Yasukatsu Oshima

Diarrhetic shellfish toxins

Abstract As the causative agents of a new type of shellfish poisoning, named diarrhetic shellfish poisoning, okadaic acid, 35(S)-methylokadaic acid, 7-O-acyl derivatives of 35(S)-methylokadaic acid, two novel polyether lactones named pectenotoxin-1 and -2 have been isolated and had their structures determined. Three pectenotoxin analogues were also present. In addition to the previously identified Dinophysis fortii, D. acuminata was newly suggested as a source of toxins.

The first evidence of paralytic shellfish toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii, isolated from Brazil

The blooms of toxic cyanobacteria (blue-green algae) are causing problems in many countries. During a screening of toxic freshwater cyanobacteria in Brazil, three strains isolated from the State of Sao Paulo were found toxic by the mouse bioassay. They all were identified as Cylindrospermopsis raciborskii by a close morphological examination. Extracts of cultured cells caused acute death to mice when injected intraperitoneally after developing neurotoxic symptoms which resembled to those caused by paralytic shellfish toxins. The analysis of the sample by HPLC-FLD postcolumn derivatization method for paralytic shellfish toxins resulted in the detection of several saxitoxin analogs. To avoid being misled by false peaks, the sample was reanalyzed after purification and also under the different postcolumn derivatizing conditions. Finally, the newly developed LC-MS method for paralytic shellfish toxins was applied to unambiguously identify the toxins. One isolate produced neosaxitoxin predominantly with saxitoxin as a minor component. The other two showed identical toxin profiles containing saxitoxin and gonyautoxins 2/3 isomers in the ratio of 1:9. This is the first evidence of paralytic shellfish toxins in this species and also the occurrence of the toxin producing cyanobacterium in South American countries.

Two analogs of azaspiracid isolated from mussels, Mytilus edulis, involved in human intoxication in Ireland.

Two new analogs of azaspiracid, azaspiracid-2 and azaspiracid-3, were isolated from mussels collected at Arranmore Island, Ireland in 1997 as additional causes of human intoxication. Their structures were determined to be 8-methylazaspiracid and 22-demethylazaspiracid, respectively by NMR and negative ion FAB CID MS/MS experiments.

New saxitoxin analogues from the freshwater filamentous cyanobacterium Lyngbya wollei.

Along with decarbamoylsaxitoxin and decarbamoylgonyautoxin-2 and -3, six new saxitoxin analogues were isolated from the freshwater mat-forming filamentous cyanobacterium Lyngbya wollei collected from Guntersville Reservoir on the Tennessee River in Alabama. Their structures were determined by electrospray ionization mass spectrometry and several NMR techniques. Five of the toxins contain an acetyl moiety attached to the side chain, which is the first report of these saxitoxin analogues. In three of the toxins a hydrated ketone at C-12 was reduced to alpha-alcohol. The presence of acetate in the side chain resulted in a sevenfold to 17-fold times decrease in mouse toxicity compared to their carbamoyl counterparts, while the reduction at C-12 resulted in a complete loss of mouse toxicity.

Comparative study on paralytic shellfish toxin profiles of the dinoflagellate Gymnodinium catenatum from three different countries

Paralytic shellfish toxin profiles of the dinoflagellate Gymnodinium catenatum Graham were investigated as a possible biochemical marker to distinguish different geographic populations of this species. Isolates obtained between 1986 and 1988 from Japan, Tasmania (Australia) and Galicia (Spain) were cultured under similar conditions and the toxins produced were analyzed using HPLC. Variations in temperature, salinity, and nitrate and phosphate levels in the culture medium had no significant effect on the toxin profile, suggesting that toxins can be used as a stable biochemical marker for this dinoflagellate. All the isolates produced mainly toxins of the N-sulfocarbamoyl group (C1–C4, gonyautoxins 5 and 6) but their relative abundance differed according to their geographic origin. Furthermore, only the Australian population produced the newly found 13-deoxydecarbamoyl toxins, and these could readily be used to distinguish the Australian populations from those of the other two countries.

Dinoflagellate Gymnodinium catenatum as the source of paralytic shellfish toxins in Tasmanian shellfish

Paralytic shellfish toxins in both cultured cells and natural phytoplankton blooms of the dinoflagellate Gymnodinium catenatum from inshore Tasmanian waters (Australia) were analyzed by high performance liquid chromatography, thin layer chromatography and electrophoresis techniques. The dinoflagellate toxins were dominated by low potency sulfocarbamoyl saxitoxin derivatives (98-99 mole% in total), including gonyautoxin VIII (C2) and its epimer (C1) and sulfocarbamoyl gonyautoxins I and IV (C3 and C4). Mussels and oysters contaminated by the dinoflagellate showed similar toxins, but contained larger proportions of C3 (40-57 mole%) and more potent carbamate toxins (7-23 mole% total).

