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.

Photochemistry and Photophysics of Shinorine Dimethyl Ester.

The photostability and photophysical properties of the dimethyl ester of the mycosporine-like amino acid shinorine have been experimentally evaluated in aqueous solution and in the presence of direct micelles prepared with a cationic or an anionic detergent, respectively. In comparison with shinorine, the ester molecule increases the photostability, the fluorescence quantum yield and the fluorescence lifetime in water as well as in the micellar solutions. The effects are more pronounced in sodium dodecyl sulfate solutions and suggest that the electrostatic attractions with the micellar interface contribute to limit the movement of the molecules and influence the relative rate of their deactivation channels. However, the predominance of the nonradiative decay is maintained together with the UV photoprotective ability of this atypical mycosporine species.

Characterization of mycosporine-serine-glycine methyl ester, a major mycosporine-like amino acid from dinoflagellates: a mass spectrometry study.

Several unknown mycosporine-like amino acids (MAAs) have been previously isolated from some cultured species of toxic dinoflagellates of the Alexandrium genus (Dinophyceae). One of them, originally called M-333, was tentatively identified as a shinorine methyl ester, but the precise nature of this compound is still unknown. Using a high-resolution reversed-phase liquid chromatography mass spectrometry analyses (HPLC/MS), we found that natural populations of the red tide dinoflagellate Prorocentrum micans Ehrenberg showed a net dominance of M-333 together with lesser amounts of other MAAs. We also documented the isolation and characterization of this MAA from natural dinoflagellate populations and from Alexandrium tamarense (Lebour) Balech cultures. Using a comparative fragmentation study in electrospray mass spectrometry between deuterated and non-deuterated M-333 compounds and synthesized mono and dimethyl esters of shinorine, this novel compound was characterized as mycosporine-serine-glycine methyl ester, a structure confirmed by nuclear magnetic resonance. These isobaric compounds can be differentiated by their fragmentation patterns in MS(3) experiments because the extension and the specific site of the methylation changed the fragmentation pathway.

Mycosporine-like amino acids: relevant secondary metabolites. Chemical and ecological aspects.

Taxonomically diverse marine, freshwater and terrestrial organisms have evolved the capacity to synthesize, accumulate and metabolize a variety of UV-absorbing substances called mycosporine-like amino acids (MAAs) as part of an overall strategy to diminish the direct and indirect damaging effects of environmental ultraviolet radiation (UVR). Whereas the enzymatic machinery to synthesize MAAs was probably inherited from cyanobacteria ancestors via the endosymbionts hypothesis, metazoans lack this biochemical pathway, but can acquire and metabolize these compounds by trophic transference, symbiotic or bacterial association. In this review we describe the structure and physicochemical properties of MAAs, including the recently discovered compounds and the modern methods used for their isolation and identification, updating previous reviews. On this basis, we review the metabolism and distribution of this unique class of metabolites among marine organism.

Analyses of photoprotective compounds in red algae from the Brazilian coast

Qualitative and quantitative studies of mycosporine-like amino acids (MAAs) in three species of the genus Gracilaria Greville (G. birdiae, G. domingensis and G. tenuistipitata) were performed. A simple and efficient extraction procedure based on ethanol was described. HPLC, UV and mass spectrometry experiments revealed different profiles between extracts obtained from one species cultivated in the laboratory (G. tenuistipitata) and two species collected in their natural environment (G. birdiae and G. domingensis). The levels detected in the latter two species were approximately 150 times higher than in the species cultivated in vitro. This study revealed that G. birdiae and G. domingensis present a potential source for economical exploration of MAAs.

Toxin variability in cultured and natural populations of Alexandrium tamarense from southern South America - Evidences of diversity and environmental regulation.

