Chemical and toxicological investigation of the toxic dinoflagellate, Karenia brevisulcata (2012)

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. K. brevisulcata cultures (total 936 litres unlabelled; 492 litres ¹³C-labelled using added NaH¹³CO₃) 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 are weakly toxic to mice with intraperitoneal (i.p.) LD₅₀ of > 1500 μg kg⁻¹ 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 toxic to mice (i.p. LD₅₀ of 32-40 μg kg⁻¹ bw), strongly haemolytic, and highly cytotoxic to 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.