Sea Anemone Toxins: A Structural Overview.

Sea anemones produce venoms of exceptional molecular diversity, with at least 17 different molecular scaffolds reported to date. These venom components have traditionally been classified according to pharmacological activity and amino acid sequence. However, this classification system suffers from vulnerabilities due to functional convergence and functional promiscuity. Furthermore, for most known sea anemone toxins, the exact receptors they target are either unknown, or at best incomplete. In this review, we first provide an overview of the sea anemone venom system and then focus on the venom components. We have organised the venom components by distinguishing firstly between proteins and non-proteinaceous compounds, secondly between enzymes and other proteins without enzymatic activity, then according to the structural scaffold, and finally according to molecular target.

Capillary electrophoresis-tandem mass spectrometry for multiclass analysis of polar marine toxins.

Polar marine toxins are more challenging to analyze by mass spectrometry-based methods than lipophilic marine toxins, which are now routinely measured in shellfish by multiclass reversed-phase liquid chromatography-tandem mass spectrometry (MS/MS) methods. Capillary electrophoresis (CE)-MS/MS is a technique that is well suited for the analysis of polar marine toxins, and has the potential of providing very high resolution separation. Here, we present a CE-MS/MS method developed, with use of a custom-built interface, for the sensitive multiclass analysis of paralytic shellfish toxins, tetrodotoxins, and domoic acid in seafood. A novel, highly acidic background electrolyte (5 M formic acid) was designed to maximize protonation of analytes and to allow a high degree of sample stacking to improve the limits of detection. The method was applied to a wide range of regulated and less common toxin analogues, and exhibited a high degree of selectivity between toxin isomers and matrix interference. The limits of detection in mussel tissue were 0.0052 mg/kg for tetrodotoxins, 0.160 mg/kg for domoic acid, and between 0.0018 and 0.120 mg/kg for paralytic shellfish toxins, all of which showed good linearity. Minimal ionization suppression was observed when the response from neat and mussel-matrix-matched standards was corrected with multiple internal standards. Analysis of shellfish matrix reference materials and spiked samples demonstrated good accuracy and precision. Finally, the method was transferred to a commercial CE-MS/MS system to demonstrate its widespread applicability for use in both R & D and routine regulatory settings. The approach of using a highly acidic background electrolyte is of broad interest, and can be considered generally applicable to simultaneous analysis of other classes of small, polar molecules with differing pKa values. Graphical abstract ᅟ.

Unraveling cyanobacteria ecology in wastewater treatment plants (WWTP).

Cyanobacteria may be important components of wastewater treatment plants' (WWTP) biological treatment, reaching levels of 100% of the total phytoplankton density in some systems. The occurrence of cyanobacteria and their associated toxins in these systems present a risk to the aquatic environments and to public health, changing drastically the ecology of microbial communities and associated organisms. Many studies reveal that cyanotoxins, namely microcystins may not act as antibacterial compounds but they might have negative impacts on protozoans, inhibiting their growing and respiration rates and leading to changes in cellular morphology, decreasing consequently the treatment efficacy in WWTP. On the other side, flagellates and ciliates may ingest some cyanobacteria species while the formation of colonies by these prokaryotes may be seen as a defense mechanism against predation. Problems regarding the occurrence of cyanobacteria in WWTP are not limited to toxin production. Other cyanobacterial secondary metabolites may act as antibacterial compounds leading to the disruption of bacterial communities that biologically convert organic materials in WWTP being fundamental to the efficacy of the process. Studies reveal that the potential antibacterial capacity differs according to cyanobacteria specie and it seems to be more effective in Gram (+) bacteria. Thus, to understand the effects of cyanobacterial communities in the efficiency of the waste water treatment it will be necessary to unravel the complex interactions between cyanobacterial populations, bacteria, and protozoa in WWTP in situ studies.

Purification of five azaspiracids from mussel samples contaminated with DSP toxins and azaspiracids.

