Phycotoxins: Chemistry and Biochemistry [2 ed.] 1118500369, 9781118500361

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Phycotoxins

Phycotoxins Chemistry and Biochemistry EDITED BY

Luis M. Botana and Amparo Alfonso University of Santiago de Compostela Department of Pharmacology Veterinary Faculty Lugo-Spain

SECOND EDITION

This edition first published 2015 © 2015 by John Wiley & Sons, Ltd. Registered office: John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030-5774, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell. The right of the author to be identified as the author of this work has been asserted in accordance with the UK Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. Limit of Liability/Disclaimer of Warranty: While the publisher and author(s) have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. It is sold on the understanding that the publisher is not engaged in rendering professional services and neither the publisher nor the author shall be liable for damages arising herefrom. If professional advice or other expert assistance is required, the services of a competent professional should be sought.

Library of Congress Cataloging-in-Publication Data Phycotoxins: chemistry and biochemistry. – 2nd edition / Luis M Botana and Amparo Alfonso [editors]. pages cm Includes bibliographical references and index. ISBN 978-1-118-50036-1 (cloth) 1. Algal toxins. I. Botana, Luis M. II. Alfonso, Amparo, 1965RA1242.A36P48 2015 615.9′ 45–dc23 2014037287 A catalogue record for this book is available from the British Library. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Cover image: Prorocentrum minimum image (Scanning Electron Microscope (SEM) Leica, S440, ×7500). Picture from Editors. Typeset in 9.5/11.5pt MeridienLTStd by Laserwords Private Limited, Chennai, India 1 2015

Contents

List of contributors, vii Preface, xiii 1

Analysis of marine toxins: gaps on food safety control of marine toxins, 1 Paz Otero & Carmen Alfonso

2

Pharmacology of ciguatoxins, 23 Carmen Vale, Álvaro Antelo & Víctor Martín

3

Chemistry of pinnatoxins, 49 Phillip Mabe & Armen Zakarian

4

Chemistry and analysis of PSP toxins, 69 Ana Botana & Verónica Rey López

5

Chemistry of palytoxin and its analogues, 85 Patrizia Ciminiello, Carmela Dell’Aversano & Martino Forino

6

Pharmacology of palytoxins and ostreocins, 113 ˜ M. Carmen Louzao, María Fraga & Natalia Vilarino

7

Recent insights into anatoxin-a chemical synthesis, biomolecular targets, mechanisms of action and LC-MS detection, 137 Custódia Fonseca, Manuel Aureliano, Feras Abbas & Ambrose Furey

8

Therapeutics of marine toxins, 181 Eva Alonso & Juan A. Rubiolo

9

Marine toxins as modulators of apoptosis, 203 Amparo Alfonso, Andrea Fernández-Araujo & Mercedes R. Vieytes

10

Cyanobacterial toxins, 225 Vitor Vasconcelos, Pedro Leão & Alexandre Campos

11

Marine toxins and climate change: the case of PSP from cyanobacteria in coastal lagoons, 239 Antonella Lugliè, Silvia Pulina, Milena Bruno, Bachisio Mario Padedda, Cecilia Teodora Satta, & Nicola Sechi

12

Microalgae as a source of nutraceuticals, 255 Sushanta Kumar Saha, Edward McHugh, Patrick Murray & Daniel J. Walsh

13

The marine origin of drugs, 293 André Horta, Celso Alves, Susete Pinteus & Rui Pedrosa

v

vi

Contents

14 Pharmacology of cylindrospermopsin, 317

Juan A. Rubiolo, Diego Alberto Fernández, Henar López & M. Carmen Louzao 15 Pharmacology of the cyclic imines, 343

˜ Sara F. Ferreiro, Andrés Crespo & José Gil Natalia Vilarino, 16 Diversity of organic structures of marine microbial origin with drug potential, 361

Marcel Jaspars, Rainer Ebel & Hai Deng 17 Polyketides as a source of chemical diversity, 381

Tanya Beletskaya, Catherine Collins & Patrick Murray 18 Ichthyotoxins, 407

John W. La Claire II & Schonna R. Manning 19 Pathological clues of phycotoxin ingestion, 463

Manuel Cifuentes, Andrés Crespo & Roberto Bermúdez Index, 513

List of contributors

Feras Abbas Mass Spectrometry Research Centre (MSRC) and PRTOEOBIO Research groups, Department of Chemistry, Cork Institute of Technology (CIT), Bishopstown, Cork, Ireland. Amparo Alfonso Department Pharmacology, Veterinary School, Campus de Lugo, University of Santiago de Compostela, 27002 Lugo, Spain. Carmen Alfonso Cifga Laboratory, Campus Universitario, Pl. Santo Domingo, 20, 5a , 27001 Lugo, Spain. Eva Alonso Department of Pharmacology, Veterinary School, Campus de Lugo, University of Santiago de Compostela, 27002 Lugo, Spain. Celso Alves Grupo de Investigação em Recursos Marinhos, Escola Superior de Turismo e Tecnologia do Mar, Instituto Politécnico de Leiria, Campus 4 – Santuário N.a Sra. dos Remédios, 2520–641 Peniche, Portugal. Álvaro Antenlo Cifga Laboratory, Campus Universitario, Pl. Santo Domingo, 20, 5a , 27001 Lugo, Spain. Manuel Aureliano Department of Biological Sciences and Bioengineering, Marine Science Center, Faculty of Science and Technology, Algarve University, Faro 8005–139 Portugal. Tanya Beletskaya Shannon Applied Biotechnology Centre, Limerick Institute of Technology, Moylish Park, Limerick, Ireland. Roberto Bermúdez Department of Anatomy and Animal Production, University of Santiago de Compostela, Lugo 27002, Spain. Ana M. Botana López Department of Analytical Chemistry, Faculty of Sciences, University of Santiago de Compostela. C/ Alfonso X s/n, 27002 Lugo, Spain.

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viii

List of contributors

Milena Bruno Istituto Superiore di Sanità, Department of Environment and Primary Prevention, V.le Regina Elena 299, 00161 Rome, Italy. Alexandre Campos Interdisciplinary Center of Marine and Environmental Research, CIIMAR, and Faculty of Sciences, University of Porto, Porto, Portugal. Manuel Cifuentes Department of Anatomy and Animal Production, University of Santiago de Compostela, Lugo 27002, Spain. Patrizia Ciminiello Department of Pharmacy, University of Napoli Federico II, Via Domenico Montesano 49, Napoli 80131, Italy. John W. La Claire II University of Texas at Austin, Department of Molecular Biosciences, 205 W. 24th St., Austin, TX 78712-1240, USA. Catherine Collins Shannon Applied Biotechnology Centre, Limerick Institute of Technology, Moylish Park, Limerick, Ireland. Andrés Crespo Vieira Department of Pharmacology, Veterinary School, University of Santiago de Compostela, 27002 Lugo, Spain. Sara F. Ferreiro Department of Pharmacology, Veterinary School, University of Santiago de Compostela, 27002 Lugo, Spain. Rainer Ebel Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Old Aberdeen, AB24 3UE, Scotland, UK. Hai Deng Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Old Aberdeen, AB24 3UE, Scotland, UK. Carmela Dell’Aversano Department of Pharmacy, University of Napoli Federico II, Via Domenico Montesano 49, Napoli 80131, Italy. Diego Alberto Fernández Department of Pharmacology, Veterinary School, Campus de Lugo, University of Santiago de Compostela 27002 Lugo, Spain.

List of contributors

ix

Andrea Fernández-Araujo Department of Pharmacology, Veterinary School, Campus de Lugo, University of Santiago de Compostela, 27002 Lugo, Spain. Custódia Fonseca Department of Chemistry and Pharmacy, Marine Science Center, Faculty of Science and Technology, Algarve University, Faro 8005–139 Portugal. Martino Forino Department of Pharmacy, University of Napoli Federico II, Via Domenico Montesano 49, Napoli 80131, Italy. María Fraga Department of Pharmacology, University of Santiago de Compostela, Spain. Ambrose Furey Mass Spectrometry Research Centre (MSRC) and PRTOEOBIO Research groups, Department of Chemistry, Cork Institute of Technology (CIT), Bishopstown, Cork, Ireland. José Gil Department of Pharmacology, School of Pharmacy, University of Santiago de Compostela, Campus Sur, Praza Seminario de Estudos Galegos, s/n., 1782 Santiago de Compostela, Spain. André Horta Grupo de Investigação em Recursos Marinhos, Escola Superior de Turismo e Tecnologia do Mar, Instituto Politécnico de Leiria, Campus 4 - Santuário N.a Sra. dos Remédios, 2520 – 641 Peniche, Portugal. Marcel Jaspars Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Old Aberdeen, AB24 3UE, Scotland, UK. Sushanta Kumar Saha Shannon Applied Biotechnology Centre, Limerick Institute of Technology, Moylish Park, Limerick, Ireland (ROI). Pedro Leão Interdisciplinary Center of Marine and Environmental Research, CIIMAR, University of Porto, Porto, Portugal. Henar López Alonso Department of Pharmacology, Veterinary School, Campus de Lugo, University of Santiago de Compostela, 27002 Lugo, Spain. M Carmen Louzao Department of Pharmacology, Veterinary School, Campus de Lugo, University of Santiago de Compostela, 27002 Lugo, Spain.

x

List of contributors

Antonella Lugliè University of Sassari, Department of Sciences for Nature and Environmental Resources, Via Piandanna 4, 07100 Sassari, Italy. Phillip Mabe Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA. Schonna R. Manning University of Texas at Austin, Department of Molecular Biosciences, 205 W. 24th St., Austin, TX 78712-1240, USA. Víctor Martín Department of Pharmacology, Veterinary School, University of Santiago de Compostela, Campus Universitario, 27002 Lugo, Spain. Edward McHugh Algae Health Ltd., Rooaunmore Lodge, Rooaunmore, Claregalway, Galway, Ireland (ROI). Patrick Murray Shannon Applied Biotechnology Centre, Limerick Institute of Technology, Moylish Park, Limerick, Ireland (ROI). Alexei Novikov Department of Chemistry, University of North Dakota, Grand Forks, ND 58201. Paz Otero Department of Pharmacology, Veterinary School, University of Santiago de Compostela, 27002 Lugo, Spain. Susete Pinteus Grupo de Investigação em Recursos Marinhos, Escola Superior de Turismo e Tecnologia do Mar, Instituto Politécnico de Leiria, Campus 4 - Santuário N.a Sra. dos Remédios, 2520 – 641 Peniche, Portugal. Bachisio Mario Padedda University of Sassari, Department of Sciences for Nature and Environmental Resources, Via Piandanna 4, 07100 Sassari, Italy. Rui Pedrosa Grupo de Investigação em Recursos Marinhos, Escola Superior de Turismo e Tecnologia do Mar, Instituto Politécnico de Leiria, Campus 4 - Santuário N.a Sra. dos Remédios, 2520 – 641 Peniche, Portugal. Silvia Pulina University of Sassari, Department of Sciences for Nature and Environmental Resources, Via Piandanna 4, 07100 Sassari, Italy.

List of contributors

xi

Cecilia Teodora Satta University of Sassari, Department of Sciences for Nature and Environmental Resources, Via Piandanna 4, 07100 Sassari, Italy. Verónica Rey López Department of Analytical Chemistry, Faculty of Sciences, University of Santiago de Compostela. C/ Alfonso X s/n, 27002 Lugo, Spain. Juan A. Rubiolo Department of Pharmacology, Veterinary School, Campus de Lugo, University of Santiago de Compostela, 27002 Lugo, Spain. Nicola Sechi University of Sassari, Department of Sciences for Nature and Environmental Resources, Via Piandanna 4, 07100 Sassari, Italy. Carmen Vale Department of Pharmacology, Veterinary School, University of Santiago de Compostela, Campus Universitario, 27002, Lugo, Spain. Vitor Vasconcelos Interdisciplinary Center of Marine and Environmental Research, CIIMAR, and Faculty of Sciences, University of Porto, Porto, Portugal. Mercedes R. Vieytes Department of Physiology, Veterinary School, Campus de Lugo, University of Santiago de Compostela, 27002 Lugo, Spain. Natalia Vilariño Department of Pharmacology, Veterinary School, University of Santiago de Compostela, 27002 Lugo, Spain. Daniel J. Walsh Shannon Applied Biotechnology Centre, Limerick Institute of Technology, Moylish Park, Limerick, Ireland (ROI). Armen Zakarian Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA.

Preface

The reason for this second edition of Phycotoxins: Chemistry and Biochemistry is to update the information that relates to the field of marine toxins and marine compounds. Although marine toxins are usually associated with risks in food safety, there is much more to the subject than this. On the one hand, marine toxins are an excellent source of drug leads, since their structures are very diverse, ranging from very simple (domoic acid) to extremely complex (maitotoxin), and they are also a growing indicator of ecological changes caused by a changing climate. A third aspect of marine compounds and marine toxins is the vast number of physiological targets they have, which makes their study extremely interesting for research purposes once their mechanism of action is understood. With the intention of offering a wide view of all these aspects, this book covers several topics which are of growing concern in several fields of research. Chapter 1 describes the current technical situation of the analysis of marine toxins for their control and monitoring as a food risk. Chapters 2 to 6, 14 and 15 describe mechanistic and chemical aspects of toxins of particular interest, as their presence and chemical profile may not be not well understood, or is changing in certain geographical areas. Chapters 7, 10 and 11 describe interesting aspects of toxins from freshwater, in some cases with equivalencies to marine toxins, and possible influence of climate. The chemical diversity of marine compounds, and their mechanism of action, as well as their diversity as possible drug leads, is covered in chapters 8, 12, 13, 16 and 17. Specifically, chapter 17 describes how the elaboration of these toxins (and their stereochemistry) is so complex. Finally, chapters 18 and 19 describe special toxins in fish, and how to identify the damage caused by the marine toxins. This book could not have been written without the generosity, talent and dedication of specialists in each field who contributed enthusiastically, giving up a large number of days to prepare each of the chapters. As editors, we wish to acknowledge their efforts and give thanks for the support they have given to this book. Without their generosity, this type of book would not be possible. So we offer a sincere ‘thank you’ to them all. Finally, we wish to thank Wiley-Blackwell for believing in this project. Amparo Alfonso and Luis M Botana

xiii

CHAPTER 1

Analysis of marine toxins: gaps on food safety control of marine toxins Paz Otero1 & Carmen Alfonso2 1 Department 2 Cifga

of Pharmacology, University of Santiago de Compostela, Spain Laboratory, Pl. Santo Domingo, Spain

Analysis of marine toxins and gaps on food safety control The field of marine toxins has been deeply studied in recent decades, but there are a lot of variables that need to be understood. These relate to the occurrence of harmful algal blooms, the production of different analogues of the same group of toxins, their accumulation and biotransformation in shellfish or in fish, and the associated hazards for human consumers, both from acute and long-term exposure. Different factors are related to the proliferation of a determined alga in a zone: physical considerations, such as temperature or light, chemical parameters like nutrients, oxygen or pollutants, and biological relationships between kinds of algae and shellfish. These proliferations can develop suddenly, due to the germination of cysts from ocean sediments under appropriate environmental conditions, (Camacho et al., 2007) and are referred to as harmful algal blooms (HABs) if marine toxins are detected during them. Human poisoning during HABs has occurred in the past, modifying habits of populations in coastal and tropical areas. For example, Native Americans from the west coast of North America did not eat shellfish when bioluminescence was observed in the sea, because this phenomenon was related to a toxic algal bloom (Hallegraeff, 2004). Nowadays, it has been described an increase in the frequency, the intensity and the duration of HABs, and also an occurrence in areas where they had not been described in the past (Hallegraeff, 2004). For example, ciguatoxins and palytoxins have appeared in temperate waters some years ago (Perez-Arellano et al., 2005; Ciminiello, 2006; Aligizaki et al., 2008; Boada et al., 2010; Amzil et al., 2012), while tetrodotoxin was described as the source of a human intoxication in Europe (Rodriguez et al., 2008) and azaspiracids were detected in Morocco or Portugal (Vale et al., 2008; Elgarch et al., 2008). The factors involved in these changes are not completely known. Human activity and climate change have influenced, through the eutrophication of coastal waters (due to fertilizers, pollutants, organic matter), global warming and the transport of toxic algae from endemic to non-endemic areas in ship ballast waters or by new marine

Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

1

2

Chapter 1

currents. Also, contaminated bivalves can harbour viable cells or cysts when they are transported to non-contaminated areas (Camacho et al., 2007; Hallegraeff, 2004; Van Dolah, 2000; Moore et al., 2008). Prediction of HABs is not possible at the moment, this task being one important objective for many investigation groups around the world (Hallegraeff, 2010). Another problem for controlling HABs is the continuous detection of new analogues and toxins, followed by the description of new toxigenic algal species. This happened with Azadinium spinosum some years ago (Krock et al., 2009; Tillmann et al., 2009). Marine toxins are natural metabolites produced during HABs. Their functions in the producer organisms have not yet been determined, but they develop very specific activities in mammals, such as binding to different cation or anion channels. Much hypotheses have been postulated relating these molecules to the interaction with prey or predators, or the gain of territories (Botana et al., 1996). Different classifications of marine toxins have been established, based on their chemical structures, their physical properties or their mechanisms of action. However, the continuous isolation and description of new analogues and new groups of toxins makes the establishment of a definitive classification difficult. Initially, marine toxins were classified in different groups depending on their toxic symptoms in humans, defining five groups of toxins: paralytic, neurotoxic, diarrheic, ciguateric and amnesic. Since then, new toxins with other symptomatology have been described, like azaspiracids, cyclic imines, yessotoxins, pectenotoxins, tetrodotoxins or palytoxin, so other classifications (see Table 1.1) have been established. Contamination of shellfish and fish due to HABs generates problems for human health and economic losses, due to ecological damage and closures of harvesting sites. To reduce these impacts, monitoring programs have been implemented in many countries, with sampling of shellfish and phytoplankton, and specific legislation has been established, with regulatory limits for each toxin based on the available scientific data (EU Reg 853/2004; EU Commission Reg 786/2013). Although the consumption of contaminated seafood is the most important human exposure route, inhalation and skin contact during recreational or occupational activities must be considered in certain cases (Backer et al., 2005; Fleming et al., 2005), mainly for brevetoxins and palytoxins. Such effects usually appear when algal scum is observed. Risk assessment of shellfish toxins is required to establish regulatory limits in shellfish for human consumption. In this sense, some considerations can be listed, such as Table 1.1

Example of classification of marine toxins.

Group

Toxins

Paralytic toxins

Saxitoxins, Gonyautoxins, N-sulfocarbamoyl-11 hidroxysulfatotoxins Okadaic acid, dinophysistoxins Pectenotoxins, yessotoxins Domoic acid and analogues Ciguatoxins, maitotoxins, brevetoxins, cyclic imines, policavernosides, gambierol Palytoxins, ostreocins, ovatatoxins Azaspiracids Tetrodotoxins

Diarrheic toxins Lipophilic toxins Amnesic toxins Neurotoxic toxins Palytoxins Azaspiracids Other toxins

Analysis to marine toxins

3

the presence of many analogues of the same toxin group in the contaminated samples, the metabolization of these toxins by predators generating other toxic molecules (Bricelj & Shumway, 1998; Suzuki et al., 2005; Jauffrais et al., 2012; Abraham et al., 2012), the effect of the presence of one group of toxins in the toxicity of another one, or the fact that some compounds toxic to rodents have never been recorded as toxic to humans (Botana, 2012). The contribution of each component of a toxic mixture to the total toxicity must be studied, establishing toxicity equivalence factors (TEFs) (Aune et al., 2007), together with the study of the absorption and distribution of toxins after an oral exposition. Moreover, the effects of toxin consumption at low levels (sub-regulatory) in long-term exposure must be determined. Processing of shellfish samples must be studied, because it can produce changes in the levels or the distribution of toxins (CONTAM, 2009). In some cases, these changes can be associated to water losses, and they can be different for various analogues of the same group of toxins (McCarron et al., 2008). In risk assessment, another important parameter is the standard portion, which had been fixed at 100 g. In recent years it has been determined that a portion size of 400 g would be more appropriate, to include high consumers (CONTAM, 2010), resulting in more restrictive regulatory limits (Paredes et al., 2011). Legislation must be revised when new toxins are detected in a specific region and they are not regulated (Perez-Arellano et al., 2005; Aligizaki et al., 2008; Rodriguez et al., 2008), and also if the scientific data suggests that regulated toxins do not pose a risk for human health (EU Commission Reg 786/2013). There are many methods available for detection and/or quantification of marine toxins, with different advantages and drawbacks, depending on the kind of samples to be analyzed (microalgae, seafood, sea water, etc.), the scope of the analysis (identification or quantification) or the number of samples to be analyzed. Mouse bioassays (MBAs) have been used for many years for the detection and quantification of marine toxins in samples (AOAC, 1980). These assays vary depending on the kind of toxin to be analyzed, but they consist of the intraperitoneal injection of white mice with extracts obtained from the samples (Hess et al., 2006; Botana, 2008). The death of two of the three mice tested in a determined time period means a positive result. This method has a number of disadvantages: its detection limits are near the regulatory limits; it is not specific (which means that each toxin from the sample cannot be individually identified and quantified); and non-toxic interferences can appear (Holland, 2008). From a practical point of view, these methods are expensive, because animals must achieve determined characteristics, such as a given weight, which means that they require housing. In addition, there are ethical issues related to animal welfare, because animals used are sacrificed at the end of the assay and suffer different cruel symptoms. To guarantee a higher level of safety in commercial products, and to implement early warning systems, these bioassays must be substituted by alternative methods, which allow the identification and quantification of each one of the analogues of each group of toxins. Nevertheless, it must be considered that the mouse bioassay determines the overall toxicity of the compounds in the sample, whereas the alternative methods quantify each toxin group or even each analogue individually (Otero et al., 2011). In the future, the mouse bioassays will be done for detecting new or unknown marine toxins, not for routine monitoring (EU Commission Reg 15/2011, 2011). Development of these alternatives has been, and continues to be slow, mainly due to the scarcity of reference materials and standards, and the need for determining the

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Chapter 1

toxicity of each analogue and/or its mechanism of action. However, there are a lot of methods designed for the detection and/or quantification of each toxin group, each having advantages and disadvantages (CONTAM, 2008a, b, c; 2009a, b, c, d, e; 2010a, b, c). Using these alternative methods can produce gaps in the analysis of emerging toxins, since they only detect their targets, not the unexpected toxins that can appear in the samples. This was the case of the first tetrodotoxin episode in Spain, with samples tested negative using Lawrence et al. (1995) and Oshima (1995) HPLC methods for PSP toxins (Rodriguez et al., 2008). Nowadays, another important objective is the development of methods to screen many samples in the same assay, with the simultaneous detection of various toxin groups (Gago-Martinez et al., 1996). The initial need of substituting the bioassays (Hess et al., 2006) was solved with the design of methods using liquid chromatography separation of the toxins, and fluorescence, UV or absorbance detection, which provide an analytical quantification of the toxins in a sample. If the target compound did not show this kind of signal, it was derivatizated or followed various purification procedures. Some years ago, this initial option was completed and improved using mass spectrometry detection, which allows the quantification of each individual analogue (Quilliam, 2003) if reference materials and standards are available. This is not the current situation in many cases, so the concentration of one analogue is estimated with a reference material or another analogue of the same group of toxins (EU-RL-MB, 2011). This temporary solution can lead to false quantifications in some cases, since the signal obtained in mass spectrometry depends on the chemical structure of the compounds and can differ between analogues (Otero et al., 2011). After this quantification, the total toxicity of the sample is calculated using the TEF of each analogue (Botana et al., 2010), which is not available for all the toxic compounds described, due to the lack of standards. In spite of the limitations of liquid chromatography-mass spectrometry, it has been selected as the official monitoring method for marine lipophilic toxins in shellfish samples in Europe (EU Commission Reg 15/2011, 2011). An example of this method for basic and acid mobile phases using HPLC appears in Table 1.2. The MS/MS detection is performed in multiple reaction monitoring (MRM) mode using two transitions per toxin: OA and DTX-2 (m/z 803.5/255.0, m/z 803.5/113.0), DTX-1 (m/z 817.5/255.0, m/z 817.5/113.0), YTX (m/z 1141.5/1061.7, m/z 1141.5/855.0), 45-OH-YTX (m/z 1157.5/1077.7, m/z 1157.5/871.5), homoYTX (m/z 1155.5/1075.5, m/z 1155.5/869.5), 45-OH-homo-YTX (m/z 1171.5/1091.5, m/z 1171.5/869.5), PTX-1 (m/z 892.5/821.0, m/z 892.5/213.2), PTX-2 (m/z 876.5/823.4, m/z 876.5/213.2), AZA-1 (m/z 842.5/824.5, m/z 842.5/806.5), AZA-2 (m/z 856.5/838.5, m/z 856.5/820.5), AZA-3 (m/z 828.5/810.5, m/z 828.5/792.5). With the exception of OA and YTX toxins groups, which are ionized in negative mode, the remaining lipophilic toxins are preferably ionized in positive mode. The transition with the highest intensity is used for quantification, while the transition with the lowest intensity is used for confirmatory purposes. According to the legislation (EU Reg 853/2004; EU Commission Reg 786/2013), the toxin amounts found in shellfish for human consumption must be lower than 3.75 mg eq YTX/kg, 0.16 mg eq AZA-1 and 0.16 mg eq OA/kg (for OA and PTX toxin group). Alternatives to this analytical methodology have been designed, based on the biological receptors of the toxins (i.e. functional methods), or on the binding with antibodies. These kinds of methods do not identify analogues; they only indicate the presence of a group of toxins. However, these methods are easy to use and are cheaper than

Analysis to marine toxins

5

Table 1.2

Chromatographic conditions of the multi-toxin LC-MS/MS method to detect the lipophilic marine toxins using acid and basic mobile phases. Chromatography with acid mobile phase

Chromatography with basic mobile phase

Flow Injection volume Column T

BDS-Hypersil C8, 50 mm × 2 mm, 3 μm particle size 0.2 mL/min 5 μL 25 ∘ C

X-Bridge C18, 150 mm × 3 mm, 5 μm particle size 0.25–0.4 mL/min 5–10 μL 40 ∘ C

Mobile phase

A: Water

A: Water

Column

B: ACN (95%)

Gradient

(both containing 2 mM ammonium formate and 50 mM formic acid)

B: ACN (90%)

(both containing 6.7 mM ammonium hydroxide) (pH 11)

Time (min)

Mobile phase A (%)

Mobile phase B (%)

Time (min)

Mobile phase A (%)

Mobile phase B (%)

0 8 11 11.5 17

70 10 10 70 70

30 90 90 30 30

0-1 10 13 15 19

90 10 10 90 90

10 90 90 10 10

liquid chromatography with mass detection, and functional assays also supply information about the toxicity of samples. Functional assays use the mechanism of action of toxins for their detection and quantification. To design these assays, it is necessary to identify the receptor targets for all the toxins (Botana et al., 2009), and to design the best detection method for the interaction between the receptor and the toxin, both in solution (Vieytes et al., 1997; Alfonso et al., 2005; Fonfria et al., 2010; Otero et al., 2011) or on some kind of surface (Pazos et al., 2004; Fonfría et al., 2007). One drawback of this methodology is the availability of the receptor, and the need to assure its stability during the assays. In this sense, the synthesis of cloned receptors would be useful (Campbell et al., 2011). Assays based on toxin recognition by an antibody can be used for the routine detection of marine toxins. The most important disadvantage of these assays is the need for large quantities of pure toxins to obtain anti-toxin antibodies (Hirama, 2005). These molecules show high specificity and sensitivity and, depending on its specificity, an antibody may be able to detect only one toxin or various members of a group of toxins. Nowadays, other methodology is being studied, such as the use of aptamers (oligonucleotides or peptides that bind to specific targets), which has many advantages compared to antibodies (Jayasena, 1999), and the same applications (Handy et al., 2013). In the field of detection methods, the main tasks that need to be solved are the validation of the alternative methods described, the multi-detection of several groups of toxins in a single assay (Fraga et al., 2013; McNamee et al., 2013), and the miniaturization and the portability for the on-site testing.

6

Chapter 1

Gaps on food safety control for marine toxins by chemical methods Marine toxins pose a significant food safety risk when they accumulate in shellfish, and adequate testing for biotoxins is required to ensure public safety and the long-term viability of commercial shellfish markets. In developed countries, there is a legislated specific requirement for regular and controlled analysis of seafood for the presence of phycotoxins. In Canada, South America and Japan, MBA is the method used for the official control of marine toxins, while in New Zealand, chromatographic methods are used for their monitoring programmes (Gerssen et al., 2011). From July 2014, in Europe, it is no longer possible officially to analyze for the presence of lipophilic marine toxins in shellfish by MBA (EU Commission Reg 15/2011, 2011). New regulations replaced the MBA for lipophilic marine toxin detection, which had been used up to now as the reference method, by the LC-MS/MS approach. This change has resulted in supporter groups and other groups against this law. There is also controversy and debate about whether the chemical method is an effective method to protect human health. Since this technique has been evaluated, and it is considered to be successful by many laboratories (Gerssen et al., 2009; Blay et al., 2011), this section highlights some gaps in the food safety control of marine toxins when they are monitored by chemical methods. The legislated group of lipophilic marine toxins consists of four different chemical groups: okadaic acid (OA) and its derivatives, yessotoxins (YTXs) azaspiracids (AZAs) and pectenotoxins (PTXs). These are the most frequently toxins that have appeared along European coasts, together with the spirolides (SPXs), for which no legislation has yet been established. Only few analogues of each group are legislated, and their quantity has to be referred to a predominant compound of the group, called the reference compound (RC). For instance, in the case of the AZA group, results should be expressed as milligrams of AZA-1 equivalents per kg of whole flesh, using TEFs for AZA-2 and AZA-3, and the other AZA analogues are considered of low relevance. To be able to determine the toxicity of a seafood sample by LC-MS/MS, the use of TEFs is necessary. Similarly, the use of TEFs requires the knowledge of the toxicity of each analogue present in a sample in order to translate analytical data into total toxicity. However, the toxicity for some toxins varies considerably, depending on the administration route, oral or i.p. administration (Tubaro et al., 2003; Munday et al., 2008). Correct TEF values are necessary since, if the TEFs are not applied properly, this can greatly affect the final results. Oral toxicity studies provide information that compare better to human food poisoning, because they take into account the ratio of the absorption of the toxic molecule in the digestive system of mammalians. However, given the lack of sufficient information available in regard to the oral route, the European Food Safety Authority (EFSA) panel proposed TEFs based on acute effects after i.p. administration (CONTAM, 2009b). The limited toxicological information does not allow the setting of TEFs for the oral route for any of the toxin groups, and EFSA mentions that the TEF values should be revised when studies on acute oral toxicity data for the relevant analogues of each toxin group become available. Despite the fact that the chemical method for lipophilic marine toxin detection has been developed as a multi-toxin detection method (Gerssen et al., 2009; Blay et al., 2011), it is only used for limited number of toxins, four analogues of YTX toxin group,

Analysis to marine toxins

7

three analogues of each OA and AZA toxin group and two of PTX toxin group (11 or 12 toxins at most). This is one of the main drawbacks of the LC-MS/MS method in order to protect human health. The number of compounds that can be analyzed in a single run is limited. LC–MS/MS technology is a targeted method, and the target toxins should be selected before the run. The toxins usually selected for LC-MS/MS analysis comprise the 12 regulated toxins, but outside of the monitoring there may be many additional analogues which may also be of toxicological relevance. For example, the OA toxin group exists outside of OA, DTX-1 and DTX-2; these toxins can also be present in the form of fatty acid-containing esters (Rossignoli et al., 2011). The AZA group and PTX group both consist out of over 20 possible analogues (Gerssen et al., 2010). For YTX group toxins, over 36 natural derivative analogues have been identified, and more than 90 analogues have been described (Miles et al., 2005). However, for routine monitoring control, experts on biotoxins considered that is not practical to fully determine the presence of all toxin levels in shellfish. If no toxic adequate information about a analogue is available, it is proposed that new compounds present in shellfish at less than five percent of the RC should not be regulated (FAO/IOC/WHO, 2011). The identification and quantification of marine toxins in order to protect human health has became the main part of the work based in HPLC, by coupling detection systems which measure the different physical and chemical properties of the molecule (Ciminiello et al., 2011). The most common detection systems for lipophilic marine toxins are quadrupole (Q) and triple quadrupole (TQ) mass spectrometers (Otero et al., 2011; These, et al., 2011), ion trap (IT) (Rodriguez et al., 2008), time-of-flight (ToF) (Meisen et al., 2009) or hybrid instrument (IT-ToF; Q-ToF; IT-LC/ESI-MS/MS) (Ferranti et al., 2009). Toxins are ionized in negative or positive mode, then separated by the m/z ratio and, finally, detected and registered in the chromatograms by converting the ion flow in an electric signal. However, this signal is not always the same between different analyzers and MS methods. This fact can give rise to different toxin concentrations when a calibration curve is used (Otero et al., 2011). As shown in a comparative study, the quantification of three lipophilic toxins – OA, DTX-1 and DTX-2 – was affected by several parameters, such as MS detection method, mobile-phase-solvent brands and equipment (Otero et al., 2011). The study demonstrated that OA, DTX-1 and DTX-2 toxin amount was increased or decreased, depending on the MS detection method used to quantify. Two MS methods were used; one with the specific transitions for OA and DTXs (four transitions), while the other method included the transitions for six lipophilic toxins (a total of 10). Quantities analyzed by LC-MS/MS using the MS method with 10 transitions were considerably lower than those obtained by the method with four transitions. This meant that the number of transitions included in the MS methods, not fixed in the reference method (EU-RL-MB, 2011), affected the toxin quantification. Figure 1.1 shows the OA quantification by a TQ instrument using three mobile phases composed by three acetonitrile brands and two MS methods. As can be observed, the toxin amount can be increased up to 42% or decreased up to 40%, depending on the ACN and mass method used. These results show that this method may result in large errors in the concentration of a toxic sample. For example, a sample containing 200 μg∕kg would be detected as 120 μg∕kg, hence negative. By contrast, a sample containing 120 μg∕kg of OA, considered as negative by EU regulations, can be quantified as 174.4 μg∕kg. From the perspective of public health protection, the worst case is in which a positive sample for EU legislation, and

Chapter 1

M2 (10 transitions) M3 (4 transitions) Real magnitude

LC-MS/MS 80

M2 (10t)

250

M3 (4t)

45 ng/mL OA

Concentration (ng/mL)

60 50 40 30 20

Concentration (ng/mL)

70

500

160 ng/mL OA

200

150

100

50

320 ng/mL OA

400

Concentration (ng/mL)

8

300

200

100

10 0

MR P

MP S

MP M

Real magnitude

0

MP P

MP S

MP M

0 Real magnitude

64 ng/mL

42 % highest (a)

MP P

MP S

MP M

Real magnitude

189 ng/mL

Theoretical concentration (b)

40 % lowest (c)

Effect of acetonitrile (ACN) from the mobile phase on the quantification of OA by LC-MS/MS. MP P: mobile phase containing ACN from Panreac. MP S: mobile phase containing ACN from Sigma. MP M: mobile phase containing ACN from Merk. Each toxin was quantified using two MS methods: an MS method that includes 10 toxin transitions; and an MS method that includes four toxin transitions. Mean ± SEM of n = 3 experiments. Each graphic represent the concentrations in methanol for OA at 45 ng/mL: (a) at 160 ng/mL; (b); and 320 ng/mL (c). Black column: theoretical concentration of toxin. Open columns: toxin concentration obtained by each MS method.

Figure 1.1

Analysis to marine toxins

9

thus toxic, reaches the market and intoxicates the consumers. This happened recently in Ireland, where several illnesses associated with DSP were reported following the consumption of frozen mussel meats. The implicated batches were removed from sale (https://www.fsai.ie/news_centre/food_alerts/SDLM_mussels_recall.html). The use of LC-MS/MS method to monitor the lipophilic marine toxin has been frequent in Ireland since 2001. An important issue to have into account in the toxin monitoring is the LOD of the detection systems. Studies using four analyzers showed variability in the LODs for PTX-2 and OA, TQ-MS being the most sensitive mass analyzers (Gerssen et al., 2008). In fact, the determination of some lipophilic toxins using IT-ToF detectors can be a problematic. Figure 1.2 shows the analysis of OA at different concentrations, using an ultra-high liquid chromatography (UPLC) method coupled to IT-TOF system. The separation was performed in a Waters Acquity UPLC® BEH C18 (100 mm × 2.1, 1.7 μm) with an acid mobile phase, composed of water and acetonitrile 95% (both containing 2 mM ammonium formate and 50 nM formic acid) at a flow of 0.4 mL/min. This is a column, an acid mobile phase and a gradient usually employed for these toxins. As it can be observed in the chromatogram of Figure 1.2a, OA standard at a concentration of 250 ng/mL eluted in 3.9 minutes. However, when 10 ng/mL OA is injected (Figure 1.2b), it is not possible determinate the peak, since it is confused with the noise. Also, despite the fact that the chromatographic conditions and method parameters were optimized, 1 ng/mL OA (Figure 1.2c) is not observed.

(x1,000,000) 1.5 1:MIC1

2:MIC1 1:803.4512 (1.00)

803.45 m/z 255.12 m/z

OA standard 250 ng/mL

1.0 2:255.1204 (1.00) 0.5 0.0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

(a) (x10,000) 5.0

1:MIC1 2:MIC1 1:803.4512 (1.00) 2:255.1204 (1.00)

OA (10 ng/mL)

0.0 0.0

0.5

1.0

1.5

2.0

2.5

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4.0

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(b) (x10,000) 1:MIC1

5.0 2:MIC1

No peak

1:803.4512 (1.00) 2:255.1204 (1.00)

OA (1 ng/mL)

2.5 0.0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

(c)

Figure 1.2 Chromatogram of OA standard at different concentrations: 250 ng/mL (a), 10 ng/mL (b) and 1 ng/mL (c), obtained in the LCMS-IT-TOF system from Shimadzu (Kyoto, Japan). The column used for the identifications was a Waters Acquity UPLC® BEH C18 (100 mm × 2.1, 1.7 μm) with a mobile phase, composed of water (a) and acetonitrile 95% (b), both containing 2 mM ammonium formate and 50 nM formic acid. The mobile phase flow rate was 0.4 mL/min and the injection volume was 5 μL. Intensity units in counts per second (cps).

10

Chapter 1

LODs and LOQs of the mass analyzers are important considerations, since the lipophilic marine toxin detection protocol is based in weighting 2.00 g ± 0.05 g of tissue homogenate and, after an extraction procedure, the resulted supernatant has to be combined and made up to 20 mL of 100% methanol. With this protocol, the original sample is highly diluted and, therefore, sensitive methods are necessary in order to protect human health.

Use of standards An important problem in the field of marine toxins is the lack of standards, needed in toxicological studies and also in routine quantification of shellfish and phytoplankton samples. Due to this, different kinds of materials are used in research and quantification, without a stated traceability, and sometimes obtaining unreliable data. An example of this need appears in the European legislation (EU Commission Reg 15/2011, 2011), which establishes the use of a validated technique of liquid chromatography coupled to mass spectrometry for the routine detection of certain lipophilic toxins, ‘both for the purposes of official controls at any stage of the food chain and own-checks by food business operators’. In this document, it is mentioned that other methods could be applied for this purpose if they fulfil the criteria stipulated by the European Union Reference Laboratory on marine biotoxins, but they should be intra-laboratory validated and tested under recognized proficiency test schemes. These activities could only be done using reference standards which guarantee the traceability of the final results. Also, contradictory results in toxicology studies of marine toxins can be related to the use of different standards from various sources, or to the instability of these compounds in the solutions used during this kind of studies. For example, production of pectenotoxins by the same dinoflagellate as okadaic acid and dinophysistoxins led to the study of the toxicology of pectenotoxins using contaminated solutions (Miles et al., 2004; Munday, 2008). Due to this, pectenotoxins were considered diarrheic compounds for many years. Other examples are different lethal doses obtained for the same compounds by different authors, using toxins obtained from various suppliers (Otero et al, 2012; Munday et al., 2012). The terminology associated with reference materials and standards is large and confusing. Different names are used for different kinds of materials with various applications (see Table 1.3), but all of them can be used to guarantee the quality of the results obtained in a laboratory. This characteristic is influenced by several parameters, one of these being the traceability of the results, which is needed to establish their reliability and to compare measurements from different methods and places. These materials are also used for quality control, calibration of equipments and method validation. Certified reference materials (CRMs) must be used for the complete development and validation of analytical methods (ILAC-G9, 2005) for the quantification of marine toxins. These kinds of materials guarantee the traceability of the measurements (ISO Guide 35, 2006), and their accuracy and must be produced and certified according to strict procedures that fulfil the requirements of ISO Guide 34 (2009) and 35 (2006), or similar. Characteristics such as stability, homogeneity or uncertainty, associated to the certified value, must be clearly described in the certificate of the material (ISO Guide 31, 2000) supplied by the manufacturer. Other parameters from the certificate must

Analysis to marine toxins

Table 1.3

11

Definitions associated to reference materials and standards.

Reference material (RM): ‘material, sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurement process’ (ISO Guide 30 (2008)). Certified reference materials (CRMs): ‘reference materials characterized by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability’ (ISO Guide 30 (2008)). Primary standard: ‘standard that is designated or widely acknowledged as having the highest metrological qualities and whose value is accepted without reference to other standards of the same quantity, within a specified context’ (ISO Guide 30 (1992)). Secondary standard: ‘standard whose value is assigned by comparison with a primary standard of the same quantity’ (ISO Guide 30 (1992)). Measurement standard (calibrant): ‘realization of the definition of a given quantity, with stated quantity value and associated measurement uncertainty, used as reference’ (VIM, 2008). Working standards: standards ‘used routinely to calibrate or verify measuring instruments or measuring systems’ (VIM, 2008).

be considered for obtaining reliable results, like period of validity, conditions of storage or intended use. There are two types of certified reference materials of marine toxins: solutions of one/more toxins in appropriate solvents with a determined concentration; and tissues contaminated with one/more toxins (matrix reference materials). Matrix reference materials are used to perform recovery studies for complete method validations, to study sample pre-treatments (extractions, partitions, derivations, evaporations, clean-ups, etc.) and for quality control purposes. Their composition and their characteristics are similar to routine tested shellfish samples, so that the behaviour of the target analyte would be the same in both materials. Due to this, matrix reference materials must be used to calibrate methods sensitive to different types of matrix (ISO Guide 32, 1997), and their use provides an estimation of the percentage of recovery of a target analyte at the end of a series of procedures. These kinds of materials are scarce, since their production is difficult. They can be obtained from contaminated natural tissues, or from non-contaminated natural tissues spiked with the target toxins. Once the initial material has been selected, homogeneity and stability of the toxin in the matrix must be assured, which is a difficult task due to the characteristics of the tissues. Another problem is the amount of materials needed, since all possible shellfish tissues (mussels, clams, oysters, cockles, etc.) can contain different groups and analogues of marine toxins. Also, the comparability of naturally contaminated and spiked tissues must be assured, since the availability of the native analyte during different procedures such as extractions can be different to the availability of the spiked analyte, which is free on the surface of the tissue. If CRMs for a group of toxins are not available, purified materials can be used for certain applications. These materials have been characterized by different methods (fluorescence, absorbance, mass spectrometry, NMR, etc.), which guarantee their purity and quantity but, usually, other parameters, such as their traceability, homogeneity or the stability, have not been completely studied due to their limited availability.

12

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Also, control materials must be used to guarantee the quality of the measurements performed by each laboratory. These materials must be measured in every batch of routine samples and fulfil some requirements: an assigned value for the target toxin, a similar physical form than the problem samples, a demonstrated stability during large time periods and a high availability (Thompson & Wood, 1995). These materials can be prepared by each laboratory, matching the characteristics needed for a determined sample pre-treatment or quantification procedure, but they do not guarantee the traceability of the measurements performed, so their uses can be in quality assurance, recovery studies or checking of the stability of a system or an instrument (EURACHEM, 1998). Commercial availability of marine toxins reference materials and standards is limited, due to the laborious procedures needed to obtain them. In a first step, toxins can be purified from contaminated shellfish or fish samples or from cultures of the producer microorganism, through a large chain of steps controlled by appropriate quantification procedures. Once the target compound is obtained, it must be characterized using complex analytical and statistical procedures that guarantee the traceability of the results, defining its quantity, purity, homogeneity, stability, the uncertainty associated to the measured values, and so on. Later, materials must be handled and stored using adequate methods and equipments, to maintain their characteristics. All of these activities need high technical knowledge and are time-consuming, so that suppliers of marine toxins are limited. Some years ago, only the National Research Council of Canada and the Japan Food Research Laboratory could provide these materials, but at present there is also a European supplier company, Laboratorio CIFGA, from Spain. These suppliers develop the activities previously listed and maintain adequate post-distribution services to guarantee that all of the information related to the production, characterization and use of the materials is available for their clients.

New risks in the EU Marine algal toxins and the associated phytoplankton species are responsible for >60, 000 toxicity incidents worldwide per year, with a mortality rate of 1.5% (Bourne, et al., 2010). In Europe, episodes of human intoxication due to the consumption of marine toxins have been common in recent years, due to toxins which were not regularly monitored (Rodriguez et al., 2008). Climatic change is expected to affect food and feed safety, including seafood production. Climatologists and other experts in atmospheric and biological sciences point out that global temperatures are expected to increase between 1.8–5.8 ∘ C by the late 21st century (Cáceres, 2012), and the highest temperature increases will be at high northern latitudes (Liu et al., 2013). These predictions have been confirmed by long-term observations in European seas, where sea surface temperature rates have increased around 0.01 ∘ C per year since the 1860s (Sarmento et al., 2010). Moreover, the distribution of precipitation is expected to change, resulting in an increase in the number of extreme precipitation events, and even areas with decreasing precipitation (Liu et al., 2013). It is likely that such changes will deeply affect different aspects of the structure and functioning of marine ecosystems. Blooms of dinoflagellates are estimated to occur more often (van der Fels-Klerx et al., 2012). Dinophysis spp. dinoflagellate, which produces OA toxins, has been studied, and it was observed the occurrence of this dinoflagellate is increasing (van der Fels-Klerx et al., 2012). If the

Analysis to marine toxins

13

behaviour of Dinophysis spp. group is similarly to other dinoflagellates in the future, then the frequency of harmful algae blooms may also increase. This is also the case with Alexandrium ostenfedii (A. ostenfeldii) dinoflagellate. This organism, which produces SPXs toxins, was originally considered a cold-water species (Gribble et al., 2005), and was only described as a producer of neurotoxins associated with PSP (MacKinnon et al., 2004). However, A. ostenfeldii has been present in recent years in temperate waters throughout the world. The presence of SPX in molluscs has increased considerably in Europe (Rundberget et al., 2011). Figure 1.3 shows the lipophilic analysis by LC-MS/MS of mussels purchased from markets originating in Galicia, Spain. Figure 1.3(a) and figure 1.3(b) show the chromatograms of the OA and SPX standards, respectively. As can be observed in Figure 1.3(c), the analysis of the mussel sample showed OA at 8.16 minute and SPX-1 (Figure 1.3.(d)) at 5.70 minutes at levels of 12.5 μg OA/kg and 41 μg SPX-1/kg. In the case of the OA, this amount is considerably lower than the maximum level

Xic of –MRM

Xic of +MRM

8.16 OA standard 250 ng/mL

160

700

SPX-1 standard

600 Intensity, cps

Intensity, cps

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500 400 300 200

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Time, min

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Time, min

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(b) Xic of +MRM 180

8.16

65 60

Mussel from market

160 OA

140 Intensity, cps

Intensity, cps

50 40 30 20

5.70 Mussel from market SPX-1

120 100 80 60 40

10 0

20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Time, min (c)

0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Time, min (d)

Figure 1.3 Chromatograms in negative mode of OA standard (a) and mussel from market (c). Chromatograms in positive mode of SPX-1 standard (b) and mussel from market (d). The equipment used was a HPLC system, from Shimadzu (Kyoto, Japan), coupled to a QTRAP LC/MS/MS system from Applied Biosystems (USA), which integrate a hybrid quadrupole-linear ion trap mass spectrometer equipped with an ESI source. The column used for the analysis was a BDS-Hypersil C8 (50 mm × 2 mm, 3 μm) with a mobile phase, composed by water (a) and ACN 95% (b), both containing 50 mM formic acid and 2 mM ammonium formate. The mobile phase flow rate was 0.2 mL/min and the injection volume was 5 μL. Intensity units in counts per second (cps).

14

Chapter 1

allowed (160 μg OA∕kg) – about 13 times lower – and, as SPX are not legislated, there is therefore no maximum level. In this analysis, the homogenate of mussel was extracted following the official method and, in total, the lipophilic toxin amount in the mussel sample was 53.5 μg∕kg. This sample is not a threat for consumers in the short term, since the toxin amount is amply below the official limit. However, a matter of concern is that the legislation allows each toxin to be quantified regardless of the presence of other lipophilic toxins. This means that a sample up to 1 mg eq YTX∕kg + up to 160 μg eq AZA-1+ up to 160 μg eq OA/kg (for OA and PTX toxin group) is allowed and, therefore, it could be available at the markets. This is another drawback of the new chemical method for lipophilic marine toxin detection with respect to the MBA. In the case of several toxin groups being present in a sample, the MBA detects all toxins together, and the chemical approach allows that a sample with higher levels of several lipophilic marine toxin groups is available at the markets, while the same sample detected by MBA is not available. The most striking observation is the fact that toxic compounds such as Ciguatoxins (CTXs), Tetrodotoxins (TTXs) and Palitoxins (PlTXs) are appearing in molluscs and gastropods in Europe, and these toxins do not have official methods for their detection. This is clearly a matter for concern, since this new phenomenon is probably to be attributed to a potential ecological change due to increased warm temperatures. TTX is one of the most potent neurotoxins, and is known to block the sodium ion channels responsible for nerve and muscle excitability (Kawatsu et al., 1999). Unlike other biotoxins which are produced by dinoflagellates, the TTX is not produced by microalgae. Symbiotic bacteria have been involved in TTX genesis for marine animals (Shewanella algae, S. putrefaciens, Vibrio sp., Pseudomonas sp., and Alteromonas tetraodonis) (Croci et al., 2006). TTX food poisonings are typical in Asian tropical countries, and is caused by the consumption of blowfish or small gastropods contaminated with the toxin (Noguchi & Arakawa, 2008). However, a food poisoning incident resulting from the ingestion of a trumpet shell (species Charonia lampas lampas) contaminated with TTX involved a single person in the south of the Iberian Peninsula in October 2007 (Rodriguez et al., 2008). The patient was a 49-year-old man, and he had bought the shell for personal consumption in a market in Malaga, Spain. The symptoms began minutes after ingestion of the mollusc, and included abdominal pain with nausea and vomiting, weakness, difficulty in articulating words and keeping the eyelids open, and difficulty breathing (Fernandez-Ortega et al., 2010). Later on, TTX and analogues were again found in three species of Monodonta liniata, Charonia lampas and Gibbula umbilicalis collected in different locations in the coast of Portugal (Vila Nova de Milfontes, Angeiras and Memória) between 2009 and 2010 (Silva et al., 2012). Despite these findings, detection methods for TTX are still not included in the European regulation. PlTXs are produced by Ostreopsis genera dinoflagellates (Ramos & Vasconcelos, 2010). This dinoflagellate is also typical from tropical waters, and it was first detected in Mediterranean worm water in the 1970s (Taylor, 1979). However, it was in more recent years when the big HABs were recorded in the European Mediterranean coast and North Africa, in the Atlantic coast of Portugal (Penna et al., 2010; Parsons et al., 2012) and also in Spain (Barroso Garcia et al., 2008). Toxic outbreaks by Ostreopsis spp. represent a completely new problem, affecting mainly the Mediterranean (Penna et al., 2005). It caused great concern after the 2005 episode occurring near Genoa (Liguria, Tyrrhenian Sea), when 209 people needed medical care (Brescianini

Analysis to marine toxins

15

et al., 2006; Durando et al., 2007). Therefore, the regulation of palitoxins in EU should be considered. CTXs are potent polyether toxins produced by Gambierdiscus species of dinoflagellate (Satake et al., 1993, 1997, 1998). CTXs open the sodium channels in excitable cells, and more than 50 analogues have been characterized to date. These compounds have high toxicity and may pose a high risk to consumers, since 1 ng/g are toxic to humans (Lehane & Lewis, 2000). CTXs have previously been only linked to Caribbean, Pacific and Indian areas, causing a human food-borne intoxication called ciguatera fish poisoning (CFP) (Dickey & Plakas, 2009; Dickey, 2008). In the last decade, Gambierdiscus have been recorded in the East Mediterranean. The first record of the presence of Gambierdiscus in this area was in Crete in 2003 (Aligizaki et al., 2008). Subsequently, in January 2004, a family of fishermen had symptoms after the ingestion of 26 Kg of a fish species, Seriola rivoliana, collected along the coast of the Canary Islands. A sample of 150 g of the fish was kept and was analyzed by an in vitro assay for specific toxins that act on sodium channels and LC-MS/MS methodology. The results were positive and the content of CTX in the sample was 1.0 mg/kg (Perez-Arellano et al., 2005). Later, the presence of organisms that produce CTXs was found in the Mediterranean, near Israel (Bentur & Spanier, 2007), and again in the Canary Islands (Boada et al., 2010). In 2010, it was confirmed the presence of CTX from different origins, in two species of Seriola dumerilli and Seriola fasciata captured in waters belong to Madeira Archipelago, Portugal (Otero et al., 2010). The detection of CTXs requires very sensitive analytical methods, since intoxications can occur at very low concentrations of the toxin, and a method for routine testing is not available worldwide as yet. All of these facts show that climate change should not be ignored in food safety management and research, particularly by Spanish and Portuguese regulatory agencies. The presence of new analogues and new toxins from other localizations are issues that must be taken into account when any analytical method is used.

References Abraham, A., Wang, Y., El Said, K.R. and Plakas, S.M. (2012) Characterization of brevetoxin metabolism in Karenia brevis bloom-exposed clams (Mercenaria sp.) by LC-MS/MS. Toxicon, 60 (6), 1030–40. Alfonso, C., Alfonso, A., Vieytes, M.R. et al. (2005) Quantification of yessotoxin using the fluorescence polarization technique and study of the adequate extraction procedure. Analytical Biochemistry, 344 (2), 266–74. Aligizaki, K., Katikou, P., Nikolaidis, G. and Panou, A. (2008) First episode of shellfish contamination by palytoxin-like compounds from Ostreopsis species (Aegean Sea, Greece). Toxicon, 51 (3), 418–27. Amzil, Z., Sibat, M., Chomerat, N. et al. (2012) Ovatoxin-a and palytoxin accumulation in seafood in relation to Ostreopsis cf. ovata blooms on the French Mediterranean coast. Marine Drugs, 10 (2), 477–96. AOAC (1980) Paralytic shellfish poison biological method, in Official methods of analysis, Washington DC, pp. 289–99. Aune, T., Larsen, S., Aasen, J.A. et al. (2007) Relative toxicity of dinophysistoxin-2 (DTX-2) compared with okadaic acid, based on acute intraperitoneal toxicity in mice. Toxicon, 49 (1), 1–7. Backer, L.C., Kirkpatrick, B., Fleming, L.E. et al. (2005) Occupational exposure to aerosolized brevetoxins during Florida red tide events: effects on a healthy worker population. Environmental Health Perspectives, 113 (5), 644–9.

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Barroso Garcia, P., Rueda de la Puerta, P., Parrón Carreño, T. et al. (2008) An epidemic outbreak with respiratory symptoms in the province of Almeria (Spain) due to toxic microalgae exposure. Gaceta Sanitaria, 22, 578–584. Bentur, Y. and Spanier, E. (2007) Ciguatoxin-like substances in edible fish on the eastern Mediterranean. Clinical Toxicology (Philadelphia, PA), 45 (6), 695–700. Blay, P., Hui, J.P., Chang, J. and Melanson, J.E. (2011) Screening for multiple classes of marine biotoxins by liquid chromatography-high-resolution mass spectrometry. Analytical and Bioanalytical Chemistry, 400, 577–585. Boada, L.D., Zumbado, M., Luzardo, O.P. et al. (2010) Ciguatera fish poisoning on the West Africa Coast: An emerging risk in the Canary Islands (Spain). Toxicon, 56 (8), 1516–9. Botana, L.M. (2008). The mouse bioassay as a universal detector. In: Botana, L.M. (Ed). Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection, pp. 149–61. CRC Press (Taylor and Francis Group): Boca Raton, FL. Botana, L.M. (2012) A perspective on the toxicology of marine toxins. Chemical Research in Toxicology, 25 (9), 1800–4. Botana, L.M., Rodriguez-Vieytes, M., Alfonso, A. et al. (1996). Phycotoxins: paralytic shellfish poisoning and diarrhetic shellfish poisoning. In: Nollet, L.M.L. (Ed). Handbook of food analysis – residues and other food component analysis, Marcel Dekker, Inc.: New York. p. 1147–69. Botana, L.M., Alfonso, A., Botana, A. et al. (2009) Functional assays for marine toxins as an alternative, high-throughput screening solution to animal tests. Trends in Analytical Chemistry, 28 (5), 603–11. Botana, L.M., Vilariño, N., Alfonso, A. et al. (2010) The problem of toxicity equivalent factors in developing alternative methods to animal bioassays for marine-toxin detection. Trends in Analytical Chemistry, 29 (11), 1316–25. Bourne, Y., Radic, Z., Aráoz, R. et al. (2010) Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism. Proceedings of the National Academy of Sciences of the United States of America, 107 (13), 6076–6081. Brescianini, C., Grillo, C., Melchiorre, N. et al. (2006). Ostreopsis ovata algal blooms affecting human health in Genova, Italy, 2005 and 2006. Euro surveillance 11, E060907.3. Bricelj, M. and Shumway, S.E. (1998) Paralytic shellfish toxins in bivalve molluscs: occurrence, transfer kinetics, and biotransformation. Reviews in Fisheries Science, 6 (4), 315–83. Cáceres, S. (2012). Climate change and animal diseases: making the case for adaption. Cambridge University Press 13(2), 209–222. Camacho, F.G., Rodríguez, J.G., Mirón, A.S. et al. (2007) Biotechnological significance of toxic marine dinoflagellates. Biotechnology Advances, 25 (2), 176–94. Campbell, K., Vilariño, N., Botana, L.M. and Elliott, C.T. (2011) A European perspective on progress in moving away from the mouse bioassay for marine-toxin analysis. Trends in Analytical Chemistry, 30 (2), 239–53. Ciminiello, P., Dell’Aversano, C., Fattorusso, E. et al. (2006) The Genoa 2005 outbreak. Determination of putative palytoxin in Mediterranean Ostreopsis ovata by a new liquid chromatography tandem mass spectrometry method. Analytical Chemistry, 78 (17), 6153–6159. Ciminiello, P., Dell’Aversano, C., Dello Iacovo, E. et al. (2011) Palytoxin in seafood by liquid chromatography tandem mass spectrometry: investigation of extraction efficiency and matrix effect. Analytical and Bioanalytical Chemistry, 401 (3), 1043–50. Commission Regulation (EU) No 15/2011 of 10 January 2011 amending Regulation (EC) No 2074/2005 as regards recognised testing methods for detecting marine biotoxins in live bivalve molluscs. 2011. Official Journal of the European Union. Commission Regulation (EU) No 786/2013 of 16 August 2013 amending Annex III to Regulation (EC) No 853/2004 of the European Parliament and of the Council as regards the permitted limits of yessotoxins in live bivalve molluscs. 2013. Official Journal of the European Union. Croci, L., Cozzi, L., Suffredini, E. et al. (2006) Characterization of microalgae and associated bacteria collected from shellfish harvesting areas. Harmful Algae, 5, 266–274.

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Dickey, R.W. (2008). Ciguatera Toxins: Chemistry, Toxicology, and Detection. In: Botana, L.M. (Ed). Seafood and Freshwater Toxins, p. 479–500. New York. Dickey, R.W. and Plakas, S.M. (2009) Ciguatera: A public health perspective. Toxicon, 56, 123–136. Durando, P., Ansaldi, F., Oreste, P. et al. (2007) Ostreopsis ovata and human health: epidemiological and clinical features of respiratory syndrome outbreaks from a two-year syndromic surveillance, 2005–06, in north-west Italy. Eurosurveillance, 12 (23). EFSA Panel on Contaminants in the Food Chain (CONTAM) (2008a) Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European Commission on marine biotoxins in shellfish – azaspiracids. EFSA Journal, 723, 1–52. EFSA Panel on Contaminants in the Food Chain (CONTAM) (2008b) Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European Commission on marine biotoxins in shellfish – okadaic acid and analogues. EFSA Journal, 589, 1–62. EFSA Panel on Contaminants in the Food Chain (CONTAM) (2008c) Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European Commission on marine biotoxins in shellfish – yessotoxin group. EFSA Journal, 907, 1–62. EFSA Panel on Contaminants in the Food Chain (CONTAM) (2009a) Statement of the Panel on Contaminants in the Food Chain on a request from the European Commission on the Influence of processing on the levels of lipophilic marine biotoxins in bivalve molluscs. EFSA Journal, 1016, 1–10. EFSA Panel on Contaminants in the Food Chain (CONTAM) (2009b) Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European Commission on marine biotoxins in shellfish – domoic acid. EFSA Journal, 1181, 1–61. EFSA Panel on Contaminants in the Food Chain (CONTAM) (2009c) Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European Commission on Marine Biotoxins in Shellfish – Summary on regulated marine biotoxins. EFSA Journal, 1306, 1–23. EFSA Panel on Contaminants in the Food Chain (CONTAM) (2009d). Scientific Opinion on marine biotoxins in shellfish – Palytoxin group. EFSA Journal 1393, [38 pp]. EFSA Panel on Contaminants in the Food Chain (CONTAM) (2009e) Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European Commission on marine biotoxins in shellfish – pectenotoxin group. EFSA Journal, 1109, 1–47. EFSA Panel on Contaminants in the Food Chain (CONTAM) (2009f) Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European Commission on Marine Biotoxins in Shellfish – Saxitoxin Group. EFSA Journal, 1019, 1–76. EFSA Panel on Contaminants in the Food Chain (CONTAM) (2010a) Statement on further elaboration of the consumption figure of 400 g shellfish meat on the basis of new consumption data. EFSA Journal 8(8), [20 pp]. EFSA Panel on Contaminants in the Food Chain (CONTAM) (2010b) Scientific Opinion on marine biotoxins in shellfish – Emerging toxins: Brevetoxin group. EFSA Journal 1677, [29 pp]. EFSA Panel on Contaminants in the Food Chain (CONTAM) (2010c) Scientific Opinion on marine biotoxins in shellfish – Emerging toxins: Ciguatoxin group. EFSA Journal 1627, [38 pp]. EFSA Panel on Contaminants in the Food Chain (CONTAM) (2010d) Scientific Opinion on marine biotoxins in shellfish – Cyclic imines (spirolides, gymnodimines, pinnatoxins and pteriatoxins). EFSA Journal 1628, [39 pp]. Elgarch, A., Vale, P., Rifai, S. and Fassouane, A. (2008) Detection of diarrheic shellfish poisoning and azaspiracid toxins in Moroccan mussels: comparison of the LC-MS method with the commercial immunoassay kit. Marine Drugs, 6 (4), 587–94. European Union Reference Laboratory for Marine Biotoxins (EU-RL-MB) (2011) EU-harmonised standard operating procedure for determination of lipophilic marine biotoxins in molluscs by LC-MS/MS. Version 4 (EU-RL-MB). FAO/IOC/WHO (2011) Summary of the FAO/IOC/WHO Expert Consultation on biotoxins in bivalve molluscs, in Assessment and management of biotoxin risks in bivalve molluscs. FAO Fisheries and Aquaculture technical paper: Rome. p. 271–281.

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Fernandez-Ortega, J.F., Morales-de los Santos, J.M., Herrera-Gutiérrez, M.E. et al (2010) Seafood Intoxication by Tetrodotoxin: First Case in Europe. Journal of Emergency Medicine, 39 (5), 612–617. Ferranti, P., Fabbrocino, S., Nasi, A. et al. (2009) Liquid chromatography coupled to quadruple time-of-flight tandem mass spectrometry for microcystin analysis in freshwaters: method performances and characterisation of a novel variant of microcystin-RR. Rapid Communications in Mass Spectrometry, 23 (9), 1328–1336. Fleming, L.E., Kirkpatrick, B., Backer, L.C. et al. (2005) Initial evaluation of the effects of aerosolized Florida red tide toxins (brevetoxins) in persons with asthma. Environmental Health Perspectives, 113 (5), 650–7. Fonfría, E.S., Vilariño, N., Campbell, K. et al. (2007) Paralytic shellfish poisoning detection by surface plasmon resonance-based biosensors in shellfish matrixes. Analytical Chemistry, 79 (16), 6303–11. Fonfria, E.S., Vilariño, N., Molgó, J. et al. (2010) Detection of 13,19-didesmethyl C spirolide by fluorescence polarization using Torpedo electrocyte membranes. Analytical Biochemistry, 403 (1–2), 102–7. Fraga, M., Vilariño, N., Louzao, M.C. et al. (2013) Multidetection of paralytic, diarrheic, and amnesic shellfish toxins by an inhibition immunoassay using a microsphere-flow cytometry system. Analytical Chemistry, 85 (16), 7794–802. Gago-Martinez, A., Rodriguez-Vazquez, J.A., Thibault, P. and Quilliam, M.A. (1996) Simultaneous occurrence of diarrhetic and paralytic shellfish poisoning toxins in Spanish mussels in 1993. Natural Toxins, 4 (2), 72–9. Gerssen, A., Mulder, P., van Rhijn, H. and de Boer, J. (2008) Mass spectrometric analysis of the marine lipophilic biotoxins pectenotoxin-2 and okadaic acid by four different types of mass spectrometers. Journal of Mass Spectrometry, 43 (8), 1140–7. Gerssen, A., McElhinney, M.A., Mulder, P.P. et al. (2009) Solid phase extraction for removal of matrix effects in lipophilic marine toxin analysis by liquid chromatography-tandem mass spectrometry. l. Analytical and Bioanalytical Chemistry, 394 (4), 1213–26. Gerssen, A., Pol-Hofstad, I.E., Poelman, M. et al. (2010) Marine toxins: chemistry, toxicity, occurrence and detection, with special reference to the Dutch situation. Toxins (Basel), 2 (4), 878–904. Gerssen, A., Mulder, P.P. and de Boer, J. (2011) Screening of lipophilic marine toxins in shellfish and algae: development of a library using liquid chromatography coupled to orbitrap mass spectrometry. Analytica Chimica Acta, 685 (2), 176–85. Gribble, K., Keafer, B.A., Quilliam, M.A. et al. (2005) Distribution and toxicity of Alexandrium ostenfeldii (Dinophyceae) in the Gulf of Maine, USA. Deep-Sea Research II, 52, 2745–2763. Hallegraeff, G.M. (2004). Harmful algal blooms: a global overview. In: Hallegraeff, G.M. Anderson, D.M. and Cembella, A.D. (eds). Manual on Harmful Marine Microalgae, p. 25–49. UNESCO: Paris. Hallegraeff, G.M. (2010) Ocean climate change, phytoplankton community responses, and harmful algal blooms: a formidable predictive challenge. Journal of Phycology, 46, 220–35. Handy, S.M., Yakes, B.J., DeGrasse, J.A. et al. (2013) First report of the use of a saxitoxin-protein conjugate to develop a DNA aptamer to a small molecule toxin. Toxicon, 61, 30–7. Hess, P., Grune, B., Anderson, D.B. et al. (2006) Three Rs approaches in marine biotoxin testing. The report and recommendations of a joint ECVAM/DG SANCO Workshop (ECVAM Workshop 54). Alternatives to Laboratory Animals, 34 (2), 193–224. Hirama, M. (2005) Total synthesis of ciguatoxin CTX3C: a venture into the problems of ciguatera seafood poisoning. The Chemical Record, 5 (4), 240–50. Holland, P.T. (2008). Analysis of marine toxins-techniques, method validation, calibration standards, and screening methods. In: Botana, L.M. (Ed). Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection, p. 21–49. CRC Press (Taylor and Francis Group): Boca Raton, FL. https://www.fsai.ie/news_centre/food_alerts/SDLM_mussels_recall.html, 2014. ILAC-G9 (2005). Guidelines for the selection and use of reference materials. International vocabulary of metrology – basic and general concepts and associated terms (VIM). 2008, JCGM. ISO Guide 30 (1992). Terms and definitions used in connection with reference materials.

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ISO Guide 30 (2008).Terms and definitions used in connection with reference materials. Amendment 1 Revision of definitions for reference material and certified reference material. ISO Guide 31 (2000). Reference materials – Contents of certificates and labels. ISO Guide 32 (1997). Calibration in analytical chemistry and use of certified reference materials. ISO Guide 34 (2009). General requirements for the competence of reference material producers. ISO Guide 35 (2006). Reference materials – General and statistical principles for certification. Jauffrais, T., Marcaillou, C., Herrenknecht, C. et al. (2012) Azaspiracid accumulation, detoxification and biotransformation in blue mussels (Mytilus edulis) experimentally fed Azadinium spinosum. Toxicon, 60 (4), 582–95. Jayasena, S.D. (1999) Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clinical Chemistry, 45 (9), 1628–50. Kawatsu, K., Shibata, T. and Hamano, Y. (1999) Application of immunoaffinity chromatography for detection of tetrodotoxin from urine samples of poisoned patients. Toxicon, 37, 325–333. Krock, B., Tillmann, U., John, U. et al. (2009) Characterization of azaspiracids in plankton size-fractions and isolation of an azaspiracid-producing dinoflagellate from the North Sea. Harmful Algae, 8, 254–63. Lawrence, J.F., Menard, C. and Cleroux, C. (1995) Evaluation of prechromatographic oxidation for liquid chromatographic determination of paralytic shellfish poisons in shellfish. Journal of AOAC International, 78, 514–20. Lehane, L. and Lewis, R.J. (2000) Ciguatera: recent advances but the risk remains. International Journal of Food Microbiology, 61 (2–3), 91–125. Liu, C., Hofstra, N. and Franz, E. (2013) Impacts of climate change on the microbial safety of pre-harvest leafy green vegetables as indicated by Escherichia coli O157 and Salmonella spp. International Journal of Food Microbiology, 163 (2–3), 119–28. MacKinnon, S.L., Cembella, A.D., Quilliam, M.A. et al. (2004). The Characterization of Two New Spirolides Isolated from Danish Strains of the Toxigenic Dinoflagellate Alexandrium ostenfeldii. In: Steidinger, K.A., Landsberg, J.H., Tomas, C.R. et al., (eds). Harmful Algae 2002, p. 186–188. McCarron, P., Kilcoyne, J. and Hess, P. (2008) Effects of cooking and heat treatment on concentration and tissue distribution of okadaic acid and dinophysistoxin-2 in mussels (Mytilus edulis). Toxicon, 51 (6), 1081–9. McNamee, S.E., Elliott, C.T., Delahaut, P. et al. (2013) Multiplex biotoxin surface plasmon resonance method for marine biotoxins in algal and seawater samples. Environmental Science and Pollution Research International, 20 (10), 6794–807. Meisen, I., Distler, U., Müthing, J. et al. (2009) Direct coupling of high-performance thin-layer chromatography with UV spectroscopy and IR-MALDI orthogonal TOF MS for the analysis of cyanobacterial toxins. Analytical Chemistry, 81 (10), 3858–66. Miles, C.O., Wilkins, A.L., Munday, R. et al. (2004) Isolation of pectenotoxin-2 from Dinophysis acuta and its conversion to pectenotoxin-2 seco acid, and preliminary assessment of their acute toxicities. Toxicon, 43 (1), 1–9. Miles, C.O., Samdal, I.A., Aasen, J.A.G. et al. (2005) Evidence for numerous analogs of Yessotoxin in Protoceratium reticulatum. Harmful Algae, 4, 1075–1091. Moore, S.K., et al. (2008). Impacts of climate variability and future climate change on harmful algal blooms and human health. Environ Health 7 Suppl 2, S4. Munday, R. (2008). Toxicology of the pectenotoxins. In: Botana, L.M. (Ed). Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection, pp. 371–80. CRC Press (Taylor and Francis Group): Boca Raton, FL. Munday, R., Aune, T. and Rossini, G.P. (2008). Toxicology of Yessotoxins. In: Botana, L.M. (Ed). Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection, pp. 581–594. CRC Press (Taylor and Francis Group): Boca Raton, FL. Munday, R., Quilliam, M.A., LeBlanc, P. et al. (2012) Investigations into the toxicology of spirolides, a group of marine phycotoxins. Toxins (Basel), 4 (1), 1–14.

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Noguchi, T. and Arakawa, O. (2008) Tetrodotoxin-distribution and accumulation in aquatic organisms, and cases of human intoxication. Marine Drugs, 6 (2), 220–242. Oshima, Y. (1995) Postcolumn derivatization liquid chromatographic method for paralytic shellfish toxins. Journal of AOAC International, 78 (2), 528–32. Otero, P., Pérez, S., Alfonso, A. et al. (2010) First toxin profile of ciguateric fish in Madeira Arquipelago (Europe). Analytical Chemistry, 82 (14), 6032–9. Otero, P., Alfonso, A., Alfonso, C. et al. (2011a) Effect of uncontrolled factors in a validated liquid chromatography-tandem mass spectrometry method question its use as a reference method for marine toxins: major causes for concern. Analytical Chemistry, 83 (15), 5903–11. Otero, P., Alfonso, A., Alfonso, C. et al. (2011b) First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples. Analytica Chimica Acta, 701 (2), 200–8. Otero, P., Alfonso, A., Rodríguez, P. et al. (2012) Pharmacokinetic and toxicological data of spirolides after oral and intraperitoneal administration. Food and Chemical Toxicology, 50 (2), 232–7. Paredes, I., Rietjens, I.M., Vieites, J.M. and Cabado, A.G. (2011) Update of risk assessments of main marine biotoxins in the European Union. Toxicon, 58 (4), 336–54. Parsons, M.L., Aligizaki, K., Bottein, M.Y.D. et al. (2012) Gambierdiscus and Ostreopsis: Reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology. Harmful Algae, 14, 107–129. Pazos, M.J., Alfonso, A., Vieytes, M.R. et al. (2004) Resonant mirror biosensor detection method based on yessotoxin-phosphodiesterase interactions. Analytical Biochemistry, 335 (1), 112–8. Penna, A., Vila, M., Fraga, S. et al. (2005). Characterization of Ostreopsis and Coolia (Dinophyceae) isolates in the Western Mediterranean Sea based on morphology, toxicity and internal transcribed spacer 5.8S rDNA sequences. Journal of Phycology 2005. 41, 212–225. Penna, A., Fraga, S., Battocchi, C. et al. (2010) A phylogeography study of the toxic benthic genus Ostreopsis Schmidt. Journal of Biogeography, 37, 830–841. Perez-Arellano, J.L., Luzardo, O.P., Pérez Brito, A. et al. (2005) Ciguatera fish poisoning, Canary Islands. Emerging Infectious Diseases, 11 (12), 1981–2. Quilliam, M.A. (2003). Chemical methods for lipophilic shellfish toxins. In: Hallegraeff, G.M. Anderson, D.M. and Cembella, A.D. (eds). Manual on harmful marine microalgae, pp. 211–45. UNESCO: Paris. Ramos, V. and Vasconcelos, V. (2010) Palytoxin and analogs: biological and ecological effects. Marine Drugs, 8 (7), 2021–2037. Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for food of animal origin. (2004). Official Journal of the European Union. Rodriguez, P., Alfonso, A., Vale, C. et al. (2008) First toxicity report of tetrodotoxin and 5,6,11-trideoxyTTX in the trumpet shell Charonia lampas lampas in Europe. Analytical Chemistry, 80 (14), 5622–9. Rossignoli, A.E., Fernández, D., Regueiro, J. et al. (2011) Esterification of okadaic acid in the mussel Mytilus galloprovincialis. Toxicon, 57 (5), 712–20. Rundberget, T., Aasen, J.A., Selwood, A.I. and Miles, C.O. (2011) Pinnatoxins and spirolides in Norwegian blue mussels and seawater. Toxicon, 58 (8), 700–11. Sarmento, H., Montoya, J.M., Vázquez-Domínguez, E. et al. (2010) Warming effects on marine microbial food web processes: how far can we go when it comes to predictions? Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 365 (1549), 2137–49. Satake, M., Ishibashi, Y., Legrand, A. et al. (1997) Isolation and structure of ciguatoxin-4A, a new ciguatoxin precursor, from cultures of dinoflagellate Gambierdiscus toxicus and parrotfish Scarus gibbus. Bioscience, Biotechnology, and Biochemistry, 60, 2103–2105. Satake, M., Fukui, M., Legrand, A.-M. et al. (1998) Isolation and structures of new ciguatoxin analogs, 2,3-dihydroxyCTX3C and 51-hydroxyCTX3C, accumulated in tropical reef fish. Tetrahedron Letters, 39, 1197–1198.

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Satake, M., Murata, M. and Yasumoto, T. (1993) The structure of CTX3C, a ciguatoxin congener Isolated from cultured gambierdiscus toxicus. Tetrahedron Letters, 34, 1975–1978. Silva, M., Azevedo, J., Rodriguez, P. et al. (2012) New gastropod vectors and tetrodotoxin potential expansion in temperate waters of the Atlantic Ocean. Marine Drugs, 10 (4), 712–26. Suzuki, T., Igarashi, T., Ichimi, K. et al. (2005) Kinetics of diarrhetic shellfish poisoning toxins, okadaic acid, dinophysistoxin-1, pectenotoxin-6 and yessotoxin in scallops Patinopecten yessoensis. Fisheries Science, 71 (4), 948–55. Taylor, F.J.R. (1979). A Description of the Benthic Dinoflagellate Associated with Maitotoxin and Ciguatoxin, Including Observations on Hawaiian Material. In: Taylor, D.L. and Seliger, H.H. (Eds). Toxic Dinoflagellate Blooms, pp. 71–77. Elsevier North-Holland: New York. EURACHEM. (1998). The fitness for purpose of analytical methods. A laboratory guide to method validation and related topics. These, A., Klemm, C., Nausch, I. and Uhlig, S. (2011) Results of a European interlaboratory method validation study for the quantitative determination of lipophilic marine biotoxins in raw and cooked shellfish based on high-performance liquid chromatography-tandem mass spectrometry. Part I: collaborative study. Analytical and Bioanalytical Chemistry, 399 (3), 1245–56. Thompson, M. and Wood, R. (1995). Harmonized guidelines for internal quality control in analytical chemistry laboratories (Technical report). Resulting from the Symposium on Harmonization of Internal Quality assurance Systems for Analytical Laboratories, Washington DC, USA, 22–23 July 1993. Pure and Applied Chemistry 67(4), 649–66. Tillmann, U., Elbrächter, M., Krock, B. et al. (2009) Azadinium spinosum gen. et sp. nov. (Dinophyceae) identified as a primary producer of azaspiracid toxins. European Journal of Phycology, 44, 63–79. Tubaro, A., Sosa, S., Carbonatto, M. et al. (2003) Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice. Toxicon, 41 (7), 783–92. Vale, P., Bire, R. and Hess, P. (2008) Confirmation by LC-MS/MS of azaspiracids in shellfish from the Portuguese north-western coast. Toxicon, 51 (8), 1449–56. van der Fels-Klerx, H.J., Olesen, J.E., Naustvoll, L.-J. et al. (2012) Climate change impacts on natural toxins in food production systems, exemplified by deoxynivalenol in wheat and diarrhetic shellfish toxins. Food Additives & Contaminants. Part A, Chemistry, Analysis, Control, Exposure & Risk Assessment, 29 (10), 1647–59. Van Dolah, F.M. (2000) Marine algal toxins: origins, health effects, and their increased occurrence. Environmental Health Perspectives, 108 (Suppl. 1), 133–41. Vieytes, M.R., Fontal, O.I., Leira, F. et al. (1997) A fluorescent microplate assay for diarrheic shellfish toxins. Analytical Biochemistry, 248 (2), 258–64.

CHAPTER 2

Pharmacology of ciguatoxins Carmen Vale1 , Álvaro Antelo2 & Víctor Martín1 1 Department 2 Cifga

of Pharmacology, University of Santiago de Compostela, Spain Laboratory, Campus Universitario s/n, Spain

Chemical structure of ciguatoxins Ciguatoxins (CTX) originally derive from precursors found in the dinoflagellate Gambierdiscus toxicus, which produces lipid-soluble CTX and water-soluble maitotoxins (MTX). While all strains produce maitotoxins, only some produce ciguatoxins (Holmes et al., 1991). These dinoflagellates produce less polar and less potent toxins, formerly known as gambiertoxins (Holmes et al., 1991), that are biotransformed in the liver of fish by oxidative metabolism and spiroisomerization, becoming increasingly polar and toxic compounds called ciguatoxins (Nicholson & Lewis, 2006; Yasumoto & Murata, 1993). Ciguatoxins are a family of heat-stable, highly oxygenated, lipid-soluble cyclic polyethers, with molecular weights ranging from ≈1000–1150 Da (Hamilton et al., 2002a, 2002b; Lewis, 1992; Pottier et al., 2003). The most notable feature, common to all CTX, is the long semi-rigid backbone of about 3 nm length, so-called huge ladder-shaped polyether (LSP), composed of a highly oxygenated long chain in which most of the oxygen atoms appear in cyclic oxoether linkages (Figure 2.1). The number of rings varies between 13 and 14, ranging from five- to nine-membered, forming a rigid structure (Yasumoto, 2001). They have structures that are similar to brevetoxins (PbTx), another family of lipid-soluble polyether toxins, produced by the marine dinoflagellate Karenia brevis (formerly Gymnodinium breve and Ptychodiscus brevis (Lee et al., 1986; Shimizu et al., 1986)). Ciguatoxins have been found to be relatively inert and heat stable molecules that remain toxic after cooking (Lewis, 2006). The heat stability property of ciguatera toxins is used to improve purification by accelerated solvent extraction (ASE) system (Wu et al., 2011). Ciguatoxins are soluble in organic solvents such as chloroform, diethyl ether, methanol, ethanol, 2-propanol or acetone. Several different CTX have been isolated from biodetritus containing wild Gambierdiscus toxicus, from toxic strains of cultured dinoflagellates isolated from different parts of the world (Holmes et al., 1991; Satake et al., 1996) or from various ciguateric fish (Chanteau et al., 1976; Lewis & Endean, 1983; Lewis & Jones, 1997; Lewis & Sellin, 1993; Scheuer et al., 1967; Vernoux & Lewis, 1997). While CTX from different geographical areas are similar in structure, differences have been determined in the symptoms caused by these toxins in humans, so a letter code prefix has been proposed to indicate CTX homologues isolated from the Pacific Ocean (P-CTX), Indian Ocean (I-CTX) and Caribbean Sea (C-CTX), with several congeners in each group

Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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24

H

HO

H

O 11

5 1 HO

A OH

H

H OH

32 H

H H O D

H O

E O H H

O H 17

O G

F

O H H

39 H O H OH I 41 H K O O O H H H O 51 M H H OH

H

P-CTX-1B (P-CTX-1, CTX1)

OH

7.3 Å

7.7 Å

31.6 Å

H

O

OH HO

H

H OH

H H O

O

H 32 H O O H H

O

H

H

HO

HO OH

O H H

O H H

HO H

OH H O O H

O

OH

P-CTX-2A

H

H H H O

H OH

O

H H O

H

H O O H H

O H H

OH H O H

O O

H

52 S

P-CTX-4B P-CTX-4A

HO H H O

1

52 R

O H H

OH

H

OH H O O

H On

H O H H OH O H H O H OH

E

F

G

H

H O OH H

H

O

H

OH H O H

OH H O O 49

HO

O H H

P-CTX-3C

51-hydroxy P-CTX-3C

H H O O O 49

HO 52

H H O D

A H 1 O H

H O

H

O H

K O H G H H O HO O H H H H F E O H H

O H

OH O H O N

H O

OH 66

56 R

HO

C-CTX-1

OH H O HO 1 2 OH HO

2,3-dihydroxy P-CTX-3C

O OH OH 49 M

H N O OH 66

56 S

HO

M-seco P-CTX-3C

C-CTX-2

Figure 2.1

2D structure model of some ciguatoxins. P-CTX-1B is the most identifiable structure within the whole ciguatoxin family. Besides the P-CTX-1B 2D model, MLP surface is depicted in order to point out lipophilicity. This surface was created using a colour ramp (gradient) in which the red colour and the black colours are the boundaries of the more lipophilic and less lipophilic moiety, respectively. Structure size is shown in the ball and stick model. Other ciguatoxin structures are shown, and the backbone difference is highlighted. I-CTXs are not shown, due to their structure still not being solved.

(Caillaud et al., 2010). Despite structural differences in the carbon backbones and their varying oxidation levels, the ciguatoxin congeners share common FG-ring structures (Yasumoto et al., 2000). P-CTX-1 was one of the first CTX compounds identified. It is the principal and most toxic CTX isolated from ciguatoxic carnivorous fish in the Pacific, and is believed to be the main cause of ciguatera food poisoning (CFP). Examination of the chemical structure of CTX suggests that P-CTX-1, extracted from the moray eel, Gymnothorax javanicus, arises from acid-catalyzed spiroisomerization and oxidative modification in the liver of carnivorous fish, of P-CTX-4A produced by Gambierdiscus toxicus (Lewis & Holmes, 1993; Murata et al., 1990; Nicholson & Lewis, 2006; Yasumoto & Murata,

Pharmacology of ciguatoxins

25

1993). Using fragmentation patterns of the known structures of P-CTX-1 and CTX-4B as templates, approximately 29 pacific ciguatoxin congeners have been identified (Yasumoto, 2001). P-CTX-1 is a white solid that comprises 60 carbon, 86 hydrogen and 19 oxygen atoms, with five double bonds equivalents. P-CTX-1 has 13 ether rings, these being seven- or six-membered rings, except from ring F and ring I, which are nine-membered and eight-membered ether rings, respectively. Ring I bears a β-methyl at C39. All ring fusions have trans-orientation, with angular protons and axial orientation of Me-56. C33 and C52 are quaternary carbons and C39 bears a methyl group. Hydroxyls and methyls have 11-OH equatorial, and H-51 has diequatoral orientations of Me-59 and Me-60. The hydroxyl group at C-54 is a β-substituent. Moreover, the dihedral angle formed by H-32/C-32 and C-33/C56 is near 180∘ , and Me-58 1,3 has a diaxial-like interaction on the methyl only for the β-orientation (Murata et al., 1990). P-CTX-4A is a steroisomer of P-CTX-4B at C52 and generates both P-CTX-4B and P-CTX-2 that are also white amorphous solids (Satake et al., 1996). The former, P-CTX-4B, is identical to P-CTX-1 except from the two terminal parts. It is a precursor of P-CTX-1 and is less oxidized than this molecule. It has a trans-butadiene moiety at one terminus and lacks a hydroxyl group at C54 at the other end. C52 is a quaternary carbon and H-51 is a double quartet (Murata et al., 1990). These congeners present slight modifications of their lipophilicity; P-CTX-4A (nonanomeric configuration of the LM spiroketal) and -4B (anomeric configuration of the LM spiroketal) are less polar than P-CTX-1. Theoretical calculated LogP values from MM2 minimized structures of P-CTX1 and P-CTX-4B are 10.25 and 13.87, respectively (Pedretti et al., 2003). The mouse lethality of P-CTX-4A (2 μg/kg) was comparable with that of P-CTX-4B (4 μg/kg). The symptoms observed in mice were also indistinguishable from those caused by P-CTX-4B. Thus, the effect of epimerization of P-CTX-4B at C-52 on toxicity seems to be insignificant. However, the ease of epimerization of P-CTX-4B under acidic conditions or in chlorinated solvents should be taken into account during the purification of ciguatera toxins (Murata et al., 1990; Satake et al., 1996). P-CTX-2 differs from P-CTX-1 by 16 mass units and is one of the less oxygenated analogues which arise from a different precursor than P-CTX-1 (P-CTX-4A). The hydroxyl at C-54 is absent and it has one oxygen less than P-CTX-1. Apart from that, rings K, L, M differ in structure (stereochemical nature) from P-CTX-1 (Lewis et al., 1991). P-CTX-3 is also a white amorphous solid that differs from P-CTX-1 by six mass units, and it is a less oxygenated analogue. The hydroxyl at C-54 is absent and has one oxygen less that P-CTX-1. P-CTX-3 also undergoes rearrangement of the spiro rings in the presence of hydrochloric acid. MM2 calculations on spiro rings L/M indicate that the 49R configuration is more stable than the 49S configuration (Yasumoto et al., 2000). P-CTX-3 has been suggested to be an intermediate in the oxidation of a gambiertoxin to P-CTX-1 (Lewis et al., 1991). 2,3-dihydroxy P-CTX-3C is larger than P-CTX-3C by 2 hydroxyl groups. Configurations of C2-OH and C3-OH are β and α respectively. 51-hydroxy P-CTX-3C is a colourless amorphous solid and it is larger than CTX-3C by one oxygen atom. The stereostructures of rings L and M are identical to those of P-CTX-1 (Satake et al., 1998). M-seco analogues are precursors of P-CTX-3C, P-CTX-4B and P-CTX-2 where spiroketal moiety is open. Their primary hydroxyl groups should be useful for preparing antigens by conjugation with career protein (Yasumoto, 2001).

26

Chapter 2

Caribbean CTX-1 and 2, like the P-CTX-group, possess a flexible nine-membered ring. Like P-CTX-3C, C-CTX-1 does not possess the aliphatic side chain seen on ring A of P-CTX-1. However, CTX-1 has a longer continuous carbon backbone (57 vs. 55 carbons for P-CTX-1), one extra ring and a hemiketal in ring N, but no spiroketal as found in the P-CTX group. It also has a primary hydroxyl group. The minor analogue, C-CTX-2, is an epimer of C-CTX-1 at the C-56 acetal carbon, due to the propensity of hemiketals moieties to undergo interconversion in solution (Vernoux & Lewis, 1997). The mouse lethality of C-CTX-1 (3.6 μg∕kg) is lower than that of C-CTX-2 (1μg/kg) (Lewis et al., 1998; Vernoux & Lewis, 1997). In 2002, Pottier et al. identified 10 additional C-CTX analogues or isomers (Pottier et al., 2002). The toxins identified were C-CTX-2 (a C-CTX-1 epimer), three additional isomers of C-CTX-1 or-2, five ciguatoxin congeners (C-CTX-1127, C-CTX-1143 and its isomer C-CTX-1143a, and C-CTX-1157 and its isomer C-CTX-1157b). The isolation and initial characterization of Indian Ocean CTX (I-CTX) from reef fish was reported in 2002 (Hamilton et al., 2002b). Later, the authors clarified that instead of one I-CTX, four closely related Indian CTX-group toxins (I-CTX-1, I-CTX-2, I-CTX-3 and I-CTX-4) were actually identified; I-CTX-1 and I-CTX-2 being the major ones, and I-CTX-3 and I-CTX-4 being the minor I-CTX-group of ciguatera toxins (Hamilton et al., 2002a). Furthermore, it was found that the I-CTX-1 and I-CTX-2 have the same molecular mass as C-CTX-1, suggesting that these compounds have closely related chemical structures (Hamilton et al., 2002a). During the last decade, liquid chromatography and advances in mass spectrometry (LC/MS) techniques have allowed deeper investigation of the structural profiles of ciguatoxins in different strains of Gambierdiscus spp (Roeder et al., 2010). Using the fragmentation patterns of known structures as templates, most structures of congeners could be identified. Thanks to this, advances on the knowledge of the chemistry of the ciguatoxins were accompanied by studies on synthesis of the most important toxins. Total synthesis of CTX3C was achieved by the Hirama group in 2001, based on highly convergent and efficient strategy for the assembly of structural fragments (Hirama, 2005; Hirama et al., 2001). In the next section, the structure and function of the main cellular targets of ciguatoxins, the voltage-dependent sodium channels, are reviewed.

Voltage-gated sodium channels Although differences in the biological activity of ciguatoxins between group members have been described, ciguatoxins are known to bind to site V of sodium-dependent voltage channels, causing cell membrane depolarization and sodium entry into the cell, which increases depolarization and gives rise to spontaneous action potentials (Bidard et al., 1984). Furthermore, as a consequence of membrane depolarization, ciguatoxins increase neurotransmitter release in excitable cells (Molgo et al., 1990). Although, so far, few reports have compared the effects of the different ciguatoxins on voltage-dependent potassium channels, some ciguatoxins also inhibit potassium channels, an effect that will contribute to increase cell depolarization (Hidalgo et al., 2002; Schlumberger et al., 2010). In this section, the main characteristics of voltage-dependent sodium channels, as the pharmacological targets of ciguatoxins, are summarized. Voltage-dependent sodium channels are constituted by one α subunit of approximately 260 KDa, associated to one or more β subunits (β1, β2 and/or β3, β4) of

Pharmacology of ciguatoxins

27

33–36 KDa. So far, nine different α subunits (Nav1.1 to Nav1.9) have been functionally characterized, and a tenth isoform (Nax) is also believed to constitute functional sodium channels (Yu & Catterall, 2003). The primary structure of the channel indicates that each α subunit consists of four similar domains (I–IV), each containing six transmembrane segments (S1–S6). In each domain, the S4 segment contains the voltage sensor with positively charged amino acid residues every third position. These residues move across the membrane to initiate channel activation in response to depolarization of the membrane. A re-entrant transmembrane loop between helices S5 and S6 forms the ion-selective filter located at the extracellular end of the pore. The intracellular end of the pore is formed by the four S6 segments. Small extracellular loops connect the transmembrane segments, and several intracellular loops link the four homologous domains. Large amino-terminal and carboxy-terminal tail domains also contribute to the internal face of the sodium channel. The short intracellular loop connecting homologous domains III and IV serves as the inactivation gate, folding into the channel structure and blocking the pore from the inside during sustained depolarization of the membrane (Yu & Catterall, 2003). Sodium channels in the adult central nervous system and the heart contain β1 through β4 subunits, whereas sodium channels in adult skeletal muscle have only the β1 subunit. The pore-forming α subunit is sufficient for functional expression, but the kinetics and voltage dependence of channel gating are modified by the β subunits, and these auxiliary subunits are involved in channel localization and interaction with cell adhesion molecules, extracellular matrix, and intracellular cytoskeleton (Yu & Catterall, 2003). The transmembrane structure of voltage-dependent sodium channels is indicated in Figure 2.2 (Yu & Catterall, 2003). Sodium channel inactivation appears to be associated with the intracellular loop connecting domains III and IV. The mechanism proposed for sodium channel inactivation is the hinged-lid mechanism, in which the intracellular loop connecting domains III and IV of the sodium channel forms a hinged lid with a critical residue of phenylalanine (F1489) within a key hydrophobic sequence motif, IFM motif (Ile-Phe-Met), that occludes the mouth of the pore during the inactivation process (Yu & Catterall, 2003). Among voltage-gated sodium channels, NaV 1.1, NaV 1.2, NaV 1.3, and NaV 1.7 are the most closely related group. They are broadly expressed in neurons and show high tetrodotoxin sensitivity. Their genes are all located on human chromosome 2. The tetrodotoxin resistant sodium channels NaV 1.5, NaV 1.8, and NaV 1.9 show a 64% amino acid sequence homology to the four sodium channels encoded on chromosome 2. These sodium channels are tetrodotoxin-resistant to varying degrees, due to changes in amino acid sequence at a single position in domain I. They are highly expressed in heart and dorsal root ganglion neurons, and their genes are located on human chromosome 3 (Catterall et al., 2007). The isoform NaV 1.4 is expressed primarily in skeletal muscle, while the isoform NaV 1.6 is expressed predominantly in the central nervous system. Due to their distant phylogenetic relationship, both of these isoforms are set apart from the other two closely related groups of sodium channel genes, although their amino acid sequences are more than 84% identical to the group of sodium channels whose genes are located on chromosome 2. The main characteristics of human sodium channels summarized from the IUPHAR/BPS Guide to Pharmacology are summarized in Tables 2.1 and 2.2 (Catterall et al., 2013).

Chapter 2

28

β2

α NH3+

II

III

+H3N

IV

ΨΨ

I

β1

Out

+ 12345 +

In

-

+ 12345 +

6

-

6

+ 12345 +

-

6 h

–O2C

Voltage sensing

+ 12345 + P

6

CO2–

Pore Inactivation

P

+H3N

CTX binding site P P

CO2–

P P

Modulation Figure 2.2

Transmembrane structure of voltage-gated sodium channels. Schematic representation of the sodium-channel subunits, as illustrated by Yu and Catterall (2003). The α subunit of the Nav 1.2 channel is illustrated, together with the β1 and β2 subunits; the extracellular domains of the β subunits are shown as immunoglobulin-like folds, which interact with the loops in the α subunits as shown. Roman numerals indicate the domains of the α subunit; segments 5 and 6 (shown in green) are the pore-lining segments and the S4 helices (yellow) make up the voltage sensors. The proposed neurotoxin site 5, the CTX binding site, is marked by the red arrows. Source: Yu and Catterall 2003.

All of the agents acting on voltage-dependent sodium channels bind to specific sites located in the alpha subunit. The receptor sites at which neurotoxins affect channel gating are allosterically coupled, suggesting that conformational changes induced by neurotoxin binding alter the equilibrium between the open and the closed/inactivated states and also alter the conformation and toxin binding affinity at other neurotoxin receptor sites (Catterall et al., 2007; Cestele & Catterall, 2000). Until now, six different binding sites for drugs acting on sodium channels (neurotoxins and local anaesthetics) have been identified. Non-peptidic blockers of sodium channels such as saxitoxin (binds to domains IIIS2-S6, IVS2-S6) and tetrodotoxin (binds at domains IS2-S6, IIS2-S6), as well as the peptide blocker μ-conotoxin, act on neurotoxin receptor site I of the sodium channel. All of these toxins act from the extracellular side of the membrane and, by binding to receptor site I, these toxins block sodium conductance (Cestele & Catterall, 2000). This receptor site is formed by two rings of amino acid residues localized in segment SS2 on the N-terminal side of the S6 transmembrane segment in each of the four domains of sodium channels. The first ring of amino acids involved in the formation of the tetrodotoxin/saxitoxin binding site is formed by the acidic residues glutamic

2q22–23

2q23–24

17q23–25

3p21

12q13

2q24

3p22–21

3p24–21

hNav1.2 2005

hNav1.3 2000

hNav1.4 1836

hNav1.5 2016

hNav1.6 1980

hNav1.7 1977

hNav1.8 1956

hNav1.9 1791

β1, β2

β1, β2, β3, β4

β1, β2, β3, β4

β1

β1, β2

β1, β2

β1, β2

SCN11A

SCN10A β1, β2, β3

SCN9A

SCN8A

SCN5A

SCN4A

SCN3A

SCN2A

SCN1A

−32

−15.7

−26

25.9 (Cs aspartate) −44 (CsF) −28.7

−33

−24

−19.6 −13.7 −33 (CsF) −7.5 (CsAspartate) −24 (CsF)

Chromosome Gene Auxiliary V0.5 symbol subunits activation (mV)

2q24.3

AA

−31

−30.9

−67

60.4 (Cs aspartate) −87 (CsF) −71.9

−76

−69.9

−41 −37 −72 (CsF) −37,6 (Cs Aspartate) −63 (CsF)

V0.5 inactivation (mV)

Veratridine, Batrachotoxin

Grayanatoxin, veratridine, batrachotoxin, β-scorpion toxin TiTXγ Aconitine, Batrachotoxin, veratridine β-scorpion toxin Cn2, Batrachotoxin, veratridine Batrachotoxin, veratridine

β-scorpion toxin Css IV, batrachotoxin, aconitine, veratridine Batrachotoxin, veratridine

Batrachotoxin Veratridine

Activators

ATX-II, AFT-II, Bc-III N-Me-aminopyrimidinone 9

α-scorpion toxin, ATX-II, AFT-II, Bc-III

AFT-II Bc-III ATX-II AFT-II Bc-III ATX-II

α-scorpion toxin, ATX-II, Bc-III, AFT-II

ATX-II Bc-III AFT-II

Gating inhibitors

XEN 907, Tetrodotoxin, Pyrrolopyrimidine 48, lidocaine, Cd2+ A-803467, tetrodotoxin (IC50 63μM), lidocaine Tetrodotoxin (IC50 39 μM)

Saxitoxin, tetrodotoxin, μ-conotoxin GIIIA, μ-conotoxin PIIIA, mexiletine, lidocaine Saxitoxin, tetrodotoxin, lidocaine, amiodarone, quinidine Tetrodotoxin, 4,9-anhydrotetrodotoxin

Tetrodotoxin, saxitoxin, etidocaine, lidocaine, phenytoin, lamotrigine Tetrodotoxin

Tetrodotoxin

Pore blockers

Human sodium channel subunits and their electrophysiological and pharmacological properties. Based on data from Catterall et al. 2013.

hNav1.1 1998

Table 2.1

Pharmacology of ciguatoxins 29

Brain, unmyelinated axons and developing premyelinated axons, neuronal cell bodies and dendrites Spinal cord (unmyelinated axons) and motor neurons Spinal cord, thalamus, amygdala, cerebellum, adult and foetal whole brain and heart Skeletal muscle Hearth

hNav1.2

Myenteric plexus neurons, C-type dorsal root ganglion neurons, trigeminal neurons

hNav1.9

hNav1.8

hNav1.7

Peripheral nervous system: dorsal root ganglia, nodes of Ranvier of sensory and motor neurons Central nervous system: cerebellum, cerebral cortex, hippocampus, cerebellar Purkinje cells, brainstem, astrocytes (somatodendritic distribution) Dorsal root ganglion neurons, sympathetic neurons, Schwann cells and neuroendocrine cells. Nociceptors and myelinated A-fibres, small and medium diameter dorsal root ganglion neurons

hNav1.6

hNav1.4 hNav1.5

hNav1.3

Spinal neurons primarily cell bodies, brain neurons, cardiac myocytes (sinoatrial node, atrial myocytes and transverse tubules of ventricular myocytes)

Tissue distribution

Neuropathic pain, heat pain after burning injuries Depolarizing phase of action potential in dorsal root ganglion neurons supports repetitive firing in depolarized neurons Contributes a depolarizing influence at resting potential and amplifies and prolongs slow sub-threshold

Conduction of action potentials, sodium persistent current

Rapid recovery from inactivation supporting repetitive firing Action potential initiation and propagation Action potential generation and conduction

Action potential conduction in transverse tubule of ventricular myocytes. Action potential initiation and repetitive firing in central neurons Conduction of action potentials

Physiological function

Mutations lead to an insensitivity to pain (without peripheral nerve pathology). Small fibre neuropathy, upregulated within cerebellar Purkinje neurons in multiple sclerosis, functional dyspepsia Inflammatory pain

Neuropathic pain is ameliorated by shRNA-knock down, epilepsy Potassium aggravated myotonia Atrial fibrillation familial 10, Brugada syndrome, long QT syndrome type 3, Sick sinus syndrome 1, Lenègre’s Disease, Dilated cardiomyopathy, susceptibility to ventricular fibrillation during myocardial infarction Cerebellar ataxia, Motor Endplate disease, Severe Epileptic Encephalopathy

Benign familial neonatal infantile seizures

Generalized epilepsy with febrile seizures

Pathophysiology and clinical relevant mutations

Tissue distribution, physiological functions and pathologies related to human sodium channels. Based on data from Catterall et al. 2013.

hNav1.1

Table 2.2

30

Chapter 2

Pharmacology of ciguatoxins

31

acid 387 in domain I, glutamic acid 945 in domain II, aspartic acid 1426 in domain III and aspartic acid 1717 in domain IV. The second ring is formed by the acidic residues aspartic acid 384 and glutamic acid 942 in domains I and II, respectively, by the basic residue lysine 1422 in domain III and the neutral residue alanine 1714 in domain IV (Cestele & Catterall, 2000). These amino acid residues are located in the pore loop and are supposed to form the ion selectivity filter (Catterall et al., 2007). The lipid-soluble toxins grayanotoxins, the alkaloids veratridine, aconitine and batrachotoxin, bind to receptor site 2 of the sodium channel. These toxins bind preferentially to the activated state of the sodium channel, causing persistent activation of the channel at resting membrane potentials by allosteric mechanisms that shift the activation of sodium channels to more negative potentials and block their inactivation. Photolabelling and mutagenesis experiments have shown the involvement of the S6 transmembrane region of domain I and domain IV in the formation of receptor site 2 (Cestele & Catterall, 2000). Neurotoxin site 3 of the voltage-dependent sodium channels is the site of action of several polypeptide toxins, which include α-scorpion toxins, sea-anemone toxins and some spider toxins. The site of action of these toxins has been located in the extracellular loop connecting the S3 and S4 segments in domain IV. Since the S4 segments of the sodium channel serve as voltage sensors, moving outward when the membrane is depolarized, toxins acting on site 3 slow sodium channel inactivation, preventing the outward movement involved in fast inactivation. Neurotoxin site 4 of voltage-gated sodium channels is the site of action of β-scorpion toxins. This site is composed by specific amino acid residues in the extracellular loops connecting the S1-S2 and S3-S4 segments in domain II. Binding of toxins to site 4 of sodium channels causes a depolarizing shift in sodium channel activation and a decrease in peak sodium current amplitude. Site 5 of voltage-dependent sodium channels is assumed to be the site of action of two lipid-soluble phycotoxins: brevetoxins and ciguatoxins. These toxins enhance sodium channel activity by activating sodium channels at negative potentials and blocking channel inactivation. Neurotoxin site 5 includes the transmembrane segments IS6 and IVS5, as determined by photoaffinity labelling experiments (Trainer et al., 1994; Trainer et al., 1991). Receptor site 6 is the site of action for δ-conotoxins, which slow channel inactivation through a mechanism though to be similar to that of the α-scorpion toxins. Site 6 comprises amino acid residues in the IVS4 segment, near receptor site 3 (Leipold et al., 2005).

Neurological symptoms of ciguatera Although the neurological symptoms of ciguatera were initially believed to be the direct consequence of the interaction of CTX with voltage-gated sodium channels (Lewis, 2001), the exact mechanisms for the long-term neurological secuela of these compounds have not yet been identified. In humans, the onset of ciguatera usually

32

Chapter 2

manifests with gastrointestinal problems such as nausea, vomiting, diarrhoea and abdominal pain within 12 hours of eating a toxic fish. Moreover, a combination of bradycardia and hypotension may be present during this initial period. From a few hours to two weeks after eating a toxic fish, subjective neurological complaints may develop. Among the neurological alterations, paresthesias (numbness and tingling of perioral region and extremities) and disturbance of temperature sensation are considered pathognomic symptoms of ciguatera food poisoning (Dickey, 2008). Some of the neurological alterations produced by ciguatoxin, such as temperature reversal (paradoxical dysaesthesia), intense pruritus and increased nociception, as a result of small-fibre peripheral neuropathy, may last for long periods of time, and the patient may experience neurological alterations for months or even years after the toxic fish ingestion. Other subjective symptoms of ciguatera are metallic taste, arthralgia, myalgias (mainly in the legs) and sensations of loose teeth. Tremors and headache may also be present. While alterations in the cranial and peripheral nerve fibres appear to be responsible for the majority of the neurological symptoms, some authors indicate that the paralysis, ataxia, stupor and confusion that appear in the most severe cases are indicative of central nervous system disturbances (Dickey & Plakas, 2010). Although these effects may be explained by the long persistence of these toxins in the body, it has also been pointed out that the related polycyclic ether toxins brevetoxins activate the thermosensitive transient receptor potential cation channel subfamily V member 1 (TRPV1), and it is suggested that this effect can mediate the pain sensation produced by these toxins (Cuypers et al., 2007). Increased age and weight have also been related to the long persistence of the neurological symptoms of ciguatera (Dickey & Plakas, 2010). In this sense, the toxicokinetic parameters of P-CTX-1 after oral, intraperitoneal and intravenous administration to rats have been described recently. In these studies, the authors found that, after oral and intraperitoneal administration, the ciguatoxin exhibited a rapid systemic absorption, reaching maximum concentrations in blood at 1.97 and 0.43 hours after the oral and intraperitoneal dosage, respectively. Moreover, the toxin suffered slow elimination from blood, which was estimated at 82 hours for oral and 112 hours for intraperitoneal administration. Ciguatoxin levels remained detectable in liver, muscle and brain even 96 hours after intraperitoneal and oral administration (Bottein et al., 2011). Further studies indicated that P-CTX-1 bioavailability was 39% after oral administration and 75% by intraperitoneal dosage (Ledreux & Ramsdell, 2013). Differences in ciguatera symptoms in the Pacific Ocean, Indian Ocean, and Caribbean Sea regions have been attributed to the different ciguatoxins congeners identified in each region. In the Caribbean, gastrointestinal symptoms and signs are characteristic in the acute phase, and are followed closely by peripheral neurologic alterations while, in the Pacific and Indian Ocean regions, the neurological symptoms and signs are more pronounced in the acute phase, with occasional reports of more severe neurologic effects, including coma. Onset of symptoms and signs typically begins within 0.5–12 hours of eating a toxic fish, and the acute phase often remits within 24 hours. As mentioned above, cardiovascular alterations, including

Pharmacology of ciguatoxins

33

bradycardia and hypotension, may appear during this acute period. In the Pacific and Indian Oceans, there are reports of rapid progression to respiratory distress, coma and, occasionally, death (Dickey & Plakas, 2010). Specific clinical treatments for ciguatera are absent, and the treatment for this disease is very limited and symptomatic in nature. Recently, the pathognomonic symptom of ciguatera, cold allodynia (characterized by intense stabbing and burning pain in response to mild cooling) was attributed to a TRPA1-dependent calcium influx activated after sodium channel activation by P-CTX-1 (Vetter et al., 2012). However, the limited efficacy of sodium channel inhibitors to reverse ciguatoxin-induced cold allodynia in vivo led to the suggestion that this ciguatera symptom could be more complex than sodium channel activation, especially since other sodium channel activators, such as veratridine and batrachotoxin, do not induce cold allodynia (Zimmermann et al., 2013). The toxicological database for the CTX-group of toxins is limited and comprises mostly studies on their acute toxicity following intraperitoneal administration. Based on the existing studies, the Food Contaminant panel of the European Food Safety Authority (EFSA) established the following toxicity equivalency factors (TEFs) for the CTX-group of toxins based on their acute intraperitoneal lethal dose 50 (LD50 ) in mice: P-CTX-1 = 1; P-CTX-2 = 0.3; P-CTX-3 = 0.3; P-CTX-3C = 0.2; 2,3-dihydroxy P-CTX-3C = 0.1; 51-hydroxy P-CTX-3C = 1; P-CTX-4A = 0.1; P-CTX-4B = 0.05; C CTX-1 = 0.1; and C-CTX-2 = 0.3 (EFSA, 2010).

Physiological effects of ciguatoxin Notwithstanding the small quantities of pure ciguatoxins available, the physiological effects of several CTX have been evaluated in a wide variety of preparations. The different ciguatoxin analogues employed in each study, and the variability in the experimental models, make it difficult to establish a common mechanism that could explain the long-lasting neurological effects of ciguatera food poisoning in humans. In this section, we provide a review of the different effects of ciguatoxins in several experimental models. Pharmacological studies have revealed that Pacific CTX activates voltage-sensitive sodium channels at low nanomolar concentrations, causing cell depolarization, spontaneous nerve firing, elevation of the intracellular free Ca2+ concentration, and oedema of Schwann cell and axons present on excitable membranes (Benoit et al., 1996; Lewis, 2000; Molgo et al., 1993a). Research on other ciguatoxins is less extensive, mainly due to difficulties in obtaining purified toxin and, in consequence, there is scarce information on the biological activity of different ciguatoxins in the same cellular model. Thus, P-CTX-1 had no effect on the kinetics of sodium currents, but it decreased the peak current amplitude in mammalian sensory neurons and cultured rat myotubes, causing a hyperpolarizing shift in the voltage-dependence of sodium channel activation (Hidalgo et al., 2002). Moreover, different potencies of P-CTX-1B and P-CTX-4B to affect sodium and potassium channels have also been recently reported on myelinated axons (Schlumberger et al., 2010).