Cell content and composition of paralytic shellfish toxins of 10 cultured strains and 6 natural populations of Alexandrium tamarense from the Argentine sea, were analyzed. These data were compiled with previously published data into a comprehensive view of the toxin composition of the complex A. tamarense/Alexandrium catenella from southern South America. The N-sulfocarbamoyl derivatives C1,2 were predominant in almost all the cultured strains. The second major derivatives were GTX1,4, although the GTX1,4/C1,2 ratio varied largely. Some strains contain relatively high amounts of GTX2,3 (up to 29%) and/or neoSTX (up to 24%). In all strains STX was a minor component (0-4.4%) whereas GTX5 was present only in Alexandrium catenella isolates. Similarity analysis based upon toxin profiles showed that cultured strains from Argentine, Brazil, Chile and Uruguay clustered together. However, whereas some strains from the same geographic area exhibited different toxin profiles, and consistently fell out in separate subgroups, strains from Chile are grouped in a unique subgroup. In contrast to cultured strains, C1,2 were minor components among field populations. The highly toxic GTX1,4 were predominant in all spring field populations (69.1-93.6%). Moreover, their toxin cell content (163.9-261.4 fmol cell(-1)) and toxicity (68.2-93.0 pg STX equiv. cell(-1)) were several times higher that showed by the cultured strains. Field populations are more closely related to one another than to the cultured strains. However a less toxic and morphologically distinctive autumn population, contained GTX2,3 as the quasi unique (88.5%) toxin derivative clustered separately. Variability in toxin content and composition of A. tamarense field populations were well correlated with in situ temperature and nitrate concentration. Whereas toxin cell content and GTX1,4 (mol %) increased following saturation functions, GTX2,3 (mol %) decrease exponentially with the increase of the in situ nitrate concentration.

Mycosporine-like amino acid content in the sea anemones Aulactinia marplatensis, Oulactis muscosa and Anthothoe chilensis.

The occurrence of mycosporine-like amino acids (MAAs) in the sea anemones Aulactinia marplatensis (Zamponi, 1977), Oulactis muscosa (Drayton in Dana, 1846) and Anthothoe chilensis (Lesson, 1830), from the rocky intertidal habitats on the coast of Mar del Plata, Argentina, was assessed by HPLC. The pattern of MAAs in the mussel Brachidontes rodriguezi, main component of the diet for A. marplatensis and O. muscosa, was as well determined. The results were comparatively analyzed together and with previously reported MAA content in species mainly of the genus Anthopleura. The correlation between the MAA concentration and light availability of their habitats is in line with the photoprotective role assigned to the compounds. The high proportion of mycosporine-taurine in the three species and the results for the evaluation of MAAs in the mussels point to a non-dietary origin or a regulated biotransformation metabolism of dietary MAAs and/or their precursors that is common to sea anemones.

Palythine-threonine, a major novel mycosporine-like amino acid (MAA) isolated from the hermatypic coral Pocillopora capitata.

Using a high-resolution reverse-phase liquid chromatography method we found that the tissues of the hermatypic coral Pocillopora capitata (collected in Santiago Bay, Mexico) contain a high diversity of primary and secondary mycosporine-like amino acids (MAAs) typical of some reef-building coral species: mycosporine-glycine, shinorine, porphyra-334, mycosporine-methylamine-serine, mycosporine-methylamine-threonine, palythine-serine, palythine and one additional novel predominant MAA, with an absorbance maximum of 320 nm. Here we document the isolation and characterization of this novel MAA from the coral P. capitata. Using low multi-stage mass analyses of deuterated and non deuterated compounds, high-resolution mass analyses (Time of Flight, TOF) and other techniques, this novel compound was characterized as palythine-threonine. Palythine-threonine was also present in high concentrations in the corals Pocillopora eydouxi and Stylophora pistillata indicating a wider distribution of this MAA among reef-building corals. From structural considerations we suggest that palythine-threonine is formed by decarboxylation of porphyra-334 followed by demethylation of mycosporine-methylamine-threonine.

In vitro cis-trans photoisomerization of palythene and usujirene. Implications on the in vivo transformation of mycosporine-like amino acids.

The in vitro photoinduced reactions of the mycosporine-like amino acids (MAA) usujirene and palythene were studied by monochromatic stationary irradiation at 366 nm. High-performance liquid chromatography analysis of the irradiated aqueous solution of usujirene indicated a low photoreactivity on the basis of the observed photodecomposition quantum yield of phi(-U) = (2.86 +/- 0.80) x 10(-5), which can be partially accounted for by the cis-trans photoisomerization of usujirene to palythene (phi(U-->P) = [1.71 +/- 0.13] x 10(-5)). However, palythene in aqueous solution showed a higher photostability than did usujirene under equivalent conditions, establishing a photostationary mixture of cis-trans isomers with a relative composition of palythene-usujirene (11:1). These results may explain the preferential in vivo accumulation of palythene relative to that of usujirene observed in several dinoflagellate species.