Human intoxications during toxic episodes in shellfish are a very important concern for public health, as well as for economic interests of producer regions. Although initially each toxin appeared in a determined geographical zone, nowadays many of them are found in multiple places worldwide. In addition, more toxic compounds (new toxins or new analogs of known toxins) are being isolated and identified, which bring about new risks for public health. An example of this situation is the group of azaspiracids (AZAs). Initially these toxins were concentrated in Irish coasts but today appear in many different geographic locations; in the first toxic episode only three analogs were isolated, but now it is known that the group is comprised of at least eleven identified compounds. A substantial problem associated with all these new toxins is the extreme difficulty associated with the study of their toxic effects and mechanisms of action due to the very small quantities of purified toxin available. Therefore, the study of procedures to isolate them from contaminated shellfish or to synthesize them is of tremendous importance. In this paper we design a complete procedure to obtain AZAs analogs from mussels contaminated with DSP toxins and azaspiracids by means of three consecutive steps: an extraction procedure to remove toxins from shellfish, a solid phase extraction (SPE) to clean the samples and separate DSP toxins and AZAs, and a preparative HPLC to isolate each analog. In all the steps LC/MS is used to detect and quantify the toxins. Large amounts of AZA1, AZA2, AZA3, AZA4 and AZA5 were obtained by use of this procedure, which can be utilized in future studies relating to the toxins such as the production of certified materials and standards.

How the marine biotoxins affect human health.

Several marine microalgae produce dangerous toxins very damaging to human health, aquatic ecosystems and coastal resources. These Harmful Algal Blooms (HABs) in recent decades seem greatly increased regarding frequency, severity and biogeographical level, causing serious health risks as a consequence of the consumption of contaminated seafood. Toxins can cause various clinically described syndromes, characterised by a wide range of symptoms: amnesic (ASP), diarrhoetic (DSP), azaspirazid (AZP), neurotoxic (NSP) and paralytic (PSP) shellfish poisonings and ciguatera fish poisoning. The spread of HABs is probably a result of anthropogenic activities and climate change, that influence marine planktonic systems, including global warming, habitat modification, eutrophication and growth of exogenous species in response to human pressures. HABs are a worldwide matter that requests local solutions and international cooperation. This review supplies an overview of HAB phenomena, and, in particular, we describe the major consequences of HABs on human health.

New SPE-LC-MS/MS method for simultaneous determination of multi-class cyanobacterial and algal toxins.

Cyanobacterial and algal toxins comprise a large group of harmful metabolites, belonging to different chemical classes, with a variety of chemical structures, physicochemical properties and toxic activities. In this study, a fast, simple and sensitive analytical method was developed for the simultaneous determination of multi-class cyanobacterial and algal toxins in water. The target compounds were: Cylindrospermopsin, Anatoxin-a, Nodularin, 12 Microcystins ([D-Asp3]MC-RR, MC-RR, MC-YR, MC-HtyR, [D-Asp3]MC-LR, MC-LR, MC-HilR, MC-WR, MC-LA, MC-LY, MC-LW and MC-LF), Okadaic acid and Domoic acid. Analytes were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A dual Solid Phase Extraction (SPE) cartridge assembly was applied for the extraction of target compounds from water. Optimized SPE parameters included cartridge material, initial sample pH, sequence of the cartridges in the SPE assembly as well as composition and volume of the elution solvent. The method was validated, providing acceptable mean recoveries and reproducibility for most analytes. Limits of detection were at the ngL-1 level. The method was successfully applied in real lake water samples from Greece, where a wide range of Microcystins were detected for the first time, at concentrations ranging from 0.034 to 63µgL-1.

Peptide toxins in sea anemones: structural and functional aspects.