34

Chapter 2

Although the scarcity of CTX has hampered quantitative evaluations of their effects on voltage-dependent sodium and potassium channels, the achievement of chemical synthesis of the ciguatoxin congener CTX-3C (Hirama et al., 2001; Inoue et al., 2002) has allowed detailed patch-clamp analysis of the effects of this toxin to be performed. Thus, CTX-3C markedly affected the operation of sodium channels in several experimental models such as mouse taste cells and cerebellar granule cells (Ghiaroni et al., 2006; Perez et al., 2011), as well as in differentiated human neuroblastoma cells. As shown in Figures 2.3(a) and 2.3(b), human neuroblastoma SH-SY5Y cells were differentiated in the presence of retinoic acid for seven days in culture and exposed to different concentrations of ciguatoxin CTX-3C added to the bath solution. In this cellular model, the toxin caused the activation of voltage-gated sodium channels at hyperpolarized potentials, and decreased peak sodium channel amplitude at the higher concentrations of toxin evaluated (10 and 20 nM), while it did not modify the amplitude of sodium currents (INa ) at the lowest concentrations employed (0.1 and 1 nM). In addition, as shown in Figures 2.4(a) and 2.4(b), even the lower concentrations of toxin employed (0.1 nM) affected sodium channel inactivation in this cellular model. However, the effect of CTX-3C on voltage-gated potassium channels appears to depend on the type of cells employed, since the toxin did not affect voltage-gated potassium currents in mouse taste cells (Ghiaroni et al., 2006) and mature cortical neurons (Martín et al., 2014), however it decreased potassium current amplitude in primary cultures of cerebellar granule cells (Perez et al., 2011). In differentiated human neuroblastoma SH-SY5Y cells, CTX-3C at 10 nM did not significantly affected voltage-gated potassium currents, as shown in Figure 3.5, probably indicating that the ciguatoxin effect depends on the channel subtype expressed in each system. Moreover, CTX-3C at much higher concentrations (1 μM) has been shown to activate tetrodotoxin-sensitive sodium channels (Nav1.2, Nav1.4 and Nav1.5) by accelerating their activation kinetics and shifting the activation curve towards hyperpolarization (Yamaoka et al., 2004). In contrast, the toxin at 100 nM preferentially affected the activation process of the tetrodotoxin-resistant Nav1.8 channel, compared with those of Nav1.2 and Nav1.4 (Yamaoka et al., 2004). As suggested by these authors, the high toxin concentration needed to affect voltage-gated sodium channels transiently expressed in HEK cells could be due to the absence of the sodium channel β subunit in this system. This suggestion is in agreement with the large effect of the same toxin in other experimental models, at low nanomolar doses, as shown previously by us in primary cultures of cerebellar granule cells (Perez et al., 2011) and human neuroblastoma cells (Figures 2.3 and 2.4). Besides the lack of ciguatoxin reference material, chemical identification of the ciguatoxin profile in a recent ciguatera case in Europe (Otero et al., 2010), has allowed us to evaluate the biological activity of the combination of six different ciguatoxins in different proportions, and to analyze in the same preparation the biological effects of synthetic CTX-3C, 51-OH CTX-3C and purified P-CTX-1B. In this study, six samples, extracted from two species of fish (Seriola dumerili and Seriola fasciata caught at Salvagen Islands, part of the Madeira Archipelago, Portugal) were analyzed (Perez et al., 2011). In this ciguatera outbreak, 51-OH-CTX-3C was the most abundant toxin in all samples, followed by C/I-CTX1/2 and CTX-4A/B. CTX-1B and CTX-3C were present at very low levels (Otero et al., 2010). The biological activity of different extracts containing this ciguatoxin combination in varying amounts, and its effect on voltage-gated sodium and potassium channels, was analyzed and compared with the biological activity of synthetic 51-OH CTX-3C,

Pharmacology of ciguatoxins

35

Vh (mV) –40 –30 –20 –10 0

10 20 30 40 50

–0.0

I/Imax

–0.2

Control 0.1 nM CTX-3C 1n M CTX-3C

–0.4 –0.6

10 nM CTX-3C

–0.8 –1.0

(a) Vh (mV) –40 –30 –20 –10 0

10 20 30 40 50

–0.0

I/Imax

–0.2 –0.4

Control 20 nM CTX-3C (5 min) 20 nM CTX-3C (10 min) 20 nM CTX-3C (15 min)

–0.6 –0.8 –1.0

(b) Figure 2.3 Effect of CTX 3C on the activation of voltage-gated sodium channels in human SH-SY5Y neuroblastoma cells. Voltage-dependent sodium currents were elicited in SH-SY5Y cells by applying a series of 25 ms depolarizing pulses (voltage steps) from –50 mV to 50 mV, in 5 mV increments, from a holding potential of –100 mV. (a): Current-voltage (I-V) relationship for the effect of different concentrations of CTX-3C on sodium currents in SH-SY5Y. (b): I-V relationship for the effect of 20 nM CTX-3C on sodium currents measured at 5, 10 and 15 minutes after bath application of the toxin. Note that the effect of 20 nM CTX3C was not dependent on the time, and that CTXC-3C decreased the peak sodium current (INa ) and shifted the activation threshold of INa towards a more negative value. Each point represents the mean ± SEM of 3–5 measurements. (See plate section for colour version.)

CTX-3C and purified P CTX-1B in cerebellar granule cells, using the perforated patch-clamp technique. In this system, the toxins affected both voltage-gated sodium and potassium channels, although with different potencies. CTX-3C was the most active toxin blocking the peak inward sodium currents, followed by P-CTX-1B and 51-OH-CTX-3C. In contrast, P-CTX-1B was more effective in blocking potassium currents. Moreover, the analysis of contaminated fish samples, in which a ciguatoxin analogue of mass 1040.6, not identical with the standard 51-OH CTX-3C, was the most prevalent compound, indicated an additive effect of the different ciguatoxins present in the samples (Perez et al., 2011). As mentioned above, the research on the mechanism of action of ciguatoxins has, until now, been hampered by the scarcity of toxin. After initial reports on the effect of ciguatoxins on sodium channels (Rayner, 1972; Rayner & Szekerczes, 1973;

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Prepulse mV –100 –90 –80 –70 –60 –50 –40 –30 –20 –10 0 0.00 –0.25 I/Imax

Control –0.50

0.1 nM CTX-3C 1n M CTX-3C

–0.75

10 nM CTX-3C

–1.00 (a) Prepulse mV –100 –90 –80 –70 –60 –50 –40 –30 –20 –10 0 0.00

I/Imax

–0.25 Control

–0.50

20 nM CTX-3C 5 min 20 nM CTX-3C 10 min

–0.75

20 nM CTX-3C 15 min –1.00 (b) Figure 2.4

Effect of CTX-3C on the voltage dependence of the steady state inactivation of voltage-gated sodium channels in human SH-SY5Y neuroblastoma cells. Steady-state inactivation was determined using a two-pulse protocol. A 500 ms conditioning pre-pulse from –100 mV to –10 mV was stepped in 10 mV increments and was followed by a 50 ms test pulse to –20 mV. (a): I-V relationship for the effect of different concentrations of CTX-3C on the steady-state inactivation of sodium currents in SH-SY5Y. Note that CTX-3C altered the steady-state inactivation at all concentrations tested. (b): I-V relationship for the effect of 20 nM CTX-3C on the steady-state inactivation of sodium currents in SH-SY5Y cells measured at 5, 10 and 15 minutes after bath application of the toxin. Note that the effect of 20 nM CTX-3C on steady-state inactivation was not dependent on the time. All currents were normalized to the maximum control current. Each point represents the mean ±SEM of 3–5 measurements. Curves were fitted by the Boltzman equation. (See plate section for colour version.)

Setliff et al., 1971), in 1982, Ohizumi et al. described that purified ciguatoxin induced supersensitivity in guinea-pig vas deferens, due to an increased Na+ permeability across the TTX sensitive Na+ channels of smooth muscle cell (Ohizumi et al., 1982). In 1984, it was described that partially purified ciguatoxin extracted from muscle of Gymnothorax javanicus at 0.1–10 ng/ml inhibited the net accumulation of gamma-aminobutyric acid (GABA) and dopamine by rat brain synaptosomes, and that this effect was completely inhibited by nanomolar concentrations of the sodium channel blocker tetrodotoxin, thus suggesting that the toxin activated voltage-dependent

Pharmacology of ciguatoxins

37

sodium channels (Bidard et al., 1984). The same report indicated that ciguatoxin induced a membrane depolarization on neuroblastoma cells, which was prevented by tetrodotoxin and due to an increase in Na+ permeability. Moreover, the toxin produced spontaneous oscillations in the membrane potential and repeated action potentials firing. However, it did not affect sodium uptake in neuroblastoma or muscle cells, but it increased the sodium uptake produced by veratridine, batrachotoxin and sea anemone toxins. This effect was attributed to the activation of voltage-dependent sodium channels, since it was blocked by tetrodotoxin (Bidard et al., 1984). At the same time, Lewis et al. described that ciguatoxin isolated from the Spanish mackerel, Scomberomorus commersoni, caused a sustained contraction of the guinea pig ileum and vas deferens which was blocked by atropine, tetrodotoxin and low Na+ solutions. This response was attributed to an increase in acetylcholine release from the nerve terminals produced by sodium dependent depolarization, which was followed by nerve blockade, suggesting a further depolarization produced by the toxin (Lewis & Endean, 1984). Further reports indicated that purified ciguatoxins induced spontaneous action potentials at the node of Ranvier of frog-isolated nerve fibres, and that this effect was reversible upon removal of the toxin. Moreover, the toxin in this preparation did not modify leakage and capacitative currents or potassium current, but reversibly induced a maintained late inward current that was dependent on membrane potential (Benoit et al., 1986). At the guinea-pig hearth, purified ciguatoxin induced positive inotropic responses in atrial and papillary muscles. Moreover, CTX induced a TTX-sensitive depolarization of atrial cells therefore indicating that its effect was mediated through the opening of voltage-dependent Na+ channels without affecting the channel inactivation process (Lewis & Endean, 1986). The effect of ciguatoxin on sodium channels was confirmed by further studies indicating that ciguatoxins and brevetoxins share the same binding site in the sodium channel, since both toxins increased batrachotoxin binding to brain membranes, and the binding of brevetoxin was blocked by ciguatoxin. However, it was not affected by other drugs acting on sodium channels, such as veratridine and tetrodotoxin (Lombet et al., 1987). At high doses, ciguatoxin induces arritmia and negative inotropic effects associated with cell depolarization in guinea-pig atria and these effects are also reverted by tetrodotoxin and lidocaine (Lewis, 1988). Two years later, it was demonstrated that ciguatoxin (1–2.5 nM) extracted for Gymnothorax javanicus moray-eel liver induced spontaneous fibrillations of the muscle fibres on frog-isolated neuromuscular preparations, which were suppressed by 1 μM tetrodotoxin (Molgo et al., 1990). Moreover, in the same experimental model, CTX (2.5 nM) increased the frequency of miniature endplate potentials – an effect that was also blocked by 1 μM tetrodotoxin, but not by calcium channel blockers, therefore indicating that ciguatoxin increased miniature end plate potential frequency through activation of voltage-dependent sodium channels that could lead to the release of calcium from internal stores and the consequent increase in neurotransmitter release. Thus, it had both pre- and postsynaptic effects at the neuromuscular junction. After the initial characterization of the structure of ciguatoxins present in the moray eel Gymnothorax javanicus (Murata et al., 1990), further studies identified the ciguatoxins present in moray eels (Lycodontis javanicus) as CTX-1 (MH + m∕z = 1111) and the less polar ciguatoxins, CTX-2 and CTX-3, and established the intraperitoneal toxicities of these toxins, which were 0.25, 2.3 and 0.9 micrograms/kg, for CTX-1, CTX-2 and CTX-3 respectively (Lewis et al., 1991). Although these ciguatoxins elicited similar signs of toxicity in mice, CTX-2 and CTX-3 induced hind-limb paralysis that was absent with

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CTX-1. However, the three ciguatoxins were active after oral administration, and they competitively inhibited the binding of [3 H]brevetoxin-3 to voltage-dependent sodium channels, indicating again that ciguatoxins and brevetoxins bind to the same site in voltage-dependent sodium channels. Further studies demonstrated that CTX-1B, extracted from moray eel at concentrations ranging from 0.22–1.12 nM, induced spontaneous action potentials at the frog node of Ranvier, suppressed by 50 μM lidocaine, which resulted from a toxin-induced late inward Na+ current (Benoit & Legrand, 1992). The determination of the activity of purified CTX-1, CTX-2 and CTX-3 on isolated guinea-pig left atria and ilea showed that, at low concentrations, each ciguatoxin caused transient positive inotropy, whereas moderate concentrations induced transient and sustained positive inotropic phases. The transient positive inotropic phase was inhibited by tetrodotoxin or atenolol, indicating that this phase was due to an indirect effect of the ciguatoxins via the stimulation of intrinsic adrenergic nerves. Moreover, the effects of these ciguatoxins on neurons were ten (ciguatoxin-1 and -2) to 100 times higher (ciguatoxin-3) than those directly on the myocardium, thus indicating differences in the affinity of ciguatoxins for the different sodium channels of each tissue (Lewis & Hoy, 1993). The complexity of the effects of ciguatoxins in different cellular models is increased by the variety of experimental in vitro systems used to evaluate these toxins. Thus, studies using purified ciguatoxins indicated that these compounds caused a sodium-dependent calcium mobilization, increasing the intracellular calcium concentration by either altering the operation of the Na/Ca exchanger in Torpedo cholinergic synaptosomes, or affecting the inositol 1,4,5-trisphosphate-releasable Ca2+ store in NG108-15 neuroblastoma x glioma hybrid cells (Molgo et al., 1993a; Molgo et al., 1993b). In the smooth muscle, purified CTX-1 at 10 pM increased the frequency of spontaneous excitatory junction potentials and elicited marked depolarizations at 100–400 pM, while propagated impulses were blocked at CTX concentrations of 100 pM. In addition, all the effects of CTX-1 were abolished by tetrodotoxin (0.3 μM) and were reduced by calcium channel blockers (Brock et al., 1995). A similar excitatory action of purified CTX-1 has been reported in autonomic ganglia at low nanomolar concentrations of the toxin (Hamblin et al., 1995). Moreover, an excitatory action of ciguatoxin, associated with an increase in c-fos expression in brain regions associated with thermoregulatory responses, was also described, thus suggesting that ciguatoxins modify immediate early genes expression in the brain, at least in regions associated with visceral and thermoregulatory responses (Peng et al., 1995). In the frog nodes of Ranvier, CTX-1B at 10 nM increased the nodal volume in a tetrodotoxin-sensitive manner and activated voltage-dependent sodium channels at resting membrane potentials decreasing their inactivation (Benoit et al., 1996), an effect similar to that elicited in the same preparation by CTX-4B at concentrations of 20 and 30 nM (Mattei et al., 1997). In rat parasympathetic neurons, CTX-1 at 1-10 nM caused gradual membrane depolarization and tonic action potential firing, which ceased with membrane depolarizations of about –35 mV and was reverted by application of 300 nM tetrodotoxin (Hogg et al., 1998). However, in this case, the finding of different responses of individual neurons to ciguatoxin led the authors to suggest that the compound could differently affect distinct types of voltage-dependent

Pharmacology of ciguatoxins

39

sodium channels or either alter factors such as phosphorylation of voltage-dependent Na+ channels by intracellular mediators, thus altering channel gating and contribute to the variation in the concentration dependent effects of CTX-1 between neurons. Later, the differential effects of Pacific CTX-1, isolated from the viscera of moray eels (Lycodontis javanicus) collected in the central Pacific Ocean, on sodium channel subtypes was investigated in mammalian sensory neurons (Strachan et al., 1999). To accomplish this, isolated dorsal root ganglia neurons which express both tetrodotoxin-sensitive voltage-gated sodium currents (blocked by 0.3 nM tetrodotoxin or 0.5 nM saxitoxin) and tetrodotoxin-resistant voltage-gated sodium currents (blocked by 100 μM tetrodotoxin or 10 μM saxitoxin) were employed. In this experimental system, P-CTX-1 at concentrations ranging from 0.2 to 20 nM had no effect on the kinetics of tetrodotoxin-sensitive or tetrodotoxin-resistant sodium channel activation and inactivation, but decreased the peak current amplitude in both channel types. In tetrodotoxin-sensitive sodium channels, 5 nM P-CTX-1 caused a hyperpolarizing shift of 13 mV in the activation voltage of sodium channels and a 22 mV shift in their steady-state inactivation, as well as a rapid rise in the membrane leakage current which was tetrodotoxin-sensitive. In contrast, at the same concentration, the toxin increased the rate of recovery from sodium channel inactivation in tetrodotoxin-resistant sodium channels without significant changes in the threshold for sodium channel activation or steady-state inactivation of tetrodotoxin-resistant sodium channels (Strachan et al., 1999). The low dose of toxin employed in this study is in contrast with the concentration used for CTX-3C in transiently expressed tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels (Yamaoka et al., 2004), suggesting that the β-subunit of sodium channels is probably a requisite for the high activity of this group of toxins. Further analysis of the physiological effects of purified P-CTX-1 (extracted from Gymnothorax javanicus moray eel liver) in cultured rat myotubes confirmed that the toxin has a time-dependent effect on voltage-dependent sodium currents. Thus, at short times of exposure, the toxin caused a moderate increase in voltage-dependent sodium currents, while prolonged exposure to the toxin increased peak Na+ current amplitude, as well as leakage currents, tail currents and outward Na+ currents, causing intracellular Na+ accumulation in a tetrodotoxin-sensitive manner. In addition, the authors described for the first time that low to moderate concentrations of P-CTX-1B (2–5 nM) partially blocked potassium currents in a manner that was dependent on the membrane potential (i.e. potassium channel blockade was higher at more depolarizing potentials without affecting the kinetics of voltage-gated potassium currents (Hidalgo et al., 2002)). Moreover, P-CTX-1B at concentrations of 10 nM caused a tetrodotoxin-sensitive transient increase of intracellular inositol 1,4,5-trisphosphate levels, as well as a transient increase in cytoplasmatic and nuclear calcium levels. So far, very few studies have compared the biological activity of Pacific and Caribbean or Indic ciguatoxins in the same experimental model. The first of these studies described the actions of Pacific and Caribbean ciguatoxins, P-CTX-1 and C-CTX-1, on isolated parasympathetic neurons from rat intracardiac ganglia, using patch-clamp recording techniques. Under current-clamp conditions, both ciguatoxins caused a gradual depolarization of the membrane potential, accompanied by

40

Chapter 2

membrane oscillations, leading to tonic action potential firing after bath application of P-CTX-1 (1–10 nM) or C-CTX-1 (10–30 nM). These effects were tetrodotoxin-sensitive and were also inhibited by tetracaine or Zn2+ , while they were not affected by potassium channel blockade with tetraethylammonium or 4-aminopyridine, nor by the calcium channel blockers Cs+ , Cd2+ , Ba2+ , or by the chloride channel blocker, 1,4,4′ diothiocyanato−2,2′ -stilbenedisulphonic acid (DIDS), or by the sodium phosphate ATPase inhibitor ouabain (Hogg et al., 2002). In summary, the differential pattern of expression of voltage-gated sodium channels between species, tissues and during development, suggests that sodium channel isoforms have unique characteristics and may be differentially affected by ciguatoxins. Therefore, not only the ciguatoxin type, but also the experimental preparation employed, may influence the physiological effect of these toxins. One of the initial studies on the affinity of ciguatoxins for the different sodium channel isoforms characterized the effect of P-CTX-3C on [3 H]brevetoxin binding using rat brain membranes and Nav 1.4 or Nav 1.5 α-subunit sodium channels, stably expressed in HEK cells. In these preparations, P-CTX-3C competed for [3 H]PbTx-3 binding on muscle (Nav 1.4) and cardiac (Nav 1.5) sodium channels and rat brain membranes, showing an IC50 for inhibition of [3 H]PbTx-3 binding of 0.48 nM in rat brain sodium channels, 0.8 nM in Nav1.4 channels and 1.10 nM in human Nav1.5 channels (Bottein Dechraoui & Ramsdell, 2003). This effect is in contrast to the activity of the synthetic ciguatoxin CTX-3C on rat voltage-dependent sodium channel isoforms transiently expressed in HEK293 cells. In this case, high doses of CTX-3C (1 μM) were needed to shift the activation potential in a hyperpolarizing direction by about 30 mV for all Na channel isoforms. Moreover, the toxin, at the same concentration, accelerated the time-to-peak current only in the rNav1.2 isoform, but higher doses of the toxin (3–10 μM) additionally decreased time-to-peak current in rNav1.4 and rNav1.5 isoforms. In addition, in the three sodium channel isoforms, the toxin shifted the inactivation potential of sodium channels in the negative direction by 15–18 mV and decreased the peak sodium current amplitude in a concentration-dependent manner (Yamaoka et al., 2004). In view of these results, the authors suggested that CTX-3C exerted multimodal effects on sodium channels, with simultaneous stimulatory and inhibitory aspects. These were attributed to the large molecular size (3 nm in length) and lipophilicity of the toxin (Yamaoka et al., 2004). However, in a later study, the authors used 0.1 nM CTX-3C to activate Nav1.2 and Nav1.4 channels present in HEK cells at less negative membrane potentials (Yamaoka et al., 2009), therefore indicating that the effect of the toxin on sodium channel is also voltage-dependent. The same study demonstrated that CTX-3C preferentially affected the activation process of the tetrodotoxin-resistant Nav1.8 channel, compared with those of the Nav1.2 and Nav1.4 channels, inducing a larger leakage current in cells expressing Nav1.8. In addition, the authors determined that the molecular domains of Nav1.8 responsible for conferring higher sensitivity to CTX-3C were associated to the N-terminal domains D1 or D2 of Nav 1.8 (Yamaoka et al., 2009). Although differences in the ciguatoxin structure of CTX-3C and CTX-1B might affect their potency. CTX-1B has almost the same ether ring skeleton as that of CTX-3C (a small difference is found at E-ring; CTX-1B has a seven-membered E-ring instead of an eight-membered one for CTX-3C), with the M-ring hydroxylated at the 51st position, and has a dihydroxylated extra-alkyl chain attached at position 1. The hydroxylation of the M-ring of CTX-3C, resulting in 51-hydroxy-CTX3C, had no

Pharmacology of ciguatoxins

41

significant effect on its potency (Yamaoka et al., 2009), although the authors suggested that hydroxy residues near the A-ring could be significant for ciguatoxin binding activity. The authors also suggested that the different potencies of ciguatoxins may also be explained for chemical modifications of the functionality of sodium channels, such as phosphorylation, or even by the differences in the internal pipette solution employed. Further studies have demonstrated that C-CTX-1 at concentrations between 10–120 nM increases asynchronous muscle contractions, nerve excitability and both spontaneous and evoked release of acetylcholine from motor nerve terminals. These neuromuscular effects were abolished by tetrodotoxin, providing evidence again that C-CTX-1 acts through an activation of voltage-gated sodium channels. This study further demonstrated that C-CTX-1, at the same nanomolar concentrations, targets the skeletal neuromuscular junction both pre- and post-synaptically. Post-synaptically, C-CTX-1 produced tetrodotoxin- or μ-conotoxin GIIIA-sensitive muscle contractions. Pre-synaptically, at the neuromuscular junction, C-CTX-1 increased both spontaneous and Ca2+ -dependent evoked quantal acetylcholine release, and this caused repetitive post-synaptic action potentials which triggered spontaneous and repetitive muscle contractions post-synaptically (Mattei et al., 2010). In addition, nerve terminals were morphologically affected by C-CTX-1, since the toxin caused a significant swelling of nerve branches, suggesting that ciguatoxin induced water influx in response to the entry of Na+ ions into nerve terminals. Besides voltage-gated sodium channels, in some experimental models ciguatoxins also inhibited voltage-gated potassium channels, although it is not clear yet whether this is a direct effect of the toxin or an indirect response of the channel to the CTX-evoked membrane depolarization. Although very few reports have investigated the effect of ciguatoxins in voltage-dependent potassium currents, it has been reported that purified P-CTX-1B at 20 nM inhibited both the fast-activating delayed-rectifier potassium channels (KDR ), which function to limit the duration of action potentials by remaining open for as long as depolarization occurs, thereby promoting the onset of repolarization, and the transient ‘A-type’ potassium channels (KA ) that acts to modulate action potential firing frequency and slow the rate of depolarization by altering the duration of the afterhyperpolarization. This blockade of voltage-gated potassium channels by P-CTX-1B was suggested to contribute to the increase in neuronal excitability produced by ciguatoxins (Birinyi-Strachan et al., 2005). In contrast, synthetic CTX-3C, at toxin concentrations of 100 nM, was ineffective on voltage-dependent potassium currents (Ghiaroni et al., 2006). However, CTX-3C at 10 nM completely suppressed both transient and delayed potassium currents in primary cultures of cerebellar granule cells, while its analogue 51-OH-CTX-3C was more potent, inhibiting the delayed potassium current in the same preparation, while P-CTX-1B inhibited potassium currents at concentrations of 5–10 nM (Perez et al., 2011). So far, although the inhibition of potassium channels by ciguatoxins may increase the CTX-evoked membrane depolarization, the effect of CTX-3C on potassium channels seems to be dependent on the experimental model and probably the intra- and extracellular solutions employed for the recording. In this sense (despite the fact that we have described an inhibitory effect of CTX-3C in primary cultures of cerebellar granule cells, at the same concentration), the toxin caused only a minor effect in potassium currents in neuroblastoma cells (Figure 2.5) or in primary cultured cortical neurons (Martín et al., 2014).

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Figure 2.5

Effect of bath application of different concentrations of CTX-3C on isolated potassium currents in human SH-SY5Y neuroblastoma cells. Voltage-gated potassium currents were pharmacologically isolated by the addition of 0.3 μM tetrodotoxin to the bath solution. Each point represents the mean ± SEM of five measurements.

In summary, in spite of the fact that all the ciguatoxin congeners known so far cause the activation of voltage-gated sodium channels at negative potentials, and decrease their inactivation, the lack of standards for this family of toxins, together with the variety of ciguatoxins and experimental models employed to evaluate their effects, makes it difficult to establish a unique hypothesis to explain the long-term neurological symptoms produced by these toxins in humans.

Ciguatoxin neurotoxicity Although a common method to test ciguatoxins is the evaluation of their toxicity in neuroblastoma cells, adding the toxin in the presence of ouabain and veratridine, ciguatoxin by itself is not toxic in neuroblastoma cells (Boydron-Le Garrec et al., 2005) or in primary cultures of cortical neurons (Figure 2.6). In fact, we have recently demonstrated that exposure of cortical neurons to up to 10 nM CTX-3C for 96 hours in the culture medium does not produce a cytotoxic effect in the neurons, nor an alteration in cell morphology, as indicated in Figure 2.6, which shows tubulin staining of cortical neurons exposed from 0 to 4 days in culture to 10 nM CTX-3C. This effect is in contrast with the neurotoxic effect elicited both in vivo and in vitro by P-CTX-1B (Braidy et al., 2014; Zhang et al., 2013).

Ciguatoxins, neurological perspectives Despite the fact that the neurological symptoms observed in clinical cases of ciguatera poisoning are believed to be consistent with the direct interaction of ciguatoxins with voltage-gated sodium channels (Lehane & Lewis, 2000; Vetter et al., 2014), some authors have argued that the unique effects of ciguatoxins on voltage-gated sodium channels does not fully explain the spectrum of symptoms elicited by ciguatoxins

Pharmacology of ciguatoxins

Control

43

5 nM CTX-3C 96 hours in culture

Figure 2.6

Confocal microscopy image showing β-tubulin staining of primary cortical neurons grown for four days in culture in the absence (left) or presence of 5 nM CTX-3C (right). Scale bar is 20 μm. (See plate section for colour version.)

(Kumar-Roine et al., 2008). In this sense, it is noteworthy that neuronal homeostasis plays an essential role in the formation, maintenance and modification of neuronal circuits, and enables neurons to adapt to changes in a reliable way in terms of activity (Burrone et al., 2002; Turrigiano, 2007). Neuronal changes in intracellular sodium concentration play a key role in controlling action potential generation, and are good candidates for mediating forms of synaptic plasticity that depend on neuronal firing (George et al., 2012; Rose et al., 1999). In fact, marine toxins activating voltage-gated sodium channels, such as brevetoxins, have been reported to be capable of mimicking the activity-dependent control of neuronal development by up-regulating N-Methyl-D-aspartate (NMDA) receptor signalling pathways that influence neuronal growth and plasticity in immature neurons (George et al., 2012, 2009). In this, sense, recent studies in our laboratory indicate that CTX-3C at 5 nM did not affect neuron viability, nor the morphology of primary rat cultured cortical neurons exposed to the toxin for 96 hours, as shown in Figure 2.6. However, bath application of 5 nM CTX-3C rapidly altered spontaneous synaptic transmission in cortical neurons, increasing the amplitude of miniature postsynaptic inhibitory currents (GABAergic mIPSCs) and decreasing the amplitude of postsynaptic excitatory currents (mEPSCs), affecting mainly the NMDA component of excitatory currents (Martín et al., in preparation). A crucial issue to understand the long clinical consequences of ciguatera is to establish the role that changes in activity represent to neurons. It is necessary to point out that small perturbations in activity can conceivably alter the functional properties of an entire network of cells which can, in turn, lead to further modifications elsewhere. Since the neurological secuela of ciguatera fish poisoning in humans usually last for months, or even years, it is not preposterous to hypothesize that those neurological effects may be related to alterations in the activity of neurons that may be reflected by dramatic changes in synaptic strength or neurotransmitter function in neurons. In this sense, alterations in GABAercic inhibitory transmission are known to manifest with

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Chapter 2

altered body temperature and ataxia (Chiu et al., 2005), both observed in ciguatera (Jones, 1980; Vetter et al., 2012). Moreover, fatigue, weakness and depression, which result from decreased excitatory activity, increased inhibitory activity or both (Zlott & Byrne, 2010) are also common in ciguatera food poisoning (Pearn, 1997). Furthermore, several drugs, including benzodiazepine and diazepam, apart from activating GABAA receptors, also block voltage-gated sodium channels and shift the curve of inactivation in the hyperpolarizing direction (Backus et al., 1991; Tallman et al., 1978; Wakamori et al., 1989). Therefore, it is not necessarily strange to suggest that ciguatoxins could target not only voltage-gated sodium channels, as has previously been assumed, but also alter other ligand-gated receptor channels, such as neurotransmitter receptors, regulating different signalling pathways in a manner dependent or independent of sodium channel activation. Therefore, it is obvious that ciguatoxins may have a key role to play in future studies facing synaptic and neurotransmitter implications, and therefore may represent a novel pharmacologic strategy to evaluate the regulation of neuronal homeostasis.

References Backus, K.H., Pflimlin, P. and Trube, G. (1991) Action of diazepam on the voltage-dependent Na+ current. Comparison with the effects of phenytoin, carbamazepine, lidocaine and flumazenil. Brain Research, 548, 41–49. Benoit, E. and Legrand, A.M. (1992) Purified ciguatoxin-induced modifications in excitability of myelinated nerve fibre. Bulletin de la Société de pathologie exotique, 85, 497–499. Benoit, E., Legrand, A.M. and Dubois, J.M. (1986) Effects of ciguatoxin on current and voltage clamped frog myelinated nerve fibre. Toxicon, 24, 357–364. Benoit, E., Juzans, P., Legrand, A.M. and Molgo, J. (1996) Nodal swelling produced by ciguatoxin-induced selective activation of sodium channels in myelinated nerve fibers. Neuroscience, 71, 1121–1131. Bidard, J.N., Vijverberg, H.P., Frelin, C. et al. (1984) Ciguatoxin is a novel type of Na+ channel toxin. Journal of Biological Chemistry, 259, 8353–8357. Birinyi-Strachan, L.C., Gunning, S.J., Lewis, R.J. and Nicholson, G.M. (2005) Block of voltage-gated potassium channels by Pacific ciguatoxin-1 contributes to increased neuronal excitability in rat sensory neurons. Toxicology and Applied Pharmacology, 204, 175–186. Bottein Dechraoui, M.Y. and Ramsdell, J.S. (2003) Type B brevetoxins show tissue selectivity for voltage-gated sodium channels: comparison of brain, skeletal muscle and cardiac sodium channels. Toxicon, 41, 919–927. Bottein, M.Y., Wang, Z. and Ramsdell, J.S. (2011) Toxicokinetics of the ciguatoxin P-CTX-1 in rats after intraperitoneal or oral administration. Toxicology, 284, 1–6. Boydron-Le Garrec, R., Benoit, E., Sauviat, M.P. et al. (2005) Ability of some plant extracts, traditionally used to treat ciguatera fish poisoning, to prevent the in vitro neurotoxicity produced by sodium channel activators. Toxicon, 46, 625–634. Braidy, N., Matin, A., Rossi, F. et al. (2014) Neuroprotective effects of rosmarinic Acid on ciguatoxin in primary human neurons. Neurotoxicity Research, 25, 226–234. Brock, J.A., McLachlan, E.M., Jobling, P. and Lewis, R.J. (1995) Electrical activity in rat tail artery during asynchronous activation of postganglionic nerve terminals by ciguatoxin-1. British Journal of Pharmacology, 116, 2213–2220. Burrone, J., O’Byrne, M. and Murthy, V.N. (2002) Multiple forms of synaptic plasticity triggered by selective suppression of activity in individual neurons. Nature, 420, 414–418.

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Caillaud, A., de la Iglesia, P., Darius, H.T. et al. (2010) Update on methodologies available for ciguatoxin determination: perspectives to confront the onset of ciguatera fish poisoning in Europe. Marine Drugs, 8, 1838–1907. Catterall, W.A., Cestele, S., Yarov-Yarovoy, V. et al. (2007) Voltage-gated ion channels and gating modifier toxins. Toxicon, 49, 124–141. Catterall, W.A., Goldin, A.L. and Waxman, S.G. (2013). Voltage-gated sodium channels. http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=578. Cestele, S. and Catterall, W.A. (2000) Molecular mechanisms of neurotoxin action on voltage-gated sodium channels. Biochimie, 82, 883–892. Chanteau, S., Bagnis, R. and Yasumoto, T. (1976) Purification of ciguatoxin from the loach, Epinephelus microdon (Bleecker). Biochimie, 58, 1149–1151. Chiu, C.S., Brickley, S., Jensen, K. et al. (2005) GABA transporter deficiency causes tremor, ataxia, nervousness, and increased GABA-induced tonic conductance in cerebellum. Journal of Neuroscience, 25, 3234–3245. Cuypers, E., Yanagihara, A., Rainie, J.D. and Tytgat, J. (2007) TRPV1 as a key determinant in ciguatera and neurotoxic shellfish poisoning. Biochemical and Biophysical Research Communications, 361, 214–217. Dickey, R.M. (2008). Ciguatera toxins: chemistry, toxicology and detection. In Botana, L.M. (ed). Seafood and Freshwater toxins. Pharmacology, physiology and detection, pp 479–500. Boca Raton, FL: CRC Press. Dickey, R.W. and Plakas, S.M. (2010) Ciguatera: a public health perspective. Toxicon, 56, 123–136. EFSA (2010) Scientific Opinion on marine biotoxins in shellfish – Emerging toxins: Ciguatoxin group. EFSA Journal, 8. George, J., Dravid, S.M., Prakash, A. et al. (2009) Sodium channel activation augments NMDA receptor function and promotes neurite outgrowth in immature cerebrocortical neurons. Journal of Neuroscience, 29, 3288–3301. George, J., Baden, D.G., Gerwick, W.H. and Murray, T.F. (2012) Bidirectional influence of sodium channel activation on NMDA receptor-dependent cerebrocortical neuron structural plasticity. Proceedings of the National Academy of Sciences of the United States of America, 109, 19840–19845. Ghiaroni, V., Fuwa, H., Inoue, M. et al. (2006) Effect of ciguatoxin 3C on voltage-gated Na+ and K+ currents in mouse taste cells. Chemical Senses, 31, 673–680. Hamblin, P.A., McLachlan, E.M. and Lewis, R.J. (1995) Sub-nanomolar concentrations of ciguatoxin-1 excite preganglionic terminals in guinea pig sympathetic ganglia. Naunyn-Schmiedeberg’s Archives of Pharmacology, 352, 236–246. Hamilton, B., Hurbungs, M., Jones, A. and Lewis, R.J. (2002a) Multiple ciguatoxins present in Indian Ocean reef fish. Toxicon, 40, 1347–1353. Hamilton, B., Hurbungs, M., Vernoux, J.P. et al. (2002b) Isolation and characterisation of Indian Ocean ciguatoxin. Toxicon, 40, 685–693. Hidalgo, J., Liberona, J.L., Molgo, J. and Jaimovich, E. (2002) Pacific ciguatoxin-1b effect over Na+ and K+ currents, inositol 1,4,5-triphosphate content and intracellular Ca2+ signals in cultured rat myotubes. British Journal of Pharmacology, 137, 1055–1062. Hirama, M. (2005) Total synthesis of ciguatoxin CTX3C: a venture into the problems of ciguatera seafood poisoning. The Chemical Record, 5, 240–250. Hirama, M., Oishi, T., Uehara, H. et al. (2001) Total synthesis of ciguatoxin CTX3C. Science, 294, 1904–1907. Hogg, R.C., Lewis, R.J. and Adams, D.J. (1998) Ciguatoxin (CTX-1) modulates single tetrodotoxin-sensitive sodium channels in rat parasympathetic neurones. Neuroscience Letters, 252, 103–106. Hogg, R.C., Lewis, R.J. and Adams, D.J. (2002) Ciguatoxin-induced oscillations in membrane potential and action potential firing in rat parasympathetic neurons. European Journal of Neuroscience, 16, 242–248.

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Holmes, M.J., Lewis, R.J., Poli, M.A. and Gillespie, N.C. (1991) Strain dependent production of ciguatoxin precursors (gambiertoxins) by Gambierdiscus toxicus (Dinophyceae) in culture. Toxicon, 29, 761–775. Inoue, M., Uehara, H., Maruyama, M. and Hirama, M. (2002) Practical total synthesis of ciguatoxin CTX3C by improved protective group strategy. Organic Letters, 4, 4551–4554. Jones, H.R. Jr., (1980) Acute ataxia associated with ciguatera-type (grouper) tropical fish poisoning. Annals of Neurology, 7, 491. Kumar-Roine, S., Matsui, M., Chinain, M. et al. (2008) Modulation of inducible nitric oxide synthase gene expression in RAW 264.7 murine macrophages by Pacific ciguatoxin. Nitric Oxide, 19, 21–28. Ledreux, A. and Ramsdell, J.S. (2013) Bioavailability and intravenous toxicokinetic parameters for Pacific ciguatoxin P-CTX-1 in rats. Toxicon, 64, 81–86. Lee, M.S., Repeta, D.J., Nakanishi, K. and Zagorski, M.G. (1986) Biosynthetic origins and assignments of carbon 13 NMR peaks of brevetoxin B. Journal of the American Chemical Society, 108, 7855–7856. Lehane, L. and Lewis, R.J. (2000) Ciguatera: recent advances but the risk remains. International Journal of Food Microbiology, 61, 91–125. Leipold, E., Hansel, A., Olivera, B.M. et al. (2005) Molecular interaction of delta-conotoxins with voltage-gated sodium channels. FEBS Letters, 579, 3881–3884. Lewis, R.J. (1988) Negative inotropic and arrhythmic effects of high doses of ciguatoxin on guinea-pig atria and papillary muscles. Toxicon, 26, 639–649. Lewis, R.J. (1992) Ciguatoxins are potent ichthyotoxins. Toxicon, 30, 207–211. Lewis, R.J. (2000) Ion channel toxins and therapeutics: from cone snail venoms to ciguatera. Therapeutic Drug Monitoring, 22, 61–64. Lewis, R.J. (2001) The changing face of ciguatera. Toxicon, 39, 97–106. Lewis, R.J. (2006) Ciguatera: Australian perspectives on a global problem. Toxicon, 48, 799–809. Lewis, R.J. and Endean, R. (1983) Occurrence of a ciguatoxin-like substance in the Spanish mackerel (Scomberomorus commersoni). Toxicon, 21, 19–24. Lewis, R.J. and Endean, R. (1984) Mode of action of ciguatoxin from the Spanish Mackerel, Scomberomorus commersoni, on the guinea-pig ileum and vas deferens. Journal of Pharmacology and Experimental Therapeutics, 228, 756–760. Lewis, R.J. and Endean, R. (1986) Direct and indirect effects of ciguatoxin on guinea-pig atria and papillary muscles. Naunyn-Schmiedeberg’s Archives of Pharmacology, 334, 313–322. Lewis, R.J. and Holmes, M.J. (1993) Origin and transfer of toxins involved in ciguatera. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 106, 615–628. Lewis, R.J. and Hoy, A.W. (1993) Comparative action of three major ciguatoxins on guinea-pig atria and ilea. Toxicon, 31, 437–446. Lewis, R.J. and Jones, A. (1997) Characterization of ciguatoxins and ciguatoxin congeners present in ciguateric fish by gradient reverse-phase high-performance liquid chromatography/mass spectrometry. Toxicon, 35, 159–168. Lewis, R.J. and Sellin, M. (1993) Recovery of ciguatoxin from fish flesh. Toxicon, 31, 1333–1336. Lewis, R.J., Sellin, M., Poli, M.A. et al. (1991) Purification and characterization of ciguatoxins from moray eel (Lycodontis javanicus, Muraenidae). Toxicon, 29, 1115–1127. Lewis, R.J., Vernoux, J.-P. and Brereton, I.M. (1998) Structure of Caribbean Ciguatoxin Isolated from Caranx latus. Journal of the American Chemical Society, 120, 5914–5920. Lombet, A., Bidard, J.N. and Lazdunski, M. (1987) Ciguatoxin and brevetoxins share a common receptor site on the neuronal voltage-dependent Na+ channel. FEBS Letters, 219, 355–359. Martín, V., Vale, C., Antelo, A. et al. (2014). Differential effects of ciguatoxin and maitotoxin in primary cultures of cortical neurons. Chemical Research in Toxicology (submitted manuscript). Martín, V., Vale, C., Hirama, M. et al. (2014). Ciguatoxin causes a rapid homeostatic scaling in inhibitory and excitatory synapses. (manuscript in preparation). Mattei, C., Benoit, E., Juzans, P. et al. (1997) Gambiertoxin (CTX-4B), purified from wild Gambierdiscus toxicus dinoflagellates, induces Na(+)-dependent swelling of single frog myelinated axons and motor nerve terminals in situ. Neuroscience Letters, 234, 75–78.

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Mattei, C., Marquais, M., Schlumberger, S. et al. (2010) Analysis of Caribbean ciguatoxin-1 effects on frog myelinated axons and the neuromuscular junction. Toxicon, 56, 759–767. Molgo, J., Comella, J.X. and Legrand, A.M. (1990) Ciguatoxin enhances quantal transmitter release from frog motor nerve terminals. British Journal of Pharmacology, 99, 695–700. Molgo, J., Gaudry-Talarmain, Y.M., Legrand, A.M. and Moulian, N. (1993a) Ciguatoxin extracted from poisonous moray eels Gymnothorax javanicus triggers acetylcholine release from Torpedo cholinergic synaptosomes via reversed Na(+ )-Ca2+ exchange. Neuroscience Letters, 160, 65–68. Molgo, J., Shimahara, T. and Legrand, A.M. (1993b) Ciguatoxin, extracted from poisonous morays eels, causes sodium-dependent calcium mobilization in NG108-15 neuroblastoma x glioma hybrid cells. Neuroscience Letters, 158, 147–150. Murata, M., Legrand, A.M., Ishibashi, Y. et al. (1990) Structures and configurations of ciguatoxin from the moray eel Gymnothorax javanicus and its likely precursor from the dinoflagellate Gambierdiscus toxicus. Journal of the American Chemical Society, 112, 4380–4386. Nicholson, M. and Lewis, R. (2006) Ciguatoxins: Cyclic Polyether Modulators of Voltage-gated Iion Channel Function. Marine Drugs, 4, 82–118. Ohizumi, Y., Ishida, Y. and Shibata, S. (1982) Mode of the ciguatoxin-induced supersensitivity in the guinea-pig vas deferens. Journal of Pharmacology and Experimental Therapeutics, 221, 748–752. Otero, P., Perez, S., Alfonso, A. et al. (2010) First toxin profile of ciguateric fish in Madeira Archipelago (Europe). Analytical Chemistry, 82, 6032–6039. Pearn, J.H. (1997) Chronic fatigue syndrome: chronic ciguatera poisoning as a differential diagnosis. Medical Journal of Australia, 166, 309–310. Pedretti, A., Villa, L. and Vistoli, G. (2003) Atom-type description language: a universal language to recognize atom types implemented in the VEGA program. Theoretical Chemistry Accounts, 109, 229–232. Peng, Y.G., Taylor, T.B., Finch, R.E. et al. (1995) Neuroexcitatory actions of ciguatoxin on brain regions associated with thermoregulation. Neuroreport, 6, 305–309. Perez, S., Vale, C., Alonso, E. et al. (2011) A comparative study of the effect of ciguatoxins on voltage-dependent Na+ and K+ channels in cerebellar neurons. Chemical Research in Toxicology, 24, 587–596. Pottier, I., Vernoux, J.P., Jones, A. and Lewis, R.J. (2002) Characterisation of multiple Caribbean ciguatoxins and congeners in individual specimens of horse-eye jack (Caranx latus) by high-performance liquid chromatography/mass spectrometry. Toxicon, 40, 929–939. Pottier, I., Hamilton, B., Jones, A. et al. (2003) Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda (Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding. Toxicon, 42, 663–672. Rayner, M.D. (1972) Mode of action of ciguatoxin. Federation proceedings, 31, 1139–1145. Rayner, M.D. and Szekerczes, J. (1973) Ciguatoxin: effects on the sodium-potassium activated adenosine triphosphatase of human erythrocyte ghosts. Toxicology and Applied Pharmacology, 24, 489–496. Roeder, K., Erler, K., Kibler, S. et al. (2010) Characteristic profiles of Ciguatera toxins in different strains of Gambierdiscus spp. Toxicon, 56, 731–738. Rose, C.R., Kovalchuk, Y., Eilers, J. and Konnerth, A. (1999) Two-photon Na+ imaging in spines and fine dendrites of central neurons. Pflügers Archiv, 439, 201–207. Satake, M., Ishibashi, Y., Legrand, A.M. and Yasumoto, T. (1996) Isolation and structure of ciguatoxin-4A, a new ciguatoxin precursor, from cultures of dinoflagellate Gambierdiscus toxicus and parrotfish Scarus gibbus. Bioscience, Biotechnology, and Biochemistry, 60, 2103–2105. Satake, M., Fukui, M., Legrand, A.-M. et al. (1998) Isolation and structures of new ciguatoxin analogs, 2,3-dihydroxyCTX3C and 51-hydroxyCTX3C, accumulated in tropical reef fish. Tetrahedron Letters, 39, 1197–1198. Scheuer, P.J., Takahashi, W., Tsutsumi, J. and Yoshida, T. (1967) Ciguatoxin: isolation and chemical nature. Science, 155, 1267–1268. Schlumberger, S., Mattei, C., Molgo, J. and Benoit, E. (2010) Dual action of a dinoflagellate-derived precursor of Pacific ciguatoxins (P-CTX-4B) on voltage-dependent K(+) and Na(+) channels of single myelinated axons. Toxicon, 56, 768–775.

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CHAPTER 3

Chemistry of pinnatoxins Phillip Mabe & Armen Zakarian Department of Chemistry and Biochemistry, University of California, USA

Introduction Pinnatoxins belong to a family of marine toxins that contain a unique structural element – spirocyclic imine moiety. This larger family of cyclic imine natural products also includes pteriatoxins, gymnodimines, spirolides and a few others, such as spiro-prorocentimine and portimine (Figure 3.1). Besides the spirocyclic imine functionality (ring A), pinnatoxins are characterized by the presence of other key structural elements. These include two polyketal fragments – the dispiro 6,5,6 tricyclic ketal at C12–C23 (rings B, C, D), and bridged bicyclic ketal at C25–C30 (rings E, F), cycloxene ring G, and the all-carbon 27-membered macrocyclic ring spanning C5–C31. Due to the presence of both basic and acidic functionality, all pinnatoxins, except for pinnatoxins F and G, are amphoteric and exist as zwitter-ions in solution at a near neutral pH. Pteriatoxins and spirolides are closely related to pinnatoxins. Pteriatoxins share an identical macrocyclic framework with pinnatoxins A and G, only differing by the side-chain composition. They are thought to be derived biosynthetically from pinnatoxin G, via epoxidation of the terminal monosubstituted double bond, followed by opening of the epoxide with cysteine. Spirolides have a very similar spiroimine fragment, differing only in an extra methyl substituent in the cyclohexene ring. Spirolides A and B also lack a methyl at C3. Like pinnatoxins, spirolides consist of an all-carbon macrocycle with ketal functionality. However, the ketal fragment is significantly altered, with the bicyclic ketal completely absent. Notably, spirolides E and F contain a hydrolyzed form of the imine. The structural relation of pinnatoxins to portimine, gymnodimines and spiro-prorocentrimineis is more distant, although still retaining the common outline of a spiroimine bicycle fused to an all-carbon macrocycle. In prorocentrolide, the spiroimine module is changed to a fused hexahydroisoquinoline bicyclic structure, and the commonality with symbioimine does not go far beyond the cyclic imine functionality.

Isolation The first report related to pinnatoxins dates back to 1990. Following a mass seafood poisoning event, an unknown toxic substance, dubbed ‘pinnatoxin’, was detected in the extracts of bivalve mollusc Pinna attenuate (Zheng et al., 1990). Five years later, Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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in 1995, pinnatoxin A was isolated from a related Okinawan shellfish, Pinna muricata, and its two-dimensional structure was determined by Uemura et al. (Uemura et al., 1995). The relative configuration of the compound was established soon after by NMR analysis (Chou et al., 1996a), followed immediately by a report of isolation and structure determination of pinnatoxin D (Chou et al., 1996b). Pinnatoxins B and C were mentioned at this point as the components of the same extract, but their structure was reported later, in 2001 (Takada et al., 2001).

PINNATOXINS

SPIROLIDES

PTERIATOXINS

R1 1

A

HN

5

10

H 30 O F OE

O O

HO

G

31

12

B

N

25

C

O

34

R4

HO O

HO R 3

R3

R2

R4

O

O

Y=

D

R1

Z=

O

23

O O

O

R2

Pinnatoxins

Spirolides A-D

A-C, G: R1,R 2=H, R 3=OH

A, B:R1,R 2=H, R 3=CH3

A: R 4=CO 2- B: R 4=(S)-CH(NH 2)CO 2-

A: R 4= Y B: R 4=Z

C: R 4=(R)-CH(NH 2)CO 2- G: R 4=CHCH 2

C,D:R1=CH3, R 2=H, R 3=CH 3 C:R 4=Y D: R 4=Z 27-hydroxy-13-desmethyl C:

D, E, F: R1=CH3,R 2=OH, R 3=H D: R 4=COCH 2CH 2CO 2-

R1 =CH3,R 2=OH, R 3=CH3, R 4=Y, unassigned stereo

E: R 4=(S)-CH(OH)CH 2CH 2CO 2-

27-hydroxy-13,19-didesmethyl C: R1 =CH3,R 2=OH, R 3=H, R 4=Y, unassigned stereo

F: R 4= O

O

H 2N Pteriatoxins

O

A, B,C: R1,R 2=H, R 3=OH A: R 4=

CO 2-

S OH

B: R 4=

+H

O

3N

CO 2

R1

HO

OH S

N

-

HO

O

HO R O 1

O

NH 3+

C: R 4=

O

OH

O O

O

OH S

CO2NH 3+

Spirolides E,F

Spirolide G: R1=H

E:R1=Y

20-methyl Spirolide G: R1=CH 3

F:R1 =Z

HO

N H O

N O

R1

O

O

OH HO

I: R1 =Z

OH

Portimine

Figure 3.1

Spirolides H, I H:R1=Y

O

Cyclic imine marine natural products.

Chemistry of pinnatoxins

51

GYMNODIMINES O

O

O

O

OSO3

N

N R1

H

R2

N

H OH

HO

HO

O

O

H

Symbioimine

R3

Gymnodimine A

Gymnodimines B:R1=OH, R 2=H, R 3=H C:R1=H, R 2=OH, R 3=H 18-deoxy B: R1,R 2=H, R 3=H 12-methyl: R1,R 2=H, R 3=CH 3 stereo unassigned

OH OH O OH N

OH

OH NH

HO O O OH

O

H OH

H

OH

O OH

OH O

OSO3

O

HO OH OH

O

Prorocentrolide

Spiro-prorocentrimine

Figure 3.1

(continued)

The absolute stereochemistry of pinnatoxin A was established in 1998, upon the completion of its total synthesis by the group of Kishi, when the absolute configuration was found to be the opposite of that initially assigned (McCauley et al., 1998). The structures of pinnatoxins B and C were confirmed eight years later, in 2006, with the completion of Kishi’s second synthesis of pinnatoxins (Matsuura et al., 2006a). Finally, in 2010, pinnatoxins E, F and G were isolated from the digestive glands of pacific oyster Pinna bicolor, collected in South Australia and New Zealand (Selwood et al., 2010). No new pinnatoxins have been identified since, although the already known pinnatoxins, as well as their fatty acid acylated metabolites (McCarron et al., 2012), have been found in mussels in Eastern Canada (McCarron et al., 2012), in clams in Mediterranean (Hess et al., 2013), and in mussels in Norway (Rundberget et al., 2011), indicating a wide distribution of the pinnatoxin-producing organisms and a potential global risk of exposure.

Bioactivity Pinnatoxins are fast-acting neurotoxins, leading to rapid death in mice, typically within an hour after intraperitoneal injection (LD99 = 180 μg⋅kg–1 for pinnatoxin A (Uemura et al., 1995), 22 μg⋅kg–1 for a 1 ∶ 1 mixture of pinnatoxins B and C (Takada et al., 2001), and 400 μg⋅kg–1 for pinnatoxin D (Chou et al., 1996b), LD50 = 57, 13 and 48 μg⋅kg–1 for pinnatoxins E, F, and G, respectively (Munday et al., 2012)). Oral toxicity was

52

Chapter 3

observed for pinnatoxins F and G, with LD50 only four and eight times lower than by intraperitoneal injection (Munday et al., 2012; Rhodes et al., 2010). Pinnatoxin D also showed significant cytotoxicity against the murine leukaemia cell line P388 (IC50 = 2.5 μg⋅mL–1 ) (Kita and Uemura, 2005). Notably, synthetic (–)-pinnatoxin A, which is the enantiomer of natural (+)-pinnatoxin A, was found to be inactive in doses up to 100 μg (5000 μg⋅kg–1 ) (McCauley et al., 1998). It was initially suggested that the mode of action of pinnatoxins involved calcium channel activation (Zheng et al., 1990). However, recently they were proven to be competitive antagonists of nicotinic acetylcholine receptors (nAChRs) (Aráoz et al., 2011). In respect to other aspects of biochemistry of pinnatoxins, limited data is available. As mentioned previously, acylated fatty acid metabolites of pinnatoxin were isolated (McCarron et al., 2012). The isolation studies and structural analysis appear to indicate that pinnatoxins F and G are produced independently in different species, and serve as biosynthetic precursors to pinnatoxins D, E, and A–C, respectively (Selwood et al., 2010). Recently, a peridinoid dinoflagellate, Vulcanodinium rugosum, was isolated in Northland, New Zealand, and was found to be a producer of pinnatoxins E and F (Rhodes et al., 2010). Strains of this organism producing these and other pinnatoxins were found in Japan (Smith et al., 2011) and China (Zeng et al., 2012) after it was discovered in France (Nézan & Chomérat, 2011; Hess et al., 2013).

Detection While pinnatoxins have not been explicitly linked to any seafood poisoning events, their high acute toxicity, including oral toxicity, combined with confirmed presence in sea organisms around the world, calls for monitoring of their levels. The current general methods used for detection of pinnatoxins, among variety of other marine toxins, include LC/MS analysis and mouse bioassay. Recently, following a discovery of the mode of action of pinnatoxins, methods based on protein target binding also became available. The LC/MS analysis (liquid chromatography coupled with mass spectrometry) is a general and powerful method that is widely applicable for the detection of known marine toxins. The use of pure standards is highly desirable for reliable identification of the detected toxins. It has been noted that this method may have introduced some misattributions as, for example, the molecular composition of pinnatoxin G, one of the main algal metabolites, is equivalent to that of spirolide B and 13-desmethylspirolide D (Hess et al., 2013). The mouse bioassay involves injection of the extract into mice intraperitoneally and monitoring of the mice after injection. Its use is effective in cases of fast-acting cyclic imine toxins that cause the onset of symptoms and death in as little as five minutes (Munday, 2008). It is the reference method for paralytic shellfish poisoning, and is invaluable for the detection of unanticipated and unknown toxins. Its use, however, is limited by new regulations, and it is being discontinued for some applications, requiring a reliable replacement (Commission Regulation (2011)). Other criticisms of the test suggested the possibility of false positive results (Hess et al., 2013). Owing to the development of a scalable and reproducible total synthesis of pinnatoxins, which provided pinnatoxin A in significant amounts, the target of pinnatoxin A was identified to be nicotinic acetylcholine receptors (nAChRs) (Aráoz et al., 2011). This opened a possibility for the development and application of selective and sensitive

Chemistry of pinnatoxins

53

detection methods based on protein binding. A typical approach in this case is the use of the protein target along with another competitive inhibitor, modified to be easily detectable. In the absence of the analyte, the full signal of the inhibitor is detected. In the presence of the analyte, some of the inhibitor is released and removed, due to competition in binding, leading to a diminished signal. Radioactive (Aráoz et al., 2011), fluorescent (Vilariño et al., 2009; Fonfría et al., 2010), and colorimetric (Aráoz et al., 2012) detection have also been employed. Most recently, a method for rapid detection of nanomolar concentrations of neurotoxins was developed by this approach, using immobilized Torpedo electrolyte membranes rich in nicotinic acetylcholine receptors as the target, with biotinylated-α-bungarotoxin as the detectable competitive inhibitor in a microplate array (Aráoz et al., 2012). Since this method does not distinguish between different cyclic imine toxins, combining it with other techniques, such as mass spectroscopy, is necessary in order to identify the particular toxin responsible for the response signal.

Total chemical synthesis The structure of pinnatoxins provides variety of challenges to synthetic chemists. Besides the overall complexity of the polycyclic structure, with 14 or more chiral centres, particular challenges include the quaternary and tertiary chiral centre dyad (C5, C31) in the cyclohexene G ring, the 27-membered all-carbon macrocycle, and control of formation of the multiple ketal chiral centres. Additionally, the assembly of the spiroimine has proved to be a challenge. Not surprisingly, this unique and challenging structure has attracted significant attention from the synthetic community. Despite the complexity of the structure, six total syntheses of pinnatoxins by four research groups have been completed, the first a mere two years after the report establishing the structure of pinnatoxin A. The group of Kishi completed the synthesis of the proposed structure of pinnatoxin A, which turned out to be the enantiomer of the natural compound, in 1998 (McCauley et al., 1998). The key strategic decision in this approach, mirroring the biosynthetic proposal (Uemura et al., 1995), was the use of intramolecular macrocyclic Diels-Alder addition to assemble the G ring, the all-carbon macrocycle, and set the quaternary-tertiary centre dyad at the same time (see Scheme 3.1). The requisite diene substrate for the Diels-Alder cycloaddition, 2, was prone to self-dimerization by the intermolecular Diels-Alder reaction of the diene onto itself. Upon its generation from mesylate 1, it had to be maintained in a dilute solution to prevent dimerization. Heating of this dilute solution triggered the desired intramolecular Diels-Alder cyclization, producing three out of eight possible products – the desired product, 3, and its two diastereomers. Remarkably, no regioisomeric addition products were observed. The product ratio changed slightly, depending on the solvent. Under the best identified conditions, a combined yield of 78% of the three products was observed, with the ratio 1 ∶ 0.9 ∶ 0.4, providing desired macrocycle 3 in 33% yield from mesylate 1. The cyclic imine, ring A, was assembled as the penultimate step in the synthesis in a transformation that proved to be quite challenging. Common acidic conditions failed to induce the cyclization of aminoketone 5. At increased temperatures, side-reactions predominated, for example acetylation of the amine in presence of acetic acid as a reagent.

54

Chapter 3

AllocHN AllocHN O

O

H O O

O O

Et3SiO

CO2tBu

0.2 mM in dodecane, 70 oC, 24 h 78%

4 steps

CO2tBu

H O

O

O O

Et3SiO

O O

HO

major in a 1.0 : 0.9 : 0.4 mixture total exo:endo 5:1

Scheme 3.1

O-

H O

O

51% OTBS

O

OTBS

O

+HN

G

O

OH

O

ent-pinnatoxin A

Intramolecular Diels-Alder and endgame in Kishi’s synthesis of pinnatoxin A.

The desired imine formation was ultimately achieved by heating aminoketone 5 at 200 ∘ C in vacuum. Subsequent removal of the tert-butyl protecting group from the acid afforded pinnatoxin A, which was found to be identical to the natural compound by NMR data, but had the opposite sign of optical rotation and no toxicity, indicating that it is the enantiomer of the natural product. Modified diene 7 was used in Kishi’s second generation synthesis of pinnatoxins and pteriatoxins reported in 2006 (Matsuura et al., 2006a, 2006b) (Scheme 3.2). The ester functionality on the diene was replaced with protected 1,2-diol for a more convenient conversion to the side-chains of pteriatoxins and pinnatoxins B–C. As the protected 1,2-diol, the diene was no longer prone to the self-dimerization reported in Kishi’s first generation synthesis. The optimization study found that dibenzoylation of the diol significantly improved the ratio of the Diels-Alder products in favour of the desired isomer, giving, in the case of the di-p-methoxylbenzoyl ester, an 88% combined yield of the cycloaddition products, with 51% isolated yield of 8 (Scheme 3.2). Additionally, in the second generation synthesis, milder conditions for the formation of the imine were developed, as the substrate for the preparation of pteriatoxins did not survive the initially used thermal conditions (Matsuura et al., 2006b). Revisiting the carboxylic acid-catalyzed condition revealed the possibility of using a sterically hindered carboxylic acid in order to suppress amine acylation. Thus, heating the aminoketone in xylene with triethylammonium 2,4,6-triisopropylbenzoate at 80 ∘ C effectively led to closing of the imine ring, which provided, after CF3 CO2 H-catalyzed removal of the Boc and diphenylmethyl protecting groups, pinnatoxin C in 89% yield over two steps. Pinnatoxin B was obtained using a similar sequence from the independently prepared C34 epimer. The physical data of synthetic pinnatoxins B and C established the configurational assignment of the natural products. The diene substrates for the intramolecular Diels-Alder reaction were prepared using a convergent approach outlined in Scheme 3.3 for the second generation

Ireland-Claisen rearrangement F3C Ph O

BnO

O

OMOM

O

+HN

O O

Et

[3,3]-shift 25 °C

R 94%

BnO OPMB

via BCD-ketal coupling and RCM

BnO TMSO

N

THF, Me3SiCl, -78 °C

O

TIPSO

NLi

Ph

PMBO R= TIPSO

Ph O O

HO Ph O 31 O

5

OPMB H O

O H OMOM

CO2 O

HO

O

O

TIPSO single isomer

(+)-pinnatoxin A (> 40 mg)

MOMO

Scheme 3.2

OH

Kishi’s second generation synthesis of pinnatoxins and pteriatoxins.

Chemistry of pinnatoxins

PMBO

6

I

6

NHAlloc

+

10 steps

O

NHAlloc

O

O

HO

11

O

TESO

55

O O

OMe

O

O O

I S

S

+

OTBS TBSO

10

OTBS 12

Scheme 3.3

O O

OR1 OR1

7 OTBS R1 = p-methoxybenzoyl Dithiane alkylation Nozaki–Hiyama–Kishi coupling

Synthesis of the substrate for the intramolecular Diels-Alder reaction.

synthesis. Substrate 7 was assembled from three subunits: triketal 10, iodides 11, and 12. Subunit 12 was first attached to 10 by dithiane alkylation, followed by protection and functional group manipulations to set up the Nozaki-Hyama-Kishi Ni(II)/Cr(II) coupling of vinyl iodide, 11, with aldehyde at C6 to attach the third subunit. Synthetic building blocks 10, 11, and 12 were independently prepared using the previously developed procedures (Scheme 3.4). The C29,C30 protected diol fragment of subunit 12 was derived from 2-deoxy-D-ribose. The diene fragment, derived from diacetate 13, was installed by another Nozaki-Hyama-Kishi Ni(II)/Cr(II) coupling followed by elimination. The remaining two chiral centres (C27 and C28) were installed by Sharpless asymmetric epoxidation, followed by an epoxide opening with dimethylcuprate. The vinyl iodide was derived from the Diels-Alder adduct of isoprene and dimenthyl fumarate (Furuta et al., 1986). The stereochemistry of the C12 and C23 stereocentres in the spiroketal fragment originated from the starting epoxides, 14 and 15, while the tertiary alcohol and the adjacent ketone were installed by Sharpless asymmetric dihydroxylation, followed by Swern oxidation. The ketalyzation step produced a mixture of ketal isomers. Curiously, C19 epimer isomerized to the desired configuration in the course of the following TES protection. Other undesired isomers were recycled by resubjection to the ketalyzation conditions, which resulted in conversion of 16 to tricyclic dispiroketal 17 in 70% yield. Subunit 10 was further prepared from the obtained dispiroketal. The overall synthesis consisted of 39 steps in the longest linear sequence, with the 0.16% overall yield of pinnatoxin A from 1-pentynol for the first-generation synthesis. In the second-generation effort, pinnatoxin C was prepared in 0.12% overall yield and 51 steps in the longest linear sequence from 2-deoxy-D-ribose. The second synthesis of pinnatoxin A, which is a formal synthesis, was reported by the Hirama and Inoue groups in 2004 (Sakamoto et al., 2004). In their approach, an intramolecular opening of the epoxide by an anion of a nitrile to construct the dyad of tertiary and quaternary stereocentres was utilized, assembling the cyclohexene ring in the same step (Scheme 3.5). To achieve this transformation, mesylate 18 was treated sequentially with 2.5, and then 1.5, equivalents of potassium hexamethyldisilazide to first generate the requisite epoxide and then the anion of the nitrile. Intramolecular attack of the anion onto the epoxide led to cyclohexene product 20 in 72% yield as a single stereo- and

Chapter 3

56

2-deoxy-D-ribose

OMe

+

O

28 steps

I

Br

Sharpless epoxidation, epoxide opening

O

28 27

Nozaki–Hiyama–Kishi coupling, then elimimination

OTBS

OAc OAc

OTBS

12

13

Lipase deacetylation

OTBS 15 steps CO2Mentyl I

+

NHAlloc

CO2Mentyl

11 From 15 From 14 Sharpless dihydroxylation, Swern O

OBn O

14

12 23

O

O 12 steps

OTBS

O

+ BnO O

OH 16

15

O

HO

OBn 13 steps

O recycling of undesired isomers

12

OTBS

CSA, MeOH

19

70%

23

10 O OH

17  

Scheme 3.4

Synthesis of subunits 10, 11, and 12.

regioisomer. The stereoselectivity likely results from the sterically undemanding cyano group occupying the pseudo-axial position in transition state 19 (Scheme 3.5). The obtained product was converted to key intermediate 21 over 22 steps, to be used in the coupling with tricyclic ketal fragment 22. This coupling was achieved via a sequence of dithiane alkylation and a ring-closing metathesis. In order for the metathesis to proceed successfully, the silyl protecting groups had to be removed prior to the reaction, likely due to the steric hindrance they imposed. The macrocyclic intermediate was advanced to azido keto acid 24 via series of steps, involving reduction of the endocyclic double bond, methylenation of the ketone, and final functional group transformations. Notably, formation of the bridged bicyclic E,F-ketal proceeded under acidic conditions without any epimerization of the bispirocyclic ketal fragment. At this point, an attempt was made to form the cyclic imine by the aza-Witting reaction (Scheme 3.6). However, the cyclic imine ring did not form under the reaction conditions (60 ∘ C, THF), and only the amino ketone resulting from the reduction of the azide was isolated. The authors then opted for the preparation of amino ketone 5, previously employed as an intermediate in Kishi’s synthesis of pinnatoxin A, therefore accomplishing the formal synthesis of pinnatoxin A. Preparation of the necessary key intermediates, 18 and 22, was achieved via the following sequences (Scheme 3.7). The precursor for dispiroketal 26 and ketol 25 was assembled from geranyl acetate and (R)-malic acid. Geranyl acetate provided the stereodefined trisubstituted double bond for Sharpless dihydroxylation that set up the hydroxyketone functionality, while malic acid lent itself to establishing of the stereochemistry at C23. The remaining chiral centre at C12 was set by a sequence of

Chemistry of pinnatoxins

TIPSO

57

TIPSO KHMDS, THF 2.5 eq

NC MsO OH

HO

N

O O

22 steps

NC HO

R1 O

then 1.5 eq

72%

OMOM R2

MOMO

H O

HO

Ph

O

Ph

19

18

OMOM

20 TIPSO

TIPSO

HO

OH 1) t-BuLi TMSO

O

THF/HMPA

O

3) Grubbs 2nd gen

S

I

O

TESO

2) TBAF

H OMOM OTMS

OH

O O

63% over 3 steps

S 21

O

O O

+

O

OTES

O

TESO

S S

23

22

Scheme 3.5

OMOM

Key steps in Hirama and Inoue synthesis of pinnatoxin A.

N3

23

H O

O

CO2H

HO

OH

HO

O

24

H O

O

CO2H O

O O

N

PMe3, THF 60 °C

O 10 steps

O

OH

O

Pinnatoxin A 2 steps

Scheme 3.6

5

Imine ring formation.

Sharpless epoxidation and reductive epoxide opening with Red-Al. The optimization of ketalyzation conditions found that the switch to a less polar solvent, toluene, improved the ratio of the isomeric spiroketals in favour of the desired one, 26, to permit its preparation in 84% in one step. The selectivity was attributed to intramolecular hydrogen bonding, stabilizing ketal 26, which is enhanced in the less polar solvent. Later reports suggested that the anomeric effect, also enhanced in a less polar solvent, is responsible for the selectivity, as intramolecular hydrogen bonding can be imagined that stabilizes the other ketal anomer as well (Lu & Zakarian, 2007). Alcohol protection and the use of dithiane alkylation provided building block 22. The substrate for the intramolecular nitrile alkylation, 18, was prepared from D-glucose and commercially available methyl (S)-3-hydroxyl-2-methyl-propionate, 27. The C27-C31 protected polyol terminus of the molecule originated from D-glucose, the C3 chiral centre was derived from (S)-3-hydroxyl-2-methyl-propionate, and the

58

Chapter 3

23

OAc

OH 15 steps 12

+

O

O

COOH

12

CSA MeOH

OH

O

HO O

O 25

OH HOOC

O O

Sharpless dihydroxylatio, Swern oxidation

then toluene 84%

O 26

23

OH

From malic acid 6 steps

Sharpless epoxidation, epoxide reduction

22 TIPSO

2

D-glucose

3

14 steps + OH

MeO2C

NC MsO

Sharpless epoxidation,

31

OMOM

OH

HO

O 27

27

18

Scheme 3.7

O

trimethylaluminum epoxide opening From 27 Horner-Wadsworth-Emmons

Ph  

Preparation of subunits.

methyl group at C2 was installed by a sequence of Sharpless epoxidation and epoxide opening with trimethylaluminum catalyzed by n-butyllithium (Pfaltz & Mailenberger, 1982). Stereoselective assembly of the trisubstituted double bond was accomplished via Horner-Wadsworth-Emmons reaction. In 2008, two syntheses of pinnatoxin A were published in rapid succession. First came the report by the group of Zakarian (Stivala & Zakarian, 2008). This initial report was followed three years later by an improved second-generation synthesis that permitted preparation of significant amounts of pinnatoxins A and G, which were used to study the mechanism of action of the toxins (Aráoz et al., 2011) and perform the development of a method for their detection (Aráoz et al., 2011). In Zakarian’s synthesis, the key step was the installation of the quaternary and tertiary centre dyad by an uncommon case of Ireland-Claisen rearrangement in the course of the assembly of the spiroimine subunit of pinnatoxin. While the Ireland-Claisen rearrangement is one of the most well-developed methods in organic synthesis, its application in this case required stereoselective generation of an α-branched enolate, a precursor to a tetrasubstituted TMS-enol ether, which is notoriously difficult to achieve. For this purpose, a methodology for stereoselective generation of α-branched enolates was developed (Qin et al., 2007), relying on deprotonation of an enantiopure substrate with a chiral base to achieve the diastereoselective transformation. Application of this methodology to requisite substrate 28 produced the desired stereodefined TMS-enol ether, 30, which rearranged to silyl carboxylic acid ester, 31, with excellent yield and stereoselectivity. Thus obtained acid 32 was converted to aldehyde 33, relying on intramolecular aldol-crotonic cyclocondensation to construct the six-membered ring G (Scheme 3.8). The completed subunit was coupled with independently prepared tricyclic ketal fragment 34 (Scheme 3.9). It was achieved by addition of the alkyllithium reagent, generated from iodide 34 by treatment with tert-butyllitium, with aldehyde 33.

Chemistry of pinnatoxins

Ph O

F3C

NLi N

Ph

O

OTMS

29 TIPSO

OSEM

O

O

59

R1 R2

O TMSCl, THF -78 °C

(CH2)4 OPMB

OTMS

25 °C, 6 h 94%

R1 R2

O

(CH2)4 OPMB

PMBO 30 28

BnO

BnO

14 steps

O

Ph

TIPSO

Scheme 3.8

O O

OPMB

HO O O

31

OBn

Ph O OSEM 32

OMOM H OSEM

Intramolecular aldol-crotonic condensation

33

Key Claisen-Ireland rearrangement in Zakarian’s synthesis of pinnatoxin A.

Scheme 3.9

Completion of the synthesis of pinnatoxins A and G.

Removal of the triisopropylsilyl (TIPS) protecting group, Dess-Martin reagent oxidation of both free alcohols to the corresponding keto aldehyde, followed by selective addition of vinylmagnesiumbromide to the aldehyde, produced metathesis substrate 35. Ring-closing metathesis catalyzed by the second-generation Grubbs catalyst successfully formed the macrocycle in 75% yield, with only a minor by-product (6%) resulting from the competitive reaction of the exo-methylene double bond. Finally, oxidation of the allylic alcohol and conjugate addition of methylcyanocuprate to the resulting α, β-unsaturated ketone completed the installation of all structurally critical functionality and chiral centres, leaving assembly of

60

Chapter 3

the remaining bicyclic ketal and protecting and functional group manipulation to complete the synthesis of pinnatoxin A. During these efforts, an attempt to construct the imine ring by aza-Witting reaction was made. However, as in the previous attempt by Hirama, it was unsuccessful, even under forcing conditions (130 ∘ C), leading only to decomposition. Kishi’s method of imine construction was employed to complete the synthesis. In the later report, pinnatoxin G was also prepared from intermediate 36 (Aráoz et al., 2011). Scheme 3.10 shows the assembly of subunits 28 and 34. The tricyclic ketal fragment in 34 was obtained divergently, relying on enzymatic resolution of racemic tert-butyl 3-hydroxy-4-pentenoate, 37, and utilizing both of the enantiomeric products, 38 and 39, to construct the opposite termini of the structure. The ketalyzation step was efficiently performed by a solvent switch to cyclohexane after the protecting group removal was complete (Lu & Zakarian, 2007). The use of a nonpolar solvent increased the ratio of the diastereomeric ketals to 9.8 ∶ 1, providing the desired ketal, 41, in 78% isolated yield in one iteration. The Claisen-Ireland substrate, ester 28, was prepared from (S)-citronellic acid for the carboxylic acid fragment 42, and D-ribose for the alcohol fragment 43. Overall, the first-generation synthesis was completed in 44 steps from citronellic acid, and 44 steps from acrolein, with the overall yields 0.3% and 0.4%, respectively. In the second-generation synthesis, the longest linear sequence was 43 steps from either (S)-citronellic acid or acrolein, with the yields improved to 1.02%. With the developed synthesis, over 40 mg of pinnatoxin A were prepared and used for biological studies.

OH Sharpless dihydroxylation, the Swern

t-BuO2C

From 39

37 Lipase

From 38

CO 2t-Bu

OH THPO

t-BuO2C

CO 2t-Bu

16 steps 38

O HO

49%, >95%ee

CSA, MeOH HO the cyclohexane

O

O

O

78%

O

10 steps

O

34 O

+ 40

OAc

41

OH

t-BuO2C

39 44%, >95%ee OBn Alkylation of Evans oxazalidinone

10 steps (S)-cironellic acid

HO

OPMB O

42

EDC, DMAP, DMF

Ph O

10 steps D-Ribose

28

94%

O

TIPSO

43

OSEM

Scheme 3.10

OH

Stereoselective catalytic E 2Zn addition

Preparation of subunits 28 and 34.

Chemistry of pinnatoxins

61

Later in 2008, the total synthesis of pinnatoxin A by the Nakamura and Hashimoto groups was published (Nakamura et al., 2008). This synthesis followed a more incremental approach, sequentially adding on smaller fragments to build up the entire structure. Ring G was assembled using intermolecular, rather than intramolecular, Diels-Alder cycloaddition. Accordingly, the initially prepared pentacyclic ketal fragment, 44 (Scheme 3.11), was advanced to diene 45, the substrate for the Diels-Alder reaction, with exocyclic diene 46 to construct ring G. As in the previous work by Kishi, the Diels-Alder cycloaddition proceeded with complete regioselectivity. The stereoselectivity, however, was modest. Four diastereomers were obtained, with a small preference towards desired 47, which was

OTBDPS

OBn OBn

6 steps

TBDPSO

O

O

O O

TBSO

O

TBSO

O

O

O

O

O

OTBS

OTBS

46 45

44

TBSO

OBn

O

p-xylene 160 °C, 12 h

O

6 steps

OTBDPS O O

TMSO

O

H

O

O

O

OTBDPS

H

O TBSO

O

+ O

TBSO

OTBS

O

O

O

OTBS

48

47 + other isomers 81% combined, 35% of 47 OTBS

H 2N

[CpRu(MeCN) 3]PF6 acetone, 50 °C, 15 min

H O

O

79% TBSO

OTBDPS

COOH O

OTBS

TBSO

O

49

O

O

50

N

chlorobenzene 120 °C, 18 h

H O

O

84% (90% conversion)

H O

O O

O

O

12 steps

TMSO

COOH O

TBSO

O

OTBS

HF-MeCN, 12 h Pinnatoxin A 91%

O

51

Scheme 3.11

Nakamura and Hashimoto’s synthesis of pinnatoxin A.

OTBS

62

Chapter 3

isolated in 35% yield. This product was further elaborated to enyne 48, a substrate for the ruthenium-catalyzed cycloisomerization (Trost et al., 2005). The cycloisomerization proceeded readily, giving macrocyclic product 49 in 79% yield, establishing the complete set of stereochemistry and functionalization required for completing the pinnatoxin A structure. Removal of the protecting groups and functional group transformations produced the natural product. Notably, while thermal conditions were used for the cyclic imine formation directly from amino ketone, the cyclization occurred under significantly milder conditions (120 ∘ C, chlorobenzene), evidently due to catalysis by the unprotected carboxylic acid present in the substrate. The pentacyclic ketal, 44, was prepared by a convergent synthesis from building blocks 52 and 53 (Scheme 3.12). The protected C28-C31 polyol of 52 was inherited from arabinose, with inversion of one of the chiral centres via oxidation/reduction (Schmidt & Gohl, 1979). The tertiary chiral centre at C27 was set by a diastereoselective conjugate addition of a methylcuprate. Fragment 53, in turn, was assembled in a noteworthy fashion by using both enantiomers of malic acid. The C12 chiral centre was

8 steps

O

O 27

L-arabinose

O

28

OTBS

Diastereoselective

31

TBSO O

OTBDPS

52

cuprate conjugate addition

OBn OH

7 steps O

(R)-Malic acid

O

O O

TBSO PMBO

15

16

19 steps

12

OTES

S

(S)-Malic acid

OTBS

OTBDPS

54

OBn S

O

Aldol, elmination

53

From (S)-malic acid Gringard addition From (R)-malic acid OBn TBDPSO O

LiOMe, THF-MeOH 77% (+14% isomers)

O O

TBSO

O

TBSO O

CSA

CSA, MeOH

44

O

MeOH

62% 62%

55

2 steps

OBn

N

OEt O

56

1) MeMgBr, CuBr·DMS OBn 2) NaHMDS; MeI

Ph OBn

O

Ph

O

57

Scheme 3.12

10 steps N

O

76% over 2 steps

O

58

Preparation of the subunits.

O O O O

46

Chemistry of pinnatoxins

63

derived from (R)-malic acid, while the chirality in (S)-malic acid was used to set up the C15 tertiary alcohol centre. It was accomplished by preparation of an α-alkoxy ketone, followed by a chelation-controlled addition of methylmagnesium bromide. Fragments 52 and 53 were stitched together by a sequence of aldol condensation and elimination, setting up the substrate for preparation of dispiroketal 54. The formation of dispiroketal was performed, unlike in all other approaches, under basic conditions, relying on trapping of the hemiketal alkoxide by α, β-unsaturated ketone as the Michael acceptor. The ketalyzation proceeded to provide the expected ketal, 55, in 77% yield, along with 14% of isomeric products. Notably, the C23 stereogenic centre was also set in this transformation. The bicyclic ketal was subsequently constructed, along with concomitant removal of acetonide under the usual acidic conditions upon treatment with camphorsulfonic acid in methanol. The dienophile for the Diels-Alder reaction was prepared from oxazalidinone 57 via a sequence of stereoselective 1,4-cuprate addition and enolate methylation (Scheme 3.12), producing oxazalidinone, 58, which incorporates the desired vicinal chiral centre dyad. Chain elongation, lactonization, and methylenation set up the rest of the structure. Overall, the synthesis was completed in 55 steps for the longest linear sequence, with 0.2% overall yield of pinnatoxin A starting from (S)-malic acid. The Murai group reported a series of synthetic studies towards pinnatoxins that culminated in an impressive preparation of the pentacyclic ketal part, 60, in a single ketalyzation step from acyclic precursor 59 (Scheme 3.13). The transformation was achieved by fine-tuning of the ketalyzation conditions to affect the complete removal of the protecting group prior to the cyclization, while minimizing acid-catalyzed decomposition of the substrate. The substrate for this transformation was assembled from three fragments (Scheme 3.14). Two of these fragments, 62 and 64, were derived from acetylenic alcohols, relying on Sharpless epoxidation, followed by epoxide opening by appropriate nucleophiles to set up the requisite stereocentres. The third fragment, 61, was derived from aspartic acid. The coupling of the fragments was performed by addition of anion of sulfone to aldehyde, followed by oxidation of the resulting alcohol to ketone, and SmI2 -mediated desulfonation. The 1,2-diketone functionality in 59 was introduced by ruthenium dioxide catalyzed alkyne oxidation. Thus, the synthetic studies resulted in successful preparation of pinnatoxins A–C and G, which led to complete assignment of their structures. Significant amounts of

TESO

OSEM O

BnO MPMO TBSO

OMPM HF·Py, MeCN

TESO O

OBn

O O

O

OTBS

83% after 1 recycle

O

O

O

O

OSEM

O 59

Scheme 3.13

60

Murai’s formation of the pentacyclic ketal in one step.

Chapter 3

64

10 steps aspartic acid

Sulfone anion to aldehyde addition /oxidation/desulfonation

OTBS SO2Ph

MMTrO

61 OTBS

O

5 steps OH

OTBS

OPMB

O

63

OPMB

11 steps +

62

O

Sharpless epoxidation, Red-Al epoxide opening

OH

TESO

OSEM O

BnO PMBO OH

OTBS

17 steps

TESO

OSEM

BnO

4 steps

TBSO

TESO O

O

OTBS

SO 2Ph TESO

O

64 Sharpless epoxidation, cuprate epoxide opening Sharpless epoxidation, epoxide opening

Scheme 3.14

59

Sulfone anion to aldehyde addition /oxidation/desulfonation Ru mediated alkyne oxidation

Preparation of 59.

pinnatoxins A and G were also prepared and used for biological studies. Several aspects of the chemistry of pinnatoxin structure also came to light during these studies, most notably thermodynamic stability of the polyketal core and the unexpected difficulty in formation of the imine.

Chemical stability The integrity of the imine functionality in pinnatoxins has been demonstrated to be critical for biological activity, as the open ring amino ketone analogues have been inactive in toxicity tests (Aráoz et al., 2011), as is the case with spirolides (Bourne et al., 2010). The susceptibility of imines to hydrolysis, therefore, has important implications for biological activity of pinnatoxins. Other functionality in pinnatoxin structure, such as polyketal and the cyclohexene ring, can be susceptible to transformation as well, leading to loss of biological activity. During the first isolation studies it was noted that pinnatoxin A exists as a stable imine under aqueous acidic conditions (Uemura et al., 1995). In the synthetic studies that followed, the cyclic imine of pinnatoxin A was found to be uncharacteristically difficult to form. The initial attempts to close the imine using typical acidic conditions were unsuccessful, leading to competing reactions at harsher conditions, such as acetylation of the amine if acetic acid were used. The transformation could only be accomplished by thermolysis at 200 ∘ C in high vacuum (McCauley et al., 1998). Later studies found that the presence of a free carboxylic acid in the molecule facilitates the transformation, letting it proceed at significantly milder conditions, at 120 ∘ C for 18 hours (Nakamura et al., 2008). A similar effect was seen with the addition of an

Chemistry of pinnatoxins

100°C, 24 h pH 7.3 20% conversion

HN H O

O O

H2N

100°C, 72h pH 4.1

O

CO2 O

HO

H O

OH

100°C, 72h pH 4.1 or 7.3

O

CO 2 O

complete decomposition

OH

65

Pinnatoxin A

100°C, 24 h pH 7.3

65

N CO 2 O O

Scheme 3.15

OH

Study of stability of pinnatoxin A in water.

external acidic catalyst although, even then, the reaction required elevated temperatures and extended reaction times. In the toxicity studies, pinnatoxins, despite their lower toxicity in intraperitoneal injection studies than spirolides, were significantly more toxic by the oral route. For instance, pinnatoxin F was only four times less toxic by voluntary intake, compared to 35 times for spirolide A, > 80 for gymnodimine, and 145 times for 13-desmethyl spirolide C (Munday et al., 2012). It was also found that pinnatoxins, unlike spirolides, retain toxicity after basic treatment in aqueous methanol (Rundberget et al., 2011). All of the above observations point to an increased hydrolytic stability of pinnatoxins, compared to other cyclic imine toxins. This prompted a specific study of the stability of the pinnatoxin imine ring, using the synthetic pinnatoxin A (Jackson et al., 2012). In this study, pinnatoxin A and its hydrolyzed form, aminoketone 65, were treated in water under physiologically relevant neutral and acidic pH. As expected, pinnatoxin A displayed notable stability, not showing any signs of hydrolysis or decomposition at 40 ∘ C in water at pH 4.0 for 48 hours, and at pH 1.5 for 24 hours, and only slowly undergoing hydrolysis at 100 ∘ C at pH 7.3. Curiously, while hydrolysis of pinnatoxin A was observed under these conditions, no formation of pinnatoxin A occurred from aminoketone 65 under these or any other examined aqueous conditions. Harsher conditions (100 ∘ C, pH 4.1) resulted in decomposition of 65. This indicates that the lack of hydrolysis is due to kinetic, not thermodynamic, factors. Also noteworthy is that no retro Diels-Alder ring opening was observed (Scheme 3.15). Notably, while the imine ring is resistant to transformations under acidic conditions, slow rearrangement of the spiroketal fragment of pinnatoxins E and F was observed on treatment in solutions of formic and trifluoroacetic acids (Selwood et al., 2010). Pinnatoxin G, however, would not undergo this rearrangement, most likely due to absence of the hydroxyl group at C22. The lactone ring in pinnatoxin F was also very sensitive

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to hydrolysis under basic conditions, as was the similar fragment in gymnodimine (Kong et al., 2011).

Conclusions Since their first isolation in 1995, pinnatoxins have been the subject of keen interest from both toxicologists and synthetic chemists, due to their acute toxicity and their intricate structure. They have been found in marine organisms around the world, raising concerns of possible risks associated with them and necessitating the monitoring of their levels. Important advances have been made in understanding the occurrence, transformations and biological activity of pinnatoxins. Dinoflagellate Vulcanodinium rugosum have been identified to be a producer of pinnatoxins. Synthetic studies have revealed key aspects of chemistry of pinnatoxins, and have provided the conclusive confirmation of their chemical structure. As a result of synthetic efforts, significant amounts of these compounds have become available, leading to identification of nicotinic acetylcholine receptors (nAChRs) as the protein target of pinnatoxins. New methods for detection of pinnatoxins have become available as a result of these findings. Studies of chemical stability have provided a likely explanation for an increased oral toxicity of pinnatoxins compared to other members of cyclic imine toxin family. The studies of pinnatoxins are continuing, and will undoubtedly bring new insights into the chemistry, biological activity and biotransformations of these fascinating compounds.

References Aráoz, R., Servent, D., Molgó, J. et al. (2011) Total Synthesis of Pinnatoxins A and G and Revision of the Mode of Action of Pinnatoxin A. Journal of the American Chemical Society, 133, 10499–10511. Aráoz, R., Ramos, S., Pelissier, F. et al. (2012) Coupling the Torpedo Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins. Analytical Chemistry, 84, 10445–10453. Bourne, Y., Radic, Z., Aráoz, R. et al. (2010) Structural Determinants in Phycotoxins and AChBP conferring High Affinity Binding and Nicotinic AChR Antagonism. Proceedings of the National Academy of Sciences of the United States of America, 107, 6076–6081. Chou, T., Haino, T., Kuramoto, M. and Uemura, D. (1996a) Isolation and Structure of Pinnatoxin D, a New Shellfish Poison from the Okinawan Bivalve. Pinna muricata. Tetrahedron Letters, 37, 4027–4030. Chou, T., Kamo, O. and Uemura, D. (1996b) Relative Stereochemistry of Pinnatoxin A, a Potent Shellfish Poison from Pinna muricata. Tetrahedron Letters, 37, 4023–4026. Commission Regulation (2011). Official Journal of the European Union 54, 3−7. Fonfría, E.S., Vilariño, N., Molgó, J. et al. (2010) Detection of 13,19-didesmethyl C Spirolide by Fluorescence Polarization using Torpedo Electrocyte Membranes. Analytical Biochemistry, 403, 102–107. Furuta, K., Iwanaga, K. and Yamamoto, H. (1986) Asymmetric Diels-Alder Reaction. Cooperative Blocking Effect in Organic Synthesis. Tetrahedron Letters, 27, 4507–4510. Hess, P., Abadie, E., Hervé, F. et al. (2013) Pinnatoxin G is Responsible for Atypical Toxicity in Mussels (Mytilus Galloprovincialis) and Clams (Venerupis Decussata) from Ingril, a French Mediterranean Lagoon. Toxicon, 75, 16–26. Jackson, J.J., Stivala, C.E., Iorga, B.I. et al. (2012) Stability of Cyclic Imine Toxins: Interconversion of Pinnatoxin Amino Ketone and Pinnatoxin A in Aqueous Media. Journal of Organic Chemistry, 77, 10435–10440.

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Kita, M. and Uemura, D. (2005) Iminium Alkaloids from Marine Invertebrates: Structure, Biological Activity, and Biogenesis. Chemistry Letters, 34, 454–459. Kong, K., Moussa, Z., Lee, C. and Romo, D. (2011) Total Synthesis of the Spirocyclic Imine Marine Toxin (–)-Gymnodimine and an Unnatural C4-Epimer. Journal of the American Chemical Society, 133, 19844–19856. Lu, C. and Zakarian, A. (2007) Studies toward the Synthesis of Pinnatoxins: The B,C,D-Dispiroketal Fragment. Organic Letters, 9, 3161–3163. Matsuura, F., Hao, J., Reents, R. and Kishi, Y. (2006a) Total Synthesis and Stereochemistry of Pinnatoxins B and C. Organic Letters, 8, 3327–3330. Matsuura, F., Peters, R., Anada, M. et al. (2006b) Unified Total Synthesis of Pteriatoxins and Their Diastereomers. Journal of the American Chemical Society, 128, 7463–7465. McCarron, P., Rourke, W.A., Hardstaff, W. et al. (2012) Identification of Pinnatoxins and Discovery of Their Fatty Acid Ester Metabolites in Mussels (Mytilus edulis) from Eastern Canada. Journal of Agricultural and Food Chemistry, 60, 1437–1446. McCauley, J.A., Nagasawa, K., Lander, P.A. et al. (1998) Total Synthesis of Pinnatoxin A. Journal of the American Chemical Society, 120, 7647–7648. Munday, R. (2008). Toxicology of Cyclic Imines: Gymnodimine, Spirolides, Pinnatoxins, Pteriatoxins, Prorocentrolide, Spiro-Prorocentrimine, and Symbioimines. In Botana, L.M. (ed.). Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection, 2nd edition (Chapter 27), pp. 581–594. Munday, R., Selwood, A.I. and Rhodes, L. (2012) Acute Toxicity of Pinnatoxins E, F and G to Mice. Toxicon, 60, 995–999. Nakamura, S., Kikuchi, F. and Hashimoto, S. (2008) Total Synthesis of Pinnatoxin A. Angewandte Chemie International Edition, 47, 7091–7094. Nézan, E. and Chomérat, N. (2011) Vulcanodinium Rugosum gen. et sp. nov. (Dinophyceae), un Nouveau Dinoflagellé Marin de la Côte Méditerranéenne Française. Cryptogamie Algologie, 32, 3–18. Pfaltz, A. and Mailenberger, A. (1982) Regioselective Opening of alpha- and beta-Alkoxyepoxides with Trimethylaluminum. Angewandte Chemie International Edition, 21, 71–72. Qin, Y., Stivala, C.E. and Zakarian, A. (2007) Acyclic Stereocontrol in the Ireland-Claisen Rearrangement of alpha-Branched Esters. Angewandte Chemie International Edition, 46, 7466–7469. Rhodes, L., Smith, K., Selwood, A. et al. (2010) Production of Pinnatoxins by a Peridinoid Dinoflagellate Isolated from Northland, New Zealand. Harmful Algae, 9, 384–389. Rundberget, T., Aasen, J.A.B., Selwood, A.I. and Miles, C.O. (2011) Pinnatoxins and Spirolides in Norwegian Blue Mussels and Seawater. Toxicon, 58, 700–711. Sakamoto, S., Sakazaki, H., Hagiwara, K. et al. (2004) A Formal Total Synthesis of (+)-Pinnatoxin A. Angewandte Chemie International Edition, 43, 6505–6510. Schmidt, R.R. and Gohl, A. (1979) 2-O-Benzyl-D-ribose und 2 ’-O-Benzyladenosin. Chemische Berichte, 20, 1689–1704. Selwood, A.I., Miles, C.O., Wilkins, A.L. et al. (2010) Isolation, Structural Determination and Acute Toxicity of Pinnatoxins E, F and G. Journal of Agricultural and Food Chemistry, 58, 6532–6542. Smith, K.F., Rhodes, L.L., Suda, S. and Selwood, A.I. (2011) A Dinoflagellate Producer of Pinnatoxin G, Isolated from Sub-tropical Japanese Waters. Harmful Algae, 10, 702–705. Stivala, C.E. and Zakarian, A. (2008) Total Synthesis of (+)-Pinnatoxin A. Journal of the American Chemical Society, 130, 3774–3776. Takada, N., Umemura, N., Suenaga, K. et al. (2001) Pinnatoxins B and C, the Most Toxic Components in The Pinnatoxin Series from The Okinawan Bivalve Pinna Muricata. Tetrahedron Letters, 42, 3491–3494. Trost, B.M., Frederiksen, M.U. and Rudd, M.T. (2005) Ruthenium-Catalyzed Reactions – A Treasure Trove of Atom-Economic Transformations. Angewandte Chemie International Edition, 44, 6630–6666. Uemura, D., Chou, T., Haino, T. et al. (1995) Pinnatoxin A: A Toxic Amphoteric Macrocycle from the Okinawan Bivalve Pinna muricata. Journal of the American Chemical Society, 117, 1155–1156.

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Vilariño, N., Fonfría, E.S., Molgó, J. et al. (2009) Detection of Gymnodimine-A and 13-Desmethyl C Spirolide Phycotoxins by Fluorescence Polarization. Analytical Chemistry, 81, 2708–2714. Zeng, N., Gu, H., Rhodes, L. et al. (2012) The First Report of Vulcanodinium Rugosum (Dinophyceae) from the South China Sea with a Focus on the Life Cycle. New Zealand Journal of Marine and Freshwater Research, 46, 511–521. Zheng, S.Z., Huang, F.L., Chen, S.C. et al. (1990) The Isolation and Bioactivities of Pinnatoxin. Chinese Journal of Marine Drugs, 33, 33–35.

CHAPTER 4

Chemistry and analysis of PSP toxins Ana Botana & Verónica Rey López Department of Analytical Chemistry, University of Santiago de Compostela, Spain

Introduction Poisoning after ingestion of bivalves is a syndrome known from ancient times, the most common being paralytic shellfish poisoning (PSP) after shellfish intake. PSP are the most world wide spread group of toxins, affecting all Europe, South Africa, India, Morocco, the eastern Asiatic coast and also North and South America. Toxicity relies on many factors, such as the type of affected shellfish. Mussels are the most toxic ones, being responsible for most poisoning episodes. Although mussels are the best indicators for toxicity, they are not the only ones such, as it also affects clams, scallops and cockles. It is well known that PSPs regularly accumulate in shellfish through direct ingestion of toxic phytoplankton, or by the consumption of zooplankton or other organisms that have fed on toxic algae or that have been indirectly exposed to toxins (Sato et al., 1993; Landsberg, 2002; Jester et al., 2009). Recent studies detected the presence of toxins in fish samples (Petitpas et al., 2013; Fire et al., 2012; Berry et al., 2012; Costa et al., 2011). PSP toxins are a group of tetrahydropurines, all of them analogues to Saxitoxin (STX), the first typified and most studied one (it is the representative compound); 57 other analogues have also been researched (Wiese et al., 2010). Traditionally, STX analogues have been composed only of hydrophilic compounds and have been classified into three subgroups, taking into account the side-chains of substituents: 1 Carbamate (STX, gonyautoxins (GTX 1-4) and neoSTX). 2 Decarbamoyl (dcSTX, dcneoSTX, dcGTX1-4). 3 N-sulfo-carbamoyl (GTX5-6, C1-4). These groups present different toxicities, with the carbamoyl analogues being the most toxic, followed by decarbamoyl analogues with intermediate toxicity, and N-sulfocarbamoyl analogues the least toxic (Vale et al., 2008a, 2008b). Figure 4.1 shows the chemical structure of the more commonly found compounds in toxic dynoflagellates and poisoned seafood. All are derivatives from STX, and Table 4.1 shows their structures with names and synonims. Other hydrophilic STX analogues have been found, with a side-chain of acetate in R4 (Figure 4.1), in fresh water filamentous cyanobacterium, Lyngbya wollei, but not in the marine environment (Onodera et al., 1997). More recently, analogues from a new family of PSP toxins were isolated containing a hydrophobic side-chain. They were characterized from Gymnodinium catenatum Australian strains and named as GC1-GC3 (Negri et al., 2003).

Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

69

70

Chapter 4

R4

H

R1

NH N

NH2+ NH

N

NH2+

OH OH

Benzoate

Decarbamoyl

N-sulfocarbamoyl

Carbamate

R2

STX GTX2 GTX3 neoSTX GTX1 GTX4 GTX5 C1 C2 GTX6 C3 C4 dcSTX dcGTX2 dcGTX3 dcneoSTX dcGTX1 dcGTX4 GC1 GC2 GC3 GC4 GC5 GC6

R1 H H H OH OH OH H H H OH OH OH H H H OH OH OH H H H OH OH OH

Figure 4.1

R2 H H OSO3¯ H H OSO3¯ H H OSO3¯ H H OSO3¯ H H OSO3¯ H H OSO3¯ H OSO3¯ H H OSO3¯ H

R3

R3 H OSO3¯ H H OSO3¯ H H OSO3¯ H H OSO3¯ H H OSO3¯ H H OSO3¯ H OSO3¯ H H OSO3¯ H H

R4 O

NH2

O

O

O

O3SNH

OH¯

O O

OH

STX structure and its derivatives.

PSP toxins block specifically the excitation current in nerves and muscle cells by means of position one in the sodium channel (Messner & Caterall, 1986); therefore, accumulation of PSP toxins in molluscs is a serious problem for public health, and affects fisheries industries. The monitoring of these compounds is unavoidable and biological assays, specifically the rat or mouse bioassay (MBA), have been used for a long period of time to establish sample toxicity, either in a qualitative or quantitative way. Nevertheless, these tests have attracted a lot of controversy, due to ethical reasons regarding animal research. Modern analytical techniques, such as immunoassays, non-animal bioassays and methods that use analytical instrumentation, have been investigated thoroughly, to replace animal bioassays in an accurate way and get reliable results in routine monitoring programs. These methods have proved in many cases to be more rapid, sensitive

Chemistry and analysis of PSP toxins

Table 4.1

71

STX and their derivative structures.

Saxitoxin (STX) Neosaxitoxin (neoSTX) Decarbamoyl saxitoxin (dcSTX) Decarbamoyl neosaxitoxin (dcneoSTX) Gonyautoxin1 (GTX1) Gonyautoxin2 (GTX2) Gonyautoxin3 (GTX3) Gonyautoxin4 (GTX4) Gonyautoxin5 (GTX5) B1 Gonyautoxin6 (GTX6) B2 Decarbamoyl gonyautoxin1 (dcGTX1) Decarbamoyl gonyautoxin2 (dcGTX2) Decarbamoyl gonyautoxin3 (dcGTX3) Decarbamoyl gonyautoxin4 (dcGTX4) C1 C2 C3 C4

R1

R2

R3

R4

H OH H OH OH H H OH H OH OH H H OH H H OH OH

H H H H OSO3– OSO3– H H H H OSO3– OSO3– H H OSO3– H OSO3– H

H H H H H H OSO3– OSO3– H H H H OSO3– OSO3– H OSO3– H OSO3–

CONH2 CONH2 H H CONH2 CONH2 CONH2 CONH2 CONHSO3– CONHSO3– H H H H CONHSO3– CONHSO3– CONHSO3– CONHSO3–

and specific, and to provide a wider spectrum of information about the presence of different toxins. These alternative methods must be able to deal with complex matrices and be capable of fully differentiating toxins under study from toxins of other groups or nontoxic compounds (Thompson et al., 2002; Anon., 2006). There is an important factor to take into account, however, namely the use of certified reference materials that some of the analytical instrumentation methods need. There are no standards for all of the existing toxins and, although their availability has been expanded in recent years, there is still an important lack of these materials. Official control methods must be previously validated (Anon., 2004), and for quantitative methods it is necessary to know the following parameters: limit of detection (LOD); limit of quantitation (LOQ); accuracy; precision; repeatability; reproducibility; applicability (matrix and concentration range); recovery; selectivity; sensitivity; linearity; and ruggedness (Thompson et al., 2002). One instrumental approach includes the use of high-performance liquid chromatography (HPLC), being the most common type that uses reversed phase, where the stationary phase is nonpolar (typically a bonded C18 chain) and the mobile phase is usually prepared from a mixture of aqueous buffers and organic solvents. Other modes include ion-pairing and ion-exchange chromatography, which both use a separation based on charged functional groups (Turner, 2014). Hydrophilic interaction liquid chromatography (HILIC) has also been used for polar toxins. All of these techniques are based on the separation of toxins before they reach the detector, where they are characterized based on their retention time, giving sensitive and specific analyses. One specific type of detection is mass spectrometry (MS), which originates an important group of methods when coupled to HPLC. Liquid chromatography with mass spectrometry detection (LC-MS) has become an

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important research tool in the field of marine biotoxins, due to its high sensitivity and specificity.

Methods of analysis Sommer and Meyer (1937) were the first to develop a method to determine PSP toxins. Their mouse bioassay method (MBA) established the basis and is the internationally accepted reference method to quantitate PSP toxicity, used all over the world in monitoring programmes. The method has several drawbacks, including low sensitivity, poor reproducibility, and ethical problems due to the use of a huge number of live animals. It was developed more than 50 years ago and afterwards refined and developed by AOAC to produce quick and reasonably accurate data about total PSP toxins content (Hollingworth & Wekell, 1990). It has been the regulatory method for approximately 50 years and is the official method of AOAC International. It is also the reference method in the European Union (EU) (Commission Regulation (EU), 2011), which establishes that total PSP content must not exceed 80 μg STX eq∕100 g of tissue. The time of exposure to death is used in mouse bioassay (MBA) to estimate the amount of toxin present in molluscs, with a detection limit for the method of 40 μg STX eq∕100 g. Although the MBA method has been shown to be very reliable, there is international pressure to reduce or eliminate assays that make use of animals. MBA gives little information about the toxic profile, but has the advantage of informing about total toxicity of the sample. The toxicity of the sample is calculated based on dose-response curves established for STX standards and expressed in mouse units (MU). The disadvantages are: detection limit of the method depends on the species; time to death related to toxin level is not linear; it is very difficult to calculate exactly the time to death; and implies the sacrifice of a great number of animals. If we compare MBA and HPLC methods, we find that, while MBA determines sample toxicity directly, HPLC methods provide qualitative information on individual toxins. As sample toxicity can only be accurately known by having data on toxicity equivalence factors (TEFs) that describe the toxicity for each toxin related to the most potent analogue of the group, the parent STX, up to now some studies have been published that calculate relative toxicities for PSPs based on MBA toxicity studies (Table 4.2). The values reported by Oshima (Oshima, 1995) have been widely used and, more recently, the European Food Standards Agency (EFSA) published the revised TEFs for the most commonly occurring STX analogues (EFSA, 2009). The importance of these TEFs values is crucial because toxicity varies for each quantitative instrumental analysis when applying different TEFs for toxicity calculation (Turner et al., 2009), as we can see in Table 4.2. The Community Reference Laboratory for Marine Biotoxins (CRLMB, 2009) recommended the EFSA TEFs, which values are similar to Oshima’s, although there is a remarkable difference in TEF for dcSTX, twofold higher, and data for C1 and C3 toxins is absent. The fact that dcSTX TEF is so different will affect for those toxins converted in it. The results can be different when dcSTX is present, depending on the value taken, and also for those species that exhibit enzymatic transformation of carbamate and N-sulfocarbamoyl toxins to their decarbamoyl counterparts (Artigas et al., 2007).

Chemistry and analysis of PSP toxins

Table 4.2

73

Toxicity equivalent factors (TEFs).

Toxin STX GTX1 GTX2 GTX3 GTX4 GTX5 GTX6 NEO dcNEO dcSTX dcGTX2 dcGTX3 C1 C2 C3 C4 11-hydroxy-STX

Oshima

EFSA

1.0000 0.9940 0.3592 0.6379 0.7261 0.0644 – 0.9243 – 0.5131 0.1538 0.3766 0.0060 0.0963 0.0133 0.0576 –

1.0 1.0 0.4 0.6 0.7 0.1 0.1 1.0 0.4 1.0 0.2 0.4 – 0.1 – 0.1 0.3

Therefore, it was very convenient to reduce or eliminate completely the bioassay for PSPs in a regulatory environment. The European Union has established the end of 2014 as the deadline to use mouse bioassay as the official method for the analysis of PSP toxins (Commission Regulation (EU), 2011).

Chemical methods Different approximations to replace MBA have been explored; chemical methods used to determine PSP toxins have been fluorimetric assays, HPLC with fluorimetric detection (either pre-column or post-column), and liquid chromatography-mass spectrometry (LC/MS). Alkaline oxidation of PSP toxins produce fluorescent compounds, allowing the determination by fluorimetric techniques (Bates & Rapoport, 1975; Bates et al., 1978). However, these techniques are exposed to certain variability: pH adjustment during extraction and before oxidation is critical; an ionic exchange column is required to isolate fluorescent co-extracts; and the presence of several metals can affect oxidation and ulterior fluorescent yield. As toxins do not have all the same fluorescence, it happens that fluorescence is weak for some carbamate toxins. Fluorescence assay results can be one order of magnitude higher than mouse bioassay, and colorimetric assay is slightly more sensitive (Mosley et al., 1985). The more used chemical methods are liquid chromatography with pre- or post-column oxidation, followed by fluorescence detection. This instrumental technology can examine samples as well as supplying detailed information about toxic profile, because actually there is a variety of calibration solutions.

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Liquid chromatography Prior to HPLC development, other chromatographic techniques arose, such as thin layer chromatography (Buckley et al., 1976) and column liquid chromatography (Buckley et al., 1978). The first steps in HPLC were performed at the beginning of the 1980s (Boyer, 1980; Rubinson, 1982). All of these techniques lacked the ability either to efficiently separate or to detect low levels of toxins. Lately, an HPLC method was developed that partially resolved these problems (Sullivan & Iwaoka, 1983), and it was studied for its correlation with mouse bioassay (Sullivan et al., 1983a). While results for this study appeared to be promising, correlation with mouse bioassay was only 0,76, possibly due partially to the poor sensitivity for some PSP toxins. Till now, HPLC methods are the most widely used to quantify PSP toxins present in seafood, and it is also useful to know the PSP profiles, due to their characteristic of identifying compounds. Many efforts have been employed to develop automatized liquid chromatography methods for routine analysis of PSP toxins. These toxins only have a weak chromophore group and they have to be modified before detection. When they are oxidized in alkaline solution, a purine is formed that is fluorescent at acidic pH. This reaction can be either pre-column or post-column, and purines obtained are monitored with a fluorescence detector. Bates and Rapoport (1975) were the first authors who studied the alkaline oxidant conditions of PSP toxins to obtain fluorescent compounds; later, Buckley et al. (1978) incorporated them into their method and established the basis of the HPLC method for these compounds with post-column reaction. Late modifications (Sullivan et al., 1984) were able to improve separation and sensitivity for all PSP toxins. These authors could separate non-derivatized PSP toxins by means of ion-pair chromatography, adding alkylsulphonic acids followed by post-column oxidation with periodic acid. A drawback of the method was the coelution of STX and dcSTX, resulting in a wrong total PSP toxicity, as STX toxicity is twice that of dcSTX. In 1984, Oshima et al. proposed a method based on alkaline oxidation to yield highly fluorescent derivatives that had some problems in separating toxins such as GTX1 from GTX4 and GTX3 from GTX5. Later, a method was described (Oshima et al., 1989) with a complete separation for toxins. Depending on acidity, three groups of PSP toxins were determined by isocratic elution in three stages: • group 1: C1-C4 • group 2: GTX1-GTX4, dcGTX1-dcGTX4, GTX5 and GTX6 • group 3: Neo, dcSTX and STX A RP-C8 column was applied as stationary phase. Tetrabutylammonium phosphate was used (eluent A) for separation of toxins C1–C4, and eluents B and C contained n-heptanesulphonic acid as ion-pair precursors for the separation of carbamate and decarbamoyl toxins. This method is expensive, because three chromatographic elutions have to be made in order to quantify all PSP toxins in one sample. Later it was optimized (Oshima, 1995) to separate and quantify all toxins in small-sized samples. This study showed that the main reason for discrepancy with bioassay is the lack of accuracy for the latter. It is well known that bioassay shows significant errors (McFarren, 1959; Park et al., 1986), especially for low toxicity samples. However, this chromatographic method requires three different isocratic elutions, and it is time-consuming and laborious. For this reason, other methods were proposed, and it is worth highlighting the development of two different methods, based on both preand post-column oxidation, which have been developed since the 1990s.

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Pre-column HPLC-FLD Pre-column oxidation involves the derivatization of sample extracts with chemical oxidation prior to chromatographic analysis; the extraction solvent is acetic acid. Lawrence et al. (1991) proposed a liquid chromatography method, with prechromatographic oxidation using hydrogen peroxide and periodic acid. N1-hydroxilated toxins (NEO, dcNEO, GTX1 and GTX4, and C3 and C4) formed fluorescent products after oxidation with periodate at ≈pH = 8, 7, but they did not form fluorescent derivatives with peroxide oxidation. Non-N1-hydroxilated toxins (STX, GTX5, GTX2, GTX3, C1 and C2) formed highly fluorescent derivatives, either for peroxide oxidation or for periodate oxidation. The addition of ammonium formiate for periodate oxidation greatly increased the yield of N1-hydroxilated toxins (Lawrence & Menard, 1991). These highly fluorescent polar oxidation products were amenable to reverse-phase HPLC utilizing gradient elution with low proportions of organic solvent. Oxidation of GTX2 and GTX3, STX, GTX5, and C1 and C2 produced single oxidation products; the epimeric toxins (GTX2 and GTX3, GTX1 and GTX4, C1 and C2, C3 and C4, dcGTX2 and dcGTX3) yielded identical oxidation products. Also, secondary oxidation products from the N1-hydroxylated toxins were found to be either identical to, or similar in chromatographic retention to, the primary oxidation products formed following peroxide oxidation of the non-N1-hydroxylated toxins. The method was modified, changing chromatographic conditions to reduce analysis time and improve separation (Lawrence et al., 1995). In 2006, this method, known as the ‘Lawrence method’ (Lawrence & Menard, 1991; Cleroux et al., 1995; Lawrence & Niedzwiadek, 2001; Lawrence et al., 2005) and formally validated as AOAC 2005.06 OM (Anon., 2004), was adopted by European Commission legislation (Anon., 2006). The method includes extraction of seafood homogenates in acetic acid, followed by several clean-up steps: • a solid-phase extraction (SPE) cleanup step, using C18-bonded cartridges to remove interferences from naturally fluorescent matrix co-extractives; • a second SPE cleanup step, involving a weak ion exchange to separate extracts into three different fractions; • one derivatization step of the sample, including oxidation of the extracts, as was mentioned in the previous paragraph, to produce oxidation products of fluorescent toxins. In this way, it was possible to separate, prior to oxidation, NEO from GTX6, and C3 and C4 from GTX1 and GTX4. In Figure 4.2, four chromatograms are shown for toxins analysis as an example of Lawrence method separation. The Lawrence et al. method has been subjected to an interlaboratory study (Lawrence et al., 2004) and to a collaborative study (Lawrence et al., 2005), and was adopted by AOAC as the first analytical alternative to MBA (Official Methods of Analysis, 2005). It is based on pre-column oxidation of PSP toxins with hydrogen peroxide and sodium periodate, followed by fluorimetric detection, and it was validated for determination of STX, NEO, GTX2,3, GTX1,4, dcSTX, GTX5, C1,2 and C3,4 in molluscs (mussel, clam, oyster and scallop). In 2009, a validation study was undertaken that allowed the extension of the method to two more toxins: dcNEO and dcGTX2,3 (Turner et al., 2009). More recently, studies have extended the validation to dcGTX2 and dcGTX3 in mussels and clams, confirming the levels of reproducibility described in the original collaborative trial (Lawrence et al., 2005), but giving evidence for poor reproducibility for some toxins including dcNEO (Ben-Gigirey et al., 2012).

