biotoxins Positive for regulated marine biotoxins Sampling Seafood producer wishes to export its products Chapter 1 1 Figure 1.2. Steps required prior to commercializing seafood: example for the European market. As the food business operator is responsible for the products that reach the market it is common that these businesses perform
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