Sea anemones are a rich source of two classes of peptide toxins, sodium channel toxins and potassium channel toxins, which have been or will be useful tools for studying the structure and function of specific ion channels. Most of the known sodium channel toxins delay channel inactivation by binding to the receptor site 3 and most of the known potassium channel toxins selectively inhibit Kv1 channels. The following peptide toxins are functionally unique among the known sodium or potassium channel toxins: APETx2, which inhibits acid-sensing ion channels in sensory neurons; BDS-I and II, which show selectivity for Kv3.4 channels and APETx1, which inhibits human ether-a-go-go-related gene potassium channels. In addition, structurally novel peptide toxins, such as an epidermal growth factor (EGF)-like toxin (gigantoxin I), have also been isolated from some sea anemones although their functions remain to be clarified.

[Biosafety. Phycotoxins].

General groups of phycotoxins are discussed. Classification of phycotoxins depending on their source organisms and syndromes of poisoning is presented. Domoic acid--a member of ASP-group is described in details. The chemical structure and properties of toxin, its source organisms and occurence in food web, mechanism of action, toxicity, tolerable contaminant levels in food and clinical presentation of poisoning are presented.

Toxinas Algales en el Mar Argentino: nuevos hallazgos, nuevos desafíos / Algal Toxins in the Argentine Sea: new findings, new challenges

Resumen Las floraciones de algas nocivas son un problema cada vez más frecuente a nivel mundial que ocasiona severos daños sobre la salud pública, pérdidas económicas en acuicultura, perjuicios al turismo y episodios de mortalidad de poblaciones naturales de peces, aves y mamíferos marinos. Las toxinas son producidas por el fitoplancton y se acumulan en moluscos bivalvos que se alimentan por filtración del agua siendo estos los principales vectores de intoxicación humana. En el Mar Argentino, se han reportado toxinas marinas de origen microalgal asociadas con cuatro síndromes de intoxicación por moluscos. Los síndromes más graves por su extensión, frecuencia, toxicidad y organismos afectados, son los originados por el dinoflagelado Alexandrium cate-nella responsable de la Intoxicación Paralizante por Moluscos la cual ha ocasionado numerosas muertes humanas. Seguidamente, la más leve, en cuanto a gravedad y frecuencia, ha sido la Intoxicación Diarreica por Moluscos. En contraste, el ácido domoico, conocido como toxina amnésica de moluscos, no ha producido hasta ahora intoxicaciones humanas. Recientemente, se amplió el rango de toxinas para la región al registrarse las toxinas y los dinoflagelados productores de la Intoxicación Azaspirácidos por Moluscos. Además, se han detectado las potencialmente tóxicas Yessotoxinas y Espirolidos, cuyos mecanismos de acción y toxicidad están siendo aún evaluados a nivel mundial. Estas toxinas emergentes para la región, representan un riesgo potencial para la salud e inconvenientes socioeconómicos por el cierre de los sitios de explotación de moluscos. Ciertamente presentan un nuevo desafío, pues la detección y cuantificación sólo puede realizarse por medio de métodos basados en HPLC - espectrometría de masas, lo cual dificulta el monitoreo en laboratorios regionales en el país. La herramienta clave de manejo es la prevención, a través de políticas, regulaciones y sistemas de monitoreo y control de cada grupo de toxinas. A través de estas mejoras, se anticipa que no sólo disminuirá el número de afectados por estas intoxicaciones, si no que se podrán realizar vedas más eficientes, asegurando un equilibrio que proteja tanto la salud pública como el desarrollo de la industria pesquera.