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Although it matches the main safety criteria regarding MBA equivalency, it also has several drawbacks when applied in a regulatory environment. The main impediment is the amount of time needed to process samples containing significant concentrations of PSPs. The Lawrence method also cannot distinguish between isomer toxins that have significantly different toxicities. Other studies have been accomplished to improve automation and efficiency for those laboratories using this method routinely (DeGrasse et al., 2011; Janecek et al., 1993; Flynn & Flynn, 1996).

Post-column HPLC-FLD Post-column methods are based on the original Oshima method and were developed because the Lawrence method did not fulfil the requirements to completely separate and quantitate PSP toxins. HPLC with post-column oxidation (PCOX) separates toxins, and then oxidation takes place online through a continuous flow reaction system. After elution of PSPs from the end of the HPLC column, the toxins mix with an oxidant, such as periodate, before passing through a reaction coil, where derivatization takes place. The derivatized compounds elute from the end of the reaction coil, and the reaction mixture is quenched with a strong acid, generating the formation of stable oxidation products. Each PSP toxin subsequently reaches the fluorimetric detector. Oshima described some modifications to his post-column oxidation method (Oshima, 1995), using a longer C8 reverse-phase HPLC column, modified reaction coil dimensions and three separate analytical runs per sample. The main changes also included an optimization in pH and mobile phase concentrations, the oxidant composition was changed, and a C18-SPE cleanup step was used to prevent the occurrence of false-positive analyses. dcNEO was the exception in separation, because it could not be separated from NEO, while the majority of toxins were adequately separated. Thomas et al. (2006) reduced the number of injections to two, but separation of GTXs and STXs took 60 minutes and used a trinary mobile phase system. To overcome these handicaps, a method to determine PSP toxins was developed (Rourke et al., 2008) that improved some of the problems of the latter method; a binary gradient elution system was employed that allowed to reduce elution system to 24 minutes. All GTX and STX toxins studied were completely resolved except GTX5, which was 50% resolved. C toxins were completely resolved and quantified in less than 15 minutes, with an isocratic system similar to Oshima’s (Oshima, 1995). Differences between the new post-column method for C toxins and Oshima’s included a different cleaning-up procedure, a different concentration of tetrabutylammonium phosphate, a different LC column, and different oxidation conditions. dcNEO was not included in this study due to its coelution with NEO. In Figure 4.3, PCOX analysis is shown as an example to illustrate separation of standards. The refined rapid version of the Oshima method developed in Canada was tested to prove its performance (Rourke et al., 2008) based on the analysis of HCl extracts of shellfish. Results indicated that linearity was very good for all toxins investigated over a wide linear range. They also proved that detection limits, as well as toxin recoveries, were acceptable. After this work, a full single-laboratory validation was undertaken, focusing on the method performance characteristics in blue mussels, soft-shell clams, American oysters and sea scallops (van de Riet et al., 2009). Given the good results of the single-laboratory validation, the method was subjected to a full collaborative study (van de Riet et al., 2011). Method performance was done for GTX1-5, NEO,

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STX, dcSTX, dcGTX2 and dcGTX3, and C1 and C2. As the availability of materials containing adequate amounts of C1 and C2 was not good, the results of reproducibility and repeatability for these toxins were poor. Also, the number of materials containing GTX5, dcSTX, and dcGTX2 and dcGTX3 was relatively limited. However, the results demonstrated that the method is suitable for official control testing, and it was accepted by the AOAC in 2011 as an alternative method (Official Method, 2011.02; Anon., 2011). The PCOX method was also approved as a limited use method in the USA by the Interstate Shellfish Sanitation Conference (ISSC) and National Shellfish Sanitation Program (NSSP) (NSSP, 2011).

Liquid chromatography-mass spectrometry Pre- and post-column HPLC methods present several benefits, which include a high sensitivity for low concentrations and less variability for results. However, they have several drawbacks that should be sorted out. Hydrophobic analogues are retained by C18 resins (Negri et al., 2003), so therefore HPLC methods will not allow their presence in monitoring programmes (Vale, 2008b; Vale et al., 2009). LC-MS methods are

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actually being developed to get a good characterization of these compounds, so it is recommended that the presence of PSP toxins should be confirmed by mass spectrometry. However, the use of reversed phase conditions, which generally consist of some organic solvent and non-volatile salts, is not suitable for LC-MS; mobile phases with phosphate content, as well as ion-pair formers, are a handicap for an efficient application of the LC-MS technique. Therefore, Jaime et al. (2001) proposed the application of ionic exchange chromatography, with eluents containing only volatile compounds to quantify PSP toxins either with fluorometric or mass spectrometry detection. A HPLC method was developed to determine PSP that allows direct coupling of HPLC with mass spectrometry (Kirschbaum et al., 1995). In the case of parallel fluorimetric and HPLC-MS detection, post-column electrochemical derivatization was suggested to avoid contamination of the ionic source with chemical oxidation compounds. Electrospray Ionization-MS (ESI-MS) is effective to detect polar PSP toxins (Quilliam, 2003; Dell’Aversano et al., 2005). However, variations in retention times were observed for PSP toxins in different seafood matrices. It has not yet been possible to achieve a complete resolution of all PSP toxins, and sensitivity for MS detection methods is not good enough to control toxin levels present in seafood at established regulatory limits (Diener et al., 2007). The most promising approach is the use of ionic liquid chromatography with hydrophilic interaction (HILIC), which can be applied for the separation of polar and charged compounds. The first HILIC-MS/MS method for a wide variety of PSP toxins was developed by Quilliam et al., and studied in depth by Dell’Aversano et al. (Quilliam et al., 2001; Dell’Aversano et al., 2005). On the other hand, as PSP toxins are a wide family of compounds with about 50 structural analogues of STX with different toxicities, most of them being produced by dinoflagellates, once they accumulate in shellfish, their relative proportions can change due to biotransformations. These changes include the hydrolysis of the carbamoyl group in C17 (N-sulfocarbamoyl > carbamoyl > decarbamoyl), the reduction of group R1 to N1 (−OH > −H), and epimerization of group 11-hydroxisulphate (Negri et al., 2003). Recently, some new analogues, named M1–M5, have been identified in mussels (Mytilus edulis and M. trossulus) contaminated by Alexandrium tamarense blooms on the east coast of Canada (Dell’Aversano et al., 2008). Presumably, they were metabolites with relatively low toxicity, because they were present only in shellfish, not in the precursor algae. Although there have been many studies about biotransformations of PSP in shellfish using feed transfer experiments, there is a lack of discussion about these new metabolites M1–M5, because they have not been detected in most studies. Their response is very low in fluorescent oxidation derivatives-based HPLC methods, and they can only be measured using LC-MS/MS. Most biotransformation studies are focused on hydrolysis of the N-sulfocarbamoyl moiety and epimerization of 11-hydroxisulphate (Samsur et al., 2007; Oikawa et al., 2008). PSP components in toxic algae exist almost exclusively as β-epimers (GTX3, GTX4, C2 and C4), while α-epimers (GTX1, GTX2, C1 and C3) predominate in shellfish profile, with epimer α : β ratio close to 3 : 1 at equilibrium (Asakawa et al., 2005; Choi et al., 2006). Although PSP analysis provides the composition profile for a specific toxic episode, the use of surface plasmon resonance biosensors has reached quite a good level of development (Campbell et al., 2007). The high sensibility of this approach allows for a quick yield, because sample extraction and detection is quite simple, and chips can be used many times (Fonfría et al., 2007). For this technology, a method based on

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antibodies was described and was validated in a single laboratory study (Campbell et al., 2010), and also in an interlaboratory study (van den Top et al., 2011). The drawbacks in LC-MS/MS are several, the first being the in-source fragmentation, resulting in the loss of SO3 for the PSP toxins with a sulfonic acid in the α-orientation on the C-11 group. This can be a problem for identification because, for example, gonyautoxin-2 predominantly gives a precursor in ESI which is the same as the one for neo-saxitoxin. According to Turrell et al., the sensitivity for some of the PSP toxins is poor, and matrix effects occur that have an effect on sensitivity and retention time stability (Turrell et al., 2008). In summary, until now, LC-MS is being used at a research level, but the methods are not developed to be employed at a regulatory level.

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Petitpas, C.M., Turner, J.T., Deeds, J.R. et al. (2013). PSP toxin levels and plankton community composition and abundance in size-fractionated vertical profiles during spring/summer blooms of the toxic dinoflagellate Alexandrium fundyense in the Gulf of Maine and on Georges Bank, 2007, 2008, and 2010: 2. Plankton community composition and abundance. Deep Sea Research Part II: Topical Studies in Oceanography. (In press). Quilliam, M.A. (2003) The role of chromatography in the hunt for red tide toxins. Journal of Chromatography A, 1000, 527–48. Quilliam, M.A., Hess, P., Dell’Aversano, C. (2001). Recent developments in the analysis of phycotoxins by liquid chromatography-mass spectrometry. In: De Koe, W.J., Samson, R.A., Van Egmond, H.P., Gilbert, J. and Sabino, M. (eds). Mycotoxins and Phycotoxins in Perspective at the Turn of the Century. The Netherlands: Wageningen, Ponsen and Looijen. Rourke, W.A., Murphy, C.J., Pitcher, G. et al. (2008) Rapid postcolumn methodology for determination of paralytic shellfish toxins in shellfish tissue. Journal of AOAC International, 91, 589–97. Rubinson, K.A. (1982) HPLC separation and comparative toxicity of saxitoxin and its reaction products. Biochimica et Biophysica Acta, 687, 315–20. Samsur, M., Takatani, T., Yamaguchi, Y. et al. (2007) Accumulation and elimination profiles of paralytic shellfish poison in the short-necked clam Tapes japonica fed with the toxic dinoflagellate Gymnodinium catenatum. Journal of the Food Hygienic Society of Japan, 48, 13–18. Sato, S., Ogata, T., Kodama, M. (1993). Wide distribution of toxins with sodium channel blocking activity similar to tetrodotoxin and paralytic shellfish toxins in marine animals. In Smayda, T.J., Shimizu, Y. (eds). Toxic phytoplankton blooms in the sea. Amsterdam: Elsevier. Sommer, H.A. and Meyer, K.F. (1937) Paralytic shellfish poisoning. Archives of Pathology, 24, 560–598. Sullivan, J.J. and Iwaoka, W.T. (1983) High pressure liquid chromatographic determination of toxins associated with paralytic shellfish poisoning. Journal of the Association of Official Analytical Chemists, 66, 297–303. Sullivan, J.J., Iwaoka, W.T. and Liston, J. (1983) Enzymatic transformation of PSP toxins in the littleneck clam (Protothaca staminea). Biochemical and Biophysical Research Communications, 114, 465–72. Sullivan, J.J. and Wekell, M.M. (1984) Determination of paralytic shellfish poisoning toxins by high pressure liquid chromatography, in Seafood toxins (ed E.P. Ragelis), American Chemical Society, Washington DC. Thomas, K., Chung, S., Ku, J. et al. (2006). In: Henshilwood, K., Deegan, B., McMahon, T., Cusack, C., Keaveney, S., Silke, J., O’Cinneide, M., Lyons, D. and Hess, E. (eds). Molluscan Shellfish Safety, pp. 132–138. The Marine Institute, Galway, Ireland. Thompson, M., Ellison, S.L.R. and Wood, R. (2002) Harmonized guidelines for single laboratory validation of methods of analysis (IUPAC technical report). Pure and Applied Chemistry, 74, 835–855. Turner, A. (2014). Validation of HPLC detection methods for marine toxins. In: Botana, L.M. (ed.). Seafood and Freshwater Toxins: Pharmacology, Physiology and Detection. Boca Ratón: CRC Press. Turner, A.D., Norton, D.M., Hatfield, R.G. et al. (2009) Refinement and extension of AOAC Method 2005.06 to include additional toxins in mussels: single-laboratory validation. Journal of AOAC International, 92, 190–207. Turrell, E., Stobo, L., Lacaze, J.P. et al. (2008) Optimization of hydrophilic interaction liquid chromatography/mass spectrometry and development of solid-phase extraction for the determination of paralytic shellfish poisoning toxins. Journal of AOAC International, 91, 1372–86. Vale, C., Alfonso, A., Vieytes, M.R. et al. (2008) In vitro and in vivo evaluation of paralytic shellfish poisoning toxin potency and the influence of the pH of extraction. Analytical Chemistry, 80, 1770–6. Vale, P. (2008) Complex profiles of hydrophobic paralytic shellfish poisoning compounds in Gymnodinium catenatum identified by liquid chromatography with fluorescence detection and mass spectrometry. Journal of Chromatography A, 1195, 85–93. Vale, P., Rangel, I., Silva, B. et al. (2009) Atypical profiles of paralytic shellfish poisoning toxins in shellfish from Luanda and Mussulo bays, Angola. Toxicon, 53, 176–83.

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van de Riet, J.M., Gibbs, R.S., Chou, F.W. et al. (2009) Liquid chromatographic post-column oxidation method for analysis of paralytic shellfish toxins in mussels, clams, scallops, and oysters: single-laboratory validation. Journal of AOAC International, 92, 1690–704. van de Riet, J., Gibbs, R.S., Muggah, P.M. et al. (2011) Liquid chromatography post-column oxidation (PCOX) method for the determination of paralytic shellfish toxins in mussels, clams, oysters, and scallops: collaborative study. Journal of AOAC International, 94, 1154–76. van den Top, H.J., Elliott, C.T., Haughey, S.A. et al. (2011) Surface plasmon resonance biosensor screening method for paralytic shellfish poisoning toxins: a pilot interlaboratory study. Analytical Chemistry, 83, 4206–13. Wiese, M., D’Agostino, P.M., Mihali, T.K. et al. (2010) Neurotoxic alkaloids: saxitoxin and its analogs. Marine Drugs, 8, 2185–211.

CHAPTER 5

Chemistry of palytoxin and its analogues Patrizia Ciminiello, Carmela Dell’Aversano & Martino Forino Department of Pharmacy, University of Napoli Federico II, Italy

Introduction Palytoxins constitute a class of complex, extremely potent non-proteic marine biotoxins, first identified in a tropical Cnidarian zoanthid, a type of colonial anemone (Moore & Scheuer, 1971). This organism was known by native Hawaiians, who used it for its unique toxic properties some centuries prior to the discovery of palytoxin, and a bit of mythology surrounded it. According to an ancient Hawaiian legend, reported by a Hawaiian scholar in the 19th century (Malo, 1951), on the island of Maui near the harbour of Hana, in Mu’olea, there was a village of fishermen haunted by a curse. Every now and then, upon their return from the sea, the fishermen would find out that one of them was missing. The angry and desperate fishermen placed the blame on a lone, hump-backed farmer living on a cliff nearby. The villagers assaulted him but, as they ripped off the cloak from the hermit, they uncovered rows of sharp and triangular teeth within huge jaws. They had caught a shark god. It was clear that the missing villagers had been eaten by the shark god on their journeys to the sea. The fishermen killed the loner, burned him and threw his ashes into a tidepool nearby the harbour of Hana. Afterwards, a deadly moss started lining the walls of the tidepool, and the fishermen learnt that the moss, if used on their spears, was able to cause instant death to their victims. This way, the evil of the god was perpetuated. The moss growing in the cursed tidepool became known as ‘limu-make-o-Hana’, which literally means ‘seaweed of death from Hana’. Over the centuries, the pool became ‘kapu’ (taboo) to the local Hawaiians, who were convinced that an ill fate would befall anyone who disturbed the sacred site collecting the deadly seaweed. Nonetheless, in the early 1960s, thanks to some local informers, Philip Helfrich, from the University of Hawaii in Oahu, accompanied by the graduate student John Shupe, tracked down the fabled pool, from where they collected some samples of the toxic moss. Coincidentally (or maybe not!), on that very day, a fire engulfed their laboratory located in the main building of the Hawaii Marine Laboratory in Oahu, destroying all of their specimens, records, books, and equipment. Some years later, during his readings Professor P. Scheuer, a pioneer of the chemistry of marine metabolites, came across a reference to the famous cursed moss and took on the challenge of substantiating the ancient legend scientifically. Propelled by his profound interest in marine toxins, Scheuer, in collaboration with R. Moore from Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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the University of Hawaii, investigated some toxic samples of the legendary seaweed. Their studies ascertained that the sample collected from the Hana tidepool was not a seaweed, as commonly believed, but an unknown type of soft coral, related to a sea anemone, belonging to the family Zoanthidae, order Zoantharia and phylum Coelenterata (Moore & Scheuer, 1971). This zoanthid was subsequently identified as Palythoa toxica, a rare species sparingly detected across the Hawaiian Islands (Walsh & Bowers, 1971), after which the molecule responsible for its high toxicity was termed palytoxin (Figure 5.1). Following the first report on palytoxin in, 1971, many research groups from around the world have undertaken scientific studies aimed at investigating this fascinating molecule from a wide range of perspectives. Efforts carried out by proficient chemists have significantly contributed to identify many palytoxin analogues. Quite a number of aspects related to palytoxin still present many blind spots – first and foremost, its biogenesis. In fact, palytoxin itself was primarily isolated from zoanthids belonging to the genus Palythoa, but significant levels of palytoxin and/or closely related compounds have also been detected in other living organisms, including the red alga Chondria crispus (Halstead, 2002), some crabs (Yasumoto et al., 1986; Alcala et al., 1988) and fish (Fukui et al., 1987), and even bacteria belonging to the Bacillus

O

OH

OH

O O

OH

OH

HO

H2N

OH

OH O

OH

OH Me OH

HO

R6

OH

O HO

( )n

R1

O

N H

Me

OH

OH Me

Me

O

9 9

OH

OH OH

OH 8

HN

OH

OH

A moiety

OH OH

OH

O

HO B moiety

OH

HO OH O

R2

O O

R3

OH R4 O

HO

Me

OH

OH OH

OH R5

OH

OH

OH

OH n

R1

R2

R3

R4

R5

R6

Palytoxin

1

Me

OH

Me

H

OH

OH

Homopalytoxin

2

Me

OH

Me

H

OH

OH

Bishomopalytoxin 3

Me

OH

Me

H

OH

OH

Neopalytoxin

1

--

OH

Me

H

OH

OH

Deoxypalytoxin

1

Me

OH

Me

H

OH

H

Ostreocin-D

1

H

H

H

OH

H

OH

O

Figure 5.1

A moiety = HO

( )n

N H

O HN

Structures of palytoxin, ostreocin-D and other analogues.

O

Chemistry of palytoxin and its analogues

87

cereus group or identified as Brevibacterium, Acinetobacter (Seemann et al., 2009), and Pseudomonas (Carballeira et al., 1998). Additionally, Vibrio sp. and Aeromonas sp. have been characterized as producers of molecules antigenically related to palytoxin (Frolova et al., 2000). Nowadays, the most accredited hypothesis is that benthic dinoflagellates belonging to the genus Ostreopsis are the real producers of palytoxins (Usami et al., 1995; Taniyama et al., 2003; Lenoir et al., 2004, 2006; Ciminiello et al., 2008, 2010). The first important consequence of this finding is that, on account of the global geographical distribution of Ostreopsis, palytoxins can now be considered present virtually everywhere (Rhodes, 2011). Indeed, palytoxin and/or its analogues have been found from the south-eastern Pacific shores, including Australia and New Zealand (Hallegraeff, 2002; Heimann et al., 2009; Shears & Ross, 2009), as far North as Japan (Sagara, 2008); from the Caribbean Sea (Faust, 1999) and northern (Parsons & Preskitt, 2007) and southern American latitudes (Akselman et al., 2008) to areas located south of the African Horn (Lenoir et al., 2004, 2006), up to the Mediterranean Sea (Mangialajo et al., 2011). This could be partly due to more intensive and widespread samplings, as well as to the improvement of microorganism identification technologies, such as phylogenetic analysis. However, it is worth underlining that a clear increase of Ostreopsis spp. blooms, such as the O. siamensis blooms in the Northern New Zealand seawater (Rhodes et al., 2000) and the massive O. ovata proliferation across the Mediterranean Sea (Mangialajo et al., 2011), has also been registered in those coastal waters that have long since been subjected to regular monitoring. As these blooms have often caused human illness and environmental suffering, there has been a steady upsurge in studies on the Ostreopsis genus in recent years. Respiratory illness in humans associated to Ostreopsis proliferation has been recorded in the Mediterranean (Durando et al., 2007), but health-related risks due to consumption of contaminated seafood seem to be quite low. However, since the sequestration of palytoxins at different levels of the food chain is proven (Aligizaki et al., 2008, 2011;, Ciminiello et al., 2011; Amzil et al., 2012), seafood eaters are usually discouraged from consuming seafood in concomitance with Ostreopsis blooms. Hence, an ever growing number of countries have implemented environmental monitoring programs with the purpose of protecting public health by early detection of palytoxins. The present chapter will describe the chemistry of palytoxin and its analogues.

Palytoxin Palytoxin was first isolated from samples of a soft coral assigned to the genus Palythoa by Professor Paul Scheuer, in 1971 (Moore & Scheuer, 1971). Since then, toxins seemingly identical to palytoxin have been identified in other Palythoa species from around the world, such as P. vestitus from Hawaii (Wiles et al., 1974), P. caribaeorum from the West Indian islands (Gleibs et al., 1995), as well as in additional unidentified Palythoa spp. from Tahiti and Japan (Wiles et al., 1974). Palytoxin appears as a white amorphous solid only soluble in polar solvents such as water, dimethyl sulfoxide, methanol and ethanol. The toxin is quite resistant to heat charring at 300 ∘ C. It possesses a complex chemical architecture, consisting of a long, alternating sequence of pyranose rings and linear segments with extended 1,2or 1,3-oxymethine systems, together with two amide functionalities and a primary amino group accounting for the palytoxin’s basicity (Figure 5.1). This impressive

88

Chapter 5

molecule features 64 asymmetric carbons and seven stereogenic double bonds. As a chiral molecule, palytoxin shows optical activity, with a specific rotation of +26∘ ± 2∘ in water. A monumental study of nearly 11 years was required for defining the complex chemical architecture of palytoxin. In 1981, two research groups – one at the University of Hawaii, led by R. Moore, and the other at Nagoya University, led by Y. Hirata – independently and almost simultaneously came up with two slightly different planar structures for palytoxin. In particular, Hirata, who had worked on a molecule isolated from an Okinawan P. tuberculosa, located a hydroxyl group at position 44 and a hemiketal functionality at C47 (Uemura et al., 1981a, 1981b); while Moore identified a ketal bond between C44 and C47 in a palytoxin isolated from a Tahitian Palythoa spp (Moore & Bartolini, 1981). In 1982, a number of scientists, among others Hirata and Moore, succeeded in elucidating the stereochemistry of palytoxin isolated from Palythoa spp. (Klein et al., 1982; Ko et al., 1982; Fujioka et al., 1982; Cha et al., 1982; Moore et al., 1982). The proposed stereochemistry was eventually proved correct a few years later through a chain of elaborate synthetic studies (Armstrong et al., 1989). These studies on palytoxin’s stereo-structure were mainly carried out by NMR techniques. However, NMR spectra of palytoxin appear highly congested, due to large magnetic resonances overlap. Thus, any structural investigation on palytoxin has usually been performed by analyzing smaller degradation products of the entire toxin obtained by treatment with periodate oxidation and/or ozonolysis. Around the turn of the new millennium, with the advent of more sensitive multidimensional NMR techniques, it became feasible to carry out stereostructural studies on the palytoxin(s) without being forced to implement degradation procedures (Table 5.1; Kan et al., 2001). Since its isolation in 1971, the scientific interest in palytoxin has never faded away. Numerous aspects related to such a toxin have been investigated, including chemistry and detection, ecobiology and origin, metabolism, mechanism of action and toxicology as well. Notwithstanding the more than 40 years of studies, the understanding of how palytoxin interacts with biological systems has yet to be fully determined. Na+ , K+ -ATPase pump constitutes a molecular receptor for palytoxin that is able to convert the pump into an open channel, with consequent loss of cellular K+ and remarkable rise of cytosolic Na+ levels (Bottinger et al., 1986; Wu, 2009; Rossini & Bigiani, 2011; Pelin et al., 2013). In addition, a slight permeability to Ca2+ is even detected when palytoxin binds to the pump (Artigas & Gadsby, 2004). On account of the widely accepted recognition of the dependence of important cellular events on calcium ion concentration, the possible coordination of Ca2+ by palytoxin in aqueous solution has been recently investigated. By extensive NMR-driven analysis supported by molecular modelling techniques, two specific regions of palytoxin where calcium is preferentially coordinated have been identified (Ciminiello et al., 2014a). This study is a further step toward the understanding of the toxin bioactivity at a molecular level.

Palytoxin’s analogues from Palythoa spp. A number of analogues have been identified in a wide range of Palythoa spp. In 1985, Uemura et al. isolated four minor palytoxin-like compounds, alongside palytoxin, from

Chemistry of palytoxin and its analogues

89

and13 C NMR chemical shift data (CD3 OD) of palytoxin (Kan et al., 2001) and ovatoxin-a. Based on data from Kan et al., 2001; Ciminiello et al., 2012b. Table 5.1

No

1H

palytoxin

ovatoxin–a

No

𝟏𝟑 C

𝟏H

𝟏𝟑 C

175.92 75.70 34.73 13.99

– 4.09 2.17 0.88

175.93 75.12 34.54 13.94

– 4.09 2.14 0.86

60 61 62 63

70.18 76.57 73.11 36.77

4

41.73

41.31

71.77

66.62 131.85

66.40 131.51

1.39 1.79 4.51 5.49

64

5 6

1.40 1.77 4.50 5.49

65 66

72.20 37.01

7 7-Me 8 9 10

138.28 13.17 80.91 72.34 29.23

– 1.72 3.92 3.81 2.12

138.31 13.22 80.90 70.68 27.95

67 68 69 70 71

77.22 76.04 79.74 75.85 77.08

11

76.19

4.18

77.15

– 1.74 3.91 3.92 1.80 2.11 4.11

72

41.51

12 13 14 15 16 17

73.88 75.17 71.68 72.91 71.28 71.68

3.64 3.54 3.60 3.62 4.03 4.04

74.26 74.55 71.32 74.55 74.34 45.60

73 74 75 76 77 78

64.99 133.47 130.04 128.87 133.88 38.64

18 19 20 21

73.27 71.35 71.11 27.38

68.43 69.90 68.10 29.00

79 80 81 82

71.20 76.29 73.04 34.35

22

26.93

83

23

35.03

24

28.44

3.54 3.79 3.87 1.39 1.48 1.35 1.47 1.55 1.64 1.36

3.65 3.57 3.58 3.58 4.04 1.33 1.52 3.87 4.05 3.88 1.32 1.63 1.40

25

39.72

1.26

40.12

26

29.70

1.67

30.01

1.23 1.58 1.22 1.47 1.14 1.30 1.61

26-Me

19.30

0.92

21.41

0.92

1 2 3 3-Me

27.11 34.82 30.43

𝟏H

palytoxin 𝟏𝟑 C

𝟏H

ovatoxin–a 𝟏𝟑 C

3.85 3.15 3.74 1.70 1.96 3.68

68.82 75.70 76.75 29.55

3.76 1.53 1.53 3.44 3.12 3.36 3.09 3.44

73.57 40.68

1.43 2.04 4.84 5.37 6.00 6.46 5.78 2.42

41.11

35.20

76.72 75.57 79.31 75.49 76.70

64.86 133.10 129.85 128.44 133.58 38.54 72.60 76.03 72.74 34.00

𝟏H

3.82 3.14 3.42 1.52 2.03 1.50 1.71 3.86 1.55 1.93 3.45 3.10 3.35 3.09 3.41 1.42 2.03 4.82 5.37 5.99 6.43 5.78 2.40

130.18

3.93 3.27 3.63 2.39 2.75 5.69

129.91

3.93 3.26 3.69 2.37 2.75 5.68

84

132.64

5.95

132.27

5.94

85

146.73

–

146.33

85’

114.86

114.74

86

34.30

87

33.13

4.94 5.07 2.25 2.34 1.59 1.72

34.01 32.77

– 4.92 5.05 2.23 2.32 1.59 1.73

(continued overleaf)

90

Chapter 5

Table 5.1 No

(continued) palytoxin 𝟏𝟑

C

27

40.78

28 29 29-Me 30

80.17 82.31 21.01 45.74

31

25.55

31-Me 32

21.89 43.74

𝟏

ovatoxin–a H

0.91 1.47 3.97 – 1.18 1.14 1.70 2.04

𝟏𝟑

C

39.35 79.94 82.20 20.70 45.44 25.25

33 34 35 36 37 38 39

109.23 38.64 23.98 30.98 30.93 30.81 31.29

0.91 1.09 1.67 – 1.60 1.41 1.31 1.31 1.31 1.36

40

39.20

1.48

34.81

41 42

69.26 39.37

71.95 77.73

43 44

64.86 73.88

3.80 1.44 1.86 4.39 3.65

45 46 47

74.28 68.25 101.24

3.95 3.67 –

41.95 72.40 44.07 16.58

51 52 53 54

48 49 50 50-Me

21.61 44.10 109.00 39.32 24.11 30.50 30.50 31.71 28.50

63.11 34.49

𝟏

No

𝟏𝟑

H

0.78 1.60 3.97 – 1.18 1.14 1.71 2.06 0.91 1.08 1.65 – 1.60 1.43 1.27 1.27 1.35 1.27 1.68 1.20 1.87 3.66 3.05

palytoxin C

𝟏

ovatoxin–a H

𝟏𝟑

C

𝟏

H

88

74.19

3.71

73.92

3.70

89 90 91 91-Me

74.02 77.82 33.00 15.65

3.50 3.35 1.89 0.91

73.56 77.59 32.71 15.38

3.50 3.35 1.88 0.90

92

27.86

27.61

93 94

74.83 73.04

1.30 2.21 4.03 3.65

74.51 72.58

1.28 2.20 4.03 3.64

95 96 97 98 99 100 101

74.73 76.01 69.71 132.43 135.28 71.90 71.77

3.61 3.15 4.32 5.55 5.71 4.36 3.68

74.42 75.71 69.35 132.11 135.08 71.64 71.50

3.61 3.16 4.31 5.54 5.70 4.36 3.67

102

40.21

1.58

39.79

1.57

103 104

68.39 40.53

4.22 1.38 1.74 4.51 1.78 1.84 4.21 4.35 1.67 1.78 1.47 3.89 4.27 1.86 2.10 4.36 2.87 2.99 7.79 5.95

68.12 40.21

4.21 1.37 1.73 4.51 1.76 1.85 4.21 4.35 1.64 1.78 1.48 3.89 4.27 1.87 2.11 4.38 2.91 3.05 7.80 5.94

105 106

76.14 36.83

69.81 71.52 101.16

4.63 1.63 2.01 4.28 3.41 –

107 108 109

79.62 82.74 26.59

1.83 3.94 2.26 1.03

40.34 72.49 43.55 16.82

1.74 3.82 2.29 1.05

110 111 112 113

32.30 83.81 73.27 39.78

134.46 134.74

5.62 5.51

134.51 134.14

5.62 5.47

114 115

75.31 45.13

74.06 34.93

4.05 1.61 1.77

73.92 34.82

4.01 1.57 1.76

2’ 3’

134.82 106.82

75.91 36.48 79.40 82.51 26.33 32.11 83.64 72.92 39.46 74.46 44.49 134.81 106.82

Chemistry of palytoxin and its analogues

Table 5.1 No

91

(continued) palytoxin 𝟏𝟑 C

55

27.79

56 57 58 59

73.11 72.81 74.19 33.05

𝟏H

1.46 1.69 3.74 3.85 3.87 1.66 2.27

ovatoxin–a 𝟏𝟑 C

27.18 72.75 71.67 70.91 32.72

No

𝟏H

1.43 1.63 3.66 3.87 3.86 1.65 2.27

palytoxin

ovatoxin–a

𝟏𝟑 C

𝟏H

𝟏𝟑 C

𝟏H

4’

169.66

–

169.39

–

6’ 7’ 8’

37.42 33.28 60.40

3.33 1.74 3.60

37.53 33.19 60.40

3.34 1.73 3.59

some Okinawan P. tuberculosa extracts. The new palytoxin analogues, successfully characterized as homopalytoxin, bishomopalytoxin, neopalytoxin, and deoxypalytoxin, featured little structural differences in comparison with their parent compound as shown in Figure 5.1 (Uemura et al., 1985). In comparison to palytoxin, homopalytoxin and bishomopalytoxin feature one and two more methylenes, respectively, in the N-substituent of the amide functionality at position 1. Neopalytoxin shows an additional 5-member ether ring deriving from a dehydration process involving the hydroxyl groups at C2 and C5 of palytoxin, respectively. Finally, deoxypalytoxin lacks a hydroxyl functionality at position 73. More recently, other analogues from Palythoa spp. have been isolated and stereostructurally characterized (Ciminiello et al., 2009, 2014b). In 2009, Ciminiello and co-workers analyzed two toxic samples of Hawaiian P. tuberculosa and P. toxica, provided by the US Army Medical Research Institute of Infectious Diseases at Fort Detrick, Maryland (Raybould, 1991). LC/MS-based analysis on the two samples highlighted the occurrence of a putative palytoxin, together with a much more abundant new palytoxin analogue. Full 1D- and 2D-NMR investigation allowed the characterization of the planar structure of this new analogue as 42-hydroxy palytoxin (Ciminiello et al., 2009). Successive NMR-based stereostructural studies disclosed that the two 42-hydroxy palytoxins isolated from P. toxica and P. tuberculosa were indeed diastereoisomers, with inverted configurations at C50 (Figure 5.2, Table 5.2). More precisely, the stereoisomer from P. toxica turned out to be 42S-hydroxy-50S-palytoxin, while the one from P. tuberculosa 42S-hydroxy-50R-palytoxin (Ciminiello et al., 2014b). Some preliminary toxicological studies have been carried out on the two 42-hydroxy derivatives. Interestingly, the cytotoxicity of 42S-hydroxy-50R-palytoxin (from P. tuberculosa) toward skin HaCaT keratinocytes appeared approximately two orders of magnitude lower than that of palytoxin and one order of magnitude lower than that of 42S-hydroxy palytoxin (from P. toxica). Seemingly, a single configurational change plays a major role on the cytotoxicity of palytoxins. It can be hypothesized that the configurational inversion at C50 causes the derivative from P. tuberculosa to undergo conformational changes that ultimately reduce the potency of the toxin against the HaCaT keratinocytes (Pelin et al., 2013; Ciminiello et al., 2014b; Del Favero et al., 2014).

Chapter 5

92

OH

O OH

O

OH

O

OH

HO

H2N

OH

OH O

OH

OH Me OH

HO

OH OH

OH OH O HO

Me

O

N H

OH

Me HO

HN

HO

OH O

Me

Me

OH

OH

O

Me OH

42

O

OH HO

OH

OH

2

OH

50 OH

50 OH

HO

Me

OH

Me

O

OH

1

OH

HO

OH

O

50

OH OH

OH

O OH

Me

OH

OH OH

OH

1

42S-hydroxy-50R-palytoxin from P. toxica

2

42S-hydroxy-50S-palytoxin from P. tuberculosa

Figure 5.2

Stereostructures of 42S-hydroxy-50R-palytoxin (from Palythoa toxica) and 42S-hydroxy50S-palytoxin (from Palythoa tuberculosa).

Additional detection of palytoxins have recently multiplied in Palythoa spp. used as popular decorations in marine aquaria. The animals easily propagate and their colonies quickly cover large parts of the aquarium. However, staff of marine aquarium exhibits, as well as home aquarium owners, should be made aware of the potentially high risks carried by these species. In fact, some case reports of palytoxin-exposure from incidental contact with marine aquarium zoanthids have been recently reported (Hoffmann et al., 2008; Deeds & Schwartz, 2010). In these reports, marine aquarium hobbyists, in the attempt to remove palytoxin-producing zoanthids from their home aquarium with boiling water, have experienced severe respiratory reactions following inhalation of steam and/or skin injuries while handling the animals. In some cases, the presence of palytoxin and 42-OH palytoxin in the infesting zoanthids was confirmed by LC-MS and/or haemolysis assay (Deeds & Schwartz, 2010; Wieringa et al., 2014; Ciminiello et al., 2014c).

Palytoxin’s analogues from Ostreopsis spp. A number of authors have reported the presence of palytoxin-like compounds in Ostreopsis species. Ostreopsis spp. are benthic and epiphytic dinoflagellates distributed worldwide. They are important components of both subtropical and tropical marine coral reef-lagoonal environments and, during recent decades, they have also been regularly detected in temperate regions of the world. The presence of potentially toxic Ostreopsis spp. (O. cf. ovata and, less frequently, O. cf. siamensis) has been repeatedly reported along the Mediterranean and the Atlantic coasts (Mangialajo et al., 2011). In certain areas of Italy, Spain, France, Croatia and Algeria, this phenomenon has been

Chemistry of palytoxin and its analogues

93

Table 5.2

List of selected NMR spectroscopic data (CD3 OD) for 42S-hydroxy-50R-palytoxin (from P. toxica) and 42S-hydroxy50S-palytoxin (from P. tuberculosa). Based on data from Ciminiello et al., 2014b.

No.

42-hydroxy palytoxin from P. toxica

42-hydroxy palytoxin from P. tuberculosa

𝛅C

𝛅H

𝛅C

𝛅H

34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

38.69 24.23 30.84 30.84 30.84 31.21 38.69 71.92 77.34 66.10 74.08 74.28 67.75 101.59 41.92

38.72 24.15 30.87 30.87 30.87 31.40 38.68 71.81 74.16 69.68 74.00 74.28 67.75 101.28 41.86

49 50 50-Me 51 52 53 54

72.45 44.41 16.72 134.59 134.69 74.06 35.09

55

27.89

56 57 58 59

73.15 72.86 74.11 33.16

1.60 1.43 1.29 1.29 1.29 1.35 1.58 3.65 3.70 4.32 3.91 3.91 3.78 – 1.80 1.96 3.92 2.25 1.03 5.64 5.50 4.06 1.61 1.78 1.46 1.69 3.75 3.86 3.87 1.66 2.26

1.60 1.43 1.33 1.33 1.33 1.38 1.61 3.70 3.71 4.34 3.92 3.91 3.78 – 1.59 1.60 3.82 2.34 1.08 5.66 5.53 4.09 1.63 1.83 1.50 1.64 3.76 3.87 3.87 1.69 2.28

72.98 42.24 16.80 133.86 134.98 74.11 35.09 26.98 73.15 72.77 74.18 33.22

related to toxicity in shellfish (Aligizaki et al., 2011; Ciminiello et al., 2011; Amzil et al., 2012), as well as to an unusual incidence of respiratory syndromes in people exposed to marine aerosols (Tubaro et al., 2011). Some Ostreopsis species turned out to be toxin producers, such as O. siamensis (Usami et al., 1995), O. ovata (Ciminiello et al., 2006), O. mascarenensis (Lenoir et al., 2004), O. lenticularis (Tindall et al., 1990), and O. heptagona (Norris et al., 1985). For some others (O. labens, O. marinus, O. belizeanus, and O. caribbeanus), toxicity data are not available (Faust, 1999).

94

Chapter 5

Ostreocins from O. siamensis Among various palytoxin-like molecules, ostreocins were the first compounds to be isolated from biogenetic sources other than Palythoa spp. In, 1995, Usami and co-workers isolated the major ostreocin (ostreocin-D) from dinoflagellates belonging to the genus Ostreopsis (Dinophyceae), namely O. siamensis, thus providing the first evidence that this species could be one of the biogenetic sources of palytoxins. O. siamensis is an epiphytic dinoflagellate that was first isolated by Schmidt in the Gulf of Siam (Thailand) in 1901 (Schmidt, 1901), but it was largely overlooked till the late 1970s (Faust & Gulledge, 2002). To date, its presence has been reported in the coastal waters of Japan, New Zealand, Australia, East Africa, Mexico, Caribbean, Spain, Italy, Greece and Tunisia (Mangialajo et al., 2011; Rhodes, 2011). Yasumoto’s group was the first to characterize O. siamensis as a toxin-producer (Nakajima et al., 1981) and to report on its lethality and haemolytic activity a few years later (Yasumoto et al., 1987). The major component of O. siamensis toxin profile, ostreocin-D, accounted for 90% of the total extract toxicity. The ostreocin-D structure (Figure 5.1) was definitely elucidated by Ukena et al. (2001a) by NMR (Table 5.3). Ostreocin-D is a colourless, amorphous solid, optically active, with a specific rotation of +16.6 in water. Its UV spectrum in water exhibits two maxima at 234 (ε 35 000) and 263 nm (ε 22 000). Both UV and NMR evidence indicated that ostreocin-D presents conjugated diene and carbonyl functionalities similar to palytoxin. Positive ion MS spectrum of ostreocin-D, obtained through fast atom bombardment mass spectrometry (FAB-MS), displayed cluster ions with a centroid at m∕z 2636.51. The elemental composition C127 H220 N3 O53 (MH+ calculated 2636.47) was assigned to the molecule. Ions at different charge states were observed in electrospray ionization (ESI) mass spectra of ostreocin-D at m∕z 2636.7, 1330.4, and 894.4 that were assigned to [M + H]+ , [M + Na + H]2+ , and [M + 2Na + H]3+ respectively. The difference in elemental composition (C2 H4 O) between palytoxin and ostreocin-D was attributed to the lack of two methyls and one hydroxyl group in the latter. Severe signal overlap in NMR spectra hampered a complete analysis of the gross structure of the toxin. In order to reduce the size of the molecule, ozonolysis, followed by treatment with NaBH4 , was performed. The obtained fragments were chromatographically separated and individually analyzed by ESI-MS and NMR. This approach reduced the signal overlap in the NMR spectra and clarified the entire structure of the molecule. The complete NMR assignment of ostreocin-D was done in 0.2% CD3 COOD∕D2 O, which demonstrated that ostreocin-D, compared to palytoxin, lacks two methyls at C-3 and C-26 and two hydroxyls at C-19 and C-44, while presenting an additional hydroxyl at C-42. Finally, ostreocin-D was identified as 42-hydroxy-3,26-didemethyl-19,44-dideoxypalytoxin. In, 2002, Ukena et al. confirmed the structure of ostreocin-D by application of negative ion FAB collision-induced dissociation (CID) tandem MS. A negative charge was introduced to either the terminal amino group or to the primary alcohol at the terminal 3-aminopropanol of the molecule by reaction with 2-sulfobenzoic acid cyclic anhydride. The negative ion FAB-CID-MS/MS spectra of the two derivatives – containing ions due to charge remote fragmentations from two different charge sites – provided crucial details on the ostreocin-D structure.

Chemistry of palytoxin and its analogues

Table 5.3 1 H and13 C NMR chemical shift data of ostreocin-D in 0.2% CD3 COOD/D2 O. Based on data from Ukena et al., 2001. No.

Ostreocin-D 𝟏𝟑

C

𝟏

No.

Ostreocin-D 𝟏𝟑

H

C

𝟏

H

1 2

175.5 71.2

– 4.31

62 63

71.4 35.5

3

29.8

64

70.3

4

31.8

65

70.4

3.77

5

67.3

1.69 1.88 1.59 1.75 4.51

3.68 1.56 2.09 3.65

66

35.6

67 68 69 70 71 72

75.9 75.7 74.0 74.1 77.4 39.2

73 74 75 76 77 78 79 80 81

63.8 131.2 130.1 127.7 133.4 36.9 69.9 75.2 71.5

1.49 2.09 3.48 3.51 3.21 3.22 3.49 1.55 2.12 4.86 5.43 6.14 6.49 5.86 2.42 3.94 3.41 3.79

82

32.1

6 7 7-Me 8 9 10

129.9 137.9 12.1 79.7 70.5 27.9

11 12 13 14 15 16 17 18 19

74.5 71.2 73.2 70.1 72.6 74.9 68.7 67.3 40.6

20

68.2

5.46 – 1.71 3.96 3.86 1.72 2.13 4.23 3.72 3.64 3.62 3.67 3.62 4.02 4.01 1.50 1.80 3.85

21 22 23 24

24.9 29.0 29.0 29.0

1.52 1.35 1.35 1.35

83 84 85 85’

128.8 132.1 145.1 114.4

25

29.0

1.35

86

32.7

26

29.0

1.35

87

31.3

27 28 29 29-Me 30

31.8 81.6 81.9 19.9 43.8

1.34 3.95 – 1.24 1.19 1.77

88 89 90 91 91-Me

72.6 72.9 76.1 31.7 14.3

2.66 5.69 6.04 – 5.14 2.27 2.32 1.61 1.75 3.76 3.60 3.47 1.84 0.92

(continued overleaf)

95

96

Chapter 5

Table 5.3

(continued)

No.

Ostreocin-D 𝟏𝟑

C

𝟏

No.

Ostreocin-D 𝟏𝟑

H

C

31

23.9

2.00

92

25.9

31-Me 32

20.8 41.9

93 94

73.6 71.2

95 96 97 98 99 100 101 102

72.6 74.3 68.2 130.9 134.8 70.9 70.3 38.5

𝟏

H 1.34 2.16 4.17 3.78

33 34 35 36 37 38 39 40

108.5 36.8 22.3 29.0 29.0 29.0 29.0 37.4

0.92 1.16 1.74 – 1.66 1.40 1.35 1.35 1.35 1.35 1.50

41 42

71.1 75.1

3.72 3.40

103 104

67.4 38.5

43 44

63.4 33.5

105 106

75.1 35.3

45 46 47 48

68.9 69.7 100.0 40.5

4.39 1.78 2.05 4.25 3.51 – 1.85 1.92 3.94 2.34 1.04

107 108 109 110

78.4 81.6 30.4 24.8

111 112 113

82.3 71.9 37.9

5.62 5.56 4.14 1.62 1.77 1.44 3.83 4.04 3.84 1.72 2.30 3.93 3.21

114 115 2’ 3’

73.4 43.2 133.4 106.5

3.70 3.32 4.29 5.62 5.78 4.35 3.66 1.58 1.62 4.21 1.48 1.79 4.64 1.83 1.93 4.33 4.42 1.52 1.66 1.76 3.94 4.37 1.98 2.21 4.45 3.16 7.71 5.95

4’ 6’ 7’ 8’

168.9 36.4 31.2 59.3

– 3.34 1.79 3.68

49 50 50-Me

71.1 42.3 15.4

51 52 53 54

134.5 132.6 72.8 33.1

55 56 57 58 59

32.4 71.0 71.7 71.9 31.8

60 61

68.5 75.1

Chemistry of palytoxin and its analogues

97

These small structural differences between ostreocin-D and palytoxin significantly affect their toxicity. In fact, ostreocin-D, compared to palytoxin, presents lower cytotoxicity against P388 cells (2.5 pM versus 0.2 pM) and lower haemolytic potency (39.5 nM versus 1.5 nM) (Usami et al., 1995). Recently, Ito & Yasumoto (2009) have reported that both toxins are toxic by intra-peritoneal injection – with ostreocin-D less potent (5 μg/kg) than palytoxin (1.5 μg/kg) – and by intra-tracheal administration, causing bleeding and alveolar destruction in the lung and resultant death at 2 μg/kg of palytoxin and 11 μg/kg of ostreocin-D. Besides ostreocin-D, other minor ostreocins were detected in the Japanese O. siamensis culture extract in amounts too low for a complete NMR-based structure elucidation. A tentative structure of ostreocin-B was proposed by Ukena on the basis of NMR data (Ukena, 2001b). Compared to ostreocin-D, ostreocin-B presents an additional hydroxyl at C-44, thus being the 42-hydroxy-3,26-didemethyl-19-deoxypalytoxin. Recent studies on European O. cf. siamensis have suggested that the Mediterranean/Atlantic O. cf. siamensis strains do not possess any appreciable toxicity. Although Cagide et al. (2009) reported that the effects of a Spanish O. cf. siamensis strain on viability, membrane potential and intracellular calcium of neuroblastoma cells were similar to those induced by palytoxin standard, neither palytoxin nor any of its analogues were detected by LC-MS analyses. In a recent study on qualitative determination of various phycotoxins in dinoflagellate whole cells based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), Paz et al. (2011) did not detect any ostreocins in a Spanish O. cf. siamensis cultured strain. Similarly, Ciminiello et al. (2013), in a LC-HRMS study on O. cf. siamensis strains from Italy and Portugal, ascertained that such strains do not produce any detectable amounts of ostreocins or other known palytoxin analogues. Only sub-fg levels of palytoxin on a per cell basis were detected in the Italian strain alone. These findings suggest that, in the European area, O. cf. siamensis does not represent a real risk to human health.

Mascarenotoxins from O. mascarenensis In 1996, during a survey of benthic dinoflagellates in the Mascareignes archipelagos (Southwest Indian Ocean), a benthic bloom of Ostreopsis mascarenensis (Quod, 1994) was observed near Rodrigues Island. O. mascarenensis is the largest species of the genus Ostreopsis; its cells are large and oval in apical view, tapering ventrally and compressed anteroposteriorly. In 2004, Lenoir et al. described the purification and the MS-based characterization of two palytoxin analogues from O. mascarenensis that were named mascarenotoxin-A and -B. Preliminary screening of O. mascarenensis crude methanolic extract revealed the presence of the compound causing neurotoxic symptoms in mice similar to those induced by palytoxin, such as prostration, progressive limp paralysis, dyspnoea, convulsions and death after 20 minutes from the i.p. injection. An LD50 of 0.9 mg∕kg was calculated for the methanolic extract. A screening of the haemolytic activity, typical of palytoxins, was carried out on the purified mascarenotoxins versus palytoxin. Under the used conditions, palytoxin induced complete haemolysis after five hours of incubation at 50 ng∕mL, while haemolytic activities of mascarenotoxin −A (800 ng∕ml) and −B (200 ng∕ml) were estimated to be lower than palytoxin. The obtained results are in agreement with the observation by Usami et al. (1995), according to which, small changes in the structure of palytoxin-like compounds affect their haemolytic potency.

98

Chapter 5

The structure of mascarenotoxins has not been elucidated, and only some observations on their chromatographic and MS behaviour have been reported so far (Lenoir et al., 2004). HPLC-DAD analyses of the extracts containing mascarenotoxins revealed that they eluted very close to the palytoxin standard and had the same UV spectrum, characterized by two maxima at 233 and 263 nm. MS studies on mascarenotoxin-A and -B were carried out on a nano-ESI hybrid quadrupole-time of flight (Q-TOF) MS instrument. Both compounds presented an MS behaviour and fragmentation pattern similar to that of a palytoxin standard, in having major peaks corresponding to bi- and tri-charged ions, several ions due to losses of water molecules and a diagnostic fragment ion at m∕z 327. Mascarenotoxin-A and -B showed dominant [M + 2H]2+ ions at m∕z 1295.5 and 1304.3, respectively, and tri-charged [M + 3H]3+ ions at m∕z 836.9 and 836.2, respectively. Ion assignments of triply and doubly charged ions were not unambiguous and, in the lack of the pseudo-molecular ion [M + H]+ , it was postulated that mascarenotoxin-A and -B have similar molecular masses likely in the range of 2500–2535 Da. In 2010, Rossi et al. reported the presence of monoisotopic ion peaks [M + H]+ at m/z 2589.3441 and at m∕z 2629.2854 in a Mediterranean O. cf. ovata extract (strain D483). The authors assigned the elemental formulae C127 H222 N3 O50 and C129 H222 N3 O51 to the two ions, respectively, and labelled the peak at m∕z 2589.3441 as mascarenotoxin-A, by interpreting the MS data reported by Lenoir et al. (2004), and the peak at m∕z 2629.2854 as the novel analogue mascarenotoxin-C. However, errors in the ion assignments exceeded the commonly accepted values (≤ 5 ppm), and they were done in absence of a reference sample of mascarenotoxins.

Ovatoxins from O. cf. ovata Ostreopsis ovata is a toxic benthic dinoflagellate, first described by Fukuyo in 1981. Since then, it has been found to be widely distributed, such as in areas of the Pacific Ocean (Fukuyo, 1981; Yasumoto et al., 1987), the Caribbean Sea (Besada et al., 1982; Faust, 1999) and the Brazilian Atlantic coasts (Granéli et al., 2002; Nascimento et al., 2012). More recently, the attention to O. ovata has been dramatically renewed by a series of cases of human illness in the Mediterranean, in particular along the Italian coastlines (Gallitelli et al., 2005; Durando et al., 2007). It has to be noted that resolving the taxonomy of Ostreopsis species based only on morphology is difficult (Penna et al., 2005), and none of the original Ostreopsis isolates from tropical areas has been sequenced yet for their genotype assignment. For these reasons, the designation O. cf. (confronta) ovata is used when referring to isolates from the Mediterranean Sea. Even though a number of O. cf. ovata blooms have been reported across the Italian seas since the late 1990s (Tognetto et al., 1995; Sansoni et al., 2003), only in the summer of 2005 the so-called ‘Ostreopsis phenomenon’ broke out with alarming proportion, drawing the attention of both national and international media (Gallitelli et al., 2005; Brescianini et al., 2006). Hundreds of people required medical attention during recreational or working activities on the beach and promenade of Genoa, Italy. The symptoms shown by the intoxicated people included high fever associated with serious respiratory distress such as watery rhinorrhea, dry or mildly productive cough, bronchoconstriction with mild dyspnoea and wheezes, while conjunctivitis was observed only in some cases. The human suffering was attributed to inhalation of aerosols allegedly containing cells or toxins of O. cf. ovata concurrently blooming

Chemistry of palytoxin and its analogues

99

in seawater. At the same time, various adverse effects were observed in benthic organisms, both sessile (cirripeds, bivalves, gastropods) and mobile (echinoderms, cephalopods, fish). Other respiratory illness in humans attributed to O. cf. ovata occurred in Spain (Barroso Garcia et al., 2008), Algeria (Iddir-Ihaddaden et al., 2009), France (Tichadou et al., 2010), and Croatia (Pfannkuchen et al., 2012). Plankton samples collected off Genoa during the 2005 toxic outbreak were investigated by applying a purposely set-up method based on liquid chromatography tandem mass spectrometry specific for palytoxins (Ciminiello et al., 2006). This analysis brought to light the occurrence of a putative palytoxin in the extracts. The detected compound was referred to as putative palytoxin since, due to the complex stereo-structure of palytoxin, the possibility that the compound was indeed a palytoxin isomer could not be ruled out. Successive HR LC-MS analysis of a number of both field and cultured samples of O. cf. ovata revealed that putative palytoxin was, indeed, a very minor component of the toxin profile, with a number of new palytoxin analogues being produced by the alga, including ovatoxin -a, -b, -c, -d, -e (Ciminiello et al., 2008, 2010) and -f (Ciminiello et al., 2012a). In most of the O. cf. ovata strains analyzed so far, ovatoxin-a (Figure 5.3) represents the major component of both field and cultured algal toxin profiles, as opposed to the other minor ovatoxins that may vary from qualitative and/or quantitative standpoints (Ciminiello et al., 2010, 2011, 2012a, 2012b; Pfannkuchen et al., 2012; Pistocchi et al., 2012). Very recently, O. cf. ovata isolates from the Central Adriatic Sea have been found to have peculiar toxin profiles; an isolate from Numana (OOAN0816) was found not to produce OVTX-b and –c (Ciminiello et al., 2012b); and an isolate from Portonovo (CBA2-122) was dominated by a new ovatoxin, designated ovatoxin-f, which accounted for 50% of the total toxin content (Ciminiello et al., 2012a).

O

OH

OH

O

OH

O

OH

HO

H2N

OH

OH O

OH

OH Me OH

HO

OH OH

OH OH O HO

N H

Me

O

OH

HO OH

Me

9

HN OH Me

Me

OH OH O

OH

OH OH

OH

O 17

64

OH

26

OH O

OH

O O

Me

OH 42 OH

OH O 44

HO

Me

OH

57

OH

OH OH OH

OH

OH

Figure 5.3

Stereostructure of ovatoxin-a, the main analogue produced by Ostreopsis cf. ovata.

100

Chapter 5

Environmental parameters were found to have a role in cellular growth and toxin production, but they do not seem to affect the relative abundance of individual components of the toxin profile (Guerrini et al., 2010; Pezzolesi et al., 2012; Vanucci et al., 2012). Extensive LC-HRMSn and NMR studies have been carried out on the major component of the toxin profile, ovatoxin-a. The complex fragmentation pattern of palytoxin contained in its HR LC-MSn spectra was interpreted and used as template to gain detailed structural information on ovatoxin-a (Figure 5.4). A key role in its interpretation was played by the finding that most of the triply-charged ions of palytoxin are due to adducts with divalent cations (Ca2+ , Mg2+ , etc.), with the highest affinity being observed for calcium (Ciminiello et al., 2012c). Ovatoxin-a (C129 H223 N3 O52 ), compared to palytoxin (C129 H223 N3 O54 ), presented an additional hydroxyl group at C-42 and lacked three hydroxyl groups, at C-17, C-44 and in the region stretching from C53 to C78 (Figure 5.4; Ciminiello et al., 2012c). An extensive NMR study (Table 5.1) carried out on the pure compound confirmed the above structural hypotheses and unequivocally allowed identification of the exact position within the C53-C78 region where the hydroxyl group was lacking. Hence, ovatoxin-a was characterized as a 42-hydroxy-17,44,64-trideoxy derivative of palytoxin (Figure 5.3; Ciminiello et al., 2012b). Further NMR studies led to fully define the configuration of all of the 69 ovatoxin-a’s stereocenters, which include seven stereogenic double bonds and 62 asymmetric carbons. Such a study was performed through the evaluation of both homo-nuclear and hetero-nuclear coupling constants, supported and complemented by an in-depth analysis of proton-proton dipolar couplings. Ovatoxin-a turned out to share the same absolute configuration as palytoxin at all of the asymmetric carbon atoms but at C9, C26, and C57, which showed inverted configurations (Figure 5.4) (Ciminiello et al., 2012d). The NMR-based stereostructural determination of the other ovatoxins was barred because of their low concentrations in the analyzed samples. Nonetheless, some structural hypotheses have been gained for all of them on the basis of their HRMSn behaviour. In fact, ovatoxins all share with palytoxin a characteristic MS fragmentation pattern (Ciminiello et al., 2011, 2012c). A particularly diagnostic fragmentation derives from the cleavage between C-8 and C-9, which divides each molecule in two moieties, A and B (Figure 5.4). Based on the HRMS/MS data deriving from this most favoured fragmentation, ovatoxin-b (C131 H227 N3 O53 ) presents C2 H4 O (potentially a hydroxyl and two methylene groups) more than ovatoxin-a in the A moiety, whereas part structure B is identical, at least in the elemental composition. Ovatoxin-c (C131 H227 N3 O54 ) presents C2 H4 O2 more than ovatoxin-a, with additional C2 H4 O atoms in the A moiety and an extra oxygen atom in the B moiety. Ovatoxin-d and -e (C129 H223 N3 O53 ) are isobaric compounds, both presenting one oxygen atom more than ovatoxin-a. While in ovatoxin-d, this extra oxygen atom is contained in the B moiety, it appears in the A moiety of ovatoxin-e (Ciminiello et al., 2010). Ovatoxin-f (C131 H227 N3 O52 ) presents C2 H4 more than ovatoxin-a in the B-moiety, residing in the region between C-95 and C-102, as suggested by in-depth HR MSn study (Ciminiello et al., 2012a). Table 5.4 reports the principal mono-, bi-, and tri-charged ions of palytoxin and of all the ovatoxins so far known, together with elemental formulae assigned to each compound (M) and to their relevant A and B moieties, which derive from the favoured fragmentation between carbon 8 and 9 of palytoxin molecules (Figure 5.4).

Chemistry of palytoxin and its analogues

101

#26 105

O

110

O

B side H2N

#25

O

OH

103

#20

OH

OH

100

#27

OH

HO 90

O

Me OH #21 #22

95

OH

#5 OH

#4 #1 O

A side HO

Me

O

OH

1'

N H

8'

#2

8

3

HN 1

HO OH

Me

OH #11

Me

31

#3 OH

Palytoxin

OH

H

OH

R4

OH

H

OH

H (or OH)

#8 OH

20

OH

Me

HO

HO

O

42 44

R3

#9

OH #10

#12R2 41

45

OH

#18

#16 Me

OH

49 50

46

R4

#13

OH OH

#7

18

#14 Ovatoxin-a

65

19

37

R3

17

R1

O

OH

16

O

O O

R2

OH

11

9

OH OH 70

OH #6

13

26

Me

R1

73

#19

78

OH

OH

#23 HO

OH

81 80 79

OH

#28

115

#24

OH OH

#17

O 60

HO

OH 52

53

56

OH OH

Region 53-78

#15

OH (or H)

Figure 5.4 Dashed lines highlight the complex fragmentation pattern of palytoxin contained in its HR LC-MSn spectra. It was used as template to gain detailed structural information on ovatoxin-a.

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Recently, three ovatoxins have been detected in a toxic clone of O. ovata (IK2) collected from Ikei Island, Okinawa Prefecture, Japan, by using liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOFMS) (Uchida et al., 2013). The three compounds showed the same molecular formulae as the Mediterranean ovatoxin-a, ovatoxin-d and ovatoxin-e, but differed from them by their chromatographic behaviour. In order to distinguish the Japanese ovatoxins from the Mediterranean ones, they were named ovatoxin-a IK2, ovatoxin-d IK2 and ovatoxin-e IK2, after the clone’s code, ‘IK2’. Some insights into the structure of these ovatoxins were obtained by complementary use of positive and negative ion LC/QTOFMS. The conjugated polyene structures observed in positive ion spectra allowed the identification of the positions of hydroxyl groups. The extension of conjugation was facilitated by dehydration of a hydroxyl residing on a carbon that was either α or β to the last sp2 carbon. Should a hydroxyl be lost or dislocated, the conjugation is likely to stop there. The negative ion spectra allowed assignments of cleavage sites of C-C bonds. Thus, ovatoxin-a IK2, ovatoxin-d IK2, and ovatoxin-e IK2 were tentatively identified as 42-hydroxy-17,44,70-trideoxypalytoxin, 42-hydroxy-17,70-dideoxypalytoxin and 42,82-dihydroxy-17,44,70-trideoxypalytoxin, respectively. As mentioned above, based on the coincidence between O. cf. ovata blooms, respiratory illness in people, and detection of palytoxin complex (palytoxin and ovatoxins) in algal samples, inhalation of toxic aerosols was postulated to be the cause of human illness. A first study on the marine aerosol composition was reported in 2013 by Casabianca et al., who succeeded in quantifying O. cf. ovata cells in marine aerosols collected along the Spanish Mediterranean coasts by a molecular qPCR assay. Palytoxins were not detected in this study, either by haemolytic assay or by liquid chromatography with fluorescence detection (LC-FLD), probably due to the detection limits of the employed methods. Very recently, a small-scale monitoring study of marine aerosol collected along the Tuscan coast (Italy) concurrently to O. cf. ovata blooms has been carried out based on molecular assay and LC-HRMS. The obtained results, besides confirming the presence of O. cf. ovata cells, demonstrated for the first time the occurrence of ovatoxins in the aerosols (Ciminiello et al., 2014d). These results are a first step toward a more comprehensive understanding of the Ostreopsis-related respiratory syndrome. Toxicological evaluation aimed at determining the acute reference dose of both palytoxin and ovatoxins by inhalation exposure are needed. Ovatoxins have also been detected in seafood collected along the coasts of Italy (Ciminiello et al., 2011), Greece (Aligizaki et al., 2011) and France (Amzil et al., 2012), thus carrying a further potential risk to human health.

Detection of palytoxins Detection and quantitation of palytoxins can be accomplished by both biological assays and chemical methods. Bioassays measure a single biological or biochemical response, which derives from the activity of all toxin congeners contained in a sample. As in the case of most phycotoxins, the simplest way to detect palytoxin is the mouse bioassay. This consists of an intra-peritoneal (i.p.) injection of a contaminated sample into a mouse and successive measurement of the death time (Yasumoto et al., 1978). The mouse responds to palytoxin by exhibiting several characteristic symptoms within 15 minutes after i.p. injection, such as stretching of hind limbs, lower backs and concave curvature of the spinal column (Riobó et al., 2008). For survival times less than

M

C129 H223 N3 O54 C129 H223 N3 O52 C131 H227 N3 O53 C131 H227 N3 O54 C129 H223 N3 O53 C129 H223 N3 O53 C131 H227 N3 O52

Toxin

Palytoxin Ovatoxin-a Ovatoxin-b Ovatoxin-c Ovatoxin-d Ovatoxin-e Ovatoxin-f

2679.4893 2647.4979 2691.5233 2707.5173 2663.4905 2663.4905 2675.5054

[M+H]+ 1331.2417 1315.2480 1337.2595 1345.2566 1323.2439 1323.2439 1329.2606

[M+2H-H2 O]2+ 906.4851 895.8255 910.4976 915.8286 901.1533 901.1533 905.1616

[M+H+Ca]3+ C16 H28 N2 O6 C16 H28 N2 O6 C18 H32 N2 O7 C18 H32 N2 O7 C16 H28 N2 O6 C16 H28 N2 O7 C16 H28 N2 O6

A moiety C113 H195 NO48 C113 H195 NO46 C113 H195 NO46 C113 H195 NO47 C113 H195 NO47 C113 H195 NO46 C115 H199 NO46

B moiety

327.1919 327.1919 371.2181 371.2181 327.1919 343.1869 327.1908

[A moiety-H2 O]+

1169.1112 1153.1194 1153.1189 1161.1173 1161.1157 1153.1179 1167.1304

[B moiety-2H2 O]2+

Elemental formulae and mono-isotopic peaks of palytoxin and ovatoxins. Elemental formulae of A and B moieties deriving from the cleavage between carbon 8 and 9 of each compound are also reported.

Table 5.4

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one hour, the mouse also shows weakening of forelimbs, ataxia, convulsions, gasping for breath and finally death; for survival times greater than one hour, death time can vary considerably. LD50 for palytoxin in the mouse by i.p. injection is less than 1 μg/kg (Munday, 2011). Alternative biological assays take advantage of palytoxins functional properties and include in vitro cytotoxicity (Bellocci et al., 2008a, 2008b; Ledreux et al., 2009; Espina et al., 2009), delayed haemolysis (Habermann et al., 1981), antibody-based haemolysis neutralization tests (Levine et al., 1988; Bignami et al., 1992; Aligizaki et al., 2008; Garet et al., 2010) and the use of biosensors (Vilarino et al., 2009; Yakes et al., 2011; Zamolo et al., 2012). Very recently, an indirect sandwich ELISA, based on a mouse antipalytoxin monoclonal antibody (capture antibody) and a rabbit antipalytoxin polyclonal antibody (detection antibody), has been developed and characterized by an intralaboratory study (Boscolo et al., 2013). The assay is able to detect PLTX and 42-hydroxy-PLTX in a similar way. It is sensitive, specific, accurate and precise, with limits of detection (LOD) and quantitation (LOQ) suitable for monitoring seafood, below the limit suggested by the European Food Safety Authority (EFSA, 2009). Although biological methods have the advantage of being highly sensitive, and some of them are suitable for regulatory use (Bignami, 1993), they are unable to identify individual components of the toxin profile. Thus, chemical determination methods are needed for confirmation and unambiguous identification of the involved toxins, and for an accurate quantitation. A pre-column derivatization method followed by LC-FLD has been proposed for the separation and quantification of palytoxin by Riobò et al. (2006). This method shows an instrumental limit of detection (LOD) of 0.75–25 ng of palytoxin standard injected. It has been applied to detection of palytoxin in plankton samples, but data are lacking on its application to seafood analysis. LC-UV (Lenoir et al., 2004; Riobò et al., 2006; Yasumoto et al., 1986; EFSA, 2009) has been largely used for detection of palytoxins in plankton. This technique is 1000-fold less sensitive than LC-FLD and heavily affected by matrix interferences, which make it unsuitable for the detection of palytoxins in seafood. Mass spectrometry has demonstrated great potential for rapid and sensitive identification of palytoxins in contaminated material. Fast-atom bombardment (FAB) has been used to detect palytoxin and ostreocin-D, both in positive (Usami et al., 1995) and negative (Ukena et al., 2002) ion modes, and matrix assisted laser desorption ionization combined to time-of-flight mass spectrometry (MALDI TOF MS) to analyze a toxic extract in comparison to palytoxin standard (Onuma et al. (1999). More recently, the capability of ESI of producing multiply-charged molecules under mild conditions has allowed the detection of high molecular weight compounds such as palytoxin (MW 2679) and its congeners by extending the mass range for m/z-limited mass spectrometers (Ciminiello et al., 2011). ESI MS spectra of palytoxins contain a very complex mixture of ionized species, including multiply-charged states (+1, +2, and + 3), mixed cationizing species (H+ , NH4 + , Na+ , K+ , Ca2+ , Mg2+ , Fe2+ ), and a number of ions due to multiple losses of water molecules. This may lead to many uncertainties in ion assignment, but it also represents a fingerprint of palytoxin-like compounds, useful also for the identification of new congeners. Due to the complexity of ESI mass spectra, the use of a reference palytoxin standard is mandatory for the identification of palytoxin and its congeners. The use of additives able to increase adduct ion formation versus ions due to dehydration reactions can be of some help in ion assignment (Ciminiello et al., 2009, 2012c). HR MS is required

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105

for gaining structural information on new palytoxin-like compounds (Ciminiello et al., 2008, 2010, 2012a, 2012c). A cross-check of elemental formulae of all mono-, bi-, and tri-charged ions of each compound is needed for assigning the correct elemental formulae to these new congeners. In fact, due to the complexity of the molecules, several elemental formulae would be possible for each mass, even if a 100.000 resolution setting, a mass tolerance of 5 ppm, and the isotopic pattern of each ion cluster were considered. The capability of ESI MS to detect palytoxins has paved the way to the development of LC-MS methods that have been used for identification of palytoxin and its analogues in algal samples (Lenoir et al., 2004; Penna et al., 2005; Riobò et al., 2006; Ciminiello et al., 2006, 2008, 2010, 2012a, 2012b, 2012c, 2013) in seafood (Aligizaki et al., 2011; Amzil et al., 2012; Ciminiello et al., 2011), and in aerosols (Ciminiello et al., 2014d). Selwood et al. (2012) developed a sensitive assay for palytoxins in seafood using LC-MS/MS analysis of substructures generated by micro-scale oxidative cleavage of vicinal diol groups contained in the intact toxin. Oxidation of palytoxins, ovatoxins and ostreocins using periodic acid generates two nitrogen-containing aldehyde fragments – an amino aldehyde common to all of these toxins, and an amide aldehyde that may vary depending on toxin type. The method LOD was reported to be 1 ng∕ml, which corresponds to 10 μg∕kg in shellfish or fish tissue. The LC-MS technique has been indicated by EFSA (2009) as a valuable tool for monitoring palytoxins for regulatory purposes, since it is rapid, sensitive, can be automated, and permits screening and quantitation of palytoxins in various matrices (plankton, seafood, etc.). However, there are still some drawbacks to overcome before such a technique can be used in routine analysis of real samples. An important issue is represented by the lack of certified reference standards of palytoxin and its analogues; they are difficult to obtain in acceptable amounts, unless a large-scale culturing of Ostreopsis spp. and efficient isolation procedures of the toxins are developed. Currently, quantitation is carried out by assuming that palytoxin-like compounds show the same molar response as palytoxin itself. However, it must be kept in mind that even limited structural features in large molecules like palytoxins could significantly impact their ionization efficiency. In addition, none of the developed LC-MS methods (Ciminiello et al., 2006, 2008; Amzil et al., 2012; Selwood et al., 2012) has been currently validated, and the recent discovery of ovatoxin-b, -c, -d, -e and -f poses the need to update the above methods, both in terms of LC separation and MS detection.

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Wiles, J.S., Vick, J.A. and Christensen, M.K. (1974) Toxicological evaluation of palytoxin in several animal species. Toxicon, 12, 427–433. Wu, C.H. (2009) Palytoxin: membrane mechanisms of action. Toxicon, 54, 1183–1189. Yakes, B.J., DeGrasse, S.L., Poli, M. and Deeds, J.R. (2011) Antibody characterization and immunoassays for palytoxin using an SPR biosensor. Analytical and Bioanalytical Chemistry, 400, 2865–2869. Yasumoto, T., Oshima, Y. and Yamaguchi, M. (1978) Occurrence of a new type of shellfish poisoning in Tohoku district. Bulletin of the Japanese Society of Scientific Fisheries, 44, 1249–1255. Yasumoto, T., Yasumura, D., Ohizumi, Y. et al. (1986) Palytoxin in two species of xanthid crab from the Philippines. Agricultural and Biological Chemistry, 50, 163–167. Yasumoto, T., Seino, N., Murakami, Y. and Murata, M. (1987) Toxins produced by benthic dinoflagellates. Biological Bulletin, 172, 128–131. Zamolo, V.A., Valenti, G., Venturelli, E. et al. (2012) Highly Sensitive Electrochemiluminescent Nanobiosensor for the Detection of Palytoxin. ACS Nano, 6, 7989–7997.

CHAPTER 6

Pharmacology of palytoxins and ostreocins M. Carmen Louzao, María Fraga & Natalia Vilariño Department of Pharmacology, University of Santiago de Compostela, Spain

Introduction Chemistry of palytoxins, ovatoxins, ostreocins and mascarenotoxins In the 1960s, an ancient Hawaiian legend led to Moore and Scheuer to compile information in order to localize a tide pool with lethal properties. Some polyps belonging to the genus Palythoa were collected in Hana (Maui). Those samples were used for the first isolation and description of the PLTX (Moore & Scheuer, 1971). Palytoxins (PLTXs), ovatoxins, ostreocins and mascarenotoxins share a common and complex chemical scaffold, characterized by the presence of a long continuous chain of carbon atoms. PLTX is the representative and the most studied toxin of this group and, actually, the first attempt to estimate its molecular weight and formula was performed more than 40 years ago. However, the complexity of that molecule hampered the obtainment of its exact chemical structure. Two independent research groups worked for almost 11 years to elucidate the planar structure, chemical formula and molecular weight of the PLTX (Moore & Bartolini, 1981; Uemura et al., 1981) although both groups needed one year more to disclose its stereochemistry (Cha et al., 1982; Moore et al., 1982). Currently, PLTX isolated from Palythoa tuberculosa is considered to be 2678 Da, corresponding to the chemical formula C129 H223 N3 O54 , which includes 64 chiral centres and eight double bonds. PLTX theoretically can have more than 1021 stereoisomers, based on the number of chiral centres and the fact that the double bonds exhibit cis/trans-isomerism (Ramos & Vasconcelos, 2010). PLTX has two chromophores displaying ultraviolet absorption maxima at 263 and 233 nm (Moore & Scheuer, 1971; Moore et al., 1975; Moore et al., 1978). The introduction of PLTX in the research field promoted deeper studies, which finally led to the identification of different analogues. Thereby, in 1985, four minor toxins were isolated from Palythoa tuberculosa, chemically characterized and named as homopalytoxin, bishomopalytoxin, neopalytoxin and deoxypalytoxin (Uemura et al., 1985). All four toxic components displayed closer chemical structures to that observed for the PLTX, therefore formulae were similar for homopalytoxin (C130 H225 N3 O54 ), bishomopalytoxin (C131 H227 N3 O54 ), neopalytoxin (C129 H221 N3 O53 )

Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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and deoxypalytoxin (C129 H223 N3 O53 ). Recently, a new analogue, found in Palythoa tuberculosa, was assigned the chemical formula C129 H223 N3 O55 and consequently called 42-hydroxy-palytoxin. These analogues possess a characteristic pattern of fragmentation between C-8 and C-9 that divides the molecules in moiety A and B, which introduces a key for their recognition when using mass spectrometry techniques. The elemental formula of the A-moiety, C16 H28 N2 O6 , after undergoing an additional loss of one molecule of water finally generates an ion at m/z 327 (Ciminiello et al., 2010, 2012). These PLTXs were isolated from the genus Palythoa besides Zoanthus and different dinoflagellates belonging to the genus Ostreopsis. Nonetheless, major toxins produced by these dinoflagellates are known as ovatoxins, ostreocins or mascarenotoxins, depending on the Ostreopsis species involved. The progress in scientific knowledge about these toxins has increased the number of analogues that comprise each group, conserving in all of them a chemical likeness with PLTX. The toxin profile of Ostreopsis ovata has revealed the presence of several PLTX-analogues. Currently, few analogues have been isolated from different strains of this dinoflagellate species: ovatoxin-a, -b, -c, -d, -e, -f and some isomers called -a AC, -b AC, -d AC, -e AC, -a IK2, -d IK2 and -e IK2. Ovatoxins-a, -d, -e IK2 and -f share the same A-moiety as PLTX. However, the A-moiety suggested for the detection of all ovatoxins-b and -c is related to the presence of the ion at m/z 371, corresponding to C18 H32 N2 O7 . For ovatoxin-e, the formula of the A-moiety was C16 H28 N2 O7 relative to the ion at m/z 343 (Ciminiello et al., 2008, 2010, 2012; Suzuki et al., 2012; Uchida et al., 2013). The molecular formulae of the analogues isolated from the Mediterranean strain are C129 H223 N3 O52 for ovatoxin-a, C131 H227 N3 O53 for ovatoxin-b, C131 H227 N3 O54 for ovatoxin-c, C129 H223 N3 O53 for ovatoxin-d and -e and C131 H227 N3 O52 for ovatoxin-f (Ciminiello et al., 2010, 2013). The recent discovery of this group of toxins pointed to the necessity of looking more deeply into their chemical, biological and toxicological characteristics. PLTX has another analogue, named ostreocin-D after the name of its producer, the dinoflagellate Ostreopsis siamensis. A Japanese strain of this dinoflagellate was used to purify ostreocin-D. Five compounds were detected, but ostreocin-D was the major constituent, showing 70% of total toxicity (Usami et al., 1995). Those molecules were determined as PLTX analogues, based on the mass spectrum profile and fragmentation pattern of ostreocin-D. Further studies, using advanced methods as nuclear magnetic resonance (NMR) and negative-ion fast-atom bombardment collision-induced dissociation tandem mass-spectrometry (FAB-CID-MS/MS), confirmed those analogue structures (Ciminiello et al., 2013; Ukena et al., 2001, 2002; Usami et al., 1995). The cleavage between the C-8 and C-9 generated an A-moiety correspondent to C15 H26 N2 O6 . Molecular formulae differ just in one atom of oxygen, ostreocin-D being C127 H219 N3 O53 and ostreocin-b C127 H219 N3 O54 . Two new PLTX-analogues were found in Ostreopsis mascarenensis and named mascarenotoxin-a and -b. Their ion and mass spectra displayed similarities with the PLTX, identical UV spectra and A-moiety. Their molecular weight was estimated between 2500 and 2535 Da; however, chemical formulae were not established (Lenoir et al., 2004). Ostreotoxin-1 and -3 were extracted from Ostreopsis lenticularis, but ostreotoxin-3 was demonstrated as a sodium-channel activating toxin (de Motta et al., 1996; Meunier et al., 1997).

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Origin and producing organisms Zoanthids, dinoflagellates and at least one species of cyanobacteria were identified as PLTX-like molecules producers. However, the biological origin of this group of toxins remains unclear. Regarding zoanthids, the presence of PLTX-like molecules was demonstrated in many species belong to Palythoa and few species of Zoanthus. PLTX was isolated for the first time by Moore and Scheuer in 1971 from Palythoa toxica, the Hawaiian soft coral (Moore & Scheuer, 1971). The study of the toxin profile of Palythoa displayed the co-production of different PLTX-analogues besides PLTX. Homopalytoxin, bishomopalytoxin, neopalytoxin and deoxypalytoxin were isolated as minor toxins from Palythoa tuberculosa, as well as 42-hydroxy-palytoxin (Uemura et al., 1985; Ciminiello et al., 2009). Recently, Palythoa toxica and Palythoa helodiscus were also demonstrated to synthesize respectively the analogues named 42-hydroxy-palytoxin and deoxypalytoxin as well as PLTX (Ciminiello et al., 2009; Deeds et al., 2011). In the case of dinoflagellates, in 1995 Dr. Takeshi Yasumoto’s group isolated and characterized PLTX-like compounds from Ostreopsis siamensis but the biosynthetic pathways are still unknown (Usami et al., 1995). The most studied species related to the synthesis of PLTX-like molecules are Ostreopsis siamensis, Ostreopsis ovata, Ostreopsis mascarenensis and Ostreopsis lenticularis. They are unicellular epiphytic benthic microalgae that originally colonized only tropical and sub-tropical areas, but nowadays they are being detected more frequently in temperate seas. PLTX has been detected in Ostreopsis siamensis at very low levels (lower than fg) (Ciminiello et al., 2013), although this dinoflagellate is known to produce ostreocins, mostly, ostreocin-D (Ciminiello et al., 2013; Usami et al., 1995). In the Mediterranean region, appearance and proliferation of Ostreopsis spp. were first reported in the late 1970s and 1980s (Taylor, 1979). Since then, the presence of Ostreopsis spp has been registered several times along the coastline (Penna et al., 2010). Ostreopsis cf. ovata has bloomed across the Mediterranean Sea, affecting the marine environment (Ciminiello et al., 2006; Aligizaki et al., 2008). In Ostreopsis ovata, PLTX represented a minor toxin, being predominantly six ovatoxins, from -a to -f, as well as some of their isomers found in closer strains collected in different geographical areas (Ciminiello et al., 2008, 2010, 2012; Suzuki et al., 2012; Uchida et al., 2013). The toxin profiles of Ostreopsis mascarenensis demonstrated to contain mascarenotoxin-a, -b and -c (Lenoir et al., 2004) and Ostreopsis lenticularis displayed the presence of ostreotoxin-1 and -3(de Motta et al., 1996). Recently, PLTX and 42-hydroxy-palytoxin have been identified in samples of Trichodesmium spp. collected from a cyanobacterial bloom (Kerbrat et al., 2011).

Toxin distribution and ecological aspects Bioaccumulation From an ecological point of view, the dangerous consequences of natural marine toxins can occur through phenomena of bioaccumulation along the food chain (Ramos & Vasconcelos, 2010). PLTX-like molecules have been detected in marine species belonging to different trophic levels, which demonstrate not just bioaccumulation but also biomagnification. Additionally, producer organisms of these toxins have increased

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their geographical distribution from Indo-Pacific to Mediterranean and Atlantic areas. The entrance of potentially toxic dinoflagellates into the ecosystem can have impact at sanitary and economic levels, often tightly connected. In this context, PLTX and their analogues become in a global hazard, magnified through their accumulation along the trophic chain, which can reach human beings. Human fatalities have been reported and associated with the consumption of fish or shellfish contaminated with PLTX-like compounds (Alcala et al., 1988; Onuma et al., 1999; Yasumoto et al., 1986). They can be found in sea urchins, bivalve molluscs such as mussels, (EFSA-Panel, 2009) cockles, oysters, scallops, crabs and fish (Ramos & Vasconcelos, 2010; Paredes et al., 2011). The presence of these toxins in marine animals is associated with the contact with or predation of producer organisms. During the development of an experimental work, mussels (Perna canaliculus), scallops (Pecten novaezealandiae) and oysters (Crassotrea gigas) were fed, under the same conditions, with Ostreopsis siamensis. Afterwards, the detection of PLTX-like molecules using haemolysis neutralization assays displayed differences in the toxin content between species. The highest concentration level was displayed in the hepatopancreas of scallop. The lack of toxin in mussels indicated that either the toxin is not taken up by the shellfish, or that it is converted after uptake to non-toxic material (Rhodes et al., 2002). A bioaccumulation of PLTX-like molecules was found in mussels (Mytilus galloprovincials) and sea urchins (Paracentrotus lividus) on the French Mediterranean coast. In this case, the highest accumulation of toxin was found in the digestive tract of sea urchins (Amzil et al., 2012). However, two or three weeks after the end of the toxic episode, PLTX-like compounds were not detected in shellfish (Aligizaki et al., 2008; Amzil et al., 2012). The fact that these animals contain the active PLTX-like molecules in their tissues provides evidence for the introduction of these toxins into the marine food web. PLTXs have been detected in copepods, crustaceans (Platypodiella spectabilis, Lophozozuymus pictor, Demania alcalai and D. reynudii), fishes (Chaetodon caistratus, C. sedentarius, Melchtys vidua, Herklotsichthys quadrimaculatus, etc.), molluscs (Mytilus galloprovincials, Perna canaliculus, Pecten novaezealandiae, Crassotrea gigas, Octopus sp., etc.) and echinoderms (Acanthaster planci, Paracentrotus lividus) (Alcala et al., 1988; Aligizaki et al., 2008, 2011; Amzil et al., 2012; Deeds et al., 2011; Espina et al., 2009; Fukui et al., 1987; Furlan et al., 2013; Gleibs & Mebs, 1999; Gleibs et al., 1995; Lau et al., 1995; Onuma et al., 1999). The accumulation of the toxins in several edible marine species opens the possibility of repeated human exposure through contaminated seafood collected in the same area (Del Favero et al., 2012). Also, the recognition that marine organisms accumulate PLTX-group toxins without being killed by these compounds ( Bellocci et al., 2008b) would require further knowledge about possible mechanisms that might sequester PLTX or inhibit its activity (Gleibs & Mebs, 1999).

Pharmacological target of PLTXs Mammalian sodium pump The original studies on the toxicity of PLTX revealed that animal exposed to the toxin had neuromuscular symptoms (Habermann et al., 1981), prompting investigations on neuronal and muscular tissues. However, in erythrocytes, PLTX elicited a concentration-, temperature- and pH-dependent haemolytic response (Habermann

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et al., 1981). Further in vitro studies on excitable cells contributed to elucidation of PTX effects at functional level and to the better characterization of its mechanism of action (Del Favero et al., 2012). These basic findings led to the recognition that sodium potassium-ATPase (Na+ ∕K+ -ATPase) is the PLTX receptor. This is a plasma membrane pump, whose physiological activity is of crucial importance for eukaryotic cells. The pump maintains trans-membrane ionic gradients in animal cells carrying ions against their concentration gradients at the expense of chemical energy (ATP), which are critical to cellular function (Deeds & Schwartz, 2010). With regard to possible advancements in the molecular PLTX mechanism of action, several new areas are being actively investigated. Particular attention has been given to the secondary effects elicited by the disruption of Na+ ∕K+ -ATPase functioning. The PLTX action involves its binding to the extracellular portion of alpha subunit of this plasma membrane protein, which converts the pump into a non-selective pore for monovalent cations, with a passive flow of ions following their concentration gradients. More recent research have indicated that PLTX would interfere with the normal coupling between the inner and outer gates of the pump, allowing the gates to be simultaneously open (Rossini & Bigiani, 2011). Artigas & Gadsby (2003) showed that the inner and outer gates of the PLTX-bound pump are still able to respond to physiological ligands, implying that the toxin does not distort the structure of the Na+ ∕K+ -ATPase (Artigas & Gadsby, 2003). The potassium efflux is accompanied by the cytosolic loading of sodium. The increased [Na+ ]i is counterbalanced by the action of two different processes: (a) the Na+ ∕H+ antiporter extruded the sodium from the cell with a consequent intracellular acidification (Yoshizumi et al., 1991); and (b) the sodium is exchanged for calcium through the Na+ ∕Ca2+ exchanger (Ishii et al., 1997; Ozaki et al., 1983). PLTX binds to the sodium pump and as a result it cause membrane depolarization in excitable cells (Cagide et al., 2009; Louzao et al., 2006), and contraction of muscle cells. Moreover, membrane depolarization due to the rising in [Na+ ]i would lead to open voltage-gated calcium channels, (VGCC) driving Ca2+ entry into the cells (Ishii et al., 1997). Most of the available data on the effects the toxin exerts in biological systems can be explained by either primary or secondary events due to the alteration that PLTX induces in the Na+ ∕K+ -ATPase (Wu, 2009). However, PLTX interaction with Na+ ∕K+ -ATPase might play some signalling role, in addition to the conversion of the pump into a channel (Rossini & Bigiani, 2011). It was suggested the capacity of PLTX to phosphorylate proteins (Wattenberg, 2007; Sala et al., 2009), and delay cytotoxic responses (Habermann et al., 1981; Bellocci et al., 2011), might call into question mechanisms of signal transduction that trigger several adverse biological reactions (Tubaro et al., 2011b).

Palytoxin toxicology Toxicity of palytoxin and analogues in humans The effects of PLTX on human health should not be underestimated. In fact, PLTX was considered among the most toxic compound of natural origin based on the first toxicological characterization of PLTX in the 1970s (Wiles et al., 1974) and the cases of

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lethal human poisonings ascribed to the toxin (Ito & Yasumoto, 2009). For instance, human fatalities associated with consumption of crab and sardines suspected to be contaminated with PLTX have been reported in the Philippines (Alcala et al., 1988) and Madagascar (Onuma et al., 1999). PLTX is thought to be the cause of clupeotoxism, a severe toxic syndrome linked with the consumption of clupeoid fish (Deeds & Schwartz, 2010) with maculo papular and/or erythematous dermatitis, myalgia, fever, respiratory and cardiac problems, even lethality, depending on the exposure route and the toxin concentration. The main target organs for systemic PLTX toxicity could be all the excitable tissues (muscular, cardiac and nervous). This hypothesis is consistent with the mechanism of action of PLTX affecting the sodium potassium pump (Wu, 2009). In spite of this, the actual in vivo toxicity of the molecule has yet to be fully evaluated, because the case reports are often scarce (Tubaro et al., 2011b). The lack of toxicokinetic data on PLTX is a challenge for the comprehension of the hazards associated with this family of toxins in the food web (Del Favero et al., 2012). Therefore, data about PLTX passage through the human intestinal barrier can help to achieve a better understanding of oral PLTX poisoning. Experimental procedures are based on a well-characterized and accepted in vitro model of intestinal permeability that uses differentiated Caco-2 cell monolayers and measures the effect of PLTX on TEER and intestinal cells absorption (Fernandez et al., 2013). Transepithelial electrical resistance (TEER) is a measure of the ionic movement across the paracellular pathway as an assessment of cellular tight junctions. PLTX induces a dose-dependent decrease in the TEER, indicating a clear disturbance of the monolayer stability and integrity. However, the PLTX that pass through Caco-2 monolayer is minimal, suggesting that this group of toxins would be poorly transported to blood, which may explain its lower oral toxicity (Table 6.1). These data confirmed the limited oral toxicity compared to toxicity upon intravenous or intraperitoneal administration (Deeds & Schwartz, 2010; Fernandez et al., 2013). The most frequent symptoms after oral exposure to contaminated seafood include gastrointestinal alterations, occasionally associated with a metallic and/or bitter taste of seafood, myalgia, weakness, cardiac dysfunctions, respiratory problems, cyanosis and possibly fever, but the whole clinical manifestation is not clear yet (EFSA-Panel, 2009). The scarcity of data concerning occurrence in Europe made to the European Food Safety Authority (EFSA) estimates the exposure to PLTXs by means of the worst case scenario (EFSA-Panel, 2009). EFSA’s Contaminants in the Food Chain (CONTAM) Panel established an oral acute reference dose (ARfD) of 0.2 mg/kg bw (body

Table 6.1

Permeability of differentiated Caco-2 cells to PLTX after 10 hours incubation. Trans epithelial electric resistance (TEER) values are expressed as the percentage respect to control (100%). Absorbed PLTX is expressed as the percentage of toxin that passed the cell monolayer with respect to the initial PLTX.

PLTX treatment (nM)

TEER 10h (%)

Absorbed PLTX (%)

0.135 1.35 13.5

94.207 ± 7.629 30.813 ± 12.654 7.677 ± 1.760

3.484 ± 0.702 6.052 ± 1.024 15.906 ± 1.185

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weight), which applies to the sum of PLTX and its analogue ostreocin-D, because of their equipotency and similar mechanism of action (Ares et al., 2009; Cagide et al., 2009). About other routes of human exposure, the characterization of cutaneous toxicity will help the prevention of illness for people exposed to PLTX, either professionally or recreationally. Erythematous dermatitis was observed in patients exposed to marine aerosol during Ostreopsis blooms along the Italian coasts (Gallitelli et al., 2005). Case reports of poisonings through cutaneous exposure to PLTX while handling toxic zoanthid coral are also characterized from a clinical point of view. However the presence of PLTX and/or analogues is not always confirmed (Nordt et al., 2011). The severity of the symptoms after cutaneous exposure depends on the length of the contact and the presence of skin lesions that could lead to systemic effects. A PLTX dermotoxicity such as local skin irritation, oedema, erythema, urticarial rush and pruritus that persisted several days, is sometimes accompanied by neurologic symptoms such as paresthesia, dysguesia and dizziness. Additionally, the systemic symptoms could be general weakness and myalgia, irregularities in the electrocardiogram (ECG) and rhabdomyolysis in more severe cases (Tubaro et al., 2011b). Since the late 1990s, a respiratory syndrome has been repetitively observed in humans during Ostreopsis blooms in the Mediterranean area. Indeed, in Italy people exposed to Ostreopsis ovata bloom aerosols during recreational or working activities had respiratory problems. Symptoms included fever, watery rhinorrhea, pharyngeal pain, dry or mildly productive cough, headache, nausea/vomiting and bronchoconstriction, with mild dyspnoea and wheezes (Durando et al., 2007). On the basis of the observed association between Ostreopsis ovata blooms, respiratory illness in people, and detection of PLTX complex in algal samples, the cause of those human illness are postulated to be toxic aerosols containing Ostreopsis cells and/or the toxins they produce (Ciminiello et al., 2014). The chemical properties of PLTX seem to exclude its volatility. Nevertheless, it is possible that molecular fragments and/or other compounds are responsible for the reported respiratory affections (Tubaro et al., 2011b). In addition, an irritative/inflammatory potential of this molecule could explain the febrile syndrome after its inhalational exposure, as well as the leukocytosis and the enhancement of C-reactive protein observed in several cases (Tubaro et al., 2011b). The symptoms recorded after exposure to this sea-spray aerosol and while cleaning zoanthid-containing aquaria suggest that these illnesses have a common origin. In the latter case, marine aquarium hobbyists who remove infesting zoanthids from their aquarium with boiling water have experienced a severe respiratory reaction following inhalation of steams. The presence of PLTX was confirmed in the zoanthids by LC-MS and/or haemolysis assay (Deeds & Schwartz, 2010).

Toxicity of palytoxin and analogues in animals The high toxicity of PLTXs has been also displayed in a broad range of different animal species, including rats, guinea pigs and rabbits (Bellocci et al., 2011). A study by Wiles et al. (1974) originally suggested the existence of a species-specific susceptibility to PLTX, and that its potency is strictly related to the administration route (Wiles et al., 1974; Munday, 2011). For instance, the LD50 for rodents by intravenous injection is in a range of 10–100 ng/kg (Vick & Wiles, 1975). Nevertheless, the toxicity studies were performed on a semi-purified preparation of the molecule.

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Some recent data are available in mice using pure PLTX administered by intravenous, intraperitoneal injection or oral (Ito & Yasumoto, 2009; Munday, 2011; Tubaro et al., 2011a). Ito and Yasumoto confirmed the high PLTX toxicity by intravenous (LD50 = 0.15 μg∕kg) and intraperitoneal (LD50 = 1.5 μg∕kg) administration. In addition, they demonstrated that PLTX causes bleeding and alveolar destruction in lungs and death at the dose of 2 μg∕kg by intratracheal administration. Ostreocin-D caused death at 11 μg∕kg. In vivo acute toxicity studies in mice allowed to propose the skeletal muscle as one of the main targets of PLTX-like molecules, in agreement with human symptoms. Those targets were subsequently confirmed by the ultra-structural changes observed (Del Favero et al., 2012). PLTX analogues have low LD50 values in rat and mouse, with an acute toxic effect on specific organs, such as lungs, gastro-intestines, and the kidney (Ito & Yasumoto, 2009). Repeated oral administration of PLTX caused lethality and toxic effects, with an estimated No-Observed-Adverse-Effect Level (NOAEL) equal to 3 mg/kg/day (Del Favero et al., 2013). Macroscopic alterations at gastrointestinal level were observed in mice dead during the treatment period. Histological analysis highlighted hypereosinophilia and fibre separation in myocardium. PLTX inhibited the motility of sperm in hamsters, guinea pigs, rabbits, cattle, humans and the invertebrate sea urchins (Morton et al., 1982). Its manifestation is characterized as a loss in flagellar-bend amplitude, which may be accompanied with an increase in beat frequency. Some other potential physiological and developmental effects of PLTX in vertebrates are inferred from studies using the amphibian model Xenopus laevis (Ramos & Vasconcelos, 2010). The toxicological effects that PLTX displayed in the amphibian include a significant increase in mortality, a number of malformations, and delays in the growth of the embryo (Franchini et al., 2010). However, little is known about the effect of PLTX and its analogues in marine vertebrates and invertebrates. Animals living near or among zoanthid colonies may incorporate PLTX (Gleibs & Mebs, 1999) either by filtering like sponges and mussels, or by other, unknown mechanisms, like gorgonians and soft corals. The PLTX content of some soft corals and mussels clearly exceeds the maximum PLTX concentration of the Palythoa species. Much higher PLTX values were detected in predators of zoanthids such as polychaete worms, the crown-of-thorns starfish, and in the intestines of some Chaetodon fish. The fact that these organisms contain the active toxin in their tissues provides evidence for the accumulation of PLTX in marine food chains (Mebs, 1998). This is further substantiated by the observation that some fish had no detectable amounts of PLTX in their intestines, but exhibited high toxin concentrations in skin, muscle or liver. As mentioned above, currently there are insufficient studies to determine the effect that PLTX has in marine organisms. During some blooms of Ostreopsis in the Mediterranean coasts, many marine organisms, especially invertebrates, were greatly impacted. Chemical analyses confirmed that all samples were contaminated with PLTX (Lopes et al., 2013). Outbreaks of Ostreopsis ovata were pointed as responsible for the detriment or death of mobile and sessile epibenthos and macroalgae populations (Ciminiello et al., 2006; Faimali et al., 2012; Sansoni, 2003). In order to evaluate the toxicity of those episodes, the sensitivity of different animal models was tested using laboratory cultures and natural samples of Ostreopsis ovata. It was observed that the increase of temperature during the treatments incremented the mortality of Tigriopus fulvus, larvae of Amphibalanus amphitrite and juveniles of

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Dicentrarchus labrax (Faimali et al., 2012). Artemia salina was demonstrated to be the most sensitive model, even when testing the toxicity of Ostreopsis siamensis, which also cause morbidity in Haliotis virginea larvae (Faimali et al., 2012; Rhodes et al., 2000). The general health status of mussels exposed to Ostreopsis ovata indicated a non-specific response (Gorbi et al., 2012), but PLTX resulted cytotoxic when applied to mantle cells from mussels (Mytilus galloprovincials) (Louzao et al., 2010). The toxin also has an affect on neurotoxic responses and on the phagocytic activity, implying changes on immunological and lysosomal mechanisms. However, the recovery of control levels led to propose the existence of a compensatory process to counteract these alterations (Gorbi et al., 2012; Malagoli et al., 2008). In general, marine animals seem to tolerate high PLTX-concentrations that are lethal to terrestrial vertebrates. However, the mechanisms of masking or inhibiting the toxin’s activity while it is transferred and stored in the body of many marine organisms are still unknown (Gleibs & Mebs, 1999).

Toxicity of palytoxin and analogues in vitro PLTX has a high ability of interaction with biological systems, due to its mix of hydrophilic and hydrophobic features (Rossini & Hess, 2010; Bellocci et al., 2011). The investigations showed the toxin’s capability to trigger a broad variety of effects, including cell rounding (Valverde et al., 2008a) and swelling (Amir et al., 1997; Falciola et al., 1994; Mullin et al., 1991), cell lysis and loss of membrane integrity resulting in leakage of cytosolic enzymes (Bellocci et al., 2008b; Sheridan et al., 2005; Louzao et al., 2010), together with the activation of caspases (Valverde et al., 2008a, 2008b), the contraction of vascular smooth muscle and cardiac cells (Ito et al., 1976, 1977, 1979), as well as membrane depolarization (Castle & Strichartz, 1988; Dubois & Cohen, 1977; Kudo & Shibata, 1980; Pichon, 1982; Louzao et al., 2006). The cutaneous toxicity was characterized using an in vitro approach. The high toxicity of PLTX on skin keratinocytes (Pelin et al., 2011) raises valid concerns about potential human exposure to PLTX-related toxins in seawater. It was found that the PLTX concentrations inducing cytotoxicity on human skin keratinocytes seem to be lower than those of cutaneous human exposure during Ostreopsis ovata blooms, indicating the harmful potential of the toxin. Transmission electron microscopy (TEM) images allowed ultrastructural observations of the changes triggered by PLTX in Caco-2 monolayers (Fernandez et al., 2013). In those cells, the first cellular target seems to be the mitochondria, inducing an electron-dense appearance that could be due to ion accumulation (Vale et al., 2007). Monolayers show important dose-dependent alterations, such as disappearance of microvilli; however, desmosomes are unaffected (Figure 6.1). In any case, the PLTX dose-response disruption of the intestinal barrier could lead to chronic effects after repeated exposures. After repeated oral exposure of palytoxin in mice, histological analysis showed alterations at the gastrointestinal level, but also fibre separation in myocardium. This cardiac damage was supported by the in vitro effect of the toxin on cardiomyocytes, indicating a severe and irreversible impairment of their electrical properties. Electrophysiological recordings detected a progressive cell depolarization, arrest of action potentials and beating (Del Favero et al., 2013). In vitro toxicological studies on ovatoxins are currently in progress, and preliminary biochemical results have demonstrated that they significantly increased the levels of mRNAs encoding inflammation-related proteins in immune cells (i.e., monocyte derived human macrophages; Ciminiello et al., 2012, 2014).

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BB

D BB

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N N

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Figure 6.1

Transmission electron microscopy (TEM) of differentiated Caco-2 cells treated with PLTX. (a) Two Caco-2 control cells in monolayer configuration, showing standard morphology. (b) Three adjacent cells in monolayer configuration treated with 270 nM PLTX for 0.5 hours. The following structures are indicated in the images: Brush border (BB), desmosomes (D), filopodia (F), amorphous cytoplasmic inclusions (I), mitochondria (M), nucleus (N) and nucleolus (No).

Cytotoxic effect of palytoxin and analogues Different studies have been performed to expand the knowledge concerning the cytotoxic effect of PLTX. The early observation of PLTX’s haemolytic effect (Habermann et al., 1981) with a large loss of potassium shifted the attention of scientists to its cytotoxic activity in experimental systems other than neuronal or muscular cells (Lauffer et al., 1985; Louzao et al., 2008, 2010). PLTX is a highly cytotoxic compound for many cellular models. Cell death in vitro is frequently recorded in isolated cells exposed to PLTX (Ledreux et al., 2009), where the toxin induces rupture of plasma membrane and the release of cellular contents. Therefore, cytolytic and cytotoxic responses to PLTX often represent complementary aspects of the same process in isolated cells, as cell lysis is a consequence of collapsed cell functioning and represents the outcome of a toxic effect. Cytolysis has been considered the end-point of a cascade of events that originates from the interaction between PLTX and Na+ ∕K+ -ATPase pump. Existing data would indicate that cell death consequent to PLTX treatment involves a necrosis (Majno & Joris, 1995). For instance, the determination of the lactate deshydrogenase (LDH) released into culture medium is an indicator of the final cytotoxic effect induced by PLTX in smooth muscle cells. On other hand, the metabolic capacity of cells is a good indicator of the viability of cell cultures (Fernandez et al., 2013). Cytotoxic studies showed that differentiated Caco-2 cells treated with PLTX had a time and dose-dependent decrease in cell viability (Figure 6.2). Some observations obtained in both excitable and non-excitable cells demonstrated the ability of PLTX to induce programmed cell death. This could be supported by the studies on activation of JNK and p38 mitogen-activated protein kinase (MAPK) phosphorylation cascades (Li & Wattenberg, 1999), the accumulation of phosphorylated hsp 27 and oxidized isoforms of DJ1 cell stress protein (Sala et al., 2009), as well as the activation of proteases and nucleases and the breakdown of mitochondrial membrane potential (Ledreux et al., 2009; Valverde et al., 2008a; Bellocci et al., 2011).

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Figure 6.2 Cell viability of differentiated Caco-2 cells incubated with PLTX for 24 hours. Values are indicated as the percentage of viability respect to control (100%).

Furthermore, cytotoxicity and cytolysis have been exploited in most of the cell-based assays for the detection and quantification of PLTX group toxins developed so far (Cañete & Diogène, 2008; Cagide et al., 2009; Espina et al., 2009; Ledreux et al., 2009). In the processes recorded in vitro, cellular conditions and experimental parameters play key roles in the cellular fate of the biological system after PLTX treatment. Thus, the identification of the mechanistic links among the series of events triggered by PLTX, and further studies will be essential for a full understanding of the molecular mechanisms of PLTX-induced cell death and its toxicity pathway.

Palytoxin and analogues effect on cytoskeleton The alteration of ion distribution across the plasma membrane consequent to PLTX action would be the driving force of secondary effects as, for instance, MAPK activation, changes in cell morphology and/or shape, and also the alteration of the cytoskeletal reorganization. The cytoskeleton is a dynamic structure essential for a wide variety of normal cellular processes, including the maintenance of cell shape and morphology, volume regulation, membrane dynamics and signal transduction. It is organized into microtubules, actin meshwork and intermediate filaments. Actin microfilaments have been identified as a major target for destruction during the apoptosis processes, and they are also important under pathological conditions such as cancer (Louzao et al., 2011). Cytoskeleton alterations were recently detected in different cellular models (Ares et al., 2005, 2009; Louzao et al., 2007; Franchini et al., 2010; Silvestre & Tosti, 2010; Bellocci et al., 2011). Fluorescence microscopy showed that PLTX caused disassembly of cytoskeletal actin microfilaments, with subsequent cytomorphologic changes such as cell rounding (Ares et al., 2005). This effect on the actin cytoskeleton indicates that

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PLTX is a potent natural actin depolymerizing compound and is probably one of the causes of the detachment of Caco-2 monolayers from the substrate (Valverde et al., 2008b; Fernandez et al., 2013). Likewise, the data obtained with ostreocin-D (reduction in 47% in the actin content) suggest an action mechanism targeting intestinal cells, similar to that of the parent compound, PLTX (Ares et al., 2005). This is consistent with in vivo toxicological data showing erosion and oedema in the small intestine of mice after intratracheal and oral (gavage) administration of PLTX. The activity of PLTX on the actin cytoskeleton of intestinal cells seems to be partially modulated by a signalling pathway involving Ca2+ influx. It could be possible that PLTX activates Ca2+ permeable channels (Wu, 2009), which may lead to gelsolin-induced severing of actin. If this were the case, cytoskeleton-associated proteins, with diverse biological functions would be regulated in response to PLTX. There is a cross-talk between the cytoskeleton and Na+ ∕K+ -ATPase. In a similar manner to ion channels, the Na+ ∕K+ -ATPase activity is regulated, in part, by cytoskeleton. On the other hand, PLTX acts on Na+ ∕K+ -ATPase and affects cytoskeleton dynamics by targeting microfilaments.

Tumour promotion by palytoxin and analogues Tumour promotion is an early, prolonged and reversible phase of carcinogenesis. The development of cancer involves a series of genetic changes that occur at different stages of tumourigenesis, starting with early changes in cell behaviour, proceeding to the development of benign tumours, and then progressing to the development of malignant tumours. In general, tumour promoters do not damage DNA, but instead contribute to carcinogenesis by disrupting the regulation of cellular signalling (Wattenberg, 2011). PLTX was defined in the 1980s as a skin tumour promoter that does not activate protein kinase C (Fujiki et al., 1983). PLTX is a skin irritant and a tumour promoter in the multistage mouse skin assay (Fujiki et al., 1986). Subsequent cell culture studies provided further evidence that PLTX stimulates signalling pathways that do not require protein kinase C {Wattenberg, 1987 371}. Therefore, PLTX has proven to be a useful tool for exploring alternative mechanisms of modulating key signal transduction pathways in carcinogenesis (Wattenberg, 2007). Sodium influx plays an important role in the ability of PLTX to stimulate the down modulation of the epidermal growth factor (EGF) receptor under non-cytotoxic conditions. A search for the PLTX target led to the identification of mitogen-activated protein (MAP) kinases as mediators of PLTX-stimulated signalling (Wattenberg, 2007). MAP kinases are a family of serine/threonine kinases that relay a variety of signals to the cellular machinery regulating the enzyme activity, gene expression, proliferation and apoptosis. This function may help to explain how PLTX can stimulate the wide range of effects that are characteristic of tumour promoters. AP-1, a dimer made up of Jun and Fos family members, may act as a type of ‘master switch’ that regulates downstream targets important for tumour promotion. PLTX can modulate AP-1 through different mechanisms that involve MAP kinases. The interaction of palytoxin with the Na+ ∕K+ -ATPase pump triggers a change in ion flux, which then stimulates the activation of protein kinase cascades that ultimately activate p38, ERK5 and JNK, (Li & Wattenberg, 1999; Charlson et al., 2009). Phosphorylation of c-Jun by JNK then modulates AP-1 transcriptional activity (Karin et al., 1997). On the other hand, PLTX action results in the down modulation of MKP-3. The loss of this negative regulator of ERK1/2, in cells that express oncogenic Ras, results in the accumulation of the phosphorylated active form of ERK1/2. PLTX stimulation

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of ERK1/2 and ERK5 activation results in an increase in c-Fos gene expression, which in turn can alter the composition and function of AP-1 dimers (Warmka et al., 2002; Charlson et al., 2009).

Detection methods Legislation Currently, there are no regulated legal limits or validated detection methods for the group of PLTXs. However, the maximum content of PLTX in seafood products destined for consumption must be controlled. Different cases of human intoxications were associated with the ingestion of contaminated fish or shellfish or the contact with marine aerosols during toxic episodes of harmful algal blooms, revealing PLTX-like molecules as a potential sanitary hazard (Alcala et al., 1988; Deeds & Schwartz, 2010; Hoffmann et al., 2008; Onuma et al., 1999; Tubaro et al., 2011b; Yasumoto et al., 1986). Therefore, the Scientific Opinion from the European Food Safety Authority (EFSA) CONTAM Panel has recommended establishing an upper limit for the sum of PLTX and its analogue ostreocin-D of 30 μg∕kg of shellfish meat, in order to ensure the consumer protection (EFSA-Panel, 2009). Nowadays, in spite of the lack of certified standard materials for the group of PLTXs, many types of detection methods have been developed. Since the discovery of PLTX, bioassays have been used to test its toxicity. Nevertheless, in order to avoid ethical issues and other technical disadvantages, different molecular and analytical assays were developed.

Bioassay The mouse bioassay (MBA) is a detection method which has been widely used in the field of marine toxins. From the initial description of PLTX-like molecules, MBA was employed to study their presence in real samples and confirmed by chemical methods. The MBA allowed establishing as characteristics some symptoms caused by PLTX-like molecules, which depend on the dose and route of administration. The most toxic administration is intravenous, followed by the intraperitoneal injection and oral administration being the least toxic (Munday, 2011; Riobo et al., 2008). The MBA for PLTXs is based on their neurotoxic effects so, therefore, initial symptoms of injected mice clearly differ from the more common marine toxins. The intraperitoneal administration of PLTX in mice shortly provokes ataxia and piloerection, followed by the progressive paralysis of the whole animal, which implies reduction of the respiration rate. Similar symptoms were observed along different works when extracts of Ostreopsis were intraperitoneally injected in mice (Aligizaki et al., 2008; Lenoir et al., 2004; Rhodes, 2011; Usami et al., 1995). In mice, as well as in other animal models like dogs or cats, the intravenous administration of PLTX also affected the cardiac function (Munday, 2011). The utility of the routine MBA is based in the fact that the initial 15-minute symptoms could identify the presence of PLTX in the sample. It could be used as semiquantitative estimation of PLTX presence. The LD50 value ranges between 150 and 720 ng∕μL, and the observation time in mice could be up to 48 hours (Riobo & Franco, 2011). However, the dose-survival time or dose-death time relationships generate serious ambiguities (Riobo et al., 2008). Also, the MBA cannot be automated – it requires specialized animal facilities and expertise.

