www.marine.ie Marine Environment & Health Series No. 28 2007 Isolation and purification of AZAs from naturally contaminated materials, and evaluation of their toxicological effects (ASTOX) Philipp Hess Pearse McCarron Nils Rehmann Jane Kilcoyne Terry McMahon Gavin Ryan Michael P. Ryan Michael J.Twiner Gregory J. Doucette Masayuki Satake Emiko Ito Takeshi Yasumoto ISSN 1649 0053 HEADQUARTERS & LABORATORIES MARINE INSTITUTE REGIONAL OFFICES www.marine.ie MARINE INSTITUTE MARINE INSTITUTE MARINE INSTITUTE Rinville 80 Harcourt Street Furnace Oranmore Dublin 2 Newport Co. Galway Tel: +353 1 4766500 Co. Mayo Tel: +353 91 387 200 Fax: +353 1 4784988 Tel: +353 98 42300 Fax: +353 91 387 201 Fax: +353 98 42340 Email: [email protected] Isolation and purification of AZAs from naturally contaminated materials, and evaluation of their toxicological effects (ASTOX) May 2007 Philipp Hess*, Pearse McCarron, Nils Rehmann, Jane Kilcoyne, Terry McMahon. Marine Institute, Rinville, Oranmore, Co. Galway, Ireland. Gavin Ryan, Michael P. Ryan. University College Dublin, School of Biomedical & Biomolecular Sciences, Conway Institute, Belfield, Dublin 4, Ireland. Michael J. Twiner, Gregory J. Doucette. Center for Coastal Environmental Health and Biomolecular Research, Marine Biotoxins Program - NOAA/NOS/NCCOS 219 Fort Johnson Road; Charleston, SC 29412-9110 USA. Masayuki Satake. Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumi-dori, Amamiya, Aoba-ku Sendai 981-8555, Japan. Emiko Ito. Research Centre for Pathogenic Fungi & Microbial Toxicoses, Chiba University, 1-8-1, Inohanam, Chuo-ku, Chiba 260-8673, Japan. Takeshi Yasumoto. Japan Food Research Laboratories, Tama Laboratory, 6-11-10, Nagayama, Tama-shi, Tokyo 2060025 Japan. * corresponding author’s contact: Fax: intl. + 353 91 387246 [email protected] Acknowledgements This project (Grant Aid Agreement no. ST/02/02) was carried out with the support of the Marine Institute and the Marine RTDI Measure, Productive Sector Operational Programme, National Development Plan 2000 – 2006. Firstly, the authors would like to thank the Irish shellfish industry for whose benefit this study was carried out. In particular, we would like to express our gratitude to Bantry Bay Seafoods, John McCloskey, John Harrington and William and Richard Murphy who have contributed to the success of this study through their supply of mussels and dissection services at industrious speed, when times were difficult for the industry and at a premium for the researchers involved in this project. We also acknowledge the perseverance of Mr. Richie Flynn (Secretary, Irish Farmers Association - Aquaculture) in outlining to stakeholders and the public the problems caused by shellfish toxins to professional shellfish farmers; without public knowledge of the difficulties it would not be possible to scientifically address the issues. These difficulties have been taken from the political stage and translated into constructive project euros with the significant input of Mr. Micheal O’Cinneide, Director of the Marine Environment and Food Safety Services at the Marine Institute. We also acknowledge the strategic support from Mr. Alan Reilly, Deputy CEO of the Food Safety Authority Ireland, through the organisation of the stakeholder forum: the Molluscan Shellfish Safety Committee; the discussions and challenges from this forum are invaluable in formulating our applied research. Finally, we would like to thank all our colleagues from the Biotoxin Chemistry Team and the Shellfish Safety Section who had an input into this study, our special thanks go to Gearoid Nicholson for administrative support and Dr. Ronel Biré who has contributed with great enthusiasm through his discussions, organisational support and hands-on approach to getting things done. Table of Contents Page No. List of abbreviations 1 1. Executive Summary 3 1.1. Background 3 1.2. Project design 3 1.3. Retrieval of shellfish tissues and preparation of reference materials 3 1.4. Isolation and purification studies 4 1.5. Toxicology studies 4 1.6. Mechanistic studies 4 1.7. Risk assessment 5 1.8. Scientific publication output 5 2. Introduction 6 2.1. History of occurrence of AZA and DTX2 poisoning 6 2.2. Previous lack of reference materials and gaps in toxicological knowledge 9 3. Study Design 11 3.1. Aims of study 11 3.2. Modular design and timeframe 11 3.3. Interdependence of work packages and project risks 12 4. Toxins & Reference Materials – Tools for Analysis & Toxicology 15 4.1. Retrieval of contaminated materials 15 4.1.1. Retrieval and storage of materials prior to ASTOX project 15 4.1.2. Retrieval of materials during ASTOX 15 4.1.3. Bulk processing of shellfish 17 4.1.4. Storage conditions and options 17 4.2. Isolation of AZA1, -2, -3, and DTX2 18 4.2.1. Introduction 18 4.2.2. Early developmental work 18 4.2.2.1. Isolation of AZA1 18 4.2.2.2. Isolation of AZA2, -3, -4 and -5 18 4.2.3. Implementation of previously developed protocol 19 4.2.3.1. Isolation 19 4.2.3.2. Clarification 20 4.2.4. Improved isolation procedure at MI 21 4.2.5. NRC-IMB isolation procedure 22 4.2.6. Comparison of MI/TU and NRC-IMB schemes 26 4.2.7. Isolation of DTX2 26 4.2.8. Characterisation by NMR 27 I 4.2.8.1. Purity assessment of AZAs by NMR 27 4.2.8.2. Purity assessment of DTX2 by NMR and LC-MS 29 4.2.9. Obtained materials 29 4.2.10. Alternative ways to standards 30 4.2.11. Provision of purified materials for additional studies 31 4.2.11.1. AZA provided to NRC-IMB, Halifax, Canada 31 4.2.11.2. Provision of AZA1 for toxicological research 31 4.2.11.3. Provision of DTX2 for toxicological research 31 4.2.11.4. Provision of AZA and DTX2 for matrix effect studies 31 4.2.11.5. Provision of material to the European CRL 31 4.2.12. Gaps in current studies 31 4.3. Preparation and characterisation of tissue reference materials 33 4.3.1. Introduction 33 4.3.2. Parameters affecting homogeneity 34 4.3.2.1. Assessing homogeneity 34 4.3.2.2. Optimising methodology in terms of homogeneity 35 4.3.2.3. Homogeneity on a large scale 37 4.3.3. Parameters affecting stability 37 4.3.3.1. Heat stabilisation 38 4.3.3.2. Freeze drying 40 4.3.3.3. Additives 43 4.3.3.4. Irradiation 43 4.3.3.5. Additional steps to improve stability of materials 45 4.3.3.6. General discussion on stabilisation techniques 45 4.3.4. Standardisation of procedure 46 4.3.5. Overview of materials produced at MI 48 4.3.6. Overview of materials produced at NRC-IMB 49 4.3.7. Use of materials produced 49 4.3.7.1. Single laboratory validation 49 4.3.7.2. Collaborative studies 50 4.3.7.3. Proficiency testing and interlaboratory studies 50 4.3.7.4. Feasibility of certification and candidate CRMs 51 4.3.8. Further developments 51 4.3.8.1. Gaps in current studies 51 4.3.8.2. Sustainable supply of tissue reference materials 51 4.4. Synopsis of progress on AZA standards and reference materials 52 5. Mechanisms Underlying the Toxicity of AZAs 54 5.1. Cellular functions affected by AZAs 54 5.1.1. Introduction 54 5.1.2. Cytotoxic effects of AZA in different cell types 55 5.1.3. Membrane integrity: effect of AZA in lymphocytes 58 5.1.4. TEER: an in vitro model of the gastrointestinal tract 59 5.1.4.1. AZA 59 5.1.4.2. Other toxins in the in vitro model of the gastrointestinal tract 61 5.1.4.3. Combinations 63 5.1.5. Cell morphology and cytoskeletal effects in lymphocytes 64 I I 5.1.6. Signalling pathways and junctional regulation in human 67 gastrointestinal cells 5.1.7. Neuro-inhibitory effects 70 5.1.7.1. Neuronal networks 70 5.1.7.2. Whole-cell patch clamp recordings following exposure to AZA1 72 5.1.8. Teratogenic effects in fish 73 5.1.9. Protein phosphatase 2A inhibition 77 5.2. In vitro gene expression studies 78 5.2.1. Gene microarray studies on Caco-2 cells 78 5.2.2. Gene chip studies on lymphocyte T cells 83 5.2.3. Gene expression summary 86 5.3. Design of in vivo studies 87 5.3.1. Defining the objectives of in vivo studies 87 5.3.2. Requirements for in vivo studies 87 5.4. Synopsis of knowledge gained on AZA toxicology 88 5.4.1. Cellular functions 88 5.4.2. Genetic interactions 89 5.4.3. Comparison of in vitro and in vivo studies 89 5.4.4. Functional assays as a non-animal method to detect AZA 89 5.4.5. Functional assays as a validation tool for other non-animal methods 89 5.4.6. Gaps and future developments 90 6. Risk Assessment of AZAs 91 6.1. Risk assessments prior to ASTOX 91 6.1.1. FSAI risk assessment 2001 91 6.1.2. EU review in 2001 91 6.1.3. FAO/IOC/Codex expert consultation 2004 91 6.1.4. CRLMB working group on toxicology 2005 92 6.2. Risk assessment supported by ASTOX 92 6.2.1. FSAI risk assessment 2006 92 7. Additional Research and Potential Outcomes 94 7.1. Toxicity of DTX2 relative to OA 94 7.2. Effects of heat treatments on AZA concentration of mussels in shell 95 7.3. Effects of heat treatments on DA concentration of mussels in shell 96 7.4. Feasibility and production of certified reference materials for AZAs 97 7.4.1. NRC-IMB 97 7.4.1.1.