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Chemical and Toxicological Investigation of the Toxic Dinoflagellate, Karenia Brevisulcata

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Chemical and Toxicological Investigation of the Toxic Dinoflagellate, Karenia Brevisulcata Lincoln University Digital Thesis Copyright Statement The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. Chemical and toxicological investigation of the toxic dinoflagellate, Karenia brevisulcata A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University by Feng Shi Lincoln University 2012 Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Ph.D. Abstract Chemical and toxicological investigation of the toxic dinoflagellate, Karenia brevisulcata by Feng Shi A severe harmful algal bloom (HAB) devastated marine life in the Wellington Harbour in the late summer 1998. A novel species of Karenia dinoflagellates was isolated from this HAB and named Karenia brevisulcata (Chang). Toxins produced by this alga were categorised into i) brevisulcatic acids (BSXs), a group of six polycyclic ethers with molecular weights 800-950; ii) brevisulcenals (acronym KBTs from a previous name), a complex of 6-8 lipophilic compounds with molecular weights 1900-2200. This PhD thesis reports the isolation, purification and chemical investigation of BSXs, analytical and toxicological studies of BSXs and KBTs, and the toxicity evaluation of three Karenia species. LC-MS provides a powerful micro-analytical technology to detect novel toxins, determine structural characteristics, guide chemical isolation and provide quantitative data on levels of toxins in cultures. An LC-MS (SIR method) with six channels for BSXs and one channel for brevetoxin-2 (calibrant) was developed to guide BSX isolation. A more sensitive method based on solid phase extraction (SPE) and LC-MS (MRM) was validated for determination of 6 BSXs and 4 KBTs in cultures with LOQs for each toxin of ca 0.15 µg L-1 in seawater. Barrels, carboys and a photobioreactor were used to optimise cell growth and toxin production of a K. brevisulcata isolate collected by Cawthron Institute in 1998. Carboys were the most efficient in batch culture mode with the highest growth rate (μ=0.086 day-1 versus 0.050-0.057 day-1 for barrels or photobioreactor) and the highest cell yield for total BSXs (0.77 pg cell-1 versus 0.29-0.38 pg cell-1 for barrel or photobioreactor). However, the large volume automated photobioreactor operated in continuous culture mode was more suited to large-scale toxin production from dinoflagellates. ii K. brevisulcata cultures (total 936 litres unlabelled; 492 litres 13C-labelled using added 13 NaH CO3) were grown at Cawthron Institute. The toxins were extracted from the bulk cultures by cell disruption and absorption using HP20 polymeric resin followed by acetone extraction. Solvent partitioning of crude acetone extracts was used to prepare neutral fractions containing KBTs and acidic fractions containing BSXs. Semi-purification of BSXs from the acidic fractions used desalting by reverse phase SPE and defatting by normal phase SPE. + + BSX-1 (C49H72O16, [M+H] 917.5) and BSX-2 (C47H68O15, [M+H] 873.5) were separated by normal phase column chromatography and were purified in mg quantities using preparative + C18 high performance liquid chromatography (HPLC). BSX-4 (C49H70O15, [M+H] 899.5) + and BSX-5 (C47H66O14, [M+H] 855.5) are the lactones which were produced from BSX-1 and BSX-2 respectively by acid-catalysed ring closure for analytical, structural and toxicological studies. The neutral fractions of crude extracts were sent to the University of Tokyo (Prof. Masayuki Satake) where four KBTs (-F, -G, -H & -I) were isolated. The chemical structures of BSXs and KBTs were characterised by spectroscopic (UV, MS-MS) and micro-derivatisation techniques (methylation, acetylation, hydrolysis). BSXs are ladder-frame polycyclic ethers with close similarities to brevetoxins. A probable structure for BSX-4 has been determined based on partial NMR spectra obtained from Massey University. BSX-4 has structural similarities to brevetoxin-1 but with some differences in the polyether rings and a longer, carboxylated side chain. However, the full structures of BSXs have not been completely elucidated because of missing NMR peaks due to conformation changes in the time frame of the NMR pulse sequences. Missing signals, even at low temperatures, probably involve three flexible 7-, 8- and 9-membered ether rings in the central part of the molecule with one double bond and a tertiary methyl group. NMR studies are continuing at the University of Tokyo. + The complex structure of KBT-F (C107H160O38, [M+H] 2054.1) has been elucidated at the University of Tokyo using NMR and TOF-TOF MS. KBT-F is a novel ladder frame polycyclic ether compound with a 2-methyl-2-butenal side chain that