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Cytotoxicity assays Cellular alterations induced by PLTX-like compounds have been employed to yield alternative detection methods to the MBA. The measurement of different cellular metabolites synthesized in presence of these toxins has allowed the development of cytotoxic detection methods.

Haemolysis assay The capability of PLTX and its analogues to transform Na+ ∕K+ -ATPase into a non-specific ion channel produces a disturbance on the ionic equilibrium in erythrocytes and leads to a delayed haemolytic effect. This effect was employed to develop a detection method based on the use of blood cells. When PLTX-like compounds are responsible for the haemolysis, it can be suppressed by the addition of ouabain, a poisonous glycoside, able to inhibit the sodium pump. PLTX and ouabain binding sites are not identical, but share some structural determinants for binding (Artigas & Gadsby, 2003, 2006). The sensitivity of the haemolytic assay depends on the origin species of the erythrocytes and on the physiological features of the animal, as well as on the incubation time and temperature of the assay (Bignami, 1993; Habermann et al., 1981; Riobo et al., 2008). An alternative method was employed for the determination of the haemolytic activity of bacteria isolated from zoanthids. For this screening, assay blood agar plates and human erythrocytes were utilized (Seemann et al., 2009). Cytotoxic assays with neuroblastoma cells Different neuroblastoma lines have been used in conjugation with an ouabain pre-treatment for the detection of PLTX-like compounds. The increasing concentration of the toxin is indirectly related to the cell viability, which can be measured through the mitochondrial oxido-reductase activity. The assessment of the mitochondrial oxido-reductase activity in Neuro-2a cells in the presence of PLTX-like molecules was performed with 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT). This method was also explored on the NG108-15 cell line, formed by fusing mouse neuroblastoma with rat glioma cells, obtaining similar results (Cañete & Diogène, 2008; Ledreux et al., 2009). Alternatively, the mitochondrial oxido-reductase activity of neuroblastoma BE(2)-M17 cells was quantified using the cell viability dye alamar blue (Espina et al., 2009). In this case, the dose-dependent decrease of the viability induced by PLTX was specifically inhibited by ouabain. Therefore, in a sample, it can be differentiated from the cytotoxic effect induced by PLTX-like compounds from other toxins. The use of microplates enables the analysis of multiple samples in a single run. The sensitivity (as low as 0.2 ng/ml) and the simplicity of this assay has allowed the estimation of PLTX-like molecules in further research works (Ares et al., 2009; Cagide et al., 2009; Fernandez et al., 2013). The assay also has many advantages, such as the wide detection range and the possibility of simultaneous testing of many samples in microtitre plates. Other cytotoxic assays The cytotoxic effect of PLTX-like molecules has been studied in several cytological subtypes. Hela (Lau et al., 1995) and rat 3Y1 cells (Oku et al., 2004) have been used to

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evaluate the presence of the PLTX using dyes which fluorescence is measured in a spectrophotometer. In this case, the toxin cell damage can be quantified. A cytolytic assay could detect PLTX by the use of the human breast cancer cell line MCF-7. Cytotoxicity is evaluated by the measurement of the released cytosolic lactate deshydrogenase (LDH) to the culture supernatant. Cells exposed to samples containing PLTX or ostreocin-D registered a dose-dependent cytolysis measured as an increase in LDH. The cytolytic response induced by PLTX and ostreocin-D is specific, as it is prevented by ouabain. This assay enables discernment between PLTX-like molecules and other marine toxins with a different target. PLTXs can be detected at concentrations around 10 ng per kg of shellfish (Bellocci et al., 2008a, 2008b). Rat hepatocytes showed to be more sensitive to PLTX than human neuroblastoma cells. The difference in the species origin previously demonstrated was suggested as a possible factor to explain unlike sensitivities, due to the expression of a α1 isoform of the Na+ ∕K+ -ATPase in rodents, which is 100-fold less sensitive to ouabain (Espina et al., 2009; Habermann et al., 1981). The proliferating Caco2 cells (human intestinal cell line) appears to be very susceptible to the cytotoxic effects of PLTX (Pelin et al., 2012). However, differentiated Caco2 cells were demonstrated to be sufficiently resistant to perform in vitro absorption studies. The differentiated monolayer of Caco2 was proved to avoid chiefly the permeability of toxin (Fernandez et al., 2013). In skin keratinocytes HaCaT, the cytotoxic effect of PLTX seems to be stronger than the cutaneous damage, which suggests the hazard that cutaneous damage represents at cellular level (Pelin et al., 2011). Additionally, the existence of two PLTX binding sites were proposed – one sensitive and one insensitive to the presence of ouabain (Pelin et al., 2013).

Immunoassays Monoclonal and polyclonal anti-PLTX antibodies have been employed for the development of different immunodetection techniques. Initially, PLTX was recently detected by a radioimmunoassay (Levine et al., 1988). In order to avoid the hazards and technical disadvantages of radioactivity, different enzyme-linked immunosorbent assays (ELISAs) have been developed. Five ELISA models performed during the same work displayed sensitivities with inhibition concentrations at 20% (IC20 ) between 0.3 and 3.1 ng/mL (Bignami et al., 1992). Surface plasmon resonance (SPR) biosensors are suited for quantitative immunoassay detection. Monoclonal anti-PLTX antibody was characterized through a SPR biosensor, displaying limits of detection (LOD) of 0.5 ng/mL when using buffer solution. However, the LOD increased more than five-fold when using 1 : 10 dilutions of grouper or clam matrixes, respectively (Yakes et al., 2011). In a posterior developed indirect sandwich ELISA, interferences of matrix of mussels, microalgae and seawater samples were avoided with a 1 : 10 dilution, showing recoveries close to 100% except for seawater. The limit of quantification was 11 ng/ml for mussel extracts. This work also demonstrated the ability of the antibodies to detect two stereoisomers of the analogue 42-hydroxy-palytoxin although the cross-reactivity, highly varied, depended on the origin of the producer species of the molecule and the tested antibody (Boscolo et al., 2013). Other common marine toxins (okadaic acid, domoic acid, saxitoxin, brevetoxin-3 and yessotoxin) do not cross-react

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in this assay. Recently, monoclonal anti-PLTX antibody was employed to detect PLTX using an electrochemiluminescence-based sensor (ECL) with a LOD of 0.07 ng/mL, which did not show matrix interferences with 1 : 10 diluted extracts of mussel and microalgae (Zamolo et al., 2012).

Liquid chromatography-based methods Liquid chromatography (LC) based methods have mostly been used for the study of PLTX-like compounds. Since the discovery of PLTX, the LC methodology has been applied to analyze the toxin content in corals, Ostreopsis and crabs (Lenoir et al., 2004; Moore & Scheuer, 1971; Riobó et al., 2006; Usami et al., 1995; Yasumoto et al., 1986). The presence of two chromophores in the PLTX-like molecules boosted the development of LC methods coupled to ultraviolet (UV) detection. There are no reports on high-performance liquid chromatography coupled to UV (HPLC-UV) methods for quantitative determination of PLTX and analogues in shellfish samples, probably due to interference present in the biological matrix, which diminish the sensitivity of the method. A HPLC method with pre-column derivatization, using fluorescent (HPLC-FLD) detection, was developed to detect PLTX-like molecules in Ostreopsis samples in the range of 0.75–25 ng (Riobó et al., 2006). The principal advantages of the HPLC-FLD method are that is simple, low-cost, and can be automated. Different electrospray ionization (ESI) sources and tandem mass spectrometry instruments (MS/MS) have been employed for the identification and quantification of PLTX-like molecules (Ciminiello et al., 2008; Rossi et al., 2010; Suzuki et al., 2012; Uchida et al., 2013). The resultant MS spectra for these group of toxins is complex, due to the formation of multiply-charged ions (with charge states of +1, +2 and/or +3), which can present losses of water molecules and/or different adducts (K+ , Na2+ , Ca2+ , etc) (Ciminiello et al., 2011). Currently, the increase in published information about MS spectra of PLTX-like molecules has allowed to identify some ions and transicions for the characterization of this group of toxins. The presence and the structural characterization of different analogues and isomers of PLTX have been discovered and investigated owing to these detection methods (Ciminiello et al., 2006, 2008, 2009, 2010, 2012; Lenoir et al., 2004; Rossi et al., 2010; Suzuki et al., 2012; Uchida et al., 2013; Usami et al., 1995). FAB and matrix-assisted laser desorption ionization, coupled to time of flight (MALDI-TOF) MS, have been also used for the detection and NMR for the elucidation of the chemical structure of some PLTX-like molecules (Ciminiello et al., 2009; Onuma et al., 1999; Uemura et al., 1985; Ukena et al., 2001, 2002; Usami et al., 1995). The study about matrix interferences and toxin recoveries performed with LC-MS/MS using mussels, sea-urchins and anchovies displayed a variable ion enhancement (3–24%), depending on the matrix employed, and recoveries around 90% for mussels and sea-urchins being lower for anchovies (Ciminiello et al., 2013). In general, LC-MS methods are fast, can be sensitive, they may screen and measure PTLXs individually and can be automated. However, they require costly equipment and highly trained personnel. The improvement and advance in these techniques has permitted obstacles derived from the chemical similarities of these toxins to be overcome, in order to achieve an efficient chromatographic separation and to discover new analogues. However, it is necessary to improve the knowledge of the spectra pattern of all of those toxins in order to detect the presence of new analogues.

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Future perspectives There has been increasing consensus in the scientific community regarding the microalgae Ostreopsis as at least one producer of PLTX, even though the biosynthetic pathways are still unclear and represent a field of open and ongoing research. Sanitary problems related to Ostreopsis spp. could be due to the entrance of previously absent toxins into the food web and to human exposure to marine aerosols and/or seawater during large algal blooms. A multidisciplinary approach to the problem needs to be further adopted for their complete characterization. Studies are urgently needed to evaluate the acute effects of these toxins and also their effects after repeated oral exposure. Another crucial point is that the number of PLTX analogues is constantly increasing, requiring additional efforts for oral and inhalatory toxicity characterization. Future laboratory work will focus on the purification of the analogues in order to assess their activity compared with PLTX in a panel of biological tests. These data will help in analyzing the health impact for assessing the risk, and establishing alert and health safety thresholds, for PLTX-like molecules. Related to that, a revision of the regulated marine biotoxins in the EU legislation could be considered, as some non-regulated toxins have been shown to be harmful and/or to occur in the EU, while other compounds have low toxicity. There is an urgent need to study PLTXs and their effects, due also to the risks for environmental and ecological hazards related to global warming, and for their likely application in pharmaceuticals.

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CHAPTER 7

Recent insights into anatoxin-a chemical synthesis, biomolecular targets, mechanisms of action and LC-MS detection Custódia Fonseca1 , Manuel Aureliano2 , Feras Abbas3 & Ambrose Furey3 1 Department

of Chemistry and Pharmacy, Algarve University, Portugal of Biological Sciences and Bioengineering, Algarve University, Portugal 3 Mass Spectrometry Research Centre (MSRC) and PRTOEOBIO Research groups, Department of Chemistry, Cork Institute of Technology (CIT), Ireland 2 Department

Anatoxin-a and analogues Anatoxin-a (ANTX-a) is a toxin well known to induce neurotoxicity. Several species of cyanobacteria including Anabena sp. [A. flos-aqua (Carmichael et al., 1975 ; Devlin et al., 1977; Furey et al., 2003b; Gallon et al., 1994; Harada et al., 1993; Kangatharalingam & Priscu, 1993; Sivonen et al., 1989), A. circinalis (Sivonen et al., 1989), A. planctonica (Bruno et al., 1994), A. mendotae (Rapala et al., 1993)); Oscillatoria sp. (Araóz et al., 2005) (O. agardhii (Sivonen et al., 1989), O. formosa (Araóz et al., 2005; Skulberg et al., 1992)]; Microcystis sp. [M. aeruginosa (Harada et al., 1993)]; Raphidiopsis sp. [R. mediterranea (Namikoshi et al., 2003; Watanabe et al., 2003)]; Planktothrix [P. rubescens (Viaggiu et al., 2004)]; Arthrospira sp. [A. fusiformes (Ballot et al., 2004)]; Nostoc sp. [N. carneum (Ghassempour et al., 2005)]; Phormidium sp. [P. favosum (Gugger et al., 2005)] and Aphanizomenon sp. (Sivonen et al., 1989) produce ANTX-a in different quantities, depending on the genetic characteristic of the strains and/or the environmental conditions (Araóz et al., 2005; Ballot et al., 2005; Bumke-Vogt et al., 1999; Gugger et al., 2005; James et al., 2008; Namikoshi et al., 2003; Osswald et al., 2007; Park et al., 1993; Viaggiu et al., 2004). It is difficult to isolate these toxins from biological material (Furey et al., 2003a; Haugen et al., 1994; Meriluoto, 1997; Mitrovic et al., 2004) because it is almost impossible to get a live monoculture that produces ANTX-a at high concentrations, and the isolation from natural samples is hampered by the fact that the toxin ANTX-a degrades to non-toxic compounds, dihydroanatoxin-a and epoxyanatoxin-a. Hence, it is imperative to synthesis these molecules (Armesto Arbella et al., 2007). ANTX-a is a low molecular weight alkaloid (165 Da) with a pKa 9.4. This bicyclic secondary amine has been established as 2-acetyl-9-azabicyclo[4.2.1]non-2-ene, C10 H15 NO; λmax 227 nm, EI-MS m/z [M]+ 165, and it has a methylene homologue,

Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

137

138

Chapter 7

O

HN

NH

HN

R

N O

HO O 1, R = CH3 2, R = C2H5 Figure 7.1

N

P O 3

Structures of ANTX-a, 1; HANTX-a. 2; ANTX-a(S),3.

homoanatoxin-a (HANTX-a), 2-(propan-1-oxo-1-yl)-9-azabicyclo[4.2.1]non-2-ene), C11 H18 NO, λmax 230 nm, EI-MS m/z [M]+ 179, (1 and 2, Figure 7.1). Another neurotoxin structurally very different from ANTX-a, but with a similar name, is anatoxin-a(S) (3, Figure 7.1). Anatoxin-a(S) is the unique natural organophosphate known and it is characterized by a λmax 220 nm, HRFABMS m/z [M + H]+ 253, and a formula C17 H17 N4 O4 P. There have been several synthetic pathways to obtain ANTX-a, and these are briefly described in the present chapter.

Chemical synthesis It is possible to separate into categories the different total synthesis approaches to ANTX-a, according to the methodology used: (a) Synthesis of ANTX-a via ring expansion of tropanes (b) Synthesis of ANTX-a via transannular cyclization of cyclooctenes (c) Synthesis of ANTX-a via iminium salts (d) Synthesis of ANTX-a via an enol (e) Synthesis of ANTX-a via nitrones (f) Synthesis of ANTX-a via allene (g) Synthesis of ANTX-a via tandem reactions (h) Synthesis of ANTX-a via enine metathesis

Synthesis of ANTX-a via ring expansion of tropanes Tropane is the common name of (1R, 5S)-8-methyl-8-azabicyclo[3.2.1]octane and it is the structural base of alkaloids. Chemically, it is an amine bicyclic ring (Figure 7.2) with a pyrrolidine (five-member ring) and a piperidine ring (six-member ring), which share a nitrogen and two carbon atoms. Many natural products have compounds where the tropane ring appears in the structure forming esters. One example is (−)-cocaine, 4, the starting material in the synthesis of ANTX-a by the Edwards group (Figure 7.3). In this synthesis, the strategy

1 N

3

R

5 Figure 7.2

Tropane – (1R, 5S)-8-methyl-8-azabicyclo[3.2.1]octane (R = H).

Recent insights into anatoxin-a (ANTX-a)

Me N

Me N a

CO2CH3 4

Me N

O

5

6

c

f

(+)-1

O

b

OCOPh

e

139

Me N

Me N

O

O

d

8

Br 7

Figure 7.3

Ring extension of (−)-.cocaine. a) HCl 37% reflux, CH3 OH; LiOH.H2 0, CH3 Li; NaH 50%, DMSO, (CH3 )3 SI. b) Li∕NH3 , 83%. c) Ac2 O, HBr, 80 ∘ C; Br2 , CHCl3 , 49%. d) LiBr, DMF, Δ. e) DEAD, benzene, Δ; HCl dil, Δ, 29%. f) hν, H2 O, 46%. Source: Based on data from Campbell et al. 1977.

used is the extension of the piperidine ring from six to seven carbons in the tropane ring. This approach represents the first documented total synthesis of ANTX-a (refer to Figure 7.3 for the route (Campbell et al., 1977)). In this sequential synthesis, the first important step is to convert the (−)-cocaine into endo-cyclopropane, 5 (Zirkle et al., 1962). The opening of the endo-cyclopropane ring leads to the extension of the ring from seven to eight carbon atoms, and this is the key step in most of the proposed synthesis. Two experimental procedures can be made to achieve ring extension: reductive fission, using lithium in liquid ammonia; or photolytic cleavage. The first approach produces the N-methyl dihydroanatoxin derivative, 6, which can be transformed into the N-methyl ANTX, 8, by the bromination/elimination processes. The second procedure, the photolytic cleavage, produces the α, β-unsaturated ketone, 8, directly with a 46% yield (Figure 7.3). Another synthesis using the same starting material, (−)-cocaine, was proposed by Steiz’s group (Wegge et al., 2000). In this eight-stage synthesis, there are two key steps (Figure 7.4): one is the ring expansion of (+)-2-tropinone, 9, to 9-azabicyclo[4.2.1]nonanone, 11, and another is the introduction of the required methyl ketone side chain in masked form, achieved by reacting the corresponding enol triflate with ethyl vinyl ether/Pd(OAc)2 under Heck reaction conditions (Figure 7.4). In this process, initially the (−)-cocaine was transformed into (+)-2-tropinone, 9, by two steps with an overall yield of 57% (Zhang et al., 1997). The insertion of one methylene group in (+)-2-tropinone was achieved regioselectively with trimethylsilyldiazomethane (TMSCHN2 ) catalyzed by the organoaluminium Lewis acid, Al(CH3 )3 forming the ring enlargement: the trimethylsilyl enol ether. This is then transformed to the 9-azabicyclo[4.2.1]nonan-2-one, 11, by acidolysis with trifluoroacetic acid. In order to get the methyl ketone side chain, it is necessary to transform compound 11 in the enol triflate (Comins & Deghani, 1992; Luker et al., 1997) using KHMDS in toluene at −78 ∘ C to give the corresponding potassium enolate, which then reacts with Comins’N-(5-chloro-2-pyridyl)triflimide to furnish the ketone-derived enol triflate at

140

Chapter 7

Me N

EtO2C N

Me N a

CO2CH3

O

O

b 10

OCOPh 4

9 c

EtO2C N

EtO2C N

O d

e

(+)-1

12

O

11

Figure 7.4

Ring extension of (−)-cocaine. a) HCl 37% reflux; (PdO)2 P(O)N3 , DMAP, Na2 CO3 ; HCl 1N reflux 80%. b) ClCO2 Et, 71%. c) TMSCHN2 ∕Al(CH3 )3 , 94%; CF3 CO2 H∕H2 O, 94%. d) KHMDS, toluene, –78 ∘ C, 2N(Tf2 )-5-chloro-pyridine, 67%; CH2 = CHOEt∕Pd(OAc)2 , DMSO, N(Et)3 ; SiO2 , 87% e) Me3 SiI∕CHCl3 ; CH3 ONa∕CH3 OH. Source: Based on data from Wegge et al., 2000.

about 67% yield. This intermediate reacts with alkyl vinyl ethers in the presence of a palladium catalyst (8% palladium acetate), using triethylamine as a base in DMSO (Heck reaction conditions), to generate the 2-ethoxydiene in a high yield, establishing the second key step of this ANTX-a approach, the N-protected (+)-ANTX, 12. Deprotection of 12 using Me3 SiI in chloroform is followed by treatment with CH3 ONa∕CH3 OH to generate the natural (+)-ANTX (Figure 7.4).

Synthesis of ANTX-a via transannular cyclization of cyclooctenes Another strategy for the formation of bicyclic ring structures is transannular cyclization (Figure 7.5). As ANTX-a is a chemical compound with this type of structure, it is possible to use this synthesis approach. The key compound is a suitably substituted cyclooctene. Figure 7.6 represents the key stages in the transannular cyclization reaction, while Table 7.1 shows the chronological order with the respective bibliographic reference of the synthesis reactions. Campbell and collaborators carried out two different methodologies in order to synthesize the 9-azabicyclo[4.2.1]nonane structure. In both approaches, the starting material was 1,5-cyclooctadiene, which was transformed into the methyl amine, 13, using Bastable (Bastable et al., 1972) conditions, hypobromous acid (generated with perchloric acid and N-bromosuccinimide (Figure 7.6a); or using Barelle (Barrelle & Apparu, 1976, 1977) methodology to convert to the aminoalcohol 14 using mercuric (II) acetate, followed by reduction with sodium borohydride in basic media to give the desired bicycle 15.

n

m Figure 7.5

n Transannular cyclization.

m

Recent insights into anatoxin-a (ANTX-a)

R

Me N

NHMe

OH

X b

a

c

Me N

141

O

HO HO 15

13 R = H 14 R = OH

H3N

H N

Br d

HO

17, X = O 18, X = NMe

16

MeO2C N

NHCO2Me Br

19

e

OTs

O

Br

22

21

20

NHTs f

MeO2C

OCO2Me 24

OMe 23 Ts N

O OCH3 25

Transannular cyclization in ANTX-a synthesis. a) 13: NBS, HClO4 , dimethylether, H2 O, 82%; 15: Hg(OAc)2 , THF; NaBH4 , NaOH, H2 O,quant. b) CrO3 , H2 SO4 , acetone, 49%. c) MeNH3 aq, TsOH, 100 ∘ C, 82%; HBr.Py.Br2 , AcOH, 115 ∘ C, 56%. d) Et3 N, CH3 CN, Δ, 32%. e) PdCl2 , CuCl2 , press. CO, MeOH, rt, 61%. f)[Pd(dba)3 ](S,S)- chiral ligand, CHCl3 , 0 ∘ C, 94%, 88% ee.

Figure 7.6

This common intermediate 15 of both pathways was oxidized under Jone’s conditions affording the generation of the racemic ketone, 26 (Figure 7.7). This ketone was then converted into the epoxysulfoxide, 27, and by thermal rearrangement produced the α, β-ketone 8. Eventually, deprotection of the N-methyl group by oxidation using ethyl azodicarboxylate (DEAD), followed by acid hydrolysis, provided the (±)-ANTX-a as its hydrochloride salt in a 35% yield (Campbell et al., 1979). Danheiser synthesized the required azabicyclic structure using a two-step process (Figure 7.8) (Danheiser et al., 1985) by electrophylic cleavage of 28 with silver tosylate in excess obtaining the trans-cyclooctenes, 29. Subsequent photoisomerization of the trans double bond to the cis-conformation (20) and transannular cyclization were then achieved in a single operation to give the azabicyclononene, 30. The final stage consisted of the conversion of 30, previously reprotected as tBoc, to ANTX. The strategy involved the interchange by bromide-lithium and posterior treatment with an acylating agent (N-methoxy-N-methylamide) (Nahm & Weinreb, 1981) furnishing the N-tBoc ANTX. Posterior deprotection with trifluoroacetic acid, and treatment with chlorohydric acid, produced the (±)-ANTX-a as a hydrochloride salt. As a result, they achieved the racemic synthesis of ANTX in seven steps, with a 17% overall yield, beginning with 4-cycloheptenone. This synthesis can be done in the gram scale. In the Wiseman synthesis, there is a common intermediate, ketone 19, generated by the Campbell method (Campbell et al., 1979), obtained by the cyclization of 1,5-cyclooctanediol (16, Figure 7.6) (Wiseman & Lee, 1986). This transformation involved the Jones oxidation of 16 to give the hemiketal 17, which was transformed into 9-methyl-9-azabicycle[3.3.1]nonan-1-ol, 18, by treatment with methylamine

142

Chapter 7

Table 7.1 Year 1979

Summary of the ANTX-a synthesis using transannular cyclization reaction as key step. Key intermediate

R

NHMe

Reference (Barrelle and Apparu, 1976, 1977; Bastable et al., 1972; Campbell et al., 1979)

13, R = H 14, R = OH 1985

H3N

(Danheiser et al., 1985)

Br

OTs Br 20 1986

OH

HO 1998

(Ferguson et al., 1995; Lindgren et al., 1987; Quinn and Wiseman, 1973; Stjernlöf et al., 1989; Wiseman and Lee, 1986)

16 NHCO2Me

(Ham et al., 1997)

22 1999

NHTs

MeO2C

(Trost, 2004; Trost et al., 1976; Trost and Oslob, 1999)

OCO2Me 24

(Quinn & Wiseman, 1973). This azabicycle[3.3.1]none skeleton was then reconverted into 9-azabicyclo[4.2.1]nonane by bromination-reorganization with pyridinium bromide perbromide in hot acetic acid, directly in one step (Stjernlöf et al., 1989). This constitutes the key step of this synthesis, producing ketone 19 in a 56% yield. The conversion of amino ketone 19 into N-methyl ANTX, 8 (Figure 7.9), is accomplished through the enonitrile intermediate 31 (D’Incan & Seyden-Penne, 1975; Wroble & Watt, 1976). N-Demethylation of compound 8 is achieved with diethyl azodicarboxylate, followed by hydrolysis, which completes the synthesis of

Recent insights into anatoxin-a (ANTX-a)

Me N

Me N

Me N

Me N c

b

a HO

O

O

O

Me O

15

143

26

S Ph 8

27

Partial synthesis of ANTX-a a) CrO3 , H2 SO4 , acetone, AcOH, 52%. b) chloroethyl phenyl sulfoxide, LDA, THF, –78 ∘ C, 90%; KOtBu, THF, –20 ∘ C, 69%. c) Benzene, Δ, 41%. Source: Based on data from Campbell et al., 1979.

Figure 7.7

NH2

H3N

H

H N

Br

b

a NH2

Br

OTs

OTs Br

Br Br 28

Br

c

29

30

20

Figure 7.8

Partial synthesis of ANTX-a a) Excess AgOTs, CH3 CN, Δ, 60%.b) HBr, Benzene, hν. c) Et3 N, CH3 CN, Δ, 32%. Source: Based on data from Danheiser et al., 1985.

Me N

Me NC N

O a

19

31

Me CH3 O

O

N b

1 8

Figure 7.9 Partial synthesis of ANTX-a a) NaNH2 , (EtO)2 P(O)CH(CH2 )CN, THF, 20 ∘ C, 64%. b) LDA, THF/HMPA, O2 ; Na2 SO3 , H2 O; NaOH, H2 O, 43% (overall the last three steps). Source: Based on data from D’Incan and Seyden-Penne, 1975; Wroble and Watt, 1976.

racemic ANTX-a. Subsequently, alternative methods for introducing the acyl moiety and different side chains in the ketone intermediate 19 were developed by Stjernlöf (Ferguson et al., 1995). The Ham group used an intramolecular aminocarbonylation, catalyzed by palladium (II) chloride in the presence of copper (II) chloride, as an oxidant, in order to obtain the bicyclic ring skeleton present in ANTX (Figure 7.6, 22–23) (Oh et al., 1998). The starting reagent was 4-cyclooctenone, which was converted to the intermediate 23 in a 27% overall yield. Then the azabicyclo α-23 was treated with potassium hydroxide to furnish the acid, followed by subsequent reaction with the N,O-dimethylhydroxylamine to give the amide (Nahm & Weinreb, 1981). Reaction with methylmagnesium bromide generated a ketone, N-acetyl dihydroanatoxin,

144

Chapter 7

O

O NH HN

N PPh2 32 Figure 7.10 The chiral ligand used in Trost synthesis of ANTX. Source: Based on data from Trost, 2004; Trost and Oslob, 1999.

in a 72% yield, which was converted into (±)-ANTX-a following the Rapoport methodology (Sardina et al., 1989). Trost also applied a palladium catalyst, but in the presence of a chiral phosphine ligand, for intramolecular asymmetric allylic alkylation of the intermediates 24–25 (Figure 7.6), in the synthesis of ANTX (Trost, 2004; Trost & Oslob, 1999). The best results were obtained with tosylamine, 24. The cyclization product was produced with a [Pd(dba)3 ] catalyst in the presence of the chiral ligand (S, S)-32 (Figure 7.10), giving the bicycle (+)-25. Transformation of the ester moiety into the ketone (Arisawa et al., 1997), and reductive desulphonylation (Somfai & Åhman, 1992; Trost et al., 1976), generated (−)-ANTX-a in an 88% yield. The natural (+)-enantiomer would be equally accessible simply by switching the chirality of the ligand. A concise route to the synthesis of a possible intermediate of ANTX, the N-tosylanatoxin was described by Ho & Zinurova (2006). Transannular heterocyclization was the key step in this synthesis, using a cycloadduct produced from reaction of cycloocta-1,5-diene N-chlorosulfonyl isocyanate, catalyzed by Pd(II).

Synthesis of ANTX-a via iminium salts Iminium ions (33) are important reactive species in organic synthesis for the construction of carbon-carbon and carbon-heteroatom bonds, due to their electrophilic character. Generally speaking, these chemical processes are α-aminoalkylation reactions, with the iminium ion serving as a defining reactive element. Depending on the N- protective group (R), their reactivity can be modulated (Figure 7.11). The asymmetric approaches leading to the construction of enantiopure 9-azabicyclo[4.2.1]nonane skeleton are mostly concerned with the intramolecular cyclization of a pyrrolidine iminium ion (35). They differ significantly in the

R2 R3

R1 N R

R = alkyl, acyl, tosyl 33

R4

R1 N R R4 = OR,CO2H

N R

34

35

R'

Figure 7.11 Structures of generic iminium ion (33), pyrrolidine iminium ion (35), and its pyrrolidine precursor (34).

Recent insights into anatoxin-a (ANTX-a)

145

preparation of substituted pyrrolidine (34) precursors and in overall yield for the construction of this basic skeleton. Using this concept, the Rapoport group achieved a total synthesis of optically active (+)-ANTX-a and its enantiomer from D and L-glutamic acid respectively (Bates & Rapoport, 1979; Petersen et al., 1984). Several improvements were subsequently made to this synthesis (Sardina et al., 1989; Sardina et al., 1990). In this approach, the L-glutamic acid was transformed into the L-benzyl pyroglutamate (36, Figure 7.12) in two steps (Quitt et al., 1963; Stadler, 1978). The alkyl chain introduction on the C-5 pyrrolidine position was achieved using a sulfide contraction reaction (Fischli & Eschenmoser, 1967; Sakurai et al., 1994; Shiosaki & Rapoport, 1985), constituting one of the determinant steps in this synthesis. Thus, treatment of the amino acid 37 with oxalyl chloride gave its acyl chloride, which directly generated the iminium intermediate 38 and, after heating, produced the desired product 39, 2,5-difuncionalized homotropane, as a mixture of diastereomers at C-2 and C-5. (Arisawa et al., 1997; Dean et al., 1976; Koskinen & Lousnasmaa, 1983; Tramontini, 1973; Weinstein & Craig, 1976). Conversion of the N-benzyl group to N-tert-butyl carbamate was reached directly in one step by the neutral hydrogenolysis conditions of the mixture of 39 in methanol and in the presence of di-tert-butyl dicarbonate, affording 38 in an overall yield of 43% (all stereoisomers). The product 40 is the common intermediate for the synthesis of both enantiomers of ANTX-a. Due to the formation of all stereoisomers at C-2 and C-5, this synthetic strategy requires transforming all four diastereomers into the desired products. With the aim of obtaining the (+)-ANTX-a, deoxygenation of the ketone group (C-5) of the common intermediate 40, and a one carbon side-chain extension (C-2), was the pathway chosen to reach the t-Boc-dihydroanatoxin, 41. Conversion of the t-Boc-dihydroanatoxin into t-Boc-ANTX, 42, was one of the

a HO2C

N Bn

HO2C

O

N Bn

CO2Me

N Bn

O

-

36

37

CO2Me

O

Cl 38 b

Boc N (+)-1

O

e

Boc N

R N

OTBDMS

d

(1R)-42

CO2CH3

c O

41

39 R = Bn 40 R = Boc

Synthesis of ANTX-a from D-glutamic acid a) Oxalyl chloride, CH2 Cl2 , –15 ∘ C to 3 ∘ C. b) ∘ Heating 60 C, Toluene, 91%; (tBoc)2 O, Pd/C, H2 , MeOH, 98%. c) NaH, THF; TBMSCl, Et3 N, THF, 98%. d) PhSeCl, THF, −78 ∘ C; MCPBA, THF, 0 ∘ C, 84%. d) TFA, CH Cl ; HCl, EtOH, 97%. Source: Based on

Figure 7.12

2

data from Bates and Rapoport, 1979; Petersen et al., 1984.

2

Chapter 7

146

important transformations of this synthesis, which was a significant improvement on earlier approaches. Eventually, the reaction of the tert-butyldimethylsylil enol ether, 41, with phenylselenenyl chloride (PhSeCl), followed by oxidation with m-chloroperoxybenzoic acid (MCPBA), produced (1R)-tBoc-anatoxin (42) at 84% yield. Cleavage of the tBoc protecting group with trifluoroacetic acid proceeded quantitatively, providing (+)-ANTX-a as its hydrochloride salt in an 18% overall yield (Figure 7.12) (Sardina et al., 1989; Sardina et al., 1990). Speckamp and collaborators proposed a similar approach to ANTX synthesis, using N-acyliminium chemistry (Speckamp & Hiemstra, 1985; Melching et al., 1986). In their first published synthesis, the α-carbon of the α, β-unsaturated ketone, 44, acts as a nucleophilic centre, with the N-acyliminium ion providing the corresponding N-acylANTX-a, 46 (Figure 7.13). The cyclization was obtained by reacting compound 44 with HCl in MeOH at −50 ∘ C, producing a mixture of compounds 45 and 46 with 11% and 47% yields respectively. The chloride compound under reflux in toluene in the presence of DBN was converted into compound (+)-46 with a 60% yield. After N-deprotection using iodotrimethylsilane in refluxing acetonitrile (55% yield), ANTX was obtained. The overall unoptimized yield from succinimide was between 3–4% (Melching et al., 1986; Wijnberg & Speckamp, 1981). The second proposal by Speckamp used an allylsilane as a nucleophile for an N-acyliminium cyclization reaction (Figure 7.13) (Esch et al., 1987). The β-effect of the silicon atom is a powerful determinant of the regiochemistry of allylsilane reactions with electrophiles, so the new carbon-carbon bond is formed at the vinyl carbon distal to silicon, that is, at the γ-position (Hiemstra et al., 1984; Hiemstra et al., 1985). MeO2C N

O a EtO

N CO2Me 44

O

N H

MeO2C N

O

O

+ Cl 45

46

O

43 SiMe3 HO

N CO2Me 47

Figure 7.13

MeO2C N

MeO2C N

b

H 48

O

c

49

Cyclization of an N-acyliminum ion with an α, β-unsaturated ketone or an allysilane a) HCl, MeOH, −50∘ to 20 ∘ C; DBN, toluene, reflux, 60%. b) HCOOH, rt, 73% (exo : endo, 19 : 1). c) PdCl2 , CuCl2 , O2 , DMF∕H2 O, 64%. Source: Based on data from Esch et al., 1987; Hiemstra et al., 1984; Hiemstra et al., 1985; Speckamp and Hiemstra, 1985: Melching et al., 1986; Wijnberg and Speckamp, 1981.

Recent insights into anatoxin-a (ANTX-a)

147

The allylsilane 47 was obtained by reacting succinimide, 43, with the corresponding Grignard alkylsilane. After being isolated, compound 47 was dissolved in formic acid at room temperature, and the cyclization product 48 was obtained in a clean, fast and irreversible process. The Wacker oxidation of 48 afforded the dihydroanatoxin derivative 49 (Figure 7.13). The nitrogen deprotection step with iodotrimethylsilane furnished produced the (±)-dihydroanatoxin, which was reprotected with tBoc and, applying the previous methodology from Rapoport’s group (Sardina et al., 1989), this produced the racemic (±)-ANTX. The overall yield of this synthesis was 7% from the succinimide, which constitutes a small increase in comparison with the previous synthesis (Esch et al., 1987). Other authors have used the same strategy for the cyclization step. As described previously, Somjai achieved the synthesis of ANTX using compounds with the N-sulphonyl group (Åhman & Somfai, 1992; Somfai & Åhman, 1992). The Shono (Shono et al., 1987), Skrinjar (Skrinjar et al., 1992) and Tanner (Hjelmgaard et al., 2005) groups also deployed the same type of intermediate as Speckamp. In order to reach a general structure of the 2,5-disubstitued pyrrolidine (54, Figure 7.14) for generating the acyliminium ion and to generate the required bicycle skeleton (55), Muniz and collaborators used one of the key steps in a sequential synthesis of ANTX-a [2 + 2] cycloaddition of a chiral enol ether, 51, with the in situ generated dichloroketene to generate dichlorocyclobutanone, 52 (Muniz et al., 2005). This adduct was submitted to a Beckmann ring expansion (Tamura et al., 1977), and a subsequent dechlorination (Johnston et al., 1985) of the resultant α, α-dichlorolactam gave the lactam 53. This lactam was transformed into the corresponding precursor of an acyl iminium intermediate, 54, to achieve the cyclization. Thus, 56 was N-protected, and the formation of the allylsilane was achieved through cross-metathesis, using a second generation Grubbs’ catalyst (Scholl et al., 1999), giving the key intermediate 54, equivalent to the Speckamp 47 (Figure 7.13).

OH

Cl a

O

O

Cl O

51

50

52 b

Ar

MeO2C

Ar

O

N

O

d

c O

Ar

O 55

Figure 7.14

N CO2Me 54

O SiMe3

N H 53

Cyclization of an N-acyliminum ion to form a bicyclic structure a) Zn-Cu, Cl3 CCOCl, diethylether. b) NH2 OSO2 C6 H2 (CH3 )3 , CH2 Cl2 ; Al2 O3 , MeOH; Zn-Cu, NH4 Cl, MeOH, (36% from 46). c) n-C4 H9 Li, THF; NCCO2 CH3 , 91%; LiB(C2 H5 )3 H, THF, 92%; CH2 = CHCH2 Si(CH3 )3 , 2nd Generation Grubbs’ catalyst, CH2 Cl2 , 74%. d) HCOOH, CH2 Cl2 , 77%. Ar = 2, 4,6-triisopropylphenyl. Source: Based on data from Muniz et al., 2005.

148

Chapter 7

Ph MeO2CN

Ph Ph

Ph N Li O

56

LiN 61 a

MeO2C N OSiMe3

57

b

MeO2C N MeO2C N

MeO2C N

OSiMe3 c

d

58

O

O 60

59

Partial synthesis of ANTX-a based on enantioselective enolization a) 61, THF, Me3 SiCl, −100 ∘ C, 84% ee. b) Et2 Zn, ICH2 Cl, ClCH2 CH2 Cl, 99%. c) FeCl3 , DMF, NaOAc, MeOH, 71%. d) [CH2 = C(OEt)]2 Cu(CN)Li2 , THF, Et2 O; 2-triflimido-5-chloropyridine, 65%; Pd(OAc)2 (PPh3 )2 , Bu3 N, HCO2 H, DMF; HCl, MeOH, 64%. Source: Based on data from Cox and Simpkins, 1991; Newcombe and Simpkins, 1995; Simpkins, 1996.

Figure 7.15

The cyclization reaction was performed under the Speckamp conditions using formic acid to reach the required bicycle 55. The ether group (C-7) was transformed into the alcohol, and then removed through its transformation into the iodide, and a posterior reduction gave the compound (1R)-48, previously obtained as a racemic mixture. Conversion into the (+)-ANTX-a was done using the same Speckamp approach (Esch et al., 1987).

Synthesis of ANTX-a via enol Simpkins synthesized ANTX-a introducing the chirality by an asymmetric catalytic process using chiral lithium amide bases, which are a chiral variant of the commonly used lithium amide base LDA (lithium diisopropylamide) (Cox & Simpkins, 1991; Newcombe & Simpkins, 1995; Simpkins, 1996). This ANTX-a synthesis is based on a enantioselective enolization reaction with a (±)3-tropinone (56, Figure 7.15) as a substrate and a chiral lithium amide base as a catalyst (61), and a subsequent cyclopropanation/ring expansion reaction to give the enol 59 (Figure 7.15). The tropane ring expansion was achieved via a cycloprapanation reaction with chloroiodomethane and diethylzinc (58, Figure 7.15) (Denmark & Edwards, 1991). Subsequent reaction with FeCl3 under mildly basic conditions (Ito & Saegusa, 1976), produces the enol (59), with > 99% ee, obtained by induced crystallization in cold storage conditions. Easy transformation into the deconjugated system, 60, and subsequent isomerization to α, β-unsaturated ketone in an 80% yield was achieved using RhCl3 (Grieco et al., 1976). The total synthesis of unnatural (−)-ANTX was accomplished by deprotection using Me3 SiI (Jung & Lyster, 1978). The natural (+)-ANTX could be synthesized by using an antipode of the chiral base 61. The racemic synthesis of ANTX was obtained using the same procedure, but using LDA as a catalyst.

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Aggarwall uses the same strategy as Simpkins but with a base derived-(R,R)-61. HCl and two equivalents of butyllithium at 100 ∘ C are used to obtain the enantioselective deprotonation of the starting material, an eight-membered ring ketone (62, Figure 7.16) (Aggarwal et al., 1999). It was reported that cyclooctanones exist in conformations, having as few trans-annular non-bonded repulsions and high-energy torsional arrangements as possible (Allinger et al., 1972; Still & Galynker, 1981) and, at low temperature, one conformation should be largely favoured, leading to the possibility of high asymmetric induction. This is fundamental for the key step, enantioselective deprotonation of ketone 62, depicted in Figure 7.16. The key desymmerisation step was carried out with the Simpkins base (R, R)-63 (Cain et al., 1990; Majewski et al., 1995) at −100 ∘ C, which allowed the enantiomerically enriched lithium enolate intermedium to be made. This reacted with diphenyl chlorophosphate, producing the enol phosphate 64 in 89% ee and an 89% yield. The Stille coupling reaction of 64 (Nicolaou et al., 1997) with ethoxyvinyltributyltin (CH2 = CH(OEt)SnBu3 ) gave a vinyl ether, which was hydrolyzed in acidic conditions, giving the masked ketone moiety (intermediate 65). This intermediate simultaneously undergoes the deprotection of the amine group and the intramolecular conjugated addition, generating directly in one step the required azabicyclic structure. The alteration of the benzyl group for tert-butoxycarbonyl in one step under Rapoport conditions gave the ketone (1R)-45. This ketone was converted into the (+)-ANTX-a by the procedure described by Rapoport’s group (Sardina et al., 1989). Eventually, the enantiomerically enriched (+)-ANTX-a was obtained in a 34% overall yield.

O

Ph

N H 63

O O P(PPh)2

Ph a

Bn N

Bn N

OCOCH2Ph

OCOCH2Ph 64

62 b Boc

O

N c O Bn NH 66

65

Figure 7.16 Partial synthesis of ANTX-a based on enantioselective enolization a) (R, R)-63 n-BuLi, (PhO)2 POCl, THF, −100 ∘ C, 89%; 89% ee. b) [Pd(PPh3 )4 ], CH2 = CH(OEt)SnBu3 , LiCl, THF, Δ, 84%. c)HBr/AcOH, 95%. d) Pd/C, H2 , MeOH, (tBoc)2 O, 89%. Source: Based on data from Aggarwal et al., 1999.

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Synthesis of ANTX-a via nitrones Nitrone is a functional group with the general structure R1 R2 C=N+ R3 -O where R3 is different from hydrogen. Thus, it is a 1,3-dipole used frequently in organic chemistry in 1,3-dipolar cycloadditions (Figure 7.17). In this synthesis, this approach it is used to make azabicyclic structures (Black et al., 1999). Tufariello and co-workers used, in the sequential synthesis of a racemic ANTX-a, two cycloadditions of a nitrone to C=C (Figure 7.18) (Tufariello, 1984; Tufariello et al., 1984, 1985). The nitrone, 67, was formed when a solution of 1-hydroxypyrrolidine in benzene reacted with mercury (II) oxide. The cycloaddition of this nitrone system to the diene 68 produced the pyrrolo[1,2-b]isoxazole derivative, 69. By allylic oxidation with manganese(IV) oxide, the cycloadduct was converted to an enone, which was treated with meta-chloroperoxybenzoic acid. The N-O bond of the isoxazoline ring underwent cleavage, giving the nitrone 70, which was induced, under reflux

R1 R2

R1

N O R3

R1

N O

R2

R2

R3

(a)

N O R3 (b)

Figure 7.17 a) Nitrone represented by two resonance structures. b) Nitrone in 1,3-dipolar cycloaddition.

HO

N O 67

H a

H b

Me

O N

HO

N O

O

OH H

69

68

70 c Boc N

OH

Me d O

O

O N

Me 72

71

Partial synthesis of ANTX-a via nitrone a) C6 H6 ,, 3h, 70%. b) MnO2 , CH2 Cl2 , 96%; m-CPBA, CH2 Cl2 , 20 min. c) K2 CO3 , Δ, 18.5 h, 71%; (CH2 OH)2 , TsOH.H2 O, C6 H6 , 97%; MeSO2 Cl, Et3 N, CH2 Cl2 , 94%; NiCl2 , THF, LiAlH4 , −78 ∘ C. d) TsOH, Me2 CO, Δ; NaHCO3 , Boc2 O, CHCl3 , 42.5%. Source: Based on data from Tufariello, 1984; Tufariello et al., 1984, 1985.

Figure 7.18

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151

in dichloromethane, to form an intramolecular cyclization to produce the required tricycle, 71. The carbonyl group was protected as the [1,3]dioxolane derivative and the hydroxyl group was activated by formation of the mesylate derivative. When this mesylate was subjected to reduction with lithium aluminium hydride (Ashby & Lin, 1978), and nickel(II) chloride in tetrahydrofuran at −78 ∘ C, cleavage of the N-O bond took place, and one of the products formed, 72, containing the enone group, which was the major component of the mixture. After ketalization and reprotection of nitrogen as t-butyl carbamate, tBoc-anatoxin-a, 72, was formed. Hydrolytic removal of the tBoc group gave the (±)-ANTX-a hydrochloride.

Synthesis of ANTX-a via allene Gallagher and co-workers proposed for ANTX-a synthesis the cyclization of an allenic aminoester (73, Figure 7.19) (Huby et al., 1991a; Smadja, 1983; Vernon & Gallagher, 1987). As depicted in Figure 7.19, the first key step in this synthesis was the high cis stereoselectivity cyclization of the allene 73, giving the cis-pyrrolidine 74 with a 98% yield. This compound was then treated in order to obtain the bicyclic ketone (77). Thus, with hydroboration/oxidation, the primary alcohol was obtained (74).

a

EtO O

b

EtO

NH Ts

O

73

N Ts

EtO

H

OH

N Ts

O

74

75 c

H N O Me S O

O

N Ts

O

1

Br

76 d

g

Boc N

Ts

Ts N

N e

f O

MeO

78

O

SO2Me

77

79

Partial synthesis of ANTX-a via allene a) AgBF4 , CH2 Cl2 , rt, 98%. b) BF3 .OEt2 , NaBH4 , (MeO(CH2 )2 )2 O, THF; NaOH, H2 O, H2 O2 , 90%. c) Me2 SO2 , BuLi; THF, 63% Ph3 P, Br2 , THF, 78%. d) NaH, DMSO, 40 ∘ C, 2h, 83%. e) Al-Hg, THF, H2 O, 60 ∘ C, 92%. f) LDA, PO(Ph)2 CH2 OH, DME, −78 ∘ C; NaH, THF, 97%; Li, NH3 , −78 ∘ C, Boc2 O, MeOH, 55%. g) PhSeCl, CH2 Cl2 , −30 ∘ C, m-CPBA, CH2 Cl2 , −78 ∘ C, 61%; MeMgI, ether, −78 ∘ C to −40 ∘ C; PCC, CH2 Cl2 , 37%; HCl, EtOAc, 98%. Source: Based on data from Huby et al., 1991a; Smadja, 1983; Vernon and Gallagher, 1987.

Figure 7.19

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The addition of LiCH2 SO2 Me to its ester moiety and subsequent alcohol bromination gave the β-ketosulphone (76), which was ready to undergo the cyclization step. The primary bromide was treated with sodium hydride amalgam to generate the bicyclic ketone 77 (Cooke & Magnus, 1976). The conversion of compound 77 to ANTX-a was achieved by reductive cleavage of the sulphone and gave the bicyclic ketone 78, with a 34% overall yield. Many modifications have been performed previously using this kind of intermediate (78), with the aim of introducing a typical α, β-unsaturated methyl ketone moiety of the ANTX-a. The new alternative here was the transformation of the bicyclic ketone 78 in the enol ethers (79), using carbanions stabilized by phosphine oxide derivatives, with subsequent easy modifications of this intermediate to produce (±)-ANTX-a. The enantioselective synthesis of (±)-ANTX-a was obtained using enzymatic resolution with chynotripsin I of the (±)-(1)-allene to obtain the (R)-(1)-allene (Jones & Beck, 1976).

Synthesis of ANTX-a via tandem reactions In general terms, it is possible to define a tandem reaction as a reaction in which several intermediate bonds are formed in sequence without isolating the intermediates, changing the reagents or the conditions of the reaction. Thus, it is a quick way to build more complex structures. The total synthesis of ANTX-a, based on the tandem reactions of anions, was published by Parsons and co-workers (Parsons et al., 1995; Parsons et al., 1996). The key step in this approach involved a tandem methyl lithium-induced β-lactam ring opening (82 to 83, Figure 7.20), with subsequent intramolecular cyclization to set up the bridged bicyclic framework of the natural product (84) with the methyl ketone moiety, in a one-step operation. This reaction was possible due to the strain of the four membered-ring and the lack of an amide type resonance in β-lactam rings, which give an electrophilic character to the carbonyl group, which is susceptible to nucleophilic attack by the methyl anion. The β-lactam ring (Figure 7.20) was obtained after the product was hydrolyzed in a reaction between the cycloctadiene and the chlorosulphonyl isocyanate. This compound was alkylated with benzyl bromide under catalytic phase transfer conditions to get compound 81. Treatment of the β-lactam 81 with MCPBA gave the epoxide 82, in the required anti disposition with respect to the lactam for the intramolecular cyclization. The reaction took place by adding methyl lithium dropwise at −25 ∘ C to the epoxide 82 in THF, providing the alcohol 84 in a 40% yield. Posterior reprotection of nitrogen with tBoc group and dehydration of the hydroxyl group, produced the tBoc-dihydroanatoxin, 86, in an 85% yield. Finally, the total synthesis was completed using the Rapoport approach to provide (±)-ANTX-a (Sardina et al., 1989; Sardina et al., 1990). Stockman and Roe published a ten-step total synthesis of ANTX-a with a 27% overall yield and also published a second total synthesis of homoanatoxin, with a 15% overall yield (Roe & Stockman, 2008; Roe et al., 2009). The first total synthesis of homoanatoxin was made by Gallagher and collaborates (Wannacott et al., 1992). The starting material, 3-butenyl bromide (87, Figure 7.21) was transformed to a Grignard reagent and reacted with ethyl formate to yield the alcohol, which was then coupled with N-tosyl-tert-butyl carbamate and, following, deprotected with TFA give the amine, 88. A ring-closing metathesis was carried out using the first generation of a Grubb’s catalyst (Trnka & Grubbs, 2001) (6,8 mol %) to convert compound 88 into the cyclic alkene, 89, followed by ozonolysis and reductive cleavage work-up

Recent insights into anatoxin-a (ANTX-a)

Bn

O

Bn

N a

153

O N

N Bn

b

c

O

Me O

80

O 82

81

83

Bn N

O

OH 84 d H N

O f

1

Boc N

86

O e

Boc N

O

I 85

Figure 7.20

Partial synthesis of ANTX-a via tandem reaction a) Chlorosulfonyl isocyanate, Na2 CO3 , CH2 Cl2 , 0 ∘ C, 2h, RT, Na2 SO3 , Na2 HPO4 , 48%; solution of NaOH at 50%, CH2 Cl2 , Bu4 NHSO4 , benzyl bromide, 95%. b) MCPBA, CH2 Cl2 , RT, 24h, 84%. c) Methyllithium, THF, −25 ∘ C, 1 h, 40%. d) H2 , 10% Pd/C, MeOH, Boc2 O, Ph3 P, I2 , imidazole, CH2 Cl2 , RT, 1h, 86%. e) Bu3 SnH, AIBN, toluene, reflux, 30 min, 86%. f) NaH, THF, MeOH, RT, 7h, TBDMSCl, Et3 N, THF, −15 ∘ C, RT; Pd(OAc)2 , MeCN, RT, 48 h, 94% or PhSeCl, THF, −78 ∘ C, 2 h, MCPBA, 0 ∘ C; TFA, CH2 Cl2 , 1h, 81%. Source: Based on data from Parsons et al., 1995; Parsons et al., 1996.

to give the aldehyde 90 (Figure 7.21). Treatment of the crude aldehyde, 90, with diethylphosphono-acetone and sodium hydride in THF, gave a mixture of diastereoisomers of 91. The use of TMSI in dichloromethane at −78 ∘ C in the first step, and the use of DBU in the second step, stirred in toluene at room temperature, gave a 70% yield of 92 from 91. N-Tosyl ANTX-a (92) was deprotected using methanol at −40 ∘ C in a phosphate buffer to give (±)-ANTX-a (Åhman & Somfai, 1992; Somfai & Åhman, 1992; Trost & Oslob, 1999).

Synthesis of ANTX-a via enines metathesis Metathesis is a very useful tool for the carbon-carbon bond-forming process, which leads to 1,3-dienes from alkenes and alkynes (Diver & Giessert, 2004). During the process, the carbon structure changes and, in the case of ANTX-a synthesis, occurs via the ring-closing metathesis. The key step in this synthesis strategy is the ring-closing enzyme metathesis (Figure 7.22) (Villar et al., 2007).

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Ts

Ts NH a

Br

NH

b

87

88 89

c

1

f

Ts N

MeO

O

MeO

Ts e

O

91

92

Ts

d

O

90

Partial synthesis of ANTX-a via tandem reaction a) Mg, EtO2 CH3 , Et2 O reflux, 76%; TsNHBOC, DIAD, Ph3 P, THF, 93%; TFA, CH2 Cl2 , 100%. b) Grubbs I, CH2 Cl2 , rt; 88%. c) O3 , MeOH, −78 ∘ C; DMSO, NaHCO3 ; 100%. d) (EtO)2 POCH2 COCH3 , NaH, THF, 76%. e) TMSI, CH2 Cl2 , −78 ∘ C; DBU, tol, rt, 70%. f) Somfai reference (Åhman & Somfai, 1992; Somfai & Åhman, 1992), 76%. Source: Based on data from Roe and Stockman, 2008, 2009.

Figure 7.21

R1 R2

R1

Enyne metathesis

R Figure 7.22

R2 R

General enyne ring-closing metathesis.

Two enantiospectific syntheses of ANTX-a using a ring-closing enyne metathesis were published almost simultaneously by two separate research groups: the Mori group (Brenneman et al., 2004; Brenneman & Martin, 2004; Mori et al., 2004) and the Martin group (Dess & Martin, 1983). Mori’s group developed the synthesis of tosylanatoxin-a by metathesis of an anyne as a substituent on a 2,5-cis-pyrrolidine (93, Figure 7.23) obtained from L-pyroglutamic acid. The methathesis was carried out with a second-generation Grubbs’s catalyst, 94, after previous protection of the alkyne with a silyl group. The product, the bicycle compound 95, was obtained with a 85% yield. The desilylation process occurred during the reaction. The α, β-unsaturated ketone moiety was the result of oxymercuration of compound 95, followed by treatment with sodium borohydride. The resultant alcohol was subject to a Dess-Martin oxidation (Dess & Martin, 1983) to afford the N-tosyl-anatoxin-a, 96, which was then transformed into (+)-ANTX-a using a previously developed procedure (Åhman & Somfai, 1992; Somfai & Åhman, 1992). The ANTX synthesis by Martin’s group differs with that of Mori’s with respect to the strategy for achieving the cis-2,5-disubstituted pyrrolidine, 97 (Figure 7.24). The starting product in this case was D-methyl pyroglutamate, which was converted in five sequential steps to the propynyl intermediate 97.

Recent insights into anatoxin-a (ANTX-a)

N Ts 93

Ts

Ts N

a

155

O

N b

NMes

MesN

96

95

Cl Ru Ph Cl PCy3 94

Partial synthesis of ANTX-a via enine metathesis a) BuLi, TMSCl, 95%; 2nd generation Grubb’s catalyst, 94, CH2 Cl2 , 85% b) Hg(OAc)2 ; NaBH4 , 42%; Dess-Martin oxidation, 86%. Source: Based on data from Brenneman et al., 2004; Brenneman and Martin, 2004; Mori et al., 2004.

Figure 7.23

Cbz N

a N Cbz 97

MesN

b

OH OH

Cbz N

NMes 100

99

Cl Ru Ph Cl PCy3 98

c

H N

Cbz N

O

O

d

(+)-1

101

Partial synthesis of ANTX-a via enine metathesis a) 2nd generation Grubb’s catalyst, 86, CH2 Cl2 , 87%. b) OsO4 , Et3 N, THF, −78 ∘ C to RT, then aq. NaHSO3 , Δ, 76%. c) NaIO4 , THF∕H2 O, rt, 95%. d)TMSI, CH3 CN, −10 ∘ C, 99%. Source: Based on data from Dess and Martin, 1983.

Figure 7.24

After exposure of 97 to a second-generation Grubb’s catalyst at room temperature, the 9-azabicyclo[4.2.1]nonane, 99 was obtained in a 87% yield. The terminal olefin in 99 was dihydroxylated using the complex osmium tetroxide and triethylamine, followed by reduction of the intermediate osmate ester to give the diol 100. The cleavage of the diol unit with the periodate ion delivered 101. Removal of the N-carbamoyl function was affected with TMSI at −10 ∘ C to form ANTX-a (1) (Brenneman et al., 2004; Brenneman & Martin, 2004).

Anatoxins’ biomolecular targets and mechanisms of action A great number of toxins are produced from cyanobacteria. These organisms are also associated with the origin of life and the appearance of oxygen on earth, although they

156

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have been found even in extreme environmental conditions not normally associated with supporting life (Valério et al., 2010; Yadav et al., 2011). Toxins can be classified according to their chemical structure – for example, alkaloids, cyclic peptides or lipopolysaccharides – and can also be classified according to their biological targets, such as neurotoxins, hepatotoxins or dermatoxins (Ferrão-Filho & Kozlowsky-Suzuki, 2011). Toxins have been found in freshwater habitats in many continents, including Europe, Asia, the United States and Australia (Araóz et al., 2005; Ballot et al., 2010b; Ballot et al., 2005; Bumke-Vogt et al., 1999; Gugger et al., 2005; Namikoshi et al., 2003; Park et al., 1993; Viaggiu et al., 2004). They have been considered a major public health and environmental issue, and several investigations have been conducted for the detection of these toxins. Studies have been undertaken to understand the factors that lead to their production in nature and in culture media (Cadel-Six et al., 2009; Gagnon & Pick, 2012). Anatoxins, the main focus of this chapter, are known as neurotoxins. In this section, we will analyze the mechanism of action of the two types of anatoxins and compare their action with the mechanisms of other toxin groups. Several in vitro and in vivo studies will be referred to, in order to describe recent advances and methodologies in the study of the biological effects, biomolecular targets and mechanisms of action of the anatoxins (Chiu et al., 2011; Ferrão-Filho & Kozlowsky-Suzuki, 2011; Yadav et al., 2011; Zegura et al., 2011).

Biological effects and mechanisms of action of anatoxins and anatoxin-a(S) As described above, anatoxins are also known as toxins that induce a ‘very fast and quick death’, not only due to their toxic potency, but also due to their main biological target and mechanism of action. It should be noted that anatoxin-a(S) is not an analogue of ANTX-a but, in fact, anatoxin-a(S) is both chemically and physiologically different from anatoxin-a. Anatoxin-a(S) is a unique natural organophosphate which is also a neurotoxin. It is produced by certain strains of cyanobacteria, including Anabaena flos-aquae NRC 525-17 (Froscio et al., 2003). It produces many of the same symptoms as anatoxin-a, which is the reason why these compounds have a similar name. However, the letter ‘s’ was appended because, unlike ANTX-a, anatoxin-a(s) caused vertebrates to salivate excessively (Kühlbrandt, 2004; Scholl et al., 1999). Anatoxin-a(S) is relatively more toxic than anatoxins with LD50 values from 10 μg∕Kg to 10 mg/Kg, depending on the mode of exposure (oral toxicity data is scarce or nonexistent for some anatoxins). However, it is considered the most toxic of algal-borne toxins (Ferrão-Filho & Kozlowsky-Suzuki, 2011; Valério et al., 2010). As neurotoxins, anatoxins affect the synaptic transmission of the neuromuscular junction by targeting post-synaptic nicotinic receptors of acetylcholine (Aronstam & Witkop, 1981). Besides being an agonist of the neurotransmitter, it binds irreversibly to these receptors, inducing membrane depolarization and hyperstimulation of respiratory muscle cells, which can lead to death by asphyxia (Valério et al., 2010). ANTX-a is a potent nicotinic agonist of nicotinic acetylcholine receptors (nAChRs), well known to have an important role in modulating the release of various neurotransmitters such as noradrenalin from different brain areas (Barik & Wonnacott, 2006). As potent agonists, anatoxins has been used as a tool to expand the understanding of several processes involved in neurotransmitter cross-talk. Tetrodotoxin has also been used to study these phenomena (Barik & Wonnacott, 2006).

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ANTX-a(S) is a cyclic peptide containing phosphate (Figure 7.1), similar structurally to pesticides such as malathion, a well-known potent insecticide. Its mode of toxicity is similar to this class of pesticide: ANTX-a(S) is a potent acetylcholine esterase inhibitor. In effect, ANTX-a(S) prevents the termination of the action of the neurotransmitter and induces stimulation of muscle contraction by maintaining the opening of the Na+ channels (Valério et al., 2010). Although the mechanism of action described for ANTX-a(S) is different from the anatoxins, the symptoms are similar and, in addition, it also causes lacrimation and salivation upon exposition (Fawell et al., 1999; Ferrão-Filho & Kozlowsky-Suzuki, 2011; Humpage, 2008; Mankiewicz et al., 2003; Valério et al., 2010; Yadav et al., 2011). Many in vivo and in vitro studies have studied the effects of ANTX-a, in several animal species such as fish, amphibian and mouse, targeting several types of cells, tissues and biological processes (Bownik et al., 2012; Campos et al., 2007; Fawell et al., 1999; Rogers et al., 2005). The biological targets of both anatoxins and anatoxin-a(S) are located at the neuromuscular junction and effect the post synaptic nicotinic receptors of acetylcholine or acetylcholine esterase activity. By targeting the receptor, ANTXs maintain the opening of the ion channel, inducing membrane depolarization and muscle stimulation whereas, by inhibiting acetylcholine hydrolysis, it does not stop the neurotransmitter action upon release into the synaptic cleft. The binding of acetylcholine to the acetylcholine receptor, which is also an ion channel, induces the influx of ions and, therefore, the same cellular event as the nicotinic receptor, although with different features.

Mechanisms of action of toxins: comparison with anatoxins and anatoxin-a(S) Anatoxin-a(S) and the saxitoxins are neurotoxins but, contrary to ANTX-a, saxitoxins are antagonists of the voltage dependent Na+ and Ca2+ channels, promoting the blockage of the nervous impulse across the axon and concomitantly preventing the release of Ca2+ and the initiation of muscle contraction (Valério et al., 2010; Yadav et al., 2011). Saxitoxins, also known as the ‘seafood toxin’, which have presented in contaminated crabs and shellfish, induce paralytic shellfish poisoning (PSP). This toxin is included in the classes of chemical weapons such as the gas sarin, mustard gas and ricin (Valério et al., 2010). Another neurotoxin, β-metilamine-alanine, which is responsible for inducing neurodegenerative diseases, is a precursor of carbamate known to be a glutamate agonist at the metabotropic and/or ionotropic receptors, and causes the stimulation of glutaminergetic neurons, therefore increasing Na+ and Ca2+ intracellular concentrations. It can induce mitochondrial dysfunction, reactive oxygen species (ROS) formation and glutathione (GSH) depletion, in addition to blocking the cysteine/glutamate antiporter (Ferrão-Filho & Kozlowsky-Suzuki, 2011; Zegura et al., 2011). Other cyanotoxins, including the microcystins (MCs) and nodularins (Nods), cause the inhibition of the protein tyrosine phosphatase (PTP), at the cellular signalling pathway, thus affecting several processes involved in cell metabolism, cell death and proliferation, in addition to other effects. Contrary to the actions of the MCs and Nods, anatoxins and saxitoxins target the hepatocytes, inducing GSH depletion and ROS formation, although the mode of action and/or the biological effects are not totally defined (Alverca et al., 2009; Bouaïcha & Maatouk, 2004; Menezes et al., 2013; Weng et al., 2007).

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Chapter 7

Cylindrospermopsin, another hepatoxin (Moreira et al., 2013), acts to inhibit protein synthesis, possibly by preventing protein translation (López-Alonso et al., 2013; Schneider-Poetsch et al., 2010) and, through cytochrome P450 biometabolism, to induce toxicity (Froscio et al., 2003 ; Moreira et al., 2013). Potential synergistic effects of ANTX-a with the alga toxin microcystin were carried out in in vitro embryotoxicity studies (Rogers et al., 2005). These studies at the developmental stage were undertaken to evaluate the toxicity of ANTX-a. It is believed that the effects and the toxin biomolecular targets are dependent, not only on the doses or the concentration of toxin used, but on many other experimental parameters, such as the mode of administration and the exposure time (Aureliano, 2013). These observations recall the words written in the 16th Century by Theophrastus Philippus Aureolus Bombastus von Hohenheim (1493–1541), ‘Alle Ding’ sind Gift, und nichts ohn’ Gift; allein die Dosis macht, daß ein Ding keinGift ist,’ which can be translated to; 1) ‘All substances are poison’; 2) ‘There is nothing without poison’; and 3) ‘Only the doses made the poison’ – or, in Latin, ‘Sola dosis facit venenum’ (Aureliano, 2014).

Putative biomolecular targets of neurotoxins As described above, the targets of anatoxins and anatoxin-a(S) include the nicotinic acetylcholine receptors or the acetylcholine esterase enzymes. Therefore, the biological targets described above for the cyanotoxins are mainly ion channels or some specific enzymes. However, besides the ion channels, the ion pumps, known as P-type ATPases and/or E1 E2 -ATPases, namely Na+ ∕K+ -ATPase, Ca2+ -ATPase and H+ ∕K+ -ATPase, involved in ion homeostasis, may also be the targets of toxins (Gadsby et al., 2012; Kühlbrandt, 2004). These E1 E2 transport ATPases were described as potential biological targets for several drugs and polyoxometalates in the treatment of heart failure and ulcers, among other effects (Yatime et al., 2009; Aureliano et al., 2013). Studies of toxins on these pumps are scarce. Some of these studies have described the effect of palytoxin, a marine toxin, on Na+ ∕K+ -ATPase (Yatime et al., 2011). In fact, it was observed that palytoxin induced K+ efflux upon exposition of the enzyme, which was firstly attributed to the formation of a membrane pore by the toxin (Rouzaire-Dubois & Dubois, 1990). Other studies suggested that palytoxin might uncouple the ion pump and induce the formation of channels (Habermann, 1989). It was observed that the K+ efflux was prevented by ouabain, a known Na+ ∕K+ -ATPase inhibitor, and therefore blocks the toxin to induce the formation of an ion channel. It was suggested that the toxin palytoxin acts throught the ion pump (Habermann, 1989). It was proposed that the interaction of palytoxin with the ion pump may change the mechanism of ion transport by the pump, inducing the formation of ion channels (Habermann, 1989). Therefore, both drugs and toxins can be associated with ion pumps, although some aspects of the mechanisms of action have yet to be clarified (Pelin et al., 2013). Moreover, the ability of transforming an ion pump into a channel is an interesting mechanistic process, with putative biological and pharmacological applications of increasing interest. It is believed that the understanding of the mode of action of several types of toxins, as well as drugs and others compounds, in targeting ion pumps and channels, will be of increasing interest in future. This is particularly so after the observation that these ion transport systems may be involved in several cellular processes besides the ones related with ion transport, such as cell motility, cell-cell contact, cell proliferation and apoptosis, opening new therapeutic and biochemical applications for these toxins (Yatime et al., 2009; Yatime et al., 2011).

Recent insights into anatoxin-a (ANTX-a)

159

Pre-synaptic ACE

ANTx

ANTx

Ch

ACh

?

ANTx-S

Ca2+

Post-synaptic

?

Na+/K+ NCx

nAChR

↑ Ca2 +

Na+ or Ca2+

↑ Na+ Ca2+

Contractile system

Figure 7.25 Scheme of proposed anatoxins (ANTX) and anatoxin-a(S) (ANTX-S) molecular targets. ANTX is the strongest agonist for nicotinic acetylcholine receptor (nAChR), leading to muscle contraction stimulation. On the contrary, anatoxin-a(S) also targets acetylcholine esterase (ACE), preventing acetylcholine (Ach) hydrolysis to choline (Ch). A putative target for ANTX would be also the ion pumps, such as Ca2+ or Na+ ∕K+ -ATPases. ANTX P-type ATPase inhibition, for example, for Na+ ∕K+ -ATPase, will increase the intracellular Na+ and, consequently, the increase of Ca2+ through Na+ ∕Ca2+ exchanger (NCx) that will trigger muscle contraction.

Putting it all together, in terms of the mechanism of action for the majority of the established toxins, the ion channels are the main direct or indirect biological targets. However, we cannot exclude others systems which may be involved in ion homeostasis, such as ion pumps (Figure 7.25).

LC-MS detection Many analytical methods have been developed to determine the anatoxins. Previously, the mouse or rat bioassay was applied to establish the presence of ANTX-a in samples and extracts; the toxin or an extract of the toxin was administered intraperitoneally (i.p.) to the animal, and the presence of ANTX-a was decided by the observation of a specific set of symptoms in the test animal (Adeyemo & Siren, 1992; Al-Layl et al., 1988; Astrachan & Archer, 1981; Astrachan et al., 1980; Baker & Humpage, 1994; Lahti et al., 1995; Stevens & Krieger, 1988; Watanabe et al., 2003). These tests were problematic for many reasons, including the inability to quantitate the amount of toxin present, the lack of accuracy, and an inability to detect the presence of co-occurring toxins and toxin analogues. In addition, the large number of animals needed for testing routine samples was regarded by many as ethically questionable when qualitative and quantitative

160

Chapter 7

instrumental-based methods became available (Al-Layl et al., 1988; Stevens & Krieger, 1988). Nowadays, analytical methods have largely superseded animal bioassays. GC-MS was one of the first mass spectrometric based methods used to detect ANTX-a. Devlin et al. (1977) detected the toxins by first converting them to their more volatile N-acetyl derivatives. Other groups also applied and modified this approach in determining the anatoxins (Himberg, 1989; Ross et al., 1989; Smith & Lewis, 1987). LC-UV methods were developed to determine ANTX-a (Harada et al., 1989), but this approach was inhibited by the presence of interferences in samples, and also by the fact that the degradation compounds of ANTX-a are not UV-active (Edwards et al., 1992; Furey et al., 2003b; Harada et al., 1989; James et al., 2008; Namikoshi et al., 2003; Powell, 1997; Rapala et al., 1994; Wong & Hindin, 1982; Zotou et al., 1993). Liquid chromatography with fluorescence detection (LC-FLD) offers a good approach to the analysis of ANTX-a, HANTX-a and its main analogues (epoxyANTX-a, epoxyHANTX-a, dihydroANTX-a and dihydroHANTX-a), but the method requires that the targets are first derivatized with 4-fluoro-7-nitro-2,1,3-benzoadiazole (NBD-F) (Furey et al., 2004; James & Furey, 2000; James et al., 1997a, 1997b, 1998; James & Sherlock, 1996; Rawn et al., 2005; Rellán et al., 2007). The LC-FLD approach proved to be sensitive, selective and reproducible, but it could not identify the presence of co-occurring toxins such as microcystins, nodularin, cylindrospermopsin, saxitoxin or anatoxin-a(S). A recent GC-MS based method, used in conjunction with a PCR investigation, was used to study the occurrence of HANTX-a from mats of Hydrocoleum lyngbyaceum, a marine cyanobacterium, in New Caledonia. Previously there had been an intoxication of giant clams during a ciguatera fish poisoning outbreak; HANTX-a was also found in the clams (Laurent et al., 2008). The presence of HANTX-a may indicate that the clams had been grazing on the Hydrocoleum lyngbyaceum (Mejean et al., 2010). GC-, with complimentary LC-FLD, was used to screen commercially available dietary cyanobacteria-based supplements (for human and animal) for ANTX-a. Both methods involved derivatization of ANTX-a to facilitate analysis: the acetyl derivative for GC analysis, and the NBD-F derivative for FLD detection. One of the samples destined for human consumption had small quantities of ANTX-a, as did two samples destined for fish and bird supplementation. This confirms the need for toxin screening of cyanobacterial-based products (Rellán et al., 2009). Without doubt, LC-MS is the best approach for the analysis of ANTX-a and its associated compounds (Dahlmann et al., 2003; Harada et al., 1993; Hormazábal et al., 2000; Poon et al., 1993; Takino et al., 1999). Furey et al. (2003a) studied the mass fragmentation of ANTX-a and HANTX-a using positive ion mode. Multiple tandem MS (MSn ) was conducted using an ion-trap mass (QIT) spectrometer (Table 7.2). The strength of this method lay in the ability of the ion-trap MS instrument to switch between full-scan MS and MSn scan modes without the loss of sensitivity (Furey et al., 2002). The molecular-ion, [M+ H]+ , at m/z 166 (AN) and at m/z 180 (HMAN), were used as the precursor ions for multiple MSn experiments. For anatoxin-a, MS2 gave major fragment ions at m/z 149 [166-NH3 + H]+ and MS3 displayed fragment ions for AN at m/z 131 [166-NH3 -H2 O+ H]+ , m/z 121, 107, 105, 91, 81 and 79. MS4 experiments, achieved by trapping and subsequent fragmentation of the ion at m/z 131, generated a product ion at m/z 116, 91. Fragmentation of the MS3 ion at m/z 107, generated a single product ion at m/z 79 in the MS4 spectrum (Table 7.2). A similar fragmentation process was observed for homoanatoxin-a: MS2 gave dominant ions at m/z 163 [180-NH3 + H]+ , m/z 145 [180-NH3 -H2 O+ H]+ , m/z 135, 120, 107 and 91. MS3 spectra were obtained

Recent insights into anatoxin-a (ANTX-a)

161

Table 7.2

Mass spectral ions detected in MS1-MS5 experiments on anatoxin-a and homoanatoxin-a. Source: Furey et al., 2003a. Reproduced with permission of Wiley and Sons Ltd. MS𝟐 AN m/z

MS𝟑 AN m/z

MS𝟒 AN m/z

MS𝟒 AN m/z

166

MS𝟐 HMAN m/z

MS𝟑 HMAN m/z

MS𝟒 HMAN m/z

MS𝟒 HMAN m/z

MS𝟓 HMAN m/z

180

149

149

163

163

–

–

145

145

131

131

131

135

135

–

121

121

–

120

121

–

–

–

–

116

–

–

116

–

145 135

–

–

117

–

–

117

–

107

107

–

107

107

107

–

107

105

105

–

–

105

105

105

–

–

91

91

91

–

91

91

91

91

–

81

81

–

–

81

80

–

81

–

79

79

–

79

80

–

–

79

79

107

Ions selected for trapping and fragmentation are shown in shaded boxes.

by fragmenting the ion at m/z 163 and MS4 utilised the ions, m/z 145 and 135. The ions at m/z 107 [M-NH3 -COCHR]+ and m/z 91 [C7 H7 ]+ were common to both toxins in the MS3 and MS4 spectra. The MS5 spectrum of HMAN, targeting the ion at m/z 107, gave a product ion at m/z 79 (Table 7.2). Thus, the fragmentation pathways for these anatoxins could be mapped, Figure 7.26 (Furey et al., 2003a). However, one of the main obstacles encountered in the development of LC-MS based methods was the presence of the ubiquitous amino acid phenylalanine (Phe), which has the same nominal mass as ANTX-a (AMU = 165) and can also have a very similar retention time under reverse phase chromatographic conditions. Several methods have been developed to eliminate this interferent. In particular, Furey et al and James et al devised LC- quadrupole ion trap (QIT) MS3 and LC-quadrupole time-of-flight (QqTOF) MS methods to identify fragmentation ion patterns which can distinguish the ANTX-a from the Phe (Furey et al., 2005; Furey et al., 2003a; James et al., 2005). Using the selected ion trapping process, QIT MSn allows an unambiguous assignment of reactant/product sequences, and signals are remarkably free from interferences typically present in complex biological matrices. A study comparing the MSn fragmentation pathways of AN and Phe facilitated the selection of ions to enable a distinction to be made between the compounds (Figure 7.27). High mass accuracy fragmentation data obtained on a QqTOF mass spectrometer helped to confirm the assigned fragment structures (Figures 7.28 and 7.29) Dimitrakopoulos et al. (2010) developed an LC-MS based method, where ANTX-a was chromatographically resolved from Phe. This method used Phe-d5 as an internal standard to develop a sensitive LC-ESI-MS/MS on a TQS quantum discovery triple quadrupole mass spectrometer (Table 7.3). LOD and LOQ were determined as 0.65 and 1.96 ng/L (Dimitrakopoulos et al., 2010). Furey et al. devised a strategy to minimize ion suppression effects commonly encountered in LC-MS methods, which can be problematic in the analysis of trace analytes in complex matrices, which could be

162

Chapter 7

+

H2 N

O

+

R

R

[M–NH3+H]+ m/z = 149 (R = CH3) [M–NH3+H]+ m/z = 163 (R = C2H5)

[M+H]+ m/z = 166 (R = CH3) [M+H]+ m/z = 180 (R = C2H5)

–RCH=C=O

+

R

[M–NH3–H2O+H]+ m/z = 131 (R = CH3) [M–NH3–H2O+H]+ m/z = 145 (R = C2H5)

42 R = CH3 56 R = C2H5

+

[M–NH3–COCHR+H]+ m/z = 107 +

[C2H7]+ m/z = 91

Figure 7.26 Proposed CID fragmentation pathways, showing the typical losses and structures of product ions from protonated anatoxin-a and homoanatoxin-a, as determined by ion-trap MSn experiments. Source: Furey et al., 2003a. Reproduced with permission of Wiley and Sons Ltd.

applied to improve the quantitation of ANTX-a in biological matrices (Furey et al., 2013). Since their initial inception for the analysis of ANTX-a, published LC-MS based methods have been increasing at an unprecedented rate and have been implemented in the determination of a myriad of trace analytes. Table 7.3 summaries some of the most recent LC-MS methods used to investigate the anatoxins. Of important note is the development of a MALDI-TOF-MS method by Araoz et al., which was used to investigate ANTX-a and HANTX-a in axenic Oscillatoria strains. Interestingly, this group induced the matrix-suppression effect to improve the quality of the ANTX-a and HANTX-a signals. They took this approach to counteract the range of low molecular mass ions that were generated by the matrix (Araóz et al., 2008a). Wood et al. applied a solid phase adsorption toxin trapping (SPATT) approach to capturing and pre-concentrating samples in situ in a river before analysis; the target toxins, ANTX-a and HANTX-a were then extracted and analyzed by LC-MS/MS (Wood et al., 2011). Lemoine and co-workers applied ultrafast analysis of ANTX-a using a laser diode thermal desorption-atmospheric pressure chemical ionization interface (LDTD-APCI-MS/MS), coupled to a triple quadrupole MS, to detect ANTX-a. The method was optimized to enhance the signal of the target analyte while removing the interfering signal from Phe (Lemoine et al., 2013).

Recent insights into anatoxin-a (ANTX-a)

163

x5 100 Relative Abundance

Phe 7.95

131.0

AN 91.0

80 60 40 20

106.8

105.1 121.0 81.0 79.8 93.0 70.8

MS3 166 30% 149 30%

132.0 122.3 149.2

0 60

80

100

120 140 m/z

50

160

180

200

(b) 100 Relative Abundance

Relative Abundance

100

AN 7.47

0

93.0

MS3

Phe

166

80

24%

120 32%

60 103.2 40 120.0 20

79.3

0 1

2

3

4

5

6

Time (min) (a)

7

8

9

10

60

80

100

120 140 m/z

160

180

200

(c)

Figure 7.27 LC-MS3 chromatogram of a water sample containing standard AN (7.47 min.) and Phe (7.95 min.), obtained using QIT MS detection. The selected ions and relative collision energies for fragmentation at each MS stage are shown as inserts. Chromatographic conditions: Luna column (C18, 5 μm, 250 × 3.2 mm, 35∘ C); 5 μl injection; mobile phase was acetonitrile/water (15/85); flow rate = 0.4 ml/min. Positive CID product ion spectra of MS3 for (b) AN-a and (c) Phe. The selected ions for trapping and fragmentation, together with the optimized RCE, are shown as inserts. Source: Furey et al., 2005. Reproduced with permission of Elsevier.

Many co-occurring toxins can be present in a single sample; to address this problem several research teams have recently developed LC-MS based multi-target methods (Table 7.3). Oehrle et al. developed an ultra-performance (UP) LC-ESI-MS/MS method to analyze a range of cyanotoxins (clylindrospermopsin, ANTX-a, and several microcystins) in under eight minutes, using Nodularin as an internal standard. The authors found that the smaller column medium, together with the selectivity of the MS/MS approach, improved signal response to the target analytes (Oehrle et al., 2010). In 2011, Yen and co-workers used a solid phase extraction (SPE) protocol prior to analysis by LC, hyphenated via an ESI interface to quadrupole MS with on line UV detection, to determine nine cyanotoxins (six microcystins, nodularin, ANTX-a and cylindrospermopsin) in drinking water samples (Yen et al., 2011).

164

Chapter 7

+ +

+

H2 O N

+

C10H7 91.0564 (2)

C10H11 131.0849 (9)

O

C9H13 121.0995 (18) C10H16NO 166.1232

C10H13O 149.0943 (16)

H+

+

C6H7 79.0549 (1)

C10H11 107.0872 (11)

+

O

O OH H3N

C9H7O 131.0482 (11) +

+

NH3

C9H12NO2 166.0868 C8H10N 120.0803 (9)

H+ C8H7 103.0530 (17)

Figure 7.28

Proposed fragmentation pathways for: (a) Anatoxin-a, and (b) Phenylalanine, elucidated by multiple tandem MS experiments using QIT MS, sowing the typical loses and structures of product ions. High mass accuracy data from QqTOF MS are presented to support the assignments with error values (ppm) in parenthesis. Source: Furey et al., 2005. Reproduced with permission of Elsevier.

Chen et al. used an LC-ESI-triple quadrupole-MS/MS method to detect twelve toxins simultaneously in water and algae samples. The toxins analyzed by this method were ANTX-a, cylindrospermopsin, dinophysistoxin-1, nodularin, okadaic acid and several microcystins (Chen et al., 2012). Al-Sammak et al. devised a LC-FLD method and a complementary LC-QIT-MS/MS based method to simultaneously detect microcystins, the neurotoxic non-protein amino acids β-methylamino-L-alanine (BMAA), 2,4-diaminobutyric (DABA) and ANTX-a in a range of environmental samples (Al-Sammak et al., 2013). LC-MS analysis has also been useful for identification of ANTX-a in clinical samples, following the deaths of three dogs in the Netherlands, after they had been swimming in a lake contaminated with the cyanobacterium Phormidium. Two bird deaths in the vicinity were also suspected to be due to ANTX-a poisoning. Several algae samples were taken from the region, and post-mortem samples were taken from one of the dogs and the two birds (including stomach contents, and organ tissues). ANTX-a and their main degradation products dihydro ANTX-a and HANTX-a, as well as algae filaments, were found in the intestines of the dog, and ANTX-a was also confirmed in the algae mat.

Recent insights into anatoxin-a (ANTX-a)

165

43.0170 131.0827

Relative Intensity

91.0567 149.0970 166.1232

79.0535

56.0519 68.0495 45.0340 40

50

60

70

107.0900 105.0742 93.0710

80.0503 81.0694 95.0562 80

90

100

110

121.0936

148.1130

120

140

130

150

160

170

m/z (a) 120.0803

Relative Intensity

103.0530

77.0356 91.517 79.0348

93.0671 107.0481 102.0466

40

50

60

70

80

90

100

166.0868

131.0482

118.0627 110

120

130

140

150

160

170

m/z (b)

Figure 7.29 Spectra of a) AN, and b) Phe, obtained using positive nano-electrospray with a hybrid quadrupole time-of-flight (QqTOF) mass spectrometer. Source: Furey et al., 2005. Reproduced with permission of Elsevier.

It was established that the birds had probably died from other causes (Faassen et al., 2012). These diverse studies demonstrate the strength and applicability of LC-MS/MS to the analysis of a wide range of sample types with very challenging matrices.

Conclusions and perspectives Anatoxin and its analogues have been the subject of scientific research in different areas, such as chemistry, biochemistry, biology and pharmacology, due to the particular characteristics of their structure, including their mechanism of action, as well as

× 2.1 mm; 1.7

SPE

dHANTX-a, eHANTX-a,

5 min 5%B, 10 min 10%B, 15–24 min

0.1% FA) 50% B.

dANTX-a

Gradient: 0–1 min 0%B,

5 mM AA

ACN/H2 O

3μm)

(10:90) +

ANTX-a, HANTX-a,

content

LC-MS/MS

elution with

B: 90% MeOH +

A: 1% MeOH + 5 mM AA

dog’s stomach

(150 × 2.1mm,

ANTX-a, Phe

SPE (ZIC-HILIC,

Atlantis T3

& 166.1/120.0 (Phe)

166.1/149.2 (AN-a)

MRM transitions:

LC-MS/MS

benthic mat &

H2 O + 0.1% FA

hexane, FA)

Phormidium

(50 × 2.1 mm,

200 (acetone,

1.8 μm)

+ 20 mM AF

Resolution

extraction – ASE

H2 O & MeOH + 0.1% FA

Rapid

Accelerated solvent

MCs

OA

Nod

B: MeOH/ACN (4:6)

ANTX-a CYN DTX-1

LC-MS/MS (SRM)

ANTX-a

AABA (IS) with AQC

DABA

BMAA

Derivatised: ANTX-a

Analytes

A: 0.1% formic acid

Linear gradient

issatschenkoi

Zorbax SB-C18

μm)

C18 (100 mm

Hypersil Gold

followed by C18

Acid extraction

dry.

MS/MS

well and heated at 30 ∘ C until

Laser diode thermal

HPLC-FD

LC-ion trap MS/MS

Method

desorption-APCI-

none

in MeOH)

(0.5 g/L in H2 O/0.5 g/L

Ammonium formate

Mobile phase

spotted (2 μL) in

none

2.1 mm; 5μm)

(250 mm ×

Hypurity C18

Column

Aphanizomenon

samples

Algae & water

preparation

Bloom matrix

samples

Samples were

SPE, Oasis-MCX

Water, fish &

aquatic plant

Extraction

(HANTX-a)

56 fmol/inj

(ANTX-a)

61 fmol/inj

LOD

column.

LOQ = 3.5 pg on

column.

LOD = 1 pg on

LOQ 0.04-0.6 ng/mL

LOD < 0.20 ng/mL

LOQ = 3 μg/L

LOD = 1 μg/L

5–7 μg/L

0.8–3.2 μg/L

LOD and LOQ

Some of the most recent LC-MS methods used to investigate the anatoxin-a (ANTX-a) and its analogues.

Samples

Table 7.3

NR

NR

1–500 ng/ml

3–250 μg/L

0.01–1.0 ml/L

Linear Range

2012)

(Faassen et al.,

2012)

(Gagnon & Pick,

2012)

(Chen et al.,

2013)

(Lemoine et al.,

et al., 2013)

(Al-Sammak

Reference

166

Chapter 7

Water samples

Sediments

μm)

2.1 mm, 1.8

(50 mm ×

column

resolution C18

XDB rapid

0.2 ml/min.

166 → 91

Agilent Zorbax

166 → 107

(v/v) formic acid,

μm)

166 → 131

166 → 149

LC-MS/MS (SRM)

both containing 0.1%

MeOH/H2 O (7:93 v/v)

1 ml/min.

0.1% TFA,

λmax = 227 nm

4.6 mm, 3.5

(75 mm ×

column

resolution C18

SPE

acidified with

LC-UV

166.1/43

10–20 min: 100%A ACN/H2 O (5:95 v/v) both

166.1/91

8–10 min: 30%A,

XDB rapid

166.1/131

50%A, 8 min: 30%A,

Agilent Zorbax

166.1/149

0–1min: 100% A, 7min:

μm)

carbon (PGC)

SRM transitions:

LC-MS/MS

LC-MS (SIM)

B : ACN + 0.1% FA

A : H2 O + 0.1% FA

60–70 min : 5%.

60 min: 5%,

40-50 min:70%,

70%A,

0–10 min: 5%A, 40 min:

0.01% HFBA

B: H2 O + 0.01M TFA +

0.01% HFBA

A: ACN + 0.01M TFA +

UPLC-MS/MS

× 2.1 mm, 3.5

Porous graphite

centrifugation

Filtrations,

90% MeOH)

XTerra C18 (100

4.6 mm,)

cartridges,

elution with

RP-18e (100 ×

Carbon

water

Performance

Chromolith

SPE (C18 &

Ultrasonic probe +

Pure & Reservoir

50% A.

0–1min: 100% A, 3min:

B: ACN + 0.1% FA

1.7 μm)

A: H2 O + 0.1% FA

Acquity BEH-C18

(50 × 1.0 mm,

(drinking)

SPATT

River water

(internal standard)

Phe and Phe-d5

ANTX-a,

ANTX-a

CYN.

RR, YR, LW, LF),

ANTX-a + MCs (LR,

dHANTX-a.

dANTX-a,

ANTX-a HANTX-a,

LOQ = 1.96 ng/L

LOD = 0.65 ng/L

LC-MS/MS

0.01 μg/L

LOD

water)

210 ng/L (reservoir

46 ng/L (Pure water)

LODs

NR

μg/L

0.5–2,000

LC-MS/MS

NR

40–2000 ng/L

NR

(continued overleaf )

et al., 2010)

(Dimitrakopoulos

2011)

(Klitzke et al.,

(Yen et al., 2011)

2011)

(Wood et al.,

Recent insights into anatoxin-a (ANTX-a) 167

HANTX-a

MALDI-TOF MS

capillary

purified by HPLC

of axenic

strains

Oscillatoria

from acid extracts.

0.25 μm)

x 0.25 m ×

column (30 m

ANTX-a,

ANTX-a and GC-MS

Lyophilized and

NMR

136, 122

ions at m/z 164, 150,

m/z 179 and fragment

HANTX-a

MS fused silica

Helium

HANTX-a were

GC-MS: Rtx-5 Sil

0.25 μm)

(30m × 0.25mm,

capillary

bleed/MS

CP-Sil 8 CB low

ANTX-a & HANTX-a

C1, C2

(Dell’Aversano et al.,

GC-MS

dcGTX-3,

formica cid (pH 3.5) 2004)

GTX 1–5,

formate & 3.6 mM

5 μm.

fresh filaments

elution)

SPE (C18, MeOH

maxima)

clam)

& Giant clam

50mM AA

(60:40) (giant

lyngbyaceum)

(Tridacna

MeOH/H2 O

MeOH &

membrane filter.

μm) RC 55

dcSTX NEO,

2004)

2.0 mM ammonium

filtered (0.45

2 × 250 mm,

column

CYN, STX,

ANTX-a

CYN

166/149

LC-MS/MS

-LR, -LA, -LY & -LW,

ANTX-a MC-RR, -YR,

-YR & -LR

ANTX-a MC-RR,

Analytes

166/43

UPLC-MS/MS

166.1/148.8/130.6

UPLC-MS/MS

Method

(Dell’Aversano et al.,

Water and ACN-water

70%,

0–0.8 min: 2%B, 9.8 min

B: ACN + 0.1% FA

A: H2 O + 0.1% FA

B : ACN + 0.1% FA

A : H2 O + 0.1% FA

Mobile phase

both containing

TSKgel Amide-80

1.8 μm)

(100 × 2.1 mm,

HSS T3

Acquity UPLC

1.7 μm)

(100 × 2.1 mm,

BEH C18

Acquity UPLC

Column

samples were

150–300 ml

Filtration

(Hydrocoleum

Benthic mat

Water samples

(Lakes & river)

Water samples

SPE (Oasis HLB)

Filtration

Water + surface

layer samples

Extraction

(continued)

Samples

Table 7.3

NR

NR

NR

0.13 μg/L

NR

LOD and LOQ

NR

NR

NR

0.5–100 μg/L

NR

Linear Range

2008a)

(Araóz et al.,

2010)

(Mejean et al.,

2010a)

(Ballot et al.,

2010)

(Oehrle et al.,

(Li et al., 2010)

Reference

168

Chapter 7

4.6 mm, 5 μm)

(250 mm ×

Luna C18(2)

× 4.6 mm i.d.)

technique

WCX SPE

C18 (250 mm

dispersion

containing 0.05% TFA.

ACN:H2 O (15:85)

B: aqueous 10 mmol/L FA.

A: 10 mmol/L FA in ACN;

2004)

LC-MS1–5

DAN 159/142

166/91

166/107

166/131

ANTX-a 166/149

HMANTX-a

ANTX-a

LC-MS3

LOD = 0.6 μg/L

LOD = 8 ng/L (water) LOD = 0.2 ng/g (fish)

dCYN

dHANTX-a 182.2/57.1

(Dell’Aversano et al.,

DAN (IS)

CYN

HANTX-a 180.2/163.15

Isocratic 95%A – 5%B

ANTX-a

dCYN, MC).

dHANTX-a

dANTX-a 168.15/91.15

ammonia

LC-MS/MS

each toxin (CYN,

HANTX-a

B: 112mM FA + 40 mM

A: 90% MeOH

ANTX-a dANTX-a

LODs ca. 2 μg/kg for

LOD 1.2 × 10–7 M

ANTX-a 166.15/149.1

ANTX-a

LC-MS/MS (MRM)

LC-MS (SIR mode)

5–1000 μg/L

0.02–200 ng

2003a)

(Furey et al.,

2006)

(Bogialli et al.,

2007)

(Wood et al.,

2008b)

10–4 M

NR

(Araóz et al.,

10–7 – 2 ×

Abbreviations ANTX-a = Anatoxin-a; BMAA = β-N-methylamino-L-alanine; DABA = DL-2,4-diaminobutyric acid hydrochloride; AABA = DL-2-Aminobutyric acid; AQC = 6-aminoquinolyl-N-hydroxysuccinimidyl; HANTX-a = Homoanatoxin-a; CYN = Cylindrospermopsin; STX = saxitoxin; NEO = Neosaxitoxin; dcSTX = decarbamoylsaxitoxin; dcNEO = decarbamoylneosaxitoxin; GTX 1-5 = Gonyautoxin 1-5; dcGTX-3 = decarbamoylgonyautoxin; C1, C2 = N-sulfogonyautoxin-1, -2; dANTX-a = Dihydroanatoxin-a; dHANTX-a = Dihydrohomoanatoxin-a; eHANTX-a = Epoxyhomoanatoxin-a; dCYN = deoxycylindrospermopsin; DAN = 1,9-diaminononane; IS = Internal Standard; LOD = Limit of Detection; LOQ = Limit of Quantitation; ACN = Acetonitrile; MeOH = Methanol; FA = Formic acid; AA = Acetic Acid; TFA = Trifluoroacetic acid; AF = Ammonium Formate; NR = Not reported; i.d. = internal diameter; Ext = Extraction; WCX = Weak cation exchange; C18 = reverse-phase chromatography column; PGC = Porous graphite carbon ; MCX = chromatograph phase involving a combination of reversed-phase and cation exchange for bases

Drinking water

Cyanobacteria,

Fish muscle

Lake water,

Alltima HP 5 μm

5μm)

stomach

Matrix solid phase

(150 × 2 mm,

& dog

samples.

Amide-80

ACN & FA

22-27 min: 80%B, 27–28 min: 80–0%B

20-22 min:0–80%B,

elution)

TSK gel

(150 × 4.6 mm) 0–20 min: 0%B,

A: H2 O + 0.1% FA B: 80% ACN + 0.1% FA

SunFire C18

C18, MeOH

SPE (Bakerbond

50mM AA.

Ext: 50% MeOH or

(from 5 rivers)

Benthic mat

Water samples

Bloom (Mat) &

Recent insights into anatoxin-a (ANTX-a) 169

170

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their target site. The biological activity of these compounds results from their structural similarity with nicotine, namely the possession of a nitrogen atom and the π system (the conjugate double bond with the carbonyl group), and also with the spatial arrangement of the molecule, which has a rigid bicyclic skeleton that makes the distance between the nitrogen atom and the π system accurate. These factors determine the linkage to nicotinic acetylcholine receptors and, in the case of anatoxin-a(S), to acetylcholine esterase, which affects several biologic processes. There are several ways to synthesize the azabicyclo [4.2.1] and ANTX-a or its analogues, as described in this chapter, which facilitate the production of compounds in sufficient quantities to allow studies to be conducted on the structure/activity relationship (Brough et al., 1992; Huby et al., 1991b; Thomas et al., 1994; Wonnacott et al., 1991, 1992). As ANTX-a is a useful core structure for nicotinic drug design, this could be an excellent starting point to devise a drug treatment strategy for several brain diseases. In terms of the mechanism of action for the majority of the established toxins, the ion channels are the main direct or indirect biological targets, but we cannot exclude others systems which may be involved in ion homeostasis, such as ion pumps (Figure 7.25).

Acknowledgements The authors acknowledge the funding obtained under the EU/INTERREG IIIB Atlantic Area Programme for projects titled ATLANTOX ‘Advanced Tests about New Toxins Occurring in the Atlantic Area due to Climate Change’ and PHARMATLANTIC ‘Knowledge Transfer Network for Prevention of Mental Diseases and Cancer in the Atlantic Area’. The Higher Education Authority (Programme for Research in Third-Level Institutions, Cycle 4 (PRTLI IV) National Collaboration Programme on Environment and Climate Changes: Impacts and Responses is also acknowledged, along with the European Cooperation in Science and Technology, COST Action ES 1105 ‘CYANOCOST-Cyanobacterial blooms and toxins in water resources: Occurrence, impacts and management’ for adding value to this study through networking and knowledge sharing with European experts and researchers in the field.

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CHAPTER 8

Therapeutics of marine toxins Eva Alonso & Juan A. Rubiolo Department of Pharmacology, Campus de Lugo, Spain

Introduction Scientific knowledge gathered during the past decades allowed a deeper understanding of the pathology of several diseases and enabled the design and synthesis of drug molecules for specific targets, shifting the attention from the ‘natural pharmacy’ to purely synthetic drugs (Houghten, 1994; Ortholand & Ganesan, 2004). This approach was furthered strengthened with the development of high-throughput screening technologies. The ability to test a large number of chemical entities at the same time in a certain assay required a larger and faster supply of compound libraries, which was fulfilled by combinatorial chemistry that appeared most promising for drug discovery. However, this approach proved not to be the right one for our chiral world, and led to a 20-year low in the number of new chemical entities (Class, 2002). Natural products are often structurally complex compounds that possess a welldefined spatial orientation. This type of molecules evolved to interact efficiently with their biological targets, representing a validated starting point for drug discovery. In addition, the marine environment presents harsh growth conditions, which trigger novel techniques and biochemical pathways. Almost all of the current natural product-derived therapeutics have terrestrial origins, but mining the marine environment will surely deliver new chemical and biological novelties. The great diversity and richness of marine natural compounds have been a strong desire for researchers and pharmaceutical companies who are fully aware of the enormous difficulties presented by this vast medium. The oceans cover almost 70% of the earth’s surface and 34 of the 36 phyla of life inhabit marine environments. Among this vast collection of life forms in the ocean, such different species as bryozoans, tunicates, soft corals, sponges, macro and microalgae and cyanobacterias are interesting sources for new natural compounds with therapeutic potential. There is scientific evidence showing that marine natural products are superior to the terrestrial ones in terms of chemical novelty. The analysis comparing molecular scaffolds reported in the Dictionary of Natural Products to those in the Dictionary of Marine Natural Products showed that approximately 71% of the molecular scaffolds in the Dictionary of Marine Natural Products were exclusively utilized by marine organisms (Kong et al., 2010). Marine organisms also show higher incidence of significant bioactivity compared to those of terrestrial origin. For example, a pre-clinical screen conducted by the National Cancer Institute showed that approximately 1% of the tested marine samples showed anti-tumour potential, versus 0.1% of the tested terrestrial samples (Munro et al., 1999). Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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In recent years, more than 1000 new marine compounds were reported, and the number is increasing each year (Blunt et al., 2012). The estimate of known marine species was recently amplified from approximately 230 000 to 250 000 after completion of the first Census of Marine Life (2000-2010), with focus on waters closer to the coastline in the explored countries. Extrapolation of these results calculates at least a million marine species, and tens to hundreds of millions of microbial species. However, despite this big diversity, only a few marine compounds have been able to reach the pharmaceutical market, with a large gap between them. It was in 1969 when the US Food and Drug Administration (FDA) approved the first drug of marine origin, Ara-C (Cytarabine), an anti-leukaemia agent originated from the isolation of spongothymidine from the marine sponge Tethya crypta. This was followed by the approval of the antiviral agent Ara-A (Vidarabine), after which a huge gap of 34 years occurred until ziconotide (Prialt) was approved as a chronic pain treatment (Klotz, 2006; Newman et al., 2009) (Figure 8.1). Nowadays, there are seven FDA-approved drugs (four anticancer, one antiviral, one pain control and one for hypertriglyceridaemia treatment). Among these, there are three that are sponge-derived, one from cone snail, one from fish, one from tunicate and a last one isolated from marine cyanobacteria (Table 8.1). These successful hits have had to undertake the reverses which have strongly influenced the marine pharmacology pipeline development (Figure 8.2). Glaser et al. gathered the opinions of experts in the field of marine discovery and emphasized as common concerns eight points which summarized why, since the beginning of the marine search, and with such a great diversity of molecules discovered in our oceans, so few drugs have reached the pharmaceutical market. Among these considerations are: 1 the isolation of compounds and elucidation of their complicated chemical structures; 2 the study of their precise mechanism of action, and how to fit these investigation into the high throughput screenings (HTS) used by big pharmaceutical companies; 3 the large quantities of compound needed to reach the clinical phases and 4 the need to improve the whole process from a natural compound to a synthetic drug to fulfil the pharmaceutics requirements (Glaser & Mayer, 2009). Some of these questions have been partially addressed, as the acceptance of marine microorganisms (bacteria, fungi, dinoflagellates, etc.) as the real producers of several of these marine compounds isolated from molluscs, sponge, soft corals and so on that are, indeed, secondary metabolites. The standardization of culture methods for these microorganisms can allow the production of the necessary quantities to perform the whole process of drug development. Moreover, as the experts state, the cooperation between academic labs, small biotechnology companies and larger pharmacological companies is essential to improve the marine compounds pipeline, in which the appearance of new biotechnology companies born from academic sources is having a key role. Several factors account for the uniqueness of marine natural products as sources of pharmaceuticals. Among these are: • that they are valid starting points to drug discovery, since they occupy biologically relevant chemical space; • as previously mentioned, the marine environment occupies over 70% of the earth’s surface and is characterized by unique growth conditions, with massive biodiversity; • the increasing need of novel therapeutics to treat what today are considered incurable diseases, as well as emerging microbial resistance; • their chemodiversity provides a wider opportunity for discovering new therapeutic agents with novel mechanisms of action.

Therapeutics of marine toxins

H

H

NH2

HO OH

N N

HO O

HO

H N

N N

H

H

N

Vidarabine (Vira-A®)

Cytarabine (Cytosar®)

O H N H

N H

O

H

H N

N H N

S

O

H

S

H N H H

N H O O

N

O

O

O

CH2

O

H

H N

H N

H3C

OH

N H

H

N

H

OH

N

H2C

H

Eribulin Mesylate (Halaven®)

H O

N H O H3C

O O

CH3

OH O

O

O

O H N N H

H

O H

S

H H N H O O S

O

O

S

O

OH

Ziconotide (Prialt®)

H 3C O

S

OH

H

N

OH O

H2N

N H O

S H

H H N

O

O H

O

CH3

O O

N H

N H

O

HO H N O

H N H N

H3C

N H H N

O

N

H

OH

O

HO

N

O

H H3C N H N

H3C H S H C N 3

O

O

N

O

HO

H

N H

N H

N H O

H N

183

O H3C

HO H3C

NH

O

O

O

O O

O

S

H

N O

O

cAC10

O S

N O

N

O N H

H N

O

O N H

O

CH3 H

O

O

CH3

H

H3C H 3C

H3C

CH3

HO

N Me O

H N

O N Me OMe O

H N

N OMe O

NH H2N

O

OH

CH3

O

Omega-3-acid ethylester (Lovaza®) Figure 8.1

Trabectedin (Yondelis®)

Brentuximab vedotin (SGN-35) (Adcetris®)

Chemical structures of FDA approved drugs with marine origin up to October 2013.

OH

H

Cytarabine (Ara-C) Vidarabine (Ara-A) Ziconotide Eribulin Mesylate (E7389) Omega-3-acid ethyl esters

FDA-approved

Plitidepsin Tetrodotoxin

DMXBA (GTS-21[1]) PM00104 PM01183 CDX-011

Phase III

Phase II

Trabectedin (ET-743) (EU registered-only) Brentuximab vedotin (SGN-35)

Compound name

Clinical Status

Alkaloid Alkaloid Alkaloid Antibody drug conjugate (MM auristatin E)

Worm Mollusc Tunicate Mollusc/ Cyano-bacteria

Depsipeptide Guanidinium alkaloid

Antibody drug conjugate (MM auristatin E)

Mollusc/ Cyano-bacteria

Tunicate Puffer fish

Alkaloid

Peptide Macrolide Omega-3 fatty acids

Cone snail Sponge Fish Tunicate

Nucleoside Nucleoside

Chemical class

Sponge Sponge

Marine organism

α7 nicotinic acetylcholine receptor DNA-binding Minor groove of DNA Glycoprotein NMB & microtubules

Rac1 & JNK activation Sodium channel

CD30 & microtubules

N-Type Ca channel Microtubules Triglyceride-synthesizing enzymes Minor groove of DNA

DNA polymerase Viral DNA polymerase

Molecular target

Cancer Cancer Cancer

Schizophrenia

Cancer Pain

Cancer

Cancer

Pain Cancer Hypertri-glyceridemia

Cancer Antiviral

Disease area

Zalypsis® NA NA

NA

Aplidin® Tectin®

Adcetris®

Yondelis®

Prialt® Halaven® Lorvaza®

Cytosar-U® Vira-A®

Trademark

List of compounds approved by FDA or in clinical phases with marine origin up to October 2013. Source: Adapted from Gerwick and Moore 2012; Mayer et al., 2010; http://clinicaltrials.gov/ct2/show/NCT00725114.

Table 8.1

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ASG-5ME

Marizomib (Salinospor-amide A; NPI-0052) PM060184 Bryostatin SGN-75

Gerwick & Moore, 2012. et al., 2010.

† Mayer

∗

Phase I

Polyketide Macrolide Antibody drug conjugate (MM auristatin F) Antibody drug conjugate (MM auristatin E)

Sponge Bryozoan Mollusc/ Cyano-bacteria Mollusc/

Beta-lactone-gamma lactam

Bacterium

ASG-5 & microtubules

Minor groove of DNA Protein kinase C CD70 & microtubules

20S proteasome

Cancer

Cancer Cancer Cancer

Cancer

NA

NA NA NA

NA

Therapeutics of marine toxins 185

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Marine Sampling ∗ Extracts Preparation ∗

Speed up processes

High-troughput Screening Positive hits

∗ Chemical Structure Elucidation and Synthesis

ADME

Lead Molecules

Clinical trials

Pharmaceutical Industry Figure 8.2

General procedure of new drug-obtaining protocol from the drugs’ natural origin to pharmaceutical companies and the needed of improve strategic steps.

Also, innovations in several fields, such as sampling techniques, nanomole structure determination, as well as genome sequencing and mining, have increased the efficiency of exploring marine samples for novel therapeutics. Finally, techniques are evolving to tackle the supply problem; these include total chemical synthesis, microbial fermentation, and molecular biology tools.

Marine toxins as a source of therapeutic compounds Natural toxins produced by plants, fungi, marine organism or animals, mainly with defensive purposes, have led directly or indirectly to the development of new drugs, either through new therapeutic compounds or through important tools for pharmacology researches (Kapoor, 2010). Among these natural toxins there is a large group of marine toxins, which includes different and chemically complex molecules produced by algae, cyanobacteria, bacteria or sponges among others, enclosed as secondary metabolites (Proksch et al., 2002). The first investigations around marine toxins were, and are, a public health matter, and therefore focused in the development of new and fast detection methods and in the study of the mechanism of action of these toxins. However, it has been the deeper understanding about their operation, and the availability of commercial certified standards, that has made us realize that some of these marine toxins hit

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important targets involved in different diseases such as cancer, diabetes, pain disorders or neurodegenerative diseases.

Present marine toxins and derived compound uses Conotoxins, with the synthetic MVII conotoxin ziconotide as a banner, are one of the most investigated groups and the only one that produced a clinical drug. This group of molecules, isolated from marine cone snails from the genus Conus, are currently under research in various areas as chronic pain treatment, epilepsy, cardiovascular diseases, and psychiatric and movement disorders. The genus Conus involves more than 500 different species that produce a potent cocktail of peptide toxins. These venom peptides are used to immobilize prey due to their specific and potent inhibition of voltageand ligand-gated channels in the nervous system. The great structural conservation of these channels across higher eukaryotes also allows conotoxins to act in mammalian channels (Livett et al., 2004). The 25-amino acid peptide ziconotide (Prialt®) is the synthetic ω-conotoxin MVIIA, isolated from Conus magus (Olivera et al., 1987). Ziconotide was approved by FDA and EMEA (European Medicines Agency) for the treatment of severe chronic pain in patients for whom intrathecal therapy is needed, and who are intolerant to other alternatives such as systemic analgesics or morphine. Ziconotide blocks N-type calcium channels in a reversible way. This blockage of pre-synaptic N-type calcium channels elicits a reduction in the release of neurotransmitters (McGivern, 2006). However, the variety of targets of these compounds means that different pharmacological targets selectively affected by conotoxins are under investigation for their therapeutical potential. α-, αA- and Ψ conotoxins target the nicotinic acetylcholine ligand-gated ion channel receptor, whereas ω-conotoxins selectively target voltage-gated ion channels such as the N-type voltage-gated calcium channel. Other conotoxins bind to sodium and potassium voltage-gated channels, μ-, μO- and δact through sodium channels and K-, KA-, KM and KViTx act through potassium channels (Livett et al., 2004). Other examples of conopeptides are conantokins and contulakins. Conantokin G, for example, isolated from Conus geographus, is a N-methyl-D-aspartate receptor antagonist which have showed effect over epileptic seizures (Jimenez et al., 2002), whereas Contulakin G, isolated from the same Conus species, has been screened as a novel therapeutic agent for pain treatment as a neurotensin subtype 1 receptor agonist (Allen et al., 2007). This vast variety of cellular targets makes the conotoxins family a large group of compounds with high therapeutical potential that is still under research. Some marine toxins are not a source of new therapeutic drugs but, instead, they have been, and currently are, important pharmacological tools widely used in biochemical investigations. Tetrodotoxin (TTX) and saxitoxin (STX) are probably the two most widely used tools in electrophysiological researches. TTX binds to receptor site 1 on sodium channels, with the consequent pore blocking (Hille, 1975). This marine biotoxin has been used to identify and classify the voltage-dependent sodium channel (Nav ) subtypes with respect to their TTX sensitivity (Catterall et al., 2005). Consequently, sodium channels are classified in TTX-resistant subtypes, the cardiac subunit Nav 1.5 and Nav 1.8 and Nav 1.9 (implicated in neuropathic pain states (Wood et al., 2004)) and TTX-sensitive subtypes, Nav 1.7, 1.3, 1.2 and 1.1, which are implicated in inflammation, epilepsy or neuropathic pain.

