We have already shown the presence of guanidine neurotoxins in calcareous red algae and mussels collected in the São Sebastião channel (State of São Paulo, Brazil). It is known that these neurotoxins comprise more than 25 analogues such as tetrodotoxin (TTX) and derivatives plus the paralytic shellfish toxins (PST) found in a variety of marine, freshwater and amphibious species. Filter feeding animals generally possess large amounts of these neurotoxins. The tunicates are sessil marine animals with a high rate of sea water filtration. The tunics and siphons of 50 specimens of Phallusia nigra were dissected and the visceral organs were immersed in methanol containing acetic acid 0.02 N pH 5.0. The extract was prepared by homogenization, filtration and the methanolic phase was concentrated under reduced pressure and defatted with chloroform. The polar phase was evaporated and the residue dissolved in deionized water for further purification in ionic-exchange resin column (Bio-Gel P-2) and HPLC analysis. The extract showed paralytic effects on mouse assay (26.9 MU/100mg) and on crustacean isolated nerve preparations. The chemical analysis for TTX and PST revealed toxins with retention times similar to gonyautoxins, saxitoxins and TTX. These findings are important to explain future toxin envenoming outbreaks on the Brazilian coast.
Phallusia nigra; tunicate; paralytic shellfish toxins; tetrodotoxin
OCCURRENCE OF TETRODOTOXIN AND PARALYTIC SHELLFISH TOXINS IN Phallusia nigra (TUNICATA, ASCIDIACEA) FROM THE BRAZILIAN COAST.
J. C. FREITAS, T. OGATA , C.H. VEIT , M. KODAMA
1 Department of General Physiology and Marine Biology Center, Institute of Biosciences, University of São Paulo, State of São Paulo, Brazil, 2 Department of Marine Biochemistry, School of Fisheries Sciences, Kitasato University, Iwate 022-01, Japan.
ABSTRACT: We have already shown the presence of guanidine neurotoxins in calcareous red algae and mussels collected in the São Sebastião channel (State of São Paulo, Brazil). It is known that these neurotoxins comprise more than 25 analogues such as tetrodotoxin (TTX) and derivatives plus the paralytic shellfish toxins (PST) found in a variety of marine, freshwater and amphibious species. Filter feeding animals generally possess large amounts of these neurotoxins. The tunicates are sessil marine animals with a high rate of sea water filtration. The tunics and siphons of 50 specimens of Phallusia nigra were dissected and the visceral organs were immersed in methanol containing acetic acid 0.02 N pH 5.0. The extract was prepared by homogenization, filtration and the methanolic phase was concentrated under reduced pressure and defatted with chloroform. The polar phase was evaporated and the residue dissolved in deionized water for further purification in ionic-exchange resin column (Bio-Gel P-2) and HPLC analysis. The extract showed paralytic effects on mouse assay (26.9 MU/100mg) and on crustacean isolated nerve preparations. The chemical analysis for TTX and PST revealed toxins with retention times similar to gonyautoxins, saxitoxins and TTX. These findings are important to explain future toxin envenoming outbreaks on the Brazilian coast.
The neurotoxins responsible for paralytic poisoning of shellfish have been identified as saxitoxin (STX) and its analogues. These poisons are particularly dangerous due to their causing acute paralysis. Several authors report that filter feeding marine animals such as the high rate filtration ascidians develop toxins during blooms of toxic dinoflagellates.
Methanolic extracts obtained from the macroalgae Arthrocardia gardineri and Jania rubens from the State of São Paulo coast, Brazil, caused neurotoxic effects including the blocking of the action potentials of isolated nerves (1). Such effects could be ascribed to gonyautoxins (GTX), neo-saxitoxin (neo-STX), STX and tetrodotoxin (TTX) as detected by HPLC fluorometry analysis of the samples (2). Other papers have reported that some bacteria produce neurotoxins and/or transform them by enzymatic conversion and such toxigenic strains have been isolated from soil, freshwater and also from a number of marine organisms including red tide dinoflagellates, rodophyta macroalgae, crabs, tunicates and fish (7,8,9,10,12,13,17,18). We have also ascertained that the shellfish Perna perna from the Brazilian coast had GTX 4,1,5,3,2, STX, neo-STX and the isolated bacterial strains of the mussel digestive glands contained GTXs 4,1,3,2, STX, neo-STX and trace amounts of TTX (3). In this paper we have extended the occurrence of paralytic shellfish toxins (PST) to the tunicate Phallusia nigra another species from the Brazilian coast, although there is no confirmation of the presence of toxic dinoflagellates in this region.
MATERIAL AND METHODS
MATERIALS: Samples of Phallusia nigra were collected on the rocky shores of the São Sebastião channel, close to the Marine Biology Center, University of São Paulo, Brazil. The tunics and siphons of each of the 50 specimens of ascidians were dissected and visceral organs extract were prepared by homogenization and filtration. The methanolic phase was concentrated under reduced pressure. The residue was dissolved in deionized water for further mouse bioassay, nerve preparation tests, purification in ionic exchange resin column and HPLC analysis.
