Shellfish poisons isolation

Other Lethal Agents. There are a number of substances, many found in nature, which are known to be more toxic than nerve agents (6). None has been weaponized. Examples of these toxic natural products include shellfish poison, isolated from toxic clams puffer fish poison, isolated from the viscera of the puffer fish the active principle of curare "heart poisons" of the digitaUs type the active principle of the sea cucumber active principles of snake venom and the protein ricin, obtained from castor beans (See Castor oil). 

Three classes of polyethers, okadaic acid derivatives, pectenotoxins, and yessotoxin were isolated from bivalves in connection with diarrhetic shellfish poisoning. The etiology of the toxins, toxicological properties, and determination methods are described. 

Heredia-Tapia, A., et al.. Isolation of Prorocentrum lima (S5U1. Exuviaella lima) and diarrheic shellfish poisoning (DSP) risk assessment in the Gulf of California, Mexico, Toxicon, 40, 8, 1121, 2002. 

Murata, M., Isolation and structural elucidation of the causative toxin of the diarrhetic shellfish poisoning, Bull. Jpn. Soc.Sci. Fish, 48, 549, 1982. 

Murata, M., Isolation and structure of yessotoxin, a novel polyether compound implicated in diarrhetic shellfish poisoning, Tet. Lett., 28, 5869, 1987. 

Nozawa, A., Tsuji, K. and Ishida, H., Implication of brevetoxin B1 and PbTx-3 in neurotoxic shellfish poisoning in New Zealand by isolation and quantitative determination with liquid chromatography-tandem mass spectrometry, Toxicon, 42, 1, 91, 2003. 

Domoic acid (Fig. 28,80) (263) is a neuro-phycotoxin responsible for the mortality of wildlife and for amnesic shellfish poisoning (ASP) of humans during algal bloom. Domoic acid was first isolated from the red alga Chondria armata ( domoi in Japanese), and it is produced also by diatoms, such as Pseudo-nitzschia spp. For the latter, evidence has been presented that it is involved in iron acquisition (307). 

The main genera responsible for freshwater toxic blooms are Microcystis, Anabaena, Aphanizomenon and Oscillatoria. Toxins produced include 1. anatoxins, alkaloids and peptides of Anabaena 2. the peptide microcystin and related peptides of Microcystis 3. aphantoxins, compounds of Aphanizomenon with properties similar to some paralytic shellfish poisons. Properties of Oscillatoria toxin suggest they are peptides similar to those of Microcystis. Microcystis toxins are peptides (M.W. approx. 1200) which contain three invariant D-amino acids, alanine, erythro-3-methyl aspartic and glutamic acids, two variant L-amino acids, N-methyl dehydro alanine and a 3-amino acid. Individual toxic strains have one or more multiples of this peptide toxin. The one anatoxin characterized is a bicylic secondary amine called anatoxin-a (M.W. 165). The aphantoxin isolated in our laboratory contains two main toxic fractions. On TLC and HPLC the fractions have the same characteristics as saxitoxin and neosaxitoxin. 

Earlier research had already suggested that certain blooms Aph. flos-aquae could produce paralytic shellfish poisons. These studies used water blooms collected from Kezar Lake, New Hampshire (25,30). In 1980 Carmichael isolated a neurotoxic strain of Aph. flos-aquae from a small pond in New Hampshire. These strains have also been shown to produce toxins similar to saxitoxin and neo- axitoxin (23) and are the ones used in the studies presented here. 

The neurotoxins isolated from Aph. flos-aquae were shown to have similar chemical and biological properties to paralytic shellfish poisons (PSP) (25,29,38) Sawyer et al. in 1968 (25) were the first to demonstrate that the crude preparation of aphantoxins behave like saxitoxin, the major paralytic shellfish poison. They showed that the toxins had no effect on the resting membrane potential of frog sartorius muscle blocked action potential on de-sheathed frog sciatic nerve and also abolished spontaneous contractions in frog heart. Sasner et al. (1981) (29) using the lab cultured strain reported similar results. 

Yessotoxin (214) is a polyether from the scallop Patinopecten yessoensis and has been implicated in diarrhetic shellfish poisoning (DSP). The structure and partial stereochemistry of yessotoxin were deduced from spectral data [219]. The relative stereochemistry of yessotoxin and the structures of two new analogues, 45-hydroxyyessotoxin (215) and 45,46,47-trinoryessotoxin (216) were also established [220]. The absolute stereochemistry of yessotoxin (214) was determined by NMR spectroscopy using a chiral anisotropic reagent [221]. The absolute configuration at C45 in 45-hydroxyyessotoxin (215), isolated from P. yessoensis, was determined by the use of a modified Mosher s method [222]. 

The cockle, Austrovenus stutchburyi from New Zealand contained brevetoxin Bi (225) [230] and the greenshell mussel, Pema canaliculus contained brevetoxin B3 (226) [231]. A further brevetoxin analogue, brevetoxin B2 (227) was isolated from the hepatopancreas of P. canaliculus [232], while the major toxin in neurological shellfish poisoning (NSP) associated with P. canaliculus was identified as brevetoxin B4 (228) [233]. 