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Several currently used drugs bind to the α-subunit of Nav , such as anti-epileptics, anti-arrhythmics and local anaesthetics (Ragsdale et al., 1996). Moreover, mutation in the genes encoding voltage-gated sodium channels (known as channelopathies) are directly related to diseases in heart, muscle, brain and peripheral nerves (Amin et al., 2010; Kullmann & Waxman, 2010; Shi et al., 2009). Okadaic acid is another example of a marine toxin widely used as a pharmacological or biochemical tool. This diarrheic shellfish toxin has been an important tool for the study of cellular signalling. This compound is a potent and selective inhibitor of protein phosphatase type I and type IIa (Bialojan & Takai, 1988), a property that turn it into a reference tool in differential studies of these proteins. Therefore, okadaic acid is the best known and more studied phosphatase inhibitor, and it has had a key role in the research into the implication of this group of proteins in cellular signalling.

Future of marine toxins and derived compounds uses The marine compounds isolated since 1998 with anti-tumour, antibacterial, antidiabetic, antifungal, anti-inflammatory, antiprotozoal, antituberculosis or antiviral properties, or affecting the immune system or the nervous system, have been thoroughly reviewed (Mayer et al., 2007, 2009, 2011, 2013; Mayer & Gustafson, 2003, 2004, 2006, 2008; Mayer & Hamann, 2002, 2004, 2005; Mayer & Lehmann, 2001). These manuscripts, together with others, make clear the potential and variety of marine compounds. Many of these compounds, besides the mentioned therapeutic application derived from their chemical novelty, also have novel targets and mechanisms of action. Examples of these types of molecules among marine toxins are described in this section. Acetylcholine receptors (AchRs) have been described as the target of several marine and freshwater toxins as cyclic imines, anatoxin-a, anabaseine and α-conotoxins, among others. Cyclic imines, toxins such as 13-desmethyl spirolide-C and gymnodimine, in vitro and in vivo, act at the nicotinic receptor level (Gill et al., 2003; Wandscheer et al., 2010). These toxins, produced by dinoflagellates from the genus Alexandrium ostenfeldii and peruvianum and Karenia selliformis respectively, have a characteristic cyclic imine moiety in their macrocyclic chemical structure (Cembella et al., 1999; Miles et al., 2003). These two groups of toxins have showed a strong affinity for the nicotinic α7 and α2β4 receptors and for the muscarinic neuronal receptors in binding assays and antagonism assays (Hauser et al., 2012), pointing to cholinergic receptors as their main cellular target. A freshwater toxin that also targets the acetylcholine receptors is anatoxin-a, a potent neurotoxin produced by Anabaena spp cyanobacteria. This alkaloid is a strong agonist of nicotinic acetylcholine receptors (Sanchez et al., 2014). Anatoxin-a binds with higher affinity than nicotine to rat brain [3 H]-nicotine and [3 H]-α-Bungarotoxin sites. However, new synthetic analogues with lower toxicity are been researched for potential studies in pathologies related with these receptors (Simoni et al., 2011). Nicotinic receptors (nAchRs) are widely distributed in the central nervous system. These ligand-gated ion channels consist of five subunits that form a central cation-permeant channel that opens following the binding of the neurotransmitter acetylcholine. The three most abundant nAchRs in the brain are the α7, α4β2 and α3β4. Vulnerable neurons in neurodegenerative disorders have shown high levels of nAchRs, specifically those with the α7 subunit. Moreover, a loss of nAchRs has

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been observed in Alzheimer’s disease post-mortem brains, a disorder characterized by an important cholinergic deficit. Several observations suggest that modulation of nAchRs acts in the amyloidogenic pathway involved in this neurodegenerative disorder through amyloid precursor protein processing, which leads to the formation of the pathological Amyloid β (Aβ). The cholinergic hypothesis has been one of the most investigated in the last two decades in studies of Alzheimer’s disease. This hypothesis states that Aβ acts on, or interferes with nAchRs, thus affecting the synaptic function which leads to the cholinergic neurons’ death. Different nicotinic agonist and antagonist have proved to have positive effects over this route, pointing not to the activation or inactivation of the receptor but to the binding and, therefore, a particular conformation or a desensitization of the receptor, which leads to a positive regulation of the Aβ formation (Buckingham et al., 2009; Mousavi & Hellstrom-Lindahl, 2009). 13-desmethyl spirolide-C and gymnodimine, which have proved their affinity for nAchRs, have showed beneficial effects in an in vitro model of Alzheimer’s disease. In these studies, both toxins elicited a decrease of tau hyperphosphorylation and Aβ production in primary cortical neurons of triple transgenic mice (3xTg-AD), with the involvement of kinases that take part in the phosphorylation of the tau protein, such as GSK-3β and ERK1/2. Moreover, both compounds also produced neuroprotection against glutamate-induced neurotoxicity in the same neuronal model. It has been described that Aβ accumulation can increase the sensitivity of neurons to glutamate toxicity in these diseases (Crews & Masliah, 2010). The same animal model, 3xtg-AD, was used to corroborate the effect of spirolide in vivo. In this study it was found that 13-desmethyl Spirolide-C crosses the brain-blood barrier and reaches the brain in only two minutes after intraperitoneal injection, remaining even after 24 hours. This compound has been able to diminish neurodegeneration markers observed by nuclear magnetic resonance in vivo in 3xTg-AD mice, and to decrease the specific markers of Alzheimer’s disease as it previously did in vitro (Alonso et al., 2011a, 2011b, 2013a). These results lead to patent gymnodimine and spirolides against neurodegenerative disorders with the implication of Aβ and Tau protein (Botana et al., 2010). Another example of marine toxins that target these receptors is the previously mentioned α-conotoxins. The α-Conotoxin PelA[S9H,V10A,E14N] selectively inhibits the α6β2β3 nAchRs, widely expressed in dopaminergic neurons and related to Parkinson’s disease and nicotine dependence (Hone et al., 2012). Following with nAChRs-related compounds, we have Anabeseine. This alkaloid was first detected in Amphihorus lactifloreus (Kem et al., 1976). Later, an analogue, 3-(2,4-dimethoxybenzylidene)-anabaseine (DMBXA), was synthesized for its high antagonism over α7 nAchRs. Several trials in mammals with rats, rabbits and monkeys proved its effect on improvement of cognition and learning. Currently, it is under clinical study at the Taiho Pharmaceutical Company for Alzheimer’s disease treatment (Zawieja et al., 2012). Marine compounds that modulate voltage-gated channels are also important milestones for pharmaceutical pipelines. Potassium and sodium voltage-gated channels (Kv and Nav ) have important considerations and potential uses in several human diseases. Mutations in voltage-gated sodium channels are related to several diseases, such as epilepsy, pain-related syndromes (congenital insensitivity to pain, primary eythromelalgia and paroxysmal extreme pain disorder) and cardiac arrhythmias (as long QT syndrome, Brugada syndrome, progressive cardiac conduction defect and atrial fibrillation, among others). Moreover, an up-regulation of these channels has been associated

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with aggressive metastatic carcinoma of prostate and breast (Savio-Galimberti et al., 2012). Therefore, marine toxins that modulate Nav could be used as antiepileptic, analgesics and antiarrhythmic drugs. As we have previously mentioned, several marine toxins have Nav channels as their cellular target. TTX is under study for pain treatment. Some of these studies have reached clinical trial, but further investigations are needed (Nieto et al., 2012). In the case of conotoxins, the great subtype selectivity of this group of compounds makes them interesting leads in the search of new analgesics. There are four conotoxins families that specifically bind to Nav channels sites with different effects: μ-, μO-, δ- and í-conotoxins. μ- and μO-conotoxins inhibit VGSCs, whereas δ- and í-conotoxins activate the channel (Knapp et al., 2012). Brevetoxins have been also classified as neurotoxins whose main effect is the activation of Nav channels. Brevetoxins bind to Nav receptor site 5 and produce its activation (Lombet et al., 1987). This effect is the responsible of the study of brevetoxins with pulmonary-related diseases such as cystic fibrosis or asthma (Baden, 2005). This group of neurotoxins has also been patented for the possible treatment of neurodegenerative diseases, due to the enhancement of neurite growth that they can produce (Taupin, 2009). Another ion channel targeted by the pharmaceutical industry is the potassium channels, which are also involved in different diseases of the nervous and immune system. Several K+ modulators are already used for the treatment of type 2 diabetes and arrhythmia, but they are also under study due to their potential use in neuronal disorders (epilepsy, memory disorders, chronic pain and brain ischemia), or as immunosuppressors (Wulff & Zhorov, 2008). These channels are formed by four individual α subunits arranged around a central pore, as a homo-or heterotetramer. In both excitable and non-excitable cells, K+ channels play a key role in important cellular processes as Ca2+ signalling, cellular volume regulation, cellular proliferation and migration and secretion. Wulff and co-workers reviewed the role of K+ modulators in autoimmune and neurological disorders, with several examples of specific K+ channels and their implications in these diseases (Wulff & Zhorov, 2008). In this sense, the widely used K+ blockers, tetraethylammonium (TEA) or 4-Aminopyridine (4-AP) have proved to have beneficial effects in staurosporine or Aβ treatments in vitro, effects that can be potentially useful for the treatment of neurodegenerative disorders as Alzheimer’s disease (Yu et al., 1998). Gambierol, a marine polycyclic ether produced by the dinoflagellate Gambierdiscus toxicus, previously showed that is a potent modulator of K+ channels in different cellular models as cortical and cerebellar neurons and transfected Xenopus oocytes, where it produced a potent blockade of these channels (Cuypers et al., 2008; Perez et al., 2012; Alonso et al., 2012). It has been recently described that this ladder-shaped toxin binds with high affinity to the resting state of the channel and, moreover, gambierol immobilizes the gating machinery in the closed state. This state can be only reverted by strong depolarization, which releases the toxin from its binding site. However, the required depolarizations are higher than the physiological ones (Kopljar et al., 2013). Taking advantage of the effect over K+ channels, the therapeutic potential over neurodegenerative diseases was studied, and the compound showed beneficial effects in a triple transgenic mouse model of Alzheimer’s disease, modulating the two main hallmarks of this neurodegenerative disorder, tau and Aβ. Moreover, this marine compound showed a decrease in the Kv 3.1 upregulation observed in this

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cellular model (Alonso et al., 2012). These data suggest that Gambierol could be an interesting compound in autoimmune processes, where Kv 1,3 has been proposed as a therapeutic target, as in rheumatoid arthritis or type 1 diabetes mellitus (Beeton et al., 2006). The full synthesis of this marine compound allows the search and creation of related structures with lower toxicity and with the same effects as Gambierol (Alonso et al., 2012; Fuwa et al., 2002, 2004), as well as guaranteeing a source of synthetic compound. Another example of an interesting marine compound with a novel target and mechanism of action is Apratoxin A, isolated from a marine cyanobacterium. Apratoxin A is a potent cytotoxin (Luesch et al., 2001) that reversibly inhibits the secretory pathway for several cancer-associated receptors by interfering with cotranslational translocation (Liu et al., 2009). This was the first reported compound to act by this mechanism. Yessotoxin (YTX), a polycyclic ether compound produced by dinoflagellates (Draisci et al., 1999; Satake et al., 1997), has shown to be quite pleiotropic when it comes to the effects produced on animal and human cells. Since research on this molecule started, it has been shown to: • increase cytosolic calcium in human lymphocytes activating phosphodiesterase (Alfonso et al., 2003; de la Rosa et al., 2001; Perez-Gomez et al., 2006); • disrupt cell adhesion at low concentrations of MCF-7 human breast cancer cells (Pierotti et al., 2003); • inhibit cell proliferation of epithelial cells (Ronzitti et al., 2004); • affect the mitochondrial permeability transition pore in hepatoma cells (Bianchi et al., 2004); • induce apoptosis with mitochondria involvement in L6 and BC3H1 skeletal muscle myoblasts (Korsnes et al., 2006); • induce apoptosis in several cell types (Korsnes & Espenes, 2011); • induce paraptosis-like cell death in BC3H1 (Korsnes et al., 2011); • ameliorate Tau and β-amyloid pathology (Alonso et al., 2013b); and • induce endoplasmic reticulum stress (ER-stress), altering lipid metabolism, with the consequent activation of the unfolded protein response and, finally, autophagic cell death in the SF295, SF539 and SNB75 glioma cell lines (Rubiolo et al., 2014b). Due to the effects observed for YTX, it has been postulated as a potential drug candidate or lead molecule. YTX, which has been shown to induce apoptosis in several tumour cell lines, was patented as a tumour cell growth inhibitor. The authors point to a mechanism of action through an increase in phosphodiesterases activity. In this patent, it is postulated that since cAMP is related to cytotoxicity and resistance to drugs, the increase in phosphodiesterase activity could be a good tool for the treatment of cancer (Alfonso et al., 2003; Botana et al., 2011b). In the same direction, YTX has been patented for the treatment of glioma. This patent points to the induction of autophagy as an important mechanism for tumour cell death induction when the apoptosis-inducing pathways are not functional in this cell type. In glioma, the canonic apoptotic pathways are inactivated, with consequent resistance to quimiotherapeutic drugs and radiation. It is known that autophagy promotes differentiation and radio-sensitization of glioma-initiating cells, implying that YTX could also be used as a co-treatment with known therapeutic approaches (Botana & Rubiolo, 2012a; Rubiolo et al., 2014b).

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Transcriptomic data from this cell type has been also used to protect the use of YTX for the treatment and/or prevention of metabolic diseases. YTX increases the expression of the insulin response sustrate-2 (IRS-2), which plays a crucial role in the insulin sensitivity in the liver and skeletal muscle. As a consequence, YTX induces the expression of enzymes involved in the synthesis of fatty acid synthase (a key enzyme in the synthesis of lipids) and acelil-CoA carboxilasa alpha (which provides malonyl-CoA substrate for the biosynthesis of fatty acids). It also increases the expression of glycolitic enzymes hexokinases 1 and 2, which phosphorylate hexoses, and fructose-2, 6-bisphosphatase 4, which regulates the steady-state concentration of fructose 2,6-bisphosphate, an activator of a key regulatory enzyme of glycolysis phosphofructokinase. YTX represses the expression of gluconeogenic enzymes phosphoenolpyruvate carboxykinase-2, which catalyzes the rate-controlling step of gluconeogenesis, and of glucose-6-phosphatase, which catalyzes the hydrolysis of glucose 6-phosphate to glucose and phosphate in the last step of the gluconeogenic and glycogenolytic pathways. This response is what is normally observed in responsive cells when exposed to insulin, but is negatively affected in people with resistance, in which case YTX could be useful. Besides the mentioned genes, YTX also induces the expression of glucose transporters (SLC2A13, SLC2A14, SLC2A3), whose down-regulation is one of the causes of hyperglycaemia in the case of metabolic syndrome (Botana et al., 2012b). Another patent exists for the use of this molecule in the treatment of allergic and asthmatic processes. This claim also relies on the activation of phosphodiesterase by YTX. Since immunological activation of mastocytes and basophils requires a temporary increase in cAMP, which is indispensable, phosphodiesterase activation could cancel this cAMP peak, inhibiting cell activation. This effect could be used as an anti-allergic and anti-asthmatic therapeutic strategy, considering that mast cells play a predominant role in both (Botana et al., 2011a). Finally, YTX and its analogues have also been patented for the treatment and/or prevention of neurodegenerative diseases linked to tau and beta-amyloid. These claims are supported by the fact that YTX prevents the increase in beta-amyloid and tau phosphorylation in cultures of transgenic mice that over-express these proteins and are a model for Alzheimer’s disease (Botana et al., 2011c). The information presented in this section highlights the new interest on marine compounds as leads for drug development. More molecules of this type are expected to be approved for clinical use in the coming years. This will be further boosted by continuous improvements in the sampling, structure determination, target identification, production, gene sequencing and genome mining methods. The next section discusses the bottlenecks and breakthroughs in these methods.

Problems and advancements in drug discovery from the seas Sampling techniques One of the main reasons preventing ocean exploration is that sampling requires more specific techniques and equipment. While numerous compounds have been reported from near-shore areas, which are easy to access, hard-to-access regions remain to be

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intensively explored. An example of the potential of hard-to-access regions comes from deep sediments research that, after obtaining samples from the sea bottom at depths greater than 2000 m, allowed the discovery of novel actinomycete strains. One of these (Salinispora tropica) was the source of the potent proteosome inhibitor salinosporamide A (Fenical & Jensen, 2006; Fenical et al., 2009).

Structure determination Until few years ago, an NMR molecular study required more than a micromole of a selected compound to completely elucidate its structure. Recent advances in NMR, however, show that structure determination is now possible starting with minor amounts of natural products, at the nanomole (Molinski, 2010), or even picomole (Fellenberg et al., 2010) scale. Added to this is the recent introduction of atomic force microscopy, and enhanced imaging techniques for organic structure determination, that produce clear molecule images that can aid in the determination of the correct structure of unknown organic molecules (Gross et al., 2010). While this method is now restricted to planar structures, advances are expected that will eliminate this limitation.

Target identification While phenotypic screening gained academic as well as industrial attention to discover drug leads that modulate biological pathways, the identification of their cellular targets remains a bottleneck for their development into drugs. The mechanistic knowledge is crucial to anticipate potential side-effects and to avoid costly clinical failures, to allow lead optimization and to provide biomarkers for preclinical and clinical trials. Several developments are now available that allow or increase the chances of target identification. Target identification approaches can be divided in direct and indirect methods. Direct methods include affinity chromatography, expression cloning and protein microarrays. Indirect approaches include global profiling techniques based on genomics, proteomics or metabolomics. The combination of these techniques allow the identification of the mechanism of action of compounds of marine origin. For example, the mechanism of action of the antifungals Theonellamides A–F was determined through chemical-genomic profiling and fluorescent labelling (Nishimura et al., 2010). Apratoxin A’s mechanism of action was studied employing functional genomics, followed by biochemical and proteomic approaches (Luesch et al., 2006; Shen et al., 2009). Transcriptomic, biochemical, and molecular biology tools provided insights in the cytotoxic mechanism of action of yessotoxin and crambescidines on tumour cells (Rubiolo et al., 2014a, 2014c).

The supply issue This is one of the major challenges at the time of development of marine natural products into drugs. Some solutions have emerged that provide relief to this problem. Synthetic chemistry, when possible, ensures a large-scale supply of the bioactive compound. It also allows lead optimization, which can be enhanced if the mechanism of action and target are known. Knowledge of the mechanism of action and target permits the identification of simplified analogues, more easily produced by synthesis, with concomitant cost reduction. An example of this is the synthesis of gambierol’s tetracyclic and heptacyclic analogues.

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Marine polycyclic ether natural compounds have been a captivating question for chemists, due to their complex and diverse molecular structure. The structure characterization and isolation of gambierol was fundamental to overcome the scarcity of the natural compound, which hampered the investigation into its mechanism of action. The total syntheses of gambierol and, thus, the availability of higher quantities of synthetic compound, not only allowed investigation about its in vitro and in vivo effects, it also permitted structure-activity relationship studies with synthetic analogues about the long-questioned statement of whether the complete ladder-shaped polycyclic ether skeleton was imperative for its activity. Finally, the construction of two analogues, a heptacyclic and a tetracyclic one, clarified that the biological activity of gambierol resides in the right domain of the gambierol skeleton. Additionally, the synthesis of stable analogues opens the possibility of searching new and less toxic compounds with the same biological effects of gambierol (Alonso et al., 2012; Furuta et al., 2009, 2010; Fuwa et al., 2002, 2003, 2004). However, due to the complexity of most of the marine-derived compounds, chemical synthesis is sometimes not possible, or is economically prohibitive for drug development. Fermentation represents another potential solution to the supply issue. Despite most marine microbes being hard to cultivate in the laboratory, some strains have already produced significant bioactive compounds that are on the market or in clinical trials. Salinosporamide A production, for the ongoing clinical trials, relies on large-scale saline fermentation for continuous drug supply. Trabectedin production employs initial large-scale fermentation to obtain the starting cyanosafracin B from Pseudomonas fluorescents, which is then modified by semi-synthetic steps to obtain the final drug (Cuevas et al., 2000). Marine microbial studies have shown that the expression of genes involved in secondary bioactive metabolite synthesis depends on the environmental conditions, and can become silent under unnatural laboratory conditions. Mixed fermentation has shown to be effective in activating this type of gene clusters (Pettit, 2009). Examples of this are the production of libertellenones A–D (Oh et al., 2005), marinamides A and B (Zhu & Lin, 2006), pyocyanin (Angell et al., 2006), and emericellamides (Oh et al., 2007).

Biotechnology As the cost of genome sequencing decreases, mining for the genes responsible for the synthesis of already known compounds is within reach. Also, genome mining has allowed the putative chemical structures of previously unobserved natural products to be predicted (Lane & Moore, 2011; Challis, 2008). Genome sequencing has made possible the characterization of several biosynthetic gene clusters and pathways, ultimately allowing for their manipulation (Lane & Moore, 2011). The identification of these gene clusters has prompted the generation of microbial chemical factories, which are now being studied for the more easy and efficient biosynthesis of fully synthesized compounds. Two approaches are now emerging for drug development and optimization. One involves biosynthetic genetic engineering of organisms for the production of the desired compound (Lane & Moore, 2011; Kittendorf & Sherman, 2006), while the other uses an in vitro multi-enzyme synthesis with the same objective (Roessner & Scott, 1996; Cheng et al., 2007). An example of the potential of genome sequencing in the identification of genes involved in secondary metabolites synthesis comes from the first fully sequenced marine actinomycete, S. tropica. The results revealed a complex secondary metabolome,

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with approximately 10% of the genome dedicated to natural product assembly. A remarkably high diversity of polyketide synthases (PKS), and nonribosomal peptide synthetases (NRPSs), with high numbers of biosynthetic pathways, was also observed. This pointed to the existence of unknown compounds that could be synthesized by S. tropica in addition to the known salinosporamides, sporolides, and lymphostin (Udwary et al., 2007). Heterologous expression of microbial natural products’ biosynthetic pathways, together with advanced DNA engineering, enables optimization of product yields, functional elucidation of cryptic gene clusters and generation of novel derivatives. This approach is based on the expression of partial or complete biosynthetic gene clusters, for example PKS and NRPS gene clusters, in heterologous hosts, to obtain complex novel compounds or known compounds out of reach for chemical synthesis (Ongley et al., 2013). Another possibility is chemienzymatic chemistry, which has already proved its efficiency in total synthesis of bioactive natural products (Mortison & Sherman, 2010). In vitro multi-enzyme synthesis has also proved to be a powerful alternative to conventional chemical synthesis and metabolic engineering (Roessner & Scott, 1996). For example, the assembling of the first complete in vitro type II PKS enzymatic pathway that totally synthesized enterocin polyketide was reported in 2007 (Cheng et al., 2007).

Conclusions Advances in technologies such as nano-scale NMR, sampling strategies, chemical synthesis, biosynthesis, and genetic engineering will determine the success of marine natural products as drug leads. These innovations, and others to come, provide grounds for optimism for marine drug discovery and development.

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CHAPTER 9

Marine toxins as modulators of apoptosis Amparo Alfonso1 , Andrea Fernández-Araujo1 & Mercedes R. Vieytes2 1 Department 2 Department

of Pharmacology, University of Santiago de Compostela, Spain of Physiology, University of Santiago de Compostela, Spain

Introduction Apoptosis, or type I programmed cell death (PCD), is a complex process that includes many biochemical pathways. The term was first adopted by Kerr and co-workers in 1972 to describe a common type of cell death, repeatedly observed in several tissues, that could be activated or inhibited by a variety of environmental physiological or pathological stimuli (Kerr et al., 1972). Since them, many different signaling pathways have been related with apoptosis being the caspases family mostly the central executioner of the apoptotic pathway (Hengartner, 2000). However, before caspases activation, several pro-apoptotic or anti-apoptotic processes, implicating different pathways, are activated. Apoptotic processes play important roles in cell development, proliferation-homeostasis, differentiation, regulation of the immune system and in the elimination of harmful and defective cells (Tan et al., 2009). There are two main apoptotic pathways, the intrinsic or mitochondrial-mediated pathway, and the extrinsic or death receptor-mediated pathway. In addition, under the term PCD, autophagy (or type II PCD) and programmed necrosis (or type III PCD) can be also included (Ouyang et al., 2012; see Schemes 9.1 and 9.2).

Intrinsic apoptotic pathway The intrinsic pathway is mediated by the mitochondria. In response to some stimuli, several pro-apoptotic molecules, cytochrome c, direct inhibitor of apoptosis (IAP)-binding protein (DIABLO, also known as second mitochondria-derived activator of caspases, SMAC) or endonuclease G, are released. This release can be mediated by changes in the mitochondrial potential, due to the opening of the Permeability Transition Pore (PTP). In these conditions, swelling and fragmentation of the mitochondrial outer membrane and the release of the pro-apoptotic factors to the cytosol is observed (Javadov & Karmazyn, 2007). PTP can be opened by the inactivation or decrease of the anti-apoptotic Bcl-2 family member after leaving the outer mitochondrial membrane, where it performs its function (Carlucci et al., 2008). However, the Bcl-2 family member may also act at the endoplasmic reticulum level. In this localization, Bcl-2 keeps Ca2+ homeostasis, and any

Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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Apoptosis

Signs

*Cell shrinkage *Nuclear fragmentation *Mitochondrial dysfunction (respiratory chain inhibition, loss of inner mitochond membrane potential, increased mitochond membrane permeability) *Plasma membrane blebbing *Apoptotic bodies and phagocyte removal attracted by PS (phosphatidylserine)

Triggered by Earliest steps

*Formation of multi protein complexes, DISC (death-inducing signaling complex) and apoptosome, that *Recruit and activate caspases 8 (extrinsic) or 9 (intrinsic), that converge in activation of the executor caspases 3, 6 and 7 *Beclin 1 interacts with antiapoptotic Bcl-2 and inhibits autophagy * BNIP3 is a BH3-only protein that can trigger apoptosis by sequestering antiapoptotic

Caspase activation

Role Caspase 8

*Intrinsic stimuli: mitochondrial release of cytochrome C by cell stress and regulation by Bcl-2 family proteins (Bcl-2 are antiapoptotic) *Extrinsic stimuli: activation of cell death receptors (e.g. Fas, TNFR (tumor necrosis factor receptor))

Bcl-2 family proteins and promoting Bax/Bad dependent mitochondrial release of proapoptotic mediators * Once activated, apoptosis effector molecules may suppress autophagy; i.e., Beclin 1 may be cleaved and inactivated by caspases during activation of apoptosis

Morphological and physiological cell changes by Cleavage of cellular targets: PARP (poly(ADP-ribose)polymerase) endonucleases proteases Caspases are modulators of autophagy and programmed necrosis (by regulation of signals involved in both pathways)

*Formation of death receptor complex DISC recruited by the adapter proteins FADD (Fas-associated protein with death domain) and/or *TRADD (TNFalphaR1 associated death domain protein) *Formation of ripoptosome (death receptor-independent cytosolic complex) containing kinases of RIP (receptor interacting protein), FADD and cFLIP (FLICE-like inhibitory protein) composition of ripoptosome

*Caspase-8-mediated cleavage of RIP1, 3 triggers caspase activation cascade and induces apoptosis *Differential caspase-8 regulation by cFLIP triggers apoptosis or necroptosis: expression of cFLIP binds to caspase-8 and suppresses the ability of caspase-8 to trigger apoptosis, and stimulates necroptosis *cFLIP is anti-apoptotic and it is a negative regulator of autophagy through inhibition of of ATG3 (autophagy-related gene 3), that regulates formation of authophagosomes

Scheme 9.1 Main features of apoptosis. Source: Based on data from Ryter et al., 2014; Galluzzi et al., 2012; Cerella et al., 2014; Korsnes, 2012; Korsnes et al., 2006a.

disorder of this protein could activate the Ca2+ liberation to the cytosol and the opening of PTP in the mitochondria (Javadov & Karmazyn, 2007; Korsnes & Espenes, 2011). The apoptogenic factors released from the mitochondria continue the apoptotic cascade through caspase-dependent or independent pathways, with different results. Cytocromo C binds to apoptotic protease-activating factor 1 (Apaf-1) and to caspase 9, making the apoptosome complex that needs ATP to recruit and activate procaspases 3 and subsequently activate caspases 2 and 6. Furthermore, SMAC/DIABLO, together

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Other types: Regulated cell death

Pyroptosis Necroptosis

inhibitor Autophagy

Apoptosis inducer Catastrophic cell death

Necrosis

Necrosis

inhibitor

Caused by non physiological cell injury Cell swelling Cell lysis and constituents leakage Does not exclude apoptosis (necrosis a higher doses) ATP content decline determines cell to switch to necrosis

Necroptosis *Fas-dependent by caspase independent *It is a form of regulated necrosis *Ligand binding death receptor (i.e. TNFR1 (tumor necrosis factor receptor 1)) promotes formation of necrosome, that involves RIP (receptor-interacting protein) kinase 1 and 3, that initiate necrosis

Pyroptosis *Requires caspase-1, and may require caspase-7

*Process similar to necrosis: formation of membranous pore, cell swelling, leakage of cytosolic content *May show, as in apoptosis, DNA fragmentation and nuclear condensation * It may be a particular case of caspase-dependent intrinsic apoptosis Apoptosis

see scheme on apoptosis

Autophagy

see scheme on autophagy

Scheme 9.2

Main features of cell death types. Source: Based on data from Ryter et al., 2014; Galluzzi et al., 2012; Cerella et al., 2014; Korsnes, 2012; Korsnes et al., 2006a.

with high temperature requirement protein A2 (HtrA2), triggers caspases activation by neutralization of IAP. On the other hand, apoptosis-inductor factor (AIF) and endonuclease G (EndoG) are translocated to the nucleus after release from the mitochondria to induce DNA fragmentation and chromatin condensation without caspases mediation (Fulda & Debatin, 2006; Korsnes & Espenes, 2011).

Extrinsic apoptotic pathway Extrinsic apoptotic pathway is activated after the binding of a ligand with the death receptor (DR). DR belongs to the tumour necrosis factor receptor (TNFR) family and the best characterized are Fas (also called Apo1 or CD95) and TNFR-1 or TNF-related apoptosis-inducing-ligand (TRAIL) (Korsnes & Espenes, 2011). The TRAIL receptor is

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divided into agonistic receptors (TRAIL-R1 and TRAIL-R2) and antagonistic receptors (TRAIL-R3 to TRAIL-R5) (Fulda & Debatin, 2006). After the binding of the stimuli (Fas-L, CD95L, TNFα) to the receptor, a death complex is formed. This complex recruits a death domain-containing protein as FADD or TNF-receptor associated protein, and activates pro-caspases 8 and 10 after interaction with the adaptor molecules. The whole structure that includes DR, adaptor molecules and procaspases is called the death-inducing signalling complex (DISC) (Korsnes & Espenes, 2011; Ouyang et al., 2012). After pro-caspase 8 recruitment, the active caspase 8 continues the apoptotic pathway by activating caspase 3. There are two types of caspase 8 activation and, therefore, two cell types (Galluzzi et al., 2012). In type I cells, the quantity of caspase 8 active is enough to activate caspase 3 and continue the apoptosis. In type II cells, the caspase 8 generated by DISC is insufficient to activate caspase 3 and, in this case, the mitochondrial apoptotic pathway, through the generation of a mitochondrion-permeabilizing fragment, is necessary to activate the rest of the caspases (Fulda & Debatin, 2006; Galluzzi et al., 2012). In addition to caspase 8, caspase 10 seems to have a relevant role in response to DR activation (Galluzzi et al., 2012).

Phycotoxins involved in apoptotic processes Marine toxins can be classified according to several criteria – by the symptoms they cause in humans, the most common or abundant toxin of each group, their chemical characteristics or the mechanism of action. The best classification is one that considers the common mechanism of action of chemically related structures. Hence, the groups should be: • those targeting the voltage-dependent channels: saxitoxins (STX), ciguatoxins (CTX), gambierols, brevetoxins (PbTx) (Catterall & Morrow, 1978; Wingerd et al., 2012) • phosphatase inhibitors: okadaic acid (OA) and dinophysistoxins (DTX) (Bialojan & Takai, 1988) • kainate receptor agonists: domoic acid (DA) group) (Vale-Gonzalez et al., 2006) • actin inhibitors pectenotoxins (PTX) (Allingham et al., 2007) • Na-K ATPase inhibitors: palytoxins, ovatoxins, ostreocins (Alfonso et al., 2014) • calcium pore formation: maitotoxins (de la Rosa et al., 2001b) • cholinergic receptor blockers: cyclic imines (CI) (Otero et al., 2011) • spirolides, pinnatoxins, gymnodimines, spiro-prorocentrimines, pteriatoxins, symbioimines, prorocentrolides)) and yessotoxin (YTX) and azaspiracids (AZA), which are not classifiable yet. In terms of apoptosis and cell death, some of these toxins are clearly considered as apoptotic inductors, while others are involved in cellular toxic processes without being related to apoptosis (Draisci et al., 1996; Zheng et al., 1996).

Okadaic acid (OA) OA belongs to diarrheic shellfish poisoning (DSP) and is mainly synthesized by dinoflagellates of the genus Prorocentrum and Dynophysis. This toxin is a potent inhibitor of eukaryotic serine/threonine protein phosphatases type 1 and 2A (PP1 and PP2A) (Franchini et al., 2010; Valdiglesias et al., 2013). These enzymes play a

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critical role in phosphorylation/dephosphorylation processes in eukaryotic cells and, therefore, OA has the potential to disturb the equilibrium of these processes, which are important for nearly all regulatory routes in the cell. OA was first described as an antiproliferative drug, but apoptosis activation, after toxin incubation, has been extensively described in many cellular systems (Bøe et al., 1991; Ishida et al., 1992; von Zezschwitz et al., 1997; Leira et al., 2001, 2002a; Lerga et al., 1999; Tubaro et al., 2008; Jayaraj et al., 2009). Several apoptosis hallmarks have been described after OA treatment, a summary of some known nd relevant data is next shown. The effect of OA on apoptosis activation has been observed by a decrease in the mitochondria membrane potential, the activation of multiple caspase isoforms, the release of cytochrome c, the DNA fragmentation or the production of oxidative stress species, among others (Leira et al., 2001, 2002a; Jayaraj et al., 2009; Rossini et al., 2001; Ravindran et al., 2011). All of these processes have been described in HeLa cells, indicating that caspase-dependent and caspase-independent pathways are involved in OA apoptosis activation in this cell line (Jayaraj et al., 2009). Besides apoptotic pathways, OA is also involved in the inhibition of cell proliferation (Gehringer, 2004; Valdiglesias et al., 2013). In the HL60 cell line (acute promyelocytic leukaemia cells), after 24 hours of OA treatment, a decrease in transcription levels of Bcl-2 and apoptosis was observed (Riordan et al., 1998), although a functional decrease of Bcl-2 had been previously postulated as the mitochondrial apoptosis trigger (Haldar et al., 1995). Also, OA activity through the Bcl-2 protein was pointed out in the K-562 leukaemia cells after the treatment with the toxin. In this case, OA apoptotic action was attenuated by the enforced expression of Bcl-2 protein. In these cells, however, the decrease in cell viability was due both to apoptosis activation and mitotic arrest. Therefore, mitotic arrest and apoptosis resulted as independent pathways activated by OA (Lerga et al., 1999). Furthermore, a time-dependent decrease of cyclins A and B related with p53-dependent apoptosis was observed in K-562 cells after OA incubation (Gartel & Tyner, 2002; Meikrantz & Schlegel, 1995). In caco-2, a human colonic epithelial cell line, caspase-3, caspase-9 and changes in mitochondrial membrane potential were described after OA incubation. Also, lactate dehydrogenase release was detected, indicating apoptosis and other cellular death, dose- and time-dependent, activated by OA, that led to a decrease in cellular proliferation (Lago et al., 2005). In summary, from the wide bibliography about OA apoptotic and anti-proliferation events, it seems that all them are related to the effect over PP inhibition. In fact, when this ability was modified, apoptosis was not observed (von Zezschwitz et al., 1997; Kiguchi et al., 1994). Microcystin (MC) is a toxin produced by several genera of cyanobacteria, which is also a potent inhibitor of PP1 and PP2A and is, as a consequence, a known pro-apoptotic drug (Gehringer, 2004). From all these data, and since the modulation of PP2A decreases cell viability, this enzyme has been postulated as a potentially useful target in the treatment of patients with adult T cell leukaemia, due to the apoptosis activation induced by PP2A inhibitors (Mori et al., 2013). However, both OA and MC are involved in tumour promotion, and the development of primary liver cancer in China has been associated with long-term chronic exposure to MC (Ueno et al., 1996; Harada et al., 1996; Yu, 1995). This conflicting response obtained in the presence of these toxins is probably due to the variety of processes where serine/threonine residues are involved. OA activates mitogen-activated protein kinases (MAPK), which may inhibit apoptosis and increasing cell proliferation

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(Gehringer, 2004), but proliferation can also be increased by increasing phosphorylations, or if critical dephosphorylations are prevented, which is happening when PP2A inhibitors are present.

Yessotoxin (YTX) One of the main groups of toxins often related with apoptosis is the YTX group. These are produced by dinoflagellates of the genus Protoceratium (P. reticulatum), Lingulodinium (L. polyedrum) and Gonyaulax (G. spinifera). The mechanism of action of YTX is totally different from that of okadaic acid (OA), since YTX does not inhibit the protein phosphatases as occurs with the OA group (Bialojan & Takai, 1988; Ogino et al., 1997). Although YTX, with up to 100 different analogues described, is considered a marine toxin, there has never been a report of human toxicity associated with this group (Panel, 2008b). It was described that YTX modulates cytosolic calcium (Ca2+ ) levels through an increase in the entry of extracellular Ca2+ in human lymphocytes, and inhibits the capacitative Ca2+ entry (de la Rosa et al., 2001a, 2001b). Later, phosphodiesterases (PDEs) were described as the target of the YTX and, through this binding, PDE is activated by decreasing the cAMP levels in human lymphocytes (Alfonso et al., 2003; Pazos et al., 2005, 2006; Tobío et al., 2012). However, the first specific mention of apoptosis caused by YTX was reported by Leira et al (2002a). Several papers have since shown that treatment with YTX develops typical apoptotic hallmarks in different cell lines types (Pang et al., 2011, 2012; Korsnes et al., 2006a, 2006b). A significant difference between apoptosis induced by YTX and that elicited by the OA group is that YTX is much slower (12 hours versus one hour for OA), either to the decrease of mitochondrial membrane potential, cell detachment, total nuclei acid content, DNA fragmentation, plasma membrane integrity, caspase 3 activation, phospholipid asymmetry (Leira et al., 2002a), caspase 3 and 7 activation, PARP cleavage and DNA fragmentation (Malaguti et al., 2002). The evidence for the apoptotic effect of YTX includes many cell types and apoptosis markers, such as cerebellar neurons (Perez-Gomez et al., 2006), L6 and BC3H1 myoblasts (Korsnes et al., 2006a, 2006b) (membrane blebbing, nuclear shrinkage, and chromatin condensation, but only in L6, caspase 3 and 9 activation, release of cytochrome c and Smac/DIABLO, PARP cleavage, opening of PTP, F-actin cytoskeleton disassembly and tensin cleavage), mouse fibroblasts NIH3T3 (Malagoli et al., 2006) (lysosomal damage, which may suggest autophagy), Caco-2 and MCF-7 (Ronzitti & Rossini, 2008; Callegari & Rossini, 2008) (accumulation of E-cadherin fragment ECRA100), HepG2 (Young et al., 2009), and Bel7402 and HL7702 human hepatoma (Pang et al., 2011) and liver cells (Pang et al., 2012), respectively. Calcium plays a key role, since it is required for the activation of endonucleases that cleave the DNA (McConkey & Orrenius, 1997). In the HL7702 liver cell line and the Bel7402 human hepatoma cell line, YTX decreased cell proliferation, and this effect was explained by the activation of apoptotic pathways. Chromatin condensation, DNA fragmentation and activation of caspase 3 were observed after treatment with the toxin. These events were associated with loss in the mitochondrial transmembrane potential (MTP), as it was described previously for the intrinsic apoptotic pathway, and this was reported in different cell lines, such as in neuroblastoma cell line BE(2)-M17 (Pang et al., 2011, 2012; Leira et al., 2002a). The opening of the permeability transition pore (PTP) decreases the MTP and releases different compounds to the cytosol that finally activate caspase 3 through the caspase 9 activation (Korsnes & Espenes, 2011).

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The mitochondrial apoptotic pathway activated by YTX is Ca2+ -dependent, since the opening of PTP in isolated mitochondria after the toxin treatment needs Ca2+ (Bianchi et al., 2004). In the L6 cell line from primary cultures of rat thigh muscle, the same caspase activation and DNA fragmentation was observed after 100 nM YTX treatment for 72 hours. Activation of caspases 9 and 3 after the toxin administration was accompanied by the cleavage of poly-ADP-ribose polymerase (PARP) (Korsnes et al., 2006b). PARP is a chromatin-associated protein that repairs DNA and maintains its stability, and it is also a typical target of caspase cleavage in apoptosis (O’Brien et al., 2001; Trucco et al., 1998). In HeLa S3 cells, activation of the caspases 3, 7 and 2 was observed after 0.25 nM YTX treatment for 48 hours, accompanied by PARP cleavage with 1 nM YTX (Malaguti et al., 2002). The YTX effect through the mitochondria is being elucidated through new studies and observations. In 2006, a report showed that the incubation of L6 cell line with 100 nM YTX for long-term periods caused swelling of the mitochondria, changes in the permeability of the outer mitochondrial membrane and pro-apoptotic factors cytochrome c and smac/DIABLO increased in the cytosol (Korsnes et al., 2006a). In this sense, the complex A-kinase anchor proteins (subtype 149 AKAP), subtype 4A PDE (PDE4A) and protein kinase A (PKA) (AKAP149-PKA-PDE4A) were described in the outer mitochondrial membrane. The structural protein AKAP 149 that anchors PKA and PDE4A to the outer mitochondrial membrane seems to play an important role in YTX activation of mitochondrial apoptotic pathway (Carlucci et al., 2008). In the human leukaemia K-562 cell line, a short term treatment with 5 μM YTX for 10 minutes decreased the expression of AKAP 149 protein, and a decrease in cell viability in this cell line was detected. However, in fresh human lymphocytes under the same conditions, the toxin increased these protein levels and no effect on cell viability was observed after the toxin incubation (Tobío et al., 2012). Therefore, more investigations need to be done over the mechanism of action of the YTX and how the mitochondrial apoptotic pathway is activated. Regarding this pathway, that includes the AKAP 149-PKA-PDE4A complex, a decrease was observed in the whole cytosolic complex after long-term incubations for 24 and 48 hours with 30 nM YTX. This complex decreased in cytosol (anchored to the mitochondria) but increased in the plasma membrane after 24 hours, while 48 hours after incubation, its levels increased in the nucleus. These new findings pointed out the localization of the complex as a key role in different cell death since, when the complex is in the plasma membrane, apoptosis is activated, while the migration of the complex to the nucleus activates a different cell death pathway (Fernández-Araujo et al., 2014). Figure 9.1 shows a theoretical pathway activated by YTX in K-562 cell line. Even though YTX has been reported to induce apoptosis in many cell types, as shown later for azaspiracids, there is a large difference in response, depending on the cell type used for the experiments (e.g. freshly isolated rabbit enterocytes are inert to YTX-effect on F-actin) (Vilarino et al., 2006). Also, the sequence of events triggered by YTX is more complex than just causing apoptosis, since lysosomal damage (Malagoli et al., 2006), paraptosis and authophagy by different routes were also reported (Korsnes, 2012). A recent article by Rubiolo et al., (2014), using the experimental approach of gene expression with RNA microarrays, concludes that the mechanism of action of YTX is mostly mediated by autophagy triggered by endoplasmic reticulum stress after

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YTX

DR

4A

Procaspase 8

E - cadherin disruption

AKA

PDE

P149

Caspase 8

X

YT

PDE4A

A - and B - type lamins depolaryzation

c-AMP

AK AP 14

PP

14 9

9

AP

1

AK

Bcl-2 MP

Procaspase 9 Cytocrome c AP AF 1

NUCLEUS

Caspase 9

Caspase 3 Procaspase 3

Figure 9.1

Model of activation after YTX incubation. Possible model of the pathways activated after incubation with YTX for 24 hours in the human cancer K-562 cell line. YTX binds to PDE4A in the AKAP 149-PKA-PDE4A complex in the outer mitochondrial membrane. After 24 hours, the complex migrates from the outer mitochondrial membrane to the plasma membrane (black fonts and lines). In the plasma membrane the complex could disrupt E-cadherin fragments [?] and activate DR [??] (grey fonts and lines). After the DR activation, caspase 8 is activated, leading to the extrinsic apoptotic pathway. The absence of the complex in the mitochondrial outer membrane decreases the anti-apoptotic Bcl-2 levels in the mitochondrial membrane and the MP is opened. Cytochrome c is released to the cytosol from the intermembrane space and recruits APAF1 and caspase 9 to activate caspase 3, leading to the intrinsic apoptosic pathway. In the nucleus, AKAP 149 levels are decreased, and the nuclear envelope integrity is lost after the A- and B-type lamins’ depolarization by the lack of PP1 in the nuclear envelope [???]. Black arrows show protein levels in each localization. Grey color shows pathways described in the literature. R and C are subunits of PKA. PP1: protein phosphatase 1. DR: death receptor. MP: mitochondrial pore. APAF1: apoptotic protease-activating factor 1.

48 hours; the mechanistic target of rapamycin (mTOR) activated with EGF protects glyoma cells from the effect of YTX. This observation by Rubiolo et al after 48 hours does not reconcile with those results that describe evident apoptotic effects elicited by YTX in the first 24 hours (Fernández-Araujo et al., 2014), which suggest that the mechanism of action of YTX is not only very complex, but not understood yet. Figure 9.1 shows the cellular pathways related with YTX apoptosis activation.

Azaspiracid (AZA) AZAs are produced by species of the genus Azadinium (Tillmann et al., 2009) and Amphidoma (Krock et al., 2012). There are close to 30 different analogues, of which only

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AZA1, 2 and 3 are abundant in shellfish, and they were reported to be toxic to humans on several occasions, with gastrointestinal symptoms (Panel, 2008a). The mechanism of action of azaspiracids is as yet unknown, and several targets have been proposed to explain their mode of action. Initially, AZAs were reported to be non-apoptotic toxins (Roman et al., 2002) in human lymphocytes and neuroblastoma cells, reducing F-actin levels and increasing cAMP and cytosolic calcium. However, this scenario was soon shown to far more complex. Subsequent studies with BE(2)-M17 neuroblastoma reported activation of caspases 1, 3 and 9 at nanomolar concentrations (Vilarino et al., 2007). The onset of the effect required only 2 minutes’ incubation with the toxin, and this effect would last for 48 hours, suggesting an irreversible binding to some target, and the initial disarrangement of the cytoskeleton was not related to caspase activation, suggesting a dual effect. Further evidence accumulated to support a dual effect of AZAs, with atypical apoptosis and necrosis, at least in neocortical neurons (Cao et al., 2010), where they induce caspase 3 activation, and nuclear condensation after eight hours of exposure. More recently, the group was shown to be aopototic in several cell lines (Jurkat T lymphocytes, intestinal epithelial caco-2 and BE(2)-M17 neuroblastoma), with effect on caspases 1 to 9, cytochrome c release from the mitochondria, and DNA fragmentation (Twiner et al., 2012b) and, although laddering was observed after 48 hours, again it was reported as an independent process for apoptosis and for changes in morphology. Experimental observation allows the conclusion that caspase activation by AZAs, which is an early sign of apoptosis (Carragher et al., 2001), happens after morphological changes triggered by the toxin (Vilarino et al., 2007). In neuroblastoma, fibroblasts, Jurkat T or human breast cancer cells MCF-7, AZA cause cell detachment (Ronzitti et al., 2007), reduction in membrane pseudopodia (Twiner et al., 2012b) and concentration of focal adhesion contacts and actin microfilament bundles (Vilarino et al., 2006). The toxin group does not have any effect on microtubules. A rather remarkable experimental observation frequently reported with this toxin group, is the extreme difference in response depending on the cellular model being studied, i.e. AZAs do not affect Jun amino-terminal kinase (JNK) phosphorylation in neurocortical neurons (Cao et al., 2010), but increase notably JNK activation and nuclear translocation in primary neuron cultures (Vale et al., 2007). In primary neurons, the inhibition of JNK activation prevents the cytotoxic effect of AZAS, and this observation defines a potential mode of action of the toxin group. There is a striking difference in the response of cerebellar granule neurons to AZA, depending of the age of the cells, as young cells (2–3 days) suffer activation of JNK and hyperpolarization, while older cells (two weeks) do not show any change. Experimental data obtained in human lymphocytes show discrepancies in the response to different AZA structures with regard to cAMP and cytosolic calcium levels, suggesting different mechanisms for some of the AZA analogues. AZA1 and 2 induce release of calcium from internal pools, while AZA3 does not cause any change (Alfonso et al., 2006; Roman et al., 2004); on the other hand, AZA3 increases cAMP levels twofold if compared with AZA1 or AZA2. Another interesting difference was reported for AZA4 with regard to AZA1–3, as AZA4 is an inhibitor of the store-operated plasma membrane calcium channels, this effect being cAMP-independent. In addition, AZA5 is inert to modify cytosolic calcium concentration (Alfonso et al., 2005). These toxin are described as a human ether-a-go-go-related gene (hERG) potassium channel inhibitors (Twiner et al., 2012a), and this effect has been proposed to explain the arrythmogenicity of AZA2 (Ferreiro et al., 2014). The effect of AZA on

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ion channels has been linked to chloride, since cell volume regulation is related to JNK activation, and the volume decrease mediated by K+ and Cl– efflux was completely reverted by the anion channel blocker DIDS or by amiloride. Additionally, the activation of JNK is secondary to this decrease in cell volume (Vale et al., 2010).

Brevetoxins (PbTx) This type of toxin is produced by the dinoflagellate Karenia brevis and it belongs to Neurotoxic Shellfish Poisoning (NSP). PbTx binds to site 5 in the voltage-gated sodium channel (VGSC) and induces Na+ ion influx through the channel (Watkins et al., 2008). First, in human lymphocytes, DNA damage after PbTx incubation was observed, pointing to the ability of this toxin to induce apoptotic cell death (Sayer et al., 2005). The activation of intrinsic apoptotic pathway was then described in human Jurkat cell line after incubation with several PbTx analogues (Walsh et al., 2008). Later, the effect of PbTx2, 3, 6 and 9 was checked in Jurkat cells, and inhibition of proliferation and DNA damage were described (Murrell & Gibson, 2009). Therefore, from all these data, an apoptotic mechanism of death activated by PbTx, either by activation of intrinsic or extrinsic apoptotic mechanisms, was postulated (Murrell & Gibson, 2009; Murrell & Gibson, 2011).

Pectenotoxin (PTX) PTXs are synthesized by algae from Dinophysis genus (Draisci et al., 1996). PTX’s mechanism of action involves interaction with the actin cytoskeleton. (Ares et al., 2007; Espiña et al., 2008, 2010; Leira et al., 2002b; Spector et al., 1999;, Zhou et al., 1994) To date, more than 20 PTX analogues have been isolated and characterized. Many of these analogues modify the actin cytoskeleton and show high toxicity, while others show low toxicity and no cytoskeletal effect (Ares et al., 2007; Miles et al., 2004). In this sense, the lactone ring is essential for the action of PTXs on actin cytoskeletal dynamics (Allingham et al., 2007; Ares et al., 2005, 2007). PTX-2, the major compound of this group, displays selective and potent cytotoxicity against human cells of lung, colon and breast cancer and human leukaemia cells (Kim et al., 2008). PTX-2 triggers cell death by apoptosis, on the basis of morphological changes, a loss of mitochondrial membrane potential, an increase in the level of cytoplasmic cytochrome C and SMAC/DIABLO, and the activation of caspase-3 and caspase-9 (Fladmark et al., 1998; Chae et al., 2005; Kim et al., 2008; Shin et al., 2008). In addition, apoptosis was described in p53-deficient cells after PTX-2 treatment, showing enhanced cytotoxicity as compared with normal cells (Chae et al., 2005). Activation of caspases 3, 8 and 9 was also observed after PTX incubation in human hepatocarcinoma cell lines (Shin et al., 2008). Down-regulation of anti-apoptotic protein Bcl-cL and up-regulation of pro-apoptotic Bax was observed after PTX-2 administration in leukaemia cells (Kim et al., 2011). Moreover, Bcl-2 proteins mediated in the apoptosis activated by PTX-2 in p53-deficient cells (Chae et al., 2008) and in these cells, phosphorylation of p53 protein was induced by actin-damage, but through different signalling pathways than DNA damage (Chae et al., 2012). It has been reported that the mechanism whereby PTX-2 induces cell death and cell cycle dysfunction in leukaemia cells is related to G2/M phase arrest, endoreduplication, and apoptosis through the signal-regulated kinase (ERK) and the JNK signal pathway via actin depolymerization (Moon et al., 2008). This toxin also increases DR4 and DR5 levels accompanied by the activation of caspase 8 (Kim et al., 2011). Therefore,

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both intrinsic and extrinsic apoptotic pathways seem to be activated by this toxin after F-actin depolymerization. On the other hand, apoptosis was not detected after PTX-6 incubation, while a significant reduction in the actin cytoskeleton was observed (Leira et al., 2002b; Ares et al., 2005). Therefore, differences in terms of cytoxicity were further studied. Espiña et al., had tested the effect of PTXs on actin cytoskeleton and cell viability in two hepatic cellular models (Espiña et al., 2008). Interesting results point to different effects of PTXs in immortal and primary cellular cultures (Espiña & Rubiolo, 2008). The higher sensitivity of immortal cells to PTXs in comparison to normal hepatocytes could be related to the fact that the cell lines share cytoskeletal and morphological characteristics with cancerous cells. PTX-2, the main compound, oxidizes progressively to PTX-1, PTX-3, and PTX-6 in the Japanese scallop. In this context, Espiña and co-workers have demonstrated that PTX-1, PTX-6 and PTX-9 induce different dose-dependent damage in the actin cytoskeleton and in the cell viability of primary cultured rat hepatocytes (Espiña et al., 2010). In Clone 9 rat hepatocytes, PTX-1 and PTX-9 also affect the morphology of cells but, surprisingly, no effect was observed in the presence of PTX-6. In accordance with this lack of activity, the actin cytoskeleton of the epithelial cell line CaCo-2 was not affected by PTX-6. In conclusion, the order of cytotoxicity of the analogues was PTX-2 > PTX-1 > PTX-6 > PTX-9 – that is, the increase in the level of oxidation of the PTX molecule decreases its cytotoxicity. Furthermore, PTX-6 is not able to induce effects on immortal cells, while retaining its toxicity against primary cultured cells, whereas PTX-9 is active in both cellular models. The different cytotoxicity exerted by PTX-6 on cell lines and primary cells could be determined by the presence of a carboxylic acid group on C43 of the PTX molecule (Espiña et al., 2010). Therefore, the cyclic structure is important for both toxicity and activity effect.

Domoic Acid (DA) DA is the main component of Amnesic Shellfish Poison (ASP) (Jeffery et al., 2004). The first DA producer discovered was the macro red algae Chondria armata, and later on the marine diatoms of the genus Pseudo-nitzschia were postulated as DA producers (Jeffery et al., 2004; Pulido, 2008). This toxin acts at glutamate receptor subtypes: N-methyl-D-aspartate (NMDA), (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid (AMPA) and Kainate Receptor (KARs), widely localized in the brain with different roles in synaptic transmission (Vale-Gonzalez et al., 2006; Giordano et al., 2007; Jeffery et al., 2004; Matute, 2011). In cerebellar granule neurons (CGN), DA increases cytosolic Ca2+ , and this influx is through NMDA receptors, L-type VSCC and reversed mode of operation of the Na+ ∕Ca2+ exchanger (Berman et al., 2002). In CGN and cardiomyocytes, apoptosis was observed after an increase in cytosolic Ca2+ levels (Gao et al., 2007; Giordano et al., 2007). Cardiomyocytes showed activation of the apoptotic pathway via mitochondria, with increase in ROS and caspase 3 activation after NMDA activation with DA (Gao et al., 2007). In CGNs, apoptosis was shown in the presence of 0.1 μM DA, while in the presence of 10 μM DA, necrotic response was observed. The apoptotic pathway activated follows all the steps of intrinsic apoptotic pathway: loss of membrane mitochondrial potential, increasing of mitochondrial oxidative stress, opening PTP, and subsequent cytochrome c release, caspase 3 activation and PARP cleavage (Giordano et al., 2007). Later, the importance of oxidative stress-activated

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JNK and p38 MAP kinase pathways in apoptosis induced by DA in these cells was also pointed out (Giordano et al., 2008). This effect was avoided in the presence of muscarinic agonists (Giordano et al., 2009). However, the exposure of slice cultures of hippocampus cells to DA for 24 hour-induced dose-dependent neuronal toxicity was independent of activation of classic apoptotic markers (Perez-Gomez & Tasker, 2012). On the other hand, a decrease in cell viability, direct DNA damage and apoptosis through upregulation of Bcl-2 in caco-2 cells was observed after DA incubation (Pinto-Silva et al., 2008). Tubular necrosis, apoptosis and renal tubular cell desquamation, with toxic vacuolization and mitochondrial swelling as hallmarks of the cellular damage, were also observed after DA administration to mice (Funk et al., 2014).

Palytoxin Palytoxin is a large molecule (m.w. 3300) with extreme toxicity (i.v. LD50 is as low as 25 ng/Kg (Wiles et al., 1974)), although oral toxicity is much lower (Tubaro et al., 2014; Munday, 2014) and the acute reference dose proposed by the EFSA Panel on Contaminants was 30 μg∕Kg shellfish meat (Panel, 2009; Munday, 2014). Although there are many possible isomers (up to 1021), few analogues have been found in nature (Katikou & Vlamis, 2014). It is produced by zoanthids that belong to the genus Palythoa, and by dinoflagellates of the genus Ostreopsis (Katikou and Vlamis, 2014). Its mechanism of action is shared with several analogues, such as ovatoxins, mascarenotoxins or ostreocins (Tubaro et al., 2014), and it is a blockade of the Na+ ∕K+ -ATPase (Bottinger & Habermann, 1984; Alfonso et al., 2014), which becomes a non-selective cationic channel, with a cytolitic imbalance of ionic homeostasis, increase of cytosolic calcium, decrease of cytosolic pH and cell death (Tubaro et al., 2014; Rodrigues et al., 2009; Vale-Gonzalez et al., 2007). Although the effect of palytoxin is well established, its links with possible apoptotic routes are not well understood. Vale-Gonzalez et al., (2007) reported that palytoxin cytotoxicity in neurons was reduced by blockade of PKC isozymes, of JNK, p38, mitogen-activated protein kinases (MAPKs) and MEK. Even though the mechanism is not yet understood, the cytotoxicity of palytoxin is completely blocked by inhibition of the ERK 2 signal (Vale et al., 2006). Despite these observations, what is clear is that the palytoxin mechanism of action has been studied in several cell types (caco-2 (Fernandez et al., 2013), immune cells, skin (Tubaro et al., 2014)) and the toxic effect is always linked to cation imbalance. However, along with the blockade of the Na+ ∕K+ -ATPase that triggers an ionic imbalance, palytoxin causes actin filaments distortion at low doses, with an increase of cell detachment correlated with cell rounding and F-actin depolymerization (Louzao et al., 2011), through a mechanism that Prandi et al., (Prandi et al., 2011) proposed as a second step after osmotic imbalance. Nevertheless, these effects do not seem to be a cause for apoptosis as the classical markers, such as NA fragmentation chromatin condensation, DNA fragmentation or caspases activation, are not modified (Valverde et al., 2008a, 2008b).

Maitotoxin Maitotoxin is a very large molecule (m.w. 3422) produced by the genus of dinoflagellage Gambierdiscus (Takahashi et al., 1982; Miyahara et al., 1979). Maitotoxin, in a similar fashion to palytoxin, causes cell death by a sustained increase of cytosolic calcium, and this effect is mediated by several transduction signals, none of which seem

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to be related to apoptosis (McGinnis et al., 2003; de la Rosa et al., 2001b). Therefore, this toxin, although extremely potent, seems to act by non-apoptotic mechanisms, and its main mechanism is interaction with the phospholipids of the plasma membrane to allow calcium influx (Konoki et al., 1999; Reyes et al., 2014).

Non-apoptotic cytotoxicity of phycotoxins Apoptosis is a programmed cell death, but other types of programmed cell death can be activated independent of caspase activation. These types of cell death include paraptosis, autophagy and mitotic catastrophe (Ouyang et al., 2012; Galluzzi et al., 2012). In addition, anoikis, parthanatos, pyroptosis, entosis and entosis are also considered as other cell death modalities, Scheme 9.2 (Galluzzi et al., 2012). Necrosis is a regulated cell death that can be activated by several triggers as DNA damage, toxins or DR activation. This death, with an important role in pathological and physiological processes, is often activated when caspases (caspase 8) are inhibited, while RIP1 (receptor-interacting protein kinase) and its homologue RIP3 are functional (Galluzzi et al., 2012). In this kind of death, apoptosis is not excluded. Necroptosis has been used as a synonym of necrosis, but it is a more restrictive nomenclature used to define a regulated necrosis activated by tumour necrosis factor receptor 1 (TNFR1) ligation. This term is reserved for RIP1 and/or RIP3- dependent necrosis (Galluzzi et al., 2012). Autophagy, or type II programmed cell death (Ouyang et al., 2012), is a process where different portions of cytoplasm are sequestered in lysosomes to degrade proteins or to eliminate damage mitochondria or functional redundant organelles. This death can be activated after nutrient deprivation and is a mechanism of cell survival (Codogno & Meijer, 2005). The degradation of cytoplasmic components by lysosomes can occurs through microautophagy, chaperone-mediated autophagy (CMA) or macroautophagy (Klionsky & Emr, 2000; Mijaljica et al., 2011; Dice, 2007). In CMA, the cytosolic proteins are translocated directly into the lysosomes while, in micro- and macroautophagy, these components are firstly sequestered in double membrane vesicles, called autophagosomes, then fuse with lysosomes to form the phagolysosomes (Klionsky & Emr, 2000; Galluzzi et al., 2012). However, this process can degenerate into what is known as Type II cell death, (Type I cell death is apoptosis) or autophagic cell death, when a massive autophagic vacuolization is developed (Galluzzi et al., 2007). The main regulator of macroautophagy is the mTOR. This protein blocks autophagy and, after its inhibition through rapamycin or nutrient deprivation, the autophagy is activated (Populo et al., 2012). After mTOR inhibition, cytoplasmic components are involved in autophagosomes with microtubule-associated protein 1 and light chain 3 (LC3) protein inside these lipid membranes (Lazova et al., 2012). These two proteins are typical hallmarks to detect autophagy. A detailed flow diagram with signs, earliest steps and regulation of autophagy is shown in Scheme 9.3. On the other hand, a mitochondrial failure or microtubules, stabilizing or destabilizing agents and DNA damage, can trigger the mitotic catastrophe. This type of programmed cell death essentially differs from apoptosis because caspase inhibition or Bcl-2 overexpression do not prevent mitotic catastrophe (Broker et al., 2005). The process is initiated by perturbations in the mitotic apparatus during M phase of the cell

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Autophagy

*Protective role an pro-survival response at initial stages of cancer development *Formation of autophagosomes by involved ATG (autophagy-related genes) *Formation of an isolation membrane or phagophore that forms a doublemembrane vesicle with the accumulation of LC-3 II (light chain protein) to entrap cellular components to be degraded *It is an alternative cell death mechanism *As apoptosis, requires high levels of ATP and energy supply * May have partial chromatin condensation, but no DNA fragmentation

Signs

* Autophagy can switch to apoptosis by caspase or calpain-mediated cleavage of mediators in mitochondria such as Beclin-1 or ATG5 * Bcl-2 (B-cell lymphoma) domain of Beclin-1 inhibits anti-apoptotic Bcl-2/Bcl-XL proteins, and activates pro-apoptotic Bax and Bak * Starvation induces autophagy through inhibition of mTOR (mechanistic target of rapamycin) which resides in a multi protein complex, mTORC1 * Coregulated by a multiprotein complex with Beclin 1, that associates with class III PI3K (phosphatidylinositol-3-kinase) VPS34, and other regulatory proteins (ATG12L, UVRAG, Ambral, Rubicon). * The production of PI3P (phosphatydilinositol-3-phosphate) by this complex regulates autophagosome formation * Beclin 1 complex is negatively regulated by PI3K/Akt pathway and Bcl-2 proteins * After phagophore formation, elongation of autophagosome membrane requires two ubiquitin-like conjugation systems: Atg5-Atg12 and LC3-Atg8 (microtubule-associated protein light chain 3) * Atg4B converts proform of LC3B to its cytosolic free form (LC3-I). * Conversion of LC3-I (and other Atg8 homologues) to its PE (phosphatidylethanolamine)-conjugated and autophagosome-membrane associated form (i.e. LC3-II) initiates autophagy

Earliest steps

mTOR regulation

activation *In stimulation by nutrients or growth factors mTORC1 negatively regulates a macromolecular complex that includes ULK1 (mammalian uncoordinated-51-like protein kinase), ATG13, ATG101 and FIP200, which results in autophagy supression *PI3K/Akt signal pathway activates mTOR in response to growth factors and phosphorylates Beclin 1 inhibition Energy depletion, that stimulates autopaghy, inhibits mTORC1 through activation of AMPPK (AMPdependent protein kinase) leading to activation of ULK1, an step that initiates autophagy

Scheme 9.3

Main features of autophagy. Source: Based on data from Ryter et al., 2014; Galluzzi et al., 2012; Cerella et al., 2014; Korsnes, 2012; Korsnes et al., 2006a.

cycle, is parallel with some mitotic arrest and triggers cell death or senescence (Galluzzi et al., 2012). Finally, paraptosis is a type of non-apoptotic cell death characterized by cytoplasm vacuolization, together with mitochondrial and ER swelling (Sun et al., 2010). Paraptosis activation is mediated by mitogen-activated protein kinases (MAPK), which can be triggered by the TNF death receptor TAJ/TROY and the insulin-like growth factor I receptor (Broker et al., 2005). In 2011, cytoplasmic vacuolation, mitochondria and endoplasmic reticulum swelling, uncondensed chromatin and lack of DNA fragmentation were observed in BC3H1 myoblast cells after the treatment with 100 nM YTX. These apoptotic hallmarks

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were detected in a caspase-independent activation, and the death was described as paraptosis or paraptosis-like cell death (Korsnes, 2012; Korsnes & Espenes, 2011). However, the ability of YTX to activate autophagy was unclear until 2012 (Korsnes, 2012), but a recent study showed that YTX is also involved in this non-apoptotic cell death in three human glioma cell lines, and autophagy was proposed as the death induced by YTX after 48 hours (Rubiolo et al., 2014). OA is related to the activation of MAPK through the PPs inhibition. Thus, after OA treatment, paraptosis could be activated. In addition, the implication of OA in autophagy activation was observed in rat neurons through the activation of mTOR, increased LC3 levels and autophagosomes formation (Yoon et al., 2008). The ability of Palytoxin to induce autophagy remains unclear. However, it has been observed that after conversion of the Na+ ∕K+ exchanger into a channel, the cytosolic K+ concentration increases. As a consequence, Palytoxin treatment in human HaCat cells phosphorylates the eukaryotic translation initiation factor alpha (eIF2a). In this sense, eIF2a in the phosphorylated state is considered as an alternative activation of the autophagic pathway (Kloft et al., 2010). Therefore, probably, Palytoxin may induce autophagy in different cell lines besides other types of cell death.

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C H A P T E R 10

Cyanobacterial toxins Vitor Vasconcelos, Pedro Leão & Alexandre Campos Interdisciplinary Center of Marine and Environmental Research, CIIMAR,University of Porto,Portugal

Introduction Cyanobacteria produce a large variety of secondary metabolites, and those with toxic effects – cyanotoxins – are widely studied all over the world. Cyanotoxins began to be studied because it was found that they could cause livestock death if animals consumed cyanotoxin-contaminated water. The first record was by Francis, who reported a fatal poisoning of sheep that drank water contaminated with cyanobacteria in Lake Alexandrina (Francis, 1878). Later, in the early 1950s, some human health intoxications that occurred in the USA were attributed to toxins present in the water (McLeod & Bondar, 1952). The development of chemical methods led to more accurate and specific approaches, and the isolation of the toxins and their characterization became possible, anatoxin-a being the first cyanotoxin to be fully characterized (Devlin et al., 1977). Later, the hepatotoxic microcystins were elucidated, being described as heptapeptides, and soon it was found that there was a high diversity of these peptides among cyanobacteria species (Botes et al., 1985). Molecular methods, specially those using primers designed to target gene clusters involved in cyanotoxin production, are now well developed. They can be used to predict the potential production of selected cyanotoxins, being multiplex PCR methods, suitable for the detection of the potential production of several toxins in a single run (Saker et al., 2007).

Chemistry of cyanotoxins Cyanotoxins are diverse not only in their modes of action, but also in terms of chemical structures. Until now, more than one hundred different molecules have been described, microcystins being the most diverse until now (Table 10.1).

Anatoxin-a Anatoxin-a is an alkaloid with isomers such as homoanatoxin-a (Figure 10.1) and will be more comprehensively described in chapter 7 of this book. It has a wide occurrence, every year being described in more countries in Europe (Finland, France, Germany, Greece, Ireland, Italy, Portugal, Spain, UK) (Devlin et al., 1977; Edwards et al., 1992; Rapala et al., 1993; Bruno et al., 1994; Bumke-Vogt et al., 1999; Furey et al., 2003; Gugger et al., 2005; Vardaka et al., 2005; Carrasco et al., 2007; Osswald et al., 2009). Although high anatoxin-a production has been reported as measured by its Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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Table 10.1 Chemodiversity of cyanotoxins, mode of action in mammals, and main producer cyanobacteria. Cyanotoxin

Chemical entity

Mode of action

Anatoxin-a ∗

Alkaloid

N

Anatoxin-a(s)

Organophosphate

N

BMAA

Amino acid

N

Cylindrospermopsin∗

Alkaloid

C

LPS

Lipopolysaccharide

D

Lyngbyatoxin∗

Alkaloid

N

Microcystin∗

Cyclic peptide

H

Moorea producens (formerly Lyngbya majuscula) Microcystis spp.

Nodularin ∗

Cyclic peptide

H

Nodularia spumigena

N

Trichodesmium spp.

N

Several species of Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, Planktothrix

Palytoxin∗ Saxitoxin∗

∗

Alkaloid

Cyanobacteria

Reference

Anabaena, Aphanizomenon, Cylindrospermum, Microcystis, Planktothrix, Oscillatoria Anabaena flos-aquae, A. lemmermani, Most species

Devlin et al., 1977

Cylindrospermopsis raciborskii, Aphanizomenon ovalisporum, Anabaena spp., Raphidiopsis curvata, Umezakia natans Most species

analogues are described; N – neurotoxin, H – hepatotoxin, C – cytotoxin, D – dermatotoxin.

H N

H N

O CH3

Anatoxin-a Figure 10.1

O CH3

Homoanatoxin-a

Structures of anatoxin-a and homoanatoxin-a.

Matsunaga et al., 1989 Cox et al. 2003 Ohtani et al., 1992

Martin et al., 1989 Cardellina et al., 1979 Botes et al., 1985 Sivonen & Jones, 1999 Kerbrat et al., 2011 Negri et al., 1995

Cyanobacterial toxins

N HN

N +

H2N

Figure 10.2

–

O

227

CH3

CH3 O P O

O

CH3

Structure of anatoxin-a(s).

concentration per g of dry weight cyanobacteria, there are not many reports indicating actual values in nature (Osswald et al., 2007). Values up to 34 μg∕l of homoanatoxin-a have been recorded in nature (Furey et al., 2003), but its low persistence and low bioaccumulation rates (Rellán Piñeiro et al., 2007; Osswald et al., 2008) may minimize the negative consequences of its occurrence under natural conditions in the plankton. If present in the benthic environment, cyanobacteria may pose more acute problems if consumed by wild or domestic animals.

Anatoxin-a(s) Anatoxin-a(s) is the only natural organophosphate described so far (Figure 10.2) and has few reports worldwide, in spite of its high toxicity. Blooms containing measurable levels of this toxin have been reported from the USA, Denmark, & Brazil (Mahmood & Carmichael, 1986; Onodera et al., 1997; Becker, et al., 2010). It has been reported from species of Anabaena, but its worldwide occurrence may be underestimated due to the fact that there are no analytical methods developed for its accurate detection and quantification. The use of an acethylcholinesterase inhibition assay may be an alternative, taking into account that this is its mode of action (Becker et al., 2010).

BMAA The methylated amino acid β-N-methylamino-L-alanine (BMAA) is a non-protein amino acid (Figure 10.3). BMAA was firstly isolated from the seeds of the cycad tree (Cycas sp.) in 1967, in Guam (Vega & Bell, 1967). Later, biomagnification of BMAA through trophic chains was first proposed for the Guam ecosystem, and showed the presence of BMAA from the endosymbiotic Nostoc sp. (on the coralloid roots of Cycas) (Cox et al., 2003). Recently, it was reported that a large number of cyanobacteria strains could produce BMAA, either as symbionts or as free-living species in marine, brackish

O H2N OH H HN CH3 Figure 10.3

Structure of β-N-methylamino-L-alanine (BMAA).

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H N

O OH

O NH

O S O O H

H

H

N

NH

OH +

NH Figure 10.4

Structure of cylindrospermopsin.

or freshwater biota (Cox et al. 2005; Banack et al., 2007; Esterhuizen & Downing 2008; Metcalf et al. 2008; Cianca et al., 2012a). BMAA can be taken up into food chains and can reach humans by consumption of fish and shellfish. The biomagnification of BMAA in food webs of the North Atlantic has been shown, from cyanobacteria, to zooplankton, to invertebrates, and to fish, with increasing BMAA concentration within higher trophic levels (Brand et al. 2010; Jonasson et al. 2010). The characterization of BMAA showed a 1H NMR spectrum characteristic of an optically active compound, with the pKa of the two amino groups being 6.5 and 9.8 (Nunn & O’Brien, 1989). L-2,4-diamino-n-butyric acid (DAB) is a structural isomer of BMAA that can co-elute in some detection methods with BMAA. BMAA can occur free or bound to protein so, to detect this last fraction, we need to hydrolyze in 6 M HCl in a vacuum (Cianca et al., 2012b).

Cylindrospermopsin Cylindrospermopsin (Figure 10.4) is an alkaloid having some isomers, such as 7-epi-cylindrospermopsin and 7-deoxy-cylindrospermopsin and it will be more comprehensively described in chapter 14 of this book. CYN has a wide occurrence, being reported every year in more countries, and new species of cyanobacteria as its producers. Cylindrospermospsin is known to be produced by other cyanobacterial species: Umezakia natans (Harada et al., 1994), Aphanizomenon ovalisporum (Banker et al., 1997), Anabaena bergii (Schembri et al., 2001), Raphidiopsis curvata (Li et al., 2001b) and Anabaena lapponica (Spoof et al., 2006) and Aphanizomenon flos-aquae (Preußel et al., 2006).

Lipopolysaccharides Cyanobacteria LPS (Figure 10.5) are similar to those of other Gram-negative bacteria (Martin et al., 1989), although they seem to be less toxic (Bernardová et al., 2008). The main risk associated with their occurrence in waters for recreational or dialysis purposes. LPS from cyanobacterial blooms have been associated with outbreaks of flu-like diseases both in Scandinavia & Zimbabwe (Annadotter et al, 2005). These compounds can also be involved in human intoxication if present in water used for dialysis, so special care has to be taken when water is used for this purpose.

Lyngbyatoxin The marine toxin lyngbyatoxin (with its variants A, B and C) (Figure 10.6) was first reported from a mat-forming Moorea producens – formerly Lyngbya majuscula (Engene et al., 2012) – growing in shallow water in Kahala Beach, Oahu, Hawaii

Cyanobacterial toxins

OH

O HO P HO O

229

O

O O

O O

HO NH O O

O

O

O

O

O

O P

HO OH

HO

O

Figure 10.5

NH O

HO

Structure of a lipopolysaccharide.

(Cardellina et al., 1979). The discovery of this molecule was prompted by investigations of ’swimmers’ itch’ reported by individuals who had come into direct contact with the cyanobacterium (Cardellina et al., 1979). Lyngbyatoxin A is structurally related to the teleocidins (teleocidin A-1 is equivalent to it and teleocidin A-2 is the C-19 epimer of lyngbyatoxin A), secondary metabolites produced by Streptomyces bacteria (Cardellina et al., 1979; Sakai et al., 1986). A decade after its discovery, Aimi and co-workers (1990) were able to isolate and structurally characterize two other congeners, lyngbyatoxins B and C, from the same cyanobacterium. M. producens is a well-known rich source of secondary metabolites, found in tropical areas (Engene et al., 2012). Lyngbyatoxin A is an indole alkaloid featuring an indolactam V moiety with a pending lynalyl group (Figure 10.6). Its structural elucidation was carried out through

H N

N

19

OH

H N

N

O

O

N H

N H

OH

H N

N O N H

OH

OH Iyngbyatoxin A

Figure 10.6

Iyngbyatoxin B

Iyngbyatoxin C

Structures of lyngbyatoxin A, B and C.

OH

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a combination of spectroscopic techniques, most prominently 1 H and 13 C NMR (Cardellina et al., 1979). The stereochemistry of the indolactam moiety was determined by comparison of its optical rotation data with that of the fully characterized teleocidin B (Cardellina et al., 1979). The remaining stereochemical assignment, C-19 (R) in the lynalyl group, was achieved by Sakai et al. (1986). Lyngbyatoxins B and C (Aimi et al., 1990) share the indolactam substructure with their ‘A’ congener, but have modified (hydroxylated) lynalyl moieties.

Microcystin Microcystins were firstly described as being produced by Microcystis aeruginosa (Botes et al., 1985), but a growing number of reports showed that MC may be produced by many genera and species of cyanobacteria (Sivonen & Jones, 1999). Most of these reports of MC occurrence are related to freshwater environments (Vasconcelos et al., 1996), estuaries (D’ors et al., 2013) or symbioses (Kaasalainen et al., 2012). Nevertheless, work done in the salt lake Salton Sea showed that MC can also occur in saline environments (Carmichael & Li, 2006). Black band disease, a pathology of marine sponges caused by cyanobacteria, has been associated with MC produced by Leptolyngbya and Geitlerinema (Gantar et al., 2009). More recently, there have been reports of the occurrence of MC in coastal areas in the Pacific (Miller et al., 2010) and the Mediterranean (Vareli et al., 2012), causing animal intoxication, although in these cases the origin is continental. Nevertheless, it has shown that freshwater cyanotoxins may persist in the marine environment and, due to its uptake by aquatic organisms, may subsequently increase their concentrations to levels that may be harmful. MC are cyclic heptapeptides with a general chemical structure as cyclo (DAla-L-X-Derythro-b-methyl-isoAsp-L-Y-Adda-D-isoGlu-N-methyldehydroAla), where X and Y are variable L-amino acids (Figure 10.7). The most distinctive amino acid of MC and NOD is the (2S,3S,8S,9S,4E,6E)-3-amino-9-methoxy-2,6,8-trimethyl10-phenyl-4,6-decadienoic acid (ADDA). There are more than 80 variants or isoforms of MC, with molecular weights ranging from 800 to 1,100 Da, the most common being MC-LR (Vasconcelos et al., 1996,; Sivonen & Jones, 1999; Saker et al., 2005).

Nodularin Nodularin is a pentapeptide similar to microcystins, also having ADDA as a distinctive amino acid (Figure 10.8). Nodularins are produced mainly by the brackish water

7

6

R2

H COOH HN H3C H

H OCH3

5

CH2 H3 C

O

O

H R1

NH H3C H

O

N

Z

CH3 4

1

O X

NH O

NH H

2

H COOH 3

Figure 10.7

General structure of microcystin (X and Y are the variable amino acids).

Cyanobacterial toxins

O

OH

CH3 N

HN O

CH3

H3C

NH CH3

O

CH3 NH

O

OH NH

CH3

CH3

O

O

231

O

O

HN HN

Figure 10.8

NH2

Structure of nodularin.

cyanobacterium Nodularia spumigena. The main reservoir of NOD is the Baltic Sea because, due to its eutrophication, low salinity and adequate physical conditions, it produces heavy blooms of N. spumigena every year (Sivonen & Jones, 1999). NOD are similar to MC, but are pentapeptides that also have ADDA as the distinctive amino acid. Due to their ring structure, MC and NOD are very stable, resistant even to boiling, freezing and other physical treatments (Morais et al., 2008).

Palytoxin Palytoxin was isolated by Moore & Scheuer (1971) and, only two decades later, two groups published the full structure and stereochemistry (Cha et al., 1982; Moore et al., 1982). Palytoxins are large (> 2500 Da) molecules featuring a polyhydroxylated and partially unsaturated aliphatic chain, punctuated by cyclic ether moieties (Figure 10.9) (Ciminiello et al., 2011). These molecules also bear three nitrogen atoms, present as one primary amino group and two amide moieties. Over a dozen members of this group of natural products are currently known (Ciminiello et al., 2011), the most recent being ovatoxin-f (Ciminiello et al., 2012c). The high degree of hydroxylation and the olefin moieties confer to these molecules an elevated stereogenic complexity. Palytoxin, for example, has 271 possible stereoisomers (64 stereogenic centres and seven double bonds with geometrical isomerism). Apart from palytoxin, the only analogue tentatively produced by cyanobacteria is 42-hydroxy-palytoxin (Kerbrat et al., 2011). The structural elucidation of this analogue benefited from previous knowledge on other palytoxins, and was achieved by high-resolution LC/MS analyses, coupled with NMR experiments, avoiding having to resort to degradation studies (Ciminiello et al., 2009). Still, for other analogues with less homology to fully characterized palytoxins, structural elucidation remains a demanding task, particularly due to the stereogenic centres, as exemplified by ovatoxin-a (Ciminiello et al., 2006, 2012a, 2012b).

Saxitoxin Saxitoxins and analogues, also designated as PSP toxins are alkaloids with many analogues (Figure 10.10), and are more comprehensively described in Chapter 4 of this

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OH

H2 N

O O

O

OH

OH

OH

HO

OH O

OH OH

HO

OH

OH OH

OH OH OH

OH O HO

N H

O N H

HO OH

OH

OH O

OH

H HO

OH

O

O

OH OH

OH OH HO OH

O

OH R

HO O

O

OH OH

OH OH

OH HO

palytoxin R = H 42-Hydroxy-palytoxin R = OH

H

OH OH

OH

OH

Figure 10.9

Structure of palytoxin.

O R4 H NH

R1N

NH2+ NH OH

H2N

R5 R2 Figure 10.10

R3

General structure of saxitoxins.

book. They have a wide occurrence, being reported every year in more countries, and new species of cyanobacteria assigned as its producers (Negri et al., 1995; Carmichael et al., 1997; Lagos et al., 1999; Dias et al., 2002).

Distribution of cyanotoxins Cyanotoxin production was initially associated with planktonic freshwater cyanobacteria, specially when present in high densities, or blooms. Nevertheless, with the increasing studies performed all over the world, and the more accurate, precise

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and sensitive methods to detect and quantify the toxins, other habitats have been studied and cyanotoxins have been found in a more diverse type of taxa, ecology and geography. Planktonic bloom forming cyanobacteria are still the most studied, but cyanotoxins have also been found in benthic-dwelling organisms. The occurrence of sheep death due to ingestion of lake clear water in Australia have shown that mat-forming Lyngbya, producing high amounts of saxitoxins and analogues, were the causative organisms (Negri et al., 1995). These benthic forms of cyanobacteria, such as L. wollei, have also been found to produce paralytic shellfish toxins in Florida Springs (Foss et al., 2012). From an environmental health risk assessment, these occurrences are very important, since they are found in clear river waters without signs of eutrophication. Aboal et al. (2005) found microcystins in clear water rivers in Spain, and they attributed their production to benthic forms of cyanobacteria. Oudra et al. (2009) also have found microcystis-producing Nostoc species in clear rivers in the High Atlas mountains in Morocco. Izaguirre et al. (2007) also found benthic Oscillatoriaceae-producing MC-LR. Faassen et al., (2012) reported the occurrence of dog neurotoxicosis due to the ingestion of benthic cyanobacteria in the Netherlands producers of homoanatoxin-a. Concerning their habitat, cyanotoxins are usually assigned to temperate or tropical areas of our planet, but high-latitude areas and deserts can also host cyanotoxins. Work in Antarctica has shown the occurrence of cyanotoxins in cyanobacteria mats from ponds (Hitzfeld et al., 2000). Recently, MC were also found in desert crusts from Qatar and were measured using ELISA and HPLC-PDA (Metcalf et al., 2012). Brackish and marine environments may also be prone to cyanotoxin occurrence. The most well known example is the Baltic Sea, a brackish water sea that, due to intense eutrophication, develops intense cyanobacteria blooms that are so large they can be seen from space. Nodularia is the most common genera but others, such as Aphanizomenon, Anabaena and Synechococcus, may also occur (Stal et al., 2003). In fact, bioaccumulation of cyanotoxins in aquatic trophic chains is quite well studied in the Baltic, using nodularin as an example (Karajalainen et al., 2008). In many areas of the world, estuaries belonging to eutrophic rivers can host toxinproducing cyanobacteria and expose local organisms, such as invertebrates and fish that are used for human consumption, to hazardous levels of toxins. Since bivalves and crustaceans may take up high concentrations of cyanotoxins without being harmed, this is a very important problem to be assessed and monitored. Unfortunately, cyanotoxins are not considered in the legislation concerning bivalves’ safety control, so this kind of toxin route is not currently assessed. In marine environments, not many of the known cyanotoxins have been reported, but toxins produced inland may be exported to coastal ecosystems, as recently reported (Miller et al., 2010; Vareli et al., 2012). In these ecosystems, cyanobacterial toxins have more seldomly been associated with human and animal intoxications, except with the cases of Moorea producens (formerly Lyngbya majuscula) -associated toxins that may cause the ‘swimmer’s itch’ (Cardellina et al., 1979). The main toxin associated with this species is the lyngbyatoxin with severe toxicity to humans including tumour promotion. More recently, the study of blooms of the marine cyanobacteria Trichodesmium spp. obtained in New Caledonia revealed the occurrence of the toxin palytoxin (Kerbrat et al., 2011). This toxin was first described as being produced by a coral, latter by marine dinoflagellates and now by marine cyanobacteria.

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Recent studies confirm that brackish water and marine cyanobacteria produce the amino acid β-Nmethylamino-L-alanine (BMAA), associated with neurodegenerative diseases such as Amyotrophic Lateral Syndrome (ALS). BMAA is accumulated in food webs of the North Atlantic, from cyanobacteria, to zooplankton, to invertebrates, and to vertebrates (fish), with increasing BMAA concentration within higher trophic levels (Brand et al. 2010; Jonasson et al. 2010). Free-living cyanobacteria, from freshwater, brackish water or marine environments, are not the only potential producers of cyanotoxins. BMAA is produced by cyanobacteria living symbiotically in a plant – Cycas spp. – although free-living cyanobacteria strains can also produce this neurotoxic amino acid (Cianca et al., 2012b). Other terrestrial symbiosis have been studied, and Kaasalainen et al. (2012) found that lichen symbiotic cyanobacteria –Nostoc spp. – have the gene clusters responsible for the production of microcystins and nodularin, and also detected the toxins by chemical methods. Since cyanobacteria have a wide range of habitats, and can colonize and also establish a wide range of associations with many other species in aquatic and terrestrial ecosystems, it will be interesting to unravel the potential spread of those toxins and to study the phylogenetic relationship among those species. This might be helpful, and even provide clues to understanding the reason why cyanobacteria produce these toxic metabolites.

Acknowledgments The authors wish to thank the European Cooperation in Science and Technology, COST Action ES 1105 ‘CYANOCOST – Cyanobacterial blooms and toxins in water resources: Occurrence, impacts and management’ for adding value to this study through networking and knowledge sharing with European experts and researchers in the field. This research was partially funded by the European Regional Development Fund (ERDF) through the project PharmAtlantic – Atlantic Area Operational Programme (Interreg IVB transnational grant 2009-1/117); by the ERDF through the COMPETE – Operational Competitiveness Programme; by national funds under the project ‘PEst-C/MAR/LA0015/2013’; by the project MARBIOTECH (reference NORTE-07-0124-FEDER-000047), co-financed by the North Portugal Regional Operational Programme (ON.2 – O Novo Norte) under the National Strategic Reference Framework (NSRF), through the ERDF.

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C H A P T E R 11

Marine toxins and climate change: the case of PSP from cyanobacteria in coastal lagoons Antonella Lugliè1 , Silvia Pulina1 , Milena Bruno2 , Bachisio Mario Padedda1 , Cecilia Teodora Satta1 & Nicola Sechi1 1 Department 2 Department

of Architecture, Planning and Design, University of Sassari, Italy of Environment and Primary Prevention, Istituto Superiore di Sanità, Italy

Introduction The Millennium Ecosystem Assessment (2003) defines the coastal ecosystem category as the region of ‘Interface between ocean and land, extending seawards to about the middle of the continental shelf and inland to include all areas strongly influenced by the proximity to the ocean’. The region covers a wide typology of aquatic ecosystems (e.g. wetland, marshes, estuaries, rias, fjords, coastal lagoons) and also artificial environments, such as harbours (Perthuisot & Guelorget, 1992). Coastal ecosystems are reported as brackish, estuarine, paralic and transitional in relation to differing historical perspectives and scientific points of view (Tagliapietra et al., 2009). Modification of food web structure and functioning has direct socio-economic implications, because coastal waters are highly productive areas in terms of primary, secondary and exploited-resources production (Costanza et al., 1997). It is known that the pressures of human activities on coastal ecosystems have been dramatically increased in the last few decades, and this process will continue and evolve, especially in developed countries (Viaroli et al., 2007). Only a relatively limited number of studies have analyzed the vulnerability of coastal zones in the light of global climate change (Box 11.1). Furthermore, a very few studies have evaluated the effect of global climate change on the appearance or magnification of eutrophication in coastal systems. Worsening eutrophication was recently assessed as one of the reasons behind the worldwide increase of harmful algal blooms (HABs; Heisler et al., 2008; Box 11.2). In the most degraded ecosystems, microalgae communities become dominated by autotrophic cyanobacteria species (Scheffer et al., 1997; Abrantes et al., 2006). Consequently, a rise of new scenarios in respect to the impacts of their harmful blooms (CyanoHABs; Carmichael, 2008) should be considered. However, our limited understanding of ecosystem responses to the numerous drivers of climate change, and poor knowledge of the adaptive genetic and phenotypic responses of algae and cyanobacteria, make it very difficult to make predictions (Hallegraeff, 2010).

Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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Box 11.1 Global climate change.

‘Climate change refers to any change in climate over time, whether due to natural variability or as a result of human activity,’ is the Intergovernmental Panel on Climate Change (IPCC) usage, whereas climate change refers to ‘a change of climate that is attributed directly or indirectly to human activity that alters the composition of the global atmosphere and that is in addition to natural climate variability observed over comparable time periods,’ in the usage of the Framework Convention on Climate Change (IPCC, 2007). Anthropogenic emissions caused by the burning of fossil fuels and modification of land use – particularly deforestation – are the most important human activities in increasing greenhouse gases in the atmosphere (IPCC, 2007), and this has a causal link to global climate change. Climate strongly influences biological ecosystem structure (i.e. distribution and diversity of animals and plants) and ecological processes in the biosphere (Stenseth et al., 2002) and, consequently, ecosystem functions. Changes in climatic conditions may alter the structure and dynamics of pelagic ecosystems (IPCC, 2007), and plankton are good indicators of those climate changes (Hays et al., 2005). The effects of climatic change on coastal waters may include an increase in sea water temperature, a rise in sea level, changes in the hydrodynamic of water masses, changes in water salinity, an increase in dissolved carbon dioxide, an increase in the frequency of extreme weather events and an increase in the appearance of eutrophication processes (IPCC, 2001; Rahmstorf, 2007). Ultraviolet B radiation is expected to increase in European regions by about 10% in coming years, particularly during spring (WMO, 2007).

Box 11.2 Phytoplankton and harmful algal blooms (HABs).

Plankton ‘refers to the collective of organisms that are adapted to spend part or all of their lives in apparent suspension in the open water of the sea, of lakes, ponds and rivers.’ (Reynolds, 2006). Phytoplankton includes the planktonic microscopic organisms that are able to photosynthesize. Phytoplankton is the most important primary producer in aquatic ecosystems, and it is at the base of the trophic chains and biogeochemical cycles (Kaiser et al., 2005). Phytoplankton consists of a broad variety of taxa, including Cyanobacteria, Prochlorophyceae, Chlorophyceae, Euglenophyceae, Prasinophyceae, Dinophyceae, Bacillariophyceae, Chrysophyceae, Cryptophyceae, Rhaphidophyceae and Prymnesiophyceae. These organisms may be eukaryotic or prokaryotic (i.e. Cyanobacteria, including Prochlorophyta), and they may lead a solitary or colonial existence (Lee, 2008). Numerous factors act on the growth and seasonal successions of phytoplankton and, in particular, physical-chemical (i.e. light intensity, temperature, and nutrient availability), climatic, and biological factors (i.e. the interactions with the other organisms) (Harris et al., 1986; Reynolds, 2006). Phytoplankton has a fundamental role in the food chain in aquatic environments, and its natural ability to give rise to large proliferations (blooms) is considered a benefit for the secondary production, both in natural environments and aquatic farms. Nevertheless, in some instances, these events can cause negative impacts on humans (both on health and economic activities), on aquatic organisms, and on the whole ecosystem. These types of blooms are known as harmful algal blooms (HABs). This phenomenon is known from ancient times, but in recent decades it has been showing a strong increase, at least apparently (e.g. Hallegraeff, 1993). The expansion of HABs is correlated with different factors, such as eutrophication (Heisler et al., 2008).

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Definition of coastal lagoons and main ecosystem characteristics Transitional aquatic ecosystems are physically located inside the shoreline and in ‘transition’ between the continental and marine biomes, such as lagoons (Tagliapietra et al., 2009). Lagoons are inshore water bodies where marine waters and freshwaters penetrate (Bramanti, 1988). They are shallow aquatic ecosystems, characterized by different physical and ecological boundaries and gradients – between water and sediment, pelagic and benthic assemblages, lagoon and marine, freshwater, terrestrial systems and with the atmosphere (Pérez-Ruzafa et al., 2011a). Consequently, they can be defined as ecotones among freshwater, marine and terrestrial biotopes, so their abiotic structure is a consequence of coastal geomorphological and hydrological processes, natural vegetation and land use in the watershed (Basset et al., 2008 and references therein) (Figure 11.1). The exchange of water masses of different origins (i.e. freshwater and marine) is crucial to the natural functioning of lagoons (Figure 11.2). The water mixing controls ecological processes (Piccini et al., 2006), with salinity spanning from freshwater to hyperhaline conditions, depending on the water balance (Kjerfve, 1994). Due to their shallow depth, light usually arrives at the sediment-water interface intensively, hydrodynamic are influenced by bottom topography, and wind acts on the whole water column, determining the re-suspension of materials, nutrients and small organisms

Figure 11.1 Schematic representation of a coastal lagoon and its main features and uses. Source: Adapted from Troussellier 2007.

(a)

(b)

(c)

Figure 11.2 Lagoon typologies according to Kjerfve (1986), based on the degree of water exchange with the adjacent sea. From left to right: (a) leaky; (b) restricted; (c) choked. Source: according to Kjerfve, 1986.

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from the surface of the sediment. Lagoons are characterized by high sediment surface area to water volume ratios. Therefore, processes occurring within the sediment, and at the water-sediment interface, can strongly influence both water quality and the biota (Castel et al., 1996; Viaroli et al., 2004). The synergy of all the physical and biological characters makes coastal lagoons highly dynamic ecosystems, considered among the most productive in the exosphere. Moreover, these ecosystems are populated by a large variety of organisms, and this permits their classification as environments with maximum value of biodiversity (Tomàs Vives, 1996). Contemporaneously, lagoons are extremely fragile ecosystems and, consequently, they will be particularly affected by future changes (Vidussi et al., 2011).

Ecosystem goods and services and human exploitation of coastal lagoons Coastal lagoons were early sites for human settlement, and continue to the present day as important centres for economic, social and cultural development (Barraqué et al., 1998). Lagoons provide key ecosystem goods and services (see Box 11.3), such as water quality improvement, reducing pollutant loads transported by rivers to the coastal marine areas, fisheries resources and recreational areas for human populations and habitats and food for migratory and resident animals (Levin, 2001; Figure 11.1). Box 11.3 Ecosystem functions, goods and services.

Ecosystem functions are ‘the capacity of natural processes and components to provide goods and services that satisfy human needs, directly or indirectly’ (De Groot, 1992). Consequently, the concept of goods and services imply human values, because they are the benefits that human populations obtain, directly or indirectly, from ecosystem functions (Costanza et al., 1997) of both natural and human-modified ecosystems (Millennium Ecosystem Assessment, 2003). On the other hand, ecosystem functions depend on ecosystem structures (e.g., plant and animal species composition) and processes (e.g., photosynthesis), which result from the complex relationships between abiotic and biotic components of the ecosystems. Thus, ecosystem goods and services are the translations of basic ecological structures and processes into value-laden entities (De Groot et al., 2002). Ecosystem functions can be grouped into four primary categories (De Groot et al., 2000): • Regulation functions with services such as clean air, water and soil, and biological control; • Habitat functions, with services such as refuge and reproduction habitat to wild plants and animals, contributing to the conservation of biological and genetic diversity and evolutionary processes; • Production functions with ecosystem goods for human consumption, such as food and raw materials to energy resources and genetic material; • Information functions with services such as opportunities for reflection, spiritual enrichment, cognitive development, recreation and aesthetic experience for humans.

The ability to provide goods and services has led to an over-exploitation of these ecosystems by humans and, consequently, to major structural changes to optimize direct anthropogenic activities. In addition, lagoons also suffer indirect human pressure resulting from intense activities on their watersheds (e.g. intensive agriculture and breeding, industry), as well as global climate changes (Lloret et al., 2008). Climatic variations intersect with or override anthropogenic changes, and affect biota and all trophic levels in lagoons. In relation to their exploitation, and because they are highly

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vulnerable to terrestrial inputs, coastal lagoons are considered naturally stressed environments, subject to frequent fluctuations and alterations (UNESCO, 1981; Kjerfve, 1994). Lagoons are extensively exploited for fishing and aquaculture (Pérez-Ruzafa et al., 2011b). Aquaculture practices are increasing worldwide, due to progressive impoverishment of natural fish populations and increasing demand for fish-associated proteins (FAO, 2000). Farming activities have a strong negative effect on natural fish populations, because of the large taking of fish from natural stocks to be converted in farmed fish feed (Naylor et al., 2000). Moreover, fish farms amplify the deterioration of coastal areas as a consequence of the high loads of organic matter and nutrients introduced (Hall et al., 1992; Christensen et al., 2000; Bartoli et al., 2005). In contrast, the extensive cultivation of filter-feeding molluscs is considered to have lower impact compared to fish farming. Shellfish production is dependent upon in situ phytoplankton productivity, not on inputs of feeds (Naylor et al., 2000). The subsequent shellfish harvesting allows nutrients to be removed from the system, limiting phytoplankton growth and reducing eutrophication (Newell, 2004). These affirmations are not valid in all lagoons, and largely depend on the farming intensity and typology, climate and geographic localization, hydrodynamic and morphology of the considered ecosystems (Cloern, 2001). For example, in shallow lagoons, characterized by low tidal exchanges, such as those in the Mediterranean Sea, shellfish cultivation may result in the formation of ‘hot spots’ of eutrophication and nutrient recycling (Souchu et al., 2001; Nizzoli et al., 2005). In fact, the production of faeces and pseudo-faeces by the farmed molluscs and their deposition (biodeposition) on the bottom increases inputs of labile organic matter to the superficial sediment (Mesnage et al., 2007). Consequently, oxygen demand increases, due to both the deposited organic matter and the cultivated organisms and their epiphytic community. In addition, nutrients are rapidly recycled to the water column as inorganic nutrients, stimulating phytoplankton growth (Nizzoli et al., 2005).

Eutrophication and climate change in coastal lagoons Eutrophication is the enrichment of nutrients, in particular nitrogen and phosphorus, in aquatic ecosystems. It is one of the most important processes of degradation of lagoons, often due to the use of lagoons as outlets for civil and industrial wastes, and for the agricultural and zootechnical activities in the catchments (Vicente & Miracle, 1992; Pastres et al., 2004). Direct human activities in the lagoons, such as aquaculture, also influence trophic status (see section 11.3 above). One of the first biological effects of nutrient enrichment is the proliferation of primary producers, increasing the produced biomass and, consequently, the demand for oxygen needed for biological degradation processes (Menéndez & Comin, 2000). This may, under the concomitance of different conditions (such as the increase of temperature and the lack of wind), cause anoxia and lead to dystrophic crises, with a strong loss of biodiversity for the extended demise of fish and other aquatic organisms (Bachelet et al., 2000; Nizzoli et al., 2005; Carlier et al., 2007). The general patterns described when a strong enrichment of nutrients is observed include the substitution of macrophytes by macroalgae at the benthic level in a first phase, and then a shift to a phytoplankton-based system (Box 11.2) with anoxic events (Niehnius, 1992). Lagoons exclusively dominated by phytoplankton, particularly

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cyanobacteria, are considered the final stage of eutrophication in coastal lagoons (Viaroli et al., 2010). Moreover, one of the effects of eutrophication is the development and persistence of HABs, caused by both toxic and nuisance algae (Ferreira et al., 2007). Global climate change, together with environment degradation due to eutrophication, can modify community composition by promoting tolerant species and reducing sensitive ones (Vinebrooke et al., 2004). Variability in climatic conditions can significantly affect phytoplankton biomass and composition on a wide range of spatial and temporal scales (Hays et al., 2005). The factors that most commonly and immediately impact phytoplankton dynamics in coastal lagoons are wind (as it relates to mixing and circulation) and rainfall (in terms of inputs of freshwater, nutrients, and turbidity from the watershed and atmosphere). The influence of climate is not limited to local conditions, but originates with shifts in large-scale climate (Cloern et al., 2007; Borkman & Smayda, 2009), which can influence coastal circulation (retention/dispersion) or transport of nutrient-rich upwelled water or toxic algae blooms into marine areas adjacent to lagoons. It is clear that the impact of changes in temperature, rain and wind patterns on phytoplankton structure and abundance may vary greatly from one coastal system to another (Zingone et al., 2010). Recent studies have shown that long-term climate variability may induce alterations in the magnitude and position of phytoplankton blooms, and changes in their composition and phenology, that may be propagated to higher trophic levels (Barbosa et al., 2010 and references therein).

Cyanobacteria in coastal lagoons It is well established that eutrophication enhances the risk of cyanobacterial blooms (O’Neil et al., 2012). Toxic CyanoHABs are known occurring in eutrophic river estuaries and harbours, in addition to freshwater ecosystems. More recently, the occurrence of CyanoHABs in coastal waters has been predicted to increase in the future, due to the interaction between eutrophication and climate change (Paerl & Huisman, 2008, 2009). It has been shown that an increase in surface water temperatures due to changing global climate could play an important role in the proliferation of cyanobacterial blooms (Peperzak, 2003; Paerl & Huisman, 2008; Paul, 2008). Specifically, cyanobacteria dominate phytoplankton assemblages under higher temperatures, due to both physiological (e.g. more rapid growth) and physical factors (e.g. enhanced stratification), with individual species showing different temperature optima (O’Neil et al., 2012). Beyond the direct effects on cyanobacterial growth rates, rising temperatures will change many of the physical characteristics of aquatic environments, in ways that may be favourable for cyanobacteria (e.g. decreasing in water viscosity, increasing in nutrient diffusion towards the cell surface) (Peperzak, 2003). Significantly less is known regarding how increasing carbon dioxide (CO2 ) concentrations will affect cyanobacteria, although some evidence suggests several genera of cyanobacteria are well-suited to bloom under high pH and low concentrations of CO2 (Oliver & Ganf, 2000; Qui & Gao, 2002). Climate change may also affect the salinity of coastal waters, due to rising sea levels, an increase in drought frequency, and duration or increases in precipitation due to storms. Although many eukaryotic phytoplankton cannot tolerate changes in salinity, a number of cyanobacterial species have very euryhaline tolerances (O’Neil et al.,

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2012 and references therein). There are several genera of euryhaline cyanobacteria that bloom in estuarine environments with a range of salinities. Therefore, changes in salinity may affect the composition of community species and potential toxin concentrations and distribution (Laamanen et al., 2002; Orr et al., 2004; Tonk et al., 2007). A recent study on wild Mitilus galloprovincialis from the Amvrakikos Gulf (Greece) showed microcystin accumulation, due to local toxic blooms of the euryhaline cosmopolitan species Synechococcus sp. and Synechocystis sp. (Vareli et al., 2012). Padedda et al. (2012) reported a cyanobacteria affirmation in a Mediterranean lagoon, Cabras Lagoon, just after heavy rainfall (November, 2001), and a consequent sharp decrease of salinity and increase in nutrient availability. Pulina et al. (2011) highlighted a drastic variation at the order level (Chroococcales-Oscillatoriales-Nostocales) in the Cabras Lagoon, in response to another high rainfall event (November, 2008) after a relatively long period of stability. Both Padedda et al. (2012) and Pulina et al. (2011) indicated temperature, salinity and nutrients as the environmental parameters that most influenced the phytoplankton composition and dynamics. Benthic cyanobacteria can also undergo intense growth in habitats with differing salinity. Species of some benthic cyanoHAB genera, including Lyngbya and Oscillatoria, are well adapted to freshwater or saline (including hypersaline) conditions (Paerl & Fulton, 2006).

Paralytic shellfish poisoning and cyanobacteria in coastal lagoons A significant aspect concerning the intense occurrence of cyanobacteria in transitional and marine ecosystems is related to their potential toxicity, which could also affect human health (Box 11.4). In general, cyanobacteria produce, as secondary metabolites, a wide variety of toxins, known as cyanotoxins, which can be classified into categories that reflect their biological effects on the systems and organs that they affect most strongly: hepatotoxins, neurotoxins, cytotoxins and dermatotoxins (Sivonen & Jones, 1999; Carmichael, 2002; Codd et al., 2005). Paralytic shellfish toxins (PSTs), responsible for paralytic shellfish poisoning (PSP), pose one of the most serious threats, due to the extreme toxicities of the compounds involved (see Box 11.5). PSTs are better known from marine dinoflagellates, even if more recently these toxins have also been found in freshwater and brackish bloom-forming cyanobacteria belonging to the orders Oscillatoriales, Nostocales and, more recently, Chroococcales (Table 11.1). Up to now, all documented human PSP cases have been caused by toxic marine dinoflagellates (Deeds et al., 2008), although Pitcher et al. (2001) postulated that cyanobacteria may be the source of the PSP toxicity in the abolone Haliotis midae along the west coast of South Africa in 1999. The available data still does not enable the complete clarification of environmental factors that influence toxin production and mechanisms of toxin release into extracellular medium (Batoréu et al., 2005). However, Soto-Liebe et al. (2012) demonstrated that a strain of Raphidiopsis brookii was capable of releasing PSTs into the extracellular medium as a protective mechanism against salt variation in the environment. This trend could enhance the fitness and adaptability of cells, and explain the invasive capacity of PSP toxin-producing cyanobacteria within a broad range of aquatic ecosystems (Soto-Liebe et al., 2012).

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Box 11.4 Cyanobacteria in marine ecosystems.

Cyanobacteria comprises unicellular prokaryotes that possess chlorophyll a, allowing to perform oxygenic photosynthesis (Castenholz & Waterbury, 1989). The prominent habitats of cyanobacteria are freshwater and marine environments, growing in water that is salty, brackish or fresh, in cold and hot springs, and in environments where no other microalgae can exist (Mur et al., 1999). The prevalence of cyanobacterial blooms in aquatic environments generally follows the hierarchy of freshwater > estuarine/brackish > marine systems (Fristachi & Sinclair, 2008). The intense proliferation of cyanobacteria in coastal eutrophic ecosystems at all latitudes is well ¯ documented in several studies (Kanoschina et al., 2003; Sorokin et al., 1996; Gasiunaite et al., 2005). The marine site of the most widely studied cyanobacterial blooms is the Baltic Sea, one of the largest brackish water bodies in the world (Edler, 1979; Stal et al., 2003). The primary bloom-forming species are Aphanizomenon flos-aquae, Nodularia spumigena, and Anabaena spp. Until a short time ago, it was believed that the toxicity of cyanobacterial blooms in the Baltic Sea was attributed exclusively to the species N. spumigena (O’Neil et al., 2012 and references therein) but, recently, strains of the genus Anabaena producing the hepatotoxin microcystin were reported in the Gulf of Finland (Halinen et al., 2007). Regarding purely marine ecosystems, the most conspicuous marine cyanobacterial bloom formers are members of the genera Lyngbya, Trichodesmium, and Synechococcus (O’Neil et al., 2012). L. majuscula is the most commonly reported species of the genus, and is found mainly in tropical waters. There have been several detailed reviews of toxins associated with L. majuscula and related deleterious effects on humans (Osborne et al., 2001 and references therein). Trichodesmium is the most abundant bloom-forming cyanobacteria in the marine pelagic environment, with a global distribution in oligotrophic waters of tropical and subtropical oceans (Capone et al., 1997). Trichodesmium blooms have caused various human health problems in many geographical areas worldwide, due to different toxins (O’Neil et al., 2012 and references therein), among which is also saxitoxin (Proença et al., 2009). Synechococcus is a cosmopolitan open ocean cyanobacterium which recently has also been increasingly reported in brackish and eutrophic environments (Paoli et al., 2007). The discovery of a haline strain of microcystin-producing Synechococcus is significant, as production of this toxin has previously only been described in freshwater strains (O’Neil et al., 2012 and references therein).

Box 11.5 Paralytic shellfish poisoning and cyanobacteria in coastal marine ecosystems.

Paralytic shellfish poisoning (PSP) is a very well known human syndrome caused by acute intoxication after the ingestion of seafood contaminated with paralytic shellfish toxins (PSTs). PSTs are a family of heterocyclic guanidines (tricyclic tetrahydropurine derivatives), which comprise saxitoxin and at least 25 derivatives (STXs; see Table 11.2), potent water-soluble neurotoxins acting in the mammalian nerve cells by binding to sodium channels, responsible for the flux of sodium in nerve and muscle cells (van Apedoorn et al., 2007). This prevents the conductance of signals along the neuron, with the final result being muscular paralysis. Symptoms range from nausea, vomiting, numbness of the lips and tongue and muscle paralysis, to death by cardio-respiratory arrest. Filter feeders such as mussels, cockles, oysters and scallops feed on toxic phytoplankton, transferring the PSTs from the gills to digestive organs, where they become concentrated (Bricelj & Shumway, 1998). If the contaminated filter feeders are ingested, the toxic seafood-related syndrome is known in humans as paralytic shellfish poisoning (PSP). Most humans who experience PSP have consumed toxic bivalves but, occasionally, fisheries closures and human STXs intoxications have also been documented in several non-filter-feeding vectors, such as toxic gastropods and crustaceans and, rarely, toxic fish (Deeds et al., 2008). The intensive presence of shellfish farms activities in coastal marine areas, and the wide human consumption of molluscs, makes this issue extremely relevant. Since the first episodes, described in the 18th century, many reports of PSP associated with human severe intoxication and death have become available worldwide (Batoréu et al., 2005). As a result, legislation controls the allowable

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concentration of saxitoxins in shellfish harvested for human consumption, and there is a substantial monitoring programme in many countries. This includes the monitoring of PSTs in shellfish by mouse bioassay (and toxin identification by HPLC-FLD), and a shellfish harvesting prohibition if toxin levels exceed 800 μg STX eqv/1000 g edible tissue (Batoréu et al., 2005). In, 1993, the estimated incidence was 2000 PSP cases per year, with 15% of mortality (Hallegraeff, 1993). The successful implementation of monitoring programs for the presence of both STX-producing microalgae and the presence of STXs in shellfish in many countries has led to minimize public health risks (Deeds et al., 2008). Nowadays, death by PSP episodes occurs mainly in those countries where prevention programs were not still adopted (Batoréu et al., 2005).

Table 11.1

Cyanobacteria species producing Paralytic shellfish toxins (PSTs).