Abstract Harmful algal blooms are an increasingly common problem worldwide, causing severe damage to public health, economic losses in aquaculture, damage to tourism and mortality events of natural populations of fish, birds and marine mammals. The toxins are produced by phytoplankton and accumulated in bivalve molluscs that feed on water filtration, being these main vectors of human intoxication. In the Argentine Sea marine toxins of microalgal origin have been reported associated with four shellfish poisoning syn-dromes. The most serious due to their extension, frequency, toxicity and affected organisms are those caused by the dinoflagellate Alexandrium catenella responsible for the Paralytic shellfish poisoning that has caused numerous human deaths. Then, the mildest, in severity and frequency, is the Diarrhetic shellfish poisoning. In contrast, domoic acid, known as Amnesic shellfish toxin, has not produced human intoxications yet. Recently, toxins and dinoflagellate species causing Azaspiracid shellfish poisoning have been re-corded, expanding the range of toxins for the region. In addition, the potentially toxic Yessotoxins and Spirolides have been detected, whose mechanism of action and toxicity is still being evaluated worldwide. These emerging toxins represent a potential risk to public health and socioeconomic activities due to the eventual closure of mollusc exploitation sites. They certainly present a new challenge, since detection and quantification can only be carried out using methods based on HPLC - mass spectrometry, which makes monitor-ing in regional laboratories difficult. Prevention through policies, regulations, and monitoring and control systems of each toxin group is the key management tool. These preventive measures are expected to contribute to reducing the number of poisonings and to ap-plying more efficient fisheries closures, ensuring a balance that protects both public health and the development of the fishing industry.

Detection of five new hydroxyl analogues of azaspiracids in shellfish using multiple tandem mass spectrometry.

The polyether dinoflagellate toxins, azaspiracids, are responsible for azaspiracid poisoning (AZP), a new human toxic syndrome arising from the consumption of shellfish. To date, five azaspiracids have been isolated and fully structurally elucidated, including, AZA1, its 8-methyl and 22-demethyl analogues, AZA2 and AZA3, respectively, and two hydroxyl derivatives of AZA3, named AZA4 and AZA5. Using a recently developed method involving liquid chromatography with multiple tandem mass spectrometry (LC-MS(n)), five new azaspiracids, AZA7-AZA11, have been found in mussels (Mytilus edulis). AZA6 is a positional isomer of AZA1 and four of the new compounds are isomers with a mass of 857.5 amu. AZA7 and AZA8 are hydroxyl analogues of AZA1 while AZA9 and AZA10 are hydroxyl analogues of AZA6. AZA11 is a hydroxyl analogue of AZA2. The separation of all 11 azaspiracids was achieved using isocratic reversed phase liquid chromatography using a combination of eluent additives, trifluoroacetic acid and ammonium acetate. The ion-trap MS experiments, with electrospray ionisation, involved the fragmentation of the protonated molecule [M+H](+), trapping and fragmenting the product ions due to the loss of a water molecule [M+H-H(2)O](+), together with mass spectral data analysis that included the characteristic A-ring fragmentation for each compound.

Isolation of 45-hydroxyyessotoxin from mussels of the Adriatic Sea.

The diarrhetic shellfish toxin composition in the hepatopancreas of mussels from the northern Adriatic sea was investigated. The major toxins were shown to be yessotoxin (YTX), homoyessotoxin (homoYTX) and 45-hydroxyyessotoxin (45-OHYTX), identified by comparison of their chromatographic and spectral properties with those reported in the literature.

The chemistry of brevetoxins: a review.

The structure of the unique 'red tide' dinoflagellate neurotoxin, brevetoxin-B is presented and the experimental data supporting the chemical structure is discussed. A brief account of the other brevetoxins and their structural relationships is also presented. A biosynthetic scheme for the natural formation of the brevetoxin skeleton is proposed. Studies of the most toxic of the three pure brevetoxins, brevetoxin-A, indicate a skeleton differing from that of brevetoxin-B.

Liquid chromatography--multiple tandem mass spectrometry for the determination of ten azaspiracids, including hydroxyl analogues in shellfish.