Additionally, a number of marine toxins with medical and toxicological importance have been isolated from marine flora and fauna. Okadaic acid, Fig. (62) is the main toxin produced by dinoflagellates, which can accumulate in the hepatopancreas of mussels and caused diarrhetic shellfish poisoning in consumers [505,506], However, this toxin is also a tumor promoter and a specific potent inhibitor of protein phosphatases which may provokes mitotic arrest and apoptosis of leukemia cells [507-509], These types of compounds have been reported in shellfish and phytoplankton, and more recently, in Spanish mussels [510], Portuguese bivalves [511], and the diatom Thalassiosira weissflogii [512],... 

Chou, T., Haino, T., Kuramoto, M., and Uemura, D., Isolation and structure of pinnatoxin D, a new shellfish poison from the Okinawan bivalve Pinna muricata, Tetrahedron Lett., 37, 4027, 1996. 

After the 1992-93 outbreak of neurotoxic shellfish poisoning (NSP) in New Zealand, several metabolites of brevetoxins were isolated from seafood . NSP is a term applied to an illness resulting from the ingestion of shellfish exposed to blooms of dinoflagellate K. brevis < 1965MI111, 1991MI471>. 

Morohashi, A., Satake, M., Naoki, H., Kasper, H.F., Oshima, Y, Yasumoto, T. 1999. Brevetoxin B4 isolated from greenshell mussels Pema canaliculus, the major toxin involved in neurotoxic shellfish poisoning in New Zealand. Natural Toxins 7, 45 8. 

Draisci, R., Giannetti, L., Lucentini, L., Marchiafava, C., James, K.J., Bishop, A.G., Healy, B.M., and Kelly, S.S. 1998. Isolation of a new okadaic acid analogue from phytoplankton implicated in diarrhetic shellfish poisoning. J. Chromatogr. A, 798, 137-145. 

In 1995, a report of human illness with diarrhetic shellfish poisoning (DSP)-like symptoms in the Netherlands was eventually found to result from the consumption of poisoned mussels (Mytilus edulis) harvested from Killary Harbour, Ireland (McMahon 1996). Yasumoto, Satake, and co-workers eventually isolated and proposed a stracture for the causative agent of this condition azaspiracid-1 (la. Fig. 16.1). The unique polyether stracture of azaspiracid-1 (la) is characterized by several spiro-cyclic systems, including an azaspiro ring fused to a 2,9-dioxabicyclo[3.3.1]nonane system and a terminal carboxylic acid. In total, there are nine rings and twenty stereogenic centers within the structure proposed by Yasumoto and co-workers in 1998 (Satake 1998). This stracture was based primarily on NMR spectroscopic data and did not include absolute stereochemistry, nor did it specify relative stereochemistry between the ABCDE and FGHI domains. 

The sponge Halichondria okadai contains a polyether metabohte named okadaic acid (16) that was also isolated as a causative agent of diarrhetic shellfish poisoning (DSP) from mussels and other bivalves (14). However, the real producers are dinoflagellates of the genus Dinophysis. It is a potent cancer promoter that was found to be caused by inhibition of protein phosphatases 1 and 2A at nanomolar levels. Pectenotoxins are also involved in DSP and are produced by Dinophysis spp. pectenotoxin 2 (17) inhibits actin polymerization (14). 

Azaspiracids [azaspiracid-1 (18)] are another class of highly unusual polyketide polyethers originally isolated from Irish mussels that caused azaspiracid shellfish poisoning (5). They are produced by the dinoflagellate Protoperidinium crassipes. A similar class of polyether toxins named pinnatoxins [pinnatoxin A (19)] were reported from the bivalve Pinna pectinata a closely related species P. attenuata is known to cause food poisoning in China. Pinnatoxins are likely of dinoflagellate origin and activate Ca channels (15). 

Okadaic acid 11 is a potent and specific inhibitor of protein phosphatases produced by the dinoflagellates Prorocentrum lima, Dinophysisfortii, and Dirwphysis accuminata. It accumulates in bivalves and is one of the main toxins responsible for diarrhetic shellfish poisoning (DSP) (11). It has a highly unusual biosynthesis that has generated a lot of speculation because of the presence of isolated acetate chain methyl carbon atoms (10). Okadaic acid is a potent inhibitor of protein phosphatase A (11). 

Diarrheic shellfish poisoning caused by okadaic acid is a gastrointestinal illness without chronic se-quellae. There is no evidence of neurotoxicity and no fatal cases have ever been reported. Diarrhea is the most commonly reported symptom, closely followed by nausea and vomiting with onset 30 min to 12 h from ingestion of contaminated shellfish. Complete clinical recovery is seen even in severe cases within 3 days. DSP has been reported predominantly in Japan and Europe. The toxin has also been isolated from P. lima cultures from the Gulf of California, Mexico. The incidence of DSP in this location as a result of this is unknown. 

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