Shellfish isolation

Polycychc aromatic hydrocarbons (PAHs) are carcinogens produced by the thermal breakdown of organic materials. These are widely distributed in both food and the environment, and are some of the principal carcinogens in cigarette tar and air pollution. Of over 20 PAHs isolated, benzopyrene and quinoline compounds are the most commonly encountered in foods, particularly those which are broiled or fried (111). Shellfish living in petroleum contaminated waters may also contain PAHs (112). 

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. 

Cyanobacterial neurotoxins are small ringed alkaloids and have dramatic effects on various components of vertebrate neurons. They are all water soluble and are synthesized by several cyanobacterial genera (Table 5.1). The most commonly isolated neurotoxins are the paralytic shellfish toxins, although several other potent neurotoxic alkaloids are synthesized by freshwater cyanobacteria (Table 5.1). 

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). 

Shellfish tissue (mussel) Extract with NaOH isolate fractions with column chromatography inject fractions to GC GC or GC/MS NR 34-120 (GC) 36-87 (GC/MS) Farrington et al. 1982a... 

Our method consisted principally of incubating sediment from shellfish beds and observing the growth of Protogonyaulax. In many cases we subsequently grew cultures of clonal isolates and demonstrated their toxicities. 

The transport of cysts via shellfish transplants or relays is even more difficult to evaluate. Not only is it possible that the sediment on the shells of seed shellfish contains cysts, but ingested cysts may even survive ingestion and germinate following defecation. Many cysts fed to soft-shell clams and mussels are viable following isolation from fecal pellets (35), but experiments have yet to be performed that mimic the conditions associated with prolonged residence in the intestines of shellfish during inter or intra-state transport. 

Despite this progress, the evidence linking cysts to shellfish toxicity remains circumstantial and care should be exercised before attributing toxin increases to this mechanism. The major problem is that it has yet to be demonstrated that shellfish can remove toxin from cysts. The feeding studies mentioned earlier (which do not yet include scallops 35) indicate that many viable G. tamarensis cysts can be isolated from the fecal pellets of mussels and soft-shelled clams fed cyst suspensions. There is certainly some cyst mortality as well (Figure 5), but whether this is also associated with toxin retention by the shellfish has yet to be demonstrated. It is reasonable to expect that the assimilation of toxin from cysts will not be a highly efficient process. 

Isolation of toxins. The digestive glands of shellfish were extracted with acetone at room temperature. After removal of the acetone by evaporation, the aqueous suspension was extracted with diethyl ether. The ether soluble residue was successively chromatographed twice over silicic acid columns with following solvents benzene to benzene-methanol (9 1), and diethyl ether to diethyl ether-methanol (1 1). To avoid degradation of dinophysistoxin-3 by contaminant acid, the silicic acid was washed with dilute sodium hydroxide solution and then with water prior to activation at 110 C. Toxic residue obtained in the second eluates was separated into two fractions... 

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. 

The major allergen of molluscan shellfish is tropomyosin, a muscle protein. The term major allergen is used to define proteins that elicit IgE binding in the sera of half or more of patienfs wifh allergies to the specific source (Metcalfe et ah, 1996). Tropomyosin is a ubiquitous muscle protein in all animals. Tropomyosin is a 34- to 36-kDa protein that is highly water soluble and heat stable as evidenced by the fact that tropomyosin can be isolated from fhe water used to boil shrimp (Daul et ah, 1994). Tropomyosin can actually be found in bofh muscle and many nonmuscle cells in animals. In muscle cells, tropomyosin is associated with the thin filaments in muscle and plays a role in the contractile activity of muscle cells. In nonmuscle cells, tropomyosin is found in microfilaments but its fimction is less well imderstood. Tropomyosins are present in all eukaryotic cells. Different isoforms of tropomyosin are found in different types of muscle cells (skeletal, cardiac, smooth), brain, fibroblasts, and other nonmuscle cells. While these tropomyosins are highly homologous, small differences do exist in their... 

In their telephone-based survey of individuals with seafood allergies, Sicherer et al. (2004) identified 67 individuals with self-reported allergy to molluscan shellfish. The inquiries were isolated to clam, scallop, oyster, and mussel which all belong in the bivalve class. Of these 67 individuals, 34 (51%) reported reactions to only 1 species, 13 (19%) to 2 species, 5 (8%) to 3 species, and 15 (22%) to all 4 species. Obviously, the interpretation of this observation is limited because diagnostic confirmation of survey... 

Lagos, N. Onodera, H. Zagatto, P.A. Andrinolo, D. Azeveth), S.M.F.Q. Oshima Y. (1999) The first evidaice of paralytic shellfish toxins in the fieshwata cyanobactaium Cylindrospermopsis raciborskii, isolated firnn Brazil. Toxicon, 37,1359-73. 

Battison, A. L., Summerfield, R., and Patrzykat, A. (2008). Isolation and characterization of two antimicrobial peptides from haemocytes of the American lobster Homarus americanus. Fish Shellfish Immunol. 25,181-187. 

Zhang, J., Yan, Q., Ji, R., Zou, W., and Guo, G. (2009). Isolation and characterization of a hepcidin peptide from the head kidney of large yellow croaker, Pseudosciaena crocea. Fish Shellfish Immunol. 26, 864-870. 

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],... 

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