Paralytic shellfish toxins in the freshwater cyanobacterium Aphanizomenon flos-aquae, isolated from Montargil reservoir, Portugal

Montargil reservoir, located in a dry flat area in the centre of Portugal, was filled in 1958 to fulfil agricultural, electric and industrial requirements. In May 1996, an intensive bloom of phytoplankton was detected. The algal community was strongly dominated by cyanobacteria with predominance of Aphanizomenon flos-aquae from May to June and Microcystis aeruginosa from July to August. Extracts of samples collected during the bloom period showed high toxicity by mouse bioassay. During the M. aeruginosa predominance period, the toxicity was ascribed to the presence of hepatotoxins, but clear symptoms of paralytic shellfish poison were observed when A. flos-aquae was the dominant species. In order to confirm the production of neurotoxins a strain of A. flos-aquae was isolated and established in culture. In this manuscript, we show the morphological characteristics and confirm paralytic shellfish toxins production by the strain isolated and maintained in culture. Identification of the saxitoxin analogs was achieved using high performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD) and liquid chromatographic mass spectrometry technique (LC-MS). The toxins found in the culture extract were GTX5 (64.5 mol%), neoSTX (23.0 mol%), dcSTX (6.1 mol%), STX (5.4 mol%) and GTX6 (1.1 mol%). This is, to our knowledge, the first report of unambiguous evidence of paralytic shellfish toxins produced by freshwater cyanobacteria in Portugal. The toxin profile is rather different from the previously reported PSP producing A. flos-aquae and demonstrates its diversity in terms of toxin production.

THE FRESHWATER CYANOBACTERIUM PLANKTOTHRIX SP. FP1: MOLECULAR IDENTIFICATION AND DETECTION OF PARALYTIC SHELLFISH POISONING TOXINS

A filamentous cyanobacterium, belonging to the Order of Oscillatoriales, was found to be responsible for a toxic algal bloom in Lake Varese, Italy, during the summer of 1997. Morphological characters, as well as near complete 16S rRNA gene sequencing, revealed that the dominant species of the bloom was most closely related to the genus Planktothrix. In addition, genetic analysis of the phycocyanin operon of Planktothrix sp. FP1 revealed a novel primary structure, previously undescribed within the cyanobacteria, which was used as a genetic marker for rapid detection and identification of this toxic strain. The occurrence of saxitoxin (STX), a principal toxin in paralytic shellfish poisoning (PSP), was confirmed in the natural bloom sample by both pre‐column and post‐column derivatization high‐performance liquid chromatography (HPLC) analyses, and eventually by liquid chromatography/mass spectrometry (LC/MS). The toxicity of this field sample was also revealed by electrophysiological assays in which the extract inhibited 90% of the voltage‐dependent Na+ current in human neuroblastoma cells at the STX concentration of 80 nM. The cultured strain showed a lower physiologic activity than the bloom sample (67% blockage of Na+ current at a toxin concentration of 200 nM), and STX was detected only by pre‐column HPLC, indicating the presence of a compound structurally close to STX. Chemical and molecular genetic analyses performed here add Planktothrix sp. FP1 to the growing list of diverse cyanobacterial species capable of synthesizing STX and its related compounds.

Gymnocin-a, a cytotoxic polyether from the notorious red tide dinoflagellate, gymnodinium mikimotoi

A new cytotoxic polyether, gymnocin-A, was isolated from the notorious red tide dinoflagellate, Gymnodinium mikimotoi, and its structure consisting of 14 contiguous ether rings and a 2-methyl-2-butenal side-chain was determined by NMR and CID MS/MS experiments.

Toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii (Cyanophyceae) isolated from Tabocas reservoir in Caruaru, Brazil, including demonstration of a new saxitoxin analogue

Abstract Cyanobacteria can produce biotoxins that are significant hazards to humans. After the intoxication incident in 1996 at the city of Caruaru, Brazil, a phytoplankton-monitoring programme was established at its main water supply, the Tabocas reservoir. Data obtained during 1997 and 1998 revealed the dominant species at Tabocas to be Cylindrospermopsis raciborskii, which was responsible for a massive bloom observed in July–October 1998. Laboratory cultures of isolate ITEP-018 demonstrated highly toxic properties, mice inoculated with this strain exhibited the same symptoms as those of paralytic shellfish poisoning, with an acute lethal effect of 9.3 mouse units mg−1 of dry cells. Several saxitoxin analogues were identified in these cultures, specifically saxitoxin (3.3 mol% total toxin content), gonyautoxin 6 (6.4 mol%), decarbamoyl-saxitoxin (8.5 mol%), neosaxitoxin (17.1 mol%), and a new saxitoxin analogue, which proved to be the major product of the Tabocas strain, accounting for 64.6 mol% of the toxin present in the sample analysed. Additionally, decarbamoylneo-saxitoxin was detected by liquid chromatography-mass spectrometry. Cylindrospermopsis raciborskii strain ITEP-018 thus produces at least five saxitoxin analogues, including the most toxic ones as assessed by mouse bioassay.