PIRE: Advancing global strategies and understanding on the origin of ciguatera fish poisoning in tropical oceans: The Norwegian contribution

Ciguatera is the name of a disease in humans associated with the consumption of tropical fish that has accumulated a specific type of toxin from microscopic algae, called ciguatoxins (CTX). The microscopic algae belong to the genus Gambierdiscus or Fukuyoa and live on other macroscopic algae, seagrass and corals in reefs. Fish that consume the algae change the chemical structure of the toxins, which possibly increases their toxicity. Ciguatera poisoning has become a global problem due to international trade, as well as the spread of the poison-producing algae to more temperate waters. The RCN-funded project is linked to a larger NSF-funded project, CiguaPIRE. CiguaPIRE is led by the University of Southern Alabama, which is also our most important partner in the project in addition to the National Research Council of Canada and the University of Oslo. The first part of our contribution to CiguaPIRE was to map and tentatively identify different variants of Caribbean CTX (C-CTX) in toxic fish from the Gulf of Mexico. For this we used modern analytical methods such as high-resolution mass spectrometry. The work resulted in the clarification of the chemical structure of two new variants of C-CTX that are present in toxic fish samples in addition to the already known major toxins (C-CTX-1 and -2). Furthermore, we demonstrated the presence of additional toxin variants in the fish samples. However, these other variants were likely present in substantially lower concentrations. The major breakthrough came towards the end of the project when the group identified a ciguatoxin in a Gambierdiscus species, which was shown to be the precursor toxin to those that are found in fish. This toxin was named C-CTX-5 and is very similar in structure to C-CTX-1. By using liver enzymes from fish, we demonstrated that C-CTX-5 can be converted into C-CTX-1 in fish. The discovery of C-CTX-5 in algae is paving the way for the production of toxin reference materials as purifying toxins from algae is by far more straightforward than it is to isolate the toxin from fish muscle as was done earlier. CTX are most often detected with a cell-based bioassay, which is a sensitive but not very selective method. To detect the toxins in a more selective way, one must use chemical methods, such as mass spectrometry. Unfortunately, CTX is a molecule that is not easy to analyze with this technique, and we have therefore developed methods that makes it easier to detect C-CTX-1 and -2 by mass spectrometry. Changing the molecules structure by a simple chemical reaction resulted in about 40-times lower detection limits. In another effort led by the National Research Council of Canada, C-CTX-1 was labelled with oxygen-18. Changing the molecule’s structure by isotope-labelling enables the more selective and accurate detection and quantification in all types of samples. Our partners at the University of Southern Alabama, collected fish from the Gulf of Mexico that were used to prepare enzyme fractions from their livers (so-called microsomes) for the purpose of studying how C-CTX is bio-transformed in fish. This work has resulted in new knowledge that is essential for CTX toxicology. We have demonstrated that both fish from the Gulf of Mexico, as well as Atlantic salmon, have the ability to detoxify CTX by conjugating them with glucuronic acid. We could not observe such a biotransformation reaction when the toxins were incubated with liver or human preparations. It may therefore seem that the ability to detoxify the toxins is a general trait in fish, while the ability to detoxify CTX via the same mechanism is lacking in mammals. In that case, the findings could partially explain the (high) potency of the toxins.

1) The project has extented the knowledge on existing ciguatoxin variants and related neurotoxins considerably. This work culminated in the discovery of the algal precursor of Caribbean-CTX-1, the main analogue of the suite of Caribbean and Atlantic ciguatoxins in fish. 2) The project has increased the understanding of the chemical reactivity of Caribbean ciguatoxins, which is a prerequisite for improving detection methods and understanding biotransformation pathways. 3) Within the project, new methods have been developed that enable the detection of ciguatoxins with higher selectivity and accuracy. 4) The project has identified ciguatoxin-glucuronic acid biotransformation products in fish and shown that this type of biotransformation reactions does probably not occurr in humans, which could in part be the reason for the potency of the toxins in humans.

Ciguatera is caused by the consumption of reef fish that have accumulated ciguatoxins (CTXs). Precursors of CTX are produced by benthic dinoflagellates (Dinophyceae) in the genus Gambierdiscus that live on the marine substrates (e.g., on macroalgae, seagrasses) in coral reef ecosystems. These toxic precursors (secondary metabolites) are incidentally consumed by herbivorous grazers (marine invertebrates and fish) and the compounds are subsequently converted to more potent tertiary metabolites known as CTXs following bioaccumulation and metabolism within an organisms and further converted following trophic transfer through the food web. These mechanisms of biotransformation and food-web pathways are poorly understood but their elucidation is critical to developing monitoring approaches for public health protection. Given the extensive impacts that CFP has in affected communities and the global reach of this public health concern (via global trade), the resolution of these deficiencies and development of predictive capabilities are major research priorities. A better understanding of toxin production and flux into the food web needs to be acquired before proactive management actions can be established. The Norwegian tasks within the larger PIRE project are to contribute in the characterisation of the CTX metabolome of Gambierdiscus species as well as to participate in the structure elucidation of key algal and fish metabolites of CTXs. The Norwegian team has also the knowledge and capabilities to perform in vitro toxicokinetic trials using liver microsome preparations from practically any animal. Such in vitro approaches will be applied to study the biotransformation of algal CTXs in various fish species. Furthermore, the group at the Norwegian Veterinary Institute is among the world-leading teams regarding the design and preparation of antibodies for marine toxins. Such anti-CTX antibodies will be raised within the project and applied in different assay formats.