TOXICITY BIOASSAY AND NERVE PREPARATION: The mouse bioassay for toxicity was made by intraperitoneal injections (male mice of BALB/CxAJ/F1, 17-23 g), according to the standard method of A.O.A.C. (5) and expressed as mouse unit (MU) where 1.0 MU is a dose of toxin to kill a 20 g-mouse in 15 minutes.
Nerve preparation was performed using crab leg sensory nerve. A walking leg was isolated from an adult blue crab (Callinectes danae) and its nerve exposed by cutting the arthrodial membranes, condylic articulations and muscle apodemes. The leg segments were removed leaving the nerve attached to the distal segment (dactylus). The preparation was mounted in a chamber which permitted mechanical stimulation of the dactyl mechanorreceptors by sea water drops falling from 10 cm in height, so that the conducting trains of action potentials could be recorded by a suction electrode located in the nerve proximal end. The action potentials were amplified by a P-15 A-C pre-amplifier (Grass Instruments) integrated (constant time 0.2 second) and recorded with a R411 Dynograph Beckmann (Figure 1). The preparation was kept in physiological solution in the chamber for at least two hours. The integrated amplitude of the action potential trains was recorded before, during and after addition of the test extract or toxin standards to the sensory nerve, as previously reported (11).
Electrophysiological set up diagram of the crab leg sensory nerve preparation. AM = audio monitor; OA = oscilloscope amplifier; IA = integrator amplifier; PR = pen recorder; PA = preamplifier; FSW = filtered sea water bottle; WD = water drop; LC = laboratory clamp; IE = indifferent electrode; SE = suction electrode; RW = rubber wall; PD = dactylus; PS = physiological solution; NC = nerve chamber; FC = Faraday cage. The arrow shows the place for drug addition.
The extract was partially purified with Bio-Gel P-2 chromatography. The Toxins adsorbed by the gel were eluted with 0.03 N AcOH. The toxic fraction was analyzed by HPLC -fluorometric analysis according to Oshima et al. (14).
Data on the different samples of mouse bioassay have shown a toxicity ranging from 6.2 MU/ml up to 53.9 MU/ml extract. The symptoms observed in mice were paralysis and respiratory failure, similar to those caused by STX and TTX.
The effect of Phallusia nigra methanolic extract on the crab leg sensory nerve preparation is shown in Figure 2. In all representative recordings a dose dependence was noticed. The inhibition of the action potential discharges could be fully reversed by repetitive washings.
FIGURE 2. Representative sample recordings of the effects of the
extract on the crab leg sensory nerve preparation. W = washings .
The HPLC analysis carried out with the Pallusia nigra purified extracts showed the presence of GTX 1,3 and 2, STX, neo-STX and TTX. GTX 4 and GTX 5 were undetectable in all samples and the presence of TTX was seen only in some samples (Figure 3). Table 1 shows the mean concentrations in MU/ml and the mean retention times of each GTX standard and GTXs found in the purified extract of Phallusia nigra, after HPLC analysis.
Top: standard toxins. Bottom: purified extract.
. Mean concentrations and retention times in HPLC of standard gonyautoxins and gonyautoxins component of the purified extract of Phallusia nigra.
Some ascidians such as Phallusia nigra have chemical defense systems, a high vanadium content and an acidic tunic protection from predators (16). In addition, other species of tunicates are also a source of important anti-viral, anti-tumor or anti-leukemic compounds (6,15).
Halocynthia roretzi, an ascidian species edible in Japan, is known to cause occasional food poisoning with no lethality. The HPLC analysis of H. roretzi demonstrated the presence of GTXs and STXs (4). A previous study reported that bacterial strains isolated from H. roretzi revealed the ability to convert GTXs 1,2 and 3 into STX by reductively eliminating N-1 hidroxyl and C-11 hydroxysulphate groups (10). In regard to P. nigra, the chemical analysis of the partially purified extracts for TTX and PSTs showed toxins with retention times similar to GTXs, STXs and TTX. However, TTX was found only in one of the analyzed samples in very small amounts. The toxins may be exogenous and it is probable that the fact that some toxins have not been detected derives from seasonal variation of the causative plankton species and/or microorganisms (3) on the Brazilian coast.
As far as we know, human consumption of ascidians occurs in Japan and in Chile. Thus, with regard to public health, it is important to know about the occurrence of paralytic toxins in this group of filter feeding marine animals.
J.C. FREITAS, Departamento de Fisiologia Geral e Centro de Biologia Marinha, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Travessa 14, nº 321, Cidade Universitária, Caixa Postal 11461, CEP 05422-970, São Paulo, Brasil.
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Publication in this collection
11 Oct 2001
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