Species

Toxins

References

Anabaena circinalis

C1, C2, gonyautoxin 2, gonyautoxin 3, saxitoxin, gonyautoxin 5, dc-saxitoxin, dc-gonyautoxin 2, dc-gonyautoxin 3 saxitoxin, neosaxitoxin saxitoxin neosaxitoxin, saxitoxin, gonyautoxin 2, gonyautoxin 3 LWtoxins 1, 2, 3, 5, 6 gonyautoxin 1, gonyautoxin 2, gonyautoxin 3, gonyautoxin 4 saxitoxin, dc-saxitoxin

Humpage et al., 1984

Aphanizomenon gracile Aphanizomenon issatschenkoj Cylindrospermopsis raciborskii Lyngbya wollei Microcystis aeruginosa Raphidiopsis brookii

Ledreux et al., 2010 Li et al., 2003 Lagos et al., 1999 Onodera et al., 1997 Sant’Anna et al., 2011 Yunes et al., 2009

Conclusions There is increasing social awareness concerning the need to protect and manage coastal lagoons, because of human interests and the vulnerability of those aquatic ecosystems (Pérez-Ruzafa et al., 2011b). Understanding the interactions of global change with environment degradation due to eutrophication is essential for identifying future possible impacts on coastal lagoons, and for establishing effective measures of coastal planning and management (Lloret et al., 2008). This is of crucial importance, especially where human health is involved, as in the case PSP toxicity. Phytoplankton, as the basis of the food web, constitutes the biological community on which scientific attention is focused when a management plan is needed or an assessment of the ecosystem health is required (Sin et al., 1999; Gameiro et al., 2007). Investigations are needed to elucidate the extent of the occurrence and distribution of PSTs and cyanobacterial producers in nature. Further insights must be obtained on their diversity and their fundamental biology, in addition to those on the biosynthesis of PSTs, their metabolic and eco-physiological function, and on the role of chemical transformation of the different toxins in shellfish and the environment (Weise et al., 2010). These studies appear particularly relevant, considering the recent affirmation of cyanobacterial potential producers of PSTs in eutrophic coastal lagoons or in brackish waters worldwide in recent decades.

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Table 11.2 Structures of saxitoxins STXs (van Apedoorn et al., 2007. Reproduced with permission of Wiley and Sons Ltd.). Name of toxin

STX GTX2 GTX3 GTX5 (B1) C1 (epiGTX8) C2 (GTX8) C3 C4 neoSTX GTX1 GTX4 GTX6 (B2) dcSTX dcneoSTX dcGTX1 dcGTX2 dcGTX3 dcGTX4 LWTX1 LWTX2 LWTX3 LWTX4 LWTX5 LWTX6

Variable chemical groups in toxins R1

R2

R3

R4

R5

H H H H H H OH OH OH OH OH OH H OH OH H H OH H H H H H H

H H OSO3 – H H OSO3 – H OSO3 – H H OSO3 – H H H H H OSO3 – OSO3 – OSO3 – OSO –

H OSO3 – H H OSO3 – H OSO3 – H H OSO3 – H H H H OSO3 – OSO3 – H H H H OSO –

CONH2 CONH2 CONH2 CONHSO3 CONHSO3 CONHSO3 CONHSO3 CONHSO3 CONH2 CONH2 CONH2 CONHSO3 H H H H H H COCH3 COCH3 COCH3 H COCH3 COCH3

OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH H OH OH H OH H

3

H H H H

3

H H H

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C H A P T E R 12

Microalgae as a source of nutraceuticals Sushanta Kumar Saha1 , Edward McHugh2 , Patrick Murray1 & Daniel J. Walsh1 1 Shannon 2 Algae

Applied Biotechnology Centre, Limerick Institute of Technology, Ireland (ROI) Health Ltd, Rooaunmore, Galway, Ireland (ROI)

Introduction The term ‘nutraceutical’ is derived from ‘nutrition’ and ‘pharmaceutical’. Therefore, nutraceuticals may be loosely defined as a food or food products with a nutritional attribute, as well as a physiological benefit providing protection from and amelioration to disease. The nutraceutical concept was coined by DeFelice in 1989, who founded the Foundation for Innovation in Medicine (FIM). Depending on geographical location, the definitions, regulations and regulatory authorities pertaining to nutraceuticals vary. Terms such as dietary supplements, food supplements, foods for special dietary use, foods for specific health use and health foods differentially classify ‘nutraceuticals’ in the United States, the European Union, India, Japan and China, respectively (Frost & Sullivan, 2011). Nutraceutical products may include isolated nutrients, dietary supplements, nutritious biomass, herbal products, genetically modified food, fortified foods with essential vitamins, minerals, amino acids, antioxidants, essential fatty acids and high value-added bioactive molecules (Table 12.1). Nutraceutical-containing foods may be a single natural component, either in powder, tablet, capsule, or in liquid form. However, no nutraceutical product is marketed as a medicine with claims to potentiate, antagonize or otherwise modify a physiological function as a pharmacologically active substance. They are essentially neither complete foods nor medicines. However, a nutraceutical may be an enriched fraction of a known natural bioactive molecule incorporated into a suitable food matrix (e.g., ACE inhibitory peptides in a yogurt based beverage). Each nutraceutical product is expected to possess specific health benefits, due to the presence of either antioxidant compounds, signalling-pathway modulators or other bioactive molecules. Microalgae are the most suitable resource for several known bioactive molecules, such as: antioxidant pigments chlorophyll, β-carotene, xanthophylls, and phycobilipigments; polyphenols and α-tocopherols; polyunsaturated fatty acids (PUFAs); and ascorbate, glutathione and ergothioneine. Thus, microalgae have gained attention for their use for health benefits. There are several nutraceutical products available in the market from cyanobacteria and microalgae, such as Spirulina platensis, Arthrospira maxima, Aphanizomenon flos-aquae, Chlorella vulgaris, Dunaliella salina, Haematococcus pluvialis and Nannochloropsis oculata. Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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Table 12.1 Nutraceutical products, divided into 11 categories with typical commercial products outlined. The potential of microalgae to offer a competitive commercial alternative source to some of these products is a reality. The extraction of multiple high value-added biomolecules from microalgal biomass may promote a more sustainable, cost-effective process, leading to the better utilization of microalgae. (Adapted from Frost and Sullivan, 2011) Nutraceutical category

Example

Amino acid and (poly)-peptide

Arginine, bioactive peptides, digestive enzymes, glutamate, immunoglobulins, isoleucine, lactoferrin, leucine, lysine, valine β-glucan, digestion resistant starch and maltodextrin, gum, insoluble fibre, inulin, pectin, polydextrose Calcium, iron, magnesium, potassium, selenium, sodium, zinc Isoflavone, isothiocyanate, lignan, phytosterol, polyphenol, tocotrienol Astaxanthin, β-carotene, lutein, lycopene, zeaxanthin Isomalt, lactitol, maltitol, xylitol Arachidonic acid, conjugated linoleic acid, docosahexaenoic acid, eicosapentaenoic acid, gamma linoleic acid Fructooligosaccharides, glucooligosaccharides, inulin, polydextrose, soya bean oligosaccharides, xylooligosaccharides Living organisms and/or bacterial cultures A, B1 , B2 , B6 , B12 , Biotin, C, D, E, Folic acid, K, niacin, pantothenic acid Chondroitin, coenzyme Q10 , glucosamin, inositol, lipoic acid

Fibres Mineral Phytochemical Pigment and carotenoid Polyol Polyunsaturated fatty acid Prebiotic Probiotic Vitamin No set category

Algae (both macro- and micro- in nature) have long been used by indigenous populations around the world especially in China, Japan and the Republic of Korea. Coastal communities of Indonesia and Malaysia are known to eat fresh macroalgae as a daily dietary staple. Interestingly, migration of people from these countries has disseminated the culture of alga consumption. While not taxonomically microalgae, a few species of cyanobacteria, such as Anabaena, Nostoc and Spirulina, have been used as human food in Chile, China, Mexico, Peru and Philippines. ‘Nostoc Balls’ (Nostoc commune) and ‘Fat Choy’ (Nostoc flagelliforme) are two terrestrial cyanobacteria which are very popular in Chinese cuisine from a nutritional and traditional health perspective. ‘Nostoc balls’ are available in Asian markets as dried cell biomass for use in a variety of meals, for their nutritional, organoleptic and functional (thickening) properties. ‘Fat Choy’ is used during the Chinese Lunar New Year. ‘Fat choy’ is the black, hair-like strands of the cyanobacterium Nostoc flagelliforme, native to northern China and other regions. The cyanobacterium Nostoc was first used by Chinese people 2000 years ago to survive from nutrient deprivation (Spolaore et al., 2006). Likewise, the Kanembu people near Lake Chad in Africa used to harvest the algal blooms to make the sun-dried cakes called ‘Dihé’, which they used to eat for a healthy life. During the 1960s, this bloom-forming miracle alga was identified as Spirulina, which is nowadays considered as ‘Super Natural Food’. According to an Algae Industry Magazine report, ‘more than 250 tons of Spirulina dry biomass is produced per year by the women around Chad Lake as the highest volume of Spirulina with the lowest production cost’. The evolution of marine algae in competitive harsh environments has resulted in their development of specialized defence strategies, comprising chemically

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and structurally diverse compounds associated with different metabolic pathways (Balandrin et al., 1985). Microalgae represent a very large group of phototrophic and heterotrophic organisms contributing to this diversity, as they are ubiquitously distributed throughout the biosphere. Due to their predominantly phototrophic life, microalgae are exposed to high oxygen and free radical stresses, which has led to the evolution of efficient protective mechanisms against oxidative damage (Becker, 1994). Microalgae are a natural reservoir, playing a crucial nutritional role in the biosphere of supplying both aquatic and terrestrial food chains. The use of microalgae for their physiological health benefit is increasing, in response to market demands which are geographically dependent (Frost & Sullivan, 2011). Due to advanced, widely available electronic and communication technologies, consumers are more informed about scientific advances than before, and are becoming increasingly more health-conscious. Foods that enhance health are thus increasing in demand. Two key drivers for increased nutraceutical demand were identified in a Frost & Sullivan marketing report in 2011. Specifically, the last decade has seen an increase in the number of scientific studies publicized, linking nutraceutical use with the prevention of chronic illness development and progression. Also, healthcare costs are increasing, and both the aging population and younger generations are choosing alternative preventative measures to avoid the costs associated with conventional treatments which may be experienced at some point in the future (Frost & Sullivan, 2011).

Microalgal taxa Microalgae can be simply defined as microscopic algae with either eukaryotic or prokaryotic types of cellular organization. Microalgae represent the taxa of Chlorophyceae, Eustigmatophyceae, Bacillariophyceae, Cyanobacteria (aka blue-green algae) and few species of Rhodophyceae. • Microalgae belonging to Chlorophyceae are green, contain chlorophyll-a and chlorophyll-b light harvesting pigments, and are flagellated at certain stages of growth. • Microalgae belonging to Eustigmatophyceae are mostly smaller in cell size (2–4 μm) than the former, have uni- or bi-flagellate, coccoid to spherical shaped cells, are pale-green in colour containing chlorophyll-a, and have cell walls containing polysaccharides. • Microalgae belonging to Bacillariophyceae are known as diatoms. They contain chlorophyll-a and chlorophyll-c, have silicate cell walls and are mostly unicellular, but may form colonies of various shapes (e.g., zigzag, filaments, fans, ribbon and stars). • Cyanobacteria are prokaryotic microalgae possessing unicellular, multicellular or colonial cell structures, contain chlorophyll-a, are blue-green in colour due to chlorophyll-a and phycocyanin, and have mucilaginous cell walls or sheaths. • Only a few species of the taxa Rhodophyceae are microalgae. Rhodophyceae are red in colour due to a high phycoerythrin content, contain chlorophyll-a and chlorophyll-c, have double-layered cell walls with inner cellulose and outer pectin layers, and use starch as their major storage carbohydrate.

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World biodiversity of microalgae The world biodiversity of algae in general, and microalgae in particular, is uncertain as, to date, none of the estimates are in agreement when compared. This would require a very comprehensive study covering live species, fossil species and herbarium species. A recent study using a conservative approach estimated the biodiversity to be 72 500 algal species. Of these, 44 000 species have been reported and 33 248 names have been processed up to June 2012 (Guiry, 2012). The outcome of this study is available as a taxonomic online database known as AlgaeBase (http://www.algaebase.org), which has information on up to one million algal species. As per the recent study, 18 755 microalgal species representing cyanobacteria and microalgae belonging to Porphyridiophyceae, Rhodellophyceae, Chlorophyceae, Pavlovophyceae, Bacillariophyceae and Eustigmatophyceae are detailed. Using a conservative approach, and adding the members of dinoflagellates and Euglenoid flagellates to these species, the estimate increases to 24 025 species (Guiry, 2012).

Microalgae in culture collections and under commercial cultivation There is no absolute figure on the number of microalgal isolates that are cultivable and stored in culture collections. Considering known culture collections around the world, it has been estimated that 14 953 strains of microalgae are available in pure culture form. The considered culture collections are: 1 CCAP, Culture Collection of Algae and Protozoa, UK (1510 microalgae). 2 UTEX, The culture collection of Algae at the University of Texas at Austin, USA (1008 microalgae). 3 PCC, The Pasteur Culture Collection of Cyanobacteria, France (750 cyanobacteria). 4 ATCC, The American Type Culture Collection, USA (168 microalgae). 5 NFMC, National Facility for Marine Cyanobacteria at Bharathidasan University, India (336 marine cyanobacteria). 6 SCCAP, Scandinavian Culture Collection of Algae and Protozoa at the University of Copenhagen, Denmark (229 microalgae). 7 CPCC, Canadian Phycological Culture Centre at the University of Waterloo, Canada (320 microalgae). 8 SAG, Culture Collection of Algae at Göttingen University, Germany (2400 microalgae). 9 NBRC, National Institute of Technology and Evaluation’s Biological Resource Centre, Japan (270 microalgae). 10 ANACC, Australian National Algae Culture Collection at the Hobart laboratories of CSIRO Marine and Atmospheric Research, Australia (300 microalgae). ´ 11 CCBA, Culture Collection of Baltic Algae at the University of Gdansk, Poland (194 microalgae). 12 CCCM, Canadian Centre for the Culture of Microorganisms at the University of British Columbia, Canada (156 microalgae). 13 NCMA, National Centre for Marine Algae and Microbiota at Bigelow Laboratory for Ocean Sciences, USA (562 microalgae). 14 CICCM, Cawthron Institute’s Culture Collection of Micro-algae, New Zealand (281 microalgae).

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15 Algobank Caen, Microalgal Culture Collection at the University of Caen Basse-Normandie, France (469 microalgae). 16 ACOI, Coimbra collection of Algae at the University of Coimbra, Portugal (4000 microalgae). 17 RCC, Roscoff Culture Collection at the Station Biologique, France (2000 microalgae). At least 5% of the above estimated isolates are common across the selected culture collections. However, it is plausible that the total number of purified microalgae available around the world would certainly be much higher than the above estimate, as the regional isolates (belonging to specific research organizations, colleges and university research laboratories) are not deposited in systematic culture collections with online details in some cases.

Commercial use of microalgae as nutraceuticals The applications of microalgae in aquaculture (indirect human nutrition), food or food supplements, high value-added biomolecules (HVABs), bioremediation and renewable energy sectors has gained momentum. Microalgae under commercial cultivation for specific bioactive molecule production are morphologically diverse (Figure 12.1). At least 15 microalgae species are presently cultivated commercially, while several others with alternative potential are only at entry-level (Table 12.2). New microalgae species are either under commercial growth optimization or are awaiting technology transfer from academia and research centres to industry for commercial evaluation. Microalgal biotechnology, like most industries, is market-driven and subject to sustainable economic and cost competitive production. To date, microalgae are mostly used as whole-biomass as health supplements. Microalgal species used in whole biomass form include Aphanizomenon flos-aquae, Chlorella vulgaris and Spirulina platensis. These species are recommended for consumption in whole-biomass form due to their lack of toxicity and reported nutritional benefits, such as high protein, essential vitamins and minerals, dietary fibre and antioxidant pigment contents. Additionally, specific biomolecules have also been extracted from microalgae for use as natural food colourants and as nutrient additives for use in a variety of food matrices (Murray et al., 2013; Priyadarshani & Rath, 2012). Such fractionated biomolecules, in the majority of cases, either possess high antioxidant activities (in the case of astaxanthin from Haematococcus pluvialis; β-carotene from Dunaliella salina; fucoxanthin from Phaeodactylum tricornutum; lutein from Chlorella protothecoides; C-phycocyanin from Spirulina platensis or B-phycoerythrin from Porphyridium cruentum) or high essential fatty acids (in the case of EPA from Phaeodactylum tricornutum; DHA from heterotrophic microalga Crypthecodinium cohnii). Microalgae are indirectly contributing to the human diet, as they serve as an aquaculture feed additive for a wide variety of farmed marine and freshwater primary food organisms. The use of suitable and recognized microalgae species which are toxin-free and contain high lipid contents rich in long chain polyunsaturated fatty acids, is crucial for the success of aquaculture hatcheries and nurseries. Common microalgae used for aquaculture are Chaetoceros calcitrans, Chlorella spp., Dunaliella spp., Isochrysis galbana, Nannochloropsis spp., Diacronema lutheri, Pseudoisochrysis paradoxa, Skeletonema costatum, Spirulina spp., Tetraselmis suecica and Thalassiosira spp. (Adarme-Vega et al., 2012).

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(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Figure 12.1 Light micrographs showing morphological diversity of commercially potential microalgae for nutraceutical applications. (a) Dunaliella salina (the source for β-carotene) at actively growing green-phase of growth (b), transition-phase of growth (c) and complete stress-phase of growth, with accumulation of carotenoid β-carotene (d). Haematococcus pluvialis (the source for astaxanthin) at actively growing green-phase of growth (e) and complete stress-phase of growth, with accumulation of carotenoid astaxanthin (f). Nannochloropsis salina (the source for essential fatty acid EPA) from active phase of growth (g). Porphyridium cruentum, the red microalga (source for antioxidant and anti-cancer pigment phycoerythrin and bioactive polysaccharides), from active phase of growth (h). Phaeodactylum tricornutum (the source for essential fatty acid EPA) from active growth phase (i). Lyngbya majuscula, the cyanobacterium with several bioactive molecules, including immuno-modulatory compounds. (See plate section for colour version.)

Categories of nutraceuticals from microalgae Microalgae may be regarded as one of the richest and natural sources of nutrients supporting life, especially in marine environments (Nagarkar et al., 2004). Microalgae, including cyanobacteria, are the primary food source of various microphagous grazers and zooplanktons, both in marine and freshwater environments. Microalgae are used as nutritional sources of bivalves, crustacean and fish in aquaculture. Previously, the nutritional value of microalgae was based on their carbohydrate, lipid and proteins content. However, commercial demand was low, as better alternative sources existed in the market. Two decades ago, microalgae with a low amount of the omega-3 fatty acids EPA and DHA were simply considered to be a poor source of food. However extensive nutritional research on microalgae since then has discovered

Health supplements Health supplements; food-colourant; cosmeceutical; aquaculture, poultry and animal feed Nutrition, pharmaceuticals Carotenoid zeaxanthin Carotenoid zeaxanthin

Aphanizomenon flos-aquae Spirulina platensis

Health supplement

Health supplement Health supplement Food fortification, pharmaceuticals, aquaculture, poultry and animal feed Food colour, food supplement, pharmaceuticals, aquaculture, poultry and animal feed Infant formulations, health supplement

Coelastrella sp. F50

Scenedesmus almeriensis Muriellopsis sp. Dunaliella salina

Parietochloris incisa

Haematococcus pluvialis

Chlorella minutissima Chlorella zofingiensis Chlorella protothecoides Coelastrella striolata

Health supplement Health supplements; food colourant; aquaculture, poultry and animal feed Health supplement Health supplement Health supplement Health supplement

Oscillatoria sp. CCAC M1944 Chlorella vulgaris

Lyngbya majuscula Microcystis aeruginosa Synechocystis sp. PCC 6803

Applications

Essential fatty acid ARA

Astaxanthin, Lipid, spent-biomass

Essential fatty acid EPA, spent-biomass Astaxanthin and canthaxanthin, spent-biomass Lutein, spent-biomass Carotenoid cocktails of canthaxanthin, β-carotene and astaxanthin, essential fatty acid ALA Carotenoid cocktails of astaxanthin, β-carotene and lutein, Essential fatty acid ALA Lutein, spent-biomass Lutein, spent-biomass β-carotene, spent-biomass, Essential fatty acid EPA

Fractions with immuno-modulatory activities Purified fractions with antioxidant activities Purified fractions with antioxidant activities, spent biomass Dietary antioxidant ergothioneine Chlorophyll, whole-biomass, spent-biomass

Whole biomass Phycocyanin, whole-biomass, spent-biomass

End products

List of promising microalgae for commercial exploitation as a source of nutraceuticals.

Microalgae

Table 12.2

(continued overleaf)

Chlorophyceae

Chlorophyceae

Chlorophyceae Chlorophyceae Chlorophyceae

Chlorophyceae

Trebouxiophyceae Trebouxiophyceae Trebouxiophyceae Chlorophyceae

Cyanobacteria Trebouxiophyceae

Cyanobacteria Cyanobacteria Cyanobacteria

Cyanobacteria Cyanobacteria

Taxa

Microalgae as a source of nutraceuticals 261

Essential fatty acids EPA and DHA, bioactive peptides Essential fatty acids DHA, EPA and carotenoid fucoxanthin Essential fatty acid EPA

β-carotene, spent-biomass β-carotene, spent-biomass Essential fatty acid EPA, Fucoxanthin Essential fatty acid EPA Fucoxanthin Phycoerythrin, polysaccharides, Essential fatty acid ARA Coenzyme Q10

Health supplement Infant formulations, health supplement, animal nutrition Health supplement

Health supplement Health supplement Health supplement Health supplement

Health supplement Food colour, pharmaceuticals, cosmetics, nutrition

Health supplement

Diacronema lutheri Isochrysis galbana

Nannochloropsis oculata N. oceanica N. salina Eustigmatos cf. polyphem Vischeria stellata Phaedactylum tricornutum Nitzschia cf ovalis Nitzschia laevis Odontella aurita Porphyridium cruentum

Porphyridium purpureum

End products

Applications

(continued)

Microalgae

Table 12.2

Rhodophyceae

Bacillariophyceae Rhodophyceae

Eustigmatophyceae Eustigmatophyceae Bacillariophyceae Bacillariophyceae

Eustigmatophyceae

Pavlovophyceae Coccolithophyceae

Taxa

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263

a range of additional criteria to be assessed to determine whether a particular microalga is nutritionally important. Criteria that can be considered include nutritionally significant biomolecules such as essential fatty acids, carotenoids and other molecules with antioxidant activities, vitamins, minerals, essential amino acids, carbohydrates as dietary fibres and bioactivities, antimicrobial compounds and bioactive peptides. One of the main commercial exploitations of microalgae is associated with their antioxidant attributes, which has allowed microalgae to develop into the nutrition and health markets. Two notable examples are β-carotene from Dunaliella salina and astaxanthin from Haematococcus pluvialis. The carotenoid astaxanthin is a multifunctional high value-added biomolecule, as it displays multiple activities and functionalities, encompassing a colourant and an antioxidant preservative with applications in food, pharmaceuticals and cosmetics. At present, DHA is produced commercially from microalga Crypthecodinium cohnii by heterotrophic fermentation, while few photoautotrophic microalgal strains are in the pipeline for the production of other essential fatty acids, such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA) and γ-linolenic acid (GLA) (Table 12.3).

Essential fatty acids Essential fatty acids (EFA) are unsaturated fatty acids that cannot be synthesized de novo by the body and, as such, must be obtained from the diet. EFAs have structural roles in tissues, and are vitally important to the proper formation and functioning of plasma membranes. Membranes associated with the brain, retina and other neuronal tissues are rich in such EFAs. While EFAs are required throughout life, they are especially required during the early stages of development (Uauy et al., 2000). Two human EFAs include the omega-3 fatty acid α-linolenic acid (ALA, 18:3n-3) and the omega-6 fatty acid linoleic acid (LA, C18:2n-6 cis). Other unsaturated fatty acids sometimes referred to as essential are merely ‘conditionally essential’, as their deficiency is associated with either developmental disorders or the progression of a diseased state such as diabetes. Conditional EFAs include eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 20:6n-3), γ-linolenic acid (GLA, C18:3n-6) and arachidonic acid (ARA, C20:4n-6). Under normal circumstances, the body is able to synthesize both EPA and DHA from α-linolenic acid, but this places stress on the body’s systems and limits the use of ALA elsewhere in the body. Microalgae offer the potential to serve as a feedstock for ‘vegetarian’ sources of the above mentioned polyunsaturated fatty acids (Martins et al., 2013). The type and quantity of PUFAs present in microalgae does, however, vary with respect to species type and culture conditions. Studies have determined that DHA is one of the major fatty acids of the brain and retina. DHA has been referred to as an IQ-associated fatty acid, due to its involvement with brain development and functioning throughout life, especially in relation to visual and cognitive development (Auestad et al., 2003). Studies have shown that EPA and DHA assist in the prevention of coronary heart disease, stroke, hypertension, dementia, Alzheimer’s and depression (Kris-Etherton et al., 2002; Das, 2008). GLA intake has been linked with lower low-density lipoprotein levels in previously diagnosed hypercholesteromic patients, alleviation of pre-menstrual syndrome symptoms and in amelioration of atopic eczema (Horrobin, 2000). GLA was also shown to slow or stop transformed cell growth under in vitro laboratory studies. Dietary GLA (mostly consumed as ALA) contributes to the de novo synthesis and regulation of prostaglandins in vivo, an activity in which the American Cancer Society deems essential towards the fight against cancer (American Cancer Society,

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Table 12.3

EPA

DHA

Essential fatty acids, health benefits, organisms and references. Health benefits

% of TFAs

Microalgae

References

EPA is one of the essential fatty acids for human health that acts as a precursor for prostaglandin-3, and helps prevent coronary heart disease, stroke, hypertension, dementia, depression and Alzheimer’s disease.

39.9 21.4 23.4 28 25 29 27.7 39 15 1.6 7 26.7 75.9 16.6 4.7

Chlorella minutissima Dunaliella salina Nannochloropsis oceanica Nannochloropsis salina Nannochloropsis sp. Diacronema lutheri Isochrysis galbana Phaeodactylum tricornutum UTEX 640 Thalassiosira pseudonana Porphyridium cruentum P. cruentum 1380 la Nitzschia cf ovalis Nitzschia laevis Thalassiosira sp. Tetraselmis sp.

Seto et al., 1984 Bhosale et al., 2010 Patil et al., 2007 Wagenen et al., 2012 Hu & Gao, 2003 Guihéneuf et al., 2009 Fidalgo et al., 1998 Yongmanitchai & Ward, 1991 Cobelas & Lechado, 1989 Fuentes et al., 2000 Cohen, 1990 Pratoomyot et al., 2005 Chen et al., 2007 Pratoomyot et al., 2005 Pratoomyot et al., 2005

Plays an important role in the brain development of infants and retinal health of all age. It helps prevent cardiovascular, cancer and Alzheimer’s diseases. It also helps to lower triacylglycerol level and increases good cholesterol (HDL) levels. DHA sourced from algae was approved for infant formulas and have several applications, such as in fruit juices, milk, soymilk, cooking oil, sauces and tortillas.

19.2 14 4.2 1.3

Diacronema lutheri Isochrysis galbana Nitzschia cf ovalis Thalassiosira sp.

Guihéneuf et al., 2009 Fidalgo et al., 1998 Pratoomyot et al., 2005 Pratoomyot et al., 2005

Microalgae as a source of nutraceuticals

Table 12.3

(continued) Health benefits

% of TFAs

Microalgae

References

ALA

ALA is one of the essential omega-3 fatty acids, dietary supplements of which acts as precursors for EPA and DHA biosynthesis in humans.

4.8 9.3

Coelastrella striolata Coelastrella sp. F50

Abe et al., 2007 Hu et al., 2013

GLA

GLA is one of the 31.2 essential omega-6 27.4 fatty acids that help to lower low-density lipoprotein in hypocholesteromic patients. It relieves from symptoms of pre-menstrual syndrome and helps in treating atopic eczema. It has pro-inflammatory and anti-inflammatory properties.

Arthrospira platensis M9108 Arthrospira platensis PCC9108

Gang-Guk et al., 2008 Gang-Guk et al., 2008

ARA

Biogenetic precursor of prostaglandin and leucotrienes, which play major roles in circulatory and central nervous systems. Important for visual sharpness. Valuable ingredient in baby food formulations.

Porphyridium cruentum 1380 la Porphyridium cruentum Parietochloris incisa Thalassiosira pseudonana Nitzschia cf ovalis Tetraselmis sp.

Cohen, 1990

Note: TFAs, total fatty acids.

40 1.7 43 14 4.4 1.1

Fuentes et al., 2000 Bigogno et al., 2002 Cobelas & Lechado, 1989 Pratoomyot et al., 2005 Pratoomyot et al., 2005

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http://www.cancer.org/treatment/treatmentsandsideeffects/complementaryand alternativemedicine/pharmacologicalandbiologicaltreatment/gamma-linolenic-acid, last accessed on 4th November 2013). One of the primary sources of omega-3 and 6 fatty acids is fish but, due to such an unsustainable reliance on diminishing fish stocks, with associated ecological and environmental concerns, alternative sustainable supply sources are required. One such environmental health concern is the reported accumulation of methyl mercury, polychlorobiphenyls and other toxins in fish fat tissue, which may end up in the human food chain (Dickey & Plakas, 2010). Extensive research is ongoing globally to address these concerns, including SME-facilitated research funded by the European Commission through a Framework 7 Knowledge Based Bio-Economy BAMMBO Project (Murray et al., 2013). Currently, there is a growing demand for vegetarian sources of essential fatty acids. Recognized microalgal sources of essential fatty acids are: the cyanobacterium Arthrospira platensis for GLA; the red microalga Porphyridium sp. for ARA; the dinoflagellate microalga Crypthecodinium cohnii and the heterotrophic microalga Schizochytrium sp., and the microalga Isochrysis galbana for DHA; the green microalga Nannochloropsis spp. and the diatoms Phaeodactylum tricornutum and Nitzschia sp. for EPA (Spolaore et al., 2006). These PUFAs have been commercially used as components within infant formulations. According to Frost & Sullivan (2011), omega-3 market revenues in 2010 were estimated to be US$ 1,668 million worldwide and US$ 308 million in Europe. These market values relate to all sources of omega-3, and not solely to microalgae. According to a market analysis report by Project Blue Biotechnology, there is a lack of omega-3 products in the market, which may be met by commercial production of microalgae (Egardt et al., 2013). The current demand for omega-3 fatty acids is straining diminishing fish stocks, which currently contribute some 90% of the revenues associated with these products. Only a small number of microalgal species have currently demonstrated commercial potential for production of polyunsaturated fatty acids. The main companies involved in the production of omega-3 polyunsaturated fatty acids for nutraceutical use are Aurora Algae (USA), Algae Biosciences (Canada), Blue Biotech International GmBH (Germany), GCI Nutrients (USA), Ingrepro BV (The Netherlands), Live Fuels (USA), Lonza (Switzerland), Martek Biosciences (USA), Photonz (New Zealand), Qualitas Health (USA) and Solazyme (USA). Using the European Patent Office, a total of 1190 patents were identified on the worldwide patent database up to December 2013, when the advanced search terms of microalgae and fatty acid were input. Of these patents, only 15 were related to fatty acids.

Carotenoids/antioxidants Certain species of microalgae are known to accumulate high levels of highly effective anti-oxidative scavenger complexes, e.g., carotenoids (β-carotene, lycopene, astaxanthin, zeaxanthin and lutein). These bio-molecules are all produced by microalgal species, and have been proven to remediate UV-oxidation damage in the skin and retina. Carotenoids are an important class of natural fat soluble pigments commonly found in many plants, algae and photosynthetic bacteria. Over 620 carotenoids have been identified and characterized, but only a few were commercially exploited (Guerrin et al., 2003).

Microalgae as a source of nutraceuticals

267

Carotenoids are accessory photosynthetic pigments of microalgae. Carotenoids can be yellow, orange or red in colour, providing microalgae with the ability to trap light energy. Carotenoids offer protection to microalgae from intense irradiation damage, because of their anti-oxidative nature. Such a photoprotective role was suggested for the carotenoid canthaxanthin in the genetically engineered cyanobacterium Synechococcus sp. (Albrecht et al., 2001). Carotenoids may be divided into two categories: i) hydrocarbon-based carotenoids; and ii) oxygen-containing xanthophyll derivatives. Total carotenoid content varies between the microalgal taxa, and may be up to 12% of their dry weight biomass (Table 12.4). Cyanobacteria contain β-carotene (80% of total carotenoids) as the major carotenoid, followed by zeaxanthin, while the richest commercial source of β-carotene is the green microalga Dunaliella salina (Ben-amotz et al., 1982; Sajilata et al., 2008). Haematococcus pluvialis, a green microalga, is the richest known natural source of carotenoid astaxanthin, which has antioxidant activities much higher than β-carotene and Vitamin E (Kang et al., 2007). Environmental growth conditions, especially the quality and quantity of light, have a dramatic influence on the carotenoid content of microalgae (Vesk & Jeffery, 1977; Lakatos et al., 2001; Lakatos et al., 2001; Saha et al., 2003, 2013; Pérez-López, et al., 2014). From a commercial point of view, microalgal carotenoids have long been used as natural colouring materials. Aquatic feed containing β-carotene is known to enhance the flesh colour of salmon. Fowl feed enriched in β-carotene is also used to enhance the colour and appeal of egg yolks and, overall, enhance the nutritional and health benefit of the egg. β-carotene incorporated into feed has also been reported to contribute to the improved health and fertility of cattle (Thajuddin & Subramanian, 2005). Carotenoids have also been reported to be physiologically important to a variety of metabolic functions in humans. One molecule of β-carotene can be enzymatically converted into two molecules of vitamin A within the body. This leads to all the health benefits of vitamin A being acquired, such as improved eyesight, enhanced immune response, and the protection against certain cancers through the vitamin’s ability to scavenge damaging free radicals (see review: Paiva & Russell, 1999). Astaxanthin and β-carotene have gained increased attention due to their health-benefiting attributes and their ‘natural’ or non-synthetic classification when derived from microalgae. Synthetic forms of astaxanthin are considered by some to be inferior as they contain three stereoisomers, while natural forms, containing only one stereoisomer, currently dominate the commercial market as an aquaculture feed additive. Natural astaxanthin is the only form commercially sold in the human supplement market. Synthetic forms of astaxanthin may be cheaper than the naturally derived ones from microalgae, but their incorporation into products that make specific health and food claims are limited. The requirement for antioxidants to maintain human health may be satisfied by a balanced intake of food and a regular diet. In some instances, a demand for antioxidant supplementation may be required due to physiological, as well as a perceived, needs. Over the last decade, the market pull is for natural antioxidants either naturally present in intact foods, or extracted from foods and other biological sources, for inclusion as an ingredient or fortifier. All living organisms have some form or forms of antioxidant mechanisms to defend against reactive oxygen species (ROS). ROS are destructive, but an inevitable product of aerobic respiration and in microalgae. Additionally, ROS are produced as a by-product of photosynthesis (Blokhina et al., 2003; Saha et al., 2003). ROS include

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Table 12.4

Carotenoid pigments, health benefits, organisms, references.

Carotenoids

Health benefits

% DW

Microalgae

References

Astaxanthin

Richest natural antioxidant pigment that helps delaying aging, prevents degenerative diseases and cancers, and stimulates the immune system by detoxifying toxic free-radicals.

7.7 9.8

Haematococcus pluvialis Haematococcus pluvialis

4 0.7 1.8 0.15

Chlorella zofingiensis Chlorococcum sp. Coelastrella sp. F50 Coelastrella striolata

Kang et al., 2007 Domínguez-Bocanegra et al., 2004 Del Campo et al., 2004 Ma & Chen, 2001 Hu et al., 2013 Abe et al., 2007

𝛃-carotene

β-carotene is the precursor of vitamin A, which is produced by human body for good eye health and vision, healthy skin and mucus membranes, our immune and antioxidant systems.

12 10 6.1 1.4 0.7 5.9

Dunaliella salina Dunaliella salina Eustigmatos cf. polyphem Coelastrella sp. F50 Coelastrella striolata Vischeria stellata

Del Campo et al., 2007 Ben-amotz et al., 1982 Li et al., 2012a Hu et al., 2013 Abe et al., 2007 Li et al., 2012b

Lutein

Helps maintain a normal visual function. Lutein absorbs and attenuates blue light striking the retina.

4.6 0.7 4.5 4.3

Chlorella protothecoides Coelastrella sp. F50 Scenedesmus almeriensis Muriellopsis sp.

Shi et al., 2006 Hu et al., 2013 Del Campo et al., 2007 Del Campo et al., 2007

Canthaxanthin Canthaxanthin is a potent 4.8 radical scavenger that 2.1 helps protect skin from rash, itch, and eczema due to harsh sunlight.

Coelastrella striolata Chlorella zofingiensis

Abe et al., 2007 Hua-Bin et al., 2006

Fucoxanthin

Fucoxanthin acts as a 1.6 non-stimulatory fat loss agent and helps correct 1.8 abnormal glucose 1.7 metabolism in muscle tissue of diabetic patients. It also possesses anti-carcinogenic properties.

Phaeodactylum tricornutum Isochrysis aff. galbana Isochrysis sp.

Kim et al., 2012a Kim et al., 2012b Crupi et al., 2013

Zeaxanthin

Reduces the risk for age-related macular degeneration. Zeaxanthin absorbs and attenuate blue light striking the retina.

Dunaliella salina Microcystis aeruginosa Spirulina sp. Synechocystis sp. PCC 6803

Jin et al., 2003 Chen et al., 2005 Liao et al., 1993 Lagarde et al., 2000

0.6 – – –

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269

singlet oxygen, super oxide radical anion, hydrogen peroxide and other species of molecules. An imbalance in favour of ROS over-production at the expense of antioxidative counter-defence systems results in oxidative stress. This, in turn, results in abnormal cell functioning, leading to various pathological conditions (Blokhina et al., 2003). Microalgae exist in relatively harsh environmental conditions, so it is not surprising that they have evolved various strategies for their survival in extreme environments, especially when excess amounts of ROS are produced. As a result, microalgae are one of the best natural sources of antioxidants. The antioxidative defence mechanisms of microalgae may be classified as: 1 enzymatic-based utilizing superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX)] enzymes; 2 non-enzymatic-based utilizing molecules, such as ascorbic acid (vitamin C), reduced glutathione (GSH), carotenoids (astaxanthin, β-carotene, lutein, canthaxanthin, zeaxanthin), tocopherols and polyphenolic bio-molecules. Apart from their antioxidant role, microalgae possess other bio-molecules associated with photosynthesis with reported health benefits. The chemical structure of microalgal chlorophyll resembles the haemoglobin molecule, and is considered as an effective source of nutrients for humans because of its predicted antioxidant activities and other health benefits (Bai et al., 2011; Simonich et al., 2007, 2008). In animal studies, dietary consumption of chlorophyll was reported to prevent multi-organ carcinogenesis in rainbow trout and rats (Simonich et al., 2007, 2008). Interestingly, consumption of chlorophyll-rich microorganisms is neither a new concept, nor one driven by a marketing campaign initially, as natives of China, Chile, Mexico, Peru and the Philippines have long been using Spirulina, Anabaena and Nostoc as food. Spirulina platensis is an excellent source for B vitamins, vitamin E, antioxidants and co-enzyme activities (Plavsic et al., 2004). Spirulina platensis is also a source of water-soluble phycocyanin (a blue phycobilipigment), with reported antioxidant activity (Cohen et al., 1993; Benedetti et al., 2004; Romay et al., 2003). The red microalga Porphyridium cruentum produces water-soluble phycoerythrin (a red phycobilipigment), with known antioxidant activities (Spolaore et al., 2006; Minkova et al., 2011). One of the applications of phycobilipigments is as colouring agents, with the additional benefit of antioxidant activity for use in food, cosmetic and pharmaceutical products. Cylindrotheca closterium was identified as a producer of a pigment which, when solubilized in ether, yielded a blue-coloured fluorescent solution. That compound was identified to be fucoxanthin, and is a member of the xanthophylls group of bio-molecules. Fucoxanthin is one of the most naturally abundant carotenoids, and it has been reported to be effective at inducing apoptosis in human leukaemic and colon cancer cells. In one specific case, an in vitro study reported a dose-dependent inhibition of Graffi myeloid tumour cell proliferation in response to fucoxanthin (Minkova et al., 2011). Almost all cyanobacteria contain a group of low molecular weight water-soluble mycosporine-like amino acids (MAAs) with antioxidant and anti-inflammatory activities, and UV-A absorbing properties. The UV-A absorbing properties have applications for use in skin care products to prevent premature ageing (Ferroni et al., 2010). Therefore, cyanobacteria can be explored for MAAs as natural antioxidant molecules, as food fortifiers and as cosmetic functional ingredients. A report on the global market for carotenoids was published by BCC Research LLC in 2011, summarizing the global market for carotenoids up to 2011, with market

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projections up to 2018. The report outlined the global carotenoid market to be worth an estimated US$ 1.2 billion in 2010 and it was projected to reach US$ 1.4 billion by 2018. Individual global carotenoid market values for 2010 with projections to 2018 were β-carotene (US$ 261 and 334), lutein (US$ 233 and 309), astaxanthin (US$ 226 and 253), canthaxanthin (US$ 87 and 74), lycopene (US$ 66 and 84) and zeaxanthin (US$ 16 and 18). The majority of currently produced carotenoids are manufactured by chemical synthesis. Due to the growing demand for natural forms of such compounds, the market for natural carotenoids will grow, but the rate of growth will depend on the cost of such natural forms, especially those from microalgae. Using the European Patent Office, only three patents were identified on the worldwide patent data base up to December 2013 using the advanced search terms of ‘microalgae’ and ‘carotenoids’. Of these patents, all three were related to carotenoid production and extraction. Separate searches identified four patents relating to production of astaxanthin from microalgae, two patents relating to microalgae and lutein and one related to microalgae and fucoxanthin. Major and developing companies involved in the production of microalgal derived astaxanthin are Algae Health (Ireland), AlgaeTech (Malaysia), AlgaTechnologies (Israel), Contract Biotics (USA), Cyanotech (USA), Fuji Health Services (Japan), Parry Nutraceutical (India and USA) and Valensa International (USA).

Coenzyme Q10 Coenzyme Q10 is a vital component of the mitochondrial electron transport chain, whose main function is cellular energy production. The reduced form of coenzyme Q10 (CoQ10) is known as ubiquinol, and it is prevalent in most organisms. It is one of the most effective antioxidants in human cells. CoQ10 is a membrane-bound redox molecule that is involved in protein disulphide bond formation, redox flux regulation, regeneration of Vitamin E, cellular signalling and gene regulation (Jeya et al., 2010). CoQ10 is a well-known and actively promoted bioactive molecule, widely used in cosmetics. In health shops, it is sold as a nutritional supplement for its reported antioxidant benefit and for its reported ability to retard progression of neurodegenerative disorders such as Parkinson’s disease and certain encephalomyopathies (Bonakdar & Guarneri, 2005). Other studies have claimed CoQ10 to improve heart health, favourably moderate blood sugar levels, and help manage high blood pressure in diabetic patients (Dhanasekaran & Ren, 2005; Hodgson et al., 2002). Recently, a study showed a decreased amyloid pathology and improved behaviour in transgenic Alzheimer’s disease-suffering mice when they were administered a naturally occurring form of CoQ10 (Dumont et al., 2011). Dietary staples contain only a small amount of CoQ10 (Mattila & Kumpulainen, 2001) and, as such, most consumers turn to supplements as an additional source of CoQ10. Commercial CoQ10 formulations are often poorly absorbed from the gut and are deemed to have limited bioavailability (Liu & Artmann, 2009). As such, the matrix or formulations containing CoQ10 need to be considered when creating such a product purporting to have this high value-added bio-molecule. CoQ10 can be produced by microalgae by selecting a suitable strain and optimizing the growth conditions. In one study, the red microalga Porphyridium purpureum produced 141 μg CoQ10 g−1 dry weight biomass at a 120 litre culture scale (Klein et al., 2012). The global market for CoQ10 is dominated by non-algal derived and synthetic forms of this antioxidant. The market for CoQ10 peaked between 2004 and 2005, when

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prices were high, as was the demand, which surpassed supply. Currently, one of the main algal based producers of CoQ10 is MayPro Industries, USA. Using the European Patent Office, 822 patents were identified on the worldwide patent database up to December 2013 using the advanced search terms for ‘Coenzyme Q10’. However, further searches for relatively recent patents within this under the search criteria of Coenzyme Q10 and algae, and/or microalgae, failed to show any record of such patent filings. As such, the freedom to operate in this space would appear to be inviting towards the commercial exploitation of CoQ10.

Ergothioneine L-ergothioneine is a naturally occurring dietary antioxidant which was first discovered in the ergot fungus in 1909. This bioactive molecule is synthesized by only a few organisms, such as filamentous fungi, actinobacteria and cyanobacteria. Ergothioneine accumulates in oxidative-stress susceptible areas of the body, such as the lens of the eye, the liver, bone marrow and in seminal fluid. Ergothioneine is not synthesized by higher plants or animals, but is taken up from the soil by some plants and, ultimately, by animals through their diet (Cheah & Halliwell, 2012). Ergothioneine is a form of amino acid derived from thiourea and containing components associated with histidine, and it mainly exists in the thione form when in an aqueous environment. Ergothioneine is a relatively stable antioxidant with its own unique properties. It is not auto-oxidized at physiological pH, and does not promote hydroxyl radical generation from H2 O2 and Fe2+ ions. In fact, ergothioneine may scavenge the oxidizing species that are not free radicals (Chaudiere & Ferrari-Iliou, 1999). Therefore, the antioxidant chemistry of ergothioneine differs from known sulphur-containing antioxidants (glutathione or lipoic acid) and antioxidant ascorbate. Until recently, mushrooms were regarded as the only dietary source of ergothioneine. However, studies have shown that some cyanobacteria produce higher levels of ergothioneine than the best known species of mushroom. Pfeiffer et al., (2011) showed higher content of ergothioneine in cyanobacterium, Oscillatoria sp. CCAC M1944 (0.8 mg g−1 dry weight biomass) compared to King oyster mushrooms, Pleurotus eryngii (0.4 mg g−1 dry weight biomass). Recognized ergothioneine-producing species are Spirulina platensis, Arthrospira maxima, Aphanizomenon flos-aquae, Scytonema sp. CCAC M3193 and Oscillatoria sp. CCAC M2010. Most cyanobacteria synthesize ergothioneine de novo (Pfeiffer et al., 2011). Red microalga Porphyridium purpureum SAG 1380-1c was found to produce only a small amount of ergothioneine (0.1 mg g−1 dry weight biomass) (Pfeiffer et al., 2011). It appears, therefore, that available health products based on Spirulina platensis, Arthrospira maxima or Aphanizomenon flos-aquae containing high amounts of ergothioneine would be ideal dietary source of natural ergothioneine. There is little to no market information available pertaining to ergothioneine. Production of this antioxidant is currently primarily from terrestrial microbial and plant sources. Using the European Patent Office, 57 patents were identified on the worldwide patent database up to December 2013 using the advanced search term of ‘ergothioneine’. However, further searches for relatively recent patents within this, under the search criteria of ergothioneine and algae and/or microalgae, failed to show any record of such patent filings. As such, the freedom to operate in this space would appear to be inviting towards the commercial exploitation of ergothioneine. From a review of the literature, it would appear that further scientific studies supporting the nutraceutical

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claims relating to the action and benefit of ergothioneine are needed. It is likely that such investigations are ongoing, and this is a possible explanation for the lack of patent filings observed on the European Patent System search.

Amino acids and vitamins The nutritional quality of microalgal biomass is mainly determined by the content of the major macronutrients (proteins, carbohydrates and lipids) and micronutrients (vitamins, antioxidants, minerals and other bioactive molecules). The nutritional quality also considers the presence of anti-nutritional compounds and, also, the type and quantity of toxic substances. Sustainable market supply of nutritionally important amino acids and vitamins through microalgae requires a consistent product standard of known predictable composition. This is because of the fact that most microalgae exhibit large variations in cellular protein, carbohydrate and lipid contents, each ranging from 15–50% of dry weight biomass (Becker, 2007), and the same may be the case with small bioactive molecules of nutritional importance. Over five decades ago, Chlorella and Spirulina were recommended for human and animal dietary consumption, because of the high nutritional quality of their proteins in terms of essential amino acid content. Microalgal proteins favourably compare to conventional vegetable proteins in terms of their nutritional attributes and digestibility. In 1973, the WHO/FAO recommended microalgae consumption based on the balance of their essential amino acids (Becker, 2007). Based on rat nutritional requirement studies, ten amino acids, including arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine were considered as essential amino acids for human and animal nutrition. Interestingly, all these ten essential amino acids can be sourced from microalgae protein derived from Chlorella vulgaris, Dunaliella bardawil, Scenedesmus obliquus, Arthrospira maxima and Spirulina platensis. Of commercial interest is the fact that the market for algal protein has started to develop over the last five years, as scientific studies reporting on the nutritional, functional and nutraceutical properties of algal proteins have increased. Microalgae contain an array of vitamins including A, B1 , B2 , B3 (Niacin), B6 , B12 , C, E, Folic acid and pantothenic acid, which have been reported to be present at high levels compared with most routine foods (Becker, 2004). The water soluble B-complex vitamins have wide-ranging functions that ultimately facilitate the processes involved in yielding energy from carbohydrates. B-complex vitamins are essential for healthy skin, hair, eyes, and the proper functioning of the liver and nervous systems. Spirulina platensis, Chlorella pyrenoidoisa and Scenedesmus quadricauda are among the richest sources of A, E, B1 , Vit B2 , B3 , B6, Folic acid, B12 , C and pantothenic acid vitamins (Becker, 2004). A diet or supplement containing biomass from a combination of carefully selected microalgal species could provide vitamins of high bio-availability. The strongest sub-sectors of the nutraceutical market over the last five years are amino acid, mineral and vitamin related. According to a report from Frost & Sullivan (2011), the world and European market revenues for amino acids were estimated to be worth US$ 2572 million and US$ 253 million, respectively. In the same report, the world market for vitamin E was estimated to be US$ 83.4 million. Interestingly, the European vitamin and mineral market was very strong, with revenues in 2010 estimated to be US$ 2.05 billion. For such a strong market, the number of new patents filed which seek to protect the use of microalgae as sources of amino acids, minerals and vitamins are relatively small.

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Using the European Patent Office, there were no patents identified on the worldwide patent database up to December 2013 using the advanced search terms of ‘microalgae’ and ‘amino acid’. However, a further search using the terms of ‘algae’ and ‘amino acid’ returned seven patents in the area of production and application of such amino acids for use in animal feeds. Similarly, 12 patents were identified on the use of algae as sources of minerals for a variety of applications. Only four patents were identified on the worldwide patent web database up to 2013, using the advanced search terms of ‘algae’ and ‘vitamin’. A search of the World Wide Web for suppliers of microalgal-derived amino acids, minerals and vitamins returned an extensive list of suppliers of such materials. The majority of suppliers were Asian-based. In the majority of cases, the amino acids, minerals and vitamins were available in mixed form as intact biomass of enriched extracts.

Carbohydrates from microalgae The carbohydrate content of microalgae ranges between 20–45 % of their biomass dry weight. Microalgal carbohydrates may be in the form of starch, glucose, sugars, and simple-to-complex polysaccharides. The chemical composition of microalgal polysaccharides depends on the species and their genetic make-up, physiological factors and growth conditions. Production of simple carbohydrates such as glucose, fructose and galactose may have practical significance from a nutritional perspective. From a functional food perspective, polysaccharides are gaining attention due to their water-holding and bio-active properties (de Jesus Raposo et al., 2013). The overall digestibility value of microalgal carbohydrates is high and, therefore, even whole biomass is recommended for use as food or feeds. Microalgae Porphyridium cruentum and Botryococcus braunii are regarded as a potential industrial source of good source for exo-polysaccharides (EPS). Several species of microalgae produce sulphated EPS with reported antiviral, antioxidant and anti-inflammatory activities. Sulphated polysaccharides have been reported to have potential as bone joint bio-lubricants (de Jesus Raposo et al., 2013). Sulphated polysaccharides of marine microalgae possess anti-adhesive properties and, thus, have the potential to retard colonization or act as an anti-adhesive towards pathogenic organisms for medical use. It was demonstrated that sulphated polysaccharides inhibited the adhesion of Helicobacter pylori to HeLa S3 cell lines and, also, the adhesion of three fish pathogens to sand bass gills, gut, and skin cultured cells. Microalgal EPS might have use in prophylactic therapy to prevent microbial infections, as EPS have the ability to block microbial cytoadhesion to host cells as well as inhibit the growth of Salmonella enteritidis (de Jesus Raposo et al., 2013). Red microalgae are the richest source of sulphated polysaccharides, as their cell walls contain no cellulosic microfibrils, and the cells are actually encapsulated within the gel matrix of sulphated polysaccharides. EPS of red microalga Porphyridium sp. possessed antioxidant activity against the auto-oxidation of linoleic acid, and protected 3T3 cells from oxidative damage (Tannin-Spitz et al., 2005). The above antioxidant activities were directly proportional to the dose of EPS, and were positively correlated to the sulphate content of the EPS (Tannin-Spitz et al., 2005). Later, in another study, the EPS from Porphyridium cruentum was fragmented into various sizes by time-dependent microwaving, and the higher antioxidant activities were obtained by low-molecular-weight fragments (6.55 kDa), with better protection of mouse cells and tissues from oxidative damage (Sun et al., 2009). Also, the crude sulphated exopolysaccharide from red microalga Rhodella reticulata showed

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dose-dependent antioxidant activity, which was twice as strong as α-tocopherol (Chen et al., 2010). Microalgal sulphated polysaccharides are also showing promise for prevention of coronary heart disease, due to their hypolipidaemic and hypoglycaemic promoting properties. However, this area has not been as extensively explored for microalgae as that of sulphated polysaccharides from macroalgae. Promising data demonstrating a reduction of serum cholesterol and triglyceride levels was obtained when rats were fed Porphyridium sp. and R. reticulata biomass containing dietary fibres in the form of sulphated polysaccharides. Also, the levels of hepatic cholesterol were improved and the levels of VLDL were considerably reduced without any toxic effects in the animals tested (see de Jesus Raposo et al., 2013). A feeding study with chickens using EPS-containing biomass of Porphyridium correlated found reduced serum and yolk cholesterol levels, desirable fatty acid profile and improved carotenoid content of egg yolk, with that of EPS intake (see de Jesus Raposo et al., 2013). Rats fed Rhodella biomass were reported to contain lower levels of insulin and glucose in serum. Likewise, sulphated polysaccharides of Porphyridium were also reported to significantly reduce serum glucose levels of diabetic mice without any change to β-Islet pancreatic cells function, fibrosis or cellular haemorrhagic necrosis (see de Jesus Raposo et al., 2013). Little to no research has been conducted on the commercial potential of microalgae to produce β-glucan (polysaccharides). The market for β-glucan has seen a dramatic increase over the last 20 years, as the health benefits of the carbohydrate have been realized. The majority of β-glucan industrially produced is derived from baker’s yeast (and brewer’s yeast). In Ireland and most likely other countries, yeast is a resource which is widely available as a waste by-product.

Bioactive peptides Bioactive peptides are short protein fragments with specific amino acid sequences that have an impact on cellular metabolism and ultimately on health conditions (Kitts & Weiler, 2003; Walsh & FitzGerald, 2004). Most bioactive peptides are produced and stored in an inactive form, which is then subsequently activated when needed. It is well recognized that, apart from their basic nutritional role, many food proteins contain, encrypted within their primary structures, peptide sequences capable of modulating specific physiological functions (FitzGerald et al., 2004). Some food peptides are latent within a protein or peptide molecule until liberated by the action of proteinases and/or peptidases. Some peptides are specific in action, or may possess multi-functional domains conferring them with wide and varied activities (Danquah & Agyei, 2012). Depending on their activities, these peptides may have anti-microbial, anti-thrombotic, anti-hypertensive, opioid, immuno-modulatory, mineral binding and/or antioxidative properties (Walsh & FitzGerald, 2004). They also act as signalling molecules and play important roles in physiological functions and pathogenesis. Bioactive peptides were defined as peptides with hormone- or drug-like activity that eventually modulate physiological function through binding interactions to specific receptors on target cells, leading to induction of physiological responses (Fitzgerald & Murray, 2006). A potent antioxidative peptide of amino acids sequence VECYGPNRPQF was isolated from the green microalga Chlorella vulgaris protein hydrolysates. This peptide was found to be active against the damaging effects of free radicals, such as the

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hydroxyl radical, superoxide radical, peroxyl radical, DPPH radical and ABTS radicals (Sheih et al., 2009a). The above peptide was resistant to gastrointestinal digestion and showed no cytotoxicity when tested against human lung fibroblasts cell lines (WI-38) in vitro (Sheih et al., 2009a). The same peptide, from microalga Chlorella vulgaris, was also reported to possess angiotensin I-converting enzyme (ACE) inhibitory activities (Sheih et al., 2009b) and anti-proliferative activities (Sheih et al., 2010). Other peptides (IVVE, AFL, FAL, AEL and VVPPA) of microalga Chlorella vulgaris were earlier reported to possess ACE-I inhibitory activities (Suetsuna & Chen, 2001). Likewise, peptides (IAE, FAL, AEL, IAPG and VAF) of Spirulina platensis were also reported to possess ACE-I inhibitory activities (Suetsuna & Chen, 2001). The tetramer EDKR, from the diatom Navicula incerta, was shown to possess antioxidative activities against DPPH, hydroxyl and superoxide radicals (Kang et al., 2011), while other peptides, such as NIPP-1 (PGWNQWFL) and NIPP-2 (VEVLPPAEL), isolated from the diatom Navicula incerta were shown to possess hepatic fibrosis inhibitory effect on TGF-β1 induced activation of LX-2 human hepatic stellate cells (Kang et al., 2013). A peptide MPGPLSPL, obtained from proteins of microalga Diacronema lutheri after fermentation with Candida rugopelliculosa, was shown to induce myofibroblasts differentiation in human dermal fibroblasts (Ryu, 2011). The peptides referred to as Lyngbyastatins, from cyanobacteria Lyngbya spp., were reported to contain anti-elastase (Matthew et al., 2007; Taori et al., 2007) and anti-chymotrypsin (Matthew et al., 2007) activities. Other peptides, named Kempopeptins A and B, isolated from a marine cyanobacterium, Lyngbya sp., were found to possess anti-elastase and anti-chymotrypsin activities (Taori et al., 2008). A new cyclic peptide, Pompanopeptins A, isolated from a marine cyanobacterium, Lyngbya confervoides, was found to possess anti-trypsin activities (Matthew et al., 2008).

Anti-microbial biomolecules Microalgae are a morphologically and genetically diverse group of photosynthetic organisms with both prokaryotic and eukaryotic cellular organizations. They survive different environmental extremes by using different survival strategies, including the production of secondary metabolites with potential pharmacological applications. Some of these bioactive secondary metabolites have antibacterial, antifungal, anti-parasitic and antiviral activities. These bioactive compounds are mostly lipopeptides with unusual oxidation, methylation and halogenation properties. Also, other compounds of microalgae, such as alkaloids, peptides, fatty acids, indoles, terpenes, phenols and volatile halogenated hydrocarbons, were reported to show antimicrobial activities (Table 12.5). The alkaloids, named as Ambiguines H and I, isolated from cyanobacterium Fischerella sp. were reported to possess antibacterial activities against Staphylococcus albus and Bacillus subtilis (Raveh & Carmeli, 2007). The exploration of microalgae for anti-microbials has yet to yield a life-saving drug, but the search is ongoing. The anti-microbial compound ‘chlorellin’ was identified from Chlorella vulgaris in the 1940s (Metting & Pyne, 1986), but progress on others has been slow ,and studies are still in their infancy from a commercial point of view. In relation to anti-viral activity, the intracellular polysaccharide ‘calcium spirulan’, extracted from Arthrospira platensis, was reported to inhibit in vitro viral replication. EPS from A. platensis and Porphyridium purpureum displayed antiviral activities in vitro and in vivo against Vaccinia and Ectromelia viruses (see de Jesus Raposo et al.,

Ethanolic extract Pheophorbide α-, β-like compounds Polysaccharide-rich fraction Sulphated polysaccharides Sulphated polysaccharide Sulpholipids Sulpholipids

Any biomolecule or synthetic agent that kills a virus or prevents viral replication from causing the symptoms. Microalgal antiviral compounds might be taken as a food supplement, as some plant-based foods are good source of dietary antiviral molecules.

Anti-viral

Pure-fraction/ Enriched fractions/ whole organism Carbamidocyclophanes A–C Crossbyanol B Lyngbyazothrins A–D Scytoscalarol Chlorellin Eicosapentaenoic acid Butanoic acid and methyl lactate Long-chain saturated and unsaturated fatty acids Organic extracts Organic extracts Organic extracts Acetone extract Ethanolic extract Ambiguines H and I

Health benefits

Antimicrobials from microalgae, activities, organisms, references.

Anti-bacterial The biomolecules, semi-synthetic or synthetic molecules that inhibit bacterial growth or kill bacteria during or after infection. These molecules may be active only against Gram-positive or Gram-negative bacteria, or both types of bacteria. Many of our plant-based foods are good source of dietary antibacterial molecules. Hence, microalgal antibacterial compounds may be fortified with our food or consumed as food supplements.

Antimicrobials

Table 12.5

Santoyo et al., 2012 Ohta et al., 1998 Santoyo et al., 2012 Fábregas et al., 1999 Lee et al., 2006 Gustafson et al., 1989 Gustafson et al., 1989

Ghasemi et al., 2007 Ghasemi et al., 2007 Das et al., 2005 Abedin & Taha, 2008 Abedin & Taha, 2008 Raveh & Carmeli, 2007 Chlamydomonas reinhardtii Chlorella vulgaris Euglena viridis Spirulina platensis Anabaena oryzae Fischerella sp. Haematococcus pluvialis Dunaliella primolecta Dunaliella salina Chlorella autotrophica Navicula directa Lyngbya lagerheimii Phormidium tenue

Bui et al., 2007 Choi et al., 2010 Zainuddin et al., 2009 Mo et al., 2009 Pratt et al., 1944 Desbois et al., 2009 Santoyo et al., 2009 Naviner et al., 1999

References

Nostoc sp., (CAVN 10) Leptolyngbya crossbyana Lyngbya sp. 36.91 Scytonema sp. UTEX 1163 Chlorella spp. Phaeodactylum tricornutum Haematococcus pluvialis Skeletonema costatum

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Any biomolecule or chemical agent that selectively eliminates pathogenic fungal growth from the host. Microalgae with antifungal properties might be consumed as a food source, as that of several herbs consumed for keeping fungal diseases away.

Any natural metabolite or synthetic agent that kills or has strong growth-inhibitory potential against disease-causing parasites such as protozoans, amoebas, roundworms, flatworms or tapeworms. Microalgae with antiparasitic properties might be consumed as a food source, as that of several traditional foods available around the world.

Anti-fungal

Anti-parasite

Calothrixin A and B Lagunamides A and B Lagunamide C Venturamides A and B Gallinamide A. Carmabin A, Dragomabin and Dragonamide A Symplostatin 4

Dragonamide E Nostocarboline Viridamides A and B

Hectochlorin Lobocyclamides A–C Scytoscalarol Tanikolide Methanolic extracts Methanolic extracts Butanoic acid and methyl lactate Ethanolic extract Ethanolic extract

Rickards et al., 1999 Tripathi et al., 2010 Tripathi et al., 2011 Linington et al., 2007 Linington et al., 2009 McPhail et al., 2007 Stolze et al., 2012 Symploca sp.

Balunas et al., 2010 Barbaras et al., 2008 Simmons et al., 2008

Marquez et al., 2002 MacMillan et al., 2002 Mo et al., 2009 Singh et al., 1999 Ghasemi et al., 2007 Ghasemi et al., 2007 Santoyo et al., 2009 Abedin & Taha, 2008 Abedin & Taha, 2008

Lyngbya majuscula Nostoc 78-12A Oscillatoria nigro-viridis OSC3L Calothrix spp. Lyngbya majuscula Lyngbya majuscula Oscillatoria sp. Schixothrix sp. Lyngbya majuscula

Lyngbya majuscula Lyngbya confervoides Scytonema sp. UTEX 1163 Lyngbya majuscula Chlamydomonas reinhardtii Chlorella vulgaris Haematococcus pluvialis Chlorella pyrenoidosa Scenedesmus quadricauda

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2013). Importantly, cell-free extracts of microalgae may be added to food and feed formulations as a natural source of anti-microbials thereby reducing the need for synthetic anti-microbial compounds. A lipopeptide termed Dragomabin from Lyngbya majuscula (McPhail et al., 2007), and Venturamide A and B from Oscillatoria sp. (Linington et al., 2007), were found to display anti-malarial activity against the Plasmodium falciparum W2 strain.

Anti-spasmodic compounds Any biological molecule or drug that helps suppress spasms and reduces muscular tension may be considered to be an anti-spasmodic. The action of anti-spasmodics may be specific to an organ and muscle type, or it may generally ease muscle cramps throughout the body. Many herbal antispasmodics act on the central nervous system as well, and can help relax psychological tension. There are only minimal literature articles available in the public domain on microalgal-derived anti-spasmodic compounds. One such pertains to hexane extracts of the Mexican cyanobacteria Blennothrix ganeshii, Microcoleous lacustris and Oscillatoria limnetica which, when tested on guinea-pig ileum, were found to possess both anti-cholinergic and anti-histaminic properties. The cyanobacterial extracts displayed their relaxant effects on the ileum by preventing contractions induced by known spasmogens such as acetylcholine, histamine and BaCl2 (Pérez Gutiérrez & Solís, 2007, 2009). The market value for natural anti-spasmodic compounds could not be identified from searches. However, there are strong demands for pharmacological compounds to treat specific ailments associated with spasms. Using the European Patent Office search facility, a total of 549 patents were identified on the worldwide patent web register which related to the generation and application of a variety of anti-spasmodic compounds. None of these patent filings were associated with compounds derived from microalgae.

Hypotensive, immunomodulatory and antiproliferative compounds Hypertension is a controllable risk factor associated with cardiovascular disease, and the renin-angiotensin-aldosterone system is a target for blood pressure control (FitzGerald et al., 2004). Cleavage of angiotensinogen by renin produces angiotensin I, which is subsequently hydrolyzed by angiotensin-I-converting enzyme (ACE) to angiotensin II (a potent vasoconstrictor). Research has demonstrated that the ACE enzyme is subject to inhibition by peptides possessing the appropriate amino acid sequence. Several peptides from the Chlorella vulgaris were reported to possess angiotensin I-converting enzyme (ACE) inhibitory activities (Sheih et al., 2009b; Suetsuna & Chen, 2001). Also, peptides from Spirulina platensis were reported to show ACE-I inhibitory activities (Suetsuna & Chen, 2001). In some instances, ACE-I inhibitory compounds may also act as immunomodulators, as the inhibition of ACE results in the formation of bradykinin, which mediates the acute inflammatory process (FitzGerald et al., 2004). Chicken and mice fed Spirulina biomass demonstrated increased phagocytic activity and increased natural killer cell-mediated antitumor activities (Singh et al., 2011). In vitro studies with human blood cells incubated with Spirulina extracts showed elevated interferon (13.6-fold) and interleukin (IL)-1β and IL-4 (three-fold) levels (Singh et al., 2011). Other studies with cyanobacterial extracts containing

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microcystin demonstrated enhanced murine immunosuppressive activities and cytokine modulation in vivo (Singh et al., 2011). A screening programme for new anticancer bioactives from cyanobacteria resulted in the isolation and characterization of several potential anti-cancer compounds, termed Apratoxin A (Lyngbya sp.), Curacin A (Lyngbya majuscula), Cryptophycin 1 (Nostoc sp. GSV224), Dolastatin 10 (Symploca sp.), Dolastatin 15 (Lyngbya sp.), Somocystinamide A (Lyngbya majuscula and Schizothrix sp. Assemblage) and Tolyporphin (Tolypothrix nodosa) and were active against various types of cancer cell lines (Singh et al., 2011). Other studies that also identified cyanobacterial bioactive metabolites with antiproliferative activities are: Ankaraholide A (Geitlerinema sp.), Antillatoxin (Lyngbya majuscula), Aurilides B (1) and C (2) (Lyngbya majuscula), Bisebromoamide and Biselyngbyaside (Lyngbya sp.), Calothrixin A and B (Calothrix sp.), Carmabin A, Dragomabin, Dragonamide A and Caylobolide A (Lyngbya majuscula), Caylobolide B (Phormidium spp.), Cocosamides A and B (Lyngbya majuscula), Coibamide A (Leptolyngbya sp.), Gallinamide A (Schizothrix sp.), Guineamides B and C (Lyngbya majuscula), Koshikalide (Lyngbya sp.), Hectochlorin (Lyngbya majuscula) (Nagarajan et al., 2011). A murine in vivo study demonstrated that extracellular sulphated polysaccharides from Porphyridium cruentum showed both immune enhancing activity and the inhibition of S180 tumour cell proliferation (Sun et al., 2012). The peptide VECYGPNRPQF, derived from protein hydrolysates of Chlorella vulgaris, was also found to possess anti-proliferative activities (Sheih et al., 2010). Using the European Patent Office search facility, two patents were identified on the worldwide patent web register which related to the generation and application of hypotensive agents using the search terms of ‘algae’ and ‘hypotensive’. One patent related to the generation of hypotensive agents from seaweed ash, while the other detailed information on the production and application of the carbohydrate mannan from algal sources. In relation to immunomodulatory, anti-proliferative and anti-cancer agents, no patents were identified on the worldwide patent register when searched using the fields of ‘algae’, ‘microalgae’, ‘immunomodulation’, ‘anti-proliferative’ and ‘anti-cancer’ individually.

Calcium-binding compounds Dietary calcium is important for maintaining healthy plasma cholesterol levels and in the prevention of dental caries, osteoporosis, hypertension and anaemia. Microalgal proteins, peptides and polysaccharides have been reported to bind and make available calcium. A novel sulphated polysaccharide named ‘Calcium-spirulan’ from Spirulina platensis consists of calcium ions, sulphate, uronic acids, rhamnose, 3-O-methylrhamnose (acofriose), 2,3-di-O-methylrhamnose, 3-O-methylxylose, O-rhamnosyl-acofriose and O-hexuronosyl-rhamnose (aldobiuronic acid) (Mišurcová et al., 2012) Calcium-spirulan has been reported to help prevent atherosclerosis, largely due to its antithrombogenic, fibrinolytic, and anti-atherogenic properties (Mišurcová et al., 2012). Microalgal anionic exo-polysaccharides are currently being explored as a means to bind and make available essential minerals and nutrients when ingested. Polysaccharides from two freshwater microalgae, Palmella texensis (UTEX 1708) and Cosmarium turpinii (UTEX LB 1042), were shown to bind calcium ions to varying degrees, depending on pH. Palmella texensis polysaccharides possessing one calcium binding site were less efficient at calcium binding, compared to polysaccharides of Cosmarium turpinii

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possessing two calcium binding sites (Ha et al., 1989). Microalgae-bound calcium with improved bioavailability has potential application in dairy-free functional food and beverage products for people with lactose intolerance. Using the European Patent Office search facility, two patents were identified on the worldwide patent web register which related to the generation and application of algal derived products which had applications for increasing mineral bioavailability. In one case, algae powder was utilized as a seafood preparation for specialty dietary uses for hypomnesia patients (Wang, 2011a). In another patent, an algal powder was described with the application to act as a special dietary seafood containing minerals, amongst other components, for postabortal and postpartum women (Wang, 2011b).

Cholesterol-lowering activity Cholesterol is a lipid molecule with a steroid nucleus and an alcohol moiety. It is an essential structural component of cell membranes that helps maintaining membrane permeability and fluidity. Cholesterol performs multiple important functions in the human body, central of which is as a steroid hormone precursor. The body obtains cholesterol by de novo synthesis and from the diet. Cholesterol is not soluble in the bloodstream and must be transported by lipoproteins. A lipoprotein protein profile defines the levels of various lipoproteins in blood of an individual, and includes high-density lipoproteins (HDL), low-density lipoproteins (LDL) and other lipoprotein level information. The ratio of HDL to LDL is an important parameter associated with the development of atherosclerosis and coronary heart disease. The balance between LDL and HDL levels is mostly an inherited characteristic but, in a number of cases, this ratio can be managed by lifestyle adjustments. Plant-based functional foods, including sterols and fibres, are widely recommended as an alternative non-pharmacological approach to lower plasma cholesterol levels. A recent study has shown that lipid and powdered biomass from Schizochytrium sp. possessed cholesterol-lowering activities, which was associated with the down-regulation of hepatic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, LDL-receptor (LDLR) expression and intestinal Niemann-Pick C1-like 1 (NPC1L1) transporter expression. Hence, the mechanisms underlying the above activities were understood to be due to enhanced steroid excretion and decreased cholesterol absorption and synthesis (Chen et al., 2011). Interestingly, the fatty acid profile of Schizochytrium sp. revealed a high content of long-chain polyunsaturated fatty acids, including DHA and EPA (Chen et al., 2011). Earlier, a study with hypercholesterolemic patients supplemented with Spirulina tablets presented a reduction in cholesterol and improved lipid profiles following a Spirulina feeding trial (Ramamoorthy & Premakumari, 1996). A cholesterol-lowering activity was also reported in hamsters following a feeding trial with Chlorella pyrenoidosa biomass powder. (Cherng & Shih, 2005). It was proposed that water-soluble fibres, vegetable protein, phospholipids, Vitamin C, Vitamin E and β-carotene were the possible active ingredients involved. Of note is that the ratio of arginine to lysine for Chlorella protein is deemed better than proteins from soya bean. Plant proteins with low lysine contents have been reported as advantageous towards lowering serum cholesterol levels (Cherng & Shih, 2005). A hypothesis was also proposed from a separate study that the Advanced Glycation End product-inhibitory property of Chlorella prevents atherosclerosis in vivo (Yamagishi et al., 2005).

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Using the European Patent Office search facility, six patents were identified on the worldwide patent web register which related to the generation and application of cholesterol-lowering agents using the search terms of ‘algae’ and ‘cholesterol lowering’. However, the most relevant patent described the methodology to generate and application of microalgal protein-derived hydrolysates as cholesterol lowering agents. The patent application covered the microalgae belonging to Chlorophyta, Phaeophyceae, Rhodophyta, Cyanobacteria, Bacillariophyceae, Dinophyta and/or Haptophyta (KR20120130024, 2012).

Potential upcoming microalgae or their alternate use Microalgal research is at higher pace today compared to a few decades ago, due to scientific discoveries which have highlighted the commercial potential of microalgae. A recent study by Goiris et al. (2012) screened the antioxidant capacity of 32 microalgal biomass extracts using three different antioxidant assay methodologies. This study identified some industrially cultivated microalgae, including Botryococcus braunii, Chlorella vulgaris, Isochrysis sp., Neochloris oleoabundans, Phaeodactylum tricornutum and Tetraselmis suecica, which were considered to represent a potentially new source of natural antioxidants. Another study recommended the exploitation of a new thermotolerant microalga Coelastrella sp. F50 for antioxidant pigment production for human consumption (Hu et al., 2013). Although several bioactive metabolites of pharmaceutical importance were discovered in several microalgae cultured for bio-diesel production, none has been used commercially as yet, mostly due to lack of success stories. In the case of nutraceutical and/or pharmaceutical product exploitation from a new microalgae source, the generation would need to be of a compound with proven competitive efficacy in vivo that has received appropriate approval and which can be made sustainably and cost-effectively. This process is a slow one and, as such, the chances are that new microalgae may only be commercially relevant in the short term if they generate previously known bioactive and/or nutraceutical molecules which are currently marketed. Such an approach is being followed as part of the BAMMBO project mentioned previously (Murray et al., 2013). Commercial utilization of microalgae for nutraceutical production may be achieved through a conventional, a genetic and/or an enhanced photobioreactor culture process. Advancement beyond the current state of the art in relation to microalgal metabolites with anti-cancer, anti-bacterial, anti-fungal, anti-viral and anti-parasite properties has been minimally explored, and needs further development for natural health benefits to be realized. Some promising microalgae to be explored for pharmaceutical and nutraceutical benefits include Lyngbya spp., Porphyridium cruentum, Nostoc sp., (CAVN 10), Leptolyngbya crossbyana, Scytonema sp. UTEX 1163, Chlorella spp., Phaeodactylum tricornutum, Haematococcus pluvialis, Skeletonema costatum, Chlamydomonas reinhardtii, Euglena viridis, Spirulina platensis, Anabaena oryzae, Dunaliella primolecta, D. salina, Navicula directa, and Scenedesmus quadricauda. Microalgae suitable for omega-3 fatty acid production are from the genera of Phaeodactylum, Nannochloropsis, Thraustochytrium and Schizochytrium, as they accumulate EPA and/or DHA up to 40% of total fatty acids. Microalgae Diacronema lutheri and Isochrysis galbana might also be explored for DHA and EPA production, while the red microalga Porphyridium cruentum might be explored for arachidonic acid (ARA) production.

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Genetically modified (GM) microalgae Several microalgae, as well as selected cyanobacteria, have been genetically modified for enhanced nutraceutical and bio-fuels production. The genetic tools required for cyanobacterial genetic improvement are well developed, while the genetic tools for other microalgae are still largely in development. About 75 microalgal genome sequences are currently available, at least in draft form, and many new genome sequencing projects are on-going (DOE Joint Genome Institute, http://genome.jgipsf.org/; Radakovits et al., 2012; GOLD database, http://www.genomesonline.org/; Cyanobase, http://genome.kazusa.or.jp/cyanobase). The nuclear genome sequencing of several microalgae, such as Chlorella vulgaris C-169, Chlamydomonas reinhardtii, Cyanidioschyzon merolae, Galdieria sulphuraria, Ostreococcus lucimarinus, O. tauri, Phaeodactylum tricornutum, Thalassiosira pseudonana, and Volvox carteri f. nagariensis, have been done. The mitochondrial and plastid genomes of Dunaliella salina have also been sequenced. Other ongoing microalgal sequencing projects include Aureococcus anophageferrens CCMP1984, Botryococcus braunii, Fragilariopsis cylindrus, Micromonas pusilla CCMP1545, Pseudo-Nitschzia multiseries CLN-47 and Thalassiosira rotula. The growing information on the genome sequences will allow comparisons of gene distribution and synteny among various microalgal strains for further genetic modifications. For example, a recently completed European Commission project, with the acronym GIAVAP, created a transgenic Phaeodactulum tricornutum with acyl-CoA dependent Δ6-desaturase gene from a marine unicellular green microalga Ostreococcus tauri. The transgenic strain of P. tricornutum overexpresses EPA. Another P. tricornutum strain, with elevated levels of fucoxanthin, was created out of the same project by genetic engineering. Microalgae C. reinhardtii and P. tricornutum were successfully transformed to express four genes encoding high value proteins, including human growth hormone. (http://cordis.europa.eu/search/index .cfm?fuseaction=lib.document&DOC_LANG_ID=EN&DOC_ID=138235201&q=). The green microalga Haematococcus pluvialis was successfully transformed with the mutagenized pds (phytoene desaturase gene) for accelerated astaxanthin biosynthesis (Steinbrenner & Sandman, 2006). The microalga C. reinhardtii has recently been used successfully for several biomolecules overproduction, including recombinant peptides, proteins and CTB-D2 gene for human oral vaccines (Specht et al., 2010). Tocopherols are important antioxidants that act as effective oxygen radical scavengers in lipophilic environments, such as oils and the lipid bilayer of biological membranes. The tocopherols biosynthetic pathway of the cyanobacterium Synechocystis sp. strain PCC 6803 was successfully improved by engineering the strain with inducible nirA promoter from Synechococcus sp. strain PCC 7942 to drive the expression of the gene for p-hydroxyphenylpyruvate dioxygenase of Arabidopsis thaliana. The nirA promoter is induced by nitrite and repressed by ammonium. The enzyme p-hydroxyphenylpyruvate dioxygenase catalyzes the formation of homogentisic acid from p-hydroxyphenylpyruvate. The heterologous expression of the above gene, under the control of nirA promoter, yielded a five-fold increase of total tocopherols, and about 20% of tocopherols were accumulated as tocotrienols (Qi et al., 2005). Zeaxanthin is one of the Xanthophyll pigments with antioxidant activities, and it has gained commercial interest. It was experimentally demonstrated that a genetically modified strain of Synechocystis sp. strain PCC 6803 can produce 2.5-fold zeaxanthin compared to the wild-type strain. For this genetic optimization, they have used strong

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psbAII promoter to overexpress yeast isopentenyl diphosphate isomerase (ipi) and Synechocystis genes coding for β-carotene hydroxylase (crtR), phytoene desaturase (crtP) and phytoene synthase (crtB) by replacing the coding sequence of the psbAII (Lagarde et al., 2000). Production of recombinant enzymes, including α-galactosidase, have been demonstrated in the marine microalga Dunaliella tertiolecta. The gene for α-galactosidase was obtained from the fungus Umbelopsis vinacea and was successfully transformed in the chloroplasts and regulated by endogenous D. tertiolecta promoters and UTR elements. The enzyme α-galactosidase has medicinal applications, such as the treatment of Fabry’s disease and prevention of digestive disorders (Georgianna et al., 2013). Photosynthetic microalgae, including cyanobacteria, have higher growth rates compared to land plants, and their biomass production needs no arable lands. The metabolic engineering of microalgae with the advancement of synthetic biology and genetic manipulations has attracted this group of organisms for the bio-production of natural active biomolecules in sustainable and economic ways. However, there would be a requirement of initial risk assessment of genetically modified (GM) microalgae for large-scale outdoor cultivation. The potential threat of the accidental escape of GM microalgae to the public health and ecosystem cannot be ruled out, as microalgae are at the base of aquatic food chains. However, the recent request by the company Algenol for large-scale cultivation of GM cyanobacteria in outdoor enclosed photobioreactor for ethanol production has been approved (Henley et al., 2013).

Concluding remarks Microalgae, as a potential source of nutraceuticals, are the fast growing segment of the health and nutrition industry today, although the traditional use of microalgae can be traced back to several decades. The changes in the trend of microalgae use are due to: 1 our present day lifestyle; 2 microalgae as a natural source of multiple nutrients/bioactive compounds; 3 recent enormous research on health and nutritional benefits of microalgae; 4 the development of industrial cultivation and optimization of down-stream technologies; and 5 few successful stories of microalgae cultivation and their available commercial products. The unlimited resource of microalgae in nature in general, and in culture collections in particular, needs detailed evaluation for best possible exploration of microalgae for mankind, and is a huge challenge. Although the microalgal biotechnology sector is growing faster than ever, the benefits from upcoming microalgae are unpredictable, and are totally dependent on industrial interests and technological developments. In fact, the present demand on biofuels from microalgae provides an opportunity to explore every possible microalga through biorefinery concept. Although genetic modification of few microalgae for specific biomolecule production has been improved in laboratory scale, the commercial production of genetically modified microalgae is still in infancy because: 1 the genetic tools for all potential microalgae are not optimized; 2 genetically modified microalgae may not be suitable for open pond/raceway cultivation as per specific countries’ legislations; 3 there is no protocol to estimate the potential threat of the accidental escape of GM microalgae to the public health and ecosystem.

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Therefore, it appears that selection of the best microalgae strains, and their conventional growth optimization depending on local weather and available technologies, are the benchmarks for microalgal biotechnology at present for nutraceutical production.

Acknowledgments The research leading to the present review has received funding from the European Union Seventh Framework Programme FP7-KBBE-2010-4 SP1-Cooperation – Collaborative Project Small or medium-scale focused research project – under grant agreement No. 265896- BAMMBO.

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C H A P T E R 13

The marine origin of drugs André Horta, Celso Alves, Susete Pinteus & Rui Pedrosa Grupo de Investigação em Recursos Marinhos,Instituto Politécnico de Leiria,Portugal

Introduction Natural products have been looked as the main source of drug discovery for pharmacological applications, mainly as disease therapeutic agents. The truth is that natural products have proved to be a major source for drug development and, consequently, a large number of plants, animals and bacteria have been examined for drug discovery. However, the marine ecosystems are much more biologically diverse than terrestrial ecosystems. It is not surprising the view that, in the last 50 years, considerable research efforts have been done on these unique organisms, namely for the structure and functionality of the marine compounds origin (Blunt et al., 2014; Gerwick & Moore, 2012; Mayer et al., 2010; Sithranga Boopathy & Kathiresan, 2010; Horta et al., 2014). Moreover, the marine environment has already proved to be an extraordinary source of natural compounds, which exhibit structural and chemical features not found in the terrestrial environment, with many applications in human health (Dhivya et al., 2012; Hussain et al., 2012; Murray et al., 2013; Pangestuti & Kim, 2011). The incredible diversity of marine life can be translated into a wide range of useful marine drug applications on therapeutic, pharmacology, cosmetic and nutraceutic areas. This marine environment is a complex ecosystem, with an enormous diversity of different life forms that establishing close associations, revealing it as an outstanding source for natural products settled by the diversity of high value added bioactive molecules that have been isolated. Actually, some of those are nowadays associated to therapeutic use and are linked to marine research success stories, for example Cytarabine (derivative marine drug), Eribulin Mesylate (derivative marine drug) and Ziconotide (marine drug) (Gerwick & Moore, 2012). From 1965 to the present day, more than 16000 marine compounds have been isolated and more than 300 patents were approved (Blunt et al., 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014; Gerwick & Moore, 2012; Mayer et al., 2010; Sithranga Boopathy & Kathiresan, 2010). Actually, the scientific and strategic policy efforts to identify drugs from the sea have been started after the 1960s. However, during the two decades from 1965 to 1985 less than 2000 new marine isolated compounds were described (Figure 13.1). After 1985, the number of new marine compounds increased radically, not only as a result of a more systematic research, but also by the use of new identification and purification tools, as well new bioassays pipelines (Figure 13.1). Moreover, in the last decade, the number of new isolated marine compounds has almost doubled when compared with the last similar period (Figure 13.1). In line with these findings, the number of the Food and Drug Administration (FDA)-approved Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd. 293

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FDA-approved marine drugs 1

Marine Isolated Compounds (proximate number/10 years)

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Figure 13.1 Approximate number (tendency growth) of new marine isolated compounds identified in the last 47 years (1975–2012). The numbers inside the top circles represents the marine natural or marine derived drugs FDA-approved in the correspondent period. Source: Based on data from Blunt et al., 2007; Blunt et al., 2008; Blunt et al., 2009; Blunt et al., 2012; Blunt et al., 2013; Blunt et al., 2014; Blunt et al., 2003; Blunt et al., 2004; Blunt et al., 2005; Blunt et al., 2006; Blunt et al., 2010; Blunt et al., 2011; Gerwick and Moore, 2012; Mayer et al., 2010; Sithranga Boopathy and Kathiresan, 2010.

marine or marine derived drugs has started to growth in the last years, and five of the seven FDA-approved marine drugs were approved in the last 17 of 50 years (Figure 13.1). Additionally, it is expected that the number of FDA-approved drugs to increase in the next few years, because of the number of new marine compounds that have mainly been described in the last 17 years (>11 000 new marine compounds), as well the view that eleven marine or marine derived drugs are in clinical trials (two marine molecules – phase III; four marine molecules – phase II; five marine molecules – phase I). One of the main reasons of interest in marine natural products knowledge was motivated by the need to detect and understand the chemical basis of various marine intoxications produced by phycotoxins, such as okadaic acid (OA) or tetrodotoxin (TTX) (Bentur et al., 2008; Hanifin, 2010; Medina et al., 2013). Moreover, the isolation and identification of some of these marine toxins opened new opportunities for marine drug development, both in the therapeutic and pharmacological areas of fundamental research. However, the marine origin of drugs from dependent or non-dependent phycotoxins sources for therapeutic use is a long journey, with many bottlenecks to be overpassed and solved. This idea is clear from the example of TTX (Box 13.1), which was isolated for the first time in 1950 (Yooko, 1950) from the puffer fish, and is now on a phase III drug clinical trials development (Tectin) for the pain area application (Hagen et al., 2007, 2008). This was only possible after understanding the pharmacological effects of TTX, which block sodium currents in excitable membranes. It is now understood that TTX binds and blocks voltage-gated sodium (Na+ ) channels with remarkably high specificity, thereby preventing the influx of Na+ (Hanifin, 2010).

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Some of the marine organisms that produce phycotoxins, like puffer fish, are proven potent sources of drugs. However, are these marine organisms the real producers of marine drugs? It is now believed that microbial flora present in marine vertebrates or invertebrates could be the true marine drug producers. In line with this view, it is clear that one of the main reasons of this marine drug origin is related to their response to ecological pressure, since these organisms live in the same environment over a long evolutionary period of time. This is particular evident for marine microflora and microalgae, which alone constitute more than 90% of the oceanic biomass (Sithranga Boopathy & Kathiresan, 2010). This huge biological resource is considered one of the main producers of the marine drugs. Moreover, several studies on marine natural products showed evidences that many of the bioactive compounds previously found in marine animals and plants were, in fact, produced or metabolized by their associated microorganisms (Zheng et al., 2005). The study of the secondary metabolites produced by microorganisms and their ecological associations with macro-organisms started to be investigated more recently. Some recent studies have shown that associated bacteria can be a new source of marine drugs, because these microorganisms have the capacity to quickly adapt and respond to their environment, competing for defence and survival through the generation of unique secondary metabolites. These compounds are produced in response to stress, and many have shown high value in biotechnological or pharmaceutical applications. Nonetheless, other of the marine bacteria competitive factors for the production of unique marine drugs is related with the biosynthesis of diverse organohalogen compounds, such as the polybrominated organic compounds (Gerwick & Moore, 2012; Molinski et al., 2009; Sashidhara et al., 2009; Villa & Gerwick, 2010; Winter & Moore, 2009). Essentially, the capacity of ocean and marine organism for buffering pollution agents, such as halogenated organic compounds, enhances the conditions for the generation of unique molecules that could have pharmacological and/or therapeutic interest. Box 13.1 The TTX marine drug origin.

TTX is a very potent neurotoxin found mainly in the Tetraodontidae fish family, particularly in the puffer fish or ‘fugu’ fish (Bentur et al., 2008). The majority of reported TTX intoxication cases are linked to Asian countries, mainly associated to puffer fish consumption in Japan, which is very relevant to the Japanese traditional gastronomy. TTX toxin is mainly found on tropical waters, but this marine toxin has also been found in European coastal waters (Katikou et al., 2009). For several years it was assumed that TTX was only present in the puffer fish, and that TTX production was directly linked to this fish. Since 1965, however, after the identification of TTX on eggs of Taricha torosa, the presence of TTX has been found in several animals, including arthropods and molluscs, among others (Miyazawa & Noguchi, 2001; Mosher et al., 1964; Noguchi & Arakawa, 2008). Moreover, the TTX toxin has also been found in dinoflagellates, specifically in the Alexandrium tamarense species (Sato et al., 2000). Since 1980, several studies have been conducted for the identification of the primary origin of TTX on the food chain process. Because it was observed that TTX was present on several marine bacteria (i.e. Vibrio alginolyticus, Shewanella alga, Alteromonas tetraodonis, etc.), both free-living and symbiotic, the prevailing theory is that TTX is produced by several species of bacteria. These kinds of bacterial TTX producers have been isolated from puffer fish, crustaceous, echinoderms and algae (Simidu et al., 1987). Moreover, this marine organism has shown a huge resistance to TTX, which is compatible with the presence of a biological defence pathway (Ikeda et al., 2006; Jeon et al., 2008; Noguchi et al., 2006).

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Marine chemical ecology and the origin of marine drugs The oceans are one of the most important resources of the earth, due to the vast area they represent and the ecosystem services they provide that are fundamental to our planet (Leal et al., 2012). The physics, chemistry and biology of the oceans are key elements in the functioning of the earth system (Karsenti et al., 2011). The oceans cover more than 70% of our planet’s surface and offer a unique environment or ecosystem for the growth of life, rendering marine organisms unique characteristics and adaptation mechanisms that are generally not present in terrestrial life forms (Demunshi & Chugh, 2009; Dias et al., 2012). As has previously been mentioned, the marine environment is extremely complex and contains a huge variety of life forms, making it a rich source of biological and chemical diversity (Debbab et al., 2010; Zheng et al., 2011; Horta et al., 2014). In terms of biodiversity, the marine environments are among the richest and most complex ecosystems, not only for the diversity of species, but also the genetic and ecosystems diversity (Hay & Fenical, 1996; Rocha et al., 2011). Harsh chemical and physical conditions enable production of quite specific and potent active molecules (Aneiros & Garateix, 2004; Rocha et al., 2011). This is the result of the complexity of interactions in the marine ecosystems that depend on many different factors, including the number of species and the population structure, variation in food and energy supply and the geography of the habitat (Raes & Bork, 2008). Sometimes, marine organisms face the dilemma of how to allocate the limited resources available. In general terms, resources can be used to increase growth and reproduction, or to counteract stress (e.g., predators, parasites, maintenance of unfouled surfaces, decay, competitive ability, paralyzing their prey, and unfavourable environmental conditions such as ultraviolet light, temperature, nutrient concentrations, high pressure, salinity, oxygen content and light). Thus, substantial investments into stress defence or avoidance strategies may result in a reduction in growth and reproduction, and vice versa (McClintock & Baker, 1997; Winter et al., 2010; Hu et al., 2011). All of these factors promote the production of chemical defences to vary even intra-species and inter-species, depending on the community or the season, pointing to the important role of biotic and abiotic factors (Cebrian et al., 2003). The natural products synthesized by marine organisms with the purpose of acting as chemical defences are normally designed as secondary metabolites. These molecules have an important role in the regulation of biology, co-existence and co-evaluation, without participating directly in their primary metabolism. They act as key mediators in the interaction between organisms and their environment. In contrast to primary metabolites, these compounds are characterized by their wide heterogeneity, as well by their restricted distribution, occurring only in some groups or species (Avila et al., 2008; Ivanisevic et al., 2011; Montaser & Luesch, 2011). Predominantly, the production of these types of compounds occur in sessile or slow-moving organisms (e.g. algae, sponges, cnidarians, tunicates and bryozoans) without effective escape mechanisms or structural protection, which are likely to be chemically defended (Noyer et al., 2011). However, the production of these compounds could occur by other pathways, since some organisms have the capacity to sequester compounds and then derivatize them to more or less toxic forms, or have acquired these molecules by association with other organisms. As mentioned before, sharing a common environment over a long evolutionary period has allowed

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the establishment of well-balanced associations between many marine organisms (Pennings & Paul, 1993; Thoms et al., 2006; Graça et al., 2013). This fact is patent in the relationship between the marine eukaryotes and microorganisms. The surfaces of marine eukaryotes provide a unique habitat for colonization by microorganisms, and competition between members of these communities and chemically mediated interactions with their host are thought to influence both microbial diversity and function, as well as promoting the production of various kinds of secondary metabolites that defend the host from natural enemies (Hay & Fenical, 1996; Penesyan et al., 2009; Horta et al., 2014). For all these reasons, marine organisms are producers of different kinds of secondary metabolites (e.g. terpenoids, alkaloids, polyketides, peptides, shikimic acid derivates, sugars, steroids and a large mixture of biogenesis metabolites) (Aneiros & Garateix, 2004; Rocha et al., 2011; Graça et al., 2013). To date, many unique chemically compounds of marine origin with various biological activities (antimicrobial, antitumor, anticoagulant, antioxidant, anti-inflammatory and antiviral, antimalarial, antituberculosis, antifouling and antiprotozoal) have been isolated. Some of these are under investigation and are being used to develop new pharmaceuticals (Abou-Elela et al., 2009; Mayer et al., 2013; Blunt et al., 2013). It is widely accepted that marine natural products provide unusual and unique chemical structures, upon which molecular modelling and chemical synthesis of new drugs can be based with greater efficacy and specificity for the treatment of many human diseases. Therefore, marine organisms are ideal candidates for novel sources of both pre-existing and unrecognized high value-added biomolecules, with potential for providing sustainable, economic and human benefits (Jha & Zi-rong, 2004; Stonik, 2009; Ebada et al., 2010; Murray et al., 2013).

Marine or marine-derived drugs sources In the 1970s, as researchers were able to access more remote marine habitats, invertebrates, plants and algae became the focus of natural product chemists who were searching for novel compounds with potent bioactivity. The marine environment covers a wide thermal range (from below-freezing temperatures in Antarctic waters, to about 350 ∘ C in deep hydrothermal vents), pressure range (1–1000 atm), a wide nutrient range (oligotrophic to eutrophic), and has extensive photic and non-photic zones. This extensive variability has facilitated extensive speciation at all phylogenetic levels, from microorganisms to mammals (Jha & Zi-rong, 2004). The sessile nature of invertebrates such as sponges, cnidarians, ascidians and algae may have favoured the evolution of structurally diverse natural products. They have developed various defence systems against predators, the larvae of other sessile organisms and pathogenic microorganisms. Since marine invertebrates do not produce antibodies, their defence mechanisms are based primarily on phagocytosis by leukocytes, aided by producing and exuding secondary metabolites (Abad & Bermejo, 2001). With the discovery of Ara-C, a potent anticancer drug based on compounds isolated from a marine sponge in the 1950s, the scientists have increased the search for bioactive compounds in marine ecosystem. Until 2009, a total of 11 phyla, 6 subphyla, 20 classes, 20 subclasses, 74 orders, 253 families, 569 genera and 1354 species recorded new marine natural products from invertebrates. Among them, phylum Porifera recorded 48.8% of all natural products

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Porifera Cnidaria Echinodermata Chordata Mollusca Other Figure 13.2 New natural products from marine invertebrate phyla. Cumulative number of new natural products discovered from different marine invertebrate phyla from 1990 to 2009. Group ‘other’ includes Annelida, Arthropoda, Brachiopoda, Hemichordata, Platyhelmintes and Bryozoa. Source: Based on data from Leal et al., 2012.

discovered since 1990, while Cnidaria comprised 28.6%. Echinodermata, Chordata and Mollusca represented 8.2%, 6.9% and 5.8%, respectively. The remaining 1.7% were documented by the following phyla in decreasing order of importance: Annelida, Bryozoa, Platyhelmintes, Hemichordata, Brachiopoda and Arthropoda (Figure 13.2) (Leal et al., 2012). Although only a few marine-derived products are currently being used as pharmacological agents, several are now in the clinical pipeline and in clinical development. In the history of marine drugs research, sponges have played a crucial role, being the source of the first compounds used for clinical purposes. Back in the 1950s, Bergmann & Feeney (1950) isolated several nucleosides from the caribbean sponge Cryptotethya crypta (Tethylidae) (Bergmann & Feeney, 1950). Two of these, spongothymidine and spongouridinecontained, are rare arabinose sugar rather than ribose, which is a quite ubiquitous sugar in nucleosides. This discovery led researchers to synthesize analogues, Ara-A (Vidarabine®, Vidarabin Thilo®) and Ara-C (Cytarabine, Alexan®, Udicil®), which nowadays are used as antiviral and antitumor drugs, respectively (Kijjoa & Sawangwong, 2004). More details related to Ara-C success drug story will be given later on in this chapter. In 1986, two Japanese chemists isolated a naturally-occurring compound from the marine sponge Halichondria okadai (Hirata & Uemura, 1986). The compound was named Halichondrin B, and it immediately began to generate great excitement when it was realized that it was extremely potent at killing certain types of cancer cells in small-scale tests. As a result of this discovery, it was immediately given top priority to be tested against a wide range of other cancers. Eribulin mesylate is a halichondrin B analogue, a nontaxane inhibitor of microtubule dynamics, and is currently available commercially for cancer treatment under the name Halaven® (Pal et al., 2012). The Eribulin mesylate success story is detailed later on this chapter. In fact, seven marine derived drugs are FDA-approved, and three had origin in marine sponges (Porifera). Although sponges have demonstrated to be the most promising source of new marine compounds with therapeutic application, other marine organisms have also revealed to produce molecules with therapeutic usage. The cone snail Conus magnus produces a 25-aminoacid peptide in its venom, the Ziconotide, that acts by binding to and inhibiting pre-synaptic calcium channels,

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thereby preventing neurotransmitter release (Olivera et al., 1985). It is commercialized to treat chronic pain under the name Prialt®. The Ziconotide success story will be detailed later on this chapter. From the marine tunicate Ecteinascidia turbinata was isolated an anticancer molecule, Trabectedin, which is now produced synthetically. Trabectedin is a tetrahydroisoquinoline alkaloid and is the first marine anticancer agent to be approved in the European Union for patients with soft tissue sarcoma (STS) and those with relapsed platinum-sensitive ovarian cancer (Wright et al., 1990). The mechanism of action of trabectedin involves the binding by a covalent reversible bond of the DNA minor groove interacting with different binding proteins of the Nucleotide Excision Repair (NER) system. Although other known DNA-interacting agents require a deficient NER mechanism to exert their activity, trabectedin needs a proficient NER system to exert its cytotoxic activity (Mayer et al., 2010). Another success case is Brentuximab vedotin. This consists of the anti-CD30 antibody cAC10 (SGN 30), conjugated to monomethyl auristatin E (MMAE), a synthetic analogue of dolastatin 10 which acts as a potent anti-tubulin agent. Dolastatin 10, first reported in 1987 by the Pettit group, was obtained from the sea hare Dolabella auricularia (Pettit et al., 1987). However, it was later found to originate from the gastropod’s cyanobacteria (Symploca sp.) (Luesch et al., 2001). To date, a dolastatin 10-analogue, auristatin E, has been formulated as an antibody drug conjugate, brentuximab vedotin, and approved for the treatment of Hodgkin lymphoma and anaplastic large cell lymphoma. In fact, at least three antibody drug conjugates (ADC) based on synthetic analogues of marine cyanobacterial compounds are currently in the clinical pipeline as anticancer agents (Tripathi et al., 2012). Among marine organisms, not only the invertebrates have contributed for the development of new drugs with clinical relevance. Fish have also revealed their importance as a source of polyunsaturated fatty acids. Among fish, salmon, herring, mackerel and anchovies are rich sources of polyunsaturated fatty acids (Simopoulos, 1991). Numerous studies have shown that the long-chain omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid are effective for lowering triglycerides (TG) and non-high-density lipoprotein (HDL) cholesterol, as well as reducing the risk of coronary arteriosclerosis (Gerwick & Moore, 2012; Maki et al., 2010). The reduction of serum triglyceride levels has been shown to be caused by decreased very low-density lipoprotein triglyceride secretion rates in kinetic studies in humans. Triglyceride synthesis can be reduced by n-3 fatty acids in three general ways: reduced substrate (i.e. fatty acids) availability, which could be secondary to increase in β-oxidation; decreased free fatty acids delivery to the liver; decreased hepatic fatty acids synthesis; increased phospholipid synthesis or decreased activity of triglyceride-synthesizing enzymes (diacylgylcerol acyltranferase or phosphatidic acid phosphohydrolase) (Harris & Bulchandani, 2006). The FDA approved prescription fish oil, Lovaza®, which is composed of the ethyl esters of n-3 fatty acids obtained from fish, mainly comprised of EPA (20 : 5n3) and DHA (22 : 6n3), administered as 400 mg/day (Gerwick and Moore, 2012). Above, we described the seven FDA-approved cases in the marine drug discovery for clinical purposes. Following is a brief resume of molecules that are revealing promising results in clinical trials, phases III, II and I. Table 13.1 summarizes all the marine drugs or marine derived drugs approved by FDA, as well all the marine compounds under clinical trials evaluations (phases III, II and I).

Sponge

Sponge

Cone snail

Sponge

Vidarabine (Ara-A)

Ziconotide

Eribulin Mesylate (E7389)

Marine organism

Cytarabine (Ara-C)

Compound name

Bacterium

Mollusc

Bacterium

Bacterium

Predicted biosynthetic source

Derivative

Natural Product

Derivative

Derivative

Natural product or derivative

Macrolide

Peptide

Nucleoside

Nucleoside

Chemical class

Microtubules

N-Type Ca chanel

Viral DNA polymerase

DNA polymerase

Molecular target

Cancer

Pain

Antiviral

Cancer

Disease area

FDA-approved marine or marine derived drugs, as well as marine drugs on phase I, II and III of clinical trials.

FDA-Approved

Table 13.1

(Gerwick & Moore, 2012, Martins et al., 2014, Hirata & Uemura, 1986, Pal et al., 2012)

(Gerwick & Moore, 2012, Mayer et al., 2010, Martins et al., 2014, Olivera et al., 1985)

(Gerwick & Moore, 2012, Mayer et al., 2010, Martins et al., 2014, Bergmann & Feeney, 1950, Kijjoa & Sawangwong, 2004)

(Gerwick & Moore, 2012, Mayer et al., 2010, Martins et al., 2014, Bergmann & Feeney, 1950, Kijjoa & Sawangwong, 2004)

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Phase III

Puffer fish

Tetrodotoxin

Mollusc/ cyanobacterium

Brentuximab vedotin (SGN-35)

Tunicate

Tunicate

Trabectedin (ET-743)

Plitidepsin

Fish

Omega-3-acid ethyl esters

Bacterium

Bacterium

Cyanobacterium

Bacterium

Microalgae

Natural Product

Natural Product

Derivative

Natural Product

Derivative

Guanidinium alkaloid

Depsipeptide

Antibody drug conjugate (MM auristatin E)

Alkaloid

Omega-3 fatty acids

Soduim Channel

Rac1 & JNK activation

CD30 & microtubules

Minor groove of DNA

Trygliceridesynthesizing enzymes

Pain

Cancer

Cancer

Cancer

Hypertriglyceridemia

(continued overleaf)

(Noguchi et al., 1986, Yasumoto et al., 1986, Newman & Cragg, 2014)

(Gerwick & Moore, 2012, Martins et al., 2014, Le Tourneau et al., 2010, Muñoz-Alonso et al., 2013, Rinehart et al., 1981, Rinehart et al., 1990)

(Luesch et al., 2001, Gerwick & Moore, 2012, Martins et al., 2014, Pettit et al., 1987)

(Gerwick & Moore, 2012, Mayer et al., 2010, Martins et al., 2014, Wright et al., 1990)

(Gerwick & Moore, 2012, Martins et al., 2014, Simopoulos, 1991, Maki et al., 2010)

The marine origin of drugs 301

Phase II

Table 13.1

Worm

Mollusc

Tunicate

Mollusc/ cyanobacterium

Zalypsis (PM00104)

Lurbinectedin (PM01183)

Glembatumumab vedotin (CDX-011)

Marine organism

DMXBA (GTS-21)

Compound name

(continued)

Cyanobacterium

Bacterium

Bacterium

Worm?

Predicted biosynthetic source

Derivative

Derivative

Derivative

Derivative

Natural product or derivative

Antibody drug conjugate (MM auristatin E)

Alkaloid

Alkaloid

Alkaloid

Chemical class

Glycoprotein NMB & microtubules

Minor groove of DNA

DNA-binding

α7 nicotinic acetylcholine receptor

Molecular target

Cancer

Cancer

Cancer

Schizophrenia

Disease area

(Gerwick & Moore, 2012, Martins et al., 2014, Tse et al., 2006)

(Newman & Cragg, 2014, Martins et al., 2014, Rinehart et al., 1990, Mayer et al., 2010)

(Ocio et al., 2009, Gerwick & Moore, 2012, Mayer et al., 2010, Martins et al., 2014, Newman & Cragg, 2014)

(Gerwick & Moore, 2012, Mayer et al., 2010, Martins et al., 2014, Kem et al., 1971, Kitagawa et al., 2003)

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Phase I

Bacterium

Sponge

Bryozoan

Mollusc/ cyanobacterium

Mollusc/ cyanobacterium

Marizomib (Salinosporamide A; NPI-0052)

PM060184

Bryostatin

SGN-75

ASG-5ME

Cyanobacterium

Cyanobacterium

Bacterium

Bacterium

Bacterium

Derivative

Derivative

Natural product

Natural product

Natural product

Antibody drug conjugate (MM auristatin E)

Antibody drug conjugate (MM auristatin F)

Macrolide Iactone

Polyketide

Beta-lactonegamma lactam

ASG-5 & microtubules

CD70 & microtubules

Protein kinase C

Minor groove of DNA

20S proteasome

Cancer

Cancer

Cancer

Cancer

Cancer

(Gerwick & Moore, 2012, Mayer et al., 2010, Martins et al., 2014, Bergmann & Feeney, 1950, Kijjoa & Sawangwong, 2004)

(Gerwick & Moore, 2012, Martins et al., 2014, Alley et al., 2009, Newman & Cragg, 2014)

(Jha & Zi-rong, 2004, Gerwick & Moore, 2012, Mayer et al., 2010, Sudek et al., 2007)

(Martins et al., 2014, Martín et al., 2013)

(Gerwick & Moore, 2012, Mayer et al., 2010, Martins et al., 2014, Feling et al., 2003, Chauhan et al., 2005)

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In clinical trials of phase III currently running, two compounds are being investigated: plitidepsin and tetrodotoxin: • Plitidepsin (Aplidin®) is a cyclic depsipeptide identified from the Mediterranean marine tunicate Aplidium albicans. Plitidepsin was identified as a new generation of didemnin that showed better therapeutic indexes with markedly higher antitumor activity than didemnin B at non-toxic concentrations (Le Tourneau et al., 2010). This marine compound is intended to be used in combination with dexamethasone as third-line therapy for the treatment of patients with multiple myeloma (MM). Plitidepsin induces rapid and persistent activation of apoptosis in tumour cells by induction of early c-Jun N-terminal kinases (JNK) and p38 MAPK, leading eventually to mitochondrial cytochrome C release that initiates the apoptosis cascade by means of caspase cascade activation. It inhibits Vascular Endothelial Growth Factor (VEGF) secretion and down-regulates VEGF receptor-1 in leukaemia cell lines (MOLT-4), blocking an essential loop for cell proliferation (Le Tourneau et al., 2010; Muñoz-Alonso et al., 2013). • Tetrodotoxin (Tectin®) as previously mentioned (see Box 13.1), acts on the central and peripheral nervous system by blocking the sodium ion channels vital for cellular signalling pathways (e.g. transmission of impulses and maintenance of cell functions), eventually leading to paralysis and even death. TTX is 10 000 times deadlier than cyanide. Although tetrodotoxin is not a formal anti-tumour agent, it is in fact in Phase III trials as an agent (Tectin®) against inadequately controlled pain related to cancer, by WEX Pharmaceuticals in the USA, together with a Phase II trial under the same company, again in the USA, against the neuropathic pain resulting from chemotherapy-induced peripheral neuropathy (Newman & Cragg, 2014). In clinical trial phase II are currently four compounds, DMXBA (GTS-21), PM00104, PM01183 and CDX-011: • DMXBA (GTS-21) (3-(2,4-dimethoxybenzylidene)-anabaseine-GTS-21) is a synthetic derivative of anabaseine, an alkaloid present in several species of marine worms (phylum Nemertea), but it was first isolated from Paranemertes peregrina (Kem et al., 1971). DMXBA (GTS-21) is a drug that acts as a partial agonist at neural nicotinic acetylcholine receptors. It binds to both the α4β2 and α7 subtypes, but activates only the α7 to any significant extent. Both GTS-21 itself and its demethylated active metabolite, 4-OH-GTS-21, display nootropic and neuroprotective effects (Kitagawa et al., 2003; Meyer et al., 1998), and GTS-21 is being investigated for the treatment of Alzheimer’s disease (Kem, 2000), nicotine dependence and, most significantly, for schizophrenia (Martin et al., 2004). • PM-10450 (Zalypsis®) is a synthetic dimeric isoquinoline alkaloid derived from jorumycin, a natural compound isolated from the skin and mucin of the nudibranch Joruna funebris, from renieramycin J, isolated from a species of the marine sponge Netropsia and tunicates (Newman & Cragg, 2014), from safracins and saframycins isolated from bacterial sources and marine sponges (Ocio et al., 2009), and from ecteinascidins isolated from marine tunicates (Leal et al., 2009). In pre-clinical trials, Zalypsis demonstrated strong in vitro and in vivo antitumor activity in a wide variety of solid and haematological tumour cell lines and human transplantable breast, gastric, prostate and renal xenografted tumours. Zalypsis also demonstrated a manageable and reversible preclinical toxicology profile. It binds to DNA and is cytotoxic, but it does not activate the ‘DNA damage checkpoint’ response. Thus, Zalypsis has cytotoxic effects dependent on DNA binding that are not associated with DNA damage (Guirouilh-Barbat et al., 2009).

