IMPORTANCE OF THE BRAZILIAN COASTAL BIOME FOR TOXINOLOGY

JOSÉ CARLOS DE FREITAS(1)

(1)Departamento de Fisiologia, IB, USP, São Paulo, Brazil; Center for Applied Toxinology, CAT/CEPID- FAPESP, Instituto Butantan, São Paulo, Brazil; Email: jfreitas@usp.br Phone: 55-11-3091-7522. FAX: 55-11-3031-5028 Biosciences Institute,USP

During the last 15 years, our group have intensively collected species of marine organisms occurring along the Brazilian coast. We have been searching for toxins in a variety of ecological sites, as plankton, benthos and nekton. About 35 compounds were isolated and already identified, and some were novels, mainly from sponges, tunicates and sea-anemones found in São Sebastião channel, São Paulo State. Various bioassays are currently used, including hemolytic, anti-mitotic, neurotoxic, anti-inflammatory and cytotoxic. The evaluation, in some cases, follow up to binding assays and human cell line assays. By using four different techniques, as mouse bioassay, ELISA using T20G10 monoclonal antibody, HPLC and mass spectrometry, the toxicity in the extracts of Colomesus asellus, a freshwater pufferfish from the rivers in the Amazonian rain forest, was analysed and identified for the first time. The components responsible for its toxicity were only  Paralytic shellfish Poisons (PSPs) Saxitoxin (STX), Gonyautoxin 2 (GTX 2) and GTX 3. These data were also confirmed by electrospray ionization mass spectrometry. An indirect competitive enzyme immunoassay using tetrodotoxin (TTX) showed very low affinity for  C. asellus extracts, indicating that TTX and its analogues are not the main toxic components of these extracts. The antibody was efficient in detecting presence of TTX in a total extract of Sphoeroides spengleri, which is one of the most toxic puffer fish found in the Atlantic coast. Studies dealing with the venomous mollusk Conus regius from Fernando de Noronha Archipelagous (PE) and sea anemones Bunodosoma caissarum and B. cangicum, from São Paulo coast have shown a variety of biologically active peptides. We have  determined the sequences of novel conotoxins from the Brazilian cone snail, Conus regius, and peptide neurotoxins from the sea anemones, B. caissarum and B. cangicum, by MS and NMR analyses.  LAS390 is a low molecular weight and non-peptidic compound purified from B.cangicum venom that induces analgesia in rats. The characterization of this analgesic effect and  the mechanisms involved in this effect is evaluated in pain threshold using the rat paw pressure test, applied before and at different times after treatments. LAS390 induces analgesic effect that is mediated by histamine and serotonin receptors and involves activation of voltage-gated K+ channels. A new hemolytic toxin named caissarolysin I was isolated from the Brazilian sea anemone B. caissarum and identified as a protein of MW 19757, which lacks PLA2 activity but is recognized by antiserum against Equinatoxin II, a toxin isolated from Actinia equina of Mediterraneum Caissarolysin I has high hemolytic activity in human erythrocytes (ED50 = 0.270 g/ml) and was inhibited by pre-incubation with sphingomyelin. Another study dealing with the dinoflagellate Prorocentrum mexicanum have shown that its organic extract (polar) blocked action potentials of crustacean isolated nerves. Also there are evidences of apoptotic events induced by P. mexicanum polar extract in T47D Cells. These type of activity have been observed for okadaic acid and yessotoxin, known cytotoxins found in dinoflagellates from the Pacific coast. This coming years we are going to extend our collections of marine organisms in São Pedro and São Paulo Rocks (RN), Fernando de Noronha Archypelagous (PE) and Antartic ocean to follow up these studies.

ACKNOWLEDGEMENTS: FAPESP, CNPq.

CORRESPONDENCE TO:Departamento de Fisiologia, IB, USP, São Paulo, Brazil; Center for Applied Toxinology, CAT/CEPID- FAPESP, Instituto Butantan, São Paulo, Brazil; Phone: 55-11-3091-7522. FAX: 55-11-3031-5028 Biosciences Institute,USP, Email: jfreitas@usp.br