ALGETOX

Chemistry and chemical biology of lipophilic algal toxins

Objective: Improved methods will be developed for isolation of lipophilic marine algal toxins (azaspiracids, pectenotoxins, yessotoxins and okadaic acid analogues) and their metabolites from algae and shellfish. Derivatives such as labelled analogues will also be produced. These materials will be used in collaborative studies to perform toxicology, identify toxin mode-of-action, and develop improved or validated analytical methods (e.g. ELISA, immunoaffinity cleanup, LC-MS). Information obtained will lead to improved regulatory levels for algal toxins and reduced use of animals (3Rs principal) for routine testing of algal toxins in shellfish.

Results

  • The EU-funded ‘Chemistry and chemical biology of lipophilic algal toxins’ (Algetox) project has improved ways of identifying and isolating toxins such as azaspiracids, okadaic acid, and pectenotoxin and yessotoxin groups, from algae. A researcher based at the National Veterinary Institute, Oslo, has investigated how shellfish metabolise and excrete these algal toxins.
  • The way the toxins operate at the cellular and molecular levels has also been studied to provide greater insights into their effects. A chemical analysis technique has been developed for detecting a range of algal toxins in shellfish.
  • Project partners have isolated a new fast-acting toxin found in Australian shellfish using nuclear magnetic resonance spectroscopic and mass spectroscopic analysis. Chemical compounds with similar properties are known as analogues; one known and five new chemical pinnatoxin analogues have been successfully identified.
  • Analogues have also been produced and labelled with radioactive material for use in biochemical experiments to reveal the toxin’s mode of action. This has been achieved by determining the metabolism and excretion of these compounds in mammals.
  • Results from the Algetox project will be used to develop or validate analytical techniques for identifying toxins. The information obtained will help to improve regulatory levels for algal toxins in shellfish. Furthermore, the methodologies developed can replace or reduce the use of animals for the testing of toxins in shellfish.
  • A range of algal and shellfish samples from Ireland, Germany, Japan, Norway and New Zealand have been analysed by a range of LC-MS techniques, and a series of novel toxin analogues have been identified.
  • The fellow is supervising a PhD student in Ireland developing improved methods for isolating azaspiracids, for which there is an acute shortage for use both in toxicological experiments and as an analytical standard.
  • A viable method for labelling azaspiracids was developed, and then optimised with Canadian collaborators. Labelling methodology could permit development of new chemical and biochemical analytical methods for azaspiracids.
  • Experiments to determine the source of azaspiracid-3 and -6 in shellfish revealed that these are primarily generated via heat-induced decarboxylation of metabolites (azaspiracid-17 and -19) produced in shellfish, and efficient isotopic labelling of azaspiracid-3 and -6 was achieved by a simple chemical reaction of these metabolites.
  • A range of yessotoxin analogues were provided to a collaborator for toxicity testing. Structure-activity data from this has provided a basis for developing labelled derivatives that are expected to be biologically active.
  • Two metabolites of brevetoxins have been prepared and characterised by chemical and biochemical methods. Radiolabelled versions of these metabolites were prepared during a visit to collaborators in the USA in 2009. These radiolabelled analogues are being used in toxicological biochemical experiments and to assess adsorption, metabolism and excretion of these compounds in mammals.
  • NMR analysis was performed on a known and on a new pectenotoxin analogue in collaboration with Japanese researchers. Full structural analysis of the known pectenotoxin, which is currently regulated, had not previously been reported.
  • An array of microcystin metabolites were provided to a Norwegian collaborator for quantitative evaluation with a protein phosphatase 2A (PP2A) assay (the molecular target of the microcystins). The structures of new fast-acting toxins, isolated by collaborators in New Zealand from Australian shellfish, were determined by NMR spectroscopic and mass spectrometric analysis. One known and five new pinnatoxin analogues were identified.
  • LC-MS/MS analysis of shellfish and sea water samples from around the coast of Norway was initiated using analytical standards of pinnatoxins available from this work. Although pinnatoxins had not previously been reported in Europe, we found pinnatoxins in nearly 70 % of shellfish samples from late 2009 (some of which contained high levels) and in 100 % of stored sea water extracts from an earlier trial in 2007.

Publications and Presentations

Project partners: Norwegian Veterinary Institute, Marine Institute in Ireland and National Research Council of Canada

Project periode: 2008–2010

Funding: Specific programme “People” implementing the 7th Framework Programme of the European Community for research, technological development and demonstration activities (Grant ID: 221117)

Project page at Cordis: https://cordis.europa.eu/project/id/221117