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• Lurbinectedin is derived from the marine tunicate Ecteinascidia turbinate (Rinehart et al., 1990) and is a new synthetic tetrahydroisoquinoline alkaloid that is currently in phase II clinical development for the treatment of solid tumours (Mayer et al., 2010). This compound is another variation on the basic structure of the dimeric isoquinoline alkaloids, but has a tetrahydro-β-carboline moiety instead of the tetrahydroisoquinoline present in ring C, and binds in the DNA minor groove. The compound has shown to have different pharmacokinetics in patients and, also like trabectedin, to attenuate nuclear excision repair (NER). It also demonstrated synergy with platinum-based agents in vitro, thus suggesting a possible treatment regimen, since it also demonstrated activity against platinum-resistant cell lines (Newman & Cragg, 2014). • Glembatumumab Vedotin (CDX-011) is an derivate of Dolastatin 10, one molecule found in the sea hare, Dolabella auricularia. It is also known as CDX-011 and CR011-vcMMAE (Martins et al., 2014). It is an ADC that targets cancer cells expressing transmembrane glycoprotein NMB (GPNMB), which is overexpressed by multiple tumour types, including breast cancer and melanoma. It has been shown to be associated with the ability of the cancer cell to invade and metastasize, and to correlate with reduced time to progression and survival in breast cancer (Tse et al., 2006). CDX-011 was targeted against patients with unresectable melanomas at stage III or IV who have failed one cytotoxic chemotherapy regimen, and its use has expanded to include metastatic breast cancer as well (Newman & Cragg, 2014). In clinical trials phase I, at the moment are five compounds: Marizomib, PM060184, Bryostatin, SGN-75 and ASG-5ME: • Marizomib®Z (Salinosporamide A) was discovered from the marine actinomycete Salinispora tropica and Salinispora arenicola, which are found in ocean sediment (Feling et al., 2003). Salinosporamide A belongs to a family of compounds known collectively as salinosporamides, which possess a densely functionalized γ-lactam-β-lactone bicyclic core. It has been studied as a potent proteasome inhibitor and is currently being evaluated in patients with multiple myeloma (Chauhan et al., 2005). The unique functionalization of the core bicyclic ring structure of salinosporamide A appears to have resulted in a molecule that is a significantly more potent proteasome inhibitor than omuralide (Feling et al., 2003). • PM060184 is a new synthetic tubulin-binding agent, originally a polyketide isolated from the marine sponge Lithoplocamia lithistoides. PM060184 and its analogue PM050489 have shown subnanomolar in vitro activity in human cancer cell lines and potent antimitotic activity, as well a new biochemical mechanism of interaction with tubulin and a potent in vivo activity in different animal models. PM60184 is currently being tested in clinical trials for safety and efficacy (Martín et al., 2013). • Bryostatin: the bryostatins are complex polyketides isolated from the marine bryozoan Bugula neritina, but some studies indicate that they are produced by the uncultured symbiotic bacterium Candidatus Endobugula sertula (E. sertula) (Sudek et al., 2007). It shows remarkable selectivity against human leukaemia, renal cancer, melanoma and non-small cell lung cancer cell lines. This compound modulates the signal transduction enzyme protein kinase-C (PKC). Bryostatin was originally described on the basis of the inhibiting growth in murine P388 lymphocytic leukaemia cells at subnanomolar concentrations. A range of properties have subsequently been described, including activation of T-cells, immunomodulation, and stimulation of haematopoietic progenitor cells. Bryostatin has been found to bind to protein kinase C with high affinity, which may be the mechanistic basis

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for both observed anti-cancer and immunostimulating activities. Bryostatin is not effective in cancer treatment by itself, but it seems to enhance the activity of such chemotherapies as taxol and cisplatin. It may be used in tandem with cancer treatments that respond to taxol, such as breast, ovarian and lung cancers (Kijjoa & Sawangwong, 2004; Jha & Zi-rong, 2004). • Vorsetuzumab Mafdotin (SGN-75) is a dolastatin analogue and is composed of the anti-CD70 antibody h1F6 conjugated to monomethylauristatin F through a non-cleavable maleimidocaproyl linkage (Alley et al., 2009). It has monomethylauristatin F linked to the humanized anti-CD70 monoclonal antibody 1F6 through a maleimidocaproyl linker that is non-cleavable, so its release has to rely upon invagination and then proteolytic digestion. This ADC is currently being evaluated in phase I trials against relapsed and refractory non-Hodgkin’s lymphoma, and also metastatic renal cancer where the cancers express the CD70 epitope (Newman & Cragg, 2014). • ASG-5ME is an ADC, like CDX-011. The natural molecule of its origin is Dolastatin 10, found in Dolabella auricularia (Martins et al., 2014). ASG-5ME is an antibody with specificity for SLC44A4, an ion transporter expressed in more than 90% of pancreatic ductal adenocarcinomas. It comprises a fully human antibody covalently linked, with a protease-cleavable linker, to the microtubule-disrupting agent monomethylauristatin E (MMAE). Upon binding to SLC44A4 and internalization, MMAE is released by proteolytic cleavage, binds to tubulin, and subsequently induces cell cycle arrest and apoptosis. Treatment with ASG-5ME represents a novel therapeutic approach for patients with pancreatic ductal adenocarcinoma, because it uses selective targeting of tumour cells by an ADC (Dimou et al., 2013).

From marine origin to therapeutics use: the success stories of Cytarabine, Ziconotide and Eribulin Mesilate As was mentioned previously, exploration of the marine environment in the past 50 years (algae, sponges, ascidians, tunicates and bryozoans) has been incredible, resulting in the isolation of thousands of structurally unique bioactive marine natural products (Blunt et al., 2003–2014; Mayer et al., 2010, 2013). This exploration became possible due to modern snorkelling, the introduction of SCUBA (1970s), the use of manned submersibles (1980s) and, more recently, due to the appearance of remotely operated vehicles (ROVs) (1990s). Although many isolated molecules do not always have pharmaceutical applications, however, as was referred before, seven marine or marine derived molecules had been approved by FDA. Some examples with major pharmacology success include Ziconotide (Prialt®, Elan Corporation), a first peptide discovered in a tropical cone snail; Eribulin Mesylate (E7389) (Halavenn®, Eisai Corporation), a macrolide isolated from the marine sponge Halichondria okadaiwas, used for the treatment of patients with metastatic breast cancer, and Cytarabine (Ara-C) (Cytosar-U ®, Upjohn Company), a nucleoside from sponge Cryptotethia crypta, for the treatment of cancers of white blood cells such as acute myeloid leukaemia, approved in FDA since 1969.

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Cytosine arabinoside (1-𝛃-D-arabinofuranosylcytosine, Cytarabine, Ara-C) In 1945, the instruments and resources for exploration of the marine environment and organisms were practically nonexistent, compared with the present day. However, Werner Bergmann (1945) collected sponges in the shallow waters off Elliot Key, Florida and, on close examination, found that they belonged to a previously unidentified species (Sneader, 2005; Bergmann & Feeney, 1951). A few years later, the same species of sponge was identified and named Cryptotethia crypta by Dr MW de Laubenfels and Werner Bergmann. Cryptotethia crypta revolutionized marine exploration, beginning a new era in drug discovery. In the first years of the 1950s, Werner Bergman and co-workers isolated and established the structure of two new compounds, spongothymidine and spongouridin (Bergmann & Feeney, 1951; Bergmann & Burke, 1955; Sneader, 2005). In 1959, Richard Walwick and co-workers synthesized one analogue, cytosine arabinoside (Ara-C) (Walwick et al., 1959). In 1961, the Upjohn group described the potential of Ara-C as antileukaemic substance (Cohen, 1977). The mechanism of action for Ara-C is clearly linked to DNA synthesis inhibition. Inside of a cell, Ara-C is converted to a cytotoxic triphosphate derivative (Ara-CTP) and enters newly formed DNA by internucleotide linkage, interfering with the activity of DNA polymerases, leading to chain termination (Godefridus, 2007; Dias et al., 2012). The Ara-C at present it is the most effective agent in the treatment of acute myeloid leukaemia, as well as being active in other haematological malignancies, including acute lymphoblastic leukaemia and non-Hodgkin’s lymphoma (Godefridus, 2007).

Ziconotide The cone snails are a large genus of gastropod molluscs, all of which are believed to be actively venomous predators. The venom of this organism is synthesized in a long tubular duct, from which it is squeezed by a muscular bulb and injected into the prey through a hollow radular tooth (Olivera et al., 1985). These disposable teeth are like barbed needles making it difficult for a prey to dislodge. Furthermore, the venom of some species is toxic for humans, causing serious injury and, in extreme situations, it may lead to death. This behaviour sparked the interest of researchers and Endean and co-workers in 1974 studied the crude venoms from several Conus species and found a rather complex pharmacological picture that suggested the presence of more than one active component (Endean et al., 1974). Different studies demonstrated that the venoms of several Conus species are constituted by different toxins that act in the nervous systems of organisms. One of the most interesting molecules is the ω-conotoxin peptide, which is one of the most potent marine toxins found in venomous marine snails (Olivera et al., 1985). Ziconotide (Prialt®) is a synthetic form of the ω-conotoxin peptide which acts by blocking the nerve impulses in a key region of the spinal cord, where pain fibres from the body connect with the nerve cells that send pain to the brain. This is why Prialt, which is 50 times more potent than morphine, is so exquisitely precise and does not cause the adverse effects of opiates. It stops pain messages from getting through, while allowing the rest of the nervous system to function normally (Kijjoa & Sawangwong,

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2004). Elan Corporation developed into an artificially manufactured drug and, since 2004, FDA has approved its utilization for intrathecal treatment of severe chronic pain (Schmidtko et al., 2010).

Eribulin mesylate In 1986, Hirata and Uemura identified Halichondrin B from the marine sponge Halichondria okada, which was a common, widely distributed sponge from the Pacific coast of Japan (Hirata & Uemura, 1986). Halichondrin B is a large polyether macrolide and has high anti-tumour capacity. This molecule inhibits the polymerization of purified tubulin and inhibits microtubule assembly dependent on microtubule-associated proteins (Hirata & Uemura, 1986). These promising results led the authors to believe that Halichondrin B could be a novel anticancer therapeutic, and in 1992 it was accepted for preclinical development by the United States National Cancer Institute. However, the low yield of natural molecule extraction slowed the progress of these tests. In 1998, Yoshito Kishi and co-workers (Melvin et al., 2005) managed to develop a complete synthetic method for Halichondrin B production, and the drug again received a new attention focus (Towle et al., 2001). In the same study, they also found that its activity resides in the macrocyclic lactone C1-C38 moiety, so the way was now open for development of a simplified synthetic analogue (Towle et al., 2001). One of the synthetic analogues from Halichondrin B is E7389 (eribulin mesylate), produced in 1998. Like its parent natural compound, eribulin mesylate interferes with microtubule dynamics by inhibiting the growth of microtubules and by sequestering tubulin into non-productive aggregates. This leads to G2/M cell cycle arrest, disruption of mitotic spindles and apoptotic cell death after prolonged mitotic blockage (Huyck et al., 2011). Eribulin mesylate is used to treat patients with metastatic breast cancer. However, this drug is only used in patients who have received at least two prior chemotherapy regimens for late-stage disease; in other words, it is used only in the third line of treatment (Wozniak et al., 2013). Eribulin mesylate (Halaven®) is produced by Eisai Company, and has been approved by the FDA since 2010.

Marine phycotoxin as a tool for signal transduction pathways analysis: the success story of okadaic acid As mentioned before, natural toxins derived from animals, plants, fungi, snails and marine organisms can be valuable tools for biochemical and pharmacological research, playing a significant role directly or indirectly in drugs development. They can serve as lead compounds in new drugs development, thus transferring toxicity into potential benefit (Camargo, 2005; Kapoor, 2010). In the specific case of marine toxins, they are some of the most popular research tools and have already contributed much to our understanding of biological processes and disease mechanisms. These toxins are potent bioactive compounds with a large diversity of chemical structures, biochemical targets, and biological effects (Fusetani, 2009). One of the most interesting groups of marine toxins, from structural and pharmacological points of view, are polyether toxins, which generally present a great diversity in size and potent biological activities. An example of these toxins is okadaic acid (OA) (Fernandez et al., 2002).

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OA, first described in 1981, was isolated from the sponges Holichondria okadai and Holichondria melanodocia (Tachibana et al., 1981). However, it was later discovered that marine dinoflagellates from the genus Prorocentrum (mainly P. lima, but also P. concavum) and Dynophysis (mainly D. acuta, D. acuminate, and D. fortii) were its genuine producers (Cruz et al., 2013; Valdiglesias et al., 2013). OA is one of the major substances among diarrheic shellfish poisoning toxins (DSP). Like other phycotoxins, OA is accumulated by shellfish – mainly bivalve molluscs such as mussels, scallops, oysters, or clams – and several fishes, through phytoplankton intake, which consequently causes important consequences in human health and the fishery industry (Cruz et al., 2013; Valdiglesias et al., 2013; Vieira et al., 2013). The importance of OA as a biological research tool started in the late 1980s, when a study of the cellular regulatory mechanisms underpinning muscle contraction produced by OA showed that it was mediated by inhibition of the phosphatase that dephosphorylates myosin light chains (Takai et al., 1987; Bialojan & Takai, 1988). As a complement to this research, the seminal work developed by Bialojan & Takai (1988) allowed them to compare the effects of OA in different protein phosphatases, helping to distinguish between them. This result was considered a milestone in cell signalling pathways research. In fact, it has been demonstrated that OA is a highly selective inhibitor of protein phosphatases type 1 (PP1) and 2A (PP2A), subsequently causing dramatic increases in phosphorylation of numerous proteins, as well as being a potent tumour promoter (Fernandez et al., 2002; Malagoli et al., 2008). More recently, OA has been suggested as tool to generate biological models for the study of Alzheimer’s disease (AD), since all altered functions induced by OA are contributory factors for development of AD-like pathology. It is evident from the shared information that AD-like disease induced by OA builds up a similar condition to the clinical neuropathology of AD patients. These evidences indicated that OA may be a novel tool to study Alzheimer’s disease, and helpful for the development of new therapeutic approach (Kamat et al., 2013). Taken together, it is not surprising that the OA has been an invaluable pharmacological tool in the study of cellular signalling pathways. The great affinity of this compound for its targets, together with its high specificity to inhibit certain protein phosphatases, enables the differential study of these proteins (Cruz et al., 2013). It is an extremely useful tool for studying the cellular processes regulated by reversible phosphorylation of proteins, such as signal transduction, cell division and memory (Cohen et al., 1990; Fernandez et al., 2002). Thus, the marine compound OA is perhaps the most well-known member of a diverse array of secondary metabolites that have emerged as valuable probes for studying the roles of various cellular protein Ser/Thr phosphatases, and it has become one of the world’s most widely used marine natural product in biological research (Medina et al., 2013). The marine compounds show not only high potential as new drugs, but also as tools for the biological research.

Conclusions Hard research work has been done in the last 50 years, resulting in the increase of outstanding knowledge related to marine organisms as producers of high value-added biomolecules. This chapter tries to review some of the main topics relating to marine drug origin, with a particular emphasis on the unique marine chemical ecology environmental as critical conditions for marine drug production. Additionally, some of the

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most relevant marine drug success stories associated with their therapeutic use, or use as a research pharmacology tools, has also been described, including the marine toxin drugs (e.g. tetradotoxin, okadaic acid, Cytarabine, Eribulin mesylate and Ziconotide). The huge variety of natural marine products, isolated from algae, marine animals and marine microbes, are provably the main sources for bioactive molecules. Within the last 50 years, more than 16000 compounds have been isolated, seven marine or marine-derived molecules have been approved by the FDA for therapeutics proposes, and eleven molecules are on clinical trials evaluation. The origins of marine drug have been found on algae, animals and bacteria marine organisms. However, the predicted biosynthetic origin of the FDA-approved compounds, as well the marine drugs on phase I, II and III of clinical trials, are mainly associated to marine bacteria synthesis. This would agree with the idea that the next generation of marine drug discovery must be essentially marine bacteria-oriented. Moreover, increased efforts must be made to identify the intracellular pathways associated to the marine drug synthesis, particularly relating to the enzymes involved in the synthesis of high value-added biomolecules. On the other hand, the existence of genomic and proteomic new tools will certainly enhance the efficacy of marine bacteria-oriented screenings as marine drug producers, as well as for the identification of molecular pathways linked to marine drug synthesis. Furthermore, the cellular synthetizes pathway identification must be looked at as an opportunity for a generation of marine drugs generated by genetically modified microorganisms to solve some of the main bottlenecks connected to the marine drug’s industrial production. The marine drugs’ origin is a huge opportunity for researchers, companies and overall society.

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C H A P T E R 14

Pharmacology of cylindrospermopsin Juan A. Rubiolo, Diego Alberto Fernández, Henar López & M. Carmen Louzao Department Pharmacology, Campus de Lugo, Spain

Introduction Cylindrospermopsin was discovered while trying to elucidate the cause of a massive intoxication occurred during 1976 in Palm Island, Queensland, Australia. During this episode, 138 inhabitants were hospitalized, suffering various symptoms of gastroenteritis. The intoxication was initially attributed to poor water quality and diet but, with inconclusive results, the illness remained as the ‘Palm Island Mystery Disease’ (Byth, 1980). It was later shown that the origin of the intoxication was a compound isolated from the cyanobacteria Cylindrospermopsis raciborskii that was named cylindrospermopsin (Ohtani et al., 1992). While this organism is still considered the main producer of cylindrospermopsin, the toxin has also been reported in Anabaena bergii, Anabaena lapponica, Anabaena planctonica, Aphanizomenon ovalisporum, Aphanizomenon flos-aquae, Aphanizomenon gracile, Raphidiopsis curvata, Raphidiopsis mediterranea, Umezakia natans and Lyngbya wollei (Blahova et al., 2009; Pearson et al., 2010). It is important to note that not all the species listed above produce the same quantity of cylindrospermopsin, that some strains are associated with other toxins, and that another strains do not produce cylindrospermopsin at all. Toxin production relies on the expression of genes involved in toxin synthesis that can be induced or repressed by different environmental stimuli. Environmental factors include, but are not limited to, light intensity and time of exposure; competition for resources; water conditions (movement, temperature and salinity); herbivory and grazing; nutrient concentration; cell division; and growth rate (Merel et al., 2013). From C. raciborskii and Aphanizomenon ovalisporum, two analogues of cylindrospermopsin have been isolated. These are 7-deoxy-cylindrospermopsin and 7-epicyclindrospermopsin, missing an oxygen and with an opposite orientation of the hydroxyl group at position C7 respectively.

Chemical and physical properties Cylindrospermopsin structure elucidation was performed with a combination of mass spectrometry and nuclear magnetic resonance. The molecule is a tricyclic guanidine moiety combined with a hydroxymethyluracil (Figure 14.1). Its molecular formula is C15 H21 N5 O7 S, with a molecular weight of 415.43 Da. The structure originally proposed (Ohtani et al., 1992), with the hydroxyl group in the opposite orientation than that shown in Figure 14.1, was later corrected, showing that the Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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Figure 14.1 Chemical structure of cylindrospermopsin. R and S indicate the stereocentres within the structure. Carbons 12 and 7 are indicated for identification purposes. Marked with a red circle is the residue modified in the analogues 7-deoxy-cylindrospermopsin and 7-epi-cyclindrospermopsin.

originally proposed structure corresponded to 7-epi-cyclindrospermopsin. The other natural analogue (deoxycylindrospermopsin) lacks this hydroxyl group (Figure 14.1). 7-epi-cylindrospermopsin structure was determined after isolation from a culture of Aphanizomenon ovalisporum from Lake Kinneret in Israel (Banker et al., 2000). The chemical synthesis of cylindrospermopsin was achieved in the year 2000. The molecule was obtained in 20 steps from 4-methoxy-3-methylpyridine, with a 3.5% yield (Xie et al., 2000). Being a zwitterion (i.e. a molecule with localized positive and negative electrical charges), cylindrospermopsin is highly soluble in water (Chiswell et al., 1999; Shaw et al., 2000), and it is also soluble in DMSO and methanol. It is stable to sunlight, at high temperatures and through a wide range of pH values (Chiswell et al., 1999).

Producing genera/species Cyanobacteria are prokaryote organisms with excellent adaptation capabilities to climate changes and varying environments. They can be found in almost every terrestrial and aquatic habitat: oceans, fresh water, damp soil, moistened rocks in deserts, bare rock and soil, and even in Antarctic rocks. These organisms are one of the earliest life forms known to date. Evidences have dated the appearance of cyanobacteria about 3.5 billion year ago (Schopf, 2000; Olson, 2006). Cyanobacteria can grow as single cells, forming colonies or in filaments. Some of these filamentous species contain heterocysts specialized in fixing nitrogen. There are certain species of cells whose colonies are packed into a mucilaginous sheath. Usually, the filamentous species grow as dense mats or strands floating freely in the water. Many other cyanobacteria have gas vacuoles that allow them to regulate their position in the water column and obtain advantage over other organisms. The aquatic cyanobacteria species can multiply rapidly, creating highly visible green-coloured blooms in marine and fresh water environments. Cyanobacterial blooms that produce cyanotoxins are given the name cyanobacterial harmful algal blooms (cyanoHABs). Conditions like high concentrations of nutrients, increased levels of nitrogen and phosphorus, warmer temperatures (>25 ∘ C), carbon availability and slow-flowing or eutrophication of water, may induce the blooms. Some other parameters of the water body, such as turbidity, pH, salinity, conductivity and received sunlight, can also play a role. This phenomenon occurs mainly in late summer and early autumn, causing mass reproduction of certain cyanobacteria species for several

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days, weeks or even months (van Apeldoorn et al., 2007; Poniedzialek et al., 2012; Merel et al., 2013). Most cyanobacteria are capable of producing metabolites that vary structurally and biochemically, with most of these variations having unknown function. Some of these molecules include terpenoids, metal chelators, lactones, protease inhibitors, alkaloids, taste and odour compounds and, most important from the public health point of view, potent toxins known as cyanotoxins. These compounds are hepatotoxic, neurotoxic and acutely dermatotoxic. The effects of poisoning include acute diarrhoea, skin irritation, liver and nervous system damage in humans and domestic animals, and even death in wild animals (Paerl et al., 2011). The most commonly identified cyanotoxins are microcystins, cylindrospermopsin, anatoxins and saxitoxin. A lot of research has been done on microcystins, as they are the most predominant toxins in freshwater bodies. Nowadays, cylindrospermopsin is also becoming an important toxin worldwide, due to the health impact it has when found in reservoirs destined for drinking water or recreational purposes (Kinnear, 2010). Generally, cyanotoxins can be enclosed between the cell walls of the producing organism, can accumulate in the cytoplasm, or can be released to the extracellular medium by excretion, or cell lysis. In the case of cylindrospermopsin, when the cell population is in the phase of exponential active growth, about 80–90% of cylindrospermopsin remains in the intracellular space whereas, in the post-exponential (stationary) growth phase, the toxin is naturally released from the cyanobacterial cells (50% of the total amount). Moreover, when cell growth slows dramatically and the population begins to enter senescence, a larger proportion of intracellular toxin is released into the water (Griffiths & Saker, 2003). It is important to note that, even if the producing cells are in the exponential growth phase, in the event of cell wall breakage, the intracellular cylindrospermopsin content can be spilled into the water. This matter is of vital importance in health risk assessment plans, as many monitoring and water-treatment programs used for sanitizing drinking water use chemical compounds or physical treatments that induce cell lysis (Hitzfeld et al., 2000; Ho et al., 2012).

Cylindrospermopsin biosynthesis Based on the conserved nature of cyanobacterial DNA sequences coding for polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS; Neilan et al., 1999), putative cylindrospermopsin biosynthesis genes (aoa/cyr) were first identified in C. raciborskii (Schembri et al., 2001). Later, an amidotransferase gene, probably involved in the formation of the guanidinoacetic acid precursor of cylindrospermopsin, was identified (Shalev-Alon et al., 2002). In 2008, the complete gene cluster (aoa/cyr) was indentified in C. raciborskii. This was followed by the identification of the gene cluster of Aphanizomenon sp. 10E6 (Stüken & Jakobsen, 2010), Oscillatoria sp. PCC6506 (Mazmouz et al., 2010), and R. curvata CHAB1150 (Jiang et al., 2012). The C. raciborskii cyr locus spans 42 kb, encoding 15 open reading frames (Figure 14.2a). The predicted pathway showed that a number of genes encoding unusual catalytic domains and enzymes were present in cylindrospermopsin biosynthesis. This pathway was proposed by (Mihali et al. 2008; Figure 14.2b) and later confirmed by the biochemical characterization of some of its enzymes

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(a)

(b)

(a) C. Raciborskii cylindrospermopsin gene cluster. Light grey: PKS; white: NRPS; dark grey: precursor biosynthesis genes, tailoring genes putatively involved in uracil ring formation; black: genes putatively involved in regulation and transport and gene transposition. (b) Representation of the biosynthetic pathway of cylindrospermopsin. In grey appear the enzymes that have been biochemically characterized. The other proposed reactions are based on bioinformatic predictions. Abbreviations: PKS domains, KS: ketoacyl synthase, AT: acyltransferase, DH: dehydratase, KR: ketoreductase, ACP: acyl carrier protein, CM: C-methyltransferase; NRPS domains, A: adenylation domain, PCP: peptidyl carrier protein. Figure 14.2

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(Muenchhoff et al., 2010; Mazmouz et al., 2011). This scheme does not account for the formation of the two cylindrospermopsin analogues, 7-deoxycylindrospermopsin and 7-epicylindrospermopsin, whose relative abundance varies in different cyanobacterial species (Mazmouz et al., 2011). It has been suggested that CyrI (a 2-oxoglutarate-dependent iron oxygenase), as a consequence of relaxed stereospecificity, performs the last step of cylindrospermopsin biosynthesis from 7-deoxy-CYN (Mazmouz et al., 2011). 7-deoxy-cylindrospermopsin is a minor product in C. raciborskii and Raphidiopsis curvata (Li et al., 2001), but a major one in Lyngbya wollei (Seifert et al., 2007), implying that the levels of CyrI or its activity in various cylindrospermopsin-producing cyanobacteria should be examined to account for the variability in the abundance of 7-deoxy-cylindrospermopsin compared to cylindrospermopsin. Besides the genes coding for cylindrospermopsin synthesis, the cluster also encodes genes for toxin transport and regulation. CyrK, similar to sodium ion-driven multidrug and toxic compound extrusion protein, is thought to be the transporter for cylindrospermopsin. CryO shows homology to WD repeat proteins (proteins which share two common features: the domain folds into a beta propeller; and the domains form a platform without catalytic activity, on which multiple protein complexes assemble reversibly) that have diverse regulatory and signal transduction roles. It also show homology to the AAA family proteins (ATPases Associated with diverse cellular Activities that share a common conserved module of approximately 230 amino acids residues), which perform chaperone-like functions (Mihali et al., 2008). The screening of cylindrospermopsin producers and nonproducing bacteria for the presence of genes encoding the sulfotransferase CyrJ, the NRPS/PKS hybrid CyrB and the PKS CyrC has shown that the genes encoded in the cyr locus are exclusively present in toxin producers (Mihali et al., 2008; Schembri et al., 2001). This explains why the presence of a potential cylindrospermopsin producing organism does not imply the presence of the toxin, regardless the environmental conditions. Two hypotheses try to explain the known distribution and organization of the cyr gene clusters, which vary depending of the species. The first one considers that there is an ancient origin for the cyr genes which were the common ancestors of all actual cylindrospermopsin-producing genera (Kellmann et al., 2006; Yilmaz & Phlips, 2011; Jiang et al., 2012). This idea implies that the present-day distribution of cylindrospermopsin-producing and -non-producing strains would, therefore, be a consequence of loss or inactivation of the cyr gene cluster in nonproducing species. The second hypothesis involves horizontal gene transfer in the sporadic distribution of the cyr gene clusters. This hypothesis was recently amended after a report of cylindrospermopsin genes from Oscillatoria sp. PCC6506 in which divergence with other cylindrospermopsin gene clusters does not support the horizontal gene transfer (Mazmouz et al., 2010). It is now suggested that recent horizontal gene transfer may explain the sequence similarity between the cyr gene clusters of some of the cylindrospermopsin producers, and that they do not have a single evolutionary pathway (Jiang et al., 2012; Yilmaz & Phlips, 2011). Maximal cylindrospermopsin synthesis in C. raciborskii occurs under light intensity higher than required for optimal growth (Dyble et al., 2006), suggesting that the redox state of the cells is involved. The culture status is also critical to the rate of toxin formation, since cylindrospermopsin accumulation is linear with the growth rate in both diazotrophic and nitrate supplied log phase cultures, but decoupling between the two parameters is observed at stationary phase (Hawkins et al., 2001). In various

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C. raciborskii strains, differential effects of temperature on growth and toxin production have been observed (Saker & Griffiths, 2000). All these data show that little is known about the cellular mechanisms involved in cylindrospermopsin synthesis and accumulation, and their interaction with abiotic conditions. As previously mentioned, since cylindrospermopsin synthesis depends on the presence of toxin synthesis-related genes in a particular strain, not all of the cyanobacteria species that have been reported to produce cylindrospermopsin are toxin producers. This is not exactly true for A. ovalisporum. With one exception (Ballot et al., 2011), all the A. ovalisporum isolated so far are cylindrospermopsin producers. The toxin was detected under all culture conditions assayed, including different nutrient settings (Bacsi et al., 2006), temperature and light intensity (Cirés et al., 2011).

Distribution and bioaccumulation C. raciborskii and other cylindrospermopsin-producing bacteria occupy a tropical habitat. Even so, it has been shown to be tolerant to a wide range of climates and has been observed to reach high biovolume and phytoplankton dominance at temperatures as low as 11.2 ∘ C (Bonilla et al., 2012). In recent decades, cylindrospermopsin producers have spread to more temperate latitudes. The increasing contamination in the aquatic ecosystems and earth climate change, which creates habitats with more favourable conditions for these organisms (Bonilla et al., 2012), reinforces the hypothesis pointing to a relation between climate change and the appearance of these microorganisms in more northern regions (Gugger et al., 2005; Stüken et al., 2006). Cylindrospermopsis blooms have increased in the tropics (Fabbro & Duivenvoorden, 1996). The spreading of this organism to subtropical and temperate latitudes has been shown by several authors (Fastner et al., 2003; Tóith & Padisák, 1986). Currently, Cylindrospermopsis distribution reaches as far as northern Europe (Wiedner et al., 2002), New Zealand (Ryan et al., 2003) and North America (Hill, 1970). In South America, the organism is well known in Brazil (Huszar et al., 2000) and has been detected in southern latitudes (Torgan & García, 1989; Vidal & Kruk, 2008). The increase in the detection of cylindrospermopsin-producing organisms have been attributed to several reasons. The improvement in water quality monitoring is one, since new locations are been inspected for the presence of these organisms. Also, the combination of climate change, increased eutrophication (Chonudomkul et al., 2004; Briand et al., 2004; Wiedner et al., 2007), and the species’ own adaptability could be behind the observed expansion. Regarding the species adaptability, it has been shown that C. raciborsckii can travel long river courses, survive swampy or slightly saline conditions, and produce resistant akinetes, which contributes to the global expansion of this species (Padisák, 1997). The first study on cylindrospermopsin bioaccumulation determined that the toxin could be detected in both, muscle and hepatopancreatic tissues of crayfish (Cherax quadricarinatus), after specimens were collected from an aquaculture pond containing 589 μg/L cylindrospermopsin. The study showed that bioaccumulation could occur within fourteen days of toxin exposure. In rainbow fish (Melanotaenia eachamensis), the toxin was also recovered from the visceral tissue (Saker & Eaglesham, 1999). Since then, cylindrospermopsin bioaccumulation has been shown for a range of invertebrate and vertebrate organisms including: Melanoides tuberculata and Tegogolos snails; Anodonta, Alathyria and Corbiculina mussels; Cherax crayfish, Melanota enia rainbow fish

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and Bufo marinus tadpoles (Berry & Lind, 2010; White et al., 2006; White et al., 2007; Saker et al., 2004). In the swan mussel Anodonta cygnea exposed to cylindrospermopsin, the toxin was detected in haemolymph, viscera, mantle, foot and gonad. The relative distribution of cylindrospermopsin in the tissues changed over the trial period, although bioaccumulation generally occurred within two days’ exposure. After a fourteen-day depuration period, almost 50% of the toxin remained in the tissues, showing a bi-phasic depuration marked by small increases in tissue toxin concentrations pointing to mobilization of tissue-bound cylindrospermopsin (Saker et al., 2004). In mice, after cylindrospermopsin intraperitoneal injection, accumulation was observed in kidneys and liver within six hours of dosing. Accumulation progressively decreased over five to seven days. Within 12 hours, 73% of toxin was excreted in urine and/or faeces (Norris et al., 2001). In plants, accumulation of cylindrospermopsin, from the uptake of aqueous toxin only, was studied in two types of duckweed (Lemna and Spirodela) and in Hydrilla. Only Lemna actually concentrated the toxin (Seifert, 2007; Kinnear et al., 2008; White et al., 2005). Cylindrospermopsin accumulation is highly variable in different aquatic animals. Apparently, a pattern exists, showing that lower-level organisms accumulate greater concentrations of the toxin than other, more biologically complex, animals. Current evidence points to a general order of bioaccumulation capacity, being gastropods > bivalves > crustaceans > amphibians > fish. The reverse relationship appears to be true for the susceptibility of organisms to cylindrospermopsin toxicity (Kinnear et al., 2009). Grazer species appear to be the most tolerant, suggesting that the toxic effects imparted during cylindrospermopsin exposure may be related to the ability of organisms to accumulate the toxin (Smith et al., 2008). The reasons for this could be that fish and other aquatic vertebrates, with highly advanced toxin-metabolism systems, are at greater risk of secondary cylindrospermopsin toxicity. Meanwhile, lower organisms, such as aquatic snails, can accumulate high levels of the toxin without harm (Shaw et al., 2000; Runnegar et al., 1994; Kinnear et al., 2007).

Human and animal intoxications Cylindrospermopsin has been linked to several toxic episodes involving animals (cattle, fish, etc.) (Carmichael, 1994; Saker et al., 1999; Thomas et al., 1998). Cyanobacterial toxins accumulate in aquatic organisms such as shellfish, prawns and fish, which has resulted in restrictions on the collection of these organisms (Meriluoto & Spoof, 2008; Van Buynder et al., 2001). In many cases, the toxicity is sub-lethal to these aquatic species, allowing the animals to survive long enough to transfer the toxins along the food chain (Meriluoto and Spoof, 2008). This accumulation can lead to cyanobacterial toxins concentrations at which human consumption of contaminated shellfish, fish, and so on should be discouraged (Kuiper-Goodman et al., 1999). Human poisoning with cylindrospermopsin has been previously recorded, but the lack of toxin exposure information makes impossible to determine a toxic dose. On the other hand, several publications provide information on cylindrospermopsin oral and intraperitoneal toxicity in laboratory animals (see Section 14.7, Cylindrospermopsin toxicity). As mentioned in the introduction for this chapter, the first documented case of human intoxication by cylindrospermopsin was the so-called ‘Palm Island Mystery

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Disease’ (Byth, 1980), which occurred in 1979 in Palm Island, located in tropical northern Queensland, Australia. In November of that year, 138 children between the ages of 2 to 16, and ten young adults from the Aboriginal families of the zone presented to the local clinic with symptoms that included malaise, unusual hepatoenteritis, acute tender liver enlargement, constipation, vomiting and headache. The disease was then followed by bloody diarrhoea that persisted up to three weeks, and loss of protein, electrolytes, glucose and ketones through the urine, with different grades of dehydration. About 50 of the cases were treated with supportive care at the clinic, while the more severe cases (more than 80) were transferred to Townsville General Hospital to receive intensive care with intravenous therapy. All of the patients recovered, and no deaths were reported. Posterior epidemiological investigations (Bourke et al., 1983) determined that all of the patients drank water from the town local reticulated network (piped-network) that came from the Solomon Dam reservoir. A few days before the incident, this reservoir experienced a dense algal bloom, and people complained about the bad taste and noticeable odour of the water. Local authorities decided to treat the algal bloom with 1 ppm of copper sulphate, an algaecide that probably lysed the cyanobacterial cells and released the intracellular toxins into the water. Five days after the treatment, the first case of hepatoenteritis occurred. There were also reports of a group of about 50 people who drank water from a shallow well outside the reticulated water system and showed no symptoms. The investigation discarded the possibility of a communicable disease, and pointed to the direction of toxins produced by the cyanobacteria of the algal bloom as the causative agent of the observed outbreak. A study conducted between 1981 and 1984 revealed that the Solomon Dam reservoir experienced harmful cyanobacterial blooms periodically, and that the predominant species were Anabaena circinalis and Cylindrospermopsis raciborskii, this last one not previously found in Australian waters (Hawkins et al., 1985). Toxicity studies of the lyophilizates obtained from this two species concluded that only the ones from Cylindrospermopsis raciborskii were toxic and produced hepatotoxicity in mice. Comparison of the symptoms of people from Palm Island with animal studies indicated that these cyanobacteria could be one of the possible causes of the disease. The second reported case of human intoxication produced by cylindrospermopsin occurred in a dialysis centre in Caruaru (Brazil) in 1996 (Carmichael et al., 2001). The lack of reverse osmosis filtration in the water supply resulted in the presence of microcystins and cylindrospermopsin, which led to 52 deaths. After a routine haemodialysis treatment, 116 of 131 people experienced visual disturbances, nausea, and vomiting, 100 of them developed acute liver failure soon after, and 76 died. The 52 deaths attributed to the toxic effects of both toxins, led to it being dubbed the ‘Caruaru syndrome’. The examination of phytoplankton from the water sources and analysis of cyanotoxins in the clinic’s water treatment system, plus serum and liver tissue samples from the patients, led to the identification of the two groups of toxins. No other cases of human intoxication have been reported so far. However, since many cases may have not been identified, due to lack of awareness and knowledge of this toxin and detection procedures, the number of human poisonings involving cylindrospermopsin could be greater.

Symptomatology As stated above, the main symptoms of cylindrospermopsin intoxication are varied, and the initial stages include malaise, anorexia, vomiting, headache, abdominal pain

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and fever. This could progress to later stages with bloody diarrhoea, dehydration, painful hepatomegaly and acute kidney disease that results in electrolyte loss, ketonuria, glycosuria and hematuria. Liver failure, with elevated serum liver enzymes, was also reported.

Exposure routes Humans may be exposed to cylindrospermopsin through several routes, which include oral, dermal, inhalation and even accidental parenteral route (Merel et al., 2013; Poniedzialek et al., 2012; Moreira et al., 2012). By far, the most frequent exposure route for humans is the oral one (Zegura et al., 2011), which occurs mainly through the ingestion of contaminated drinking water or the accidental swallowing of water during bathing, showering or recreational activities and sports practised in contaminated waters during harmful algal blooms. Moreover, in these activities, dermal contact or inhalation of the toxin are also likely to occur. The ingestion of contaminated food is another important source of intoxication, as cylindrospermopsin can bio-accumulate in animals, such as fish or crabs, that have been exposed to it. Even eating vegetables that were irrigated with contaminated water can cause problems on human health (Kinnear, 2010). Another potentially source of cyanotoxins intoxication are dietary supplements sold as blue-green algae supplements (BGAS), which are very popular in Western countries due to their presumed benefits to human health (Dietrich & Hoeger, 2005; Saker et al., 2005). These are mostly produced from the cyanobacteria Spirulina spp. and Aphanizomenon flos-aquae, which are both capable of producing cyanotoxins when harvested from natural lakes. Even though these food products have been found to be cylindrospermopsin-free so far, contamination with this toxin cannot be entirely ruled out, and requires thorough research.

Cylindrospermopsin toxicity Cylindrospermopsin has been demonstrated to be toxic to many species of bacteria, protozoa, plants, invertebrates and vertebrates including humans. There is also evidence that it can bioaccumulate at various levels of the food chain, affecting implicated organisms (Kinnear, 2010). In vivo laboratory studies have been performed primarily in mice using lyophilized samples of cyanobacteria producing cylindrospermopsin, cell extracts, and also purified cylindrospermopsin. These mouse bioassays showed that, while the toxin affects the kidneys, lungs, spleen, heart and intestine, the main target is the liver, regardless of the intoxication route. For this reason, it is considered a hepatotoxin (Bernard et al., 2003; Shaw et al., 2000). The first symptoms upon oral administration were anorexia, diarrhoea and dyspnoea. Nescroscopic inspection evidenced hemorrhagic tissue in liver as the principal lesion. Damage was also detected in lungs, kidneys, small intestine and adrenal glands. Microscopically, hepatocyte necrosis, lipidosis, fibrine thrombus in liver and lungs, and necrotic foci in nephronaa were observed (Hawkins et al., 1985; Saker et al., 1999). In agreement with the abovementioned studies, some authors put cylindrospermopsin in the cytotoxin category, given the variety of organs affected besides the liver (Bazin et al., 2012; Falconer et al., 1999; Hawkins et al., 1985; Terao et al., 1994). Besides the diversity of organs affected by cylindrospermopsin, the toxicity of this molecule is variable, depending on the animal model used, and there even appears to

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be considerable variability between individuals of the same species (Kinnear, 2010). This is evidenced by the variable LD50 s obtained (Falconer & Humpage, 2006; Seawright et al., 1999). The lowest lethal dose after oral administration reported so far is 4.4 mg/kg. On the other hand, the highest non-lethal dose reported is 6.9 mg/kg (Seawright et al., 1999). It should be noted that these studies were performed with extracts, implying that an effect of any of the extract components cannot be ruled out. By intraperitoneal injection, using purified cylindrospermopsin, the LD50 in mice was reported to be 2 mg/kg after 24 hours and 0.2 mg/kg if administered for 4–6 days (Ohtani et al., 1992). These data indicate that repeated low doses for several days are more toxic than a single dose, even when this single dose is higher than the addition of all the doses administered for six days. In humans, apart from gastroenteritis due to the injury of the gut epithelium, the ingestion of cylindrospermopsin can lead to hepatotoxicity (Poniedzialek et al., 2012; Duy et al., 2000; van Apeldoorn et al., 2007; Bernard et al., 2003; Reisner et al., 2004; Humpage & Falconer, 2003; Oliveira et al., 2012). Hepatocytes experience a reduction of nuclei size and detachment of ribosomes from the membranes with accumulation in the cytoplasm; proliferation of agranular membranes because of lipid peroxidation induced by the decrease of cytochrome P450 levels; and accumulation of fat droplets in the central portion of hepatic lobules. Finally, a severe necrosis of the liver occurs. While neurotoxicity studies are scarce, a recent one (Zagatto et al., 2012) revealed that the toxin caused trembling, ataxia, convulsion and death by respiratory arrest in a couple of minutes, all typical neurotoxic symptoms. Another study (Schoeb et al., 2002) found that alligators exposed to the toxin showed depressed clinical responses, reduced velocity on nerve conduction, axonal degeneration and even necrosis in the midbrain. Several studies support that cylindrospermopsin is toxic to foetuses of mice after intraperitoneal injection, showing an elevated number of early births, reduced litter size, elevated number of foetal deaths and also reduced weight and an increase of the incidence of anomalies such as blood in the gastrointestinal tract and hematomas in the tips of the tails in survivors (Rogers et al., 2007). This all could be related to the toxin effect on the progesterone/oestrogen relation (Young et al., 2008). The effect of cylindrospermopsin on pregnancy in humans is still a matter for research. Dermatotoxic effects of cylindrospermopsin have also been reported. For example, Balb/c mice that were treated by topical application of cylindrospermopsin in the abdomen presented a dried skin in that area, yellow and brown crusts, desquamation, and blood and serous fluid suppurating from the exposed skin (Stewart et al., 2006a). However, the presence of secondary metabolites and lipopolysaccharides in the cell walls of cylindrospermopsin producers could also cause dermal effects on humans under direct contact such as in bathing or showering, so not all the effects should be attributed to cylindrospermopsin itself (Stewart et al., 2006b; Rzymski & Poniedziałek, 2012). Even though further research is still needed to determine the inmunotoxicity of cylindrospermopsin, some signs of this type of effect have been reported. For example, in ICR mice that were intraperitoneally injected with cylindrospermopsin (Terao et al., 1994), a massive lymphocytes necrosis in the cortical layer of the thymus was detected soon afterwards. In other studies with MF1 (Seawright et al., 1999) and Quackenbush mice (Shaw et al., 2000) that were treated orally and intraperitoneally with the toxin,

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a degeneration and necrosis of cortical lymphocytes, as well as lymphophagocytosis in the lymphoid tissue of the spleen, was observed. So far, only one study reported tumour initiation in mice after exposure to cylindrospermopsin (Falconer & Humpage, 2001). In that study, 53 mice were treated up to three times orally with a Cylindrospermopsis raciborskii extract containing cylindrospermopsin. After 30 weeks, tumours were found in five of the 53 cylindrospermopsin-treated mice, while none were found in the 27 controls. The genotoxic effects of cylindrospermopsin have been demonstrated mainly with cell lines studies, also animal models have been used. For example, mice injected with a single intraperitoneal dose of cylindrospermopsin showed that the toxin (or its metabolites) binds to nucleic acids (Shaw et al., 2000). In addition, in Balb/c mice, DNA strand breakage was observed in hepatocytes after 24 hours of a single dose of cylindrospermopsin (Shen et al., 2002). A recent study (Chernoff et al., 2011) reported altered expression profiles of genes involved in the ribosomal biogenesis, xenobiotic and lipid metabolism, inflammatory response and oxidative stress on CD-1 mice (Swiss-Webster). In vitro studies show that cylindrospermopsin is more cytotoxic for primary hepatocytes when compared with results observed in other cell lines (Shaw et al., 2000). For example, after 72 hours, a LD50 of 96.15 nM (40 ng/ml) was reported for primary hepatocytes exposed to cylindrospermopsin, while it was 200 ng/ml for the cervical adhenocarcinoma cell line KB after the same period (Chong et al., 2002). In vitro, as with many other toxic molecules, cylindrospermopsin induces necrosis or apoptosis depending on the dose employed and the exposure time. Using 5 μM cylindrospermopsin, lactate dehydrogenase release to the media experiments showed that the toxin induced primary hepatocytes necrosis (Froscio et al., 2003; Humpage et al., 2005). At this concentration, apoptosis cannot be ruled out, since no experiments were performed to determine its presence. On the other hand, lower cylindrospermopsin concentrations (90–300 nM) has been shown to induce apoptosis in primary rat hepatocytes. While clear apoptosis was observed after 24 and 48 hours of toxin exposure, longer incubation times showed lactate dehydrogenase release to the medium, pointing to a late necrosis (Lopez-Alonso et al., 2013; Majno & Joris, 1995). This observation is related to the appearance of secondary necrosis during the late phases of apoptosis, which has been termed ‘apoptotic necrosis’, as has been observed on other in vitro cellular models (Majno & Joris, 1995). In vitro, non-toxic doses of cylindrospermopsin are genotoxic if exposition is prolonged. The molecule induced DNA strand breakage and loss of chromosomes (aneuploidy) affecting the kinetochore/spindle function in a lymphoblastoid cell line (Humpage et al., 2000), In SHE cells (in vitro model to evaluate the carcinogenic potential of a chemical, physical of biological agent; Maire et al., 2010), it induced and increased the morphological cell transformation. In hepatic cells (primary and tumour derived), cylindrospermopsin induced DNA strand breaks, also observed in a lymphoblastoid cell line (Humpage et al., 2005; Straser et al., 2011). Genotoxicity has also been observed in vivo after toxin administration by gavage to mice (Bazin et al., 2012). Contrary to that observed between protein synthesis and CYP450 derived metabolites, genotoxicity appears to be linked to these, implying that cylindrospermopsin phase I metabolism is responsible for the genotoxicity of this molecule (see next section) (Humpage et al., 2005).

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The neurotoxic activity of cylindrospermopsin is quite controversial, due to the few studies using human cell lines and the lack of neurotoxic effects (Kiss et al., 2002).

Mechanism of action Acute toxicity of cylindrospermopsin is believed to be related to the toxin metabolites generated by the cytochrome P450 (CYP450) which, during xenobiotic metabolism, modifies cylindrospermopsin to generate a more toxic molecule, as observed in experiments where cylindrospermopsin toxicity was evaluated in the presence of CYP450 inhibitors. Inhibition of CYP450 with alpha-naphthoflavone was enough to partially cytoprotect hepatocytes from cylindrospermopsin toxicity. Cylindrospermopsin genotoxicity was also blocked by CYP450 inhibitors. In vivo, the CYP450 inhibitor piperonyl butoxide inhibited death induced by the toxin seven days after dosing (Humpage et al., 2005; Norris et al., 2002; Runnegar et al., 1995). This molecule exerts protein synthesis inhibition which is considered the long-term toxic effect (Runnegar et al., 2002; Terao et al., 1994; Humpage et al., 2005; Runnegar et al., 1995). Protein synthesis inhibition is irreversible, and a concentration of 360 nM has been shown to completely inhibit protein synthesis after 24 hours in primary hepatocytes, while lower concentrations only partially inhibited protein synthesis (Froscio et al., 2003; Lopez-Alonso et al., 2013). As observed in the presence of a CYP450 inhibitor, there is a dissociation between the involvement of CYP450 generated metabolites in the toxicity process and the effect on protein synthesis. In the presence of the inhibitor, cylindrospermopsin is still capable of protein synthesis inhibition (Froscio et al., 2003). Cylindrospermopsin also inhibits glutathione synthesis, so the toxic effect of reactive oxygen species was considered as a potential component of the toxicity. Initially, using necrotizing concentrations of the toxin, oxidative stress appeared not to be involved in the toxic effect as observed after lipid peroxidation determination and glutathione reductase inhibition. Lipid peroxidation was not elevated in the presence of the toxin, and no difference in cell death was observed between cells treated with cylindrospermopsin and cells treated with the toxin plus a glutathione reductase inhibitor (Humpage et al., 2005). It was later shown that apoptotic concentrations of cylindrospermopsin also induced oxidative stress and, contrary to what was observed in the previous case, it added to cylindrospermopsin induced cell death. The loss of cell viability observed after toxin treatment was lowered in the presence of a reactive oxygen species scavenger (Lopez-Alonso et al., 2013). This raises the possibility that, at high concentrations (necrotic), the toxicity is of such a magnitude that the effect of reactive oxygen species becomes negligible, since cell death occurs rapidly, while it is responsible for part of the toxicity of this molecule at lower concentrations (apoptotic).

Detection methods A wide range of methods exists to detect cylindrospermopsin and its analogues. However, each method requires specific steps of sample preparation before the analysis. The mouse bioassay was one of the first methods used to determine toxicity, as it is relatively fast to implement. Even this, it can only differentiate the class of a toxin (hepatotoxin, neurotoxin etc), not identify the toxin itself; furthermore, the

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sensitivity is low. Immunoassays using specific antibodies have a high sensitivity and are affordable, but they cannot differentiate between toxin analogues. Chromatographic methods also have high sensitivity and can identify and differentiate toxins and analogues; however, results are not related to toxicity of the sample, and require extra steps of sample separation and expensive equipment. In broad terms, the methods to detect and analyze cylindrospermopsin and related compounds can be classified in two groups: biological and physico-chemical methods.

Biological approach methods Morphological methods These methods allow the identification of the cyanobacteria species that can potentially produce cylindrospermopsin by using a variety of microscopic techniques (light microscopy, transmission and electron microscopy, confocal and laser scan microscopy, etc.). The identification is mainly based on the morphological characteristics of such species. While these techniques are accessible and relatively inexpensive, they are time-consuming and require extensive training and an excellent knowledge of the taxonomic classification of cyanobacteria by their differential attributes. In addition, the morphological characteristics can be also influenced by the environmental conditions and the phase of their life cycle at the moment of sample analysis (Moore et al., 2004; Gugger & Hoffmann, 2004). This can be a negative handicap for the researchers. Furthermore, these methods cannot provide evidence of the presence of the toxin since there are strains of cyanobacteria that in some situations are unable to produce cylindrospermopsin. Thus, while microscopic methods can help to identify the producing organisms, they can not determine the actual presence of the toxin in samples.

In vivo assays One of the earliest and most extended methods to determine the presence of cylindrospermopsin is the mouse bioassay. This method comprises the intra peritoneal (ip) injection of bloom materials (extracts of lysed cells) into a minimum of three mice, followed by clinical observation and necropsy of the animals after 24 hours of exposure (Hawkins et al., 1985; Falconer, 1993). Observing the different symptoms that the animals have, (hepatotoxic, neurotoxic etc.), or the damage to certain organs or cell types (increased liver size and damage to hepatic cells in the case of cylindrospermopsin), we can determine the class of the toxin but not exactly which one it is. Despite its low sensitivity, the method is considered a semi-quantitative method, because the extent of the lesions in animals can be compared to others exposed to pure cylindrospermopsin standard. However, the ethical implications, and the development of faster and more sensitive and specific methods, restrict the mouse bioassay mostly to toxicological studies. Apart from the mouse bioassay, alternative animals and organisms have been used to detect cylindrospermopsin. For example, bioassays with the larvae of certain crustacean species, such as Daphnia magna (Nogueira et al., 2004), Artemia salina (Metcalf et al., 2002) or Thamnocephalus platyurus, can detect cyanotoxins. There are even commercial kits like ThamnoToxkit or RapidToxkit, which utilize those Thamnocephalus larvae that have a good correlation with mouse bioassay (Törökné, 1999; Törökné et al., 2007). The background of this kind of assays is simple and consists of incubating the larvae in a growth medium, adding the unknown toxin samples and comparing the larvae mortality with the effects that a standard of cylindrospermopsin exerts in

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the same larvae. These methods are easy and fast to implement, but are unspecific to cylindrospermopsin, and the results can vary due to the matrix effect of the tested samples. Other animals such as the insect Locusta migratoria can also be used instead of the mouse bioassay, mainly because it is more sensitive and cheaper (Hiripi et al., 1998). Even certain plants, like mustard, Sinapis alba, are susceptible to the inhibitory effect of cylindrospermopsin on growth and metabolism, so a test using etiolated seedlings has been developed (Vasas et al., 2002). Guinea pigs have also been used to study the potential allergenic effects of cylindrospermopsin to humans due to the similar sensitivity (Törökné et al., 2001). Helix pomatia and Lymnaea stagnalis, two species of snails, were employed to test cylindrospermopsin toxicity, and it was found that the effects were mainly neurotoxic, due to the inhibition of acetylcholine responses in neurons, in contrast with the hepatotoxic effects that the toxin has on vertebrates (Kiss et al., 2002).

Cell line based assays Different cell lines assays can be useful for detecting cylindrospermopsin, and as an alternative for replacing the traditional mouse bioassays. The use of cell lines, in contrast with primary cultures such as primary hepatocytes, eliminates the variations that could occur in the isolation procedures to obtain those cells. Basically, this kind of assay consists of incubating the cells with known amounts of cylindrospermopsin (or standards), to obtain a cytotoxicity curve that can be used to extrapolate values of samples with unknown toxin concentration. The first cell line used to assay the genotoxicity of cylindrospermopsin was the ovary-derived CHO K1 (Fessard & Bernard, 2003). Since then, other cell lines have been used in cylindrospermopsin cytotoxicity assays. Examples include: HepG2, C3A and Clone-9 (liver), Vero (kidney), MNA (brain), BE-2 (bone marrow), HDF (dermis), NCI-N87 (stomach), HCT-8 (ileum), HuTu-80 (duodenum) and Caco-2 (colon) (Froscio et al., 2009b; Bain et al., 2007; Neumann et al., 2007; Lankoff et al., 2007; Gutierrez-Praena et al., 2012). Moreover, it has been demonstrated that hepatic cell lines are more sensitive to the effects of cylindrospermopsin, while intestinal cell lines such as Caco-2 appears to be more resistant. Vero cells showed variable results (Froscio et al., 2009a). Immunological assays Specific antibodies for detecting cylindrospermopsin have been developed and are commercially available as components of ELISA (enzyme-linked immunosorbent assay) kits in 96-plate format from various suppliers. ELISA has the disadvantage of being non-selective for cylindrospermopsin derivatives such as 7-epicylindrospermopsin or deoxy-cylindrospermopsin. Moreover, it is also susceptible of matrix interferences (Blahova et al., 2009; Graham et al., 2010; Yilmaz et al., 2008; Berry & Lind, 2010). Immunoassays are promising future approaches to the monitoring of cylindrospermopsin, based on their low detection limit range (0.05 to 2 μg L–1 ) and that they are relatively easy and fast to use. Molecular assays Molecular assays are based mainly on the detection of genes involved in the production of cylindrospermopsin. So far, there are least 11 genes involved in this biosynthesis that have already been described (Schembri et al., 2001; Shalev-Alon et al., 2002; Kellmann

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et al., 2006; Mihali et al., 2008; Glowacka et al., 2011; Mazmouz et al., 2011). The principal strategy of these methods is the simultaneous amplification of several of these genes by using different PCR techniques, for example multiplex PCR, real-time PCR (Fergusson & Saint, 2003; Rasmussen et al., 2008; Moreira et al., 2011). Those assays may be applied to determine whether certain populations of cyanobacteria have the ability to produce the toxin. These methods are useful for monitoring potential cylindrospermopsin-producing cyanobacteria, but are not indicative of active production and, therefore, cannot detect the toxin itself.

Biochemical assays Using the potent inhibition of protein synthesis properties of cylindrospermopsin, a cell-free assay was developed using a commercially available rabbit reticulocytes lysate system (Froscio et al., 2001, 2008). This method is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts, but can detect the toxin within the 200–1200 μg L−1 range, having less sensitivity than immunoassays. However, it obtained good correlations when compared to LC/MS-MS or HPLC-PDA chemical methods.

Physico-chemical approach methods Chemical methods to detect and quantify cylindrospermopsin in cyanobacterial cultured species and in environmental samples of fresh and brackish water are based on the chemistry of cylindrospermopsin. Techniques to monitor this cyanotoxin could require previous steps of extraction, separation and isolation of the sample components, and a later analysis and identification of the toxins. The most common separation techniques for cylindrospermopsin analysis are liquid chromatography (LC), gas chromatography (GC) and capillary electrophoresis (CE). Liquid chromatography is often employed using a reversed phase C18 or HILIC column with a mobile phase of methanol/water or water/acetonitrile. This separation technique is widely used for separating cyanotoxins due to its flexibility and adaptability, as it can be easily coupled with UV absorbance, fluorescence or mass spectrometry detection methods. Gas chromatography can also be employed to separate cyanotoxins, but it can be difficult to use with large and not very volatile molecules. This technique requires extra steps and a more complex process of sample preparation making its use not as extensive as liquid chromatography. Finally, capillary electrophoresis separates compounds according to their mass and charge (Vasas et al., 2004). When coupled with mass spectrometry or fluorescence, it can provide sufficient sensitivity. However, a recent study has revealed that this method is not yet robust enough to be used in routine analysis of cylindrospermopsin (Kaushik & Balasubramanian, 2012). The detection techniques most commonly used for the analysis of cylindrospermopsin can be classified into ultraviolet absorbance (UV), fluorescence or mass spectrometry techniques.

UV Absorbance methods Several studies have revealed that cylindrospermopsin has a specific UV spectra maximum at 262 nm. This can be used to detect this toxin after a step of separation by liquid chromatography (LC) or even high performance liquid chromatography (HPLC)

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(Ohtani et al., 1992; Harada et al., 1994; Hawkins et al., 1997) for better results. These methods are quantitative and can detect all kind of toxins and distinguish analogues, but they cannot provide their exact identity. Moreover, the sensitivity is low (when compared to mass spectrometry), and the specificity can be reduced when using samples with complex matrices, such as bloom waters (Merel et al., 2010, 2013).

Fluorescence methods Detection by fluorescence is often used after LC separation as an alternative method to UV absorbance ones. This technique generally improve the sensitivity, but it needs derivatization steps during the sample preparation to incorporate fluorescent elements, since cylindrospermopsin and its derivatives do not have natural fluorescence properties (Harada et al., 1997; Kaushik & Balasubramanian, 2012). Mass spectrometry methods Mass spectrometry (MS) detection techniques are available for both LC and gas chromatography (GC) separation processes. In these methods, the detection of the compounds is based on their mass and charge reducing the interferences and improving the selectivity. These MS techniques to detect cylindrospermopsin are often more used coupled with LC than coupled with GC, because the latter requires extra steps, such as sample oxidation and post-treatment to remove some of the reagents used in the process. Therefore, LC-MS and the sensitive and specific LC coupled to tandem mass spectrometry (LC-MS/MS) (Eaglesham et al., 1999; Stirling & Quilliam, 2001; Bogialli et al., 2006) are good techniques to determine small amounts of cylindrospermopsin and other toxins, in water samples. In addition, there are techniques that allow detecting cylindrospermopsin with no need of previous chromatographic separation by using time-of-flight (TOF) spectrometers such as MALDI-TOF ones (Kaushik & Balasubramanian, 2012). All of these methods based on mass spectrometry offer high sensitivity. Like UV and fluorescence methods, they can also detect other toxins (apart from cylindrospermopsin) but, in addition, they are able to distinguish between its derivatives, providing their identity. However, the equipment and materials required to perform the analysis are usually very. Also the vality of this approach as a toxicity method has been questioned, since all the analytical results can not be converted in equivalent toxic values due to the lack of toxin standards.

Cylindrospermopsin elimination In the presence of algal extract, cylindrospermopsin decomposes rapidly when exposed to sunlight, with a half-life of 1.5 hours. On the other hand, sunlight produces no decomposition when the toxin is dissolved in pure water. This difference is thought to be due to the presence of pigments in the algal extracts, such as chlorophyll α, β-carotene, xanthophylls and phycobiliproteins, that promote the rapid decomposition of cylindrospermopsin. Temperatures ranging from 4–50 ∘ C produce a slow decomposition of cylindrospermopsin at pH 7. A degradation of 57% of the toxin was observed after ten weeks at 50 ∘ C. Boiling the toxin for 15 minutes produced no significant degradation (Chiswell et al., 1999).

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The increasing occurrence of cyanobacterial blooms in drinking water reservoirs has led to the development of more effective water treatment technologies for the elimination of the toxins produced by these organisms. Dissolved cyanotoxins can be partially eliminated by activated carbon treatment during water processing, but a residual often remains. Due to this, several alternatives have been assayed for the elimination of cylindrospermopsin. Oxidants and disinfectants typically used in waterworks can eliminate the toxin. Chlorination has been used to remove the toxin from water at pH values of 6–9 (Senogles et al., 2000). Oxidation of this molecule by chlorine produced 5-chloro-cylindrospermopsin, or a truncated carboxylic acid derivative of cylindrospermopsin, which are both non-toxic to mice. Chloramine and chlorine dioxide were found to be less effective (Banker et al., 2001). Another method proven to be effective for cylindrospermopsin elimination is photocatalytic degradation using titanium dioxide and UV irradiation (Senogles et al., 2001). Ozone, which reacts with the deprotonated amine moieties of cylindrospermopsin, is also effective for its destruction (Onstad et al., 2007).

Legislation Cyanotoxins are a major concern for the public health, due to the use of water for drinking, recreational or agricultural purposes. For this reason, many countries have adopted regulatory measures following safety guideline values for these toxins. While the majority of the countries have a regulation for microcystins (MCs), only a few have established safety levels for cylindrospermopsin. Analyzing water for cyanotoxins is expensive and cannot be done on a regular basis. Due to this, several risk managements schemes based on the number of cyanobacteria cells present in the water have been adopted by some countries. The World Health Organization (WHO) proposed two alert levels – when the count reaches 20 000 cells/mL and when it reaches 100 000 cells/mL (Chorus & Bartram, 1999). These are the points when the analysis of the cyanotoxins begins and when safety measures (drinking or bathing prohibited respectively) are applied. A study conducted by Humpage & Falconer (2003) proposed the reference guideline value of 1 μg∕L of cylindrospermopsin in drinking water when they estimated the no observed adverse effect level (NOAEL). This value was adopted taking into account the average body of a human adult (60 kg), the average daily water consumption (2 L) and the tolerable daily intake of 0.02 μg∕kg body weight per day, calculated based on acute toxicity studies in mice. This resulted in 0.9 μg∕L of toxin intake. At the time of writing this chapter, only four countries have specific regulations for cylindrospermopsin. Australia and New Zealand have adopted the 1 μg∕L limit in drinking water. On the other hand, Brazil is much more permissive and has established a limit of 15 μg∕L, while Germany has the most restrictive regulation for cylindrospermopsin, with 0.1 μg∕L of cylindrospermopsin in drinking water (Chorus, 2012). Unfortunately, for recreational or bathing waters, there are no guidelines adopted yet by any country. The establishment of regulations for cylindrospermopsin is a matter of vital importance, and all national governments should develop programs for monitoring both drinking and recreational water.

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C H A P T E R 15

Pharmacology of the cyclic imines Natalia Vilariño, Sara F. Ferreiro, Andrés Crespo & José Gil Department of Pharmacology, University of Santiago de Compostela, Spain

Introduction Cyclic imines are a class of marine toxins characterized by the presence in their chemical structure of a cyclic imine moiety. This group comprises several cyclic imine subclasses: spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs) and pteriatoxins (PtTXs), prorocentrolides, spiro-prorocentrimine and symbioimines, discovered during the last 20 years. The SPXs were described for the first time in 1991, due to the appearance of an unusual toxicity during routine monitoring of bivalve molluscs for the presence of lipophilic (formerly diarrheic) marine toxins along the Nova Scotia shore, Canada (Hu et al., 1995). Since their discovery 14 different structures within this group have been reported. These toxins have worldwide distribution (Hu et al., 1995; Aasen et al., 2005; MacKinnon et al., 2006; Villar Gonzalez et al., 2006; Amzil et al., 2007; Álvarez et al., 2010) and they are produced by marine dinoflagellates of the genus Alexandrium, mainly Alexandrium ostenfeldii and Alexandrium peruvianum (Cembella et al., 1999, 2000; Touzet et al., 2008). GYMs are also phytoplanctonic toxins produced by the marine dinoflagellate Karenia selliformis (Haywood et al., 2004). They were discovered as a consequence of unusual neurological toxicity when performing lipophilic toxin detection by the mouse bioassay in New Zealand (Mackenzie, 2004). GYMs have been detected at different coastal locations all over the world (Stirling, 2001; Takahashi et al., 2007; Krock et al., 2009; Marrouchi et al., 2010). Currently there are four known GYMs. PnTXs were named after the bivalve Pinna attenuata, from which the first PnTX was isolated in 1990, following an episode of human poisoning (Zheng et al., 1990). However, currently there is no evidence of a causal relationship between the presence of PnTXs and human intoxication. PtTXs are usually classified in the same group as PnTXs, due to their similar chemical structure. The dinoflagellate Vulcanodinium rugosum is the only PnTX producer identified so far (Rhodes et al., 2011). Similarly to the cyclic imines mentioned above, PnTXs have appeared in coastal areas of several continents (Zheng et al., 1990; Rhodes et al., 2011; Rundberget et al., 2011; McCarron et al., 2012). Prorocentrolides, spiro-prorocentrimine and symbioimines are the less well-known members of this class. Prorocentrolide was isolated in 1988 from Prorocentrum lima cultures originally collected at Sesoko Island, Okinawa, Japan (Torigoe et al., 1988). Later, the other member of this group, prorocentrolide B, was isolated from Caribbean Prorocentrum maculosum (Hu et al., 1996b). Spiro-prorocentrimine was purified and Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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characterized in 2001 from Prorocentrum cultures originally obtained from the coral reef in Taiwan (Lu et al., 2001). Symbioimine and neosymbioimine were discovered in a symbiotic marine dinoflagellate belonging to the genus Symbioiminium isolated from an Okinawan marine flatworm (Kita et al., 2004, 2005). Marine organisms are a huge source of bioactive compounds in the search for new pharmacological tools with a possible therapeutic application. Although still far from clinical applications, cyclic imines from marine origin constitute a promising toxin class with interesting biological activities. SPXs, GYMs and PnTXs have been proposed as primary molecules for the development of new nicotinic acetylcholine receptor (nAChR) antagonists, with possible uses as neuromuscular blockers or anti-Alzheimer’s disease drugs (Bourne et al., 2010; Alonso et al., 2011a, 2011b; Aráoz et al., 2011). Symbioimines, on the other hand, have been suggested for the treatment and prevention of osteoporosis and for the generation of new anti-inflammatory drugs (Kita et al., 2004, 2005).

Overview of cyclic imine chemical structure SPXs A, B, C, D, 13-desmethyl SPX C (13-desMe SPX C), 13,19-didesmethyl SPX C (13,19-didesMe SPX C), 27-hydroxy-13-desmethyl SPX C, 27-hydroxy-13,19didesmethyl SPX C, 27-oxo-13,19-didesmethyl SPX C and 13-desmethyl SPX D are macrocyclic compounds consisting of a 5:5:6-bis-spiroketal moiety or spiro-linked tricyclic ether ring system and a seven-membered spiro-linked cyclic imine included in the 23-membered macrocycle (Figure 15.1) (Hu et al., 2001; MacKinnon et al., 2006; Ciminiello et al., 2007, 2010). SPX G and 20-methyl SPX G (20-Me SPX G) also have a spiro-linked cyclic imine but contain a 5:6:6-bis-spiroketal moiety (Figure 15.1) (Aasen et al., 2005; MacKinnon et al., 2006). SPXs H and I have been classified as a SPX subclass, due to the presence of only two cycles (5:6) in the spiroketal ring system (Figure 15.1) (Roach et al., 2009). SPXs E and F have been also included in this group, although these compounds contain a keto amine moiety in their structure instead of a cyclic imine, due to reduction or opening of the C28-N bond (Hu et al., 1996a). SPXs E and F can be obtained by acid hydrolysis of SPXs A and B respectively (Figure 15.1) (Hu et al., 1996a). GYMs have a six-membered spiro-linked cyclic imine (Figure 15.2) (Seki et al., 1995; Stewart et al., 1997; Miles et al., 2000, 2003). Instead of a bis-spiroketal moiety, GYMs contain a tetrahydrofuran moiety as part of their 16-membered macrocycle. Recently, 12-methyl GYM has been characterized as a member of the GYM group produced by Alexandrium peruvianum, which is a known SPX-producing organism, suggesting an interesting biogenetic link among SPXs and GYMs (Van Wagoner et al., 2011). PnTXs and PtTXs are also macrocyclic compounds with a seven-membered spiro-linked cyclic imine moiety (Figure 15.2) (Uemura et al., 1995; Chou et al., 1996a, 1996b; Takada et al., 2001a; Selwood et al., 2010). These toxins share a common backbone structure containing a 6:5:6 spiro-linked tricyclic ether ring system and a 5:6-bicyclo ring moiety embedded in the 27-membered macrocycle. Prorocentrolides and spiro-prorocentrimine contain a cyclic imine ring, and they are also characterized by the presence of two macrocycles in their chemical structure (Figures 15.1 and 15.2) (Torigoe et al., 1988; Hu et al., 1996b; Lu et al., 2001). Spiro-prorocentrimine has a six-membered spiro-linked cyclic imine as part of its bigger macrocycle (Figure 15.2). However, the cyclic imine of the prorocentrolides is not a spiro-linked moiety (Figure 15.1).

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Figure 15.1 Structure of SPXs and prorocentrolides. Δ indicates the presence of a double bond, between C2 and C3 and between C27 and oxygen at the R4 position.

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Figure 15.2 Structure of GYMs, PnTXs and PtTXs spiroprorocentrimines and symbioimines. Δ indicates the presence of a double bond.

Symbioimines contain in their structure a 6:6:6 tricyclic iminum ring structure, unique in this class of marine toxins (Kita et al., 2004, 2005). Other important differences versus the cyclic imines mentioned above are the presence of an aryl sulphate moiety and the absence of a macrocycle (Figure 15.2).

In vivo effects of cyclic imines All the groups classified as cyclic imines display ‘fast acting’ toxicity when tested by the mouse bioassay for lipophilic toxins, except for symbioimine, for which in vivo acute toxicity data are not available. This ‘fast acting’ toxicity is characterized

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by the appearance of neurological symptoms and death within 20 minutes after intraperitoneal (i.p.) administration. After 20 minutes, if mice survive, they recover completely. Most of the data concerning in vivo effects of these marine toxins have been obtained by i.p. administration of the toxin to mice. The signs of i.p. toxicity are similar for GYMs, SPXs and PnTXs. These symptoms include hyperactivity, jumping, hyperextension of the back, arching of the tail towards the head, paralysis of the hind limbs, convulsions, dyspnoea and death (Richard et al., 2001; Biré et al., 2002; Gill et al., 2003; Munday et al., 2004). The i.p. LD50 values published until now vary greatly among different studies, probably due to variations in the source of the toxin, purity or mouse strain, among others (Table 15.1). The i.p. LD50 and/or LD99 for several analogues of these groups are remarkably low (Table 15.1). GYM toxicity by subcutaneous and intracerbrovetricular Table 15.1 Lethal doses of cyclic imines in mice. MLD: minimum lethal dose. I.p.: intraperitoneal. Values in italics correspond to LD99 or voluntary intake. Toxin

I.p. LD50 , LD99 (𝛍g∕kg)

Oral LD50 gavage, voluntary intake (𝛍g∕kg)

References

GYM A

80–96

GYM B SPX A

800 37

Munday et al., 2004; Kharrat et al., 2008 Kharrat et al., 2008 Munday et al., 2012a

SPX B Dihydro SPX B SPX C

99 >1000 (MLD) 8

13-desMe SPX C

6.9 27.9 32.2 250 (LD100 ) >1000 (MLD) >1000 (MLD) 8

750 >7500 – 550 1300 440 440 180 780 130 1000 – – – – 88 630 630 –

13,19-didesMe SPX C SPX D SPX E SPX F 20-Me SPX G

PnTX B:C 1:1 PnTX D PnTX E PnTX F

>2000 180 135 22 400 57 12.7

PnTX G

48

PtTX A PtTX B:C 1:1

100 8

SPX H PnTX A

– – 2800 25 50 150 400 – –

Hu et al., 1995 Hu et al., 1995 Munday et al., 2012a Munday et al., 2012a; Otero et al., 2012 Otero et al., 2012 Hu et al., 1995 Hu et al., 1996a Hu et al., 1996a Munday et al., 2012a Roach et al., 2009 Uemura et al., 1995; McCauley et al., 1998 Takada et al., 2001a Chou et al., 1996a Munday et al., 2012b Munday et al., 2012b Munday et al. 2012b Takada et al., 2001b Takada et al., 2001b

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injections in mice displayed similar symptoms, although the intracerebroventricular LD50 was much lower – about 30 times compared with i.p. LD50 in the same study (Kharrat et al., 2008). The SPXs were also shown to elicit similar signs of toxicity in rats (Gill et al., 2003). Interestingly, in vivo toxicity of GYM and SPXs was reduced by previous administration of neostigmine or physostigmine (Richard et al., 2001; Munday et al., 2004), two acetylcholinesterase inhibitors, supporting the critical role of acetylcholine receptors in cyclic imine toxicity. At necropsy, cyclic imine-treated rodents (rats and mice) did not show macroscopic lesions in skeletal muscle, peripheral nerves, brain, heart, liver, kidney, spleen, adrenal glands, lungs, GI tract and retina (Gill et al., 2003). Histological damage was also not detected in these organs, except for the brain of treated mice, where neuronal damage was described in the hippocampus and brain stem (Gill et al., 2003). In rats, in spite of the absence of histological alterations, an increase of early neuronal injury markers, such as c-jun and HSP-72, appeared in the brain tissue of treated animals, which died two minutes after toxin administration (Gill et al., 2003). In the same time frame, gene expression of muscarinic acetylcholine receptors (mAChR) 1, 4 and 5 and nAChRα2 and nAChRβ4 were upregulated (Gill et al., 2003). These results may reflect the relevance of AChRs for the toxicity of the SPXs, considering the incredibly fast kinetics for up-regulation of the expression of these genes. Additional in vivo evidence of cyclic imines targeting acetylcholine receptors is the inhibition of the tail muscle contraction in anesthetized mice, elicited by stimulation of the caudal motor nerve, following local administration of GYM A and 13-desMeSPX C (Marrouchi et al., 2013). The results indicated that both compounds inhibit neuromuscular transmission without altering motor nerve excitability. As in systemic toxicological studies, 13-desMeSPX C displayed a much higher potency than GYM A, in this case being 300-fold higher. Cyclic imines have not been unequivocally related to human poisoning. No effects have been reported in humans due to SPXs, GYMs, procentrolides, spiroprorocentrimine or symbioimines (Munday, 2008). Regarding PnTXs, references can be found in the literature to human poisoning outbreaks in Japan and China. However, these references are misleading, because the toxic episodes in Japan were later related to Vibrio contamination (Munday et al., 2012b), and the PnTX-contaminated samples collected in China were chronologically not coincident with the human intoxication events (McNabb et al., 2012).

Pharmacodynamics: in vitro evidence of spirolides, pinnatoxins and gymnodimines targeting nicotinic acetylcholine receptors Initial investigations into the pharmacology of cyclic imines suggested that at least the SPXs and GYMs targeted cholinergic receptors (Richard et al., 2001; Gill et al., 2003). Some symptoms caused by these toxins were clearly neurological, suggesting a nervous system-related target, and were similar to the signs elicited by antagonists of cholinergic receptors. Additionally, the higher toxicity of intracerebrovetricular administration pointed to a central nervous system location for the toxin target (Kharrat et al., 2008). In the last decade, several studies have contributed evidence that demonstrates that cyclic imines bind with high affinity to nAChRs and block their activity.

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Table 15.2 Binding affinities of cyclic imines for different nAChR subtypes by radioligand binding assays. Ki (apparent affinity constant), nM. Receptor

GYM A

13-desMe SPX C

PnTX A

References

Torpedo marmorata α12 βγδ

0.23

0.08

2.8

Human α12 βγδ α7 α7-5HT3 α6β3β4α5 α3β2

440 1 – 1 0.24

31 0.7 – 2 0.021

– – 0.35 – 9.4

α3β4 α4β4 Human α4β2

30 36 0.62 70

47 43 0.58 96

– – 15.6 (partial)

Bourne et al., 2010 Aráoz et al., 2011 Hauser et al., 2012 Hauser et al., 2012 Aráoz et al., 2011 Hauser et al., 2012 Bourne et al., 2010 Aráoz et al., 2011 Hauser et al., 2012 Hauser et al., 2012 Bourne et al., 2010 Hauser et al., 2012 Aráoz et al., 2011

The first information regarding a high affinity interaction of cyclic imines with nAChR referred to GYM A. Competition-binding experiments performed with radiolabelled ligands demonstrated high binding affinities for the α12 βγδ muscle-type nAChR and a quimeric α7-5HT nAChR (Table 15.2; Kharrat et al., 2008). The affinities for the neuronal α4β2 and α3β2 nAChR subtypes were significantly lower. In the same study, GYM was shown to block neuromuscular transmission in vitro in mouse hemidiaphragm preparations by inhibition of nerve stimulation-elicited twitch responses. Additionally, GYM A also blocked endplate potentials in mouse and frog neuromuscular preparations and acetylcholine-elicited muscle-type nAChR currents in vitro. Moreover, GYM A inhibited the neuronal subtype homomeric human α7 nAChR currents induced by acetylcholine. Overall, this evidence indicates that GYM A targets both muscle and neuronal nAChRs. Subsequently, nAChR blocking activity has been described for other members of the cyclic imine group, including 13-desMe SPX C, PnTX A, PnTXs E and F and PnTX G (Bourne et al., 2010; Aráoz et al., 2011; Hellyer et al., 2011). Blocking efficiency varies greatly, depending on receptor subtype and cyclic imine (Table 15.3). The most striking difference is related to the human muscle nAChR subtype. This receptor is blocked by 13-desMe SPX C at concentrations tenfold lower than GYM A, while both molecules block α7 receptors with similar potency (Table 15.3). PnTX A is also an efficient antagonist of both muscle and α7 subtypes (Aráoz et al., 2011). PnTXs E and F block neuromuscular transmission in vitro in mouse hemidiafragm preparations, but do not affect hippocampal gamma oscillations, suggesting blockade of muscle nAChRs and no effect on the α7 subtype (Hellyer et al., 2011). High binding affinities have been reported for 13-desMe SPX C and PnTX A for several nAChR subtypes (Table 15.2). Interaction of 13,19-didesMe SPX C, 20-Me SPX G and PnTX G with Torpedo α12 βγδ nAChRs has also been described, although binding affinities were not characterized by radioligand binding assays. Apparent affinity constants (Ki ) were, however, determined by a colorimetric microplate-receptor

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Table 15.3

Antagonism of nAChR subtype response. IC50 , nM. GYM A

T. marmorata α12 βγδ current (ACh-evoked, EC50 ) Human α12 βγδ-elicited calcium response α7 elicited calcium response α7 ACh-evoked current Nicotine induced dopamine release in rat striatal synaptosomes Human ganglion nAChR elicited calcium response α4β4 elicited calcium response α4β2 α4β2 current Low sensitivity human α4β2 elicited calcium response High sensitivity human α4β2 elicited calcium response

2.8 357 2

13-desMe SPX C

PnTX A

0.51

5.53

PnTX G

References

Bourne et al., 2010 Aráoz et al., 2011 Hauser et al., 2012

11 0.4

0.3

0.2

Hauser et al., 2012 Aráoz et al., 2011 Hauser et al., 2012

1

3

Hauser et al., 2012

8 0.5 0.9

22 0.7 3.9

30.4

0.5

0.7

0.7

Hauser et al., 2012 Hauser et al., 2012 Bourne et al., 2010 Aráoz et al., 2011 Hauser et al., 2012

4

9

9

0.107

5.06

9

Hauser et al., 2012

binding assay (Aráoz et al., 2012). Ki values estimated by this method were 0.8 nM for 13,19-didesMe SPX C, 1.77 nM for 13-desMe SPX C, 5.2 nM for PnTX G, 6.6 nM for 20-Me SPX G, 19.3 nM for GYM A and 23 nM for PnTX A. Direct interaction between 13-desMe SPX C and GYM A and the nAChR has been demonstrated by X-ray crystallography, using an acetylcholine-binding protein (AChBP) from the inverterbrate Aplysia californica, a soluble AChBP commonly used as a surrogate of nAChR, due to the similarity of their binding pocket. Positioning of 13-desMe SPX C and GYM A in the binding pocket of AChBP favours the establishment of hydrogen bond interactions involving the cyclic imine group and Trp-147, and a remarkable complementarity between binding pocket and toxin conformations, with several additional interactions contributing to the high affinity binding (Bourne et al., 2010). Later modelling of 13-desMe SPX and GYM A interactions with the α7 nAChR suggests that additional hydrophobic interactions may also contribute to a high affinity, virtually irreversible interaction (Hauser et al., 2012). The predicted interactions of PnTX A with human α7 (seven hydrogen bonds), human α4β2 (five hydrogen bonds) and Torpedo α12 βγδ (five hydrogen bonds plus hydrophobic interactions) by computational modelling correlate with the experimental binding affinities obtained for these receptors. Although no covalent bond is established between the toxin and the receptor, experimental results indicate that GYM A and 13-desMe SPX C blockade of some nAChR subtypes is virtually irreversible (Bourne et al., 2010; Hauser et al., 2012). The inhibition of α7 receptor function persists even in the presence of increasing concentrations of agonist for both GYM A and 13-desMe SPX C. Additionally, 13-desMe SPX C blockade of the Torpedo muscle type and the α4β2 persists after washout, as well

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351

Contraction (% of control)

Contraction (% of control)

120 100 80 60 40 20 0

100 80 60 40 Control SPX SPX, 1 h washout

20 0

–8

–7 –6 –5 log [acetylcholine] (M) (a)

–4

–8

–5 –7 –6 log [acetylcholine] (M)

–4

(b)

Figure 15.3 Effect of 13-desMe SPX C and 13,19-didesMe SPX C on acetylcholine-induced contraction of rat ileum in an organ bath. Acetylcholine-induced contraction was abolished completely by 10 μM atropine, even at high concentrations of ACh. Epibatidine did not have any effect in this preparation. (a) 3 μM 13-desMe SPX C. (b) 1 μM 13,19-didesMe SPX C. Mean ± sem, n = 3, *p < 0.05 vs. control by t-test).

as PnTX A blockade of α7 receptors. However, GYM A antagonism of Torpedo muscle type and the α4β2 neuronal subtype is reversed shortly after toxin removal (Bourne et al., 2010). Muscarinic AChRs were initially proposed as a possible target of cyclic imines, due to toxicity symptoms and upregulation of mAChR in vivo (Richard et al., 2001; Gill et al., 2003). Later, in vitro evidence suggested functional blockade and interaction of 13-desMe SPX C with mAChR in a neuroblastoma cell culture, although, in these experiments, the potency of this toxin for mAChR-related effects seemed lower than that reported for nAChRs (Wandscheer et al., 2010). Additional experiments showed that acetylcholine-elicited contraction of rat ileum, a mAChR-dependent effect, was not efficiently inhibited by 13-desMe SPX C or 13,19-didesMe SPX C, with only a 20% reduction in the presence of 3 μM and 1 μM concentrations respectively (Figure 15.3). Although the inhibition was statistically significant, at these concentrations, potent antagonists of mAChRs would display an almost complete inhibition of ACh-induced contraction. Moreover, 13-desMe SPX C and 13,19-didesMe SPX C, at concentrations of 0.5 μM and lower, did not inhibit binding of [3H]N-methyl scopolamine to mAChRs M1, M3 and M5 expressed transiently in CHO cells (unpublished results). Similar results were reported for PnTX A that did not show significant binding to M1-5 receptors at concentrations up to 1 μM (Aráoz et al., 2011), and for GYM and 13-desMe SPX C that did not displace radioligand binding to M1-3 (Hauser et al., 2012). Overall, the SPXs, PnTXs and gymodimine characterized so far are potent antagonists of nAChRs, but are not selective among nAChR subtypes. On the other hand, cyclic imines have not been demonstrated to bind to mAChRs with high affinity. The biological targets of prorocentrolides and spiro-prorocentrimine have not yet been elucidated. The scarce information available about symbioimine indicates that it can inhibit in vitro-induced differentiation into osteoclasts of a murine monocyte cell line (EC50 = 120 μM) and cyclooxygenase-2 (COX-2) activity (32% inhibition at 10 μM) (Kita et al., 2004, 2005). However, the potency of

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symbioimine to produce both effects is low, and further studies should be performed to clarify symbioimine biological target and activity.

Structure-activity relationship of cyclic imines The discovery of SPXs E and F, which lack a cyclic imine, and their reported absence of toxicity, evidenced the relevance of this moiety for the toxicity of the SPXs (Hu et al., 1996a). The reduction of GYM to gymnodamine, in which the cyclic imine moiety is transformed to a secondary amine, is also responsible for a remarkable loss of toxicity (>4 mg∕kg displayed no signs of toxicity) (Stewart et al., 1997). Very recently, the relevance of the cyclic imine ring for the activity of PnTX A was also demonstrated by the extreme decrease in affinity for nAChRs shown by PnTX A amino ketone analogue (Aráoz et al., 2011). The cyclic imine moiety has been referred to as the pharmacophore of this toxin class in many texts. More recently, the characterization of the crystal structures of 13-desmethyl SPX C and GYM bound to an acetylcholine binding protein (AChBP), a nAChR surrogate, demonstrated the involvement of the cyclic imine group in the interaction with the acetylcholine binding site of the nAChR. The protonated nitrogen of the cyclic imine is responsible for hydrogen bonding with the carbonyl oxygen of Trp147 located at loop C of the AChBP structure, anchoring the toxin within the binding pocket (Bourne et al., 2010). Additionally, the bis-spiroacetal ring system of the SPX and the tetrahydrofuran of GYM are responsible for several additional interactions with loop C amino acids, including hydrogen bonds, that contribute to the high affinity binding of these molecules to the receptor (Bourne et al., 2010). Cis stereochemistry of the bis-spiroacetal three-ring system, which is the preferred conformation of the SPX, provides a nice complement for the apolar environment of the apical side of the ACh binding site. The butyrolactone is also implicated in the establishment of weak hydrogen bonds deep in the ACh binding pocket. Cation-π and van der Waals interactions, and the hydrophobic environment of the binding pocket, also contribute to the high-affinity, virtually irreversible, binding of these cyclic imines (Bourne et al., 2010; Hauser et al., 2012). This study demonstrated the absence of relevant interactions with the more variable loop F, which would account for the low selectivity of these toxins for nAChR subtypes, compared with other antagonists (Bourne et al., 2010). Homology modelling and docking studies have been used to explore the interaction of 13-desMe SPX C and GYM with the α7 nAChR binding site (Hauser et al., 2012). The prediction for α7 nAChR binding to these cyclic imines is similar to the interactions described for Aplysia-AChBP by X-ray crystallography. The scarcity of information related to some members of this group of toxins makes it difficult to explore structure-activity relationships. However, for most compounds, the i.p. lethal dose is one of the first parameters studied in order to determine the toxicity of the molecule, and it can be used to infer structure-activity relationships. As mentioned above, the loss of the cyclic imine moiety is clearly related to a significant loss of toxicity. The most toxic compounds of this group are 13-desMe SPX C, SPX C and 20-Me SPX G, followed by PnTx F and B/C. SPXs A and B display a significant reduction of toxicity, being five and 13 times less toxic than SPX C respectively, and this points to a relevant role of the C31 methyl group of the cyclic imine ring present in SPX Cs and G for toxin activity (Figure 15.1). The other PnTXs are clearly less toxic

Pharmacology of the cyclic imines

353

than SPX Cs and G. SPXs and PnTXs, with their closely related chemical structures displaying the highest toxicity within this group. Finally, the less toxic members of this toxin class are GYMs, GYM B being considerably less toxic than GYM A. GYMs lack the methyl moieties present at the cyclic imine ring of SPXs and PnTXs. Although the cyclic imine is critical for toxicity, the variation of small moieties along the common backbone structure of these subgroups may affect their toxic potency and their affinity for nAChRs. Actually, SPX H, which conserves the cyclic imine ring but has a 5:6 spiroketal ring system instead of the three-ring system of the other SPXs, showed a considerably reduced toxicity (see Table 15.1), indicating that the cyclic imine ring is not the only part of the molecule responsible for toxicity, which is supported by the toxin-receptor interaction data and models. No information about the structure-activity relationship for prorocentrolides, spiro-prorocentrimine and symbioimines has been produced so far.

Involvement of nAChR antagonism in in vivo and in vitro effects of cyclic imines In regards to in vivo effects, blockade of muscle nAChRs can certainly explain some of the symptoms observed after i.p. and oral administrations of these toxins to mice – mainly the skeletal muscle paralysis and the death by respiratory arrest, as it has often been described. Other symptoms, such as tremors and hyperactivity, would be probably related to effects on the central nervous system. GYM A is highly toxic by intracerebroventricular administration, inducing similar neurologic symptoms and death at concentrations 60 times lower than by i.p. injection (Kharrat et al., 2008). It seems evident that blockade of neuronal nAChRs centrally has also an important implication in the toxic signs of this class of toxins which, from their lipophilic nature, have a high probability of traversing the blood-brain barrier. Most in vitro studies performed with cyclic imines have been aimed at the identification of their biological target. As we mentioned above, the remarkably high affinity for nAChRs and their potent nAChR antagonism currently point to these receptors as the main biological targets of SPXs, PnTXs and GYMs. SPXs B and D have been shown to have no effect on NMDA, AMPA, kainate receptors, voltage-dependent sodium channels or protein phosphatases PP1 and PP2A activity (Hu et al., 1995). SPXs B and D have also been reported to weakly activate L-type calcium channels (Hu et al., 1995) and 13-desMe SPX C to decrease, also with low potency, acetylcholine-elicited mAChR-dependent calcium responses in neuroblastoma cells (Wandscheer et al., 2010). Additionally, cyclic imines do not display potent cytotoxicity in vitro against several cell models, the most potent being PnTXs D, with an IC50 of 3 μM in P38 mouse leukaemia cells (Seki et al., 1995; Alonso et al., 2011a, 2011b, 2013). So far, the absence of effect or low potency on other macromolecules, and the fair correlation of nAChR antagonism with the toxic symptoms observed for these three subclasses of cyclic imines, reinforce their specificity for nAChRs and their blockade as mechanism of action. GYM and 13-desMe SPX C induced a reduction of Alzheimer’s disease markers in an in vitro model of this pathology (Alonso et al., 2011a, 2011b). The reduction of intracellular amyloid-β and the decreased level of hyper-phosphorylated isoforms of tau, two well-recognized markers of this neurodegenerative disease, could be related to

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an increase of inactive glycogen synthase 3β (GSK-3β) and a reduction of ERK phosphorylation levels by these toxins. The levels of acetylcholine in this neuronal model obtained from triple transgenic mice for Alzheimer’s disease are elevated after SPX or GYM treatment. These toxins also reduced glutamate-induced neurotoxicity by a glutamate-receptor independent mechanism, as well as 13,19-didesMe SPX C (Alonso et al., 2011a, 2011b, 2013). Similar effects have been reported for other nAChR antagonists (Laudenbach et al., 2002; Mousavi & Hellstrom-Lindahl, 2009; Livingstone et al., 2010), suggesting that blockade of nAChRs present in this cell model might explain these observations. The potency of the SPX to produce the reduction of Alzheimer’s disease markers would help to evaluate the involvement of nAChRs. However, these data were obtained using western blot, which is not a quantitative technique, and they did not allow the accurate calculation of an EC50 . Interestingly, 20-Me SPX G did not display a significant neuroprotective activity, which might be the consequence of a different preference for nAChR subtypes. Later, in vivo studies with an Alzheimer’s disease mouse model showed a decrease of amyloid-β levels (again evaluated by western blot) in the hippocampus, but not in the cortex, of mice treated intraperitoneally with 13-desMe SPX C (Alonso et al., 2013). In the same model, this toxin induced an increase of cortex/hippocampus N-acetylaspartate in vivo, using proton magnetic resonance spectroscopy (Alonso et al., 2013). N-acetylaspartate is typically reduced in Alzheimer’s disease. These experiments were performed six hours after two doses of SPX were administered in a one week interval, and it is the first report of a long-term effect of SPXs, which have been considered ‘fast acting’ toxins with a rapid full recovery. It is interesting that 13-desMe SPX C did not cause any signs of toxicity in these mice, since the average dose used (11.9 μg∕kg) is fairly close to the reported LD50 of this toxin (6.9–27.9, Table 15.1), which might reflect a different sensitivity to SPX toxicity of this mouse model. The role of nAChRs in Alzheimer’s disease treatment and prevention and other neurodegenerative diseases is not yet perfectly known. Understanding the effect of these pan-nicotinic antagonists is complicated by different roles of nAChR subtypes in these diseases, and by the dual action of nAChR agonists, which can both activate and desensitize nAChRs (Posadas et al., 2013). Further studies will be necessary to evaluate the potential of cyclic imines for the control of clinical signs of Alzheimer’s disease and for unwanted side-effects.

Pharmacokinetics of cyclic imines The pharmacokinetic characteristics of cyclic imines are critical for understanding the potential toxicity to humans. The high affinity of cyclic imines for nAChRs, and the essential role of these receptors in the central nervous system and neuromuscular junction, would suggest high in vivo toxicity. However, in spite of a high i.p. toxicity, the toxic potency of these compounds by oral exposure is much lower. A few studies have been conducted to clarify cyclic imine fate after oral administration. The information available is very limited and involves only the SPX group. Trans-epithelial permeability through a human intestinal epithelium model suggests that the SPXs 13-desMeSPX C and 13,19-didesMeSPX would be readily absorbed in the intestine. The passage of these two molecules through an intact Caco-2 epithelial barrier allows the calculation of apparent permeability coefficients

Pharmacology of the cyclic imines

355

(Papp ) of 18 and 12 × 10−6 for 13-desMeSPX C and 13,19-didesMeSPX respectively (Espina et al., 2011). These values were used to predict a fraction of human intestinal absorption of 80% for 13-desMeSPX C and 50% for 13,19-didesMeSPX. Actually, considering the data available regarding the permeability coefficients in Caco-2 monolayers and absorption after oral administration in humans for several drugs, it would be logical to suggest that the fraction of toxin absorbed after oral exposure in humans would be fairly similar for both SPXs, and probably higher than 80%. The lipophilic nature of the SPXs (Hauser et al., 2012), and the fact that Caco-2 monolayers are actually considered a good model for passive transcellular absorption (Artursson et al., 2001), indicates that SPX absorption probably occurs through this mechanism. Another important issue regarding the potential oral toxicity of cyclic imines is the possibility of degradation in the acidic environment of the stomach, or enzymatic degradation by digestive enzymes. Additionally, the toxicity of parental compounds generated by phytoplankton may also be modified by transformation in shellfish. SPXs A and B are transformed into SPXs E and F by oxalic acid hydrolysis (oxalic acid, 60 ∘ C), which points to a possible detoxification by the acid of the stomach (Hu et al., 2001). By contrast, SPXs C and D are resistant to oxalic acid hydrolysis (Hu et al., 2001), and this resistance seems related to the presence of two vicinal methyl groups in the cyclic imine ring (Figure 15.1). It has also been suggested that the lack of oral toxicity of GYM, with no methyl moiety in the six-member imine ring, is due to degradation in the intestinal tract (Hu et al., 2001). Further studies demonstrated that hydrolysis of SPX B did not occur after 24 hours in pH 3 aqueous hydrochloric acid at 37 ∘ C, conditions that are closer to the physiological stomach acidic environment, indicating that degradation in the stomach would not be responsible for the lower-than-i.p. oral toxicity. PnTXs are also stable in pH 1.5 aqueous hydrochloric acid at 40 ∘ C for 24 hours (Jackson et al., 2012). In shellfish, several metabolites of cyclic imines have been reported. One of the routes for cyclic imine transformation is shellfish acylation of hydroxyl groups to form fatty acid esters. Acyl esters have been described for the 20-Me SPX G and PnTX G in mussels, and for GYMs A, B and C in clams (Aasen et al., 2006; de la Iglesia et al., 2012; McCarron et al., 2012). This reaction does not imply a complete loss of toxicity after i.p. administration but, rather, a delay in the manifestation of toxic symptoms (Aasen et al., 2006), which probably start to be evident as ester hydrolysis progresses in the organism. The other metabolism route in shellfish involves the opening of the cyclic imine ring to form biologically inactive keto-amine derivatives. SPXs E and F, which do not display toxic activity, are thought to be generated by enzymatic hydrolysis from SPXs A and B (Hu, 1996; Christian, 2008). Conversely, SPXs 13-desMe SPX C, 13,19-didesMe SPX C and 20-Me SPX G do not seem sensitive to enzymatic hydrolysis in shellfish (Christian et al., 2008). Overall, the evidence reported above indicates that at least some parental compounds of the cyclic imine group generated in phytoplankton would not be detoxified in shellfish or the GI tract, and would be efficiently absorbed. Another important step after oral administration of a compound is metabolism in the liver before it reaches systemic circulation, also called first-pass effect. In vitro studies regarding biotransformation of 13-desMeSPX C by the liver microsome enzymatic system have demonstrated the generation of at least nine metabolites as a product of oxidative processes that included hydroxylation, dihydroxylation, oxidation of methyl groups to carboxylic acid, and dehydrogenation and hydroxylation, all of them phase

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I reactions (Hui et al., 2012). Phase II reactions, such as glucuronidation were not detected for this SPX, and the metabolism of the toxin fitted a first-order clearance process. The estimated value of intrinsic clearance calculated from the kinetics of the SPX degradation was 41 μL∕min∕mg (Hui et al., 2012). This high clearance rate is responsible for the metabolism of more than 90% of the toxin in the first hour and more than 50% in the first 17 minutes in vitro. The metabolites generated by the human microsomal system show modification of the bis-spiroacetal ring system (Hui et al., 2012), which structure is critical for the interaction with nAChRs (Bourne et al., 2010) and, therefore, microsomal metabolism probably implies a loss of toxic activity. This high metabolism rate and the subsequent inactivation of the SPXs may have two implications in vivo. First, the low oral toxicity of these toxins could be explained by an intense first pass effect that greatly limits their passage to systemic circulation. Second, the characteristic ‘fast acting’ toxicity following i.p. administration, meaning that the toxic effects are displayed within 20–30 minutes or do not appear at all, could also be partly due to the high rate of hepatic elimination. No information is available regarding metabolism of other cyclic imines. The data available about in vivo pharmacokinetics is scarce. There is only one study for oral administration performed with 13-desMeSPX C and 13,19-didesMeSPX. Both SPXs were detected in blood and urine after oral administration, indicating absorption from the GI tract (Otero et al., 2012). However, as the authors state, the amount detected in blood was very small for both compounds. The total amount absorbed might be underestimated, since only three time points were sampled after toxin administration and the concentrations were too close to the limit of quantification reported in this study. A higher number of time points and higher doses would help to clarify the rate of absorption for these compounds. The amounts in urine of 13-desMeSPX C and 13,19-didesMeSPX were also very small. The low concentrations in blood and urine in this study could be influenced by the low dose of toxin administered and by intense metabolism of the toxins. Additionally, 13-desMe SPX C has been detected in the brain of mice after i.p. injection (Alonso et al., 2013), also at low concentrations, similar to those detected in blood in the first study. These results indicate that the SPX crosses the blood-brain barrier, as initially predicted based on its lipophilic nature. Overall, the pharmacokinetic data available seem to indicate that at least the SPXs are probably absorbed and metabolized at a high rate in the liver, which would be responsible for low concentrations in plasma and tissues and the lack of toxic effects in humans at former exposure levels.

Conclusions Cyclic imines are promising compounds for future drug development. SPXs, PnTXs and GYMs are potent antagonists of nAChRs that can be used as starting point for the development of new therapeutic alternatives for neuromuscular block, and for neurodegenerative diseases such as Alzheimer’s disease. The molecular targets of prorocentrolides, spiro-prorocentrimines and symbioimines have not yet been discovered. However, although the evidence is still very scarce, symbioimines could have a potential as anti-osteoporotic drugs and as COX-2 inhibitors.

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References Aasen, J., MacKinnon, S.L., LeBlanc, P. et al. (2005) Detection and identification of spirolides in norwegian shellfish and plankton. Chemical Research in Toxicology, 18, 509–15. Aasen, J.A., Hardstaff, W., Aune, T. and Quilliam, M.A. (2006) Discovery of fatty acid ester metabolites of spirolide toxins in mussels from Norway using liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 20, 1531–7. Alonso, E., Vale, C., Vieytes, M.R. et al. (2011a) 13-Desmethyl spirolide-C is neuroprotective and reduces intracellular Abeta and hyperphosphorylated tau in vitro. Neurochemistry International, 59, 1056–65. Alonso, E., Vale, C., Vieytes, M.R. et al. (2011b) The cholinergic antagonist gymnodimine improves Abeta and tau neuropathology in an in vitro model of Alzheimer disease. Cellular Physiology and Biochemistry, 27, 783–94. Alonso, E., Otero, P., Vale, C. et al. (2013) Benefit of 13-desmethyl spirolide C treatment in triple transgenic mouse model of Alzheimer disease: beta-amyloid and neuronal markers improvement. Current Alzheimer Research, 10, 279–89. Álvarez, G., Uribe, E., Avalos, P. et al. (2010) First identification of azaspiracid and spirolides in Mesodesma donacium and Mulinia edulis from Northern Chile. Toxicon, 55, 638–641. Amzil, Z., Sibat, M., Royer, F. et al. (2007) Report on the first detection of pectenotoxin-2, spirolide-a and their derivatives in French shellfish. Marine Drugs, 5, 168–79. Aráoz, R., Servent, D., Molgó, J. et al. (2011) Total synthesis of Pinnatoxins A and G and revision of the mode of action of Pinnatoxin A. Journal of the American Chemical Society, 133, 10499–10511. Aráoz, R., Ramos, S., Pelissier, F. et al. (2012) Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins. Analytical Chemistry, 84, 10445–53. Artursson, P., Palm, K. and Luthman, K. (2001) Caco-2 monolayers in experimental and theoretical predictions of drug transport. Advanced Drug Delivery Reviews, 46, 27–43. Biré, R., Krys, S., Frémy, J. M., Dragacci, S., Stirling, D. and Kharrat, R. (2002). First evidence on occurrence of gymnodimine in clams from Tunisia. Journal of Natural Toxins 11, 269–275. Bourne, Y., Radic, Z., Aráoz, R. et al. (2010) Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism. Proceedings of the National Academy of Sciences of the United States of America, 107, 6076–6081. Cembella, A.D., Lewis, N.I. and Quilliam, M.A. (1999) Spirolide composition of micro-extracted pooled cells isolated from natural plankton assemblages and from cultures of the dinoflagellate Alexandrium ostenfeldii. Natural Toxins, 7, 197–206. Cembella, A.D., Lewis, N.I. and Quilliam, M.A. (2000) The marine dinoflagellate Alexandrium ostenfeldii (Dinophyceae) as the causative organism of spirolide shellfish toxins. Phycologia, 39, 67. Chou, T., Haino, T., Kuramoto, M. and Uemura, D. (1996a) Isolation and structure of Pinnatoxin D, a new shellfish poison from the Okinawan bivalve Pinna muricata. Tetrahedron Letters, 37, 4027–4030. Chou, T., Kamo, O. and Uemura, D. (1996b) Relative stereochemistry of pinnatoxin A, a potent shellfish poison from Pinna muricata. Tetrahedron Letters, 37, 4023–4026. Christian, B., Below, A., Drebler, N. et al. (2008) Are spirolides converted in biological systems? – A study. Toxicon, 51, 934–40. Ciminiello, P., Dell’Aversano, C., Fattorusso, E. et al. (2007) Spirolide toxin profile of Adriatic Alexandrium ostenfeldii cultures and structure elucidation of 27-hydroxy-13,19-didesmethyl spirolide C. Journal of Natural Products, 70, 1878–83. Ciminiello, P., Dell’aversano, C., Iacovo, E.D. et al. (2010) Characterization of 27-hydroxy-13desmethyl spirolide C and 27-oxo-13,19-didesmethyl spirolide C. Further insights into the complex Adriatic Alexandrium ostenfeldii toxin profile. Toxicon, 56, 1327–33.

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de la Iglesia, P., McCarron, P., Diogene, J. and Quilliam, M.A. (2012) Discovery of gymnodimine fatty acid ester metabolites in shellfish using liquid chromatography/mass spectrometry. Rapid Communications in Mass Spectrometry, 27, 643–53. Espina, B., Otero, P., Louzao, M.C. et al. (2011) 13-Desmethyl spirolide-c and 13,19-didesmethyl spirolide-c trans-epithelial permeabilities: human intestinal permeability modelling. Toxicology, 287, 69–75. Gill, S., Murphy, M., Clausen, J. et al. (2003) Neural injury biomarkers of novel shellfish toxins, spirolides: a pilot study using immunochemical and transcriptional analysis. Neurotoxicology, 24, 593–604. Hauser, T.A., Hepler, C.D., Kombo, D.C. et al. (2012) Comparison of acetylcholine receptor interactions of the marine toxins, 13-desmethylspirolide C and gymnodimine. Neuropharmacology, 62, 2239–50. Haywood, A.J., Steidinger, K.A., Truby, E.W. et al. (2004) Comparative morphology and molecular phylogenetic analysis of three new species of the genus Karenia (Dinophyceae) from New Zealand. Journal of Phycology, 40, 165–179. Hellyer, S.D., Selwood, A.I., Rhodes, L. and Kerr, D.S. (2011) Marine algal pinnatoxins E and F cause neuromuscular block in an in vitro hemidiaphragm preparation. Toxicon, 58, 693–9. Hu, T., Curtis, J.M., Oshima, Y. et al. (1995) Spirolides B and D, two novel macrocycles isolated from the digestive glands of shellfish. Journal of the Chemical Society, Chemical Communications, 2159–2161. Hu, T., Curtis, J.M., Walter, J.A. and Wright, J.L.C. (1996a) Characterization of biologically inactive spirolides E and F: Identification of the spirolide pharmacophore. Tetrahedron Letters, 37, 7671–7674. Hu, T., deFreitas, A.S.W., Curtis, J.M. et al. (1996b) Isolation and structure of Prorocentrolide B, a fast-acting toxin from Prorocentrum maculosum. Journal of Natural Products, 59, 1010–1014. Hu, T., Burton, I.W., Cembella, A.D. et al. (2001) Characterization of spirolides A, C, and 13-desmethyl C, new marine toxins isolated from toxic plankton and contaminated shellfish. Journal of Natural Products, 64, 308–12. Hui, J.P., Stuart Grossert, J., Cutler, M.J. and Melanson, J.E. (2012) Strategic identification of in vitro metabolites of 13-desmethyl spirolide C using liquid chromatography/high-resolution mass spectrometry. Rapid Communications in Mass Spectrometry, 26, 345–54. Jackson, J.J., Stivala, C.E., Iorga, B.I. et al. (2012) Stability of cyclic imine toxins: interconversion of pinnatoxin amino ketone and pinnatoxin A in aqueous media. Journal of Organic Chemistry, 77, 10435–40. Kharrat, R., Servent, D., Girard, E. et al. (2008) The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity. Journal of Neurochemistry, 107, 952–63. Kita, M., Kondo, M., Koyama, T. et al. (2004) Symbioimine exhibiting inhibitory effect of osteoclast differentiation, from the symbiotic marine dinoflagellate Symbiodinium sp. Journal of the American Chemical Society, 126, 4794–5. Kita, M., Ohishi, N., Washida, K. et al. (2005) Symbioimine and neosymbioimine, amphoteric iminium metabolites from the symbiotic marine dinoflagellate Symbiodinium sp. Bioorganic & Medicinal Chemistry, 13, 5253–8. Krock, B., Pitcher, G.P., Ntuli, J. and Cembella, A. (2009) Confirmed identification of Gymnodimine in Oysters from the west coast of South Africa by Liquid Chromatography-Tandem Mass Spectrometry. African Journal of Marine Science, 31, 113–118. Laudenbach, V., Medja, F., Zoli, M. et al. (2002) Selective activation of central subtypes of the nicotinic acetylcholine receptor has opposite effects on neonatal excitotoxic brain injuries. The FASEB Journal, 16, 423–5. Livingstone, P.D., Dickinson, J.A., Srinivasan, J. et al. (2010) Glutamate-dopamine crosstalk in the rat prefrontal cortex is modulated by Alpha7 nicotinic receptors and potentiated by PNU-120596. Journal of Molecular Neuroscience, 40, 172–6. Lu, C.-K., Lee, G.-H., Huang, R. and Chou, H.-N. (2001) Spiro-prorocentrimine, a novel macrocyclic lactone from a benthic Prorocentrum sp. of Taiwan. Tetrahedron Letters, 42, 1713–1716.

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Mackenzie, L. (2004). Production of spirolides by New Zealand isolates of Alexandrium ostenfeldii and some new observations on Heterocapsa spp. in New Zealand. In: Proceedings, 21st Marine biotoxin science workshop. New Zealand Food Safety Authority, Wellington, New Zealand. MacKinnon, S.L., Walter, J.A., Quilliam, M.A. et al. (2006) Spirolides isolated from Danish strains of the toxigenic dinoflagellate Alexandrium ostenfeldii. Journal of Natural Products, 69, 983–7. Marrouchi, R., Dziri, F., Belayouni, N. et al. (2010) Quantitative determination of gymnodimine-A by high performance liquid chromatography in contaminated clams from Tunisia coastline. Marine Biotechnology (New York.), 12, 579–85. Marrouchi, R., Rome, G., Kharrat, R. et al. (2013) Analysis of the action of gymnodimine-A and 13-desmethyl spirolide C on the mouse neuromuscular system in vivo. Toxicon, 75, 27–34. McCarron, P., Rourke, W.A., Hardstaff, W. et al. (2012) Identification of pinnatoxins and discovery of their fatty acid ester metabolites in mussels (Mytilus edulis) from eastern Canada. Journal of Agricultural and Food Chemistry, 60, 1437–46. McCauley, J.A., Nagasawa, K., Lander, P.A. et al. (1998) Total Synthesis of Pinnatoxin A. Journal of the American Chemical Society, 120, 7647–7648. McNabb, P.S., McCoubrey, D.J., Rhodes, L. et al. (2012) New perspectives on biotoxin detection in Rangaunu Harbour, New Zealand, arising from the discovery of pinnatoxins. Harmful Algae, 13, 34–39. Miles, C.O., Wilkins, A.L., Stirling, D.J. and MacKenzie, A.L. (2000) New analogue of gymnodimine from a Gymnodinium species. Journal of Agricultural and Food Chemistry, 48, 1373–1376. Miles, C.O., Wilkins, A.L., Stirling, D.J. and MacKenzie, A.L. (2003) Gymnodimine C, an isomer of gymnodimine B, from Karenia selliformis. Journal of Agricultural and Food Chemistry, 51, 4838–4840. Mousavi, M. and Hellstrom-Lindahl, E. (2009) Nicotinic receptor agonists and antagonists increase sAPPalpha secretion and decrease Abeta levels in vitro. Neurochemistry International, 54, 237–44. Munday, R., Towers, N.R., Mackenzie, L. et al. (2004) Acute toxicity of gymnodimine to mice. Toxicon, 44, 173–8. Munday, R. (2008). Toxicology of cyclic imines: gymnodimine, spirolides, pinnatoxins, pteriatoxins, prorocentrolide, spiro-prorocentrimine and symbioimines. In: Botana, L. M. (ed). Seafood and freshwater toxins: pharmacology, physiology and detection, 2nd edition, pp. 581–594 CRC Press, Boca Raton, FL. Munday, R., Quilliam, M.A., LeBlanc, P. et al. (2012a) Investigations into the toxicology of spirolides, a group of marine phycotoxins. Toxins (Basel), 4, 1–14. Munday, R., Selwood, A.I. and Rhodes, L. (2012b) Acute toxicity of pinnatoxins E, F and G to mice. Toxicon, 60, 995–9. Otero, P., Alfonso, A., Rodriguez, P. et al. (2012) Pharmacokinetic and toxicological data of spirolides after oral and intraperitoneal administration. Food and Chemical Toxicology, 50, 232–7. Posadas, I., Lopez-Hernandez, B. and Cena, V. (2013) Nicotinic receptors in neurodegeneration. Current Neuropharmacology, 11, 298–314. Rhodes, L., Smith, K., Selwood, A. et al. (2011) Dinoflagellate Vulcanodinium rugosum identified as the causative organism of pinnatoxins in Australia, New Zealand and Japan. Phycologia, 50, 624–628. Richard, D., Arsenault, E., Cembella, A. and Quilliam, M. A. (2001). Investigations into the toxicology and pharmacology of spirolides a novel group of shellfish toxins. In: Hallegraeff, G. M., Blackburn, S. I., Bolch, C. J. and Lewis, R. J. (eds). Harmful Algal Blooms 2000: Proceedings of the Ninth International Conference on Harmful Algal Blooms. Paris, Intergovernmental Oceanographic Commission of UNESCO. Roach, J.S., Leblanc, P., Lewis, N.I. et al. (2009) Characterization of a Dispiroketal Spirolide Subclass from Alexandrium ostenfeldii. Journal of Natural Products, 72, 1237–40. Rundberget, T., Aasen, J.A.B., Selwood, A.I. and Miles, C.O. (2011) Pinnatoxins and spirolides in Norwegian blue mussels and seawater. Toxicon, 58, 700–711. Seki, T., Satake, M., Mackenzie, L. et al. (1995) Gymnodimine, a new marine toxin of unprecedented structure isolated from New Zealand oysters and the dinoflagellate, Gymnodinium sp. Tetrahedron Letters, 36, 7093–7096.