Azaspiracids (AZAs) are a group of polyether toxins that cause food poisoning in humans. These toxins, produced by marine dinoflagellates, accumulate in filter-feeding shellfish, especially mussels. Sensitive liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS(n)) methods have been developed for the determination of the major AZAs and their hydroxyl analogues. These methods, utilising both chromatographic and mass resolution, were applied for the determination of 10 AZAs in mussels (Mytilus edulis). An optimised isocratic reversed phase method (3 microm Luna-2 C18 column) separated 10 azaspiracids using acetonitrile/water (46:54, v/v) containing 0.05% trifluoroacetic acid (TFA) and 0.004% ammonium acetate in 55 min. Analyte determination using MS3 involved trapping and fragmentation of the [M + H]+ and [M + H - H2O]+ ions with detection of the [M + H - 2H2O]+ ion for each AZA. Linear calibrations were obtained for AZA1, using spiked shellfish extracts, in the range 0.05-1.00 microg/ml (r2 = 0.997) with a detection limit of 5 pg (signal : noise = 3). The major fragmentation pathways in hydroxylated azaspiracids were elucidated using hydrogen/deuterium (H/D) exchange experiments. An LC-MS3 method was developed using unique parent ions and product ions, [M + H - H2O - CgH10O2R1R3]+, that involved fragmentation of the A-ring. This facilitated the discrimination between 10 azapiracids, AZA1-10. Thus, this rapid LC-MS3 method did not require complete chromatographic resolution and the run-time of 7 min had detection limits better than 20 pg for each toxin.

The mouse bioassay for diarrhetic shellfish poisoning: a gross misuse of laboratory animals and of scientific methodology.

The UK shellfish industry has recently been affected by the statutory closure of several cockle beds, following the detection of samples causing rapid and severe reactions in the regulatory approved test for diarrhetic shellfish poisoning (DSP) toxins, the mouse bioassay (MBA). It is contended that these so-called atypical results are due to procedural artefacts of the MBA; so far, several studies have failed to identify their cause. This paper critically assesses the development, regulatory use and methodological deficiencies of the MBA. It also discusses how testing for DSP toxins could and should have been improved and made more humane by applying the Three Rs concept of Reduction, Refinement and Replacement, and by the proper validation of the test method used. It is concluded that the MBA should not have been developed for the routine screening of shellfish samples, as it has a substantially severe endpoint and is not used as part of a tiered-testing strategy with non-animal methods. Moreover, during the UK monitoring programme for DSP toxins, the assay has been used without an optimised and universal protocol, and apparently without due regard to the principles of basic scientific methodology. In view of this, the atypical results obtained for cockle samples cannot be relied on as evidence of a human health hazard. It is recommended that the use of the MBA should be discontinued as soon as possible, in favour of other methods, especially those involving non-animal techniques. In the short-term, these methods should be based on analytical chemical detection systems and the essential availability of the relevant pure toxin standards. The lack of any known toxins in samples should be taken as evidence of lack of contamination. The suitability of the existing non-animal methods needs to be assessed as a matter of urgency. It is crucial that all new methods should be properly validated, and that their acceptability for their stated purposes should be endorsed by recognised criteria and validation centres, before being recommended to, or required by, regulatory agencies. In this way, the possibility that scientifically unsuitable methods will once again be used for monitoring for the contamination of shellfish with toxins can be avoided. This gross misuse of laboratory animals and ill-judged application of science should never be allowed to occur again.

Characterizing the evolution and functions of the M-superfamily conotoxins.

Conotoxins from cone snails are valuable in physiology research and therapeutic applications. Evolutionary mechanisms of conotoxins have been investigated in several superfamilies, but there is no phylogenetic analysis on M-superfamily conotoxins. In this study, we characterized identical sequences, gene structure, novel cysteine frameworks, functions and evolutionary mechanisms of M-superfamily conotoxins. Identical M-superfamily conotoxins can be found in different Conus species from the analysis of novel 467 M-superfamily conotoxin sequences and other published M-superfamily conotoxins sequences. M-superfamily conotoxin genes consist of two introns and three exons from the results of genome walking. Eighteen cysteine frameworks were identified from the M-superfamily conotoxins, and 10 of the 18 may be generated from framework III. An analysis between diet types and phylogeny of the M-superfamily conotoxins indicate that M-superfamily conotoxins might not evolve in a concerted manner but were subject to birth-and-death evolution. Codon usage analysis shows that position-specific codon conservation is not restricted to cysteines, but also to other conserved residues. By analysing primary structures and physiological functions of M-superfamily conotoxins, we proposed a hypothesis that insertions and deletions, especially insertions in the third cysteine loop, are involved in the creation of new functions and structures of the M-superfamily conotoxins.