is the same as that in gymnocin-A. It has the most contiguous ethers rings amongst the known polycyclic ethers. Acute toxicity, haemolytic activity and cytotoxicity of BSXs and KBTs were studied by mouse bioassay, Bignami assay, and in vitro mammalian cell bioassays respectively. BSXs -1 are weakly toxic to mice with intraperitoneal (i.p.) LD50 of > 1500 μg kg body weight (bw), and weakly haemolytic. Neuro2a cell assays revealed that BSXs are brevetoxin-like voltage gated Na channel (VGSC) agonists but with lower activity. KBT-F and KBT-G are strongly -1 toxic to mice (i.p. LD50 of 32-40 μg kg bw), strongly haemolytic, and highly cytotoxic to iii P388, Vero and Neuro2a cells, but are unlikely to be VGSC agonists. Further cytotoxicity assays using multiple end-points e.g. LDH/MTS and LDH/caspase-3/7 revealed that damage to P388 cells by KBT-F involved effects on membranes but apoptotic mechanisms were not involved. In vivo bioassays using marine biota (two fish species and larvae of six marine invertebrate species) were used to investigate the toxicity and potential modes-of-action of three toxic Karenia species. The bioassays confirmed field observations on the very high ecotoxic risks posed by blooms of K. brevisulcata. A provisional trigger of 1 x 104 cells L-1 is recommended to provide an early warning of a K. brevisulcata bloom. The mechanism for fish and invertebrate kills by K. brevisulcata is likely to depend on release of toxins from algal cells into seawater with KBTs being the main toxic agents. Keywords: Wellington Harbour toxin, Toxic dinoflagellate, Karenia brevisulcata, Brevisulcatic acid, Brevisulcenal, Brevetoxins, BSX, KBT, LC-MS iv Acknowledgements Wow, I did it! I still cannot believe that I have finished all my experiments and the writing of my PhD thesis in three years. It was a difficult three years, but also inspiring, exciting and a challenging experience. This wonderful journey was made possible by many people who supported, guided and helped me. I wish to convey my deep gratitude to all of them. First and foremost, I would like to acknowledge Lincoln University and Cawthron Institute who gave me an opportunity to carry out this research. I would like to thank the Ministry for Science and Innovation (MSI) and Lincoln University for supporting my studies at Cawthron Institute. My sincere thanks go to my supervisor Associate Professor Dr Ravi Gooneratne, Lincoln University for introducing me to the field of toxicology. His constant source of guidance, encouragement and criticism helped me to stay persistent, focused and motivated. His persistence, faith and kindness were essential to my success. I would like to take this opportunity to especially thank my co-supervisor Dr Patrick Holland, Cawthron Institute, for assistance during every step of my research project. His constant guidance, support, and criticism made this work possible. I would like to thank my associate supervisor Dr Sue Mason, Lincoln University, for her encouragement and valuable advice. I would like to thank my scientific collaborators: Professor Masayuki Satake (the University of Tokyo) for further isolation and chemical studies of KBTs as well as assistance with interpretation of NMR spectra for BSXs, Dr Rex Munday (AgResearch) for toxicological studies using mouse bioassay, Dr Penny Truman (ESR) for Neuro2a assays and invaluable guidance on establishment of in vitro cell bioassays, Dr Lynn Briggs (AgResearch) who carried out Bignami assays, and Dr Patrick Edwards (Massey University) for NMR studies of BSXs. I would like to thank the Cawthron Institute, Nelson for welcoming me and allowing me to conduct all my research there. Particular thanks go to all the staff in the R & D team: Paul McNabb, Dr Patrick Holland, Dr Tim Harwood, Andy Selwood, Roel van Ginkel, David White, Donato Romanazzi and Anne Cargill. Their support and making me feel a valuable member of the team really was essential to me. Special appreciation goes to Paul McNabb v who, as team manager, made this study possible, Dr Tim Harwood who helped with my thesis writing, and Andy Selwood and Roel van Ginkel for technical guidance and assistance. I would also like to thank the collaborators in the Biosecurity Division, Cawthron Institute. Special thanks go to Veronica Beuzenberg for her invaluable instructions and assistance on algal culturing and harvesting. I would also like to thank Lincoln Mackenzie for his help and advice on my research, Dr Lesley Rhodes for assistance with fish and invertebrate larvae bioassays, and invaluable advice on my experiments and thesis, Janet Adamson and Dr Krystyna Ponikla for their specific advice and assistance on algal culturing, and Dr Doug Mountfort for useful advice. I would also like to thank staff in the Natural Toxin Lab, Cawthron Institute.
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