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Selwood, A.I., Miles, C.O., Wilkins, A.L. et al. (2010) Isolation, structural determination and acute toxicity of pinnatoxins E, F and G. Journal of Agricultural and Food Chemistry, 58, 6532–6542. Stewart, M., Blunt, J.W., Munro, M.H.G. et al. (1997) The absolute stereochemistry of the New Zealand shellfish toxin gymnodimine. Tetrahedron Letters, 38, 4889–4890. Stirling, D.J. (2001) Survey of historical New Zealand shellfish samples for accumulation of gymnodimine. New Zealand Journal of Marine and Freshwater Research, 35, 851–857. Takada, N., Umemura, N., Suenaga, K. et al. (2001a) Pinnatoxins B and C, the most toxic components in the pinnatoxin series from the Okinawan bivalve Pinna muricata. Tetrahedron Letters, 42, 3491–3494. Takada, N., Umemura, N., Suenaga, K. and Uemura, D. (2001b) Structural determination of pteriatoxins A, B and C, extremely potent toxins from the bivalve Pteria penguin. Tetrahedron Letters, 42, 3495–3497. Takahashi, E., Yu, Q., Eaglesham, G. et al. (2007) Occurrence and seasonal variations of algal toxins in water, phytoplankton and shellfish from North Stradbroke Island, Queensland, Australia. Marine Environmental Research, 64, 429–42. Torigoe, K., Murata, M. and Yasumoto, T. (1988) Prorocentrolide, a toxic nitrogenous macrocycle from a marine dinoflagellate, Prorocentrum lima. Journal of the American Chemical Society, 110, 7876–7877. Touzet, N., Franco, J.M. and Raine, R. (2008) Morphogenetic diversity and biotoxin composition of Alexandrium (Dinophyceae) in Irish coastal waters. Harmful Algae, 7, 782–797. Uemura, D., Chou, T., Haino, T. et al. (1995) Pinnatoxin A: a toxic amphoteric macrocycle from the Okinawan bivalve Pinna muricata. Journal of the American Chemical Society, 117, 1155–1156. Van Wagoner, R.M., Misner, I., Tomas, C.R. and Wright, J.L.C. (2011) Occurrence of 12-methylgymnodimine in a spirolide-producing dinoflagellate Alexandrium peruvianum and the biogenetic implications. Tetrahedron Letters, 52, 4243–4246. Villar Gonzalez, A., Rodriguez-Velasco, M.L., Ben-Gigirey, B. and Botana, L.M. (2006) First evidence of spirolides in Spanish shellfish. Toxicon, 48, 1068–74. Wandscheer, C.B., Vilarino, N., Espina, B. et al. (2010) Human muscarinic acetylcholine receptors are a target of the marine toxin 13-desmethyl C spirolide. Chemical Research in Toxicology, 23, 1753–61. Zheng, S., Huang, F., Chen, S. et al. (1990) The isolation and bioactivities of pinnatoxin. Chinese Journal of Marine Drugs, 9, 33–35.

C H A P T E R 16

Diversity of organic structures of marine microbial origin with drug potential Marcel Jaspars, Rainer Ebel & Hai Deng Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, UK

Introduction Although soil bacteria have proven to be prolific as producers of antibiotics and other bioactive natural products, the rate of discovery of useful natural products from this source has recently declined at an alarming rate (Li & Vederas, 2009). More than 70% of our planet’s surface is covered by oceans. The living conditions to which marine bacteria had to adapt during evolution range from extremely high pressure and anaerobic conditions at low temperatures on the deep sea floor, to highly acidic conditions at temperatures of over 100 ∘ C near hydrothermal vents at the mid-ocean ridges. The ocean is rich in biodiversity, and the microflora and microalgae alone constitute more than 90% of oceanic biomass (Sithranga Boopathy & Kathiresan, 2010). It is estimated that, despite constituting less than 1% of the earth’s surface, the narrow ocean fringe and the deep sea vent communities are home to a majority of the world’s species, and thus they constitute the most species-rich and biologically productive regions of the world. Accordingly, it is likely that this is also reflected in the genetic and metabolic diversity of marine bacteria, which may produce a structurally elaborate profile of natural products with drug potential (Gulder & Moore, 2009). Compounds isolated from marine invertebrates and marine microorganisms often show startlingly novel carbon skeletons, different from those found in terrestrial biota (Grabowski et al., 2008). Chemoinformatic approaches estimate that 71% of molecular scaffolds found are unique to the marine environment, and that these cover only 30% of marine natural products, with the remainder appearing only once (Kong et al., 2010). However, the marine environment is a virtually untapped source of novel bacterial diversity and, therefore, of new metabolites. The presence of natural products in the oceans remains largely unexplored (Lam, 2006). Some of these products have been approved for clinical use in the treatment of cancer, and many others are in clinical trials. Ecteinascidin-743 (1), isolated from the Caribbean ascidian Ecteinascidia turbinata, is now marketed as Yondelis by Pharmamar SA of Spain, as a treatment for soft tissue sarcoma. Halichondrin B (2), obtained from the Japanese sponge Halichondria okadai, was extremely promising in animal trials, but its structure was too complex for an economically viable total synthesis, and analogue synthesis eventually gave rise to a

Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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OH HO

H3N

MeO

HO

O

Me

OAc O

S

H

Me

H

O

O

O

H O

O

O

Me

H

O

H

O O

H

S

N

H

O

O

H N

O

O

OMe

NH

OH

ecteinascidin-743 1 marketed as Yondelis ®

halaven 3

H H HO

O

H

H

O O

H O OH H

H

O O H

H

H

O O

O H

H

O

O

OH halichondrin B 2

H

O

O

O

O

O

simpler molecule, now marketed as Halaven (3) by Eisai of Japan, for the treatment of solid tumours (Mayer et al., 2010).

Marine bacterial natural products In the past decade, studies have shown that marine bacteria produce potential drug-like natural products with great structural diversity (Newman & Cragg, 2004). These bacteria include marine Streptomyces, Salinispora, Micromonospora, Verrucosispora, α-proteobacterium Tistrella mobilis and γ-proteobacterium Pseudoalteromonas (Fenical & Jensen, 2006). Recently, it has been shown that the range of habitats in which marine microorganisms can be found extends below the sea floor, with a large diversity of metabolically rich microorganisms thriving in the deep biosphere (Orsi et al., 2013). Work on bacteria residing in the marine sponge Theonella swinhoei has delineated a new bacterial phylum, the Tectomicrobia, whose only representative species, Entotheonella palauensis, has a large genome (>9 Mb) containing a vast range of biosynthetic pathways (Wilson et al., 2014). It is therefore clear that the chemistry originally associated with this sponge must, in fact, be attributed to this new bacterial species, and it is believed that most other sponge natural products originate in this way. The investigation of marine microorganisms from a range of different marine habitats is, therefore, likely to be a productive approach to the discovery of novel chemical entities with potent and selective biological activity. Varied habitats to

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explore include, but are not limited to, marine invertebrates, marine sediments, sub seafloor, hot vents, cryogenic environments and cold seeps.

Marine bacterial natural products with anticancer drug potential Salinosporamide A (NPI-0052, 4) is a novel β-lactone-γ-lactam isolated from a fermentation broth of a new obligate marine actinomycete, Salinispora tropica (Feling et al., 2003). Salinosporamide A is an orally active proteasome inhibitor that induces apoptosis in multiple myeloma cells. Its mechanisms are distinct from the commercial proteasome inhibitor anticancer drug bortezomib (5) (Chauhan et al., 2005). NPI-0052 has been in clinical studies for treatment of cancer in humans since 2006, and salinosporamide A represents the first clinical candidate for the treatment of cancer produced by saline fermentation of an obligate marine actinomycete. Difficulties were encountered with the fermentation process under GMP conditions, but were solved by IRL-Biopharm of New Zealand.

HO H N O

O

O N

O

N H

N

H N

OH B

OH

O

Cl salinosporamide A 4

bortezomib 5

Thiocoraline (6) is a thiodepsipeptide, isolated from a marine actinomycete, Micromonospora sp. L-13-ACM2-092, collected off the Mozambique coast (Romero et al., 1997). It exhibits exceptionally potent activity in the L1210 mouse leukaemia cytotoxic assay (IC50 = 200 pM) (Erba et al., 1999), and inhibits elongation activity of DNA α polymerase by binding to double-stranded DNA through intercalation of the two aromatic chromophores (Negri et al., 2007). Thiocoraline is currently in late preclinical development for the treatment of cancer. H3CS O

O N

O N

N S

N H OH

O

H N

S O O S

N H

S N

N

O

O

thiocoraline 6

HO H N

N O

O SCH3

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Diazepinomicin (ECO-4601, 7) is a unique farnesylated dibenzodiazepinone produced by a sponge-associated Micromonospora strain (Charan et al., 2004). It was discovered through DECIPHER technology, Thallion’s proprietary drug discovery platform. The compound was shown to have broad cytotoxic activity in the low micromolar range when tested in the NCI 60 cell line panel. It has demonstrated in vivo activity against glioma, breast and prostate cancer in mouse models (Gourdeau et al., 2008). The preclinical development of ECO-4601 as an anticancer agent has been completed, and it is currently in clinical trials in Canada (Mason et al., 2012). OH

HO N

HN

O

HO

diazepinomicin 7

Lomaiviticin A (8) was first isolated from the marine-derived actinomycete Micromonospora lomaivitiensis, a microbial symbiont from the marine ascidian Polysyncraton lithostrotum (He et al., 2001). Lomaiviticin A is a powerful antibiotic, with N OH O N OH

O

N+

O O

C-

H

H

OH

O HO O

OH O

O

O

O

O

OH

O

OH

H

H O

CO

O

N+ N

O HO N

lomaiviticin A 8

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minimum inhibitory concentrations (MICs) of 6–25 ng∕spot, and Lomaiviticin A is an exceptionally potent anticancer agent, with half-maximal inhibitory concentration (IC50 ) values in the 72 nM to 7.3 pM range against 24 cancer cell lines. Evidence suggests that lomaiviticin A may operate by a novel mechanism of action, compared to the known DNA-damaging anticancer drugs such as adriamycin and mitomicin C, and lomaiviticin A cleaved double-stranded DNA under reducing conditions. The marine environment consists of highly complex microbial communities, and many invertebrates are associated with large numbers of microbes (Grozdanov & Hentschel, 2007). Didemnins are cyclic depsipeptide compounds isolated from a tunicate (sea squirt) of the genus Trididemnum that was originally collected in the Caribbean Sea. The most potent analogue, didemnin B (9) was the first marine natural product to enter clinical trials as an anti-cancer agent, and it showed strong activity against murine leukaemia cells (Egan et al., 2008). Didemnin B was advanced to clinical trials and completed phase II human clinical trials against adenocarcinoma of the kidney, advanced epithelial ovarian cancer and metastatic breast cancer. Unfortunately, the compound exhibited cardiac and neuromuscular toxicities, and trials were terminated. OCH3

N

N O O

O O O

R N

NH

N H

NH O

O

O O

OH

O

O

N O didemnin B 9 (R = OH) aplidine 10 (R = =O)

A closely related compound, dehydrodidemnin B (aplidine, 10), was isolated from the Mediterranean tunicate Aplidium albicans and was shown to be more potent and less toxic, despite the minor structural change. Aplidine has since replaced didemnin B in clinical trials, and is currently in multiple phase II and III trials for the treatment of various cancers. Due to their cyclic depsipeptide structures, with highly modified amino acid residues, didemnins have long been suspected to be microbial products assembled by a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) enzymatic pathway. The recent study demonstrated that the α-proteobacteria Tistrella mobilis and Tistrella bauzanensis also produce the didemnins through a unique post-assembly activation of lipoglutamine congeners (Xu et al., 2012), consistent with previous suggestions that microbes could be the actual producers of many molecules originally isolated from invertebrate tissues.

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O H N

N

H N

N N OCH3 O

O

S N

OCH3 O

dolastatin 10 11

O H N

N O

H N

N N OCH3 O

OCH3 O

TZT-1027 12

Dolastatin 10 (11) is a pentapeptide natural product, isolated from the sea hare, Dolabella auricularia, in the 1970s. The structural elucidation of dolastatin 10 took nearly 15 years to complete, due to the extremely low abundance of the active component (≈ 1.0 mg∕100k g of collected organism) (Pettit et al., 1987). Dolastatin 10 was later also isolated from the marine cyanobacterium Symploca sp. (Luesch et al., 2001). Dolastatin 10 is one of the most potent antiproliferative agents known, with an ED50 = 4.6 × 10−5 μg∕mL against murine PS leukaemia cells, and it entered clinical trials in the 1990s. Unfortunately, it was dropped from clinical trials as a single agent, due to the development of neurocytotoxicity in patients. Nevertheless, dolastatin 10 offered a logical starting point for SAR studies and synthetic drug design, ultimately leading to the analogue TZT-1027 (12), which has been now entered into phase II clinical trials (Riely et al., 2007).

Marine actinomycete natural products with antimicrobial drug potential Antibiotics have saved countless millions of lives. Most clinically used antibiotics originated from natural products discovered between the late 1940s and 1970, the time period called the golden age of antibiotic development. From the 1980s, however, antibiotic development has slowed alarmingly, and no new class of antibiotic was launched between 1980 and 2000 (Fischbach & Walsh, 2009). The industry has largely abandoned new antibiotic development, as the risk : reward ratio has been considered unattractive, and the drug pipeline is poorly stocked. This is against a backdrop where there is an urgent need for new antibiotics, particularly to tackle the rise of antibiotic-resistant bacteria. It is crucial that the research community should find new sources of drugs and, in this respect, marine-derived bacteria are emerging as an attractive option for discovery of new antibiotic molecules.

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Abyssomicin C (13) is a novel polycyclic polyketide antibiotic produced by a marine Verrucosispora strain (Bister et al., 2004). It is a potent inhibitor of p-aminobenzoic acid biosynthesis and, therefore, inhibits the folic acid biosynthesis at an earlier stage than the well-known synthetic sulfa drugs. Abyssomicin C possesses potent activity against Gram-positive bacteria, including clinical isolates of multiple-resistant and vancomycin-resistant Staphylococcus aureus. Abyssomicin C or its analogues have the potential to be developed as antibacterial agents against drug-resistant pathogens.

O

O

O O

O

H OH abyssomicin C 13

Marinispora is the new family of obligate marine actinomycete species. The first Marinispora strain to be subjected to chemical study, strain CNQ-140, was isolated from a sediment sample collected at a depth of 56 m offshore of La Jolla, CA (Kwon et al., 2006). A bioassay-guided approach led to the isolation of a new class of bis-salicylate-containing polyene macrodiolides, including marinomycin A (14). Marinomycin A possesses impressive antibiotic activities, with MIC values of 0.1–0.6 μM, against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF).

OH

HO O

O

OH

OH

OH

OH

OH

OH

O

O OH

HO marinomycin A 14

The first total synthesis of marinomycin A was achieved in 2006 (Nicolaou et al., 2006), while a recent triply convergent synthetic approach of 14 used the salicylate as a novel molecular switch for the chemoselective construction of the macrodiolide, which is the first stepping stone for the future structure-based discovery (SBD) on this unique 44-membered macrocyclic antibiotic (Evans et al., 2012).

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Anthracimycin (15) is a structurally unique polyketide antibiotic natural product isolated from Streptomyces sp. CNH365, collected from near-shore marine sediments near Santa Barbara, California, USA (Jang et al., 2013). Anthracimycin is a potent antibiotic against Gram-positive genera such as Staphylococci, Enterococci, and Streptococci, but it either lacked activity or was weakly active against the Gram-negative species. More importantly, it shows significant antibacterial activity against Bacillus anthracis (strain UM23C1-1), the etiologic agent of anthrax in livestock and occasionally in humans, with a minimum inhibitory concentration (MIC) of 0.031 μg mL−1 in a broth microdilution assay. While the mechanism of action remains to be fully defined, it has been observed that anthracimycin inhibits DNA/RNA synthesis in metabolic labelling experiments. A thorough examination of the efficacy of anthracimycin is currently ongoing. H H H O

O

O

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Pseudoalteromonas is a genus of marine Gram-negative bacterium. Those Pseudoalteromonas species isolated before 1995 were originally part of the Alteromonas genus. Pseudoalteromonads are frequently known to be bioactive, and are often found in association with higher eukaryotes or marine surfaces. In 1993, a Japanese group reported the fermentation and isolation of thiomarinol A from a marine Gram-negative bacterium, Pseudoalteromonas sp nov. SANK 73390, isolated from seawater (Shiozawa et al., 1993). Thiomarinol A (16) is a hybrid compound that consists of a dithiolopyrrolone moiety attached via an amide linkage to a pseudomonic acid analogue, an esterified unusual fatty acid component connected to the monic acid. Monic acid is an important polyketide moiety of an anti-methicillin resistant Staphylococcus aureus (MRSA) antibiotic mupirocin (Fukuda et al., 2011). Thiomarinol A is a potent antibiotic, with a significant MIC value of

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PbTx-3 > PbTx-2 > PbTx-6 (Berman & Murray, 1999), representing a > 90% pure culture of glutamatergic neurons. This cell toxicity is mediated by NMDA receptors activated secondarily to brevetoxin-induced Na+ channel activation and resultant release of glutamate. The neurotoxicity observed in vitro would be related to pathology observed in animals.

Cellular and sub-cellular effects Cytotoxic and genotoxic effects have been described for brevetoxin, predominantly in immune cells. Brevetoxin B (also known as PbTx2) has been found to induce apoptosis in the Jurkat human T-lymphocyte line (Walsh et al., 2008). Additional studies have shown that brevetoxin B causes a depletion of glutathione in a U-937 human monocyte line, suggesting that brevetoxin-exposed immune cells are more likely to be subjected to oxidative stress (Walsh et al., 2009). A comet assay of human lymphocytes treated in vitro with brevetoxins showed clastogenic properties by exhibiting both single DNA and double strand breaks (Sayer et al., 2005). These effects of brevetoxin are clearly clastogenic, in that they caused breaks in chromosomes, and they are also consistent with increased oxidative stress. Brevetoxin may promote antioxidant stress in lymphocytes by activation of voltage-gated Na+ channels (Fraser et al., 2004) or depletion of the antioxidant glutathione (Walsh et al., 2008). Brevetoxins bind with high affinity to receptor site 5 on the voltage-gated sodium channel (VGSC), and induce a channel-mediated Na+ ion influx (Dechraoui, 1999, 2005). Neuroexcitation results from nerve membrane depolarization and spontaneous firing. In some cases, only the nerve is depolarized, but both nerve and muscle depolarization have been noted (Hughes & Merson, 1976; Baden, 1983; Huang, 1984; Poli et al., 1986; Sakamoto et al., 1987; Wu & Narahashi, 1988). Although considerable information has been gathered on the cellular mechanisms of action in excitable tissues and the effects of brevetoxins in intact organisms, little is known concerning the mechanisms by which brevetoxins affect the CNS, or what the neuroanatomic targets may be. Brevetoxins were distributed widely to all organs in the body, and they have been shown to reach significant levels within were the CNS when, administered orally to rats, given their lipid solubility, passing through cell membranes, including the blood-brain barrier (Cattet & Geraci, 1993). Peng et al. (1995) demonstrated that acute brevetoxin and ciguatoxin-induced thermoregulatory disturbances correlate closely with neuroexcitation and c-fos mRNA induction in a variety of hypothalamic and brainstem regions. These data indicate that brevetoxins can reach concentrations in the brain sufficient to produce functional alterations in CNS neurons. Moreover, because brevetoxins have been shown to stimulate the release of excitatory amino acid neurotransmitters from cortical synaptosomes (Risk et al., 1982), this raises the question as to whether brevetoxins might stimulate excitatory neurons to release glutamate in regions of the CNS vulnerable to excitotoxic cell death. Brevetoxins are rapidly absorbed and distributed throughout the body, being metabolized in the liver (Poli et al., 1990; Cattet & Geraci, 1993). They are primarily removed in the bile within the first 48 hours, but urinary excretion plays a key role after this time, as well. Chronic and sub-chronic toxicity of brevetoxins remain unknown, as mammalian studies have explored only acute effects.

Symptoms and toxicological effects in animals NSP toxins have produced several important events which have led us to understand their mechanism of action and the pathological effects of their ingestion.

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In 1996, 149 manatee deaths were attributed to Florida red tide. In previous instances, manatee deaths had been primarily associated with the consumption of brevetoxin-contaminated tunicates based on the examination of manatee stomach contents and timing of death (Bossart et al., 1998). Different tissues of 25 of these dead manatees were collected in order to do necropsy studies. Consistent gross lesions were present in the nasopharyngeal tissues, trachea, bronchi, lungs, liver, kidneys, and brain of all manatees. The nasopharyngeal tissues were characterized by severe diffuse mucosal erythema, oedema and congestion. The lungs had severe, diffuse congestion with a diffuse red mottled appearance, while their margins were bright red to pink. Copious amounts of blood and serosanguinous fluid oozed from all cut lung surfaces. The tracheal and bronchial mucosas were less congested, oedematous and erythematous, and occasionally contained adherent, blood-tinged, ropey mucus. Additionally, the meninges and choroid plexi were congested. Microscopically, 21 (84%) of the manatees had moderate to severe, multifocal to diffuse pulmonary congestion, haemorrhage and oedema. Mild to severe catarrhal inflammation of the nasal mucosa, trachea, bronchi and/or larger bronchioles was present. This inflammation was typically characterized by multifocal sub-mucosal infiltrates of lymphocytes and plasma cells with sparse neutrophils. Sub-mucosal congestion and haemorrhage were frequently seen. The nasal mucosal inflammatory lesions were severe and diffuse, often with an accompanying excessive catarrhal exudate, congestion, and haemorrhage that resulted in sub-mucosal thickening. Occasionally, intramucosal vesicles contained proteinaceous fluid, a heterogeneous coccobacillary gram-negative bacterial population, and neutrophils. Hemosiderosis (a disorder implying the accumulation of hemosiderin) was confirmed by positive Prussian blue staining, and was present in multiple tissues in 21 (84%) of the manatees. Hepatic CNS, and increased splenic deposits of hemosiderin were present. The hepatic and splenic hemosiderosis was generally moderate and multifocal. Low numbers of perivascular siderophages were present in the cerebrum, cerebellum, meninges and spinal cord. In 13 (52%) of the manatees, the CNS hemosiderosis was also associated with mild haemorrhage and congestion. In 12 (48%) manatees, a mild, multifocal, nonsuppurative leptomeningitis was present. This lesion, which primarily affected the cerebellar meninges, was characterized by multifocal lymphocytic infiltrates with mild haemorrhage. Another important intoxication event happened in 1998, when several manatees exposed to brevetoxins displayed severe nasopharyngeal congestion, oedema and haemorrhage (Bossart et al., 1998). Immunohistochemical staining showed brevetoxin-positive staining of lymphocytes and macrophages in the lung, liver and secondary lymphoid tissues (Bossart et al., 1998). In addition, lymphocytes and macrophages associated with the inflammatory lesions of the nasal mucosa and meninges were brevetoxin-positive and also stained for interleukin-1beta-converting enzyme (ICE). ICE converts the pro-inflammatory cytokine, interleukin-1beta, into the active form, which may play a role in apoptosis induction (Bossart et al., 1998). Dolphin deaths have also been reported, most notably the significant mortality event of 107 bottlenose dolphins in 2004 (Flewelling, 2005; Fire et al., 2007). Necropsy showed gut contents of menhaden (Brevoortia spp.) with greatly elevated brevetoxin concentrations (Flewelling, 2005). Menhaden were the primary stomach content in these dolphins, and the fish samples were found to be highly contaminated with brevetoxins. Subsequent experimentation demonstrated that the brevetoxins accumulate in the viscera and muscles of both omnivorous and planktivorous fish.

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In addition, tissue samples (lungs, liver, blubber, kidneys) from dolphin carcasses categorized as not recently exposed to a red tide bloom revealed detectable levels of brevetoxins. This may imply toxin retention in these dolphins and/or continued exposure through the consumption of finfish which had accumulated and retained brevetoxins (Fire, 2007). Experimental procedures with brevetoxins in rodents showed principally respiratory and neuronal pathological effects. In animal studies with inhaled brevetoxins, suppressed splenic antibody production was observed among Sprague-Dawley rats inhaling aerosols of crude K. brevis extract four hours/day for one and four weeks. No toxicity to the nervous, respiratory, or hematopoietic systems was noted (Benson et al., 2003). The extract contained primarily brevetoxins 2 and 3, but also contained brevenal, a newly identified compound in K. brevis having pharmacologic activity antagonistic to brevetoxin-induced neurotoxicity and bronchoconstrictor activities (Bourdelais et al., 2003; Abraham et al., 2005). Brevetoxin-induced suppression of splenic antibody production was confirmed in rats inhaling pure brevetoxin 3 at 500 μg∕m3 for 0.5 hours and two hours/day for five consecutive days (Benson et al., 2004). Antibody production was suppressed by >70% in the low-exposure (0.5 hours exposure/day) and high-exposure (two hours exposure/day) groups. Small numbers of splenic and peribronchiolar lymphoid tissue macrophages stained positive for brevetoxin. Benson et al. (2005) reported that the only lesion observed in the brevetoxinexposed rats was alveolar macrophage hyperplasia of minimal severity, characterized by a slight increase in the number of alveolar macrophages in the lung and occasional alveoli (Figure 19.7). Macrophages were also found in the lumens of terminal bronchioles, an atypical location for these cells. The cytoplasm of the macrophages appeared normal and was not vacuolated, enlarged, or pigmented. No evidence of neuronal damage or loss was detected in sections of the hippocampus and cerebellar cortex. Occasional neurons or groups of neurons were crenated and darkly stained. However, there were no tissue changes associated with these cells, such as oedema,

(a)

(b)

Figure 19.7 Increased numbers of alveolar macrophages in the alveoli of a rat exposed to high-brevetoxin concentration, a typical presentation. (a): Bar = 50 μm. (b): Bar = 25 μm. Arrows indicate macrophages. Inhalation Toxicity of Brevetoxin 3 in Rats Exposed for Twenty-Two Days. Source: Benson, JM. et al., (2005). Inhalation Toxicity of Brevetoxin 3 in Rats Exposed for Twenty-Two Days. Environ Health Perspect. 113, 626–631. Reproduced with permission of Environmental Health Perspectives. (See plate section for colour version.)

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inflammatory cell infiltrates, macrophages, nor staining alterations of the neutrophil indicating degeneration of nerve fibres. Animal studies have demonstrated that brevetoxins distribute to the rat and mouse brain following acute administration by several routes (Cattet & Geraci, 1993; Tibbetts et al., 2006). These brain concentrations of brevetoxins range from 2.12 nM (Benson et al., 1999) to 6.8 nM (Cattet & Geraci, 1993), and are therefore sufficient to produce significant fractional occupancy (42–70%) of VGSCs (Poli et al., 1986). Yan et al. (2006) examined the neurotoxic effects of sub-acute exposure of PbTx-3 via inhalation in a mouse model. They observed a similar restricted pattern of PbTx-3-induced neural degeneration in the posterior cingulate/retrosplenial cortices.

Symptoms and toxicological effects in humans A recent evaluation of human toxicity to establish an acute reference dose (acute RfD) (Joint FAO/WHO/IOC ad hoc Expert Consultation on Biotoxins in Molluscan Bivalves) concluded there was insufficient human toxicity data to complete a risk assessment, and that further research on brevetoxins and their metabolites (particularly their oral potencies) needs to be performed before an acute RfD could be established (Toyofuku, 2006). Nevertheless, we can cite several common symptoms observed after NSP events in population. NSP causes a range of signs and symptoms, both neurological and gastrointestinal. Most individuals report multiple symptoms. Mild to moderate nausea, vomiting and diarrhoea are often reported, although these may not be the chief presenting complaints. Victims of NSP most frequently describe numbness and tingling in the lips, mouth and face, throat tightness and chest heaviness, as well as numbness and tingling in the extremities. These paresthesias may be minor to fairly severe, and have been described as feeling like the ‘nerves are on fire or ants are crawling and biting all over’. Ataxia, overall loss of coordination, and partial limb paralysis may also occur. The reversal of hot and cold sensation has been reported as well, a symptom shared with ciguatera poisoning (Friedman et al., 2008). Slurred speech, headache, pupil dilation and overall fatigue are also commonly reported, with some victims appearing disoriented. Throat tightness and chest heaviness have also been reported. A few individuals have had respiratory discomfort and distress, with a handful of cases requiring ventilatory support. The characteristic cluster of both gastrointestinal and neurological symptoms occurs at approximately the same time, with the neurological symptoms lasting longer than the gastrointestinal discomfort (Music et al., 1973; Hughes & Merson, 1976; Baden & Mende, 1982; Sakamoto et al., 1987; Wu & Narahashi, 1988; Noble, 1990; Martin et al., 1996; Poli et al., 2000; Fleming, 2001). The largest and best documented outbreak in the United States occurred in North Carolina (Morris, 1991). The most prevalent symptoms reported were: paresthaesia (81%); vertigo (61%); malaise (50%); abdominal pain (48%); nausea (44%); diarrhoea (33%); motor weakness (31%); and ataxia (27%) (Morris, 1991). The temperature reversal was also reported in 17% of the intoxicated people. Mean time to onset was three hours (range from 15 minutes to 18 hours), and a dose response was associated with meal size. Nearly all cases had multiple symptoms, and most had more than one neurological symptom. Only one case in the outbreak was admitted to the hospital for severe neurological effects – bilateral carpopedal tremor, myalgias, total body paresthaesia, ataxia, and vertigo. There were no cases involving respiratory distress in the North Carolina outbreak. Morris (1991) reported a mean duration of illness of 17 hours (range 1–72 hours).

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This outbreak remains the best clinical and epidemiological description of an NSP outbreak reported in the peer-reviewed literature. Routine monitoring of shellfish beds for K. brevis has now been established in North Carolina, based in part on the success in reducing the number of cases after the bloom was identified and warnings issued. A more recent outbreak occurred in south-western Florida in 2006, during a prolonged K. brevis red tide bloom. To date, 20 cases have been reported to Florida health officials, all of them having reported multiple symptoms similar to the North Carolina outbreak. In addition to the more common symptoms mentioned above, some individuals reported spasms of uncontrollable muscle contractions and psychotic-like outbursts (n = 6). Only one case in this Florida series reported temperature reversal sensations. Differences in reported symptoms between the Florida and the North Carolina outbreaks may be due, in part, to variations in the methods used to document symptoms, and to the longer lag time between disease and interview for some of the later reported Florida cases. Variations in the composition of the brevetoxin mixture between these two red tide events may also be responsible for symptom differences, although this theory cannot be tested retrospectively (Watkins et al., 2008).

Azaspiracid shellfish poisoning In November 1995, several people in the Netherlands became ill after eating mussels (Mytilus edulis) cultivated at Killary Harbour (Ireland), which had previously been passed as safe for consumption by the DSP mouse bioassay (McMahon & Silke, 1996). The symptoms observed in the patients included nausea, vomiting, severe diarrhoea and stomach cramps, resembling those of diarrheic shellfish poisoning (Ito et al., 1998, 2000; Satake et al., 1998). However, mouse symptoms induced by i.p. injection of acetone extracts of mussels hepatopancreas were distinctly different from those normally associated with DSP toxins, showing prominent neurological symptoms, such as respiratory difficulties, spasms, paralysis of the limbs and death (Ito et al., 2000). It was then that azaspiracid (formerly called Killary Toxin-3 or KT3) was identified, and the new toxic syndrome was called ‘azaspiracid poisoning’ (AZP). Azaspiracids (AZAs) accumulate in bivalve molluscs (mainly mussels, but also oysters, scallops and clams) and crab (Torgersen et al., 2008) that feed on toxic microalgae (Furey et al., 2002, 2003; Magdalena et al., 2003). They were detected mainly in Ireland, and to a lesser extent throughout Europe (De Schrijver et al., 2002; Magdalena et al., 2003; Vale et al., 2008), north-western Africa (Taleb et al., 2006) and Chile (López-Rivera et al., 2010). To date, more than 20 naturally occurring analogues of the toxin have been described (Rehmann et al., 2008). Recently, a marine algae designated as Azadinium spinosum was found to produce AZA1 (Krock et al., 2008; Tillmann et al., 2009).

Mechanism of action Several analogues of AZA1 (formerly KT3) were reported in the literature (Ofuji et al., 1999a, 2001; Diaz Sierra et al., 2002; James et al., 2003; Blay et al., 2003). They have spiral ring assemblies, a cyclic amine and a carboxylic acid, and these characteristics make them unique within the nitrogen-containing marine toxins (Stake et al., 1998). Acute oral administration of AZA in mice reported toxic effects in the gastrointestinal tract (necrosis in the lamina propia of the small intestine), in lymphoid tissues such as thymus, spleen and the Peyer’s patches (with injuries in lympthocytes B and T), and

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fatty changes in the liver (Ito et al., 2000). Chronic exposure to the toxin was associated with the development of lung tumours (Ito et al., 2002). AZA1 was shown to be cytotoxic to all cell lines including kidney, lung, neuronal, pituitary and immune-type cell cultures tested in a concentration and time-dependent manner (Furey et al., 2010). The concentration of AZA1 inducing the half-maximal effect (EC50 ) relating to cell viability was within the narrow range of 1.1–7.9 nM after 24 hours exposure. While the Jurkat T lymphocytes and the GH4C1 rat pituitary cell lines were found to be most sensitive, the AZA cytotoxic effects appeared to be broad-spectrum in action. The entire AZA1 molecule and its unique architecture are necessary for imparting toxicity (Ito et al., 2006). Furthermore, unlike other phycotoxins, AZA1 requires an unusually long exposure time (>24 hours) to cause complete cytotoxicity and induce cell death (Ito et al., 2002).

Cellular and sub-cellular effects AZA toxic effects in culture can be resumed in five points: changes in cellular architecture; affectation of cellular adhesion mechanisms; alterations of calcium and cAMP levels; cytotoxicity; and, finally, tumorigenic activity. AZAs can induce alterations to the actin cytoskeleton of human neuroblastoma cells (BE(2)-M17) and human T lymphocytes (Jurkat cells), inducing a time- and dose-dependent decrease in F-actin levels when cells are exposed to nanomolar concentrations of the toxin, and resulting in reduced cell proliferation (Roman et al., 2002; Leira et al., 2001; Twiner et al., 2005). Moreover, the cytoskeletal damage is irreversible after toxin withdrawal. However, as reported by Vale et al. (2007a), the concentration required to decrease F-actin content is a thousand-fold higher than that needed to decrease cell viability. In these studies, the cytotoxic effects of AZA1 include alterations in the cytoskeleton, but F-actin should not be a target of the toxin. Nanomolar concentrations of AZA1 reduced MCF-7 epithelial cell viability after exposure to the toxin for 24 hours (Belloci et al., 2010). The reduced cell proliferation has been attributed to the impaired cell-cell adhesion ability of the tissue, owing to fragmentation of the E-cadherin protein, resulting in the accumulation of the E-cadherin fragment, ECRA100 (Ronzitti et al., 2007), similar to that noted by yessotoxin induced toxicity (Pierotti et al., 2003). Direct evidence that AZA1 inhibits endocytosis was obtained by showing that AZA1 blocked the intracellular transfer of E-cadherin-bound antibody in MCF-7 cells (Bellocci et al., 2010). AZA1 affects cell adhesion in both epithelial cells and fibroblasts, yet the toxin only affects the cadherin protein of the epithelial cells (E-cadherin), and not that of fibroblasts (N-cadherin), indicating tissue specificity in relation to molecular mechanism. Considering that the alterations to E-cadherin structure have previously been linked to the spread/metastasis of tumours in human cancers (Ronzitti et al., 2007), this toxic effect could be related to possible oncogenic activity of AZAs. Studies of the effects of AZA1 to AZA5 in calcium, cAMP and pH levels showed a high disparity of results. While AZA1 and AZA2 both increased intracellular calcium concentration and cAMP levels by activation of calcium release from internal stores and calcium influx (Roman et al., 2002), AZA3 increased both intracellular calcium and cAMP levels by calcium influx, together with an increase of pH level. AZA4 inhibited stores operated channels, while inhibiting the basal pH increase (Alfonso et al., 2005). Finally, AZA5 did not modify intracellular calcium levels, and had no effect on intracellular pH (Alfonso et al., 2006).

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The varied effects of the toxins on the modulation of intracellular calcium and pH structure have been attributed to the difference in chemical structure of AZAs toxins, indicating that the presence of either a methyl or hydroxyl groups can affect the activation or inhibition of calcium influx (Roman et al., 2004; Alfonso et al., 2006). The increase of cAMP is an effect modulated by the adenylyl cyclase pathway. Adenylyl cyclase is a membrane enzyme which catalyzes the transformation of ATP to cyclic AMP and releases it into the cytoplasm. As the adenylyl cyclase-cAMP pathway responds to membrane receptor activation, the results suggest that the target of the AZAs toxins is a membrane protein. Cytotoxic effects of AZAs were performed principally in AZA1, but also in AZA2. AZA1 inhibited synaptic transmission in spinal cord neurons in a manner independent of sodium and calcium channels (Kulagina et al., 2006), suggesting the possibility of a neurotoxic effect. Vale et al. (2007b) found that AZA1 and AZA2 caused the accumulation of active C-Jun-N-terminal protein kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) signalling pathway, in the nucleus of neuron cells. MAPK signal transduction pathways are among the most widespread mechanisms of eukaryotic cell regulation, being implicated in cell differentiation, embryonic development, cell movement and apoptosis (Whitmarsh & Davis, 1998). Cao et al. (2010) found that AZA-1 exposure in murine neocortical neurons increased lactate dehydrogenase (LDH) efflux and nuclear condensation, and stimulated caspase-3 activity. These data indicate that AZA1 triggers neuronal death in neocortical neurons by both necrotic and apoptotic mechanisms, and that the observed neurotoxicity is dependent on a caspase signalling pathway. The caspase activity was previously reported in human neuroblastoma cells (Vilariño et al., 2007), after 48 hours of exposure to the toxin. The induction of apoptosis by AZAs after two days of exposure concurs with the pathological findings of Ito et al. (2002). Twiner et al. (2012) demonstrated that AZA1 was highly cytotoxic to T lymphocytes, intestinal and neuroblastoma cells, T lymphocytes being the most sensible cell type. In this study, levels of caspase-3/7 were higher in all cell types treated with AZA1, and cells exposed to the toxin underwent atypical apoptosis, possibly in conjunction with necrotic cytotoxicity. Cytotoxicity was also observed in primary neuronal cultures, where AZA1 produced a complete cytotoxic effect after eight hours of exposure to the toxin, with a half maximal inhibitory concentration (IC50 ) in the low nanomolar range (Vale et al., 2007a). Finally, although oncogenic activity of AZA has not been studied profoundly, Colman et al. (2005) reported the potential teratogenic effects of AZA1 on microinjected embryos of Japanese medaka (Orzias latipes). Concentrations of ≥40 pg∕egg of AZA1 resulted in morphological and physiological affects approximately four days after fertilization. AZAs have just recently been identified as open-state blockers of human ether-àgo-go related gene (hERG) potassium K+ channels. hERG K+ channels are transcriptionally expressed in a broad array of cell/tissue types, including heart, brain, liver, kidney, breast, pancreas, and colon, with the highest levels of expression in heart and brain tissue. Expression is cell cycle-dependent, involved in apoptosis, and commonly up-regulated in cancerous cells. AZA1 physically blocks the K+ conductance pathway of hERG1 channels by occluding the cytoplasmic mouth of the open pore (Twiner et al., 2012). Therefore, Twiner and co-workers concluded that future in vivo studies testing

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AZAs should assess potential cardiotoxic effects of these toxins (i.e. electrocardiogram, molecular markers, etc.).

Symptoms and toxicological effects in animals The symptoms observed in mice exposed to AZAs differed from those that were exposed to diarrhetic shellfish poisoning (DSP) toxins. Mice injected with low doses of AZA did not develop diarrhoea (Ito et al., 2002) but, within a few hours, the mice developed progressive paralysis, had difficulty in breathing and sat listless in the corners of their cages; all died within 1.5 hours (Satake et al., 1998). Acute pathological changes caused by per os (p.o.) administration of pure AZA1 in vivo experiments were reported, employing three groups of mice at different dose levels (Ito et al., 2000). A concentration of 150 μg∕kg was found to be the minimum lethal dose in this study. AZA1 caused fluid accumulation in the small intestine, and the injuries caused by the toxin were most prominent in the upper part of the small intestine. Eight hours after treatment with AZA1 at 600 or 700 μg∕kg, villi became shorter by losing upper parts, and the injury progressed to lamina propria and epithelial cells. Arrangements and shapes of the holes in the crypts became irregular. After 24 hours, epithelial cells showed signs of recovery, but lamina propria lagged in recuperation. Interestingly, AZA1 caused no prominent changes in stomach mucosa (Figure 19.8) (Ito et al., 2000).

Figure 19.8

Section of villi from mice intestine following treatment with azaspiracid at 300 μg∕kg. Vacuole degeneration in the epithelial cells (circle) and atrophy of lamina propria (LP) are shown. After shrinkage of lamina propria, spaces (*) are left between epithelial cells and lamina propria. Source: Ito et al., 2000. Reproduced with permission of Elsevier.

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The spleen became atrophic and contained many degenerating cells in both the red and the white pulp. Cells in the marginal zone were markedly decreased, compared with the normal state. The total number of non-granulocytes was reduced to two-thirds in the control group. In the control mice, cells stained with antibodies against the type B cells, type T cells, or cytotoxic/suppressor T cells, were observed mainly in the white pulp of the spleen. Only a few cells were stained in the red pulp. All of the cells, including the stained ones, had a distinctly round shape. In contrast, the effect of AZA on B and T lymphocytes was not limited to those inside the white pulp. Stained cells were observed in both the red and white pulp in the spleens of mice treated with AZA (Ito et al., 2000, 2002). In mice treated with AZA1, onset of the accumulation of fat droplets was microscopically confirmed at one hour. The colour of the liver changed from dark red to pinkish red at four hours and this change was macroscopically apparent. After 24 hours, the weights of the livers had increased by 38% in the mice treated with 500 μg∕kg, compared with livers of control mice (Ito et al., 2000, 2002). Data generated by Ito et al. (2002), on the chronic effects on mice of orally administered AZA1 at low doses (20–50 μg∕kg), suggested that the toxin caused shortening of the small intestinal villi as a consequence of erosion. Moreover, hyperplastic changes in the mucosa of the stomach were observed. Long term AZA1 exposure also exerted an effect on the lungs, and all of the mice used in the studies developed interstitial pneumonia. Mild inflammation of the liver was also observed. These authors also reported a low recovery of injured organs, so that damage in these organs was still observed three months after withdrawal. Nevertheless, the most relevant result evolved in this study was the revelation of a tumorigenic property of AZA, since there was a 20% incidence of lung cancer among the mice administered with the toxin (four out 20 mice in the 20 and 50 μg∕kg group). The tumours were evident only 2–3 months into the post-administration recovery period and histological characterization was unsuccessful (Ito et al., 2002). Studies employing a single sub-lethal dose of AZA1 (100, 200 and 300 μg∕kg bw) were carried out in mice, in order to find the lowest oral dose associated with pathological changes in the gastrointestinal tract, as well as to determine the distribution of the toxin and the speed of recovery of the lesions (Aasen et al., 2010). In this study, the absorption and accumulation of the toxin in the different organs was dose-dependent. The highest concentration of AZA1 was found in the kidneys and spleen, closely followed by the lungs, while lower concentrations were found in liver and heart. In the brain, only trace amounts of AZA1 were detected. After 24 hours, the total amount of AZA in the internal organs was estimated to be only about 2% of the total amount given. After seven days, toxin concentration had dropped in all examined organs except the kidneys. In previous studies (Ito et al., 2002), very long persistence of damage to the gastrointestinal tract by repeated exposures to AZA toxins had been reported. However, Aasen and co-workers reported full recovery from the pathological changes as soon as seven days after a single exposure to AZA1 (Aasen et al., 2010).. The authors concluded that there seems to be a pronounced difference in recovery times between single and double exposures of AZA. Sub-lethal lower doses led to a more inflammatory response, with infiltration of neutrophils in the lamina propria, whereas the higher dose seemed to be more toxic and led to more severe necrosis and loss of cells in the lamina propria. The effects of AZA1 may be mediated through a direct effect on the intestinal mucosa, since the toxic effects on the small intestine were found only in the upper segment, where the concentration of toxin is highest (Figure 19.9).

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(a)

(b)

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Figure 19.9 Representative micrographs of duodenum from mice orally exposed to either vehicle (a) or various doses of azaspiracid-1: 100 μg∕kg (b), 200 μg∕kg (c) or 300 μg∕kg (d), 24 hours prior to sampling. Mice exposed to toxin showed shortened villi, elongated crypts, slight detachment of epithelium in tips of villi (arrow) and accumulation of inflammatory cells in lamina propria (arrowheads). The changes were less pronounced in mice exposed to 100 μg∕kg (b). H&E. Bar: 100 mm. Source: Aasen et al., 2010. Reproduced with permission of Elsevier.

Lack of pathology in organs outside the gastrointestinal tract was also observed, which agreed with the low levels of toxin found in those organs. Injuries were only seen in duodenum (Figure 19.9), although stomach was exposed to a higher AZA dose, but it seemed to be resistant to the toxin effects (Aasen et al., 2010). Studies about the possible combined toxic effects of AZA and other marine toxins were performed in mice via the oral route (Aasen et al., 2011; Aune et al., 2012). When AZA1 and okadaic acid were given together, there was a considerably reduced absorption for both toxins, because of competition for the simple diffusion of weak organic acids across the membranes of the gastrointestinal tract. Furthermore, combined exposure to AZA1 and OA did not lead to enhance acute oral toxicity, compared to the toxic effects from exposure to the same toxins alone. Consequently, regulation of toxins, based on maximum tolerance levels for each group, seems to be valid (Aune et al., 2012). Additionally, studies on combined oral toxicity of AZA1 and yessotoxin in mice demonstrated that the oral toxicity of yessotoxin is not enhanced in the presence of sub-lethal levels of AZA1 (Aasen et al., 2011).

Symptoms and toxicological effects in humans Unlike many of the other well-described marine phycotoxins, relatively little is known about AZA. Similar to DSP toxins, human consumption of AZA-contaminated shellfish

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can result in severe acute symptoms that include nausea, vomiting, diarrhoea, and stomach cramps (Ito et al., 2000, 2002). The toxicity of the mussels was estimated to be 0.15 mouse units (MU)∕g (equivalent to 0.6 mg AZA∕g) (EU/SANCO, 2001). A higher toxin content of 1.4 mg AZAs∕g of meat (0.4 MU/g of meat) was reported by Ofuji et al. (1999b). Human toxicity was seen between 6.7 (5%) and 24.8 (95%) mg∕person, with a mean value of 15 mg∕person. However, McCarron et al. (2009) found that levels of AZA3 increased significantly during heating in the absence of water. The same team found that AZA3 also tended to increase in concentration in shellfish tissue during storage at temperatures as low as 4 ∘ C; nevertheless, the concentrations of AZA1 and AZA2 remained unchanged. Therefore, the recalculated range of the LOEL is 23–86 mg per person, with a mean value of 51.7 mg∕person (EU/SANCO, 2001). Due to the limited data available from many of the AZA events, nearly all information regarding AZA toxicology has been obtained from controlled in vitro and in vivo experiments. Many of these efforts have been directed towards assessing the risk of AZA consumption in contaminated shellfish and, in turn, identifying the molecular target(s) of AZA, which is currently unknown.

Ciguatera shellfish poisoning Ciguatera fish poisoning (CFP) is a food-borne disease endemic to tropical and subtropical coral reef regions of the world. Is the most common type of marine food poisoning worldwide, with an estimated 10 000 to 500 000 people suffering from the disease annually, constituting a global health problem (Fleming et al., 1998; Dickey & Plakas, 2010). It is contracted by consumption of finfish that have accumulated lipid-soluble toxins produced by microalgae (dinoflagellates) of the genus Gambierdiscus. CFP causes a clinical syndrome that comprises gastrointestinal, neurological and cardiovascular symptoms. Variation in symptoms patterns have been observed in the Pacific Ocean, Indian Ocean and Caribbean Sea regions, and they have been attributed to the different suites of ciguatoxins (CTX) identified from those regions (P-CTX, I-CTX, C-CTX, respectively) (Bagnis et al., 1979; Lawrence et al., 1980; DeFusco et al., 1993; Habermehl et al., 1994; Lewis, 2000). There are currently no reliable biomarkers that can be used to confirm exposure to CTX in humans. At present, therefore, CFP diagnosis is based on the presenting symptoms and time course, the history of having eaten a reef fish and, importantly, the exclusion of other diagnoses that could account for the symptoms (Friedman et al., 2008).

Mechanism of action CTX are one of the most potent natural toxins known, with an LD50 of 0.45 μg∕kg (mouse i.p. studies). This dose corresponds to only 2–5 g of flesh from a toxic fish. Symptoms of ciguatera were long recognized as indicative of central and peripheral nervous system injury. CTX cause and enhance spontaneous and evoked action potentials by lowering activation thresholds and delayed repolarization of voltage-gated sodium channels (Molgo et al., 1990; Cameron et al., 1991; Baden et al., 1995; Benoit et al., 1996; Nicholson & Lewis, 2006). Although the neurological symptoms observed in clinical cases of CFP are believed to be consistent with the direct interaction of ciguatoxins with voltage-gated sodium channels, Kumar-Roine et al. (2008) argued

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that the unique effects of ciguatoxins on voltage-gated sodium channels did not fully explain the spectrum of symptoms elicited by CTX. Moreover, other mechanisms, such as elevation of intracellular calcium levels (Lewis & Endean, 1986; Molgo et al., 1990), stimulation of spontaneous and evoked neurotransmitter release from synaptosomes and motor nerve terminals (Molgo et al., 1990; Brock et al., 1995; Hamblin et al., 1995), axonal and schwannn cell oedema (Allsop et al., 1986; Benoit et al., 1996; Mattei et al., 1999); induction of tetrodotoxin-sensitive leakage current in dorsal root ganglion neurons (Strachan et al., 1999) and blockade of voltage-gated potassium channels (Hidalgo et al., 2002; Birinyi-Strachan et al., 2005a, 2005b) were also reported. Recently, evidence of the implication of inducible nitric oxide synthase (iNOS) in CFP in an in vitro mammalian model was provided, opening a possible way for new therapies. On the contrary, Ryan et al. (2010) observed that many genes, differentially expressed in mice administered with CTX, figured prominently in protection against neuroinflammation. Therefore, anti-inflammatory processes were at work, creating a systemic anti-inflammatory environment to protect against the initial cellular damage caused by the toxin.

Cellular and sub-cellular effects One of the clinical signs characteristic of CFP is the reversal of temperature perception. Temperature sensations are generated in C-polymodal nociceptor fibres in skin and deep structures, and the intensity of these sensations, depends on the intensity of discharge in these fibres. Ciguatoxin can cause persistent sodium channel opening in nerve membrane, resulting in oscillations in membrane potentials and runs of spontaneous discharges. Studies on ciguatera victims, in which their hands were immersed in water baths ranging from 0 − 50 ∘ C, suggest that the paradoxical sensory discomfort experienced is, most likely, a result of an exaggerated and intense nerve depolarization occurring in peripheral small A-delta myelinated and, in particular, C-polymodal nociceptor fibres. CTX induces a rapid decrease in core body temperature that persists for several hours. A study employing c-fos transitional product, a biomarker for neuroexcitability, demonstrated that CTX caused neuroexcitatory actions on brain stem regions receiving vagal afferents and ascending pathways associated with visceral and thermoregulatory responses (Peng et al., 1995). Zhang et al. (2013) observed that intracerebroventricular administration of Pacific ciguatoxin 1 (P-CTX-1) in rats produced a dose-dependent increase in anterior cingulate cortex neuronal firings and medial thalamus-anterior cingulate cortex synaptic transmission, as well as activation of astrocytes at these levels. CTX-invoked brain cortex neuronal excitotoxicity in vivo was demonstrated, and a possible role of neuron and astroglia in acute ciguatera poisoning was suggested. Recently, the role of the nitric oxide radical (NO) in the swelling of frog red blood cells challenged with CTX was proved (Sauviat et al., 2006).

Symptoms and toxicological effects in animals In mice, the LD50 was calculated to be 87 mg∕kg and the range of CTX doses between 0% and 100% lethality was narrow (62.5–112.5 mg∕kg). Studies in mice showed an almost immediate and transient decrease in motor activity and temperature after i.p. injection with CTX, and subsequent long-lasting thermoregulatory dysfunction (Peng et al., 1995; Bottein Dechraoui et al., 2008; Ryan et al., 2010). Hypothermia is not a typical feature of human intoxications, but reports of temperature dysregulation are

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not unusual in CFP (Ryan et al., 2010). The hypothermic response and the reduced activity were enhanced with a second exposure, and the greater neurological response in the second exposition was associated with elevated concentrations of CTX in the blood, which was measurable in blood up to three days after the first exposure (Bottein Dechraoui et al., 2008). In addition to the neurotoxic effects, other symptoms commonly observed in mice were diarrhoea and cyanosis, whereas breathing difficulties and convulsions occurred more often at higher doses (Hoffman et al., 1983). Continuous erection of the penis was also observed in a reduced percentage of mice (Terao et al., 1991; Ito et al., 2003). By light and electron microscopy, marked swelling and focal necrosis of cardiac muscle were observed, as well as degeneration of cells in the medulla of the adrenal glands. Although severe diarrhoea was brought about by the administration of these phycotoxins, no morphological alterations were seen in the mucosa and muscle layers of the small intestine except in autonomic nerve fibres and synapses (Terao et al., 1991, 1992). Cardiovascular effects were observed after repeated i.p. and oral administrations of CTX in mice, resulting in marked swelling of cardiac cells and endothelial lining cells of blood capillaries in the heart. This swelling caused narrowing of the lumen and accumulation of blood platelets in capillaries, which resulted in multiple single-cell necroses of cardiomyocites, followed by diffuse interstitial fibrosis and bilateral ventricular hypertrophy. Strikingly, single doses of the toxin caused no discernible pathological changes. Gambierol, isolated from Gambierdiscus toxicus and causing CFP, was observed to produce injuries in lung tissue, and secondary injuries in heart tissue, resulting in systemic congestion. Another toxic effect was seen in the stomach, inducing hypersecretion and ulceration. In this study, the authors suggested that the death of gambierol-treated mice was mainly due to dyspnoea (Ito et al., 2003). Furthermore, CTXs produced interesting effects in other species. Embryos of medaka (Oryzias latipis), microinjected with 0.1-0.9 pg∕egg (ppb) of CTX into the egg yolk, exhibited the adverse effects of ciguatoxin on fish embryos. Embryos microinjected with 0.1-0.9 pg∕egg (ppb) of ciguatoxin exhibited cardiovascular, muscular and skeletal abnormalities, and those injected with higher levels (1.0 − 9.0 pg∕egg) exhibited significantly reduced hatching success (Edmunds et al., 1999). Studies in chicken revealed that this species is two to five time at least more sensitive to CTX than mouse, pointing out to its convenience for laboratory studies in ciguatera research. In chicken, administration of CTX gave rise to hypersalivation, acute motor ataxia, low rectal temperature and arrested growth (Vernoux & Lahlou, 1986).

Symptoms and toxicological effects in humans In CFP, the onset of the first symptoms can be as short as 30 minutes for severe intoxications while, in milder cases, onset may be delayed for up to 24–48 hours after consumption of fish. CFP symptoms typically last for some weeks to several months. In a small percentage of cases, certain symptoms may persist for a number of years, although such protracted complaints should be studied further to address potentially confounding psychiatric and medical explanations for them (Lewis, 2001; Ting & Brown, 2001; Achaibar, 2007; Friedman et al., 2008; Boada et al., 2010; Kumar-Roiné et al., 2011). On the other hand, the severity, number and duration of CFP symptoms reflect a combined influence of dose, toxin profile and individual susceptibility (Lewis, 2001). Neurological symptoms predominate in the Pacific Ocean while, in the Caribbean Sea, gastrointestinal symptoms are a dominant feature of the disease. In the Indian Ocean,

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a cluster of symptoms reminiscent of hallucinatory poisoning was reported (Quod & Turquet, 1996; Lewis, 2001). Pharmacokinetic and subtle pharmacological differences are likely to be involved in the different pattern of symptoms of CFP (Lewis, 2001). The typical clinical features of CFP include acute gastrointestinal symptoms (nausea, vomiting, diarrhoea and abdominal pain) within 0.5–12 hours of contaminated fish ingestion (Hokama, 1988; Dickey & Plakas, 2010). This is followed within 12–72 hours by several neurologic symptoms, comprising circumoral and limb paresthaesia and dysesthaesia, including pathognomonic paradoxical apparent temperature sensation reversal (where hot objects feel cold and cold objects feel hot). During this stage, musculoskeletal features such as myalgia, arthralgia, cramps and weakness, as well as pruritus, sweating and dental pain, may be present (Cameron & Capra, 1993; Chan & Kwok, 2001; Friedman et al., 2008; Boada et al., 2010; Dickey & Plakas, 2010). Neuropsychiatric symptoms such as anxiety, depression, memory loss, hallucinations, giddiness, ataxia and coma, have been reported, frequently associated to CFP in Indian and Pacific Ocean regions (Quod & Turquet, 1996; Friedman et al., 2007). In severe cases, cardiovascular disorders have been signalled, occurring generally only in the early stage of the disease process. Ciguatoxins strongly altered the morphology of cardiac tissue inducing swelling of the cells and alterations of cellular organelles. These toxins impair the conduction of cardiac nerves and increase the opening probability of Na+ channels in intracardiac ganglions. Depending on the concentration applied, the substances exerted either a fast positive inotropic effect or a negative inotropic effect on the contraction of mammalian atrial and ventricular cardiac muscle. These effects were attributed to a release of noradrenaline and acetylcholine from neural terminals of the autonomic nervous system present in cardiac tissue (Marquais & Sauviat, 1999).The cardiac signs may include hypotension and bradycardia, and may necessitate urgent medical care (Friedman et al., 2008). Hematologic or biochemical abnormalities were rarely reported in the literature (Chan & Kwok, 2001; Perez-Arellano et al., 2005). CFP is rarely fatal. However, death may occur in severe cases due to severe dehydration, cardiovascular shock during the initial illness period, or respiratory failure resulting from paralysis of the respiratory musculature, especially in areas where ventilatory support and emergency medical care are unavailable (Friedman. et al., 2008). The low fatality rate appears to arise because fish rarely accumulate sufficient levels of CTX to be lethal at a single meal, perhaps because fish succumb to the lethal effects of higher CTX levels (Lewis, 1992). There are reports of sensitization to ciguatoxins in CFP patients; that is, individuals who previously suffered from CFP have been reported to experience recurrence of CFP symptoms after eating a potentially ciguateric fish that did not produce symptoms in other individuals (Gillespie et al., 1986; Ruff & Lewis, 1994). Moreover, anecdotal reports indicate that some patients experience recurrence of neurological CFP symptoms upon consuming alcohol, any type of fish, and certain other foods (fish, peanuts, chicken and pork), even years after the initial exposure (Glaziou & Martin, 1993; Ting & Brown, 2001; Lewis, 2006). Such reports of recurrence have not been noted for cardiac or gastrointestinal symptoms. One theory to explain the recurrence of neurologic symptoms is that ingested CTX may be stored in the adipose tissue, and that any activity involving increased lipid metabolism may result in CTX re-entering the bloodstream, with subsequent re-emergence of CFP symptoms (Nicholson & Lewis, 2006). Alternatively, symptom

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recurrence may be related to immunologically mediated sensitization to CTX after initial exposure (Ting & Brown, 2001; Lewis, 2006).

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Wang, J., Wang, Y.Y., Lin, L. et al. (2012) Quantitative proteomic analysis of okadaic acid treated mouse small intestines reveals differentially expressed proteins involved in diarrhetic shellfish poisoning. Journal of Proteomics, 75, 2038–2052. Wang, J.X., Salata, J.J. and Bennett, P.B. (2003) Saxitoxin is a gating modifier of HERG K1 Channels. Journal of General Physiology, 121, 583–598. Watkins, S.M., Reich, A., Fleming, L.E. and Hammond, R. (2008) Neurotoxic shellfish poisoning. Marine Drugs, 6, 431–55. White, A.W. (1981) Sensitivity of marine fishes to toxins from the red-tide dinoflagellate Gonyaulax excavata and implications for fish kills. Marine Biology, 65, 255–260. Whitmarsh, A.J. and Davis, R.J. (1998) Structural organisation of MAP-kinase signalling modules by scaffold proteins in yeast and mammals. Trends in Biochemical Science, 12, 481–485. Work, T.M., Barr, B., Beale, A. et al. (1993) Epidemiology of domoic acid poisoning in brown pelicans (Pelecanus occidentalis) and Brandt’s cormorants (Phalacrocorax penicillatus) in California. Journal of Zoo and Wildlife Medicine, 24, 54–62. Wright, J.L., Boyd, R.K., De Freitas, A.S.W. et al. (1989) Identification of domoic acid, a neuroexcitatory amino acid, in toxic mussels from eastern Prince Edward Island. Canadian Journal of Chemistry, 67, 481–490. Wu, C.H. and Narahashi, T. (1988) Mechanism of action of novel marine neurotoxins in ion channels. Annual Review of Pharmacology and Toxicology, 28, 141–161. Xi, D., Peng, Y.G. and Ramsdell, J.S. (1997) Domoic acid is a potent neurotoxin to neonatal rats. Natural Toxins, 5, 74–79. Yan, X., Benson, J.M., Gomez, A.P. et al. (2006). Brevetoxin-induced neural insult in the retrosplenial cortex of mouse brain. Inhalation Toxicology 18, 1109–1116. Yan, Y., Shay, J.W., Wright, W.E. and Mumby, M.C. (1997) Inhibition of protein phosphatase activity induces p53-dependent apoptosis in the absence of p53 transactivation. Journal of Biological Chemistry, 272, 15220–15226. Yang, C., Kao, C.Y. and Oshima, Y. (1992) Actions of decarbamoylosaxitoxin and decarbamoylneosaxitoxin on the frog skeletal muscle fiber. Toxicon, 30, 645–652. Yasumoto, T. and Murata, M. (1993) Marine toxins. Chemical Reviews, 93, 1897–1909. Yasumoto, T., Oshima, Y. and Yamaguchi, M. (1978) Occurrence of a new type of shellfish poisoning in Tohoku district. Bulletin of the Japanese Society of Scientific Fisheries, 44, 1249–1255. Yasumoto, T., Murata, M., Oshima, Y. et al. (1984) Diarrhetic. xxxxshellfish poisoning. In: Ragelis, E.P. (ed.). Seafood Toxins, ACS Symposium Series 262, pp. 207–214. American Chemical Society, Washington D.C. Yasumoto, T., Murata, M., Oshima, Y. et al. (1985) Diarrhetic shellfish toxins. Tetrahedron, 41, 1019–1025. Yasumoto, T., Igarashi, T. and Satake, M. (2001) Chemistry of phycotoxins-Structural elucidation, in Mycotoxins and Phycotoxins in Perspective at the Turn of the Millennium. Proceedings of the X International IUPAC Symposium on Mycotoxins and Phycotoxins (eds W.J. De Koe, R.A. Samson, H.P. Van Egmond et al.), Ponsen and Looyen, Wageningen, the Netherlands, pp. 465–474. Zabka, T.S., Goldstein, T., Cross, C. et al. (2009) Characterization of a degenerative cardiomyopathy associated with domoic acid toxicity in California sea lions (Zalophus californianus). Veterinary Pathology, 46, 105–119. Zatorre, R.J. (1990). Memory loss following domoic acid intoxication from ingestion of toxic mussels. Canada Diseases Weekly Report 16, 101–103. Zhang, X., Cao, B., Wang, J. et al. (2013) Neurotoxicity and reactive astrogliosis in the anterior cingulate cortex in acute ciguatera poisoning. NeuroMolecular Medicine, 15, 310–323.

Index

A Abramis brama, see Bream; Common bream Abudefduf saxatilis, see Sergeant major Acanthaster, 116 Acanthochromis polycanthis, see Damselfish Acanthopagrus schlegelii schlegelii, see Japanese black porgy Acanthostracion quadricornis, see Scrawled cowfish Acanthurus xanthopterus, see File fish Acetylcholine, 37, 41, 47, 156–159, 188, 278, 330, 348, 350–352, 354, 431, 465, 496 binding protein, 53, 350–352 esterase, 157–159, 170, 407 receptor, 52, 53, 66, 156–159, 170, 184, 188, 302, 304, 344, 348, 349, 353 Achirus lineatus, see Lined sole Acinetobacter, 87 Acipenser baeri, see Sturgeon hybrid gueldenstaedtii, see Sturgeon hybrid Aconitine, 29, 31 Adda (3-amino-9-methoxy-2-6,8-trymethyl-10phenyldeca-4,6-dienoic acid), 230, 231, 397–399 Aeromonas, 87 Alburnus alburnus, see Bleak Alcohol, 55, 57, 59, 60, 63, 94, 140, 148, 151, 152, 154, 173, 280, 383, 395, 496 Alexandrium, 395, 427, 428 andersonii, 427 catenella, 426–428, 467 excavatum, 427, 428 fundyense, 427 hiranoi, 394 leei, 427 lusitanicum, 427 minutum, 427, 428 ostenfeldii, 13, 188, 343, 394, 427 peruvianum, 188, 343, 344, 427 tamarense, 79, 295, 426–428, 436 tamarense var. excavate, 427 tamiyavanichii, 427 Alosa mediocris, see Hickory shad Alteromonas, 14, 295, 368 Aluterus schoepfii, see Orange filefish Ambigol, 431 Ambiguine, 275–276 Ameiurus melas, see Black bullhead American eel, 430 American harvestfish, 412

American Pollock, 428 Aminoalcohol, 140 Aminotransferase, 395, 399 Amnesic, 2, 213, 433, 475, 482 Amphibalanus, 120 Amphidinium, 436 carterae, 431, 433 klebsii, 431 Amphidinolide, 393 Amphiprion percula, see Orange clownfish Anabaena, 227, 233, 256, 269, 397 arnoldii, 417 bergii, 228, 317, 408 catenula, 417 circinalis, 247, 324, 395, 408, 427, 464 farciminiformis, 408 flos-aquae, 156, 226, 407, 408, 417, 427 lapponica, 228, 317 lemmermannii, 408, 464 macrospora, 408 mendotae, 408 oryzae, 276, 281 planctonica, 317, 408 spiroides, 408 spp., 188, 226, 246, 408, 417 variabilis, 408 Anacystis marina, 438 Analysis cytotoxicity, 126 ELISA, 104, 127, 233, 330 fungal strain, 369 gaps, 1, 6–10 histological, 120 LC-MS/MS, 7 market, 266 mass spectrometry anatoxin, 160–165 azaspiracid, 1, 2 ciguatoxin, 35, 39 cyanotoxins, 324 cylindrospermopsin, 328, 331, 332 domoic acid, 476 microcystin, 160, 163, 164 okadaic acid, 10, 308, palytoxin, 91, 104, 105, 121 ostreocin, 94 ovatoxin, 99, 100 pectenotoxin, 6, 10 pinnatoxin, 52

Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

513

514

Index

Analysis (continued) PSP, 69–80, 395, 396 spirolides, 13 yessotoxin, 6 molecular scaffolds, 181 mussel homogenate, 14 NMR, 50, 88 Neuropathological, 482 patch-clamp, 34 phylogenetic, 87 scope, 3 seafood, 6 shellfish, 475 Analytical instrumentation, 70, 71, Anatoxin-a, 137–170, 188, 225–227, 233, 407–410 Anchoa mitchilli, see Bay anchovy Angelichthys ciliaris, see Blue angelfish Anguilla anguilla, see European eel japonica, see Japanese eel rostrata, see American eel Ankaraholide A, 279 Anthracimycin, 368 Antibacterial, 188, 275, 276, 367, 368 Anticancer, 182, 260, 279, 281, 297, 299, 306, 308, 363, 364 Antillatoxin, 279, 431 Aphanizomenon, 137, 319, 396, 408, 417, 427 flos-aquae, 228, 246, 255, 259, 261, 271, 317, 325, 407, 408, 427, 428, 432, 436, 464 gracile, 247, 317, 432, 464 issatschenkoi, 166, 226, 464 ovalisporum, 228, 317, 318, 432 Aplidin, 184, 304, 365–366 Apoptosis, 158, 203–207, 215–217 azaspiracid, 209–211, 489 brevetoxin, 212, 213, 483, 484 cylindrospermopsin, 327 domoic acid, 213, 214, 476, 477 fucoxanthin, 269 maitotoxin, 214, 215 microcystin, 420, 421 monomethyl auristatin E (MMAE), 306 nodularin, 436 okadaic acid, 206–208, 433, 468–470 ophiobolins, 373 palytoxin, 123, 124, 214 pectenetoxin, 212 plinabulin, 370 plitidepsin, 304 salinosporamide A, 363 yessotoxin, 191, 208–210 Apratoxin, 191, 193, 279 Archosargus probatocephalus, see Sheepshead Argentinian silverside, 418 Ariopsis felis, 413 (see also Hardhead catfish; Sea catfish) Aristichthys nobilis, see Bighead carp Artemia, 121, 329 Arthrospira, 137 fusiformis, 417 maxima, 255, 271, 272 platensis, 265, 266, 275

Asian swamp eel, 428 Aspergillus, 370, 372, 373 Astaxanthin, 256, 259–261, 263, 267–270, 282 Astroscopus y-graecum, see Southern stargazer Atlantic bumper, 412 Atlantic croaker, 430 Atlantic herring, 428 Atlantic mackerel, 412, 428, 465 Atlantic menhaden, 413, 416, 430 Atlantic midshipman, 411 Atlantic needlefish, 412 Atlantic salmon, 413, 417, 418, 425, 428, 433, 449, 450 Atlantic spadefish, 412 Atlantic thread herring, 411, 412 Aureococcus anophageferrens, 282 Aurilides, 279 Auristatin, 184–185, 299, 301–306 Autophagy, 191, 203–205, 208, 209, 215–217 Azadinium, 2, 210, 487 Azaspiracid, 206, 209–211, 391, 392, 431, 471, 487, 490, 492 Azaspiro, 372 B β-carotene, 255, 256, 259–263, 266–270, 280, 283, 332 β-ketosulphone, 152 β-methylamino-L-alanine (BMAA), 164, 166, 169, 226–228, 234 Bacillus, 86, 275, 368 Back crappie, 409, 430 Bagre marinus, see Gafftopsail catfish Bairdiella chrysoura, see Silver perch Balistes capriscus, see Turbot Balistes vetula, see Queen triggerfish Balloonfish, 411, 428 Banded grouper, 428 Bank cusk-eel, 411 Barbel steed, 428 Bascanichthys scuticaris, see Sooty eel Bathygobius fuscus, see Dusky frillgoby Batrachotoxin, 31, 37 Bay anchovy, 411, 416 Belica, 418, 423 Belted sandfish, 412 Bermuda chub, 412 Bighead carp, 418–420, 425 Bigmouth buffalo, 409 Biosynthesis, 192, 194, 195, 247, 265, 282, 295, 319–321, 330, 367, 374, 381, 382, 384, 389, 391, 393, 395, 399–401 Bisebromoamide, 279 Biselyngbyaside, 279 Bis-salicylate, 367 Bis-spiroacetal, 352, 356 Bis-spiroketal, 344 Black bullhead, 409, 425 Blackcheek tonguefish, 412 Black drum, 412, 437 Black goby, 416 Black grouper, 412, 430

Index

Black sea bass, 411, 414 Bleak, 425 Blennius gattorugine, see Blenny Blenny, 416 Blicca bjoerkna, see Silver bream Bloom Alexandrium tamarense, 79 algae, 1, 13, 125, 129, 239, 240, 244, 256, 324, 325, 407, 412, 463, 465 Aphanizomenon, 408 Chatonella marina, 413 cyanobacterial, 115, 170, 228, 232–234, 244, 246, 318, 324, 333 Cylindrospermopsis, 322 dinoflagellates, 12, 437 Euglena sanguinea, 443 Heterosigma akashiwo, 414 Mycrocistis, 419 natural, 126, 423, 435 Nodularia spinifera, 231, 398 Ostreopsis, 87, 97, 98, 102, 115, 119–121 Planktothrix, 419 Prorocentrum minimum, 432 red tide, 485, 487 toxic, 245, 421, 436 Blue angelfish, 412 Blue catfish, 425 Bluegill sunfish, 409, 430 Blue tilapia, 430, 433 Bluntnose minnow, 409 Bond, 152, 299, 391 C-C, 102, 146 disulphide, 270 double, 25, 49, 56, 58, 59, 88, 100, 113, 141, 170, 231, 345, 346, 389, 391, 394, 400 hydrogen, 350, 352 N-O, 150, 151 Bortezomib, 363 Botryococcus braunii, 273, 281, 282 Bream, 416, 418, 425 Brentuximab, 183–184, 299, 301 Brevetoxin, 2, 23, 31, 32, 38, 40, 127, 190, 206, 212, 384, 385, 387, 410, 411, 414, 482–487 Brevibacterium, 87 Brevoortia patronus, see Gulf menhaden tyrannus, see Atlantic menhaden Brown trout, 409, 418–420 Buffalo, 425 Bungarotoxin, 53, 188 Burbot, 425 Butanoic acid, 276, 277 C Cabrilla sea bass, 414 Calamus, see Porgy Calcium, 97, 100, 117, 191, 208, 211, 214, 256, 279, 280, 488 channel, 37, 38, 40, 52, 117, 187, 299, 353, 465, 489 influx, 33, 215, 488, 489 levels, 39, 88, 475, 488, 494 mobilization, 38

515

pore, 206 response, 350, 353 spirulan, 275, 279 Calothrix, 277, 279 Calothrixins, 277, 279 Calpain, 216 Cancer, 123, 124, 170, 184, 185, 187, 191, 216, 263, 264, 267, 268, 298, 300–308, 361, 363, 364, 366, 370, 469, 472, 488 Candida, 275 Candidatus, 305 Canthaxanthin, 261, 267–270 Capelin, 428 Caranx crysos, see Hardtail hippos, see Crefalle jack Carassius auratus, see Goldfish auratus gibelio, see Gibel carp carassius, see Crucian carp Carbamate, 69, 72–74, 145, 151, 152, 157, 395, 396, 464 Carbamidocyclophane, 276 Carmabin, 277, 279 Carp, 409, 416, 418, 421–426, 428 Carpoides carpio, see Carp sucker Carp sucker, 425 Carteraol, 431 Caspase, 121, 203, 205, 207, 209, 211, 214–217, 304, 489 caspase-1, 205 caspase-2, 204, 209 caspase-3, 204, 206–213, 479, 489 caspase-6, 204 caspase-7, 204, 205, 209, 489 caspase-8, 204, 206, 210, 212, 215 caspase-9, 204, 207–212 caspase-10, 206 Catalyst, 65, 148, 383 Grubbs, 59, 147, 152, 154, 155 palladium, 140, 144 Catfish, 416, 420, 426 Catostomus commersonnii, see White sucker Caylobolide, 279 C1-C4, 71, 73, 75, 78, 79, 108, 248, 464 Cell adaptability, 245 adhesion, 27, 191, 468, 488 aerosol, 98 algal, 410, 435 alteration, 126 assay, 123 atrial, 37 β-Islet pancreatic, 274 behavior, 124 blood, 126, 306, 494 bloom, 436 C3A, 330 Caco-2, 118, 122, 127, 207, 214, 330 calcium, 117, 190 cancer, 269, 298, 305, 364, 489 cardiac, 495 cerebellar granule (CGC), 34, 35, 41

516

Index

Cell (continued) cervical adenocarcinoma, 327 CHO, 351 clone 9, 330 cyanobacterial, 319, 324, 333, 423 cycle, 305, 308, 370, 420 Cylindroespermopsis raciborskii, 432 contact, 158 culture, 213, 273, 407, 408, 437 damage, 214, 273 death, 122, 123, 157, 191, 204–206, 209, 212, 214–217, 308, 328, 409, 424, 469, 483 depolarization, 26, 33, 37, 121 detachment, 208 differentiation, 489 dinoflagellate, 426 division, 309, 317 dried, 256 endothelial, 495 epithelial, 191, 207, 211, 213, 354, 364, 427, 470, 488, 490 excitable, 15, 26, 117, 464 extract, 325, 407, 422, 432 eukaryotic, 117, 207 fibroblasts, 275, 420 functioning, 269 Gambierdiscus toxicus, 414 gill chloride, 422, 427, 433, 434 glioma, 126, 191, 210, 217 glioma hybrid, 38 growth, 100, 263, 319 HaCat, 217 HEK, 34, 40 HeLa, 207, 209, 273 hematopoietic progenitor, 305 hepatoma, 191, 208 hepatic, 329 HepG2, 330 hippocampus, 214, 479 HL60, 207, 372 human, 191, 212, 270 human breast cancer, 127, 191, 211 human hepatic stellate, 275 human neuroblastoma SH-SY5Y, 34–36 human T, 305, 370, 488 immune, 121, 483 inflammatory, 492 ingestion, 410 intestinal, 118, 124, 420, 469 Jurkat, 212, 488 K-562 leukaemia, 207, 209, 210 Karlodinium corsicum, 415 kidney, 330, 488, 489 leukaemia, 212, 305, 353 L6, 209 lamina propria, 491 line, 207, 209, 211–213, 279, 304, 305, 327, 328, 330, 418, 419, 421, 488 liver, 208, 421, 428, 429, 438 lymphocytes, 209 lymphoblastoid, 327 lymphoma, 299

lyophilized, 431 lysis, 121, 122, 319, 410, 415, 419, 423, 424, 427 lung, 212, 275, 305, 306, 370, 485, 488 macrophages, 485 MCF-7, 488 mammalian, 436 membrane, 26, 280, 483 microalgae, 115, 257, 269, 275, 278 Microcystis spp. 419–423 metabolism, 157, 274 model, 33, 34, 122, 123, 191, 213, 327, 353, 354 monolayer, 118 morphology, 42 motility, 158, mouse, 34, 273 murine leukaemia, 52, 364, 366 murine monocyte, 351 muscle, 37, 70, 117, 156, 246 mussel, 121 myoblast, 216 myocardial, 474 natural killer, 278 network, 43 Neuro-2a, 126 neuroblastoma, 34, 37, 41, 42, 97, 126, 127, 211, 353, 488, 489 neuroendocrine, 30 neuronal, 30, 489 nerve, 246, 307 NG108-15, 126 non-excitable, 122, 190 Ostreopsis, 97, 102, 119 organization, 257, 275 P388, 97 permeability, 426 plasma, 484 programmed death, 122, 191, 203, 215 (see also Apoptosis) proliferation, 157, 158, 190, 191, 207, 208, 269, 279, 409, 488 Prorocentrum spp., 432 Purkinje, 30, 474 pyramidal, 477, 479, 482 rat 3Y1, 126 Schwann, 30, 33, 494 SHE, 327 single, 318, 423 size, 257 smooth muscle, 37, 122 solid, 429 spinal neurons primarily, 30 squamous, 472 stress, 122, 416 structure, 257 surface, 244 swelling, 415, 427, 474, 496 target, 26, 121, 187, 188, 190, 274 toxic, 410 toxicity, 483 topminnow PLHC-1, 418, 420 type, 34, 157, 191, 192, 206, 208, 209, 214, 329 tumour, 193, 304, 306

Index

viability, 122, 123, 126, 207, 213, 214, 328, 488 viable, 2 volume, 190, 212 wall, 257, 273, 319, 326 Centropomus undecimalis, see Common snook Centropristis striata, see Black sea bass Cephalimysin, 371 Cephalosporin, 369, 396 Cephalosporium, 373 Ceratium furca, 438 Chaetodipterus faber, see Atlantic spadefish Chaetodon, 116, 120 Chaetomorpha minima, 433, 434 Channel calcium, 37, 38, 40, 52, 117, 187, 211, 299, 353, 465, 488, 489 inactivation, 27–31, 34, 37–42, 44, 189 potassium, 34, 35, 39, 41, 187, 190, 211, 489, 494 sodium, 15, 26–44, 70, 114, 184–189, 212, 246, 294, 304, 353, 414, 464–466, 483, 489, 493, 494 voltage-gated, 26, 32–36, 39–44, 117, 187–189, 212, 294, 410, 431, 437, 464, 483, 493, 494 Channel catfish, 409, 418, 425, 430, 433, 438 Chanodichthys erythropterus, see Predatory carp Charonia, 14 Chattonella, 437, 482 marina, 413, 434 verruculosa, 413 Checkered puffer, 412 Chilomycterus schoepfii, see Striped burrfish Chinook salmon, 413, 419 Chlamydomonas reinhardtii, 276, 277, 281–282 Chlorella, 259, 272, 281 autotrophica, 276 minutisima, 261, 264 protothecoides, 259, 261, 268 pyrenoidoisa, 272, 280 vulgaris, 255, 261, 272, 274–282 zofingiensis, 261, 268 Chlorellin, 276 Chlorophyte, 434 Chloroscombrus chrysurus, see Atlantic bumper Chlrorellin, 276 Cholesterol, 264, 274, 279–281, 299, 420 Chondria, 86, 481 Chondroitin, 256 Chroococcales, 245 Chrysochromulina sp., 424, 483 Chrysophyte, 434 Cichlasoma cyanoguttatum, see Cichlid Cichlid, 425 Ciguatoxin, 1, 2, 23–27, 29, 31–44, 206, 384, 386, 414, 436, 493–496 Cirrhinus molitorella, 425 Cleavage, 100–105, 114, 139, 141, 146, 150–155, 204, 208, 209, 213, 216, 278, 306, 384 Clupea harengus, see Atlantic herring Cobia, 412 Cocoa damselfish, 412 Cocosamides, 279

517

Cod, 415, 416, 428, 438 Coelastrella sp., 261, 265, 268, 281 Coenzyme Q10, 256, 262, 270, 271, 381 Coho salmon, 438 Coibamide, 279 Common bream, 425 Common halfbeak, 412 Common snook, 411, 413, 437 Conotoxin, 29, 32, 41, 187–190, 307 Conus, 187, 298, 307 Coregonus lavaretus, see Whitefish Corethron criophilum, 438 Corydoras paleatus, see Peppered cory Crambescidines, 193 Crappie, 425 Crassotrea, 116 Crefalle jack, 411, 412 Crimson spotted rainbowfish, 419 Crossbyanol, 276 Crucian carp, 425, 428 Cryptotethia, 298 Ctenolabrus rupestris, see Wrasse Ctenopharyngodon idellus, see Grass carp Cuspidothrix issatschenkoi, 408 Cyanidioschyzon merolae, 282 Cyanobium bacillare, 417 Cyanotoxin, 157–158, 163, 225–226, 230–234, 245, 318–319, 324–333, 397 Cyclic imine, 2, 49, 50, 52, 53, 56, 60, 62, 64, 66, 188, 206, 343–356, 394 Cylindrospermopsin, 158, 163, 164, 169, 226, 228, 317–333, 399, 400, 432 Cylindrospermopsis catemaco, 432 philippinensis, 432 raciborskii, 226, 247, 317, 324, 395, 400, 427, 432 Cynoscion arenarius, see Spotted sea trout nebulosus, see Spotted sea trout Cyprinodon variegatus, see Sheepshead minnow; Variegated minnow Cyprinus carpio, see Carp Cytarabine, 182–184, 293, 298, 300–310 Cytotoxin, 191, 226, 245, 319, 326 D Damselfish, 412, 428 Danio rerio, see Zebra fish Daphnia, 329 dcGTX, 69, 71, 73–76, 78, 168, 169, 248, 464 dcNeo, 69, 73, 75–77, 169, 248, 464 dcSTX, 69, 71–76, 78, 168, 169, 248, 464 Decarbamoyl, 69, 71, 72, 74, 79, 169, 395, 396, 426, 464 Demania, 116 Dermatotoxin, 226, 245, 319, 326 2,4-Diaminobutyric (DABA), 164, 166, 169 Diastereomers, 53, 60, 61, 91, 145, 153 (see also Stereoisomers) Diazepinomicin, 363–364 Dicentrarchus labrax, see European sea bass Didemnin, 304, 364–366

518

Index

Dinophysis, 12, 13, 212, 468 fortii, 395 acuminata, 432 Dinophysistoxin, 2, 10, 164, 206, 389, 390, 400, 468 Diodon holocanthus, 411, 428 Diplectrum formosum, 413 (see also Sand perch) DL-2,4-diaminobutyric acid (AABA), 169 Dogfish, 416 Dolastatin, 279, 299, 305–306, 366 Dolichospermum, circinale, 408, 417 farciminiforme, 417 Domoic acid, 2, 127, 206, 213, 433, 475–482 Dorosoma, see Shad cepedianum, see Gizzard shad petenense, see Threadfin shad Dragomabin, 277–279 Dragonamide, 277–279 Dorosoma, see Shad cepedianum, see Gizzard shad petenense, see Threadfin shad Dragomabin, 277–279 Drechslera, 373 Dunaliella, 259 bardawil, 272 primolecta, 276, 281 salina, 255, 259–261, 263, 264, 267, 268, 281, 282 tertiolecta, 283 Dusky frillgoby, 428 E Eastern mosquitofish, 430 Echeneis naucrates, see Sharksucker Ecteinascidia, 299, 305 Ecteinascidin, 304, 361–362 Eicosapentaenoic acid (EPA), 256, 263, 276 Elops saurus, see Ladyfish Emericellamides, 194 Emericella variecolor, 373 Engraulis japonica, see Japanese anchovy epiGTX8, 248 Epinephelus amblycephalus, see Banded grouper itajara, see Goliath grouper morio, see Sand perch Ergothioneine, 255, 261, 271–272 Eribulin, 183–184, 293, 298, 300, 306–310 Esox lucius, see Northern pike Etheostoma graham, see Rio Grande darter Eucinostomus gula, see Silver jenny Euglena sanguinea, 433 viridis, 276, 281 Euglenophycin, 433 Eurasian ruffe, 418, 425 European chub, 418 European eel, 425 European perch, 418, 425, 436 European sea bass, 121, 415, 416, 432, 434 European smelt, 418 Eustigmatos cf. polyphem, 262, 268 Exotoxin, 429

F Fathead minnow, 409, 425, 434 Fatty acids, 184, 192, 255, 259–266, 274–276, 280, 281, 299, 301, 384, 433–435 File fish, 438 Fischerella, 275, 276, 417 ambigua, 431 Flathead catfish, 425 Flathead mullet, 372, 411, 412, 425, 427, 428, 430 Florida pompano, 411 Floridichthys carpio, see Goldspotted killifish Flucoxanthin, 259, 262, 268–270, 282 Fragilariopsis cylindrus, 282 French angelfish, 412 Fucoxanthin, 259, 262, 268–270, 282 Fundulus grandis, see Gulf killifish; Killifish heteroclitus, see Killifish similis, see Longnose killifish Fusarium, 370 G Gadus morhua, see Cod pollachius, see Pollack Gafftopsail catfish, 412 Galdieria sulphuraria, 282 Gallinamide, 277, 279 Gambierdiscus, 15, 26, 214, 493 excentricus, 414 polyniensis, 414 toxicus, 23, 24, 190, 384, 414, 436, 495 Gambierol, 2, 190, 191, 193, 194, 206, 495 Gambusia affinis, see Mosquito fish holbrooki, see Eastern mosquitofish Gamma-aminobutyric acid (GABA), 36 Geitlerinema, 230, 279 Gibbula, 14 Gibel carp, 419 Gill, 246, 273, 408, 410, 415, 422, 426, 427, 433, 435, 437, 438, 465 Gilthead sea bream, 438 Gizzard shad, 409, 416, 425, 438 Glembatumumab, 302, 305 Glenodimin, see Tetrodotoxin Gloeotrichia echinulata, 417 Glucosamin, 256 Glutamate, 157, 171, 189, 213, 256, 354, 433, 466, 475, 476, 483 Gobiosoma bosc, see Naked goby Gobius, see Goby niger, see Black goby virescens, see Round goby Goby, 416 Golden shiner, 409 Goldfish, 408, 409, 418–423, 428, 431, 435 Goldspotted killifish, 411 Goliath grouper, 412 Goniodomine, 393 Gonyaulax monilata, 427 spinifera, 208, 386

Index

Gonyautoxin, 2, 69, 71, 80, 169, 247, 426, 464 Grass carp, 418, 425 Grayanotoxin, 31 Gray snapper, 411 Greenback flounder, 427, 428 Green sunfish, 438 Grunt, 412 GTX, 69, 71, 73–79, 168, 169, 248, 396, 397, 464 Guineamides, 279 Gulf flounder, 411 Gulf killifish, 418 Gulf kingfish, 412 Gulf menhaden, 411, 412 Gulf toadfish, 411 Guppy, 409, 411, 428, 430, 437 Gymnocephalus cernua, see Eurasian ruffe Gymnocin, 435 Gymnodimines, 49, 51, 206, 343, 348 Gymnodinium, 395 breve, 23, 410, 482 catenatum, 69, 427, 464 nagasakiense, 434 Gymnothorax javanicus, 24, 36, 37 vicinus, see Purplemouth moray Gymnothorax moringa, see Spotted moray eel H Haematococcus pluvialis, 255, 259–261, 263, 267, 268, 276, 277, 281, 282 Haemulon aurolineatum, see Tomtate parra, see Sailor’s choice plumierii, see Grunt sciurus, see Bluestriped grunt Halaven, see Eribulin Halfbeak, 411 Halichondria, 298, 306, 361 Halichondrin, 308, 361–362 Halimide, 369 Halorosellinia oceanica, 373 Halorosellinic acid, 373 Hapalosiphon hibernicus, 417 Haptophyte, 434, 438 Hardhead catfish, 411, 418, 437 Hardtail, 412 Harengula jaguana, see Scaled sardine Harmful algal blooms, see Bloom algal Hectochlorin, 277, 279 Hemibarbus labeo, see Barbel steed Hemiramphus, see Halfbeak Herklotsichtys, 116 Hermitamide, 435 Heteropneustes fossilis, see Stinging catfish Heterosigma akashiwo, 413, 414 Hickory shad, 416 High-hat, 412 Hog choker, 412, 430 Holacanthus ciliaris, see Queen angelfish Homoanatoxin-a, 138, 160–162, 169, 225–227, 233, 408, 410 Hormothammin, 435

519

Horse mackerel, 438 HPLC, 4, 7, 13, 71–75, 77–79, 98, 128, 166, 168, 233, 247, 331, Hydrocoleum lyngbyaceum, 160, 168 Hypophthalmichthys molitrix, see Silver carp Hyporhamphus unifasciatus, see Common halfbeak I Ichthyotoxicity, 410, 414, 426, 429, 433, 437 Ichthyotoxins, 407–439 Ictalurus furcatus, see Blue catfish punctatus, see Channel catfish Ictiobus, see Buffalo Inland silverside, 411 Inshore lizardfish Instrumental, 104, 160 Inulin, 256 Isochrysis, 259, 268 galbana, 262, 266, 268 J Jamaicamide, 435 Japanese anchovy, 428 Japanese black porgy, 428 Japanese eel, 428 Jenynsia multidentata, see One-sided livebearer K Karenia brevis, 482 brevisulcata, 482 digitata, 438 mikimotoi, 434, 482 papilionacea, 482 selliformis, 482 Karlodinium, 415 veneficum, 427 Karlotoxins, 415 Killifish, 409–412, 416, 418, 428, 430, 431, 433, 434, 437 Kinase, 122, 124, 185, 189, 204–216, 303–305, 374, 476, 489 Koshikalide, 279 Kyphosus sectatrix, see Bermuda chub L Lactoferrin, 256 Ladyfish, 412 Lagodon rhomboids, see Pinfish Lagunamide, 277 Largemouth bass, 414, 416, 425, 430 Lateolabrax japonicus, see Sea bass Leatherjack, 412 Lebistes reticulatus, see Guppy Leiostomus xanthurus, see Spot Leopard searobin, 412 Lepisosteus osseus, see Longnose gar Lepomis, see Sunfish auritus, see 430 cyanellus, see Green sunfish gibbosus, see Pumpkinseed

520

Index

Lepomis, see Sunfish (continued) humilis, see Orange-spotted sunfish macrochirus, see Bluegill sunfish microlophys, 430 Leptolyngbya, 230 boryana, 417 crossbyana, 276, 281 Lesser weaver, 416 Leucaspius delineates, see Belica Libertellenones, 194 Lignan, 256 Limnothrix redekei, 417 Lined sole, 411 Lipopolysaccharides, 156, 228, 326 Loach, 418, 419, 422 Lobocyclamides, 277 Lomaiviticin, 364 Longnose batfish, 413 Longnose gar, 411, 425 Longnose killifish, 411 Lophozozuymus, 116 Lota lota, see Burbot Lurbinectedin, 305 Lutein, 256, 259, 261, 266, 268–270 Lutjanus griseus, see Gray snapper Lycopene, 266, 270 Lymphostin, 195 Lyngbya, 233, 245, 246, 275, 279, 281, 437 aerugineo-coerulea, 438 confervoides, 277 majuscula, 261, 277–279, 431, 435 wollei, 69, 247, 317, 321, 395, 427, 432, 464 Lyngbyastatins, 275 Lyngbyatoxin, 226, 228–230, 233 Lyngbyazothrins, 276 M Macrocyclic, 49, 53, 56, 62, 188, 308, 344, 367, 394, 468 Macrolide, 184–185, 300, 303, 306, 308, 381, 393–395, 400 Maitotoxin, 2, 23, 206, 214, 384–385, 400, 436 Malotus villosus, see Capelin Maltitol, 256 Maltodextrin, 256 Malyngamide, 435 Marinomycin, 367 Marizomib, see Salinosporamide Mascarenotoxin, 97–98, 113–115, 214, 393 Megastomatobus cyprinella, see Bigmouth buffalo Melanotaenia duboulayi, see Crimson spotted rainbowfish Menidia beryllina, see Inland silverside Menticirrhus littoralis, see Gulf kingfish Microcystin, 157–164, 207, 225–230, 233, 234, 319, 333, 397–400, 416–418, 420–423, 436–437 Micromonas, 282 Micromonospora, 362–364 Micropogonias undulates, see Atlantic croaker Micropterus salmoides, see Largemouth bass Microtubule, 184–185, 211, 215–216, 298, 300–303, 306, 308, 369, 468, 479 Misguruns mizolepis, see Loach

Monodonta, 14 Monopterus albus, see Asian swamp eel Morone saxatilis, see Striped bass Mosquito fish, 411, 426, 431, 438 Mozambique tilapia, 418, 420, 422, 430 Mud carp, 425 Mueggelone, 436 Mugil cephalus, see Flathead mullet; Striped mullet Mycteroperca bonaci, see Black grouper Mytilus, 79, 116, 121, 481, 487 N Naked goby, 430 Neurotoxin, 13– 14, 28, 31, 51, 53, 156–158, 188, 190, 226, 245–246, 295, 328, 395, 397, 407, 414, 426, 431, 437, 464, 476 Niacin, 256, 272 Nodularin, 160, 163, 164, 226, 230–231, 233, 234, 397–400, 436–437 Northern pike, 425 Nostocarboline, 277 Notemigonus crysoleucas, see Golden shiner O Odontesthes bonariensis, see Argentinian silverside Ogcocephalus vespertilio, see Longnose batfish Oligoplites saurus, see Leatherjack Oncorhynchus kisutch, see Coho salmon tshawytscha, see Chinook salmon One-sided livebearer, 419 Ophichthus, 411 Ophidion, 411 Ophidion holbrookii, see Bank cusk-eel Ophiobolin, 372 Ophiobolus, 372 Opisthonema oglinum, see Atlantic thread herring Opsanus beta, see Gulf toadfish Orange clownfish, 430 Orange filefish, 411 Orange-spotted sunfish, 409 Oreochromis aureus, see Blue tilapia mossambicus, see Mozambique tilapia Oscillatoria, 137, 162, 168, 226, 233, 245, 261, 271, 277, 278, 319, 321, 397, 408, 417, 432 Osmerus eperlanus, see European smelt Ostreococcus, 282 Ostreopsis, 14, 87, 92–94, 97–99, 102, 105, 114–116, 119–121, 125, 128, 129, 214, 414 Ostreotoxin, 114–115 Ovatatoxin, see Ovatoxin Ovatoxin, 15, 89–91, 98–103, 105, 113–115, 121, 206, 214, 231 Oxosorbiquinol, 370, 371 P Pahayokolide, 437 Palythoa, 86–88, 91, 92, 94, 113–115, 120, 214, 393 Palytoxin, 2, 85–105, 113–124, 158, 206, 214, 393 Paracentrotus, 116 Paralichthys albigutta, see Gulf flounder

Index

Paranemertes, 304 Pareques acuminatus, see High-hat Patellamide, 374–375 Pecten, 116 Pectenotoxin, 2, 6, 10, 206, 212, 393–395, 468–469, 473 Pectin, 256–257 Peppered cory, 419, 422 Peprilus paru, see American harvestfish Peptide, 5, 28, 187, 255, 279, 282, 297–298, 300, 307, 374, 420 bioactive, 262–263, 274–275 cyclic, 156–157, 226, 275, 415, 435–437 depsi, 184, 301, 306, 363, 365 hepta, 225, 230, 366, 397, 401 lipo, 275, 278, 431, 435 penta, 230–231, 398 poly, 31, 256, 381, 400 Perca fluviatilis, see European perch Perna, 116 Phaeocystis sp., 424, 438 Phaeodactylum, 259–260, 264, 266, 268, 276, 281–282 Phomactin, 372 Phycocyanin, 257, 259, 261, 269 Pimephales notatus, see Bluntnose minnow promelas, see Fathead minnow Pinfish, 411, 413, 430 Pinnatoxin, 49–66, 206, 343, 348, 393 Planktothrix agardhii, 408, 417, 438 Platypodiella, 116 Plitidepsin, 184, 301, 304 Poecilia reticulate, see Guppy Pogonias cromis, see Black drum Policavernosides, 2 Pollachius virens, see American pollock Pollack, 416 Polonicumtoxins, 437 Polybromine, 295 Polychaete, 120 Polychlorobiphenyl, 266 Polyclonal, 104, 127 Polycyclic, 32, 53, 190–194, 367, 385, 388, 391, 392, 400 Polydextrose, 256 Polyene, 102, 367 Polyether, 15, 23, 308, 384–395, 410, 414, 424, 432, 435, 468, 482 Polyhydroxyl, 231, 431 Polyketide, 185, 195, 297, 303, 305, 319, 365, 367–368, 381–401, 410, 424 Polymerase, 184, 204, 209, 300, 307, 363 Polymerization, 308 Polymodal, 494 Polyol, 57, 62 Polyoxometalate, 158 Polyp, 113 Polypeptide, 31 Polyphenol, 255, 269 Polysaccharide, 156, 226, 228, 229, 257, 260–262, 273–276, 279, 326 Polysyncraton, 364 Polyunsaturated, 255, 259, 280, 299, 433, 438

521

Pomacanthus paru, see French angelfish Pomoxis, see Crappie nigro-maculatus, see Black crappie Porgy, 411 Porichthys porossimimus, see Atlantic midshipman Predatory carp, 419, 428 Prionotus scitulus, see Leopard searobin Prorocentrolide, 51, 206, 343–345, 351, 353, 393 Prymnesin, 424–426, 435 Prymnesium sp., 424, 435, 438 Pseudomonas, 14, 87, 194 Pseudo-nitzschia, 213, 282, 433, 475–478, 482 Pseurotin, 371–372 Pteriatoxin, 49–50, 54, 206, 343, 394 Puffer, 184, 294–295, 301, 412–413, 428, 437 Pumpkinseed, 409, 416, 419 Purplemouth moray, 413 Pyocyanin, 194 Q Queen angelfish, 413 Queen triggerfish, 413 R Rachycentron canadus, see Cobia Raphidiopsis curvata, 226, 228, 317, 321, 432 meditteranea, 408, 432 Receptor acetylcholine, see Acetylcoline brevetoxin, 190 cancer-associated, 191 carotid, 467 conotoxin, 189 death-mediated, 203–207, 209, 210 dopamine, 466 epidermal growth factor (EGF), 124 GABAA , 44 glutamate, 213, 354, 475 insulin-like growth factor, 216 interacting protein kinase, 215 ionotropic, 157 kainate, 213, 353, 478 low density lipoprotein (LDL), 280 membrane, 489 neurotensin, 187 nicotinic, 52–53, 66, 156–159, 170, 184, 187–189, 302, 344, 348, 354, 407 (see also Acetylcholine) N-methyl-D-aspartate (NMDA), 43, 187, 213, 476, 483 palytoxin, 88, 117 platelet activating factor (PAF), 372 tetrodotoxin, 187 tumor necrosis factor (TNF), 215–216 vascular endothelial growth factor (VEGF), 304 sodium channel site 1, 28 sodium channel site 2, 31 sodium channel site 3, 31 sodium channel site 6, 31 Redbreast sunfish, 430 Redear sunfish, 430 Red grouper, 411

522

Index

Rhombosolea tapirina, see Greenback flounder Ribosome, 326 Rio Grande darter, 425 Round goby, 415, 416 S Sailor’s choice, 413 Salinispora, 193, 305, 362–363 Salinosporamide, 193–195, 305, 363 Salmo salar, see Atlantic salmon Salmo trutta, see Brown trout Sand perch, 411, 413 Sand seatrout, 411 Saxitoxin, 28–29, 39, 69, 71, 80, 127, 157, 160, 169, 187, 206, 226, 231–233, 246–248, 319, 395–397, 400, 437, 464 Scaled sardines, 411, 413 Schizothrix calcicola, 438 Scomber scombrus, see Atlantic mackerel Scrawled cowfish, 413 Scyllium canicula, see Dogfish Scytoscalarol, 276–277 Sea bass, 434, 438 Sea catfish, 413 Sergeant major, 413 Serranus subligarius, see Belted sandfish Shad, 425 Sharksucker, 411 Sheepshead, 409, 413, 430 Sheepshead minnow, 409, 416, 428, 430, 433, 437, 438 Shewanella, 14, 295 Shikimic acid, 297 Signalosa mexicana, see Threadfin shad Silver bream, 425 Silver carp, 418, 419, 421–423, 425, 428 Silver jenny, 413 Silver perch, 412, 413 Sooty eel, 412 Sorbicillactone, 370–371 Southern stargazer, 412 Sparus auratus, see Gilthead sea bream Sphoeroides testudineus, see Checkered puffer Spirociclic imine, 49, 53, 58, 391–392, 431 Spiroisomerization, 23–24 Spiroketal, 25–26, 55–57, 65, 344, 353 Spirolide, 6, 49, 50, 52, 64, 188–189, 206, 343, 348–352, 393–394 Spiroprorocentrimine, 346, 348 Spot, 411, 413, 416 Spotted moray eel, 413 Spotted sea trout, 413, 416, 430 Squalius cephalus, see European chub Staphylococci, 368 Stegastes variabilis, see Cocoa damselfish Stereoisomers, 91, 113, 145, 391, 400 Sterol, 256, 264, 280 Stinging catfish, 418, 420 Streptococci, 368 Streptomyces, 229, 362, 368, 372 Striped bass, 416, 425, 430, 433

Striped bass hybrid, 416, 430 Striped burrfish, 413 Striped mullet, 430 Strongylura marina, see Atlantic needlefish Sturgeon hybrid, 419 Sunfish, 416, 425 Symbioimine, 49, 51, 343–344, 346, 348, 351–353, 356 Symphurus plagiusa, see Blackcheek tonguefish Symploca, 277, 279, 299, 366 Symplostatin, 277 Synodus foetens, see Inshore lizardfish Synthase, 192, 195, 293, 319–320, 354, 365, 371, 381–382, 389, 395, 438, 465, 476, 494 Synthetase, see Synthase T Takifugu, 428 Tanikolide, 277 Tetrodotoxin, 2, 4, 14, 27–29, 34, 37–42, 156, 184, 187, 294, 301, 304, 437, 464, 494 Thalassiosira, 259, 264–265, 282 Thalassoma, 414 Thamnocephalus, 329 Thiomarinol, 368 Threadfin shad, 413, 419 Tigriopus, 120 Tistrella, 362, 365 Tocopherol, 269, 274, 282 Tomtate, 412 Trabectedin, 183–184, 194, 299, 301, 305 (see also Yondelis) Trachinotus carolinus, see Florida pompano Trachinus vipera, see Lesser weaver Trachurus japonicas, see Horse mackerel Trichodermanone, 370, 371 Trichodesmium, 115, 226, 233, 246, 417 Trinechtes maculatus, see Hog choker Turbot, 412 U Umezakia natans, 226, 228, 432 V Variegated minnow, 412, 413 Venturamide, 277–278 Veratridine, 29, 31, 33, 37, 42 Verrucosispora, 362, 367 Vibrio, 14, 87, 295, 348 Viridamide, 277 Vitamin, 255–256, 259, 263, 267–270, 272–273, 280, 469 Volvox, 282 Vorsetuzumab, 306 W Whitefish, 419, 421, 423 White grunt, 412 White sucker, 409 Worm, 14, 120, 184, 277, 302, 304, 344 Wrasse, 416, 437

Index

X Xylitol, 256 Xylooligosaccharide, 256 Y Yessotoxin, 2, 6, 127, 191, 193, 206, 208–210, 384–385, 388, 468, 488, 492 Yondelis, 183–184, 361–362 (see also Trabectedin)

523

Z Zalypsis, 184, 302, 304 Zeaxanthin, 256, 261, 266–270, 282 Zebra fish, 409, 415, 416, 418–423, 429, 431–433, 436–437 Ziconotide, 182–184, 187, 293, 299–300, 306–307, 310 Zoanthus, 114–115

Vh (mV) –40 –30 –20 –10 0

10 20 30 40 50

–0.0

I/Imax

–0.2

Control 0.1 nM CTX-3C 1n M CTX-3C

–0.4 –0.6

10 nM CTX-3C

–0.8 –1.0

(a) Vh (mV) –40 –30 –20 –10 0

10 20 30 40 50

–0.0

I/Imax

–0.2 –0.4

Control 20 nM CTX-3C (5 min) 20 nM CTX-3C (10 min) 20 nM CTX-3C (15 min)

–0.6 –0.8 –1.0

(b) Figure 2.3 Effect of CTX 3C on the activation of voltage-gated sodium channels in human SH-SY5Y neuroblastoma cells. Voltage-dependent sodium currents were elicited in SH-SY5Y cells by applying a series of 25 ms depolarizing pulses (voltage steps) from −50 mV to 50 mV, in 5 mV increments, from a holding potential of −100 mV. (a): Current-voltage (I-V) relationship for the effect of different concentrations of CTX-3C on sodium currents in SH-SY5Y. (b): I-V relationship for the effect of 20 nM CTX-3C on sodium currents measured at 5, 10 and 15 minutes after bath application of the toxin. Note that the effect of 20 nM CTX3C was not dependent on the time, and that CTXC-3C decreased the peak sodium current (INa ) and shifted the activation threshold of INa towards a more negative value. Each point represents the mean ± SEM of 3–5 measurements.

Phycotoxins: Chemistry and Biochemistry, Second Edition. Edited by Luis M. Botana and Amparo Alfonso. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

Prepulse mV –100 –90 –80 –70 –60 –50 –40 –30 –20 –10 0 0.00 –0.25 I/Imax

Control –0.50

0.1 nM CTX-3C 1n M CTX-3C

–0.75

10 nM CTX-3C

–1.00 (a) Prepulse mV –100 –90 –80 –70 –60 –50 –40 –30 –20 –10 0 0.00

I/Imax

–0.25 Control

–0.50

20 nM CTX-3C 5 min 20 nM CTX-3C 10 min

–0.75

20 nM CTX-3C 15 min –1.00 (b) Figure 2.4

Effect of CTX-3C on the voltage dependence of the steady state inactivation of voltage-gated sodium channels in human SH-SY5Y neuroblastoma cells. Steady-state inactivation was determined using a two-pulse protocol. A 500 ms conditioning pre-pulse from −100 mV to −10 mV was stepped in 10 mV increments and was followed by a 50 ms test pulse to –20 mV. (a): I-V relationship for the effect of different concentrations of CTX-3C on the steady-state inactivation of sodium currents in SH-SY5Y. Note that CTX-3C altered the steady-state inactivation at all concentrations tested. (b): I-V relationship for the effect of 20 nM CTX-3C on the steady-state inactivation of sodium currents in SH-SY5Y cells measured at 5, 10 and 15 minutes after bath application of the toxin. Note that the effect of 20 nM CTX-3C on steady-state inactivation was not dependent on the time. All currents were normalized to the maximum control current. Each point represents the mean ±SEM of 3–5 measurements. Curves were fitted by the Boltzman equation.

Control

Figure 2.6

5 nM CTX-3C 96 hours in culture

Confocal microscopy image showing β-tubulin staining of primary cortical neurons grown for four days in culture in the absence (left) or presence of 5 nM CTX-3C (right). Scale bar is 20 μm.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Figure 12.1 Light micrographs showing morphological diversity of commercially potential microalgae for nutraceutical applications. Dunaliella salina (the source for β-carotene) at actively growing green-phase of growth (b), transition-phase of growth (c) and complete stress-phase of growth, with accumulation of carotenoid β-carotene (d). Haematococcus pluvialis (the source for astaxanthin) at actively growing green-phase of growth (e) and complete stress-phase of growth, with accumulation of carotenoid astaxanthin (f). Nannochloropsis salina (the source for essential fatty acid EPA) from active phase of growth (g). Porphyridium cruentum, the red microalga (source for antioxidant and anti-cancer pigment phycoerythrin and bioactive polysaccharides), from active phase of growth (h). Phaeodactylum tricornutum (the source for essential fatty acid EPA) from active growth phase (i). Lyngbya majuscula, the cyanobacterium with several bioactive molecules, including immuno-modulatory compounds.

Figure 19.3 Hepatic histology of rats given OA intravenously. Rats were given a single i.v. injection of DMSO/NaCl (control, (a)), or OA at 0.4 μg/g body wt (b). After 24 hours, paraffin sections from the livers were prepared, and stained H&E Notice extensive congestion in (b). A. B. × 600. Source: Berven et al., 2001. Reproduced with permission of Elsevier.

(a)

(b)

Figure 19.6 Hippocampal sections of control rat (a) and DOM-treated rat (b) staining with MAP-2 antibody. Note the lack of MAP-2 staining in B, with an important cytoskeletal alteration. A, B. × 40.

(a)

(b)

Figure 19.7 Increased numbers of alveolar macrophages in the alveoli of a rat exposed to high-brevetoxin concentration, a typical presentation. (a): Bar = 50 μm. (b): Bar = 25 μm. Arrows indicate macrophages. Inhalation Toxicity of Brevetoxin 3 in Rats Exposed for Twenty-Two Days. Source: Benson, JM. et al., (2005). Inhalation Toxicity of Brevetoxin 3 in Rats Exposed for Twenty-Two Days. Environ Health Perspect. 113, 626–631. Reproduced with permission of Environmental Health Perspectives.

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