Characterization, phylogenetic analysis and cDNA cloning of natterin-like gene from the blood of lamprey, Lampetra japonica.

Lamprey as a "living fossil" of immunological origin and "rich treasure" of biological pharmaceutical development has caused attention of scholars. The cDNA library construction and EST sequencing of blood had been done previously in our lab, and bioinformatics analysis provided a gene fragment which is highly homologous with natterin family, named natterin-like. To elucidate the characterization and phylogeny of natterin-like genes in early evolution, we cloned the full-length cDNA of natterin-like gene from the blood of Lampetra japonica. The open reading frame of this sequence contained 942bp and encoded 313 amino acids, including a lectin-like domain and a pore-forming toxin-like domain. Using reverse transcription PCR, natterin-like mRNA was also detected in lamprey blood, kidney, heart, liver, medullary, gonad, but absent in lamprey intestine and gill. Our results suggested that in lampreys and most of other species, there might be only one natterin-like gene, which was fused by certain sequences during evolution and encoded proteins with more functions. It is similar between C terminal of natterin-like protein and Aerolysin in space structure and the lectin-like domain of natterin-like equivalent to glycoprotein binding motif of Aerolysin in function. We also propose that the defense mechanism against specific predators in historical evolution of lamprey. Our findings may provide insights into the function and characterization of natterin-like genes as well as other gene families in vertebrates and provide a foundation for identification and structural, functional, and evolutionary analyses of more natterin-like genes and other gene families.

[Structures and functions of animal toxins]. / Structures et fonctions de toxines animales.

This brief review is devoted to the presentation of the major toxic proteins found in venoms of animals from five phyla. It is shown that various groups of venomous animals, including scorpions and snakes, produce toxins that exert different functions although they adopt a similar structure, suggesting that these toxins result from a divergent evolution. On the opposite, it is shown that toxins produced by animals from different phyla can exert similar functions even though they adopt unrelated structures, suggesting a convergent evolution. Finally, this review describes, at the molecular level, the functional and structural properties, including the dynamical characteristics, of a snake toxin which binds to the peripheral nicotinic acetylcholine receptor.

Toxins from sea cucumbers (holothuroids): chemical structures, properties, taxonomic distribution, biosynthesis and evolution.

Studies on structures, biological activities, chemical properties, taxonomic distribution, biosynthesis, and evolution of toxins from sea cucumbers (the phylum Echinodermata, the class Holothurioidea) were reviewed with special emphasis on recent results from our laboratory. These toxins are triterpene oligoglycosides having very often one or several sulfate groups in carbohydrate moieties. Their aglycones belong to lanostane derivatives and sometimes contain shortened side chains. Many aglycones are labile in the acid medium. There is a relationship between structures of the glycosides and taxonomic positions of corresponding animals, producers of these toxins. Toxins from sea cucumbers act on delta 5-sterol-containing biological membranes with the formation of glycoside-sterol complexes followed by the disturbance of membrane permeability and inhibition of activities of some membrane enzymes. The presence of the toxins causes the alterations in membrane sterol compositions of toxic sea cucumbers in comparison with non-toxic species. These alterations include the change of delta 5-sterols for those having 7(8)- and 9(11)-double bonds as well as biotransformation of a part of free sterol fractions into sterol sulfates and sterol xylosides.